Light Chain Determination from Peripheral Blood Samples



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


Bovine lyophilized NfL is obtained from UmanDiagnostics. Calibrators were serially diluted in standard diluent (10,000, 2,000, 1,000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125 pg/ml, and blank containing only diluent) (see Notes 4 and 5 ).



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.

 

2.

Add 30 μl per well (see Note 6 ).

 

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).

1.

Rinse plate three times with 200 μl of wash buffer per well (see Note 7 ). Tap plate dry against absorbent paper.

 

2.

Add 100 μl of blocking buffer per well (see Note 8 ).

 

3.

Incubate plate for 1 h (see Note 9 ).

 


3.3 Adding Calibrators and Samples




1.

Rinse plate three times with 200 μl of wash buffer per well. Tap plate dry against absorbent paper (see Note 10 ).

 

2.

Add 25 μl of sample diluent to each well.

 

3.

Add 25 μl of standard or sample in duplicate (see Note 11 ).

 

4.

Incubate plate for 2 h.

 


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.

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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Jul 12, 2017 | Posted by in NEUROLOGY | Comments Off on Light Chain Determination from Peripheral Blood Samples
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