Preparation Of Monoclone Antibody And Establishment Of ELISA Rapid Test Method Of Chlorothalonil

Chlorothalonil is a kind of systemic broad-spectrum organochlorine fungicide.When chlorothalonil in soil is transformed into hydroxyl chlorothalonil by light,enzyme or microorganism, its toxicity and stability increases and can dissolve in water,which poses a more serious threat to environmental and food safety. Studies haveshown that chlorothalonil is a kind of chemicals which can make plant and animalcells genetic mutation. Research, therefore, to establish rapid, sensitive and effectivedetection method for chlorothalonil residues has vital significance. Immunologicaldetection methods have the advantages of sensitive, accurate, rapid, specific, and hasbecome a powerful supplement for traditional physical and chemical test methods.Based on analysis immunology characteristics of CTN, monoclonal antibody againstCTN was prepared using monoclonal antibody technology, and the indirectcompetitive CTN-kit was assembled. The main contents and results of this study wereas follows:1. Synthesis and identification of the artificial antigen for ChlorothalonilThe derivative of chlorothalonil, called as CTN-OH, was synthesized byethanediol method. Two complete antigens of chlorothalonil （CTN-BSA, CTN-OVA）were prepared by N, N’-carbonyldiimidazole method （CDI）. The characterization ofcomplete antigen CTN-BSA was examined by two methods, ultraviolet UV, SDS-PAGE respectively. BALB/c mice were immunized with CTN-BSA, the titer ofpolyclonal antibody（pAb）was detected by indirect ELISA, the sensitivity andspecificity of pAb was identified by blocking ELISA. The results of UV and SDS-PAGE show that CTN-BSA has been coupled with success. The titer of pAb for mouse2is the highest; Its IC₅₀was93.593ng/mL. The high-titer, sensitive anti-CTN pAb hadbeen produced.2. Preparation and immunological characteristics identification of chlorothalonilmonoclonal antibody The titre and sensitivity of polyclonal antibody were detected by indirect ELISAand blocking ELISA, so as to select the mouse used in cell fusion. CTNmAb wasprepared by hybridoma technology. The titer, affinity, sensitivity, specificity andsubtype of the mAb were characterized. There hybridoma cell lines of4A31E8、4A31F4、4A32A10and4A32E2were screened for specificity to CTN. The indirectELISA titer of the mAb was1:6.0×10²¹:6×10³in supernatant.4A31E8hybridomacells induced mice to produce CTNmAb, its titer is1:5.12×10⁵. CTNmAb is identifiedas IgG1monoclonal antibody subtypes and the CTN monoclonal antibody affinityconstant is3.1×10¹⁰L/mol. The CTNmAb of4A31E8showed good sensitivity withIC₅₀of21.6685ng/mL to CTN. The rate of cross reaction of CTNmAb with quintozeneis1.98%, and there was no cross-reactivity to other compounds.3. Self-assemble of CTN indirect competitive ELISA detection kitThe final concentration of envelope antigen is2μg/mL by ELISA method. The dilutionratio of CTNmAb is1:64000. So indirect competitive ELISA is established for therapid detection. At the same time, chlorothalonil indirect competitive ELISA rapiddetection kit is preparated. After the test evaluation, the data show that the property isvery good. The high correlation coefficient of the linear regression equation of theCTN-Kit standard curve is0.9932, and IC₅₀of CTN-Kit is29.44ng/mL. Through theexperimental result, CTN-Kit has high sensitivity, good specificity and stability andcan save more than six months without invalidation.