Fig 1: Study overview.(A) From an initial study employing 4595 antibodies on 16 melanoma cases and 16 non-diseased controls, antibodies against 48 and 82 protein targets were selected for further analysis in 149 sera from melanoma patients. Antibody selection was based on results from the initial study, as well as candidates from immunohistochemical staining on tissue material from matched individuals, literature searches, RNAseq data on melanoma tissue or antibody performance in Western blots with plasma. (B) The 149 patients were analyzed using a direct-labeling assay with 78 antibodies on suspension bead array (SBA1, Assay 1). The performance of the most significant set of 25 antibodies was studied further in a new bead array (SBA2, 160-plex) for repeated analysis of the previously prepared 149 samples (Assay 2). In addition, a new sample labeling and analysis with SBA2 was performed two years later (Assay 3). Antibody profiles for candidate proteins RGN, S100A6, STX7 and MTHFD1L displayed reproducible associations to T-stage or recurrence. (C) Candidate proteins were further evaluated for binding selectivity of antibodies using epitope mapping, immuno-capture mass spectrometry, Western blot, sandwich immunoassays and immunohistochemistry.