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.