NR AORQ
AU Dawn,D.N.; Murdoch,H.; Carr,J.; Buswell,C.M.; Dickinson,J.; Sutton,J.M.; Raven,N.D.H.
TI Development of novel immunoassays for the detection of TSE agent on surgical instruments and other biological samples
QU International Conference - Prion diseases: from basic research to intervention concepts - TSE-Forum, 08.10.-10.10.2003, Gasteig, München - Poster session - DG-33
PT Konferenz-Poster
AB
Routine surgery and transplant / transfusion remain potential routes for the transmission of Creutzfeldt-Jakob disease (CJD) and its new variant form (vCJD). To reduce the likelihood of this happening, new methods are required to detect the presence of prion material in complex biological samples, such as tissue or blood, and on the surface of surgical instruments. The method described here has been designed as a flexible format for the detection of prion material in such samples.
The method uses antibody conjugated to an adenylate kinase (AK) marker enzyme to generate ATP. The ATP generated is then coupled with luciferin/luciferase detection reagents to provide an ultrasensitive bioluminescent detection method. A thermostable AK was cloned, expressed and purified. The enzyme was conjugated via a cleavable linker to the commercially available anti-prion antibody 6H4, and a model assay developed for recombinant PrP (recPrP). Cleavage of the AK enzyme after antibody binding allows transfer of the remainder of the assay to standard laboratory equipment. This enables the method to be applied to large surgical equipment without the need for complex apparatus. The assay format uses heat denaturation steps to eliminate any contaminating enzymatic activity by utilising the thermostability of the AK. We have been able to detect recPrP at a concentration of 6pM in a background of mouse brain homogenate. We have also demonstrated that the assay can detect recPrP in complex biological samples such as blood and in a model of prion contamination on surgical steel. The potential of the assy to allow ultrasensitive detection of the TSE agents in a variety of samples will be discussed.
AD D.N. Dawn, H. Murdoch, J. Carr, C.M. Buswell, J. Dickinson, J.M. Sutton, N.D.H. Raven, HPA-Porton Down, UK
SP englisch
PO Deutschland