Dual-label fluoroimmunoassay for simultaneous determination of primidone and phenobarbital

A dual-label magnetizable solid-phase fluoroimmunoassay for direct determination of serum levels of primidone and its main metabolite, phenobarbital, in a single tube was developed and optimized. Fluorescein isothiocyanate was used to label phenobarbital and a rhodamine 101 derivative (XRITC) to label primidone; these could be independently quantitated with no fluorimetric "cross-talk." The dual-label assay employs mixed immunochemical reagents but is otherwise similar in performance to conventional, single-drug, magnetizable solid-phase fluoroimmunoassays. The ability to measure simultaneously two related analytes, such as a drug and its metabolite, represents a useful extension of immunoassay. This was illustrated in the present work by assay of sera from patients on primidone therapy. Dual-label assay results correlated well with those by a conventional enzymoimmunoassay (for primidone) and by a polarization fluoroimmunoassay (for phenobarbital).