一种基于链霉素沉淀的PrPSc检测方法的建立

建立一种新的基于链霉素沉淀的PrPSc的Western blot检测方法,用终浓度为60 mmol/L的链霉素处理蛋白酶K消化的PrPSc贮存液,通过离心沉淀PrPSc,用Western blot对PrPSc的链霉素富集效果进行检测.结果显示,链霉索能够与PrPSc结合形成高分子量复合物,但不影响糖基化形式.此外,基于链霉素沉淀的Western blot,无论是在低浓度或是大容积的条件下,均可显著地提高对PrPSc检测的敏感性.基于链霉素沉淀的Western blot试验是一种敏感、特异、快速及灵活的检测方法,有潜力用于脑组织、外周组织及体液中低水平的PrPSc的检测.

Establishment of an Assay for PrPSc Detection Based on Streptomycin Precipitation

To establish a new Western blotting assay for PrPSc detection, we optimized the Western blotting assay with a precipitation procedure of streptomycin sulfate. After digestion with PK, 10% scrapie infected hamster brain homogenates were incubated with 60 mmol/L streptomycin and the precipitated PrPSc was recovered by centrifugation. The enrichment of PrPSc by streptomycin sulfate precipitation was evaluated using Western blotting assay. The results showed streptomycin could bind to PK-treated PrPSc, forming high molecular masses, but not influence the glycosylated patterns on SDS-PAGE. Western blot assay revealed that the detective sensitivity of the streptomycin-precipitation PrPSc was remarkably improved. As a sensitive, specific, rapid and flexible protocol for PrPSc, the protocol in this study has the potential utility, alone or combined with other techniques, for the detection of low level PrPSc in the specimens from central nerve system, or from peripheral organs or body fluids.