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

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 t...

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Bibliographic Details
Published in:Analytical biochemistry Vol. 352; no. 2; pp. 208 - 221
Main Authors: Katsamba, Phinikoula S., Navratilova, Iva, Calderon-Cacia, Maria, Fan, Linsey, Thornton, Kevin, Zhu, Mingde, Bos, Tim Vanden, Forte, Carla, Friend, Della, Laird-Offringa, Ite, Tavares, Gisele, Whatley, John, Shi, Ergang, Widom, Angela, Lindquist, Kevin C., Klakamp, Scott, Drake, Andrew, Bohmann, David, Roell, Marina, Rose, Larry, Dorocke, Jill, Roth, Bruce, Luginbühl, Béatrice, Myszka, David G.
Format: Journal Article
Language:English
Published: United States Elsevier Inc 15-05-2006
Subjects:
Antibodies, Monoclonal - chemistry
Antibody–antigen interactions
Antigen-Antibody Reactions
Biosensing Techniques - instrumentation
Biosensing Techniques - methods
Biosensing Techniques - standards
Humans
Kinetics
Ligands
Molecular interactions
Prostate-Specific Antigen - analysis
Prostate-Specific Antigen - chemistry
Prostate-Specific Antigen - immunology
Protein Binding
Protein–protein interactions
Reproducibility of Results
Sensitivity and Specificity
Surface plasmon resonance
Surface Plasmon Resonance - instrumentation
Surface Plasmon Resonance - methods
Surface Plasmon Resonance - standards
Time Factors
Surface plasmon resonance
Protein–protein interactions
Antibody–antigen interactions
Molecular interactions
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Summary: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.
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content type line 23
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2006.01.034