Enzyme Allergosorbent Test Inhibition Research Articles

Food-Cooking Processes Modulate Allergenic Properties of Hen’s Egg White Proteins

Background and Objective: Reducing the allergenicity of food allergens can suppress the clinical symptoms of food allergy. The objective of the present study was to investigate the effects of processing on the allergenic properties of hen’s egg white proteins. Methods: Eggs were processed by traditional Chinese cooking, including steaming, water boiling, frying, spicing and tea boiling. The contents of processed egg protein were assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis; the allergenicity was evaluated by Western blotting, enzyme-linked immunosorbent assay and enzyme allergosorbent test inhibition. Circular dichroism spectrum analysis of four major egg allergens from various egg products was performed as well. A mouse model of food allergy was developed to test the allergenicity of processed egg protein in vivo. Results: Protein degradation was significant following tea boiling and spiced-tea boiling. The total allergenic potential of water-boiled egg and fried egg was relatively higher than that of steamed egg, spiced egg and tea-boiled egg. Challenge with proteins from raw egg, water-boiled egg and fried egg induced skewed T-helper 2 pattern responses (Th2 responses) in the intestine of mice sensitized to egg proteins; however, when the mice sensitized to egg proteins were challenged with proteins from steamed egg, spiced egg and tea-boiled egg, respectively, only weak Th2 responses were induced in their intestine. Conclusion: Processing by steaming, spicing, or tea boiling can weaken the allergenicity of egg proteins.

The Russian Thistle (Salsola kali) Pollen Major Allergen, Sal k 1, Can Be Quantified in Allergenic Extracts and Airborne Pollen

Background: Russian thistle (Salsola kali) pollen is an important cause of pollinosis in areas where rainfall is not abundant. Our aim was to develop an ELISA for quantification of the major allergen of S. kali extracts, Sal k 1, and to assess the correlation of this allergen content with the allergenic activity of extracts. Methods: Sal k 1 was purified by ion exchange and gel permeation chromatography and identified by mass spectrometry. Monoclonal antibody 4C11 was used for capture at 5 µg/ml and biotin-labeled specific antiserum at 0.25 µg/ml served for detection. The allergenic activity of the pollen extracts was measured by enzyme allergosorbent test inhibition. Results: Sal k 1 reacted to 85% of sera from 40 S. kali-allergic patients and was able to inhibit 92% of the IgE-binding capacity of patients’ serum pool to the whole extract. The ELISA had a lineal range between 1.25 and 20 ng/ml of purified Sal k 1. The intra- and interassay coefficients of variation were lower than 5 and 10%, respectively. The assay was very sensitive since it had a detection limit of 0.08 ng/ml. No reactivity was found outside the Amaranthaceae family where only Kochia and Salicornia sp. gave significant reactivity. A good correlation (Spearman’s ρ = 0.92) was obtained between Sal k 1 content of different S. kali extracts and their IgE-binding activity. Conclusions: The results proved the usefulness of the two-site sandwich ELISA for aeroallergen control and for the standardization of S. kali pollen extracts intended for clinical use.

Characterization of a Hypoallergenic Recombinant Bet v 1 Variant as a Candidate for Allergen-Specific Immunotherapy

Background: Recombinant allergens and especially their hypoallergenic variants are promising candidates for a more effective and safer specific immunotherapy. Methods: Physicochemical and immunological characteristics of a folding variant of recombinant Bet v 1 (rBet v 1-FV) were investigated in comparison to natural Bet v 1 (nBet v 1) and the correctly folded recombinant Bet v 1 (rBet v 1-WT) by SDS-PAGE, size exclusion chromatography, multi-angle light scattering, circular dichroism, immunoblotting and enzyme allergosorbent test inhibition assay for detection of IgE reactivity and ELISA with Bet v 1-specific monoclonal antibodies. The functional IgE reactivity of the different Bet v 1 proteins was investigated using basophil activation in terms of CD203c expression and histamine release. T cell reactivity was investigated using T cell lines raised from birch pollen-allergic subjects against nBet v 1. Immunogenicity was investigated in mice. Results: Physicochemical characterization revealed purity, homogeneity and monomeric properties of rBet v 1-FV. Unlike nBet v 1 and rBet v 1-WT, rBet v 1-FV showed almost no IgE binding in immunoblots. The reduction of allergenicity was further proved by IgE-binding inhibition assays, basophil activation and histamine release. T cell reactivity was completely conserved, as demonstrated by proliferation of Bet v 1-specific T cell lines with multiple epitope specificities. rBet v 1-FV showed strong immunogenicity in mice. Conclusions: Due to its reduced IgE reactivity and decreased capacity to activate basophils, but retained T cell reactivity and strong immunogenicity, rBet v 1-FV proved to be a very promising candidate for specific immunotherapy in birch pollen-allergic subjects.

A sensitive monoclonal antibody‐based enzyme‐linked immunosorbent assay to quantify Parietaria judaica major allergens, Par j 1 and Par j 2

Parietaria pollen is one of the most important causes of pollinosis in Mediterranean countries. Parietaria judaica pollen extract presents two major allergens, Par j 1 and Par j 2, that belong to the lipid transfer protein family. To develop an ELISA for quantification of both major allergens of P. judaica pollen extracts, and to assert correlation of these allergens content with the allergenic activity of extracts. Natural Par j 1-Par j 2 allergens were purified by gel filtration, ion exchange, and affinity chromatography and identified by mass spectrometry. Rabbit antisera were obtained using this protein preparation as antigen and used for immunoaffinity purification of nPar j 1-Par j 2. BALB/c mice were immunized with the immunopurified nPar j 1-Par j 2 and after fusion and screening by direct ELISA, 5D4 monoclonal antibody was selected as capture antibody to develop a quantitative two-site ELISA. Bound proteins were detected by a biotinylated Par j 1-Par j 2-specific polyclonal antibody. The optimized ELISA was developed from 25 to 8000 pg/mL of purified Par j 1-Par j 2, and a linear portion of 200-1000 pg/mL. The intraassay and interassay coefficients of variation were lower than 7% and 14% respectively. The assay was very sensitive and specific as it had a detection limit of 25 pg/mL and did not detect reactivity with the same family plants, as Urtica. Par j 1-Par j 2 allergens content was measured in 14 P. judaica and two P. officinalis pollen extracts showing a significant correlation with their allergenic activity measured by enzyme allergosorbent test inhibition. The results proved the usefulness of the two-sandwich ELISA for the standardization of Parietaria pollen extracts intended for clinical use, because of its good correlation with allergenic potency.

Biological characterization of glutaraldehyde-modified Parietaria judaica pollen extracts.

Allergoids are widely used in specific immunotherapy (SIT) for the treatment of IgE-mediated allergic diseases, but all techniques for standardization of conventional allergic extracts may not be appropriate for standardization of a glutaraldehyde (GA)-modified extract because of the unique characteristics of these extracts. To assess an accurate methodology for standardization of chemically modified extracts. GA-modified extracts from Parietaria judaica pollen were purified by diafiltration. Biochemical properties were investigated by determination of amino groups, chromatography, and SDS-PAGE. The IgE-binding activity was determined by skin prick test, enzyme allergosorbent test inhibition, basophil activation, and histamine release tests. Peripheral blood mononuclear cells (PBMCs) from P. judaica pollen-allergic subjects were stimulated with either native or allergoid extracts, and proliferation was measured. Biochemical data indicated a high degree of allergen polymerization resulting in extract components higher than 100 kDa. IgE-binding activity, both in vivo and in vitro, was reduced by more than 99.8%. Both allergen and allergoid induced PBMC proliferation and synthesis of blocking IgG antibodies at similar rates. Moreover, no evidence of introduction of new determinants by chemical modification was found. The preparation of GA-modified extracts by diafiltration is faster and more reliable than previous chromatographic methods. These modified extracts have drastically reduced their allergenicity while maintaining their immunogenicity, and therefore they can be used in safer and shortened schedules of SIT.

Soybean allergy in patients allergic to birch pollen: Clinical investigation and molecular characterization of allergens

Background Allergic reactions to legumes are generally thought to be acquired by means of primary sensitization through the gastrointestinal tract. Recently, Gly m 4 (starvation-associated message 22), a Bet v 1–related pathogenesis-related protein 10 from soy, was suggested to be an allergen in patients with allergic reactions to a dietary product containing a soy protein isolate. Objective We sought to evaluate the clinical relevance of Gly m 4 in subjects allergic to birch pollen with soy allergy and to assess the risk for subjects allergic to birch pollen to acquire soy allergy. Methods Twenty-two patients allergic to birch pollen with soy allergy confirmed by means of positive double-blind, placebo-controlled food challenge results (n = 16) or a convincing history (n = 6) were investigated for IgE reactivity to birch pollen and soy allergens by using the Pharmacia CAP system and immunoblot analysis. Cross-reactivity was assessed by means of enzyme allergosorbent test inhibition. Ninety-four patients with birch pollen allergy were interviewed to assess soy tolerance and screened for IgE reactivity to Gly m 4 by means of immunoblotting. The Gly m 4 content in soy foods and soybean varieties was investigated by means of quantitative evaluation of immunoblots. Results During double-blind, placebo-controlled food challenge, 10 patients experienced symptoms localized to the oral cavity, and 6 patients had a more severe reaction. CAP analysis revealed Gly m 4–specific IgE in 96% (21/22) of the patients. All patients had Bet v 1–specific IgE antibodies, and 23% (5/22) had positive Bet v 2 results. In IgE immunoblotting 25% (6/22) of the patients recognized soy profilin (Gly m 3), and 64% (14/22) recognized other soy proteins. IgE binding to soy was at least 80% inhibited by birch pollen and 60% inhibited by rGly m 4 in 9 of 11 sera tested. Seventy-one percent (67/94) of highly Bet v 1–sensitized patients with birch pollen allergy were sensitized to Gly m 4, and 9 (9.6%) of those patients reported soy allergy. The Gly m 4 content in soy products ranged between 0 and 70 ppm (milligrams per kilogram). Conclusions Our results confirm that soybean is another birch pollen–related allergenic food. Gly m 4 is the major soy allergen for patients allergic to birch pollen with soy allergy. The content of Gly m 4 in soy food products strongly depends on the degree of food processing.

Immediate-type hypersensitivity reaction to ingestion of mycoprotein (Quorn) in a patient allergic to molds caused by acidic ribosomal protein P2

Background: Quorn is the brand name for a line of foods made with so-called “mycoprotein,” which springs from the mold Fusarium venenatum . Since the introduction on the food market, there have been complaints from consumers reporting adverse gastrointestinal reactions after ingestion of mycoprotein. To date, it is not clear whether the reported symptoms are IgE-mediated. Objective: The aim of the study was to describe for the first time a case history of an asthmatic patient with severe hypersensitivity reactions to ingested mycoprotein and to identify and characterize the potential allergen that might be responsible for this. Methods: The sensitization pattern of the asthmatic subject was characterized, and food allergy to mycoprotein was assessed by double-blinded placebo-controlled food challenge. Afterward, specific IgE antibodies of the serum of this patient were used to screen a Fusarium culmorum cDNA expression library. The coding sequence of one enriched cDNA-clone was expressed in Escherichia coli to produce a recombinant protein that was further purified and immunologically characterized. Results: The patient showed high sensitization to many known aeroallergens but apart from Quorn not to any other tested food samples. The deduced amino acid sequence of the enriched cDNA-clone (Fus c 1) showed large identity to the 60S acidic ribosomal protein P2 which is highly conserved among several species and also described as minor allergen in other mold species. The frequency of IgE reactivity of sera from F culmorum -sensitized subjects to rFus c 1 was approximately 35%. By enzyme allergosorbent test inhibition, we found 65% inhibition of mycoprotein IgE reactivity by rFus c 1 . On the opposite we found reduced IgE reactivity of rFus c 1 of 68% by using mycoprotein as inhibitor. Conclusions: Sensitization to mold allergens by the respiratory tract and subsequent oral ingestion of cross-reactive proteins may lead to severe food-allergic reactions. Thus, the 60S acidic ribosomal protein P2 of F venenatum probably is the reason for the described severe hypersensitivity reactions of the patient to Quorn-mycoprotein because of its potential cross-reactivity to the F culmorum allergen Fus c 1. (J Allergy Clin Immunol 2003;111:1106-10.)

Identification of hazelnut major allergens in sensitive patients with positive double-blind, placebo-controlled food challenge results

Background: The hazelnut major allergens identified to date are an 18-kd protein homologous to Bet v 1 and a 14-kd allergen homologous to Bet v 2. No studies have reported hazelnut allergens recognized in patients with positive double-blind, placebo-controlled food challenge (DBPCFC) results or in patients allergic to hazelnut but not to birch. Objective: We characterized the hazelnut allergens by studying the IgE reactivity of 65 patients with positive DBPCFC results and 7 patients with severe anaphylaxis to hazelnut. Methods: Hazelnut allergens were identified by means of SDS-PAGE and IgE immunoblotting. Further characterization was done with amino acid sequencing, evaluation of the IgE-binding properties of raw and roasted hazelnut with enzyme allergosorbent test inhibition, assessment of cross-reactivity with different allergens by means of immunoblotting inhibition, and purification by means of HPLC. Results: All the sera from the patients with positive DBPCFC results recognized an 18- and a 47-kd allergen; other major allergens were at molecular weights of 32 and 35 kd. Binding to the 18-kd band was inhibited by birch extract, indicating its homology with the birch major allergen, and abolished in roasted hazelnut. The 47-kd allergen is a sucrose-binding protein, the 35-kd allergen is a legumin, and the 32-kd allergen is a 2S albumin. Patients with severe anaphylactic reactions to hazelnut showed specific IgE reactivity to a 9-kd allergen, totally inhibited by purified peach lipid-transfer protein (LTP), which was heat stable and, when purified, corresponded to an LTP. Conclusions: The major allergen of hazelnut is an 18-kd protein homologous to Bet v 1, and the 9-kd allergen is presumably an LTP. Other major allergens have molecular weights of 47, 32, and 35 kd. (J Allergy Clin Immunol 2002;109:563-70.)

Determination of the allergenicity of various hazelnut products by immunoblotting and enzyme allergosorbent test inhibition

Although allergic reactions to hazelnuts are common especially in Europe, there are only a few investigations with regard to the influence of processing on the IgE-binding potency of hazelnut proteins. In this study the allergenicity of different hazelnut products, such as chocolate, nougat products, croquant or cookies, was examined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), immunoblotting and enzyme allergosorbent test (EAST) inhibition experiments using sera of 17 hazelnut-allergic individuals. In only a few cases did the immunoblotting experiments yield positive results as regards the allergenicity of the investigated products. By means of EAST inhibition a residual IgE-binding potency could be detected in almost all of the product extracts. Therefore hazelnuts are a potential hazard to allergic people even as an ingredient of processed foods.