德国小蠊变应原Bla g 2单克隆抗体的制备与鉴定

Production and identification of monoclonal antibodies against cockroach allergen Bla g 2

目的制备德国小蠊(Blattella germanica)变应原Bla g 2的单克隆抗体并鉴定其特异性。方法以纯化的0.2 mg/ml Bla g 2蛋白溶液为抗原免疫雌性BLAB/c小鼠6只,免疫4次,每次间隔1周。免疫后第5天取小鼠尾静脉血,制备小鼠血清,第6天加强免疫1次,第7天处死小鼠取脾脏,制备脾细胞悬液。测定小鼠抗血清滴度,运用杂交瘤技术将免疫脾细胞与骨髓瘤细胞融合,采用有限稀释法对阳性孔杂交瘤细胞进行克隆化。蛋白质印迹(Western blotting)检测单克隆抗体特异性,间接ELISA测定单克隆抗体效价,双抗夹心ELISA鉴定单克隆抗体的亚型。结果在小鼠血清稀释至1∶16 000时A450值仍>0.100,判断为免疫成功。通过杂交瘤技术获得2株能稳定分泌Bla g 2单克隆抗体的杂交瘤细胞,标记为Bla g 2-1、Bla g 2-2。Western blotting结果表明,Bla g 2-1和Bla g 2-2单克隆抗体对Bla g 2蛋白均具有高度特异性。间接ELISA测得2株单克隆抗体的效价分别为Bla g 2-1(1∶16 000)、Bla g 2-2(1∶8 000)。双抗夹心ELISA结果表明单克隆抗体蛋白亚类均为免疫球蛋白G1(IgG1)。结论制备的德国小蠊变应原单克隆抗体Bla g 2能与Bla g 2蛋白发生特异性结合。

Objective To produce anti-cockroach Blattella germanica allergen （Bla g 2） monoclonal antibody （mAb） and identify the specificity. Methods Six BLAB/c female mice were immunized with Bla g 2 protein every other week, for 4 times. Serum was obtained at 5 days after immunization, and an immune boosting was performed on the next day. On day 7, the mice were sacrificed and spleens were obtained to prepare the splenic cell suspension. The antiserum titer was determined and hybridoma cells were obtained by fusing the splenoeytes with myeloma cells. The positive hybridoma cells were cloned by the limited dilution method. The specificity, titer, and subtype of monoclonal antibody were detected by Western blotting, indirect ELISA, and EL1SA, respectively. Results The A450 value was 〉 0.100 when the mouse serum was diluted at 1 ： 16000, which suggested the successful immunization. Two hybridoma cell lines that were capable of stably secreting Bla g 2 mAbs were obtained, named Bla g 2-1 and Bla g 2-2. Western blotting demonstrated that both Bla g 2-1 and Bla g 2-2 mAbs specifically binded the Bla g 2 protein. The titers of mAbs from Bla g 2-1 and Bla g 2-2 were 1 ： 16 000 and 1 ： 8 000, respectively. ELISA results showed that both mAbs belonged to the IgG1 subtype. Conclusion Two monoclonal cell subtypes expressing Bla g 2 mAbs have been obtained, which can specifically bind the Bla g 2 protein.