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

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) x 10(4) M-1 s(-1) and (4.5 +/- 0. 6) x 10(-5) s(-1), respectively. Overall, the experimental standard errors in the rate constants were only similar to 14%. Based on the kinetic rate constants, the affinity (K-D) of the PSA/mAb interaction was 1.1 +/- 0.2nM. (c) 2006 Elsevier Inc. All rights reserved.