ACPA Levels in Rheumatoid Arthritis Synovial Fluid

ACPA IgA and IgG levels were determined in synovial fluids of RA patients according to the protocol previously described (20 (link)) with minor adaptations. Microtiter ELISA plates (Nunc MaxiSorp™ flat-bottom plates; ThermoFisher) were coated with 50 μl of 1 μg/ml streptavidin (ThermoFisher) and incubated overnight at 4°C after which 50 μl of 1 μg/ml biotinylated CCP2-cittruline or CCP2-arginine (kindly provided by Dr. J.W. Drijfhout, Department of Immunohematology and Blood Transfusion (Leiden University Medical Center)) was added for 1 hour at RT. Synovial fluid (1:10 diluted) was added for 1 hour at 37°C. Wells were washed and incubated with F(ab’)2 goat anti-human IgA-HRP (1:4000, ThermoFisher) or F(ab’)2 goat anti-human IgG-HRP (1:4000, ThermoFisher) for 1 hour at 37°C. Fifty μl of 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate was added after which the reaction was stopped with 50 μl of sulfuric acid (10% H₂SO₄). Absorbance was measured with a microplate reader (Bio-Rad, Berkeley, CA) at 450nm.