Human melanoma-associated antigens: analysis of antigenic heterogeneity by molecular, serologic and flow-cytometric approaches.
The relationship of antigenic heterogeneity to the epitope recognized by an antibody was examined with monoclonal antibodies to human melanoma-associated antigens. Expression of the human melanoma-associated antigens, 250-Kd glycoprotein/proteoglycan and p97, was examined quantitatively by flow cytometry on fresh cell suspensions of human melanoma. Percent positive cells and mean fluorescence intensity were consistently higher with antibody 9.2.27 to the 250-Kd glycoprotein/proteoglycan than with antibody to p97. In addition, assessment of percent positive cells in multiple skin lesions biopsied from individual patients indicated that in 26 of 30 lesions, greater than 90% of the cells stained positively with 9.2.27. This relative lack of antigenic heterogeneity with antibody 9.2.27 contrasted with previous reports which showed considerable antigenic heterogeneity with other antibodies to the 250-Kd glycoprotein/proteoglycan. The explanation for this distinction was sought by quantitative flow cytometric and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) techniques. Comparison by flow cytometry and immunoperoxidase of three antibodies, which recognized distinct epitopes of the 250-Kd glycoprotein/proteoglycan, indicated that 9.2.27 reacted more intensely with cultured cells and tissue sections than other antibodies to the same antigen. Examination by SDS-PAGE indicated that 9.2.27 could immunoprecipitate a larger proportion of 250-Kd glycoprotein molecules than other antibodies. In addition, immunodepletion experiments in gels indicated that the 9.2.27 determinant was present on a higher proportion of 250-Kd glycoprotein molecules than PG-2 antibody to a separate determinant. It is likely that 9.2.27 antibody displays less antigenic heterogeneity because its epitope is represented on a higher proportion of the antigen molecules. Thus, not only the nature of the antigen but also the epitope recognized by an antibody influences the degree of antigenic heterogeneity.
A C
AC
Morgan
C
C
Woodhouse
R
R
Bartholemew
R
R
Schroff
D000911:Antibodies, Monoclonal / Q000276:immunology; D000951:Antigens, Neoplasm / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis; D005434:Flow Cytometry; D006801:Humans; D007124:Immunoenzyme Techniques; D008545:Melanoma / Q000276:immunology*; D012878:Skin Neoplasms / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D000939:Epitopes