Ethics and Consent to participate
The study was approved by the Local Committees of all participating centers. Informed and written consent was obtained from all study participants at enrolment.
Blood and CSF samples were obtained from participants of the European Spinocerebellar ataxia type-3/Machado-Joseph disease Initiative (ESMI) cohort. To test whether the newly developed assay discriminates between mutation carriers and healthy controls, we measured plasma ataxin-3 concentrations in an exploratory cohort of 9 healthy controls and 10 ataxic SCA3 mutations carriers (= exploratory cohort). For validation and more in-depth analysis, we performed plasma measurements in a cohort of 15 healthy controls, 11 pre-ataxic mutation carriers, and 45 ataxic SCA3 mutation carriers (= validation cohort). For a subset of 17 mutation carriers (4 pre-ataxic and 13 ataxic mutation carriers) parallel measurements in CSF were performed. CSF samples from 18 age and sex-matched healthy controls were provided by the biobank of the Donor Institute for Brain, Nijmegen. Longitudinal plasma assessments were available for 37 participants of the validation cohort (4 healthy controls, 5 pre-ataxic, 28 ataxic mutation carriers), with follow-up plasma samples collected one year after the first visit (table 1).
Biosamples were collected under highly standardized protocols at all participating centers. Briefly, blood collection was performed by standard venipuncture. Plasma was separated using cell preparation tubes (CPT) from BD Biosciences. Directly after blood collection, CPT tubes were inverted 8 to 10 times and centrifuged within 2 hours for 30 min at 1,700 RCF. Plasma was collected from the tubes without disturbing the mononuclear cell layer. After processing, all plasma samples were immediately frozen at -80ºC.
CSF collection was performed by lumbar puncture. Samples were processed within 2 hours. First, samples were centrifuged at 1,100 RCF for 10 minutes, afterwards divided in aliquots and immediately frozen at -80ºC.
Age at ataxia onset (AAO) in ataxic mutation carriers was defined as the reported age at onset of gait difficulties. In the pre-ataxic mutation carriers, predicted AAO was calculated on the basis of age at recruitment and CAG repeat length . The scale for the assessment and rating of ataxia (SARA) was used to assess the severity of ataxia . Mutation carriers were classified either as pre-ataxic (SARA ˂ 3 points) or ataxic (score ≥ 3). The inventory of non-ataxia signs (INAS) count was used to assess the degree of non-cerebellar involvement . The CAG repeat length of the expanded allele was determined using PCR-based fragment length analysis (CEQ8000 capillary sequencer, Beckman Coulter).
For Single Molecule Counting (SMCTM) assay development, two monoclonal antibodies were used: (I) mouse antibody MW1 specifically binding to disease-associated polyQ stretches of HTT, whose generation has been previously described  and (II) the mouse anti-spinocerebellar ataxia type 3 antibody, clone 1H9, with the epitope mapped to amino acids E214-L233 of the human ataxin-3 protein, obtained from Millipore (MAB5360). Graphical illustration of epitope binding sites is shown in Figure 1A.
Recombinant purified proteins
Recombinant human ataxin-3 proteins with a polyQ lengths of 15Q and 62Q, respectively, were purified as described elsewhere . Recombinant proteins were pre-diluted to a concentration of 10 µg/mL in artificial CSF (aCSF: 300 mM NaCl; 6 mM KCl; 2.8 mM CaCl2 x 2H2O; 1.6 mM MgCl2 x 6 H2O; 1.6 mM Na2HPO4 x 2H2O; 0.4 mM NaH2PO4 x H2O), supplemented with 10% Tween-20 and cOmplete protease inhibitor (Roche) and stored at -80 °C. On assay day, recombinant proteins were further diluted to finally 400 pg/mL as a starting concentration for the preparation of standard dilutions. Afterwards, standard was generated by a 1:4 fold serial dilution series for usage as a calibrator standard in the assay.
Sample preparation for SMCTM assay
Plasma and CSF from SCA3 mutation carriers and healthy controls were diluted in aCSF (supplemented with 10% Tween-20 and cOmplete protease inhibitor(PI)) in a ratio of 1:4 (plasma) or 1:5 (CSF) before assay application.
Ataxin-3 SMCTM assay
The assay employs the Single Molecule Counting (SMCTM) technology that affords ultra-sensitivity and a wide linear detection . Specific detection of polyQ-expanded ataxin-3 in this assay occurs by bead-based immunoreaction with antibody combination 1H9 and MW1: the 1H9 antibody was labeled and coated to magnetic particles (MP) according to SMCTMCapture Labeling Kit manufacturer’s instructions by coupling 25 µg of labeled 1H9 antibody per mg of magnetic particles (MP). MP-1H9 suspension was stored at 10 mg/mL in Coated-bead-buffer at 2-8° C. The MW1 antibody was labeled as detection antibody with fluorophore Alexa according to SMC™ Detection Antibody Labeling Kit manufacturer’s instructions.
In the assay, capture and detection antibodies were used at final concentrations of 50 ng/well or 5 ng/well, respectively. 50 μL/well of Blocking buffer containing 6% BSA, 0.8% Triton X-100, 750 mM NaCl, and cOmplete protease inhibitor; and 150 μL/well of standard or human sample (CSF prediluted 1:5; plasma prediluted 1:4 in aCSF+1%Tween+PI) were added to a 96-conical assay plate (Axygen). MP-1H9 antibody (100 μL/well) diluted to 1:1000 with Assay/Discovery Buffer (Merck Millipore) was added, and the plate was sealed and incubated under shaking (400 rpm) at room temperature for 1 hour. All washing steps described here were performed with the HydroFlexTM microplate washer (Tecan). The plate was first placed on a magnetic stand and then washed once with 200 µl 1x SMCTM Wash Buffer with ProClin (Merck Millipore). Washing buffer was removed, and 20 μL/well of 5 ng/µl diluted labeled MW1-antibody (1:4000 diluted in Assay/Discovery Buffer and filtered) was added to the plate. The plate was sealed and incubated under shaking (700 rpm) at room temperature for 1 hour. After 4 washes with 200 μl 1x SMCTM Wash Buffer with ProClin and aspiration, 11 µl of elution buffer (acidic glycine solution, 0.1 M, pH 2.7) were added to the plate, and the plate was incubated under shaking (700 rpm) for 6 minutes. The plate was placed on the magnetic stand for 2 min, 11 µl of buffer D (neutralization buffer Tris, 1 M, pH 9) were added. 20 µl of eluate were transferred to an Aurora plate, 384-well, F-bottom, transparent (Merck Millipore), the plate was shaken for 2 min and then spun down (2 min, 400g) to eliminate foaming and bubble formation. The analysis plate was subsequently measured with the SMCxProTM platform. All assays were performed by operators blinded to genotype and clinical state of the participant.
The assay´s limit of detection (LOD) was defined as the concentration corresponding to the signal 2.5 SDs above the background (zero calibrator), lower limit of quantification (LLoQ) as the concentration corresponding to the signal 10 SDs above the background, and upper limit of quantification (ULoQ) as the concentration corresponding to the signal 2.5 SDs above the highest calibration standard on the calibration curve.
Analyte distribution were tested for normality using Shapiro-Wilk test. Non-parametric group analyses were performed using two-sided Mann-Whitney test with Bonferroni correction for multiple comparison. For linear correlation we used partial Spearman correlation. Data were adjusted for age and CAG-repeat length. CAG repeat adjustment was included as MW1 antibody can bind 16 or more polyQ repeats with increasing intensity for longer repeats.7 Both, age and expanded CAG repeat length were identified as covariable in our dataset and as independent modifier of SCA3 disease severity [10, 11]. Multivariate analyses revealed that age at onset, disease duration and sex did not correlate with our datasets. Therefore, all statistical analyses were only corrected for age and expanded CAG repeat length. Correlation analyses of plasma and CSF ataxin-3 levels were performed on z-transformed datasets. Effect sizes (r) were calculated as Cohen´s d. Intraclass variation (ICC) was performed to analyze the stability of the analyte ataxin-3 at the longitudinal study design. To test the quality of classification of the cohort into healthy controls and mutation carriers, we calculated receiver operating characteristic (ROC) curves and determined the area under the curve (AUC).
Data are presented as median and interquartile range [IQR]. Statistical significance is demonstrated by P-values (< 0.05 (*), < 0.01 (**), < 0.001 (***).
All statistical and graphical evaluations were performed with GraphPad prism 8.0. For linear regression, we used IBM SPSS Statistics version 27.