NR AXTJ
AU Nagata,T.; Yokoyama,T.; Sekiya,S.; Nishikawa,S.; Noda,K.
TI Femtograms-Detection of PrPsc in Biological Samples using Chemically Synthesized RNA-Aptamer
QU International Conference - Prion 2007 (26.-28.9.2007) Edinburgh International Conference Centre, Edinburgh, Scotland, UK - Book of Abstracts: Epidemiology, Risk Assessment and Transmission P04.103
IA http://www.prion2007.com/pdf/Prion Book of Abstracts.pdf
PT Konferenz-Poster
AB
For the safety of biological products, it is one of our major concerns to reduce the TSErisk of cattle-blood derived materials such as serum and plasma. For the detection of possibly contaminated abnormal isoform of prion protein (PrPsc) in the biological samples, it is indispensable to develop a highly sensitive PrP detection procedure. Here, we have developed an aptamer-beads PrP-concentration procedure by using RNA-aptamer 60-3 which binds to recombinant mouse PrP with high affinity (Kd = 5.6 nM) (1).
The RNA-aptamer 60-3 was chemically synthesized employing a novel RNA synthetic method with a 2'-O-(2-cyanoethoxymethyl) protecting group (2), with 2'OMepyrimidine modification for RNase resistance, and conjugated with biotin. The aptamer was then bound to streptavidin-coated magnetic beads (60-3 aptamer-beads) and used for pull-down assays. The pulled-down PrPsc was analyzed by Western blotting.
The 60-3 aptamer-beads demonstrated the enrichment of PrPsc from the 20-milion times diluted scrapie-infected mouse brain (50ml of 50ng brain equivalent /ml). Comparing to phosphotungstic acid (PTA) concentration method, the 60-3 aptamerbeads revealed more than 100 times efficiency in concentrating PrPsc spiked in bovine serum. Moreover, the 60-3 aptamer-beads showed binding ability to PrPsc in highly diluted BSE-infected bovine brain.
The present Aptamer-beads pull-down procedure enables us to perform a femtograms-detection of PrP. The procedure was also proven to be applicable to BSE-PrPsc. The present aptamer-beads system could serve as a resource for prionremoval column and serum prion assays, and potentially achieve the safety of the blood derived biological products.
References
(1)S. Sekiya, K. Noda, F. Nishikawa, T. Yokoyama, P.K.R. Kumar and S. Nishikawa, J. Biochem. 139, 383-390, 2006. (2)T. Ohgi, Y. Masutomi, K. Ishiyama, H. Kitagawa, Y. Shiba and J. Yano, Org. Lett. 7, 3477-3480, 2005.
AD T. Nagata, K. Noda, National Veterinary Assay Laboratory, Japan; T. Yokoyama, National Institute for Animal Health, Japan; S. Sekiya, S. Nishikawa, National Institute of Advanced Industrial Science and Technology (AIST), Japan
SP englisch
PO Schottland