96 well elisa plates

Human IgG1 antibody levels in serum and BAL fluid were determined by IgG1 Ready-SET-Go! ELISA (Affymetrix, eBioscience) according to the manufacturer’s instructions. After heat inactivation (56^°C for 30 min) samples were diluted 1:40 (serum) and 1:2 (BAL fluid). Influenza-specific human Ab titers in serum and BAL fluid were determined by ELISA as previously described (20 (link)) with the following modifications. The IgG ELISA was performed in 96-well ELISA plates (BD Biosciences) coated with 1 × 10⁶ PFU/ml of A/swine/England/1353/09 over night at 4°C. Twofold dilutions of BAL fluid samples or serum (heat inactivated for 30 min at 56°C) were added, starting from 1:2 or 1:10 dilution, respectively. Binding of influenza-specific Abs was detected using a monoclonal anti-human IgG (hIgG) (Fc) (Biorad) and 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (BioLegend). Optical density (OD) readings were taken at 450 and 570 nm (wavelength correction). Ab values were expressed as endpoint titers defined as the highest dilution at which the OD was higher than twice the background OD.

Estimation of cytokines in conditioned media was performed according to the manufacturer’s instructions. Briefly, 96-well ELISA plates (BD Bioscience) were coated overnight with capture antibody at 4°C. Next day, plates were washed with 0.1% Tween-20 containing PBS and blocked with 10% FBS for 1 h. Following blocking, wells were washed and incubated with 100 μL of test samples (conditioned media) for 2 h at room temperature. Subsequently, plates were washed and incubated with detection antibody and enzyme reagent for 1 h at room temperature. TMB (Sigma) was used as a substrate and reactions were stopped with 2 N H₂SO₄. Absorbance was measured at 450 nm wavelength and the concentration of cytokines was interpolated from a standard curve.