Competitive Binding Assay for Antibody Characterization

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).