Inexpensive double-antibody fluoroimmunoassay for aminoglycoside antibiotics, phenytoin, and theophylline in serum.

We describe simple, clinically useful double-antibody fluoroimmunoassays for amikacin, gentamicin, tobramycin, theophylline, and phenytoin. The fluorescent tracers were prepared by conjugation to fluorescein isothiocyanate; the antisera were raised in rabbits. A simple filter fluorometer and disposable culture tubes are used. The tracer, sample, and first and second antibodies are combined and incubated at room temperature for 30 min. A precipitation-acceleration buffer is added, the samples are centrifuged, and the fluorescence of the supernate is measured directly in the assay tube without decantation. Interferences, usually negligible, can be corrected for by use of a sample blank. Results compare favorably in performance with various commercially available RIA and enzyme immunoassays.