Tetramethylbenzidine

Sera from immunized and control mice were analyzed for PPRV-specific antibodies (IgG, IgG1, and IgG2a) by indirect ELISA. Briefly, 96-well flat-bottomed plates were coated with 100µLof purified PPRV whole-virus antigen at a concentration of 2 µg/mL per well in 0.1 M carbonate/bicarbonate coating buffer (pH 9.6),overnight at 4°C, and then the plates were blocked with 5% skim milk in PBST, for 2 h at 37°C. Serum samples, serially diluted in PBST, were added to the wells, in duplicate, and incubated at 37°C for 1 h. After washing with PBST three times, each well received 100µL of a 1/4000-diluted HRP-labeled goat anti-mouse IgG, IgG1, and IgG2a (Southern Biotech, Birmingham, AL, USA) and incubated at 37°C for 1 h. Finally, after washing with PBST three times, 100 µL of freshly prepared tetramethylbenzidine (Tiangen, Beijing, China) solution was added to each well and color was allowed to develop for 10 min, before stopping the reaction with 100µL of 2 M H₂SO_4.Optical density (OD) values were measured at a wavelength of 450 nm using an ELISA microplate reader (Multiskan Ascent; Thermo, Vantaa, Finland). The mean titers were calculated from log₁₀ of the highest serum dilution that yielded a two-fold higher OD value compared to that of sera from PBS-vaccinated mice.

On days 28 and 70 after postimmunization, serum was collected from common carp according to a previously reported method (49 ). The ELISAs for CD80/86, TNF-α, MHC-I, and MHC-II expression in treated fish were analyzed by using fish CD80/86, TNF-α, MHC-I, and MHC-II ELISA kits (Renjiebio, China), respectively. The expression of serum antigen-specific IgM was analyzed by ELISA according to a previous method (53 ). Briefly, the 96-well ELISA plate was coated with SVCV at 4°C for 12 h, followed by incubation with 5% skimmed milk in PBS (pH 7.4) for 2 h at 37°C. Each well was washed with PBST and incubated with serum samples (diluted at 1:50) as the primary antibody, followed by incubation at 37°C for 2 h and then washing with PBST. After that, horseradish peroxidase-conjugated anti-common carp (Cyprinus carpio carpio)/koi carp (Cyprinus carpio koi) IgM monoclonal antibody (Aquatic Diagnostics, Ltd., England) diluted at 1:1,000 was used as the secondary antibody. Tetramethylbenzidine (Tiangen, China) was used as a colorimetric substrate, and the color was developed. The absorbance at 450 nm was measured by using a microplate reader (Molecular Devices Corp., Palo Alto, CA).