WO1995009187A1 - Monoclonal antibody and assay of denatured dna by using the same - Google Patents

Monoclonal antibody and assay of denatured dna by using the same Download PDF

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Publication number
WO1995009187A1
WO1995009187A1 PCT/JP1994/001644 JP9401644W WO9509187A1 WO 1995009187 A1 WO1995009187 A1 WO 1995009187A1 JP 9401644 W JP9401644 W JP 9401644W WO 9509187 A1 WO9509187 A1 WO 9509187A1
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Prior art keywords
dna
monoclonal antibody
antibody
denatured
ultraviolet
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PCT/JP1994/001644
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French (fr)
Japanese (ja)
Inventor
Hiroaki Kohno
Yukiko Noguchi
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Kyowa Medex Co., Ltd.
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Publication of WO1995009187A1 publication Critical patent/WO1995009187A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • 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 a monoclonal antibody that specifically binds to a denatured portion of DNA generated by irradiating DNA with ultraviolet light, and a method of using the antibody.
  • a labeling method there is a method in which DNA used as a probe is irradiated with ultraviolet light to form a certain degree of denaturation of the nucleic acid present in the DNA, and the DNA itself is haptenized. Those formed between thymines are particularly useful.
  • a method for detecting the probe it has been considered to use an antibody obtained by immunizing DNA irradiated with ultraviolet rays [Science, 153, 1666-1667 (1966) and Eurobian Journal. 'Ob' Biochemistry Story (Bur. J. Biochem.), 142, 313-316 (1984)] 0
  • the polyclonal antibody produced by the former method specifically reacts with the hydrolyzed DNA denatured by ultraviolet irradiation
  • the monoclonal antibody produced by the latter method produces ultraviolet light. Reacts specifically with single-stranded DNA denatured by irradiation. Therefore, using these antibodies, it is possible to detect a denatured double-stranded DNA generated in vivo by receiving ultraviolet light or a double-stranded DNA obtained by hybridizing a probe denatured with ultraviolet light. S difficult.
  • the present invention provides a monoclonal antibody (hereinafter, also referred to as anti-ultraviolet-denatured DNA) that binds to DNA having thymine dimer (hereinafter, referred to as denatured DNA) generated by irradiating the DNA with ultraviolet light, and denaturation using the monoclonal antibody.
  • the present invention relates to an immunochemical assay for DNA and an immunoassay for DNA or RNA using the denatured DNA as a probe.
  • the monoclonal antibody of the present invention is a monoclonal antibody that binds to both single-chain DNA and double-chain DNA having a thymine dimer.
  • the thymine dimer may be a continuous thymine-thymine dimer or a thymine-thymine dimer.
  • Examples of the binding type include cyclobutane-type thymine dimer and (6-4) photoproduct-type thymine dimer.
  • the cyclobutane-type thymine dimer is formed by irradiating DNA containing two or more thymines with ultraviolet light having a wavelength of 254 ⁇ m or 313 ⁇ m in the presence of acetate phenone, resulting in a cis-syn structure. And trans-syn structure [mola, AA, Photochem. P hotobiol., 9 ⁇ 291-2J4 (196i))] D
  • Photoproduct-type thymine dimer is produced by irradiating DNA containing 254 wavelengths of 254 nm to thymine-containing DNA. It changes into another substance [Taylor, JS. & Cohrs, MP, J. Am. Chem. Soc. O.2834-2835 (1987)]. Cyclobutane thymine dimer and
  • IgG 1 A yields to class, has an isoelectric point of (J.5 ⁇ ⁇ .5, molecular weight of 1). (] 5 ⁇ 0 0 Sat 1 ⁇ ⁇ ⁇ , showing physicochemical properties, IgG 1 ⁇ class, isoelectric point G. G ⁇ 5. ...
  • the monoclonal antibody of the present invention is produced, for example, from hybridoma cells created by the cell fusion method.
  • the antibody-producing cells and the myeloma cells immunized with the DNA denatured by ultraviolet rays are fused with myeloma cells to form hybridoma cells, the hybridoma cells are monocloned, and the DNA denatured by ultraviolet rays (below, ultraviolet denatured DNA) It is produced by selecting a clone that produces an antibody with high specificity.
  • UV-denatured D ⁇ As an immunogen, This immunogen is produced by irradiating D ⁇ with ultraviolet light.
  • the DNA to be irradiated with ultraviolet light may be obtained by synthesis or from biological material, and is preferably DNA having at least 10 bases, more preferably at least 100 bases. Is used. Both the method of obtaining by synthesis and the method of obtaining from biological materials are known methods [Nucleic acid ⁇ (New Biological Experiment Lecture ME 2), 253, 1992, edited by The Biochemical Society of Japan, ed. 6, 1990, Kodansha] can be used.
  • UVB ultraviolet irradiating device
  • UVC ultraviolet lamp
  • UV-denatured DNA Even at an irradiation dose of 1 JZm 2 or less in the irradiation of UV light.For sufficient antigenicity, irradiate at a dose of 100 JZm 2 or more. It is desirable.
  • the UV-denatured DNA used for the immunogen may be prepared by irradiating the DNA with ultraviolet light, and then purified.
  • the UV-denatured DNA may be purified by irradiating the purified DNA with UV light. It may be made;
  • UV-modifying DNA may be used as an immunogen as it is, but in order to obtain sufficient immunogenicity in vivo, it has been combined with the so-called carrier K and used.
  • the carrier K include proteins in living tissues such as albumin, albumin, globulin, keyhole rinsin mosinin, and thyroglobulin, and synthetic polymers such as ⁇ .
  • UV-modified DNA and the amino group of serum albumin can be linked by a divalent crosslinking agent.
  • UV-denatured DNA can be adsorbed to methylated glucose serum albumin and used as an immunogen.
  • mice As animals immunized with the immunogen, mice, rats, hamsters, rabbits, guinea pigs, goats, sheep, and the like are used, and mice and rats are preferably used.
  • an immunization method for example, a method is used in which the immunogen is emulsified into a complete or incomplete adjuvant of a font and administered intraperitoneally, subcutaneously or intramuscularly several times at regular intervals at regular intervals.
  • Sources for collecting antibody-producing cells include spleen, lymph node, and peripheral blood of immunized animals.
  • cells obtained by direct immunization in vitro of antibody-producing cells from spleen, lymph nodes, peripheral blood, etc. of non-immunized animals [Arai, Ota, Experimental Medicine J1. , 43 (1988)].
  • the myeloma cells used for cell fusion between the antibody-producing cells and the myeloma cells are not particularly limited, but it is preferable to use a cell line derived from an animal of the same species as the antibody-producing cells. In order to efficiently select only cells that have been appropriately subjected to cell fusion, those having a specific drug marker are preferable.
  • 8-azaguanine-resistant myeloma cells cannot grow in a medium containing hypoxanthine, aminopterin and thymidine (HAT medium), but cells in which these cells and positive cells are fused cannot be grown in H HT medium. It is preferable because it can grow and can be distinguished from the fused bone marrow femoral cells.
  • Cell fusion is performed by the method established by Kofi 1 er and Mi 1 sin “NaI.urc, impart)-7 (1975)].
  • antibody-producing cells and bone marrow cells can be expressed in 1 ⁇ to 3: 1.
  • the fusion method using electric pulses [Okochi
  • the cells that have undergone cell fusion are suspended in a selection medium, placed in a culture container such as a 96-microtiter plate, and cultured under conditions that allow only the fused cells to selectively grow. At the stage where only the fused cells have selectively grown, select only those cells that are producing antibodies against UV-denatured DNA.
  • a selection method there is used a method of examining the presence or absence of a target antibody in the culture supernatant of the fused cells using, for example, a method such as Enzyme Immunoassay or Radioimmunoassay.
  • the selected cells are monocloned using, for example, the limiting dilution method and the soft agar method, and the above selection method is repeated to obtain a hybridoma cell line that produces an anti-UV-denatured DNA monoclonal antibody. it can.
  • hybridoma cell lines producing anti-ultraviolet-denatured DNA monoclonal antibody include hybridomas KTM-51 and KTM-53.
  • Hypri-Dorma KTM-51 and KTM-53 have been deposited as FERM BP-4422 and FERM BP-4423 on September 2, 1993 with the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology.
  • the monoclonal antibody of the present invention can be obtained by culturing a hybridoma cell line in an appropriate medium and collecting the culture solution, or by transplanting the cell line into the peritoneal cavity of an animal and growing it in ascites, and then collecting the ascites. Then, it can be obtained from the obtained culture solution or ascites. Antibodies obtained from the culture solution or ascites can be purified and used as necessary. Examples of the purification method include ammonium sulfate fractionation, ion exchange chromatography, gel filtration chromatography, affinity chromatography using protein A and protein G, affinity chromatography using a gel on which an antigen is immobilized, and the like. Is used.
  • a secondary antibody is labeled with a substance such as a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or a metal, and is detected by the labeled substance.
  • a substance such as a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or a metal
  • the monoclonal ⁇ -nal antibody of the present invention is used for detecting DNA having an ultraviolet denaturation site.
  • any DNA such as single-stranded DNA, double-stranded or triple-stranded DNA may be used as long as it is ultraviolet-denatured DNA, but double-stranded DNA is preferably used.
  • the ultraviolet-denatured DNA used as the antigen may be thymine dimer by irradiating ultraviolet rays to DNA, for example, DNA obtained by synthesis, DNA present in an organism, or DNA extracted from an organism. Any DNA may be used as long as it is a DNA that has caused the above.
  • the detection of ultraviolet-denatured DNA using the monoclonal antibody of the present invention can be performed by any method as long as it is an immunological measurement method, but preferably, a competition method and a sandwich method are used.
  • the competitive methods include: (1) a method in which the labeled antigen competes with the antigen for binding to the antigen in the sample or the standard substance; and (2) the binding of the labeled antibody is derived from the sample in the liquid phase or the antigen of the standard substance. And (3) a method in which the binding to the immobilized antibody competes with the labeled antigen in the sample or the antigen of the standard substance.
  • the primary antibody solid phase in which the antibody is bound to an appropriate solid phase such as beads, tubes, and plates reacts with the standard substance ⁇ the antigen in the sample, and then the antigen bound to the solid phase antibody.
  • an appropriate solid phase such as beads, tubes, and plates reacts with the standard substance ⁇ the antigen in the sample, and then the antigen bound to the solid phase antibody.
  • a secondary antibody, and a method of detecting the formation of a formed ternary complex of the solid phase antibody, the antigen and the secondary antibody is generally used.
  • DNA or RNA having a specific base sequence or DNA or RNA containing a specific base sequence can be qualitatively or quantitatively measured. That is, first, ultraviolet rays are applied to D ⁇ ⁇ or R ⁇ ⁇ of the specific nucleotide sequence to be detected or DN ⁇ ⁇ ⁇ having a sequence complementary to D ⁇ ⁇ or RN ⁇ ⁇ containing the specific nucleotide sequence. Prepare UV-denatured DNA and use it as a probe. Next, the DNA or RNA to be detected and the probe are combined due to the affinity of the sequence E due to the non- ⁇ / ⁇ property, and the D ⁇ -probe or R ⁇ -probe complex is formed into a probe. The immunochemical detection can be carried out in the same manner as described above using a monoclonal antibody against UV-denatured DNA.
  • the DNA and RNA containing a specific nucleotide sequence detected using the monoclonal antibody of the present invention may be obtained by synthesis or from biological material.
  • the DNA and RNA those existing in an electrophoresis gel, on a tissue section, in a cell, or on a plotting membrane after performing dot blotting, southern blotting, or northern blotting, etc. You may. BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1 shows a calibration curve of a Rij contact competitive enzymimnoassy of Example 3.
  • ⁇ indicates the case of using the monoclonal antibody KTM-51
  • ⁇ indicates the case of using the monoclonal antibody KTM-53
  • ⁇ indicates the case of using the anti-ultraviolet-denatured DNA poly- ⁇ antibody.
  • UZmg ultraviolet denatured DNA in the figure, defining the amount of ultraviolet denatured DNA to generate when irradiated with 1 0 4 UV J / m 2 to 1 mg of the standard normal DNA as 10 3 U Zmg.
  • FIG. 2 shows the relationship between the amount of ultraviolet irradiation and the amount of ultraviolet-denatured DNA by the fill contact competitive enzymimnoassy of Example 3.
  • ⁇ indicates the use of the monoclonal antibody KTM-51
  • ⁇ indicates the use of the monoclonal antibody KTM-53
  • ⁇ indicates the use of an anti-ultraviolet denatured DNA polyclonal antibody.
  • the definition of UZmg of the UV-denatured DNA in the figure is the same as that in Fig. 1.
  • FIG. 3 shows a calibration curve of a sandwich-type enzyme in the case of irradiating ultraviolet rays to intracellular DN in Example 4.
  • ⁇ shows the results obtained using the monoclonal antibody KTM-51, shows the results obtained using the monoclonal anti-vaccine KTM-53, and ⁇ shows the results obtained using the anti-UV denatured DNA polyclonal antibody.
  • the definition of UZ mg for UV-denatured DNA in the block is the same as that in Fig. 1.
  • FIG. 4 shows the relationship between the amount of ultraviolet irradiation and the amount of ultraviolet-denatured DNA in the case where the intracellular DNA of Example 3 was irradiated with ultraviolet light.
  • ⁇ indicates that the monoclonal antibody KTM-51 was used, and ⁇ ⁇ indicates that the monoclonal antibody KTM-53 was used.
  • the definition of UZmg of the UV-denatured DNA in the figure is the same as the definition in FIG. The best form to carry out the framing
  • Example 1 Preparation of anti-ultraviolet-denatured D N ⁇ monoclonal ⁇ -null antibody
  • the antigen for immunization was prepared as follows using purified salmon sperm DN ⁇ .
  • This UV-denatured DNA was used as standard UV-denatured DNA, and immediately stored in a light-shielded condition at 140 ° C.
  • G-male male B1bZc mice were subcutaneously injected into the back of the mouse with lml of an emulsion mixed with an equal amount of immunogen and Freund's complete juvant (Seikagaku Corporation), and then twice every three weeks thereafter.
  • 0.5 ml _ of an emulsion mixed with an equal amount of an immunogen and an incomplete fin fin adjuvant (Seikagaku Kogyo Co., Ltd.) was administered.
  • PBS buffer (1) was injected from the tail vein.
  • the immunogen dissolved in PH 7.4) was administered to 0.1 mg mgZ animals.
  • mouse mye n-ma cells P3U1 were cultured in GIT medium (Wako Pure Chemical Industries) until the logarithmic growth phase, and the obtained cells were collected three times in serum-free RPMI-164 medium. Washed by centrifugation.
  • Antibody-producing cells and mouse mye ⁇ -ma cells P 3 U1 were mixed at a ratio of 10: 1, centrifuged at 1200 rpm for 5 minutes, and the supernatant was removed. After centrifugation, add 1% 1 of 50% polyethylene glycol 150 solution (Boehringer's Mannheim) to the cell residue slowly, and add serum-free RPM1-1-640 medium 5 Om 1 gradually. And the cells were suspended.
  • the suspension 1 2 0 0 r pm at removing the supernatant for 5 minutes and centrifuged, the cell residue HAT medium (1 x 1 0- 4 M hypoxanthine, 4 x 1 0 one 7 M Aminoputerin, 2 X 1 0- 5 M thymidine was suspended so as to be GIT medium) ⁇ This 1 X 1 0 6 cells Zm 1 comprising, by 2 0 0 1 Z ⁇ Le this into 9 6 ⁇ Weru microtiter first plate dispensing did. The cells were cultured as they were in a carbon dioxide gas incubator at 37: in air containing 5% carbon dioxide. When observed 10 days later, colonies of hybridomas were observed in all the pellets.
  • Cells containing cells producing the target antibody were selected by the following method.
  • the plate was washed five times with a PBS buffer containing Tween 20 at 0.05%, and then 50 ⁇ P ⁇ D-labeled anti-mouse immunoglobulins 100 egret IgG (Dakosha Co., Ltd.) ) was added and reacted at room temperature for 1 hour.
  • the plate was washed 5 times with a PBS buffer containing Tween 20 at 0.05%, and MCDP (10-N-methylcarbamoyl-3,7-dimethylamino-10H-phenothiazine) was added.
  • the absorbance at 660 rim was measured for each well using a microplate reader (Corona, Inc., 120). As a result, the number of gels with strong reactivity (the ones showing absorbance of 1.0 or more) was 8 ⁇ out of 576 ⁇ ⁇ .
  • Cloning was performed by the limiting dilution method.
  • D diluting the strong reactivity of the cells of the resulting the in Weru shown in section so as to be 0.5 or Zm 1 in GIT medium containing 1 x 1 0 7 or Zm 1 thymocytes.
  • the solution was dispensed into a 96-well microtiter plate at a rate of 200 ⁇ ⁇ Z-well, and cultured in a carbon dioxide incubator in air containing 37: 5% carbon dioxide. 10 to 14 days after the start of culture, each pinil was observed.
  • One colony was selected as a growing colony, and the culture supernatant of the selected cell was measured by the method described in section (d).
  • a well containing the antibody-producing cell line was selected. The same procedure was repeated twice to establish a monoclonal antibody-producing cell line that stably produces the target antibody. A total of eight cell lines were obtained.
  • the molecular weight was determined by SDS-polyacrylamide gel electrophoresis (Pharmacia, First System) using a molecular weight marker of Bi0-Rad, and the isoelectric point was determined by isoelectric focusing (Pharmacia).
  • the first isoelectric point marker was used for the isoelectric point marker and the immunoglobulin subclass was measured using the Zymmet monoclonal antibody typing kit.
  • the binding constant of the cyclobutane-type thymine dimer was determined by irradiating 100000 JZm 2 with UV light of 250 nm, referring to the method of Hoylaerts (J. Immuno.Methods, 126, 253 (1990)). during DN a 1 mg 1 X 1 0 - was determined as thymine dimer of 7 mo 1 s is present.
  • the monoclonal antibody KTM-51 produced by the hybridoma KTM-51 and the monoclonal antibody KTM-53 produced by the hybridoma KTM-53 had the following properties.
  • Irradiation method is a method of irradiating only 254 nm ultraviolet light, a method of irradiating with 254 nm ultraviolet light, and an additional irradiation of 313 nm ultraviolet light, 1 () mM acetophenone is included in the DNA solution, Three methods of irradiating 3 13 nm ultraviolet light were used, and Poly [!
  • the mixture was dispensed at 0 ° C. at 4 ° C. overnight. After washing the plate three times with PBS buffer, dispens a 250 ⁇ l buffer solution containing 5% skim milk at room temperature. Then, each well was washed three times with a PBS buffer to prepare a reaction ffl plate. Dispense the above-mentioned UV-denatured DN ⁇ or unirradiated DNA diluted to 1 ⁇ 0 ⁇ g Zm 1 with TE buffer or TE buffer into 50 ⁇ Z ⁇ ⁇ of the reaction river plate, and further dispose the plate.
  • Monoclonal antibody while stirring include 100 ng / m 1 of KTM-51 or KTM-53 0.1 Add 0.1% BSA-PBS buffer at 5 ⁇ 1 / ⁇ , mix and mix at 4 t overnight or at room temperature At 3 o'clock] ⁇
  • the plate was washed 5 times with BS mild solution containing Tween 2 2 (5) .5%, and then peroxidase (PD ⁇ ) -labeled anti-mouse immunoglobulin maize IgG ( (Dako Co., Ltd.) was added in 5 51 Zwell portions, and reacted at room temperature for 1 hour.
  • the plate was washed five times with a PBS buffer containing Tween 20% 5%, and then the MCDP solution was added in 50 nl Z-wells, and the reaction was performed for 3 minutes at room temperature.
  • the stop solution was added in 501 ml portions, and the absorbance at 660 nm was measured using a microplate reader. The relative absorbance was calculated as a percentage of the obtained absorbance when the absorbance when using only the TE buffer was 100%.
  • the antibody solution contains 0.1% of anti-UV denatured DNA polyclonal IgG obtained in specific examples, 0.1 ng Zm1, 0.1% of methylated BSA and 0.1% of normal DNA.
  • the same procedure was followed except that the POD-labeled anti-mouse immunoglobulin's egret IgG was replaced with a POD-labeled anti-mouse immunoglobulin's goat antibody (Dako) in PBS buffer.
  • Dako POD-labeled anti-mouse immunoglobulin's goat antibody
  • the DNA that had been irradiated with UV light of 2 ⁇ ) nm was strongly irradiated with DNA that had been additionally irradiated with UV light of 3 nm13 nm.
  • the reaction was even stronger than that of DN ⁇ , and the purple light produced by irradiating the D ⁇ wave with 1 () mM of cetophenone with 313 Tim ultraviolet light. Strongly anti J wick in the line denatured DNA also:.
  • the first ultraviolet irradiation polynucleotide there was reactive with monochromator Naru anti rest of KiKiwamu ⁇ was only 1 o I y I path "! .
  • UV irradiation rat liver sections ultraviolet in order to prevent drying non-irradiated rat liver sections was added dropwise distilled water traces, 3 ultraviolet radiation 0. 5 mWZcm 2 using a commercially available germicidal UV lamp (Toshiba) 0 It was produced by irradiation with 0 J / m 2 . The ultraviolet intensity was measured with an ultraviolet dosimeter (Topcon Corporation).
  • a PBS buffer solution containing various concentrations of the monoclonal antibody KTM-51 or KTM-53 and a PBS solution containing 0.1% BSA was added dropwise to the slides on which the liver sections of the UV-irradiated and non-irradiated rat were fixed, respectively. For 3 hours. After the reaction, the slide glass was immersed and washed 5 times with a PBS gentle S solution, washed, and then a POD-labeled anti-mouse immunoglobulin sperm antibody was dropped on the slide glass and reacted at room temperature for 1 hour. After After the reaction, the slide Douglas was 5 times immersed AraiKiyoshi with PBS buffer, the di ⁇ amino benzidine a 0.
  • the antibody solution was prepared using the anti-UV denatured DNA polyclonal antibody obtained in Comparative Example. 0.1% of each IE concentration and methylated BS ⁇ and 0.1% of standard positive DNA in 0.1% and 0.1% BSA combined with PBS buffer, P ⁇ D-labeled anti-mouse immunoglobulin Principe IgG Liver sections were stained using the same method except that POD-labeled anti-Pseudosagiminog prince-goat antibody was used, and the liver sections were observed. The results are shown in Table 2. Table 2
  • the plate was washed five times with a PBS buffer containing Tween 20 at 0.05%, and then peroxidase (POD) -labeled anti-mouse immunoglobulin zoo- egret IgG (Dako) was added to the plate. Then, the mixture was added at room temperature and reacted for 1 hour at room temperature. After the reaction, the plate was washed five times with a PBS buffer solution containing Tween 20 at 0.05%, and the MCDP solution was added at a volume of 501 ⁇ , and allowed to react at room temperature for 30 minutes. The reaction was terminated by adding 50 ⁇ l of the reaction stop solution at a time.
  • POD peroxidase
  • the absorbance at 660 nm was measured for each gel using a microplate reader.
  • the relative absorbance was calculated as a percentage of the obtained absorbance when the absorbance when using only the TE buffer was 100%.
  • an antibody solution containing 100 ng / 1 of anti-ultraviolet-denatured DNA polyclonal IgG obtained from a specific antibody solution ( ⁇ 0.1% methylated BS ⁇ , 0.1% normal DNA Containing 0.1% BS (PBS buffer solution) instead of POD-labeled anti-mouse immunoglobulin zoo egret IgG
  • Fig. 1 shows the calibration curve
  • Fig. 2 shows the relationship between the amount of UV irradiation and the amount of UV-modified DNA measured by this measurement system.
  • UV denatured DNA can be quantified using the monoclonal antibody of the present invention.
  • the use of the monoclonal antibody of the present invention produced UV-denatured DNA in proportion to the amount of UV light irradiated to the DNA, while a calibration curve was prepared using an anti-UV-denatured DNA monoclonal antibody. In this case, it was shown that a 10-fold or more amount was required to obtain sensitivity equivalent to that of the monoclonal antibody of the present invention.
  • the anti-UV denatured DNA-Egret polyclonal antibody was used, no linear correlation was observed between the amount of irradiated UV and the amount of UV-modified DNA based on the calibration curve.
  • A431 cells were monolayer cultured on a 5 cm diameter culture dish in a D-MEM medium (Gibco) containing 10% FCS. The medium was removed, the cells were washed three times per dish Te in PBS buffer, placed in the dish PBS buffer so that the depth 2 mm, and the ultraviolet 0. 3 mWZ cm 2 was irradiated for various times . Immediately after the ultraviolet irradiation, the PBS buffer was removed, and the cells were frozen together with the dish using liquid nitrogen. Thaw the frozen cells at room temperature, and add a lysate [1 OmM Tris Z-HCl buffer (pH 8.0) containing 1 mM ethylenediaminetetraacetic acid and 1% SDS] to this.
  • a lysate [1 OmM Tris Z-HCl buffer (pH 8.0) containing 1 mM ethylenediaminetetraacetic acid and 1% SDS]
  • the cell lysate was collected in a tube with a microphone opening. DNA was purified and recovered from the recovered cell lysate using SepaGene Kit (Sanko Junyaku). The DNA solution obtained by dissolving the collected DNA in TE buffer is quantified based on the measured absorbance at 26 O nm, and is frozen and stored at 180 ° C until it is used for the assay. did.
  • the antibody and the POD solution recovered from the column were concentrated to lrnl with a centrifugal concentrator (Centricon 30, Amicon), and both dissolved waves were stirred and mixed and reacted at 3 for 30 minutes. After the reaction, the antibody was passed through a G-150 column (1.5 X G0 cm) equilibrated with 0.1 M phosphate buffer (pH 7.0) to collect a PD ⁇ -labeled antibody fraction. The recovered P ⁇ D-labeled anti-ultraviolet-denatured DNA mono- ⁇ -nal antibody was added to BSA at 0.1% and then frozen and stored at 140 until use.
  • the absorbance at 660 nm was measured for each gel using a microplate reader.
  • the relative absorbance was calculated as a percentage of the obtained absorbance when the absorbance when using only the TE buffer was set to 100%.
  • the antibody solution was replaced with 0.1 M carbonate buffer (pH 9.5) containing 100 ⁇ g;
  • a sandwich-type enzyme immunoassay was performed using the same method except that the P0D-labeled anti-ultraviolet-denatured DNA monoclonal antibody prepared in step 1 was replaced with a P0D-labeled anti-ultraviolet-denatured DNA monoclonal antibody. The relative absorbance was calculated.
  • Fig. 3 shows the calibration curve
  • Fig. 4 shows the relationship between the irradiation UV dose and the amount of UV-modified DNA for DNA recovered from cells irradiated with ultraviolet light.
  • a sandwich-type enzyme immunoassay system can be constructed by using the monoclonal antibody of the present invention, and that the amount of intracellular ultraviolet-denatured DN protein can be quantified by using the monoclonal antibody of the present invention.
  • lymphocyte layer was recovered in PBS buffer.
  • the collected lymphocyte fluid was further centrifuged at 1500 rpm for 5 minutes, and the supernatant was removed to obtain lymphocytes.
  • DNA in lymphocytes was purified using Sepagene Kit (Sanko Junyaku). The purified DNA was dissolved in 2001 buffer solution, and the DNA concentration was quantified by measuring the absorbance at 260 ⁇ .
  • the 51 samples were electrophoresed on a 1% agarose gel (Sigma, 90 mM Tris borate buffer pH 8.0 containing 2 mM ethylenediaminetetraacetic acid, type IIZ2) at 60 V constant voltage for 2 hours.
  • the gel after electrophoresis is shaken in an aqueous denaturant solution (1.5 M sodium chloride, 0.5 Na ⁇ H) for 45 minutes, washed with ice, and neutralized with 1.5 M sodium chloride.
  • P ATK 105 Dissolve 1 Mg in 25 ⁇ 1 TE buffer and place in a quartz chamber. UVC (ultraviolet light whose main component is 254 nm) was irradiated with 50,000 JZm 2 to denature the ultraviolet light to obtain a probe. Next, the nitrocellulose membrane prepared in section (c) was placed in a hybridization bag, and a hybridization solution (0.9 M sodium chloride, 0.09 M sodium citrate, 0.1% sodium citrate) was added.
  • a hybridization solution 0.9 M sodium chloride, 0.09 M sodium citrate, 0.1% sodium citrate
  • the nitrocellulose membrane was washed twice with 2 x SSC containing 0.1% SDS by shaking at room temperature for 30 minutes, and then 0.535 (: solution containing 0.1% SDS) After washing with shaking for 65 hours for 1 hour, blocking was performed for 1 hour at room temperature in PBS buffer containing 3% BSA, and blocking was performed.
  • ngZm 1 of the KTM-53 antibody of the present invention and 0. BSA were allowed to react in a 1% PBS buffer solution with shaking 4: overnight, and then Tween 20 was added to 0.05. After washing, the membrane was reacted for 3 hours at room temperature with an 85 buffer solution containing P0D-labeled anti-mouse immunoglobulin- ⁇ sagi IgG and 0.1% 838.8.
  • Example 5 A solution containing 2 g 1 of the UV-denatured probe prepared in section (d) was treated at 100 t for 3 minutes to make the DNA single-stranded, and then rapidly cooled in ice. This was mixed with a hybridization solution of 20 to 501 (40% formamide, 0.6 M sodium chloride, 1 mM ethylenediaminetetraacetic acid, 10% dextran sulfate, 0.01% BSA. 10% Tris Z HCl buffer (pII containing 0.01% solution, 0.01% polyvinylpyrrolidone, 125 jugZm 1 salmon sperm DNA and 250 ⁇ ugZm 1 yeast tRNA 7.3)] and added dropwise to the slide glass prepared in (a).
  • a hybridization solution 20 to 501 (40% formamide, 0.6 M sodium chloride, 1 mM ethylenediaminetetraacetic acid, 10% dextran sulfate, 0.01% BSA. 10% Tris Z HCl buffer (pII
  • the monoclonal antibody KTM-53 was replaced with the anti-ultraviolet ray-modifying DNA polyclonal antibody obtained in the comparative example, and POD-labeled anti-mouse immunoglobulin zoo- egret IgG was replaced with POD-labeled anti-sagyeminoglobulin.
  • In situ hybridization was performed using the same method except that the antibody was replaced with a goat antibody.
  • Methylated BSA was prepared with 10 mM carbonate buffer (pH 8.5) to 1 OmgZm1 and washed with 10 mM carbonate buffer ( ⁇ 8.0) to 1 OmgZm1.
  • An equal amount of CNBr-activated Sepharose was stirred and mixed, and allowed to react by gentle stirring and mixing at room temperature for 6 hours. After the reaction, the gel is collected on a filter, washed with 0.1 M Tris Z hydrochloric acid buffer (pH 8.5), suspended in the same buffer, and gently mixed by mixing at room temperature for 2 hours. And the locking operation was performed.
  • the antiserum prepared in (a) was diluted 3-fold with binding buffer (3M sodium chloride, 1.5M glycine pH 8.9) and passed through a protein A column equilibrated with binding buffer. Tower. After the column was washed with PBS buffer, the antibody was eluted with 50 mM glycine Z buffer (pH 2.5). The eluted antibody solution was immediately neutralized with 1 M phosphate buffer (PII7.5). The collected antibody solution was dialyzed sufficiently against PBS buffer, passed through a methylated BSA column equilibrated with the PBS buffer prepared in section (b), and further passed through the same fraction as PBS buffer. Normal DNA equilibrated with binding buffer (3M sodium chloride, 1.5M glycine pH 8.9) and passed through a protein A column equilibrated with binding buffer. Tower. After the column was washed with PBS buffer, the antibody was eluted with 50 mM glycine Z buffer (pH 2.5). The eluted antibody solution was immediately neutralized with
  • a monoclonal antibody that specifically binds to a denatured portion of DNA produced by irradiating DN ⁇ with ultraviolet light, a method for immunologically measuring denatured DNA using the monoclonal antibody, and a method for measuring denatured DNA An immunoassay for DNA or RNA used as a probe is provided.

Abstract

A monoclonal antibody belonging to the IgG1λ class and recognizing specifically the cyclobutane type thymine dimer or (6-4) photoproduct formed between the thymine residues of a DNA; a simple high-sensitivity method of immunoassaying a denatured DNA by using the above antibody; and a method of immunoassaying a DNA or RNA having a specified sequence.

Description

明 細 書 モノ クロ一ナル抗体およびこれを用し、る変性 D N Aの測定法 技術分野  Description Monoclonal antibody and method for measuring denatured DNA using it
本発明は D N Aに紫外線を照射することによって生じる DNAの変性部に特異 的に結合するモノクローナル抗体および該抗体の利用法に関する。 背景技術  The present invention relates to a monoclonal antibody that specifically binds to a denatured portion of DNA generated by irradiating DNA with ultraviolet light, and a method of using the antibody. Background art
生物が紫外線の照射を受けると生体内で様々な変化を生じる。これは紫外線が生 物体の構成成分に化学的な変性を引き起こす結果として生じると考えられている 。とりわけ、 D N Aは紫外線の照射を受けて変性を受けやすく、 D N Aに紫外線が 照射されると DNAは変性を受け、 その結果としてピリ ミジンフォトプロダクト 〔フォ トケミス ト リー ' アンド · フォ トバイオロジー （Photochetn. Photobiol. ) ,42,265-273(1985)3 またはプリンフォ トプロダク ト 〔フォ トケミストリー ·ァ ンド · フォ トバイオロジー (Photochem. Photobiol. ), 48.35-39 (1988)3 などを 生じることが知られている。  When a living organism is irradiated with ultraviolet rays, various changes occur in the living organism. This is believed to be the result of ultraviolet radiation causing chemical denaturation of the constituents of the living body. In particular, DNA is susceptible to denaturation when exposed to ultraviolet light, and DNA is denatured when exposed to ultraviolet light, resulting in pyrimidine photoproducts [Photochemistry and Photobiology (Photochetn). Photobiol.), 42, 265-273 (1985) 3 or purine photoproducts (Photochemistry and Photobiol.), 48.35-39 (1988) 3. I have.
DN Aの変性が生物へ及ぼす害作用は深刻なものであり、 突然変異、 細胞癌化 および細胞死の原因の一つと考えられている。  The detrimental effects of DNA degeneration on organisms are severe and are thought to be one of the causes of mutation, cell carcinogenesis and cell death.
DN Aの変性を測定する方法として、 D N Λを加水分解した後、 クロマトグラ フィ一で分離 ·定量する方法 〔バイォケミカル ' アンド♦バイオフィジカル · リ サーチ ·コミュニケーションズ（Biochem. Biopys. Res. Co画 un. )， 105, 1215(198 2)] または紫外線エンドヌクレア一ゼによつて損傷特異的な D N A切断を入れた 後、 DNAがどの程度まで低分子化するかを測定する方法 〔ミューテ一シヨン · リサーチ（Mutat. Res. ),19, 245 (1973)] などが用いられている。しかし、これらの 方法は感度が低く、測定するために多くの検体が必要である。また、ク σマトグラ フィ一による方法では変性した DNAを検出するために放射性物質で標識化する 必要があり、 酵素による方法では間接的かつ相対的な測定法であることから、 D Ν Αの変性度を正確に定量することができない。  As a method for measuring the denaturation of DNA, a method for separating and quantifying DNA by hydrolysis after DNΛ hydrolysis [Biochemical 'and ♦ Biophysical Research Communications (Biochem. Biopys. Res. Co. ), 105, 1215 (198 2)] or a method of measuring the extent to which DNA is degraded after DNA damage specific to DNA has been cut by ultraviolet endonuclease [mutation research. (Mutat. Res.), 19, 245 (1973)]. However, these methods have low sensitivity and require many samples to measure. In the method using chromatographic chromatography, it is necessary to label the DNA with a radioactive substance in order to detect denatured DNA, and the enzyme method is an indirect and relative measurement method. The degree cannot be accurately determined.
1  1
訂正きれた用紙 (規則 91) 一方、 特定の塩基配列を含む D N Λまたは R N Λを定性的または定量的に測 定する技術は、 遺伝子を扱う遺伝子工学、 細菌学、 ウィルス学、 臨床検査など様 々な分野で必要不可欠な技術である。 目的の D N Aまたは R N Aとハイブリダィ ズする D N Aを何らかの形で標識化してプローブとし、 このプローブと目的の D N Λまたは R N Λとを反応させ、 形成された複合体のプ口一ブの標識を検出する ことにより、 目的の DN Aまたは RN Aを定量的に測定することができる。 Corrected form (Rule 91) On the other hand, technology for qualitatively or quantitatively measuring DNΛ or RNΛ containing a specific nucleotide sequence is indispensable in various fields such as genetic engineering, bacteriology, virology, and clinical testing for genes. It is. DNA that hybridizes with the target DNA or RNA is labeled in some way to form a probe, and this probe is reacted with the target DN Λ or RN 、 to detect the label at the end of the formed complex. This makes it possible to quantitatively measure the target DNA or RNA.
標識化の方法として、 プローブとして用いる D N Aに紫外線を照射して D N A 中に存在する核酸に一定の変性を形成させプ n—ブ DNA自身をハプテン化する 方法があり、 紫外線による変性がチミ ン -チミン間に形成されたものが特に有用 とされている。 該プローブの検出方法としては、 紫外線を照射した DN Aを免役 することにより得られる抗体を用いることが考えられている 〔サイエンス（Scien ce), 153, 1666- 1667 (1966)およびユーロビアン · ジャーナル 'ォブ 'バイオケミ ス ト リー（Bur. J. Biochem.),142, 313-316 (1984)] 0 As a labeling method, there is a method in which DNA used as a probe is irradiated with ultraviolet light to form a certain degree of denaturation of the nucleic acid present in the DNA, and the DNA itself is haptenized. Those formed between thymines are particularly useful. As a method for detecting the probe, it has been considered to use an antibody obtained by immunizing DNA irradiated with ultraviolet rays [Science, 153, 1666-1667 (1966) and Eurobian Journal. 'Ob' Biochemistry Story (Bur. J. Biochem.), 142, 313-316 (1984)] 0
紫外線照射によって変性した DN Aを検出するために、 チミン溶液に紫外線を 照射し、 チミ ンダイマー分子を作製してこれを免疫用抗原とする方法 〔ユーロピ 了ン · ジャーナル ·ォブ .バイオケミス ト リー（Eur. J. Biochem.), 142, 313-316 (1984)3 、 またはテトラヌクレオシドである GTTGに紫外線を照射してこれを 免疫用抗原とする方法 〔フォ トケミス ト リー · アンド · フォ トバイオロジー （Ph otochem. Photobiol. ), 8.627-633 (1988)] を用いることによるチミ ンダイマー に対する抗体が作製されている。  In order to detect DNA denatured by ultraviolet irradiation, a method of irradiating a thymine solution with ultraviolet light to produce a thymine dimer molecule and use this as an antigen for immunization [Europe Journal, Biob. Eur. J. Biochem.), 142, 313-316 (1984) 3, or a method of irradiating ultraviolet rays to GTTG, a tetranucleoside, to use this as an antigen for immunization [photochemical tree and photobiology. (Photochem. Photobiol.), 8.627-633 (1988)], and an antibody against thymine dimer has been produced.
しかしながら、 前者の方法によって作製されたポリクローナル抗体は紫外線照 射によつて変性した D N Aを加水分解したものに特異的に反応し、 また後者の方 法によつて作製されたモノクロ一ナル抗体は紫外線照射によつて変性した D N A のうち 1本鎖のものに特異的に反応する。 このため、 これらの抗体を用いて生体 内で紫外線を受けて生じた二本鎖の変性 D N Λまたは紫外線により変性させたプ ローブをハイプリダイズさせて得られる二本鎖 DN Aを検出することが S難であ 。  However, the polyclonal antibody produced by the former method specifically reacts with the hydrolyzed DNA denatured by ultraviolet irradiation, and the monoclonal antibody produced by the latter method produces ultraviolet light. Reacts specifically with single-stranded DNA denatured by irradiation. Therefore, using these antibodies, it is possible to detect a denatured double-stranded DNA generated in vivo by receiving ultraviolet light or a double-stranded DNA obtained by hybridizing a probe denatured with ultraviolet light. S difficult.
一方、 紫外線により変性を受けた DN Aを検出する方法として、 紫外線照射を 受けた DN Aに対するポリクローナル抗体が作製されている 〔フォ トケミス ト リ  On the other hand, as a method for detecting DNA that has been denatured by ultraviolet light, polyclonal antibodies against DNA that have been irradiated with ultraviolet light have been produced (photochemistry).
2  Two
訂正きれた用紙 (規則 91) — ' アンド · フォ トバイオロジー (Photochem. Photohiol. ), 0, 743-748 (198Ί) 、 およびフオ トケミス ト リ一 ' 了ンド ' フォ トバイオロジー （PhoLocliem. Photo biol.),45, 485-491(1987)3 。 Corrected form (Rule 91) — 'And Photobiology (Photochem. Photohiol.), 0, 743-748 (198Ί), and Photochemistry (PhoLocliem. Photo biol.), 45, 485- 491 (1987) 3.
しかしながら、 紫外線照射によつて生じる D N Λの変性には多種多様なものが あり、 目的の抗原認識部位に特異的に結合する抗体のみを取得することは極めて 因難である。  However, there are various types of denaturation of DNΛ caused by ultraviolet irradiation, and it is extremely difficult to obtain only antibodies that specifically bind to the target antigen recognition site.
このように、 チミンダイマ一を有する一本鎖 DN Aおよび二本鎖 DN Aの両方 の D N Aを認識するモノクローナル抗体は知られていない。  Thus, there is no known monoclonal antibody that recognizes both single- and double-stranded DNA having a thymine dimer.
発明の開示 Disclosure of the invention
本発明は、 D N Aに紫外線を照射することによって生じるチミンダイマ一を有 する DNA (以下、 変性 DNAという。 ） と結合するモノクローナル抗体 （以下 、 抗紫外線変性 DNAともいう。 ） および該モノクローナル抗体を用いる変性 D N Aの免疫化学的測定法ならびに該変性 DNAをプローブとして用いる DNAま たは R N Aの免疫学的測定法こ関する。  The present invention provides a monoclonal antibody (hereinafter, also referred to as anti-ultraviolet-denatured DNA) that binds to DNA having thymine dimer (hereinafter, referred to as denatured DNA) generated by irradiating the DNA with ultraviolet light, and denaturation using the monoclonal antibody. The present invention relates to an immunochemical assay for DNA and an immunoassay for DNA or RNA using the denatured DNA as a probe.
本発明のモノクローナル抗体は、 チミンダイマ一を有する一本鉞 D N Λおよび 二本鎖 D N Aの両方の D N Λに結合するモノクローナル抗体である。  The monoclonal antibody of the present invention is a monoclonal antibody that binds to both single-chain DNA and double-chain DNA having a thymine dimer.
チミンダイマ一としては、 連続したチミンーチミン問のダイマーでも、 離れた チミン一チミン間のダイマーでもよい。 その結合型としては、 例えばサイクロブ タン型チミ ンダイマー、 （6— 4〉 フォ トプロダク ト型チミ ンダイマ一などが挙 げられる。  The thymine dimer may be a continuous thymine-thymine dimer or a thymine-thymine dimer. Examples of the binding type include cyclobutane-type thymine dimer and (6-4) photoproduct-type thymine dimer.
サイクロブタン型チミンダイマ一は、 チミンが 2個以上連続する DNAにァセ トフエノ ン存在下 2 5 4 π mまたは 3 1 3 π mの波長を含む紫外線を照射するこ とにより生じ、 cis- syn構造と trans- syn構造をとる [ mola, A. A.， Photochem. P hotobiol.,9^291-2J4(196i))]D The cyclobutane-type thymine dimer is formed by irradiating DNA containing two or more thymines with ultraviolet light having a wavelength of 254 πm or 313 πm in the presence of acetate phenone, resulting in a cis-syn structure. And trans-syn structure [mola, AA, Photochem. P hotobiol., 9 ^ 291-2J4 (196i))] D
(0 -4) フォ トプロダク ト型チミンダイマ一は、 チミンが 2個以上速続する DNAに 2 5 4 nmの波長を含む紫外線を照射することにより生じ、 3 1 3 nm の波長を含む紫外線照射により別物質に変化する [Taylor, J-S. & Cohrs, M. P.， J. Am. Chem. Soc. O .2834-2835 (1987) ] 。 サイクロブタン型チミンダイマーおよび (0 -4) Photoproduct-type thymine dimer is produced by irradiating DNA containing 254 wavelengths of 254 nm to thymine-containing DNA. It changes into another substance [Taylor, JS. & Cohrs, MP, J. Am. Chem. Soc. O.2834-2835 (1987)]. Cyclobutane thymine dimer and
(6 -4) フォ トプロダク ト型チミンダイマ一の構造を下記に示す。
Figure imgf000006_0001
cis-syn trans-syn サイクロブ夕ン型チミ ンダイマー (6-4) The structure of the photoproduct type thymine dimer is shown below.
Figure imgf000006_0001
cis-syn trans-syn cyclobutene-type thymidine dimer
Figure imgf000006_0002
Figure imgf000006_0002
( 6 - 4 ) フォ トプロダク 卜型チミ ンダイマ一 本発明のモノク口一ナル抗休としては、 I g G 1 A:クラスに屈し、 等電点が (J . 5 ± ϋ . 5、 分子量が 1 (] 5 ϋ 0 0土 1 ϋ ϋ ϋの理化学的性質を示すもの、 I gG 1 κクラスに厲し、 等電点が G. G土 ϋ. 5、 分子量が 1 7 50 0 0 ± 5 0 ϋ ()、 変性 DNAに対する結合: ίϋ数が ϋ. 1〜5. 0 X 1 03ZMの理化学的性 質を示すものがあげられる。 具体的に奵-適な例としては、 前者ではハイプリ ドー マ ΚΤΜ—5 1および後者ではハイプリ ドーマ ΚΤΜ—53がそれぞれ産生する モノク口一ナル抗休があげられる。 ハイブリ ドーマ ΚΤΜ— 5 1が産生するモノ クロ一ナル抗体は （G— 4) フォ トプロダク ト型チミ ンダイマーに対して特異的 に反応し、 ハイプリ ドーマ ΚΤΜ— 5 3が産生するモノクローナル抗体はサイク ロブタン型チミンダイマーに対して特異的に反応する。 本発明のモノ クローナル抗体は、 例えば細临融合法によって作り出されたハイ プリ ドーマ細胞から製造される。 すなわち、 紫外線により変性した DNAで免役 した抗体産生細胞と骨髄腫細胞とを融合させてハイプリ ドーマ細胞を形成させ、 このハイプリ ドーマ細胞をモノ クローン化し、 紫外線により変性した DNA (Ρ 下、 紫外線変性 DNAという。 ） に高い特異性を示す抗体を産生するク α—ンを 選択することによって製造される。 (6-4) Photoproduct-type thymidine dimer As the monoclonal anticancer of the present invention, IgG 1 A: yields to class, has an isoelectric point of (J.5 ± ϋ.5, molecular weight of 1). (] 5 ϋ 0 0 Sat 1 ϋ ϋ 理, showing physicochemical properties, IgG 1 κ class, isoelectric point G. G 土 5. ... ϋ (), bind to denatured DNA: ίϋ number ϋ 1~5 0 X 1 0 3 shows the physicochemical properties quality of ZM can be mentioned specifically奵- as suitable examples, the former High Priestess Monoclonal antibodies produced by Hypri-Doma III-53 and the latter are exemplified by the monoclonal antibodies produced by Hybridoma Doma III-53, respectively. (G-4) Photoproduct The monoclonal antibody produced by hybridoma ト -53 reacts specifically with React specifically to Ndaima. The monoclonal antibody of the present invention is produced, for example, from hybridoma cells created by the cell fusion method. That is, the antibody-producing cells and the myeloma cells immunized with the DNA denatured by ultraviolet rays are fused with myeloma cells to form hybridoma cells, the hybridoma cells are monocloned, and the DNA denatured by ultraviolet rays (below, ultraviolet denatured DNA) It is produced by selecting a clone that produces an antibody with high specificity.
本発明のモノク口ーナル抗体を製造するためには、 免疫原として紫外線変性 D Ν Λを取得しなければならない。 この免疫原は D Ν Λに紫外線を照射することに よって作製する。 紫外線を照射する DNAは、 合成することにより取得してもよ いし、 生物材料から取得してもよく、 奵-ましくは 1 0塩基以上の、 さらに好まし くは 1 0 0塩基以上の DNAが用いられる。 合成により取得する方法、 生物材料 から取得する方法はともに公知の方法 〔核酸 ΙΠ (新生物学実験講 ME 2 ) , 253， 1992, 日本生化学会編， 柬京化学同人、 バイオテクノロジー実験操作入門， 6, 1 990， 講談社〕 が使用できる。 照射する紫外線の種類は大別して1^八（320〜38 Onm 、 365nmが主成分） 、 U V B (280~320nm 、 313nmが主成分） 、 UVC (280 以下、 254nm が主成分） の 3種があるが、 このうち U V Bまたは U V C：、 とくに UVCが好ましい。 紫外線を照射する装置としては市販の紫外線照射装置 （アト 一社等） が、 殺菌灯としては市販の紫外線ランプ （東芝社等） がそれぞれ用いら れる。 紫外線の照射に隙して、 1 JZm2 以下の照射量でも紫外線変性 DNAを 作製することは可能である力 充分な抗原性をもたせるためには 1 0 0 0 JZm 2 以上の照射量で照射することが望ましい。 In order to produce the monoclonal antibody of the present invention, it is necessary to obtain UV-denatured DΝ as an immunogen. This immunogen is produced by irradiating DΝ with ultraviolet light. The DNA to be irradiated with ultraviolet light may be obtained by synthesis or from biological material, and is preferably DNA having at least 10 bases, more preferably at least 100 bases. Is used. Both the method of obtaining by synthesis and the method of obtaining from biological materials are known methods [Nucleic acid ΙΠ (New Biological Experiment Lecture ME 2), 253, 1992, edited by The Biochemical Society of Japan, ed. 6, 1990, Kodansha] can be used. The types of ultraviolet rays to be irradiated are roughly classified into three types: 1 ^ 8 (320 to 38 Onm, 365 nm is the main component), UVB (280 to 320 nm, 313 nm is the main component), and UVC (280 or less, 254 nm is the main component). Of these, UVB or UVC: UVC is particularly preferred. As a device for irradiating ultraviolet rays, a commercially available ultraviolet irradiating device (Ato, etc.) is used, and as a germicidal lamp, a commercially available ultraviolet lamp (Toshiba, etc.) is used. It is possible to produce UV-denatured DNA even at an irradiation dose of 1 JZm 2 or less in the irradiation of UV light.For sufficient antigenicity, irradiate at a dose of 100 JZm 2 or more. It is desirable.
免疫原に用いられる紫外線変性 DNAとしては、 DNAに紫外線を照射し紫外 線変性 DNAを作製し、 これを精製して川いてもよいし、 精製した DNAに紫外 線を照射して紫外線変性 DNAを作製し、 これを； ijいてもよい。 また、 紫外線変 性 DNAはそのまま免疫原として川いてもよいが、 生体内において充分な免疫原 性を獲得.するためには、 いわゆるキヤリア一物 Kと結合させ、 これを用いること が了-ましい。 キヤリ了一物 ¾としては、 ゥシ血沾アルブミ ン、 グ ブリン、 キー ホールリ ンぺッ トへモシァニン、 サイログロブリ ンなどの生休内蛋白質や ΜΛΡ などの合成高分子などが用いられる。 例えば、 ゥシ血清アルブミ ンをキャ リア一 物質とした場合には、 紫外線変性 DN Λとゥシ血清アルブミ ンのァミノ基とを 2 価性架橋剤で結合することができる。 また、 抗 DNA抗体を取得する場合に用い られる方法を応用し、 メチル化ゥシ血清アルブミンに紫外線変性 DNAを吸着さ せて、 これを免疫原とすることもできる。 As the UV-denatured DNA used for the immunogen, the UV-denatured DNA may be prepared by irradiating the DNA with ultraviolet light, and then purified.The UV-denatured DNA may be purified by irradiating the purified DNA with UV light. It may be made; In addition, UV-modifying DNA may be used as an immunogen as it is, but in order to obtain sufficient immunogenicity in vivo, it has been combined with the so-called carrier K and used. No. Examples of the carrier include proteins in living tissues such as albumin, albumin, globulin, keyhole rinsin mosinin, and thyroglobulin, and synthetic polymers such as ΜΛΡ. For example, transfer serum albumin to a carrier When a substance is used, the UV-modified DNA and the amino group of serum albumin can be linked by a divalent crosslinking agent. In addition, by applying the method used to obtain an anti-DNA antibody, UV-denatured DNA can be adsorbed to methylated glucose serum albumin and used as an immunogen.
免疫原で免疫する動物としては、 マウス、 ラッ ト、 ハムスター、 ゥサギ、 モル モッ ト、 ャギ、 ヒッジなどが用いられるが、 好ましくはマウスおよびラッ トが用 いられる。  As animals immunized with the immunogen, mice, rats, hamsters, rabbits, guinea pigs, goats, sheep, and the like are used, and mice and rats are preferably used.
免疫方法としては、 例えば免疫原をフ イントの完全または不完全了ジュバン トにェマルジヨン化し、 腹腔内、 皮下または筋肉内に一定の時 Π1間隔をおいて数 回以上投与する方法が用いられる。  As an immunization method, for example, a method is used in which the immunogen is emulsified into a complete or incomplete adjuvant of a font and administered intraperitoneally, subcutaneously or intramuscularly several times at regular intervals at regular intervals.
抗体産生細胞の採取源としては、 免疫した動物の脾蔵、 リ ンパ節、 末梢血液な どが挙げられる。 また、 免疫を行っていない動物の脾臓、 リンパ節、 末梢血液な どより取り出した抗体産生細胞に、 イ ンビトロ（in vitro)で直接免疫を行って得 られた細胞 〔新井、 太田、 実験医学 J1, 43(1988) 〕 を用いてもよい。  Sources for collecting antibody-producing cells include spleen, lymph node, and peripheral blood of immunized animals. In addition, cells obtained by direct immunization in vitro of antibody-producing cells from spleen, lymph nodes, peripheral blood, etc. of non-immunized animals [Arai, Ota, Experimental Medicine J1. , 43 (1988)].
抗体産生細胞と骨髄腫細胞との細胞融合を行う際に使用する骨髄腫細胞には特 に限定はないが、 抗体産生細胞と同種の動物 ώ来の細胞株を使用するのが好まし い。 また、 適切に細胞融合が行われた細胞のみを効率よく選択するために、 特定 の薬物マーカ一を有するものが好ましい。 例えば、 8—ァザグァニン耐性の骨髄 腫細胞はヒポキサンチン、 ァミノプテリンおよびチミジンを舍有した培地 （H A T培地） 中では生育できないが、 この細胞と正^細胞とが融合した細胞は H Λ T 培地中で生育できるようになり、 夫融合の骨髄腿細胞と区別できることから好ま しい。 具体的には、 Ρ 3 χ ΰ 3— A g. 8. G 5 3 ( G 5 3) [J.！闘 unology, 1 ,_1548 (1979)] . Γ 3 x β 3 - Λ g . 8. U 1 (P 3 -U 1 ) [Current Topics in Microbiology and Immunology, 81, 1 (1078)], S p/O-A g 1 4 (S P- 2 ) [Natiire， m_2Gi)(li378):iなどが げられる。  The myeloma cells used for cell fusion between the antibody-producing cells and the myeloma cells are not particularly limited, but it is preferable to use a cell line derived from an animal of the same species as the antibody-producing cells. In order to efficiently select only cells that have been appropriately subjected to cell fusion, those having a specific drug marker are preferable. For example, 8-azaguanine-resistant myeloma cells cannot grow in a medium containing hypoxanthine, aminopterin and thymidine (HAT medium), but cells in which these cells and positive cells are fused cannot be grown in H HT medium. It is preferable because it can grow and can be distinguished from the fused bone marrow femoral cells. Specifically, Ρ 3 χ ΰ 3—A g. 8. G 53 (G 53) [J.! U 3 x β 3-Λg. 8. U 1 (P 3 -U 1) [Current Topics in Microbiology and Immunology, 81, 1 (1078)], S p / OA g 14 (SP-2) [Natiire, m_2Gi) (li378): i.
細胞融合は Kofi 1 erと M i 1 s i n「Na l.urc,„ )- 7 ( 1975) ]によつて確立された 方法、 例えば抗体産生細胞と骨髄腿細^を 1 ϋ〜3 ： 1の割合で混合し、 3 ϋ〜 5 0 %のポリエチレングリコール （平均分子量 1 5 0 0〜 6 0 0 0 ) を融合剤に 用いて処理する方法など力、'用いられる。 また、 電気パルスによる融合法 〔大河内  Cell fusion is performed by the method established by Kofi 1 er and Mi 1 sin “NaI.urc,„)-7 (1975)]. For example, antibody-producing cells and bone marrow cells can be expressed in 1ϋ to 3: 1. Mixing at a ratio of 3 to 50% of polyethylene glycol (average molecular weight of 1500 to 600) as a fusion agent. The fusion method using electric pulses [Okochi
G ら、 実験医学 J, 50 (1988) 〕 を用いてもよい。 G For example, Experimental Medicine J, 50 (1988)] may be used.
細胞融合を行った細胞は選択培地に懸濁し、 これを 9 6マイクロタイタープレ 一トなどの培養容器に入れて融合細胞のみが選択的に生育する条件下で培養する 。 融合細胞のみが選択的に生育した段階で、 紫外線変性 D N Aに対する抗体を産 生している細胞のみを選択する。 選択方法としては、 融合細胞の培養上清中の目 的抗体の有無を、 例えばェンザィ厶ィ厶ノアッセィやラジオィムノアッセィなど の方法を用いて調べる方法などが用いられる。 選択された細胞を、 例えば限界希 釈法ゃ軟寒天培地法などの方法を用いてモノクローン化し、 さらに上記の選択方 法を繰り返すことにより、 抗紫外線変性 D N Aモノクローナル抗体産生ハイブリ ドーマ細胞株が取得できる。  The cells that have undergone cell fusion are suspended in a selection medium, placed in a culture container such as a 96-microtiter plate, and cultured under conditions that allow only the fused cells to selectively grow. At the stage where only the fused cells have selectively grown, select only those cells that are producing antibodies against UV-denatured DNA. As a selection method, there is used a method of examining the presence or absence of a target antibody in the culture supernatant of the fused cells using, for example, a method such as Enzyme Immunoassay or Radioimmunoassay. The selected cells are monocloned using, for example, the limiting dilution method and the soft agar method, and the above selection method is repeated to obtain a hybridoma cell line that produces an anti-UV-denatured DNA monoclonal antibody. it can.
抗紫外線変性 D N Aモノク口ーナル抗体産生ハイブリ ドーマ細胞株の一例とし ては、 ハイプリ ドーマ K T M— 5 1および K T M— 5 3が挙げられる。 ハイプリ ドーマ K TM— 5 1および K T M— 5 3は、 平成 5年 9月 2 日付けで工業技術 院生命工学工業技術研究所にそれぞれ FERM BP-4422. FERM BP-4423として寄託さ れている。  Examples of hybridoma cell lines producing anti-ultraviolet-denatured DNA monoclonal antibody include hybridomas KTM-51 and KTM-53. Hypri-Dorma KTM-51 and KTM-53 have been deposited as FERM BP-4422 and FERM BP-4423 on September 2, 1993 with the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology.
本発明のモノクローナル抗体は、 ハイプリ ドーマ細胞株を適当な培地で培養し た後にその培養液を回収するか、 あるいは細胞株を動物の腹腔内に移植して腹水 中で増殖させた後に腹水を回収し、 得られた培養液または腹水から得ることがで きる。 培養液または腹水中から得られた抗体は必要に応じて精製して使用するこ とができる。 精製方法としては例えば、 硫安分画、 イオン交換クロマトグラフィ ―、 ゲル濾過クロマトグラフィ一、 プロティン Aやプロティン Gを用いた了フィ 二ティクロマトグラフィー、 抗原を固相化したゲルを用いるァフィ二ティクロマ トグラフィ一などが用いられる。  The monoclonal antibody of the present invention can be obtained by culturing a hybridoma cell line in an appropriate medium and collecting the culture solution, or by transplanting the cell line into the peritoneal cavity of an animal and growing it in ascites, and then collecting the ascites. Then, it can be obtained from the obtained culture solution or ascites. Antibodies obtained from the culture solution or ascites can be purified and used as necessary. Examples of the purification method include ammonium sulfate fractionation, ion exchange chromatography, gel filtration chromatography, affinity chromatography using protein A and protein G, affinity chromatography using a gel on which an antigen is immobilized, and the like. Is used.
本発明のモノク π—ナル抗体を検出する方法としては、 二次抗体に放射性同位 体、 酵素、 蛍光体、 発光体、 金属などの物質で標識をし、 この標識物質によって を該抗体を検出する方法が用いられる。  As a method for detecting a monoclonal π-nal antibody of the present invention, a secondary antibody is labeled with a substance such as a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or a metal, and is detected by the labeled substance. A method is used.
これらの方法は、 溶液内に存在する抗原を測定する場合に用いられる方法であ るが、 組織中または細胞内もしくはニトロセル口一ス膜上またはナイ口ン膜上な ど固体の内外に存在する紫外線変性 D N Aを定性的または定量的に測定すること  These methods are used to measure antigens present in a solution, but they are present inside or outside a solid such as in a tissue or in a cell or on a nitrocellular membrane or on a nitrocellular membrane. Qualitative or quantitative measurement of UV denatured DNA
7  7
訂正された S紙 (規則 91 もできる。 Corrected S-paper (Rule 91 Can also.
本発明のモノク π—ナル抗体は、 紫外線変性部位を有する DNAの検出に用い られる。  The monoclonal π-nal antibody of the present invention is used for detecting DNA having an ultraviolet denaturation site.
モノクローナル抗体によって検出する抗原としては、 紫外線変性 DN Aであれ ば一本銷、 二本鎖または三本鎖などいずれの DNAを用いてもよいが、 好ましく は二本鎖の DNAが用いられる。 また、 該抗原として用いる紫外線変性 DN Aと しては、 DNA、 たとえば合成することにより得られた DNAもしくは生物体内 に存在する D N Aまたは生物体から取り出された D N Aに紫外線を照射すること によってチミンダイマーを生じた DN Aであればいずれを用いてもよい。  As the antigen to be detected by the monoclonal antibody, any DNA such as single-stranded DNA, double-stranded or triple-stranded DNA may be used as long as it is ultraviolet-denatured DNA, but double-stranded DNA is preferably used. The ultraviolet-denatured DNA used as the antigen may be thymine dimer by irradiating ultraviolet rays to DNA, for example, DNA obtained by synthesis, DNA present in an organism, or DNA extracted from an organism. Any DNA may be used as long as it is a DNA that has caused the above.
本発明のモノク ϋーナル抗体を用いた紫外線変性 DNAの検出は、 免疫学的な 測定方法であればいずれの方法を用いて行うことができるが、 好ましくは競合法 およびサンドイ ッチ法が用いられる。 競合法としては、 ①標識された抗原により 、 サンプル中あるいは標準物質の抗原と抗体の結合を競合させる方法、 ②標識さ れた抗体の結合を液相中のサンプル由来の、 あるいは標準物質の抗原と固相化さ れた抗原とで競合させる方法、 ③固相化抗体への結合を標識された抗原とサンプ ル中の、 あるいは標準物質の抗原とで競合させる方法などがある。 サンドイ ッチ 法としては、 ビーズやチューブ、 プレートなどの適当な固相に抗体を結合させた 一次抗体固相と標準物質ゃサンプル中の抗原と反応させ、 次に固相抗体と結合し た抗原と二次抗体とを反応させ、 形成した固相抗体一抗原一二次抗体の三者複合 体の形成を検出する方法が一般的に用いられる。  The detection of ultraviolet-denatured DNA using the monoclonal antibody of the present invention can be performed by any method as long as it is an immunological measurement method, but preferably, a competition method and a sandwich method are used. . The competitive methods include: (1) a method in which the labeled antigen competes with the antigen for binding to the antigen in the sample or the standard substance; and (2) the binding of the labeled antibody is derived from the sample in the liquid phase or the antigen of the standard substance. And (3) a method in which the binding to the immobilized antibody competes with the labeled antigen in the sample or the antigen of the standard substance. In the sandwich method, the primary antibody solid phase in which the antibody is bound to an appropriate solid phase such as beads, tubes, and plates reacts with the standard substance 標準 the antigen in the sample, and then the antigen bound to the solid phase antibody. And a secondary antibody, and a method of detecting the formation of a formed ternary complex of the solid phase antibody, the antigen and the secondary antibody is generally used.
さらに、 本発明のモノクローナル抗体を用いることにより、 特定の塩基配列の DNAまたは RNAもしくは特定の塩基配列を含む DNAまたは RNAを定性的 または定量的に測定することができる。 すなわち、 まず、 検出しょうとする特定 の塩基配列の D Ν Λまたは R Ν Λもしくは特定の塩基配列を含む D Ν Αまたは R N Λに対して相補的な配列を持つ D N Λに紫外線を照射して紫外線変性 D N Aを 作製し、 これをプローブとする。 次に、 検出しょうとする DNAまたは RNAと プ u—ブとがその相 的 E列の親不 ·π性により結合して形成した D Ν Λ—プローブ または R Ν Α—プローブ複合体を、 プローブである紫外線変性 D N Λに対するモ ノクローナル抗体を利用して上記と同様に免疫化学的に検出することができる。  Furthermore, by using the monoclonal antibody of the present invention, DNA or RNA having a specific base sequence or DNA or RNA containing a specific base sequence can be qualitatively or quantitatively measured. That is, first, ultraviolet rays are applied to D Ν Λ or R の 特定 of the specific nucleotide sequence to be detected or DN を 持 つ having a sequence complementary to D Ν Α or RN 含 む containing the specific nucleotide sequence. Prepare UV-denatured DNA and use it as a probe. Next, the DNA or RNA to be detected and the probe are combined due to the affinity of the sequence E due to the non-π / π property, and the DΝ-probe or RΝ-probe complex is formed into a probe. The immunochemical detection can be carried out in the same manner as described above using a monoclonal antibody against UV-denatured DNA.
8  8
訂正きれた用紙 (規則 91) 本発明のモノク π—ナル抗体を用いて検出する特定の塩基配列を含む D N Λお よび RNAとしては、 合成することにより取得してもよいし、 生物材料から取得 してもよい。 また、 これら DNAおよび RNAとしては、 電気泳動ゲル内、 組織 切片上、 細胞内、 ドッ トブロッティング、 サザンプロッティングまたはノーザン ブロティングなどを行った後のプロッティング膜上に存在するものなどいずれを 用いてもよい。 図面の簡単な説明 Corrected form (Rule 91) The DNA and RNA containing a specific nucleotide sequence detected using the monoclonal antibody of the present invention may be obtained by synthesis or from biological material. As the DNA and RNA, those existing in an electrophoresis gel, on a tissue section, in a cell, or on a plotting membrane after performing dot blotting, southern blotting, or northern blotting, etc. You may. BRIEF DESCRIPTION OF THE FIGURES
第 1図は、 実施例 3の Rij接競合型ェンザィムィムノアッセィの検量線を示す。 〇はモノクロ一ナル抗体 KTM— 5 1、 △はモノクローナル抗体 KTM— 53、 □は抗紫外線変性 DNAポリク π—ナル抗体を用いた場合をそれぞれ示す。 なお 、 図中の紫外線変性 DNAの UZmgは、 1 mgのスタンダード正常 DNAに 1 04 J/m2 の紫外線を照射し場合に生成する紫外線変性 DNAの量を 103 U Zmgとして定義した。 FIG. 1 shows a calibration curve of a Rij contact competitive enzymimnoassy of Example 3. 〇 indicates the case of using the monoclonal antibody KTM-51, Δ indicates the case of using the monoclonal antibody KTM-53, and □ indicates the case of using the anti-ultraviolet-denatured DNA poly-π antibody. Incidentally, UZmg ultraviolet denatured DNA in the figure, defining the amount of ultraviolet denatured DNA to generate when irradiated with 1 0 4 UV J / m 2 to 1 mg of the standard normal DNA as 10 3 U Zmg.
第 2図は、 実施例 3の fill接競合型ェンザィムィムノアッセィによる紫外線照射 量と紫外線変性 DN A量との関係を示す。 〇はモノクローナル抗体 KTM— 51 、 △はモノクローナル抗体 KTM— 53、 □は抗紫外線変性 DNAポリクローナ ル抗体を用いた場合をそれぞれ示す。 なお、 図中の紫外線変性 DNAの UZmg の定義は、 第 1図における定義と同じ。  FIG. 2 shows the relationship between the amount of ultraviolet irradiation and the amount of ultraviolet-denatured DNA by the fill contact competitive enzymimnoassy of Example 3. 〇 indicates the use of the monoclonal antibody KTM-51, △ indicates the use of the monoclonal antibody KTM-53, and □ indicates the use of an anti-ultraviolet denatured DNA polyclonal antibody. The definition of UZmg of the UV-denatured DNA in the figure is the same as that in Fig. 1.
第 3図は、 実施例 4の細胞内の D N Λに紫外線を照射した.場合のサンドイッチ 型ェンザィムィムノアツセィの検量線を示す。 〇はモノク口ーナル抗体 KTM— 51、 厶はモノクローナル抗休 KTM— 53、 ロは抗紫外線変性 DNAポリク口 ーナル抗体を用いた場合をそれぞれ示す。 なお、 図屮の紫外線変性 DNAの UZ mgの定義は、 第 1図における定義と同じ。  FIG. 3 shows a calibration curve of a sandwich-type enzyme in the case of irradiating ultraviolet rays to intracellular DN in Example 4. 〇 shows the results obtained using the monoclonal antibody KTM-51, shows the results obtained using the monoclonal anti-vaccine KTM-53, and ロ shows the results obtained using the anti-UV denatured DNA polyclonal antibody. The definition of UZ mg for UV-denatured DNA in the block is the same as that in Fig. 1.
第 4図は、 実施例 3の細胞内の D N Λに紫外線を照射した場合の紫外線照射量 と紫外線変性 DN A量との関係を示す。 〇はモノクローナル抗体 KTM— 51、 △はモノク n—ナル抗怵 KTM— 53を川いた場合をそれぞれ示す。 なお、 図中 の紫外線変性 DNAの UZmgの定義は、 第 1図における定義と同じ。 発叨を実施するための最良の形態 FIG. 4 shows the relationship between the amount of ultraviolet irradiation and the amount of ultraviolet-denatured DNA in the case where the intracellular DNA of Example 3 was irradiated with ultraviolet light. 〇 indicates that the monoclonal antibody KTM-51 was used, and モ ノ indicates that the monoclonal antibody KTM-53 was used. The definition of UZmg of the UV-denatured DNA in the figure is the same as the definition in FIG. The best form to carry out the framing
以下、 実施例および比蛟例により本発叨をより詳細に説叨する。 実施例 1 抗紫外線変性 D N Λモノク π—ナル抗体の調製  Hereinafter, the present invention will be described in more detail with reference to examples and examples. Example 1 Preparation of anti-ultraviolet-denatured D N Λmonoclonal π-null antibody
(a) 免疫用抗原の作製  (a) Preparation of antigen for immunization
免疫用抗原は、 精製サケ精子 DN Λを川いて以下のようにして作成した。  The antigen for immunization was prepared as follows using purified salmon sperm DNΛ.
市販のサケ精子 D N Λ (シグマ社） を T E緩衝液 （ 1 mM エチレンジァミ ン 四酢酸を含む 1 0 ト リス Z塩酸 p II 8. 0 ) に溶解した後、 通常のフェ ノール Zクロロフォルム変性ーェタノール沈澱法 （バイォテクノ πジー実験操作 入門、 柴 忠義 著、 講談社） にて精製し、 2 6 0 8 0 11111比が1. 8 Commercially available salmon sperm DNΛ (Sigma) is dissolved in TE buffer (10 Tris-Z hydrochloric acid pII 8.0 containing 1 mM ethylenediaminetetraacetic acid), and the normal phenol-Z-chloroform-modified ethanol precipitation method is used. (Introduction to biotech π-G experiment operation, written by Tadayoshi Shiba, Kodansha), and the ratio of 2608011111 was 1.8.
5を示す高純度に精製された DNAを取得し、 スタンダード正常 DNAとした。 スタンダード正常 DNAを l irigZm l となるように T E緩術液で希釈し、 希釈 液を直 ί圣 1 0 cmのプラスチックシャーレに深さ 1. 5 mmになるように入れ、 市販の殺菌用紫外線ランプ （東芝） を用いて (). mW/cm2 の紫外線を 1 0 0 0 0 J/m2 となるように照射し、 紫外線変性 DNAを作製した。 なお、 紫外 線強度は紫外線線量計 （トプコン社） で測定した。 Highly purified DNA showing 5 was obtained and used as standard normal DNA. Dilute standard normal DNA with TE buffer to make lirigZm l, put the diluted solution into a 10 cm plastic Petri dish to a depth of 1.5 mm, and use a commercially available UV lamp for sterilization. (Toshiba) was used to irradiate (). MW / cm 2 ultraviolet rays at 1000 J / m 2 to produce ultraviolet-denatured DNA. The UV intensity was measured with an ultraviolet dosimeter (Topcon Corporation).
この紫外線変性 DNAをスタンダード紫外線変性 DNAとし、 直ちに一 4 0 で遮光保存した。  This UV-denatured DNA was used as standard UV-denatured DNA, and immediately stored in a light-shielded condition at 140 ° C.
(b) 免疫  (b) Immunity
1 m g Zm 1のメチル化したゥシ血清アルブミン （ B S Λ). 水溶液 （シグマネ J: ) と l mgZm lの （a) 項で作製したスタンダード紫外線変性 D N Λを等量混 合し、 免疫原とした。  1 mg Zm 1 methylated serum albumin (BS Λ). An equal amount of an aqueous solution (Sigmane J :) and 1 mg Zml of the standard UV-denatured DNΛ prepared in (a) were mixed and mixed with the immunogen. did.
G週令雄 B 1 b Z cマウスの背中皮下に免疫原とフロイ ンドの完全了ジュバ ント （生化学工業） とを等量混合したェマルジヨンを l m l 匹投与し、 以後 3 週間おきに 2回、 免疫原とフ□イ ンドの不完全了ジュバン ト （生化学工業） とを 等量混合したェマルジヨンを 0. 5m l _ 匹投与し、 さらに 3週問後、 尾静脈よ り P B S緩衝液 （ 1 4 0 mM お ί化ナト リゥムを含む 1 ϋ リン酸緩衝液 G-male male B1bZc mice were subcutaneously injected into the back of the mouse with lml of an emulsion mixed with an equal amount of immunogen and Freund's complete juvant (Seikagaku Corporation), and then twice every three weeks thereafter. 0.5 ml _ of an emulsion mixed with an equal amount of an immunogen and an incomplete fin fin adjuvant (Seikagaku Kogyo Co., Ltd.) was administered. After 3 weeks, PBS buffer (1) was injected from the tail vein. 40 mM phosphate buffer containing 40 mM sodium phosphate
P H 7. 4 ) に溶解した免疫原を 0. S mgZ匹投与した。 The immunogen dissolved in PH 7.4) was administered to 0.1 mg mgZ animals.
(c ) ハイプリ ドーマ作製法 免疫原の最終投与日の 3日後、 マウスから脾臓を無菌的に摘出し、 血清を含ま ない RPM I— 1 6 4 0培地 （日水製薬） 中に懸濁し、 1 0 0メッシュの網を通 過させて単独細胞化した。 これを低張液中に懸濁して赤血球を溶解した後、 血清 含まない RPM I— 1 64 0培地で 3回遠心洗浄し、 得られた細胞を抗体産生細 胞とした。 (c) Hypri-doma production method Three days after the last day of immunogen administration, the spleen was aseptically removed from the mouse, suspended in serum-free RPMI-164 medium (Nissui Pharmaceutical), and passed through a 100 mesh mesh. To make it a single cell. This was suspended in a hypotonic solution to lyse the erythrocytes, and then centrifugally washed three times in a serum-free RPMI-1640 medium, and the obtained cells were used as antibody-producing cells.
一方、 マウスミエ n—マ細胞 P 3 U 1を G I T培地 （和光純薬） で対数増殖期 まで培養し、 得られた細胞を回収して血清を含まない RPM I -1 6 4 0培地で 3回遠心洗浄した。  On the other hand, mouse mye n-ma cells P3U1 were cultured in GIT medium (Wako Pure Chemical Industries) until the logarithmic growth phase, and the obtained cells were collected three times in serum-free RPMI-164 medium. Washed by centrifugation.
抗体産生細胞とマウスミエ σ—マ細胞 P 3 U 1を 1 0 ： 1の比率で混合し、 1 2 0 0 r pmで 5分間遠心分離し、 上清を取り除いた。 遠心分離後、 細胞残査に 5 0%ポリエチレングリコール 1 5 0 0液 （ベーリンガー 'マンハイム社） を 1 m 1ゆつくり加え、 さらに血清を含まない RPM 1 - 1 64 0培地を 5 Om 1徐 々に加えて細胞を懸濁させた。 懸濁液を 1 2 0 0 r pmで 5分間遠心分離して上 清を取り除き、 細胞残査を HAT培地 （ 1 x 1 0—4Mヒポキサンチン、 4 x 1 0 一7 Mァミノプテリン、 2 X 1 0— 5Mチミジンを含む G I T培地） 〖こ 1 X 1 06細 胞 Zm 1となるようよう懸濁し、 これを 9 6ゥヱルマイクロタイタ一プレートに 2 0 0 1 Zゥ ルずつ分注した。 細胞はこのままの状態で炭酸ガスィンキュベ —ターにて 5%炭酸ガスを含む空気中で 3 7 :で培養した。 1 0日後に観察した ところ、 すべてのゥヱルにハイプリ ドーマのコロニーが観察された。 Antibody-producing cells and mouse mye σ-ma cells P 3 U1 were mixed at a ratio of 10: 1, centrifuged at 1200 rpm for 5 minutes, and the supernatant was removed. After centrifugation, add 1% 1 of 50% polyethylene glycol 150 solution (Boehringer's Mannheim) to the cell residue slowly, and add serum-free RPM1-1-640 medium 5 Om 1 gradually. And the cells were suspended. The suspension 1 2 0 0 r pm at removing the supernatant for 5 minutes and centrifuged, the cell residue HAT medium (1 x 1 0- 4 M hypoxanthine, 4 x 1 0 one 7 M Aminoputerin, 2 X 1 0- 5 M thymidine was suspended so as to be GIT medium) 〖This 1 X 1 0 6 cells Zm 1 comprising, by 2 0 0 1 Z © Le this into 9 6 © Weru microtiter first plate dispensing did. The cells were cultured as they were in a carbon dioxide gas incubator at 37: in air containing 5% carbon dioxide. When observed 10 days later, colonies of hybridomas were observed in all the pellets.
(d) スク リ一二ング  (d) Screening
以下に示す方法で目的抗体を産生している細胞を含むゥ ルを選択した。 Cells containing cells producing the target antibody were selected by the following method.
9 6ウェルマィクロタイタープレートに 1 0 gZm 1になるよう 4 X S SC9 4 x SSC to 10 gZm 1 in 6-well microtiter plate
( 1 X S S C= 1 5 0 m 塩化ナト リウム、 1 5mM クェン酸ナト リウム PH 7. 4) で希釈したスタンダード正常 DNAまたはスタンダード紫外線変性 DNA溶液を 2 5 1 ゥ ルずつ分注し、 4 :で一夜静置した。 プレートを P BS緩衝液で 3回洗浄した後、 BS Aを 1 %含む PBS緩衝液を 2 5 0 1 ウ ュルずつ分注して室温で 1時間静置し、 P B S緩衝液で各ゥェルを 3回洗淨して 反応用プレートとした。 反応用プレートに 0. 1 %85八を舍む？83緩衝液で 1 1倍に希釈した培養上清を 5 0 1 Zゥュルずつ入れ、 室温で 1〜 3時間静置 (1 XSSC = 150 m sodium chloride, 15 mM sodium citrate PH 7.4) Dispense 25 1 μl each of standard normal DNA or standard UV-denatured DNA solution, and overnight at 4: It was left still. After the plate was washed three times with PBS buffer, dispensed 2501 wells of PBS buffer containing 1% BSA, allowed to stand at room temperature for 1 hour, and washed each well with PBS buffer. After washing three times, a reaction plate was obtained. Do you put 0.1% 85 in the reaction plate? Add 500 ml of culture supernatant diluted 11-fold with 83 buffer, and allow to stand at room temperature for 1-3 hours
11  11
訂正された-^ & (規 91^ 反応させた。 Corrected-^ & (Regulation 91 ^ Reacted.
反応後、 プレートを Twe e n 2 0を 0. 0 5 %含む P B S緩衝液で 5回洗浄 した後、 5 0〃 1の P〇D標識抗マウスィムノグロブリンズ一ゥサギ I g G (ダ コ社） を加え、 室温で 1時 反応させた。 反応後、 プレートを Twe e n 2 0を 0. 0 5%含む PBS緩衝液で 5回洗'净した後、 MCDP (1 0—N—メチルカ ルバモイルー 3, 7—ジメチルァミノ一 1 0 H—フヱノチアジン） を 3 ju τη 1および H202を 0. 0 1 5%含む 5 OmMクェン酸緩衝液 （ρΗ 5. 0) を 5 0 1 Zゥ ルずつ加えて 3 0分室温で反応させ、 最後に反応停止液を 5 0 β 1 Ζゥエルずつ加えて反応を終了させた。 After the reaction, the plate was washed five times with a PBS buffer containing Tween 20 at 0.05%, and then 50〃 P〇D-labeled anti-mouse immunoglobulins 100 egret IgG (Dakosha Co., Ltd.) ) Was added and reacted at room temperature for 1 hour. After the reaction, the plate was washed 5 times with a PBS buffer containing Tween 20 at 0.05%, and MCDP (10-N-methylcarbamoyl-3,7-dimethylamino-10H-phenothiazine) was added. 3 ju Tauita 1 and H 2 0 2 to 0.0 1 5% containing 5 Omm Kuen acid buffer (ρΗ 5. 0) of 5 0 1 Z © Le by addition reacted with 3 0 minutes at room temperature, and finally the reaction The reaction was terminated by adding 50 β 1 gel of stop solution.
反応後、 マイクロプレートリーダー （コロナ社、 ΜΤΡ— 1 2 0) を用いて各 ゥエルについて 6 6 0 rimの吸光度を測定した。 その結果、 強い反応性の得られ たゥヱル （1. 0以上の吸光度を示したゥヱル） は、 5 7 6ゥヱル中 8ゥエルで After the reaction, the absorbance at 660 rim was measured for each well using a microplate reader (Corona, Inc., 120). As a result, the number of gels with strong reactivity (the ones showing absorbance of 1.0 or more) was 8 ゥ out of 576 ゥ ヱ.
0O -37"こ o 0O -37 "this o
(e) クローニング  (e) Cloning
クローニングは限界希釈法でおこなった。 （d) 項に示した強い反応性の得ら れたゥェル内の細胞を 1 x 1 07個 Zm 1の胸腺細胞を含む G I T培地にて 0. 5個 Zm 1になるように希釈し、 9 6ゥヱルマイクロタイタ一プレートに 2 0 0 1 Zゥヱルずつ分注し、 炭酸ガスィンキュベータ一にて 37 ：、 5%炭酸ガス を含む空気中で培養を行った。 培養開始 1 0〜1 4日後に各ゥニルを観察し、 生 育コロニーが 1個のゥ ルを選び出し、 選択されたゥ ルの培養上清を （d) 項 に示す方法にて測定し、 目的抗体を産生している細胞株を含むゥエルを選択した 。 さらに同様の操作を 2回繰り返し、 安定に目的抗体を産生するモノクローナル 抗体産生細胞株を確立した。 得られた細胞株は全部で 8株であった。 Cloning was performed by the limiting dilution method. (D) diluting the strong reactivity of the cells of the resulting the in Weru shown in section so as to be 0.5 or Zm 1 in GIT medium containing 1 x 1 0 7 or Zm 1 thymocytes, The solution was dispensed into a 96-well microtiter plate at a rate of 200 ゥ ヱ Z-well, and cultured in a carbon dioxide incubator in air containing 37: 5% carbon dioxide. 10 to 14 days after the start of culture, each pinil was observed. One colony was selected as a growing colony, and the culture supernatant of the selected cell was measured by the method described in section (d). A well containing the antibody-producing cell line was selected. The same procedure was repeated twice to establish a monoclonal antibody-producing cell line that stably produces the target antibody. A total of eight cell lines were obtained.
後述の交差反応率の検討、 結合定数の検討結果より、 確立した 8株の内もっと も優れた性質を持つ細胞株を最終的に 2株選択し、 それぞれ KTM— 5 1および 5 3と名付けた。  Based on the results of the cross-reactivity rate and binding constant studies described below, two cell lines with the most excellent properties were finally selected from the eight established strains and named KTM-51 and 53, respectively. .
( f ) 抗体の精製  (f) Antibody purification
8週令以上の雄 Ba 1 bZcマウスの腹腔内にプリスタン （2， 6, 1 0, 1 4ーテトラメチルペンタデカン） を lm l Z匹注入し、 このマウスを 2週間飼育  Male Ba 1 bZc mice over 8 weeks old were injected intraperitoneally with lmL Z pristane (2,6,10,14-tetramethylpentadecane) and bred for 2 weeks
12  12
訂正きれた用紙 (規則 91) した。 このマウスに （e) 项で確立した細胞株を 1 X I 06個 匹ずつ接種した 。 7〜 1 4曰後、 マウス腹腔に十分腹水が貯留した時点で、 腹腔内に 1 8 Gの注 射針を挿入して腹水を回収し、 3 0 0 0 r pmで 1 0分遠心分離して上清を回収 した。 この上清を結合緩衝液 （3M 塩化ナトリウム、 1. 5 M グリシン p H 8. 9 ) で 3倍に希釈し、 結合緩衝液で平衡化したプロテイ ン Aカラムに通塔 した。 カラムを P B S緩衝液で洗浄した後、 5 OmMグリシン Z塩酸緩衝液 （P H 2. 5 ) で抗体を溶出した。 溶出した抗体液は 1 M リン酸緩衝液 （ P H 7. 5) で直ちに中性化した。 回収した抗体液は P B S緩衝液に対して十分透析を行 い、 精製モノクローナル抗体を得た。 Corrected form (Rule 91) did. This mouse cell lines established in (e) paragraph was inoculated 1 XI 0 6 or animals. After 7 to 14, the ascites was sufficiently stored in the abdominal cavity of the mouse, and an 18 G injection needle was inserted into the abdominal cavity to collect the ascites fluid, and centrifuged at 300 rpm for 10 minutes. To collect the supernatant. This supernatant was diluted 3-fold with a binding buffer (3 M sodium chloride, 1.5 M glycine, pH 8.9) and passed through a protein A column equilibrated with the binding buffer. After washing the column with a PBS buffer, the antibody was eluted with 5 OmM glycine Z hydrochloride buffer (PH2.5). The eluted antibody solution was immediately neutralized with 1 M phosphate buffer (pH 7.5). The recovered antibody solution was sufficiently dialyzed against a PBS buffer to obtain a purified monoclonal antibody.
(g) 抗体の性質  (g) Antibody properties
以下に （e) 項で確立したハイプリ ドーマの産生する抗体の性質を示す。  The properties of the antibody produced by the hybridoma established in section (e) are shown below.
なお、 分子量は S DS—ポリアク リルアミ ドゲル電気泳動法 （フアルマシア社 、 ファース トシステム） で B i 0 -R a d社の分子量マーカ一を使用して、 等電 点は等電点電気泳動法 （フアルマシア社、 ファース トシステム） で等電点マーカ 一にはフアルマシア社の p I力リブレーションキッ トを用い、 免疫グロブリンサ ブクラスはザィメッ ト社のモノクローナル抗体タイピングキッ トを用いてそれぞ れ測定した。  The molecular weight was determined by SDS-polyacrylamide gel electrophoresis (Pharmacia, First System) using a molecular weight marker of Bi0-Rad, and the isoelectric point was determined by isoelectric focusing (Pharmacia). The first isoelectric point marker was used for the isoelectric point marker and the immunoglobulin subclass was measured using the Zymmet monoclonal antibody typing kit.
なお、 サイクロブタン型チミンダイマーの結合定数は、 Hoylaerts の方法 （J. Immuno. Methods, 126, 253(1990)) を参考に、 1 0 0 0 0 JZm2の 2 5 4 nmの 紫外線を照射した DN A 1 mg中には 1 X 1 0 - 7mo 1 sのチミンダイマーが存 在するものとして求めた。 The binding constant of the cyclobutane-type thymine dimer was determined by irradiating 100000 JZm 2 with UV light of 250 nm, referring to the method of Hoylaerts (J. Immuno.Methods, 126, 253 (1990)). during DN a 1 mg 1 X 1 0 - was determined as thymine dimer of 7 mo 1 s is present.
その結果、 ハイプリ ドーマ KTM— 5 1が生産するモノクローナル抗体 KTM 一 5 1およびハイブリ ドーマ KTM— 5 3が生産するモノクローナル抗体 KTM - 5 3は以下の性質を有していた。  As a result, the monoclonal antibody KTM-51 produced by the hybridoma KTM-51 and the monoclonal antibody KTM-53 produced by the hybridoma KTM-53 had the following properties.
モノクローナル抗体 KTM— 5 1 Monoclonal antibody KTM—5 1
分子量： 1 6 5 0 0 0 ± 5 0 0 0  Molecular weight: 1 6 5 0 0 0 ± 5 0 0 0
等電点： 6. 5 ± 0. 5  Isoelectric point: 6.5 ± 0.5
サブクラス ： I g G1 κ  Subclass: Ig G1 κ
モノクローナル抗体 KTM— 5 3 Monoclonal antibody KTM—5 3
13  13
訂芷された用紙 (規則 91) 分子量： 1 7 5 0 0 Π土 5 Π 0 0 Revised Form (Rule 91) Molecular weight: 1 7 5 0 0 Π soil 5 Π 0 0
等電点： G . 6 ± 0. 5  Isoelectric point: G. 6 ± 0.5
サブクラス ： I g G1 κ  Subclass: Ig G1 κ
結合定数： 0. 1〜 5. 0 x 1 0 VM  Coupling constant: 0.1 to 5.0 x 10 VM
サブクラスは両者ともフラグメントへの分解や、 標識体修飾が容易な I g Gl タイプであった (Lamoyi, Π. & Nisonnff, Λ. , J. Immunol. Methods (1983), 56, 235 ) 。 また結合定数も十分に満足できる値であった。  Both subclasses were of the Ig Gl type, which was easy to decompose into fragments and modify the label (Lamoyi, Π. & Nisonnff, Λ., J. Immunol. Methods (1983), 56, 235). The coupling constant was also a value that was sufficiently satisfactory.
(h) 交差反応率の検討  (h) Examination of cross-reactivity
特異性検討用の抗原を £1下のようにして作製した。  Antigens for specificity studies were prepared at under £ 1.
(a) 項と同様の方法で調製したスタンダード正常 DNAを 1 mg/m 1含む TE緩衝液を、 直径 1 0 cmのプラスチックシャーレに深さ 1. 5 mmになるよ うに入れ、 波長選択が可能な紫外線照射装置 （ΛΤΤΟ社） にて特定波長の紫外 線を 1 0 0 0 JZm2照射した。 照射の方法は 2 54 nmの紫外線のみを照射す る方法、 2 54 nmの紫外線照射後、 3 1 3 n mの紫外線を追加照射する方法、 1 (]mMのァセトフヱノンを DNA溶液中に含 させ、 3 1 3 nmの紫外線を照 射する方法の 3種を用いた。 また、 P o l y [ !A]、 P o l y [dC]、 P o 1 y [dT] (いずれもシグマ社） を 1 Π () gZm Ηこなるように TE緩衝液 に溶解し、 深さ 1. 5 mmになるようにプラスチックプレートに添加した。 これ に実施例 1 (a) 项と同様の装置を用いて 1 () 0 0 J 2の紫外線を照射した o .. Selectable wavelength by adding TE buffer containing 1 mg / m1 of standard normal DNA prepared in the same manner as in (a) to a plastic dish of 10 cm in diameter to a depth of 1.5 mm. Irradiation of 1000 JZm 2 with ultraviolet light of a specific wavelength was performed using a suitable ultraviolet irradiation device (Isha). Irradiation method is a method of irradiating only 254 nm ultraviolet light, a method of irradiating with 254 nm ultraviolet light, and an additional irradiation of 313 nm ultraviolet light, 1 () mM acetophenone is included in the DNA solution, Three methods of irradiating 3 13 nm ultraviolet light were used, and Poly [! A], Poly [dC], and Poly1 [dT] (all from Sigma) were replaced by 1Π ( ) gZm な る Dissolved in TE buffer so that it became a mixture, and added it to a plastic plate to a depth of 1.5 mm, using the same apparatus as in Example 1 (a) 1. Irradiated with 0 J 2 UV o ..
各種 DNAに対する抗休の反応性を Hi]接競合型工ンザィ厶ィムノアッセィで以 下のようにして測定した。 9 6ゥヱルマイク πタイタ一プレートに （a) 項で作 製したスタンダード紫外線変性 DNAを 1 0 1含む 4 X S SC溶液を 5 Anti-reactivity of various DNAs was measured by Hi] competition type enzyme immunoassay as follows. 9 A 4X SSC solution containing 101 the standard UV-denatured DNA prepared in (a) was added to a 6-micrometer π-titer plate.
0 1 ゥヱルずつ分注し、 4 °Cで一夜 ifj :した。 プレートを PBS緩衝液で 3 回洗浄した後、 スキムミルクを 5%含む 緩 ®液溶液を 2 5 0〃 1 Zゥエル ずつ分注して室温で 1
Figure imgf000016_0001
し、 PB S緩衝液で各ゥェルを 3回洗'净して反応 fflプレートとした。 反応川プレートに TE緩術液、 TE緩衝液で 1 ϋ 0 u gZm 1に希釈した上記紫外線変性 D N Λまたは紫外線照射を行つていない DNAを 5 0 】 Zゥヱルずつ分注し、 さらにプレートを撹排しながらモノクローナル抗体 KTM— 5 1または KTM— 5 3をそれぞれ 1 0 0 n g/m 1含む 0. 1 %B S A— P B S緩衝液をそれぞれ 5 ϋ 1 /ゥ ルずつ入れ、 混合して 4 tで一夜ま たは室温で 3時出]^ して反 させた。 The mixture was dispensed at 0 ° C. at 4 ° C. overnight. After washing the plate three times with PBS buffer, dispens a 250 µl buffer solution containing 5% skim milk at room temperature.
Figure imgf000016_0001
Then, each well was washed three times with a PBS buffer to prepare a reaction ffl plate. Dispense the above-mentioned UV-denatured DNΛ or unirradiated DNA diluted to 1ϋ0 μg Zm 1 with TE buffer or TE buffer into 50 μ】 Z ゥ ヱ of the reaction river plate, and further dispose the plate. Monoclonal antibody while stirring Include 100 ng / m 1 of KTM-51 or KTM-53 0.1 Add 0.1% BSA-PBS buffer at 5ϋ1 / ゥ, mix and mix at 4 t overnight or at room temperature At 3 o'clock] ^
反応後、 プレートを T w e e n 2 ϋを（) . 0 5 %含む Ρ B S緩術液で 5回洗诤 した後、 ぺルォキシダーゼ （ P〇 D ) 標識抗マウスィ厶ノグロブリンズーゥサギ I g G (ダコ社） を 5 ϋ ΛΙ 1 Zゥェルずつ加え、 室温で 1時 反応させた。 反応 後、 プレートを Tw e e n 2 0を ϋ. 0 5 %含む P B S緩衝液で 5回洗浄した後 、 MC DP溶液を 5 0 n 1 Zゥュルずつ加え、 3 ϋ分室温で反応させ、 最後に反 応停止液を 5 0 1 Ζゥヱルずつ加え、 マイクロプレート リ一ダ一で 6 6 0 n m の吸光度を測定した。 相対吸光度は、 得られた吸光度について TE緩衝液のみを 用いた場合の吸光度を 1 0 0%としたときの百分率として算出した。  After the reaction, the plate was washed 5 times with BS mild solution containing Tween 2 2 (5) .5%, and then peroxidase (PD〇) -labeled anti-mouse immunoglobulin maize IgG ( (Dako Co., Ltd.) was added in 5 51 Zwell portions, and reacted at room temperature for 1 hour. After the reaction, the plate was washed five times with a PBS buffer containing Tween 20% 5%, and then the MCDP solution was added in 50 nl Z-wells, and the reaction was performed for 3 minutes at room temperature. The stop solution was added in 501 ml portions, and the absorbance at 660 nm was measured using a microplate reader. The relative absorbance was calculated as a percentage of the obtained absorbance when the absorbance when using only the TE buffer was 100%.
なお、 抗体溶液を比蛟例で得られた抗紫外線変性 DNAポリクローナル I g G を 1 0 0 0 n gZm 1、 メチル化 B S Aを 0. 1 %および正常 D N Aを 0. 1 % 含む 0. 1 %B S A— P B S緩衝液に、 POD標識抗マウスィムノグロブリンズ 一ゥサギ I g Gを POD標識抗ゥサギィ厶ノグロブリンズ一ャギ抗体 （ダコ社） に代える以外は同様の方法を川いて lli]接競合型ェンザィムィムノアツセィを行い 、 相対吸光度を算出した。  The antibody solution contains 0.1% of anti-UV denatured DNA polyclonal IgG obtained in specific examples, 0.1 ng Zm1, 0.1% of methylated BSA and 0.1% of normal DNA. The same procedure was followed except that the POD-labeled anti-mouse immunoglobulin's egret IgG was replaced with a POD-labeled anti-mouse immunoglobulin's goat antibody (Dako) in PBS buffer. Competitive enzyme immunoassay was performed to calculate the relative absorbance.
結果を第 1表に示す。  The results are shown in Table 1.
15 され ffi 鎖 1) ^ l m 15 will be ffi chain 1 ) ^ lm
Figure imgf000018_0001
Figure imgf000018_0001
1) 25411111の¾外線を!¾&1  1) 25411111 ¾ outside line! ¾ & 1
2) 254nmの紫外線を |W射した後、 3 I 3 n の¾外 ¾¾を; EiWl!SiH  2) After irradiating 254 nm ultraviolet rays | W, 3I 3 n ¾outside¾¾; EiWl! SiH
3) 1 OmMァセトフェノン存在下 3〗 3 nmの を 1 モノクローナル抗体 KTM— [ 1は紫外線を照射していない DNA (スタンダ ―ド ΊΕΙΐ' DNA) tは反応せず、 2 5 " mを主成分とする紫外線を照射した D ΝΛとは強く反応したが、 この 2 5 /1 rimの紫外線を照射した DNAに 3 1 3 π inの紫外線を追加照射した DNAでは、 2 5 4 π mの紫外線を照射しただけの D ΝΛに比して反応、性が減弱した。 一方、 モノク —ナル抗 ί木 KTM— 5 3は紫外 線を照射していない DNAとは反応せず、 2 5 4 πττιを主成分とする紫外線を照 射した DNAとは強く反応した。 この 2 Γ) nmの紫外線を照射した DNAに 3 1 3 nmの紫外線を追加照射した DNAでは、 Γ) nmの紫外線を照射しただ けの DN Λよりさらに強く反応した。 また 1 () mMの了セトフヱノンを加えた D ΝΛ溶波に 3 1 3 Ti mの紫外線を照 して作 した紫外線変性 DNAにも強く反 J芯した。 また： 1の紫外線照 ポリヌクレオチドの内、 木究叨のモノク ーナル 抗休と反応性があったのは！ 1 o I y Iパ 」 のみであった。 3) In the presence of 1 OmM acetophenone, 3〗 3 nm of 1 monoclonal antibody KTM— [1 does not react with UV-irradiated DNA (standard ΊΕΙΐ 'DNA) t; Although the DNA reacted strongly with the UV-irradiated D ΝΛ, the DNA that had been irradiated with this UV-irradiation of 25/1 rim was further irradiated with UV-irradiation of 3 13 π in. On the other hand, the reaction and the attenuated sex were weaker than those of D の, whereas Monoc-Nal anti-Kitsuki KTM-5 3 did not react with DNA that had not been irradiated with ultraviolet light, and had a major component of 25 4 πττι. The DNA that had been irradiated with UV light of 2Γ) nm was strongly irradiated with DNA that had been additionally irradiated with UV light of 3 nm13 nm. The reaction was even stronger than that of DNΛ, and the purple light produced by irradiating the DΝΛ wave with 1 () mM of cetophenone with 313 Tim ultraviolet light. Strongly anti J wick in the line denatured DNA also:. Of the first ultraviolet irradiation polynucleotide, there was reactive with monochromator Naru anti rest of KiKiwamu叨was only 1 o I y I path "! .
抗紫外線変性 DN Λボリク "一ナル 休は、 木発 Iリ jのモノク uーナル杭休と比 蛟できる吸光度を得るためには約 1 (Ηί:ίの濃 を必¾とし、 この濃度条件下でも  Anti-ultraviolet-denatured DN Λ ク 休 約 約 を 得 を 得 を 得 を 得 を 得 を 得 を 得 を 得 を 得 を 得 を 得 を 得 を 得 を 得 を 得 を 得 を 得 を 得 を 得 を 得 を 得 を 得 を 得 を 得 を 得 を 得 を 得 を 得 を 得 を 得 を 得 を 得 を 得 1 に はBut
lfi 紫外線変性 DNAに対する結合力が劣ることが示された。 さらに、 メチル化 BS Aや正常 DNAで吸収したにも関わらず、 各種 DN Aに対して反応性を示し、 特 異性の面でモノクローナル抗体 KTM— 5 1または 5 3と比較して劣ることが示 された。 lfi It was shown that the binding ability to ultraviolet denatured DNA was poor. In addition, it shows reactivity to various DNAs, despite being absorbed by methylated BSA and normal DNA, indicating that it is inferior to the monoclonal antibody KTM-51 or 53 in specificity. Was done.
実施例 2 紫外線照射ラッ ト肝臓紐織に対する反応 Example 2 Response to UV-irradiated rat liver cord
(a) 紫外線照射ラッ ト肝臓組織切片 (肝臓 tJJ片) の作製  (a) Preparation of UV-irradiated rat liver tissue section (liver tJJ section)
ホルマリ ン固定パラフィ ン包埋ラッ ト肝臓からミ ク uト一厶にて 5 amの K片 を作製し、 ゲラチンをコートしたスライ ドグラス上に固定した。 このスライ ドグ ラスをホッ トプレートを用いて/! 5 °Cで一夜乾燥させた。 乾燥させたスライ ドグ ラスをトルエン、 エタノールに浸潰処理後、 PBS緩衝液 （1 4 OmM 塩化ナ ト リゥムを舍む 1 0 mM リン酸綏 ¾液 p 117. 4 ) に 5分ずつ 3回浸潰洗浄 した。 スライ ドグラス上にプロティナーゼ K (0. 2 U/m 1 , シグマ社） を滴 下し、 37 °Cで 1 5分処现した後、 PBS緩術液に 5分ずつ 3回浸瀆して洗浄し 、 さらに蒸留水で洗浄し、 紫外線非照射ラッ ト肝臓切片とした。  From the liver of formalin-fixed paraffin-embedded rat, K pieces of 5 am were prepared with microtome and fixed on a gelatin-coated slide glass. The slide glass was dried at / 5 ° C overnight using a hot plate. After the dried slide glass is immersed in toluene and ethanol, it is immersed three times in PBS buffer (10 mM phosphate phosphate solution containing 11 OmM sodium chloride, p117.4) for 5 minutes each. It was flushed. Drop proteinase K (0.2 U / m 1, Sigma) on the slide glass, treat at 37 ° C for 15 minutes, and then immerse the cells in PBS slow surgery solution 3 times for 5 minutes each for washing Then, it was further washed with distilled water to obtain a rat liver section not irradiated with ultraviolet rays.
紫外線照射ラット肝臓切片は、 紫外線非照射ラッ ト肝臓切片に乾燥を防止する ため微量の蒸留水を滴下し、 市販の殺菌用紫外線ランプ （東芝） を用いて 0. 5 mWZcm2 の紫外線を 3 0 0 0 J/m2 照射して作製した。 なお、 紫外線強度 は紫外線線量計 （トプコン社） で測定した。 UV irradiation rat liver sections, ultraviolet in order to prevent drying non-irradiated rat liver sections was added dropwise distilled water traces, 3 ultraviolet radiation 0. 5 mWZcm 2 using a commercially available germicidal UV lamp (Toshiba) 0 It was produced by irradiation with 0 J / m 2 . The ultraviolet intensity was measured with an ultraviolet dosimeter (Topcon Corporation).
( b ) 抗体によるラッ ト肝臓切片の染色  (b) Staining of rat liver sections with antibodies
紫外線照射および非照射ラッ ト肝臓切片をそれぞれ固定したスライ ド上にモノ クローナル抗体 K T M— 5 1または K T M— 5 3をそれぞれ各種濃度および B S Aを 0. 1 %含む PBS緩衝液をそれぞれ滴下し、 室温で 3時間静置反応させた 。 反応後、 スライ ドグラスを PBS緩 S液で 5回浸'? 洗浄した後、 スライ ドグラ ス上に P 0 D標識抗マウスィ厶ノグロブリンズーゥサギ抗体を滴下し、 室温で 1 時間反応させた。 反応後、 スライ ドグラスを PBS緩衝液で 5回浸漬洗淨した 後、 ジァミノベンチジンを 0. 2mgZrn 1および 11202を（）. ϋ 1 5 %含む 0 . 1 M酌酸緩衝液 (Ρ ΙΙ 5. 0) を滴下して室 で 5〜1 5分 ίίϋ発色させ、 水で 充分洗淨した後、 肝臓切片を顕微鏡 （Χ4 ϋ 0〜1 00 Q) で観察した。 A PBS buffer solution containing various concentrations of the monoclonal antibody KTM-51 or KTM-53 and a PBS solution containing 0.1% BSA was added dropwise to the slides on which the liver sections of the UV-irradiated and non-irradiated rat were fixed, respectively. For 3 hours. After the reaction, the slide glass was immersed and washed 5 times with a PBS gentle S solution, washed, and then a POD-labeled anti-mouse immunoglobulin sperm antibody was dropped on the slide glass and reacted at room temperature for 1 hour. After After the reaction, the slide Douglas was 5 times immersed AraiKiyoshi with PBS buffer, the di § amino benzidine a 0. 2mgZrn 1 and 11 2 0 2 (). Ϋ 1 containing 5% 0. 1 M Shakusan buffer (Ρ 5.0) was added dropwise, and the color was developed in a room for 5 to 15 minutes. After thorough washing with water, the liver section was observed under a microscope (Χ4ϋ 0 to 100 Q).
なお、 抗体溶液を比較例で得られた抗紫外線変性 DNAポリク ナル抗体を 各禾 IE濃度およびメチル化 B S Λを 0. 1 %およびスタ ンダード正 ' DNAを 0. 1 %および 0. 1%BSA合む PBS緩 »波に、 P〇 D標識抗マウスィ厶ノグロ プリンズーゥサギ I gGを POD標識抗ゥサギィムノグ プリンズ—ャギ抗体に 代える以外は同様の方法を用いて肝臓切片を染色し、 肝臓切片を観察した。 結果を第 2表に示す。 2表 抗關 The antibody solution was prepared using the anti-UV denatured DNA polyclonal antibody obtained in Comparative Example. 0.1% of each IE concentration and methylated BSΛ and 0.1% of standard positive DNA in 0.1% and 0.1% BSA combined with PBS buffer, P〇D-labeled anti-mouse immunoglobulin Principe IgG Liver sections were stained using the same method except that POD-labeled anti-Pseudosagiminog prince-goat antibody was used, and the liver sections were observed. The results are shown in Table 2. Table 2
紫外線照 W 片 紫外線非照射 K)片  UV irradiation W piece UV non-irradiation K) piece
(ng/ml) 核 細顧 細胞股 翻 核 細胞 ¾ 細胞膜 頹洞  (ng / ml) Nuclear review cell translocation nucleus cell ¾ cell membrane sinus
画 冊 a) Books a)
TH-5I 100 +++  TH-5I 100 +++
10 ++  10 ++
ft  ft
1000 ++++  1000 ++++
KTH-53 100 +++  KTH-53 100 +++
10 ++  10 ++
ιηηο ++ -/+ -h 小 +/- -/+ -/+ +/- ゥサキ 'ホ' '1ク α-ナ MgG 100 ++ +/- +/- 、卜 -/+ V- +/- V- ιηηο ++-/ + -h Small +/--/ +-/ + +/- ゥ Saki 'ho' '1k α-na MgG 100 ++ +/- +/-,--/ + V- + / -V-
10 +/- -/+ 10 +/--/ +
α) 相対反応 ¾：非 1Sに強い ( + + + + ) 、強い （-ト-ト -I ) 、 【|' TO ( + + ) 、やや弱い (+ ) 、かなり 弱い (+/-) 、非 ¾に弱い (-/+) 、 ϊβされず (一） 本 叨のモノク π—ナル抗休を使川した ¾合、 紫外線照射.ラッ ト肝臓切片の核 の部位のみで濃度依^的に反応性が認められた以外は、 細胞 κや細胞膜および類 洞の部位では反応性が認められず、 紫外線非照射ラッ ト肝臓切片では反応性がま つたく認められなかつた。 α) Relative response ¾: strong (+ + + +), strong (-to-to -I), [| 'TO (++), slightly weak (+), considerably weak (+/-), Non-weak (-/ +), not ϊβ (1) When using the pi-mononal anti-cancer of this quince, UV irradiation. Concentration-dependent only at the nucleus site in rat liver slices Except for the reactivity, no reactivity was observed in the cell κ, cell membrane, or sinusoidal sites, and no reactivity was observed in the UV-irradiated rat liver sections.
抗紫外線変性 DNAポリク π—ナル 休を使川した場合、 紫外線照射ラッ ト肝 臓切片の核の部位で反応性が認められたが、 木発 njjのモノクローナル抗体と比較 するとその反応性は く、 また細 、 細 ll 膜および類洞の部位とも弱いながら 反応性が認められ、 また紫外線非 Mij ラッ ト )] F臓 'の 部位で反応性が認めら れた。  When the anti-ultraviolet-denatured DNA polyclonal π-null suspension was used, reactivity was observed at the nucleus site of the ultraviolet-irradiated rat liver slices, but the reactivity was low when compared with the wooden antibody of njj. In addition, reactivity was observed in the thin, thin membranes and sinusoidal areas, although weakly, and in the non-Mij rat (UV) non-Mij rat)] area.
実施例 3 ΙίΠ接兢合型ェンザ'ィムィ ムノ了ッセィによる 製 DNA中の紫外線変 性 DN A量の定量 Example 3 Ultraviolet transformation in DNA produced by an integrative enzymatic Of quantitative DNA
(a) 紫外線変性 DN Aの作製  (a) Preparation of UV-modified DNA
スタンダード正常 DNAに対する紫外線の照射方法を、 0. 1〜0. 5mW/ cm2の紫外線を種々の時 lj照射することに代える以外は実施例 1 (a) と同様 の方法を用いて紫外線変性 D N Λを作製した。 Standard radiation methods of ultraviolet to normal DNA, 0. 1~0. 5mW / cm ultraviolet modified using the same method 2 of ultraviolet except for replacing to lj irradiation at various times to Example 1 (a) DN Λ was prepared.
(b) 間接競合型ェンザィムィムノアッセィ  (b) Indirect competitive enzymimnoassy
9 6ゥヱルマイクロタイタープレートに実施例 1 (a) 項で作製したスタンダ ード紫外線変性 DNAを 1 O igZm l含む 4 xS SC溶液を 5 0 β 1Ζゥエル ずつ分注し、 4 tで一夜静置した。 プレートを PBS緩衝液で 3回洗浄した後、 1 %83 ー？85緩衝液溶液2 5 0 / 1を分注して室温で 1時間静置し、 PB S緩衝液で各ゥュルを 3回洗浄して反応用プレートとした。 反応用プレートに 0 . 1 %BS A— PBS緩衝液で種々の濃度に希釈したスタンダード紫外線変性 D NAまたは （a) 項で作製した紫外線変性 DNA液を 5 0 】 ゥエルずつ分注 し、 さらにプレートを撹袢しながら 1 0 n gZm 1の KTM— 5 1または KTM 一 5 3抗体溶液 （0. 1 %BSA— PBS緩衝液） をそれぞれ 5 0 l Zゥヱル ずつ入れ、 混合して 4 t:で一夜または室温で 3時間静置反応させた。  Dispense 50 x 1 SC of 4 x SSC solution containing 1 OigZml of the standard UV-denatured DNA prepared in Example 1 (a) into a 96-well microtiter plate, and allow to stand overnight at 4 t Was placed. After washing the plate 3 times with PBS buffer, 1% 83-? An 85 buffer solution 250/1 was dispensed, allowed to stand at room temperature for 1 hour, and each plate was washed three times with a PBS buffer to prepare a reaction plate. Dispense 50 µl of standard UV-denatured DNA or UV-denatured DNA solution prepared in (a) diluted to various concentrations in 0.1% BSA-PBS buffer into the reaction plate, and plate again. While stirring, add 50 ng of KTM-51 or KTM-53 antibody solution (0.1% BSA-PBS buffer) of 10 ng Zm1 each, mix and mix at 4 t: The reaction was allowed to stand overnight or at room temperature for 3 hours.
反応後、 プレートを Twe e n 2 0を 0. 0 5 %含む P B S緩衝液で 5回洗浄 した後、 ペルォキシダーゼ （POD) 標識抗マウスィ厶ノグロブリンズーゥサギ I gG (ダコ社） を 5 0 1 Zゥ ルずつ加え、 室温で 1時間反応させた。 反応 後、 プレートを T we e n 2 0を 0. 0 5 %含む P B S緩衝液で 5回洗淨した後 、 MC DP溶液を 5 0 1 Zゥ ルずつ加え、 30分室温で反応させ、 最後に反 応停止液を 5 0 u 1 Zゥュルずつの加えて反応を終了させた。  After the reaction, the plate was washed five times with a PBS buffer containing Tween 20 at 0.05%, and then peroxidase (POD) -labeled anti-mouse immunoglobulin zoo- egret IgG (Dako) was added to the plate. Then, the mixture was added at room temperature and reacted for 1 hour at room temperature. After the reaction, the plate was washed five times with a PBS buffer solution containing Tween 20 at 0.05%, and the MCDP solution was added at a volume of 501 ゥ, and allowed to react at room temperature for 30 minutes. The reaction was terminated by adding 50 μl of the reaction stop solution at a time.
反応後、 マイクロプレート リーダ一を用いて各ゥヱル毎に 6 6 0 nmの吸光度 を測定した。 相対吸光度は、 得られた吸光度について TE緩衝液のみを用いた場 合の吸光度を 1 0 0%としたときの百分率として算出した。  After the reaction, the absorbance at 660 nm was measured for each gel using a microplate reader. The relative absorbance was calculated as a percentage of the obtained absorbance when the absorbance when using only the TE buffer was 100%.
比較のため、 抗体溶液を比蛟例で^られた抗紫外線変性 DNAポリクローナル I g Gを 1 0 0 n g / 1含む抗体溶液 （ ϋ . 1 %メチル化 B S Λ、 0. 1 %正 常 DNAを含む 0. 1%BS Α— PBS緩衝液溶液） に代え、 POD標識抗マウ スィ厶ノグロプリンズーゥサギ I g Gを P〇 D標識抗ゥサギィ厶ノグロブリンズ  For comparison, an antibody solution containing 100 ng / 1 of anti-ultraviolet-denatured DNA polyclonal IgG obtained from a specific antibody solution (ϋ0.1% methylated BSΛ, 0.1% normal DNA Containing 0.1% BS (PBS buffer solution) instead of POD-labeled anti-mouse immunoglobulin zoo egret IgG
19  19
訂正された) ¾紙 (規則 91) ーャギ抗体 （ダコ社） に代える以外は同様の方法を用いて間接競合型ェンザィム ィムノアツセィを行い、 相対吸光度を算出した。 (Corrected) Paper (Rule 91) An indirect competitive enzyme immunoassay was performed using the same method except that the antibody was replaced with a goat antibody (Dako), and the relative absorbance was calculated.
検量線を第 1図に、 本測定系で測定した紫外線照射量と紫外線変性 DN A量と の関係を第 2図に示す。  Fig. 1 shows the calibration curve, and Fig. 2 shows the relationship between the amount of UV irradiation and the amount of UV-modified DNA measured by this measurement system.
以上の結果、 本発叨のモノク π—ナル抗体を用いて紫外線変性 DNAが定量で きることが示された。 また、 本発明のモノクローナル抗体を用いることにより、 紫外線変性 D N Aが D N Aに照射する紫外線量に比例して生成することが示され 一方、 抗紫外線変性 DN Aポリク口ーナル抗体を用いて検量線を作製する場合 、 本発明のモノクローナル抗体と同等の感度を得るためには 1 0倍以上の量を必 要とすることが示された。 また、 抗紫外線変性 DN Aゥサギポリクロ一ナル抗体 を用いた場合、 検量線に基づいて照射紫外線量と紫外線変性 D N Aの生成量との 間に直線的な相関関係は認められなかった。  As a result, it was shown that ultraviolet denatured DNA can be quantified using the monoclonal antibody of the present invention. In addition, it was shown that the use of the monoclonal antibody of the present invention produced UV-denatured DNA in proportion to the amount of UV light irradiated to the DNA, while a calibration curve was prepared using an anti-UV-denatured DNA monoclonal antibody. In this case, it was shown that a 10-fold or more amount was required to obtain sensitivity equivalent to that of the monoclonal antibody of the present invention. In addition, when the anti-UV denatured DNA-Egret polyclonal antibody was used, no linear correlation was observed between the amount of irradiated UV and the amount of UV-modified DNA based on the calibration curve.
実施例 4 サンドイ ッチ型ェンザィムィムノアッセィによる細胞内紫外線変性 D Example 4 Intracellular UV Denaturation by Sandwich Enzymimnoassay D
N A量の測定  Measurement of N A amount
(a) 細胞内紫外線変性 DN Aの作製  (a) Preparation of intracellular UV-denatured DNA
A 4 3 1細胞を FCSを 1 0%含む D— MEM培地 （ギブコ社） で直径 5 cm の培養用ディッシュ一面に単層培養した。 培地を除去し、 細胞を PBS緩衝液に てディッシュごと 3回洗浄した後、 深さ 2 mmになるよう PBS緩衝液をディッ シュに入れ、 0. 3 mWZ cm2の紫外線を種々の時間照射した。 紫外線照射後 、 直ちに PBS緩衝液を除去して細胞をディッシュごと液体窒素を用いて凍結し た。 凍結した細胞を室温で融解し、 これに溶解液 [ 1 mM エチレンジァミ ン四 酢酸および SDSを 1 %舍む 1 OmM ト リス Z塩酸緩衝液 （pH 8. 0) ] をA431 cells were monolayer cultured on a 5 cm diameter culture dish in a D-MEM medium (Gibco) containing 10% FCS. The medium was removed, the cells were washed three times per dish Te in PBS buffer, placed in the dish PBS buffer so that the depth 2 mm, and the ultraviolet 0. 3 mWZ cm 2 was irradiated for various times . Immediately after the ultraviolet irradiation, the PBS buffer was removed, and the cells were frozen together with the dish using liquid nitrogen. Thaw the frozen cells at room temperature, and add a lysate [1 OmM Tris Z-HCl buffer (pH 8.0) containing 1 mM ethylenediaminetetraacetic acid and 1% SDS] to this.
0. 2m 1加えて溶解後、 細胞溶解液をマイク口チューブに回収した。 回収した 細胞溶解液からセパジーンキッ ト （三光純薬社） を用いて DN Aを精製♦回収し た。 回収した DN Aを TE緩衝液で溶解して得られた DN A溶液は 2 6 O nmの 吸光度の測定値に基づいて DN A量を定量し、 アツセィに用いるまで一 8 ϋ Τ:で 凍結保存した。 After lysis by adding 0.2 ml, the cell lysate was collected in a tube with a microphone opening. DNA was purified and recovered from the recovered cell lysate using SepaGene Kit (Sanko Junyaku). The DNA solution obtained by dissolving the collected DNA in TE buffer is quantified based on the measured absorbance at 26 O nm, and is frozen and stored at 180 ° C until it is used for the assay. did.
(b) 抗体の酵素標識  (b) Enzyme labeling of antibody
20 訂正された ¾鉱 (規則 91) KT - 5 1または KTM— 5 3抗体をぞれぞれ 5 m gZm 1含む 0. 1M ほう酸緩衝液 （P H 8. 0 ) を 3 に保ち、 これにィ ミノチオラン （ピアス社 ) を 2mgZm〗含む 0. 1 ほう酸緩衝液 （ p I- 18. 0) を撹捭しながら 1 0 0 X 1加え、 3 0 °Cで 3 0分反応させた。 反応後、 5 mM エチレンジ了ミ ン 四酢酸を含む 0. 1 Mリ ン酸緩衝液 （pH G. 0) で平衡化した G— 2 5力 ラム （ 1 X 3 0 cm) に通塔し、 抗怵画分を回収した。 20 Corrected Mines (Rule 91) KT-51 or KTM-53 antibodies each containing 5 mg Zm1 0.1 M borate buffer (PH 8.0) kept at 3, containing 2 mg Zm〗 of iminothiolane (Pierce) 0 100 × 1 was added while stirring a borate buffer (pI-18.0), and the mixture was reacted at 30 ° C. for 30 minutes. After the reaction, pass through a G-25 column (1 × 30 cm) equilibrated with 0.1 M phosphate buffer (pH G.0) containing 5 mM ethylenediaminetetraacetic acid. The anti-III fraction was collected.
1 m 1の P 0 D (柬洋紡績社） 溶液 （ 1 () τπ g 】 0. 1 リン酸緩衝 液 ρΙ-17. ϋ) を 3 0 tに保ち、 これに 4 0〃 1の EMCS (ピアス社） を 4 mg/ 2 0 0〃 1含むジメチルホルムアミ ドを撹排しながら加え、 3 0 °Cで 3 0 分反応させた。 反応後、 1 M リン酸緩衝液 （P H 6. 0) で^衡化した G— 2 5カラム （I X 3 0 cm) に通し、 I)〇D画分を回収した。  Keep the 1 m 1 P 0 D (Seyobo Co., Ltd.) solution (1 () τπ g) 0.1 phosphate buffer ρΙ-17.ϋ) at 30 t, and add 40〃 1 EMCS ( (Pierce Co., Ltd.) was added while stirring, and dimethylformamide containing 4 mg / 200〃1 was added thereto, and reacted at 30 ° C. for 30 minutes. After the reaction, the mixture was passed through a G-25 column (IX 30 cm) equilibrated with 1 M phosphate buffer (pH 6.0), and the I) 〇D fraction was collected.
カラムより回収した抗体および P 0 D溶液を遠心型濃縮器 （セントリコン 3 0 、 アミコン社） で l rn lにまで濃縮し、 両溶波を撹拌混合して 3 で 3 0分反 応させた。 反応後、 0. 1 M リン酸緩衝液 （pH 7. 0 ) で平衡化した G— 1 5 0カラム （ 1. 5 X G 0 c m) に通し、 P〇 D標識化抗体画分を回収した。 回収した P〇 D標識化抗紫外線変性 D N Aモノク π—ナル抗体は 0. 1 %になる よう BS Aを加えた後、 使用するまで一 4 0でで凍結保存した。  The antibody and the POD solution recovered from the column were concentrated to lrnl with a centrifugal concentrator (Centricon 30, Amicon), and both dissolved waves were stirred and mixed and reacted at 3 for 30 minutes. After the reaction, the antibody was passed through a G-150 column (1.5 X G0 cm) equilibrated with 0.1 M phosphate buffer (pH 7.0) to collect a PD〇-labeled antibody fraction. The recovered P〇D-labeled anti-ultraviolet-denatured DNA mono-π-nal antibody was added to BSA at 0.1% and then frozen and stored at 140 until use.
比較のため、 抗体溶液を比較例で得られた抗紫外線変性 DN Aポリク π—ナル 抗体を 5TngZm 1含む 0. 1 ほう酸緩衝液 （pH 8. 0.) に代える以外は 同様の方法を用いて POD標識化抗紫外線変性 DNAポリクローナル抗体を作製 し Λ-ο  For comparison, the same method was used except that the antibody solution was replaced with 0.1 borate buffer (pH 8.0) containing 5 TngZm1 of the anti-UV denatured DNA π-nal antibody obtained in Comparative Example. Preparation of POD-labeled anti-UV denatured DNA polyclonal antibody
( C ) サンドイ ツチ型ェンザィ厶ィムノアッセィ  (C) Sandwich-type enzymimmynoassy
9 ( ウェルマイク タイタ一プレートに KTM— 5 1または KTM— 5 3抗体 をそれぞれ 1 0 0 zgZm 1含む 0. 1 M 炭酸緩衝液 （pH 9. 5 ) を 5 0 « 1 Zゥュルずつ分注し、 4 tで一夜静置した。 プレートを PBS緩衝液で 3回洗 した後、 B S Λを 1 %含む P H S緩術液' 液を 2 0； u I Zゥニルずつ分注し て 温で 1時 HHi^iSし、 P 13 S¾»液で ゥ ルを 3 ΙΰΙ洗^して反応川プレート とした。 反応用プレートに B S Λを 0. 1 %含む P B S緩衝液で希釈した各濃度 のスタンダード紫外線変性 DNA溶液、 ならびに （a) ： ¾で作製した DNA溶波 を 5 0 1 Zゥエルずつ分注し、 4 °Cで一夜または室温で 3時問静置反応させた 。 反応後、 プレートを Tw e e π 2 0を 0. 0 5 %舍む Ρ Β S緩衝液で 5回洗浄 した後、 （b) 項で作製した P〇D標識抗紫外線変性 DNAモノクローナル抗体 を 5 0 】ノウヱルずつ加え、 室温で 1時問反応させた。 反応後、 プレートを T we e n 2 0を 0. 0 5 %含む P B S緩衝液で 5回洗浄した後、 MCDP溶液を 5 0 M 1 Zゥ ルずつ加え、 室温で 3 0分間反応させ、 最後に反応停止液を 5 0 μ 1 Ζゥュルずつ加えて反応を終了させた。 9 (KTM-51 or KTM-53 antibody containing 100 zgZm1 each in a 0.1 μM carbonate buffer (pH 9.5) was dispensed into 50 «1 Z-wells. After washing the plate three times with PBS buffer, dispensed 20% of PHS gently perfused solution containing 1% of BSΛ; ^ iS, wash the pellet 3 times with P13S¾ solution, and use it as a reaction plate. プ レ ー ト The reaction plate was diluted with PBS buffer containing 0.1% of BS ス タ ン ダ ー ド in each concentration of standard UV-denatured DNA. Solution, and (a): DNA wave created in ¾ Was dispensed at a time of 501 Zell and allowed to stand at 4 ° C overnight or at room temperature for 3 hours. After the reaction, the plate is washed with 0.05% Tween π20 for 5 times. 5 After washing the plate 5 times with S buffer, 50% of the P〇D-labeled anti-ultraviolet-denatured DNA monoclonal antibody prepared in section (b) was added. The mixture was added one by one and allowed to react at room temperature for 1 hour. After the reaction, the plate was washed five times with a PBS buffer containing Tween 20 at 0.05%, and the MCDP solution was added at 50 M / Z, and allowed to react at room temperature for 30 minutes. The reaction was terminated by adding 50 μl of the reaction stop solution at a time.
反応後、 マイクロプレート リ一ダーを用いて各ゥヱル毎に 6 6 0 nmの吸光度 を測定した。 相対吸光度は、 得られた吸光度について TE緩衝液のみを用いた場 合の吸光度を 1 00%としたときの百分率として算出した。  After the reaction, the absorbance at 660 nm was measured for each gel using a microplate reader. The relative absorbance was calculated as a percentage of the obtained absorbance when the absorbance when using only the TE buffer was set to 100%.
比較のため、 抗体溶液を比較例で得られた抗紫外線変性 DN Aポリクロ一ナル 抗体を 1 0 0 ;«gZm】含む 0. 1M炭酸緩衝液 （pH 9. 5) に代え、 （b) 項で作製した P 0 D標識抗紫外線変性 DNAモノクロ—ナル抗体を P 0 D標識抗 紫外線変性 DN Aポリク口ーナル抗体に代える以外は同様の方法を用いてサンド ィツチ型ェンザィ厶ィムノ了ッセィを行い、 相対吸光度を算出した。  For comparison, the antibody solution was replaced with 0.1 M carbonate buffer (pH 9.5) containing 100 µg; A sandwich-type enzyme immunoassay was performed using the same method except that the P0D-labeled anti-ultraviolet-denatured DNA monoclonal antibody prepared in step 1 was replaced with a P0D-labeled anti-ultraviolet-denatured DNA monoclonal antibody. The relative absorbance was calculated.
検量線を第 3図に、 紫外線を照射した細胞から回収した D N Aについて照射紫 外線量と紫外線変性 D N A量との関係を第 4図に示す。  Fig. 3 shows the calibration curve, and Fig. 4 shows the relationship between the irradiation UV dose and the amount of UV-modified DNA for DNA recovered from cells irradiated with ultraviolet light.
本発明のモノクローナル抗体を用いることによりサンドィツチ型ェンザィムィ ムノ了ッセィ系が構築でき、 また本発明のモノクロ一ナル抗体を用いることによ り細胞内の紫外線変性 DN Λ量を定量できることが示された。  It was shown that a sandwich-type enzyme immunoassay system can be constructed by using the monoclonal antibody of the present invention, and that the amount of intracellular ultraviolet-denatured DN protein can be quantified by using the monoclonal antibody of the present invention.
一方、 抗紫外線変性 DN Aゥサギポリクロ一ナル抗体を用いた場合は、 バック ダラゥンドが高く、 また精度の髙ぃ検量線を作製することができなかったため、 サンドィツチ型ェンザィ厶ィムノアツセィ系を構築することができなかった。 実施例 5 サザンプロッティ ングによるポリメラ一ゼ 'チヱ一ン · リアクション  On the other hand, when the anti-ultraviolet-denatured DNA heron polyclonal antibody was used, the back-drain was high and an accurate calibration curve could not be prepared. Did not. Example 5 Polymerization by Southern Plotting 'Chain Reaction'
(PCR) 増幅 DNA中の特定塩基配列 DNAの検出  (PCR) Detection of specific base sequence DNA in amplified DNA
( a ) 成人 T細胞白血病患者白血球からの D N Aの抽出  (a) Extraction of DNA from leukocytes of adult T-cell leukemia patients
正常人および成人 T細胞白血病患者から 5 %クェン酸緩衝液を抗凝固剤に用い て 5 m〗血液を採取し、 30 0 0 r pmで 1 0分間遠心分離を行った。 この血液 よりバフィ一コートを採取し、 等量のリューコプレップ溶液 （ベタ トンディキン  5 ml blood was collected from normal and adult T cell leukemia patients using 5% citrate buffer as an anticoagulant, and centrifuged at 300 rpm for 10 minutes. A buffy coat was collected from this blood and an equal volume of leukoprep solution (Beta Tondikin
22  twenty two
訂正された兩紙 (規則 ソン社） と混合した後、 30 0 0 r pmで 1 0分 Hi遠心分離し、 リンパ球層を P BS緩衝液中に回収した。 回収したリンパ球液をさらに 1 5 0 0 r pmで 5分遠 心分離し、 上清を取り除いてリンパ球とした。 リンパ球中の DN Aはセパジーン キッ ト （三光純薬社） を用いて精製した。 精製した DNAは 2 0 0 1の丁£緩 衝液に溶解し、 2 6 0 πτηの吸光度を測定して DNA濃度を定量した。 Corrected sheets (Rule After the mixture was centrifuged at 300 rpm for 10 minutes, the lymphocyte layer was recovered in PBS buffer. The collected lymphocyte fluid was further centrifuged at 1500 rpm for 5 minutes, and the supernatant was removed to obtain lymphocytes. DNA in lymphocytes was purified using Sepagene Kit (Sanko Junyaku). The purified DNA was dissolved in 2001 buffer solution, and the DNA concentration was quantified by measuring the absorbance at 260 πτη.
(b) PCRによる特定 DNAの増幅  (b) Amplification of specific DNA by PCR
1. 5mMの dNTP (宝酒造） 1 6 1、 （a) 項で得られた精製した DN A 1 ug, リ了クシヨン B液 （パーキンエルマーシータス社） 1 0 1、 2 5 0 Uの T a qポリメラーゼ （パーキンエルマ一シ一タス社） 0. 5 / l、 DNA 合成機で作製した 1 0 xMのプライマー 1 [Seiki, M. et al. Pro Natl. Acad . Sci. USA., 80.3618(1983) 記載の gag遺伝子の 62-82 番目の塩基配列を有する プライマー] およびプライマ一 2 (前記 gag遺伝子の 209- 229 番目の塩基配列を 有するプライマ一） をそれぞれ 2. 5 】ならびにミネラルオイリレ 1 0 0 1 を P CR用反応チューブに入れ、 パーキンエルマーシータス社の DN Αサ一マル サイクラ一 P J 2 0 0 0を用いて、 9 4。。で1分問、 5 5 :で 1分間および 7 2 でで 2分間を 1サイクルとして、 3 0サイクル繰り返した後、 7 2 で 5分間反 応させた。 反応後、 1 0 0 1のクロロホルムを加えて撹拌後、 1 0 0 0 0 r p mで 5分間遠心分離し、 氷層を回収して 4 tで保存した。  1. 5 mM dNTP (Takara Shuzo) 161, 1 ug of purified DN A obtained in (a), Ryxion B solution (Perkin Elmer Cetus) 101, 250 U of Taq polymerase (PerkinElmer Systems Inc.) 0.5 / l, 10 xM primers prepared with a DNA synthesizer 1 [Seiki, M. et al. Pro Natl. Acad. Sci. USA., 80.3618 (1983)] Primers having the 62-82th nucleotide sequence of the gag gene] and Primer 2 (primer having the 209-229th nucleotide sequence of the gag gene) 2.5 and mineral oil lily, respectively. Into a PCR reaction tube, and use a PerkinElmer Cetus DN® Thermal Cycler PJ 2000 to obtain 94. . One cycle was repeated for 1 minute, 55: 1 minute, and 72 for 2 minutes. The cycle was repeated 30 times, and then reacted for 72 minutes for 5 minutes. After the reaction, 1001 of chloroform was added and stirred, followed by centrifugation at 100,000 rpm for 5 minutes. The ice layer was collected and stored at 4 t.
( c ) サザントランスファ一  (c) Southern transfer
1 %ァガロースゲル （シグマ社、 タイプ IIZ2mM エチレンジァミ ン四酢酸 を含む 9 0 mM トリス ホウ酸緩衝液 p H 8. 0 ) で 5 1のサンプルを 6 0 V定電圧下 2時間電気泳動した。 電気泳動後のゲルを変性剤水溶液 （1. 5M 塩化ナト リウム、 0. 5 Na〇H) 中で 4 5分間振とうし、 氷洗後、 中和 液 Π . 5 M 塩化ナト リゥムを含む 1 M ト リス Z塩酸緩衝液 （ p II 7. 4) ] 中で 3 0分間、 2 0 X S S C中で 1時 R13振とうした。 2 0 x S S Cを充分含ん だヮッ トマン社 3 MM紙を水平台に置き、 この上にゲル、 ニトロセルロースフィ ルター （S&S社） 、 ヮッ トマン社 3 MM紙およびペーパータオルを重ね、 この 上に水平にガラス板をおいて、 さらに約 1 k gの重りを置いて一晩トランスファ —した。 トランスファ一後のニトロセルロース膜は濾紙にはさみ、 真空オーブン  The 51 samples were electrophoresed on a 1% agarose gel (Sigma, 90 mM Tris borate buffer pH 8.0 containing 2 mM ethylenediaminetetraacetic acid, type IIZ2) at 60 V constant voltage for 2 hours. The gel after electrophoresis is shaken in an aqueous denaturant solution (1.5 M sodium chloride, 0.5 Na〇H) for 45 minutes, washed with ice, and neutralized with 1.5 M sodium chloride. M13 Tris Z hydrochloric acid buffer (pII7.4)] for 30 minutes and 1 hour in 20 XSSC at R13. 20 x 3 x 35 mm paper containing a lot of SSC is placed on a horizontal table, and the gel, nitrocellulose filter (S & S), 3 mm paper, and a paper towel are stacked on top of this. The transfer was done overnight with a glass plate and about 1 kg of weight. Nitrocellulose membrane after transfer is sandwiched between filter paper, vacuum oven
23  twenty three
された 紙 (規則 91) で 80 °cで 2時間処理した。 Paper (Rule 91) At 80 ° C for 2 hours.
(d) ハイブリダイゼーション  (d) Hybridization
P ATK 1 0 5 〔Sekine、 S. et al.,Agric. Oiol. Chem. :52, 1267 (1988)] 1 M gを 2 5〃 1の TE緩衝液に溶解し、 石英製チャンバ一にて UVC ( 2 5 4 nm が主成分の紫外線） を 5 0 0 0 JZm2 照射して紫外線変性化して、 プローブと した。 次に、 （c) 項で作製したニトロセルロース膜をハイブリダィゼーシヨン バックに入れ、 ハイブリダイゼーシヨン液 （ 0. 9 M 塩化ナト リ ウム、 0. 0 9 Mクェン酸ナト リウム、 0. 0 1 %BSA、 0. 0 1%フイコール、 0. 0 1 %ポリビニルビ σリ ドン、 0. 5%SDS、 1 0 Q g/m 1 牛胸線 DNA) を 2 0 m 1入れて 6 5 で 1時間ィンキュベ一トした後、 ハイブリダイゼーショ ンバック中の液を廃棄した。 このハイブリダイゼ一ションバックに、 上記紫外線 変性化プローブを 1 0 で 3分間処理し、 氷中で急冷したものを 1 2. 5 M 1 含む 1 0m lのハイブリダイゼーション液を入れ、 6 5でで一晩反応させた。 反 応後、 0. 1 %SDSを含んだ 2 xS SCでニトロセルロース膜を 2回室温で 3 0分間振とうして洗浄し、 次に SDSを 0. 1 %含む 0. 5 35(：溶液で6 5 、 1時間振とうして洗浄し、 さらに BSAを 3%含む PBS緩衝液中で室温で 1時問振とうしてプロッキングを行った。 プロッキング後の二トロセルロース膜 を 1 0 n gZm 1の本発明の KTM— 5 3抗体および 0. B S Aを 1 %舍む PB S緩衝液溶液中で 4 :一晩振とうしながら反応させ、 次に Twe e n 2 0を 0. 0 5 %含む P B S緩衝液で洗浄した。 洗浄後、 膜を P 0 D標識抗マウスィムノグ ロブリンーゥサギ I gGおよび 0. 1 %83八を含む 85緩衝液と室温で3時 間反応させた。 反応後、 二トロセルロース膜を Tw e e n 2 0を 0. 0 5 %舍む PBS緩衝液で洗浄し、 ジァミノベンチジンを 0. Smg/m】および H202を 0. 0 1 5%舍む0. 11^酢酸緩¾液 （ 1-15. 0) に浸潰して発色反応を検出 した。 P ATK 105 [Sekine, S. et al., Agric. Oiol. Chem .: 52, 1267 (1988)] Dissolve 1 Mg in 25〃1 TE buffer and place in a quartz chamber. UVC (ultraviolet light whose main component is 254 nm) was irradiated with 50,000 JZm 2 to denature the ultraviolet light to obtain a probe. Next, the nitrocellulose membrane prepared in section (c) was placed in a hybridization bag, and a hybridization solution (0.9 M sodium chloride, 0.09 M sodium citrate, 0.1% sodium citrate) was added. 0.1% BSA, 0.01% Ficoll, 0.1% Polyvinyl sigma-don, 0.5% SDS, 10 Qg / m1 bovine chestline DNA) After incubation for 1 hour, the solution in the hybridization bag was discarded. The hybridization back is treated with the UV-denatured probe at 10 for 3 minutes, and 10 ml of a hybridization solution containing 12.5 M1 of the solution rapidly cooled in ice is added thereto. The reaction was performed overnight. After the reaction, the nitrocellulose membrane was washed twice with 2 x SSC containing 0.1% SDS by shaking at room temperature for 30 minutes, and then 0.535 (: solution containing 0.1% SDS) After washing with shaking for 65 hours for 1 hour, blocking was performed for 1 hour at room temperature in PBS buffer containing 3% BSA, and blocking was performed. ngZm 1 of the KTM-53 antibody of the present invention and 0. BSA were allowed to react in a 1% PBS buffer solution with shaking 4: overnight, and then Tween 20 was added to 0.05. After washing, the membrane was reacted for 3 hours at room temperature with an 85 buffer solution containing P0D-labeled anti-mouse immunoglobulin- ゥ sagi IgG and 0.1% 838.8. the cellulose membrane was washed with Tw een 2 0 at 0.0 5%舍Mu PBS buffer, di § amino benzidine a 0. Smg / m] and the H 2 0 2 0.0 1 5%舍Mu 0. 11 ^ Acetic acid buffer (1-15.0) It was detected.
その結果、 成人 T細胞白血病の患者より回収したリンパ球をサンプルとした場 合のみ、 2 G 1 b pの位置に発色バンドが観察され、 患者リンパ球中には成人 T 細胞白血病ウイルスが存在することが示された。  As a result, only when a sample of lymphocytes collected from an adult T-cell leukemia patient was used, a colored band was observed at 2 G 1 bp, indicating that adult T-cell leukemia virus was present in the patient's lymphocytes. It has been shown.
実施例 6 イ ンサイチュ （ i n s i t u) ハイブリダィゼーシヨンによる M〇 Example 6 In situ (in situ) M〇 by hybridization
24 訂正きれた用紙 (規則 91) LT- 細胞内特定塩基配列 R N Aの検出 24 Corrected Form (Rule 91) LT- Detection of specific base sequence RNA in cells
(a) 細胞の固定  (a) Cell fixation
実施例 5 (a) 項と同様の方法で取得した正常人リ ンパ球および FCSを 1 0 %含む RPM I 1 64 0培地で培養した MOLT— 4細胞（ATCC CRL-1582) を P BS緩衝液で 2回洗'净し、 PBS緩衝液で 5 0細胞 1になるように希釈した 後、 サイ トスピンを用いて、 8 0 0 r pm l 0分 00でスライ ドグラス上に細胞を 張り付けた。 室温で 1 0分間乾燥させた後、 パラホルム了ミ ドを 4%含む PBS 緩衝液で ：、 1 0分間固定し、 P B S緩衝液で洗浄した。 さらにエタノール Z 酢酸 （3 1) 液に浸漬して室温で 2 0分間固定し、 7 0%エタノール水溶液で 2回、 PBS緩衝液で 2回洗浄した後、 グリシンを 2mg/m l含む PBS緩衝 液 （PH 7. 4) に 4 tで 1 0分問浸潰した。 P B S緩衝液で 2回洗浄し、 ホル ムアミ ドを 5 0%含む 2 XS SC溶液に浸漬し、 室温で 1 0分間放置した。 Example 5 MOLT-4 cells (ATCC CRL-1582) cultured in RPMI 1640 medium containing 10% FCS and normal human lymphocytes obtained by the same method as in section (a) were added to PBS buffer. After washing twice with a PBS buffer, the cells were diluted to 50 cells 1 with a PBS buffer, and the cells were adhered to a slide glass at 800 rpm using a site spin at 800 rpm. After drying at room temperature for 10 minutes, the cells were fixed with PBS buffer containing 4% paraform medium for 10 minutes, and washed with PBS buffer. After immersion in ethanol-Z-acetic acid (31) solution, fixation at room temperature for 20 minutes, washing twice with 70% aqueous ethanol solution and twice with PBS buffer solution, PBS buffer solution containing 2 mg / ml glycine (2 mg / ml) PH 7.4) was immersed for 10 minutes at 4 t. The plate was washed twice with a PBS buffer, immersed in a 2XSSC solution containing 50% formamide, and allowed to stand at room temperature for 10 minutes.
(b) ノヽィブリダイゼ一ション (b) Noidization
実施例 5 (d) 項で作製した紫外線変性化プローブを 2 g 1含む溶液を 1 0 0 tで 3分間処理して DNAを 1本鎖にした後、 氷中で急冷した。 これを 2 0〜5 0 1のハイブリダィゼ一シヨン液 〔4 0%ホルムアミ ド、 0. 6M 塩 化ナト リウム、 1 mM エチレンジ了ミン四酢酸、 1 0 %硫酸デキストラン、 0 . 0 1 %B S A. 0. 0 1%フイ コール、 0. 0 1 %ポリビニルピロ リ ドン、 1 2 5 jugZm 1サケ精子 DN Aおよび 2 5 0 ^ugZm 1酵母 t RNAを含む1 0 m トリス Z塩酸緩衝液 （p I-I 7. 3) 〕 に添加し、 （a) 項で作製したスラ ィ ドグラスに滴下した。 湿潤箱にて 4 5 :で 6時問反応させた後、 3 7"tに温め たホルムアミ ドを 5 0%含む 2 XS SC溶液で 30分問浸漬洗浄を 4回繰り返し 、 1 XS SCでさらに 3 0分間浸漬洗浄を 2回行った。 スライ ドグラスに BS A を 5%およぴ酵 ¾ L ΚΝΛを 1 (1 0 zgZm 1合む P B S緩衝液を充分量滴下し て室温で 1時間放置し、 KTM— 5 3抗体を 1 0 n g/m l、 135八を1 %、 サ ケ精子 D N Λを 1 0 0〃 g Zm 1、 (· R Ν Λを 1 0 0〃 g Ζτη 1および N a Ν3を 0. 1 %含む PBS緩衝液を 5 0 u 1滴下して湿潤箱にて室温で一晩反応 させた。 反応後、 スライ ドグラスを PBS緩衝液で充分洗浄し、 POD標識抗マ ウスィ厶ノグロプリンズーゥサギ I gG、 83 を1 %、 サケ精子 DNAを 1 0 Example 5 A solution containing 2 g 1 of the UV-denatured probe prepared in section (d) was treated at 100 t for 3 minutes to make the DNA single-stranded, and then rapidly cooled in ice. This was mixed with a hybridization solution of 20 to 501 (40% formamide, 0.6 M sodium chloride, 1 mM ethylenediaminetetraacetic acid, 10% dextran sulfate, 0.01% BSA. 10% Tris Z HCl buffer (pII containing 0.01% solution, 0.01% polyvinylpyrrolidone, 125 jugZm 1 salmon sperm DNA and 250 ^ ugZm 1 yeast tRNA 7.3)] and added dropwise to the slide glass prepared in (a). After reacting in a wet box at 45: 6 for 6 hours, immersion and washing were repeated four times for 30 minutes in a 2 XS SC solution containing 50% formamide warmed to 37 "t, and further washed with 1 XS SC. Performed immersion washing twice for 30 minutes, and a sufficient amount of PBS buffer containing 5% BSA and 1 L of enzyme (10 zgZm1) was added dropwise to the slide glass and left at room temperature for 1 hour. , KTM-53 antibody at 10 ng / ml, 135 at 1%, salmon sperm DN 1 at 100 〃g Zm1, (R Ν 1 at 100 〃g Ζτη1 and Na Ν 50 μl of a PBS buffer containing 0.1% of 3 was added dropwise and reacted overnight at room temperature in a wet box After the reaction, the slide glass was sufficiently washed with a PBS buffer and the POD-labeled anti-mouse was removed. Roh Glo pudding Zoo © heron I g G, 83 1%, 0 1 salmon sperm DNA
25  twenty five
訂正きれた翔紙 (規貝 1191) 0 M g/m 1および酵母 t RN Aを 1 0 0 gZm〗含む P B S緩衝液を 5 0 1滴下して湿潤箱にて室温で 1時間反応させた。 P B S緩衝液で充分洗浄した後 、 ジァミノベンチジンを 0. 2mg/m 1および Η2〇2を 0. 0 1 5 %含む 0. 1 Μ酢酸緩衝液 （ ρ Η 5. ϋ ) で 5 ~ 1 5分 ΠΙ1発色させ、 水で充分洗浄した後、 顕微鏡 （X 4 0 0〜 1 0 0 0 ) で観察した。 Corrected Shogami (Kaikai 1191) A PBS buffer solution containing 0 Mg / m1 and 100 gZmII of yeast tRNA was added dropwise, and the mixture was allowed to react in a wet box at room temperature for 1 hour. After extensive washing with PBS buffer, at a di § amino benzidine 0. 2 mg / m 1 and Eta 2 〇 2 0.0 1 5% containing 0. 1 Micromax acetate buffer (ρ Η 5. ϋ) 5 After ~ 15 minutes, the color was developed, washed thoroughly with water, and observed with a microscope (X400 to 100).
比較のため、 モノクローナル抗体 KTM— 5 3を比較例で得られた抗紫外線変 性 D N Aポリクローナル抗体に代え、 P 0 D標識抗マウスィムノグロブリンズー ゥサギ I gGを POD標識抗ゥサギィムノグロプリンズ一ャギ抗体に代える以外 は同様の方法を用いてィンサイチュハイプリダイゼーションを行った。  For comparison, the monoclonal antibody KTM-53 was replaced with the anti-ultraviolet ray-modifying DNA polyclonal antibody obtained in the comparative example, and POD-labeled anti-mouse immunoglobulin zoo- egret IgG was replaced with POD-labeled anti-sagyeminoglobulin. In situ hybridization was performed using the same method except that the antibody was replaced with a goat antibody.
その結果、 モノクローナル抗体 KTM— 5 3を用いて検出を行うと MOLT— 細胞のみ鮮明に褐色の染色部分が観察され、 特定の遺伝子が MO LT-4細咆 にのみ発現されていることが示され、 M 0 L T— 4細胞が成人 T細胞白血病ゥィ ルスに感染していることが確認された。  As a result, when the detection was performed using the monoclonal antibody KTM-53, only the MOLT- cells showed a clear brown stain, indicating that the specific gene was expressed only in the MOLT-4 cells. However, it was confirmed that M0LT-4 cells were infected with adult T-cell leukemia virus.
一方、 抗紫外線変性 DNAポリクローナル抗体を用いて検出を行うと、 MOL T一 4細胞は全体に褐色の染色部分が観察され、 また正常人リンパ球でも核と思 われる部分を中心に褐色の染色部分が観察され、 MO L T— 4細胞が成人 T細胞 白血病ウィルスに感染していることを充分に検出することができなかった。 比較例 抗紫外線変性 D N Λポリクローナル抗体の調製  On the other hand, when detection was performed using an anti-ultraviolet-denatured DNA polyclonal antibody, MOL T-14 cells showed a brown stained part as a whole, and brown stained parts of normal human lymphocytes centered on the nucleus. Was observed, and it was not possible to sufficiently detect that MOLT-4 cells were infected with adult T-cell leukemia virus. Comparative Example Preparation of anti-ultraviolet-denatured D N Λ polyclonal antibody
(a) 抗血清の作製  (a) Preparation of antiserum
体重 3 k gのニュージーランドホワイ トラビッ ト （雄） の背中皮下に実施例 1 ( b ) 項と同様の方法で作製した免疫原とフロイ ンドの完全ァジュバントとを等 量混合したェマルジヨンを 2m 1ノ匹投与し、 以後 3週間おきに 3回、 免疫原と フロイ ンドの不完全アジュバントとを等量混合したェマルジョンを 2m 1 Z匹投 与し、 さらに 3週間後、 耳静脈より PBS緩衝液に溶解した免疫原を 0. 5mg Z匹投与した。 最終投与日の 3日後、 ホワイ トラビッ トの頸動脈より血液を採取 した。 採取した血液は 37 tで 1時間凝血させた後、 遠心分離を 30 0 0 r pm 1 0分間行い、 血清画分を回収した。  Emulsion with 2 m / kg of a 3 kg New Zealand White Travit (male) subcutaneously administered to the back of the back in an equal amount of an immunogen prepared in the same manner as in Example 1 (b) and complete adjuvant of Freund Thereafter, 2 ml 1 Z of an emulsion containing an equal amount of the immunogen and incomplete Freund's adjuvant was administered 3 times every 3 weeks, and 3 weeks later, immunization was performed by dissolving in PBS buffer from the ear vein. The stock was administered at 0.5 mg Z animals. Three days after the last dose, blood was collected from the carotid artery of the white travit. The collected blood was allowed to clot at 37 t for 1 hour, and then centrifuged at 300 rpm for 10 minutes to collect a serum fraction.
(b) 吸収用了フィニティカラムの作製  (b) Preparation of absorption affinity column
CNB r活性化セファロース （フアルマシア社） をフィルタ一上で ImM 塩  Immobilized CNB r-activated Sepharose (Pharmacia) on a filter
26 訂芷きれた用紙 (規貝 91) 酸を用いて十分洗浄し、 2分割した。 洗淨済み CNB r活性化セファロースを 1 OmgZm 1になるよう 1 OmM リン酸緩衝液 （pH 8. 0) で調製し、 直ち に 1 OmgZm】になるよう 1 OmM リン酸緩衝液 （pH 8. 0) で調製した 正常スタンダ一ド D N Aを等量撹袢混合し、 室温で 6時間緩やかに撹拌混合して 反応させた。 反応後、 ゲルをフィルター上に回収し、 0. 1Mトリス Z塩酸緩衝 液 （pH 8. 0) で洗浄した後、 同緩衝液に懸镯し、 室温で 2時間緩やかに撹拌 混合してプロッキング操作を行った。 プロッキング終了後、 ゲルをフィルタ一上 に回収した後、 塩化ナト リウムを 0. 5 M含む 0. 1 M炭酸緩衝液 （ p H 9. 5 ) と塩化ナトリウムを 0. 5 [含む 0. 1M酢酸緩衝液 （PH4. 0) とを用い て交互に 3回洗浄し、 最後に PBS緩衝液で十分に洗浄した後、 PBS緩衝液に 懸濁してカラムに充塡し、 正常スタンダード D N Aカラムとした。 26 Revised paper (Kaikai 91) After thoroughly washing with an acid, the mixture was divided into two parts. Prepare the cleaned CNB r-activated Sepharose with 1 OmM phosphate buffer (pH 8.0) to 1 OmgZm 1 and immediately add 1 OmM phosphate buffer (pH 8. An equal volume of the normal standard DNA prepared in step 0) was mixed with stirring, and allowed to react with gentle stirring and mixing at room temperature for 6 hours. After the reaction, the gel was collected on a filter, washed with 0.1 M Tris Z hydrochloric acid buffer (pH 8.0), suspended in the same buffer, gently stirred at room temperature for 2 hours, mixed and blocked. The operation was performed. After blocking, collect the gel on a filter and add 0.5 M sodium chloride in 0.1 M carbonate buffer (pH 9.5) and 0.5% sodium chloride in 0.1 M Washed alternately with acetate buffer (PH 4.0) three times, and finally washed thoroughly with PBS buffer, suspended in PBS buffer, and applied to the column to prepare a normal standard DNA column. .
メチル化 BS Aを 1 OmgZm lになるよう 1 0 mM炭酸緩衝液 （pH8. 5 ) で調製し、 1 OmgZm 1になるよう 1 0 mM炭酸緩衝液 （ρΗ8. 0 ) で調 製した洗净済み C N B r活性化セファロースを等量撹袢混合し、 室温で 6時間緩 やかに撹拌混合して反応させた。 反応後、 ゲルをフィルター上に回収し、 0. 1 Mトリス Z塩酸緩衝液 （ p H 8. 5) で洗'净した後、 同緩衝液に懸濁し、 室温で 2時間緩やかに撹捭混合してプロッキング操作を行った。 プロッキング終了後、 ゲルをフィルタ一上に回収し、 塩化ナトリウムを 0. 5M含む 0. 1M炭酸緩衝 液 （pH 9. 5) と塩化ナト リウムを 0: 51^含む0. 1 M酢酸緩衝液 （pH4 . 0) とを用いて交互に 3回洗淨し、 最後に PBS緩衝液で十分に洗浄した後、 P B S緩衝液に懸濁して力ラムに充塡し、 これをメチル化 B S Aカラムとした。 Methylated BSA was prepared with 10 mM carbonate buffer (pH 8.5) to 1 OmgZm1 and washed with 10 mM carbonate buffer (ρΗ8.0) to 1 OmgZm1. An equal amount of CNBr-activated Sepharose was stirred and mixed, and allowed to react by gentle stirring and mixing at room temperature for 6 hours. After the reaction, the gel is collected on a filter, washed with 0.1 M Tris Z hydrochloric acid buffer (pH 8.5), suspended in the same buffer, and gently mixed by mixing at room temperature for 2 hours. And the locking operation was performed. After blocking, collect the gel on a filter and add 0.1 M sodium chloride, 0.5 M carbonate buffer (pH 9.5) and sodium chloride, 0.1 M acetate buffer, 0.1 M sodium chloride. (PH 4.0) and then alternately three times, and then thoroughly washed with PBS buffer, suspended in PBS buffer, and applied to a force column. did.
(c) 精製 (c) Purification
(a) 項で作製した抗血清を結合緩衝液 （3M 塩化ナト リウム、 1. 5M グリ シン pH 8. 9) で 3倍に希釈し、 結合緩衝液で^衡化したプロテイ ン A カラムに通塔した。 カラムを PBS緩衝液で洗淨した後、 5 0mMグリ シン Z塩 酸緩衝液 （ p H 2. 5) で抗体を溶出した。 溶出した抗体液は 1 Mリン酸緩衝液 ( P II 7. 5) で直ちに中性化した。 回収した抗体液は PBS緩衝液に対して十 分透析を行い、 （b) 項で作製した P B S緩衝液で平衡化したメチル化 B S A力 ラムに通塔し、 さらに素通り画分を同じく P B S緩衝液で平衡化した正常 DN A  The antiserum prepared in (a) was diluted 3-fold with binding buffer (3M sodium chloride, 1.5M glycine pH 8.9) and passed through a protein A column equilibrated with binding buffer. Tower. After the column was washed with PBS buffer, the antibody was eluted with 50 mM glycine Z buffer (pH 2.5). The eluted antibody solution was immediately neutralized with 1 M phosphate buffer (PII7.5). The collected antibody solution was dialyzed sufficiently against PBS buffer, passed through a methylated BSA column equilibrated with the PBS buffer prepared in section (b), and further passed through the same fraction as PBS buffer. Normal DNA equilibrated with
27 訂正された^ >；£ (規則 91 カラムに通塔し、 素通り画分を紫外線変性 D N Aゥサギポリクローナル抗体 （ I gG) とした。 産業上の利用可能性 27 Corrected ^>; £ (Rule 91 The mixture was passed through a column, and the passed fraction was used as an ultraviolet-denatured DNA-Sagi polyclonal antibody (IgG). Industrial applicability
本発明により、 D N Λに紫外線を照射することによって生じる DNAの変性部 と特異的に結合するモノクローナル抗体おょぴ該モノクロ一ナル抗体を用いる変 性 D N Aの免疫学的測定法、 ならびに変性 D N Aをプローブとして用いる D N A または R N Λの免疫学的測定法が提供される。  According to the present invention, a monoclonal antibody that specifically binds to a denatured portion of DNA produced by irradiating DNΛ with ultraviolet light, a method for immunologically measuring denatured DNA using the monoclonal antibody, and a method for measuring denatured DNA An immunoassay for DNA or RNA used as a probe is provided.
28 28
訂正きれた用紙 (規貝 91)  Corrected paper (Kaikai 91)

Claims

請 求 の 筛 圆 請 の of request
1. チミンダイマーを有する一本鎖 DNAおよび二本鎖 DNAの両方の DN Αに 結合するモノク口一ナル抗体。 1. Monoclonal monoclonal antibody that binds to both single- and double-stranded DNA with thymine dimer.
2. DNAが紫外線を照射することによって取得できる変性 D N Λである請求項 1記載のモノクローナル抗体。  2. The monoclonal antibody according to claim 1, wherein the DNA is denatured DNA that can be obtained by irradiating ultraviolet light.
3. 照射する紫外線が、 2 5 4 nmまたは 3 1 3 n mの波長を含む紫外線である 請求項 2記載のモノク D—ナル抗体。  3. The monoclonal antibody according to claim 2, wherein the ultraviolet light to be irradiated is an ultraviolet light having a wavelength of 254 nm or 313 nm.
4. チミ ンダイマーが、 （G— 4) フォ トプロダク ト型チミ ンダイマ一である請 求項 1記載のモノク口ーナル抗体。  4. The monoclonal antibody according to claim 1, wherein the thymidine dimer is (G-4) a photoproduct-type thymidine dimer.
5. チミンダイマーが、 サイクロブタン型チミンダイマ一である請求項 1記載の モノクロ一ナル抗体。  5. The monoclonal antibody according to claim 1, wherein the thymine dimer is cyclobutane-type thymine dimer.
6. I g G 1 A:クラスに属し、 等電点が 6. 5 ± 0. 5、 分子量が 1 6 5 0 0 0 ± 5 0 0 0である請求項 4記載のモノク口ーナル抗体。  6. The monoclonal antibody according to claim 4, wherein the monoclonal antibody belongs to the class, has an isoelectric point of 6.5 ± 0.5, and has a molecular weight of 16500000 ± 500.
7. I g G 1 Λ:クラスに属し、 等電点が 6. 6土 0. 5、 分子量が 1 7 5 0 0 0 ± 5 0 0 0、 変性 DNAに対する結合定数が 0. ：!〜 5. 0 x l 03 /Mである 請求項 5記載のモノク口一ナル抗体。 7. IgG 1 Λ: belongs to class, isoelectric point is 6.6 soil 0.5, molecular weight is 1750 000 ± 500, binding constant for denatured DNA is 0. . 0 xl 0 3 / M a is claim 5, wherein the monochromator port one monoclonal antibody.
8. マウスハイプリ ドーマ KTM— 5 1が生産するモノクローナル抗体である請 求項 6記載のモノクローナル抗休。 ，  8. The monoclonal antibody according to claim 6, which is a monoclonal antibody produced by mouse hybridoma DTM KTM-51. ,
9. マウスハイプリ ドーマ KTM— 5 3が^産するモノクひ ナル抗体である請 求項 Ί記載のモノクローナル抗体。  9. The monoclonal antibody according to claim 6, which is a monoclonal antibody produced by mouse hybridoma KTM-53.
1 0. 請求项 G記載のモノク口一ナル抗休を生産するマウスハイプリ ドーマ Κ Τ Μ— 5 1。  10 0. Mouse Hypridoma producing the monoclonal anticancer described in claim G 项 Τ 5 — 51.
1 1 , 請求项 7記載のモノクローナル抗体を生産するマウスハイプリ ドーマ ΚΤ Μ- 5 3。  11. A mouse hybridoma producing the monoclonal antibody according to claim 7, ΚΤ-53.
1 2. 請求项 1記載のモノクローナル抗休を; πいるチミンダイマ一を有する D Ν Αの免疫学的測定法。  1 2. An immunoassay for DΝ with the thymine dimer according to claim 1;
1 3. チミ ンダイマーを有する DNAプローブ。  1 3. DNA probe with thymine dimer.
1 4. 請求項 1 3記載の DN Aをプローブとして用いる、 チミンダイマ一を有す る D N Λまたは R N Λの免疫学的測: di'法 ( 1 4. It has a thymine dimer, using the DNA according to claim 13 as a probe. Immunological measurement of DN Λ or RN る: di 'method (
3 Three
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WO2004015016A2 (en) 2002-08-02 2004-02-19 Rhodia Chimie Flame-retardant composition, preparation method and use thereof
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Title
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PHOTOCHEMISTRY AND PHOTOBIOLOGY, Vol. 54, No. 2, (1991), T. MORI et al., "Simultaneous Establishment of Monoclonal Antibodies Specific for Either Cyclobutane Pyrimidine Dimer or (6-4) Photoproduct from the Same Mouse Immunized with Ultraviolet-Irradiated DNA", pages 225-232. *

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Publication number Priority date Publication date Assignee Title
WO2004015016A2 (en) 2002-08-02 2004-02-19 Rhodia Chimie Flame-retardant composition, preparation method and use thereof
CN102253219A (en) * 2011-05-03 2011-11-23 深圳职业技术学院 Immunofluorecence technique based method for evaluating activities of cryptosporidium parvum and giardia

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