IMMUNO-COLLOIDAL GOLD-LABELED SURFACE REPLICA, AND ITS APPLICATION TODETECT SP56, THE EGG RECOGNITION AND BINDING-PROTEIN, ON THE MOUSE SPERMATOZOON

Using immune-colloidal gold surface replicas (IGSR), we developed a me thod to demonstrate surface antigens in a whole view of a cell at the FM level. In this study, a monoclonal antibody (MAB) 7C5 directed agai nst sp56, which is a mouse sperm protein (Mr = 56,000 daltons) and has specific affinity for the zona pellucida, was used as primary antibod y. Fixed mouse spermatozoa were incubated with primary antibody and co lloidal gold-labeled secondary antibody on a slide glass, Used gold pa rticles were 10, 20 or 30 nm in diameter. The spermatozoa were then cr itical point dried, and replicated with platinum and carbon. Replicas were examined with a transmission EM, The result suggests that sp56 is located on the outer surface of the plasma membrane covering approxim ately 2/3 of dorsal edge of the sperm head, but it is virtually absent in the tapered apical tip, When 10 and 20 nm colloidal particles were used, about 500 particles were observed on one side of the flat sperm head. Although the large 30 nm particles were easily recognized in lo w magnification figures, the attached number on the sperm head was gen erally lower than the number of 10 or 20 nm particles observed. By thi s method, we could effectively demonstrate the localization of a surfa ce antigen in a wide cell view with clear landmarks.