Fig. 1
Schematic illustration of the electrochemiluminescence NfL immunoassay. The carbon ink electrodes are coated with the capture mAB 47:3. The biotinylated detection mAB 2:1 binds to SULFO-TAG labelled streptavidin, which generates electrochemiluminescence signal
4.
Bovine serum albumin (BSA)
5.
Ethylenediaminetetraacetic disodium salt (EDTA)
6.
NaCl
7.
1× Phosphate buffered saline (PBS), pH 7.5
8.
Tris base
9.
Tween 20
10.
96-well plate including integrated screen-printed carbon ink electrodes on the bottom of the wells (96-well SECTOR standard plates, Meso Scale Discovery, Gaithersburg, MD).
2.1 Buffers
1.
10× Tris-buffered saline (TBS, 100 mM tris base and 1.5 M NaCl: 87.66 g NaCl and 12.1 g tris base per liter), adjusted to pH 7.5, using 10 N HCL as a stock solution.
On day 1 of the experiment the following buffers are prepared (amounts for one 96-well plate):
2.
Working strength TBS (50 ml 10× TBS and 450 ml H2O)
3.
Blocking buffer: 3 % BSA in TBS (375 mg BSA in 12.5 ml TBS)
4.
Wash buffer: 0.1 % Tween 20 in TBS (487.5 μl Tween 20 in 487.5 ml TBS)
5.
Sample/standard diluent: 1 % BSA, 0.1 % Tween 20 in TBS (150 mg BSA in 15 ml of wash buffer)
6.
Read buffer T (4×): 15 ml read buffer (MSD) in 15 ml H2O
2.2 Standards
3 Methods
3.1 Coating of the Plate (Day 1)
1.
Dilute mAB 47:3 (stock concentration: 1.1 mg/ml; working concentration: 1.25 μg/ml: 3,496 μl of PBS and 4 μl of mAB 47:3) in PBS.
3.
Seal wells with adhesive plate seals.
4.
Incubate overnight at 4 °C.
3.2 Blocking of Unspecific Binding Sites (Day 2)
All the following incubation steps are done on a plate shaker (800 rpm, adhesive plate seals required for each incubation).
3.3 Adding Calibrators and Samples
3.4 Adding Detection Antibody (mAB 2:1)
1.
Dilute mAB 2:1 (stock concentration: 0.58 mg/ml, working concentration: 0.5 μg/ml: 2,997.41 μl of sample/standard diluent and 2.59 μl of mAB 2:1).
2.
Rinse plate three times with 200 μl of wash buffer per well. Tap plate dry against absorbent paper.
3.
Add 25 μl of mAB 2:1 to each well.
4.
Incubate plate for 1 h
3.5 Adding MSD SULFO-TAG™ Labelled Streptavidin
1.
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Dilute MSD SULFO-TAG™ labelled streptavidin (stock concentration: 0.5 mg/ml, working concentration: 0.25 μg/ml: 5,997 μl of sample/standard diluent and 3 μl of MSD SULFO-TAG™).
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