Horseradish peroxidase hrp conjugated streptavidin

A total of 96-well plates were coated in duplicate with 100 μL of lysates at a dilution of 1:10 and supernatants at a dilution of 1:5 of VP1 DNA vaccines or control-transfected cells. EV71 VP1 protein (100 μL, Abnova, USA; working concentration 50 μg/L) was used as coating antigen to detect the immunized sera. After blocking, 100 μL of anti-EV71 VP1 monoclonal mouse antibody (Abnova) was used to determine the production of VP1 protein from the vaccines, and a rabbit anti-human IgG Fc (Jackson ImmunoResearch, West Grove, PA, USA) was performed to determine the production of VP1-hFc protein. After washing, 100 μL of biotinylated secondary antibody (Multi Sciences Biotech Co., Ltd., Hangzhou, Zhejiang, China) and 100 μL of horseradish peroxidase (HRP)-conjugated streptavidin (Southern Biotech, Birmingham, AL, USA) were added to each well. The plates were developed using a 3,3′,5,5′-tetramethylbenzidine (Sigma, St Louis, MO, USA) solution. For one step ELISA assays, HRP-conjugated goat anti-rabbit or mouse secondary antibody (Multi Sciences Biotech Co., Ltd.) was added after incubation with the appropriate primary antibody followed immediately by development. The plates were read spectrophotometrically at 450 nm, and the wells were scored as positive when the absorbance value was greater than twice the value of the negative control.