A Sandwich Enzyme-Linked Immunosorbent Assay (ELISA) for the Quantitation of Peanut in Foods

Peanuts are one of the most allergenic foods known. Strict avoidance of peanut by peanut-allergic individuals is difficult and often unsuccessful. Peanut proteins have previously been found in nonpeanut foodstuffs prepared on shared processing equipment, and such carryover contamination increases the risk of occurrence of allergic reactions. Immunoassays offer a specific, sensitive, and rapid technique to detect and quantitate small amounts of proteins in food systems. A sandwich-type, enzyme-linked immunosorbent assay (ELISA) was developed for the detection of peanut protein in foods. Selected food samples were ground and extracts prepared by overnight extraction in 0.01 M phosphate buffered saline, followed by centrifugation before analysis. Rabbit polyclonal antibodies elicited against an oil-roasted peanut extract were used as the capture antibody. The food sample extracts were then added, along with a standard extract of peanut. Goat polyclonal antibodies directed against dry-roasted peanuts were employed as detector antibodies, and the amount bound was ultimately determined using rabbit antigoat IgG conjugated to alkaline phosphatase, with subsequent substrate reaction. The ELISA described has a detection limit of approximately 2 ppm of peanut, and succeeded in detecting peanut in foods that did not have peanut in the ingredient listing

Allergenic Foods

Virtually all food allergens are proteins, although only a small percentage of the many proteins in foods are allergens. Any food that contains protein has the potential to cause allergic reactions in some individuals. However, a few foods or food groups are known to cause allergies on a more frequent basis than other foods. At a 1995 consultation on food allergies sponsored by the Food and Agriculture Organization, a group of international experts confirmed that peanuts, soybeans, crustacea, fish, cow’s milk, eggs, tree nuts, and wheat are the most common allergenic foods. These foods are responsible for more than 90% of serious allergic reactions to foods. Allergies to certain fresh fruits and vegetables are also rather common, but the allergens tend to be labile to processing and cooking and the symptoms are mild and confined primarily to the oropharyngeal area. The prevalence of allergic sensitivities to specific foods varies from one country to another depending on the frequency with which the food is eaten in that country and the typical age at its introduction into the diet. For example, peanuts are a much more frequent cause of food allergies in the United States than in most other countries. Americans eat peanuts more often and introduce peanut butter into the diet of children at an early age. The Japanese probably experience more soybean and rice allergies than some other cultures because of the frequency of these two foods in the Japanese diet. Scandinavians have a high incidence of codfish allergy for similar reasons. Table 1 provides a listing of the most common allergenic foods and food groups compiled from a thorough search of the medical literature. Table 2 provides a listing of the less common allergenic foods. Only some of the foods listed in this table have been documented to cause severe, life-threatening allergic reactions. Citations are provided to studies and/or case reports that document the allergenicity of those particular foods. The absence of a particular food on this list may not mean that it is nonallergenic but may indicate that its allergenicity has not been documented. Conversely, the presence of a specific food on the list merely indicates that it has been listed in one or more reports as a cause of food allergy and does not indicate the prevalence or potential as an allergenic food

Secretion of Food Allergen Proteins in Saliva

RATIONALE: Peanut proteins were found to be secreted in 50% of lactating women’s breast milk. We wanted to develop a testing method to predict the secretion of peanut protein in breast milk. The secretion of food protein in saliva was hypothesized to be a possible predictor of secretion of foods in breast milk following ingestion. METHODS: Non-allergic volunteers, some lactating, ingested 50 grams of either whole peanuts, peanut milk or cow’s milk and various immunoassays were utilized to analyze for the presence of peanut or cow’s milk proteins in saliva and breast milk. Saliva and breast milk samples were subjected to SDS-PAGE, Western blot and ELISA analysis with anti-raw and roasted peanut and anti-alpha-casein antibodies and pooled serum IgE from peanut allergic individuals. RESULTS: Peanut protein levels in breast milk were undetectable using Western blot analysis and inconsistent with ELISA analysis. However, peanut proteins around 20 and 30 kDa that reacted with anti-roasted peanut antibody were detected, 6-18 hours following ingestion, in saliva of different individuals. An 18 KDa band that reacts with anti-alpha casein antibody was also detected in saliva 6-18 hours following ingestion. CONCLUSIONS: Secretion of food allergen proteins or peptides in saliva several hours following ingestion may have important implications for delayed allergic reaction by sensitive patients. Also, due to the fact that these proteins or peptides survive digestive enzymes, become absorbed into the blood stream and are subsequently secreted in biological fluids may indicate that they are most likely the sensitizing or tolerizing agent within an allergic food. Funding: National Peanut Board, USD

An Evaluation of the Sensitivity of Subjects with Peanut Allergy to Very Low Doses of Peanut Protein: A Randomized, Double-Blind, Placebo-Controlled Food Challenge Study

The minimum dose of food protein to which subjects with food allergy have reacted in double-blind, placebo-controlled food challenges is between 50 and 100 mg. However, subjects with peanut allergy often report severe reactions after minimal contact with peanuts, even through intact skin. Objective: We sought to determine whether adults previously proven by challenge to be allergic to peanut react to very low doses of peanut protein. Methods: We used a randomized, double-blind, placebo-controlled food challenge of 14 subjects allergic to peanuts with doses of peanut ranging from 10 μg to 50 mg, administered in the form of a commercially available peanut flour. Results: One subject had a systemic reaction to 5 mg of peanut protein, and two subjects had mild objective reactions to 2 mg and 50 mg of peanut protein, respectively. Five subjects had mild subjective reactions (1 to 5 mg and 4 to 50 mg). All subjects with convincing objective reactions had short-lived subjective reactions to preceding doses, as low as 100 μg in two cases. Five subjects did not react to any dose up to 50 mg. Conclusion: Even in a group of well-characterized, highly sensitive subjects with peanut allergy, the threshold dose of peanut protein varies. As little as 100 μg of peanut protein provokes symptoms in some subjects with peanut allergy

Lupine Allergy: Not Simply Cross-Reactivity with Peanut or Soy

Background: Reports of lupine allergy are increasing as its use in food products increases. Lupine allergy might be the consequence of cross-reactivity after sensitization to peanut or other legumes or de novo sensitization. Lupine allergens have not been completely characterized. Objectives: We sought to identify allergens associated with lupine allergy, evaluate potential cross-reactivity with peanut, and determine eliciting doses (EDs) for lupine allergy by using double-blind, placebo-controlled food challenges. Methods: Six patients with a history of allergic reactions to lupine flour were evaluated by using skin prick tests, CAP tests, and double-blind, placebo-controlled food challenges. Three of these patients were also allergic to peanut. Lupine allergens were characterized by means of IgE immunoblotting and peptide sequencing. Results: In all 6 patients the ED for lupine flour was 3 mg or less for subjective symptoms and 300 mg or more for objective symptoms. The low ED and moderate-to-severe historical symptoms indicate significant allergenicity of lupine flour. Two patients allergic to lupine but not to peanut displayed IgE binding predominantly to approximately 66-kd proteins and weak binding to 14- and 24-kd proteins, whereas patients with peanut allergy and lupine allergy showed weak binding to lupine proteins of about 14 to 21 or 66 kd. Inhibition of binding was primarily species specific. Conclusion: Lupine allergy can occur either separately or together with peanut allergy, as demonstrated by 3 patients who are cosensitized to peanut and lupine. Clinical implications: Lupine flour is allergenic and potentially cross-reactive with peanut allergen, thus posing some risk if used as a replacement for soy flour

Clinical relevance of sensitization to lupine in peanut-sensitized adults

Background: The use of lupine in food has been increasing during the last decade and allergic reactions to lupine have been reported, especially in peanut-allergic patients. The frequency and the degree of cross-reactivity to other legumes are not known. The aim of the study was to investigate the frequency of sensitization to lupine, and in addition to pea and soy, and its clinical relevance, in peanutsensitized patients. Furthermore, to determine the eliciting dose (ED) for lupine using double-blind placebo-controlled food challenges (DBPCFC). Methods: Thirty-nine unselected peanut-sensitized patients were evaluated by skin prick tests (SPT) and ImmunoCAP to lupine, pea, and soy. Clinical reactivity was measured by DBPCFC for lupine, and by history for pea and soy. Results: Eighty-two percent of the study population was sensitized to lupine, 55% to pea, and 87% to soy. Clinically relevant sensitization to lupine, pea, or soy occurred in 35%, 29%, and 33% respectively of the study population. None of the patients was aware of the use of lupine in food. The lowest ED for lupine, inducing mild subjective symptoms, was 0.5 mg, and the no observed adverse effect level (NOAEL) was 0.1 mg. No predictive factors for lupine allergy were found. Conclusion: In peanut-sensitized patients, clinically relevant sensitization to either lupine or to pea or soy occurs frequently. The ED for lupine is low (0.5 mg), which is only five fold higher than for peanut. Patients are not aware of lupine allergy and the presence of lupine in food, indicating that education is important to build awareness

Chestnut as a Food Allergen: Identification of Major Allergens

To evaluate the clinical significance of chestnut as a food allergen in Korea, skin prick test and ELISA were done in 1,738 patients with respiratory allergies. To identify the IgE binding components, IgE-immunoblotting, 2D IgE-immunoblotting and MALDI-TOF were performed. To observe the effects of digestive enzymes and a boiling treatment, simulated gastric fluid (SGF) and simulated intestinal fluids (SIF) were incubated with chestnut extracts, and IgE-immunoblotting were then repeated. Skin prick test revealed that 56 (3.2%) patients showed more than 2+ of allergen to histamine ratio to chestnut. Among the 21 IgE binding components, 9 bands were found in more than 50% of the sera tested and the 24 kDa protein had the highest binding intensity. The amino acid sequence of the 24 kDa protein (pI 6.3) had homology with legume protein of oak tree. SGF, SIF and boiling treatment were able to suppress the IgE binding components. In conclusion, chestnut ingestion was shown to induce IgE mediated responses with a 3.2% sensitization rate. Twenty one IgE binding components and one new allergen (the 24 kDa protein) were identified. Digestive enzymes and boiling treatment were able to decrease the allergenic potency

Factors affecting the determination of threshold doses for allergenic foods: How much is too much?

Background: Ingestion of small amounts of an offending food can elicit adverse reactions in individuals with IgE-mediated food allergies. The threshold dose for provocation of such reactions is often considered to be zero. However, because of various practical limitations in food production and processing, foods may occasionally contain trace residues of the offending food. Are these very low, residual quantities hazardous to allergic consumers? How much of the offending food is too much? Very little quantitative information exists to allow any risk assessments to be conducted by the food industry. Objective: We sought to determine whether the quality and quantity of existing clinical data on threshold doses for commonly allergenic foods were sufficient to allow consensus to be reached on establishment of threshold doses for specific foods. Methods: In September 1999,12 clinical allergists and other interested parties were invited to participate in a roundtable conference to share existing data on threshold doses and to discuss clinical approaches that would allow the acquisition of that information. Results: Considerable data were identified in clinical files relating to the threshold doses for peanut, cows\u27 milk, and egg; limited data were available for other foods, such as fish and mustard. Conclusions: Because these data were often obtained by means of different protocols, the estimation of a threshold dose was very difficult. Development of a standardized protocol for clinical experiments to allow determination of the threshold dose is needed

Biosensor immunoassay for traces of hazelnut protein in olive oil

The fraudulent addition of hazelnut oil to more expensive olive oil not only causes economical loss but may also result in problems for allergic individuals as they may inadvertently be exposed to potentially allergenic hazelnut proteins. To improve consumer safety, a rapid and sensitive direct biosensor immunoassay, based on a highly specific monoclonal antibody, was developed to detect the presence of hazelnut proteins in olive oils. The sample preparation was easy (extraction with buffer); the assay time was fast (4.5 min only) and the limit of detection was low (0.08 μg/g of hazelnut proteins in olive oil). Recoveries obtained with an olive oil mixed with different amounts of a hazelnut protein containing hazelnut oil varied between 93% and 109%