Extended Data Fig. 2: Autoantibody detection from plasma samples.
(a) Immunofluorescent analysis (IFA) of plasma samples on Hep2 slides using confocal microscopy. Representative confocal microscopy images of Hep2 slides stained with HC-Ags (n = 7), pRD-N (n = 1) and pRD-Ags patients (n = 12) plasma samples and detected with anti-IgG (green). Staining for all samples were performed simultaneously in a single experiment with two technical replicates for each. Score is depicted in left bottom corner for each sample. Nucleus (DAPI, blue), cytoplasm (Evans Blue, red). Magnification: 60x. (b) Quantification of cytoplasmic and nuclear Hep2 positivity. Bars represent mean±s.e.m. with individual values from HC-Ags (n = 7), pRD-N (n = 1) and pRD-Ags patients (n = 12). Two-sided unpaired Student’s t-test. (c) Heatmap of autoantigen reactivities. Binding of plasma IgGs to a set of autoantigens (human embryonic kidney 293 cell protein extract, human M2 antigen, intrinsic factor, proliferating cell nuclear antigen, tissue transglutaminase, threonyl-tRNA Synthetase, Ro/SSA (52 kDa), thyroid peroxidase, U1-SnRNP C protein, thyroglobulin, ribosomal phosphoprotein P0, IFN-α, IFN-ω, IL-12, single and double stranded DNA were measured with ELISA in 400x dilution. Blank corrected absorbance values are shown by HC-Ags (n = 10) pRD-N (n = 1) and pRD-Ag (n = 14) subjects. Antigens, with z score above 2 in three or more patients were considered as common autoantigens and are labeled with bold (left). Statistical analysis was performed using two-sided unpaired Student’s t-test on Ag-experienced subjects (excluding pRD-N, P1). P values for differences between HC-Ags and pRD-Ag patients are shown (right) and the statistically significant differences are depicted by red. (d) Anti-cytokine auto-Ab levels. Binding of plasma IgGs to IFN-α, IFN-ω, IL-12 was measured with ELISA in 2-fold serial dilution (200–1,600-fold). (e) IgM 9G4 auto-Ab levels. Plasma 9G4 auto-Abs were measured with ELISA in 2x serial dilution (100 – 204,800-fold). Cut-off for positivity on b and c is indicated with green dashed line defined as z = 2 at each dilution. Mean values of HC-Ags for c and d (n = 11 or 4, respectively) are depicted with thick black line and dark gray circles, individual values for pRD-N (n = 1) and pRD-Ag (n = 14) patients are shown. ELISAs for c-e were performed with two technical replicates.
