Our objective is the identification, isolation and characterization of cross-related antigens shared by human milk fat globule membrane (MFGM) and breast tumor membranes. Two general areas of research are planned to accomplish this objective. The first will concentrate on the characterization of proteins/glycoproteins isolated from human MFGM, preparation of antisera to these, and clarification of recognized determinants. The second will focus on the characterization, especially with emphasis on antibody-recognized determinants, of cross-related antigens of MFGM and membrane-enriched fractions of breast tumors which are recognized by a panel of monoclonal antibodies. Major techniques of protein/glycoprotein isolation will include molecular sieving and affinity (lectin and antibody-mediated) chromatography and preparative gel electrophoresis. Characterizations will include molecular weights, quantitative amino acid and carbohydrate analyses, and partial sequencing. Identification of antigenic determinants will be performed by comparisons of antibody-binding to glycoprotein, deglycosylated glycoprotein (chemical and enzymatic carbohydrate cleavage) and glycopeptides by Western and dot blotting and ELISA. A relatively large number of mono- and polyclonal antisera have been prepared versus MFGM and membrane-enriched breast tumor fractions which detect antigens common to both. The following salient characteristics of these antigens are pertinent to our proposed studies: (1) in general, these antigens are not tissue-specific and are expressed by secretory cells of the breast as well as by some exocrine and ductal epithelial cells; (2) when determined, all antisera generated thus far detect carbohydrate determinants; (3) MGFM-related antigens are generally expressed on the luminal surface of normal cells but assume a cytoplasmic localization in less-well-differentiated tumor cells; and (4) MFGM-related antigens from breast tumor cells possess lower molecular weights than corresponding antigen(s) in MFGM. Various human trial studies have recently been reported concerning the diagnostic, prognostic and therapeutic efficacy of antisera to MFGM and related tumor antigens. It is likely that the full potential of these reagents will only be realized when their recognized determinants are identified.
