Study On The Multi-residue Detection Of Immunoassay Method Of Fluoroquinolones

Fluoroquinolone (FQs) are the third generation quinolones, and whis are one ofthe fastest-growing and most studied chemosynthetic antibiotics in recent twentyyears. It was widely used in human clinical and animal husbandry and fishery in theaquaculture industry because of its advantages such as broad antibacterial spectrum,low toxicity and high efficiency. FQs antibacterial agents could be used by bothhuman beings and animals, the drug residues in animal products enter the human bodythrough the food chain, thus directly cause harm to human health, and in subsequentlycause bacterial resistance and induce cancer. Thus, many countries had set of a higheststandard of residue limits of FQs antibacterial drugs in animal products(10-1900μg/Kg). Since there are so many different kinds of fluoroquinolone drugs,the establishment of multi-residue assay method of these drugs would have a positiveimpact on controlling the quality and safety of animal product efficiently and quickly,so the study of FQs drug residues detection methods is one of the current researchhotspots and trends. Based on this, in this study, we successfully developed FQsmulti-residue detection kit and colloidal gold immunoassay test strips by largenumbers of experiments.On the basis of analyzing the molecular structure of FQs drugs andimmunogenicity, then connected Norfloxacin (Nor, the mother nucleus structure ofmany FQs drugs) with BSA and OVA to produce Nor-BSA and Nor-OVA by themethod of DCC. The antigen was identified by UV scanning, SDS-PAGEelectrophoresis and animal immunization. The results showed that the polyclonalantibody serum (pAb) that produced by animals immunization could specific reactwith FQs, but had no cross reaction with other types of drugs. Combining with theidentification results of UV scanning and SDS-PAGE electrophoresis could prove thatNor successfully coupled to carrier proteins. This lay a foundation to the followingantibody preparation of FQs. The New Zealand rabbits were immunized with theprepared Nor-BSA. The serum was got after eight immune. The titer of pAb wasmeasured by indirect ELISA, and the sensitivity and specificity of which was assayedby blocking ELISA. The results showed that the titer of rabbit pAb were between1:1.92×105and1:7.68×105. The lowest IC50could be8.97μg/L. The pAb hadspecific reaction with FQs, but have no cross-reaction with other types of drugs. Balb/C mice were immunized with prepared Nor-BSA, The mice with high serumtiters were screened for cell fusion and establishing hybridoma cell lines. Themonoclonal antibody (mAb) was prepared and identify. The results showed that twohybridoma that with high titer and strong affinity were screened:1A3-D7and3E5-C6,the average number of chromosomes is99. The mAbs subtypes are all IgG1/κ, with2.56×105and1:1.024×106tite,3.63×109and3.86×109L/moL Ka, respectively.Furthermore, the IC50of mAb produced by3E5-C6was5.50μg/L, and the mAb onlyhad specific reaction with FQs while had no cross reaction with other types of drugs.Based on the prepared FQs mAb, the working concentration of FQs mAb, Nor-OVAand enzyme labeled antibody were determined by the checkerboard method. FQsresidue detection ELISA kit was prepared and which performance was alsodetermined. The results showed that both prepared ELISA kits only had specificreaction with FQs while had no cross reaction with other types of drugs. The IC50ofblocking ELISA kit was4.61μg/L, and the IC50of rapid blocking ELISA Kit was6.27μg/L. The accuracy, precision and consistent retention period of the two kitswere basically consistent, and also consistent with the result of LC-MS-MS detection.Based on the prepared FQs mAb, using the technique of colloidal gold labeling andmembrane Immunochromatography, FQs multi-residue detection test strip weredeveloped and which performance were also identified. The results showed that thetest strip for Nor and Pei’s visual sensitivity was50ng/mL, for Lom’s visual sensitivitywas40ng/mL. The strip for Nor’s machine readable sensitivity (B/B0=80%) was2.519ng/mL, for Lom’s was1.533ng/mL, and for Pei’s was2.394ng/mL. The stripshave no cross reaction with other antibiotics. The false positive rate of50negativesamples was0, and the false negative rate of72positive samples was0. Thecoefficients of variation of intra-assay and inter-assay of different batches of test stripswere all less than10%. The strip is valid in more than12months. The detectionresults of strip were basically consistent with the results of LC-MS-MS.