Epitope-Based Chicken-Derived Novel Anti-PAD2 Monoclonal Antibodies Inhibit Citrullination

Figure 3

Ability of anti-PAD2 antibodies to inhibit PAD2-mediated citrullination of Nα-benzoyl-L-arginine ethyl ester (BAEE). S4, S10, S24, S108, S170, and S309 antibodies were incubated with recombinant human PAD2 (rhPAD2) and buffer (20 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 1 mM dithiothreitol) in a total volume of 44 μL for 30 min at 25°C. BAEE and CaCl₂ (volume: 6 μL) were added simultaneously and the mixture was stirred well. The mixture comprised 5 nM rhPAD2, 10 mM BAEE, 1 mM CaCl₂, and various concentrations of antibodies (0.1–600 nM). The mixture was incubated at 37°C for 4 h, and 12.5 μL of 5 M perchloric acid was added to stop the reaction. The level of citrullinated BAEE was determined using a colorimetric assay system.