Volition’s Nu.Q® Discover H3.1 ELISA Research Use Only kit detects quantitative levels of circulating intact nucleosomes containing Histone H3 in plasma. Nucleosomes are repeating subunits of DNA and histone proteins that constitute human chromatin. During cellular damage, such as apoptosis or necrosis, chromatin is fragmented into oligo- or mono- nucleosomes which can be released into the blood stream.

A specific cell death process involved with immune function and results in the release of nucleosomes into biological fluids is known as NETosis.  In this process, neutrophils produce neutrophil extracellular traps (NETs), which are long web-like strands of chromatin structures containing nucleosomes and antimicrobial proteins.  NETs function to trap pathogens (viral, bacterial, cancer, etc) and are thought to be protective when properly controlled but harmful if not. Systemically elevated levels of NETs are associated with immune-mediated pathologies and poor outcomes.

Given their relation to disease, measuring NETs and the process of NETosis in human samples is important. Currently, there are several common methodologies that can directly measure NETs, including NET microscopy and NET flow cytometry. Both methods require NET formation to be captured in process and laborious sample handling. NETosis ELISA methodologies measure NET components and include dsDNA, histones, nucleosomes, citrullinated histone, granular proteins in complex with DNA, and granular proteins alone. These can be measured via a variety of NETosis ELISA kits (MPO ELISA, MPO+DNA Complex ELISA, citH3 ELISA, total nucleosome ELISA). Some of these NETosis ELISA kits are available commercially, but most are in-house developed assays. Volition’s Nu.Q® Discover H3.1 ELISA Research Use Only kit offers a set of standards composed of recombinant nucleosomes, controls and a standardized output of ng/mL and is able to be used as a surrogate measure of NETs or NETosis via ELISA.

The Nu.Q® Discover H3.1 ELISA Research Use Only kit is a powerful research tool for a wide range of research areas that study nucleosomes and neutrophil extracellular traps (NETs) such as:

  • Oncology
  • Coagulation
  • Sepsis
  • Auto-immune Diseases
  • Neurodegenerative Diseases
  • Inflammation
  • NETopathies (neutrophil extracellular trap associated diseases)
  • COVID-19

 

Assay Principle:

The Nu.Q® Discover H3.1 ELISA Research Use Only kit is a sandwich format colorimetric assay for research use only (RUO). The sample is added with the assay buffer to the well coated with anti-H3.1 antibody. After a first incubation followed by a washing step, the detection antibody coupled to HRP is added. After a second incubation and washing step, the TMB substrate is added. The absorbance is read after addition of stop solution. The signal obtained is proportional to the concentration of H3.1 nucleosome present in the sample. This colorimetric assay is provided in a 96-stripwell format, enabling high and low throughput analysis.

 

For Research Use Only. Not for use in diagnostic procedures.

 

All reagents are exclusively for Research Use Only – Not for Use in Diagnostic Procedures

Component Symbol Description Volume
Antibody coated plate

 

CP1001 12 strips of 8 wells coated with monoclonal anti-H3.1 antibody against nucleosomes and provided on a carrier tray

 

1 plate

 

Wash buffer

 

WB1001 10X concentrated Wash buffer, containing preservative ProClin®300 and Tween 20 0.5%

 

2x 1 vial (30 mL)

 

Assay buffer AS1001 Ready-to-use Assay buffer, containing CMIT/MIT preservative 1 vial (14 mL)
Standard curve

Standard A->F

 

SC2001-A

SC2001-B

SC2001-C

SC2001-D

SC2001-E

SC2001-F

Lyophilized solutions containing H3.1 nucleosome to be used for the standard curve

 

6x 1 vial (1mL)

 

Kit controls 1 & 2 KC1001-1

KC1001-2

Lyophilized solutions containing H3.1 nucleosome to be used as controls 2x 1 vial (1 mL)

 

HRP labelled detection antibody

 

DE1001 Ready-to-use solution of Anti-nucleosome antibody conjugated with horseradish peroxidase (HRP)

 

1 vial (14 mL)

 

Substrate solution

 

SU1001 Ready-to-use TMB (3,3′,5,5′- Tetramethylbenzidine) substrate

Note: the substrate solution is light sensitive

 

1 vial (14 mL)
Stop solution

 

ST1001 Hydrochloric acid 1M

Note: avoid any skin and eyes contact

 

1 vial (14 mL)

 

Sealing film N/A N/A Sealing films 3 Seals

 

 

Material required but not provided

  • Deionized water.
  • Adjustable or preset single-channel pipettes able to dispense 20μL.
  • Multi-channel pipettes able to dispense 50μL to 200μL.
  • Disposable pipette tips.
  • Disposable reagent reservoirs.
  • Vortex mixer.
  • Volumetric cylinders.
  • Container for biohazardous waste.
  • Microplate incubator with orbital shaking facility (shaker at 700 rpm).
  • Microplate reader equipped with 450nm filter
  • Absorbent paper.
  • Light-tight plate cover.

REMARK: For best assay efficiency, we recommend the use of a qualified pipettes, shaker, and reader.

Standardized

Analytically validated quantitative assays calibrated in ng/ml using recombinant nucleosome standards for use in EDTA plasma. The Nu.Q® H3.1 ELISA Assay was developed under the CLSI guidelines.

 

Quantification

Recombinant nucleosomes enable absolute quantification of levels of circulating H3.1 nucleosomes, ideal to measure apoptosis or release of histone complexes in research.

 

Detection Range

The reportable range of the Nu.Q® Discover H3.1 ELISA Assay is 22.7 ng/mL to 650 ng/mL.

 

Analytical Performance

Sensitivity: The Lower Limit of Detection is 9.1ng/mL.

Specificity: Nucleosome epitope specific antibody enables detection of only intact nucleosomes

 

Interferences

The following potential interfering substances were tested and have shown not to interfere on the assay when present below the concentrations defined in the following table:

Interfering Substance Threshold Concentration
Triglycerides 3000 mg/dL
Haemoglobin 500 mg/dL
Bilirubin, unconjugated 20 mg/dL
Bilirubin, conjugated 20 mg/dL
Protein (total) 8 g/dL
Cholesterol 300 mg/dL

 

Species Cross-Reactivity

Nucleosome structure is conserved across species. H3.1 has been evaluated in samples taken from human and multiple animal models including mouse, dogs, cats, horses, rats, and pigs.

 

Please use in conjunction with the instructions for use.

FAQ Volition Nu Q® Discover H3.1 ELISA RUO Kit

 

Does this kit measure NETs?

Nucleosomes are DNA-histone complexes that are released during the process of NETosis. Measuring nucleosomes in plasma serves as a surrogate marker of NET formation. This allows researchers to assess the extent of NETosis in various disease states, such as thrombosis, autoimmune disorders, and infectious diseases with a robust and standardized assay.  It is worth noting that nucleosomes alone may not provide a comprehensive understanding of NETosis. Additional surragate markers, such as specific NET-associated proteins (MPO or NE), citrullinated histone H3 (H3Cit), or other functional assays, are often used in conjunction with nucleosome measurements to obtain a more comprehensive view of NETosis dynamics.

My lab already measures NETs with our own assay, how is this kit adding to what I do?

Because there are not many commercial assays available for NET measurement, many labs have generated their own in-house assays.  While these in-house assays provide insightful information, they leave a gap in standardization and the data generated is difficult to compare within and between areas of study.  In addition, these processes are often VERY labor intensive and may be limited to labs that have access to cell culture methodologies for in-house production of standards.  The Volition Nu Q® Discover H3.1 ELISA RUO Kit provides continuity for high throughput analysis by including standards, controls, and data outputs in ng/mL.  The kit was developed under CLSI guidelines and has been well characterized for many analytical parameters (intra and inter-assay variability, interferences, limits of detection, etc.).

Is there a method to measure free H3?

The Nu Q® Discover H3.1 ELISA Research Use Only Kit is specific for detection of intact nucleosomes and does not detect free histones or free DNA. There are ELISA assays by other manufacturers that claim to measure H3 alone, but Volition has not done any work with them. They are typically made up of 2 H3 antibodies.

Can this kit also recognize EETs (eosinophil extracellular traps)? If so, how can we estimate the levels of NETs?

The Nu Q® Discover H3.1 ELISA Research Use Only Kit is specific for detection of intact nucleosomes. Nucleosomes are released during cell death and can include events related to NETosis. Volition has not tested out H3.1 Nu.Q assay on EETs, but if nucleosomes are intact in EETs then we would expect that we would be able to detect them.

Is it possible to stain tissue samples with this biomarker?

The Nu Q® Discover H3.1 ELISA Research Use Only is an ELISA kit. The antibodies are not available for purchase outside of the kit format. One would think that there are antibodies that could be used to stain H3.1, but it is unknown if there are antibodies that can specifically detect intact nucleosomes in tissue samples.

How popular and in use is flow cytometry for NETosis?

Flow cytometry for measuring NETosis requires specific instrumentation and needs to capture NET formation while it is occurring. These hurdles limit the widespread use of the technique, but there are certainly reports using the technique.

Can you measure NETs in the whole blood, plasma, serum?

Publications indicate that NETs have been found in a variety of body fluids and cell culture conditions including blood, sputum, synovial fluid, urine, saliva, wounds, tears, and CSF. While the Volition Nu Q® Discover H3.1 RUO ELISA kit has been characterized with human EDTA plasma samples, other sample types have been tested within Volition’s R&D. While the kit will work with serum samples, they should be avoided because the process of preparing serum can activate immune cells and produce NETs, resulting in false positives.

Does the sample have to be fresh?

No, the samples can be fresh or frozen.

Can you measure NETs from other species or is it only human nucleosomes?

The Volition Nu Q® Discover H3.1 ELISA RUO Kit has been evaluated in samples taken from human and multiple animal models including mice, dogs, cats, horses, rats, and pigs. The sample volume required is 20uL per well, making it an effective kit for small sample collections.

Nucleosome structure is conserved across species. While not every species has been tested, the conservation of H3 increases the likelihood that the Volition Nu Q® Discover H3.1 ELISA RUO Kit will work with most model systems.

Do anticoagulants affect the assay?

Preanalytical variables and sample collection conditions for clinical samples are under review by KOLs in the field. Time from collection to analysis, blood temperature, anticoagulation, etc. have all been shown to impact the measurement of NETs in human blood samples, but the extent is to be determined. Volition does not recommend the assay to be used with heparinized plasma and has some anti-coagulant data. Please reach out to have a conversation about a specific anticoagulant.

Have you tested this kit with airway samples, human sputum supernatants or bronchoalveolar lavage?

While BAL or sputum have not been tested with this kit specifically, the chances of success are high because of successes with other sample types and other neutrophil focused assays. Also, the ProAxsis ProteaseTag® Active Neutrophil Elastase Immunoassay might be an additional fit for these needs.

Has this assay been tested with in vitro generated NETs?

Volition has tested in vitro stimulated neutrophils in limited situations. If this is of interest to you, we recommend reaching out to DiaPharma to discuss the project in more detail.

H3.1 Nu.Q is not binding to H3Cit but H3, so how this is a marker for NETosis?

Measuring nucleosomes in plasma serves as a surrogate marker of NET formation as nucleosomes are severely elevated when NETs are produced. This surrogate allows researchers to correlate the extent of NETosis in various disease states with a robust and standardized assay. Nucleosomes alone may not provide a comprehensive understanding of NETosis and additional markers, such as MPO or NE, citrullinated histone H3 (H3Cit), are often used in conjunction with nucleosome measurements to obtain a more comprehensive view of NETosis dynamics.

Can H3Cit also be released from cells other than neutrophils?

Publications suggest that PAD4 is expressed in cells of hematopoietic origin and include neutrophils, eosinophils, and monocytes. H3Cit is mostly reported in the context of NETs but there are some reports that it is found in cancer as well (Zhu et al. Molecular Cancer (2021) 20:90; https://doi.org/10.1186/s12943-021-01373-z).

Nucleosomes can be from any apoptotic cells including NETosis, so do you have kit for CitH3- specific as (PAD) NETosis-specific?

A CitH3-specific kit (H3R8 Cit kit) is in development by Volition and DiaPharma will announce when the RUO version is officially available in the US and Canadian markets. Currently, there is a prototype version available for evaluation. Please inquire with DiaPharma if you have an interest in being a test site for the kit now.

Can you detect NETs in healthy subjects with your assay and if so, what are the values compared to sepsis patients for example?

Turnover rate for nucleosomes in healthy subjects is fast. Therefore, nucleosome levels seen in frozen plasma from healthy control groups are detectable, but less than 60 ng/mL. Studies with sepsis or COVID subjects are in the several hundred ng/ml and greater.

It was not clear to me how you determine nucleosomes are neutrophil specific?

This cannot be determined with the existing assay; however, Volition has performed NGS on sister samples and confirmed that the DNA is derived from neutrophils using methylation-based cell of origin analysis.