Victor2 multilabel time resolved fluorometer

Peptides were synthesized and purified by the manufacturer (Sigma). Putative citrullinated epitopes were predicted based on published binding data for anchor residues at pockets 1, 4, 6, 7, and 9 and our data about accommodation of citrulline versus arginine (17 (link)). As summarized in Supplemental Table 1, DR0401 motifs were defined by calculating the product of the coefficients for each possible combination of residues that included a citrulline and scores of 0.1 or higher were selected for synthesis.
For peptide binding assays, increasing concentrations of each non-biotinylated test peptide were incubated in competition with 0.01 µM biotinylated HA_306–318 peptide in wells coated with HLA-DR0401 protein as previously described (18 (link)). Europium-conjugated streptavidin (PerkinElmer) was used to label biotinylated peptide bound to the HLA-DR protein and was quantified using a Victor2 multilabel time resolved-fluorometer (PerkinElmer). Binding curves were fitted by non-linear regression with a sigmoidal dose response curve model using Prism software (version5.0, GraphPad Software Inc.). Peptides selected for further study based on positive binding results were re-synthesized at a higher purity by another manufacturer (Genscript).