PERSISTENCE OF BIOLOGICAL-ACTIVITY OF BIOTINYLATED HUMAN CHORIONIC-GONADOTROPIN AND ITS USE FOR VISUALIZATION OF RAT LUTEINIZING-HORMONE RECEPTORS IN TISSUE-SECTIONS

Biotinylation of antibodies is an established method for producing sys tems for detection of antigens. We currently aim to develop liposomal targeting Vectors for gene transfer into transgenic gonadal tumor cell s expressing the luteinizing hormone (LH) receptor (R). We have biotin ylated (B) human chorionic gonadotrophin (hCG) to obtain a selective t argeting molecule to be attached to biotinylated liposomes via an avid in-streptavidin bridge. The biotinylation was performed by combining b iotin isothiocyanate (BITC) and hCG in alkaline reaction buffer in a 1 00:1 (BITC:hCG) molar ratio. B-hCG maintained its ability to bind spec ifically to rat testicular membranes and was also bound to streptavidi n-coated polypropylene wells. cAMP production was induced in BLT-1 Ley dig tumor cells in vitro after stimulation with B-hCG, as a sign of pe rsistent bioactivity. Frozen sections of rat testicular and ovarian ti ssues and skeletal muscle were labeled by incubating for 2 hr at 37C w ith 10 ng/mu l B-hCG. The binding was subsequently visualized by the a vidin-biotin-peroxidase system, followed by silver enhancement of NI-D AB staining. In rat testicular and ovarian sections, labeling was obse rved in structures known to strongly express the LH-R, i.e., Leydig ce lls, corpora lutea, and blood vessels. The labeling was blocked by pre incubation with a 100-fold excess of the native hormone, and by inject ing the rats sc with a high dose of hCG (1000 IU/kg) 48 hr before sacr ifice. Skeletal muscle, used as negative control, was not labeled. The se data demonstrate that the bioactivity of hCG is relatively well pre served after biotinylation. The biotinylated gonadotropin offers a new nonradioactive alternative for visualization of bioactive LH receptor s in tissue sections.