Monoclonal Antibody Approach to the Relationship between Immunological Structure and Biological Activity of Thyrotropin

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Abstract
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 affintiy 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. mAb interference with bTSH receptor binding and stimulation of adenylate cyclase was well correlated in seven cases; in the four remaining cases inhibition of bTSH binding to the receptor was not accompanied by inhibition of adenylate cyclase stimulation. Taken together our results indicate that the receptor binding domain of TSH comprised but did not completely overlap with antigenic regions expressed by both subunits and the holomolecule. Furthermore our data are in favor of TSH microheterogeneity and suggest that mAb are of potential interest in separating active and inactive isoforms as they apparently distinguished these different molecules.

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