Identification Of IgE Sequential Epitopes Of Ovomucoid With Enzyme-linked Immunosorbent Assay

Objective:Egg allergy is a widespread disease that mainly affects children,and ovomucoid is the most important egg allergen.Allergen epitopes,especially linear epitopes,play an important role for the occurrence of food allergy.In this study,we established an ELISA method that can be used for the identification of linear epitopes of food allergens.Take ovomucoid as research object,we have analyzed its linear epitope spectrum,and the differences in epitope recognition between children and adults were compared.Analyze the relationship between epitope s Ig E antibody heterogeneity and clinical manifestations to further improve our understanding of antigen epitopes.Methods:1.Establishment of a linear epitope screening method for food allergensUse biotin-streptavidin interaction to prepare biotinylated bovine serum albumin-streptavidin universal enzyme label reaction plate.Add biotinylated allergen epitope peptide as coating antigen,and then add serum and HRP-labeled goat anti-human Ig E antibody to form epitope antigen-specific Ig E antibody-HRP-labeled goat anti-human Ig E complex.And then a liquid-phase enzyme-linked immunosorbent assay for indirect antigen coating was established.According to the reported results of ovomucoid epitope research,relevant linear epitope peptides were synthesized.And the mixed serum of egg allergy patients（P）and the healthy control（N）were used as the antibodies to be tested,and their A₄₅₀were detected respectively.We optimized the experimental conditions,including the coating concentration and coating conditions of biotinylated bovine serum albumin,blocking solution,biotinylated epitope concentration,serum dilution and incubation conditions,and enzyme-labeled antibody dilution,by calculating the ratio of A_P/A_N.And the sensitivity and repeatability of the method were evaluated.2.Identification of linear epitope of ovomucoid and applicationSera of egg allergic and non-egg allergic patients were collected,use the established method described above to detect ovomucoid epitope-specific Ig E antibody levels under optimal conditions.The response frequency of each epitope were analyzed,and the difference of characteristics of epitope recognition between children and adults were compared.The heterogeneity of epitope-specific Ig E antibody was analyzed,and its relationship with clinical manifestation was verified by basophil activation test and rat basophil leukemia cellβ-hexosaminidase release test.Results:1.This study successfully established an indirect ELISA method for linear epitope detection of food allergens.By optimizing the reaction conditions,the best coating concentration of BSA-Bio was 1μg/m L,and the best coating condition was4°C overnight.1%BSA was the most optimal blocking solution.The optimal concentration of peptide is 5μg/m L.The optimal reaction condition of serum samples were diluted 1:20 and reacted at 37°C for 2 h.The optimal dilution of enzyme-labeled antibody was 1:2000.This method was sensitive,and the coefficient of variation within and between batches were less than 10%,the repeatability was good,indicating that it was suitable for the linear epitope study of ovomucoid.2.A total of eight ovomucoid epitope peptides were synthesized,of which four peptides（AA1-10,AA9-20,AA101-114,AA113-124）had a reaction frequency higher than 50%.The reaction frequency of AA31-44 and AA175-186 were the lowest.85%of the patients recognized at least one of the peptides,while 15%of the patients did not recognize any of the peptides,and 24%of the patients could recognize all the peptides.Adults showed stronger and more frequent allergen and epitope recognition than children.The reaction frequency of children response to ovomucoid was 60%,while that of adults was 78%.The positive rate of each peptide in children was lower than 50%,while that in adults was higher than 50%.In adults,AA1-10,AA9-20,AA101-114 were mainly recognized.And in children,multiple epitopes（AA1-10,AA9-20,AA46-59,AA91-104,AA101-114,AA113-124）showed similar reaction intensity.The average number of recognition epitopes of adults was also higher than that of children.Patients with multiple systemic allergy symptoms recognized more epitopes than patients with only skin symptoms,and the heterogeneity of epitope-specific Ig E antibodies may be used as a marker of the severity of symptoms.The basophil activation test andβ-hexosaminidase release test confirmed that the serum with a large number of epitope recognition could more effectively induce effector cell responses.Conclusion:1.By preparing the biotinylated bovine serum albumin-streptavidin enzyme-labeled reaction plate,an indirect ELISA method that can be used for the screening of linear epitopes of food allergens was initially established.This method was sensitive and reproducible.2.Four important ovomucoid epitopes were identified:AA1-10,AA9-20,AA101-114,AA113-124.Children and adults had different epitope recognition characteristics.The heterogeneous intensity of the epitope-specific Ig E antibody can be used as a marker of the severity of symptoms to predict the degree of disease and the development of tolerance.