A Sensitive Solid-Phase Fluoroimmunoassay for Detection of Opiates in Urine

Abstract

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).

Recent advances in immunosensor for narcotic drug detection

“…The developed immunoassay was rapid, amenable to on site with no prerequisite of sophisticated instrumentation. 10 Similar indirect fluorocompetitive assays were also developed for other opiate drugs such as papavarine (PAP) and morphine 12 , 13 and its metabolites by using an immunoaffinity column based approaches. 14 …”

A microfluidic system utilizing molecularly imprinted polymer films for amperometric detection of morphine

“…Alternatively, immunoassays (IA) which utilize antibodyantigen affinity for detecting bio-samples are another popular method for morphine detection [11]. Enzyme-linked immunosorbent assay (ELISA) is one of the most popular methods for these immunoassays [12].…”

Specificity of the cocaine aptamer and its application in measurement of cocaine and metabolites in homogeneous samples for forensic analysis

“…False negative results due to reduced sensitivity of the test or false positives due to lack or specificity may result from co-extraction of compounds having similar physicochemical properties. The cross reactivity is often seen, due to structural similarity, with concomitant administration of opioids, barbiturates and benzodiazepines along with cocaine [22,31,32]. Specific and sensitive detection is also important because street drugs are mostly adulterated and have an uncertain composition.…”

“…20 Aptamers have been selected that recognize two drugs of abuse, which are cocaine 20 and codeine, 23 and many medicinal drugs and antibiotics including theophylline, 24 tobramycin, 25 neomycin, 26 kanamycin, 27 dopamine, 28 chloramphenicol, 29 streptomycin, 30 and tetracycline. 31 The affinities (K a ) of these aptamers are in the range of 10 5 to 10 7 M À1 .…”