Quantifying Antibody Binding Kinetics on Fixed Cells and Tissues via Fluorescence Lifetime Imaging
We present a method for monitoring spatially localized antigen-antibody binding events on physiologically relevant substrates (cell and tissue sections) using fluorescence lifetime imaging. Specifically, we use the difference between the fluorescence decay times of fluorescently tagged antibodies in free solution and in the bound state to track the bound fraction over time and hence deduce the binding kinetics. We make use of a microfluidic probe format to minimize the mass transport effects and localize the analysis to specific regions of interest on the biological substrates. This enables measurement of binding constants (