抗氟喹诺酮类抗生素单抗的研制及其检测应用  

Development and Detection Application of a Monoclonal Antibody Against Fluoroquinolone

作　　者：吴建祥[1] 王震成 金仁耀[2] 刘欢[1] 周崇盈 张少恩 

机构地区：[1]浙江大学农业与生物技术学院生物技术研究所,浙江杭州310058 [2]浙江工商大学食品与生物工程学院,浙江杭州310012 [3]杭州南开日新生物技术有限公司,浙江杭州310022

出　　处：《核农学报》2013年第12期1961-1967,共7页Journal of Nuclear Agricultural Sciences

基　　金：浙江省科技计划资助项目(2011C22078)

摘　　要：用与牛血清白蛋白交联的环丙沙星免疫的BALB/C鼠脾细胞与SP2/0鼠骨髓瘤细胞融合,经筛选、克隆,获得1株能稳定传代并分泌抗氟喹诺酮类药物的单克隆抗体(MAb)的杂交瘤细胞9H9,用杂交瘤细胞制备单克隆抗体腹水。单克隆抗体对环丙沙星、恩诺沙星、氧氟沙星、达氟沙星、诺氟沙星、依诺沙星、麻保沙星、洛美沙星、沙拉沙星、双氟沙星均有免疫反应,且50%抑制质量浓度分别为达到3.21、3.01、8.15、4.85、7.75、3.70、9.83、6.21、11.62、200.70 ng·mL-1。利用单抗建立了检测蜂蜜、鸡肉组织、牛奶中氟喹诺酮类药物残留的间接竞争酶联免疫吸附测试(CI-ELISA)和免疫胶体金试纸条法。CI-ELISA方法的IC50及IC10分别为3.21 ng·mL-1和0.42 ng·mL-1。添加试验和特异性分析表明,免疫胶体金试纸条能用于检测蜂蜜、鸡肉组织、牛奶中的氟喹诺酮类药物残留,且检测上述样品中环丙沙星残留的灵敏度分别为5、20、30 ng·mL-1。A hybridoma cell line （9H9） secreting a monoclonal antibody （MAb） against fluoroquinolone were developed by fusing mouse myeloma cells （SP2/0） with spleen cells from BALB/C mouse immunized by ciprofloxacin （CIP） conjugated with bovine serum albumin （BSA） and selecting hybridoma cell and cloning cell. Monoclonal antibody from the mouse ascites was prepared using the hybridoma cell. The MAb can specifically react with ciprofloxacin, enrofloxacin, ofloxacin, danofloxain, norfloxacin, enoxacin, marbofloxacin, lomefloxacin, sarafloxacin and difloxacin, and its 50% maximal inhibitory concentrations （IC50） for them were 3.21, 3.01, 8.15,4.85,7.75, 3.70, 9.83, 6. 21, 11.62, and 200.70 ng·mL-1, respectively. Using the MAb, an indirect competitive ELISA （CI-ELISA） and a colloidal gold -based immunochromatographic strip for fluoroquinolone residue detection in honey, chicken tissues and milk were developed. The developed CI-ELISA for CIP detection show an ICs0of 3.21 ng· mL-1 with a detection limit of 0. 42 ng · mL-1（10% inhibition）. The results of spiked and specific analyses demonstrated that the immunochromatographic strip can be applied to screening fluoroquinolone residues in honey, chicken tissues and milk samples. The CIP detection sensitivities of immunochromatographic strips for honey, chicken tissues and milk samples were 5, 20, 30 ng/mL, respectively.

关 键 词：氟喹诺酮类药物 单克隆抗体 CI-ELISA 免疫胶体金试纸条 

分 类 号：TQ465[化学工程—制药化工]