摘要
目的：
食物过敏是当今重大的卫生学问题,食物过敏原的鉴定、纯化、和标准化是食物过敏性疾病预防和治疗的中心环节。本研究以牛奶、鸡蛋中的过敏原为研究对象,以过敏患者血清中的特异性抗体为探针,对牛奶和鸡蛋中的过敏原组分进行鉴别,并设法将其纯化,作为原料制备包被抗原和酶标抗原,建立食物过敏的双抗原夹心酶免疫检测法,并对其临床应用性能进行考察,为食物过敏诊断试剂的改善和国产化提供实验依据。
方法：
1.血清收集：从天津市儿童医院检验科收集鸡蛋、牛奶过敏的阳性患者血清及正常对照血清。
2.牛奶/鸡蛋过敏原组分的鉴定：提取牛奶/鸡蛋中的总蛋白成分,经SDS-PAGE观察蛋白条带,以牛奶/鸡蛋过敏血清中的特异性抗体作为探针,通过免疫印迹实验鉴定其中的过敏原组分。
3.牛奶/蛋清过敏原组分的纯化：在鉴定工作的的基础上,通过去脂、等电点沉淀、盐析、凝胶层析、超滤等手段设法对牛奶/蛋清中的蛋白进行分离和纯化,提取出主要的过敏原组分,并进一步验证致敏性。
4.牛奶过敏特异性抗体的双抗原夹心酶免疫检测法的建立：通过ELISA考察牛奶过敏原组分的致敏性,将其进行合理配比制备包被抗原,同时对过敏原组分进行辣根过氧化物酶标记,制备酶标抗原,建立双抗原夹心法检测模式,摸索最佳工作浓度和反应条件。
5.双抗原夹心酶联免疫法检测牛奶过敏特异性抗体的初步临床应用：以目前临床应用的进口食物过敏检测试剂作为参考方法,与本实验建立的双抗原夹心法进行比较,测定同一批临床收集的血清标本,计算检测的灵敏度、特异度和阴阳性预测值,比较二者检测结果的符合率,对本研究建立的方法的临床应用效果进行考察。
结果：
1.牛奶过敏原组分的鉴定：在牛奶总蛋白粗提液中观察到约9个主要蛋白条带；鉴定出其中18kd、14kd、28kd、及70kd附近的多个条带出现阳性反应,推测依次为β-乳球蛋白、α-乳白蛋白、酪蛋白、以及包括牛血清白蛋白在内的多个大分子蛋白组分。
2.鸡蛋过敏原组分的鉴定：在蛋清总蛋白粗提液中观察到约4个主要蛋白条带,蛋黄总蛋白粗提液中观察到10个主要条带;鉴定出蛋清中分子量大约为78kd、43kd、28kd、14kd的多个条带出现阳性反应,判断为卵清转铁蛋白、卵白蛋白、卵类黏蛋白和溶菌酶：在蛋黄中检测到更多的显色条带,但在实验中出现了明显的非特异显色。
3.牛奶、蛋清过敏原组分的纯化：通过脱脂、等电点沉淀、凝胶层析、离子交换层析等手段,先后从牛奶中纯化出酪蛋白、大分子蛋白组分(P1)、β-乳球蛋白和α-乳白蛋白4个过敏原组分,建立了一套完善的纯化工艺;从蛋清中纯化出卵类黏蛋白一个单一组分及数个部分纯化组分。
4.过敏原的标记及致敏性考察：用辣根过氧化物酶标记了四种牛奶过敏原组分,并通过单一过敏原组分的酶联免疫吸附实验结果发现,双抗原夹心法的阳性率明显高于间接法,而四种过敏原中,β-乳球蛋白和P1组分的阳性率较高。
5.牛奶过敏特异性抗体的双抗原夹心酶免疫检测法的建立及条件优化：根据实验确定配比抗原中各组分的比例为P1：β-乳球蛋白：α-乳白蛋白：酪蛋白=30%：40%：20%：10%,复合酶标抗原中P1、β-乳球蛋白、α-乳白蛋白、酪蛋白的比例依次为1：1500、1：12000、1：6000和1：6000。最佳包被浓度为50μg/ml,最佳反应时间为1h,最佳血清用量为10μl。
6.双抗原夹心酶联免疫法检测牛奶特异性抗体的初步临床应用：用本实验建立的双抗原夹心法ELISA检测模式和德国MEDIWISS Analytic GmbH公司的IVT Allergen Screen食物过敏检测试剂盒同时测定一批临床血清,通过比较二者的测定结果得出该方法的灵敏度为91.304%,特异度为94.286%,阳性预测值(PPV)为91%,阴性预测值为94%,同参考方法有较高的符合率
结论：
1.鉴定出牛奶中的多个过敏原组分,并成功地从牛奶中纯化出4个过敏原组分,摸索出一套完善的牛奶过敏原纯化工艺。
2.鉴定出蛋清中的多种过敏原组分,在鉴定蛋黄中过敏原的实验中发现非特异反应,成功地从蛋清中纯化出一种单一过敏原组分,蛋清过敏原的纯化方法有待进一步改进。
3.对牛奶过敏原进行了辣根过氧化物酶标记,建立了牛奶过敏的双抗原夹心酶免疫检测方法,并确定了试剂的最佳工作浓度和反应条件,通过实验证实其检测性能优于传统的间接法酶免疫检测模式,并在初步临床应用中表现出很高的灵敏度和特异度,同临床应用的进口检测试剂有较高的符合率。
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.
Results
1. 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.
引文
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