Paraquat ELISA

The paraquat ELISA developed in our laboratory started with the synthesis of the valeric acid derivative of paraquat (I) as hapten, and took well over one man-year to develop(7) (Figure 1). It is a very good method with fractional nanogram/mL sensitivity and (once fully validated) precision slightly better than the GC method. It is applicable to air filters, clothing patches, and hand washes, and showed promise for lymph and plasma analysis. The sample throughput was greater than GC and the method could be picked up rapidly by persons not skilled in the art. (Novices frequently pick up IA much faster than veteran analysts with a history of GC and HPLC experience ). 

Van Emon et al. ° developed an immunoassay for paraquat and applied this assay to beef tissue and milk samples. Milk was diluted with a Tween 20-sodium phosphate buffer (pH 7.4), fortified with paraquat, and analyzed directly. Fortified paraquat was detected in milk at less than 1 pgkg , a concentration which is considerably below the tolerance level of 10 pg kg Ground beef was extracted with 6 N HCl and sonication. Radiolabeled paraquat was extracted from ground beef with recoveries of 60-70% under these conditions. The correlation coefficient of ELISA and LSC results for the ground beef sample was excellent, with = 0.99, although the slope was 0.86, indicating a significant but reproducible difference between the assays. 

Niewola et al. [183, 185] have described a rapid, convenient and accurate method, based upon an enzyme-based immunosorbent assay (ELISA) for the determination of Paraquat residues in soil. Polystyrene plates, coated with paraquat-keyhole limpet haemocyanin (KLH) conjugate, are incubated with the test samples and a known amount of monoclonal antibody. Residual antibody that has not reacted with free Paraquat in the sample combines with paraquat-KLH on the plate. The determination of the fixed antibody is achieved by the addition of peroxidase labelled rabbit antimouse immunoglobulin G followed by reaction with a chromogenic substrate. The enzyme activity of the solid phase is determined from the absorbance measurements, which are inversely proportional to the concentration of Paraquat. The method shows high specificity and correlates well with the traditional ion exchange-spectrophotometric method for the determination of Paraquat [178]. 

The suitability of the ELISA for soil analysis was initially tested by assaying a number of control soil samples, fortified after extraction and neutralisation with Paraquat in the range 10-300mg kgy1. The results in Table 9.19 were close to the expected values and thus confirmed that natural soil components did not interfere with the determination. These results justified the further refinement of the method for soil analysis. 

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. 

Gas-liquid chromatography following reduction of paraquat to the mono- and diunsaturated derivatives (21) is of adequate sensitivity for most work when N-selective detectors are employed. Seiber and Woodrow (22.) modified this method for assaying paraquat in air samples. The method is time consuming and labor intensive, involving acid extraction and many concentration and evaporation steps. The maximum sample output per analyst per day is 6-8 with no duplicates. The reported recovery efficiency was 75% (22). although an efficiency closer to 50% is frequently encountered in practice. A modified acid extraction combined with analysis by the ELISA provides recoveries of 75% (Figure 3). This... 

Figure 2. Standard curves for paraquat using ELISA (top), GC (middle), and colorimetry (bottom).

Figure 3. Comparison of sample preparation steps for analysis of paraquat on air filters using ELISA and gas chromatography.

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. 

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. 

Figure 1. Schematic of ELISA procedure for paraquat (Reprinted from ref.

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). 

In a recent statement of proposed research FDA angled out development and validation of ELISA for paraquat in potatoes as a target. This exemplifies the changing attitude toward IA by the regulatory community. 

Table II. Detection Limits (ppm) for Paraquat in Foodstuffs by ELISA and Spectroassay...

Molinate. The thiolcarbamate molinate also appeared to be a good target for ELISA but for different reasons than existed for paraquat. The herbicide is used extensively on rice in the Sacramento Valley and each year is detected as a contaminant in drainwaters from the field and in the Sacramento River. There are many water samples taken each year for molinate analysis and some other chemicals used for pest control on rice. Thus, an ELISA could be put to immediate use supplementing or perhaps replacing entirely the conventional method based upon extraction of water, followed by GC analysis of the extract. 

J. Van Emon, J. Seiber, B. Hammock, Application of an ELISA to Determine Paraquat Residues in Milk, Beef and Potatoes , Bull. Environ. Gontamin. Toxicol, 39, 490-497 (1987). 

Van Emon, J. Seiber, J. Hammock, B. Application of an enzyme-linked immunosorbent assay (ELISA) to determine paraquat residues in milk, beef, and potatoes. Bull. Environ. Contam. Toxicol., 39 490-7. 1987. 

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