NR AWGF
AU Guyon,D.; Simon,S.; Morel,N.; Lacroux,C.; Andreoletti,O.; Grassi,J.
TI Biochemical and immunological characterization or PrP in ewe milk
QU International Conference - Prion 2006: Strategies, advances and trends towards protection of society - 3.10.-6.10.2006, Torino, Italy, Lingotto Conference Centre - Poster sessions RA-04
PT Konferenz-Poster
AB Because of the possible presence of the BSE strain in sheep and goat flocks some concerns have long been raised about the possibility that small ruminant milk could carry some infectivity. These concerns have been reinforced recently by the demonstration that infectivity is currently reported in blood from animal or human infected by the BSE strain associated with the presence of blood related cells in milk. In addition, recent works have shown that PrPsc accumulates in mammary glands from sheep affected by scrapie and mastitis. In this context it is critical to analyse more precisely the relationship between PrP and milk components. In this study, we have analysed the PrP content of the main milk fractions (casein whey, fat and cells) using biochemical (ultracentrifugation, western-blot) and immunological (western-blot, ELISA) methods. By using a panel of monoclonal antibodies directed against different epitopes located along the PrP sequence and by combining immunoprecipitation techniques with western-blot measurements, we could demonstrate that PrP is essentially present in milk fractions (casein whey, fat and cells) as an N-terminally truncated protein. Ultracentrifugation analysis showed that PrP exists in casein whey and fat fractions as an amphiphilic protein bearing a GPI anchor. Measurements made on the different ewe milk fractions using an adapted sandwich ELISA showed that important concentration of PrP are present in casein whey (75-600 ng/ml) and fat (150-600 ng/ml) while much lower concentration are measured in cells extracts. Taking into account the proportion of each milk fraction it clearly appeared that casein whey accounts for more than 95% of the total PrP content of milk with approximately 5% for fat and less than 0.1% for cells. Measurements performed on colostrums showed that it contains about twice more PrP than milk. Finally, we have optimized immunoprecipitation methods in order to bind on magnetic beads more than 95% of the total PrP content of the different milk fractions. This was obtained by using a mixture of monoclonal antibodies including an antibody having demonstrated its capacity to immunoprecipitate aggregated PrPsc in TSE infected brain extracts. These beads will be inoculated in ovine transgenic mice in order to detect infectivity possibly associated with PrP in the three milk fractions.
AD D. Guyon, S. Simon, N. Morel, J. Grassi: CEA, Service de Pharmacologie et d'Immunologie, CEA/Saclay, 91191 Gif sur Yvette cedex, France; C. Lacroux, O. Andréoletti: UMR INRA ENVT 1225, Interactions Hôte- Agents Pathogènes, Ecole Nationale Vétérinaire de Toulouse, 23 Chemin des Capelles 31076 Toulouse, France
SP englisch
PO Italien