WO2023010647A1 - 一种cTnI抗体包被磁珠的保存液及其制备方法 - Google Patents
一种cTnI抗体包被磁珠的保存液及其制备方法 Download PDFInfo
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- WO2023010647A1 WO2023010647A1 PCT/CN2021/117122 CN2021117122W WO2023010647A1 WO 2023010647 A1 WO2023010647 A1 WO 2023010647A1 CN 2021117122 W CN2021117122 W CN 2021117122W WO 2023010647 A1 WO2023010647 A1 WO 2023010647A1
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- preservation solution
- concentration
- coated magnetic
- ctni antibody
- magnetic beads
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- 239000003761 preservation solution Substances 0.000 title claims abstract description 64
- 239000011324 bead Substances 0.000 title claims abstract description 59
- 101100537532 Rattus norvegicus Tnni3 gene Proteins 0.000 title claims abstract 16
- 238000002360 preparation method Methods 0.000 title abstract description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 53
- 239000003223 protective agent Substances 0.000 claims abstract description 35
- 239000000337 buffer salt Substances 0.000 claims abstract description 28
- 239000003755 preservative agent Substances 0.000 claims abstract description 28
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 28
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 28
- 239000004094 surface-active agent Substances 0.000 claims abstract description 28
- 230000002335 preservative effect Effects 0.000 claims abstract description 18
- 229910017053 inorganic salt Inorganic materials 0.000 claims abstract description 15
- 239000002904 solvent Substances 0.000 claims abstract description 10
- 238000000034 method Methods 0.000 claims abstract description 5
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- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 38
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 claims description 38
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 30
- 235000018102 proteins Nutrition 0.000 claims description 27
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- 235000005074 zinc chloride Nutrition 0.000 claims description 19
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 18
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 16
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- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 13
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- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical group [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims description 11
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- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 9
- 239000001103 potassium chloride Substances 0.000 claims description 8
- 235000011164 potassium chloride Nutrition 0.000 claims description 8
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 2
- 108010076119 Caseins Proteins 0.000 claims description 2
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- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 2
- 235000021240 caseins Nutrition 0.000 claims description 2
- 229960005091 chloramphenicol Drugs 0.000 claims description 2
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 claims description 2
- 229920000159 gelatin Polymers 0.000 claims description 2
- 239000008273 gelatin Substances 0.000 claims description 2
- 235000019322 gelatine Nutrition 0.000 claims description 2
- 235000011852 gelatine desserts Nutrition 0.000 claims description 2
- 229960002518 gentamicin Drugs 0.000 claims description 2
- 235000011147 magnesium chloride Nutrition 0.000 claims description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 2
- 229920000053 polysorbate 80 Polymers 0.000 claims description 2
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 16
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 238000003860 storage Methods 0.000 abstract description 5
- 238000010923 batch production Methods 0.000 abstract 1
- 102100036859 Troponin I, cardiac muscle Human genes 0.000 description 42
- 101710128251 Troponin I, cardiac muscle Proteins 0.000 description 42
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- 238000012360 testing method Methods 0.000 description 10
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 206010000891 acute myocardial infarction Diseases 0.000 description 3
- 150000001298 alcohols Chemical class 0.000 description 3
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- AFCARXCZXQIEQB-UHFFFAOYSA-N N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CCNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 AFCARXCZXQIEQB-UHFFFAOYSA-N 0.000 description 2
- 102000004903 Troponin Human genes 0.000 description 2
- 108090001027 Troponin Proteins 0.000 description 2
- 102000013394 Troponin I Human genes 0.000 description 2
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- 208000029549 Muscle injury Diseases 0.000 description 1
- NIPNSKYNPDTRPC-UHFFFAOYSA-N N-[2-oxo-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 NIPNSKYNPDTRPC-UHFFFAOYSA-N 0.000 description 1
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- 102000013534 Troponin C Human genes 0.000 description 1
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Images
Classifications
-
- 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
- G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4712—Muscle proteins, e.g. myosin, actin, protein
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present application relates to the technical field of detection reagents, in particular to a preservation solution of cTnI antibody-coated magnetic beads and a preparation method thereof.
- Troponin (troponin, Tn) complex is composed of troponin I, troponin T and troponin C, which can regulate muscle contraction.
- Cardiac troponin I (cTnI) is a isoform of troponin I present in cardiomyocytes, which has a high degree of cardiac specificity and can be used to identify clinical situations related to myocardial injury and skeletal muscle injury.
- Acute myocardial infarction is one of the major diseases threatening human life.
- myocardial infarction or ischemia damages myocardial cells, cTnI quickly enters the blood through the myocardial cell membrane. Therefore, rapid, sensitive and accurate determination of cTnI in human blood and its changing trend in the early stage of the disease has important clinical significance for the diagnosis and treatment of acute myocardial infarction.
- cTnI is the preferred marker of myocardial injury.
- the commonly used method for clinical detection is chemiluminescence immunoassay, and magnetic particle chemiluminescence is mainly used at present.
- Magnetic bead-coated antibodies play an important role in chemiluminescent detection reagents, so the stability of magnetic bead-coated antibodies lays a solid foundation for high-quality chemiluminescent reagents.
- the improvement of the stability of magnetic bead-coated antibodies mainly starts from the optimization of the magnetic bead coating process or the optimization of the formula.
- the process of the former is relatively complicated, while the latter usually uses commercial magnetic bead preservation solution, which is generally more expensive, and there is a certain batch-to-batch difference, which affects the performance of the reagent.
- the first object of the present invention is to provide a preservation solution of cTnI antibody-coated magnetic beads;
- the second purpose of the present invention is to provide the preparation method of the above-mentioned cTnI antibody-coated magnetic beads preservation solution;
- the preservation solution of cTnI antibody-coated magnetic beads provided by this application can effectively improve the stability of cTnI antibody-coated magnetic beads during storage and use without adding protective agents such as sugar, alcohol and lipids; especially adding Adding a protective agent can significantly improve reagent stability, sensitivity and linear gradient.
- the cTnI antibody-coated magnetic bead preservation solution provided by the present application has simple components, easy and convenient preparation, low cost, and is convenient for mass production.
- the pH of the preservation solution is 7.0-8.0.
- the pH of the preservation solution is 7.0-8.0.
- the buffer salt is specifically any one of Tris, MES, MOPS, HEPES; and/or,
- the concentration of the buffer salt is 50-60mmol/L.
- the protein is any one or more of bovine serum albumin, casein, and gelatin.
- the protein is bovine serum albumin, and the concentration of the bovine serum albumin is 10-15g/L.
- the surfactant is any one or more of Triton x-100, Triton x-405, Tween 20, Tween 80; and/or,
- the concentration of the surfactant is 1-2g/L.
- the inorganic salt is specifically a combination of sodium chloride, magnesium chloride, and zinc chloride; or, the ionic salt is a combination of potassium chloride, magnesium chloride, and zinc chloride; the concentration of sodium chloride is 0.1-0.3 mol/L; The concentration of magnesium chloride is 0.5-5mmol/L; the concentration of zinc chloride is 0.05-1mmol/L; the concentration of potassium chloride is 0.1-0.3mol/L.
- the concentration of sodium chloride is 0.1-0.2mol/L; the concentration of magnesium chloride is 2.5-5mmol/L; the concentration of zinc chloride is 0.1-0.5mmol/L; the concentration of potassium chloride is 0.1- 0.2mol/L.
- the inorganic salt composition used is: sodium chloride 0.15mol/L, magnesium chloride 5mmol/L, zinc chloride 0.1mmol/L;
- potassium chloride 0.15mol/L magnesium chloride 5mmol/L, zinc chloride 0.1mmol/L;
- potassium chloride 0.15mol/L magnesium chloride 2.5mmol/L, zinc chloride 00.5mmol/L.
- the preservative is specifically any one or more of sodium azide, PC300, chloramphenicol, and gentamicin; and/or,
- the concentration of the preservative is 1-2g/L.
- the pH of the preservation solution is adjusted with an inorganic acid or an inorganic base.
- the inorganic acid is preferably hydrochloric acid
- the inorganic base is preferably sodium hydroxide.
- a preparation method for a cTnI antibody-coated magnetic bead preservation solution comprising the following steps:
- buffer salt 0.5-5g/L
- concentration of protein 20-100mmol/L
- concentration of protein 2-20g/L
- concentration of surfactant is 0.5-5g/L
- concentration of inorganic salt is 0.1-0.5mol/L
- concentration of protective agent ceteareth-25 is 1-10g/L
- concentration of preservatives is 0.5-5g/L; and adjust the pH to 7.0-8.0;
- Constant volume, filter The preparation method of the preservation solution of the cTnI antibody-coated magnetic beads provided in this application does not require the sequence of adding buffer salts, proteins, surfactants, inorganic salts, protective agent ceteareth-25, and preservatives. It is only necessary to add each substance separately and mix well, then add the next one, and finally adjust the pH, constant volume, and filter.
- the preservation solution of cTnI antibody-coated magnetic beads provided by this application can effectively improve the stability of cTnI antibody-coated magnetic beads during storage and use without adding protective agents such as sugar, alcohol and lipids; especially adding Adding a protective agent can significantly improve reagent stability, sensitivity and linear gradient.
- the cTnI antibody-coated magnetic bead preservation solution provided by the present application has simple components, easy and convenient preparation, low cost, and is convenient for mass production.
- Fig. 1 is dose-response curve test result figure in the embodiment of the present invention
- first and second are used for descriptive purposes only, and cannot be interpreted as indicating or implying relative importance or implicitly specifying the quantity of indicated technical features. Thus, a feature defined as “first” and “second” may explicitly or implicitly include one or more of these features. In the description of the present application, the meanings of "plurality” and “several” are two or more, unless otherwise specifically defined.
- a preservation solution for cTnI antibody-coated magnetic beads the solvent is pure water, comprising:
- Buffer salt Tris 50mmol/L
- Protein bovine serum albumin 10g/L
- Inorganic salts including sodium chloride: 0.15mol/L; magnesium chloride: 5.00mmol/L; zinc chloride: 0.10mmol/L;
- Preservatives sodium azide 1g/L, and PC300 1g/L;
- the pH of the preservation solution is 7.4.
- the preparation method of the preservation solution of the above-mentioned cTnI antibody coated magnetic beads comprises the following steps:
- the protective agent ceteareth-25 added in the above examples is CremophorA25; the protective agent added in subsequent examples is all ceteareth-25 (CremophorA25).
- a preservation solution for cTnI antibody-coated magnetic beads the solvent is pure water, comprising:
- Buffer salt Tris 50mmol/L
- Protein bovine serum albumin 15g/L
- Inorganic salts including sodium chloride: 0.15mol/L; magnesium chloride: 2.50mmol/L; zinc chloride: 0.50mmol/L;
- Preservatives sodium azide 1g/L, and PC300 1g/L;
- the pH of the preservation solution is 7.4.
- the preparation method of the preservation solution of the above-mentioned cTnI antibody coated magnetic beads comprises the following steps:
- a preservation solution for cTnI antibody-coated magnetic beads the solvent is pure water, comprising:
- Buffer salt Tris 50mmol/L
- Protein bovine serum albumin 10g/L
- Inorganic salts including sodium chloride: 0.15mmol/L; magnesium chloride: 2.50mmol/L; zinc chloride: 0.50mmol/L;
- Preservatives sodium azide 1g/L, and PC300 1g/L;
- the pH of the preservation solution is 7.4.
- the preparation method of the preservation solution of the above-mentioned cTnI antibody coated magnetic beads comprises the following steps:
- a preservation solution for cTnI antibody-coated magnetic beads the solvent is pure water, comprising:
- Buffer salt Tris 50mmol/L
- Protein bovine serum albumin 10g/L
- Inorganic salts including sodium chloride: 0.15mol/L; magnesium chloride: 2.50mmol/L; zinc chloride: 0.50mmol/L;
- Preservatives sodium azide 1g/L, and PC300 1g/L;
- the pH of the preservation solution is 7.4.
- the preparation method of the preservation solution of the above-mentioned cTnI antibody coated magnetic beads comprises the following steps:
- a preservation solution for cTnI antibody-coated magnetic beads the solvent is pure water, comprising:
- Buffer salt Tris 50mmol/L
- Protein bovine serum albumin 10g/L
- Inorganic salts including sodium chloride: 0.15mol/L; magnesium chloride: 2.50mmol/L; zinc chloride: 0.50mmol/L;
- Preservatives sodium azide 1g/L, and PC300 1g/L;
- Protective agent glycerin 50g/L, sucrose 10g/L, polyethylene glycol 40001g/L;
- the pH of the preservation solution is 7.4.
- the preparation method of the preservation solution of the above-mentioned cTnI antibody coated magnetic beads comprises the following steps:
- a preservation solution for cTnI antibody-coated magnetic beads the solvent is pure water, comprising:
- Buffer salt Tris 50mmol/L
- Protein bovine serum albumin 10g/L
- Inorganic salts including sodium chloride: 0.15mol/L; magnesium chloride: 2.50mmol/L; zinc chloride: 0.50mmol/L;
- Preservatives sodium azide 1g/L, and PC300 1g/L;
- the pH of the preservation solution is 7.4.
- the preparation method of the preservation solution of the above-mentioned cTnI antibody coated magnetic beads comprises the following steps:
- a preservation solution for cTnI antibody-coated magnetic beads the solvent is pure water, comprising:
- Buffer salt Tris 50mmol/L
- Protein bovine serum albumin 10g/L
- Inorganic salts including sodium chloride: 0.15mol/L; magnesium chloride: 2.50mol/L; zinc chloride: 0.50mol/L;
- Preservatives sodium azide 1g/L, and PC300 1g/L;
- the pH of the preservation solution is 7.4.
- the preparation method of the preservation solution of the above-mentioned cTnI antibody coated magnetic beads comprises the following steps:
- the magnetic bead reagent is prepared by adding cTnI antibody-coated magnetic bead mother solution to the above preservation solution.
- the above reagents were divided into two parts, one part was placed at 37°C for accelerated aging for 7 days, and the other part was stored at 2-8°C. After standing for 7 days, use the reagents stored at 2-8°C as the control, and use the two reagents to test the same sample synchronously.
- the test results are as follows:
- the magnetic bead reagent is prepared by adding cTnI antibody-coated magnetic bead mother solution to the above preservation solution, and is combined with an enzyme marker to form a cTnI reagent.
- the 0-concentration sample is tested 20 times, and the blank limit is calculated. The test results are as follows:
- the magnetic bead reagent is prepared by adding cTnI antibody-coated magnetic bead mother solution to the above preservation solution, and the cTnI reagent is composed of enzyme markers, and the linear gradient test is carried out.
- the test results are as follows:
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- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
提供一种cTnI抗体包被磁珠的保存液,溶剂为水,包括:缓冲盐;蛋白质;表面活性剂;无机盐;保护剂鲸蜡硬脂醇聚醚-25;防腐剂;所述保存液的pH为7.0-8.0。还提供一种cTnI抗体包被磁珠的保存液的制备方法。提供的cTnI抗体包被磁珠的保存液,能有效地提高cTnI抗体包被磁珠在储存和使用过程中的稳定性;尤其是加入了保护剂,可显著提高试剂稳定性、灵敏度和线性梯度。并且提供的cTnI抗体包被磁珠的保存液,组份简单、制备简便、廉价、便于实现大批量生产。
Description
本申请要求于2021年08月06日提交中国专利局、申请号为202110905348.5、发明名称为“一种cTnI抗体包被磁珠的保存液及其制备方法”的中国专利申请的优先权,其全部内容通过引用结合在本申请中。
本申请涉及检测试剂技术领域,特别是涉及一种cTnI抗体包被磁珠的保存液及其制备方法。
肌钙蛋白(troponin,Tn)复合物由肌钙蛋白I、肌钙蛋白T和肌钙蛋白C组成,能调节肌肉收缩。心肌肌钙蛋白I(cardiactroponin I,cTnI)是存在于心肌细胞中的一种肌钙蛋白I异构体,具有高度的心肌特异性,可用于鉴别心肌损伤和骨骼肌损伤相关的临床情形。
急性心肌梗死(Acute Myocardial Infarction,AMI)是目前威胁人类生命的主要疾病之一。当心肌梗死或缺血而使心肌细胞受损时,cTnI迅速透过心肌细胞膜进入血液。因此,在发病初期,快速、灵敏且准确地测定人血中的cTnI及其变化趋势,对于急性心肌梗死的诊断和治疗有着重要的临床意义。
cTnI作为心肌损伤的首选标志物,目前临床检测常用的方法是化学发光免疫分析法,且目前主要采用磁微粒化学发光法。磁珠包被抗体在化学发光检测试剂中担任重要角色,所以磁珠包被抗体的稳定性为高质量的化学发光试剂奠定坚实的基础。
目前,提高磁珠包被抗体稳定性,主要是从磁珠包被工艺的优化,或者配方的优化两个方面入手。但前者工艺比较复杂,而后者通常选用商业化的磁珠保存液,一般价格比较昂贵,且存在一定的批间差,影响试剂性能。
因此,如何提供一种提高cTnI抗体包被磁珠使用及储存稳定性的保存液,提高保存稳定性、减少批间差异的同时能够降低成本,是本领域技术人员亟待解决的技术问题。
发明内容
为解决上述技术问题,本发明的第一个目的为提供一种cTnI抗体包被磁珠的保存液;本发明的第二个目的为提供上述cTnI抗体包被磁珠的保存液的制备方法;本申请提供的cTnI抗体包被磁珠的保存液,能有效地提高cTnI抗体包被磁珠在储存和使用过程中的稳定性,而无需添加糖、醇以及脂类等保护剂;尤其是加入了保护剂,可以显著提高试剂稳定性、灵敏度和线性梯度。并且本申请提供的cTnI抗体包被磁珠的保存液,组份简单、制备简便、廉价、便于实现大批量生产。
本发明提供的技术方案如下:
一种cTnI抗体包被磁珠的保存液,溶剂为水,包括:
缓冲盐;
蛋白质;
表面活性剂;
无机盐;
保护剂鲸蜡硬脂醇聚醚-25;
防腐剂;
所述保存液的pH为7.0-8.0。
优选地,包括:
缓冲盐 20-100mmol/L;
蛋白质 2-20g/L;
表面活性剂 0.5-5g/L;
无机盐 0.1-0.5mol/L;
保护剂鲸蜡硬脂醇聚醚-25 1-10g/L;
防腐剂 0.5-5g/L;
所述保存液的pH为7.0-8.0。
优选地,所述缓冲盐具体为Tris、MES、MOPS、HEPES中的任意一种;和/或,
所述缓冲盐的浓度为50-60mmol/L。
优选地,所述蛋白质具体为牛血清白蛋白、酪蛋白、明胶蛋白中的任意一 种或多种。
优选地,所述蛋白质为牛血清白蛋白,所述牛血清白蛋白的浓度为10-15g/L。
优选地,所述表面活性剂为曲拉通x-100、曲拉通x-405、吐温20、吐温80中的任意一种或多种;和/或,
所述表面活性剂的浓度为1-2g/L。
优选地,所述无机盐具体为氯化钠、氯化镁、氯化锌的组合;或者,离子盐为氯化钾、氯化镁、氯化锌的组合;氯化钠浓度为0.1~0.3mol/L;氯化镁浓度为0.5~5mmol/L;氯化锌浓度为0.05~1mmol/L;氯化钾浓度为0.1~0.3mol/L。
优选地,所述无机盐组合中,氯化钠浓度为0.1~0.2mol/L;氯化镁浓度为2.5~5mmol/L;氯化锌浓度为0.1~0.5mmol/L;氯化钾浓度为0.1~0.2mol/L。
更优选地,使用的无机盐组合物为:氯化钠0.15mol/L,氯化镁5mmol/L,氯化锌0.1mmol/L;
或,氯化钾0.15mol/L,氯化镁5mmol/L,氯化锌0.1mmol/L;
或,氯化钠0.15mol/L,氯化镁2.5mmol/L,氯化锌00.5mmol/L;
或,氯化钾0.15mol/L,氯化镁2.5mmol/L,氯化锌00.5mmol/L。
优选地,所述防腐剂具体为叠氮钠、PC300、氯霉素、庆大霉素中的任意一种或多种;和/或,
所述防腐剂的浓度为1-2g/L。
优选所述保存液使用无机酸或无机碱调节pH。其中,无机酸优选盐酸,无机碱优选氢氧化钠。
一种cTnI抗体包被磁珠的保存液的制备方法,包括以下步骤:
将缓冲盐、蛋白质、表面活性剂、无机盐、保护剂鲸蜡硬脂醇聚醚-25、防腐剂分别加入到水中混匀,使得缓冲盐的浓度为20-100mmol/L,蛋白质的浓度为2-20g/L;表面活性剂的浓度为0.5-5g/L;无机盐的浓度为0.1-0.5mol/L;保护剂鲸蜡硬脂醇聚醚-25的浓度为1-10g/L,防腐剂的浓度为0.5-5g/L;并调节pH至7.0-8.0;
定容,过滤。本申请提供的cTnI抗体包被磁珠的保存液的制备方法,缓 冲盐、蛋白质、表面活性剂、无机盐、保护剂鲸蜡硬脂醇聚醚-25、防腐剂的加入先后顺序没有要求,只需要每种物质单独加入并混匀后,再加入下一种即可,最后在调节pH、定容、过滤。
本申请提供的cTnI抗体包被磁珠的保存液,能有效地提高cTnI抗体包被磁珠在储存和使用过程中的稳定性,而无需添加糖、醇以及脂类等保护剂;尤其是加入了保护剂,可以显著提高试剂稳定性、灵敏度和线性梯度。并且本申请提供的cTnI抗体包被磁珠的保存液,组份简单、制备简便、廉价、便于实现大批量生产。
为了更清楚地说明本申请实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本申请中记载的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为本发明实施例中剂量反应曲线测试结果图;
为了使本技术领域的人员更好地理解本申请中的技术方案,下面将结合本申请实施例中的附图,对本申请实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本申请一部分实施例,而不是全部的实施例。基于本申请中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都应当属于本申请保护的范围。
需要说明的是,当元件被称为“固定于”或“设置于”另一个元件上,它可以直接在另一个元件上或者间接设置在另一个元件上;当一个元件被称为是“连接于”另一个元件,它可以是直接连接到另一个元件或间接连接至另一个元件上。
需要理解的是,术语“长度”、“宽度”、“上”、下”、“前”、“后”、“左”、“右”、“竖直”、“水平”、“顶”、“底”“内”、“外”等指示的方位或位置关系为基于附图所示的方位或位置关系,仅是为了便于描述本申请和简化描述,而不是指示或 暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作,因此不能理解为对本申请的限制。
此外,术语“第一”、“第二”仅用于描述目的,而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此,限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。在本申请的描述中,“多个”、“若干个”的含义是两个或两个以上,除非另有明确具体的限定。
须知,本说明书附图所绘示的结构、比例、大小等,均仅用以配合说明书所揭示的内容,以供熟悉此技术的人士了解与阅读,并非用以限定本申请可实施的限定条件,故不具技术上的实质意义,任何结构的修饰、比例关系的改变或大小的调整,在不影响本申请所能产生的功效及所能达成的目的下,均应仍落在本申请所揭示的技术内容得能涵盖的范围内。
本申请实施例采用递进的方式撰写。
实施例1
一种cTnI抗体包被磁珠的保存液,溶剂为纯水,包括:
缓冲盐:Tris 50mmol/L;
蛋白质:牛血清白蛋白 10g/L;
表面活性剂:曲拉通x-100 1g/L;
无机盐,包括氯化钠:0.15mol/L;氯化镁:5.00mmol/L;氯化锌:0.10mmol/L;
保护剂:鲸蜡硬脂醇聚醚-25 1g/L;
防腐剂:叠氮钠 1g/L,以及PC300 1g/L;
所述保存液的pH为7.4。
上述cTnI抗体包被磁珠的保存液的制备方法,包括以下步骤:
将缓冲盐加入到水中混匀;
向水中加入无机盐混匀;
向水中加入蛋白质混匀;
向水中加入表面活性剂混匀;
向水中加入保护剂混匀;
向水中加入防腐剂混匀;
调节pH至7.40±0.05;
定容,用0.22um滤芯过滤。
以上实施例中添加的保护剂鲸蜡硬脂醇聚醚-25,即CremophorA25;后续实施例中添加的保护剂均为鲸蜡硬脂醇聚醚-25(CremophorA25)。
实施例2
一种cTnI抗体包被磁珠的保存液,溶剂为纯水,包括:
缓冲盐:Tris 50mmol/L;
蛋白质:牛血清白蛋白 15g/L;
表面活性剂:曲拉通x-100 1g/L;
无机盐,包括氯化钠:0.15mol/L;氯化镁:2.50mmol/L;氯化锌:0.50mmol/L;
保护剂:鲸蜡硬脂醇聚醚-25 10g/L;
防腐剂:叠氮钠 1g/L,以及PC300 1g/L;
所述保存液的pH为7.4。
上述cTnI抗体包被磁珠的保存液的制备方法,包括以下步骤:
将缓冲盐加入到水中混匀;
向水中加入蛋白质混匀;向水中加入无机盐混匀;
向水中加入表面活性剂混匀;
向水中加入保护剂混匀;
向水中加入防腐剂混匀;
调节pH至7.40±0.05;
定容,用0.22um滤芯过滤。
实施例3
一种cTnI抗体包被磁珠的保存液,溶剂为纯水,包括:
缓冲盐:Tris 50mmol/L;
蛋白质:牛血清白蛋白 10g/L;
表面活性剂:曲拉通x-100 1g/L;
无机盐,包括氯化钠:0.15mmol/L;氯化镁:2.50mmol/L;氯化锌:0.50mmol/L;
保护剂:鲸蜡硬脂醇聚醚-25 5g/L;
防腐剂:叠氮钠 1g/L,以及PC300 1g/L;
所述保存液的pH为7.4。
上述cTnI抗体包被磁珠的保存液的制备方法,包括以下步骤:
将缓冲盐加入到水中混匀;
向水中加入表面活性剂混匀;
向水中加入保护剂混匀;
向水中加入无机盐混匀;
向水中加入蛋白质混匀;
向水中加入防腐剂混匀;
调节pH至7.40±0.05;
定容,用0.22um滤芯过滤。
对比例1
一种cTnI抗体包被磁珠的保存液,溶剂为纯水,包括:
缓冲盐:Tris 50mmol/L;
蛋白质:牛血清白蛋白 10g/L;
表面活性剂:曲拉通x-100 1g/L;
无机盐,包括氯化钠:0.15mol/L;氯化镁:2.50mmol/L;氯化锌:0.50mmol/L;
防腐剂:叠氮钠 1g/L,以及PC300 1g/L;
所述保存液的pH为7.4。
上述cTnI抗体包被磁珠的保存液的制备方法,包括以下步骤:
将缓冲盐加入到水中混匀;
向水中加入无机盐混匀;
向水中加入蛋白质混匀;
向水中加入表面活性剂混匀;
向水中加入防腐剂混匀;
调节pH至7.40±0.05;
定容,用0.22um滤芯过滤。
对比例2
一种cTnI抗体包被磁珠的保存液,溶剂为纯水,包括:
缓冲盐:Tris 50mmol/L;
蛋白质:牛血清白蛋白 10g/L;
表面活性剂:曲拉通x-100 1g/L;
无机盐,包括氯化钠:0.15mol/L;氯化镁:2.50mmol/L;氯化锌:0.50mmol/L;
防腐剂:叠氮钠 1g/L,以及PC300 1g/L;
保护剂:甘油 50g/L、蔗糖 10g/L、聚乙二醇 40001g/L;
所述保存液的pH为7.4。
上述cTnI抗体包被磁珠的保存液的制备方法,包括以下步骤:
将缓冲盐加入到水中混匀;
向水中加入无机盐混匀;
向水中加入蛋白质混匀;
向水中加入表面活性剂混匀;
向水中加入保护剂混匀;
向水中加入防腐剂混匀;
调节pH至7.40±0.05;
定容,用0.22um滤芯过滤。
对比例3
一种cTnI抗体包被磁珠的保存液,溶剂为纯水,包括:
缓冲盐:Tris 50mmol/L;
蛋白质:牛血清白蛋白 10g/L;
表面活性剂:曲拉通x-100 1g/L;
无机盐,包括氯化钠:0.15mol/L;氯化镁:2.50mmol/L;氯化锌:0.50mmol/L;
保护剂:鲸蜡硬脂醇聚醚-25 0.5g/L;
防腐剂:叠氮钠 1g/L,以及PC300 1g/L;
所述保存液的pH为7.4。
上述cTnI抗体包被磁珠的保存液的制备方法,包括以下步骤:
将缓冲盐加入到水中混匀;
向水中加入无机盐混匀;
向水中加入蛋白质混匀;
向水中加入表面活性剂混匀;
向水中加入保护剂混匀;
向水中加入防腐剂混匀;
调节pH至7.40±0.05;
定容,用0.22um滤芯过滤
对比例4
一种cTnI抗体包被磁珠的保存液,溶剂为纯水,包括:
缓冲盐:Tris 50mmol/L;
蛋白质:牛血清白蛋白 10g/L;
表面活性剂:曲拉通x-100 1g/L;
无机盐,包括氯化钠:0.15mol/L;氯化镁:2.50mol/L;氯化锌:0.50mol/L;
保护剂:鲸蜡硬脂醇聚醚-25 20g/L;
防腐剂:叠氮钠 1g/L,以及PC300 1g/L;
所述保存液的pH为7.4。
上述cTnI抗体包被磁珠的保存液的制备方法,包括以下步骤:
将缓冲盐加入到水中混匀;
向水中加入无机盐混匀;
向水中加入蛋白质混匀;
向水中加入表面活性剂混匀;
向水中加入保护剂混匀;
向水中加入防腐剂混匀;
调节pH至7.40±0.05;
定容,用0.22um滤芯过滤
对实施例1-3以及对比例1-4的cTnI抗体包被磁珠的保存液进行试验。
实验一:稳定性验证
磁珠试剂由上述保存液中加入cTnI抗体包被磁珠母液制得。
将上述试剂分为2份,一份放置于37℃加速老化7天,一份放置于2~8℃保存。放置7天后,以放置2~8℃试剂为对照,用两份试剂同步测试相同样本。测试结果如下:
表1 稳定性测试结果
从实验例1-3结果可知,本发明保存液稀释cTnI抗体包被磁珠得到的工 作液,在37℃加速7天,发光值降幅在3.0%以内;鲸蜡硬脂醇聚醚-25浓度在1-10g/L范围内对稳定性无明显影响。从对比例1可知,未添加保护剂发光值降幅在20.0%以上。从比对例2可知,保存液中添加醇类、糖类、聚合物等保护剂,发光值降幅15.0%左右。从对比例3-4可知,鲸蜡硬脂醇聚醚-25浓度过多或过少,均影响试剂稳定性。
实验二:灵敏度验证
磁珠试剂由上述保存液中加入cTnI抗体包被磁珠母液制得,搭配酶标记物组成cTnI试剂,测试0浓度样本20次,计算空白限,测试结果如下:
表2 灵敏度测试结果
从实验例1-3结果可知,本发明保存液稀释cTnI抗体包被磁珠得到的工作液,空白限为0.002。从对比例1-2可知,不添加鲸蜡硬脂醇聚醚-25保护剂或者添加醇类、糖类、聚合物等其他保护剂,对空白限影响均较大。从对比例3-4可知,鲸蜡硬脂醇聚醚-25浓度过多或过少,均不利空白限测试。
对实施例1-3和对比例1-4的浓度与RLU关系作图,得到剂量反应曲线,如图1所示。
实验三:线性梯度验证
磁珠试剂由上述保存液中加入cTnI抗体包被磁珠母液制得,搭配酶标记物组成cTnI试剂,进行线性梯度测试,测试结果如下:
表3 线性梯度测试结果
从实验例1-3结果可知,本发明保存液稀释cTnI抗体包被磁珠得到的工作液,线性梯度明显,0~50ng/mL范围内,呈明显上升趋势。从对比例1-2可知,不添加鲸蜡硬脂醇聚醚-25保护剂或者添加醇类、糖类、聚合物等其他保护剂,线性梯度明显下降。从对比例3-4可知,鲸蜡硬脂醇聚醚-25浓度过多或过少,均不利线性梯度的测试。
对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (10)
- 一种cTnI抗体包被磁珠的保存液,溶剂为水,其特征在于,包括:缓冲盐;蛋白质;表面活性剂;无机盐;保护剂鲸蜡硬脂醇聚醚-25;防腐剂;所述保存液的pH为7.0-8.0。
- 根据权利要求1所述的cTnI抗体包被磁珠的保存液,其特征在于,包括:缓冲盐 20-100mmol/L;蛋白质 2-20g/L;表面活性剂 0.5-5g/L;无机盐 0.1-0.5mol/L;保护剂鲸蜡硬脂醇聚醚-25 1-10g/L;防腐剂 0.5-5g/L;所述保存液的pH为7.0-8.0。
- 根据权利要求1-2中任一项所述的cTnI抗体包被磁珠的保存液,其特征在于,所述缓冲盐具体为Tris、MES、MOPS、HEPES中的任意一种;和/或,所述缓冲盐的浓度为50-60mmol/L。
- 根据权利要求1-2中任一项所述的cTnI抗体包被磁珠的保存液,其特征在于,所述蛋白质具体为牛血清白蛋白、酪蛋白、明胶蛋白中的任意一种或多种。
- 根据权利要求4所述的cTnI抗体包被磁珠的保存液,其特征在于,所述蛋白质为牛血清白蛋白,所述牛血清白蛋白的浓度为10-15g/L。
- 根据权利要求1-2中任一项所述的cTnI抗体包被磁珠的保存液,其特 征在于,所述表面活性剂为曲拉通x-100、曲拉通x-405、吐温20、吐温80中的任意一种或多种;和/或,所述表面活性剂的浓度为1-2g/L。
- 根据权利要求1-2中任一项所述的cTnI抗体包被磁珠的保存液,其特征在于,所述无机盐为氯化钠、氯化镁、氯化锌的组合;或,为氯化钾、氯化镁、氯化锌的组合;其中,氯化钠浓度为0.1~0.3mol/L;氯化镁浓度为0.5-5mmol/L;氯化锌浓度为0.05-1mmol/L;氯化钾浓度为0.1~0.3mol/L。
- 根据权利要求7所述的cTnI抗体包被磁珠的保存液,其特征在于,所述无机盐组合中,氯化钠的浓度为0.1~0.2mol/L;氯化镁的浓度为2.5~5mmol/L;氯化锌0.1~0.5mmol/L;氯化钾浓度为0.1~0.2mol/L。
- 根据权利要求1-2中任一项所述的cTnI抗体包被磁珠的保存液,其特征在于,所述防腐剂具体为叠氮钠、PC300、氯霉素、庆大霉素中的任意一种或多种;和/或,所述防腐剂的浓度为1-2g/L。
- 一种cTnI抗体包被磁珠的保存液的制备方法,其特征在于,包括以下步骤:将缓冲盐、蛋白质、表面活性剂、无机盐、保护剂鲸蜡硬脂醇聚醚-25、防腐剂分别加入到水中混匀,使得缓冲盐的浓度为20-100mmol/L,蛋白质的浓度为2-20g/L;表面活性剂的浓度为0.5-5g/L;无机盐的浓度为0.1-0.5mol/L;保护剂鲸蜡硬脂醇聚醚-25的浓度为1-10g/L,防腐剂的浓度为0.5-5g/L;并调节pH至7.0-8.0;定容,过滤。
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