Monoclonal Antibody Mapping of the Antigenic Surface of Human Thyrotropin and Its Subunits

Abstract

Although the amino acid sequence of the alpha- and beta-subunits of glycoprotein hormones in various species has been deciphered, data on their tertiary structure are not abundant. This impedes correlation between structure and function. The availability of monoclonal antibodies to human TSH (hTSH) offers the opportunity to enumerate the antigenic determinants present on the surface of hTSH and its subunits and to examine their spatial relationships. Twenty-eight monoclonal antibodies to hTSH were obtained from several fusions, and screens carried out separately in the laboratories involved in this study. Affinities for hTSH ranged from 10(8)-10(11) M-1. Cross-reactivity with bovine TSH (bTSH), human gonadotropins (hLH, hFSH, and hCG), and the alpha- and beta-subunits of hTSH distinguished 10 groups of monoclonal antibodies (mAb) according to their main cross-reactions: 1) hTSH alpha, hLH, hFSH, and hCG; 2) hTSH alpha, bTSH, hLH, hFSH, and hCG; 3) hFSH; 4) bTSH and hFSH; 5) bTSH, hLH, and hFSH; 6) bTSH, hLH, hFSH, and hCG; 7) hTSH beta; 8) hTSH beta and bTSH; 9) hTSH beta and hFSH; and 10) hTSH beta, hLH, hFSH, and hCG. mAb were incorporated into 2-site binding assays to probe hTSH by a 28 X 28 matrix, the free alpha-subunit by a 4 X 4 matrix, and the free beta-subunit by a 18 X 18 matrix. Regarding intact hTSH, 12 different clusters of mAb were distinguished and interpreted as reflecting 12 distinct antigenic regions on the surface of the hTSH molecule. Two of them were localized on the alpha-subunit, and 6 on the beta-subunit; 4 were only expressed by the holo-hormone and, thus were designated conformational antigenic regions (alpha beta). Surface mapping of the free alpha- and beta-subunits was virtually identical to that observed with the holo-hormone. Modification of the operative conditions of mAb reacting only with holo-hTSH shows that they recognize the alpha-subunit, but not the beta-subunit of hTSH. These results indicate that 1) hTSH beta presents epitopes that are evolutionary conserved; 2) hTSH alpha presents several epitopes that are species specific and 2 that are not hormone specific; 3) dissociation of hTSH does not modify the antigenic surface expressed by both subunits when they are associated; and 4) some of the conformational determinants expressed only by holo-hTSH are more likely derived from the alpha-subunit than from the beta-subunit.