Epitope Mapping of Human Luteinizing Hormone Using Monoclonal Antibodies

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Abstract
To establish structure-function relationships for human (h) LH, 14 murine monoclonal antibodies (MABs) to hLH were characterized in terms of their affinity of binding (Ka), their specificity for intact glycoproteins and their subunits, their paratopic relationships, and their ability to interfere with the biological activity of hLH. The Ka values obtained ranged between 2.4 .times. 106 and 1.0 .times. 1010 for intact hLH and between 2.3 .times. 106 and 7.5 .times. 108 liters/M for the free .alpha.- and .beta.-subunits, indicating that, in general, the antibodies showed higher avidity for the intact hormone. Six MAB recognized both the intact and free .alpha.-subunit of hLH, cross-reacted with intact hCG, hFSH, and hTSH, and thus appeared to be .alpha.-directed. Four MAB were .beta.-directed, recognizing both intact hLH and its free .beta.-subunit. One of these .beta.-directed MABs also cross-reacted with intact hCG, hFSH, and hTSH, while two others recognized both intact and free .beta.-subunits of hLH and hCG. The fourth .beta.-directed remaining four MABs recognized epitopes only on the intact hormone; three recognized intact hLH and hCG, and the fourth was specific for intact hLH. Their paratopic relationships tested in competitive binding studies resulted in either mutual competition or complementarity, sometimes with cooperativity. Biointerference, defined as the ability to inhibit hLH-induced testosterone biosynthesis in dispersed rat Leydig cells, indicated that three of the .alpha.- and one of the .beta.-directed antibodies neutralized the biological response of hLH in this bioassay in a dose-responsive manner. Their ability to inhibit hLH bioactivity largely paralleled their affinity constants. Our data have allowed us to establish a tentative topographic relationship of epitopes to the biological region of the molecule of hLH, foreshadowing (in additive binding studies) some of the possible combinations of antibodies that might allow us to design two- or multiple-site immunometric assays in which measurement of immunoactive LH reflects biological activity. In addition, these studies suggest that both the .alpha.- and .beta.-subunits participate in LH receptor binding and/or biological activity.

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