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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 prepare monoclonal antibody against Bla g 2 of Blattella germanica and to identify its specificity. Methods Six female BLAB / c mice were immunized with the purified 0.2 mg / ml Bla g 2 protein solution and immunized four times with one week interval. On the 5th day after the immunization, the tail vein blood of the mice was taken, the mouse serum was prepared, the immunity was boosted on the 6th day, and the mice were sacrificed on the 7th day to take the spleen to prepare the spleen cell suspension. The titer of the antiserum was determined. The immunized spleen cells were fused with myeloma cells by the hybridoma technique. The positive hybridoma cells were cloned by the limiting dilution method. The specificity of the monoclonal antibody was detected by Western blotting, the titer of monoclonal antibody was measured by indirect ELISA, and the subtype of monoclonal antibody was identified by double-antibody sandwich ELISA. Results When the serum of mice was diluted to 1: 16 000, the A450 value was still> 0.100, and the immunization was successful. Two hybridoma cells that can stably secrete Bla g 2 monoclonal antibody were obtained by the hybridoma technique and labeled as Bla g 2-1 and Bla g 2-2. Western blotting showed that the monoclonal antibodies against Bla g 2-1 and Bla g 2-2 were highly specific to Bla g 2 protein. Indirect ELISA measured two monoclonal antibodies were Bla g 2-1 (1:16 000), Bla g 2-2 (1: 8000). Double-antibody sandwich ELISA results showed that the monoclonal antibody protein subclasses were immunoglobulin G1 (IgG1). Conclusion The prepared Bla g2 allergen monoclonal antibody Bla g 2 can specifically bind Bla g 2 protein.