Department, Pharmaceuticals Division, Ciba-Geigy Limited, 4002 Basle, Switzerland and Research Laboratory for Calcium Metabolism, Departments of Orthopedic Surgery and Medicine, University of Zurich, 8008 Zurich, Switzerland Research
SYNTHETIC HUMAN CALCITONIN: ANALYSIS OF ANTIBODIES OBTAINED FROM VARIOUS ANIMAL SPECIES AND DETERMINATION OF IMMUNOREACTIVE HORMONE IN HUMAN SERA
By F. M. Dietrich, W. H. Hunziker and
J. A. Fischer
ABSTRACT Antibodies to
synthetic
human calcitonin
(hCT)
were
developed
in
rabbits, goats and mice. The free peptide (32 amino-acid residues,
Mwt.
3418) was administered together with adjuvant, and the effect of various immunization procedures, as well as of different dose-levels, was evaluated comparatively. Synthetic hCT was found to be a good immunogen for the three animal species examined. The relative importance of various structural parts of the hCT molecule with regard to immunological specificity was determined by reference to the inhibition of the specific binding of 125I-hCT to antibodies by peptide fragments of hCT. All the antisera studied were directed to structural and/or conformational properties of the 11\p=n-\28or 11\p=n-\32amino acid sequence of hCT. Six different antisera from rabbits and goats were selected for radioimmunological assay of hCT on the basis of their inhibitory dose50-values and immunological specificity. To improve the sensitivity of the radioimmunoassay (RIA), we studied the preparation of radioiodinated hCT and assessed various parameters determining the sensitivity of the assay. Despite all the efforts, CT in human plasma from healthy subjects could not be determined with certainty. The difficulties encountered in the
determination of normal levels of circulating CT are discussed in terms of the sensitivity of RIA and non-specific interference of serum factors with RIA.
Human calcitonin (hCT)1) was isolated from tumour tissue from patients with medullary carcinoma of the thyroid (MCT) (Riniker et al 1968). Its aminoacid sequence was identified (Neher et al 1968) and confirmed by total syn¬ thesis (Sieber et al. 1968). Being a low-molecular-weight polypeptide (Mwt. 3418), synthetic hCT is a suitable model antigen to study the relations between chemical structure and immunological specificity. Experiments carried out to this end were reported on earlier (Dietrich Sc Rittel 1970a,b). Recently, we described a sensitive procedure that makes it possible to detect minute amounts of anti-hCT. Bacteriophage T4-hCT conjugates were found to be specifically neutralized by antisera to hCT diluted more than 106-fold (Steiner Sc Dietrich 1973). Several studies have been conducted by other investigators in an effort to develop radioimmunological assay systems sufficiently sensitive to allow CT levels to be measured accurately in the circulation of non-tumorous human subjects (e.g. Clark et al. 1969; Tashjian et al. 1970; Hackeng et al. 1970; Frölich et al. 1971; Deftos 1971; Deftos et al. 1971; Habener et al. 1972; Samaan et al 1973; Heynen Sc Franchimont 1974). These efforts have, how¬ ever, only met with limited success. Although synthetic hCT is detectable by radioimmunological means in amounts as low as a few picograms, most assay systems are not sensitive and precise enough to quantitate CT in health. Furthermore, non-specific interference of human sera with immunochemical reactions creates an additional difficulty in quantitating CT and in the inter¬ pretation of reported results. The present study was carried out to define some of the immunological con¬ ditions that have to be met by a successful radioimmunoassay of CT. To this end, we made comparative evaluations of the immune responses to synthetic hCT in rabbits, goats and mice and analyzed quantitative and qualitative properties of anti-hCT. Inhibition tests were carried out to determine the rela¬ tive importance of various structural parts of the synthetic hCT molecule with ') Abbreviations:
CT, calcitonin; hCT, pCT, sCT, human, porcine, salmon CT; iCT, immunoreactive CT; cFA, complete Freund's adjuvant; B/F, ratio of antibody-bound 1251-hCT to free i^I-hCT; BSA, HSA, bovine and human serum albumin; DCC, dextran-coated charcoal; dilution-50, dilution of antiserum yielding 50 % binding of 125I-hCT; ID-)0, inhibition dose^, the amount of inhibitor required for a 50 % reduction of the
binding
of '251-hCT to its antibody; carcinoma of the thyroid; RIA,
MCT, medullary
radioimmunoassay.
regard to immunological specificity. Antibodies exhibiting different immuno¬ logical properties were then selected for the determination of CT in human sera.
MATERIALS
Peptides. Synthetic hCT (Sieber et al. 1968) consisting of (Mwt. 3418) has the following primary structure:
32 amino acid residues
-
I 12
4
3
5
I
6
8
7
9
10
11
12
13
14
15
16
H-Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-Tyr-Thr-Gln-Asp-Phe17
18
19
21
20
22
23
24
25
26 27
28
29
30
31
32
Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro-NH2 Peptide fragments
of hCT
H-ll-32-NH22), H-1-10-OH, H-11-16-OH,
H-17-28-OH
prepared from the corresponding intermediates by removal of protecting groups. The fragments H-11-18-OH, H-19-28-OH and H-19-32-NH2 were isolated from enzymatic digests of H-11-32-OH and the peptides H-1-28-OH, H-l-31NH2 and H-1-32-OH were synthesized. All the peptides were generously provided by Drs. W. Rittel, B. Riniker, M. Brugger, B. Kamber and P. Sieber, Ciba-Geigy. Chemicals and reagents. Human serum albumin (HSA) was purchased from the Central Laboratories of the Swiss Red Cross, Berne, bovine serum albumin (BSA) from the Behringwerke, Marburg-Lahn, Germany, 2-mercaptoethanol (2-ME) from Eastman Organic Chemicals, Rochester, N. Y. and chloramine-T from Fluka AG, Buchs, Swit¬ zerland. Na125I purchased from either the Radiochemical Centre, Amersham, England or the Swiss Institute for Reactor Research, Wiirenlingen, contained 80-400 mCi/ml. QUSO G-32 (microfine precipitated silica granules) was obtained from Sorvall, New¬ ton, Conn., Bio-Gel P-6 (200-400 mesh) from Bio-Rad Laboratories, Richmond, Calif., dextran-coated charcoal (DCC) was prepared with dextran-T-70 (Pharmacia, Uppsala, Sweden) and Norit-A C-176 (Fisher Scientific Company, Fair Lawn, N.J.) (Herbert et al. 1965). Alhydrogel (2°/o aluminum hydroxide) was purchased from Superfos Export Company a/s, Copenhagen, Denmark, complete Freund's adjuvant (cFA) from Difco Laboratories, Detroit, Mich., and Bordetella pertussis vaccine (4 IO10 . per¬ tussis phase I organisms per ml) from the Swiss Institute of Sera and Vaccines, Berne. All remaining reagents were purchased from E. Merck, AG, Darmstadt, Germany or Fluka AG, Buchs, Switzerland, and were of analytical grade. and H-11-28-OH
were
-
METHODS
Trace-labelling. Synthetic hCT was radioiodinated with chloramine-T (Hunter 8c Greenwood 1962) according to a slightly modified procedure described by Tashjian (1969). Labelled peptides appeared on chromato-electrophoresis (Yalow 8c Berson 1960) to be chemically pure. Analysis of chromato-electrophoretograms revealed spe¬ -
cific activities ranging from 50 to 150 mCi/mg. Thin-layer chromatography (Avicelcellulose, solvent system n-butanol-pyridine-acetic acid-water) (Riniker et al. 1968)
2)
-NH2 denotes the carboxy-terminal amide, -OH carboxyl group.
the free
carboxy-terminal
combined with radioautography revealed, however, that radiolabelled hCT consisted of at least two components differing slightly in their migratory properties. Incubation with excess anti-hCT showed that 27 l^I-hCT preparations had an average binding of 88.1 ± 2.4 "lo (sd), whereas in 30 preparations 91.4 ± 4.6 °/o of the radioactivity was adsorbed onto DCC. Specific binding to anti-hCT was tested by incubating 0.1 ml of a high-titer antiserum diluted 1:500, with 0.4 ml of the labelled preparation in 0.1 M Tris buffer pH 7.5 containing 2.5 mg HSA/ml for 20 h at 4°C, followed by DCCseparation of antibody-bound and free radioactivity. Adsorption onto DCC was assessed by briefly shaking on a Vortex mixer 0.1 ml of the labelled peptide and 1 ml of DCC suspension, followed by centrifugation and determination of the sedi¬ ment-associated radioactivity. 125I-hCT preparations were shown to retain their capa¬ city to bind to antibody and to adsorb onto DCC for more than 40 days. For all the antigen-binding studies in a given experiment, the same labelled preparation was used, or preparations with comparable properties and antibody-binding profiles were selected. To improve the immunological properties of radioiodinated hCT, the standard iodination procedure was modified by varying the amounts of Na123I (0.2 to 4.0 mCi) and chloramine-T (7.5-75 µg). Furthermore, QUSO-purified 125I-hCT was additionally gel-filtrated. All these attempts, however, were unsuccessful. As shown in Fig. 1, peak fractions of 125I-hCT re-purified on Bio-Gel P-6 did not exhibit increased binding to antibody. Fig. 1 illustrates that the immunological properties of 125I-hCT are more readily detectable with highly diluted antibody than with antibody in large excess. As can be seen from the results presented in Fig. 2, gel filtration following purifica¬ tion by QUSO did not offer any additional advantage as regards sensitivity in de¬ tecting hCT. IDr,o-values (see below) of gel-filtrated trace-labelled hCT were compar¬ able to the IDj0 obtained with a preparation which had only been purified with
QUSO.
Immunization and quantitation of antibodies. Synthetic hCT was dissolved in acetic acid, admixed either to activated charcoal (Norit A) or to Alhydrogel and incorporated into cFA with the aid of a Sorvall Omnimixer (Newton, Conn.). Antibodies to hCT were raised in male rabbits (1.5-2.0 kg), in goats of either sex (25-30 kg) and in male Swiss albino mice (20-22 g). The antigen doses, routes of injection and immunization schedules are indicated under 'Results". In some ex¬ periments the animals received B. pertussis vaccine subcutaneously. The rabbits were repeatedly bled from the ear and the final bleedings were carried out by cardiac puncture. The goats were bled from the jugular vein and the mice by retro-orbital puncture. The sera were heat-inactivated (56°C, 30 min) and kept at -20°C until use. The antibodies were quantitated on the basis of their capacity to bind trace-labelled antigen. The total incubation volume was 0.5 ml containing the diluted serum, and 125I-hCT in 0.1 M Tris buffer pH 7.5 containing HSA (0.25 mg/ml). Incubations were carried out for 20 h at 4°C. Control tubes contained non-immune serum or diluent only. Antibody-bound hCT was separated from free hCT by addition of 1 ml DCC suspension (Herbert et al. 1965). The tubes were briefly shaken on a Vortex mixer and centrifuged. Radioactivity was determined in sediments, and later in the study all determinations, including those of CT in human sera, were made in both sediments and supernatants. Calculations of specifically bound 12äI-hCT were made and the values corrected for non-antibody-associated radioactivity present in the supernatant. Dilution-50 values, which are defined as the dilutions of antisera yielding 50 "lo specific binding of the radioiodinated antigene), were determined graphically. 0.01
3) All antisera dilutions indicated in this the final incubation mixture of 0.5 ml.
-
publication
refer to
serum
concentration in
O m
UJ
o HI
a.
EFFLUENT VOLUME (ml)
Binding A: i^I-hCT B: 12iI-hCT The column
Fig. 1. synthetic hCT to antibody charcoal (DCC). QUSO (see Methods).
of radioiodinated
and dextran-coated
purified with purified with QUSO and Bio-Gel P-6. (1x50 cm) was equilibrated and eluted with 0.2 m ammonium acetate (pH 4.7) containing 2.5 mg HSA/ml. The void volume (V0) (determined with Dextran blue, Pharmacia, Uppsala) and the elution volume of Na125I are indicated by arrows (top row). The flow rate was 4.8 ml/h and 0.8 ml fractions were collected. The eluted radioactivity is denoted by closed circles (·). Na125I represents less than 0.2% of the total radioactivity. Fractions containing 4000 cpm were incubated with DCC and anti¬ bodies as described under "Methods". The arrows a and b (bottom row) denote frac¬ tions which were used to prepare standard curves (see Fig. 2). DCC-associated radioaciviy ( ); specifically bound 125I-hCT to antibody (goat 6A, Day 143) diluted 1:500 ( ) and 1:80 000 (O). The incubation medium consisted of 0.1 m Tris (pH 7.5) containing 10% DCC-treated normal human serum.
100
80
60 < H
40
20
)
100
1000 pg hCT
Fig. 2. using 125I-hCT purified with QUSO, with and without additional gel filtration. l^I-hCT purified with QUSO only (O), iMI-hCT purified with QUSO and additionally gel-filtered: fraction a (see Fig. 2) (A) and fraction b (·). The antiserum (goat 6A, Day 143) was diluted 1:80 000. Ordinate: per cent initial ratio of antibody-bound and free ligand (B/F) without hCT standard
curves
added inhibitor.
Qualitative properties of antibodies and radioimmunoassay of hCT in human sera. Inhibition of the primary interaction between antibodies and trace-labelled hCT by various peptide fragments of synthetic hCT and analogues was determined with
-
dilutions of antisera yielding 30-40 "lo specific binding in the absence of inhibitor. The optimal duration of pre-incubation of antiserum with graded amounts of hCT and incubation after addition of the trace-labelled antigen were determined for each anti¬ serum used in a radioimmunoassay. Pre-incubation was carried out for periods of time ranging from 0.5 to 115 h whereas incubation lasted for 22 h, 46 h or 118 h. The IDjQ-values depend mainly on the duration of pre-incubation. They were generally lowest, i. e. the sensitivity when quantitating hCT was greatest, after pre-incubation for 1 to 2 days. Given a fixed pre-incubation time, the IDsQ-values were lowest when incubation lasted for 1 day. Routinely, graded amounts of inhibitors were pre-incubated for 1 to 2 days at 4°C with diluted antisera (volume 0.3-0.4 ml). The diluent con¬ taining 123I-hCT was added to make up the volume to 0.5 ml and the mixture in¬ cubated for another 20 to 24 h. The amount of radioactivity specifically bound to antibody in the presence of inhibitors was compared with the control to which no inhibitor had been added. Each standard or unknown B/F ratio was expressed as the percentage of the initial B/F ratio. The amounts of inhibitors required to reduce specific binding to 50% (ID50) were determined graphically. Immunoreactive hCT was estimated in peripheral human sera obtained from sur¬ gically verified MCT and phaeochromocytoma patients and from control subjects. The control subjects had normal serum concentrations of calcium (total and ionized), phosphorus, alkaline phosphatase, creatinine and immunoreactive parathyroid hor¬ mone (Fischer et al. 1974 and unpublished results). Undiluted or diluted sera in quantities of 0.2, 0.1 and 0.05 ml were added to the incubation system (total volume: 0.5 ml). Control tubes in quadruplicate with and without antiserum containing 0.2, 0.1 and 0.05 ml DCC-absorbed normal human serum or serum from totally thyroidecto¬ mized patients were included in the assay. This control serum did not influence the B/F ratios. The diluent consisted of 0.1 M Tris buffer (pH 7.5) supplemented with 10% serum (DCC-treated normal human serum or serum from thyroidectomized patients). Intra-assay and inter-assay coefficients of variation amounted to 8 and 15%, respectively (Diem Sc Lentner 1968). Under optimal experimental conditions and using our most potent antisera, which exhibited ID^-values of hCT as low ai 50-100 pg, the limit of sensitivity in detecting hCT added to undiluted human sen was found to be 100 pg/ml.
RESULTS
Quantity
and
quality of rabbit antibodies to synthetic hCT In an earlier study, synthetic hCT adsorbed onto charcoal and incorporated into cFA was shown to induce rabbit antibodies having specificities directed to amino-acid sequences located in the carboxy-terminal parts of the molecule, whereas the amino-terminus was structurally irrelevant in this respect (Dietrich Sc Rittel 1970a,b). In the present investigation, additional experiments were carried out to increase the quantity of antibodies, characterize their qualitative properties and, in addition, compare them with anti-hCT produced in goats and mice. The quantity and quality of the antibodies obtained after multiple injections totalling 12.6, 3.6 and 2.6 mg hCT are shown in Fig. 3 A, and C, respectively. It is evident that the antibody titers were inversely related to the
——. .
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1989
%
^ ·
20
t
t t
40
t
0.1 0.5 1.0 10
80
t 5.0
100 120
140
t
5.0
20
t
t
40
60
80
!
t
t
100
120
140
160
180
20
t
t
40
60
t
t
BO
K>0
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(40
20
40
60
80
TOO DAYS
t
HUMAN
CALCITONIN INJECTED (mg)
005
Fig. 3. quantity and quality of rabbit antibodies to synthetic hCT. Reciprocal dilutions-r,0 indicating serum concentrations which bind 50 % of 123I-hCT are given in the top panels. ID5{)-values indicating a 50 % reduction of the specific binding of 125I-hCT to anti-hCT are given in the bottom panels. The abscissa denotes days after the first injection of the antigen. Antigen administrations are indicated by arrows. In experiments A and (except for the last two injections in A) hCT was adsorbed onto a tenfold amount of Norit A and incorporated into cFA. In C and D, hCT was adsorbed onto Alhydrogel and incorporated into cFA. In A, and C, the first in¬ jection (0.2 ml of adjuvant mixture) was made directly into the inguinal lymph nodes, the second (1.0 ml) im, the third (2.0 ml) into four foot pads, and the fourth and the fifth (Group B) sc at multiple sites. In experiment A, the last two injections were made iv; in this case, hCT was dissolved in 0.5 ml 0.01 m phosphate-buffered (pH 7.2) NaCI (0.14 m). Finally, in experiment D, 0.5 ml adjuvant mixture was injected into 6-8 intradermal sites. Simultaneously, the rabbits received 1 ml B. pertussis vaccine sc. Time-course of
Rabbits 152, 153 and 156
were
immunized with 0.5 mg hCT and rabbits 155, 157 and 158 with 0.05 mg hCT.
dose. The highest dilutions-50 were seen in group C around Day 140, after four injections totalling 2.6 mg hCT. Judged from the ID30-values, several antisera of group C also exhibited the greatest affinity for hCT. A single injection of 0.5 or 0.05 mg hCT still induced the formation of anti¬ bodies (Fig. 3), whereas 0.005 mg was not immunogenic. The antibody titers obtained after a single dose of submilligram amounts of hCT were rather low. Reciprocal dilutions-50 ranged from 50 to 180. On the other hand, the ID5o obtained with serum from rabbit 152 was remarkably low, decreasing progres¬ sively from flOO pg on Day 54 to 140 pg 103 days after immunization with 0.5 mg hCT. Table 1 summarizes the results of immunochemical analysis of 6 high-titer antisera from rabbits immunized with total doses of 3.6 and 2.6 mg hCT (see
antigen
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lOCOTf|iOCOqcO ) 75. Eisen . . Sc Siskind G. W.: Biochemistry 3 (1964) 996. Fischer J. ., Binswanger U. Sc Dietrich F. M.: J. clin. Invest. 54 (1974) 1382. Frolich M., Kassenaar A. A. H. Sc Smeenk D.: Horm. Metab. Res. 3 (1971) 297. Gelzer J.: Immunochemistry 5 (1968) 23. Goidl . ., Paul W. E., Siskind G. W. & Benacerraf B.: J. Immunol. 100 (1968) 371. Goltzman D., Potts J. T. Jr., Ridgway C E. Se Maloof F.: New Engl. J. Med. 290
(1974)
1035.
L. J. & Potts J. T. Jr.: Clin. chim. Acta 39 (1972) 407. Hackeng W. H. L., Schellekens A. P. M. & Schopman W'.: Horm. Metab. Res. 2 (1970) 311. Hennessy J. F., Wells S. A. Jr., Ontjes D. A. 8c Cooper C. W'.: J. clin. Endocr. 39 (1974) 487. Herbert V., Lau K. S., Gottlieb C. W. 8c Bleicher S. J.: J. clin. Endocr. 25 (1965) 1375. Hesch R. D., Woodhead J. S., Hüfner M. Se Dietrich F. M.: Acta endocr. (Kbh.) Suppl. 773 (1973) 161. Hcynen G. Se Franchimont P.: Europ. J. clin. Invest. 4 (1974) 213. Hüfner M. Se Hesch R. D.: Klin. Wschr. 49 (1971) 1149. Hunter W. M. Se Greenwood F. C: Nature (Lond.) 194 (1962) 495. Kaplan E. L., Sizemore G., Hill B. J. 8e Peskin G. W.: Clin. Res. 20 (1972) 724 (Abstract). Kraus L. M.: J. Immunol. 99 (1967) 894. Lee M. R., Deftos L. J. Sc Potts J. T. Jr.: Endocrinology 84 (1969) 36. Lequin R. M., Hackeng W. H. L. Sc Schopman W.: J. Endocr. 44 (1969) 283. Melvin K. E. W., Tashjian A. H. & Miller H. H.: Recent Progr. Hormones Res. 28 (1972) 399. Milhaud G., Tharaud D., Julienne A. 8c Moukhtar M. S. In: Taylor S., Ed. Endo¬ crinology 1971. W. Heinemann Medical Books, London (1972c?) 380. Milhaud G., Calmette C, Julienne ., Tharaud D., Bloch-Michel H., Cavaillon J. P., Colin R. 8e Moukhtar M. S. In: Talmage R. V. and Munson P. L., Eds. Calcium, Parathyroid Hormone and the Calcitonins. Internat. Congr. Series, Vol. 243. Excerpta med. (Amst.) (1972&) 56. Milhaud G., Calmette C, Taboulel J., Julienne A. Se Moukhtar M. S.: Lancet 1 (1974) Habener
J. F., Deftos
462.
Moukhtar M. S., Julienne ., Tharaud D., Taboulet J. Se Milhaud G.: C. R. Acad. Sci. (Paris) 276 (1973) 3445. Neher R., Riniker B., Rittel W. Se Zuber H.: Helv. chim. Acta 51 (1968) 1900. Parthemore J. G., Bronzert D., Roberts G. Se Deftos L. ].: J. clin. Endocr. 39 (1974) 108.
Pento
J. T.,
Glick S. M. Sc
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.: Metabolism 22
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Potts J. T. Jr., Keulmann H. T., Deftos L. J. Se Niall H. D. In: Laude S., Ed. Progress in Peptide Research, Vol. 2. Gordon and Breach, New York (1972) 93. Potts J. T. Jr., Niall H. D., Keutmann H. T., Deftos L. J. Se Parsons J. A. In: Taylor S. and Foster G. V., Eds. Calcitonin 1969. W. Heinemann Medical Books, London (1970) 56. Riniker ., Nehcr R., Maier R., Kalint F. W., Byfield P. G. H., Gudtnundsson T. V.. Galante L. Sc Maclntyre I.: Helv. chim. Acta 51 (1968) 1738. Rose J. G. 8c Newsome H. H. Jr.: J. clin. Endocr. 35 (1972) 469. Rosenquist G. L. 8c Holmquist A. M.: Immunochemistry 11 (1974) 489. Samaan . ., Hill C. S., Beceiro J. R. Se Schultz P. N.: J. Lab. clin. Med. 81 (1973) 671. Sieber P., Brugger M., Kamber B., Riniker B. 8- Ritte! W'.: Helv. chim. Acta 51 (1968) 2057. Silva O. L., Snider R. H. & Becker K. L.: Clin. Chem. 20 (1974 ) 337. Silva O. L., Becker K. L., Primack ., Doppman J. Se Snider R. H.: New Engl. J. Med. 290 (19746) 1122. Siskind G. W.: Pharmacol. Rev. 25 (1973) 319. Siskind G. W. Se Benacerraf B.: Advanc. Immunol. 10 (1969) 1. Siskind G. W., Dunn P. 8c Walker J. G.: J. exp. Med. 127 (1968) 55. Sizemore G. W'., Go V. L. W'., Kaplan E. L., Sanzenbacher L. J., Hollermuller . H. 8- Arnaud C. D.: New Engl. J. Med. 288 (1973) 641. Steiner A. W. 8c Dietrich F. M.: Z. immun. Forsch. 145 (1973) 275. Tashjian A. H. Jr.: Endocrinology 84 (1969) 140. Tashjian A. H. Jr., Howland B. G., Melvin K. E. W. Sc Hill C S.: New Engl. J. Med. 283 (1970) 890. Tashjian A. H. Jr. 8c Voelkcl E. F. In: Jaffe B. M. and Behrman H. R., Eds. Methods of Hormone Radioassay. Academic Press, New York (1974) 199. Tashjian A. H. Jr., Wolfe H. J. Se Voelkel E. F.: Amer. J. Med. 56 (1974) 480. Vaitukaitis J., Robbins J. B., Nieschlag E. Se Ross G. T.: J. clin. Endocr. 33 (1971) 988. Vallotton M. B. In: Page I. H. and Bumpus F. M., Eds. Handbuch exp. Pharm., Vol. 37. Springer Verlag, Berlin (1974) 185. Voelkel E. F., Tashjian A. H. Jr., Davidoff F. F., Cohen R. B., Pedia C. P. Sc Wurtmann R. ].: J. clin. Endocr. 37 (1973) 297. Werblin T. P., Kim Y. T., Quagliata F. 8c Siskind G. W.: Immunology 24 (1973) 477. Yalow R. S. Sc Berson S. .: J. clin. Invest. 39 (1960) 1157.
Received
on
December 3rd, 1974.
SYNTHETIC HUMAN CALCITONIN: ANALYSIS OF ANTIBODIES OBTAINED FROM VARIOUS ANIMAL SPECIES AND DETERMINATION OF IMMUNOREACTIVE HORMONE IN HUMAN SERA
By F. M. Dietrich, W. H. Hunziker and
J. A. Fischer
ABSTRACT Antibodies to
synthetic
human calcitonin
(hCT)
were
developed
in
rabbits, goats and mice. The free peptide (32 amino-acid residues,
Mwt.
3418) was administered together with adjuvant, and the effect of various immunization procedures, as well as of different dose-levels, was evaluated comparatively. Synthetic hCT was found to be a good immunogen for the three animal species examined. The relative importance of various structural parts of the hCT molecule with regard to immunological specificity was determined by reference to the inhibition of the specific binding of 125I-hCT to antibodies by peptide fragments of hCT. All the antisera studied were directed to structural and/or conformational properties of the 11\p=n-\28or 11\p=n-\32amino acid sequence of hCT. Six different antisera from rabbits and goats were selected for radioimmunological assay of hCT on the basis of their inhibitory dose50-values and immunological specificity. To improve the sensitivity of the radioimmunoassay (RIA), we studied the preparation of radioiodinated hCT and assessed various parameters determining the sensitivity of the assay. Despite all the efforts, CT in human plasma from healthy subjects could not be determined with certainty. The difficulties encountered in the
determination of normal levels of circulating CT are discussed in terms of the sensitivity of RIA and non-specific interference of serum factors with RIA.
Human calcitonin (hCT)1) was isolated from tumour tissue from patients with medullary carcinoma of the thyroid (MCT) (Riniker et al 1968). Its aminoacid sequence was identified (Neher et al 1968) and confirmed by total syn¬ thesis (Sieber et al. 1968). Being a low-molecular-weight polypeptide (Mwt. 3418), synthetic hCT is a suitable model antigen to study the relations between chemical structure and immunological specificity. Experiments carried out to this end were reported on earlier (Dietrich Sc Rittel 1970a,b). Recently, we described a sensitive procedure that makes it possible to detect minute amounts of anti-hCT. Bacteriophage T4-hCT conjugates were found to be specifically neutralized by antisera to hCT diluted more than 106-fold (Steiner Sc Dietrich 1973). Several studies have been conducted by other investigators in an effort to develop radioimmunological assay systems sufficiently sensitive to allow CT levels to be measured accurately in the circulation of non-tumorous human subjects (e.g. Clark et al. 1969; Tashjian et al. 1970; Hackeng et al. 1970; Frölich et al. 1971; Deftos 1971; Deftos et al. 1971; Habener et al. 1972; Samaan et al 1973; Heynen Sc Franchimont 1974). These efforts have, how¬ ever, only met with limited success. Although synthetic hCT is detectable by radioimmunological means in amounts as low as a few picograms, most assay systems are not sensitive and precise enough to quantitate CT in health. Furthermore, non-specific interference of human sera with immunochemical reactions creates an additional difficulty in quantitating CT and in the inter¬ pretation of reported results. The present study was carried out to define some of the immunological con¬ ditions that have to be met by a successful radioimmunoassay of CT. To this end, we made comparative evaluations of the immune responses to synthetic hCT in rabbits, goats and mice and analyzed quantitative and qualitative properties of anti-hCT. Inhibition tests were carried out to determine the rela¬ tive importance of various structural parts of the synthetic hCT molecule with ') Abbreviations:
CT, calcitonin; hCT, pCT, sCT, human, porcine, salmon CT; iCT, immunoreactive CT; cFA, complete Freund's adjuvant; B/F, ratio of antibody-bound 1251-hCT to free i^I-hCT; BSA, HSA, bovine and human serum albumin; DCC, dextran-coated charcoal; dilution-50, dilution of antiserum yielding 50 % binding of 125I-hCT; ID-)0, inhibition dose^, the amount of inhibitor required for a 50 % reduction of the
binding
of '251-hCT to its antibody; carcinoma of the thyroid; RIA,
MCT, medullary
radioimmunoassay.
regard to immunological specificity. Antibodies exhibiting different immuno¬ logical properties were then selected for the determination of CT in human sera.
MATERIALS
Peptides. Synthetic hCT (Sieber et al. 1968) consisting of (Mwt. 3418) has the following primary structure:
32 amino acid residues
-
I 12
4
3
5
I
6
8
7
9
10
11
12
13
14
15
16
H-Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-Tyr-Thr-Gln-Asp-Phe17
18
19
21
20
22
23
24
25
26 27
28
29
30
31
32
Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro-NH2 Peptide fragments
of hCT
H-ll-32-NH22), H-1-10-OH, H-11-16-OH,
H-17-28-OH
prepared from the corresponding intermediates by removal of protecting groups. The fragments H-11-18-OH, H-19-28-OH and H-19-32-NH2 were isolated from enzymatic digests of H-11-32-OH and the peptides H-1-28-OH, H-l-31NH2 and H-1-32-OH were synthesized. All the peptides were generously provided by Drs. W. Rittel, B. Riniker, M. Brugger, B. Kamber and P. Sieber, Ciba-Geigy. Chemicals and reagents. Human serum albumin (HSA) was purchased from the Central Laboratories of the Swiss Red Cross, Berne, bovine serum albumin (BSA) from the Behringwerke, Marburg-Lahn, Germany, 2-mercaptoethanol (2-ME) from Eastman Organic Chemicals, Rochester, N. Y. and chloramine-T from Fluka AG, Buchs, Swit¬ zerland. Na125I purchased from either the Radiochemical Centre, Amersham, England or the Swiss Institute for Reactor Research, Wiirenlingen, contained 80-400 mCi/ml. QUSO G-32 (microfine precipitated silica granules) was obtained from Sorvall, New¬ ton, Conn., Bio-Gel P-6 (200-400 mesh) from Bio-Rad Laboratories, Richmond, Calif., dextran-coated charcoal (DCC) was prepared with dextran-T-70 (Pharmacia, Uppsala, Sweden) and Norit-A C-176 (Fisher Scientific Company, Fair Lawn, N.J.) (Herbert et al. 1965). Alhydrogel (2°/o aluminum hydroxide) was purchased from Superfos Export Company a/s, Copenhagen, Denmark, complete Freund's adjuvant (cFA) from Difco Laboratories, Detroit, Mich., and Bordetella pertussis vaccine (4 IO10 . per¬ tussis phase I organisms per ml) from the Swiss Institute of Sera and Vaccines, Berne. All remaining reagents were purchased from E. Merck, AG, Darmstadt, Germany or Fluka AG, Buchs, Switzerland, and were of analytical grade. and H-11-28-OH
were
-
METHODS
Trace-labelling. Synthetic hCT was radioiodinated with chloramine-T (Hunter 8c Greenwood 1962) according to a slightly modified procedure described by Tashjian (1969). Labelled peptides appeared on chromato-electrophoresis (Yalow 8c Berson 1960) to be chemically pure. Analysis of chromato-electrophoretograms revealed spe¬ -
cific activities ranging from 50 to 150 mCi/mg. Thin-layer chromatography (Avicelcellulose, solvent system n-butanol-pyridine-acetic acid-water) (Riniker et al. 1968)
2)
-NH2 denotes the carboxy-terminal amide, -OH carboxyl group.
the free
carboxy-terminal
combined with radioautography revealed, however, that radiolabelled hCT consisted of at least two components differing slightly in their migratory properties. Incubation with excess anti-hCT showed that 27 l^I-hCT preparations had an average binding of 88.1 ± 2.4 "lo (sd), whereas in 30 preparations 91.4 ± 4.6 °/o of the radioactivity was adsorbed onto DCC. Specific binding to anti-hCT was tested by incubating 0.1 ml of a high-titer antiserum diluted 1:500, with 0.4 ml of the labelled preparation in 0.1 M Tris buffer pH 7.5 containing 2.5 mg HSA/ml for 20 h at 4°C, followed by DCCseparation of antibody-bound and free radioactivity. Adsorption onto DCC was assessed by briefly shaking on a Vortex mixer 0.1 ml of the labelled peptide and 1 ml of DCC suspension, followed by centrifugation and determination of the sedi¬ ment-associated radioactivity. 125I-hCT preparations were shown to retain their capa¬ city to bind to antibody and to adsorb onto DCC for more than 40 days. For all the antigen-binding studies in a given experiment, the same labelled preparation was used, or preparations with comparable properties and antibody-binding profiles were selected. To improve the immunological properties of radioiodinated hCT, the standard iodination procedure was modified by varying the amounts of Na123I (0.2 to 4.0 mCi) and chloramine-T (7.5-75 µg). Furthermore, QUSO-purified 125I-hCT was additionally gel-filtrated. All these attempts, however, were unsuccessful. As shown in Fig. 1, peak fractions of 125I-hCT re-purified on Bio-Gel P-6 did not exhibit increased binding to antibody. Fig. 1 illustrates that the immunological properties of 125I-hCT are more readily detectable with highly diluted antibody than with antibody in large excess. As can be seen from the results presented in Fig. 2, gel filtration following purifica¬ tion by QUSO did not offer any additional advantage as regards sensitivity in de¬ tecting hCT. IDr,o-values (see below) of gel-filtrated trace-labelled hCT were compar¬ able to the IDj0 obtained with a preparation which had only been purified with
QUSO.
Immunization and quantitation of antibodies. Synthetic hCT was dissolved in acetic acid, admixed either to activated charcoal (Norit A) or to Alhydrogel and incorporated into cFA with the aid of a Sorvall Omnimixer (Newton, Conn.). Antibodies to hCT were raised in male rabbits (1.5-2.0 kg), in goats of either sex (25-30 kg) and in male Swiss albino mice (20-22 g). The antigen doses, routes of injection and immunization schedules are indicated under 'Results". In some ex¬ periments the animals received B. pertussis vaccine subcutaneously. The rabbits were repeatedly bled from the ear and the final bleedings were carried out by cardiac puncture. The goats were bled from the jugular vein and the mice by retro-orbital puncture. The sera were heat-inactivated (56°C, 30 min) and kept at -20°C until use. The antibodies were quantitated on the basis of their capacity to bind trace-labelled antigen. The total incubation volume was 0.5 ml containing the diluted serum, and 125I-hCT in 0.1 M Tris buffer pH 7.5 containing HSA (0.25 mg/ml). Incubations were carried out for 20 h at 4°C. Control tubes contained non-immune serum or diluent only. Antibody-bound hCT was separated from free hCT by addition of 1 ml DCC suspension (Herbert et al. 1965). The tubes were briefly shaken on a Vortex mixer and centrifuged. Radioactivity was determined in sediments, and later in the study all determinations, including those of CT in human sera, were made in both sediments and supernatants. Calculations of specifically bound 12äI-hCT were made and the values corrected for non-antibody-associated radioactivity present in the supernatant. Dilution-50 values, which are defined as the dilutions of antisera yielding 50 "lo specific binding of the radioiodinated antigene), were determined graphically. 0.01
3) All antisera dilutions indicated in this the final incubation mixture of 0.5 ml.
-
publication
refer to
serum
concentration in
O m
UJ
o HI
a.
EFFLUENT VOLUME (ml)
Binding A: i^I-hCT B: 12iI-hCT The column
Fig. 1. synthetic hCT to antibody charcoal (DCC). QUSO (see Methods).
of radioiodinated
and dextran-coated
purified with purified with QUSO and Bio-Gel P-6. (1x50 cm) was equilibrated and eluted with 0.2 m ammonium acetate (pH 4.7) containing 2.5 mg HSA/ml. The void volume (V0) (determined with Dextran blue, Pharmacia, Uppsala) and the elution volume of Na125I are indicated by arrows (top row). The flow rate was 4.8 ml/h and 0.8 ml fractions were collected. The eluted radioactivity is denoted by closed circles (·). Na125I represents less than 0.2% of the total radioactivity. Fractions containing 4000 cpm were incubated with DCC and anti¬ bodies as described under "Methods". The arrows a and b (bottom row) denote frac¬ tions which were used to prepare standard curves (see Fig. 2). DCC-associated radioaciviy ( ); specifically bound 125I-hCT to antibody (goat 6A, Day 143) diluted 1:500 ( ) and 1:80 000 (O). The incubation medium consisted of 0.1 m Tris (pH 7.5) containing 10% DCC-treated normal human serum.
100
80
60 < H
40
20
)
100
1000 pg hCT
Fig. 2. using 125I-hCT purified with QUSO, with and without additional gel filtration. l^I-hCT purified with QUSO only (O), iMI-hCT purified with QUSO and additionally gel-filtered: fraction a (see Fig. 2) (A) and fraction b (·). The antiserum (goat 6A, Day 143) was diluted 1:80 000. Ordinate: per cent initial ratio of antibody-bound and free ligand (B/F) without hCT standard
curves
added inhibitor.
Qualitative properties of antibodies and radioimmunoassay of hCT in human sera. Inhibition of the primary interaction between antibodies and trace-labelled hCT by various peptide fragments of synthetic hCT and analogues was determined with
-
dilutions of antisera yielding 30-40 "lo specific binding in the absence of inhibitor. The optimal duration of pre-incubation of antiserum with graded amounts of hCT and incubation after addition of the trace-labelled antigen were determined for each anti¬ serum used in a radioimmunoassay. Pre-incubation was carried out for periods of time ranging from 0.5 to 115 h whereas incubation lasted for 22 h, 46 h or 118 h. The IDjQ-values depend mainly on the duration of pre-incubation. They were generally lowest, i. e. the sensitivity when quantitating hCT was greatest, after pre-incubation for 1 to 2 days. Given a fixed pre-incubation time, the IDsQ-values were lowest when incubation lasted for 1 day. Routinely, graded amounts of inhibitors were pre-incubated for 1 to 2 days at 4°C with diluted antisera (volume 0.3-0.4 ml). The diluent con¬ taining 123I-hCT was added to make up the volume to 0.5 ml and the mixture in¬ cubated for another 20 to 24 h. The amount of radioactivity specifically bound to antibody in the presence of inhibitors was compared with the control to which no inhibitor had been added. Each standard or unknown B/F ratio was expressed as the percentage of the initial B/F ratio. The amounts of inhibitors required to reduce specific binding to 50% (ID50) were determined graphically. Immunoreactive hCT was estimated in peripheral human sera obtained from sur¬ gically verified MCT and phaeochromocytoma patients and from control subjects. The control subjects had normal serum concentrations of calcium (total and ionized), phosphorus, alkaline phosphatase, creatinine and immunoreactive parathyroid hor¬ mone (Fischer et al. 1974 and unpublished results). Undiluted or diluted sera in quantities of 0.2, 0.1 and 0.05 ml were added to the incubation system (total volume: 0.5 ml). Control tubes in quadruplicate with and without antiserum containing 0.2, 0.1 and 0.05 ml DCC-absorbed normal human serum or serum from totally thyroidecto¬ mized patients were included in the assay. This control serum did not influence the B/F ratios. The diluent consisted of 0.1 M Tris buffer (pH 7.5) supplemented with 10% serum (DCC-treated normal human serum or serum from thyroidectomized patients). Intra-assay and inter-assay coefficients of variation amounted to 8 and 15%, respectively (Diem Sc Lentner 1968). Under optimal experimental conditions and using our most potent antisera, which exhibited ID^-values of hCT as low ai 50-100 pg, the limit of sensitivity in detecting hCT added to undiluted human sen was found to be 100 pg/ml.
RESULTS
Quantity
and
quality of rabbit antibodies to synthetic hCT In an earlier study, synthetic hCT adsorbed onto charcoal and incorporated into cFA was shown to induce rabbit antibodies having specificities directed to amino-acid sequences located in the carboxy-terminal parts of the molecule, whereas the amino-terminus was structurally irrelevant in this respect (Dietrich Sc Rittel 1970a,b). In the present investigation, additional experiments were carried out to increase the quantity of antibodies, characterize their qualitative properties and, in addition, compare them with anti-hCT produced in goats and mice. The quantity and quality of the antibodies obtained after multiple injections totalling 12.6, 3.6 and 2.6 mg hCT are shown in Fig. 3 A, and C, respectively. It is evident that the antibody titers were inversely related to the
——. .
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1CW6 1990
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1989
%
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20
t
t t
40
t
0.1 0.5 1.0 10
80
t 5.0
100 120
140
t
5.0
20
t
t
40
60
80
!
t
t
100
120
140
160
180
20
t
t
40
60
t
t
BO
K>0
120
(40
20
40
60
80
TOO DAYS
t
HUMAN
CALCITONIN INJECTED (mg)
005
Fig. 3. quantity and quality of rabbit antibodies to synthetic hCT. Reciprocal dilutions-r,0 indicating serum concentrations which bind 50 % of 123I-hCT are given in the top panels. ID5{)-values indicating a 50 % reduction of the specific binding of 125I-hCT to anti-hCT are given in the bottom panels. The abscissa denotes days after the first injection of the antigen. Antigen administrations are indicated by arrows. In experiments A and (except for the last two injections in A) hCT was adsorbed onto a tenfold amount of Norit A and incorporated into cFA. In C and D, hCT was adsorbed onto Alhydrogel and incorporated into cFA. In A, and C, the first in¬ jection (0.2 ml of adjuvant mixture) was made directly into the inguinal lymph nodes, the second (1.0 ml) im, the third (2.0 ml) into four foot pads, and the fourth and the fifth (Group B) sc at multiple sites. In experiment A, the last two injections were made iv; in this case, hCT was dissolved in 0.5 ml 0.01 m phosphate-buffered (pH 7.2) NaCI (0.14 m). Finally, in experiment D, 0.5 ml adjuvant mixture was injected into 6-8 intradermal sites. Simultaneously, the rabbits received 1 ml B. pertussis vaccine sc. Time-course of
Rabbits 152, 153 and 156
were
immunized with 0.5 mg hCT and rabbits 155, 157 and 158 with 0.05 mg hCT.
dose. The highest dilutions-50 were seen in group C around Day 140, after four injections totalling 2.6 mg hCT. Judged from the ID30-values, several antisera of group C also exhibited the greatest affinity for hCT. A single injection of 0.5 or 0.05 mg hCT still induced the formation of anti¬ bodies (Fig. 3), whereas 0.005 mg was not immunogenic. The antibody titers obtained after a single dose of submilligram amounts of hCT were rather low. Reciprocal dilutions-50 ranged from 50 to 180. On the other hand, the ID5o obtained with serum from rabbit 152 was remarkably low, decreasing progres¬ sively from flOO pg on Day 54 to 140 pg 103 days after immunization with 0.5 mg hCT. Table 1 summarizes the results of immunochemical analysis of 6 high-titer antisera from rabbits immunized with total doses of 3.6 and 2.6 mg hCT (see
antigen
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lOCOTf|iOCOqcO ) 75. Eisen . . Sc Siskind G. W.: Biochemistry 3 (1964) 996. Fischer J. ., Binswanger U. Sc Dietrich F. M.: J. clin. Invest. 54 (1974) 1382. Frolich M., Kassenaar A. A. H. Sc Smeenk D.: Horm. Metab. Res. 3 (1971) 297. Gelzer J.: Immunochemistry 5 (1968) 23. Goidl . ., Paul W. E., Siskind G. W. & Benacerraf B.: J. Immunol. 100 (1968) 371. Goltzman D., Potts J. T. Jr., Ridgway C E. Se Maloof F.: New Engl. J. Med. 290
(1974)
1035.
L. J. & Potts J. T. Jr.: Clin. chim. Acta 39 (1972) 407. Hackeng W. H. L., Schellekens A. P. M. & Schopman W'.: Horm. Metab. Res. 2 (1970) 311. Hennessy J. F., Wells S. A. Jr., Ontjes D. A. 8c Cooper C. W'.: J. clin. Endocr. 39 (1974) 487. Herbert V., Lau K. S., Gottlieb C. W. 8c Bleicher S. J.: J. clin. Endocr. 25 (1965) 1375. Hesch R. D., Woodhead J. S., Hüfner M. Se Dietrich F. M.: Acta endocr. (Kbh.) Suppl. 773 (1973) 161. Hcynen G. Se Franchimont P.: Europ. J. clin. Invest. 4 (1974) 213. Hüfner M. Se Hesch R. D.: Klin. Wschr. 49 (1971) 1149. Hunter W. M. Se Greenwood F. C: Nature (Lond.) 194 (1962) 495. Kaplan E. L., Sizemore G., Hill B. J. 8e Peskin G. W.: Clin. Res. 20 (1972) 724 (Abstract). Kraus L. M.: J. Immunol. 99 (1967) 894. Lee M. R., Deftos L. J. Sc Potts J. T. Jr.: Endocrinology 84 (1969) 36. Lequin R. M., Hackeng W. H. L. Sc Schopman W.: J. Endocr. 44 (1969) 283. Melvin K. E. W., Tashjian A. H. & Miller H. H.: Recent Progr. Hormones Res. 28 (1972) 399. Milhaud G., Tharaud D., Julienne A. 8c Moukhtar M. S. In: Taylor S., Ed. Endo¬ crinology 1971. W. Heinemann Medical Books, London (1972c?) 380. Milhaud G., Calmette C, Julienne ., Tharaud D., Bloch-Michel H., Cavaillon J. P., Colin R. 8e Moukhtar M. S. In: Talmage R. V. and Munson P. L., Eds. Calcium, Parathyroid Hormone and the Calcitonins. Internat. Congr. Series, Vol. 243. Excerpta med. (Amst.) (1972&) 56. Milhaud G., Calmette C, Taboulel J., Julienne A. Se Moukhtar M. S.: Lancet 1 (1974) Habener
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Pento
J. T.,
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Received
on
December 3rd, 1974.
