Elisa stop solution

The SARS-CoV-2 (Wuhan-Hu-1 strain) S1 protein (Sanyou Biopharmaceuticals Co., Ltd., Shanghai, China) was added at a concentration of 0.1 μg/100 μL/well to 96-well ELISA plates (Corning, NY, USA). Subsequently, serum was added to the wells using 2-fold multiplicative dilution and then incubated at 37 °C for 1 h. Following this, the wells underwent five washes using PBST before 100 μL of horseradish peroxidase-conjugated goat anti-mouse IgG (Thermo Fisher, Waltham, MA, USA) was applied to each well and incubated again at 37 °C for a duration of 40 min. During the serum IgG subtype detection experiments, the antibodies were replaced with horseradish peroxidase-conjugated goat anti-mouse IgG1 and horseradish peroxidase-conjugated goat anti-mouse IgG2a (Abcam, Cambridge, MA, USA). The plate was then washed five times with PBST buffer. Then, 100 μL of TMB substrate (3,3′,5,5′-tetramethylbenzidine) was added for 10 min to allow for color development. Finally, 50 μL of ELISA stop solution (Solarbio, Beijing, China) was added to terminate color development. The results were read using an ELISA plate reader (Gene Company, Hong Kong, China) at a wavelength of 450 nm. The terminal dilution criterion was met when the OD value in the sample wells exceeded 2.1 times that of the blank wells.

Purified NiV and HeV soluble G proteins were coated in 96-well ELISA plates using 50 mM carbonate buffer (pH=9.6) at a concentration of 1 μg/mL and incubated overnight at 4°C. After washing with PBST (PBS with 0.1% Tween20), all wells were blocked for 1 h at 37°C by adding 100 μL of PBS with 2% BSA. After the supernatant was discarded, the plate was washed three times with PBST. Subsequently, diluent (PBS with 0.2% BSA) and serum were added and serially diluted with two replicates per dilution, before incubation at 37°C for 1 h. The plate was washed three times with PBST after discarding the supernatant and adding a 1:50,000 dilution of HRP-conjugated goat anti-mouse IgG (Abcam, UK) at 100 μL/well. This was incubated for 45 min at 37°C. After discarding the supernatant, the plate was washed three times with PBST, and TMB solution (Solarbio Life Sciences, China) was added at 100 μL/well. ELISA Stop Solution (Solarbio Life Sciences, China) was then added at 50 μL/well after 5 minutes of color development, as the final enzyme marker. The absorbance was measured at 450 nm.

Sera were collected on day 14 after primary and booster immunization, respectively. Blood samples were incubated at 37 °C for 2 h and centrifuged for serum. The sera were stored at −20 °C for later detection. The 96-well MICROLON ELISA plates were coated with 100 μL protein diluent (5 μg/mL rEsxAB or rHla_m protein, extracted in the previous study), and placed at 4 °C overnight. Then, the plates were washed 3 times with PBST (PBS with 0.05% v/v Tween-20) solution, blocked with 5% BSA at 37 °C for 2 h, and further washed 3 times with PBST solution. Corresponding to the coated protein, 100 μL/well diluted sera (1:100 dilution with PBS solution) were added, and the plates were incubated at 37 °C for 2 h. After washing with PBST solution 5 times, HRP-conjugated goat anti-mouse IgG (Proteintech) solution (1:2000 dilution with 1% BSA) was added to the wells, and the plates were incubated at 37 °C for 2 h. Finally, after washing another 5 times with PBST solution, 100 μL/well TMB single-component substrate (Solarbio, Beijing, China) solution was added, with the incubation at 37 °C for 2 h. Then, 50 μL/well ELISA stop solution (Solarbio, Beijing, China) was added, and absorbance was measured at OD₄₅₀.

Orbital blood sampling was used to collect serum from mice at 7, 14, and 21 days after immunization. Indirect ELISA was then used to detect IgG antibody levels in mouse sera. ELISA coating solution (Solarbio, China) was used to dilute the antigen to a working concentration; and FC (4.5 ng/ml), GS (2.6 ng/ml), and FCGS (3.0 ng/ml) were added to 96-well plates and kept overnight at 4°C. Each well was washed three times with PBST, 5% skim milk was added, and the plates incubated at 37°C for 2 h. The liquid in each well was then discarded, wells were washed with PBST, and mouse serum (1:2,000) was added and again incubated at 37°C for 1 h. After being washed with PBST, rabbit anti-mouse IgG H&L (HRP) (1:5,000) (Abcam, Cambridge, UK) was added to each well and incubated at 37°C for 1 h. After being washed with PBST, a one-component TMB substrate color developing solution (Solarbio, China) was added, and plates were maintained at room temperature in the dark for 15 min. An ELISA stop solution (Solarbio, China) was then added to each well, and the absorbance at 450 nm (OD value) was measured with a microplate reader within 5 min.

96-well microplates (Corning) were coated with the expressed recombinant viral protein at 4 °C overnight and blocked with 5% skim milk in PBST for 1 h at 37 °C after washing. The plates were then incubated with the diluted serum for 1h at 37 °C and washed again. Each well was incubated with HRP-conjugated anti-human IgG or IgA antibody for 1 h at 37 °C. After washing with PBST, TMB substrate was added to the wells, and the reaction was stopped by adding ELISA Stop Solution (Solarbio). After briefly shaking the plates, the absorbance at 450 nm was read. Anti-EBV VCA-IgA antibodies in serum samples were tested with commercial ELISA kits (EUROIMMUN), which employed native VCA proteins as antigens.

The conditions of the iELISA, including the concentrations of coated antigen, blocking solution, sera, and HRP-conjugated goat anti-cat IgG and their incubation times, were optimized according to the P/N value. The best reaction conditions were as follows: ELISA plate (Costa, Corning, NY, USA) wells were coated with 4 μg/mL of purified His-tagged FCoV-SP protein in 0.05 mol/L of carbonate buffer (pH 9.6) and incubated overnight at 4 °C. After washing with phosphate-buffered saline tween (PBST) three times, 100 µL of 5% skimmed milk was added to each well and incubated at 37 °C for 1 h. After washing with PBST three times, 50 µL of serum at a dilution of 1:400 was added to the wells and incubated at 37 °C for 1.5 h. After washing with PBST three times, HRP-conjugated goat anti-cat IgG diluted 1:20,000 in PBST was added and allowed to incubate at room temperature (RT) for 1 h. After adding 100 µL of 3,3,5,5-tetramethylbenzidine (TMB) substrate solution and incubating at RT for 6 min, the reaction was stopped by adding 100 µL of ELISA Stop Solution (Solarbio, Beijing, China), and the OD₄₅₀ was measured.

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.