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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 bridge analysis (ELBA) system based on nucleic acid hybridization and ligase and endonuclease was established for the quantitative analysis of antisense oligonucleotide (ODN) drugs in biological matrices. The conditions of avidin coating, capture probe / detection probe concentration, amount of T4 DNA ligase and S1 nuclease, reaction time of alkaline phosphatase substrate in ELBA system were optimized to confirm the use of neutravidin Coated ELISA plate, capture probe and detection probe concentration ratio of 2: 3, S1 enzyme 40U / well, color 15min was the optimal conditions, and investigated the biological matrix effect and dilution effect on the method, The antisense oligonucleotide in plasma of macaque monkey was established and quantified by methodological method. The quantitative range of the method was 0.10 ~ 6.25nmol / L. The method is successfully applied to the pharmacokinetic study of antisense oligonucleotide oligonucleotide in low dose (1mg / kg) intravenous drip, which has become an effective method for the quantitative analysis of oligonucleotide in biological matrix.