NR ATJW
AU Leon,F.; Segarra,C.S.; Coste,J.C.
TI Detection of infectious prion protein by protein misfolding cyclic amplification technology (PMCA)
QU International Conference - Prion 2005: Between fundamentals and society's needs - 19.10.-21.10.2005, Congress Center Düsseldorf - Poster Session: Diagnosis DIA-08
PT Konferenz-Poster
AB
Since the publication of two cases of probable transmission to British patients of the variant of Creutzfeldt-Jakob Disease by blood transfusion, it is likely that infectious prion protein (PrPsc) is present in human blood. Studies on rodent indicate that estimated sensitivity level of assays needs to reach a minimum of 1 femtomolar (0.1pg/ml or 10 IU/ml) in order to detect PrPsc in the blood of patients in the pre-clinical phase of the disease. The most sensitive test actually available doesn't reach this level of detection. So, an amplification of the PrPsc by the PMCA technology, may allow detection of very low concentrations present in the blood.
The aim of our laboratory is to develop a screening test for PrPsc in human blood components. The first phase consists in the reproduction and optimization of the PMCA technology described by C. Soto on hamster brain, in order to in a second phase adapt the method to hamster blood and finally to human blood.
PMCA allows accelerated production of infectious prions by successive incubation and sonication steps. During incubation, aggregates of PrPsc proteins are produced from low quantity of PrPsc. This template initiates transconformation of the normal prion protein (PrPc) into PrPsc. Sonication of the aggregates leads to numerous small amplification units, each one allowing new conversions. Amplified PrPsc is then detected by Western Blot.
PMCA can amplify 40 folds the initial input of PrPsc. Optimization of PMCA can be enhanced by addition of cofactors and by increasing the number of cycles (to reach a min 500x). PMCA has been adapted on hamster and on human leukocytes. This method is reproducible and specific.
PMCA technology can be adapted for PrPsc detection in the blood. In order to achieve the estimated threshold, we plan to improve the sensitivity of the manual PMCA by its adaptation on an automated sonicator allowing increased number of amplification cycles with a high throughput.
AD Fa Leon, Christiane Segarra (christiane.segarra@efs.sante.fr), Joliette Coste, Etablissement Français du Sang, Pyrénées-Méditerranées, Montpellier, FRANCE
SP englisch
PO Deutschland