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.

 

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.