Immunochemical Analysis of the Determinant Recognized by a Monoclonal Antibody (MBr1) Which Specifically Binds to Human Mammary Epithelial Cells

TLDR: A monoclonal antibody raised against a membrane preparation of a human breast cancer line (MCF-7) and characterized as mammary gland epithelium associated, was used to biochemically define and partially purify its target antigen.

Abstract: A monoclonal antibody (MBr1) raised against a membrane preparation (CM) of a human breast cancer line (MCF-7) and characterized as mammary gland epithelium associated (S. Menard, E. Tagliabue, S. Canevari, G. Fossati, and M. I. Colnaghi. Generation of monoclonal antibodies reacting with normal and cancer cells of human breast. Cancer Res., 43:1295-1300, 1983), was used to biochemically define and partially purify its target antigen. The antigenic activity recognized by MBr1 was unaffected by treatment of MCF-7 cells with trypsin, protease K, or Vibrio cholerae neuraminidase and by heating at 100 degrees but was abolished by treatment with methanol. Since this behavior suggested a glycolipid nature of the MBr1-defined antigen, total lipids were obtained by chloroform:methanol or tetrahydrofuran:phosphate buffer extractions from crude membrane preparations of MCF-7 cells and of breast cancer surgical specimens. Total absorption of MBr1 activity was found by breast cancer lipid extracts, whereas no absorbing capability was detected with a series of highly purified acid and neutral glycolipids or with normal and neuraminidase-treated red blood cells of human, ox, and sheep species. The same pattern of inhibition of MBr1-binding activity was obtained with total lipid extract and both phases after diethyl ether partition. However, when the three extracts were chromatographed on diethylaminoethyl-Sepharose, the antigenic activity was recovered only in the neutral glycolipid fractions. Periodate oxidation of MCF-7 crude membrane preparation abolished MBr1-binding activity, suggesting that the carbohydrate portion of the molecule may constitute the antigenic determinant.