Preparation And Application Of The Generic Monoclonal Antibody For Fluoroquinolone Drugs

Fluoroquinolone drugs is a class of synthetic antibacterial drugs that has several merits:broad antimicrobial spectrum, strong antibacterial ability, quick action, compeleteabsorption, and low toxicity. Therefore, this class of drugs is widely used in human andveterinary clinic. As the wide application, their residues in animal derived foods produceda great harm to human health and induced the widespread concern. The purpose of thisstudy was to develop a multi-residue immunoassay for determination of the residualfluoroquinolone drugs in foods of animal origin.In this study, two novel haptens of ciprofloxacin and two novel haptens of sarafloxacincontaining a free amidogen group on the piperazinyl ring were synthesized. The fourhaptens were coupled with bovine serum albumin and ovalbumin respectively to producefour immunogens and four coating antigens by using of glutaraldehyde method. Thehapten/carrier coupling ratios in the eight conjugates were in the range of12:l-17:1. Thefour immunogens were used to immunize Balb/c mice. During multiple immunizations,serum titers of these mice were determined by indirect ELISA method to evaluate differentcoating antigen/antibody ombinations. The results showed that the two immunogens ofciprofloxacin had better immunogenicity. Then the spleen cells from the mice with thehigheset titer were fused with SP2/0myeloma cells to produce hybridomas. The positivehybridoma cells were screened by using indirect competitive ELISA and then subcloned byusing the limited dilution method. Finally, two hybridoma cell lines secreting themonoclonal antibody were obtained. The ascites from the mice were purified by acrylicacid-ammonium sulfate precipipitation method to produce the monoclonal antibody. Theobtained antibody simultaneously recognized twelve fluoroquinolone drugs (ciprofloxacin,enrofloxacin, norfloxacin, sarafloxacin, diflocaxin, danofloxacin, ofloxacin, marbofloxacin,pefloxacin, lomefloxacin, amifloxacin and enofloxacin). The crossreactivities to the tweledrugs were in the range of23%-120%, the IC50values were in the range of7.6-39.5ng/mL,and the limits of detection were in the range of1.0-4.5ng/mL. Then the antibody was usedto develop a heterologous indirect competitive ELISA to determine the twelve drugs. Eightfluoroquinolone drugs licensed as veterinary drugs in China were fortified into blank chicken for analysis. The recoveries were in the range of61.5%-82.5%with coefficients ofvariation in the range of7.5%-15.2%.Therefore, this method could be used as a rapid screen tool for routine monitoring theresidues of these fluoroquinolone drugs in animal derived foods to ensure the safety ofanimal-original food.