To evaluate the presence and estimate the frequency of tumorreactive antibodies, we selected wells with OD values above 75% of the OD of the positive control antibody. To compare the percentage of positive cultures across patients, OD values were normalized against the positive control. For these evaluations, the mean positive control OD was assigned a relative absorbance of 1 for each plate and B cell cultures were converted from OD units to relative absorbance, and culture wells with relative absorbance values greater than 0.75 to melanoma cells but not melanocytes were selected. These criteria were also applied in limiting dilution assays to estimate the percentage of non-reactive B cell culture well. In these limiting dilution assays, B cells were plated at different densities (ranging from 125 to 2,500 B cells) and the percentage of non-reactive cultures was calculated for different patients and cell lines as a way to approximate the frequency of B cells producing melanoma-reactive antibodies using Poisson distribution.