NR AXPP
AU Kamali-Moghaddam,M.; Le Hir,G.; Englund,H.; Schallmeiner,E.; Darmanis,S.; Ekholm Pettersson,F.; Comoy,E.; Sehlin,D.; Lannfelt,L.; Deslys,J.P.; Landegren,U.
TI Proximity Ligation-based Detection of Biomarkers of Protein Folding Disorders
QU International Conference - Prion 2007 (26.-28.9.2007) Edinburgh International Conference Centre, Edinburgh, Scotland, UK - Book of Abstracts: Protein Misfolding P01.66
IA http://www.prion2007.com/pdf/Prion Book of Abstracts.pdf
PT Konferenz-Poster
AB
The development of sensitive and specific techniques for protein detection could provide opportunities for early detection and therapeutic intervention in protein folding disorders, for instance Prions- and Alzhiemer's diseases. We aim to develop extremely sensitive and specific molecular tools for quantitative measurement of soluble protein oligomers at very low concentration, using proximity ligation assay.
Proximity ligation assay (PLA) is a recently developed method in which specific proteins are analyzed by converting detection reactions to DNA sequences. In this method target molecules are recognized by two or more proximity probes that are prepared by attaching DNA strands to affinity binders. When a pair of such probes bind to a common target molecule, the free ends of the probes are brought in proximity and can be hybridized to a connector oligonucleotide, allowing the ends to be joined by enzymatic DNA ligation. The ligation products are then amplified by PCR and distinguished from unreacted probes.
Here, we present the application of PLA for sensitive identification of oligomers and aggregated prion proteins, and Aß peptide. The abnormal prion proteins can be detected and distinguished from normal proteins by PLA without requirement of proteinase K treatment. Soluble Aß oligomers, which have been shown to mediate neurotoxicity, may be used as a candidate biomarker for diagnosis of Alzheimer's disease at early stages.
The combination of efficient PCR amplification and the use of two or more binding reagents provide very high sensitivity and specificity of detection, surpassing the conventional protein detection methods. Furthermore, the proximity ligation technique can be carried out as in the homogenous assay - requiring very small amount of materials to be tested -, or in a heterogeneous format in which the target molecules to be detected are immobilized on a surface using affinity probes, while other materials are washed away. Proximity ligation can, therefore, provide a powerful molecular tool for detection and study of the biology of protein folding disorders.
AD M. Kamali-Moghaddam, E. Schallmeiner, S. Darmanis, U. Landegren, Uppsala University, Dept. of Genetics and Pathology/Molecular Medicine, Sweden; G. Le Hir, E. Comoy, J.-P. Deslys, CEA, DSV/DRM/GIDTIP, France; H. Englund, F. Ekholm Pettersson, D. Sehlin, L. Lannfelt, Uppsala University, Dept. of Public Health and Caring Sciences, Sweden
SP englisch
PO Schottland
EA pdf-Datei und Poster (Änderung in der Reihung der Autoren, zusätzlicher Autor: O. Söderberg)