Monoclonal antibody approach to the relationship between immunological structure and biological activity of thyrotropin.

In order to locate the domains involved in the biological activity of TSH and to get some insight in the relationship between immunological and biological properties of TSH, 24 monoclonal antibodies (mAb) to 11 different antigenic regions of hTSH were tested for both binding to hTSH and inhibition of hTSH stimulation of adenylate cyclase in human thyroid membranes. These mAb were also investigated for binding to bovine TSH (bTSH), and interference with bTSH binding to the receptor and stimulation of adenylate cyclase. Radioiodinated human TSH (hTSH) was incubated with increasing concentrations of mAb. Maximum hTSH binding by the various mAb ranged from 15-75% and was not related to the apparent affinity of the mAb for hTSH. Maximum inhibition by the mAb of hTSH stimulation of adenylate cyclase ranged from 3-92%. As compared to the antigenic map of hTSH, it was observed that mAb reacting with the same antigenic regions might display varying inhibition of hTSH. Nevertheless, it was clearly shown that the most potent inhibitors of hTSH stimulatory activity interacted with epitopes located on the alpha- and beta-subunits or expressed only by holo hTSH. Only 11 of the 24 mAb cross-reacted significantly with bTSH. Seven exhibited the same inhibition of hTSH and bTSH stimulatory activity; the four remaining mAb rather than to inhibit adenylate cyclase stimulation as observed with hTSH, did not interfere or even increased adenylate cyclase stimulation by bTSH.(ABSTRACT TRUNCATED AT 250 WORDS)

Production of monoclonal antibodies complementary to an antibody-antigen complex. Use in an immunoradiometric assay for follitropin.

Monoclonal antibodies were prepared against human follitropin by fusion of the myeloma cell line P3-X63-Ag8-653 with spleen cells of mice immunized with either human follitropin or follitropin bound to an anti-beta hFSH monoclonal antibody. The latter immunization procedure permits shielding of the immunodominant specific site and allows the production of numerous specific monoclonal antibodies to human follitropin which are complementary to the monoclonal antibody used in the immunogen. In this investigation two specific monoclonal antibodies were used in a two site immunoradiometric assay which was highly specific, rapid, with one incubation step and demonstrated a sensitivity level of 0.1 mIU/ml. It was possible to differentiate between prepubertal and adult levels of follitropin and to recognize individuals with hyposecretory states.

Immunization with immune complexes: characterization of monoclonal antibodies against a TSH-antibody complex.

Monoclonal antibodies (MAb) to human thyrotropin (hTSH) were prepared by immunization of mice and rats according to different procedures. We have previously demonstrated that a specific antigenic region on the surface of the hTSH molecule was highly immunogenic; in order to produce specific MAb to weakly immunogenic regions of hTSH, we immunized mice and rats with a complex composed of hTSH and an anti-beta hTSH MAb 27 directed against the highly immunogenic region. Monoclonal antibodies elicited by this immunization procedure were highly specific and a high percentage was found complementary to the MAb 27 used in the immunogen. We did not search for anti-MAb 27 antibodies, however one hybridoma produced antibody that preferentially reacted with the immune complex. This antibody, called 515, is an IgG1 that binds the complex with 100-fold greater affinity than it does to the anti-beta hTSH MAb 27 alone. This enhancement was also observed with the Fab fragment of the MAb suggesting that the epitope recognized by this anti-complex MAb is displayed in a very different way when hTSH is bound to the first MAb.

Monoclonal antibody mapping of the antigenic surface of human thyrotropin and its subunits.

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.