Reliable enzyme-linked immunosorbent assay for the determination of walnut proteins in processed foods

Among food allergens of tree nuts, walnuts are a frequent cause of adverse food reactions in allergic patients. A novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of walnut soluble proteins in processed foods was developed. The sandwich ELISA is highly specific for walnut soluble proteins. The recovery ranged from 83.4 to 123%, whereas the intra- and interassay coefficients of variation were less than 8.8 and 7.2%, respectively. This study showed that the proposed method is a reliable tool for detection in the presence of hidden walnut proteins in processed foods.     

Reliable enzyme-linked immunosorbent assay for the determination of soybean proteins in processed foods

Among allergenic foods, soybean is known as a food causing adverse reactions in allergenic patients. To clarify the validity of labeling, the specific and sensitive detection method for the analysis of the soybean protein would be necessary. The p34 protein, originally characterized to be p34 as an oil-body associated protein in soybean, has been identified as one of the major allergenic proteins and named Gly m Bd 30K. A novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of the soybean protein in processed foods was developed using polyclonal antibodies raised against p34 as a soybean marker protein and the specific extraction buffer for extract. The developed sandwich ELISA method was highly specific for the soybean protein. The limit of detection (LOD) and the limit of quantification (LOQ) of the developed ELISA were 0.47 ng/mL (equivalent to 0.19 microg/g in foods) and 0.94 ng/mL (equivalent to 0.38 microg/g in foods), respectively. The recovery ranged from 87.7 to 98.7%, whereas the intra- and interassay coefficients of variation were less than 4.2 and 7.5%, respectively. This study showed that the developed ELISA method is a specific, precise, and reliable tool for the quantitative analysis of the soybean protein in processed foods.     

Glucuronidase assay in a rapid MPN determination for recovery of Escherichia coli from selected foods

Glucuronidase is present in most strains of Escherichia coli but absent in most other enteric microorganisms; therefore, an assay for this enzyme is useful for determining the presence of the organism. The substrate 4-methylumbelliferyl beta-D-glucuronide (MUG) is incorporated into either lauryl tryptose (LT) broth or EC medium; the inoculated tubes are then incubated under specified conditions and examined under longwave UV light for the presence of a fluorogenic glucuronidase end product. When compared with the 10-day most probable number (MPN) procedure of AOAC, the LT-MUG and the EC-MUG tests required 24 and 96 h, respectively, and gave comparable mean log MPN values for samples of crabmeat, sunflower kernels, and walnut pieces. However, false-positive and false-negative reactions were observed with foods tested by both of these rapid methods. Overall, method sensitivity was not compromised by using the LT-MUG rather than the EC-MUG method. Incorporation of 25 micrograms MUG/mL into LT broth resulted in diminished fluorescence of positive reactions, whereas MUG concentrations of 50 and 100 micrograms/mL provided decisive fluorogenic reactions.     

Reliable enzyme-linked immunosorbent assay for the determination of coconut milk proteins in processed foods

This study was designed to develop a novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of coconut milk proteins in processed foods. The developed sandwich ELISA was able to detect coconut milk proteins from various coconut milk products and did not show any cross-reactivity with 41 of 42 kinds of popularly used food ingredients, thus reflecting great specificity for coconut milk proteins. In addition, the established ELISA is highly sensitive and allowed the detection of 0.31 μg/g of coconut milk protein in complex food matrices. This proposed assay could serve as a useful tool for the detection of the presence of hidden coconut milk proteins in processed foods.     

A review of bromine determination in foods.

Organic compounds containing bromine, including methyl bromide, ethylene dibromide, and 1,2-dibromo-3-chloropropane, have been used extensively for the fumigation of foods, or soils in which foods grow, making it necessary to determine residues of bromine and bromine-containing organic compounds. A large number of methods for the determination of bromine in foods, as organic, inorganic, and combined total bromide, have been developed. In methods for organic bromide, the bromine is converted to the inorganic form for measurement by titration, photometry, or other means. In recent years, instrumental methods have been developed in which the total bromine in the sample is determined, regardless of the state in which it exists. X-ray fluorescence and neutron activation analysis are the 2 instrumental methods used most widely. Residue data are presented for some typical bromine-containing samples.     

Sample homogenization procedure for determination of lead in canned foods.

To improve the homogeneity of canned foods before Pb determination, composited samples or single can contents (150--500 g) are treated with an equal weight of dilute nitric acid, and then thoroughly blended in a high efficiency, probe-type Polytron homogenizer; subsamples are then either dry-ashed or wet-ashed in preparation for analysis. Within-can variability is reduced from the 20--50% coefficient of variation range obtained with conventional blending procedures to a coefficient of variation of less than 5% by using the proceudre described. Precision data for Pb, using both the probe-type and conventional blade-type homogenizers, are compared. The procedure described for Pb determination was used to analyze a number of canned products. Data indicate that the Polytron plus dilute acid procedure improves overall Pb homogeneity for subtle as well as severe initial Pb heterogeneity.     

Quantitative determination of FD&C colors in foods.

A published procedure using Amberlite LA-2 (a liquid anion exchange resin) solutions to extract colors from food and the salt solution cellulose chromatographic technique to separate colors from each other were updated and submitted to collaborative study. Three batches of cookies and 4 purchased products were analyzed by 13 collaborators. The 3 batches of cookies contained different color mixtures requiring the selection of various procedures. The 4 purchased products were selected principally to include a variety of foods. Some results were partially deficient. The deficiencies reflect certain inadequacies in the directions as written rather than basic flaws in the method. Some improvements were suggested by the collaborators. The Assocaite Referee recommends that the method be further revised to correct the present inadequacies and to include some improvements, and that the revised method be submitted to another collaborative study.     

Application of a tri-enzyme extraction for total folate determination in foods

A tri-enzyme digestion procedure using chicken pancreas conjugase, alpha-amylase, and Pronase was evaluated to determine its usefulness in the microbiological quantitation of total folate in foods. Folate values obtained by traditional conjugase digestion were compared to those obtained by the tri-enzyme method for 12 food products that represent diverse matrixes. The tri-enzyme treatment increased measurable folate from most foods when compared to levels found after conjugase digestion. Largest increases were noted for tuna fish (51%) and yogurt (33%) after tri-enzyme digestion. For the 12 foods, a mean increase of 19% in measurable folate was obtained with tri-enzyme treatment. The study shows that traditional conjugase treatment does not completely free folate from complex food matrixes before microbiological analysis. Further, as other investigations have suggested, current accepted methods for folate analysis may be underestimating folate levels in foods.     

Automated fluorometric determination of vitamin C in foods.

Two automated fluorometric methods were compared with the official AOAC methods for determining vitamin C in fortified ready-to-eat cereals, fruits, vegetables, baby foods, flour products, pet foods, meats, frozen dinners, juices, and nutritional health bars. Each sample was analyzed in duplicate on 2 days and included a recovery study on all products. Egberg's semiautomated method (Method 1) and Roy's automated method (Method 2) were compared with the manual AOAC tritrimetric and fluorometric methods. The correlation factor for Methods 1 and 2 were 0.999 and 0.979, respectively, when compared with the AOAC methods. The recovery study showed average recoveries of 97.8% for Method 1, 99.3% for Method 2, and 100.6% for the AOAC methods. The results suggest that Method 1 is the method of choice for the majority of the products analyzed.     

Turbidimetric assay for tetracyclines in feeds using a microtiter plate system

The microtiter plate system for turbidimetric assay of chlortetracycline (CTC) and oxytetracycline (OTC) levels in feeds uses a 96 well microtiter plate, a multichannel pipette, and an ELISA reader to measure turbidity. Feeds are extracted for both tetracyclines using AOAC extraction systems. For CTC, the range of the standard curve is 0.001-0.005 microgram CTC/mL; for OTC, the range is 0.004-0.016 microgram OTC/mL. Repeatability of CTC assays, as shown by the coefficient of variation (CV), ranged from 0.54 to 5.65% for same-day assays and from 2.01 to 9.39% for assays on different days. For OTC, CVs ranged from 2.69 to 10.01% for same-day assays and 3.24 to 9.08% for different-day assays. Average recoveries for CTC were 108.7% for same-day assays and 106.8% for different-day assays; for OTC, average recoveries were 112.4% and 106.5% for same-day and different-day assays, respectively.     

Gas-liquid chromatographic determination of total cholesterol in multicomponent foods.

A method is described for the determination of total cholesterol in multicomponent foods and also other products such as nonfat dry milk, dried whole egg solids, and certain candy bars. The lipid is extracted from the sample by a mixed solvent and saponified. The unsaponifiable fraction which contains the cholesterol and other sterols is extracted with benzene. An aliquot is evaporated to dryness and the residue is dissolved in dimethylformamide. The sterols are derivatized to form trimethylsilyl (TMS) ethers. The TMS-cholesterol derivative is quantitatively determined by gas-liquid chromatography, using 5alpha-cholestane as an internal standard. Nine laboratories participated in a collaborative study of the determination of total cholesterol in deviled ham sandwich spread, vegetable beef stew, corned beef hash, frozen chicken pot pie, pizza pepperoni, fish sticks, breaded shrimp, chocolate-covered candy bars, dried whole egg solids, and nonfat dry milk and the results are reported here. The coefficient of variation ranged from 5.64 to 23.2%, with an average coefficient of variation of 14.8%.     

Collaborative study for the comparison of two methods for the determination of total cholesterol in multicomponent foods.

The gas chromatographic method for determining total cholesterol in multicomponent foods, collaboratively studied by the AOAC in 1974 (Method 1), has been evaluated by 9 collaborating laboratories and compared with the Interim Methodology Instructions No. 2 modified method (Method 2). The 5 samples selected for collaboration were deviled ham sandwich spread, vegetable beef stew, frozen chicken pot pie, frozen fish sticks, and mayonnaise. The recovery data were obtained from a sample of wheat germ oil spiked with 0.297% cholesterol as cholesteryl palmitate. Collaborators performed 2 replicate analyses on all samples by both methods. The statistical evaluation of the results showed that Method 1 is superior to Method 2. Average recoveries from the spiked wheat germ oil samples were 91.4% (9 laboratories) and 85.8% (7 laboratories) with coefficients of variation of 12.5 and 14.4%, respectively. Based on the collaborative results and statistical evaluation, Method 1 has been adopted as official first action.     

Development of a fluorescence polarization assay for the determination of aflatoxins in grains

Aflatoxins produced by Aspergillus flavus are commonly found in human and animal foods including grains, cereals, peanut products, sorghum, and soy seeds. Exposure to aflatoxins has been associated with carcinogenicity. This paper reports a simple, portable, and rapid fluorescence polarization (FP) assay for aflatoxin determination in grains. This immunoassay is field portable, homogeneous, and without any washing and cleaning steps. The assay is based upon the competition between free aflatoxin and an aflatoxin-fluorescein tracer for an aflatoxin-specific monoclonal antibody in solution. A series of naturally contaminated corn, sorghum, peanut butter, and peanut paste samples were analyzed by FP and compared with HPLC results. Similarly, spiked popcorn samples were analyzed by FP. FP results of naturally contaminated samples correlated well with HPLC (r (2) = 0.97). FP analysis of spiked popcorn samples (with a mixture of B(1)/B(2)/G(1)/G(2), 7/1/3/1, w/w) gave a good correlation with spiked values (r (2) = 0.99). However, FP consistently underestimated the aflatoxin contents. This was perhaps due to low cross-reactivity of the antibody used toward B(2), G(1), and G(2) aflatoxins. These results combined with the portability and simplicity of the assay suggest that the assay can be used for screening total aflatoxin in grains.     

Comparative studies of nucleic acid hybridization assay for Listeria in foods

A nucleic acid hybridization assay has been developed for Listeria spp. in dairy foods and environmental samples. The assay is based on detection of unique Listeria 16S rRNA sequences by using a 32P-labeled synthetic DNA probe. Inclusivity and exclusivity of the probe were confirmed with 139 Listeria isolates representing all known species, and 73 non-Listeria bacterial strains. In this paper, we present results from our preliminary studies comparing the hybridization assay with conventional culture on a total of 575 specimens that represent a variety of inoculated and uninoculated foods and environmental samples. The assay, which is done in a filter manifold format after 2 days of cultural enrichment, requires a total assay time of less than 2.5 days. The false-negative rate for all sample groups tested using the GENE-TRAK hybridization assay was less than the rate for culture. Thus, the new assay allows rapid screening of the indicated product groups and provides reliable numerical results.     

Quantitative sandwich ELISA for the determination of tropomyosin from crustaceans in foods

The ubiquitous muscle protein tropomyosin has been identified as the major shrimp allergen and is suggested to be a cross-reacting allergen. Previously, only a few methods for the detection of tropomyosin in food have been published. A quantitative sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of tropomyosin from crustaceans in foods has been developed and validated. A polyclonal rabbit antitropomyosin capture antibody and the biotinylated conjugate of the same antibody for detection were the basis for the ELISA, which was specific for crustaceans. The ELISA was able to quantitate tropomyosin in various food matrixes, had a detection limit of 1 microg/g, and cross-reacted to some extent with cockroach. Recoveries ranged from 63 to 120%, and the intra and interassay coefficients of variation were <6 and <14%, respectively.     

Sandwich immunoassays for the determination of peanut and hazelnut traces in foods

People suffering from food allergies are dependent on accurate food labeling, as an avoidance diet is the only effective countermeasure. Even a small amount of allergenic protein can trigger severe reactions in highly sensitized patients. Therefore, sensitive and reliable tests are needed to detect potential cross-contamination. In this paper two fast sandwich immunoassays are described for the determination of peanut (Arachis hypogaea) and hazelnut (Corylus avellana) traces in complex food matrices. Mouse monoclonal antibodies were used as capture antibodies, and labeled rabbit polyclonal antibodies were used as detection antibodies in both assays. The assay time was 30 min in total, and cross-reactivities against a variety of fruits and seeds were found to be in the low 10(-4)% (ppm) level or in some cases not detectable. The recoveries in all tested food matrices ranged from 86 to 127%, and the limits of detection were in the range of 0.2-1.2 mg/kg (ppm) in food for both peanut and hazelnut, respectively.     

Simplified method for determination of total dietary fiber in foods

A simplified method, based on the same principles as the AOAC enzymatic-gravimetric method for determining total dietary fiber (TDF) (43.A14-43.A20), has been tested on 12 food samples which had been used in other collaborative studies. TDF values obtained in our laboratory for these 2 methods were in good agreement (y = 0.96x + 0.39; r = 0.999). The simplified method uses a single incubation period and only 1 enzyme (amyloglucosidase), and thus yields smaller blank and ash corrections but a higher protein correction.     

Improved method for determination and identification of serotonin in foods

A previously described method to identify and quantitate serotonin in foods has been improved. The extraction and separation of serotonin from interfering substances has been improved, and the scope of material to which the method may be applied has been widened. The relative standard deviation (RSD) for repeated determinations of serotonin in canned fried tomato purée and the average recovery of serotonin added to the same sample was 6.25 and 89.9%, respectively. The method showed the presence of serotonin in apricots, cherries, and peaches.     

Validation of a monoclonal enzyme immunoassay for the determination of carbofuran in fruits and vegetables

The N-methylcarbamate pesticide carbofuran is a very important insecticide used worldwide. In the present work, the validation of a monoclonal antibody-based enzyme immunoassay (ELISA) to determine this compound in fruits and vegetables is described. The immunoassay is a competitive heterologous ELISA in the antibody-coated format, with an I(50) value for standards in buffer of 740 ng/L and with a dynamic range between 200 and 3100 ng/L. For recovery studies, peppers, cucumbers, strawberries, tomatoes, potatoes, oranges, and apples were spiked with carbofuran at 10, 50, and 200 ppb. After liquid extraction, analyses were performed by ELISA on extracts purified on solid-phase extraction (SPE) columns and crude, nonpurified extracts. Depending on the crop, mean recoveries in the 43.9--90.7% range were obtained for purified samples and in the 90.1--121.6% range for crude extracts. The carbofuran immunoassay performance was further validated with respect to high-performance liquid chromatography (HPLC) with postcolumn derivatization and fluorescence detection (EPA Method 531.1). Samples were spiked with carbofuran at several concentrations and analyzed as blind samples by ELISA and HPLC after SPE cleanup. The correlation between methods was very good (y = 0.90x + 2.66, r(2)() = 0.958, n = 25), with HPLC being more precise than ELISA (mean coefficients of variation of 4.1 and 11.5%, respectively). The immunoassay was then applied to the analysis of nonpurified extracts of the same samples. Results also compared very well with those obtained by HPLC on purified samples (y = 1.02x + 10.44, r(2)() = 0.933, n = 29). Therefore, the developed immunoassay is a suitable method for the quantitative and reliable determination of carbofuran in fruits and vegetables even without sample cleanup, which saves time and money and considerably increases the sample throughput.     

Multiresidue method for quantitative determination of organophosphorus pesticides in foods

A multiresidue method for the quantitative determination of organophosphorus pesticides in foods from the Food and Drug Administration's Total Diet Study is described. The organophosphorus pesticides are separated on the basis of polarity and determined in both fatty and nonfatty foods with a minimum of interferences. The foods analyzed included raw and cooked individual foods as well as combination dishes, water, and whiskey. Recoveries of 17 organophosphorus pesticides in 41 foods ranged from about 80 to 118%.