A sensitive solid-phase fluoroimmunoassay for detection of opiates in urine

An automated flow fluorometer designed for kinetic binding analysis was ada pted to develop a solid-phase competitive fluoroimmunoassay for urinalysis of opiates. The solid phase consisted of polymer beads coated with commerci al monoclonal antibodies (MAbs) raised against morphine. Fluorescein-conjug ated morphine (FL-MOR) was used as the fluorescein-labeled hapten. The diss ociation equilibrium constant (K-D) for the binding of FL-MOR to the anti-M OR MAb was 0.23 nM. The binding of FL-MOR to the anti-MOR MAb reached stead y state within minutes and was displaced effectively by morphine and other opiates. Morphine-3-glucuronide (M3G), the major urinary metabolite of hero in and morphine, competed effectively with FL-MOR in a concentration-depend ent manner for binding to the antimorphine MAb and was therefore used to co nstruct the calibration curve. The sensitivity of the assay was 0.2 ng/mL f or M3G. The assay was effective at concentrations of M3G from 0.2 to 50 ng/ mL, with an IC50 of 2 ng/mL. Other opiates and heroin metabolites that show ed >50% crossreactivity when present at 1 mu g/mL included codeine, morphin e-6-glucuronide, and oxycodone. Methadone showed very low crossreactivity ( <5%), which is a benefit for testing in patients being treated for opiate a ddictions. The high sensitivity of the assay and the relatively high cutoff value for positive opiate tests allows very small sample volumes (e.g., in saliva or sweat) to be analyzed. A double-blind comparison using 205 clini cal urine samples showed good agreement between this single-step competitiv e assay and a commercially performed enzyme multiplied immunoassay techniqu e for the detection of opiates and benzoylecgonine (a metabolite of cocaine ).