Immunopurification, characterization, and nature of membrane association of human melanoma-associated oncofetal antigen gp87 defined by monoclonal antibody 140.240.
A melanoma-associated oncofetal antigen, gp87 (a p97-like molecule), defined by the monoclonal antibody (MoAb) 140.240 has been purified to homogeneity from the spent medium of cultured melanoma cells by a two-step immunoadsorbent procedure. The first immunoadsorbent step using glutaraldehyde-insolubilized MoAb 140.240 (ascites fluid) resulted in a 13-fold enrichment with 93% recovery in the bound material. In the second immunoadsorbent step constructed by the purified IgG2a of MoAb 140.240 (culture fluid) coupled to CNBr-activated Sepharose 4B the bound material from the first step was further purified resulting in a 330-fold purification with 90% recovery. SDS-polyacrylamide gel electrophoretic analysis of the final purified material revealed a single band migrating as a polypeptide with an approximate molecular weight of 87 Kd, consistent with the size of the molecule immunoprecipitated by MoAb 140.240 from lysates of radiolabelled melanoma cells. Preliminary amino acid analysis indicates a particularly high proportion of phenylalanine in gp87. We have also compared gp87 with two well defined antigens, HLA-A,B,C (integral membrane protein) and "94K" melanoma/carcinoma-associated antigen (peripheral membrane protein) with respect to antigen extractability from melanoma cells using phosphate-buffered saline, 0.1 M urea, 3 M NaCl, or nonionic detergent (NP-40). The results showed that whereas 94K antigen was extractable by each of the four different solutions, gp87, similar to HLA-A,B,C antigens, could only be extracted with NP-40, strongly suggesting that gp87 is an integral melanoma cell component.
D000596:Amino Acids / Q000032:analysis; D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm / Q000276:immunology / Q000302:isolation & purification*; D002460:Cell Line; D002462:Cell Membrane / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007163:Immunosorbent Techniques; D008545:Melanoma / Q000276:immunology*; D008565:Membrane Proteins / Q000276:immunology; D008970:Molecular Weight
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D008565:Membrane Proteins; C031192:oncofetal antigens