WO2002076377A2 - Mononucleosome et son procede de production, methode d'analyse d'un anticorps specifique a un nucleosome, methode de diagnostic d'une maladie auto-immune, procede de production d'un adn nucleosomique, plaque d'adn et procede de production d'une plaque d'adn - Google Patents

Mononucleosome et son procede de production, methode d'analyse d'un anticorps specifique a un nucleosome, methode de diagnostic d'une maladie auto-immune, procede de production d'un adn nucleosomique, plaque d'adn et procede de production d'une plaque d'adn 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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Japanese (ja)
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WO2002076377A3 (fr
WO2002076377A1 (fr
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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/ja
Priority to AU2002238994A priority patent/AU2002238994A1/en
Publication of WO2002076377A2 publication Critical patent/WO2002076377A2/fr
Publication of WO2002076377A1 publication Critical patent/WO2002076377A1/fr
Publication of WO2002076377A3 publication Critical patent/WO2002076377A3/fr

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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

L'invention concerne un procédé permettant de produire facilement et efficacement un mononucléosome a pureté élevée. Plus particulièrement, l'invention concerne un procédé de production d'un mononucléosome consistant à capturer et recueillir un nucléosome contenu dans un échantillon au moyen d'un anticorps spécifique à ce nucléosome, à dissocier le nucléosome ainsi capturé de l'anticorps et à recueillir ce nucléosome, puis à isoler et purifier un mononucléosome à partir du nucléosome recueilli en fonction du poids moléculaire.
PCT/JP2002/002664 2001-03-23 2002-03-20 Mononucleosome et son procede de production, methode d'analyse d'un anticorps specifique a un nucleosome, methode de diagnostic d'une maladie auto-immune, procede de production d'un adn nucleosomique, plaque d'adn et procede de production d'une plaque d'adn WO2002076377A1 (fr)

Priority Applications (2)

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JP2002574893A JP4276843B2 (ja) 2001-03-23 2002-03-20 モノヌクレオソーム及びその製造方法、ヌクレオソームに特異的な抗体の測定方法、自己免疫病診断方法、ヌクレオソームｄｎａ製造方法、ｄｎａプレート、ｄｎａプレート製造方法、並びに抗ｄｎａ抗体測定法
AU2002238994A AU2002238994A1 (en) 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

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JP2001-84480 2001-03-23
JP2001084480 2001-03-23
JP2001-291182 2001-09-25
JP2001291182 2001-09-25

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WO2002076377A2 true WO2002076377A2 (fr) 2002-10-03
WO2002076377A1 WO2002076377A1 (fr) 2002-10-03
WO2002076377A3 WO2002076377A3 (fr) 2002-11-21

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WO2005040798A1 (fr) * 2003-10-29 2005-05-06 Eisai Co., Ltd. Procede de diagnostic de la maladie d'alzheimer
JP4832291B2 (ja) * 2004-03-31 2011-12-07 一男 新家 ラベル用物質とキメラ物質、これらの物質の作製方法、並びに該ラベル用物質を用いて生体物質を捕捉、構造解析又は／及び同定する方法
KR20140078638A (ko) * 2011-09-01 2014-06-25 싱가폴 볼리션 피티이 리미티드 히스톤 변이체를 함유하는 뉴클레오솜의 검출 방법
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999047924A1 (fr) * 1998-03-18 1999-09-23 Roche Diagnostics Gmbh Detection de produits apoptotiques

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999047924A1 (fr) * 1998-03-18 1999-09-23 Roche Diagnostics Gmbh Detection de produits apoptotiques

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 *

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JP4832291B2 (ja) * 2004-03-31 2011-12-07 一男 新家 ラベル用物質とキメラ物質、これらの物質の作製方法、並びに該ラベル用物質を用いて生体物質を捕捉、構造解析又は／及び同定する方法
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AU2002238994A1 (en) 2002-10-08
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