Figure 2

Anti-replication protein A (RPA) antigen-capture ELISA. (a) Effects of NaCl concentration of the cell extracts on the reactivity of anti-RPA human sera. ELISAs were performed as described in the Materials and methods section with mAbs against RPA32 or RPA70 to coat ELISA plates and to capture RPA from K562 cell extracts, which were prepared in buffer containing either 0.15 M or 0.5 M NaCl. Sera diluted to 1:500 in 0.5 M NaCl NET/Nonidet P40 were tested. (b) Effects of single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), or double-stranded RNA (dsRNA) on the reactivity of human anti-RPA autoantibodies. Affinity-purified RPA on a microtiter plate was incubated for 30 minutes with ssDNA, dsDNA, or dsRNA (poly I:C) at concentrations of 0.01 to 100 μg/ml or with buffer alone. Wells were then incubated with 1:2,000 diluted anti-RPA-positive sera followed by ALP mouse mAb anti-human IgG, and developed. The percentage reactivity compared with RPA incubated with buffer alone (100%) is shown. ssDNA or dsDNA, but not dsRNA, inhibited the human anti-RPA binding in a dose-dependent manner. (c) Correlation between levels of anti-RPA by antigen-capture ELISA with mAbs against RPA32 and against RPA70. The reactivity of eight anti-RPA-positive human autoimmune sera in ELISA with mAbs against RPA32 and against RPA70 was compared. Spearman r = 0.9524, p = 0.0011. (d) Titration curves of anti-RPA-positive human sera. Titration curves of nine anti-RPA-positive autoimmune sera and four normal human sera (NHS) were created by ELISA with mAb against RPA32. K562 cell extracts in 0.5 M NaCl NET/Nonidet P40 buffer were used and sera were serially diluted 1:5 starting from 1:500. (e) Screening of anti-RPA antibodies in sera from patients with various systemic rheumatic diseases by ELISA. Sera from SLE (n = 276), rheumatoid arthritis (RA; n = 35), SSc (n = 47), PM/DM (n = 43), SjS (n = 40), and normal control (NHS, n = 30) were tested at 1:2,500 dilutions by ELISA with mAb against RPA32. SLE (p < 0.001 versus RA, p < 0.05 versus SSc, p < 0.01 versus PM/DM, p < 0.001 versus NHS) and SjS (p < 0.001 versus RA or NHS, p < 0.05 versus SSc, p < 0.01 versus PM/DM) showed high reactivity. RA versus SSc, p < 0.05; SSc versus NHS, p < 0.05; all other pairs were not significant. All comparisons were made with the Kruskal–Wallis test with Dunn's multiple comparison test. Open symbols, immunoprecipitation negative; filled symbols, immunoprecipitation positive. SjS, Sjögren syndrome; SSc, systemic sclerosis.