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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 p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Co lumb ia , I a g r e e t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and S tudy. I f u r t h e r a g ree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department or by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f P h y s i o l o g y The U n i v e r s i t y o f B r i t i s h Co lumbia Vancouver 8, Canada Date O c t o b e r 6 t h 1970 This thesis is dedicated to my wife, Lynne, for her love, understanding, encouragement and invaluable help. i i ACKNOWLEDGMENT I would l i k e to express my s i n c e r e s t a p p r e c i a t i o n to Dr. Harold Copp, whose p a t i e n c e , guidance and continued support 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 to 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 the pre p a r a t i o n of 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 are extended to Dr. John Ledsome, Dr. C. Owen Parkes and Dr. Ralph 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 indebted to Mr Kurt Henze f o r h i s help throughout the d u r a t i o n of my stay i n the Department of Physiology and f o r the p r e p a r a t i o n of i l l u s t r a t i o n s . In a d d i t i o n , I wish to express my g r a t i t u d e to Miss Kathy Finch f o r her t e c h n i c a l a s s i s t a n c e and Mrs Frances News'ome f o r her help i n the e d i t i n g of t h i s manuscript. F i n a l l y , I would l i k e to accord r e c o g n i t i o n to the Medical Research C o u n c i l of Canada f o r f i n a n c i a l support. i i i 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 is 50 - 60 pg/ml of incubate. The dis-appearance of synthetic salmon calcitonin in 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 . .- 56 v LIST OF TABLES Table Page I Protocol for Radioimmunoassay 16 II Labelling Variation and Effect of Dowex on the Stored Products- 21 125 III Effect of Dowex .2-X8 on Freshly Prepared SCT-I 28 125 IV 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. 22 125 6. An Autoradiograph of Chromatographed SCT-I Before and After Purification by Method A 23 7. Purification Method A: Elution Profile of the Cellulose Column 24 125 8. An Autoradiograph of Chromatographed SCT-I Before and After Purification by Method B 25 125 9. An Autoradiograph of Chromatographed SCT-I Stored for 2 Weeks: Before and After Treatment With Dowex 2-X8 27 125 10. Adsorption of SCT-I Onto Tubes During Incubation 30 v i i Figure Page 11. Concentration of Coated Charcoal Suspension 31 Versus Counts in the Bound Fraction. 125 12. Binding and Damage of SCT-I as a Function of the Time of Incubation. . 33 125 13. 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 in Rainbow Trout 41 18. Disapp earance of Synthetic SCT in Rainbow Trout 42 19. . Disappearance of Synthetic SCT: Log % Zero Time Concentration Versus Time 43 v i i i ABBREVIATIONS AND SYMBOLS Ab - antibody Ag - antigen As - antiserum Agt2 - angiotensin II 125 125 Agt2~I - - iodine labelled angiotensin II c.p.m. - counts per minute -2 g - 9.8m. sec MCi - millicurie M.W. - molecular weight NRP - normal rabbit plasma SCT - salmon calcitonin [SCT] - concentration of salmon calcitonin 125 125 SCT-I - iodine labelled salmon calcitonin TCA - trichloroacetic acid A - index of precision Sy.x - standard deviation for the regression line y on x %B - percentage binding Weight arid Volume _3 mg - milligram (10 gm) —6 ug - microgram (10 gm)' -9 ng - nanogram (10 gm) -12 pg - picogram (10 gm) - 3 ul - microlitre (10 1) ix • INTRODUCTION The h i s t o r y of radioimmunoassay began i n 1955 when Berson et dl 131 were studying the metabolic f a t e of I l a b e l l e d i n s u l i n . A f t e r 131 intravenous a d m i n i s t r a t i o n of i n s u l i n - I i t was found that the 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 the plasma of s e v e r a l p a t i e n t s which had re c e i v e d i n s u l i n therapy, was composed of two f r a c t i o n s , p a r t that could and part that could not be s a l t e d out w i t h Sodium Sulphate. Zone e l e c t r o p h o r e s i s of these plasma samples revealed two peaks of r a d i o a c t i v i t y , one at the o r i g i n r e p r e s e n t i n g f r e e l a b e l l e d i n s u l i n and one m i g r a t i n g w i t h the gamma-globulins. Presoaking of 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 of 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 occurrence of i n s u l i n a n t i b o d i e s i n the plasma of i n s u l i n t r e a t e d people disproved 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 . Berson and Yalow (Berson et dl. , 1956; Yalow and Berson, 1960) l a t e r reported the development of a radioimmunoassay method f o r the measurement of i n s u l i n concentrations i n body f l u i d s . This e a r l y work began a new era i n endocrinology, a l l o w i n g the accurate measurement of p h y s i o l o g i c a l l e v e l s of many polypeptide hormones. The p r i n c i p l e of radioimmunoassay i s based on the a b i l i t y of a l a b e l l e d antigen to compete e q u a l l y - w i t h u n l a b e l l e d antigen f o r the 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 antigen-antibody r e a c t i o n s 1 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 radio-immunoassay 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. All 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 Labelled Antigen -Antigen Antibody Antibody Complex Ag + Ab Ag - Ab + Ag Unlabelled Antigen Ag — Ab Unlabelled Antigen - Antibody Complex FIGURE 1 4 Group C - Adsorption of Free Antigen. 1. Ion Exchange 2. Charcoal 3. Silica 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 precipit-ation 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, will adsorb molecules ranging widely in molecular size. However, i f the charcoal is first coated with an agent such as dextran (M.W. 70,000) i t will 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 This d i s c r i m i n a t o r y a b i l i t y i s presumed to be due to the pore s i z e on the surface of each charcoal p a r t i c l e , the pores being created by the l a t t i c e w o r k of adsorbed c o a t i n g molecules. Thus, a f t e r a p e r i o d of i n c u b a t i o n , the f r e e antigen may be separated from the s o l u t i o n by adsorption onto the coated c h a r c o a l . This i s shown d i a g r a m a t i c a l l y i n Figure 2. The r a t i o of r a d i o a c t i v i t y i n the charcoal ( f r e e antigen) to r a d i o a c t i v i t y i n the supernatant (bound antigen) v a r i e s w i t h c o n c e n t r a t i o n of u n l a b e l l e d antigen. This system was f i r s t a p p l i e d to the assay of i n t r i n s i c f a c t o r (Herbert et a l . , 1964) and has subsequently found a p p l i c a t i o n i n the radioimmunoassay of many polypeptide hormones such as i n s u l i n , (Herbert et at., 1965) growth hormone,(Lau, G o t t l i e b and Herbert, 1966) and angiotensin.(Waxman, Gopdfriend and Herbert, 1967). The s e n s i t i v i t y of the radioimmunoassay i s g r e a t l y dependent on the immunoreactivity and s p e c i f i c a c t i v i t y of the t r a c e r . The amount of t r a c e r present i n the i n c u b a t i o n should be l e s s than the minimum conc e n t r a t i o n of hormone to be measured. I f t h i s i s not the case, small changes i n the concentration of the u n l a b e l l e d antigen w i l l not be detected. Damage to the t r a c e r during i n c u b a t i o n r e s u l t s i n a l o s s of 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 the a f f i n i t y of the antibody f o r the antigen. A n t i s e r a from d i f f e r e n t animals of the same species may vary g r e a t l y i n t h e i r s e n s i t i v i t y f o r d e t e c t i o n of the antigen (Yalow and Berson, 1968). In p r i n c i p l e the s p e c i f i c i t y of the radioimmunoassay i s dependent upon the homogeneity of the p r o t e i n which i s l a b e l l e d and antibody s p e c i f i t y (Hunter, 1969a:);7. As r e l a t i v e l y crude m a t e r i a l i s of t e n used f o r immunization, 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 will be obtained, some or a l l of which may react in 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 antigen-antibody reaction as well as the system to separate the bound and free hormone. This is especially apparent when assaying physiological fluids such as urine. 13X X23 The antigen can be labelled with I 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 is found in 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 sensit-ivity to detect basal endogenous levels of calcitonin in rabbits and pigs respectively. Tashjian (1969) was not so fortunate. All 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 in 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 106) of high antigenicity. The large 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-dimethyl-aminopropyl) carbodiimide was the coupling agent. The coupled material was then purified by gel filtration 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 intra-muscularly 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 first 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 first 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 pre-servative, at a concentration of 0.1%. A stock solution of the antiserum currently in 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 yl of 0.4 M phosphate buffer pH 7.5 125 2. approximately 2 MCi. Nal (New England Nuclear) contained in 10-20 yl of solution 3. 2.5 yg synthetic salmon calcitonin in 10 yl (stock solution stored at -15°C, contained 250 yg/ml in 0.05 M acetic acid) 4. 65 yg chloramine-T (oxidant) in 10 yl 0.25 M phosphate buffer pH 7.5 5. 130 yg sodium metabisulfite (reductant) in 20 yl 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 yl 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 in volume and washed with 0.05 M veronal buffer. Fractions of 0.5 ml were coll-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 radio-o 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 silica (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 silica. 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 super-natant 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 effect-iveness 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, fill 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-I12 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 (dil-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 yl Hamilton syringe. 3. Standards (stock solution stored at -15°C at a concen-tration of 10 yg/ml in diluent) or unknowns, contained in 50 yl, 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 yl 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 (Exclud-ing the "total counts" tubes). This mixture was conr stantly stirred on a magnetic mixer to prevent settling 15 o f t h e s u s p e n s i o n . 3. Each t ube was capped and m i xed by i n v e r s i o n f o r about 10 s e c o n d s . The t u b e s were c e n t r i f u g e d a t 1000 g f o r 10 m i n u t e s . 4 . S u p e r n a t a n t a l i q u o t s o f 3 m l were p l a c e d i n c o u n t i n g t ube s and c o u n t e d f o r enough t i m e t o a c c u m u l a t e a t l e a s t 10 ,000 c o u n t s . T h i s r e d u c e d t h e c o u n t i n g e r r o r t o 1%. 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 i n t h e 3 m l v o l -ume t o e n s u r e 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 o n l y a p o r t i o n o f t h e s u p e r n a t a n t was c o u n t e d , t h e dange 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 . C o u n t i n g o f t h e p e l l e t was n o t a t t e m p t e d on a r o u t i n e b a s i s 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 t o d e c a n t a l l o f 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 t h e c h a r c o a l . " C a l c i t o n i n - F r e e " P l a s m a S t a n d a r d c u r v e s i n a s s a y s c o n t a i n i n g a s i g n i f i c a n t c o n c e n -t r a t i o n o f p l a s m a ( e . g . 12.5%) 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 " b y 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 g r a n u l e s a t a c o n c e n t r a t i o n o f 10 mg/ml o f p l a s m a . D i s a p p e a r a n c e o f S y n t h e t i c Sa lmon C a l c i t o n i n E a ch o f 3 r a i n b o w " t r o u t we re i n j e c t e d w i t h 5 y g o f s y n -t h e t i c s a lmon c a l c i t o n i n th rough , a c a n n u l a s e c u r e d i n t h e d o r s a l a o r t a . B l o o d samp le s we re t a k e n 5, 2 0 , 40 and 60 m i n u t e s a f t e r i n j e c t i o n . 50 y l s amp le s o f d i l u t e d p l a s m a ( 1 : 6 ) we re a s s a y e d i n d u p l i c a t e . Table 1 Protocol for Radioimmunoassay Sample Buffer (ml) Antiserum Dilution (ml) Standard or Unknown Amount SCT(yl)-SCT-I 1 2 5 (yl) -Charcoal -Dextran : (miy Unknown or 3.4 0.5 50 Pre- 50 Incubate 2 Mix and Standard incubate at 4°C centri-at 4°C for fuge at for 24 hours 1000 g Total 24 hours for 10 Bound 3.45 0.5 '— • 50 2 minutes. Counts Decant 3 ml of 0.5 of normal super-Control 3.4 non-immune 50 50 2 natant in--'. serum. Dilution to counting similar to As. tubes and count radio-Total 5.95 — — 50 — activity. Counts t - 1 RADIOIMMUNOASSAY SCHEDULE FOR SALMON CALCITONIN Ab + SCT Ab-SCT SCT ' 24 hr. Pre-lncubation Centrifuqafion »-SCT - Ab S C T I ' " - Ab Ab + SCT 1L A b SCT % Ab-SCT Ab-SCT Charcoal-Dextrqn Suspension i . 24 hr. Incubation Free SCT +• SCT x in Charcoal Pellet / 3 cc Aliquot for Counting Dispersion of Charcoal in Solution Discard Counting Tube with Sample FIGURE 3 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 to Free R a t i o of R a d i o a c t i v i t y (B/F) bound counts _ c.p.m. - av. c o n t r o l counts f r e e counts av. t o t a l counts - c.p.m. b) Percentage B i n d i n g % b i n d i n g bound counts av. t o t a l counts - av. c o n t r o l counts c) L i n e a r Regression and Confidence L i m i t s 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 c a l c -u l a t e d f o r standard curves (Snedecor, 1956). The form of the 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% confidence l i m i t s were c a l c u l a t e d f o r the r e g r e s s i o n l i n e s (Snedecor, 1956) . The 95% confidence l i m i t s f o r a pre-d i c t e d [SCT] (from the standard curve) were c a l c u l a t e d by de t e r -mining the x values ( l o g [SCT]) on the r e g r e s s i o n l i n e f o r the 95% confidence l i m i t values of y (%B)*. This i s shown g r a p h i c a l l y i n Figure 4. In t h i s i l l u s t r a t i o n , the lower and upper confidence l i m i t s f o r X q are "-xj and-xg r e s p e c t i v e l y . The index of 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 l i n e . ^ _ standard d e v i a t i o n slope The s m a l l e r the value of X the greater i s the inherent p r e c i s i o n of the assay method ( B l i s s , 1952). * Dr. P. L a r k i n , Department of Zoology, Univ. of B.C. supported t h i s method f o r confidence l i m i t c a l c u l a t i o n s (personal 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 1 = value of log [SCT] on the regression line at Y 2 > lower confidence limit for XQ = value of log [SCT] on the regression line at Y^, upper confidence limit for X„ • .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 silica (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 its 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 Total Counts in Assay within 0-1 days of labelling Total Counts in Assay After Re-Treatment with Dowex No. of Days After Labelling When Dowex Treated June 8 June 17 June 29 5648 5888 4058 4230 5176 6 21 9 July 13 July 15 4452 July 20 4550 4142 2 Aug. 4 Aug. 5 14206 AN AUTORADIOGRAPH OF CHROMATOGRAPHED SCT-I BEFORE AND AFTER PURIFICATION BY METHOD A FIGURE' 6 125 1 Before p u r i f i c a t i o n of SCT-I 125 i i A f t e r p u r i f i c a t i o n of SCT-I S3 PURIFICATION METHOD A : ELUTION PROFILE OF THE CELLULOSE COLUMN 2.4 2.0 H O-O / I / * I I I <o 1.6 H O x G \ O I E CL d 2 H 0.8 H \ o o 0.4 H T 1 1 1 1 -6 12 Fraction No. I o-o-o i r T r T 18 f 24 *— Beginning of Elution -i 28 FIGURE 7 1 OS AN AUTORADIOGRAPH OF CHROMATOGRAPHED SCT- I BEFORE AND AFTER PURIF ICATION BY . a__ . . _ . METHOD B 0 R I G I N V N N 1 1 i i i FIGURE 8 125 i B e f o r e p u r i f i c a t i o n o f SCT- I i i A f t e r p u r i f i c a t i o n o f S C T - I 1 2 5 i i i R e - p u r i f i c a t i o n N J 125 assays suggested b e t t e r Immunoreactivity w i t h SCT-I p u r i f i e d by Method B. Two such assays were s i m i l a r i n a l l respects except that 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 other. The maximal B/F*.tatios were 0.21 and 0.51 r e s p e c t i v e l y . 125 An attempt was made to f u r t h e r p u r i f y the SCT-I . The product of method B was 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 of r a d i o a c t i v i t y at the o r i g i n as compared to that at the solvent f r o n t d i d not seem to be reduced (Figure 8 i i i ) • b) Storage of Tracer I t was c o n s i s t e n t l y found that treatment w i t h Dowex 2-X8 125 anion exchange r e s i n of SCT-I st o r e d f o r more than 1 or 2 days, r e s u l t e d i n a r e d u c t i o n of counts f o r a constant a l i q u o t (Table I I ) . 125 Figure 9> i ) i s an autoradiograph of chromatographed SCT-I a f t e r a 2 week storage p e r i o d . The l a r g e accumulation of i o d i d e seen at the o r i g i n was removed a f t e r treatment w i t h Dowex (Figure 9 i i ) . T h i s decrease i n counts was not marked i n samples test e d 1 or 2 days a f t e r l a b e l l i n g (Table I I I ) i n d i c a t i n g l i t t l e o r no adsorption of S C T - I 1 2 5 . Immunoreactivities of a l l preparations were s u f f i c i e n t l y reduced a f t e r 2 weeks to n e c e s s i t a t e frequent l a b e l l i n g . Several products only 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 of use. The maximal B/F value 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 decreased,often to-such an extent that the d i f f e r e n c e s i n b i n d i n g were no longer d e t e c t a b l e at hormone concentrations 125 r o u t i n e l y used i n the assay. Table IV i l l u s t r a t e d t h i s p o i n t . SCT-I l 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 A l l s t a t e d B/F values represent 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 i i FIGURE 9 i Before Dowex treatment i i 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 6765 - July 20 6460 6640 TABLE.-IV, Effect of Storage on Immunoreactivity of SCT-I As Dilution Unlabelled SCT Date of Assay B/F 1:400 May 13 0.77 1:400 • - May 26 0.52 1:800 - May 13 0.48 1:800 1 ng/ml May 13 0.27 1:800 - May 26 0.29 1:800 1 ng/ml May 26 0.19 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 minim-125 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 albumin-125 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 gm% over which binding is effected very l i t t l e . Outside this range, 30 ADSORPTION OF SCT-I 1 2 5 ONTO TUBES DURING INCUBATION Uncoated * 80.3 78.6 • 52.5 Albumin coated ¥ 98.7 89.6 Gelatin coated 86.6 80.7 100 O 80 g 3 h 60 a> 3 o 40 5-(O 20 0 Siliconized Siliconized Acid rinsed Acid rinsed ^Albumin O.I N HCL + Albumin 64.1 57.7 coated 77.1 71.0 V7. m 52.2 471 w coated B5.I 70.2 Disposable Glass Tubes Soft Glass Tubes I Plastic Tubes r 100 O 80 g 3 W 60 -, 3 Q 40 5 ' 3 20 0 FIGURE 10 CONCENTRATION OF COATED CHARCOAL SUSPENSION VERSUS COUNTS. IN THE BOUND FRACTION 8 0 0 - i 6 0 0 -E CL 6 c ZD o m 4 0 0 -2 0 0 --o— —o. / / / © © 12.5% N.R.R • • Buffer 1 1 1 0 . 2 5 0 . 5 0 . 7 5 gm % Coated Charcoal Suspension - r — 1 . 0 FIGURE 11 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 first 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 initially .present, which:was 14% in this experiment. Assessment of Antisera All antisera tested demonstrated the presence of antibodies to SCT, However, the titres were variable (Figure 13). Rabbit(R-2) had the best titre. 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 48 Hours of Incubation FIGURE 12 (A) Binding of SCT-I (B) Damage of SCT-I1 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, is 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 in Figure 14 the standard curve flattens out at 1:1600 dilution of antiserum with the result that there is 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 sub-sequent 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 ( A ) A s D i l u t i o n U 4 0 0 5 0 i 4 0 -3 CQ 3 0 -c 3 O O 2 0 -10 -n = 0 . 0 5 5 S y x = 2 . 1 6 9 \ y = 1 2 5 . 8 - 3 0 . 7 x 125 2 5 0 P g / m l I n c u b a t e - i 1 5 0 0 7 5 0 ( B ) A s " D i l u t i o n P 8 0 0 3 0 - 1 o 2 0 m o IO H k = 0 . 0 6 1 S y - x = I . 4 4 8 y = 7 0 . 1 - 2 3 . 6 x - i — 125 250 500 750 P g / m l I n c u b a t e 3 O o (C) •o c 15-o CO 1 0 -5 -0 -fib A s D i l u t i o n 1 : 1 6 0 0 h = 0 . 1 5 0 S y x = I . 3 8 4 , y = 2 9 . 6 - 9 . 2 7 x 125 — I 2 5 0 1 1 5 0 0 7 5 0 I / m l I n c u b a t e FIGURE 15 of time. The slope of the regression line for the standard curve in Figure 16 was not significantly different from that in Figure 15b (p = 0.05, t = 1.25 for n-4 =18) even though the assays were separated by a period of 1 ^ 2 months. 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 in 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) is shown in Figure 16a. The slope of the regression line of the semilogarithmic plot (Figure 16b) was compared to that for synthetic SCT shown in 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=— . .. T ' — , 1 Micro Units partially purified 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 in Figure 17. A B/F value was cal-culated 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 in the disappearance of synthetic SCT, Ta11'2 = 12.5 minutes and Tb 1^ = 59 minutes. The data is summ-arized in Table V. Detection Of Endogenous Calcitonin in 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 is 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 = 0.055 S y x = 1.40 y = 87.3 -25.1 x FIGURE 17 DISAPPEARANCE OF SYNTHETIC SCT IN RAINBOW TROUT FIGURE 18 DISAPPEARANCE OF SYNTHETIC SCT: LOG % ZERO TIME CONCENTRATION VERSUS TIME FIGURE 19 TABLE V The Disappearance of Synthetic SCT in Rainbow Trout 44 Fish Time in Minutes SCT ng/ml Incubate With 95% Confidence Limits Percentage of Zero Time Extrapolated Value 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 Zero Time Extrapolated Value A = 1295 B = 1230 C = 1025 Disappearance Values Tal/2 = 12.5 Minutes Tb1/? = 59 Minutes Average Percentage Difference Between Duplicate Sample Values Fish A - 7.7 Fish B -13.3 Fish C - 9.8 DISCUSSION Since the radioimmunoassay is 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 is due to differences in isotope shipments (Berson and Yalow, 1966). Theoretically, a tracer of highest possible specific activity is desirable for maximal, sensitivity. There is a practical limit to the specific activity that can be achieved since a loss of immunoreactivity and stability occurs with iod-ination 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 I- has a 57 day half-life, the useful l i f e of a labelled hormone often is 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 in the assay at a dilution of 1:800. The higher titre obtained by Deftos et at was probably due to the larger number of booster injections. An example of the benefit of numerous booster injections is 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 in 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 is included in the incubation system, fewer binding sites on the antibody molecules are available and therefore fewer tracer molecules will 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 in-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 will 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 silica 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 fifth 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) in 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 in the incubation medium. Donald (1968), applying the charcoal method to the radioimmunoassay of a ACTH, also found this effect and felt that plasma may addition-ally coat the charcoal such that i t is a more effective "molecular sieve", hence standards and unknowns must be assayed under similar conditions. Variation in the amount of dextran used has no effect on separation as long as the amount used is sufficient to saturate the charcoal. This amount is about 20% (Herbert, 1969) of the weight of charcoal used. The disappearance of synthetic SCT in the plasma of trout, as determined radioimmunologically, showed at least two components ( T a i , = 12.5, Tbiy =59 minutes). Shorter time intervals between 12. 12 samples would be needed for a more definite statement. Watts and McGowan (1970b), using the bioassay, found the half-life of partially purified SCT to be 27 minutes. However, they took samples only at 30 minute intervals which would obscure the inital 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 in mind that loss of biological activity does not necess-arily mean loss of immunological activity, since the two components often reside in 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. There-fore 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 Little-dike (1970) were 300 and 500 pg/ml respectively. There is no . reason to believe that the concentration of calcitonin in salmon should be less than that found in mammals. Assuming a basal con-centration 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 in 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 is acute hypercalcemia (Copp et al., 1962). For example, Lee, Deftos and Potts (1969) showed radioimmunologically, a four fold increase in calcitonin levels in rabbits after infusing a 2% calcium sol-ution for 40 minutes. Therefore the radioimmunoassay for salmon calcitonin could be used to measure such things as SCT concentra-tions in 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) is 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. Proc, 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 insulin-treated 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 in 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 in the radioimmunoassay of human growth hormone in 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 in 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 reliability of radioimmunoassay methods (precision, accuracy, sensitivity, specificity). Acta Endocrinologica, 63, 163. Niall, 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 in 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 in man. J. Clin. Invest., 39_, 1157. Yalow, R.S., and Berson, S.A. 1966. Purification of -^-'-I-parathyroid hormone with microfine granules of precipitated sil 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 CH3C6H4S04NClNa (oxidant) + HOH C H3 C6 H4 S°2 N H2 + N a 0 C 1 125 _ CIO + H20 + 2 I CI + I £ + 2 OH Substitution + 2 tyrosine •— 2 tyrosine-! 1^ 5 + 2H+ Reduction I„ + 2 NaoSo0- (reductant) Na„S,0^ + 2 Nal I 1 1 3 2 4 6 * Brewster, R.G. In Organic Chemistry. - Prentice Hall, 2nd Ed. p.532 

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