Lund Christoffer, Olsen Christel Moraeus, Tveit Heidi, Tranulis Michael A
Department of Basic Sciences and Aquatic Medicine, Norwegian School of Veterinary Science, P.O. Box 8146 Dep., N-0033 Oslo, Norway.
J Neurosci Methods. 2007 Sep 30;165(2):183-90. doi: 10.1016/j.jneumeth.2007.06.005. Epub 2007 Jun 13.
The monoclonal antibody (MAb) 3F4 has for nearly two decades been one of the most commonly used tools in prion research. This MAb has contributed significantly to our understanding of the normal cell biology of the prion protein (PrP(C)), as well as the disease related abnormalities occurring in prion diseases. The 3F4 antibody binds strongly to human and hamster PrP, with a specific requirement of two Met residues at positions 109 and 112 in the human PrP. Other species in which PrP lack one of the Met residues, like cattle and sheep, or both, like rat and mouse, do not react with the 3F4 antibody. These and other observations have led to the commonly accepted notion that the 3F4 epitope consists of the tetra-peptide Met-Lys-His-Met. In this study, we have identified the minimal epitope for 3F4 by studying its binding to synthetic peptides and by analysis of mutated ovine PrP::GFP constructs expressed in cell culture. We have found that the 3F4 epitope consists of a hepta-peptide (Lys-Thr-Asn-Met-Lys-His-Met), which in sheep encompass residues 109-115. We found that Lys 109 is critically important for 3F4 binding, as omission, or substitution of this residue to Ala resulted in no binding. We also demonstrate that the hepta-peptide constituting the minimal 3F4 epitope, can be used as a discrete, moveable high-affinity molecular tag. Thus, the 3F4 antibody can find its use beyond prion research.
近二十年来，单克隆抗体（MAb）3F4一直是朊病毒研究中最常用的工具之一。这种单克隆抗体对我们理解朊病毒蛋白（PrP(C)）的正常细胞生物学以及朊病毒疾病中出现的与疾病相关的异常情况做出了重大贡献。3F4抗体与人和仓鼠的PrP强烈结合，对人PrP中第109和112位的两个甲硫氨酸残基有特定要求。PrP缺乏其中一个甲硫氨酸残基的其他物种，如牛和羊，或两者都缺乏，如大鼠和小鼠，不会与3F4抗体发生反应。这些以及其他观察结果导致了一个普遍接受的观点，即3F4表位由四肽甲硫氨酸-赖氨酸-组氨酸-甲硫氨酸组成。在本研究中，我们通过研究其与合成肽的结合以及分析在细胞培养中表达的突变绵羊PrP::GFP构建体，确定了3F4的最小表位。我们发现3F4表位由一个七肽（赖氨酸-苏氨酸-天冬酰胺-甲硫氨酸-赖氨酸-组氨酸-甲硫氨酸）组成，在绵羊中包含第109-115位残基。我们发现赖氨酸109对3F4结合至关重要，因为省略该残基或将其替换为丙氨酸会导致不结合。我们还证明，构成最小3F4表位的七肽可以用作离散的、可移动的高亲和力分子标签。因此，3F4抗体的用途可能不限于朊病毒研究。
