Lateral flow inspection device

Abstract

The present invention relates to a lateral flow analysis apparatus for detecting target nucleosomes in a sample, as well as a method and system comprising the apparatus.

Description

TECHNICAL FIELD

[0001] (Field of the Invention) The present invention relates to a lateral flow test device for detecting target nucleosomes in a sample, which may be useful in biological applications, for example, in the fields of medical, environmental and veterinary diagnostics.

BACKGROUND ART

[0002] (Background of the Invention) Cellular DNA exists as a protein-nucleic acid complex called chromatin. Nucleosomes are the basic unit of chromatin structure, consisting of DNA wrapped around a protein complex. DNA is wrapped around successive nucleosomes in a structure often described as resembling "beads on a string", which forms the basic structure of open chromatin or euchromatin. In condensed chromatin or heterochromatin, this string is coiled and supercoiled into a closed, complex structure.

[0003] Each nucleosome in chromatin consists of a protein complex of eight highly conserved core histones (composed of a pair of each of histones H2A, H2B, H3, and H4). Around this complex, approximately 145 base pairs (bp) of DNA are wrapped. Another histone, H1, which can be located on the nucleosome outside the core histones, binds to an additional 20 bp of DNA to produce a nucleosome (or chromatosome) containing about 165 bp of DNA. Histone H1 is said to act as a linker histone, and the additional DNA is often referred to as "linker DNA", that is, the DNA that connects one nucleosome to another in a chromosome. The linker DNA separating two nucleosomes in a chromosome may be longer than 20 bp, and may be up to 80 bp in length.

[0004] Holdenrieder et al., Int. J. Cancer (2001) 95: 114-120, previously described the detection of nucleosome levels in serum samples from patients with benign and malignant diseases. The epigenetic composition of circulating cell-free nucleosomes has also been investigated as a blood-based biomarker in cancer in terms of its histone modifications, histone variants, DNA modifications, and adduct content. See WO 2005 / 019826, WO 2013 / 030577, WO 2013 / 030579, and WO 2013 / 084002.

[0005] Nucleosomes are released into circulation when chromatin fragments during cell death. Many infections, such as viral infections, initiate cell death through various mechanisms (cell binding and entry, endosomal TLR3 activation, and gene expression), thereby increasing the number of circulating nucleosomes in the blood (Danthi et al., Annu. Rev. Virol. (2016) 3: 533-53). Furthermore, infection can induce NETosis, which promotes the decondensation of chromatin released into circulation as a first-line response to infection by post-translational histone modifications such as acetylation or hypercitrullination of histones H3 and H4 (Wang Y et al., J. Cell Biol. (2009) 184(2): 205-213). However, extracellular nucleosomes and neutrophil extracellular traps (NETs) can cause severe complications if not rapidly removed. For example, nucleosome binding to the glomerular membrane is associated with renal injury in lupus (Kalaaji et al., Kidney Int. (2007) 71(7): 665-672), while NETs have been shown to enhance lung injury during viral pneumonia (Ashar et al., Am. J. Pathol. (2018) 188(1): 135-148). In fact, host-targeted NET toxicity is associated with respiratory distress, airway obstruction, endothelial and epithelial cell damage, inflammatory responses, as well as thrombosis and other pathologies (Marcos et al., Nat. Med. (2010) 16: 1018-23; Hoeksema et al., Future Microbiol. (2016) 11: 441-53). The usefulness of free nucleosomes as a biomarker in patients with sepsis and NETosis-related conditions such as COVID-19 has been previously described (see WO 2021 / 1186037).

[0006] Point-of-care (POC) testing is essential for the rapid detection of analytes near the patient, which facilitates better disease diagnosis, monitoring, and management. This allows for diagnosis at a very early stage, enabling rapid medical decisions and improved patient health outcomes by allowing for earlier initiation of treatment. This is particularly important in diseases with rapid deterioration. For example, in sepsis, each hour's delay in antimicrobial agent administration is associated with an average 7.6% decrease in survival rate (Anand et al. (2006) Critical Care Medicine 34(6): 1589-1596). Furthermore, POC testing allows patients to monitor their condition without needing to travel to a clinic or hospital for testing. There remains a need in this technology for providing POC testing that detects nucleosomes to enable real-time patient diagnosis and monitoring.

[0007] Lateral flow testing (LFT) is a very well-known type of test used in point-of-care (POC) environments, but a brief explanation of its background is provided. An LFT typically consists of the following elements: a sample pad, a conjugate release pad, a substrate with an immobilized capture reagent, and an absorbent pad. The components of the strip are usually fixed to an inert support material. The sample pad acts as a sponge, positioned to receive the sample liquid and also hold any excess. After the sample pad is saturated with the sample liquid, the sample liquid moves to the conjugate pad, which contains a labeled detection reagent, usually an antibody, which is released into the sample as it flows through the pad. The labeled detection reagent is specific to the target molecule, so as the sample flows across the conjugate pad, if the target molecule is present, the reagent will bind to the target molecule. The label is usually a colored or fluorescent nanoparticle bound to the detection reagent, making the diagnostic results of the test visible to the user. The sample liquid then moves to a substrate (usually made of nitrocellulose) on which a specific capture reagent (mostly an antibody or antigen) is immobilized in a line. Typically, there are at least two such lines. The test line indicates the presence or absence of the target molecule in the sample. The control line indicates that the test returned a valid result and that the test has not deteriorated, for example, due to improper storage. The properties and positions of the reagents on the substrate vary depending on the type of target and LFT. The final component of the LFA is the absorbent pad. This simply absorbs excess liquid, while the sample continues to be drawn through the nitrocellulose substrate by capillary action.

[0008] This invention describes an LFT device for nucleosome detection. To the best of the inventors' knowledge, no such LFT has been described previously. This invention provides a solution particularly suitable for proof-of-concept (POC) applications. [Overview of the project]

[0009] (Summary of the invention) According to a first aspect of the present invention, a lateral flow analysis device for detecting target nucleosomes in a sample, (i) Substrate including inspection area containing capture reagent; and (ii) A conjugate pad upstream of a substrate, comprising a labeled detection reagent, the labeled detection reagent being able to move to the test site along with the liquid sample and / or further liquid to generate a detectable signal; A lateral flow analysis device is provided, characterized in that both the labeled detection reagent and the capture reagent contain : and bind to the target nucleosome or its components.

[0010] According to a further aspect of the present invention, a method for detecting the presence of a target nucleosome in a sample, a) A step of bringing a liquid sample into contact with an inspection apparatus as described herein, wherein the liquid sample is applied to a part of the inspection apparatus upstream of the inspection site; and b) A step of evaluating detectable signals at the test site to determine the presence, absence, and / or quantity of target nucleosomes in the sample. A method is provided that includes:

[0011] According to a further aspect of the present invention, a system for evaluating a sample, a) Testing apparatus described herein; and b) A reader for detecting detectable signals A system including the following is provided. [Brief explanation of the drawing]

[0012] (Brief explanation of the drawing) [Figure 1] Figure 1 shows an embodiment of the lateral flow inspection apparatus according to the present invention. [Figure 2] Figure 2 shows an embodiment of the lateral flow inspection apparatus according to the present invention, in which the inspection apparatus further includes pre-removal areas. [Figure 3] Figure 3 shows the correlation between results obtained from the lateral flow analyzer according to the present invention and the Nu.Q® H3.1 immunoassay performed with the IDS-i10 automated immunoassay analyzer system. [Figure 4] Figure 4 shows the correlation between results obtained from the lateral flow analyzer according to the present invention and the Nu.Q® H3.1 immunoassay performed on ELISA plates. Samples from 122 patients from three different groups (healthy controls / COVID-19 / sepsis) were tested. [Figure 5] Figure 5 shows identification box plots for results obtained from three groups of patient samples (healthy individuals / COVID-19 / sepsis) examined with the lateral flow analyzer according to the present invention. [Modes for carrying out the invention]

[0013] (Detailed description of the invention) A detailed description of one or more embodiments of the claimed subject matter is provided below, along with accompanying drawings illustrating the principles of the claimed subject matter. While the claimed subject matter is described in relation to such embodiments, it is not limited to any particular embodiment. It should be understood that the claimed subject matter can be embodied in a variety of forms and encompasses numerous alternatives, modifications, and equivalents.

[0014] (Lateral flow testing device) According to a first aspect of the present invention, a lateral flow analyzer for detecting target nucleosomes in a sample, comprising: (i) Substrates including inspection sites containing capture reagents; and (ii) A conjugate pad upstream of the substrate, comprising a labeled detection reagent, the labeled detection reagent moving with the liquid sample and / or further liquid to the test site to generate a detectable signal. : includes, A lateral flow analysis device is provided, characterized in that both a labeled detection reagent and a capture reagent bind to a target nucleosome or its components.

[0015] Lateral flow test (LFT) per se is well known, and has four main elements: a target molecule, a labeled detection reagent, a capture reagent, and a target molecule-detection conjugate complex. Despite these main elements being well established, the terms used by those skilled in the art are not necessarily consistent, and different terms may refer to the same element. It will be understood that the present invention encompasses the functional elements, however they may be referred to herein and used interchangeably. For example, the target molecule is also referred to as an analyte, an antigen, an antigen molecule, a target analyte, or a biomarker. The labeled detection reagent is also referred to as a labeled reagent, a conjugate, an (analyte) tag, a tagging particle, a chemical partner, a (sample) conjugate mixture, a bioactive particle, or a conjugate receptor. The capture reagent is also referred to as a binder, an antibody, a receptor, a chemical partner, or a capture molecule. The target molecule-detection conjugate complex is a combination of an antigen and a conjugate. This complex is also referred to as a tagged analyte, or a particle on which an analyte molecule is immobilized.

[0016] Described herein is an LFT for the detection of nucleosomes. It will be understood that reference to "nucleosome" herein may refer to "free nucleosome" when detected in a body fluid sample. It will be understood that the term free nucleosome is intended throughout this document to include any free chromatin fragment comprising one or more nucleosomes.

[0017] In one embodiment, the test device further comprises a sample pad upstream of the substrate and in fluid communication with the substrate. In one embodiment, the test device further comprises an absorbent pad downstream of the substrate and in fluid communication with the substrate. In a preferred embodiment, the test device comprises or consists of the following elements: a sample pad, a conjugate pad, a substrate, and an absorbent pad, which are sequentially mounted on a solid support material.

[0018] This test device can further include a housing that covers at least a portion of the test device, wherein the housing includes a sample application port for enabling application of a sample upstream of or to the test site, and an optical aperture around the test site for enabling signal detection at the test site. In some embodiments, the housing covers the entire test device. In other embodiments, at least a portion of the sample receiving portion of the substrate or the sample application element is not covered by the housing. The housing may comprise any suitable material. For example, the housing may comprise a plastic material.

[0019] (Sample pad) In one embodiment, the device comprises a sample application element that is upstream of the substrate and in fluid communication with the substrate. The sample pad facilitates uniform and controlled distribution of the sample to the conjugate pad. This can also control the rate at which liquid enters the conjugate pad and prevent flooding of the device. When impregnated with components such as proteins, detergents, viscosity enhancers, and buffer salts, the sample pad increases the viscosity of the sample (adjusts flow characteristics), enhances the ability of the sample to solubilize the detection reagent, prevents non-specific binding of the conjugate and analyte to any of the downstream materials, modifies the chemistry of the sample to be compatible with immune complex formation at the test line, and can also be used to promote uniform flow of the sample along the substrate.

[0020] The presence of added proteins (e.g., albumin) as well as detergents and surfactants (e.g., very low concentrations of SDS or TWEEN® 20) can promote resolubilization of the conjugate, reduce non-specific binding of the conjugate, and optionally minimize adsorption of the analyte to the substrate.

[0021] Any suitable material can be used in the sample pad of the present device. For example, the sample pad may comprise cellulose fiber filters and / or woven mesh.

[0022] (Conjugate pad) This testing device includes a conjugate pad containing a labeled detection reagent, which can be moved to the test site by a liquid sample and / or further liquid to generate a detectable signal. The conjugate pad (sometimes referred to as a conjugate element) can be placed in any suitable location. For example, the conjugate pad can be placed downstream of the sample pad.

[0023] This testing device may include any suitable labeled detection reagent that binds to the target nucleosome or its components.

[0024] Any suitable label can be used. For example, the label may be a soluble label, such as a colorimetric, radioactive, enzymatic, luminescent, fluorescent, or magnetic label. In another example, the label may be a particle or particulate label, such as a direct particulate label or a colored particle label. Exemplary particle or particulate labels include colloidal gold labels, latex particle labels, nanoparticle labels, and quantum dot labels.

[0025] In one embodiment, the labeled detection reagent comprises a single compound capable of both binding to a target nucleosome or its components and generating a detectable signal. For example, in one embodiment, the labeled detection reagent comprises a DNA-binding dye. In one embodiment, the DNA-binding dye is a DNA intercalator dye. The DNA intercalator dye can be used to preferentially bind to chromatin fragments containing linker DNA. DNA bound to nucleosomes is relatively difficult for the DNA intercalator dye to bind. Unbound DNA between nucleosomes (linker DNA) is more strongly intercalated by the DNA intercalator dye (see, for example, Bosire et al., PLoS One (2019) doi:10.1371 / journal.pone.0224936). In an alternative embodiment, the DNA-binding dye is a groove-binding dye, e.g., a minor groove-binding dye or a major groove-binding dye.

[0026] The labeled reagent can be dried in the presence of a material that a) stabilizes the labeled reagent; b) facilitates the solubilization or resuspension of the labeled reagent in liquid; and / or c) facilitates the mobility of the labeled reagent. Any suitable material can be used. For example, the material may be a protein, e.g., casein or BSA, a peptide, a polysaccharide, a sugar, a polymer, e.g., polyvinylpyrrolidone (PVP-40), a gelatin, or a detergent, e.g., Tween-20.

[0027] In one embodiment, the labeled detection reagent is colloidal gold nanoparticles conjugated to an antibody that binds to a target nucleosome or its components.

[0028] (base material) Any suitable substrate can be used in this apparatus. For example, the substrate may include nitrocellulose, glass fiber, polypropylene, polyethylene (preferably very high molecular weight), polyvinylidene fluoride, ethylene vinyl acetate, acrylonitrile, and / or polytetrafluoroethylene.

[0029] The substrate can be any suitable form. For example, the substrate may be in the form of a strip or a circle. The substrate may be a single element or may consist of multiple elements.

[0030] This inspection device may further include a sample application element located upstream of the substrate and in fluid communication with the substrate.

[0031] At least a portion of the substrate can be supported by a solid support material. In some embodiments, the entire substrate can be supported by a solid support material.

[0032] (Inspection site) The substrate of the present invention includes a test site, which contains a capture reagent that binds to a target nucleosome or its components. The test site may also be referred to as a test line.

[0033] The capture reagent can be any suitable substance. For example, the capture reagent can be a peptide or a protein. In some embodiments, the protein can be an antigen or antibody, such as an antibody that specifically binds to a nucleosome or its components, including the DNA associated with it.

[0034] In one embodiment, the capture reagent directly binds to the target nucleosome. In another embodiment, the capture reagent binds indirectly to the target nucleosome, for example, by binding to another binding reagent that binds to the target nucleosome, or by being an analyte that competes with the target nucleosome in the sample for binding to the labeled detection reagent.

[0035] Preferably, the testing device is configured as a sandwich-type LFT for detecting target nucleosomes in a sample, where the target nucleosomes are first labeled with a labeled detection reagent that binds to the target nucleosomes or their components. The target nucleosome-labeled detection reagent complex then moves to a test site, which contains a capture reagent that binds to the target nucleosomes or their components at a site different from the labeled detection reagent. The test site then generates a detectable signal by the concentration of the labeled detection reagent, thus confirming the presence of target nucleosomes.

[0036] This testing device can also be configured as a competitive LFT, where the target nucleosome is first labeled with a labeled detection reagent that binds to the target nucleosome or its components. The target nucleosome-labeled detection reagent complex then moves to the test site, which contains the target nucleosome or a capture reagent that is an analogue of the target nucleosome competing for binding to the labeled detection reagent. If the target nucleosome is not present in the sample, the unbound labeled detection reagent binds to the capture reagent, resulting in a detectable signal at the test site. Conversely, if the target nucleosome is present in the sample, it binds to the labeled detection reagent, preventing it from binding to the immobilized capture reagent at the test site, and therefore, no detectable signal is present.

[0037] (Nucleosome binding agent) In the present invention, both the labeled detection reagent and the capture reagent bind to the target nucleosome or its components.

[0038] As used herein, the terms “binding” or “specific binding” refer to the specificity of a binder, such as an antibody, that preferentially binds to a target, such as a polypeptide antigen. When referring to a binding partner, such as a protein, nucleic acid, antibody, or other affinity capture reagent, “specific binding” may include the binding reaction of two or more binding partners having high affinity and / or complementarity to ensure selective hybridization under specified assay conditions. Typically, specific binding is at least three times the standard deviation of the background signal. Therefore, under specified conditions, the binding partner binds to its specific target molecule and not to other molecules present in the sample in significant amounts. Recognition of a specific target by the binder or antibody in the presence of other potential interfering substances is one characteristic of such binding. Preferably, a binder, antibody, or antibody fragment that is specific to or specifically binds to a target binds to the target with higher affinity than binding to other non-target substances. Also preferably, a binder, antibody, or antibody fragment that is specific to or specifically binds to a target avoids binding to a significant proportion of non-target substances, such as non-target substances present in the test sample. In some embodiments, the binders, antibodies, or antibody fragments of the Disclosure avoid binding to more than about 90% of the non-target substance, although a higher percentage is clearly intended and preferred. For example, the binders, antibodies, or antibody fragments of the Disclosure avoid binding to about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, and more than about 99% of the non-target substance. In other embodiments, the binders, antibodies, or antibody fragments of the Disclosure avoid binding to about 10%, 20%, 30%, 40%, 50%, 60%, or more than 70%, or more than about 75%, or more than about 80%, or more than about 85% of the non-target substance.

[0039] The labeled detection and capture reagents may contain any suitable substance that binds to the target nucleosome or its components. For example, the substance that binds to the target nucleosome or its components may be a peptide or a protein. In some embodiments, the protein may be an antigen or antibody, such as an antibody that specifically binds to the nucleosome or its components, including the associated DNA.

[0040] In this specification, the term “antibody” is used in its broadest sense and includes polyclonal and monoclonal antibodies, which include intact antibodies and functional (antigen-binding) antibody fragments, which include fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments including single-chain variable fragments (scFv), and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term also encompasses genetically modified and / or otherwise modified forms of immunoglobulins, such as intrabody, peptide-body, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific antibodies, such as bispecific antibodies, diabody, triabody, and tetrabody, tandem di-scFv, tandem tri-scFv, etc. Unless otherwise specified, the term “antibody” should be understood to include its functional antibody fragments. This term also includes intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses IgM, IgE, IgA, and IgD.

[0041] The "class" of an antibody refers to the type of constant domain or constant region in its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0042] In another embodiment, the substance that binds to the target nucleosome or its components is an aptamer.

[0043] In one embodiment, the aptamer is a nucleic acid aptamer. In one embodiment, the nucleic acid aptamer is a DNA aptamer. In an alternative embodiment, the nucleic acid aptamer is an RNA aptamer. In one embodiment, the nucleic acid aptamer comprises one or more modified nucleotides such as 2'-O-methyl-; 2'-O-(2-methoxyethyl); 2-O-alkyl; 2-O-allyl; 2'-S-alkyl; 2'-S-allyl; 2'-fluoro-; 2'-amino; 2'-halo or 2-azidolibose, carbocyclic sugar analogs, α-anomeric sugars; epimeric sugars (where the epimeric sugar is arabinose, xylose, or lyxose), pyranose sugars, furanose sugars, or sedoheptulose; 2'-fluoro-β-D-arabinonucleotide (FANA) modification; or loc nucleic acid (LNA).

[0044] In an alternative embodiment, the aptamer is a peptide aptamer.

[0045] It has been previously shown that when the higher-order structure of an anti-nucleosome aptamer is removed through a thermal denaturation process, the aptamer functions similarly to commercially available anti-nucleosome antibodies (see WO 2014 / 131841). Therefore, in one embodiment, the aptamer is a denatured aptamer from which the higher-order structure has been removed. Such denaturation may typically involve a thermal cycle of heating at 95°C for 1 minute followed by cooling for 10 minutes.

[0046] In one embodiment, the labeled detection reagent and / or capture reagent binds to the nucleosome itself. The reference to “nucleosome itself” refers to all nucleosomes present in the sample, regardless of any epigenetic features that may or may not be present in the nucleosome. Therefore, a reagent that binds to the nucleosome itself is a reagent that can bind to nucleosomes (preferably intact nucleosomes) regardless of their epigenetic features. Detection at the whole nucleosome level usually involves detecting a histone protein common to all nucleosomes, such as a core histone protein, such as histone H4. Therefore, in one embodiment, the labeled detection reagent and / or capture reagent binds specifically to a histone protein. In one embodiment, the histone is a histone common to all nucleosomes, such as histone H4.

[0047] The detection of nucleosomes themselves may also involve the detection of epitopes that are present in intact nucleosomes but not on isolated (free) histone or DNA nucleosome components. Because these types of epitopes require the native three-dimensional structure of the target nucleosome to be intact, they are sometimes referred to herein as “structural nucleosome epitopes.” Binders may also recognize numerous structural epitopes formed by interactions between DNA and core histones, and as a result, the binder is “nucleosome-specific,” that is, it binds to nucleosomes but not to individual components of the nucleosome complex, such as histones or dsDNA. In one embodiment of the present invention, the antibody is directed to bind to structural nucleosome epitopes present in intact nucleosomes containing a histone octameric core complexed with DNA. Therefore, in some embodiments, the labeled detection reagent and / or capture reagent specifically binds to intact nucleosomes, including DNA and histone cores, via structural epitopes present only on intact nucleosomes. In one embodiment, the labeled detection reagent and / or capture reagent is an anti-nucleosome antibody that specifically binds to epitopes present in intact nucleosomes. The anti-nucleosome antibody does not bind (or weakly binds) to free histone octamer complexes, free histones (i.e., without DNA), free DNA, or free histones. In a preferred embodiment, the anti-nucleosome antibody specifically binds to structural nucleosome epitopes. In a preferred embodiment, the capture reagent specifically binds to structural nucleosome epitopes.

[0048] Circulating nucleosomes are not a homogeneous population of protein-nucleic acid complexes. Rather, they are a heterogeneous population of chromatin fragments derived from the digestion of chromatin during cell death, and contain a vast variety of epigenetic structures, including specific histone isoforms (or variants), post-translational histone modifications, nucleotides or modified nucleotides, and protein adducts. It will be apparent to those skilled in the art that an increase in nucleosome levels is associated with an increase in several subsets of circulating nucleosomes containing specific epigenetic signals, including nucleosomes containing specific histone isoforms (or variants), nucleosomes containing specific post-translational histone modifications, nucleosomes containing specific nucleotides or modified nucleotides, and nucleosomes containing specific protein adducts.

[0049] In one embodiment, the components of a free nucleosome include the epigenetic features of the free nucleosome. The level of a nucleosome can be measured by detecting the epigenetic features of the nucleosome. The terms “epigenetic signal structure” and “epigenetic features” will be understood to be used interchangeably herein. These refer to specific features of a nucleosome that can be detected. In one embodiment, the epigenetic features of a nucleosome are selected from the group consisting of post-translational histone modifications, histone isoforms (or variants), modified nucleotides, and / or proteins bound to nucleosomes in nucleosome-protein adducts.

[0050] The structure of a nucleosome can vary due to post-translational modifications (PTMs) of histone proteins. Histone protein PTMs typically occur at the tail of the core histone, 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, and many others. Many histone modifications are known in the art, and their number is increasing as new modifications are identified (Zhao and Garcia (2015), Cold Spring Harb Perspect Biol, 7: a025064). Therefore, in one embodiment, the epigenetic features of a free nucleosome may be histone post-translational modifications (PTMs).

[0051] In one embodiment, a labeled detection reagent and / or capture reagent specifically binds to a unique epitope formed by post-translational modification. The histone PTM may be present on a coanucleosome histone (e.g., H2A, H2B, H3, or H4) or a linker histone (e.g., H1 or H5). The histone PTM may be a histone PTM of a coanucleosome, e.g., H3, H2A, H2B, or H4, and in particular, H3, H2A, or H2B. In particular, the histone PTM is a histone H3 PTM. Examples of such PTMs are described in WO 2005 / 019826 and WO 2017 / 068359.

[0052] For example, post-translational modifications include acetylation, methylation (which may be monomethylation, dimethylation, or trimethylation), phosphorylation, ribosylation, citrullination, ubiquitination, hydroxylation, glycosylation, nitrosylation, glutamation, and / or isomerization (see Ausio's reference (2001) Biochem Cell Bio 79: 693). In one embodiment, the histone PTM is methylation of a lysine residue. In a further embodiment, the methylation is methylation of a histone 3-lysine residue. In yet another embodiment, the histone PTM is selected from H3K4Me, H3K4Me2, H3K9Me, H3K9Me3, H3K27Me3, or H3K36Me3. In one embodiment, the histone PTM is acetylation of a lysine residue. In yet another embodiment, the acetylation is acetylation of a histone 3-lysine residue. In a further embodiment, the histone PTM is selected from H3K9Ac, H3K14Ac, H3K18Ac, or H3K27Ac. In another embodiment, the histone PTM is H4PanAc. In one embodiment, the histone PTM is phosphorylation of a serine residue. In a further embodiment, the phosphorylation is phosphorylation of isoform X of the histone 2A(H2AX) serine residue or phosphorylation of a histone 3 serine residue. In another embodiment, the histone PTM is selected from pH2AX or H3S10Ph. In one embodiment, the histone PTM is selected from citrullination or ribosylation. In another embodiment, the histone PTM is citrullinated H3(H3cit) or citrullinated H4(H4cit). In another embodiment, the histone PTM is citrullination of the histone 3 arginine residue. In yet another embodiment, the histone PTM is H3R8Cit. In one embodiment, the histone PTM is selected from the group consisting of H3K4Me, H3K4Me2, H3K9Me, H3K9Me3, H3K27Me3, H3K36Me3, H3K9Ac, H3K14Ac, H3K18Ac, H3K27Ac, ​​H4PanAc, pH2AX, H3S10Ph, and H3R8Cit.

[0053] In one embodiment, the histone PTM is selected from citrullinated or ribosylated. In a further embodiment, the histone PTM is H3 citrulline (H3cit) or H4 citrulline (H4cit). In yet another embodiment, the histone PTM is H3cit.

[0054] In one embodiment, the histone PTM is ribosylation, also known as ADP-ribosylation. Post-translational histone ADP-ribosylation of nucleosomes occupying promoters of inflammatory response markers in macrophages is stimulated by exposure to lipopolysaccharides, leading to increased transcription and potentially possessing antiviral properties. Therefore, changes in the level of circulating ADP-ribosylated nucleosomes released from macrophages are expected to be useful in the method of the present invention.

[0055] It is also possible to detect a group or class of related histone post-translational modifications (rather than a single modification). Typical examples, though not limited to them, involve two-site immunoassays utilizing one antibody or other selective binder directed to bind to a nucleosome and another antibody or other selective binder directed to bind to the group of histone modifications in question. Examples of such antibodies directed to bind to a group of histone modifications, though not limited to them, include, for illustrative purposes, anti-panacetylated antibodies (e.g., panacetyl H4 antibodies [H4panAc]), anti-citrullinated antibodies, or anti-ubiquitin antibodies.

[0056] In a preferred embodiment, the labeled detection reagent specifically binds to unique epitopes formed by post-translational modifications of core histones, and the capture reagent specifically binds to structural nucleosome epitopes.

[0057] In one embodiment, the epigenetic features of a nucleosome include one or more histone variants or isoforms. The epigenetic features of a free nucleosome may be histone isoforms of the core nucleosome, particularly histone isoforms such as the histone H3 isoform. The terms “histone variant” and “histone isoform” can be used interchangeably herein. The structure of a nucleosome may also differ by the inclusion of alternative histone isoforms or variants that are different genes or splice products and have different amino acid sequences. Many histone isoforms are known in the art. Histone variants can be classified into several families, which are further subdivided into individual types. Numeric acid sequences of many histone variants are known and are publicly available, for example, in the National Human Genome Research Institute's 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 Databank of Japan (DDBJ). For example, histone H2 variants include H2A1, H2A2, mH2A1, mH2A2, H2AX, and H2AZ. In another example, histone isoforms of H3 include H3.1, H3.2, H3.3, and H3t.

[0058] In one embodiment, the antibody specifically binds to an epitope present in histone H3.1 ("anti-histone H3.1 antibody"). The amino acid sequence of histone H3.1 is publicly known in the art and is described in UniProt accession number P68431. In mammals, histone H3 variants include: H3.1, H3.2, H3.3, H3t, and centromere protein A (CENP-A). Human H3.1 and H3.2 are 99% identical (different by only one amino acid), histone H3t is 96% identical to H3.1 (different at four amino acid positions), and histone H3.3 is 96% identical to H3.1 (different at five amino acid positions). CENP-A shares only 46% identity with H3.1. In one embodiment, the antibody selectively binds to histone H3.1, H3.2, and H3t. In this embodiment, it will be understood that the antibody does not bind to other histone H3 isoforms such as histone H3.3 and CENP-A.

[0059] Nucleosomes undergo clipping, a regulated proteolytic degradation of histone tails, which is physically and irreversibly removed by clipping. Furthermore, histone degradation has been shown to be involved in the formation of NETs (see Papayannopoulos et al. (2010) J. Cell Biol. 191(3): 677-691). In histone H3, clipping has been reported to occur around amino acid position 21 (Yi and Kim (2018) BMB Reports, 51(5): 211-218). Therefore, in one embodiment, the antibody specifically binds to an epitope located higher than amino acid position 21. This allows for the capture of both clipped and unclipped nucleosomes.

[0060] The amino acid sequence of histone H3.1 at positions 27-36 is: [ka] Therefore, the amino acid sequence at positions 29-35 does not generally contain post-translationally modified amino acids (e.g., lysine, serine, or arginine). Consequently, an antibody directed to bind to this epitope (i.e., amino acid positions 29-35) is unaffected, or minimally affected, by the post-translational modification status of the nucleosome and binds to all or most nucleosomes containing histone H3.1, regardless of the PTM structure. In one embodiment, an anti-histone H3.1 antibody binds to amino acids 30-33 of histone H3.1. [ka] It specifically binds to epitopes containing [specific amino acids]. In another embodiment, the anti-histone H3.1 antibody binds to amino acids 28-32 of histone H3.1. [ka] The antibody specifically binds to epitopes containing [specific components]. In particular, these epitopes are located at or around amino acid 31 of histone H3, which is alanine in H3.1 and serine in H3.3. Binding to these regions ensures that both intact and clipped nucleosomes are reliably captured by the antibody, regardless of their PTM state. This maximizes the capture of H3.1 nucleosomes.

[0061] In one embodiment, the labeled detection reagent and / or capture reagent specifically binds to histone H3.1 and / or H3.2 and / or H3t. In a further embodiment, the labeled detection reagent and / or capture reagent is an anti-histone H3.1 antibody. The anti-histone H3.1 antibody may be directed to bind to an epitope around amino acids 30-33 of the histone H3.1 protein in order to capture clipped and unclipped nucleosomes. In a preferred embodiment, the labeled detection reagent specifically binds to histone H3.1 and / or H3.2 and / or H3t, and the capture reagent binds to the nucleosome itself, for example, by binding to a structural nucleosome epitope.

[0062] In one embodiment, the labeled detection reagent and / or capture reagent specifically bind to histone H3.1 and / or citrullinated histone H3R8 (H3R8Cit). In a preferred embodiment, the labeled detection reagent specifically binds to histone H3.1 and / or citrullinated histone H3R8 (H3R8Cit), and the capture reagent binds to the nucleosome itself, for example, by binding to a structural nucleosome epitope.

[0063] In a more preferred embodiment, the labeled detection reagent specifically binds to histone H3.1, and the capture reagent specifically binds to the three-dimensional nucleosome epitope.

[0064] In one embodiment, the labeled detection reagent and / or capture reagent specifically bind to histone H3.3. In a preferred embodiment, the labeled detection reagent specifically binds to histone H3.3, and the capture reagent binds to the nucleosome itself, for example, by binding to a stereochemical nucleosome epitope.

[0065] In a preferred embodiment, the labeled detection reagent and / or capture reagent specifically bind to the linker DNA.

[0066] (Comparison section) In one embodiment, the testing device further includes a control site. The control site indicates that the test returned a valid test result and that the test has not deteriorated, for example, due to improper storage. The control site may also be referred to as a control line. In one embodiment, the control site includes a control reagent that binds to a labeled detection reagent. For example, if the labeled detection reagent is a labeled anti-nucleosome mouse antibody, the control reagent would be an anti-mouse antibody.

[0067] (Absorbent pad) In one embodiment, the apparatus further includes an absorbent pad located downstream of the substrate and in fluid communication with the substrate. The main function of the absorbent pad is to increase the total amount of sample entering the test strip. Any suitable material can be used for the absorbent pad of the apparatus. For example, the absorbent pad may include cellulose and / or glass fiber material.

[0068] (pre-removal area) One problem reported with LFT is the occurrence of false-positive and false-negative interference, most likely due to anti-species antibodies in clinical plasma samples. For example, human anti-mouse antibodies (HAMA) or other heterophile antibodies present in patient samples can bind to mouse antibodies used as components of the immunoassay, potentially either i) blocking the mouse monoclonal antibody from binding to the target marker, resulting in a false-negative result, or ii) forming crosslinks between pairs of mouse monoclonal antibodies, generating a false-positive signal. If a patient is misdiagnosed due to the presence of HAMA, the consequences can be severe. At least 34 cases of hCG false-positive testing in the United States between 1999 and 2004 resulted in patients undergoing chemotherapy or surgery, including 10 hysterectomies, for false cancer (Human Chorionic Gonadotropin (hCG), Laurence A. Cole, Stephen A. Butler. Elsevier. 2010).

[0069] Rheumatoid factor (Rf) has also been reported to be quite reactive to antibodies used in immunoassays. Rheumatoid factor is a disease-associated autoantibody that targets regions of host IgG and generates immune complexes that contribute to disease pathology. RF has been associated with falsely elevated results in immunoassays used in clinical care and research (see Literature, Rheumatoid factor and falsely elevated results in commercial immunoassays: data from an early arthritis cohort. Rheumatol Int. 2021 Sep;41(9):1657-1665). Similarly, RF has been reported to be able to directly bind to nucleosomes, which is particularly important when immunoassays directly or indirectly report nucleosome levels (Hannestad and Stollar, Nature (1978) 275:671-673).

[0070] Assay manufacturers are attempting to develop assays to minimize interference from HAMA, for example, by adding an excess of mouse immunoglobulin to the assay buffer. When this is done, HAMA present in the patient sample can bind to the excess mouse immunoglobulin rather than the reagents used in the assay. An alternative solution to control HAMA interference is to use alternative species-derived monoclonal antibodies in the immunoassay instead of mouse monoclonal antibodies. Rabbit and sheep monoclonal antibodies are candidates here. However, this is not a guaranteed solution, as some individuals in the population produce human anti-rabbit and anti-sheep antibodies as well as antibodies with interspecies cross-reactivity. It has been reported that endogenous antibodies (including interfering antibodies) can be removed from samples by, for example, applying them to a spin column packed with an immunoaffinity matrix to remove immunoglobulins. It has also been reported that excess albumin in samples can be conveniently removed by using an albumin-affinity spin column (Montage® Albumin Deplete kit, Millipore). However, such approaches are cumbersome and will be understood as inappropriate in the context of POC.

[0071] In one embodiment, the apparatus includes a blocking system for removing or blocking one or more interfering substances from a sample. The blocking system may include a removal reagent capable of removing one or more interfering substances from a sample or blocking one or more interfering substances from interfering with the binding of target nucleosomes to the capture reagent. The removal reagent can be applied to the sample before or during application to the testing apparatus.

[0072] In a preferred embodiment, the testing apparatus further includes a pre-removal site located upstream of the testing site, wherein the pre-removal site includes a removal reagent capable of removing one or more interfering substances from the sample or blocking one or more interfering substances from interfering with the binding of target nucleosomes to the capture reagent. One advantage of the pre-removal site is that sample pretreatment is not required to remove or block interfering substances.

[0073] In one embodiment, the pre-removed portion is located upstream of the conjugate pad. In another embodiment, the pre-removed portion is located between the sample pad and the conjugate pad. In yet another embodiment, the pre-removed portion forms part of the sample pad.

[0074] In one embodiment, a removal reagent capable of removing one or more interfering substances is immobilized at the pre-removal site so that the interfering substances are not transported to the test site by the liquid sample. For example, the removal reagent may be coated on beads (e.g., latex beads) that are large enough not to move through the substrate to the test site.

[0075] In one embodiment, the testing device further includes a false-positive site. The false-positive site indicates that the testing device did not return a valid test result. For example, a false positive may be caused by the test detecting the presence of an interfering substance rather than the presence of a target (i.e., a nucleosome). The false-positive site is sometimes referred to as a false-positive line. In one embodiment, the false-positive site includes a reagent that binds to one or more interfering substances. This embodiment is particularly useful in combination with a blocking system (e.g., a pre-removal site as described herein) because a signal detected at the false-positive site may indicate that the blocking system was not effective in removing the interfering substance.

[0076] (Non-assay-related antibodies) In one embodiment, one or more interfering substances include antibodies unrelated to the assay (non-assay-related). At any stage of immunoassay development, it is important to consider how to control interfering antibodies. Interfering antibodies need to be blocked using specific antibodies and / or non-assay-related antibodies derived from the same species as the assay. For some assays, passive blocking can be utilized by adding purified normal immunoglobulins or subunits derived from the same species as the detection and capture antibodies. Blocking strategies typically rely on the addition of high, several-milligram levels of polyclonal IgG, unrelated mouse monoclonal antibodies, or IgG fragments. This overwhelms the interfering antibody, reducing its probability of binding to the detection or capture antibody. Often, passive approaches may be insufficient due to factors such as the presence of high concentrations or high-affinity antibodies that can overwhelm the blocking system, forcing a more targeted approach. In this case, an active blocking strategy using targeted antibodies that bind to and neutralize specific components of the potential interference may be required. Anti-human immunoglobulins can be added to the sample to agglutinate interfering factors, which can then be removed by centrifugation before performing the assay. However, it is clear that including pre-incubation and centrifugation steps for the sample does not contribute to a rapid and simple point-of-care assay (POC).

[0077] Interference in immunoassays by anti-animal antibodies and other antibodies represents a true antigen / antibody reaction. It is the specific reactivity of these antibodies with the antibody in the immunoassay that can lead to false-positive or false-negative results. This invention utilizes such interactions at a pre-removal site placed before the conjugate zone, so that the sample passes through the pre-removal zone before encountering the conjugate. Advantageously, this arrangement avoids sample pre-treatment and enables a small, relatively inexpensive, disposable device that provides reliable test results.

[0078] In a further embodiment, the non-assay-related antibody is selected from non-assay-related anti-animal antibodies, such as human anti-animal antibodies (HAAA), rheumatoid factor (RF), and / or heterophilic antibodies.

[0079] Human anti-animal antibodies (HAAAs) arise from a genuine immune response that occurs after exposure to animal proteins. The most widely recognized and perhaps most common interference within this group is human anti-mouse antibody (HAMA). However, human antibodies are produced against a variety of species, including rabbits, dogs, goats, sheep, cattle, or rats, and HAAAs often cross-react between species. HAMAs can cause interference in sandwich assays consisting of mouse monoclonals, and other HAAAs can interfere with assays designed using monoclonal or polyclonal antibodies from other species. While the use of capture and detection antibodies from multiple species can mitigate the problem, the tendency for cross-reactivity between species means that the problem may not be completely avoided. Many factors influence the development of these HAAAs or HAMAs in an individual, including exposure to animal species through food handling, pets, or the care of livestock and laboratory animals. This can also occur as a direct result of the administration of monoclonal antibodies for imaging or therapeutic purposes. These antibodies can be high-affinity and reactive to various immunoglobulin domains. Because antibodies are highly conserved across species, antibodies against immunoglobulins of one species can cross-react with immunoglobulins from another species. In this invention, a strategy for controlling assay interference caused by HAAA involves using purified antibody classes of the same species and class used in the lateral flow immunoassay in the pre-removal zone. Thus, interfering factors are isolated from the sample via a binder in the pre-removal zone that targets common epitopes in the interfering factors that are not present in the target analyte. Consequently, the interfering factors are substantially removed from the sample before they can enter the conjugate zone and interact with the detection antibody on the conjugate.

[0080] While most studies on interference in immunoassays relate to human medical research, recent studies have shown that interfering antibodies are also associated with immuno-based laboratory tests, such as endocrine tests, used in veterinary clinical practice. The prevalence of anti-mouse antibodies is estimated to be 9% in dogs, 5% in cats (Bergman et al. (2018) Veterinary Clinical Pathology 47(2):205-212), and 9% in horses (Dong et al. (2021) Acta Veterinaria Scandinavica 63).

[0081] The term heterophilic antibody may be used in the art to broadly refer to human interfering antibodies, such as HAAAs, that have the ability to bind to animal antibodies, antigens, or certain autoantigens. However, references to “heterophilic antibodies” in this specification are used specifically to describe multireactive antibodies to poorly defined or unknown antigens. As described in the literature by Kaplan and Levinson (1999) Clin. Chem. 45(5), if there is no history of medical treatment with animal immunoglobulins or other well-defined immunogens, and the interfering antibody may be shown to be multispecific (reacting with immunoglobulins from two or more species) or to exhibit innate rheumatoid factor activity, then the interfering antibody should be called heterophilic. In contrast, if there is a history of treatment with animal immunoglobulins and immunoglobulins from the same species are used in the examination, then the antibody should be called HAAA.

[0082] Heterophilic antibodies are found in approximately 40% of the population. While typically low affinity and weakly reactive, they can still crosslink detection and capture antibodies in the absence of analytes, effectively competing with analytes for binding sites. Interference can be minimized by adding purified, unrelated antibodies from the same species used in the assay to a pre-removal zone. These unrelated antibodies isolate the heterophilic antibodies from the sample, preventing interference.

[0083] Rheumatoid factor (RF) is an autoantibody that recognizes the Fc region of IgG, primarily of the IgM isotype, but also includes other isotypes. RF can bind not only to human IgG but also to immunoglobulins from other species. It is found in approximately 70% of rheumatoid arthritis patients, some normal populations, and other patients with various autoimmune diseases. An effective approach to controlling the effects of RF is to include unrelated IgG antibodies or IgG Fc regions as components of the pre-removal zone. Alternatively, for example, an anti-RF antibody targeting the entire IgM subclass of antibodies present in the sample can be included in the pre-removal zone.

[0084] Therefore, in one embodiment, the removal reagent at a pre-removal site capable of removing one or more interfering substances from the sample or blocking one or more interfering substances from interfering with the binding of the target analyte to the capture reagent is a removal reagent capable of removing or blocking non-assay-related antibodies.

[0085] In one embodiment, a removal reagent capable of removing or blocking non-assay-related antibodies is: (i) non-immune serum; (ii) Species-specific polyclonal IgG (iii) Anti-human IgG (iv) Polymerized mouse IgG; (v) Non-immune mouse monoclonal; (vi) species-specific IgG fragments (e.g., Fc, Fab, F(ab')2); (vii) Heterophilic blocking reagent (HBR); (viii) Immunoglobulin inhibitor reagents (IIRs) (ix) Protein A; and / or (x) Protein G Includes a removal reagent selected from:

[0086] In a further embodiment, the removal reagent capable of removing or blocking non-assay-related antibodies includes a blocking agent derived from the same species as the capture reagent.

[0087] (Non-assay-related nucleosomes) Nucleosome levels and epigenetic composition may be useful in determining the presence and subtype of cancer, inflammation, or sepsis; however, the identification of specific tumor-derived nucleosomes may be confused with the presence of non-tumor-derived nucleosomes, for example, those arising from tumor and non-tumor-related inflammation or normal hematopoietic cell turnover. Therefore, in one embodiment, one or more interfering substances include nucleosomes.

[0088] In a further embodiment, the nucleosome is a nucleosome containing linker DNA. Each nucleosome in the chromosome of a living cell consists of a protein complex of eight highly conserved core histones (each pair consisting of histones H2A, H2B, H3, and H4). Approximately 145–147 base pairs (bp) of DNA are wrapped around this complex. Another histone H1 can be located on the nucleosome outside the core histones and bind to an additional 20 bp of DNA to create a nucleosome (or chromatosome) containing approximately 165 bp of DNA. This additional DNA is referred to in the art as linker DNA, i.e., DNA that connects one nucleosome to another. The DNA separating two nucleosomes in a chromosome may be longer than 20 bp, and may be up to 80 bp in length, for example, if both adjacent nucleosomes contain H1.

[0089] It has been reported that free nucleosomes derived from tumors can be separated based on nucleosome features related to the presence or absence of linker DNA. It has been stated that most circulating free nucleosomes derived from non-tumor sources can be considered to contain linker DNA, while circulating free nucleosomes derived from tumor sources can be considered to not contain linker DNA. In WO2021 / 038010, the inventors reported that they found that separation can occur by (i) using a histone feature (e.g., H1) to bind to linker DNA, (ii) using a (non-histone) chromatin-binding protein to bind to linker DNA, (iii) using an antibody or other binder to bind to a chromatin-binding protein that binds to linker DNA, or (iv) using an antibody or other binder to bind to nucleosome features related to linker DNA. The apparatus of the present invention can be used in such a manner.

[0090] Therefore, in one embodiment, the removal reagent at a pre-removal site capable of removing one or more interfering substances from a sample or blocking one or more interfering substances from interfering with the binding of the target analyte to the capture reagent is a removal reagent capable of removing or blocking nucleosomes containing linker DNA.

[0091] In one embodiment, a removal reagent capable of removing or blocking nucleosomes containing linker DNA is: (a) Linker DNA associated with nucleosomes; (b) chromatin-binding proteins that bind to linker DNA; or (c) Coanucleosome features associated with linker DNA Contains a binder for bonding.

[0092] According to another aspect of the present invention, a method is provided for separating linker DNA-free circulating free nucleosomes from a biological fluid sample using the apparatus of the present invention, wherein the method is as follows: (i) The sample (a) Linker DNA associated with nucleosomes; (b) chromatin-binding proteins that bind to linker DNA; or (c) Coanucleosome features associated with linker DNA The process of bringing the : into contact with a binder; and (ii) A step of analyzing nucleosomes derived from the sample that were not bound to the binder in step (i). : includes and Here, the binder (a), (b), or (c) forms part of the pre-removed area.

[0093] Surprisingly, the inventors hereby demonstrate that linker DNA-containing nucleosomes can be bound to and removed from plasma samples using the protein histone H1 (itself) immobilized on a solid support as an immunoadsorbent. Furthermore, linker DNA-containing nucleosomes can be bound to and removed from serum, plasma, or other bodily fluid samples using other binding proteins that bind to linker DNA-containing nucleosomes in a similar manner. Linker DNA-containing nucleosomes can also be bound to and removed from serum, plasma, or other bodily fluid samples using antibodies or other binders directed to bind to molecules that bind to linker DNA-containing nucleosomes in a similar manner.

[0094] In one embodiment, the binding agent that binds to the linker DNA associated with the nucleosome is a histone protein selected from histone H1, macroH2A, or fragments or modified analogs thereof.

[0095] It will be understood that the method of the present invention may be carried out using histone H1, or a fragment or modified analog thereof. Histone H1 has several isoforms known in the art. In one embodiment, histone H1 is histone H1.0.

[0096] Similar to histone proteins, linker DNA can be bound using many (non-histone) chromatin-binding proteins. Such proteins include, but are not limited to, chromodomain helicase DNA-binding proteins (CHDs), DNA (cytosine-5)-methyltransferases (DNMTs), high-mobility or high-mobility box proteins (HMGs or HMGBs), poly[ADP-ribose] polymerases (PARPs), proteins containing methyl CpG-binding domains (MBDs), such as MECP2, and certain transcription factors that bind to nucleosome linker DNA in a sequence-independent manner, such as the transcription factor p53.

[0097] In one embodiment, the binder binds to a chromatin-binding protein that binds to a nucleosome containing linker DNA. The reference to “chromatin-binding protein” that binds to a nucleosome in the presence of linker DNA refers to a protein that binds to (i.e., becomes an adduct to) the linker DNA portion of a free nucleosome. Therefore, a chromatin-binding protein that preferentially or selectively binds only in the presence of linker DNA can be used as a marker to determine whether linker DNA is present in a free nucleosome. Examples of such proteins, though not limited to, include proteins of the MBD, CHD, DNMT, and PARP families. It will be understood that these protein families include multiple members. For example, there are at least nine CHD family members, CHD1–CHD9. The term “chromatin” will be understood to refer to the complex formed from DNA and histone proteins when DNA is packaged within a cell. Therefore, histones are components of chromatin, and the term “chromatin-binding protein” does not include the histone proteins themselves. Therefore, histone proteins, such as H1, H2A, H2B, H3, and H4, are not chromatin-binding proteins; in other words, chromatin-binding proteins are not histone proteins.

[0098] In a further aspect of the present invention, a binder is used that binds to core nucleosome features associated with linker DNA. The reference to “core nucleosome features” associated with linker DNA refers to the features of core nucleosome particles (e.g., core histone proteins, one of H2A, H2B, H3, or H4, including their post-translational modifications and isoforms) that occur only in or primarily in nucleosomes containing linker DNA. In some cases, the nucleosome features can bind to linker DNA, stabilize it, or protect it from nuclease digestion.

[0099] Nucleosomes containing the histone isoform macroH2A are thought to stabilize linker DNA (Bowerman and Wereszczynski (2016) Biophys. J. 110: 327-337). The inventors have found that this type of histone isoform is more preferentially associated with free nucleosomes containing linker DNA and can therefore be used as a marker for separating healthy nucleosomes from biological samples. Accordingly, in one embodiment, the core nucleosome feature associated with linker DNA is the histone isoform macroH2A.

[0100] In one embodiment, a nucleosome containing DNA of approximately 150 base pairs or longer refers to a nucleosome that holds linker DNA. The nucleosome core particle (i.e., a histone octamer composed of each pair of histone H2A, H2B, H3, and H4) consists of approximately 150 bp of DNA, for example, 145-150 bp of DNA, particularly 147 bp of DNA. Therefore, nucleosomes containing DNA larger than 150 bp (e.g., DNA larger than 155 bp, 160 bp, or 165 bp) contain linker DNA. In one embodiment, the binder binds to nucleosomes consisting of at least 165 base pairs of DNA. In one embodiment, the binder does not bind to nucleosomes containing DNA of approximately 150 bp or less. That is, the binder selectively binds to nucleosomes containing linker DNA.

[0101] In one embodiment, the binder binds to a region within the linker DNA. In this embodiment, the binder binds (directly) to a region of DNA that is not in contact with the histone (core) octamer, i.e., to the linker DNA.

[0102] (Detection method) According to a further aspect of the present invention, a method for determining the presence, absence, and / or quantity of target nucleosomes in a sample, a) A step of bringing a liquid sample into contact with an inspection apparatus as described herein, wherein the liquid sample is applied to a part of the inspection apparatus upstream of the inspection site; and b) A step of evaluating detectable signals at the test site to determine the presence, absence, and / or quantity of target nucleosomes in the sample. A method is provided that includes:

[0103] In one embodiment, the presence of a detectable signal at the test site indicates the presence of target nucleosomes in the sample. In an alternative embodiment, the absence of a detectable signal at the test site indicates the presence of target nucleosomes in the sample.

[0104] In some embodiments, the sample is applied to a conjugate pad. In some embodiments, the sample is applied to a pre-removal site. In some embodiments, the sample is applied to a sample pad.

[0105] As used herein, the term “sample” refers to anything that may contain the analyte from which the analyte assay is desired. In some embodiments, the sample is or includes a biological sample. As used herein, “biological sample” includes any sample obtained from a biological or viral (or prion) source or other source of macromolecules and biomolecules, and includes any cell type or tissue of an object from which nucleic acids, proteins, and / or other macromolecules can be obtained. A biological sample may be a sample obtained directly from a biological source or a processed sample. For example, isolated and amplified nucleic acids constitute a biological sample. Biological samples include, but are not limited to, bodily fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat, animal and plant-derived tissue and organ samples, and processed samples derived therefrom. In some embodiments, the sample may be derived from tissue or body fluid, such as connective tissue, epithelial tissue, muscle tissue, or nerve tissue; tissue selected from the group consisting of the brain, lungs, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidneys, gallbladder, stomach, intestines, testes, ovaries, uterus, rectum, nervous system, glands, and blood vessels in the body; or body fluid selected from the group consisting of blood, cerebrospinal fluid (CSF), sputum, urine, saliva, bone marrow, semen, cystic fluid (e.g., pancreatic cystic fluid), synovial fluid, and ascites, and their subtractions, such as serum or plasma. In certain embodiments, the body fluid is selected from blood, serum, or plasma. In some embodiments, the sample is a liquid sample, such as a biological liquid sample.

[0106] References to actions performed on bodily fluid samples "obtained" from a subject are intended to include actions performed only on bodily fluid samples already obtained or "available" from the subject, and vice versa.

[0107] The detectable signal can be evaluated by any suitable means. For example, the detectable signal can be evaluated by visual inspection by a user. In another example, the detectable signal can be evaluated by a reader. Any suitable reader can be used. In some embodiments, the detectable signal is a fluorescent signal, and the fluorescent signal is evaluated by a fluorescent reader. Any suitable fluorescent reader can be used. For example, the fluorescent reader may be a laser-based or light-emitting diode (LED)-based fluorescent reader. The reader comprises one or more photodetectors.

[0108] The detected signal can be used to quantify the amount of analytes present in the sample. Therefore, in one embodiment, a detectable signal at the test site is evaluated to determine the amount (i.e., quantity) of target nucleosomes in the sample. Commercial instruments for quantifying the signal detected by lateral flow analysis, such as the ESEQuant LR3 or ESEQuant Flex from DIALUNOX GmbH, are available in the art.

[0109] Similar to conventional lateral flow testing formats, the present invention is also suitable for use with adapted versions of this format. For example, the device may be formatted to capture multiple analytes at once, for example, in a multiplex format. The most frequently used structure for achieving multiplex detection of biomarkers in LFIA is a test strip with multiple test sites, where each test site can detect a specific biomarker. For example, a single strip can be used to test a single patient for many different biomarkers in the same run. An alternative structure used for multiplex detection of biomarkers is a multi-channel structure with multiple test strips, where each test strip has one test site for detecting a specific biomarker. One example of a multiplex system is the BlueDiver Dot system, which consists of 24 nitrocellulose strips coated with an antigen in a dot-like format that can be read by a specific instrument. Similarly, the LightDeck MINI system is an automated fluorescence analyzer. Planar waveguides with printed microarrays are assembled into disposable plastic cartridges. Each spot within the microarray measures a biomarker or a built-in control. Light is focused through an integrated lens, creating an evanescent field just above the waveguide surface. This evanescent field excites only the fluorescent dye bound by the capture antibody on the waveguide.

[0110] In one embodiment, the lateral flow apparatus is a multiplexed lateral flow apparatus. The multiplexed apparatus can provide parallel detection of various combinations of biomarkers. For example, the multiplexed apparatus may be applicable to the parallel detection of histone isoforms (e.g., H3.1 and / or H3.3) and linker DNA. Furthermore, non-nucleosome biomarkers may also be useful in the multiplexed apparatus. For example, a multiplexed apparatus for use in the diagnosis and monitoring of sepsis may also detect procalcitonin (to distinguish between bacterial and viral infections), myxovirus resistance protein A (MxA) (to further elaborate on viral and bacterial infections), and / or proadrenomedullin (MRproADM) (as a severity marker).

[0111] (Detection of free nucleosomes) The present invention is applicable to the detection of (free) nucleosomes themselves in a sample. Accordingly, according to a further aspect of the present invention, the target nucleosome is a free nucleosome, and one or both of the labeled detection reagent and / or capture reagent bind to the nucleosome itself.

[0112] (Detection of post-translational modifications of histones in free nucleosomes) The present invention is applicable to the diagnosis of disease conditions such as cancer and autoimmune diseases by analyzing free nucleosomes containing histone post-translational modifications (e.g., as reported in WO2005 / 019826).

[0113] Therefore, according to a further aspect of the present invention, the target nucleosome is a nucleosome comprising a histone post-translational modification, and the labeled detection reagent and / or capture reagent binds to the histone post-translational modification.

[0114] In one embodiment, the labeled detection reagent specifically binds to the target histone post-translational modification, and the capture reagent binds to the nucleosome itself, for example, by binding to a three-dimensional nucleosome epitope.

[0115] (Detection of circulating tumor-derived nucleosomes) The present invention is applicable, for example, to the detection of tumor-derived free nucleosomes from bodily fluid samples, as described in WO2016 / 067029. In particular, this embodiment relates to the use of histone H3.1 and / or H3.2 and / or H3t binders in the apparatus of the present invention for detecting tumor-derived nucleosomes and / or circulating tumor DNA (ctDNA) from biological samples.

[0116] Therefore, according to one aspect of the present invention, the target nucleosome is a tumor-derived nucleosome, and the labeled detection reagent and / or capture reagent specifically binds to histones H3.1, H3.2 and / or H3t.

[0117] In one embodiment, the labeled detection reagent and / or capture reagent binds to H3.1. In a further embodiment, the labeled detection reagent and / or capture reagent is an anti-histone H3.1 antibody. The anti-histone H3.1 antibody may be directed to bind to an epitope around amino acids 30-33 of the histone H3.1 protein in order to capture clipped and unclipped nucleosomes.

[0118] In a preferred embodiment, the labeled detection reagent specifically binds to histones H3.1, H3.2, and / or H3t, and the capture reagent specifically binds to the nucleosome itself, for example, by binding to a structural nucleosome epitope. In a further preferred embodiment, the labeled detection reagent specifically binds to histone H3.1, and the capture reagent specifically binds to a structural nucleosome epitope.

[0119] (Detection of neutrophil extracellular traps (NETs)-derived nucleosomes) The present invention is applicable to the detection of NET-derived nucleosomes as described in WO2021 / 186037.

[0120] Therefore, according to one aspect of the present invention, the target nucleosome is a part of or a free nucleosome derived from a neutrophil extracellular trap. In one embodiment, the labeled detection reagent and / or capture reagent specifically binds to H3.1. In another embodiment, the labeled detection reagent and / or capture reagent specifically binds to an epigenetic epitope of a nucleosome associated with NETs (e.g., a citrullinated nucleosome).

[0121] Many proteins are generated in NETs that are directly or indirectly attached to nucleosomes. These proteins include, but are not limited to, myeloperoxidase (MPO), neutrophil elastase (NE), lactotransferrin, azulocidine, 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 β, actin γ, alpha-actin, plastin-2, cytokeratin-10, catalase, alpha-enolase, and transketolase (Urban et al. (2009) PLOS Pathogens. 10: e1000639). Any nucleosome-protein adduct generated in NETs is a useful adduct for detecting elevated levels of NETs in the method of the present invention. For example, C-reactive protein (CRP) can also be added to nucleosomes in NETs, ​​and therefore, nucleosome-CRP adducts are useful adducts for detecting elevated levels of NETs in the method of the present invention. Accordingly, a lateral flow analyzer may include an antibody directed to bind to CRP. This can be used in conjunction with an anti-nucleosome antibody (optionally labeled, for example, as a labeled detection reagent) directed to bind to a structural nucleosome epitope.

[0122] Citrullinated nucleosomes are also associated with NETs. Therefore, lateral flow analyzers may include antibodies directed to bind to citrullinated nucleosomes, for example, nucleosomes citrullinated with arginine 8 of histone H3 (anti-H3R8Cit antibodies). This can be used in conjunction with anti-nucleosome antibodies directed to bind to structural nucleosome epitopes (optionally labeled, for example, as labeled detection reagents).

[0123] (Detection of nucleosomes derived from non-dividing cells) In most cells and tissues, the majority of present histone H3 consists of histone isoforms H3.1 and H3.2. Histone isoform H3.3 is found in open chromatin regions and constitutes a minor component. Two major mechanisms for histone H3 uptake into nucleosomes are described in the literature: “replication coupled” (RC) and “replication independent” (RI) histone uptake. In 2002, Ahmad and Henikoff reported that histone variant H3.3 is uptaken into chromatin throughout the entire cell cycle without any corresponding DNA replication and is RI, while the uptake of (canonical H3.1 and H3.2) H3 is limited to DNA during replication and is RC (Ahmad and Henikoff (2002) PNAS 99: 16477-16484).

[0124] Brain tissue contains dividing cells in fetuses, but non-dividing (e.g., very slowly dividing) cells in adult animals. In 2015, Maze et al. studied the histone isoform composition of fetal and adult brains in mice and humans using mass spectrometry (Maze et al. (2015) Neuron 87: 77-94). They reported that the histone isoform composition of dividing brain cells in fetuses mainly consists of canonical histone H3 isoforms H3.1 and H3.2. When fetal brain cell division stops, RC uptake of histone H3.1 and H3.2 stops, but RI uptake of histone H3.3 continues, leading to the accumulation of histone isoform H3.3 in both neurons and glial cells in the brain. It was found that histone H3 present in primary mouse neurons contains less than 20% isoform H3.3. However, at 24 months of age, histone H3.3 accumulated to constitute 94% of the total histone H3 pool in the adult mouse brain, becoming the dominant H3 isoform, if not exclusively expressed. Similar results have been reported in humans. Histone isoform H3.3 was found to constitute approximately 31% of the total H3 pool in the (postmortem) fetal human brain. However, H3.3 gradually accumulated to constitute over 93% of the total H3 pool in the brains of human individuals aged 14 to 72 years.

[0125] The brain is not the only organ that contains long-lived cells with long cell turnover times. Subsequent research has confirmed the findings of Maze et al., finding that other slowly dividing somatic cell tissues, including the liver, kidneys, and heart, also undergo similar age-dependent, genome-wide accumulation of histone isoform H3.3 (Tvardovskiy et al. (2017) Nucleic Acids Res. 45: 9272-9289).

[0126] Therefore, the present invention is applicable to the detection of nucleosomes derived from non-dividing cells. In this specification, the term "non-dividing cells" includes slow-dividing cells. In general terms, slow-dividing cells refer to cells with a turnover of 200 days or more. Such cells include those derived from adipose tissue, the adrenal glands, bone marrow, cardiac muscle, keratinocytes, kidneys, liver, lungs, skeletal muscle, and thyroid gland. Non-dividing cells, such as slow-dividing cells, also include cells of the central nervous system (CNS), including brain cells such as neurons and glial cells. Such CNS cells, including brain cells, are particularly derived from adolescents and adults. Adolescence can be defined as the stage of life between childhood and adulthood, from age 10 to 19.

[0127] Therefore, according to one aspect of the present invention, the target nucleosome is a free nucleosome released into circulation or other bodily fluids from a slowly dividing or non-dividing cell. In one embodiment, the labeled detection reagent and / or capture reagent specifically binds to H3.3.

[0128] (Methods of diagnosis and monitoring) A further aspect of the present invention relates to a method for diagnosing a disease or disorder in a subject, (i) A step of determining the presence or absence of a target nucleosome in a sample using a method defined herein; (ii) A process in which the presence of a target nucleosome is used as an indicator of the presence of a disease or disorder in the subject. A method is provided that includes:

[0129] A further aspect of the present invention relates to a method for monitoring the progression of a disease or disability in a subject, (i) A step of determining the presence, absence, and / or amount of target nucleosomes in a body fluid sample obtained from a subject using a method defined herein; (ii) A process that repeats process (i) once or more times; and (iii) A process of monitoring the progression of a disease or disorder in a subject using any change in the presence, absence, and / or quantity of a target nucleosome. A method is provided that includes:

[0130] A further aspect of the present invention is a method for assigning a risk of adverse outcomes to a subject, (i) the step of determining the presence, absence, and / or amount of target nucleosomes in a body fluid sample obtained from a subject using a method defined herein; and (ii) A step of assigning potential adverse outcomes to the subject using the presence, absence, and / or quantity of the target nucleosome. : includes, Here, a method is provided for assigning subjects identified as being at high risk of adverse outcomes to medical intervention.

[0131] References to "subject," "individual," or "patient" are used interchangeably herein. A subject may be a human or an animal subject. In one embodiment, the subject is human. In one embodiment, the subject is a (non-human) animal.

[0132] In some embodiments, the present invention encompasses animal subjects (wild or domesticated). In some embodiments, the present invention relates to veterinary uses, including those for livestock and companion animals, such as cats, dogs, horses, sheep, goats, pigs, deer, llamas, cows, and cattle.

[0133] Detection and / or quantification can be compared to a cutoff level. The cutoff value can be predetermined by analyzing results from multiple patients and controls and determining a suitable value for classifying subjects as having or not having the disease or disorder (sometimes referred to as “disease”). For example, for a biomarker level being higher in patients suffering from the disease, a detected level higher than the cutoff indicates the patient is suffering from the disease. Alternatively, for a biomarker level being lower in patients suffering from the disease, a detected level lower than the cutoff indicates the patient is suffering from the disease. Advantages of using a simple cutoff value include ease of understanding the test for clinicians and the elimination of the need for software or other aids in interpreting the test results. The cutoff level can be determined using methods available in the art.

[0134] Detection and / or quantification may also be compared with a control. It will be apparent to those skilled in the art that control subjects can be selected according to various criteria, and may include, for example, subjects known to be free of the disease, or subjects with different diseases (e.g., for differential diagnosis). “Control” may include healthy subjects and / or subjects without the disease. A control may be a subject with the disease, but showing no symptoms or only mild symptoms. Mild symptoms may include manageable symptoms that do not require hospital intervention and / or intensive medical treatment.

[0135] In one embodiment, a subject showing a positive reaction by the method of the present invention is suffering from the disease and will suffer further or continue to suffer from further medical complications. In contrast, a control subject may also be suffering from the disease, but will not suffer from or continue to suffer from medical complications. Comparison with controls is well known in the field of diagnosis. The range of values ​​observed in the control group can be used as normal, healthy, or a reference range, and the values ​​observed in the test subject can be compared to this.

[0136] Therefore, in one embodiment, the method further includes comparing the level of target nucleosomes with one or more controls. For example, the method may include comparing the level of target nucleosomes present in a sample obtained from a subject with the level of target nucleosomes present in a sample obtained from a normal subject. The controls may be healthy subjects.

[0137] In one embodiment, the level of the target nucleosome is elevated compared to the control.

[0138] It will be understood that it is not necessary to measure control levels every time for comparative purposes. For example, once a “normal range” is established for healthy controls, it can be used as a benchmark for all subsequent tests. The normal range can be established by taking samples from multiple control subjects who do not have the disease and examining the levels of the biomarker. Then, the results (i.e., biomarker levels) of subjects suspected of having the disease can be examined to see if they are within or outside their respective normal ranges. The use of “normal ranges” is standard practice for disease detection.

[0139] Holdenrieder et al. (2001) Int. J. Cancer 95: 114-120 previously described the detection of nucleosome levels in serum samples from patients with benign and malignant diseases. Thus, in one example, the target nucleosome is the nucleosome itself.

[0140] Several studies have shown that cfDNA increases with both high-intensity and endurance exercise (Vittori et al. (2019) Methods Mol Biol. 1909:183-197). Since free DNA levels correlate with nucleosome levels in the blood, this method could potentially be used to monitor overtraining during exercise.

[0141] The epigenetic composition of circulating free nucleosomes is also being investigated as a blood-based biomarker in cancer, in terms of its histone modifications, histone variants, DNA modifications, and adduct content. See WO 2005 / 019826, WO 2013 / 030577, WO 2013 / 030579, and WO 2013 / 084002.

[0142] Therefore, in one example, the target nucleosome is a tumor-derived nucleosome, and the disease is a cancer such as lung cancer, small cell lung cancer, gastrointestinal cancer, intestinal cancer, colon cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, or melanoma.

[0143] The usefulness of free nucleosomes as a biomarker in patients with sepsis and NETosis-related diseases such as COVID-19 has also been previously described (see WO 2021 / 1186037).

[0144] Therefore, in one embodiment, the target nucleosome is a free nucleosome that is part of or derived from a neutrophil extracellular trap, and the disease or disorder is a NETosis-related disease or disorder. In one embodiment, the NETosis-related disease or disorder is an infection. In one embodiment, the infection is a viral, bacterial, fungal, or microbial infection. Bacterial infections may include mycobacteria, pneumococci, and Haemophilus influenzae infections, e.g., infections caused by Streptococcus pneumoniae, Escherichia coli, Mycobacterium tuberculosis, Haemophilus influenzae, and Staphylococcus aureus (e.g., pneumonia). In a further embodiment, the infection is a viral infection. Viral infections may include infections caused by respiratory syncytial virus (RSV), influenza A, influenza B, and coronaviruses (e.g., COVID-19).

[0145] An infectious disease can be defined by the tissue affected by the disease. For example, a disease may affect the heart, brain, kidneys, liver, pancreas, lungs, and / or blood, and an infectious disease may be a bacterial, viral, fungal, or microbial infection that is generally known to affect such tissues or organs. In one embodiment, the infectious disease is a respiratory infection. According to this embodiment, the infectious disease affects the lungs, upper respiratory tract, and / or lower respiratory tract.

[0146] Other tissues that may be affected by the disease include peripheral tissues such as the 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 artery failure, and other tissues or organs affected by the disease may include the kidneys and renal system and / or the brain (e.g., stroke). In yet another example, the disease may affect the lungs, or the infection may be a respiratory infection, and other tissues or organs affected may include the heart, coronary artery system, and / or the brain (e.g., heart failure, myocardial infarction, and / or stroke).

[0147] In one embodiment, the respiratory infection is selected from influenza, pneumonia, and severe acute respiratory syndrome (SARS). SARS is a respiratory infection caused by the SARS coronavirus (SARS-CoV), and other related coronaviruses are known (e.g., COVID-19 (also known as SARS-CoV-2, and formerly as 2019-nCoV)). It is known to cause fever, flu-like symptoms, cough, and malaise, and can lead to pneumonia (e.g., direct viral pneumonia or secondary bacterial pneumonia).

[0148] In one embodiment, this method may be performed by a subject for the purpose of self-monitoring.

[0149] (sepsis) Sepsis is a life-threatening condition characterized by one or all of the following: low blood pressure, accelerated heart rate, pain, fever and chills with sweating, shortness of breath, and disorientation or confusion. A patient with sepsis may rapidly deteriorate within hours, potentially leading to septic shock with low blood pressure, stroke, respiratory failure, heart failure, or multiple organ failure. Without emergency treatment, sepsis can be fatal and is a leading cause of death worldwide. Therefore, point-of-care tests, such as LFT described herein, are crucial in monitoring patients suspected of having sepsis.

[0150] Immunologically, sepsis is characterized by an extreme immune response to infection or other invasion, including elevated cytokine release and elevated production and release of NETs. We have previously observed that sepsis results in a high level of circulating free nucleosomes containing histone isoform H3.1, and that these levels correlate with the SOFA score (Sequential Organ Failure Assessment Score) in septic shock (Morimont et al., (2022) Biomolecules 12: 1038). Therefore, the LFT described herein can be used in methods for monitoring and diagnosing sepsis.

[0151] The methods described herein are particularly useful in patients suffering from systemic inflammatory response syndrome (SIRS). SIRS is an inappropriate bodily response to an invasive event, characterized by an inappropriately high level of NETosis. The invasive event may be infectious or non-infectious, and may include trauma, surgery, acute inflammation, ischemia, reperfusion, malignancy, and many others. In one embodiment, the subject is suffering from (or suspected of suffering from) SIRS. In one embodiment, the subject is suffering from (or suspected of suffering from) an infection. In one embodiment, the subject is suffering from (or suspected of suffering from) sepsis or septic shock.

[0152] (Organ transplant and prosthesis malfunctions) Extremely high levels of NETs have been observed in in vivo organs (Dengu et al. (2022), Annual Congress 2022, Int Liver Transplant Society, Istanbul, May 4-7, 2022). Upon removal from the donor's body, in vitro donor organs rapidly become inflamed, accompanied by a large co-release of NETs. This inflammation causes organ damage with loss of function, and such organs are discarded as unviable for transplantation. The NETs generated in in vitro organs can be measured, for example, as an increase in the level of extra vivo nucleosomes (e.g., those containing histone H3 isoform H3.1) in the perfusion fluid of the perfusion circuit containing the organ. Therefore, the LFT described herein can be used in methods for monitoring or evaluating the health status of donor organs or tissues.

[0153] Once a donor organ is transplanted into a recipient in need of the organ, the recipient must be further monitored for the health of the transplanted organ and for organ rejection. This is currently done using slow, low-throughput, and very expensive cfDNA testing methods. Briefly, a blood sample is taken from the recipient, and then cfDNA is extracted from the sample and sequenced to search for any sequences corresponding to the donor DNA. Since any donor DNA sequence originates from the transplanted organ, the presence of cfDNA of the donor sequence indicates the possibility of cell death and rejection of the donated organ. In contrast to these cfDNA methods, the method of the present invention is low-cost, requires only a fraction of the blood volume required for cfDNA sequencing, and can be performed quickly to provide information for patient management. Therefore, the LFT described herein can be used in a method for predicting or assessing the risk of rejection of a donor organ or tissue transplanted into a subject.

[0154] Therefore, in one embodiment, the donor organ is selected from the kidney, liver, heart, lung, pancreas, stomach, or intestine. In one embodiment, the donor tissue is selected from whole blood, plasma, platelets, cornea, bone, tendon, skin, pancreatic islets, heart valves, nerves, veins, bone marrow, or stem cells. In one embodiment, the donor tissue is a limb such as a hand, arm, or foot.

[0155] As described regarding transplant rejection, subjects who have received medical implants or prostheses also require monitoring in case of rejection. Elevated or high levels of NETs or inflammatory markers can be used as warning signs that the implant or prosthesis has been rejected. Therefore, the LFT described herein can be used in a method for predicting or assessing the risk of failure (or rejection) of medical implants or prostheses in subjects.

[0156] In one embodiment, the prosthesis is an orthopedic implant such as a hip or knee joint prosthesis. In another embodiment, the medical implant is a cardiovascular implant (e.g., a defibrillator, pacemaker, stent, or catheter), a cochlear implant, a cosmetic implant, a dental implant, or an intraocular implant (e.g., an artificial lens).

[0157] Furthermore, high levels of NETs are pathological and cause tissue damage and cell death. However, since cells derived from the liver, lungs, kidneys, and heart are all or primarily slow-growing tissues containing nucleosomes with histone isoform H3.3, this damage cannot be detected by measuring nucleosomes containing histone H3 isoform H3.1. Therefore, the method of the present invention can be used to measure nucleosomes containing histone isoform H3.3 as an indicator of tissue damage and cell death in in vitro organs, implants, or prostheses. Dual measurement of both nucleosomes containing histone H3 isoform H3.1 and nucleosomes containing H3.3 can be used to determine both the level of NETs present and the level of tissue damage caused.

[0158] (system) According to a further aspect of the present invention, a system for evaluating a sample, a) Testing apparatus described herein; and b) A reader for detecting detectable signals A system including the following is provided.

[0159] In one embodiment, the reader comprises a light source and a photodetector. In a further embodiment, the reader further comprises a barcode detector and / or a radio frequency identification (RFID) detector.

[0160] It will be understood that all embodiments described herein may apply to all aspects of the present invention. Other features and advantages of the present invention will become apparent from the description provided herein. However, it should be understood that this description and examples, while illustrating preferred embodiments, are given only as examples, as various changes and modifications will become apparent to those skilled in the art.

[0161] The present invention will be illustrated by the following embodiments. [Examples]

[0162] (Example 1: Comparative study of H3.1 LFT and IDS-i10 automated immunoassay analyzer system) Lateral flow assays (LFTs) were prepared for nucleosomes containing histone isoform H3.1, which includes the following components: • Sample pad - 18mm Ahlstrom 222 sample pad • Absorbent pad - 18mm Ahlstrom 222 absorbent pad • Substrate - Unisart® CN 95 nitrocellulose membrane with support (25mm; Sartorius catalog number 1UN95E) • Support material - 6.1cm support card • Conjugate pads - Ahlstrom 8964 10mm pads Conjugate pad processing buffer - Dipotassium hydrogen phosphate (1.4 g / L); Potassium dihydrogen phosphate (0.31 g / L); Bovine serum albumin (5 g / L); Surfactant 10 g (1 ml / L); Deionized water. • Capture reagent - Anti-nucleosome antibody diluted to 0.1 mg / ml in 50 mM KPO4 (applied as a striping at 8 μl / cm). • Labeled detection reagent - Anti-H3.1 antibody conjugated to 40 nm colloidal gold.

[0163] Samples were prepared in Candor Assay Defender Buffer and applied to LFT. Results were read using a Dialunox lateral flow reader.

[0164] The results obtained by LFT were compared with the Nu.Q® H3.1 immunoassay performed using the IDS-i10 automated immunoassay system. For the immunoassay, the test samples were analyzed at the nucleosome level containing histone isoform H3.1 (H3.1-nucleosome) using an automated immunoassay system. The assay measurements were performed using an automated immunoassay system. Briefly, the calibrant or sample was incubated with acridinium ester-labeled anti-nucleosome antibody and assay buffer at 37°C for 1800 seconds. Magnetic beads coated with anti-histone H3.1 antibody were added, and the mixture was incubated for a further 900 seconds. The magnetic beads were then isolated, washed three times, and the acridinium ester bound to the magnetic beads was determined by luminescence output over 7000 milliseconds.

[0165] Figure 3 shows a good correlation between results obtained using LFT and IDS-i10 immunoassay in 40 K2EDTA plasma samples from human subjects.

[0166] Furthermore, the results obtained by LFT were compared with those obtained from the Nu.Q® H3.1 immunoassay performed using an ELISA plate (Volition NuQ® H3.1 ELISA kit). Figure 4 shows the correlation between LFT and ELISA results in samples from 122 patients from three different groups (healthy controls / COVID-19 / sepsis). The level ranges obtained with the H3.1 ELISA (Nu.Q(registered trademark) H3.1) kit were (95% confidence intervals) 24.63 ng / ml (20.50~30.60), minimum: 8.30 ng / ml to maximum: 160 ng / ml; 2490.79 ng / ml (1780.91~3316.48); minimum: 76.02 ng / ml to maximum: 5272.61 ng / ml; 823.23 ng / ml (66.35~1085.65), minimum: 48.62 ng / ml to maximum: 23056.41 ng / ml.

[0167] Figure 5 shows a discriminant box plot illustrating the significant difference in H3.1 levels detected by LFT between the healthy control group and the COVID-19 / sepsis group.

[0168] (Example 2: H1 removal) A 65-year-old male smoker with a smoking history of over 30 years and confirmed pulmonary nodules by low-dose CT was initially identified as H3.1 nucleosome-positive by lateral flow assay involving a conjugate pad containing gold beads coated with anti-H3.1 antibody and a test line striped with anti-nucleosome antibody. Lumos levels from the lateral flow reader were above 200 ng / mL. To pre-remove nucleosomes with linker DNA, the patient was retested using a second lateral flow assay with histone 1 globular domains striped in front of the conjugate pad. Lumos levels decreased to a nucleosome concentration corresponding to approximately 100 ng / mL.

[0169] (Example 3: HAMA removal) A 75-year-old male smoker with a smoking history of over 30 years is initially identified as H3.1 nucleosome-positive by lateral flow assay involving gold beads coated with anti-H3.1 antibody in a conjugate pad and a test line striped with anti-nucleosome antibody. Lumos levels from the lateral flow reader are above 350 ng / mL. This patient is screened by low-dose CT, but no nodules are confirmed. The patient sample is incubated with a commercially available HAMA removal reagent and retested, and the lumos levels are below detectable levels, indicating that the above was a false positive reading due to human anti-mouse antibody (HAMA). The patient is retested using a second lateral flow assay with mouse IgG striped before the conjugate pad to pre-remove HAMA. No visible test line is observed, but a positive control line is present. Lumos levels from the lateral flow reader are below detectable levels.

[0170] (Example 4: Rheumatoid factor removal) A 70-year-old male smoker with a smoking history of over 30 years and being treated with methotrexate for rheumatoid arthritis is initially identified as H3.1 nucleosome-positive by lateral flow assay involving a conjugate pad containing anti-H3.1 antibody-coated gold beads and a test line striped with anti-nucleosome antibodies. Lumos levels from the lateral flow reader are above 500 ng / mL. The patient is screened by low-dose CT, but no nodules are confirmed. To pre-clear rheumatoid factor (RF), the patient is retested using a second lateral flow assay with human IgG Fc regions striped in front of the conjugate pad. No visible test line is observed, but a positive control line is present. Lumos levels from the lateral flow reader are below detectable levels.

[0171] (Example 5: Removal of rheumatoid factor using beads) A 70-year-old male smoker with a smoking history of over 30 years, treated with methotrexate for rheumatoid arthritis with identified rheumatoid factor, and negative lung nodule screening was examined. The patient was tested for H3.1 nucleosomes by a lateral flow assay involving a conjugate pad containing gold beads coated with anti-H3.1 antibody and a test line striped with anti-nucleosome antibody. This assay included human IgG Fc regions coated on 10 μm latex beads embedded in a sample pad prior to the conjugate pad. Lumos values ​​were below detectable levels, and a positive control line was observed. Repeating the lateral flow without the IgG Fc coated beads revealed a false-positive test line in addition to the control line.

[0172] (Example 6: Removal of heterophilic antibodies from canine samples) An 8-year-old male Labrador is identified as being at high risk of cancer by lateral flow assays including gold beads coated with anti-H3.1 antibodies in a conjugate pad and a test line striped with anti-nucleosome antibodies, due to the evident presence of H3.1 nucleosomes exceeding 100 ng / mL. Lumos values ​​from the lateral flow reader show levels exceeding 300 ng / mL. Because the animal is asymptomatic and appears healthy, it is retested using a second lateral flow assay. This assay included a combination of canine and mouse IgG striped in front of the conjugate pad to pre-clear heterophile antibodies (HF). No visible test line is identified, but a positive control line is present. Lumos values ​​from the lateral flow reader are below detectable levels.

[0173] (Example 7: Removal of heterophilic antibodies from dog samples using beads) An 8-year-old male Labrador Retriever, previously identified as having heterophilic antibodies at levels that produce a false-positive signal in a lateral flow-based cancer screening test involving anti-H3.1 antibody-coated gold beads in a conjugate pad and a test line striped with anti-nucleosome antibodies, is re-tested using a modified H3.1 nucleosome lateral flow containing a combination of canine and mouse IgG coated on 10 μm latex beads embedded in a sample pad prior to the conjugate pad. No visible test line is observed, but a positive control line is present. Lumos values ​​from the lateral flow reader are below detectable levels.

Claims

1. A lateral flow analyzer for detecting target nucleosomes in a sample, (i) Substrate including inspection area containing capture reagent; and (ii) A conjugate pad located upstream of the substrate, comprising a labeled detection reagent, the labeled detection reagent moving toward the test site along with the liquid sample and / or further liquid to generate a detectable signal; The lateral flow analyzer comprising: a labeled detection reagent and a capture reagent, wherein both the labeled detection reagent and the capture reagent bind to the target nucleosome or its components.

2. The inspection apparatus according to claim 1, wherein the labeled detection reagent and / or capture reagent binds to the nucleosome itself.

3. The inspection apparatus according to claim 1 or 2, wherein the labeled detection reagent and / or capture reagent specifically bind to a stereochemical nucleosome epitope.

4. The inspection apparatus according to any one of claims 1 to 3, wherein the labeled detection reagent and / or capture reagent specifically bind to unique epitopes formed by post-translational modifications of core histones.

5. The inspection apparatus according to any one of claims 1 to 4, wherein the labeled detection reagent and / or capture reagent specifically binds to histone H3.1, H3.2, and / or H3t.

6. The inspection apparatus according to any one of claims 1 to 5, wherein the labeled detection reagent and / or capture reagent specifically binds to histone H3.1 and / or citrullinated histone H3R8 (H3R8Cit).

7. The inspection apparatus according to claim 1, wherein the labeled detection reagent specifically binds to histone H3.1, and the capture reagent specifically binds to a stereochemical nucleosome epitope.

8. The inspection apparatus according to any one of claims 1 to 7, wherein the capture reagent directly binds to the target nucleosome.

9. The inspection apparatus according to any one of claims 1 to 7, wherein the capture reagent indirectly binds to the target nucleosome, for example, the capture reagent binds to another binding reagent that binds to the target nucleosome, or is a reagent that competes for binding of the target nucleosome to the labeled detection reagent.

10. The testing apparatus according to any one of claims 1 to 9, wherein the capture reagent comprises an antigen or an antibody.

11. The inspection apparatus according to any one of claims 1 to 10, wherein the labeled detection reagent includes a label that is soluble, for example, a fluorescent label.

12. The inspection apparatus according to any one of claims 1 to 10, wherein the labeled detection reagent includes a label, such as a particle label, gold or latex particle label.

13. The inspection apparatus according to any one of claims 1 to 10, wherein the labeled detection reagent comprises a DNA intercalator dye.

14. An inspection apparatus according to any one of claims 1 to 13, further comprising a comparison area.

15. The inspection apparatus according to any one of claims 1 to 14, further comprising a sample pad located upstream of the substrate and in fluid communication with the substrate.

16. The inspection apparatus according to any one of claims 1 to 15, further comprising an absorbent pad located downstream of the substrate and in fluid communication with the substrate.

17. The inspection apparatus according to any one of claims 1 to 16, further comprising a pre-removal site located upstream of the inspection site, wherein the pre-removal site is capable of removing one or more interfering substances from the sample or blocking one or more interfering substances from interfering with the binding of the target nucleosome to the capture reagent.

18. The inspection apparatus according to claim 17, wherein the pre-removed portion is located upstream of the conjugate pad.

19. The inspection apparatus according to claim 17 or claim 18, wherein the pre-removed portion is located between the sample pad and the conjugate pad.

20. The inspection apparatus according to claim 17 or claim 18, wherein the pre-removed portion forms part of the sample pad.

21. The inspection apparatus according to any one of claims 17 to 20, wherein a removal reagent capable of removing one or more interfering substances is fixed to the pre-removal site so as not to be transported to the inspection site by the liquid sample.

22. The inspection apparatus according to any one of claims 17 to 20, wherein a removal reagent capable of blocking one or more interfering substances from interfering with the binding of the target nucleosome to the capture reagent is not immobilized on the pre-removal site.

23. The inspection apparatus according to any one of claims 17 to 22, wherein the one or more interfering substances are non-assay-related interfering substances.

24. The inspection apparatus according to any one of claims 17 to 23, wherein the one or more interfering substances are interfering substances capable of interfering with the binding of the target nucleosome and the capture reagent.

25. The testing apparatus according to any one of claims 17 to 24, wherein one or more interfering substances are selected from non-assay-related antibodies and / or nucleosomes.

26. The testing apparatus according to any one of claims 17 to 25, wherein the one or more interfering substances include non-assay-related nucleosomes.

27. The testing apparatus according to any one of claims 17 to 26, wherein the non-assay-related nucleosome is a nucleosome containing linker DNA.

28. A removal reagent capable of removing or blocking nucleosomes containing linker DNA is available. (a) Linker DNA associated with nucleosomes; (b) chromatin-binding proteins that bind to linker DNA; or (c) Coanucleosome features associated with linker DNA The inspection apparatus according to claim 27, comprising a binder that binds to the object.

29. The testing apparatus according to claim 28, wherein the binder binds to linker DNA associated with nucleosomes.

30. The testing apparatus according to claim 29, wherein the binder that binds to the linker DNA associated with the nucleosome is a histone protein selected from histone H1, macroH2A, or fragments or modified analogs thereof.

31. The testing apparatus according to any one of claims 17 to 25, wherein the one or more interfering substances include non-assay-related antibodies.

32. The testing apparatus according to claim 31, wherein the non-assay-related antibody is selected from non-assay-related anti-animal antibodies, such as human anti-animal antibodies (HAAA), rheumatoid factor (RF), and / or heterophilic antibodies.

33. A removal reagent capable of removing or blocking the non-assay-related antibody is, (i) non-immune serum; (ii) Species-specific polyclonal IgG (iii) Anti-human IgG (iv) Polymerized mouse IgG; (v) Non-immune mouse monoclonal; (vi) Specific IgG fragments (e.g., えば, Fc, Fab, F(ab')) 2 ); (vii) Heterophilic blocking reagent (HBR); (viii) Immunoglobulin inhibitor reagents (IIRs) (ix) Protein A; and / or (x) Protein G The inspection apparatus according to claim 31 or claim 32, comprising a removal reagent selected from:

34. The testing apparatus according to any one of claims 31 to 33, wherein the removal reagent capable of removing or blocking the non-assay-related antibody includes a removal reagent derived from the same species as the capture reagent.

35. A method for determining the presence, absence, and / or quantity of target nucleosomes in a sample, a) The step of bringing a liquid sample into contact with an inspection apparatus according to any one of claims 1 to 34, wherein the liquid sample is applied to a part of the inspection apparatus upstream of the inspection location; and b) A step of evaluating the detectable signal at the test site to determine the presence, absence, and / or quantity of target nucleosomes in the sample. The method including:

36. The method according to claim 35, wherein the detectable signal is evaluated by a reader.

37. The method according to claim 36, wherein the detectable signal is a fluorescent signal, and the fluorescent signal is evaluated by a fluorescence reader.

38. The method according to claim 37, wherein the fluorescent reader is a laser-based or light-emitting diode (LED)-based fluorescent reader.

39. The method according to any one of claims 36 to 38, wherein the reader comprises one or more photodetectors.

40. The method according to any one of claims 35 to 39, wherein the liquid sample is a blood, serum, or plasma sample.

41. A system for evaluating samples, a) The inspection apparatus according to any one of claims 1 to 34; and b) A reader for detecting detectable signals The system comprising:

42. The system according to claim 41, wherein the reader comprises a light source and a photodetector.

43. The system according to claim 42, wherein the reader further comprises a barcode detector and / or a radio frequency identification (RFID) detector.

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