Preparation of soluble infectious samples from scrapie-infected brain: a new tool to study the clearance of transmissible spongiform encephalopathy agents during plasma fractionation.
BACKGROUND
Concern about the safety of blood, blood components, and plasma-derived products with respect to prions has increased since the report of two blood-related infections of variant Creutzfeldt-Jakob disease in the United Kingdom. Efforts were directed toward the development of procedures able to remove or inactivate prions from blood components or plasma-derived products with brain fractions of transmissible spongiform encephalopathy (TSE)-infected rodents as spiking materials. These spiking materials, however, are loaded with pathological prion protein (PrP(TSE)) aggregates that are likely not associated to blood infectivity. The presence of these aggregates may invalidate these studies.


METHODS
Brains from 263K scrapie-infected hamsters were suspended in 10 percent phosphate-buffered saline. After low-speed centrifugation, the supernatant was collected and ultracentrifuged at 220,000 x g at 25 degrees C for 30 minutes. The high-speed supernatants (S(HS)) and pellets were collected; the proteinase-resistant PrP(TSE) was measured by Western blot and infectivity by intracerebral inoculation into weanling hamsters.


RESULTS
A substantial amount of prion infectivity (more than 10(5) LD(50) per mL of a 10% suspension of brain tissues) is present in the S(HS) fraction of 263K scrapie-infected hamster brains. Concomitantly, this fraction contains none or only traces of PrP(TSE) in its aggregate form.


CONCLUSIONS
This study describes a simple and fast protocol to prepare infectious material from 263K scrapie-infected brains that is not contaminated with PrP(TSE) aggregates. This S(HS) fraction is likely to be the most relevant material for endogenous spiking of human blood in validation experiments aimed at demonstrating procedures to remove or inactivate TSE infectious agents.
Degenerative and Inflammatory Neurological Diseases Unit, Department of Cell Biology and Neurosciences, Italian National Institute of Health, Rome.
Vito Angelo
VA
Berardi
Franco
F
Cardone
Angelina
A
Valanzano
Mei
M
Lu
Maurizio
M
Pocchiari
D000818:Animals; D016913:Blood Component Transfusion / Q000009:adverse effects*; D001921:Brain / Q000473:pathology*; D006224:Cricetinae; D008647:Mesocricetus; D023421:Models, Animal; D018620:PrPSc Proteins / Q000302:isolation & purification*; D017096:Prion Diseases / Q000097:blood* / Q000517:prevention & control* / Q000635:transmission*; D011328:Prions / Q000302:isolation & purification
D018620:PrPSc Proteins; D011328:Prions