How specific are the conformation-specific α-synuclein antibodies? Characterization and validation of 16 α-synuclein conformation-specific antibodies using well-characterized preparations of α-synuclein monomers, fibrils and oligomers with distinct structures and morphology
How specific are the conformation-specific α-synuclein antibodies? Characterization and validation of 16 α-synuclein conformation-specific antibodies using well-characterized preparations of α-synuclein monomers, fibrils and oligomers with distinct structures and morphology
复制标题
DOI：
10.1016/j.nbd.2020.105086
复制
发表时间：
2020-12-01
影响因子：
6.1
通讯作者：
Lashuel, Hilal A.
中科院分区：
文献类型：
--
作者：
Kumar, Senthil T.;Jagannath, Somanath;Lashuel, Hilal A.
越来越多的证据表明，α-突触核蛋白（α-syn）寡聚体是参与α-syn纤维化和路易体（LB）形成的途径中的专性中间体，也可能在帕金森病（PD）和其他突触核蛋白病的LB内积累。因此，开发检测和定量α-syn寡聚体的工具和方法对于了解其在PD中的作用以及开发PD和其他突触核蛋白病的新诊断方法和疗法的机制研究变得越来越重要。这些工具和方法中的大多数主要依赖于聚集状态特异性或构象特异性抗体的使用。考虑到使用这些抗体产生的数据和知识对形成α-syn和PD研究的基础和方向的影响，至关重要的是，这些抗体被彻底表征，并测试和验证它们捕获不同α-syn物种的特异性或能力。在本文中，我们描述了一种抗体表征和验证管道，其允许使用各种α-syn物质的明确定义和明确表征的制备物系统地研究α-syn抗体的特异性，所述α-syn物质包括单体、原纤维和不同的寡聚体制备物，其特征在于不同的形态学、化学和二级结构特性。该管道用于表征18种α-syn抗体，其中16种已被报道为构象或寡聚体特异性抗体，使用一系列技术，包括免疫印迹分析（狭缝印迹和Western印迹），使用单分子阵列技术和表面等离子体共振的数字ELISA测定。我们的结果显示：i）所测试的抗体中没有一种对一种特定类型的α-syn种类具有特异性，包括单体、寡聚体或原纤维; ii）所有被报道为寡聚体特异性的抗体也识别纤维状α-syn;以及iii）少数抗体显示出对寡聚体和原纤维的高特异性，但不结合单体。这些发现表明，绝大多数α-syn聚集体特异性抗体不能区分寡聚体和原纤维，因此强调了在解释使用这些抗体获得的结果时谨慎行事的重要性。我们的研究结果还强调了在将抗体用于机理研究以及作为诊断工具或治疗剂之前对其进行表征和验证的至关重要性。这不仅将提高研究的质量和可重复性并降低成本，还将减少临床治疗抗体失败的数量。
Increasing evidence suggests that alpha-synuclein (alpha-syn) oligomers are obligate intermediates in the pathway involved in alpha-syn fibrillization and Lewy body (LB) formation, and may also accumulate within LBs in Parkinson's disease (PD) and other synucleinopathies. Therefore, the development of tools and methods to detect and quantify alpha-syn oligomers has become increasingly crucial for mechanistic studies to understand their role in PD, and to develop new diagnostic methods and therapies for PD and other synucleinopathies. The majority of these tools and methods rely primarily on the use of aggregation state-specific or conformation-specific antibodies. Given the impact of the data and knowledge generated using these antibodies on shaping the foundation and directions of alpha-syn and PD research, it is crucial that these antibodies are thoroughly characterized, and their specificity or ability to capture diverse alpha-syn species is tested and validated. Herein, we describe an antibody characterization and validation pipeline that allows a systematic investigation of the specificity of alpha-syn antibodies using well-defined and well-characterized preparations of various alpha-syn species, including monomers, fibrils, and different oligomer preparations that are characterized by distinct morphological, chemical and secondary structure properties. This pipeline was used to characterize 18 alpha-syn antibodies, 16 of which have been reported as conformationor oligomer-specific antibodies, using an array of techniques, including immunoblot analysis (slot blot and Western blot), a digital ELISA assay using single molecule array technology and surface plasmon resonance. Our results show that i) none of the antibodies tested are specific for one particular type of alpha-syn species, including monomers, oligomers or fibrils; ii) all antibodies that were reported to be oligomer-specific also recognized fibrillar alpha-syn; and iii) a few antibodies showed high specificity for oligomers and fibrils but did not bind to monomers. These findings suggest that the great majority of alpha-syn aggregate specific antibodies do not differentiate between oligomers and fibrils, thus highlighting the importance of exercising caution when interpreting results obtained using these antibodies. Our results also underscore the critical importance of the characterization and validation of antibodies before their use in mechanistic studies and as diagnostic tools or therapeutic agents. This will not only improve the quality and reproducibility of research and reduce costs but will also reduce the number of therapeutic antibody failures in the clinic.