番茄细菌性叶斑病菌单克隆抗体的制备与鉴定

Preparation and identification of monoclonal antibody against Pseudomonas syringae pv.tomato

用纯培养的番茄细菌性叶斑病菌(Pseudomonas syringae pv.tomato,Pst)免疫BALB/c小鼠,取免疫好的小鼠脾细胞与SP2/0骨髓瘤细胞融合,采用有限稀释和间接ELISA法,筛选得到了1株稳定分泌Pst单克隆抗体的杂交瘤细胞株Pst 10号,经鉴定其分泌的免疫球蛋白亚类为Ig G1型,轻链为κ型。间接ELISA显示该单克隆抗体的效价为1∶1.02×10^(5),亲和常数为9.65×10^(8)。用碱性磷酸酯酶标记单克隆抗体所建立的双抗体夹心酶联免疫吸附(DAS-ELISA)测定可有效地检测出Pst的3个菌株,而与其他8种细菌不产生反应。检测灵敏度为10-5CFU/mL。

BALB/c mice were immunized by cultured Pseudomonas syringae pv.tomato(Pst).The splenocytes of immunized mice were fused with SP2/0 cells.The positive hybridoma cells were screened by finite dilution and indirect ELISA.One hybridoma cell strain stably secreting monoclonal antibody(McAb)was obtained and named as Pst 10.The subclass of the McAb was Ig G1 with Kappa light chain.The titers of ascites were 1∶1.02×10^(5) and the affinity constants were 9.65×10^(8)by indirect ELISA.DAS-ELISA method was developed by labeled the McAb with alkaline phosphatase.DAS-ELISA results showed that three isolates of Pst could be specifically detected and eight other bacterial strains got negative reaction.Sensitiviy of the method was 105 CFU/mL for detecting cultured Pst.