Hrp conjugated goat anti mouse igg1 igg2a

Flat bottom 384‐well MediSorp plates (Sigma‐Aldrich) were coated with 0.4 μg/mL (20 µL/well) of HEK293T‐produced E1E2 antigen in PBS and incubated overnight at 4 °C. After extensive washing with PBS containing 0.05% Tween 20 (Sigma‐Aldrich), plates were blocked for 1 h, at RT with PBS containing 5% nonfat dry milk (BioRad) and then washed as before. Six binary dilutions, starting from 1:200, of serum in PBS containing 5% nonfat dry milk were added to the antigen‐coated plates (40 μL/well) and incubated for 2 h, at RT. The plates were washed, incubated with HRP‐conjugated goat anti‐mouse IgM/IgG (Southern Biotech, AL), diluted 6000‐fold or with HRP‐conjugated goat anti‐mouse IgG1/IgG2a (Southern Biotech) diluted 1000‐fold in PBS containing 5% nonfat dry milk, for 2 h, at RT. Enzymatic reactions were developed following the addition of the TMB substrate (R&D Systems), for 20 min at RT then stopped with 20 μL/well of 1 m H₂SO₄. Absorbance was measured at 450 nm using a Tecan Infinite M1000 microplate reader. Normalization across ELISA plates was performed by pooling all the immune sera from day 49 which served as an internal standard. Serum pool dilution starting from 1:100 was used as an internal standard on each plate and a 4‐parameter logistic regression model was fitted to the measured values.