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目的制备和鉴定鼠抗鸡蛋主要过敏原卵类黏蛋白的单克隆抗体。方法利用鸡蛋卵类黏蛋白(ovm)为免疫原,免疫Balb/c小鼠,取免疫小鼠脾细胞与小鼠骨髓瘤NS-1细胞融合。采用半固体培养基法和有限稀释法相结合的方法快速筛选获得稳定分泌的特异性杂交瘤细胞。用杂交瘤细胞株诱生小鼠腹水,应用蛋白A亲和层析法进行抗体纯化。采用Ig类与亚类鉴定试剂盒鉴定该单克隆抗体的Ig亚型;通过间接ELISA、Western blotting鉴定该单克隆抗体的特性和交叉性。利用双抗体夹心ELISA法检测食品中的鸡蛋过敏原。结果获得4株可稳定分泌鼠抗卵类黏蛋白的单克隆抗体,分别命名为1G8,2H5,3A7,4G4,其Ig亚型均为IgG1。且4株单抗效价均在10-8以上。ELISA和Western blotting分析表明该4株单抗均能特异性识别卵类黏蛋白,并且建立双抗体夹心ELISA的方法可以准确的检测出食品中鸡蛋过敏原的存在。结论成功制备了4株高效价的鼠抗卵类黏蛋白的单克隆抗体,为建立卵类黏蛋白检测及纯化方法奠定了基础,也可为食品中鸡蛋过敏原的检出提供依据。
OBJECTIVE To prepare and identify monoclonal antibodies against mouse egg against egg major mucin. Methods Balb / c mice were immunized with the ovum protein of ovum as immunogen, and the spleen cells of immunized mice were fused with the mouse myeloma NS-1 cells. Semi-solid medium method and limited dilution method were combined to rapidly screen and obtain stable and secreted specific hybridoma cells. Mouse ascites was induced by hybridoma cell line and purified by protein A affinity chromatography. The Ig subtype of the monoclonal antibody was identified by using the Ig class and subclass identification kit. The characteristics and crossings of the monoclonal antibody were identified by indirect ELISA and Western blotting. Detection of Egg Allergens in Food by Double Antibody Sandwich ELISA. Results Four monoclonal antibodies that could stably secrete mouse anti-ovomucin were obtained and named as 1G8, 2H5, 3A7 and 4GG, and their Ig subtypes were both IgG1. And 4 monoclonal antibody titers were above 10-8. ELISA and Western blotting analysis showed that the four monoclonal antibodies could specifically recognize the egg mucins, and the establishment of double antibody sandwich ELISA method can accurately detect the presence of egg allergens in food. Conclusion Four monoclonal antibodies against murine anti-ovomucin were successfully prepared, which laid the foundation for the establishment of the method for the detection and purification of egg mucins. It also provided the basis for the detection of egg allergens in food.