Kinetic analysis of a protein antigen-antibody interaction limited by mass transport on an optical biosensor.
Using BIAcore technology, we determined the rate constants for a protein antigen-antibody interaction that was mass transport limited on the optical biosensor. The antigen consisted of a soluble form of the human T-cell receptor CD4 (two amino terminal domains, D1D2) and the antibody was an anti-CD4 monoclonal from monkey engineered with the constant domains from human IgG1. High quality response data were obtained for this interaction by orienting the attachment of the antibody on the sensor surface and correcting for instrument artifacts with control experiments. Using numerical integration and global fitting, we demonstrate that a mass transport limited reaction was the only model of those tested that described well D1D2 binding to three different surface densities of the antibody. Statistical profiling techniques showed that the error space and correlation for the parameters in the non-linear model were essentially linear, but only when the model was simultaneously fitted to data from multiple surface densities. The "on" and "off" rate constants (1.2 x 10(-6) M-1 s-1 and 2.9 x 10(-4) s-1) determined from the kinetic analysis predict an equilibrium dissociation constant (KD = 0.24 +/- 0.01 nM) that agrees with the value measured in solution by titration calorimetry (KD = 0.2 +/- 0.1 nM). The results indicate that, although the D1D2-antibody reaction is partially controlled by mass transport on the optical biosensor, by optimizing the experimental design and analyzing data from multiple surface densities it is possible to determine accurate estimates of the intrinsic equilibrium and kinetic rate constants.
Myszka
D G
DG
Department of Molecular Immunology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406, USA. dmyszka@genetics.utah.edu
Morton
T A
TA
Doyle
M L
ML
Chaiken
I M
IM
D000911:Antibodies, Monoclonal / Q000276:immunology; D000937:Antigen-Antibody Reactions; D015374:Biosensing Techniques; D015704:CD4 Antigens / Q000276:immunology; D006801:Humans; D007700:Kinetics
D000911:Antibodies, Monoclonal; D015704:CD4 Antigens