ACPA reactivity with PAD2- and PAD4-citrullinated fibrinogen.

(A-D) Microtiter plates were coated with native fibrinogen or fibrinogen citrullinated by either PAD2 or PAD4 (cit-Fib) at various concentrations. Pooled synovial fluid (SF) from 6 anti-CCP-positive RA patients was applied at different dilutions ranging from 1:50 to 1:800, and the binding of ACPAs was detected using HRP-anti-human IgG and OPD substrate. Shown are optical density (OD) values from citrullinated minus native fibrinogen. (E) The pooled SF was also tested in dilutions of 1:5 and 1:15 using fibrinogen citrullinated by 0.62 μg PAD per mg protein. Average and range of duplicate measurements are shown. (F) Western blotting showing the binding of ACPAs from pooled SF (diluted 1:100) to native fibrinogen and fibrinogen citrullinated by PAD2 or PAD4 (0.62 μg PAD per mg protein). Coomassie staining is shown as a loading control. The three bands show α-chain, β-chain and γ-chain of fibrinogen. (G) Anti-modified citrulline (AMC) western blot to detect citrullinated fibrinogen, citrullinated by 0.62 or 10 μg PAD per mg protein, respectively, (top) and brilliant blue stained gel to detect total protein (bottom). (H) The density of the visible citrullinated protein bands in the AMC blot was normalized to corresponding protein signal. Mean and SEM are shown for triplicate blots with *p<0.05, **p<0.01.