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

NEUROBIOLOGY OF DISEASE

卷:146

Article

Kumar, Senthil T.¹  Jagannath, Somanath¹  Francois, Cindy²  Vanderstichele, Hugo^2,3  Stoops, Erik²  Lashuel, Hilal A.¹ 

[1] Ecole Polytech Fed Lausanne, Brain Mind Inst, Lab Mol & Chem Biol Neurodegenerat, Lausanne, Switzerland

[2] ADx NeuroSci, Technol Pk 94, Ghent, Belgium

[3] Biomarkable, Ghent, Belgium

关键词: alpha-Synuclein;    Parkinson's disease;    Conformational antibodies;    Oligomers;    Binding specificity;   

DOI  :  10.1016/j.nbd.2020.105086

来源: Elsevier

【 摘 要 】

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.