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Table 1 Analytical characteristics of the β-syn ELISAs

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

  1. The analytical performance of the ELISAs was assessed based on sensitivity, precision, parallelism, dilution linearity and recovery. The sensitivity was determined by calculating the detection limits of each assay. We utilized a commercially available recombinant truncated human β-syn (residues 1–122, UniProt: P37840), lacking the 12 C-terminal amino acids of the full-length 134-residue protein. The recombinant protein was untagged. The functional LLoD was calculated as the mean signal of 16 blanks plus 10 times the SD multiplied by the dilution factor of four times. Quality control (QC) samples were made of remnant CSF samples. The mean intra- and inter-assay variation was determined by measuring the QC panels over three independent runs on three days. The 160 clinical samples were measured in duplicate. Precision was evaluated by calculating the intra-assay and inter-assay coefficients of variation (CV%) of these measurements. Parallelism was evaluated to ensure the consistency of assay performance across different sample dilutions, and dilution linearity confirmed the linear response of the assays to diluted samples. For parallelism, four samples were measured after being serially diluted four times 2-fold, starting with a 2-fold dilution that reached 32a-fold). For dilution linearity, three samples were spiked with 1000 pg/mL recombinant β-syn and subsequently measured undiluted and diluted 256-fold. Recovery tests were used to assess the accuracy of the assays in quantifying known amounts of β-syn. PL: polynomial; LLoD: lowest limit of detection; QC: quality control; CV: coefficient of variation; %L: % linearity; %R: % recovery, F/T: freeze / thaw cycles