Chapter 21
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A Paramagnetic Particle-Based Enzyme-Linked Immunosorbent Assay for the Quantitative Determination of 3,5,6-Trichloro-2-pyridinol in Water 1
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Jeanne A. Itak , William A. Day , Angel Montoya , Juan J. Manclús , Amy M . Phillips , Dwayne A. Lindsay , and David P. Herzog 3
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Ohmicron Corporation, 375 Pheasant Run, Newtown, PA 18940 Laboratorio Integrado de Bioingenieria, Universidad Politécnica de Valencia, Apdo. de Correos 22012, Camino de Vera s/n, 46071 Valencia, Spain DowElanco, 9330 North Zionsville Road, Indianapolis, IN 46268
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A competitive enzyme-linked immunosorbent assay (ELISA) for the quantitation of 3,5,6-trichloro-2-pyridinol (TCP), the major biological and environmental degradation product of chlorpyrifos and triclopyr, was developed. Magnetic particles were used as the solid phase to attach monoclonal anti-TCP antibodies. The ELISA has an estimated least detectable dose of 0.25 parts per billion (ppb; ng/mL) in water. Specificity studies indicate that the assay is specific for TCP and can distinguish it from the parent compounds as well as many other related and unrelated agricultural compounds. Results compare favorably with GC/MSD measurements (r = 0.959). Interest in monitoring pesticide residues has increased in recent years as a result of concerns over the potential for water, soil and food contamination. Some attention has also focused on the monitoring of certain pesticide metabolites as indicators of contamination or exposure, to study metabolic pathways under field use conditions or to monitor for degradation of parent pesticides during shipment or storage of environmental samples. An example is 3,5,6-trichloro-2-pyridinol (TCP), the major biological and environmental degradation product of the insecticide chlorpyrifos and the herbicide triclopyr in soil, water and plant surfaces (7,2). The toxicity of TCP itself has been defined for several species (3-5). At neutral pH, TCP is ionic and appears to be more leachable than either parent compound and hence may serve better as an indicator of water contamination. Traditionally, TCP analysis requires specific and labor intensive sample preparation steps. This paper describes the development of a paramagnetic particlebased ELISA which can be used for the quantitation of TCP in environmental water samples at detection levels in the sub-parts per billion (ppb; ng/mL) without any sample preparation. Magnetic particle-based ELISAs for the quantitation of other © 1997 American Chemical Society In Immunochemical Technology for Environmental Applications; Aga, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
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3,5,6-Trichloro-2-pyridinol (TCP)
pesticide residues have been described previously (6-9). In these systems, the antibody is covalently coupled to the paramagnetic-particle solid phase. Because the antibody is covalently bound, sensitivity and precision problems associated with passive adsorption to polystyrene surfaces are eliminated. These problems include desorption or leaching of proteins passively adsorbed to microtiter plates and coated tubes, and well-to-well variability within microtiter plates (10-13). With magnetic particle-based assays, the paramagnetic particles are uniformly dispersed throughout the reaction mixture, allowing for precise addition of antibody and rapid reaction kinetics. The TCP immunoassay presented takes less than one hour to perform. Photometric determination of the final colored product is done with a specially designed microprocessor-controlled photometer with extensive data reduction capability that directly reports parts per billion (ppb, ng/mL, ug/L) concentrations of TCP in the sample ?). Materials and Methods Amine terminated superparamagnetic particles were obtained from PerSeptive Diagnostics, Inc. (Cambridge, M A ) . Glutaraldehyde and horseradish peroxidase (HRP) were obtained from Sigma Chemical (St. Louis, MO) and the T M B Microwell Peroxidase Substrate was obtained from Kirkegaard & Perry Labs (Gaithersburg, MD). 3,5,6-Trichloro-2-pyridinol, chlorpyrifos, chlorpyrifos-methyl, clopyralid, fluroxypyr, fluroxypyr-2-pyridinol, 2-methoxy-3,5,6-trichloropyridine, picloram and triclopyr were obtained from DowElanco (Indianapolis, IN). Other pesticides and metabolites were purchased from ChemService (West Chester, PA) and Crescent Chemical Co., Inc. (Hauppauge, NY). All other reagents were reagent grade or chemically pure as supplied by commercial sources. The following apparatus were utilized: Magnetic Separation Unit, RPA-I Analyzer™ and vortex mixer obtained from Ohmicron Corporation, Newtown, PA;
In Immunochemical Technology for Environmental Applications; Aga, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
Downloaded by STANFORD UNIV GREEN LIBR on October 6, 2012 | http://pubs.acs.org Publication Date: May 5, 1997 | doi: 10.1021/bk-1997-0657.ch021
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ITAKETAL.
ELISA for 3,5,6- Trichloro-2-pyridinol in Water
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adjustable precision pipette, Gilson P-1000 (Rainin; Woburn, M A ) ; and repeating pipette (Eppendorf; Hamburg, Germany). Water samples were characterized using Hach Test Kits for nitrate/nitrite (cat. #14081-00), salinity (cat. #24218-00) and chlorine, hardness, iron and pH (cat. #2230-02). The TCP monoclonal antibody, LIB-MC2, was derived from a hapten prepared by hydrolysis of the thiophosphate ester of a chlorpyrifos hapten, as previously described (14,15). The procedure for coupling anti-TCP antibody to magnetic particles was also detailed in prior literature (9). The TCP-HRP conjugate was synthesized according to methods outlined previously for a chlorpyrifos hapten and hapten-protein conjugates (14). The immunoassay procedure required adding 250 uL of standard or sample with 250 uL of horseradish peroxidase labeled TCP analog and 500 uL of anti-TCP antibody coupled paramagnetic particles to disposable test tubes. The tubes were vortexed and incubated for 20 min at room temperature. The reaction mixture was separated by placing the tube rack into the magnetic base and the supernatant was decanted. Particles were washed twice with 1 mL washing solution (preserved deionized water). The peroxidase substrate solution, peroxide/TMB (3,3',5,5-tetramethylbenzidine) was added, 500 uL per tube, and allowed to develop for 20 min. The color reaction was stopped with 500 uL of 0.5% sulfuric acid. To ensure accurate results, a calibration curve (0.0, 0.5, 2.5 and 6 ppb TCP), in duplicate, was included with every run. The concentrations of TCP for each sample were determined using the RPA-I Analyzer set at 450 nm. The RPA-I compares the observed sample absorbances to a regression line using a log-logit standard curve derived from the calibrator absorbances and reports parts per billion TCP in the sample. Residue analysis for TCP was performed by capillary gas chromatography with mass selective detection. The method required that 25 mL water samples be extracted twice with 1.0 mL of 2.0 N hydrochloric acid, 10 g sodium chloride and 5.0 mL of 1-chlorobutane and shook for 30 minutes on a reciprocating shaker. Samples were centrifuged for 5 minutes at 2500 rpm and the 1-chlorobutane layers were combined and concentrated to less than 1 mL (not to dryness) on an evaporator set at 35°C with a flow rate of 200 mL/min. Sample volume was adjusted to 1 mL with 1-chlorobutane. Then, 100 uL of MTBSTFA (7V-^r/-butyldimethylsilyl)-A^-methyltrifluoro-acetamide) derivatizing reagent was added and the samples were vortexed or sonicated for 10-15 seconds. Samples were allowed to react in an oven at 60°C for 1 hour. After cooling, the samples were analyzed by capillary gas chromatography/mass spectrometry using a J&W D B 1701 capillary column and an HP 5971 mass selective detector. Results and Discussion The TCP immunoassay described uses a competitive assay format. Since the enzymelabeled TCP competes with the unlabeled (sample) TCP for the antibody sites, the color developed is inversely proportional to the concentration of TCP in the sample. To describe color inhibition, it is common to report displacement in terms of a B/Bo
In Immunochemical Technology for Environmental Applications; Aga, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
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measurement, defined as the absorbance observed for a sample or standard (B) divided by the absorbance at a zero analyte concentration (Bo). Figure 1 illustrates the mean standard curve for the TCP calibrators, collected over 50 assay runs, linearly transformed using a log/logit curve fit with error bars representing 2 standard deviations. The range of the immunoassay calibration curve is 0.5 to 6 ppb TCP with an estimated least detectable dose (LDD) of 0.25 ppb. The assay L D D , defined as the lowest concentration that can be distinguished from zero, was based on an average 90% B/Bo estimation from 50 assay runs (16). The 0.25 ppb L D D estimate is greater than 4 standard deviations from the "true" zero, determined by calculating the standard deviation and mean absorbance value for three sets of twenty replicates of the zero standard. Quantitation with the immunoassay should be limited to within the range of the standard curve. To analyze samples with higher TCP concentrations, water samples were diluted in the zero standard for analysis and samples concentrations calculated by multiplying results by the appropriate dilution factor. Table I summarizes a precision study that was conducted with four concentrations of TCP in four environmental water samples: two wells, a spring and a creek. The water samples are described in Table II. TCP was added at 1, 2, 4 and 5 ppb. Five replicates of each level were assayed within a single run for each of 5 days. The within and between day and total variation was determined by the method of Bookbinder and Panosian (17) using Statistical Analysis Software (18). Coefficients of variation (CVs) were less than 10% for all levels tested. The accuracy of the TCP assay was assessed by evaluating the same four environmental water samples each spiked with known amounts of TCP at four levels.
Table I. Precision of TCP Measurement by Immunoassay Concentration Level (ppb) Sample
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N Mean ppb % C V within assay % C V between assay % C V total
25 1.00 9.1 1.1 9.2
25 2.24 6.6 3.4 7.3
25 4.34 6.6 1.1 6.6
25 5.18 5.7 1.1 5.6
Table II. Characterization of Water Samples Used in Precision and Accuracy Experiments Sample
iron, mg/L
nitrate, mg/L
nitrite, mg/L
pH
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