WO2001055724A1 - Technique de quantification applicable a un anticorps de maladie auto-immune, reactif de quantification et trousse de quantification - Google Patents

Technique de quantification applicable a un anticorps de maladie auto-immune, reactif de quantification et trousse de quantification Download PDF

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Publication number
WO2001055724A1
WO2001055724A1 PCT/JP2000/000394 JP0000394W WO0155724A1 WO 2001055724 A1 WO2001055724 A1 WO 2001055724A1 JP 0000394 W JP0000394 W JP 0000394W WO 0155724 A1 WO0155724 A1 WO 0155724A1
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WO
WIPO (PCT)
Prior art keywords
antibody
quantification
autoimmune disease
reagent
mouse
Prior art date
Application number
PCT/JP2000/000394
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English (en)
Japanese (ja)
Inventor
Tatsuyuki Hachisu
Masaaki Kojima
Tadahiro Kikukawa
Original Assignee
Shibayagi Co., Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shibayagi Co., Ltd filed Critical Shibayagi Co., Ltd
Priority to PCT/JP2000/000394 priority Critical patent/WO2001055724A1/fr
Publication of WO2001055724A1 publication Critical patent/WO2001055724A1/fr

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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9

Definitions

  • the present invention relates to a novel bacteriostatic and bactericidal composition and a method for using the same, and more particularly to a bacteriostatic and bactericidal composition capable of exhibiting a bactericidal action by mixing substances having a bacteriostatic action. It is not only safe to add directly to foods and drinks, but also safe for dishes, kitchen utensils, cleaning around toilets, and antibacterial bottles.
  • the present invention relates to a bacteriostatic and bactericidal composition that can be used and a method for using the same. Background art
  • autoimmune diseases There are a wide variety of autoimmune diseases, the main examples being autoimmune diseases of the thyroid gland, SLE (systemic erythrothematosus), rheumatoid arthritis, glomerulonephritis, autoimmunity Sexual blood disease.
  • SLE systemic erythrothematosus
  • rheumatoid arthritis rheumatoid arthritis
  • glomerulonephritis autoimmunity Sexual blood disease.
  • the same individual ⁇ suppresses the immune response to the autoantigen in the same individual.
  • abnormal lymphocyte tissue hyperplasia occurs, the immune response becomes abnormal, and the autoreactor reacts with the autoantigen.
  • Autoantibodies are detected.
  • Autoimmune diseases are diseases caused by the immune response to autoantigens.
  • the etiology of the autoimmune disease remains unclear, and elucidation of the mechanism is awaited.
  • An object of the present invention is to administer a drug candidate or a functional food candidate to an autoimmune disease animal and quantify the amount of an antibody such as a rheumatoid factor or an autoantibody in the serum of the administered animal. ing.
  • an object of the present invention is to provide an autoimmune disease antibody quantification method capable of quantifying a rheumatoid factor autoantibody in the serum of a laboratory animal.
  • the present invention relates to an autoimmune disease antibody capable of quantifying the amount of an antibody such as a rheumatoid factor or an autoantibody in the serum of an administered animal using a standard antibody curve. It is intended to provide a measurement system.
  • Another object of the present invention is to provide a quantification reagent that can be used in a method for quantifying an autoimmune disease antibody in a laboratory animal.
  • the present invention provides, as one aspect, a method for quantifying an antibody in an autoimmune disease.
  • the method for quantifying an autoimmune disease antibody according to the present invention is characterized by using a reagent for quantifying an autoimmune disease antibody in a mammalian autoimmune disease animal excluding a human.
  • the autoimmune disease antibody quantification reagent in the method for quantifying an autoimmune disease antibody according to the present invention, includes a mouse rheumatoid factor quantification reagent or a mouse autoimmune disease quantification reagent. It is characterized by using a reagent for quantifying the self antibody.
  • the self is characterized in that a reagent for quantifying anti-mouse single-strand (singlestrand) DNA or a reagent for quantifying anti-mouse double-strand (doub 1 estrand) DNA is used as the reagent for quantifying the self-antibody.
  • a drug candidate or a functional food candidate is administered to an autoimmune disease animal, and
  • a standard antibody curve for the amount of autoimmune disease antibodies is created and the antibody values are quantified.
  • the present invention provides a reagent for quantifying an autoimmune disease antibody, which can be used in the method for quantifying an autoimmune disease antibody.
  • a reagent for quantifying a mouse rheumatoid factor or a reagent for quantifying a mouse autoantibody is provided as the reagent for quantifying an autoimmune disease antibody according to the present invention. I have.
  • the reagent for quantifying a mouse rheumatoid factor is a reagent for quantifying a mouse Ig G type rheumatoid factor or a reagent for quantifying a mouse Ig M type rheumatoid factor.
  • the mouse autoantibody quantification reagent is a mouse anti-ssDNA quantification reagent or a mouse anti-ds DNA quantification reagent.
  • the present invention provides a kit for quantifying an autoimmune disease antibody, which can be used in the above-described method for quantifying an autoimmune disease antibody.
  • the kit for quantifying an autoimmune disease antibody according to the present invention comprises an immoglobulin fraction-immobilized plate, and a mouse for detecting a mouse autoantibody.
  • Mouse consisting of a labeled antibody (second antibody), a standard antibody, a labeling substance, a coloring substance, and a coloring reaction terminator Kit for quantifying antibodies for autoimmune diseases, or mouse anti-double-stranded DNA solid phase Plate, mouse It is characterized in that it is a mouse autoantibody quantification kit comprising a labeled antibody for detection of self-antibody (second antibody), a standard antibody, a label-forming substance, and a coloring reaction terminator.
  • the kit for quantifying an antibody for an autoimmune disease is a mouse IgG type rheumatoid factor, a mouse IgM type rheumatoid factor, a mouse anti-ssDNA, a mouse anti-ssDNA. It is an ELISA kit for anti-ds DNA antibody quantification.
  • FIG. 1 shows a standard antibody curve for quantification of mouse IgG type rheumatoid factor and Ig M type rheumatoid factor.
  • FIG. 2 shows a standard antibody curve for quantification of mouse anti-dsDNA.
  • An autoimmune disease antibody quantification method comprises administering a drug candidate or a functional food candidate to an autoimmune disease animal, and measuring the amount of antibody such as rheumatoid factor or autoantibody in the serum of the administered animal. In quantifying the antibody, a standard antibody curve is created and the antibody value is quantified.
  • the experimental animals, experimental reagents, experimental methods, and the like used in the present invention are those conventionally used to produce antibodies by administering drug candidates or functional food candidates to autoimmune disease animals. Since the description does not change, those skilled in the art are familiar with the description, and the description is omitted here to simplify the description of the specification.
  • an antibody for a standard antibody curve is an MRL / ipr mouse at the age of 20 weeks. Mix the sera collected from two or more animals.
  • a standard antibody curve can be created by creating a dilution series for each positive antibody, determining a unit for each dilution standard, measuring each diluted standard antibody with UV, and obtaining its OD value. it can.
  • a standard antibody curve is created by displaying the OD value of the measurement reagent on the vertical axis and the antibody titer (unit display) on the horizontal axis. I can do it.
  • the antibody titer of the sample can be determined from the standard antibody curve, the antibody titer of the sample can be quantified for each sample. This makes it possible to compare pharmacological tests between facilities, and is useful, for example, in the evaluation of rheumatic pharmacological tests.
  • Example 2 Preparation of standard antibody curve for quantification of mouse IgG type R rheumatoid factor and results of cyclophosphamide administration test.
  • tetramethyltilbenzidine ( ⁇ ⁇ +) + substrate chromogen was added in a quantity of 1001 to each well, and reacted at room temperature for 20 minutes.
  • a reaction stop solution (1 ⁇ sulfuric acid) was added to each well at 100 ⁇ l to stop the reaction.
  • the absorbance of the reaction solution in each well was measured at 450 nm / 600 nm to obtain the average value of the amount of IgG type rheumatoid factor in the mouse serum of each group.
  • Cyclophosphamide (code number: 034-12951, a product of Wako Pure Chemical Industries, Ltd.), which is recognized as an anti-inflammatory substance, was administered in MRL / ipr mouse (Nippon Chills River, Inc.). ), 12 animals, subcutaneously administered 25 mg / kg (lmg / 0.5 ml physiological saline) once a week from the age of 8 weeks, and the mouse IgG G type rheumatism at the age of 20 weeks. Factors were quantitatively measured.
  • Example 3 Preparation of standard antibody curve for quantification of mouse Ig M type M rheumatoid factor and results of cyclophosphamide administration test (see Fig. 1).
  • the immobilized 96-ml plate was washed three times with a washing solution (Twin 20). Each sample and 100 ⁇ l of the standard antibody obtained in Example 1 were added to physiological saline, and this solution was added to each well, and the plate was allowed to react at room temperature for a predetermined time. Then, it was washed three times with the washing solution. Next, HRPconjugatted anti-mouse IgM was added to each well at a rate of 100 z1 and reacted at room temperature for a predetermined time. After the reaction, each well was washed three times with a washing solution.
  • TMB tetramethyltilbenzidine + substrate-chromogen
  • a reaction stop solution (1 ⁇ sulfuric acid) was added to each well in an amount of 100 ⁇ l to stop the reaction.
  • the absorbance of the reaction solution in each well was measured at 450 nm / 600 nm, and the average value of the IgM-type rheumatoid factor in mouse serum of each group was obtained.
  • Cyclophosphamide (code number: 034-12951, manufactured by Wako Pure Chemical Industries, Ltd.), which is recognized as an anti-inflammatory substance, was administered in MRL / ipr mice (Japan 12 mice, 25 mg / kg once a week (8 mg ZO. 5 ml saline) administered once a week from the age of 8 weeks. ⁇ Quantitative measurement of gusset factor was performed.
  • Example 4 Preparation of a standard antibody curve for mouse anti-ds DNA quantification (see FIG. 2) A 96-ml plate with immobilized ds DNA fraction was washed three times with a washing solution (Tween 20). 100 ⁇ l of the standard antibody obtained in Example 1 was added to physiological saline, this solution was added to each well, and the plate was allowed to react at room temperature for a predetermined time. After washing three times with the washing solution, HRP conjugated anti-mouse IgM was added to each well in a volume of 100 ⁇ l, and the mixture was reacted at room temperature for a predetermined time.
  • a washing solution Tween 20
  • the ELISA kit for mouse IgG type R rheumatoid factor quantification has the following composition.
  • the mouse anti-dsDNA quantification ELISA kit has the following composition.
  • the anti-mouse I g G (H + L) turbocharger formic serum was purified by P rotei n G, resulting anti-mouse I g G (H + L) turbocharger formic I g G a HRP (Horse La Defense Mesh Perot Oxidase) to give anti-mouse IgG (H + L) goat IgG—HRP.
  • HRP Hase La Defense Mesh Perot Oxidase
  • the method for quantifying an autoimmune disease antibody includes a reagent for quantifying an autoimmune disease antibody in a mammal with an autoimmune disease excluding human, such as mouse IgG G Rheumatoid factor quantification reagents, such as rheumatoid factor quantification reagents or mouse Ig M type rheumatoid factor quantification reagents.
  • a mouse autoantibody quantification reagent such as a mouse anti-ssDNA quantification reagent or a mouse anti-dsDNA quantification reagent
  • the amount of autoimmune disease antibody is determined using a standard antibody curve. This allows for comparative quantification. Therefore, since the amount of autoimmune disease antibody can be quantified, it is possible to eliminate or reduce the fluctuation of the value between laboratories, and objectively judge the pharmacological experiment for autoimmune disease. It has the advantage of being able to do so.
  • the quantification reagents and quantification kits that can be used in the above-described autoimmune disease antibody quantification method have the effects described above, and are simple to use. It is extremely effective for efficient implementation.
  • the autoimmune disease antibody quantification method and the quantification reagent and the quantification kit according to the present invention having the above-mentioned constitution include, for example, ANA antibody, RNP antibody, Sm antibody, Scl-70 antibody, Mitochondrial antibody, neutrophil cytoplasmic antibody, smooth muscle antibody, skeletal muscle antibody, gastric parietal cell antibody, epidermal component autoantibody, pituitary antibody, cardiolipin antibody, 33–80 antibody , Ss—B / La antibody, ⁇ 0-1 Antibody, centromeric antibody, DNA antibody, thyroglobulin antibody, adrenocortical antibody, ribosome antibody, etc. It can be applied to quantitative measurement of autoimmune disease antibodies.

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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Hematology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Rheumatology (AREA)
  • Rehabilitation Therapy (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
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Abstract

Cette invention concerne un système de dosage d'anticorps de maladie auto-immune, qui consiste à administrer un médicament candidat une nourriture fonctionnelle candidate destiné à un animal de laboratoire porteur d'une maladie auto-immune et à exprimer un niveau d'anticorps ( tel qu'un facteur rhumatoïde dans un sérum ou un auto-anticorps) en utilisant une courbe anticorps standard. L'utilisation de ce système de dosage permet de quantifier facilement et efficacement un anticorps ( tel qu'un facteur rhumatoïde dans un sérum ou un auto-anticorps) dans le sérum d'un animal auquel est administré un médicament candidat ou une nourriture fonctionnelle candidate.
PCT/JP2000/000394 2000-01-27 2000-01-27 Technique de quantification applicable a un anticorps de maladie auto-immune, reactif de quantification et trousse de quantification WO2001055724A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102323404A (zh) * 2011-05-18 2012-01-18 高翔 一种自身免疫抗体检测试剂盒及检测方法
CN107525924A (zh) * 2017-08-28 2017-12-29 张庆丰 一种自身免疫肝病抗体检测试剂盒

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS61114165A (ja) * 1984-11-09 1986-05-31 Yamasa Shoyu Co Ltd 免疫複合体の測定法
JPS63255662A (ja) * 1987-04-13 1988-10-21 Nitsusui Seiyaku Kk 免疫複合体の測定法
JPH01296163A (ja) * 1987-12-28 1989-11-29 Univ California ＩｇＧリウマトイド因子試験
JPH01316662A (ja) * 1988-03-30 1989-12-21 Hoechst Japan Ltd 抗核抗体測定用器具の製法
JPH08289780A (ja) * 1995-04-20 1996-11-05 Japan Organo Co Ltd 微生物等の検出方法、この検出方法に用いる抗体、及びこの抗体を産生する新規ハイブリドーマ

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS61114165A (ja) * 1984-11-09 1986-05-31 Yamasa Shoyu Co Ltd 免疫複合体の測定法
JPS63255662A (ja) * 1987-04-13 1988-10-21 Nitsusui Seiyaku Kk 免疫複合体の測定法
JPH01296163A (ja) * 1987-12-28 1989-11-29 Univ California ＩｇＧリウマトイド因子試験
JPH01316662A (ja) * 1988-03-30 1989-12-21 Hoechst Japan Ltd 抗核抗体測定用器具の製法
JPH08289780A (ja) * 1995-04-20 1996-11-05 Japan Organo Co Ltd 微生物等の検出方法、この検出方法に用いる抗体、及びこの抗体を産生する新規ハイブリドーマ

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Masamitsu KANAI ed., "Rinsho Kensaho Teiyou the 30th ed.", Kanehara Shuppan K.K. (1993), page 977. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102323404A (zh) * 2011-05-18 2012-01-18 高翔 一种自身免疫抗体检测试剂盒及检测方法
CN107525924A (zh) * 2017-08-28 2017-12-29 张庆丰 一种自身免疫肝病抗体检测试剂盒

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