Immunochemical mapping of gonadotropins

As a glycoprotein hormone, human chorionic gonadotropic (hCG) is not a single molecular entity but this term rather comprises an array of molecular variants such as hCG, hCG beta, hCGn, hCG beta n, hCG beta cf, -CTPhCG, hCG beta CTP, deglyhCG, asialohCG, hCGav and the closely related molecules hLH, hLH beta and hLH beta ef. The advent of monoclonal antibodies (MCA), the availability of ultrasensitive detection systems and the recent determination of the crystal structure of hCG, made it possible to design special purpose diagnostic and clinical research immunoassays for hCG-like molecules. For more than a decade we and others have tried to refine epitope maps for hCG and related molecules by means of a large panel of MCA, naturally occurring metabolic variants of hCG (hCGn, hCG beta, hCG alpha, hCG beta cf, hCG beta CTP), homologous hormones and subunits of various species (e.g. hLH, hLH beta, hFSH, hTSH, oLH, rLH beta), chemically modified molecules (deglyhCG, asialohCG, tryptic and chymotryptic hCG beta and hCG alpha fragments) and synthetic peptides (octapeptides and longer). It appeared that all epitopes on molecular hCG-variants recognized by our MCA are determined by the protein backbone. Except for the two major epitopes on hCG beta CTP and parts of two antigenic domains on hCG alpha, epitopes on hCG-derived molecules are determined by the tertiary and quarternary structure. Operationally useful descriptive epitope maps were designed including information on assay suitability of antigenic determinants. On this basis we established ultrasensitive time-resolved fluoroimmuno-assays for hCG, hCG and hCGn, hCG beta and hCG beta n and hCG beta cf, hCG alpha and additional assays recognizing different spectra of hCG-variants. Such assay have been applied by us and others to the detection of pregnancy, early pregnancy loss, choriocarcinoma, testicular cancer, other cancers and prenatal diagnosis. However, as the molecular structure of many epitopes utilized in immunoassays of different laboratories was not resolved, comparability of results was not satisfactory. Consequently, attempts were made to compare schematic epitope maps from different research institutions. The situation has been much improved by solving the three-dimensional (3D) structure of hCG. It has been shown that hCG is a member of the structural superfamily of cystine knot growth factors like NGF, PDGF-B and TGF-beta. Each of its subunits is stabilized in its topology by three disulfide bonds forming a cystine knot. Moreover, it turned out that the disulfide bridges in their majority have previously been wrongly assigned. Computer molecular modeling of crystallographic coordinates of hCG and subsequent selective combined--PCR-based and immunological--mutational analyses of hCG beta expressed via the transmembrane region of a MHC molecule made it possible to more precisely localize epitopes on hCG-derived molecules. Although the entire surface of hCG has to be regarded as potentially immunogenic there seems to be hot spots where epitopes are clustered in antigenic domains. These are located on the first and third loops protuding from the cystine knots of both subunits and are possibly centered around the knot itself. Ultimate answers on epitope localizations will be given by the crystal structure determination of hCG complexed with different Fabs.