Quantification of rHuEPO-α by magnetic beads-based aptameric real-time PCR assay.
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.
Tang
Jijun
J
Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China.
Guo
Lei
L
Shen
Rui
R
Yu
Tao
T
Xu
Hua
H
Liu
Hezhu
H
Ma
Xiaoxi
X
Xie
Jianwei
J
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
D052158:Aptamers, Peptide; D011994:Recombinant Proteins; D004921:Erythropoietin