title: Quantification of rHuEPO-α by magnetic beads-based aptameric real-time PCR assay.
abstract: Recombinant human erythropoietin (rHuEPO) is one kind of important hematopoietic growth factor, which is widely used in anaemia treatment as well as sometimes abused by endurance athletes. Based on a set of anti-rHuEPO-α aptamers successfully in vitro isolated in our laboratory, we herein describe a novel magnetic beads-based aptameric real-time PCR assay for the accurate quantification of rHuEPO-α, which combined the specific recognition with amplification capability of aptamers. Two detection strategies, termed 'recognition-after-hybridization' and 'recognition-before-hybridization' respectively, were constructed and compared. Strategy B, i.e.'recognition-before-hybridization', was finally adopted as the preferred one to measure rHuEPO-α. A limit of detection (LOD) of 1 pmol/L rHuEPO-α and a wide dynamic range from 6 pmol/L to 100 nmol/L were obtained for physiological buffer. Furthermore, a LOD of 6 pmol/L was achieved for more complicated matrix-half diluted artificial urine. These results indicate that the anti-rHuEPO-α aptamer fits the high sensitive detection of rHuEPO-α very well. The use of the aptamer with magnetic beads-based real-time PCR allows a direct and novel assay for rHuEPO-α.
authors.affiliation: Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China.
authors.forename: Jijun
authors.initials: J
authors.lastname: Tang
authors.forename: Lei
authors.initials: L
authors.lastname: Guo
authors.forename: Rui
authors.initials: R
authors.lastname: Shen
authors.forename: Tao
authors.initials: T
authors.lastname: Yu
authors.forename: Hua
authors.initials: H
authors.lastname: Xu
authors.forename: Hezhu
authors.initials: H
authors.lastname: Liu
authors.forename: Xiaoxi
authors.initials: X
authors.lastname: Ma
authors.forename: Jianwei
authors.initials: J
authors.lastname: Xie
mesh_terms: D052158:Aptamers, Peptide / Q000737:chemistry*; D004921:Erythropoietin / Q000235:genetics* / Q000652:urine*; D006801:Humans; D008280:Magnetics; D011994:Recombinant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D052158:Aptamers, Peptide; D011994:Recombinant Proteins; D004921:Erythropoietin