Use of sperm viability and acrosomal status assays in combination with immunofluorescence technique to ascertain surface expression of sperm antigens.

Sperm antigens are being sought for the development of prototype contraceptive vaccines. For vaccine-induced antisperm antibodies to inhibit the function of viable sperm, target antigens must be expressed on the sperm surface before, during or after the acrosome reaction. The purpose of this study was to develop an approach to detect accurately surface binding of antibodies to viable acrosome-intact or acrosome-reacted sperm, and to confirm sperm surface binding characteristics of a panel of antisperm monoclonal antibodies submitted to the World Health Organization Antifertility Vaccines Task Force. Monoclonal antibodies were tested using a combination of carboxyl-fluorescein diacetate (CoFDA) sperm viability, and rhodamine immunofluorescence assays to ascertain surface binding to viable membrane-intact human sperm, and, in parallel studies, with a combination of fluorescein isothiocyanate (FITC) conjugated Pisum sativum agglutinin (PSA) and rhodamine immunofluorescence assays to ascertain the acrosomal status of antibody-positive sperm. Only 14 out of 32 antibodies that were previously considered to be surface-reactive were confirmed to be positive with these methods. Five were positive on unfixed viable (CoFDA-labelled) fresh sperm in this study, and 9 different antibodies were positive on unfixed acrosome-reacted sperm. We conclude that this multifaceted approach is more accurate and informative than previous techniques used for detection of sperm surface antigens such as radioimmunoassay, standard immunofluorescence, sperm agglutination assays and enzyme-linked immunosorbent assays, which can give false positive results due to the presence of contaminating non-sperm cells, insoluble seminal plasma material, or permeabilized non-viable sperm. Furthermore, the combination of assays employed in this study does not depend on sperm motility, as do the immunobead and sperm immobilization assays, and provides information about the acrosomal status of antibody-positive cells.