- Email: [email protected]
Utilization of synthetic peptides for the study of calcitonin and biosynthetic precursors for calcitonin
Nucl.
Int.
Med.
Bial.
J. Radiat.
Printed
Vo1.14,
No.4.
pp.289-294,
1987
in Great
Pergamon
$3.00~0.00 Journals
Britain
UTILIZATION
OF SYNTHETIC PEPTIDFS FOR THE STUDY OF CALCITONIN
BIOSYNTHETIC
PhilipFe
0883-2897/87
Appl. Instrum.Part B
Vott6,
PRECURSORS
Pascal Vauzelle, GilLes Alberici, Malika Ait-Abdellar, DominiqL,e Ballet
Unite d'Immunochimie,
AND
FOR CALCITONIN
Inatitut Gustave-Roussy,
Claude Eohuon,
94805 Villejuif, France
APSTRACT -synthetic peptides and a libr?#ry of moncclonal anti-peptide antibodies, By using we have developed a pare1 of techniques that allow the dissection of circulating immunoreactive calcitonin in the serum. C Cells of the thyroid were found to release both and biosynthetic intermediates in the circulation. mature calcitonin Finally, these products were found to circulate as heterogenous molecular species. A methodology for the standardization of the measurement of calcitonin is proposed in the form of a two-site immunoradiometric assay specific for mature calcitonin.
INTRODUCTION
Amidated polypeptide hormones usually derive biosynthetically from the processing The sequence of the precursor comprises specific sites that are of a preprohormone. More recognized by specific enzymes resulting in proteolytic cleavage of the molecule. particularly, a cleavage amidation signal X-Gly-Y-Y (in which X will be the amino acid residue at the C-terminus of the mature hormone, and Y is a basic amino acid such as Lys or Arg) is found at the C-terminus of the hormone backbone inside the prohormone molecule. This signal is an efficient precursor for amidated polypeptides and the active hormone will bear X-CONHg as the C-terminal residue (Gomez, 19841. (CT) in C cells of the thyroid follows such a The biosynthesis of calcitonin sequence is pathway (Gkonos, 1986). From the N- to the C-terminus, the preprocalcitonin composed of: (i) a N-terminal region terminated by a Lys-Arg site, (ii) the 32 amino acidlong calcitonin sequence separated by the signal Gly-Lys-Lys-Arg from (iii) the katacalcin (KC) sequence that comprises 21 amino acids (Le Moullec, 1984). The post-translational events leading to the release of CT take place in various segments of the endoplasmic Thyroid C cells will secrete reticulum, the Golgi apparatus, and the secretory granules. calcitonin in response to hypercalcemia or to exogenous stimulation, e.g. the injection of twenty-five was discovered a dose of pentagastrin (Austin, 1981‘1. Although calcitonin were socn available (Tashjian, years ago, and radioimmunoassays 19701, the precise composition of the secretory products released by C cells in the circulation remained to be characterized: the immunoreactive profile of calcitonin in the serum is typically molecular weight are detected defined as he'cerogenous since molecular species of various by polyclonal antisera (Bikle, 1983). In an attempt to identify these products, we have peptides and monoclonal antideveloped several techniques based on the use of synthetic peptide antibodies.
MATERIAL AND METHODS p--
Synthetic
peptides, -and hapten conjugates.
analogous to various fragments of calcitonin and A panel of synthetic peptides preprocalcitonin, either native or modified sequence, were synthesized by the solid phase method (Merrifield, 1986) using an Applied Biosystems (Foster-City, peptide CA) The Calcitonin 1-32 was a gift from Ciba-Geigy (Basel, Switzerland). synthesizer.
289
Ltd
P. Mott6 -. et al
290
sequences of these peptides are depicted in table 1. Some of these peptides conjugated to tetanus toxoid (TT) using glutaraldehyde as a coupling agent.
Table 1: ~~~~ acid sequences -_ One letter code was used.
of the CT- related synthetic
peptides
used
were
in this study.
CT l-32 CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP CT l-10 CGNLSTCMLG TYTQDFNKFHTFP CT 11-23 NKFHTFPQTAIGVGAP CT 17-32 QTAIGVGAP CT 24-32 AIGVGAP CT 26-32 QTAIGVGAPGKKRDMSSDLERDHRPHVSMPQNAN PQN-34 TYTQDFNKFHTFPQTAIGVGAPGKKRDMSSDLERDHRPHVSMPQNAN PTN-47 YDMSSDLERDHRPHVSMPQNAN Tyr-KC DMSSDLERDHRPHVSMPQNAN 1-21 KC DMSSDLERDHR l-11 KC PHVSMPQNAN KC 12-21
Production and characterization of monoclonal anti-peptide antibodies. Splenocytes from mice immunized using calcitonin 1-32, katacalcin l-21, and PQN-34 as haptens were utilized for the obtaining of monoclonal antibodies. For this purpose, NSI myeloma cell line was chosen as the fusion partner and hybridoma production was performed as previously described (Bellet, 1983). The culture supernatant of hybridoma was tested for the presence of specific antibodies in an enzyme-linked immunosorbent assay (ELISA) as previously described (Bidart, 1985). After cloning of the cell lines, monoclonal antibody (mab) expansion was performed by inoculation into Nude mice. Mabs were purified from the ascites fluids using protein-A affinity chromatography, and the isotype of the monoclonal immunoglobulins was determined as previously reported (Manil, 1986). The affinity constant of monoclonal antibodies for 125I-CT or 125I-Tyrosyl-KC (Matte, 1986) was evaluated from F binding data according to the method of Van Heyningen (1983).
Epitope mapping of monoclonal antibodies. The antigenic determinant recognized by the monoclonal antibodies was determined using two different but complementary techniques. First, the binding of monoclonal antibodies to synthetic peptides was tested in a direct binding assay by using a solid phase ELISA and CT-, PQN-34-, and PTN-47- coated plates; the antibody binding was detected using peroxidase labeled rabbit anti-mouse antiserum and o-phenylene diamine as a substrate. Second, hapten inhibition experiments were performed. In this technique, the binding Of a monoclonal antibody to a synthetic peptide linked to a solid phase support was inhibited by various related amino acid sequences. Percent inhibition of binding was calculated as previously reported (Bidart, 1985), and from these data, the inhibiting concentration 50% (IC50, i.e. the concentration of competitor peptide inhibiting 50% of the antibody binding to the immobilized peptide) was determined (Matte, 1987a). Development of multisite immunoradiometric m
_-with predetermined specificity,
We have constructed several multisite immunoradiometric assay in a format currently used for sensitive and specific assays in our laboratory (Ballet, 1984, 1986). A specific "capture" solid phase was prepared by coating polystyrene beads with a 7:500 dilution of mab-containing ascites fluid overnight at room temperature. Then, this solid phase was incubated simultaneously with the sample (standard or serum) and a second monoclonal antibody ("indicator antibody") radiolabeled with 1251. The reaction was allowed to sit overnight at room temperature and the radioactivity bound to the solid phase was measured in a gamma Counter. The specificity of the assay was dependent on the mab combination used. Several types of assay were developed that were specific for different circulating species (see results).
RESULTS
Production of monoclonal antibodies From the
fusions
performed with splenocytes from mice immunized with CT-TT, PQN-
VlIth InternationalSymposium on Radioimnunology
291
34-TT or KC-TT, 1430 hybridoma ware grown, and among them we selected a library of antibodies that recognized distinct epitopes. Two epitopes were found to be expressed on the molecule of calcitonin (Mot&, 1985), and two epitopes were localized on the molecule of katacalcin. Several antibodies ware produced against these epitopes and this report will describe the results obtained with the highest affinity antibodies. The table 2 describes the immunochemical characteristics of these antibodies.
Table 2: Immunochemical -study. Monoclonal Antibody
characteristics
of the monoclonal
Affinity constant (M-1)
Isotype
selected
in
this
Antibody binding site CT 26-32 CT 11-17 KC 1-11 KC 12-21
0.9xlO~O
IgG2 IgCl IgGl IgGl
CT07 CT08 KC0 1 KC04
antibodies
::;::$" 3.0~108
The binding of these antibodies to peptide-linked solid phase was assessed using the ELISA technique. The results of such experiments are depicted in table 3.
-1
: Binding of monoclonal
antibodies
to synthetic
peptide-coated
plates
in
the
ELISA. Monoclonal Antibody
Coated
cT 1-32
CT07 CT08 KC01 KC04
1.805 1.950
peptide
PQN-34
PTN-47
KC I-21 < 0.010
Results are expressed as absorbance at 492 nm after subtraction background. Monoclonal antibodies were tested at a I:104 dilution specific supernatant.
of the non of culture
From these results, we concluded that the CT 11-17, KC l-11, and KC 12-21 epitopes for antibody binding both to mature calcitonin (CT 17-17) or free were accessible katacalcin (KC 1-11, KC 12-211, and to peptides with sequence analogous to biosynthetic precursors for calcitonin. In contrast, the CT 26-32 epitope was found to be expressed analogous to calcitonin only on mature calcitonin and silent on peptides with sequence 1987b) that this selectivity was biosynthetic precursors. We have demonstrated (Matte, attributable mainly to the presence of a carboxamide group at the C-terminus of calcitonin that is absent when the calcitonin sequence is included in the larger molecular weight Then, monoclonal CT07 antibody could be used as a tool for the specific precursors. recognition of the mature calcitonin molecule in the presence of other species. Monoclonal
immunoradiometric
assays.
Based on these results, we have developed several types of assay. For the specific detection of mature calcitonin, we used CT07 as a capture mab and CT08 as an indicator mab (combination CT07-CT08) in the format described in the Material and Methods section. According to the construction of this assay, no fragments of calcitonin could be detected. The limit of detection of this assay was 10pglml and gel filtration analyses showed that only mature calcitonin was detected in the serum of patients bearing medullary carcinoma of the thyroid (Matte, 1987c). However, these studies established that mature calcitonin as heteregenous species in the serum and that these species were itself circulates composed of calcitonin polymerized and/or bound to irrelevant proteins. We have developed specific methods for the detection of calcitonin precursors in the biosynthetic pathway for calcitonin indicates that the the circulation. Indeed, katacalcin sequence is cleaved from the CT sequence only when calcitonin is amidated, i.e. during the final processing step. Then, methods based on the detection of the simultaneous
292
P. f4otte eta).
presence of a calcitonin and katacalcin epitope should have a predetermined specificity for calcitonin intermediates of biosynthesis. We have used several assays based on such involving the use of CT08 as an anticombinations and finally selected two constructions CT mab and KC01 as an anti-KC mab. Similar results were obtained by using CT06 as a capture mab and KC01 as an indicator (combination CT08-KCOl) or by using KC01 as a capture These assays have been found to mab and CT08 as an indicator mab (combination KCOI-CT08). detect two molecular species in the circulation (data not shown): OXIt? with a molecular weight around 14kD in agreement with the expected molecular weight for procalcitonin and a the intermediate second with a molecular weight around 8kD that could represent of 57 amino acid composed of calcitonin linked to the katacalcin sequence. These species were also found to circulate as heterogenous species either polymerized or linked to irrelevant proteins and displayed higher apparent molecular weight. The limit of detection of these assays was 5pgiml of synthetic PTN-47 peptide. Effect of pentagastrin --
stimulation
m
p atients with medullary carcinoma -of the
thyroid. In some patients with medullary carcinoma of the thyroid, we have studied the effect of pentagastrin injection. We have found that this stimulation resulted in the release of mature calcitonin and of biosynthetic intermediates in the serum. However, the amount of precursor released in the serum was far less than the amount of mature calcitonin.
DISCUSSION
The assay for calcitonin is the most reliable method for establishing the diagnosis of medullary carcinoma of the thyroid (Milhaud, 1968; Melvin, 1968). Moreover, as the calcitonin level may be increased before any clinical evidence of the tumor, the measurement of calcitonin for the detection of medullary carcinoma of the thyroid at a preclinical stage is one of the best example of a Laboratory test in preventative medicine. Measurements of calcitonin have been found different among laboratories because each antiserum recognizes different species bearing a molecular calcitonin immunoreactivity in the serum of the patients. In other respects, the level of circulating calcitonin is very low (in the pg/ml range) and sensitive methods are needed. In an attempt to standardize the assay method for calcitonin and to identify circulating species Of calcitonin, we have developed a library of monoclonal antibodies directed against calcitonin and katacalcin binding site by using synthetic and mapped their epitope peptides analogous to different fragments of calcitonin, katacalcin and procalcitonin. This approach enabled us to develop an assay specific for mature,calcitonin. This assay uses mab CT07 as the solid phase capture antibody. That antibody binds to an antigenic determinant that is only expressed on the mature form of calcitonin. This assay is sensitive (lOpg/ml) and provides a useful tool for the early diagnosis of medullary carcinoma of the thyroid (Mott.6, 1987c). It was noteworthy that this assay detects mature calcitonin as different molecular forms in the serum. The sequence of calcitonin includes two cystein residues that may be used for inter-molecule disulfide linkage. Indeed, high molecular weight products may be resolved into monomeric calcitonin by reduction and alkylation. These high molecular products are also bound to irrelevant proteins. It was not possible from our experiments to conclude whether these forms are stored in secretory granules or result from chemical transformation in the serum. However, these data show that monomeric calcitonin does not represent mature calcitonin in the serum. Then, extraction methods (Body, 1983) that enrich monomeric calcitonin in the serum sample by using a complex chromatography procedure evaluate only a fraction of mature calcitonin in the serum. As mature CT is the active form of calcitonin since the C-terminal prolinamide residue is mandatory for biological activity, the CT07-CT08 assay might approach the measurement of biologically active CT in the serum. It was then necessary to identify forms of calcitonin the other circulating detected by classical radioimmunoassays. The biosynthesis of calcitonin involves several proteolytic steps that result in the release of mature CT from its precursor. The Cterminal flanking peptide katacalcin (KC) is removed only during the final step when the C-terminal proline residue of CT is amidated. it was conceivable to develop assay Then, techniques for CT biosynthetic precursors based on the simultaneous identification of a calcitonin epitope and a katacalcin epitope. We have developed such techniques and selected the CTOE-KC01 and KCCl-CT06 combinations because of their efficient binding to the PTN-47 peptide. These assays were found to detect molecular species in the serum with molecular weight in agreement with the hypothesis of a detection of biosynthetic precursors for calcitonin. Again, these precursor intermediates were found to circulate as h&erogenous species resulting form disulfide polymerization and Linkage to irrelevant
VIlth !~teroatlanal jymposiumon Padlo-mmunology
293
proteins. In conclusion, C cells release mature calcitonin as well as biosynthetic precursors for calcitonin in the serum. As pentagastrin stimulation raises the level of circulating biosynthetic precursors in the serum, all these forms are likely to be stored in the secretory granules. We have demonstrated that mature CY and precursors circulate as heterogenous species resulting fora i;he protein-protein interactions. It is not clear at present time whether tnese high molecular species are only released fron C cells or are also synthesized in the serum. By using monoclonal antibodies we tave dissected the and syntketic peptides, immunoreactive circulating calcitonin in the serum of patients with medullary carcinoma of the thyroid and we propose a technique for the measurement of calcitonin that is practicabie and sensitive. This methodolsgy should help the standardization of serum CT measurements among laboratories.
REFESENCES
Austin LA. Heath HIII.
(1981) Calcitonin,
physiology
and pathclegy,
N Engl J e,
33il:269
H, Troalen F, Bohuon C. Wands ;F. Bidart JM, Ozturk ?1, Bellet D, Jolivet M, Gras-Masse (1985) Identification of epitopes associated with hCG and :he p-hCG carboxyl terminus by monoclonai antibodies produced against a synthetic peptide. J Immunol, '34:"57 Bikle DD. (1983) Assay of caicium regulating -. hormones. Bellet D, Schlumberger M, Bidart Z., Assicot M, i r. vitro utilization (1983) Production and thyroglobulin. -J Clin Endocrinoi Metab, 58:530
Springer Verlag
(New- York)
Caillou B, Mott& P, Vignal A, Bonuon C. antibodies human of monoclonal :o
Bell& D, Wands JFC, Isselbacher K, Bohuon C. (1984) Serum alpha-fetoprotein radioimmunoassay. human disease: perspective from a highly specific monoclonal Acad Sci ('&A), al:3869 ---
levels in Proc Nat1 -__
Bellet D, Ozturk M. Bidart JM, Bohuon C, Wands J9. (1986) Sensitive and specific assay for and anti-hCG monoclonal gonadotropin (hCG) based on anti-peptide charionic human 63:1319 antibodies: construction and clinical implications. -__ J Clin Endocrincl -3 Metab cf circulating monomeric calcitonin: Body JJ, Heath H III. (
physiological
Gkonos PL, Born W, Jones BN, Petermann JB, Keutir.annHT. Birnbaum RS, Fisher ,'A, Roos BA. (1986) Biosycthesis of calcitonin-gene related peptide and calcitonin by a human medullary carcinoma cell line. --_--..--! J Biol Chem 261:14386 JY, Milhaud F, Guliana Le Moullec JM, Ju1ienr.e A, Chenais J, Lasmoles (1984) The complete sequence of human preprocaicitonin. FEBS lett, 767:93
G.
Manil L, Mot'& P, Pernas P, Troalen F, Eohuon C, Bellet D. (1986) Evaiuation for purification of mouse monoclonal anzibodies. J Immurol gethods , 90:'25 Melvin KEW, Tashjian AH. 11968) The syndrome of excessive medullarJi carinoma of the thyroid &vc _--_) Nat1 Acad Sci (USA) Merrifield
F,N, Solid phase synthesis.
(1960) Science,
thyrocalcitonin 59:1216
Moukhtar M.
of protocols
produced
by
232:341
Milhaud G, Tubiana M, Parmentier C, Coutris G. (1966) Epithblioma de la thyrocalcitonine. ---A CR Acad Sci [Paris) 266:608
de la thyroide secr6tant
M, Pernas P, Bohuon C, Bellet D. Mott.6 P, Vauzelle P, Caillou 3, Troalen ?', Moukhtar (1985) Production of monoclonal antibodies directed against distinct epitopes of human 43:59h calcitonin. ----..---I Ann Biol Clin D. (1986) Fadioiodination Mot& P, Vauzelle P, Alberici G, Troalen F, Bohuon C, Bell& human calcitonin using the Iodogen reagent. -~ J Immunol Methods, 87:223
cf
P, Bohuon C, Bellet D. (1987~1) Mottd P. VZlUZelie P, Ait-Rbdellah M, Alberici G, Gardet port&s Application i la des d6terminants antig&iques Etude Par la calcitonine. construction d'un dosage immunoradiom&trique -~ Act Pharm Eioi Clin, in press
294
P. Mott; et al -.
Matte P, Alberici G, Ait-Abdellah M, Bellet D. (1987b) Monoclonal antibodies distinguish synthetic peptides which differ in one chemical group. J Immunol, in press Matte P, Ait-Abdellah M, Vauzelle P, Gardet P, Bohuon C, Bellet D. (1987~) A two-site immunoradiometric assay for serum calcitonin using monoclonal anti-peptide antibodies. Henri Ford Hasp Med J, in press -Tashjian AH, Howland BG, Melvin KEW, Stanton-Hill C. (1970) Immunoassay of human J Med calcitonin and studies in patients with medullary carcinoma of the thyroid. N Engl _-1 283:890 Van Heyningen V, Brock DJH, Van Heyningen S. (1983) A simple .~ affinities of monoclonal antibodies. -~ J Immunol L Methods 62:147
method
for
ranking
the
Correspondence and requests for reprints should be addressed to Philippe Motti, Ph.D., present address: Gastrointestinal Unit, Massachusetts General Hospital, Boston (MA 02114)
Int.
Med.
Bial.
J. Radiat.
Printed
Vo1.14,
No.4.
pp.289-294,
1987
in Great
Pergamon
$3.00~0.00 Journals
Britain
UTILIZATION
OF SYNTHETIC PEPTIDFS FOR THE STUDY OF CALCITONIN
BIOSYNTHETIC
PhilipFe
0883-2897/87
Appl. Instrum.Part B
Vott6,
PRECURSORS
Pascal Vauzelle, GilLes Alberici, Malika Ait-Abdellar, DominiqL,e Ballet
Unite d'Immunochimie,
AND
FOR CALCITONIN
Inatitut Gustave-Roussy,
Claude Eohuon,
94805 Villejuif, France
APSTRACT -synthetic peptides and a libr?#ry of moncclonal anti-peptide antibodies, By using we have developed a pare1 of techniques that allow the dissection of circulating immunoreactive calcitonin in the serum. C Cells of the thyroid were found to release both and biosynthetic intermediates in the circulation. mature calcitonin Finally, these products were found to circulate as heterogenous molecular species. A methodology for the standardization of the measurement of calcitonin is proposed in the form of a two-site immunoradiometric assay specific for mature calcitonin.
INTRODUCTION
Amidated polypeptide hormones usually derive biosynthetically from the processing The sequence of the precursor comprises specific sites that are of a preprohormone. More recognized by specific enzymes resulting in proteolytic cleavage of the molecule. particularly, a cleavage amidation signal X-Gly-Y-Y (in which X will be the amino acid residue at the C-terminus of the mature hormone, and Y is a basic amino acid such as Lys or Arg) is found at the C-terminus of the hormone backbone inside the prohormone molecule. This signal is an efficient precursor for amidated polypeptides and the active hormone will bear X-CONHg as the C-terminal residue (Gomez, 19841. (CT) in C cells of the thyroid follows such a The biosynthesis of calcitonin sequence is pathway (Gkonos, 1986). From the N- to the C-terminus, the preprocalcitonin composed of: (i) a N-terminal region terminated by a Lys-Arg site, (ii) the 32 amino acidlong calcitonin sequence separated by the signal Gly-Lys-Lys-Arg from (iii) the katacalcin (KC) sequence that comprises 21 amino acids (Le Moullec, 1984). The post-translational events leading to the release of CT take place in various segments of the endoplasmic Thyroid C cells will secrete reticulum, the Golgi apparatus, and the secretory granules. calcitonin in response to hypercalcemia or to exogenous stimulation, e.g. the injection of twenty-five was discovered a dose of pentagastrin (Austin, 1981‘1. Although calcitonin were socn available (Tashjian, years ago, and radioimmunoassays 19701, the precise composition of the secretory products released by C cells in the circulation remained to be characterized: the immunoreactive profile of calcitonin in the serum is typically molecular weight are detected defined as he'cerogenous since molecular species of various by polyclonal antisera (Bikle, 1983). In an attempt to identify these products, we have peptides and monoclonal antideveloped several techniques based on the use of synthetic peptide antibodies.
MATERIAL AND METHODS p--
Synthetic
peptides, -and hapten conjugates.
analogous to various fragments of calcitonin and A panel of synthetic peptides preprocalcitonin, either native or modified sequence, were synthesized by the solid phase method (Merrifield, 1986) using an Applied Biosystems (Foster-City, peptide CA) The Calcitonin 1-32 was a gift from Ciba-Geigy (Basel, Switzerland). synthesizer.
289
Ltd
P. Mott6 -. et al
290
sequences of these peptides are depicted in table 1. Some of these peptides conjugated to tetanus toxoid (TT) using glutaraldehyde as a coupling agent.
Table 1: ~~~~ acid sequences -_ One letter code was used.
of the CT- related synthetic
peptides
used
were
in this study.
CT l-32 CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP CT l-10 CGNLSTCMLG TYTQDFNKFHTFP CT 11-23 NKFHTFPQTAIGVGAP CT 17-32 QTAIGVGAP CT 24-32 AIGVGAP CT 26-32 QTAIGVGAPGKKRDMSSDLERDHRPHVSMPQNAN PQN-34 TYTQDFNKFHTFPQTAIGVGAPGKKRDMSSDLERDHRPHVSMPQNAN PTN-47 YDMSSDLERDHRPHVSMPQNAN Tyr-KC DMSSDLERDHRPHVSMPQNAN 1-21 KC DMSSDLERDHR l-11 KC PHVSMPQNAN KC 12-21
Production and characterization of monoclonal anti-peptide antibodies. Splenocytes from mice immunized using calcitonin 1-32, katacalcin l-21, and PQN-34 as haptens were utilized for the obtaining of monoclonal antibodies. For this purpose, NSI myeloma cell line was chosen as the fusion partner and hybridoma production was performed as previously described (Bellet, 1983). The culture supernatant of hybridoma was tested for the presence of specific antibodies in an enzyme-linked immunosorbent assay (ELISA) as previously described (Bidart, 1985). After cloning of the cell lines, monoclonal antibody (mab) expansion was performed by inoculation into Nude mice. Mabs were purified from the ascites fluids using protein-A affinity chromatography, and the isotype of the monoclonal immunoglobulins was determined as previously reported (Manil, 1986). The affinity constant of monoclonal antibodies for 125I-CT or 125I-Tyrosyl-KC (Matte, 1986) was evaluated from F binding data according to the method of Van Heyningen (1983).
Epitope mapping of monoclonal antibodies. The antigenic determinant recognized by the monoclonal antibodies was determined using two different but complementary techniques. First, the binding of monoclonal antibodies to synthetic peptides was tested in a direct binding assay by using a solid phase ELISA and CT-, PQN-34-, and PTN-47- coated plates; the antibody binding was detected using peroxidase labeled rabbit anti-mouse antiserum and o-phenylene diamine as a substrate. Second, hapten inhibition experiments were performed. In this technique, the binding Of a monoclonal antibody to a synthetic peptide linked to a solid phase support was inhibited by various related amino acid sequences. Percent inhibition of binding was calculated as previously reported (Bidart, 1985), and from these data, the inhibiting concentration 50% (IC50, i.e. the concentration of competitor peptide inhibiting 50% of the antibody binding to the immobilized peptide) was determined (Matte, 1987a). Development of multisite immunoradiometric m
_-with predetermined specificity,
We have constructed several multisite immunoradiometric assay in a format currently used for sensitive and specific assays in our laboratory (Ballet, 1984, 1986). A specific "capture" solid phase was prepared by coating polystyrene beads with a 7:500 dilution of mab-containing ascites fluid overnight at room temperature. Then, this solid phase was incubated simultaneously with the sample (standard or serum) and a second monoclonal antibody ("indicator antibody") radiolabeled with 1251. The reaction was allowed to sit overnight at room temperature and the radioactivity bound to the solid phase was measured in a gamma Counter. The specificity of the assay was dependent on the mab combination used. Several types of assay were developed that were specific for different circulating species (see results).
RESULTS
Production of monoclonal antibodies From the
fusions
performed with splenocytes from mice immunized with CT-TT, PQN-
VlIth InternationalSymposium on Radioimnunology
291
34-TT or KC-TT, 1430 hybridoma ware grown, and among them we selected a library of antibodies that recognized distinct epitopes. Two epitopes were found to be expressed on the molecule of calcitonin (Mot&, 1985), and two epitopes were localized on the molecule of katacalcin. Several antibodies ware produced against these epitopes and this report will describe the results obtained with the highest affinity antibodies. The table 2 describes the immunochemical characteristics of these antibodies.
Table 2: Immunochemical -study. Monoclonal Antibody
characteristics
of the monoclonal
Affinity constant (M-1)
Isotype
selected
in
this
Antibody binding site CT 26-32 CT 11-17 KC 1-11 KC 12-21
0.9xlO~O
IgG2 IgCl IgGl IgGl
CT07 CT08 KC0 1 KC04
antibodies
::;::$" 3.0~108
The binding of these antibodies to peptide-linked solid phase was assessed using the ELISA technique. The results of such experiments are depicted in table 3.
-1
: Binding of monoclonal
antibodies
to synthetic
peptide-coated
plates
in
the
ELISA. Monoclonal Antibody
Coated
cT 1-32
CT07 CT08 KC01 KC04
1.805 1.950
peptide
PQN-34
PTN-47
KC I-21 < 0.010
Results are expressed as absorbance at 492 nm after subtraction background. Monoclonal antibodies were tested at a I:104 dilution specific supernatant.
of the non of culture
From these results, we concluded that the CT 11-17, KC l-11, and KC 12-21 epitopes for antibody binding both to mature calcitonin (CT 17-17) or free were accessible katacalcin (KC 1-11, KC 12-211, and to peptides with sequence analogous to biosynthetic precursors for calcitonin. In contrast, the CT 26-32 epitope was found to be expressed analogous to calcitonin only on mature calcitonin and silent on peptides with sequence 1987b) that this selectivity was biosynthetic precursors. We have demonstrated (Matte, attributable mainly to the presence of a carboxamide group at the C-terminus of calcitonin that is absent when the calcitonin sequence is included in the larger molecular weight Then, monoclonal CT07 antibody could be used as a tool for the specific precursors. recognition of the mature calcitonin molecule in the presence of other species. Monoclonal
immunoradiometric
assays.
Based on these results, we have developed several types of assay. For the specific detection of mature calcitonin, we used CT07 as a capture mab and CT08 as an indicator mab (combination CT07-CT08) in the format described in the Material and Methods section. According to the construction of this assay, no fragments of calcitonin could be detected. The limit of detection of this assay was 10pglml and gel filtration analyses showed that only mature calcitonin was detected in the serum of patients bearing medullary carcinoma of the thyroid (Matte, 1987c). However, these studies established that mature calcitonin as heteregenous species in the serum and that these species were itself circulates composed of calcitonin polymerized and/or bound to irrelevant proteins. We have developed specific methods for the detection of calcitonin precursors in the biosynthetic pathway for calcitonin indicates that the the circulation. Indeed, katacalcin sequence is cleaved from the CT sequence only when calcitonin is amidated, i.e. during the final processing step. Then, methods based on the detection of the simultaneous
292
P. f4otte eta).
presence of a calcitonin and katacalcin epitope should have a predetermined specificity for calcitonin intermediates of biosynthesis. We have used several assays based on such involving the use of CT08 as an anticombinations and finally selected two constructions CT mab and KC01 as an anti-KC mab. Similar results were obtained by using CT06 as a capture mab and KC01 as an indicator (combination CT08-KCOl) or by using KC01 as a capture These assays have been found to mab and CT08 as an indicator mab (combination KCOI-CT08). detect two molecular species in the circulation (data not shown): OXIt? with a molecular weight around 14kD in agreement with the expected molecular weight for procalcitonin and a the intermediate second with a molecular weight around 8kD that could represent of 57 amino acid composed of calcitonin linked to the katacalcin sequence. These species were also found to circulate as heterogenous species either polymerized or linked to irrelevant proteins and displayed higher apparent molecular weight. The limit of detection of these assays was 5pgiml of synthetic PTN-47 peptide. Effect of pentagastrin --
stimulation
m
p atients with medullary carcinoma -of the
thyroid. In some patients with medullary carcinoma of the thyroid, we have studied the effect of pentagastrin injection. We have found that this stimulation resulted in the release of mature calcitonin and of biosynthetic intermediates in the serum. However, the amount of precursor released in the serum was far less than the amount of mature calcitonin.
DISCUSSION
The assay for calcitonin is the most reliable method for establishing the diagnosis of medullary carcinoma of the thyroid (Milhaud, 1968; Melvin, 1968). Moreover, as the calcitonin level may be increased before any clinical evidence of the tumor, the measurement of calcitonin for the detection of medullary carcinoma of the thyroid at a preclinical stage is one of the best example of a Laboratory test in preventative medicine. Measurements of calcitonin have been found different among laboratories because each antiserum recognizes different species bearing a molecular calcitonin immunoreactivity in the serum of the patients. In other respects, the level of circulating calcitonin is very low (in the pg/ml range) and sensitive methods are needed. In an attempt to standardize the assay method for calcitonin and to identify circulating species Of calcitonin, we have developed a library of monoclonal antibodies directed against calcitonin and katacalcin binding site by using synthetic and mapped their epitope peptides analogous to different fragments of calcitonin, katacalcin and procalcitonin. This approach enabled us to develop an assay specific for mature,calcitonin. This assay uses mab CT07 as the solid phase capture antibody. That antibody binds to an antigenic determinant that is only expressed on the mature form of calcitonin. This assay is sensitive (lOpg/ml) and provides a useful tool for the early diagnosis of medullary carcinoma of the thyroid (Mott.6, 1987c). It was noteworthy that this assay detects mature calcitonin as different molecular forms in the serum. The sequence of calcitonin includes two cystein residues that may be used for inter-molecule disulfide linkage. Indeed, high molecular weight products may be resolved into monomeric calcitonin by reduction and alkylation. These high molecular products are also bound to irrelevant proteins. It was not possible from our experiments to conclude whether these forms are stored in secretory granules or result from chemical transformation in the serum. However, these data show that monomeric calcitonin does not represent mature calcitonin in the serum. Then, extraction methods (Body, 1983) that enrich monomeric calcitonin in the serum sample by using a complex chromatography procedure evaluate only a fraction of mature calcitonin in the serum. As mature CT is the active form of calcitonin since the C-terminal prolinamide residue is mandatory for biological activity, the CT07-CT08 assay might approach the measurement of biologically active CT in the serum. It was then necessary to identify forms of calcitonin the other circulating detected by classical radioimmunoassays. The biosynthesis of calcitonin involves several proteolytic steps that result in the release of mature CT from its precursor. The Cterminal flanking peptide katacalcin (KC) is removed only during the final step when the C-terminal proline residue of CT is amidated. it was conceivable to develop assay Then, techniques for CT biosynthetic precursors based on the simultaneous identification of a calcitonin epitope and a katacalcin epitope. We have developed such techniques and selected the CTOE-KC01 and KCCl-CT06 combinations because of their efficient binding to the PTN-47 peptide. These assays were found to detect molecular species in the serum with molecular weight in agreement with the hypothesis of a detection of biosynthetic precursors for calcitonin. Again, these precursor intermediates were found to circulate as h&erogenous species resulting form disulfide polymerization and Linkage to irrelevant
VIlth !~teroatlanal jymposiumon Padlo-mmunology
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proteins. In conclusion, C cells release mature calcitonin as well as biosynthetic precursors for calcitonin in the serum. As pentagastrin stimulation raises the level of circulating biosynthetic precursors in the serum, all these forms are likely to be stored in the secretory granules. We have demonstrated that mature CY and precursors circulate as heterogenous species resulting fora i;he protein-protein interactions. It is not clear at present time whether tnese high molecular species are only released fron C cells or are also synthesized in the serum. By using monoclonal antibodies we tave dissected the and syntketic peptides, immunoreactive circulating calcitonin in the serum of patients with medullary carcinoma of the thyroid and we propose a technique for the measurement of calcitonin that is practicabie and sensitive. This methodolsgy should help the standardization of serum CT measurements among laboratories.
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Correspondence and requests for reprints should be addressed to Philippe Motti, Ph.D., present address: Gastrointestinal Unit, Massachusetts General Hospital, Boston (MA 02114)