Characterization of antibody-antigen interactions for biosensor applications

There has been considerable interest in the use of optical fibers for remote spectroscopic sensing of biological molecules. The potential application of these biosensors is very broad, including patient bedside monitoring during intensive care, the diagnosis of human disease, biotechnology process control, and therapeutic drug monitoring. We are interested in developing a remote, continuous or at least semicontinuous optical fiber immunosensor by using the principle of total internal reflection fluorescence (TIRF).;An electromagnetic field generated at a silica-liquid interface by total internal reflection is used to excite fluorescence of molecules present at the interface. Antibody molecules immobilized at the surface of a quartz optical element bind their complementary antigen from solution permitting analysis of antigen concentration in the bulk solution. If the bound antigen is fluorescent, a fluoroimmunoassay can be performed. The dynamic range of such a sensor is enhanced in a competitive immunoassay mode. The sensor can also be used to detect solution antibody by prior antigen immobilization.;The sensitivity of TIRF immunoassay is maximized by using antibodies with high binding constants, which will result in a slow response time to permit continuous or semicontinuous measurements. In order to reuse such a device in a reasonable time, there must be a means to increase dissociation rate of the antibody-antigen complex without sacrificing the sensitivity. Thus, it would be important to be able to regulate the binding constant between each measurement.;In this dissertation, first, TIRF immunoassay was demonstrated by using the well-defined flat-plate single-reflection geometry. Second, different surfaces used to couple Abs were studied and binding properties between immobilized Abs and Ags were characterized by the radioisotope technique. Third, two approaches based on the results of a thermodynamic study of Ab-Ag binding in solution were suggested to regulate the binding constant. Fourth, one of these approaches, using photoresponsive water-soluble polymers, was investigated to evaluate the feasibility.