抗百菌清单克隆抗体的研制与鉴定

doi:10.3864/j.issn.0578-1752.2013.07.022

摘要/Abstract

摘要： 【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。

关键词: 百菌清 , 人工抗原 , 单克隆抗体 , 免疫学特性

Abstract: 【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. The preparation 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/c 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 1﹕1.6×103, 1﹕6×102, 1﹕6×102, and 1﹕6×102 in supernatant and 1﹕5.12×105 in ascites. The special mAb showed good sensitivity with IC50 of 21.6685 ng•mL-1 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.

Key words: chlorothalonil , artificial antigen , monoclonal antibody , immunological trait

王玲玲, 职爱民, 杨艳艳, 宋春美, 王坤, 柴书军, 侯玉泽, 邓瑞广, 张改平. 抗百菌清单克隆抗体的研制与鉴定[J]. 中国农业科学, 2013, 46(7): 1509-1515.

WANG Ling-Ling, ZHI Ai-Min, YANG Yan-Yan, SONG Chun-Mei, WANG Kun, CHAI Shu-Jun, HOU Yu-Ze, DENG Rui-Guang, ZHANG Gai-Ping. Preparation and Identification of the Monoclonal Antibody Against Chlorothalonil[J]. Scientia Agricultura Sinica, 2013, 46(7): 1509-1515.

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