Spatially resolved detection of antibody-antigen reaction on solid/liquid interface using total internal reflection...

Spatially-resolved detection of antibody-antigen reactions at the solid/liquid interface was investigated by total internal reflection excited fluorescence from large area flat surfaces. Anti-HSA immunoglobulin G (IgG) antibody was immobilized at four spatially distinct spots. Binding of fluorescein-labeled human serum albumin (HSA) from the solution to immobilized antibody was detected by a cooled charge-coupled device (CCD) as a charge in the fluorescence intensity. A two-dimensional representation of the fluorescence was obtained during the binding reaction time of 25 mins. The contributions from bound and free antigen to the total signal were evaluated. The influence of the scattered excitation light and the normalization of fluorescence signal with respect to the two-dimensional incident light intensity distribution are discussed.