Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, Ohio 4410, USA.
J Biol Chem. 2010 Apr 30;285(18):13874-84. doi: 10.1074/jbc.M109.088831. Epub 2010 Mar 1.
The epitope of the 3F4 antibody most commonly used in human prion disease diagnosis is believed to consist of residues Met-Lys-His-Met (MKHM) corresponding to human PrP-(109-112). This assumption is based mainly on the observation that 3F4 reacts with human and hamster PrP but not with PrP from mouse, sheep, and cervids, in which Met at residue 112 is replaced by Val. Here we report that, by brain histoblotting, 3F4 did not react with PrP of uninfected transgenic mice expressing elk PrP; however, it did show distinct immunoreactivity in transgenic mice infected with chronic wasting disease. Compared with human PrP, the 3F4 reactivity with the recombinant elk PrP was 2 orders of magnitude weaker, as indicated by both Western blotting and surface plasmon resonance. To investigate the molecular basis of these species- and conformer-dependent preferences of 3F4, the epitope was probed by peptide membrane array and antigen competition experiments. Remarkably, the 3F4 antibody did not react with MKHM but reacted strongly with KTNMK (corresponding to human PrP-(106-110)), a sequence that is also present in cervids, sheep, and cattle. 3F4 also reacted with elk PrP peptides containing KTNMKHV. We concluded that the minimal sequence for the 3F4 epitope consists of residues KTNMK, and the species- and conformer-dependent preferences of 3F4 arise largely from the interactions between Met(112) (human PrP) or Val(115) (cervid PrP) and adjacent residues.
3F4 抗体最常用于人类朊病毒病诊断的表位被认为由残基 Met-Lys-His-Met（MKHM）组成，对应于人类 PrP-(109-112)。这一假设主要基于以下观察结果：3F4 与人类和仓鼠 PrP 反应，但不与来自小鼠、绵羊和鹿的 PrP 反应，其中残基 112 处的 Met 被 Val 取代。在这里，我们报告说，通过脑组织印迹，3F4 不会与表达麋鹿 PrP 的未感染转基因小鼠的 PrP 反应；然而，它在感染慢性消耗性疾病的转基因小鼠中表现出明显的免疫反应。与人类 PrP 相比，3F4 与重组麋鹿 PrP 的反应性弱 2 个数量级，这一点通过 Western blot 和表面等离子体共振都可以得到证实。为了研究 3F4 这种物种和构象依赖性偏好的分子基础，我们通过肽膜阵列和抗原竞争实验探测了表位。值得注意的是，3F4 抗体不会与 MKHM 反应，但会与 KTNMK（对应于人类 PrP-(106-110)）强烈反应，该序列也存在于鹿、绵羊和牛中。3F4 还与含有 KTNMKHV 的麋鹿 PrP 肽反应。我们得出结论，3F4 表位的最小序列由残基 KTNMK 组成，3F4 的物种和构象依赖性偏好主要来自于 Met(112)（人类 PrP）或 Val(115)（鹿 PrP）与相邻残基之间的相互作用。
