Purification and partial characterization of a shed 66 kDa melanoma-associated antigen identified by autologous antibody

Abstract

We have previously reported the isolation of a 66 kDa melanoma-associated antigen, identified by autologous antibody, in serum and unfractionated spent tissue culture media by Western blot analysis. The antigen, detected by autologous serum S150, was found to be broadly represented on melanoma, glioma, renal cell carcinoma, neuroblastoma and head and neck carcinoma cell lines. S150 did not react with bladder or colon carcinoma, fetal fibroblasts, pooled platelets, lymphocytes and red blood cells, autologous cultured lymphocytes or fetal calf serum. To further characterize the antigen, spent tissue culture media, obtained from autologous melanoma cell line, Y-Mel 84:420, was separated by an isoelectric focusing column. Unabsorbed control serum S150 was noted to have a maximum titer of 1:2040 against autologous melanoma cells as measured by protein A hemadsorption. Following isoelectric focusing the greatest decrease in autologous antibody titer (30-fold) occurred with fractions having a pI between 2 and 3. Further resolution of the antigen was accomplished with high-pressure ion-exchange chromatography. One of these fractions showed a significantly higher concentration of antigen and was distinctly resolved from bulk serum albumin. Subsequent Western blot analysis, with autologous antibody, of the isolated antigen-containing fraction, confirmed the presence of a single 66 kDa band. Exposure of the antigen, purified by high-pressure ion-exchange chromatography, to neuraminidase ablated recognition by autologous antibody and suggests that sialic acid is present on the protein and may be part of the antigenic epitope. Binding of antigen, obtained following DEAE anion exchange chromatography, was noted to lectins derived from Triticum vulgaris, Dolichos biflorus and Lycopersicon esculentum. Preparative purification of the antigen was accomplished by anion exchange followed by lectin affinity chromatography with a Dolichos biflorus column. Antigen obtained following lectin affinity chromatography subjected to SDS-PAGE and silver stain revealed a single band at 66 kDa. We conclude that a melanoma-associated antigen detected by autologous antibody in spent tissue culture media is an unusually acidic glycoprotein (pI 2-3).