WO2002076377A2 - Mononucleosome and process for producing the same, method of assaying antibody specific to nucleosome, method of diagnosing autoimmune disease, process for producing nucleosome dna, dna plate, process for producing dna plate and method of assaying anti-dna antibody - Google Patents

Mononucleosome and process for producing the same, method of assaying antibody specific to nucleosome, method of diagnosing autoimmune disease, process for producing nucleosome dna, dna plate, process for producing dna plate and method of assaying anti-dna antibody Download PDF

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WO2002076377A2
WO2002076377A2 PCT/JP2002/002664 JP0202664W WO02076377A2 WO 2002076377 A2 WO2002076377 A2 WO 2002076377A2 JP 0202664 W JP0202664 W JP 0202664W WO 02076377 A2 WO02076377 A2 WO 02076377A2
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mononucleosome
dna
nucleosome
antibody
producing
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PCT/JP2002/002664
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French (fr)
Japanese (ja)
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WO2002076377A1 (en
WO2002076377A3 (en
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Yoshiyuki Kanai
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Yoshiyuki Kanai
Ct For Advanced Science & Tech
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Priority to JP2002574893A priority Critical patent/JP4276843B2/en
Priority to AU2002238994A priority patent/AU2002238994A1/en
Publication of WO2002076377A2 publication Critical patent/WO2002076377A2/en
Publication of WO2002076377A1 publication Critical patent/WO2002076377A1/en
Publication of WO2002076377A3 publication Critical patent/WO2002076377A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6875—Nucleoproteins

Definitions

  • the present invention relates to a mononucleosome and a nucleosomal DNA suitable for diagnosing an autoimmune disease, and a diagnostic method using any of the mononucleosome and the nucleosomal DNA.More specifically, the present invention relates to the mononucleosome and the nucleosome DNA.
  • Mononucleosomes produced by the method for producing mononucleosomes and suitable for diagnosis and the like, kits for producing mononucleosomes capable of effectively producing the mononucleosomes, and analysis of mononucleosomes A useful histone test method, a method for measuring an antibody specific to a nucleosome including the above-mentioned mononucleosome, a simple and reliable method for diagnosing an autoimmune disease, a kit for diagnosing an autoimmune disease excellent in handleability, A DNA plate having the nucleosomal DNA and suitable for autoimmune disease diagnosis and the like, Method for efficiently producing serial DNA plate, and to a suitable anti-DN A antibody assay in autoimmune diseases diagnosis.
  • Background art
  • a nucleosome is a unit structure that forms the basic structure of chromatin (chromatin).
  • the nucleosome is composed of a nucleosomal core consisting of a histone octamer, which is an aggregate of two molecules of histones H2a, H2b, H3, and H4, each of which has 1.75 turns of DNA around the histone. Consists of one molecule of H 1 histone bound to the beginning and end of the winding. H2a, H2b, H3, and H4 histones interact mainly at the C-terminal side from the center of the molecule, and ionically bind to DNA at the N-terminal region. Each nucleosome is linked to each other by linker DNA, and is regularly arranged.
  • nucleosomes When a nuclease isolated from a cell is treated with nuclease, the linker DNA is cleaved, and the nucleosome is used as a unit of monomer (ie, Non-nucleosomes), and multimers such as dimers and trimers are cut out.
  • monomeric linker DNA When the monomeric linker DNA is completely cleaved, HI histones are released, containing an aggregate of eight other four histone molecules and 146 base pair DNA. It becomes a 5,5 nm cylindrical nucleosome core. It is thought that the nucleosomes are linked to form a fiber (nucleofilament) with a diameter of about 1 nm, and a higher order structure to form a fiber with a diameter of 30 nm.
  • anti-nucleosomal antibodies along with anti-DNA antibodies, have a very important clinical significance in the early diagnosis and diagnosis of systemic lupus erythematosus (Coristsidis, GN et al.
  • Glomerular uptake Glomerular uptake of nucleosomes: evidence for receptor mediated mesangial cell binding, Kidney Int, 47: 1258-1265 (1995); Burlingam, RW et al., Antichromatin in murine SLE
  • the origin and production of autoantibodies are determined by T cell-dependent immunity with autoantigens [Genesis and evolution of antichromat in autoant ibod ees in murine lupus implicates T-dependent immunization with self antigen], J. Clin. Invest., 91: 1687-1696 (1993); and And Amoura, Z.
  • nucleosomes derived from apoptosis are ideal for accurate detection of anti-nucleosome autoantibodies.
  • the nucleosome obtained as a result of apoptosis occurring naturally or in a pathological state and the nucleosome obtained by artificially cleaving chromatin having a normal structure with endonuclease are described in detail. It is considered that the structure is different.
  • nucleosomes obtained by apoptosis are variously modified and are likely to acquire so-called autoantigenicity.
  • modified nucleosomes serve as antigens.
  • the modified nucleosome itself may be a core histone modification, for example, phosphorylation, acetylation, methylation, ADP-ribosylation, glycosylation, ubiquitination and the like. If the modified nucleosomes can be isolated with high purity, the modified state can be retrieved from changes in mobility by 15–17% SDS-PAGE or 0.5% agarose gel electrophoresis.
  • the F arr method has a reputation as an accurate measurement method, but the F arr method requires that DNA antigens be labeled with radioactive substances, which is an advanced method. There is a disadvantage that it cannot be measured unless it is a medical facility.
  • the ELISA method is frequently used at the laboratory level. Whereas the Farr method performs an antigen-antibody reaction in liquid form, the ELISA method involves attaching an antigen to a plastic plate or the like. In order to efficiently attach DNA antigens to a solid phase, a method is generally used in which a plastic plate is previously coated with a basic protein, for example, poly-L-lysine (PLL), and then DNA is attached. .
  • PLL poly-L-lysine
  • nucleosomes derived from apoptosis are used as autoantigens for the production of anti-DNA antibodies.
  • the theory of action has been noticed (Amoura, Z. et al, Arthritis Rheum. 42: 833-843, 1999).
  • anti-DNA antibody measurement uses calf thymus-derived DNA or recombinant DNA as an antigen instead of human nucleosomal DNA.
  • accurate diagnosis was not always possible. This is because it is not easy to prepare human nucleosome DNA as an antigen.
  • An object of the present invention is to solve the above-described various problems and achieve the following objects. That is, the present invention provides a mononucleosome which is suitable for various uses including autoimmune disease diagnosis and the like, and which can maintain morphological stability, can be efficiently obtained by a simple operation, and can be obtained with high purity.
  • An object of the present invention is to provide a DNA production method, a DNA plate having high performance and excellent handling properties, an efficient production method of the DNA plate, and an anti-DNA antibody measurement method suitable for autoimmune disease diagnosis and the like. . Disclosure of the invention
  • the present inventor has earnestly studied to solve the above-mentioned problems, and as a result, has obtained the following knowledge. That is, the nucleosome is adsorbed to a protein A column or the like via an antibody specific to the nucleosome, and then eluted with a Tris buffer solution containing a high concentration of sodium chloride, and the nucleosome assembly is micrococcal Finding that mononucleosomes can be produced simply, efficiently and with high purity by recovering only mononucleosomes by HPLC etc. after treating them with nucleases etc. It is.
  • the present invention is based on the above findings by the present inventors, and the means for solving the above problems are as follows. That is,
  • ⁇ 1> a capture and collection step of capturing and collecting nucleosomes contained in the sample using an antibody specific to the nucleosome;
  • ⁇ 2> The method for producing a mononucleosome according to ⁇ 1>, wherein the antibody is at least one selected from an anti-nucleosome antibody, an anti-DNA antibody, and an anti-histone antibody.
  • ⁇ 3> The method for producing a mononucleosome according to ⁇ 1> or ⁇ 2>, wherein the antibody has an antigen-binding ability at a salt concentration of 14 O mM.
  • the antibody has the amino acid sequence of the variable region of 2C10 or an amino acid sequence in which 1 to 20 amino acids have been deleted, substituted or added in the amino acid sequence,
  • ⁇ 5> The method for producing a mononucleosome according to ⁇ 4>, wherein the antibody is 2C10.
  • ⁇ 6> The method according to any one of ⁇ 1> to ⁇ 5>, wherein in the capture collection step, the antibody is bound to a solid phase having an affinity for the antibody so that the antibody captures nucleosomes. This is a method for producing a nucleosome.
  • the antibody is previously bound to a solid phase, and the sample is brought into contact with the solid phase, whereby the antibody captures nucleosomes. And a method for producing a mononucleosome.
  • a fraction having a molecular weight of 200,000 to 250,000 is collected using a gel filtration column to isolate and purify the mononucleosome.
  • a method for producing the described mononucleosome is described.
  • ⁇ 10> The method for producing a mononucleosome according to any one of ⁇ 1> to ⁇ 9>, wherein the nucleosome is treated with a nuclease that can be cleaved into mononucleosome units before the capture and collection step.
  • ⁇ 12> The method for producing a mononucleosome according to ⁇ 10> or ⁇ 11>, wherein the nuclease is Micrococcal 'nuclease.
  • ⁇ 13> a release step in which cells are disrupted in a low-salt solution and nucleosomes are released into the solution;
  • ⁇ 15> The method for producing a mononucleosome according to ⁇ 13> or ⁇ 14>, wherein in the isolation and purification step, a fraction having a molecular weight of 200,000 to 250,000 is separated using a gel filtration column. .
  • the mononucleosome according to any one of ⁇ 1> A mononucleosome manufactured by a manufacturing method.
  • ⁇ 18> an antibody specific to the nucleosome for capturing and collecting nucleosomes contained in the sample
  • a test histone is recovered from a mononucleosome obtained from a test sample by the method for producing a mononucleosome according to any one of ⁇ 1> to ⁇ 15>, and an electrophoresis pattern of the test histone is obtained.
  • a method for testing a histone comprising comparing an electrophoretic pattern of a control histone with the electrophoretic pattern.
  • a method for measuring an antibody specific to a nucleosome comprising:
  • ⁇ 22> The method for measuring an antibody specific to a nucleosome according to ⁇ 21>, wherein the mononucleosome is an apoptosis-derived nucleosome.
  • ⁇ 23> The method for measuring an antibody specific to a nucleosome according to ⁇ 21> or ⁇ 22>, wherein the test sample is serum or plasma of an autoimmune disease patient.
  • the immobilized mononucleosome is blocked with a blocking solution containing at least skim milk.
  • the sample is diluted with a reaction solution containing at least skim milk, and then the sample is The method for measuring an antibody specific to a nucleosome according to any one of the above ⁇ 21> to ⁇ 23>, wherein the antibody is reacted with the immobilized mononucleosome.
  • a method for diagnosing an autoimmune disease comprising:
  • a kit for diagnosing an autoimmune disease comprising:
  • ⁇ 29> a capture and collection step of capturing and collecting a nucleosome contained in the sample by using an antibody specific to the nucleosome;
  • a method for producing a nucleosomal DNA is provided.
  • ⁇ 30> a mononucleosome production process comprising the method for producing a mononucleosome according to any one of ⁇ 1>
  • nucleosome DNA produced by the DNA production method which is a DNA plate characterized in that a human-derived nucleosomal DNA is immobilized on c.
  • ⁇ 32> The mononucleosome according to ⁇ 16> or ⁇ 17>, wherein the nucleosome DNA isolated and purified from human-derived mononucleosomes is immobilized on a ⁇ 32> plate. This is a DNA plate.
  • nucleosome DNA according to ⁇ 31> or ⁇ 32>, wherein the nucleosome DNA is a double-stranded DNA having a nucleosome structure, and the average length of the nucleosome DNA is 145 bp or more and 200 bp or less. It is a DNA plate.
  • ⁇ 34> The DNA plate according to any one of ⁇ 31> to ⁇ 33>, wherein the nucleosomal DNA is directly immobilized on a plate.
  • ⁇ 35> The DNA plate according to any one of ⁇ 31> to ⁇ 34>, wherein the plate comprises polystyrene.
  • ⁇ 36> The method for producing a mononucleosome according to any one of the above ⁇ 1> to ⁇ 15>, wherein the sample is a human-derived sample, a mononucleosome production step, and the nucleosome DNA is obtained from the mononucleosome.
  • ⁇ 37> In the immobilization step, dissolve nucleosome DNA on a plate in either Tris buffer or boric acid-caseisoder buffer containing 0.1M or more and 1.0M or less NaC1.
  • ⁇ 36> The method for producing a DNA plate according to ⁇ 36>, wherein the DNA plate is added.
  • a human-derived nucleosome DNA which is a nucleosome DNA produced by the method for producing nucleosome DNA described in ⁇ 29> or ⁇ 30>, is used at 0.1 M or more.
  • a method for producing a DNA plate comprising dissolving and adding to either a Tris buffer solution or a boric acid monosodium buffer containing 0 M or less NaCl to solidify the DNA.
  • a measuring step of measuring a specific antibody that binds to the nucleosome DNA BRIEF DESCRIPTION OF THE FIGURES
  • Figure 1 is a photograph showing the results of a 2% agarose gel electrophoresis of a DNA extract obtained by concentrating the washing solution of the protein A column in which 2C10-nucleoprotein complex has been captured.
  • Fig. 2 is a graph showing the nucleosomal profile (before micrococcal nuclease treatment) analyzed by condensing the eluate of 2C10-protein A column and analyzing by Superdex 200-HP LC chromatography. is there.
  • FIG 3 shows the profile of Superdex 200-HPLC chromatography after treatment of the sample of Figure 2 with micrococcal 'nuclease (MN) (mononucleosomes and linker DNA cut with MN). Is clearly understood).
  • MN micrococcal 'nuclease
  • FIG. 4 is a graph showing the results of rechromatography of a portion (1) corresponding to the mononucleosome in FIG. 3 by Superdex 200-HPLC.
  • FIG. 5 is a graph showing the results of rechromatography (typical example) of nucleosomes obtained from cultured cells or peripheral lymphocytes by Snperdex200-HPLC.
  • FIG. 6 is a graph showing the results of rechromatography of the portion (1) corresponding to the mononucleosome of FIG. 5 by Superdex 200-HPLC.
  • FIG. 7 is a photograph showing the results of fractionation of mononucleosomes derived from cultured cells having undergone apoptosis and mononucleosomes derived from normal cultured cells by 0.5% agarose electrophoresis.
  • FIG. 8 is a photograph showing the result of separating DNA constituting a mononucleosome by agarose gel electrophoresis.
  • Figure 9 shows that the core histones that make up the mononucleosome were converted to 15% SDS-PAG 4 is a photograph showing the results of fractionation and analysis by E.
  • FIG. 10 is a photograph showing an electrophoresis image of all histones (including H1) derived from normal cells by 15% SDS-PAGE.
  • FIG. 11 is a graph showing the results of measurement of antibodies specific to mononucleosomes by ELISA.
  • FIG. 12 is a graph showing the results of measurement of antibodies specific to mononucleosomes by subclass by ELISA.
  • FIG. 13 is a photograph showing the result of agarose electrophoresis of nucleosomal DNA.
  • FIG. 14 is a graph showing the immune response of SLE patient sera to DNA plates using nucleosome DNA, genomic DNA and calf thymus DNA as DNA antigens, respectively.
  • FIG. 15 is a graph showing the inhibitory effect of the anti-nucleosomal DNA antibody reaction on a plate without PLL pretreatment.
  • FIG. 16 is a graph showing the inhibitory effect of anti-nucleosome DNA antibody reaction on a plate pretreated with PLL.
  • FIG. 17 shows antibody responses of SLE patient sera to Immulon 2HB plates coated with nucleosomal DNA without PLL pretreatment and uncoated plates.
  • FIG. 18 is a diagram showing antibody responses of SLE patient sera to a plate coated with nucleosomal DNA and a plate not coated with PLL pretreated on Immulon 2 HB plate.
  • FIG. 19 shows the relationship between the presence or absence of PLL pretreatment and the anti-nucleosomal DNA antibody titer.
  • FIG. 20 is a graph showing the results of measuring the effect of NaC1 concentration when coating human nucleosome DNA on Immulon 2 HB plate by anti-DNA antibody titer.
  • FIG. 21 is a diagram showing the results of measuring the anti-DNA antibody reaction of a DNA plate prepared at a NaCl concentration of 0.14M.
  • FIG. 22 is a diagram showing the results of measuring the anti-DNA antibody reaction of a DNA plate prepared at a NaCl concentration of 0.25M.
  • FIG. 23 is a diagram showing the results of measuring the anti-DNA antibody reaction of a DNA plate prepared at a 0.5 M NaCl concentration.
  • the method for producing a mononucleosome according to the present invention comprises the steps of: capturing and collecting nucleosomes contained in a sample with an antibody specific to the nucleosome; dissociating and recovering the captured nucleosomes from the antibody. A recovery step, and an isolation and purification step of isolating and purifying mononucleosomes from the recovered nucleosomes based on molecular weight.
  • nucleosome means not only a monomer (mononucleosome) having a nucleosome as a unit, but also a multimer such as a dimer or a trimer, a mixture thereof, or a mixture thereof.
  • a sample containing a nucleosome ie, a nucleosome source is also meant.
  • nucleosome-containing sample examples include those obtained by separating cell nuclei from cells directly separated from human organs or tissues, those obtained by separating cell nuclei from general cultured cells, and cultured cells secreting nucleosomes ( KML! -7 cells (Kanai, Y. et al. Purification of a novel B cell growth and differentiation factor associated with lupus Syndrome, Immunol. Lett, 32: 43-48, 1992) or HL-60 cells (Collins, SJ et al .: Culture supernatant of Continuous growth and differentiation of human myeloid cells in suspension culture. Nature, 270: 347-349, 1997).
  • the “mononucleosome” refers to a nucleosomal monomer comprising a double-stranded DNA of about 146 base pairs and core histones (H3, H2B, H2A and H4).
  • the capture and collection step is a step of capturing and collecting nucleosomes contained in a sample using an antibody specific to the nucleosome.
  • antibody specific to a nucleosome refers to an antibody that exhibits a specific reaction to a nucleosome that is a complex of DNA and histone. As long as it can form an immune complex with the nucleosome, it has affinity for any of the nucleosome itself, the double-stranded DNA that forms the nucleosome, and the histone that forms the nucleosome. Or a specific reaction with a nucleosome and a reaction with a single-stranded double-stranded DNA or a single histone.
  • Antibodies specific to the nucleosomes also include known antibodies that were believed to be specific for double-stranded DNA, but rather were found to have higher affinity for nucleosomes.
  • the antibody specific to the nucleosome one that reacts more strongly with the nucleosome than with double-stranded DNA is preferable, and is adsorbed to an affinity column such as a protein A column via the Fc of the antibody. What can be done is preferred.
  • the affinity column examples include, in addition to Protein A, Pharmacia Biotech (PB) Hitrap afiiniticol mn, and the like. Specifically, Hitrap NHS- A 1 ml or 5 ml column of activated is preferably used.
  • the method for adsorbing (coupling) the antibody specific to the nucleosome to the affinity column is not particularly limited.
  • coupling buffer one (0. 2MNaHC0 3 0. 5M sodium chloride, p H 8. 3) at 0.5 to 1. adjust 2 C 10 so as to Omg / ml can and this done, adsorption
  • the operation after the coupling can be performed according to the protocol specified for each affinity column.
  • the antibody specific to the nucleosome is not particularly limited and may be appropriately selected depending on the intended purpose.An antibody capable of binding to an antigen even at a high salt concentration is used in a high salt solution. Monoclonal, which is an autoantibody produced by BRL hybridoma derived from MRL / 1 pr mouse, which is preferable because proteins that are nonspecifically bound to the column can be removed by washing. Antibody 2C 10 (belonging to IgG2b) is particularly preferred in that it has an antigen-binding ability even at a high salt concentration. The monoclonal antibody 2C10 is specific for double-stranded DNA (see Kubota, T. et al., Immunol. Lett.
  • the monoclonal antibody having the antigen specificity of 2C10 can be a human antibody having a variable region of 2C10, or one to 20 variable regions of 2C10, more easily. A person skilled in the art can easily obtain a mutant by deleting, substituting, or adding one or several amino acids. Antibodies specific to nucleosomes including the mononucleosome also include the mutant. The method for producing the monoclonal antibody 2C10 is briefly described below.
  • the spleen of a 6-month-old MRL / 1pr mouse was minced with an ophthalmic scalpel in a plastic dish containing culture solution (DMEM), and then the minced section was pressed with the frosted surfaces of two slide glasses. It crushes and releases lymphocytes from connective tissue. After a few minutes, centrifuge the floating cells. Immediately, cell fusion between mouse myeloma cells (SP 2) and lymphocytes is performed for 2 minutes in the presence of 44.4% by mass polyethylene glycol (PEG).
  • DMEM ophthalmic scalpel
  • SP 2 mouse myeloma cells
  • PEG polyethylene glycol
  • the monoclonal antibody 2C10 reacts with the double-stranded portion in the single-stranded DNA, but shows a strong affinity for ⁇ 174 plasmid, which is a template of the double-stranded DNA, and It is an excellent anti-double-stranded DNA antibody that prefers the base sequence AT.
  • the monoclonal antibody 2C10 was previously thought to be specific for double-stranded DNA.However, in studies conducted before reaching the present invention, purification of anti-double-stranded DNA antibody using a protein A column was performed. In the process, it was found that the DNA has strong affinity not only for double-stranded DNA but also for nucleosomes.
  • the “Protein A column” refers to a column in which protein A, a protein with a molecular weight of 42,000 derived from the bacterial wall of Staphylococcus aureus, is immobilized. It was done. Since the protein A binds to the constant region (Fc fragment) of IgG, it can bind to the immune complex without interfering with the antigen-antibody reaction. Utilizing this property, the protein A column method of capturing an immune complex with protein A is a method established as a method for purifying IgG. Examples of commercially available protein A columns that can be used in the present invention include Hittrap (Hitrap) Protein A column (manufactured by Amersham Pharmacia Biotech).
  • the antibody serving as a medium for adsorbing a nucleosome to a column such as a protein A column may be present in a free form in a solution such as a culture solution containing nucleosomes, or may be a protein A column. It may be immobilized in advance on a column such as.
  • the column may be a carrier column other than the protein A column.
  • the nucleosome and the antibody (immune complex) are contained in the culture supernatant or the like, it is preferable to concentrate the supernatant by a means such as ultrafiltration before pouring the supernatant into the protein A column.
  • a means such as ultrafiltration
  • the concentration is carried out by ultrafiltration, for example, Diaflo membrane (PH30) (Millipore) having a molecular weight of 30,000 cut-off can be used.
  • the dissociation and recovery step is a step of dissociating and recovering the nucleosomes captured and collected in the capture and collection step from the antibody.
  • the dissociation and recovery can be performed by dissociating the nucleosome-specific antibody with the nucleosome using a high-concentration sodium chloride solution or the like. As a result, only the nucleosome can be separated.
  • the eluate used for dissociating the nucleosome can be appropriately selected according to the purpose.
  • the concentration of sodium chloride is about 0.8 to 1.2 M, preferably 0.6 to 1.2 M. 0.8 M, particularly preferably 1.2 M, and a buffer having a pH of about pH 4.5 to 9.0, preferably pH 6 to 8, and particularly preferably pH 7 to 7.5.
  • a Tris buffer having a concentration of about 25 to 10 OmM, preferably 25 to 75 mM, and particularly preferably 25 to 5 OmM is preferred. .
  • the kind of the salt and the kind of the buffer in the eluate are not particularly limited and can be appropriately selected.
  • examples include a phosphate buffer and a carbonate buffer.
  • the washing solution used at this time is, for example, Tris buffer (25 mM Tris, 14 OmM sodium chloride, pH 7.4), etc., and the concentration, type of salt, pH, etc. may be appropriately adjusted depending on various conditions. it can. Whether or not the non-specific binding protein has been completely removed can be confirmed, for example, by the absorbance at 26 Onm being 0.00.
  • the isolation and purification step is a step of isolating and purifying mononucleosomes from the mononucleosomes recovered in the dissociation and recovery step based on the molecular weight.
  • a protein A column eluate containing nucleosomes is desirably used.
  • Nucleases that can cleave nucleosomes into mononucleosome units This is a process in which mononucleosomes are purified from the recovered nucleosomes based on their molecular weights after digestion with Nuclease (MN).
  • the purification based on the molecular weight can be performed, for example, by using high performance liquid chromatography (HPLC) to recover only the mononucleosome in a high yield, or using a gel filtration column to have a molecular weight of 200,000.
  • HPLC high performance liquid chromatography
  • the HP LC column is preferably a column capable of separating a molecular weight of 10,000 to 1,000,000, for example, a Superdex 200 column (manufactured by Amersham Pharmacia Biotech). Superdex 100 column (manufactured by Amersham Pharmacia Biotex) and the like.
  • the developing solvent for the HPLC has a sodium chloride concentration of about 140 to 120 OmM, and preferably 25 OmM; a concentration of sodium azide or the like is about 0.02 to 0.1 mass%, preferably 25-75 mM, preferably 25 mM, Tris buffer (pH 6.8-8.3, preferably pH 7,4) which is 0.04% by mass.
  • concentration, pH and the like can be appropriately selected according to the purpose.
  • the protein concentration of the mononucleosome for preservation is about 100 to 1 OOO zg / ml when measured with a BCA protein assay kit (manufactured by PIERCE) using serum albumin as a standard. Preferably, it is more preferably 500 gZml.
  • Mononucleosomes were detected by 15% SDSPAGE electrophoresis except for H1 except for core histones H3, H2a, H2b, H4 and their modified products, and the size of DNA extracted from nucleosomes was 150--20. This can be done by confirming that the probe is mounted only at the point of Obp by 1-2% agarose gel electrophoresis.
  • the protein A column eluate can be subjected to HP LC Before the non-nucleosome can be recovered, it is preferred that the eluate be treated (digested) with Micrococcal 'Nuclease (MN) prior to that. As a result, the recovery of mononucleosomes can be significantly increased by decomposing the polymerized nucleosomes into mononucleosomes.
  • MN Micrococcal nuclease
  • the concentration of the solution containing the nucleosome to be digested is preferably about 1 to 100 units / ml, and more preferably about 5 to 50 units Zml.
  • the sample containing the nucleosome may be treated (digested) with micrococcal nuclease before the capture and collection step. This makes it possible to increase the recovery of mononucleosomes as in the case of treating (consuming) before the isolation / purification step.
  • the purity of the mononucleosome obtained by the present invention is usually 95% by mass or more, preferably 99% by mass or more.
  • the mononucleosome contained in the obtained mononucleosome peak fraction was determined to be a mononucleosome by the method of Ishizaka et al. (Ishizaka et al., Nucleic Acid Res., 19: 5792, 1991).
  • the extracted DNA was detected in a concentration of 150-200 base pairs by 2% by mass agarose gel electrophoresis, and the core histones (histone H3, H2B, Only H2A and H4) are detected, and histone H1 is not detected.
  • the separation of mononucleosomes is extremely high.
  • the purity of DNA isolated from the obtained nucleosome is high, which can contribute to the efficiency of cloning for obtaining genetic information.
  • the method for producing mononucleosomes of the present invention can produce modified nucleosomes that have attracted attention in autoimmune diseases and the like, and can contribute to elucidation of the mechanisms of those diseases and the physiological significance of apoptosis. It has great academic significance. Also, as part of the genome project, elucidation of disease susceptibility and drug resistance is being pursued on a large scale in terms of single-nucleotide polymorphism (SNP).
  • SNP single-nucleotide polymorphism
  • the method for producing a mononucleosome of the present invention can greatly contribute.
  • a cell is crushed in a solution having a low salt concentration, and a releasing step of releasing nucleosomes into the solution; a collecting step of collecting the nucleosomes
  • the release step releases the nucleosomes into the solution and then converts the mononucleosomes into monolayers. You only have to separate them.
  • the nuclease treatment step can be performed in the same manner as the treatment (digestion) using nuclease, and the isolation and purification step can be performed as described above.
  • the mononucleosome of the present invention is produced by the method for producing a mononucleosome of the present invention.
  • the mononucleosome of the present invention is produced under strict conditions, its purity is usually as high as 95% by mass or more, preferably as high as 99% by mass or more.
  • the nucleosome maintains its original form well (excellent in form retention). Therefore, it can be suitably used for various measurements and diagnoses.
  • the mononucleosome of the present invention can be stored stably for a long period of time.
  • tris N a C 1 added Tris buffer consisting of N a C 1 and N a N 3 in (TBS), or 1 wt% bovine serum albumin (BSA), 0. 4% by weight
  • TBS Tris buffer
  • BSA bovine serum albumin
  • the mononucleosome maintained its antigenicity even after being stored at 4 ° C for 2 months in Chofu 83 containing skim milk, 10% by mass Block Ace, and ImM 0-cho.
  • Nucleosomes generally have low stability and are destroyed when frozen, so the stability is a great advantage.
  • a mononucleosome derived from apoptosis retains the modification by apoptosis and easily acquires autoantigenicity, so that it can be particularly preferably used for antibody measurement (autoimmune disease diagnosis) of an autoimmune disease patient. .
  • the kit for producing a mononucleosome of the present invention comprises an antibody specific to the nucleosome for capturing and collecting the nucleosome contained in a sample,
  • the kit for producing mononucleosomes of the present invention in the method for producing mononucleosomes of the present invention, it is suitable for various uses including mononucleosome analysis, autoimmune disease diagnosis, etc., and storage stability. It is possible to easily and efficiently produce a mononucleosome with excellent form stability, high efficiency, and high purity.
  • a test histone is recovered from a mononucleosome obtained by the method for producing a mononucleosome of the present invention, and the electrophoretic pattern of the test histone is compared with the electrophoretic pattern of a control histone.
  • a nucleosome modified by apoptosis is considered to be an antigen, and the main body of the modified nucleosome is considered to be cohistone modification. Therefore, by examining the modification of the core histone of the mononucleosome, the state of the modification of the nucleosome can be grasped and the self- It can be used for diagnosis and elucidation of epidemics.
  • a mononucleosome obtained from a test sample by the method for producing a mononucleosome of the present invention is analyzed by SSD-PAGE.
  • the results of this analysis can be performed by comparing the results of SDS-PAGE analysis of histones obtained from normal cells.
  • a mononucleosome produced by the method for producing a mononucleosome of the present invention is used.
  • This mononucleosome has a high purity of usually 95% by mass or more, preferably 99% by mass or more, and retains the form of the mononucleosome well (excellent in shape stability). Therefore, it is suitable as an antigen against a nucleosome-specific antibody.
  • apoptosis-derived mononucleosome obtained by the method for producing a mononucleosome of the present invention retains apoptotic modification and easily acquires self-antigenicity. Particularly preferred.
  • the buffer for immobilizing the mononucleosome is not particularly limited and can be appropriately selected as long as it does not change the form of the mononucleosome.
  • a 5 OmM carbonate buffer pH 9 6 can be used.
  • the solid phase used in the solid phase immobilization step is not particularly limited as long as it can immobilize mononucleosomes, and can be appropriately selected.
  • a polystyrene micro-titer plate Immu 1 on 2 HB (Dynex Technologies, Chantilly, VA, manufactured by Dynex Technology, Inc.) or the like can be suitably used.
  • the conditions for the immobilization of the mononucleosome can be appropriately selected according to the type of the solid phase and the like.
  • the polystyrene micro tie plate is fixed.
  • poly-L-lysine (PLL) (1 ⁇ ) which has been conventionally used for adsorbing ds DNA antigen to a plate is used.
  • PLL poly-L-lysine
  • the solid phase immobilization step after the mononucleosome is immobilized on the solid phase, the solid phase is blocked with a blocking solution in order to shield the non-immobilized solid phase of the mononucleosome and prevent nonspecific reaction.
  • a blocking solution in order to shield the non-immobilized solid phase of the mononucleosome and prevent nonspecific reaction.
  • the blocking can be performed by reacting the solid phase with the blocking solution and then washing.
  • the blocking solution preferably a solution containing skim milk even without low, Tris, the NaC l and NaN 3 or Ranaru Na C 1 added Tris buffer (TB S), 0. 1 ⁇ 0. 3 % by weight of skim Solutions containing milk are particularly preferred.
  • reaction step If the reaction step is not started immediately after the solid phase immobilization step, add TBS or the reaction solution described below to each well, seal with a plate seal, and store at 4 ° C.
  • the reaction step is a step in which a test sample is reacted with the immobilized mononucleosome.
  • the test sample is preferably serum or plasma of a healthy person or a patient, and is preferably an autoimmune patient. Serum or plasma of a patient suspected of having an autoimmune disease is particularly preferable because it can be used for diagnosis of diseases such as diseases.
  • the method of reacting the sample with the mononucleosome is not particularly limited and may be appropriately selected as long as the method allows an antibody specific to the nucleosome in the sample to bind to the mononucleosome.
  • the method can be carried out by diluting the sample with a reaction solution, reacting the sample with shaking, removing the sample by suction after the reaction, and washing.
  • the reaction solution for diluting the sample preferably contains skim milk from the viewpoint of preventing the non-specific reaction, and includes 1% by mass bovine serum albumin (BSA), 0.4% by mass skim milk, and 10% by mass block.
  • BSA bovine serum albumin
  • Ace and TBS containing ImM EDTA are particularly preferred.
  • the BSA was obtained from Albumin Fraction V (Boehnnger Mannheim, manufactured by Boehringer Mannheim, Inc.). Germany) is preferable, the block ace is preferably manufactured by Dainippon Pharmaceutical Co., Ltd., and the EDTA is preferably a disodium salt of EDTA (manufactured by Dojindo).
  • the measurement step is a step of measuring a specific antibody that binds to the mononucleosome, but is not particularly limited as long as the antibody that binds to the mononucleosome can be measured. You can choose.
  • a secondary antibody that recognizes an antibody that binds to the mononucleosome may be measured instead of the antibody.
  • an alkaline phosphatase (AP) conjugated anti-human IgG antibody (goat) diluted solution is added to each well, and the mixture is shaken. After washing, a reagent such as P-nitrophenyl phosphate (PNP) (Sigma, St.
  • the antibody titer can be expressed as the actual measured value of the absorbance, it is preferable to prepare a standard curve in advance with a high-titer serum and display the antibody titer in the sample in units of unit by comparison with the standard curve. Good.
  • the measurement step may also include a step of reacting an anti-human antibody recognizing an IgG subclass as a secondary antibody, and in this case, measurement of an antibody for each subclass is particularly preferable. .
  • IgG antibodies there are four subclasses of IgG antibodies, IgGl, IgG2, IgG3 and IgG4, and the deviation of the subclass of the antibody specific to the nucleosomal possessed by the patient is as follows: It may characterize the disease pathology. Therefore, if the measurement of antibodies by subclass is possible, the disease pathology can be better understood.
  • a biotin-labeled anti-human subclass antibody (mouse) (Zymed, San Francisco, CA) is used instead of the alkaline phosphatase (AP) conjugated anti-human IgG antibody (goat).
  • AP alkaline phosphatase conjugated anti-human IgG antibody
  • the method for measuring an antibody specific to a nucleosome uses a mononucleosome having high purity and a good original form, so that when a self-antigen is positive, an extremely high antibody titer to a nucleosome can be obtained. This is an extremely accurate measurement method that always shows a low antibody titer when the autoantigen is negative. Also, if necessary, the composition of the blocking agent and the reaction solution can be adjusted to prevent a non-specific reaction and reduce the background threshold. Further, if necessary, the condition of an autoimmune disease patient such as SLE can be analyzed in more detail by measuring antibodies by subclass.
  • the method for measuring an antibody specific to a nucleosome according to the present invention can measure an antibody specific to a nucleosome with extremely high accuracy, and can be used to diagnose autoimmune diseases, particularly SLE, lupus nephritis, vasculitis, CNS lupus, Very effective in diagnosing pediatric SLE. (Autoimmune disease diagnosis method)
  • the method for diagnosing an autoimmune disease of the present invention includes a solid-phase immobilizing step of immobilizing the mononucleosome produced by the method for producing a mononucleosome of the present invention,
  • a reaction step of reacting the sample with the immobilized mononucleosome a measurement step of measuring a specific antibody that binds to the mononucleosome; and an evaluation step of evaluating the antibody titer.
  • an antibody specific for a nucleosome is measured by the method described in the method for measuring an antibody specific for a nucleosome, and the measured antibody titer is evaluated.
  • the evaluation of the antibody titer for example, a positive value or a negative value can be evaluated based on whether or not a predetermined threshold value is exceeded, by, for example, three times the average value + standard deviation.
  • nucleosome-specific antibodies were measured for each IgG subclass, and the antibody titer of each was determined to be that of IgG. It is also possible to evaluate whether or not each predetermined threshold set for each subclass is exceeded. It can also be evaluated by analyzing the antibody titer pattern for each subclass. The assessment by subclass is more representative of the condition. (Kit for diagnosing autoimmune diseases)
  • the kit for diagnosing an autoimmune disease of the present invention comprises a solid phase obtained by immobilizing the mononucleosome produced by the method for producing a mononucleosome of the present invention, a buffer, and one of a plate and a column.
  • the autoimmune disease diagnostic kit is produced by the method for producing a mononucleosome of the present invention and contains a mononucleosome having excellent morphological stability in high purity, so that an antibody specific to the nucleosome is extremely purified. It can be measured with high accuracy and is extremely effective in diagnosing autoimmune diseases, especially SLE. Further, the autoimmune test kit can be hermetically sealed by filling with a buffer solution, and is excellent in storage stability because the storage stability of the nucleosome is high.
  • the kit for diagnosing an autoimmune disease may further include a reaction solution, a diluting solution, a washing solution, a secondary antibody, and the like, if necessary. Further, the secondary antibody may be an anti-human subclass antibody.
  • the kit for diagnosing an autoimmune disease can be prepared, for example, by the method described in the immobilization step of the method for measuring an antibody specific to a nucleosome.
  • the method for producing a nucleosome DNA of the present invention comprises the steps of producing a mononucleosome comprising the method for producing the mononucleosome of the present invention
  • nucleosome DNA isolation and purification step of isolating and purifying the nucleosome DNA from the mononucleosome.
  • the nucleosomal DNA isolation and purification step for isolating and purifying the nucleosomal DNA from the mononucleosome can be performed by appropriately selecting from known methods (Kanai, Y et al: Induction and natural occurrence of serum nucleosomal). DNA in autoimmune MRL / lpr / lpr mice: its relation to apoptosis in the thymus. Immunol Lett. 46: 207-214, 1995). For example, an aliquot of purified nucleosome is suspended in Tris-EDTA buffer containing 1% SDS and 0.5 mg / ml Proteinase K and treated at room temperature for 60 minutes.
  • RNA solubilized by this procedure is precipitated with 50% isopropyl alcohol, and the precipitated DNA is dissolved in the above Tris-EDTA buffer, a small amount of RNase is added, and RNA that may be present may be mixed.
  • a decomposition method can be used.
  • the nucleosomal DNA produced by the method for producing nucleosomal DNA is extracted from the mononucleosome having a high purity and a good shape as described above, so that the purity derived from the mononucleosome is high. DNA.
  • DNA According to agarose gel electrophoresis of the nucleosome DNA produced by one example of the method for producing the nucleosome DNA, there is a strong band around 150 bp, which is considered to be mononucleosome double-stranded DNA. .
  • the average chain length of these nucleosome DNAs is preferably between 145 bp and 20 Obp, and may contain as little as 320-400 bp of DNA.
  • Nucleosome DNA produced by the above nucleosome DNA production method can produce human-derived nucleosomal DNA if produced from a human-derived sample. It is superior in that it can eliminate non-specific reactions caused by using DNA, and that it is a nucleosome DNA that has a strong possibility of becoming a self-antigen for the production of anti-DNA antibodies, as noted in recent years.
  • the method for producing nucleosome DNA of the present invention provides a method for easily and accurately producing such nucleosome DNA, which has not always been easy to produce until now. A nucleosomal DNA suitable for diagnosis can be provided.
  • the DNA plate of the present invention is not particularly limited, except that it is a nucleosomal DNA produced by the above-described nucleosomal DNA production method, and a human-derived nucleosomal DNA is immobilized on the plate. Further, the DNA plate of the present invention is also a DNA plate, wherein the nucleosome DNA isolated and purified from the human-derived mononucleosome is immobilized on the plate. Good. These DNA plates have attracted attention in recent years for using human DNA as an antigen for determining pathological conditions such as SLE, which can eliminate non-specific reactions caused by using heterologous DNA. It is excellent in that it uses a nucleosome DNA that has a strong possibility of becoming an autoantigen for producing anti-DNA antibody.
  • nucleosome DNA is directly immobilized on the plate.
  • An unknown antigen structure formed by an antibody against PLL and a PLL-DNA complex of a conventional plate to which DNA is attached via a basic protein such as poly-L-lysine (PLL) This is preferable because problems due to an antibody reaction to the above can be eliminated.
  • the material of the plate is preferably a material containing polystyrene because DNA can be directly attached to the plate.Micro-titer plates Immu1on2HB, Immulon4HB and ImmulonHB (Dynex Technology Co., Ltd. Technologies, Chantilly, VA) are particularly preferred.
  • the DNA plate of the present invention reduces the background of normal serum, thereby increasing the reliability of the antibody titer of the target disease serum, and measuring the anti-DNA antibody reaction for determining the pathological condition such as SLE. Can be used very suitably.
  • the method for producing a DNA plate of the present invention is the method for producing a mononucleosome, wherein the sample is a human-derived sample;
  • a nucleosomal DNA isolation and purification step for isolating and purifying the nucleosome DNA from the mononucleosome isolating and purifying the nucleosome DNA from the mononucleosome
  • the method includes a step of immobilizing the nucleosome DNA on a plate.
  • the nucleosome DNA is directly attached to the plate without a pretreatment such as PLL.
  • the buffer for immobilizing the mononucleosome is not particularly limited and may be appropriately selected as long as it does not change the form of the nucleosomal DNA.
  • the immobilization buffer is preferably a Tris buffer or a borate monophosphate buffer containing 0.1 to 1.0 M of NaC 1, and 0.11 or more. Tris buffers containing 1.0 M or less NaCl are more preferred. It is particularly preferable that the concentration of NaC1 is 0.25 M or more from the viewpoints of coating efficiency and anti-DNA antibody reactivity of the prepared DNA plate.
  • the solid phase used in the solid phase immobilization step is not particularly limited as long as it can immobilize the nucleosome DNA, and can be appropriately selected. From the viewpoint of directly attaching the chromosomal DNA to the plate, a polystyrene microphone plate is preferred, and Immulon2HB (Dynex Technologies, Chantilly, VA) is particularly preferred.
  • the conditions for the immobilization of the nucleosomal DNA can be appropriately selected according to the type of the solid phase and the like.
  • the immobilization include the polystyrene microphone opening plate When using as a solid phase, nucleosomal DNA is dissolved at a concentration of 0.5 ⁇ / 1111 in Tris buffer containing 0.25 M NaCl and adsorbed overnight at 4 ° C. Preferably, a method of once removing the excess antigen by suction and washing with a Tris buffer solution containing NaCl is preferably used.
  • Blocking can be performed according to the case of the immobilization of the nucleosome.o
  • the method for producing a DNA plate of the present invention comprises, on a plate, the nucleosomal DNA produced by the method for producing a nucleosomal DNA and the human-derived nucleosomal DNA having a concentration of 0.1 M or more and 1.0 M or less.
  • the solid phase may be formed by dissolving and adding NaCl in either Tris buffer or boric acid-caustic soda buffer.
  • the method for measuring anti-DNA antibody of the present invention comprises using the above-mentioned DNA plate, and reacting a test sample with nucleosome DNA immobilized on the DNA plate;
  • a measurement step of measuring a specific antibody that binds to the nucleosome DNA includes: a measurement step for measuring a specific antibody that binds to the nucleosome DNA. can do.
  • An autoantibody 2C10 (belonging to IgG2b) specific to double-stranded DNA, produced by a B cell hybridoma derived from the MR L / lpr mouse known as an autoimmune disease model, Separation from the culture supernatant of the above hybridoma using the protein A column method already established as a purification method, and analyzing the separated antibodies by SDS-polyacrylamide gel (PAGE) electrophoresis.
  • PAGE SDS-polyacrylamide gel
  • DNA binding proteins nucleoprotein complexes
  • histones histones
  • the protein A column on which the 2C10-nucleoprotein complex was captured was washed with a 25 mM Tris buffer containing 1.2 M sodium chloride (hereinafter, a 25 mM Tris buffer containing 1.2 M sodium chloride). Was called "buffer A") as the eluent, and the eluate was concentrated to extract DNA.
  • buffer A a 25 mM Tris buffer containing 1.2 M sodium chloride
  • C10 hybridomas are cultured in large amounts (about 1 liter) in a serum-free medium (the medium may be DMEM or RPMI normal medium supplemented with 5% by mass fetal serum), and the culture supernatant is subjected to a molecular weight of 30,000 cells.
  • the solution was concentrated 20-fold by ultrafiltration using a Diaflo membrane PM30 (manufactured by Millipore) of Tooff.
  • the concentrated solution is poured into the above-mentioned Hittrap Protein A column (5 ml) (manufactured by Amersham Pharmacia Biotech) to capture the nucleoprotein complex, and then to a 25 mM Tris buffer containing 14 OmM sodium chloride.
  • the squirrel buffer was washed with “buffer C” (pH 7.4) to completely remove non-specifically bound proteins. Removal of non-specifically bound proteins was complete when the absorbance at 260 nm was 0.00.
  • buffer C pH 7.4
  • nucleosomes were eluted with buffer A. This eluate contained a mononucleosome and an oligo- or polynucleosome in which two or more mononucleosomes were linked.
  • a Superdex 2000 column manufactured by Amersham Pharmacia Biotech
  • a Superdex 2000 column capable of separating molecules having a molecular weight of 10,000 to 1,000,000 is used.
  • HPL C high performance liquid chromatography
  • a 25 mM Tris buffer containing 25 OmM sodium chloride and 0.04% by mass of sodium azide hereinafter referred to as a 25 mM Tris buffer containing 25 OmM sodium chloride and 0.04% by mass of sodium azide
  • the solution was called “buffer B” (pH 7.4).
  • Figure 2 shows the absorbance profile of the eluted fraction. After the large peak of the polynucleosome fraction, the peak of the mononucleosome fraction was detected at a molecular weight of 200,000 to 250,000.
  • polynucleosomes were digested into mononucleosome units. Specifically, C a 2 + a 2. 5 mM, was added by Uni comprising a micro Cocker Le Nuclear Ichize to 0.5 units / ml and 200/1 to eluate obtained, 37 ° C For 45 minutes. Immediately after the completion of the incubation, the reaction was stopped by adding HGT A to a concentration of 5 mM. Insolubles were removed with a microfuge (15 k rpm).
  • a protease inhibitor cocktail complete TM and EDTA free (from Boehringer Mannheim) were used as prescribed to prevent protein degradation.
  • a serine protease inhibitor AEBSF 4- (2-aminoethyl) -benzenesulfonylfluoride) (manufactured by Sigma) was added at 100 / M. The same applies to the following embodiments.
  • the method for producing a mononucleosome of the present invention is also applicable to general established cells that do not produce double-stranded DNA and / or antibodies specific to nucleosomes or cells directly isolated from animal tissues including humans (primary cultured cells). it can.
  • KML i-7 (Kanai et al., Intl. Archs. Allergy Appl. I recitation unol., 81: 92-94, 1986), a cell line derived from MRL / lpr mouse, which is an autoimmune disease model Regardless of the method, it is known that nucleosomes are secreted into the culture supernatant without stimulation. Therefore, this culture supernatant can be used as a source of nucleosome as it is.
  • Nucleosomes can also be released into the culture supernatant by adding an apoptosis-inducing agent to various commercially available cancer cell lines or primary culture cells that have been established. Can be used as a source. After concentrating the culture supernatant to an appropriate concentration (10- to 20-fold), it was treated (digested) with micrococcal nuclease (MN), and the monoclonal antibody 2C10 was immobilized. Pour over a Protein A column.
  • MN micrococcal nuclease
  • the protein A column is washed with buffer C to remove non-specifically bound proteins, and then eluted with buffer A. Immediately dialyze the eluate against buffer B and concentrate. This concentration can be carried out by ultrafiltration using the Diaflo membrane PM30 if the volume is large, and it can be carried out using the ultrafree if the volume is small. Can be.
  • Example 2 the above concentrated solution was subjected to HPLC using a Superdex 200 column to recover mononucleosomes.
  • the fractionation pattern by HPLC was the same as in FIGS. 3 and 4.
  • the immobilization of the monoclonal antibody 2C10 was performed as follows.
  • the protein A column was equilibrated with the Tris buffer described in paragraph No. 0060, and 2C 10 (0.5 to 1.0 mg / ml) prepared with the Tris buffer was changed.
  • the flow rate through the column is 5 ml / min and the cycle is two times (the second time the adsorption is perfect). After adsorption and immobilization, the column was again equilibrated with Tris buffer. (Example 3)
  • Hypotonic buffer (5 Omm Tris, 50 mM potassium chloride, 0. 5 mM chloride magnetic Shiumu, 0. 15mM2- ME, 0. 25M sucrose, 0. 2 mM AEBSF, 0. 04 wt% sodium azide (NaN 3 ), PH 7.4)
  • washed nuclei were suspended in 0.5 ml of a buffer having the same composition as that described above except that sucrose was not contained, and 1.25 U / ml of micrococcal nuclease (MN) and 5 mM Ca2 + was added, and the enzyme was digested in the same manner as in Example 1, and the reaction was stopped by adding EGTA to 5 mM. Centrifugation was performed at 13k for 5 minutes. Buffer B was added to the resulting precipitate, and the soluble fraction was collected by 13k centrifugation.
  • MN micrococcal nuclease
  • mononucleosome fraction when the obtained mononucleosome fraction was applied again to a Superdex 200 column, it was purified as a substantially single beak shown in FIG.
  • mononucleosomes could also be purified by directly applying them to a Superdex 200 column without using an antibody 2C10 binding column and fractionating fractions having a molecular weight of 200,000 to 250,000. Purification without using a 2C10 binding column is slightly inferior to purification using a 2C10 binding column. Similar effects were obtained. (Example 4)
  • Example 4 a comparative analysis was performed between the mononucleosome derived from the cultured cells having undergone apoptosis obtained in Example 2 above and the mononucleosome derived from normal cultured cells obtained in Example 3. went.
  • nucleosomes were fractionated by 0.5% agarose electrophoresis. As shown in FIG. 7, nucleosomes from cells that underwent apoptosis compared to mononucleosomes from normal cells migrated as broad bands.
  • each of the core histones and DNAs constituting the mononucleosome was analyzed.
  • Fig. 9 shows the results.
  • the histones derived from normal cells exhibited the same fractionation pattern as the normal pattern of H3, H2b, H2a and H4 shown in FIG.
  • abnormalities were observed in the fractionation pattern of core histones derived from apoptotic cells. That is, the band corresponding to H 2 b decreased, and two new bands appeared sandwiching the H4 band.
  • nucleosomes were added to the support, 2 g / ml via poly-L-lysine (PLL) (Sigma, St. louis, MO, manufactured by Sigma) (lg / ml distilled water) conventionally used for the adsorption of ds DNA antigen to the plate. It was adsorbed at a concentration of ml at 4 ° C.
  • PLL poly-L-lysine
  • TBS NaCl-containing Tris buffer
  • 100 ⁇ 1 blocking solution (TBS containing 2% by mass skim milk (Difco, Detroit, MI) containing 2% by weight of Difco, Inc.) was added to each well, and the mixture was allowed to react for one hour to shield the site where no antigen was attached.
  • an antigen-attached plate was prepared by washing four times with TBS in the same manner as the washing after the antigen adsorption. Until use, add 100 ⁇ l of TBS to each well, seal tightly with a plate seal, and store at 4 ° C.
  • the anti-nucleosome antibody was measured in the sera of 12 SLE patients and 26 healthy subjects during treatment.
  • Serum was collected from 12 SLE patients and 26 healthy subjects. Reaction solution [1% by mass bovine serum albumin (BSA), 0.4% by mass skim milk, 1 The serum was diluted 100-fold with TBS containing 0% by mass Block Ace and ImM EDTA, and added to each well of a microtiter plate at 50: 1.
  • BSA is albumin fraction V (Albumin, pronounced V, Boehringer Mannheim, Germany)
  • the block ace is Dainippon Pharmaceutical Co., Ltd.
  • the EDTA is EDTA disodium salt (Dojindo). It was used.
  • the microplate was allowed to react at room temperature for 30 minutes with gentle shaking on a horizontal shaker. Immediately after the reaction, the reaction solution was removed by suction, and the plate was washed four times with TBS containing 0.05% by mass of Tween 20 in the same manner as the plate after the antigen was attached.
  • the mixture was diluted 2000-fold, added to each well, and reacted with shaking at room temperature for 30 minutes as in the case of the serum. After washing in the same manner as in the case of the serum, the substrate of AP adjusted to 1 mg / m1 with 2.5 mM Mg 2+ -added carbonate buffer (50 mM, pH 9.8) was added. 100 ⁇ l of ditrophenyl phosphate (PNP) (Sigma, St. Louis, M0) reagent was added to each well, and the mixture was shaken at room temperature for 30 minutes in the same manner as in the case of the serum.
  • PNP ditrophenyl phosphate
  • the degree of color development was measured at an absorption wavelength of 405 nm using an autoreader.
  • the control or blind test was the absorbance obtained when serum was not added in the above series of reaction systems, and the value obtained by subtracting the absorbance was used as the actual measurement location.
  • the antibody titer was represented by absorbance.
  • the nucleosome-specific antibody can be measured with extremely high accuracy by the measurement method of the present invention, and the nucleosome-specific antibody measurement method of the present invention is extremely effective for SLE diagnosis. This has been proven.
  • IgG antibodies There are four subclasses of IgG antibodies, IgG1, IgG2, IgG3 and IgG4.
  • the bias of the patient's subclass of nucleosomal-specific antibodies may characterize the disease state. Therefore, measurement of antibodies by subclass is important for understanding disease pathology.
  • Mononucleosomes were produced from human promyelocytic leukemia cell culture strain HL-60 chromatin in the same manner as in Example 1, and nucleosomal DNA was further extracted. Nucleosome DNA was extracted by a known method (Kanai, Yetal: Induction and natural occurrence of serum nucleosomal DNA in autoimmune MRL / lpr / lpr mice: its relation to apoptosis in the thymus. Immunol Lett. 46: 207-214, 1995). Specifically, a fixed amount of the purified nucleosome was suspended in a Tris-EDTA buffer containing 1% SDS and 0.5 mg / ml Proteinase K, and incubated at room temperature for 60 minutes.
  • FIG. 13 shows the agarose gel electrophoresis pattern of the nucleosome DNA. A single band was observed around 150 bp, indicating that the mononucleosomal DNA was extracted.
  • the nucleosome DNA obtained in Example 8 its mother genomic DNA of HL-60 cells, and calf thymus DNA conventionally used were immobilized on an ELISA plate.
  • Genomic DNA of HL-60 cells was extracted from the chromatin of the cells according to the method for extracting DNA from the nucleosomes.
  • the size of the genomic DNA was approximately 20 kbp, whereas the nucleosome DNA was approximately 150 bp.
  • the DNA was added to the support at 0.5 ⁇ / It was dissolved in Tris buffer (pH 7.4) containing 0.25 M NaCl at a concentration of 1111, added at 25 ng / we11, and adsorbed at 4 ° C overnight. After the adsorption, the excess antigen was once removed by suction, and then washed four times with NaCl-containing Tris buffer (25 mM Tris, 140 mM NaCl, 0.04% by mass NaN 3 , pH 7.4) (TBS).
  • Tris buffer pH 7.4
  • TBS TBS containing 2 mass% skim milk (Difco, Detroit, MI) containing skim milk (Difco, Inc., Difco, Detroit, MI)
  • 100/1 blocking solution TBS containing 2 mass% skim milk (Difco, Detroit, MI) containing skim milk (Difco, Inc., Difco, Detroit, MI)
  • an antigen-attached plate was prepared by washing the plate four times with TBS in the same manner as the washing after the antigen adsorption. Until use, add 100/1 TBS to each well, seal with a plate seal, and store at 4 ° C.
  • the serum of one SLE patient was subjected to the measurement of the anti-DNA antibody using the microtiter plate of Example 9 in the same manner as the measurement of the anti-nucleosomal antibody of Example 6.
  • Figure 14 shows the measurement results.
  • the patient's immune response to human nucleosomal DNA was found to be much stronger than to calf thymus DNA. Furthermore, it was found that the nucleosome DNA had a stronger immune response than the human genome DNA. This tendency was also observed in the other three SLE patients with high anti-DNA antibody titers.
  • an antibody in the serum was previously absorbed in a solution using an antigen, and then an anti-nucleosomal DNA antibody activity was measured in a suppression experiment.
  • antigens nucleosomal DNA, genomic DNA and calf thymus DNA were used.
  • Figure 15 shows the results of the suppression experiment. The amount of calf thymus DNA required to inhibit anti-nucleosome DNA antibody activity by 40% was three times that of nucleosome DNA, indicating the weak antigenicity of the heterologous DNA.
  • FIG. 16 shows the results of the suppression experiment.
  • the amount of calf thymus DNA required to inhibit anti-nucleosome DNA antibody activity by 40% is 1.3 times that of nucleosomal DNA, which is lower than that of a plate not pre-treated with PLL. And the difference between them becomes inconspicuous. This is thought to be due to the modification of the DNA antigen structure by the PLL. That is, since the antibody activity measured by the DNA plate pretreated with PLL includes a reaction against an unknown antigen newly formed by DNA and PLL, the suppression effect of the original DNA antigen is inconspicuous. It is thought that it became.
  • the difference in antigenicity between the plate with DNA attached without PLL pretreatment and the plate with DNA pretreated with PLL was further investigated.
  • the sera of 24 SLE patients and the sera of 24 healthy subjects were pretreated with nucleosome DNA without PLL pretreatment and nucleosome DNA was pretreated with PLL to attach nucleosome DNA.
  • the anti-DNA antibody reaction was measured with the plate.
  • the anti-DNA antibody reaction was also measured on each plate to which no nucleosomal DNA antigen was attached.
  • Figure 17 shows the measurement results on the plate to which nucleosome DNA was attached without pretreatment with PLL
  • Fig. 18 shows the measurement results on the plate to which nucleosome DNA was attached with PLL pretreatment.
  • the reactivity of the serum of the SLE patient to the plate on which the nucleosomal DNA antigen was not attached was high.
  • the reactivity of the serum of a healthy subject to the plate (+ Ag in the figure) to which the nucleosomal DNA antigen was attached was also high. This indicated that non-specific reactions could not be excluded in plates pre-treated with PLL.
  • FIG. 19 shows the result of comparing the measured values of the SLE patients as the normal range.
  • the positive rate in the former was 41.6% (10/24), while the positive rate in the latter was 16.6% (4/24), indicating that nucleosomal DNA was obtained without pretreatment with PLL.
  • the attached plates proved to be significantly better at diagnosing SLE.
  • the DNA plate not pre-treated with PLL that is, the DNA plate attached without the PLL, can measure the immune response to the DNA antigen more accurately, It can be used for various diagnoses.
  • the coating efficiency ie, the serum of one SLE patient with a high anti-nucleosomal DNA antibody titer It investigated using.
  • a Tris buffer containing 0.14M, 0.251 and 0.5M NaCl The anti-DNA antibody was measured when nucleosome DNA (0.5 / g / ml) was dissolved and adsorbed on the plate.
  • Fig. 20 shows the measurement results. It can be seen that the case of 0.25 M has the highest anti-DNA antibody reactivity and the highest coating efficiency.
  • anti-DNA antibodies were measured on 24 SLE patients and 24 healthy subjects on plates prepared at the above three concentrations.
  • Figure 21 shows the measurement results for the plate made at 0.14M NaCl concentration
  • Figure 22 shows the measurement results for the plate made at 0.25M NaCl concentration
  • Figure 23 shows the measurement results. It was found that the plate prepared at a NaCl concentration of 0.25M or more was particularly excellent in detecting anti-DNA antibodies.
  • a method for producing a mononucleosome which can obtain nucleosomes suitable for various uses including autoimmune disease diagnosis and the like while maintaining morphological stability, efficiently by simple operations, and with high purity

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Abstract

It is intended to provide a process whereby a highly pure mononucleosome can be conveniently and efficiently produced. Namely, a process for producing a mononucleosome involving: the step of capturing and collecting a nucleosome contained in a sample by using an antibody specific to the nucleosome; the step of dissociating and collecting the thus captured nucleosome from the antibody; and the step of isolating and purifying a mononucleosome from the collected nucleosome depending on molecular weight.

Description

明 細 モノヌクレオゾーム及びその製造方法、 ヌクレオゾームに特異的な抗体の測定方 法、 自己免疫病診断方法、 ヌクレオソ一ム DNA製造方法、 DNAプレート、 D N Aプレート製造方法、 並びに抗 DN A抗体測定法  Details Mononucleosomes and methods for producing the same, methods for measuring antibodies specific to nucleosomes, methods for diagnosing autoimmune diseases, methods for producing nucleosomal DNA, methods for producing DNA plates, DNA plates, and methods for measuring anti-DNA antibodies
技術分野 Technical field
本発明は、 自己免疫病診断に好適なモノヌクレオソーム及びヌクレオソ一ム D N A、 並びに前記モノヌクレオソーム及び前記ヌクレオソーム DNAのいずれか を利用する診断方法に関し、 更に詳しくは、 前記モノヌクレオソ一ム及びヌクレ ォソーム DN Aの効率的な製造方法、 前記モノヌクレオソオームの製造方法によ り製造され診断等に好適なモノヌクレオソーム、 前記モノヌクレオソームを効果 的に製造し得るモノヌクレオソ一ム製造用キッ ト、 モノヌクレオソームの分析等 に有用なヒストン検査方法、 前記モノヌクレオソ一ムを含むヌクレオソ一ムに特 異的な抗体の測定方法、 簡便かつ確実な自己免疫病診断方法、 取扱性に優れた自 己免疫病診断用キット、 前記ヌクレオゾーム DNAを有し自己免疫病診断等に好 適な DNAプレート、 前記 DNAプレートの効率的な製造方法、 及び、 自己免疫 病診断等に好適な抗 DN A抗体測定法に関する。 背景技術  The present invention relates to a mononucleosome and a nucleosomal DNA suitable for diagnosing an autoimmune disease, and a diagnostic method using any of the mononucleosome and the nucleosomal DNA.More specifically, the present invention relates to the mononucleosome and the nucleosome DNA. Mononucleosomes produced by the method for producing mononucleosomes and suitable for diagnosis and the like, kits for producing mononucleosomes capable of effectively producing the mononucleosomes, and analysis of mononucleosomes A useful histone test method, a method for measuring an antibody specific to a nucleosome including the above-mentioned mononucleosome, a simple and reliable method for diagnosing an autoimmune disease, a kit for diagnosing an autoimmune disease excellent in handleability, A DNA plate having the nucleosomal DNA and suitable for autoimmune disease diagnosis and the like, Method for efficiently producing serial DNA plate, and to a suitable anti-DN A antibody assay in autoimmune diseases diagnosis. Background art
ヌクレオソ一ム （nuc l eo s ome) とは、 クロマチン （染色質） の基本 構造をなす単位構造体である。 ヌクレオソ一ムは、 ヒストン H2 a、 H2b、 H 3及び H 4各 2分子ずつの会合体であるヒストン 8量体の周りに DN Aが 1. 7 5卷回してなるヌクレオソ一ムコアと、 DN Aの卷き始めと巻き終わりの部位に 結合した 1分子の H 1ヒストンとで構成されている。 H2 a、 H2b、 H3及び H4ヒストンは、 主として分子中央から C末端側で相互作用し、 N末端領域で D NAとイオン結合している。 各ヌクレオソ一ムは、 リンカ一 DNAにより互いに 連結され、 規則的に配置している。 細胞より単離した核をヌクレアーゼ処理する と、 リンカ一 DNAが切断され、 ヌクレオソ一ムを単位として単量体 （即ち、 モ ノヌクレオソ一ム）、 及び二量体、 三量体等の多量体が切り出される。 前記単量体のリンカ一 D N Aを完全に切断すると、 H Iヒストンが離脱し、 そ の他の 4種のヒストン 8分子の会合体と 1 4 6塩基対 D N Aとを含む、 直径 1 1 n m、 高さ 5、 5 n mの円筒形のヌクレオソームコアとなる。 ヌクレオソームは 連なって直径約 1 n mの繊維 （ヌクレオフィラメント） を形成し、 更に高次構造 をとることにより直径 3 0 n mの繊維を形成していると考えられている。 A nucleosome is a unit structure that forms the basic structure of chromatin (chromatin). The nucleosome is composed of a nucleosomal core consisting of a histone octamer, which is an aggregate of two molecules of histones H2a, H2b, H3, and H4, each of which has 1.75 turns of DNA around the histone. Consists of one molecule of H 1 histone bound to the beginning and end of the winding. H2a, H2b, H3, and H4 histones interact mainly at the C-terminal side from the center of the molecule, and ionically bind to DNA at the N-terminal region. Each nucleosome is linked to each other by linker DNA, and is regularly arranged. When a nuclease isolated from a cell is treated with nuclease, the linker DNA is cleaved, and the nucleosome is used as a unit of monomer (ie, Non-nucleosomes), and multimers such as dimers and trimers are cut out. When the monomeric linker DNA is completely cleaved, HI histones are released, containing an aggregate of eight other four histone molecules and 146 base pair DNA. It becomes a 5,5 nm cylindrical nucleosome core. It is thought that the nucleosomes are linked to form a fiber (nucleofilament) with a diameter of about 1 nm, and a higher order structure to form a fiber with a diameter of 30 nm.
前記ヌクレオソームの概念及び実体は、 Kornberg, R. D. ： クロマチン構造： ヒス 卜ン及び D N Aの反復単位 ( Chromatin structure； a repeating unit of histories and DNA) ， Science, 184: 868-871 ( 1974) によって明確にされ、 ここ からヌクレオソーム研究の歴史がスタートした。  The concept and identity of the nucleosomes are clarified by Kornberg, RD: Chromatin structure: Chromatin structure; a repeating unit of histories and DNA, Science, 184: 868-871 (1974). This is where the history of nucleosome research began.
ヌクレオゾームの分離 '精製については、 Kornberg, R. D. ら ；ヌクレオソ一 ム及びクロマチンの調製 (Preparation of nucleosomes and chromatin) ， Method Enzymol . 170 : 3-14 ( 1989) に記載されている。 これは、 臓器又は組織から細胞 核を分離し、 マイクロコッカル 'ヌクレアーゼ (microccocal nuclease；以下、 M Nと略称する） で消化し、 ショ糖濃度勾配 ·超遠心法によってモノヌクレオソ ームからポリヌクレオソームまでを時間をかけて分離するという古典的な方法で ある。  Separation of nucleosomes' Purification is described in Kornberg, R. D. et al .; Preparation of nucleosomes and chromatin, Method Enzymol. 170: 3-14 (1989). This involves separating the cell nucleus from an organ or tissue, digesting it with microccocal nuclease (hereinafter abbreviated as MN), sucrose concentration gradient, and ultracentrifugation to convert the mononucleosome to polynucleosome. This is the classic method of separating over time.
前記 Kornbergらの方法を簡易化したものとしては、 Widlund, H. R. ら ；ゲノ ムヌクレオゾーム · ポジショニング配列の同定と解析 （ Identification and characterization of genomic nucleosome-positioning sequences) , J. Mo. Biol . , 267: 807-817 ( 1997) に記載されている。  As a simplified version of the method of Kornberg et al., Widlund, HR et al .; Identification and characterization of genomic nucleosome-positioning sequences, J. Mo. Biol., 267: 807- 817 (1997).
しかし、 この方法は、 クロマチンからヒストン H 1を除去し、 M Nで切断され たヌクレオソ一ムをァガロースゲル電気泳動で分離した後、 ゲルを切り出してヌ クレオソームを抽出することが必要であり、 操作が煩雑な上に時間も要するとい う問題があった。 また、 ヌクレオゾームの回収率も純度も低く、 リンカ一 D N A を回収することも不可能であるという問題があった。  However, this method requires removal of histone H1 from chromatin, separation of MN-cleaved nucleosomes by agarose gel electrophoresis, and then cutting out the gel to extract nucleosomes, which is complicated. In addition, there was a problem that it took time. In addition, the recovery rate and purity of nucleosomes were low, and it was impossible to recover linker DNA.
なお、 従来、 二本鎖 D N Aに特異的であると信じられていた 4種類のモノクロ ーナル抗体を詳細に解析した結果、 その 2種類が二本鎖 D N Aよりもヌクレオソ ームに強い親和性を有する、 抗ヌクレオソ一ム抗体であったことが明らかにされ た （Christine Ste匪 er ら ；ニ本鎖 D N A及びヒストン H 3の短いセグメントに 対する幾つかのモノクロ一ナル抗ヌクレオソ一ム自己抗体の二重活性 （Dual reactivity of several monoclonal anti-nucleosome autoantibodies for double-stranded DM and a short segment of histone H3) , J. Biol. Chem.， 271 : 21257-21261 ( 1996) 参照）。 A detailed analysis of four types of monoclonal antibodies that were previously believed to be specific for double-stranded DNA revealed that the two types had a stronger affinity for nucleosomes than double-stranded DNA It was revealed that it was an anti-nucleosomal antibody (Christine Ste bander et al .; Dual reactivity of several monoclonal anti-nucleosome autoantibodies for double-stranded DNA and a short segment of histone H3. stranded DM and a short segment of histone H3), J. Biol. Chem., 271: 21257-21261 (1996)).
Franek, F. 及び Dornicova, J. , タンパク質を含まないハイブリ ドーマ細胞培 養上清中に生じるヌクレオソ一ム ： プログラムされた細胞死の実証 （Nucleosome occurrling in protein-free hybriaoma cell-culture: evidence for progra腿 ea cell death) ， FEBS lett. 284: 285-287 ( 1991 ) には、 抗 D N Aモノクローナル 抗体産生ハイプリ ドーマの培養液中にヌクレオソ一ムとモノクローナル抗体との 免疫複合体が形成されることが報告されている。  Franek, F. and Dornicova, J., Nucleosome generated in protein-free hybridoma cell culture supernatant: Demonstration of programmed cell death (Nucleosome occurrling in protein-free hybriaoma cell-culture: evidence for progra Ea cell death), FEBS lett. 284: 285-287 (1991) reported that an immune complex between nucleosomes and monoclonal antibodies was formed in cultures of anti-DNA monoclonal antibody-producing hybridomas. Have been.
最近、 膠原病である全身性エリテマトーデス （S L E ) の病態形成に重要と考 えられている抗クロマチン抗体産生に関連して、 ヌクレオソ一ムの形態を保持し たまま細胞が崩壊してゆくアポトーシスとの関係が注目されている。 生体は血液 細胞を例に取り上げても、 一分間に少なくとも百万細胞単位で生死を繰り返して いる。 その一方の死がアポトーシスである。 このようなアポト一シスに由来する ヌクレオゾームが抗ヌクレオゾーム抗体産生及び抗 D N A抗体産生のための自己 抗原として作用しているという説が注目されるようになった （総説としては、 Amoura, Z. ら， S L Eにおけるヌクレオソ一ムの重要な役割 [The key role of nucleosomes in lnpus], Arthritis Rheum. , 42:833-843 ( 1999) 参照）。  Recently, in connection with the production of anti-chromatin antibodies, which are considered to be important for the pathogenesis of systemic lupus erythematosus (SLE), which is a collagen disease, apoptosis in which cells collapse while retaining the nucleosomal morphology The relationship has attracted attention. Even in the case of blood cells, living organisms are living and dying at least one million cells per minute. One death is apoptosis. The theory that nucleosomes derived from such apoptosis are acting as autoantigens for anti-nucleosomal antibody production and anti-DNA antibody production has attracted attention (for review, see Amoura, Z. et al. , The key role of nucleosomes in lnpus, Arthritis Rheum., 42: 833-843 (1999)).
また、 全身性エリテマトーデスの早期診断 ·病態把握の上で、 抗 D N A抗体と 並んで抗ヌクレオソ一ム抗体が非常に重要な臨床的意義を有することが報告され ている （Coristsidis, G. N. ら、 ヌクレオゾームの糸球体取り込み： レセプ夕一 仲介メサンギゥム細胞結合の証拠 [Glomerular uptake of nucleosomes: evidence for receptor mediated mesangial cell binding], Kidney Int, 47: 1258-1265 ( 1995) ； Burlingam, R. W. ら、 ネズミ S L Eにおける抗クロマチン自己抗体の 起源と産生は自己抗原による T細胞依存性免疫による [Genesis and evolution of ant i chromat inautoant ibod ι es in murine lupus implicates T - dependent immunization with self antigen] , J. Clin. Invest. , 91 : 1687- 1696( 1993) ;及 び Amoura, Z. ら、 S L Eモデルである M R L/ l p r及びその対照として使わ れる M R L/nが蛋白尿を呈する場合、 抗ヌクレオソーム抗体は抗 D N A抗体や 抗ヒス 卜ン抗体に先んじて検出される [Nucleosome- restricted antibodies are detected before anti-dsDNA and/ or antihistone antibodies in serum of MRL-Mp lpr/lpr and +/+ mice with proteinuria] , Arthritis Rheum. , 37: 1684-1688 ( 1994) 参照）。 In addition, it has been reported that anti-nucleosomal antibodies, along with anti-DNA antibodies, have a very important clinical significance in the early diagnosis and diagnosis of systemic lupus erythematosus (Coristsidis, GN et al. Glomerular uptake: Glomerular uptake of nucleosomes: evidence for receptor mediated mesangial cell binding, Kidney Int, 47: 1258-1265 (1995); Burlingam, RW et al., Antichromatin in murine SLE The origin and production of autoantibodies are determined by T cell-dependent immunity with autoantigens [Genesis and evolution of antichromat in autoant ibod ees in murine lupus implicates T-dependent immunization with self antigen], J. Clin. Invest., 91: 1687-1696 (1993); and And Amoura, Z. et al., When the SRL model MRL / lpr and its control MRL / n exhibit proteinuria, anti-nucleosome antibodies are detected prior to anti-DNA and anti-histon antibodies [ Nucleosome-restricted antibodies are detected before anti-dsDNA and / or antihistone antibodies in serum of MRL-Mplpr / lpr and + / + mice with proteinuria], Arthritis Rheum., 37: 1684-1688 (1994)).
また、 抗ヌクレオソーム自己抗体の正確な検出にはアポトーシス由来のヌクレ ォゾームが理想とされる。 自然又は病的状態で生じるアポトーシスの結果得られ るヌクレオソ一ムと、 正常な構造を保持しているクロマチンを人工的にェンドヌ クレア一ゼで切断して得られたヌクレオソームとでは、 詳細な点で構造が異なつ ていると考えられる。 自己免疫現象を考える場合、 アポトーシスによって得られ たヌクレオソ一ムは種々の修飾を受け、 いわゆる自己抗原性を獲得し易くなると 考えられる。 したがって、 代表的自己免疫疾患である S L Eで最近注目されてい る抗ヌクレオソ一ム抗体の出現は、 このような修飾ヌクレオゾームが抗原になつ ていると考えられる。 修飾ヌクレオゾームの本体は、 コアヒストンの修飾、 例え ば、 リン酸化、 ァセチル化、 メチル化、 A D P—リボシル化、 グリコシル化、 ュ ビキチン化などが考えられる。 修飾ヌクレオソームが高純度で単離できれば、 修 飾の状態が 1 5〜 1 7 % S D S— P A G Eあるいは 0 · 5 %ァガロースゲル電気 泳動などによる移動度の変化から検索可能である。  In addition, nucleosomes derived from apoptosis are ideal for accurate detection of anti-nucleosome autoantibodies. The nucleosome obtained as a result of apoptosis occurring naturally or in a pathological state and the nucleosome obtained by artificially cleaving chromatin having a normal structure with endonuclease are described in detail. It is considered that the structure is different. When considering the autoimmune phenomenon, it is thought that nucleosomes obtained by apoptosis are variously modified and are likely to acquire so-called autoantigenicity. Therefore, the emergence of anti-nucleosomal antibodies that have recently attracted attention in SLE, which is a typical autoimmune disease, suggests that such modified nucleosomes serve as antigens. The modified nucleosome itself may be a core histone modification, for example, phosphorylation, acetylation, methylation, ADP-ribosylation, glycosylation, ubiquitination and the like. If the modified nucleosomes can be isolated with high purity, the modified state can be retrieved from changes in mobility by 15–17% SDS-PAGE or 0.5% agarose gel electrophoresis.
このような、 自己免疫疾患のメカニズムの解明のためには、 自己抗原となり得 る修飾ヌクレオソームを高純度 ·高収率で単離することができる方法が必要とさ れ、 従来のヌクレオソームの単離 ·精製方法では不十分であるという問題があつ た o  In order to elucidate the mechanism of such autoimmune diseases, a method that can isolate high-purity and high-yield modified nucleosomes that can serve as autoantigens is required. There was a problem that the purification method was not sufficient o
一方、 全身性自己免疫疾患、 なかでも全身性エリテマトーデス （S L E ) の診 断並びにその病態把握のための抗 D N A抗体の測定法としては、 従来から多くの 手法が試みられてきた （Kanai, : 代謝、 2 0卷： 253- 261， 1983)。 その一覧を表 1 に示した。 抗 DN A抗体測定法 On the other hand, many methods have been attempted for the diagnosis of systemic autoimmune diseases, especially systemic lupus erythematosus (SLE), and for the measurement of anti-DNA antibodies to understand their pathology (Kanai, Metabolism). 20th volume: 253-261, 1983). The list is shown in Table 1. Anti-DNA antibody assay
1) 沈降法  1) Sedimentation method
a) 定量的沈降反応  a) Quantitative sedimentation reaction
b) 免疫拡散法  b) Immunodiffusion method
c) カウン夕一免疫電気泳動法  c) Yuichi Kaun immunoelectrophoresis
2) 受身 （赤） 血球凝集反応  2) passive (red) hemagglutination
3) 定量的補体結合反応  3) Quantitative complement fixation reaction
4) 液相ラジオィムノアッセィ  4) Liquid phase radioimnoassy
a) ミリポアフィル夕一法  a) Milliporefil Yuichiho
b) グラスファイバ一フィル夕一法  b) Glass fiber-filling method
c )硫安沈殿法 （ F a r r )  c) Ammonium sulfate precipitation method (F a r r)
d) 二抗体法  d) Two antibody method
e) プロテイン A法  e) Protein A method
f) ポリエチレングリコール法  f) Polyethylene glycol method
5) 固相ラジオィムノアッセィ  5) Solid-state radioimnoassy
a) 二抗体  a) Two antibodies
b) プロテイン A法  b) Protein A method
6) 固相酵素抗体法 （EL I SA)  6) Enzyme-linked immunosorbent assay (ELISA)
a) 二抗体法  a) Two antibody method
b) プロティン A法  b) Protein A method
7) 蛍光抗体法  7) Fluorescent antibody method
a) DNAスポット法  a) DNA spot method
b ) Chrithidia 1 u c a e¾ これらの手法の中でも、 正確な測定法として定評のあるのが F a r r法である が、 F a r r法においては DNA抗原を放射性物質で標識することが必要とされ、 高度の医療施設でないと測定できない欠点がある。 これに対して、 現在特に実験 室レベルで頻用されているのが E L I S A法である。 Far r法が液状で抗原抗 体反応を行うのに対し、 E L I S A法は抗原をプラスチックプレートなどに付着 させて行うものである。 DNA抗原を固相に効率よく付着させるために、 一般的 には塩基性タンパク質、 例えばポリ— L—リジン （PLL) で予めプラスチック プレートを被覆してから DN Aを付着させる手法が用いられている。  b) Chrithidia 1 uca e¾ Among these methods, the F arr method has a reputation as an accurate measurement method, but the F arr method requires that DNA antigens be labeled with radioactive substances, which is an advanced method. There is a disadvantage that it cannot be measured unless it is a medical facility. On the other hand, the ELISA method is frequently used at the laboratory level. Whereas the Farr method performs an antigen-antibody reaction in liquid form, the ELISA method involves attaching an antigen to a plastic plate or the like. In order to efficiently attach DNA antigens to a solid phase, a method is generally used in which a plastic plate is previously coated with a basic protein, for example, poly-L-lysine (PLL), and then DNA is attached. .
しかし、 この方法で抗体を測定した場合には、 測定された抗体の中に、 PLL と D N Aのィォン結合によって形成されるヌクレオソ一ム様構造や人工的な未知 の抗原に対する抗体が含まれる可能性を否定できないという欠点がある。 However, when antibodies are measured by this method, PLL is included in the measured antibodies. However, there is a drawback that the possibility of inclusion of a nucleosomal-like structure formed by ion bonding between DNA and DNA or the possibility of including an antibody against an artificially unknown antigen cannot be ruled out.
正確な抗 D N A抗体の測定に P L Lのようなスぺ一サ一を用いずに直接プレー 卜に付着させる方法の開発がなされたが、 まだ実用には至っていない。  For accurate measurement of anti-DNA antibody, a method of attaching directly to a plate without using a probe such as PLL was developed, but it has not yet been put to practical use.
前述したように、 S L Eの病態形成に重要と考えられている抗 D N Aを主とす る抗クロマチン抗体産生に関連して、 アポトーシスに由来するヌクレオソ一ムが 抗 D N A抗体産生のための自己抗原として作用しているという説が注目されるよ うになつた (Amoura, Z. et al,： Arthritis Rheum. 42:833-843, 1999)。  As described above, in connection with the production of anti-chromatin antibodies, mainly anti-DNA, which is considered to be important for the pathogenesis of SLE, nucleosomes derived from apoptosis are used as autoantigens for the production of anti-DNA antibodies. The theory of action has been noticed (Amoura, Z. et al, Arthritis Rheum. 42: 833-843, 1999).
このように、 ヒトのヌクレオソ一ムが自己抗原であると指摘されていながら、 現在抗 D N A抗体の測定には、 ヒトのヌクレオソ一ム D N Aではなく、 仔牛胸腺 由来 D N Aや組換え D N Aが抗原として使用されており、 必ずしも正確な診断が できない問題点があった。 これは、 ヒトヌクレオソーム D N Aを抗原として調製 することが容易でないことに起因している。  Thus, although it has been pointed out that human nucleosome is an autoantigen, at present, anti-DNA antibody measurement uses calf thymus-derived DNA or recombinant DNA as an antigen instead of human nucleosomal DNA. However, accurate diagnosis was not always possible. This is because it is not easy to prepare human nucleosome DNA as an antigen.
本発明は、 従来における、 前記諸問題を解決し、 以下の目的を達成することを 課題とする。 即ち、 本発明は、 自己免疫病診断等をはじめ各種用途に好適なヌク レオソ一ムを、 形態安定性を維持し、 簡便な操作で効率よく、 しかも高純度で得 ることができるモノヌクレオソ一ムの製造方法、 前記モノヌクレオソオームの製 造方法により製造されたモノヌクレオソーム、 取扱性に優れたモノヌクレオソ一 ム製造用キッ ト、 モノヌクレオソ一ム分析、 自己免疫病診断等に好適なヒストン 検査方法、 前記モノヌクレオゾームを含む、 ヌクレオゾームに特異的で各種診断 等に好適な抗体の測定方法、 簡便かつ確実な自己免疫病診断方法、 高性能で取扱 性に優れた自己免疫病診断用キッ ト、 自己免疫病診断等をはじめ各種用途に好適 なヌクレオゾーム D N Aを、 簡便な操作で効率よく、 しかも高純度で得ることが できるヌクレオソ一ム D N A製造方法、高性能で取扱性に優れた D N Aプレート、 前記 D N Aプレートの効率的な製造方法、 及び、 自己免疫病診断等に好適な抗 D N A抗体測定法を提供することを目的とする。 発明の開示  An object of the present invention is to solve the above-described various problems and achieve the following objects. That is, the present invention provides a mononucleosome which is suitable for various uses including autoimmune disease diagnosis and the like, and which can maintain morphological stability, can be efficiently obtained by a simple operation, and can be obtained with high purity. A mononucleosome produced by the method for producing a mononucleosoam, a kit for producing a mononucleosome with excellent handling properties, a mononucleosome analysis, a histone inspection method suitable for autoimmune disease diagnosis, etc., A method for measuring an antibody specific to a nucleosome containing the above mononucleosome and suitable for various diagnoses, etc .; a simple and reliable method for diagnosing an autoimmune disease; a high-performance, easy-to-handle autoimmune disease diagnostic kit; Nucleosome that can obtain nucleosomal DNA suitable for various uses including immunological disease diagnosis with high efficiency and efficiency by simple operation An object of the present invention is to provide a DNA production method, a DNA plate having high performance and excellent handling properties, an efficient production method of the DNA plate, and an anti-DNA antibody measurement method suitable for autoimmune disease diagnosis and the like. . Disclosure of the invention
本発明者は、前記課題を解決すべく鋭意検討を重ねた結果、以下の知見を得た。 即ち、 ヌクレオゾームを、 前記ヌクレオゾームに特異的な抗体を介してプロティ ン Aカラム等に吸着させた後、 高濃度の塩化ナトリゥムを含むトリス緩衝液等を 用いて溶出させ、 ヌクレオソーム集合体をマイクロコッカル ·ヌクレア一ゼ等で 処理してモノヌクレオソ一ムにした後、 H P L C等によってモノヌクレオソ一ム のみを回収することによって、 モノヌクレオソームを簡易にかつ効率よく、 しか も高純度で製造することができるという知見である。 The present inventor has earnestly studied to solve the above-mentioned problems, and as a result, has obtained the following knowledge. That is, the nucleosome is adsorbed to a protein A column or the like via an antibody specific to the nucleosome, and then eluted with a Tris buffer solution containing a high concentration of sodium chloride, and the nucleosome assembly is micrococcal Finding that mononucleosomes can be produced simply, efficiently and with high purity by recovering only mononucleosomes by HPLC etc. after treating them with nucleases etc. It is.
本発明は、 本発明者による前記知見に基づくものであり、 前記課題を解決する ための手段は以下の通りである。 即ち、  The present invention is based on the above findings by the present inventors, and the means for solving the above problems are as follows. That is,
< 1 > 試料中に含まれるヌクレオソームを、 該ヌクレオゾームに特異的な抗 体によつて捕獲収集する捕獲収集工程と、  <1> a capture and collection step of capturing and collecting nucleosomes contained in the sample using an antibody specific to the nucleosome;
捕獲したヌクレオソームを前記抗体から解離回収させる解離回収工程と、 回収したヌクレオゾームからモノヌクレオソ一ムを分子量に基づいて単離 ·精 製する単離精製工程と、  A dissociation and recovery step of dissociating and recovering the captured nucleosomes from the antibody, and an isolation and purification step of isolating and purifying a mononucleosome from the recovered nucleosomes based on molecular weight.
を含むことを特徴とするモノヌクレオソ一ムの製造方法である。 And a method for producing a mononucleosome.
< 2 > 抗体が、 抗ヌクレオソーム抗体、 抗 D N A抗体、 及び抗ヒストン抗体 から選択される少なくとも一種である前記 < 1 >に記載のモノヌクレオソ一ムの 製造方法である。  <2> The method for producing a mononucleosome according to <1>, wherein the antibody is at least one selected from an anti-nucleosome antibody, an anti-DNA antibody, and an anti-histone antibody.
< 3 > 抗体が、 1 4 O mMの塩濃度において抗原との結合能を有する前記 < 1 >又は < 2 >に記載のモノヌクレオソ一ムの製造方法である。  <3> The method for producing a mononucleosome according to <1> or <2>, wherein the antibody has an antigen-binding ability at a salt concentration of 14 O mM.
< 4 > 抗体が、 2 C 1 0の可変域のアミノ酸配列、 又は、 該アミノ酸配列に おいて 1以上 2 0以内のアミノ酸が欠失、 置換若しくは付加されたアミノ酸配列 を有し、 2 C 1 0の抗原特異性を有する前記く 1 >からく 3 >のいずれかに記載 のモノヌクレオゾームの製造方法である。  <4> the antibody has the amino acid sequence of the variable region of 2C10 or an amino acid sequence in which 1 to 20 amino acids have been deleted, substituted or added in the amino acid sequence, The method for producing a mononucleosome according to any one of <1> to <3>, having an antigen specificity of 0.
< 5 > 抗体が、 2 C 1 0である前記 < 4 >に記載のモノヌクレオソ一ムの製 造方法である。  <5> The method for producing a mononucleosome according to <4>, wherein the antibody is 2C10.
< 6 > 捕獲収集工程において、 抗体を該抗体と親和性を有する固相に結合さ せることにより、 該抗体にヌクレオソ一ムを捕獲させる前記 < 1 >から < 5 >の いずれかに記載のモノヌクレオゾームの製造方法である。  <6> The method according to any one of <1> to <5>, wherein in the capture collection step, the antibody is bound to a solid phase having an affinity for the antibody so that the antibody captures nucleosomes. This is a method for producing a nucleosome.
< 7 > 固相が、 プロテイン Aカラムである前記 < 6 >に記載のモノヌクレオ ゾームの製造方法である。 <7> The mononucleotide according to <6>, wherein the solid phase is a protein A column. This is a method for producing a some.
< 8 > 捕獲収集工程において、 抗体を予め固相に結合させておき、 試料を該 固相と接触させることにより、 該抗体にヌクレオソ一ムを捕獲させる前記 < 1 > から < 5 >のいずれかに記載のモノヌクレオソ一ムの製造方法である。  <8> In the capture and collection step, the antibody is previously bound to a solid phase, and the sample is brought into contact with the solid phase, whereby the antibody captures nucleosomes. And a method for producing a mononucleosome.
< 9 > 単離精製工程において、 ゲルろ過カラムを用いて分子量 2 0万から 2 5万の分画を回収してモノヌクレオソームを単離 ·精製する前記 < 1 >から < 8 >のいずれかに記載のモノヌクレオゾームの製造方法である。  <9> In the isolation and purification step, a fraction having a molecular weight of 200,000 to 250,000 is collected using a gel filtration column to isolate and purify the mononucleosome. A method for producing the described mononucleosome.
< 1 0 > 捕獲収集工程前にヌクレオソ一ムをモノヌクレオソ一ム単位に切断 し得るヌクレアーゼで処理する前記 < 1 >から < 9 >のいずれかに記載のモノヌ クレオソ一ムの製造方法である。  <10> The method for producing a mononucleosome according to any one of <1> to <9>, wherein the nucleosome is treated with a nuclease that can be cleaved into mononucleosome units before the capture and collection step.
< 1 1 > 解離回収工程後、 単離精製工程前にヌクレオソームをモノヌクレオ ソ一ム単位に切断し得るヌクレアーゼで処理する前記 < 1 >から < 9 >のいずれ かに記載のモノヌクレオソ一ムの製造方法である。  <11> After the dissociation and recovery step, the method for producing a mononucleosome according to any one of <1> to <9>, wherein the nucleosome is treated with a nuclease that can be cleaved into mononucleosome units before the isolation and purification step. It is.
< 1 2 > ヌクレアーゼが、 マイクロコッカル 'ヌクレア一ゼである前記く 1 0 >又はく 1 1 >に記載のモノヌクレオゾームの製造方法である。  <12> The method for producing a mononucleosome according to <10> or <11>, wherein the nuclease is Micrococcal 'nuclease.
< 1 3 > 細胞を低塩濃度の溶液中で破砕させ、 溶液中にヌクレオゾームを放 出させる放出工程と、  <13> a release step in which cells are disrupted in a low-salt solution and nucleosomes are released into the solution;
前記ヌクレオゾームを収集する収集工程と、 A collection step of collecting the nucleosome,
収集した前記ヌクレオゾームを低塩濃度の溶液に懸濁し、 モノヌクレオゾーム単 位に切断し得るヌクレァ一ゼで処理するヌクレァ一ゼ処理工程と、 A nuclease treatment step of suspending the collected nucleosomes in a solution having a low salt concentration and treating with a nuclease which can be cleaved into mononucleosome units;
前記ヌクレアーゼ処理後の溶液からモノヌクレオソ一ムを、 該ヌクレオソ一ムに 特異的な抗体を用いて、 及び/又は分子量に基づいて製造する単離精製工程と、 を含むことを特徴とするモノヌクレオゾームの製造方法である。 An isolation and purification step of producing a mononucleosome from the solution after the nuclease treatment, using an antibody specific to the nucleosome, and / or based on the molecular weight. It is a manufacturing method of.
< 1 4 > 単離精製工程で用いる抗体が、 2 C 1 0である前記 < 1 3 >に記載 のモノヌクレオソ一ムの製造方法である。  <14> The method for producing a mononucleosome according to <13>, wherein the antibody used in the isolation and purification step is 2C10.
< 1 5 > 単離精製工程において、 ゲルろ過カラムを用いて分子量 2 0万から 2 5万の分画を分離する前記 < 1 3 >又はく 1 4 >に記載のモノヌクレオソーム の製造方法である。  <15> The method for producing a mononucleosome according to <13> or <14>, wherein in the isolation and purification step, a fraction having a molecular weight of 200,000 to 250,000 is separated using a gel filtration column. .
< 1 6 > 前記く 1 >からく 1 5 >のいずれかに記載のモノヌクレオソ一ムの 製造方法により製造されたことを特徴とするモノヌクレオソ一ムである。 <16> The mononucleosome according to any one of <1> A mononucleosome manufactured by a manufacturing method.
< 1 7 > 純度が 9 8 %以上である前記 < 1 6 >に記載のモノヌクレオソ一ム である。  <17> The mononucleosome according to <16>, having a purity of 98% or more.
< 1 8 > 試料中に含まれるヌクレオゾームを捕獲収集するための該ヌクレオ ソ一ムに特異的な抗体と、  <18> an antibody specific to the nucleosome for capturing and collecting nucleosomes contained in the sample,
捕獲収集したヌクレオソ一ムから分子量に基づきモノヌクレオソ一ムを単離する ためのカラムと、 A column for isolating mononucleosomes from the captured and collected nucleosomes based on molecular weight;
を含むことを特徴とするモノヌクレオソ一ム製造用キットである。 And a kit for producing a mononucleosome.
< 1 9 > 前記抗体が、 2 C 1 0である前記く 1 8 >に記載のモノヌクレオソ ーム製造用キットである。  <19> The kit for producing a mononucleosome according to <18>, wherein the antibody is 2C10.
< 2 0 > 前記 < 1 >からく 1 5 >のいずれかに記載のモノヌクレオソームの 製造方法により被検試料から得たモノヌクレオソームより被検ヒストンを回収し、 該被検ヒストンの電気泳動パターンと対照ヒストンの電気泳動パターンとを比較 することを含むことを特徴とするヒストン検査方法である。  <20> A test histone is recovered from a mononucleosome obtained from a test sample by the method for producing a mononucleosome according to any one of <1> to <15>, and an electrophoresis pattern of the test histone is obtained. A method for testing a histone, comprising comparing an electrophoretic pattern of a control histone with the electrophoretic pattern.
< 2 1 > 前記く 1 >からく 1 5 >のいずれかに記載のモノヌクレオソームの 製造方法により製造されたモノヌクレオソームを固相化する固相化工程と、 被検試料を、 固相化された該モノヌクレオゾームと反応させる反応工程と、 前記モノヌクレオソ一ムに結合する特異的な抗体を測定する測定工程と、 を含むことを特徴とするヌクレオゾームに特異的な抗体の測定方法である。  <21> A solid phase immobilizing step of immobilizing the mononucleosome produced by the method for producing a mononucleosome according to any one of <1> to <15>, and the test sample is immobilized. A reaction step of reacting with the mononucleosome, and a measuring step of measuring a specific antibody that binds to the mononucleosome. A method for measuring an antibody specific to a nucleosome, comprising:
< 2 2 > モノヌクレオソ一ムが、 アポトーシス由来のヌクレオソ一ムである 前記 < 2 1 >に記載のヌクレオゾームに特異的な抗体の測定方法である。  <22> The method for measuring an antibody specific to a nucleosome according to <21>, wherein the mononucleosome is an apoptosis-derived nucleosome.
< 2 3 > 被検試料が、 自己免疫病患者の血清又は血漿である前記 < 2 1 >又 は < 2 2 >に記載のヌクレオソ一ムに特異的な抗体の測定方法である。  <23> The method for measuring an antibody specific to a nucleosome according to <21> or <22>, wherein the test sample is serum or plasma of an autoimmune disease patient.
< 2 4 > 固相化工程において、 固相化したモノヌクレオソ一ムを少なくとも スキムミルクを含むブロッキング液でブロッキングさせ、 反応工程において、 試 料を少なくともスキムミルクを含む反応液で稀釈してから該試料を、 前記固相化 されたモノヌクレオソ一ムと反応させる前記 < 2 1 >からく 2 3 >のいずれかに 記載のヌクレオソ一ムに特異的な抗体の測定方法である。  <24> In the solid phase immobilization step, the immobilized mononucleosome is blocked with a blocking solution containing at least skim milk.In the reaction step, the sample is diluted with a reaction solution containing at least skim milk, and then the sample is The method for measuring an antibody specific to a nucleosome according to any one of the above <21> to <23>, wherein the antibody is reacted with the immobilized mononucleosome.
< 2 5 > 反応液が、 トリス、 N a C l、 N a N 3、 血清アルブミン、 スキム ミルク、 及び E D T Aを含有する前記く 2 4 >に記載のヌクレオゾームに特異的 な抗体の測定方法である。 <2 5> reaction solution, Tris, N a C l, N a N 3, serum albumin, skim 24. The method for measuring a nucleosome-specific antibody according to the above item 24> containing milk and EDTA.
< 2 6 > 測定工程において、 I g Gサブクラスを認識する抗ヒト抗体を二次 抗体として反応させる前記く 2 1 >から < 2 5 >のいずれかに記載のヌクレオソ ームに特異的な抗体の測定方法である。  In the <26> measuring step, the antibody specific to the nucleosome according to any one of <21> to <25>, wherein an anti-human antibody recognizing an IgG subclass is reacted as a secondary antibody. It is a measuring method.
< 2 7 > 前記く 1 >からく 1 5 >のいずれかに記載のモノヌクレオソ一ムの 製造方法により製造されたモノヌクレオソームを固相化する固相化工程と、 被検試料を、 固相化された該モノヌクレオソームと反応させる反応工程と、 前記モノヌクレオゾームに結合する特異的な抗体を測定する測定工程と、 その抗体価を評価する評価工程と、  <27> A solid-phase immobilizing step of immobilizing the mononucleosome produced by the method for producing a mononucleosome according to any one of <27> to <17>, wherein the test sample is immobilized. A reaction step of reacting with the obtained mononucleosome, a measurement step of measuring a specific antibody that binds to the mononucleosome, and an evaluation step of evaluating the antibody titer,
を含むことを特徴とする自己免疫病診断方法である。 A method for diagnosing an autoimmune disease, comprising:
< 2 8 > 前記く 1 >からく 1 2 >のいずれかに記載のモノヌクレオゾームの 製造方法により製造したモノヌクレオゾームを固相化してなる固相と、緩衝液と、 プレート及びカラムのいずれかと、 を含むことを特徴とする自己免疫病診断用キ ッ トである。  <28> A solid phase obtained by immobilizing the mononucleosome produced by the method for producing a mononucleosome according to any one of <1> to <16>, a buffer, a plate, and a column. A kit for diagnosing an autoimmune disease, comprising:
< 2 9 > 試料中に含まれるヌクレオソ一ムを、 該ヌクレオソ一ムに特異的な 抗体によって捕獲収集する捕獲収集工程と、  <29> a capture and collection step of capturing and collecting a nucleosome contained in the sample by using an antibody specific to the nucleosome;
捕獲したヌクレオソームを前記抗体から解離回収させる解離回収工程と、 回収したヌクレオソームからモノヌクレオソ一ムを分子量に基づいて単離 ·精製 する単離精製工程と、 A dissociation and recovery step of dissociating and recovering the captured nucleosomes from the antibody, an isolation and purification step of isolating and purifying a mononucleosome from the recovered nucleosomes based on the molecular weight,
単離 ·精製されたモノヌクレオゾームからヌクレオソーム D N Aを単離 '精製す るヌクレオゾーム D N A単離精製工程と、 Isolation and isolation of nucleosome DNA from purified mononucleosomes
を含むことを特徴とするヌクレオソ一ム D N A製造方法である。 A method for producing a nucleosomal DNA.
< 3 0 > 前記く 1 >からく 1 5 >のいずれかに記載のモノヌクレオソ一ムの 製造方法からなるモノヌクレオゾーム製造工程と、  <30> a mononucleosome production process comprising the method for producing a mononucleosome according to any one of <1>
モノヌクレオソ一ムからヌクレオゾーム D N Aを単離精製するヌクレオソ一ム D N A単離精製工程とを含むことを特徴とするヌクレオゾーム D N A製造方法であ る o A nucleosomal DNA isolation and purification step for isolating and purifying the nucleosome DNA from the mononucleosome.
< 3 1 > プレート上に、 前記 < 2 9 >又はく 3 0 >に記載のヌクレオソ一ム DNA製造方法により製造されたヌクレオソ一ム DNAであって、 ヒト由来のヌ クレオソ一ム DN Aが固相化されてなることを特徴とする DN Aプレートである cOn the <31> plate, the nucleosome according to <29> or <30> Nucleosome DNA produced by the DNA production method, which is a DNA plate characterized in that a human-derived nucleosomal DNA is immobilized on c.
< 32 > プレート上に、 前記く 16>又はく 17 >に記載のモノヌクレオソ —ムであって、 ヒト由来のモノヌクレオソームから単離精製されたヌクレオソー ム DN Aが固相化されてなることを特徴とする DN Aプレートである。 <32> The mononucleosome according to <16> or <17>, wherein the nucleosome DNA isolated and purified from human-derived mononucleosomes is immobilized on a <32> plate. This is a DNA plate.
< 33 > ヌクレオソーム DN Aが、ヌクレオソ一ム構成 2本鎖 DN Aであり、 ヌクレオソ一ム DN Aの平均鎖長が、 145 bp以上 200 bp以下である前記 < 31 >又は < 32 >に記載の DNAプレートである。  <33> The nucleosome DNA according to <31> or <32>, wherein the nucleosome DNA is a double-stranded DNA having a nucleosome structure, and the average length of the nucleosome DNA is 145 bp or more and 200 bp or less. It is a DNA plate.
<34> ヌクレオソ一ム DNAが、 プレート上に直接固相化されてなる前記 <31>からく 33 >のいずれかに記載の DN Aプレートである。  <34> The DNA plate according to any one of <31> to <33>, wherein the nucleosomal DNA is directly immobilized on a plate.
< 35 > プレートが、 ポリスチレンを含んでなる前記く 31 >からく 34 > のいずれかに記載の DN Aプレートである。  <35> The DNA plate according to any one of <31> to <34>, wherein the plate comprises polystyrene.
< 36 > 前記く 1 >からく 15 >のいずれかに記載のモノヌクレオソ一ムの 製造方法であって、試料がヒト由来の試料であるモノヌクレオソーム製造工程と、 モノヌクレオソームからヌクレオソ一ム DN Aを単離精製するヌクレオソ一ム D NA単離精製工程と、  <36> The method for producing a mononucleosome according to any one of the above <1> to <15>, wherein the sample is a human-derived sample, a mononucleosome production step, and the nucleosome DNA is obtained from the mononucleosome. A nucleosomal DNA isolation and purification step for isolation and purification;
前記ヌクレオソ一ム DN Aをプレート上に固相化する固相化工程と、 Immobilizing step of immobilizing the nucleosome DNA on a plate,
を含むことを特徴とする DN Aプレート製造方法である。 And a method for producing a DNA plate.
< 37 > 固相化工程において、 プレート上に、 ヌクレオソ一ム DN Aを 0. 1M以上1. 0M以下の NaC 1を含む、 トリス緩衝液及びホウ酸—カセイソ一 ダ緩衝液のいずれかに溶解させて添加する前記 < 36 >に記載の DNAプレート 製造方法である。  <37> In the immobilization step, dissolve nucleosome DNA on a plate in either Tris buffer or boric acid-caseisoder buffer containing 0.1M or more and 1.0M or less NaC1. <36> The method for producing a DNA plate according to <36>, wherein the DNA plate is added.
< 38 > プレート上に、 前記 < 29 >又はく 30 >に記載のヌクレオソ一ム DN A製造方法により製造されたヌクレオソーム DN Aであってヒト由来のヌク レオソーム DN Aを、 0. 1M以上1. 0M以下の NaC 1を含む、 トリス緩衝 液及びホゥ酸一力セイソ一ダ緩衝液のいずれかに溶解させて添加することにより 固相化することを特徴とする DN Aプレート製造方法である。  On a <38> plate, a human-derived nucleosome DNA, which is a nucleosome DNA produced by the method for producing nucleosome DNA described in <29> or <30>, is used at 0.1 M or more. A method for producing a DNA plate, comprising dissolving and adding to either a Tris buffer solution or a boric acid monosodium buffer containing 0 M or less NaCl to solidify the DNA.
< 39 > 前記く 31 >からく 35 >のいずれかに記載の DNAプレートを用 いてなり、 被検試料を、 該 DNAプレー卜の固相化されたヌクレオソ一ム DNAと反応させ る反応工程と、 <39> The DNA plate according to any of <31> to <35>, A reaction step of reacting the test sample with nucleosomal DNA immobilized on the DNA plate;
前記ヌクレオゾーム DN Aに結合する特異的な抗体を測定する測定工程と、 を含むことを特徴とする抗 DN A抗体測定法である。 図面の簡単な説明 A measuring step of measuring a specific antibody that binds to the nucleosome DNA. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 2 C 10—核タンパク質複合体が捕獲されているプロティン Aカラム の洗浄液を濃縮し、 そこから得た DNA抽出物を、 2%ァガロースゲル電気泳動 した結果を示す写真である。  Figure 1 is a photograph showing the results of a 2% agarose gel electrophoresis of a DNA extract obtained by concentrating the washing solution of the protein A column in which 2C10-nucleoprotein complex has been captured.
図 2は、 2 C 10—プロテイン Aカラム溶出液を濃縮し、 スーパーデックス 2 00— HP LCクロマトグラフィ一にて解析したヌクレオソ一ムのプロファイル (マイクロコッカル ·ヌクレア一ゼ処理前） を示すグラフである。  Fig. 2 is a graph showing the nucleosomal profile (before micrococcal nuclease treatment) analyzed by condensing the eluate of 2C10-protein A column and analyzing by Superdex 200-HP LC chromatography. is there.
図 3は、 図 2のサンプルをマイクロコッカル 'ヌクレア一ゼ （MN) で処理し た後のスーパ一デックス 200— HPLCクロマトグラフィ一のプロファイル (モノヌクレオゾームと、 MNで切断されたリンカ一 DN Aの形成がよくわかる） を示すグラフである。  Figure 3 shows the profile of Superdex 200-HPLC chromatography after treatment of the sample of Figure 2 with micrococcal 'nuclease (MN) (mononucleosomes and linker DNA cut with MN). Is clearly understood).
図 4は、 図 3のモノヌクレオソ一ムに相当する部分 （一） の、 スーパ一デヅク ス 200— HPLCによるリクロマトグラフィーの結果を示すグラフである。 図 5は、 培養細胞又は末梢リンパ球から得られたヌクレオソ一ムの Snp e r dex200— HPLCによるリクロマトグラフィー （代表例） の結果を示すグ ラフである。  FIG. 4 is a graph showing the results of rechromatography of a portion (1) corresponding to the mononucleosome in FIG. 3 by Superdex 200-HPLC. FIG. 5 is a graph showing the results of rechromatography (typical example) of nucleosomes obtained from cultured cells or peripheral lymphocytes by Snperdex200-HPLC.
図 6は、 図 5のモノヌクレオソームに相当する部分 （一） の、 スーパーデヅク ス 200— HPLCによるリクロマトグラフィーの結果を示すグラフである。 図 7は、 アポト一シスを起こした培養細胞由来のモノヌクレオゾームと、 正常 な培養細胞由来のモノヌクレオソームを 0. 5 %ァガロース電気泳動により分画 した結果を示す写真である。  FIG. 6 is a graph showing the results of rechromatography of the portion (1) corresponding to the mononucleosome of FIG. 5 by Superdex 200-HPLC. FIG. 7 is a photograph showing the results of fractionation of mononucleosomes derived from cultured cells having undergone apoptosis and mononucleosomes derived from normal cultured cells by 0.5% agarose electrophoresis.
図 8は、 モノヌクレオソ一ムを構成する DN Aをァガロースゲル電気泳動で分 祈した結果を示す写真である。  FIG. 8 is a photograph showing the result of separating DNA constituting a mononucleosome by agarose gel electrophoresis.
図 9は、 モノヌクレオゾームを構成するコアヒストンを 15%SDS— PAG Eにより分画して解析した結果を示す写真である。 Figure 9 shows that the core histones that make up the mononucleosome were converted to 15% SDS-PAG 4 is a photograph showing the results of fractionation and analysis by E.
図 10は、 正常な細胞由来の全ヒストン （H 1を含む） の 15%SDS— PA G Eによる泳動像を示す写真である。  FIG. 10 is a photograph showing an electrophoresis image of all histones (including H1) derived from normal cells by 15% SDS-PAGE.
図 11は、 EL I S Aによるモノヌクレオソ一ムに特異的な抗体の測定結果を 示すグラフである。  FIG. 11 is a graph showing the results of measurement of antibodies specific to mononucleosomes by ELISA.
図 12は、 EL I SAによるモノヌクレオゾームに特異的な抗体のサブクラス 別測定結果を示すグラフである。  FIG. 12 is a graph showing the results of measurement of antibodies specific to mononucleosomes by subclass by ELISA.
図 13は、 ヌクレオソ一ム DNAをァガロース電気泳動した結果を示す写真で ある。  FIG. 13 is a photograph showing the result of agarose electrophoresis of nucleosomal DNA.
図 14は、 ヌクレオソーム DNA、 ゲノム DNA及び仔牛胸腺 DNAをそれそ れ DN A抗原とする DN Aプレートに対する、 S LE患者血清の免疫応答を示す グラフである。  FIG. 14 is a graph showing the immune response of SLE patient sera to DNA plates using nucleosome DNA, genomic DNA and calf thymus DNA as DNA antigens, respectively.
図 15は、 P L L前処理なしのプレートにおける抗ヌクレオソ一ム DNA抗体 反応の抑制効果を示すグラフである。  FIG. 15 is a graph showing the inhibitory effect of the anti-nucleosomal DNA antibody reaction on a plate without PLL pretreatment.
図 16は、 PLL前処理したプレートにおける抗ヌクレオソーム DNA抗体反 応の抑制効果を示すグラフである。  FIG. 16 is a graph showing the inhibitory effect of anti-nucleosome DNA antibody reaction on a plate pretreated with PLL.
図 17は、 Immul on 2 H Bプレートに P L L前処理なしでヌクレオソ ーム DNAをコートしたプレート及びコ一トしないプレートに対する S LE患者 血清の抗体反応を示す図である。  FIG. 17 shows antibody responses of SLE patient sera to Immulon 2HB plates coated with nucleosomal DNA without PLL pretreatment and uncoated plates.
図 18は、 Immul on 2 HBプレートに P L L前処理してヌクレオソ一 ム DNAをコートしたプレート及びコートしないプレートに対する S LE患者血 清の抗体反応を示す図である。  FIG. 18 is a diagram showing antibody responses of SLE patient sera to a plate coated with nucleosomal DNA and a plate not coated with PLL pretreated on Immulon 2 HB plate.
図 19は、 P L L前処理の有無と抗ヌクレオソ一ム DNA抗体価の関係を示す 図である。  FIG. 19 shows the relationship between the presence or absence of PLL pretreatment and the anti-nucleosomal DNA antibody titer.
図 20は、 Immul on 2 H Bプレートにヒトヌクレオゾーム D N Aをコ 一ティングする場合の N a C 1濃度の効果を抗 DNA抗体価により測定した結果 を示すグラフである。  FIG. 20 is a graph showing the results of measuring the effect of NaC1 concentration when coating human nucleosome DNA on Immulon 2 HB plate by anti-DNA antibody titer.
図 21は、 0. 14Mの Na C 1濃度で作製した DNAプレートの抗 DNA抗 体反応の測定結果を示す図である。 図 22は、 0. 25Mの Na C 1濃度で作製した DNAプレートの抗 DNA抗 体反応の測定結果を示す図である。 FIG. 21 is a diagram showing the results of measuring the anti-DNA antibody reaction of a DNA plate prepared at a NaCl concentration of 0.14M. FIG. 22 is a diagram showing the results of measuring the anti-DNA antibody reaction of a DNA plate prepared at a NaCl concentration of 0.25M.
図 23は、 0. 5Mの NaCl濃度で作製した DNAプレートの抗 DNA抗体 反応の測定結果を示す図である。 発明を実施するための最良の形態  FIG. 23 is a diagram showing the results of measuring the anti-DNA antibody reaction of a DNA plate prepared at a 0.5 M NaCl concentration. BEST MODE FOR CARRYING OUT THE INVENTION
(モノヌクレオソ一ムの製造方法）  (Production method of mononucleosome)
本発明のモノヌクレオゾームの製造方法は、 試料中に含まれるヌクレオソ一ム を、 該ヌクレオゾームに特異的な抗体によって捕獲収集する捕獲収集工程と、 捕獲したヌクレオソ一ムを前記抗体から解離回収させる解離回収工程と、 回収したヌクレオソームからモノヌクレオゾームを分子量に基づいて単離 ·精 製する単離精製工程と、 を含む。  The method for producing a mononucleosome according to the present invention comprises the steps of: capturing and collecting nucleosomes contained in a sample with an antibody specific to the nucleosome; dissociating and recovering the captured nucleosomes from the antibody. A recovery step, and an isolation and purification step of isolating and purifying mononucleosomes from the recovered nucleosomes based on molecular weight.
本発明において、 「ヌクレオゾーム」 とは、 ヌクレオゾームを単位とする単量体 (モノヌクレオソ一ム） のみならず、 二量体、 三量体等の多量体、 これらの混合 物、 更には、 総てのヌクレオソ一ムを含有する試料 （即ち、 ヌクレオソ一ム供給 源） をも意味する。  In the present invention, the term “nucleosome” means not only a monomer (mononucleosome) having a nucleosome as a unit, but also a multimer such as a dimer or a trimer, a mixture thereof, or a mixture thereof. A sample containing a nucleosome (ie, a nucleosome source) is also meant.
前記ヌクレオソーム含有試料としては、 例えば、 ヒトゃ動物の臓器又は組織か ら直接分離した細胞から細胞核を分離したもの、 一般の培養細胞から細胞核を分 離したもの、 ヌクレオソ一ムを分泌する培養細胞 （KML!-7細胞 (Kanai, Y. et al. Purification of a novel B cell growth and differentiation factor associated with lupus Syndrome, Immunol. Lett, 32: 43-48, 1992)あるいは H L— 60細 胞 (Collins, S. J. et al.： Continuous growth and differentiation of human myeloid cells in suspension culture. Nature, 270: 347-349， 1997)) の培養 上清、 等が挙げられる。  Examples of the nucleosome-containing sample include those obtained by separating cell nuclei from cells directly separated from human organs or tissues, those obtained by separating cell nuclei from general cultured cells, and cultured cells secreting nucleosomes ( KML! -7 cells (Kanai, Y. et al. Purification of a novel B cell growth and differentiation factor associated with lupus Syndrome, Immunol. Lett, 32: 43-48, 1992) or HL-60 cells (Collins, SJ et al .: Culture supernatant of Continuous growth and differentiation of human myeloid cells in suspension culture. Nature, 270: 347-349, 1997).
本発明において、 「モノヌクレオソ一ム」 とは、 約 146塩基対の二本鎖 DN A とコアヒストン （H3、 H2B、 H2A及び H4) とからなる、 ヌクレオソ一ム の単量体をいう。  In the present invention, the “mononucleosome” refers to a nucleosomal monomer comprising a double-stranded DNA of about 146 base pairs and core histones (H3, H2B, H2A and H4).
前記捕獲収集工程は、 試料中に含まれるヌクレオゾームを、 該ヌクレオソ一ム に特異的な抗体によって捕獲収集する工程である。 本発明において、 「ヌクレオソ一ムに特異的な抗体 （以下、 単に 「抗体」 という ことがある）」とは、 DNAとヒストンとの複合体であるヌクレオソ一ムに特異的 な反応を示す抗体を意味し、 ヌクレオゾームと共に免疫複合体を形成できるもの であればよく、 ヌクレオゾーム自体、 ヌクレオソームを構成している二本鎖 DN A、 及びヌクレオソ一ムを構成しているヒストンのいずれに親和性を有するもの であってもよいし、 ヌクレオソ一ムと特異的な反応を示すと共に二本鎖 DN Aの 単体やヒストン単体とも反応を示すものであってもよい。 前記ヌクレオゾームに 特異的な抗体の中には、 二本鎖 DN Aに特異的であると信じられていたが、 むし ろヌクレオゾームにより高い親和性を有することが判明した公知の抗体も含まれ る。 前記ヌクレオソ一ムに特異的な抗体としては、 二本鎖 DNAよりも、 ヌクレ ォゾームにより強く反応するものが好ましく、 また、 その抗体の Fcを介してプ ロティン Aカラム等のァフィ二ティーカラムに吸着できるものが好ましい。 The capture and collection step is a step of capturing and collecting nucleosomes contained in a sample using an antibody specific to the nucleosome. In the present invention, the term “antibody specific to a nucleosome (hereinafter sometimes simply referred to as“ antibody ”)” refers to an antibody that exhibits a specific reaction to a nucleosome that is a complex of DNA and histone. As long as it can form an immune complex with the nucleosome, it has affinity for any of the nucleosome itself, the double-stranded DNA that forms the nucleosome, and the histone that forms the nucleosome. Or a specific reaction with a nucleosome and a reaction with a single-stranded double-stranded DNA or a single histone. Antibodies specific to the nucleosomes also include known antibodies that were believed to be specific for double-stranded DNA, but rather were found to have higher affinity for nucleosomes. As the antibody specific to the nucleosome, one that reacts more strongly with the nucleosome than with double-stranded DNA is preferable, and is adsorbed to an affinity column such as a protein A column via the Fc of the antibody. What can be done is preferred.
なお、 前記ァフィ二ティーカラムとしては、 プロテイン A以外に、 Pharm a c i a B i ot e ch (PB) Hi t rap af i ini ty co lli mnなどが挙げられ、 具体的には、 Hi t rap NHS— ac t ivat ed の 1 ml又は 5 mlのカラムが好適に挙げられる。 前記ヌクレオソ一ムに特異的 な抗体の前記ァフィ二ティ一カラムへの吸着 （カップリング） の方法としては、 特に制限はないが、 該抗体として後述の 2 C 10を使用する場合には、 例えば、 カップリングバッファ一 （0. 2MNaHC03 0. 5M塩化ナトリウム、 p H 8. 3) で 0. 5〜1. Omg/mlになるように 2 C 10を調整して行うこ とができ、 吸着カップリング後の操作はァフィ二ティーカラム毎に指定されたプ 口 トコ一ルに準じて行うことができる。 Examples of the affinity column include, in addition to Protein A, Pharmacia Biotech (PB) Hitrap afiiniticol mn, and the like. Specifically, Hitrap NHS- A 1 ml or 5 ml column of activated is preferably used. The method for adsorbing (coupling) the antibody specific to the nucleosome to the affinity column is not particularly limited. When 2C10 described later is used as the antibody, for example, coupling buffer one (0. 2MNaHC0 3 0. 5M sodium chloride, p H 8. 3) at 0.5 to 1. adjust 2 C 10 so as to Omg / ml can and this done, adsorption The operation after the coupling can be performed according to the protocol specified for each affinity column.
前記ヌクレオソ一ムに特異的な抗体としては、 特に制限はなく、 目的に応じて 適宜選択することができるが、 高塩濃度においても抗原との結合能を有するもの が、 高塩濃度溶液での洗浄によりカラムに非特異的に結合したタンパク質を除去 することができる点で好ましく、 自己免疫疾患モデルとして知られている MRL /1 p rマウス由来 B細胞ハイプリ ドーマの産生する自己抗体であるモノクロ一 ナル抗体 2C 10 (I gG2bに属する） が、 高塩濃度においても抗原との結合 能を有する点で、 特に好ましい。 前記モノクローナル抗体 2 C 10は、二本鎖 DN Aに特異的であり（Kubota， T. ら， Immunol. Lett. 14: 53-58, 1986 を参照されたい）、 本発明者らのグループ によって作製されたものであり、その製造方法については、前記 Immunol lett. を 参照することができ、 詳細な性質等については、 Kubota, T, ら、 2つの抗ニ本鎖 D N A抗体の結合部位による D N Aの酸化的開裂の促進 （Enhabcement of oxidative cleavage of DNA by the binding sites of two anti - double - stranded DNA antibodies) ,J. Biol. Chem. , 271:6555-6561 (1996) に記載されている。 また、 前記モノクローナル抗体 2 C 10の軽鎖可変域のアミノ酸配列は、 以下 の通 り 同定されている （Jang YJ， Stollar BD 他、 Mol Immunol 1998 Dec;35(18):1207-1217)o The antibody specific to the nucleosome is not particularly limited and may be appropriately selected depending on the intended purpose.An antibody capable of binding to an antigen even at a high salt concentration is used in a high salt solution. Monoclonal, which is an autoantibody produced by BRL hybridoma derived from MRL / 1 pr mouse, which is preferable because proteins that are nonspecifically bound to the column can be removed by washing. Antibody 2C 10 (belonging to IgG2b) is particularly preferred in that it has an antigen-binding ability even at a high salt concentration. The monoclonal antibody 2C10 is specific for double-stranded DNA (see Kubota, T. et al., Immunol. Lett. 14: 53-58, 1986) and is produced by the group of the present inventors. For the production method, it is possible to refer to the above-mentioned Immunol lett., And for detailed properties and the like, Kubota, T, et al., Et al. Enhancement of oxidative cleavage (Enhabcement of oxidative cleavage of DNA by the binding sites of two anti-double-stranded DNA antibodies), J. Biol. Chem., 271: 6555-6561 (1996). Further, the amino acid sequences of the light chain variable region of monoclonal antibody 2 C 10, the following passage Ri have been identified (Jang YJ, Stollar BD others, Mol Immunol 1998 Dec; 35 ( 18): 1207-1217) o
ETTVTQSPASLSVATGEKVTIRCITNTDIDDDMNWYQQKPGEPPKLLISEGNTLRPGVPSRFSSSGYGTDFV FTIENTLSEDVADYCCLQSDNMPLTFGAGT (配列番号 1 )  ETTVTQSPASLSVATGEKVTIRCITNTDIDDDMNWYQQKPGEPPKLLISEGNTLRPGVPSRFSSSGYGTDFV FTIENTLSEDVADYCCLQSDNMPLTFGAGT (SEQ ID NO: 1)
前記モノクローナル抗体 2 C 10の抗原特異性を有するものは、 2 C 10の可 変域を有するヒト型抗体等や、 前記 2 C 10の可変域の 1個から 20個程度の、 より容易には 1又は数個の、 アミノ酸を欠失、 置換、 若しくは付加した変異型を 作製することにより、 当業者であれば容易に得ることができる。 前記モノヌクレ ォゾームを含むヌクレオソームに特異的な抗体には、 前記変異型も含まれる。 前記モノクローナル抗体 2 C 10の製造方法を以下に簡単に述べる。  The monoclonal antibody having the antigen specificity of 2C10 can be a human antibody having a variable region of 2C10, or one to 20 variable regions of 2C10, more easily. A person skilled in the art can easily obtain a mutant by deleting, substituting, or adding one or several amino acids. Antibodies specific to nucleosomes including the mononucleosome also include the mutant. The method for producing the monoclonal antibody 2C10 is briefly described below.
6ヶ月齢の MRL/1 p rマウスの脾臓を培養液 （DMEM) の入ったプラス チックデッシュの中で眼科せん刀を用いて細切した後、 2枚のスライ ドグラスの フロスト面で細切を圧挫し、 結合組織からリンパ球を遊離させる。 数分後に浮遊 細胞を遠心分離する。 直ちに 44. 4質量％ポリエチレングリコール （PEG) 存在下でマウスミエローマ細胞 （SP 2) とリンパ球との細胞融合を二分間かけ て行う。 融合後、 直ちに培養液で PEGを稀釈 '除去し、 最後に選別培地である HATに置き換え、 200 1ずつ 96穴のマイクロタイ夕一プレートに加え、 37°Cの 002ィンキュペータ一（ 5質量％ CO 2存在下）で培養する。二週間後、 コロニーの増殖した各ゥエルの培養上清の抗 D N A抗体価を E L I S A (enzyme- linked immunosorbent assay) にて測疋し、 該 ί几 D Ν Αί几体価の高レヽゥ エル中の 2 C 10ハイプリ ドーマを選択し、 該 2 C 10ハイプリ ドーマを適宜培 養することにより前記モノクローナル抗体 2 C 10が得られる （T. Kubota他、 Immuno lett., 14: 53-58, 1986/1987)。 The spleen of a 6-month-old MRL / 1pr mouse was minced with an ophthalmic scalpel in a plastic dish containing culture solution (DMEM), and then the minced section was pressed with the frosted surfaces of two slide glasses. It crushes and releases lymphocytes from connective tissue. After a few minutes, centrifuge the floating cells. Immediately, cell fusion between mouse myeloma cells (SP 2) and lymphocytes is performed for 2 minutes in the presence of 44.4% by mass polyethylene glycol (PEG). After fusion, immediately PEG was diluted 'removed in culture, replacing the last is a selective medium HAT, in addition to the microtiter evening first plate of 96 by 200 1, of 37 ° C 00 2 Inkyupeta one (5 wt% (In the presence of CO 2 ). Two weeks later, the anti-DNA antibody titer of the culture supernatant of each colony in which the colonies grew was measured by ELISA (enzyme-linked immunosorbent assay), and the amount of the anti-DNA antibody in the high-level of the geometric D value was measured. Select the 2C10 hybrid dome and culture the 2C10 hybrida as appropriate. The monoclonal antibody 2C10 can be obtained by culturing (T. Kubota et al., Immunolett., 14: 53-58, 1986/1987).
次に、 前記モノクローナル抗体 2 C 10の性質を以下に簡単に述べる。  Next, the properties of the monoclonal antibody 2C10 will be briefly described below.
前記モノクローナル抗体 2 C 10は、 一本鎖 DN A中の二本鎖部分と反応する が、 二本鎖 DN Aの雛型である、 ΦΧ 174プラスミ ド DN Aに対し強い親和性 を示し、 かつ塩基配列の A · Tを好む、 優れた抗ニ本鎖 DNA抗体である。 なお、 前記モノクローナル抗体 2 C 10は、 これまで二本鎖 DN Aに特異的で あると考えられていたが、 本発明に到達するまでの研究において、 プロテイン A カラムによる抗ニ本鎖 D N A抗体精製過程で、 二本鎖 D N Aと並びヌクレオソー ムに対しても強い親和性を有することが判明した。 このことから、 本発明のモノ ヌクレオゾームの製造方法において、 二本鎖 DN Aに特異的な抗体のみならず、 ヌクレオソームに特異的な抗体や、 二本鎖 DN A及びヌクレオソ一ムの両者に特 異的な抗体を用いることができると考えられる。  The monoclonal antibody 2C10 reacts with the double-stranded portion in the single-stranded DNA, but shows a strong affinity for ΦΧ174 plasmid, which is a template of the double-stranded DNA, and It is an excellent anti-double-stranded DNA antibody that prefers the base sequence AT. The monoclonal antibody 2C10 was previously thought to be specific for double-stranded DNA.However, in studies conducted before reaching the present invention, purification of anti-double-stranded DNA antibody using a protein A column was performed. In the process, it was found that the DNA has strong affinity not only for double-stranded DNA but also for nucleosomes. From the above, in the method for producing a mononucleosome of the present invention, not only an antibody specific to double-stranded DNA, but also an antibody specific to nucleosome, and both a double-stranded DNA and a nucleosome are different. It is thought that a specific antibody can be used.
前記 「プロティン Aカラム」 とは、 黄色ブドウ球菌 （St aphyl o co c cus au r eu s) 細菌壁に由来する分子量 42， 000のタンパク質であ るプロティン Aを固相化したものをカラムに充填したものである。 前記プロティ ン Aは、 I gGの定常領域 （Fcフラグメント） に結合するので抗原抗体反応を 妨害せずに、 免疫複合体と結合することができる。 この性質を利用して、 前記プ ロティン Aによって免疫複合体を捕獲するプロティン Aカラム法は、 I gGの精 製法として確立された手法である。 本発明で用いることができる市販のプロティ ン Aカラムとしては、 例えば、 ヒッ トラップ （H i t r ap) プロテイン Aカラ ム （アマシャムフアルマシアバイオテック社製） 等が挙げられる。  The “Protein A column” refers to a column in which protein A, a protein with a molecular weight of 42,000 derived from the bacterial wall of Staphylococcus aureus, is immobilized. It was done. Since the protein A binds to the constant region (Fc fragment) of IgG, it can bind to the immune complex without interfering with the antigen-antibody reaction. Utilizing this property, the protein A column method of capturing an immune complex with protein A is a method established as a method for purifying IgG. Examples of commercially available protein A columns that can be used in the present invention include Hittrap (Hitrap) Protein A column (manufactured by Amersham Pharmacia Biotech).
本発明において、 ヌクレオソ一ムをプロティン Aカラム等のカラムに吸着させ る媒介物となる抗体は、 ヌクレオソームを含む培養液等の液中に遊離の形で存在 していてもよいし、 プロティン Aカラム等のカラムに予め固定化されていてもよ い。なお、前記カラムは、前記プロティン Aカラム以外に、担体カラムでもよい。 前記抗体が遊離の形で液中に存在する場合は、 液中で免疫複合体を形成した後、 形成された免疫複合体をプロティン Aカラム等のカラムに流すことにより、 該抗 体は、 その F C部分を介してプロテイン Aカラムに吸着される。 前記抗体がプロ ティン Aカラム又は適当な担体カラムに固定化されている場合には、 ヌクレオソ ームを含む液をカラムに流すことによって、 プロティン Aカラムに固定化されて いる抗体を介してヌクレオソームを吸着することができる。 In the present invention, the antibody serving as a medium for adsorbing a nucleosome to a column such as a protein A column may be present in a free form in a solution such as a culture solution containing nucleosomes, or may be a protein A column. It may be immobilized in advance on a column such as. The column may be a carrier column other than the protein A column. When the antibody is present in a liquid in a free form, an immune complex is formed in the solution, and the formed immune complex is passed through a column such as a protein A column, whereby the antibody is It is adsorbed on the protein A column via the FC part. The antibody is a pro When immobilized on a Tin A column or a suitable carrier column, nucleosomes can be adsorbed through the antibody immobilized on the Protein A column by flowing a solution containing nucleosomes through the column. it can.
前記抗体をプロティン Aカラムに固定化する方法としては、 従来公知の方法を 用いることができる。 具体的には、 プロテイン Aカラムを pH 6. 8〜8. 3ま でのレンジで T r i sを主体とした緩衝液で平衡化し、 予め同じ緩衝液で平衡化 した抗体又は抗体ーヌクレオソ一ム複合体をそれに流入し、 それを同カラムに数 回循環させた後、 抗体又は抗体—免疫複合体を含まない T r i s緩衝液でカラム を洗浄する。 洗浄後のカラム通過液は A26。=0. 0が好ましい。 As a method for immobilizing the antibody on the protein A column, a conventionally known method can be used. Specifically, the protein A column was equilibrated with a buffer mainly composed of Tris in the range of pH 6.8 to 8.3, and the antibody or antibody-nucleosome complex previously equilibrated with the same buffer was used. After washing the column several times through the same column, wash the column with Tris buffer without antibody or antibody-immune complex. The solution passing through the column after washing is A 26 . = 0.0 is preferred.
ヌクレオソーム及び抗体 （免疫複合体） が培養液上清等に含まれている場合に は、 限外濾過等の手段によって上清をプロティン Aカラムに注ぐ前に濃縮してお くことが好ましい。 濃縮を行うことによって、 より効率的なモノヌクレオソ一ム の製造が達成できる。 限外濾過によって濃縮を行う場合、 例えば、 分子量 30, 000カツトオフのダイアフロ一膜（PH 30) (ミリポア社製）等を用いること ができる。  When the nucleosome and the antibody (immune complex) are contained in the culture supernatant or the like, it is preferable to concentrate the supernatant by a means such as ultrafiltration before pouring the supernatant into the protein A column. By performing the concentration, more efficient production of mononucleosomes can be achieved. When the concentration is carried out by ultrafiltration, for example, Diaflo membrane (PH30) (Millipore) having a molecular weight of 30,000 cut-off can be used.
前記解離回収工程は、 前記捕獲収集工程において捕獲収集されたヌクレオソー ムを前記抗体から解離回収させる工程である。 前記解離回収は、 ヌクレオゾーム に特異的な抗体とヌクレオソ一ムとの結合を高濃度塩化ナトリゥム液等を用いて 解離させることにより行うことができ、 その結果、 ヌクレオゾームのみを分離す ることができる。  The dissociation and recovery step is a step of dissociating and recovering the nucleosomes captured and collected in the capture and collection step from the antibody. The dissociation and recovery can be performed by dissociating the nucleosome-specific antibody with the nucleosome using a high-concentration sodium chloride solution or the like. As a result, only the nucleosome can be separated.
ヌクレオソ一ムを解離させるために用いられる溶出液としては、 目的に応じて 適宜選択することができるが、 例えば、 塩化ナトリウム濃度が 0. 8〜1. 2M 程度であり、 好ましくは 0. 6〜0. 8Mであり、 特に好ましくは 1. 2Mであ り、 pHが、 pH4. 5〜9. 0程度であり、 好ましくは pH6〜8であり、 特 に好ましくは pH7〜7. 5である緩衝液などが好適に挙げられ、 具体的には、 濃度が 25〜10 OmM程度であり、 好ましくは 25〜75mMであり、 特に好 ましくは 25〜5 OmMであるトリス緩衝液が好適に挙げられる。  The eluate used for dissociating the nucleosome can be appropriately selected according to the purpose.For example, the concentration of sodium chloride is about 0.8 to 1.2 M, preferably 0.6 to 1.2 M. 0.8 M, particularly preferably 1.2 M, and a buffer having a pH of about pH 4.5 to 9.0, preferably pH 6 to 8, and particularly preferably pH 7 to 7.5. A Tris buffer having a concentration of about 25 to 10 OmM, preferably 25 to 75 mM, and particularly preferably 25 to 5 OmM is preferred. .
前記溶出液中の塩の種類や緩衝液の種類としては、 特に制限はなく、 適宜選択 でき、 上記以外には、 例えば、 リン酸緩衝液や、 炭酸緩衝液などが挙げられる。 なお、 プロティン Aカラムなどの担体からヌクレオソームを解離させる前に、 カラムに非特異的に結合しているタンパク質を洗浄によって完全に除去すること が不可欠である。 その際用いる洗浄液としては、 例えば、 トリス緩衝液 （25m Mトリス、 14 OmM塩化ナトリウム、 pH7. 4 ) 等が挙げられるが、 種々の 条件によって濃度、 塩類の種類、 pH等を適宜調整することができる。 非特異結 合タンパク質が完全に除去されたか否かは、例えば、 26 Onmでの吸光度が 0. 00であることによって確認することができる。 The kind of the salt and the kind of the buffer in the eluate are not particularly limited and can be appropriately selected. In addition to the above, examples include a phosphate buffer and a carbonate buffer. Before dissociating nucleosomes from a carrier such as a protein A column, it is essential to completely remove the proteins non-specifically bound to the column by washing. The washing solution used at this time is, for example, Tris buffer (25 mM Tris, 14 OmM sodium chloride, pH 7.4), etc., and the concentration, type of salt, pH, etc. may be appropriately adjusted depending on various conditions. it can. Whether or not the non-specific binding protein has been completely removed can be confirmed, for example, by the absorbance at 26 Onm being 0.00.
プロテイン Aに結合した 2 C 10 · クロマチン複合体 （2 C 10ハイプリ ドー マ自身がこれに該当する） 又は 2 C 10を予め結合したプロティン Aカラムある いは 2 C 10を共有結合で固定化したァフィ二ティ一カラムに結合した培養上清 中の総てのヌクレオゾーム （モノマーからオリゴ又はポリマーまで） は、 1. 2 Mの塩化ナトリウムを含むトリス緩衝液 （ 25mM Tr i s， 0. 04質量％ NaN3， pH 7. 4) (以後、 これを緩衝液 Bと呼ぶ） を溶出液として用いて、 解離させることができる。 この溶出液をウルトラフリー膜 （分子量 3万カツ トォ フ、 ミリポア社） を用いて濃縮、 それを 0. 25 M塩化ナトリウム添加トリス緩 衝液 A (以下、 これを 「緩衝液 C」 と呼ぶ） で平衡化したスーパ一デックス 20 0のカラムを用いた HP L Cによりモノヌクレオゾームのみを分離することがで きる。しかし、培養上清中にはそれよりもオリゴマーやポリマーが圧倒的に多く、 効率良くモノマ一を回収するには緩衝液 Aでの溶出液 （濃縮後） をマイクロコヅ カル .ヌクレア一せ （MN) で消化し、 できるだけモノマーにしてから反応を停 止する。 このモノマーを前記 HP L Cにより分離するのが好ましい。 その際、 従 来のショ糖濃度勾配超遠心法やゲル切り出し法では、 回収することはできなかつ たヌクレオゾームを繋いでいるリンカ一 DNAも、 本クロマトグラフィ一で分子 量約 10 K位の所に溶出される （図 3)。 この切断されたリンカー DNAは、 遺伝 子情報を知るためのクローニングに最適である。 2C10-chromatin complex bound to protein A (2C10 hybridoma itself) or protein A column pre-bound to 2C10 or 2C10 immobilized covalently All nucleosomes (from monomer to oligo or polymer) in the culture supernatant bound to the affinity column were converted to Tris buffer containing 1.2 M sodium chloride (25 mM Tris, 0.04% by mass NaN 3 , pH 7.4) (hereinafter referred to as buffer B) can be used for dissociation. The eluate was concentrated using an ultra-free membrane (Molecular weight 30,000 Kattov, Millipore), and concentrated with 0.25 M sodium chloride-added Tris buffer A (hereinafter referred to as “buffer C”). Only mononucleosomes can be separated by HP LC using an equilibrated Superdex 200 column. However, the culture supernatant contains much more oligomers and polymers than that. To recover the monomer efficiently, the eluate (after concentration) in buffer A must be collected using microcoal.nuclease (MN). Digest to make monomer as much as possible and stop the reaction. Preferably, the monomers are separated by the HP LC. At this time, linker DNA that cannot be recovered by the conventional sucrose gradient ultracentrifugation method or gel excision method and that is linked to nucleosomes is also eluted at a molecular weight of about 10 K by this chromatography. (Figure 3). This cleaved linker DNA is most suitable for cloning to know gene information.
前記単離精製工程は、 前記解離回収工程において回収したモノヌクレオゾーム からモノヌクレオゾームを分子量に基づいて単離精製する工程であり、 具体的に は、 ヌクレオソームを含むプロテイン Aカラム溶出液を、 所望によりヌクレオソ ームをモノヌクレオソ一ム単位に切断し得るヌクレアーゼ （例えば、 マイクロコ ッカル 'ヌクレアーゼ （MN) など） で消化した後、 回収したヌクレオソームか らモノヌクレオソームを分子量に基づいて精製する工程である。 The isolation and purification step is a step of isolating and purifying mononucleosomes from the mononucleosomes recovered in the dissociation and recovery step based on the molecular weight. Specifically, a protein A column eluate containing nucleosomes is desirably used. Nucleases that can cleave nucleosomes into mononucleosome units This is a process in which mononucleosomes are purified from the recovered nucleosomes based on their molecular weights after digestion with Nuclease (MN).
前記分子量に基づく精製は、 例えば、 高速液体クロマトグラフィー（HPLC) を用いて、 モノヌクレオソ一ムのみを高収率に回収することにより行うことがで き、 又は、 ゲルろ過カラムを用いて分子量 20万から 25万の分画を回収するこ とにより行うことができる。  The purification based on the molecular weight can be performed, for example, by using high performance liquid chromatography (HPLC) to recover only the mononucleosome in a high yield, or using a gel filtration column to have a molecular weight of 200,000. By collecting 250,000 fractions from
前記 HP L C用カラムとしては、 分子量 10， 000〜1， 000, 000ま でを分離できるものが好ましく、 例えば、 スーパ一デヅクス 200 (Super d ex 200 ) カラム （アマシャムフアルマシアバイオテック社製） ス一バーデ ックス 100 (Superdex l O O) カラム （アマシャムフアルマシアバイ ォテックス社製） 等が挙げられる。  The HP LC column is preferably a column capable of separating a molecular weight of 10,000 to 1,000,000, for example, a Superdex 200 column (manufactured by Amersham Pharmacia Biotech). Superdex 100 column (manufactured by Amersham Pharmacia Biotex) and the like.
前記 HPLCの展開溶媒としては、 塩化ナトリウム濃度が 140〜120 Om M程度であり、 好ましくは 25 OmMであり ； アジ化ナトリウム等の濃度が 0. 02〜0. 1質量％程度であり、 好ましくは 0. 04質量％である、 25~75 mM、 好ましくは 25mMのトリス緩衝液 （pH6. 8〜8. 3、 好ましくは p H7， 4) が挙げられる。 なお、 前記濃度、 pH等は、 目的に応じて適宜選択す ることができる。  The developing solvent for the HPLC has a sodium chloride concentration of about 140 to 120 OmM, and preferably 25 OmM; a concentration of sodium azide or the like is about 0.02 to 0.1 mass%, preferably 25-75 mM, preferably 25 mM, Tris buffer (pH 6.8-8.3, preferably pH 7,4) which is 0.04% by mass. The concentration, pH and the like can be appropriately selected according to the purpose.
H P L Cカラムを上記展開溶媒で平衡化した後、 プロテイン Aカラム溶出液を 流し、 モノヌクレオソ一ムのビーク画分を集めると、 単離 ·精製されたモノヌク レオソームが得られる。保存用としてのモノヌクレオゾームのタンパク質濃度は、 ゥシ血清アルブミンをスタンダードとして、 B C Aプロテインアツセィキット（ピ —ス （P IERCE)社製） で測定した場合、 100〜1 O O O zg/ml程度 が好ましく、 500 gZmlであることがより好ましい。  After equilibrating the HPLC column with the above-mentioned developing solvent, the eluate of the protein A column is allowed to flow, and the beak fraction of the mononucleosome is collected to obtain an isolated and purified mononucleosome. The protein concentration of the mononucleosome for preservation is about 100 to 1 OOO zg / ml when measured with a BCA protein assay kit (manufactured by PIERCE) using serum albumin as a standard. Preferably, it is more preferably 500 gZml.
モノヌクレオゾームの検出は、 15%SDS · PAGE電気泳動で H 1を除く コアヒストン H 3、 H2 a、 H2b、 H 4及びその修飾産物の同定並びにヌクレ ォゾームから抽出した DNAの大きさが 150〜20 Obpの点にのみ集蔟して いることを 1〜 2 %ァガロースゲル電気泳動にて確かめることによって行うこと ができる。  Mononucleosomes were detected by 15% SDSPAGE electrophoresis except for H1 except for core histones H3, H2a, H2b, H4 and their modified products, and the size of DNA extracted from nucleosomes was 150--20. This can be done by confirming that the probe is mounted only at the point of Obp by 1-2% agarose gel electrophoresis.
なお、 プロティン Aカラム溶出液をそのまま HP L Cにかけてもある程度のモ ノヌクレオソ一ムを回収できるが、 その前に、 溶出液をマイクロコッカル 'ヌク レア一ゼ （MN) で処理 （消化） することが好ましい。 これによつて、 重合して いるヌクレオソ一ムをモノヌクレオソ一ムに分解することによって、 モノヌクレ ォソームの回収率を顕著に高めることができる。 溶出液の消化に用いることがで きるマイクロコッカル 'ヌクレア一ゼ （MN) としては、 例えば、 マイクロコッ カル .ヌクレアーゼ EC 3. 131. 1 (シグマ社製）、 等が挙げられる。 消化さ れるヌクレオソ一ムを含む溶液の濃度としては、 1〜 100ュニッ卜/ ml、 程 度が好ましく、 5〜50ユニット Zmlがより好ましい。 It should be noted that the protein A column eluate can be subjected to HP LC Before the non-nucleosome can be recovered, it is preferred that the eluate be treated (digested) with Micrococcal 'Nuclease (MN) prior to that. As a result, the recovery of mononucleosomes can be significantly increased by decomposing the polymerized nucleosomes into mononucleosomes. Examples of micrococcal nuclease (MN) that can be used for digestion of the eluate include Micrococcal nuclease EC 3.13.1 (Sigma). The concentration of the solution containing the nucleosome to be digested is preferably about 1 to 100 units / ml, and more preferably about 5 to 50 units Zml.
マイクロコッカル ·ヌクレア一ゼによる消化は、 溶出液に C a2 +を 1〜 1 Om M程度、 好ましくは 2. 5mM、 マイクロコッカル ·ヌクレア一ゼ（MN)を 0. 25〜： LU、 好ましくは 0. 5U添加し、 30〜40°C、 好ましくは 37°Cで、 45- 120分間、好ましくは 60分間保温することによって行うことができる。 反応容量は適宜設定することができ、 例えば 200 1とすることができる。 保 温終了後、 直ちに 5 mM (E GT A) を添加してマイクロコッカル ' ヌクレア一 ゼを失活させ反応を停止させる。 不溶物をマイクロヒュ一ジ （ 13Krpm、 5 分） で除去し、 上清を HP LCにかけ単離精製工程を行う。 Digestion with micro Cocker Le Nuclear one Ze is, C a 2 + a. 1 to 1 Om M about the eluate, preferably 2. 5 mM, 0.. 25 to the micro Cocker Le Nuclear Ichize (MN): LU, Preferably, 0.5 U is added, and the temperature is maintained at 30 to 40 ° C, preferably 37 ° C, for 45 to 120 minutes, preferably 60 minutes. The reaction volume can be appropriately set, for example, 2001. Immediately after the incubation, add 5 mM (EGT A) to inactivate micrococcal nuclease and stop the reaction. Remove the insoluble material with a micro-huge (13K rpm, 5 minutes) and apply the supernatant to HP LC for isolation and purification.
なお、 抗体がプロテイン Aカラムに固定化されている場合、 前記捕獲収集工程 の前に、 ヌクレオソ一ムを含むサンプルをマイクロコッカル 'ヌクレア一ゼによ つて処理 （消化） してもよい。 これによつて、 前記単離精製工程の前に処理 （消 化） するのと同様にモノヌクレオソ一ムの回収率を高めることができる。  When the antibody is immobilized on the protein A column, the sample containing the nucleosome may be treated (digested) with micrococcal nuclease before the capture and collection step. This makes it possible to increase the recovery of mononucleosomes as in the case of treating (consuming) before the isolation / purification step.
本発明によって得られたモノヌクレオソ一ムの純度は、 通常 95質量％以上、 好ましくは 99質量％以上である。 なお、 得られたモノヌクレオソ一ムのピーク 画分に含まれるものがモノヌクレオソームであることは、 I shi z akaらの 方法 (Ishizaka ら、 Nucleic Acid Res., 19: 5792, 1991) によって該画分から 抽出された DN Aが、 2質量％ァガロースゲル電気泳動で 150〜200塩基対 のサイズに集中して検出されること、 及び 15質量％SD S— PAGEでコアヒ ストン （ヒストン H 3、 H 2 B、 H 2 A、 及び H 4) のみが検出され、 ヒストン H 1は検出されないことによって証明される。  The purity of the mononucleosome obtained by the present invention is usually 95% by mass or more, preferably 99% by mass or more. The mononucleosome contained in the obtained mononucleosome peak fraction was determined to be a mononucleosome by the method of Ishizaka et al. (Ishizaka et al., Nucleic Acid Res., 19: 5792, 1991). The extracted DNA was detected in a concentration of 150-200 base pairs by 2% by mass agarose gel electrophoresis, and the core histones (histone H3, H2B, Only H2A and H4) are detected, and histone H1 is not detected.
本発明のモノヌクレオソームの製造方法は、 モノヌクレオゾームの分離が極め て厳格であるため、得られたヌクレオソ一ムから分離される D N Aの純度が高く、 その遺伝子情報入手のためのクロ一ニングの能率化に寄与することができる。 本発明のモノヌクレオゾームの製造方法は、 自己免疫疾患等で注目されている 修飾ヌクレオソ一ムを製造することができるため、 それらの疾患のメカニズム、 アポトーシスの生理的意義等の解明に貢献でき、学術的にも大きな意義を有する。 また、 ゲノムプロジェクトの一環として、 疾患感受性、 薬剤抵抗性の解明が、 単一ヌクレオチド多形態 （single- nucleotide polymorphism; SNP) の点から大規 模に進められている。 しかし、 それは全ゲノムが対象で、 膨大な人力と資金を要 する。 これに対し、 ヌクレオソ一ム単位での S N Pの解析は、 より低コストで且 つ効率が良いと考えられる。 したがって、 ヌクレオゾーム単位での S N P解析を 行うに当たって、 本発明のモノヌクレオソ一ムの製造方法は大きく貢献できる。 本発明のモノヌクレオソームの製造方法の他の態様としては、 細胞を低塩濃度 の溶液中で破砕させ、 溶液中にヌクレオソームを放出させる放出工程と、 前記ヌクレオソームを収集する収集工程と、 In the method for producing a mononucleosome of the present invention, the separation of mononucleosomes is extremely high. As a result, the purity of DNA isolated from the obtained nucleosome is high, which can contribute to the efficiency of cloning for obtaining genetic information. The method for producing mononucleosomes of the present invention can produce modified nucleosomes that have attracted attention in autoimmune diseases and the like, and can contribute to elucidation of the mechanisms of those diseases and the physiological significance of apoptosis. It has great academic significance. Also, as part of the genome project, elucidation of disease susceptibility and drug resistance is being pursued on a large scale in terms of single-nucleotide polymorphism (SNP). However, it covers the entire genome and requires a great deal of manpower and money. In contrast, SNP analysis on a nucleosomal basis is considered to be lower cost and more efficient. Therefore, in performing SNP analysis in units of nucleosomes, the method for producing a mononucleosome of the present invention can greatly contribute. In another embodiment of the method for producing a mononucleosome of the present invention, a cell is crushed in a solution having a low salt concentration, and a releasing step of releasing nucleosomes into the solution; a collecting step of collecting the nucleosomes
収集した前記ヌクレオゾームを低塩濃度の溶液に懸濁し、 モノヌクレオソ一ム 単位に切断し得るヌクレアーゼで処理するヌクレアーゼ処理工程と、  A nuclease treatment step of suspending the collected nucleosomes in a solution having a low salt concentration and treating with a nuclease that can be cleaved into mononucleosome units;
前記ヌクレアーゼ処理後の溶液からモノヌクレオソ一ムを、 該ヌクレオソーム に特異的な抗体を用いて、及び/又は分子量に基づいて製造する単離精製工程と、 を含む。  An isolation and purification step of producing a mononucleosome from the solution after the nuclease treatment using an antibody specific to the nucleosome and / or based on the molecular weight.
通常の状態ではヌクレオソ一ムを培養液中に放出しない細胞からモノヌクレオ ソ一ムを製造する場合には、 前記放出工程により、 溶液中にヌクレオソ一ムを放 出させてから、 モノヌクレオソ一ムを単離すればよい。 前記ヌクレアーゼ処理工 程は、 前記ヌクレア一ゼを用いた処理 （消化） と同様に行うことができ、 前記単 離精製工程は、 上述したとおり行うことができる。  When producing mononucleosomes from cells that do not release nucleosomes into the culture medium under normal conditions, the release step releases the nucleosomes into the solution and then converts the mononucleosomes into monolayers. You only have to separate them. The nuclease treatment step can be performed in the same manner as the treatment (digestion) using nuclease, and the isolation and purification step can be performed as described above.
(モノヌクレオソ一ム）  (Mononucleosome)
本発明のモノヌクレオゾームは、 本発明のモノヌクレオゾームの製造方法によ り製造される。  The mononucleosome of the present invention is produced by the method for producing a mononucleosome of the present invention.
本発明のモノヌクレオソームは、 厳格な条件により製造されるため、 純度が通 常 9 5質量％以上、 好ましくは 9 9質量％以上と高く、 保存安定性に優れ、 モノ ヌクレオゾーム本来の形態をよく維持している （形態維持性に優れる）。 したがつ て、 各種測定乃至診断に好適に用いることができる。 Since the mononucleosome of the present invention is produced under strict conditions, its purity is usually as high as 95% by mass or more, preferably as high as 99% by mass or more. The nucleosome maintains its original form well (excellent in form retention). Therefore, it can be suitably used for various measurements and diagnoses.
また、 本発明のモノヌクレオソ一ムは長期間安定に保存することができる。 具 体的には、 トリス、 N a C 1及び N a N 3からなる N a C 1添加トリス緩衝液（T B S ) 中で、 又は、 1質量％牛血清アルブミン （B S A )、 0 . 4質量％スキムミ ルク、 1 0質量％ブロックエース、及び I mM 0丁八を含む丁8 3中で、 4 °C で 2ヶ月間保存しても前記モノヌクレオソームは、 なおかつ抗原性を維持した。 ヌクレオソ一ムは、 一般的に安定性が低く、 また、 凍結すると破壊されるため、 前記安定性は大きな利点である。 Further, the mononucleosome of the present invention can be stored stably for a long period of time. In concrete terms, tris, N a C 1 added Tris buffer consisting of N a C 1 and N a N 3 in (TBS), or 1 wt% bovine serum albumin (BSA), 0. 4% by weight The mononucleosome maintained its antigenicity even after being stored at 4 ° C for 2 months in Chofu 83 containing skim milk, 10% by mass Block Ace, and ImM 0-cho. Nucleosomes generally have low stability and are destroyed when frozen, so the stability is a great advantage.
また、 アポト一シス由来のモノヌクレオソ一ムは、 アポトーシスによる修飾を 保持しており、 自己抗原性を獲得し易いため、 自己免疫病患者の抗体測定 （自己 免疫病診断） に、 特に好適に利用できる。  In addition, a mononucleosome derived from apoptosis retains the modification by apoptosis and easily acquires autoantigenicity, so that it can be particularly preferably used for antibody measurement (autoimmune disease diagnosis) of an autoimmune disease patient. .
(モノヌクレオソ一ム製造用キッ ト）  (Kit for manufacturing mononucleosomes)
本発明のモノヌクレオソ一ム製造用キッ トは、 試料中に含まれるヌクレオソ一 ムを捕獲収集するための該ヌクレオソ一ムに特異的な抗体と、  The kit for producing a mononucleosome of the present invention comprises an antibody specific to the nucleosome for capturing and collecting the nucleosome contained in a sample,
捕獲収集したヌクレオソ一ムから分子量に基づきモノヌクレオソームを単離する ためのカラムと、 を含む。 A column for isolating mononucleosomes from the captured and collected nucleosomes based on molecular weight.
本発明のモノヌクレオソ一ム製造用キッ トを本発明のモノヌクレオソ一ムの製 造方法に使用することにより、 モノヌクレオソ一ム分析、 自己免疫病診断等をは じめ各種用途に好適で、 保存安定性に優れたモノヌクレオソ一ムを、 形態安定性 よく、 簡易にかつ効率よく、 しかも高純度で製造することができる。  By using the kit for producing mononucleosomes of the present invention in the method for producing mononucleosomes of the present invention, it is suitable for various uses including mononucleosome analysis, autoimmune disease diagnosis, etc., and storage stability. It is possible to easily and efficiently produce a mononucleosome with excellent form stability, high efficiency, and high purity.
(ヒストン検査方法）  (Histone inspection method)
本発明のヒストン検査方法は、 本発明のモノヌクレオゾームの製造方法により 得たモノヌクレオソ一ムより被検ヒストンを回収し、 該被検ヒストンの電気泳動 パターンと対照ヒストンの電気泳動パターンとを比較することを含む。  According to the histone testing method of the present invention, a test histone is recovered from a mononucleosome obtained by the method for producing a mononucleosome of the present invention, and the electrophoretic pattern of the test histone is compared with the electrophoretic pattern of a control histone. Including.
自己免疫疾患における、 自己免疫現象は、 アポト一シスにより修飾されたヌク レオソ一ムが抗原となっていると考えられ、 この修飾ヌクレオゾームの本体はコ ァヒストンの修飾であると考えられる。 したがって、 モノヌクレオソ一ムのコア ヒストンの修飾を検査することで、 ヌクレオゾームの修飾状態を把握し、 自己免 疫疾患の診断、 解明に利用することができる。 In an autoimmune phenomenon in an autoimmune disease, a nucleosome modified by apoptosis is considered to be an antigen, and the main body of the modified nucleosome is considered to be cohistone modification. Therefore, by examining the modification of the core histone of the mononucleosome, the state of the modification of the nucleosome can be grasped and the self- It can be used for diagnosis and elucidation of epidemics.
前記ヒストン検査方法は、 具体的には、 本発明のモノヌクレオソームの製造方 法により被検試料から得られるモノヌクレオソ一ムを、 S D S— P A G Eにより 解析する。 この解析結果を、 正常細胞から得られるヒストンの S D S— P A G E による解析結果と比較することにより行うことができる。  In the histone test method, specifically, a mononucleosome obtained from a test sample by the method for producing a mononucleosome of the present invention is analyzed by SSD-PAGE. The results of this analysis can be performed by comparing the results of SDS-PAGE analysis of histones obtained from normal cells.
(ヌクレオソ一ムに特異的な抗体の測定方法）  (Method for measuring antibodies specific to nucleosomes)
本発明のモノヌクレオゾームの製造方法により製造されたモノヌクレオソ一ム を固相化する固相化工程と、  Immobilizing step of immobilizing the mononucleosome produced by the method for producing a mononucleosome of the present invention,
被検試料を、 固相化された該モノヌクレオソ一ムと反応させる反応工程と、 前記モノヌクレオソームに結合する特異的な抗体を測定する測定工程と、を含む。 前記固相化工程においては、 本発明のモノヌクレオゾームの製造方法により製 造されたモノヌクレオソームを用いる。 このモノヌクレオゾームは、 純度が通常 9 5質量％以上、 好ましくは 9 9質量％以上と高く、 モノヌクレオソ一ムの形態 をよく保持している （形状安定性に優れる）。 したがって、 ヌクレオソームに特異 的な抗体に対する抗原として好適である。 A reaction step of reacting a test sample with the immobilized mononucleosome; and a measurement step of measuring a specific antibody that binds to the mononucleosome. In the solid-phase immobilization step, a mononucleosome produced by the method for producing a mononucleosome of the present invention is used. This mononucleosome has a high purity of usually 95% by mass or more, preferably 99% by mass or more, and retains the form of the mononucleosome well (excellent in shape stability). Therefore, it is suitable as an antigen against a nucleosome-specific antibody.
また、 本発明のモノヌクレオゾームの製造方法により得られる、 アポトーシス 由来のモノヌクレオソームは、 アポトーシスによる修飾を保持しており、 自己抗 原性を獲得し易いため、 自己免疫病患者の抗体測定に、 特に好適である。  In addition, apoptosis-derived mononucleosome obtained by the method for producing a mononucleosome of the present invention retains apoptotic modification and easily acquires self-antigenicity. Particularly preferred.
前記モノヌクレオゾームの固定化緩衝液としては、 モノヌクレオソ一ムの形態 を変化させないものであれば、 特に制限はなく、 適宜選択することができるが、 例えば、 5 O mM炭酸緩衝液 （p H 9 . 6 ) を使用することができる。  The buffer for immobilizing the mononucleosome is not particularly limited and can be appropriately selected as long as it does not change the form of the mononucleosome. For example, a 5 OmM carbonate buffer (pH 9 6) can be used.
前記固相化工程に用いられる固相としては、 モノヌクレオソームを固定化でき るものであれば特に制限はなく、 適宜選択することができるが、 例えばポリスチ レン製のマイクロタイ夕一プレート I mm u 1 o n 2 H B (ダイネックステクノ ロジ一社製 Dynex Technologies, Chantilly, VA) 等が、 好適に使用することが できる。 前記モノヌクレオゾームの前記固相化の条件としては、 固相の種類等に 応じて適宜選択することができ、 前記固相化の具体例としては、 前記ポリスチレ ン製マイクロタイ夕一プレートを固相として使用する場合には、 従来から d s D N A抗原のプレートへの吸着に用いられているポリ— L—リジン（P L L ) ( 1〃 g/ml蒸留水） を介して、 l〃g/mlの濃度で 4°Cで一晩吸着させ、 過剰な 抗原を一度吸引除去した後、 NaCl添加トリス緩衝液洗浄する方法等が、 好適 に挙げられる。 The solid phase used in the solid phase immobilization step is not particularly limited as long as it can immobilize mononucleosomes, and can be appropriately selected.For example, a polystyrene micro-titer plate Immu 1 on 2 HB (Dynex Technologies, Chantilly, VA, manufactured by Dynex Technology, Inc.) or the like can be suitably used. The conditions for the immobilization of the mononucleosome can be appropriately selected according to the type of the solid phase and the like. As a specific example of the immobilization, the polystyrene micro tie plate is fixed. When used as a phase, poly-L-lysine (PLL) (1〃) which has been conventionally used for adsorbing ds DNA antigen to a plate is used. g / ml of distilled water) at 4 ° C overnight at a concentration of l〃g / ml to remove excess antigen once, followed by washing with NaCl-added Tris buffer. No.
前記固相化工程においては、 モノヌクレオソームの固定化されていない固相を 遮蔽し、 非特異性反応を防止するため、 モノヌクレオゾームの固相への固定化後 に固相をプロヅキング液でブロッキングすることが好ましい。 ヌクレオソ一ムは 複雑な高次構造を有するため、 特に非特異性反応の影響が大きく、 ブロッキング の役割は大きい。 前記ブロッキングは、 前記固相とブロッキング液とを反応させ た後、 洗浄することにより行うことができる。 前記ブロッキング液としては、 少 なくともスキムミルクを含む溶液が好ましく、 トリス、 NaC l及びNaN3か らなる Na C 1添加トリス緩衝液 （TB S) に、 0. 1〜0. 3質量％のスキム ミルクを含む溶液が特に好ましい。 In the solid phase immobilization step, after the mononucleosome is immobilized on the solid phase, the solid phase is blocked with a blocking solution in order to shield the non-immobilized solid phase of the mononucleosome and prevent nonspecific reaction. Is preferred. Because nucleosomes have a complex higher-order structure, they are particularly affected by nonspecific reactions and play a large role in blocking. The blocking can be performed by reacting the solid phase with the blocking solution and then washing. As the blocking solution, preferably a solution containing skim milk even without low, Tris, the NaC l and NaN 3 or Ranaru Na C 1 added Tris buffer (TB S), 0. 1~0. 3 % by weight of skim Solutions containing milk are particularly preferred.
固相化工程の終了後、 直ちに反応工程を開始しない場合には、 各ゥエルに TB S又は後述の反応液を加え、 プレートシールで密閉し、 4°Cで保存することがで きる。  If the reaction step is not started immediately after the solid phase immobilization step, add TBS or the reaction solution described below to each well, seal with a plate seal, and store at 4 ° C.
前記反応工程は、 被検試料を、 固相化された該モノヌクレオソ一ムと反応させ る工程であるが、 該被検試料としては、 健常者又は患者の血清又は血漿が好まし く、 自己免疫病等の病気の診断に利用できる点で、 自己免疫病の疑いのある患者 の血清又は血漿が特に好ましい。  The reaction step is a step in which a test sample is reacted with the immobilized mononucleosome. The test sample is preferably serum or plasma of a healthy person or a patient, and is preferably an autoimmune patient. Serum or plasma of a patient suspected of having an autoimmune disease is particularly preferable because it can be used for diagnosis of diseases such as diseases.
前記試料を前記モノヌクレオゾームと反応させる方法については、 前記試料中 のヌクレオゾームに特異的な抗体が前記モノヌクレオソ一ムに結合できる方法で あれば特に制限はなく、 適宜選択することができる。 例えば、 前記マイクロタイ 夕一プレートを用いた場合には、試料を反応液で稀釈し、振盪しながら反応させ、 前記反応後吸引除去し、 洗浄する等の方法により行うことができる。  The method of reacting the sample with the mononucleosome is not particularly limited and may be appropriately selected as long as the method allows an antibody specific to the nucleosome in the sample to bind to the mononucleosome. For example, when the micro tie plate is used, the method can be carried out by diluting the sample with a reaction solution, reacting the sample with shaking, removing the sample by suction after the reaction, and washing.
なお、試料を稀釈する反応液としては、前記非特異性反応を防止する観点から、 スキムミルクを含むことが好ましく、 1質量％牛血清アルブミン （B S A)、 0. 4質量％スキムミルク、 10質量％ブロックエース、 ImM EDTAを含む T B Sであることが特に好ましい。 前記 B S Aは、 アルブミン フラクション V (Albumin, Fraktion V， ベ一リンガ一マンノヽィム社製 Boehnnger Mannheim, Germany)が好ましく、前記プロックエースは、大日本製薬株式会社製が好ましく、 前記 EDTAは EDTAニナトリウム塩 （同仁化学製） が好ましい。 The reaction solution for diluting the sample preferably contains skim milk from the viewpoint of preventing the non-specific reaction, and includes 1% by mass bovine serum albumin (BSA), 0.4% by mass skim milk, and 10% by mass block. Ace and TBS containing ImM EDTA are particularly preferred. The BSA was obtained from Albumin Fraction V (Boehnnger Mannheim, manufactured by Boehringer Mannheim, Inc.). Germany) is preferable, the block ace is preferably manufactured by Dainippon Pharmaceutical Co., Ltd., and the EDTA is preferably a disodium salt of EDTA (manufactured by Dojindo).
前記測定工程は、 前記モノヌクレオソ一ムに結合する特異的な抗体を測定する 工程であるが、 前記モノヌクレオゾームに結合する抗体を測定することができれ ば特に制限はなく、 目的に合わせて適宜選択することができる。 なお、 前記測定 工程においては、 前記抗体の代りに、 前記モノヌクレオソ一ムに結合する抗体を 認識する二次抗体を測定してもよい。 例えば、 前記マイクロタイ夕一プレートを 用いた場合には、 前記洗浄後、 直ちに、 アルカリホスファターゼ （AP) コンジ ユゲー卜抗ヒト I gG抗体 （ャギ） 稀釈液を、 各ゥエルに加え、 振盪反応させ、 洗浄した後、 P—二トロフエニル ホスフェイ ト（PNP) (シグマ社製 Sigma， St. Louis, MO)試薬を各ゥエルに添加し、 再び振盪反応させ、 発色させる等の方法に より行うことができる。なお、前記発色は、 反応直後に発色の度合（吸光度）を、 オートリーダーを用いて、 吸光波長 405 nmで測定する。 対照又は盲検は、 前 記一連の反応系で、 血清又は血漿を加えなかった時に得られる吸光度とし、 各資 料における吸光度からこれを差し引いた値が実測値となる。 抗体価は、 前記吸光 度の実測値として表すことも可能であるが、 高力価の血清で予め標準曲線を作成 し、 それとの対比により試料における抗体価をュニッ ト単位で表示することが好 ましい。  The measurement step is a step of measuring a specific antibody that binds to the mononucleosome, but is not particularly limited as long as the antibody that binds to the mononucleosome can be measured. You can choose. In the measuring step, a secondary antibody that recognizes an antibody that binds to the mononucleosome may be measured instead of the antibody. For example, in the case of using the microtiter plate, immediately after the washing, an alkaline phosphatase (AP) conjugated anti-human IgG antibody (goat) diluted solution is added to each well, and the mixture is shaken. After washing, a reagent such as P-nitrophenyl phosphate (PNP) (Sigma, St. Louis, MO, manufactured by Sigma) is added to each well, and the mixture is shaken again to develop color. . The color is measured immediately after the reaction by measuring the degree of color development (absorbance) at an absorption wavelength of 405 nm using an auto-reader. The control or blind test is the absorbance obtained when serum or plasma is not added in the above series of reaction systems, and the value obtained by subtracting this from the absorbance of each material is the measured value. Although the antibody titer can be expressed as the actual measured value of the absorbance, it is preferable to prepare a standard curve in advance with a high-titer serum and display the antibody titer in the sample in units of unit by comparison with the standard curve. Good.
前記測定工程は、 また、 I gGサブクラスを認識する抗ヒト抗体を二次抗体と して反応させる工程を含んでもよく、 この場合にはサブクラス別の抗体の測定が 可能となる点で、 特に好ましい。  The measurement step may also include a step of reacting an anti-human antibody recognizing an IgG subclass as a secondary antibody, and in this case, measurement of an antibody for each subclass is particularly preferable. .
即ち、 I gG抗体には、 I gG l、 I gG 2、 I gG3及び I gG4の四種類 のサブクラスがあり、 患者の有する、 ヌクレオソ一ムに特異的な抗体の、 サブク ラスの片寄りが、 疾患病態を特徴付けることがある。 したがって、 サブクラス別 抗体の測定が可能であれば、 疾患病態をよりよく把握することができる。  That is, there are four subclasses of IgG antibodies, IgGl, IgG2, IgG3 and IgG4, and the deviation of the subclass of the antibody specific to the nucleosomal possessed by the patient is as follows: It may characterize the disease pathology. Therefore, if the measurement of antibodies by subclass is possible, the disease pathology can be better understood.
前記サブクラス別抗体の測定には、 例えばピオチン標識した抗ヒトサブクラス 抗体 （マウス） （Zymed, San Francisco, CA) を前記アルカリホスファターゼ （A P)コンジユゲート抗ヒト I gG抗体（ャギ）の代りに使用して反応及び洗浄後、 アルカリホスファタ一ゼ標識ストレプトアビジン （Zymed， San Francisco, CA) を反応させ、 前記と同様に基質の P N Pを加えて、 発色させ、 オートリーダーで 吸光度を測定することができる。 For the measurement of the antibodies by subclass, for example, a biotin-labeled anti-human subclass antibody (mouse) (Zymed, San Francisco, CA) is used instead of the alkaline phosphatase (AP) conjugated anti-human IgG antibody (goat). After reaction and washing, alkaline phosphatase labeled streptavidin (Zymed, San Francisco, CA) Is reacted, and PNP as a substrate is added in the same manner as described above to cause color development, and absorbance can be measured with an auto-reader.
本発明のヌクレオゾームに特異的な抗体の測定方法は、 純度が高く、 本来の形 態をよく保持したモノヌクレオソ一ムを用いることにより、 自己抗原が陽性の場 合にはヌクレオソームに対する極めて高い抗体価を示し、 自己抗原が陰性の場合 には常に低い抗体価しか示さない、 極めて精度の良い測定方法である。 また、 必 要に応じて、 ブロッキング剤、 及び反応液の組成を調整することによつても、 非 特異性反応を防ぎバックグラウンドの閾値を低くすることができる。 更に必要に 応じて、 サブクラス別に抗体を測定することにより、 S L E等の自己免疫病患者 病態をより詳しく分析することができる。  The method for measuring an antibody specific to a nucleosome according to the present invention uses a mononucleosome having high purity and a good original form, so that when a self-antigen is positive, an extremely high antibody titer to a nucleosome can be obtained. This is an extremely accurate measurement method that always shows a low antibody titer when the autoantigen is negative. Also, if necessary, the composition of the blocking agent and the reaction solution can be adjusted to prevent a non-specific reaction and reduce the background threshold. Further, if necessary, the condition of an autoimmune disease patient such as SLE can be analyzed in more detail by measuring antibodies by subclass.
本発明のヌクレオゾームに特異的な抗体の測定方法は、 ヌクレオソ一ムに特異 的な抗体を極めて精度良く測定することができ、自己免疫病、特に S L Eの診断、 ループス腎炎、血管炎、 C N Sループス、小児 S L Eの診断に極めて有効である。 (自己免疫病診断方法）  The method for measuring an antibody specific to a nucleosome according to the present invention can measure an antibody specific to a nucleosome with extremely high accuracy, and can be used to diagnose autoimmune diseases, particularly SLE, lupus nephritis, vasculitis, CNS lupus, Very effective in diagnosing pediatric SLE. (Autoimmune disease diagnosis method)
本発明の自己免疫病診断方法は、 本発明のモノヌクレオゾームの製造方法によ り製造されたモノヌクレオソームを固相化する固相化工程と、  The method for diagnosing an autoimmune disease of the present invention includes a solid-phase immobilizing step of immobilizing the mononucleosome produced by the method for producing a mononucleosome of the present invention,
試料を、 固相化された該モノヌクレオゾームと反応させる反応工程と、 前記モノヌクレオゾームに結合する特異的な抗体を測定する測定工程と、 その抗体価を評価する評価工程と、 を含む。 A reaction step of reacting the sample with the immobilized mononucleosome; a measurement step of measuring a specific antibody that binds to the mononucleosome; and an evaluation step of evaluating the antibody titer.
前記自己免疫病診断方法は、 前記ヌクレオソームに特異的な抗体の測定方法に おいて記載した方法により、 ヌクレオソ一ムに特異的な抗体を測定し、 その測定 された抗体価を評価する。 前記抗体価の評価は、 例えば、 平均値 +標準偏差の 3 倍などにより、 予め設定した所定閾を超えるか否かにより陽性か陰性かを評価す ることができる。  In the method for diagnosing an autoimmune disease, an antibody specific for a nucleosome is measured by the method described in the method for measuring an antibody specific for a nucleosome, and the measured antibody titer is evaluated. In the evaluation of the antibody titer, for example, a positive value or a negative value can be evaluated based on whether or not a predetermined threshold value is exceeded, by, for example, three times the average value + standard deviation.
また、 I g Gサブクラスを認識する抗ヒト抗体を二次抗体として使用して、 ヌ クレオソームに特異的な抗体を I g Gのサブクラスごとに測定し、 それそれの抗 体価が I g Gのサブクラスごとに予め設定した各々の所定閾を超えるか否かを評 価することもできる。 また、 サブクラス別の抗体価のパターンを分析して評価す ることもできる。 サブクラスごとの評価は、 病態をより詳しく表すものである。 (自己免疫病診断用キッ ト） In addition, using an anti-human antibody that recognizes the IgG subclass as a secondary antibody, nucleosome-specific antibodies were measured for each IgG subclass, and the antibody titer of each was determined to be that of IgG. It is also possible to evaluate whether or not each predetermined threshold set for each subclass is exceeded. It can also be evaluated by analyzing the antibody titer pattern for each subclass. The assessment by subclass is more representative of the condition. (Kit for diagnosing autoimmune diseases)
本発明の自己免疫病診断用キットは、 本発明のモノヌクレオソームの製造方法 により製造したモノヌクレオソームを固相化してなる固相と、 緩衝液と、 プレー ト及びカラムのいずれかを含む。  The kit for diagnosing an autoimmune disease of the present invention comprises a solid phase obtained by immobilizing the mononucleosome produced by the method for producing a mononucleosome of the present invention, a buffer, and one of a plate and a column.
前記自己免疫病診断用キットは、 本発明のモノヌクレオソームの製造方法によ り製造され、 形態安定性に優れたモノヌクレオソ一ムを高純度で含むため、 ヌク レオソ一ムに特異的な抗体を極めて精度良く測定することができ、 自己免疫病、 特に SLEの診断に極めて有効である。 また、 前記自己免疫検査キッ トは、 緩衝 液を満たして密封シールすることができ、 前記ヌクレオゾームの保存安定性が高 いことからその保存安定性に優れている。 前記自己免疫病診断用キッ トは、 必要 に応じて、反応液、稀釈液、洗浄液、 二次抗体等を更に含んでいてもよい。 また、 前記二次抗体が抗ヒトサブクラス抗体であってもよい。  The autoimmune disease diagnostic kit is produced by the method for producing a mononucleosome of the present invention and contains a mononucleosome having excellent morphological stability in high purity, so that an antibody specific to the nucleosome is extremely purified. It can be measured with high accuracy and is extremely effective in diagnosing autoimmune diseases, especially SLE. Further, the autoimmune test kit can be hermetically sealed by filling with a buffer solution, and is excellent in storage stability because the storage stability of the nucleosome is high. The kit for diagnosing an autoimmune disease may further include a reaction solution, a diluting solution, a washing solution, a secondary antibody, and the like, if necessary. Further, the secondary antibody may be an anti-human subclass antibody.
前記自己免疫病診断用キットは、 例えば、 前記ヌクレオソ一ムに特異的な抗体 の測定方法の固相化工程で説明した方法等により作製することができる。  The kit for diagnosing an autoimmune disease can be prepared, for example, by the method described in the immobilization step of the method for measuring an antibody specific to a nucleosome.
(ヌクレオソ一ム DN A製造方法）  (Nucleosome DNA production method)
本発明のヌクレオソーム DNA製造方法は、 前記本発明のモノヌクレオソーム の製造方法からなるモノヌクレオソーム製造工程と、  The method for producing a nucleosome DNA of the present invention comprises the steps of producing a mononucleosome comprising the method for producing the mononucleosome of the present invention,
モノヌクレオソームからヌクレオソ一ム DN Aを単離精製するヌクレオソーム D N A単離精製工程とを含むこと以外は特に制限はない。 There is no particular limitation except that it includes a nucleosome DNA isolation and purification step of isolating and purifying the nucleosome DNA from the mononucleosome.
モノヌクレオソ一ムからヌクレオソ一ム DN Aを単離精製するヌクレオソ一ム DNA単離精製工程は、公知の方法から適宜選択して行うことができる（Kanai， Y et al： Induction and natural occurrence of serum nucleosomal DNA in autoimmune MRL/lpr/lpr mice: its relation to apoptosis in the thymus . Immunol Lett.46:207- 214， 1995)。 例えば、 一定量の精製ヌクレオソームを、 1%SDS 及び 0. 5mg/ml Proteinase Kを含むトリス— E D T A緩衝液に懸濁し、 室温で 60分間処理する。 次に、 6Mの Nal (ヨウ化ナトリウム） を含む変性 剤を 3倍量加え、 60°Cで 15分間加熱する。 この操作で可溶化した DNAを 5 0%ィソプロピルアルコールで沈殿させ、 沈殿した DNAを上記トリス— EDT A緩衝液に溶解し、 少量の RNa s eを添加し、 混在する可能性のある RN Aを 分解する方法を用いることができる。 The nucleosomal DNA isolation and purification step for isolating and purifying the nucleosomal DNA from the mononucleosome can be performed by appropriately selecting from known methods (Kanai, Y et al: Induction and natural occurrence of serum nucleosomal). DNA in autoimmune MRL / lpr / lpr mice: its relation to apoptosis in the thymus. Immunol Lett. 46: 207-214, 1995). For example, an aliquot of purified nucleosome is suspended in Tris-EDTA buffer containing 1% SDS and 0.5 mg / ml Proteinase K and treated at room temperature for 60 minutes. Next, add 3 times the amount of denaturant containing 6M Nal (sodium iodide), and heat at 60 ° C for 15 minutes. The DNA solubilized by this procedure is precipitated with 50% isopropyl alcohol, and the precipitated DNA is dissolved in the above Tris-EDTA buffer, a small amount of RNase is added, and RNA that may be present may be mixed. To A decomposition method can be used.
前記ヌクレオソ一ム DN A製造方法により製造されたヌクレオソ一ム DN Aは、 前述したような純度が高くかつ形態をよく保持したモノヌクレオソ一ムから抽出 されるため、 モノヌクレオソ一ムに由来する純度の高い DNAである。 前記ヌク レオソーム DN A製造方法の一例により製造されたヌクレオソーム DN Aのァガ ロースゲル電気泳動によると、 150 b p付近に強いバンドがあり、 これはモノ ヌクレオソ一ムの 2本鎖 D N Aであると考えられる。 これらのヌクレオソ一ム D N Aの平均鎖長は、 145 bp〜20 Obpであることが好ましく、 320〜4 00 b pの DN Aをわずかに含んでいることもある。  The nucleosomal DNA produced by the method for producing nucleosomal DNA is extracted from the mononucleosome having a high purity and a good shape as described above, so that the purity derived from the mononucleosome is high. DNA. According to agarose gel electrophoresis of the nucleosome DNA produced by one example of the method for producing the nucleosome DNA, there is a strong band around 150 bp, which is considered to be mononucleosome double-stranded DNA. . The average chain length of these nucleosome DNAs is preferably between 145 bp and 20 Obp, and may contain as little as 320-400 bp of DNA.
前記ヌクレオソーム DN A製造方法により製造されたヌクレオソ一ム DN Aは、 ヒ ト由来の試料から製造すればヒト由来のヌクレオソーム DNAが得られるため、 S LE等の病態判断のための抗原として、 異種の DNAを用いることに起因する 非特異的反応を排除できる点、 近年注目されているように抗 DN A抗体産生の自 己抗原となる可能性が強いヌクレオソームの DN Aである点で優れている。 本発 明のヌクレオゾーム DN A製造方法は、 今まで必ずしも製造が容易ではなかった このようなヌクレオソーム DNAの製造を容易に、 かつ、 精度よく行う方法を提 供したものであり、 これにより S L E等の診断に好適なヌクレオソ一ム DN Aを 提供することができる。  Nucleosome DNA produced by the above nucleosome DNA production method can produce human-derived nucleosomal DNA if produced from a human-derived sample. It is superior in that it can eliminate non-specific reactions caused by using DNA, and that it is a nucleosome DNA that has a strong possibility of becoming a self-antigen for the production of anti-DNA antibodies, as noted in recent years. The method for producing nucleosome DNA of the present invention provides a method for easily and accurately producing such nucleosome DNA, which has not always been easy to produce until now. A nucleosomal DNA suitable for diagnosis can be provided.
(DNAプレート）  (DNA plate)
本発明の DNAプレートは、 プレート上に、 前記ヌクレオソーム DNA製造方 法により製造されたヌクレオソ一ム D N Aであって、 ヒト由来のヌクレオゾーム DNAが固相化されてなること以外は特に制限はない。 また、 本発明の DNAプ レートは、 プレート上に前記ヒト由来のモノヌクレオソ一ムから単離精製された ヌクレオソ一ム DN Aが固相化されてなることを特徴とする DN Aプレートであ つてもよい。 これらの DNAプレートは、 SLE等の病態判断のための抗原とし て、 ヒトの DNAを用いることにより、 異種の DNAを用いることに起因する非 特異的反応を排除できる点、 近年注目されているように抗 DN A抗体産生の自己 抗原となる可能性が強いヌクレオゾームの DN Aを用いる点で優れている。  The DNA plate of the present invention is not particularly limited, except that it is a nucleosomal DNA produced by the above-described nucleosomal DNA production method, and a human-derived nucleosomal DNA is immobilized on the plate. Further, the DNA plate of the present invention is also a DNA plate, wherein the nucleosome DNA isolated and purified from the human-derived mononucleosome is immobilized on the plate. Good. These DNA plates have attracted attention in recent years for using human DNA as an antigen for determining pathological conditions such as SLE, which can eliminate non-specific reactions caused by using heterologous DNA. It is excellent in that it uses a nucleosome DNA that has a strong possibility of becoming an autoantigen for producing anti-DNA antibody.
前記 DNAプレートは、 ヌクレオソーム DNAがプレート上に直接固相化され てなることが、 ポリ一 L-リジン （PLL) 等の塩基性タンパク質を介して DN Aを付着させた従来プレートが有する、 P LLに対する抗体及び PL L— DNA 複合体が形成する未知の抗原構造に対する抗体反応による問題点を排除できる点 で好ましい。 プレートの材質は、 ポリスチレンを含むことが DNAを直接付着さ せやすい点で好ましく、 マイクロタイ夕一プレート I mmu 1 o n 2 HB、 I m mu l on4HB及び Immu l onHB (ダイネックステクノロジ一社製 Dynex Technologies, Chantilly, VA) が特に好ましい。 In the DNA plate, nucleosome DNA is directly immobilized on the plate. An unknown antigen structure formed by an antibody against PLL and a PLL-DNA complex of a conventional plate to which DNA is attached via a basic protein such as poly-L-lysine (PLL) This is preferable because problems due to an antibody reaction to the above can be eliminated. The material of the plate is preferably a material containing polystyrene because DNA can be directly attached to the plate.Micro-titer plates Immu1on2HB, Immulon4HB and ImmulonHB (Dynex Technology Co., Ltd. Technologies, Chantilly, VA) are particularly preferred.
このように、 本発明の DNAプレートは正常血清のバックグランドが減少する ため、 目的とする疾患血清の抗体価の信頼性が高くなり、 SLE等の病態判断の ための抗 DN A抗体反応の測定に極めて好適に用いることができる。  Thus, the DNA plate of the present invention reduces the background of normal serum, thereby increasing the reliability of the antibody titer of the target disease serum, and measuring the anti-DNA antibody reaction for determining the pathological condition such as SLE. Can be used very suitably.
(DNAプレート製造方法）  (DNA plate manufacturing method)
本発明の DN Aプレート製造方法は、 前記モノヌクレオソ一ムの製造方法であ つて、 試料がヒト由来の試料であるモノヌクレオソーム製造工程と、  The method for producing a DNA plate of the present invention is the method for producing a mononucleosome, wherein the sample is a human-derived sample;
モノヌクレオソームからヌクレオゾーム DN Aを単離精製するヌクレオソ一ム D NA単離精製工程と、 A nucleosomal DNA isolation and purification step for isolating and purifying the nucleosome DNA from the mononucleosome,
前記ヌクレオソーム DN Aをプレート上に固相化する固相化工程とを含むこと以 外は特に制限はない。 There is no particular limitation except that the method includes a step of immobilizing the nucleosome DNA on a plate.
前記固相化工程は、 PLL等の前処理なしに、 プレート上に、 直接ヌクレオソ ーム DN Aを付着させることが好ましい。  In the solid phase immobilization step, it is preferable that the nucleosome DNA is directly attached to the plate without a pretreatment such as PLL.
前記モノヌクレオソ一ムの固定化緩衝液としては、 ヌクレオソ一ム D N Aの形 態を変化させないものであれば、特に制限はなく、適宜選択することができるが、 PLL等の前処理なしに、 ヌクレオソーム DNAを付着させる観点から、 固定化 緩衝液は、 0. 11\以上1. 0M以下の NaC 1を含む、 トリス緩衝液又はホウ 酸一カセイソ一ダ緩衝液であることが好ましく、 0. 11\以上1. 0M以下の N aClを含むトリス緩衝液がより好ましい。 NaC 1の濃度は 0. 25 M以上で あることがコ一ティング効率、 及び製造された DN Aプレートの抗 DN A抗体反 応性の面から特に好ましい。  The buffer for immobilizing the mononucleosome is not particularly limited and may be appropriately selected as long as it does not change the form of the nucleosomal DNA. From the viewpoint of adhering, the immobilization buffer is preferably a Tris buffer or a borate monophosphate buffer containing 0.1 to 1.0 M of NaC 1, and 0.11 or more. Tris buffers containing 1.0 M or less NaCl are more preferred. It is particularly preferable that the concentration of NaC1 is 0.25 M or more from the viewpoints of coating efficiency and anti-DNA antibody reactivity of the prepared DNA plate.
前記固相化工程に用いられる固相としては、 ヌクレオソーム DN Aを固定化で きるものであれば特に制限はなく、 適宜選択することができるが、 前記、 ヌクレ ォソーム DNAを直接プレートに付着させる観点から、 ポリスチレン製のマイク 口夕イタ一プレートであることが好ましく、 Immul on2HB (ダイネック ステクノロジ一社製 Dynex Technologies, Chantilly, VA) が、 特に好ましい。 前記ヌクレオソ一ム DNAの前記固相化の条件としては、 固相の種類等に応じて 適宜選択することができ、 前記固相化の具体例としては、 前記ポリスチレン製マ イク口夕イタ一プレートを固相として使用する場合には、 ヌクレオソ一ム DNA を 0. 5〃 /1111の濃度で0. 25 Mの NaClを含むトリス緩衝液に溶解し た溶液で、 4°Cで一晩吸着させ、 過剰な抗原を一度吸引除去した後、 NaCl添 加トリス緩衝液洗浄する方法等が、 好適に挙げられる。 The solid phase used in the solid phase immobilization step is not particularly limited as long as it can immobilize the nucleosome DNA, and can be appropriately selected. From the viewpoint of directly attaching the chromosomal DNA to the plate, a polystyrene microphone plate is preferred, and Immulon2HB (Dynex Technologies, Chantilly, VA) is particularly preferred. The conditions for the immobilization of the nucleosomal DNA can be appropriately selected according to the type of the solid phase and the like. Specific examples of the immobilization include the polystyrene microphone opening plate When using as a solid phase, nucleosomal DNA is dissolved at a concentration of 0.5〃 / 1111 in Tris buffer containing 0.25 M NaCl and adsorbed overnight at 4 ° C. Preferably, a method of once removing the excess antigen by suction and washing with a Tris buffer solution containing NaCl is preferably used.
ブロッキングは、 前記ヌクレオソームの固定化の場合に準じて行うことができ る o  Blocking can be performed according to the case of the immobilization of the nucleosome.o
本発明の DNAプレート製造方法は、 プレート上に、 前記ヌクレオソ一ム DN A製造方法により製造されたヌクレオゾーム DN Aであってヒト由来のヌクレオ ソ一ム DNAを、 0. 1M以上1. 0M以下の NaC 1を含む、 トリス緩衝液及 びホウ酸—カセイソーダ緩衝液のいずれかに溶解させて添加することにより固相 化するものであってもよい。  The method for producing a DNA plate of the present invention comprises, on a plate, the nucleosomal DNA produced by the method for producing a nucleosomal DNA and the human-derived nucleosomal DNA having a concentration of 0.1 M or more and 1.0 M or less. The solid phase may be formed by dissolving and adding NaCl in either Tris buffer or boric acid-caustic soda buffer.
(抗 DN A抗体測定法）  (Anti-DNA antibody measurement method)
本発明の抗 DN A抗体測定法は、 前記 DN Aプレートを用いてなり、 被検試料を、 該 DN Aプレートの固相化されたヌクレオソーム DN Aと反応させ る反応工程と、  The method for measuring anti-DNA antibody of the present invention comprises using the above-mentioned DNA plate, and reacting a test sample with nucleosome DNA immobilized on the DNA plate;
前記ヌクレオゾーム DN Aに結合する特異的な抗体を測定する測定工程と、 を含むものであれば、 特に制限はなく、 前記測定工程は、 前記ヌクレオソームに 特異的な抗体の測定法に準じて適宜選択することができる。 実施例 A measurement step of measuring a specific antibody that binds to the nucleosome DNA, as long as the method includes: a measurement step for measuring a specific antibody that binds to the nucleosome DNA. can do. Example
以下、 本発明の実施例を説明するが、 本発明はこれらの実施例により何ら限定 されるものではない。 以下の実施例に示す材料、 使用量、 割合、 処理内容、 処理 手順等は、 本発明の効果を害しない限り適宜変更することができる。  Hereinafter, examples of the present invention will be described, but the present invention is not limited to these examples. The materials, amounts used, ratios, treatment details, treatment procedures, and the like shown in the following examples can be appropriately changed as long as the effects of the present invention are not impaired.
(実施例 1) 抗ニ本鎖 DN A抗体産生ハイプリ ドーマ培養細胞上清からのモノヌクレオソ一ム の製造 (Example 1) Production of mononucleosomes from supernatants of cultured hybridoma cells producing anti-double-stranded DNA antibodies
自己免疫疾患モデルとして知られている MR L/l p rマウス由来 B細胞ハイ プリ ドーマの産生する、 二本鎖 DNAに特異的な自己抗体 2 C 10 ( I gG 2 b に属する） を、 I gG抗体の分離 '精製法として既に確立されているプロテイン Aカラム法を用いて上記ハイプリ ドーマの培養上清から分離し、 分離した抗体を SDS—ボリァクリルアミ ドゲル（PAGE)電気泳動で、その成分を調べると、 ヒストンをはじめとする DNA結合タンパク質 （核タンパク質複合体） を含んで いることが知られている （金井ほか：ハイプリ ドーマモノクローナル抗体からの 自己抗原の分離とその応用、 厚生省特定疾患 混合性結合組織病調査研究班 平 成 5年度研究報告書 pp56— 59、 1994)。  An autoantibody 2C10 (belonging to IgG2b) specific to double-stranded DNA, produced by a B cell hybridoma derived from the MR L / lpr mouse known as an autoimmune disease model, Separation from the culture supernatant of the above hybridoma using the protein A column method already established as a purification method, and analyzing the separated antibodies by SDS-polyacrylamide gel (PAGE) electrophoresis. It is known to contain DNA binding proteins (nucleoprotein complexes) including histones (Kanai et al .: Separation of self-antigen from hybridoma antibodies and its application, Ministry of Health and Welfare-specific disease Mixed connective tissue disease Investigative Research Group FY2005 Research Report pp56-59, 1994).
前記 2 C 10—核タンパク質複合体が捕獲されているプロテイン Aカラムを、 1. 2 Mの塩化ナトリウムを含む 25 mMトリス緩衝液 （以下、 この 1. 2 M塩 化ナトリウム含有 25 mMトリス緩衝液を 「緩衝液 A」 という） を溶出液として 用いて解離溶出させ、 その溶出液を濃縮し、 DNAを抽出した。 得られた DNA 抽出物を、 2 %ァガロースゲル電気泳動で調べたところ、 モノヌクレオソーム D NA (約 150塩基対） 及びその重合体が検出された （図 1)。  The protein A column on which the 2C10-nucleoprotein complex was captured was washed with a 25 mM Tris buffer containing 1.2 M sodium chloride (hereinafter, a 25 mM Tris buffer containing 1.2 M sodium chloride). Was called "buffer A") as the eluent, and the eluate was concentrated to extract DNA. When the obtained DNA extract was examined by 2% agarose gel electrophoresis, mononucleosome DNA (about 150 base pairs) and its polymer were detected (FIG. 1).
一方、 タンパク質を SDS— PAGH ( 15%) で調べたところ、 コアヒスト ン、 即ち、 ヒストン H 3、 H2b、 H 2 a及び H 4のみが検出された。 これらの 結果から、 この溶出液中には、 モノヌクレオソーム及びその種々の重合度の多量 体が存在することが明らかになった。  On the other hand, when the protein was examined by SDS-PAGH (15%), only core histones, that is, histones H3, H2b, H2a and H4 were detected. From these results, it was revealed that mononucleosomes and multimers of various degrees of polymerization existed in the eluate.
上記事実に基づき、 スケールを上げてモノヌクレオソ一ムの回収を試みた。 2 Based on the above facts, we tried to recover mononucleosomes on a larger scale. Two
C 10ハイプリ ドーマを無血清培地 （培地は、 5質量％ゥシ胎児血清添加 DME M、 RPMI通常培地でもよい） で大量培養 （約 1リットル） し、 培養上清を分 子量 30, 000カッ トオフのダイアフロ一膜 PM30 (ミリポア社製） を用い た限外濾過法により 20倍に濃縮した。 この濃縮液を上記ヒッ トラップ （Hi t rap)プロテイン Aカラム （5ml) (アマシャムフアルマシアバイオテック社 製） に注いで核タンパク質複合体を捕獲し、 14 OmM塩化ナトリウムを含有す る 25mMトリス緩衝液 （以下、 この 14 OmM塩化ナトリウム含有 25 mMト リス緩衝液を 「緩衝液 C」 という） （pH 7. 4 ) で洗浄し、 非特異的結合タンパ ク質を完全に除去した。 非特異的結合タンパク質の除去は、 2 60 nmでの吸光 度が 0. 00となったときに完了とした。 次に、 緩衝液 Aでヌクレオソームを溶 出させた。 この溶出液には、 モノヌクレオソ一ム、 及びモノヌクレオソ一ムが 2 個以上連なつたオリゴ乃至ポリヌクレオゾームが含まれていた。 C10 hybridomas are cultured in large amounts (about 1 liter) in a serum-free medium (the medium may be DMEM or RPMI normal medium supplemented with 5% by mass fetal serum), and the culture supernatant is subjected to a molecular weight of 30,000 cells. The solution was concentrated 20-fold by ultrafiltration using a Diaflo membrane PM30 (manufactured by Millipore) of Tooff. The concentrated solution is poured into the above-mentioned Hittrap Protein A column (5 ml) (manufactured by Amersham Pharmacia Biotech) to capture the nucleoprotein complex, and then to a 25 mM Tris buffer containing 14 OmM sodium chloride. Solution (hereinafter referred to as 25 mM toluene containing 14 OmM sodium chloride) The squirrel buffer was washed with “buffer C” (pH 7.4) to completely remove non-specifically bound proteins. Removal of non-specifically bound proteins was complete when the absorbance at 260 nm was 0.00. Next, nucleosomes were eluted with buffer A. This eluate contained a mononucleosome and an oligo- or polynucleosome in which two or more mononucleosomes were linked.
次に、 モノヌクレオソームのみを単離するため、 分子量 10， 000〜1 , 0 00 , 000までの分子を分離できるスーパ一デヅクス 2 00 (Sup e r d e x 2 00) カラム （アマシャムフアルマシアバイオテック社製） を用いた高速液 体クロマトグラフィー （HPL C) を導入した。 展開溶媒としては、 2 5 OmM 塩化ナトリウム及び 0. 04質量％アジ化ナトリウムを含む 2 5mMトリス緩衝 液 （以下、 この 2 5 OmM塩化ナトリウム及び 0. 04質量％アジ化ナトリウム を含む 2 5mMトリス緩衝液を 「緩衝液 B」 という） （pH7. 4) を使用した。 溶出画分の吸光度のプロファイルを図 2に示す。 ポリヌクレオゾーム画分の大 きなピークに遅れてモノヌクレオソーム画分のピークが分子量 20万から 2 5万 の位置に検出された。  Next, in order to isolate only mononucleosomes, a Superdex 2000 column (manufactured by Amersham Pharmacia Biotech) capable of separating molecules having a molecular weight of 10,000 to 1,000,000 is used. ) Was introduced for high performance liquid chromatography (HPL C). As a developing solvent, a 25 mM Tris buffer containing 25 OmM sodium chloride and 0.04% by mass of sodium azide (hereinafter referred to as a 25 mM Tris buffer containing 25 OmM sodium chloride and 0.04% by mass of sodium azide) The solution was called “buffer B” (pH 7.4). Figure 2 shows the absorbance profile of the eluted fraction. After the large peak of the polynucleosome fraction, the peak of the mononucleosome fraction was detected at a molecular weight of 200,000 to 250,000.
モノヌクレオゾームの収率を上げるために、 ポリヌクレオソームをモノヌクレ ォソ一ム単位に消化することを行った。 具体的には、 得られた溶出液に C a2 +を 2. 5 mM、 マイクロコッカル ·ヌクレア一ゼを 0. 5ユニット/ mlになるよ うに添加して 200 / 1とし、 37 °Cで 45分間保温した。 保温終了後、 直ちに HGT Aを 5 mMになるように添加して反応を停止した。 不溶物をマイクロヒュ —ジ （ 1 5 k rpm) で除去した。 To increase the yield of mononucleosomes, polynucleosomes were digested into mononucleosome units. Specifically, C a 2 + a 2. 5 mM, was added by Uni comprising a micro Cocker Le Nuclear Ichize to 0.5 units / ml and 200/1 to eluate obtained, 37 ° C For 45 minutes. Immediately after the completion of the incubation, the reaction was stopped by adding HGT A to a concentration of 5 mM. Insolubles were removed with a microfuge (15 k rpm).
得られた上清を、 緩衝液 Bで平衡化したスーパーデックス 200カラムを用い た HPL C (3 2 1ポンプ、 ギルソン （Gilson) 社製を使用） にかけた。 溶出画 分の吸光度のプロファイルを図 3に示す。 ゲルろ過カラムにかける前にヌクレア ーゼ処理することにより、モノヌクレオゾームの画分を増加させることができた。 この画分を再度クロマトグラフィーにかけた結果を図 4に示す。 ほぼ単一なピー クとして精製することができた。 一方、 この方法を用いることにより同時にモノ ヌクレオソ一ムの間をつなぐリンカー DN Aも回収することができた （図 3)。 モノヌクレオソームのビーク画分をウルトラフリー （ULTRAFREE) (5 O kカヅ トオフ）（ミリポア（Millipore)社製）で濃縮した。濃縮液のタンパク質濃度を、 B C Aプロテインァヅセィキッ ト （ピース （PIERCE) 社製） で測定したところ、 3 0 0〜5 0 0 g/m lであった。 The obtained supernatant was applied to HPLC (321 pump, manufactured by Gilson) using a Superdex 200 column equilibrated with buffer B. Figure 3 shows the absorbance profile of the eluted fraction. By performing nuclease treatment before applying to the gel filtration column, the fraction of mononucleosomes could be increased. FIG. 4 shows the result of re-chromatography of this fraction. It could be purified as almost a single peak. On the other hand, by using this method, a linker DNA linking between mononucleosomes could be simultaneously recovered (Fig. 3). Ultra-free beak fraction of mononucleosome (ULTRAFREE) (5 Ok (Millipore). The protein concentration of the concentrate was measured using a BCA protein assay kit (manufactured by Peerce) and found to be 300 to 500 g / ml.
なお、 濃縮前の細胞培養液以外は総て、 タンパク質分解を防止するためにプロ デアーゼ阻害剤カクテル、 コンプリート （complete) T M , E D T Aフリ一 （ベ —リンガーマンハイム （Boehringer Mannheim) 社製） を処方通り添加し、 更にセ リンプロテア一ゼ阻害剤 A E B S F ( 4 - ( 2—アミノエチル） 一ベンゼンスル ホニルフルオラィ ド） （シグマ社製） を 1 0 0 / Mになるように添加した。 以下の 実施例でも同様である。  In addition, except for the cell culture solution before the concentration, a protease inhibitor cocktail, complete ™ and EDTA free (from Boehringer Mannheim) were used as prescribed to prevent protein degradation. Then, a serine protease inhibitor AEBSF (4- (2-aminoethyl) -benzenesulfonylfluoride) (manufactured by Sigma) was added at 100 / M. The same applies to the following embodiments.
(実施例 2 )  (Example 2)
一般培養細胞上清からのモノヌクレオゾームの製造 Production of mononucleosome from general culture cell supernatant
本発明のモノヌクレオソームの製造方法は、 二本鎖 D N A及び/又はヌクレオ ゾームに特異的な抗体を生産しない一般の樹立細胞又はヒトを含む動物組織から 直接分離した細胞 （一次培養細胞） にも適用できる。  The method for producing a mononucleosome of the present invention is also applicable to general established cells that do not produce double-stranded DNA and / or antibodies specific to nucleosomes or cells directly isolated from animal tissues including humans (primary cultured cells). it can.
自己免疫疾患モデルである M R L / l p rマウス由来株価細胞 K M L i - 7 (Kanai ら、 Intl. Archs. Allergy Appl . I誦 unol.， 81 : 92— 94, 1986) は、 血清添加培地及び無血清培地の如何に係わらず、 無刺激でその培養上清中にヌク レオゾームを分泌することが知られている。 したがって、 この培養上清がそのま まヌクレオソ一ムの供給源として使用することができる。  KML i-7 (Kanai et al., Intl. Archs. Allergy Appl. I recitation unol., 81: 92-94, 1986), a cell line derived from MRL / lpr mouse, which is an autoimmune disease model Regardless of the method, it is known that nucleosomes are secreted into the culture supernatant without stimulation. Therefore, this culture supernatant can be used as a source of nucleosome as it is.
また、 樹立されている市販の各種癌細胞株又は一次培養細胞でもアポトーシス 誘導剤の添加培養によって培養上清中にヌクレオソ一ムを放出させることができ、 上記と同様に、培養上清をヌクレオゾームの供給源として使用することができる。 この培養上清を適切な濃度 （ 1 0〜2 0倍） まで濃縮した後、 マイクロコッカ ル .ヌクレア一ゼ （M N ) で処理 （消化） し、 モノクローナル抗体 2 C 1 0が固 定化されたプロティン Aカラムに流す。  Nucleosomes can also be released into the culture supernatant by adding an apoptosis-inducing agent to various commercially available cancer cell lines or primary culture cells that have been established. Can be used as a source. After concentrating the culture supernatant to an appropriate concentration (10- to 20-fold), it was treated (digested) with micrococcal nuclease (MN), and the monoclonal antibody 2C10 was immobilized. Pour over a Protein A column.
前記プロティン Aカラムを、 緩衝液 Cで洗浄して非特異的結合タンパク質を除 去した後、 緩衝液 Aで溶出する。 溶出液を直ちに緩衝液 Bに対して透析した後、 濃縮を行う。 この濃縮は、 大容量であれば前記ダイアフロ一膜 P M 3 0を用いた 限外濾過で行うことができ、 少量であれば前記ウルトラフリーを用いて行うこと ができる。 The protein A column is washed with buffer C to remove non-specifically bound proteins, and then eluted with buffer A. Immediately dialyze the eluate against buffer B and concentrate. This concentration can be carried out by ultrafiltration using the Diaflo membrane PM30 if the volume is large, and it can be carried out using the ultrafree if the volume is small. Can be.
以下、 実施例 1と同様にして、 上記濃縮液をスーパーデックス 200カラムを 用いた HPLCにかけ、 モノヌクレオソ一ムを回収した。 HPLCによる分画パ 夕一ンは図 3及び図 4と同様であった。  Hereinafter, in the same manner as in Example 1, the above concentrated solution was subjected to HPLC using a Superdex 200 column to recover mononucleosomes. The fractionation pattern by HPLC was the same as in FIGS. 3 and 4.
なお、 モノクローナル抗体 2 C 10の固定化は、 次のようにして行った。  The immobilization of the monoclonal antibody 2C10 was performed as follows.
段落番号 0060に記したトリス緩衝液でプロティン Aカラムを平衡化しておき、 それにトリス緩衝液で調製した 2 C 10 (0. 5〜1. Omg/ml) をチヤ一 ジした。 カラム中の流速は 5 ml/分で、 サイクルは 2回である （2回目で吸着 は完璧となる）。 吸着 ·固定化の後、 再度カラムは、 トリス緩衝液で平衡化した。 (実施例 3) The protein A column was equilibrated with the Tris buffer described in paragraph No. 0060, and 2C 10 (0.5 to 1.0 mg / ml) prepared with the Tris buffer was changed. The flow rate through the column is 5 ml / min and the cycle is two times (the second time the adsorption is perfect). After adsorption and immobilization, the column was again equilibrated with Tris buffer. (Example 3)
培養細胞又は末梢血単核球からのヌクレオソームの製造 Production of nucleosomes from cultured cells or peripheral blood mononuclear cells
低張緩衝液 （ 5 OmMトリス、 50mM塩化カリウム、 0. 5 mM塩化マグネ シゥム、 0. 15mM2— ME、 0. 25Mショ糖、 0. 2 mM AEBSF、 0. 04質量％アジ化ナトリウム （NaN3)、 pH 7. 4) 10ml当たり 2 x 108個の細胞を懸濁し、 ポッター 'テフロン（R)製ホモジナイザー（20スト口 —ク） で手動でホモジナイズする。 これを 500 gで 7分間遠心分離し、 細胞核 を沈降させた。 細胞核を上記緩衝液にて一度洗浄して不純物を除去した。 Hypotonic buffer (5 Omm Tris, 50 mM potassium chloride, 0. 5 mM chloride magnetic Shiumu, 0. 15mM2- ME, 0. 25M sucrose, 0. 2 mM AEBSF, 0. 04 wt% sodium azide (NaN 3 ), PH 7.4) Suspend 2 x 10 8 cells per 10 ml and homogenize them manually using a Potter 'Teflon ( R) homogenizer (20 strokes). This was centrifuged at 500 g for 7 minutes to sediment the cell nuclei. Cell nuclei were washed once with the above buffer to remove impurities.
次いで、 洗浄した核を、 ショ糖を含まない以外は上記緩衝液と同じ組成の緩衝 液 0. 5mlに懸濁し、 マイクロコッカル ·ヌクレア一ゼ （MN) 1. 25 U/ ml及び 5mMCa2 +を添加し、 実施例 1と同様に酵素消化し、 EGTAが 5m Mになるように添加して反応を停止させた。 13kで 5分間還心分離し、 得られ た沈渣に緩衝液 Bを添加し、 可溶性画分を 13 kの遠心分離で回収した。 前記可 溶性画分を直ちにモノクローナル抗体 2 C 10結合プロティン Aカラムにかけ、 結合したヌクレオソ一ムのみを上記溶出液（緩衝液 A)にて選択的に溶出させた。 得られた溶出液を濃縮後、 実施例 2と同様のスーパーデックス 200カラムを用 いた HPLCにかけ、 モノヌクレオソームを単離した。 HPLCによるモノヌク レオゾームの単離の結果を図 5に示す。 Next, the washed nuclei were suspended in 0.5 ml of a buffer having the same composition as that described above except that sucrose was not contained, and 1.25 U / ml of micrococcal nuclease (MN) and 5 mM Ca2 + Was added, and the enzyme was digested in the same manner as in Example 1, and the reaction was stopped by adding EGTA to 5 mM. Centrifugation was performed at 13k for 5 minutes. Buffer B was added to the resulting precipitate, and the soluble fraction was collected by 13k centrifugation. The soluble fraction was immediately applied to a monoclonal antibody 2C10-binding protein A column, and only the bound nucleosome was selectively eluted with the above eluate (buffer A). After concentrating the obtained eluate, the eluate was subjected to HPLC using the same Superdex 200 column as in Example 2 to isolate mononucleosomes. Figure 5 shows the results of the isolation of mononucleosomes by HPLC.
更に、 ここで得られたモノヌクレオソ一ム画分を再度、 スーパ一デヅクス 20 0カラムにかけると図 6に示すほぼ単一なビークとして精製された。 一方、 モノヌクレオソームは、 抗体 2 C 10結合カラムを用いずに、 直接ス一 パーデックス 200カラムにかけ、 分子量 20万から 25万の画分を分画するこ とによっても精製することができた。 2 C 10結合カラムを用いずに精製した場 合は、 2 C 10結合カラムを用いて精製した場合に比して若干純度が劣るが、 再 度 HPLCにかけると 2 C 10カラムの場合とほぼ同様の効果が得られた。 (実施例 4) Further, when the obtained mononucleosome fraction was applied again to a Superdex 200 column, it was purified as a substantially single beak shown in FIG. On the other hand, mononucleosomes could also be purified by directly applying them to a Superdex 200 column without using an antibody 2C10 binding column and fractionating fractions having a molecular weight of 200,000 to 250,000. Purification without using a 2C10 binding column is slightly inferior to purification using a 2C10 binding column. Similar effects were obtained. (Example 4)
本実施例 4では、 上記実施例 2で得られたアポトーシスを起こした培養細胞由 来のモノヌクレオソ一ムと、 実施例 3で得られた正常な培養細胞由来のモノヌク レオソ一ムとの比較解析を行った。  In this Example 4, a comparative analysis was performed between the mononucleosome derived from the cultured cells having undergone apoptosis obtained in Example 2 above and the mononucleosome derived from normal cultured cells obtained in Example 3. went.
両ヌクレオソ一ムを 0. 5 %ァガロース電気泳動により分画した。 図 7に示す ように、 正常な細胞由来のモノヌクレオソ一ムに比較してアポトーシスを起こし た細胞由来のヌクレオソ一ムは、 ブロードなバンドとして泳動された。  Both nucleosomes were fractionated by 0.5% agarose electrophoresis. As shown in FIG. 7, nucleosomes from cells that underwent apoptosis compared to mononucleosomes from normal cells migrated as broad bands.
更に、 これらモノヌクレオソームを詳細に分析するために、 モノヌクレオソ一 ムを構成するコアヒストン、 DNAをそれそれ解析した。  Furthermore, in order to analyze these mononucleosomes in detail, each of the core histones and DNAs constituting the mononucleosome was analyzed.
DN Aについては、 ァガロースゲル電気泳動によりその泳動パターン、 位置な どを比較したところ、 大きな差異は観られなかった （図 8)。  No significant differences were observed for DNA, when its electrophoresis pattern and position were compared by agarose gel electrophoresis (Figure 8).
一方、 コアヒストンについては 15%SDS— PAGEにより画ヒストン成分 を分画して解析した。 結果を図 9に示す。 正常な細胞由来のヒストンは、 図 10 に示す H3、 H2b、 H 2 a及び H 4の通常のパターンと同様の分画パターンが 示された。 しかし、 一方のアポトーシスを起こした細胞由来のコアヒストンは、 分画パターンに異常が観られた。 即ち、 H 2 bに相当するバンドが減少し、 H4 のバンドを挟むように 2本の新たなバンドが現れた。  On the other hand, core histones were analyzed by fractionating histone components by 15% SDS-PAGE. Fig. 9 shows the results. The histones derived from normal cells exhibited the same fractionation pattern as the normal pattern of H3, H2b, H2a and H4 shown in FIG. However, on the other hand, abnormalities were observed in the fractionation pattern of core histones derived from apoptotic cells. That is, the band corresponding to H 2 b decreased, and two new bands appeared sandwiching the H4 band.
このようにモノヌクレオソ一ム単位の製造が簡便になったことから、 従来のそ の単離が困難であったヒストンの分離も簡便となった。 更には上記のようなヒス トンに基づいた生物現象の解析や疾患の診断や解明をも簡便に行うことが可能と なる。  Since the production of mononucleosomal units has been simplified in this way, the separation of histones, which was conventionally difficult to isolate, has also been simplified. Furthermore, analysis of biological phenomena and diagnosis and clarification of diseases based on the above-mentioned histos can be easily performed.
(実施例 5)  (Example 5)
ヌクレオソームの ELI S Aプレートへの固相化 Immobilization of nucleosomes on ELI S A plate
自己抗原に対する免疫応答を調べる場合の E L I S Aプレートへの抗原の固相 化条件を検討した。 まず、 抗原付着用溶媒の検討を行った。 経験上、 抗原のプレ 一卜への固相化には、 5 OmM炭酸緩衝液 （pH9. 6) が使用される場合が多 いことから、 該炭酸緩衝液が、 本プレートの抗原付着用溶媒として適するかを、 該緩衝液によるヌクレオゾームの形態的変化の有無により調べた。 具体的には、 ヌクレオゾームの溶媒である 0.5MNaC 1を該 50 mM炭酸緩衝液に置換し、 当該緩衝液で平衡化したスーパーデックス 200カラムを用いた HPLCにより 調べた。 その結果、 溶液の置換に伴う溶出位置及びパターンに変化は観られなか つた。 これにより、 当該緩衝液を、 抗原付着用溶媒として用いることができるこ とが判った。 Solid phase of antigen on ELISA plate when examining immune response to self antigen Conditions were examined. First, the solvent for antigen attachment was examined. Experience has shown that 5 OmM carbonate buffer (pH 9.6) is often used to immobilize antigen on a plate. Was examined by the presence or absence of morphological changes in nucleosomes caused by the buffer. Specifically, 0.5 M NaCl, which is a nucleosome solvent, was replaced with the 50 mM carbonate buffer, and the mixture was examined by HPLC using a Superdex 200 column equilibrated with the buffer. As a result, no change was observed in the elution position and pattern accompanying the replacement of the solution. This proved that the buffer could be used as a solvent for antigen attachment.
次に、 抗原の支持体としてポリスチレン製のマイクロ夕イタ一プレート Imm u 1 o n 2 HB (ダイネヅクステクノロジ一社製 Dynex Technologies, Chant illy, VA) を用い、 ヌクレオソームを、 該支持体に、 従来から d s DNA抗原のプレー トへの吸着に用いられているポリ— L—リジン（ P L L ) (シグマ社製 Sigma, St. louis, MO) (l g/ml蒸留水） を介して、 2 g/m 1の濃度で 4 °Cでー晚吸 着させた。 吸着後、 過剰な抗原を一度吸引除去した後、 NaC l添加トリス緩衝 液 （25mM ト リス、 140mM NaC l、 0. 04質量％ NaN3、 p H 7. 4) (TBS)で、 4回洗浄した。 1プレート当たり約 10mlの TB Sを 使用した。 洗浄後、 各ゥエル当たり 100〃1のブロッキング溶液 （2質量％ス キムミルク （ディフコ社製 Difco， Detroit, MI) 含有 TBS) を加えて、 一時 間反応させ、 抗原の付着していない部位を遮蔽した。 最後に先の抗原吸着後の洗 浄と同様に TB Sで 4回洗浄することにより、 抗原付着プレートを作製した。 使 用に供するまで、 各ゥエルに TB Sを 100〃 1ずつ加え、 プレートシールで密 閉し、 4 °Cで保存した。 Next, using a polystyrene microplate Immu 1 on 2 HB (Dynex Technologies, Chantilly, VA, Dynex Technologies, Inc.) as a support for the antigen, nucleosomes were added to the support, 2 g / ml via poly-L-lysine (PLL) (Sigma, St. louis, MO, manufactured by Sigma) (lg / ml distilled water) conventionally used for the adsorption of ds DNA antigen to the plate. It was adsorbed at a concentration of ml at 4 ° C. After adsorption, the excess antigen is once removed by suction, and then washed four times with NaCl-containing Tris buffer (25 mM Tris, 140 mM NaCl, 0.04% by mass NaN 3 , pH 7.4) (TBS). did. About 10 ml of TBS was used per plate. After washing, 100〃1 blocking solution (TBS containing 2% by mass skim milk (Difco, Detroit, MI) containing 2% by weight of Difco, Inc.) was added to each well, and the mixture was allowed to react for one hour to shield the site where no antigen was attached. . Finally, an antigen-attached plate was prepared by washing four times with TBS in the same manner as the washing after the antigen adsorption. Until use, add 100 μl of TBS to each well, seal tightly with a plate seal, and store at 4 ° C.
(実施例 6)  (Example 6)
患者血清中の抗ヌクレオゾーム抗体測定 Anti-nucleosomal antibody measurement in patient serum
加療中の 12例の SLE患者及び 26例の健常者の血清を対象として抗ヌクレ ォソーム抗体の測定を行つた。  The anti-nucleosome antibody was measured in the sera of 12 SLE patients and 26 healthy subjects during treatment.
血清は、 12人の SLE患者及び 26人の健常者からそれそれ血清を採取した。 反応液 [1質量％牛血清アルブミン （BSA)、 0. 4質量％スキムミルク、 1 0質量％ブロックエース、 ImM EDTAを含む TBS] にて血清を 100倍 に稀釈し、 マイクロタイ夕一プレートの各ゥエルに 50〃 1ずつ加えた。 前記 B SAは、 アルブミン フラクション V (Albumin, Fraktion V, ベ一リンガーマ ンハイム社製 Boehringer Mannheim, Germany)、 前記ブロックエースは、 大日本 製薬株式会社製、 前記 EDTAは EDTAニナトリウム塩 （同仁化学製） を使用 した。 Serum was collected from 12 SLE patients and 26 healthy subjects. Reaction solution [1% by mass bovine serum albumin (BSA), 0.4% by mass skim milk, 1 The serum was diluted 100-fold with TBS containing 0% by mass Block Ace and ImM EDTA, and added to each well of a microtiter plate at 50: 1. The BSA is albumin fraction V (Albumin, Fraktion V, Boehringer Mannheim, Germany), the block ace is Dainippon Pharmaceutical Co., Ltd., and the EDTA is EDTA disodium salt (Dojindo). It was used.
前記マイクロ夕イタ一プレートを、 水平振盪器上で軽く振盪しながら、 室温で 30分反応させた。 反応後、 直ちに反応液を吸引除去し、 今度は 0. 05質量％ Twe en20を含有した TBSで、 抗原付着後のプレートの洗浄と同様の手技 で、 プレートを 4回洗浄した。  The microplate was allowed to react at room temperature for 30 minutes with gentle shaking on a horizontal shaker. Immediately after the reaction, the reaction solution was removed by suction, and the plate was washed four times with TBS containing 0.05% by mass of Tween 20 in the same manner as the plate after the antigen was attached.
前記洗浄後、 直ちに、 アルカリホスファタ一ゼ （AP) コンジュゲート抗ヒト Immediately after the washing, alkaline phosphatase (AP) conjugated anti-human
1 gG抗体 （ャギ） （ジムド社製 Zymed, San Francisco, CA) を前記反応液にて1 gG antibody (goat) (Zymed, San Francisco, CA, manufactured by Jimdo) in the above reaction mixture
2000倍稀釈し、 各ゥエルに加え、 前記血清の場合と同様に室温で 30分振盪 反応させた。 前記血清の場合と同様に洗浄した後、 2. 5mM Mg2+添加炭酸 緩衝液 （50mM, pH 9. 8 ) で 1 mg/m 1になるように調整した APの基 質である、 p—二トロフエニル ホスフェイ ト （PNP) (シグマ社製 Sigma, St. Louis, M0) 試薬を 100〃 1ずつ各ゥエルに添加し、 前記血清の場合と同様に、 室温で 30分振盪反応させた。 The mixture was diluted 2000-fold, added to each well, and reacted with shaking at room temperature for 30 minutes as in the case of the serum. After washing in the same manner as in the case of the serum, the substrate of AP adjusted to 1 mg / m1 with 2.5 mM Mg 2+ -added carbonate buffer (50 mM, pH 9.8) was added. 100 μl of ditrophenyl phosphate (PNP) (Sigma, St. Louis, M0) reagent was added to each well, and the mixture was shaken at room temperature for 30 minutes in the same manner as in the case of the serum.
反応直後に発色の度合 （吸光度） を、 オートリーダーを用いて、 吸光波長 40 5 nmで測定した。 なお、 対照又は盲検は、 前記一連の反応系で、 血清を加えな かった時に得られる吸光度とし、 これを差し引いた価を実測地とした。 抗体価は 吸光度で表した。  Immediately after the reaction, the degree of color development (absorbance) was measured at an absorption wavelength of 405 nm using an autoreader. The control or blind test was the absorbance obtained when serum was not added in the above series of reaction systems, and the value obtained by subtracting the absorbance was used as the actual measurement location. The antibody titer was represented by absorbance.
測定結果を、 図 11に表した。 加療中の SLE患者から採取した 12例の血清 うち、 2例の血清が、 ヌクレオゾームに対する極めて高い抗体価を示し、 陽性と 判定された。 健常者から採取した 26例の血清は、 いずれも低い抗体価しか示さ ず、 これらの平均値 +3倍の標準偏差（m+ 3 SD)で定義したバックグラウンド の閾値は二例の陽性血清の抗体価よりも極めて低かった。 なお、 2例の陽性と判 定された患者以外の S LE患者からの血清が、 高い力価を示さないのは、 加療中 処方されている薬剤の影響により既に治癒に近い状態である等の理由により、 既 に血清中にヌクレオゾームに特異的な抗体が多く存在しないことを示すものであ る。 The measurement results are shown in FIG. Out of 12 sera collected from patients undergoing treatment with SLE, 2 sera showed extremely high nucleosome antibody titers and were determined to be positive. All 26 sera collected from healthy subjects showed low antibody titers, and the background threshold defined by the mean + 3 times the standard deviation (m + 3 SD) was the antibody threshold of the two positive sera. It was much lower than the value. The sera from SLE patients other than the two positive patients did not show high titers due to the effect of the prescribed drug during treatment. For some reason, This shows that there are not many nucleosome-specific antibodies in the serum.
以上の結果から、 本発明の測定方法により、 ヌクレオソームに特異的な抗体を 極めて精度良く測定することができ、 本発明のヌクレオゾームに特異的な抗体の 測定方法が S LEの診断に極めて有効であることが実証された。  From the above results, the nucleosome-specific antibody can be measured with extremely high accuracy by the measurement method of the present invention, and the nucleosome-specific antibody measurement method of the present invention is extremely effective for SLE diagnosis. This has been proven.
(実施例 7)  (Example 7)
抗体サブクラス別の測定法 Measurement method for each antibody subclass
I gG抗体には、 I gG l、 I gG 2、 I gG 3及び I gG 4の四種類のサブ クラスがある。 患者の有する、 ヌクレオソ一ムに特異的な抗体のサブクラスの片 寄りが、 疾患病態を特徴付けることがある。 したがって、 サブクラス別抗体の測 定が疾患病態を把握する上で重要である。  There are four subclasses of IgG antibodies, IgG1, IgG2, IgG3 and IgG4. The bias of the patient's subclass of nucleosomal-specific antibodies may characterize the disease state. Therefore, measurement of antibodies by subclass is important for understanding disease pathology.
実施例 6の図 1 1でヌクレオソーム抗体の最高値を呈した症例について、 サブ クラス別抗体の測定を行った。  For the case showing the highest value of the nucleosome antibody in FIG. 11 of Example 6, the antibody by subclass was measured.
前記サブクラス別抗体の測定には、ピオチン標識した抗ヒトサブクラス抗体（マ ウス） （Zymed, San Francisco, CA) を実施例 6のアルカリホスファタ一ゼ （AP) コンジュゲート抗ヒト I gG抗体 （ャギ） の代りに使用した。 反応様式 '反応時 間は上記と同様に行った。 次に、 アルカリホスファタ一ゼ標識ストレブトァビジ ン （Zymed, San Francisco, CA) を反応させ、 最後に実施例 6と同様に基質の P NPを加えて、 発色させ、 オートリーダ一で吸光度を測定した。  For the measurement of the antibodies by subclass, a biotin-labeled anti-human subclass antibody (mouse) (Zymed, San Francisco, Calif.) Was used in conjunction with the alkaline phosphatase (AP) conjugated anti-human IgG antibody of Example 6. G: Used in place of Reaction mode 'The reaction time was the same as above. Next, alkaline phosphatase-labeled streptavidin (Zymed, San Francisco, CA) was reacted, and finally, the substrate PNP was added as in Example 6 to cause color development, and the absorbance was measured with an auto-reader. .
測定結果を図 12に表した。  The measurement results are shown in FIG.
(実施例 8)  (Example 8)
ヌクレオゾーム DN Aの製造 Manufacture of nucleosome DNA
ヒト前骨髄性白血病細胞培養株 HL— 60クロマチンから、 実施例 1と同様に モノヌクレオソ一ムを製造し、 さらに、 ヌクレオソーム DNAを抽出した。 ヌク レオソ一ム D N Aの抽出は、公知の方法により行った（Kanai， Yetal: Induction and natural occurrence of serum nucleosomal DNA in autoimmune MRL/lpr/lpr mice: its relation to apoptosis in the thymus . Immunol Lett. 46:207-214, 1995)。 具体的には、 一定量の精製ヌクレオソームを、 1%SDS及び 0. 5mg /ml Proteinase Kを含むトリス— E D T A緩衝液に懸濁し、 室温で 60分間 処理した。次に、 6MのNaI (ヨウ化ナトリウム）を含む変性剤を 3倍量加え、 60°Cで 15分間加熱した。 この操作で可溶化した DNAを 50%イソプロピル アルコールで沈殿させた。 沈殿した D N Aを上記トリス— E D T A緩衝液に溶解 し、 少量の RNa s eを添加し、 混在する可能性のある RN Aを分解した。 Mononucleosomes were produced from human promyelocytic leukemia cell culture strain HL-60 chromatin in the same manner as in Example 1, and nucleosomal DNA was further extracted. Nucleosome DNA was extracted by a known method (Kanai, Yetal: Induction and natural occurrence of serum nucleosomal DNA in autoimmune MRL / lpr / lpr mice: its relation to apoptosis in the thymus. Immunol Lett. 46: 207-214, 1995). Specifically, a fixed amount of the purified nucleosome was suspended in a Tris-EDTA buffer containing 1% SDS and 0.5 mg / ml Proteinase K, and incubated at room temperature for 60 minutes. Processed. Next, a three-fold amount of a denaturant containing 6 M NaI (sodium iodide) was added, and the mixture was heated at 60 ° C. for 15 minutes. The DNA solubilized by this procedure was precipitated with 50% isopropyl alcohol. The precipitated DNA was dissolved in the above Tris-EDTA buffer, and a small amount of RNase was added to decompose RNA that might be present.
このヌクレオソーム DNAのァガロースゲル電気泳動パターンを図 13に示し た。 150 b p付近に単一のパンドが見られ、 モノヌクレオソ一ムの DN Aが抽 出されたことがわかる。  FIG. 13 shows the agarose gel electrophoresis pattern of the nucleosome DNA. A single band was observed around 150 bp, indicating that the mononucleosomal DNA was extracted.
(実施例 9)  (Example 9)
ヌクレオゾーム DNAの EL I SAプレー卜への固相化 Immobilization of nucleosome DNA on ELISA plate
実施例 8で得られたヌクレオソーム DNA、 その母体である HL— 60細胞の ゲノム DNA、 及び、 従来使用されてきた仔牛胸腺 DNAを EL I S Aプレート に固相化した。 HL— 60細胞のゲノム DNAは、 前記細胞のクロマチンから、 前記ヌクレオゾームからの DN Aの抽出法に準じて抽出した。 ゲノム DN Aの大 きさは、 ヌクレオソーム DN Aがおよそ 150 b pであるのに対して、 およそ 2 0 kb pであった。  The nucleosome DNA obtained in Example 8, its mother genomic DNA of HL-60 cells, and calf thymus DNA conventionally used were immobilized on an ELISA plate. Genomic DNA of HL-60 cells was extracted from the chromatin of the cells according to the method for extracting DNA from the nucleosomes. The size of the genomic DNA was approximately 20 kbp, whereas the nucleosome DNA was approximately 150 bp.
次に、 抗原の支持体としてポリスチレン製のマイクロタイ夕一プレート Imm ul on2HB (ダイネックステクノロジ一社製 Dynex Technologies, Chantilly, VA) を用い、 前記 DNAを、 該支持体に、 0. 5〃 /1111の濃度で0. 25M の Na C 1を含むトリス緩衝液 （pH 7. 4) に溶解し、 それを 25 ng/we 11で添加し、 4°Cで一晩吸着させた。 吸着後、 過剰な抗原を一度吸引除去した 後、 NaC 1添加トリス緩衝液（25mM トリス、 140mM NaCl、 0. 04質量％ NaN3、 pH7. 4) (TBS) で、 4回洗浄した。 1プレート当 たり約 10mlの TB Sを使用した。 洗浄後、 各ゥエル当たり 100 /1のプロ ッキング溶液 （2質量％スキムミルク （ディフコ社製 Difco， Detroit, MI)含 有 TBS) を加えて、 一時間反応させ、 抗原の付着していない部位を遮蔽した。 最後に先の抗原吸着後の洗浄と同様に TB Sで 4回洗浄することにより、 抗原付 着プレートを作製した。 使用に供するまで、 各ゥエルに TBSを 100 /1ずつ 加え、 プレートシールで密閉し、 4 °Cで保存した。 Next, using the polystyrene micro-tie plate Immulon2HB (Dynex Technologies, Chantilly, VA) made of polystyrene as a support for the antigen, the DNA was added to the support at 0.5 、 / It was dissolved in Tris buffer (pH 7.4) containing 0.25 M NaCl at a concentration of 1111, added at 25 ng / we11, and adsorbed at 4 ° C overnight. After the adsorption, the excess antigen was once removed by suction, and then washed four times with NaCl-containing Tris buffer (25 mM Tris, 140 mM NaCl, 0.04% by mass NaN 3 , pH 7.4) (TBS). About 10 ml of TBS was used per plate. After washing, add 100/1 blocking solution (TBS containing 2 mass% skim milk (Difco, Detroit, MI) containing skim milk (Difco, Inc., Difco, Detroit, MI)) for each well, and react for 1 hour to shield the site without antigen. did. Finally, an antigen-attached plate was prepared by washing the plate four times with TBS in the same manner as the washing after the antigen adsorption. Until use, add 100/1 TBS to each well, seal with a plate seal, and store at 4 ° C.
(実施例 10) DN Aプレートによる抗 DN A抗体の測定 (Example 10) Measurement of anti-DNA antibody using DNA plate
S L E患者 1症例の血清について、 実施例 9の各マイクロタイタープレートに より、 実施例 6の抗ヌクレオソ一ム抗体測定と同様に、 抗 DN A抗体の測定を行 つた。 測定結果を図 14に表す。 前記患者のヒトヌクレオソ一ム DN Aに対する 免疫応答は、 仔牛胸腺 DNAに対するより遥かに強いことが分かった。 さらに、 ヒトゲノム DN Aと比較してもヌクレオゾーム DN Aの方が免疫応答が強いこと が分かった。 この傾向は抗 DN A抗体価の高い他の 3例の S LE患者症例でも認 められた。  The serum of one SLE patient was subjected to the measurement of the anti-DNA antibody using the microtiter plate of Example 9 in the same manner as the measurement of the anti-nucleosomal antibody of Example 6. Figure 14 shows the measurement results. The patient's immune response to human nucleosomal DNA was found to be much stronger than to calf thymus DNA. Furthermore, it was found that the nucleosome DNA had a stronger immune response than the human genome DNA. This tendency was also observed in the other three SLE patients with high anti-DNA antibody titers.
(実施例 11)  (Example 11)
抗体の抑制実験 Antibody suppression experiment
実施例 10で用いた血清につき、 予め血清中の抗体を、 抗原を用いて溶液中で 吸収した後、 抗ヌクレオソ一ム D N A抗体活性の測定を行う抑制実験を行つた。 抗原には、 それそれ、 ヌクレオゾーム DNA、 ゲノム DNA及び仔牛胸腺 DNA を用いた。 抑制実験の結果を図 15に表す。 抗ヌクレオソーム DN A抗体活性を 40%阻害するのに要する仔牛胸腺 DN Aの量はヌクレオソーム DN Aの 3倍で あったことから、 異種 D N Aの抗原性の弱さが明確になった。  For the serum used in Example 10, an antibody in the serum was previously absorbed in a solution using an antigen, and then an anti-nucleosomal DNA antibody activity was measured in a suppression experiment. As antigens, nucleosomal DNA, genomic DNA and calf thymus DNA were used. Figure 15 shows the results of the suppression experiment. The amount of calf thymus DNA required to inhibit anti-nucleosome DNA antibody activity by 40% was three times that of nucleosome DNA, indicating the weak antigenicity of the heterologous DNA.
同様の抑制実験を、 マイクロ夕イタ一プレートを P L Lで前処理してからヌク レオソ一ム DNA ( 500 ng/ml) をマイクロ夕イタ一プレートに添加して 作製したプレートについて行った。抑制実験の結果を図 16に表す。この場合は、 抗ヌクレオソーム DN A抗体活性を 40%阻害するのに要する仔牛胸腺 DN Aの 量はヌクレオソ一ム DNAの 1. 3倍と P L Lで前処理しないプレートの場合に 比べて低く、 異種 DNAとの差が目立たなくなる。 このことは、 PLLによる D NA抗原構造の修飾によるものと考えられる。 即ち、 PLLで前処理した DNA プレートにより測定された抗体活性が、 DNAとPL Lとで新たに形成された未 知の抗原に対する反応を含むため、 本来の DN A抗原による抑制効果が目立たな くなったものと考えられる。  Similar suppression experiments were performed on plates prepared by pretreating microplates with PLL and then adding nucleosomal DNA (500 ng / ml) to the plate. Figure 16 shows the results of the suppression experiment. In this case, the amount of calf thymus DNA required to inhibit anti-nucleosome DNA antibody activity by 40% is 1.3 times that of nucleosomal DNA, which is lower than that of a plate not pre-treated with PLL. And the difference between them becomes inconspicuous. This is thought to be due to the modification of the DNA antigen structure by the PLL. That is, since the antibody activity measured by the DNA plate pretreated with PLL includes a reaction against an unknown antigen newly formed by DNA and PLL, the suppression effect of the original DNA antigen is inconspicuous. It is thought that it became.
(実施例 12)  (Example 12)
P L Lで前処理せずに DNAを付着させたプレー卜と P L Lで前処理して DN Aを付着させたプレートとの抗原性の差についてさらに調べた。 24例の S L E患者の血清と、 24例の健常者の血清とについて、 PLLで前 処理せずにヌクレオソーム DN Aを付着させたプレー卜と P L Lで前処理してヌ クレオソーム DN Aを付着させたプレートとにおいて抗 DN A抗体反応を測定し た。 また、 それそれのプレートでヌクレオソ一ム DNA抗原を付着させないプレ 一トにおいても抗 D N A抗体反応を測定した。 P L Lで前処理せずにヌクレオソ ーム D N Aを付着させたプレートにおける測定結果を図 17に、 P L Lで前処理 してヌクレオソ一ム DN Aを付着させたプレートにおける測定結果を図 18に表 す。 The difference in antigenicity between the plate with DNA attached without PLL pretreatment and the plate with DNA pretreated with PLL was further investigated. The sera of 24 SLE patients and the sera of 24 healthy subjects were pretreated with nucleosome DNA without PLL pretreatment and nucleosome DNA was pretreated with PLL to attach nucleosome DNA. The anti-DNA antibody reaction was measured with the plate. The anti-DNA antibody reaction was also measured on each plate to which no nucleosomal DNA antigen was attached. Figure 17 shows the measurement results on the plate to which nucleosome DNA was attached without pretreatment with PLL, and Fig. 18 shows the measurement results on the plate to which nucleosome DNA was attached with PLL pretreatment.
P L Lで前処理したプレートでは、 ヌクレオソ一ム DNA抗原を付着させない プレート （図中一 Ag) に対する SLE患者の血清の反応性が高かった。 また、 ヌクレオソ一ム DNA抗原を付着させたプレート （図中 +Ag) に対する健常者 の血清の反応性も高かった。 このことから、 P L Lで前処理したプレートにおい ては、 非特異的反応が排除できないことが分かった。  In the plate pretreated with PLL, the reactivity of the serum of the SLE patient to the plate on which the nucleosomal DNA antigen was not attached (1 Ag in the figure) was high. In addition, the reactivity of the serum of a healthy subject to the plate (+ Ag in the figure) to which the nucleosomal DNA antigen was attached was also high. This indicated that non-specific reactions could not be excluded in plates pre-treated with PLL.
前記 P L Lで前処理せずにヌクレオソ一ム DN Aを付着させたプレートと P L Lで前処理してヌクレオソ一ム DNAを付着させたプレートとにおいて、 各々で 測定された健常者の平均 + 3 SDを正常域として、 S LE患者の測定値を比較し た結果を図 19に表す。 前者における陽性率が 41. 6% ( 10/24) であつ たのに対し、 後者における陽性率は 16. 6% (4/24) であり、 PLLで前 処理せずにヌクレオソ一ム DNAを付着させたプレートが S L Eの診断に関して 格段に優れていることが明確になった。  In the plate to which nucleosome DNA was attached without pretreatment with the PLL and the plate to which nucleosome DNA was attached with PLL pretreatment, the average + 3 SD of healthy subjects measured in each was calculated. FIG. 19 shows the result of comparing the measured values of the SLE patients as the normal range. The positive rate in the former was 41.6% (10/24), while the positive rate in the latter was 16.6% (4/24), indicating that nucleosomal DNA was obtained without pretreatment with PLL. The attached plates proved to be significantly better at diagnosing SLE.
これらのことから、 P L Lで前処理しない DNAプレート、 即ち、 PLLを介 さずに付着された DN Aプレートがより正確に DN A抗原に対する免疫応答を測 定することができ、 S LE等の正確な診断に用いることができることが明らかに なった。  From these facts, the DNA plate not pre-treated with PLL, that is, the DNA plate attached without the PLL, can measure the immune response to the DNA antigen more accurately, It can be used for various diagnoses.
(実施例 13)  (Example 13)
ヌクレオソ一ム DNAをプレートに直接吸着 （コーティング） する場合の溶媒 中の N a C 1濃度と抗体反応度、 つまり、 コーティング効率について、 抗ヌクレ ォソーム DN A抗体価の高い 1例の S L E患者の血清を用いて調べた。  In the case of direct adsorption (coating) of nucleosomal DNA to a plate, the serum concentration of NaC1 in the solvent and the antibody reactivity, that is, the coating efficiency, ie, the serum of one SLE patient with a high anti-nucleosomal DNA antibody titer It investigated using.
0. 14M、 0. 251 及び0. 5Mの NaC 1を含むトリス緩衝に、 それそ れヌクレオソーム DNA (0. 5 /g/ml) を溶解してプレートに吸着させた 場合について、 抗 DNA抗体の測定を行った。 測定結果を図 20に表す。 0. 2 5 Mの場合が最も抗 DN A抗体の反応性がよく、 コ一ティング効率がよいことが 分かる。 In a Tris buffer containing 0.14M, 0.251 and 0.5M NaCl, The anti-DNA antibody was measured when nucleosome DNA (0.5 / g / ml) was dissolved and adsorbed on the plate. Fig. 20 shows the measurement results. It can be seen that the case of 0.25 M has the highest anti-DNA antibody reactivity and the highest coating efficiency.
さらに、 24例の SLE患者及び 24例の健常者を対象とした前記 3種の濃度 で作製されたプレートにおける抗 DNA抗体の測定を行った。 0. 14Mの Na C 1濃度で作製したプレートの測定結果を図 21に、 0. 25Mの NaCl濃度 で作製したプレートの測定結果を図 22に、 0. 5Mの NaC 1濃度で作製した プレートの測定結果を図 23にそれそれ表す。 0. 25M以上の Na C 1濃度で 作製したプレートにおいて、 特に抗 DN A抗体の検出に優れていることが分かつ た。  Furthermore, anti-DNA antibodies were measured on 24 SLE patients and 24 healthy subjects on plates prepared at the above three concentrations. Figure 21 shows the measurement results for the plate made at 0.14M NaCl concentration, and Figure 22 shows the measurement results for the plate made at 0.25M NaCl concentration. Figure 23 shows the measurement results. It was found that the plate prepared at a NaCl concentration of 0.25M or more was particularly excellent in detecting anti-DNA antibodies.
本発明によると、 自己免疫病診断等をはじめ各種用途に好適なヌクレオソーム を、 形態安定性を維持し、 簡便な操作で効率よく、 しかも高純度で得ることがで きるモノヌクレオゾームの製造方法、 前記モノヌクレオソオームの製造方法によ り製造されたモノヌクレオソ一ム、 取扱性に優れたモノヌクレオソ一ム製造用キ ッ ト、 モノヌクレオソーム分析、 自己免疫病診断等に好適なヒストン検査方法、 前記モノヌクレオソ一ムを含む、 ヌクレオゾームに特異的で各種診断等に好適な 抗体の測定方法、 簡便かつ確実な自己免疫病診断方法、 高性能で取扱性に優れた 自己免疫病診断用キッ ト、 自己免疫病診断等をはじめ各種用途に好適なヌクレオ ソ一ム DNAを、 簡便な操作で効率よく、 しかも高純度で得ることができるヌク レオソーム DNA製造方法、 高性能で取扱性に優れた DN Aプレート、 前記 DN Aプレートの効率的な製造方法、 及び、 自己免疫病診断等に好適な抗 DNA抗体 測定法を提供することができる。  According to the present invention, a method for producing a mononucleosome, which can obtain nucleosomes suitable for various uses including autoimmune disease diagnosis and the like while maintaining morphological stability, efficiently by simple operations, and with high purity, A mononucleosome produced by the method for producing a mononucleosome, a kit for producing a mononucleosome with excellent handling properties, a mononucleosome analysis, a histone inspection method suitable for autoimmune disease diagnosis, etc .; Nucleosome-specific antibody measurement methods suitable for various diagnostics, etc., simple and reliable method for diagnosing autoimmune disease, high-performance and easy-to-handle autoimmune disease diagnostic kit, autoimmune disease Nucleosome DNA suitable for various applications including diagnostics can be obtained efficiently and with high purity by simple operations. Arm DNA production methods, DN A plate excellent in handling property at high, efficient production method of the DN A plate, and can provide a suitable anti-DNA antibody assay in autoimmune diseases diagnosis.

Claims

請 求 の 範 囲 The scope of the claims
1 . 試料中に含まれるヌクレオソームを、 該ヌクレオゾームに特異的な抗体に よって捕獲収集する捕獲収集工程と、 1. a capture collection step of capturing and collecting nucleosomes contained in the sample using an antibody specific to the nucleosome.
捕獲したヌクレオソ一ムを前記抗体から解離回収させる解離回収工程と、 回収したヌクレオソ一ムからモノヌクレオソームを分子量に基づいて単離 ·精 製する単離精製工程と、  A dissociation and recovery step of dissociating and recovering the captured nucleosome from the antibody, and an isolation and purification step of isolating and purifying mononucleosomes from the recovered nucleosome based on molecular weight.
を含むことを特徴とするモノヌクレオソ一ムの製造方法。 A method for producing a mononucleosome, comprising:
2 . 抗体が、 抗ヌクレオソ一ム抗体、 抗 D N A抗体、 及び抗ヒストン抗体から 選択される少なくとも一種である請求の範囲第 1項に記載のモノヌクレオソ一ム の製造方法。  2. The method for producing a mononucleosome according to claim 1, wherein the antibody is at least one selected from an anti-nucleosomal antibody, an anti-DNA antibody, and an anti-histone antibody.
3 . 抗体が、 1 4 O mMの塩濃度において抗原との結合能を有する請求の範囲 第 1項又は第 2項に記載のモノヌクレオソ一ムの製造方法。  3. The method for producing a mononucleosome according to claim 1 or 2, wherein the antibody has an ability to bind to an antigen at a salt concentration of 14 O mM.
4 . 抗体が、 2 C 1 0の可変域のアミノ酸配列、 又は、 該アミノ酸配列におい て 1以上 2 0以内のアミノ酸が欠失、 置換若しくは付加されたアミノ酸配列を有 し、 2 C 1 0の抗原特異性を有する請求の範囲第 1項から第 3項のいずれかに記 載のモノヌクレオゾームの製造方法。  4. The antibody has the amino acid sequence of the variable region of 2C10, or an amino acid sequence in which 1 to 20 amino acids have been deleted, substituted or added in the amino acid sequence, and the antibody has an amino acid sequence of 2C10. 4. The method for producing a mononucleosome according to any one of claims 1 to 3, which has antigen specificity.
5 . 抗体が、 2 C 1 0である請求項 4に記載のモノヌクレオゾームの製造方法。 5. The method for producing a mononucleosome according to claim 4, wherein the antibody is 2C10.
6 . 捕獲収集工程において、 抗体を該抗体と親和性を有する固相に結合させる ことにより、 該抗体にヌクレオソ一ムを捕獲させる請求の範囲第 1項から第 5項 のいずれかに記載のモノヌクレオソ一ムの製造方法。 6. The mononucleosome according to any one of claims 1 to 5, wherein in the capturing and collecting step, the antibody is bound to a solid phase having an affinity for the antibody so that the antibody captures a nucleosome. Production method.
7 . 固相が、 プロテイン Aカラムである請求の範囲第 6項に記載のモノヌクレ ォゾームの製造方法。  7. The method for producing a mononucleosome according to claim 6, wherein the solid phase is a protein A column.
8 . 捕獲収集工程において、 抗体を予め固相に結合させておき、 試料を該固相 と接触させることにより、 該抗体にヌクレオソ一ムを捕獲させる請求の範囲第 1 項から第 5項のいずれかに記載のモノヌクレオゾームの製造方法。  8. The method according to any one of claims 1 to 5, wherein in the capturing and collecting step, the antibody is previously bound to a solid phase, and the sample is brought into contact with the solid phase so that the antibody captures a nucleosome. A method for producing a mononucleosome according to the above.
9 . 単離精製工程において、 ゲルろ過カラムを用いて分子量 2 0万から 2 5万 の分画を回収してモノヌクレオソ一ムを単離精製する請求の範囲第 1項から第 8 項のいずれかに記載のモノヌクレオゾームの製造方法。 9. In the isolation and purification step, any of claims 1 to 8 wherein the fraction having a molecular weight of 200,000 to 250,000 is recovered using a gel filtration column to isolate and purify the mononucleosome. 2. The method for producing a mononucleosome according to item 1.
1 0 . 捕獲収集工程前にヌクレオソ一ムをモノヌクレオソーム単位に切断し得 るヌクレア一ゼで処理する請求の範囲第 1項から第 9項のいずれかに記載のモノ ヌクレオソ一ムの製造方法。 10. The method for producing a mononucleosome according to any one of claims 1 to 9, wherein the nucleosome is treated with a nuclease capable of cleaving into mononucleosome units before the capturing and collecting step.
1 1 . 解離回収工程後、 単離精製工程前にヌクレオゾームをモノヌクレオソー ム単位に切断し得るヌクレアーゼで処理する請求の範囲第 1項から第 9項のいず れかに記載のモノヌクレオゾームの製造方法。  11. The mononucleosome according to any one of claims 1 to 9, wherein the nucleosome is treated with a nuclease capable of cleaving into mononucleosome units after the dissociation recovery step and before the isolation and purification step. Manufacturing method.
1 2 . ヌクレアーゼが、 マイクロコッカル ·ヌクレア一ゼである請求の範囲第 1 0又は第 1 1項に記載のモノヌクレオゾームの製造方法。  12. The method for producing a mononucleosome according to claim 10 or 11, wherein the nuclease is micrococcal nuclease.
1 3 . 細胞を低塩濃度の溶液中で破砕させ、 溶液中にヌクレオソ一ムを放出さ せる放出工程と、  13. Disrupting cells in a low salt solution to release nucleosomes into the solution;
前記ヌクレオゾームを収集する収集工程と、 A collection step of collecting the nucleosome,
収集した前記ヌクレオソ一ムを低塩濃度の溶液に懸濁し、 モノヌクレオソ一ム単 位に切断し得るヌクレアーゼで処理するヌクレアーゼ処理工程と、 A nuclease treatment step of suspending the collected nucleosomes in a solution having a low salt concentration and treating with a nuclease which can be cleaved into mononucleosome units;
前記ヌクレア一ゼ処理後の溶液からモノヌクレオソ一ムを、 該ヌクレオソ一ムに 特異的な抗体を用いて、 及び/又は分子量に基づいて製造する単離精製工程と、 を含むことを特徴とするモノヌクレオソ一ムの製造方法。 An isolation and purification step of producing a mononucleosome from the solution after the nuclease treatment using an antibody specific to the nucleosome, and / or based on the molecular weight. Production method.
1 4 . 単離精製工程で用いる抗体が、 2 C 1 0である請求の範囲第 1 3項に記 載のモノヌクレオソ一ムの製造方法。  14. The method for producing a mononucleosome according to claim 13, wherein the antibody used in the isolation and purification step is 2C10.
1 5 . 単離精製工程において、 ゲルろ過カラムを用いて分子量 2 0万から 2 5 万の分画を分離する請求の範囲第 1 3項又は第 1 4項に記載のモノヌクレオソー ムの製造方法。  15. The production of the mononucleosome according to claim 13 or 14, wherein a fraction having a molecular weight of 200,000 to 250,000 is separated using a gel filtration column in the isolation and purification step. Method.
1 6 . 請求の範囲第 1項から第 1 5項のいずれかに記載のモノヌクレオソ一ム の製造方法により製造されたことを特徴とするモノヌクレオソ一ム。  16. A mononucleosome, which is produced by the method for producing a mononucleosome according to any one of claims 1 to 15.
1 7 . 純度が 9 8 %以上である請求の範囲第 1 6項に記載のモノヌクレオソー ム。  17. The mononucleosome according to claim 16, having a purity of 98% or more.
1 8 . 試料中に含まれるヌクレオソームを捕獲収集するための該ヌクレオソー ムに特異的な抗体と、  18. An antibody specific to the nucleosome for capturing and collecting the nucleosome contained in the sample,
捕獲収集したヌクレオゾームから分子量に基づきモノヌクレオソ一ムを単離する ためのカラムと、 を含むことを特徴とするモノヌクレオソーム製造用キッ ト。 A column for isolating mononucleosomes from captured and collected nucleosomes based on molecular weight; A kit for producing mononucleosomes, comprising:
1 9 . 前記抗体が、 2 C 1 0である請求の範囲第 1 8項に記載のモノヌクレオ ソーム製造用キッ ト。  19. The kit for producing mononucleosomes according to claim 18, wherein the antibody is 2C10.
2 0 . 請求の範囲第 1項から第 1 5項のいずれかに記載のモノヌクレオソ一ム の製造方法により被検試料から得たモノヌクレオソームより被検ヒストンを回収 し、 該被検ヒストンの電気泳動パターンと対照ヒストンの電気泳動パターンとを 比較することを含むことを特徴とするヒストン検査方法。  20. A test histone is recovered from a mononucleosome obtained from a test sample by the method for producing a mononucleosome according to any one of claims 1 to 15, and electrophoresis of the test histone is performed. A method for testing a histone, comprising comparing a pattern with an electrophoretic pattern of a control histone.
2 1 . 請求の範囲第 1項から第 1 5項のいずれかに記載のモノヌクレオソーム の製造方法により製造されたモノヌクレオソームを固相化する固相化工程と、 被検試料を、 固相化された該モノヌクレオソームと反応させる反応工程と、 前記モノヌクレオソ一ムに結合する特異的な抗体を測定する測定工程と、 を含むことを特徴とするヌクレオゾームに特異的な抗体の測定方法。  21. A solid phase immobilizing step of immobilizing the mononucleosome produced by the method for producing a mononucleosome according to any one of claims 1 to 15; A method for measuring an antibody specific to a nucleosome, comprising: a reaction step of reacting with the obtained mononucleosome; and a measuring step of measuring a specific antibody that binds to the mononucleosome.
2 2 . モノヌクレオゾームが、 アポト一シス由来のヌクレオソ一ムである請求 の範囲第 2 1項に記載のヌクレオゾームに特異的な抗体の測定方法。  22. The method for measuring a nucleosome-specific antibody according to claim 21, wherein the mononucleosome is a nucleosome derived from apoptosis.
2 3 . 被検試料が、 自己免疫病患者の血清又は血漿である請求の範囲第 2 1項 又は第 2 2項に記載のヌクレオゾームに特異的な抗体の測定方法。  23. The method for measuring a nucleosome-specific antibody according to claim 21 or 22, wherein the test sample is serum or plasma of an autoimmune disease patient.
2 4 . 固相化工程において、 固相化したモノヌクレオソームを少なくともスキ ムミルクを含むブロッキング液でブロッキングさせ、 反応工程において、 試料を 少なくともスキムミルクを含む反応液で稀釈してから該試料を、 前記固相化され たモノヌクレオソームと反応させる請求の範囲第 2 1項から第 2 3項のいずれか に記載のヌクレオソ一ムに特異的な抗体の測定方法。  24. In the solid phase immobilization step, the immobilized mononucleosome is blocked with a blocking solution containing at least skim milk. The method for measuring an antibody specific to a nucleosome according to any one of claims 21 to 23, wherein the method is reacted with the phased mononucleosome.
2 5 . 反応液が、 トリス、 N a C l、 N a N 3、 血清アルブミン、 スキムミル ク、 及び E D T Aを含有する請求の範囲第 2 4項に記載のヌクレオソームに特異 的な抗体の測定方法。 2 5. The reaction solution, Tris, N a C l, N a N 3, serum albumin, Sukimumiru click, and specific method for measuring the antibody nucleosomes according to the second item 4 claims containing EDTA.
2 6 . 測定工程において、 I g Gサブクラスを認識する抗ヒト抗体を二次抗体 として反応させる請求の範囲第 2 1項から第 2 5項のいずれかに記載のヌクレオ ゾームに特異的な抗体の測定方法。  26. The nucleosome-specific antibody according to any one of claims 21 to 25, wherein an anti-human antibody recognizing an IgG subclass is reacted as a secondary antibody in the measurement step. Measuring method.
2 7 . 請求の範囲第 1項から第 1 5項のいずれかに記載のモノヌクレオソ一ム の製造方法により製造されたモノヌクレオソ一ムを固相化する固相化工程と、 被検試料を、 固相化された該モノヌクレオソ一ムと反応させる反応工程と、 前記モノヌクレオソームに結合する特異的な抗体を測定する測定工程と、 その抗体価を評価する評価工程と、 27. A solid phase immobilizing step of immobilizing the mononucleosome produced by the method for producing a mononucleosome according to any one of claims 1 to 15; A reaction step of reacting the test sample with the immobilized mononucleosome, a measurement step of measuring a specific antibody that binds to the mononucleosome, an evaluation step of evaluating the antibody titer,
を含むことを特徴とする自己免疫病診断方法。 A method for diagnosing an autoimmune disease, comprising:
28. 請求の範囲第 1項から第 15項のいずれかに記載のモノヌクレオソーム の製造方法により製造したモノヌクレオソ一ムを固相化してなる固相と、 緩衝液 と、 プレート及びカラムのいずれかとを含むことを特徴とする自己免疫病診断用 キット。  28. A solid phase obtained by immobilizing the mononucleosome produced by the method for producing a mononucleosome according to any one of claims 1 to 15; a buffer; and a plate or column. A kit for diagnosing an autoimmune disease, comprising:
29. 試料中に含まれるヌクレオソームを、 該ヌクレオソ一ムに特異的な抗体 によって捕獲収集する捕獲収集工程と、  29. a capture collection step of capturing and collecting nucleosomes contained in the sample with an antibody specific to the nucleosome;
捕獲したヌクレオゾームを前記抗体から解離回収させる解離回収工程と、 回収したヌクレオゾームからモノヌクレオソームを分子量に基づいて単離 ·精製 する単離精製工程と、 A dissociation and recovery step of dissociating and recovering the captured nucleosomes from the antibody, an isolation and purification step of isolating and purifying mononucleosomes from the recovered nucleosomes based on molecular weight,
単離 '精製されたモノヌクレオソ一ムからヌクレオソ一ム DN Aを単離 '精製す るヌクレオソ一ム DN A単離精製工程と、 Isolation 'Isolating nucleosome DNA from purified mononucleosome'
を含むことを特徴とするヌクレオソーム DN A製造方法。 A method for producing a nucleosome DNA, comprising:
30. 請求の範囲第 1項から第 15項のいずれかに記載のモノヌクレオソーム の製造方法からなるモノヌクレオソ一ム製造工程と、  30. A mononucleosome production process comprising the method for producing a mononucleosome according to any one of claims 1 to 15;
モノヌクレオソ一ムからヌクレオソ一ム DN Aを単離精製するヌクレオソーム D NA単離精製工程と、 A nucleosome DNA isolation and purification step for isolating and purifying the nucleosome DNA from the mononucleosome,
を含むことを特徴とするヌクレオゾーム DN A製造方法。 A method for producing nucleosome DNA, comprising:
31. プレート上に、 請求の範囲第 29項又は第 30項に記載のヌクレオソー ム DN A製造方法により製造されたヌクレオソ一ム DN Aであって、 ヒト由来の ヌクレオソ一ム DN Aが固相化されてなることを特徴とする DN Aプレート。 31. A nucleosome DNA produced by the method for producing a nucleosome DNA according to claim 29 or claim 30, wherein the nucleosome DNA is derived from human and immobilized on a plate. A DNA plate characterized by being made.
32. プレート上に、 請求の範囲第 16項又は第 17項に記載のモノヌクレオ ソームであって、 ヒト由来のモノヌクレオソームから単離精製されたヌクレオソ —ム DN Aが固相化されてなることを特徴とする DN Aプレート。 32. The mononucleosome according to claim 16 or 17, wherein the nucleosome is isolated and purified from a human-derived mononucleosome, and the mononucleosome according to claim 16 is immobilized on the plate. Features DN A plate.
33. ヌクレオソ一ム DNAが、 ヌクレオソ一ム構成 2本鎖 DNAであり、 ヌ クレオソーム DN Aの平均鎖長が 145 b p以上 200 b p以下である請求の範 囲第 31項又は第 32項に記載の DNAプレート。 33. The claim wherein the nucleosome DNA is a double-stranded nucleosome DNA and the average length of the nucleosome DNA is 145 bp or more and 200 bp or less. Item 33. The DNA plate according to Item 31 or 32.
34. ヌクレオソーム DNAが、 プレート上に直接固相化されてなる請求の範 囲第 31項から第 33項のいずれかに記載の DNAプレート。  34. The DNA plate according to any one of claims 31 to 33, wherein the nucleosome DNA is directly immobilized on the plate.
35. プレートが、 ポリスチレンを含んでなる請求の範囲第 31項から第 34 項のいずれかに記載の DN Aプレート。  35. The DNA plate of any one of claims 31 to 34, wherein the plate comprises polystyrene.
36. 請求の範囲第 1項から第 15項のいずれかに記載のモノヌクレオソーム の製造方法であって、 試料がヒト由来の試料であるモノヌクレオソーム製造工程 と、  36. The method for producing a mononucleosome according to any one of claims 1 to 15, wherein the sample is a human-derived sample;
モノヌクレオソ一ムからヌクレオソ一ム DN Aを単離精製するヌクレオソ一ム D NA単離精製工程と、 A nucleosomal DNA isolation and purification step for isolating and purifying the nucleosomal DNA from the mononucleosome,
前記ヌクレオソ一ム DN Aをプレート上に固相化する固相化工程と、 Immobilizing step of immobilizing the nucleosome DNA on a plate,
を含むことを特徴とする DN Aプレート製造方法。 A method for producing a DNA plate, comprising:
37. 固相化工程において、 プレート上に、 ヌクレオゾーム DN Aを 0. 1M 以上 1. 0M以下の NaC 1を含む、 トリス緩衝液及びホウ酸—カセイソーダ緩 衝液のいずれかに溶解させて添加する請求の範囲第 36項に記載の DN Aプレー ト製造方法。  37. In the immobilization step, nucleosome DNA is added to the plate by dissolving it in either Tris buffer or boric acid-caustic soda buffer containing 0.1M to 1.0M NaC1. Item 36. The method for producing a DNA plate according to Item 36.
38. プレート上に、 請求の範囲第 29項又は第 30項に記載のヌクレオソー ム DN A製造方法により製造されたヌクレオソ一ム DN Aであってヒト由来のヌ クレオソーム DN Aを、 0. 1M以上1. 0M以下の NaC 1を含む、 トリス緩 衝液及びホウ酸—カセィソーダ緩衝液のいずれかに溶解させて添加することによ り固相化することを特徴とする DN Aプレート製造方法。  38. A human-derived nucleosome DNA, which is a nucleosome DNA produced by the method for producing a nucleosome DNA according to claim 29 or 30, which is 0.1 M or more, on a plate. 1. A method for producing a DNA plate, comprising dissolving and adding to a Tris buffer solution or boric acid-cassette buffer solution containing 0 M or less NaC1 and solidifying the solution.
39. 請求の範囲第 31項から第 35項のいずれかに記載の DNAプレートを 用いてなり、 39. The DNA plate according to any one of claims 31 to 35,
被検試料を、 該 DN Aプレートの固相化されたヌクレオソーム DN Aと反応させ る反応工程と、 A reaction step of reacting the test sample with the nucleosome DNA immobilized on the DNA plate;
前記ヌクレオゾーム DN Aに結合する特異的な抗体を測定する測定工程と、 を含むことを特徴とする抗 DN A抗体測定法。 A measuring step of measuring a specific antibody that binds to the nucleosome DNA.
PCT/JP2002/002664 2001-03-23 2002-03-20 Mononucleosome and process for producing the same, method of assaying antibody specific to nucleosome, method of diagnosing autoimmune disease, process for producing nucleosome dna, dna plate, process for producing dna plate and method of assaying anti-dna antibody WO2002076377A1 (en)

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Cited By (33)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005040798A1 (en) * 2003-10-29 2005-05-06 Eisai Co., Ltd. Method of diagnosing alzheimer’s disease
JP4832291B2 (en) * 2004-03-31 2011-12-07 一男 新家 Labeling substances and chimeric substances, methods for producing these substances, and methods for capturing, analyzing and / or identifying biological substances using the labeling substances
KR20140078638A (en) * 2011-09-01 2014-06-25 싱가폴 볼리션 피티이 리미티드 Method for detecting nucleosomes containing histone variants
WO2015048535A1 (en) * 2013-09-27 2015-04-02 Natera, Inc. Prenatal diagnostic resting standards
US9639657B2 (en) 2008-08-04 2017-05-02 Natera, Inc. Methods for allele calling and ploidy calling
US9677118B2 (en) 2014-04-21 2017-06-13 Natera, Inc. Methods for simultaneous amplification of target loci
US9695477B2 (en) 2005-11-26 2017-07-04 Natera, Inc. System and method for cleaning noisy genetic data from target individuals using genetic data from genetically related individuals
CN106987629A (en) * 2017-03-31 2017-07-28 上海市第妇婴保健院 A kind of method that nucleosome arrangement on genome is detected on individual cell level
EP2751572B1 (en) * 2011-09-01 2017-12-27 Belgian Volition SPRL Method for detecting nucleosomes
US10011870B2 (en) 2016-12-07 2018-07-03 Natera, Inc. Compositions and methods for identifying nucleic acid molecules
US10017812B2 (en) 2010-05-18 2018-07-10 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US10061890B2 (en) 2009-09-30 2018-08-28 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US10081839B2 (en) 2005-07-29 2018-09-25 Natera, Inc System and method for cleaning noisy genetic data and determining chromosome copy number
US10083273B2 (en) 2005-07-29 2018-09-25 Natera, Inc. System and method for cleaning noisy genetic data and determining chromosome copy number
US10113196B2 (en) 2010-05-18 2018-10-30 Natera, Inc. Prenatal paternity testing using maternal blood, free floating fetal DNA and SNP genotyping
US10179937B2 (en) 2014-04-21 2019-01-15 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US10262755B2 (en) 2014-04-21 2019-04-16 Natera, Inc. Detecting cancer mutations and aneuploidy in chromosomal segments
US10316362B2 (en) 2010-05-18 2019-06-11 Natera, Inc. Methods for simultaneous amplification of target loci
US10526658B2 (en) 2010-05-18 2020-01-07 Natera, Inc. Methods for simultaneous amplification of target loci
US10577655B2 (en) 2013-09-27 2020-03-03 Natera, Inc. Cell free DNA diagnostic testing standards
US11111544B2 (en) 2005-07-29 2021-09-07 Natera, Inc. System and method for cleaning noisy genetic data and determining chromosome copy number
US11111543B2 (en) 2005-07-29 2021-09-07 Natera, Inc. System and method for cleaning noisy genetic data and determining chromosome copy number
US11306357B2 (en) 2010-05-18 2022-04-19 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US11322224B2 (en) 2010-05-18 2022-05-03 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US11326208B2 (en) 2010-05-18 2022-05-10 Natera, Inc. Methods for nested PCR amplification of cell-free DNA
US11332785B2 (en) 2010-05-18 2022-05-17 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US11332793B2 (en) 2010-05-18 2022-05-17 Natera, Inc. Methods for simultaneous amplification of target loci
US11339429B2 (en) 2010-05-18 2022-05-24 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US11408031B2 (en) 2010-05-18 2022-08-09 Natera, Inc. Methods for non-invasive prenatal paternity testing
US11479812B2 (en) 2015-05-11 2022-10-25 Natera, Inc. Methods and compositions for determining ploidy
US11485996B2 (en) 2016-10-04 2022-11-01 Natera, Inc. Methods for characterizing copy number variation using proximity-litigation sequencing
US11525159B2 (en) 2018-07-03 2022-12-13 Natera, Inc. Methods for detection of donor-derived cell-free DNA
US11939634B2 (en) 2010-05-18 2024-03-26 Natera, Inc. Methods for simultaneous amplification of target loci

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999047924A1 (en) * 1998-03-18 1999-09-23 Roche Diagnostics Gmbh Detection of apoptotic products

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999047924A1 (en) * 1998-03-18 1999-09-23 Roche Diagnostics Gmbh Detection of apoptotic products

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CHIU M.L. ET AL.: 'Absence of histone H2B in nucleosomes containing histone TH2B and interaction of immunoglobulin with nucleosomes' ARCH. BIOCHEM. BIOPHYS. vol. 244, no. 1, January 1986, pages 42 - 49, XP002953859 *
KOUTOUZOV S. ET AL.: 'Binding of nucleosomes to a cell surface receptor: redistribution and endocytosis in the presence of lupus antibodies' EUR. J. IMMUNOL. vol. 26, no. 2, February 1996, pages 472 - 486, XP002953860 *
KUBOTA T. ET AL.: 'A monoclonal anti-double stranded DNA antibody from an autoimmune MRL/Mp-1pr/1pr mouse: specificity and idiotype in serum immunoglobulins' IMMUNOL. LETT. vol. 14, no. 1, 17 November 1986, pages 53 - 58, XP000601123 *
MENDELSON E. ET AL.: 'Immunofractionation of chromatin regionsassociated with histone H1o' EUR. J. BIOCHEM. vol. 160, no. 2, 15 October 1986, pages 253 - 260, XP002953864 *
STEMMER C. ET AL.: 'Dual reactivity of several monoclonal anti-nucleosome autoantibodies for double-stranded DNA and a short segment of histone H3' J. BIOL. CHEM. vol. 271, no. 35, 30 August 1996, pages 21257 - 21261, XP002953863 *
WILLIAMS R.C. ET AL.: 'Detection of nucleosome particles in serum and plasma from patients with systemic lupus erythematosus using monoclonal antibody 4H7' J. RHEUMATOL. vol. 28, no. 1, January 2001, pages 81 - 94, XP002953861 *

Cited By (82)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005040798A1 (en) * 2003-10-29 2005-05-06 Eisai Co., Ltd. Method of diagnosing alzheimer’s disease
JP4832291B2 (en) * 2004-03-31 2011-12-07 一男 新家 Labeling substances and chimeric substances, methods for producing these substances, and methods for capturing, analyzing and / or identifying biological substances using the labeling substances
US10081839B2 (en) 2005-07-29 2018-09-25 Natera, Inc System and method for cleaning noisy genetic data and determining chromosome copy number
US11111544B2 (en) 2005-07-29 2021-09-07 Natera, Inc. System and method for cleaning noisy genetic data and determining chromosome copy number
US11111543B2 (en) 2005-07-29 2021-09-07 Natera, Inc. System and method for cleaning noisy genetic data and determining chromosome copy number
US10392664B2 (en) 2005-07-29 2019-08-27 Natera, Inc. System and method for cleaning noisy genetic data and determining chromosome copy number
US10266893B2 (en) 2005-07-29 2019-04-23 Natera, Inc. System and method for cleaning noisy genetic data and determining chromosome copy number
US10260096B2 (en) 2005-07-29 2019-04-16 Natera, Inc. System and method for cleaning noisy genetic data and determining chromosome copy number
US10227652B2 (en) 2005-07-29 2019-03-12 Natera, Inc. System and method for cleaning noisy genetic data from target individuals using genetic data from genetically related individuals
US10083273B2 (en) 2005-07-29 2018-09-25 Natera, Inc. System and method for cleaning noisy genetic data and determining chromosome copy number
US11306359B2 (en) 2005-11-26 2022-04-19 Natera, Inc. System and method for cleaning noisy genetic data from target individuals using genetic data from genetically related individuals
US9695477B2 (en) 2005-11-26 2017-07-04 Natera, Inc. System and method for cleaning noisy genetic data from target individuals using genetic data from genetically related individuals
US10711309B2 (en) 2005-11-26 2020-07-14 Natera, Inc. System and method for cleaning noisy genetic data from target individuals using genetic data from genetically related individuals
US10597724B2 (en) 2005-11-26 2020-03-24 Natera, Inc. System and method for cleaning noisy genetic data from target individuals using genetic data from genetically related individuals
US10240202B2 (en) 2005-11-26 2019-03-26 Natera, Inc. System and method for cleaning noisy genetic data from target individuals using genetic data from genetically related individuals
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US10522242B2 (en) 2009-09-30 2019-12-31 Natera, Inc. Methods for non-invasive prenatal ploidy calling
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US11306357B2 (en) 2010-05-18 2022-04-19 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US10174369B2 (en) 2010-05-18 2019-01-08 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US11482300B2 (en) 2010-05-18 2022-10-25 Natera, Inc. Methods for preparing a DNA fraction from a biological sample for analyzing genotypes of cell-free DNA
US10113196B2 (en) 2010-05-18 2018-10-30 Natera, Inc. Prenatal paternity testing using maternal blood, free floating fetal DNA and SNP genotyping
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US11339429B2 (en) 2010-05-18 2022-05-24 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US10526658B2 (en) 2010-05-18 2020-01-07 Natera, Inc. Methods for simultaneous amplification of target loci
US11326208B2 (en) 2010-05-18 2022-05-10 Natera, Inc. Methods for nested PCR amplification of cell-free DNA
US10538814B2 (en) 2010-05-18 2020-01-21 Natera, Inc. Methods for simultaneous amplification of target loci
US10557172B2 (en) 2010-05-18 2020-02-11 Natera, Inc. Methods for simultaneous amplification of target loci
US10793912B2 (en) 2010-05-18 2020-10-06 Natera, Inc. Methods for simultaneous amplification of target loci
US11286530B2 (en) 2010-05-18 2022-03-29 Natera, Inc. Methods for simultaneous amplification of target loci
US10590482B2 (en) 2010-05-18 2020-03-17 Natera, Inc. Amplification of cell-free DNA using nested PCR
US11746376B2 (en) 2010-05-18 2023-09-05 Natera, Inc. Methods for amplification of cell-free DNA using ligated adaptors and universal and inner target-specific primers for multiplexed nested PCR
US11939634B2 (en) 2010-05-18 2024-03-26 Natera, Inc. Methods for simultaneous amplification of target loci
US10597723B2 (en) 2010-05-18 2020-03-24 Natera, Inc. Methods for simultaneous amplification of target loci
US10017812B2 (en) 2010-05-18 2018-07-10 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US10655180B2 (en) 2010-05-18 2020-05-19 Natera, Inc. Methods for simultaneous amplification of target loci
US11111545B2 (en) 2010-05-18 2021-09-07 Natera, Inc. Methods for simultaneous amplification of target loci
US11332785B2 (en) 2010-05-18 2022-05-17 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US10731220B2 (en) 2010-05-18 2020-08-04 Natera, Inc. Methods for simultaneous amplification of target loci
US10774380B2 (en) 2010-05-18 2020-09-15 Natera, Inc. Methods for multiplex PCR amplification of target loci in a nucleic acid sample
US10184945B2 (en) 2011-09-01 2019-01-22 Belgian Volition Sprl Method for detecting nucleosomes containing histone variants
EP2751572B1 (en) * 2011-09-01 2017-12-27 Belgian Volition SPRL Method for detecting nucleosomes
KR102130855B1 (en) * 2011-09-01 2020-07-09 벨지언 볼리션 에스피알엘 Method for detecting nucleosomes containing histone variants
KR20140078638A (en) * 2011-09-01 2014-06-25 싱가폴 볼리션 피티이 리미티드 Method for detecting nucleosomes containing histone variants
CN104053993A (en) * 2011-09-01 2014-09-17 新加坡意志私人有限公司 Method for detecting nucleosomes containing histone variants
JP2014529405A (en) * 2011-09-01 2014-11-13 シンガポールボリション ピーティーイーリミテッド Histone variant-containing nucleosome detection method
JP2017215338A (en) * 2011-09-01 2017-12-07 ベルジアン ボリション エスピーアールエル Method of detecting nucleosomes containing histone variants
US10577655B2 (en) 2013-09-27 2020-03-03 Natera, Inc. Cell free DNA diagnostic testing standards
WO2015048535A1 (en) * 2013-09-27 2015-04-02 Natera, Inc. Prenatal diagnostic resting standards
US11390916B2 (en) 2014-04-21 2022-07-19 Natera, Inc. Methods for simultaneous amplification of target loci
US11371100B2 (en) 2014-04-21 2022-06-28 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US11319595B2 (en) 2014-04-21 2022-05-03 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US10179937B2 (en) 2014-04-21 2019-01-15 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US10597708B2 (en) 2014-04-21 2020-03-24 Natera, Inc. Methods for simultaneous amplifications of target loci
US11319596B2 (en) 2014-04-21 2022-05-03 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US10351906B2 (en) 2014-04-21 2019-07-16 Natera, Inc. Methods for simultaneous amplification of target loci
US11486008B2 (en) 2014-04-21 2022-11-01 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US10597709B2 (en) 2014-04-21 2020-03-24 Natera, Inc. Methods for simultaneous amplification of target loci
US11530454B2 (en) 2014-04-21 2022-12-20 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US11408037B2 (en) 2014-04-21 2022-08-09 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US11414709B2 (en) 2014-04-21 2022-08-16 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US9677118B2 (en) 2014-04-21 2017-06-13 Natera, Inc. Methods for simultaneous amplification of target loci
US10262755B2 (en) 2014-04-21 2019-04-16 Natera, Inc. Detecting cancer mutations and aneuploidy in chromosomal segments
US11479812B2 (en) 2015-05-11 2022-10-25 Natera, Inc. Methods and compositions for determining ploidy
US11946101B2 (en) 2015-05-11 2024-04-02 Natera, Inc. Methods and compositions for determining ploidy
US11485996B2 (en) 2016-10-04 2022-11-01 Natera, Inc. Methods for characterizing copy number variation using proximity-litigation sequencing
US11519028B2 (en) 2016-12-07 2022-12-06 Natera, Inc. Compositions and methods for identifying nucleic acid molecules
US10533219B2 (en) 2016-12-07 2020-01-14 Natera, Inc. Compositions and methods for identifying nucleic acid molecules
US10577650B2 (en) 2016-12-07 2020-03-03 Natera, Inc. Compositions and methods for identifying nucleic acid molecules
US11530442B2 (en) 2016-12-07 2022-12-20 Natera, Inc. Compositions and methods for identifying nucleic acid molecules
US10011870B2 (en) 2016-12-07 2018-07-03 Natera, Inc. Compositions and methods for identifying nucleic acid molecules
CN106987629A (en) * 2017-03-31 2017-07-28 上海市第妇婴保健院 A kind of method that nucleosome arrangement on genome is detected on individual cell level
CN106987629B (en) * 2017-03-31 2021-01-15 上海市第一妇婴保健院 Method for detecting nucleosome arrangement on genome at single cell level
US11525159B2 (en) 2018-07-03 2022-12-13 Natera, Inc. Methods for detection of donor-derived cell-free DNA

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