In situ citrullination of human fibrinogen. (A) Fibrinogen (Fib) or citrullinated fibrinogen (cFib) was immobilized to enzyme-linked immunosorbent assay (ELISA) plates at a concentration of 1 μg/ml. Increasing concentrations of the anti-cFib monoclonal antibody (mAb; clone 20B2) were added, followed by addition of enzyme-conjugated rabbit anti-mouse immunoglobulin antibodies and o-phenylenediamine substrate. All data points represent means and ranges of duplicate measurements of optical density at 490 nm. (B) ELISA plates were coated with 1.0 μg/ml human fibrinogen and incubated with citrullination buffer including recombinant human peptidylarginine deiminase (rhPAD) enzyme, ModiQuest (MQ) PAD2 (8 mU/ml), rhPAD2 (42 ng/ml), MQPAD4 (49 mU/ml) or rhPAD4 (1,500 ng/ml). Shown is the activity as a percentage of the maximal activity obtained for each enzyme, corresponding to the activity at 4 hours. Anti-cFib mAb was used at a concentration of 0.5 μg/ml, as determined from the graph shown in (A). Symbols and error bars represent means and ranges of duplicate measurements.