Eric A. E. Garber

Detection of melamine using commercial enzyme-linked immunosorbent assay technology.

Recent cases of adulteration with melamine have led to the need for rapid and reliable screening methods. To meet this need, commercial enzyme-linked immunosorbent assay (ELISA) test kits for the detection of triazines were evaluated. The recently released Melamine Plate kit (Abraxis, Warminster, Pa.) displayed a limit of detection of 9 ng/ml for melamine in phosphate-buffered saline (PBS) and approximately 1 microg/ml for melamine added to dog food. An atrazine ELISA test kit produced by Abraxis required 0.2 mg/ml to generate a response more than four times the standard deviation from background. In contrast, with the EnviroGard Triazine Plate kit (Strategic Diagnostics, Inc., Newark, Del.), 1.5 mg/ml melamine in PBS generated a signal only one standard deviation from background, which was insufficient to define a limit of detection. Extraction based on dilution with 105 mM sodium phosphate/75 mM NaCl/2.5% nonfat milk/0.05% Tween 20 (UD) enabled detection of fivefold less melamine in dog food than did use of the procedure recommended by the manufacturer, which entailed extraction into 60% methanol, sonication, centrifugation, filtration, and further dilution into 10% methanol/PBS. Using the Abraxis Melamine ELISA, both extraction protocols yielded identical results with a dog food sample adulterated with melamine. The recovery of melamine spiked into gravy from dog food using UD was 74% +/- 4%. In conclusion, the recently released Abraxis ELISA for melamine proved to be a useful alternative to more cumbersome methods.

Feasibility of Immunodiagnostic Devices for the Detection of Ricin, Amanitin, and T-2 Toxin in Food

Qualitative and quantitative comparisons were conducted of commercially available immunodiagnostic devices for the detection of three select agents with oral LD50 values > or = 0.1 mg/kg of body weight. Ricin (oral LD50 > 1 mg/kg), amanitin (oral LD50 approximately 0.1 mg/kg), and T-2 toxin (oral LD50 > 1 mg/kg) were spiked into beverages, produce, dairy, and baked goods and assayed using commercially available enzyme-linked immunosorbent assays (ELISAs) and lateral flow devices. In all cases, the commercial diagnostic kits successfully detected all three select agents at concentrations below what might be a health concern. The considerable difference between the limit of detection of the immunodiagnostic devices employed (typically < or = 0.020 microg/g) and the amount of the select agent necessary to pose a health threat in a single serving of food facilitated the design of protocols for the high throughput screening of food samples. These protocols entailed simple extraction methods followed by sample dilution. Lateral flow devices and sandwich ELISAs for the detection of ricin had no significant background problems due to the food matrices. Competitive ELISAs, which typically have unacceptably high background reactions with food samples, successfully detected amanitin and T-2 toxin.

Toxicity and detection of ricin and abrin in beverages.

The oral and intraperitoneal (i.p.) toxicities to female BALB/c mice of ricin and abrin in phosphate-buffered saline (PBS), spring water, apple juice, and half-and-half (only oral) were examined after brief (2 h) and prolonged (11 to 13 days) storage. The ricin and abrin samples prepared in PBS had oral toxicities consistent with those previous studies, indicating oral and i.p. 50% lethal doses of > 1 mg/kg of body weight and between 2 and 20 microg/kg of body weight, respectively. The toxicities of ricin and abrin in PBS were greater than those in apple juice and water. The oral toxicity of ricin and abrin in half-and-half appeared comparable to or less than that observed for the toxins in water. Spiked samples stored for a maximum of 11 days (13 for the abrin samples) at 4 degrees C induced similar numbers of fatalities as did samples stored for only 2 h. Enzyme-linked immunosorbent assays of the samples administered by i.p. injection indicated a decrease in detectable toxin at 0.5 microg/ml.

Detection of abrin in food using enzyme-linked immunosorbent assay and electrochemiluminescence technologies.

Abrin is a toxic ribosome-inactivating protein present in beans of Abrus precatorius, also known as rosary peas. The possibility that abrin could be used to adulterate food has made the development of assays for the detection of abrin a priority. Rabbit-derived polyclonal antibodies and mouse monoclonal antibodies were prepared against a mixture of abrin isozymes. The specificity and cross-reactivity of the antibodies were evaluated against a challenge library of 40 grains, nuts, legumes, and foods. An enzyme-linked immunosorbent assay (ELISA) and an electrochemiluminescence (ECL)-based assay were assembled and optimized. Polyclonal (capture) and polyclonal (detection) ELISAs, polyclonal and monoclonal ELISAs, and polyclonal and monoclonal ECL assays had limits of detection (LODs) of 0.1 to 0.5 ng/ml for abrin in buffer. The LOD for abrin dissolved into juices, dairy products, soda, chocolate drink, and condiments and analyzed with the ECL assay ranged from 0.1 to 0.5 ng/ml in the analytical sample. In contrast, the LODs for the ELISAs ranged from 0.5 to 10 ng/ml in the analytical sample.

Enzyme-Linked Immunosorbent Assay Detection of Melamine in Infant Formula and Wheat Food Products

The adulteration of food products with melamine to inflate the nitrogen content necessitates the establishment of analytical methods that can distinguish between proteinaceous ingredients and such adulterants. The specificity and ability to detect melamine by two commercial enzyme-linked immunosorbent assay (ELISA) kits were evaluated along with three protocols for sample preparation. Both ELISAs displayed cross-reactivity with ammeline, but neither was able to detect ammelide or cyanuric acid, indicating either a requirement for the 4,6-diamino-1,3,5-triazine structure or inability to bind 1,3,5-triazine-4,6-diones. The limits of detection for melamine in powder infant formula ranged from 0.2 to 3 microg/g depending on the ELISA kit and the method used to prepare the sample. The limits of detection for melamine in liquid infant formula and wheat products were <1 microg/ml and <2.5 microg/g, respectively. The ELISA kits provide an effective alternative for the analysis of samples suspected of containing melamine without relying on extensive sample preparation or expensive instrumentation.

Presence of Undeclared Food Allergens in Cumin: The Need for Multiplex Methods

Beginning in the autumn of 2014, millions of dollars of food and over 675 products were recalled in the United States due to the presence of undeclared peanut, attributed to cumin used in the manufacture of the products. Initial analyses also indicated the presence of almond. Subsequent research showed that the presence of peanut and almond did not fully explain the analytical results for the cumin samples. Using a combination of mass spectrometry, DNA-based methods (i.e., PCR and Sanger DNA Sequencing), microscopy, and antibody-based technologies (i.e., ELISA, Western blot analysis, and a novel xMAP multiplex assay) the presence of peanut was confirmed. Screening for secondary sources of adulteration (e.g., tree nuts, mahleb, peach, and cherry) supported the assessment that the cumin contained multiple contaminants. These results demonstrate the limitations of single analyte-specific assays and the need for orthogonal multiplex methods to detect food allergens irrespective of varietal or other differences.

Application of deadenylase electrochemiluminescence assay for ricin to foods in a plate format.

A recently developed bead-based deadenylase electrochemiluminescence assay for ricin is simple and sensitive in its ability to detect ricin, based on the catalytic activity of the toxin subunit, ricin A chain. The assay was modified to work in a 96-well plate format and evaluated by using juice samples. The plate-based assay, unlike the bead-based assay, includes wash steps that enable the removal of food particles. These steps minimize matrix effects and improve the signal-to-noise ratios and limits of detection (LOD). The LOD values for ricin in apple juice, vegetable juice, and citrate buffer by using the bead-based assay were 0.4, 1, and 0.1 microg/ml, respectively. In contrast, the LOD values for ricin by using the plate-based assay were 0.04, 0.1, and 0.04 microg/ml in apple juice, vegetable juice, and citrate buffer, respectively. The plate-based assay displayed three- to 10-fold lower LOD values than did the bead-based assay. Signal-to-noise ratios for the plate-based assay were comparable to those for the bead-based assay for ricin in citrate buffer, but 2- to 4.5-fold higher when the plate-based assay was used for analysis of juice samples.

Identification of the RNA N-glycosidase activity of ricin in castor bean extracts by an electrochemiluminescence-based assay

A simple electrochemiluminescence-based assay for RNA N-glycosidase activity has been modified to permit its use with authentic extracts of Ricinus communis (castor beans) and Abrus precatorius (jequirity seeds)--the natural sources of ricin and abrin. Modifications include the addition of an RNase inactivator to the reaction mixture, elimination of a signal-enhancing monoclonal antibody, and optimization of the incubation temperature. Concurrent testing with two substrates provides a diagnostic tool enabling castor bean toxins to be differentiated from a larger selection of N-glycosidase toxins than was previously examined.

Cross-reactivity profiles of legumes and tree nuts using the xMAP(®) multiplex food allergen detection assay.

AbstractThe homology between proteins in legumes and tree nuts makes it common for individuals with food allergies to be allergic to multiple legumes and tree nuts. This propensity for allergenic and antigenic cross-reactivity means that commonly employed commercial immunodiagnostic assays (e.g., dipsticks) for the detection of food allergens may not always accurately detect, identify, and quantitate legumes and tree nuts unless additional orthogonal analytical methods or secondary measures of analysis are employed. The xMAP® Multiplex Food Allergen Detection Assay (FADA) was used to determine the cross-reactivity patterns and the utility of multi-antibody antigenic profiling to distinguish between legumes and tree nuts. Pure legumes and tree nuts extracted using buffered detergent displayed a high level of cross-reactivity that decreased upon dilution or by using a buffer (UD buffer) designed to increase the stringency of binding conditions and reduce the occurrence of false positives due to plant-derived lectins. Testing for unexpected food allergens or the screening for multiple food allergens often involves not knowing the identity of the allergen present, its concentration, or the degree of modification during processing. As such, the analytical response measured may represent multiple antigens of varying antigenicity (cross-reactivity). This problem of multiple potential analytes is usually unresolved and the focus becomes the primary analyte, the antigen the antibody was raised against, or quantitative interpretation of the content of the analytical sample problematic. The alternative solution offered here to this problem is the use of an antigenic profile as generated by the xMAP FADA using multiple antibodies (bead sets). By comparing the antigenic profile to standards, the allergen may be identified along with an estimate of the concentration present. Cluster analysis of the xMAP FADA data was also performed and agreed with the known phylogeny of the legumes and tree nuts being analyzed. Graphical abstractThe use of cluster analysis to compare the multi-antigen profiles of food allergens

Detection of Gluten during the Fermentation Process To Produce Soy Sauce

Advances have been made to provide people with celiac disease (CD) access to a diverse diet through an increase in the availability of gluten-free food products and regulations designed to increase label reliability. Despite advances in our knowledge regarding CD and analytical methods to detect gluten, little is known about the effects of fermentation on gluten detection. The enzyme-linked immunosorbent assay (ELISA) and lateral flow devices routinely used by analytical laboratories and regulatory agencies to test for the presence of gluten in food were examined for their ability to detect gluten during the fermentation processes leading to the production of soy sauce, as well as in finished products. Similar results were observed irrespective of whether the soy sauce was produced using pilot-plant facilities or according to a homemade protocol. In both cases, gluten was not detected after moromi (brine-based) fermentation, which is the second stage of fermentation. The inability to detect gluten after moromi fermentation was irrespective of whether the assay used a sandwich configuration that required two epitopes or a competitive configuration that required only one epitope. Consistent with these results was the observation that ELISA, lateral flow devices, and Western immunoblot analyses were unable to detect gluten in commercial soy sauce, teriyaki sauce, and Worcestershire sauce. Although reports are lacking on problems associated with the consumption of fermented soy-containing sauces by consumers with CD, additional research is needed to determine whether all immunopathogenic elements in gluten are hydrolyzed during soy sauce production.