UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Relative allergenicity of modified bovine milk proteins Jang, Colin B. 1993

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_1993_fall_jang_colin.pdf [ 5.46MB ]
Metadata
JSON: 831-1.0098822.json
JSON-LD: 831-1.0098822-ld.json
RDF/XML (Pretty): 831-1.0098822-rdf.xml
RDF/JSON: 831-1.0098822-rdf.json
Turtle: 831-1.0098822-turtle.txt
N-Triples: 831-1.0098822-rdf-ntriples.txt
Original Record: 831-1.0098822-source.json
Full Text
831-1.0098822-fulltext.txt
Citation
831-1.0098822.ris

Full Text

RELATIVE ALLERGENICITY OF MODIFIED BOVINE MILK PROTEINSbyCOLIN BARRY JANGB.Sc., The University of British Columbia, 1990A THESIS SUBMITTED IN PARTIAL FULFILMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIESDEPARTMENT OF FOOD SCIENCEWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAAugust 1993© Colin Barry Jang, 1993In 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 Food ScienceThe University of British ColumbiaVancouver, CanadaDate October 12, 1993DE-6 (2/88)iiABSTRACTCow's milk contains a variety of proteins which are capableof eliciting an allergic reaction. The casein fraction andS-lactoglobulin of the whey fraction are recognized as the mostpotent allergens in cow's milk. It is not known, however,whether the individual sub-fractions of casein possess equalallergenicity. In this study, the relative allergenicities ofce81 and S casein were determined. The relative allergenicitiesof both caseins after enzymic dephosphorylation were alsoinvestigated. In addition, the relative allergenicity of wheyafter the partial removal of S-lactoglobulin by FeCl3precipitation was studied. The relative allergenicity of themilk proteins was determined by the passive cutaneousanaphylaxis (PCA) assay, which utilized antisera obtained frommice that were exposed to the test proteins using separate oraland intraperitoneal experimental protocols. In addition, anenzyme linked immunosorbent assay (ELISA) was used to determinethe relative concentrations of antigen specific immunoglobulinsG and E in the antisera.Mice which received the test proteins by oraladministration were not sensitized against the proteins. Incontrast, results obtained from mice exposed to the testproteins by intraperitoneal injection, revealed thatdephosphorylated ces, casein and S casein were allergenic whilenative a81 casein was not. The dephosphorylation of S-casein didiiinot significantly affect allergenicity. The partial removal ofg-lactoglobulin did not significantly reduce the allergenicityof whey. The proteins which remain in the whey after treatmentwith FeCl3 (primarily u-lactalbumin and a residual amount of 0-lactoglobulin) are equal to untreated whey in their ability toproduce an allergic reaction.ivTABLE OF CONTENTSPAGEAbstract^ iiTable of Contents^ ivList of Tables viiList of Figures^ xiiiList of Appendices xviiiAcknowledgement^ xxIntroduction 1Objectives^ 3Literature Review^ 4A. Immune Response to Antigens^ 4A.1 Cells of the Immune Response 4A.2 Thymus Dependent/Thymus IndependentAntigens^ 4A.3 The Humoral Immune Response^ 5B. Allergic Reactions^ 9C. Physical and Chemical Properties of FoodAllergens^ 11C.1 Host and Environmental Factors AffectingSensitization to Allergens^ 12C.2 Antigenic Determinants/Epitopes 14C.3 Molecular Size^ 15C.4 Stability 16C.5 Foreigness^ 16VD. Cow's Milk Allergens^ 18E. Assays of Relative Allergenicity^ 21E.1 Animal Models^ 21E.2 Passive Cutaneous Anaphylaxis^ 23Materials and Methods^ 27A. Modification of Proteins^ 27A.1 Dephosphorylation of Caseins^ 27A.2 Preparation of Acid Whey 27A.3 Ferric Chloride Precipitation ofS-lactoglobulin^ 28B. Protein Determination 28C. Phosphorus Determination^ 29D. E-lactoglobulin Determination by SDS-PAGE^30E. Immunisation Protocol^ 30E.1 Oral Administration of Proteins^ 31E.2 Intraperitoneal Injection of Proteins^31F. Preparation of Antisera^ 32G. Determination of Relative Antigenicity^32H. Determination of Relative Allergenicity 34H.1 Relative IgE Concentration by ELISA^34H.2 Passive Cutaneous Anaphylaxis^ 34I. Statistical Analysis^ 35Results/Discussion^ 36A. Experiment 1: Orally Administered Proteins^36A.1 Relative Antigenicity^ 36A.2 Relative Allergenicity 39viB. Experiment 2: Intraperitoneally InjectedProteins^ 43B.1 Relative Antigenicity 43B.2 Relative Allergenicity^ 46Conclusions^ 57References 58vi iLIST OF TABLESTable 1. Comparison of the Mean Relative IgGValues for Mice Orally AdministeredPAGECasein Proteins. 37Table 2. Comparison of the Mean Relative IgGValues for Mice Orally AdministeredWhey Proteins. 38Table 3. Mean Passive Cutaneous AnaphylaxisTitres for Mice Orally AdministeredCasein Proteins. 40Table 4. Mean Passive Cutaneous AnaphylaxisTitres for Mice Orally AdministeredDephosphorylated Casein Proteins andChallenged with Potato Acid Phosphatase. 41Table 5. Mean Passive Cutaneous AnaphylaxisTitres for Mice Orally AdministeredWhey Proteins. 41Table 6. Comparison of the Mean Relative IgGValues for Mice IntraperitoneallyInjected with Casein Proteins. 44Table 7. Comparison of the Mean Relative IgGValues for Mice IntraperitoneallyInjected with Whey Proteins. 47viiiTable 8. Comparison of the Mean Relative IgEValues for Mice IntraperitoneallyInjected with Casein Proteins. 48Table 9. Comparison of the Mean Relative IgEValues for Mice IntraperitoneallyInjected with Whey Proteins. 51Table 10. Mean Passive Cutaneous AnaphylaxisTitres for Mice IntraperitoneallyInjected with Casein Proteins. 52Table 11. Mean Passive Cutaneous AnaphylaxisTitres for Mice IntraperitoneallyInjected with Dephosphorylated CaseinProteins and Challenged with PotatoAcid Phosphatase. 54Table 12. Mean Passive Cutaneous AnaphylaxisTitres for Mice IntraperitoneallyInjected with Whey Proteins. 56Table 13. Protein Concentration of OvalbuminStandard. 66Table 14. Protein Concentration of Stock CaseinSolutions. 66Table 15. Protein Concentration of Stock WheySolutions. 67Table 16. Phosphorus Concentration of CaseinSamples. 69ixTable 17. g-Lactoglobulin Concentration ofWheys. 71Table 18. Composition of Milk-Free Rodent Diet. 76Table 19. IgG Equations of the Lines and r2 Valuesfor Individual Mice Orally AdministeredNative Casein Proteins. 96Table 20. IgG Equations of the Lines and r2 Valuesfor Individual Mice Orally AdministeredDephosphorylated Casein Proteins. 97Table 21. IgG Equations of the Lines and r2 Valuesfor Individual Mice Orally AdministeredWhey Proteins. 98Table 22. IgG Equations of the Lines and r2 Valuesfor Individual Mice IntraperitoneallyInjected with Native Casein Proteins. 99Table 23. IgG Equations of the Lines and r2 Valuesfor Individual Mice IntraperitoneallyInjected with Dephosphorylated CaseinProteins. 100Table 24. IgG Equations of the Lines and r2 Valuesfor Individual Mice IntraperitoneallyInjected with Whey Proteins. 101Table 25. IgE Equations of the Lines and r2 Valuesfor Individual Mice IntraperitoneallyInjected with Native Casein Proteins. 102Table 26. IgE Equations of the Lines and r 2 Valuesfor Individual Mice IntraperitoneallyxInjected with Dephosphorylated CaseinProteins. 103Table 27. IgE Equations of the Lines and r 2 Valuesfor Individual Mice IntraperitoneallyInjected with Whey Proteins. 104Table 28. Relative IgG Values for IndividualMice Orally Administered Native CaseinProteins. 105Table 29. Relative IgG Values for IndividualMice Orally AdministeredDephosphorylated Casein Proteins. 106Table 30. Relative IgG Values for IndividualMice Orally Administered WheyProteins. 107Table 31. Relative IgG Values for IndividualMice Intraperitoneally Injected withNative Casein Proteins. 108Table 32. Relative IgG Values for IndividualMice Intraperitoneally Injected withDephosphorylated Casein Proteins. 109Table 33. Relative IgG Values for IndividualMice Intraperitoneally Injected withWhey Proteins. 110xiTable 34. Relative IgE Values for IndividualMice Intraperitoneally Injected withNative Casein Proteins. 111Table 35. Relative IgE Values for IndividualMice Intraperitoneally Injected withDephosphorylated Casein Proteins. 112Table 36. Relative IgE Values for IndividualMice Intraperitoneally Injected withWhey Proteins. 113Table 37. Passive Cutaneous Anaphylaxis Titresfor Individual Mice OrallyAdministered Native Casein Proteins. 114Table 38. Passive Cutaneous Anaphylaxis Titresfor Individual Mice Orally AdministeredDephosphorylated Casein Proteins. 115Table 39. Passive Cutaneous Anaphylaxis Titresfor Individual Mice Orally AdministeredDephosphorylated Casein Proteins andChallenged with Potato Acid Phosphatase. 116Table 40. Passive Cutaneous Anaphylaxis Titresfor Individual Mice Orally AdministeredWhey Proteins. 117Table 41. Passive Cutaneous Anaphylaxis Titresfor Individual Mice IntraperitoneallyInjected with Native Casein Proteins. 118xiiTable 42. Passive Cutaneous Anaphylaxis Titresfor Individual Mice IntraperitoneallyInjected with Dephosphorylated CaseinProteins.^ 119Table 43. Passive Cutaneous Anaphylaxis Titres forIndividual Mice IntraperitoneallyInjected with Dephosphorylated CaseinProteins and Challenged with Potato AcidPhosphatase.^ 120Table 44. Passive Cutaneous Anaphylaxis Titres forIndividual Mice IntraperitoneallyInjected with Whey Proteins.^ 121LIST OF FIGURESPAGEFigure 1. The Humoral Antibody Response. 7Figure 2. Ovalbumin Standard Curve. 68Figure 3. Phosphorus Standard Curve. 71Figure 4. S-lactoglobulin Standard Curve. 74Figure 5. SDS-PAGE of Wheys andg-lactoglobulin Standards. 75Figure 6. Determination of us, Casein SpecificIgG in Mice Orally Administered usiCasein. 78Figure 7. Determination of us, Casein SpecificIgG in Control Mice(Oral Administration). 78Figure 8. Determination of g Casein SpecificIgG in Mice Orally Administered gCasein. 79Figure 9. Determination of g Casein SpecificIgG in Control Mice(Oral Administration). 79Figure 10. Determination of Dephosphorylatedc ^Specific IgG in MiceOrally Administered Dephosphorylatedus/ Casein.^ 80Figure 11. Determination of Dephosphorylatedc ^Specific IgG in ControlMice (Oral Administration).Figure 12. Determination of Dephosphorylated0 Casein Specific IgG in Mice OrallyAdministered Dephosphorylated gCasein. 81Figure 13. Determination of Dephosphorylatedfl Casein Specific IgG in Control Mice(Oral Administration). 81Figure 14. Determination of Whey Specific IgG inMice Orally Administered Whey. 82Figure 15. Determination of Whey Specific IgG inControl Mice^(Oral Administration). 82Figure 16. Determination of FeC13 Treated WheySpecific IgG in Mice OrallyAdministered FeC13 Treated Whey. 83Figure 17. Determination of FeCl3 Treated WheySpecific IgG in Control Mice(Oral Administration). 83Figure 18. Determination of c ^SpecificIgG in Mice IntraperitoneallyInjected with us, Casein. 84Figure 19. Determination of o ^SpecificIgG in Control Mice(Intraperitoneal Injection). 84xiv80XVFigure 20. Determination of g Casein SpecificIgG in Mice IntraperitoneallyInjected with 0 Casein. 85Figure 21. Determination of g Casein SpecificIgG in Control Mice(Intraperitoneal Injection). 85Figure 22. Determination of Dephosphorylatedus, Casein Specific IgG in MiceIntraperitoneally Injected withDephosphorylated us„ Casein.^ 86Figure 23. Determination of Dephosphorylatedus, Casein Specific IgG in ControlMice (Intraperitoneal Injection). 86Figure 24. Determination of Dephosphorylatedg Casein Specific IgG in MiceIntraperitoneally Injected withDephosphorylated g Casein. 87Figure 25. Determination of Dephosphorylatedg Casein Specific IgG in Control Mice(Intraperitoneal Injection). 87Figure 26. Determination of Whey specific IgG inMice Intraperitoneally Injected withWhey. 88Figure 27. Determination of Whey Specific IgG inControl Mice^(Intraperitoneal Injection). 88Figure 28. Determination of FeC13 Treated WheySpecific IgG in Mice Intraperitoneallyxviinjected with FeC13 Treated Whey. 89Figure 29. Determination of FeC13 Treated WheySpecific IgG in Control Mice(Intraperitoneal Injection). 89Figure 30. Determination of us, Casein SpecificIgE in Mice IntraperitoneallyInjected with us, Casein. 90Figure 31. Determination of as, Casein SpecificIgE in Control Mice(Intraperitoneal Injection). 90Figure 32. Determination of g Casein SpecificIgE in Mice IntraperitoneallyInjected with g Casein. 91Figure 33. Determination for g Casein SpecificIgE in Control Mice(Intraperitoneal Injection). 91Figure 34. Determination of Dephosphorylateda ^Specific IgE in MiceIntraperitoneally Injected witha^ 92Figure 35. Determination of Dephosphorylatedas, Casein Specific IgE in ControlMice (Intraperitoneal Injection).^92xviiFigureFigure36.37.Determination of Dephosphorylatedg Casein Specific IgE in MiceIntraperitoneally Injected withg Casein.Determination of Dephosphorylatedie Casein Specific IgE in Control93Mice^(Intraperitoneal Injection). 93Figure 38. Determination of Whey Specific IgEin Mice Intraperitoneally Injectedwith Whey. 94Figure 39. Determination of Whey Specific IgEin Control Mice(Intraperitoneal Injection). 94Figure 40. Determination of FeC13 Treated WheySpecific IgE in Mice IntraperitoneallyInjected with FeC13 Treated Whey. 95Figure 41. Determination of FeC13 Treated WheySpecific IgE in Control Mice(Intraperitoneal Injection). 95LIST OF APPENDICESPAGEAppendix 1. Protein Determination.^ 66Appendix 2. Phosphorus Determination. 69Appendix 3. g-lactoglobulin Determination.^ 72Appendix 4. Rodent Diet.^ 76Appendix 5. ELISA Buffer Compositions.^ 77Appendix 6. ELISA Absorbance vs. Time Graphs.^78A. ELISA IgG Determinations^ 78A.1 Experiment 1: Orally AdministeredProteins^ 78A.2 Experiment 2: IntraperitoneallyInjected Proteins^ 84B. ELISA IgE Determinations^ 90Appendix 7. Equations of the Lines and r2 Valuesfor Individual Mice.^ 96A. ELISA IgG Determinations 96A.1 Experiment 1: Orally AdministeredProteins^ 96A.2 Experiment 2: IntraperitoneallyInjected Proteins^ 99B. ELISA IgE Determinations^ 102xixAppendix 8. IgG Values for Individual Mice.^105A. Experiment 1: Orally Administered Proteins^105B. Experiment 2: Intraperitoneally InjectedProteins^ 108Appendix 9. IgE Values for Individual MiceIntraperitoneally Injected withProteins.^ 111Appendix 10. PCA Titres for Individual Mice.^114A. Experiment 1: Orally Administered Proteins^114B. Experiment 2: Intraperitoneally InjectedProteins^ 118ACKNOWLEDGEMENTI would like to thank all the members of my researchcommittee for their help and comments: Dr. D. Kitts, Dr. E.Li-Chan, Dr. B. Skura and Dr. J. Vanderstoep. I would also liketo thank Sherman Yee, Val Skura, Angela Kummer, Donna Smith, andDr. L. Kastrukoff and his staff for their assistance during thecourse of my thesis. A very special thank you goes to EmmanuelAkita for his help above and beyond the call of duty.XX1INTRODUCTIONFood provides the human body with nutrients which areessential for maintaining life. However, foods can also haveharmful effects on the human body. Under certain circumstances,certain individuals may develop adverse reactions to particularfoods. Adverse reactions to foods can occur by severaldifferent mechanisms; reactions that are immunologically basedare categorized as food allergies. Food allergies are definedas food induced immune reactions that are harmful to the tissuesor disruptive of the physiology of the host. A food allergen isthat specific component of food which takes part in the immunereaction that results in allergy. In the majority of clinicalcases, protein is the food component responsible for elicitingan allergic response.The incidence of food allergies in the overall populationis estimated at less than ]A (Taylor, 1985). Only a fewindividuals will develop the allergen specific immunoglobulinisotype (IgE) and an allergic response after exposure to apotentially allergenic food (Aas, 1978). Genetic andenvironmental factors, as well as the physical and chemicalproperties of the protein itself will affect the likelihood ofallergic sensitization to the allergen.Cow's milk is a common source of food related allergies,particularly among children and infants. Due to the complexmixture of proteins in cow's milk, a number of differentproteins may act as allergens. However, those individuals2allergic to cow's milk often show sensitivity to more than oneprotein (Baldo, 1984). The proteins which are the most commonlyimplicated in individuals allergic to cow's milk areS-lactoglobulin and the casein fraction. Modification orremoval of these proteins may reduce the likelihood of allergicsensitization to milk.The purpose of the thesis, was to determine if an infantformula could be produced with reduced allergenicity. Cow'smilk can be made similiar to human milk by manipulating itsprotein composition and acid clotting properties. Human milkdoes not contain the whey protein P-lactoglobulin. SinceP-lactoglobulin is recognized as one of the most potentallergens in cow's milk, its removal may reduce allergenicity.In addition, human milk does not contain us, casein. Althoughthe casein fraction is also considered to be one of the mostpotent allergens in cow's milk, the relative allergenicities ofthe individual sub-fractions have not been determined. Humanand bovine milks clot differently in the stomachs of infants(Nakai and Li-Chan, 1987; Pildes et al., 1980; Cavell, 1979).Bovine casein which has been dephosphorylated with potato acidphosphatase, forms a fine dispersion with a microstructuresimiliar to that of human casein when acidified to pH 4 (Li-Chanand Nakai, 1989). However, the effect of dephosphorylation onallergenicity is unknown.3OBJECTIVESThere were four objectives of the thesis:1.) To determine the relative allergenicities of native as„and 8 casein.2.) To determine the relative allergenicity of us, caseinfollowing enzymatic dephosphorylation.3.) To determine the relative allergenicity of 8 caseinfollowing enzymatic dephosphorylation.4.) To determine the relative allergenicity of whey afterthe removal of g-lactoglobulin.4LITERATURE REVIEWA. Immune Response to AntigensA.1 Cells of the Immune ResponseAll the cells of the blood, including those that governimmune reactions, are derived from a common precursor called astem cell. Stem cells are generated in the bone marrow, andundergo a process known as hematopoeisis to differentiate intothe various cell types (Pestka and Witt, 1985). The cell lineresponsible for specific immunity are the lymphocytes. Thelymphocytes can be divided into two types: T-cells and B-cells,and are named after the organ in which they differentiate.Thus, stem cells which migrate to and mature in the thymusbecome T-cells. Stem cells which mature in the bursalequivalent (bone marrow in humans) become B-cells. T-cells arefurther subdivided into various subpopulations, depending on thefunction that each T-cell performs. B-cells can be divided intoplasma and memory cells. T-cells regulate the immune responseand are also involved in cell mediated immunity. B-cells areresponsible for humoral or antibody mediated immunity.A.2 Thymus Dependent/Thymus Independent AntigensAntigens can be of two types: thymus dependent or thymusindependent. Thymus dependent antigens require a synergisticcooperation between both B and T-cells in order to initiate ahumoral response (Kimball, 1983). Thymus independent antigensrequire only B-cells for the production of antibodies. Inhumans, T-cells are required in order for the antigen to provoke5an immune response (Kimball, 1983). Therefore, all antigens inhumans are probably thymus dependent. Thymus dependent antigensinduce the production of antibodies of the IgM, IgG, IgA and IgEclass, while thymus independent antigens initiate an IgMresponse (Sweeney and Klotz, 1987).A.3 The Humoral Immune ResponseImmune responses to food antigens occur in thegastrointestinal tract. The gastrointestinal tract containslymphoid tissue capable of mounting an immune response toantigens which cross the mucosal epithelial barrier. Gutassociated lymphoid tissue is found primarily in "Peyer'spatches", a group of nodules found in the ileum of the smallintestine (Anderson and Sogn, 1984). Overlying the Peyer'spatches is a single layer of epithelium containing M-cells,which regulate antigen uptake (Owen and Nemanic, 1978). Antigenabsorption in the gut by M-cells is an important access routefor ingested antigens to reach lymphoid tissues and therebystimulate the local and distant immune system (Walker, 1987).The remainder of the intestine is also a site for antigensampling, and is probably of even more significance than theM-cell, since the surface area of the M-cell is small comparedto the total surface area of the intestine (Kleinman, 1992).When the antigen penetrates the epithelial mucosa, the firststructure to respond immunologically is a macrophage (Breneman,1987) (See Figure 1). The macrophage engulfs the antigen intophagocytic vacuoles, which fuse with lysosomes and digest the6antigen with proteases and other degradative enzymes. However,not all the antigen is degraded; some of it is retained inimmunogenic form and localized on the surface membrane of themacrophage in association with an antigen encoded by the majorhistocompatability complex (MHC) (Kimball, 1983).The macrophage presents the antigen fragment/MHC antigen toa particular subset of T-cell known as a T-helper (TH) cell. AllTH cells express a single type of receptor specific for aparticular antigen fragment/MHC encoded antigen. Thespecificity of the receptor is determined prior to contact withthe antigen. Once the TH cell binds to the antigen fragment/MHCencoded antigen, the macrophage secretes interleukin-1 whichactivates the TH cell. The activated T-helper cell synthesizesand secretes interleukin-2 and produces its own receptors forthis chemical. The binding of interleukin-2 with its receptorscauses the T-helper cell to undergo mitosis and proliferate intoclones with the same antigen fragment/MHC encoded antigenreceptor specificity. The clones produced do not express theinterleukin-2 receptor, therefore, the clones must interact withthe appropriate macrophages in order for clonal proliferation tocontinue.B-cells have membrane bound immunoglobulins on theirsurface which act as antigen receptors. The receptors consistof IgD and monomers of IgM. Both isotypes exhibit the sameantigen binding specificity. The specificity of the receptorsis determined prior to contact with the antigen. When antigenFigure 1. The Humoral Antibody Response. (1) Macrophage engulfs and digeststhe food antigen. (2) Food antigen is presented on the surface of the macrophage in association with MHCencoded antigen. (3) T. cell with receptor for food/MHC antigens binds to macrophage. (4) Macrophagesecretes IL-1. (5)T. cell secretes IL-2. (6) T. cell undergoes mitosis. (7) B-cell binds to food antigen.(8) Food antigen endocytosed by B-cell. (9) Food antigen presented on the surface of the B-cell inassociation with MHC encoded antigen. (10) T. cell blnds to B-cell. (11) T. cell secretes BCGF and BCDF.(12) B-cells proliferate and differentiate. (13) Plasma cells secrete antigen specific antibodies.8binds to the immunoglobulins on the surface of the B-cell, theyare moved to one pole of the cell where they form a cap. Theantigen is then taken into the cell by endocytosis. Fragmentsof the antigen are returned to the B-cell surface in associationwith the same MHC encoded antigen which was found on the surfaceof the macrophages. The antigen fragment/MHC encoded antigen onthe surface of the B-cell serves as a bridge to physically linkthe B-cell with T, cells that have receptors of the samespecificity. Interleukin-1 secreted by the macrophages andB-cell growth factors secreted by TH cells cause the B-cell toundergo mitosis and generate a clone with the same antigenbinding specificity. The ability of the B-cell to respond tointerleukin-1 and the B-cell growth factors is believed to bemediated by the production of specific receptors for each ofthese factors, once the B-cell has bound antigen and linked toa T„ cell. The TH cell also secretes B-cell differentiationfactors which transform the proliferating B-cells into plasmacells. The ability of the proliferating cells to respond to theB-cell differentiation factors is also probably governed by theproduction of receptors specific for these factors.Plasma cells are capable of synthesizing and secretingantibodies. The secreted immunoglobulins may be of any isotype(IgM, IgG, IgA, or IgE), and will have the same antigen bindingspecificity as the original B-cell receptor. Usually, plasmacells can synthesize only one isotype at any one time. Clonesof the B-cell may also become memory cells. Memory cells9possess antibodies on their surface which function as receptors.These antibodies have the same antigen binding specificity asthe original B-cell, however, the receptors may be of otherisotypes (IgG, IgA or IgE). Memory cells allow for an enhancedimmune response upon subsequent exposure to the antigen. Uponreexposure, the magnitude of the humoral response is greater,the lag time between exposure to the antigen and the productionof antibodies is reduced, and antibody production is extendedfor a greater length of time. Isotype switching also occurs;initial contact with the antigen produces IgM while subsequentexposure to the antigen produces IgG, IgA, and IgE. Inaddition, affinity maturation occurs. Over the course of theimmune response, B-cell clones with higher affinity receptorswill dominate the response and secrete antibodies with increasedaffinity for the antigen (Kimball, 1983).B. Allergic ReactionsAll allergic reactions can be classified on the basis offour different immunological mechanisms, as first described byCoombs and Gell (1975). However, the four mechanisms are notmutually exclusive; more than one mechanism may operate at thesame time (Bahna, 1987).The Type I reaction is also known as immediate or acutehypersensitivity and is characterized by a rapid onset ofsymptoms (Taylor, 1985). Type I reactions to foods are widelydocumented and accepted, and it is the only allergic reactionthat is relatively well understood (Kniker, 1987). The Type I10reaction involves antigen specific IgE. IgE is capable ofbinding to the surface of mast cells and basophils via the Fcregion of the antibody. Both mast cells and basophils possesssurface receptors specific for IgE. Each basophil can carry upto a few thousand to one million receptors for IgE on itsmembrane (Aas, 1978). When antigen cross links two IgEmolecules on the surface of the mast cell or basophil, the celldegranulates and releases vasoactive mediators. These mediatorsare capable of causing smooth muscle contraction and increasedvascular permeability, which results in the clinical symptoms ofallergy (Pestka and Witt, 1985). While over 40 substances havebeen identified as mediators, histamine is responsible for themajority of the immediate effects (Taylor et al., 1989).For a Type II allergic reaction to occur, the antigen mustpassively attach to the cells of the host. The Fc region of acell surface antigen-antibody complex (with IgM or IgG) iscapable of activating the complement cascade via the classicalpathway (Pestka and Witt, 1985). When antigen specific IgM orIgG binds to the antigen, complement is activated and theantigen along with the host cells that the antibody has attachedto are destroyed. Type II reactions have not been shown tooccur with foods (Taylor and Cumming, 1985).Type III allergic reactions occur when antigen binds tospecific IgG or IgM and forms an immune complex. Immunecomplexes which circulate in the fluid of the vascular systemcan become trapped within tight vascular tufts and settle11(Breneman, 1987). The antigen-antibody complex can activatecomplement, and the complex along with the host cells in whichit has become trapped are destroyed. The circulation of immunecomplexes containing food antigen and specific antibodies is acommon and normal phenomenon, and whether such circulation anddeposition of complexes contributes much to food-related diseaseis difficult to prove (Kniker, 1987). Both uncertainty andcontroversy exist regarding the importance of Type III reactionsto food (Haddad et al., 1983).Type IV allergic reactions are also known as delayed typehypersensitivity, and are characterized by a delayed onset (> 6hr. after ingestion of the offending food) of symptoms (Taylor,1985). Type IV reactions are mediated by a subset of sensitizedT-cells known as T-Delayed Type Hypersensitivity (T DTH) cells.In order for a Type IV reaction to occur, the antigen must bebound to a cell surface (Pestka and Witt, 1985). When a TDTH cellwith specific receptors binds to the antigen, it releaseslymphokines which cause non-specific cell killing. Type IVreactions can involve food, but the precise mechanism of thesereactions is poorly understood at the molecular level (Taylorand Cumming, 1985).C. Physical and Chemical Properties of Food AllergensIn food allergy, the role of antibodies other than those ofIgE is poorly understood (Aas, 1989). Type I or IgE mediatedfood reactions are the only type of responses for which it ispossible to demonstrate a clear relationship with the symptoms12of food allergies (Pastorello et al., 1987). All known foodallergens have been implicated in IgE mediated reactions (Tayloret al., 1987a). This review of the physical and chemicalproperties of food allergens will be restricted to antigenscapable of eliciting a Type I response.In order to be an allergen, the offending food constituentmust be capable of stimulating the production of IgE. All foodallergens capable of initiating an IgE mediated allergicresponse are naturally occurring proteins or glycoproteins (Aas,1989). Very little is known about the characteristics of thosefood proteins which can function as allergens (Taylor, 1992).Proteins that can function as allergens are found predominantlyin foods of plant and marine origin; with the exception of cow'smilk and chicken egg, proteins of animal origin are rarelyimplicated in food allergies (Taylor, 1992).C.1 Host and Environmental Factors Affecting Sensitizationto AllergensHost factors will contribute to, or determine, whether acertain molecule will act as an allergen (Aas, 1978). Hostfactors are equally as important as the molecular structure ofthe allergen (Aas, 1976). Studies have indicated that while thedisposition to become allergic is inherited, the type of diseasemanifestation and the sensitization to a particular allergenusually are not, although they can be (Marsh, 1975). Thelikelihood of allergic sensitization to the proteins in food isheavily dependent on the immune responsiveness of the individual13(Taylor et al., 1987b). Only those individuals whose geneticcode is strongly programmed to produce IgE under the normalconditions of exposure to food, are likely to become sensitizedto food proteins (Aas, 1978).Several environmental factors will influence the likelihoodof allergic sensitization to particular food proteins (Taylor etal., 1987b). The degree and frequency of exposure to theantigen is critical (Taylor et al., 1987b). Some experimentswith animals have suggested that low dose stimulation withantigen favours the production of IgE, whereas high dosestimulation favours the production of IgG (Ishizaka, 1976). Theage of the individual at the time of exposure to the antigen isalso critical (Taylor et al., 1987b). Food allergy is much morecommon among children than adults (Fries, 1959). Although thepotential exists to develop allergies at any age, infants arethe most susceptible (Taylor and Cumming, 1985), since the gutof the infant is more permeable to dietary antigens than the gutof adults (Udall et al., 1981a; Walker and Bloch, 1983).Therefore, if exposure to an antigen occurs at an early age, thelikelihood of allergic sensitization to that protein isincreased (Taylor et al., 1987b). Age is a less importantdeterminant of intestinal permeability than the state ofmaturation of the intestinal barrier (Udall et al., 1981b).Infants with food allergies tend to lose their sensitivity asthey grow older (Taylor, 1985) as a result of gut closure.Other factors which affect gut permeability, such as intestinal14injury, will also enhance the likelihood of allergicsensitization to food proteins (Taylor et al., 1987b).C.2 Antigenic Determinants/EpitopesAn antigenic determinant or epitope is that specific areaor site on a protein molecule to which an antibody or receptorof an immunocompetent lymphocyte can bind (Aas, 1989). Sinceall naturally occurring allergens are proteins, epitopes can beof two types; these being sequential or conformational.Sequential epitopes consist of a number of amino acid residuesas found in the primary sequence of the protein. Conformationalepitopes result from the steric folding of the polypeptidebackbone chain, which brings together amino acid residues thatwere originally found at different sites on the primary sequence(Aas, 1989). In proteins, antigenic determinants are usuallycomposed of between five and ten amino acid residues (Sweeneyand Klotz, 1987). Most epitopes are conformational (Aas, 1989).To be an allergen, the protein must have at least two epitopesin order to be able to crosslink the IgE molecules once theantibodies have bound to the surface of the mast cell orbasophil (Aas, 1978). Although two epitopes are required forbridging to occur, there is no evidence to conclude that thoseepitopes must be identical (Aas, 1978). In addition, epitopesmust be on the surface of the antigen in order for IgE producingcells and antibodies to react with it (Atassi, 1975; Crumpton,1974). Therefore, the factors that may determine the potency ofa particular allergen are: (1) the number of epitopes that are15accessible for specific antibodies, and (2) the binding dynamicsbetween these epitopes and antibodies (Aas, 1989).C.3 Molecular SizeThe ideal size (molecular weight) of food allergens rangesbetween 10,000 to 70,000 daltons (Taylor, 1992). The size offood allergens appears to be dictated by at least three factors:(1) the bridging requirement, (2) ability of the protein toinitiate an immune response, and (3) intestinal permeability(Taylor, 1992). In order for the basophil or mast cell todegranulate, the allergen must cross link two IgE molecules onthe cell membrane surface. Therefore, the allergen must havethe appropriate molecular size or dimensions to allow bridgingto occur. More correctly, there must be two or more epitopeslocated along the protein molecule at distances equivalent tothose between IgE molecules on the surface of the basophils ormast cells (Aas, 1978). Thus, bridging may be less dependent onmolecular size than on molecular shape (Taylor et al., 1987a).The allergen must also be capable of initiating theproliferation of immunocompetent lymphocytes and trigger thesynthesis of specific antibodies. Small proteins are lesslikely to be antigenic (Taylor et al., 1987b). However, smallpeptides can act as haptens and become allergenic if theyaggregate with larger proteins (Taylor et al., 1987b). A haptenis a molecule that by itself will not initiate an immuneresponse, but may serve as an antigenic determinant when coupledto another antigenic molecule (Kimball, 1983). Proteins with16molecular weights in excess of 70,000 daltons are less likely tobe efficiently absorbed through intestinal mucosal membranes andobtain access to IgE producing cells (Taylor, 1987a). However,larger molecules may induce IgE production following parenteraladministration (Aas, 1978).C.4 StabilityAllergenic food proteins are capable of inducing anallergic reaction after various food processing treatments(Taylor, 1986). Therefore, it is reasonable to assume thatthese food proteins are comparatively stable to heat and acidtreatments (Taylor, 1992). Food allergens must also survive thedigestive process, and thus, are resistant to pH mediatedchanges and peptic-tryptic digestion (Taylor et al., 1987a).Denaturation of food proteins with heat or chemical treatmentscan substantially reduce the allergenicity of proteins withconformational allergenic determinants, since such treatmentscould irreversibly alter the three-dimensional structure of theepitope (Taylor et al., 1987b). However, protein denaturationmay have no effect on allergenicity if the allergenicdeterminant resides on the primary sequence of the proteinmolecule (Taylor et al., 1987b).C.5 ForeignessProteins must be able to stimulate the production ofspecific IgE in order to be an allergen. The ability of aparticular protein to induce the production of antibodies isrelated to the protein's perceived degree of foreigness to the17host (Crumpton, 1974). However, this concept of foreigness innot well understood, especially as related to the production ofIgE (Taylor, 1992). In general, the more foreign or chemicallydifferent an antigen is from the normal tissues of the host, thegreater its antigenicity (Sweeney and Klotz, 1987). Proteinsthat are similiar in nature to proteins existing in the hostoften have little or no antigenicity within that host (Sweeneyand Klotz, 1987). An example is an experiment that wasperformed with the pituitary hormone adrenocorticotrophin(ACTH). Dayhoff and Eck (1969) studied ACTH in various animalspecies. ACTH consists of 39 amino acid residues, 24 of whichwere shared by the animals in their study. Species specificityto ACTH occurred in the amino acid regions between 25 and 33.When antibodies were raised against ACTH, the antibodies werefound to react only with the areas of variability, not withthose areas that were common to the various species. Thus,antibodies are formed only against particular areas of a foreignprotein that are not shared by the host (Crumpton, 1974).It has not been possible to determine any physicochemicalfeature that is characteristic for allergens, apart from beingproteins with a molecular weight between 10,00 to 70,000 daltons(Aas, 1989). Although the amino acid composition or sequenceand the three dimensional structure of most food allergens areunknown, they do not appear to be unusual proteins in any wayother than their allergenicity (Taylor et al., 1987a). Theallergenicity of these proteins is unlikely to be due to the18existence of unique compositional features (Taylor et al.,1987a). In addition, the general structure and chemicalproperties of an antigen alone cannot be used to predict thetype of reaction one might observe in populations (Sweeney andKlotz, 1987).D. Cow's Milk AllergensThe most common allergenic food among children and infantsis cow's milk (Bock and Martin, 1983; Bock et al., 1978). Cow'smilk allergy (CMA) is a hypersensitivity reaction that is theresult of an abnormal immunologic response to one or moreproteins found in cow's milk (Taylor, 1986). Manifestations ofCMA can result in a number of gastrointestinal, respiratory anddermatologic symptoms. The least commonly observed (Lebenthal,1975), but most serious manifestation of CMA is anaphylacticshock, which can result in death (Taylor, 1986). The occurrenceof Type I or IgE mediated responses has been confirmed in CMA(Taylor, 1986). Type III and Type IV allergic reactions mayplay a role in some forms of CMA, but further evidence isrequired to establish this possibility (Taylor, 1986).Cow's milk contains between 28 to 41 grams protein perlitre (Bahna and Heiner, 1980) and can be divided into two mainprotein components: casein and whey. Milk also containsnumerous minor protein fractions as well as several enzymes(Taylor, 1986). Casein (csn) constitutes approximately 809s ofthe proteins in milk, while the whey fraction makes up theremaining 20-96- (Swaisgood, 1985). Based on their order of19decreasing electrophoretic mobility, the casein fraction can befurther subdivided into the ce (1-csn and us2-csn), S, 1, and Kcaseins (Bahna and Heiner, 1980). Of the caseins, u81-csn isfound in the largest concentration, and constitutesapproximately 34% of the total proteins in milk and 42.5% of theproteins in the casein fraction (Swaisgood, 1985). The secondlargest casein fraction in terms of concentration is S-csn,which constitutes approximately 25% of the total proteins inmilk and 31.3% of the proteins in casein (Swaisgood, 1985). Thewhey fraction is composed of S-lactoglobulin, a-lactalbumin andthe blood proteins (serum albumin and immunoglobulins)(Swaisgood, 1985). Of the proteins in the whey fraction,S-lactoglobulin is found in the largest concentration, andconstitutes approximately 9% of the total proteins in milk and45% of the proteins in whey (Swaisgood, 1985). Individualprotein constituents will vary quantitatively with the stage oflactation, the breed of cattle and the feed used, but withlittle qualitative variation (McMeekin, 1954).Cow's milk contains between 18 to 25 proteins which can actas antigens (Bahna and Gandhi, 1983; Baldo, 1984; Hanson andMansson, 1961; Savilahti, 1981). However, not all antigens inmilk are allergenic. In addition, those proteins which areallergenic do not possess the same allergenicity (Bahna andHeiner, 1980). The allergenicity of individual milk proteinshas been evaluated by skin tests and oral challenges.S-lactoglobulin was found to produce the strongest response when20skin tests were performed with persons known to be allergic tocow's milk (Bleumink and Young, 1968; Goldman et al., 1963b).Oral challenge studies with isolated protein fractions showedS-lactoglobulin and casein to be the two most common allergensin cow's milk (Davidson et al., 1965; Goldman et al., 1963b;Kuitunen et al., 1975; Liu et al., 1967; Visakorpi and Immonen,1967). u-lactalbumin was less frequently incriminated as anallergen in oral challenges, and reactions to bovine serumalbumin and immunoglobulins were rare (Taylor, 1986). Nochallenge studies have been performed using the individualsub-fractions of casein (Taylor, 1986). IgE antibodies toseveral minor proteins such as lactoferrin, lactoperoxidase,alkaline phosphatase and catalase have been identified in a fewpatients with cow's milk allergy (Baldo, 1984), however, thesignificance of these proteins as allergens has yet to bedetermined (Taylor, 1992). It should also be noted, thatindividuals with cow's milk allergy may react to more than oneprotein in cow's milk (Taylor, 1992).The enzymatic digestion of milk proteins may also affectallergenicity. Epitopes must be expressed on the surface of theprotein in order to interact with immunocompetent lymphocytes.When a protein is digested, epitopes which were hidden withinthe molecule can become exposed. Ishizaka et al. (1960)presented evidence that new antigenic determinants were exposedwhen bovine serum albumin was partly degraded with pepsin. Thisphenomenon has also been reported with casein, S-lactoglobulin21and a-lactalbumin when treated in vitro with pepsin and trypsin(Spies et al., 1970).Neither milk fat nor lactose has been shown to be antigenic(Bahna and Heiner, 1980), however, both components may affectthe allergenicity of milk. Poulsen et al. (1987) demonstratedthat the homogenization of milk increases the ability of themilk to induce a systemic anaphylactic reaction in sensitizedmice when challenged intravenously. This phenomenon has alsobeen confirmed in children allergic to cow's milk upon oralchallenge (Host and Samuelsson, 1988). The allergenicity ofcow's milk proteins might also be enhanced by the non-enzymaticor Maillard browning reaction with lactose (Smith, 1976;Bleumink and Berrens, 1966; Bleumink and Young, 1968; Lietze,1969). When the Maillard browning reaction occurred betweeng-lactoglobulin and lactose, the amount of antigen required toelicit a positive intradermal reaction in subjects allergic tocow's milk was decreased by a factor of 100 (Bleumink, 1970;Bleumink and Berrens, 1966).E. Assays of Relative AllergenicityE.1 Animal ModelsAnimal models provide comparative indications on theallergenicity of proteins from various sources and on the impactof different technologies affecting immunoreactivity in vivo (Pahud et al., 1988). However, the extent to whichallergenicity in any laboratory animal model correlates withhuman allergenicity is unknown (Kleinman, 1992). The validity22of these model systems is restricted by the limited informationconcerning the prevailing epitopes in humans and animals (Aas,1978).Laboratory animals may be sensitized by parenteralimmunization using adjuvant (Williams and Chase, 1967; Humn andChantler, 1980). Two species which have been used to assess theallergenicity of milk by this method are guinea pigs and mice(Ratner et al., 1958; Poulsen et al., 1990). When mice are usedin allergenicity studies, a critical factor to consider is theantigen dose. Vaz et al. (1971) found that immunizing mice withminute doses of 0.1 Ag of ovalbumin in aluminum hydroxide gelinduced high titres and the persistent synthesis of IgEantibodies. Immunization with high doses of 100 Ag of antigeninduced weak and transient IgE formation.The route of immunization affects the concentration ofantigen and the types of cells with which the antigen willinteract (Sweeney and Klotz, 1987). When a protein is ingested,it is exposed to digestive enzymes and the acid conditions ofthe stomach. The protein must filter through the mucous layerand intestinal cell wall before encountering immunocompetentcells (Guesry et al., 1989). These processes dramaticallyreduce the amount of foreign protein that reaches theimmunological system (Guesry et al., 1989). Therefore, animalmodels which utilize oral sensitization are more desirable,since they more accurately represent the normal route ofsensitization to food proteins. Anderson et al. (1979)23demonstrated that guinea pigs could be sensitized to cow's milkproteins by gavage via the oral route. Use of the oral guineapig model for assessing the allergenicity of milk proteins aftervarious treatments is well documented (Heppel et al., 1984; Jostet al., 1987; Kilshaw et al., 1982; McLaughlan et al., 1981;Pahud et al., 1985), and is an accepted in vivo experimentalapproach for comparing the effect of different treatments onprotein allergenicity (Jost et al., 1987). Mice can also besensitized to milk proteins by the oral route (Poulsen et al.,1990; Poulsen et al., 1987; Nielsen et al., 1989). A studyconducted by Paulsen and Hau (1987) indicated that guinea pigscould be replaced by mice when assessing the allergenicity ofvarious compounds by PCA. The sensitivity of the two species issimiliar, but the husbandry and handling of mice is moreconvenient and they are less expensive (Poulsen and Hau, 1987).E.2 Passive Cutaneous AnaphylaxisIn vivo assays such as anaphylactic shock models andpassive cutaneous anaphylaxis are widely employed andrecommended when assessing the allergenicity of variouscompounds (Poulsen and Hau, 1988). Anaphylactic shock modelsemploy test animals which have been immunized against theproteins. Following intravenous challenge with the antigen, theanimals are observed to see if fatal anaphylaxis occurs.Anaphylactic shock scores have also been used to evaluate thedegree of anaphylaxis. The scores are assigned according to theseverity of the anaphylactic reaction observed after injection24of the antigen (Paulsen et al., 1987). An alternative in vivoapproach is passive cutaneous anaphylaxis. PCA corresponds inevery respect to systemic anaphylaxis (Ovary et al., 1963),however, it is easier to quantitate than systemic anaphylaxis(Watanabe and Ovary, 1977) and there is less suffering on thepart of the animals. To perform the PCA test, serum collectedfrom an immunized animal is injected intracutaneously in asuitable recipient. The IgE in the sera binds to mast cells inthe skin via its Fc fragment. Fc fragments vary according toantibody class, and it is for this reason that not all isotypesare capable of binding to the mast cells in the skin (Watanabeand Ovary, 1977). After a specific time period to allow bindingto occur, the allergen and a non-toxic dye are intravenouslyinjected into the recipient animal. When the allergencrosslinks two IgE molecules on the surface of the mast cell,the mast cell degranulates and releases vasoactive substances.The release of the vasoactive substances increases thepermeability of the capillaries, which allows the dye to diffuseinto the surrounding tissue and cause the skin around thereaction sites to change colour. For precise readings, theanimal should be killed, skinned and the reaction measured onthe inside of the skin (Watanabe and Ovary, 1977). PCAreactions can be quantitated by the size of the reaction, or bythe endpoint technique (Watanabe and Ovary, 1977). The size ofthe reaction is roughly proportional to the antibody content(within certain limits), and can be quantitated by measuring the25diameter of the reaction (Watanabe and Ovary, 1977). Thistechnique is seldom used since it is much more demandingtechnically than the endpoint technique. The endpoint techniqueemploys a series of diluted antisera. The reciprocal of thehighest dilution to give a positive reaction is used as theendpoint or titre.The standard assay for quantitating antigen specific mouseIgE has been the PCA reaction in rats (Watanabe and Ovary,1977). The antisera from mice can be titred in both homologousand heterologous species. In homologous species (eg. mice-mice), both IgE and IgG, participate in the PCA reaction. Inheterologous species (eg. mouse-rat) only IgE is involved in thereaction (Ovary et al., 1975).Only in vivo studies on animal models or human beings cangive precise information on the relative allergenicity of agiven protein after processing (Guesry et al., 1989). In vitro tests should only be used when the results have been confirmedby in vivo studies (Guesry et al., 1989). Theradioallergosorbent test (PAST) and enzyme linked immunosorbentassay (ELISA) are two in vitro techniques which have been usedto measure the levels of a particular immunoglobulin class tospecific antigens in the serum. To perform a RAST, the antigenis bound to a solid phase. The test serum is added to theantigen, and incubated to allow binding to occur between theantigen and antibodies. A radiolabeled anti-Ig antibodyspecific for the isotype under study is added (Chandra and26Jeevanandam, 1984) and the amount of bound radioactivity ismeasured, giving an estimate of the antigen specific Ig in theserum (Aas, 1978). A similiar principle is used for the ELISA,however, instead of using a radiolabeled anti-Ig antibody, anenzyme coupled to the anti-Ig antibody is substituted and asubstrate is added. The enzyme acts on the substrate whichcauses it to change colour or become fluorescent (Anderson,1990). The amount of colour or fluorescence is measured, whichprovides an estimate of the antigen specific Ig in the serum.In vitro methods such as RAST and ELISA are routinely used tomeasure antigen specific IgE. Alone, these tests cannot be usedto assess relative allergenicity, but must be employed inconjunction with an in vivo method. The RAST and ELISA methodsmeasure free IgE in the serum, not cell-bound IgE (Chandra andJeevanadam, 1984). In addition, they are unable to evaluate theextent of antigen induced mediator release (Conroy and Adkinson,1977). The drawback with in vitro tests such as the RAST andELISA, is that they are designed to measure a single isolatedevent (eg. antigen-IgE binding), and fail to take into accountother physiological events in the allergic reaction (Poulsen andHau, 1987).27MATERIALS AND METHODSA. Modification of ProteinsA.1 Dephosphorylation of Caseinsa81-csn (donated by Guillermo Arteaga, PhD candidate,Department of Food Science, The University of British Columbia,and prepared by the method of Zittle and Custer (1963), minimum90% us,-csn), and S-csn (Sigma, St. Louis, MO, USA, minimum 90%g-csn) were enzymatically dephosphorylated according to theprocedure of Li-Chan and Nakai (1989). a51-csn or g-csnsolutions (0.5%) at pH 7.0 were treated with 50 mg acidphosphatase (Sigma, St. Louis, MO, USA)/g protein and held at37°C for 2 hours. Following incubation, the samples weredialysed (regenerated cellulose membrane, MWCO 6,000 - 8,000)against distilled, deionized water adjusted to pH 7 at 4°C for65 hours.Native casein samples were dialyzed against distilled,deionized water at 4°C for 48 hours.A.2 Preparation of Acid WheyUnpasteurized milk was obtained from The University ofBritish Columbia Dairy Unit. Butterfat was removed from themilk by centrifugation at 5,000 x g for 30 minutes at 4°C. Thecasein fraction was separated from the whey by isoelectricprecipitation; the milk was heated to 37°C and the pH adjustedto 4.6 with 1N HC1 (Li-Chan and Nakai, 1988). The precipitatedcasein was removed by centrifugation at 10,000 x g for 20minutes at 4°C. The whey was recentrifuged at 15,000 x g for 1028minutes at 4°C to remove residual casein. The acid whey wasdialyzed against distilled, deionized water for 48 hours at 4°C.A.3 Ferric Chloride Precipitation of S-lactoglobulinS-lactoglobulin was removed from the acid whey by themethod of Kaneko et al. (1985). An aliquot of 1M FeC13 (BDHInc., Toronto, ON, Canada) was added to undialyzed acid wheywhich had been adjusted to pH 4.2, to give a final concentrationof 7.5 mM FeCl3. The pH was maintained at 4.2 throughout theprocedure by the simultaneous dropwise addition of a 3N NaOHsolution. The whey was incubated in an ice water bath for 2hours, and the precipitated S-lactoglobulin was removed bycentrifugation at 10,000 x g for 15 minutes at 4°C. Excess Fe3'was removed by adjusting the pH to 9.0 and adding 2 mg/mlNa2HPO4. The pH was readjusted back to 9.0 and the whey allowedto stand for 1 hour. The precipitated iron salts were removedby centrifugation at 15,000 x g for 10 minutes at 4°C. The wheywas dialyzed against distilled, deionized water at 4°C for 48hours.B. Protein DeterminationThe protein concentration of the sample stock solutionswere determined by the Biuret method using Sigma Diagnostics(St. Louis, MO, USA) procedure number 541. The procedure wasmodified for use as a microassay using microtitre plates.Solutions (60 Al) of the samples or a diluted chicken eggalbumin (Sigma, St. Louis, MO, USA) standard were mixed with 250Al of total protein reagent in the wells of an ELISA microtitre29plate (Dynatech Laboratories Inc., Chantilly, VA, USA), and theabsorbance read immediately at 550 nm using a SLT LabinstrumentsEAR 400 plate reader (Salzburg, Austria). A standard curve wasconstructed using the SlideWrite Plus program (Advanced GraphicsSoftware Inc., Sunnyvale, CA, USA) and the proteinconcentrations of the samples determined from the standard curve(See Appendix 1). The standards were assayed in quadruplicateand the samples in triplicate. No blank wells were included inthe assay to correct for defects in the plate which mightcontribute absorbance.C. Phosphorus DeterminationDetermination of the phosphorus content of a1-csn and g-csnbefore and after dephosphorylation were performed by a modifiedprocedure of Morrison (1964). The casein samples or standard(NaH2PO4 x 2H20 solution containing 200 Ag phosphorus/ml) weredried in glass test tubes calibrated at 5 ml. Sulfuric acid(0.03 ml) was added to each tube, and heated in a boiling hotwater bath for approximately 15 seconds. Aliquots of hydrogenperoxide (10 pl, 30% w/v) were added until the acid becameclear. The tubes were heated in a boiling hot water bath for 1minute, then allowed to cool. Water (3 ml) was used to washdown the inside walls of each tube, and 0.1 ml of a 33% (w/v)sodium sulfite solution was added. An ammonium paramolybdatesolution (1.0 ml, 2% w/v) was added to each tube, followed by0.5 ml of a 2% (w/v) ascorbic acid solution which was preparedimmediately before use. The tubes were heated at 100°C for 1030minutes, then cooled. The volume was adjusted to 5.0 ml withdistilled deionized water, and the absorbance measured at 822 nmusing a Shimadzu UV-160 spectrophotometer (Kyoto, Japan). Astandard curve was constructed using the SlideWrite Plus program(Advanced Graphics Software Inc., Sunnyvale, CA, USA) and thephosphorus concentrations of the samples determined from thestandard curve (See Appendix 2). All samples and standards weredone in triplicate.D. Z-lactoglobulin Determination by SDS-PAGEThe S-lactoglobulin concentration of the whey before andafter precipitation with FeC13 was determined using the PharmaciaPhastSystem (Pharmacia LKB Biotechnology, Uppsala, Sweden) on a12.5% (w/v) gel with Coomassie Brilliant Blue staining. Thesamples or a diluted g-lactoglobulin (Sigma, St. Louis, MO, USA)standard were adjusted to approximately pH 8 with 1.5M Tris-HC1.An aliquot of a 25% w/v sodium dodecyl sulfate solution and2-mercaptoethanol was added to give a final concentration of 2%for each reagent respectively. An aliquot of a 1% w/v chickenegg albumin (Sigma, St. Louis, MO, USA) internal standard wasalso added to each sample and standard to give a finalconcentration of 0.1%. The protein samples were heated in aboiling hot water bath for 5 min. and allowed to cool before 0.5ill of each sample was applied to the gel. The proteins werequantified using a Pharmacia PhastImage Gel Analyzer (PharmaciaLKB Biotechnology, Uppsala, Sweden). A standard curve wasconstructed using the SlideWrite Plus program (Advanced Graphics31Software Inc., Sunnyvale, CA, USA) and the concentrations ofS-lactoglobulin in the samples were determined from the standardcurve (See Appendix 3). The g-lactoglobulin concentrations ofthe samples and standard were determined from a single gel. Theprotein concentration of the g-lactoglobulin standard wasdetermined by the Biuret method as described in section B.E. Immunisation ProtocolTwo separate methods were used to expose Balb/C mice to theproteins; the proteins were administered by oral gavage or byintraperitoneal injection. Mice were obtained either fromCharles River Canada Inc. (St.-Constant, PQ, Canada), or from amating with mice from The University Of British Columbia AnimalUnit. Mice of both sexes, aged 6-8 weeks old, and maintained ona milk free diet (See Appendix 4) were used in all experiments.E.1 Oral Administration of ProteinsMice were orally administered the test proteins accordingto the method of Poulsen et al. (1990). Protein (10 Ag) in 100Al of sterile diluent (H20) was administered on day 1. Oralbooster doses of 1 Ag of protein in 100 Al of sterile diluentwere administered on days 11 and 22.E.2 Intraperitoneal Injection of ProteinsThe mice were intraperitoneally injected with the testproteins using a modified procedure of Poulsen et al. (1990).Test protein (10 gig) mixed with 1 mg of aluminum hydroxideadjuvant (Pierce, Rockford, IL, USA) in 200 Al of sterilediluent^was^intraperitoneally^injected^on^day^1.32Intraperitoneal booster doses of 1 Ag of protein mixed with 1 mgaluminum hydroxide adjuvant in 200 pl of sterile diluent wereadministered on days 11 and 22.F. Preparation of AntiseraThe mice were bled 29 days after initial exposure to thetest proteins. Antiserum was prepared using a modifiedprocedure of Garvey et al. (1977). Whole blood was kept at roomtemperature for 2 hours to induce clot formation, then stored at4°C for 24 hours to allow the clot to contract. Antiserum wasseparated from the clot by centrifugation at 2,000 x g for 30minutes at 4°C. The antiserum was diluted 5x with PBS and storedat -70°C.G. Determination of Relative AntigenicityThe relative concentrations of antigen specific IgG inmurine serum were determined by an indirect ELISA and used as ameasure of antigenicity. Immulon 2 microtitre ELISA plates(Dynatech Laboratories Inc., Chantilly, VA, USA) were coatedwith 50 pl of 0.01% w/v antigen in carbonate buffer (seeAppendix 5) overnight at 4°C. After removing the excess antigen,the plates were washed 3x with PBS/ TWEEN (see Appendix 5) and200 pl of a 0.25% ovalbumin (Canadian Lysozyme, Aldergrove, BC,Canada) blocking buffer (see appendix 5) was applied. Themicrotitre plates were incubated at 35°C for 30 minutes, and theblocking removed. The wells of the plates were coated with 50pl of diluted antisera and incubated at 35°C for 1 hour. Afterthe antisera was removed, the plates were washed 3x with33PBS/TWEEN and coated with 50 Al of 0.001% w/v anti-mouse IgGalkaline phosphatase conjugate (Sigma, St. Louis, MO, USA) for1 hr. at 35°C. The conjugate was removed, and the plates washed3x with PBS/TWEEN, and once with distilled deionized water.P-nitrophenyl phosphate substrate (50 Al) (Sigma, St. Louis, MO,USA) in diethanolamine buffer (see Appendix 5) was added and theplates incubated at 35°C.^All samples were analyzed intriplicate. The absorbance at 405 nm was measured (using areference filter at 620 nm) using a SLT Labinstruments EAR 400plate reader (Salzburg, Austria) over time and subtracted fromblanks. Absorbance values above 1.50 were excluded from theplot. The rate of colour development is proportional to theamount of conjugate which has reacted. This in turn is relatedto the amount of antibody being assayed (Voller, 1980). Theabsorbances were plotted over time and the slopes of the graphs(multiplied by 1.0 x 10') were calculated using the SlideWritePlus program (Advanced Graphics Software Inc., Sunnyvale, CA,USA).^The slopes were used as estimates of the relativeconcentrations of antigen specific IgG in the sera and expressedas a relative IgG values. Absorbance points measured after asingle designated time were not used to determine the relativeconcentrations of antigen specific IgG, because the ELISA platereader was incapable of reading all the wells of the platesimultaneously. Therefore, wells containing identical amountsof antibody could have different absorbances. By using theslope of the graph, this error was eliminated.^Also, any34defects in the wells of the plate which might contribute to theabsorbance were also eliminated. In addition, plotting theabsorbance values over time allows for the identification ofoutlying groups of measurements, while measuring the absorbanceat a single designated time does not.H. Determination of Relative AllergenicityH.1 Relative IgE ConcentrationsThe relative concentrations of antigen specific IgE in thesera were determined by an indirect ELISA. The same procedurefor determining the relative concentration of IgG (section G)was used for determining IgE, except that an anti-mouse IgEalkaline phosphatase conjugate (Pharmingen, San Diego, CA, USA)was substituted to detect the appropriate isotype.H.2 Passive Cutaneous AnaphylaxisHeterologous passive cutaneous anaphylaxis (PCA) wasperformed according to the procedure of Akita and Nakai (1990).Male CD rats (Charles River Canada Inc., St.-Constant, PQ), 9-11weeks old, were intradermally injected with 100 Al of doublingdilutions of antisera. After 4 hours, 1.0 mg of antigen(protein) diluted in 1.0 ml of 2.%, (w/v) Evans Blue dye (Sigma,St. Louis, MO, USA) was administered intravenously via thepenile vein. PCA reactions were evaluated 45 minutes afterinjection of the antigen/dye. The titre value equalled thereciprocal of the highest dilution of antiserum to give apositive reaction. A positive reaction was considered acircular blue spot larger than 5 mm in diameter. All antisera35were tested in duplicate.I. Statistical AnalysisDifferences between the protein treatments and the negativecontrols were determined using t-tests at a significance levelof p < 0.05 as described by Ott (1988).36RESULTS/DISCUSSIONSix protein samples and a negative control (water) wereused in each experiment. The casein samples consisted of: usi-csn, dephosphorylated a81-csn, S-csn, and dephosphorylated S-csn.The results of the phosphorus determination showed 66.5% and84.1% dephosphorylation for cesi-csn and S-csn respectively (SeeAppendix 2). These results are lower than those obtained byLi-Chan and Nakai (1989). Li-Chan and Nakai (1989) obtained 70%and 99% dephosphorylation for usl-csn and S-csn respectively.The remaining two protein samples consisted of whey and FeCl3treated whey. The results of the S-lactoglobulin determinationindicated that at least 85.5% of the S-lactoglobulin had beenremoved from the FeCl3 treated whey (See Appendix 3). Theconcentration of g-lactoglobulin in the FeC13 treated whey wasbelow the minimum detection limit of the PhastImage program.Therefore, the lowest concentration used for constructing the g-lactoglobulin standard curve was substituted to determine theminimum amount of g-lactoglobulin that had been removed from thewhey. The actual amount of g-lactoglobulin removed from theFeC13 treated whey exceeded this value.A. Experiment 1: Orally Administered ProteinsA.1 Relative AntigenicityThe relative IgG values for mice orally administered thetest proteins are given in Tables 1 and 2. T-tests wereperformed on the data to detect significant differences (p <0.05) between the samples. The results indicate that the mice37Table 1. Comparison of the Mean Relative IgG Values for MiceOrally Administered Casein Proteins.ANTIGEN ANTISERAMEAN ABSORBANCE+^S.E.M.bMEAN IgG VALUE ± S.E.M.(abs.c/min.)x103as1 0.110^+^0.005e 0.63^+^0.04CASEIN CASEINNEGATIVE 0.130^+^0.007e 0.76^+^0.05CONTROLS CASEIN S CASEIN 0.018^+^0.003f 0.13^+^0.02NEGATIVE 0.023^+^0.002f 0.16^+^0.01CONTROLDEPHOS DEPHOSd0.281^+^0.050e 1.42^+^0.35CASEIN CASEINNEGATIVE 0.134^+^0.019e 0.76^+^0.06CONTROLDEPHOSgdDEPHOSgd0.045^+^0.006f 0.28^+^0.03CASEIN CASEINNEGATIVE 0.041^+^0.003f 0.23^+^0.02CONTROL'Antisera diluted 1/25.bS.E.M. = Standard Error of the Mean.'AbsorbancedaephosphorylatedeAbsorbance measured after 150.41 minutes of incubation at 37°C.fAbsorbance measured after 149.05 minutes of incubation at 37°C.38Table 2. Comparison of the Mean Relative IgG Values for MiceOrally Administered Whey Proteins.ANTIGEN ANTISERAaMEAN ABSORBANCE^MEAN IgG VALUE ± S.E.M.+ S.E.M.b^(abs.c/min.)x103WHEY WHEY 0.065^+^0.003d 0.26^+^0.01NEGATIVE 0.062^+^0.003d 0.26^+^0.02CONTROLFeC13 FeC13 0.070^+^0•003d 0.29^+^0.02TREATED TREATEDWHEY WHEYNEGATIVE 0.091^+^0.011d 0.35^+^0.07CONTROL'Antisera diluted 1/25.bS.E.M. = Standard Error of the Mean.cAbsorbancedAbsorbance measured after 205.91 minutes of incubation at 37°C.3 9were not sensitized against any of the test proteins.A.2 Relative AllergenicityThe relative IgE concentrations were not measured, sincethe IgG data suggested that the mice were not sensitized againstthe proteins, ie. there was no antigen specific IgG in the sera.It should be noted, that the antigenic epitopes which give riseto IgG may not be the same epitopes which produce IgE (Taylor,1989 et al.). A food protein that induces an IgG response inanimal models, even humans, will not necessarily induce an IgEresponse (Taylor, 1989 et al.). IgE is much harder to detectthan IgG, because it is normally found in extremely lowconcentrations in the serum. IgE is the least prevalent of thefive antibody classes (Metcalfe et al., 1991). In the normalhuman subject, IgE comprises approximately 0.002 96 of the totalserum immunogobulins (Metcalfe et al., 1991). The concentrationof IgE in normal human serum is approximately 1/40,000 that ofIgG (Kimball, 1983). Since the results indicated that noantigen specific IgG was in the antisera, it is improbable thatIgE would be detected.The IgG data indicated that the test proteins were notantigenic when administered orally. Since a protein must beantigenic in order to be allergenic, it would be reasonable toassume that the mice which had been orally administered the testproteins, would exhibit no allergenicity towards the proteins.Tables 3, 4, and 5 show the PCA titres for the mice orallyadministered the proteins. There was no response at the lowestTable 3. Mean Passive Cutaneous Anaphylaxis Titresfor Mice Orally Administered CaseinProteins.ANTIGEN^ANTISERA^MEAN TITRE + S.E.M.aNRbCASEIN^CASEINNEGATIVEc^NRCONTROLS CASEIN^S CASEIN^NRNEGATIVE NRCONTROLDEPHOSPHORYLATED DEPHOSPHORYLATED^NRus,CASEIN^CASEINNEGATIVE^NRCONTROLDEPHOSPHORYLATED DEPHOSPHORYLATED^NRS CASEIN^S CASEINNEGATIVE^NRCONTROLaS.E.M. = Standard Error of the Mean.bAll negative controls consisted of a pooled antiserumsample.cNR = No Response at the lowest dilution tested (1/5).40Table 4. Mean Passive Cutaneous Anaphylaxis Titresfor Mice Orally Administered DephosphorylatedCasein Proteins and Challenged with PotatoAcid Phosphatase.ANTIGEN^ANTISERA^MEAN TITRE + S.E.M.aPOTATO ACIDb DEPHOSPHORYLATED^NRcPHOSPHATASECASEINDEPHOSPHORYLATED^NRS CASEINNEGATIVEd^ NRCONTROLaS.E.M. = Standard Error of the Mean.bMice challenged with 0.00005 g enzyme.cITR = No Response at the lowest dilution tested (1/5).dUegative control consisted of a pooled antiserumsample.Table 5. Mean Passive Cutaneous Anaphylaxis Titresfor Mice Orally Administered Whey Proteins.ANTIGEN ANTISERAWHEYNEGATIVEcCONTROL MEAN TITRE + S.E.M.aNRbNRWHEYFeCl3^FeC13^ NRTREATED WHEY^TREATED WHEYNEGATIVE NRCONTROLaS.E.M. = Standard Error of the Mean.bINTR = No Response at the lowest dilution tested (1/5).cAll negative controls consisted of a pooled antiserumsample.4142dilution for all the protein samples tested.^This resultsupported the assumption that the mice were not sensitizedagainst the proteins.It is not known why the mice were not sensitized againstthe test proteins, since other researchers have been successfulin obtaining sensitization following oral exposure. A possibleexplanation may be the strain of mice used.^The oralimmunisation procedure and schedule of Poulsen et al. (1990) wasfollowed, however, Poulsen et al. (1990) used inbred NMRI micein their experiments. NMRI mice are not available in NorthAmerica, so Balb/C mice were substituted in this study.However, Poulsen et al. (1987) have shown that Balb/C mice canbe orally sensitized to milk proteins.^Although in theirexperiment, Poulsen et al. (1987) fed the mice ad libitum overseveral generations. Balb/C mice may not have been suitablewith the immunisation protocol used for this experiment. Itshould also be noted that Poulsen et al. (1990) performedhomologous PCA in their experiment, therefore, both IgE and IgG,would participate in the PCA reaction. It is possible that thetitre values obtained in their experiment were due solely to theinfluence of IgG, alone. Nielsen et al. (1989) compared therelative allergenicity of homogenized and non-homogenized milkusing mice orally exposed to whole milk. However, in theirexperiments, the effect of IgG, on the PCA reaction wasdetermined.^A positive IgE mediated PCA response could beobtained with homogenized milk, but not with non-homogenized43milk. The results of their experiments indicated that thephysical state of the milk fat affected the allergenicity of theproteins. The protein preparations used in our experiments werepure and devoid of fat, which may have had the same effect asusing non-homogenized milk.After experiment 1 was completed, it was not clear why themice were not sensitized against the proteins; either theproteins were not antigenic and/or the route of exposure wasinappropriate for sensitizing the mice. To resolve thisquestion, a second experiment was performed using a differentroute of exposure for the test proteins. Instead of orallyexposing the mice to the antigens, the proteins were injectedintraperitoneally in experiment 2.B. Experiment 2: Intraperitoneally Injected ProteinsB.1 Relative AntigenicityTable 6 shows the relative IgG values for the miceintraperitoneally injected with the casein proteins. T-testswere performed to determine if significant differences existedbetween the treatments. No significant difference (p < 0.05)was found between usl_csn and the negative control. This resultindicated that the mice intraperitoneally injected with a1-csnwere not sensitized against the protein. It should be notedthat the IgG response of mouse number 5 was abnormally high incomparison to the other mice administered the same protein. Forthis reason, mouse number 5 was treated as an outlier andremoved from the statistical analyses. All other casein44Table 6. Comparison of the Mean Relative IgG Values for MiceIntraperitoneally Injected with Casein Proteins.ANTIGEN ANTISERAaMEAN ABSORBANCE^MEAN IgG VALUE + S.E.M.+^S.E.M.b^(abs.'/min.)x1030.023^+^0•003d'f 0.38d +^0.08CASEIN CASEINNEGATIVE 0.019^+^0•006g 0.26^±^0.02CONTROLS CASEIN S CASEIN 0.607^+^0.058f 7.31^+^1.33NEGATIVE 0.001^+^0.000g 0.07^+^0.01CONTROLDEPHOSusl eDEPHOSusle0.597^+^0.063f 7.71^+^1.43CASEIN CASEINNEGATIVE 0.011^+^0•001g 0.25^+^0.02CONTROLDEPHOSse DEPHOSse 0.221^+^0.030f 2.86^+^0.56CASEIN CASEINNEGATIVE 0.005^+^0.001g 0.14^+^0.02CONTROLaAntisera diluted 1/1000.bS.E.M. - Standard Error of the Mean.'AbsorbancedMause #5 was treated as an outlier and not included in theaverage.eDephosphorylatedfAbsorbance measured after 59.83 minutes of incubation at 37°C.°Absorbance measured after 59.90 minutes of incubation at 37°C.4 5proteins (dephosphorylated a1-csn, S-csn and dephosphorylatedS-csn) were significantly different (p < 0.05), from thenegative control, which indicated that the mice were sensitizedagainst these proteins by this route of exposure. A significantdifference (p < 0.05) was found between untreated a1-csn anddephosphorylated usl-csn. The mean IgG value for thedephosphorylated ors,-csn was approximately 20 times greater thanthe mean for untreated usl-csn. Dephosphorylation of usl-csnsignificantly increased the antigenicity of the protein.Dephosphorylation may have exposed new antigenic determinantsthat were previously hidden, or changed the conformation of theprotein to form new conformational epitopes, although, it is notknown what effect dephosphorylation has on the tertiarystructure of the molecule. It should be noted, that theincrease in antigenicity may not be completely attributable tothe exposure or formation of new antigenic determinants on theprotein. The enzyme which was used to dephosphorylate theproteins may have also contributed to the antigenicity.Therefore, the increase in antigenicity as a result ofdephosphorylation was probably lower than what the results haveindicated.A significant difference (p < 0.05) was found between us,-csn and g-csn. The mean IgG value for g-csn was approximately19 times greater than the mean IgG value for a1-csn.^Thisresult suggests that S-csn is more antigenic than usl-csn.A significant difference (p < 0.05) was found between46untreated S-csn and dephosphorylated S-csn. The mean IgG valuefor dephosphorylated S was approximately 2.5 times lower thanthe mean for the untreated protein. Dephosphorylation of S-csnsignificantly decreased the antigenicity of the protein.Dephosphorylation may have destroyed conformational epitopes orchanged the conformation of the protein so that epitopes becameinaccessible to immunocompetent lymphocytes. It is not knownwhat effect dephosphorylation had on the tertiary structure ofthe molecule. It should be noted, that the enzyme used todephosphorylate the protein could also contribute toantigenicity. Therefore, the reduction in antigenicity as aresult of dephosphorylation was probably greater than what theresults have indicated.Table 7 shows the relative IgG values for the miceintraperitoneally injected with the whey proteins. Asignificant difference (p < 0.05) was found between each of thetreatments and the negative control, which indicated that themice were sensitized against the proteins. A significantdifference (p < 0.05) was found between the whey and the FeC13treated whey. The mean IgG value of the FeCl3 treated whey wasapproximately 1.7 times lower than the mean for the untreatedwhey. Thus, the removal of S-lactoglobulin decreased theantigenicity of the whey fraction.B.2 Relative AllergenicityTable 8 shows the relative IgE values for the miceintraperitoneally injected with the casein proteins. No47Table 7. Comparison of the Mean Relative IgG Values for MiceIntraperitoneally Injected with Whey Proteins.ANTIGEN ANTISERAaMEAN ABSORBANCE^MEAN IgG VALUE + S.E.M.+^S.E.M.b^(abs.c/min.)x103WHEY WHEY 0.505^+^0•023d 6.49^+^0.33NEGATIVE 0.009^+^0.007d 0.22^+^0.01CONTROLFeC13 FeC13 0.245^+^0.012d 3.81^+^0.26TREATED TREATEDNEGATIVE 0.020^+0.001d 0.38^+^0.02CONTROL'Antisera diluted 1/1000.bS.E.M. = Standard Error of the Mean.'AbsorbancedAbsorbance measured 50.85 minutes after incubation at 37°C.48Table 8. Comparison of the Mean Relative IgE Values for MiceIntraperitoneally Injected with Casein Proteins.ANTIGEN ANTISERAaMEAN ABSORBANCE^MEAN IgG VALUE + S.E.M.+^S.E.M.13^(abs.c/min.)x103as1 0.016^+^0•004d'E 3.80d +^1.10CASEIN CASEINNEGATIVE 0.004^+^0.001g 1.78^+^0.70CONTROLg CASEIN S CASEIN 0.100^+^0.009E 20.26^+^2.67NEGATIVE 0.027^+^0•009g 5.74^+^1.95CONTROLDEPHOSasi eDEPHOSasie0.061^+^0.004E 14.52^+^1.75CASEIN CASEINNEGATIVE 0.015^+^0.004g 3.92^+^1.18CONTROLDEPHOSse DEPHOSse 0.089^+^0.011E 19.24^+^3.02CASEIN CASEINNEGATIVE 0.031^+^0.004g 6.71^+^0.55CONTROLakntisera diluted 1/20.= Standard Error of the Mean.cAbsorbancedMouse #5 was treated as an outlier and not included in theaverage.eDephosphorylatedE.Absorbance measured after 3.93 hours of incubation at 37°C.°Absorbance measured after 3.94 hours of incubation at 37°C.49significant difference (p < 0.05) was found between a81-csn andthe negative control. Since the IgG values indicated that themice injected with u81-csn were not sensitized against theprotein, this result was expected. As observed with the IgGdata, mouse number 5 showed an abnormally high IgE response incomparison to the mice injected with the same protein.Therefore, mouse number 5 was treated as an outlier and removedfrom the statistical analyses.^All other proteins(dephosphorylated ces1-csn, S-csn, dephosphorylated S-csn) weresignificantly different (p < 0.05) than the negative control.A significant difference (p < 0.05) was found betweendephosphorylated a1-csn and untreated us,-csn.^The mean IgEvalue for dephosphorylated as, was approximately 3.8 timesgreater than the mean^for the untreated protein.Dephosphorylation^increased the ability of the protein toproduce antigen specific IgE.^Presumably, dephosphorylationexposed new antigenic determinants that were previously hidden,or induced conformational changes in the protein which resultedin the formation of new conformational epitopes. However, theincrease in antigen specific IgE cannot be completely attributedto dephosphorylation of the protein alone. The enzyme used fordephosphorylation could have also induced the production ofspecific IgE, which would inflate the results.A significant difference (p < 0.05) was found between us,-csn and S-csn. The mean IgE value for S-csn was approximately5.3 times greater than the mean for us„, which indicated that S50casein had a greater capacity for inducing the production ofantigen specific IgE.The mean IgE value of untreated S casein was almostidentical to the mean for the dephosphorylated protein. Nosignificant difference (p < 0.05) was found between thetreatments, which suggested that dephosphorylation did notaffect the ability of S-csn to elicit the production of specificIgE. Although dephosphorylation might destroy or hide theantigenic determinants responsible for the production of IgG,the epitopes for IgE appeared to be unaffected. It should benoted, that the enzyme used to dephosphorylate the protein couldinduce the production of specific IgE. Therefore,dephosphorylation may reduce the capacity of S casein to produceIgE, but the presence of the enzyme may have masked this effect.Table 9 shows the relative IgE values for miceintraperitoneally injected with the whey proteins. Asignificant difference (p < 0.05) was found between each of thetreatments and the negative control. Although the mean IgEvalue for the untreated whey was almost double the mean for theFeCl3 treated whey, no significant difference was found betweenthe two treatments. Thus, the removal of the S-lactoglobulindid not appear to significantly reduce the ability of the wheyto induce the production of specific IgE.Table 10 shows the passive cutaneous anaphylaxis titres ofthe mice intraperitoneally injected with the casein proteins.The t-test results of the PCA titres mirror the results obtained51Table 9. Comparison of the Mean Relative IgE Values for MiceIntraperitoneally Injected with Whey Proteins.ANTIGEN ANTISERAaMEAN ABSORBANCE^MEAN IgG VALUE + S.E.M.+ S.E.M.b^(abs.c/min.)x103WHEY WHEY 0.081 +^0.012d 11.30^+^2.83NEGATIVE 0.001 +^0•000d 0.42^+^0.16CONTROLFeC13 FeC13 0.041 +^0.004d 6.63^+^0.39TREATED TREATEDWHEY WHEYNEGATIVE 0.003 +^0•001d 0.61^+^0.24CONTROLakntisera diluted 1/20.bS.E.M. = Standard Error of the Mean.cAbsorbancedAbsorbance measured after 6.07 hours incubation at 37°C.Table 10. Mean Passive Cutaneous Anaphylaxis Titres of MiceIntraperitoneally Injected with Casein Proteins.ANTIGEN^ANTISERA^MEAN TITRE + S.E.M.as1 NRb''aCASEIN^CASEINNEGATIVEd^NRCONTROLS CASEIN^S CASEIN^176 + 35NEGATIVE NRCONTROLDEPHOSPHORYLATED DEPHOSPHORYLATED^84 + 19as1CASEIN^CASEINNEGATIVE^NRCONTROLDEPHOSPHORYLATED DEPHOSPHORYLATED^160 + 0S CASEIN^g CASEINNEGATIVE^NRCONTROLaS.E.M. = Standard Error of the Mean.IDNR = No Response at the lowest dilution tested (1/5).94ouse #5 was treated as an outlier and not includedin the average.dAll negative controls consisted of a pooled antiserumsample.5253by measuring the concentrations of antigen specific IgE. Therewas no response at the lowest dilution tested (1/5) for the miceinjected with a1-csn.^This indicated that a1-csn was notallergenic. Mouse number 5 produced an unusually large PCAtitre in comparison to the other mice injected with the sameprotein, and was removed from the statistical analyses.A significant difference (p < 0.05) was found betweenuntreated a1-csn and dephosphorylated a1-csn. This resultindicated that dephosphorylation increased the allergenicity ofthe protein. Table 9b shows the PCA titres for the mice whenchallenged with the enzyme which was used to dephosphorylate thecaseins. The results indicate that the enzyme contributes to,but is not wholly responsible for, allergenicity. Thus, the PCAtitre reflects the increase in allergenicity due todephosphorylation of the protein, and the inherent allergenicityof the enzyme.A significant difference (p < 0.05) was found betweenu81-csn and 8-csn. Thus, g-csn is more allergenic than usl-csn.The mean PCA titres for untreated g-csn anddephosphorylated 13-csn were similiar, and no significantdifference (p < 0.05) was found between the two treatments.This result indicates that dephosphorylation has no significanteffect on the allergenicity of this protein. The results fromTable 11 shows that the enzyme used to dephosphorylate theprotein contributes to, but is not completely responsible for,Table 11. Mean Passive Cutaneous Anaphylaxis Titresfor Mice Intraperitoneally Injected withDephosphorylated Casein Proteins andChallenged with Potato Acid Phosphatase.54ANTIGEN^ANTISERA^MEAN TITRE + S.E.M.aPOTATO ACIDb DEPHOSPHORYLATED^27 + 7PHOSPHATASECASEINDEPHOSPHORYLATED^81 + 21S CASEINNEGATIVE^ NRc'dCONTROLaS.E.M. = Standard Error of the Mean.bMice challenged with 0.00005 g enzyme.cNR = No Response at the lowest dilution tested (1/5).dNegative control consisted of a pooled antiserumsample.55the observed allergenicity. Dephosphorylation may reduce theallergenicity of the protein, but the presence of the enzymeinflates the PCA titre which may have masked this effect. Ifthe enzyme could be immobilized, its influence on allergenicitywould be removed. The enzyme used in these experiments wasderived from potatoes. An acid phosphatase from a source lessforeign to mice and humans could be substituted, which mightreduce its effects on allergenicity.Table 12 shows the PCA titres for the miceintraperitoneally injected with the native and modified wheyproteins. Although the mean titre for the FeCl3 treated whey wasalmost half the mean titre for the untreated whey, nosignificant difference was found between the two treatments, dueto the large range in variation of titres for the mice exposedto the untreated whey. Therefore, the removal ofZ-lactoglobulin does not appear to significantly reduce theallergenicity of whey. It is important to note that whey iscomposed of a variety of proteins. The next largest wheyfraction after g-lactoglobulin is cy-lactalbumin, which is alsothe second most incriminated allergenic whey protein in oralchallenge studies (Lebenthal, 1975). The proteins whichremained in the whey after treatment with FeC13 (primarilya-lactalbumin with a residual amount of 0-lactoglobulin) appearto equal untreated whey in their ability to elicit an allergicreaction.Table 12. Mean Passive Cutaneous Anaphylaxis Titresof Mice Intraperitoneally Injected withWhey Proteins.ANTIGEN ANTISERA^MEAN TITRE + S.E.M.aWHEY 608 + 171NEGATIVE^NRbCONTROLWHEYFeC13^FeC13 320 + 78TREATED WHEY^TREATED WHEYNEGATIVEc^NRCONTROLaS.E.M. = Standard Error of the Mean.IDNR = No Response at lowest dilution tested (1/5).eNegative control consisted of a pooled antiserumsample.5657CONCLUSIONThe milk proteins that were administered orally to micefailed to sensitize the animals. The mice could, however, besensitized to the same proteins (except usi) whenintraperitoneally injected. Intraperitoneal administration ofthe proteins removes the molecular size and stability(resistance to digestive enzymes and acid conditions)constraints on the antigens, which normally exist when a food isingested. These factors can reduce the amount of antigen whichcomes into contact with the immune system. The mice were notsensitized against a51-csn by either route of exposure (oraladministration or intraperitoneal injection). These resultsstrongly indicate that as,-csn is non-allergenic.Dephosphorylation of as/-csn with acid phosphatase increased theallergenicity of the protein. S-csn was more allergenic thana81-csn. Dephosphorylation of S-csn with acid phosphatase didnot significantly affect the allergenicity of the protein. Thepotato acid phosphatase used to dephosphorylate the caseinscontributed to allergenicity.Partial removal of the S-lactoglobulin from whey did notsignificantly reduce the allergenicity. The proteins thatremain in the whey after treatment with FeCl3 (primarily a-lactalbumin and a residual amount of g-lactoglobulin) are equalto untreated whey in their ability to produce an allergicreaction.58REFERENCESAas, K. 1976. Common characteristics of major allergens. In"Molecular and Biological Aspects of the Acute AllergicReaction", S.G.O. Johansson, K. Standberg and B. Uvnas(Ed.), P. 3. Plenum Press, New York, New York.Aas, K. 1978. What makes an allergen an allergen. Allergy33:3.Aas, K. 1989. Chemistry of food allergens. In "FoodIntolerance in Infancy: Allergology, Immunology andGastroenterology", R.N. Hamburger (Ed.), P. 9. Raven PressLtd., New York, New York.Akita, E.M. and Nakai, S. 1990. Lipophilization ofbeta-lactoglobulin: effect on digestibility andallergenicity. J. Food Sci. 55:718.Anderson, J.A. 1990. Diagonsis of food allergy - part 1: thephysician's perspective. In "Food Allergies and AdverseReactions", J.E. Perkin (Ed.), P.15. Aspen PublishersInc., Gaithersburg, Maryland.Anderson, J.A. and Sogn, D.D.(Ed.). 1984. Fate of ingestedantigens in the intestinal Tract. Ch.3. In "AdverseReactions to Foods", p.27. NIH Publication no. 84-2442.Anderson, K.J., Mclaughlan, P., Devey, M. and Coombs, R.R.A.1979. Anaphylactic sensitivity of guinea-pigs drinkingdifferent preparations of cow's milk and infant formulae.Clin. Exp. Immunol. 35:454.Atassi, M.Z. 1975. Antigenic structure of myoglobin: thecomplete immunochemical anatomy of a protein andconclusions relating to antigenic structures of proteins.Immunochem. 12:423.Bahna, S.L. 1987. Handling reactions to foods and foodadditives. Postgrad. Med. 82:195.Bahna, S.L. and Heiner, D.C. 1980. Composition of cow's milk.Ch. 3. In "Allergies to Milk", p. 11. Grune andStratton Inc., New York, New York.Bahna, S.L. and Gandhi, M.D. 1983. Milk hypersensitivity. I.pathogenesis and symptomology. Annals of Allergy50:218.Baldo, B.A. 1984. Milk allergies. Aust. J. Dairy Technol.39:120.59Bleumink, E. 1970. Food allergy: the chemical nature of thesubstances eliciting symptoms. World Rev. Nutr. Diet.12:505.Bleumink, E. and Berrens, L. 1966. Synthetic approaches to thebiological activity of S-lactoglobulin in human allergy tocow's milk. Nature 212:541.Bleumink, E. and Young, E. 1968. Identification of the atopicallergen in cow's milk. Int. Arch. Allergy 34:521.Bock, S.A., Lee, W.Y., Remigio, L.K. and May, C.D. 1978.Studies of hypersensitivity reactions to foods in infantsand children. J. Allergy Clin. Immunol. 62:327.Bock, S.A. and Martin, M. 1983. The incidence of adversereactions to foods - a continuing study. J. Allergy Clin.Immunol. 71:98.Breneman, J.C. 1987. Immunology of food allergy. Ch. 1. In"Handbook of Food Allergies", J.C. Breneman (Ed.), p.l.Marcel Dekker Inc., New York, New York.Cavell, B. 1979. Gastric emptying in preterm infants. ActaPaed. Scand. 68:725.Chandra, R.K. and Jeevanandam, S. 1984. Diagnostic approach.In "Food Intolerance", R.K. Chandra (Ed.), P.103. ElsevierScience Publishing Co. Inc., New York, New York.Conroy, M.C. and Adkinson, N.F. 1977. Assessment of theinfluence of irrelevant IgE on allergic sensitivity to twoindependent allergens. J. Allergy Clin. Immunol. 63:15.Coombs, R.R.A. and Gell, P.H.G. 1975. Classification ofallergic reactions responsible for clinicalhypersensitivity and disease. In "Chemical Aspects ofImmunology", P.G.H. Gell, R.R.A. Coombs and P.J. Lachmann(Ed.), P.761. Blackwell Scientific, Lippincott,Philadelphia, PA.Crumpton, M. 1974. Protein antigens: the molecular bases ofantigenicity and immunogenicity. Ch.l. In "The Antigens- Vol. II", M. Sela (Ed.), p. 1. Academic Press, New York,New York.Davidson, M., Burnstine, R.C., Kugler, M.M. and Bauer, C.H.1965. Malabsorption defect induced by ingestion ofS-lactoglobulin. J. Pediatr. 66:545.60Dayhoff, O.M. and Eck, R.V. 1969. "Atlas of Protein Sequenceand Structure". National Biomedical Research Foundation,Silver Spring, Maryland.Fries, J.H. 1959. Factors influencing clinical evaluation offood allergy. Pediat. Clin. N. Am. 6:87.Garvey, J.S., Cremer, N.E. and Sussdorf, D.H. 1977. Basicmethods. Pt. 1. In "Methods in Immunology", 3rd ed. P.1.W.A. Benjamin, Inc., Reading, Mass.Goldman, A.S., Anderson, D.W., Sellers, W.A., Saperstein, S.,Kniker, W.T. and Halpern, S.T. 1963a. Milk allergy. I.oral challenge with milk and isolated milk proteins inallergic children. Pediatr. 32:425.Goldman, A.S., Sellers, W.A., Halpern, S.R., Anderson, D.W.,Furlow, T.E. and Johnson, C.H. 1963b. Milk allergy. II.skin testing of allergic and normal children with purifiedmilk proteins. Pediatr. 32:572.Guesry, P.R., Secretin, M.C., Jost, R., Pahud, J.J. and Monti,J.C. 1989. Hypoallergenic formulas. In "Food Intolerancein Infancy: Allergology, Immunology, and Gastroenterology",R.N. Hamburger (Ed.), P.253. Raven Press Ltd., New York,New York.Haddad, Z.H., Veter, M., Friedmann, J., Sainz, C., and Brunner,E. 1983. Detection and kinetics of antigen-specific IgEand IgG immune complexes in food allergy. Annals ofAllergy 42:368.Hanson, L.A. and Mansson, I. 1961. Immune electrophoreticstudies of bovine milk and milk products. Acta Pediatr.50:484.Heppel, L.M., Cant, A.J. and Kilshaw, P.J. 1984. Reduction inthe antigenicity of whey proteins by heat treatment: apossible strategy for producing a hypoallergenic infantmilk formula. Br. J. Nutr. 51:29.Host, A. and Samuelsson, E.G. 1988. Allergic reactions to raw,pasteurized and homogenized/pasteurized cow milk: acomparison. Allergy 43:113.Hum, B.A.L. and Chantler, S.M. 1980. Production of reagentantibodies. Meth. Enzymol. 70:104.Ishizaka, K. 1976. Cellular events in the IgE antibodyresponse. Adv. Immunol. 23:1.61Ishizaka, T., Campbell, D.H. and Ishizaka, K. 1960. Internalantigenic determinants in protein molecules. Proc. Soc.Exper. Biol. Med. 103:5.Jost, R., Monti, J.C. and Pahud, J.J. 1987. Whey proteinallergenicity and its reduction by technological means.Food Technol. 41(10):118.Kaneko, T., Wu, B.T. and Nakai, S. 1985. Selectiveconcentration of bovine immunoglobulins and u-lactalbuminfrom acid whey using FeC13. J. Food Sci. 50:1531.Kilshaw, P.J., Heppel, L.M.J. and Ford, J.E. 1982. Effects ofheat treatment of cow's milk and whey on the nutritionalquality and antigenic properties. Arch. Dis. Child.57:842.Kimball, J.W. 1983. "Introduction to Immunology", 2nd ed.MacMillan Publishing Co., New York, New York.Kirschenbaum, D.M. 1976. "Handbook of Biochemistry andMolecular Biology", Vol. 2, 3rd ed. G.D. Fasman (Ed.),P.383. CRC Press, Cleveland, Ohio.Kleinman, R.E. 1992. Immune response to dietary antigens andthe development of hypoallergenic formulas. Nutr. Res.12:151.Kniker, W.T. 1987. Immunologically mediated reactions to food:state of the art. Annals of Allergy 59 (part II):60.Kuitunen, P., Visakorpi, J.K., Savilahti, E. and Pelkonen, P.1975. Malabsorption syndrome with cow's milk intolerance.Clinical findings and course in 54 Cases. Arch. Dis.Child. 50:351.Kummer, A., Kitts, D.D., Li-Chan, E., Losso, J.N., Skura, B.J.and Nakai, S. 1992. Quantification of bovine IgG in milkusing enzyme-linked immunosorbent assay. Food and Agric.Immunol. 4:93.Lebenthal, E. 1975. Cow's milk protein allergy. Pediatr.Clin. North Am. 22:827.Li-Chan, E. and Nakai, S. 1988. Rennin modification of bovinecasein to simulate human casein composition: effect on acidclotting and hydrolysis by pepsin. Can. Inst. Food Sci.Technol. J. 21:200.Li-Chan, E.C. and Nakai, S. 1989. Enzymic dephosphorylation ofbovine casein to improve acid clotting properties anddigestibility for infant formula. J. Dairy Res. 56:381.62Lietze, A. 1969. Laboratory research in food allergy. I. foodallergens. J. Asthma Res. 7:25.Liu, H.Y., Tsao, M.U., Moore, B. and Giday, Z. 1967. Bovinemilk protein induced intestinal malabsorption of lactoseand fat in infants. Gastroenterol. 54:27.Marsh, D.G. 1975. Allergens and the genetics of allergy. In"The Antigens - Vol. III", M. Sela (Ed.), P. 271. AcademicPress, New York, New York.McLaughlan, P., Anderson, K.J., Widdowson, E.M. and Coombs,R.R.A. 1981. Effect of heat treatment on the anaphylacticsensitizing capacity of cow's milk, goat's milk, andvarious infant forumulae fed to guinea pigs. Arch. Dis.Child. 56:165.McMeekin, T.L. 1954. Milk proteins. Ch. 16. In "TheProteins: Chemistry, Biological Activity, and Methods - VolII, Part A", H. Neurath and K. Bailey (Ed.), P. 389.Academic Press, New York, New York.Metcalfe, D.D., Sampson, H.A. and Simon, R.A. (Ed.). 1991.Mast cells, basophils, and immunoglobulin E. Ch. 2. In"Food Allergy: Adverse Reactions to Foods and FoodAdditives", p. 13. Blackwell Scientific Publications,Cambridge, Massachusetts.Morrison, W.R. 1964. A fast simple and reliable method for themicrodetermination of phosphorus in biological materials.Anal. Biochem. 7:218.Nakai, S. and Li-Chan, E. 1987. Effect of clotting in stomachsof infants on protein digestibility of milk. FoodMicrostructure 6:161.Nielsen, B.R., Poulsen, O.M. and Hau, J. 1989. Reaginproduction in mice: effect of subcutaneous and oralsensitization with untreated bovine milk and homogenizedbovine milk. In Vivo 3:271.Ott, L. 1988. Inferences about Al - ii2• Ch. 5. In "AnIntroduction to Statistical Methods of Data Analysis", 3rded. p. 170. PWS-Kent Publishing Co., Boston, Mass...Owen, R.L. and Nemanic, P. 1978. Antigen processing structuresof the mammalian intestinal tract: an SEM study oflymphoepithelial organs. Scanning Electron Microscopy.2:367.63Ovary, Z., Benacerraf, B. and Bloch, K.S. 1963. Properties ofguinea pig 7S antibodies. II. identification of antibodiesinvolved in passive cutaneous and systemic anaphylaxis. J.Exp. Med. 117:951.Ovary, Z., Caizza, S.S. and Kojima, S. 1975. PCA reactionswith mouse antibodies in mice and rats. Int. Archs.Allergy Appl. Immun. 48:16.Pahud, J.J., Monti, J.C. and Jost, R. 1985. Allergenicity ofwhey proteins: its modification by tryptic in-vitrohydrolysis of the protein. J. Pediatr. Gastroenterol.Nutr. 4:408.Pahud, J.J., Schwartz, K. and Granato, D. 1988. Control ofhypoallergenicity by animal models. In "Food Allergy", E.Schmidt (Ed.), p. 199. Raven Press Ltd., New York, NewYork.Pastorello, E.A., Pravettoni, V., Bigi, A, Qualizza, R.Vassellatti, D., Schilke, M.L., Stocchi. L., Tedeschi, A.,Anasaloni, R. and Zanussi, C. 1987. IgE-mediated foodallergy. Ann. of Allergy 59 (part II):82Pestka, J.J. and Witt, M.F. 1985. An overview of immunefunction. Food Technol. 39(2):83.Pildes, R.S., Blumenthal, I. and Ebel, A. 1980. Stomachemptying in the newborn. Pediatr. 66:482.Poulsen, O.M. and Hau, J. 1987. Murine passive cutaneousanaphylaxis test (PCA) for the "all or none" determinationof allergenicity of bovine whey proteins and peptides.Clin. Allergy 17:75.Poulsen, 0.M., Hau, J. and Kollerup, J. 1987. Effect ofhomogenization and pasteurization on the allergenicity ofbovine milk analysed by a murine anaphylactic shock model.Clin. Allergy 17:449.Poulsen, O.M. and Hau, J. 1988. Murine passive cutaneousanaphylaxis test (PCA) for the "All or None" determinationof allergenicity of bovine whey proteins and peptides. In"New Developments in Biosciences: Their Implications forLaboratory Animal Science", A.C. Beyen and H.A. Solleveld(Ed.), P. 87. Martinus Nijhoff, Dordrecht, Netherlands.Poulsen, 0.M., Neilsen, B.R., Basse, A. and Hau, J. 1990.Comparison of intestinal anaphylactic reactions insensitized mice challenged with untreated bovine milk andhomogenized bovine milk. Allergy 45:321.64Ratner, B., Dworetzky, M., Oguri, S. and Aschheim, L. 1958.Effect of heat treatment on the allergenicity of milk andprotein fractions from milk as tested in guinea pigs byparenteral sensitization and challenge. Pediatr. 22:648.Savilahti, E. 1981. Cow's milk allergy. Allergy 36:73.Sigma Diagnostics. 1989. Total protein - procedure no. 541.Sigma Chemical Co., St. Louis, Mo.Smith, G. 1976. Whey protein. World Rev. Nutr. Diet. 24:88.Spies, J.R., Stevan, M.A., Stein, W.J. and Coulson, E.J. 1970.The chemistry of allergens. XX. new antigens generated bypepsin hydrolysis of bovine milk proteins. J. Allergy45:208.Swaisgood, E. 1985. Characteristics of edible fluids of animalorigin: milk. Ch. 13. In "Food Chemistry", 2nd ed., 0.Fennema (Ed.), p.791. Marcel Dekker Inc., New York, NewYork.Sweeney, M.J. and Klotz, S.D. 1987. Immunology of foodantigens. Ch. 2. In "Handbook of Food Allergies", J.C.Breneman (Ed.), p.13. Marcel Dekker Inc., New York, NewYork.Taylor, S.L. 1985. Food allergies. Food Technol. 39(2):98Taylor, S.L. 1986. Immunologic and allergic properties ofcow's milk proteins in humans. J. Food Prot. 49:239.Taylor, S.L. 1992. Chemistry and detection of food allergens.Food Technol. 46(5):146.Taylor, S.L and Cumming, D.B. 1985. Food Allergies andsensitivities. Food Technol. 39(9):65.Taylor, S.L., Lemanske, R.F., Bush, R.K. and Busse, W.D. 1987a.Food allergens: structure and immunologic properties.Ann. of Allergy 59 (part II):93.Taylor, S.L., Lemanske, R.F., Bush, R.K. and Busse, W.W. 1987b.Chemistry of food allergens. Ch. 3. In "Food Allergy",R.K. Chandra (Ed.), P.21. Nutrition Research EducationFoundation, St. John's, Newfoundland.Taylor, S.L., Nordlee, J.A. and Rupnow, J.H. 1989. Foodallergies and sensitivities. Ch. 10. In "Food Toxicology- A Perspective on the Relative Risks", S.L. Taylor andR.A. Scanlan (Ed.), p.255. Marcel Dekker Inc., New York,New York.65Udall, J.N., Pang, K., Fritze, L., Kleinman, R. and Walker, W.A.1981a. Development of the gastrointestinal mucosalbarrier. I. the effect of age on intestinal permeability tomacromolecules. Pediatr. Res. 15:241.Udall, J.N., Colony, P., Fritze, L., Pang, K., Trier, J.S. andWalker, W.A. 1981b. Development of gastrointestinalmucosal barrier. II. the effect of natural versusartificial feeding on intestinal permeability tomacromolecules. Pediatr. Res. 15:245.Vaz, E.M., Vaz, N.M. and Levine, B.B. 1971. Persistentformation of reagins in mice injected with low doses ofovalbumin. Immunol. 21:11.Visakorpi, J.K. and Immonen, P. 1967. Intolerance to cow'smilk and wheat gluten in the primary malabsorption syndromein Infancy. Acta Paed. Scand. 56:49.Voller, A. 1980. Heterogeneous enzyme-immunoassays and theirapplications. Ch. 9. In "Enzyme-Immunoassay", E.T. Maggio(Ed.), P. 181. CRC Press Inc., Boca Raton, Florida.Walker, W.A. 1987. Pathophysiology of intestinal uptake andabsorption of antigens in food allergy. Ann. of Allergy59 (part II):7.Walker, W.A. and Bloch, K.J. 1983. Gastrointestinal transportof macromolecules in the pathogenesis of food allergy.Ann. of Allergy 51:240.Watanabe, N. and Ovary, Z. 1977. Antigen and antibodydetection by in vivo methods; a reevaluation of passivecutaneous anaphylaxis reactions. J. Immun. Met. 14:381.Williams, C.A. and Chase, M.W. (Ed.). 1967. "Methods inImmunology and Immunochemistry", Academic Press, New York,New York.Zittle, C.A. and Custer, J.H. 1963. Purfication and some ofthe properties of us-casein and K-casein. J. Dairy Sci.46:1183.66APPENDIX 1PROTEIN DETERMINATIONTable 13. Protein Concentration of Ovalbumin Standard.MEANABSORBANCEa ABSORBANCE PROTEIN'SAMPLE^REPLICATE #^(280 nm)^+ S.E.M.b (mg/ml) OVALBUMIN^1 0.69^0.69 + 0.00^9.92^0.693 0.70aStandard diluted 1/10.bS.E.M. = Standard Error of the Mean.'Extinction Coefficient of Ovalbumin (Kirschenbaum, 1976) = 7Table 14.^Protein Concentration of Casein Stock Solutions.SAMPLE^REPLICATE #ABSORBANCE(550 nm)MEANABSORBANCE PROTEIN+ S.E.M.a^(mg/ml)ces1 1 0.49 0.51 +^0.09 8.452 0.523 0.53S 1 0.24 0.24 +^0.00 3.832 0.243 0.25DEPHOSPHORYLATED 1 0.17 0.18 +^0.00 2.72us, 2 0.183 0.19DEPHOSPHORYLATED 1 0.17 0.18 +^0.01 2.73S 2 0.193 0.18aS.E.M. = Standard Error of the Mean.Table 15. Protein Concentration of Whey Stock Solutions.SAMPLE REPLICATE #ABSORBANCE(550nm)MEANABSORBANCE PROTEIN+ S.E.M.a^(mg/ml)WHEY 1 0.16 0.17 + 0.00^2.522 0.173 0.17FeC13 1 0.06 0.06^+^0.00^0.76TREATED WHEY 2 0.063 0.06aS.E.M. . Standard Error of the Mean.670.000.000.750.600.450.300.15Y = 0.058 X + 0.021- r2 = 0.996-^_I^ I2.20 4.40 6.60 8.80 11.0068PROTEIN (mg/m1)Figure 2. Ovalbumin Standard Curve. The Biuret assay was used to determine theprotein concentration of the fl-lactoglobulin standards.69APPENDIX 2PHOSPHORUS DETERMINATIONTable 16. Phosphorus Concentration of Casein Samples.SAMPLEPROTEIN(mq)REP'#ABSORBANCE(822 nm)MEANABSORBANCE^PHOSPHORUS+ S.E.M.b^(q/mg protein)as l 0.380 1 0.84 0.92 +^0.04^13.812 0.903 1.01DEPHOS 0.999 1 0.79 0.82 +^0.01 4.62asic 2 0.81CASEIN 3 0.85S 0.383 1 0.45 0.46 +^0.01 6.762 0.473 0.47DEPHOS 1.093 1 0.22 0.22 +^0.00 1.07iy 2 0.21CASEIN 3'ReplicatebS.E.M. = Standard Error of the Mean.cDephosphorylated70CALCULATIONS FOR DETERMINING % DEPHOSPHORYLATION% Dephosphorylation = 1 - Pf  X 100 PiWhere: Pi = phosphorus concentration before dephosphorylationPf = phosphorus concentration after dephosphorylation4.62 yg phosphorus % Dephosphorylation c = 1 - ^mg protein ^x 100 = 66.5%13.81 aq phosphorus mg protein1.07 yg phosphorus % Dephosphorylation S = 1 - ^mg protein ^x 100 = 84.1%6.76 yq phosphorus mg protein0.000.002.001.601.200.800.40Y = 0.175 X + 0.011- r2 = 0.999__2.20 4.40 6.60 8.80 11.0071PHOSPHORUS (MICROGRAMS)Figure 3. Phosphorus Standard Curve. Phosphorus concentrations of thestandards were determined using the method of Morrison (1964).WHEYFeC13 TREATED WHEY2.341^1.167_ a _APPENDIX 3S-LACTOGLOBULIN DETERMINATION72Table 17. S-Lactoglobulin Concentration of Wheys.SAMPLEOPTICAL^S-LACTOGLOBULINDENSITY^CONCENTRATION (mg/ml) aBelow the minimum detection limit of the PhastImage program.73CALCULATIONS FOR DETERMINING 96 S-LACTOGLOBULIN REMOVED% S-lactoglobulin removed = 1 -^x 100SiWhere: Si = S-lactoglobulin concentration of wheygf = S-lactoglobulin concentration of FeCl3 treatedwheySince the concentration of S-lactoglobulin in the FeC13 treatedwhey was below the minimum detection limit of the PhastImageprogram, the lowest concentration used for constructing theS-lactoglobulin standard curve was substituted.^This valuerepresents the minimum % of S-lactoglobulin removed. Pleasenote, that the actual amount of S-lactoglobulin removedexceeds this value.Minimum % S-lactoglobulin = 1 - 0.169 mg/ml x 100 = 85.5%removed^ 1.167 mg/m14 74Y = 2.156 X - 0.175r2 = 0.99900^ 1^ 2BETA-LACTOGLOBULIN ClughnOFigure 4. P-lactoglobulin Standard Curve.1 2 3 4 5 6 7 8Figure 5. SDS-PAGE Gel of Wheys and fl-lactoglobulin Standards.(1) fl-lactoglobulin standard; 1.69 mg/ml and internal standard, (2) P-lactoglobulin standard; 1.27 mg/mland internal standard, (3) P-lactoglobulin standard; 0.85 mg/ml and internal standard, (4) FeCl, TreatedWhey and internal standard, (5) Untreated Whey and internal standard, (6) 0-lactoglobulin standard; 0.34mg/ml and internal standard, (7) P-lactoglobulin standard; 0.17 mg/ml and internal standard, (8) Ovalbumininternal standard.7576APPENDIX 4RODENT DIETThe following diet is based on the American Instituteof Nutrition AIN-76 semi-purified diet' for rats and mice.Table 18.^Composition of Milk-Free Rodent DietINGREDIENT % LEVEL OF INGREDIENT (w/w)Sucrose' 50.0Soy Protein Isolateb 20.0Corn Starchc 15.0Canola Oild 5.0Non-Nutritive Fiber' 5.0Mineral Mixf 3.5Vitamin Mixg 1.0D-L Methioninec 0.3Choline bitartratec 0.2'B.C. Sugar (Vancouver, B.C., Canada.)b921 protein (ICN Nutritional Biochemicals, Cleveland, OH, USA).cICN Nutritional Biochemicals, Cleveland, OH, USA.dSunfrie (Vancouver, B.C., Canada).eAlphacel; composed of finely ground alpha-cellulose (ICNNutritional Biochemicals, Cleveland, OH, USA).LAIN Mineral Mixture 76 (ICN Nutritional Biochemicals, Cleveland,OH, USA)gAIN Vitamin Mixture 76 (ICN Nutritional Biochemicals, Cleveland,OH, USA)'ICN Biomedicals Catalog/Animal Research Diets. 1991/1992.p. 1036.APPENDIX 5ELISA BUFFER COMPOSITIONSThe following compositions are based on the ELISA buffersdescribed by Kummer et al. (1992).1. Carbonate Coating Buffer (pH 9.6)1.59 g Na2CO32.93 g NaHCO30.50 g NaN31.00 1 H2O2. Phosphate Buffered Saline (PBS) (pH 7.4)8.00 g NaCl0.20 g KH2PO41.15 g Na2HPO40.20 g KC11.00 1 11203. PBS/TWEEN- as described for PBS (composition 2), in addition to0.5 ml Tween 20.4. Blocking Buffer- as described for PBS (composition 2), in addition to2.50 g chicken egg albumin.5. Diethanolamine Substrate Buffer (pH 9.8)0.10 g MgC12 x 6 H2O0.20 g NaN397.00 ml Diethanolamine1.00 1 H2077Mouse 1 + ^Mouse 2 A Mouse 3 0 - - - - -Mouse 4 0 -----Mouse 5 A ---- --Mouse 6 • ----Mouse 7 • ---Mouse 1 +Mouse 2 A^Mouse 3 0 - - - - -Mouse 4 0 -----APPENDIX 6ELISA ABSORBANCE VS. TIME GRAPHSA. ELISA IgG Determinations780.280.210.140.07A.1 Experiment 1: Orally Administered Proteins0.350.000^75^150^225^300^375TIME (MINUTES)Figure 6. Determination of as, Casein Specific IgG in MiceOrally Administered as, Casein.0.350.280.210.140.070.0075^150^225^300^375TIME (MINUTES)Figure 7. Determination of us, Casein Specific IgG in ControlMice (Oral Administration).65 130 195 260 3250.90Mouse 1 +Mouse 2 A^Mouse 3 0 ---Mouse 4 0 -----0.0000.72Mouse 1 +Mouse 2 A ^Mouse 3 0 - - - - -Mouse 4 0 -----Mouse 5 A ---Mouse 6 • ----0.900.72sr)0.54WUZ0.36000^0.18-40.000^65^130^195^260^325TIME (MINUTES)Figure 8. Determination of 13 Casein Specific IgG in Mice OrallyAdministered 0 Casein.TIME (MINUTES)79Figure 9. Determination of 0 Casein Specific IgG in Control Mice(Oral Administration).75 225150 300 3751.251.000.750.500.250.00075 150 225 300 3751.251.000.750.500.250.000TIME (MINUTES)Figure 10. Determination of Dephosphorylated as1 CaseinSpecific IgG in Mice Orally AdministeredDephosphorylated as1 Casein.TIME (MINUTES)Figure 11. Determination of Dephosphorylated as, CaseinSpecific IgG in Control Mice (Oral Administration).80Mouse 1 +Mouse 2 A ^Mouse 3 0 - - - - -Mouse 4 0 -----Mouse 5 A-----Mouse 1 + Mouse 2 A Mouse 3 0 - - - -Mouse 4 0 -----Mouse 1 + ^Mouse 2 A ^- Mouse 3 0 - - - - -Mouse 4 0 -----0.2475 150 300225 3750.300.180.120.060.0000.300.240.18 81Mouse 1 +^ AMouse 2 A ^Mouse 3 0 - - - - - AMouse 4 0 -----Mouse 5 A ---^AMouse 6 0 ---Mouse 7 • ---^A0.120.060.000^75^150^225^300^375TIME (MINUTES)Figure 12. Determination of Dephosphorylated A Casein SpecificIgG in Mice Orally Administered Dephosphorylated ACasein.TIME (MINUTES)Figure 13. Determination of Dephosphorylated g Casein SpecificIgG in Control Mice (Oral Administration).0.110.050.000 90 180 270 360 4500.27Mouse 1 +Mouse 2 A ^Mouse 3 0 - - - - -Mouse 4 0 -----Mouse 5 A ---Mouse 6 • ----0.220.16Mouse 1 +Mouse 2 A^Mouse 3 0 ---Mouse 4 0 -----0.2290 180 270 360 4500.270.160.110.050.000TIME (MINUTES)Figure 14. Determination of Whey Specific IgG in Mice OrallyAdministered Whey.TIME (MINUTES)Figure 15. Determination of Whey Specific IgG in Control Mice(Oral Administration).82Mouse 1 +Mouse 2 A ^Mouse 3 0 - - - - -Mouse 4 o -----Mouse 5 A -----Mouse 6 • ----Mouse 7 • ---0.220.160.1 10.050.000 90 180 270 360 4500.27Mouse 1 + ^Mouse 2 A^- Mouse 3 0 - - -Mouse 4 0 -----02290 180 450270 3600.270.160.1 10.050.000TIME (MINUTES)Figure 16. Determination of FeC13 Treated Whey Specific IgG inMice Orally Administered FeC13 Treated Whey.TIME (MINUTES)Figure 17. Determination of FeC13 Treated Whey Specific IgG inControl Mice (Oral Administration).83A.2 Experiment 2: Intraperitoneally Injected Proteins^1.50 ^Mouse 1 +^4/Mouse 2 A ^/A--d^ -1.20a Mouse 3 0 — - - - ,/,/41^Mouse 4 a -----0.90^kr/0 Mouse 5 A ----- /I...,^- t/al /Z-4 0.60^/.,1'mM A".0^/cnal 0.30 -0.00 165 .......^..^......'^2"dkifILL4L-A^^0^55^110^220^275TIME (MINUTES)Figure 18. Determination of ce81 Casein Specific IgG in MiceIntraperitoneally Injected with asi Casein.1.50 ^Mouse 1 +Mouse 2 A ^1.20 - Mouse 3 0 — ---Mouse 4 0 -----Mouse 5 A -----0.900.600.300.000^55^110^165^220^275TIME (MINUTES)Figure 19. Determination of ce81 Casein Specific IgG in ControlMice (Intraperitoneal Injection).841.501.200.900.600.300.0085t/Mouse 1 +Mouse 2 A ^Mouse 3 0 - - - - -Mouse 4 0 -----Mouse 5 A ----120=0.900•4^0.600.300.000 551.50Mouse 1 + Mouse 2 A Mouse 3 0 - - - -Mouse 4 0 -----Mouse 5 A -----165110 2752200^55^110^165^220^275TIME (MINUTES)Figure 20. Determination of g Casein Specific IgG in MiceIntraperitoneally Injected with Casein.TIME (MINUTES)Figure 21. Determination of 13 Casein Specific IgG in ControlMice (Intraperitoneal Injection).,, ,, A ,, ...„-..„--,.0 ,, /^'-^---........„7. A• /121^,^,i ....„...„-^..„,,.. ,^„ .7•- /e .7^.Bli/^7// / .1 e....^...."0 •.Y-"/-"/^*7,,,,., '...,_,-/K/%IN- A,,,;-,-AN." e/861.501.20a 0.9044 0.600• 0.30Mouse 1 +Mouse 2 A ^Mouse 3 0 - - - - -Mouse 4 o -----Mouse 5 A -----0.000 55 110 165 220 275TIME (MINUTES)Figure 22. Determination of Dephosphorylated ces1 CaseinSpecific IgG in Mice Intraperitoneally Injected withDephosphorylated as, Casein.1.501.200.90.4 0.600• 0.3044Mouse 1 +Mouse 2 A ^Mouse 3 0 -- ---Mouse 4 0 -----Mouse 5 A -----0.000 55 110 165 220 275TIME (MINUTES)Figure 23. Determination of Dephosphorylated us, CaseinSpecific IgG in Control Mice (IntraperitonealInjection).0 55Mouse 1 +Mouse 2 ^Mouse 3 0 - - - - -Mouse 4 0 -----Mouse 5 A0.901.500.600.300.001.20165110 220 2750 55Mouse 1 + Mouse 2  Mouse 3 0 - - - - -Mouse 4 0 -----Mouse 5 A -----0.00165110 2752201.501.200.900.600.30TIME (MINUTES)Figure 24. Determination of Dephosphorylated A Casein SpecificIgG in Mice Intraperitoneally Injected withDephosphorylated 0 Casein.TIME (MINUTES)Figure 25. Determination of Dephosphorylated A Casein SpecificIgG in Control Mice (Intraperitoneal Injection).87881.50=^1.20•Nt. 0.9004t 0.600.300.000^42^84^126^168^210TIME (MINUTES)Figure 26. Determination of Whey specific IgG in MiceIntraperitoneally Injected with Whey.1.501.200.900.600.300.000^42^84^126^168^210TIME (MINUTES)Mouse 1 +Mouse 2 A ^Mouse 3 0 - - - - -Mouse 4 0 - - - - -- Mouse 5 A ----•  Figure 27. Determination of Whey Specific IgG in Control Mice(Intraperitoneal Injection).891.501.200.900.600.300.00Mouse 1 +Mouse 2 A ^Mouse 3 0 - - - - -Mouse 4 0 -----Mouse 5 A -----Mouse 1 + Mouse 2 A Mouse 3 0 - - - - -Mouse 4 0 -----Mouse 5 A -----1.50'-do 1.200.900< 0.6000.300.00 - 6""a6='=ill'4"^I^42^84^126^168^210TIME (MINUTES)Figure 28. Determination of FeC13 Treated Whey SpecificIgG in Mice Intraperitoneally injected with FeC13Treated Whey.0^42^84^126^168^210TIME (MINUTES)Figure 29. Determination of FeC13 Treated Whey Specific IgG inControl Mice (Intraperitoneal Injection).B. ELISA IgE Determinations0.42Mouse 1 +0.34 - Mouse 2 A^ .A'.^Mouse 3 0 --- -- .....-'A,-.----Mouse 4 0 --0.25^--- )k-.Mouse 5 • -----^••• ..,..,.t.0.17 -^•#--',---±---o,0.08^,^ 8-Ar ------ 0.- 0 ----- 9-------- 6 --------- __---^.-Q ------- 5_....- A ------ ---------------- cr0.00^- =^------- t--74^ '4^^-------  0.00^2.50^5.00^7.50^10.00^12.50TIME (HOURS)Figure 30. Determination of us, Casein Specific IgE in MiceOrally Intraperitoneally Injected with a90•0.42Mouse 1 +Mouse 2 A0.34 Mouse 3 o - -Mouse 4 0 -----Mouse 5 • -----0.250.170.080.0004-^----- ----------------- -----0.00^2.50^5.00 1-2.500°---^ WTIME (HOURS)Figure 31. Determination of a ^Specific IgE in ControlMice (Intraperitoneal Injection).0.170.080.000.00 2.50 5.00 7.50 10.00^12.50Mouse 1 + Mouse 2 A Mouse 3 0 - - - - -Mouse 4 0 -----Mouse 5 A -----0250.340.42Mouse 1 +Mouse 2 A^Mouse 3 0 - -Mouse 4 0 -----Mouse 5 A -----•••^0•0.420.340.250.170.0891TIME (HOURS)Figure 32. Determination of g Casein Specific IgE in MiceIntraperitoneally Injected with g Casein.•00••••••••••• -^•••^ --------------- -------^A------2.50^5.00^7.50^10.00^12.50TIME (HOURS)Figure 33. Determination of 0 Casein Specific IgE in ControlMice (Intraperitoneal Injection).0.000.00AA0^A0.42Mouse 1 + Mouse 2 A Mouse 3 0 - - - - -Mouse 4 0 -----Mouse 5 A -----0.340.25•4^0.1700.080.42DeterminationSpecificaTIME (HOURS)of Dephosphorylated as/IgE in Mice IntraperitoneallyMouse 1 +Mouse 2 A^0.34 Mouse 3 0 -- ---Mouse 4 0 -----Mouse 5 A -----0.250.170.08Figure 34. CaseinInjected with92TIME (HOURS)Figure 35. Determination of Dephosphorylated as/ CaseinSpecific IgE in Control Mice (IntraperitonealInjection).0.000.00Mouse 1 + ^Mouse 2 A ^- Mouse 3 - - - - -Mouse 4 0-----Mouse 5 A -----0.170.08A^•^•7.502.50 5.00 10.00 12.500.420.34025Mouse 1 +Mouse 2 A - Mouse 3 0 - -Mouse 4 o -----Mouse 5 A -----0.340.250.420.170.080.000.00 5.002.50 7.50 12.5010.0093TIME (HOURS)Figure 36. Determination of Dephosphorylated 13 Casein SpecificIgE in Mice Intraperitoneally Injected with g Casein.TIME (HOURS)Figure 37. Determination of Dephosphorylated g Casein SpecificIgE in Control Mice (Intraperitoneal Injection).Mouse 1 +Mouse 2 A ^Mouse 3 0 - - - - -Mouse 4 0 -----Mouse 5 • ------0.280.210.350.140.070.000.00 7.502.50 5.00 10.00 12.50TIME (HOURS)Figure 38. Determination of Whey Specific IgE in MiceIntraperitoneally Injected with Whey.94 0.350280210.140.07Mouse 1 +Mouse 2 A ^- Mouse 3 0 - - - - -Mouse 4 0 -----Mouse 5 • -----0.000.00^2.50^5.00^7.50^10.00^12.50TIME (HOURS)Figure 39. Determination of Whey Specific IgE in Control Mice(Intraperitoneal Injection).0.350.280210.1495TIME (HOURS)Mouse 1 +Mouse 2 A ^Mouse 3 0 - - - - -Mouse 4 0 -----Mouse 5 A -----Mouse 1 +Mouse 2 A ^Mouse 3 0 - - - - -Mouse 4 0 -----Mouse 5 A -----0.210280350.140.070.000.00 7.502.50 5.00 12.501000Figure 40. Determination of FeCl3 Treated Whey Specific IgE inMice Intraperitoneally Injected with FeC13 TreatedWhey.TIME (HOURS)Figure 41. Determination of FeC13 Treated Whey Specific IgE inControl Mice (Intraperitoneal Injection).96APPENDIX 7EQUATIONS OF THE LINES AND r2 VALUES FOR INDIVIDUAL MICEA. ELISA IgG DeterminationsA.1 Experiment 1: Orally Administered ProteinsTable 19. IgG Equations of the Lines and r2 Values for IndividualMice Orally Administered Native Casein Proteins.ANTIGEN ANTISERA MOUSE #EQUATION OFTHE LINE r2 VALUEus, us, 1 Y = 6.50X x 10-4 + 4.44 x 10-3 0.99CASEIN CASEIN 2 Y = 6.10X x 10-4 + 4.20 x 10-3 0.933 Y = 6.20X x 10-4 +^5.44 x 10-3 0.894 Y = 8.40X x 10-4 +^1.02 x 10-2 0.905 Y = 5.24X x 10-4 -^4.43 x 10-4 0.956 Y = 6.48X x 10-4 +^9.67 x 10-3 0.987 Y = 4.97X x 10-4 +^2.62 x 10-2 0.67NEGATIVE 1 Y = 8.23X x 10-4 +^6.89 x 10-3 0.87CONTROL 2 Y = 7.93X x 10-4 +^1.08 x 10-2 0.933 Y = 5.70X x 10-4 +^3.24 x 10-3 0.984 Y = 8.42X x 10-4 +^1.22 x 10-2 0.98S CASEIN J3 CASEIN 1 Y = 1.00X x 10-4 -^2.23 x 10-3 0.652 Y = 9.50X x 10-5 -^2.63 x 10-3 0.703 Y = 1.09X x 10-4 -^3.17 x 10-3 0.374 Y = 8.20X x 10-5 -^2.20 x 10-3 0.875 Y = 2.06X x 10-4 -^8.39 x 10 -4 0.586 Y = 2.12X x 10-4 -^3.51 x 10-4 0.97NEGATIVE 1 Y = 1.23X x 10-4 -^7.16 x 10-4 0.86CONTROL 2 Y = 1.60X x 10-4 +^8.70 x 10-5 0.753 Y = 1.64X x 10-4 +^2.99 x 10-3 0.964 Y = 1.86X x 10-4 -^1.65 x 10-3 0.9697Table 20. IgG Equations of the Lines and r2 Values for IndividualMice Orally Administered Dephosphorylated CaseinProteins.ANTIGEN ANTISERA MOUSE #EQUATION OFTHE LINE r2 VALUEDEPHOS DEPHOS 1 Y = 7.02X x 10-4 +^8.92 x 10-3 0.86,,,^ausi asia 2 Y = 8.45X x 10-4 +^1.67 x 10-2 0.47CASEIN CASEIN 3 Y = 1.06X x 10-3 +^1.88 x 10-2 0.374 Y = 2.84X x 10-4 +^1.42 x 10-1 0.975 Y = 1.67X x 10-3 + 4.93 x 10-2 0.75NEGATIVE 1 Y = 9.27X x 10-4 +^1.51 x 10-2 0.37CONTROL 2 Y = 6.79X x 10-4 +^8.96 x 10-3 0.963 Y = 6.03X x 10-4 +^8.68 x 10-3 0.554 Y = 8.36X x 10-4 +^1.68 x 10-2 0.43DEPHOS DEPHOS 1 Y = 3.58X x 10-4 +^3.07 x 10-3 0.64ga ga 2 Y = 4.35X x 10-4 +^6.45 x 10-3 0.29CASEIN CASEIN 3 Y = 2.13X x 10-4 + 2.94 x 10-3 0.724 Y = 2.59X x 10-4 +^5.48 x 10-3 0.745 Y = 2.68X x 10-4 +^7.33 x 10-3 0.906 Y = 2.34X x 10-4 -^4.54 x 10-3 0.707 Y = 1.58X x 10-4 +^2.38 x 10-3 0.81NEGATIVE 1 Y = 2.22X x 10-4 +^5.49 x 10-3 0.97CONTROL 2 Y = 2.71X x 10-4 +^7.35 x 10-3 0.993 Y = 2.28X x 10-4 +^7.36 x 10-3 0.474 Y = 1.80X x 10-4 +^5.52 x 10-3 0.96aDephosphorylated98Table 21. IgG Equations of the Lines and r2 Values for IndividualMice Orally Administered Whey Proteins.ANTIGEN ANTISERA MOUSE #EQUATION OFTHE LINE r2 VALUEWHEY WHEY 1 Y = 2.96X x 10 -4 +^5.14 x 10-3 0.972 Y = 2.64X x lo-4 -^4.81 x 10-4 0.953 Y = 2.42X x 10 -4 +^2.75 x 10-3 0.914 Y = 2.55X x lo-4 +^3.92 x 10-3 0.945 Y = 2.01X x 10 -4 +^4.08 x 10-3 0.946 Y = 2.95X x lo-4 +^7.03 x 10-3 0.93NEGATIVE 1 Y = 2.58X x 10-4 +^5.74 x 10-3 0.90CONTROL 2 Y = 3.05X x 10-4 +^8.23 x 10-3 0.923 Y = 2.21X x 10-4 +^3.82 x 10-3 0.944 Y = 2.52X x 10-4 +^9.78 x 10-3 0.96FeC13 FeC13 1 Y = 2.67X x 10-4 + 4.57 x lo-3 0.95TREATED TREATED 2 Y = 3.32X x 10-4 +^6.36 x 10-3 0.95WHEY WHEY 3 Y = 2.42X x 10-4 +^8.06 x lo-3 0.964 Y = 3.49X x 10-4 +^1.23 x 10 -2 0.965 Y = 3.15X x 10-4 +^9.38 x 10-3 0.976 Y = 2.38X x 10-4 +^4.85 x 10 -3 0.987 Y = 2.70X x 10-4 +^8.77 x 10-3 0.98NEGATIVE 1 Y = 2.16X x 10-4 +^8.36 x 10-3 0.97CONTROL 2 Y = 4.16X x 10-4 +^1.40 x 10-2 0.863 Y = 2.26X x 10-4 +^1.15 x 10-2 0.964 Y = 5.28X x 10-4 +^2.57 x 10-2 0.9599A.2 Experiment 2: Intraperitoneally Injected ProteinsTable 22. IgG Equations of the Lines and r2 Values for IndividualMice Intraperitoneally Injected with Native CaseinProteins.ANTIGEN ANTISERA MOUSE #EQUATION OFTHE LINE r2 VALUEasi ces1 1 Y = 2.06X x 10-4 -^1.38 x 10-3 0.91CASEIN CASEIN 2 Y = 6.36X x 10-4 -^7.58 x 10-4 0.993 Y = 2.24X x 10-4 -^1.15 x 10-3 0.874 Y = 4.66X x 10-4 +^9.57 x 10-4 0.775 Y = 8.59X x 10-3 +^1.34 x 10-1 0.78NEGATIVE 1 Y = 2.87X x 10-4 -^5.70 x 10-3 0.99CONTROL 2 Y = 2.71X x 10-4 -^3.90 x 10-3 0.993 Y = 3.07X x 10-4 -^4.53 x 10-3 0.984 Y = 2.03X x 10-4 -^4.28 x 10-3 0.965 Y = 2.28X x 10-4 -^4.17 x 10-3 0.91S CASEIN S CASEIN 1 Y = 1.24X x 10-2 +^2.63 x 10-1 0.952 Y = 5.79X x 10-3 +^1.69 x 10-1 0.933 Y = 5.00X x 10-3 +^1.80 x 10-1 0.824 Y = 4.45X x 10-3 +^1.06 x 10-1 0.975 Y = 8.96X x 10-3 +^1.31 x 10-1 0.98NEGATIVE 1 Y = 1.23X x 10-4 -^4.79 x 10-3 0.94CONTROL 2 Y = 5.50X x 10-5 -^1.86 x 10-3 0.523 Y = 8.50X x 10-5 -^2.71 x 10-3 0.464 Y = 3.80X x 10-5 -^2.19 x 10-3 0.745 Y = 4.50X x 10-5 -^1.18 x 10-3 0.58100Table 23.^IgG Equation of the Lines and r2 Values forIndividualMice Intraperitoneally Injected with DephosphorylatedCasein Proteins.ANTIGEN^ANTISERA MOUSE #EQUATION OFTHE LINE r2 VALUEDEPHOSacesi DEPHOS°Isla 12Y =Y =2.74X1.20Xxx10-310-2+^2.73+^1.04xx10 -210-10.950.99CASEIN CASEIN 3 Y = 7.95X x 10-3 +^6.32 x 10-2 0.994 Y = 9.82X x 10-3 +^1.74 x 10-1 0.965 Y = 6.02X x 10-3 +^1.61 x 10-1 0.97NEGATIVE 1 Y = 2.98X x 10-4 -^3.36 x 10-3 0.99CONTROL 2 Y= 2.12X x 10-4 -^5.59 x 10-3 0.903 Y = 3.16X x 10-4 -^2.92 x 10-3 0.934 Y = 1.92X x 10-4 -^2.56 x 10-3 0.965 Y = 2.22X x 10-4 -^1.91 x 10-3 0.73DEPHOS DEPHOS 1 Y = 1.13X x 10-3 +^3.37 x 10-3 0.96ga ga 2 Y = 3.87X x 10-3 +^7.28 x 10-2 0.98CASEIN CASEIN 3 Y = 4.24X x 10-3 +^9.25 x 10-2 0.994 Y = 1.58X x 10-3 +^6.59 x 10-3 0.995 Y = 3.48X x 10-3 +^6.19 x 10-2 0.98NEGATIVE 1 Y= 1.36X x 10-4 -^2.97 x 10-3 0.95CONTROL 2 Y= 1.91X x 10 -4 -^3.95 x i0- 0.733 Y= 1.79X x 10-4 -^3.65 x 10-3 0.984 Y= 1.32X x 10-4 -^2.57 x 10-3 0.915 Y = 8.40X x 10-5 -^1.00 x 10-3 0.77aDephosphorylated101Table 24. IgG Equations of the Lines and r2 Values for IndividualMice Intraperitoneally Injected with Whey Proteins.ANTIGEN ANTISERA MOUSE #EQUATION OFTHE LINE r2 VALUEWHEY WHEY 1 Y = 6.08X x 110-3 +^1.40 x 10-1 0.972 Y = 7.40X x icr3 +^1.90 x 10-1 0.963 Y = 5.51X x lo-3 +^1.02 x 10-1 0.964 Y = 6.12X x icy3 +^1.33 x 10-1 0.985 Y = 7.32X x lo-3 +^2.24 x 10-1 0.97NEGATIVE 1 Y = 1.94X x 10-4 +^2.11 x 10-3 0.67CONTROL 2 Y = 2.15X x 10-4 -^1.61 x 10-3 0.883 Y = 2.49X x 10-4 -^1.46 x 10-3 0.944 Y = 2.03X x 10-4 -^1.63 x 10-3 0.805 Y = 2.42X x 10-4 -^1.59 x 10-3 0.89FeC13 FeC13 1 Y = 3.28X x 10-3 +^2.88 x 10-2 0.95TREATED TREATED 2 Y = 4.42X x 10-3 +^6.58 x 10-2 0.95WHEY WHEY 3 Y = 3.09X x 10-3 +^3.58 x 10-2 0.964 Y = 4.50X x 10-3 +^6.41 x 10-2 0.965 Y = 3.76X x 10-3 + 4.00 x 10-2 0.97NEGATIVE 1 Y = 3.49X x 10-4 -^1.14 x 10-3 0.97CONTROL 2 Y = 3.72X x 10-4 +^8.64 x 10-4 0.863 Y = 4.74X x 10-4 + 4.12 x 10-3 0.964 Y = 3.53X x 10-4 -^6.95 x 10-2 0.955 Y = 3.27X x 10-4 -^5.30 x 10-5 0.95102B. ELISA IgE DeterminationsTable 25. IgE Equations of the Lines and r2 Values for IndividualMice Intraperitoneally Injected with Native CaseinProteins.EQUATION OFANTIGEN ANTISERA MOUSE # THE LINE r2 VALUEasi as, 1 Y = 2.59X x 10-3 -^7.78 x 10" 0.36CASEIN CASEIN 2 Y = 1.15X x 10-3 -^1.91 x 10-3 0.503 Y = 4.45X x 10-3 +^6.38 x 10" 0.834 Y = 7.01X x 10-3 +^1.99 x 10-3 0.705 Y = 2.74X x 10-2 +^1.80 x 10-2 0.97NEGATIVE 1 Y = 1.39X x 10-3 -^2.47 x 10-3 0.40CONTROL 2 Y = 0.00X x 100 +^0.00 x 100 0.003 Y = 4.61X x 10-3 -^4.09 x 10-3 0.514 Y = 2.09X x 10-3 -^2.46 x 10-3 0.415 Y = 8.23X x 10" -^1.95 x 10-3 0.45g CASEIN 8 CASEIN 1 Y = 1.96X x 10-2 +^1.67 x 10-2 0.972 Y = 2.90X x 10-2 +^2.43 x 10-2 0.983 Y = 1.06X x 10-2 + 4.65 x 10-3 0.924 Y = 2.30X x 10-2 +^1.52 x 10-2 0.985 Y = 1.91X x 10-2 +^1.07 x 10-2 0.96NEGATIVE 1 Y = 2.48X x 10-3 -^1.46 x 10" 0.40CONTROL 2 Y = 0.00X x 100 +^0.00 x 100 0.003 Y = 1.06X x 10-2 +^3.81 x 10-2 0.574 Y = 4.72X x 10-3 -^1.25 x 10-3 0.875 Y = 1.09X x 10-2 + 3.61 x 10-3 0.29103Table 26. IgE Equations of the Lines and r2 Values for IndividualMice Intraperitoneally Injected with DephosphorylatedCasein Proteins.ANTIGEN ANTISERA MOUSE #EQUATION OFTHE LINE r2 VALUEDEPHOS DEPHOS 1 Y = 9.65X x 10-3 +^1.22 x 10-3 0.91asia asia 2 Y = 2.13X x 10-2 -^1.01 x 10-2 0.91CASEIN CASEIN 3 Y = 1.35X x 10-2 +^4.36 x 10-3 0.954 Y = 1.58X x 10-2 +^7.39 x 10-3 0.745 Y = 1.24X x 10-2 +^3.64 x 10-3 0.96NEGATIVE 1 Y = 3.05X x 10-3 -^3.44 x 10-3 0.48CONTROL 2 Y = 2.95X x 10-3 -^3.21 x 10-3 0.303 Y= 8.84X x 10-3 -^2.44 x 10-3 0.764 Y = 3.87X x 10-3 -^5.97 x 10-4 0.355 Y = 8.82X x 10-4 -^8.76 x 10-4 0.31DEPHOS DEPHOS 1 Y = 2.60X x 10-2 +^1.89 x 10-2 0.75sa sa. 2 Y = 2.13X x 10-2 -^1.01 x 10-2 0.91CASEIN CASEIN 3 Y = 2.10X x 10-2 +^1.36 x 10-2 0.814 Y = 2.17X x 10-2 +^1.51 x 10-2 0.935 Y = 6.23X x 10-3 +^2.32 x 10-3 0.87NEGATIVE 1 Y = 6.84X x 10-3 -^1.17 x 10-4 0.95CONTROL 2 Y = 8.08X x 10-3 -^4.53 x 10-4 0.733 Y = 7.05X x 10-3 + 4.41 x 10-4 0.984 Y = 7.19X x 10-3 -^3.56 x 10-4 0.915 Y = 4.40X x 10-3 +^3.12 x 10-3 0.77aDephosphorylated104Table 27. IgE Equations of the Lines and r2 Values for IndividualMice Intraperitoneally Injected with Whey Proteins.ANTIGEN ANTISERA MOUSE #EQUATION OFTHE LINE r2 VALUEWHEY WHEY 1 Y = 1.11X x 10-2 -^3.49 x 10-3 0.822 Y = 2.26X x 10-2 +^1.62 x 10-2 0.953 Y = 6.56X x 10-3 +^1.18 x 10-3 0.964 Y = 4.21X x 10-3 +^9.97 x 10-3 0.835 Y = 1.20X x 10-2 +^1.07 x 10-2 0.75NEGATIVE 1 Y = 1.38X x 10-4 + 4.25 x 10-4 0.05CONTROL 2 Y = 3.53X x 10-4 -^1.02 x 10-3 0.213 Y = 1.01X x 10-4 -^1.75 x 10-3 0.644 Y = 0.00X x 100 +^0.00 x 10° 0.005 Y = 6.21X x 10-4 -^2.33 x 10-3 0.49FeCl3 FeCl3 1 Y = 6.03X x 10-3 -^3.95 x 10-3 0.79TREATED TREATED 2 Y = 7.71X x 10-3 -^2.36 x 10-3 0.62WHEY WHEY 3 Y = 6.75X x 10-3 +^5.18 x 10-4 0.564 Y = 5.32X x 10-3 -^5.84 x 10-4 0.855 Y = 7.36X x 10-3 -^2.86 x 10-3 0.85NEGATIVE 1 Y = 7.21X x 10-4 +^1.59 x 10-3 0.07CONTROL 2 Y = 1.28X x 10-4 +^8.33 x 10-4 0.033 Y = 1.53X x 10-3 -^3.16 x 10-4 0.404 Y = 0.00X x 10° +^0.00 x 10° 0.005 Y = 6.53X x 10-4 -^2.05 x 10-3 0.31105APPENDIX 8IgG VALUES FOR INDIVIDUAL MICEA. Experiment 1: Mice Orally Administered ProteinsTable 28. Relative IgG Values for Individual Mice OrallyAdministered Native Casein Proteins.MEAN ABSORBANCE^IgG VALUEANTIGEN^ANTISERAa^MOUSE #^+ S.E.M.b^(abs./min.)x103as, us, 1 0.114^±^0.000c 0.65CASEIN CASEIN 2 0.105^+^0.006 0.613 0.121^+^0.016 0.624 0.146^+^0.011 0.845 0.084^+^0.004 0.526 0.117^+^0.002 0.657 0.098^±^0.015 0.50NEGATIVE 1 0.140^±^0.013c 0.82CONTROL 2 0.137^+^0.008 0.793 0.097^+^0.003 0.574 0.146^+^0.003 0.84S CASEIN S CASEIN 1 0.011^+^0.003d 0.102 0.011^+^0.003 0.103 0.012^+^0.005 0.114 0.010^+^0.000 0.085 0.029^+^0.006 0.216 0.031^+^0.003 0.21NEGATIVE 1 0.018^+^0•003d 0.12CONTROL 2 0.022^+^0.004 0.163 0.028^+^0.001 0.164 0.025^+^0.003 0.19'Antisera diluted 1/25.bS.E.M.^Standard Error of the Mean.'Absorbance measured after 150.41 minutes of incubation at 37°C.dAbsorbance measured after 149.05 minutes of incubation at 37°C.Table 29. Relative IgG Values for Individual Mice OrallyAdministered Dephosphorylated Casein Proteins.ANTIGEN ANTISERAa MOUSE #MEAN ABSORBANCE IgG VALUE(abs./min.)x103DEPHOS DEPHOS 1 0.128^+^0•011d 0.70usic usic 2 0.153^±^0.041 0.85CASEIN CASEIN 3 0.188^+^0.061 1.064 0.618^±^0.016 2.845 0.320^+^0.046 1.67NEGATIVE 1 0.164^+^0.054d 0.93CONTROL 2 0.117^+^0.005 0.683 0.105^+^0.023 0.604 0.149^+^0.042 0.84DEPHOS DEPHOS 1 0.059^+^0.011e 0.36gc gc 2 0.074^+^0.031 0.44CASEIN CASEIN 3 0.035^+^0.006 0.214 0.044^+^0.007 0.265 0.047^+^0.004 0.276 0.030^+^0.004 0.237 0.025^+^0.002 0.16NEGATIVE 1 0.039^+^0.001e 0.22CONTROL 2 0.048^+^0.001 0.273 0.043^+^0.010 0.234 0.032^+^0.001 0.18'Antisera diluted 1/25.bS.E.M. = Standard Error of the Mean.cDephosphorylateddAbsorbance measured after 150.41 minutes of incubation at 37°C.eAbsorbance measured after 149.05 minutes of incubation at 37°C.106107Table 30. Relative IgG Values for Individual Mice OrallyAdministered Whey Proteins.ANTIGEN ANTISERA' MOUSE #MEAN ABSORBANCE+ S.E.M.bIgG VALUE(abs./min.)x103WHEY WHEY 1 0.073^±^0.009c 0.302 0.054^+^0.003 0.263 0.056^±^0.005 0.244 0.061^+^0.004 0.265 0.047^+^0.003 0.206 0.072^+^0.006 0.30NEGATIVE 1 0.060^+^0.005c 0.26CONTROL 2 0.076^+^0.005 0.313 0.049^+^0.003 0.224 0.064^+^0.002 0.25FeC13 FeC13 1 0.062^+^0.005c 0.27TREATED TREATED 2 0.077^+^0.004 0.33WHEY WHEY 3 0.060^+^0.002 0.244 0.090^+^0.004 0.355 0.078^+^0.003 0.326 0.055^+^0.002 0.247 0.067^+^0.001 0.27NEGATIVE 1 0.056^+^0.002c 0.22CONTROL 2 0.105^+^0.010 0.423 0.060^+^0.003 0.234 0.142^+^0.011 0.53'Antisera diluted 1/25.bS.E.M. = Standard Error of the Mean.cAbsorbance measured after 205.91 minutes of incubation at 37°C.108B. Experiment 2: Mice Intraperitoneally Injected With ProteinsTable 31. Relative IgG Values for Individual MiceIntraperitoneally Injected with Native Casein Proteins.ANTIGEN ANTISERA' MOUSE #MEAN ABSORBANCE+^S.E.M.bIgG VALUE(abs./min.)x103usl as, 1 0.011^+^0.061c 0.21CASEIN CASEIN 2 0.034^+^0.001 0.643 0.013^+^0.001 0.224 0.030^+^0.004 0.475 0.693^+^0.016 8.59NEGATIVE 1 0.014^+^0•002d 0.29CONTROL 2 0.012^+^0.000 0.273 0.012^+^0.001 0.314 0.008^+^0.001 0.205 0.013^+^0.001 0.23S CASEIN S CASEIN 1 0.712^+^0.255c 12.362 0.485^+^0.050 5.793 0.485^+^0.050 5.004 0.377^+^0.016 4.455 0.711^+^0.011 8.96NEGATIVE 1 0.001^+^0.000' 0.12CONTROL 2 0.000^+^0.000 0.063 0.001^+^0.001 0.094 0.000^+^0.000 0.045 0.001^+^0.000 0.05'Antisera diluted 1/1000.bS.E.M. = Standard Error of the Mean.cAbsorbance measured after 59.83 minutes of incubation at 37°C.dAbsorbance measured after 59.90 minutes of incubation at 37°C.109Table 32. Relative IgG Values for Individual MiceIntraperitoneally Injected with DephosphorylatedCasein Proteins.ANTIGEN ANTISERAa MOUSE #MEAN ABSORBANCE+^S.E.M.13IgG VALUE(abs./min.)x103DEPHOS DEPHOS 1 0.190^+^0.013c 2.74usic cesic 2 0.864^+^0.013 12.03CASEIN CASEIN 3 0.568^+^0.009 7.954 0.823^+^0.023 9.825 0.541^+^0.007 6.02NEGATIVE 1 0.014^+^0•001d 0.30CONTROL 2 0.007^+^0.001 0.213 0.013^+^0.002 0.324 0.009^+^0.001 0.195 0.010^+^0.002 0.22DEPHOS DEPHOS 1 0.069^+^0.004c 1.13gc gc 2 0.306^+^0.013 3.87CASEIN CASEIN 3 0.356^+^0.005 4.244 0.103^+^0.003 1.585 0.273^+^0.005 3.48NEGATIVE 1 0.003^+^0.001d 0.14CONTROL 2 0.007^+^0.003 0.193 0.006^+^0.000 0.184 0.006^+^0.001 0.135 0.003^±^0.001 0.08'Antisera diluted 1/1000.bS.E.M. = Standard Error of the Mean.cAbsorbance measured after 59.83 minutes of incubation at 37°C.dAbsorbance measured after 59.90 minutes of incubation at 37°C.110Table 33. Relative IgG Values for Individual MiceIntraperitoneally Injected with Whey Proteins.ANTIGEN ANTISERAa MOUSE #MEAN ABSORBANCE^IgG VALUE+ S.E.M.b^(abs./min.)x103WHEY WHEY 1 0.327^+^0.115c 6.082 0.602^+^0.017 7.403 0.391^+^0.013 5.524 0.452^+^0.002 6.125 0.614^+^0.004 7.32NEGATIVE 1 0.011^+^0.003c 0.19CONTROL 2 0.009^+^0.001 0.223 0.009^+^0.001 0.254 0.008^+^0.003 0.205 0.010^+^0.002 0.24FeC13 FeC13 1 0.201^+^0.008c 3.28TREATED TREATED 2 0.297^+^0.006 4.42WHEY WHEY 3 0.196^+^0.001 3.094 0.301^+^0.008 4.505 0.232^+^0.006 3.76NEGATIVE 1 0.016^+^0.001c 0.35CONTROL 2 0.020^+^0.002 0.373 0.028^+^0.001 0.474 0.017^+^0.002 0.355 0.017^+^0.001 0.33aAntisera diluted 1/1000.bS.E.M. = Standard Error of the Mean.cAbsorbance measured after 50.85 minutes of incubation at 37°C.111APPENDIX 9IgE VALUES FOR INDIVIDUAL MICE INTRAPERITONEALLY INJECTED WITHPROTEINSTable 34. Relative IgE Values for Individual MiceIntraperitoneally Injected with Native Casein Proteins.MEAN ABSORBANCE^IgG VALUEANTIGEN^ANTISERAa^MOUSE #^+ S.E.M.b^(abs./min.)x103us, as, 1 0.008^+^0.005' 2.59CASEIN CASEIN 2 0.002^+^0.001 1.153 0.020^+^0.003 4.454 0.032^+^0.006 7.015 0.136^+^0.003 27.41NEGATIVE 1 0.002^+^0•001d 1.39CONTROL 2 0.000^+^0.000 0.003 0.012^+^0.002 4.614 0.006^+^0.003 2.095 0.001^+^0.001 0.8213 CASEIN S CASEIN 1 0.100^+^0.003c 19.562 0.116^+^0.022 28.993 0.050^+^0.004 10.614 0.113^+^0.004 23.025 0.089^+^0.004 19.10NEGATIVE 1 0.014^+^0•005 d 2.48CONTROL 2 0.000^+^0.000 0.003 0.048^+^0.015 10.624 0.024^+^0.004 4.725 0.049^+^0.028 10.88'Antisera diluted 1/20.bS.E.M. = Standard Error of the Mean.'Absorbance measured after 3.93 hours of incubation at 37°C.dAbsorbance measured after 3.94 hours of incubation at 37°C.112Table 35. Relative IgE Values for Individual MiceIntraperitoneally Injected with Dephosphorylated CaseinProteins.ANTIGEN ANTISERAa MOUSE #MEAN ABSORBANCE+^S.E.M.bDEPHOS DEPHOS 1 0.043^+^0•003dusic usic 2 0.072^+^0.008CASEIN CASEIN 3 0.062^+^0.0034 0.072^+^0.0135 0.038^+^0.012NEGATIVE 1 0.008^+^0.004eCONTROL 2 0.008^+^0.0053 0.034^+^0.0064 0.019^+^0.0075 0.005^+^0.002DEPHOS DEPHOS 1 0.129^+^0•023dgc gc 2 0.072^+^0.008CASEIN CASEIN 3 0.103^+^0.0154 0.110^+^0.0095 0.030^+^0.003NEGATIVE 1 0.029^+^0.011eCONTROL 2 0.035^+^0.0153 0.031^+^0.0014 0.032^+^0.0075 0.030^+^0.012IgG VALUE(abs./min.)x1039.6521.2613.5115.8112.383.052.958.843.870.8825.9721.2621.0321.736.236.848.087.057.194.40'Antisera diluted 1/20.bS.E.M. = Standard Error of the Mean.eDephosphorylateddAbsorbance measured after 3.93 hours of incubation at 37°C.eAbsorbance measured after 3.94 hours of incubation at 37°C.113Table 36. Relative IgE Values for Individual MiceIntraperitoneally Injected with Whey Proteins.ANTIGEN ANTISERA' MOUSE #MEAN ABSORBANCE+ S.E.M.bIgG VALUE(abs./min.)x103WHEY WHEY 1 0.069^+^0.009 11.082 0.165^+^0.008 22.593 0.044^+^0.001 6.564 0.040^+^0.004 4.215 0.089^+^0.013 12.03NEGATIVE 1 0.001^+^0.001 0.14CONTROL 2 0.000^+^0.000 0.353 0.003^+^0.000 1.014 0.001^+^0.001 0.005 0.000^+^0.000 0.62FeC13 FeC13 1 0.035^+^0.006 6.03TREATED TREATED 2 0.047^+^0.011 7.71WHEY WHEY 3 0.044^+^0.010 6.754 0.032^+^0.003 5.325 0.045^+^0.005 7.36NEGATIVE 1 0.006^+^0.004 0.72CONTROL 2 0.000^+^0.000 0.133 0.007^+^0.003 1.534 0.000^+^0.000 0.005 0.001^+^0.001 0.65aAntisera diluted 1/20.bS.E.M. = Standard Error of the Mean.'Absorbance measured after 6.07 hours of incubation at 37° C.APPENDIX 10PCA TITRES FOR INDIVIDUAL MICEA. Experiment 1: Orally Administered ProteinsTable 37. Passive Cutaneous Anaphylaxis Titresfor Individual Mice Orally AdministeredNative Casein Proteins.ANTIGEN ANTISERA MOUSE # TITRE1 NRaCASEIN CASEIN 2 NR3 NR4 NR5 NR6 NR7 NRNEGATIVE 1-4b NRCONTROL13 CASEIN g CASEIN 1 NR2 NR3 NR4 NR5 NR6 NRNEGATIVE 1-4 NRCONTROLaNR = No Response at the lowest dilution tested (1/5).bPooled sample.114Table 38. Passive Cutaneous Anaphylaxis Titresfor Individual Mice Orally AdministeredDephosphorylated Casein Proteins.ANTIGEN^ANTISERA^MOUSE #^TITRE^DEPHOSPHORYLATED DEPHOSPHORYLATED 1^NRacis,^ as,^ 2 NRCASEIN CASEIN 3^NR4 NR5^NRNEGATIVE^1-4b^NRCONTROLDEPHOSPHORYLATED DEPHOSPHORYLATED 1^NRS CASEIN^S CASEIN^2 NR3^NR4 NR5^NR6 NR7^NRNEGATIVE^1-4^NRCONTROL1TR = No Response at the lowest dilution tested (1/5).bPooled sample.115Table 39. Passive Cutaneous Anaphylaxis Titresfor Individual Mice Orally AdministeredDephosphorylated Casein Proteins andChallenged with Potato Acid Phosphatase.ANTIGEN ANTISERA MOUSE # TITREPOTATO ACIDa DEPHOSPHORYLATED 1 NRbPHOSPHATASE usi 2 NRCASEIN 3 NR4 NR5 NRDEPHOSPHORYLATED 1 NRS CASEIN 2 NR3 NR4 NR5 NR6 NR7 NRNEGATIVE 1-4' NRCONTROLaMice challenged with 0.00005 g enzyme.biNTR = No Response at the lowest dilution tested (1/5).'Pooled sample.116Table 40. Passive Cutaneous Anaphylaxis Titresfor Individual Mice Orally AdministeredWhey Proteins.ANTIGEN ANTISERA MOUSE # TITREWHEY WHEY 1 NRa2 NR3 NR4 NR5 NR6 NRNEGATIVE 1-4b NRCONTROLFeCl3 FeCl3 1 NRTREATED WHEY TREATED WHEY 2 NR3 NR4 NR5 NR6 NR7 NRNEGATIVE 1-4 NRCONTROLaNR = No Response at the lowest dilution tested (1/5).bPooled Sample.117B. Experiment 2: Intraperitoneally Injected ProteinsTable 41. Passive Cutaneous Anaphylaxis Titresfor Individual Mice IntraperitoneallyInjected with Native Casein Proteins.ANTIGEN ANTISERA MOUSE # TITREus, usl 1 NRaCASEIN CASEIN 2 NR3 NR4 NR5 320NEGATIVE 1-5b NRCONTROLg CASEIN g CASEIN 1 1602 3203 804 1605 160NEGATIVE^1-5^NRCONTROLaITR. = No Response at the lowest dilution tested (1/5).bPooled Sample.118Table 42. Passive Cutaneous Anaphylaxis Titresfor Individual Mice IntraperitoneallyInjected with Dephosphorylated CaseinProteins.ANTIGEN^ANTISERA^MOUSE #^TITRE^DEPHOSPHORYLATED DEPHOSPHORYLATED 1^20as, asl^ 2 80CASEIN^CASEIN 3^1604 805^80NEGATIVE^1-5a^NRbCONTROLDEPHOSPHORYLATED DEPHOSPHORYLATED 1^160S CASEIN^0 CASEIN^2 1603^1604 1605^160NEGATIVE^1-5^NRCONTROLaNR = No Response at the lowest dilution tested (1/5).bPooled Sample.119Table 43. Passive Cutaneous Anaphylaxis Titresfor Individual Mice IntraperitoneallyInjected with Dephosphorylated CaseinProteins and Challenged with PotatoAcid Phosphatase.ANTIGEN ANTISERA MOUSE # TITREPOTATO ACIDa DEPHOSPHORYLATED 1 5PHOSPHATASE as, 2 40CASEIN 3 404 405 10DEPHOSPHORYLATED 1 80S CASEIN 2 803 804 55 160NEGATIVE^1-4b^NRcCONTROL'Mice challenged with 0.00005 g enzyme.bPooled Sample.cl\TR = No Response at the lowest dilution tested (1/5).120Table 44. Passive Cutaneous Anaphylaxis Titresfor Individual Mice IntraperitoneallyInjected with Whey Proteins.ANTIGEN^ANTISERA^MOUSE #^TITREWHEY WHEY 1^6402 12803^3204 1605^640NEGATIVE^1-5a^NRbCONTROLFeCl3^FeC13^1^320TREATED WHEY^TREATED WHEY^2 1603^1604 6405^320NEGATIVE^1-5^NRCONTROL'Pooled sample.13NR = No Response at lowest dilution tested (1/5).121

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0098822/manifest

Comment

Related Items