ANALYSIS OF A CONFORMATION-SPECIFIC EPITOPE OF THE ALPHA-SUBUNIT OF HUMAN CHORIONIC-GONADOTROPIN - STUDY USING MONOCLONAL-ANTIBODY PROBES

Monoclonal antibodies (MAbs) have been used extensively for identifica tion of sequence-specific epitopes using either the ELISA or/and IRMA methods, However, attempts to use MAbs for identification of conformat ion-specific epitopes have been very few as they are considered very l abile. We have investigated the stability of conformation-specific epi topes of human chorionic gonadotropin (hCG) using a quantitative solid -phase radioimmnunoassay (SPRIA) technique. Several epitopes are stabl e to mild modification (chemical and proteolytic) conditions, and epit opes show differential stability for these modifications. Based on the se observations, a monoclonal antibody (MAb 16) for an a-subunit-speci fic epitope of hCG has been used to monitor changes at the epitopic si te (identified as epitope 16) on modification of hCG, using SPRIA with immobilized MAb 16. Modifications of amino groups, hydroxyl group of tyrosine as well as carboxyl group of Asp/Glu all bring about sufficie nt changes in the epitope integrity. Peptide bond hydrolysis at lysine residues damages the epitope, but not at arginine residues, Hydrolysi s at tyrosine does not affect the epitope, though modification of the side-chain of tyrosine inactivates the epitope. Destruction of the epi tope occurs on reduction of the disulphide bonds. Partial retention of the epitope activity is seen on modification of carboxyl or the epsil on-amino groups of lysine. Based on these results four to six amino ac ids have been identified to be at the epitopic site, and the data sugg est that two peptide segments are brought together by the disulphide b ond Cys10-Cys60 to form the epitope.