JP2005315859A - CANINE INTERFERON-gamma MEASURING METHOD - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a measuring method capable of accurately and speedily measuring canine interferon-γ. <P>SOLUTION: Canine interferon-γ is measured by an enzyme immunoassay by a sandwich method using a reference canine interferon-γ solution; an insoluble carrier; an anti-canine interferon-γ antibody; an anti-canine interferon-γ antibody bonded with a labeling agent; an enzyme complex; a substrate liquid; a reaction stop liquid; and a coloring agent to be used if needed. It is thereby possible to accurately and speedily measure canine interferon-γ. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for measuring canine interferon-γ by a sandwich method using a monoclonal antibody using an enzyme immunoassay (ELISA method) to measure canine interferon-γ. .

  In recent years, immunological measurement methods have been increasingly used as methods for detecting antigenic substances in body fluids such as serum and measuring the concentration of the antigenic substances. This method is a well-known method, for example, an antigenic substance to be measured by labeling an antigenic substance or antibody with a radioactive substance, forming a complex between the antigenic substance and the antibody, and measuring the labeling component. Is detected and the concentration is measured.

  In addition, useful proteins such as cytokines present in the living body that are promising for the treatment of various diseases have been discovered one after another due to recent advances in gene manipulation techniques, and research on the development of uses as pharmaceuticals has been actively conducted. Among them, interferon (IFN) is a physiologically active substance having an immunomodulating action and an antiviral action, and is attracting attention for its pharmaceutical use. Genetic engineering technology enables mass production of not only human IFN but also animals such as cattle, horses, cats, etc. As a result, research on the development of uses of IFN as a therapeutic agent for viral diseases and tumors is being conducted. There are also things.

  For dogs, α, β, and γ types of IFN have been reported. Dogs are known for numerous tumors such as breast tumors, numerous viral diseases such as parvovirus infection, distemper infection, and numerous skin diseases such as atopic dermatitis. Since it is considered that canine IFN-γ is particularly effective as such a therapeutic agent, the inventors have been developing canine IFN-γ as an animal drug.

  In developing canine IFN-γ as a veterinary drug, naturally it is necessary to measure canine IFN-γ. Conventionally, as a method for measuring canine IFN-γ, measurement of protein concentration using a BCA reagent or the like, measurement of antiviral activity by cytopathic effect, and the like are used.

On the other hand, as a method for measuring canine insulin, Patent Document 1 discloses a method for measuring by a sandwich enzyme immunoassay method.
JP-A-11-626322

  However, in the case of protein concentration measurement, it is necessary to purify canine IFN-γ in the measurement sample with high purity. Further, the measurement of antiviral activity has the disadvantages that the operation is complicated and it takes a long time to measure, and that measurement errors are likely to occur. Therefore, a simpler, faster and more sensitive measurement method is desired as a method for measuring canine IFN-γ.

  In view of such circumstances, the present inventors have intensively studied a method for measuring canine IFN-γ, and as a result, a monoclonal antibody obtained by immunizing canine IFN-γ to a mouse, and a labeling agent-bound canine IFN-γ monoclonal antibody. The present inventors have found that canine IFN-γ can be measured accurately and rapidly by an ELISA method using a sandwich method. Therefore, the present invention is “a method for measuring canine IFN-γ, wherein canine IFN-γ is measured by an ELISA method using a sandwich method using an anti-canine IFN-γ antibody and a labeling agent-bound anti-canine IFN-γ antibody”.

  The method for measuring canine IFN-γ according to the present invention can measure canine IFN-γ more accurately and quickly than the measurement of antiviral activity known as a conventional method for measuring IFN-γ.

  As described above, the method for measuring canine IFN-γ in the present invention is the sandwich ELISA method using an anti-canine IFN-γ antibody and a labeling agent-bound anti-canine IFN-γ antibody. Details of the invention will be described below.

Preparation of Standard Canine IFN-γ Solution The canine IFN-γ standard solution used in the method for measuring canine IFN-γ of the present invention can be prepared by techniques well known to those skilled in the art. For example, canine IFN-γ DNA can be prepared by a method of integrating a silkworm polynuclear virus into a recombinant vector that can be expressed in a silkworm cell and producing it in a silkworm cell (Japanese Patent Laid-Open No. 9-234085). A standard canine IFN-γ solution is prepared using the thus prepared canine IFN-γ. A standard dog IFN-γ solution can be obtained by diluting the stock solution with a buffer.

Production of anti-canine IFN-γ monoclonal antibody The anti-canine IFN-γ monoclonal antibody used in the present invention is prepared according to a conventional method, for example, according to the Kohler-Milstein method (Nature, vol. 256, pp. 495-497, 1975). It can be produced from a fused cell (hybridoma) obtained by cell fusion of a spleen cell of an immunized animal immunized with canine IFN-γ as an immunizing antigen and a myeloma cell derived from the same species. Examples of immunized animals that immunize canine IFN-γ as an immunogen used in the present invention include mice, nude mice, and rats. The immunization amount of the immunogen is appropriately determined depending on the type of immunized animal, the site of immunization, etc. For example, in the case of mice, the immunization amount per mouse contains 0.1 μg to 5 mg of immunogen. It is preferable to make an immunization injection. The immunogen is preferably injected by adding a known adjuvant such as Freund's complete adjuvant or Freund's incomplete adjuvant. After the first immunization of the immunized animals in this way, the immunogen was boosted 2 to 6 times. As in the case of the first immunization, the booster injection is preferably performed by adding and mixing known adjuvants. After the first immunization, the antibody titer in the sera of the immunized animal was repeatedly measured by ELISA or the like. When the antibody titer reached a plateau, the immunogen was dissolved in physiological saline (0.9% sodium chloride aqueous solution). An aqueous solution of the immunogen is injected into the immunized animal for final immunization. After an appropriate time has passed after the final immunization, cells having antibody-producing ability such as spleen cells, lymph node cells or peripheral lymphocytes of the immunized animal are obtained.

  The cells having the antibody-producing ability thus obtained are fused with myeloma cells (myeloma cells) derived from allogeneic mammals. Examples of such myeloma cells include enzyme-deficient cell lines such as hypoxanthine, guanine, phosphoridyl transferase (GPRT), and thymine kinase (TK). This cell fusion can be performed using a fusion promoter such as polyethylene glycol of various molecular weights, liposomes, Sendai virus, or by electrofusion. When the myeloma cell is derived from a GPRT-deficient strain or a TK-deficient cell line, by using a selection medium (HAT medium) containing hypoxanthine, aminopterin, and thymidine, cells having antibody-producing ability, myeloma cells, Only the fused cells (hybridoma) can be selectively cultured and proliferated. By measuring the culture supernatant of the hybridoma thus obtained by immunoassay methods such as ELISA and Western blotting, it was not bound to unmodified or modified canine IFN-γ, Hybridomas that produce antibodies that specifically bind to denatured or modified canine IFN-γ can be selected and used in the present invention by combining this method with known screening methods such as limiting dilution. A monoclonal antibody-producing cell line can be isolated and obtained. This monoclonal antibody-producing cell line is cultured in an appropriate medium such as a serum-free medium, and for example, the monoclonal antibody used in the present invention can be obtained from the culture supernatant by a conventional method.

Production of Labeling Agent-Binding Anti-Canine IFN-γ Antibody Subsequently, the anti-IFN-γ monoclonal antibody separately produced as described above is bound to the labeling agent. The binding method can be carried out according to a conventional method, for example, according to the method described in “Second Life Chemistry Laboratory Lecture 5 Immunobiochemical Research Method” (published by Tokyo Kagaku Dojin: 1986, pages 102-112). Examples of the labeling agent used include enzymes such as β-galactosidase, peroxidase, alkaline phosphatase, and glucose oxidase, coenzymes such as biotin and heme, and prosthetic groups.

Immobilization of anti-canine IFN-γ monoclonal antibody The anti-canine IFN-γ monoclonal antibody obtained as described above is then immobilized on an insoluble carrier to produce a solid phase reagent. Examples of insoluble carriers used include polyvinyl chloride, polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, styrene-divinylbenzene copolymer, styrene-maleic anhydride copolymer, nylon, polyvinyl alcohol, polyacrylamide, and fluorine. Examples thereof include polymers, polymer materials such as crosslinked dextran, polysaccharides, paper, glass, metal, agarose, and combinations thereof. Further, the shape of such an insoluble carrier is not particularly limited, and examples thereof include a plate shape such as a microtiter plate and a disk, a particle shape such as a bead, a tube shape such as a test tube, a fiber shape, a film shape, and a latex particle. Various shapes such as fine particles, cells, and the like can be used, and can be appropriately changed depending on measurement conditions. In addition, methods well known to those skilled in the art can be used for obtaining a solid phase reagent by immobilizing the anti-canine IFN-γ monoclonal antibody on the insoluble carrier.

Generation of antigen-antibody complex by reaction of antigen and solid phase reagent Next, the solid phase reagent prepared as described above is used as a specimen or a standard dog IFN-γ solution and a labeling agent-conjugated anti-canine IFN-γ monoclonal antibody. React. By this reaction, canine IFN-γ as an antigen present in the specimen or standard canine IFN-γ solution is bound to the immobilized reagent labeling agent-bound anti-canine IFN-γ monoclonal antibody, and a sandwich type antigen-antibody complex is obtained. Generate.

Production of antibody-labeling substance complex The antigen-antibody complex produced as described above is further reacted with a labeling substance at a constant temperature for a certain period of time to produce an antibody-labeling substance complex. As the labeling substance, an enzyme, a luminescent substance, a fluorescent substance or the like is generally used. Examples of the enzyme include peroxidase (HRP), alkaline phosphatase, β-D-galactosidase and the like. Examples of the luminescent substance include isolucinol and lucigenin, and examples of the fluorescent substance include fluorescein isothiocyanate and phycobiliprotein.

  When an enzyme is used as a labeling substance, it is preferable to use a substrate in order to measure its activity, but a color former can also be used if necessary. For example, when HRP is used as an enzyme, hydrogen peroxide, peroxidase avidin, or the like is used as a substrate, 2,2′-azinodi [3-ethylbenzthiazolinesulfonic acid] ammonium salt (ABTS) as a color former, 4-Aminoantipyrine, 3,3 ′, 5,5′-tetramethyl-benzidine and the like are preferably used. When alkaline phosphatase is used as the enzyme, it is preferable to use θ-nitrophenyl phosphate as the substrate, and when β-D-galactosidase is used as the enzyme, p- -Nitrophenyl-β-D-galactopyranoside, fluorescein-di- (β-D-galactopyranoside), 4-methylumbelliferyl-β-D-galactopyranoside, etc. Is preferred.

  Next, the substrate solution is reacted with the antibody-labeling substance complex obtained as described above. A reaction terminator is used to stop the reaction after the reaction has been performed for a predetermined time and the reaction has proceeded. As the reaction terminator to be used, for example, an agent known to those skilled in the art such as an acid such as sulfuric acid can be used. The absorbance of the obtained reaction solution is measured at a predetermined wavelength, and the activity of the enzyme as the labeling substance in the antibody-labeling substance complex is measured. For example, a streptavidin complex is allowed to act on the antigen-antibody complex obtained as described above to form an antibody-enzyme complex, and p-nitrophenyl-β-D-galactopyrano is formed on the complex. Β-D-galactosidase activity in the complex can be measured by reacting the side and colorimetrically determining the liberated p-nitrophenol at a wavelength of 415 nm. Similarly, when the peroxidase avidin conjugate is reacted with the complex, the peroxidase activity in the complex is measured by colorimetric determination at 412 nm for θ-phenylenediamine and 450 nm for tetramethylbenzidine. can do.

Preparation of calibration curve of standard canine IFN-γ concentration The standard canine IFN-γ solution concentration was calculated by calculating the canine IFN-γ concentration (ng / ml) on the X-ray (log side) of the logarithmic graph paper and the Y axis, respectively. A standard curve is prepared by plotting the obtained absorbance against the standard canine IFN-γ concentration, and by reading the canine IFN-γ concentration corresponding to the absorbance of the specimen from this standard curve, The IFN-γ concentration can be calculated.

  Therefore, the canine IFN-γ measuring method according to the present invention includes, as described above, a standard canine IFN-γ solution, an insoluble carrier, a labeling agent-conjugated anti-canine IFN-γ antibody, and an enzyme complex as described above. And a substrate solution, a reaction stop solution, and a color former used as necessary. In order to measure canine IFN-γ by the method for measuring canine IFN-γ of the present invention, an antigen-antibody reaction is used. Various techniques for measuring proteins such as IFN-γ using this antigen-antibody reaction have been developed, but such measurement techniques that can be used in the present invention are not particularly limited and are employed for measuring proteins. Any method may be used, and it is particularly preferable to use the enzyme immunization method (EIA method). The EIA method includes a competitive method and a non-competitive method, and any of the measurement methods according to the present invention can be used.

  Next, the ELISA method using the sandwich method used for the measurement method of the present invention will be described. Obviously, the measurement method of the present invention is not limited to the measurement method exemplified below. First, the purified anti-IFN-γ antibody is diluted to a constant volume with a buffer solution, and a certain amount of this antibody solution is added to each well of a 96-well microtiter plate and reacted at a constant temperature for a certain period of time. Remove the antibody solution. Thereafter, a constant amount of a phosphate buffered saline buffer (PBS) containing bovine serum albumin (BSA), for example, is added to each well of the plate, and the mixture is allowed to react at a constant temperature for a certain period of time. A solid phase plate can be obtained. A fixed amount of a sample diluted with PBS or a standard IFN-γ solution is added to each well of the anti-IFN-γ solid-phase plate and reacted at a constant temperature for a fixed time. After completion of the reaction, the solution in the well is removed and washed with a washing solution. Thereafter, a certain amount of a labeling agent-conjugated anti-IFN-γ antibody PBS-containing solution such as a biotin-conjugated anti-IFN-γ antibody is added to each well and reacted at a certain temperature for a certain time. After this reaction is completed, the solution in the well is removed and washed with a washing solution. After washing, constant enzyme solution and substrate solution such as peroxidase avidin conjugate and 3,3 ', 5,5'-tetramethyl-benzidine (TMB) hydrochloride and hydrogen peroxide mixture in each well Add a quantity and react at a constant temperature for a certain time. In order to stop this reaction, after reacting for a certain period of time, a reaction stop solution such as sulfuric acid is added. The absorbance of the reaction solution thus obtained is measured at a predetermined wavelength using a spectrophotometer. By plotting this measurement value on a calibration curve of a standard IFN-γ solution prepared separately, the IFN-γ concentration contained in the sample is measured.

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

Preparation of standard dog IFN-γ solution A standard dog IFN-γ solution was prepared according to the method described in the above literature. The standard dog IFN-γ solution was obtained by diluting the stock solution with PBS to a predetermined concentration.

Preparation of anti-canine IFN-γ antibody Mice were immunized with canine IFN-γ antigen, and monoclonal antibodies were prepared by a conventional method of limiting dilution as described below. Canine IFN-γ antigen was dissolved in sterilized distilled water to prepare an antigen solution having an IFN-γ concentration of 10 mg / ml. An equal amount of Freund's complete adjuvant was mixed with this solution to obtain an oily emulsion. The BALB / c mice (7 weeks old, female) were subcutaneously injected with 0.2 ml each as a first immunization. After the first immunization, booster immunization was performed on the 7th and 16th days, and the antigen solution was injected intraperitoneally at 0.25 mg / 0.2 ml as the final immunization 3 days before cell fusion. Three days after the final immunization, the spleen of the mouse was removed, the spleen cells were mixed with myeloma cells at a ratio of 10: 1, and the cells were fused using 50% polyethylene glycol 4000, and HAT medium was used. Selected using. On day 14 after cell fusion, the activity against canine IFN-γ in the culture supernatant was measured by the EIA method. That is, 200 μl of cell fusion culture solution was added to each well of a 96-well EIA plate in which canine IFN-γ was immobilized at a protein concentration of 100 μl, and reacted at 37 ° C. for 1 hour. After the reaction, the plate was washed and 200 μl of peroxidase-labeled anti-mouse IgG (1: 500) was added. After washing, 200 μL of a mixed solution of 0.1Mθ-phenylenediamine and 0.012% hydrogen peroxide solution was added to each well as a substrate solution and reacted at room temperature for 15 minutes. Thereafter, 4 N sulfuric acid was added to each well in an amount of 50 μL to stop the enzyme reaction. Next, the absorbance value at 492 nm was measured, and a clone positive only for canine IFN-γ was cloned twice by the limiting dilution method. Monoclonal antibodies 1D6 strain, 1H10 strain, 5E1 strain and the like obtained as ascites by this cloning were purified as follows. In other words, each 1 ml of the ascites obtained was diluted 2-fold with 1 ml of PBS, 2 ml of saturated ammonium sulfate was added dropwise, allowed to stand at 4 ° C. for 4 hours, then centrifuged at 3000 rpm for 20 minutes, and the resulting precipitate was obtained. The suspension was dialyzed with 2 ml of PBS, filtered through a 0.2 μm filter, and the immunoglobulin subclass of the purified anti-canine IFN-γ antibody clone was found to be IgG1.

(Example 1)
Preparation of antibody-immobilized plate In each well of a 96-well plate, the anti-canine IFN-γ antibody 1D6 strain obtained as described above was diluted 5000 times with sodium bicarbonate and dispensed in 100 μl aliquots. And it left still at 4 degreeC overnight, and wash | cleaned with the washing | cleaning liquid. Thereafter, 100 μl of PBS containing BSA was added to each well, allowed to stand at room temperature for 1 to 2 hours, and washed with a washing solution.

Generation of antigen-antibody complex 100 μl of canine IFN-γ-containing specimen diluted with PBS was added to each well of a 96-well plate on which the anti-canine IFN-γ antibody 1D6 strain was immobilized as described above and coated. After reacting for 1 hour at room temperature, it was washed with a washing solution. Thereafter, 100 μl of biotin-labeled anti-canine IFN-γ antibody 5E1 strain diluted 2000 times with PBS was added to each well, coated, reacted at room temperature for 1 hour, and washed with a washing solution.

Production of antibody-enzyme complex 100 μl of peroxidase-avidin conjugate was added to each well of the antibody-immobilized plate that had been reacted and washed as described above, and reacted at room temperature for 1 hour. And washed with a washing solution.

Measurement of Absorbance 100 μl of a mixture of TMB hydrochloride and hydrogen peroxide as a color developing solution was added to each well of the antibody-immobilized plate and allowed to react at room temperature for 5 to 30 minutes. Thereafter, 100 μl of a mixture of equal amounts of 6N sulfuric acid and 2N hydrochloric acid was added to each well as a reaction stop solution to stop the reaction. The wavelength of 492 nm was measured for the obtained reaction liquid with a plate reader.

Preparation of calibration curve of standard canine IFN-γ curve The standard canine IFN-γ concentration was calculated by taking the canine IFN-γ concentration on the X-ray (log side) of the semilogarithmic graph paper and the absorbance on the Y axis, respectively. A standard curve was prepared by plotting the obtained absorbance against the γ concentration. FIG. 1 shows a standard curve of standard canine IFN-γ concentration. From this standard curve, the concentration of canine IFN-γ was calculated by reading the canine IFN-γ concentration corresponding to the absorbance of the specimen.

(Example 2)
Preparation of antibody-immobilized plate In each well of a 96-well plate, the anti-canine IFN-γ antibody 1H10 strain obtained as described above was diluted 1000-fold with sodium bicarbonate and dispensed in 100 μl aliquots. And it left still at 4 degreeC overnight, and wash | cleaned with the washing | cleaning liquid. Thereafter, 100 μl of PBS containing BSA was added to each well, allowed to stand at room temperature for 1 to 2 hours, and washed with a washing solution.

Generation of antigen-antibody complex 100 μl of canine IFN-γ-containing specimen diluted with PBS was added to each well of a 96-well plate on which the anti-canine IFN-γ antibody 1H10 strain was immobilized as described above and coated. After reacting for 1 hour at room temperature, it was washed with a washing solution. Thereafter, 100 μl of biotin-labeled anti-canine IFN-γ antibody 5E1 strain diluted 1000 times with PBS was added to each well, coated, reacted at room temperature for 1 hour, and washed with a washing solution.

Production of antibody-enzyme complex 100 μl of peroxidase-avidin conjugate diluted 2000-fold was added to each well of the antibody-immobilized plate that had been reacted and washed as described above. After reacting for a time, it was washed with a washing solution.

Measurement of Absorbance 100 μl of a mixture of TMB hydrochloride and hydrogen peroxide as a color developing solution was added to each well of the antibody-immobilized plate and allowed to react at room temperature for 5 to 30 minutes. Thereafter, 100 μl of a mixture of equal amounts of 6N sulfuric acid and 2N hydrochloric acid was added to each well as a reaction stop solution to stop the reaction. The wavelength of 492 nm was measured for the obtained reaction liquid with a plate reader.

Preparation of calibration curve of standard canine IFN-γ curve The standard canine IFN-γ concentration was calculated by taking the canine IFN-γ concentration on the X-ray (log side) of the semilogarithmic graph paper and the absorbance on the Y axis. A standard curve was prepared by plotting the obtained absorbance against the γ concentration. FIG. 2 shows a standard curve of standard canine IFN-γ concentration. From this standard curve, the concentration of canine IFN-γ was calculated by reading the canine IFN-γ concentration corresponding to the absorbance of the specimen.

1 is a diagram showing a standard curve of a standard canine IFN-γ solution obtained in Example 1. FIG. 2 is a diagram showing a standard curve of a standard canine IFN-γ solution obtained in Example 2. FIG.

Claims (4)

A method for measuring canine interferon-γ, comprising measuring canine interferon-γ by an enzyme immunoassay using a sandwich method. The dog according to claim 1, wherein a standard canine interferon-γ solution, an insoluble carrier, an anti-canine interferon-γ antibody, a labeling agent-bound anti-canine interferon-γ antibody, an enzyme complex, a substrate solution and a reaction stop solution are used. Interferon-γ measurement method. The method for measuring canine interferon-γ according to claim 2, further comprising using a color former. 3. The anti-canine interferon-γ antibody and the labeling agent-conjugated anti-canine interferon-γ antibody, wherein the anti-canine interferon-γ antibody is a monoclonal antibody obtained by immunizing a mouse with canine interferon-γ. 3. The method for measuring canine interferon-γ according to 3.

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