Assessment of anti-Fc antibody responses
SECONDARY, ALDRICH
Anti-human Fc antibodies were measured by an in-house ELISA. Flat-bottomed maxisorp nunc immune plates (Thermofisher, 439,454) were coated with 100 μl of human IgG Fc fragment (Arotec Diagnostics, ATF01) at 2 μg/mL in PBS buffer overnight at 4 °C. Plates are blocked with PBS 1% BSA (Sigma–Aldrich, A3059) and subsequently incubated with human serum (diluted to 1:20 and 1:100) for 2 h at 37 °C. Well-characterised patient sera with IgG, IgA, and IgM anti-Fc antibodies, respectively, were included as positive controls. After washing plates four times with PBS 0.05% Tween 20, anti-human IgG (Fab specific; Sigma-A8542), anti-human IgA (Sigma, A3400) or anti-human IgM (Invitrogen-AH10605) secondary antibodies were used to detect individual anti-human Fc antibody isotypes. Following 1-h incubation with secondary antibodies, plates were washed six times with PBS 0.05% Tween 20 and phosphatase substrate (Sigma, S0942) is added. Optical density at 405 nm is measured by a plate reader (Spectramax Id5) at 30 min of incubation.