AlphaLISA detection of alpha-synuclein in the cerebrospinal fluid and its potential application in Parkinson’s disease diagnosis
Parkinson’s disease (PD) is the second most prevalent neurodegenerative disorder affecting about 1% of the worldwide population over the age of 60 (El-Agnaf, 2003; Majbour, Vaikath et al., 2016). Motor symptoms, which is currently the major trait for PD diagnosis, appear when 50%–60% of dopaminergic neurons in the substantia nigra (SN) and 70%–80% of dopaminergic terminals in the striatum are lost (Kim, Paik et al., 2014; Landeck, Hall et al., 2016), making PD extremely challenging to treat when diagnosed. Thus, PD biomarkers other than the motor symptoms may provide additional help in PD diagnosis, making earlier PD treatment possible to help the management and treatment of the disease. One of the pathological hallmarks of PD is the presence of Lewy bodies (LBs) and Lewy neurites in surviving PD neurons (Delenclos, Jones et al., 2016; Majbour, Vaikath et al., 2016). LBs contain a large amount of accumulated alpha-synuclein proteins (α-syn), which is a 14.6 kDa lipid-binding protein and encoded by the SNCA gene (De Genst, Guilliams et al., 2010, Bartels, Choi et al., 2011). The accumulation and aggregation of α-syn is likely contributing to PD progression. The α-syn variant A53T (53 A→T) found in human patients has no effect on osmotic stress-induced phosphorylation, but increases oligomerization and exacerbates disease progression; suggesting a causal relationship between α-syn oligomerization and PD (Berrocal, Vasquez et al., 2014). Consistently, recent work suggested that monomeric α-syn level is likely reduced in cerebrospinal fluid (CSF) of the PD patients, while the oligomeric α-syn may increase (Aasly, Johansen et al., 2014). Meanwhile, the results have been controversial possibly due to lack of sensitive and reliable detection methodologies for CSF α-syn detection, especially for the α-syn oligomers.
Here we established a robust and reproducible AlphaLISA assay that allows sensitive and reliable measurements of the relative levels of oligomeric versus total α-syn directly in the microliter wells. AlphaLISA signals are generated only when the acceptor-bead conjugated antibody and the donor-bead conjugated biotinylated antibody bind to the same target protein molecule, which brings the acceptor and donor beads to close proximity (<200 nm) so that the single O2 molecules could be transferred from the donor to the acceptor (Bielefeld-Sevigny, 2009). We utilized the Life/Nb antibody pair (see supplement for antibody information) to detect the total α-syn. These antibodies bind with different α-syn epitopes, and thus both the monomeric and the oligomeric α-syn can bind with the two antibodies simultaneously, generating AlphaLISA signals (Fig. S1A). For α-syn oligomer detection, we utilized the Life/Life antibody pair. In this case, the same antibody (Life) was conjugated with both the acceptor and the donor beads. A single monomeric α-syn molecule has only one binding epitope for the Life antibody and thus could not bind with both the acceptor- and the streptavidin donor- conjugated Life antibodies at the same time, and thus cannot generate any AlphaLISA signals (Fig. S1B). Meanwhile, oligomeric α-syn protein molecules can generate AlphaLISA signals by providing multiple epitopes for the Life antibody (Fig. S1B).
To determine whether the Life/Life antibody pair detects oligomeric α-syn specifically, we purified different forms of wild-type and A53T α-syn proteins by size-exclusion chromatography (SEC) (Fig. 1D and 1E), and validated the separation by electrophoresis under native conditions (Fig. S2C and S2D). The a10 fraction mainly contains oligomeric α-syn at high molecular weight (Fig. S2C, lane 2) whereas the a12 and a16 fraction mainly contain monomer (Fig. S2C, lanes 3 and 4). Similarly, A10 fraction mainly contain oligomeric A53T α-syn (Figs. 1E and S2D, lane 2), whereas A12 and A16 were mainly monomer (Fig. S2D, lanes 3 and 4). The native PAGE gel takes less charge so that the apparent molecular weight could be deviated from the predicted molecular weight.
We then tested different α-syn forms from the above SEC samples with both AlphaLISA and TR-FRET assays. Consistent with our design, in which the Life/Nb antibody pair detects total α-syn, AlphaLISA signals from all the fractions tested were detected using this antibody pair (Fig. 1G for AlphaLISA and Fig. S2F for TR-FRET). In comparison, when using the Life/Life antibody pair, only the oligomeric α-syn containing samples including a10, and A10 gave detectable signals while the monomeric fractions a16, A16 generated essentially no signal (Fig. 1F for AlphaLISA and Fig. S2E for TR-FRET), confirming that the Life/Life antibody pair specifically detects oligomeric α-syn. To further exclude the influence of the protein loading, we calculated the ratio between Life/Life versus Life/Nb signals (O/T), as an indicator of oligomerization. It is clear that a10 and A10 fractions have the highest O/T in AlphaLISA assays (Fig. 1H for AlphaLISA and Fig. S2G for TR-FRET), consistent with our SEC and native electrophoresis results showing that they have highest oligomerization. The data above confirm that the Life/Life antibody pair detects oligomeric α-syn, and the ratio between the Life/Life signal and the Life/Nb signals (O/T) could be used as an indicator for α-syn oligomerization.
While the AlphaLISA results are consistent with the TR-FRET results, AlphaLISA has a higher sensitivity and can detect lower concentration of α-syn (Fig. S3). The detection limit is about 50 ng/mL by TR-FRET using our antibody pair (Fig. S3A and S3B), whereas AlphaLISA can detect as low as 3.15 ng/mL (Fig. S3C and S3D). In addition, the oligomerization signals (O/T) clearly has a larger signal window when using AlphaLISA (Fig. 1H). Finally, AlphaLISA has been reported to be more tolerable to contaminants in clinical samples such as CSF, plasma, etc. (Bielefeld-Sevigny 2009), whereas TR-FRET failed to detect α-syn oligomers in CSF samples. Thus, AlphaLISA assay is likely superior than TR-FRET assay used for quantification of oligomerization.
We further tested the potential of using the O/T detected by AlphaLISA to provide auxiliary information for PD diagnosis. We plotted the receiver operating characteristic (ROC) curve using the data above obtained from PD and control patients, and the area under the ROC curve is 0.701, P = 0.012 (Fig. 2D). The cut-off levels of 1.155 for CSF O/T ratio yielded a sensitivity of 61.5% and a specificity of 67.9%.
In the present study, we established novel bead-based AlphaLISA assays to measure the relative levels of oligomeric and total α-syn in CSF samples, which is potentially useful for the diagnosis of patients with PD and MSA. Our data suggest that the O/T ratio of α-syn was significantly higher in patients with typical PD. The O/T ratio of α-syn may be served as an auxiliary biomarker for PD diagnosis (Fig. 2) and provides important information in the clinical practices. Using the O/T ratio of CSF α-syn, a sensitivity and specificity of 61.5% and 67.9% for the discrimination of patients with PD from controls could be reached based on our data in the current study.
The authors wish to thank Chinese Ministry of Science and Technology (2016YFC0905100, 2016YFC1306500) and National Natural Science Foundation of China (Grant Nos. 81371413, 31371421, 31422024, 81571232, 91649105, and 31470764) for funding.
The authors have filed a Chinese patent application based on some of the data of this study (Patent No. CN 201510772846).
For studies with human subjects, all procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000. Informed consent was obtained from all patients for being included in the study.
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