A quantitative assessment of heterogeneity for surface-immobilized proteins.

Many biotechnological applications use protein receptors immobilized on solid supports. Although, in solution, these receptors display homogeneous binding affinities and association/dissociation kinetics for their complementary ligand, they often display heterogeneous binding characteristics after immobilization. In this study, a fluorescence-based fiber-optic biosensor was used to quantify the heterogeneity associated with the binding of a soluble analyte, fluorescently labeled trinitrobenzene, to surface-immobilized monoclonal anti-TNT antibodies. The antibodies were immobilized on silica fiber-optic probes via five different immobilization strategies. We used the Sips isotherm to assesses and compare the heterogeneity in the antibody binding affinity and kinetic rate parameters for these different immobilization schemes. In addition, we globally analyzed kinetic data with a two-compartment transport-kinetic model to analyze the heterogeneity in the analyte-antibody kinetics. These analyses provide a quantitative tool by which to evaluate the relative homogeneity of different antibody preparations. Our results demonstrate that the more homogeneous protein preparations exhibit more uniform affinities and kinetic constants.

A computational reaction-diffusion model for the analysis of transport-limited kinetics.

Optical, evanescent wave biosensors have become popular tools for quantitatively characterizing the kinetic properties of biomolecular interactions. Analyzing data from biosensor experiments, however, is often complicated when mass-transfer influences the detection kinetics. We present a computational, transport-kinetic model that can be used to analyze transport-limited biosensor data. This model describes a typical biosensor experiment in which a soluble analyte diffuses through a flow chamber and binds to a receptor immobilized on the transducer surface. Analyte transport in the flow chamber is described by the diffusion equation while the kinetics of analyte-surface association and dissociation are captured by a reactive boundary condition at the sensor surface. Numerical integration of the model equations and nonlinear least-squares fitting are used to compare model kinetic data to experimental results and generate estimates for the rate constants that describe analyte detection. To demonstrate the feasibility of this model, we use it to analyze data collected for the binding of fluorescently labeled trinitrobenzene to immobilized monoclonal anti-TNT antibodies. A successful analysis of this antigen-antibody interaction is presented for data collected with a fluorescence-based fiber-optic immunoassay. The results of this analysis are compared with the results obtained with existing methods for analyzing diffusion-limited kinetic data.