WO1995009187A1 - Anticorps monoclonal et dosage d'adn denature a l'aide de celui-ci - Google Patents

Anticorps monoclonal et dosage d'adn denature a l'aide de celui-ci 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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dna
monoclonal antibody
antibody
denatured
ultraviolet
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PCT/JP1994/001644
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English (en)
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/fr

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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.

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Abstract

Anticorps monoclonal de la classe IgG1λ reconnaissant spécifiquement le dimère de thymine de type cyclobutane ou le photoproduit (6-4) formé entre les restes de thymine d'un ADN; procédé simple de haute sensibilité pour le dosage immunologique d'ADN dénaturé à l'aide de l'anticorps ci-dessus ; et procédé de dosage immunologique d'un ADN ou d'un ARN possédant une séquence précise.
PCT/JP1994/001644 1993-09-30 1994-09-30 Anticorps monoclonal et dosage d'adn denature a l'aide de celui-ci WO1995009187A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004015016A2 (fr) 2002-08-02 2004-02-19 Rhodia Chimie Composition ignifugeante, procede de preparation et utilisation de cette composition
CN102253219A (zh) * 2011-05-03 2011-11-23 深圳职业技术学院 一种基于免疫荧光技术的隐孢子虫和贾第虫活性评价方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ACTA PATHOLOGICA JAPONICA, Vol. 40, No. 11, (1990), K. TAKEHIKO et al., "Localization In Situ of Specific mRNA Using Thymine-Thymine Dimerized DNA Probes", pages 793-807. *
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. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004015016A2 (fr) 2002-08-02 2004-02-19 Rhodia Chimie Composition ignifugeante, procede de preparation et utilisation de cette composition
CN102253219A (zh) * 2011-05-03 2011-11-23 深圳职业技术学院 一种基于免疫荧光技术的隐孢子虫和贾第虫活性评价方法

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