Identification, Purification And Application Research Of Allergens From Milk And Hen's Egg

Objectives:Food allergy is one of the major public health issues today, and the identification, purification and standardization of food allergens are critical for prevention and treatment of anaphylactic diseases. Milk and hen's egg are investigated in this study. Specific antibodies from food allergic patients serum are used as probe. After identifying, the allergen of milk and eggs are attempted to be separated and purified. And then, the separated allergens are used as coating antigen and enzyme conjugated antigen to establish a double antigen sandwich enzyme linked immunosorbent assay (DAS ELISA) for food allergy detection. The clinical application performance of the method is inspected finally, aiming to provide experiment data for the methodology improvement and domestically production of food allergy diagnostic kits.Methods:1.Sample collection:The positive serums from milk allergic patients, hen's egg allergic patients and control serums from normal people were collected from clinical laboratory in children hospital of Tianjin.2. Identification of allergen components in milk/hen's egg:The total protein in milk/hen's egg was extracted. SDS-PAGE and Western Blot were used to differentiate and identify the protein composition and allergens.3. Allergen purification:The identified allergen components were separated and purified by degreasing, isoelectric precipitation, salting-out, gel chromatography, ultrafiltration and other possible methods. And the purified allergens were verified by Western Blot.4. The establishment and optimization of the DAS ELISA for milk allergy specific antibody detection:The separated milk allergen components were regrouped to prepare the coating antigen based on respective allergenicity inspected by ELISA. Horseradish peroxidase (HRP) were labeled with antigen to prepare enzyme conjugate antigen. Then the DAS ELISA for milk allergy detection was established and the working dilution and conditions were optimized.5. Preclinical application of DAS ELISA for milk allergy specific antibody detection: The new method was compared with commercialized kit (IVT Allergen Screen, Germany) by detecting clinical serum samples. The related reference including sensitivity, specificity, positive/negative predictive value, etc. were calculated to evaluate the application effect of the self-established method.Results1. Identification of allergen components in milk:9 protein bands were visible in the total protein ingredients of milk, among which the proteins with the molecular mass of 18kDa,14kDa,28kDa and around 70kDa reacted with positive serum. According to result, we conclude they areβ-lactoglobulin (BLG), a-lactoalbumin (ALA), caseins (CN) and several macromolecule proteins including Bovine serum albumin (BSA).2. Identification of allergen components in hen's egg:there were about 4 and 10 major protein bands visible in the total protein ingredients of egg white and egg yolk respectively. The allergens with the molecular mass of about 78kDa,43kDa,28kDa and 14kDa were confirmed with Western Blot in egg white, and were considered to be Ovotransferrin (OVT), Ovalbumin (OVA), Ovomucoid (OVM) and Lysozyme (Lys) respectively. There were more protein bands reacted positively in egg yolk but most of which proved to be nonspecific reaction.3. Purification of allergen components in milk/hen's egg:CN, macromolecule proteins (P1), BLG and ALA were successfully purified from milk, a range of sophisiticated methods for milk purification was established. One single protein OVM and several partly separated components were obtained from egg white.4. The allergenicity inspection and HRP labeling of allergens:4 milk allergen components were labeled with HRP. The results of ELISA coating with single component proved that the positive rate of DAS ELISA was obviously higher than traditional indirect ELISA. Among the 4 components, BLG and P1 had higher positive rates.5. The establishment and optimization of the DAS ELISA for milk allergy specific antibody detection:The proportion of P1, BLG, ALA and CN in the compound antigen fixed by ELISA was 30%,40%,20% and 10% respectively. The optimum dilution of the 4 allergen-HRP in the compound enzyme labeled antigen was 1:1500, 1:12000,1:6000 and 1:6000 for P1-HRP, BLG-HRP, ALA-HRP and CN-HRP respectively. The optimum coating concentration, reaction time and serum reacting volume was 50μg/ml, 1h and 10μl respectively.6. Preclinical application of DAS ELISA for milk allergy specific antibody detection: Patients serums were detected by this self-established method and the IVT Allergen Screen kit (MEDIWISS Analytic GmbH, Germany). The sensitivity, specificity and positive/negative predictive value of the method were 91.304%,94,286% 91% and 94% respectively, indicating favorable coincidence with the commercialized kit.Conclusion:1. Several allergen components from milk were identified. Furthermore, a range of sophisticated methods for milk purification was established and 4 components were successfully purified.2. Several allergen components were identified from egg white, nonspecific reaction was discovered when identifying egg yolk allergens. One single allergen was purified from egg white. But the methods for egg white purification needed further investigation.3. Milk allergen components were labeled with HRP. The DAS ELISA for milk allergy detection was established and the working dilution and reaction conditions were optimized. The performance of this new method was proved better than traditional indirect ELISA. According to the preclinical investigation, the specificity and sensitivity of this method were satisfactory. In addition, this method coincided well with the imported commercialized kit.