3 3 5 5 tetramethylbenzidine liquid substrate system

Three recombinant proteins were respectively diluted in phosphate-buffered saline (PBS) to a final concentration of 0.5 μg/mL for coating a 96-well microtiter plate (No. 3590; Corning, Corning, NY, United States) overnight at 4 °C. The antigen-coated wells were blocked with 10% fetal bovine serum (FBS) at 37 °C for 1 h. Human serum diluted 1:200 was incubated in the antigen-coated wells for 60 min. Horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Zhongshan Golden Bridge Biological Technology Co Ltd, Beijing, China) as a secondary antibody was diluted 1:10000 for coating (1 h) followed by washing with PBS containing 0.1% Tween 20 (PBST). The 3,3’,5,5’-Tetramethylbenzidine Liquid Substrate System (Solarbio Science & Technology Co Ltd, Beijing, China) was used as the detecting agent.
The optical density (OD) value of all wells was read at 450 nm, and the cut-off value for defining a positive reaction was designated as the mean OD value of the 59 normal sera plus three standard deviations (SDs). Each microtiter plate included 10 NHC samples representing a range of absorbance values above and below the mean of 59 NHC samples, and the average OD value of 10 NHC samples was used to normalize all OD values to the standard mean of the 59 NHC samples. Each sample was tested in triplicate.