Quantitative recovery of scrapie agent with minimal protein from highly infectious cultures.
There are few reports on the isolation, quantitative recovery, and relative purification of infectious particles that cause scrapie, Creutzfeldt-Jakob disease (CJD) and epidemic bovine spongiform encephalopathy (BSE). Because pure prion protein (PrP) has failed to show significant infectivity, it is critical to find other molecules that are integral agent components. Only complex diseased tissues such as degenerating brain have been fractionated, and agent recoveries have been quite low in concentrated abnormal prion protein (PrP-res) preparations. To simplify the purification of infectious particles, we evaluated a monotypic cell line that continuously produced high levels of the 22L scrapie agent (N2a-22L). A new rapid and accurate GT1 culture assay was used to titrate infectivity in six representative sucrose gradients. We developed a streamlined approximately 3-h procedure that yielded full recovery of starting infectivity in fractions with only a few selected protein bands (representing <1% of starting protein). Infectious particles reproducibly sedimented through >30% sucrose steps, whereas PrP and PrP-res sedimentation varied depending on the conditions used. Both normal and abnormal PrP could be largely separated from infectivity in a single short centrifugation. Because no foreign enzymes were added to achieve reasonably purified infectious particles, these preparations may be used to elicit diagnostic antibodies to foreign agent proteins.
Sun
Ru
R
Section of Neuropathology, Yale Medical School, New Haven, Connecticut 06510, USA.
Liu
Ying
Y
Zhang
He
H
Manuelidis
Laura
L
D000818:Animals; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D051379:Mice; D009447:Neuroblastoma / Q000378:metabolism*; D018620:PrPSc Proteins / Q000302:isolation & purification* / Q000378:metabolism / Q000472:pathogenicity; D012608:Scrapie / Q000378:metabolism*
D018620:PrPSc Proteins
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