From: Novel CSF β-synuclein-specific assays signal early synaptic degeneration in Alzheimer’s disease
N-terminus β-syn | Mid-region β-syn | C-terminus β-syn | |||||||
---|---|---|---|---|---|---|---|---|---|
Assay | Platform | ELISA | ELISA | ELISA | |||||
Status | Prototype | Prototype | Prototype | ||||||
Developers | Ulm University Hospital | Amsterdam UMC / ADx NeuroSciences | Amsterdam UMC / ADx NeuroSciences | ||||||
Biofluid | CSF | CSF | CSF | ||||||
Fold sample dilution | 4 | 4 | 4 | ||||||
Calibration curve | Type | Recombinant | Recombinant | Recombinant | |||||
No. of calibrator points | 10 | 10 | 10 | ||||||
Range, pg/mL | 4–1000 | 4–1000 | 4–1000 | ||||||
Curve fit | 1/y2-weighted 5PL | 1/y2-weighted 5PL | 1/y2-weighted 5PL | ||||||
Antibodies | Capture | EP1646Y | ADx- β-syn1 | ADx- β-syn2 | |||||
Detector | EP1537Y | EP1537Y | EP1537Y | ||||||
Clinical samples measurements (Amsterdam Dementia Cohort) | Number | 160 | 160 | 160 | |||||
Range concentration, pg/mL | 60.0–976.0 | 13.2–252.8 | 22.7–636.9 | ||||||
Range, CV% | 0–33.0% | 0–26.0% | 0–32.6% | ||||||
Average CV% | 4.9% | 3.9% | 7.5% | ||||||
n measured < LLOQ | 0 | 0 | 0 | ||||||
n measured > 20%CV | 2 | 1 | 11 | ||||||
Analytical validation results | |||||||||
Sensitivity | Functional LLoD, pg/mL | 2.15 | 0.74 | 2.42 | |||||
Concentrations of QC panels | QC1: high, pg/mL | NA | 57.8 | 23.8 | |||||
QC2: intermediate, pg/mL | 90.3 | 179.8 | |||||||
QC3: low, pg/mL | 154.0 | 278.6 | |||||||
Precision of QCs | Average Intra-assay %CV | NA | 7.2 | 16.7 | |||||
Average Inter-assay %CV | 31.8 | 17.4 | |||||||
Parallelism | Average slope of samples | NA | 0.78 | 0.70 | |||||
Range of slopes of samples | 0.87–70 | 0.64–0.76 | |||||||
Average slope of calibrator | 0.82 | 0.67 | |||||||
Parallelism, % | 94% | 105% | |||||||
Dilution linearity | Spiked concentration, pg/ml | 1000 | 1000 | ||||||
Df (x) | Mean %L | Mean %L | Mean %L | ||||||
Dilution linearity factor with mean %Linearity | 1 | NA | - | - | |||||
4 | 144 | 58 | |||||||
16 | 103 | 99 | |||||||
64 | 139 | 96 | |||||||
256 | 87 | 73 | |||||||
Recovery | Spiked concentration (pg/mL) With mean %Recovery | Spike | Mean %R | Spike (pg/mL) | Mean %R | Spike (pg/mL) | Mean %R | ||
NA | 500 | 67 | 500 | 63 | |||||
125 | 64 | 250 | 64 | ||||||
32 | 67 | 125 | 57 | ||||||
Stability | Freeze/ thaw cycles | Average response (%) | Standard deviation | Average normalized response (%) | Standard deviation | Average response (%) | Standard deviation | ||
0 F/T | NA | 100.0 | 2 | 100.0 | 15 | ||||
1 F/T | NA | 100.0 | 2 | 100.0 | 10 | ||||
2 F/T | NA | 100.0 | 4 | 105.2 | 15 | ||||
3 F/T | NA | 100.0 | 2 | 100.0 | 11 |