Homogeneous immunoassay of phenobarbital by phase-resolved fluorescence spectroscopy

A new technique for determination of phenobarbital by homogeneous fluoroimmunoassay is described. Simultaneous determination of free and antibody-bound fluorescein-labelled phenobarbital is accomplished by making phase-resolved fluorescence measurements, in which selectivity is provided by the difference between the fluorescence lifetimes of the two species, as well as by the difference in fluorescence intensities. The technique requires no specialized reagents other than those inherent in immunoassay (antibody, antigen and labelled antigen). Problems due to non-specific binding of labelled antigen to sample matrix components can be alleviated by addition of a relative excess of albumin.