小麦GLB 1蛋白单克隆抗体的制备及鉴定

Preparation and Application of Monoclonal Antibodies Against Allergen Glb- 1

以小麦主要过敏原Glb-1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。采用细胞融合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株,用杂交瘤细胞株诱生小鼠腹水并用蛋白A亲和层析法纯化抗体后检测。采用间接ELISA法鉴定该单克隆抗体的IgG亚型;通过间接ELISA鉴定该单克隆抗体的特性和交叉性。利用双单抗夹心ELISA法检测单抗的抗原表位特异性。结果表明:共获得4株可稳定分泌小鼠抗小麦主要过敏原Glb-1蛋白的单克隆抗体,分别命名为1C4、4H5、1A9、4F5,经检测其Ig亚型均为IgG1,且4株单抗效价均在10-9以上。ELISA结果分析表明该4株单抗均能特异性识别小麦主要过敏原Glb-1蛋白且和其他常见食物无交叉反应性。将4株单抗两两配对进行ELISA实验,结果发现1C4与4H5可能有不同的抗原表位,以此建立的双抗夹心ELISA系统可以检测小麦Glb1-G3蛋白。实验成功制备了鼠抗小麦主要过敏原Glb-1蛋白抗原的单克隆抗体,并且建立了双单抗夹心ELISA检测系统,为小麦主要过敏原蛋白的检测奠定了基础。

BALB/c mice were immunized with G1 b -1 protein,the main allergen from wheat.The splenocytes of the immunized mice were fused with NS-1 myeloma cells.Cell fusion and limited dilution assay were used to screen hybridoma with stable secretion character and mice ascites were induced by the selected bybridoma.Monoclonal antibodies （McABs）were purified using affinity chromatography and further tested by their specificity,subtype,titers and cross-reactivity.The antigen epitope specificity of the McABs was examined by ELISA method.Four cell lines secreting McABs against wheat G1 b-1 pro-tein were obtained,named 1C4,4H5,1A9 and 4F5,respectively.The four McABs belong to IgG1subtype and their titer was above 9 -10 .Moreover,all the four McABs specially recognized wheat G1 b-1 protein and had no cross-reaction with other familiar foods.In order to establish a double-antibody sandwich ELISA system to detect wheat GIB1 proteins,we paired off the four monoclonal antibodies for ELISA assay and found 1 C4 and 4h5 might have different epitopes.Four mice anti-wheat G1 b-1 protein McABs were prepared successfully and a double-antibody sandwich ELISA method was set up in this work,which may facilitate other wheat allergen protein detection.