Hrp conjugated goat anti mouse igg antibody

ELISA immunoplates (JET BIOFIL, Guangzhou, China) were coated with 200 ng of rtNA protein antigens per well in 0.05 M of sodium bicarbonate buffer (pH 9.6), and incubated overnight at 4 °C. On the second day, the immunoplates were washed three times with PBS, and then 300 μL of the blocking buffer containing 5% skim milk in PBS supplemented with 0.5% Tween 20 (PBST) was added per well to block the immunoplates for 1 h at room temperature. The serum samples were 3-fold serially diluted in a buffer containing 50% skim milk solution and 50% PBST, starting from a dilution factor of 100, and then were added to the immunoplates for 1 h incubation at 37 °C for binding to antigens. After washing three times with PBST, the immunoplates were incubated with the horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (ABclonal Technology Co., Ltd., Wuhan, China) for 1 h incubation at room temperature. Then, the immunoplates were washed with PBST three times, 50 µL of 3,3′,5,5′-Tetramethylbenzidine (TMB; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) was added per well, and the immunoplates were incubated at 37 °C for 15 min for catalyzing subtracts. Finally, 50 μL of 2 M H₂SO₄ was added per well to stop the reaction. Absorbance was read at 450 nm using a microplate reader (DLJ-200, DLJ Bio-Tech, Nanjing, China).