Cucumber mosaic virus 2b-Deficient Mutant Causes Limited, Asymptomatic Infection of Bell Pepper.

Cucumber mosaic virus Fast New York strain (CMV-Fny) containing a mutated 2b protein (CMV-FnyΔ2b) was evaluated for the ability to infect 'Calwonder' bell pepper (Capsicum annuum) plants in comparative tests with the parent virus, CMV-Fny. Plants inoculated with CMV-FnyΔ2b did not develop local or systemic symptoms of infection, whereas CMV-Fny-infected plants developed systemic chlorosis by 7 days post inoculation (dpi), followed by mosaic and leaf deformation. Virus accumulation, determined by enzyme-linked immunosorbent assay (ELISA), revealed that CMV-FnyΔ2b accumulated in inoculated Calwonder leaves and inconsistently infected some noninoculated leaves at a low titer but was not detected in the youngest, noninoculated leaves. Immuno-tissue blot tests did not detect CMV-FnyΔ2b in the stems of infected plants, whereas CMV-Fny accumulated throughout the length of the stems of inoculated plants. In two experiments, protoplasts were isolated from Calwonder leaves, inoculated with viral RNAs of CMV-Fny or CMV-FnyΔ2b, and tested by ELISA for infection. In both experiments, less CMV-FnyΔ2b than CMV-Fny accumulated in protoplasts. These results suggest that the CMV 2b protein is needed for systemic infection of Calwonder pepper plants and for accumulation of the virus in inoculated protoplasts.

Case Report: Allergic Reactivity to Mahaleb (Prunus mahaleb) Spice in a Subject With Almond and Other Tree Nut Allergies.

BACKGROUND: Mahaleb is an aromatic spice prepared from the fruit stone of the St. Lucie Cherry that is used as a flavoring agent in traditional Turkish and Middle Eastern baking. Immunodiagnostic kits for almond, which are based on polyclonal almond-specific IgG antibodies, have been shown to demonstrate considerable cross-reactivity with mahaleb as was incidentally discovered during a cluster of allergen-related food recalls in 2015. OBJECTIVE: Though acute allergy to almond is somewhat common, allergies to mahaleb have not been previously documented. However, based on antigenic similarity observed with almond-specific IgG, it is predicted that mahaleb nut proteins would exhibit some level of cross-reactivity with almond-specific IgE and may therefore potentiate acute allergic symptoms in individuals with food allergy to almond.Case Presentation: Herein, we report on a 40-year old Caucasian female with longitudinal history of multiple tree nut allergies including allergy to almond, presenting with moderate pruritus and oropharyngeal swelling shortly following ingestion of mahaleb seed kernels. METHODS AND RESULTS: Skin-prick testing using extracts compounded from pistachio, almond, and mahaleb revealed positive wheals measuring 8, 3, and 7 mm respectfully. Indirect enzyme-linked immunosorbent assay (ELISA) using plate-bound antigens prepared from pistachio, almond, and mahaleb revealed IgG positive responses to all three targets. ELISA and Western blot analysis performed using goat anti-almond polyclonal IgG demonstrated significant cross-reactivity between almond and mahaleb, but not to pistachio. CONCLUSION: This is the first documented case of acute allergy to mahaleb, co-occurring in the context of plural tree nut allergies, providing novel evidence that mahaleb may pose a risk to nut-allergic individuals and indicating a need for awareness of spice contamination with nut and mahaleb residues.

Quantitative Detection of Chicken and Turkey Contamination in Cooked Meat Products by ELISA.

Background: Concerns about the contamination of meat products with undeclared meats and new regulations for the declaration of meat adulterants have established the need for a rapid test to detect chicken and turkey adulteration. Objective: To address this need, Microbiologique, Inc. has developed an ELISA that can quantify the presence of chicken and turkey down to 0.1% (w/w) in cooked pork, horse, beef, goat, and lamb meats. Results: This chicken/turkey authentication ELISA has an analytical sensitivity of 0.000037% and 0.000048% (w/v) for cooked and autoclaved chicken, respectively, and an analytical range of quantitation of 0.025-2% (w/v), in the absence of other meats. The assay cross-reacts with cooked duck and pheasant but does not demonstrate any cross-reactivity with cooked pork, horse, beef, goat, and lamb meats, egg, or common food matrixes. Conclusions: The assay is rapid, can be completed in 70 min, and can detect a 0.1% level of meat adulteration. Highlights: The Microbiologique Cooked Chicken/Turkey ELISA can quantitate cooked chicken/turkey in the presence of pork, horse, chicken, goat, or sheep meat to 0.1% (w/w) and is not affected by common food matrixes.

Quantitative Detection of Beef Contamination in Cooked Meat Products by ELISA.

Background: Concerns about the contamination of meat products with undeclared meats, and new regulations for the declaration of meat adulterants have established the need for a rapid test to detect beef adulteration to 0.1% sensitivity. Objective: To address this need, Microbiologique, Inc. has developed an ELISA that can quantify the presence of beef down to 0.1% (w/w) in cooked pork, horse, chicken, goat, and sheep meat. Results: The beef-authentication ELISA has an analytical sensitivity of 0.00022 and 0.00012% (w/v) for cooked and autoclaved beef, respectively, and an analytical range of quantitation of 0.025 to 2% (w/v), in the absence of other meats. Moreover, the assay is specific for cooked beef and does not cross react with common food matrixes. Conclusions: The assay is rapid, can be completed in 70 min, and can detect a 0.1% level of meat adulteration. The assay is an improvement over a previous U.S. Department of Agriculture's tested assay, which is sensitive to 1% adulteration and takes 2.5-3 h to complete. Highlights: The Microbiologique Cooked Beef ELISA can quantitate cooked beef in the presence of pork, horse, chicken, goat, and sheep meat to 0.1% (w/w) and is not affected by common food matrixes.

Quantitative Detection of Pork Contamination in Cooked Meat Products by ELISA.

Recent news of many cases of adulteration of meats with pork has bolstered the need for a way to detect and quantify the unwanted contamination of pork in other meats. To address this need, Microbiologique, Inc. has produced a sandwich ELISA assay that can rapidly quantify the presence of pork in cooked horse, beef, chicken, goat, and lamb meats. We carried out a validation study and showed that this assay has an analytical sensitivity of 0.00014 and 0.00040% (w/v) for cooked and autoclaved pork, respectively, and an analytical range of quantitation of 0.05-3.2% (w/v) in the absence of other meats. The assay can measure pork contamination down to 0.1% (w/w) in the presence of cooked horse, beef, chicken, goat, and lamb meats. The assay is quick and can be completed in 1 h and 10 min.

Quantitative Detection of Horse Contamination in Cooked Meat Products by ELISA.

Concerns about the contamination of meat products with horse meat and new regulations for the declaration of meat adulterants have highlighted the need for a rapid test to detect horse meat adulteration. To address this need, Microbiologique, Inc., has developed a sandwich ELISA that can quantify the presence of horse meat down to 0.1% (w/w) in cooked pork, beef, chicken, goat, and lamb meats. This horse meat authentication ELISA has an analytical sensitivity of 0.000030 and 0.000046% (w/v) for cooked and autoclaved horse meat, respectively, and an analytical range of quantitation of 0.05-0.8% (w/v) in the absence of other meats. The assay is rapid and can be completed in 1 h and 10 min. Moreover, the assay is specific for cooked horse meat and does not demonstrate any cross-reactivity with xenogeneic cooked meat sources.

Assessment of Avenins from Different Oat Varieties Using R5-Based Sandwich ELISA.

Gluten derived from wheat and related triticeae cereals possesses distinct amino acid sequences that provoke the immunopathogenic features of celiac disease (CD) in genetically susceptible individuals. However, the role of oat-derived gluten, or avenins, in CD pathogenesis remains a disputed matter, as evidenced by a lack in harmonized legislation regarding gluten classification in relation to gluten-free labeling. In this study, we have analyzed a panel of pure oat cultivars using a sandwich ELISA based on the R5 monoclonal antibody (mAb), which binds to canonical epitopes occurring within celiagenic peptides present in triticeae-derived gluten but reportedly not present in avenins. We have identified three varieties of oats that reproducibly bind R5 antibodies and levels indicating the presence of gluten at more than the 20 ppm gluten regulatory threshold. Nested assessment using Western blot analysis and alternative gluten detection systems corroborated these results. Collectively, these data suggest that select oat varieties may prove problematic to patients with CD and to food companies and regulatory agencies and will extend our basic understanding of current gluten detection systems.

A Novel Immunoassay Test System for Detection of Modified Allergen Residues Present in Almond-, Cashew-, Coconut-, Hazelnut-, and Soy-Based Nondairy Beverages.

A growing number of plant-based milk substitutes have become commercially available, providing an array of options for consumers with dietary restrictions. Though several of these products rival cow's milk in terms of their nutritional profiles, beverages prepared with soy and tree nuts can be a significant concern to consumers because of potential contamination with food allergens. Adding to this concern is the fact that allergen residues from plant-based beverages are modified during manufacturing, thereby decreasing the sensitivity of antibody-based detection methods. Consequently, many commercially available allergen detection kits are less effective for allergens derived from nondairy milk substitutes. To address this limitation, we developed a panel of polyclonal antibodies directed against the modified proteins present in almond, cashew, coconut, hazelnut, and soy milks and incorporated them into rapid lateral flow immunoassay tests configured in both sandwich and competitive format. The tests had robust detection capabilities when used with a panel of various brand-name products, with a sensitivity of 1 ppm and selectivity values of 3 to 5 ppm in nondairy beverages. Minimal cross-reactivity to extracts prepared from common commodities was observed. The development of a highly sensitive and rapid test specifically designed to detect trace quantities of highly modified allergen residues in plant-based, dairy-free beverages will aid food manufacturers and regulatory agencies in monitoring products for these modified allergens when testing environmental and food samples.

Novel Monoclonal Antibody-Based Immunodiagnostic Assay for Rapid Detection of Deamidated Gluten Residues.

Gluten derived from wheat and related Triticeae can induce gluten sensitivity as well as celiac disease. Consequently, gluten content in foods labeled "gluten-free" is regulated. Determination of potential contamination in such foods is achieved using immunoassays based on monoclonal antibodies (mAbs) that recognize specific epitopes present in gluten. However, food-processing measures can affect epitope recognition. In particular, preparation of wheat protein isolate through deamidation of glutamine residues significantly limits the ability of commercial gluten testing kits in their ability to recognize gluten. Adding to this concern, evidence suggests that deamidated gluten imparts more pathogenic potential in celiac disease than native gluten. To address the heightened need for antibody-based tools that can recognize deamidated gluten, we have generated a novel mAb, 2B9, and subsequently developed it as a rapid lateral flow immunoassay. Herein, we report the ability of the 2B9-based lateral flow device (LFD) to detect gluten from wheat, barley, and rye and deamidated gluten down to 2 ppm in food as well as its performance in food testing.

Development and Validation of a Lateral Flow Immunoassay Test Kit for Dual Detection of Casein and ß-Lactoglobulin Residues.

Allergies to cow's milk are very common and can present as life-threatening anaphylaxis. Consequently, food labeling legislation mandates that foods containing milk residues, including casein and/or ß-lactoglobulin, provide an indication of such on the product label. Because contamination with either component independent of the other can occur during food manufacturing, effective allergen management measures for containment of milk residues necessitates the use of dual screening methods. To assist the food industry in improving food safety practices, we have developed a rapid lateral flow immunoassay test kit that reliably reports both residues down to 0.01 µg per swab and 0.1 ppm of protein for foods. The assay utilizes both sandwich and competitive format test lines and is specific for bovine milk residues. Selectivity testing using a panel of matrices with potentially interfering substances, including commonly used sanitizing agents, indicated reduction in the limit of detection by one-to fourfold. With food, residues were easily detected in all cow's milk-based foods tested, but goat and sheep milk residues were not detected. Specificity analysis revealed no cross-reactivity with common commodities, with the exception of kidney beans when present at high concentrations (> 1%). The development of a highly sensitive and rapid test method capable of detecting trace amounts of casein and/or ß-lactoglobulin should aid food manufacturers and regulatory agencies in monitoring for milk allergens in environmental and food samples.