Improvement of PrPSc-detection in mouse spleen early at the preclinical stage of scrapie with collagenase-completed tissue homogenization and Sarkosyl-NaCl extraction of PrPSc
Improvement of PrPSc-detection in mouse spleen early at the preclinical stage of scrapie with collagenase-completed tissue homogenization and Sarkosyl-NaCl extraction of PrPSc
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DOI：
10.1007/bf01718200
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发表时间：
1996-01-01
影响因子：
2.7
通讯作者：
Shinagawa, M
中科院分区：
文献类型：
--
作者：
Grathwohl, KUD;Horiuchi, M;Shinagawa, M
羊瘙痒症最近再次成为控制措施和根除计划的目标。这些措施的有效性的关键是在漫长和潜在危险的潜伏期内发现受感染的绵羊。然而，常规诊断大多限于疾病变得明显时的临床检查和尸检。通过免疫印迹法检测羊瘙痒症特异性的π-离子蛋白（PrPSc）亚型在脾脏和淋巴结从羊瘙痒症感染的小鼠和绵羊，我们已经表明，在临床前阶段的诊断是可能的。我们在这里介绍一种改进的方法来诊断感染后不久的小鼠瘙痒症。通过匀浆程序，其中包括胶原酶消化步骤，并通过提取和盐析的PrPSc Sarkosyl和NaCl，分别，我们能够检测到PrPSc在腹腔内感染的小鼠脾组织感染后7天。此外，新协议使样品处理更容易，并减少了动手时间。我们还成功地富集PrPSc从脾组织通过固定化金属亲和层析（IMAC），然而，在感染的最早阶段的诊断，提取PrPSc Sarkosyl和NaCl是更有效的。
Scrapie in sheep has recently become again a target of control measures and eradication programs. Crucial for the effectiveness of these measures is the detection of infected sheep during the long and potentially hazardous incubation period. However, routine-diagnosis is mostly limited to clinical examinations when disease: becomes apparent, and to postmortem investigations. Through the detection of the scrapie-specific isoform of the pi-ion protein (PrPSc) by Western blot in the spleen and lymph nodes from scrapie-infected mice and sheep, we have shown previously that diagnosis during the preclinical stage is possible. We introduce here an improved method for the diagnosis of mouse scrapie shortly after infection. Through a homogenization procedure that includes a collagenase digestion step, and through extraction and salting-out of PrPSc by Sarkosyl and NaCl, respectively, we were able to detect PrPSc in spleen tissue of intraperitoneally infected mice seven days postinfection. Moreover, the new protocol makes sample-handling easier and reduces the hands-on time. We also successfully enriched PrPSc from spleen tissue through immobilized metal affinity chromatography (IMAC); however, for the diagnosis at the earliest stage of infection, extraction of PrPSc by Sarkosyl and NaCl was more effective.