喹乙醇单克隆抗体制备及其免疫学快速检测方法建立-preparation of monoclonal antibody against olaquindox and establishment of its immunological rapid detection method.docxVIP

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喹乙醇单克隆抗体制备及其免疫学快速检测方法建立-preparation of monoclonal antibody against olaquindox and establishment of its immunological rapid detection method.docx

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喹乙醇单克隆抗体制备及其免疫学快速检测方法建立-preparation of monoclonal antibody against olaquindox and establishment of its immunological rapid detection method

摘要swimming faster than OLA-BSA, the OLA-BSA molecular weight greater than BSA, This further shows that OLA-BSA has been coupled with success. The titer of pAb for 3# mouse is the highest, its IC50 was 58.923ng/mL, cross reaction rate to carbadox was 1.841%, and no cross reaction to other antibiotics. The high-titer, sensitive and specific anti-OLA pAb had been produced.Preparation of monoclonal antibodies and immunological characterization of OlaquindoxThe titre and sensitivity of polyclonal antibody was detected by indirect ELISA and blocking ELISA, so as to select the mouse used in cell fusion. OLAmAb was prepared by hybridoma technology. The titer, affinity, sensitivity, specificity and subtype of the mAb were characterized. Massive OLAmAb were induced from invivo method. There hybridoma cell lines of 2B1、4F5、4E12、5B5 and 5E5 werescreened for specificity to OLA, all the isotypes of the mAb were IgG1. The indirect ELISA titer of the mAb were 1:3.0×102 ~1:1.28×103 in supernatant, 1:5.12×105 of 4E12 in ascites, and the affinity constant(Ka) was 3.75×1010L/moL, the mAb of 4E12 showed good sensitivity with IC50 of 1.66ng/mL to OLA. The rate of cross reaction of OLAmAb with carbadox was 5.19%, and there was no cross-reactivity to other compounds. OLAmAb of high-titer, sensitivity and specificity had beengenerated, it is possible to establish immunoassay of OLA residues in animal food.Development of rapid determination of Difloxacin residue methodBased on the enzyme-linked immunosorbent assay principle, a competitive ELISA kit for determination of OLA (OLA-Kit) was developed with OLAmAb. The calibration curve of OLA-Kit with standard OLA inhibitor was typical sigmoid curve fitted to the four parameters logistic equation, the detection limit for OLA was 1ng/mL and IC50 was1.66ng/mL. The precision and accuracy of the assay as determined by inter-assay and intra-assay coefficient variation were below 15%. The recoveries from pig liver and pork were 77.6%