Ergang Shi | PubMed Health

PubMed2006

Monoclonal antibody classification based on epitope-binding using differential antigen disruption.

Currently, classifying a population of specific antigen-reactive monoclonal antibodies (mAbs) according to their epitope-binding properties has been limited to competition assays. Such assays are time consuming, labor intensive and restricted to the number of mAbs in the experiment. To overcome this problem, a differential antigen disruption-based antibody profiling procedure was developed. This procedure rapidly classifies specific antigen-reactive mAbs into epitope-related groups by measuring the binding signal of the antibodies to a set of structurally disrupted antigens and then clustering the antibodies according to the similarity of their binding profiles. The clustering results generated by differential antigen disruption showed a significant concordance with those generated by competition experiments. Therefore, differential antigen disruption method opens an opportunity to assess the entire population of antigen-reactive mAbs according to their epitope-binding properties. In doing so, a set of representative antibodies can be drawn to describe the epitope complexity for systematically exploring their functions.

Animals↗

PubMed2006

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 1h. 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 approximately 14%. Based on the kinetic rate constants, the affinity (K(D)) of the PSA/mAb interaction was 1.1+/-0.2 nM.

Antibodies, Monoclonal↗