Histone combination, histone antibody detection reagent, kit and application of histone combination and histone antibody detection reagent

Abstract

The invention provides a histone combination, a histone antibody detection reagent, a kit and application of the histone combination, the histone combination comprises multiple histone proteins, the multiple histone proteins comprise histone H1, histone H2A, histone H2B, histone H3 and histone H4, the multiple histone proteins exist in the form of first polymers and / or in the form of free monomers, and the histone antibodies exist in the form of second polymers and / or in the form of free monomers. The fifth lysine of the histone H2A has acetylation modification; the fifth, twelfth, fifteenth and twenty sites of lysine of the histone H2B have acetylation modification; the 9th, 14th, 18th, 23rd, 56th, 64th and 122nd lysine of the histone H3 has acetylation modification, and the 27th lysine of the histone H3 has methylation modification; and the fifth, eighth, twelfth and sixteenth lysine of the histone H4 has acetylation modification. By utilizing the histone combination, the detection rate of the histone antibody can be effectively improved, so that the detection result is more accurate.

Description

Technical Field

[0001] This application relates to the field of biology, specifically to histone assemblies, histone antibody detection reagents, kits, and their applications. Background Technology

[0002] Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that can affect multiple organs and tissues throughout the body. The various autoantibodies and immune complexes produced can affect organs such as the heart, kidneys, and liver. The etiology and pathogenesis of SLE are complex, and the production of autoantibodies is a major characteristic of SLE. In clinical examinations, anti-histone antibodies (AHAs) have been found to be specific antibodies for diagnosing SLE.

[0003] Currently, the detection methods for anti-histone antibodies have a low detection rate, which poses a significant challenge to the diagnosis of SLE. There is an urgent need for a detection reagent to improve the detection rate of SLE. Summary of the Invention

[0004] This application aims to at least partially address the technical problems existing in the prior art. To this end, this application proposes histone combinations, histone antibody detection reagents, histone antibody detection kits, and their applications in histone antibody preparation and detection products. The histone combinations of this application can accurately detect anti-histone antibodies with a high detection rate, which helps improve the accuracy of systemic lupus erythematosus (SLE) detection and is suitable for widespread application.

[0005] It should be noted that this application is based on the inventor's discovery and understanding of the following facts and problems:

[0006] Chromatin is based on nucleosomes as its basic building blocks. Each nucleosome consists of an octamer of histone (two molecules of H2A-H2B dimer, two molecules of H3 and two molecules of H4) and about 146 bp of DNA wrapped around it with 1.75 turns. Nucleosomes are linked to each other by 40-60 bp of DNA. Histone H1 binds to the DNA between nucleosomes.

[0007] The three-dimensional structure of histone octamers is globular, while the amino terminus of the histone monomers protrudes, forming the N-terminal tail. Many residues on the N-terminal tail can be covalently modified, such as through methylation, acetylation, phosphorylation, ubiquitination, SUMOylation, and ADP ribosylation. Studies have shown that histone modifications are related to the pathogenesis of systemic lupus erythematosus (SLE), and various modifications, such as methylation and acetylation, exist on histone amino acid residues. The differences in histone modifications between SLE patients and healthy individuals pose a significant challenge to SLE diagnosis, necessitating a diagnostic reagent to improve the detection rate of SLE.

[0008] In view of this, to restore the true antigenic epitopes in SLE patients and further improve the detection rate, the inventors modified histone monomers. The modification sites include acetylation of lysine 5 in H2A, acetylation of lysines 5, 12, 15, and 20 in H2B, acetylation of lysines 9, 14, 18, 23, 56, 64, and 122 in H3, methylation of lysine 27, and acetylation of lysines 5, 8, 12, and 16 in H4. Histone monomer H1, in combination with the above-mentioned modified histone monomers, in free monomer form, polymer form, or a combination of both, can effectively detect histone antibodies in samples. The high detection rate of histone antibodies makes the test results more accurate, thereby achieving accurate diagnosis of systemic lupus erythematosus.

[0009] Therefore, in one aspect of this application, a histone combination is proposed. According to an embodiment of this application, the histone combination comprises multiple histones, including histone H1, histone H2A, histone H2B, histone H3, and histone H4, wherein the multiple histones exist as a first polymer and / or as free monomers respectively; wherein: histone H2A has an acetylated lysine residue at position 5; histone H2B has acetylated lysine residues at positions 5, 12, 15, and 20; histone H3 has acetylated lysine residues at positions 9, 14, 18, 23, 56, 64, and 122, and a methylated lysine residue at position 27; and histone H4 has acetylated lysine residues at positions 5, 8, 12, and 16.

[0010] The inventors of this application combine four histone monomers with histone H1 as antigens, which can be used to detect histone antibodies. This helps to improve the detection rate of histone antibodies and make the test results more accurate, thereby achieving accurate diagnosis of systemic lupus erythematosus.

[0011] According to embodiments of this application, the above-described histone combination may also have the following additional technical features:

[0012] According to embodiments of this application, the multiple histones exist in the form of free monomers, and the mass ratio of histone H1, histone H2A, histone H2B, histone H3, and histone H4 is 1:(2-5):(2-5):(2-5):1; or, the multiple histones exist simultaneously in the form of a first polymer and free monomers, wherein histone H2A, histone H2B, histone H3, and histone H4 in the first polymer are modified, and histone H2A, histone H2B, histone H3, and histone H4 in the free monomers may or may not be modified, and the mass ratio of the first polymer, histone H1, histone H2A, histone H2B, histone H3, and histone H4 is (2-6):1:(2-5):(2-5):(2-5):1.

[0013] According to an embodiment of this application, the various histones are coated on a solid support; the coating ratio of the solid support to the total amount of the various histones is (60-150):1.

[0014] According to an embodiment of this application, the first polymer and / or free monomer are coated on a solid support and then mixed.

[0015] In another aspect of this application, a histone antibody detection reagent is provided. According to an embodiment of this application, the histone antibody detection reagent includes a first reagent comprising the aforementioned histone combination.

[0016] According to embodiments of this application, the first reagent further comprises at least one of the following: Tris-HCl, sodium chloride, bovine serum albumin, Tween-20, mannitol, Proclin 300, and sodium hydroxide.

[0017] According to embodiments of this application, the various histones are coated on a solid support, and based on the total volume of the first reagent, the concentration of the solid support is 50 ng / mL to 150 ng / mL, and / or the concentration of Tris-HCl is 30 mM to 50 mM, and / or the concentration of sodium chloride is 5 g / L to 15 g / L, and / or the concentration of bovine serum albumin is 1 g / L to 10 g / L, and / or the concentration of Tween-20 is 1 g / L to 5 g / L, and / or the concentration of mannitol is 5 g / L to 15 g / L, and / or the concentration of Proclin 300 is 0.01 g / L to 1 g / L.

[0018] According to an embodiment of this application, the pH value of the first reagent is 6.0 to 6.2.

[0019] In another aspect of this application, a histone antibody detection kit is provided. According to embodiments of this application, the histone antibody detection kit comprises: the aforementioned histone combination or histone antibody detection reagent.

[0020] According to an embodiment of this application, the kit further includes a second reagent comprising at least one of the following: disodium hydrogen phosphate-potassium dihydrogen phosphate, sodium chloride, Tween-20, Proclin 300, and sodium hydroxide.

[0021] According to an embodiment of this application, the pH value of the second reagent is 8.5 to 9.5.

[0022] According to an embodiment of this application, the pH value of the second reagent is 9.5.

[0023] According to embodiments of this application, based on the total volume of the second reagent, the concentration of disodium hydrogen phosphate-potassium dihydrogen phosphate is 50mM to 150mM, and / or the concentration of sodium chloride is 5g / L to 15g / L, and / or the concentration of Tween-20 is 1g / L to 5g / L, and / or the concentration of Proclin 300 is 0.01g / L to 1g / L.

[0024] According to an embodiment of this application, the kit further includes a third reagent comprising an antibody labeled with a signal generator.

[0025] According to embodiments of this application, the antibody is selected from histone antibodies or anti-human IgG antibodies.

[0026] According to embodiments of this application, the third reagent further comprises at least one of the following: Tris-HCl, sodium chloride, bovine serum albumin, Tween-20, mannitol, Proclin 300, and sodium hydroxide.

[0027] According to embodiments of this application, based on the total volume of the third reagent, the concentration of the antibody labeled with the signal generator is 50 ng / mL to 150 ng / mL, and / or the concentration of Tris-HCl is 40 mM to 60 mM, and / or the concentration of sodium chloride is 5 g / L to 15 g / L, and / or the concentration of bovine serum albumin is 1 g / L to 10 g / L, and / or the concentration of Tween-20 is 1 g / L to 5 g / L, and / or the concentration of mannitol is 5 g / L to 15 g / L, and / or the concentration of Proclin 300 is 0.01 g / L to 1 g / L.

[0028] According to an embodiment of this application, the pH value of the third reagent is 7.4 to 7.6.

[0029] According to an embodiment of this application, the kit further includes: an excitation substrate.

[0030] According to embodiments of this application, the excitation substrate includes NaOH and H2O2.

[0031] In another aspect of this application, the application of the aforementioned histone combination, histone antibody detection reagent, and histone antibody detection kit in the preparation of detection products is proposed. According to embodiments of this application, the detection product is used for diagnosing systemic lupus erythematosus, or the detection product is used to detect histone antibodies that specifically bind to the aforementioned histone combination.

[0032] In another aspect of this application, the application of histone antibody preparation in a detection product is proposed. According to an embodiment of this application, the detection product is used to diagnose systemic lupus erythematosus; the histone antibody can specifically bind to the aforementioned histone combination.

[0033] In another aspect of this application, a method for detecting histone antibodies is proposed, wherein the histone antibodies can specifically bind to the aforementioned histone combination. According to an embodiment of this application, the method includes: (1) incubating the aforementioned histone combination with a sample to be tested in a first incubation to obtain a first reactant; (2) incubating the first reactant with a second antibody in a second incubation to obtain a second reactant, wherein the second antibody can specifically bind to the histone antibody and is labeled with a signal generator; (3) incubating the second reactant with an excitation substrate in a third incubation, wherein the signal generator reacts with the excitation substrate to generate a detectable signal; and (4) determining the histone antibody content in the sample to be tested based on the detectable signal.

[0034] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Detailed Implementation

[0035] The embodiments of the technical solution of this application are described in detail below. The following embodiments are only used to illustrate the technical solution of this application more clearly, and are therefore only examples, and should not be used to limit the scope of protection of this application.

[0036] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0037] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60-120 and 80-110 are listed for a specific parameter, it is also expected that ranges of 60-110 and 80-120 are also included. Furthermore, if minimum range values ​​1 and 2 are listed, and if maximum range values ​​3, 4, and 5 are listed, then the following ranges are all expected: 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5. In this application, unless otherwise stated, the numerical range "ab" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers, and ranges defined in this way can include endpoints a and b. For example, the numerical range "0-5" indicates that all real numbers between "0-5" have been listed in this article; "0-5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0038] Unless otherwise specified, all embodiments and optional embodiments of this application can be combined to form new technical solutions.

[0039] Unless otherwise specified, all technical features and optional technical features of this application may be combined to form new technical solutions.

[0040] Unless otherwise specified, all steps in this application may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the mention that the method may also include step (c) indicates that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0041] This application discloses histone combinations, histone antibody detection reagents, histone antibody detection kits, and their applications in the preparation of detection products, as well as their applications in the preparation of detection products using histone antibodies. These will be described in detail below.

[0042] histone combination

[0043] Therefore, in one aspect of this application, a histone combination is proposed. According to an embodiment of this application, the histone combination includes multiple histones, including histone H1, histone H2A, histone H2B, histone H3, and histone H4, which exist as a first polymer and / or as free monomers respectively; the first polymer in this application is a natural polymer formed by histone H1, histone H2A, histone H2B, histone H3, and histone H4, wherein: the lysine at position 5 of histone H2A is acetylated; the lysines at positions 5, 12, 15, and 20 of histone H2B are acetylated; the lysines at positions 9, 14, 18, 23, 56, 64, and 122 of histone H3 are acetylated, and the lysine at position 27 is methylated; and the lysines at positions 5, 8, 12, and 16 of histone H4 are acetylated.

[0044] The inventors of this application combine four histone monomers with histone H1 as an antigen, which can be used to detect histone antibodies. The aforementioned histones can exist in various forms as follows:

[0045] (1) Monomer combinations formed by unmodified monomer H1 and modified monomers (H2A, H2B, H3 and H4) coexist;

[0046] (2) The polymer formed by the combination of monomers in (1) above;

[0047] (3) The aforementioned (2) and unmodified monomers (H1, H2A, H2B, H3 and H4) coexist.

[0048] (4) Both (1) and (2) mentioned above exist.

[0049] In this process, histone monomers may partially mask antigenic epitopes, resulting in a slightly lower detection rate for histone antibodies. Therefore, by employing the methods described above (1), (3), and (4), the antigenic epitopes of histones are fully exposed, which helps to improve the detection rate of histone antibodies, making the test results more accurate and thus enabling an accurate diagnosis of systemic lupus erythematosus.

[0050] The rules for describing histone modifications are: histone structure + amino acid name + amino acid position + modification type. For example, H3K14ac represents the acetylation of lysine 14 in the H3 histone, and K2AK5me represents the methylation of lysine 5 in the H2A histone.

[0051] According to embodiments of this application, the various histones exist in the form of free monomers, and all histones except histone H1 have corresponding modifications. The mass ratio of histone H1, histone H2A, histone H2B, histone H3, and histone H4 is 1:(2-5):(2-5):(2-5):1, preferably 1:(3-5):(3-5):(3-5):1. This can further improve the detection rate of histone antibodies.

[0052] According to embodiments of this application, the multiple histones exist simultaneously in the form of a first polymer and free monomers. The histones H2A, H2B, H3, and H4 in the first polymer have corresponding modifications. The histones H2A, H2B, H3, and H4 in the free monomers may or may not have corresponding modifications. The first polymer, histone H1, histone H2A, histone H2B, and histone H4... 3. The mass ratio of histone H4 is (2-6):1:(2-5):(2-5):(2-5):1, preferably (3-6):1:(2-5):(2-5):(2-5):1, (4-6):1:(2-5):(2-5):(2-5):1, (3-6):1:(3-5):(3-5):(3-5):1, (4-6):1:(3-5):(3-5):(3-5):1. This can further improve the detection rate of histone antibodies.

[0053] According to embodiments of this application, the various histones are coated onto a solid support. Exemplarily, the solid support includes magnetic beads.

[0054] It should be noted that the aforementioned multiple histones can be co-coated on the same solid support or immobilized on different solid supports. Multiple proteins can be fully or partially coated on the solid support; for example, the polymer is coated on the solid support while the monomer exists in free form; or the polymer exists in free form while the monomer is coated on the solid support. In some embodiments, the first polymer and / or free monomer are coated on the solid support separately before being mixed.

[0055] According to embodiments of this application, the coating ratio of the solid-phase carrier to the total amount of the various histones is (60-150):1, for example, 60:1, 80:1, 100:1, 120:1, 140:1, or 150:1. Therefore, the coating amount meets the above conditions, which is beneficial for the specific binding of the histone combination to histone antibodies, reducing non-specific binding or weak detection signals, improving the efficiency, specificity, and accuracy of immunoassay, while reducing detection costs.

[0056] Histone antibody detection reagents and kits

[0057] In another aspect, this application provides a histone antibody detection reagent. According to an embodiment of this application, the histone antibody detection reagent includes a first reagent comprising the aforementioned histone combination. Therefore, the histone antibody detection reagent of this application can accurately detect anti-histone antibodies with a high detection rate, helping to improve the accuracy of systemic lupus erythematosus (SLE) detection and is suitable for widespread application.

[0058] It should be noted that the term "first reagent" in this application should be interpreted broadly, referring to a reagent containing a histone combination. This histone combination can exist within the same reagent, in which case "first reagent" refers to a reagent simultaneously containing that histone combination; or it can exist independently in different reagents, in which case any reagent containing any one of the histones in that histone combination can be considered a first reagent. These first reagents are used in combination during detection. Furthermore, in the first reagent, the histones in the histone combination can be coated onto a solid support or provided in free form, and then coated onto the solid support during detection.

[0059] According to embodiments of this application, the first reagent further comprises at least one of the following: Tris-HCl, sodium chloride, bovine serum albumin (BSA), Tween-20, mannitol, Proclin 300, and sodium hydroxide. Tris-HCl maintains a suitable pH value, sodium chloride adjusts the ionic strength of the reagent, bovine serum albumin (BSA) reduces nonspecific binding, Tween-20 acts as a surfactant to reduce foaming and aid dissolution, mannitol acts as a stabilizer to prevent magnetic bead aggregation, Proclin 300 acts as a preservative to prevent microbial contamination, and sodium hydroxide is used to adjust the pH value of the solution. Thus, the above components work together to improve the stability and long-term shelf life of the first reagent, facilitating specific binding of histone combinations and histone antibodies.

[0060] According to embodiments of this application, based on the total volume of the first reagent, the concentration of the solid support is 50 ng / mL to 150 ng / mL, and / or the concentration of Tris-HCl is 30 mM to 50 mM, and / or the concentration of sodium chloride is 5 g / L to 15 g / L, and / or the concentration of bovine serum albumin is 1 g / L to 10 g / L, and / or the concentration of Tween-20 is 1 g / L to 5 g / L, and / or the concentration of mannitol is 5 g / L to 15 g / L, and / or the concentration of Proclin 300 is 0.01 g / L to 1 g / L, and the pH value of the first reagent is 6.0 to 6.2. Therefore, the biocompatibility and stability of the first reagent can be further improved, non-specific binding can be reduced, thereby improving the sensitivity and specificity of detection.

[0061] In another aspect, this application provides a histone antibody detection kit. According to embodiments of this application, the kit comprises either the histone combination described above or the histone antibody detection reagent described above. Therefore, the kit of this application can accurately detect anti-histone antibodies with a high detection rate, which helps improve the accuracy of systemic lupus erythematosus (SLE) detection and is suitable for widespread application.

[0062] According to embodiments of this application, the kit further comprises a second reagent, which includes at least one of the following: disodium hydrogen phosphate-potassium dihydrogen phosphate, sodium chloride, Tween-20, Proclin 300, and sodium hydroxide. Disodium hydrogen phosphate-potassium dihydrogen phosphate maintains a suitable pH value, sodium chloride adjusts the ionic strength of the reagent, Tween-20 acts as a surfactant to reduce foaming and aid dissolution, Proclin 300 acts as a preservative to prevent microbial contamination, and sodium hydroxide is used to adjust the pH value of the solution. Thus, the above components facilitate the specific binding of histone combinations and histone antibodies, improving titer and sensitivity, and reducing false negatives.

[0063] According to embodiments of this application, based on the total volume of the second reagent, the concentration of the disodium hydrogen phosphate-potassium dihydrogen phosphate is 50 mM to 150 mM, and / or the concentration of the sodium chloride is 5 g / L to 15 g / L, and / or the concentration of the Tween-20 is 1 g / L to 5 g / L, and / or the concentration of the Proclin 300 is 0.01 g / L to 1 g / L, and the pH value of the second reagent is 8.5 to 9.5. Therefore, the detection potency and sensitivity can be further improved, and the false negatives in samples can be reduced.

[0064] According to embodiments of this application, the kit further includes a third reagent comprising an antibody labeled with a signal generator (also referred to herein as a "second antibody"). In some embodiments, the antibody is selected from histone antibodies or anti-human IgG antibodies.

[0065] According to embodiments of this application, the signal generator includes at least one of isoluminol or its derivatives, luminol, horseradish peroxidase, and alkaline phosphatase. Exemplarily, the signal generator includes N-(4-aminobutyl)-N-ethylisoluminol (ABEI).

[0066] According to embodiments of this application, the third reagent further comprises at least one of the following: Tris-HCl, sodium chloride, bovine serum albumin (BSA), Tween-20, mannitol, Proclin 300, and sodium hydroxide. Tris-HCl maintains a suitable pH value, sodium chloride adjusts the ionic strength of the reagent, bovine serum albumin (BSA) reduces nonspecific binding, Tween-20 acts as a surfactant to reduce foaming and aid dissolution, mannitol acts as a stabilizer to prevent magnetic bead aggregation, Proclin 300 acts as a preservative to prevent microbial contamination, and sodium hydroxide is used to adjust the pH value of the solution. Thus, the above components work together to improve the stability and long-term shelf life of the third reagent, facilitating the specific binding of histone antibodies and antibodies labeled with signal generators.

[0067] According to embodiments of this application, based on the total volume of the third reagent, the concentration of the antibody labeled with the signal generator is 50 ng / mL to 150 ng / mL, and / or the concentration of Tris-HCl is 40 mM to 60 mM, and / or the concentration of sodium chloride is 5 g / L to 15 g / L, and / or the concentration of bovine serum albumin is 1 g / L to 10 g / L, and / or the concentration of Tween-20 is 1 g / L to 5 g / L, and / or the concentration of mannitol is 5 g / L to 15 g / L, and / or the concentration of Proclin 300 is 0.01 g / L to 1 g / L; the pH value of the third reagent is 7.4 to 7.6. This further improves the stability and long-term shelf life of the third reagent, facilitating the specific binding of histone antibodies and antibodies labeled with the signal generator.

[0068] According to embodiments of this application, the kit further includes an excitation substrate. This substrate can then react with a label on a second antibody to generate a detectable signal. In some embodiments, the excitation substrate includes NaOH and H₂O₂.

[0069] It should be noted that the characteristics and advantages described above for histone combinations also apply to this histone antibody detection reagent and kit, and will not be repeated here.

[0070] Applications and methods for detecting histone antibodies

[0071] In another aspect of this application, the application of the aforementioned histone combination, histone antibody detection reagent, and histone antibody detection kit in the preparation of detection products is proposed. According to embodiments of this application, the detection product is used for diagnosing systemic lupus erythematosus, or the detection product is used to detect histone antibodies that specifically bind to the aforementioned histone combination.

[0072] In another aspect of this application, the application of histone antibody preparation in a detection product is proposed. According to an embodiment of this application, the detection product is used to diagnose systemic lupus erythematosus; the histone antibody can specifically bind to the aforementioned histone combination.

[0073] As mentioned earlier, patients with systemic lupus erythematosus (SLE) will have modified undissociated histones or histone monomers from the aforementioned histone combination, which in turn leads to the production of corresponding histone antibodies. Therefore, by utilizing the property that this histone combination can specifically bind to histone antibodies in the body, the purpose of diagnosing SLE or detecting histone antibodies can be achieved.

[0074] In another aspect of this application, a method for detecting histone antibodies is proposed, wherein the histone antibodies can specifically bind to the aforementioned histone combination. According to an embodiment of this application, the method includes: (1) incubating the aforementioned histone combination with a sample to be tested to obtain a first reactant; (2) incubating the first reactant with an antibody labeled with a signal generator to obtain a second reactant, wherein the antibody labeled with the signal generator can specifically bind to the histone antibody; (3) incubating the second reactant with an excitation substrate to generate a detectable signal; and (4) determining the histone antibody content in the sample to be tested based on the detectable signal.

[0075] In step (1), the histone complex can bind to the histone antibody in the sample to be tested, forming a histone antigen-histone antibody complex. In step (2), the histone antigen-histone antibody complex can bind to the second antibody, forming a histone antigen-histone antibody-second antibody complex. The second antibody has a signal generator, which can react with the excitation substrate in step (3) to generate a detectable signal. The intensity of this signal is proportional to the concentration of the target antibody, thereby achieving quantitative detection of the target antibody concentration. Based on this detectable signal, the histone content in the sample to be tested can be determined. Exemplarily, this method can be used for diagnostic purposes, such as in the diagnosis of systemic lupus erythematosus; it can also be used for non-diagnostic purposes, such as in the detection of histone antibodies and the screening of drugs for the prevention or treatment of systemic lupus erythematosus.

[0076] According to an embodiment of this application, the method is implemented using the aforementioned kit; step (1) includes: incubating the first reagent, the second reagent, and the sample to be tested with the first reagent; step (2) includes: incubating the first reactant with the third reagent with the second reagent.

[0077] According to an embodiment of this application, the mass ratio of the sample to be tested to the first reagent is 1:(1-10). This facilitates a specific reaction between the histone antibodies in the sample to be tested and the histone antigens in the first reagent, reducing non-specific binding or weak detection signals, thus improving the efficiency, specificity, and accuracy of the immunoassay, while simultaneously reducing detection costs.

[0078] According to an embodiment of this application, the mass ratio of the sample to be tested to the second reagent is 1:(1-20). This facilitates a specific reaction between the histone antibody and the histone antigen, reduces non-specific binding or weak detection signals, improves the efficiency, specificity, and accuracy of the immunoassay, and simultaneously lowers the detection cost.

[0079] According to an embodiment of this application, the first incubation time is 5 to 15 minutes. This allows for sufficient reaction and binding between the histone antibody in the test sample and the histone antigen combination in the first reagent.

[0080] According to an embodiment of this application, the mass ratio of the test sample to the third reagent is 1:(10-30). This ensures that the histone antibody in the test sample reacts and binds sufficiently with the second antibody in the third reagent.

[0081] According to an embodiment of this application, the second incubation time is 5 to 15 minutes. This allows the histone antibody in the test sample to fully react and bind with the second antibody in the third reagent.

[0082] It should be noted that the features and advantages described above for histone combinations, histone antibody detection reagents and kits also apply to this application and method, and will not be repeated here.

[0083] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0084] The histone antigen modification methods used in the following examples are as follows:

[0085] 1. Source of modifying enzymes

[0086] P300 / KAT3B enzyme was purchased from Biomol (catalog number BPS-50071).

[0087] Ezh2 enzyme (GeneID: 2146) was purchased from Biomol (catalog number BPS-51003).

[0088] 2. Preparation of human histone monomers and human histone octamers

[0089] (1) Recombinant expression and purification of human histone monomers

[0090] Human histones H1 (UniProt sequence number: P07305), H2A (UniProt sequence number: P04908), H2B (UniProt sequence number: P62807), H3 (UniProt sequence number: P84243), and H4 (UniProt sequence number: P62805).

[0091] Based on the amino acid sequences of each histone in UniProt, corresponding DNA coding sequences were designed and synthesized, and codons were optimized to suit the *E. coli* expression system. Each gene was cloned into a pET series expression vector containing a His6 tag and transformed into *E. coli* BL21(DE3) competent cells. Positive clones were picked and inoculated into LB medium, cultured at 37°C until OD600 ≈ 0.6-0.8, then 0.1-1 mM IPTG was added to induce expression, and the cells were cultured at 16-20°C for 16-20 hours. After cell collection, the cells were sonicated, centrifuged, and the supernatant was purified by nickel affinity chromatography. Further purification was performed using ion exchange chromatography (e.g., SP or Q column) and molecular sieve chromatography (e.g., Superdex 75). Finally, the cells were dialyzed or ultrafiltered to a storage buffer (e.g., 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM DTT, 10% glycerol), aliquoted, and stored at -80°C. High-purity histone monomers were obtained.

[0092] (2) Extraction of natural octamer

[0093] Natural octamer histone was extracted from HeLa cells using the extraction method described in the literature by O'Neill, TE et al. [1] The process involves culturing and collecting HeLa cells under experimental conditions, lysing the collected cells under hypotonic conditions, and collecting intact cell nuclei. Micrococcal nuclease digestion is followed by overnight dialyzing, removal of the H1 component by hydroxyapatite column chromatography, elution, and overnight dialyzing. Repeating this step yields high-purity natural octamer histone.

[0094] 3. Modification of human recombinant histone monomers and natural octamer histones

[0095] (1) Modification of H2A

[0096] In a buffer containing 50 mM Tris-HCl (pH 8.0) (5% glycerol, 10 mM sodium butyrate, 0.1 mM EDTA, 1 mM MTT, and 1 mM MPMSF), 0.1 mg of P300 / KAT3B enzyme, 10 mM / L acetyl-CoA, and 0.1 mg of H2A monomer were added and incubated at 37 °C for 30 min to acetylate the K5 of H2A. The mixture was then purified by gel filtration.

[0097] (2) Modification of H2B

[0098] In a buffer containing 50 mM Tris-HCl (pH 8.0) (5% glycerol, 10 mM sodium butyrate, 0.1 mM EDTA, 1 mM MTT, and 1 mM PMSF), 0.1 mg of P300 / KAT3B enzyme, 10 mM / L acetyl-CoA, and 0.1 mg of H2B monomer were added and incubated at 37°C for 30 min to acetylate K5, K12, K15, and K20 of H2B. The mixture was then purified by gel filtration.

[0099] (3) Modification of H3

[0100] In a 50 mM Tris-HCl (pH 8.5) buffer (5 mM MgCl2, 4 mM DTT), 0.1 mg of histone methyltransferase Ezh2, 3 mg of S-adenosylmethionine, and 0.1 mg of H3 monomer were added and incubated at 37 °C for 30 min to methylate K27 of H3. The modified recombinant H3 monomer was purified by gel filtration. In a 50 mM Tris-HCl (pH 8.0) buffer (5% glycerol, 10 mM sodium butyrate, 0.1 mM EDTA, 1 mM DTT, and 1 mM PMSF), 0.1 mg of P300 / KAT3B enzyme and 10 mM / L acetyl-CoA were added and incubated at 37 °C for 30 min to acetylate K9, K14, K18, K23, K56, K64, and K122 of H3. The monomer was then purified by gel filtration.

[0101] (4) Modification of H4

[0102] In a buffer containing 50 mM Tris-HCl (pH 8.0) (5% glycerol, 10 mM sodium butyrate, 0.1 mM EDTA, 1 mM MTT, and 1 mM PMSF), 0.1 mg of P300 / KAT3B enzyme, 10 mM / L acetyl-CoA, and 0.1 mg of H4 monomer were added and incubated at 37°C for 30 min to acetylate K5, K8, K12, and K16 of H4. The mixture was then purified by gel filtration.

[0103] (5) Modification of natural histones

[0104] In a 50 mM Tris-HCl (pH 8.5) buffer (5 mM MgCl2, 4 mM DTT), 0.1 mg of histone methyltransferase Ezh2, 3 mg of S-adenosylmethionine, and 0.1 mg of native histone antigen were added. The mixture was incubated at 37°C for 30 min to methylate K27 of H3. The modified native histone antigen was purified by gel filtration. Subsequently, in a 50 mM Tris-HCl (pH 8.0) buffer (5% glycerol, 10 mM sodium butyrate, 0.1 mM EDTA, 1 mM DTT, and 1 mM MPMSF), 0.1 mg of... P300 / KAT3B enzyme and 10 mM / L acetyl-CoA were incubated at 37°C for 60 min to acetylate K5 of H2A, K5, K12, K15, and K20 of H2B, K9, K14, K18, K23, K56, K64, and K122 of H3, and K5, K8, K12, and K16 of H4. The mixture was then purified by gel filtration.

[0105] Example 1

[0106] Experimental Group 1: Unmodified histone polymer-coated magnetic beads

[0107] 1. Magnetic bead coating: Unmodified histone polymers were used for coating at a ratio of 1 mg (magnetic bead mass): 7.5 μg (antigen mass). The coated magnetic beads were prepared according to Table 1 to obtain reagent R1.

[0108] Table 1. Composition of Reagent R1

[0109]

[0110]

[0111] 3. Prepare reagent R2 according to Table 2.

[0112] Table 2 Composition of Reagent R2

[0113] Element concentration Disodium hydrogen phosphate-potassium dihydrogen phosphate 100mM Sodium chloride 9g / L Twain-20 2g / L Proclin 300 0.1g / L Sodium hydroxide Adjust the pH to 7.30.

[0114] 2. ABEI labeling: Anti-human IgG was labeled with ABEI, and then reagent R3 was prepared according to Table 3.

[0115] Table 2 Composition of Reagent R3

[0116] Element concentration Anti-human IgG labeled with ABEI 100ng / mL Tris-HCl 50mM Sodium chloride 9g / L BSA 5g / L Twain-20 2g / L Mannitol 10g / L Proclin 300 0.1g / L Sodium hydroxide Adjust the pH to 7.40 using sodium hydroxide.

[0117] 4. After the reagents are prepared, test the sample on the New Industries fully automated chemiluminescence analyzer according to the following steps:

[0118] (1) Mix 10 μL of plasma sample with 20 μL of reagent R2 and 100 μL of reagent R1 simultaneously, react for 10 min, and then wash with washing buffer.

[0119] (2) Add 200 μL of reagent R3, continue the reaction for 10 min, and wash;

[0120] (3) Add the luminescent substrate NaOH and measure the luminescence value RLU using the New Industrial Chemiluminescence Automated Analyzer.

[0121] Experimental group 2: Modified histone polymer antigen-coated magnetic beads

[0122] Modified histone polymers were used to coat the magnetic beads at a ratio of 1 mg (magnetic bead mass) to 7.5 μg (antigen mass). The coated magnetic beads were prepared according to Table 1 to obtain reagent R1. The remaining reagent components were the same as in Example 1. Clinical samples were tested according to the detection method in Example 1. The results are shown in Table 4.

[0123] Experimental group 3: Unmodified histone monomer antigen-coated magnetic beads

[0124] Unmodified histone monomers H1, H2A, H2B, H3, and H4 were used for coating at a ratio of 1 mg (magnetic bead mass) to 1.5 μg (antigen mass). The five coated magnetic beads were mixed and prepared according to Table 1 to obtain reagent R1. The remaining reagents were the same as in Example 1. Clinical samples were tested according to the detection method in Example 1, and the results are shown in Table 4.

[0125] Experimental group 4: Histone monomers coated with magnetic beads

[0126] Histone monomer H1, modified monomer H2A, modified monomer H2B, modified monomer H3, and modified monomer H4 were used for coating at a ratio of 1 mg (mass of magnetic beads) to 1.5 μg (mass of antigen). The five coated magnetic beads were mixed and prepared according to Table 1 to obtain reagent R1. The remaining reagents were the same as in Example 1. Clinical samples were tested according to the detection method in Example 1, and the results are shown in Table 4.

[0127] Experimental group 5: Unmodified histone aggregates + unmodified histone monomers mixed antigen-coated magnetic beads

[0128] Magnetic beads were coated with unmodified histone monomers H1, H2A, H2B, H3, H4 and histone polymers, with the mass ratio of histone polymers to monomers being: polymer:H1:H2A:H2B:H3:H4 = 5:1:1:1:1:1. That is, natural histones (polymers) were coated at a ratio of 1 mg (mass of magnetic beads): 7.5 μg (mass of antigen), and histone monomers were coated at a ratio of 1 mg (mass of magnetic beads): 1.5 μg (mass of antigen). After the magnetic beads were coated, R1 was prepared according to Table 1.

[0129] Experimental group 6: Modified histone aggregates + unmodified histone monomers mixed antigen-coated magnetic beads

[0130] Modified histone aggregates and unmodified histone monomers H1, H2A, H2B, H3, and H4 were used to coat magnetic beads according to the coating ratio in Example 5. That is, unmodified histone monomers were coated at a ratio of 1 mg (magnetic bead mass): 1.5 μg (antigen mass), and modified histone aggregates were coated at a ratio of 1 mg (magnetic bead mass): 7.5 μg (antigen mass). The six coated magnetic beads were mixed and prepared according to Table 2 to obtain reagent component R1. The remaining reagent components were the same as in Example 1. Clinical samples were tested according to the detection method in Example 1, and the results are shown in Table 4.

[0131] Experimental group 7: Modified histone aggregate antigen + modified histone monomer mixed antigen coated magnetic beads

[0132] Using the modified histone first-mer antigen and the modified histone monomers H1, H2A, H2B, H3, and H4, magnetic beads were coated according to the coating ratio in Example 5. Specifically, the modified histone polymers were coated at a ratio of 1 mg (magnetic bead mass): 7.5 μg (antigen mass), and the modified monomer antigens were coated at a ratio of 1 mg (magnetic bead mass): 1.5 μg (antigen mass). The six coated magnetic beads were mixed and reagent R1 was prepared according to Table 1. The remaining reagents were the same as in Example 1. Clinical samples were tested according to the detection method in Example 1, and the results are shown in Table 4.

[0133] The above-described embodiments were used to test 50 patients diagnosed with SLE, and the results are shown in Table 4.

[0134] Table 4. Detection results of SLE patients

[0135]

[0136]

[0137]

[0138] The cutoff values ​​for histone detection in the above kits are all 0–20 RU / mL. The detection results are higher than the normal reference range, indicating that histone antibody detection is positive. The data in the table above show that in 50 patients with systemic lupus erythematosus, the kits using this method have a higher positive detection rate than those using unmodified histone aggregates or histone monomers.

[0139] Based on the results of Examples 1 to 7, it can be seen that Example 7, which uses a mixture of modified histone aggregate antigen and modified histone monomer antigen coated with magnetic beads, achieved a significantly higher positive detection rate for the 50 SLE patient samples compared to the other examples.

[0140] Another 50 patients with drug-induced SLE were tested using Examples 5 and 7. The specific test results are shown in Table 5.

[0141] Table 5. Detection results in patients with drug-induced lupus.

[0142] Drug-induced SLE patients Example 5 Example 7 Negative 5 1 Positive 45 49 Positive detection rate 90％ 98％

[0143] The results showed that using magnetic beads coated with a mixture of modified histone aggregate antigen and modified histone monomer antigen improved the detection rate compared to using unmodified antigen-coated magnetic beads.

[0144] Example 7 was used to test 40 patients with ankylosing spondylitis and 200 healthy individuals during physical examinations. The specific test results are shown in Tables 6 and 7.

[0145] Table 6. Detection results of patients with ankylosing spondylitis and healthy individuals.

[0146] Ankylosing spondylitis patients Example 7 Health checkup sample Example 7 Negative 40 Negative 198 Positive 0 Positive 2 Positive detection rate 0％ Positive detection rate 1％

[0147] The results showed that the positive detection rate of magnetic beads coated with a mixture of modified histone aggregate antigen and modified histone monomer antigen was 0% in patients with ankylosing spondylitis and 1% in healthy physical examination samples, indicating that this method has high specificity for lupus.

[0148] Example 2: Detection of mixed histone antigens with different mass ratios

[0149] According to the mass ratio of each group of protein antigens in Table 5, the magnetic balls were coated with magnetic balls at a ratio of 1 mg (mass of magnetic balls): 15 μg (mass of total antigens). The remaining reagents were the same as in Example 7. The aforementioned SLE patient samples were tested according to the detection method in Example 1. The results are shown in Table 6.

[0150] Table 5. Mass ratio of protein antigens in different groups

[0151]

[0152] Table 6 Detection Rate Data

[0153] Detection rate Detection rate Experimental group 8 48％ Experimental group 13 56％ Experimental group 9 46％ Experimental group 14 52％ Experimental group 10 50％ Experimental group 15 52％ Experimental group 11 52％ Experimental group 16 62％ Experimental group 12 54％ Experimental group 17 62％

[0154] The data above shows that further adjusting the mass ratio between coating antigens significantly improves the detection rate of positive samples.

[0155] Example 3: Detection of reagent R2 at different pH values

[0156] R2 reagents with different pH values ​​were prepared according to Table 7. The other reagent components were the same as in Example 7. Ten normal samples and ten SLE positive samples were tested according to the detection method in Example 1, and the average deviation of the samples was calculated. The results are shown in Table 8.

[0157] Table 7 Composition of Reagent R2

[0158]

[0159] Table 7. Effect of different pH values ​​on reagent R2

[0160]

[0161] The data above shows that when the pH of reagent R2 is between 5.5 and 9.5, the titer of SLE negative samples gradually decreases while the titer of positive samples gradually increases with increasing pH, meaning the differentiation between SLE negative and positive samples gradually increases. However, if the pH of reagent R2 is further increased to 10.5, the titer of positive samples decreases further, and there is no further effect on increasing the differentiation between positive and negative samples. In summary, the optimal pH range for reagent R2 is 8.5–9.5, with pH 9.5 being the most effective.

[0162] References:

[0163] [1] O'Neill, TE, Roberge, M. & Bradbury, EMNucleosome arrays inhibit both initiation and elongation of transcripts by bacteriophage T7RNApolymerase. J Mol Biol 223, 67-78 (1992).

[0164] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A histone combination, characterized in that, It includes multiple histones, including histone H1, histone H2A, histone H2B, histone H3 and histone H4, which exist as a first polymer and / or as free monomers respectively; Wherein: the 5th lysine residue of histone H2A is acetylated; The histone H2B has acetylated lysine residues at positions 5, 12, 15, and 20. The histone H3 has acetylated lysine residues at positions 9, 14, 18, 23, 56, 64, and 122, and methylated lysine residue at position 27. The histone H4 has acetylated lysine residues at positions 5, 8, 12, and 16.

2. The histone combination according to claim 1, characterized in that, The various histones exist in the form of free monomers, and the mass ratio of histone H1, histone H2A, histone H2B, histone H3, and histone H4 is 1:(2-5):(2-5):(2-5):1; Alternatively, the multiple histones may exist simultaneously in the form of a first polymer and free monomers, wherein histone H2A, histone H2B, histone H3, and histone H4 in the first polymer are modified, and histone H2A, histone H2B, histone H3, and histone H4 in the free monomers may or may not be modified, and the mass ratio of the first polymer, histone H1, histone H2A, histone H2B, histone H3, and histone H4 is (2-6):1:(2-5):(2-5):(2-5):

1.

3. The histone combination according to claim 1 or 2, characterized in that, The various histones are coated on a solid support; The coating ratio of the solid carrier to the total amount of the various histones is (60-150):1; Optionally, the first polymer and / or free monomer are coated on a solid support and then mixed.

4. A histone antibody detection reagent, characterized in that, Includes a first reagent, which comprises the histone combination according to any one of claims 1-3.

5. The histone antibody detection reagent according to claim 1, characterized in that, The first reagent further comprises at least one of the following: Tris-HCl, sodium chloride, bovine serum albumin, Tween-20, mannitol, Proclin 300, and sodium hydroxide; Optionally, the various histones are coated on a solid support, and based on the total volume of the first reagent, the concentration of the solid support is 50 ng / mL to 150 ng / mL, and / or the concentration of Tris-HCl is 30 mM to 50 mM, and / or the concentration of sodium chloride is 5 g / L to 15 g / L, and / or the concentration of bovine serum albumin is 1 g / L to 10 g / L, and / or the concentration of Tween-20 is 1 g / L to 5 g / L, and / or the concentration of mannitol is 5 g / L to 15 g / L, and / or the concentration of Proclin 300 is 0.01 g / L to 1 g / L; Optionally, the pH value of the first reagent is 6.0 to 6.

2.

6. A histone antibody detection kit, characterized in that, include: The histone combination according to any one of claims 1-3 or the histone antibody detection reagent according to claim 4 or 5.

7. The reagent kit according to claim 6, characterized in that, Further including a second reagent, The second reagent includes at least one of the following: disodium hydrogen phosphate-potassium dihydrogen phosphate, sodium chloride, Tween-20, Proclin 300, and sodium hydroxide; Optionally, the pH value of the second reagent is 8.5 to 9.5; Optionally, the pH value of the second reagent is 9.5; Optionally, based on the total volume of the second reagent, the concentration of the disodium hydrogen phosphate-potassium dihydrogen phosphate is 50 mM to 150 mM, and / or the concentration of the sodium chloride is 5 g / L to 15 g / L, and / or the concentration of the Tween-20 is 1 g / L to 5 g / L, and / or the concentration of the Proclin 300 is 0.01 g / L to 1 g / L.

8. The reagent kit according to claim 6, characterized in that, It further includes a third reagent, which comprises an antibody labeled with a signal generator; Optionally, the antibody is selected from histone antibodies or anti-human IgG antibodies; Optionally, the third reagent further comprises at least one of the following: Tris-HCl, sodium chloride, bovine serum albumin, Tween-20, mannitol, Proclin 300, and sodium hydroxide; Optionally, based on the total volume of the third reagent, the concentration of the antibody labeled with the signal generator is 50 ng / mL to 150 ng / mL, and / or the concentration of Tris-HCl is 40 mM to 60 mM, and / or the concentration of sodium chloride is 5 g / L to 15 g / L, and / or the concentration of bovine serum albumin is 1 g / L to 10 g / L, and / or the concentration of Tween-20 is 1 g / L to 5 g / L, and / or the concentration of mannitol is 5 g / L to 15 g / L, and / or the concentration of Proclin 300 is 0.01 g / L to 1 g / L; Optionally, the pH value of the third reagent is 7.4 to 7.

6.

9. The reagent kit according to claim 6, characterized in that, Further includes: Excite the substrate; Optionally, the excitation substrate includes NaOH and H2O2.

10. The use of the histone combination according to any one of claims 1-3, the histone antibody detection reagent according to claim 4 or 5, and the histone antibody detection kit according to any one of claims 6-9 in the preparation of detection products, characterized in that, The detection product is used to diagnose systemic lupus erythematosus, or the detection product is used to detect histone antibodies, wherein the histone antibodies can specifically bind to the histone combination according to any one of claims 1-3.

11. Application in histone antibody preparation and detection products, characterized in that, The testing product is used to diagnose systemic lupus erythematosus; The histone antibody can specifically bind to the histone combination described in any one of claims 1-3.

Images