Immunoassays for Residue Analysis - American Chemical Society

Chapter 21

Immunoassays for Detecting Insect Contamination of Food Products Downloaded by UNIV OF CALIFORNIA SAN DIEGO on December 9, 2015 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1996-0621.ch021

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G. Barrie Kitto , Pei Wang Thomas , Jim Lemburg , Bob Brader , and Wendell Burkholder 3

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Department of Chemistry and Biochemistry, University of Texas, Austin, TX 78712 Biotect, Inc., P.O. Box 500068, Austin, TX 78750 U.S. Department of Agriculture Stored Product Insects Laboratory, University of Wisconsin, Madison, WI 53706 2

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Federal standards for regulating the amount of insect contamination in a wide variety of foodstuffs make it imperative that accurate and reliable analytical techniques be available for measuring such contamination. Present analytical methods such as the insect fragment count are time consuming, costly and show wide variability. New analytical techniques for insect detection, based on immunological ELISA assays, have been developed which offer simplicity of use, low cost and excellent accuracy and reliability. One class of ELISA assay, based on the insect muscle protein myosin, is designed to detect a broad range of insect pests in a wide range of stored grains, milled grain products, spices, nuts and dried fruits. This assay is available in several formats, ranging from highly quantitative microwell plate assays for laboratory analysis to simple qualitative dipstick assays for on-site screening. Other insect immunoassays have been developed which are species specific for both beneficial and deleterious insects and also for detecting Tephritidfruitflies,such as the medfry, in import and exportfruits.Work is in progress on additional immunoassays for the detection of insect material in cooked processed foods and for rodent contamination of foodstuffs. Immunoassays have the capability of combining exceptional selectivity with high precision and a relatively low cost in easy to use formats. For this reason they have become a mainstay of clinical laboratory testing. By contrast with their acceptance in the medical fields, immunoassays have been slower to be adopted for use in agriculture, although such testing for pesticide residues and fungal toxins is now becoming more widespread. One area of agriculture which has a potential for introduction of immunoassays is in the testing for insects and insect remains in stored products. The current tests for insect contamination of grain and milled grain products were first introduced many decades ago and have seen few improvements. 0097-6156/96/0621-0281$15.00/0 © 1996 American Chemical Society In Immunoassays for Residue Analysis; Beier, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on December 9, 2015 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1996-0621.ch021

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For whole grains these tests include the insect-damaged kernel (JDK) analysis (7,2), and insect exit hole counts and crack and float techniques (3-5). For milled grain products, such as wheat flour, the primary method is the insect fragment count (6). Each of these methodologies employs visual inspection as part of the procedure and this factor, in addition to other limitations, results in considerable variation between analysts (see for example 7). X-ray analysis of whole grain kernels (8) offers good precision for detecting hidden insects, but the need to provide radiation sources and the considerable training needed to interpret the X-ray films has hampered widespread adoption of this procedure. Other techniques, such as the acoustical detection of live insects (9JO) offer considerable potential but are still in the experimental stages. Immunoassays offer considerable advantages over the present methodologies for detecting insect contamination of food products. First, they have the potential of providing a single type of detection that can be used all the way through the food processing chain, from raw material to processed food. Second, the same basic type of assay can be utilized for analyzing large numbers of samples in a laboratory, or for testing individual samples on site, such as silos, loading docks, or even in the field. Third, simple and rapid immunoassays can be developed which meet the precision and reliability standards required for assessing insect contamination levels of food products. We present here an overview of the immunoassay methodologies that we have developed over the past several years for testing a wide range of commodities including grains, milled grain products, spices, nuts, and dried and fresh fruit. Materials and Methods An insect myosin ELISA assay we developed for the detection of insects in grain (77) has served as the basis for the development of other methodologies for insect detection. The reliability of this procedure and its application to the detection of different insect life stages is described in Schatzki et al. (12). The use of a modified version of this assay for detecting larval through adult insects in fruit, as well as an ELISA procedure for detecting insect eggs in fruit is provided in Proske (73). Monoclonal antibody based immunoassay procedures for insect species-specific tests are described in Chen (14) and Chen and Kitto (15). The isolation and characterization of unique Africanized bee proteins and their use in immunoassays for distinguishing between Africanized and European honeybees is given in Davidson et al. (16) and Verdel (17). Immunoassay kits for insect detection, based on this work, are available from Biotect, Inc., Austin, Texas. Results and Discussion The Insect Myosin Immunoassays. A prerequisite to developing an immunoassay which would be capable of detecting a variety of insects in a broad array of fruit flies was to select a suitable insect antigen. Ideally such an antigen should be present in large quantities in all the suspected insect pests, and in all major life stages. Additionally, the antigen should differ very little in structure from insect to insect and be readily solubilized from samples of different food products. The insect muscle protein myosin, specifically the "heavy chain," meets these exacting criteria. Myosin

In Immunoassays for Residue Analysis; Beier, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


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Detecting Insect Contamination of Food Products

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is present in relatively large quantities in all insects and in all life stages from larva to adult. Insect myosin also is a slowly evolving protein so that antibodies raised against one insect species should cross-react with a large selection of insects. This proved to be a fortuitous choice of antigen. Rabbit antisera prepared against myosin heavy chain from crickets (Acheta domesticus) crossreacts with all of the common stored product insect pests (11). Insect myosin is readily extracted from food samples using a high salt buffer. In order to provide an immunoassay that would require minimal setup time, we developed an insect myosin ELISA assay in a sandwich format, which is illustrated schematically in Figure 1. To test a food product for insect contamination, the sample is ground in a common household blender and then extracted in high salt buffer in the same blender. The extraction procedure takes less than 5 min. Duplicate aliquots of the extract are then placed in wells of microtiter plates or strips, along with a range of myosin standards. The wells of microtiter plates or strips are precoated with capture antibody. After a forty-five minute incubation with antigen, the plates are rinsed three times with a wash buffer (0.1 M potassium phosphate, p H 7.0; 0.1% bovine sereum albumin, 0.05% Tween-20). A secondary antibody solution (50 pL) consisting of rabbit antimyosin IgG conjugated to horseradish peroxidase is then added to the wells and allowed to incubate for 30 min. The wells are then emptied by inversion of the plate and rinsed three times, as above. Enzyme substrate (2,2*-azino-di-3-ethyl benzthiazoline sulfonic acid [ABTS]; 100 pL) is added to the wells and color development is allowed to proceed for 25 min in the dark. Color develops in the wells in proportion to the amount of antigen present. After this time, 100 uL of stop solution (0.5 M oxalic acid) is added to each well and the plates are then read at 414 nm in a standard ELISA reader. Up to 24 samples can conveniently be tested in duplicate at one time with this procedure, which takes approximately 2Vi h. The myosin ELISA assay gives an excellent linear response to the amount of myosin present and also correlates extremely well with the number of insects measured in food samples using the X test (Figure 2). This simple assay procedure is highly reproducible both for reassays of a single test sample (Figure 3), and with a variety of industrial wheat mill samples with varying degrees of insect contamination that were assayed 2 or 3 times over a 3 week period, as shown in Figure 4. The myosin ELISA assay detects all of the common stored insect pests and extensive testing has been carried out with all of the major commercial grains including wheat, oats, corn, barley, soybeans, sorghum and rice (19). In addition to performing well in in-house and industrial grain mill trials, this procedure has been very favorably tested by the Federal Grain Inspection Service of the U.S. Department of Agriculture (18). The myosin assay works well with both whole grain and milled grain products. Collaborative trials with grain mills have established that, for a given mill, the distribution of any insect material in the whole grain is distributed in a consistent fashion into the flour, shorts and bran fractions as illustrated in Figure 5. Thus, ELISA testing of raw material is an excellent indicator of how much insect contamination will turn up in the finished product. In a similar fashion, the myosin immunoassay provides a quantitative tool for the blending of clean and dirty grain to meet regulatory sanitary standards (Figure 6).




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Figure 1: A schematic representation of the sandwich E L I S A procedure. Reproduced with permission from reference (19), C A B International, 1994.


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Figure 3: Replicability of the insect myosin ELISA. A test sample of contaminated grain was assayed multiple times on the same day.


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Figure 5: Use of the insect myosin ELISA to follow distribution of insect contamination throughout wheat processing. Assays of whole grain (left) showed the initial level of contamination and assays of the milledflour,shorts and bran fractions (right) indicated how insect material fractionated. In Immunoassays for Residue Analysis; Beier, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


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Much of the early work was restricted to the analysis of grain products, but we have recently shown that the insect myosin assay is applicable to a broader range of food products, such as peanuts, almonds, Brazil nuts,filberts,cashews and walnuts; dried fruits such as dates, figs, and raisins; and an extensive group of spices. Some of these materials require slight modifications in the extraction procedure, although in general the same sensitivity and linearity of the assay is obtained as with the grain analysis procedure (19; Behrens, P.; Heller, I; Trieff, S.; Stephenson, J.; Kitto, G. B., The University of Texas at Austin, unpublished results). The insect myosin ELISA assay requires only a very modest outlay for equipment, the major expense being an ELISA reader costing in the range of $2,000-6,000 U.S. dollars. Technical personnel can be trained to high proficiency in one or two days. In cases where large numbers of samples need to be analyzed the procedure can readily be automated using off the shelf equipment employed in clinical laboratories. While the assays described above provide a reliable quantitative means for checking food products for insect contamination in the laboratory, there are many occasions where it also would be desirable to have available very simple immunoassays that could be applied to single samples in a short period of time. Such tests could be used in loading and unloading docks, for spot-checking during processing, at warehouses or even in the field. For these reasons we have developed a rapid test strip procedure which can be completed within 15 min with the results being read using a low cost hand-held spectrophotometer. This test strip assay is in the final stages of laboratory development, with field trials planned for the near future. The transport of fruit, either within a country or through import and export, carries with it the threat of introducing noxious insect pests. This is true, for example, of the import offruitfrom Hawai into the mainland United States, with the potential for bringing in the OrientalfruitflyBactrocera dorsalis or the Mediterranean fruitfly Ceratitis capitata (20,21). Similarly the export of stone fruits and apples to Japan from the U.S. is hampered by concerns about insect introductions (22). In addition to the use of certified fumigation procedures, inspection offruitalso is carried out at ports of entry. Typically such inspection involves slicing samples of the fruit and carrying out a visual inspection for insect larval and pupal stages. It would be desirable to have more quantitative biochemical techniques available which had the capabilities of being automated and could effectively test large numbers of samples. Because the insect myosin ELISA procedure can detect an exceptionally broad range of insects we reasoned that it might be adapted for checkingfruitfor insect infestation. The myosin assay works well for detecting a range of Tephritid insect pests, including Rhagoletis, Dacus, Ceratitis, Paracantha, Orellia, Aciurina and Neotephrita species in a variety of fruits including grapefruit, oranges, lemons, limes, mangos, pears, tomatoes and watermelon. Life stagesfromfirstinstar larvae through adults could be detected. Modifications to the extraction procedures of the fruit insect ELISA assays have to be made for each major type of fruit, to ensure neutralization of the fruit acids which can denature the insect antigen (13; Proske, P.; Lemburg, J.; Kitto, G. B., The University of Texas at Austin, unpublished results). Detecting Insects in Fruit.



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Although the insect myosin assay can detect the majority of fruit insect life stages it cannot detect insect eggs. For this purpose we have devised an immunoassay based on vitellin, a major protein in insect eggs. Rabbit polyclonal antisera were prepared against purified vitellin from Anastrepha suspensa eggs. In this case a sandwich ELISA using these antibodies proved highly specific for eggs from Anastrepha species, with little crossreactivity shown to Dacus and Ceratitis flies (73). This is most likely due to the fact that vitellin is a fairly rapidly evolving protein. While the specificity of the A. suspensa vitellin antibodies precludes the use of this particular assay as a general tool for detecting insect eggs, such species specific assays could be very helpful in identifying the particular type of insect involved in infestations (see for example 20). Antibody Based Immunoassays. Polyclonal antibodies are advantageous for immunoassay development since they can readily be prepared in large quantities at low cost. However, polyclonals typically exhibit a broad range of specificity. On the other hand monoclonal antibodies can be elicited which show exceptionally narrow specificity. We have taken advantage of monoclonal antibodies to develop a species specific test for the granary weevil Sitophilus granarius, a major stored grain pest (75). In this case the antigen was a specific, stable 60,000 dalton protein isolated from adult granary weevils. As shown in Figure 7, when used in combination with the myosin ELISA for estimating the total insect load of a food sample, the species-specific assay can accurately estimate the proportion of insect contamination contributed by granary weevils. In a similar manner a species-specific assay has been devised for the Kaphra beetle (23). With increasing restrictions being applied to the use of fumigants and pesticides for controlling insect infestations of stored food materials alternate methods of control are being pursued with renewed vigor. Among the alternative techniques is the use of beneficial insects for reducing damage to grain and other foodstuffs which harbor destructive insect pests within the product (such as grain kernels or peanuts). The beneficial insects either directly kill or parasitize the destructive pests but are themselves easily removed from the product by airstream cleaning, since they are external feeders. We and our colleagues have developed monoclonal antibody based, species-specific immunoassays for several of the beneficial insects including Bracon Monoclonal

hebetor, Laelius pedatus, Xylocoris flavipes and Trichogramma pretiosum (24;

Lemburg, J.; Kedzieski, R.; Ross, C; Kitto, G. B.,The University of Texas at Austin, unpublished results). These assays should prove useful for studying the dynamics of beneficial/destructive insect interactions and could be used in conjunction with the myosin assay (to measure total insect load) for appropriate timing of beneficial insect introductions. Africanized bees first moved into the United States from Mexico in 1990 and since that time have moved into the southern states from Texas to California. These bees, because of their aggressive defensive behavior, pose a considerable threat to apiculture, particularly for managed pollination. The Africanized bees bear an extremely close physical resemblance to the common European bee, which makes the task of distinguishing between them extremely difficult. The primary identification methodologies employed today involve dissection of suspect bees, mounting of the


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Figure 6: Illustration of how the insect ELISA procedure could be used for grain blending. Samples of clean and insect contaminated grain were mixed in varying proportions and assayed by the insect myosin ELISA. 10

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Figure 7: Use of the insect myosin and species-specific ELISA procedures to determine the proportion of insect contamination contributed by a given insect. Clean grain was spiked with the mixture of insects shown at the left. Total insect contamination was measured by the myosin ELISA (center) and the number of granary weevils was estimated by a monoclonal antibody based ELISA. The contaminated grain contained 5 granary weevils/50 g grain. Reproduced with permission from reference (19), CAB International, 1994. In Immunoassays for Residue Analysis; Beier, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


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parts of the bee on slides and projecting these slides so that measurements of the body parts can be made. These measurements are then analyzed by a computerized statistical procedure (25,26). Such testing services are available at only a few sites in the U.S.A. We are engaged in the development of simple immunoassay procedures which should allow for ready discrimination of Africanized and European bees. Three related proteins which are unique to Africanized bees have been identified (77) and these have been used to prepare monoclonal antibodies. These antibodies have been employed for devising a sandwich ELISA procedure which, in the laboratory, provides the appropriate distinction between the bee species. Work is in progress to convert the ELISA procedure to a rapid test-strip format, using a simple bee squash, that would allow for identification of Africanized bees in the field. Summary As illustrated here and in other papers in the symposium immunoassays are finding increasing use for analytical purposes in agriculture for tasks rangingfrompesticide residue detection to insect species identification. For many purposes these immunoassays provide major advantages over traditional wet chemistry procedures in terms of ease of use, sensitivity and replicability. Along with techniques such as highperformance liquid chromatography, mass spectrometry, capillary electrophoresis and gamma-ray spectrophotometry, immunological methods arefindinga solid home in the armamentarium of the modern agricultural analyst. Acknowledgments This work was supported in part by grantsfromthe U.S. Department of Agriculture, Millers' National Federation, the American Spice Trade Association, and the Texas Advanced Technology Program. We thank our many industry and U.S. Department of Agriculture colleagues for providing samples, participating in collaborative trials, offering valuable feedback and above all for their encouragement in developing these immunoassays. We also thank Susan Fullilove for her assistance in preparing this manuscript. This article reports the results of research only. Mention of a proprietary product does not constitute an endorsement or a recommendation for its use by USDA. References 1.

Anonymous. FDA technical bulletin number 5, microanalytical procedures manual; Assoc. Off. Anal. Chem.: Arlington, VA, 1994. 2. Russell, G. E. J. Food Protect. 1988, 51, 547-533. 3. Trauba, R. L. J. Assoc. Of. Anal Chem. 1981, 64, 1408-1410. 4. Parker, P. E.; Brauwin, G. R.; Ryan, H. L. In Storage of Cereal Grains and Their Products, Am. Assoc. Cereal Chem.: St. Paul, MN, 1982. 5. Arteman, R. L. USDA Fed. Grain Insp. Serv. Rep. No. 81-22-1; USDA Fed. Grain Insp. Service: Washington, D.C., 1982.


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8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

20. 21. 22. 23. 24. 25. 26.


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U.S. Food and Drug Administration Compliance Policy Guides, Section 7104.06; Office of Enforcement, Division of Compliance Policy, Food and Drug Administration: Washington, D.C. Kurtz, O'D.; McCormack, T. H. J. Assoc. Off. Anal. Chem. 1965, 48, 554558. Schatzki, T. F.; Fine, T. A. Cereal. Chem. 1988, 65, 233-239. Webb, J. C. Ph.D. Dissertation, University of Tennessee: Knoxville, TN, 1973. Webb, J. C.; Slaughter, D. C.; Litzkow, C. A. Florida Entomol. 1988, 71, 492504. Quinn, F. Α.; Burkholder, W.; Kitto, G. Β. J. Econ. Entomol. 1992, 85, 14631470. Schatzki, T. F.; Wilson, Ε. K.; Kitto, G. Β.; Heller, I. J. Econ. Entomol. 1993, 86, 1584-1589. Proske, P. A. Masters' Thesis, University of Texas at Austin, Austin, TX, 1993. Chen, W.-M. Masters' Thesis, University of Texas at Austin, Austin, TX, 1992. Chen, W.-M.; Kitto, G Β. Food Agric. Immunol. 1993, 5, 165-175. Davidson, F. I.; Udagawa, T.; Verdel, E.; Kitto, G. Β. Bee Sci. 1992, 2, 193199. Verdel, E. F. O. Masters' Thesis, University of Texas at Austin, Austin, TX, 1994. Cook, C.; Kao, C. Final Report QUARD-RD-12; USDA Fed. Grain Insp. Service, 1994 Kitto, G. Β.; Quinn, F. Α.; Burkholder, W. Ε. Stored Product Protection: Proc. 6th Internatl. Working Conf. Stored-Product Protection; Highey, E.; Wright, E. J.; Banks, H.J.;Champ, B. R., eds.; CAB International: Wallingford, UK, 1994; pp 415-420. Liquido, N.J.;Chan, H. T.; McQuate, G. T. J. Econ. Entomol. 1995, 88, 8596. Lance, D. R.; Gates, D. B. J. Econ. Entomol. 1994, 87, 1377-1383. Yokoyama, V. K.; Miller, G. T.; Hartsell, P. L. J. Econ. Entomol. 1993, 87, 730-735. Stuart, M. K.; Barak, A.V.; Burkholder, W. E. J. StoredProd.Res. 1994, 30, 9 -16. Stuart, M. K.; Burkholder, W. E. Biol. Control 1991, 1, 302-308. Daly, H. V.; Balling, S. S. J. KansasEntomol.Soc. 1978, 51, 857-869. Sylvester, Η. Α.; Rinderer, T. E. Am. Bee J. 1987, 127, 511-515.

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Immunoassays for Residue Analysis - American Chemical Society

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