We developed and employed a novel cell-based ELISA to identify melanoma-reactive antibodies. Adherent cells of interest were plated at 3610 5 cells per in 200 mL of appropriate media well on 96-well flat bottom tissue culture plates (Corning, Corning, NY) and were grown in a monolayer at 37uC and 5% CO 2 to 80-100% confluence. Cells were then lightly fixed in 0.5% formaldehyde/ Hank's Buffered Salt Solution. Plates were then wrapped in foil and placed in a -80uC freezer until the day of the assay. On the day of the assay, plates were thawed for 30 minutes, washed 3 times with PBS and then blocked with a 5% non-fat milk/PBS solution for 2 hours. After removal of the blocking solution, 50 mL of culture supernatants or tumor-specific antibodies were diluted 1:2 in 1% non-fat milk/PBS solution and then added to each well, and plates were incubated for 90 minutes at room temperature on an orbital shaker. Plates were then washed 4 times with PBS/ 0.05%Tween (PBS-T). The binding of antibodies to cell surface proteins was detected following a 45 minute incubation with a goat anti-human horseradish peroxidase-labeled F(ab)' 2 Fc-specific antibody (Jackson ImmunoResearch, West Grove, PA) diluted 1:250 in 1% milk/PBS-T at room temperature on an orbital shaker. Wells were then washed 4 times with PBS-T. The color reaction was developed for 15 minutes with OPD (Sigma) and OD was measured in an ELISA reader (BMG Labtech, Offenbury, Germany) at 492 nm (reference wavelength, 650 nm). Each plate contained triplicate wells of a positive control antibody, Trastuzumab (Genentech, South San Francisco, CA), and a negative control antibody, non-specific human IgG 1 (Jackson Immunoresearch) at a concentration of 250 ng/mL both diluted in RPMI-1640 media supplemented with 10% fetal calf serum. Binding of Trastuzumab to cells and background OD values for the negative non-specific human IgG control antibody formed the criteria for inclusion of readouts in the study. Since we were limited by the volume of culture supernatants for each culture, assays were repeated only when sufficient culture supernatants were available to confirm reproducibility of readouts.