Structural characterization of human melanoma-associated antigen p97 with monoclonal antibodies.
Monoclonal antibodies were used to study p97, a human melanoma-associated antigen (MAA). Four hybridomas, designated 4.1, 96.5, 118.1, and 8.2, were obtained by fusing mouse myeloma cells with spleen cells from mice immunized with human melanoma cells. Antibodies 4.1 and 8.2 were IgG1; antibodies 96.5 and 118.1 were IgG2a. Sequential immunoprecipitation (IP) and sodium-dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that all 4 antibodies recognized the same 97 kilodalton (kD) protein. Binding studies with 125I-labeled antibody showed that antibodies 4.1 and 96.5 bound the same epitope, p97a. Antibodies 118.1 and 8.2 defined epitopes p97b and p97c, respectively. Six monoclonal antibodies (M17, L1, L10, R10, I12, and K5) specific for gp95, a kD melanoma cell surface glycoprotein were also tested. Sequential IP showed that these antibodies bound p97; p97 and gp95 are thus identical. Binding studies showed that antibody m17 bound epitope p971, and antibodies L1, L10, and R19 bound epitope p97c. Antibodies I12 and K5 defined 2 other epitopes, p97d and p97e, respectively. SDS-PAGE under nonreducing conditions indicated that p97 is monomeric, probably with intrachain disulfide bonds. Cell-surface labeling of sialic acid residues and neuraminidase digestion showed that p97 is a sialoglycoprotein. Digestion of p97 with papain or trypsin produced a stable 40 kD fragment, which expressed epitopes p971, p97b, and p97c, but not p97d or p97e.
D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D000951:Antigens, Neoplasm; D002459:Cell Fusion; D011232:Chemical Precipitation; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis; D008545:Melanoma / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008808:Mice, Inbred CBA; D009439:Neuraminidase / Q000494:pharmacology; D010447:Peptide Hydrolases / Q000494:pharmacology
D000906:Antibodies; D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D000939:Epitopes; D007074:Immunoglobulin G; D009439:Neuraminidase; D010447:Peptide Hydrolases