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.
Center for Biomolecular Interaction Analysis, School of Medicine, University of Utah, Salt Lake City, 84132, USA.
Phinikoula S
PS
Katsamba
Iva
I
Navratilova
Maria
M
Calderon-Cacia
Linsey
L
Fan
Kevin
K
Thornton
Mingde
M
Zhu
Tim Vanden
TV
Bos
Carla
C
Forte
Della
D
Friend
Ite
I
Laird-Offringa
Gisele
G
Tavares
John
J
Whatley
Ergang
E
Shi
Angela
A
Widom
Kevin C
KC
Lindquist
Scott
S
Klakamp
Andrew
A
Drake
David
D
Bohmann
Marina
M
Roell
Larry
L
Rose
Jill
J
Dorocke
Bruce
B
Roth
Béatrice
B
Luginbühl
David G
DG
Myszka
D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000937:Antigen-Antibody Reactions; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D006801:Humans; D007700:Kinetics; D008024:Ligands; D017430:Prostate-Specific Antigen / Q000032:analysis / Q000737:chemistry* / Q000276:immunology; D011485:Protein Binding; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D020349:Surface Plasmon Resonance / Q000295:instrumentation / Q000379:methods / Q000592:standards; D013997:Time Factors
D000911:Antibodies, Monoclonal; D008024:Ligands; D017430:Prostate-Specific Antigen