Analysis of the male-specific H-Y antigen is difficult, in part, because of the tendency of “male-specific” antisera to bind cell surface components found in male and in female cells. Some insight as to the nature of that difficulty is provided by biochemical evaluation of H-Y antibody. In this study, purification of a monoclonal H-Y antibody with ammonium sulfate or Protein-A Sepharose, revealed the possibility of microheterogeneity. Despite evidence of multiple subtypes, Protein-A elution profiles suggested that the male-specific activity of the antibody resided in an IgG2a moiety. This was borne out by decreased activity after absorption of the IgG2a subtype with male cells, and by reaction of the monoclonal antibody with mouse subtype-specific antisera in an ELISA. Combined analysis using biological (absorption), biochemical (EF and PAGE) and immunological (ELISA) methods could find applicability in other complex systems.
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