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利用大豆主要过敏原Gly m Bd 30K蛋白抗原表位蛋白为免疫原免疫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。利用双单抗夹心ELISA法检测大豆过敏原。结果表明:获得6株可稳定分泌鼠抗大豆主要过敏原Gly m Bd 30K蛋白的单克隆抗体,分别命名为1C10,1D12,2D1,4B4,5F9,6B12,其Ig亚型除1D12和4B4为IgG2a外,其余均为IgG1,且6株单抗效价均在10-5以上。ELISA和Western Blotting分析表明该6株单抗均能特异性识别大豆主要过敏原Gly m Bd 30K蛋白,并且建立双单抗夹心ELISA的方法可以准确检测出大豆过敏原的存在。鼠抗大豆主要过敏原Gly m Bd 30K蛋白抗原表位区蛋白的单克隆抗体的成功制备,以及双单抗夹心ELISA检测系统的建立,为大豆主要过敏原蛋白的检测奠定了基础,也可以为食品中大豆过敏原的检出提供依据。
BALB / c mice were immunized with Gly m Bd 30K protein epitope protein, a major soybean allergen, and splenocytes from immunized mice were fused with mouse myeloma NS-1 cells. Semi-solid medium method and limiting dilution method were combined to obtain the stable secreted specific hybridoma cells. The mouse ascites was induced by hybridoma cell line and the protein was 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 Soybean Allergens by Dual Monoclonal Antibody Sandwich ELISA. The results showed that six monoclonal antibodies that could stably secrete Gly m Bd 30K protein, which is the key allergen of mouse anti-soybean, were obtained and named 1C10, ID12, 2D1, 4B4, 5F9 and 6B12 respectively. The Ig subtypes except IgD and IgG4 , The rest are IgG1, and 6 monoclonal antibody titer are above 10-5. ELISA and Western Blotting analysis showed that the six monoclonal antibodies could specifically recognize Gly m Bd 30K protein, a soybean major allergen, and the establishment of double-antibody sandwich ELISA method can accurately detect the presence of soybean allergens. The successful preparation of the monoclonal antibody against the mouse anti-soybean major allergen Gly m Bd 30K protein epitope protein and the establishment of the double-antibody sandwich ELISA test system laid the foundation for the detection of the major allergen protein in soybeans, Food allergen detection of soy provide the basis.