We screened for tumor-reactive antibodies from patient B cell cultures and approximated frequency and specificity of selected cultures to tumor cells. For this we screened B cell culture supernatants against a stringent comparator using a positive control monoclonal antibody, Trastuzumab, that recognizes the HER2/neu tumor antigen, expressed on breast cancer cells and on some melanoma cells [38] . Trastuzumab was selected as a positive control because of comparable binding across melanoma cell lines and melanocytes, as shown by mean fluorescence intensities of antibody binding against a range of these cells (Figure 4, A) . Previous studies screening for tumor-specific antibodies have selected wells greater than the mean negative control optical density (OD) + three standard deviations as criteria for positive tumor-reactive antibodies [39] . Due to the inherent variability of cell-based assays, and the potential identification of false positive cultures, we chose more stringent criteria for antibody screening, by comparing antibodies produced by B cells to a positive control antibody (. 75% OD of positive control). Based on this antibody selection criteria (.75% OD of positive control), we estimate that 28% of B cell cultures (n = 1,800) derived from 21 patients, each arising from 500 B cells, produced antibodies that recognized metastatic melanoma cells, compared to 2% (n = 600) of cultures derived from 10 healthy volunteers (Figure 4 , B). From these 10 healthy volunteers, 2 individuals had one reactive culture (out of 60 cultures), 1 individual had 10 reactive cultures, and the rest of the cohort had no reactive cultures.