Chapter 2
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Adapting Immunoassays to the Analysis of Food Samples Scott W. Jourdan, Adele M . Scutellaro, Mary C. Hayes, and David P. Herzog Ohmicron, 375 Pheasant Run, Newtown, PA 18940
Immunoassays are powerful tools which augment current monitoring and measurement capabilities in residue testing of food samples. Immunoassay technology can minimize cleanup in analysis and provide a rapid screen for the analysis of large sample loads when conventional analytical methods are too costly or cumbersome. Many immunoassay test kits are sensitive enough to directly analyze crude extractions with few or no steps involved in extract cleanup. Often extracted samples can be diluted to remove matrix interferences in the immunoassay. To adapt an immunoassay to food samples, potential problems can be systematically investigated. Studies can be easily performed to evaluate the matrix interferences and method performance. In this paper, common problems encountered in this process will be reviewed using examplesfromthe literature and our experience. The application of immunoassay to foods has been widely reported and reviewed (1-3). Over the past five years our group has developed over 25 immunoassays and, in addition to using them to detect and measure residues in water and soil, they have been adapted for whole fruits and vegetables, juices, oils, dairy products and grains. This paper will review the approach we have been taking to the application of immunoassay to foods. Based on our experience the most important factors when developing a method are: 1) the required detection levels, 2) the choice of an extraction technique, and 3) matrix effects. We hope that sharing our experience will aid other investigators in their application of immunoassays to foods.
Detection Levels When applying an immunoassay to a food matrix, detection levels required for the testing and achieved by the immunoassay must be determined. This may sound trivial, 0097-6156/96/0621-0017$15.00/0 © 1996 American Chemical Society In Immunoassays for Residue Analysis; Beier, Ross C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
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but this consideration probably dictates the ultimate method more than any other factor. The first issue to consider is the concentration required for detection. A number of sources may dictate this requirement or can provide guidance. They include tolerances published by the US EPA in the Federal Register, the regulations of the European Community, the Codex Commission of the World Health Organization, or internal organizational requirements. Next, it is important to understand the sensitivity of the immunoassay. Typically, immunoassays are more sensitive than is required. However, performance aspects of an immunoassay differ across its working range. Detecting analytes at the least detectable dose (LDD) is not generally the most reliable way of employing immunoassays since the optimum performance of these methods is found in the center of their working range. The point of optimum performance is estimated to be at the estimated dose at 50% B/Bo (ED ). The E D or inhibitory concentration at 50% (IC ) in a small molecule assay is the concentration of analyte required to inhibit the signal in the assay 50% compared to the signal at zero analyte concentration. Based on the detection requirements and the sensitivity of the immunoassay the degree to which dilution or concentration step(s) will be required can be calculated. It should be recognized that, while dilution steps will dilute potential interferences, concentrations will often concentrate these inferences. Consequently it is advantageous to begin with an immunoassay with greater sensitivity than will be required. 50
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Extraction Techniques Once the detection level required is understood, an extraction technique can be developed. There is an obvious need to extract the residue from the food stuff since the antibody-antigen reaction is typically optimum in an aqueous environment. The current literature is rich with examples of extraction methods. To fully capitalize on the value of an immunoassay it is important that the investigator recognize that the immunoassay can often tolerate much less cleanup than traditional chromatographic methods due to the selectivity of antibodies. In some cases the residue is known to be confined to the surface of a food. In these cases the dislogible foliar residues (DFRs) can be obtained by simple methods which wash the surface of the food. This technique also has been useful in monitoring worker reentry into pesticide treated fields (4-5). Washes often employ solvents or water-based detergents. When the residue is found throughout the food, it becomes necessary to blend or homogenize the food and extract the residue in an organic solvent. The methods of Luke (6) and Mills (7) are examples of these techniques that were developed for traditional chromatographic methods. Sometimes the residue is more difficult to remove from the food tissue and requires more strenuous extraction. An example is paraquat which can be extracted by acid digestion aided by refluxing or sonication. (*)· Another extraction technique that has been applied to immunoassay is supercritical fluid extraction (SFE) (9-10; see chapters by King and Nam, and Lopez-Avila et al, this volume). This technique is based on the solvating properties
In Immunoassays for Residue Analysis; Beier, Ross C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
2. JOURDAN ET AL.
Adapting Immunoassays to Analysis of Food Samples
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of a compound such as carbon dioxide used in its liquid state by applying it under increased pressure. SFE is becoming more widely applied as equipment becomes more commonly available. Examples of applying this extraction method to immunoassays for foods is described in greater detail by King and Nam, and by Lopez-Avila et al. in this volume.
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Evaluating Matrix Effects Once an extraction method is chosen, it is important to evaluate the effect of the extracted sample on the immunoassay. All immunoassays rely on antibodies as the critical analytical reagent. Anything in the sample presented to the immunoassay that effects the antibody binding event can have a negative effect on the accuracy of the immunoassay. Antibodies are large protein molecules known as immunoglobulins. The properties of antibodies vary widely and a concentration of organic solvent that has no effect on one immunoassay can dramatically increase or decrease the sensitivity of other immunoassays. One step in determining potential matrix effects is to examine the effect of the extraction solvent on the results by validating that the addition of known quantities of the analyte can be accurately recovered. Potential solvent effects can be further investigated by validating that dilutions of positive samples behave as expected. Once the development of the method is complete it is always important to fully validate it including correlation to a traditional method.
Solvent Tolerance. The simplest approach to determining solvent tolerance in an immunoassay is to evaluate the effect of the solvent on the calibration curve. A n enzyme-linked immunosorbent assay (ELISA) based on a magnetic particle solid phase has been used in our work. Further details of this technique have been given elsewhere (11-13). This type of immunoassay is known as a competitive heterogeneous immunoassay where the antibody is immobilized. The "bound" or antibody fraction of the enzyme label is measured. Consequently the resulting color produced is inversely related to concentration. Figure 1 illustrates the effect of methanol on an atrazine immunoassay (11). In our experience, methanol is generally one of the best tolerated solvents in immunoassays. In this case as the methanol concentration is increased, the calibration curve is depressed, i.e., the methanol seems to inhibit the antigen-antibody binding. This phenomenon is particularly pronounced at low atrazine concentrations. Figure 2 shows the same atrazine immunoassay in the presence of various concentrations of acetone. These data demonstrate that in this system acetone is less tolerated than methanol. This is consistent with our overall experience. Figure 3 demonstrates the effect of acetonitrile on the calibration curve of the same atrazine immunoassay. It should be noted that at a concentration of 2.5% acetonitrile the calibration curve is nearly identical to the control containing no acetonitrile. In most cases, it would be recommended to dilute out the acetonitrile to this concentration before introducing the sample into the immunoassay. At 10% acetonitrile there is still an adequate dose response to distinguish atrazine concentrations but at higher concentrations, 25-50%, the response is unusable.
In Immunoassays for Residue Analysis; Beier, Ross C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
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IMMUNOASSAYS FOR RESIDUE ANALYSIS
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