Monoclonal Antibodies Directed to Human and Equine Chorionic Gonadotropins as Probes for the Topographic Analysis of Epitopes on the Human α-Subunit*

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Abstract
To improve our knowledge of the structural features of the .alpha.-subunit of hCG we have studied the antigenic site recognized by monoclonal antibody (MAb) ECG01 raised against equine CG (eCG) which binds to hormones and .alpha.-subunits from human and equine species. We have also delineated regions of hCG.alpha. comprising the epitope species. We have also delineated regions of hCG.alpha. comprising the epitope recognized by HT13 which was raised against hCG and binds to hCG and hCG.alpha.. To define the residues involved in the antigenic sites recognized by ECG01 and HT13, we have studied the reactivities of these two MAbs with native or chemically modified LH and CG with subunits from equine, human, or ovine (o) species or with synthetic peptides analogous to various portions of hCG.alpha.. We have also compared these reactivities with those displayed by MAbs AHT20 and FA 36, whose epitopes have been previously described; anti-hCG.alpha. MAb AHT20 is specific for the free .alpha.-subunits of various species and recognizes residues localized to the 36-41 region of hCG.alpha., whereas antipeptide MAb FA36 binds to the 87-92 carboxyl-terminal part of hCG.alpha.. Our results show that the epitopes of HT13 and ECG01 are 1) probably discontinuous, as these MAbs did not bind to the reduced and S-carboxymethylated hCG.alpha.; and 2) constituted by residues borne on the 1-35 and 52-86 sequences, as they do recognize the hCG.alpha. core missing the 36-51 portion, yet do not recognize (87-92) region recognized by FA36. The comparative studies performed with specific two-site immunoradiometric assays to determine the interspecies cross-reactivities of the MAbs allow us hypothetical assignment of residues on the primary structure of the hCG.alpha.. The antigenic site recognized by ECG01 might include two to six amino acids, four of these residues being located at inverted places compared to those of oLH.alpha. (Asp6/Gly22 and Arg67/Lys75). These residues present important charged functional groups highly conserved among evolutionarily related variants, and it is likely that they are located on the surface of both the intact hormone and its .alpha.-subunit. Three peptidic portions of hCG.alpha., 16-17, 64-66, and 73-76, respectively, might be involved in the epitope recognized by HT13, although we could not rule out the possibility that other residues were also involved in the antigenic site. These observations allows us to identify several residues as potentially constituting the epitopes recognized by two MAbs on both hCG and hCG.alpha.