Double-antibody homogeneous fluorescence immunoassay of phenytoin

A homogeneous fluorescence immunoassay suitable for quantifying 5–25 mg l^?1 phenytoin (5,5-diphenyl-2,4-imidazolidenedione) in serum is described. The fluorophor-labeled phenytoin and unlabeled phenytoin (analyte) compete for a limited number of anti-phenytoin antibody binding sites in the presence of anti-fluorophor antibodies. The label employed is a sulfonamido derivative of 2-naphthol-8-sulfonic acid (2-8) which, at neutral pH, undergoes excited-state proton transfer. Binding of anti 2-8 antibodies to the drug-fluorophor conjugate results in quenching of the conjugate base emission at 480 nm. The relative standard deviations are about 5%.