Hrp conjugated anti mouse igg

IL-17 and IFN-γ ELISAs were done using kits following the manufacturer’s instructions (BD Bioscience, San Diego, CA). 30 µg/ml of sonicated C. rodentium protein was coated on 96-well plates as the capture antigen to quantitate C. rodentium-specific IgA and IgG in the samples and used HRP-conjugated anti-mouse IgA and HRP-conjugated anti-mouse IgG, respectively (Bethyl Laboratories, Montgomery, TX). Values are reported as C. rodentium -specific IgG or IgA relative to pooled serum used to generate a standard curve.

ELISA plates were pre-coatedprecoated overnight with pseudotyped HIVluc-MERS virus at 4 °C. The coated plates were washed three times with PBS-T (PBS containing 0.1% Tween 20) and then blocked with blocking buffer (5% skim milk in PBS-T) for 2 h at 25 °C. Plates were incubated with the twofold serial dilution of mouse serum starting from 1:100 and incubated at 25 °C for 2 h. Then, the plates were washed four times with PBS-T and incubated with HRP-conjugated anti-mouse IgG (Bethyl Laboratories, 1:1000), IgG1 (Bethyl Laboratories, 1:1000), or IgG2a (Bethyl Laboratories, 1:1000) at 25 °C for 1 h. After four washes with PBS-T, the plates were incubated with a 0.03% 3,3′,5,5′-tetramethylbenzidine solution (Koma Biotech) for 15 min at 25 °C. The reaction was stopped with 1 N H₂SO₄. The optical density (OD) at a wavelength of 450 nm was measured using an ELISA plate reader (Bio-Rad). The endpoint titer was defined as the last serum dilution at which the OD value was above the average of negative-control wells (secondary antibody only).