TITLE: ELISA for assessing the burden of Neutrophil Extracellular Traps (NETs) in clinical serum samples.
AUTHORS: kathryn.hally 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol steps through the ELISA procedure for detecting three NET-specific biomarkers: 1) myeloperoxidase-DNA (MPO-DNA) complexes, 2) neutrophil elastase-DNA (NE-DNA) complexes, and 3) citrullinated histone H3 (H3(cit)). </div></div>

[STEPS]
?. [Solutions to be prepared for ELISAs.]
For Day 1:Coating buffer:
[1x PBS]
?. [ELISA day 2.]
Thoroughly remove the capture antibody by flicking off the solution and banging each plate face down on paper towels. Add   to each well and incubate for  . Flick and bang again. Repeat another four times (five washes in total). Add  to each well and incubate for . Flick and bang again. Add  to each well, cover and incubate for  at .
[wash buffer]
[blocking buffer]
0 Room temperature
?. [Solutions to be prepared for ELISAs.]
For Day 3:Stop solution (1.8M H2SO4):  + Substrate buffer (0.11M acetate buffer):  +  , adjust to  with 100% acetic acid. Can be stored for up to two weeks refrigerated. 6 mg/mL TMB solution:  +  . Protect against light exposure. Can be stored for up to one month at room temperature. 3% hydrogen peroxide:  +  . Can be stored for up to one week refrigerated. TMB substrate solution:  +  +  . Protect against light exposure.
[sulfuric acid (H2SO4)]
[1x PBS]
[sodium acetate]
[ddH2O]
[TMB]
[DMSO]
[30% hydrogen peroxide]
[ddH2O]
[substrate buffer]
[TMB solution]
[3% hydrogen peroxide]
?. [Solutions to be prepared for ELISAs.]
For Day 2:Wash buffer (0.1% Tween 20 in 1X PBS):  + , adjust to Assay diluent (1% BSA in 1X PBS):   +  , filter before useBlocking buffer (5% BSA in 1X PBS):   +  , filter before use
[Tween 20]
[1X PBS]
[BSA powder]
[1x PBS]
[BSA powder]
[1x PBS]
?. [ELISA day 1.]
Label three 96-well ELISA plates as follows: MPO-DNA, NE-DNA, H3(cit).  Add    to each well and incubate for  . Following this, flick off coating buffer and bang each plate face down on paper towels. Add   to each well and incubate for .
[coating buffer]
?. [ELISA day 1.]
ABCDEF1Type of ELISACapture antibodyDilutionAntibody amountCoating buffer amountFinal volume2MPO-DNAMPO1:30035 µL10,465 µL10,500 µL3NE-DNAElastase1:30035 µL10,465 µL10,500 µL4H3(cit)Histone1:100105 µL10,395 µL10,500 µL[Anti-histone antibody (clone H11-4) is supplied as a part of the Cell Death Detection ELISA]Add  to each well in the ELISA plate.Seal each plate with a cover slip and incubate  at  .
ABCDEF1Type of ELISACapture antibodyDilutionAntibody amountCoating buffer amountFinal volume2MPO-DNAMPO1:30035 µL10,465 µL10,500 µL3NE-DNAElastase1:30035 µL10,465 µL10,500 µL4H3(cit)Histone1:100105 µL10,395 µL10,500 µL
[appropriate diluted capture antibody]
4 °C
?. [Preparing ELISA standards.]
Supernatant from PMA-stimulated neutrophils will be used as the standard for the MPO-DNA and H3(cit) ELISAs. Carry out the following to obtain NET-rich supernatant. Isolating granulocytes:From a healthy volunteer, draw  into EDTA blood collection tubes. To two 15 mL centrifuge tubes, add .Carefully layer  on top of each Polymorphprep layer.Centrifuge these tubes .Following centrifugation, two cell layers will have formed which are distinct from the packed red blood cell fraction. Aspirate of and discard the first cell layer (PBMCs).Carefully aspirate off the remaining granulocyte layer and pool in a 50 mL centrifuge tube and add 1x PBS to  .Centrifuge at  .Resuspend the granulocyte pellet in    . This will lyse any contaminating red blood cells. After  , add  . Centrifuge at  . Resuspend in   , count and adjust cell count to 2 x 106 cells/mL. Cell culture media used here: 10% FCS, 2 mM L-glutamate, 100 U/mL penicillin, 100 μg/mL streptomycin, 0.01M HEPES buffer, 0.1% β-mercaptoethanol, 0.01 nM non-essential amino acids. Diluting phorbol 12-myristate 13-acetate (PMA):PMA is provided as a lyophilized powder that is reconstituted in DMSO. Dilute PMA to a final concentration of   using cell culture media. Ensure that the concentration of DMSO does not exceed 0.1% in this solution.  PMA stimulation:Add isolated granulocytes with diluted PMA in a 1:1 ratio (50 µL: 50 µL) into wells within a 96-well non-tissue culture treated plate. Incubate for   at  .After incubation, centrifuge at  .Aspirate off the NET-rich supernatant, aliquot and store at    until required. Be careful not to disturb the neutrophil monolayer, only collect cell-free supernatant.Repeat:Repeat this protocol for a total of 10 times. Each time, draw blood from a new healthy donor. Carefully aspirate off the remaining granulocyte layer and pool in a 50 mL centrifuge tube and add 1x PBS to .Centrifuge at .Resuspend the granulocyte pellet in . This will lyse any contaminating red blood cells. After , add . Centrifuge at . Resuspend in  , count and adjust cell count to 2 x 106 cells/mL. Cell culture media used here: 10% FCS, 2 mM L-glutamate, 100 U/mL penicillin, 100 μg/mL streptomycin, 0.01M HEPES buffer, 0.1% β-mercaptoethanol, 0.01 nM non-essential amino acids. Diluting phorbol 12-myristate 13-acetate (PMA):PMA is provided as a lyophilized powder that is reconstituted in DMSO. Dilute PMA to a final concentration of  using cell culture media. Ensure that the concentration of DMSO does not exceed 0.1% in this solution. PMA stimulation:Add isolated granulocytes with diluted PMA in a 1:1 ratio (50 µL: 50 µL) into wells within a 96-well non-tissue culture treated plate. Incubate for   at  .After incubation, centrifuge at  .Aspirate off the NET-rich supernatant, aliquot and store at    until required. Be careful not to disturb the neutrophil monolayer, only collect cell-free supernatant.Repeat:Repeat this protocol for a total of 10 times. Each time, draw blood from a new healthy donor. Incubate for  at .After incubation, centrifuge at .Aspirate off the NET-rich supernatant, aliquot and store at   until required. Be careful not to disturb the neutrophil monolayer, only collect cell-free supernatant.Repeat:
[blood]
[Polymorphprep]
[undiluted blood]
Centrifuge: 650 34, 30 min, 25 10
25 mL
Centrifuge: 100 34, 20 min, 4 10
[ice-cold ddH2O]
on ice
[1x PBS]
Centrifuge: 100 34, 20 min, 4 10
[cell culture media]
[and 5% CO2]
Centrifuge: 400 34, 20 min, 25 10
-80 °C
?. [ELISA day 2.]
During this incubation, dilute all unknown clinical serum samples with assay diluent as follows: Also during this incubation, dilute NET standards with assay diluent as follows:  ABCD1ELISADilution (% of final volume)Amount of serumAmount of assay diluent2MPO-DNA5%6 µL114 µL3NE-DNA40%48 µL72 µL4H3(cit)20%24 µL96 µL  ABCDEFG1ELISAType of standardDilution of Standard 1 (% of final volume)Amount of standardAmount of assay diluentSerial dilutionConcentration of standards2MPO-DNASupernatant from PMA-stimulated neutrophils5%12 µL228 µL1:15.00, 2.50, 1.25, 0.63, 0.31, 0.163NE-DNAPooled clinical serum samples with high endogenous levels of target antigen50%120 µL120 µL1:150.00, 25.00, 12.50, 6.25, 3.13, 1.56, 0.784H3(cit)Supernatant from PMA-stimulated neutrophils40%96 µL144 µL1:140.00, 20.00, 10.00, 5.00, 2.50, 1.25
ABCD1ELISADilution (% of final volume)Amount of serumAmount of assay diluent2MPO-DNA5%6 µL114 µL3NE-DNA40%48 µL72 µL4H3(cit)20%24 µL96 µL
ABCDEFG1ELISAType of standardDilution of Standard 1 (% of final volume)Amount of standardAmount of assay diluentSerial dilutionConcentration of standards2MPO-DNASupernatant from PMA-stimulated neutrophils5%12 µL228 µL1:15.00, 2.50, 1.25, 0.63, 0.31, 0.163NE-DNAPooled clinical serum samples with high endogenous levels of target antigen50%120 µL120 µL1:150.00, 25.00, 12.50, 6.25, 3.13, 1.56, 0.784H3(cit)Supernatant from PMA-stimulated neutrophils40%96 µL144 µL1:140.00, 20.00, 10.00, 5.00, 2.50, 1.25
All sample dilutions should be empirically established according to spike-and-recovery and parallelism experiments. Likewise, the concentrations for the standard should be empirically established to ensure saturation and acceptable recovery. We provide these dilutions as a starting point.
?. [ELISA day 2.]
Add   or   to each well, and ensure that each standard/serum sample is run in duplicate. Cover and incubate  at .
[standard]
[diluted serum]
4 °C
?. [ELISA Day 3.]
ABCDEF1Type of ELISADetection antibodyDilutionAntibody amountCoating buffer amountFinal volume2MPO-DNA and NE-DNADNA-Peroxidase1:200105 µL20,895 µL21,000 µL3H3(cit)H3(cit)1:40002.5 µL9,997.5 µL10,000 µL[Anti-DNA-peroxidase (clone MCA-33) is supplied as a part of the Cell Death Detection ELISA]Wash each plate five times with wash buffer and add  to each well in the ELISA plate.Cover and incubate for  at  in the dark.
ABCDEF1Type of ELISADetection antibodyDilutionAntibody amountCoating buffer amountFinal volume2MPO-DNA and NE-DNADNA-Peroxidase1:200105 µL20,895 µL21,000 µL3H3(cit)H3(cit)1:40002.5 µL9,997.5 µL10,000 µL
All antibody dilutions should be empirically established to ensure maximal signal-to-noise ratio, but we provide these dilutions as a starting point.
[diluted detection antibody]
0 Room temperature
?. [ELISA Day 3.]
During this incubation, make up the second detection antibody for the H3(cit) ELISA using assay diluent, as follows:   Wash each plate five times with wash buffer.  ABCDEF1Type of ELISADetection antibodyDilutionAntibody amountCoating buffer amountFinal volume2H3(cit)Goat anti-rabbit IgG-HRP1:25004.2 µL10,495.8 µL10,500 µL
ABCDEF1Type of ELISADetection antibodyDilutionAntibody amountCoating buffer amountFinal volume2H3(cit)Goat anti-rabbit IgG-HRP1:25004.2 µL10,495.8 µL10,500 µL
All antibody dilutions should be empirically established to ensure maximal signal-to-noise ratio, but we provide these dilutions as a starting point.
?. [ELISA Day 3.]
FOR MPO-DNA and NE-DNA ELISA plates:Add  to each well, cover and incubate for  at  in the dark. Stop the colour change reaction by adding .
[TMB substrate solution]
0 Room temperature
[stop solution]
?. [ELISA Day 3.]
For the H3(cit) ELISA plate:Add   to each well, cover and incubate for  at  in the dark.Wash the plate five times with wash buffer. Add   to each well, cover and incubate for   at   in the dark. Stop the colour change reaction by adding  .Read the optical density at 450 nm.Add  to each well, cover and incubate for  at  in the dark. Stop the colour change reaction by adding .
[second detection antibody]
0 Room temperature
[TMB substrate solution]
0 Room temperature
[stop solution]
?. [Preparing ELISA standards.]
For the NE-DNA ELISA, a number of unknown clinical serum samples should be screened to determine a number of samples (preferably > 5) that show the highest level of endogenous NE-DNA complexes. The NE-DNA ELISA procedure described below can be used to determine these samples.
?. [Preparing ELISA standards.]
Preparation and storage of NET standards:For MPO-DNA and H3(cit) ELISAs.For each of 10 healthy volunteers, NET-rich supernatant from PMA-stimulated neutrophils has been aliquoted and stored. Before proceeding with NET ELISAs, thaw an aliquot from each healthy volunteer and pool. Aliquot out pooled NET-rich supernatant in   and store at    until required. For NE-DNA ELISA. For each of 10 healthy volunteers, NET-rich supernatant from PMA-stimulated neutrophils has been aliquoted and stored. Before proceeding with NET ELISAs, thaw an aliquot from each healthy volunteer and pool. Aliquot out pooled NET-rich supernatant in  and store at   until required. Having identified a number of clinical serum samples with high endogenous levels of NE-DNA complexes, thaw an aliquot from each sample and pool. Aliquot out pooled serum in  and store at   until required.
[aliquots]
-80 °C
[aliquots]
-80 °C