Advantage
NF-light®
102258
SUMMARY AND EXPLANATION OF THE NF-LIGHT® TEST
Neurofilament light (NF-L) is a 68 kDa cytoskeletal intermediate filament protein that is expressed in neurons. It associates with the 125 kDa Neurofilament medium(NF-M) and
the 200 kDa Neurofilament heavy (NF-H) to form neurofilaments. They are major components of the neuronal cytoskeleton, and are believed to function primarily to provide
structural support for the axon and to regulate axon diameter. Neurofilaments can be released in significant quantity following axonal damage or neuronal degeneration. NF-L
has been shown to associate with traumatic brain injury, multiple sclerosis, frontotemporal dementia and other neurodegenerative diseases. The Simoa NF-light® assay is a
digital immunoassay for the quantitative determination of NF-L in serum, plasma and CSF. The antibodies (Uman Diagnostics, Umeå Sweden) also cross react with murine,
bovine, and macaque NF-L epitopes, and the assay can be used for research with these species.
GENERAL CHARACTERISTICS OF THE NF-LIGHT® TEST
AEB
%CVdose
NF-L, pg/mL
Healthy donors
Figure 1:
Simoa NF-light® immunoassay calibration
curve. Four-parameter curve fit parameters are
depicted.
EDTA
plasma
NF-L, pg/mL
NF-L, pg/mL
Figure 2: Dose CV profile. Diluted serum was
Serum
Heparin
plasma
CSF
Mouse
serum/plasma
Figure 3: [NF-L] in EDTA plasma (n = 20), matched serum (n =
assayed in reps of 3 over multiple days and run
(90 determinations).
Concentrations are
uncorrected for pre-dilutions. Intersection with
power fit line at 20% CV was 0.0483 pg/mL.
20), heparin plasma (n = 20), cerebral spinal fluid (CSF, n = 20),
and mouse serum and plasma (n = 10 each) from nonmedicated, non-immunized mice. Error bars depict median and
interquartile ranges.
Table 1: General characteristics of NF-light® immunoassay
Calibration range
0-500 pg/mL
Dynamic range1
2000 pg/mL
Lower limit of detection (2.5 SD; 3 reps x 12 runs across 3 instruments, 2 reagent lots, 2 cal lots; mean LoD2)
0.038 pg/mL
Lower limit of quantification3 (12 runs across 2 reagent lots, 3 instruments, mean LoQ)
0.174 pg/mL
Spike-recovery: serum/plasma (NF-L spiked into 4 serum and 2 plasma samples at 10, 100, 1000 pg/mL, mean4)
90.7%
Spike-recovery: CSF (NF-L spiked into 6 CSF samples at 100, 1000, and 10000 pg/mL,
118.5%
mean5)
Linearity (high NF-L plasma sample fractionally admixed with low NF-L plasma sample, mean of 10 levels6)
97.0%
Dilution linearity: serum/plasma (diluted 2X serially from MRD to 128 MRD w/ Sample Diluent; mean
100.7%
recovery7)
Dilution linearity: CSF (2 CSF samples diluted 2X serially from MRD to 128 MRD w/ Sample Diluent; mean recovery8)
100.4%
Inter lot CV (Pool of CVs from 5 samples9 tested with 2 reagent lots across 2 runs x 3 instruments)
1.74%
Inter instrument CV (Pool of CVs from 5 samples9 tested with 3 instruments across 2 runs x 2 reagent lots)
2.07%
Specificity: healthy normal CSF and plasma (5 of each type depleted with capture antibody, grand mean)
99.0%
Specificity: ALS CSF and plasma (5 of each type depleted with capture antibody, grand mean)
99.6%
Typical serum/plasma sample volume (Includes dead volume; see Package Insert for details)
CSF sample volume (Includes dead volume; See Package Insert for details)
The Simoa NF-light®
assay reliably
quantifies NF-L in
serum, plasma and
CSF in healthy
subjects, with
median NF-L values
well above the
sensitivity limits.
46 µL
1.84 µL
1Samples auto-diluted 4X; note: ranges may vary between calibrator lots. 2SD 0.020 pg/mL; range 0.003-0.079 pg/mL.
3≤20%CV, 80-120% recovery; pooled CV 14.7%, mean
recovery 109.9%; not corrected for pre-dilutions; 4X LoQ 0.696 pg/mL. 4Recovery range 70.3-119.7%. 5Recovery range 89.1-147.2%. 6Recovery range 88.2-105.1%. 7Recovery
range 85.1-178.2%; minimum recommended dilution (MRD) 1:4. 8Recovery range 94.1-106.3; MRD 1:100. 9Range 6.41-256 pg/mL.
Simoa HD-1 Analyzer
Representative data. Individual results may vary depending upon samples tested and protocols followed.
Assay designed by Dandan
For Research Use Only. Not for use in diagnostic procedures.
DS-0077 01 (184) 24MAR2017
Advantage
NF-light®
Page 2
102258
NF-L, pg/mL
NF-L, pg/mL
REPRODUCIBILITY & REPEATABILITY CHARACTERISTICS OF THE NF-LIGHT® TEST
Mean NF-L, pg/mL
Mean NF-L, pg/mL
Figure 5:
Figure 4:
Reproducibility across instruments. Five
native and spiked serum and plasma NF-L samples
tested across 2 runs x 2 reagent lots each instrument.
Reproducibility across reagent lots. Five
native and spiked serum and plasma NF-L samples
tested across 2 runs x 3 instruments each lot.
Table 2: Reproducibility precision of the NF-light® immunoassay across instruments and reagent lots. Reproducibility was
determined with guidance from CLSI Protocol EP5-A. Five samples, consisting of serum/plasma panels and NF-L controls, were assayed in
replicates of three for two runs on each of three instruments and two reagent lots. Analysis of variance (nested ANOVA) results are
summarized in the following table.
Between
Instrument
Mean NF-L
Within Run
Between Run
Between Lot
pg/mL
%CV, n=36
%CV, n=12
%CV, n=2
Control 1
Control 2
6.44
283
6.5
4.7
4.8
5.9
1.4
0.0
0.0
6.4
Serum Panel 1
Serum Panel 2
Serum Panel 3
6.62
21.2
257
7.5
6.7
9.3
3.8
4.1
4.3
0.0
0.0
0.0
3.8
0.0
0.4
Sample
%CV, n=3
Table 3: Repeatability precision of the NF-light® immunoassay.
Repeatability was determined in a
separate study from Table 2 with guidance from CLSI Protocol EP5-A. Five samples, consisting of serum/plasma
panels and NF-L controls, were assayed in replicates of three at two separate times per day for five days using a
single lot of reagents and calibrators. Analysis of variance (nested ANOVA) results are summarized in the following
table.
Sample
Control 1
Control 2
Serum Panel 1
Serum Panel 2
Serum Panel 3
Mean NF-L
Within Run
Between Run
Between Day
pg/mL
%CV, n=30
%CV, n=10
%CV, n=5
6.41
255
6.2
21.7
256
6.6
3.5
6.1
6.8
7.3
0.0
3.1
5.3
1.9
5.3
0.90
7.7
2.7
1.8
0.0
Simoa
HD-1 Analyzer
Representative data. Individual results may vary depending upon samples tested and protocols followed.
Assay designed by Dandan
For Research Use Only. Not for use in diagnostic procedures.
DS-0077 01 (184) 24MAR2017