Detection and characterization of antibodies against mouse melanoma MB-16 by immunofluorescence

Tumor-specific antibodies to mouse melanoma MB-16 were detected by Immunofluorescence in the sera of C57BL/6J mice, the inbred host strain. Fixed tumor cells grown in tissue culture on cover-slips were employed as target cells for immunofluorescent tests. Fluorescence of the cytoplasm of tumor cells was an indication of a positive reaction. Antibody was detectable on the 5th post-challenge day in mice injected (6 x 10[5]) s.c. with MB-16 cells isolated from subcutaneous transplants. The antibody reached a maximum titer of 1:4 on day 10 and this titer persisted until the 21st day following the initial injection. Tumors formed on tails of mice injected s.c. with 6 x 10[5] viable tumor cells were removed by tail amputation 35 days post-challenge. Prior to tumor removal, titers gradually increased from 1:4 to 1:16. The incidence of antibody also increased from 80% after 1 week to 100% in the 5th week after the challenge dose. Following tumor removal, titers fell to 1:4 and then gradually declined. Antibody was still detectable in the serum of mice 19 weeks following removal of the tumor. The antibody persisted in the presence of overt metastatic lesions as well as in apparent absence of tumor. Five of nine (56%) of sera from human patients with malignant melanoma were reactive at low titers (1:2) on MB-16 target cells. Six of nine (78%) melanoma sera were positive on human melanoma cells. The antibody reached titers of 1:8. With on exception, the serum from a patient with choriocarcinoma, 26 sera from patients with disorders unrelated to malignant melanoma, and 20 blood bank sera were either non-reactive or non-specific for human melanoma target cells. The specificity of the fluorescent staining reaction was determined by absorption as sera with heterologous antigens (normal and neoplastic cells), one-step inhibition procedures, and test target cells from different tissue culture cell lines. Absorption of sera with surface membrane preparations from MB-16 cells indicated the absence of antibodies to surface antigens.