Autoimmune antibody decline in Parkinson’s disease and Multiple System Atrophy; a step towards immunotherapeutic strategies

All PD and MSA patients were followed by a movement disorder specialist at the Movement Disorders Clinic, Department of Neurology, Bispebjerg-Frederiksberg Hospital, Copenhagen. The clinical diagnosis for PD was defined according to UK Parkinson’s Disease Society Brain Bank clinical diagnostic criteria [30]. The clinical diagnosis of MSA was performed according to the accepted clinical diagnostic criteria [31] and with initial inclusion of cases with both possible and probable diagnoses (either the parkinsonian or the cerebellar subtype). Patients were included consecutively and a clinical follow-up was performed for all patients. At follow-up only those patients fulfilling the diagnostic criteria for a probable or definite MSA (n = 18) or definite PD (n = 46) diagnosis were accepted and included in the study. All PD and MSA patients received levodopa treatment. Healthy control volunteers (n = 41) were recruited through general announcements or advertisements at the hospital and were free of conditions that might affect the central nervous system.

Moreover, the following inclusion criteria were applied: Male or female aged 18 years or older at screening, able to cooperate with consent procedures, able to participate in study activities including all required clinical assessments and biological donations. The exclusion criteria comprised the following: Unable to participate in consent procedures, current treatment with anti-coagulants, unable to participate in biological specimen collection due to a medical condition or medication status, parkinsonism of different nature than idiopathic, such as other forms of atypical or vascular parkinsonism and severe dementia.

The Ethics Committee for the Copenhagen Regional Area gave approval for this protocol (H-1-2011-093), and all participants gave written informed consent. For summarized patient and control demographic characteristics see Table 1.

Levels and avidity of anti-α-synuclein NAbs were measured by competitive enzyme-linked immunosorbent assay (ELISA). 96-well polystyrene microtiter plates (Nunc MaxiSorp® flat-bottom 96 well plate) were coated with either 10 μg/mL or 5 μg/mL recombinant α-synuclein monomer (rPeptide, #S-10001-2) in ice-cold 0.1 M carbonate buffer (pH 8.5) overnight (>12 h) at 4 °C. The plates were emptied and blocked for 2 h at room temperature (RT) with 3% bovine serum albumin (BSA) and 0.1% NP-40 in phosphate-buffered saline (PBS, pH 7.4100 μL per well) and washed 5 times with PBS + 0.05% Tween-20. Next, 50 μL portions of plasma (diluted 1:100, 1:200, and 1:400 in PBS + 0.1%BSA) were transferred to the coated plates and incubated for 1 h at RT. For the competition reaction, plasma samples were incubated before transfer onto plates for 1 h with α-synuclein monomer at a range of concentrations: (1000, 250, 62.5, 15.5, 3.9, 0.97 and 0.24 nM for competition curve with plasma pools, and 1000, 50 and 2 nM for individual samples (the range of α-synuclein monomer concentrations was decided after completing preliminary experiments). After five washes with PBS + 0.05% Tween 20, 50 μL of peroxidase-labeled polyclonal goat anti-human IgG (Fc fragment specific; Abcam #ab98567: 1:20,000 dilution) was added to each well and incubated at RT for 2 h. After five more washes, tetramethylbenzidine (TMB) Liquid Peroxidase Substrate (50 μL; Sigma-Aldrich #T8665) was added, followed by incubation in the dark at RT for 30 min. The reaction was then stopped with 50 μL of 0.5 N H₂SO₄, and the absorbance at 450 nm was measured on a Fisher Scientific™ Multiskan™ FC Microplate Reader.

The α-synuclein concentration in plasma samples was measured with commercially available Human α-Synuclein Kit from MSD (Meso Scale Discovery®; MULTI-ARRAY Assay Systems, #K151TGD). The assay was performed as per the manufacturer’s instructions, with 1:50 dilution of plasma in Diluent-35, as provided by the manufacturer. For measurement of α-synuclein–anti-α-synuclein antibody complexes, samples were incubated with Sulfo-TAG anti-human IgG (MSD, #R32AJ-1). α-Synuclein/NAbs complexes were quantified with reference to an α-synuclein standard curve. The plates were read using an MSD Sector Imager S600 instrument, and the data analyzed using Discover Workbench 4.0 software.

To validate our ELISA results we have optimized an MSD electrochemiluminescence assays to measure anti-α-synuclein NAbs in plasma samples. MSD assays present several advantages over traditional ELISAs, including increased sensitivity and low background [32]. 96-well microtiter plates (Standard MSD bind plate #L15XA-1) were coated with either 5 ng/mL or 0.5 ng/mL of recombinant α-synuclein monomer (rPeptide, #S-10001-2) in ice-cold 0.1 M carbonate buffer (pH 8.5) overnight (>12 h) at 4 °C. The plates were washed 3 times with washing buffer (PBS + 0.05% Tween 20) and blocked for 1 h at RT with 3% BSA fraction V and 0.1% NP-40 in PBS, pH 7.4, 150 μL per well on a shaker set at 800 rpm. The plates were washed 3 times with PBS + 0.05% Tween-20. The 25 μL portions of plasma (diluted 1:500 in PBS + 0.1%BSA fraction V) were transferred to the coated plates and incubated for 1 h at RT on the shaker. Before transfer onto plates, plasma pools were incubated for 1 h with α-synuclein monomer at a range of concentrations 8000, 2000, 500, 125, 31.2, 7.8, 1.95, 0.48, 0.122, 0.03 and 0.007 nM). The range of α-synuclein monomer and plasma concentrations were decided after preliminary experiments. Wells were then washed 3 times with PBS + 0.05% Tween 20, and 25 μL of MSD Sulfo Tag Goat anti-human IgG antibody (MSD #R32AJ-1; 1:500 dilution in PBS + 0.1% BSA fraction V) was added to each well, with incubation at RT for 1 h on the shaker. After three more washes, MSD Read Buffer at 1:2 concentration diluted in MilliQ water was added to the wells. Immediately afterwards, the plates were read using an MSD Sector Imager S600 instrument.

Levels and avidity of anti-β/γ-synuclein NAbs were measured by competitive enzyme-linked immunosorbent assay (ELISA). 96-well polystyrene microtiter plates (Nunc MaxiSorp® flat-bottom 96 well plate) were coated with either 10 μg/mL recombinant β-synuclein monomer (rPeptide,GA,USA, #S-1003-2) or 10 μg/mL recombinant γ-synuclein monomer (rPeptide,GA,USA, #S-1007-1) in ice-cold 0.1 M carbonate buffer (pH 8.5) overnight (>12 h) at 4 °C. The plates were emptied and blocked for 2 h at room temperature (RT) with 3% bovine serum albumin (BSA) and 0.1%NP-40 in phosphate-buffered saline (PBS, pH 7.4, 100 μL per well) and washed 5 times with PBS + 0.05% Tween-20. Fifty μL portions of plasma diluted 1:400 in PBS + 0.1%BSA were transferred to the coated plates and incubated for 1 h at RT. For the competition reaction, before transfer onto plates, samples were incubated for 1 h with either β-synuclein or γ-synuclein monomer at a 2-fold dilution range of concentrations: 1000-2 nM for competition curve with plasma pools, and 1000, 100 and 10 nM for individual samples (the range was decided after completing preliminary experiments). After five washes with PBS + 0.05% Tween-20, 50 μL of peroxidase-labeled polyclonal goat anti-human IgG (Abcam, UK: 1:20,000 dilution) was added to each well and incubated at RT for 2 h. After five more washes, tetramethylbenzidine (TMB) Substrate (50 μL; Sigma-Aldrich,MO,USA) was added. After 30 min the reaction was then stopped with 50 μL of 0.5 N H2SO4, and the absorbance at 450 nm was measured on a Fisher Scientific™ Multiskan™ FC Microplate Reader,MA,USA.

Alpha-synuclein (Abcam #ab51189) was phosphorylated using PLK 2 (Invitrogen Cat# PV4204) at a concentration of 1.44 mg/ml (100 μM). The phosphorylation reactions were carried out in the presence of 1.09 mM ATP, 1× reaction solution (2 mM HEPES, 10 mM MgCl₂, 2 mM DDT, pH 7.4) and 1 μg of PLK/144 μg/ml of α-syn at 30 °C for 24 h. The reaction was quenched with 25 mM EDTA. After quenching the sample was desalted on a G25 column (HiTrap Desalting, GE healthcare #G-25 17–1408-01) into DPBS (Invitrogen #14190–094).

The sample was analyzed by LC-MS. Briefly; the sample was separated on a C4 2.1 × 50 mm BEH300 column run in FA/ACN and introduced to a XEVO QTOF Mass spectrometer (Waters). The multicharged signal obtained from the ion trace was deconvoluted and the mass identified to be a mixture of a non-phosphorylated 14,459 Da α-synuclein (minor component) and the major 14,539 Da phosphorylated species corresponding to a + 80 Da change caused by phosphorylation on serine 129.

We have optimized an MSD electrochemiluminescence assays to measure binding properties of anti-P-α-synuclein NAbs in plasma samples. 96-well microtiter plates (Standard MSD bind plate #L15XA-1) were coated with either 5 ng/mL recombinant α-synuclein monomer (rPeptide, #S-10001-2) or 5 ng/mL P-α-synuclein in ice-cold 0.1 M carbonate buffer (pH 8.5) overnight (>12 h) at 4 °C. The plates were washed 3 times with washing buffer (PBS + 0.05% Tween 20) and blocked for 1 h at RT with 3% BSA fraction V and 0.1% NP-40 in PBS, pH 7.4, 150 μL per well on a shaker set at 800 rpm. The plates were washed 3 times with PBS + 0.05% Tween-20. The 25 μL portions of plasma (diluted 1:400 in PBS + 0.1%BSA fraction V) were transferred to the coated plates and incubated for 1 h at RT on the shaker. Before transfer onto plates, plasma samples were incubated for 1 h with α-synuclein monomer at 1 μM and a range of P-α-synuclein concentrations 10, 1, and 0.1 nM. For competition curve, plasma pools (diluted 1:400 in PBS + 0.1%BSA fraction V) were incubated for 1 h with P-α-synuclein at a 3-fold dilution range of concentrations 100 nM-0.3pM. The range of P-α-synuclein and plasma concentrations were decided after preliminary experiments.

Wells were then washed 3 times with PBS + 0.05% Tween 20, and 25 μL of MSD Sulfo Tag Goat anti-human IgG antibody (MSD #R32AJ-1; 1:500 dilution in PBS + 0.1% BSA fraction V) was added to each well, with incubation at RT for 1 h on the shaker. After three more washes, MSD Read Buffer at 1:2 concentration diluted in MilliQ water was added to the wells. Immediately afterwards, the plates were read using an MSD Sector Imager S600 instrument.

To obtain the two site inhibition curves, ten plasma samples from each group were pooled and incubated in 1:400 dilution with increasing concentration of α-, β-, and γ-synuclein monomers at a 2-fold dilution range of concentrations 1000-2 nM, in combinations: α- and β-synuclein; α- and γ-synuclein, β- and γ-synuclein, with subsequent measurement of free NAbs by ELISA (as described above) on plates coated with 10 μg/ml of α-synuclein. For individual samples (the range was decided after completing preliminary experiments) 1000, 100 and 10 nM concentrations of α-, β-, and γ-synuclein monomers were used in the same combinations as for the inhibition curves. To obtain relative maximum of inhibition, each sample has been preincubated with high concentration (1000 nM) of mixed together α-, β-, and γ-synuclein.

The relative binding of anti-α-, β-, and γ-synuclein NAbs is expressed as a percentage of maximum binding observed in the assay for each sample; the competition reactions with 1000 nM α-, β- or γ-synuclein monomer were defined as representing 0% binding (unspecific binding), and reactions without competition are taken to indicate 100% (maximum) of binding in the displacement curves. For the inhibition assays, results are expressed as % inhibition calculated as [(OD₄₅₀ of non-inhibited sample) ÷ (OD₄₅₀ of inhibited sample)] × 100/OD₄₅₀ of non-inhibited sample, where OD₄₅₀ is the optical density at 450 nm.

Analysis of the competition binding curves was performed according to the one- and two-site models using computer-assisted curve fitting Fit logIC50 model (GraphPad Software Inc., La Jolla, CA)

Polyclonal plasma antibodies against a particular antigen potentially comprise a mixture of antibodies with different epitope specificities and affinities. We employed the competitive ELISA method to measure relative binding properties of the composite of anti-α-synuclein NAbs in plasma, exploiting the stronger antibody-antigen interactions of high affinity antibodies. By incubating plasma samples with increasing concentrations of free α-synuclein monomer prior to measurement of free antibody titre on plates with immobilized α-synuclein monomer we were able to separate anti-α-synuclein NAbs into a low and high affinity/avidity pools. The schematic diagram of the assay setup is presented in Fig. 1.

For the initial series of experiments, 10 randomly selected plasma samples from each group were pooled. The pooled samples were used to determine the appropriate plasma dilutions suitable for inhibition assays, as well as the concentrations of the coating antigen required to test the individual samples in the later part of the study. Fig. 2a, b present examples of the binding curves obtained in competitive ELISA, showing distinct apparent affinity profiles of anti-α-synuclein plasma NAbs from PD, MSA patients and normal controls. These displacement curves are expressed as percentage of the maximal binding measured in the absence of free α-synuclein monomers. High affinity/avidity is indicated by efficient inhibition of antibody binding in the presence of low (1–10 nM) concentrations of α-synuclein, whereas low affinity/avidity binding type is revealed by inhibition at a high concentration (10–1000 nM). The inhibition curve distinctly follows a two-site model for plasma from normal controls, indicating that a substantial fraction (approximately 50%) of total antibody binding is characterized by antibodies with high apparent affinity/avidity. The remaining part of total antibody binding indicates antibodies of low affinity, as well as the contribution of unspecific binding. Displacement curves from PD patient plasma samples also supported a two-affinity state model but, with a reduced fraction of high affinity antibodies. In contrast, the MSA plasma required high α-synuclein concentration for inhibition indicating an absence of high affinity antibodies. These results were further supported by using the MSD assay platform. By applying the MSD assay we were able to reduce the concentration of coating antigen from 10 μg/mL and 5 μg/mL to 5 ng/mL (Fig. 2c) and 0.5 ng/ml (not shown), due to sensitivity of the assay, and to test the pooled plasma samples at 1:500 dilutions. The binding curves obtained by MSD were in accordance with those obtained using ELISA, in that we could further discern the two site binding model comprising a high affinity component at low nM range and a low affinity group with approximate affinity around 1 μM.

To evaluate the specificity of the binding of anti-α-synuclein plasma NAbs, control competition assays were conducted on the pooled plasma samples using antigens unrelated to synucleinopathies. Here we performed ELISA assay using either α-synuclein monomer or amyloid β1–40 (Aβ1–40) peptides in the fluid inhibition phase and plates coated with α-synuclein at 5μg/mL (Figs. 3a, b). The α-synuclein monomer were able to inhibit plasma NAbs binding in all tested groups (Fig. 3a), whereas there was no inhibition detected with Aβ1–40 (Fig. 3b) suggesting the specificity of anti-α-synuclein plasma NAbs.

Consequently, we proceeded with the ELISA set-up to test the binding properties of the anti-α-synuclein NAbs in the individual plasma samples from all 46 PD and 18 MSA patients, and the 41 controls. . Each plasma sample was diluted 1:100 and 1:200 with PBS and preincubated with low 2 nM, 50 nM (Fig. 4a–d respectively) and high concentrations (1 μM) of free α-synuclein monomer to saturate the inhibition reaction, followed by incubation on plates coated with 5 μg/ml of immobilized α-synuclein. The data show that in the presence of 50 nM free α-synuclein monomer a binding of approximately 70–80% of the maximal binding is obtained for anti-α-synuclein NAbs in plasma from MSA patients. The corresponding values for PD and normal controls are significantly lower 50–55% and 40–45% respectively (Fig. 4a–b), indicating that these plasma samples contain a significant larger proportion of high affinity anti-α-synuclein NAbs compared to MSA patients. In the same experiment performed in the presence of 2 nM free α-synuclein monomer a binding of approximately 90–95% of the maximal binding is obtained for anti-α-synuclein NAbs in plasma from MSA patients. The corresponding values for PD and normal controls are approx. 80% and 70–75% respectively, (Fig. 4c-d). Under these conditions both MSA and PD are significant different from healthy controls, meaning that both MSA and PD patients contains a significant lower amount of high affinity anti-α-synuclein NAbs in plasma compared to controls. These are the key findings in the study.

We furthermore tested the ability of α-synuclein monomer to inhibit binding to α-synuclein coated plates of NAbs from individual samples, using the same conditions as described for maximal binding in the experiments above. We expressed the percent inhibition relative to that in the unblocked plasma, i.e. samples with no free antigen added. Both 1:100 (Fig. 5a) and 1:200 (Fig. 5b) diluted plasma samples from MSA patients had significantly reduced binding capability for α-synuclein monomer in comparison with plasma samples from PD patients and healthy controls. Only with a low plasma concentration (1:200) and high antigen levels (1 μM) could we achieve comparable inhibition in all groups (Fig. 5b). This further supports our finding of absence of high affinity anti-α-synuclein NAbs in plasma samples from MSA patients.

There is evidence suggesting that the general changes in the humoral immune responses with age are qualitative rather than quantitative, i.e. it is the affinity and specificity of the antibodies that change, rather than the quantity of antibodies produced [33]. Due to the difference in mean age of our control subjects (Table 1), we tested if the maximal binding to α-synuclein of NAbs from elderly controls (>50 years old) differed from that of younger controls (<50 years old). There was no significant difference in binding properties of anti-α-synuclein NAbs from plasma of controls stratified by age, either at a dilution of 1:100 (Fig. 6a, b) or 1:200 (Fig. 6d, c).

In addition to measuring the individual binding properties of the plasma NAbs, we also measured the concentration of plasma α-synuclein and α-synuclein/NAbs complexes. The mean (±SD) plasma concentration of α-synuclein was significantly higher in the control group (127 ± 8.3 ng/ml) than for PD (100.2 ± 3.2 ng/ml; p = 0.015) and MSA (98.6 ± 3.1 ng/ml; p = 0.024) patients (Fig. 7a). To measure α-synuclein/NAbs complexes in plasma, the α-synuclein detection antibody in the human α-synuclein MSD kit had been substituted with an equivalent amount of an anti-human IgG antibody, while the nature of the capture antibody was unchanged. The results here show a significantly higher α-synuclein/NAbs complex concentration in controls (423 ± 31.2 pg/ml) than in PD (350 ± 21.6 pg/ml; p = 0.042) and MSA (217 ± 34.7 pg/ml; p = 0.0001) patients (Fig. 7b). The plasma α-synuclein/NAbs complex concentration was significantly lower in MSA than PD patients (p = 0.002).

The synuclein family consists of α-, β-, and γ-synucleins, that are 55–62% identical in sequence with most of the similarity lying within the N-terminus of the proteins (aa 1–100) and with more variability in sequence located toward the C-terminus. Because NAbs are often polyreactive, and may exhibit cross-reactivity, we tested if the binding properties of the anti-α-synuclein NAbs would be reflected in anti-β and -γ-synuclein NAbs. Figures. 8a and 9a present examples of the binding curves obtained in competitive ELISA on pooled samples (n = 10) for each disease and controls, showing similar apparent affinity profiles of anti-β-synuclein and anti-γ-synuclein, respectively, for plasma NAbs from PD, MSA patients and normal controls. Further, we proceeded with the ELISA set-up to test the binding properties of the anti-β and anti-γ-synuclein NAbs in the individual plasma samples from all 46 PD and 18 MSA patients, and the 41 controls. Each plasma sample was diluted 1:400 with PBS and preincubated with low (10 nM and 100 nM) and high concentrations (1 μM) of either free β-synuclein (Fig. 8b, c) or γ-synuclein monomer (Fig. 9b, c), followed by incubation on plates coated with 10 μg/ml of immobilized β- or γ-synuclein respectively. The data show that in the presence of 10 nM free β- or γ-synuclein monomer a binding of approximately 50–70% of the maximal binding is obtained for anti-β- and anti-γ-synuclein NAbs in plasma from all three groups. The corresponding values for 100 nM free β- or γ-synuclein monomer are also similar in all tested groups and are in the range of 40–45%. These data suggest that there are no differences in the levels of high and low affinity components of anti-β- and anti-γ-synuclein NABs in plasma from PD, MSA patients and normal controls.

To evaluate cross-reactivity of anti-α-synuclein high and low affinity plasma NAbs with β- and γ-synuclein monomers, we have performed cross-binding ELISA set-ups. The inhibition curves for the scenario where α-synuclein is present simultaneously with either β- or γ synuclein distinctly follows a two-site model seen before with free α-synuclein (Fig. 10a and b). Accordant to the α-synuclein experiments plasma from normal controls, is characterized by antibodies with high apparent affinity/avidity. PD plasma samples also supported a two-affinity state model, but with a reduced fraction of high affinity antibodies, and to a lesser extent in comparison to when α-synuclein was present alone. MSA plasma samples required high α−/β-synuclein and α−/γ-synuclein concentrations for inhibition indicating an absence of high affinity antibodies. In the simultaneous presence of increasing concentrations of free β- and γ-synuclein monomers prior to measurement of free antibody titre on plates coated with α-synuclein monomer (Fig. 10c) plasma samples from normal individuals still supported a two-affinity state model but to a lower extend than in the previous set-ups, whereas plasma samples from PD and MSA patients required high β−/γ-synuclein concentrations for maximum of inhibition. These data indicate that anti-α-synuclein NAbs from normal controls cross-react with β- and γ-synuclein monomers whereas such cross-binding is not found in PD and MSA patients.

Subsequently, each individual plasma sample was diluted 1:400 with PBS and preincubated with low (10 nM or 100 nM) and high concentrations (1 μM) of free α-, β-, and γ-synuclein monomers in combinations analogous to the inhibition curve experiments, followed by incubation on plates coated with 10 μg/ml of immobilized α-synuclein. The data are presented as percent of inhibition relative to 100% where high levels (1 μM) of each α-, β-, and γ-synuclein were added simultaneously to the reaction, and to the unblocked plasma, i.e. samples with no free antigen added. The data show that in the presence of 100 nM free proteins an inhibition of approximately 60–70% is obtained for combinations α−/β-synuclein and α−/γ synuclein in plasma from normal controls. The corresponding values for PD and MSA patients are significantly lower 50% and 30% respectively for α−/β-synuclein (Fig. 10d), and 60% and 30% respectively for α−/γ synuclein. Notably in the presence of α−/γ synuclein we did not observe significant differences between healthy individuals and PD patients. In the presence of 10 nM concentrations of free α−/β-synuclein (Fig. 10e) and α−/γ synuclein (Fig. 10g) significant differences in the percentage of inhibition were observed in plasma from controls and PD patients vs. MSA patients, whereas no difference was found between PD patients and control individuals.

The inhibition levels of anti-α-synuclein NAbs by simultaneous presence of 100 nM β- and γ-synuclein monomers were decreased in comparison to the previous approach in normal controls and PD patients (approximately 40–45%), but still significantly higher than in MSA patients (Fig. 10h). No differences between the groups were observed with 10 nM of free β- and γ-synuclein monomers (Fig. 10i).

These data suggest the high affinity anti-α-synuclein plasma components, seen in healthy individuals, are selective for physiologically relevant epitopes and probably react solely with C-terminal epitopes of α-synuclein.

Posttranslational modifications of α-synuclein, particularly phosphorylation at serine 129, may be critical in pathogenesis of PD and MSA [34]. The neuropathological diagnosis of PD and MSA is often based on specific regional distribution of P-α-synuclein in glial cells and neurons of the CNS, respectively. Therefore, we tested if the binding properties of the anti-α-synuclein NAbs would also be reflected in anti-P-α-synuclein NAbs. For that, an α-synuclein monomer preparation was phosphorylated with a serine⁄threonine kinase PLK2. PLK2 is a principle contributor to α-synuclein phosphorylation at Ser-129 in neurons and directly phosphorylates α-synuclein at Ser-129 in in vitro biochemical assays [35].

To test binding properties of the anti-P-α-synuclein NAbs, each plasma sample was diluted 1:400 with PBS and preincubated with low 1 nM, 0.1 nM (Fig. 11a–d, respectively) and high concentrations (10 nM) of free P-α-synuclein to saturate the inhibition reaction, followed by incubation on plates coated with 10 ng/ml of immobilized either α-synuclein (11a, b) or P-α-synuclein (11c, d).

The data show that in the presence of 1 nM free P-α-synuclein monomer binding to plates coated with either α-synuclein (Fig. 11a) or P-α-synuclein (Fig. 11c) of approximately 35%–45% and 50–60% of the maximal binding is obtained for anti-P-α-synuclein NAbs in plasma from PD and MSA patients respectively. The corresponding values for normal controls are significantly lower, 15–20%, indicating that these plasma samples contain a significant larger proportion of high affinity anti-P α-synuclein NAbs compared to MSA patients. In the same experiment performed in the presence of 0.1 nM free P-α-synuclein a binding of approximately 80–90% and 70–80% of the maximal binding is obtained for anti-P-α-synuclein NAbs in plasma from PD and MSA patients, respectively. The corresponding values for normal controls are 40–50%% on plates coated with either α-synuclein (Fig. 11b) or P-α-synuclein (Fig. 11d). Under these conditions both MSA and PD are significantly different from healthy controls, meaning that plasma samples from MSA and PD patients contain a significantly lower amount of high affinity anti-P-α-synuclein NAbs compared to normal controls.