Chang Binggong, Cheng Xin, Yin Shaoman, Pan Tao, Zhang Hongtao, Wong Poki, Kang Shin-Chung, Xiao Fan, Yan Huimin, Li Chaoyang, Wolfe Lisa L, Miller Michael W, Wisniewski Thomas, Greene Mark I, Sy Man-Sun
Institute of Pathology School of Medicine, Case Western Reserve University, Cleveland, Ohio 44107-1712, USA.
Clin Vaccine Immunol. 2007 Jan;14(1):36-43. doi: 10.1128/CVI.00341-06. Epub 2006 Nov 1.
We have developed a sensitive in vitro assay for detecting disease-associated prion aggregates by combining an aggregation-specific enzyme-linked immunosorbent assay (AS-ELISA) with the fluorescent amplification catalyzed by T7 RNA polymerase technique (FACTT). The new assay, named aggregation-specific FACTT (AS-FACTT), is much more sensitive than AS-ELISA and could detect prion aggregates in the brain of mice as early as 7 days after an intraperitoneal inoculation of PrP(Sc). However, AS-FACTT was still unable to detect prion aggregates in blood of infected mice. To further improve the detection limit of AS-FACTT, we added an additional prion amplification step (Am) and developed a third-generation assay, termed Am-A-FACTT. Am-A-FACTT has 100% sensitivity and specificity in detecting disease-associated prion aggregates in blood of infected mice at late but still asymptomatic stages of disease. At a very early stage, Am-A-FACTT had a sensitivity of 50% and a specificity of 100%. Most importantly, Am-A-FACTT also detects prion aggregates in blood of mule deer infected with the agent causing a naturally occurring prion disease, chronic wasting disease. Application of this assay to cattle, sheep, and humans could safeguard food supplies and prevent human contagion.
我们通过将聚集特异性酶联免疫吸附测定（AS-ELISA）与T7 RNA聚合酶催化的荧光扩增技术（FACTT）相结合，开发了一种用于检测疾病相关朊病毒聚集体的灵敏体外检测方法。这种新的检测方法名为聚集特异性FACTT（AS-FACTT），比AS-ELISA灵敏得多，并且早在腹腔接种PrP(Sc)后7天就能检测到小鼠脑中的朊病毒聚集体。然而，AS-FACTT仍然无法检测到感染小鼠血液中的朊病毒聚集体。为了进一步提高AS-FACTT的检测限，我们增加了一个额外的朊病毒扩增步骤（Am），并开发了第三代检测方法，称为Am-A-FACTT。Am-A-FACTT在检测感染小鼠处于疾病晚期但仍无症状阶段血液中的疾病相关朊病毒聚集体时具有100%的灵敏度和特异性。在疾病非常早期阶段，Am-A-FACTT的灵敏度为50%，特异性为100%。最重要的是，Am-A-FACTT还能检测感染导致自然发生的朊病毒疾病——慢性消耗病的病原体的骡鹿血液中的朊病毒聚集体。将这种检测方法应用于牛、羊和人类可以保障食品供应并预防人类传染。
