F ab 2 goat anti human iga hrp

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.

RF IgA levels were determined in synovial fluid of RA patients. Microtiter ELISA plates (ThermoFisher) were coated with 50 μl of 50 μg/ml human serum IgG (Sigma Aldrich) and incubated overnight at 4°C after which wells were blocked with 1% PBSA for 1h at 37°C. 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) for 1 hour at 37°C. Fifty μl of TMB substrate was added after which the reaction was stopped with 50 μl of 10% H₂SO₄. Absorbance was measured with a microplate reader (Bio-Rad, Berkeley, CA) at 450nm.

Anti-Carp IgA and IgG were determined in synovial fluids of RA patients according to protocols previously described (7 (link)) with minor adaptations. Microtiter ELISA plates (ThermoFisher) were coated with 50 μl of 10 μg/ml carbamylated bovine serum albumin (Ca-BSA) or non-modified BSA and incubated overnight at 4°C after which wells were blocked with 1% PBSA for 6 hours at 4°C. Synovial fluid (1:10 diluted) was added for 1 hour overnight at 4°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 TMB substrate was added after which the reaction was stopped with 50 μl of 10% H₂SO₄. Absorbance was measured with a microplate reader (Bio-Rad, Berkeley, CA) at 450nm.