Quantifying Myeloperoxidase-DNA and Neutrophil Elastase-DNA Complexes from Neutrophil Extracellular Traps by Using a Modified Sandwich ELISA

Podsumowanie

We present a protocol for a modified sandwich enzyme-linked immunosorbent assay technique to quantitatively measure two components of neutrophil extracellular trap remnants, myeloperoxidase conjugated-DNA and neutrophil elastase conjugated-DNA complexes,derived from activated neutrophils.

Streszczenie

Certain stimuli, such as microorganisms, cause neutrophils to release neutrophil extracellular traps (NETs), which are basically web-like structures composed of DNA with granule proteins, such as myeloperoxidase (MPO) and neutrophil elastase (NE), and cytoplasmic and cytoskeletal proteins. Although interest in NETs has increased recently, no sensitive, reliable assay method is available for measuring NETs in clinical settings. This article describes a modified sandwich enzyme-linked immunosorbent assay to quantitatively measure two components of circulating NETs, MPO-DNA and NE-DNA complexes, which are specific components of NETs and are released into the extracellular space as breakdown products of NETs. The assay uses specific monoclonal antibodies for MPO or NE as the capture antibodies and a DNA-specific detection antibody. MPO or NE binds to one site of the capture antibody during the initial incubation of samples containing MPO-DNA or NE-DNA complexes. This assay shows good linearity and high inter-assay and intra-assay precision. We used it in 16 patients with COVID-19 with accompanying acute respiratory distress syndrome and found that the plasma concentrations of MPO-DNA and NE-DNA were significantly higher than in the plasma obtained from healthy controls. This detection assay is a reliable, highly sensitive, and useful method for investigating the characteristics of NETs in human plasma and culture supernatants.

Wprowadzenie

This article outlines a method to quantify neutrophil extracellular trap (NET) formation in biological fluids by using sandwich enzyme-linked immunosorbent assay (ELISA) to detect complexes of myeloperoxidase (MPO) and neutrophil elastase (NE) with DNA^1,2. NETs are composed of a DNA backbone decorated with antimicrobial proteases originating from neutrophil granules^3,4. Both MPO-DNA and NE-DNA complexes are important and specific components of NETs and are released into the extracellular space as breakdown products of NETs^3,4.

Besides their important physiological role in antimicrobial defense³, NETs also have various pathological effects^4,5, including the promotion of thrombogenesis⁶ and the worsening of sepsis⁷. Accordingly, NETs have been gaining attention recently. Nevertheless, the in vivo quantification of NETs has proven challenging because of the lack of a sensitive, reliable quantitative assay method.

A few methods are available, including the direct measurement of NETs by fluorescence microscopy^8,9 and flow cytometry¹⁰ and the indirect measurement of circulating cell-free DNA, nucleosomes, and citrullinated histone H3, but each method has its own advantages and limitations¹¹. Although the immunofluorescence microscopic method is specific to NETs and clearly shows the localization and degree of NET formation, samples are limited to biopsy tissue and secreted materials. Moreover, this method needs to be performed by skilled researchers and requires a long time for results to be obtained. Measuring circulating levels of NET-related components by flow cytometry is easy and provides results quickly; however, the method is not specific to NETs¹².

We¹³ and others^1,2 have developed a highly sensitive and reliable assay to measure the circulating NET components, MPO-conjugated or NE-conjugated DNA, in human plasma with a modified ELISA technique that uses specific antibodies for MPO or NE as the capture antibodies and a DNA-specific detection antibody. This assay can also be used ex vivo to identify NET components in cell culture supernatants released by activated neutrophils in response to phorbol 12-myristate 13-acetate (PMA) stimulation.

Protokół

This study was conducted in conformity with the Declaration of Helsinki and was approved by the institutional review boards of Aichi Medical University (2017-H341, 2019-H137). Written informed consent was obtained from each participant.

1. Reagent preparation

NOTE: To perform the sandwich ELISA assay, the reagents are prepared as described below.

1. Coating buffer:
   1. To make a 0.1 mol/L carbonate-bicarbonate buffer, weigh 10.6 g of anhydrous sodium carbonate (molecular weight, 106 g/mol) and 8.4 g of sodium bicarbonate (molecular weight, 84 g/mol).
   2. Add it to approximately 900 mL of distilled water in a beaker.
   3. Adjust the pH of the buffer to 9.6 by adding the required quantity of diluted hydrochloric acid or sodium hydroxide.
   4. Check the pH with a pH meter.
   5. Then, add the required volume of distilled water to achieve a total volume of 1,000 mL.
   6. Store this 0.1 mol/L carbonate-bicarbonate buffer in the refrigerator at 4 °C.
2. Blocking buffer:
   1. Add approximately 250 µL of 20% sodium azide and 1 g of bovine serum albumin (BSA) to 70 mL of phosphate-buffered saline (PBS) composed of 137 mmol/L NaCl, 8.1 mmol/L Na₂HPO₄, 2.68 mmol/L KCl, and 1.47 mol/L KH₂PO₄ (pH 7.4), and mix gently.
   2. Add distilled water to achieve a total volume of 100 mL. The final concentrations of bovine serum albumin and sodium azide are 1% and 0.05%, respectively. Wait for 10 min, and then the solution is ready to use.
3. Washing solution: Prepare 0.5% t-octylphenoxypolyethoxyethanol, polyethylene glycol tert-octylphenyl ether (Triton X-100) in distilled water.
   1. Dilute 100% Triton X-100 to 10% with distilled water, and allow the 10% solution to stand for 1 day before use.
   2. Then, re-dilute the 10% Triton X-100 solution 20-fold with distilled water to achieve a concentration of 0.5%.
4. Antibody dilution:
   1. Dilute the capture antibody (anti-MPO antibody [1 mg/mL] or anti-NE antibody [1 mg/mL]) to 1:2,000 in the coating buffer (pH 9.6) before use on day 1 of the assay (see below) and the detection antibody (peroxidase-conjugated anti-DNA antibody) to 1:40 with PBS before use on day 3 of the assay (see below).
      NOTE: For the preliminary experiment, dilute the iso-type antibodies (IgG rabbit [5 mg/mL] and IgG1 mouse clone Ci4 [0.5 mg/mL]) to 1:10,000 and 1:1,000 in the coating buffer (pH 9.6).
5. ABTS substrate solution: Depending on the number of samples, dissolve one, two, or three 2,2'-azino-bis(3 ethylbenzothiazoline-6-sulfonic acid (ABTS) tablets in 5 mL, 10 mL, or 15 mL of ABTS buffer (containing sodium perborate, citric acid, and disodium hydrogen phosphate); 100 µL is needed per sample. Store the prepared solution at 2-8 °C protected from light. The solution is stable for up to 3 months.

2. Sample collection and storage

1. Plasma isolation:
   1. Collect 4 mL of peripheral blood samples into a lithium heparin blood collection tube by venipuncture with a 22 G syringe, and centrifuge at 1,600 x g for 10 min at 4 °C.
   2. Transfer the supernatant into a 2 mL safe-lock sample tube.
   3. Re-centrifuge the supernatant at 16,000 x g for 10 min at 4 °C to remove any residual cells.
   4. Collect the supernatant into a new safe-lock sample tube without disturbing the pellet at the bottom of the tube.
   5. Store the obtained plasma at −80 °C until further use.
      NOTE: To obtain high-throughput results with this assay, good-quality samples are required. If plasma samples are used, perform a second centrifugation to remove any residual cells. Avoid repeated thawing of the samples. Use heparin as the anticoagulant because EDTA affects the DNase activity.
2. Isolation of polymorphonuclear neutrophils
   1. Collect the peripheral blood samples into a lithium heparin blood collection tube by venipuncture with a 22 G syringe.
   2. Layer 6 mL of blood onto 6 mL of one-step polymorphs, and centrifuge the tubes at 1,000 x g for 45 min at room temperature (RT).
   3. Harvest the third buffy coat layer containing neutrophils, and wash it three times in phenol red-free RPMI-1640 at 400 x g for 10 min at RT.
   4. Resuspend the neutrophils in phenol red-free RPMI-1640 containing 2 mM L-glutamine supplemented with 6% heat-inactivated fetal bovine serum, and count the cells in the microscope field with a hemocytometer.
      NOTE: This method yields 96% to 98% purity with greater than 95% viability, as assessed by trypan blue dye exclusion.
3. Activation of polymorphonuclear neutrophils and NETosis in vitro
   1. Dilute freshly isolated polymorphonuclear neutrophils to 1 × 10⁵ cells/mL in phenol red-free RPMI-1640 containing 2 mM L-glutamine supplemented with 6% heat-inactivated fetal bovine serum, and seed them in 35 mm culture dishes.
   2. To induce NETs, stimulate polymorphonuclear neutrophils with 25 nM PMA for 4 h at 37 °C in 5% CO₂/95% air.
   3. After 4 h of incubation, partially digest the NETs by adding 0.6 µg/mL DNase I directly to the culture dishes for 15 min at RT, and stop the DNase I activity by adding 5 mM EDTA.
   4. Collect the medium containing the synthesized NETs, and centrifuge at 400 x g for 10 min at 4 °C to remove the cell debris.
   5. Obtain the supernatants from four healthy controls, mix, and store at −80 °C until use.
      ​NOTE: DNase-digested PMA-stimulated neutrophils obtained from four healthy controls are used as the NET-standard^1,14,15. Generate a calibration curve from a serially diluted NET-standard with PBS, and assign the optical density (OD) values obtained from an undiluted NET-standard as 100% NET.

3. Assay method

NOTE: The steps for performing the assay are described in detail below.

1. Day 1
   1. Apply a total of 100 µL of diluted anti-MPO or anti-NE antibody containing 0.05 µg of antibody to each well of the plate, including the blank well.
      NOTE: The coating buffer must not contain any kind of detergent because, otherwise, the antibodies may not bind equally and smoothly to the walls of each well. To prevent the hook effect, the coating protein concentration must not be more than 20 µg/mL, because concentrations above that level will saturate most of the available sites on the microtiter plate. The typical concentration range of protein coating solutions is 2-10 µg/mL. For the preliminary experiment, use diluted iso-type control antibody IgG rabbit for coating instead of specific monoclonal antibodies against MPO. Use diluted iso-type control antibody IgG1 mouse for coating instead of specific monoclonal antibodies against NE.
   2. Cover the plate with an adhesive plastic cover to prevent the evaporation of the sample, and incubate overnight at 4 °C to allow the binding of the capture antibodies.
2. Day 2
   1. Discard the diluted antibody solution from the wells, and then pipette 300 µL of wash solution per well, and repeat this washing procedure three to four times.
   2. Remove excess PBS by tapping the plate dry on a paper towel.
   3. Block each well of the ELISA plate with 200 µL of the blocking buffer.
   4. Cover the plate closely with an adhesive plastic cover, and incubate it for 1.5-2 h at RT to obstruct the wells.
   5. Discard the blocking solution completely from the wells, and then pipette 300 µL of wash solution per well, and repeat this washing procedure three to four times.
   6. Remove excess PBS by tapping the plate dry on a paper towel.
   7. Apply a total of 25 µL of plasma or medium to each well except the blank well, and add 75 µL of PBS as a diluent to make the final volume 100 µL; add 100 µL of PBS to the blank well.
   8. Then, mix the samples at RT for 10 s by placing the plate on a shaker at 250 rpm.
   9. Apply 2 µL of 100-fold diluted DNase I (0.03 mg/mL) to each well, including the blank well (final concentration of DNase I in the reaction mixture: 0.6 µg/mL).
   10. Seal the plate with an adhesive plastic cover, and thoroughly mix the samples at RT for 10 s by placing the plate on a shaker at 250 rpm.
       NOTE: To assess the optimum conditions for DNA digestion, when developing the assay, we incubated samples containing MPO-associated DNA from patients with COVID-19 with 0-0.9 µg/mL DNase I for 15 min at RT and used plates coated with specific capture antibody for MPO or a species-matched iso-type control antibody.
   11. Incubate the samples for 15 min at RT.
   12. Remove the adhesive plastic cover, and apply 1 µL of 0.5 M EDTA to each well to stop the DNase reaction.
   13. Reseal the plate with an adhesive plastic cover, and shake it at RT for 15 s by placing it on a shaker at 250 rpm.
   14. Then, incubate the plate overnight at 4 °C to allow the protein components of the NETs to attach to the capture antibodies.
3. Day 3
   1. Remove the adhesive plastic cover, and discard the solution from the wells.
   2. Discard the solution completely from the wells, and then pipette 300 µL of the wash solution per well, and repeat this washing procedure three to four times.
   3. Apply a total of 100 µL of the diluted peroxidase-conjugated anti-DNA detection antibody to each well.
   4. Seal the plate closely with an adhesive plastic cover, and incubate it for 1.5 h at RT.
   5. Discard the solution completely from the wells, and then pipette 300 µL of wash solution per well, and repeat this washing procedure three to four times.
   6. Carefully remove the residual solution.
   7. Apply a total of 100 µL of ABTS substrate solution to each well, and cover the plate with an adhesive plastic cover.
   8. Incubate the plate in the dark at RT for 20-30 min on a shaker at 250 rpm.
      NOTE: Monitor the plate at 5 min intervals until the desired OD readings are obtained.
   9. Add a total of 50 µL of 2 M sulfuric acid to stop the reaction.
   10. Mix the liquid contents of the wells by carefully tapping the side of the plate.
   11. Switch on the microplate reader, and connect it to the computer.
   12. Open the software application on the computer.
   13. From the status bar, create a new experiment and name it.
   14. Set all the following parameters for plate reading: Read type, Absorbance; Read mode, Endpoint; Wavelengths, 2; Lm-1, 405 nm; Lm-2, 490 nm; Auto mix & Blanking before, Off; Pre-read plate, Off; Auto calibration, On; Strips, Read entire plate; Column wavelength priority, Column priority; Carriage speed, Normal; and Auto read, Off.
   15. Then, put the 96-well plate containing the samples into the drawer and close it.
   16. Last, select the Read button so that the plate is read immediately.
   17. Read the absorbance of each well at a wavelength of 405 nm by using a microplate reader (at this stage, use a wavelength of 490 nm as a reference).
   18. Perform automatic subtraction of the absorbance value of the assay medium alone from all the unknown samples, and save the data.
       ​NOTE: Use a standard curve obtained from serially diluted NET-standard to calculate the relative concentration of NET in the sample^1,14,15. Here, the final results are presented as "MPO- or NE-DNA complexes (% of NET-standard)." The limit of detection (LOD) was calculated from the standard deviation (SD) and the slope of the calibration curve (S) as follows: LOD = 3.3(SD/S).

4. Statistics

1. Perform all the statistical analyses using appropriate statistical analysis software. Here SigmaPlot v14.5 was used. Percentages and continuous variables are shown as the median with the interquartile range due to the skewed distribution of most of the parameters.
2. Compare the plasma MPO-DNA and NE-DNA levels between COVID-19 patients and healthy controls using the Wilcoxon rank sum test. Use correlation analysis to explore the relationship between optical density and the percentage of NET-standard.

Wyniki

This method used a sandwich ELISA with anti-MPO, anti-NE, and anti-DNA monoclonal antibodies to measure MPO-associated and NE-associated DNA (Figure 1). In this method, the wells of a microtiter plate were coated with an MPO-specific or NE-specific monoclonal antibody to capture DNA-associated MPO and DNA-associated NE, as well as non-DNA-associated MPO and NE. To calculate the intra-assay coefficient of variability (CV), duplicate measurements were performed within the same plate for 30 sam...

Dyskusje

We have described a sandwich ELISA method in which MPO or NE binds to one site of the capture antibody during the initial incubation of samples containing MPO-DNA or NE-DNA complexes. After washing, the "sandwich" is completed by incubating the samples with a peroxidase-associated anti-DNA monoclonal antibody. After the removal of unbound secondary antibody, the bound peroxidase conjugate is detected by the addition of a chromogenic ABTS peroxidase substrate, which yields a soluble end-product that can be read sp...

Materiały

Name	Company	Catalog Number	Comments
1-Step Polymorphs	Accurate Chemical and Scientific Corporation	AN221725	Isolation of PMN's from human blood.
96-well microtiter plate	Thermo Fisher Scientific	467466	flat bottom
ABTS buffer solution	Sigma-Aldrich Merck	11 204 530 001	Contains sodium perborate, citric acid, and disodium hydrogen phosphate.
ABTS tablets	Sigma-Aldrich Merck	11 204 521 001	Each tablet contains 5 mg ABTS substrate and 60 mg vehicle substances.
Adhesive plastic cover, Axygen	Thermo Fisher Scientific	14222348
Anti-MPO antibody	Sigma-Aldrich Merck	07-496-I	Store at 2-8 °C. stable for 1 year. Host species is rabbit.
Anti-NE antibody, clone AHN-10	Sigma-Aldrich Merck	MABS461	Store at 2-8 °C. stable for 1 year. Host species is mouse.
Bovine serum albumin	Biomedical Science	BR-220700081	Albumin from bovine fraction V. Store at 2–8 °C. stable for 2 year.
DNase I	New England BioLabs	M0303M	Store at -20 °C
IgG, rabbit, Isotype Control	GENETEX, Inc.	GTX35035	Store as concentrated solution at 2–8 °C.
IgG1, mouse Isotype Control, clone Ci4	Merck	MABC002	Store as concentrated solution at 2–8 °C.
Lithium heparin blood collection tube	Becton Dickinson and Company
Microplate mixer	As one corporation	NS-P
Microplate Reader	Molecular Devices	SpectraMax 190	Any microplate plate reader capable of reading wavelengths from 405–490 nm can use.
Microplate reader application	Molecular Devices	SoftMax pro
Peroxidase-conjugated anti-DNA antibody, Cell death Detection ELISA	Roche Diagnostics	1154467500	bottle 2. Store at 2–8 °C. stable for 1 year.
Phorbol 12-myristate 13-acetate	Sigma-Aldrich Merck	P8139	Activation of PMN's from human blood.
Phosphate buffered solution	Takara Bio	T9181	Store at room temperature. Stable for 6 months.
SigmaPlot v14.5	Systat Software Inc. San Jose, CA, USA
Sodium azide	Fujifilm Wako Chemicals	190-14901	Store at room temperature.
t-Octylphenoxypolyethoxyethanol, Polyethylene glycol tert-octylphenyl ether	Fujifilm Wako Chemicals	9002-93-1	Store at room temperature.