WO2021164109A1 - Kit de dosage immunologique à liaison enzymatique pour détecter la teneur en st2 soluble, et son utilisation - Google Patents

Kit de dosage immunologique à liaison enzymatique pour détecter la teneur en st2 soluble, et son utilisation Download PDF

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Publication number
WO2021164109A1
WO2021164109A1 PCT/CN2020/083144 CN2020083144W WO2021164109A1 WO 2021164109 A1 WO2021164109 A1 WO 2021164109A1 CN 2020083144 W CN2020083144 W CN 2020083144W WO 2021164109 A1 WO2021164109 A1 WO 2021164109A1
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variable region
soluble
chain variable
heavy chain
kit
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PCT/CN2020/083144
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English (en)
Chinese (zh)
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张学光
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苏州旭光科星抗体生物科技有限公司
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Publication of WO2021164109A1 publication Critical patent/WO2021164109A1/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
    • G01N33/68—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction

Definitions

  • the invention belongs to the field of biomedical technology, and specifically relates to an enzyme-linked immunoassay kit for detecting the content of soluble ST2 and its use.
  • Heart failure (Heart Failure, HF) is abbreviated as heart failure. It mainly refers to the failure of the systolic and diastolic function of the heart when the myocardium is damaged due to various reasons. A clinical syndrome of blood stasis and insufficient blood filling in the arterial system, which in turn causes cardiac circulatory disorders. In recent years, with the discovery of IL-33/ST2 signal transduction pathway, growth-stimulating expression gene 2 protein ST2 has attracted people's attention as a new indicator of heart failure detection.
  • ST2 Growth stimulation expression gene 2 protein ST2 (IL1RL1 or IL-1R) is a member of the interleukin 1 receptor family.
  • ST2 related to heart disease is soluble (sST2) and cross-model (ST2L).
  • sST2L cross-model
  • the concentration of sST2 is high, sST2 can competitively bind to IL-33, resulting in ST2L and IL-33.
  • the combination is reduced, thereby blocking the ST2L/IL-33 signal transduction pathway, thereby inhibiting IL-33's anti-cardiomyocyte hypertrophy, anti-atherosclerosis and anti-tissue fibrosis, reducing the function of the heart and increasing the risk of heart disease, such as Myocardial hypertrophy, decreased cardiac ejection fraction and atrial and ventricular dilatation are similar to myocardial remodeling after acute myocardial infarction or severe heart failure.
  • ST2 can also play an important role in the occurrence and development of acute coronary syndrome as an inflammatory marker reflecting the degree of coronary artery vulnerability.
  • one of the objectives of the present invention is to provide an enzyme-linked immunoassay kit for detecting the content of soluble ST2.
  • the second objective of the present invention is the detection method of the kit, and the detection method is mainly to detect the soluble ST2 content.
  • the third object of the present invention is the use of the kit.
  • the first anti-human ST2 monoclonal antibody is the ST2-4F12 antibody, which includes a heavy chain and a light chain.
  • the amino acid sequence of the heavy chain variable region (mVH) of the ST2-4F12 is the same as SEQ ID NO. 1, specifically as follows :
  • amino acid sequence of the light chain variable region (mVL) of ST2-4F12 is the same as SEQ ID NO. 2, and the details are as follows:
  • the second anti-human ST2 monoclonal antibody is an ST2-2D1 antibody, which includes a heavy chain and a light chain, and the amino acid sequence of the heavy chain variable region (mVH) of the ST2-2D1 is the same as SEQ ID NO. 3, specifically as follows :
  • amino acid sequence of the light chain variable region (mVL) of ST2-2D1 is the same as SEQ ID NO. 4, specifically as follows:
  • the kit of the present invention can effectively detect the content of soluble ST2 and play a role in subsequent quality.
  • Figure 1 is a schematic diagram showing the content of soluble ST2 in a sample detected by the kit of the present invention.
  • Figure 2 is a schematic diagram of the correlation of the test results of the commercially available kits.
  • the fusion recombinant protein was used to immunize BALB/c mice four times with an interval of 21 days. After 5-7 days after the fourth immunization, the mouse's orbital blood was taken and the titer was determined. Strengthen immunity.
  • mice 1.3.1 The immunized mice were sacrificed by cervical dislocation and placed in a beaker filled with 75% alcohol for 2 minutes.
  • the method for determining the variable regions of the heavy and light chains of an anti-human ST2 monoclonal antibody includes the following steps:
  • RNA is extracted from the obtained hybridoma cells, and the obtained RNA is reverse transcribed into cDNA by RT-PCR technology.
  • the heavy chain variable region (mVH) and light chain variable region (mVL) of the hybridoma cells were cloned by PCR using specially designed upstream and downstream primers.
  • the heavy chain variable region (mVH) and light chain variable region (mVL) are respectively connected to the cloning vector (pJET cloning vector), and the connected product is transformed into competent bacteria (DH5a), because the pJET cloning vector contains the ampicillin resistance gene (Amp+), so the transformed bacterial solution can be evenly spread on Amp-resistant LB solid medium, placed in an incubator, and cultivated overnight at 37°C.
  • the candidate light and heavy chain variable region sequences are retained, and the light chain and heavy chain variable region sequences that can be connected to the expression vector are cloned again by PCR, and the PCR product is linearly expressed with the double enzyme digestion treatment in advance
  • the vector is ligated, and the ligated product is transformed into DH5a. Since the expression vector contains the kanamycin resistance gene (Kana+), the transformed bacterial solution can be evenly spread on the Kana-resistant LB solid medium, and placed Incubate overnight at 37°C in an incubator.
  • the present invention extracts the variable regions of the heavy and light chains of the antibody from the hybridoma cells expressing the anti-human ST2 monoclonal antibody, retains the candidate light and heavy chain variable region sequences according to the sequencing results, and amplifies and expresses them through PCR
  • the light and heavy chain variable region sequences matched by the vector are ligated with the PCR product and the expression vector pretreated by double enzyme digestion, and then transformed into competent bacteria DH5a.
  • the expression vector linked to the light chain and heavy chain variable region of the target monoclonal antibody was co-transfected into the eukaryotic expression cell line 293, and the supernatant after culture was collected and contained the target antibody, indicating that the obtained heavy chain and light chain variable region sequences were correct.
  • the ELISA kit of the present invention includes the ST2 coating antibody (ST2-2D1) prepared in Example 1 coated on an enzyme-labeled plate, a biotin-labeled ST2 detection antibody (ST2-4F12), and a soluble ST2 protein standard product (R&D Systems), Horseradish Peroxidase (HRP), Sample Diluent, Washing Solution (PBST), Chromogenic Solution (TMB) and Stop Solution.
  • ST2-2D1 the ST2 coating antibody prepared in Example 1 coated on an enzyme-labeled plate
  • ST2-4F12 biotin-labeled ST2 detection antibody
  • R&D Systems soluble ST2 protein standard product
  • HRP Horseradish Peroxidase
  • PBST Washing Solution
  • TMB Chromogenic Solution
  • the coating solution composed of Na2CO3 and NaHCO3 dilutes the coating antibody ST2-2D1 (1ug/ml), and adds 100ul each to the 96-well microtiter plate at 4°C overnight to make the coating antibody tightly bind to the microtiter plate.
  • the kit of the present invention can reach the following indicators:

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  • 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)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Food Science & Technology (AREA)
  • Pathology (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne un kit de dosage immunologique à liaison enzymatique pour détecter la teneur en ST2 soluble, et son utilisation. Le kit comprend deux anticorps monoclonaux anti-ST2 humain, le premier anticorps monoclonal anti-ST2 humain étant un anticorps anti-ST2-4F12 et comprenant une chaîne lourde et une chaîne légère, et la séquence d'acides aminés de la région variable de chaîne lourde (mVH) de ST2-4F12 est identique à SEQ ID NO : 1 et la séquence d'acides aminés de la région variable de chaîne légère (mVL) de ST2-4F12 est identique à SEQ ID NO : 2; le second anticorps monoclonal anti-ST2 humain est un anticorps anti-ST2-2D1 et comprend une chaîne lourde et une chaîne légère, et la séquence d'acides aminés de la région variable de chaîne lourde (mVH) de ST2-2D1 est identique à SEQ ID NO : 3 et la séquence d'acides aminés de la région variable de chaîne légère (mVL) de ST2-2D1 est identique à SEQ ID NO : 4. Le kit peut détecter efficacement la teneur en ST2 soluble et a pour effet de garantir une qualité de détection ultérieure.
PCT/CN2020/083144 2020-02-21 2020-04-03 Kit de dosage immunologique à liaison enzymatique pour détecter la teneur en st2 soluble, et son utilisation WO2021164109A1 (fr)

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CN202010108213.1A CN111308086B (zh) 2020-02-21 2020-02-21 一种检测可溶性st2含量的酶联免疫检测试剂盒及用途
CN202010108213.1 2020-02-21

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CN113980128B (zh) * 2020-07-27 2023-06-16 中元汇吉生物技术股份有限公司 一种抗人甘胆酸单克隆抗体及其应用
CN117024595B (zh) * 2023-10-08 2024-01-26 江西乐成欣生生物技术研究有限责任公司 抗人st2的单克隆抗体及其应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001070817A1 (fr) * 2000-03-21 2001-09-27 Medical & Biological Laboratories Co., Ltd. Anticorps monoclonal et methode et kit d'immunodosage de st2 humaine soluble l'utilisant
US20130210040A1 (en) * 2010-08-04 2013-08-15 Isis Innovation Limited Diagnostic method
CN106556705A (zh) * 2016-11-30 2017-04-05 天津康尔克生物科技有限公司 一种可溶性st2的酶联免疫检测试剂盒及其制备方法
CN107991485A (zh) * 2017-12-12 2018-05-04 北京普恩光德生物科技开发有限公司 用于检测可溶性st2蛋白的试剂盒
CN108152486A (zh) * 2017-12-22 2018-06-12 太原瑞盛生物科技有限公司 一种人可溶性st2的磁微粒化学发光检测试剂盒及制备方法
CN108267594A (zh) * 2017-11-27 2018-07-10 南京天纵易康生物科技股份有限公司 一种基于双分子荧光互补技术的st2检测试剂盒、制备及使用方法
CN110357963A (zh) * 2019-07-31 2019-10-22 北京智仁美博生物科技有限公司 抗人st2抗体及其用途

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SI2556090T1 (sl) * 2010-04-09 2016-07-29 Critical Care Diagnostics, Inc. Protitelesa in testi topnega človeškega ST-2
AR091069A1 (es) * 2012-05-18 2014-12-30 Amgen Inc Proteinas de union a antigeno dirigidas contra el receptor st2
CN105259353B (zh) * 2015-10-15 2017-03-22 北京市心肺血管疾病研究所 一种主动脉瘤和/或主动脉夹层患者血液中可溶性st2的检测试剂盒及检测方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001070817A1 (fr) * 2000-03-21 2001-09-27 Medical & Biological Laboratories Co., Ltd. Anticorps monoclonal et methode et kit d'immunodosage de st2 humaine soluble l'utilisant
US20130210040A1 (en) * 2010-08-04 2013-08-15 Isis Innovation Limited Diagnostic method
CN106556705A (zh) * 2016-11-30 2017-04-05 天津康尔克生物科技有限公司 一种可溶性st2的酶联免疫检测试剂盒及其制备方法
CN108267594A (zh) * 2017-11-27 2018-07-10 南京天纵易康生物科技股份有限公司 一种基于双分子荧光互补技术的st2检测试剂盒、制备及使用方法
CN107991485A (zh) * 2017-12-12 2018-05-04 北京普恩光德生物科技开发有限公司 用于检测可溶性st2蛋白的试剂盒
CN108152486A (zh) * 2017-12-22 2018-06-12 太原瑞盛生物科技有限公司 一种人可溶性st2的磁微粒化学发光检测试剂盒及制备方法
CN110357963A (zh) * 2019-07-31 2019-10-22 北京智仁美博生物科技有限公司 抗人st2抗体及其用途

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KUROIWA K, ET AL.: "CONSTRUCTION OF ELISA SYSTEM TO QUANTIFY HUMAN ST2 PROTEIN IN SERA OF PATIENTS", HYBRIDOMA, LIEBERT, NEW YORK, NY, US, vol. 19, no. 02, 1 January 2000 (2000-01-01), US, pages 151 - 159, XP008013066, ISSN: 0272-457X, DOI: 10.1089/02724570050031194 *

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