Profiling Antibody Polyreactivity Using PubArray

All experiments were performed at 4°C using ProtoArray Human Protein Microarrays (Thermo Fisher Scientific). Microarrays were blocked for 1 h in blocking solution (Thermo Fisher Scientific), washed, and incubated for 1 h 30 min with IgG antibodies at 2.5 µg/ml as previously described (Planchais et al., 2019 (link)). After washings, arrays were incubated for 1 h 30 min with AF647-conjugated goat anti-human IgG antibodies (at 1 µg/ml in PBS; Thermo Fisher Scientific), and revealed using GenePix 4000B microarray scanner (Molecular Devices) and GenePix Pro 6.0 software (Molecular Devices) as previously described (Planchais et al., 2019 (link)). Fluorescence intensities were quantified using Spotxel software (SICASYS Software GmbH), and mean fluorescence intensity signals for each antibody (from duplicate protein spots) were plotted against the reference antibody mGO53 (non-polyreactive isotype control) using GraphPad Prism software (v8.1.2, GraphPad Prism Inc.). For each antibody, Z-scores were calculated using ProtoArray Prospector software (v5.2.3, Thermo Fisher Scientific), and deviation (σ) to the diagonal, and polyreactivity index (PI) values were calculated as previously described (Planchais et al., 2019 (link)). Antibodies were defined as polyreactive when PI >0.21.