核酸杂交酶联桥接分析系统定量分析寡核苷酸

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、T4 DNA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40 U/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0.10～6.25 nmol/L.本方法成功应用于反义硫代寡核苷酸药物在低剂量(1 mg/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.

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

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.