Preparation And Identification Of Monoclonal Antibody Against Norfloxacin

Norfloxacin(NFLX) belongs to the fluoroquinolones which are synthetic antibiotics. It isused in a wide range of gastrointestinal, urinary and respiratory tract infections, ocular and skininfections as well as in patients with intraabdominal infections in combination with antianaerobicagents. It is used both in human and veterinary medicine. The administration ofFluoroquinolones to animals, which are destined for human consumption can result in thepresence of residues in food products. These residues represent a potential hazard for theconsumer and are a concern due to the emergence of drug-resistant bacteria. Because of thepossibility of NFLX trace residues in edible tissue, it is very important to establish a kind ofcredible, sensitive and quick assay method.In this study, two kinds of NFLX artificial antigen were synthesized by conjugating NFLXto either bovine serum albumin(BSA) or ovalbumin(OVA) by two different chemical approaches.The immunogen, NFLX-BSA was synthesized by carbordiimide method. The coating antigen,NFLX-OVA was prepared by isobutyl chlorofomate method. The two artificial antigens werepurified by dialysis and gel filtration and identified by UV absorption method and animalimmunization test. The results demonstrated that the two artificial antigens were successfullysynthesized and could be used to prepare the monoclonal antibody against NFLX. Theconjugating ratios were calculated by Bradford and UV absorbance method and the ratios ofnorfloxacin connected with either BSA or OVA were about 9.7 and 8.2, respectively.NFLX-BSA was used as immunogen to immunize ten healthy female 4-6-week-oldBALB/c mice intraperitoneally and subcutaneouly. The mice were immunized with the emulsivemixture of NFLX-BSA and CFA adjuvant at day 1. Immunized with the emulsive mixture ofNFLX-BSA and IFA adjuvant at day 21, 35, 49, 63 and so on. Seven days after booster, serumtiters were determined by indirect ELISA and indirect competitive ELISA(ci-ELISA).The splenocytes from the immunized mouse which exhibited the highest titer were fusedwith SP2/0 myeloma cells using PEG-3350. The hybridoma cells were selected with HAT andHT medium. Hybridoma cells from the 3E9 well were selected using NFLX-OVA by indirect ELISA, expanded and subcloned by limited dilution method for two times. The hybridlma cellswere injected in mice intraperitoneally to produce ascites. The titers of cultivate medium andascites were 1:16 and 1:1.6×10~3 respectively. The ascites was purified by caprylic-ammoniumsulfate method roughly and detected by SDS-PAGE. SDS-PAGE showed that 3E9 monoclonalantibody had two bands in about 50 kDa and 25 kDa respectively. But there was still anunwanted band in about 67 kDa can not be eliminated. The monoclonal antibody was analysizedby ci-ELISA, and the result suggested that it had no cross-reaction with six otherfluoroquinolones antibiotics, named, sarafloxacinhydrochlride, ciprofloxacinhydrochlride,difloxacinhydrochlride, pefloxacinmsylate, enrofloxacin, danofloxacinmsylate.