Kinetics of biological binding studied by flow injection fluorescence microscopy

An automated system for performing rapid immunoassay, kinetic measurements, and affinity ranking of biomolecular interactions using fluorescence labeled ligands is described. Its distinctive feature is the automated renewal of solid phase for each measurement, which avoids the need for regeneration of the sensing surface. This system--Flow Injection Renewable Surface Immunoassay (FIRSI)--is used for the first time here for determination of rate constants for an antibody/antigen interaction and for affinity ranking of several related antigens against one antibody. The performance of the system is compared with a commercial BIAcore system that uses surface plasmon resonance for monitoring biomolecular interactions. While the values of association and dissociation rate constants for human serum albumin (HSA) with monoclonal anti HSA antibody obtained by these techniques were comparable, it is shown that the FIRSI technique requires simpler, less expensive instrumentation, handles a broader size range of analytes, and does not suffer from disturbances caused by changes in the refractive index.;Furthermore, a novel method for real-time measurement of ligand binding to live cells is described. Two approaches are compared. The first approach applies a new flow chamber that fits onto a fluorescence microscope and connects a conventional microscope cell culture chamber with a flow injection system. In the other approach, the live cells grow on microcarrier beads that are introduced to the microscope via the flow injection system. Measurements based on the second approach are shown to be superior due to enhanced mass transport and enhanced signal to noise ratio. Rate constants for the binding of the monoclonal antibody 14.4.4S to the murine MHC class II receptor I-E;This work demonstrates that real-time kinetic measurements of biospecific interactions can be made at a moderate cost and describes, for the first time, a generally applicable method for performing measurements of kinetics of fast interactions at the surface of live cells.