Demonstration of Two Distinct Antigens in Spent Tissue Culture Medium of a Human Malignant Melanoma Cell Line

Spent tissue culture medium was used to define and isolate antigens associated with cultured human malignant melanoma cells (UCLA-SO-M14) grown in a serum-free, chemically defined medium (CDM). Antigenic activities of the spent CDM (M14-CDM) were monitored by complement fixation (CF) and Immune adherence. Allogeneic melanoma sera containing antibodies to oncofetal antigen (OFA) and tumor-associated antigen (TAA) were the source of specific antisera. The crude concentrated M14-CDM contained OFA and TAA. Extraction of partially purified M14-CDM with chloroform: methanol (C:M) separated OFA and TAA into C:M-insoluble and CM-soluble fractions, respectively. When tested by CF, 82% (74/90) of the melanoma sera reacted against the crude concentrated M14-CDM. In contrast, only 40% (36/90) and 29% (26/90) of these melanoma sera were reactive to the C:M-insoluble and C:M-soluble fractions, respectively. Absorption of the 90 melanoma sera with cultured lymphoblastoid cells from the melanoma serum donor (M14 melanoma cell line) reduced the positive incidence against concentrated M14-CDM to 58% (52/90), whereas reactions against the C:M fractions were almost unaffected. Of the 90 absorbed melanoma sera, 21% (19/90) reacted against the CM-insoluble (OFA) fraction but not against the C:M-soluble (TAA) fraction. Conversely, only 10% (9/90) of the sera reacted against TAA but not against OFA. About 19% (17/90) of the sera contained antibodies to both antigens. These results suggest that two distinct antigens, OFA and TAA, were separated from spent tissue culture medium of M14 cells.