A new immunoassay based on fluorescence excitation by internal reflection spectroscopy

A new immunoassay technique is described which uses totally internally reflected light to excite the fluorescence of fluorescein labeled antibody which has become bound to a hapten--protein conjugate absorbed on a quartz-plate in the antibody solution. The presence of any free hapten in solution reduces the amount of antibody free to bind to the surface and thus reduces the fluorescence signal. Measurement of the decrease of the fluorescent signal then gives a measure of the concentration of free hapten present. The technique is simple, fast and has high intrinsic sensitivity and specificity. It has been demonstrated for phenyl arsonic acid and morphine. Free morphine at a concentration of 2 X 10(-7) M is readily detected.