O phenylenediamine dihydrochloride

Ribose (30 mM) was mixed with 2 mg/mL gelatin in 200 mM NaPB in the presence of AMG (0.1, 1, 10 and 100 µM) or TBE (0.01, 0.1, 1, 10, 100 µg/mL), and incubated at 37 °C for 7 days. MG-gelatin was prepared by incubating 2 mg/mL gelatin with 100 µM MG in PBS at 37 °C for 3 days in the presence of AMG or TBE. MG-H1 formation was measured by ELISA as previously described [37 (link)]. In brief, for noncompetitive ELISA, each well of a 96-well immune plate (Thermo Fisher Scientific, Waltham, MA, USA) was coated with 0.1 mL of the 1 µg/mL sample in PBS and blocked with 0.5% gelatin hydrolysate in PBS. The wells were incubated for 1 h with 0.1 mL of 1 µg/mL MG-H1 antibody [19 (link)]. Antibodies bound to the wells were detected using horseradish peroxidase-conjugated anti-mouse IgG antibody (Thermo Fisher Scientific, Waltham, MA, USA). Then, stained with 100 μL of 500 µg/mL O-phenylenediamine dihydrochloride (Fuji Film Wako Pure Chemical, Japan) in citrate-phosphate buffer (pH 5.0) containing 5.9 mM hydrogen peroxide for 3 min. The reaction was terminated with 100 μL of 1.0 M sulfuric acid, and the absorbance was measured at 492 nm using a Sunrise RAINBOW THERMO RC system (TECAN, Männedorf, Switzerland).

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).