Department of Molecular Biochemistry and Pharmacology, Istituto di Ricerche Farmacologiche Mario Negri, 20156 Milan, Italy.
J Biol Chem. 2012 Aug 10;287(33):27796-805. doi: 10.1074/jbc.M111.334979. Epub 2012 Jun 26.
Soluble oligomers of the amyloid-β (Aβ) peptide play a key role in the pathogenesis of Alzheimer's disease, but their elusive nature makes their detection challenging. Here we describe a novel immunoassay based on surface plasmon resonance (SPR) that specifically recognizes biologically active Aβ oligomers. As a capturing agent, we immobilized on the sensor chip the monoclonal antibody 4G8, which targets a central hydrophobic region of Aβ. This SPR assay allows specific recognition of oligomeric intermediates that rapidly appear and disappear during the incubation of synthetic Aβ(1-42), discriminating them from monomers and higher order aggregates. The species recognized by SPR generate ionic currents in artificial lipid bilayers and inhibit the physiological pharyngeal contractions in Caenorhabditis elegans, a new method for testing the toxic potential of Aβ oligomers. With these assays we found that the formation of biologically relevant Aβ oligomers is inhibited by epigallocatechin gallate and increased by the A2V mutation, previously reported to induce early onset dementia. The SPR-based immunoassay provides new opportunities for detection of toxic Aβ oligomers in biological samples and could be adapted to study misfolding proteins in other neurodegenerative disorders.
可溶性淀粉样蛋白-β(Aβ)肽的低聚物在阿尔茨海默病的发病机制中起关键作用,但由于其难以捉摸的性质,检测具有挑战性。在这里,我们描述了一种基于表面等离子体共振(SPR)的新型免疫测定法,该方法特异性识别具有生物活性的 Aβ低聚物。作为捕获剂,我们将针对 Aβ的中心疏水区的单克隆抗体 4G8 固定在传感器芯片上。这种 SPR 测定法允许特异性识别在合成 Aβ(1-42)孵育过程中迅速出现和消失的低聚物中间体,将其与单体和更高阶聚集体区分开来。SPR 识别的物质在人工脂质双层中产生离子电流,并抑制秀丽隐杆线虫的生理咽收缩,这是一种测试 Aβ低聚物毒性潜力的新方法。通过这些测定法,我们发现表没食子儿茶素没食子酸酯抑制了具有生物学相关性的 Aβ低聚物的形成,而先前报道可诱导早发性痴呆的 A2V 突变则增加了其形成。基于 SPR 的免疫测定法为在生物样本中检测有毒 Aβ低聚物提供了新的机会,并可用于研究其他神经退行性疾病中的错误折叠蛋白。