花生主要过敏原Ara h1单克隆抗体的制备与应用

发布时间：2017-12-31 01:35

本文关键词：花生主要过敏原Ara h1单克隆抗体的制备与应用　出处：《免疫学杂志》2017年01期 　论文类型：期刊论文

【摘要】：目的制备与鉴定抗花生主要过敏原Ara h1单克隆抗体,建立双单抗ELISA法,检测食品中花生过敏原。方法以花生主要过敏原Ara h1为抗原免疫Balb/c小鼠,融合免疫鼠脾细胞和小鼠骨髓瘤细胞NS-1。半固体培养基法结合有限稀释法筛选稳定分泌抗体的杂交瘤细胞株后诱生小鼠腹水,采用蛋白A亲和层析法获得纯化抗体。利用Ig类与亚类鉴定试剂盒鉴定该单克隆抗体的Ig亚型。间接ELISA法和Western blot鉴定抗体效价和特异性以及与其他过敏源的交叉反应性。建立双单抗夹心ELISA法检测食品中的花生过敏原。结果共获得抗Ara h1细胞株4株,分别命名为2G9、5G4、5B5、2C7,效价均高于1∶106。经抗体亚型鉴定,4株抗体均为Ig G1型。Western blot的结果表明4株抗体均能识别Ara h1,其中2G9与5G4结合能力较强。在特异性检测实验中,2G9和5G4与其他种类过敏原无交叉反应。通过建立双单克隆抗体夹心ELISA法,发现花生Ara h1蛋白的检出低限为:5 ng/ml,标准曲线在~80 ng/ml范围内线性良好。利用此法检测了10种食品,结果显示在5种含有花生成分的食品中均检测到了花生过敏原。结论获得高效价抗体4株,建立了高效、高特异性的食品中花生过敏原的检测方法,为食品中花生过敏原的检测提供了依据。
[Abstract]:Objective to prepare and identify monoclonal antibodies against Ara h1, a major peanut allergen, and to establish a double monoclonal antibody ELISA method. Methods the main allergen of peanut, Ara H1, was used as antigen to immunize Balb/c mice. Fusion immunized mouse spleen cells and mouse myeloma cells NS-1.The semi-solid medium combined with limited dilution method was used to screen hybridoma cell lines secreting antibody stably and then induce ascites in mice. Purified antibody was obtained by affinity chromatography of protein A. Ig subtype of monoclonal antibody was identified by Ig and subclass identification kit. Indirect ELISA and Western were used to identify the monoclonal antibody. Blot was used to identify antibody titers, specificity and cross-reactivity with other allergens. A double monoclonal antibody sandwich ELISA method was established for the detection of peanut allergens in food. H1 cell line 4. They were named as 2G9, 5G4, 5B5, 2C7, and their titers were higher than 1: 106. The antibody subtypes were identified. The results showed that all the four antibodies could recognize Ara H1. The binding ability of 2G9 to 5G4 was stronger. In the specific test, there was no cross reaction between 2G9 and 5G4 with other allergens. A sandwich ELISA method with double monoclonal antibodies was established. It was found that the detection limit of peanut Ara H1 protein was 1: 5 ng / ml, and the standard curve was linear in the range of 80 ng/ml. Ten foods were detected by this method. The results showed that peanut allergens were detected in 5 kinds of foods containing peanut ingredients. Conclusion High titer antibodies of 4 strains were obtained and a high efficiency and high specificity method was established for the detection of peanut allergens in food. It provides the basis for the detection of peanut allergen in food.
【作者单位】：深圳大学过敏反应与免疫学研究所;
【基金】：国家自然科学基金(81271950) 广东省工程技术研究开发中心项目(2013158925) 广东省对外科技合作项目(2013B051000088) 深圳市科技计划国际科技合作项目(GJHZ20130408174112021) 深圳市科技计划基础研究项目(JCYJ20140418095735604)
【分类号】：R392
【正文快照】： concentration from 5 ng/ml to 80 ng/ml and the detection limit is 5 ng/ml of peanut allergen protein.Ten foodproducts were tested using this method,and 5 products were found contain the peanut allergen protein,which wereconsistent with the food allergen

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