Application of immunoassay in the food industry.

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Application of immunoassay in the food industry a

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U. Samarajeewa , C. I. Wei , T. S. Huang & M. R. Marshall

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Department of Food Science and Technology, University of Peradeniya, Peradeniya, Sri Lanka b

University of Florida, Gainesville, FL, 32611–0163

Version of record first published: 29 Sep 2009

To cite this article: U. Samarajeewa, C. I. Wei, T. S. Huang & M. R. Marshall (1991): Application of immunoassay in the food industry, Critical Reviews in Food Science and Nutrition, 29:6, 403-434 To link to this article: http://dx.doi.org/10.1080/10408399109527535

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Food Science and Nutrition

Application of Immunoassay inthe Food Industry

spectrometry. Microbiological examinations involve enrichment, cultivation, and isolation procedures, followed by morphological examination and biochemical testing. Again the testing procedures are time consuming and costly; the analysis in many cases requires highly skilled personnel. Thus, the use for routine surveillance to maintain food quality and safety during food production and processing is limited. Since the analyses must be performed in laboratories, special storage and transport of the samples is necessary. Immunoassays can be used in the food industry to determine the presence of chemical compounds and microorganisms. They have advantages of being low in cost, fast to operate, and having increased sensitivity and specificity compared to conventional detection methods. In addition, immunoassay kits can be used conveniently in the field or within the food-processing facility, thus eliminating the need to transport the samples to laboratories.

U. Samarajeewa, C. I. Wei, T. S. Huang, and M. R. Marshall

Downloaded by [University of Arizona] at 11:57 21 July 2012

ABSTRACT Immunoassay techniques using the highly specific and sensitive nature of immunological reactions have been developed and applied in the food industry for detecting the naturally occurring constituents, antibiotics, pesticide residues, microorganisms, and fragments of microbial constituents related to food analysis, food production, food processing, and food safety. Both polyclonal and monoclonal antibodies are employed for the development of the various immunoassay systems, including enzyme-linked immunoassay (ELISA) and radioimmunoassay (RIA). Immunoassay techniques provide complementary and/or alternate approaches in reducing the use of costly, sophisticated equipment and analysis time, but still maintaining reliability and improved sensitivity. Immunoassay techniques in their most simple forms provide excellent screening tools to detect adulteration and contaminations qualitatively. The application of immunoassay techniques contributes tremendously to the quality control and safety of our food supply.

A. Principles of Immunoassay Immunoassay makes use of the inherent mechanisms in living systems to produce specific antibodies that interact with foreign substances (immunogenic antigens). The entry of antigens into a living system triggers the production of antibodies by lymphoid tissues following recognition of the antigenic characteristics. The specificity of the antigen-antibody interaction (referred to as immunological reaction) is extremely useful for clinical use in infectious disease diagnostics and therapeutic drug monitoring, and, recently, for identification and quantification of food constituents, agricultural chemicals, and microbiological products. A variety of food constituents belonging to the groups carbohydrates, lipids, proteins, and nucleic acids are antigenic and can be estimated by immunoassays through interactions with specific antibodies. However, small molecules such as pesticides, mycotoxins, and antibiotics having molecular weights below 1000 Da rarely act as antigens. Such molecules can act as haptens, and after conjugation with large protein molecules such as bovine serum albumin, they become antigenic and are capable of inducing antibody production in test animals with specific recognition capacity for the hapten-carrier combination, or the hapten only. Thus by using antibodies that bind only to haptens, many of the pesticides, mycotoxins, and therapeutic agents present in foods can be detected.

I. INTRODUCTION The large number of chemical compounds present in foods can either be the natural constituents including carbohydrates, proteins, fats, vitamins, minerals, flavor, color, and even antinutritional components, or additives introduced intentionally to enhance processing. Foods may also contain residues of pesticides, drugs, and hormones used to treat disease or promote growth. Microorganisms are used in food processing for fermentation or preservation. They may introduce nutritionally beneficial as well as harmful compounds into foods. Mycotoxins and enterotoxins are some of the toxic compounds produced by microbes in infected foods. Food chemicals are present over a wide range of concentrations. Although some compounds exist only at picogram levels per gram of food, they may contribute significantly to the characteristics of foods. Detection of these compounds and microorganisms is important for the assessment of the nutritional value and processing characteristics, as well as the quality and safety of foods. Many of the conventional physical and chemical analysis methods for food constituents compiled in the Official Methods of Analysis1 involve cumbersome extraction, concentration, and separation protocols followed by identification and quantitation using sophisticated tools such as chromatography and

C.-I. Wei (corresponding author), B.S., M.S., Ph.D., M. R. Marshall, B.S., M.S., Ph.D., and T.-S. Huang, University of Florida, Gainesville, FL 32611-0163. U. Samarajeewa, B.S., Ph.D., Department of Food Science and Technology, University of Peradeniya, Peradeniya, Sri Lanka.

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Critical Reviews In B. Immunological Methods In the development of immunoassays, food chemical constituents and microbial toxins or cellular structures to be estimated are injected in their pure forms or in conjugation to a relevant protein such as bovine serum albumin, into test animals such as mice, rabbits, or goats to generate the respective specific antibodies. Antibodies used in immunoassays can be polyclonal produced in the sera of immunized animals that are reactive for several antigenic sites; or monoclonal antibodies prepared after fusion of splenic B-lymphocytes obtained from immunized mice with myeloma cells. This is then followed by screening of those clones that secrete antibody of "predefined" specificity (monoclonal).2"4 Antibodies are employed to react with antigens of interest in food. From the specificity of the immunological reaction, the identity of the antigen is determined. Two types of immunological reactions are noted: precipitation is formed when soluble food constituents or microbial toxins are reacted with specific antibodies; agglutination occurs with cellular antigens, including pathogenic microorganisms, viruses, and treated RBCs, following reaction with their respective specific antibodies. Various gel diffusion techniques, including single-diffusion tube (Oudin), microslide double diffusion, microOuchterlony slide, and electroimmunodiffusion are used for detection and quantitation of bacterial toxins, enterotoxins, and soluble food constituents including enzymes. The enrichment serology (ES) technique is used to detect salmonella in food samples using antisera to agglutinate bacteria after selective enrichment.5 The hemagglutination inhibition (HI) and reverse passive hemagglutination (RPH) tests utilize the antigen-antibody agglutination reaction to determine the identity of antigens that in many cases are microbial toxins. Other immunological methods applicable for analysis of foodborne pathogens include the lysis inhibition test (LIT),6 passive immune hemolysis (PIH), 7 latex particle agglutination test (LPAT),8 or the Biken test,9 which detects strains of Escherichia coli that produce heat-labile enterotoxins. The aggregate hemagglutination test was also used to detect enterotoxins of Bacillus cereus.I0 The principles of the specific immunological reaction are applied in the development of immunoassays for quantitation of flavor constituents, hormones, microbial contaminants, microbial and plant toxins, pesticide and drug residues, anabolic agents, enzyme activities, environmental contaminants, adulterants, and misbranding of foods. For convenience and to determine the extent of the immunological reaction (quantitation), antibodies are usually covalently attached to marker molecules, including fluorophores, radionuclides, or enzymes. From the determination of fluorescence intensity of the immunological complex in the fluorescent antibody technique (FA) using a UV-light microscope or fluorescence spectroscopy, or the measurement of radioactivity of the immunological complex in a radioimmunoassay (RIA) using a counter, the

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antigen content in a food sample is determined. In enzymelinked immunosorbent assay (ELISA), antibody is labeled with an enzyme such as peroxidase. Following the antigen-antibody interaction, the substrate for the enzyme is added; from the measurement of the color development using a spectrophotometer, the antigen content is again determined. The enzyme immunosorbent assay (EIA), the enzyme-multiplied immunoassay technique (EMIT), and the indirect enzyme-linked antibody technique (ELAT) are modifications of this immunoassay technique. The chemiluminescence assay uses the antibody bound to a chemiluminescent compound. The overall advantages of the immunological methods over conventional analytical methods are well established.11-12 The reduction in assay time to a few hours or less per estimation compared with several days needed for conventional analyses has allowed greater productivity. This allows increased sampling and rapid movement of food between production and processing, thus further reducing spoilage and quality losses. Sample size can be reduced to a few grams, as opposed to a few hundred grams in conventional analyses. In addition, the laborious cleaning steps required in chemical assays can be eliminated, which further diminishes assay time and expense. The reduced requirement of chemicals and sophisticated equipment has also helped bring down the assay cost by up to 90%. The high recovery rate and reproducibility, and the increased sensitivity at nanogram or picogram levels have made immunoassays substantially more effective than conventional analyses. This article reviews the application of immunoassay methods in the food industry for detecting changes in food constituents during food production and processing, and in assessing food safety through the estimation of levels of environmental contaminants, additives, and pathogenic microorganisms or their toxins.

II. FOOD CONSTITUENTS Immunoassays have been used in the estimation of naturally occurring low-molecular-weight plant constituents that may contribute to the flavor, toxicity, and nutritional characteristics of the food. The techniques can also be used to study the distribution of the constituents inedible tissues during growth. Through the understanding of the production and distribution of the flavor constituents in fruits, a better manipulation of their production and processing could be achieved. Such information may allow alteration to improve quality characteristics of food products. A. Flavor Constituents Limonin is a major compound that contributes to bitterness and an occasional objectionable taste to grapefruit juice. 13 RIA has been shown to detect limonin in Citrus parodist at 0.7 parts per billion (ppb, ng/g). Using RIA, limonin was shown

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Food Science and Nutrition rapidly screen large numbers of potato tubers that contain glycoalkaloids above the tolerance limit of 20 mg/100 g of fresh tubers, as well as for breeding new potato varieties. The ELISA method was shown to give results consistent with HPLC and colorimetric methods in detecting the alkaloids, a-solanine, and ot-charconine in six commercial potato varieties.26 Yams and other root crops are important carbohydrate sources for people living in South American, African, and South Asian countries. However, their use is limited by the presence of the toxic alkaloids, saponins.27 RIA has been applied for detection of saponin ginsenoside RG, in the roots of Panax ginseng.2* This method to quantify saponins may find application in other food crops. RIA has proven useful in estimating the toxic alkaloid scopolamine found in Datura species. The technique detects scopolamine at nanogram levels in contrast to the microgram quantities estimated by TLC and GC methods.29-30 Retronecine and monocrotaline have been used to raise antibodies for their quantitation using ELISA.31 The steroids andrestenone, androgen, estrogen, and progesterone have been studied widely in animals. However, systematic screening of these compounds in plants had not been achieved until the recent development of a RIA method that detects steroids at nanogram levels.32 Of 128 plant species screened, more than half of the species were found to contain at least one of the four steroids. Although the role of the steroids in plants is as yet speculative, their possible contribution as "hormones" in flowering and fruit production, as protective agents in repelling insects, and their relationship to seasonal breeding of animals cast important implications in food production.

to be present at low concentrations in the juice compared with the seeds and embryos.14 This method can thus be used to rapidly screen for trees that produce low levels of limonin naturally, as well as for evaluating the quality (degree of bitterness) of juice. The use of a peroxidase-linked solid phase immunoassay further improves the detectability of limonin to picomole concentrations.15 A comparative study conducted by four laboratories using thin-layer chromatography (TLC), highperformance liquid chromatography (HPLC), RIA, and EIA to quantitate limonin levels in processed orange juice revealed that EIA was more advantageous than chromatographic methods.16 RIA was shown to have good correlation with HPLC for quantification of the flavanone hesperidin in oranges. Hesperidin and its derivatives were detected by RIA at the 2 ppb level. Barthe et al." applied this method to determine the distribution of hesperidin in tissues of mature fruits and found that hesperidin was present in high levels in albedo, membranes, and pith, but in much lower concentrations in juice vesicles and seeds. The distribution of hesperidin in tissues of C. sinensis" was found using RIA to be remarkably similar to that of naringin.18 Naringin is the most abundant flavonoid imparting bitterness to grapefruit products in C. paradisi.18 The results of these two studies using RIA indicated that the distribution of hesperidin and naringin might be almost identical in the two species of Citrus, although this occurrence was, for the most part, mutually exclusive. RIA has been applied in cell culture studies to quantitate isopentenyl adenosine from anise {Pimpinella anisum L.). RIA showed good agreement with GC-MS estimations of the compound.19 Miraculin is responsible for the sweetness of the miracle fruit, Richadella dulcifera. The compound could be detected at the 2 ppb level in fruit extracts, by using ELISA with antibodies raised in rabbits.20

C. Nutritionally Important Constituents RIA has been used to quantitate serum folate levels in rats to determine the bioavailability of folates in foods.33 Because RIA is relatively inexpensive and rapid, and can be used to determine the tissue distribution of folate after ingestion, this method is of physiological significance. RIA has been used to identify food sources rich in pantothenic acid,34 and to determine the pantothenic acid content in nursing home diets.35 In these studies and others,36 adults living in nursing homes were found to consume pantothenic acid at levels lower than the recommended dosage of 4 mg/d. The RIA37 and ELISA38 method for pantothenic acid estimation in foods has been validated against the standard microbiological method.

B. Undesirable Compounds Occurring Naturally in Foods There are at least 6000 plant species worldwide that produce alkaloids.21 Of over 200 individual pyrrolizidine compounds identified, many are shown to exert carcinogenic, hepatotoxic, teratogenic, and mutagenic effects.22 Because humans consume a variety of these plant components, it is important to have rapid methods available for quantitation of these undesirable compounds. Potato varieties contain different amounts of toxic glycoalkaloids. Because these glycoalkaloids are resistant to baking, boiling, frying, and microwave heating,23 potato varieties having the least content of antinutritional glycoalkaloids are selected for food production. RIA has been used to estimate solanidine, the common aglycone unit of potato glycoalkaloids in human plasma at 0.33 ppb.24 The horseradish peroxidaseELISA shown to be the most effective for estimating potato glycoalkaloid content25 has been used by the food industry to

III. FOOD PRODUCTION AND PROCESSING Postharvest problems such as senescence in fruits and vegetables, chemical/biochemical degradation of fruits, vegetables and meat products, and microbial spoilage of foods are major concerns for food production and processing. The prevention

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Critical Reviews In


of these deleterious processes from happening will improve food availability and reduce food prices. Immunoassays have been used to detect and quantitate plant growth substances, enzymes, spoilage microorganisms, and constituents that contribute to quality problems important for food production and processing. A. Plant Growth Substances The understanding of the functional action of plant growth substances in growth and development, in manipulating the quality characteristics of plant products, and in controlling of senescence is of vital importance to fruit and vegetable production and processing. Weiler39 has reviewed the application of immunoassays for determining plant growth regulators; antibodies were first used for such detections in 1972.40 Cytokinins at picomolar levels were detected by RIA after plant extracts were separated by HPLC.41 The HPLC-RIA combination has been applied to elucidate the roles played by endogenous and exogenous cytokinins, as well as contributory factors in controlling bud dormancy in potato tubers.42 Cytokinins, zeatin-riboside, isopentenyl adenosine, 2-(methylthio)isopentenyl adenosine, and several other closely related compounds in Valencia oranges and Red Ruby grapefruit have been detected at 50 pg levels using the HPLC-EIA technique. Both HPLC-RIA and HPLC-EIA were used successfully to monitor changes in citrus cytokinin content during flowering and fruit development.43 Gibberellins A 1; A3, A6, A7, A,, and A26 at picograms per milligram from Hyoscyamus niger L. have been estimated using RIA.44 The use of solid phase EIA further improves the detectability of gibberellins at subpicogram quantities.45 RIA has been used to detect indole-3-acetic acid (IAA) at nanogram quantities46 in a variety of food crops, including maize, broad bean,47 pea,48 and sunflower.49 The RIA method for IAA quantitation in corn shoot extract has been validated by comparing with gas chromatography-selected ion monitoring-mass spectrometry (GC-SIM-MS).50 RIA was shown to provide a reliable estimate of the free, but not the total (free plus ester) IAA content, due to interference. After removal of the interfering compounds from extracts by chromatography, RIA estimates of total IAA in the etiolated corn shoots agreed with that of GC-SIM-MS. Sagee et al.51 developed an ELISA method using polyclonal antibodies that were raised against IAA conjugated to bovine serum albumin (BSA) via indolic nitrogen (IAA-N r BSA), and compared the sensitivity and specificity of these antibodies to those of polyclonal and monoclonal antibodies raised against IAA conjugated to BSA via C| of the carboxylic group (IAAC,.-BSA). The sensitivity of the assay was shown to improve in the following order: monoclonal antibodies > antibodies to IAA-C,.-BSA > antibodies to IAA-N,-BSA. When ELISA methods were used to quantitate IAA in citrus (Citrus sinensis

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L.) shoot tips, the IAA values obtained with all three antibodies were similar. However, for leaf tissues that contained lower amounts of IAA compared with shoot tips, the use of monoclonal antibodies in ELISA gave higher IAA values than using polyclonal antibodies.51 Weiler et al.22 used ELISA to quantify IAA methyl ester at 10 to 20-fmol levels, and IAA at 3 to 4 pg in maize, oat, and avena coleoptiles. The growth inhibitor abscisic acid (ABA) has been detected at picogram levels in wheat leaves using the combined HPLCRIA.52 Walton et al.53 used antibodies obtained from rabbits immunized with ABA bound via its carboxyl group to bovine serum albumin, and developed a RIA that detected ABA at nanogram levels per gram of fresh bean leaves (Phaseolus vulgaris and Vicia faba). Using this RIA, ABA esters were shown to have complete cross-reactivity with ABA, while transABA phaseic acid, and dihydrophaseic acid had much lower but significant cross-reactivities. Overestimation of ABA in plant extracts by RIA could occur due to the presence of the cross-reacting compounds. However, the partition of the free ABA from the interfering cross-reacting compounds by HPLC would enhance the sensitivity and accuracy of the assay. Recently, Quarrie et al.54 applied monoclonal antibody in RIA to detect ABA at 100 to 4000 pg quantities in aqueous extracts of wheat, maize, and lupin leaves. RIA analysis of these samples gave very similar ABA concentrations to those determined by GC after TLC purification, or by GC-MS after HPLC purification. The levels of free ABA and its conjugates in pepper and apple extracts are affected by the physiological condition of the plants. ABA conjugates are present at significantly lower levels than free ABA in unstressed pepper plants, but increase rapidly with stress. Thus, the studies on ABA levels would provide useful information for early prediction of stress on the plants that might affect crop production.55 Studies on the distribution of ABA and abscisyl-B-D-glucopyranoside in citrus fruit (Citrus sinensis) at nanomole levels per gram fresh tissue during growth were achieved using ELISA.56 ABA content was shown to increase slightly in exocarp during fruit maturation. However, the corresponding change in glucopyranoside was shown to increase by 12-fold. This information could be used to help manipulate fruit maturation process. ELISA was also used to determine the levels of cis ( + )-abscisic acid in plants.39 The acid and flavonoid levels in citrus could be affected by manipulating plant hormone levels using tissue culture models.57 The use of highly sensitive immunoassays to detect plant growth regulators and flavor constituents at picogram levels will further help the manipulation process. EIA in general was found to be 100 to 200 times more sensitive than RIA,45 and 100 to 1000 times more sensitive than GC-MS.58 The potential for applying immunoassays in future biotechnology is thus immense.

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Food Science and Nutrition ony count methods to determine mold counts in nuts and spices, Notermans et al.72 found that the values obtained by ELISA were higher because this method estimated both viable and nonviable organisms through the reaction with antigenic polysaccharides. Immunocounting methods could thus be used to provide information on changes of microbial populations in food prior to processing, and for detection of heat-stable toxins.

B. Estimation of Food Constituents That Provide Processing Characteristics Immunoassays are widely used to determine food constituents that impart important processing and quality characteristics to foods. The sandwich inhibition ELISA was used to quantitate in rye the water-soluble pentosans that contribute to good dough characteristics.59 Globulin content in developing Avena sativa L. was determined using RIA; the content increased from 0.32 to 2.37 mg per seed from 2 to 21 d postanthesis, and then maintained at that level up to 30 d.60 Enzyme-conjugated monoclonal antibodies were used to differentiate gluten-containing products such as baked cereal bread or cookie,61-62 and to study the accumulation of gliadin and glutenin polypeptides during development of normal and sulfur-deficient wheat seeds.63 The changes of protein compositions during maturation of wheat grain,64 during germination of soybean seeds,65 and during brewing of barley66 can be monitored using immunoelectrophoresis. Ewart67 employed immunoelectrophoresis and demonstrated that the baking characteristics of the different wheat varieties were related to their protein constituents.

2. Foodborne Bacteria and Molds Immunoassays are used as diagnostic tools to identify spoilage microorganism in foods and food crops; the conventional methods still provide certain advantages over immunoassays. Many foodborne fungi could be identified by examining the morphological characteristics of the spores and mycelia using light microscopy. Because the antibodies raised against the fungal hyphae or the culture supernatants lack the specificity important for identification of the microorganism, more specific antigens such as enzymes, toxins, or purified extracellular polysaccharides are used for antibody production. Dewey73 has reviewed the recent developments of fungal immunodiagnostic methods for plant pathogens. Table 1 summarizes some of the immunoassays applicable for identification of spoilage microorganisms in foods and food crops.

C. Spoilage of Foods and Food Crops Food spoilage due to microbial activities and parasitic organisms during preharvest, harvest, and postharvest stages of food production can sometimes account for 20% losses of the food. Food spoilage is induced by a variety of molds and bacterial species. Viral infections of food crops can lead to losses and/or reduced productivity. The presence of parasites in animal foods makes the flesh unsuitable for consumption and adds to food losses. Immunoassays can be used to help control and prevent food spoilage by early detection of the causative organisms and by estimating their populations. These harmful organisms cannot be easily detected by physical examination, especially at their initial stage of growth.

Table 1 Immunoassay for Detection and Identification of Spoilage Microorganisms in Food Crops Microorganism Erwinia spp. Fusarium spp. Humicola lanuginosa Legionella pneumophila Ophiostoma ulmi Phytophihora megasperma Pseudocerocosporella herpotrichoids Pseudomonas syringae Rhizoctonia solani Xanthomonas campestris

1. Mold Counts Antigenic compounds present in mold extracts can be used to raise antibodies for immunofluorescent assays68 or ELISA69 to determine mold counts. The ELISA test applied for mold counting in tomato puree was 100-fold more sensitive than the HPLC method.69 Although the constituents of the tomato puree interfered with the ELISA test, prior washing of the puree would help eliminate the interference.70 The ELISA test only took 5 to 10 h to operate when compared with a few days needed for conventional culture methods. The test time could be reduced further to 10 to 20 min using the latex agglutination assay. However, this method was five- to tenfold less sensitive than ELISA. Agglutination method was shown to detect all Aspergillus and Penicillium sp. with the exception of P. rubrum.11 In a comparison study using ELISA and three different col-

•

Method

Crop

Ket

Western blotting ELISA ELISA Immunoblotting ELISA ELISA ELISA

Potato Cultures' Rice Water Cultures Soybean Wheat

74 297 77 80 298 78 73

ELISA ELISA ELISA Dot-immunobinding

Pea Soybean Citrus Sorghum

79 78 299 76

Tested with microbial cultures grown in synthetic media.

Monoclonal antibody raised against the pectate lyase of Erwinia sp. was very specific and did not cross-react with the pectate lyase obtained from other common foodborne microorganisms belonging to Aspergillus, Xanthomanas, and Pseudomonas species.74 However, immunoglobulin G prepared from the rabbit antiserum against the extracellular heat-stable proteinase from P. florescens AH-70 was found to interact with 8 of the 26 psychrotrophic Pseudomonas spp. isolated from Spanish refrigerated raw milk using various immunoassays.75 Thus, if immunoassays are to be used to detect and quantify

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Critical Reviews In proteolytic proteinases in refrigerated raw milk, antibody with broader specificity should be used. Dot-immunobinding assay was used to identify the infectious agent of "bacterial streak". Using this method, the Xanthomonas sp. responsible for "bacterial streak" can be distinguished from the Pseudomonas sp. that causes in sorghum "bacterial stripe", a disease with similar visual symptoms as "bacterial streak".76 ELISA, dot-blotting (DOT-BLOT) and dip-stick method have been applied to detect yellowing disease in rice during drying and storage.77 Miller et al.78 used the double antibody sandwich ELISA tests to detect Phytophthora megasperma f. sp. glycinea in roots and stems of artificially inoculated and field-grown, naturally infected soybean plants and Rhizoctonia solani in naturally infected soybean stems and roots. These immunoassay kits can be used to diagnose fungal diseases in crop plants and provide information about pathogen identity; therefore, disease management inputs such as fungicide application can be selected and timed more effectively. The use of immunoassays to determine the setting of Erwinia carotovora subsp. carotovora at an early stage of potato tuber decays,74 and Pseudomonas syringae for pea blight in pea seeds79 was not completely successful unless prior enrichment was applied. Selective media were used to inhibit the interfering bacteria in water before monoclonal antibodies were employed for Legionella pneumophila detection.80 The presence of sulfate-reducing bacteria in canned vegetables and molasses can also be detected by ELISA.81 3. Viruses Once viral infection occurs, very little can be done to treat the food crops. Early diagnosis of viruses in seeds thus helps in avoiding food losses. Table 2 summarizes some of the im-

munological methods that can be used for viral detection in plants. The methods can be used to study the taxonomic status of viruses capable of infecting several crops and to help understand the interrelationships among viral strains of different geographic and plant origins.82 4. Diseases in Animal Foods Meat and fish are potential sources of pathogenic microorganisms. Animal foods could harbor parasites that use the animal or the human as an intermediate host in completing their life cycles. Immunoassays using the relevant antibodies can be employed to detect the presence of such undesirable organisms in animal foods prior to slaughter. The presence of the parasite Toxoplasma gondii in sheep and rabbit was detected by carbon immunoassay,83'84 and in cattle and pig by ELISA.85 EIA was used to detect Trichinella spiralis in swine86 and in meat.87 Immunoassays are applied to detect Mycobacterium paratuberculosis in goats,88 Brucella in cattle,89 and Stephanuras dentatus in pigs.90 Early diagnosis of the unhealthy condition in animal foods could avoid potential health hazards to humans and economic losses to the meat production industry. D. Food Processing Immunoassays are used to identify important constituents and microorganisms encountered during food processing. Microorganisms play important roles in imparting the desirable organoleptic and nutritional characteristics to fermented foods. Immunoelectrophoresis and immunoadsorption are used to elucidate the surface structure of the slime-forming encapsulated Streptococcus cremoris used in fermented milk, villi. An understanding of the surface structure and thereby the characterization of the microorganism helps in the development of the

Table 2 Immunoassay for Detection of Viruses in Food Crops Method

Virus Beet necrotic yellow vein virus Cauliflower mosaic virus Citrus Tresteza virus Cucumber mosaic virus Elongated potato virus Isometric plant viruses

ELISA RIA Immunodiffusion Immunodiffusion Immunodiffusion Immunodiffusion

Pea seed bome mosaic virus Plant viruses Potyviruses

Immunodiffusion EBRIA Immunoblotting

Soybean mosaic virus Zucchini yellow mosaic virus

RIA Immunodiffusion

Note: EBRIA — electro-blot radioimmunoassay.

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Food

Ref.

Beet Cauliflower Citrus Tobacco Potato Cowpea Cucumber Bean Squash Pea Plants Maize Sorghum Sugar cane Soybean Squash

300 301 302 303 304 305

306 307 82

308 309


Food Science and Nutrition detect low concentrations of enzymes encountered in industrial fermentation.99

more reliable starter cultures for fermentation and the establishment of resistance in fermented milk against the growth of spoilage and pathogenic bacteria.91 Immunosasays are used to detect immunoglobulins that react specifically with pathogenic microorganisms. These immunoglobulins can be introduced to processed foods to enhance resistance parameters. Shimizu et al.92 suggested the incorporation of an anti-£. coli immunoglobulin isolated from immunized chicken into human infant formulae. Immunoblotting was applied to study the effects of heating, roasting, and blanching on the allergic constituents in almonds. The binding ability of the high-molecular-weight proteins (>50,000 Da) to sera from eight allergic individuals was affected after heat treatment.93 Immunoassay can thus be used to determine processing heat parameters required to eliminate food allergens. Immunoassays are employed to rapidly check the activities of enzymes used for processing in modern food production technology (Table 3). The specificity of the immunological reaction has become useful for distinguishing isoenzymes of the mitochondrial or cytoplasmic malate dehydrogenase of yeasts,94 polyphenoloxidases in different varieties of beans,95 and Kunitz trypsin inhibitor in soybeans.96 Immunoassays are employed in understanding the enzymatic inactivation of malate dehydrogenase,94 in recognizing the denatured and native forms of p-amylase from mustard,97 and in locating lipase activity in germinating grape (Brassica napus L. var. Mikado) seeds.98 Immunostaining is employed after rocket immunoelectrophoresis to detect low concentrations of Aspergillus oryzae ot-amylase used to convert starch to glucose during brewing. This technique detects the precipitates that cannot be revealed by Coomassie brilliant blue stain; it can be used to

IV. FOOD SAFETY Immunoassays have been explored extensively for application in detecting and estimating food additives and contaminants that render food unsafe. Immunoassays are also used for detection of pathogenic and toxigenic microorganisms, their toxins, residues of pesticides, antibiotics, anabolic agents, and food adulterants. A. Pathogenic Microorganisms and Their Toxins The change in food consumption patterns as well as the shift to novel foods that receive minimal heat treatments have raised greater concern on the safety of food in relation to the presence of pathogenic and toxigenic microorganisms. The conventional methods applied for identification of the microorganisms or toxins associated with foodborne infections and intoxications employ test animals. The results obtained using test animals are less uniform due to biological variability of animal models. The methods are protracted, involve high cost, and are open to objections resulting from the growing concerns of animal rights groups. Immunoassays overcome these limitations and objections almost totally and meet the required sensitivity. 1. Clostridium perfringens Enterotoxins The sensitivity of some of the immunoassays applicable for estimation of C. perfringens enterotoxins is listed in Table 4. Among them, the counter immunoelectrophoresis (CIEP) carried a higher sensitivity of 0.2 fig/ml compared with the electroimmunodiffusion or the single cell differentiation method.100

Table 3 Application of Immunoassay for Assessment of Enzyme/ Inhibitor Activity in Foods Enzyme/inhibitor

Method

a-Amylase p-Amylase Catalase inhibitor Chymotrypsin Debranching enzyme Lipase Malate dehydrogenase Papain Pepsin Polyphenoloxidase

Immunoelectrophoresis Immunodiffusion RIA Immunodiffusion Immunodiffusion ELISA Agglutination

Brew Mustard Maize Rennet Barley Rape seed Yeast

99 97 310 311 312 98 94

RIA Immunodiffusion Immunoblotting

313 311 95

Proteolytic Trypsin Trypsin inhibitor

Immunodiffusion RIA ELISA

Beer Rennet Soybean, pea Mung bean Bush bean Protein hydrolysate Human milk Soybean

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Ref

314 315 96

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Critical Reviews In Table 4 Application and Sensitivity of Immunoassay for Clostridium Toxins

Method


Toxin

RPPH PH

Sheep, cattle Sheep, cattle Meat extract Foods Stools Foods Feces Fish Cultures Cultures Foods Foods

C. botulinum toxin-G C. botulinum toxin-A

EUSA Amplified EUSA

Human tissue Salmon, beef

C. botulinum toxin A C. botulinum toxin-A C. botulinum toxin-A

Amplified EUSA Amplified EUSA RIA

Pork Pork Can-fish

C. botulinum toxin-A

RIA

Pure toxin

C. perfringens enterotoxin E C. perfringens enterotoxin E C. perfringens enterotoxin A C. perfringens enterotoxin A C. perfringens enterotoxin A C. perfringens enterotoxin A C. perfringens enterotoxin A C. botulinum toxin C. botulinum toxin-A C. botulinum toxin-A, B, E, F C. botulinum toxin-A C. botulinum toxin-A, B, E

RPPH

Specimen

SRI CIEP

RIA EUSA EUSA RPLA CPID

GI GI

Minimum detection level

Ref

6 pg/ml 0.1 mg/ml 0.2 ng/ml 1.0 ng/ml — 5 ng/g 2 ng/g 2.5 x MLD Presence Presence 27 x mouse LDso/ml 1-few X mouse LDx/ml 1 i.p. mouse LD M 5—10 x mouse

106 106 100 103 104 105 316 110 107 108 111 112

33 x mouse LDVml 20 x mouse IDs/ml 1 ng/ml = 80 x mouse LD^/ml 100 x MLD

114 116 119

117 113

118

Note: CIEP — counter immunoelectrophoresis, CPID — capillary tube immunodiffusion, ELISA — enzyme-linked immunosorbent assay, GI — gel immunodiffusion, MLD — mouse lethal dose, PH — passive hemagglutination, RIA — radioimmunoassay, RPLA—reverse phase latex agglutination, RPPH — reverse phase passive hemagglutination, and SRI — single radial immunodiffusion.

Although CIEP did not meet the sensitivity of the rabbit ileal loop test that detected the enterotoxin at 1 to 2 ng/ml, this method could be applied to study the relationship among growth, sporulation, and toxin production by C. perfringens.101 The higher sensitivity of the RIA to detect enterotoxin at 1 to 2 ng/ ml made the method useful for distinguishing the toxigenic from the nontoxigenic strains and thus help identify toxic foods.102 RIA was applied to show that only one toxic compound is produced by C. perfringens.IM ELISA tests with high specificity and sensitivity were developed to detect C. perfringens enterotoxins in human stools associated with gastroenteritis.104 An ELISA test that detected the toxins at 5 ng/g of feces was applied by Bartholomew et al.105 to check for toxins in food. The reverse phase passive hemagglutination and the single radial immunodiffusion were used to estimate epsilon-antigen of C. perfringens type D in cattle and sheep.106 2. Clostridium botulinum Toxins Although a variety of immunoassays are developed for detecting C. botulinum enterotoxins (Table 4), many of them do not carry the sensitivity comparable with that of the mouse bioassay. Immunoassays detect specifically the botulinum toxin

410

alone, while the mouse assay determines toxicity that may not be related exclusively to botulinum toxin. The immunodiffusion method used to detect toxigenic colonies of C. botulinum type A107 was improved and used to detect neurotoxins A, B, E, and F, either individually or collectively.108 The improved method was less cumbersome and more rapid compared with the mouse neutralization test or the anaerobic culture techniques. However, the low correlation between the size of the precipitation rone and the toxicity of the samples made immunodiffusion method less reliable for toxin quantitation. Immunodiffusion can be used to screen C. botulinum colonies in mixed cultures.I09 Although the capillary tube immunodiffusion method detected botulinum toxins in canned fish at 2.5 to 5 times the minimum lethal dose (MLD); cross-reaction of the antibody by nontoxigenic sample extracts made the method less dependable.110 However, this method is still useful for initial screening of toxic foods. The reversed passive hemagglutination technique (RPHA) is reliable; it shows excellent correlation with the mouse assay in detecting botulinum type A toxin in foods, although its sensitivity is only equivalent to 27-fold mouse LD50/ml.IH Passive hemagglutination test had a sensitivity equivalent to a few mouse LD50112 (Table 4).

Volume 29, Issue 6


Food Science and Nutrition Table 5 Application and Sensitivity of Immunoassay in Detecting Staphylococcal Enterotoxins in Foods

Since botulinum toxin is very potent, immunoassays used for detection should be highly sensitive. The amplified ELISA test employing monoclonal antibodies detects the toxins at levels 5- to 10-fold mouse LD5O/ml;113 it shows no cross-reactivity with C. botulinum toxins B or E or several other bacterial toxins examined. The assay time is only 6 h. Gibson et a l . m applied the amplified ELISA method to check 144 samples (including 45 cultures and 95 slurries). The results were in very good agreement with that of mouse assay; no false positives were found in this study. The amplified ELISA method using a monoclonal antibody raised against type B toxin115 showed disagreements with mouse lethal assay only at toxin concentrations below the 20-fold mouse LD50 of toxin B per milliliter.116 An ELISA test was used to detect C. botulinum enterotoxin G in five cadavers of humans with sudden unexplained deaths.117 The method detected the toxin at less than one mouse LD50 and was more sensitive than the ELISA tests used to assay toxin types A, B, and C. The ELISA method may be applicable to detect botulinum toxin G in foods. RIA 118119 was comparably less sensitive than ELISA; it is 80- to 100-fold less sensitive than the mouse bioassay. RIA is used less frequently for detecting botulinum toxins in foods (Table 4). C. botulinum toxins are sensitive to irradiation at dosages higher than that normally used in foods. Tranter et al.12° studied the effects of irradiation on the loss of biological and irnmunological activity of C. botulinum neurotoxins and found that the treated toxins still retained high immunological activity, although the biological activity was almost totally lost. The use of immunoassays to assess residual immunological activity of the irradiated enterotoxins will provide a better margin of safety than the determination of the loss of biological activity alone.

Toxin type B B B B A B C D E A A B A, B, C, E A A, B, C, D A B

Method

Specimen

Minimum detection limit

RIA RIA RIA RIA RIA RIA RIA RIA RIA RIA ELISA ELISA ELISA ELISA ELISA ELISA MEIA

Pure toxin Milk, cheese Nonfat dry milk Hamburger Foods Boiled egg Pudding Foods Cheese Foods Food extracts Custard Minced meat Foods Foods Sausages Foods

1—5 ng/ml 1 ng/ml or g 2.2 ng/ml 6.3 ng/g 0.66—1.2 ng/g 0.63 ng/g 1.3 ng/g 0.67—0.84 ng/g 1.0 ng/g 0.3 ng/ml 2 ng/ml 1 ng/g < 5 ng/g 0.4—3.2 ng/g 0.1 ng/ml 0.025 ng/g 1 ng/g

Ref 122 309 126 126 127 127 127 127 128 128 130 317 318 131 133 141 136

Note: MEIA — magnetic enzyme immunoassay.

proach, enterotoxin as little as 1 ng/g of food can be detected in food extracts using RIA. Dickie and Akhtar128 used the better defined tracers and different incubation conditions and improved the detectability of the RIA to 0.3 ng/ml. However, a collaborative study conducted in low laboratories using 378 samples showed considerable interlaboratory imprecision; the sensitivity of the RIA was found to be at 2 ng.129 The ELISA test capable of detecting staphylococcal enterotoxins A, B, C, and E in aqueous extracts from minced meat, a 40 to 80% recovery with no false positives or false negatives was noted in spiked samples. However, the recovery was reduced to only 0.14% when the same extraction procedure was applied for heat-treated minced meat; gelatin in minced meat appeared to interfere with enterotoxin detection. The sensitivity of the ELISA test can be improved using double-antibody solid phase assay.131 The application of indirect double sandwich ELISA using monoclonal antibodies was shown to increase the detection range to include toxin types A, B, C, and D in foods.132 In a comparative evaluation study among the competitive ELISA with polystyrene spheres, the sandwich ELISA with labeled antibody, the inhibitor test with labeled antibody, and the inhibitor test with unlabeled antibody in estimating enterotoxins A, B, C, and D in foods, Fey et al.133 showed that the sandwich ELISA with labeled antibody was the best, having a sensitivity of 0.1 ng/ml. They also showed that for sandwich ELISA, the use of monoclonal antibody was better than polyclonal antibody in giving more reliable results for spiked sausages. Freed et al.134 and Thompson et al.' 35 also showed that the monoclonal antibody was a better substitute

3. Staphylococcus aureus Enterotoxins Among the various immunoassays used for detection of S. aureus enterotoxins in food, RIA and ELISA are used more frequently. Table 5 lists the sensitivities of the RIA and ELISA in estimating staphylococcal enterotoxins in various foods. RPHA121 and solid phase RIA122123 were used earlier; however, a comparison of these two methods revealed that RPHA was unreliable.124-123 Niyomvit et al.126 also showed that affinity RIA was 20 to 60 times more sensitive than the single gel diffusion tube, double gel diffusion tube, Laurell electrodiffusion, and microslide techniques for detecting staphylococcal enterotoxins. Bergdoll and Reiser127 used low concentrations of staphylococcal enterotoxin, chloramine-T, and 125I for iodination of the toxin. Antigen-antibody complex was separated from the unreacted enterotoxin by antibodies adsorbed onto tubes or bromoacetyl-cellulose or by precipitation of the complex with a second antibody or protein A cells (formaldehyde-treated and heat-inactivated S. aureus cowan 1 strain). Using this ap-

1991

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Critical Reviews In for polyclonal antibody in ELISA tests. The monoclonal antibodies in RIA, however, did not perform as well when compared with polyclonal antibodies.78 Immunoassays are further improved for higher sensitivity and convenience. Polyacrylamide agarose beads with entrapped iron oxide particles for ease of separation were used in magnetic enzyme immunoassay (MEIA). Although the method is highly sensitive (Table 5), variation in recovery rate makes the estimation semiquantitative.136 Nonenterotoxin specific antibodies were used prior to the MEIA to eliminate the interfering protein A and prevent false positives. Protein A can also be eliminated by passing the enterotoxin extract through an affinity column with rabbit immunoglobulins coupled to a solid matrix.137 Enzyme amplified ELISA was shown to have increased sensitivity for staphylococcal enterotoxin.138 Monoclonal antibody-based immunoassays have demonstrated improved sensitivity and detectability. Monoclonal antibodies capable of interacting with all the five serotypes of staphylococcal enterotoxins139 and detecting enterotoxins in heat-processed foods140 are developed. Immunoassays appeared reliable enough in helping the control of suspicious raw materials and food products.141 This diagnostic kit has been used to help in understanding the relationships between the biochemical characteristics and enterotoxigenicity of staphylococcal strains isolated from sheep milk.142 However, further studies are needed to clarify the discrepancies observed in detection of enterotoxins in aviation meals using ELISA and microslide immunodiffusion test,143 and the low agreement noted with ELISA and reversed passive latex agglutination (RPLA) in detecting enterotoxins.144 The emetic effects observed in monkey test with extracts of S. aureus strains, which were found to be negative for enterotoxins when tested by ELISA, suggests the possible presence of a new enterotoxic factor.145 The modification of the single radial immunodiffusion technique as an economical method for detection of toxigenic strains merits further studies.146 4. Salmonella The development of immunoassays for Salmonella detection has received much attention in the last 2 decades. Salmonella are human pathogens living in the alimentary canal of humans and animals. Being a fecal microorganism, they are able to contaminate foods of animal origin and thus pose major public health problems. The conventional culture methods for Salmonella estimation are cumbersome; they involve several enrichment steps for selection and enhancement of the microorganism to detectable levels. The procedures take up to 4 d. Since the bacteria grow fast in perishable animal food, and storage of suspicious food for clearance of safety is costly, it is prudent to develop rapid identification methods for Salmonella in food. The high demand for rapid, inexpensive, and reliable assay systems for Salmonella detection has resulted in worldwide research on development of immunoassays. Several

412

reviews have highlighted the early research on this development.147150 EIA techniques for Salmonella detection have received the most attention. A comparison of the efficiency for Salmonella detection using immunoassay and conventional culture methods is presented in Table 6. The replacement of the final detection step in conventional culture method by direct EIA reduced the total test time from 5 to 7 d to 3 d. Table 6 A Comparison of Immunoassay Methods with Cell Culture Methods for Detection of Salmonella in Foods

Method/kit" Direct EIA EIMA RIMA and EIMA Enzabead" Enzabead" Enzabead" Enzabead" Enzabead* ELISA1 Bactelisa" TECRA" TECRA" 1-2 Test1 1-2 Test" 1-2 Test' Modified 1-2 Test"

Number of samples tested Immunoassay Cell culture Ref. 48 82 235 120

26 45 91 1289 45 46 572 173 220 186 196 314

22 24 20 63 17 35 76 594 28 45 60 104 94—102 45 26 82

23 24 19 50 24 3241 570 32 44 59 85 125 43 34 81

157 151 160 166 319 155 153 320 155 319 285 154 156 286 287 287

Note: EIMA = enzyme-immunometric assay; RIMA = radioimmunometric assay. • Commercially available immunoassay kits.

Immunoassays are used to examine bacterial cultures at preenrichment, selective-enrichment, and postenrichment stages, and the results correlated with those obtained by culture methods. Erroneous results, mostly arising from false positives, are observed when enzyme immunometric assay (EIMA)151 or Bio-Enzabead kit152153 is used to check for cultures at preenrichment stage, or for cultures enriched for only 6 to 8 h using the TECRA test kit.154 Immunoassays when used to check for cultures at the late selective enrichment stage does not appear to improve the detection accuracy of immunoassays in nonfat dry milk.155-156 The elimination of the centrifugation step for EIA simplifies test procedures.157-158 Modification of the immunoassay kit itself would improve the efficiency and sensitivity of the assay. The coating of proteins onto the plate, the labeling of the antibody, and the introducing of accelerators for antibody-antigen reactions and enzyme-substrates reactions all improve the efficiency of im-

Volume 29, Issue 6


Food Science and Nutrition and Canada174 have developed monoclonal antibody-based immunoassays for detecting Listeria monocytogenes. The ELISA system developed in the U.S., named Listeria-tek, has been tested with milk and meat. The EIA method from Canada175 is reported to be under scrutiny for commercial exploitation, while the direct immunofluorescence test (DEFT) from the U.K. is used to check Listeria in cheese. These immunoassays reduce the testing duration from 1 month to about 2 d. The validity of the methods is as yet to be established.

munoassays.159 Using the best combinations, Salmonella at 105 to 106 per milliliter of a 24-h enrichment culture can be detected using an ELISA. Most Salmonella immunoassays in general detect Salmonella at about 106 cells per milliliter. Salmonella at concentrations of 105 per milliliter or less always yielded negative results using a direct ELISA test.157 The use of elevated incubation temperatures is also shown to yield better results when the immunoassay kit is used.155 The use of titanous hydroxide as the solid phase immobilizer for interfering microorganisms improved the sensitivity by 100- to 160-fold;160 titanous hydroxide has been used as the immobilizing agent in EIMA and RIMA.161 Mattingly and Genie162 also described horseradish peroxidase as a more convenient enzyme than alkaline phosphatase for color detection in sandwich ELISA for Salmonella. Of the two types of Salmonella antigens used to raise antibodies, the flagella extracts appear to produce more specific antibodies than cell wall polysaccharides.150 Emswiler et al.163 used a monoclonal antibody developed by Robison et al.164 to detect Salmonella in meat products; the results agreed better with that of the culture method. A monoclonal antibody that would recognize the lipopolysaccharide extract of Salmonella has been developed recently.165 Several commercial kits have been examined extensively for their reliability in detecting and estimating Salmonella in different foods (Table 6). When spectrometric methods were employed to determine immunoassay results, it was found that various foods would give different baseline values.135166 Validation of EIA methods and the test kits against culture methods to standardize the baseline values for at least similar food groups is thus important. Extensive collaborative studies have been performed to evaluate the efficiency of the various immunoassays in detecting Salmonella in hundreds of food samples. Fluorescent EIA is recommended for rapid screening in food,167 immunodiffusion for motile Salmonella in food,168 EIA169 and the abbreviated EIA158 for Salmonella in low-moisture foods. It only takes 4 h to conduct the abbreviated EIA test. An abbreviated enrichment followed by a rapid immunodiffusion method has been shown to provide close agreement with that obtained by culture methods.156 However, further examination of this method is needed to determine the applicability to a variety of foods. Reasonably good agreements are achieved using immunoassays and cultural methods to detect Salmonella (Table 6). However, the occurrence of false positives and false negatives continues to be a limitation.170 The 76 positive samples detected in crustaceans using EIA were not confirmed by culture methods; cross-reacting microorganisms are probably present.171 Further studies are needed to improve the reliability of immunoassays for estimating Salmonella in foods originating from varying ecological environments.

6. Other Microorganisms Immunoassays are used to detect other pathogenic microorganisms in foods. These include the application of EIA to detect Escherichia coli enterotoxins,176 RIA for E. coli enterotoxins and cholera toxins,177 ELISA for Yersinia enterocolitica in milk, 178 the immunoassay employing monoclonal antibodies to detect Enterobacteriaceae in drinking water,179 the enzyme capture assay for E. coli in oysters,180 and the biotype and serotype correlations of Campylobacter jejuni and E. coli in chicken and in patient stool samples.181 7. Mycotoxins Conventional analyses of mycotoxins are time consuming and tedious; these include extraction, separation, and clean-up steps followed by estimations based mostly on chromatography. Immunoassays, by eliminating some of these steps, decrease assay time to 10 to 15 min. ELISA is shown to be sixfold faster compared with HPLC analysis.182 Special cartridges can be used when required for rapid cleanup of extracts. The cost of using ELISA to quantitate mycotoxins has been estimated to be only 6 and 2% of gas chromatography and HPLC, respectively. Mycotoxins are small molecules and are therefore nonimmunogenic by themselves. They require conjugation with a relevant protein to elicit antibody production. Two recent reviews highlight the principles about immunoassay development183 and its application for mycotoxin detection in food.184 Both RIA and ELISA detect mycotoxins at parts per billion levels. ELISA has been shown to be slightly more sensitive than RIA in estimating the levels of several mycotoxins in foods (Table 7), and to provide more consistent results and better accuracy in estimating aflatoxin I*! (AFB,) in corn, wheat, and peanut butter.185 The recoveries of spiked AFB, are low in wheat and corn when RIA is employed for analysis.186 The cleanup of milk extract led to increased sensitivity of RIA analysis of AFB, in whole milk.187 Since coumarins, caffeic acid, chlorogenic acid, and vanillin at parts per thousand (ppt) levels appear to simulate AFB, at parts per billion concentrations in RIA tests,188 false positive results could occur and contribute to erroneous high estimates of AFB, as noted with coffee beans. Excellent agreement has been observed between ELISA and HPLC or TLC in estimating aflatoxins in many foods.189"191 Interlaboratory collaborative studies have led

5. Listeria monocytogenes Three independent groups residing in the U.S., 172 U.K.,173

1991

413

Critical Reviews In Table 7 Application and Sensitivity of Immunoassay for Mycotoxins in

Foods

Mycotoxin


Aflatoxin b, Aflatoxin b, Aflatoxin b, Aflatoxin b, Aflatoxin b, Aflatoxin b, Aflatoxin B + G, Aflatoxin B + G, Aflatoxin B, diol Aflatoxin M, Aflatoxin M, Aflatoxin M, Aflatoxin Q, Ochratoxin A Ochratoxin A . Ochratoxin A Ochratoxin A Ochratoxin A Ochratoxin A Ochratoxin A Ochratoxin A Ochratoxin A Ochratoxin A Ochratoxin A Ochratoxin A Ochratoxin A T-2 toxin T-2 toxin T-2 toxin T-2 toxin T-2 toxin T-2 toxin T-2 toxin T-2 toxin T-2 toxin T-2 toxin T-2 toxin T-2 toxin 3'-OH-T-2 toxin T-2 tetraoltetracetate HT-2 toxin Group A Tnchothecenes Group A Tnchothecenes Roridin A Diacetoxyscirpenol Diacetoxyscirpenol Deoxynivalenol Deoxynivalenol 3-Acetyl deoxynivalenol Deoxyverrucarol Zearalenone Zearalenone Zearalenone Zearalenone

414

Method ELISA RIA • RIA ELISA ELISA ELISA ELISA RIA ELISA ELISA RIA ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELISA RIA RIA RIA RIA ELISA ELISA ELISA RIA ELISA ELISA . RIA ELISA EIA ELISA RIA RIA ELISA RIA RIA RIA RIA ELISA EIA EIA ELISA RIA ELISA ELISA RIA ELISA ELISA ELISA ELISA

Sample Com, wheat, peanut butter Corn, wheat, peanut butter Peanut butter < Com, cottonseed Barley Peanut butter Extract Extract Milk Milk Milk Urine Wheat Animal tissue Meat, wheat, plasma Barley Barley Biologic fluids Pig kidney Barley Serum Kidney Extracts Barley Wheat Corn, wheat Urine, milk Wheat flour Culture extract Corn, wheat Urine, milk Culture extracts Com Urine, serum Culture extracts Barley Animal tissue Urine Urine Pure compound Pure compound Feeds Culture extracts Wheat Wheat, corn Extract Rice Culture extracts Com, wheat, feed Grain-based foods Com Com

Volume 29, Issue 6

Minimum detection limit 3ng/g 5.8 ng/g 0.5 ng/assay 2.5 ng/g 0.5 ng/ml 0.1 ng/ml 0.25 ng/g 1 ng/ml 1 pmol/assay 0.25 ng/g 0.50 ng/g 0.25 ng/g 2 ng/ml 1—2 ng/g 12.5 ng/ml 1 ng/g 5 ng/g 0.06 ng/g 1—2 ng/g 25 pg/assay 0.5 ng/g 0.5 ng/g 0.4 ng/g 0.2 ng/g 1—2 ng/ml 1 ng/ml 2.5 ng/g 5 ng/g 2.5 ng/ml 0.5 ng/g 0.1 ng/g 2.5; 0.1 ng/g 0.2—1 ng/g 50 ng/assay 50 ng/g 10 ng/ml 1—20 ng/ assay 10 ng/ml 0.1 ng/assay 0.5 ng/assay 0.1 ng/assay 0.1 ng/assay 0.05 ng/assay 5 ng/ml 16 ng/ml 300 ng/g 20 ng/g 0.2 |xg/ml 1 ng/g 25 ng 1 ng/ml 2.5 ng/ml 0.5 ng/g 1 ng/ml

Ref. 185 185 186 189 190 321 322 196 194 187 187 190 195 323 324 202 203 325 326 200 327 328 204 201 326 321 329 329 207 205 330 331 332 208 333 334 335 321 206 212 212 210 210 336 211 337 215 209 338 339 340 213 341 342



Table 7 (continued) Application and Sensitivity of Immunoassay for Mycotoxins in Foods

Mycotoxin

Method

Sample


Zearalenone Zearalenone Sterigmatocystin Sterigmatocystin Rubratoxin B PR toxin

RIA ELISA ELISA ELISA RIA RIA

Sera Urine Culture extract Barley Culture extract Cheese

5 ng/ml 10 ng/ml 25 ng/ml 10 pg/well 0.1 fig 50—100 jJLg/g

Ref. 343 344 345 346 347 348

clonal antibodies, and cross-reactivity still occurred. Crossreaction was applied to simultaneously estimate T-2 and HT2 toxins in spiked urine using ELISA test.212 Of the immunoassays developed for zearalenone, sterigmatocystin, and PR toxin (Table 7), the assay for zearalenone has been employed in a study to survey grains.213 In validation studies, agreement has been observed between immunoassay and radiochromatography in estimating T 2 toxins in rat tissues,214 and between TLC and RIA in estimating deoxynivalenol in com and wheat.215 In general, the use of immunoassay for mycotoxin analysis will reduce the test time to 10 to 15 min and increase the sensitivity to parts per billion levels. The cleanup of the food extracts usually increases the assay sensitivity. The success of the collaborative studies, and the acceptance of several immunoassay methods as official first action by the AOAC, indicate high prospects of applying immunoassays to quantify mycotoxins in foods.

to the acceptance of ELISA as an official method by the AOAC191 and for commercial use as a test kit.192 Many commercial kits are available for aflatoxin estimation in foods. The development of monoclonal antibodies for aflatoxins has increased the sensitivity of the ELISA test. 19° A monoclonal antibody raised against AFB, has been used in affinity columns to clean up the extract; the purified mycotoxin was detectable at 0.5 ng by HPLC or 5 ng in a visual spot test. This screening method only takes 10 min.193 Antibodies raised against aflatoxin dihyrodiol and AFQ, are used in ELISA to trace AFB, degradation products in processed foods or aflatoxin metabolites in biological systems.194-195 A generic type of antibody raised against AFB3 was capable of detecting AFB, and AFG, together in RIA at 1 to 100 ng/ ml concentrations.196 Since AFB, and AFG, in foods were estimated simultaneously, the cost and duration of analysis were reduced. The use of nylon beads or terasaki plates to bind antibodies was shown to be more economical than the use of microtitre plates.197 The indirect immunoperoxidase localization method used for microscopic detection of alfatoxin contents in rat liver may find application for studying the distribution of alfatoxins in grains.198 Since versicolorin, the aflatoxin precursor, is immunogenic,199 the detection of versicolorin in foods using immunoassays will provide useful information for early control of aflatoxin production. Ochratoxin A at parts per billion ranges has been estimated in foods and tissues by RIA and ELISA using ochratoxin Aspecific monoclonal antibody (Table 7).200-203 Validation studies using RIA and HPLC to analyze ochratoxin A in purified extracts showed very good agreement.204 Trichothecenes are a group of structurally related mycotoxins produced by several fungal species.205 They are metabolized into other toxic derivatives in animal tissues.206 Several immunoassays have been developed for analysis of trichothecenes, with greater emphasis on T-2 toxins (Table 7). RIA analysis of trichothecenes appeared to carry the same sensitivity as HPLC or GC-MS, but at a much lower cost and ease of operation when applied to biological fluids.207 Although several monoclonal antibodies have been raised against trichothecenes,2051208"211 they did not meet the sensitivity of the poly-

8. Mushroom Poisonings Mushroom poisoning still occurs worldwide. RIA has been applied to determine amatoxin from Amanita mushrooms as well as in body fluids of the accidentally poisoned individuals.216 The development of a simple ELISA test to identify poisonous mushrooms will find useful application in food production. 9. Algal and Seafood Toxins Several species of marine dinoflagellates have been reported to produce toxins. The toxins pose a threat to human health by transfer through marine food sources. Antibodies raised against two of the neurotoxins, brevetoxin T17 and T34, were used in an ELISA to detect the toxins at 600-pg levels.217 The RIA applied for ciguatoxin assessment showed moderate correlations with the mouse bioassay and the guinea pig atrium assay.18 Good agreement was observed between the RIA and EIA for quantitating ciguatoxins using the same antibody. EIA has a sensitivity of 0.01 to 0.05 ng,219 thereby allowing application in the laboratory for routine tests or in the field for screening of large numbers of fish samples.220 A collaborative study on the use of a stick enzyme immunoassay is underway 1991

415


Critical Reviews In for establishing the applicability of the method for detecting ciguatoxins and the related polyether toxins.221 The RIA method applied to estimate saxitoxin, the causative agent of paralytic shellfish poisoning, detected the toxin at picomole quantities. However, the RIA estimates of the toxin in clam extracts were less than 33% of that estimated by mouse assay.222 These discrepancies could arise from cross-reactivities of the antibodies with structurally related compounds. Chu and Fan223 used the antibodies obtained from rabbits immunized with saxitoxin-bovine serum albumin to develop indirect ELISA for saxitoxin detection. Although the rabbit antibodies recognize neosaxitoxin, the ELISA for this toxin was not as sensitive as that for saxitoxin. The detection limit for saxitoxin by this method was about 50 to 100 ppb in clams or mussels spiked with the toxin. Currently, an ELISA test kit employing a polyclonal antibody is available commercially for detection of saxitoxins and other gonyautoxins.221 Levine et al.224 developed a monoclonal antibody-based RIA to detect okadoic acid in fish. Usgawa et al.225 also used a monoclonal antibody ELISA to quantitate okadoic acid in fish extract at the 10-ng/ml level. Foodborne poisoning resulting from consumption of a variety of fish has been reported in different parts of the world. Immunoassays could be used to quickly screen seafood and fish samples that may contain toxins and reduce human exposure to potent seafood toxins. Once immunoassays are well established and verified, they can be used to replace the mouse assay system currently used for toxicity determination. Mouse assays are time consuming and expensive. B. Pesticides Pesticides are used to improve crop harvest by reducing pestinduced damage and spoilage. Since the general public is very concerned about the health effects of pesticide residues on foods, monitoring of pesticide residues will always be needed. Assessment of pesticide residues in foods is carried out by chemical analyses using GC, HPLC, or GC/MS. Recently, immunoassays have been employed for pesticide analyses. ELISA and RIA are shown to detect pesticides at levels as small as picograms or nanograms per gram of food product or extract (Table 8). However, due to the problems listed below, the applicability of such methods for pesticide residue monitoring in foods needs further investigation. Pesticide residues in foods may undergo degradation or transformation due to enzymatic action, environmental effects, and processing treatment. Such conversion may also occur during pesticide extraction from foods as noted with the conversion of benomyl to methyl-2-benzimidazolecarbamate.226 Pesticide derivatives may sometimes retain toxicity; they may also retain the structural similarity with the parent compound and will thus interact with the antibody used in immunoassays. When the toxic derivatives begin to lose the structural similarity as the parent compounds, they will not be detected accurately

416

using immunoassays. In this case, the valuation of these immunoassay results in assessing the health risks of total pesticides residues will be misleading and may provide completely different interpretations on the health hazards. The inhibition of the immunological interaction due to coextractives from the foods or pesticide derivatives had been observed in immunoassays of cyanozine, 227 endosulfan, 228 metalaxyl,229 paraoxon,230 parthion,231 and diflubezuron." Antibody raised against dichlorfop-methyl or 2,4-dichlorophenoxy acetic acid, respectively, was shown to cross-react with several compounds carrying the phenoxy (2,4-dichlorophenoxy) moiety or 2,4,5-trichlorophenoxy acetic acid moiety.232 Some compounds that exhibit more than 20% cross-reactivity with pesticides of interest in foods are shown in Table 9. The occurrence of cross-reactivity limits the applicability of immunoassays in estimating some specific pesticides. In such situations, chromatographic methods should be applied to help separate the chemicals. However, immunoassays remain useful in estimating the total pesticide content taking advantage of the cross-reactivities. Although cross-reactivities due to food constituents may occur, it is not commonly encountered in immunoassay of pesticides. Thus a pesticide-specific antibody could be employed in estimating the pesticide in several foods as seen in the quantitation of benomyl in orange, grapes, lemon, apple, tomato, grapefruits, peach, and cucumber;226 dichlorfop-methyl in sugarbeet, soybean, wheat, and milk;232 metalaxyl in avocado, potato, cucumber, squash, and tomato;229 and triadimefon in apple, pear, pineapple, and grape.233 More than 85% recovery has been achieved with immunoassays for paraquat,234 diflubenzuron," and triadimefon233 using foods spiked with these pesticides. Validation studies using chromatographic techniques showed good agreement with immunoassay estimations. The sensitivity of immunoassays in detecting pesticide residues can be improved by modifying the operation process. Insoluble endosulfan is converted into a soluble amino derivative for enhanced interaction with the peroxidase.228 The use of collagenase-hyaluronidase to digest sheep fat helped the extraction of oxfendazole235 and has enhanced the sensitivity of the immunoassay. The recent efforts to develop immunoassays for rodenticide flocoumafen and insecticide flufenoxuron, and the development of monoclonal and polyclonal antibodies against cypermethrin,227 further indicate the future prospects in applying immunoassays for pesticide analyses. C. Anabolic Agents In animal production, hormones and related compounds are used to increase animal growth. Because some of the anabolic agents are potentially carcinogenic or may seriously affect the hormonal status in humans,236 it is important to apply highly sensitive methods to estimate residual anabolic agents at nanogram or picogram levels for regulatory measures. Currently,

Volume 29, Issue 6

Food Science and Nutrition Table 8 Application and Sensitivity of Immunoassays for Pesticides and Their Metabolites in Foods


Pesticide or derivative Aldrin Alachlor Atrazine Atrazine Bay SIR 8514 Benomyl S-Bioallethrin Chlorsulfuron Cyanazine Dieldrin Diflubenzuron Diclorfop-methyl

2,4-Dichlorophenoxy acetic acid Endosulfan Iprodione Methyl-2-benzimidazole carbamate Metalaxyl Oxfendazole Paraquat

Parathion Paraoxon 3-Phenoxybenzoic acid Terbutym Triadimefon 2,4,5-Trichlorophenoxy acetic acid Warfarin


Ref.

Sample

Method

Water Water Water Water Fruits Plasma Soil Water — Milk Beetroot Milk Soybeans Wheat Water

RIA ELISA EIA ELISA ELISA RIA RIA ELISA ELISA RIA ELISA EIA EIA EIA EIA RIA

700 pg 0.2 ng/ml 1.5 pg 50 pg/ml

Water Foods Fruits

EIA ELISA RIA

3 ng/ml 0.2 ng 5 ng/ml

228 356 226

Foods Sheep fat Plasma Serum Washings Lettuce Serum Tea Water Fruits Water

ELISA RIA RIA ELISA ELISA RIA ELISA ELISA ELISA ELISA RIA

100 ng/g 3 ng/g 0.6 ng/ml 0.8 ng/ml 0.1 ng/ml 10 ng 28 pg/ml 50 ng/g 25 ng/g 500 ng/g 0.02 mg/ml

229 235 357 358 234 231 230 227 359 233 355

Plasma

RIA

25 ng/ml

360

5 ng/ml 1.5 nmol/assay 0.4—1.2 ng/g 0.5 ng/g 150 pg 1 ng/ml 1150 ng/g 230 ng/g 115 ng/g 230 ng/g 0.1 ng/ml

349 350 351 352 11 226

353 354 227 349 11 232 232 232 232 355

for analysis since anabolic agents are metabolized in liver and are present in higher concentrations there. The analysis of anabolic agents should include their derivatives and metabolic products in the animal tissues. Trenbolone acetate is converted to trenbolone immediately after administration; the accurate detection of the compounds can be achieved by RIA using two antibodies, one specific for trenbolone and the other nonspecific.242 Trenbolone is also known to bind strongly to tissues through covalent bonds, making solvent extraction inefficient. In the assay of diethylstilbestrol and its derivatives in bovine liver, diethyl stilbesterol monogluconide was hydrolyzed enzymatically first.243 Immunoassays have become useful in meeting most of the analytical requirements. Of the different methods available, RIA has been applied more widely to a variety of anabolic agents in animal tissues (Table 10). Chemiluminescence im-

hexoestrol, medroxyprogesterone, and diethylstilbestrol are banned for use at least in the European Economic Community due to health concerns (EEC/81/602 directive). The estimation of anabolic compounds in foods has always been difficult; it poses problems not always encountered for other food components. Some of the anabolic agents used in animals are already present as endogenous hormones in animal tissues at concentrations varying among animals,237 and between tissues.238-239 In addition, the age, sex, and reproductive status of the animals also affect the hormonal levels,240 thus making it difficult to differentiate the exogenous from the endogenous origin and establishing tolerance limits. Since proper treatment of animals with endogenous sex steroids would not lead to increased hormonal levels in edible tissues,241 this further complicates the assessment as to whether the animals had been subjected to such treatment. Liver is the major organ used

1991

417

Critical Reviews In Table 9 Cross-Reactivities Observed in Immunoassays of Pesticides and Validation of Immunoassay by Comparing with Other Analytical Methods


Pesticides

Number of compounds tested

Alachlor

14

Aldrin and dieldrin Atrazinc Bay SIR 8514 and diflubenzuron

8 27 24

Benomyl and methyl-2 benzimidazole carbamate S-Bioallethrin Dichlorfop and dichlorfop methyl 2,3,-D and 2,4,5-T Endosulfan Metalaxyl Oxfendazole Paraquat Parathion Triadimefon Cyanazine

7

8 28 6 4 8 4 4 12 5 3

Compounds showing cross-reactivity*

Other method

Methyl thioalachlor, mercapturic acid None Propazine 2'-Hydroxyflubenzuron and 3 '-hydroxyflubenzuion 2'-Benzimidazolyl urea and 2-aminobenzimidazone

GC/MS

None None None Endrin Metolachlor None None Reduced parathion Triademenol Desethyl cyanazine

— HPLC HPLC CELC

— LSCR — — GC HPLC GC — GLC GC

Note: CELC — catron exchange liquid chromatography, GC — gas chromatography, HPLC — high performance liquid chromatography, and LSCR — liquid scintillation counting of radioactivity. " Only compounds showing more than 20% cross-reactivity are listed here.

munoassay is also used, while ELISA methods have yet to be developed to their full capabilities. The antibodies used for RIA tests exhibit insignificant crossreactivities in most estimations except in the case of medroxyprogesterones, where 31% cross-reactivity occurred with megesterol acetate.244 Also, two commercial antibodies 6024 and 6139 showed 89 and 45% cross-reactivity with diethylstilbestrol, respectively, in the assay for hexoestrol.245 Chemiluminescence immunoassay for zeranol showed high interactions with a variety of structurally related compounds.238 When RIA and ELISA tests are used, which have much higher specificities than chemiluminiscence assay, only a-zeralenol exhibited crossreactivity. The degradation of the mycotoxin zearalenone to zeronal in foods thus gave false values in assaying the anabolic agent. The use of monoclonal antibodies in RIA to determine zeranol246 and hexestrol245 made estimates more reliable. The use of radioactive (125I) instead of (3H) in RIA has been shown to be less costly and retain the same precision and specificity in assaying progesterone.247 False positives have been noted using RIA to determine stilbene in animals that had undergone therapeutic treatment with fluorinated corticosteroids.248 The quantitative estimates of the nonhormonal blanks and solvents used in the assay of steroid hormones in food-producing animals at 7 to 9 pg further 418

introduced inaccuracies for estimation.249 Thus, in future development of immunoassays for anabolic agents, it is essential to overcome the interference arising from the broad spectrum of compounds. The use of a second antibody in ELISA doubled the sensitivity of assay compared to RIA.250 The use of a second antibody in RIA was claimed to reduce the assay time of bovine prolactin from 7 h to 15 min.251 D. Therapeutic Agents Antibiotics are used to prevent the occurrence of and treat disease in food animals. They normally are injected directly into animals, mixed with the feed lot, or added to water. The presence of residual antibiotics in animal foods could lead to selection and development of drug-resistant microorganisms that may pose harmful health effects to humans. It is therefore necessary to monitor antibiotic residues in animal foods. Both EIA and RIA were employed for detection of antibiotic residues in animal tissues (Table 11). Most of the antibodies developed for assays have been shown to be extremely specific toward target compounds with insignificant cross-reactivities occurring either with structurally or functionally related compounds (Table 11). A few exceptions reported were 127 and 23% cross-reactivities shown by cephaloglycin and cephalothin, respectively, toward the antibody for cephalexin; 52%

Volume 29, Issue 6



Table 10 Application and Sensitivity of Immunoassays for Anabolic Agents in Food Animals Animal

Tissue

Minimum detectable level

Ref

Anabolic agent

Method

17p-Estradiol 17p-Estradiol Estrone Testosterone 17-a Methyltestosterone Progesterone Trenbolone Trenbolone acetate Trenbolone acetate

RIA RIA RIA RIA Chemiluminiscence

Cattle Cattle Cattle Cattle Cattle

F, K, L, M Sera F, K, L, M F, K, L, M M, U

200 pg 5 Pg 200 pg 10 pg 8pg

241 272 241 241 361

RIA RIA RIA RIA

Cattle Cattle Cattle Cattle

F Plasma F, K, L, M F, K, L, M

241 362 241 242

17P-OH-Trenbolone

RIA

Cattle

F, K, L, M

17ot-Trenbolone Diethylstilbestrol Diethylstilbestrol Hexoestrol Hexoestrol Hexoestrol

RIA RIA RIA RIA RIA RIA

F, K, L F, K, F, K, Urine B,K,

Medroxyprogesterone acetate Zeranol

RIA

Cow Cattle Cattle Cattle, sheep Cattle Cattle U, Fe Cattle

< 5 ng/g lOOpg/ml 70 pg 53, 26, 25, 24 pg/g, respectively 92, 46, 193, 81 pg/g, respectively 5 pg/g 30 pg/g 50 pg/g

RIA

Cattle

Zeranol Zeranol

RIA RIA

Cattle Cattle

Zeronal Zeronal

Chemiluminiscence EIA

Cattle Cattle

L, M L, M L, M L,M

Adipose tissue K, M, U, Fe F, K, L, M B, K, L , U , Fe Urine Urine

0.6 pg/ml 1 pg/g or ml 10 ppt

242 363 243 364 365 16 366 244

1 ng/g

367

1 Pg/g Ipg/g

246 237

2—5 pg/tube 10 pg/well

238 250

Note: F = fat, K = kidney, L = liver, M = muscle, B = bile, U = urine, and Fe = feces.

be examined according to the types of antibiotics used, and the duration after antibiotic treatment. The EIA described recently to estimate the macrolide antibiotics in fermentation systems using a polyclonal antibody may find application in detecting antibiotic production in many fermented foods.257

cross-reactivity shown by laidlomycin in the assay of monensin; and 12% cross-reactivity by sulfamerazine in the assay of sulfamethazine. Immunoassays were shown to have more than 85% recovery rate in detecting antibiotics in spiked samples. Validation experiments indicated good agreement between RIA and microbiological assay for hygromycin B, 252 and between RIA and GC estimations for chloramphenicol.253 Sometimes specific reagents are used to extract antibiotics from food samples for immunoassay quantitation. Cephalexin and colistin were extracted using 5% trichloroacetic acid254 and 5% acetic acid,255 respectively. The use of competitive EIA for sulfamethazine quantitation was used to eliminate the extraction step, thus reducing the assay time to 2 h.256 Tissue distribution of antibiotic residues differed markedly. Higher concentrations of colistin were found in kidney than in muscle of rainbow trout.255 Although cephalexin was present in yolk, it did not get transferred to egg white.255 Thus, when immunoassays are used to monitor antibiotic residues in animal foods, it is important to decide the type of animal tissues to

E. Adulterants The mixing of more expensive foods with less expensive substitutes for greater profit needs to be regulated for health, religious, and economic reasons. Sometimes the cheaper foods are misbranded as expensive products. In most of these instances the adulterants are so close in appearance, chemical composition, and organoleptic properties as their substituted products, that identification remains a problem even to the analyst. The specificity of the immunological reaction makes immunoassay a suitable method for identifying food adulterants. The agar gel immunodiffusion and ELISA are two major immunoassays used for assessing the presence of the less desirable or objectionable species in meat products (Table 12).

1991

419

Critical Reviews In Table 11 Application of Immunoassay.in Detection and Estimation of Therapeutic Agent Residues in Animal Tissues and Feeds Therapeutic agent


Cephalexin Chloramphenicol Colistin Gentamicin Hygromycin B Monensin Sulfamethazine

Method EIA R1A EIA EIA RIA EIA EIA

Animal

Tissue

Chick and cow Chick and cow Rainbow trout Swine Animal feeds Horse Swine

Eggs, tissue, milk Egg, meat, milk Muscle Muscle, sera — Sera Plasma

By using the agar gel immunodiffusion, Swart and Wilks258 identified products that contained 5 to 10% adulterants. The sensitivity of this technique was improved to detect as low as 2% adulterants when RBCs were removed from the antigenic inoculum used for antibody production.259 However, the technique only identified 49% of the samples correctly when it was used to survey frozen commercial meats. The accuracy in identifying the same meat samples containing both mixed and single species of meat was increased to 99% when counter immunoelectrophoresis was used.260 In addition, the test duration was reduced from 72 to 26 to 36 h using this method. The improved immunodiffusion using stabilized reagent paper discs and a printed template enabled detection of pork,261 beef,262 and poultry263 at 3 to 5% concentrations in blended meat products. The method has been accepted as the action test by the AOAC for detection of meat adulterants at 5*10% levels. ELISA was used to improve the detection limit to parts per million levels (Table 12) and expand the detection range to cover a wider variety of meats, including phylogenetically similar pairs of animal species such as donkey and horse, goat and sheep, and cattle and buffalo.264 The shift to utilize muscle proteins instead of sera as antigens for raising antibodies, and the use of polyclonal antibodies in ELISA further improved the minimum detection limits.265266 Further improvement in the ELISA test was achieved using monoclonal antibodies.266-267 Muscle proteins changed structurally after heat treatment, thus posing a limitation in the development of immunoassay. This was overcome by using thermostable proteins for antibody production.268 The antibodies developed this way have been used in immunodiffusion to detect adulteration of goat meat by beef in canned curries,269 and for speciation determination of cooked and autoclaved meat by ELISA.270 Partially purified antigens from chicken and pig were used to raise antisera to be used for speciation determination of cooked meats.267 The method was found to be applicable for speciation determination of heat-treated meat at 120°C for 15 min. When immunoassays are employed to detect adulteration of meat products for regulatory purposes, only the qualitative estimation of an undeclared meat in the product would be 420


Ref.

30 ng/ml or g 2pg/g 30 ng/ml 2.3 ng/ml 13.6 n-g/g 2 ng/ml 1 ng/ml

254 253 255 368 252 369 256

sufficient. The increased sensitivity of the assay is not a major concern in this case since meat adulterants are usually added in higher percentages. The inexpensive and highly nutritious soya proteins are common adulterants used in meat products. ELISA tests showing insignificant cross-reactivity with other plant and egg proteins have been developed for estimating soya proteins in meat products (Table 12).271-272 The sensitivity of the assay was improved after antisoya protein serum was purified by affinity chromatography prior to application in ELISA.273 The use of sodium dodecyl sulfate to denature soya proteins prior to reaction with the specific antibodies also enhanced the sensitivity of the immunoblotting test.274 Collaborative studies have confirmed that ELISA can be applied to estimate soya proteins in meat products.275 The results obtained by ELISA are more accurate than those obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).276 Immunodotting has also been employed in detecting soya proteins in milk replacers and infant formulae at levels as low as 25 ng,277 and in detecting cows' milk for ewes' milk and cows' milk in cheese.278 Heat denaturation of whey proteins in milk causes problems when antibodies used for immunoassays are raised against the whey proteins. In a study using radioimmunoassay to detect bovine milk added to ovine or caprine-milk, Calvo et al.279 demonstrated that reliable results were obtained with the high temperature-short time (HTST, 76°C for 30 s) treated milk, but not with milk treated at 90°C for 30 s. However, the use of thermostable casein as the immunogen has enabled the application of immunoassay to check heat-treated milk products. Wheat, rye, barley, or oat proteins in processed meat and baked foods were detected and quantitated by spot tests using enzyme conjugated-monoclonal antibody for gluten, following the immobilization of the food extracts on nitrocellulose paper62 or by ELISA.61 Immunoassay could find useful application for seafood species identification. A monoclonal antibody specific for rock shrimp was developed.280 It showed no cross-reactivity with the aqueous extracts of 12 other common shrimp species and lobsters. The monoclonal antibody was shown to be very

Volume 29, Issue 6

Food Science and Nutrition Table 12 Application of Immunoassay in Interspecies Meat and Adulterant Identification


Food item* Beef Beef Beef Beef Beef, sheep Beef, sheep Meat products Meat products Meat products Meat products Meat products Meat products Beef, pig Heat processed Fermented meat products Beef •B, Bu, C, G, H, K, P, S Beef Pig Goat Horse Sheep Beef Beef products •B, Bu, C, C, G, H, K, S Sausages Meat products Processed meat

Adulterant*

Method

H K, S H + K H + S K K P P T T Ch Ch Ch Ch/T P

Immunodiffusion Immunodiffusion Immunodiffusion Immunodiffusion Immunodiffusion Immunodiffusion Immunodiffusion Immunodiffusion Immunodiffusion Immunodiffusion Immunodiffusion Immunodiffusion ELISA ELISA ELISA

Pi Each other

ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELISA Immunoblotting ELISA

Bu W I D G Bu Pi Each other Soya Soya Pork

Minimum % or level content required for detection11 Ref 5 20 5, 20 5, 15 10, 15 2—10 3 5

3 5 10 7.5 1—30 126 ppm 250 ppm —50 —10 —10 —10 0.1 0.1 1 0.5 1 0.25 0.5 1

258 258 258 258 258 259 261 261 263 263 263 263 265 267 267 265 370 270 270 270 264 264 264 371 372 273 274 373

B = beef, Bu = buffalo, C = camel, Ch = chicken, D = donkey, G = goat, H = horse, I = impala, K = red kangaroo, P = pork, Pi = pig, S = sheep, T = turkey, and W = warthog. The percent level or concentration of adulterant above which the presence of the adulterant could be detected.

Immunoassays have also been applied to determine the authenticity or presence of undesirable levels of constituents in soft drinks (Table 13) and in the estimation of contamination or adulteration of sunflower butter by other vegetable oils.281 The potential for applying immunoassay for food regulatory purposes is great; the assay methods would reduce cost and time considerably.

effective and sensitive in blind studies to identify rock shrimp, and to detect the presence of rock shrimp in samples containing mixtures of diverse seafoods or other nonseafood meats using the ELISA test. In addition, the detection of rock shrimp proteins in various seafood and meat sample mixtures was achieved at a level as low as 4.3 ng on the average using the optimal ELISA testing conditions. Monoclonal antibodies such as this can be used in fish species identification and detection of adulteration in seafood products. The deviscerated, cleaned fish fillets cannot be identified easily by the physical appearance or a simple chemical test.

F. Immunodiagnostic Kits Immunodiagnostic methods differ from the conventional analytical methods in that specific antibodies are used. Antibodies

1991

421

Critical Reviews In


Table 13 Application of Immunoassays to Detect Authenticity and Objectionable Constituents in Soft Drinks Component examined for

Method

Orange juice Quinine Drosphila melanogaster egg Caffeine

Immunodiffusion ELISA ELISA RIA

are structurally more complicated than the chemicals used in conventional analysis. Thus it is more difficult, if not impossible, to generate antibodies in the same scale as chemicals. Immunoassay kits are less cumbersome for operation and less expensive. A test kit designed for detection of viruses in potatoes is quoted to carry the capacity of testing 1000 to 5000 samples at a cost of only 25 cents per sample.282 Some of the commercially available assay kits useful for detection of specific food constituents are listed in Table 14. Precision and accuracy of the test results have been the major concerns for applying test kits in detection and quantitation of food constituents. Many validation studies have been conducted to evaluate the precision and accuracy of the test kit results against the conventional analytical procedures; the results of these studies are helpful for further improvement of the test kits. Immunoassay kits have been used widely to detect and estimate mycotoxins, especially aflatoxins. The assays are easy to operate, convenient, and require less technical skills compared with chemical assays. Although several commercial test kits are available for aflatoxins, the testing of the other mycotoxins such as ochratoxin and deoxynivalenol by assay kits still needs further investigation. With increased numbers of test kits becoming available on the market, it has become necessary to establish guidelines for users. The Official Methods Board Task Force on Test Kits and Proprietary Methods (of the Association of the Official Analytical Chemists) has commented on the following requirements pertaining to test kits: 1.

2.

3. 4.

422

The need to include specifications for proprietary reagents, components, and appropriate controls to the users to assure them that the kits are performing according to validated specifications The need to inform the AOAC through the general referee any changes and modifications done in the kits by manufactureres so that the protocols could be reevaluated and approved The need to consider false positive/negative results, sensitivity, and specificity in relation to intended use The necessity to lay no limits on the number of methods to be approved by AOAC, as long as proper collaborative studies are carried out

5.

Minimum detection limit Ref. 2.5% 1 pg/ml 0.03 egg/ml At |xg/ml

374 375 376 377

The necessity of having a specific approach, including titles283

Very good agreement has been observed with Oxoid Salmonella Rapid Test,284 Tecra® Salmonella Visual Immunoassay, 154 - 285 and Bio-Enzabead® enzyme immunoassay technique153 when compared individually with the standard culture methods for Salmonella in foods. Salmonella Bio-Enzabead® Screen Test and ELISA Screening Test for Salmonella are reported as useful alternatives to the selective enrichment methods.155 Poor agreement was noted with the 1-2 Test®, when compared with the culture methods, for detection of Salmonella in foods.286-287 The presence of the competing microflora after enrichment apparently interferes with Salmonella detection. Collaborative studies were conducted on the Salmonella BioEnzabead® Screen Test158-167-288 and the Salmonella 1-2 Test®;168 these two assay kits have been accepted as official first action by the AOAC for analysis of Salmonella in foods. Assay kits based on RPLA, when compared with ELISA, did not show high sensitivity in detecting Clostridium perfringens and Staphylococcus aureus enterotoxins, although the assay kits were easy to operate.289-290 Fujikawa and Igarashi291 used the high-density latex particles to increase the sensitivity and rapidity of the RPLA assay. Windemann et al.138 also suggested the use of microtiter plates to overcome limitations associated with the polystyrene. Cox et al.292 reviewed the results of several studies in which both the commercially available kits and the conventional methods were used to detect foodborne bacteria and found that over 90% correlation was achieved. Medina et al.293 after comparing three immunochemical test kits for detecting sulfamethazine in milk suggested that by manipulating the test systems, the detection can be optimized at 10 ppb levels. The specificity of the immunological reaction can be utilized to help in the cleanup process. The antibody binds specifically to the compound of interest and allows "everything else" to pass through the column, thus demonstrating the distinct advantages over most physicochemical separation methods. Commercialization of this immunoseparation technique has resulted in development of cleanup columns for specific food constituents. Several such columns are available commercially for separating aflatoxin in foods.182-294 This process can be used

Volume 29, Issue 6

Food Science and Nutrition Table 14 Application of Immunoassay Kits in Analysis of Foods


Analyte

Sensitivity

Kit

Manufacturer"

Duration

Method EUSA Affinity column ELISA ELISA ELISA ELISA ELISA Affinity column Affinity column ELISA Selective absorption Selective absorption ELISA Affinity column ELISA Affinity column ELISA EUSA ELISA ELISA EUSA Selective absorption ELISA Chemiluminiscence ELISA ELISA Selective absorption EUSA

CITE-PROBE aflatoxin AflaQuick EZ-SCREEN*:aflatoxin Afla-20 Afla-10 IDEXX-AFB AgriScreen Aflatest P Total aflatoxins Aflatoxin test SAM-A SAM-AZ EZ-SCREEN®:aflatoxin Aflatest-P AgriScreen Aflatoxin M, Aflatoxin M, test CITE®:aflatoxin M, test EZ-SCREEN®:ochratoxin EZ-SCREEN®:T-2 toxin EZ-SCREEN®:zearalenone SAM-AZ PathoScreen® Lumi-Phage® Tecra® BioEnzabead 1—2 Test® QTrol®

I V EDS IDS IDS

ELISA

K OT O

Salmonella

Presence

3 min 5 min 7 min 4 min 4 min 45 min 12 min 7 min 30 min 30 min 10 min 10 min 7 min 7 min 12 min 30 min 40 min 10 min 10 min 5 min 5 min 10 min 2d 19 h' 2h 40—52 h' 32 h 92 samples per 2—5 h 72 h'

Salmonella Staphylococcus aureus toxin Staphylococcus aureus Campylobacter Listeria monocytogenes Listeria monocytogenes Limonin Hormones Sulfadimethoxine Sulfamethazine Sulfamethazine Sulfamethazine

Presence 2 ng/ml

72 h° 24 h

ELISA RPLA

Salmonella antigen screening test Salmonella-TEK® Oxold Set RPLA

15 s

Agglutination

Staphyloslide®

BB

Agglutination ELISA

Campyloslide® Tecra®

BB 3M

EUSA

Listeria-TEK®

OT

Bitterdetek® Phytodetek®-Dh2R EZ-SCREEN®:sulfadimethoxine LacTek® CITE®:sulfamethazine SIGNAL® milk test EZ-SCREEN®:sulfamethazine SPOT TEST Penzyme®lll LacTek®

Id Id EDS

AFB,, B2 AFB,, B2 AFB,, B 2 , G, AFB,, B,, G, AFB,, B 3 , G, AFB,, B 2 , G, AFB,, B 2 , G, AFB,, B 2 , G,, G2 AFB,, B 2 , B,, G2 AFB,, B 2 , G,, G2 AFB,, B 2 , G,, G 2 . AFB,, B 2 , G,, G2 AFB,, B 2 , G,, G2 AFM, AFM, AFM, AFM, AFM, Ochratoxin A T-2 toxin Zearalenone Zearalenone Vims/bacteria Salmonella Salmonella Salmonella Salmonella Salmonella

Sulfamathazine B-Lactam antibiotics* B-Lactam antibiotics'" B-Lactam antibiotics0

20 p.g/g 2p.g/g 5 ng/g 20 H-g/g

10 ng/g 3 H.g/g

2ng/g 1 M-g/g 0.001 (JLg/g 0.01 |Ag/g

0.01 ng/g 20 ng/g

0.1 ng/g 0.2 |xg/g 0.1 jig/g lOpg/g 0.25 ng/g 20 ng/g 50 ng/g 200 ng/g

0.5 ng/g 1 organism per 25 g 1 organism per 25 g 1—2 organism per 25 g

3—5 min 3h 48 h ppm 0.02 pmol 100 ng/g 10 ng/g

The infant-food industry.

The infant food industry.

The infant-food industry.

The infant-food industry.

Principles and application of high pressure-based technologies in the food industry.

Microbial transglutaminase and its application in the food industry. A review.

Potential for change in the food industry.

Phage therapy in the food industry.

Infant-food industry.

Diversity of glycosyl hydrolase enzymes from metagenome and their application in food industry.

Production of Lipopeptide Biosurfactant by a Marine Nesterenkonia sp. and Its Application in Food Industry.

Food industry perspectives on the mature market.

Application of evidence on probiotics, prebiotics and synbiotics by food industry: a descriptive study.

Listeria monocytogenes Biofilms in the Wonderland of Food Industry.

Salt: the dying echoes of the food industry.

Response to "Salt: the dying echoes of the food industry".

Framework for managing mycotoxin risks in the food industry.

Bacteriophages as Weapons Against Bacterial Biofilms in the Food Industry.

Nanotechnology -New Lifeline For Food Industry.

Food waste in the Swiss food service industry - Magnitude and potential for reduction.

Improved Functional Characteristics of Whey Protein Hydrolysates in Food Industry.

Applications of sonochemistry in Russian food processing industry.

Effect of Food Regulation on the Spanish Food Processing Industry: A Dynamic Productivity Analysis.

Preventing type 2 diabetes: Changing the food industry.