WO2003075013A1 - Method of measuring the activity of dna-binding protein - Google Patents

Method of measuring the activity of dna-binding protein Download PDF

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Publication number
WO2003075013A1
WO2003075013A1 PCT/JP2003/002407 JP0302407W WO03075013A1 WO 2003075013 A1 WO2003075013 A1 WO 2003075013A1 JP 0302407 W JP0302407 W JP 0302407W WO 03075013 A1 WO03075013 A1 WO 03075013A1
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WIPO (PCT)
Prior art keywords
protein
ahr
arnt
antibody
activity
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PCT/JP2003/002407
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French (fr)
Japanese (ja)
Inventor
Hitoshi Ashida
Yutaka Hatanaka
Itsuko Fukuda
Yoshiyuki Yabushita
Masashi Mizuno
Rie Kodoi
Shin Nishiumi
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The New Industry Research Organization
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Priority to JP2003573421A priority Critical patent/JPWO2003075013A1/en
Priority to AU2003211623A priority patent/AU2003211623A1/en
Publication of WO2003075013A1 publication Critical patent/WO2003075013A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6875—Nucleoproteins

Definitions

  • the present invention relates to a method for measuring the activity of a DNA-binding protein. More specifically, the present invention relates to a method for measuring the activity of a DNA-binding protein, which has been developed by further modifying a method in which an ELISA method is combined with a probiotic technique called Southwestern chemistry. The present invention relates to a method for screening a protein activity agent and an activation inhibitor, and a kit for measuring the activity of a DNA-binding protein.
  • a DNA-binding protein that exerts various functions by being activated by a reaction with a chemical substance and generating a binding ability to a specific DNA sequence (consensus sequence) and binding to the sequence is generated. There are many mechanisms.
  • polycyclic aromatic hydrocarbon compounds such as 2,3,7,8-tetraclodin dibenzodioxin (TCDD) and 3-methylcholanthrene (3-MC) Compound
  • TCDD 2,3,7,8-tetraclodin dibenzodioxin
  • 3-MC 3-methylcholanthrene
  • AhR Aryl hydrocarbon receptor
  • Arnt Arnt Nuclear Trans-locator
  • DNG Dioxins Response Element
  • GCMS gas chromatography-mass spectrometry
  • dioxin - AhR- Arnt complex comprising a DRE sequences radiolabeled with pre 3 2 P-ATP DNA Reacts with the probe to form a complex of dioxin-AhR-Arnt and the DNA probe. This complex is separated by electrophoresis and analyzed.
  • dioxin analysis by this method is poor in quantitativeness because it uses the mobility and concentration of band as indices, and the results are generally qualitative or semi-quantitative analysis.
  • accurate quantification not only requires expensive equipment to measure the amount of radioisotopes in the band, but also treats and analyzes many types of test substances at once, as in the GCMS method. It is difficult to do.
  • hapten labeling using biotin or the like may be used instead of labeling with 32P-ATP, multiple types of test substances can be processed and analyzed at a time in the same manner as with the method using radioisotopes. It is still difficult. Also, this In such a method, since enzymatic coloring is finally used, there is a problem in that sensitivity is reduced.
  • a DNA antibody is preliminarily immobilized on a microtiter plate to bind dioxins via AhR-Arnt, and a primary antibody that specifically reacts with AhR-Arnt and a secondary antibody that produces a color reaction
  • a dioxin bound to AhR-Arnt is quantitatively analyzed by sequentially adding a coloring reagent and measuring the degree of coloring by absorbance.
  • streptavidin-biotin is considered to be used for immobilization of the DNA probe and the carrier, streptavidin-biotin is used to improve sensitivity (sensitization) at the final color development stage. Therefore, even if a chemical substance having dioxin-like activity is detected by this method, its sensitivity is not satisfactory.
  • protein-containing samples were prepared from tissues containing a large amount of endogenous biotin, such as liver, kidney, and brain, or cultured cells or cell lines derived from these tissues, and these were used as DNA probes for the streptavidin-biotin reaction. When added to the measurement system used for immobilization, the effect of the biotin contained in the sample is of concern. Disclosure of efforts
  • the present inventors have developed anti-hapten antibodies that have been conventionally applied to biochemical and histochemical detection methods for haptenated molecules, such as Southern plotting methods that do not use radioisotopes. Good detection sensitivity can be obtained by using it for immobilizing DNA on a carrier. Since sensitization with biotin is possible, the activity of any DNA-binding protein is simple, rapid, and efficient by using a protein-activating substance and a binding DNA sequence specific to the target DNA-binding protein. The present inventors have found that it is possible to simultaneously measure a large number of specimens with high accuracy, and that it is possible to screen not only the protein but also the activating agent and the activation inhibitor.
  • the activated protein-containing sample of 1) is reacted with the haptenated DNA probe of 3) to form an activated protein / haptenated DNA probe complex
  • a method for measuring the activity of a DNA-binding protein (hereinafter referred to as the SW-ELISA method), a method for screening an activator and an activation inhibitor for the protein, and a kit for measuring the activity of a DNA-binding protein Is provided.
  • Figure 1 shows the results of confirming the specificity of the anti-FITC antibody and its applicability to ELISA.
  • FIG. 2 is a diagram showing confirmation of applicability of a specific antibody to AhR-Arnt in the SW-ELISA method.
  • FIG. 3 is a diagram showing a difference in sensitivity between the one-stage detection and the two-stage detection.
  • FIG. 4 shows the results of comparing the concentration dependence and the quantification range of AhR-Arnt activation by TCDD by the SW-ELISA method and the gel shift assay method.
  • A Gel shift assay
  • B SW-ELISA.
  • Arrow A indicates a specific band of the complex of activated AhR-Arnt and DRE.
  • FIG. 5 shows the result of screening of a compound having an inhibitory activity against AhR-Arnt activity.
  • A represents a half inhibitory concentration of each compound (IC 5 values.) To the active I inhibit the first nM TCDD AhR- Arnt.
  • White bars are by gel shift assay, black bars are by SW-ELISA.
  • B Shows the correlation between the gel shift assay method and the SW-ELISA method.
  • FIG. 6 is a diagram showing screening results of a liquid food having an inhibitory activity on AhR-Arnt activation by a SW-ELISA method and a gel shift assay.
  • FIG. 7 is a diagram showing the expansion of the quantification range with an increase in the concentration of activated protein by TCDD.
  • FIG. 8 is a diagram showing a comparison of detection limits of specific antibodies to AhR-Arnt activity by TCDD.
  • FIG. 9 is a diagram showing a comparison of AhR-Arnt activation by TCDD in various cells.
  • FIG. 10 shows the time dependence of AhR-Arnt activation by TCDD in Hepa-lclc7 cells. A is detected using an anti-AhR antibody, and B is detected using an anti-Arnt antibody.
  • FIG. 11 shows the concentration dependence of AhR-Arnt activation by TCDD in Hepa-lclc7 cells. A is detected using an anti-AhR antibody, and B is detected using an anti-Arnt antibody.
  • FIG. 12 is a diagram showing AhR-Arnt activation by an AhR ligand compound in Hepa-lclc7 cells. '
  • FIG. 13 is a view showing the inhibitory effect of Moroheiya extract on AhR-Arnt activation in Hepa_lclc7 cells.
  • FIG. 14 is a view showing the effect of the vegetable extract on AhR-Arnt activation in Hepa-lclc7 cells.
  • FIG. 15 shows the time dependence of AhR-Arnt activation in the liver of rats treated with 3-MC.
  • A SW-ELISA method
  • B Gel shift assay method.
  • the arrow in B indicates a specific band of the complex of AhR-Arnt and DRE.
  • FIG. 16 shows the activation of AhR-Arnt in guinea pig liver treated with TCDD.
  • A SW-ELISA method
  • B Gel shift atsey method.
  • the arrow in B indicates a specific band of the complex of AhR-Arnt and DRE.
  • FIG. 17 shows the effect of green tea and black tea on the suppression of AhR-Arnt activity in animals.
  • A SW-ELISA method
  • B Gel shift assay method.
  • the arrow in B indicates a specific band of the complex of AhR and DRE.
  • the method of the present invention is applicable to any protein-containing sample found in a natural environment such as animal or plant tissues, cells, or extracts thereof, or water or soil, as long as it contains a DNA-binding protein. Is for samples that have been subjected to artificial manipulation, such as foods containing the DNA-binding protein. Can be applied for
  • Such proteins include proteins involved in dioxin toxic expression, specifically AhR-Axnt, proteins related to endocrine disruption, such as estrogen receptor (ER), and immune function. Transcription factors, specific examples include Nuclear factor-kappa B (NF-KB), transcription factors involved in carcinogenesis, specific examples include Activator protein-1 (AP-l), etc.
  • AhR-Arnt-containing samples should be obtained by preparing cytoplasmic fractions of tissues and cell lysates prepared from various mammals and their derived cultured cells and cell lines.
  • a dioxin such as TCDD or a polycyclic aromatic hydrocarbon such as 3-MC, indigo, indirbin and its structural analog isodindigo as an activator
  • it can be a ligand for AhR. It becomes an active form having DNA binding ability.
  • a liver extracted from a decapitated rat is used as a sample, which is perfused with ice-cold phosphate-buffered saline (PBS) to remove blood, and then the liver is doubled in volume.
  • PBS ice-cold phosphate-buffered saline
  • HEDG buffer After crushing with a tissue disruption buffer (25 mM HEPES, pH 7.4, 1.5 raM EDTA, 1.0 mM dithiothreitol, 10% glycerol; hereafter referred to as HEDG buffer), 105,000 The AhR-Arnt force can be obtained by centrifuging at xg for 70 minutes at 4 ° C and obtaining the supernatant (cytoplasmic fraction). It can be stored at -85 ° C if needed.
  • tissue disruption buffer 25 mM HEPES, pH 7.4, 1.5 raM EDTA, 1.0 mM dithiothreitol, 10% glycerol
  • AhR- Arnt a 4 to 30 mg / ml preferably prepared to a protein concentration of 20 mg / ml, 0 to thereto: ⁇ TCDD of (final concentration), 0 to 5 0 nM of (final concentration) 3- MC Or 0-50 nM (final concentration) of dimethyl sulfoxide such as indigo
  • DMSO dimethyl sulfoxide
  • a non-activated protein-containing sample serving as a control in the case of AhR-Arnt, an AhR-Arnt treated with only the solvent DMS0 was prepared, and the subsequent treatment was activated. Perform in the same way as Arnt.
  • AhR-Arnt activated in vivo can be converted into the nuclear protein fraction of animal tissues. It is also possible to obtain by preparing In this case, a solvent such as corn oil can be administered to any animal to obtain AhR-Arnt as a control.
  • guinea pigs in which TCDD corn oil solution was intraperitoneally administered to 1 ⁇ g / Kg body weight, or rats in which 3-MC corn oil solution was intraperitoneally administered to 10 mg / Kg body weight
  • TCDD corn oil solution was intraperitoneally administered to 1 ⁇ g / Kg body weight
  • 3-MC corn oil solution was intraperitoneally administered to 10 mg / Kg body weight
  • liver extracted from, as a sample, it is perfused with ice-cold phosphate buffered saline (PBS) to remove blood.
  • PBS ice-cold phosphate buffered saline
  • tissue disruption buffer (10 niM HEPES VH 7.9, 0.5 mM dithiothreitol, 10 mM KC1, 1.5 mM MgCl 2 , 5 ⁇ g / ml
  • tissue disruption buffer 10 niM HEPES VH 7.9, 0.5 mM dithiothreitol, 10 mM KC1, 1.5 mM MgCl 2 , 5 ⁇ g / ml
  • the anti-hapten antibodies used in the SW-ELISA method are hapten molecules used in general immunological techniques, such as biotin, digoxigenin (DIG), full-aged resin isothiocyanate (FITC), dinitrophenol ( DNP) is processed according to the usual method (A. Kambegawa, Nippon Rinsho, Vol. 53 (9), p2160-p2167 (1995)), and can be either polyclonal or monoclonal. You may.
  • polyclonal antibodies can be used for serum albumin (BSA; molecular weight 66 kD), human serum albumin (HAS; molecular weight 58 kD), egret serum albumin (RSA; molecular weight 68 kD), and goat serum albumin (GSA; molecular weight).
  • BSA serum albumin
  • HAS human serum albumin
  • RSA egret serum albumin
  • GSA goat serum albumin
  • Hapten molecules are used for carrier proteins such as serum albumin of mammals such as 68 kD), chicken ovalbumin (OVA; molecular weight 45 kD), and keyhole limpet hemocyanin (KLH; molecular weight of about 1000 kD).
  • the bound conjugate can be obtained as an immunogen (antigen) according to a conventional immunization method (WH Newsome et al., Assoc. Off. Anal. Chem., Vol. 70, pl025-pl027 (1987)).
  • Animals used for immunization include mammals such as rabbits, mice, rats, goats, and dogs. Blood collected from animals is centrifuged; precipitation using ammonium sulfate or polyethylene dalicol A polyclonal antibody can be obtained through separation and purification by a conventional method such as gel filtration chromatography, ion exchange chromatography, chromatography such as affinity chromatography, and the like.
  • Monoclonal anti-hapten antibodies having high specificity and affinity have been used to isolate a plurality of B cells that have acquired the ability to produce antibodies against the immunogen from the mammals immunized as described above, and Continuously and indefinitely cleave cells.Fused with tumor cells such as myeloid cells that can proliferate. After that, hybridoma cells that efficiently produce the desired antibody are isolated and cloned from a plurality of fusion cells (hybridoma cells) to be produced, and are produced by culturing them in a test tube or animal body. Can be.
  • a polyclonal anti-hapten antibody obtained using FITC as the hapten molecule is preferable, and a polyclonal anti-hapten antibody derived from egrets is particularly preferable.
  • a carrier used for immobilization of the anti-hapten antibody a carrier generally used in the ELISA method can be used.
  • a carrier generally used in the ELISA method can be used.
  • These carriers may have various shapes according to the intended purpose, for example, dish, sphere, hole, plate, small rod, cell, small tube, small bottle. Shape, fiber shape, net shape and the like.
  • the carrier include a transparent plastic material, for example, a microtiter plate made of polychlorinated butyl or polystyrene, a small ball made of polystyrene and polystyrene latex, a tube or a rod, and the like.
  • a transparent plastic material for example, a microtiter plate made of polychlorinated butyl or polystyrene, a small ball made of polystyrene and polystyrene latex, a tube or a rod, and the like.
  • polystyrene 96-well microtiter plates are particularly preferred because they are easy to handle.
  • the carrier surface is activated with dartartaldehyde or cyanogen bromide, which is usually used in ELISA, and a solution containing the anti-hapten antibody is added thereto.
  • the reaction can be carried out, but is not limited to this method.
  • a carrier a polychlorinated rubber or polystyrene is used.
  • 50 mM sodium bicarbonate buffer pH 9.6
  • the adsorption of the antibody to the carrier is 1 to 10 g / mL.
  • the final concentration of the anti-hapten antibody be in the range of 0.1 to 10 g / mL.
  • the anti-hapten antibody-containing solution thus obtained can be used, for example, in a hole-shaped microtiter plate at a volume of about 0.1 mL / well, and after reacting at about 4 ° C for about 6 to 24 hours, washing Unreacted anti-hapten antibody is removed using a buffer solution (for example, phosphate-buffered saline containing 0.05% Tween TM 20 ; hereinafter referred to as PBST), whereby the anti-hapten is added to the carrier.
  • PBST phosphate-buffered saline containing 0.05% Tween TM 20
  • V. Use a high-concentration solution of a protein that does not show reactivity with any of the antibodies, such as a 1% BSA solution, 3% skim milk solution, or a commercially available blocking reagent as a blocking reagent for nonspecific binding. It is desirable to wash the carrier by using. The resulting carrier with immobilized anti-hapten antibody can be stored refrigerated or frozen as necessary.
  • a haptenized DNA probe having a consensus sequence is added to the anti-hapten antibody-immobilized carrier.
  • Such a DNA probe may be a double-stranded DNA containing one to several consensus sequences, and its production method and the like are not limited, and its 5 'or 3' end is chemically or enzymatically labeled with a hapten. Thus, a haptenized DNA probe can be obtained.
  • a single-stranded sense strand consisting of a 26-base sense strand containing the sequence is included.
  • DNA 5, -GAT CCG GAG TTG CGT GAG AAG AGC CA-3 '
  • single-stranded antisense strand DNA 5, -GAT CTG GCT CTT CTC ACG C CTC CG-3'
  • the haptenized DNA probe obtained as described above binds to the activating protein which has been treated with a protein activating substance to generate DNA binding ability via its consensus sequence, and the activated protein / haptenized DNA Form a complex of DNA probes.
  • a previously prepared protein sample containing active AhR-Arnt is mixed with 10 ⁇ L of HEDG buffer containing 150 mM potassium chloride (final concentration). This mixture is added to a microtiter plate on which anti-haptenized antibody and haptenized DNA probe are immobilized, and reacted at about 4 ° C for 2 hours to activate activated AhR-Arnt / haptenated DNA. A complex of probes is formed on the carrier.
  • a specific antibody that specifically recognizes the complex is added to the complex of the activated protein / haptenated DNA probe.
  • the specific antibody may be any specific antibody against the constituent molecules of the activation protein, and the range and the like are not limited.
  • AhR-Arnt it must be an antibody against the whole AhR protein or its partial peptide, or the whole Arnt protein or its partial peptide, and furthermore, it is a dimer of AhR-Arnt It is also necessary that the antibody be capable of recognizing the body.
  • Antibodies can be obtained. Specifically, a specific antibody obtained by immunization by synthesizing a peptide constituting the C-terminal part of the Arnt protein or the N-terminal part of the AR protein is preferable, and particularly, the protein derived from human or mouse is preferable. Are preferred.
  • immunized animal species producing the anti-hapten antibody and the immunized animal species producing the specific antibody be the same.
  • the specific antibody is detected by the enzyme-labeled secondary antibody for coloring. Therefore, the secondary antibody must be one that does not show reactivity with the anti-hapten antibody but shows reactivity only with the specific antibody, and uses the same animal species as that of the animal from which the anti-hapten antibody is derived. It is desirable to prepare in the same manner as the hapten antibody.
  • biotin-streptavidin reaction in two steps.
  • a combination of a solution containing a biotinylated secondary antibody and a solution containing an enzyme-labeled streptavidin is used.
  • a polymer reagent having a structure in which a large number of antibodies and streptavidin and a color-forming enzyme are bonded together in a molecule can be used in each step.
  • Enzymes for color development include peroxidase, alkaline phosphatase, ⁇ -D-galactosidase, g / recose oxidase, gnorecoamylase, carbonic anhydrase, aceti / recolin esterase, lysozyme, maleate dehydrogenase, and gnorecose. 6-phosphate dehydrogenase and the like.
  • the specific antibody reacted with the secondary antibody can be obtained by a general method.
  • peroxidase hydrogen peroxide and 0-phenylenediamine (0PD) or 3, 3 ', 5, 5'-tetramethylbenzidine ( ⁇ )
  • ⁇ 0-phenylenediamine
  • ⁇ 3, 3 ', 5, 5'-tetramethylbenzidine
  • It can be measured as absorbance using a measuring device such as the one described above. It is preferable to measure the absorbance after stopping the color reaction by, for example, adding a 1N aqueous sulfuric acid solution.
  • the activity level of a DNA-binding protein can be determined by comparing the absorbance measured as described above with the absorbance obtained for a sample containing a non-activated protein.
  • a standard curve is created from the obtained absorbance values, and the concentration of the activator of the unknown protein in the sample is determined as the concentration equivalent to the known activator. be able to.
  • the quantification range of the substance in the SW-ELISA method can be clarified.
  • the SW-ELISA method according to the present invention is the same as the gel shift assay method when the protein concentration is low, for example, when the protein concentration is 4 mg / ml.
  • the quantification range and detection limit are shown to a certain extent, when the concentration is high, for example, 20 mg / ml, the quantification range is extended while maintaining the detection limit.
  • the SW-ELISA method according to the present invention can easily, rapidly and accurately measure the activity of a DNA-binding protein in multiple samples simultaneously with the conventional method. It is also possible to screen the active inhibitor.
  • test chemical substance is used for protein treatment in the same manner as the activation treatment with a known protein activator, and the test chemical substance is used with a known protein activator.
  • the activating effect of the test chemical substance can be assayed.
  • test chemical contains a chemical whose effect on the target protein is to be examined, a solution in which the purified compound is dissolved; a liquid food, an extract from solid gold or soil, or a water quality test Any of a non-purified solution containing a multi-component multi-chemical substance represented by a sample or the like may be used.
  • the AhR-Arnt-containing sample is treated in advance with the liquid food itself, or the sample is treated with an AhR-Arnt activator such as TCDD.
  • AhR-Arnt activator such as TCDD.
  • a compound that antagonizes AhR-Arnt activation by simultaneously treating both liquid foods and comparing the resulting absorbance with the absorbance obtained by treatment with the AhR-Arnt-activating substance alone, that is, AhR-Arnt activity inhibitors can be tested for inclusion in liquid foods.
  • the solid food when examining the presence or absence of the activity of inhibiting AhR-Arnt in a solid food, the solid food must be extracted using a solvent such as ethanol in advance. The resulting extract is concentrated and dried, and then dissolved or suspended in a solvent such as water.
  • a carrier on which the anti-hapten antibody obtained as described above is immobilized, a haptenated DNA probe, a specific antibody that specifically recognizes a complex of the probe and the active protein, a detection antibody A kit for measuring the activity of a DNA-binding protein, comprising a reagent and a coloring reagent, is provided.
  • the carrier may have a haptenated DNA probe bound to an anti-hapten antibody.
  • the detection reagent refers to a reagent involved in a secondary antibody reaction with a specific antibody and, if desired, an enzyme labeling reaction.
  • a reagent containing a biotinylated secondary antibody for example, a biotinylated secondary antibody itself
  • a streptavidin-containing reagent such as a polymer reagent having a large number of streptavidin bound in the molecule.
  • kits are involved in the expression of dioxin toxicity when the protein activator is a dioxin such as TCDD or a polycyclic aromatic hydrocarbon such as 3-MC, indigo, indilbin, and isindigo. It can be used for measuring the activity of a protein to be used.
  • a dioxin such as TCDD
  • a polycyclic aromatic hydrocarbon such as 3-MC, indigo, indilbin, and isindigo. It can be used for measuring the activity of a protein to be used.
  • a haptenized DNA probe containing 7 bases of TNGCGTG (N is any base) and a peptide constituting the C-terminal part of Arnt protein or the N-terminal part of AhR protein are synthesized and immunized.
  • the kit including the obtained specific antibody the activity level of the DNA-binding protein AhR-Arnt can be measured.
  • AhR-Arnt preliminary tests and Examples the present invention will be described in more detail with reference to AhR-Arnt preliminary tests and Examples. However, the present invention is not limited to only the following Examples, but with ordinary changes in the technical field. Are also included in the scope of the present invention.
  • a 96-well microtiter plate (Nalgenunc International) was immobilized with an anti-hapten antibody, anti-FITC ⁇ sagi polyclonal antibody (Dako).
  • the blocking solution is diluted to 0.01-fold with a buffer (antibody dilution buffer) diluted 10-fold with PBST; the FITC-labeled antibody was diluted to a concentration of 1000 ng / mL.
  • Munoglobulin G (IgG) protein was diluted, and 100 ⁇ L was added to each well to react at room temperature for 2 hours.
  • an anti-goat IgG antibody (manufactured by CAPPEL) labeled with horseradish-derived peroxidase (HRP) was then added to the antibody dilution buffer at 1.5 ⁇ g / The mixture was diluted to a concentration of rnL (1: 1000, v / v), added to each well in an amount of 100 ⁇ l, and allowed to react at room temperature for 2 hours. After washing each well of the plate four times, add 100 ml of TMB color reagent (manufactured by Dako) to each well to start the color reaction, and stop color development after 30 minutes by adding 50 of 1N sulfuric acid. I let it.
  • TMB color reagent manufactured by Dako
  • Activated AhR-Arnt was added to a rat liver cytoplasmic fraction prepared by adding a 1.61 ng / mL (5 nM) TCMS DMS0 solution to a final protein concentration of 15 mg / mL, and then activated at 20 ° C. It was prepared in advance by reacting in the dark for hours. As a control, a rat hepatocyte fraction to which only the same amount of DMS0 had been added was also reacted at 20 ° C for 2 hours.
  • DRE probe a DNA probe containing a previously synthesized FITC-labeled DRE sequence (hereinafter referred to as DRE probe; sense strand; 5'-GAT CCG GAG TTG CGT GAG AAG AGC CA —3, antisense strand; 5'-one GAT CTG GCT CTT CTC ACG CAA CTC CG-3 ') diluted with PBST to 25 fmols / 100 ⁇ L, add 100 ⁇ to each well and add 2 hours at room temperature The reaction was performed, and each well was washed three times with 100 ⁇ ⁇ of PBST.
  • DRE probe sense strand
  • Antibody (anti-AR MA1-514) (manufactured by affiliate Bioregent) and (d) a peptide constituting the N-terminal portion of mouse AhR protein was synthesized and immunized.
  • Antibody (anti-AhR B100-128) (Novas. Biologicals), (a) and (b) are 1: 10000, (c) is 1: 1500, and (d) is 1 : Diluted to 2000, added 100 L to each well, and reacted at room temperature for 1 hour.
  • a biotin-labeled anti-goat IgG antibody (Jackson ImnoResearch 'Laboratory') or an anti-mouse IgG antibody (Jackson ImnoResearch Lab. Diluted with antibody dilution buffer to a concentration of 0.34 ⁇ g / mL (1: 5000) and 0.33 ⁇ g / mL (1: 4000), respectively. The mixture was added and reacted at room temperature for 1 hour.
  • PBST PBST
  • a concentration of 0.33 ⁇ g / mL (1: 2000) of a labeled polymer reagent (manufactured by Dako) bound with both HRP and streptavidin was added using PBST. And added to each well in an amount of 100 ⁇ 100, and reacted at room temperature for 30 minutes.
  • Example 2 Confirmation of difference in detection sensitivity due to difference in detection process
  • the two-step detection of Example 1 using biotin-labeled anti-goat IgG antibody and HRP / streptavidin-labeled polymer reagent was performed at a concentration of 1.5 ⁇ g / mL (1: 1000) with antibody dilution buffer.
  • the gel shift assay was performed by mixing the cytoplasmic fraction containing AhR-Arnt with HEDG buffer containing 150 mM potassium chloride (final concentration) and using the method described previously (H. Ashida et al., FEBS Lett.). , Vol. 476, p213-217 (2000)).
  • the same DNA sequence as the DRE probe described in Example 1 was used as the DNA probe, and the unlabeled (non-FITC-labeled) DRE probe was synthesized using 32 P-ATP and T4 polynucleotide kinase. The ends were subjected to 32 P labeling, and 0.5 L of a DRE probe solution was added per reaction volume (12 ⁇ ).
  • the AhR-Arnt / DRE conjugate was exposed to X-ray film (FIG. 4A).
  • TCDD DMS0 solution 0-75 fmols of TCDD DMS0 solution was added to the rat liver cytoplasmic fraction adjusted to 4 mg / mL in protein amount, and activated by reacting at 20 ° C for 2 hours in the dark.
  • AhR-Arnt was prepared and the SW-ELISA method of Example 1 was performed using an anti-Arnt C-19 antibody as a specific antibody (FIG. 4B).
  • test substances included the flavonoids apigenin, hesperetin, myricetin (Sigma), galandin, kaempferonore, tangeretin (both from EXTRASY THESE), quercetin, rutin (both from Wako Pure Chemical Industries), naringenin 20 (manufactured by Nacalai Tester) and added so that the final concentration of each compound-containing solution in AhR-Arnt would be 0.1 to 50501.
  • the reaction mixture was treated with 1 nM TCDD at 20 ° C for 2 hours.
  • the measurement results of the SW-ELISA method were calculated assuming that the absorbance of AhR-Arnt treated only with TCDD was 100% and that of DMS0 alone was 0%.
  • the results of the gel shift assay were exposed to X-ray film, the band density of AhR-Arnt was measured with an imaging analyzer, then the band density of the cells treated with TCDD only was set to 100%, and only DMS0 was used. At that time, it was calculated as 0%. Also to calculate the half-inhibitory concentration (IC 5. Value) for the active I inhibit the AhR-Arnt of 1 nM TCDD from% at each concentration of the test substance in any of the methods (Fig. 5A).
  • SW-ELISA method Example 1
  • Genoleshift Assy method Example 3
  • the inhibitory effect on the activation of AhR-Arnt of the 16 commercial beverages obtained by the SW-ELISA method showed a good correlation with the results obtained by the gel shift assay. That is, Chinese tea, oolong tea, two kinds of black tea, coffee, milk cocoa, milk, and wine almost completely suppress the activation of AhR-Arnt by TCDD, and orange juice, apple juice, vegetable juice, aloe juice, and beans The inhibitory effect was weak, and the inhibitory effect of green tea was found to vary widely between products.
  • SW-ELISA method can be applied not only to the inhibitory effect of the test chemical substance shown in Example 4, but also to the evaluation of the inhibitory effect of an unpurified solution such as a liquid food.
  • Activated AhR-Arnt was added to a rat liver cytoplasmic fraction adjusted to a protein concentration of 20 mg / mL with a final concentration of 1 nM TCDD in DMS0, and then reacted at 20 ° C for 2 hours in the dark. And was prepared in advance. 0.99 mM salt Ihikariumu the activation AhR-Arnt 40 L of HEDG buffer 10 x L containing (final concentration) were mixed respectively, which was (800 mu ⁇ as Tan Park mass) added in 50 / i L per each well The reaction was performed at room temperature for 2 hours. Subsequent operations were performed according to Example 1, and the results in FIG. 7 were obtained.
  • the quantification range of TCDD was 0 compared with Example 3 (0.3 to 75 fmols (about 0.1 to 20 pg)) using activated AhR_Arnt at a concentration of 4 mg / mL as a protein. Expanded to 4-400 fmols (approximately 0.13--130 pg).
  • the three antibodies (anti-Arnt C-19 antibody, anti-AhR MAI-514 antibody and anti-AhR BlOO-128 antibody) having good detection sensitivity in Example 1 were used to determine the TCDD concentration. The detection limits of the resulting AhR-Arnt activities were compared.
  • Activated AhR-Arnt was detected after adding various concentrations of TCDD to the cytoplasmic fraction of rat liver and allowing it to act at 20 ° C for 2 hours (Fig. 8).
  • anti-Arnt antibody an anti-Arnt C-19 antibody (hereinafter, referred to as anti-Arnt antibody) or an anti-AhR MAI-514 antibody (hereinafter, referred to as anti-AhR antibody) was used.
  • anti-Arnt antibody an anti-Arnt C-19 antibody
  • anti-AhR MAI-514 antibody an anti-AhR MAI-514 antibody
  • SW-ELISA method of the present invention can be applied to a test using cells was examined.
  • liver-derived cell lines (mouse liver cells) were used. Tupa-derived Hepa-lclc7 cells, rat normal liver-derived RL-34 cells, and human liver tumor-derived HepG2 cells (distributed from Takeda Foods Co., Ltd., purchased from JCRB Cell Bank, distributed by Professor Ikura, Kyoto Institute of Technology, respectively) ) Were treated with DMS0 as a negative control or 1 nM TCDD as a positive control at 37 ° C for 2 or 2 hours to examine the sensitivity of each cell to TCDD.
  • the time dependence of AhR-Arnt activation in Hepa-lclc7 cells was determined in the same manner as above except that the treatment time with TCDD was set to 0, 1, 2, 4, 8, and 24 hours.
  • Hepa-lclc7 cells were treated with 0, 0.001, 0.01, 0.1, 0.2, 0.5, 1, and 5 nM TCDD to confirm the concentration dependence of AhR-Arnt activation. And acted at 37 ° C for 2 hours.
  • the active AhR-Arnt contained in the nuclear extract was detected in the same manner as above, and when using either antibody, it was 0.01-0.5 nM.
  • the linearity was confirmed, and the detection limit when using an anti-AhR antibody was lpM TCDD (Fig. 11). From the above, it was confirmed that the SW-ELISA method of the present invention was able to detect TCDD in the test using cells.
  • the SW-ELISA method of the present invention can evaluate not only the activation of AhR-Arnt by dioxins but also the AhR-Arnt activating ability of AhR ligand compounds.
  • Pretreatment was performed by impregnating Hepa-lclc7 cells with 1 or 2 mg / ml of Morohaea extract obtained by alcohol extraction using 90% ethanol in the presence of 0.05% acetic acid for 20 minutes, 2 hours, and 24 hours. Then, 500 pM TCDD was allowed to act on this for 2 hours, and activated AhR-Arnt in the nuclear extract was detected with an anti-Arnt antibody.
  • capochia was tested.
  • Each vegetable extract was used at 2 mg / ml, and pretreatment was performed for 20 minutes or 2 hours (24 hours for Morroja).
  • Capochia, carrot, and parsley extracts could only suppress AhR-Arnt activation by 500 pM TCDD by about 20%, whereas Moroheiya extract was pre-treated for 2 hours, about 50%, 24 hours before The treatment suppressed about 75%, and it was found that the suppression effect was the strongest among the four kinds of vegetables (Fig. 14).
  • SW-ELISA method was performed using experimental animals.
  • Example 12 Examination of AhR-Arnt activity suppression effect of liquid food in animals Evaluation of AhR-Arnt activation suppression effect when using animals was examined.
  • AhR-Arnt activity was induced by 3-MC in the water intake group, whereas activation was reduced in the green tea and black tea intake groups.
  • the present invention provides a method for measuring an activity, a method for screening an activator for an activator of the protein, and a kit for measuring an activity of a DNA-binding protein.

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Abstract

It is intended to provide a method of measuring the activity of a DNA-binding protein which comprises: 1) treating a protein-containing sample with a protein activator to thereby give a sample containing an activated protein capable of binding to DNA; 2) solidifying an anti-hapten antibody on a carrier; 3) bonding a haptenized DNA probe to the anti-hapten antibody; 4) reacting the activated protein-containing sample obtained in 1) with the haptenized DNA probe obtained in 3) to thereby form a complex of the activated protein/the haptenized DNA probe; 5) adding a specific antibody and allowing it to specifically recognize the complex; 6) adding a secondary antibody labeled with an enzyme for coloration; 7) adding a coloration reagent to induce a coloration reaction and measuring the degree of the coloration based on absorbance; and 8) comparing the absorbance with the absorbance of an inactivated protein-containing sample to thereby determine the activity level of the protein.

Description

明 細 書  Specification
DNA結合性タンパク質の活性測定方法 技術分野 Method for measuring DNA binding protein activity
本発明は、 DNA結合性タンパク質の活性測定方法に関する。詳しくは、 本発明は、 サウスウェスタン ·ケミストリーと称される生ィヒ学的手法に ELISA法を組合せた方法をさらに改変することにより開発された、 DNA結 合性タンパク質の活性測定方法に関し、 該タンパク質の活性ィヒ剤およぴ 活性化抑制剤のスクリ一ユング方法ならびに DNA結合性タンパク質の活 性測定用キットに関する。 背景技術  The present invention relates to a method for measuring the activity of a DNA-binding protein. More specifically, the present invention relates to a method for measuring the activity of a DNA-binding protein, which has been developed by further modifying a method in which an ELISA method is combined with a probiotic technique called Southwestern chemistry. The present invention relates to a method for screening a protein activity agent and an activation inhibitor, and a kit for measuring the activity of a DNA-binding protein. Background art
生物には、 化学物質との反応によって活性化され、 特定の DNA配列（コ ンセンサス配列）に対する結合能を生じて該配列に結合することにより、 種々の作用を発揮する DNA結合性タンパク質の介在する機序が多く存在 する。  In living organisms, a DNA-binding protein that exerts various functions by being activated by a reaction with a chemical substance and generating a binding ability to a specific DNA sequence (consensus sequence) and binding to the sequence is generated. There are many mechanisms.
例えば、 2， 3, 7， 8 -テトラクロ口ジべンゾ ダイ才キシン (TCDD)および 3-メチルコランスレン（3- MC)などの多環性芳香族炭化水素化合物 （ァリ ール炭化水素化合物） は、 DNA結合性タンパク質であるァリール炭化水素 受容体 (Aryl hydrocarbon receptor,以下 AhRと称する、分子量約 100 kD) と結合し、 これがさらに Arnt (AhR Nuclear Trans-locator, 分子量約 90 kD)と結合することで、 これらのタンパク質のコンフオメーシヨンを 変化させてダイォキシン- AhR- Arntの複合体を形成し、 TNGCGTG(Nは任意 の塩基）の 7塩基からなる DRE (Dioxins Response Element)配列を含む DNAに対する結合能を生じて、前記複合体をこの DNA配列に結合させるこ とで毒性を発現することが知られている。 For example, polycyclic aromatic hydrocarbon compounds (aryl hydrocarbons) such as 2,3,7,8-tetraclodin dibenzodioxin (TCDD) and 3-methylcholanthrene (3-MC) Compound) binds to the Aryl hydrocarbon receptor (hereinafter referred to as AhR, molecular weight of about 100 kD), which is a DNA-binding protein, and further binds to Arnt (AhR Nuclear Trans-locator, molecular weight of about 90 kD). By binding, the conformation of these proteins is changed to form a dioxin-AhR-Arnt complex, which contains a DNG (Dioxins Response Element) sequence consisting of 7 bases of TNGCGTG (N is any base) It is known that toxicity is exhibited by causing the complex to bind to the DNA sequence by generating a binding ability to DNA.
従来、 ダイォキシン類の分析には、 ガスクロマトグラフィー '質量分 析法 (GCMS法）が公定法として定められている。 この方法によれば、 ダイ ォキシン類が試料中に低濃度で存在する場合でも、 その分析結果が感度 および精度の両面において満足できる。  Conventionally, gas chromatography-mass spectrometry (GCMS) has been defined as the official method for the analysis of dioxins. According to this method, even when dioxins are present in a sample at a low concentration, the analysis results are satisfactory in both sensitivity and accuracy.
しかしながら、 この方法では、 既知物質のみが分析の対象となり、 ダ • ィォキシン様の生理活性、 すなわちダイォキシンに似た毒性を有する新 規な化学物質を分析できない。 また、 この方法では、 試料に対して非常 に複雑な化学的前処理が必要であり、 しかも分析機器が高価であるとい う難点がある。  However, according to this method, only known substances are analyzed, and a new chemical substance having a dioxin-like physiological activity, that is, a toxicity similar to dioxin cannot be analyzed. In addition, this method requires very complicated chemical pretreatment of the sample, and has the disadvantage that the analytical equipment is expensive.
DNA結合性タンパク質 AhR-Arntの性質を利用する分析方法としては、 ゲルシフトアツセィ法があり、 ダイォキシン- AhR- Arnt複合体を、 予め 32P-ATPで放射性同位元素標識した DRE配列を含む DNAプローブと反応さ せて、 ダイォキシン- AhR- Arntと DNAプローブとの複合体を形成させ、 こ の複合体を電気泳動で分離し解析する。 As an analytical method utilizing the nature of the DNA binding protein AhR-Arnt, there are gel shift Atsu Si method, dioxin - AhR- Arnt complex, comprising a DRE sequences radiolabeled with pre 3 2 P-ATP DNA Reacts with the probe to form a complex of dioxin-AhR-Arnt and the DNA probe. This complex is separated by electrophoresis and analyzed.
し力 し、 この方法によるダイォキシンの解析は、 パンドの移動度およ び濃度を指標とするため定量性に劣り、 その結果は、 一般的に定性もし くは半定量解析に留まる。 また、 正確に定量を行うためにはバンドの放 射性同位元素量を測定するための高価な機器が必要となるのみならず、 GCMS法と同様に多種類の被験物質を一度に処理、 解析することが困難で ある。  However, dioxin analysis by this method is poor in quantitativeness because it uses the mobility and concentration of band as indices, and the results are generally qualitative or semi-quantitative analysis. In addition, accurate quantification not only requires expensive equipment to measure the amount of radioisotopes in the band, but also treats and analyzes many types of test substances at once, as in the GCMS method. It is difficult to do.
32P-ATPでの標識に代えて、 ビォチン等を用いてハプテン標識を行う方 法もあるが、 放射性同位元素を用いる方法と同様に多種類の被験物質を —度に処理、 解析することが困難であることは変わりがない。 また、 こ のような方法では、 最終的に酵素発色を用いるため、 感度が低くなる面 において問題がある。 Although hapten labeling using biotin or the like may be used instead of labeling with 32P-ATP, multiple types of test substances can be processed and analyzed at a time in the same manner as with the method using radioisotopes. It is still difficult. Also, this In such a method, since enzymatic coloring is finally used, there is a problem in that sensitivity is reduced.
最近では、 Ahィムノアッセィ法と呼ばれる新たな分析方法が実用化さ れている（産業と環境、 平成 12年 4月号 (29卷 4号))。  Recently, a new analytical method called the Ah Imnoassay method has been put into practical use (Industry and Environment, April 2000 (Vol. 29, No. 4)).
この方法は、 DNAプローブを予めマイクロタイタープレートに固定ィ匕す ることにより AhR - Arnt を介してダイォキシン類を結合させ、 AhR - Arnt に特異的に反応する一次抗体、 発色反応を生じる二次抗体および発色試 薬を順次添加して、 発色の度合いを吸光度で測定することにより、 AhR- Arntに結合したダイォキシン類を定量分析するものである。  In this method, a DNA antibody is preliminarily immobilized on a microtiter plate to bind dioxins via AhR-Arnt, and a primary antibody that specifically reacts with AhR-Arnt and a secondary antibody that produces a color reaction A dioxin bound to AhR-Arnt is quantitatively analyzed by sequentially adding a coloring reagent and measuring the degree of coloring by absorbance.
この方法では、 ストレプトァビジン -ピオチンが DNAプローブと担体と の固定ィ匕に用いられていると考えられることから、 最終的な発色段階で ストレプトアビジン -ビォチンを感度向上（増感）に用いることができな いため、 この方法によってダイォキシン様の活性を有する化学物質が検 出されたとしても、その感度は満足できるものではない。 さらに、肝臓、 腎臓、 脳など内在性ビォチンを多く含む組織、 あるいはこれら組織に由 来する培養細胞や株化細胞などよりタンパク質含有試料を作製し、 これ らを、ストレプトアビジン-ビォチン反応を DNAプローブの固定ィヒに利用 した測定系に添加した場合、 試料中に含まれるピオチンによる影響が懸 念される。 努明の開示  In this method, since streptavidin-biotin is considered to be used for immobilization of the DNA probe and the carrier, streptavidin-biotin is used to improve sensitivity (sensitization) at the final color development stage. Therefore, even if a chemical substance having dioxin-like activity is detected by this method, its sensitivity is not satisfactory. Furthermore, protein-containing samples were prepared from tissues containing a large amount of endogenous biotin, such as liver, kidney, and brain, or cultured cells or cell lines derived from these tissues, and these were used as DNA probes for the streptavidin-biotin reaction. When added to the measurement system used for immobilization, the effect of the biotin contained in the sample is of concern. Disclosure of efforts
このような状況下、 本発明者らは、 従来は放射性同位元素を使用しな いサザンプロッティング法などのハプテン化分子の生化学的および組織 化学的検出法に適用されている抗ハプテン抗体を DNAの担体への固定化 に用いることにより良好な検出感度が得られ、特にス ピオチンによる増感が可能になることから、 目的の DNA結合性タンパク 質に特有のタンパク質活性ィ匕物質および結合 DNA配列を用いることによ り、 いずれの DNA結合性タンパク質の活性も簡便、 迅速かつ高精度に多 検体を同時に測定できること、 また、 当該タンパク質のみならず、 その 活性ィ匕剤および活性化抑制剤のスクリ一ユングも可能であることを見出 し、 この発明を完成した。 Under these circumstances, the present inventors have developed anti-hapten antibodies that have been conventionally applied to biochemical and histochemical detection methods for haptenated molecules, such as Southern plotting methods that do not use radioisotopes. Good detection sensitivity can be obtained by using it for immobilizing DNA on a carrier. Since sensitization with biotin is possible, the activity of any DNA-binding protein is simple, rapid, and efficient by using a protein-activating substance and a binding DNA sequence specific to the target DNA-binding protein. The present inventors have found that it is possible to simultaneously measure a large number of specimens with high accuracy, and that it is possible to screen not only the protein but also the activating agent and the activation inhibitor.
したがって、 この発明によれば、 次の工程：  Therefore, according to the present invention, the following steps:
1) タンパク質含有試料をタンパク質活性化物質で処理して DNA結合能 を有する活性ィ匕タンパク質含有試料を得て、  1) treating a protein-containing sample with a protein activator to obtain a DNA-binding ability-active protein-containing sample;
2) 担体に抗ハプテン抗体を固相化し、  2) Immobilize anti-hapten antibody on the carrier,
3) ハプテン化 DNAプローブを抗ハプテン抗体に結合させ、  3) binding the haptenized DNA probe to the anti-hapten antibody,
4) 1)の活性化タンパク質含有試料を 3)のハプテン化 DNAプローブと反 応させて、活性化タンパク質/ハプテン化 DNAプローブの複合体を形成し、 4) The activated protein-containing sample of 1) is reacted with the haptenated DNA probe of 3) to form an activated protein / haptenated DNA probe complex,
5) 特異抗体を加えて、 複合体を特異的に認識させ、 5) Add a specific antibody to specifically recognize the complex,
6) 発色用酵素標識二次抗体を加え、  6) Add enzyme-labeled secondary antibody for color development,
7) 発色試薬を加えて発色反応を生じさせ、 発色の度合いを吸光度によ り測定し、  7) Add a coloring reagent to cause a coloring reaction, measure the degree of coloring by absorbance,
8) 非活性ィヒタンパク質含有試料について測定される吸光度との比較か ら、 タンパク質の活性レベルを測定すること  8) To determine the activity level of the protein by comparing it with the absorbance measured for the sample containing inactive protein.
からなる、 DNA結合性タンパク質の活性測定方法 (以降、 SW- ELISA法と称 する）、該タンパク質の活性化剤ならびに活性化抑制剤のスクリーユング 方法およぴ DNA結合性タンパク質の活性測定用キットが提供される。 図面の簡単な説明 A method for measuring the activity of a DNA-binding protein (hereinafter referred to as the SW-ELISA method), a method for screening an activator and an activation inhibitor for the protein, and a kit for measuring the activity of a DNA-binding protein Is provided. BRIEF DESCRIPTION OF THE FIGURES
図 1は、抗 FITC抗体の特異性と ELISA法への適用性の確認結果を示す 図である。 Figure 1 shows the results of confirming the specificity of the anti-FITC antibody and its applicability to ELISA. FIG.
図 2は、 SW-ELISA法における AhR- Arntに対する特異抗体の適用性の確 認を示す図である。  FIG. 2 is a diagram showing confirmation of applicability of a specific antibody to AhR-Arnt in the SW-ELISA method.
図 3は、一段階検出と二段階検出による検出の感度差を示す図である。 図 4は、 TCDDによる AhR- Arnt活性化の濃度依存性およぴ定量範囲を、 SW-ELISA法とゲルシフトアツセィ法で比較した結果である。 A：ゲルシ フトアツセィ法、 B ： SW- ELISA法。 Aの矢印は、 活性化 AhR- Arntと DRE の複合体の特異的なバンドを示している。  FIG. 3 is a diagram showing a difference in sensitivity between the one-stage detection and the two-stage detection. FIG. 4 shows the results of comparing the concentration dependence and the quantification range of AhR-Arnt activation by TCDD by the SW-ELISA method and the gel shift assay method. A: Gel shift assay, B: SW-ELISA. Arrow A indicates a specific band of the complex of activated AhR-Arnt and DRE.
図 5は、 AhR- Arntの活性ィ匕に対し抑制活性を有する化合物のスクリ一 ニングの結果である。 A： 1 nM TCDDの AhR- Arntの活性ィヒに対する各化合 物の半阻害濃度 (IC5。値）を示す。 白い棒はゲルシフトアツセィ法、黒い棒 は SW - ELISA法による。 B ：ゲルシフトアツセィ法と SW-ELISA法それぞ れの相関性を示す。 FIG. 5 shows the result of screening of a compound having an inhibitory activity against AhR-Arnt activity. A: represents a half inhibitory concentration of each compound (IC 5 values.) To the active I inhibit the first nM TCDD AhR- Arnt. White bars are by gel shift assay, black bars are by SW-ELISA. B: Shows the correlation between the gel shift assay method and the SW-ELISA method.
図 6は、 SW- ELISA法とゲルシフトアツセィ法による、 AhR- Arntの活性 化に対し抑制活性を有する液状食品のスクリ一エング結果を示す図であ る。  FIG. 6 is a diagram showing screening results of a liquid food having an inhibitory activity on AhR-Arnt activation by a SW-ELISA method and a gel shift assay.
図 7は、 TCDDによる活性ィ匕タンパク質濃度の増加に伴う定量範囲の拡 大を示す図である。  FIG. 7 is a diagram showing the expansion of the quantification range with an increase in the concentration of activated protein by TCDD.
図 8は、 TCDDによる AhR- Arnt活性ィ匕に対する特異性抗体の検出限界の 比較を示す図である。  FIG. 8 is a diagram showing a comparison of detection limits of specific antibodies to AhR-Arnt activity by TCDD.
図 9は、 様々な細胞における TCDDによる AhR- Arnt活性化の比較を示 す図である。  FIG. 9 is a diagram showing a comparison of AhR-Arnt activation by TCDD in various cells.
図 1 0は、 Hepa - lclc7細胞における TCDDによる AhR - Arnt活性化の時 間依存性を示す図である。 Aは抗 AhR抗体、 Bは抗 Arnt抗体を用いて検 出している。 図 1 1は、 Hepa- lclc7細胞における TCDDによる AhR- Arnt活性化の濃 度依存性を示す図である。 Aは抗 AhR抗体、 Bは抗 Arnt抗体を用いて検 出している。 FIG. 10 shows the time dependence of AhR-Arnt activation by TCDD in Hepa-lclc7 cells. A is detected using an anti-AhR antibody, and B is detected using an anti-Arnt antibody. FIG. 11 shows the concentration dependence of AhR-Arnt activation by TCDD in Hepa-lclc7 cells. A is detected using an anti-AhR antibody, and B is detected using an anti-Arnt antibody.
図 1 2は、 Hepa- lclc7 細胞における AhR リガンド化合物による AhR-Arnt活性化を示す図である。 '  FIG. 12 is a diagram showing AhR-Arnt activation by an AhR ligand compound in Hepa-lclc7 cells. '
図 1 3は、 Hepa_lclc7細胞におけるモロヘイヤ抽出物の AhR- Arnt活 性化抑制効果を示す図である。  FIG. 13 is a view showing the inhibitory effect of Moroheiya extract on AhR-Arnt activation in Hepa_lclc7 cells.
図 1 4は、 Hepa- lclc7細胞における野菜抽出物の AhR-Arnt活性化抑制 効果を示す図である。  FIG. 14 is a view showing the effect of the vegetable extract on AhR-Arnt activation in Hepa-lclc7 cells.
図 1 5は、 3 - MCを処理したラットの肝臓中における AhR- Arnt活性化 の時間依存性を示す図である。 A： SW-ELISA法、 B ：ゲルシフトアツセ ィ法。 Bの矢印は、 AhR-Arntと DREの複合体の特異的なパンドを示して いる。  FIG. 15 shows the time dependence of AhR-Arnt activation in the liver of rats treated with 3-MC. A: SW-ELISA method, B: Gel shift assay method. The arrow in B indicates a specific band of the complex of AhR-Arnt and DRE.
図 1 6は、 TCDDを処理したモルモッ トの肝臓における AhR- Arntの活性 化を示す図である。 A： SW-ELISA法、 B ：ゲルシフトアツセィ法。 Bの 矢印は、 AhR- Arntと DREの複合体の特異的なバンドを示している。  FIG. 16 shows the activation of AhR-Arnt in guinea pig liver treated with TCDD. A: SW-ELISA method, B: Gel shift atsey method. The arrow in B indicates a specific band of the complex of AhR-Arnt and DRE.
図 1 7は、動物における緑茶と紅茶の AhR-Arnt活性ィヒ抑制効果を示す 図である。 A： SW- ELISA法、 B ：ゲルシフトアツセィ法。 Bの矢印は、 AhRと DREの複合体の特異的なパンドを示している。 発明を実施するための最良の形態  FIG. 17 shows the effect of green tea and black tea on the suppression of AhR-Arnt activity in animals. A: SW-ELISA method, B: Gel shift assay method. The arrow in B indicates a specific band of the complex of AhR and DRE. BEST MODE FOR CARRYING OUT THE INVENTION
本発明の方法は、 DNA結合性タンパク質を含むものであれば、動物もし くは植物の組織、 細胞もしくはそれらの抽出物、 または水もしくは土壌 などの自然環境中に見出されるあらゆるタンパク質含有試料、 さらには 該 DNA結合性タンパク質を含む食品などの人為的操作が加わった試料に ついて適用することができる。 The method of the present invention is applicable to any protein-containing sample found in a natural environment such as animal or plant tissues, cells, or extracts thereof, or water or soil, as long as it contains a DNA-binding protein. Is for samples that have been subjected to artificial manipulation, such as foods containing the DNA-binding protein. Can be applied for
そのようなタンパク質としては、 ダイォキシン類の毒'性発現に関与す るタンパク質、 具体的には AhR- Axnt、 内分泌攪乱作用に関するタンパク 質、 具体例としてェストロジェン受容体 (ER)、 免疫機能に関与する転写 因子、 具体例として Nuclear factor- kappa B (NF-KB)、 発がんに関与する 転写因子、 具体例として Activator protein- 1 (AP-l)などが挙げられ、 それぞれに適当なタンパク質活性ィヒ物質で当該タンパク質、 または当該 タンパク質を含む細胞、 組織、 動物個体などを処理することにより、 コ ンセンサス配列に対する DNA結合能を生じさせることができる。  Such proteins include proteins involved in dioxin toxic expression, specifically AhR-Axnt, proteins related to endocrine disruption, such as estrogen receptor (ER), and immune function. Transcription factors, specific examples include Nuclear factor-kappa B (NF-KB), transcription factors involved in carcinogenesis, specific examples include Activator protein-1 (AP-l), etc. By treating the protein or a cell, tissue, animal individual, or the like containing the protein, DNA binding ability to a consensus sequence can be generated.
例えば、 AhR- Arnt含有試料は、 様々な哺乳動物おょぴそれらに由来す る培養細胞や株化細胞などから調製される組織およぴ細胞溶解液の細胞 質画分を調製して得ることができ、 その活性化物質として TCDDなどのダ ィォキシン類、 または AhRのリガンドとなりえる、 3- MC、 インディゴ、 ィンディルビン及びその構造類縁体であるィソィンディゴなどの多環性 芳香族炭化水素を用いることにより DNA結合能を有する活性型となる。 具体例としては、 断頭屠殺したラットから摘出した肝臓を試料として 用い、 これを予め氷冷したリン酸緩衝化生理食塩水 (PBS)で灌流して脱血 し、次いでこの肝臓を 2倍量の組織破碎用緩衝液 (25 mM HEPES、 pH 7. 4、 1. 5 raM EDTA、 1· 0 mMジチオスレィトール、 10% グリセロール；以下 HEDG 緩衝液と記す）で破碎処理した後、 105, 000 x g、 70分間、 4°Cで遠心分離 し、上清 (細胞質画分）を得ることにより、 AhR - Arnt力得られる。これは、 必要に応じて - 85°Cで保存することができる。  For example, AhR-Arnt-containing samples should be obtained by preparing cytoplasmic fractions of tissues and cell lysates prepared from various mammals and their derived cultured cells and cell lines. By using a dioxin such as TCDD or a polycyclic aromatic hydrocarbon such as 3-MC, indigo, indirbin and its structural analog isodindigo as an activator, it can be a ligand for AhR. It becomes an active form having DNA binding ability. As a specific example, a liver extracted from a decapitated rat is used as a sample, which is perfused with ice-cold phosphate-buffered saline (PBS) to remove blood, and then the liver is doubled in volume. After crushing with a tissue disruption buffer (25 mM HEPES, pH 7.4, 1.5 raM EDTA, 1.0 mM dithiothreitol, 10% glycerol; hereafter referred to as HEDG buffer), 105,000 The AhR-Arnt force can be obtained by centrifuging at xg for 70 minutes at 4 ° C and obtaining the supernatant (cytoplasmic fraction). It can be stored at -85 ° C if needed.
次に、 AhR- Arntを 4〜30mg/ml、 好ましくは 20mg/mlのタンパク質濃度 に調製し、 これに 0〜： ΙΟηΜ (終濃度）の TCDD、 0〜50nM (終濃度）の 3- MCあ るいは 0〜50nM (終濃度）のインディゴなどのジメチルスルホキシド (DMSO)溶液を加え、 20°Cで 2 時間暗所にて反応させることにより、 in vitroで活性ィ匕 AhR - Arntを得ることができる。 Next, AhR- Arnt a 4 to 30 mg / ml, preferably prepared to a protein concentration of 20 mg / ml, 0 to thereto: ΙΟηΜ TCDD of (final concentration), 0 to 5 0 nM of (final concentration) 3- MC Or 0-50 nM (final concentration) of dimethyl sulfoxide such as indigo By adding a (DMSO) solution and reacting at 20 ° C. for 2 hours in a dark place, it is possible to obtain an activated AhR-Arnt in vitro.
また、 上記の活性ィ匕と同時に、 対照となる非活性化タンパク質含有試 料、 AhR- Arntの場合には溶媒の DMS0のみで処理した AhR- Arntを調製し、 以降の処理を活性化 AhR-Arntと同様に行う。  Simultaneously with the above-mentioned activation, a non-activated protein-containing sample serving as a control, in the case of AhR-Arnt, an AhR-Arnt treated with only the solvent DMS0 was prepared, and the subsequent treatment was activated. Perform in the same way as Arnt.
TCDDなどの AhR- Arnt活性ィ匕物質の溶液をコ一ンオイルなどの溶媒で 作製して任意の動物に投与することにより、 in vivo で活性化した AhR-Arntを、 動物組織の核タンパク質画分を調製して得ることも可能で ある。 この場合、 コーンオイルなどの溶媒を任意の動物に投与して対照 となる AhR-Arntを得ることができる。  By preparing a solution of AhR-Arnt-activating substance such as TCDD in a solvent such as cone oil and administering it to any animal, AhR-Arnt activated in vivo can be converted into the nuclear protein fraction of animal tissues. It is also possible to obtain by preparing In this case, a solvent such as corn oil can be administered to any animal to obtain AhR-Arnt as a control.
具体例としては、 TCDDのコーンオイル溶液を 1 μ g/Kg体重となるよう に腹腔内投与したモルモット、 あるいは 3- MC のコーンオイル溶液を 10 mg/Kg体重となるように腹腔内投与したラットから摘出した肝臓を試料 として用い、 これを予め氷冷したリン酸緩衝化生理食塩水 (PBS)で灌流し て脱血する。次いで、この肝臓を 10倍量の組織破砕用緩衝液 (10 niM HEPESV H 7. 9、 0. 5 mMジチォスレイ トール、 10 mM KC1、 1. 5 mM MgCl2、 5 β g /ml ァプロチュン、 0. 5 mMフッ化フエ二ルメチルスルホニル、 SO g/mlロイ ぺプチン）で破砕処理した後、 l，500 x g、 10分間、 4°Cで遠心分離し、 得 られた沈殿の 2倍量の核抽出用緩衝液 (20 mM HEPES、 pH 7. 9、 0. 2 mM EDTA、 0, 5 mMジチオスレィ トール、 25%グリセロール、 0. 42 M NaCl、l. 5 mM MgCl2、 5 i g/mlァプロチニン、 0. 5 mMフッ化フエ-ルメチルスルホ -ル、 20 /mlロイぺプチン）で懸濁する。 さらに、 得られた溶液を 1時間回転攪拌 した後、 18， 000 X g、 20分間、 4°Cで遠心分離し、 上清 (核タンパク質画 分）を得ることにより、 in vivoで活性化した AhR- Arntが得られる。 これ は、 必要に応じて- 85°Cで保存することができる。 SW-ELISA法で使用される抗ハプテン抗体は、一般的な免疫学的手法に 用いられるハプテン分子、 例えばビォチン、 ジゴキシゲニン (DIG)、 フル 才レセィン ·ィソチオシァネート (FITC)、 ジニトロフエノール (DNP) を、 通常の方法 （A. Kambegawa, Nippon Rinsho, Vol. 53 (9) , p2160-p2167 (1995) ) に従って処理して得られる抗体であり、 ポリクロ一ナルまたは モノクローナノレのいずれであってもよい。 Specific examples include guinea pigs in which TCDD corn oil solution was intraperitoneally administered to 1 μg / Kg body weight, or rats in which 3-MC corn oil solution was intraperitoneally administered to 10 mg / Kg body weight Using the liver extracted from, as a sample, it is perfused with ice-cold phosphate buffered saline (PBS) to remove blood. Then, the liver was diluted with 10 volumes of a tissue disruption buffer (10 niM HEPES VH 7.9, 0.5 mM dithiothreitol, 10 mM KC1, 1.5 mM MgCl 2 , 5 βg / ml After crushing with 5 mM phenylmethylsulfonyl fluoride, SO g / ml leptin), centrifuged at 1,500 xg for 10 minutes at 4 ° C. nuclear extraction buffer (20 mM HEPES, pH 7. 9 , 0. 2 mM EDTA, 0, 5 mM Jichiosurei torr, 25% glycerol, 0. 42 M NaCl, l. 5 mM MgCl 2, 5 ig / ml Apurochinin , 0.5 mM fluormethylsulfol fluoride, 20 / ml leptin). Furthermore, the obtained solution was rotated and stirred for 1 hour, and then centrifuged at 18,000 X g for 20 minutes at 4 ° C to obtain a supernatant (nuclear protein fraction), which was activated in vivo. AhR-Arnt is obtained. It can be stored at -85 ° C if needed. The anti-hapten antibodies used in the SW-ELISA method are hapten molecules used in general immunological techniques, such as biotin, digoxigenin (DIG), full-aged resin isothiocyanate (FITC), dinitrophenol ( DNP) is processed according to the usual method (A. Kambegawa, Nippon Rinsho, Vol. 53 (9), p2160-p2167 (1995)), and can be either polyclonal or monoclonal. You may.
例えば、 ポリクローナル抗体は、 ゥシ血清アルブミン （BSA；分子量 66 kD)、 ヒ ト血清アルブミン （HAS；分子量 58 kD)、 ゥサギ血清アルブミ ン （RSA；分子量 68 kD)、 ャギ血清アルブミン （GSA；分子量 68 kD) な どの哺乳動物の血清アルブミン、 ニヮトリ卵白アルプミン （OVA；分子量 45 kD)、 キーホール · リンぺット ·へモシァニン （KLH；分子量約 1000 kD) などのキャリア ·プロテインにハプテン分子を化学結合させた結合体を 免疫原 （抗原） として、 通常の免疫感作法 (W. H. Newsomeら、 Assoc. Off. Anal. Chem. , Vol. 70, pl025-pl027 (1987) ) に従って得ることが できる。  For example, polyclonal antibodies can be used for serum albumin (BSA; molecular weight 66 kD), human serum albumin (HAS; molecular weight 58 kD), egret serum albumin (RSA; molecular weight 68 kD), and goat serum albumin (GSA; molecular weight). Hapten molecules are used for carrier proteins such as serum albumin of mammals such as 68 kD), chicken ovalbumin (OVA; molecular weight 45 kD), and keyhole limpet hemocyanin (KLH; molecular weight of about 1000 kD). The bound conjugate can be obtained as an immunogen (antigen) according to a conventional immunization method (WH Newsome et al., Assoc. Off. Anal. Chem., Vol. 70, pl025-pl027 (1987)).
免疫感作に用いられる動物としては、ゥサギ、マウス、ラット、ャギ、 ィヌ等の哺乳動物が挙げられ、 動物から採取した血液を、 遠心分離；硫 酸アンモニゥムまたはポリエチレンダリコールを用いる沈殿法；ゲル濾 過クロマトグラフィー、 イオン交換クロマトグラフィー、 ァフィ二ティ 一クロマトグラフィ一のようなクロマトグラフィーなどの通常の方法に よる分離'精製を経てポリクローナル抗体とすることができる。  Animals used for immunization include mammals such as rabbits, mice, rats, goats, and dogs. Blood collected from animals is centrifuged; precipitation using ammonium sulfate or polyethylene dalicol A polyclonal antibody can be obtained through separation and purification by a conventional method such as gel filtration chromatography, ion exchange chromatography, chromatography such as affinity chromatography, and the like.
高度の特異性おょぴ親和性を有するモノクローナルの抗ハプテン抗体 は、 上記の免疫感作を受けた哺乳動物から、 免疫原に対する抗体の産生 能を獲得した B細胞を複数分離し、 該細胞を連続的かつ無限的に細胞分 裂 ·増殖し得るミエ口一マ細胞のような腫瘍細胞とそれぞれ融合させた のち、 生成する複数の融合細胞 （ハイプリドーマ細胞） の中から所望の 抗体を効率的に産生するハイプリ ドーマ細胞を単離 ·クローン化し、 試 験管内または動物生体内で培養することにより製造することができる。 上記のようにして得られる抗ハプテン抗体のなかでも、 ハプテン分子 に FITCを用いて得られるポリクローナル抗ハプテン抗体が好ましく、 ゥ サギ由来のポリクローナル抗ハプテン抗体が特に好ましい。 Monoclonal anti-hapten antibodies having high specificity and affinity have been used to isolate a plurality of B cells that have acquired the ability to produce antibodies against the immunogen from the mammals immunized as described above, and Continuously and indefinitely cleave cells.Fused with tumor cells such as myeloid cells that can proliferate. After that, hybridoma cells that efficiently produce the desired antibody are isolated and cloned from a plurality of fusion cells (hybridoma cells) to be produced, and are produced by culturing them in a test tube or animal body. Can be. Among the anti-hapten antibodies obtained as described above, a polyclonal anti-hapten antibody obtained using FITC as the hapten molecule is preferable, and a polyclonal anti-hapten antibody derived from egrets is particularly preferable.
抗ハプテン抗体の固相化に使用される担体としては、 ELISA法において 通常用いられる担体を使用することができ、 たとえば、 マイクロタイタ 一プレート、 試験管のブラスティック表面、 ポリスチレン、 ポリプロピ レン、 ポリ塩ィ匕ビュル、 ガラスもしくはプラスティック等からなるビー ズ表面、 デキストラン、 セルロース、 ニトロセノレロースもしくはその他 タンパク質結合性素材の細片の表面等が挙げられる。 これらの担体は、 意図された目的に応じて種々の形状を有していてもよく、例えば、皿状、 球状、 穴状、 プレート状、 小型ロッド状、 セル状、 小型チューブ状、 小 型ボトル状、 ファイバー状、 ネット状などの形状であってもよい。  As a carrier used for immobilization of the anti-hapten antibody, a carrier generally used in the ELISA method can be used. For example, a microtiter plate, a plastic surface of a test tube, polystyrene, polypropylene, polysalt Surface of a bead made of glass, plastic, or the like, or the surface of a strip of dextran, cellulose, nitrosenololose, or other protein-binding material. These carriers may have various shapes according to the intended purpose, for example, dish, sphere, hole, plate, small rod, cell, small tube, small bottle. Shape, fiber shape, net shape and the like.
担体の具体的な例としては、 透明プラスティック材料、 例えばポリ塩 化ビュルまたはポリスチレンからなるマイクロタイタープレート、 ポリ スチレンおよびポリスチレンラテックスからなる小球、 チューブまたは ロッド等が挙げられる。 これらのうち、 ポリスチレンの 96穴マイクロタ イタ一プレートは、 取り扱いが容易であり、 特に好ましい。  Specific examples of the carrier include a transparent plastic material, for example, a microtiter plate made of polychlorinated butyl or polystyrene, a small ball made of polystyrene and polystyrene latex, a tube or a rod, and the like. Of these, polystyrene 96-well microtiter plates are particularly preferred because they are easy to handle.
抗ハプテン抗体の担体への固相化は、 ELISA法において通常用いられる ダルタルアルデヒドまたは臭化シアンなどにより担体表面を活性ィ匕し、 これに抗ハプテン抗体を含有する溶液を添カ卩し、 反応させることにより 行うことができるが、 この方法に限定されるものではない。  For immobilization of the anti-hapten antibody on the carrier, the carrier surface is activated with dartartaldehyde or cyanogen bromide, which is usually used in ELISA, and a solution containing the anti-hapten antibody is added thereto. The reaction can be carried out, but is not limited to this method.
例えば、 担体としてポリ塩ィ匕ビュルまたはポリスチレンからなるマイ クロタイタープレートを使用する場合は、 抗ハプテン抗体を 50 mM炭酸 水素ナトリゥム緩衝液 (pH 9. 6) とともに担体に添加するのが最適であ り、 担体に対する抗体の吸着は 1〜10 g/mLの濃度範囲で平衡化するこ と力、ら、 抗ハプテン抗体の最終濃度を 0. 1〜： 10 g/mLの範囲内にするの が望ましい。 こうして得られる抗ハプテン抗体含有溶液は、 例えば穴状 のマイクロタイタープレートでは約 0. 1 mL/穴の容量で用いることがで き、 約 4°Cで約 6〜24時間反応させた後、 洗浄用緩衝液 （例えば 0. 05% Tween (商標) 20 を含むリン酸緩衝化生理食塩水；以下 PBST と記す） を 用いて未反応の抗ハプテン抗体を除去することにより、 担体に抗ハプテ ン抗体を固相化することができる。 For example, as a carrier, a polychlorinated rubber or polystyrene is used. When using crotiter plates, it is best to add the anti-hapten antibody to the carrier together with 50 mM sodium bicarbonate buffer (pH 9.6), and the adsorption of the antibody to the carrier is 1 to 10 g / mL. It is desirable that the final concentration of the anti-hapten antibody be in the range of 0.1 to 10 g / mL. The anti-hapten antibody-containing solution thus obtained can be used, for example, in a hole-shaped microtiter plate at a volume of about 0.1 mL / well, and after reacting at about 4 ° C for about 6 to 24 hours, washing Unreacted anti-hapten antibody is removed using a buffer solution (for example, phosphate-buffered saline containing 0.05% Tween ™ 20 ; hereinafter referred to as PBST), whereby the anti-hapten is added to the carrier. The antibody can be immobilized.
固相化後の担体表面にタンパク質等に対する吸着能が残存している場 合は非特異的結合の生じる恐れがあるため、 本発明の方法で使用される In the case where the adsorption ability for proteins and the like remains on the surface of the carrier after immobilization, non-specific binding may occur, so that the carrier is used in the method of the present invention.
V、ずれの抗体とも反応性を示さないタンパク質の高濃度溶液、例えば 1% BSA溶液、 3% スキムミルク溶液、市販のブロッキング専用試薬を非特異 結合のブロッキング試薬として用い、 ブロッキング操作終了後は、 PBST により担体を洗浄するのが望ましい。 得られた抗ハプテン化抗体固相化 担体は、 必要に応じて冷蔵または冷凍での保存が可能である。 V. Use a high-concentration solution of a protein that does not show reactivity with any of the antibodies, such as a 1% BSA solution, 3% skim milk solution, or a commercially available blocking reagent as a blocking reagent for nonspecific binding. It is desirable to wash the carrier by using. The resulting carrier with immobilized anti-hapten antibody can be stored refrigerated or frozen as necessary.
抗ハプテン抗体固相化担体には、 次いで、 コンセンサス配列を有し、 ハプテン化された DNAプ口ーブを添加する。  Next, a haptenized DNA probe having a consensus sequence is added to the anti-hapten antibody-immobilized carrier.
かかる DNAプローブは、コンセンサス配列を 1〜数ケ所含む 2本鎖 DNA であればよく、 その製造法等は限定されず、 その 5'や 3'末端を化学的あ るいは酵素的にハプテン標識してハプテン化 DNAプローブとすることが できる。  Such a DNA probe may be a double-stranded DNA containing one to several consensus sequences, and its production method and the like are not limited, and its 5 'or 3' end is chemically or enzymatically labeled with a hapten. Thus, a haptenized DNA probe can be obtained.
例えば、 AhR- Arntのコンセンサス配列は前記のとおり 7塩基の DRE配 列からなるため、 当該配列を含む 26塩基からなるセンス鎖一本鎖ォリゴ DNA (5，—GAT CCG GAG TTG CGT GAG AAG AGC CA— 3' )、 およびその相補 配列であるアンチセンス鎖一本鎖オリゴ DNA (5， -GAT CTG GCT CTT CTC ACG C CTC CG— 3' ) を合成した後、 それぞれを化学的に FITC標識し、 精製し、 次いで一本鎖オリゴ DNAをアニーリングし、 二本鎖 DNAプロ一 ブを作製することにより、 AhR-Arnt用のハプテン化 DNAプローブを得る ことができる。 こうして得たハプテン化 DNAプローブは、 担体に結合さ せた状態で、 必要に応じて冷蔵または冷凍での保存が可能である。 For example, since the consensus sequence of AhR-Arnt consists of a 7-base DRE sequence as described above, a single-stranded sense strand consisting of a 26-base sense strand containing the sequence is included. DNA (5, -GAT CCG GAG TTG CGT GAG AAG AGC CA-3 ') and its complementary sequence, single-stranded antisense strand DNA (5, -GAT CTG GCT CTT CTC ACG C CTC CG-3') After synthesizing the haptenized DNA probe for AhR-Arnt, each was chemically labeled with FITC, purified, and then annealed with single-stranded oligo DNA to produce a double-stranded DNA probe. Obtainable. The haptenated DNA probe thus obtained can be stored in a refrigerated or frozen state, if necessary, in a state of being bound to a carrier.
上記のようにして得られるハプテン化 DNAプローブは、 そのコンセン サス配列を介して、 タンパク質活性化物質で処理されることにより DNA 結合能を生じた活性化タンパク質と結合し、活性化タンパク質/ハプテン 化 DNAプロ一ブの複合体を形成する。  The haptenized DNA probe obtained as described above binds to the activating protein which has been treated with a protein activating substance to generate DNA binding ability via its consensus sequence, and the activated protein / haptenized DNA Form a complex of DNA probes.
例えば、 AhR-Arntの場合、予め調製された活性ィヒ AhR - Arntを含むタン パク質試料 40 μ Lを、 150 mM塩ィ匕カリウム（終濃度）を含む HEDG緩衝液 10 μ Lと混合させ、 この混合液を坑ハプテン化抗体とハプテン化 DNAプ 口一ブを固相化したマイクロタイタープレートに添加し、約 4°Cで 2時間 反応させることにより、活性化 AhR - Arnt/ハプテン化 DNAプローブの複合 体が担体上で形成される。  For example, in the case of AhR-Arnt, 40 μL of a previously prepared protein sample containing active AhR-Arnt is mixed with 10 μL of HEDG buffer containing 150 mM potassium chloride (final concentration). This mixture is added to a microtiter plate on which anti-haptenized antibody and haptenized DNA probe are immobilized, and reacted at about 4 ° C for 2 hours to activate activated AhR-Arnt / haptenated DNA. A complex of probes is formed on the carrier.
次いで、 活性ィヒタンパク質/ハプテン化 DNAプローブの複合体に対し、 複合体を特異的に認識する特異抗体をさらに加える。  Next, a specific antibody that specifically recognizes the complex is added to the complex of the activated protein / haptenated DNA probe.
特異抗体は、 活性化タンパク質の構成分子に対する特異抗体であれば よく、 その範囲等は限定されない。 例えば、 AhR- Arnt の場合は、 AhRタ ンパク質の全体やその部分ペプチド、 または Arntタンパク質の全体やそ の部分ぺプチドのいずれかに対する抗体であること、 さらにはそれが AhR-Arntの二量体を認識することのできる抗体でもあることが要件とな り、 これら構成分子を利用する以外は抗ハプテン抗体と同様にして特異 抗体を得ることができる。 具体的には、 Arntタンパク質の C末端部分ま たは A Rタンパク質の N末端部分を構成するぺプチドを合成し、 免疫感 作にて得られる特異抗体が好ましく、 なかでもヒトまたはマウス由来の 該タンパク質に対する抗体が好ましい。 The specific antibody may be any specific antibody against the constituent molecules of the activation protein, and the range and the like are not limited. For example, in the case of AhR-Arnt, it must be an antibody against the whole AhR protein or its partial peptide, or the whole Arnt protein or its partial peptide, and furthermore, it is a dimer of AhR-Arnt It is also necessary that the antibody be capable of recognizing the body. Antibodies can be obtained. Specifically, a specific antibody obtained by immunization by synthesizing a peptide constituting the C-terminal part of the Arnt protein or the N-terminal part of the AR protein is preferable, and particularly, the protein derived from human or mouse is preferable. Are preferred.
なお、 抗ハプテン抗体を産生する免疫動物種と特異抗体を産生する免 疫動物種とが同種であることは望ましくない。  It is not desirable that the immunized animal species producing the anti-hapten antibody and the immunized animal species producing the specific antibody be the same.
次に、 特異抗体を発色用酵素標識二次抗体に.より検出する。 したがつ て、 二次抗体は、 抗ハプテン抗体に反応性を示さず特異抗体のみに反応 性を示すものでなければならず、 抗ハプテン抗体の由来動物と同種の動 物種を用いて、 抗ハプテン抗体と同様にして調製することが望ましい。  Next, the specific antibody is detected by the enzyme-labeled secondary antibody for coloring. Therefore, the secondary antibody must be one that does not show reactivity with the anti-hapten antibody but shows reactivity only with the specific antibody, and uses the same animal species as that of the animal from which the anti-hapten antibody is derived. It is desirable to prepare in the same manner as the hapten antibody.
また、 増感のため検出工程を二段階に分けて行うことにより、 良好な 検出感度を得ることができる。 この場合、 特にピオチン一ストレプトァ ビジン反応を二段階に分けて用いるのが好ましく、 例えば、 ピオチン化 二次抗体含有溶液および酵素標識ストレプトアビジン含有溶液を組み合 わせて使用するか、 あるいはビォチン化二次抗体およびストレプトアビ ジンと発色用酵素が共に分子内に多数結合した構造をもつポリマー試薬 を各段階で利用することができる。  In addition, by performing the detection step in two steps for sensitization, good detection sensitivity can be obtained. In this case, it is particularly preferable to use the biotin-streptavidin reaction in two steps.For example, a combination of a solution containing a biotinylated secondary antibody and a solution containing an enzyme-labeled streptavidin is used. A polymer reagent having a structure in which a large number of antibodies and streptavidin and a color-forming enzyme are bonded together in a molecule can be used in each step.
発色用の酵素としては、パーォキシダーゼ、アル力リホスファターゼ、 β - D-ガラクトシダーゼ、 グ /レコースォキシダーゼ、 グノレコアミラーゼ、 炭酸アンヒドラーゼ、 ァセチ /レコリンエステラーゼ、 リゾチーム、 マレ ートデヒドロゲナーゼ、 グノレコース- 6-ホスフェートデヒドロゲナーゼな どが挙げられる。  Enzymes for color development include peroxidase, alkaline phosphatase, β-D-galactosidase, g / recose oxidase, gnorecoamylase, carbonic anhydrase, aceti / recolin esterase, lysozyme, maleate dehydrogenase, and gnorecose. 6-phosphate dehydrogenase and the like.
二次抗体と反応させた特異抗体は、 一般的な方法、 例えば、 発色用酵 素にパーォキシダーゼを用いる場合には基質として過酸化水素と 0 フエ 二レンジァミン (0PD)あるいは 3, 3'， 5， 5' -テトラメチルベンチジン (ΤΜΒ) との組み合わせを最終容量 100 Lになるように添加し、 室温で約 20〜 30分間反応させることによって発色させることができ、 その度合いは、 450〜490nm、 好ましくは 450nmの波長で、 マルチプレートリーダーのよ うな測定装置を用いて吸光度として測定することができる。 吸光度の測 定は、 1N硫酸水溶液を添加するなどして、 発色反応を停止させてから行 つた方が好ましい。 The specific antibody reacted with the secondary antibody can be obtained by a general method. For example, when peroxidase is used as a color-forming enzyme, hydrogen peroxide and 0-phenylenediamine (0PD) or 3, 3 ', 5, 5'-tetramethylbenzidine (ΤΜΒ) Can be developed by adding the mixture to a final volume of 100 L and reacting at room temperature for about 20 to 30 minutes, at a wavelength of 450 to 490 nm, preferably 450 nm, and a multiplate reader. It can be measured as absorbance using a measuring device such as the one described above. It is preferable to measure the absorbance after stopping the color reaction by, for example, adding a 1N aqueous sulfuric acid solution.
DNA結合性タンパク質の活性レベルは、上記のようにして測定した吸光 度を、 非活性化タンパク質含有試料について得られる吸光度と比較する ことにより判定することができる。 また、 既知のタンパク質活 '性化物質 を用いて、 得られた吸光度値から標準曲線を作成し、 試料中の未知のタ ンパク質の活性化物質濃度を既知の活性化物質相当濃度として定量する ことができる。 さらに、 タンパク質活性化物質を種々の濃度で使用して SW-ELISA法を実施することにより、当該物質の SW-ELISA法における定量 範囲を明らかにすることができる。  The activity level of a DNA-binding protein can be determined by comparing the absorbance measured as described above with the absorbance obtained for a sample containing a non-activated protein. In addition, using a known protein activator, a standard curve is created from the obtained absorbance values, and the concentration of the activator of the unknown protein in the sample is determined as the concentration equivalent to the known activator. be able to. Furthermore, by performing the SW-ELISA method using various concentrations of the protein activator, the quantification range of the substance in the SW-ELISA method can be clarified.
本発明者らの実験によれば、 タンパク質が AhR-Arntである場合、 本発 明による SW-ELISA法は、 タンパク質濃度が低い値、 例えば 4mg/mlの場 合にはゲルシフトアツセィ法と同程度の定量範囲および検出限界値を示 すが、 濃度が高い値、 例えば 20mg/ml の場合には、 検出限界値を維持す る一方で、 定量範囲を拡大する。  According to the experiments of the present inventors, when the protein is AhR-Arnt, the SW-ELISA method according to the present invention is the same as the gel shift assay method when the protein concentration is low, for example, when the protein concentration is 4 mg / ml. Although the quantification range and detection limit are shown to a certain extent, when the concentration is high, for example, 20 mg / ml, the quantification range is extended while maintaining the detection limit.
したがって、 本発明による SW- ELISA法は、 従来法と比較して、 簡便、 迅速かつ高精度に DNA結合性タンパク質の活性を多検体について同時に 測定することができ、 さらには当該タンパク質の活性ィ匕剤おょぴ活性ィ匕 抑制剤をスクリ一ユングすることも可能である。  Therefore, the SW-ELISA method according to the present invention can easily, rapidly and accurately measure the activity of a DNA-binding protein in multiple samples simultaneously with the conventional method. It is also possible to screen the active inhibitor.
活性化抑制剤のスクリ一ユングの場合には、 タンパク質の活性化の際 に、 比較検討の対象となる、 既知タンパク質活性化物質とは異なる被験 化学物質で処理した後に、 既知のタンパク質活性化物質を用いて活性ィ匕 処理を行うか、 または被験ィヒ学物質をタンパク質活性化物質と同時に用 いて活性化処理することにより、 被験ィヒ学物質の活性化抑制作用を検定 することができる。 さらには、 既知のタンパク質活性化物質を用いて活 性化処理した後に、 被験化学物質を処理することで活性化抑制効果を評 価することも可能である。 In the case of screening, which is an activation inhibitor, a different test from known protein activators to be compared when activating the protein After the treatment with a chemical substance, an activation treatment is performed using a known protein activator, or an activation treatment is performed using a test activator at the same time as the protein activator. The activity of the substance to suppress activation can be assayed. Furthermore, it is also possible to evaluate the activation inhibitory effect by treating the test chemical substance after activating with a known protein activator.
また、 活性ィ匕剤のスクリーニングの場合には、 被験化学物質を、 既知 のタンパク質活性化物質による活性化処理と同様にしてタンパク質の処 理に用い、 これを、 既知のタンパク質活性化物質を用いて行った活性ィ匕 処理と比較することにより、 被験化学物質の活性化作用を検定すること ができる。  In addition, in the case of screening for an activator, the test chemical substance is used for protein treatment in the same manner as the activation treatment with a known protein activator, and the test chemical substance is used with a known protein activator. By comparing with the activation treatment performed in the above, the activating effect of the test chemical substance can be assayed.
被験化学物質は、 目的となるタンパク質に対する作用を検討したい化 学物質が含まれているものであれば、 精製化合物を溶解した溶液；液状 食品、 固形金品や土壌からの抽出液；あるいは水質調査用試料などに代 表される多成分ノ多化学物質を含む未精製溶液などのいずれであっても よい。  If the test chemical contains a chemical whose effect on the target protein is to be examined, a solution in which the purified compound is dissolved; a liquid food, an extract from solid gold or soil, or a water quality test Any of a non-purified solution containing a multi-component multi-chemical substance represented by a sample or the like may be used.
例えば、 AhR- Arntに対する活性化抑制作用の有無を液状食品について 検討する際には、 AhR- Arnt含有試料を液状食品自体で予め処理するか、 あるいは試料を TCDDなどの AhR-Arnt活性化物質と液状食品の双方で同 時に処理し、得られる吸光度を該 AhR-Arnt活性ィ匕物質単独で処理するこ とで得られる吸光度と比較することによって、 AhR- Arnt活性化に拮抗す る化合物、すなわち AhR-Arntの活性ィヒ抑制剤が液状食品中に含まれてい るかどうかを検定することができる。  For example, when examining the presence or absence of AhR-Arnt activation inhibitory effects on liquid foods, the AhR-Arnt-containing sample is treated in advance with the liquid food itself, or the sample is treated with an AhR-Arnt activator such as TCDD. A compound that antagonizes AhR-Arnt activation by simultaneously treating both liquid foods and comparing the resulting absorbance with the absorbance obtained by treatment with the AhR-Arnt-activating substance alone, that is, AhR-Arnt activity inhibitors can be tested for inclusion in liquid foods.
さらに、 AhR-Arntに対する活性ィ匕抑制作用の有無を固形食品について 検討する際には、 予め固形食品をエタノールなどの溶媒を用いて抽出し 得られた抽出液を濃縮乾燥後、 水などの溶媒にて溶解もしくは懸濁しFurthermore, when examining the presence or absence of the activity of inhibiting AhR-Arnt in a solid food, the solid food must be extracted using a solvent such as ethanol in advance. The resulting extract is concentrated and dried, and then dissolved or suspended in a solvent such as water.
AhR-Arnt含有試料に対して上記と同様に処理することにより、 AhR-Arnt の活性化抑制剤が固形食品中に含まれているかどうかを検定することが できる。 By treating the AhR-Arnt-containing sample in the same manner as described above, it can be determined whether or not the AhR-Arnt activation inhibitor is contained in the solid food.
また、 本発明によれば、 前記のようにして得られる抗ハプテン抗体を 固相化した担体、 ハプテン化 DNAプローブ、 プローブと活性ィヒタンパク 質との複合体を特異的に認識する特異抗体、 検出用試薬および発色試薬 からなる、 DNA結合性タンパク質の活性測定用キットが提供される。 担体は、 抗ハプテン抗体にハプテン化 DNAプローブを結合させていて もよい。 また、 検出用試薬とは、 特異抗体に対する二次抗体反応、 およ ぴ所望の場合には、 酵素標識反応に関わる試薬を意味し、 ビォチン化二 次抗体含有試薬、 例えばピオチン化二次抗体自体、 およぴストレプトァ ビジンが分子内に多数結合したポリマー試薬のようなストレプトァビジ ン含有試薬が含まれていてもよい。  Further, according to the present invention, a carrier on which the anti-hapten antibody obtained as described above is immobilized, a haptenated DNA probe, a specific antibody that specifically recognizes a complex of the probe and the active protein, a detection antibody A kit for measuring the activity of a DNA-binding protein, comprising a reagent and a coloring reagent, is provided. The carrier may have a haptenated DNA probe bound to an anti-hapten antibody. The detection reagent refers to a reagent involved in a secondary antibody reaction with a specific antibody and, if desired, an enzyme labeling reaction. A reagent containing a biotinylated secondary antibody, for example, a biotinylated secondary antibody itself And a streptavidin-containing reagent such as a polymer reagent having a large number of streptavidin bound in the molecule.
このようなキットは、 タンパク質活性化物質が TCDDのようなダイォキ シン類または 3- MC、 ィンディゴ、 ィンディルビン、 ィソィンディゴのよ うな多環性芳香族炭化水素である場合に、 ダイォキシン毒性の発現に関 与するタンパク質の活性測定に用いることができる。  Such kits are involved in the expression of dioxin toxicity when the protein activator is a dioxin such as TCDD or a polycyclic aromatic hydrocarbon such as 3-MC, indigo, indilbin, and isindigo. It can be used for measuring the activity of a protein to be used.
具体的には、 TNGCGTG(Nは任意の塩基）の 7塩基を含むハプテン化 DNA プローブ、および Arntタンパク質の C末端部分または AhRタンパク質の N末端部分を構成するぺプチドを合成し免疫感作にて得られる特異抗体 を備えるキットにより、 DNA結合性タンパク質 AhR- Arntの活性レベルを 測定することができる。 実施例 以下、 AhR- Arntに関する予備試験および実施例によって本発明を更に 詳細に説明するが、 本発明は以下の実施例のみに限定されるものではな く、 当該技術分野における通常の変更を加えたものも本発明の範囲に含 まれる。 Specifically, a haptenized DNA probe containing 7 bases of TNGCGTG (N is any base) and a peptide constituting the C-terminal part of Arnt protein or the N-terminal part of AhR protein are synthesized and immunized. With the kit including the obtained specific antibody, the activity level of the DNA-binding protein AhR-Arnt can be measured. Example Hereinafter, the present invention will be described in more detail with reference to AhR-Arnt preliminary tests and Examples. However, the present invention is not limited to only the following Examples, but with ordinary changes in the technical field. Are also included in the scope of the present invention.
予備試験：抗ハプテン抗体の特異性確認 Preliminary test: Confirmation of specificity of anti-hapten antibody
96穴マイクロタイタープレート （ナルジェヌンクインターナショナル 社製） に抗ハプテン抗体である抗 FITCゥサギポリクローナル抗体 （ダコ 社製） の固相化を行った。  A 96-well microtiter plate (Nalgenunc International) was immobilized with an anti-hapten antibody, anti-FITC ゥ sagi polyclonal antibody (Dako).
すなわち、 抗 FITC抗体を 50 mM炭酸水素ナトリゥム緩衝液 (pH 9. 6)で 0. 46 ^ g/mL (1 : 5000， v/v)に希釈したのち、 プレート内の各穴へ 100 μ Lずつ分注し、 4°Cで 8時間反応させた。該抗 FITC希釈液を除去し、 100 μ Lの PBSTで各穴を 3回洗浄した。プロッキング溶液（ナカライテスタ製） を PBSTで 5倍に希釈し、 プレート内の各穴に 100 μ Lずつ加えて室温で 2時間ブ口ッキングを行つた。  That is, after diluting the anti-FITC antibody to 0.46 ^ g / mL (1: 5000, v / v) with 50 mM sodium bicarbonate buffer (pH 9.6), add 100 μL to each well in the plate. The reaction was carried out at 4 ° C for 8 hours. The anti-FITC dilution was removed and each well was washed three times with 100 μL of PBST. The blocking solution (manufactured by Nacalai Tester) was diluted 5-fold with PBST, and 100 μL of the solution was added to each well in the plate, followed by booking at room temperature for 2 hours.
100 し の PBSTで各穴を 3回洗浄した後、 ブロッキング溶液を PBST で 10倍に希釈した緩衝液 （抗体希釈用緩衝液） で 0. 01〜； 1000 ng/mLの 濃度に FITC標識ャギィムノグロブリン G (IgG) タンパク質を希釈し、各 穴に 100 μ Lずつ加え室温で 2時間反応させた。 100 Lの PBSTで各穴を 3回洗浄した後、 次いで西洋わさび由来パーォキシダーゼ （HRP) を標識 した抗ャギ IgG抗体 （CAPPEL社製） を抗体希釈用緩衝液で 1. 5 ^ g/rnL (1 : 1000, v/v)の濃度に希釈し、 各穴へ 100 ^u Lずつ添加し、 室温で 2時 間反応させた。 プレート内の各穴をそれぞれ 4回ずつ洗浄した後、 各穴 に TMB発色試薬 （ダコ社製） を 100 ずつ加えて発色反応を開始し、 30分後に 1N硫酸を 50 し加えることで発色を停止させた。  After washing each well three times with 100 PBST, the blocking solution is diluted to 0.01-fold with a buffer (antibody dilution buffer) diluted 10-fold with PBST; the FITC-labeled antibody was diluted to a concentration of 1000 ng / mL. Munoglobulin G (IgG) protein was diluted, and 100 μL was added to each well to react at room temperature for 2 hours. After washing each well three times with 100 L of PBST, an anti-goat IgG antibody (manufactured by CAPPEL) labeled with horseradish-derived peroxidase (HRP) was then added to the antibody dilution buffer at 1.5 ^ g / The mixture was diluted to a concentration of rnL (1: 1000, v / v), added to each well in an amount of 100 μl, and allowed to react at room temperature for 2 hours. After washing each well of the plate four times, add 100 ml of TMB color reagent (manufactured by Dako) to each well to start the color reaction, and stop color development after 30 minutes by adding 50 of 1N sulfuric acid. I let it.
プレートを攪拌後、 マルチプレートリーダー （ヮラック社製） を用い て 450 nmの吸光度を測定し、 抗 FITC抗体の特異性と ELISA法における 固相化用抗体としての適用性を確認した （図 1)。 After stirring the plate, use a multiplate reader (manufactured by Lack Co., Ltd.) The absorbance at 450 nm was measured to confirm the specificity of the anti-FITC antibody and its applicability as an antibody for immobilization in ELISA (Fig. 1).
実施例 1 ： SW- ELISA法における特異抗体の適用性確認 Example 1: Confirmation of applicability of specific antibody in SW-ELISA method
活性化 AhR-Arntは、終濃度として 1. 61ng/mL (5nM)の TCDDの DMS0溶液 を、 タンパク質量として 15mg/mLに調製したラット肝細胞質画分に添加 した後、 20°C下で 2時間暗所にて反応させることにより予め調製した。 また、 コント口ールとして、 同量の DMS0のみを添加したラット.肝細胞質 画分を同様に 20°C下で 2時間喑所にて反応させた。  Activated AhR-Arnt was added to a rat liver cytoplasmic fraction prepared by adding a 1.61 ng / mL (5 nM) TCMS DMS0 solution to a final protein concentration of 15 mg / mL, and then activated at 20 ° C. It was prepared in advance by reacting in the dark for hours. As a control, a rat hepatocyte fraction to which only the same amount of DMS0 had been added was also reacted at 20 ° C for 2 hours.
96穴マイクロタイタープレートへの抗 FITC ゥサギポリクローナル抗 体の固相化およぴブロッキングは、 予備試験に記載のように行った。  The immobilization and blocking of the anti-FITC / Egret polyclonal antibody on a 96-well microtiter plate were performed as described in the preliminary test.
100 /z Lの PBSTで各穴を 3回洗浄した後、 あらかじめ合成した FITC 標識 DRE配列を含む DNAプローブ（以下、 DREプローブとする。センス鎖； 5' -GAT CCG GAG TTG CGT GAG AAG AGC CA—3，、 アンチセンス鎖； 5'一 GAT CTG GCT CTT CTC ACG CAA CTC CG—3' ) を PBSTで 25 fmols/100 μ L に希釈し、 各穴に 100 μ ΐずつ加えて室温で 2時間反応させ、 100 μ ΐ の PBSTで各穴を 3回洗浄した。 さらに上述の活性化 AhR - Arnt 40 1と 150mM塩ィヒカリゥム（終濃度)含有 HEDG緩衝液 10 μ 1との混合液を各穴あ たり 50 μ ΐずつ添力 Π (タンパク質量として 120 μ g)し、 室温で 2時間反 応させた。  After washing each well three times with 100 / z L of PBST, a DNA probe containing a previously synthesized FITC-labeled DRE sequence (hereinafter referred to as DRE probe; sense strand; 5'-GAT CCG GAG TTG CGT GAG AAG AGC CA —3, antisense strand; 5'-one GAT CTG GCT CTT CTC ACG CAA CTC CG-3 ') diluted with PBST to 25 fmols / 100 μL, add 100 μΐ to each well and add 2 hours at room temperature The reaction was performed, and each well was washed three times with 100 μ μ of PBST. Further, add a mixture of activated AhR-Arnt 401 described above and 10 μl of HEDG buffer containing 150 mM salt solution (final concentration) to each well 50 μΐ per hole (120 μg as protein). The reaction was performed at room temperature for 2 hours.
100 /z Lの PBSTで各穴を 4回洗浄した後、 抗体希釈用緩衝液で特異抗 体 4種；すなわち（a) ヒト Arntタンパク質の C末端部分を構成するぺプ チドを合成し免疫感作にて得られる抗体 (抗 -Arnt C- 19)、 (b)マウス Arnt タンパク質の C末端部分を構成するペプチドを合成し免疫感作にて得ら れる抗体（抗 -Arnt M-20) (いずれもサンタクルーズ ·バイオテクノロジー 社製)、 （c) マウス AhRタンパク質の N末端部分を構成するペプチドを合 成し免疫感作にて得られる抗体（抗- A R MA1-514) (ァフイエティー ·バ ィォリージェント社製)及ぴ (d) マウス AhRタンパク質の N末端部分を構 成するペプチドを合成し免疫感作にて得られる抗体（抗 -AhR B100-128) (ノーバス.バイオロジカルス社製）を、（a)及び (b)は 1 : 10000、 (c)は 1 : 1500、 （d)は 1 : 2000に希釈し、 100 Lずつを各穴に加えて室温 で 1時間反応させた。 After washing each well four times with 100 / zL PBST, four specific antibodies are diluted with antibody dilution buffer; that is, (a) the peptide constituting the C-terminal portion of human Arnt protein is synthesized and immunized. (Anti-Arnt C-19), (b) An antibody (anti-Arnt M-20) obtained by synthesizing a peptide constituting the C-terminal part of mouse Arnt protein (anti-Arnt M-20) ( (All manufactured by Santa Cruz Biotechnology, Inc.), (c) Peptide constituting the N-terminal part of mouse AhR protein was synthesized. Antibody (anti-AR MA1-514) (manufactured by Affiliate Bioregent) and (d) a peptide constituting the N-terminal portion of mouse AhR protein was synthesized and immunized. Antibody (anti-AhR B100-128) (Novas. Biologicals), (a) and (b) are 1: 10000, (c) is 1: 1500, and (d) is 1 : Diluted to 2000, added 100 L to each well, and reacted at room temperature for 1 hour.
100 の PBSTで各穴を 3回洗浄した後、 次いで二次抗体としてビォ チン標識した抗ャギ IgG抗体 （ジャクソン ·ィムノリサーチ 'ラボラト リー社製） あるいは抗マウス IgG抗体 (ジャクソン ·ィムノリサーチ · ラボラトリ一社製）を抗体希釈用緩衝液でそれぞれ 0. 34 μ g/mL (1： 5000)、 0. 33 μ g/mL (1 :4000)の濃度に希釈し、 各穴へ 100 // Lずつ添加し、 室温 で 1時間反応させた。  After washing each well three times with 100 PBST, a biotin-labeled anti-goat IgG antibody (Jackson ImnoResearch 'Laboratory') or an anti-mouse IgG antibody (Jackson ImnoResearch Lab. Diluted with antibody dilution buffer to a concentration of 0.34 μg / mL (1: 5000) and 0.33 μg / mL (1: 4000), respectively. The mixture was added and reacted at room temperature for 1 hour.
100 μ Lの PBSTで各穴を 3回洗浄した後、 さらに PBSTで HRPとストレ プトァビジンの両方を結合した標識ポリマー試薬 （ダコ社製） を 0. 33 μ g/mL (1 : 2000)の濃度に希釈し、 各穴へ 100 μ ΐずつ添加し、 室温で 30 分間反応させた。  After washing each well three times with 100 μL of PBST, a concentration of 0.33 μg / mL (1: 2000) of a labeled polymer reagent (manufactured by Dako) bound with both HRP and streptavidin was added using PBST. And added to each well in an amount of 100 μ 100, and reacted at room temperature for 30 minutes.
プレート内の各穴をそれぞれ 4回ずつ洗浄した後、 各穴に ΤΜΒ発色試 薬 （ダコ社製） を 100 μ ΐずつ加えて発色反応を開始し、 30分後に 1N 硫酸を 50 z L加えることで発色を停止させた。  After washing each well of the plate four times each, add a color reagent (manufactured by Dako) 100 μ 穴 to each well to start the color reaction. After 30 minutes, add 50 zL of 1N sulfuric acid. To stop color development.
プレートを攪拌後、 マルチプレートリーダー （ヮラック社製） を用い て 450 nmの吸光度を測定し、 図 2に示す結果を得た。  After stirring the plate, the absorbance at 450 nm was measured using a multiplate reader (manufactured by Perak), and the results shown in FIG. 2 were obtained.
この結果、 Arntタンパク質では C末端部分、 AhRタンパク質では N末 端部分を構成するぺプチドに対して調製された抗体が AhR-Arntの活性ィ匕 を良好に捉えることが確認された。  As a result, it was confirmed that the antibody prepared for the peptide constituting the C-terminal part of the Arnt protein and the N-terminal part of the AhR protein could well capture the activity of AhR-Arnt.
実施例 2 ：検出工程の違いによる検出の感度差の確認 ビォチン標識した抗ャギ IgG抗体と HRP ·ストレプトァビジン標識ポリ マー試薬とを用いる実施例 1の二段階検出を、 抗体希釈用緩衝液で 1. 5 μ g/mL (1： 1000)の濃度に希釈した HRP標識抗ャギ IgG抗体 (CAPPEL社製） のみを使用する一段階検出と比較した。 Example 2: Confirmation of difference in detection sensitivity due to difference in detection process The two-step detection of Example 1 using biotin-labeled anti-goat IgG antibody and HRP / streptavidin-labeled polymer reagent was performed at a concentration of 1.5 μg / mL (1: 1000) with antibody dilution buffer. Comparison with one-step detection using only an HRP-labeled anti-goat IgG antibody (manufactured by CAPPEL) diluted in water.
特異抗体として抗- Arnt C-19抗体のみを用いる以外は実施例 1に準じ て行い、 一段階検出よりもビォチン -ストレプトアビジンによる二段階 増感検出の感度が良好であることを確認した （図 3)。  The procedure was performed in the same manner as in Example 1 except that only the anti-Arnt C-19 antibody was used as the specific antibody, and it was confirmed that the sensitivity of the two-step sensitization detection using biotin-streptavidin was better than the one-step detection. 3).
実施例 3 ： SW - ELISA法とゲルシフトアツセィ法との相関性の確認 Example 3: Confirmation of correlation between SW-ELISA method and gel shift assay method
ゲルシフトアツセィ法は、 活性ィ匕 AhR- Arntを含む細胞質画分を 150 mM 塩化カリウム （終濃度） を含む HEDG緩衝液と混合し、 これまでに報告し た方法（H. Ashidaら、 FEBS Lett. , Vol. 476, p213 - 217 (2000) )に従い行 つた。 なお、 DNAプローブには、実施例 1に記載する DREプローブと同一 の DNA配列を用い、 未標識 （FITC標識していないもの） DREプローブを 32P-ATPと T4ポリヌクレオチドキナーゼを用いて 5'末端を 32Pラベノレし、 一反応液量（12 μ ί)あたり 0. 5 L の DRE プローブ溶液を添加した。 AhR-Arnt/DRE複合体は、 X線フィルムに露光させた（図 4A)。 The gel shift assay was performed by mixing the cytoplasmic fraction containing AhR-Arnt with HEDG buffer containing 150 mM potassium chloride (final concentration) and using the method described previously (H. Ashida et al., FEBS Lett.). , Vol. 476, p213-217 (2000)). The same DNA sequence as the DRE probe described in Example 1 was used as the DNA probe, and the unlabeled (non-FITC-labeled) DRE probe was synthesized using 32 P-ATP and T4 polynucleotide kinase. The ends were subjected to 32 P labeling, and 0.5 L of a DRE probe solution was added per reaction volume (12 μί). The AhR-Arnt / DRE conjugate was exposed to X-ray film (FIG. 4A).
一方、 0〜75 fmolsの TCDDの DMS0溶液を、タンパク質量として 4 mg/mL に調製したラット肝細胞質画分に添加した後、 20°C下で 2時間暗所にて 反応させることにより活性化 AhR-Arntを調製し、特異抗体として抗- Arnt C- 19抗体を用いて、 実施例 1の SW- ELISA法を行った（図 4B)。  On the other hand, 0-75 fmols of TCDD DMS0 solution was added to the rat liver cytoplasmic fraction adjusted to 4 mg / mL in protein amount, and activated by reacting at 20 ° C for 2 hours in the dark. AhR-Arnt was prepared and the SW-ELISA method of Example 1 was performed using an anti-Arnt C-19 antibody as a specific antibody (FIG. 4B).
この結果、 ゲルシフトアツセィ法では、 活性ィ匕 AhR- Arntのパンドの濃 さが 25 fmols まで濃度依存的に増加し、 TCDD の定量範囲が 0. 25〜25 fmolsであることが示された（矢印）。 また、 SW-ELISA法では、 AhR - Arnt の活性化が TCDDの濃度に依存して増し、 定量範囲が 0. 3〜75 fmols (約 0. l〜20pg)であった。 SW - ELISA法による検出限界値 (0. 3 fmols)は非常に 高感度であり、 本方法は、 ゲルシフトアツセィ法と同程度の検出感度を 有することが確認された。 As a result, it was shown that in the gel shift assay, the concentration of the band of Activated AhR-Arnt increased to 25 fmols in a concentration-dependent manner, and the quantification range of TCDD was 0.25 to 25 fmols ( Arrow). In the SW-ELISA method, AhR-Arnt activation increased depending on the concentration of TCDD, and the quantification range was 0.3 to 75 fmols (approximately 0.1 to 20 pg). The detection limit (0.3 fmols) by SW-ELISA is very high The method was highly sensitive, and it was confirmed that this method had the same level of detection sensitivity as that of the gel shift assay.
実施例 4 ： AhR-Arntの活性ィヒを抑制する化合物のスクリーニングにおけ る SW-ELISA法とゲルシフトアツセィ法との相関性の確認 Example 4: Confirmation of the correlation between the SW-ELISA method and the gel shift assay in screening for compounds that inhibit the activity of AhR-Arnt
1 nMの TCDDによつて誘導される A R - Arntの活性化に対し抑制効果を 有する化合物のスクリ一ユングを目的として、 SW - ELISA法 (実施例 1 )お よぴゲルシフトアツセィ法 (実施例 3 )をそれぞれ行つた。  In order to screen compounds having an inhibitory effect on AR-Arnt activation induced by 1 nM TCDD, the SW-ELISA method (Example 1) and the gel shift assay method (Example 3) went to each.
被験物質には、 フラボノイドであるァピゲニン、 ヘスペレチン、 ミリ セチン (シグマ社製）、 ガランジン、 ケンフェローノレ、 タンゲレチン (共 に EXTRASY THESE社製）、ケルセチン、ルチン（共に和光純薬工業社製）、 ナリンゲニン （ナカライテスタ社製） を用い、 AhR- Arntにおける各化合 物含有溶液の終濃度が 0. 1〜50 ¾1となるように添加し、 20。Cで 10分間 作用させ、 次いで、 該反応混液に 1 nM TCDDを 20°Cで 2時間処理した。  The test substances included the flavonoids apigenin, hesperetin, myricetin (Sigma), galandin, kaempferonore, tangeretin (both from EXTRASY THESE), quercetin, rutin (both from Wako Pure Chemical Industries), naringenin 20 (manufactured by Nacalai Tester) and added so that the final concentration of each compound-containing solution in AhR-Arnt would be 0.1 to 50501. The reaction mixture was treated with 1 nM TCDD at 20 ° C for 2 hours.
SW-ELISA法の測定結果は、 TCDDのみで処理した AhR-Arntの吸光度を 100%、 DMS0のみの時のそれを 0%として算出した。 一方、 ゲルシフトァ ッセィ法の結果は X線フィルムに露光させ、イメージング'アナライザー で AhR- Arntのパンド密度を測定した後、同様に、 TCDDのみで処理した細 胞質のバンド密度を 100%、 DMS0のみの時のそれを 0%として算出した。 いずれの方法においても被験物質の各濃度における％から 1 nM TCDDの AhR-Arntの活性ィヒに対する半阻害濃度 (IC5。値）を算出した （図 5A)。 その結果、 SW- ELISA法の測定結果は、 ゲルシフトアツセィ法の結果と よく一致し、 両法で得られた半阻害濃度の間に正の相関 (r=0. 950)が得 られた （図 5B)。 The measurement results of the SW-ELISA method were calculated assuming that the absorbance of AhR-Arnt treated only with TCDD was 100% and that of DMS0 alone was 0%. On the other hand, the results of the gel shift assay were exposed to X-ray film, the band density of AhR-Arnt was measured with an imaging analyzer, then the band density of the cells treated with TCDD only was set to 100%, and only DMS0 was used. At that time, it was calculated as 0%. Also to calculate the half-inhibitory concentration (IC 5. Value) for the active I inhibit the AhR-Arnt of 1 nM TCDD from% at each concentration of the test substance in any of the methods (Fig. 5A). As a result, the measurement results of the SW-ELISA method were in good agreement with the results of the gel shift assay method, and a positive correlation (r = 0.950) was obtained between the half-inhibitory concentrations obtained by both methods (r = 0.950) (Figure 5B).
これらの結果から、 SW - ELISA法は、被験ィ匕学物質の AhR- Arnt活性ィ匕抑 制効果の評価に適用できることが確認できた。 実施例 5 ： AhR- Arntの活性化に対し抑制活性を有する液状食品のスクリ 一二ング From these results, it could be confirmed that the SW-ELISA method can be applied to the evaluation of the AhR-Arnt activity of the test substance. Example 5: Screening of liquid foods having inhibitory activity on AhR-Arnt activation
1 nMの TCDDによって誘導される AhR- Arntの活性化に対し、 抑制活性 を示す液状食品のスクリーニングを目的として、 SW- ELISA法 (実施例 1 ) およぴゲノレシフトアツセィ法 (実施例 3 )をそれぞれ行つた。  For the purpose of screening for liquid foods that show inhibitory activity against AhR-Arnt activation induced by 1 nM TCDD, the SW-ELISA method (Example 1) and the Genoleshift Assy method (Example 3) were used. ).
被験食品には、市販飲科 16種 (緑茶 3種、 中国茶、烏龍茶、紅茶 2種、 オレンジジュース、 リンゴジュース、 野菜ジュース、 アロエジュース、 コーヒー、 ミルクココア、 牛乳、 豆乳、 ワイン）を用い、 AhR- Arntの体積 に対し 5%の濃度となるよう、 1 nMの TCDDと同時に添カロした。  As test foods, 16 kinds of commercial drinks (3 kinds of green tea, Chinese tea, Oolong tea, 2 kinds of black tea, orange juice, apple juice, vegetable juice, aloe juice, coffee, milk cocoa, milk, soy milk, wine) were used. Caroten was added simultaneously with 1 nM TCDD to give a concentration of 5% based on the volume of AhR-Arnt.
SW-ELISA法およびゲルシフトァッセィ法の結果は、 実施例 4と同様に して得た（図 6)。  The results of the SW-ELISA method and the gel shift assay method were obtained in the same manner as in Example 4 (FIG. 6).
その結果、 SW - ELISA法で得られた巿販飲料 16種の AhR- Arnt活性化に 対する抑制効果は、 ゲルシフトアツセィ法で得られた結果と良い相関を 示した。 すなわち、 中国茶、 烏龍茶、 紅茶 2種、 コーヒー、 ミルクココ ァ、 牛乳、 ワインは TCDDによる AhR- Arnt活性化をほぼ完全に抑制し、 オレンジジュース、 リンゴジュース、 野菜ジュース、 アロエジュース、 豆轧は、 抑制効果が弱く、 緑茶の抑制効果は製品間のばらつきが大きい ことがわかった。  As a result, the inhibitory effect on the activation of AhR-Arnt of the 16 commercial beverages obtained by the SW-ELISA method showed a good correlation with the results obtained by the gel shift assay. That is, Chinese tea, oolong tea, two kinds of black tea, coffee, milk cocoa, milk, and wine almost completely suppress the activation of AhR-Arnt by TCDD, and orange juice, apple juice, vegetable juice, aloe juice, and beans The inhibitory effect was weak, and the inhibitory effect of green tea was found to vary widely between products.
これらのことから、 SW-ELISA法は、 実施例 4で示した被験化学物質の 抑制効果のみならず、 液状食品のような未精製溶液の抑制効果の評価に も適用できることが確認できた。  From these results, it was confirmed that the SW-ELISA method can be applied not only to the inhibitory effect of the test chemical substance shown in Example 4, but also to the evaluation of the inhibitory effect of an unpurified solution such as a liquid food.
実施例 6 ：活性ィ匕タンパク質濃度の増加による定量範囲の拡大 Example 6: Expansion of quantification range by increasing concentration of active protein
活性化 AhR- Arntは、 終濃度 1 nMの TCDDの DMS0溶液を、 タンパク質 量として 20 mg/mLに調製したラット肝細胞質画分に添加した後、 20°C下 で 2時間暗所にて反応させることにより予め調製した。 活性化 AhR-Arnt 40 Lに 150 mM塩ィヒカリウム（終濃度）を含む HEDG 緩衝液 10 x Lをそれぞれ混合し、 これを各穴あたり 50 /i Lずつ添加（タン パク質量として 800 μ §)し、室温で 2時間反応させた。以降の操作は実施 例 1に準じて行い、 図 7の結果を得た。 Activated AhR-Arnt was added to a rat liver cytoplasmic fraction adjusted to a protein concentration of 20 mg / mL with a final concentration of 1 nM TCDD in DMS0, and then reacted at 20 ° C for 2 hours in the dark. And was prepared in advance. 0.99 mM salt Ihikariumu the activation AhR-Arnt 40 L of HEDG buffer 10 x L containing (final concentration) were mixed respectively, which was (800 mu § as Tan Park mass) added in 50 / i L per each well The reaction was performed at room temperature for 2 hours. Subsequent operations were performed according to Example 1, and the results in FIG. 7 were obtained.
これによると、 TCDDの定量範囲は、 タンパク質として 4 mg/mL濃度で 活性化 AhR_Arntを用いた実施例 3 (0. 3〜75 fmols (約 0. l〜20pg) )と比 較して、 0. 4〜400 fmols (約 0. 13〜： 130 pg)に拡大した。  According to this, the quantification range of TCDD was 0 compared with Example 3 (0.3 to 75 fmols (about 0.1 to 20 pg)) using activated AhR_Arnt at a concentration of 4 mg / mL as a protein. Expanded to 4-400 fmols (approximately 0.13--130 pg).
つまり、 活性化タンパク質濃度を増加させると、 検出限界値は 0. 4 fmolsで実施例 3の場合とほとんど変わらな 、高感度を保つ一方、定量範 囲が拡大されることが示された。  In other words, it was shown that, when the concentration of the activated protein was increased, the detection limit was 0.4 fmols, which was almost the same as that of Example 3, while maintaining high sensitivity and expanding the quantification range.
実施例 7 ：特異性抗体の検出限界の比較 Example 7: Comparison of detection limits of specific antibodies
実施例 1で良好な検出感度を有した 3種の抗体 (抗- Arnt C- 19抗体、抗 - AhR MAI- 514抗体及ぴ抗- AhR BlOO- 128抗体）を用いて、 TCDD濃度に依 存した AhR-Arnt活性ィ匕の検出限界を比較した。  The three antibodies (anti-Arnt C-19 antibody, anti-AhR MAI-514 antibody and anti-AhR BlOO-128 antibody) having good detection sensitivity in Example 1 were used to determine the TCDD concentration. The detection limits of the resulting AhR-Arnt activities were compared.
ラット肝臓の細胞質画分にさまざまな濃度の TCDDを添加して 20°Cで 2 時間作用させた後、 活性化 AhR - Arntを検出した（図 8 )。  Activated AhR-Arnt was detected after adding various concentrations of TCDD to the cytoplasmic fraction of rat liver and allowing it to act at 20 ° C for 2 hours (Fig. 8).
TCDDの濃度依存性は、 抗 -Arnt C- 19抗体と抗- AhR MAI- 514抗体を用 いた場合に特によく確認でき、 また、 これらの抗体はベースラインが低 い点で優れていた。  The concentration dependence of TCDD was particularly well confirmed when anti-Arnt C-19 antibody and anti-AhR MAI-514 antibody were used, and these antibodies were excellent in that they had a low baseline.
このため、 以降の実験では、 抗- Arnt C- 19抗体（以降、 抗 Arnt抗体と する）または抗- AhR MAI- 514抗体 (以降、 抗 AhR抗体とする）を用いた。 実施例 8 ：細胞系における SW-ELISA法の適用  Therefore, in the subsequent experiments, an anti-Arnt C-19 antibody (hereinafter, referred to as anti-Arnt antibody) or an anti-AhR MAI-514 antibody (hereinafter, referred to as anti-AhR antibody) was used. Example 8: Application of SW-ELISA method in cell line
本発明の SW- ELISA法が細胞を用いた試験にも適用できるか否かを検討 した。  Whether the SW-ELISA method of the present invention can be applied to a test using cells was examined.
まず、 用いる細胞を選択するため、肝臓由来の 3種の株細胞（マウス肝 腫瘍由来 Hepa- lclc7細胞、 ラット正常肝由来 RL-34細胞およびヒト肝腫 瘍由来 HepG2細胞（それぞれ、武田食品工業株式会社より分与、 JCRB Cell Bankより購入、京都工芸繊維大学伊倉教授より分与）に陰性コント口ール として DMS0または陽性コントロールとして 1 nM TCDDを 37°Cで 2または 2 時間作用させ、 各細胞に対する TCDDの感受性を調べた。 First, in order to select the cells to be used, three liver-derived cell lines (mouse liver cells) were used. Tupa-derived Hepa-lclc7 cells, rat normal liver-derived RL-34 cells, and human liver tumor-derived HepG2 cells (distributed from Takeda Foods Co., Ltd., purchased from JCRB Cell Bank, distributed by Professor Ikura, Kyoto Institute of Technology, respectively) ) Were treated with DMS0 as a negative control or 1 nM TCDD as a positive control at 37 ° C for 2 or 2 hours to examine the sensitivity of each cell to TCDD.
活性ィヒ AhR- Arntは核内に移行するため、それぞれの細胞から核タンパ ク質を抽出し、 活性ィヒ AhR-Arntを、 抗 Arnt抗体を用いて検出し、 陰性 コントロールの 450nmにおける吸光度に対する陽性コントロールの吸光 度の比を算出し、 平均値土標準偏差 (n=3)であらわした（図 9 )。  Since activated AhR-Arnt translocates into the nucleus, nuclear protein is extracted from each cell, and activated AhR-Arnt is detected using an anti-Arnt antibody. The ratio of the absorbance of the positive control was calculated and expressed as the average soil standard deviation (n = 3) (FIG. 9).
2時間、 24時間ともに、 Hepa- lclc7細胞において活性化 AhR - Arntが最 も顕著に検出された。  Activated AhR-Arnt was most prominently detected in Hepa-lclc7 cells at both 2 and 24 hours.
そこで、 TCDDでの処理時間を 0、 1、 2、 4、 8、 24時間とする以外は上 記と同様にして Hepa-lclc7細胞中での AhR- Arnt活性化の時間依存性を 抗 Arnt抗体または抗 AhR抗体を用いて検出し、 450nmにおける吸光度を 求め、 平均値士標準偏差 (n=3)を算出した。  Therefore, the time dependence of AhR-Arnt activation in Hepa-lclc7 cells was determined in the same manner as above except that the treatment time with TCDD was set to 0, 1, 2, 4, 8, and 24 hours. Alternatively, detection was performed using an anti-AhR antibody, the absorbance at 450 nm was determined, and the mean standard deviation (n = 3) was calculated.
その結果、どちらの抗体を用いた場合でも 1〜2時間において AhR-Arnt の活性ィヒは最大となり、 4時間以降は最大値の半分程度に低下して横ばい となった（図 1 0 )。  As a result, the activity of AhR-Arnt became maximal in 1 to 2 hours with both antibodies, and after 4 hours, decreased to about half of the maximum and leveled off (Fig. 10).
また、 AhR- Arnt活性化の濃度依存性を確認するため、 Hepa- lclc7細胞 に 0、 0. 001、 0. 01、 0. 1、 0. 2、 0. 5、 1、 5 nM TCDDを処理して 37°Cで 2 時間作用させ、上記と同様にして核抽出液に含まれる活性ィヒ AhR - Arntを 検出したところ、 どちらの抗体を用いた場合でも 0. 01〜0. 5 nMにおいて 直線性が確認でき、 抗 AhR抗体を用いた場合の検出限界は lpM TCDDであ つた（図 1 1 )。 以上のことから、 細胞を用!/、た試験でも本発明の SW - ELISA法は TCDD を検出できることが確認できた。 Hepa-lclc7 cells were treated with 0, 0.001, 0.01, 0.1, 0.2, 0.5, 1, and 5 nM TCDD to confirm the concentration dependence of AhR-Arnt activation. And acted at 37 ° C for 2 hours.The active AhR-Arnt contained in the nuclear extract was detected in the same manner as above, and when using either antibody, it was 0.01-0.5 nM. The linearity was confirmed, and the detection limit when using an anti-AhR antibody was lpM TCDD (Fig. 11). From the above, it was confirmed that the SW-ELISA method of the present invention was able to detect TCDD in the test using cells.
実施例 9 ： AhRリガンド化合物による AhR - Arnt活性化検出 Example 9: Detection of AhR-Arnt activation by AhR ligand compound
AhRのリガンド化合物であるィンディゴ、ィンディルビンおょぴィソィ ンディゴが AhR- Arntの活性化を検出できるか否かを検討した。  We examined whether AhR ligand compounds, indigo, indirbin and dysindigo, could detect AhR-Arnt activation.
Hepa-lclc7細胞に陰性コントロールとして DMS0、 陽性コントロールと して 1 nM TCDD, 各 ΙΟ μ Μのインディゴ、 インディルビンおょぴィソイン ディゴを処理して 37°Cで 2時間作用させ、核抽出液中の活性ィヒ AhR - Arnt を抗 Arnt抗体用いて検出し、 450nmにおける吸光度を求め、 平均値土標 準偏差 (n=3)を算出した。  Hepa-lclc7 cells were treated with DMS0 as a negative control, 1 nM TCDD as a positive control, ΙΟμΜ of each indigo and indirubin and indigo, and allowed to act at 37 ° C for 2 hours. AhR-Arnt was detected using an anti-Arnt antibody, the absorbance at 450 nm was determined, and the average soil standard deviation (n = 3) was calculated.
その結果、 これらの化合物は高濃度（10 μ Μ)において AhR- Arntの活性 化を誘導した（図 1 2 )。  As a result, these compounds induced AhR-Arnt activation at high concentrations (10 μM) (Fig. 12).
したがって、 本発明の SW-ELISA 法では、 ダイォキシン類による AhR- Arnt の活性化のみならず、 AhRのリガンド化合物についても、 その AhR- Arnt活性化能を評価できることが確認できた。  Therefore, it was confirmed that the SW-ELISA method of the present invention can evaluate not only the activation of AhR-Arnt by dioxins but also the AhR-Arnt activating ability of AhR ligand compounds.
実施例 1 0 ：細胞系における野菜抽出物の AhR- Arnt活性ィ匕抑制効果の検 討 Example 10: Examination of AhR-Arnt activity inhibition effect of vegetable extract in cell line
細胞系での SW-ELISA法の使用例として、 野菜抽出物の AhR- Arnt活性 化に対する抑制効果の評価を行った。  As an example of using the SW-ELISA method in a cell line, the inhibitory effect of vegetable extract on AhR-Arnt activation was evaluated.
0. 05%酢酸の存在下で 90%エタノールを用いるアルコール抽出により 得たモロヘイヤ抽出物 1または 2 mg/mlを Hepa- lclc7細胞に 20分、 2 時間おょぴ 24時間含浸させて前処理し、 次いでこれに 500pM TCDDを 2 時間作用させて、 核抽出液中の活性化 AhR-Arntを抗 Arnt抗体により検 出した。  Pretreatment was performed by impregnating Hepa-lclc7 cells with 1 or 2 mg / ml of Morohaea extract obtained by alcohol extraction using 90% ethanol in the presence of 0.05% acetic acid for 20 minutes, 2 hours, and 24 hours. Then, 500 pM TCDD was allowed to act on this for 2 hours, and activated AhR-Arnt in the nuclear extract was detected with an anti-Arnt antibody.
500pM TCDDの吸光度を 100%、 DMS0 (陰性コント口ール）を 0%として、 それぞれの吸光度から活性化 AhR- Arnt を％表示（平均値土標準偏差 (n=3) )したところ、 モロヘイヤ抽出物は、 2または 24時間前処理を行う ことにより、 500 pM TCDDが誘導する AhR- Arnt活性化を 30〜50%抑制す ることがわかった（図 1 3 )。 Assuming that the absorbance of 500pM TCDD is 100% and DMS0 (negative control) is 0%, Activated AhR-Arnt was expressed in% (mean soil standard deviation (n = 3)) from each absorbance. The Morohea extract was pre-treated for 2 or 24 hours, and AhR induced by 500 pM TCDD -It was found to inhibit Arnt activation by 30-50% (Fig. 13).
次に、 カポチヤ、 ニンジンおよびパセリについても同様に試験した。 各野菜抽出物は 2 mg/mlを用い、 前処理は 20分あるいは 2時間（モロ ヘイャについては、 さらに 24時間）行った。  Next, capochia, carrot and parsley were similarly tested. Each vegetable extract was used at 2 mg / ml, and pretreatment was performed for 20 minutes or 2 hours (24 hours for Morroja).
カポチヤ、 ニンジン、 パセリの抽出物が 500 pM TCDDによる AhR- Arnt 活性化を 20%程度しか抑制できなかったのに対して、 モロヘイヤ抽出物 は 2時間前処理することで約 50%、 24時間前処理では約 75%抑制し、 4 種の野菜の中では最も抑制効果が強いことがわかった（図 1 4 )。  Capochia, carrot, and parsley extracts could only suppress AhR-Arnt activation by 500 pM TCDD by about 20%, whereas Moroheiya extract was pre-treated for 2 hours, about 50%, 24 hours before The treatment suppressed about 75%, and it was found that the suppression effect was the strongest among the four kinds of vegetables (Fig. 14).
実施例 1 1 ：動物における SW - ELISA法の適用 Example 11 1: Application of SW-ELISA method in animals
本発明の方法の適用性をヒトの体内により近い環境で評価するために、 実験動物を用いて SW- ELISA法を行った。  In order to evaluate the applicability of the method of the present invention in an environment closer to the human body, a SW-ELISA method was performed using experimental animals.
SD系雄性ラット（7週齢）に、 コーンオイルに懸濁した 3- MCを 10 mg/kg 体重となるように腹腔内投与し、 AhR-Arntの活性化を誘導した。 0時間 処理としては、 溶媒であるコーンオイルのみを投与した。  To male SD rats (7 weeks old), 3-MC suspended in corn oil was intraperitoneally administered at a concentration of 10 mg / kg body weight to induce AhR-Arnt activation. As the 0-hour treatment, only corn oil as a solvent was administered.
3-MC投与 1、 3、 6、 12 時間後に肝臓を摘出し、 核抽出物に含まれる AhR- Arnt の活性化の程度を抗 AhR抗体おょぴ抗 Arnt 抗体を用いた SW-ELISA法とゲルシフトアツセィ法により検出した（図 1 5 )。  At 1, 3, 6, and 12 hours after 3-MC administration, the liver was removed, and the degree of activation of AhR-Arnt contained in the nuclear extract was determined by SW-ELISA using an anti-AhR antibody and an anti-Arnt antibody. It was detected by the gel shift assay (Fig. 15).
SW - ELISA法とゲルシフトアツセィ法の結果はほぼ一致したものであり、 実験動物を用いた試験でも SW- ELISA法が適用できることが確認できた。 次に、 雄性モルモット（7週齢）に TCDD (1 g/kg体重）またはコーンォ ィル (陰性コントロール)を腹腔内投与し、 1および 3週間後の肝臓の核抽 出物中に含まれる活性化 AhR - Arntを、 抗 AhR抗体を用いる SW-ELISA法 とゲルシフトアツセィ法により検出した。 この際、 SW - ELISA法では、 陰 性コントロールに対する TCDDの吸光度の比を平均値土標準誤差 (n=3)で 評価した（図 1 6 )。 The results of the SW-ELISA method and the gel shift assay method were almost the same, and it was confirmed that the SW-ELISA method was applicable to the test using experimental animals. Next, male guinea pigs (7 weeks old) were intraperitoneally administered TCDD (1 g / kg body weight) or corn follicle (negative control), and the activity contained in liver nuclear extracts 1 and 3 weeks later AhR-Arnt, SW-ELISA using anti-AhR antibody And the gel shift assay. At this time, in the SW-ELISA method, the ratio of the absorbance of TCDD to the negative control was evaluated based on the average value of the standard error of the soil (n = 3) (Fig. 16).
その結果、 SW - ELISA法とゲルシフトアツセィ法ともに、 1週間おょぴ 3 週間後に TCDD投与動物で活性化 AhR-Arntを検出することができた。 実施例 1 2 ：動物における液状食品の AhR-Arnt活性ィ匕抑制効果の検討 動物を用いた場合の AhR - Arnt活性化抑制効果の評価について検討した。 As a result, activated AhR-Arnt could be detected in the TCDD-administered animals 1 week to 3 weeks after both the SW-ELISA method and the gel shift assay method. Example 12: Examination of AhR-Arnt activity suppression effect of liquid food in animals Evaluation of AhR-Arnt activation suppression effect when using animals was examined.
SD系雄性ラット（5週齢）に水、 市販の緑茶または紅茶を 1週間自由摂 取させた後、 3-MCを実施例 1 1と同様に投与し、投与から 24時間後の肝 臓の核抽出物に含まれる活性化 AhR- Arnt を、 抗 Arnt 抗体を用いた SW - ELISA法とゲルシフトアツセィ法により検出した（図 1 7 )。 Male SD rats (5 weeks old) were allowed to freely take water, commercially available green tea or black tea for 1 week, and then 3-MC was administered in the same manner as in Example 11; Activated AhR-Arnt contained in the nuclear extract was detected by SW-ELISA using anti-Arnt antibody and gel shift assay (Fig. 17).
水摂取群で 3- MCにより AhR - Arnt活性ィヒが誘導されたのに対して、 緑 茶およぴ紅茶摂取群では活性化が低下していた。  AhR-Arnt activity was induced by 3-MC in the water intake group, whereas activation was reduced in the green tea and black tea intake groups.
また、 SW- ELISA法とゲルシフトアツセィ法の結果は、 ほぼ一致したも のであり、 実験動物を用いた抑制効果の試験においても、 本発明の SW-ELISA法が適用できることがわかった。 本発明によれば、 従来法の実施に必要とされていた特殊な実験設備お ょぴ煩雑な処理を要しない、 簡便、 迅速かつ高精度に多検体を同時に評 価できる、 DNA結合性タンパク質の活性測定方法、該タンパク質の活性化 剤ならぴに活性ィ匕抑制剤のスクリーニング方法おょぴ DNA結合性タンパ ク質の活性測定用キットが提供される。  Further, the results of the SW-ELISA method and the gel shift assay method were almost the same, and it was found that the SW-ELISA method of the present invention can be applied to the test of the inhibitory effect using experimental animals. According to the present invention, there is no need for special experimental equipment required for the implementation of the conventional method and complicated processing, and it is possible to evaluate multiple samples simultaneously, simply, quickly and with high accuracy. The present invention provides a method for measuring an activity, a method for screening an activator for an activator of the protein, and a kit for measuring an activity of a DNA-binding protein.

Claims

請求の範囲 The scope of the claims
1 . 次の工程： 1. Next steps:
1) タンパク質含有試料をタンパク質活性化物質で処理して DNA結合能 を有する活性ィヒタンパク質含有試料を得て、  1) treating a protein-containing sample with a protein activator to obtain a sample containing an active protein having DNA binding ability;
2) 担体に抗ハプテン抗体を固相化し、  2) Immobilize anti-hapten antibody on the carrier,
3) ハプテン化 DNAプロ一ブを抗ハプテン抗体に結合させ、  3) binding the haptenized DNA probe to the anti-hapten antibody,
4) 1)の活性化タンパク質含有試料を 3)のハプテン化 DNAプローブと反 応させて、活性化タンパク質/ハプテン化 DNAプ口ーブの複合体を形成し、 5) 特異抗体を加えて、 複合体を特異的に認識させ、  4) Reacting the activated protein-containing sample of 1) with the haptenated DNA probe of 3) to form an activated protein / haptenated DNA probe complex, 5) adding a specific antibody, Specific recognition of the complex,
6) 発色用酵素標識二次抗体を加え、  6) Add enzyme-labeled secondary antibody for color development,
7) 発色試薬を加えて発色反応を生じさせ、 発色の度合いを吸光度によ り測定し、  7) Add a coloring reagent to cause a coloring reaction, measure the degree of coloring by absorbance,
8) 非活性化タンパク質含有試料について測定される吸光度との比較か ら、 タンパク質の活性レベルを測定すること 力 らなる、 DNA結合性タンパク質の活性測定方法。  8) A method for measuring the activity of a DNA-binding protein, which comprises measuring the activity level of a protein by comparing the absorbance measured with a sample containing a non-activated protein.
2 . 6)の工程で、 ピオチンおょぴストレプトアビジンが二段階に分け て用いられる請求項 1に記載の方法。  2. The method according to claim 1, wherein in step 2.6), biotin and streptavidin are used in two stages.
3 . タンパク質含有試料がダイォキシン類の毒性発現に関与するタン パク質を含み、 タンパク質活性ィヒ物質がダイォキシン類または多環性芳 香族炭化水素である請求項 1または 2に記載の方法。  3. The method according to claim 1 or 2, wherein the protein-containing sample contains a protein involved in the expression of toxicity of dioxins, and the protein active substance is dioxins or polycyclic aromatic hydrocarbons.
4. タンパク質活性ィ匕物質が、 2, 3， 7, 8 -テトラクロ口ジベンゾ" ダイ ォキシン、 3-メチルコランスレン、 インディゴ、 インディルビン及ぴィ ソィンディゴからなる群から選択される請求項 1〜 3のいずれか 1つに 記載の方法。  4. The protein-active substance according to claims 1 to 3, wherein the substance is selected from the group consisting of 2,3,7,8-tetraclodibenzodioxin, 3-methylcholanthrene, indigo, indirubin and disindigo. The method according to any one of the above.
5 . タンパク質含有試料が AhR- Arntを含み、ハプテン化 DNAプローブ が TNGCGTG(Nは任意の塩基）の 7塩基を含み、 かつ特異抗体が Arntタン パク質の C末端部分または AhRタンパク質の N末端部分を構成するぺプ チドを合成し免疫感作にて得られる抗体である請求項 1〜 4のいずれか 1つに記載の方法。 5. The protein-containing sample contains AhR-Arnt, the haptenized DNA probe contains 7 bases of TNGCGTG (N is any base), and the specific antibody is Arnt protein. The method according to any one of claims 1 to 4, which is an antibody obtained by immunization by synthesizing a peptide constituting the C-terminal part of the protein or the N-terminal part of the AhR protein.
6. 活性化タンパク質含有試料中のタンパク質濃度が 4〜30mg/mlであ る請求項 3〜 5のいずれか 1つに記載の方法。  6. The method according to any one of claims 3 to 5, wherein the protein concentration in the activated protein-containing sample is 4 to 30 mg / ml.
7 . 請求項 1に記載される 1)の工程で、 タンパク質含有試料が、 タン パク質活性化物質での処理の前もしくは後に、 タンパク質活性化物質と は異なる被験化学物質を用いて処理されるか、 または被験化学物質とタ ンパク質活性化物質を同時に用いて処理されることにより、 被験化学物 質の活性ィヒ抑制作用を検定することからなる、 タンパク質の活性化抑制 剤のスクリーニング方法。  7. In the step (1) according to claim 1, the protein-containing sample is treated with a test chemical different from the protein activator before or after the treatment with the protein activator. Or a method for screening a protein activation inhibitor, which comprises assaying the activity of a test chemical substance for suppressing the activity thereof by simultaneously treating the test chemical substance and a protein activator.
8 . 請求項 1に記載される 1)の工程で、 タンパク質含有試料が、 タン パク質活性化物質およぴ該物質とは異なる被験化学物質それぞれを用レ、 て個別に処理され、 両者を比較することにより、 被験化学物質の活性化 作用を検定することからなる、 タンパク質の活性化剤のスクリーニング 方法。  8. In the step 1) according to claim 1, the protein-containing sample is treated separately using a protein activator and a test chemical different from the protein activator, and both are treated. A method for screening a protein activator, comprising assaying the activating effect of a test chemical by comparing.
9 . 抗ハプテン抗体を固相化した担体、 ハプテン化 DNAプローブ、 プ ローブと活性化タンパク質との複合体を特異的に認識する特異抗体、 検 出用試薬および発色試薬からなる、 DNA結合性タンパク質の活性測定用キ ッ卜。  9. DNA-binding protein consisting of a carrier on which anti-hapten antibody is immobilized, a haptenated DNA probe, a specific antibody that specifically recognizes a complex between the probe and the activating protein, a detection reagent, and a coloring reagent Activity measurement kit.
1 0 . 担体が、 抗ハプテン抗体にハプテン化 DNAプローブを結合してな る、 請求項 9に記載のキット。  10. The kit according to claim 9, wherein the carrier is formed by binding a haptenated DNA probe to an anti-hapten antibody.
1 1 . 検出用試薬として、 ビォチン化二次抗体含有試薬およびストレブ トァビジン含有試薬が含まれる請求項 9または 1 0に記載のキット。  11. The kit according to claim 9 or 10, wherein the detection reagent includes a reagent containing a biotinylated secondary antibody and a reagent containing streptavidin.
1 2 . 活性化タンパク質がダイォキシン類または多環性芳香族炭化水素 を用いて得られる、 ダイォキシン毒性の発現に関与するタンパク質の活 性を測定するための請求項 9〜 1 1のいずれか 1つに記載のキット。1 2. Activating protein is dioxin or polycyclic aromatic hydrocarbon The kit according to any one of claims 9 to 11, which is used for measuring the activity of a protein involved in the expression of dioxin toxicity, obtained using the kit.
1 3 . 活性化タンパク質が、 2, 3, 7, 8-テトラクロ口ジベンゾ- -ダイォキ シン、 3-メチノレコランスレン、 インディゴ、 インディノレビン及びイソィ ンディゴからなる群から選択される少なくとも 1つを用いて得られる、 請求項 9〜1 2のいずれか 1つに記載のキット。 1 3. The activating protein is at least one selected from the group consisting of 2,3,7,8-tetraclomouth dibenzo-dioxin, 3-methinorecolanthrene, indigo, indinolevin and isoindigo. The kit according to any one of claims 9 to 12, obtained using the kit.
1 4 . TNGCGTG(Nは任意の塩基）の 7塩基を含むハプテン化 DNAプローブ および Arntタンパク質の C末端部分または AhRタンパク質の N末端部分 を構成するぺプチドを合成し免疫感作にて得られる特異抗体が用いられ る、 AhR- Arnt の活性を測定するための請求項 9〜： 1 3のレ、ずれか 1つに 記載のキット。  14. Haptenized DNA probe containing 7 bases of TNGCGTG (N is any base) and peptide constituting C-terminal part of Arnt protein or N-terminal part of AhR protein are synthesized and obtained by immunization. The kit according to any one of claims 9 to 13, wherein the antibody is used for measuring AhR-Arnt activity.
PCT/JP2003/002407 2002-03-04 2003-03-03 Method of measuring the activity of dna-binding protein WO2003075013A1 (en)

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
WO2005116079A1 (en) * 2004-05-25 2005-12-08 Research Institute Of Innovative Technology For The Earth Antibody capable of recognizing ligand of allylhydrocarbon receptor and use thereof

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JPH06186232A (en) * 1992-03-03 1994-07-08 Sanyo Chem Ind Ltd Immunoassay method and reagent kit for measuring immunity
JPH0889296A (en) * 1994-09-30 1996-04-09 Toyota Central Res & Dev Lab Inc Method for detecting nucleic acid
JPH08114590A (en) * 1994-09-23 1996-05-07 Boehringer Mannheim Gmbh Qualitative and/or quantitative detecting method for material to be measured
JP2001503130A (en) * 1996-02-14 2001-03-06 パラセルシャン・インコーポレイテッド Detection of dioxin-like compounds by detection of modified Ah receptor / ARNT complex

Patent Citations (4)

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Publication number Priority date Publication date Assignee Title
JPH06186232A (en) * 1992-03-03 1994-07-08 Sanyo Chem Ind Ltd Immunoassay method and reagent kit for measuring immunity
JPH08114590A (en) * 1994-09-23 1996-05-07 Boehringer Mannheim Gmbh Qualitative and/or quantitative detecting method for material to be measured
JPH0889296A (en) * 1994-09-30 1996-04-09 Toyota Central Res & Dev Lab Inc Method for detecting nucleic acid
JP2001503130A (en) * 1996-02-14 2001-03-06 パラセルシャン・インコーポレイテッド Detection of dioxin-like compounds by detection of modified Ah receptor / ARNT complex

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005116079A1 (en) * 2004-05-25 2005-12-08 Research Institute Of Innovative Technology For The Earth Antibody capable of recognizing ligand of allylhydrocarbon receptor and use thereof

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