Objective
SUMMARY
In this proposed research project, we want to concentrate on existing weaknesses of validation processes:
infectious material used for spiking should always be close to the real situation, but is usually crude brain extract of infected animals because of its high titre. This is, however, in many cases quite different from natural infection, especially if we consider blood products.
We therefore want to use different infectious preparations, including suspensions of infected neuroblastoma cells, to spike plasma and simulate the industrial process of plasma fractionation, concentrating on the 3 major fractions containing clotting factor Vlll, IgG and albumin.
If a product is made in a multi-step process, consisting of several inactivating steps, each of these steps can be challenged in a spiking experiment and the overall reduction of infectivity can be calculated as the product of the inactivation results of the individual steps. This is valid, however, only, IF the steps are different (employing different modes of inactivation/removal) AND if the units of infectivity are truly independent entities. In the case of TSE this is doubtful, due to the very hydrophobic nature of their agents, leading to self-aggregation.
To test this, we want to perform a 3 step - process twice:
First, the infectious spike is added to the starting materials of all 3 steps, these steps are validated individually and the overall reduction of infectivity is calculated;
Secondly, infectious material is added only to the starting material of step 1, and the 3 steps are performed consecutively, using this material. The final product's infectivity is determined and compared to the calculated result of test 1.
We also want to evaluate existing (western blot) and emerging (DELFIA) IN VITRO ASSAYS by comparing them with the results of the IN VIVO ASSAYS used to determine infectivity in the experiments mentioned above. This could result in faster and yet sensitive alternatives to the use of laboratory animals, at least for certain applications.
KEYWORDS: INACTIVATION - VALIDATION - TSE causing agents - INFECTIVITY - SPIKING EXPERIMENTS - IN VITRO ASSAYS - WESTERN BLOT - DELFIA
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Programme(s)
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Coordinator
8045 Graz
Austria
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