Characterization and partial purification of human milk fat globule membrane antigens by polyacrylamide gel electrophoresis and immunoblotting using monoclonal antibodies.

Polyclonal and monoclonal antibodies were used to characterize human milk fat globule (HMFG) membrane antigens, using gel filtration, ion exchange chromatography and western blotting. Although originally generated against HMFG, monoclonal antibodies also reacted with skim milk. In western blotting, several antigen molecules were seen, of which some were detected by all available antibodies, and one only with monoclonal antibody III D5, previously shown to react with mammary and ovarian carcinomas bearing estrogen receptors. This or these antigens, with a molecular weight of about 53 kd, were isolated by fractionation in SDS-PAGE. A low polypeptide content was demonstrated but the antigenic structure could be stained with periodic-acid-Schiff and biotinylated peanut agglutinin, indicating the presence of galactose or N-acetylgalactosamine residues. While this 53-kd molecule is the only determinant exclusively stained by III D 5 antibody, it is suggested that it carries epitopes related to estrogen receptor activation.