To estimate the frequency of melanoma-reactive antibodyproducing B cells in melanoma patients we performed limiting dilution analysis. We selected a stage II patient, who, we predict, may have a high antibody response to melanoma, based on our findings that the antibody responses were highest in this group (Figure 3 , B). For this stage II patient (Patient 15, see Table 1 ) from our limiting dilution analysis assays using the cell-based ELISA, we estimate that one out of 1,790 peripheral blood B cells produces antibodies that bind to A-375 melanoma cells (Figure 4 , C). For this same patient, the frequency of B cells producing antibodies that react with melanocytes was also evaluated at the same B cell densities as melanoma cells. We did not observe a comparable patient antibody response to melanocytes as we did to melanoma cells, suggesting a much lower frequency of antibodies that bind to normal cells of the same origin ( Figure 4 , C left). Limiting dilution analysis against two additional metastatic (SK-MEL-28, A-2058) and one primary (WM-115) melanoma cell lines for the same patient yielded different but comparable frequencies to A-375 for the metastatic cell lines (SK-MEL-28, 1 out of 1,650 B cells; A-2058, 1 out of 1,170 cells), and a much lower frequency of antibodies that bind to the primary melanoma line WM-115 which was similar to that observed with primary melanocytes (Figure 4 , C right). For this patient, the data suggest detectable circulating B cell humoral response frequency against metastatic melanoma cells and lower frequency for normal human melanocytes or primary melanoma cells. To further confirm the frequency observations for the patient-derived circulating B cell repertoire, we performed additional limiting dilution assays for another stage II patient (Patient 21, Table 1 ). For Patient 21, we estimate 1 out of 2,430 B cells that produces antibodies bind to the same melanoma cell line tested for Patient 15 (Figure 4 , D), suggesting lower but comparable frequency to those estimated for B cells from Patient 15. In summary, applying the above methodology, these results suggest that tumor-reactive antibodies from circulating B cells are more frequent in melanoma patients than healthy volunteers and more frequent against a range of metastatic melanoma cells compared to normal melanocytes.