IMMUNOCHEMICAL ANALYSIS OF THE DETERMINANT RECOGNIZED BY A MONOCLONAL-ANTIBODY (MBR1) WHICH SPECIFICALLY BINDS TO HUMAN MAMMARY EPITHELIAL-CELLS

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 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.degree. 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. When the 3 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.