The Heterogeneity of Ovomucoid-Specific IgE Idiotype Is Associated With Egg Allergy Symptom Severity.

Immunoglobulin E (IgE)-mediated egg allergy presents as one of the most common food allergies. The level of specific IgE (sIgE) antibody is widely used as an important in vitro diagnostic indicator. However, sIgE antibody levels are often inconsistent with the clinical manifestations of patients. The heterogeneity of egg-specific IgE idiotypes (sIgE-IDs) may help reflect clinical egg allergy severity. Eight peptides were synthesized, corresponding to the linear epitopes of ovomucoid (OVM). The sIgE-IDs of egg-allergic patients were detected by enzyme-linked immunosorbent assay. Fresh peripheral blood was collected from patients with different heterogeneity strength of sIgE-ID, and egg extract was used as a stimulus to the basophil activation test (BAT). RBL-2H3 cells were sensitized with serum with different strength of sIgE-ID heterogeneity and the release rate of ß-hexosaminidase was calculated. Among 75 patients with egg allergy, 24% had sIgE for all epitopes and 85% had sIgE for at least one epitope. Analysis of individual patients revealed differences in epitope recognition patterns among the patients, that is, heterogeneity in sIgE-ID. More importantly, the number of IgE-positive peptides had a strong correlation with allergic symptoms in egg-allergic patients (r = 0.706). BAT and RBL-2H3 cell degranulation confirmed that higher sIgE-ID heterogeneity strength was more effective in inducing effector cell responses. Our results suggest that the greater the heterogeneity strength of OVM-sIgE-ID, the more severe the allergic symptoms.

A Simple and Rapid Light-Initiated Chemiluminescence Assay for Quantitation of Artemisia-Specific Immunoglobulin E.

BACKGROUND: Light-initiated chemiluminescence assay (LICA) is a homogeneous assay that has been successfully used for the quantitation of food allergen-specific immunoglobulin E (sIgE), but not inhaled allergen-sIgE. Simultaneously, current assays used to detect allergen-sIgE are serum consuming and/or time consuming. Hence, we established a method for the quantitation of Artemisia-sIgE based on LICA and verified its performance according to the clinical guideline documents, laying a foundation for the quantitation of inhaled and food allergen-sIgE in parallel on LICA. METHODS: The assay was established after optimizing the first incubation time and the dilutions of Artemisia-coated chemibeads, biotinylated goat anti-human IgE, and serum. In order to quantitate Artemisia-sIgE, the calibration curve was established with a high positive serum of known concentration. The assay performance was confirmed per the clinical guideline documents. In addition, the correlation between the results of LICA and capture enzyme-linked immunosorbent assay was evaluated. RESULTS: The developed LICA's coefficients of variation of repeatability and intermediate precision were 3.20%, 2.14%, and 3.85% and 4.30%, 4.00%, and 4.40%, respectively. The limit of detection was 0.10 kUA/L, and the limit of quantitation was 0.11 kUA/L. The range of linearity was from 0.27 kUA/L to 97.53 kUA/L (r = 0.9968). The correlation coefficient (r) for the correlation analysis between results of LICA and capture ELISA was 0.9087. This assay was successfully applied in 64 human serum samples, showing good sensitivity (82.20%) and specificity (100%). CONCLUSION: An Artemisia-sIgE quantitation assay based on LICA was successfully established. Its performance satisfied the clinical requirements and could be widely used in clinical laboratories.

Component resolved diagnosis of egg yolk is an indispensable part of egg allergy

Introduction and objectives It was urgent to explain the role of egg yolk allergen sensitization to the egg allergic population and we would evaluate the diagnostic value of allergen components in whole eggs, including egg white and egg yolk. Materials and methods Firstly, we collected 99 positive and 21 negative sera against egg allergy. Then we used modified enzyme linked immunosorbent assay (ELISA) to survey specific IgE (sIgE) to all-proven and single component in eggs, Ovomucoid (Gal d 1), Ovalbumin (Gal d 2), Ovotransferrin (Gal d 3), Lysozyme C (Gal d 4), Serum Albumin (Gal d 5), and YGP42(Gal d 6) in allergic and non-allergic populations. Last but not least, we studied the sIgE reactivities to egg allergen components by receiver operating characteristic (ROC) analysis. Results Among egg-allergic individuals, nearly 10% were sensitized to five of six egg allergen components, and the cross-reaction frequency between two egg yolk allergens with Gal d 1 was about 30% in the groups diagnosed with egg allergy or non-allergy. The best component-combination diagnosis in egg allergy of Gal d 1+ Gal d 6 demonstrated the largest area under curve (AUC) of 0.994. Conclusions Our results suggested that there were individual differences in allergenicity of different egg allergen components, especially in the samples negative to egg allergy diagnosed but sensitive to egg yolk components. It was indicated that component resolved diagnosis of egg yolk improved the value for egg allergy management indispensably (AU)

IgE and IgG4 responses to shrimp allergen tropomyosin and its epitopes in patients from coastal areas of northern China.

Sensitization to allergens and their peptides varies among patients due to geographical or ethnic differences. The present study aimed to investigate immunoglobulin (Ig)E and IgG4 responses to tropomyosin and its peptides in shrimp allergic patients from northern China. A total of 92 subjects were studied, including 35 shrimp allergic patients, 29 patients with house dust mite (HDM) and/or cockroach allergic patients and 28 healthy volunteers. Serum IgE and IgG4 antibodies to recombinant shrimp tropomyosin (rPen a 1) and its peptides were measured by means of a light­initiated chemiluminescent assay. A total of 9 major sequential epitopes of Pen a 1 reported in the literature were synthesized. Of 35 shrimp allergic patients, 25 (71.4%) had positive Pen 1­specific IgE (sIgE) antibodies and 22 (62.9%) contained measurable rPen a 1­specific IgG4 (sIgG4) antibodies. A strong IgG4 response accompanied the presence of IgE to Pen a 1. None of the patients with HDM and/or cockroach allergy demonstrated IgE reactivity to rPen a 1. The reaction frequency of IgE binding epitope was 20­48%, while that of IgG4 binding epitope was 63.6­90.9%. The IgE and IgG4 recognition patterns of the tropomyosin peptides demonstrated high interpatient heterogeneity. Diversity of IgE binding epitopes was positively correlated with Pen a 1 sIgE levels. In the study population, tropomyosin was a major allergen recognized by the majority of shrimp allergic patients, which is consistent with previous reports. However, none of the 9 epitopes are major (reaction frequency >50%) IgE­binding regions, indicating the epitopes profile may be different in other regions.

A light-initiated chemiluminescent assay for rapid quantitation of allergen-specific IgG4 in clinical samples.

BACKGROUND: An increase in allergen-specific IgG4 (sIgG4), which serves as a blocking antibody, is associated with acquisition of immune tolerance after immunotherapy. In this study, we developed a rapid, sensitive, and homogeneous immunoassay based on the light-initiated chemiluminescent assay (LICA) technology for quantifying allergen sIgG4 in serum samples. METHODS: Allergen sIgG4 was measured in vitro by incubating the sample with biotinylated allergens and chemiluminescent beads coated with anti-human IgG4 antibody, followed by the addition of streptavidin-coated sensitizer beads. Multiple tests were performed to optimize the working conditions of the LICA and evaluate its performance. RESULTS: We established the optimal concentration of biotinylated allergens (250 ng/mL), the optimal dilution range (1:8 for Gal d 1, Gal d 2 sIgG4 and 1:4 for Gal d 3, Gal d 4 sIgG4), and the optimal incubation time (20 min for Gal d 1, Gal d 2 sIgG4 and 40 min for Gal d 3, Gal d 4 sIgG4). The lower limit of quantification (LLOQ) was 0.261 ng/mL. The coefficient variation (CV) of the LICA was <10%. The assay was unaffected by general interfering substances at physiological concentrations. It exhibited excellent accuracy to detect allergen-sIgG4 in human serum. Additionally, we demonstrated that the levels of Gal d 1, Gal d 2, and Gal d 3-sIgG4 were significantly higher in the egg allergy group (p < .05), but no differences were found between the groups for Gal d 4-sIgG4. CONCLUSIONS: The LICA demonstrated satisfactory performance and can be used for quantifying allergen sIgG4 in clinical practice.

Identification and characterization of ovary development-related protein EJO1 (Eri s 2) from the ovary of Eriocheir sinensis as a new food allergen.

SCOPE: Crab is a major source of shellfish allergens. Most studies have focused on allergens in crab muscle (CM) rather than on allergens in crab ovary (CO). This study aimed to identify potential allergens in CO from Eriocheir sinensis. METHODS AND RESULTS: Dot blot and immunoblotting results revealed the differential reactivity of CM and CO extracts to positive sera from patients allergic to crabs. Four CO proteins showed good specific IgE-binding activities in 2-DE/immunoblotting analysis; mass spectrometry identified the proteins as hemocyanin, vitellogenin, ovary development-related protein EJO1and EJO2. The recently identified allergen EJO1 is named 'Eri s 2' in the World Health Organization and International Union of Immunological Societies (WHO/IUIS) allergen nomenclature database. Recombinant Eri s 2 exhibited good reactivity to positive sera, and pre-incubation with recombinant Eri s 2 abrogated the reactivity of positive sera from two patients to CO in a dose-dependent manner. Moreover, co-incubation of recombinant Eri s 2 with patient basophils dose-dependently promoted basophil activation, confirming the biological activity of Eri s 2. CONCLUSION: CO tissue is an important allergen source, and Eri s 2 is a new crab allergen. This study provides insights that will be useful for component-resolved diagnostics for crab allergy.

Identification of the major allergenic epitopes of Eriocheir sinensis roe hemocyanin: A novel tool for food allergy diagnoses.

Crab meat and roe are highly nutritious delicacies in China. While extensive research has been conducted for allergens derived from crab-meat, data relevant to the allergenic potential of crab roe derived proteins, of which hemocyanin is a principal contender, are almost entirely absent. Using bioinformatics prediction and IgE-binding assays, the three principal immunodominant epitopes of hemocyanin were identified and then combined as a single recombinant fusion protein (rHc). This together with the full-length recombinant protein (Hc) were expressed in Escherichia coli and subsequently identified by SDS-PAGE and immunoblotting. Ninety-five percent of our patients were found to carry rHc-specific IgE antibodies by ELISA. Dot-blot inhibition, together with ELISA inhibition studies, showed that pre-incubation of patient sera with the recombinant epitope protein could inhibit26% to 63% (mean: 50%) of IgE binding to immobilized, full-length Hc and the dose-response curve represents as a sigmoid shape. The recombinant protein (rHc) represents a versatile biologic tool with which to diagnose and investigate therapies for E. sinensis allergy.