NR ATNU
AU Arrabal,S.; You,B.; Le Hir,G.; Aubin,J.T.; Laude,H.; Flan,B.
TI Development of an in vitro TSE Infectivity Assay and Application to Validation of Manufacturing Processes
QU International Conference - Prion 2005: Between fundamentals and society's needs - 19.10.-21.10.2005, Congress Center Düsseldorf - Poster Session: Human prions, risk of blood products, and therapy HUMAN-18
PT Konferenz-Poster
AB
Titration experiments of transmissible spongiform encephalopthies (TSE) agents, which are required for the validation of manufacturing processes, involve either time-consuming infectivity protocols, consisting in intra-cerebral inoculation of laboratory rodents, or less sensitive immunochemical PrPres detection, mostly by Western Blot (WB).
Here, we present the development of an alternative sensitive in vitro infectivity titration assay. This assay is based on the infection of TSE permissive cells, cultured in multi-well plaques, by serial dilutions of infectious material. Cells are cultured for 8 passages and the number of positive wells is estimated by WB. The infectious titre is determined by the Spearman Karber method, and expressed in tissue culture infectious doses.
The cellular assay was shown to be ~ 80 times more sensitive than WB, and comparable to bioassay. It was also shown to be suitable for evaluating the TSE infectivity removal in manufacturing processes used for the manufacture of biological products, using a 15nm nanofiltration step as a model. A reduction factor of more than 4.2 log10 was evidenced for this step, quite well correlated with previously reported data from experiments performed by bioassay or WB.
Latest developments of this tissue culture infectivity assay will be shown, and recognition by regulatory authorities will be discussed.
IN Die Autoren haben einen vergleichsweise billigen und schnellen Test für TSE-Infektiosität entwickelt, welcher die Sensitivität von Übertragungsexperimenten erreichen und die Sensitivität von Western blots um fast zwei Größenordnungen übersteigen soll. Sie infizieren dazu nicht Tiere, sondern Zellkulturen. Mit ihrer Methode ermittelten sie für bei Herstellern biologischer Produkte anscheinend verbreitete 15-nm-Nanofilter einen Abreicherungsfaktor von 4,2 log10-Stufen.
AD Samuel Arrabal, Jean-Thierry Aubin, Benoit Flan, Laboratoire du Fractionnement et des Biotechnologies, Unité de Sécurisation Biologique, Les Ulis, France; Bruno You, Gwenn Le Hir, Laboratoire du Fractionnement et des Biotechnologies, Laboratoire de Sécurité Biologique, Fontenay-aux-Roses, France; Hubert Laude, Institut National de la Recherche Agronomique, Unité de Virologie Immunologie Moléculaires, Jouy-en-Josas, France
SP englisch
PO Deutschland