克诺罗杆菌检测用单克隆抗体的制备及功效评价

Establishment and characterization of monoclonal antibody against Cronobavcter spp.

目的:选择克诺罗杆菌标准菌株中的多株代表菌株作为抗原,采用多抗原组合免疫的方法,筛选制备抗克诺罗杆菌属(Cronobacter spp.)检测用单克隆抗体(monoclonal antibody,mAb),并对获得的抗体进行各项指标评价,为进一步建立克诺罗杆菌免疫学快速检测方法创造条件。方法:采用冻融裂解、超声破碎、全颗粒三种方法制备抗原,以克诺罗杆菌标准菌株及分离菌株20株,9株非克诺罗杆菌进行筛选。筛选获得的抗体鉴定特异性,进行Ig亚类分型,测定效价及相对亲和力常数。结果:获得6株针对克诺罗杆菌的杂交瘤细胞株,腹水效价均在1∶107以上;相对亲和常数均大于1.0×1010L/mol。采用"鸡尾酒法"将多种抗体应用到胶体金试纸条制备上,可获得检测灵敏度为105CFU/mL,特异性较好的试纸条。结论:成功制备了针对克诺罗杆菌的多株单克隆抗体,抗体特异性检测表明,所制备的6株单克隆抗体针对克诺罗杆菌不同的亚种或亚型。利用单克隆抗体建立了抗原包被间接ELISA(ACP-ELISA)和胶体金试纸条检测阪崎肠杆菌的方法。为进一步开发阪崎肠杆菌的免疫检测试纸条,建立快速的检测方法创造条件。

Objective：To develop monoclonal antibody specific for Cronobacter spp.,Seven standard strains purchased from ATCC or DSM were used as antigen. Freeze thawing, ultrasonic smashing,and cell inactivation techniques were applied to prepare antigen. Methods ;Spleen cells were collected after common immunization and be used to fuse with hybridoma cell lines sp2/0.20 Cronobacter spp. Strains and 9 non-Cronobacter spp. strains inactivated were served as antigen to screen mAb by Indirect ELISA and Western blot techniques. Results：Five hybridoma cell strains that could secret Cronobacter spp. mAb stably were obtained,appraisal to its subclass,respectively was IgGl,lgG2b and I gG3,isotypes of light strains of the four MAbs all belong tok. The titers were over 1：107,and affinity constant was 1.0×10^10L/mol. All mAb were specific to few but only few of Cronobacter spp. strains. Conclusion, Cronobacter spp. was a complicated and diversity genus,It is difficult to get one mAb which can cover all species or sub-species,but multi mAb combined use may be an effective way to detect Cronobacter slop. by immunology technique. The established mAb will make a foundation for further research.