A panel immunoblot using co-incubated monoclonal antibodies for identification of melanoma cells

Antigen expression in melanoma is heterogeneous. Immunophenotyping using a panel of monoclonal antibodies may facilitate immunotherapy. An immunoblot procedure was developed to detect antigens in melanoma cells. Numerous monoclonal antibodies were tested to determine if (1) antigens were detected after transfer to membranes, (2) single bands or discrete multiple bands were obtained, (3) co-incubation of multiple monoclonal antibodies had no interference, and (4) banding patterns were non-overlapping. Antigens were selected based upon their association with melanoma and the availability of respective monoclonal antibodies. Antigens were melanoma antigen recognized by T-cells (MART-1), tyrosinase, tyrosinase-related protein 1 (TRP-1), S100, vimentin, glycoprotein 130 (gp130), a carcinoembryonic antigen (CEA)-like marker, KBA-62 and NKI-C3. Actin positive controls could be assessed simultaneously. Test samples were separated by polyacrylamide gel electrophoresis in a 4-15% polyacrylamide gradient, transferred to polyvinylidine fluoride membrane, blotted using a Fast-Blot apparatus (Pierce), and developed using diaminobenzidine/metal. Melanoma cell lines were immunophenotyped using this panel immunoblot, and were compared to a standard control and to non-melanoma cells. Up to four antigens could be detected simultaneously in a single lane of the immunoblot, using a single test sample of greater than 100000 cells.