抗百菌清单克隆抗体的研制与鉴定

Preparation and Identification of the Monoclonal Antibody Against Chlorothalonil

【目的】制备高灵敏、高特异性的百菌清(CTN)单克隆抗体,并对其免疫学特性进行鉴定。【方法】将CTN进行化学修饰后引入羟基活性基团,合成具有半抗原结构特征的百菌清衍生物(CTN-OH);采用N,N-羰基二咪唑法(CDI)将CTN-OH与牛血清白蛋白(BSA)和鸡卵清蛋白(OVA)偶联合成人工免疫抗原CTN-BSA和包被抗原CTN-OVA,用紫外分光光度法(UV)和凝胶电泳(SDS-PAGE)进行鉴定后,免疫BALB/c小鼠,利用间接ELISA和间接竞争ELISA测定其多抗血清效价和敏感性,选出效价高、敏感性好的小鼠进行细胞融合,经过多次亚克隆筛选出分泌高敏感的特异性抗CTN单克隆抗体的杂交瘤细胞株,体内诱生腹水法制备抗CTN的单克隆抗体并对其免疫学特性进行鉴定。【结果】UV和SDS-PAGE鉴定结果显示,CTN-BSA成功偶联,间接ELISA检测显示免疫的3只小鼠效价均达1﹕104以上,其中2号小鼠多抗血清敏感性最好,半数抑制浓度IC50为93.593 ng.mL-1,融合后筛选出4株杂交瘤细胞株1E8、1F4、2A10和2E2,其细胞上清效价分别为1﹕1.6×103、1﹕6×102、1﹕6×102、1﹕6×102,1E8株腹水效价为1﹕5.12×105,抗CTN单克隆抗体对CTN的IC50为21.6685 ng.mL-1,且具有较好的特异性。【结论】本试验成功合成了CTN人工抗原,并制备了敏感性高、特异性好的单克隆抗体,为CTN免疫学快速检测方法的建立奠定了基础。

[Objective] The objective of this study is to generate high sensitivity and high specificity monoclonal antibody against chlorothalonil and identification of its immunological characteristics. [Method] The active group hydroxyl was introduced to chlorothalonil （CTN） and formed CTN derivatives （CTN-OH） by chemical modification. The artificial immunogen CTN-BSA and coating antigen CTN-OVA were obtained by using the method of mixed N, N-carbonyl diimidazole （CDI） to conjugate CTN-OH with bovine serum albumin （BSA） and ovalbumin （OVA）, respectively. Ultraviolet （UV） and sodium dodecyl sulfate polycrylamide gel electropheresis （SDS-PAGE） were used to identify CTN-BSA. Then BALB/c mice were immunized. The titer and sensitivity of polyclonal antibody was detected by indirect ELISA and blocking ELISA after four times immunization. The high titer and high sensitivity mice were selected for cell fusing. High sensitivity and high specificity monoclonal antibody was prepared after many times subcloning, and immunological characterization was characterized. Thepreparation of ascites was carried out using in vivo induction method. [Result] UV and SDS-PAGE results showed that CTN-BSA artificial antigen was synthesized successfully. Indirect ELISA showed a high titer above 1 ： 104 of the antiserum of all the three BALB/e mice. The sensibility of antiserum of No.2 mouse was the best, which the IC50 was 93.593 ng.mL^-1. Four hybridoma cell lines 1E8, 1F4, 2A10, and 2E2 were screened after cell fusing, the indirect ELISA titers of the mAb were 1 ： 1.6x10^3, 1 ： 6x10^2, 1 ： 6x10^2, and 1 ： 6x10^2 in supernatant and 1 ： 5.12x10^5 in ascites. The special mAb showed good sensitivity with ICs0 of 21.6685 ng-mL^-l to chlorothalonil. [ Conclusion] The CTN artificial antigens were synthesized successfully and high sensitivity and specificity monoclonal antibodies were obtained. It laid a foundation for the CTN immunology fast detection methods.