牛奶过敏原β-乳球蛋白的单克隆抗体制备及其双抗体夹心法的建立

Preparation of monoclonal antibodies and setting-up a sandwich-ELISA system against allergen β-lactoglobulin from milk

中文关键词:  牛奶β-lactoglobulin蛋白  单克隆抗体  特性鉴定  过敏原检测

英文关键词:β-lactoglobulinprotein from milk  Monoclonal antibody  Characterization  Allergen test

中文摘要:

      目的 制备与鉴定牛奶主要过敏原β-乳球蛋白的单克隆抗体，以及建立双抗体夹心检测法。方法 以牛奶主要过敏原β-乳球蛋白（β-LG）为抗原免疫BALB/c小鼠，融合免疫鼠脾细胞和小鼠骨髓瘤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。杂交瘤细胞株诱生小鼠腹水, 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方法和WesternBlot鉴定抗体效价和特异性以及与其他过敏原的交叉反应性。建立双单克隆抗体夹心法，检测牛奶过敏原β-乳球蛋白。结果 共获得抗β-LG细胞株6株，分别命名为1H8，4A7，4C3，1F9，1G5，3D11，效价均高于20万。经抗体亚型鉴定，6株抗体均为IgG1型。Westernblot的结果表明6株抗体能识别β-LG。在特异性检测实验中，6株抗体与其他种类食物过敏原无交叉反应，而1G5和3D11与牛奶酪蛋白过敏原有交叉反应性。通过建立双单克隆抗体夹心ELISA法，发现牛奶β-LG蛋白的检出低限为：15.625 ng/mL，标准曲线在15.625~250 ng/mL 范围内线性良好。结论 获得高效价抗体6株，建立了高效、高特异性的牛奶过敏原β-LG的检测方法，为食品中牛奶过敏原的检测提供了依据。

英文摘要:

      Objective To prepare monoclonal antibodies against allergenβ-lactoglobulin from milk and to characterize,then a sandwich-ELISA system was set up to detect the presence of milk allergens. Methods BALB /c mice were immunized withβ-lactoglobulinprotein, the main allergen from peanut.Using hybridoma and limit dilution technique,the fusion cell strain secreting antibodies stably was screened.The cells were used to induce ascites in mice and monoclonal antibodies were purified using affinity chromatography on immobilized protein A.The subclass of Ig, speciality, titers and cross-reactvity of the antibdies were detected with indirect enzymelinked immunosorbent assay(ELISA) and western blot.We used a method of sandwich-ELISA to detect β-lactoglobulin allergen protein tracein milk with these antibodies. Results Six hybridoma cell lines secreting McAbs against antigenic epitope of β-lactoglobulin protein from milkwere obtained, which were denominated as 1H8,4A7,4C3,1F9,1G5,3D11.The titers ofsixmonoclonal antibodies(1H8,4A7,4C3,1F9,1G5,3D11) were higher than 1:2 00 000 ( P/N＞2.1).The six McABs belong to IgG1subtype.Results of western blot and ELISA indicated that allantibodies could bind toβ-lactoglobulinspecifically,and had no cross-reaction with other familiar foods, but 1G5 and 3D11had cross-reactivity with casein allergen from milk.A sandwich-ELISA system was set up to detect the presence of milk allergens.Our results indicated that the standard curve is linear withβ-lactoglobulin allergen protein concentration from 15.625~250 ng/mL and the detection limit is 15.625 ng/mL of β-lactoglobulin allergen protein. Conclusion These six monoclonal antibodies against of β-lactoglobulinprotein from milk were prepared successfully,and a sandwich-ELISA system was set up to detect the presence of milk allergens,which would facilitate establishing detection method of milk allergen.