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WO2003099869A1 - Anticorps capable de reconnaitre la 8-nitroguanine - Google Patents

Anticorps capable de reconnaitre la 8-nitroguanine Download PDF

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Publication number
WO2003099869A1
WO2003099869A1 PCT/JP2003/006665 JP0306665W WO03099869A1 WO 2003099869 A1 WO2003099869 A1 WO 2003099869A1 JP 0306665 W JP0306665 W JP 0306665W WO 03099869 A1 WO03099869 A1 WO 03099869A1
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Prior art keywords
antibody
bsa
antigen
nitroguanine
present
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PCT/JP2003/006665
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English (en)
Japanese (ja)
Inventor
Takaaki Akaike
Tomohiro Sawa
Kiminori Miyazaki
Eri Watanabe
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Dojindo Laboratory and Co Ltd
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Dojindo Laboratory and Co Ltd
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Priority to AU2003241827A priority Critical patent/AU2003241827A1/en
Priority to JP2004508123A priority patent/JPWO2003099869A1/ja
Publication of WO2003099869A1 publication Critical patent/WO2003099869A1/fr
Anticipated expiration legal-status Critical
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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
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

Definitions

  • the present invention relates to an antibody capable of recognizing 8-nitroguanine.
  • inducible nitric oxide synthase produces large amounts of nitric oxide, which reacts with the active oxygen superoxide to produce peroxynitrite.
  • This peroxynitrite is a powerful nitrifying agent, and is known to dinitrogenize tyrosine residues of proteins in the living body to generate 3_nitrotyrosine, which causes tissue damage.
  • peroxynitrite also reacts with nucleobases, especially nitrating guanine bases. It is known that nitroninated guanine bases are more easily eliminated from nucleotides, and the remaining abasic site (AP site: apurinic / apyrimidicscite) causes mutation and cell death.
  • An object of the present invention is to provide a means for detecting 8-ditroguanine with high sensitivity. More specifically, an antibody that recognizes 8-ditroguanine is a means for easily and surely measuring 8-ditroguanine and for clarifying the localization of 8-ditroguanine in a living body. It is an object of the present invention to provide
  • the present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, have found that a polyclonal antibody that recognizes 8-ditroguanine can be produced by using a specific antigen protein containing an 8-ditroguanosine residue. I found it. In addition, we succeeded in producing a polyclonal antibody that specifically recognizes 8-nitroguanine using the above antigen protein. The present invention has been completed based on the above findings. That is, the present invention provides an antibody that recognizes 8-two troganin.
  • a polyclonal antibody recognizing 8-ditroguanine a polyclonal antibody specifically recognizing 8-ditroguanine, and a monoclonal antibody specifically recognizing 8-nitroguanine.
  • the present invention provides a method for measuring 8-2-troginine in a biological sample using the above antibody.
  • an antigen for use in the production of an antibody recognizing 8-trogoyun which comprises a serum protein bound to 8-nitroguanosine.
  • the above-mentioned antigen wherein the serum protein is albumin, preferably pepsin albumin, and the number of 8-etroguanosine per serum protein molecule is 3 or more, preferably 3 to 10; More preferably, 4 to 6 of the above antigens are provided.
  • the present invention provides a method for producing an antibody recognizing 8-ditroguanine, which comprises a step of immunizing a mammal with the above antigen.
  • FIG. 1 is a view showing the results of direct binding of the antibody of the present invention to 8-nitroguanine as measured by ELISA.
  • N0 2 - Gu o- BSA was 8- two Toroguanoshin binding BSA
  • N0 2 - Gu o- KLH was 8- two Toro guanosine binding KLH
  • R ib- BSA is ribose Bound BSA
  • modified—BSA refers to sodium periodate-treated BSA.
  • FIG. 2 is a diagram showing the cross-reactivity of the antibody of the present invention with 8-nitroguanine analogs by the competitive ELISA method.
  • FIG. 3 is a diagram showing the cross-reactivity of the monoclonal antibody of the present invention obtained in Example 3 to various nucleic acid bases.
  • NO 2 -Gu a is .8-nitroguanine
  • NO 2 -Guo is 8-ditroguanosine
  • Gua is guanine
  • G uo is guanosine
  • OH-Gu is 8 -hydroxyguayun
  • OH-d Gu a is 8—Hydroxydoxy Guanosine
  • X anth represents xanthine.
  • FIG. 4 is a diagram showing the cross-reactivity of the monoclonal antibody of the present invention with various nucleic acid bases by the competitive ELISA method.
  • Ad e is adenine
  • Ad o is adenosine
  • T hy is thymine
  • d T hd is deoxythymidine
  • Ur a is peracil
  • U rd is peridine
  • NO2-Tyr is 3_nitrotyrosine
  • Troymidazole Cyt indicates cytidine.
  • the method for producing the antibody of the present invention is not particularly limited.
  • an antigen-antigen containing serum protein bound to 8-nitroguanosine preferably serum albumin bound to 8-nitroguanosine.
  • the method for producing this antigen is not particularly limited.
  • binding of 8-ditroguanosine to serum proteins such as serum albumin using sodium periodate and sodium cyanoborohydride in a phosphate buffer. Can be manufactured more easily.
  • 8_Nitroguanosine can be easily obtained by subjecting 8-bromoguanosine to nitro-nitrification in dimethylformamide, but it is preferable to purify it by high-performance liquid chromatography before binding to serum proteins. .
  • any protein may be used as long as it is generally a serum protein having no antigenicity.
  • serum albumin in addition to human serum albumin (HSA), serum albumin of other mammals such as human serum albumin (BSA) and Japanese heron serum albumin (RSA), ovalbumin (OVA), and keyhole rimpet hemosyanin (KLH) can be used, but is not limited to these.
  • BSA is most preferred.
  • the nucleobase 8-2-ditroguanine has a limited number of sites that can be chemically modified due to its structure. Since the easily reactable 3-position amino group is thought to play an important role as a nucleobase recognition site by hydrogen bonding, it is bound to serum albumin via the 3-position amino group of 8-ditroguanine. 8–2 It is difficult to produce antibodies that recognize loganin. On the other hand, by binding the sugar moiety of 8-nitroguanosine to serum protein using 8-nitroguanosine, which is a branidium of 8-nitroguanine, the 8-ditroguanine residue is preserved as it is. By immunizing a mammal using the antigen, an antibody recognizing 8 ---- troganin can be efficiently obtained.
  • the rate of introduction of 8-nitroguanosine into serum proteins is not particularly limited, but it is desirable that at least three or more 8-nitroguanosines per serum protein molecule be introduced.
  • the antibody of the present invention can be easily produced by dissolving the antigen in a solvent such as phosphate buffered saline (PBS) and administering the solution to an animal for immunization. If necessary, the solution may be added with an appropriate adjuvant, and then immunized with an emulsion.
  • PBS phosphate buffered saline
  • Adjuvants include water-in-oil emulsions, water-in-oil-in-water emulsions, oil-in-water emulsions, adjuvants such as liposomes and aluminum hydroxide gels, as well as proteins and peptide substances derived from biological components. .
  • adjuvants such as liposomes and aluminum hydroxide gels
  • proteins and peptide substances derived from biological components .
  • Freund's incomplete adjuvant or Freund's complete adjuvant can be suitably used.
  • the adjuvant be appropriately selected so as to enhance the desired immune response.
  • the type of animal used for immunization is not particularly limited, and for example, mouse, rat, magpie, magpie, goat, higgie, etc. can be used.
  • Immunization of the animal may be performed according to methods available in the art, for example, by injecting a solution of the antigen, preferably a mixture with an adjuvant, into the mammal subcutaneously, intradermally, intravenously, or intraperitoneally. It can be carried out. Since the immune response generally varies depending on the type and strain of mammal to be immunized, it is desirable to set the immunization schedule appropriately according to the animal used. The antigen administration is preferably repeated several times after the first immunization.
  • the first immunization is performed using an emulsion obtained by mixing a solution in which the antigen is dissolved in PBS and Freund's complete adjuvant
  • the second and subsequent immunizations are performed using a solution in which the antigen is dissolved in PBS.
  • an emulsion obtained by mixing with Freund's incomplete adjuvant it is advantageous to use Freund's complete adjuvant containing the cells to induce an immune response.
  • the second and subsequent booster immunizations are already expressed at the time of the first immunization rather than producing new antibodies. This is because it is advantageous to use Freund's incomplete adjuvant since it aims at increasing the number of antibodies stored in B cells.
  • the interval between the initial immunization and the booster immunization, and the interval between the booster immunizations and the booster immunization are not particularly limited.
  • the first immunization is for the production of antibodies, and the antibodies usually appear in the blood 4 to 5 days after the first injection of the antigen, and gradually increase in amount to reach a peak around 10 days ago. Therefore, it is preferable that the period from the first immunization to the first booster immunization be at least two weeks.
  • the antibody of the present invention can be obtained from the serum of an immunized animal, but the method is not particularly limited, and any method available to those skilled in the art may be used.
  • antibody purification can be performed by appropriately combining DEAE anion exchange chromatography, affinity chromatography, ammonium sulfate fractionation, PEG fractionation, and ethanol fractionation. it can. Whether or not the antibody recognizes 8 ---- troginin or whether or not the reaction is specific can also be easily confirmed using a method well known to those skilled in the art.
  • the examples of the present specification specifically describe the method for producing the polyclonal antibody of the present invention, a method for immunizing animals, a method for purifying antibodies, and a method for confirming the characteristics of antibodies.
  • the antibody of the present invention can be easily produced by making appropriate modifications or alterations to the method as necessary while referring to the general description and the specific methods in Examples.
  • the antibodies of the present invention include guanine, guanosine, and 8-hydroxyguanosine, which react with 8 --troguanosine, 8- ditroguanine, and a protein in which these are bound. Identifies antibodies that do not react with syn, 8-hydroxysquiguanine and other nucleobases and their derivatives, and proteins bound to them, and, for example, nitroguanine isomers with different nitro-substitution positions and 8_nitroguanine Antibodies that can be used can be preferably used. As described above, the antibody of the present invention recognizes 8-nitroguanine “specifically” when the antibody is partially or substantially completely based on 8-nitroguanine as compared with other substances.
  • a monoclonal antibody produced from a hybridoma produced using lymphocytes of an immunized animal may be used as the antibody of the present invention.
  • a polyclonal antibody having the above properties can be suitably used, and a monoclonal antibody having the above properties is also preferable.
  • the type of mammal used for the preparation of the antibody-producing cells is not particularly limited, and examples thereof include mouse, rat, magpie, magpie, goat, and sheep, and more preferably, rodents such as mouse, rat, magpie, etc. And more preferably a mouse.
  • a mouse of the BALB / C strain is preferable because a myeloma-derived cell line established in the same strain at the time of hybridoma production can be used.
  • spleen cells are excised from the immunized mammal and hybridized with a cell line derived from myeloma to produce a hybridoma.
  • a cell line derived from myeloma it is preferable to use an immunoproducing cell line having a high proliferation ability, and it is preferable that a cell line derived from myeloma be compatible with a mammal from which the immunoproducing cell to be fused is derived.
  • Cell fusion can be performed according to a method known in the art. Examples of the method include a polyethylene glycol method, a method using Sendai virus, and a method using electric current. Which can be adopted.
  • the obtained hybridomas can be grown under conditions commonly used in the art, and a desired hybridoma can be selected while confirming the properties of the antibody to be produced.
  • the cloning of the hybridoma can be performed by a known method such as the limiting dilution method ⁇ ⁇ soft agar method.
  • Hybridomas that produce monoclonal antibodies with desired properties can be confirmed by assaying the ability of the produced antibodies to bind to 8 ---- trojanin using ELISA, RIA, or fluorescent antibody methods. it can.
  • By culturing the hybridomas selected as described above in large quantities it is possible to produce a monoclonal antibody that specifically reacts with 8-2-troginin.
  • the method of mass culture is not particularly limited.For example, a method of culturing a hybridoma in an appropriate medium to produce a monoclonal antibody in the medium, or a method of injecting a hybridoma into the abdominal cavity of a mammal to grow the hybridoma. And a method for producing an antibody in ascites. Purification of the monoclonal antibody can be performed by appropriately combining DAE anion exchange chromatography, affinity chromatography, ammonium sulfate fractionation, PEG fractionation, ethanol fractionation, and the like.
  • a fragment chimera antibody of an antibody having antigen-antibody reaction activity can also be used.
  • the fragment of the antibody is preferably a functional fragment, for example, F (ab ') 2 , Fab', etc., which are obtained by treating the antibody with a protease (for example, pepsin or papain).
  • a protease for example, pepsin or papain.
  • the monoclonal antibody of the present invention can be used as an immobilized antibody fixed on an insoluble carrier such as a solid phase carrier, or as a labeled antibody labeled with a labeling substance. All such immobilized antibodies and labeled antibodies are included in the scope of the present invention.
  • an immobilized antibody can be produced by physically adsorbing or chemically bonding a monoclonal antibody to an insoluble carrier.
  • an insoluble carrier composed of a polymer base such as polystyrene resin, an inorganic base such as glass, a polysaccharide base such as cellulose agarose, and the like can be used.
  • the shape is not particularly limited, and an arbitrary shape such as a plate shape or a bead shape can be selected.
  • Examples of a labeling substance for producing a labeled antibody include enzymes, fluorescent substances, chemiluminescent substances, biotin, avidin, and radioisotopes.
  • a method for binding the labeling substance to the antibody is known to those skilled in the art. Available methods such as the dartalaldehyde method, the maleimide method, the pyridyl disulfide method, and the periodate method can be used.
  • the types of the immobilized antibody and the labeled antibody, and the methods for producing them are not limited to the above examples.
  • detecting a signal generated by reacting an enzyme, a chemiluminescent substance, biotin, and avidin with one or more other substances to detect a labeled antibody are well known to those skilled in the art. It is preferably used for detecting the antibody of the invention.
  • an enzyme a method of measuring enzyme activity using a substrate can be used, and in the case of biotin, it is common to react at least avidin or an enzyme-modified avidin.
  • 8-ditroguanine can be measured.
  • the term “measurement” must be interpreted in the broadest sense, including detection and quantification, and should not be interpreted in any limited manner.
  • the antibody of the present invention it is possible to measure 8-2-troginin contained in a biological sample separated and collected from a human.
  • the type of biological sample separated from humans is not particularly limited, and examples thereof include blood, serum, plasma, lymphocyte culture supernatant, urine, cerebrospinal fluid, saliva, sweat, ascites, amniotic fluid, or cell or organ extract.
  • the measurement target can be the strength of a liquid sample, tissue or cells excised by surgery. The measurement is performed by a known method (for example, “Clinical Pathology Special Issue, Special Issue No.
  • 8-Promoguanosine (0.5 g) and sodium nitrite (0.143 g) were dissolved in 5 ml of dimethylformamide by stirring at room temperature for 3 hours, and then at 37 ° C for 3 days. Thereafter, the reaction was further allowed to stand at room temperature for 2 weeks. 600 ⁇ l of the reaction solution was diluted with 4.04 ml of an aqueous solution containing 6% acetonitrile and 0.01% trifluoroacetic acid, and an aqueous solution containing the same 6% acetonitrile and 0.01% trifluoroacetic acid was used as the mobile phase.
  • the target substance 8 ---- troguanosine
  • the aqueous solution containing 8-nitroguanosine was freeze-dried.
  • the aromatic proton at the 8-position which appeared at 7.98 ppm in normal guanosine, had disappeared. 1 was observed.
  • the aqueous solution of this product showed a UV-VIS spectrum having an absorption maximum at 385 nm derived from the nitro group.
  • HPLC analysis performed by reducing the aqueous solution of this product with sodium dithionite and converting it to 8-aminoguanosine showed that it was consistent with commercially available 8-aminoguanosine. From the above, it was confirmed that 8-nitroguanosine could be purified as a single substance.
  • 8-Nitroguanosine (5 mg) obtained above was dissolved in 0.5 ml of ultrapure water, sodium periodate (10.7 mg) was added, and the mixture was stirred at room temperature for 1 hour. To the reaction solution 1 ⁇ l of Recall was added, and the mixture was further stirred for 30 minutes to decompose excess sodium periodate. To a solution of BSA (5 mg) in a 0.1 M phosphate buffer (H 7.0) was added the above-mentioned aqueous solution of guanosine at the two-mouthed mouth. After incubating at room temperature for 1 hour, 2.5 mg of sodium cyanoborohydride was added, and the mixture was stirred at 4 ° C as it was.
  • the reaction solution was dialyzed against PBS at 4 ° C, filtered through a gel filtration column (2 x 30 cm) packed with Sephadex G-25, eluted with PBS, and separated into 2 ml fractions. separated. The absorbance at 280 nm and 395 nm of the obtained fraction was measured, and the fraction containing the target substance was collected and dialyzed against ultrapure water to obtain the antigen of the present invention.
  • 8-nitroguanosine binding BSA; 8-NO2-Gua-BSA 8-nitroguanosine binding BSA; 8-NO2-Gua-BSA
  • Example 2 The antigen obtained in Example 1 dissolved in PBS so as to have a lmg / m1 volume of 500 ⁇ l was taken in a syringe.Freund's complete adjuvant was taken in a volume of 750 ⁇ l and connected to another syringe. The emulsions were created by moving them alternately. Two Japanese white herons were used as animals for antibody production, and this emulsion was injected subcutaneously at the back of the heron (primary immunization).
  • Example 1 Two weeks later, the antigen of Example 1 was dissolved in PBS to lmgZm 1 500 ⁇ l and incomplete Freund's adjuvant Using 750 ⁇ l, an emulsion was prepared by injecting the syringe alternately as described above, and injected to obtain a booster immunization. This boost was repeated every two weeks.
  • a small amount of blood is collected to determine whether the antibody reacts with 8- to 2-mouth guanosine-conjugated BSA (8-NO2-Gua-BSA). was confirmed by the ELISA method.
  • the production of polyclonal antibodies was constant by the above confirmation (where the production of polyclonal antibodies did not fluctuate compared to two weeks ago, that is, the antibodies confirmed one week after a certain booster immunization) And the antibody production confirmed one week after the next booster performed two weeks after the booster became the same), and blood was collected from the two herons and serum was obtained from this blood. After that, the polyclonal antibody of the present invention was obtained from the serum.
  • the procedure for obtaining the polyclonal antibody of the present invention was performed as follows. First, a large amount of blood was collected from the heart of a Japanese White Heron, kept at 37 ° C for 1 hour, and allowed to stand at 4 ° C. This was centrifuged at 3000 rpm for 10 minutes, and the obtained supernatant was passed through a sterile filter. Thereafter, the antibody was purified using an affinity column described below.
  • the bound antibody was eluted with 1M glycine-hydrochloric acid (pH 2.0) using Seikagaku Corporation's Protein A cell mouth fine (5 ml, 1 volume) and passing through serum diluted with PBS according to the company's protocol. Immediately after neutralization, an IgG fraction was obtained.
  • a BSA cell port fine column was prepared according to the company's protocol, in which BSA was bound to the company's formyl cell port fine (5 ml volume), and the anti-BSA antibody mixed with the polyclonal antibody was passed through the IgG fraction described above. Was removed by adsorption.
  • an 8-nitroguanosine cell-open fine column was prepared by binding 8-nitroguanosine to the company's aminocell-open fine (5 ml capacity), and an antibody that reacts with 8-ditroguanine was passed through the antibody. After binding, the bound antibody was eluted with 1 M glycine monohydrochloride (pH 2.0), immediately neutralized, and then dialyzed against PBS at 4 ° C. to purify the anti-8-trojanin antibody. In addition, it was bound to the cell opening fine column by elution operation with 1M glycine-hydrochloric acid (pH 2.0). This column can be used only once, since 8-nitroguanosine is cleaved by nucleobases with acid and elutes with the antibody. In addition, dialysis is required to remove 8-nitroguanine released along with the antibody.
  • N0 2 — Gu o — KLH was prepared by replacing BSA with KLH
  • Gu o — B SA was prepared by replacing 8-guanosine with troguanosine
  • Rib-BSA was prepared by replacing 8 — 2
  • the procedure was as described above, except that troguanosine was replaced by report.
  • ELISA was performed as follows. N_ ⁇ 2 - Gu o-B SA, N0 2 _Guo- K LH, Gu o- BSA, dissolved R i b_BSA, mo dified- BSA, in the untreated B SA 50 mM carbonate buffer (pH 9 7.) Each was 5 ⁇ g / m 1.
  • the polyclonal antibody of the present invention recognizes the nucleobase portion of 8--troguanosine, that is, 8-ditroginin, regardless of the type of protein, BSA and KLH, and does not react with a normal guanosine-bound protein. It has been shown. In addition, it was confirmed that the protein did not bind to the protein modified by opening the report, which is the sugar structure part, or to the side reaction modified structure by sodium periodate used for the binding.
  • the polyclonal antibody of the present invention recognizes 8-ditroguanine, which is a nucleic acid base of 8-nitroguanosine, and uses a normal guanine base, and a reagent used in a ribose II reaction, which is a cross-linking structure used for protein binding. It was confirmed that it did not recognize by-products derived from sodium periodate and ethylenic alcohol).
  • test substances include 8-nitroguanosine and 8-nitroguanine as a free form of its nucleobase, guanine as a normal nucleobase, and 8-hydroxydoxyguanosine as an indicator of gene damage caused by active oxygen.
  • 3-hydroxytrotyrosine a protein-damaging substance caused by 8-hydroxyguanine and peroxynitrite, which was released from glycerol, was used as a competitor.
  • Test Example 1 mentioned methods as well as 8 _N0 2 - Gu o- B SA 5 ⁇ g / m 1 and was the one by 1 00 mu 1 shall be added to the EL ISA plates 4 ° C De ⁇ and location And solidified. After washing 3 times with 0.4 ml of PBS containing 5% Tween 20 (PBS-T), add 5% OmM carbonate buffer (pH 9) containing 0.5% gelatin enzyme hydrolyzate to each well. 7) 200 ⁇ 1 was added, and the mixture was allowed to stand at room temperature for 1 hour, and blocked to prevent the subsequent adsorption of non-specific antibodies.
  • the polyclonal antibody of the present invention has a very high specificity for 8-ditroguanine, which is the base structure of 8-ditroguanosine, does not recognize normal guanosine, and has a DNase caused by oxidative stress. It was shown that it did not cross-react with 8-hydroxyguanine, which is a typical substance of A damage.
  • the antibody of the present invention is a new gene-damaging substance that is damaged by peroxynitrite produced by nitric oxide and active oxygen produced in large quantities by inflammation and the like in vivo, and is a new gene-damaging substance. It was proved that it can be used for measurement of
  • Immunohistochemical staining was performed using biological samples.
  • influenza infection model which is created by intranasally inoculating mice with influenza virus, a large amount of nitric oxide synthase (iNOS) is expressed in the trachea of the lung, and nitric oxide is produced.
  • iNOS nitric oxide synthase
  • peroxynitrite is produced by reacting with superoxide produced during the production of peroxynitrite. Therefore, in lung tissue, it is expected that guanine bases of DNA or RNA in cells will be converted to nitro by the peroxynitrite to produce 8-nitroguanine.
  • Lung tissues of mice infected with Influenza were removed, fixed with periodate-lysine-paraformaldehyde (PLP), and prepared and fixed to 5-6 ⁇ thick sections. The preparation was washed by immersing it in PBS at 4 ° C for 3 times for 5 minutes. In order to suppress non-specific reactions, the cells were incubated with 9.5 ml of 0.5% BSA-PBS supplemented with 0.5 ml of hidge serum for 20 minutes at room temperature. Next, 1 to 2 drops of the antibody of the present invention (15 ⁇ g / m 1 (PBS solution)) was dropped on the tissue specimen, and the ink was incubated at room temperature for 60 minutes. I did it.
  • PBS solution periodate-lysine-paraformaldehyde
  • the antigen obtained in Example 1 was dissolved in PBS so as to be 2 mg / m 1 .500 ⁇ l was placed in a syringe, and Freund's complete adjuvant 7500 ⁇ l was further taken and connected to another syringe. Were alternately moved to create an emulsion. Two female 5-week-old BALB / c mice were used as animals for preparing antibodies, and the mice were injected with the emulsion at several sites intradermally on the back (primary immunization). Two weeks later, the antigen was prepared using lmgZm1, 500 ⁇ l and Freund's incomplete adjuvant 7500 ⁇ l, and an emulsion was prepared by alternately moving the syringe as described above.
  • the reactivity of the monoclonal antibodies was tested in a competitive ELISA. 8—N ⁇ 2 — Gua— BSA is dissolved in 50 mM carbonate buffer ( ⁇ ⁇ 9.7) to make each 1 ⁇ g / ⁇ , and 0.1 ⁇ l each in a 96-well microtube. Each plate of the titer plate was incubated at room temperature for 1 hour to immobilize it. P including 0.0 5% Tween 20
  • Competitors include 8-nitroguanine, 8-nitroguanosine, guanine, guanosine, 8-hydroxyguanine, 8-hydroxydoxyguanosine, xanthine, adenine, adenosine, thymine, deoxythymidine, peracyl, ⁇ lysine, cytidine, 3-nitrotyrosine and nitroimidazole were studied.
  • the monoclonal antibody obtained in Example 3 was 8-nitroguanine, 8 —Reacted strongly with nitroguanosine, but did not react with other nucleobases at all and did not react with normal nucleobases such as guanine or guanosine (Figs. 3 and 4).
  • the monoclonal antibody of the present invention recognizes 8-ditroguanosine and its free nucleobase, 8-ditroguanine, but has normal guanine base, adenine, adenosine, thymine, thymidine, cytosine, cytidine, and active oxygen. It does not react with 8-hydroxyguanine or 8-hydroxydeoxyguanosine, which are representative indicators of gene damage caused by guanosine. In addition, it does not cross-react with 3-nitrosine, a protein nitration product, or nitroimidazole, whose chemical structure is partially similar to that of 8-ditroguanine. Recognizing the structure, it is useful for the specific detection of 8-ditroguanosine in vivo. Industrial applicability
  • the antibody of the present invention is useful as a means for simply and specifically measuring 8 ---- troguanine in a living body.

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Abstract

L'invention concerne un anticorps capable de reconnaître la 8-nitroguanine pouvant être produit à l'aide, par exemple, d'un antigène contenant de la 8-nitroguanosine liée par sa partie sucre à une protéine de sérum telle que l'albumine sérique bovine; et un procédé de mesure de 8-nitroguanine contenue dans un échantillon biologique à l'aide de l'anticorps.
PCT/JP2003/006665 2002-05-28 2003-05-28 Anticorps capable de reconnaitre la 8-nitroguanine Ceased WO2003099869A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU2003241827A AU2003241827A1 (en) 2002-05-28 2003-05-28 Antibody capable of recognizing 8-nitroguanine
JP2004508123A JPWO2003099869A1 (ja) 2002-05-28 2003-05-28 ８−ニトログアニンを認識する抗体

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP2002-153340 2002-05-28
JP2002153340 2002-05-28
JP2003021378 2003-01-30
JP2003-21378 2003-01-30

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
JP2006298869A (ja) * 2005-04-25 2006-11-02 Kumamoto Univ 抗８−ニトロサイクリックグアノシン３’，５’−一リン酸抗体
JP2007091622A (ja) * 2005-09-28 2007-04-12 Univ Nagoya 炎症反応に関連した酸化的損傷のマーカー及びその利用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SODUM K S, FIALA E S: "Analysis of peroxynitrite reactions with guanine, xanthine and adenine nucleosides by high-pressure liquid chromatography with electrochemical detection: C8-nitration and -oxidation", CHEM. RES. TOXICOL., vol. 14, no. 4, 2001, pages 438 - 450, XP002971870 *
YERMILOV V. ET AL.: "Formation of 8-nitroguanine in DNA treated with peroxynitrite in vitro and its rapid remocal from DNA by depurination", FEBS LETTERS, vol. 376, no. 3, 1995, pages 207 - 210, XP002971869 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006298869A (ja) * 2005-04-25 2006-11-02 Kumamoto Univ 抗８−ニトロサイクリックグアノシン３’，５’−一リン酸抗体
JP2007091622A (ja) * 2005-09-28 2007-04-12 Univ Nagoya 炎症反応に関連した酸化的損傷のマーカー及びその利用

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