NR AVHH
AU Fritz,M.
TI Zum Nachweis von pathogenen Prionen mittels "Kapillarelektrophorese-laserinduzierter Fluoreszenz (CE-LIF)"
QU Inaugural-Dissertation zur Erlangung der tiermedizinischen Doktorwürde der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München von Markus Fritz aus Eichstätt, München, 28. Juli 2006
PT Dissertation
AB
For the detection of PrPc and PrPsc a capillary electrophoresis laser-induced fluorescence method was established and compared with conventional techniques (ELISA, Westernblot, Dot-EIA). For CE-LIF the monoclonal antibody F89 and a fluorescent-labelled peptide (FITC-c P3) were used. The limit of detection for recombinant bovine PrPsc was 1 pg/test.
For enrichment of PrPsc from brain and cerebrospinal fluid (ultra-) centrifugation-, extraction- (HFIP) and chromatographic methods (HPLC, SPE-extraction) were evaluated separately or in combination; furthermore affinity,- or immunomagnetic separation were used for enrichment, respectively.
The detection of the PrPsc-content of selected extracts when compared to the source material using ELISA showed clear losses of substance. A selective binding of PrPsc to Plasminogen coupled Dynabeads(R) could not be detected. On the other hand an adsorption onto Dynabeads(R) coupled Plasminogen was determined. The binding capacity of PrPc or PrPsc to the beads was higher in PBS than in experiments using cerebrospinal fluid.
Extracts purified by single methods were inappropriate for analysis by capillary laser-induced fluorescence, as this caused either technical problems (blockage or damage of the capillary) or interferences in the electropherogram which made an analysis impossible. The purification by HPLC resulted in a useable preparation, but showed widely varying retention times for PrPsc so that this method was not used. The best results were obtained by using a combined method consisting of a purification by Bio-Rad(R) and SPE-cartridges. While the detection of PrPsc in brain was successful with CE-LIF no definitive result could be shown in cerebrospinal fluid of a BSE positive animal.
In principle CE-LIF is a suitable method for detecting PrPsc. However it needs to be improved due to its susceptibility to interferences and technical and methodical problems. The sensitivity can be increased by improving concentration of PrPsc in small volumes.
SP deutsch
PO Deutschland