Studies of a melanoma tumor-associated antigen detected in the spent culture medium of a human melanoma cell line by allogeneic antibody. I. Purification and development of a radioimmunoassay.

Abstract

A tumor-associated antigen (TAA) was isolated from spent culture medium (chemically defined serum-free medium) of human melanoma cell line UCLA-SO-M14 (M14). The isolation procedures included concentration, ultrafiltration, gel filtration chromatography, and chloroform:methanol (C:M) extraction. The melanoma TAA activity recovered from the organic phase of the C:M extract was subsequently fractionated by gel filtration and radiolabeled with Na125I. The radioiodinated antigen was further purified by Sephacryl S-200 gel filtration and allogeneic antibody affinity chromatography. With the use of previously characterized anti-TAA allogeneic sera from melanoma patients and 125I-labeled TAA, a radioimmunoassay (RIA) was developed. Protein A-bearing Staphylococcus aureus was used to separate bound and unbound 125I-labeled TAA. The coefficient of variation between experiments and within experiments with unlabeled melanoma TAA as the competitor in the competitive RIA ranged from 8.9 to 20.4%. These variations were consistently lower (8.9-13.6%) at high levels (6 micrograms melanoma TAA/ml) of the competitor than they were (17.3-20.5%) at low levels (0.5 microgram melanoma TAA/ml), suggesting reasonable reproducibility of the assay. A logit versus log plot of the competitive RIA data and analysis by linear regression yielded a straight line. This line represented a 5- to 1,000-ng detection range for melanoma TAA. Analysis of C:M-extracted and Sephacryl S-200-purified melanoma TAA by the competitive RIA revealed a 695-fold purification of the antigen that represented a 37.5% recovery from the spent culture medium. The greatest enrichment of the melanoma TAA was achieved by the C:M extraction step. This step separated the melanoma TAA from other antigens, e.g., fetal antigen and human leukocyte antigens.