Profiling Antibody Polyreactivity on Protein Microarrays

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), washed and incubated for 1 h 30 min with IgG antibodies at 2.5 µg/ml as previously described^{25 (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; # A-21445, Thermo Fisher Scientific), and revealed using GenePix 4000B microarray scanner (Molecular Devices) and GenePix Pro 6.0 software (Molecular Devices) as previously described^{25 (link)}. Fluorescence intensities were quantified using Spotxel® software (SICASYS Software GmbH), and mean fluorescence intensity (MFI) signals for each antibody (from duplicate protein spots) was 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^{25 (link)}. Antibodies were defined as polyreactive when PI > 0.21.