核酸杂交酶联桥接分析系统定量分析寡核苷酸  

Development of a Nucleic Acid Hybridization Enzyme-linked Bridging Assay System and Its Application to Oligonuleotides Quantification

作　　者：孙云娟[1] 付洁[1] 王清清[1] 周磊[1] 董立厚[1] 欧伦[1] 高新[1] 宋海峰[1] 

机构地区：[1]军事医学科学院放射与辐射医学研究所药理毒理研究室,药代动力学重点实验室,北京100850

出　　处：《分析化学》2012年第4期503-509,共7页Chinese Journal of Analytical Chemistry

摘　　要：建立了基于核酸杂交及连接酶和核酸内切酶的酶联桥接分析(ELBA)系统,用于生物基质中反义寡核苷酸(ODN)药物的定量分析。对ELBA系统中的亲和素包被、俘获探针/检测探针浓度、T4DNA连接酶及S1核酸酶用量、碱性磷酸酶底物反应时间等条件进行了优化,确定使用中性亲和素包被的酶标板,俘获探针与检测探针浓度比为2∶3,S1酶为40U/孔,显色15min为最优条件,并考察了生物基质效应和稀释效应对方法的影响,建立了猕猴血浆中反义寡核苷酸的定量方法,并进行方法学确证,方法学定量范围为0.10～6.25nmol/L。本方法成功应用于反义硫代寡核苷酸药物在低剂量(1mg/kg)静脉滴注后的药代动力学研究,成为生物基质中寡核苷酸类药物定量分析的有效手段。An enzyme-linked bridging assay（ELBA） system based on nucleic acid hybridization principles was developed for quantification of antisense oligodeoxynucleotide（AS-ODN） in biomatrix.Conditions of the ELBA system were elaborately optimized.NeutrAvidin coated plate was used in the experiments.The optimal capture probe and detection probe concentration ratio was 2 ∶ 3,Color deve-loping time of Phosphatase Substrate（p-nitrophenyl phosphate PNPP） was 15 min and 40 U/well S1 nuclease was used.Neither matrix nor dilution effect had influence on the quantification of ODN in plasma using ELBA.Quantitative method of the concentration of AS-ODN in monkey plasma was validated,with the quantitative linear range of 0.10-6.25 nmol/L.The method has been successfully applied to the pharmacokinetics（PK）profile of the AS-ODN following vein drip（v.d.）administration at 1 mg/kg,It was concluded that the hybridization-based ELBA system offered a powerful alternative tool for quantifying ODN drugs in biomatrice.

关 键 词：寡核苷酸 药代动力学 定量 核酸杂交 酶联桥接分析系统 

分 类 号：R965[医药卫生—药理学]