Cayman's new NET Fragment Assay Kit (MPO-DNA) (Item No. 501330) provides a straightforward, qualitative method for assessment of NETosis via the detection of MPO-DNA complexes (i.e., NET fragments) in human plasma and cell culture supernatants. The assay works as an immunometric (i.e., sandwich) assay and requires intact MPO-DNA NET fragments for simultaneous binding to both the capture and detection antibodies.

Assay Kit At-A-Glance

About NETosis

Neutrophils defend against pathogens by utilizing antimicrobial effector functions including degranulation, phagocytosis of pathogens, and the release of neutrophil extracellular traps (NETs) in a process called NETosis.^1,2 During NETosis, decondensed chromatin is released into the extracellular environment where it forms web-like structures of DNA decorated with citrullinated histone H3 (CitH3) and neutrophil granule proteins such as neutrophil elastase (NE), and myeloperoxidase (MPO) (Figure 1).^1,3 These NETs can ensnare and neutralize microbial pathogens, including bacteria, viruses, fungi, and parasites.³

Figure 1. During NETosis, extracellular web-like structures of decondensed chromatin decorated with NE, MPO, and citrullinated histones trap and neutralize pathogens.

NETosis can be triggered by stimuli including pathogen-associated molecular patterns (PAMPs), host-derived damage-associated molecular patterns (DAMPs), or pharmacological agents such as calcium ionophores (e.g., A23187, ionomycin) or phorbol 12-myristate 13-acetate (PMA).^1,3 Depending on the stimulus, NETosis may follow a lytic pathway, which involves membrane rupture and cell death, or a non-lytic pathway, also known as vital NETosis, in which neutrophilic functions such as chemotaxis and phagocytosis are preserved.^1,3,4 Once released, NETs are cleared by plasma DNase and macrophages via phagocytosis of the NET fragments.⁵ Aberrant NETosis or defects in NET clearance have been implicated in a host of inflammatory disorders and autoimmune diseases, including systemic lupus erythematosus (SLE), psoriasis, and rheumatoid arthritis.^6,7

Measuring NETosis

Several challenges exist when measuring NETosis, including a lack of consensus in the field regarding standardized biomarkers.³ While extracellular DNA is a hallmark of NETosis, it is also observed in other types of cell death. Citrullination of histones by peptidylarginine deiminase 4 (PAD4) is a more specific marker of NETosis, however it has been suggested that some forms of NETosis may not require PAD4, depending on the stimulus.^2-4 Therefore, it is recommended to measure multiple NETosis markers to improve specificity.³

Some of the most specific and objective markers of NETosis are NET fragments consisting of DNA bound to MPO or NE.^3,4 Increased levels of circulating MPO-DNA complexes (i.e., NET fragments) in various biological matrices have been associated with several diseases, including active small-vessel vasculitis, COVID-19, and cancer.^8-11

Cayman's new NET Fragment Assay Kit (MPO-DNA) detects MPO-DNA complexes and complements our highly cited Citrullinated Histone H3 (Clone 11D3) ELISA Kit to help researchers measure NETosis in a variety of physiological and pathological contexts.

About the Assay

Cayman's NET Fragment Assay Kit (MPO-DNA) is a qualitative immunometric assay that can be used for the detection of MPO-DNA complexes (i.e., NET fragments) in human plasma and cell culture supernatants. This kit includes all necessary reagents and uses a straightforward protocol that provides rapid results in under four hours.

This immunometric assay is based on a double-antibody "sandwich" technique and requires intact MPO-DNA NET fragments for simultaneous binding to both the capture and detection antibodies. Interruption of these NET fragments by DNases or other intrinsic factors may affect the readout of the assay.

Each well of the microwell plate supplied with the kit has been coated with a recombinant mouse monoclonal antibody specific for MPO (Figure 2). This antibody will bind any MPO introduced into the well. A second monoclonal antibody that recognizes DNA in the NET fragment and is conjugated to horseradish peroxidase (HRP) is added to the well forming a "sandwich". The "sandwich" is immobilized on the plate and the excess reagents are washed away. The detection of NET fragments is accomplished by measuring the enzymatic activity of HRP using the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB). After a sufficient period, the reaction is stopped with acid, forming a product with a distinct yellow color that can be measured at 450 nm. The intensity of the color is directly proportional to the amount of bound antibody-HRP conjugate, which is indicative of the NET fragments.

Figure 2. Schematic of the immunometric assay. For the complete assay protocol, please refer to the kit booklet (PDF).

Digestion of DNA as Confirmation of Assay Specificity

To confirm the specificity of the assay for intact MPO-DNA NET fragments, samples were treated with S7 nuclease to digest NET-associated DNA prior to analysis. Aliquots of human plasma (citrate and EDTA) were spiked with A23187-stimulated PMN supernatant and incubated at 37°C with 15 U/ml of S7 nuclease. At various time points, digestion was halted by adding EDTA to a final concentration of 15 mM. Samples were then diluted 1:10 with Assay Buffer prior to analysis using the NET Fragment Assay Kit (MPO-DNA). Digestion of NET-associated DNA by S7 nuclease reduced the assay signal in a time-dependent manner, confirming the specificity of the assay for intact NET fragments (Figure 3). NET-associated signal is retained longer in plasma collected with EDTA as the anti-coagulant, as EDTA inhibits nuclease through chelation of divalent metal ions required for enzymatic function.

Figure 3. Loss of signal with DNA digestion.

Measured vs. Expected Absorbance in Assay

To evaluate assay performance in complex biological matrices, Assay Buffer or plasma samples were spiked with supernatant from polymorphonuclear leukocytes (PMNs) stimulated with PMA and A23187. Samples were diluted in Assay Buffer and analyzed using the assay.

Expected absorbance values were determined from samples prepared in Assay Buffer alone, while measured absorbance values were obtained from plasma-spiked samples (Figure 4). Measured absorbance values within 80-120% of the expected absorbance were considered acceptable. Values outside this range may indicate interference from matrix components.

Figure 4. Measured vs. expected absorbance.

For additional information on the performance of this kit, including dilutional linearity and intra- and inter-assay precision, please refer to the validation data (PDF).

The Cayman Advantage

At Cayman, we have an exceptional understanding of assay development, validation, and performance. Each of our assay kits undergoes rigorous quality testing to certify high precision and accuracy. Our attention to these details ensures you will obtain reproducible results, from day to day and lot to lot, with expert technical support readily available to assist you. Learn more about the Cayman Advantage in our article, Why Cayman Assay Kits.

Our new NET Fragment Assay Kit (MPO-DNA) is complemented by our extensive and trusted line of NETosis research tools, including several additional assay kits to measure and visualize the process of NETosis and aid in the development of inhibitors or inducers:

NETosis Assay Kits

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