Measurement of Anti-TMEV Antibodies

When the mice were killed, blood was collected from the heart of TMEV-infected mice. The levels of serum anti-TMEV isotype antibodies were assessed by ELISA as described previously (43 (link)). Ninety-six-well flat-bottom Nunc-Immuno plates, MaxiSorp surface (Thermo Fisher Scientific) were coated with TMEV antigen at 4°C overnight. After blocking with 10% fetal bovine serum (FBS) and 0.2% Tween 20 in PBS, 2⁷- or 2¹¹-fold diluted serum samples were plated. Horseradish peroxidase (HRP)-conjugated anti-mouse IgG1 (Thermo Fisher Scientific), IgG2b (Thermo Fisher Scientific), IgG2c (SouthernBiotech, Birmingham, AL), or IgA (Thermo Fisher Scientific) was used to detect binding anti-TMEV isotype antibodies. The reaction was developed by adding o-phenylenediamine dihydrochloride (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), and stopped with 1N HCl. Absorbance was read at 490 nm on a Model 680 Microplate Reader (Bio-Rad Laboratories, Hercules, CA).