ELISA, pesticide analysis

Newman, J.K., T.D. Glanville, and J.L. Baker (1996). Two quahty assurance measures for pesticide analysis of wellwater Degradation in storage and GC/ELISA comparison. Trans. Amer. Soc. Agri. Eng., 39 1725-1730. 

ELISA could potentially be used advantageously in many types of exposure and monitoring situations, for paraquat and other pesticides amenable to ELISA analysis. An obvious use of ELISA is the detection of pesticide residue levels in plant and animal tissues, or food extracts. Biological specimens such as plasma and urine currently analyzed by RIA seem particularly amenable to analysis by ELISA. Portable field kits could be developed to determine safe worker re-entry times into treated fields. Environmental samples such as soil, water, and air, can be analyzed by the ELISA. Pesticide conjugates have been proposed for skin testing of individuals suspected of sensitivity to pesticides (fi.) the ELISA could be used to detect specific antibodies in these individuals and aid in exposure studies. 

Generally, paraquat represents a successful IA development, and a reasonably well accepted example of IA in trace pesticide analysis. Following the human exposure study, we provided additional applications to meat, milk, and potatoes ). In each case, the ELISA provided detection limits lower than those available from spectroassay with minimal sample preparation (Table II). 

Depending on the label, lAs are classified in different groups. Radioisotopes are used in RIAs, enzymes in enzyme-linked immunosorbent assays (ELISAs) or EIAs, fluorophores in FIAs or PFIAs and chemiluminescent compounds in CLIAs. Additional types of lA exist, but are not very common in pesticide analysis. A more detailed description of these lAs can be found in Gosling. 

The ICMs used for pesticide analysis include immimoassays (lAs) and the use of antibodies for sample preparation (e.g., for SPE and the cleanup of samples) [153], detection in flow-injection analysis, and biosensors. The earliest ICMs to be developed for pesticides analysis were lAs. There are various t) es of lAs, but the most frequently used in this context is the enzyme-linked immunosorbent assay (ELISA) [185]. ELISA is a heterogeneous assay because the antibodies or antigens are immobilized on a solid phase. Table 18.3 lists selected ELISA methods for the determination of pesticides in water samples [186-190]. Bjamason et al. have proposed an enzyme flow immunoassay (EFIA) using a protein G column for the determination of triazine herbicides in surface and wastewaters with a linear range between 0.1 and 10 pg/L [191]. 

Sample preparation techniques vary depending on the analyte and the matrix. An advantage of immunoassays is that less sample preparation is often needed prior to analysis. Because the ELISA is conducted in an aqueous system, aqueous samples such as groundwater may be analyzed directly in the immunoassay or following dilution in a buffer solution. For soil, plant material or complex water samples (e.g., sewage effluent), the analyte must be extracted from the matrix. The extraction method must meet performance criteria such as recovery, reproducibility and ruggedness, and ultimately the analyte must be in a solution that is aqueous or in a water-miscible solvent. For chemical analytes such as pesticides, a simple extraction with methanol may be suitable. At the other extreme, multiple extractions, column cleanup and finally solvent exchange may be necessary to extract the analyte into a solution that is free of matrix interference. 

Most of the analytical methods for the analysis of pesticides in food are based on instrumental approaches based on chromatography coupled to mass spectrometry. However, a great effort of development has been paid to develop rapid screening methods based on biological methods, such as, enzyme linked immunosorbent assays (ELISA). 

Muldoon, M.T., G.F. Fries, and J.O. Nelson (1993). Evaluation of ELISA for the multianalyte analysis of s-triazines in pesticide waste and rinsate. J. Agric. Food Chem., 41 322-328. 

Several qualitative and quantitative immunochemical methods and their application to the analysis of environmental samples have been described for OP insecticides, a family that includes widely used pesticides such as azinphos-ethyl/methyl, dichlorvos, fenitrothion or fenthion, malathion, mevinphos, and parathion. Mercader and Montoya202 produced monoclonal antibodies against azinphos-methyl and developed an ELISA that was used for the analysis of water samples from different sources, reaching detectability levels near 0.05 pg I. Watanabe et al.203 reported the production of polyclonal antibodies and ELISA procedures to analyze fenitrothion in river, tap, and mineral water (LOD = 0.3 pg L ). Banks et al.204 produced polyclonal antibodies against dichlorvos, an organophosphate insecticide used for stored grain, which also cross-reacts with fenitrothion. Nishi et al.205 reported the first immunoassay for malathion. Residues of this insecticide have... 

The ELISA procedure for the analysis of parathion as described above requires nearly eight hours, although many samples can be simultaneously assayed. However, incubation times can be shortened to one-half hour, in most cases, resulting in only a 10% reduction in sensitivity. Also the polystyrene microtiter plates containing bound RSA-AP can be mass produced and stored in a freezer. Since the enzyme-linked antibody can be purchased, the limiting factor of the applicability of the ELISA procedure, as well as the RIA procedures, for other pesticides is the development of the antiserum to the pesticide. 

The enzyme-linked Immunosorbent assay (ELISA) is a rapid Immunochemical procedure which can be used for trace analysis. We have applied the procedure to paraquat and other compounds difficult to analyze by the more classical methods. The Immunoassay for paraquat shows the practicality of the method for fortified and actual residue samples, and Is being compared with a gas chromatography procedure. Our work with the ELISA Illustrates that the Immunochemical technology can be used to solve problems encountered In pesticide residue analysis. 

Antibodies have been raised against representative compounds from the major classes of pesticides. Although the ELISA will be useful for individual analysis of a wide variety of compounds, if one needed to analyze several different compounds simultaneously in one matrix immunoassay may not be the method of choice, due to the large amount of controls and standards needed. However, it could be successfully used for the rapid screening of a large number of samples for the presence of specific types of pesticides and for confirmatory tests (Ji). The work reported here with paraquat,... 

Immunoassays offer much potential for rapid screening and quantitative analysis of pesticides in food and environmental samples. However, despite this potential, the field is still dominated by conventional analytical approaches based upon chromatographic and spectrometric methods. We examine some technical barriers to more widespread adoption and utilization of immunoassays, including method development time, amount of information delivered and inexplicable sources of error. Examples are provided for paraquat in relation to exposure assessment in farmworkers and food residue analyses molinate in relation to low-level detection in surface waters and bentazon in relation to specificity and sensitivity requirements built in to the immunizing antigen. A comparison of enzyme-linked immunosorbent assay (ELISA) results with those obtained from conventional methods will illustrate technical implementation barriers and suggest ways to overcome them. 

The main practical environmental application of SPR has been as an immunosensor for the detection of phytosanitary products (Mullet, 2000). Recently, SPR immunodetection of 2,4D (Kim et al., 2007), DDT (Mauriz et al., 2006a, 2007), chlorpyrifos and carbaryl pesticides (Mauriz et al., 2006b) was found ten times more sensitive than ELISA methods, with detection limits in the ppt range, and an analysis time of less than 20 min. 

Immunochemical methods are rapidly gaining acceptance as analytical techniques for pesticide residue analysis. Unlike most quantitative methods for measuring pesticides, they are simple, rapid, precise, cost effective, and adaptable to laboratory or field situations. The technique centers around the development of an antibody for the pesticide or environmental contaminant of interest. The work hinges on the synthesis of a hapten which contains the functional groups necessary for recognition by the antibody. Once this aspect is complete, immunochemical detection methods may take many forms. The enzyme-linked immunosorbent assay (ELISA) is one form that has been found useful in residue applications. This technique will be illustrated by examples from this laboratory, particularly molinate, a thiocarbamate herbicide used in rice culture. Immunoassay development will be traced from hapten synthesis to validation and field testing of the final assay. 

Applications of immunoassay to pesticide chemistry have been described which address some difficult problems in analysis by classical methods. These include stereospecific analysis of optically active compounds such as pyrethroids (38), analysis of protein toxins from Bacillus thuringiensis (5,37), and compounds difficult to analyze by existing methods, such as diflubenzuron (35) and maleic hydrazide (15 also Harrison, R.O. Brimfield, A.A. Hunter, K.W.,Jr. Nelson, J.O. J. Agric. Food Chem. submitted). An example of the excellent specificity possible is seen in assays for parathion (10) and its active form paraoxon (3). Some immunoassays can be used directly for analysis without extensive sample extraction or cleanup, dramatically reducing the work needed in typical residue analysis. An example of this is given in Figures 2 and 3, comparing the direct ELISA analysis of molinate in rice paddy water to the extraction required before GC analysis. 

This table illustrates one of the major impediments to the rapid assimilation of immunochemical technology into pesticide residue analysis labs. Because of the amount and variety of work involved, new method development costs may be high when compared to routine chromatographic methods. However, the low cost per run allows for rapid recovery of the initial investment with sufficiently high sample loads. For example, the cost of reagents and supplies for an ELISA for diflubenzuron was estimated to be 0.20/sample as compared with 4 for HPLC or 11 for GC (35). In addition to the lower reagent and supply costs, the major economic advantage of immunoassay is the dramatic decrease in labor costs. 

The sample workup necessary for pesticide residue analysis will vary with each combination of analyte and antibody, each of which may have a different tolerance for the matrix and other factors. The effects of these factors must be considered as with the development of any other analytical technique. Matrix effects for one ELISA system are summarized in Figure 4. While the effect of the matrix on the antibodies in Figure 4 is different for each antibody-solvent-matrix combination, the competitive ELISA standard curves for most of these combinations are similar when expressed as percent of the appropriate control. Some systems may not require extensive adjustment, but this must be tested with each individual system. For example, our molinate assay performs equally well in a variety of water types at high concentrations of molinate (Figure 5). The small difference seen between the buffer and water standard curves in Figure 5 was eliminated by the addition of small amounts of concentrated buffer to water samples to equalize them to the buffer composition. 

M.T. Muldoon, G.F. Fries, 1.0. Nelson, Evaluation of an ELISA for the Multianalyte Analysis of s-Triazines in Pesticide Waste and Rinsate , /. Agric. Food Chem., 41, 322-328 (1993). 

The evaluation of a number of immunoassay diagnostic kits was undertaken to determine their usefulness in a regulatory analytical laboratory environment in the food, feed and pesticide areas. Four rapid enzyme immunoassay tests for the detection of aflatoxin residues at the 20 ppb level in animal feeds were compared to the official HPLC procedure. In the pesticide area, a commercial pentachlorophenol competitive inhibition assay for residues in water was investigated as to its applicability to poultry and pork liver matrices. In addition, an ELISA screening procedure for the herbicide fusilade was developed. Modifications were incorporated into the rapid immunoband 1-2 Test procedure for the detection of motile Salmonella in various food and animal feed products resulting in quicker analysis than the standard culture method. Also, a comparative evaluation of a Quik-Card Test for sulphamethazine drug residues in pork urine, liver and muscle tissue, is described. 

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