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Radioimmunoassay of salmon calcitonin Bass, Sydney 1970

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RADIOIMMUNOASSAY OF SALMON CALCITONIN by  SYDNEY BASS B.Sc., University of Manitoba, 1966  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in the Department of Physiology  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA  Date  October. 6th. 197Q  In  presenting  this  an a d v a n c e d  degree  the  shall  I  Library  further  for  agree  scholarly  by  his  of  this  written  thesis  in  at  University  the  make  that  it  purposes  for  of  October 6th  It  financial  is  of  Columbia,  British  by  gain  Columbia  for  the  understood  Physiology  1970  of  extensive  may be g r a n t e d  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  Date  for  permission.  Department  fulfilment  freely available  permission  representatives. thesis  partial  shall  requirements I agree  r e f e r e n c e and copying  Head o f  that  not  the  of  or  that  Study.  this  thesis  my D e p a r t m e n t  copying  for  or  publication  be a l l o w e d w i t h o u t  my  This thesis i s dedicated to my wife, Lynne, for her love, understanding, encouragement and invaluable help.  ii  ACKNOWLEDGMENT  I would l i k e t o e x p r e s s my s i n c e r e s t a p p r e c i a t i o n to Dr. H a r o l d Copp, whose p a t i e n c e , guidance and c o n t i n u e d s u p p o r t have made t h i s t h e s i s p o s s i b l e . I would a l s o l i k e t o thank Dr. Benjamin L. Gordon I I f o r h i s a s s i s t a n c e i n t h e p r e p a r a t i o n o f a n t i s e r a as w e l l as f o r h i s i n v a l u a b l e  advice.  Many thanks a r e extended t o D r . John Ledsome, D r . C. Owen P a r k e s and Dr. R a l p h S p i t z e r f o r t h e i r c o n s t r u c t i v e c r i t i c i s m s and suggestions. I am g r e a t l y i n d e b t e d  t o Mr K u r t Henze f o r h i s h e l p  throughout t h e d u r a t i o n o f my s t a y i n t h e Department o f P h y s i o l o g y and  f o r the preparation o f i l l u s t r a t i o n s . I n a d d i t i o n , I wish to  e x p r e s s my g r a t i t u d e t o M i s s Kathy F i n c h f o r h e r t e c h n i c a l a s s i s t a n c e and Mrs F r a n c e s News'ome f o r h e r h e l p i n t h e e d i t i n g o f t h i s m a n u s c r i p t . F i n a l l y , I would l i k e t o a c c o r d  r e c o g n i t i o n to the Medical  C o u n c i l o f Canada f o r f i n a n c i a l s u p p o r t .  iii  Research  ABSTRACT  The development of a radioimmunoassay for salmon calcitonin 125 is described. Synthetic salmon calcitonin was iodinated with I and used as tracer. The coated charcoal method, suitably modified, was employed to separate bound and free salmon calcitonin. Antisera were raised by intradermal and intramuscular injection of calcitonin conjugated to keyhole limpet hemocyanin with carbodiimide. The sensitivity of the assay i s 50 - 60 pg/ml of incubate. The disappearance of synthetic salmon calcitonin i n rainbow trout was determined, illustrating two components, an i n i t i a l rapid decline followed by a prolonged drop in concentration.  iv  TABLE OF CONTENTS  Page  INTRODUCTION  .  .  1  MATERIALS AND METHODS  9  RESULTS .;  20  DISCUSSION  45  BIBLIOGRAPHY.  52  APPENDIX  .  .-  v  56  LIST OF TABLES  Table  I  II  Page  Protocol for Radioimmunoassay  16  Labelling Variation and Effect of Dowex on the Stored Products-  21  III  125 Effect of Dowex .2-X8 on Freshly Prepared SCT-I  28  IV  125 Effect of Storage on Immunoreactivity of SCT-I  28  V  The Disappearance of Synthetic SCT in Rainbow Trout... 44  vi  LIST OF ILLUSTRATIONS  Figure  Page  1.  Reactions Involved in Radioimmunoassays  3  2.  Separation of Free and Bound Salmon Calcitonin  by Dextran-Coated Charcoal  6  3.  Radioimmunoassay Schedule for Salmon Calcitonin..  17  4.  Method for the Calculation of Confidence Limits  19  5.  Typical Autoradiograph of an Anion - Exchange Chromatogram.  22  6.  An Autoradiograph of Chromatographed SCT-I Before and After Purification by Method A  23  Purification Method A: Elution Profile of the Cellulose Column  24  125  7.  125 8.  An Autoradiograph of Chromatographed SCT-I Before and After Purification by Method B  25 125  9.  10.  An Autoradiograph of Chromatographed SCT-I Stored for 2 Weeks: Before and After Treatment With Dowex 2-X8  27  125 Adsorption of SCT-I Onto Tubes During Incubation  30  vii  Figure  11.  Page  Concentration of Coated Charcoal Suspension Versus Counts i n the Bound Fraction.  31  125 Binding and Damage of SCT-I as a Function of the Time of Incubation. .  33  13.  125 Titres of SCT-I Antisera  34  14.  Standard Curves for Synthetic SCT.  36  15.  Regression Lines with 95% Confidence Limits for Standard Curves of Figure 14  37  16.  Immunoreactivity of Partially Purified SCT  39  17.  Standard Curve and Regression Line for the Disappearance of Synthetic SCT i n Rainbow Trout  41  18.  Disapp earance of Synthetic SCT i n Rainbow Trout  42  19. .  Disappearance of Synthetic SCT: Log % Zero Time Concentration Versus Time  43  12.  viii  ABBREVIATIONS AND SYMBOLS  Ab  - antibody  Ag  -  As  - antiserum  Agt  angiotensin II  2  Agt2~I  125  c.p.m.  antigen  125 - - iodine -  labelled angiotensin II  counts per minute -2  g  - 9.8m. sec  MCi  - millicurie  M.W.  - molecular weight  NRP  - normal rabbit plasma  SCT  - salmon calcitonin  [SCT] 125 SCT-I  - concentration of salmon calcitonin 125 - iodine labelled salmon calcitonin  TCA  -  A  - index of precision  Sy.x  - standard deviation for the regression line y on x  %B  - percentage binding  trichloroacetic acid  Weight arid Volume _3  mg  - milligram (10 gm)  ug  pg  - microgram (10 gm)' -9 - nanogram (10 gm) -12 - picogram (10 gm)  ul  - microlitre (10 1)  ng  —6  -3  ix •  INTRODUCTION  The  h i s t o r y o f radioimmunoassay began i n 1955 when Berson et dl  were s t u d y i n g  the metabolic  fate of  131  I labelled insulin. After  131 intravenous  administration of i n s u l i n -  I i t was found t h a t t h e  TCA p r e c i p i t a b l e r a d i o a c t i v i t y i n t h e plasma o f s e v e r a l p a t i e n t s w h i c h had r e c e i v e d i n s u l i n t h e r a p y , was composed o f two f r a c t i o n s , p a r t t h a t c o u l d and p a r t t h a t c o u l d n o t be s a l t e d o u t w i t h Sodium Sulphate.  Zone e l e c t r o p h o r e s i s o f t h e s e plasma samples r e v e a l e d two  peaks o f r a d i o a c t i v i t y , one a t t h e o r i g i n r e p r e s e n t i n g  free labelled  i n s u l i n and one m i g r a t i n g w i t h t h e gamma-globulins. P r e s o a k i n g o f the s t r i p s w i t h u n l a b e l l e d s o l u t i o n s of i n s u l i n decreased the r e l a t i v e amount o f r a d i o a c t i v i t y i n . t h e gamma-globulin f r a c t i o n . The  r e l a t i v e l y common o c c u r r e n c e o f i n s u l i n a n t i b o d i e s i n t h e plasma  of i n s u l i n t r e a t e d p e o p l e d i s p r o v e d  a p r e v a i l i n g hypothesis  that  i n s u l i n was r a r e l y a n t i g e n i c . B e r s o n and Yalow (Berson et dl. , 1956; Yalow and B e r s o n , 1960) l a t e r r e p o r t e d  t h e development o f a  radioimmunoassay method f o r t h e measurement o f i n s u l i n i n body f l u i d s .  concentrations  T h i s e a r l y work began a new e r a i n e n d o c r i n o l o g y ,  a l l o w i n g t h e a c c u r a t e measurement o f p h y s i o l o g i c a l l e v e l s o f many polypeptide The  hormones. p r i n c i p l e o f radioimmunoassay i s based on t h e a b i l i t y o f a  l a b e l l e d a n t i g e n t o compete e q u a l l y - w i t h u n l a b e l l e d a n t i g e n f o r t h e s p e c i f i c antibody  combining s i t e s . The k i n e t i c s i n v o l v e d i n a n t i g e n - a n t i b o d y 1  reactions  2  are governed by the Law of Mass Action. Berson and Yalow (1964) have applied this basic chemical concept to a theoretical analysis of the radioimmunoassay system. The reactions involved in a radioimmunoassay are outlined in Figure 1. The concentration .of labelled antigen is constant in a l l incubation mixtures, only the concen tration of unlabelled antigen varies. The ratio of bound to free labelled hormone (B/F) decreases as the concentration of unlabelled hormone increases. A standard curve is constructed each time an assay is performed by incubating known amounts of unlabelled antigen. A basic requirement for the radioimmunoassay is that unknown and standard hormone have the same affinity for the antibody. A l l radioimmunological methods basically differ only in the method of separation of the free antigen and the antigen antibody complex. These methods may be classified into 3 groups.  Group A - Differential Migration of Bound and Free Antigen. 1. Chromatography 2. Electrophoresis 3. Gel Filtration 4.  Chromatoelectrophoresis  Group B - Precipitation of Bound Antigen. 1. Double Antibody 2. Salt Precipitation 3. Solvent Fractionation 4. Binding to a Solid Phase  REACTIONS INVOLVED IN RADIOIMMUNOASSAYS  Labelled Antigen  Ag  Labelled Antigen Antibody Complex  Antibody  +  Ab  Ag  -  Ab  + Ag  Ag — Ab  Unlabelled  Antigen  Unlabelled Antigen - Antibody Complex  FIGURE 1  4 Group C - Adsorption of Free Antigen. 1. Ion Exchange 2. Charcoal 3. S i l i c a 4. Talc  Excellent reviews of these various methods are available.(Felber and Aubert, 1969; Raiti and Davis, 1969). The methods outlined in group A are technically involved and not applicable to large assays, although they are considered to be the most accurate. The'techniques utilizing the precipitation of bound antigen do not give complete separation of antibody - bound and free antigen. The double antibody system has the additional disadvantage of requiring a second antibody directed against the gamma globulin. Assays utilizing adsorbants are simple to perform, relatively inexpensive, quite reproducible and applicable to a large number of samples. The coated charcoal method (Herbert et at., 1965) for adsorption of free tracer was the method chosen in this thesis. Charcoal alone, w i l l adsorb molecules ranging widely in molecular size. However, i f the charcoal i s f i r s t coated with an agent such as dextran (M.W. 70,000) i t w i l l only adsorb molecules of smaller molecular weight than the dextran; hence a gamma globulin would be rejected. The suitable dextran is determined empirically. For example, Herbert (1969) found that charcoal coated with dextran of 40,000 molecular weight, rejected free insulin, whereas that coated with, dextran of 80,000 molecular weight adsorbed insulin.  5 T h i s d i s c r i m i n a t o r y a b i l i t y i s presumed t o be due t o t h e pore s i z e on t h e s u r f a c e o f each c h a r c o a l p a r t i c l e , t h e pores b e i n g c r e a t e d by t h e l a t t i c e w o r k o f adsorbed  c o a t i n g m o l e c u l e s . Thus, a f t e r a  p e r i o d o f i n c u b a t i o n , t h e f r e e a n t i g e n may be s e p a r a t e d from t h e s o l u t i o n by a d s o r p t i o n onto t h e c o a t e d c h a r c o a l . T h i s i s shown d i a g r a m a t i c a l l y i n F i g u r e 2. The r a t i o o f r a d i o a c t i v i t y i n t h e charcoal (free antigen) t o r a d i o a c t i v i t y i n the supernatant (bound a n t i g e n ) v a r i e s w i t h c o n c e n t r a t i o n o f u n l a b e l l e d a n t i g e n . T h i s system was f i r s t a p p l i e d t o t h e assay o f i n t r i n s i c ( H e r b e r t et a l . , 1964) and has s u b s e q u e n t l y  factor  found a p p l i c a t i o n i n  the radioimmunoassay o f many p o l y p e p t i d e hormones such as i n s u l i n , ( H e r b e r t et at., 1965) growth hormone,(Lau, G o t t l i e b and H e r b e r t , 1966) and angiotensin.(Waxman, G o p d f r i e n d and H e r b e r t , 1967). The s e n s i t i v i t y o f t h e radioimmunoassay i s g r e a t l y dependent on t h e i m m u n o r e a c t i v i t y and s p e c i f i c a c t i v i t y o f t h e t r a c e r . The amount o f t r a c e r p r e s e n t i n t h e i n c u b a t i o n s h o u l d be l e s s than t h e minimum c o n c e n t r a t i o n o f hormone t o be measured. I f t h i s i s n o t t h e c a s e , s m a l l changes i n t h e c o n c e n t r a t i o n o f t h e u n l a b e l l e d a n t i g e n w i l l n o t be d e t e c t e d . Damage t o t h e t r a c e r d u r i n g i n c u b a t i o n r e s u l t s i n a l o s s o f s e n s i t i v i t y . The s e n s i t i v i t y i s a l s o determined  by t h e  a f f i n i t y o f t h e a n t i b o d y f o r t h e a n t i g e n . A n t i s e r a from d i f f e r e n t a n i m a l s o f t h e same s p e c i e s may v a r y g r e a t l y i n t h e i r for  sensitivity  d e t e c t i o n o f t h e a n t i g e n (Yalow and B e r s o n , 1968). In p r i n c i p l e t h e s p e c i f i c i t y o f t h e radioimmunoassay i s  dependent upon t h e homogeneity o f t h e p r o t e i n w h i c h i s l a b e l l e d and a n t i b o d y s p e c i f i t y (Hunter, 1969a:);. As r e l a t i v e l y crude m a t e r i a l i s 7  o f t e n used f o r i m m u n i z a t i o n ,  the antiserum obtained i s q u i t e  FIGURE 2  heterogeneous. Therefore, unless a pure or almost pure preparation of the antigen is used for labelling, several radioactive species w i l l be obtained, some or a l l of which may react i n the system giving a false value. Production of an antibody highly specific for the antigen to be measured is quite desirable since cross reactivities with other proteins in the incubation medium may give a falsely high result.Fortunately, cross-reactivities, i f they do exist, are usually much weaker than the primary reaction and do not influence the results significantly. According to Hunter (1969a),other factors (extremes of pH and ionic strength) may affect specificity by influencing the rate of the antigenantibody reaction as well as the system to separate the bound and free hormone. This i s especially apparent when assaying physiological fluids such as urine. 13X The antigen can be labelled with I  X23 or I  by a subs-  titution of an iodide atom for a hydrogen atom on the benzene 125 ring of tyrosine. Most investigators prefer I because i t has 131 a much longer half l i f e than I (57 days as compared to 8 days). 125 The counting efficiency is much better for I and the isotopic 125 131 abundance is greater on arrival (I  - 99%, I  - as low as 20%).  (Freedlender, 196,9) . The principles discussed above formed the basis for the development of a radioimmunoassay for salmon calcitonin which i s found i n the ultimobranchial gland of salmon (Copp and Parkes, 1968). Calcitonin is a hypocalcemic polypeptide discovered by Copp et al in 1962.  8 Radioimmunoassays for other calcitonins have been developed. Several reports have been published on the radioimmunoassay of porcine calcitonin (Arnaud et at., 1968; Arnaud, Tsao and Littledike, 1970; Deftos, Lee and Potts, 1968; Lee, Deftos and Potts, 1969; Lequin, Hackeng and Schopman, 1969; Tashjian, 1969). The charcoal dextran method, as well as chromatoelectrophoresis have been used in the radioimmunoassay of porcine calcitonin. Deftos et aZ-.,(1968) and Lequin et at.,(1969) have achieved assays of sufficient sensitivity to detect basal endogenous levels of calcitonin i n rabbits and pigs respectively. Tashjian (1969) was not so fortunate. A l l investigators have reported attaining high titre antisera - with frequent and prolonged immunization. In a l l cases the antisera > were highly specific for calcitonin. The human hormone has only recently been synthesized, therefore there has been no substantial progress in the direction of radioimmunoassay. Nevertheless, an assay for human calcitonin has been reported (Clark et at., 1969) in which calcitonin extracted from the thyroid tissue of patients with medullary carcinoma was used for immunization. The assay could detect calcitonin in patients with medullary carcinoma but not i n normal subjects. The intention of the work presented was to develop basic techniques for the radioimmunoassay of SCT, with the hope that i t could eventually be used on a routine basis.  * Since the preparation of this thesis, Tashjian has reported the development of a sensitive radioimmunoassay for human calcitonin. (Annual Meeting of the Endocrine Society, St. Louis, June 1970).  /  MATERIALS AND METHODS  Immunization Partally purified salmon calcitonin (Armour Pharmaceutical Co.) approximately 5% pure and 300 MRC units/mg was coupled to hemocyanin, the copper-containing respiratory pigment of keyhole limpets. Hemocyanin is a large molecule (M.W.- 1 X 10 ) of high antigenicity. The large 6  protein complex induces the production of heterogeneous antibodies, some of which are directed against the calcitonin molecule. The method of Goodfriend, Levine and Fasman (1964) was used; L-ethyl-3-(3-dimethylaminopropyl) carbodiimide was the coupling agent. The coupled material was then purified by gel f i l t r a t i o n on a 1.5 X 30 cm Sephadex G-10 column in saline. The fractions containing the coupled protein were combined and emulsified with an equal volume of Freund's  complete  adjuvant. This material was injected into 3 rabbits and 3 guinea pigs, intradermally on the dorsum and into the foot pads, and intramuscularly into the hamstrings. Each animal received about 15 mg of the crude hormone preparation. At the end of 8 weeks, serum was harvested from each animal, the rabbits by ear vein bleeding and the guinea pigs by heart puncture. The sera were later tested for the presence of salmon calcitonin antibodies by their ability to bind 125 SCT-I  . Work was begun using the antiserum with the highest titre  which was from a rabbit. Eight months later, this animal was given a booster similarly coupled to the larger protein. Emulsification was done in mineral o i l . The serum was harvested 4 weeks later and used in subsequent studies. A final booster in Freund's  complete  adjuvant was given 3 months after the f i r s t and the serum was 9  collected  2 weeks after the injection. This antiserum was used  in the final stages of the experimental work. Both boosters were given intramuscularly. The f i r s t and second boosters contained about 8 mg and 4 mg respectively of the crude hormone preparation. Serums were stored at -15°C and sodium azide was added as a preservative, at a concentration of 0.1%. A stock solution of the antiserum currently i n use was prepared by diluting 1:10 in 0.01 M phosphate buffer pH 7.5 and dispensed into vials, each sufficient for one assay.  Radioiodination and Purification of Labelled Salmon Calcitonin Synthetic salmon calcitonin prepared byt-Sandoz . was _used_ for iodination by the chloramine-T method of Greenwood and Hunter (1963) with modifications (Yalow and Berson, 1969). The procedure was as follows: to a small test tube were added in sequence with microlitre Drummond pipettes, the following reagents: 1.  20 y l of 0.4 M phosphate buffer pH 7.5 125  2.  approximately 2 MCi. Nal  (New England Nuclear)  contained in 10-20 y l of solution 3.  2.5 yg synthetic salmon calcitonin in 10 y l (stock solution stored at -15°C, contained 250 yg/ml in 0.05 M acetic acid)  4.  65 yg chloramine-T (oxidant) in 10 y l 0.25 M phosphate buffer pH 7.5  5.  130 yg sodium metabisulfite (reductant) in 20 y l 0.25 M phosphate buffer pH 7.5  "  Gentle bubbling insured adequate mixing. The procedure was carried out as quickly as possible to prevent unnecessary damage of the hormone from exposure to oxidizing and reducing agents. The iodinated tracer was then purified by one of two methods methods A (Yalow and Berson, 1960) and B (Yalow and Berson, 1966). Method A was used in preliminary studies only. Method B was used for a l l results reported in this thesis. Method A Fifty y l of normal rabbit plasma was added to the iodination mixture and i t was applied to a small cellulose column (Whatman fibrous cellulose powder CF11) approximately 1 ml i n volume and washed with 0.05 M veronal buffer. Fractions of 0.5 ml were c o l l ected and counted with a Nuclear Chicago automatic scintillation well-type counter with background subtraction. The-column was washed until radioactivity in the fractions f e l l substantially and approached zero. Damaged components are adsorbed to plasma proteins. Free iodide passes through the column and the labelled product is adsorbed to the cellulose. Elution of the labelled hormone was then begun with normal serum or plasma. Fractions of 0.5 ml were collected into tubes containing the diluent. These fractions were then counted and the tubes in the peak of radioo  activity were retained, quick frozen and stored at -15 C. The remainder of the fractions were discarded. Method B One ml of normal rabbit plasma was added to the iodination vessel to elute any adsorbed hormone from its'" walls. The contents were transferred to a test tube containing 5 mg of Quso?G32 micro-  fine precipitated s i l i c a (Philadelphia Quartz Co.) and shaken on a vortex mixer. The suspension was then centrifuged at about 800 g for 10 minutes. The supernatant containing damaged components (adsorbed to the plasma proteins) and free iodide was discarded. Three ml of water* were added, the pellet was dispersed by mixing on a vortex mixer and centrifuged again. This supernatant was discarded and the pellet was resuspended in 6.5 ml of a solution of 40% acetone  and 1% acetic acid. This solution elutes the labelled  salmon calcitonin from the s i l i c a . Water (1.5 ml) was added to the tube and the sample was centrifuged. The supernatant was decanted and diluted 1:25 in 0.01 M phosphate buffer pH 7.5 containing 0.5 gm% albumin. Finally aliquots were quick frozen and stored at o  -15 C. Dowex anion exchange resin 2-X8 (200 - 400 mesh, 250 mg/ml) was suspended within the labelled hormone solution immediately before use in an assay. This resin removed free iodide which had accumulated during storage. The sample was centrifuged, the supernatant withdrawn and suitably diluted. Each batch of tracer was used for 1 - 2 weeks. Chromatography Descending chromatography was carried out on anion-exchange resin-impregnated paper (WB-2 Reeve Angel) as a check on the effectiveness of the purification methods. The solvent system used was pyridine-acetic acid-water-ethanol (65:57:278:400) (Goodfriend, Ball and Farley, 1968). Aliquots were applied to the origin and the  *A11 water used was distilled and deioriized.  solvent allowed to flow for about 2 hours. The chromatogram was dried and radioactive spots were located by placing i t in close contact with X-ray film in a dark room for 2 hours. The autoradiograph was retained as a permanent record.  Assay Procedure Tubes, prior to use in an assay were coated with albumin (bovine albumin, Armour Fraction V). This was accomplished by allowing the tubes to stand overnight at 4°C, f i l l e d with a 2.5 gm% solution of albumin. The tubes were air dried at room temperature, and then stored at 4°C. This albumin coating prevented adsorptive losses of the SCT-I  12  The coating solution was re-used several times. Samples were set up in triplicate, except unknowns, usually in duplicate. The assay procedure (as summarized in Table I and Figure 3) was performed as follows: 1.  Diluent (3.4 ml of 0.01 M phosphate buffer pH 7.5, 0.35 gm% bovine albumin) was added to 14 X 150 mm disposable glass test tubes with a 10 ml repipette. Buffer was prepared fresh for each assay from a stock solution of 0.1 M phosphate buffer, stored at 4°C.  2.  One half ml of the desired dilution of antiserum ( d i l uted to the point such that 30 - 50% of the radioactivity was bound in the absence of unlabelled hormone) was then added with a 500 y l Hamilton syringe.  3.  Standards (stock solution stored at -15°C at a concentration of 10 yg/ml in diluent) or unknowns, contained i n 50 y l , were then included with a disposable Drummond pipette. If greater sensitivity was required, a larger volume of  unknown such as 0.5 ml was added. To compensate for this increased protein concentration, a similar amount of normal rabbit plasma or hormone-free plasma was added to a l l control and standard tubes. In addition, the volume of buffer was reduced an equal amount. 4. Control tubes and "total counts" tubes were set up for the calculation of B/F values. The control tubes contained normal non-immune serum instead of antiserum and estimated damage tracer and free iodide which remains in the supernatant. The 125 "total counts" tubes contained SCT-I in buffer only. The 6 ml volume of these tubes (5.95 ml buffer + 0.05 ml 125 SCT-I solution) equalled the final volume of the assay tubes. 5.  Tubes which contained buffer (3.45 ml) antiserum (0.5 ml) and 125 SCT-I  (50 yl) measured maximal binding ( i.e. no competing  unlabelled SCT present). 6. The tubes were then preincubated for 24 hours at 4°C. 7.  After preincubation, 50 y l of tracer solution containing 5 - 10,000 c.p.m. was pipetted into the tubes.  8. The solutions were incubated for 24 hours at 4°C. Separation of Bound and Free Tracer 1.  a 0.5 gm% solution of Dextran T-70 (Pharmacia) and a 1.0 gm% suspension of Norit A neutral charcoal ( Fisher Scientific) made up in the buffer of the assay were combined in the ratio of 1:1.  2.  Two ml of this mixture was added to each tube (Excluding the "total counts" tubes). This mixture was conr stantly stirred on a magnetic mixer to prevent settling  15 of the 3.  suspension.  E a c h t u b e was c a p p e d a n d m i x e d b y i n v e r s i o n f o r 10 s e c o n d s . 10  4.  The t u b e s w e r e c e n t r i f u g e d a t 1 0 0 0  about g  for  minutes.  Supernatant tubes  aliquots  o f 3 m l were p l a c e d i n  counting  and c o u n t e d f o r enough t i m e to a c c u m u l a t e a t  10,000 c o u n t s .  This  reduced the counting e r r o r to  R a d i o a c t i v i t y was s u f f i c i e n t l y h i g h ume t o e n s u r e  i n the 3 ml  least 1%.  vol-  s a t i s f a c t o r y c o u n t i n g a c c u r a c y . When  only a portion of  t h e s u p e r n a t a n t was  counted,  the  d a n g e r o f d i s t u r b i n g t h e c h a r c o a l p e l l e t was m i n i m a l . Counting of basis of  t h e p e l l e t was n o t a t t e m p t e d o n a  s i n c e i t was e x t r e m e l y d i f f i c u l t  Standard  i n assays  (e.g.  12.5%)  containing a significant  Each of  10 mg/ml o f  aorta.  B l o o d samples  duplicate.  granules  c a l c i t o n i n through, a cannula secured i n the were t a k e n 5,  50 y l s a m p l e s  at  a  Calcitonin  3 rainbow "trout were i n j e c t e d w i t h 5 yg of  salmon  concen-  plasma.  o f S y n t h e t i c Salmon  thetic  injection.  charcoal.  c o n t a i n e d p l a s m a made " c a l c i t o n i n -  f r e e " by e x t r a c t i o n t w i c e w i t h m i c r o f i n e s i l i c a concentration of  the  all  Plasma  curves  t r a t i o n of plasma  Disappearance  to decant  t h e s u p e r n a t a n t w i t h o u t r e m o v i n g some o f  "Calcitonin-Free"  routine  2 0 , 40 a n d 60 m i n u t e s  of d i l u t e d plasma  (1:6)  syndorsal after  were assayed  in  Table 1 Protocol for Radioimmunoassay  Sample  Buffer (ml)  Antiserum Dilution (ml)  Unknown or Standard  3.4  0.5  50  Total Bound Counts  3.45  0.5  '— •  Control  3.4  Total Counts  5.95  0.5 of normal non-immune serum. Dilution similar to As.  —  SCT-I (yl)  Standard or Unknown Amount SCT(yl)-  -Charcoal -Dextran (miy  125  Preincubate at 4°C for 24 hours  50  :  Incubate at 4°C for 24 hours  2  50  2  50  50  2  —  50  —  Mix and centrifuge at 1000 g for 10 minutes. Decant 3 ml of supernatant in--'. to counting tubes and count radioactivity.  t-  1  RADIOIMMUNOASSAY SCHEDULE FOR SALMON CALCITONIN  Charcoal-Dextrqn Suspension  SCT ' Ab + SCT  Ab +  Ab  SCT  SCT  1L  i.  %  Ab-SCT Ab-SCT  Ab-SCT  24 hr. Pre-lncubation  24 hr. Incubation  / Centrifuqafion »-  Dispersion of Charcoal in Solution  Aliquot for Counting 3 cc  SCT - Ab S C T I ' " - Ab  Discard  Free SCT +• SCT in Charcoal Pellet x  FIGURE 3  Counting Tube with Sample  C a l c u l a t i o n s and S t a t i s t i c s  a)  Bound t o F r e e R a t i o o f R a d i o a c t i v i t y (B/F) bound counts f r e e counts  b)  Percentage  c.p.m. - av. c o n t r o l counts av. t o t a l counts - c.p.m.  Binding  % binding  c)  _  bound counts av. t o t a l counts - av. c o n t r o l  L i n e a r R e g r e s s i o n and C o n f i d e n c e  counts  Limits  L i n e a r r e g r e s s i o n l i n e s f o r y (%B) on x ( l o g [SCT]) were  calc-  u l a t e d f o r s t a n d a r d curves (Snedecor, 1956). The form o f t h e equations f o r the l i n e s are %B = a + b l o g [SCT] where a = y i n t e r c e p t b = slope The 95% c o n f i d e n c e l i m i t s were c a l c u l a t e d f o r t h e r e g r e s s i o n lines  (Snedecor, 1956) . The 95% c o n f i d e n c e l i m i t s f o r a p r e -  d i c t e d [SCT] (from t h e s t a n d a r d c u r v e ) were c a l c u l a t e d by d e t e r m i n i n g t h e x v a l u e s ( l o g [SCT]) on t h e r e g r e s s i o n l i n e f o r t h e 95% c o n f i d e n c e l i m i t v a l u e s o f y (%B)*. T h i s i s shown g r a p h i c a l l y i n F i g u r e 4. I n t h i s i l l u s t r a t i o n , t h e lower and upper c o n f i d e n c e limits for X  q  a r e "-xj and-xg r e s p e c t i v e l y .  The i n d e x o f p r e c i s i o n (A) was c a l c u l a t e d f o r each r e g r e s s i o n line. ^  _ standard d e v i a t i o n slope  The s m a l l e r t h e v a l u e o f X t h e g r e a t e r i s t h e i n h e r e n t p r e c i s i o n of t h e assay method ( B l i s s ,  1952).  * Dr. P. L a r k i n , Department of Z o o l o g y , U n i v . o f B.C. s u p p o r t e d t h i s method f o r c o n f i d e n c e l i m i t c a l c u l a t i o n s ( p e r s o n a l comm.).  X  Q  =  predicted log [SCT]  YQ  =  value of % B on regression line corresponding to XQ  Y^  =  lower confidence limit at XQ  Y2  =  upper confidence limit at XQ  X  =  value of log [SCT] on the regression line at Y confidence limit for XQ  =  value of log [SCT] on the regression line at Y ^ , upper confidence limit for X „  1  2 >  lower  • .20 RESULTS  Assessment of Labelled Salmon Calcitonin SCT was always successfully labelled, however the degree of labelling varied with the shipment of isotope. It was similar for a l l iodinations using a single shipment. (Table II) a)  Purification Methods A and B were evaluated by the behaviour of the  labelled material chromatographically.  A typical autoradiograph  of an anion exchange chromatograph is shown in Figure 5. Three 125 components are visible, I  at the origin since i t is an anion,  125 SCT-I  at the solvent front and damaged material about midway  between the origin and solvent front (Goodfriend, 1969). Figure 6 shows the results of a chromatogram before and after purification by Method A. Fraction 24 (Figure 7) was chromatographed. The same sequence is demonstrated in Figure 8 but s i l i c a (Method B) was used to purify the tracer. In both cases the amounts of iodide, damaged, components and contaminants were greatly reduced. Tracer purified by Method B gave a distinct migration spot (Figure 8 i i ) and the relative amount of radioactivity at the origin compared to the solvent front was small. This was not the case for the tracer purified by Method A (Figure 6 i i ) . It is reasonable to assume that Method B results in removal of almost a l l damaged components and free iodide. Purification Method B  was chosen for the major part of the  experimental work because of i t s simplicity and because i t yielded a better labelled product as shown chromatographically.  Comparative  TABLE II Labelling Variation and Effect of Dowex on the Stored Products  Isotope Lot  Date of Labelling  June 8  June 17  5648  June 29  5888  July 15  4452  July 20  4550  Aug. 5  14206  July 13  Aug. 4  Total Counts in Assay within 0-1 days of labelling  Total Counts i n Assay After ReTreatment with Dowex  No. of Days After Labelling When Dowex Treated  4058 4230 5176  6 21 9  4142  2  AN AUTORADIOGRAPH OF CHROMATOGRAPHED SCT-I  BEFORE AND AFTER PURIFICATION BY METHOD A  FIGURE' 6  125  1 B e f o r e p u r i f i c a t i o n o f SCT-I  125 i i A f t e r p u r i f i c a t i o n o f SCT-I  S3  PURIFICATION METHOD A : ELUTION PROFILE OF THE CELLULOSE COLUMN  2.4  2.0  <o O  1.6  H  O  H  I  G  x E  O-O / I / * I I I  \  2H  \  o  o  CL  d 0.8  H  0.4  H  I  T  1  6  1  1  1  12  -  i  Fraction No.  r  o-o-o 18  fT  r  T  24  *— Beginning of Elution  FIGURE 7  -i 28  1 OS  .  AN AUTORADIOGRAPH OF CHROMATOGRAPHED S C T - I a__ . . _ . METHOD B  BEFORE AND AFTER P U R I F I C A T I O N  BY  V  0 R I G I N  N  N 11  i i i  FIGURE 8  125 i i i i i i  Before p u r i f i c a t i o n of SCT-I A f t e r p u r i f i c a t i o n of S C T - I Re-purification  1 2 5  NJ  125 assays suggested b e t t e r I m m u n o r e a c t i v i t y w i t h SCT-I p u r i f i e d by Method B. Two such a s s a y s were s i m i l a r i n a l l r e s p e c t s except t h a t the t r a c e r was  p u r i f i e d by Method A i n the one'and by Method B i n  the o t h e r . The maximal B/F*.tatios were 0.21  and 0.51  respectively. 125  An attempt was p r o d u c t o f method B was  made to f u r t h e r p u r i f y the SCT-I  . The  f r e e z e - d r i e d , e l u t e d from the g l a s s w i t h  plasma and r e - p u r i f i e d . The  r e l a t i v e amount o f r a d i o a c t i v i t y a t  the o r i g i n as compared t o t h a t at the s o l v e n t f r o n t d i d n o t seem to be reduced ( F i g u r e b)  S t o r a g e of  8 iii)•  Tracer  I t was  c o n s i s t e n t l y found t h a t treatment w i t h Dowex 2-X8 125 a n i o n exchange r e s i n of SCT-I s t o r e d f o r more than 1 or 2 d a y s , r e s u l t e d i n a r e d u c t i o n o f counts f o r a c o n s t a n t a l i q u o t (Table I I ) . 125 F i g u r e 9> i ) i s an a u t o r a d i o g r a p h o f chromatographed SCT-I a  2  week s t o r a g e  the o r i g i n was  p e r i o d . The  after labelling 1 2 5  l a r g e a c c u m u l a t i o n o f i o d i d e seen a t  removed a f t e r t r e a t m e n t w i t h Dowex ( F i g u r e 9 i i ) . T h i s  d e c r e a s e i n counts was  SCT-I  after  not marked i n samples t e s t e d 1 o r 2 days  (Table I I I ) i n d i c a t i n g l i t t l e o r no a d s o r p t i o n  of  . Immunoreactivities  reduced a f t e r  2  of a l l p r e p a r a t i o n s  were s u f f i c i e n t l y  weeks t o n e c e s s i t a t e f r e q u e n t  labelling.  Several  p r o d u c t s o n l y gave s a t i s f a c t o r y r e s u l t s w i t h i n the f i r s t week o f use. The  maximal B/F  v a l u e would d e c l i n e s u b s t a n t i a l l y . S e n s i t i v i t y  would a l s o be d e c r e a s e d , o f t e n t o - s u c h an e x t e n t i n b i n d i n g were no l o n g e r d e t e c t a b l e  a t hormone  t h a t the  differences  concentrations 125  r o u t i n e l y used i n the a s s a y . T a b l e IV i l l u s t r a t e d t h i s p o i n t . SCT-I Al a b e l l e d  on May 12th gave i n i t a l B/F r a t i o s of 0.77 and 0.48 as A l l s t a t e d B/F v a l u e s r e p r e s e n t the average of t r i p l i c a t e samples.  125 AN AUTORADIOGRAPH OF CHROMATOGRAPHED SCT-I STORED FOR 2 WEEKS: BEFORE AND AFTER TREATMENT WITH DOWEX 2-X8  0  R I G I N  ii  FIGURE 9 i ii  B e f o r e Dowex treatment A f t e r Dowex treatment  ho  TABLE III Effect of Dowex 2-X8 on Freshly Prepared SCT-I 125 c.p.m. of SCT-I Before Dowex Treatment  125 c.p.m. of SCT-I After Dowex Treatment  6500 - June 9  6460  6765 - July 20  6640  TABLE.-IV, Effect of Storage on Immunoreactivity of SCT-I As Dilution  Unlabelled SCT  1:400 1:400 1:800 1:800 1:800 1:800  •  Date of Assay  B/F  May 13  0.77  -  May 26  0.52  -  May 13  0.48  May 13  0.27  May 26  0.29  May 26  0.19  1 ng/ml -  1 ng/ml  maximum values at 1:400  and 1:800  dilutions of antiserum respectively.  The same preparation used in an assay under similar conditions 13 days later, gave values of 0.52 and 0.29 respectively. The sensitivity of the assay was also reduced as testified by the diminished response to the inclusion of competing unlabelled SCT in the incubation medium  Assay Conditions A) Preparation of Incubation Tubes An experiment was designed to determine conditions which minim125 ized adsorption of SCT-I  onto incubation tubes. Tubes containing  125 buffer and SCT-I only, were incubated for 24 hours. Figure 10 shows the percentage of counts remaining in the buffer after incubation. Both varieties of soft glass tubes, when uncoated, adsorbed about 20% 125 of the SCT-I  , whereas plastic tubes adsorbed much more (50%).  Albumin (1 gm% solution) was considerably more effective than gelatin (1 gm% solution) as a coating agent. Unexpectedly, plastic albumin125 coated tubes adsorbed the least amount of SCT-I  . However, since  plastic tubes broke easily during centrifugation and assay results with them were more variable, these tubes were not used routinely. Other preparations of the glass surfaces included siliconizing and acid rinsing, both of which increased adsorption. Albumin-coated disposable soft glass tubes were used regularly. B) Charcoal Concentration The optimal charcoal concentration to separate bound and free tracer was found to be 2 ml of a 0.5 gm% coated charcoal suspension as shown in Figure 11. Also shown is a range of + 0.25 over which binding  is effected very l i t t l e . Outside this range,  gm%  30  ADSORPTION OF SCT-I  Uncoated  ONTO TUBES DURING INCUBATION  125  Albumin coated  *  ¥ 89.6  Gelatin coated  98.7 86.6  80.3 78.6  80.7  100 O  80  g  3  h 60  • 52.5  a> 3 o 40 5(O 20 0  Siliconized  57.7  64.1  Siliconized ^Albumin coated  Acid rinsed O.I N HCL  77.1 71.0  Acid rinsed + Albumin coated B5.I 70.2  r 100 O  80  V7.  W  mw 471  Disposable Glass Tubes  60 -,  52.2  Soft Glass Tubes  I  g  3  3 40 20 0 Plastic Tubes  FIGURE 10  Q  5'  3  CONCENTRATION OF COATED CHARCOAL SUSPENSION VERSUS COUNTS. IN THE BOUND FRACTION  8 0 0  -i -o—  6 0 0 -  —o.  / / /  E  CL 6  4 0 0 -  c ZD  o  m  2 0 0 -  1  0.25  ©  © 12.5% N.R.R  •  •  Buffer  1  1  0 . 5 0 . 7 5  gm % Coated Charcoal Suspension  FIGURE 11  -r— 1.0  binding drops off considerably. When 12.5% plasma is included in the incubation medium, the optimal charcoal concentration is 0.75  gm%  with a range of - 0.5 gm% over which the number of counts bound is relatively constant. C) Incubation Time It is evident from the data presented in Figure 12a that the amount of binding is directly proportional to the time of incubation, the increase being most marked in the f i r s t 24 hours of incubation. Incubation damage, estimated as the percentage of total counts in the buffer control tubes, increased linearly in this same experiment (Figure 12b). Extrapolation of the line to zero time gave an estimate of the damaged tracer i n i t i a l l y .present, which:was 14% in this experiment.  Assessment of Antisera A l l antisera tested demonstrated the presence of antibodies to SCT, However, the titres were variable (Figure 13). Rabbit(R-2) had the best t i t r e . The titres obtained after the second and third booster injections are shown, indicating  the improvement of titre  after the second booster.  Specificity of Antiserum R-2 The antiserum R-2 was tested for specificity by two means: 125 a) ability to bind angiotensin-I  , and b) ability of unlabelled  angiotensin II at high concentrations to inhibit the binding of 125 125 SCT-I . In test (a), Agtjl was shown to be immunoreactive since 45% of the counts added to incubation tubes containing antiserum directed against Agt„ were bound. Tubes containing a  125 BINDING AND DAMAGE OF SCT-I AS A FUNCTION OF THE TIME OF INCUBATION  0  24  Hours  48  of Incubation  FIGURE 12 (A) Binding of SCT-I (B) Damage of SCT-I  1  TITRES OF SCT-I  ANTISERA  FIGURE 13  35 125 Similar dilution of antiserum R-2, bound 42% of the added SCT-I but 125 not any of the added Agt-I  . In test (b) the maximal B/F value in a  SCT assay was 1.6. Tubes with 12 ng/ml of Agt^ did not inhibit the binding (B/F = 1.7), whereas tubes containing only 1.25 ng/ml greatly inhibited 125 the binding of SCT-I  (B/F = 0.8). In addition, binding was not  inhibited in assays carried out in 12.5% plasma. These observations show that the calcitonin antibodies do not bind and binding is not inhibited by non-specific polypeptides to any measureable extent. Standard Curves Standard curves obtained with three different dilutions of antiserum are shown in Figure 14. The B/F values are plotted against the concentration of synthetic SCT. The average B/F value only, i s 125 shown in the standard curves. Plotting the percentages of SCT-I bound against the logarithm of the concentration gives a straight line (Figure 15) except at the extreme ends of the concentration scale. The index of precision, standard deviation and the equation for each regression line are shown. The 95% confidence limits are indicated by a dotted line. As shown i n Figure 14 the standard curve flattens out at 1:1600 dilution of antiserum with the result that there i s a loss of accuracy (95% confidence limits for 300 pg/ml are 140 and 620 pg/ml). A  1:800  dilution instead of a 1:400 dilution of antiserum was chosen for subsequent assays to economize antiserum, since sensitivity and accuracy were comparable (95% confidence limits for 300 pg/ml were 220 and 400 pg/ml for 1:400 As, 215 and 420 pg/ml for 1:800 As). The standard curve was reproducible over an extended period  STANDARD CURVES FOR SYNTHETIC SCT  FIGURE 14  REGRESSION LINES WITH 95% CONFIDENCE LIMITS FOR STANDARD CURVES OF Figure 14  (B)  (A)  50  As  i \  Dilution n  =  0.055  =  2.169  Dilution  P 8 0 0  k =  U400  S y x  As"  Sy-x  30-1  = y=  y = 125.8-30.7 x  o m  20  o  IO  0.061 I  .448  70.1-23.6x  40-  H  3  CQ  30- i —  125  c 3  O O  250  500  Pg/ml  20-  (C)  10 -  As  Dilution  h = S y x  •o -i 125  250 P g / m l  500 Incubate  1 750  c  15-  CO  10-  3  5-  o  O  o  ,  1:1600 0.150  = I .384 y=  2 9 . 6 - 9 . 2 7  -fib  0 125  —I  250  I / ml  FIGURE 15  750  Incubate  1  500 Incubate  1  750  x  of time. The slope of the regression line for the standard curve in Figure 16 was not significantly different from that i n Figure 15b (p = 0.05, t = 1.25 for n-4 =18) even though the assays were separated by a period of 1 ^ months. 2  Sensitivity Sensitivity was defined as the smallest quantity of unlabelled hormone which could be distinguished from no hormone. This was found to be 50 - 60 pg/ml of incubate. In two separate comparable assays the decrease i n binding was 15% and 12% with 50 and 60 pg/ml of incubate respectively. However the semilogarithmic graph was not linear at this concentration. Approximately 100 pg/ml was the limit of linearity 'at this end of the regression line. Therefore the lowest concentration for which one could define 95% confidence limits was about 140 pg/ml. with 95% confidence limits of 100 and 190 pg/ml (Figure 17).  Immunoreactivity of Partally Purified SCT vs. Synthetic SCT The inhibitory curve for 5% pure SCT (same material as that used to immunize) i s shown i n Figure 16a. The slope of the regression line of the semilogarithmic plot (Figure 16b) was compared to that for synthetic SCT shown i n Figure 14b. The slopes were not significantly different (p = 0.05, t = 1.61 for n - 4 = 18) indicating that the natural and synthetic product are immunologically similar.  IMMUNOREACTIVITY OF PARTIALLY PURIFIED SCT c=—  Micro Units  . .. T '  —  partially  ,  purified  1  S C T / m l Incubate  FIGURE 16 (A) Inhibitory curve for 5% pure SCT (B) Regression line with 95% confidence limits  40  Disappearance of Synthetic SCT in Rainbow Trout The standard curve as well as the regression line of the semilogarithmic plot is shown i n Figure 17. A B/F value was calculated for each unknown. A concentration was then read from the standard curve. The average concentrations of duplicate samples were plotted against time in Figure 18. A zero time concentration for each fish was obtained by extrapolating the curve to zero. The values from the three fish were expressed as a percentage of the extrapolated zero time value and the logarithms of these percentages were plotted against time. This final treatment yielded 2 regression lines (Figure 19),J.Therefore at least two p.  components are involved i n the disappearance of synthetic SCT, Ta 1'2 = 12.5 minutes and T b ^ 1  1  = 59 minutes. The data i s summ-  arized in Table V. Detection Of Endogenous Calcitonin i n Rainbow Trout Plasma samples of rainbow trout inhibited the binding of 125 SCT-I  -), implying that trout calcitonin was competing 'for the  antibody binding sites. Since the stimulus for calcitonin release in mammals i s hypercalcemia, the same fish were placed in water containing 100 mg% Ca for 2 hours. Although plasma calciums rose ' 125 about 1 mg%, inhibition of binding of SCT-I by plasma samples from these fish was not greater. An accurate estimate of the circulating endogenous level of trout calcitonin was not attempted.  STANDARD CURVE AND REGRESSION LINE FOR THE DISAPPEARANCE OF SYNTHETIC SCT IN RAINBOW TROUT  As 1:800  R - 2 July 13/70  FIGURE 17  = 0.055 S y x = 1.40 y = 87.3 -25.1 x  DISAPPEARANCE OF SYNTHETIC SCT IN RAINBOW TROUT  FIGURE 18  DISAPPEARANCE OF SYNTHETIC SCT: LOG % ZERO TIME CONCENTRATION VERSUS TIME  FIGURE 19  44 TABLE V The Disappearance of Synthetic SCT i n Rainbow Trout  Fish  Time i n Minutes  A  5  800 >_ 1100 '<_ 1480  84.9  B  5  760 >_ 1050 <_ 1400  85.3  C  5  620  >>-875 <_ 1180  85.4  A  20  370  >_ 480  <_ 640  37.1  B  20  350  >_ 455  <_ 600  37.0  C  20  280  >_ 375  <_ 500  36.6  A  40  260  >_ 345  <_ 460  26.6  B  40  230  >_ 310  <_ 420  25.2  C  40  200  >_ 265  <_ 360  25.9  B  60  195  >_ 260  £ 350  21.1  C  60  155  >_ 205  <_ 280  20.0  SCT ng/ml Incubate With 95% Confidence Limits  Zero Time Extrapolated Value  Percentage of Zero Time Extrapolated Value  Disappearance Values  A = 1295  Ta /  B = 1230  Tb /? = 59  l  2  1  = 12.5 Minutes  Minutes  C = 1025  Average Percentage Difference Between Duplicate Sample Values Fish  A - 7.7  Fish  B -13.3  Fish  C - 9.8  DISCUSSION  Since the radioimmunoassay i s greatly dependent upon the quality of the labelled tracer, much attention has been focused in this direction. Several investigators (Berson and Yalow, 1966. Jacobs, 1969. Chard, Kitau and Landon, 1970) have found that the degree of labelling, as well as the amount of damaged hormone produced, varies with iodinatioris. There is evidence that some of this variation i s due to differences in isotope shipments (Berson and Yalow, 1966). Theoretically, a tracer of highest possible specific activity i s desirable for maximal, sensitivity. There i s a practical limit to the specific activity that can be achieved since a loss of immunoreactivity and stability occurs with iodination beyond a certain level. Greenwood, Hunter and Glover (1963) found that growth hormone labelled with 4 - 6  MCi. had a  decreased affinity for the antibody, whereas maximal sensitivity in the assay was obtained with growth hormone that was labelled with 2 MCi. of iodide. 125 It has been shown that storage of ACTH-I  (Yalow and  Berson, 1960) as well as other labelled hormones (Hunter, 1969) results in the appearance of iodide and loss of immunoreactivity. 125 This was also evidenced with. SCT-I  in the data presented.  Complete destruction of some of the labelled molecules as well as spontaneous loss of the label is probably responsible for the 125 accumulation of the iodide. Therefore, although Ihas a 57 day h a l f - l i f e , the useful l i f e of a labelled hormone often i s 125 comparatively short. SCT-I has.proved to be especially unstable  and this is probably due to the susceptibility of the disulfide bond at the amino terminus (Niall et al., 1969) to oxidation and subsequent polymerization. Potts (1969) found that decreasing the amounts of oxidizing and reducing agents helped somewhat and Tashjian (1969) found that the inclusion of 0.05 M mercaptoethanol in the iodination and storage solutions, significantly improved 125 the stability of porcine calcitonin-I Despite the small molecular size of calcitonins antigenicity has not posed any great problems. Antibody production has been elicited by calcitonin with (Tashjian, 1969) or without (Deftos, Lee and Potts, 1968) coupling to a larger molecule. Deftos, Lee and Potts (1968) immunized guinea pigs with highly purified porcine calcitonin at 2 week intervals for a period of 32 weeks. The antisera was diluted 1:50,000 for use in the assay. Rabbit-2 in this study was immunized 3 times and produced an antiserum that was used i n the assay at a dilution of 1:800. The higher t i t r e obtained by Deftos et at was probably due to the larger number of booster injections. An example of the benefit of numerous booster injections i s given by Chard, Kitau and Landon (1970). They injected oxytocin into rabbits 5 times and produced an antisera that was used at a dilution of 1:800. After 2 further boosters, the titre rose to 1:25,000. Although Chard found that the increase i n titre was associated with increased sensitivity, Berson and Yalow (1964) have shown that antisera of high titre are not necessarily highly sensitive. The sensitivity of the antiserum used in this work was quite adequate. The conditions of incubation used in this radioimmunoassay  were based on the following considerations. Hales and Randle (1963) originally showed that preincubation for 6 hours without tracer considerably increased the sensitivity of the double antibody radioimmunoassay. Poznanski and Poznanski (1969) found similar results when applying this technique to the coated charcoal assay of insulin. Preincubation allows selective binding of some of the unlabelled hormone molecules. When the tracer i s included in the incubation system, fewer binding sites on the antibody molecules are available and therefore fewer tracer molecules w i l l be bound. The preincubation times commonly chosen by other investigators are 24 to 48 hours. The slope of the standard curve, hence the sensitivity of the assay, was shown by Herbert, Lau and Gottlieb (1965) to be increased with the length of incubation ( after inclusion of the tracer), since the amount of bound hormone i n creases with time. A more dilute antiserum and a smaller amount of tracer could be used i f the incubation time were longer, because the antigen-antibody  reactions w i l l more closely approach com-  pletion. Maximum sensitivity occurs with the smallest possible amount of tracer, since less unlabelled hormone is required to cause measurable inhibition of tracer binding. However, the amount of tracer present must be sufficient to allow counting within a practical period of time (10 - 20 min./sample) with suitable accuracy (c.p.m. several times background). Incubation periods are usually 4 to 5 days. The shorter incubation period reported here (24 hours) worked well and allowed the completion of a large number of experiments. The dilution of antiserum chosen was such that maximal binding was 30 - 50%. Berson and Yalow (1964)  have shown that sensitivity is theoretically greatest when maximal binding is 33^/3%. The preparation of relatively calcitonin-free plasma is 125 supported by the following reasons: a) maximal binding of SCT-I in extracted fish plasma was greater than that in fish plasma which was not extracted,indicating removal of a competitive inhibitor. 125 b) SCT-I  was adsorbed to s i l i c a during the method of purification.  c) Martin, Melick and Cooper (1969) have reported the use of Quso G32 to extract calcitonin from the plasma of a patient with medullary carcinoma of the thyroid. Also with as l i t t l e as 1 mg Quso/ml of 125 plasma, 81% of porcine calcitonin-I  could be extracted.  Coated charcoal is the most widely used adsorptive agent to separate bound and free antigen. Optimal conditions can only be achieved by empirical means. A wide variety of conditions affect the apparent binding. The amount of charcoal required depends upon the affinity of the antigen for charcoal. One f i f t h of the amount of charcoal used in insulin assays (Herbert, Lau and Gottlieb,  1965)  and growth hormone assays (Lau, Gottlieb and Herbert, 1966) was used in SCT assays, indicating that SCT is somewhat more strongly attracted to charcoal. It has also been necessary to decrease the amount of charcoal originally used by Herbert, Lau and Gottlieb (1965) i n the radioimmunoassay of angiotensin.(Waxman, Goodfriend and Herbert, 1967). Best results were obtained with a 1/100  of that amount of charcoal  used in insulin assays. The decrease in binding with increasing amounts of charcoal probably results from dissociation of the antigen-antibody complex and/or adsorption of the entire complex  due to the strong attractive forces (Herbert, 1969). Binding seemed to be increased when plasma was included i n the incubation medium. Donald (1968), applying the charcoal method to the radioimmunoassay of a ACTH, also found this effect and felt that plasma may additionally coat the charcoal such that i t is a more effective "molecular sieve", hence standards and unknowns must be assayed under similar conditions. Variation i n the amount of dextran used has no effect on separation as long as the amount used i s sufficient to saturate the charcoal. This amount i s about 20% (Herbert, 1969) of the weight of charcoal used. The disappearance of synthetic SCT i n the plasma of trout, as determined radioimmunologically, showed at least two components ( T a i , = 12.5, Tbiy 12.  12  =59 minutes). Shorter time intervals between  samples would be needed for a more definite statement. Watts and McGowan (1970b), using the bioassay, found the h a l f - l i f e of partially purified SCT to be 27 minutes. However, they took samples only at 30 minute intervals which would obscure the i n i t a l decline. Examined on this basis, the two sets of data compared extremely well. In comparisons of biological and radioimmunological assays, i t must be borne i n mind that loss of biological activity does not necessarily mean loss of immunological activity, since the two components often reside i n different parts of the molecule. Indeed, biologically inactive fragments of a molecule are often immunologically active, though to a weaker degree than the intact molecule(Bryant, Huxster and Greenwood, 1969). The possibility therefore exists that the attenuated second component of the disappearance represents to some  extent, such fragments. The normal endogenous level of calcitonin in salmon plasma is not known. Furthermore, Watts et al (1970a) did not find a hypocalcemic response by injecting SCT into rainbow trout. Therefore i t is not possible to make a realistic prediction of the physiological concentration; however, figures are available for mammals. Deftos, Lee and Potts (1968) found the average value for calcitonin in 7 rabbits to be 140 pg/ml of plasma, the values ranging from 20 - 300 pg/ml. Porcine calcitonin concentrations as determined by Lequin et al (1969) and Arnaud, Hang and L i t t l e dike (1970) were 300 and 500 pg/ml respectively. There i s no . reason to believe that the concentration of calcitonin in salmon should be less than that found i n mammals. Assuming a basal concentration of 200 - 300 pg/ml of plasma, the radioimmunoassay for SCT would be adequate to detect such levels i f suitable assays can be performed i n 30 - 50% plasma. Concrete data has not been forthcoming concerning the physiolological role of calcitonin in fish, although i t is well known that the stimulus for calcitonin release in mammals i s acute hypercalcemia (Copp et al., 1962). For example, Lee, Deftos and Potts (1969) showed radioimmunologically, a four fold increase in calcitonin levels i n rabbits after infusing a 2% calcium solution for 40 minutes. Therefore the radioimmunoassay for salmon calcitonin could be used to measure such things as SCT concentrations i n the ultimobranchial gland and plasma of salmon subjected to varying degress of acute and chronic hypercalcemia and other stressful conditions, as well as any seasonal changes. Other useful  experiments which could be conducted are to measure the rate, or rates of secretion of SCT and the disappearance of SCT in several animals. As a final comment on this radioimmunoassay, the following statement by Midgely, Niswender and Rebar (1969) i s appropriate: " no existing radioimmunoassay for protein hormones can be stated to be absolutely accurate, and further, i t is apparent that only relative accuracy can be considered until the properties of the form of the hormone being measured are more precisely known."  52  BIBLIOGRAPHY  Arnaud, C.D., Littledike, T., Tsao, H.S., and Kaplan, E.L. 1968. Radioimmunoassay of calcitonin: a preliminary report. Mayo Clin. P r o c , 43, 496. Arnaud, CD., Tsao, H.S., and Littledike, T. 1970. Calcium homeostasis, parathyroid hormone and calcitonin: preliminary report. Mayo Clin. Proc, 45, 125. Bagenal, M. 1955. A note on the relations of certain parameters following a logarithmic transformation. J. Mar. Biol. Ass., U.K. 34, 289. Berson, S.A., Yalow, R.S., Bauman, A., Rothschild, M.A., and Newerly, K. 1956. Insulin-ll31 metabolism in human subjects: demonstration of insulin binding globulin in the circulation of insulintreated subjects. J. Clin. Invest. ,35_, 170. Berson, S.A., and Yalow, R.S. 1957. Kinetics of reaction between insulin and insulin-binding antibody. J. Clin. Invest.,36, 873. Berson, S.A., and Yalow, R.S. 1964. Immunoassay of protein hormones. Jn. The Hormones, vol. 4. Edited by Pincus, G., Thimann, K.V., and Astwood, E.B. Academic Press,Inc., New York. p. 557. Berson, S.A., and Yalow, R.S. 1966. Iodininsulin used to determine specific activity of iodine-131. Science, 152, 205. Bliss, CD. 1952. In The Statistics of Bioassay. Academic Press, Inc., New York. p. 472. Bryant, CD., Huxster, M., and Greenwood, F. 1969. Immuno-reactive fragments of endogenous protein hormone in plasma. In Protein and Polypeptide Hormones. Proc. Internat. Symp., Liege, May 19-25, 1968. Edited by Margoulies, M. Excerpta Medica Foundation, Amsterdam, p. 26. Chard, T., Kitau, M.J., and Landon, J. 1970. The development of a radioimmunoassay for oxytocin: radioiodination, antibody production and separation techniques. J. Endocr.,46, 269. Clark, M.B., Boyd, G.W., Byfield, P.G.H., and Foster, CV. 1969. A radioimmunoassay for human calcitonin. M. Lancet, J., 74. Copp, D.H., Cameron, E.C, Cheney, B.A. , Davidson, A.G.F., and Henze, K.C 1962. Evidence for calcitonin - a new hormone from the parathyroid that lowers blood calcium. Endocrinology, _70, 638.  53  Copp, D.H., and Parkes, CO. 1968. Extraction of calcitonin from ultimobranchial tissue. In Parathyroid Hormone and Thyrocalcitonin (Calcitonin). Edited by Talmage, R.V. , and Belanger, L.F. Excerpta Medica Foundation, Amsterdam. 1968. p. 74. Deftos, L.J., Lee, M.R., and Potts, J.T. 1968. A radioimmunoassay for thyrocalcitonin. Proc. N.A.S., 60_, 293. Donald, R.A. 1968. Application of the coated charcoal separation method to the radioimmunoassay of plasma corticotrophin. J. Endocr., 41, 499. Felber, J.P., and Aubert, M.L. 1969. Radioimmunoassays. General principles. J. Nucl. Biol. Med., 13, 1. Freedlender, A.E. 1969. Practical and theoretical advantages for the use of I125 in radioimmunoassay. In Protein and Polypeptide Hormones. Proc. Internat. Symp., Liege, May 19-25, 1968. Edited by Margoulies, M. Excerpta Medica Foundation, Amsterdam, p.351. Goodfriend, T.L., Levine, L., and Fasman, G.D. 1964. Antibodies to bradykinin and angiotensin. A use of carbodiimides i n immunology. Science, 144, 1344. Goodfriend, T.L., Ball,_D.L.,,and Farley, D.B. 1968 Radioimmunoassay of angiotensin. J. Lab. Clin. Med., 12_, 648. Goodfriend, T.L.;1969. Labelling and Separation: purification. In Protein and Polypeptide Hormones. Proc..Internat. Symp., Liege, May 19-25, 1968. Edited by Margoulies, M. Excerpta Medica Foundation, Amsterdam, p. 624; Greenwood. F.C., Hunter, W.M., and Glover, J.S.-1963..The preparation of I -labelled human growth hormone of high specific radioactivity. Biochem. , J. j), 114. Hales, C.N., and Randle, P.J. 1963. Immunoassay of insulin with insulin-antibody precipitate. Biochem., J. 88_, 137. Herbert, V., Gottlieb, C.W., Lau, K.S., and Wasserman, L.R. 1964. Intrinsic factor assay. Lancet, 2_, 1017. Herbert, V., Lau, K.S., Gottlieb, C.W., and Bleicher, S.J. 1965. Coated charcoal immunoassay of insulin. J. Clin. Endocr. Metab., 25, 1375. Herbert, V. 1969. Coated charcoal separation of free labelled hormone from hormone bound to antibody. In Protein and Polypeptide Hormones. Proc. Internat. Symp., Liege, May 19-25* 1968. Edited by Margoulies, M. Excerpta Medica Foundation, Amsterdam, p. 55.  54  Hunter, W.M. 1969a. Control of specificity of radioimmunoassay. In Protein and Polypeptide Hormones. Proc. Internat. Symp., Liege, May 19-25, 1968. Edited by Margoulies, M. Excerpta Medica Foundation, Amsterdam, p. 5. Hunter, W.M. 1969b. Assessment of radioiodinated hormone preparations. Acta Endocrinologica, (v3_, 134. Supp. 142. Jacobs, H.S. 1969. Use of activated charcoal i n the radioimmunoassay of human growth hormone i n plasma. J. Clin. Path., 22, 710. Lau, K.S., Gottlieb, C.W., and Herbert, V. 1966. Preliminary report on coated charcoal immunoassay of human chorionic growth hormone-prolactin and growth hormone. Proc. Soc. Exp. Biol. Med. New York., 123, 126. Lee, M.R., Deftos, L.J., and Potts, J.T. 1969. Control of secretion of thyrocalcitonin i n the rabbit as evaluated by radioimmunoassay. Endocr., 84_, 36. Lequin, R.M., Hackeng, W.H.L., Schopman, W., and Care, A.D. 1969. Comparison between the bioassay and radioimmunoassay of calcitonin in plasma of several species. J. Endocr., 45, 309. Martin, T.J., Melick, R.A., and Cooper, L. 1969. Quso G32 extraction of labelled peptide hormones. Anal. Bioch., 32^ 174. Midgley, A.R., Niswender, G.D., and Rebar, R.W. 1969. Principles for the assessment of the r e l i a b i l i t y of radioimmunoassay methods (precision, accuracy, sensitivity, specificity). Acta Endocrinologica, 63, 163. N i a l l , H.D., Keutmann, H.T., Copp, D.H., and Potts, J.T. 1969. Amino acid sequence of salmon ultimobrancial calcitonin. Proc. N.A.S., 64, 771. Potts, J.T. 1969. Comparative study of methods of separation. In Protein and Polypeptide Hormones. Proc. Internat. Symp., Liege, May 19-25, 1968. Edited by Margoulies, M. Excerpta Medica Foundation, Amsterdam, p. 26. Poznanski, N., and Poznanski, W.J. 1969. Laboratory application of the dextran-^cdated charcoal radioimmunoassay of insulin. Clin. Chem., 15, 908. Raiti, S., and Davis, W.T. 1969. The principles and application of radioimmunoassay with special reference to gonadotropins. Obstetrical and Gynecological Survey, 2A_, 289.  55  Samols, E., ans Bilkus, D. 1964. A comparison of insulin immunoassays. Proc. Soc. Exp. Med., 115, 79. Snedecor, G.W. 1956. Linear regression. In Statistical Methods, 5th ed. Iowa State University Press.,p.122. Tashjian, A.H. 1969. Immunoassay of thyrocalcitonin. I. The method and its serological specificity. Endocrinology,, J54, 40. Watts, E., Copp, D.H., Dawson, K., Hui, F., and McGowan, E. 1970a. Calcitonin i n the rainbow trout Salmo gairdnerii. In Proc. Can. Fed. Biol. Soc, Montreal, June 9-12, 1970. 13, 78. Watts, E., and McGowan, E. 1970b. Personal communications. Waxman, S., Goodfriend, T., and Herbert, V. 1967. Angiotensin assay and assessment of free iodide contamination using dilute coated charcoal. Clin. Res., 15,.457. Yalow, R.S., and Berson, S.A. 1960. Immunoassay of endogenous plasma insulin i n man. J. Clin. Invest., 39_, 1157. Yalow, R.S., and Berson, S.A. 1966. Purification of -^-'-I-parathyroid hormone with microfine granules of precipitated s i l i c a . Nature (Lond.), 212, 357. Yalow, R.S., and Berson, S.A. 1969. Topics on radioimmunoassay of peptide hormones. In Protein and Polypeptide Hormones. Proc. Internat. Symp., Liege, May 19-25, 1968. Edited by Margoulies, M. Excerpta Medica Foundation, Amsterdam, p.36. Yalow, R.S., and Berson, S.A. 1968. General principles of radioimmunoassay. In Radioisotopes in Medicine: In vitvo studies. Proc. Symp., Oakridge, Nov. 13-16, 1967. Edited by Hayes, R.L., Goswitz, F.A., and Murphy, B.P.,U.S. A.E.C. Tennessee.p. 7.  56  APPENDIX  Chemistry of Iodination *  Oxidation CH C H S0 NClNa (oxidant) + HOH 3  CIO  6  4  C H  4  125 + H0 + 2 I 2  _ CI + I £  3 6 4 °2 2 C  H  S  N H  +  N a 0 C 1  + 2 OH  Substitution + 2 tyrosine •—  2 tyrosine-! ^ + 2H 1  5  +  Reduction I„ + 2 Na S 0- (reductant) o  I  *  o  1 1 3  Na„S,0^ + 2 Nal 2 46  Brewster, R.G. In Organic Chemistry. - Prentice Hall, 2nd Ed. p.532  


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