Scientia Agricultura Sinica ›› 2013, Vol. 46 ›› Issue (7): 1509-1515.doi: 10.3864/j.issn.0578-1752.2013.07.022

• RESEARCH NOTES • Previous Articles     Next Articles

Preparation and Identification of the Monoclonal Antibody Against Chlorothalonil

 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   

  1. 1.He’nan Academy of Agricultural Sciences/Key Laboratory of Animal Immunology of the Ministry of Agriculture/He’nan Provincial Key Laboratory of Animal Immunology, Zhengzhou 450002
    2.Food and Bioengineering College, He’nan University of Science and Technology, Luoyang 471003, He’nan
    3.College of Food Science and Technology, Jiangnan University, Wuxi 214122, Jiangsu
  • Received:2012-10-26 Online:2013-04-01 Published:2013-01-24

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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

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