Pod substrate

Competitive binding assays were performed as described previously 14 (link). In brief, the NUNC plate was pre-coated with RBD-VLP 40 (link) overnight at 4ºC. After the plate was washed and blocked with 5% dried skimmed milk in PBS for 1 h at room temperature, the mixture of biotinylated antibody (EZ-Link Sulfo-NHS-LC-biotin, Life Technologies, United States) and at least ten-fold excess of competing antibody was added to the plate and incubated for 1 h. After washing, the plate was incubated with Streptavidin-Horseradish Peroxidase conjugate (Life Technologies, United States) for another one hour. After the plate was washed, the signal was developed using the POD substrate (Roche, Switzerland) and the reaction was stopped with 1M H₂SO₄. The OD450 value was measured using a Clariostar plate reader (BMG Labtech, Germany). The mean and 95% confidence interval of four replicates were calculated. Competition was measured as: (X-minimum binding/(maximum binding-minimum binding), where X is the binding of the biotinylated antibody in the presence of competing antibody. Minimum binding is the self-blocking of the biotinylated antibody or background binding. Maximum binding is binding of biotinylated antibody in the presence of non-competing antibody (anti-influenza neuraminidase antibody Z3B2).

Competitive binding assays were performed with monoclonal antibodies as described previously^{6 (link)}. Briefly, RBD-VLP were coated on 96-well microplates overnight at 4 °C, washed and blocked with dried skimmed milk in PBS for 1 h at room temperature prior to the assays. Antibody was biotinylated using EZ-Link Sulfo-NHS-LC-biotin (Life Technologies, USA) and then mixed with competing antibody (in at least 10-fold excess) and transferred to the blocked plates for 1 h. A second layer Streptavidin-HRP (Life Technologies, USA) was then added and incubated for another 1 h. Plates were then washed, and signal was developed by adding POD substrate (Roche, USA) for 5 min before stopping the reaction with 1 M sulfuric acid. Absorbance (OD450) was measured using a plate reader. The mean and 95% confidence interval of 4 replicate measurements were calculated. The competition was measured as: (X-minimum binding/(maximum binding-minimum binding), where X is the binding of the biotinylated antibody in the presence of a competing antibody. Minimum binding is the self-blocking of the biotinylated antibody or background binding. Maximum binding is the binding of a biotinylated antibody in the presence of non-competing antibody (anti-influenza N1 neuraminidase antibody).