JP4704709B2 - Anti-8-hydroxy-2'-deoxyguanosine monoclonal antibody and hybridoma producing the same - Google Patents

Description

  The present invention relates to an anti-8-hydroxy-2′-deoxyguanosine monoclonal antibody and a hybridoma producing the same. The monoclonal antibody of the present invention is 8-hydroxy-2′-deoxyguanosine (hereinafter sometimes referred to as “8-OHdG”), which is one of the oxidative degradation products of DNA, which is used as an indicator of oxidative stress. It is useful for immunoassay.

  Conventionally, 2'-deoxyguanosine (hereinafter referred to as "2dG") has been studied for various effects such as radiation exposure, drug metabolism, adult diseases, oxidative stress due to aging, and environmental pollutants and food additives. Is evaluated using as an index the oxidation to 8-OHdG.

  Conventionally, 8-OHdG in a test sample has been quantified by using a standard sample, separation analysis by thin layer chromatography, high performance liquid chromatography, gas chromatography mass spectrometry and immunoassay using a monoclonal antibody. An analysis was underway.

  The thin-layer chromatography, high-performance liquid chromatography, and gas chromatography mass spectrometer methods are used in the qualitative and quantitative analysis of natural product samples. It is also pointed out that it takes a long time for separation by a column, and that it requires relatively expensive equipment or is expensive due to the contamination of the column, etc. ing.

  As a method for avoiding the above-mentioned various chromatographic problems, it is also known to measure 8-OHdG by immunoassay using an anti-8-OHdG monoclonal antibody (Patent Document 1).

Japanese Patent No. 3091974

  In the test sample, there are various nucleosides that structurally approximate 8-OHdG, including 2dG, which is the source of 8-OHdG. Therefore, in order to accurately measure 8-OHdG, immunoassay is required. The antibody used should have a sufficiently high specificity for 8-OHdG. Known anti-8-OHdG monoclonal antibodies are not always satisfactory in their specificity for 8-OHdG.

  Accordingly, an object of the present invention is to provide an anti-8-OHdG monoclonal antibody having sufficiently high specificity for 8-OHdG and a hybridoma producing the same.

  As a result of diligent research, the inventors of the present application have shown that anti-OH-OHdG monoclonal antibodies produced by hybridomas obtained by fusing rat iliac lymph node cells and mouse myeloma cells immunized with 8-OHdG include 8- The inventors found that there are some having extremely high specificity for OHdG, and succeeded in actually creating such an anti-8-OHdG monoclonal antibody, thereby completing the present invention.

That is, the present invention is produced by a hybridoma obtained by fusing rat iliac lymph node cells immunized with 8-hydroxy-2′-deoxyguanosine and mouse myeloma cells, and 8-hydroxy-2′-deoxy. The 50% inhibitory concentration by guanosine is 1/50 of the 50% inhibitory concentration by each of all nucleosides selected from the group consisting of 2'-deoxyguanosine, 2'-deoxyadenosine, 2'-deoxyinosine, 8-bromoguanosine and guanosine. 1000 or less anti-8-hydroxy-2′-deoxyguanosine monoclonal antibody or antigen-binding fragment thereof, wherein the monoclonal antibody is produced by hybrid cell 1B1 (FERM P-20122) A binding fragment is provided. The present invention also provides a hybridoma that produces the monoclonal antibody of the present invention. The present invention also provides an antigen-antibody reaction between the anti-8-hydroxy-2′-deoxyguanosine monoclonal antibody of the present invention or an antigen-binding fragment thereof and 8-hydroxy-2′-deoxyguanosine in a test sample. Provided is an immunoassay method for 8-hydroxy-2'-deoxyguanosine in a test sample, comprising quantifying 8-hydroxy-2'-deoxyguanosine in the test sample by an immunoassay method used.

  According to the present invention, an anti-8-OHdG monoclonal antibody having a very high specificity for 8-OHdG is provided. The anti-8-OHdG monoclonal antibody of the present invention has extremely high specificity for 8-OHdG. Therefore, when 8-OHdG in a test sample is quantified by immunoassay using the anti-8-OHdG monoclonal antibody of the present invention, Accurately quantify 8-OHdG with little interference from other similar nucleosides present in the sample. In addition, the affinity column in which the anti-8-OHdG monoclonal antibody of the present invention is bound to a carrier can efficiently concentrate 8-OHdG and trace it even at a trace level. It is possible to inspect the influence on the sensor with high sensitivity.

  As described above, the monoclonal antibody of the present invention is produced by a hybridoma obtained by fusing rat iliac lymph node cells immunized with 8-OHdG and mouse myeloma cells. Immunization of rats with 8-OHdG can be performed by conventional methods. Since 8-OHdG itself is a small molecule, if it is administered alone, it may not be able to induce an immune response sufficiently.Therefore, carrier proteins such as KLH (keyhole limpet hemocyanin) and bovine serum albumin (BSA) It is preferable to use the bound one as an immunogen. Immunization itself can be performed by a conventional method using a well-known adjuvant, and specific methods are described in detail in the following Examples.

  Iliac lymph node cells are collected from the immunized rat and fused with mouse myeloma. The fusion step itself can be performed by a well-known electric cell fusion method or the like. Next, whether or not anti-8-OHdG antibody is contained in the culture supernatant of each clone of the fused cells is examined by ELISA or the like. Regarding the antibody in the culture supernatant positive for anti-8-OHdG antibody, the 50% inhibitory concentration by 8-OHdG was 2dG, 2′-deoxyadenosine (hereinafter “2dA”), 2′-deoxyinosine (hereinafter, “2dI”), 8-bromoguanosine (hereinafter referred to as “8BrG”) and guanosine (hereinafter referred to as “G”) are required to be 1/1000 or less of the 50% inhibitory concentration of each nucleoside selected from the group consisting of Screen for satisfactory monoclonal antibodies. As a result of the screening, a hybridoma clone that produces the monoclonal antibody of the present invention can be obtained by selecting a clone that satisfies the requirements of the present invention. The obtained hybridoma can be transplanted into the abdominal cavity of a nude mouse, and a large amount of monoclonal antibody can be collected from ascites.

  The 50% inhibitory concentration by 8-OHdG and the above-mentioned various nucleosides similar thereto is measured as follows. First, the monoclonal antibody is quantified by ELISA or the like in the state where these competitors are not used, and the measured value (in the case of ELISA, the absorbance may be used) is set to 100%. Next, the diluted diluted competitor and the monoclonal antibody are mixed, and the mixed monoclonal antibody is quantified by the same method. In the case of ELISA, the competitor may be added to the ELISA plate first, followed by the monoclonal antibody. If the monoclonal antibody and the competitor react with each other by an antigen-antibody reaction, free monoclonal antibodies will decrease, and the measured value will decrease accordingly. The concentration of the competitive substance at which the measured value is 50% when no competitor is used is the 50% inhibitory concentration by that substance. In the monoclonal antibody of the present invention, 50% inhibition concentration by 8-OHdG is 50% inhibition concentration by dG, 50% inhibition concentration by 2dA, 50% inhibition concentration by 2dI, 50% inhibition concentration by 8BrG, and 50% inhibition by G. It is 1/1000 or less for any concentration. In other words, the monoclonal antibody of the present invention does not substantially cross-react with any of 2dG, 2dA, 2dI, 8BrG and G.

The present invention also provides an antigen-binding fragment of the above-described monoclonal antibody of the present invention. Here, the “antigen-binding fragment” means an antibody that maintains the binding property (antigen-antibody reactivity) of the antibody to the corresponding antigen, such as an Fab fragment or F (ab ′) ₂ fragment of an immunoglobulin. Means a fragment. It is well known that such antigen-binding fragments can also be used for immunoassays. Fab fragments and F (ab ′) ₂ fragments can be obtained by treating monoclonal antibodies with proteolytic enzymes such as papain and pepsin, as is well known. The antigen-binding fragment is not limited to the Fab fragment or the F (ab ′) ₂ fragment, and may be any fragment that maintains the binding property with the corresponding antigen. It may be prepared. In addition, for example, an antibody in which a single chain fragment of variable region (scFv) is expressed in E. coli can be used by genetic engineering techniques. The method for producing scFv is also well known. The hybridoma mRNA produced as described above is extracted, single-stranded cDNA is prepared, and PCR is performed using primers specific to immunoglobulin H chain and L chain for immunization. ScFv by amplifying globulin H chain gene and L chain gene, linking them with a linker, adding an appropriate restriction enzyme site and introducing it into a plasmid vector, transforming E. coli, and recovering scFv from E. coli Can be produced. Such scFv is also included in the “antigen-binding fragment” referred to in the present invention.

  The monoclonal antibody of the present invention is produced by the hybridoma prepared as described above, and the same monoclonal antibody or antigen-binding fragment thereof produced by the hybridoma is prepared by genetic engineering techniques. Even if it was made, it is included in the scope of the present invention.

  The monoclonal antibody or antigen-binding fragment thereof of the present invention can be used for immunoassay for quantification of 8-OHdG. The immunoassay method itself is well known in this field, and the monoclonal antibody or antigen-binding fragment thereof of the present invention can be used in any known immunoassay method. That is, when classified based on the reaction format, there are a sandwich method, a competitive method, an agglutination method, etc., and when classified based on the label, there are enzyme immunoassay, radioimmunoassay, fluorescent immunoassay, etc. It is included in the term “immunoassay”. Reagents necessary for each immunoassay are also well known, and immunoassay can be performed using a normal immunoassay kit except that the monoclonal antibody or antigen-binding fragment thereof is characterized.

  When the monoclonal antibodies of the present invention or antigen-binding fragments thereof are used as the second antibody in the sandwich method, they are often labeled. Labels include fluorescent labels such as fluorescein, FITC, calmodulin, enzyme labels such as β-galactosidase, glucose oxidase, alkaline phosphatase, peroxidase, ferrocene or derivatives thereof, quinones or derivatives thereof, cobalt compounds, etc. It can be illustrated.

  It is also possible to construct an 8-OHdG measurement sensor that electrically measures an antigen-antibody reaction between the monoclonal antibody of the present invention or an antigen-binding fragment thereof and 8-OHdG by a known method. For example, β-galactosidase is bound to the anti-8-OHdG monoclonal antibody of the present invention and held on the conjugate release pad. A sample pad is formed on the upstream side, and a membrane on which 8-OHdG / KLH conjugate is immobilized is formed as a competitive reaction layer on the downstream side. Further, an electrochemical sensor is formed on the downstream side. In the measurement, first, a measurement sample is applied to the sample pad. At that time, the specimen moves to the conjugate release pad located downstream of the specimen pad, and 8-OHdG and the antibody in the specimen undergo an antigen-antibody reaction. Further, the specimen moves to the competitive reaction layer located downstream thereof, but the antigen-antibody reaction product that has undergone the antigen-antibody reaction in the conjugate release pad moves to the electrochemical sensor section. On the other hand, the unreacted antibody undergoes an antigen-antibody reaction with the immobilized antigen (8-OHdG) in the competitive reaction layer and does not migrate to the electrochemical sensor. Therefore, since the 8-OHdG-anti-8-OHdG antibody, which is proportional to the 8-OHdG concentration in the sample, reaches the electrochemical sensor section, para-aminophenyl-β-D-galactopyra, which is a substrate for β-galactosidase, is reached. By maintaining noside (PAPG) on the electrochemical sensor, paraaminophenol is produced, and since the substance is an electrochemically active substance, the oxidation current is measured. Can be converted to -OHdG concentration.

  The monoclonal antibody or antigen-binding fragment thereof of the present invention can be used not only for immunoassay of 8-OHdG but also for affinity chromatography of 8-OHdG by binding to a carrier for affinity chromatography. Affinity chromatography carriers are well known and are commercially available, and a monoclonal antibody can be bound to them by a conventional method. For example, Amino Toyopearl 650M (trade name, manufactured by Tosoh Corporation), which is a commercially available carrier for amino group binding affinity chromatography, is used as a carrier, and an anti-8-OHdG antibody with a ligand attached to the end is immobilized through an appropriate spacer. Or by using the Biotinylation Kit (Sulfo-OSu) (trade name, manufactured by Dojindo) to label the free amino group of the anti-8-OHdG antibody with biotin, and to which streptavidin was previously bound. This can be carried out, for example, by binding an anti-8-OHdG antibody to a column carrier.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

1. Preparation of immunogen
8-OHdG was dissolved in anhydrous dimethyl sulfonyl oxide (DMSO), 1.5 equivalents of succinic anhydride was added, and the mixture was reacted at room temperature for 5 days. After the reaction, HPLC measurement was performed to confirm a plurality of products. Then, a peak containing succinyl-8-hydroxydeoxyguanosine (Suc-8-OHdG) was isolated and purified to obtain Suc-8-OHdG. This Suc-8-OHdG was dissolved in dimethyl sulfoxide (DMSO), and 10 equivalents of N, N-carbonyl diimidazole (CDI) (manufactured by Tokyo Chemical Industry Co., Ltd.) was added thereto and reacted at room temperature for 2 hours. . This reaction solution was added to a BSA (Seikagaku Kogyo) aqueous solution or a KLH (Sigma) aqueous solution, respectively, and allowed to react overnight at room temperature. After the reaction, dialysis was performed. The obtained BSA conjugate was calculated to have about 6 molecules of 8-OHdG bound thereto.

2. Immunization of rats As an antigen for immunization, 150 μg / OH of 8-OHdG / KLH conjugate was administered into rats (WKY / female, 8 weeks old) hind paw footpad with Freund complete adjuvant (FCA). On the 9th day after immunization from the immunized rat, partial blood was collected from the tail vein to obtain antiserum. The antibody titer in the obtained antiserum was confirmed by ELISA.

3. Preparation of hybridoma Rat iliac lymph node cells were collected on the 21st day after immunization and fused with mouse myeloma cells P3U1 by the electric cell fusion method. After the fusion, 8-OHdG-BSA conjugate was immobilized on a plate and screened by ELISA to select wells containing antibody-producing cells. After cloning twice, the final strain was obtained. In the above-mentioned electric cell fusion method, as usual, rat iliac lymph node cells and mouse myeloma cells are mixed, a high frequency electric field is applied, the cells are contacted by dielectric electrophoresis, and then a high voltage pulse is applied. Then, cell fusion was performed. The ELISA method was specifically performed as follows. That is, 8-OHdG-BSA conjugate was placed in a well of a microplate at 5 μg / mL and 100 μL / well and allowed to stand at 4 ° C. overnight to be immobilized. After washing, 200 μL of a blocking agent (Block Ace manufactured by Dainippon Pharmaceutical Co., Ltd.) was added to each well and left at room temperature for 2 hours. After washing, 100 μL of undiluted culture supernatant was placed in a well and allowed to react at room temperature for 1 hour. After washing, 100 μL of horseradish peroxidase (HRP) -labeled anti-rat IgG antibody was added to each well and allowed to react at room temperature for 1 hour. After washing, 100 μL of HRP substrate solution (ο-phenylenediamine (OPDA, Sigma P-8287) was added to each well and allowed to react for 25 minutes at room temperature in the dark, and then 2 N sulfuric acid was added at 100 μL per well. The reaction was stopped by adding at a concentration, and the absorbance at a wavelength of 490 nm was measured.

4). Cross-reactivity Among 144 antibody-producing strains, the reactivity to 8-OHdG was examined by competitive inhibition ELISA. As a result, 20 wells showed reactivity. Here, the competitive inhibition ELISA method is the same as the ELISA method described above, except that 50 μL of an 8-OHdG solution having a concentration of 10, 1, 0.1, 0 μg / mL is added to the well before adding the culture supernatant, and then the undiluted culture top is used. This was done by adding 50 μL of clean. Further, the 20 wells were examined for cross-reactivity with 6 kinds of related compounds (dG, 2dA, 2dI, 8BrG, G and 8-hydroxyguanosine (hereinafter “8-OHG”) by the competitive inhibition ELISA method described above. Six wells were selected from the reactivity to -OHdG and the cross-reactivity to 8-OHG, and the reactivity of the monoclonal antibodies in the selected 6 wells to various nucleosides is shown in Fig. 1. As shown in Fig. 1, these monoclonal antibodies Showed virtually no cross-reactivity with 2dA, 2dG, 2dI, 8BrG and G. That is, as shown in Figure 1, the 50% inhibitory concentration was lower than 10 ng / mL with 8-OHdG In 2dA, 2dG, 2dI, 8BrG and G, the inhibition was only several percent even at a concentration of 10000 ng / mL, and the 50% inhibitory concentration by 8-OHdG was 50 by 2dA, 2dG, 2dI, 8BrG and G, respectively. It was in the range of 1/1000 or less of% inhibitory concentration. 1 to 3, “OD%” on the vertical axis represents the percentage of the measured absorbance with respect to 100 when the horizontal axis is 0. From 6 wells, 3A9 strain and 6C1 Two of the strains were selected and cloned, and the two strains were further screened to determine cross-reactivity for five similar compounds and to react with 8-OHdG and 8-OHG. Two wells were selected based on the cross-reactivity with respect to the 2H6 and 2H2 hybridomas, and screening was performed after the second cloning in the same manner, and finally the 1B1 strain was finally obtained. The 1B1 strain was deposited with the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, and its receipt number is FERM P-20122 (indication for identification given by the depositor: 80HdG 1B1) .

5. Production of anti-8-OHdG antibody derived from mouse ascites The resulting hybridoma 1B1 cells (1 × 10 ⁷ cells / mouse) were transplanted into the abdominal cavity of nude mice (female, 6 weeks old). One week later, additional inoculation (1 × 10 ⁷ cells / mouse) was performed, and ascites was collected from 1 week to 2 weeks later. Purification of monoclonal antibody 1B1 was performed using a Protein G-Sepharose 4 Fast Flow column (trade name). After purification, 1B1 antibody was refrigerated at 4 ° C. Using the obtained monoclonal antibody, competitive inhibition ELISA with 8-OHdG standard solution was performed as described above. As a result, the detection limit concentration of 8-OHdG was 1 ng / ml, and the 50% competitive inhibition concentration was 15 ng / ml.

6). Reactivity with protein The cross-reactivity of monoclonal antibody 1B1 obtained in 5 with various proteins was examined. 8-OHdG / BSA, BSA, ovalbumin (OVA) or KLH was immobilized on a plate, and a dilution series of 8-OHdG was prepared and added, followed by competition inhibition ELISA using monoclonal antibody 1B1 in the same manner as described above. The result is shown in FIG. On the other hand, 8-OHdG / BSA or BSA was immobilized on a plate, and a dilution series of BSA or HAS, which is a competitive protein, was prepared and added, followed by competition ELISA using anti-8-OHdG / BSA antibody as described above. It was. The result is shown in FIG. As is clear from these, the monoclonal antibody 1B1 of the present invention did not substantially cross-react with these proteins.

It is a figure which shows the cross-reactivity of the monoclonal antibody of this invention, the reactivity of 8-OHdG, and other various nucleosides. It is a figure which shows the cross-reactivity of the monoclonal antibody of this invention and various proteins. It is a figure which shows the cross-reactivity of the monoclonal antibody of this invention and various proteins.

Claims (3)

Produced by hybrid cells obtained by fusing rat iliac lymph node cells immunized with 8-hydroxy-2'-deoxyguanosine and mouse myeloma cells, 50% inhibition by 8-hydroxy-2'-deoxyguanosine The concentration is 1/1000 or less of the 50% inhibitory concentration of each of all nucleosides selected from the group consisting of 2'-deoxyguanosine, 2'-deoxyadenosine, 2'-deoxyinosine, 8-bromoguanosine and guanosine. An 8-hydroxy-2′-deoxyguanosine monoclonal antibody or an antigen-binding fragment thereof, wherein the monoclonal antibody is produced by hybrid cell 1B1 (FERM P-20122) . A hybrid cell producing the monoclonal antibody according to claim 1 , wherein the hybrid cell is a hybrid cell 1B1 (FERM P-20122) .   An immunoassay method using an antigen-antibody reaction between the anti-8-hydroxy-2'-deoxyguanosine monoclonal antibody or antigen-binding fragment thereof according to claim 1 and 8-hydroxy-2'-deoxyguanosine in a test sample A method for immunoassay of 8-hydroxy-2'-deoxyguanosine in a test sample, comprising quantifying 8-hydroxy-2'-deoxyguanosine in the test sample by

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