Fluorescent immunoassay employing total reflection for activation

Abstract

Receptors are employed to which one or more fluroescent molecules are bound. When the receptors are bound to the epitopic sites on the surface and light of predetermined wave length is directed toward the surface, the fluorescing molecules are activated and fluoresce. In carrying out an assay, receptor is combined with an unknown suspected of containing molecules having the same epitopic sites bound to the surface. The receptor will bind to these molecules reducing the number of receptor sites available for binding to the epitopic sites on the surface. When the assay medium is contacted with the surface, the amount of receptor which binds to the surface, will be a function of available binding sites and, therefore, to the number of the molecules present in the unknown. Upon irradiation of the surface, substantially only the fluoroescent molecules bound to the surface will fluoresce. By monitoring the fluorescence, one can determine the presence and number of molecules of interest present in the unknown. The apparatus consists of a transparent solid sheet, conveniently as part of or optically connected to a prism, a light source set at an angle to provide total internal reflection at the sheet, a cell which includes the reflecting surface as a wall, and a fluorescence detector. Various optics and filters may be employed to modify the light source beam and the fluorescence beam. This work was carried out under a grant of the National Science Foundation.