Microlon 96 wells flatbottom plates

Influenza antigen specific antibody titers were determined by ELISA. Microlon 96-wells flatbottom plates (Greiner) were coated overnight with 600 ng of A/PR8/34 subunit HA (as determined by SPR) per well at 4°C. After washing twice with 0.05% Tween80, serial two-fold dilutions of individual mouse sera in PBS, 0.5% BSA, 0.1% Tween80 were applied on the plate and incubated for 1 hour at 37°C. Plates were washed three times and subsequently incubated for 1 hour at 37°C with horseradish peroxidase-conjugated goat antibodies against mouse IgG, IgG1 or IgG2c (1∶5000, Southern Biotech). Detection of antibodies was performed with 3,3′5,5′-tetramethylbenzine (TMB) substrate buffer (0.4 mM TMB in 0.11 M sodium acetate, 0.006% H₂O_2, pH 5.5) after washing three times and incubated for 10 min at room temperature. Enzymatic reaction was stopped by adding 2 M sulfuric acid, after which the optical density (OD) was measured at a wavelength of 450 nm using a Synergy Mx platereader (BioTek). Titers are given as the reciprocal of the serum dilution corresponding to OD₄₅₀ = 0.1 after background correction.

Influenza A/PR8 and A/HKx31-specific antibody titers were determined by ELISA as described previously (15 (link)). In short, Microlon 96-wells flatbottom plates (Greiner) were coated overnight with either 600 ng (HA) of A/PR8/34 subunit or A/HKx31 virus per well at 4°C. Serial two-fold dilutions of individual mouse sera in PBS, 0.5% BSA, 0.1% Tween80 were applied on the plate and incubated for 1 h at 37°C. Subsequently, plates were incubated for 1 h at 37°C with horseradish peroxidase-conjugated goat antibodies against mouse IgG, IgG1 or IgG2c (1:5000, Southern Biotech). Detection of antibodies was performed with TMB substrate buffer (0.4 mM TMB in 0.11 M sodium acetate, 0.006% H₂O_2, pH 5.5). The reaction was stopped by adding 2 M sulfuric acid, after which the optical density (OD) was measured at a wavelength of 450 nm by using a Synergy Mx platereader (BioTek). Titers are reported as the reciprocal of the serum dilution corresponding to OD₄₅₀ = 0.2 after background correction.