NR AOTG
AU Gofflot,S.; Lachman,I.; El Moualij,B.; Amin,M.; Zorzi,D.; Thonnart,J.F.; Heinen,E.; Zorzi,W.; Osman,A.A.
TI Development of one-step real time immuno-PCR assay for prion proteins
QU International Conference - Prion diseases: from basic research to intervention concepts - TSE-Forum, 08.10.-10.10.2003, Gasteig, München - Poster session - DG-35
PT Konferenz-Poster
AB
Basis for the development of an ante mortem BSE/TSE test is the detection of very low amounts of prion protein. One of the best possibilities to detect rare proteins is the immunoquantitative-PCR* (iqPCR). Therefore we developed an iqPCR assay for the detection of prion proteins using two specific anti prion protein monoclonal antibodies. The reporter DNA was initially generated by PCR amplification using a biotinylated primer, and was bound with streptavidin to biotinylated second antibody. Human and bovine recombinant prion proteins sandwiched by monoclonal antibodies were detected by amplifing the reporter DNA using PCR. The detection limit of the immuno-PCR assay was 1ng/l, an approximately 1*102-fold improvement compared with a conventional enzyme-linked immunosorbent assay. This is the first report applying this highly sensitive technique of iqPCR for prion protein analysis. Furthermore, real time iqPCR in one step without changing the reaction vessels after enzyme immunoassay for subsequent PCR analysis allows minimizing risks of contamination and loss of sensitivity.
*Patent WO0131056, 2001-05-03: Detection method by PCR, Zorzi Willy (BE); El Moualij Benaïssa (BE); Zorzi Danièle (BE); Heinen Ernst (BE); Melen Laurence (BE).
1 Laboratory of Human Histology-CRPP, Institute of Pharmacy, Liège University, Avenue de l'Hôpital, 1, Sart Tilman, 4000, Liège, Belgium.
2 Roboscreen Gesellschaft für molekulare Biotechnologie mbH, Delitzscher Strasse 135, 04129 Leipzig, Germany
These authors contributed equally to this work.
This work is supported by the "Région Wallonne" : contract 14531, iPCRq.
AD Stéphanie Gofflot, Benaïssa El Moualij, Danièle Zorzi, Jean François Thonnart, Ernst Heinen, Willy Zorzi, Liège University, Belgium; Ingolf Lachman, Mona Amin, Awad Ahmed Osman, Roboscreen GmbH, Germany
SP englisch
PO Deutschland