Homogeneous immunoassays

Abstract

The present invention relates to detecting the concentration of chromatin fragments in a body fluid sample using a homogeneous immunoassay (HIA) method.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to homogeneous immunoassay methods for measuring extracellular chromatin fragments, as well as kits for carrying out such methods. [Background technology]

[0002] BACKGROUND OF THEINVENTION Acute organ failure due to infectious or aseptic sepsis and septic shock is a leading cause of death. Sepsis is a preventable, life-threatening condition characterized by severe organ dysfunction. According to the Global Report on the Epidemiology and Burden of Sepsis published by the World Health Organization (ISBN: 978-92-4-001078-9), it was estimated that in 2017, 49 million individuals suffered from sepsis, and sepsis was associated with approximately 11 million potentially avoidable deaths worldwide, accounting for approximately 20% of all causes of death worldwide. The largest contributors to the incidence and mortality of sepsis across all age groups were diarrheal diseases and lower respiratory tract infections, respectively. Nearly half of all sepsis-related deaths were complications of wounds and non-communicable diseases, and 41% (20 million) of all sepsis cases worldwide occurred among children under the age of 5 years.

[0003] Nucleosomes are released into the circulation after chromatin fragmentation during cell death. Many infections initiate cell death through various mechanisms (e.g., cell binding and invasion, activation of endosomal TLR3, and gene expression), thereby increasing the number of circulating nucleosomes in the blood (Danthi et al., Annu. Rev. Virol. (2016) 3: 533-53). In particular, NETosis is a form of neutrophil cell death in which neutrophils release neutrophil extracellular traps (NETs) that can capture and kill bacteria and other pathogens, preventing them from spreading. Recent studies have shown that extracellular nucleosomes and NETs are associated with the progression of several diseases, including sepsis. Although most described ETs are produced by neutrophils, other immune cells also produce ETs.

[0004] Current assays for NETs include myeloperoxidase (MPO), neutrophil elastase (NE), extracellular free DNA (cfDNA), and nucleosomes. However, none of these are used in the clinic. The reason is that patients with systemic inflammatory response syndrome or sepsis may develop multiple organ failure and deteriorate rapidly or unexpectedly, and the clinical treatment decision must be made in a timely manner. Currently available measurement methods are manual immunoassay kits that require several hours to perform. Furthermore, these assays are not performed in real time, but in assay batches up to several days after blood sample collection. Therefore, although NET levels have been shown to have potential for use as a biomarker for sepsis, currently no methods have been reported for assaying nucleosomes or other chromatin fragments for use in making clinical decisions in real time, in a low-cost near-patient method that provides results with a short turnaround time.

[0005] Previously, Holdenrieder et al., Int. J. Cancer (2001) 95: 114-120, described the detection of nucleosome levels in serum samples from patients with benign and malignant diseases. The epigenetic composition of circulating extracellular free nucleosomes in terms of their histone modifications, histone variants, DNA modifications, and adduct content has also been investigated as a blood-based biomarker for cancer. See WO 2005 / 019826, WO 2013 / 030577, WO 2013 / 030579, and WO 2013 / 084002. Levels of circulating extracellular free nucleosomes are particularly highly elevated in hematological cancers in both humans (WO2021110776) and other animals (Dolan et al., BMC Veterinary Research (2021) 17:276 and Wilson-Robles et al., BMC Veterinary Research (2021) 17:231).

[0006] There remains a need in the art to provide rapid turnaround tests necessary to diagnose and monitor patients and guide treatment in real time. Summary of the Invention

[0007] (Summary of the invention) According to a first aspect, there is provided a method for detecting the concentration of extracellular traps (ETs), chromatin fragments and / or nucleosomes in a body fluid sample, the method comprising analysing the body fluid sample using a homogeneous immunoassay (HIA) method, and using results from the analysis to determine the concentration of ETs, chromatin fragments and / or nucleosomes in the body fluid sample.

[0008] According to a further aspect of the invention there is provided a method of monitoring disease progression in a subject comprising: (i) detecting the concentration of ETs, chromatin fragments and / or nucleosomes in a body fluid sample obtained from said subject using a method defined herein; (ii) repeating step (i) on one or more occasions; and (iii) using any changes in ET, chromatin fragments and / or nucleosome concentrations to monitor the progression of the disease in the subject.

[0009] According to a further aspect of the invention there is provided a method for assigning a risk of an adverse outcome to a subject suffering from an infectious disease comprising: (i) detecting the concentration of ETs, chromatin fragments and / or nucleosomes in a body fluid sample obtained from said subject using a method defined herein; and (ii) using the detected concentrations of ETs, chromatin fragments and / or nucleosomes to assign a likelihood of an adverse outcome to the subject; The method further provides for assigning subjects identified as having a high likelihood of an adverse outcome to a medical intervention.

[0010] According to a further aspect of the invention there is provided a method for detecting a subject in need of medical treatment for sepsis or septic shock comprising: (i) detecting the concentration of ETs, chromatin fragments and / or nucleosomes in a body fluid sample obtained from said subject using a method defined herein; and (ii) using the concentration of ET, chromatin fragments and / or nucleosomes as an indicator that the subject is in need of medical treatment for sepsis or septic shock.

[0011] According to a further aspect of the invention there is provided a kit for determining the concentration of ETs, chromatin fragments and / or nucleosomes in a body fluid sample comprising: The kit includes a container suitable for use in a reagent solution comprising one or more antibodies capable of specifically binding to ET, chromatin fragments and / or nucleosomes, and a means for determining the concentration of ET, chromatin fragments and / or nucleosomes by measuring the degree of aggregation or precipitation caused upon antibody binding, and optionally includes one or more buffer solutions.

[0012] According to a further aspect of the invention there is provided the use of a kit as defined herein for identifying a subject susceptible to sepsis or septic shock or for monitoring a subject with sepsis or septic shock.

[0013] According to a further aspect of the invention there is provided the use of a kit as defined herein for identifying a subject having cancer, in particular a haematological cancer, or for monitoring a subject having cancer, in particular a haematological cancer. [Brief description of the drawings]

[0014] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] HIA nucleosome standard curve, generated using the method described in Example 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] (Detailed Description) There is a need for an effective and rapid test to diagnose and monitor acute conditions, such as sepsis. Current methods in the art generally use sandwich immunoassays that are highly analytically specific (they can detect a single analyte in a mixture of thousands) and extremely analytically sensitive (they can detect and quantify proteins present in minute amounts) to measure blood-based biomarkers. However, only manual immunoassays that require several hours to perform are commercially available. The industry leader is Roche, offering the Roche Cell Death ELISA, which takes up to two working days to complete, and the Roche Cell Death ELISAPLUS, which also requires several hours. Furthermore, the assays must be performed in batches, with one assay kit being sufficient for approximately 40 samples. Thus, samples are not analyzed in real time, but are collected, frozen, and stored until such time that sufficient samples for an analytical batch are accumulated. Manual immunoassays are expensive, cannot be performed quickly in real time, and cannot be performed near the patient.

[0016] Most immunoassays are heterogeneous and require a separation step. For example, separation may involve separating antigen-bound antibody from free antibody, or separating antibody-bound antigen from free antigen. These assays are typically highly sensitive, but as noted above, are time consuming.

[0017] Since the levels in normal humans and animals are low, analytical sensitivity is considered to be important for the assay used to measure circulating chromatin fragments.In general, the levels in most normal humans are low, with a median value of approximately 30 ng / ml, and some subjects have levels below 10 ng / ml.Therefore, it was considered that simple assays such as homogeneous immunoassays (HIA) cannot accurately and reproducibly measure clinical chromatin fragment levels in healthy subjects or subjects with mildly elevated nucleosome levels.

[0018] (Systemic Inflammatory Response Syndrome, Sepsis and Multiple Organ Failure) Recent studies have shown that NET and chromatin fragment levels are found to be significantly elevated in patients with acute conditions such as systemic inflammatory response syndrome (SIRS) or sepsis. Results indicate that an assay sensitivity of 250 ng / ml for nucleosomes (from NETs) is sufficient to effectively monitor patient response to treatment (Stanford S., 07 / 17 / 21; 326363; PB0152 ISTH Academy). Similarly, an assay sensitivity of 1 μg / ml is sufficient to effectively identify patients who require organ support or are likely to exhibit the need for organ support (or have or may develop multiple organ failure) (Rea C., 07 / 17 / 21; 326469; PB0268 ISTH Academy). These levels are within the measurable range of the HIA method. We propose to provide a low-cost, rapid, near-patient method that can be used to make clinical decisions in real time using HIA technology.

[0019] Thus, according to a first aspect of the present invention there is provided a method for detecting the concentration of extracellular traps (ETs), in particular neutrophil extracellular traps (NETs), chromatin fragments and / or nucleosomes in a body fluid sample, said method comprising analysing said body fluid sample using a homogeneous immunoassay method and using the results obtained from the analysis to determine the concentration of ETs, chromatin fragments and / or nucleosomes in said body fluid sample.

[0020] Described herein is an immunoassay method in which a solution of extracellular chromatin fragments and detection antibodies in a biological sample is subjected to an agglutination reaction and the resulting optical properties of the agglutination mixture (e.g. its absorbance or its scattered light upon irradiation) are analyzed to determine the content of chromatin fragments in the biological sample. In particular, the immunoassay is a homogeneous immunoassay in which the resulting optical properties are measured directly from the agglutination / precipitation mixture without the need to separate unbound reagents. Thus, the claimed HIA method does not involve separation of (i) antibody-bound NETs or chromatin fragments (i.e., analyte) or (ii) unbound antibodies from the examined body fluid sample.

[0021] References herein to "homogeneous immunoassay" or "HIA" refer to immunoassays that detect analytes without the need for a step to separate bound and unbound reagents. Thus, immunoassays are simple methods that can be performed in one reaction mixture. That is, the method can be performed using a single antibody incubation step. This simplicity allows for rapid assays that require only a few minutes to complete and easy automation. The instrumentation used can be located in a central laboratory or at a location near the patient. Heterogeneous immunoassays will not be discussed in detail, but require the use of a separation step to separate bound from unbound reagents for measurement.

[0022] In one embodiment, the HIA method comprises: (a) mixing the body fluid sample with a solution of one or more antibodies capable of specifically binding to ETs, chromatin fragments and / or nucleosomes and causing aggregation or precipitation upon binding; and (b) measuring the degree of aggregation or precipitation in the mixture; The measurement in step (b) is used to determine the concentration of ETs, chromatin fragments and / or nucleosomes in the body fluid sample.

[0023] One example of an HIA is immunoturbidimetry, which is a homogeneous, label-free immunoassay that measures changes in optical transmittance of a reaction mixture at a specific wavelength. Another assay, immunonephelometry, measures changes in light scattering rather than transmittance. Both methods use specific binding of antibodies to the analyte, where binding results in an agglutination reaction that causes an increase in scattering of incident light in the sample that can be measured as an increase in scattered light or a decrease in transmitted light. The change in light scattering is then compared to that produced by a standard / control sample with known analyte concentration.

[0024] For example, when a patient specimen containing a test substance is combined with a solution of a detection antibody, a complex of the test substance and the antibody is formed. When light is directed through this suspension, a portion of the light is transmitted and focused onto an optical device (e.g., a photodiode via an optical lens system). The amount of transmitted light observed by the optical device is inversely proportional to the concentration of the test substance in the patient specimen. Thus, specimens containing high concentrations of the test substance transmit less light than specimens containing low concentrations of the test substance.

[0025] A conventional HIA reagent consists of a reaction buffer and a solution of antibodies. Under optimal conditions, the antibodies form a three-dimensional lattice with the analyte in the sample being analyzed. An alternative method uses particle enhancement of the agglutination reaction. This usually involves adsorbing the antibodies to the surface of beads (e.g. latex beads, nanoparticles or colloidal gold) to obtain a stronger signal. Thus, in one embodiment, one or more antibodies are present in a suspension of antibody-coated particles. One or more antibodies can be conjugated to the surface of particles, such as latex beads or nanoparticles. The antibodies can be directly linked to the beads or particles, or indirectly linked through the use of affinity linkers or tags, such as biotin, avidin, streptavidin or His (e.g. hexa-His) tags.

[0026] The methods of the invention generally use optical properties to measure the extent of aggregation or precipitation. Thus, in one embodiment, the extent of aggregation or precipitation is measured by the absorbance, transmittance, reflectance, light scattering, fluorescence or scintillation of the mixture.

[0027] The antibody suitable for use in the immunoassay method is known in the art. Such an antibody can specifically bind to the test substance, i.e., NET, chromatin fragment and / or nucleosome. In one embodiment, one or more antibodies are monoclonal antibodies. In another embodiment, one or more antibodies are polyclonal antibodies.

[0028] It will be apparent to one of skill in the art that the terms "antibody," "binding agent," or "ligand" as used herein are intended to include, but are not limited to, any binding agent capable of binding to a particular molecule or entity, and that any suitable binding agent may be used in the methods of the present invention.

[0029] The reagent may comprise one or more ligands or binders, e.g., natural or chemically synthesized compounds, capable of specific binding to a desired target. The ligands or binders may comprise peptides, antibodies, or fragments thereof, or synthetic ligands, such as plastic antibodies, or aptamers, or oligonucleotides, capable of specific binding to a desired target. The antibodies may be monoclonal, polyclonal, or fragments thereof. It will be appreciated that when an antibody fragment is used, it retains the ability to bind to the biomarker, such that the biomarker can be detected (in accordance with the present invention).

[0030] In one embodiment, the method described herein is used to measure the concentration of extracellular traps. In particular, ETs can be neutrophil extracellular traps (NETs). NETs have been detected in many body fluid samples (see, for example, Saffarzadeh et al., (2012) PLoS ONE 7(2): e32366), and any body fluid containing NETs can be tested for NETs or chromatin fragments using the method described herein. The method and use described herein can be tested on body fluid samples, particularly blood, serum, or plasma samples. Preferably, plasma samples are used. Plasma samples can be collected in collection tubes containing one or more anticoagulants, such as ethylenediaminetetraacetic acid (EDTA), heparin, or sodium citrate, particularly EDTA.

[0031] In one embodiment, the method described herein is used to measure the concentration of chromatin fragments.In one embodiment, the chromatin fragments measured by the method of the present invention comprise one or more nucleosomes.In one embodiment, the chromatin fragments and / or nucleosomes measured by the method of the present invention comprise the epigenetic features of extracellular free nucleosomes.Therefore, one or more antibodies may be capable of specifically binding to the epigenetic features of extracellular free nucleosomes.

[0032] The nucleosome is the basic unit of chromatin structure and consists of a protein complex of eight highly conserved core histones, including pairs of each of histones H2A, H2B, H3, and H4. Approximately 146 base pairs of DNA are wrapped around this complex. Additional histones, H1 or H5, act as linkers and are involved in chromatin compaction. DNA wraps around successive nucleosomes in a structure often described as "beads on a string," which forms the basic structure of open, or euchromatin. In condensed chromatin or heterochromatin, the string coils and supercoils into a closed complex structure (Herranz and Esteller, Methods Mol. Biol. (2007) 361:25-62).

[0033] Reference to "nucleosomes" may refer to "extracellular free nucleosomes" when detected in a body fluid sample. It will be understood that throughout this document, the term extracellular free nucleosomes is intended to include any extracellular free chromatin fragment that contains one or more nucleosomes. Extracellular free nucleosomes may be mononucleosomes, oligonucleosomes, parts of larger chromatin fragments or parts of NETs, ​​or mixtures thereof.

[0034] It will be understood that the extracellular free nucleosome can be detected by binding to its component. The term "its component" as used herein refers to a part of the nucleosome, i.e. the entire nucleosome does not need to be detected. The component of the extracellular free nucleosome can be selected from the group consisting of: histone protein (i.e. histone H1, H2A, H2B, H3, or H4), histone post-translational modification, histone variant or isoform, protein bound to the nucleosome (i.e. nucleosome-protein adduct), DNA fragment associated with the nucleosome, and / or modified nucleotide associated with the nucleosome. For example, the component can be histone (isoform) H3.1 or histone H1 or DNA.

[0035] The method and use of the present invention may measure the level of (extracellular free) nucleosomes themselves. Thus, in one embodiment, the method described herein is used to measure the concentration of nucleosomes, i.e., nucleosomes themselves. Reference to "nucleosomes themselves" refers to the total level or concentration of nucleosomes present in a sample, whether or not the nucleosomes contain any epigenetic features. Detecting the total level of nucleosomes typically involves detecting histone proteins common to all nucleosomes, such as histone H4. Thus, nucleosomes themselves can be measured by detecting core histone proteins, such as histone H4. As described herein, histone proteins form structural units known as nucleosomes, which are used to compactly pack DNA in eukaryotic cells. Nucleosomes also contain DNA, and thus nucleosomes and ETs can be measured by the method of the present invention using binding agents to DNA, such as anti-DNA antibodies, alone or in combination with binding agents to other epitopes found on ETs.

[0036] Circulating nucleosomes are not a homogeneous group of protein-nucleic acid complexes. Rather, they are a heterogeneous group of chromatin fragments resulting from digestion of chromatin during cell death, and contain a wide variety of epigenetic structures, including specific histone isoforms (or variants), post-translational histone modifications, nucleotides or modified nucleotides, and protein adducts. It will be clear to those skilled in the art that the increase in nucleosome levels is associated with the increase in a subset of circulating nucleosomes that contain specific epigenetic signals, including nucleosomes that contain specific histone isoforms (or variants), nucleosomes that contain specific post-translational histone modifications, nucleosomes that contain specific nucleotides or modified nucleotides, and nucleosomes that contain specific protein adducts.

[0037] The method of the present invention can detect the level of extracellular free nucleosomes themselves and / or the epigenetic features of extracellular free nucleosomes. It is understood that the terms "epigenetic signal structure" and "epigenetic feature" are used interchangeably herein. These terms refer to a specific detectable feature of nucleosomes. In one embodiment, the epigenetic feature of nucleosomes is selected from the group consisting of: post-translational histone modifications, histone isoforms, modified nucleotides, and / or proteins that bind to nucleosomes in nucleosome-protein adducts.

[0038] In one embodiment, the epigenetic feature of nucleosome comprises one or more histone variants or isoforms. The epigenetic feature of extracellular free nucleosome can be histone isoform, for example, core nucleosomal histone isoform, particularly histone H3 isoform. The terms "histone variant" and "histone isoform" can be used interchangeably herein. Nucleosome structure can also vary by including alternative histone isoforms or variants that are different genes or splicing products and have different amino acid sequences. Many histone isoforms are known in the art. Histone variants can be classified into several families that are subdivided into individual types. Numerous histone variant nucleotide sequences are known and publicly available, for example, at the National Human Genome Research Institute NHGRI Histone Database (Marino-Ramirez et al., The Histone Database: an integrated resource for histones and histone fold-containing proteins. Database Vol. 2011. and http: / / genome.nhgri.nih.gov / histones / complete.shtml), the GenBank (NIH gene sequence) database, the EMBL Nucleotide Sequence Database, and the DNA Data Bank of Japan (DDBJ). For example, variants of histone H2 include H2A1, H2A2, mH2A1, mH2A2, H2AX, and H2AZ. In another example, histone isoforms of H3 include H3.1, H3.2, H3.3, and H3t. In one embodiment, the histone isoform is H3.1.

[0039] The structure of nucleosomes can vary depending on the post-translational modification (PTM) of histone proteins. PTM of histone proteins typically occurs at the tail of core histones, and common modifications include acetylation, methylation, or ubiquitination of lysine residues, as well as methylation or citrullination of arginine residues and phosphorylation of serine residues, among many others. Many histone modifications are known in the art, and the number is increasing as new modifications are identified (Zhao and Garcia, 2015 Cold Spring Harb Perspect Biol, 7: a025064). Thus, in one embodiment, the epigenetic feature of the extracellular free nucleosome can be a histone post-translational modification (PTM). The histone PTM can be the histone PTM of core nucleosomes, such as H3, H2A, H2B, or H4, particularly H3, H2A, or H2B. In particular, the histone PTM is histone H3 PTM. Examples of such PTMs are described in WO 2005 / 019826.

[0040] For example, post-translational modifications can include methylation, which can be acetylation, mono-, di-, or tri-methylation, phosphorylation, ribosylation, citrullination, ubiquitination, hydroxylation, glycosylation, nitrosylation, glutamation, and / or isomerization (see Ausio (2001) Biochem Cell Bio 79:693). In one embodiment, the histone PTM is selected from citrullination or ribosylation. In a further embodiment, the histone PTM is H3 citrulline (H3cit) or H4 citrulline (H4cit). In yet a further embodiment, the histone PTM is H3cit.

[0041] Groups or classes of related histone post-translational modifications (rather than a single modification) may also be detected. Examples of antibodies directed against binding to groups of such histone modifications include, by way of illustration and not limitation, anti-pan-acetylation antibodies (e.g., pan-acetyl H4 antibody [H4panAc]), anti-citrullination antibodies, or anti-ubiquitin antibodies. Antibodies against histone N-terminal domains, such as the N-terminal domains of H2A or H4, may be useful in the present invention, since they provide a multi-dentate dimension to the nucleosome assay.

[0042] In one embodiment, the epigenetic features of nucleosomes include one or more DNA modifications. In addition to epigenetic signaling mediated by the composition of nucleosomal histone isoforms and PTMs, nucleosomes also differ in their nucleotide and modified nucleotide composition. Some nucleosomes may contain more 5-methylcytosine residues (or 5-hydroxymethylcytosine residues or other nucleotides or modified nucleotides) than other nucleosomes. In one embodiment, the DNA modifications are selected from 5-methylcytosine or 5-hydroxymethylcytosine.

[0043] In one embodiment, the epigenetic features of nucleosomes include one or more protein-nucleosome adducts or complexes. A further subset of circulating nucleosomes is nucleosome protein adducts. It has been known for many years that chromatin contains many non-histone proteins bound to its component DNA and / or histones. These chromatin-associated proteins are highly diverse and have a variety of functions, including transcription regulators, transcription enhancers, transcription repressors, histone modifying enzymes, DNA damage repair proteins, and many others. These chromatin fragments, including nucleosomes and other non-histone chromatin proteins or DNA and other non-histone chromatin proteins, have been described in the art.

[0044] In one embodiment, the protein that is added to the nucleosome is selected from: a transcription regulator, a high mobility group protein, or a chromatin modifying enzyme. Reference to a "transcription regulator" refers to a protein that regulates gene expression by binding to DNA and promoting (i.e., an activator) or suppressing (i.e., a repressor) transcription. A transcription regulator contains one or more DNA binding domains (DBDs) that bind to specific sequences of DNA adjacent to the gene that it regulates. All of the circulating nucleosomes and nucleosome parts, types, or subgroups described herein may be useful in the present invention.

[0045] The chromatin exuded in NETs can contain, be attached to, or be decorated with a number of proteins, including, but not limited to, myeloperoxidase (MPO), neutrophil elastase (NE), lactotransferrin, azurocidin, cathepsin G, leukocyte proteinase 3, lysozyme C, neutrophil defensin 1, neutrophil defensin 3, myeloid cell nuclear differentiation antigen, S100 calcium binding protein A8, S100 calcium binding protein A9, S100 calcium binding protein A12, actin beta, actin gamma, alpha-actin, plastin-2, cytokeratin-10, catalase, alpha-enolase, and transketolase (Urban et al., PLOS Pathogens. (2009) 10: e1000639). Any nucleosome-protein adducts that occur in NETs are useful adducts for detecting elevated levels of NETs in the methods of the present invention.

[0046] In one embodiment, the one or more antibodies are capable of specifically binding to a neutrophil extracellular trap (NET)-associated protein. In a further embodiment, the NET-associated protein is selected from myeloperoxidase (MPO) and neutrophil elastase (NE).

[0047] Polyclonal antibodies consist of multiple antibodies (produced by multiple clones) directed to bind to different epitopes on the same target. The presence of multiple antibodies directed to bind to multiple epitopes in a NET is useful to maximize the degree of aggregation achieved. Thus, in one embodiment of the present invention, one or more polyclonal antibodies capable of binding to multiple available epitopes on ETs or nucleosomes are used as binding agents.

[0048] Similarly, the use of multiple monoclonal antibodies directed to multiple epitopes within NETs (e.g., including different histone proteins, DNA, different histone modifications, or other epigenetic features) can increase the degree of aggregation achieved. Thus, in another embodiment of the present invention, multiple (i.e., two or more) monoclonal antibodies are used as binding agents. In this embodiment, the antibodies bind to different epitopes. In one embodiment, the HIA method comprises using two or more binding agents that bind to different epitopes and cause aggregation or precipitation upon binding. In a further embodiment, the HIA method comprises: (a) mixing a body fluid sample with a solution of two or more binding agents (e.g., antibodies), the two or more binding agents being capable of specifically binding to different epitopes of ETs, chromatin fragments, and / or nucleosomes, and causing aggregation or precipitation upon binding; and (b) measuring the degree of aggregation or precipitation in the mixture, and the measurement in step (b) is used to determine the concentration of ETs, chromatin fragments, and / or nucleosomes in the body fluid sample.

[0049] Thus, multiple binding agents directed to different epitopes in NETs can be used. In one embodiment, multiple binding agents (e.g., two or more binding agents, e.g., two or more antibodies) can specifically bind to different features of extracellular free nucleosomes. In further embodiments, the different features can include one or more epigenetic features of extracellular free nucleosomes (e.g., post-translational histone modifications, histone isoforms, modified nucleotides, and / or proteins that bind to nucleosomes in nucleosome-protein adducts). For example, one of the binding agents can be directed to a histone protein epitope (e.g., histone H3 isoform H3.1), and the other binding agent is directed to a different feature (e.g., a nucleosome conformational epitope that is only present in intact nucleosomes that contain histones and DNA).

[0050] (Diagnostic and monitoring methods) According to a further aspect of the invention there is provided a method of monitoring disease progression in a subject comprising: (i) detecting the concentration of ETs (e.g., NETs), chromatin fragments and / or nucleosomes in a bodily fluid sample obtained from a subject using the methods defined herein; (ii) repeating step (i) on one or more occasions; and (iii) using any changes in ET, chromatin fragments and / or nucleosome concentrations to monitor disease progression in the subject.

[0051] As described herein, the method of the present invention can be used in situations that require rapid results to allow clinical decision making in real time.Thus, in one embodiment, the subject is in a critical care unit (also called an intensive care unit).

[0052] The methods described herein also find particular use with patients suffering from systemic inflammatory response syndrome (SIRS). SIRS is the body's inappropriate response to an injury with an inappropriately high level of nephrosis. The injury can be infectious or non-infectious, including trauma, surgery, acute inflammation, ischemia, reperfusion, malignancy, and many others. In one embodiment, the subject suffers from (or is suspected of suffering from) SIRS. In one embodiment, the subject suffers from (or is suspected of suffering from) an infection. In one embodiment, the subject suffers from (or is suspected of suffering from) sepsis or septic shock.

[0053] According to a further aspect of the invention there is provided a method for assigning a risk of an adverse outcome to a subject suffering from an infectious disease comprising: (i) detecting the concentration of ETs (e.g., NETs), chromatin fragments and / or nucleosomes in a body fluid sample obtained from a subject using the methods defined herein; and (ii) using the detected concentrations of ETs, chromatin fragments and / or nucleosomes to assign a likelihood of an adverse outcome to the subject; The method further provides for assigning subjects identified as having a high likelihood of an adverse outcome to a medical intervention.

[0054] The method of the present invention may be directed to assigning patients at risk of adverse outcomes. Adverse outcomes include death and / or acute events requiring urgent medical care, such as hospitalization (i.e., inpatient care) and / or surgery. For many patients, the infection is overcome by the person's own immune system without the need for medical intervention. However, in many patients, the infection may progress or increase in severity without being overcome by the immune system, or the patient's own immune response to the infection may lead to an adverse outcome. Similarly, patients with SIRS may be overwhelmed by the condition, potentially suffering from multiple organ failure. For example, adverse outcomes may include the development of acute coronary or cardiac events (e.g., myocardial infarction and / or stroke), acute multi-organ or single organ failure (e.g., renal failure, liver failure, and / or heart failure), debilitating acute conditions, and / or acute respiratory conditions (e.g., pneumonia, hypoventilation / bradypnea, acute respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), bronchiolitis and / or bronchitis). Thus, in one embodiment, the method described herein assigns patients or subjects at risk of developing acute respiratory conditions. In a further embodiment, the acute respiratory condition is pneumonia. In a further embodiment, the acute respiratory condition is hypoventilation / bradypnea. In yet a further embodiment, the acute respiratory condition is acute respiratory distress syndrome (ARDS) and / or severe acute respiratory syndrome (SARS). The condition of patients with sepsis is often expressed in terms of organ failure assessment score (SOFA score). SOFA is used as a measure of organ function or organ failure to track the condition of patients in critical care.

[0055] Assigning a patient at risk for an adverse outcome can assign an immediate or short-term risk, or can assign an intermediate risk. Immediate or short-term risk includes when a patient may develop an adverse outcome within 30 days, e.g., within 2 weeks or 14 days, within 1 week or 7 days, or within 5 days or 1 day or hours, of onset of symptoms or a positive diagnosis. Such immediate or short-term risk can also include when a patient may develop an adverse outcome within 30 days, e.g., within 2 weeks or 14 days, within 1 week or 7 days, or within 5 days or 1 day or hours, of implementation of the methods described herein.

[0056] Examples of acute short-term risks include the development of NET-associated complications for COVID infection or sepsis requiring hospitalization. The condition of patients with COVID or sepsis can rapidly deteriorate within hours, leading to death within 12 hours in severe acute cases. This is why patients with COVID, sepsis, SIRS, ARDS, SARS and similar conditions require the monitoring method of the present invention to be performed quickly and close to the patient.

[0057] Intermediate-term risks include cases where a patient may develop an adverse outcome more than 30 days after the onset of symptoms, a positive diagnosis, and / or the implementation of the methods described herein. An example of an intermediate-term risk is the development of so-called Long COVID, where the effects of COVID infection may persist for many months.

[0058] Thus, in one embodiment, the method described herein assigns the patient or subject with risk of developing an adverse outcome within 2 weeks or 14 days from the onset of symptoms or positive diagnosis.In a further embodiment, the method described herein assigns the risk of developing an adverse outcome within 1 week or 7 days from the onset of symptoms or positive diagnosis.In yet a further embodiment, the method described herein assigns the risk of developing an adverse outcome within 1 day from the onset of symptoms or positive diagnosis.

[0059] Even if a subject is determined not to currently have at-risk or inappropriately high levels of NETs, ​​the invention can be used to monitor disease progression for future development of medical complications. For example, if the method involves a sample from a subject determined to have a mild infection, measurements of the levels of NETs or chromatin fragments can be repeated at other time points to establish whether the levels have changed.

[0060] The term "biomarker" refers to a distinct biological or biologically derived indicator of a process, event, or condition. Biomarkers can be used in methods of diagnosis, such as clinical screening and prognostic evaluation, as well as in monitoring treatment outcomes, identifying patients most likely to respond to a particular therapeutic treatment, drug screening, and drug discovery. Biomarkers and their uses are beneficial in identifying new drug therapies and discovering new targets for drug therapy.

[0061] Detection and / or quantification can be performed directly on the purified or enriched nucleosome sample, or indirectly on an extract from the sample or a dilution thereof. Quantifying the amount of biomarker present in a sample can include determining the concentration of the biomarker present in the sample. The detection, monitoring and diagnostic uses and methods according to the present invention described herein are useful for confirming the presence of disease, monitoring the development of disease by assessing onset and progression, or assessing improvement or reversal of disease. The detection, monitoring and diagnostic uses and methods are also useful for clinical screening evaluation, prognosis, treatment selection, evaluation of treatment benefit, i.e., drug screening and drug discovery methods.

[0062] In one embodiment, the disease is a condition associated with high levels of NETs or pathological clinical complications of NETosis.

[0063] The terms "detecting" or "diagnosing" as used herein encompass the identification, confirmation, and / or characterization of a disease state. The detection, monitoring, and diagnostic methods according to the invention are useful for confirming the presence of a disease, monitoring the development of a disease by assessing onset and progression, or assessing the amelioration or reversal of a disease. The detection, monitoring, and diagnostic methods are also useful for clinical screening evaluation, prognosis, treatment selection, evaluation of treatment benefit, i.e., drug screening and drug discovery methods.

[0064] In one embodiment, the method described herein is repeated multiple times.This embodiment provides the advantage of being able to monitor the detection results over a period of time.Such a regimen provides the benefit of monitoring or evaluating the effectiveness of treating disease conditions.Such monitoring methods of the present invention can be used to monitor onset, progression, stabilization, improvement, recurrence, and / or remission.

[0065] In the monitoring method, the test sample may be taken at two or more times. The method may further include comparing the level of the biomarker(s) present in the test sample with one or more control(s) and / or one or more previous test sample(s) taken previously, e.g., from the same test subject before treatment begins and / or from the same test subject at an earlier stage of treatment. The method may include detecting changes in the nature or amount of the biomarker(s) in the test samples taken at the different times.

[0066] A change in the level of the biomarker in the test sample compared to the level in a previous test sample taken earlier from the same test subject can indicate a beneficial effect of the treatment on the disorder or suspected disorder, e.g., stabilization or amelioration. Furthermore, once treatment is completed, the methods of the invention can be repeated periodically to monitor for recurrence of the disease.

[0067] Therapeutic effectiveness monitoring methods can be used to monitor the therapeutic effectiveness of existing and new therapies in human subjects and non-human animals (e.g., animal models). These monitoring methods can be incorporated into screening of new drug substances and substance combinations.

[0068] In further embodiments, monitoring for more rapid changes due to fast acting treatments can be performed over shorter intervals of hours or days.

[0069] Diagnostic or monitoring kits (or panels) are provided for carrying out the methods of the invention. Such kits suitably contain one or more ligands for detection and / or quantification of the biomarkers, optionally together with instructions for use of the kit.

[0070] Methods involving detection and / or quantification of one or more biomarkers of the invention can be performed on benchtop equipment or can be integrated into disposable diagnostic or monitoring platforms that can be used in non-laboratory settings, such as in a physician's office or at a subject's bedside. Thus, in further aspects of the invention, the methods can be performed near the patient or as point-of-care immunoassays.

[0071] The method of the present invention allows the integration of diagnostic procedures with treatment regimens. The method provides a means of indicating treatment response, failure to respond, undesirable side effect profile, degree of compliance, and achievement of proper serum drug levels. The method can be used to provide warnings of adverse drug reactions. The method is useful in developing personalized treatments, since response assessment can be used to fine-tune dosage, minimize the number of prescribed doses, reduce delays in achieving effective treatment, and avoid adverse drug reactions. Thus, by monitoring a subject, care can be precisely tailored to meet the needs determined by the subject's disorder and pharmacological profile, and can therefore be used to adjust optimal dosage, predict positive treatment response, and identify subjects at high risk of severe side effects.

[0072] Also, methods, biosensors, point-of-care tests and kits for monitoring biomarkers are important as targeted monitoring tools to allow physicians to determine whether relapse is due to a worsening of the disorder. If pharmacological treatment is assessed as insufficient, treatment can be resumed or increased; treatment can also be appropriately altered. Because biomarkers are sensitive to the state of the disorder, they provide an indication of the impact of drug treatment.

[0073] References to "subject" or "patient" are used interchangeably herein. The subject may be a human or animal subject. In one embodiment, the subject is a human. In one embodiment, the subject is a (non-human) animal. In a further embodiment, the animal is a companion animal (also called a pet or livestock). Companion animals include, for example, dogs, cats, rabbits, ferrets, horses or cows. In particular, the companion animal is a dog or cat, in particular a dog. The panels and methods described herein may be performed in vitro or ex vivo.

[0074] The detection and / or quantification results can be compared with a cut-off level. The cut-off value can be pre-determined by analyzing the results from multiple patients and controls and determining a suitable value for classifying subjects as having or not having a disease. For example, for diseases in which the level of the biomarker is higher in patients with the disease, if the detected level is higher than the cut-off, it indicates that the patient has the disease. Alternatively, for diseases in which the level of the biomarker is lower in patients with the disease, if the detected level is lower than the cut-off, it indicates that the patient has the disease. The advantage of using a simple cut-off value is that the test is easily understood by clinicians and the need for software or other aids to interpret the test results is eliminated. The cut-off level can be determined using methods in the art.

[0075] The detection and / or quantification results can also be compared to a control. It will be clear to those skilled in the art that the control subject can be selected by various criteria, which can include, for example, subjects known to be disease-free or can be subjects with different diseases (e.g., for differential diagnosis studies). A "control" can include a healthy subject or a non-diseased subject, for example, a subject without an infectious disease. A control can also be a subject with an infectious disease that shows no symptoms or mild symptoms, for example, a subject infected with a respiratory virus that shows no symptoms or mild symptoms. Mild symptoms can include manageable symptoms that do not require hospital intervention and / or intensive medical treatment.

[0076] In one embodiment, the subject who tests positive by the method of the present invention may be infected with viral disease and further suffer from or subsequently suffer from additional medical complications.In contrast, the control subject may also be infected with viral disease, but does not suffer from and subsequently does not suffer from medical complications.Comparison with a control is well known in the field of diagnosis.The range of values ​​found in the control group can be used as the normal or healthy or reference range to which the value found for the test subject can be compared.For example, if the reference range is less than 10 units, a test value of 5 units is considered to be normal or not requiring treatment, while a value of 11 units is considered to be abnormal and indicating the need for treatment.

[0077] Thus, in one embodiment, the method further comprises comparing the concentration of ETs (particularly NETs), chromatin fragments and / or nucleosomes in the subject's body fluid sample with one or more controls.The control can be a healthy subject.In one embodiment, the concentration of ETs, chromatin fragments and / or nucleosomes is increased compared to the control.

[0078] It will be understood that it is not necessary in all cases to measure a control level for comparison purposes. For example, once a "normal range" is established for healthy / non-diseased controls, it can be used as the basis for all subsequent testing. A normal range can be established by obtaining samples from multiple control subjects who do not have an infection and testing the levels of the biomarkers. The results (i.e., biomarker levels) of subjects suspected of having an infection can then be examined to see if they are within or outside the respective normal ranges. The use of "normal ranges" is a standard practice for detecting disease.

[0079] In one embodiment, the method further comprises determining at least one clinical parameter of the patient. This parameter can be used to interpret the results. The clinical parameter can include any relevant clinical information, such as but not limited to SOFA score, body temperature, sex, weight, body mass index (BMI), smoking status, and dietary habits. Thus, in one embodiment, the clinical parameter is selected from the group consisting of: SOFA score, body temperature, age, sex, and body mass index (BMI).

[0080] In one embodiment, the methods of the invention are performed to identify subjects who are at high risk of developing a severe response to an infection and therefore in need of medical intervention, which may include one or more of the treatments described herein.

[0081] According to another aspect of the invention, there is provided the use of the binding agent in the manufacture of a kit for use in a homogeneous immunoassay (HIA) method (e.g., as described herein) for detecting the concentration of ETs (e.g., NETs), chromatin fragments and / or nucleosomes in a body fluid sample.

[0082] (infectious disease) The method of the present invention finds particular use in managing infectious disease outbreaks. Infectious diseases can be caused by a variety of pathogens and environmental factors. In one embodiment, the infectious disease is a viral, bacterial, fungal or microbial infection. Bacterial infections can include mycobacterial, pneumococcal and influenzae infections, such as infections (e.g., pneumonia) caused by Streptococcus pneumoniae, Escherichia coli, Mycobacterium tuberculosis, Haemophilus influenzae and Staphylococcus aureus. In a further embodiment, the infectious disease is a viral infection. Viral infections can include infections caused by respiratory syncytial virus (RSV), influenza A, influenza B and coronaviruses (e.g., COVID-19).

[0083] Infectious disease can be defined by the tissue affected by the disease.For example, the disease may affect the heart, brain, kidney, liver, pancreas, lungs and / or blood, and the infectious disease can be a bacterial, viral, fungal or microbial infection commonly known to affect such tissues or organs.In one embodiment, the infectious disease is a respiratory tract infection.According to this embodiment, the infectious disease affects the lungs, upper respiratory tract and / or lower respiratory tract.

[0084] Other tissues that may be affected by the disease include peripheral tissues, such as limbs, hands and feet, and the infection may be a bacterial infection (e.g., gangrene). In one embodiment, the infection and / or disease may affect multiple tissues or organs simultaneously. For example, the infection may be a bacterial infection of the limbs, hands or feet, and the disease may also affect the blood (e.g., sepsis). In one embodiment, the infection is sepsis. In another example, the disease may be heart failure or coronary insufficiency, and other tissues or organs that may be affected by the disease may include the kidney and renal system and / or the brain (e.g., stroke).

[0085] In a further embodiment, the concentration of chromatin fragments in the sample taken from the subject suffering from sepsis or septic shock is measured, in particular to assess the prognosis of the disease.Further measurements can be carried out on multiple samples taken from the subject suffering from sepsis or septic shock at intervals to monitor the progression of the disease and / or evaluate the effectiveness of treatment.

[0086] In yet further examples, the disease may affect the lungs, or the infection may be a respiratory tract infection, and other affected tissues or organs may include the heart, the coronary system, and / or the brain (e.g., heart failure, myocardial infarction, and / or stroke). In one embodiment, the respiratory tract infection is selected from: influenza, pneumonia, and severe acute respiratory syndrome (SARS). SARS is a respiratory infection caused by SARS coronavirus (SARS-CoV), and other related coronaviruses are known (e.g., COVID-19 (also known as SARS-CoV-2, formerly known as 2019-nCoV)). It is known to cause fever, flu-like symptoms, cough, and fatigue, and can lead to pneumonia (e.g., direct viral pneumonia or secondary bacterial pneumonia).

[0087] NET or NETosis-associated diseases include infectious diseases such as SIRS, sepsis, pneumonia, COVID and influenza, and other diseases associated with pathological increase in the production of NETs, ​​including, but not limited to, pneumonia of any cause, SARS or ARDS, thrombotic or microthrombotic conditions, many inflammatory disease states, and NETosis-associated complications of other diseases, including amputation and thrombotic complications of diabetes and thrombotic complications of cancer. The method of the present invention also has applicability for ex vivo assessment of viability of organs or tissues removed from donor patients for transplantation into recipient patients and monitoring of recipient patients for rejection of transplanted organs or tissues in many other NETosis-associated diseases or conditions.

[0088] Methods of Treating Subjects with Pathological Levels of NETs According to a further aspect, there is provided a method of treating a disease (particularly an infectious disease) in a subject in need thereof, comprising administering a treatment (e.g., a therapeutic agent) to a subject identified as having a different concentration of ETs (e.g., NETs), chromatin fragments and / or nucleosomes in a sample obtained from the subject compared to the concentration of ETs, chromatin fragments and / or nucleosomes in a sample obtained from a control subject. The treatment can include one or more suitable treatments for the condition, including, but not limited to, a drug (e.g., an anti-inflammatory drug, an anticoagulant or blood clotting inhibitor, a therapeutic anti-NET antibody drug, a DNase drug, a NETosis inhibitor, an antibacterial drug, or an antiviral drug), apheresis treatment, ventilator support, fluid support, or others.

[0089] In one embodiment, the treatment includes: antibiotic treatment (e.g., penicillin, cephalosporin, tetracycline, aminoglycoside, macrolide, clindamycin, sulfonamide, trimethoprim, metronidazole, tinidazole, quinolone, and / or nitrofurantoin), antimicrobial treatment (e.g., ethambutol, isoniazid, pyrazinamide, rifampicin, aminoglycosides (amikacin, kanamycin), polypeptides (capreomycin, viomycin, enviomycin), fluoroquinolones (ciprofloxacin, levofloxacin, moxifloxacin), thioamides (ethionamide, prothionamide), cycloserine (clocerin), terizidone, rifabutin, macrolides (clarithromycin), linethambutol, isoniazid, pyrazinamide, rifabutin, cyclosporine ... zolidin, thioacetazone, thioridazine, arginine, vitamin D and / or R207910), antiviral COVID treatments (e.g., remdesivir), antiviral influenza treatments (e.g., amantadine, umifenovir, moroxydine, rimantadine, umifenovir, zanamivir and neuraminidase inhibitors, cap-dependent endonuclease inhibitors, adamantane, peramivir, zanamivir, oseltamivir phosphate, and baloxavir marboxil), and antiviral treatments for other viral diseases that may result in high levels of nephrosis, and antifungal treatments (e.g., clotrimazole, econazole, miconazole, terbinafine, fluconazole, ketoconazole, and amphotericin).

[0090] In one embodiment, the treatment is an anti-inflammatory drug.Many steroidal and non-steroidal anti-inflammatory drugs are known in the art.Some examples of steroidal anti-inflammatory drugs include, but are not limited to, dexamethasone, hydrocortisone, cortisone, betamethasone, prednisone, prednisolone, triamcinolone, and methylprednisolone.Some examples of non-steroidal anti-inflammatory drugs include, but are not limited to, aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, CD24Fc (CD24 protein bound to the Fc region of immunoglobulin G), and EXO-CD24 (CD24-exosome).

[0091] In one embodiment, the treatment is DNase treatment to digest excess NETs, ​​or an inhibitor of NETosis, such as an anthracycline drug. In a further embodiment, the anthracycline drug is selected from: epirubicin, daunorubicin, doxorubicin, and idarubicin.

[0092] In one embodiment, the therapy is a therapeutic antibody drug directed to bind to a NET or a component part of a NET, including, but not limited to, a therapeutic antibody directed to bind to a nucleosome or any component part of a nucleosome. Examples include a therapeutic antibody directed to bind to a nucleosome containing histone isoform H3.1, a citrullinated histone, myeloperoxidase, or neutrophil elastase.

[0093] In one embodiment, the therapy is a nucleic acid scavenger, such as a DNA scavenger polyamidoamine, which adsorbs and / or removes nucleic acids from the circulation or from the body.

[0094] Treatments involving removal of NETs and NET degradation products from the circulation by plasmapheresis have been reported that provide a NET treatment that avoids pharmaceutical toxic side effects (see WO2019 / 053243). Thus, in one embodiment, the treatment comprises plasmapheresis treatment.

[0095] In one embodiment, the infectious disease is a respiratory infection, such as influenza or coronavirus, and the medical complication is pneumonia.Suitable treatments may include, but are not limited to, respiratory support using extracorporeal oxygen supply, respiratory support using a medical ventilator designed to provide artificial breathing of air into and out of the lungs of patients who are physically unable to breathe adequately without assistance, and / or the provision of oxygen and / or antiviral, antibacterial, or anti-inflammatory drugs.

[0096] (blood cancer) Blood cancers are types of cancer that affect the blood, bone marrow, and lymph nodes. Common blood cancers include, but are not limited to, leukemia, lymphoma, myeloma, and hemangiosarcoma, depending on the type of cells affected. Blood cancers are especially common in companion animals, such as dogs or cats. Leukemia is a cancer of blood cells that usually begins in the bone marrow and travels through the bloodstream. In leukemia, the bone marrow produces mutant cells and disseminates them in the blood. The mutant cells grow in the blood and crowd out healthy blood cells. Lymphoma disease affects cells in the lymphatic system. In lymphoma, immune cells called lymphocytes grow uncontrollably and collect in lymph nodes, spleen, other lymphatic tissues, or nearby organs. Myeloma, also known as multiple myeloma, develops in the bone marrow and affects plasma cells that produce antibodies that fight infection and disease. Hemangiosarcoma (and hemangiosarcoma in dogs) develops in the endothelium of the vasculature. Examples of blood cancers include acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL). Common blood cancers in dogs include canine lymphoma and hemangiosarcoma.

[0097] Recent studies have shown that patients with hematological cancers have a significantly elevated level of circulating extracellular free nucleosomes with median levels of 300-700 ng / ml in humans (WO2021110776) and up to 6000 ng / ml in dogs (Dolan et al., BMC Veterinary Research (2021) 17:276 and Wilson-Robles et al., BMC Veterinary Research (2021) 17:231). These results indicate that an assay sensitivity in the range of 100 ng / ml for nucleosomes is sufficient to effectively detect the presence of hematological cancer in patients or to monitor patient response to treatment. These levels are within the measurable range of the HIA method. We propose to provide a low-cost, rapid, near-patient method that can be used to make clinical decisions in real time using HIA technology.

[0098] In addition to being useful in human subjects, the methods of the invention are useful in the diagnosis and treatment of subjects of other species, particularly canine subjects, with blood cancers, in part because blood cancers are common in dogs, and in part because current methods involve scans that require the subject to remain motionless in the scanner during the scan. Non-human subjects such as dogs must be anesthetized because they cannot remain motionless during a scan if they are conscious. Thus, the methods of the invention provide a solution that is low cost, fast, accurate, and avoids the need to use general anesthesia in dogs.

[0099] Thus, according to a further aspect of the present invention there is provided a method of detecting a hematological cancer disease in a subject comprising: (i) detecting the concentration of ETs, chromatin fragments and / or nucleosomes in a body fluid sample obtained from a subject using the methods defined herein; (ii) using the concentration of ET, chromatin fragments and / or nucleosomes as an indicator of the presence of a blood cancer in the subject.

[0100] According to a further aspect of the invention, there is provided a method of detecting or monitoring the progression of a hematological cancer disease in a subject, comprising: (i) detecting the concentration of ETs, chromatin fragments and / or nucleosomes in a body fluid sample obtained from a subject using the methods defined herein; (ii) repeating step (i) on one or more occasions; and (iii) monitoring disease progression in the subject using changes in ET, chromatin fragments and / or nucleosome concentrations.

[0101] In one embodiment, a subject with a hematological cancer is monitored to determine the effectiveness of a treatment provided to the subject. Although the methods of the invention are particularly useful for identifying and monitoring patients with hematological cancers, it will be appreciated that the applicability of the methods is not limited to hematological cancers, but may be applied to any cancer.

[0102] (Methods of Treating a Subject with Cancer) According to a further aspect, there is provided a method of treating cancer, particularly hematological cancer, in a subject in need thereof, comprising administering a treatment (e.g., a therapeutic agent) to a subject identified as having a different concentration of ETs, chromatin fragments and / or nucleosomes in a sample obtained from the subject compared to the concentration of ETs, chromatin fragments and / or nucleosomes in a sample obtained from a control subject. The treatment may include one or more suitable treatments for the condition, including, but not limited to, drugs (e.g., cytotoxic drugs, chemotherapeutic drugs, immunotherapeutic drugs, epigenetic drugs, and many others), radiation therapy treatments, transfusion treatments, hemodialysis treatments, and apheresis treatments. In cases where the hematological cancer is associated with metastasis, the treatment may include surgery to remove secondary tumors.

[0103] (kit) According to a further aspect of the invention there is provided a kit for measuring the concentration of ET (e.g., NET) chromatin fragments and / or nucleosomes in a body fluid sample comprising: The kit includes a reagent solution containing one or more antibodies capable of specifically binding to ET, chromatin fragments, and / or nucleosomes, and a container suitable for use in a means for determining the concentration of ET, chromatin fragments, and / or nucleosomes by measuring the degree of aggregation or precipitation caused upon antibody binding. Such a kit may optionally include one or more buffer solutions.

[0104] Means for measuring the degree of agglutination or precipitation caused upon antibody binding are well known in the art. For example, a spectrophotometer can be used to measure the increase in turbidity (i.e., the decrease in the intensity of light transmitted through the sample) due to the increase in particle size resulting from the agglutination reaction. A nephelometer can be used in an immunonephelometric method to measure the increase in light scattering. Due to its simple one-step procedure and its short processing time, the HIA method is suitable for use in automated analyzers, especially clinical chemistry instruments.

[0105] According to a further aspect of the invention there is provided the use of a kit as defined herein for identifying a subject susceptible to sepsis or septic shock or for monitoring a subject with sepsis or septic shock. According to another aspect of the invention there is provided the use of a kit as defined herein for identifying a subject with a haematological cancer or for monitoring a subject with a haematological cancer.

[0106] (Additional biomarkers) The concentration of NETs, ​​chromatin fragments and / or nucleosomes can be detected or measured as one of the measurement panels. The panel can include different epigenetic features of nucleosomes (e.g., histone isoforms and PTMs) as described herein above. Biomarkers useful in panel testing for detection of severe respiratory infections requiring medical intervention include, but are not limited to, cytokine components (interleukins, including but not limited to IL-1, IL-6 and IL-8), C-reactive protein, myeloperoxidase, D-dimer, factor VII activating protease (FSAP), fibrinogen and fibrin / fibrinogen degradation products. In one embodiment, the panel includes C-reactive protein. In one embodiment, the panel includes one or more cytokines, such as one or more interleukins.

[0107] It will be apparent to one of skill in the art that any combination of the biomarkers disclosed herein can be used in panels and algorithms (e.g., for monitoring the progression of a disease, such as an infectious disease), and that additional markers can be added to panels that include these markers.

[0108] According to an aspect of the present invention, there is provided the use of a panel test for detecting or predicting complications with an infectious disease in a patient, said panel test comprising reagents for detecting the concentration of ETs (e.g. NETs), chromatin fragments and / or nucleosomes in a homogeneous immunoassay.

[0109] It will be understood that the embodiments described herein are applicable to all aspects of the invention, i.e., embodiments with descriptions of use are equally applicable to the methods as claimed, etc. The methods of the invention will now be illustrated by the following examples. EXAMPLES

[0110] Example 1 Nucleosome homogeneous immunoassays were performed using a Beckman Coulter AU400 analyzer.

[0111] Agglutination was achieved using a combination of two different antibodies coated onto latex particles. The first antibody was directed to bind to nucleosomes containing the histone H3 isoform H3.1 (H3.1-nucleosomes) with an epitope present near amino acid 30 of H3 and was directly coated onto latex particles (0.2 μm in size). The second antibody was directed to bind to a nucleosomal conformational epitope present only in intact nucleosomes containing histones and DNA and was indirectly bound to biotinylated, neutravidin-coated latex particles (0.3 μm).

[0112] The assay procedure was as follows: For use, nucleosome test solution (25 μL) was added to phosphate buffer (150 μL) at pH 7.2 containing KCl, NaCl, detergent (Tween 20), polyethylene glycol 100, ethylenediaminetetraacetic acid (EDTA), bovine serum albumin, and preservative (sodium azide). The mixture was incubated at 37°C for 180 s. A 0.2% suspension of latex particles in 50 mM Tris buffer (pH 8.0) containing sucrose, Tween 20, bovine serum albumin, and sodium azide (30 μL) was added and further incubated at 37°C for 36 s. The aggregation of nucleosomes bound to latex particles was then kinetically measured as an increase in absorbance of light at 700 nm over a period of 270 s at 37°C.

[0113] The results are shown in FIG. 1. The results demonstrate that the measured aggregation increased with the concentration of nucleosomes present in the sample solution. The sensitivity of the assay is at the level of 100 ng / ml, which is suitable for detection of nucleosomes in the context of diseases or conditions associated with nephrosis, including, for example, SIRS, ARDS, SARS, sepsis, pneumonia, COVID, influenza, thrombotic or microthrombotic conditions, inflammatory disease conditions, and thrombotic conditions. The sensitivity is also suitable for use in many other fields, including applications in cancer, particularly hematological cancers (myeloproliferative disorders), and also in assessing the viability of organs or tissues ex vivo for transplantation into recipient patients and monitoring recipient patients for rejection of transplanted organs or tissues.

Claims

1. A method for detecting the concentration of extracellular traps (ETs), chromatin fragments and / or nucleosomes in a body fluid sample, the method comprising analyzing the body fluid sample using a homogeneous immunoassay (HIA) method, and using the results obtained from the analysis to determine the concentration of ETs, chromatin fragments and / or nucleosomes in the body fluid sample.

2. The HIA method comprises: (a) mixing the body fluid sample with a solution of one or more antibodies capable of specifically binding to ETs, chromatin fragments and / or nucleosomes and causing aggregation or precipitation upon binding; and (b) measuring the degree of aggregation or precipitation in the mixture; The method of claim 1, wherein the measurement in step (b) is used to determine the concentration of ETs, chromatin fragments and / or nucleosomes in the body fluid sample.

3. 3. The method of claim 2, wherein the one or more antibodies are present in a suspension of antibody-coated particles.

4. 4. The method of claim 2 or 3, wherein the one or more antibodies are conjugated to the surface of latex beads or nanoparticles.

5. 3. The method of claim 2, wherein the degree of aggregation or precipitation is measured by absorbance, transmittance, reflectance, light scattering, fluorescence, or scintillation of the mixture.

6. The method of claim 2, wherein the one or more antibodies are monoclonal or polyclonal antibodies.

7. 3. The method of claim 1 or 2, comprising a single antibody incubation step.

8. 3. The method according to claim 1, wherein the HIA method is an immunoturbidimetric method or an immunonephelometric method.

9. 3. The method of claim 1 or 2, wherein the body fluid sample is blood, serum or plasma.

10. 10. The method of claim 9, wherein the blood, serum or plasma sample is obtained from a subject suffering from or suspected of suffering from sepsis or septic shock.

11. 10. The method of claim 9, wherein the blood, serum or plasma sample is obtained from a subject suffering from or suspected of suffering from a blood cancer.

12. 3. The method of claim 1 or 2, wherein the ET comprises neutrophil extracellular traps (NETs).

13. The method of claim 2, wherein the chromatin fragments and / or nucleosomes comprise epigenetic features of extracellular free nucleosomes, and the one or more antibodies are capable of specifically binding to the epigenetic features of the extracellular free nucleosomes.

14. 14. The method of claim 13, wherein the epigenetic feature is a histone isoform (e.g., a histone H3 isoform, particularly H3.1).

15. 14. The method of claim 13, wherein the epigenetic feature is a histone post-translational modification (PTM) (e.g., a core-nucleosomal histone PTM, in particular a histone H3 or H4 PTM).

16. 16. The method of claim 15, wherein the histone PTM is selected from citrullination or ribosylation.

17. 3. The method of claim 2, wherein the one or more antibodies are capable of specifically binding to a neutrophil extracellular trap (NET)-associated protein.

18. 18. The method of claim 17, wherein the NET-associated protein is selected from myeloperoxidase (MPO) and neutrophil elastase (NE).

19. 1. A method of obtaining data for monitoring disease progression in a subject, comprising: (i) detecting the concentration of ETs, chromatin fragments and / or nucleosomes in a body fluid sample obtained from said subject using the method of claim 1; (ii) repeating step (i) one or more times; and (iii) obtaining data for monitoring the progression of the disease in the subject using changes in the concentration of the ET, chromatin fragments and / or nucleosomes.

20. 1. A method of obtaining data for assigning risk of an adverse outcome to a subject suffering from an infectious disease, comprising: (i) detecting the concentration of ETs, chromatin fragments and / or nucleosomes in a body fluid sample obtained from said subject using the method of claim 1; and (ii) using the detected concentrations of ETs, chromatin fragments and / or nucleosomes to obtain data for assigning a likelihood of an adverse outcome to the subject; The method, wherein the data is used to identify subjects with a high likelihood of an adverse outcome and assign the subjects to a medical intervention.

21. 21. The method of claim 19 or 20, wherein the subject is in a critical care unit.

22. 21. The method of claim 19 or 20, wherein the subject is suffering from sepsis or septic shock.

23. 21. The method of claim 19 or 20, wherein the subject is suffering from a hematological cancer.

24. 21. The method of claim 19 or 20, wherein the subject is a human or animal subject.

25. 21. The method of claim 19 or 20, further comprising comparing the concentration of ETs, chromatin fragments and / or nucleosomes in the body fluid sample of the subject with one or more controls.

26. 21. The method of claim 19 or 20, wherein the concentration of ETs, chromatin fragments and / or nucleosomes is detected or measured as one of a panel of measurements.

27. 1. A method of obtaining data for detecting a subject in need of medical treatment for sepsis or septic shock, comprising: Detecting the concentration of ETs and / or chromatin fragments and / or nucleosomes in a body fluid sample obtained from the subject using the method of claim 1 or 2, The method, wherein the detected concentration of ETs and / or chromatin fragments and / or nucleosomes is used as an indicator that the subject needs medical treatment for sepsis or septic shock.

28. 1. A kit for measuring the concentration of ETs and / or chromatin fragments and / or nucleosomes in a body fluid sample, comprising: The kit comprises a reagent solution containing one or more antibodies capable of specifically binding to the ET and / or chromatin fragments and / or nucleosomes, and a container suitable for use in a means for determining the concentration of the ET and / or chromatin fragments and / or nucleosomes by measuring the degree of aggregation or precipitation caused upon antibody binding, and optionally one or more buffer solutions.

29. 30. The kit of claim 28, for use in identifying a subject susceptible to sepsis or septic shock or for monitoring a subject with sepsis or septic shock.

30. 30. The kit of claim 28, used to identify a subject suffering from cancer or to monitor a subject having cancer.

31. 31. The kit of claim 30, wherein the cancer is a blood cancer.

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