Autoantigen array

IgA and IgG autoantibody reactivities against a panel of 122 autoantigens were measured in 50 µL BAL using an array developed by the University of Texas Southwestern Medical Center (table S1) [8, 26]. Briefly, samples were incubated with autoantigens (listed in table S1) printed on 16-pad FAST™ microarray slides before being incubated with secondary fluorophore-conjugated anti-human IgG or IgA antibodies. Following detection, images were analysed by Genepix Pro 6.0 (Molecular Devices, USA). Analysed data underwent quality control that included filtering bad spot data and batch effect correction. Average net fluorescence intensities (NFIs) and signal/noise ratios (SNRs) were calculated by subtracting negative control (PBS) fluorescence values from sample fluorescence values. If SNR/NFI<0, SNR/NFI was set to 0.001; if NFI<20 and SNR>5, then SNR=NFI⋅0.15; and if SNR<0.05 and NFI>20, then NFI was set to 0.001. To avoid outliers in either the NFI or SNR values, antibody scores were calculated (Ab score=log₂(NFI⋅SNR+1). All data were normalised using variance stabilising normalisation.