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| Preparation of an Anti-imidacloprid Monoclonal Antibody and Its Indirect Competitive Chemiluminescence Enzyme-linked Immunoassay |
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| DOI： |
| KeyWord:imidacloprid monoclonal antibody indirect competitive chemiluminescence enzyme-linked immunoassay（ic-CLEIA） |
| Author | Institution |
| XIE Bo，LIU Xin-mei，LU Di-sha，CHENG Yong-jian，LUO Lin，XIAO Zhi-li |
Guangdong Provincial Key Laboratory of Food Quality and Safety，College of Food Science，South China Agricultural University，Guangzhou ，China |
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| Abstract: |
| An indirect competitive chemiluminescence immunoassay（ic-CLEIA） was established for the detection of imidacloprid based on monoclonal antibody．Firstly，the imidacloprid haptens were synthesized with imidacloprid and 3-mercaptopropionic acid，then the haptens were coupled with carrier protein KLH and BSA by active ester method to prepare complete antigens．The complete antigens were used to immunize Balb/c female mice，and their spleen cells were fused with myeloma cells to prepare monoclonal antibody．The conditions of the method such as dilution multiple of coating antigen and antibody，category of buffer，pH of buffer，competition reaction time of primary antibody，and dilution multiple of enzyme-labeled secondary antibody were optimized．The optimum conditions were as follows：a 62.50 ng/mL of coating antigen（dilute 16 000 times），a 103.12 ng/mL of antibody（dilute 64 000 times），a PBS buffer（pH 7.4），a 30 min of the primary antibody competition reaction time and a 7 000-fold dilution of the secondary antibody．Results showed that under the optimal conditions，the detection limit（IC10） of the method was 0.03 ng/mL，IC50 was 0.57 ng/mL，and the linear detection range（IC20－IC80） was 0.083－3.99 ng/mL．The cross-reaction rate with imidaclothiz was 2.2%，and no obvious cross reaction was observed with 5 structural analogues i.e.clothianidin，dinotefuran，acetamiprid，thiacloprid and thiamethoxam．The spiked recoveries for cucumber and apple samples ranged from 82.0% to 112%，with relative standard deviations below 15%．The detection results for real sample has good correlation with those of HPLC method（r2 = 0.989），indicating that the established ic-CLEIA method could be applied to detect imidacloprid residues in food with high specificity and sensitivity． |
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