Hrp conjugated goat anti mouse igg h l

Blood was collected from infected or control mice by the use of retro-orbital bleeds, and serum was stored at −80°C for subsequent use in the experiments. The presence of antibodies in the serum was measured by ELISA. Ninety-six-well microtiter plates (Costar; Corning) were coated overnight at 4°C with 100 μl of 0.1 M carbonate buffer (pH 9.6) containing 2 μg/ml of BPMM whole-cell lysate. After blocking with 2% bovine serum albumin (BSA) in PBS containing 0.1% Tween 20, 100 μl of serially diluted serum was added to the wells. The plates were incubated at 37°C for 1.5 h, rinsed in PBS–0.1% Tween 20, and incubated at 37°C for 1 h with 100 μl of appropriately diluted horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H+L; Sigma), HRP-conjugated goat anti-mouse IgG1 and IgG2a (Abcam), and HRP-conjugated goat anti-mouse IgM and IgA (Bio-Rad) secondary antibodies. The reaction was then developed with 100 μl of tetramethylbenzidine liquid substrate solution (catalog number T4444; Sigma) at room temperature for 30 min in the dark and stopped by the addition of 1 M sulfuric acid. The absorbance at 450 nm was measured by an ELISA plate reader (Tecan Sunrise).

The purified particles (E30, E6, E3, E11 or CVB3) were coated onto ELISA plates (Costar, Corning, USA) at 30 ng/well, followed by overnight incubation at 4 °C. The coated plates were blocked with 1% BSA in PBST (PBS plus 0.1% Tween 20) at 37 °C for 2 h. Thereafter, the plates were washed five times with PBST and 6C5-IgG or 4B10-IgG was added as primary antibody to each well at 10-fold serial dilutions resulting in a range of concentrations from 0.02 nM to 20 nM. The plates were kept at 37 °C for 1 h after which they were washed again with PBST five times. HRP-conjugated goat anti-mouse IgG H&L (# AP308P, 1/3,000 dilution) (Sigma-Aldrich, St. Louis, USA) was added as a secondary antibody and the plates were incubated at 37 °C for 0.5 h. The plates were washed with PBST five times, and 3,3′,5,5′ -tetramethylbenzidine (TMB) substrate (# P0209, Beyotime, Shanghai, China) was added to each well for 5 min at room temperature. Finally, 2 M H₂SO₄ was added to the plates to stop the reaction, and the absorbance value of each well was read at 450 nm.