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Silica based immunoassays for a covalently attached antigen Melzak, Kathryn 1993

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SILICA BASED IMMUNOASSAYSFOR A COVALENTLY ATTACHED ANTIGENByKATHRYN MELZAKB.Sc., University of British Columbia, 1986A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIESDEPARTMENT OF CHEMISTRYWe accept this thesis as conformingTHE UNIVERSITY OF BRITISH COLUMBIAAugust 1993© Kathryn MelzakIn presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of C The University of British ColumbiaVancouver, CanadaDate Cc^( 5 /DE-6 (2/88)AbstractAn immunoassay was developed using a monoclonal antibody and anantigen covalently immobilized on silica, showing that silica is apotentially useful assay substrate.Silica beads prepared from tetraethyl orthosilicate (0.78 pmdiameter) and fused quartz slides were thoroughly cleaned and modifiedwith 3-aminopropyltriethoxysilane. Amine groups on the modified slidesand beads were used for carbodiimide mediated coupling to carboxylgroups on a peptide antigen. High surface concentrations of antigen wereachieved.Antibody binding to the antigen-modified beads and slides wasmeasured using an iodinated monoclonal antibody. The equilibriumconstant for the antibody binding on the beads was ten times higher thanon the flat silica, but the maximum amount bound was lower.Immunoassays performed using a second enzyme-labelled antibodyto detect the monoclonal antibody bound to the silica permitteddetection of the antigen at 10-8 M using the modified beads and at10-7 M using the slides.1 1Table of ContentsPageAbstractTable of contentsList of tables^ viiiList of figures ixAbbreviations^ xiiiAcknowledgements xv1. Introduction^ 11.1 General Background^ 11.2 Development and applications of immunoassays: some historicalnotes^ 41.3 Immunogens, epitopes, haptens and antigens^ 71.3a Immunogens^ 71.3b Epitopes 81.3c Haptens (and carriers)^ 81.3d Antigens^ 91.3e Conjugating haptens to carrier molecules^ 91.4 Antibodies 101.4a Different classes of antibodies and their functions^ 101.4b Structure of Immunoglobulin G^ 131.4c Clonal selection and the mechanism of antibodyproduction^ 131.4d Monoclonal antibody production^ 171.5 Antigen-antibody interactions: chemistry and thermodynamics.. ^ 201.5a Binding of antigen to antibody^ 201.5b Equilibrium constants and affinities for monovalentantibodies and antigens interacting in solution^ 211.5c Equilibrium constant for a divalent antibody andmonovalent antigen in solution^ 221.5d Some comments on the antibody preparation used^ 231.5e Antibody binding to antigen on a solid surface:kinetics and equilibrium constants^ 231.5f Free antigen in solution inhibiting binding of antibodyto the surface-bound antigen: another interactioninvolved in the equilibrium^ 271.5g Experimental determination of equilibrium constants:information required and methods of calculation^ 281.6 Measuring the antibody-antigen interaction with ELISAs^ 301.6a Limitations of ELISAs^ 301.6b Calibration curves for ELISAs^ 321.6c Experimental requirements 32iii1.6d Covalent enzyme-linked immunoassays on polystyrenesusbstrates^ 341.6e Advantages of silica^ 341.7 Characterizing a modified surface^ 351.7a Information that will describe a substrate^ 351.7b Ninhydrin assays^ 371.7c X-ray photoelectron spectroscopy 371.7d Electrophoretic mobility of the beads 411.8 The silica surface^ 421.9 Characterizing the antigen-modified substrate^ 431.9a Measuring the surface concentration of antigen^ 431.9b Determining whether or not the antigen is attachedcovalently^ 431.10 Measuring antibody concentration on the surface and insolution^ 431.11 This project 442. Methods and materials: preparation and description of the antigen,antibody and modified substrate^ 462.1 Isolation of the antigen 462.2 Coupling antigen to KLH and BSA^ 522.2a Coupling procedure^ 522.2b Western Blot with ferrichrome A/KLH: an indication ofsuccess of the coupling procedure^ 522.3 Developing an ELISA prior to raising a monoclonal antibody... ^ 542.3a ELISA with serum from an immunized rabbit^ 542.3b An ELISA with blood from an immunized mouse 562.3c A second ELISA with blood from immunized mice^ 592.3d Comparing the efficiencies of skim milk and BSA asblocking reagents^ 632.3e The effect of Tween 80 on the ELISA^ 632.3f Absorbance of the substrate solution as a function ofHRP concentration, and evidence that a large excess ofHRP-conjugated antibody is used in the ELISAs 652.4 Production and purification of the monoclonal antibody, withSDS-PAGE to determine the antibody purity^ 692.4a Antibody production^ 692.4b Antibody purification 732.4c ELISA results with the monoclonal antibodies^ 752.4d SDS-PAGE procedure for the antibody AC3 752.4e SDS-PAGE results for AC3: an indication of the antibodypurity^ 782.5 Synthesis of silica beads^ 812.6 XPS measurements^ 87iv2.7 Cleaning the silica^ 882.8 Surface modification of the silica with silane^ 892.9 Ferrichrome A on beads: non-specific adsorption 902.9a Measurement of ferrichrome A adsorption^ 902.9b Effect of bead storage time on ferrichrome A adsorption ^ 912.9c Adsorption as a function of time^ 932.10 Reaction of ferrichrome A and EDC 932.10a Some possible reactions of ferrichrome A and EDC^ 932.10b Reactions of ferrichrome and EDC^ 952.10c The EDC:ferrichrome A mole ratio 952.10d Extinction coefficient for ferrichrome A and EDC^ 982.11 Modification of the silica beads with ferrichrome A and EDC ^ 982.11a The reaction procedure^ 982.11b Some comments on the procedure^ 992.11c Varying the pH of the reaction mixture^ 1002.12 The ELISA procedure^ 1002.12a The ELISA procedure for beads^ 1002.12b The ELISA procedure with flat modified silica^ 1012.12c The ELISA procedure for polystyrene plates 1022.12d ELISAs with plates: measuring the equilibriumconcentration of antibody in solution^ 1022.12e Inhibition of the ELISA with free ferrichrome A^ 1032.13 Characterization of the substrate: detection of the amines onmodified silica^ 1032.13a XPS measurements on flat silica^ 1032.13b Ninhydrin assay of amine groups on the silylated beads ^ 1032.14 Characterization of the antigen on the substrate^ 1042.14a Measuring the amount of ferrichrome A on the beads bysolution depletion^ 1042.14b BCA assay of the amount of ferrichrome A on the beads. ^ 1042.14c XPS measurements of ferrichrome A on the slides^ 1052.14d Ninhydrin assays of slides modified with acetic acidand EDC^ 1052.15 Antibody adsorption isotherms on the modified silica^ 1062.15a Labelling the antibody with 1251^ 1062.15b SDS-PAGE of the labelled antibody 1072.15c Comparing the antigen binding activity of the labelledand unlabelled antibody^ 1072.15d Measuring the antibody adsorption isotherms^ 1082.15e Inhibition of antibody binding with free antigen^ 1102.15f Calculation of the antibody-antigen affinity^ 1112.15g SEM studies with a gold-labeled secondary antibody onflat silica^ 1112.16 Aggregation of the silica beads^ 1122.17 Particle electrophoresis^ 1123. Results and Discussion^ 1143.1 Silica surface 1143.1a Cleaning the silica^ 1143.1b Differences between flat silica and beads^ 1173.1c Electrophoretic measurements on the beads 1183.2 Silylated silica^ 1183.2a Density of amine groups on the surface^ 1183.2b Charge density on the surface and surface pH:effect on EDC coupling^ 1183.2c Thickness of the amine layer^ 1213.3 Silica with ferrichrome A^ 1233.3a Some comments on ferrichrome A^ 1233.3b Effect of changing the beads area on ferrichrome Aadsorption^ 1263.3c The adsorption isotherm: ferrichrome A adsorption as afunction of solution concentration^ 1263.3d Effect of salt concentration on ferrichrome Aadsorption^ 1303.3e Reaction of ferrichrome A with EDC^ 1303.3f The amount of ferrichrome A on the beads at a constantEDC:ferrichrome A ratio^ 1333.3g The amount of ferrichrome A on the beads at a constantEDC concentration^ 1383.2h BCA assays of the amount of ferrichrome A on the beads ^ 1383.21 Ninhydrin assays of silica modified with acetic acidand EDC^ 1413.2j XPS measurements of ferrichrome A on the silica^ 1413.4 Antibody binding to the ferrichrome A-modified silica^ 1433.4a The radiolabeled antibody^ 1433.4b Non-specific adsorption 1433.4c Antibody binding to ferrichrome A on flat silica andbeads^ 1493.4d Inhibition of antibody binding with free ferrichrome A:a measurement of the solution equilibrium constant^ 1603.5 ELISAs using the beads and flat silica^ 1613.5a Reproducibility of ELISAs on the beads^ 1613.5b ELISAs on the beads: varying the surface concentrationof antigen^ 1643.5c ELISAs on the beads: inhibition of the antibody bindingwith free antigen^ 1663.5d Some comments on ELISAs on beads^ 1663.5e ELISAs on flat silica 1693.5f Quantitative aspects of ELISAs and comparisonsof ELISAs with adsorption isotherms^ 1734. Concluding discussion^ 179References^ 182Appendix 1.^Analysis of competitive inhibition of monoclonalantibody binding to surface-attached antigen^ 193Appendix 2. Estimating molecular weights from the SDS-PAGE^ 198viAppendix 3. Sample UV/VIS absorbance spectra and derivatives dA/dAfor ferrichrome A solutions and beads suspensions^ 199Appendix 4. Additional antibody binding isotherms and other data...200viiList of TablesTable^ PageI. Preparation of growth medium for Ustilago sphaerogena^ 47II. Preparation of substrate solution for ELISAs^ 57III. Preparation of Dulbecco's modified Eagle medium 70IV. Supplements for serum-free medium^ 74V. Preparation of gels and reagents for SDS-PAGE^ 79VI. Effect of bead storage time on ferrichrome A adsorption^ 92VII. Adsorption as a function of time^ 92VIII. XPS measurements on clean silica and glass slides: comparingthe efficiencies of the different cleaning procedures^ 115IX. XPS measurement of modified silica slides^ 125X. Ferrichrome A adsorption to different areas of beads^ 125XI.^Equilibrium constants for antibody binding to ferrichrome Aon the beads and flat silica^ 157List of FiguresFigure^ Page1. ELISA: indirect method for detecting antibodies^ 32. ELISA: competitive method for detecting antigen 53. The different classes of immunoglobulins^ 114. Shape of the IgG molecule^ 145. Structure of the IgG molecule 146. Antibody production in vivo^ 157. Adsorption isotherms: variation with binding affinity and numberof binding sites^ 298. Scatchard plots: variation with binding affinity and number ofbinding sites 299. Deposition of silanes on silica^ 3610. Depth profiles by XPS measurements 4011. Structure and conformation of Ferrichrome A^ 4712. The effect of additional light scattering on the derivative ofthe absorbance curve^ 4913. Determining the concentration of ferrichrome A from thederivative of the absorbance spectrum^ 5014. Western blot showing that the antigen is coupled to KLH^ 5115. ELISA procedure for microwell plates^ 5516. ELISA with ferrichrome A adsorbed to plates and rabbit serum asthe antibody^ 5817. ELISA with ferrichrome A adsorbed to plates: comparing theresults for antibodies in mouse serum and rabbit serum^ 6018. ELISA with ferrichrome A./BSA adsorbed to plates and mouse serumas the antibody^ 6119. ELISA with ferrichrome A/BSA adsorbed to plates: trying toinhibit binding of antibodies in mouse serum with freeferrichrome A^ 6220. ELISA comparing the efficiency of BSA and skim milk as blockingreagents^ 6421. The effect of Tween 80 on the ELISA^ 6622. Absorbance of the ELISA as a function of low HRP concentrations ^ 67ix23. Absorbance of the ELISA as a function of higher HRPconcentrations^ 6824. ELISA with purified monoclonal antibodies and ferrichrome A/BSAadsorbed to the plates^ 7625. ELISA with monoclonal antibodies and ferrichrome A/BSA adsorbedto the plates: inhibiting the antibody binding with freeferrichrome A^ 7726. SDS-PAGE of human plasma proteins as molecular weight standards.8027. SDS-PAGE of the monoclonal antibody AC3^ 8228. SDS-PAGE of the reduced antibody^ 8329. TEM photograph of silica beads 8530. Silica bead size distribution^ 8631. Carbodiimide coupling mechanism 9432. Structure of ferrichrome^ 9633. Choosing the ferrichrome A: EDC mole ratio^ 9734. Determining the concentration of ferrichrome A with EDC from thederivative of the absorbance spectrum^ 9735. XPS spectrum of silica cleaned with hot chromic acid, rinsedwith hydrochloric acid and water, and dried under vacuum^ 11536. Angularly resolved XPS measurements on modified silica: the S12ppeak^ 12237. Crystallographic structure of ferrichrome A, showing the facewith the three carboxyls^ 12438. Adsorption Isotherm for ferrichrome A on silylated beads atdilute solution concentrations of ferrichrome A^ 12739. Adsorption isotherm for ferrichrome A on silylated beads^ 12840. Scatchard plot corresponding to adsorption data shown inFig. 38^ 12941. The effect of NaC1 on ferrichrome A adsorption to silylatedbeads^ 13142. Absorbance (UV-Vis) of ferrichrome A mixed at different ratioswith EDC^ 13243. Coupling ferrichrome A to silylated beads at a constant EDC:ferrichrome A mole ratio^ 13444. Electrophoretic mobility of silylated beads with ferrichrome Aattached covalently at a constant EDC:ferrichrome A mole ratio. .13745. Coupling ferrichrome A to silylated beads at a constant EDCconcentration of 20 mM^ 13946. BCA assay of ferrichrome A coupled to silyiated beads^ 14047. An XPS spectrum: the Fe 2p peak of ferrichrome A on silica^ 14248a.ELISA comparing the activity of the labelled and unlabeledantibody^ 14448b.ELISA comparing the activity of the labelled and unlabeledantibody using different commercial assay plate^ 14549. Non-specific adsorption: antibody in PBS^ 14650. Non-specific adsorption: antibody in PBS-Tween^ 14751. Non-specific adsorption: antibody in 2% BSA 14852a.Antibody adsorption to silica beads with a range of surfaceconcentrations of ferrichrome A: bound antibody determined fromamount remaining after washing^ 15152b.Scatchard plot for the data shown in figure 52a^ 15253a.Antibody adsorption to flat silica, modified with differentsolution concentrations of ferrichrome A: bound antibodydetermined from amount remaining after washing^ 15353b.Scatchard plot for the data shown in figure 53a 15454. Scatchard plot for a system with two different bindingaffinities^ 15555. SEM photograph showing the distribution of a gold-labeledsecondary antibody bound to a primary antibody on ferrichrome Amodified silica^ 15956. Antibody binding on flat silica: inhibition by freeferrichrome A^ 16257. Reproducibility of ELISAs on ferrichrome A-modified beads^ 16358. ELISAs on silica beads modified with a range of surfaceconcentrations of ferrichrome A^ 16559. ELISA results for silica beads plotted as a function of surfaceconcentration of antigen^ 16760. ELISA on beads: inhibition of antibody binding with freeferrichrome A^ 16861. ELISAs on flat silica modified using a range of solutionconcentrations of ferrichrome A^ 170xi62. ELISA results for flat silica plotted as a function of surfaceconcentration of ferrichrome A on beasds modified withequivalent solution concentrations^ 17163. ELISA on flat silica: inhibition of antibody binding with freeferrichrome A^ 17264. Absorbance in the ELISA as a function of surfaceconcentration of antibody (from an assay using flat silica) ^ 17465. Absorbance in the ELISA as a function of surfaceconcentration of antibody (from an assay using a commercialassay plate)^ 17566. Determining molecular weights from the SDS-PAGE^ 19867. Sample spectra showing absorbance (or optical density)of a ferrichrome A solution, a suspension of beads in waterand a suspension of beads in a ferrichrome A solution^ 19968. The derivatives dA/dA for the sample spectra shown in Fig. 67...19969. Antibody binding to silica beads with a range of differentsurface concentrations of ferrichrome A: bound antibodydetermined from solution depletion measurements (from the sameexperiment as Fig. 52)^ 20070. Antibody binding to flat silica modified with differentsolution concentrations of ferrichrome A: bound antibodydetermined from solution depletion measurements (from the sameexperiment as Fig. 53)^ 20171. Antibody binding to silica beads with a range of differentsurface concentrations of ferrichrome A, at low solutionconcentrations of antibody: bound antibody determined fromamount remaining after washing^ 20272. The amount of bound antibody calculated by solution depletionmeasurements for the experiment described in Fig. 71^ 20373. Absorbance spectra of ferrichrome and ferrichrome with EDC^ 204xi iAbbreviationsAb^ antibodyAC3^ the name of the hybridoma cell line used to produce theantibody used in most of the experiments, and the name of theantibody produced by that cell line.Ag^ antigenBALB/c^ an inbred strain of miceBCA^ bicinchoninic acidBSA^ bovine serum albumin•Csat ^ a proposed minimum silicic acid concentration required toinitiate nucleation.CD-1^ a strain of mice^ sampling depth. In XPS measurements, this denotes the depthof the region from which the measured photoelectronsoriginate.DME^ Dulbecco's modified Eagle mediumDMSO^ dimethyl sulfoxideDNA^ deoxyribonucleic acidEb^ binding energyEDC^ 1-ethyl-3-(3-dimethylaminopropyl carbodiimide)EDTA^ehylene diamine tetraacetic acidEhv^ photon energyEkE^ electron kinetic energyELISA^ enzyme-linked immunosorbent assayFab^ the antigen binding fragment of immunoglobulins (see Fig. 3)FBS^ fetal bovine serumFc^ the crystallizable fragment of immunoglobulinsFOX-NY^ a myeloma cell line derived from BALB/c miceAG°^ the change in the standard state free energyHAT^ hypoxanthine, adenine and thymidineHRP^ horseradish peroxidaseHT^ hypoxanthine and thymidineIg^ immunoglobulin. Different classes are designated as IgA, IgD,IgE, IgG and IgM.^ equilibrium constantka^ association constantkd^ dissociation constantKLH^ keyhole limpet haemocyaninPBS^ phosphate buffered salinePBS-Tweem^ phosphate buffered saline with 0.5% Tween 80PEG^ polyethylene glycol^ the gas constantSDS^ sodium dodecyl sulfateSDS-PAGE^ sodium dodecyl sulfate polacrylamide gel electrophoresisSEM^ scanning electron microscopyTEM^ transmission electron microscopyTEOS^ tetraethyl orthosilicatetic^ thin layer chromatographyTMB^ tetramethyl benzidineTris^ tris(hydroxymethyl)aminomethaneTween 80^ polyoxyethylene sorbitan monooleateXPS^ X-ray photoelectron spectroscopya^ used to denote specificity of antibody, eg goat a-rabbit isgoat antibody against rabbit.0^ angle away from normal at which measurements are made for XPS^ surface charge densityxivAcknowledgementsI would like to thank all the people who have offered me advice andgood cheer while I have been working on this project, including:-Don Brooks, who always listened if things were going badly, and whocould usually point out that things weren't that bad after all,-my parents, who always had faith in me-Adam, who may consider that AC3 stands for Adam's company (cubed)-the meanest mouse of all, who made it all possible,-and all the people in the Brooks lab, who have made the daysbrighter and easier and altogether more cheerful.Chapter 1Introduction1.1 General backgroundImmunoassays use the sensitive and specific antibody-antigeninteraction to detect either antibodies or antigens. Detection ofantibodies is used clinically to diagnose disease, and detection ofantigens is used for a variety of clinical, agricultural and researchpurposes (1-3).Antibodies are produced in vivo as part of the immune response tomost foreign substances and will bind specifically to the antigenstimulating the response. The antibody and antigen associate reversiblyto form a complex, with an equilibrium constant K defined as shown below(4).[1] K - [Ab-Ag][Ab][Ag]where [Ab-Ag] = concentration of theantibody-antigen complex[Ab]= concentration ofunoccupied bindingsites on the antibody[Ag]= concentration of unboundantigenMeasured equilibrium constants range from less than 105 litre mo1-1 forlow affinity systems with weak binding to greater than 1012 litre mo1-1for high affinity systems (5,6). High affinity systems can be used todetect antigens at concentrations as low as pmo1/1 (7).There are many different sorts of immunoassay designs, with somecommon features that can be used for classification. Assays measureanalyte bound in an antibody-antigen complex, and must therefore be able1to differentiate between the bound and unbound analyte. One of the waysto classify assays is by the method of differentiation used: inhomogeneous assays, there is a detectable change in a reagent when theantibody antigen complex is formed (8, 9), and in the more commonheterogeneous assays (10), the bound and unbound reagent are separatedbefore quantitation. A simple way to effect the separation is to attacheither the antigen or antibody to a solid phase, so that unbound reagentcan be washed away.Assays can also be classified by the different methods used todetect the reagents. Radioactive (11, 12), enzyme (13, 14, 15),particulate (16), and fluorescent (17) labels can be detected at thelow concentrations and small volumes required in a sensitive andconvenient assay. The labels can be attached directly to the antibody orantigen being measured, or can be attached to secondary reagents.Enzyme-linked immunosorbent assays (ELISAs, 13) are a common form ofimmunoassay that use an enzyme label to determine antibodyconcentration. The antibody-antigen interaction takes place at asolid-liquid interface with one of the reagents attached to the solidsubstrate; unbound reagent left in solution can be washed away. One ofthe simplest formats of ELISAs is shown in Fig. 1. The assay shown hasan enzyme linked to a secondary antibody; this antibody binds to theprimary antibody being measured (the primary antibody is a target of thesecondary antibody).Commonly used enzyme substrates have coloured products that can bedetected spectrophotometrically. Under appropriate conditions, theIntensity of the colour in the developed assay is proportional to theamount of enzyme present, which is, in turn, related to the amount ofantibody being measured. Results from an assay such as the one described2The antigen is immobilized on a solidsupport. Commercially availableassay plates contain different wellsthat are used as separate incubationchambers.An antibody solution is added to theantigen-coated substrate. Antibodiestowards the antigen on the substratewill reach an equilibrium with a portionof the antibody on the plate and aportion in solution. Differentconcentrations of antibodies can beadded to the different wells.The unbound antibody can be washed away.The amount of antibody left on the substratewill depend on the amount of immobilizedantigen, the antigen-antibody affinity and theamount of antibody in solution.A second, enzyme-labelled antibody isincubated with the assay plates. Thesecondary antibody will bind generallyto antibodies from a particular species.Unbound enzyme-labelled antibody is washedaway. If there is an excess of the secondaryantibody, then the amount bound will not dependon the solution concentration of enzyme-labelled antibody, but will, instead, reflectthe amount of the primary antibody bound.The enzyme substrate is added in the finalassay step to determine the amount of antibodybound. Enzyme that is associated withthe surface will catalyze conversion of thesubstrate to a coloured or otherwise readilydetectable reaction product.Figure 1: An indirect ELISA for detecting antibodies.in Fig. 1 are usually presented as absorbance against the initialsolution antibody concentration applied to the assay plate. Antibodyconcentration in an unknown sample can be determined using a suitablestandard curve.Antigen concentration can be determined using a displacement assaysimilar to the assay described above (Fig. 2). A known concentration ofantibody is incubated with a sample containing an unknown concentrationof antigen prior to being added to the assay plate. If there is freeantigen present in the sample, it will compete with the antigen adsorbedto the plate for binding sites on the antibody. More antigen in solutionwill result in less antibody being bound to the plate, and in lessintense colour after the substrate is added.1.2 Development and applications of immunoassays: some historical notesApplications of early immunoassays were limited due to their lack ofsensitivity. The antigen-antibody binding could be followed byappearance of visible aggregates formed when divalent antibodiesinteracted with multivalent antigens (18), but this required largeamounts of antibody and antigen (19). Red blood cells were attached tothe antibody to make the aggregates more visible (and to decrease theamount of antibody required to form a visible aggregate (20)), but thisgave unreliable results (21). Assays measuring formation of visibleprecipitates (turbidimetric assays) are still used to measure serumproteins, where larger volumes of relatively concentrated samples areavailable (22).Immunoassays developed during the 1950s greatly increased the rangeof antigen concentration that could be measured. Latex agglutinationassays were first described in 1954 by Singer and Plotz (23) as a method4Free antigen and antibodyare incubated together. Theantibody concentration ischosen to obtain a clearresponse when there is noantigen in solution.Antigen is immobilizedon the substrate as forFig. 1The antibody-antigen solution is added to theassay plate. The antigen in solution willcompete with the antigen on the substrate forbinding sites on the antibody.Antibody not bound to the plate is washedaway. Antibody that has bound to antigen insolution will be washed away, leaving lessantibody bound to the substrate.Enzyme-labeled antibody is added and thenwashed away.The enzyme substrate is added and theresulting colour change is detectedspectrophotometrically.Figure 2: A competitive ELISA for detecting antigen5for diagnosing rheumatoid arthritis. Latex particles (0.77 pm diameter)were coated with antibody and mixed with serum from patients. If thepatients had rheumatoid arthritis, antigens present in their serumcaused the particles to aggregate. Latex agglutination assays were moreconsistent and reliable than the assays involving red blood cells, andare still widely used. One of the common applications is to test forhuman chorionic gonadotrophin as a means for diagnosing pregnancy (24).Radioimmunoassays were developed in 1959 by Berson and Yalow as aquantitative measurement for insulin in human plasma (12). Insulinlabelled with 131 I was incubated with antibody, after which bound andfree insulin were separated electrophoretically and quantitated using theiodine label. Insulin in human plasma was determined by addition of aplasma sample to the labelled insulin; insulin in the plasma competed forantigen binding sites on a limited amount of antibody, and caused adecrease in the amount of bound radiolabelled insulin measured.Radioimmunoassays are sensitive (insulin can be detected at thenormal plasma concentration of about 0.1 nmo1/1), specific (due tothe specific nature of the antigen-antibody interaction) andconceptually simple. The method is also widely applicable, since it canbe used to measure any molecule against which an antibody can be raised.Radioimmunoassays have been used to measure hormones (25, 26), drugs anddrug metabolites (27,28), serum proteins (29) and viruses (30, 31), andwere at one time a commonly used clinical assay (32). The popularity ofradioimmunoassays has declined due to problems associated withradioisotopes.Enzymes linked to antibodies gave a sensitive detection method thatdid not require the use of radioisotopes and expensive countingequipment (13, 32). Enzyme linked antibodies have been used for several6different assays (15, 33); one of the most common of these is the ELISAthat was developed and named by Engvall and Perlmann in 1971 (13).Several factors have contributed to the popularity of ELISAs: reagentsare stable, non-hazardous and readily available, commercial assay platesused as the substrate typically have 96 wells and can be used forprocessing multiple samples, and the assay results after the final stepin the procedure (see Fig. 1) can be determined using aspectrophotometer that will read all the wells in an assay platesimultaneously.Clinical uses of ELISAs include detection of viruses (33-35),bacteria (36), antibodies (37, 38) and plasma protein levels. The assaysare also popular in research laboratories: the Medline computer database of biological journals lists more than 13 000 papers that havedescribed developments or applications of ELISAs.Immunoassays today are used as a sensitive and specific means todetect antibodies and any molecule that can be used to raise anantibody. Since there is a wide range of molecules that can be used forraising antibodies (39), there is an equally wide range of immunoassayapplications: assays have been used to detect drugs and drug metabolites(40), antibiotics (41), food additives (42) and toxins (43), as well asmetabolites, infectious agents and antibodies measured for clinicaldiagnoses.1.3 Immunogens, epitopes, haptens and antigens1.3a ImmunogensImmunogens are agents that will induce an immunological response invivo (44). Whether or not a chemical structure will be immunogenic isdetermined by the mechanism of the response (section 1.5c): immunogens7must have a binding site for which an antibody is available and musthave a second region to promote the cellular interactions that comprisepart of the immune response. Immunogens have several features in common:they are perceived as foreign to the individual producing the response,are chemically complex, and have a high molecular weight (45).Healthy individuals do not respond immunologically to their owntissues. This is because the cells that would produce such a responseare removed or inactivated early in the individual's life (46).The size requirements of immunogens derive from the need for anantibody binding site and for a second region to promote cellularinteraction (47). Compounds with a molecular weight of less than3 000 g/mol are generally not immunogenic (48).Immunogenic compounds must also be chemically complex. Highmolecular weight homopolymers of amino acids will not be immunogenicunless they are chemically modified so that they are no longer aregularly repeating unit.1.3b EpitopesAlthough molecules inducing an immunological response are relativelylarge, an antibody only binds to a small region of the immunogen calledthe epitope. Epitopes have been shown to have a size of approximately7x12x35 A (49). One immunogen can have several different epitopes.1.3c Haptens (and carriers)Molecules that do not have a region that promotes the cellularinteraction necessary for the immune response are not immunogenic bythemselves but can be conjugated to carrier molecules to produce acomplex that will be immunogenic. An immunological response induced8against the conjugated complex can result in antibodies that will bindto the first molecule in the absence of the carrier. Non-immunogenicmolecules treated this way to raise antibodies are called haptens.Regions identified as possible promoters of the cellular interactionin mice are peptides with a molecular weight of 1000 to 2000 g/mol (50).A synthetic peptide with the appropriate sequence could be used byitself as a carrier, but might not be effective in the strain andspecies of animal used (51). Larger proteins are generally used ascarriers to ensure a good response.1.3d AntigensAn antigen is the material to which an antibody binds. Antigens canbe whole cells, large molecules or haptens, and can contain one or moreepitopes; antigens with several epitopes can interact with severaldifferent antibodies. Since the term "antigen" refers specifically tothe reagent that interacts with an antibody, it is usually used indiscussions of antibody binding.1.3e Conjugating haptens to carrier moleculesThe two most commonly used protein carriers are keyhole limpethaemocyanin (KLH) and bovine serum albumin (BSA) (52). Some of theresidues of the twenty different amino acids commonly found in proteinshave functional groups that can be conjugated to haptens. Useful groupsinclude amino groups (on lysine residues and the amino terminus),carboxyl groups (on glutamic and aspartic acid residues and the carboxylterminus), sulfhydryl groups (on cysteine residues) and phenolichydroxyls (on tyrosine residues).The conjugation method used depends on the groups available on the9hapten. The hapten functional group used should be chosen so thatepitopes of interest remain unmodified.Water soluble carbodiimides can be used to couple carboxyl or aminogroups on the hapten directly to the carrier, forming a peptide bond((53), section 2.10a). Various small bifunctional cross linkers can alsobe used to conjugate different pairs of the groups listed above (54).1.4 Antibodies1.4a Different classes of antibodies and their functionsAntibodies are proteins found in the blood and in varioussecretions. They are produced in response to immunogens and will bindspecifically to different epitopes on the immunogen surface. By bindingto the foreign particles or molecules, antibodies can perform a varietyof functions: they can, among other things, neutralize toxins,agglutinate microorganisms, precipitate soluble antigens, neutralizeviruses and make foreign cells targets for killer cells produced by theimmune system. In humans, there are five different classes ofantibodies, which are also known as immunoglobulins (Fig. 3). Thedifferent classes have a similarly shaped subunit, a Y-shaped tetramericprotein with two identical heavy polypeptide chains and two identicallighter chains (Fig. 3). The subunits are symmetric, with identicalbinding sites on each arm of the Y.Immunoglobulin G (IgG) is found in the blood, where it constitutesabout 15% of the total serum protein (55), and in milk. It can alsocross the placental barrier to confer immunity on the fetus. Since IgGis divalent, it can interact with multivalent antigens to form aprecipitate, which is engulfed and removed by phagocytic cells in theblood. The IgG molecule can also bind to foreign cells with the Fab10heavy chain(in secretions)..,........secretorycomponent\/ Fab portion1 Fc portion^IgGadditional peptideIgMYn serum) IgAYFigure 3: The different classes of immunoglobulins11portion (Fig. 3), and then with the Fc portion (Fig. 3) to naturalkiller cells found in the blood; the killer cells then releasesubstances that destroy the foreign cell. Immunoglobulin G bound toforeign cells can also activate the complement system resulting in celllysis, and can cause immobilization of motile microorganisms by clumpingflagella or cilia. Viruses can be inactivated by IgG bound to the regionrequired for attachment to the target cell. Toxins can also beneutralized by bound IgG.Immunoglobulin G is produced after the second and subsequentexposures to an immunogen. Elevated levels of IgG indicate presence ofan antigen, and many clinical assays test for an increase in serumconcentrations of a particular specificity of IgG in order to make adiagnosis (56, 57). Assays testing for the presence of antigencommonly use IgG because it can be isolated readily from the serum ofimmunized animals.There are subclasses of IgG with different conserved amino acidsequences on the heavy polypeptide chain. Each subclass can also havedifferent classes of light chains (58).Immunoglobulin M (IgM) is produced after the first exposure to anantigen. Elevated IgM levels can indicate recent infection or ongoingexposure to an antigen that does not cause production of IgG (59, 60).Immunoglobulin M can agglutinate antigens and can activate thecomplement pathway resulting in lysis of foreign cells.Immunoglobulin A is present in serum, where it may have severalpoorly understood functions, and in secretions such as tears andmucus, where it keeps organisms from attaching to and infecting theepithelial surface (61).Immunoglobulin D is present on the surface of cells called B12lymphocytes, and may be involved in cellular development (62).Immunoglobulin E is present on the surface of some cells circulatingIn the blood, and in low concentrations in the serum. Allergic reactionsare mediated by IgE.1.4b Structure of Immunoglobulin GImmunoglobulin G is a Y shaped glycoprotein (Fig. 4) with (whenproduced in vivo) two identical light polypeptide chains that arecombined with two identical heavy chains to form a tetramer having atotal molecular weight of about 160 000 g/mol. The light and heavychains are held together by a combination of disulfide and non-covalentbonds, in an arrangement shown in Fig. 5. Regions of the polypeptidechains with conserved amino acid sequences form globular domains (Figs.4 and 5) and give IgG its characteristic structure. Proline residues inthe hinge region disrupt the folding of the polypeptide chain, givingthe IgG molecule flexibility and permitting the Fab region (the arms ofthe Y) to open and close. The amino terminal of the light and heavychains (at the ends of the arms) contains a variable region with anamino acid sequence that is not as strongly conserved as the constantdomains. Hypervariable regions within this amino terminal domain combineto form the antigen binding site, conferring the antibody specificity.The folding of the polypeptide chains and the overall molecularshape of IgG (Fig. 4) have been determined by X-ray crystallography (63,64).1.4c Clonal selection and the mechanism of antibody productionAntibodies are produced by the combined actions of several differentsorts of cells, following a complex procedure which is briefly13hinge regionconstant ------4 carbohydratesdomainsvariable domainslight chain^ light chainheavy chain --4^J^4.--heavy chain40AFigure 4: Shape of the IgG molecule (63)Figure 5: Structure of the IgG molecule14seicrclicrivsurface boundencauthersani-iclen +ha+binctl6 toanti bold jsound an+icien isinternal imeararx:1broken downcirculatingAntigen-presdrikinacell cApcyencoun+er5anilsenan4-i gen fevirnentsMove Ji-o Surracebound 40 class ILpro-fenscorvIpler of clgas Itprol. tins and anki.3enon ourfaceoc+i va4 eciT- cell binds4o aniqaenfragrneArl- onB-cellperT-cellencountersgrrligen onC10.3.5 ertfteinan Aftsecrei-eanti bod5asmaaci-ivoired?cellhelper- T-cetlwith T-cellrec.eplor onstir-RICEmemory cellsStACCice.can be si-invAlgFurtKer encounterswi+ h an+19enFigure 6: Antibody production in vivo15summarized here and in Fig. 6. Two different sorts of cells, helper Tlymphocytes and B lymphocytes, proliferate after stimulation by anappropriate antigen and then bind to each other, with subsequentdifferentiation of the B cells into antibody producing cells. The Bcells have modified antibodies on the cell surface, with the sameantigen binding specificity as the antibody that will be produced by thedifferentiated cells. Receptors on the T cells will also recognize andbind to a specific antigen. B cells with different surface receptors andthe potential to make different antibodies are present in the spleen andother secondary lymphoid organs before contact with an immunogen, butonly cells having a receptor to which an antigen binds are stimulated toproliferate and produce antibody. A large repertoire of differentantigen binding sites is created by rearrangements of the DNA that codesfor the antibodies. Variable regions of the DNA are combined randomly toproduce the mature DNA coding for the light or heavy chains of theantibody. Light and heavy chains can also combine randomly; in mice,there are about 107 different potential antigen binding sites that canbe generated this way (65). After binding to the receptors on the Bcells, the antigen is degraded by the cell and antigen fragments arepresented on the cell surface bound to receptors called class IIproteins (these are glycoprotein dimers encoded by the genes of themajor histocompatibility complex and involved in interactions of some Tcells).Helper T cells are stimulated to proliferate by antigen fragmentsbound to class II proteins on the surface of a third group of cellscalled antigen presenting cells, which process antigen non-specifically.Receptors on the T cells recognize specific antigen-class II proteincomplexes, with the additional requirement that the antigen fragment16must contain one of a group of conserved amino acid sequences. Thestimulated T cells will also bind to the antigen-class II proteincomplex on the surface of the B cells. Contact between the B cells andT cells causes the B cells to proliferate and differentiate into plasmacells and memory cells.Plasma cells produce IgM, and cause a short lived primary responseto an antigen starting about seven to ten days after immunization. Ifthe antigen is cleared out of the body by the IgM, the plasma cells die,leaving the memory B cells and helper T cells. Second and subsequentexposures to an antigen result in memory B cells interacting with the Tcells to produce more plasma cells. Plasma cells involved in thesecondary response produce predominantly IgG with a high affinity forthe antigen. The change from IgM to IgG is effected by a rearrangementof the DNA that switches the constant region of the heavy chain thatdetermines the subclass, but leaves intact the variable region thatdetermines the specificity.Assays for IgM are used to detect the primary response againstinfection or response to antigens that do not cause class switching fromIgM to IgG (59, 60), and assays for IgG are used to detect the secondaryresponse (56, 57).1.4d Monoclonal antibody productionAntibodies isolated from the serum of immunized animals have amixture of different antigen-binding specificities. This can presentproblems in some immunoassays, since the antibody preparation used willcontain an unknown proportion of antibody binding to the antigen beingstudied, and antibodies that do bind to the antigen in question willbind to different epitopes with a range of different affinities.A homogeneous antibody preparation can be obtained by isolating17antibodies from one B cell, since each cell is only capable of producingantibody with one antigen-binding specificity. Although B cells will notgrow in culture by themselves, the terminally differentiated plasmacells that produce antibodies can be fused with myeloma cells that growand divide in culture to produce a hybridoma cell that will proliferatein culture and will continue to produce antibodies.Immunoglobulin G producing plasma cells from the secondary responsecan be obtained from the spleen of an immunized animal. After an initialinjection of immunogen, different epitopes on the antigen bind to Bcells with appropriate receptors, stimulating the B cells todifferentiate into plasma cells and memory cells. After the immunogen iscleared from the system and the IgM producing plasma cells involved inthe primary response die off, the animal can be given another injectionof immunogen. The second injection causes differentiation of the memorycells into IgG producing plasma cells which can then be fused with theimmortal myeloma cells.Bone marrow tumours called myelomas can be induced in the inbredstrain of BALB/c mice by mineral oil injected into the peritoneum. Celllines derived from BALB/c myelomas and adapted for continuous cultureare available commercially, and have become the most widely used myelomacells for monoclonal antibody production. Most of the BALB/c myelomacell lines, including the FOX-NY cell line that was used in theseexperiments, produce light chains that are not secreted by themselvesbut that are incorporated into the antibody produced by the spleencell-myeloma hybrid (66). If the light chains originating from the spleencell and the myeloma cell are incorporated randomly into the antibody,25 % of the molecules will have two light chains from the myeloma and noantigen binding site, 50 % will have one binding site, and 25 % will18have two binding sites. A myeloma cell line that does not produce lightchains has been isolated (67), but is not available from the AmericanType Culture Collection (68), which is the non-profit agency used as themost common source of cell lines in North America.Commonly used cell lines have a mutation in the gene coding for oneof the enzymes involved in a salvage pathway for production of purinebases for nucleic acids. Addition of toxic compounds such as aminopterinthat block the de novo synthesis pathway will kill the cells, since theywill not be able to use the salvage pathway for conversion of othernucleotide bases to the required product. Unfused myeloma cellsremaining after the fusion of the myeloma cells with the spleen cellscan be rapidly killed by addition of aminopterin to cell culture medium.Polyethylene glycol (PEG) is the most common cell fusing agent usedin monoclonal antibody production (69). Addition of sufficiently highconcentrations of PEG will result in fusion of the plasma cell membranesof adjacent cells and formation of hybrid cells with two or more nuclei.The first division of the hybrids gives cells with one nucleus each. Thecells initially have a large number of extra chromosomes, some of whichare lost during the early cell divisions before a stable cell line isobtained.Unfused spleen cells will not grow in culture and unfused myelomacells can be killed off by addition of aminopterin, leaving only thespleen cell-myeloma hybrids. The cell culture medium is screened forsecreted antibodies, and cells producing antibody are kept and allowedto proliferate. Positive cell lines are subcloned to ensure that thecell line originates from only one cell and thus produces only oneantibody. To subclone, the cells are diluted and added to a 96 wellplate at a calculated concentration of about one cell per well. Wells19with growing cells are inspected visually to check that the cells growfrom one colony. Cells are sometimes subcloned two or three times toensure stability and purity of the cell line.A homogeneous preparation of antibody with a unique specificity canbe isolated from the hybridoma supernatant. The hybridomas can also befrozen and stored and then thawed later to provide a constant source ofantibody.1.5 Antigen-antibody interactions: chemistry and thermodynamics1.5a Binding of antigen to antibodyThe antigen binding sites are located at the ends of the arms of theIgG molecules, in shallow pockets formed by the variable regions of thelight and heavy chains. Three polypeptide loops from each of the lightand heavy chains protrude into the pockets. The loops correspond to thehypervariable regions of DNA and can determine the antigen bindingspecificity by providing an appropriately shaped binding site andallowing for a variety of non-covalent interactions (71).Amino acid side chains can be polar, non-polar, aromatic, and atphysiological pH, can also be positively or negatively charged. Theamino acid side chains and peptide bonds of the antibody can bind to theantigen through electrostatic interactions, hydrogen bonds, Lifshitz-vander Waals interactions and hydrophobic interactions. Since theinteractions are relatively short range and are separately weak, theremust be an area of close contact between the antibody and antigen forstrong antibody-antigen interactions to occur. Crystallographic studieswith cocrystals of antibody bound to antigen have shown that the area ofcontact can be as large as 750 A2 and that binding can occur withoutdeformation of the antibody (70).201.5b Equilibrium constants and affinities for monovalent antibodies andantigens interacting in solutionThe binding of one epitope to one antigen-binding site on anantibody is reversible (71) under physiological conditions, due to thenature of the interactions involved. Antigen (Ag) and antibody (Ab) insolution can reach an equilibrium with the antigen-antibody complex,where the rate of formation of the complex is equal to the spontaneousrate of dissociation. At this point, the equilibrium constant can bedefined from the concentrations of reactants. For the simplest case,where the antigen and antibody are monovalent, the binding reaction willbe:Ag + Ab^Ag-AbThe equilibrium constant K will then be (72):K - [AR-Abl^ka^ [21[Ag][Ab]^kdwhere Ag-Ab is the antigen-antibody complex, square brackets denoteequilibrium concentration of the reagents and ka and kd are theassociation and dissociation rate constants for the reaction.The antibody affinity is defined as the negative of the standard freeenergy change of the binding reaction, and can be calculated from theequilibrium constant K:A = -AG° = RT1nK^ [3]where R is the gas constant and T the absolute temperature. Theequilibrium constant is usually used by itself to describe the strengthof the antibody-antigen reaction, with antigen-antibody systems beingreferred to generally as high or low affinity, depending on the valueof K.211.5c Equilibrium constant for a divalent antibody and monovalentantigen in solutionIntact IgG produced in vitro is divalent, meaning that a sequence oftwo antigen binding reactions can occur:^Ag + Ab^Ag-AbAg-Ab + Ag^Ag2-AbIf the antigen and antibody are both in solution, the equilibriumconstants are similar, although there may be some cooperativity (73). Ifthe constants are assumed to be the same, then the binding can bedescribed as a special case of the general multiple equilibria problem.For a macromolecule with n equivalent sites (i.e., all sites have thesame intrinsic association constant, K, for Ag) which are independent (nocooperativity) the average number of sites filled, v, at an equilibriumconcentration [Ag] of antigen, is given by (74):n K [Ag]v -  ^ [4]1 + K [Ag]where n is the total number of sites per macromolecule (74). For adivalent Ab, n = 2. The treatment which provides [4) allows thedistribution of ligand occupation to be calculated from:ci -c n! ([Ag] K)0[5](n-i)! i!where c is the concentration of the macromolecule with i of the n sitesfilled and the other symbols are as defined above.221.5d Some comments on the antibody preparation usedThe myeloma cells that were used for antibody production producedtheir own light chains, which would not have provided an antigen bindingsite for the antigen used. If the light chains were producedin equal amounts and incorporated randomly into the antibody molecules,then there would be a distribution of antibody molecules with zero, oneand two binding sites, in a 1:2:1 ratio.1.5e Antibody binding to antigen on a solid surface: kinetics andequilibrium constantsIf the antigen is adsorbed to a solid phase, the antibody-antigeninteraction will be more complex than the reactions in solution. Theantibody must be transported to the interface from the bulk phase. Thereit may also diffuse in two dimensions before associating with theantigen, and it must be transported away from the interface afterdissociation. In experiments in which the antibody solution isunstirred, the solution will become depleted of antibody near theinterface, after which the rate of reaction will be limited by the rateof diffusion of antibody from the bulk of the sample (75). If thesolution is well stirred, the rate of the antibody binding to thesurface will be limited by the intrinsic rate of the reaction (75).Reactions occurring on small spheres also tend to remain reaction-ratelimited, due to the geometry of the system (75).If the surface concentration of antigen is sufficiently high, onedivalent antibody molecule can bind to two antigen epitopes. Thereaction of the second antigen binding site on the antibody moleculewill be fast. Dissociation of the divalent antibody from the surfacewill be slow, since both binding sites would have to dissociate from the23antigen at once.Other effects are also active in decreasing the rate of dissociationof the antibody from the surface-bound antigen, since monovalentportions of antibody molecules will also show a decreased rate ofdissociation relative to free solution reactions (76). The samenon-specific forces responsible for adsorption of any protein to asurface will affect the antibody binding. Slow dissociation may also bedue to attractive forces between the antibodies or antibody fragments,which have been implied by some experiments: antibodies immobilized on aphospholipid layer have been shown to form a two-dimensional crystallattice (77) and high affinity antibodies bound to surface-immobilizedantigen have been shown to increase the apparent affinity of lowaffinity antibodies in an experiment measuring binding of a mixture oflow and high affinity antibodies (78). Lateral attractive forces are notobserved for all systems with IgG binding to a surface. For instance,IgG binding to bovine serum albumin coated latex has a large excludedarea relative to the molecular cross-sectional area (79).The maximum amount of antibody bound on the surface can also belimited by steric constraints if the antigen is smaller than theantibody and is packed sufficiently closely on the surface. Thegeometric constraints lead to what has been called the large ligandeffect (80). Obviously, if the average area of surface associated witheach molecule of antigen is much smaller than the area of the antibodywhen bound to surface-bound antigen, the molecular surface concentration(molecules per unit area) of Ab will not coincide with one half the Agsurface concentration, which would be the theoretical limit in theabsence of geometric effects.The dissociation rate for antibodies bound to surface immobilized24antigen is decreased to such an the extent that the binding has beendescribed as irreversible (81, 82). However, the off rate is not zeroand the apparent irreversibility just depends on the time scaleconsidered.A decrease in the dissociation rate constant for antibodies bound toindividual epitopes will cause an increase in the measured apparentequilibrium constant. The above discussion does not, however, imply anincrease in the magnitude of the free energy change associated withepitope interacting with the antigen binding site on the antibody.Since the system reaches equilibrium, a single apparent equilibriumconstant can usually be measured. The apparent or macroscopic constantrepresents the overall reaction of the antibody binding to the surfaceand is a function of the microscopic constants for interactions such asthose described above (including the antibody-antigen binding). Theapparent equilibrium constant cannot be interpreted in terms of theindividual reaction steps without kinetic analyses of the reactionsinvolved. Some analyses have been formulated to account for differentaspects of the problem (83-85) and to interpret the apparent constant interms of the free energy change associated with the antibody-antigeninteraction, but these will not be used here. The apparent equilibriumconstants will be calculated for the antibody interacting with thesurface bound antigen and will be discussed with respect to the variouseffects described above.The apparent equilibrium constant for monovalent antibody fragmentsbinding to antigen immobilized on the surface at a low enough density toavoid geometric effects and assuming no non-specific binding of antibodyoccurs will be given by an equation of the same form as [4] (86):25n K [Ab][Ag-Ab] -  ^ [6]1 + K [Ab]where [Ag-Ab] = surface concentration of bound Ab (mass or number ofmoles per unit area)n = maximum surface concentration of bound Ab possibleK = apparent association constant[Ab] = Ab concentration in equilibrium with surfaceFor sufficiently well separated molecules of surface-bound antigen,it would be expected that n, determined from the saturation value of theAb binding isotherm, should equal the surface concentration of Ag. Thisis generally not observed, however, because as the antigen densitydecreases, more of the underlying substrate becomes available to theantibody and non-specific adsorption of the protein results. Thisnon-specific adsorption can overwhelm the specific adsorption if thesubstrate is a good protein adsorbent (e.g., hydrophobic solid polymer).The non-specific adsorption can be minimized by adsorbing an uninvolved"blocking" protein to the substrate before exposure to the antibodysolution. Protein adsorption is sufficiently slow to reverse (87) thatnon-specific binding of antibody to the blocked areas can be greatlyreduced.For the antibody preparation used in this work, with a distributionof divalent, monovalent, and non-binding antibodies, for a surfaceantigen concentration low enough that an antibody cannot form a bridgebetween adjacent binding sites (a condition that will be referred to asdilute surface antigen concentration) the equilibrium will obey:3 n K [Ab][Ag-Ab] -  ^ [7]4 + 3 K [Ab]26since only 3/4 of the antibodies can bind and monovalent and divalentcan be assumed to behave similarly towards isolated Ags on the surface.At higher surface concentrations of antigen, the equilibrium surfaceconcentration of antibody will also tend to increase at a givenequilibrium concentration, since n increases in [7]. Formation ofbridges between adjacent epitopes and attractive lateral forces betweenthe bound antibody molecules will cause an increase in the calculatedaffinity since they will both act to decrease the desorption of theantibody. Increased surface concentrations of antigen have been shown tocause variations of several orders of magnitude in the measured apparentequilibrium constant (83).1.5f Free antigen in solution inhibiting binding of antibody to thesurface-bound antigen: another interaction involved in theequilibriumThe binding of antibody to surface-immobilized antigen can beinhibited by free antigen in solution as shown in Fig. 2. Free antibodyand antigen in solution will reach an equilibrium with concentrations offree and bound reagents that are a function of the initialconcentrations and of the equilibrium constant for the reaction. If thesolution of antibody and antigen is then applied to a surface bearingbound antigen, a new equilibrium will be reached: some of the freeantibody will bind to the surface and, since the antibody-antigenreaction in solution is reversible, some antibody will dissociate fromthe antibody-antigen complex in solution. For a fixed surfaceconcentration of immobilized antigen and a fixed initial antibodyconcentration, the amount of antibody bound on the surface will decreasewith increasing initial concentrations of free antigen.27For a system with a known apparent equilibrium constant forthe antibody binding to antigen on the surface, solution inhibitionmeasurements can be used to calculate an equilibrium constant for theantibody-antigen interaction in solution, following the procedureoutlined in Appendix 1 (D. E. Brooks, unpublished).1.5g Experimental determination of equilibrium constants: informationrequired and methods of calculationThe apparent equilibrium constant for antibody binding to antigenimmobilized on a surface can be calculated from equation [6], knowingthe surface concentration of antibody as a function of antibodyconcentration in solution. The concentrations of bound and free antibodycan be measured accurately using a radiolabelled IgG. For a fixed Agsurface density, the surface concentration of bound antibody can beplotted as a function of the equilibrium solution concentration to givea binding isotherm as shown in Fig. 7.The surface concentration of antibody binding sites, n, can becalculated from the maximum amount bound at high solution concentrationsof antibody. The equilibrium constant will affect the shape of the curveas shown in Fig. 7 and can be calculated from a direct non-linear leastsquares fit of equation [6] to the measured data. However, it is oftenmore revealing to re-cast equation [6] into a linear form known as theScatchard equation since the shape of the resulting plot can provideinformation on the binding mechanisms if the equation is not followed.It is also straightforward to obtain estimates of n and K from such aplot from linear least square fits of the data. The equation (74) is:28number of availablebinding sitesSurface concentrationof bound antibody Equilibrium solution concentrationFigure 7: Adsorption isotherms. A = high affinity and large number ofbinding sites, B = low affinity and large number of binding sites,C = high affinity and low number of binding sites, D = low affinity andlow number of binding sites.Surface concentrationof bound antibodySolution concentrationof antibodySurface concentration of antibodyFigure 8: Scatchard plots. A = high affinity and large number of bindingsites, B = low affinity and large number of binding sites, C = highaffinity and low number of binding sites.29[Ag-Ab]- -K [Ag-Ab] + K n^ [8][Ab]A plot of [Ag-Ab]/[Ab] vs [Ag-Ab] will be linear with slope -K andx-intercept n if [8] is obeyed; a plot is illustrated in Fig. 8.In the particular case of the antibody preparation with 1/4 of themolecules unable to bind, [Ab] would be taken to be 3/4 of the IgGprotein in the experiment.These calculations do not take into account the possibility ofbridging between epitopes at higher surface antigen concentrations. Thesimplest treatment of this possibility would be to assume that bindingof both valences on IgG would simply double the free energy differenceassociated with one site binding so the affinity would double and theassociation constant would be squared. However, the factors discussed inthe earlier sections suggest this would be an unwarrantedsimplification.There does not appear to be a suitable way to treat allthe complications associated with the interpretation of divalent Abbinding to surfaces of the type of interest here. Instead, since it isknown that equation [5] was capable of describing binding in relatedsystems (80) in which Ab binding to mobile surface-bound antigens wasstudied, the approach will be to estimate values of K from experimentalmeasurements employing a Scatchard plot and try to interpret theapparent values derived.1.6 Measuring the antibody-antigen interaction with ELISAs1.6a Limitations of ELISAsAlthough ELISAs can in principle be used for determining theequilibrium constants as described in section 1.5g, they are generally30used only to determine the concentration of a specific antibody orantigen in an unknown sample by comparison to standards under carefullycontrolled conditions (88). There is generally insufficient informationfor further calculations.The solid substrate used for ELISAs is usually a polymer withunknown surface characteristics. Commercially available 96 well platesand strips of detachable wells are made from polystyrene (89), polyvinylchloride (89) or other plastics not specified by the manufacturer (90).For a given brand of ELISA plate, there can be variation in the bindingcapacity in different batches or even between different wells in oneplate (92).The antigen is usually adsorbed non-specifically to the wells of theELISA plate. The surface concentration of antigen can be increased ordecreased by changing the initial concentration used for coating theplates, but is not usually measured. Protein antigens will usuallysaturate polystyrene plates at solution concentrations of less than4 gg m1-1 (93).The absorbance measured after addition of the substrate solution inthe final step of the assay is related to the amount of antibody on thesurface. The absorbance can be calibrated to give a measure of thesurface concentration of the antibody (94), but since there are severalsteps separating the binding of antibody from the production of colour inthe final assay step, the relationship between the antibodyconcentration and the absorbance may not be simple.The initial concentrations of antibody added to the different wellsin the ELISA plate is measured, rather than the equilibrium solution31concentration after some antibody has adsorbed to the wells. This makesdetermination of the concentration in an unknown sample simpler, butmakes interpretation of the binding more complex.1.6b Calibration curves for ELISAsThe calibration curves for ELISAs are obtained by plottingabsorbance after addition of the substrate against the initialconcentration of antibody or initial solution concentration of antigen.Since the interactions involved in ELISAs are not usually modeled byequations, a choice must be made about how to obtain a line from thedata points. Separate points are often connected with straight lines.Although this may cause less bias in drawing the calibration curve (95),it can cause greater error for regions in the middle of the straightline segments. Curves are also fit by eye or by splines (96), whichare continuous curves formed by connecting segments of differentpolynomials.The useful region of the calibration curve is one in which there isa change in the measured absorbance with a change in the analyte beingmeasured. At high surface concentrations of antibody, the surface willbe saturated and will not show a change in the absorbance; at lowsurface concentrations of antibody there may be insufficient change tobe distinguishable from the background.Clinically used assays are often sold as kits complete withstandards.1.6c Experimental requirementsThere must be a stable bond between the substrate and the antigen,so that the antigen does not desorb during the course of the assay.32Hydrophobic surfaces are usually used to minimize desorption of proteinantigens (93). Larger antigens will often adsorb effectively irreversiblyto surfaces because of multiple attachment points (87, 91) but smallermolecules may not bind or may desorb slowly in the presence ofmolecules competing for adsorption sites (91). Small molecules can beconjugated to larger molecules that will adsorb to the surface beingused, but this creates the possibility of cross reaction of the antibodypreparation with the protein carrier if a monoclonal antibody is notused. Small molecules can be attached covalently to plastic plates usinggluteraldehyde or other cross linking reagents (97), but this canresult in irreproducibly modified surfaces (98). In most such cases thechemistry of the coupling is obscure since the polymers involved arenot reactive with the cross linking agent (e.g., polystyrene); anyincrease in association is more likely due to stronger adsorption ofcross linked protein. It is well known that protein desorptiondecreases with increasing molecular weight (87, 91).Conditions used to attach the antigen must leave the wells opticallyclear, since the absorbance after addition of substrate is read throughthe bottom of the well. Since antigens can have different functionalgroups available for covalent attachment, it would also be useful to beable to modify the surface with different functional groups. The processused for attaching the antigen should be simple, in order to make itviable for routine use.Coupling a functional group on the antigen to the surface willgive the antigen a defined orientation, with the functional moietytowards the surface and the other portion of the antigen presentedtowards the solution. If the same functional group is used for couplingthe antigen to the surface and to the carrier molecule used during the33immunization, then the same portion of the antigen will be available forantibody binding.Reproducibility is another necessary feature of ELISAs. If there isa consistent source of antibody from a hybridoma cell line,reproducibility will be only limited by substrate characteristics. Thesubstrate should be easy to prepare and modify with antigen in verylarge batches or else with small batch to batch variation.The substrate should also have a high capacity for antigen, sincethis will increase the sensitivity of the assay. A low capacity fornon-specific binding is also necessary: if the antibody binds to thesurface in the absence of surface-immobilized antigen there will be ahigher background reading and the assay will be less sensitive. As notedabove, blocking agents are routinely used to minimize this problem.1.6d Covalent enzyme -linked immunoassays on polystyrene substratesPolystyrene and polystyrene derivative plates are available withsurface carboxyl groups (99), aldehyde groups (100), and withirradiated surfaces that will bind to carboxyl groups in the presence ofa carbodiimide (101). Coupling procedures can be used to give surfacesthat are more extensively modified than those resulting fromnon-specific adsorption, with subsequent higher assay signals (99, 101).1.6e Advantages of silicaSilica is a useful substrate for attachment of covalent antigens,because it can be functionalized with a large variety of groups (102,103). The silica surface can be modified by reaction with compounds ofthe form RnSiX(4-n), where X is a hydrolyzable group and R contains thefunctional group to be used for covalent attachment of the antigen. The34reaction occurs as shown in Fig. 9, and leaves the silica surfacecovalently modified with the organosilane used. There is a large varietyof commercially available organosilanes to provide surfaces modifiedwith different functional groups.Monodisperse silica beads can be made in large quantities fromtetraethylorthosilicate (104, 105) and can likewise be modified with asilane. Beads can be used to provide a large surface area for assays orfor characterization studies. The geometrical surface area of the beadscan be determined and used for calculations because the surface of thebead appears smooth at the level of electron microscopic images.Silica is also optically clear, resistant to solvents that could beused in any modification step and autoclavable so that the substratecan be readily sterilized. Silica beads are mechanically stable andcan be centrifuged without damage.1.7 Characterizing a modified surface1.7a Information that will describe a substrateIf a surface is modified with functional groups to be used forcovalent coupling of the antigen, it is useful to know the extent of themodification. The silica can also be characterized prior tomodification, to ensure that the surface is clean so the silane will beable to modify the surface evenly. Since pure silica contains no carbon,measuring carbon present on the surface will give an indication of thecleanliness of the surface.Silica used in these experiments was modified with3-aminopropyltriethoxy silane, to give a surface covered with aminogroups that could be used for coupling to the carboxyl groups of theantigen. The atomic percentage of nitrogen and number of amino groups on35,RSi (OR ) 3hydrolysis^3H2condensation3ROHwR-Si(OH)3IR^R^RI^I^IHO--Si --0--Si --0--Si --OHI^1^1OH OH OH+ OH^OH^OHI^I^I isilicahydrogenbondingR^1^7^7HO--Si 00^Si--OH^ISi,^. •^. ,^.H.'^%,II^ik, %).1^II.;^.o.H'00 '00 `CP1 I IR^R1  I^7HO--Si^0^Si ^0^Si--OH1 10 0 01^I^IFigure 9: Deposition of silanes on silica36the surface was measured to determine the extent of the modification.The electrophoretic mobility of the beads was also measured to allowcalculation of the electrokinetic charge density; unmodified beads havesurface silanols which will give the beads a negative charge in waterand modified beads will have a positive charge.1.7b Ninhydrin assaysNinhydrin reacts with primary amines on the beads to give a colouredcomplex that will diffuse away from the bead surface and remain insolution. The absorbance of the solution can then be measured andcombined with geometric information to calculate the surface density ofthe amines (106).1.7c X-ray photoelectron spectroscopyX-ray photoelectron spectroscopy can be used for identification andsemi-quantitative measurement of elements near the surface of a sample.The principle of the method is as follows (107, 108). Initially, anX-ray is directed onto the sample being studied and is absorbed by atomsin the surface region. When the X-ray photon is absorbed, its energy istransferred to a core electron in one of these atoms. Because the energyfrom the photon is greater than the binding energy of the electron, theelectron is ejected from the atom. The kinetic energy of thephotoelectron can be measured and used to calculate the binding energyfrom:EB = Ehv - EKE -^ [9]where Es is the binding energy, Ehv is the X-ray energy, EKE is thekinetic energy of the photoelectron and t is a constant for theparticular instrument being used.37The XPS spectrum is presented as intensity vs. binding energy, whereintensity is a measure of the number of scattered photoelectrons. Thebinding energy is characteristic of the orbital and element; therelative intensities of the different peaks can be used to determine therelative amounts of elements present.The binding energy of the core electrons varies with the molecularbonding environment and oxidation state of the element. In general, thebinding energy for electrons in a given element and orbital willincrease with increasing oxidation state and decreased interatomicdistance to the nearest neighbours (108).The intensity of the peaks is a function of the intensity of theX-ray source, the atomic density of the element in question, thecross-section of the orbital and various instrumental factors.Silica samples are non-conducting and will build up a charge afterloss of photoelectrons due to ionisation by X-rays. To maintain aconstant charge, the sample is bombarded with electrons from a lowenergy flood gun. Compensation is made for the change in binding energydue to the surface charge by assigning a reference peak in the XPSspectrum to a known binding energy and shifting the other peaksaccordingly. Since aliphatic carbon is present on the surface ofvirtually all samples as a contaminant, it is often used for a referencepeak (108).Photoelectrons can be scattered inelastically by atoms. Thisaccounts for the surface sensitivity of XPS: only photoelectronsproduced in the surface region pass out of the sample. Although a fixedsample depth is often given for XPS measurements (about 100 A fororganic samples, (108)), the sensitivity decreases exponentially withincreasing depth in the sample. Non-destructive depth profiling can be38done by changing the angle at which the photoelectrons are detected(Fig. 10). The maximum sampling depth will be obtained when thephotoelectrons are detected normal to the surface. If the photoelectronsare detected at high angle 0 away from the normal, the electrons willtravel the same distance d through the sample, but will originate fromatoms that are dcos0 away from the surface.X-ray photoelectron spectroscopy was used to determine theefficiency of cleaning procedures for the silica surfaces prior tomodification with the silane by measuring the percentage of carbon inthe surface region. The extent of modification with the silane wasdetermined by measuring the percentage of nitrogen. The Fe 2p XPS signalwas used to confirm the presence of antigen on the surface, since thesubstrate does not contain iron.The extent of coverage of the surface can be determined by followingthe disappearance of the Si 2p signal originating from the substrate. Ifthe surface is covered by a sufficiently thick overlying layer, thennone of the Si 2p signal will be detected. The thickness of an evenoverlying layer can be obtained from the relativestrengths of a characteristic signal from the substrate with and withoutthe layer present, if the mean free path length of the photoelectrons inthe layer is known (109). Information about the distribution of anoverlying layer can be obtained from angularly resolved measurements(110, 111). If the layer is continuous, at sufficiently large angles awayfrom the normal the sampling depth will be small enough (Fig. 11) thatthe signal from the substrate disappears. If the overlying layer formsdiscontinuous patches on the surface, then there will be signal from thesubstrate at all measurement angles.39X-raypeampling depth = d cos 0ejected photo electronsmeasured normal tothe surfaceX-rayI sample depth dejected photoelectrons measuredat an angle 0 away from the normalFigure 10: Depth profiles by XPS measurements401.7d Electrophoretic mobility of beadsThe mobility of particles in an electric field can be used tocalculate the electrokinetic surface charge density. Theelectrophoretic mobility u, defined as the particle velocity per unitelectric field strength, is related to cr, the surface charge density atthe shear plane (the hydrodynamic boundary of the particle) by (112):=Knu^ [10]where cr = surface charge density (esu/cm2)-1K = [8 ii Na e2 1/1,000 e k 1/2 (cm) is the Debye-HUckelparameterNa = Avogadro's numbere = electron charge2= ionic strength = 1/2 Ec ztc = molar concentration of i th ionic species in suspending mediumz = valence of i th ionic species in suspending mediumk = Boltzmann constantT = absolute temperaturee = dielectric constant of suspending mediumn = viscosity of the suspending medium (Pa-s)Equation [10] holds providing the particle radius, a, obeys (Ka > 300)and providing the surface potential is "small", i.e., providing:^(4 it o. e/K c k T) << 1^ [11]where (4mcr/Kc) = C, the zeta potential or potential at the plane ofshear. For larger surface zeta potentials the charge density can becalculated from [12] (112), where z = 1z11:^(2 c k t no)1/2^z e C^ sinh  ^[12]it 2 k T41The electrophoretic mobility can be determined experimentally for agiven electric field strength and solution viscosity. The surfacecharge density can be calculated by comparison of the mobility to thatof a well characterized particle such as a red blood cell measured inthe same system.The hydroxyl groups on the surface of the silica beads will givethe beads a net negative charge in water. As the silica surface ismodified and the silanol groups react, the charge will decrease. Sincethe beads were modified with a silane having an amine group, thesurface would be expected to be positive for the silylated beads.1.8 The silica surfaceGlass and silica surfaces that have been standing in air have asurface covered with a contaminating layer of aliphatic hydrocarbons(108). If the surface is to be modified, then the reactive groups on thesurface must be accessible to the reagents in solution, and the surfaceshould be clean. Glass and silica have been cleaned with solvents,hydrofluoric acid etching, sodium hydroxide, hot nitric acid, hotammoniacal hydrogen peroxide (113-116), and hot chromic acid (117,118).The extent of contamination left on the surface after the cleaningprocedure can be monitored by measuring the percentage of carbon in thesurface region, using a surface sensitive technique such as Auger (116)or X-ray photoelectron spectroscopy (113)(section 1.8c). The surfacewill become more hydrophobic as hydrocarbon contaminants are deposited.Water droplets will spread on a clean silica surface to givewater-silica contact angle of 0 ° , and increasing contact angles will beindicative of the hydrophobicity due to surface contamination.421.9 Characterizing the antigen-modified substrate1.9a Measuring the surface concentration of antigenThe surface concentration of antigen on the beads can be measuredindirectly from solution depletion measurements during the modificationprocedure or directly from assays that are sufficiently sensitive todetect the antigen on the beads.The antigen used for these experiments was a ferric trihydroxamatethat absorbs strongly around 436 run; the concentration of the antigen insolution was followed by absorbance measurements. The Fe3+ ion could bedetected by XPS and used to confirm the presence of the antigen on theflat silica.1.9b Determining whether or not the antigen is attached covalentlyThe presence of a covalent bond can usually only be inferredindirectly from stability of the attachment between the antigen and thesubstrate. If the antigen can be removed from the surface when adsorbednon-specifically but remains on after being attached to the surfaceunder conditions expected to produce a covalent bond, this is used asevidence for formation of the covalent bond.The clearest evidence for formation of a covalent bond between theantigen and the substrate would be detection of the bond itself by somesuitable spectroscopic technique.1.10 Measuring antibody on the surface and in solutionThe amount of antibody on the surface and in solution must bedetermined accurately for the calculations outlined in section 1.5g. Theantibody can be labelled with 1251; the protein can then be detected atlow concentrations using a 7-counter.431.11 This projectThis project was the development and detailed characterization ofassays analogous to the ELISAs shown in Figs. 1 and 2. The substrateused as a solid phase for the assays was silica, which was modified withamine groups for covalent attachment to a carboxyl containing antigen.Covalent attachment of the antigen has an advantage over conventionalELISAs using non-specifically adsorbed reagent, since a stable bond canbe formed between the small antigen used and the substrate (see section1.6c).Silica slides and beads (0.78 pm diameter) were both used for assaysbecause they could be characterized in different ways, in order toobtain additional information. The silica beads provided a high surfacearea for more accurate measurement of surface amine groups and surfaceantigen concentration and could also be used for particleelectrophoresis measurements to determine the surface charge on thebeads after different modifications. The silica slides had a smooth flatsurface and were used for XPS measurements determining the cleaningprocedure prior to silylation and the amount of nitrogen on the surfaceafter silylation. The XPS measurements were also used to detect antigenon the slides.The silica slides and beads were cleaned thoroughly to increase theaccessibility of the surface silanols to reagents in solution. The beadsand slides were both modified with 3-aminopropyltriethoxy silane, togive a surface covered with amine groups. The surface concentration ofamine groups on the silylated beads was measured using a ninhydrin assayand the surface concentration of nitrogen was measured using XPS.The antigen used in the assays was ferrichrome A, a cyclichexapeptide that chelates iron through three hydroxamate groups.44Ferrichrome A was chosen as an antigen because of several usefulfeatures: the ferric hydroxamate group absorbs at 436 nm and can be usedto determine the peptide concentration, there are three carboxyl groupsper molecule for covalent attachment to the amine-modified silica, theiron can be distinguished by XPS and the isolation results in a highyield of antigen which can be readily purified.A water soluble carbodilmide, (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, EDC) was used to promote the formation of an amide bondbetween the antigen and the silylated silica. The surface concentrationof ferrichrome A on the beads was measured as a function of solutionconcentration. Some evidence is presented for formation of a covalentbond.Ferrichrome A was coupled to a carrier protein using EDC in order toproduce an antibody. A monoclonal antibody was obtained againstferrichrome A and used in later studies so that the antibody preparationwould have only one binding specificity and the antibody bindingmeasurements would be simpler to interpret.The surface concentration of antibody bound to the antigen-modifiedbeads and flat silica was measured as a function of solutionconcentration of antibody and surface concentration of antigen, using an125I labelled antibody. The equilibrium binding constant for theantibody-antigen reaction at the substrate surface was calculated forthe different surface concentrations of ferrichrome A used. An estimateof the solution binding constant was obtained as described in Appendix 1using a series of different solution concentrations of antigen toinhibit antibody binding to flat slides modified with ferrichrome A.The slides and beads were both used for ELISAs measuring theconcentration of antibody and antigen in solution.45Chapter 2Methods and materials: preparation of the antigen, antibody and modifiedsubstrate2.1 Isolation of the antigenWhere not otherwise specified, the chemicals used in all experimentswere reagent grade products from BDH (Vancouver) or Fisher (Vancouver).Ferrichrome A is one of a large group of related compounds that aresecreted by microorganisms in order to chelate extracellular ferric ionsand transport them into the cell. Because ferrichrome A is secretedexternally to the cell, it can be isolated readily from the culturesupernatant (see Fig. 11 for structure of ferrichrome A).Ferrichrome A was isolated (119) from Ustilago sphaerogena Burrilwhich was obtained from the American Type Culture Collection (cat. no.12421), with an import permit from Agriculture Canada. The initialfreeze dried fungus was grown in 10 ml of a low iron medium(composition, Table I) in a 125 ml Erlenmeyer flask. The cell line wasmaintained in 125 ml Erlenmeyer flasks with re-inoculation of freshmedium every ten days. The fungus was grown at 30 0C in a reciprocalshaker. Larger amounts of culture used for isolation of ferrichrome Awere grown in 500 ml of medium in a 2.8 litre Fernbach flask.Three or four days after inoculation of the large flask, the cellswere removed by centrifugation. Ferric chloride was added to the culturesupernatant to produce the coloured iron hydroxamate. The supernatantwas then saturated with ammonium sulphate and extracted with benzylalcohol. After addition of diethyl ether to the organic phase, the46p-c.Z\ cc%)/CHNM \p0/ 3CW°/c^Int^co= c^R-cf a^(CM2 ) 3 ••■•^CM^110112CCMe" *C$PalNCH20HNMFigure 11: (a) Structure of ferricnrome A, where R = trans-CH=C(CH3)CH2COOH (b) Crystallographic structure of ferrichrome A (120).Table I: Preparation of growth medium for Ustilago sphaerogena1. Solution A was made in an EDTA washed test tube to minimize ironadsorbed to the glass: 5 mg thiamine hydrochloride in 5 ml H20. Freshlymade solution A was used.2. Solution B was made and stored in an EDTA washed bottle: 4.5 mgCuSO4.5H20, 32.0 mg MnSO4.41120 and 2.0 g 211504.7H20 were added to100 ml of water.3. The growth medium was then made up in a Fernbach flask with 520 ml ofH20, to which the following ingredients were added:• FinalconcentrationK2SO4 0.52 g 1.0 g/1K2HPO4 1.56 g 3.0 g/1NH4C00CH3 1.56 g 3.0 g/1sucrose 10.4 g 20.0 g/1citric acid 0.569 g 1.0 g/1solution A (thiamine) 0.52 ml 2.0 mg/1MgSO4.71120 0.422 g Mg 80.0 mg/1solution B (trace salts) 0.174 ml Cu 0.005 mg/mlMn 0.035 mg/mlZn 2.0 mg/ml4. The pH of the solution was adjusted to 6.8 with concentrated ammoniumhydroxide.5. The solution was sterilized by autoclaving.47ferrichrome A was extracted using water and was obtained as a dark redcrystalline material. The ferrichrome A was purified byrecrystallization from water.Ferrichrome A absorbs strongly in the visible region of the spectrum(Amax at 436 ma) because of the iron hydroxamate centre. Absorbance (A)was measured with a Hewlett-Packard 8450A UV/VIS spectrophotometer. Theextinction coefficient at A=436 nm was determined to bec = 2.778 ml mg-1 cm-1 .The derivative curve (dA/dA) was also used to determine theconcentration of ferrichrome A (121). This method has the advantage ofeliminating most of the effects due to low concentrations of suspendedparticles and was used in later experiments measuring adsorption offerrichrome A to silica beads, because of problems with beads in thesamples. Light scattering due to the particles will increase theabsorbance reading, but because the increase in light scattering isnearly linear with decreasing wavelength (for the particle size andwavelength range used), the difference between the derivative at any twowavelengths will remain unaffected by increased light scattering (Fig.12, sample spectra are included in Appendix 3). The difference betweenthe maximum and minimum points on the derivative curve was determined;this corresponds to the sum of the absolute values of the slopes at theinflection points on the absorbance curve at 389nm and 492nm. The sum ofthe slopes was shown to vary linearly with concentration (Fig. 13), andthe concentration of ferrichrome A in mg/ml was found to be equal to thesum divided by 0.05986±0.00017, where the error is the standarddeviation in the slope of the calibration curve (Fig. 13).48Absorbance due to ferrichrome Ain solutionInflection pointsTurbidity due to suspended particlesAThe absorbance curve measuredwill be the sum of the turbidityand absorbancewavelength A (mu)^389^436^492derivative of curve due to^dA/dAabsorbance (dA/dA)this difference is measured derivative of light scatteringcurve^ dA/dAThe derivative of the measured^dA/dAabsorbance curve will be thesum of the two derivatives above.The measured difference willremain unchanged.Figure 12: The effect of light scattering on the derivative of theabsorbance curve.A490.0250.02-0.005-0.05^ 0.1^0.15^ 0.2^0.125[Ferrichrome A] mg/ml0.3 0.35 0.4Figure 13: Determining the concentration of ferrichrome A from thederivative of the absorbance spectrum (dA/dA). The sum of the absolutevalues of the slopes at the inflection points at either side of theabsorbance peak is plotted against concentration of ferrichrome A.50start of separating gel"MITT1 2 3 4 5Figure 14: Western blot showing that the antigen is coupled to KLH.The outline on the blot marks the position of the gel. Higher molecularweight samples are near the top of the gel. The different lanes are for1) molecular weight standards 2) KLH 3) KLH mixed with ferrichrome A4) KLH coupled to ferrichrome A using EDC 5) ferrichrome A.512.2 Coupling antigen to KLH and BSA2.2a Coupling ProcedureKeyhole limpet haemocyanin (KLH, Calbiochem, San Diego) was used asa carrier molecule for raising both the rabbit polyclonal and mousemonoclonal antibodies and was coupled to ferrichrome A using1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Ferrichrome A wasdispersed in water (5 mg/lath most of the ferrichrome A dissolved afteraddition of a 34x molar excess of EDC. Keyhole limpet haemocyanin(5 mg/m1) was added and the mixture was stirred at room temperature forone hour, after which it was dialyzed against water at 4 °C for 48 hrusing dialysis tubing with a molecular weight cut off of 50 000 g/mol(Spectrapor, Spectrum Industries, Los Angeles). This left an aggregatedmass of undissolved KLH and a clear supernatant; both were orange,indicating that there was still free ferrichrome A remaining afterdialysis. Both the supernatant and the precipitate (termed collectivelyferrichrome A/KLH) were used for eliciting the antibodies.Ferrichrome A/BSA was prepared the same way as ferrichrome A/KLH,but had no precipitate because the BSA dissolved completely.2.2b Western Blot with ferrichrome A/KLH: an indication of the successof the coupling procedureA Western blot (122) on the clear supernatant from theKLH/ferrichrome A preparation using one of the monoclonal antibodiesraised as described later showed that the antibody interacted with ahigh molecular weight antigen, indicating that the ferrichrome A insolution was attached to KLH.Samples in solution were mixed at a 1:1 volume ratio with PBScontaining 2% SDS and 5% v/v g-mercaptoethanol, and boiled for two52minutes to denature and reduce the sample. The initial electrophoreticseparation of the proteins was carried out using the automated PharmaciaPhastsystem with precast 8-25% polyacrylamide gels and sodium dodecylsulfate (SDS) buffer strips. The samples were electroblotted ontonitrocellulose membrane with a 0.45 gm pore size in pH 8.3 transferbuffer with 25 mM Tris (tris[hydroxymethyl]aminomethane) 192 mM glycineand 20% methanol. The nitrocellulose membrane was incubated overnightwith supernatant from the AC3 cell line grown in serum free medium(described in section 2.4) diluted 1:15 in PBS with 0.5% Tween 20 and 3%BSA to block non-specific binding. Antibody produced by the AC3 cellline was shown to interact with ferrichrome A (section 2.4c, Fig. 25).The membrane was washed using PBS with 0.5% Tween 20 and developed withhorseradish peroxidase conjugated antibody and substrate from theBio-Rad Immun-blot kit with 4-chloro-1-naphthol as a substrate.The Western blot indicates the species to which the antibody binds.The antibody bound to the ferrichrome A/KLH but not to the KLH by itselfwith EDC, indicating that the monoclonal antibody bound to ferrichromeA. No band was seen for ferrichrome A on the initial gel or on the blot.This may be because ferrichrome A was too small to be distinguishablefrom the solvent front on the gel used or because ferrichrome A alonedoes not stain with Coomassie blue and does not adsorb to the blottingmembrane. No band was seen for ferrichrome A mixed with KU! withoutEDC, indicating that the EDC was required for the coupling.The band for the ferrichrome A/KLH is a smear, indicating that therewas a range of molecular weights for the species in solution. The KLH byitself can form aggregates that could be cross-linked by EDC to give aproduct with a molecular weight too high to run onto the gel (samplesthat do not run onto the gel can be seen as a band between the sample53application layer and the separating gel). Aggregation does not pose aproblem and can help stimulate antibody production (123).2.3 Developing an ELISA prior to raising a monoclonal antibodyAn enzyme linked immunosorbent assay (ELISA) was developed usingrabbit and mouse polyclonal antibodies, so that an assay would beavailable to detect the monoclonal antibody when it was produced.2.3a ELISA with serum from an immunized rabbitA rabbit polyclonal antibody was elicited against theferrichrome A/KLH preparation described in section 2.2a (124). One NewZealand White rabbit was injected subcutaneously at the back of the neckwith one ml of a 1:1 emulsion of ferrichrome A/KLH (containing 0.5 mg ofKLH) and Freund's complete adjuvant (Difco Laboratories, Detroit). Therabbit was given a subcutaneous booster injection one month later withone ml of a 1:1 emulsion of ferrichrome A/KLH and Freund's incompleteadjuvant (Difco). Blood was obtained from an ear vein five days afterthe second injection.An ELISA was developed with commercial plates, following the ELISAprotocol for the Vancouver Research Station of Agriculture Canada (125).Figure 15 provides a general description of the ELISA procedure.Ferrichrome A adsorbed directly to ELISA plates gave a poor assayresponse, so the ferrichrome A was cross-linked with EDC, ethylenediamine and triethylamine to give a higher molecular weight product thatwould remain adsorbed to the plates. Since the ferrichrome A has threecarboxyl groups per molecule, EDC coupling of the carboxyl groups withthe amino groups on a polyamine will result in a cross linked product.Ferrichrome A, ethylene diamine, EDC and triethylamine were mixedtogether in water at a mole ratio of 2:3:6:6. The unpurified product was54I Commercial 96-well ELISA plate (or other solid^1^phase).Add a solution of antigen to the wells of theELISA plate.I AL 1r4 PA I Incubate the plate with the antigen solution.Wash plate three times to remove unboundantigen.Block uncoated sites to minimize non-specificbinding of antibodies.Incubate plate with antibody solution, thenwash three times to remove excess antibody.Incubate plate with HRP-conjugated antibodysolution, then wash plate three times toremove unbound antibody.Add a substrate for HRP. The HRP bound to theplates will cause a colour change. Addphosphoric acid to stop the reaction.Read the absorbance of the different wells.KEYAi AntigenBlocking AgentI( Antibody76 HRP-conjugated antibodySubstrateFigure 15: The ELISA procedure55adsorbed to Immulon 2 plates (Dynatech, from Fisher, Vancouver)overnight at room temperature in 100 mM acetate buffer pH 4.5 at aconcentration equivalent to 5 gg/ml free ferrichrome A and a volume of100 gl/well. The uncoated sites were blocked with 0.2% Carnation skimmilk in phosphate buffered saline with 0.05% Tween 80 (blocking buffer)for half an hour at 37 °C, to minimize non-specific adsorption ofantibodies in subsequent steps (Tween 80 is a non-ionic detergent, poly-oxyethylene sorbitan mono-oleate). The ELISA plate was washed threetimes with PBS containing 0.05% Tween 80 (PBS-Tween) after alladsorption steps.A dilution series of the rabbit serum in blocking buffer wasadsorbed to the plates for one hour at 37 °C, and then washed off. Asecondary horseradish peroxidase conjugated goat anti-rabbit antibody(HRP-gar Ab, Cappel, Cooper Biomedical, West Chester, P.A.) was dilutedin blocking buffer and incubated with the plates for one hour at 37 °C.The plate was developed using tetramethyl benzidine (TMB) as a substratefor the horseradish peroxidase (Table II gives the composition of thesubstrate solution). Substrate solution was added to the plates(100 pl/well) and left at room temperature for two to three minutesbefore the reaction was stopped in all wells by addition of 50 Al of2.0 M phosphoric acid/well. The absorbance of each well was read at 450nm with a reference at 620 nm (where the substrate has minimalabsorbance) using an SLT Instruments plate reader.The ELISA procedure outlined above gave a clear positive response forthe rabbit serum (Fig. 16).2.3b An ELISA with blood from an immunized mouseTwo BALB/c mice were also used to raise antibodies against56Table II: Preparation of substrate solution for ELISAs (126)1. Stock solutions. The following stock solutions were made up.acetate/citrate buffer^200 mM sodium citrate was added to 200 mMsodium acetate to give a solution with afinal pH of 6.0.tetramethylbenzidine (TMB) THE (Sigma, St. Louis) was dissolved indimethylsulfoxide (DMSO) at a concentrationof 10 mg/ml.2. Substrate solution. The substrate solution was made up immediatelybefore use, with reagents added in the order listed.acetate/citrate buffer 4 mlwater^ 16 mlTMB solution^200 gl3% w/w H202 20 gl571^1 1 1 III^I^1^1^1 1 1 1 11^1^I^I^I I I I II^1^1^1^1 1 1 1 11^1^I^I^I I I II I^1^1^1 1 1 1 1 11E-06 1E-05 0.0001 0.001 0.01 0.1Dilution of Serum (whole serum = 1)Figure 16: An ELISA with ferrichrome A adsorbed to Immulon 2 plates andrabbit serum as antibody. The rabbit serum was diluted in blocking bufferwith an initial dilution of 1:25 and subsequent five-fold stepwisedilutions.58ferrichrome A/KLH. A 1:1 emulsion of ferrichrome A/KLH prepared asdescribed in section 2.2a was injected subcutaneously in the sides ofthe mice (100 pl total volume). An intraperitoneal boost offerrichrome A/KLH in PBS (0.5 ml total volume) was administered 28 daysafter the initial immunization. Blood was obtained by tail bleedingthree days after the boost.Blood from immunized mice did not give a positive reaction on theELISA (Fig. 17). Mice were later shown to produce antibodies againstferrichrome A (section 2.4), but the rabbit serum may have containedmore antibodies against ferrichrome A because of species differences inthe immune response (127).2.3c A second ELISA with blood from immunized miceAn ELISA with mouse serum as the antibody and ferrichrome A/BSAadsorbed to the ELISA plates as the antigen (adsorbed at a concentrationof 1 pg/ml BSA in PBS) gave a strong positive response (Fig. 18), but afurther ELISA showed that this response was not due to antibodies thatwere active against free ferrichrome A.Inhibition of the antibody binding by free ferrichrome A wasmeasured using antibody at a constant concentration (1:3000 dilution ofmouse blood in blocking buffer) and varying concentrations offerrichrome A. The diluted mouse blood was incubated with theferrichrome A for 30 min at 37 °C before being added to the ELISAplates.Free ferrichrome A did not inhibit the ELISA response, as would beexpected if the ferrichrome A in solution were competing with theferrichrome A on the ELISA plates for binding sites on the antibodymolecules (Fig. 19).592.5-0.5-0-^1E-07I I IIIH^1 541 I IIIH WL7F-MdM I^1E-06 1E-05^0.0001^0.001Dilution of Serum0.01^0 1Figure 17: An ELISA with ferrichrome A adsorbed to Immulon 2 plates:comparison of the ELISA results for blood from two mice (x and o) and arabbit (m).601.20.8-0.4-0.2-0^1E-071^1^1111I1^1^1^1111111^1^I1111111^11E-06 1E-05 0.0001^"01001Dilution of Serum1^1 11111111^1^11111611Figure 18: An ELISA with ferrichrome A/BSA adsorbed to Immulon 2 platesand serum from an immunized mouse as antibody.611.210.8I^I^f^I^I^I^III^I^I^I^I^I^I^III0.001 0.01[ferrichrome A] mg/ml0.40.20.0001 1I^I^I^I^I^I III^I^I^I^I^11110.1Figure 19: An ELISA with ferrichrome A/BSA adsorbed to Immulon 2 plates.The antibody used was blood from an immunized mouse diluted 1:3000.Ferrichrome A at different concentrations was pre-incubated withantibody and added to the ELISA plate (m). The absorbance resulting fromantibody with no inhibiting ferrichrome A is also shown (----), witherror limits (^ ).62Typical epitopes on proteins comprise six to eight amino acidresidues (49). Most of the antibodies produced by the mouse may beactive against the ferrichrome A together with one or more amino acidresidues on the protein. This problem could be minimized by using aspacer molecule when attaching the ferrichrome A (54).2.3d Comparing the efficiencies of skim milk and BSA as blocking reagentsThe efficiencies of skim milk powder and BSA as blocking reagentswere compared for polystyrene plates. The assays were carried out asdescribed for the previous ELISAs with BSA/ferrichrome A adsorbed to theplates, but without Tween in the PBS at any point and adding theblocking reagents only in the first 30 minute blocking step (0.2% skimmilk powder in PBS or 0.2% BSA in PBS). The antibody and HRP conjugatedantibody were made up in PBS with no Tween or blocking agent. A mouseantibody against an unrelated antigen was also used, to determinenon-specific binding. The results obtained are shown in Figure 20. Thenon-specific binding, as determined by the unrelated antibody, washigher with the BSA block. This curve also seemed to form the baselinefor the results obtained with the AC3 antibody with a BSA block in PBS.Skim milk powder was used initially in adherence to the proceduredescribed by the Agriculture Canada monoclonal antibody laboratory, andthese results were taken to justify continued use of skim milk powder asa blocking reagent.2.3e The effect of Tween 80 on the ELISAThe effect of Tween 80 in the buffer was also studied for assays onpolystyrene plates. The plates were coated as before with ferrichrome AlBSA in PBS. One plate was then washed with PBS Tween, and blocked with631 .1............^....^..................... .^.......................^....................^/if...^..........^..............^...^........11 1 111^I^I I I 1 11 11^I^I I II I I II^I^II mill^I^I I min^1E-07 1E-06 1E-05 0.0001 0.001^0.01[Antibody] mg/ml01E-081E-09Figure 20: An ELISA comparing the efficiency of 0.2% BSA and 0.2% skimmilk as blocking agents. Results are shown for an antibody toferrichrome A with BSA (m--s) and with skim milk (.---11), and for anunrelated antibody with BSA (o---a) and skim milk (o---o) as blockingagents. • = anti-ferr. A, o = other antibody, --- = BSA,--- = skim milk.640.2% BSA or skim milk in PBS Tween. Antibody and HRP conjugated antibodywere also added in PBS Tween. A second plate was treated the same way,but all washes and solutions were in PBS without Tween. The results fromthese experiments (Fig. 21) show that BSA is an effective blocking agentwhen there is detergent in the solution. The results also show thatTween seems to enhance the affinity of the antibody for the antigen.2.3f Absorbance of the substrate solution as a function ofHRP-conjugated antibody concentration and evidence that there is alarge excess of HRP-conjugated antibody used in the ELISAsDilute solutions of HRP-conjugated antibody solution were addeddirectly to the substrate to determine if the absorbance increasedlinearly with increasing enzyme concentration. A series ofHRP-conjugated antibody solutions was made up in dilutions ranging from1:100 000 to 1:1 000 of a commercial stock solution. A 20 pl volume ofeach solution was added to the substrate solution described above andleft at room temperature for three minutes. The absorbance of eachsolution was read after the enzyme reaction was stopped by addition ofphosphoric acid. The absorbance is plotted as a function of dilution ofthe stock solution in Figs. 22 and 23. In solutions having an absorbanceof up to 1.5 after three minutes, the absorbance was a linear functionof the amount of HRP-conjugated antibody present. At absorbances greaterthan 2.5, there was almost no increase in absorbance with increasingamounts of the HRP-conjugated enzyme. This is not merely a function ofresponse of the spectrophotometer. The absorbance was measured with ashorter optical path length and used to calculate the total absorbance;this gave a similarly shaped curve, with a non-linear response seen forHRP-conjugated antibody concentrations giving absorbances higher than6511/.. Ai.................. --........................... 0, .I I IIIIP I^I^I I I MI^I^1 1 1 11111^1^1^1 1 1 1111^1^1 1 1 11111^1^1^1 1 1 1111^1^1^ii arr^1E-08 1E-07 1E-06 1E-05 0.0001 0.001 0.01[Antibody] mg/mlFigure 21: The effect of Tween 80 on the ELISA. Results are shown for anantibody to ferrichrome A using BSA as a blocking agent with plateswashed with PBS-Tween (n---m) and PBS (m---m) and using skim milk as ablocking agent, with plates washed with PBS-Tween (a---o) or PBS (o---o).6621^2^3^4^6^6Dilution of HRP conjugated Ab(Times 10E-5)0 10•meas. tot.Figure 22: Absorbance of the ELISAs as a function of low BR?concentrations670calc. tot.•meas. tot.m2.5-0trr) 1.5-¢0.5-0.0001 0.00^1^102 0.0003 0.0004 0.00^105 0.0006 0.0007 0.01008 0.01009 0.001Dilution of HRP conjugated AbFigure 23: Absorbance of the ELISA as a function of higher HRPconcentrations • = direct measurement, a = absorbance for total samplecalculated from measurement with a short optical path length.681.5. The calculated value for the absorbance was slightly lower than themeasured value; this may be due to the fact that the optical path lengthwas calculated assuming a level surface of liquid, and the path lengththrough the centre of the smaller volume would have been affected moreby the shape of the meniscus.For the ELISAs, 100 Al of a 1:1000 dilution of the HRP-conjugatedantibody was added to the wells. The amount of HRP-conjugated enzymeremaining on the plates after rinsing was sufficient to cause anabsorbance of about 1. If the activity of the surface bound enzyme isthe same of that of the enzyme in solution, then about a 300-fold excessof the conjugated enzyme is added.2.4 Production and purification of the monoclonal antibody, withSOS-PAGE to determine the antibody purity2.4a Antibody productionThe monoclonal antibody was made following the procedure of theVancouver Research Station of Agriculture Canada (125).Three BALB/c mice were injected subcutaneously with ferrichrome AlKLH and complete Freund's adjuvant at zero days. The mice were injectedintraperitoneally with ferrichrome A/KLH in buffer at 30 days. Serumobtained by tail bleeding at 33 days was tested for antibodies using anELISA with ferrichrome A/BSA adsorbed to the ELISA plate. At 34 days,the spleen from the mouse with the highest serum antibody titre wasremoved and placed in a Petri dish with 10 ml of Dulbecco's modifiedEagle medium (DME from GIBCO, Grand Island; for composition see TableIII). The spleen cells were separated by covering the spleen with a pieceof gauze and squeezing the spleen with the end of a syringe plunger. TheDME, containing the spleen cells, was pipetted off, and the cells were69Table III: Preparation of Dulbecco's Modified Eagle medium1. DME was made up from powdered packets from Gibco (cat. no. 430-1600,low glucose medium with L-glutamine and sodium pyruvate). One litre ofmedium was made up at a time and contained components in the followingconcentrations (128):mg/1Inorganic saltsCaC12.21120^264.9Fe(NO3)3.91120 0.100KC1^ 400.0MgSO4 97.7NaC1 6400.0NaH2PO4^ 125.0Other componentsglucose^1000.0phenol red, sodium salt^15.0sodium pyruvate^110.0Amino acidsL-arginine.HC1^84.00L-cystine, disodium salt 56.78L-glutamine 584.0glycine^ 30.00L-histidine.HC1.H20^42.00L-isoleucine^104.8L-leucine 104.8L-lysine.HC1 146.2L-methionine^30.00L-phenylanine 66.00L-serine 42.00L-threonine^95.20L-tryptophan 16.00L-tyrosine 72.00L-valine^ 93.60VitaminsD-Ca pantothenate^4.00choline chloride 4.00folic acid^4.00i-inositol 7.00nicotinamide 4.00pyridoxal.HC1^4.00riboflavin 0.4000thiamin.HC1 4.002. The DME solution was sterilized by filtration with Falcon Easy FlowTmfiltration units (0.22 pm pore size), and stored in autoclaved bottles at4 °C.3. If the DME solution had been stored for more than ten days, additionalglutamine was added immediately before use. Glutamine was added at a1:100 dilution from a 29.2 mg/ml L-glutamine solution from Flowlaboratories.70collected by centrifugation and washed twice with fresh DME.FOX-NY myeloma cells (129)(ATCC CRL1732) frozen in DME with 20% fetalbovine serum (FBS, Hyclone, Logan) were thawed at 30 days, washed withDME, added to DME with 5% FBS, and plated out in five 15x100 mm Petridishes (Fisherbrand, Fisher Scientific, Ottawa). The cells were grownunder 10% CO2 and were collected at 34 days and washed twice with DME.PEG 4000 (Serva, Heidelberg) was used to fuse the myeloma and spleencells. Five grams of PEG 4000 were dissolved with heating in four ml ofDME. The cooled PEG solution was sterilized by filtration through a0.22 pm filter after addition of one ml DMSO (BDH Assured, BDH,Toronto) and stored at 37 °C.The spleen cells and the FOX-NY myeloma cells were suspended in DMEin a 12 ml round bottom tube, the cells were centrifuged, and all theliquid was removed. The myeloma cells and spleen cells were then fused,with careful attention to the mixing times. The bottom of the tube withcell pellet was immersed in a 37 °C water bath. One ml of the 50% PEG10% DMSO mixture was added to the cells with a 5 ml serological pipetteand the cells were mixed with the pipette tip for one minute. One ml ofDME was added over the second minute while continuing to mix the cellswith the pipette tip, two ml of DME was added over the third minute, andsix ml of DME was added over the following five minutes.The cells were washed in DME and resuspended in DME with 20% FBS andadded HAT (hypoxanthine, aminopterin and thymidine, obtained as a 100xconcentrate from GIBCO). Thymocytes were collected from the thymuses oftwo four week old CD-1 mice following the same procedure used to collectthe spleen cells. The thymocyte and the fused myeloma and spleen cellmixture was diluted to 50 ml in DME with 20% FBS and added HAT, andplated out in 96 well tissue culture plates (with 100 pl cell71suspension/well).Four days after the fusion, the cells were fed by adding 100 pi ofDME with 20% FBS and added HT (hypoxanthine and thymidine) to each well.Seven days after the fusion, 160 gl of medium was removed from eachwell and 160 pl of fresh HT medium was added.Ten days after the fusion, the wells were screened for supernatantsthat gave a positive reaction in an ELISA with adsorbed ferrichrome AlBSA and a negative reaction in an ELISA with adsorbed KLH.Positive cell lines were transferred to 24 well plates and subclonedat 18 days after the fusion. The number of viable cells/ml was countedusing a haemocytometer and erythrosin B (80 mg/ml final concentration)to stain the dead cells. The cell suspension was diluted out to 5, 20 or50 viable cells/ml. Thymocytes from one four week old CD-1 mouse wereadded and the cells were plated out at 100 pl/well in 96 well plates,with one plate for each dilution of cells. The colonies in each wellwere counted and the wells were checked for antibody production usingthe same ELISA procedure that was used after the fusion.Cells from wells that had one colony and were positive in the ELISAwere grown and then stored in liquid nitrogen. Cells from large Petridishes were diluted one or two days before freezing to ensure that theywould be in logarithmic growth phase. The cells from three or four15x100 mm petri dishes were collected by centrifugation and resuspendedin 10m1 DME (with 20% FBS and 10% DMSO) that had been equilibrated in10% CO2 at 37 °C. One ml aliquots of cell suspension were added toten freezer vials, and the vials were stored in a styrofoam box in a-70 °C freezer overnight before being transferred to liquid nitrogen.After subcloning, three positive cell lines were obtained: AC3, AA3and CC3. The isotypes were determined to be IgG2bk (AA3 and CC3) and72IgG1K and A (AC3). The AC3 cell line was subcloned two more times; aftereach cycle the supernatants from all subcloned wells with cells werepositive for both the K and A light chains.The cells were grown on a large scale in serum free medium withsupplements (see Table IV). The cells were grown in 15x100 mm Petridishes and were diluted slowly, with no more than a two-fold dilution inone day. The supernatants from the cultures were filtered with a 0.22 gmfilter to remove residual cell and cell fragments left in suspensionafter centrifugation, and were filtered with a YM-100 Amicon filter toconcentrate the antibody. The antibodies were purified as follows usinga column modified with protein A, a protein isolated from the cell wallof S. aureus that binds specifically to the Fc portion of antibodies(protein A modified column from Pharmacia, Dorval).2.4b Antibody purificationThe IgG1 from the AC3 cell line was purified (130) in high salt toincrease binding to the protein A column. The concentrated tissueculture supernatant was transferred to 3.0 M NaCl with 0.1 M sodiumborate, pH 8.9, using the Amicon filter to retain the protein while thebuffer was exchanged. The protein solution was applied to the protein Acolumn, which was then rinsed with 10 column volumes of 3.0 M NaCl with50 mM borate pH 8.9, and with another ten column volumes of 3.0 M NaClwith 10 mM borate. The bound protein was eluted with 0.5 ml aliquots of0.1 M glycine (pH 3.0). Fractions of 0.5 ml volume were collected andwere mixed immediately with 50 gl of 1M Tris pH 8.0. Theprotein-containing fractions were determined by measurement of theabsorbance at 280 nm, and were pooled and stored at -70 °C. The proteinA column was cleaned after each use by washing sequentially with 1073Table IV: Supplements for serum - free medium (131)1. Stock solutions of supplements were made up or purchased atconcentrations listed below. The protein solutions and oleic acid werestored at -20 °C, and all other solutions were stored at 4 °C.insulin^bovine insulin (Sigma) 2 mg/ml in water that had beenadjusted to pH 2.5 with HC1transferrin^Human transferrin (Sigma) 2 mg/ml in DMEethanolamine^ethanolamine (Sigma) 2mM in DMEsodium selenite Na 2 Se03 (Sigma) 200 AM in DMEoleic acid^sodium oleate (Sigma) 431 Ag/m1 in DME (to give a freeacid concentration of 400 pg/m1)pyruvate^100 mM solution from Gibcoamino acids^10 mM non-essential amino acids solution from Gibco.2. Final concentrations of supplements in serum-free medium:insulin^ 5 mg/m1transferrin 35 pg/m1ethanolamine^20 AMsodium selenite 2 pMoleic acid 4 AMpyruvate^ 1 mMnon-essential amino acids 0.1 mM74column volumes of 2M urea, 1M LiC1 and 100 mM glycine.The IgG2b from the AA3 and CC3 cell lines was purified (132) in lowsalt. The pH of the culture supernatant was adjusted to 8.0 by additionof 1/10 volume of 1.0 M Tris pH 8.0. The antibody solution was appliedto the column which was then rinsed with 10 column volumes of 100 mMTris pH 8.0 and with another ten column volumes of 10 mM Tris pH 8.0.The bound protein was eluted the same way as the IgGl.2.4c ELISA results with the monoclonal antibodiesThe three monoclonal antibodies obtained from the cell fusion werepurified in order to increase the chances of obtaining an antibody thatwould perform well in a solid phase immunoassay. Since monoclonalantibodies have only one binding specificity, it is possible forantigens in an inappropriate orientation on a surface to present noantibody binding site towards the surrounding solution. Since the threeantibodies raised seemed to have similar affinities for ferrichrome A onthe assay plates (Fig. 24), only one was chosen for use in this project.The antibody AC3 was chosen because the the other two antibodies wereof the subclass IgG2b, which tends to precipitate in low saltconcentrations (133) (it also had a slightly higher affinity forferrichrome A).The binding of the antibody AC3 was inhibited by free ferrichrome Ain solution (Fig. 25).2.4d SDS-PAGE procedure for the antibody AC3Polyacrylamide gel electrophoresis in sodium dodecylsulfate(SDS-PAGE, 134) was used to estimate the purity of the AC3 antibodypreparation. Reagents were combined as described in Table V and cast asrod gels on 125 mm long glass tubes with an inner diameter of 5 mm. An751^1^IIILIII^1^1^1^1^1 1 1 1 I^1^I^1^1^1 1 I I I^1^1^1^I^1 1 1 1 1^1^1^I^I^I I I I1E-06 1E-05 0.0001 0.001 0.01[Antibody] mg/ml1E-07Figure 24: ELISA with the purified monoclonal antibodies and ferrichromeA/BSA adsorbed to the plate. Results are shown for AC3 (m), CC3 (o) andAA3 (A).761.20.1 110.8e 30A decrecase0.40.2^I ^I^1 1 I 1111^I^I^1 [11111^I^I^1 1 11111^I^I^1 1 1 1 111^I^I^1 I 1 1111^1E-07 1E-06 1E-05^0.0001 0.001 0.01[ferrichrome A] mg/mlFigure 25: ELISA with monoclonal antibody and ferrichrome A/BSA adsorbedto the plates: inhibiting the antibody with free ferrichrome A.---- indicates absorbance for antibody with no ferrichrome A in solution(antibody concentration = 1.4x10- mg/m1).77overlay of the gel solution without acrylamide was added to ensure aflat upper surface on the gel. After the acrylamide had gelled at roomtemperature, the overlay solution was flushed away with running buffer,the samples were mounted in a Bio-Rad model 150A electrophoresischamber, and the apparatus was equilibrated at 4 °C. The antibody inbuffer was mixed with reducing or non-reducing sample preparation buffer(Table V) at a 1:1 ratio by volume and boiled for one minute. Sampleswere layered on to the gel surface and the current was set at 0.5 mA pertube until the samples entered the gel. The electrophoresis was thencarried out at 4 °C at a current of 6 mA per tube.Gels were removed from the tubes with a 2% w/v solution of glycerolin water added with a flexible hypodermic needle inserted between thegel and the tube wall. The position of the pyronin Y tracking dye wasmarked with ink and the gels were stained and fixed for one hour withCoomassie Blue solution (Table V), followed by destaining in a solutionof 30% v/v methanol and 5% v/v acetic acid (135).The gels were scanned for dye binding at a wavelength of 595 nm in aBeckman Model 25 spectrophotometer fitted with a gel scanningattachment.2.4e SDS-PAGE results for AC3: an indication of the antibody purityThe characteristic proteins in human serum were used as molecularweight standards for the gel scans (Fig. 26). Molecular weight wasplotted as a function of mobility on the gel and the calibration curvethus obtained was used to estimate the molecular weights of the proteinin the antibody samples (Appendix 2). The non-reduced IgG showed amolecular weight of about 101 000 g/mol. This is much lower than theactual molecular weight for mouse IgG of about 160 000, but mouse IgG178Table V: Preparation of gels and reagents for SDS-PAGE1. Stock solutions. The following solutions were made up for use inpreparation, running and staining of the SDS-PAGE gels:10x buffer^0.4 M Tris, 0.2 M sodium acetate, 0.02 M EDTA,pH 7.430% acrylamide/BIS 30% w/v acrylamide (Bio-Rad, Richmond CA),1.2% w/v N,N'-methylene-bis-acrylamide(Bio-Rad)The acrylamide/BIS solution was degassed beforeuse.Running buffer^100 ml 10x buffer and 50 ml 4% SDS (Bio-Rad),made up to one litre with water.Sample reagent^20 mM Tris-HC1, 2 mM EDTA, 2% w/v SDS, 14% w/vsucrose, 20 pg/m1 pyronin Y, with pH adjusted to8 using acetic acid.Reducing sample reagent as above with 50 mM 2-mercaptoethanolCoomassie blue stain/fix 0.2 g Coomassie Blue G250, 27.5 ml methanol,30.5 ml acetic acid, made up to 500 ml withwater.2. Reagents for casting gels. Reagents were combined in the order listed,to give a final acrylamide concentration of 3%.30% acrylamide/BIS^ 3 ml10x buffer^ 3 ml4% w/v SDS 1.5 ml0.5% w/v N,N,N',N'-Tetramethylethylenediamine(TEMED, Bio-Rad)^ 1.5 mlwater^ 18 ml1.5% w/v ammonium persulfate (Bio-Rad)^3 ml790.9-0.8-0.7-(3) -L0 0.6^)U)a)Ca-oC)0.3-0.2-c,0.2^0.4^0.6^0.8^1Relative mobilityFigure 26: SDS-PAGE of human plasma proteins as molecular weightstandards (a= IgM, 950 kD, b= azM, 750 kD, c= fibrinogen, 340 kD,d= IgG, 160 kD, e= transferrin, 76 kD, e= transferrin, 76 kD,f= albumin, 66 kD (136)1280has been reported to have a fast electrophoretic mobility (133) thatwould give it a low apparent molecular weight.The gel scans show one band for the non-reduced sample and twobands for the reduced sample, as expected for IgG (Figs. 27 and 28),indicating that there was no Coomassie Blue staining protein impuritypresent in greater than trace amounts. Addition of mercaptoethanolreduces the disulfide bonds that help hold the IgG tetramer (Fig. 5)together. Addition of the SDS disrupts the remainder of the bondsmaintaining the integrity of the IgG so that the heavy and light chainsseparate and run as distinct bands.2.5 Synthesis of silica beadsSilica beads were made by hydrolysis of tetraethyl orthosilicate(TEOS, Aldrich, Milwaukee) and subsequent condensation of silicic acid(104). Monodisperse beads are probably formed as a result of a limitednumber of nucleation sites being produced at the start of the reaction,when the concentration of silicic acid exceeds a critical value Csat(132). If Csat* is greater than the saturation concentration of silicicacid in solution, the silicic acid will condense on the sites that havebeen formed and eventually lower the concentration below Csat , afterwhich particle growth will continue, but no new nucleation will occur.If nucleation sites are only formed over a short period of time, thenthe final bead preparation will have a narrow size distribution.The reaction mixture contained ethanol, water and TEOS inproportions of 150:30:6 by volume (100). An ethanol/water solution(464.6 g ethanol, 117.5g water) was saturated with ammonia at 4 °C bybubbling ammonia through the solution until the solution volume stoppedincreasing. This resulted in 179.7 g of ammonia being dissolved in the810.9-0.8-0.7-u9 0.6-Crn 0.5--aTrs.1-1 0.4-0.3-0.2-0.1-0.2^0.4^0.6^0.8Relative mobilityFigure 27: SDS-PAGE of the non-reduced monoclonal antibody AC3. Arrowsindicate position of the human plasma protein standards (Fig. 26) (c=fibrinogen, 340 kD, d= IgG, 160 kD, e= transferrin, 76 kD, f= albumin,66 kD).82 0.90.80.7<ocr)0 0.6ciE 0.5a)-aTaa-ti 0.40a0.3^1^sie0.20.1€^iI^I^I0.2 0.4 0.6Relative mobility^A s•-----...---..--..I0.8^I 12^_...iFigure 28: SDS-PAGE of the reduced antibody AC3. Arrows indicate positionof the human plasma protein standards (Fig. 26) (c= fibrinogen, 340 kD,d= IgG, 160 kD, e= transferrin, 76 kD, f= albumin, 66 kD).83solution. The reaction flask was transferred to a -20 °C cold room andequilibrated in an ethylene glycol bath. Distilled TEOS (21.75 g) wasadded to a glass syringe and also equilibrated at -20 °C. The TEOS wasadded as rapidly as possible through the syringe while theethanol/water/ammonia solution was being stirred rapidly.The reaction mixture turned opalescent four minutes and 30 secondsafter addition of the TEOS, indicating formation of the nucleationsites. The reaction was allowed to proceed overnight, after which thesilica beads were collected by centrifugation.All glassware used in producing the silica beads was cleaned withchromic acid before use to minimize contamination on the surface of thebeads.Spherical beads with a fairly uniform size distribution wereobtained (Fig. 29). The bead size distribution was determined usingphotographs with a total of 314 beads. The area of each bead wasdetermined by tracing the photograph on a digitizing board and usingcommercial analytical software (Bioquant). The diameter of the beadswas calculated by assuming that images of the beads were circular. Anaverage diameter of 0.779 ±0.042 pm (± 1 s.d.) was calculated, with abead size distribution shown in Fig. 30. As can be seen from thephotograph and from the size distribution, the scatter in the beaddiameter results from bead aggregation (or from nucleated sitesaggregating before the polymerization has finished). Largernon-spherical beads can be seen in the photographs and appear to be theresult of aggregates that have formed during the polymerization.The density of the silica beads was measured by centrifugation in asulfuric acid solution. The beads were suspended in the acid solution by84IAA. 711. F••••-•-•-•Figure 29: TEM photograph of silica beads. Arrows indicate examples ofnon-spherical beads.8570605040302010-5a -4a -3a -2a -10^+1 a +20 +3a +4a +50BEAD DIAMETERFigure 30: Silica bead size distribution showing the number of beads asa function of bead diameter, where bead diameter is shown as X ± no (X =0.779 pm and r = 0.042 pm)86sonication and vortexing and then centrifuged at 5 500 x g. Inconcentrated sulfuric acid solutions, the beads floated to the top afterthe centrifugation. The density of the sulfuric acid for the isopycnicsystem was measured and found to be 1.83 g cm-3.The specific area of the beads was 4.2 x 104 CM2 g-1 .2.6 XPS measurementsX-ray photoelectron spectroscopy (XPS) measurements were done by Dr.Philip Wong using a Leybold Heraeus MAX 200 spectrometer. Silica orglass slides cleaned as described in section 2.7 were put under vacuumand were stored for up two months before analysis. The samples weremounted on the sample holder using copper strips and transferred throughair to the loading chamber of the XPS machine, which was evacuatedovernight to a final pressure of approximately 1x10-8 mbar. Thesamples were then transferred to the analysis chamber and analyzed at apressure of 8x10-9 mbar using a 2x4 mm sample area near the centreof the slide, chosen visually to avoid the copper binding strips.Samples were analyzed using an Alka 1486.6 eV X-ray source (excitationvoltage 15.0 kV, emission current 25.0 mA) or with a Mgka 1253.6 eVsource (excitation voltage 15.0 kV, emission current 20.0 mA).Ejected photoelectrons were measured normal to the surface of thesilica unless otherwise specified. The kinetic energy of thephotoelectrons was measured using a hemispherical analyzer at a constantpass energy of 192 eV. The carbon is peak at 285.0 eV (108) was usedfor charge correction of the measured binding energies.The relative amounts present were calculated for all elementsdetected with photoelectrons in the binding energy range of 100-1100 eV(Mgka X-ray source) or 100-1300 eV (Alka X-ray source). Areas under87characteristic peaks for each element were measured from narrow scans ofthe peak region, with baselines determined visually. Atomic percentagesfor the elements were calculated using the elemental sensitivity factorsprovided by the instrument manufacturer.2.7 Cleaning the silicaSilica was cleaned before modification with the silane in order toremove the contaminating surface layer of hydrocarbon. Since thereshould be no carbon present in the substrate, samples can easily bechecked for contamination by taking XPS measurements to determine theatomic % of carbon in the surface region, as described in section 2.6.Initial studies were made using glass slides (Canlab). Variouscleaning and drying procedures were tried to minimize the amount ofcarbon at the surface. Subsequent studies were carried out with clearfused quartz microscope slides (Quartz Scientific International,Fairport Harbour). The slides had trace impurities, but none insufficient quantities to be detectable by XPS (Na 1.0 ppm by weight, Ca0.6 ppm, Mg 0.1 ppm, and other elements in the ppm range, 133).Silica or glass slides were cut into 8x8 mm sections, wiped cleanwith lens paper, and placed in separate 13x100 mm glass test tubes. Allcleaning and drying procedures were carried out in these test tubes inorder to minimize sample handling.Slides were heated at 80 °C for one hour in chromic acid made withFisher Chromerge solution and concentrated sulfuric acid or with aslurry of 5 g potassium dichromate and 5 g water added to 100 ml ofconcentrated sulfuric acid. Some slides were rinsed with water(distilled then deionized using a Millipore milli-Q ion exchangeapparatus), and dried in a laminar flow cabinet equipped with a High88Efficiency Particle Air filter to reduce dust. Some slides were given anadditional brief rinse with concentrated hydrochloric acid (BDH AnalaR)and then rinsed again with water and dried in the flow hood.Some samples were then cleaned with the same procedure (chromic acidfollowed by an HC1 rinse) and dried in vacuum; this resulted in asignificantly lower amount of carbon at the surface (section 3.1).Samples for subsequent experiments were cleaned for one hour in chromicacid at 80 °C, rinsed with water, rinsed briefly with concentrated HC1,rinsed again with water, and then dried and stored under vacuum.The silica beads were cleaned by the same procedure, but werecleaned in a glass centrifuge tube so that they could be rinsed bycentrifugation and resuspension in the tube.Cleaned slides were stirred for one hour in ref luxing heptane (BDHOmniSolv, BDH, Toronto), the solvent used for the silylation reaction(section 2.8). The slides were dried, rinsed with water and dried again,following the same procedure used for the silylation to determine theeffect of the solvent.Some attempts were made to clean the slides with hot concentratednitric acid, but this gave irreproducible results with some very highoxygen to silicon ratios. This may be due to the silica being oxidized.2.8 Surface modification of the silica with silaneSilica slides and beads were modified with alkyl trialkoxysilanes,which react as shown in Figure 9. The alkoxy groups hydrolyze to formsilanols, which then react by condensation with silanols on other silanemolecules or on the silica surface. The water required for hydrolysiscan be supplied as water adsorbed to the silica or in the solvent. Thereaction is often carried out in water (137), but this can result in89polymerization of the silane in solution to form large clumps, whichthen react with the silica surface to form an uneven coating (134).All glassware was cleaned with chromic acid before use. Freshlydistilled silane was added to heptane (BDH OmniSolv) at a 1% w/v ratioand stirred briefly before addition of the silica beads or slides. Thereaction was carried out in a 250 ml round bottom flask stirred from thebottom with a magnetic stirrer. The silica beads dispersed readily inthe silane solution. The solution was heated to reflux for one hour,after which the silica was rinsed in fresh heptane, dried under vacuum,and stored overnight at room temperature (22 °C) under vacuum. Themodified silica was then sonicated in water for one minute to remove anypolymerized clumps of silane adsorbed to the surface, and dried undervacuum after removal of most of the water with a pipette.2.9 Ferrichrome A on beads: non-specific adsorption2.9a Measurement of ferrichrome A adsorptionSilica beads were modified with 3-aminopropyltriethoxy silane asdescribed above. The amount of ferrichrome A adsorbed or attached to thebeads under various conditions was determined by solution depletionmeasurements of ferrichrome A as measured from the absorbance.Modified silica beads were weighed into 1.5 ml polyethylenecentrifuge tubes. For a series of measurements with a given weight ofbeads, all samples were within 2% of the stated amount. The ferrichromeA solution was centrifuged to clarity before use to ensure that therewas no undissolved ferrichrome A in suspension, and the concentration ofthe resulting solution was determined from the absorbance. The solution(0.75 ml) was pipetted onto the bead samples, which were immediatelysuspended by sonicating and vortexing. The samples were then mixed at90room temperature, after which they were centrifuged at 11 600 xg and thesupernatants were collected. The supernatants were centrifuged againbefore the concentration of ferrichrome A in solution was measured. Theconcentration of ferrichrome A was determined from the derivative of theabsorbance curve as described in section 2.1.The amount of ferrichrome A which washed off the beads in PBS wasalso measured. All the solution on top of the pellet was removed byblotting with a piece of tissue paper. The beads were resuspended in0.75 ml PBS using a glass stir rod followed by sonication and vortexing.The amount of ferrichrome A which washed off into PBS was measured fromthe derivative of the absorbance curve, and the amount of ferrichrome Aleft on the beads after the PBS rinse was calculated.The weight of the beads was varied, as well as the adsorption time,ferrichrome A concentration, and salt concentration. The amount offerrichrome A adsorbed per unit area (specific adsorption) wascalculated from the amount lost from solution and the surface area ofthe beads. The surface area of the beads was calculated from the weightof the bead sample and the specific area of 4.2 x 10 4 CM2 g-1 (section2.5).2.9b Effect of bead storage time on ferrichrome A adsorptionIf the beads were stored after modification with the silane, theywould not suspend readily when sonicated. Some beads were left as large(easily visible) lumps that settled out rapidly, although a portion ofthe beads did suspend as usual. The total amount adsorbed decreased asthe beads were stored for longer periods of time (Table VI). Thisdecrease is presumably due to a smaller area of beads being available,and does not indicate a smaller amount per unit area. A large effect was91Table VI: The amount of ferrichrome A adsorbed as afunction of the storage time for the beads between thesilylation and the ferrichrome A adsorptionStorage time(days)amount adsorbed(mg/m2)0 2.122 2.010 1.03Table VII: The amount of ferrichrome A adsorbed as afunction of the time for adsorption (equilibriumsolution concentration of ferrichrome A = 0.27 mg/miladsorption time(min)amount adsorbed(mg/m2)1 2.202 2.2010 2.1520 2.2060 1.911200 1.1892seen if the beads were stored for a week before use. Unless otherwisespecified, experiments were carried out within two days of the beadsbeing sonicated in water and dried in the final step of themodification. Samples were checked after sonication to ensure that therewere no visible aggregates remaining.2.9c Adsorption as a function of timeThe ferrichrome A adsorbed rapidly to the beads (Table VII), with nochange in the amount adsorbed being observed between a one and twentyminute adsorption time. A decrease in the specific adsorption was seenas the beads settled out of suspension overnight (Table VII). A tenminute adsorption time was chosen for subsequent experiments2.10 Reaction of ferrichrome A and EDC2.10a Some possible reactions of ferrichrome A and EDCCarbodiimides can be used to promote the formation of peptide bonds,by a mechanism shown in Figure 31. Figure 31 also shows an interferingside reaction: the activated ester formed by reaction of the carboxylgroup with EDC can hydrolyze to give the carboxyl group as before and aurea. The rate of hydrolysis may be very rapid, with a rate constant ashigh as 2-3 s-1 at pH 4.7 (138). This would give a half life for theactivated ester of 0.28 s.Ferrichrome A could also react through the serine group to form astable o-acylurea, or possibly through the hydroxamate groups(Fig. 11). The iron is very tightly bound (the stability constant ofdesferriferrichrome A and Fe3+ is a 1032 (137)) , but will exchangefreely with iron in solution, so the hydroxamate groups could beavailable transiently to react with EDC.93H--HH2 (SI LATED SILICA)R-S°0-FERRICHROHE A^ EDC(ACTIVATED ESTER INTERMEDIATE)CH +3 >H-CH2CH2CH2--NH—C.11—ClICH 3CH3 0A‘,RHYDROLYSIS ^ AMIDE LINKAGEBACK TOCARBOXYLATE GROUPCH3>I—CH2CH2CH2-^4M-CH2CH3CH30- A CYL I SOURE AFigure 31: Carbodiimide coupling mechanism942.10b Reactions of ferrichrome and EDCFerrichrome is another metabolite of Ustilago sphaerogena that doesnot have a serine residue or any carboxyl groups (Fig. 32). Ferrichromewas kindly provided by Allen Budde, Department of Plant Pathology,University of Wisconsin. Ferrichrome and EDC were mixed together to showthat the peak that appears at 367 nm in absorbance spectrum of theferrichrome A/EDC mixture is not due to the reaction of EDC with thehydroxamate groups.2.10c The EDC:ferrichrome A mole ratioThe ratio of ferrichrome A to EDC used was chosen by determining theamount of EDC required to solubilize the ferrichrome A rapidly.Ferrichrome A is not very soluble in water, but will dissolve readilyafter reaction with EDC. Ferrichrome A was added to water at aconcentration of 4.06 mg/ml and suspended by sonication. The suspensionwas pipetted into separate centrifuge tubes, and dry EDC was added togive mole ratios of EDC:ferrichrome A ranging from 0 to 68.6. Themixtures were sonicated for ten minutes, after which the undissolvedferrichrome A was separated by centrifugation and the supernatant wasremoved. The supernatant was diluted by a factor of 10 in water and theabsorbance was measured.Figure 33 shows that increasing amounts of ferrichrome A dissolvewithin ten minutes as the EDC concentration increases. All theferrichrome A is dissolved when EDC is added at a mole ratio slightlyhigher than 40:1. Although this does not necessarily mean that all thecarboxyl groups on ferrichrome A have reacted with EDC at this moleratio, it does imply that up to a ratio of 40:1 of EDC:ferrichrome A,950(C82 ) 3ZN\O\NH(CH2 ) 3 \/H.... at Of- -^N^C •°` °NH• )(riP3*.- l I•,. or- CNH0 = C H3c Ci ^1cH3 I\^\ ,c.- ::N^( C712 ) 3^OfNHNHOfHFigure 32: Structure of ferrichrome96"E as&CLI-OBco a2s.2= mu-Ira 0.14•^:0^ioMolar excess of EDCFigure 33: Choosing the EDC: ferrichrome A mole ratio. Increasingamounts of ferrichrome A were added to a 4.06 mg/ml suspension offerrichrome A. The amount of ferrichrome A in solution after ten minuteswas measured and plotted as a function of EDC:ferrichrome A mole ratio.caw 0:1^ais cia ais 0.3 nig 0:4[fenichrome A In solution] mg/m1Figure 34: Determining the concentration of ferrichrome A from thederivative of the absorbance spectrum dA/dA for a solution with a 50:1mole ratio of EDC: ferrichrome A.97increasing the amount of EDC will increase the extent of the reactionwith ferrichrome A. A ratio of 50:1 was chosen for future experiments.2.10d Extinction coefficient for ferrichrome A with EDCConcentration of ferrichrome A in the presence of a 50:1 ratio ofEDC was determined from the derivative of the absorbance curve at 395 nmand at 492 nm. A series of different concentration solutions offerrichrome A was made up in water. Concentration of ferrichrome A wasdetermined from the absorbance of the solutions. The EDC was added tothe solutions at a 50:1 ratio, and the derivative of the absorbance wasmeasured. The difference between the derivative at 395 nm and at 492 nmwas plotted against concentration (Fig. 34); information from a linearleast squares fit of this data was used to calculate the concentrationof ferrichrome A in EDC solutions (the slope was 0.0567±0.0004, where0.0004 = 1 a.).2.11 Modification of silica beads with ferrichrome A and EDC2.11a The reaction procedureSilylated silica beads were weighed into polyethylene centrifugetubes. Ferrichrome A solutions were made in water (unless otherwisespecified), and were centrifuged to clarity before use. The ferrichromeA concentration was determined from the absorbance, and the amount ofEDC required to give a 50:1 ratio was also weighed into polyethylenecentrifuge tubes. The ferrichrome A solution was added to the EDC andmixed for 40 s before being pipetted onto the beads (0.75 ml).Immediately thereafter, the beads were sonicated and vortexed until ahomogeneous suspension was obtained. The suspension was then mixed atroom temperature (21-22 °C) for 10 min before the beads were separated98by centrifugation. The supernatant was removed and stored in anotherpolyethylene centrifuge tube and was centrifuged again to removeremaining beads before the absorbance was measured. The excesssupernatant was removed from the initial bead pellet by blotting withtissue paper, and the beads were resuspended in 0.75 ml PBS by stirringwith a glass rod followed by sonication. The PBS supernatant wascollected after centrifugation and stored in a polyethylene centrifugetube before the absorbance was measured. These samples were alsocentrifuged again before the absorbance was measured.2.11b Some comments on the procedureForty seconds was chosen as the minimum convenient mixing time. Thisis a very long time if the rate constant for hydrolysis is on the order-1of 2-3 s (138), but resulted in successful attachment of theferrichrome A to the silica (see 3.3c).Since ferrichrome A is a cyclic peptide, there are no amine groupsand no concern about the ferrichrome A cross linking to itself beforeaddition to the beads.The beads could be suspended in a small volume of EDC solution,followed by addition of ferrichrome A. This would avoid the 40 s mixingtime during which the activated ester could hydrolyze before addition tothe beads. Although this might result in more ferrichrome A attached tothe beads, the method used was chosen to simplify measuring the amountof ferrichrome A adsorbed: one ferrichrome A and EDC solution could bemade in sufficient volume to add to replicate samples of beads, withsolution left over to measure the concentration of ferrichrome A in theinitial solution directly by absorbance rather than by calculation ofdilution factors.992.11c Varying the pH of the reaction mixtureThe pH of the ferrichrome A solutions with EDC in water was about5.7. Since the activated ester may be more stable at lower pH (138), someexperiments were carried out after addition of hydrochloric acid to theferrichrome A solution to lower the pH to 4.7 after mixing with the EDC.This resulted in a 47% decrease in the amount of ferrichrome A left onthe beads after rinsing with PBS, and all subsequent experiments werecarried out in water.2.12 The ELISA procedure2.12a The ELISA procedure for beadsSilica beads (5 or 10 mg/tube) were modified with ferrichrome A inpolyethylene centrifuge tubes at a range of ferrichrome A concentrationsin water, following the procedure outlined in 2.11a. A 50:1 mole ratioof EDC: ferrichrome A was used, and the EDC and ferrichrome A were mixedfor 40 s before addition to the beads. After ten minutes, the beads wereseparated from the solution by centrifugation and washed three timeswith PBS. The beads were resuspended in one ml of PBS, followed by a1:10 dilution of the suspension in more PBS. The diluted suspension waspipetted into plastic centrifuge tubes in order to give 74 mm2 totalbead area per sample.The beads were coated in blocking buffer, rinsed, incubated withantibody, and incubated with HRP conjugated antibody in the same way asthe silica slides. The ELISAs were developed by suspending the beads in250 gl of citrate/acetate buffer (Table II), followed by addition of afurther 250 gl of substrate solution that was made up with twice the TMBand hydrogen peroxide concentrations that were used for the slides, inorder to give final concentrations that were the same. The beads had to100be suspended before adding the substrate because of the time required tosuspend the beads and the necessity of incubating the beads with thesubstrate for a precise amount of time. The reaction with the substratewas stopped by addition of 250 gl of 2.0 M phosphoric acid. Thesubstrate solution was again transferred to an ELISA plate so that allthe absorbances could be read at once, but 50 gl of solution was addedto each well instead of 150 gl because of the higher absorbances found.2.12b The ELISA procedure with flat modified silicaPieces of flat silica were modified with 3-aminopropyl silane inseveral batches until enough had been accumulated for an assay. Thesilylated silica was stored in air between modification and use. Piecesof silica (approximately 4x8 mm2) were placed in separate glass testtubes. Ferrichrome A solutions at a range of concentrations in waterwere added to EDC with a 50:1 mole ratio of EDC to ferrichrome A. TheEDC and ferrichrome A were mixed for 40 s before being added to theslides (0.5 ml/test tube). The tubes were then mixed on a orbital shakerfor ten minutes before the slides were removed and rinsed with PBS.The remainder of the ELISA steps were carried out with the modifiedslides remaining in the same test tubes, following the proceduredescribed in Fig. 2. Uncoated sites were blocked by incubation for 30 minat 37 °C in blocking buffer (0.2% Carnation skim milk and 0.05% Tween 80in PBS). Slides at different surface concentrations of ferrichrome Awere incubated with a dilution series of antibody in blocking buffer,then with an HRP conjugated antibody also diluted in blocking buffer.Both of the antibody incubation steps were done at 37 °C for one hourwith 0.5 ml solution. The slides were rinsed three times after eachincubation step by vortexing in PBS-Tween.101The slides were next transferred to clean test tubes to avoidproblems with antibody adsorbed non-specifically to the walls of thetube. The ELISAs were developed with 500 gl of the TMB solution used insection 3.3a (Table II). The reaction was stopped after three minutes byaddition of 250 gl of 2.0 M phosphoric acid and the substrate solutionwas transferred to an ELISA plate (150 gl/well) so absorbances of allthe samples could be read at once with an SLT plate reader. Theabsorbance was read at 450 nm, with a reference at 620 nm.Because the slides were not precisely 64 mm2, the area of each slidewas measured. The area was assumed to be directly proportional to thefinal absorbance reading (section 2.3f) and was used to correct thereadings to that for a sample with an area of 64 mm2. The areas weremeasured by photographing the slides on a background of 1mm2 graph paperand then digitizing and measuring the area of the photographs with theBioquant program, using the graph paper for calibration.2.12c The ELISA procedure for polystyrene platesAssays with polystyrene plates were carried out as described insection 2.3.2.12d ELISAs with plates: measuring the equilibrium concentration ofantibody in solutionThe antibody concentration given for the ELISA results is, unlessotherwise specified, the initial concentration of the antibody solutionadded to the ELISA plate or test tube. As the antibody binds to theantigen on the substrate surface, the solution concentration of antibodywill decrease. The surface concentration of bound antibody is a functionof the equilibrium concentration of antibody (85) so the equilibrium102concentration would be a more relevant parameter for binding studies.The equilibrium concentration of antibody after incubation wasmeasured using AC3 labelled with 1251 (section 2.15a).2.12e Inhibition of the ELISA with free ferrichrome AThe inhibition of antibody binding by free ferrichrome A in solutionwas also measured. The ELISAs were carried out as before, but usingonly one concentration of antibody, and incubating it with ferrichrome Abefore addition to the silica. The antibody was made up to twice thedesired final concentration in blocking buffer and pipetted into aseries of test tubes. An equal volume of ferrichrome A solution inblocking buffer was added and the antibody and ferrichrome A wereincubated for 30 minutes before being added to the silica. The finalconcentrations of ferrichrome A ranged from 5x10-2 mg/ml to5x10-8 mg/ml.2.13 Characterization of the substrate: detection of the amines onmodified silica2.13a XPS measurements on flat silicaThe silica slides were analyzed by XPS as described in section 2.6,and the percentage of nitrogen on the surface was determined.2.13b Ninhydrin assay of amine groups on the silylated beadsThe extent of modification on the beads was estimated by an assay ofthe amine groups using ninhydrin (139). A ninhydrin solution (0.5 ml 10mM ninhydrin in 100 mM sodium acetate pH 5.0) was added to 10 mg ofmodified beads. The beads were sonicated but remained in small clumpsdue to the high buffer concentration. The beads and ninhydrin were then103placed in a boiling water bath for 5 minutes, during which time thebeads turned blue. When the beads were shaken, the blue colour came offinto solution, leaving the beads white. The remainder of theamine-ninhydrin reaction product was washed off the beads with a 50 v/v%solution of ethanol in water. The supernatants from the beads and theethanol rinse were collected and diluted to one ml. The absorbance ofthis solution at 570 nm was then used to determine the amount of amineson the surface, with glycine (Fisher, Vancouver) used as a standard.2.14 Characterization of the antigen on the substrate2.14a Measuring the amount of ferrichrome A on the beads by solutiondepletionThe silylated beads used in the assays were coupled to ferrichrome Ausing EDC at a 50:1 EDC:ferrichrome A mole ratio in water (section2.11). The surface concentration of ferrichrome A on the beads wasdetermined for different equilibrium solution concentrations offerrichrome A.2.14b BCA assay of the amount of ferrichrome A on the beadsThe amount of ferrichrome A on the beads was also measured by abicinchoninic acid (BCA) assay (Pierce, Rockford, IL). The assaymeasures protein indirectly by measuring the amount of Cu2+reduced to Cu+ by protein in alkaline medium. The BCA complexes with Cu+to form a purple product that can be detected by measurement of theabsorbance at 562 nm and with an appropriate standard curve theabsorbance at 562 nm can be used to determine protein concentration.Since Fe3+ can also complex with the BCA to form a product with anabsorbance at 562 run, BCA was added to a ferrichrome A concentration104higher than that used for any of the standard curves, without additionof any Cu*. No colour change was observed.A standard curve was made up using the Pierce microassay procedurewith ferrichrome A as a standard. Ten microlitres of ferrichrome Asolution was added to a well in a 96 well ELISA plate. Pierce BCAreagent mixed according to instructions (containing BCA, Cu2+, andbuffer) was added (200 Ml/well) and the plate was mixed for 30 s beforebeing incubated at 37 °C for varying lengths of time. The absorbance wasthen read at 560 nm. The concentration of ferrichrome A on the beads wasmeasured by suspending the beads at a concentration of 50 mg/ml andtaking a 10 Ml aliquot.2.14c XPS measurements of ferrichrome A on the slidesSilica slides modified with ferrichrome A at differentconcentrations were analyzed by XPS following the procedure described insection 2.6. A slide with ferrichrome A deposited from an aqueoussolution was also measured by XPS. Successive drops of ferrichrome Asolution were placed on the slide and dried, until a clear red mark fromthe ferrichrome A could be seen on the slide.2.14d Ninhydrin assays of slides modified with acetic acid and EDCSilylated beads and slides were modified with acetic acid using EDCto promote formation of a peptide bond. The EDC was added to a 1.2 mMsolution of acetic acid in water at a 15:1 mole ratio of EDC:aceticacid. The amine groups on the beads were assayed before and aftermodification to check the efficiency of the acetylation. Acetamide(Fisher, Ottawa) was used as a control for the ninhydrin assay of theamide groups.1052.15 Antibody adsorption isotherms on the modified silica2.15a Labelling the antibody with 1251The purified AC3 was labelled with 1251 using Bio-Rad enzymobeads(Bio-Rad, Hercules, CA) which have covalently attached glucose oxidaseand lactoperoxidase. The enzymobeads are added to a protein solution withglucose and iodide. The glucose oxidase oxidizes g-D-glucose to formhydrogen peroxide. The lactoperoxidase then reacts with the peroxide andiodide to form iodine, which reacts predominantly with the phenolichydroxyls on tyrosine residues, displacing them and replacing them withan iodide group (140).One vial of enzymobeads from Bio-Rad was rehydrated by addition of0.5 ml of water. A fresh 1% glucose solution was prepared in 0.2 Msodium phosphate pH 7.2. The rehydrated enzymobead suspension (50 gl)was added to a polystyrene test tube and mixed gently. The antibody inphosphate buffer (0.2 M, pH 7.2) was added to the enzymobead suspension(200 gl of antibody solution at 0.616 mg/ml, 123 gg total). Sodiumiodide with 125I from Amersham (Oakville, Ont.) was added (5 gl at2.63x108 dpm/ml, 1.315x106 total), and the reaction was started byaddition of 50 gl of the glucose solution.The free iodide was separated from the labelled protein using aSephadex G-25 gel exclusion column. A solution of unlabelled IgG (AC3)was run through the column to minimize sticking of the labelled IgG. Thelabelled protein mixture was run through the column in the same phosphatebuffer used in the reaction and twenty drop fractions were collected.One gl aliquots were removed from each fraction using calibratedcapillary tubes which were counted with an LKB 1282 Compugamma gammacounter. The counts indicated two peaks in the eluate from the column.Since the antibody elutes in the void volume, the two highest activity106fractions from the first peak were pooled. The protein concentration wasdetermined by absorbance at 280 nm (1 OD = 0.8 mg/ml (130)), and theactivity was determined by counting ten 1 gl aliquots in capillary tubes.A specific activity of (3.83±0.10)x109 cpm/mg was determined for theprotein. The antibody solution was stored at -20 °C in 100 gl aliquots inplastic centrifuge tubes.The amount of free label in the protein was determined using thinlayer chromatography with Baker-flex silica gel 1B2-F TLC sheets. Theprotein sample in buffer was spotted onto the TLC sheet, and the TLC wasrun in water. The protein stays at the origin and the iodide moves withthe solvent front, so the proportion of free label can be determined bycutting up the TLC sheet and counting the region around the origin andthe region around the solvent front. This gave a result of 3.7% freelabel.2.15b SDS-PAGE of the labelled antibodyGel electrophoresis with tube gels was done by Dr. Johan Janzen onnative and reduced samples of the labelled and unlabelled protein,following the procedure described in section 2.4d. The gels for thelabelled protein were sliced into 100 slices using a Bio-Rad model 195electric gel slicer and each slice was counted separately in the gammacounter. The counts at different positions on the gel were then comparedto the scans for the Coomassie blue dyed unlabelled protein.2.15c Comparing the antigen binding activity of the labelled andunlabelled antibodyThe labelled and unlabelled antibodies were compared using an ELISAwith BSA/ferrichrome A adsorbed to Falcon Pro-bind polystyrene ELISA107plates (Becton Dickinson, Lincoln Park, NJ) or to Immulon ELISA stripwells (Dynatech, purchased through Fisher, Ottawa). The unlabelledantibody was treated the same way as the labelled sample: a sample wastransferred to 0.2 M phosphate buffer pH 7.2, diluted to the sameconcentration as the labelled antibody, and frozen in a 100 Al aliquotin a plastic centrifuge tube.The ELISAs were carried out as described in section 2.3, withmatching series of antibody dilutions made for the labelled andunlabelled antibody. The equilibrium solution concentration was measuredby counting samples of the labelled antibody solutions after the plateswere incubated with antibody for one hour. The activity of the labelledand unlabelled antibodies was compared by plotting the absorbancemeasured after the substrate and phosphoric acid were added against theequilibrium concentration of antibody measured for the labelled sample.Since the equilibrium concentration was assumed to be the same for boththe antibody preparations, the graphs would be displaced along the yaxis (A450 - A620) if the antibodies had different activities.The amount of labelled antibody on the plates and strip wells wascalculated from solution depletion measurements. The amount of labelledantibody left on the wells after the completion of the assay wasmeasured for the strip wells by breaking the strips apart and countingthe wells individually.2.15d Measuring the antibody adsorption isothermsThe adsorption isotherms were measured for samples of beads andslides with an area of approximately 70 mm2. The beads or slides wereplaced in polypropylene centrifuge tubes and incubated with a blockingbuffer following the same procedure used for the ELISAs (sections 2.12a108and 2.12b). Skim milk (0.2%) was used as a blocking agent for the firstisotherm measured, and 0.2% BSA (Miles Laboratories, Etobicoke, ON) wasused as blocking agent in the PBS-Tween in all subsequent experiments.The 100 pl aliquots of the labelled antibody were mixed withunlabelled antibody, and the specific activity of the new preparationwas measured. A dilution series was made up in blocking buffercontaining either 0.2% skim milk or 0.2% BSA; 100 pl aliquots of thedifferent concentrations were added to the beads and 200 pl aliquotswere added to the slides. Duplicate samples were used for eachconcentration of antibody. The larger volume for the flat silica wasnecessary to cover the entire slide. The precise amount added to eachsilica sample was determined by weighing the centrifuge tubes before andafter addition of the antibody solution. The silica samples wereincubated with the antibody solutions for approximately two hours at37 °C.The concentrations of the different solutions were determined bycounting samples with an LKB Compugamma gamma counter with a five minutecounting time. Samples were added to gamma tubes containing one ml ofwater to distribute the sample evenly in the tube and prevent geometriceffects in the counting. The concentration of the initial solutions wasdetermined from 100 or 200 pl samples, and the equilibrium antibodyconcentration was determined from 90 or 190 pl samples. The precisevolumes of the solutions counted were determined by weighing the gammatubes before and after addition of the samples.The amount of antibody left on the silica after washing the samplestwice was measured by placing the centrifuge tubes containing the beadsdirectly into a gamma tube and counting the beads and centrifuge tubetogether and by counting the flat silica and centrifuge tubes109separately.The amount of antibody adsorbed per unit area was calculated fromsolution depletion measurements and for the direct counts of samplesafter being washed. The protein bound to the walls of the tube wasaccounted for in the solution depletion calculations either bysubtracting the amount left on the tube walls after the wash or bytaking as the initial concentration the equilibrium concentration leftin an empty tube. The area of the flat silica samples was calculated asdescribed in section 2.12b.Adsorption of Na1251 was also measured using the same proceduregiven above for the antibody. The free iodide washed off the silica,leaving an amount that could not be distinguished from the backgroundradiation. Since the antibody preparation contained some free label, thespecific activity used to calculate the amount of antibody on thesurface was calculated for the protein only, using the amount of freelabel found for each experiment.The radioactivity of the samples varied with protein concentration.The solutions measured had radiation levels of 1x103 dpm to 2x106 dpmper sample.2.15e Inhibition of antibody binding with free antigenThe inhibition of antibody binding by free ferrichrome A wasmeasured for flat silica. A low initial antibody concentration waschosen so that the concentration would not be in the range where thesurface was saturated. A low solution concentration of ferrichrome A wasused to modify the slides to give a low surface concentration offerrichrome A and minimize the the difference between the initial andfinal concentrations of antibody as well as to minimize the proportion110of antibody molecules bound to two antigen molecules on the surface.The initial antibody concentration was approximately 1x10-4 mg/ml,and the concentration of ferrichrome A used to modify the slides was0.007 mg/ml. The adsorption measurements were carried out as describedin section 2.12e with BSA as a blocking agent. A dilution series offerrichrome A was made up in a solution containing blocking buffer andantibody at a concentration of lx10-4 mg/ml. The same slides used forthe adsorption measurements were used for ELISA measurements.2.15f Calculation of the antibody-antigen affinityThe antibody-antigen affinity was calculated from the measurementsof the inhibition of antibody binding on flat silica by freeferrichrome A following the procedure outlined in Appendix 1.2.15g SEM studies with a gold labelled secondary antibody on flat silicaSilica slides were cleaned, silylated and modified with ferrichromeA as described previously. The ferrichrome A coated slides wereincubated in blocking buffer for 30 min at 37 °C, and with antibody inblocking buffer at 37 °C for another hour. Gold conjugated anti-mouseIgG (30 nm gold particles, Polysciences Inc., Warrington, PA) wastransferred from a glycerol solution to blocking buffer using an Amiconmicro concentrator filtration unit with a 30 000 g/mol cutoff filter(Amicon, Beverly, MA). The antibody was diluted to 1/10 theconcentration of the initial commercial preparation, and one drop of theantibody solution was added to each of the silica slides. The slideswere incubated with the gold conjugated antibody for one hour at 37 °Cbefore being rinsed with buffer and freeze-dried.Samples were attached to the conductive sample holders for the SEM111using a colloidal carbon suspension. The suspension was painted up thesides of the silica to ensure conduction between the top surface and thesample holder. The samples were coated by sputtering with a mixture ofgold and platinum and photographed with a Hitachi S-570 scanningelectron microscope.2.16 Aggregation of the silica beadsSilica beads modified with a range of surface concentrations offerrichrome A were incubated with blocking buffer and mixed withdifferent concentrations of antibody. After the beads had been incubatedwith the antibody for one hour at 37 °C they were allowed to settle outof the suspension at room temperature. The beads were then gentlyresuspended and examined under a microscope to determine the extent ofthe aggregation.2.17 Particle electrophoresisThe electrophoretic mobility u of silica particles was measured inorder to calculate the surface charge density (section 1.6b, equation[6]). Particles were suspended in 0.15 M NaCl and the suspension wasadded to the chamber of a Rank Mark 1 particle electrophoresis apparatus(Rank, Cambridge, U.K.). A voltage (40V, electric field 4V/cm) wasapplied across the sample and the movement of particles through a narrowcylindrical chamber was viewed and measured with a microscope focussedat the stationary layer where the movement of the cells is not affectedby electroosmotic flow through the chamber (141).The electrophoretic mobility was calculated from the transit time ofthe particles across a grid:u - 1 D Le^[12]t V112where t = transit timeD = size of grid division in pmLe = electrical pathlength in cm, determined as described (141)V = applied voltageThe electrophoretic mobility was determined for silica particlesbefore and after silylation and modified with a range of surfaceconcentrations of ferrichrome A.113Chapter 3Results and Discussion3.1 Silica surface3.1a Cleaning the silicaThe silica slides and beads were cleaned prior to modification withthe silane. The optimum cleaning procedure was determined by comparisonof the amount of carbon left on the surface as measured by XPS. Sincethere should be no carbon in the silica substrate, the amount of carbonleft on the surface should be a good indication of the efficiency of thecleaning procedure. Uncleaned glass slides had 15.5% carbon on thesurface (Table VIII). Slides were cleaned with chromic acid at 80° C,followed by rinses with hydrochloric acid and water or with water only.When the slides were dried in air (in a laminar flow hood that filteredout dust particles), very little decrease in the amount of carbon on thesurface was seen. This was due to carbon from the air rapidlyrecontaminating the surface, since slides that were cleaned by the sameprocedure, with hot chromic acid followed by HC1 and water rinses, andthen stored under vacuum, had a large decrease in the amount of carbonon the surface (Table VIII). Only 4% carbon was observed on the surfacefor slides by this procedure. The slides were stored under a poor vacuumthat was initially approximately 10-3 torr but that dissipated withincreased storage time. A stronger vacuum or better sample handling withdecreased time for the samples in air might result in a cleaner measuredsurface, but the procedure used compared well with a survey of othercleaning processes.11401sSi2p0 (a)$12.40.C 1 ssato^1100^six^scs^scs^ace^assMONO ENERGY (eViFigure 35: Wide scan XPS spectrum of silica cleaned with chromic acid at80 °C, followed by rinses with HC1 and water. The sample was dried undervacuum (Alka X-ray source).Table VIII. XPS measurements on clean silica and glass slides:comparing the efficiencies of the different cleaning procedures.Atomic % of the different elements presentSi2p Ols Cis S2s Na Mg Ca33.33 66.6720.73 52.31 15.53 7.58 2.02 1.8322.15 54.82 15.35 3.99 1.56 1.47 0.6826.54 57.34 13.00 0.00 0.39 1.65 1.0924.83 64.03 6.85 3.04 0.64 0.6125.54 65.35 4.17 3.62 0.73 0.6025.50 64.64 5.92 2.45 0.88 0.6132.53 63.31 4.1633.04 63.79 3.1632.07 63.09 4.8433.66 61.64 4.6913.99 83.79 2.2213.62 84.37 2.0134.25 61.36 4.39115bulk Si02.glass slides not clean.glass slides, chromicacid cleaned, dried inflow hood.glass cleaned withchromic acid then HC1,dried in flow hood.glass cleaned withchromic acid and HC1,dried in vacuum.SIO2 cleaned withchromic acid and HC1dried in vacuum.Si02 cleaned as above,ref luxed with heptane.SIO2 cleaned withHNO3' dried in vacuum.After the initial studies with glass slides, subsequent measurementsused silica to obtain a cleaner spectrum and a surface closer to thesilica beads.Contact angle measurements of water droplets in air can be used todetermine if the sample is clean with respect to hydrophobiccontaminants, but do not give much analytical information. Water willspread on clean surfaces to give a contact angle of 0°. Clean slides canbe detected visually, since a droplet placed on a clean slide willspread off the edge of the slide, but it can be difficult to measure lowcontact angles formed by droplets on slightly contaminated glass.The XPS measurements have a further advantage over the contact anglemeasurements, since they can also detect other contamination. Sulfurfrom the sulfuric acid in the chromic acid mix was detected on the glassslides after the chromic acid wash. Hydrochloric acid removed thecontaminating sulfur and left a surface with only carbon, silicon andoxygen detected.Hot nitric acid was tried as a cleaning procedure, since it has beenreported to be more efficient than chromic acid (116). Although thenitric acid resulted in a low concentration of carbon on the surface, italso gave an oxygen: silicon ratio of about 4:1, much higher than thebulk ratio of 2:1. Although silica is generally resistant to acids(142), the results imply that the surface is being modified in some way,so nitric acid cleaning was not used.Flat silica was ref luxed in heptane, the solvent used for thereaction. This did not cause any increase in the amount of carbondetected at the silica surface (Table VIII) implying that heptanedoes not adsorb to the surface or else is sufficiently volatile todesorb in vacuum.1163.1b Differences between flat silica and beadsExperiments were carried out with silica slides and beads toincrease the amount of information that could be obtained about thesilica surface, since different experiments could be carried out withthe different systems. It is very difficult, however, to make a directcomparison of the surface properties of the beads and the slides, so theexperiments with the two geometries should be thought of as beingcomplementary rather than as being measurements on one system.The flat silica slides are fused quartz and have a density of2.2 g cm-3 (143) while the beads had a density of 1.83 g cm-3 asmeasured by centrifugation in sulfuric acid. Although the bead surfacewas smooth on a macroscopic scale (Fig. 29), the density differenceimplies a more open structure for the silica in the beads. If thesurface of the beads was rough on a molecular level, there might be agreater surface area exposed to modifying reagent in solution and agreater number of hydroxyl groups available for reaction with thesilane. Since silylations can result in all the surface hydroxyl groupsbeing modified (144), a lower number of available hydroxyl groups wouldresult in less silane on the flat silica surface than on the beads.The flat fused quartz is produced by heating of quartz withsubsequent cooling to produce an amorphous glassy state. Heating ofsilica is a well known way to remove hydroxyl groups from the surfaceand so fusion of the quartz slides may also have contributed toreducing the number of hydroxyl groups per unit area on the surface ofthe slides relative to the beads.1173.1c Electrophoretic measurements on the beadsThe cleaned beads had a negative electrophoretic mobility (in a 25 mMsodium chloride solution) of -3.28 ± 0.21 x 10-4 CM2 V-1 S-1 , with acalculated surface charge density, from equation [10] of 5.87 x 103 esuCM-2 and an area per charge (equal to e/c) of 818 A2, indicating thatthere are negative groups at the surface of the beads, probablydissociated silanol groups.3.2 Silylated silica3.2a Density of amine groups on the surfaceA ninhydrin assay was used to estimate the amine content on thesurface of the silylated beads. The surface concentration of amines wasdetermined to be (0.9±0.3)x10-5 mol -NH2/m2, corresponding to an areaper group of 18 A2. This implies a surface that has very closely packedamine groups. Since the surface area was calculated from the bead radiusassuming a smooth sphere, the true surface area may be higher and thesurface concentration of amine groups might be lower. However,modification of silica surfaces with other silanes has been shown toresult in modification of all the surface hydroxyl groups, implying thata high degree of modification of the surface can be achieved (144).The error given for the ninhydrin assay measurement is the standarddeviation for four samples. A similar error was observed for thesolution measurements of the standards.3.2b Charge density on the surface and surface pH: effect on EDCcouplingElectrophoretic measurements showed that silylated beads had apositive surface potential in 25 mM NaCl. The electrophoretic mobility118was 3.86 ±0.2 x 10-4 CM2 V-1-1 . This gives a calculated charge densityof 7.27 x 103 esu/cm2 , with a corresponding area per charge of 660 A2 .The surface charge density was lower than the value that would beexpected from the ninhydrin assay of the surface amines. This is partlydue to the effect of surface pH on ionization of the surface aminegroups discussed below. However, it likely also reflects the fact thatthe beads were stored prior to the electrophoresis measurement,permitting adsorption of negatively charged contaminants.Charged surfaces will affect the distribution of ions in solutionadjacent to the surface, giving rise to what is known as the electricdouble layer (112). Hydrogen and hydroxide ions in aqueous solutionclose to the surface will also be affected, causing a change in the pHof solutions close to a charged surface (112). The surface pH may becalculated from the bulk pH and the zeta potential, making the usualassumption that the surface and zeta potentials are equal. The relevantion distribution is just given by the Boltzmann equation, which for thepresent case may be written, for the proton concentration adjacent to asurface whose surface potential is assumed to be C:^Hs = Hb exp(-eC/kT)^ [13]where Hs = hydrogen ion concentration adjacent to surfaceHb = hydrogen ion concentration in bulk phase.In terms of surface pH this equation becomes:^pHs = pHb + eC/2.303kT^ [14]If in the calculation of surface pH the surface concentrationimplied by the ninhydrin assay is used and it is assumed each propylamine group is charged, the surface potential is calculated by invertingequation [10]. When used in [14], the value of pHs is calculated to be119about 10, close to the pKa of the group. This means the assumption thateach amine bears a positive charge is likely incorrect. If instead thezeta potential is calculated from the measured electrophoretic mobilityand used as the basis for the calculation, the surface pH associatedwith the silylated bead surface is increased above the bulk value byonly 0.94 units.The EDC coupling is affected by pH. The EDC mediated couplingbetween acetylglycine and glycine methyl ester in solution has a broadoptimum between pH 5 and 7 (145). The pH of the solution offerrichrome A and EDC was 5.6 before addition of the beads and 6.01after addition of the beads. An increase of 0.94 over 6.01 would stillleave the pH at surface within the optimum range for the reaction. Theproblems with the EDC mediated coupling on the bead surface are that theEDC-carboxylate adduct can hydrolyze before reaction with the surfaceand that the positively charged surface would repel the positivelycharged group on the EDC. The rate of hydrolysis should not be greatlyaffected by the surface pH, since most of the EDC-ferrichrome A productremains in the bulk of the solution.Electrophoretic measurements with the ferrichrome A-modified beads(section 3.3f, Fig. 44) showed that the beads had a negative charge,implying that not all the carboxyl groups on the ferrichrome A hadreacted and that the ferrichrome A-EDC product would not be repelledfrom the surface. If the antigen used had not had more than one carboxylgroup per molecule, use of a positively charged carbodiimide might haveresulted in lower amounts of antigen bound on the surface. Problems withthis could be solved by use of a negatively charged water solublecarbodiimide.1203.2c Thickness of the amine layerSilylation of silica surfaces in aqueous solutions can result indeposition of seven to ten molecular layers of the silane (137).Trihydroxysilanes can condense in solution after hydrolysis of thealkoxy groups and since the condensation is not limited to twodimensions, it can produce a thick layer of silanes which can then reactwith the surface (see Fig. 9 for general reaction of silanes withsilica). Condensation of the silane into such "lumps" might result in anunevenly modified surface, so the reaction was not carried out inaqueous solution.Heptane was chosen as a solvent for the reaction because it takes uponly a small amount of water, and could provide sufficient water forhydrolysis of the silane while minimizing the condensation of silane insolution. The 3-aminopropyltrihydroxy silane used in these experimentsappeared to coat the beads rapidly; the silica beads could be suspendedreadily in a solution of the silane in heptane but not in the heptanealone. This could be due to the hydroxyl groups on the silicainteracting with amine groups on the silane.Angularly resolved XPS measurements on a silylated silica slide (thathad also been modified with ferrichrome A) showed a shift in the Si2ppeak at higher angles away from the normal (Fig. 36). The Si2p peak forclean unmodified silica is also shown. When measurements on the modifiedsilica were taken normal to the surface to achieve a greater samplingdepth (Fig. 10), the observed peak was closer to that for the unmodifiedsilica. As the measurement angle moved away from the normal, thesampling depth became shallower and the signal from silicon in thesilane layer became enhanced relative to the signal from the underlyingsilica. When the photoelectrons were detected at 700 away from the1210-1108^106^104^102BINDING ENERGY [eV]98Figure 36: Angularly resolved measurements on silylated silica, showingthe shift in the Si2p peak away from the value for unmodified silica, asthe angle of measurement away from the normal becomes larger and thesampling depth becomes shallower (a= clean silica at 90°, normal to thesurface, b-e = the silylated surface, b= 90°, c= 45°, d= 30°, e= 20°)122normal, there seemed to be very little signal contributed from theunderlying silica layer.These measurements imply that there is a continuous layer of silane,since if the silane were present as islands on the silica surface, therewould be some of the underlying silica at the surface anddistinguishable at all takeoff angles (111). The silane layer is thinenough that signal from the underlying silica can be seen. The intensityof the signal decreases at high takeoff angles because of theferrichrome A covering the silica.X-ray photoelectron spectroscopy measurements showed only 2.4%nitrogen on the flat silica (Table IX), less than the amount of carbondetected on the cleaned surface. This low measurement is in contrast tothe high surface concentration of amines measured by the ninhydrinassay, and may reflect the fact that the amines are at the top of thesilane layer.3.3 Silica with ferrichrome A3.3a Some comments on ferrichrome AFigure 37 is a crystallographic structure of ferrichrome A showingthe face with the three carboxyl groups. If the carboxyl groups allinteracted with the positively charged amine groups on the silylatedsilica, the ferrichrome A would be adsorbed with the carboxyl face downand a molecular cross-sectional area of about 70 A2 (all numbers areobtained using Silicon Graphics and the ferrichrome A coordinates (120)from the Cambridge Data Base).123052Figure 37: Crystallographic structure of ferrichivme A, showing the facewith the three carboxyls (120) (the carboxyl groups are indicated byarrows).124Atomic Y. of the different elements presentSi2p Ols Cis Nis26.4131.5048.8651.6121.2314.543.753.35silica modified withundistilled silanesilica modified withdistilled silaneTable XI: XPS measurements on silica slides modified with distilled orundistilled 3-aminopropyltriethoxysilaneTable X: Ferrichrome A adsorption to different areas of beads. Theinitial ferrichrome A concentration was the same for each sample. Theerror listed is the standard deviation for six data points.amount of beads used for^amount adsorbedeach measurement(mg)^ (mg/m2)1 2.07±0.215^ 2.16±0.211253.3b Effect of changing the bead area on ferrichrome A adsorptionChanging the area of the beads did not change the amount offerrichrome A adsorbed per unit area (Table X). This implies that theferrichrome A does not contain a significant concentration ofaggregates in solution, since higher molecular weight aggregates wouldadsorb preferentially and would result in a higher amount adsorbed perunit area for smaller area bead samples (if the initial ferrichrome Asolutions were the same volume and concentration) (79).3.3c The adsorption isotherm: ferrichrome A adsorption as a function ofsolution concentrationThe amount of ferrichrome A adsorbed per unit area was measured fora range of dilute concentrations of ferrichrome A (using 5 mg beadsamples with six samples for each data point) and for a wider range ofconcentrations (using 10 mg bead samples, with two samples for each datapoint), see Fig. 38 and Fig. 39. Curves are fit by eye to the datapoints. The error bars at each point are the standard deviation ofmeasurements of the amount adsorbed. Figure 39 also shows the amount offerrichrome A left on the beads after washing with PBS. Theseexperiments were done on beads modified in separate silylationprocedures. Data from the two experiments agree well.A Langmuir type adsorption isotherm was obtained, with the amountadsorbed increasing rapidly with equilibrium solution concentrationbefore reaching a plateau. A Scatchard plot (Fig. 40) for the dataobtained in dilute solutions (Fig. 38) gave a maximum amount adsorbed of1.76 mg/m2. This corresponds to an area per molecule of 99 A2/molecule.The same data also gave a K value of 8.26±0.99 1 mo1-1.1261.81.61.4---- 1.2-oa)0 0.82_cE- 0.6a)U-0.40.20.01^0.02^0.03^0.04^0.05^0.06^0.07^0.08[ferrichrome Meg mg/ml0.09Figure 38: Adsorption isotherm for ferrichrome A in water on silylatedbeads at low solution concentrations C. = amount adsorbed from solution).1271.6-1.4-1.2-0.4-1.8•0.2-• •0 -0.20^0:05^0.1^0:15^0.2^0.25^0.3^0.35[Ferrichrome A]eq mg/mlFigure 39: Adsorption isotherm for ferrichrome A in water on silylatedbeads (0 = amount adsorbed from solution, o = amount left on after PBSwash).128I^I^I^I0.2 0.4 0.6 0.8 1^I^I.2 1.4^1:1 6 18[ferrichrome A] adsorbed mg/m2Figure 40: Scatchard plot for ferrichrome A binding to silylated beads(from the data shown in Fig. 38)1293.3d Effect of salt concentration on ferrichrome A adsorptionThe amount of ferrichrome A adsorbed in increasing concentrations ofsodium chloride was measured using beads that had been stored for tendays after the silylation. A series of ferrichrome A solutions was madeup in distilled water and 5, 10, 20, 50 and 100 mM NaCl. Although theinitial concentration of ferrichrome A was the same in all solutions,the equilibrium concentrations differed because different amounts offerrichrome A adsorbed to the beads. The amount bound was lower than forfreshly prepared beads (see Table VI). Data is presented in Fig. 41 asamount bound as a fraction of the amount bound in water.Increasing salt concentrations rapidly decreased the amount bound;10 mM NaCl would cause a 50% decrease in the amount bound. No detectableferrichrome A was bound at 100 mM NaCl. The chloride ions andferrichrome A would both act as counter ions to the positively chargedamines on the silylated silica, and increasing concentrations of sodiumchloride would compete with the ferrichrome A to cause a decrease in theamount of ferrichrome A bound.The decrease in amount of ferrichrome A bound may also be partly dueto the beads aggregating and decreasing the area of silica available forbinding. Aggregates could be seen under the the microscope for beads inhigher salt concentrations. The aggregates could also be detected insuspension because the beads settled out more rapidly in salt solutions.3.3e Reaction of ferrichrome A with EDCWhen ferrichrome A is reacted with EDC by adding a solution of EDCin water to dry ferrichrome A, an additional peak appears in the1300 10^20^30^4'0^g0^60[NaCI] mM100Figure 41: The effect of increasing concentrations of NaC1 on adsorptionto the silylated beads. Results are shown as a fraction of the amountbound at 0 mM NaCl.131atoa8t*1.201. 00.800.400.200.0Wavelength (mu)Figure 42: The absorbance of ferrichrome A mixed at different ratioswith EDC, where a= the lowest EDC: ferrichrome A ratio and b= thehighest ratio. Results are shown for solutions with increasingconcentrations of ferrichrome A so that they may all be shown on onefigure.132absorbance spectrum at 367 nm (Fig. 42). The peak at 367 nm increaseswith respect to the peak at 436 mu as the concentration of EDC insolution is increased (Fig. 42). This absorbance is not due to EDC. Thepeak at 367 rim is outside the range reported for typical 3:1 ferrictrihydroxamates (420 to 450 rim, 146) and seems quite narrow, where mostferric hydroxamates have a broad absorbance peak (147). The peak is alsoshifted to longer wavelengths than the maximum for ferrichrome A, unlikethe shift that occurs for ferric trihydroxamate as the hydroxamategroups dissociate. As the solution pH is lowered, ferric trihydroxamatewill dissociate and form the 1:1 ferric hydroxamate. As this occurs,the absorbance maximum shifts from a range of 425 mu to 440 mu for theferric trihydroxamate to 520 mu for the 1:1 complex, with a decrease inthe extinction coefficient (147).If the peak at 367 mu represents a different species, then at anEDC:ferrichrome A mole ratio of 50:1, it seems to account for only asmall fraction of the total ferrichrome A.Ferrichrome is another metabolite of Ustilago sphaerogena withouta serine residue or any carboxyl groups. Ferrichrome does not react withEDC to produce a peak at 367 mu, implying that the hydroxamate groupsare not involved in the reaction (Figure 73, Appendix 4).3.3f The amount of ferrichrome A on the beads at a constantEDC:ferrichrome A ratioThe EDC was mixed with ferrichrome A at a mole ratio of 50:1 inwater and then mixed with the silylated beads as described in section2.11. Figure 42 shows the amount of ferrichrome A associated with thebeads at different concentrations of ferrichrome A and the amount lefton the beads after rinsing with PBS, with all data points for the1331.21_C \ 1EasEC')-aas 0.6a)_aCo<a) _E u.42_c0-caiU-0soi^I^I^I^I^I^1^1^10^0.05^0.1^0.15^0.2^0.25^0.3^0.35[Ferrichrome Meg mg/ml_0.80.2--0.20.4 050.45Figure 43: Coupling ferrichrome A to silylated beads at a constant EDC:ferrichrome A mole ratio of 50:1 (o = amount of ferrichrome A associatedwith the beads, calculated from initial solution depletion measurements,• = ferrichrome A left on the beads after a PBS wash).134experiment shown. The amount adsorbed on the silica shows the initialsharp increase with equilibrium solution concentration that was seen forferrichrome A adsorbed in water, but then decreases as the ferrichrome Asolution concentration continues to increase. This is probably due tothe increasing concentration of EDC, which could have the same effect assodium chloride in disrupting the ionic interaction between theferrichrome A and the positively charged silica.At an equilibrium solution concentration of 0.459 mg/ml offerrichrome A, 89% of the ferrichrome A stayed on the beads afterrinsing with PBS. This is distinctly different from the negligibleamount of ferrichrome A left on the beads after the PBS rinse of theferrichrome A adsorbed in water. At higher surface concentrations offerrichrome A on the beads, the beads remained orange after the PBSwash.The absorbance spectrum of the ferrichrome A that washed off thebeads did not show any shoulder at 367 nm.At an equilibrium solution concentration of 0.18 mg/ml ferrichrome A,the initial ferrichrome A concentration was 0.226 mg/ml, and the EDCconcentration was 11 mM. From Figure 39, a 0.226 mg/ml solution offerrichrome A in water would give a ferrichrome A surface concentrationof 1.6 mg/m2. If EDC is a 1:1 electrolyte at pH 5.3, it would have anionic strength equivalent to that of a sodium chloride solution of thesame concentration, which caused a 50% decrease in the amount offerrichrome A bound (Fig. 41). This is similar to the decrease observedfor ferrichrome A bound to the beads in an EDC solution relative to theamount in water.The amount left on the beads after the PBS wash increases lessrapidly with solution concentration of ferrichrome A than the total135amount associated with the surface (Fig. 43). There is a kink in thebinding curve corresponding to a surface concentration of 0.62 mg/m 2 oran area per molecule of 270 A2 . Adsorption isotherms of proteins canshow kinks where reorientations of the the protein occurs to give a morecompact arrangement (87). The protein saturates the surface at oneorientation, but can then reorient to allow more protein to bind.If the ferrichrome A is attached covalently, then at least one ofthe carboxyl groups must be towards the surface. The other two carboxylgroups on each molecule may be originally in contact with the surface sothat the triangular face shown in Fig. 37 is towards the silica surface.At higher concentrations, the molecules could reorient so that carboxylgroups not covalently bound to the silane were exposed to the solution.Electrophoretic measurements on the same bead samples prepared forFig. 43 showed that the beads at concentrations above the kink in theferrichrome A binding curve had a large negative surface charge. Figure44 shows the electrophoretic mobility of the beads as a function ofsolution concentration of ferrichrome A. The graph has a similar shapeto the graph of the amount left on the silica after the PBS rinse as afunction of solution concentration of ferrichrome A, but with a sharperincrease after the apparent saturation point. The high electrophoreticmobility supports the idea that the carboxyl groups are oriented towardsthe solution.The specific amount of ferrichrome A bound would be expected toreach a saturation point at the concentration observed for adsorption offerrichrome A with no EDC, but the limited solubility of ferrichrome A inwater prevented measurements at higher concentrations.136-3.5-1-0.5-^1111111^0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5[ferrichrome A] eq mg/mlFigure 44: Electrophoretic mobility of silylated beads with ferrichrome Aattached covalently at a constant EDC:ferrichrome A mole ratio. Units ofelectrophoretic mobility plotted are (1O ^V ^uncertaintiesin data points are approximately the same magnitude as the size of thesymbol).1373.3g Amount of ferrichrome A on the beads at constant EDCconcentrationAnother experiment was carried out varying the concentration offerrichrome A but maintaining a constant concentration of EDC. The EDCconcentration of 20 mM was chosen to give a 50:1 mole ratio for thehighest concentration of ferrichrome A; the mole ratio of EDC toferrichrome A increased for lower concentrations of ferrichrome A. Theinitial increase in adsorbed ferrichrome A, followed by a decrease athigher equilibrium solution concentrations that was observed in Fig. 43was not seen, indicating that the increase was due to the lowconcentration of EDC in solution (Fig. 45).Since the amount of ferrichrome A left in solution was calculatedfrom the derivative of the absorbance using the same number that wasdetermined for a 50:1 ratio of EDC:ferrichrome A, the results obtainedwill be less accurate for the lower concentrations of ferrichrome A withhigher EDC:ferrichrome A ratios, but should still serve to indicate theshape of the binding curve.The binding curves showing the amount of ferrichrome A on the beadsfor a constant EDC:ferrichrome A mole ratio and a constant EDCconcentration were similar at higher ferrichrome A concentrations wherethe mole ratio of EDC:ferrichrome A was similar. At lower concentrationsof ferrichrome A, increased EDC decreased the amount initiallyassociated with the beads and the amount remaining after the PBS wash.3.3h BCA assays of the amount of ferrichrome A on the beadsThe amount of ferrichrome A left on the beads after a PBS rinse wasalso estimated directly with a BCA assay (section 2.14b). This gavevalues similar to those obtained from solution depletion (Fig. 46).1384124105^0^0.05 0.1^0.15 0.2 0.25 0.3 0.35 0.4 0.45 050.20.40.60.81.201 0•••0IT[Ferrichrome Meg mg/mlFigure 45: Coupling ferrichrome A to silylated beads at a constant EDCconcentration of 20 mM, giving an EDC:ferrichrome A mole ratio of 50:1for the highest ferrichrome A concentration used (o = ferrichrome Ainitially associated with the beads, m = ferrichrome A left on the beadsafter a PBS wash).1390.3 0.35 041.81.6-1.4-0.2-0-0.2 ^0 0.105^0.1^0.15^0.2^0.25[Ferrichrome A]eq mg/mlFigure 46: BCA determination of the amount of ferrichrome A on the beadsafter coupling ferrichrome A to the beads with a constant EDCconcentration of 20 mM and washing the beads with PBS C. = amount offerrichrome A calculated from solution measurements, o = BCA assay with30 min incubation time, A = BCA assay with 120 min incubation).1403.21 Ninhydrin assays of silica modified with acetic acid and EDCNinhydrin assays of the acetylated beads were done in search ofevidence for formation of a covalent bond on the surface. The beads weremodified with acetic acid following the same procedure used for couplingferrichrome A, with a 20:1 mole ratio of EDC:ferrichrome A. Theacetylated beads had a surface amine concentration of 2.1x10-7 mol m -2 ,as compared to the silylated beads with a surface amine concentration of1.9x10-5 mol/m2 . The decrease in the measured amount of amines on thesurface was not due only to ionic interactions of the acetic acid andthe amines on the silica, since the assay was carried out in 100 mMacetic acid. The ninhydrin assay implies that nearly all the availableamine groups on the bead surface reacted with acetic acid plus EDC.3.2j XPS measurements of ferrichrome A on the silicaAngularly resolved XPS measurements of ferrichrome A coupled to flatsilylated silica with EDC showed that the ferrichrome A covered some butnot all of the surface. The S12p signal from the underlying substratedecreased for measurements made at angles higher away from the normal,but appeared to approach a constant value at high angles (Fig. 39). ThisImplies that some of the silica is covered with a thin layer offerrichrome A, and that some of the silica remains uncovered.The ferrichrome A on the surface was identified by the iron 2p peakat 711 eV (Fig. 47).Ferrichrome A dried onto the silica from an aqueous solution with noEDC desorbed in the vacuum of the XPS machine, although the initialamount of ferrichrome A on the slide was sufficient to cause a dark redmark. This is further evidence that EDC results in formation of a stablebond between ferrichrome A and the silylated silica.14117000 ^735^730^725^720 7115^710^7105BINDING ENERGY [eV]Figure 47: An XPS spectrum showing the Fe2p peak from ferrichrome A onsilica (Mgka source)7001423.4 Antibody binding to the ferrichrome A-modified silica3.4a The radiolabelled antibodyThe purified IgG (section 2) was labelled with 1125 (2.15a). Theeffects of the labelling were determined with SDS-PAGE of the labelledantibody and an ELISA to compare the activity of the labelled andunlabelled antibody preparations (Figs. 48a and 48b).The SDS-PAGE indicated that the sample was pure and the ELISA showedthat the labelling did not affect the antibody activity.3.4b. Non-specific adsorptionThe adsorption of the antibody to unmodified and silylated silicawas measured and the effect of Tween 80 and 0.2% BSA in minimizing thenon-specific binding was measured. The BSA was used instead of skim milkas a blocking agent for the adsorption measurements with radiolabelledantibody to avoid any problems that might result from components of theskim milk being centrifuged out of suspension during the assay.Antibody adsorbed more strongly to the silylated silica than to theunmodified silica (Fig. 50). This is probably due to the positivelycharged surface, which is known to increase binding (148). Addition ofTween 80 decreased the binding to the silylated silica (Fig. 51) andaddition of 0.2 % BSA by itself as a blocking agent caused a greaterdecrease (Fig. 52). The Tween 80 and 0.2 % BSA were used together formaximum effect.A greater non-specific adsorption was seen for antibody on the beadswith no antigen than for the flat silica (Figs. 52 and 53) with noantigen. This could indicate that the beads were more difficult to washor that the beads were modified with a higher surface concentration ofamines to give a more positively charged and higher binding surface.1430 1^1^1^111111^1^1^1111111^J^1^u11111^i^i^iiirTT1E-07 1E-06 1E-05 0.0001Equilibrium concentration Ab mg/mlFigure 48a: An ELISA comparing the activity of labelled and unlabelledantibody, using commercial polymer microwells as the substrate (m= thelabelled antibody, o= the unlabelled antibody).0.0011441.41^1^I^1^1 1111^1^1^1111111^I^1^11 1 1111^I^I^I^1 11111 E-()6 1E-05 0.0001 0.001Equilibrium concentration Ab mg/mlFigure 48b: A second ELISA comparing the activity of labelled andunlabelled antibody, using a different commercial assay plate. Thelabelled and unlabelled antibody were consistently shown to have similaractivity (Fig. 48a).01E-07145S102/-NH2 + ferr. ASi02/-NH2SiO21.21.0cJE 0.80.6ctia)-2O 0.4.r)0.20.0-0.20^0.0005^0.001^0.0015^0.002^0.0025equilibrium [Ab] mg/ml0.003^0.0035Figure 49: Antibody adsorption in PBS onto clean flat silica, silylatedsilica, and silylated silica with ferrichrome A.1461.21.11.00.9E• 0.8• 0.7cocti• 0.6cci 0.5-a)-2o 0.4co-0t 0.3• 0.2-¢0.10.0S102/-NH2-0.14120^0.0005^0.001^0.0015^0.002^0.0025equilibrium [At)] mg/ml0.003 0.0035Figure 50: Antibody adsorption in PBS-Tween onto flat silica with noferrichrome A147_1.21 . 0_Si02/-NH20 . 0 ^Si02-0.2 I^I^I^I^1^10^0.0005^0.001^0.0015^0.002^0.0025equilibrium [Ab] mg/mlFigure 51: Antibody adsorption in PBS with 2% BSA onto flat silica withno ferrichrome A0.003 0.0035148Some increased adsorption was seen for the silylated silica modifiedwith ferrichrome A, but it was not greatly different from thenon-specific adsorption to silica without ferrichrome A.The non-specific adsorption could be determined by labelling anunrelated IgG and using it in the assays. This would permit thedetermination of the amount of antibody bound non-specifically to theferrichrome A modified surface. Although this was done as a control forsome ELISAs, it was not done for the protein adsorption isotherms.3.4c Antibody binding to ferrichrome A on flat silica and beadsSilica beads were prepared with a range of surface concentrations offerrichrome A. Silica slides were modified with the same range ofsolution concentrations used to modify the beads. Both the slides andbeads were used for adsorption isotherm measurements.The amount of antibody bound to the antigen coated surfaces wascalculated by solution depletion measurements and by direct counts ofthe amount of antibody left on the beads after washing. The antibodybinding was seen to be irreversible over the washing time period used,with the amount bound from solution depletion measurements being thesame or slightly greater than the amount determined from a direct count.The surface concentrations of protein appeared to be more accuratefrom the direct measurements, giving less scatter and no negativevalues for the surface concentration of antibody and were thereforeused to calculate the binding constants. The data points measured for theprotein adsorption isotherm were used directly in the Scatchard plots,although this may not be the best approach, given the scatter in thedata. Another way would have been to plot the data of the amountbound against solution equilibrium concentration, fit curves by eye and149then take data points off the graph.Results for the beads and the slides are shown in Figs. 53 and 54.The beads with no ferrichrome A on the surface showed some non-specificadsorption of antibody, which may be due to the strongly positivesurface. As the surface concentration of ferrichrome A increased, theamount of exposed silylated silica decreased; this would affect the non-specific binding and might result in lower non-specific binding athigher ferrichrome A concentrations. The shapes of the isotherms forbeads with successively higher surface concentrations of ferrichrome Aare consistent with this idea; at high solution concentrations ofantibody, the isotherms for beads with high surface concentrations showedsaturation, whereas the isotherms for beads with lower concentrations offerrichrome A did not.Additional plots included in Appendix 4 show the amount of antibodybound to the beads and flat silica as calculated from solution depletionmeasurements. Appendix 4 also shows the binding of antibody to beads atlow antibody concentrations.Information about systems having binding sites with two differentaffinities can be obtained from Scatchard plots where the data is nottoo scattered. The Scatchard plots can be used to estimate the twodifferent binding constants as shown in Fig. 55. Data for the Scatchardplot of the beads with 0.086 mg/m2 could be used to draw two lines withdifferent slopes.If there is a reorientation of ferrichrome A at higher surfaceconcentrations, as postulated in sections 3.2e and 3.2f, the antibodymight bind differently to the beads at high surface concentrations. Thiscould possibly explain the binding isotherm for antibody on the beads ata ferrichrome A surface concentration of 0.83 mg M-2;if the ferrichrome A1501.6 0.8270.5950.398--A--0.264--x--0.075-0.0450.001^0.002^0.003^0.004^0.005[Antibody]eq,solution mg/mlFigure 52a: Antibody bound to silica beads with a range of differentsurface concentrations of ferrichrome A: bound antibody determined fromthe amount remaining after washing. The legend shows th; measuredsurface concentration of antigen on the surface in mg/m1510.8270.595-4K--0.3980.2640.075-0.0452100^1900-1700-E.0.827rzi0.5950.3980.2640.075-I--0.04501500-01300-/;6.01100-0 900-,4CP700-0:0 50o-<Ab.300-X1 00"'AL10*0^0:2^0:4^0:6^0.8^1:2^1:4 ^1:6Antibody on silica mg/m22.41:8^220:2^0:4^0:6^0181:2^1:4^1:6Antibody on silica mg/m22.4Figure 52b: The Scatchard plot for the data shown in Fig. 52, shown withand without a linear regression.1320--Ar-0.2640.5950.8270.001^0.002 0.003 0.004 0.005 0.006 0.007 0.008[Antibody]eq mg/mlFigure 53a: Antibody adsorption to flat silica modified with a range ofdifferent solution concentrations of ferrichrome A: bound antibodydetermined from the amount remaining after wrhing. The legend shows thesurface concentration of ferrichrome A (mg/m ) obtained on beads modifiedwith the same solution concentration of ferrichrome A.15300.2640.5950.827800-70.5^1^1.5^2^2:5[antibody] on silica3.5^4^850—.^SOO^750—700 650—600-550•-.500—450-•400—350—300-250—200—50—00-50-kN614-440^0 O'5^1^15^ 2:5a4[antibody] on silicaFigure 53b: The Scatchard plot for the data shown in Fig. 53, shown withand without a linear regression.15400.264—ea-0.5950.827\\^4---- high affinity1\\a4\\Slow affinity\^\\ ......AC.." \‘ \\\\Surface concentrationof bound antibodySolution concentrationof antibodySurface concentration of antibodyFigure 54: Scatchard plot for a system with two different bindingaffinities. The equilibrium constant for high affinity binding can bedetermined from the slope of line a, and equilibrium constant for lowaffinity binding can be determined from the slope of line b.155were in the more densely packed mode postulated for surfaceconcentrations below the apparent saturation point at 1.1 mg m -2 , theantibody might bind less strongly, resulting in lower surfaceconcentrations of antibody than were seen for the beads with lowersurface concentrations of ferrichrome A.The antibody did not show as much non-specific binding on thesilylated flat silica (Fig. 54) as on the beads. If the non-specificbinding is due to the amine groups on the surface, as is implied by thecomparisons of binding on unmodified silica and on silylated silica(Figs. 50 and 52), the flat silica would seem to have fewer amine groupsthan the beads.No difference was seen for the adsorption isotherms onto silicamodified with the different solution concentrations of ferrichrome Athat caused variations on the binding isotherms on the beads. Thismay be because the different solution concentrations of ferrichrome A didnot in fact result in different surface concentrations. If the flatsilica has fewer amine groups than the beads, the ferrichrome Amodification would not be expected to give the same results. The surfaceconcentration of ferrichrome A on the flat silica could be limited bythe amine group density.Equilibrium binding constants for the antibody binding toferrichrome A on the beads and flat silica are summarized in Table XII.The equilibrium constants for the binding to the beads are largerthan for the binding to the flat silica (approximately-1^ -11.1 x 108 litre mol for the beads as opposed to 1.5 x 107 litre molfor the binding to the flat silica modified with higher concentrationsof ferrichrome A). If the ferrichrome A is at low surface concentrationson the slides and is distributed evenly on the surface, then the156max.area per K amount areaAg molecule in Abboundpermolec.A2 litre mol-1 -2mg m A2concentrationferrichrome Aon the surfacemg m-2Table XII: Apparent equilibrium constants for antibody binding toferrichrome A- modified beads and slides. These constants are calculatedfrom the Scatchard plots and do not take into account the distributionof light chains that would render one quarter of the antibodies inactive.Equilibrium constants K for binding to slides modified with threedifferent solution concentrations of ferrichrome A (1, 2, 3) and with noferrichrome A (4)maximum amountof Ab boundmg/m2Kin -1litre molarea per AbmoleculeA21 7.12 1.8 x 107 3 7002 8.77 1.3 x 107 3 0303 8.24 1.3 x 107 3 2244 0.68 7.2 x 106 39 000Equilibrium constants for antibody binding to beads modified with arange of ferrichrome A surface concentrations0.827 211 1.1 x 108 2.22 11 9700.595 293 1.2 x 108 1.90 13 9600.398 438 1.7 x 108 1.61 16 5030.264 661 1.9 x 108 0.99 26 5940.075 2 327 1.4 x 108 0.64 41 345157antibody would not be able to bridge between two surface bound antigenmolecules when it bound to the surface. Bridging increases theequilibrium constant as described in section 1.5e and has been reportedto increase the measured equilibrium constants by several orders ofmagnitude (83).Only binding of multivalent antibodies could be enhanced by bridgingbetween antigens. Since the antibody preparation used probably containsboth divalent and monovalent antibodies, a portion of the antibodieswould bind with enhanced affinity at higher surface concentrations ofantigen, and a portion would bind with lower affinity. The antibody alsobinds non-specifically to the silylated silica, so the observed bindingisotherm would be a function of antibody binding to the antigen-modifiedsurface in three different ways and with three different affinities.The SEM photograph in Fig. 55 shows the distribution of bindingsites for a gold-labelled secondary antibody on flat silica modifiedwith ferrichrome A and with the antibody AC3. The gold particles do notshow any large clumps, implying that the antibody binding sites areevenly distributed on the surface.Comparison of the amount of Ab predicted to be bound at saturationto beads with the measured Ag density demonstrates that the Ag densityis considerably more than twice the saturation amount of the Ab.Hence, bound Abs are presumably covering antigen molecules withoutspecifically binding to them. The ferrichrome A-modified slides have ahigher maximum number of antibody binding sites as determined by theScatchard plot than do the beads. The lower affinity constant suggeststhat little bridging of surface Ag by Ab is taking place and thereforethe surface Ag density is lower than on the bead surface. The Scatchardintercepts for Ab binding to the slides are higher than on the beads,158(a)^ (b)Figure 55: SEM photograph showing the distribution of gold labelledsecondary antibody on silica slides with immobilized ferrichrome A andadsorbed antibody. The small white dots represent gold particles (a=results obtained for no antigen on the surface and b= results obtainedfor antigen present)159however. This may indicate that singly bound Abs can pack moreefficiently near saturation, perhaps showing more potential for rotationif the ferrichrome A is attached by only a single covalent bond, than doAbs that are more rigidly associated with the surface by binding two Agsper molecule.There are also geometric considerations to be taken into accountwhen comparing the beads and the slides. Antibody binding todiffusing and sedimenting particles in suspension is less likely to belimited by kinetics than is binding to a flat surface in an unstirredsolution (75). If the binding of the antibody were limited by diffusion,then the equilibrium constant might be underestimated. The antibody wasincubated with the silica for more than two hours at 37 °C with somestirring early in the incubation. Two hours was chosen as an incubationtime as an optimum between the time required for antibody adsorption andthe time during which the beads would settle out of solution.Antibody on beads can form bridges in an additional way: thedivalent antibody can bridge between two beads, to form an aggregate.Small clumps of two or three beads were seen for samples mixed withantibodies at an initial antibody concentration of about 10 -5 to 10-7mg/ml and a low measured surface concentration of ferrichrome A.Large clumps would affect the binding of the antibody due to bridgesbetween the adjacent beads.3.4d Inhibition of antibody binding with free ferrichrome A: ameasurement of the solution equilibrium constantFerrichrome A in solution was used as a competitive inhibitor of theantibody binding to slides with immobilized ferrichrome A and themeasurements of the amount of antibody bound as a function of solution160concentration of antigen were used to calculate the solution equilibriumbinding constant as described in Appendix 1 (Fig. 56). Silica slides werechosen as a substrate rather than the beads to minimize problems withnon-specific binding. The slides were modified with a low concentrationof ferrichrome A in order to minimize possible bridging of the divalentantibody between adjacent antibody binding sites on the substrate.An equilibrium constant of 2.5 ± 0.8 x 10 7 litre mo1 -1 wascalculated for the antigen binding to antibody in solution. This valueis about half that calculated for specific binding to flat silicamodified with the same solution concentration of ferrichrome A, takingthe effect of light chain distribution into account: 7.1 x 10 7 litremol-l . This is the smallest estimate of the constant obtained in any ofthe systems examined, and is consistent with earlier discussion sincenone of the complications associated with interpretation of binding tosurface-associated Ag, most of which tend to increase the apparentassociation constant, are relevant.3.5 ELISAs using the beads and flat silica3.5a Reproducibility of ELISAs on the beadsThe ELISAs performed Using the same batch of ferrichrome A-modifiedbeads gave very consistent results (Fig. 57). Assays performed with asecond batch of beads gave results that were similar, with the greatestchange in absorbance occurring over the same concentration range ofantibody in solution.If reproducible ELISAs are desired, a large batch of beads canbe modified and used for all experiments. The 10 mg bead sample sizesused were sufficient for about 300 data points at the sample area used,1610.20.18-0.16-E 0.14-E-ac 0.12-0.00.1-0tPco▪^0.08-0E< 0.06- • ■•0.04-0.02-0 i i imm^i i Hum^i i twill^i i I 1 1 111^i i limn^I i jinni^rrrr1 E-08^1 E-07^1 E-06^1 E-05^0.0001^0.001 0.01 0 1[Ferrichrome A] in solution mg/mlFigure 56: Antibody binding on flat silica: inhibition by freeferrichrome A. (- - -) = amount antibody bound with no inhibitingferrichrome A.1622.5-1.5-0.5-^Os n^-r1 E-08^1 E-07 1 E-06^1 E-05^0.0001[Antibody] mg/ml0.010.001Figure 57: Reproducibility of ELISAs on ferrichrome A-modified beads(■, a = beads modified with ferrichrome A at one time; A = beadssilylated and then modified with ferrichrome A in a separate experiment).163and larger bead samples could easily be modified with ferrichrome A.In order to ensure reproducibility, the beads must be well suspendedin the final assay step before addition of the substrate. Since thereaction of the substrate occurs quickly there is not sufficient time tomix and suspend the beads after addition of the substrate andincompletely resuspended beads will result in lower absorbances.3.5b ELISAs on the beads: varying the surface concentration of antigenAssays were performed on beads with a range of different surfaceconcentrations of antigen (Fig. 58). There was very little differencebetween the two highest surface concentrations of antigen used. One ofthe two highest concentrations used was in the region where the kinkoccurs in the plot of the amount of ferrichrome A bound as a function ofsolution concentration and one was above it. If the ferrichrome A has infact formed a close packed layer, it is not surprising that the amountof antibody bound does not increase. The amount of antibody bound asmeasured using radiolabelled antibody showed a similar pattern, with thetwo highest surface concentrations of antigen giving similar bindingisotherms.At lower surface concentrations of antigen, the maximum absorbanceobtained decreased, and the minimum solution concentration of antibodyrequired to give a detectable signal increased.The shape of the ELISA plots changed as the surface concentration ofantigen decreased, but this is probably only due to the fact that at thehigh surface concentrations of antigen and high solution concentrationsof antibody, there is too much HRP in the samples to give a linearresponse (Fig. 23). The binding isotherms of antibody on the beads alsoshowed a saturation, but this was at higher concentrations of antibody.1642.50.8270.5950.3980.2640.075--x--0.0450.5-01E-08^1E-07^1E-06^1E-05^0.0001^0.001^0.01[Antibody] mg/mlFigure 58: ELISAs on silica beads modified with a range of surfaceconcentration of ferrichrome A. The legrd shows the surfaceconcentration of ferrichrome A in mg/ in .2-165Smaller surface areas of beads might have given a linear response due tothe smaller amount of HRP that would have been present when thesubstrate was added, but the bead surface area used was chosen and keptat a constant value for purposes of comparison with the assays on theflat silica.The ELISA results obtained are also plotted as a function of surfaceconcentration of antigen for the different initial concentrations ofantibody (Fig. 59). For a given solution concentration of antibody, theELISA signal increased linearly with the surface concentration ofantigen.3.5c ELISAs on the beads: inhibition of the antibody binding with freeantigenThe ELISA signal could be inhibited by free antigen, indicating thatthe antibody was binding specifically to the ferrichrome A.An initial antibody concentration of 10 ^was used for theinhibition assay (Fig 60), which was performed at the same time as theELISA measuring response as a function of solution concentration ofantibody (Fig. 61). The ferrichrome A inhibited the response at solutionconcentrations down to 10 -s mg/ml, or lx10-8 M. The sensitivity of theinhibition assay may be limited since there would be very little changein absorbance with decreasing HRP concentration at the initialabsorbance seen with no inhibiting antibody (Fig. 23). The assayconditions were chosen to be comparable to the beads rather than tomaximize sensitivity.3.5d Some comments on ELISAs on beadsThe beads can be modified with a very high concentration of antigen.This has the advantage of giving a very high response at high solution1660.2^0.4^0.6^0.8^1^1.2Surface [Ferrichrome A] mg/m2Able-2le-3le-4--Ar-1 e-5—x---le-6le-7Figure 59: ELISA result for silica beads plotted as a function ofsurface concentration of antigen. The legend shows the initialconcentration of antibody in mg/ml.16732.5-••■•■2-1.5-0.5-0 ^1E-081^1 1 IIIII^I^1^1 I I liii^1^1 1 11111^1E- 7 1E-06^1E-051 1 1 11111^I^I 1 1 1 1111^1^1 1 II 1111^1^I MITT0. 0 0.001 0.01 0 1[Ferrichrome A] mg/mlFigure 60: ELISA on beads: inhibition of antibody binding wIth freeferrichrome A with an initial antibody concentration of 10 mg/ml. A30% decrease in the ELISA signal was seen at a ferrichrome Aconcentration of 1.1x10 -5 mg/ml.168concentrations of antibody, with the binding of antibody likely beingenhanced by the formation of bridges between adjacent antigens. There issome difficulty defining the term "sensitivity" with respect to anELISA. The overall sensitivity is determined by the association constantof the antibody. For a given antibody, the bead assays are quitesensitive in that a very small surface area of beads gives the responsethat is equivalent to that obtained with ELISAs using other systems.When the usual practice of varying antibody concentrationslogarithmically (sequential halving dilutions) is used the beads are notmore sensitive than flat substrates in terms of minimum concentration ofantibody detected, since the response decreases to background at aboutthe same concentration of antibody. However, by optimizing conditionsit should be possible to set up an assay for soluble antigen using thebeads which would be able to reliably detect lower levels of inhibitingfree antigen than with the usual flat wells since a higher signal for agiven concentration of Ab can be provided. This is of interest since agreat many practical diagnostic assays employ inhibition of an ELISAsignal for detection and quantitation of free antigen.3.5e ELISAs on flat silicaAssays were also performed using flat silica modified with differentsolution concentrations of ferrichrome A (Figs. 61-63). The assays gavea much lower response than the equivalent experiments using beads withthe same surface area. Larger solution volumes were used in themeasurements of absorbance than for the measurements of the beads, sothe results shown should be divided by two to obtain a direct comparisonwith the beads. This means that the beads are giving more than six timesthe response of the flat silica (which is comparable to the signal169I^1^1^1 1 1 11^1^11111111^1^1^1^1^1 1 1 11^11E-05 0.0001 0.001[Antibody] mg/ml0.8270.5950.398--A--0.264K0.075-0.045Figure 61: ELISAs on flat silica modified using a range of solutionconcentrations of ferrichrome A. The2legend shows the surfaceconcentration of ferrichrome A (mg/m ) measured on beads that aremodified with the same solution concentration of ferrichrome A.1700^0.2^0.4^0.6^0.8 1.2 1.4 16Able-2—El--1 e -3le-41 e-5le-6Surface [Ferrichrome A] mg/mlFigure 62: ELISAs on flat silica modified with a range of solutionconcentrations of ferrichrome A. The absorbance in the ELISA is plottedagainst the surface concentration of ferrichrome A determined for beadsmodified with an equivalent solution concentration of ferrichrome A.1710.2-• ••.0.60.5-•••0.4-•0.1-0^11E-08I Him!^I I I mill^I^I mull^I I I 111111^I 1 III 1111^I^I II 1 1d1101 I I I 1118 1^1E-07^1E-06 1E-05^0.0001^0.001[Ferrichrome A] in solution mg/mlFigure 63: ELISA on flat silica showing inhibition by free ferrichrome A.A 30% decrease in the signal was seen with a ferrichrome A concentrationof 1.1x10-4 mg/ml.172obtained with commercial plates and adsorbed, haptenated protein).One of the problems with the low response is a simple geometricissue: the reaction occurs on a flat surface so the HRP wasnot distributed as evenly throughout the substrate solution as forexperiments with the beads. The test tubes in which the substratereaction was taking place were vortexed twice before that reaction wasstopped, but could have been mixed more thoroughly. If the slides wereallowed to sit briefly without mixing, a dark coloured layer formedadjacent to the slide.The assays with the flat silica all give results in the range wherethe substrate response varies linearly with the amount of HRP present inthe solution, that is, absorbances under 1.5. There was no saturation athigh concentrations of antibody and high surface concentrations ofantigen as was seen for the beads.3.5d Quantitative aspects of ELISAs and comparison of ELISAs withadsorption isothermsThe surface concentration of antibody was measured directly with aradiolabelled antibody used for some ELISAs. The results showed that theresponse in the ELISA is not linear with the surface concentration ofantibody (Figs. 64, 65), even though the absorbance readings obtainedwere in the range where the absorbance varies linearly with the amountof HRP present (Fig. 22). The ELISA gives a relatively higher responseat lower surface concentrations of antibody, which could be due eitherto a relatively high amount of the the HRP-conjugated antibody beingbound or to a relatively high conversion of substrate to colouredproduct. Binding of the HR?-labelled antibody could be enhanced athigher surface concentrations of antibody, however, where the secondary1730.5-0.4-0.2-0.1-0.6•• ••10.02^0.04^0.06^0.08^0.I1^0.12^0.14^0.16^0.18Amount of antibody bound mg/m2Figure 64: Absorbance in the ELISA as a function of surfaceconcentration of antibody for an assay using flat silica. The resultsshow that the absorbance in the ELISA is not directly proportional tothe amount of antibody on the surface. The amount bound was determinedfrom the amount remaining after washing and was therefore the amount ofantibody present during the ELISA.021740.8-0.4-0.2-0^-0.0051.20.005 0.01 0.e)15 0.02 0.025 0.03 0.035 0.104 0.045Equilibrium concentration Ab mg/mlFigure 65: Absorbance in the ELISA as a function of surfaceconcentration of antibody for an assay using a commercial ELISA plate.The amount bound was determined from the amount remaining after washing.175antibody would be able to form bridges between adjacent target antigens(the target antigens of the HRP-labelled antibody being the primaryantibody).The HRP-labelled antibody is specific towards the fc portion of theprimary antibody and so binding of the HRP-labelled secondary antibodywould be affected by the orientation of the primary antibody. If theprimary antibody were binding in such a manner that the fc portion wereless accessible, then low secondary antibody binding and a low ELISAreading would result. The binding of the primary antibody (AC3) isdifferent on the beads and flat silica: the equilibrium binding constantand maximum amount bound both differ (Table XII). The primary antibodymay bind to the silica beads in such a way that the binding of thesecondary antibody is enhanced, giving a stronger ELISA response on thebeads for the same amount of primary antibody bound. This could alsoexplain the increased sensitivity of the bead assays for measuring freeferrichrome A (Figs. 60, 63): if a smaller amount of primary antibody(AC3) were bound to the beads then less free antigen would be required toinhibit the antibody binding and a lower concentration of antigen wouldcause a decrease in the measured ELISA signal.Since the conversion of the substrate to the coloured product occursat a surface over a short period of time in a poorly stirred orunstirred solution, the extent of the reaction may be limited by thediffusion of the substrate to the surface-bound enzyme. At highersurface concentrations of enzyme, this effect would become morepronounced since the substrate near the surface would be depleted morerapidly. This may be the cause of the relative loss of signal as thesurface concentration of primary antibody was increased.Thorough mixing would be expected to increase the absorbance at176higher surface concentrations of antibody. This has been demonstratedusing an acoustic probe to provide mixing during all incubation steps ofan ELISA. The effect was to increase the absorbance obtained in thefinal step of the assay, in addition to decreasing the time required foreach incubation step (149).The response of the ELISA as a function of surface concentration ofantigen has been seen to vary with the affinity of the primary antibody(94); lower affinity antibodies were reported to give a relatively higherabsorbance in the ELISA but no explanation for this observation wasgiven.Direct comparisons of the absorbance from the ELISA with the surfaceconcentration of antigen were only made for the assays using cylindricalwells in commercial plates and for the flat silica. The surfaceconcentration of antibody on the beads can be compared to theabsorbance. The ELISA results for the beads might be more directlyrelated to the surface concentrations of antibody, due to thedistribution of the beads throughout the substrate solution. The shapeof the binding isotherm on the beads seems to resemble the shape of theELISA plot more closely than the shape of the binding isotherm on thecylindrical assay plate resembles the corresponding ELISA plot.If the ELISAs are to be used for determination of the solutionconcentration of antibody, then the non-linear response of theabsorbance with respect to surface concentration of antibody need nothinder the assay. The reference standards should be measured under thesame conditions used for the unknown, using antibodies that have thesame affinity as antibodies in the unknown sample to be measured. Thiscan, however, prove to be quite difficult due to the large number ofvariables involved. Changing the assay conditions can result in177different measured antibody concentrations (88).The antibody-antigen affinity can determine the minimum detectableconcentration of antibody and can also affect the accuracy of themeasurements (88) when determining concentration of an unknownantibody. Increasing the surface concentration of antigen can increasethe effective affinity of the antibody for the surface due to bridgingbetween adjacent epitopes and will minimize the dependency of the assayresults on the intrinsic affinity of the antibody for the antigen (94).178Chapter 4Concluding DiscussionSilica beads and slides can be silylated with 3-aminopropyltriethoxysilane to give a surface covered with amino groups. The silylated beadscan be modified rapidly and easily with carboxyl containing antigens ina one step reaction in water. Reproducible surface concentrations ofantigen which lead to reproducible assays can be obtained by usingfreshly silylated beads or simply by modifying at one time enough beadsto use in many assays. The high specific surface area of the beadspermits modification of a large area of beads using only a smallreaction volume.The EDC-mediated coupling produced a stable bond between thesubstrate and the small antigen used for the assays, so that the antigencould be immobilized on a surface without being coupled to a largermolecule that would adsorb non-specifically. The EDC-coupled antigen didnot wash off in buffer, and antibody bound to ferrichrome A-modifiedsilica did not desorb when washed with PBS-Tween.The concentration of antigen coupled to silica beads by the EDC wasmeasured as a function of solution concentration of antigen. Thesolution depletion measurements of the ferrichrome A on the beads andthe particle electrophoresis with the modified beads gave evidence forrearrangement of the ferrichrome A at higher surface concentrations.Presence of the ferrichrome A on modified slides was confirmed by XPSmeasurements showing the iron peak.Ferrichrome A was chosen as an antigen because of several featuresuseful for analysis but it proved to be a poor immunogen in BALB/c mice,179giving a polyclonal response much weaker than in rabbits and only lowaffinity monoclonal antibodies. This limited the potential sensitivityof the assays, and future experiments should include the use of higheraffinity antibodies.The binding of the antibody to the antigen-modified beads and slideswas different: the equilibrium constant for the antibody binding to theantigen-modified beads was higher, but the maximum amount bound on thebeads was lower. The silica beads were less dense than the slides andcould therefore have been porous or else could have contained organicmatter from the tetraethyl orthosilicate used in the bead production.The silica beads could also have had a higher surface concentration ofsilanol groups, since the heating process involved in the production ofthe fused quartz slides decreases the surface silanol concentration. Ifsilylation results in modification of all the silanol groups (144), thenthe beads could have had a greater surface concentration of amines. Theantibody binding could be affected by the surface charge due to theamine groups, by the surface concentration of antigen, and by theporosity of the silica substrate, since the antigen might be moreaccessible if it is not fixed on a flat surface.The ELISA response was much stronger for assays using the silicabeads, which could be due to the different modes of binding of theprimary antibody on the beads and on the slides affecting the binding ofthe secondary FIR?- conjugated antibody.The antigen-modified beads and slides were both used for ELISAsmeasuring the concentration of free antigen. Assays with the beads weremore sensitive and could be used to detect the ferrichrome A at onetenth the concentration that could be detected with ELISAs on theslides. 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Chaimberg, M. and Cohen, Y. Notes on the Silylation of InorganicSupports. J. Colloid Interface Sci., 134, 576- 579, 1990.191145. Means, G.E. and Feeney, R.F. Chemical Modification of Proteins,Holden-Day, Inc. San Francisco, 1971, p 144-145.146. Neilands, J.B. Hydroxamic Acids in Nature: Sophisticated LigandsPlay a Role in Iron Metabolism and possibly in Other Processes inMicroorganisms. Science. 156, 1443-1447, 1967.147. Emery, T. and Neilands, J.B. Structure of the ferrichromecompounds. J. Am. Chem. Soc. 83, 1626-1628, 1961.148. Gray, B.M. Methodology for polysaccharide antigens: protein couplingof polysaccharides for adsorption to plastic tubes. J. Immun.Methods, 28, 187-191.149. Boraker, D.K., Bugbee, S.J. and Reed, B. A. Acoustic probe-basedELISA. J. Immun. Methods, 155, p. 91-94, 1992.192Appendix 1Analysis of Competitive Inhibition of Monoclonal AntibodyBinding to Surface-attached AntigenD. E. BrooksThe analysis of inhibition by free Ag of monoclonal IgG Ab bindingto surface-attached Ag is complicated by:1. the presence of mixed light chains in the monoclonal Ab suchthat 1/4 of the IgG molecules present are expected to carry twospecific Ag binding sites, 1/2 of the molecules one site and1/4 of the molecules no specific binding sites;2. the statistical nature of Ag binding to divalent Ab in solution.Since the Ag in question is small, it can be assumed thatoccupation of one of the two sites by Ab in solution will notinhibit binding of the Ab molecule to surface-bound Ag. Hence,for any of the Ab species present in solution, only those withall binding sites filled will be inhibited from bindingspecifically to the surface.Both effects are taken into account in the following analysis.Assumptions:1. The binding isotherm for Ab to surface-bound Ag exhibits nonon-specific binding due the effectiveness of blocking protein.2. The Ab is pre-equilibrated with free Ag at variousconcentrations. Since the actual amount of each Ab speciesbound to the surface is very small compared to the solution193concentration (which is readily verifiable in the present case)it is assumed that the concentrations of Ab species thatequilibrate with the surface are those which result from theinitial equilibration with free Ag.3. The antibody is radiolabelled so the total amount of IgG and thetotal surface concentration of IgG are known but not theconcentrations of the individual species.4. The total concentration of soluble inhibiting Ag added is knownbut its distribution among the various species is not measured.Analysis:The binding equilibrium for Ab binding specifically to surface-attachedin the absence of free Ag is given by:r^rs Kg Cg/(1 + Kg Cg)^ [Al]where r, rs are the surface concentrations of Ab at equilibrium with Abconcentration C4 and at saturation, respectively and Kg is the apparentassociation constant for Ab binding to surface-attached Ag.From measurements of r as a function of C4 values for Kg and Fs areobtained. In the presence of soluble inhibiting Ab, and taking intoaccount the distribution of binding sites due to light chain variation,the total amount of Ab able to bind to surface sites, Con, is:Cqin = Cio + C20 + C21 (A2)where Cni = concentration of Ab species with i of its n specific sitesoccupied by soluble Ag, n = 0, 1 or 2. The individual species Cnj canbe expressed in terms of the association constant for Ag binding to Abin solution, Kag, and the equilibrium concentration of free Ag, Cag, by:194Cni = Cno n!^(Cag Kag ) [A3] (n-i)! I!Cno =^Cn T [A4]( 1 + Cag K.ag )where CnT is the total concentration of Ab bearing n specific sites inthe system. Note that according to complication 1:C1T = 2 C2T = 2 COT^ [A5]so the total concentration of labelled Ab in the system, CT,CT = ECIT = 4 C2T^ [A6]Once the free Ag has equilibrated with the Ab the total concentration ofsoluble Ag, Cagin, can be expressed in terms of the various Ab speciesCni as was the concentration of Ab available to react with the surfaceAg in [A2]:Cag n = Ci 1 + C21 + 2C22 + Cag^ [A7]since there are two Ag molecules bound per molecule of C22. Utilizing[A3], [A4] and [A6] and simplifying gives for Con:Cgin^(4 + 3z)—  ^[A8]C2T^(1 + Z)2and for Cagin, using [A7]:Cagin^z(4 + 3z)^Cog^ + ---^ [A9]Car^(1 + Z)2^C2Twhere z = Gag Kag. In the presence of soluble inhibiting Ag the bindingisotherm [Al] can be written, in terms of the fractional saturation 9 =r/rs:Kg(Cgin - TA)0= ^1 + (Cgin - VA)[A10]195[A6]where C4 = Cgin - TA and A = area per ml of surface in experiment.The object of the present effort is to estimate a value for thebinding constant of Ag for Ab in solution, Kag, from the inhibition ofsurface binding. The known or measured parameters are Kg, CT, 0, A andCagin; for a fixed choice of A and CT, 0 is measured as a function ofCagin. Equations [A6] and [A8] - [A10] uniquely determine Kag. Theirsimultaneous solution gives:Kag - [All]Cagin - z L C2Twhere:1^TAL= ^ —,_-1Kg C2Tku^- i)^u2Tz = Cag Kag = (3/2L - 1) ± (4L 9)1/2/21.in which the positive root is taken and:C2T = CT/4Applying this solution to the inhibition data shown in Figure 56gives the estimates for Kag in the table below. The following valuesfor the constants were used, derived from experiment:Kg = 7.1 x 107 litre/mole (this value takes into account the lightchain distribution)re = 0.565 mg m-2 = 3.5 x 10-13 moles/cm2CT = 9.25 x 10-9 moles/litreA = 3.2 cm2iM1196Cagin (M)^0^L^z^Kag (M-1)9.5 x 10-9^0.327^3.07^0.240^3.08 x 1071.9 x 10-8^0.275^2.41^0.519^3.22 x 1074.75 x 10-8^0.192^1.52^1.27^2.94x 1079.5 x 10-8^0.1 7^1.19^1.82^2.02 x 1071.9 x 10-7^0.129^0.948^2.47^1.34 x 107The five values for Kag give an average value of:Kag = (2.5 ± 0.8) x 107 M-11971E+040.0 0.8^1.01E+06relative mobility IgG0.2^0.4^0.6Relative MobilityAppendix 2Figure 66: Determining molecular weights from SDS-page of proteins inhuman serum. The proteins were identified by comparison to gels run byDr. Johan Janzen, and the peaks correspond to a= IgM (950 kD), b= azM,(750 kD), c= fibrinogen (340 kD), d= IgG (160 kD), e= transferrin (76 kD)and f= albumin (66 kD).198I 10.400.3300.3041)^0.250c.)0.10O. 0500.0aAppendix 3Wavelength (mu)Figure 67: Sample spectra showing optical density of a ferrichrome Asolution (---), a suspension of beads in water (- -) and a suspension ofbeads in a ferrichrome A solution (...). The two ferrichrome A solutionsare at the same concentration.0.00600.1313400. 00200.0—0.13020—0.13040—0. 0000Wavelength (na)Figure 68: The derivatives dAidA for the sample spectra shown in Fig. 67((---) ferrichrome A, (- -) beads in water and (...) beads in aferrichrome A solution).199Appendix 41.61.41.210-aa)• 0.8.0• 0.6.0• 0.40.200.8270.5950.3980.075-0.045-0.20^0.001^0.002^0.003^0.004[Antibody]eq,solution mg/ml0.005Figure 69: Antibody bound to silica beads with a range of differentsurface concentrations of ferrichrome A: bound antibody determined fromsolution depletion measurements (from the same experiment as Fig. 52).The legend shoys the measured surface concentration of antigen on thesilica in mg/m .200--x--0-A-0.2640.5950.8273.5-0.5-0.601^0.002 0.003 0.004 0.005 0.006 0.607 0.008[Antibody]eq mg/ml-0.50Figure 70: Antibody binding to flat silica modified with differentsolution concentrations of ferrichrome A: bound antibody determined fromsolution depletion measurements. The legend shrs the measured surfaceconcentration of antigen on silica beads (mg/m ) modified with the samesolution concentration of ferrichrome A.2010^0.0001^0.0002^0.0003^0.0004^0.0005^0.0006[Antibody]eq,siolutim mg/mlFigure 71: Antibody bound to silica beads withsurface concentrations of ferrichrome A: boundamount remaining after wash (a=0.827 mg/m ferrc= 0.636 mg/m2 , d= 0.398 mg/m2 , e= 0.264 mg/m0.022 mg/m2).a range of differentantibody determined fropichrome A, b=02.595 mg/mf= 0.075 mg/m, g=2020.450.4-0.35-(4Ecss 0.3-E(Fs0.25-as-Cl)0.2-00*.= 0.15-c0.1-0.05-0.0001^0.0002^0.0003^0.0004^0.0005^0.0006[Antibody]eq,solution mg/mlFigure 72: Antibody bound to silica beads with a range of differentsurface concentrations of ferrichrome A: concentration of antibodydetermined from solution depletion for the experiment shown in Figure 71.2031. 201. 00. 800. 01:10.400.200.0Wavelength (nm)Figure 73: Absorbance of ferrichrome in water and ferrichrome with alarge excess (>100:1 ratio) of EDC. There is no peak at 367 nm afteraddition of the EDC.204

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