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

A new solid phase fluoroimmunoassay using a fully automated flow fluoromete r adapted for urinalysis of drug metabolites is described. Fluorescein-conj ugated benzoylecgonine (FL-BE) and monoclonal antibodies (mAb) against benz oylecgonine (BE) were the reagents used for demonstration. The solid phase consisted of anti-BE mAbs immobilized on the surface of polymethyl methacry late (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 bo und by uncoated beads nor beads coated with non-specific proteins or IgG. T he 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), wi th an IC50 value of 12 ng ml(-1). The mAb showed equal affinities for BE, c ocaine, and cocaethylene, but a five order-of-magnitude lower affinity for ecgonine and ecgonine methylester. In a double-blind comparison using clini cal urine samples, the data from this single-step competitive assay had exc ellent 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, ca lculated from kinetic analysis of the time course of the on and off reactio n, was 2.25 x 10(-9) M. (C) 1999 Elsevier Science B.V. All rights reserved.