Pan Tao, Sethi Jasmeet, Nelsen Craig, Rudolph Alan, Cervenakova Larisa, Brown Paul, Orser Cindy S
Adlyfe Inc., 9430 Key West Avenue, Rockville, MD 20850, USA.
Transfusion. 2007 Aug;47(8):1418-25. doi: 10.1111/j.1537-2995.2007.01284.x.
The long-standing goal of a preclinical diagnostic test for transmissible spongiform encephalopathy (TSE) has recently become urgent because of the discovery that humans with variant Creutzfeldt-Jakob disease can transmit disease via blood transfusions.
The misfolded protein diagnostic (MPD) assay employs a pyrene-labeled palindromic sequence of prion peptides that undergoes a cascade of coil to beta-sheet conversion in the presence of the misfolded prion protein (PrP(TSE)). The ability of the assay to detect PrP(TSE) in brain, serum, and plasma was tested. The basic protocol involved a several-hour incubation of 200-microL sample volumes with the peptide reagent in 96-well plates, after which fluorescence was monitored by a fluorescence plate reader with an excitation wavelength of 350 nm and emission scanning wavelength range of 365 to 600 nm.
Target specificity for PrP(TSE) was documented by correlation of assay signal with Western blot signals in brain tissue from TSE-infected, normal, and knockout mice and negative assay signals by use of reagents with different peptide sequences. When applied to plasma or serum, the assay discriminated between samples from a variety of experimental and natural TSE infections compared to uninfected controls, with a sensitivity threshold of approximately 1 infectious dose per mL in pooled plasma from TSE-infected mice.
The MPD assay is a sensitive and specific test for the detection of PrP(TSE) that may be useful in both preclinical and clinical diagnosis of TSE diseases of animals and humans.
由于发现变异型克雅氏病患者可通过输血传播疾病，因此，针对传染性海绵状脑病（TSE）的临床前诊断测试这一长期目标最近变得十分紧迫。
错误折叠蛋白诊断（MPD）检测法采用了一种芘标记的朊病毒肽回文序列，该序列在错误折叠的朊病毒蛋白（PrP(TSE)）存在的情况下会经历一系列从卷曲到β-折叠的转变。测试了该检测法检测脑、血清和血浆中PrP(TSE)的能力。基本方案包括在96孔板中将200微升样本体积与肽试剂孵育数小时，之后用激发波长为350nm、发射扫描波长范围为365至600nm的荧光酶标仪监测荧光。
通过将检测信号与来自感染TSE的、正常的和基因敲除小鼠的脑组织中的蛋白质印迹信号进行关联，证明了对PrP(TSE)的目标特异性，并且使用具有不同肽序列的试剂得到了阴性检测信号。当应用于血浆或血清时，与未感染的对照相比，该检测法能够区分来自各种实验性和自然TSE感染的样本，在来自感染TSE小鼠的混合血浆中的灵敏度阈值约为每毫升1个感染剂量。
MPD检测法是一种检测PrP(TSE)的灵敏且特异的检测方法，可能对动物和人类TSE疾病的临床前和临床诊断都有用。
