Goat anti mouse igg2c

OVA-specific antibodies in serum samples were measured by IgG total or isotype specific ELISA. The plates were coated overnight at 4°C with OVA (2 µg/ml in 0.05 M carbonate buffer, pH 8.2) and blocked with 3% w/v bovine serum albumin (BSA) in PBS for 1 h at 37°C. Serial 2-fold dilutions of the samples were prepared in 3% w/v BSA in PBS and incubated for 2 h at 37°C, washed 6 times with 0.1% v/v Tween20 in PBS. The biotinylated detection antibodies goat, anti-mouse IgG (Sigma-Aldrich), goat anti-mouse IgG1 or goat, anti-mouse IgG2c (Southern biotech, USA), 1∶5000 in 1% w/v BSA/0.1% v/v Tween20 in PBS, were used for incubation for 2 h at 37°C. Six washes were followed by incubation with Streptavidin-horse radish peroxidase (HRP) (BD Pharmingen), 1∶1000 in 1% w/v BSA/0.1% v/v Tween20 in PBS, for 1 h at 37°C, six washes, incubation with ABTS [2,20-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)] in 0.1 M citrate-phosphate buffer (pH 4.35) and 0.03% H₂O₂ (Sigma-Aldrich), and the absorbance of light at 405 nm wave length was measured using a Synergy 2 Multi-Mode Microplate Reader (Biotek, USA). Titers were determined as the highest dilution factors of the assay samples with twice the average readout value of the blank.

ELISA plates were coated with 100 ng/well recombinant SARS-CoV-2 RBD protein (RayBiotech) overnight at 4 °C. The plates were washed twice with PBS, containing 0.05% Tween-20 (PBS-T) and then blocked with 8% FBS for 1.5 h. Sera or BAL samples were diluted 1:100 or undiluted in blocking buffer and were added for 1 h at 37 °C. Plates were washed 5 times with PBS-T. Goat anti-mouse IgG (1:2000, 1013-05, Southern Biotech, USA), or Goat anti-mouse IgG2C (1:2000, 1078-04, Southern Biotech, USA) conjugated with horseradish peroxidase (HRP) or alkaline phosphatase was added at a 1:2000 dilutions for 1 h at 37 °C followed by adding TMB (3, 3, 5, 5′- tetramethylbenzidine) peroxidase substrate (Thermo Scientific) or p-nitrophenyl phosphate (Sigma-Aldrich), and the intensity was read at an absorbance of 450 or 405 nm. To determine IgA titer, HRP -conjugated goat anti-mouse IgA (1:2000, 1040-05, Southern Biotech) was added as the secondary antibody at a 1:2000 dilution for 1 h at 37 °C, followed by adding TMB peroxidase substrate (Thermo Scientific) for about 15 min. The reactions were stopped by 1 M sulfuric acid, and the intensity was read at an absorbance of 450 nm. Binding endpoint titers were determined using a cutoff value which is negative control + 3x SD.

The sera were tested for ZIKV E protein-specific antibodies using commercially available ELISA kits (Alpha Diagnostic, USA) according to the manufacturer’s instructions. Briefly, 100 μl aliquots of diluted serum (1:50,000) were added per well. Env_M- or Env_Z-specific antibodies (IgG, IgG1, and IgG2c) in sera were determined by indirect ELISA. Microplates (Nunc-Immuno Plates; Thermo Scientific, UK) were coated with Env_M (1 μg/ml) at 4 °C overnight. Each well was washed with 0.05% Tween 20 in PBS (PBST) and then with 1% BSA in PBS and incubated at 37 °C for 2 h. The plates were washed and then diluted serum was incubated at 37 °C for 2 h. Plates were washed and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG heavy and light chain antibody (1:10,000; Bethyl Laboratories, TX, USA), goat anti-mouse IgG1 (1:10,000; Bethyl Laboratories), or goat anti-mouse IgG2c (1:8000; Southern Biotech, USA) at 37 °C for 1 h and washed with PBST. Color development was performed using tetramethylbenzidine substrate (Surmodics, USA) and stopped with 2 N H₂SO₄. The optical density of the plates was read at 450 nm in an ELISA plate reader (BioTek, Winooski, NT, USA).