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Journal of Immunological Methods 202 1997 173–182
Immunochemical approach to the mapping of an assembled
epitope of human chorionic gonadotropin: proximity of CTP-a to
the receptor binding region of the b-subunit
N. Venkatesh, G.S. Murthy
)
Primate Research Laboratory, Centre for ReproductiÕe Biology and Molecular Endocrinology, Indian Institute of Science, Bangalore 560
012, India
Received 4 June 1996; revised 21 October 1996; accepted 17 December 1996
Abstract
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A single-step solid-phase RIA SS-SPRIA developed in our laboratory using hybridoma culture supernatants has been
utilised for the quantitation of epitope–paratope interactions. Using SS-SPRIA as a quantitative tool for the assessment of
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epitope stability, it was found that several assembled epitopes of human chorionic gonadotropin hCG are differentially
stable to proteolysis and chemical modification. Based on these observations an approach has been developed for identifying
the amino acid residues constituting an epitopic region. This approach has now been used to map an assembled epitope
atrnear the receptor binding region of the hormone. The mapped site forms a part of the seat belt region and the cystine knot
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region C34–C38–C88–C90–H106 . The carboxy terminal region of the a-subunit forms a part of the epitope indicating its
proximity to the receptor binding region. These results are in agreement with the reported receptor binding region identified
through other approaches and the X-ray crystal structure of hCG.
Keywords: Monoclonal antibody; Human chorionic gonadotropin; Single-step solid-phase radioimmunoassay; Assembled epitope; Carboxy
terminal region; Epitope mapping
1. Introduction
The interaction of paratope with epitope as seen
in an antigen–antibody reaction represents a very
Abbreviations: hCG, human chorionic gonadotropin; hLH,
human luteinising hormone; SS-SPRIA, single-step solid-phase
radioimmunoassay; MAb, monoclonal antibody; TNBS, trini-
trobenzene sulphonic acid; TNM, tetranitromethane; EDAC, 1-
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ethyl-3- 3-dimethyl-aminopropyl carbodiimide; DEPC, diethylpy-
rocarbonate; PG, phenylglyoxal; CTP, carboxy terminal region
)
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Corresponding author. Tel.: q91-80 309-2490 or 3344411,
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ext. 2490; Fax: q91-80 334-1683; e-mail:
nvenkat@hamsadvani.serc.iisc.ernet.in
specific type of protein–protein interaction exclu-
sively dependent upon the three-dimensional struc-
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ture of the protein Davis et al., 1990 . This general
specificity is further increased with a monoclonal
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antibody MAb capable of interacting exclusively
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with a discrete region of the antigen Ag molecule
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epitopes or antigenic determinants Sela, 1969; Van
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Regenmortel, 1992 . Epitopes are essentially confor-
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mation-specific Barlow et al., 1986; Laver et al.,
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1990 and even those which are termed sequence-
specific are directed against the conformation of the
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identified sequence Berzofsky, 1985; Leder and
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Bosshard, 1994 . The present methods of epitope
0022-1759r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved.
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PII S0022-1759 96 00246-3