两株高特异性抗恩诺沙星单克隆抗体的研制.pdf

第27卷第1期
食品安全质量检测学报
Yol 27 No
2010年2月
Journal of Food Safety and Quality
Feb.2010
两株高特异性抗恩诺沙星单克隆抗体的研制
王晓艳23,林纪的2-3,杨利3,親23,王文2,3
张浩明23,崔迎利23,焦
唐宏
(1.中国科学院生物物理研究所北京100101:2.江苏泰州蛋白质工程研究院泰州225300;
3.江苏泰州康正生物技术有限公司泰州225300:4.广东出人境检验检疫局广州510623)
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
PEC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1.28x
103,8抗体在1g/mL时,效价达到1.024x10-°。两茠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
3.125wu/kg不和0.3?g/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醇联免疫法检测食品中残留的恩诺沙星。
关键词:恩诺沙星;人工抗原;单克隆抗体
中图分类号:R392.11文献识别码:A国家标准学科分类代码:180.1750
Development of two specific Monoclonal Antibodies against Enrofloxacin
Wang Xiaoyan2-, Lin Jiyun'-, Yang Li, Yu Jing, Wang Wen?
Thang Haoming, Cui Yingli", Jiao Hong", Tang Hong: 23*
I Institute of Biophsics hins Acdemy ofsciences, Being 00 h: 2 sti e of Protein Enin
Taishow Jiangsu, Taishou 225300, China; 3 Kangheng Biotech Co. Ltd, Taizhou 225300, Chin
4 Guangdong EntryExi nection and unt ne Buw, Guangzhou 51023, Ch)
Abstract: 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 al/c mice were immunized wh artificial antigen ENR-HSA. Antibodies were screened by limie dilution
methods after PEG fusion. Two antibodies which can secrete enrofloxacin-specific antibody were obtained( Clone
6A4 and 8E 6).The purity of Mabs was more than 95% after purified by ion exchange method. The titer of th
purified 6A4 antibody was 1.28 10, and 8E6 antibody was 1.0A x10"when the concentration of antibody
was I mg/ml. The crossreactivity of both Mabs to ciproloxacin was 1.5% and both Mabs did not react with
ot her fluorouinolones. The detection limit of MAB 6A4 is 3. 125 ug/kg and MAB 8E is 0.3 w/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 MAI
项目基金:江苏省科技支撑计划(BE200861);广州市重大科技工程项目(200671-O121
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