Hrp conjugated goat anti mouse igg or iga antibody

Genital tract mucus, intestinal mucus, nasal fluid, and fecal extracts were treated as previously described to detect SIgA antibodies [25 (link)]. In brief, all mucus samples were treated with 500 μL of PBS and mixed well (dilution not required). Feces were treated with 500 μL of EDTA-Na₂-PBS (50 mmol/L), and the supernatant was collected after centrifugation (dilution not required). Mouse serum was diluted 10-fold to detect IgG antibodies and cytokine levels (IL-4, IL-2, IL-10, IL-12, IL-17, and IFN-γ). All samples were analyzed using the ELISA commercial kit (MEIMIAN, Enzyme industry Co., Ltd., Suzhou, Jiangsu, China).
Polystyrene microtiter plates were coated with PoRV as an antigen or PBS as a negative control and incubated overnight at 4 °C (0.1 mL per well). After blocking for two hours with 5% skim milk at 37 °C, the samples were added in triplicate and co-incubated with the antigen for one hour at 37 °C. HRP-conjugated goat anti-mouse IgG or IgA antibody at a dilution of 1:5000 (Sigma) was incubated for one hour at 37 °C. After washing, the color was developed using tetramethylbenzidine (Sigma), and the absorbance was measured at OD₄₅₀.

Influenza virus antigen-specific antibodies were measured by ELISA. Briefly, polystyrene 96-well plates (Nunc, China) were coated with whole ITIV antigens (HA, 1 μg/mL per strain) in 0.1 M bicarbonate buffer (pH 9.5) and incubated overnight at 4°C. The plates were washed with PBS-0.05% Tween-20 (PBST) and then blocked with PBS-1% bovine serum albumin (BSA, Sigma-Aldrich, USA) for 2 h at 37°C. After washing with PBST, 100 μL of the samples serially diluted with PBS-0.1%BSA was added, and the plates were incubated for 2 h at 37°C. After washing with PBST, an HRP-conjugated goat anti-mouse IgG or IgA antibody (Sigma-Aldrich, USA) was added at a 1:5000 dilution and incubated overnight at 4°C. A color reaction was developed using tetra-methyl benzidine substrate solution (Sigma-Aldrich, USA) at 37°C for 1 h. The reaction was stopped by adding 0.05 mL of 0.5 M H₂SO₄ per well, and the optical densities (ODs) were read at 450 nm using a microplate reader (Thermo Scientific Multiskan GO, USA). The antibody titers were expressed as the reciprocal of the highest dilution of sample for which the OD was 5×OD_{mean background}, and the geometric mean titer (GMT) was calculated.

The levels of anti-BVDV IgG antibody in the serum and anti-BVDV sIgA antibody in the intestinal mucus, nasal fluid, genital tract fluid, and feces samples were detected by ELISA. In brief, 96-well polystyrene plates were coated with 200 TCID₅₀ (10^7.2/0.1 mL) BVDV strain ZD-2018 at 4 °C for 12 h. After washing three times with PBST (PBS containing 0.1% Tween-20), the plates were blocked with 5% skim milk at 37 °C for 2 h, followed by washing with PBST. Next, the serum sample (1:100) and other samples (1:10) used as the primary antibodies were added into the plates and kept in 37 °C for 1 h, followed by washing three times with PBST. Subsequently, HRP-conjugated goat anti-mouse IgG or IgA antibody (1:5000) (Sigma, USA) was added as the secondary antibody into the plates, and kept in 37 °C for 1 h. After washing three times with PBST, the substrate o-phenylenediamine dihydrochloride was used for color development, followed by the measurement of absorbance at 450 nm. On the 42d post primary vaccination, the levels of cytokines IL-2, IL-4, IL-10, IL-12, IL-17, and IFN-γ in the serum of these groups were analyzed by ELISA Kits (Abcam, Cambridge, MA, USA). Moreover, an IHC assay was performed, as described above, to detect the antigen-specific sIgA-secreting cells in the PPs of the mice collected from each group using the anti-E2 sIgA antibody induced by pPG-E2-ctxB/Lc W56.