Nunc medisorp elisa plates

Antibody titers against collagen type II were analyzed by coating Nunc MediSorp ELISA plates (Thermo Fisher Scientific, Waltham, MA, USA) with bovine (MD Bioscience, St. Paul, MN, USA) and murine (Chondrex Inc., Redmond, WA, USA) collagen type II at 20 μg/ml each in carbonate/bicarbonate buffer overnight at 4°C. Sera were applied at a dilution of 1:6,000 for 1.5 h at RT. After washing, the plates were incubated with diluted rabbit anti-mouse IgG for 1 h at RT. Thereafter, bound antibodies were detected as described for C. difficile IgG detection.

The 96-well Nunc MediSorp ELISA plates (Thermo Fisher, Germany) were coated with 50 µL of serum serially diluted in PBS and incubated overnight at 4°C. Each well was washed four times with PBS and blocked with 100 µL 1% BSA-PBS for 1 h at 37°C. Primary antibodies were diluted with 1% BSA-PBS (Supplementary Table S1), and 100 µL was added to the wells for 1 h at 37°C, followed by 100 µL of biotinylated secondary antibody diluted in 1% BSA-PBS (Supplementary Table S1) and incubated for a further 1 h at 37°C. The plate was washed four times, and 100 µL of VECTASTAIN ABC Reagent (Vector Laboratories, USA) was added. Staining was performed according to the manufacturer’s instructions, and the absorbance at 450 nm was measured with the FLUOstar Omega photometer (BMG Labtech, Germany).