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

An automated flow fluorometer designed for kinetic binding analysis was adapted to develop a solid-phase competitive fluoroimmunoassay for urinalysis of opiates. The solid phase consisted of polymer beads coated with commercial monoclonal antibodies (MAbs) raised against morphine. Fluorescein-conjugated morphine (FL-MOR) was used as the fluorescein-labeled hapten. The dissociation equilibrium constant (K(D)) for the binding of FL-MOR to the anti-MOR MAb was 0.23 nM. The binding of FL-MOR to the anti-MOR MAb reached steady state within minutes and was displaced effectively by morphine and other opiates. Morphine-3-glucuronide (M3G), the major urinary metabolite of heroin and morphine, competed effectively with FL-MOR in a concentration-dependent manner for binding to the antimorphine MAb and was therefore used to construct the calibration curve. The sensitivity of the assay was 0.2 ng/mL for 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 showed >50% crossreactivity when present at 1 microg/mL included codeine, morphine-6-glucuronide, and oxycodone. Methadone showed very low crossreactivity (<5%), which is a benefit for testing in patients being treated for opiate addictions. 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 clinical urine samples showed good agreement between this single-step competitive assay and a commercially performed enzyme multiplied immunoassay technique for the detection of opiates and benzoylecgonine (a metabolite of cocaine).

Assessment of an automated solid phase competitive fluoroimmunoassay for benzoylecgonine in untreated urine.

A new solid phase fluoroimmunoassay using a fully automated flow fluorometer adapted for urinalysis of drug metabolites is described. Fluorescein-conjugated benzoylecgonine (FL-BE) and monoclonal antibodies (mAb) against benzoylecgonine (BE) were the reagents used for demonstration. The solid phase consisted of anti-BE mAbs immobilized on the surface of polymethyl methacrylate (PMMA) beads. Free BE in solution competed with FL-BE and reduced bead-bound fluorescence in a concentration-dependent manner. The binding of FL-BE to the anti-BE mAb reached steady-state within minutes. FL-BE was not bound by uncoated beads nor beads coated with non-specific proteins or IgG. The signal-to-noise ratio was 33, and the sensitivity of the assay was 2 ng ml(-1) for BE. The effective concentration of BE was 1 to 100 ng ml(-1), with an IC50 value of 12 ng ml(-1). The mAb showed equal affinities for BE, cocaine, and cocaethylene, but a five order-of-magnitude lower affinity for ecgonine and ecgonine methylester. In a double-blind comparison using clinical urine samples, the data from this single-step competitive assay had excellent agreement with results obtained using a fiber-optic biosensor (FOB), and the EMIT assay performed commercially. The assay provided kinetic data rapidly and can be used to detect small analytes for which antibodies and fluorescein conjugates are available. The affinity of the mAb for FL-BE, calculated from kinetic analysis of the time course of the on and off reaction, was 2.25 x 10(-9) M.