Increased labeling of human melanoma cells in vitro using combinations of monoclonal antibodies recognizing separate cell surface antigenic determinants

A panel of mouse anti-melanoma monoclonal antibodies (MoAb) were analyzed for reactivity with human melanoma cells singly and in combination. Five MoAb, ZME-018,96.5, P94, 4.2, and 5.1, reactive with individual cell surface melanoma-associated antigens were tested with seven melanoma cell lines and seven fresh tumor biopsies. Cells were incubated with the MoAb, indirectly stained with fluorescein-conjugated goat anti-mouse immunoglobulin, and analyzed by flow cytometry. Percentage of labeled cells and relative fluorescence intensity (Fl) with individual MoAb varied with different cell lines and biopsy samples. The most reactive MoAb, ZME-018, 96.5, and P94, labeled 29-93% of the cells from cell lines with relative Fl of 2-59 units, thereby demonstrating phenotypic diversity of these cells. Similar results were obtained with cells derived from tumor biopsies, where 1-73% of cells were labeled and relative Fl ranged from 0-27. These variations were reduced by using a “cocktail” of MoAb which recognized different melanoma-associated antigens. In cell lines both the percentage of labeled cells (range, 82-95%) and relative Fl (range, 36-115) increased substantially (P < 0.025 and P < 0.005, respectively) when a “cocktail” prepared from all five MoAb rather than individual MoAb was used. A cocktail of MoAb increased the percentage of labeled tumor biopsy cells (range, 53-78%; P < 0.01) and relative Fl (range, 11-69; P < 0.025). The mean Fl obtained by incubating cells with a cocktail of suboptimal concentrations of three MoAb (ZME-018, 96.5, P94) was 48 ± 12 (SD), which was significantly increased compared to the mean Fl seen with suboptimal concentrations of MoAb alone (ZME-018, 7 ± 10; 96.5, 8 ± 7; P94, 2 ± 2; P < 0.005). These findings were confirmed by radioimmunoassay using a combination of two MoAb, ZME-018 and 96.5. The data suggest that cocktails of MoAb were more effective than single MoAb alone for melanoma tumor cell labeling in vitro and might be more effective for tumor imaging and therapy.