Foodborne Contaminants Research Unit, U.S. Department of Agriculture Agricultural Research Service, Albany, CA 94710, USA.
J Immunol. 2010 Jul 1;185(1):729-37. doi: 10.4049/jimmunol.0902930. Epub 2010 Jun 7.
Prion diseases are fatal, neurodegenerative illnesses caused by the accumulation of PrP(Sc), an aberrantly folded isoform of the normal, cellular prion protein. Detection of PrP(Sc) commonly relies on immunochemical methods, a strategy hampered by the lack of Abs specific for this disease-causing isoform. In this article, we report the generation of eight mAbs against prion protein (PrP) following immunization of Prnp-null mice with rPrP. The eight mAbs exhibited distinct differential binding to cellular prion protein and PrP(Sc) from different species as well as PrP-derived synthetic peptides. Five of the eight mAbs exhibited binding to discontinuous PrP epitopes, all of which were disrupted by the addition of 2-ME or DTT, which reduced the single disulfide bond found in PrP. One mAb F20-29 reacted only with human PrP, whereas the F4-31 mAb bound bovine PrP; the K(D) values for mAbs F4-31 and F20-29 were ~500 pM. Binding of all five conformation-dependent mAbs to PrP was inhibited by 2-ME in ELISA, Western blots, and histoblots. One conformation-dependent mAb F4-31 increased the sensitivity of an ELISA-based test by nearly 500-fold when it was used as the capture Ab. These new conformation-dependent mAbs were found to be particularly useful in histoblotting studies, in which the low backgrounds after treatment with 2-ME created unusually high signal-to-noise ratios.
朊病毒病是由朊病毒蛋白（PrP）的异常折叠异构体 PrP（Sc）积累引起的致命神经退行性疾病。PrP（Sc）的检测通常依赖于免疫化学方法，但这种策略受到缺乏针对这种致病异构体的 Abs 的限制。在本文中，我们报告了在用 rPrP 免疫 Prnp 缺失小鼠后，针对朊病毒蛋白（PrP）产生了 8 种 mAb。这 8 种 mAb 表现出对不同物种的细胞朊病毒蛋白和 PrP（Sc）以及 PrP 衍生的合成肽的不同差异结合。这 8 种 mAb 中的 5 种表现出与不连续 PrP 表位的结合，所有这些表位都被添加 2-ME 或 DTT 破坏，这会减少 PrP 中发现的单个二硫键。一种 mAb F20-29 仅与人类 PrP 反应，而 F4-31 mAb 与牛 PrP 结合；mAb F4-31 和 F20-29 的 K(D)值约为 500 pM。在 ELISA、Western blot 和组织印迹中，所有 5 种构象依赖性 mAb 与 PrP 的结合均被 2-ME 抑制。在 ELISA 基础测试中用作捕获 Ab 时，构象依赖性 mAb F4-31 可将其灵敏度提高近 500 倍。这些新的构象依赖性 mAb 在组织印迹研究中特别有用，在这些研究中，用 2-ME 处理后背景非常低，从而产生了异常高的信噪比。
