NR AOZO
AU Strom,A.; Müller,H.; Stuke,A.W.
TI Characterisation of Prion Protein (PrP)-binding Phages and Peptides
QU International Conference - Prion diseases: from basic research to intervention concepts - TSE-Forum, 08.10.-10.10.2003, Gasteig, München - Poster session - DG-44
PT Konferenz-Poster
AB
The conversion of the cellular prion protein (PrPc) is induced by the pathogenic isoform (PrPsc) and can be investigated by monitoring PrP epitopes with the aid of monoclonal and recombinant antibodies. Such antibodies may also inhibit the conversion reaction, discriminate PrP isoforms and be useful for the identification of PrP-binding proteins, e.g. receptors.
In our experiments we have isolated phage antibodies from random phage display libraries interacting with different linear and discontinuous PrP epitopes. For the selection libraries from MoBiTech (6-, 7- and 8-mers) and New England Biolabs (7- and 12-mers) were used. As antigen 15 overlapping 18-mer PrP-peptides of ovine and human origin were utilized. Biopanning was also performed with recombinant bovine PrP (rbPrP), recombinant human PrP (rhPrP) and an intact 3T3-L1-pTet/Off cell line over-expressing the human PrPFFI on the cell surface. Antigenes were directly coated onto polystyrene or nitrocellulose membranes. Through several selection rounds specifically bound phages were isolated and amplified. Phage clones were tested in ELISA with the PrP-peptides, rbPrP and rhPrP. Clones showing OD405 signals higher than 0.2 were tested in ECL blot. We finally segregated a number of clones showing strong signals in ELISA at a concentration of 10 to the 10 pfu/well and in ECL blot at a concentration of 10 to the 10 pfu/ml. The intensity of the ELISA signals was as strong as the signals obtained with a mix of monoclonal anti-PrP antibodies 12F10, 8G8 and 3B5 (each 1 µg/ml). Up to now we isolated and sequenced twenty different PrP-binding phages. The corresponding peptides of three phages were synthesized and tested in ELISA, ECL blot and flow cytometry. Phages are currently being tested in a cell culture model for their potential to inhibit PrP conversion. Furthermore, BLAST sequence comparisions may lead to the discovery of PrP-receptors with important biological functions.
AD A. Strom, A.W. Stuke, German Primate Centre (DPZ), Göttingen, Germany; H. Müller, Heinrich-Heine University Düsseldorf, Düsseldorf, Germany
SP englisch
PO Deutschland