As a first step to evaluate the tumor reactivity and specificity of patient B cell-derived IgG antibodies, we developed a mediumthroughput cell-based ELISA with melanoma cells to detect antibodies against tumor cell surface antigens (Figure 1 ). Cells were allowed to grow and adhere on to 96-well plates prior to being preserved by a light fixative (0.5% formaldehyde). While preserving the cells and allowing for storage and access to multiple plates at any one time, light fixation with formalin allows preservation of potentially-antigenic epitopes on the surface of target cells. Previous studies have reported cell-based ELISA methods to identify tumor-specific antibodies in melanoma [39, 41] , where tumor cells were preserved using strong fixatives such as glutaraldehyde, known to potentially mask antigenic epitopes, thus compromising the recognition of antigens by antibodies [42] . Furthermore, the specificity and sensitivity of such methods has not been reported using antibodies against known cell surface antigens. Although many intracellular melanoma associated antigens have been described (tyrosinase, TRP-1, TRP-2, gp100/pmel17), most are also expressed by normal melanocytes, only a few defined cell surface antigens such as the High Molecular Weight Melanoma-Associated Antigen (HMW-MAA) are reported to be expressed on the surface of melanoma cells, and other antigens show heterogeneous expression among patients [43, 44] . Thus, this ELISA constitutes an attractive tool to evaluate broad responses to any naturally-expressed antigens on the surface of melanoma cells and melanocytes in this context. This screening methodology has additional potential advantages. Unlike assays screening against a single recombinant antigen or antigenic epitope, our method enables the evaluation of antibody repertoires of patients against a multitude of cell surface antigens in their native confirmation on the surface of both primary and metastatic melanoma cells and also melanocytes, providing more comprehensive information on the broad prevalence of tumorreactive and tumor-specific antibodies. Previous studies have shown concordance of cell line-associated antigens with antigens expressed on corresponding tumors, making them a suitable platform for tumor-reactive antibody screening [26, 45] . Thus, cell lines provide a promising alternative source of multiple tumor antigens in the absence of multiple well-defined, highly expressed, and readily available recombinant antigens. Unlike flow cytometric evaluations, the cell-based ELISA does not require the use of proteolytic enzymes such as trypsin, therefore better preserving cell surface antigens. Plates of target cells can be prepared, fixed and frozen in batches, thus allowing for higher throughput screening for tumor cell-reactive antibodies. It can be applied to evaluate . 300 culture supernatants against cell lines within a few hours. In principle, numerous ELISA plates for screening a range of cell lines with multiple supernatant samples can be processed simultaneously. Additionally, this methodology may be a potential tool for immunomonitoring tumor-specific humoral responses to therapies; selecting patients most likely to benefit from immunotherapy; or as a prognostic factor in linking tumor-reactive humoral responses to clinical outcomes. This assay may also be utilized to detect surface antigens in a range of cell types, and thus may be adapted to monitor the B cell-derived antibody repertoire in different disease contexts.