Tmb 3 3 5 5 tetramethylbenzidine substrate

Microtiter plates (Corning) were coated with 2 µg/mL CHIKV E2, 10 µg/mL purified virus virions or the control protein SUMO in a carbonate buffer (CBS, pH9.6) overnight at 4 °C, and blocked with 3% BSA(Sigma-Aldrich) PBS (pH7.4) at 37 ℃. Serial tenfold dilutions of the SUMO-tagged VHHs in 3% BSA were incubated with the immobilized antigen, followed by incubation with HRP-conjugated goat anti-SMT3 (1:2000, CUSABIO). After wash, 100μL of TMB (3,3′,5,5′-tetramethylbenzidine) substrate (TIANGEN) was added to the wells, and reactions were stopped with 100μL of 1 M HCl. Absorbance was measured at 450 nm on an Epoch™ microplate reader (BioTek Instruments Inc., Winooski, VT, USA). The EDIII protein of ZIKA was used as the negative control.

Polystyrene microplates coated with Gn and Gc proteins of HTNV and SEOV were incubated overnight at 4 °C. Next, PBST containing 1% BSA was added and incubated at 37 °C for 1 h. Serial dilutions of serum samples (experimental wells) and naïve mouse serum samples (negative control) were then added and incubated at 37 °C for 1 h. HRP-goat anti-mouse IgG (Zhuang Zhi Bio, China) was added to each well and incubated at 37 °C for 1 h, followed by the addition of TMB (3,3′,5,5′-tetramethylbenzidine) substrate (TIANGEN, China). The reaction was stopped by adding ELISA stop solution (Solarbio, China) after standing at room temperature for 10 min. The absorbance value at 450 nm of each well was measured, and a P/N value greater than 2.1 was considered positive. The reciprocal of the highest dilution of the serum considered positive was used to calculate the specific antibody titers of HTNV and SEOV based on the geometric mean titer (GMT) of particular antibodies in each group of immunized mouse serum.
The Gn and Gc proteins of HTNV and SEOV were coated similarly to identify different types of immunoglobulins. Mouse serum samples were diluted, and HRP-labeled IgG was used as IgG1, IgG2a, IgG2b, IgA and IgM (Southern Biotech, USA). The absorbance at 450 nm was then measured to detect the quantity of each immunoglobulin isotype.