WO2012100599A1 - Utilisation de la peroxyrédoxine iv dans la préparation de réactifs diagnostiques in vitro pour la polyarthrite rhumatoïde - Google Patents

Utilisation de la peroxyrédoxine iv dans la préparation de réactifs diagnostiques in vitro pour la polyarthrite rhumatoïde Download PDF

Info

Publication number
WO2012100599A1
WO2012100599A1 PCT/CN2011/083637 CN2011083637W WO2012100599A1 WO 2012100599 A1 WO2012100599 A1 WO 2012100599A1 CN 2011083637 W CN2011083637 W CN 2011083637W WO 2012100599 A1 WO2012100599 A1 WO 2012100599A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
elisa kit
peroxide reductase
detecting
reductase
Prior art date
Application number
PCT/CN2011/083637
Other languages
English (en)
Chinese (zh)
Inventor
吴乔
吴玉章
Original Assignee
中国人民解放军第三军医大学
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 中国人民解放军第三军医大学 filed Critical 中国人民解放军第三军医大学
Publication of WO2012100599A1 publication Critical patent/WO2012100599A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • 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
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • 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
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints

Definitions

  • the present invention relates to the field of medical testing, and in particular to an indirect ELISA kit for detecting a peroxide reductase IV antibody in a biological specimen, and a method and use thereof.
  • Rheumatoid arthritis is a systemic autoimmune disease characterized by synovitis as a basic pathological change characterized by chronic destructive joint disease. Most of the conditions are progressive, eventually leading to joint fibrosis or bony rigidity. Severe disability, seriously affecting the quality of life of patients. Rheumatoid arthritis without proper treatment can be delayed and even cause joint deformities. As a self-immune disease, pathogenic antigen-driven autoreactive CD4+ T cell activation is a central pathway in the pathogenesis of rheumatoid arthritis.
  • RA pathological changes of RA gradually progress from cartilage tissue to cartilage to bone. If RA is not effectively treated in the early stage, it will cause joint dysfunction and even labor loss, and cause multiple organs in the body to be involved and endangered. If you can diagnose the condition of RA as soon as possible and treat it early to prevent or delay its development, it will effectively improve the treatment effect of RA and reduce its complications.
  • the detection of high sensitivity and specificity in the early stage of RA is a prerequisite for the early diagnosis and treatment of RA.
  • Rheumatoid factor has the disadvantage of low sensitivity; 53.3% of patients with early RF-negative rheumatoid arthritis are positive for anti-peripheral factor (APF), but APF antigen
  • APF antigen
  • the storage time of the tablets is short (only 1 ⁇ 2 weeks), and the detection stability is low; the positive rate of Sa antibody in rheumatoid arthritis is 40%, the specificity is 98.9%, but the early stage of rheumatoid arthritis is only about 23%. Correlation studies between peroxide reductase IV and rheumatoid arthritis have not been reported.
  • the ELISA kit for detecting a peroxide reductase IV antibody in a biological specimen according to 1, wherein the biological specimen is serum, urine, cells, tissue or plasma.
  • a second object of the present invention is to provide a use of the above kit, which can make up for the technical blank in the field of detection of a peroxide reductase IV antibody, and provides a diagnostic idea for rheumatoid arthritis and other immune diseases, and is particularly suitable for use in Detection of biological specimens.
  • a third object of the present invention is to provide a method for the content of a peroxide reductase IV antibody which is highly sensitive and simple to handle.
  • step a coating the antibody capture agent with an enzyme label, contacting the biological sample to be tested with the antibody capture agent, and keeping warm and using The blocking solution is blocked; the biological specimen is a serum dilution of no more than 200 volume dilutions, and the antibody capture agent is a dilution of less than lg/ml of the peroxide reductase IV.
  • step b The method for detecting the content of a peroxide reductase IV antibody by using an ELISA kit according to 13, in step b, separating the unbound biological specimen by washing the enzyme plate sealed in step a and blocked with a blocking solution, Antigen-antibody complex.
  • step d the substrate is added to the double-antibody complex obtained in step c, and the substrate is protected from light and then terminated.
  • the colorant sample solution is reacted, and the content of the peroxide reductase IV antibody bound to the antibody capture agent is detected by measuring the OD 45Qnm value of the color developing sample solution.
  • the kit can specifically detect the content of the peroxide reductase IV antibody, Low, sensitive; use this kit to detect the content of peroxide reductase IV antibody, and compare with the normal human peroxide reductase IV antibody level, can specifically teach high diagnostic rheumatoid arthritis Therefore, the kit can be prepared into a rheumatoid arthritis diagnostic kit, which is especially suitable for early diagnosis.
  • a human peroxidase reductase IV expression plasmid was constructed, and a recombinant antigen TRX-peroxide reductase IV fusion protein was prepared using the pET prokaryotic expression system.
  • TRX-peroxide reductase IV plasmid and the expressed peroxidase IV antigen was identified by DNA sequencing, relative molecular mass analysis of the fusion protein and Western blotting.
  • Horseradish-labeled goat anti-human IgM was purchased from Lifescience; horseradish-labeled goat anti-mouse IgG (H+L) was purchased from Beijing Zhongshan Biotechnology Co., Ltd.; mouse anti-human peroxide reductase IV monoclonal antibody was purchased from Abeam Company (potency 1:12800);
  • 1% blocking solution Add 10mL blocking solution to 90ml TBS and store at -15 ⁇ -25°C. 0.5% blocking solution: Add 5 mL of blocking solution to 95 mL of TBS and store at -15 ⁇ -25 °C. Serum dilution: The primary antibody was diluted with 0.5% blocking solution. HRP-labeled anti-mouse or human IgG was diluted 1:0.5 times with 0.5% blocking solution to obtain a secondary antibody working solution. Coating solution: 0.85M (pH 9.6) Carbonate buffer: 1.59g Na 2 C0 3 plus 2.93g NaHC0 3 , three distilled water to a constant volume of 1000mL. Washing solution: 0.01 M pH 7.4 PBS Ten Tween-20 (T) The final mass fraction was 0.05%.
  • Substrate solution Phosphate-citrate buffer (pH 5.0 ⁇ 5.5): 0.1M Citric acid 24.3mL Add 25.7ml 0.2M Na 2 HP0 4 ⁇ 12H 2 0 Add o-phenylenediamine 40.0mg, dilute to 50mL , before use, add 0.15mL volume fraction of 30% 3 ⁇ 40 2 . Terminator: 22.2 mL of sulfuric acid was added to 177.8 mL of distilled water.
  • the human peroxide reductase IV coating and the serum to be tested were serially diluted, and the square matrix test was carried out. The results are shown in Table 1.
  • the results of square array titration showed that the concentration of human peroxidase reductase IV was 50 g/ml, and the serum to be tested was diluted 1:200, the serum OD 45 () nm value of rheumatoid arthritis was greater than 1.0, negative positive serum OD 45 () nm ratio (PIN) maximum of 10.45; human peroxide reductase IV coating concentration of 20 g / ml, serum 1: 200 dilution, rheumatoid arthritis serum OD 45 () nm value is greater than 1.0, the ratio of OD 45 () nm in the positive-positive serum (P / N) was 10.08, which was only 0.37 less than the ratio of OD 450nm in the largest negative-positive serum.
  • Table 1 Determination of the optimal working concentration of human peroxidase reductase IV and human test serum. Dilution factor of human test serum. Human peroxide reductase IV coating concentration ( ⁇ g/ml)
  • AD autoimmune diseases
  • RA rheumatoid arthritis
  • NC normal people
  • the average value of OD 45Qmn value is 0.2146 0.2152 0.2336 2.4231 0.1667
  • the 2 g/ul mouse anti-human peroxide reductase IV monoclonal antibody was diluted according to the following concentration gradient (2000 ng/ml, 1000 ng/mK 500 ng/ml, 250 ng/ml, 125 ng/ml, 62.5 ng/ Ml, 31.25 ng/mU 15.625 ng/mK 7.8125 ng/ml), 100 ⁇ L/well was added to the ELISA plate, and each dilution was repeated 3 times.
  • the indirect ELISA method was established according to the established peroxide reductase IV antibody. The results are shown in Table 3 and Figure 2.
  • the OD450nm value was 1.082, which was slightly larger than the blank control value of 0.895, since lOO L was added to each enzyme standard well, therefore, in serum
  • the minimum content of the peroxide-containing reductase IV antibody was determined to be 0.78 ng.
  • the serum OD 45Q values of the above-mentioned early RA patients and normal subjects were analyzed by the GraphPad Prism software for the receiver operating curve (R0C), as shown in Fig. 3, the specific sensitivities were 76.67%, 96.67 %, respectively. .
  • the OD 45Q value of the above-mentioned patients with advanced RA and normal subjects was analyzed by the GraphPad Prism software for the receiver operating curve (R0C), as shown in Fig. 4, the specific sensitivities were 94.12%, 96.67 %, respectively. .

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Pathology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Cette invention concerne une trousse de réactifs ELISA permettant de détecter un anticorps anti-peroxyrédoxine IV dans des échantillons biologiques, comprenant un agent de capture d'anticorps, un anticorps secondaire marqué par une enzyme et un substrat. L'invention concerne également des applications de ladite trousse pour détecter des volumes d'anticorps anti-peroxyrédoxine IV et la polyarthrite rhumatoïde.
PCT/CN2011/083637 2011-01-24 2011-12-07 Utilisation de la peroxyrédoxine iv dans la préparation de réactifs diagnostiques in vitro pour la polyarthrite rhumatoïde WO2012100599A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201110025635.3 2011-01-24
CN201110025635.3A CN102346186B (zh) 2011-01-24 2011-01-24 用于检测生物标本中的过氧化物还原酶ⅳ抗体的elisa试剂盒及其方法与运用

Publications (1)

Publication Number Publication Date
WO2012100599A1 true WO2012100599A1 (fr) 2012-08-02

Family

ID=45545055

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2011/083637 WO2012100599A1 (fr) 2011-01-24 2011-12-07 Utilisation de la peroxyrédoxine iv dans la préparation de réactifs diagnostiques in vitro pour la polyarthrite rhumatoïde

Country Status (2)

Country Link
CN (1) CN102346186B (fr)
WO (1) WO2012100599A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110872582A (zh) * 2018-09-01 2020-03-10 哈尔滨工业大学（威海） 一种适冷过氧化物还原酶及其编码基因与应用

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103308674B (zh) * 2012-12-20 2018-04-06 周继蓉 过氧化物还原酶iv的循环免疫复合物及其应用
CN106370855B (zh) * 2016-09-28 2018-02-02 吉林大学 基于BSaBA信号放大系统的绵羊过氧化物氧还酶6双抗夹心ELISA试剂盒

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100451653C (zh) * 2005-05-09 2009-01-14 浙江大学 H7亚型水禽流感病毒血凝素抗体间接elisa检测试剂盒
CN1706866A (zh) * 2005-05-26 2005-12-14 江苏省农业科学院 水稻条纹病毒单克隆抗体及水稻条纹病毒的免疫学检测方法
US20120129187A1 (en) * 2009-06-16 2012-05-24 B.R.A.H.M.S. Gmbh Diagnostical use of peroxiredoxin 4

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BO, GANPING ET AL.: "Differential proteome of human synovial fibroblasts from rheumatoid arthritis patients", ACTA ACADEMIAE MEDICINAE MILITARIS TERTIAE, vol. 30, no. 2, January 2008 (2008-01-01), pages 166 - 169 *
CHANG, X. ET AL.: "Identification of proteins with increased expression in rheumatoid arthritis synovial tissues", J RHEUMATOL., vol. 36, no. 5, 2009, pages 872 - 880 *
IWATA, Y. ET AL.: "Autoantibody against peroxiredoxin I, an antioxidant enzyme, in patients with systemic sclerosis: possible association with oxidative stress", RHEUMATOLOGY, vol. 46, 2007, pages 790 - 795 *
KARASAWA, R. ET AL.: "Autoantibodies to peroxiredoxin I and IV in patients with systemic autoimmune diseases", MICROBIOL IMMUNOL., vol. 49, no. 1, 2005, pages 57 - 65 *
LI, XUEQIU ET AL.: "Development of Indirect ELISA for Peroxiredoxin Antigen of Toxoplasma gondii in Sera of Mice", CHINESE JOURNAL OF BIOLOGICALS, vol. 23, no. 3, March 2010 (2010-03-01), pages 317 - 319 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110872582A (zh) * 2018-09-01 2020-03-10 哈尔滨工业大学（威海） 一种适冷过氧化物还原酶及其编码基因与应用

Also Published As

Publication number Publication date
CN102346186A (zh) 2012-02-08
CN102346186B (zh) 2015-05-27

Similar Documents

Publication Publication Date Title
CN110850100B (zh) 一种检测血清抗Annexin A2-IgG抗体的试剂盒
CN111638332B (zh) 一种新型冠状病毒IgA/IgM/IgG抗体ELISA检测试剂盒
JP2011521216A5 (fr)
CN104764888A (zh) 一种抗环瓜氨酸肽抗体检测试剂盒
WO2012107450A1 (fr) Méthode de diagnostic d'une infection des voies urinaires
AU2010303628B2 (en) A method for diagnosing primary biliary cirrhosis (PBC) using novel autoantigens
RU2019113127A (ru) REP белок в качестве белкового антигена для диагностических анализов
Vejtorp The interference of IgM rheumatoid factor in enzyme-linked immunosorbent assays of rubella IgM and IgG antibodies
Drapała et al. A new human IgG avidity test, using mixtures of recombinant antigens (rROP1, rSAG2, rGRA6), for the diagnosis of difficult-to-identify phases of toxoplasmosis
JP7503505B2 (ja) 自己抗体の直接イムノアッセイ測定法
WO2012100599A1 (fr) Utilisation de la peroxyrédoxine iv dans la préparation de réactifs diagnostiques in vitro pour la polyarthrite rhumatoïde
EP2330420A1 (fr) Procédé de diagnostic et kit de diagnostic pour la dermatomyosite
WO2012100600A1 (fr) Utilisation d'anticorps spécifiques anti-peroxyrédoxine iv dans la préparation de réactifs diagnostiques in vitro pour la polyarthrite rhumatoïde de stade précoce
AU771535B2 (en) Method of diagnosing Sjogren's syndrome
CN108508208B (zh) 抗Kaiso蛋白抗体在制备早期强直性脊柱炎诊断试剂盒中的应用
CN114720691B (zh) 一种检测生物标志物的试剂盒及其制备方法和应用
CN110687285B (zh) 诊断试剂盒及mak16在制备系统性红斑狼疮早期诊断试剂中的应用
Saba et al. Anti-Trypanosoma cruzi cross-reactive antibodies detected at high rate in non-exposed individuals living in non-endemic regions: seroprevalence and association to other viral serologies
JP5093087B2 (ja) ポリエチレングリコールおよび尿素による免疫反応増強方法
JP2014162772A (ja) 抗シトルリン化タンパクヒトＩｇＧ抗体およびその用途
CN113607957A (zh) 一种针对SARS-CoV-2 RBD结构域的特异性中和抗体竞争法ELISA试剂盒
Pyun et al. Performance characteristic of anti-cyclic citrullinated peptide (CCP) assay on Korean rheumatoid arthritis (RA) patients and healthy controls
KR20140132410A (ko) 간섭 펩티드 및 미생물 검출 방법
Bizzaro et al. Autoantibody detection in scleroderma patients. Diagnostic and analytical performances of a new coupled particle light scattering immunoassay
Gurung et al. Development of 316v antibody enzyme-linked immunosorbent assay for detection of paratuberculosis in sheep

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11856856

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11856856

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 11856856

Country of ref document: EP

Kind code of ref document: A1