Kinetic analysis of a high-affinity antibody/antigen interaction performed by multiple Biacore users

Abstract

To explore the reliability of Biacore-based assays, 22 study participants measured the binding of prostate-specific antigen (PSA) to a monoclonal antibody (mAb). Each participant was provided with the same reagents and a detailed experimental protocol. The mAb was immobilized on the sensor chip at three different densities and a two-step assay was used to determine the kinetic and affinity parameters of the PSA/mAb complex. First, PSA was tested over a concentration range of 2.5–600 nM to obtain k a information. Second, to define the k d of this stable antigen/antibody complex accurately, the highest PSA concentration was retested with the dissociation phase of each binding cycle monitored for 1 h. All participants collected data that could be analyzed to obtain kinetic parameters for the interaction. The association and the extended-dissociation data derived from the three antibody surfaces were globally fit using a simple 1:1 interaction model. The average k a and k d for the PSA/mAb interaction as calculated from the 22 analyses were (4.1 ± 0.6) × 10 4 M −1 s −1 and (4.5 ± 0.6) × 10 −5 s −1, respectively. Overall, the experimental standard errors in the rate constants were only ∼14%. Based on the kinetic rate constants, the affinity ( K D ) of the PSA/mAb interaction was 1.1 ± 0.2 nM.