Ab97235

Enzyme‐linked immunosorbent assay (ELISA) plates were coated with individual peptides or rEg.P29 at 10 μg/ml and incubated overnight at 4°C. The plates were washed five times with 0.05% Tween 20 PBS (PBST) and blocked with 5% skim milk powder in PBST at 37°C for 1 h. After washing five times with PBST, the plates were incubated with primary serum (1:100) in PBS (with 10% FBS) for 2 h and washed five times with PBST for 2–3 min. One hundred microliters each of horseradish peroxidase (HRP)‐conjugated anti‐mouse IgM, IgG, IgA, IgG1, IgG2a, IgG2b, IgG2c, and IgG3 (Cat nos.: ab97230, ab97023, ab97235, ab97240, ab97245, ab97250, ab97255, and ab97260, respectively; Abcam), and IgE (Cat no.: PA1‐84764; Invitrogen) were added enzyme plates and incubated at 37°C for 1 h. After washing, 3,3′,5,5′‐Tetramethylbenzidine (TMB) was added for 8–10 min, and the reaction was stopped by 2 M H₂SO₄. The absorbance was measured at 450 nm within 15 min using an ELISA reader (Thermo Fisher Scientific).

Binding properties of murine sera to monomer RBD or S protein were determined by ELISA. 96-well plates (3590; Corning, USA) were coated overnight with 3 μg/mL of monomer RBD or S protein (Sino Biological, China) in 0.05 M carbonate–bicarbonate buffer, pH 9.6, and blocked in 5% skim milk in PBS. Serum or bronchoalveolar lavage fluid (BALF) samples were serially diluted and added to each well. The plates were incubated for 2 h and then washed. The plates were incubated with goat anti-mouse IgG-HRP antibody (Abcam, ab6789, for IgG titration), goat anti-mouse IgA-HRP antibody (Abcam, ab97235, for IgA titration), goat anti-mouse IgG1-HRP antibody (Abcam, ab97240, for IgG1 titration), or goat anti-mouse IgG2a-HRP antibody (Abcam, ab97245, for IgG2a titration), incubated for 1.5 h and then washed. The plates subsequently developed with 3,3’,5,5’-tetramethylbenzidine (TMB) substrate. Reactions were stopped with 2 M hydrochloric acid, and the absorbance was measured at 450 nm using a microplate reader (PerkinElmer, USA). The endpoint titres were defined as the highest reciprocal dilution of serum to give an absorbance greater than 2.5-fold of the background values. Antibody titre below the limit of detection was determined as half the limit of detection.

Antigen-specific levels of antibodies were determined by ELISA performed in 96-well microtiter plates coated overnight at 4 °C with the synthetic peptide (5 μg/mL) in PBS (pH 7.2). The plates were blocked with PBS containing 5% fetal calf serum (FCS) for 1 h at room temperature. The plates were washed 3 times in PBS containing Tween. The diluted samples were added in volumes of 100 μL per well and the plates were incubated for 1 hour at room temperature. As a conjugate, HRP-labelled goat anti-mouse IgG (ab97040, Abcam), IgG1 (ab98693, Abcam), IgG2a (ab98698, Abcam), and IgA (ab97235, Abcam) were used at 1:5000 to 1:20,000 dilutions. After adding tetramethylbenzidine substrate (BD Bioscience) and monitoring the color development, the reaction was stopped withy H₂SO₄, and the OD at 450 nm was measured on a microplate spectrophotometer.

Antigen-specific IgG and IgA antibodies in mouse serum and stool were quantified by ELISA. In brief, a 96-well plate was coated with 100 ng of KP177R, E183L, E199L, CP204L, E248R, EP402R, B602L, or B646L protein (Wuhan Keqian Biology Co., Ltd., Wuhan, China) overnight at 4°C. The plate was blocked with PBST and 5% skim milk for 2 h at room temperature. Serially diluted serum samples or fecal samples were added to each well, followed by incubation at 37°C for 2 h. HRP-conjugated goat anti-mouse IgG (Abcam, ab97023), IgG1 (Abcam, ab97240), IgG2a (Abcam, ab97245), or IgA (Abcam, ab97235) antibodies were added to the wells, followed by incubation at 37°C for 1 h. The assays were performed using 3,3′,5,5′-tetramethylbenzidine as the colorimetric substrate, and the optical density was measured at 450 nm. The cut-off value was determined by calculating the OD₄₅₀ + 3 SDs of serum samples from unvaccinated animals. The endpoint titer was calculated as the reciprocal of the highest serum dilution at which OD₄₅₀ was equal to or greater than the cut-off value.

Protein samples from BALF, plasma, lung homogenates, and culture cell supernatants were collected as described above, and the concentrations of TNF-a (ab208348), IL-1b (ab197742), IL-6 (ab222503), and IFN-γ (ab100689) were determined using ELISA kits from Abcam (Cambridge, UK) in accordance with the manufacturer’s instructions. CXCL5 (MX000), CXCL1 (MKC00B), CXCL2 (MM200), CXCL12 (MCX120), and CXCL13 (MCX130) levels were determined using ELISA kits from R&D Systems (MN, USA) in accordance with the manufacturer’s instructions. For detection of virus-specific Igs, a 96-well High-Binding Flat-Bottom Microplate (Corning, USA) was coated with H1N1 virions in PBS at 4°C overnight. Coated plates were blocked with 0.2 ml of PBS containing 5% bovine serum albumin (BSA) at room temperature for 2 h. BALFs were 2-fold serial dilutions starting with a 1:1 dilution in virion binding assays. PBS was used as a negative control. After binding, the plates were washed 4 times with ice-cold PBS containing 0.1% Tween 20 (PBST) and the plates were incubated with rabbit/goat anti-mouse antibodies to IgG and IgA (ab6728, ab97235, Abcam, UK). The plate was then incubated with 100 µl of tetramethylbenzidine (TMB) ELISA Substrate (Solarbio, China) for 15 min at room temperature. Finally, the reaction was stopped with 100 µl of 0.5 M H₂SO₄. The absorbance was determined at 450 nm using a plate reader.