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The Quest for Anti-α-Synuclein Antibody Specificity-Lessons Learnt From Flow Cytometry Analysis
Lukas Leupold1, Veronika Sigutova1, Elizaveta Gerasimova1
1Department of Stem Cell Biology, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.
Frontiers in Neurology
|August 1, 2022
Summary
This study introduces a flow cytometry method to accurately detect alpha-synuclein (aSyn) protein, crucial for understanding neurodegenerative diseases like Parkinson's. The research validates antibodies, identifying 2A7 as optimal for sensitive aSyn detection.
Area of Science:
- Neuroscience
- Cell Biology
- Immunology
Background:
- Alpha-synuclein (aSyn) accumulation defines synucleopathies, but its physiological roles and detection are hindered by antibody variability and lack of sensitive methods.
- Accurate measurement of aSyn expression is critical for understanding its role in neurodegeneration and normal cellular functions.
- Existing anti-aSyn antibodies often lack characterization, impeding reliable protein expression analysis.
Purpose of the Study:
- To develop and validate a robust flow cytometry workflow for sensitive and specific detection of alpha-synuclein (aSyn).
- To evaluate the performance of commercially available anti-aSyn antibodies (MJFR1, LB509, 2A7) across various human cell types.
- To establish a reliable method for aSyn detection in research, particularly for neurodegenerative disease studies.
Main Methods:
- Development of a flow cytometry-based workflow for alpha-synuclein (aSyn) detection and antibody validation.
- Testing of three anti-aSyn antibodies (MJFR1, LB509, 2A7) in diverse human cell types, including iPSCs, T lymphocytes, and fibroblasts.
- Application of the validated workflow to analyze aSyn expression differences in cortical organoids and neural cells from healthy controls and Parkinson's disease patients.
Main Results:
- Demonstrated unspecificity of the LB509 antibody and low sensitivity of the MJFR1 clone for aSyn detection.
- Identified the 2A7 antibody clone as optimal for sensitive and specific aSyn detection across various cell types, including low-expressing ones.
- Successfully utilized the 2A7 antibody and workflow to differentiate physiological aSyn levels in neuronal and non-neuronal cells and in Parkinson's disease models.
Conclusions:
- The developed flow cytometry workflow provides a robust platform for high-throughput aSyn analysis and antibody validation.
- Rigorous validation of anti-aSyn antibodies is essential for advancing research into aSyn physiology and pathology.
- The 2A7 antibody is a sensitive and specific tool for detecting aSyn in diverse cellular contexts, including disease models.
