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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 systems for detection of antigens. We currently aim to develop liposomal targeting vectors for gene transfer into transgenic gonadal tumor cells expressing the luteinizing hormone (LH) receptor (R). We have biotinylated (B) human chorionic gonadotrophin (hCG) to obtain a selective targeting molecule to be attached to biotinylated liposomes via an avidin-streptavidin bridge. The biotinylation was performed by combining biotin isothiocyanate (BITC) and hCG in alkaline reaction buffer in a 100:1 (BITC:hCG) molar ratio. B-hCG maintained its ability to bind specifically to rat testicular membranes and was also bound to streptavidincoated polypropylene wells. cAMP production was induced in BLT-1 Leydig tumor cells in vitro after stimulation with B-hCG, as a sign of persistent bioactivity. Frozen sections of rat testicular and ovarian tissues and skeletal muscle were labeled by incubating for 2 hr at 37C with 10 ng/μl B-hCG. The binding was subsequently visualized by the avidin-biotin-peroxidase system, followed by silver enhancement of Ni-DAB staining. In rat testicular and ovarian sections, labeling was observed in structures known to strongly express the LH-R, i.e., Leydig cells, corpora lutea, and blood vessels. The labeling was blocked by preincubation with a 100-fold excess of the native hormone, and by injecting the rats SC with a high dose of hCG (1000 IU/kg) 48 hr before sacrifice. Skeletal muscle, used as negative control, was not labeled. These data demonstrate that the bioactivity of hCG is relatively well preserved after biotinylation. The biotinylated gonadotropin offers a new nonradioactive alternative for visualization of bioactive LH receptors in tissue sections.