Modeling of immunosensors under nonequilibrium conditions : II. Experimental determination of performance characteristics

Abstract

In an attempt to optimize immunosensors operating with an immobilized antibody as binding protein and an analyte-enzyme conjugate as signal generator that is significantly larger in molecular size than the analyte, in a previous communication (Part I) ([1.] Anal. Biochem. 196) we developed mathematical models for the prodiction of performance characteristics. These models are compared in this contribution with experimentally obtained results. As an example, a monoclonal antibody to the steroid hormone progesterone has been used as binding protein, an 125I-progesterone derivative, and a progesterone-horseradish peroxidase derivative as tracers for signal generation. A minimum of parameters needs to be experimentally determined to calculate the performance: the amount of immobilized antibody, the diffusion coefficient of antigens, the thickness of the penetration layer, and the on- and off-rates for binding of the antigen to the antibody. We have described simple methods to obtain these data for the labeled antigen and for the unlabeled analyte that does not provide a signal per se. Kinetic binding curves for antigen-antibody complex formation obtained with the mathematical models correlated well with experimentally obtained results for antigens of different sizes. Although equilibrium of the antigen-antibody complex for the enzyme-labeled analyte conjugate requires about 4 h in the absence of free analyte, dose-response curves can be obtained after 5 min and the relative position of these curves does not change significantly after 30 min. Using a total volume of 200 [mu]l for the analytical procedure in microtiter wells, agitation as a means to accelerate convective diffusion during an incubation period of 30 min is not necessary with the analyte-enzyme conjugate. However, immunosensors using large analyte-enzyme conjugates as signal generators for the detection of small analytes require strict control of the incubation time if operated within short periods of time (<30 min).