A Biochemical and Biosynthetic Analysis of Human Melanoma-Associated Antigens with Monoclonal Antibodies

Abstract

The molecular profile and cellular topography of two melanoma associated antigens defined by monoclonal antibodies 9.2.27 and F11 were investigated. The 9.2.27 antibody recognizes a 250K glycoprotein which associates with a high molecular weight complex (HMW-C)1 Mr > 500K in indirect immunoprecipitation analysis of detergent extracts obtained from biosynthetically labeled melanoma cells. The HMW-C appears late in the biosynthesis of this antigenic complex as judged by pulse-chase studies and is sensitive to degradation by chondroitinase ABC lyase suggesting that it is a chondroitin sulfate proteoglycan. Direct binding analysis of iodinated 9.2.27 IgG to isolated membrane fractions of melanoma cells reveal strong antigenic activity of this antigen demonstrating its membrane association. In contrast to these results, the F11 antibody recognized three glycoproteins in detergent extracts of biosynthetically labeled melanoma cells at Mr 75, 77 and 100K. The primary antigenic activity recognized by F11, however, is in the spent media of melanoma cells. Indirect immunoprecipitation analysis of secreted spent media components revealed that the F11 antibody recognizes only the 100K component as a secreted melanoma associated antigen and a cross reactive lower molecular weight antigen in spent media of two neuroblastoma cell lines. Thus, monoclonal antibodies can be used to dissect the molecular nature and cellular topography of melanoma associated antigens through the combination of biosynthetic, enzymatic and cell fractionation techniques allowing the identification of these potentially tumor associated gene products. This information will allow the design of experiments to investigate the functional roles of these molecules in malignant melanoma.