Computational protocol: Anti-Melanoma immunity and local regression of cutaneous metastases in melanoma patients treated with monobenzone and imiquimod; a phase 2 a trial

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Protocol publication

[…] Antibodies in serum samples were detected using radioligand-binding assays (RBA), as described previously. This technique is at least as sensitive as ELISA and more sensitive than Western blotting. Briefly, plasmids pcDNA3-TH, pcDNA3-TYR, pcDNA3-PMEL17, pcDNA3-MCHR1 and pcDNA3-Melan-A (MART-1) were used according to the manufacturer in an in vitro TnTT7-coupled Reticulocyte Lysate System (Promega, Southampton, UK) with [35S]-methionine to produce radiolabeled full-length proteins TH, tyrosinase, gp100, MCHR1 and MART-1, respectively. Radiolabeled antigens were then used in RBAs with patient and healthy control (n = 20) sera at a 1:100 dilution, as described previously. An antigen-specific animal antibody was included in each RBA as a positive control. In each RBA, an antibody index for each serum was calculated as the counts per minute immunoprecipitated by tested serum divided by the mean counts per minute immunoprecipitated by 20 healthy control sera. In each experiment, the upper limit of normal was calculated as mean antibody index of controls plus 3 times the standard deviation (SD) and used as a threshold value for positivity. Increases in antibody indexes during therapy were analyzed statistically using the nonparametric Wilcoxon signed rank test of paired data (IBM SPSS Statistics version 22). [...] HLA typing of patients was performed on PBL, cultured from peripheral blood without cryopreservation, by flow cytometry using fluorescein isothiocyanate (FITC)-conjugated mouse anti-human HLA-A2-specific monoclonal antibody (mAb) (BD Biosciences, Breda, the Netherlands) and biotinylated HLA-A1/A36-specific antibody (BIH0331), biotinylated HLA-A2-specific antibody (BIH0648) or biotinylated HLA-A3-specific antibody (BIH0269, all from One Lambda Inc., Canoga Park, CA), followed by allophycocyanin (APC) conjugated streptavidin (Biolegend, ITK Diagnostics, Uithoorn, The Netherlands) to detect biotinylated antibody binding. Cells were acquired on a FACS Canto II (Beckton Dickinson, Breda, the Netherlands), and analyzed by FlowJo Software (Treestar, Ashland, OR). […]

Pipeline specifications

Software tools SPSS, FlowJo
Applications Miscellaneous, Flow cytometry
Organisms Homo sapiens
Diseases Melanoma, Vitiligo