两株高特异性抗恩诺沙星单克隆抗体的研制

Development of two specific Monoclonal Antibodies against Enrofloxacin

中文关键词:  恩诺沙星  人工抗原  单克隆抗体  

英文关键词:Enrofloxacin  artificial antigen  monoclonal antibody  

中文摘要:

      为自主研制抗恩诺沙星的高亲和力、高特异性单克隆抗体,提供动物源食品中恩诺沙星药物残留生物学快速检验方法试剂,利用碳二亚胺法(carbodiimide,EDC)制备恩诺沙星(enrofloxacin,ENR)的人工抗原,免疫Balb/c小鼠,采用PEG融合法,通过有限稀释法筛选出抗恩诺沙星的单克隆抗体,获得了两株抗恩诺沙星的单克隆抗体(克隆号:6A4和8D6)。经过离子交换法纯化得到的抗体纯度在95%以上,其中,纯化后的6A4抗体在1mg/mL时,效价达到1.28×10-5,8E6抗体在1mg/mL时,效价达到1.024×10-6。两株单克隆抗体与环丙沙星的交叉反应率为1.5%,与其他氟喹诺酮类药物不反应。在酶联免疫法(enzyme-linked immunosorbent assay,ELISA)中,6A4和8E6抗体的检测限分别为3.125μg/kg和0.3μg/kg。采用的半抗原合成以及单克隆抗体筛选的方法可以获得高灵敏度、高特异性的抗恩诺沙星的单克隆抗体,经后续研究证实单克隆抗体(8E6)可以用于免疫胶体金和酶联免疫法检测食品中残留的恩诺沙星。

英文摘要:

      To provide reagents for rapid detection methods of enrofloxacin residues in the animal-derived food,high affinity and sensitivity monoclonal antibodies(MAbs)against enrofloxacin were developed.The conjugation of enrofloxacin to human serum albumin(HSA)and ovalbumin(OVA)were achieved by carbodiimide method.The Balb/c mice were immunized with artificial antigen ENR-HSA.Antibodies were screened by limited dilution methods after PEG fusion.Two antibodies which can secrete enrofloxacin-specific antibody were obtained(Clone 6A4 and 8E6).The purity of MAbs was more than 95% after purified by ion exchange method.The titer of the purified 6A4 antibody was 1.28×10-5,and 8E6 antibody was 1.024×10-6 when the concentration of antibody was 1mg/mL.The crossreactivity of both MAbs to ciprofloxacin was 1.5% and both MAbs did not react with other fluoroquinolones.The detection limit of MAb 6A4 is 3.125 μg/kg and MAb 8E6 is 0.3 μg/kg in enzyme-linked immunosorbent assay(ELISA).The method used for hapten synthesis and MAbs screening can obtain sensitive and specific monoclonal antibodies against enrofloxacin.The subsequent work has confirmed that MAb(8E6)could be used for detection of enrofloxaxin residue in food by use of lateral flow assay and ELISA.