Detection of Human Malignant Melanoma Antigens by Immunofluorescence and Autologous Postimmune Antimelanoma Sera

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Abstract
The objectives of this study are to demonstrate the specificity of sera from melanoma patients undergoing autologous immunization and to localize melanoma antigens on a cellular level. Five melanoma patients were immunized with autologous melanoma cells and Bacillus Calmette-Guerin. This immunization program was conducted at Tulane University. Indirect immunofluorescence using both viable and fixed melanoma cells was employed. Four of five postimmune sera were reactive to five of seven melanoma cell lines. Two of the four reactive antisera showed positive binding with two additional melanoma lines obtained from other laboratories. All these sera were negative against seven nonmelanoma lines. Negative controls consisted of sera from 65 nonimmunized melanoma patients and 140 nonmelanoma patients. Membrane immunofluorescence (MIF) demonstrated sequential full MIF, capping, polarization, and extrusion of antigen-antibody complexes on the cell surface. MIF inhibition showed shedding of melanoma antigens in the culture medium. Ethanol, methanol, formalin, trichloroacetic acid, and acetone yielded sharp MIF. Isopentane and isooctane gave bright cytoplasmic fluorescence. In conclusion, this study provides suggestive evidence for the existence of common melanoma antigens as defined by the postimmune antimelanoma sera. These antigens may be localized in the membrane or within the cytoplasm.