Regional binding of human anti-sperm antibodies assessed by indirect immunofluorescence

Indirect immunofluorescence (IIF) can be a powerful tool for determining the site on spermatozoa to which antibodies bind. Human sera that contain anti-sperm antibodies are often of low titre, and may contain antibodies directed against both intracellular and surface antigens. We have developed an IIF protocol that helps to distinguish intracellular from surface labelling. The two types of labelling were differentiated by exposing the spermatozoa to Hoechst 33258, a nuclear stain of low membrane permeability, to tag the spermatozoa that had disrupted membranes. Surface labelling detected in this fashion was patchy. It was much more uniform if the spermatozoa were fixed in paraformaldehyde, or if a univalent, Fab fragment was used as the second antibody. Thus, it is likely that most of the patchy appearance is due to the bivalent second antibody cross-linking mobile antigen-antibody complexes. For some sera, patching was so pronounced that it appeared to remove the label from portions of the sperm surface, giving a misleading picture of the regions to which the antibodies were directed. Fourteen sera were used in IIF and none of them labelled spermatozoa solely on the head or on the tail.