NR AOWV
AU Meyer,R.K.; Oesch,B.; Zurbriggen,A.; Vandevelde,M.
TI An Improved Assay for TSE
QU International Conference - Prion diseases: from basic research to intervention concepts - TSE-Forum, 08.10.-10.10.2003, Gasteig, München - Poster session - DG-04
PT Konferenz-Poster
AB
In previous experiments we have shown that denaturaton of PrP by exposure to defined heat in the presence of GdnSCN enabled binding of our antibodies to both PrPc and PrPsc in an ELISA (Meyer et al., 1999, J. Virol. 73, 9398-9392). The soluble PrP fraction (about 10% of total) was detected only (Meyer et al. 2000, J. Biol. Chem. 275, 38081-38087). In this study we investigated the influence of sample treatment in detail.
In a typical experiment a brain homogenate (BSE and normal cattle, scrapie and normal sheep), containing 0.1 M GdnSCN, was divided into 30 identical samples. One was left without treatment. The rest of the samples were placed in a block heater. One sample was heated to 132 C, a second one to 134 C, a third one to 136 C, and so on. The last was heated to 188 C. All samples were cooled to room temperature and probed by ELISA.
With proteinase K digested normal brain homogenate all samples had about the same intensity. With proteinase K digested scrapie and BSE samples as well as all undigested homogenates several reproducible minima and maxima in ELISA signal intensity were observed. All normal brain homogenates showed maxima in signal intensity in the samples heated to 140 C and in those heated to 156 C. The relative high of signal intensity was significant, when compared to other samples from the same animal, e.g. those heated to 146 C (p < 0.01, paired T-test). With BSE samples (both undigested and proteinase K digested) maxima were observed in samples heated to 140 C, 158 C and to 170 - 174 C. Again, the relative high of signal intensity was significant.
The highly reproducible patterns of relative maxima and minima in ELISA signal intensity were preserved even when the values were near background, which allowed the reliable detection of < 10 ng PrP/ml homogenate. In addition, a few IHC negative cows were detected witch had maxima and minima patterns different from other negative ones but very similar to those with BSE.
AD R.K. Meyer, A. Zurbriggen, NeuroCenter, Uni. Bern, CH; B. Oesch, Prionics, Schlieren, CH; M. Vandevelde, Uni. Bern, CH
SP englisch
PO Deutschland