NR ATKW
AU Clark,J.K.; Everest,D.J.; Moorthie,S.A.; Barnard,G.; Sauer,M.J.
TI Towards an ante-mortem blood test for TSEs: evaluation of microwell PrP fluoroimmunoassay formats using europium label.
QU International Conference - Prion 2005: Between fundamentals and society's needs - 19.10.-21.10.2005, Congress Center Düsseldorf - Poster Session: Diagnosis DIA-34
PT Konferenz-Poster
AB
Blood represents the most accessible sample type for TSE diagnosis in the live animal, although a reliable test using this matrix has yet to be validated. The only accepted biomarker for TSEs is PrPsc. The challenge is to develop an assay with sufficient sensitivity to detect vanishingly low blood levels of PrPsc in infected animals.
To date the only test reported as capable of detecting proteinase K resistant PrPsc (PrPres) in blood is capillary electrophoresis immunoassay1 (ICE); a fluoroimmunoassay (FIA), utilising CE to separate free from antibody bound label (fluorescein labelled peptide). However, ICE appears to lack the robustness required for routine application. The exquisite sensitivity of this method is presumed to be derived from enhanced label detection resulting from the use of laser induced fluorescence, rather than the CE separation per se. Method transfer to a more adaptable standard microwell format can not be achieved readily though, since the concentration of fluorescein label used is incompatible with the sensitivity of current microplate readers.
Europium (Eu) has a lower limit of detection (LLOD) in the amol range and so is well suited as a label for FIAs where sensitivity may otherwise be restricted. Substituting Eu for fluorescein thus presents the possibility of developing simple FIAs with comparable performance to ICE.
We report on the use of several Eu labelling strategies (labelled peptides, antibodies, and biotin-streptavidin formats) applied to microwell FIAs for PrP. Use of model proteins, recombinant ovPrP and PrPres (aggregated and 'soluble' forms from ovine CNS tissue), enabled comparison of assay performance criteria (sensitivity, dynamic range and reproducibility etc.) with those of ICE. Preliminary data are encouraging and indicate that use of streptavidin-EU label enables a LLOD (~1 fmol/ well) similar to that for ICE to be achieved.
1. Schmerr, M.J., et al. (1999) J. Chromatog. A, 853, 207-214.
AD J.K.Clark, D.J.Everest, M.J.Sauer, Veterinary Laboratories Agency, UK; S.A.Moorthie, G.Barnard, University of Cambridge, UK
SP englisch
PO Deutschland