Directly Measured Signals as Response Variables in Fluorescence Polarization Ligand Binding Assays

The use of fluorescent dyes to label analyte In ligand binding assays has recently attained practical significance. Differences In Intensity or polarization between bound and free label have been reported as the basis for homogeneous response variables. A significantly higher polarization for bound labeled analyte is a more general phenomenon than are Intensity differences, although both may occur simultaneously. The individual orthogonal-polarized components of the emission, from which polarization Is calculated, also change significantly, but their use as potential response variables has not been examined. In this report, the fluorolmmunoassay response from the directly measured individual polarized emission components, their ratio, their difference, and polarization are evaluated and compared, In a hypothetical manner and in experimental examples. The response provided by the different variables Is found to depend upon the relative intensity of bound to free labeled analyte except for the difference signal which appeared to be generally best.