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

Identification and Analysis of the IgE Binding by Parvalbumin and Other Potential Allergens in Different Fish and Frog Species

Rationale: Serological cross-reactivity to different fish and frog species is common among fish-allergic individuals.We examined the intra- and inter-individual diversity in IgE responses of fish-allergic subjects to various fish and frog species and identified novel allergens besides parvalbumin. Methods: Sera from 38 subjects with a clinical history of fish allergy were analyzed for IgE-binding profiles to crude extracts of 26 raw fish and frog species, and purified cod and carp parvalbumin using IgE-immunoblotting. Sera of 7 subjects showing similar IgE-binding profiles in the IgEimmmunoblotting were pooled to identify potential allergens in pilchard, herring, cod, cusk, and rainbow trout using two-dimensional electrophoresis (2D) combined with IgE-immunoblotting and liquid chromatography-tandem mass spectrometry. Results: IgE-immunoblotting demonstrated great diversity among the fish-allergic individuals with respect to the IgE-binding to the parvalbumins and non-parvalbumin proteins in fish and frog species. Of the 38 individuals, 26 (68%) and 21 (55%) reacted to cod and carp parvalbumin, respectively. However, low IgE reactivity to parvalbumin from frog, mahi-mahi, and swordfish was observed. The pooled sera showed IgE-binding to parvalbumin and its corresponding isoforms separated by 2D in all 5 species. The IgE from pooled sera also recognized several novel fish allergens, including alpha actin, enolase, creatine kinase, glyceraldehyde 3-phosphate dehydrogenase, and fast myosin light chain proteins. Conclusions: The variation in IgE-binding depended on the individuals and fish species analyzed. The results suggested parvalbumin as the major cross-reactive allergens among fish species. Further characterization of the novel fish allergens is warranted at the molecular level using sera from additional fish-allergic subjects

Characterization of IgG and IgE Binding to Parvalbumin Derived from Commercially Important Fish Species

Rationale: Parvalbumin is recognized as pan-allergen in fish and frog. However, previous studies demonstrated that the IgE- and IgG-binding patterns to parvalbumins vary depending on the fish species. We aimed to use 3 anti-parvalbumin IgG and human IgE to investigate the contributing factors for the binding differences. Methods: Indirect enzyme-linked immunosorbent assay (ELISA) and IgG immunoblotting were used to determine the reactivity of the polyclonal anti-cod parvalbumin antibody and the commercially available, monoclonal anti-frog and anti-carp parvalbumin antibodies against raw muscle extracts of 25 fish species. Additionally, sera from 46 individuals with clinical history of fish allergy were analyzed for IgE reactivity to parvalbumin using indirect ELISA. Inhibition ELISAwas performed to determine the effects of heating and calcium on IgG-binding to parvalbumin. Results: The 3 IgG antibodies demonstrated varying specificity for different fish species. Polyclonal anti-cod parvalbumin antibody showed reactivity to a wider range of species, whereas the monoclonal anti-frog parvalbumin antibody showed the least cross-reactivity. The binding of the 3 IgG antibodies to parvalbumin was unaffected by heating, but the absence of calcium abolished the binding. IgE reactivity to cod parvalbumin or cod extracts were observed in \u3e 50% of individuals’ sera, whereas \u3c 0.1% of the sera showed reactivity to tuna and swordfish extracts. Both IgG and IgE antibodies showed low reactivity to tuna and swordfish that are apparently deficient in parvalbumin. Conclusions: These results suggested that the antibodies’ specificity to parvalbumins in various fish species is associated with the parvalbumin expression, its structural conformation, and the primary structure of antigenic determinations on parvalbumin

Measuring Parvalbumin Levels in Fish Muscle Tissue: Relevance of Muscle Locations and Storage Conditions

Fish is an allergenic food capable of provoking severe anaphylactic reactions. Parvalbumin is the major allergen identified in fish and frog muscles. Antibodies against fish and frog parvalbumin have been used to quantify parvalbumin levels from fish. However, these antibodies react variably with parvalbumin from different fish species. Several factors might be responsible for this variation including instability of parvalbumin in fish muscle as a result of frozen storage and differential parvalbumin expression in muscles from various locations within the whole fish. We aimed to investigate whether these factors contribute to the previously observed variable immunoreactivity of the anti-parvalbumin antibodies. Results showed the detection of parvalbumin by these antibodies was unaffected by frozen storage of muscles for 112 days. However, the parvalbumin content decreased in fish muscles from anterior to posterior positions. This factor may partially explain for the inconsistent reactivity of anti-parvalbumin antibodies to different fish species

Evaluation and Comparison of the Species-Specificity of 3 Antiparvalbumin IgG Antibodies

Parvalbumin is a pan-allergen in fish and frogs that triggers IgE-mediated reactions in fish-allergic individuals. Previous studies demonstrated that antibodies raised against fish and frog parvalbumins displayed varying specificity for different fish species, and thus, the applicability of these antibodies for potential use in immunoassays to detect fish residues were limited. We aimed to determine the specificity of 3 IgG antibodies for various fish species. Indirect enzyme-linked immunosorbent assay (ELISA) and IgG-immunoblotting were used to compare the reactivity of the polyclonal anticod parvalbumin antibody and the commercially available, monoclonal antifrog and monoclonal anticarp parvalbumin antibodies against raw muscle extracts of 29 fish species. All antibodies demonstrated varying specificities for different fish species. Of the 3 antibodies, the polyclonal anticod parvalbumin antibody is the most suitable for the detection of fish parvalbumins, as it showed reactivity to the widest range of species, including herring, pilchard, carp, pike, cod, pollock, haddock, cusk, hake, bluegill, tilapia, bass, grouper, trout, catfish, and perch, although detection was still limited for several key fish species

In Vitro Digestion and Characterization of 2S Albumin and Digestion-Resistant Peptides in Pecan

The 2S albumins are one of the major protein families involved in severe food allergic reactions to nuts, seeds, and legumes, thus potentially making these proteins clinically relevant for allergic sensitization and potential diagnostic markers. In this study, we sought to purify native 2S albumin protein from pecan to further characterize this putative allergen. The purified 2S albumin, Car i 1, from pecan was found to be resistant to digestion by pepsin in simulated gastric fluid (SGF) and comparatively stable to proteolysis by trypsin and pancreatin in simulated intestinal fluid (SIF). Digestion of purified Car i 1 in SGF and SIF resulted in formation of different digestion-resistant peptides that were capable of binding IgE antibodies from allergic individuals. Digestion stability of Car i 1 and formation of digestion-resistant antigenic peptides may explain why it is a potent sensitizing protein in pecan for susceptible individuals. The observation that digestion-resistant peptides are able to bind IgE implies that pecan can trigger systemic allergic reactions even after digestion in the stomach and small intestine

Purification of Parvalbumin from Carp: A Protocol That Avoids Heat Treatment

Parvalbumin from carp, a major allergen,was purified to homogeneity using ion exchange chromatography and size exclusion chromatography (estimated purity \u3e 95% to 98% based on SDS-PAGE and native PAGE) with a yield of 318 mg, and a number of basic biochemical characteristics were determined. The identity was confirmed by peptide-mass fingerprinting, and IgE-binding was demonstrated. The UV/Vis absorbance spectra were explained using the previously published amino acid sequences. Far UV-CD spectroscopy was used to confirm the folding character of parvalbumin. We conclude that parvalbumin from carp can be purified on a comparatively large (hundreds of milligrams) scale using a purification protocol that does not include denaturing steps. The purified protein resembles biochemical characteristics as were earlier published for carp parvalbumin, that is, a molecular weight of approximately 12 kDa, amino acid sequence identity and a secondary structure containing α-helices and β-structures. The described method provides a yield sufficient to produce and characterize antibodies to construct immunochemical methods to detect parvalbumin in food as well as for use as a standard calibrator for such assays. Practical Application: Parvalbumin is a major allergen from fish. Here,we have purified a comparatively large quantity from carp that can be used to develop antisera for use in an assay method to detect fish allergens

Comparison of Six Commercial ELISA Kits for Their Specificity and Sensitivity in Detecting Different Major Peanut Allergens

Six commercial peanut enzyme-linked immunosorbent assay kits were assessed for their ability to recover peanut from the standard reference material 2387 peanut butter and also for their specificity in detecting four major peanut allergens, Ara h 1, Ara h 2, Ara h 3, and Ara h 6. The percentage recovery of peanut from peanut butter differed across different kits as well as at different sample concentrations. The highest recovery was observed with the Romer and R-Biopharm kits, while four other kits were found to underestimate the protein content of the reference peanut butter samples. Five of the kits were most sensitive in detecting Ara h 3 followed by Ara h 1, while hardly recognizing Ara h 2 and Ara h 6. The other kit showed the highest sensitivity to Ara h 2 and Ara h 6, while Ara h 1 and Ara h 3 were poorly recognized. Although Ara h 2 and Ara h 6 are known to be heat stable and more potent allergens, antisera specific to any of these four peanut proteins/allergens may serve as good markers for the detection of peanut residues

Digestibility and IgE-Binding of Glycosylated Codfish Parvalbumin

Food-processing conditions may alter the allergenicity of food proteins by different means. In this study, the effect of the glycosylation as a result of thermal treatment on the digestibility and IgE-binding of codfish parvalbumin is investigated. Native and glycosylated parvalbumins were digested with pepsin at various conditions relevant for the gastrointestinal tract. Intact proteins and peptides were analysed for apparent molecular weight and IgE-binding. Glycosylation did not substantially affect the digestion. Although the peptides resulting from digestion were relatively large (3 and 4 kDa), the IgE-binding was strongly diminished. However, the glycosylated parvalbumin had a strong propensity to form dimers and tetramers, and these multimers bound IgE intensely, suggesting stronger IgE-binding than monomeric parvalbumin. We conclude that glycosylation of codfish parvalbumin does not affect the digestibility of parvalbumin and that the peptides resulting from this digestion show low IgEbinding, regardless of glycosylation. Glycosylation of parvalbumin leads to the formation of higher order structures that are more potent IgE binders than native, monomeric parvalbumin. Therefore, food-processing conditions applied to fish allergen can potentially lead to increased allergenicity, even while the protein’s digestibility is not affected by such processing

Peanut Allergen Threshold Study (PATS): Novel single-dose oral food challenge study to validate eliciting doses in children with peanut allergy

Background: Eliciting doses (EDs) of allergenic foods can be defined by the distribution of threshold doses for subjects within a specific population. The ED05 is the dose that elicits a reaction in 5% of allergic subjects. The predicted ED05 for peanut is 1.5 mg of peanut protein (6 mg of whole peanut). Objective: We sought to validate the predicted peanut ED05 (1.5 mg) with a novel single-dose challenge. Methods: Consecutive eligible children with peanut allergy in 3 centers were prospectively invited to participate, irrespective of previous reaction severity. Predetermined criteria for objective reactions were used to identify ED05 single-dose reactors. Results: Five hundred eighteen children (mean age, 6.8 years) were eligible. No significant demographic or clinical differences were identified between 381 (74%) participants and 137 (26%) nonparticipants or between subjects recruited at each center. Three hundred seventy-eight children (206 male) completed the study. Almost half the group reported ignoring precautionary allergen labeling. Two hundred forty-five (65%) children experienced no reaction to the single dose of peanut. Sixty-seven (18%) children reported a subjective reaction without objective findings. Fifty-eight (15%) children experienced signs of a mild and transient nature that did not meet the predetermined criteria. Only 8 (2.1%; 95% CI, 0.6%-3.4%) subjects met the predetermined criteria for an objective and likely related event. No child experienced more than a mild reaction, 4 of the 8 received oral antihistamines only, and none received epinephrine. Food allergy–related quality of life improved from baseline to 1 month after challenge regardless of outcome (η2 = 0.2, P < .0001). Peanut skin prick test responses and peanut- and Ara h 2–specific IgE levels were not associated with objective reactivity to peanut ED05. Conclusion: A single administration of 1.5 mg of peanut protein elicited objective reactions in fewer than the predicted 5% of patients with peanut allergy. The novel single-dose oral food challenge appears clinically safe and patient acceptable, regardless of the outcome. It identifies the most highly dose-sensitive population with food allergy not otherwise identifiable by using routinely available peanut skin prick test responses or specific IgE levels, but this single-dose approach has not yet been validated for risk assessment of individual patients