Nabil A. Anis

A Fiber-Optic Immunosensor for Detecting Parathion

Abstract Casein-parathion conjugates, immobilized by adsorption on quartz fibers, selectively adsorbed antiparathion rabbit antibodies raised against bovine serum albumin (BSA)-parathion conjugates from polyclonal immune sera. The presence of free parathion interfered with binding of the rabbits antiparathion Abs. Fluorescein isothiocyanate(FITC) goat antirabbit IgG were used to generate an optical signal, which was measured by a signal transducer based on total internal reflection fluorescence. Inhibition of binding of rabbit Abs by free parathion, reduced the optical signal and provided the basis for detection of parathion with this optical immununosensor. It could detect 0.3 ppb parathion and was highly specific since its detection of the oxygen analog paraoxon was possible at 100-fold higher concentration.

Reusable fiber-optic-based immunosensor for rapid detection of imazethapyr herbicide

Abstract Imazethapyr is a herbicide belonging to the imidazolinone class of compounds. It is the active ingredient of a commercial herbicide PURSUIT®. Polyclonal antibodies have been prepared in rabbits and sheep which specifically recognize this class of imidazolinone compounds. Using the immune rabbit serum, an enzyme-linked immunosorbent assay (ELISA) has been developed for the entire class of commercial imidazolinone herbicides including imazaquin, imazapyr, imazethapyr and imazamethabenz methyl. The quantitation of imidazolinones is soil requires a certain amount of sample pretreatment, thus the through-put is not ideal. A simpler immunoassay method for screening large amount of soil samples economically would be useful. Taking the sheep polyclonal immune serum, we used a fluorescent immunoassay employing optical fiber and fluorescence (US Patent 4,582,809, 1986), to assay for imazethapyr. Purified sheep antibody was immobilized on quartz fibers. A mixture of fluorescein-labelled imazethapyr analog and free imazethapyr was presented to the fiber for direct competition of the antibody binding sites or displacement of a previously fluorescein labelled fiber. The response time for the detection of imazethapyr ranged from seconds to minutes. The sensitivity of the assay was 1 nM. This binding of the fluorochrome to the fiber was reversible by washing with a phosphate buffered saline. Multiple measurements were easily processed with a single fiber over the course of several hours. Analysis of imazethapyr residue in soil can be accomplished by subjecting a clarified soil extract solution directly for analysis without further treatment. The crossreactivity data indicates that the assay is apparently specific for the imidazolinone class of compounds.

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 (KD) 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 µg/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).

Reusable, Real-Time, Immuno-Optical Protein C Biosensor

A Protein C (PC) biosensor can be used to diagnose PC deficiency, to monitor the PC level in the blood of PC deficient patients, and to measure the PC concentration in other PC-containing samples, such as PC producing animal cell culture broth or transgenic animal milk. A fully functional biosensor requires extremely high sensitivity and specificity, and real-time measurement. To satisfy these requirements, it is proposed to develop an immuno-optical fiber biosensor that utilizes PC-specific biomolecules (PC probes) tagged with fluorophores. The method involves immobilizing monoclonal antibody against PC (anti-PC) on the surface of an optical fiber. When PC in a sample is adsorbed to the anti-PC on the fiber, it can be reached with the fluorophore tagged PC-probe. The intensity of light transported through the optical fiber, therefore, can be correlated with the concentration of PC in the sample. The sensor will be designed so it can be reused, following a simple elution step, thus reducing diagnostic expense. The preliminary study shows encouraging future for the real-time optical PC biosensor.

Reusable fiber optic immunofluorosensor for rapid detection of pesticides

Quartz fibers coated with acetylcholinesterase (AChE) or antibody (Ab) are used as biosensors utilizing total reflectance fluorescence for the rapid detection of pesticides. The enzyme biosensor was constructed by immobilizing fluorescein isothiocyanate (FITC)-tagged eel electric organ AChE on quartz fibers. The fluorescent signal was generated by hydrolysis of acetylcholine (ACh) that is present in the perfusate. Organophosphate (OP) and carbamate anticholinesterase (AntiChE) insecticides inhibited AChE and reduced the fluorescent quenching resulting from AChE hydrolysis. A parathion biosensor was constructed by immobilizing casein-parathion on the quartz fibers, that bound rabbit antiparathion antibody. The optical signal was generated by perfusing the fibers with fluorescein-labeled goat antirabbit IgG. Free parathion inhibited the binding of antiparathion Abs and reduced the optical signal and provided the basis for detection of parathion. Another immunosensor developed detected the herbicide PursuitR by utilizing the reversible binding of a fluorescein-Pursuit derivative to antiPursuit Abs immobilized on the fiber. Unlabeled Pursuit competed effectively and displaced the bound fluorescent compound in a dose-dependent manner. The sensor discriminated effectively between Pursuit-like and structurally unrelated herbicides. The immunosensor offers the advantage of continuous monitoring, ease of operation, speed of detection, low cost, stability, specificity, matrix transparency, and reusability.

Reusable Fiber Optic Biosensors for Detection of Drugs and Toxicants

Use of fluorescein derivatives of specific analytes, that are bound reversibly by biological sensing elements (e.g. receptors or antibodies), proved to be a successful strategy for fiber optic biosensors. Two biosensors, one using nicotinic acetylcholine receptor (nAChR) and the other using antibodies, detected anaytes in real time and were easy to regenerate for multiple measurements.

Antibody-based bacterial toxin detection

Fiber optic evanescent fluorosensors are under investigation in our laboratory for the study of drug-receptor interactions for detection of threat agents and antibody-antigen interactions for detection of biological toxins. In a one step assay, antibodies against Cholera toxin or Staphylococcus Enterotoxin B were noncovalently immobilized on quartz fibers and probed with fluorescein-isothiocyanate (FITC)-labeled toxins. In the two-step assay, Cholera toxin or Botulinum toxoid A was immobilized onto the fiber, followed by incubation in an antiserum or partially purified antitoxin IgG. These were then probed with FITC-anti-IgG antibodies. Unlabeled toxins competed with labeled toxins or antitoxin IgG in a dose-dependent manner and the detection of the toxins was in the nanomolar range.