毒死蜱单克隆抗体制备及icELISA检测方法优化.pdf

第5卷第6期
食品安全质量检测学报
VOL 5 No 6
2014年6月
Journal of Food Salcly and Quality
um..2014
毒死蜱单克隆抗体制备及 ICELISA检测方法优化
王伟华,田木星,韩占江
(1.塔里木大学生命科学学院新疆生产建设兵团南疆特色农产品深加工重点实验室,阿拉尔84330
2.塔里木大学植物科学学院新疆生产建设兵团塔里木盆地生物资源保护利用重点实验室,阿拉尔843300
摘要:目的制备毒死蜱单克隆抗体、建立 ICELISA(间接竟争鶤联免疫吸附方法)检測方法并对其进行优
化。方法以毒死蜱为基础物质、制备半抗原CF-H1和CPF-HI2,偶联蛋白制备人工抗原,进而通过免疫、细
胞融合、筛选得到特异性的单克隆抗体.建立毒死蜱的 ICELISA检測方法、并对其分析条件进行优化。结果质
谱鉴定结果表明毒死蜱半抗原制备成功;半抗原偶联蛋白,紫外全波长扫描鉴定人工抗原制备成功;免疫动
物、细胞融合并制备单克隆抗体;建立了 ICHLISA检测方法,优化得到最佳反应条件:PBST为标准品稀释液,
包被抗原最佳稀释倍数为1:1000、抗体最佳稀释倍数为1:1000一抗反应最佳时间为40min.二抗反应最佳时
间为30min,制得毒死蜱标准曲线,毒死蜱对抗体的ICs0为73.25 na/ml,线性范围IC2w~ICso为32.52~260
ng/ml,1.OD)为19.34ngml.。结论本研究为毒死蜱快速检测技术奠定了理论基础,对保障人们的身体健康具
有重要的意义。
关键词:毒死蜱;单克隆抗体; ICELISA;优化
Preparation of chlorprifos monoclonal antibody and optimization of
ICELISA detection method
WANG Wei-hua, TIAN Mu-xing HAN Zhan-jiang
injiang Prodaction c Constrction Corps Key aboratory f Deep Processing ofgricutra/ Products in South i
jiang, College of Life Science, Iaim Universit, dlar, China; 2. Xiniang Prodction Constriction Corps er
Labora/oo/Proection ad Tilization Biolgic es in I m Basin, lege Pan Sc ence, Trim Unieily,
ABSTRACT: Objective Io prepare monoclonal antibodies o! chlorpyrifos, and establish and optimize ICE
I S (indirect competitive enyme-linked munosorbent assay)detection methods. Mcthods I lapten
CPI-IIL and CPIT-I12 were prcparcd based on chlorpyrifos, artificial antigen were prepared through conjugated
prolein, and then specific monoclonal antibodies were oblained through immunization, cellfusion and screenin
ICHI ISA of chlorpyrifos was established, and its analysis conditions were optimized. Results Mass spec
trometry results indicated that the chlorpyrifos hapten was successfully prepared; hapten conjugated proteins
UV wavelength scanning verified the successul preparation of artilicial antigen animals were immunized
cells were fusioned and monoclonal antibodies were prepared, ICELISA detection melhod was established, the
optimal reaction conditions were optimized to be: PBST was used as the standard diluent, the optimal coating
基金项目:新疆生产建设兵团博士资金项目(2012BBO12)、国家级大学生创新创业训练计划项目(2013107570010)
Fund: Supported by the Doctor Fundation of Xinjian Production and Construction Crops(2012BB012); and National College Students'
Innovation and I ntrepreneurship Raining Program(20131075
通讯作者:王伟华,博士,副教授,主要研究方向为食品质量安全。-mail:wangwenhua66880o163.com
Corresponding author: WANG Wei-hua, Ph. D, Associate Professor, College of Life Scicncc, Tarim Univcrsity. Alar 843300, China. E-mail
wangwcihua6688(a163.com