We next investigated whether specific antibody responses to melanoma could be detected from circulating B cells of patients and healthy volunteers utilizing our cell-based ELISA. Antibodysecreting B cell cultures from 10 healthy volunteers and 10 patients (n = 4 stage II, n = 4 stage III, and n = 2 stage IV) were evaluated for reactivity to both metastatic and primary melanoma cells relative to non-specific human IgG control (calculated as fold increase above the non-specific human IgG control) employing our cell-based ELISA. We found a significant (P,0.0001) increase in the mean reactivity (fold increase) of patient-derived antibody cultures (n = 600) to metastatic melanoma cells (2.5 fold increase, 95% CI = 2.4 to 2.6) compared to antibody cultures (n = 600) derived from healthy volunteers (1.1 fold increase, 95% CI = 1.1 to 1.2) (Figure 2 , A). A significant (P,0.0001) increase was also seen in the mean reactivity of patient-derived antibodies to primary melanoma cells (2.3 fold increase, 95% CI = 2.2 to 2.4) compared to antibodies from healthy volunteers (1.0 fold increase, 95% CI = 1.0 to 1.1) (Figure 2 , B). From this patient cohort, we thus observed a significantly increased reactivity to primary and metastatic melanoma cells, compared to healthy volunteers.