关键词: 德国小蠊, Bla g 2, 单克隆抗体, 蛋白质印迹

Abstract:

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 splenocytes 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 ELISA, 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.
 

Key words: Blattella germanica, Bla g 2, Monoclonal antibody, Western blotting