WO2016127322A1 - Kit de réactif de détection de facteur d'hyperaldostéronisme, et procédé de préparation et son application - Google Patents

Kit de réactif de détection de facteur d'hyperaldostéronisme, et procédé de préparation et son application Download PDF

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WO2016127322A1
WO2016127322A1 PCT/CN2015/072692 CN2015072692W WO2016127322A1 WO 2016127322 A1 WO2016127322 A1 WO 2016127322A1 CN 2015072692 W CN2015072692 W CN 2015072692W WO 2016127322 A1 WO2016127322 A1 WO 2016127322A1
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component
antibody
antigen
angiotensin
magnetic
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PCT/CN2015/072692
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English (en)
Chinese (zh)
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饶微
高莉
杜凯
李钦
徐红
罗凯
李婷华
袁锦云
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深圳市新产业生物医学工程股份有限公司
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Priority to PCT/CN2015/072692 priority Critical patent/WO2016127322A1/fr
Publication of WO2016127322A1 publication Critical patent/WO2016127322A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • 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
    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors

Definitions

  • the invention relates to a detection kit, in particular to an aldosteronism factor detection kit.
  • the invention also relates to a method of preparing the kit, and a method of using the kit to detect a concentration of a aldosteronism factor.
  • Primary aldosteronism is caused by the lesion of the adrenal cortex, which secretes excessive aldosterone, resulting in retention of water and sodium, increased blood volume, and inhibition of the activity of the renin-angiotensin system.
  • the clinical manifestations are hypertension and hypokalemia as the main features of the syndrome.
  • Factors that characterize aldosteronism include angiotensin I (A I), angiotensin II (A II), and aldosterone (ALD).
  • Patients with aldosteronism have high levels of aldosterone, low plasma rennin activity and angiotensin II, and a method of stimulating renin release, such as a standing, low-sodium diet, in the basal state (ie, under supine conditions). Or for diuretics, etc., these factors have no significant changes.
  • a I is produced by renin acting on the angiotensinogen secreted by the liver, and the rate of A I production is one of the important indicators indicating renin activity (PRA).
  • renin-angiotensin-aldosterone system is a hormonal system.
  • the kidney secretes renin, which regulates the hydrolysis of angiotensinogen to produce A I, A.
  • I is essentially free of biological activity, but cleaves two amino acid residues at the C-terminus by Angiotensin Converting Emzyme (ACE) to form A II.
  • ACE Angiotensin Converting Emzyme
  • a II has an efficient contraction of blood vessels, which increases blood pressure.
  • a II also stimulates the secretion of aldosterone from the adrenal cortex. Aldosterone can promote the reabsorption of water and sodium by the kidneys, which in turn increases body fluid volume and raises blood pressure.
  • the methods for measuring A I clinically mainly include radioactive immunoassay, enzyme-linked immunosorbent assay and the like.
  • the iodine [ 125 I]A I radioimmunoassay kit produced by Beijing North Biotechnology Research Institute uses the principle of competition to determine the A I content in plasma. After adding the sample and rabbit anti-A I antibody (blue) and 125 IA I label (red), the 125 IA I label competes with the AI in the sample for binding to the rabbit anti-AI antibody, and the anti-rabbit immune separator is added. After shaking, place at room temperature for 15 min, centrifuge at 3000 rpm for 15 min, remove the supernatant, and measure the radioactivity count (cpm) of the sedimentation tube. The A I concentration in the sample is quantified based on a Log-Logit mathematical model established from the calibrator concentration and the corresponding radioactivity count to detect the A I content in the sample.
  • radioimmunoassay and enzyme-linked immunoassay have many shortcomings.
  • the above radioactive immunization method has radioactive contamination, short half-life of the marker, radioactive damage to the operator, and is cumbersome, time consuming, low in sensitivity, narrow in detection range, and incapable of achieving full automation.
  • the traditional radioactive immunoassay or enzyme-linked immunosorbent assay has a long detection time, and mainly relies on a series of cumbersome operations such as pure manual loading, which is inefficient and easily leads to large errors in the experimental results; the enzymatic reaction is not thorough enough and is susceptible to external interference factors.
  • the effects, such as temperature, time, and material concentration are therefore low in specificity, poor in sensitivity, and narrow in detection range. Therefore, there is a need in the art for a method for improving the safety and stability of a detection reagent while improving sensitivity, and the operation is simpler, and the detection kit can be fully automated to detect the A I detection kit by means of an analytical instrument.
  • a II is formed by cleavage of two amino acid residues at the C-terminus by Angiotensin Converting Emzyme (ACE), which is an 8-peptide substance.
  • ACE Angiotensin Converting Emzyme
  • renin-angiotensin-aldosterone system is a hormonal system.
  • the kidney secretes renin, which regulates the hydrolysis of angiotensinogen to produce angiotensin.
  • I(A I) A I has substantially no biological activity.
  • a II has an efficient contraction of blood vessels, which increases blood pressure.
  • a II also stimulates the secretion of aldosterone from the adrenal cortex. Aldosterone can promote the reabsorption of water and sodium by the kidneys, which in turn increases body fluid volume and raises blood pressure.
  • the methods for clinically determining A II mainly include radioactive immunoassay, enzyme-linked immunosorbent assay, and the like.
  • the iodine [ 125 I] angiotensin II radioimmunoassay kit produced by Beijing North Biotechnology Research Institute uses the principle of competition to determine the content of A II in plasma. After adding the sample and rabbit anti-A II antibody (blue) and 125 IA II label (red), the 125 IA II label competes with the A II in the sample for binding to the rabbit anti-A II antibody, and the anti-rabbit immune isolation is added.
  • the A II concentration in the sample is quantified based on a Log-Logit mathematical model established from the calibrator concentration and the corresponding radioactivity count to detect the A II content of the sample.
  • radioimmunoassay and enzyme-linked immunoassay have many shortcomings.
  • the above radioactive immunization method has radioactive contamination, short half-life of the marker, radioactive damage to the operator, and is cumbersome, time consuming, low in sensitivity, narrow in detection range, and incapable of achieving full automation.
  • the traditional radioactive immunoassay or enzyme-linked immunosorbent assay has a long detection time, and mainly relies on a series of cumbersome operations such as pure manual loading, which is inefficient and easily leads to large errors in the experimental results; the enzymatic reaction is not thorough enough and is susceptible to external interference factors.
  • the effects, such as temperature, time, and material concentration are therefore low in specificity, poor in sensitivity, and narrow in detection range. Therefore, there is a need in the art for improving the safety and stability of a detection reagent while having high sensitivity, and the operation is simpler, and the detection kit can be fully automated to detect the A II detection kit by means of an analytical instrument.
  • Aldosterone is a mineralocorticoid synthesized and secreted by adrenal cortical spheroid cells, which is normally regulated by the renin-angiotensin system.
  • the renin-angiotensin-aldosterone system (RAAS) is a hormonal system.
  • RAAS renin-angiotensin-aldosterone system
  • a I has substantially no biological activity, but cleaves two amino acid residues at the C-terminus by Angiotensin Converting Emzyme (ACE) to form A II.
  • a II has an efficient contraction of blood vessels, which increases blood pressure.
  • a II can also stimulate the secretion of ALD from the adrenal cortex.
  • ALD can promote the reabsorption of water and sodium ions by the kidneys, which in turn increases the fluid volume and raises blood pressure.
  • ALD adrenocorticotropic hormone
  • adrenergic and dopaminergic systems also regulate the secretion of ALD.
  • blood ALD primarily binds to plasma albumin and rarely binds to corticosteroid-binding globulin (CBG) and, therefore, has a relatively short half-life (about 35 min) and a high metabolic clearance.
  • CBG corticosteroid-binding globulin
  • the urinary uncontaminated ALD accounts for about 6% of the secreted amount and has hormonal activity.
  • the methods for clinically determining ALD mainly include radioactive immunoassay, enzyme-linked immunosorbent assay, and the like.
  • the iodine [ 125 I] ALD radioimmunoassay kit produced by Beijing North Biotechnology Research Institute uses the principle of competition to determine the ALD content in plasma and urine. After adding the sample and rabbit anti-ALD antibody (blue) and 125 I-ALD label (red), the 125 I-ALD label competes with the ALD in the sample for binding to the rabbit anti-ALD antibody, and mixes at 2-8 ° C.
  • the ALD concentration in the sample is quantified based on a Log-Logit mathematical model established from the calibrator concentration and the corresponding radioactivity count to detect the ALD content in the sample.
  • radioimmunoassay and enzyme-linked immunoassay have many shortcomings.
  • the above radioactive immunization method has radioactive contamination, short half-life of the marker, radioactive damage to the operator, and is cumbersome, time consuming, low in sensitivity, narrow in detection range, and incapable of achieving full automation.
  • the traditional radioactive immunoassay or enzyme-linked immunosorbent assay has a long detection time, and mainly relies on a series of cumbersome operations such as pure manual loading, which is inefficient and easily leads to large errors in the experimental results; the enzymatic reaction is not thorough enough and is susceptible to external interference factors.
  • the effects, such as temperature, time, and material concentration are therefore low in specificity, poor in sensitivity, and narrow in detection range. Therefore, there is a need in the art for improving the detection accuracy, safety and stability of detection reagents while obtaining high sensitivity, and the operation is simpler, and the detection process can be fully automated by means of an analytical instrument.
  • kits for detecting aldosteronism factors including angiotensin I, angiotensin II and aldosterone.
  • the kit has high detection sensitivity and accuracy and high stability. Therefore, the kit for detecting aldosteronism factors can diagnose or help diagnose aldosteronism more accurately and quickly.
  • the present invention also provides a method of preparing a kit for detecting an aldosteronism factor.
  • the present invention also provides the use of the aldosteronism factor detection kit according to the present invention, in particular, the method for detecting aldosteronism factor by the fully automated chemiluminescence method, thereby reducing the operation time and reducing the human operation.
  • the error while using the specificity of the chemical tracer marker, improves detection sensitivity.
  • a aldosteronism factor detecting kit comprising a component A and a component B, the component A being an aldosteronism factor antigen or an aldosteronism factor antigen and a protein carrier a linker, component B is an aldosteronism factor antibody, any one of component A and component B is labeled with a tracer marker, and the other is coated with a magnetic sphere; wherein the aldosteronism factor It is selected from any one of angiotensin I, angiotensin II, and aldosterone.
  • the tracer label directly or indirectly labels component A or component B, and the manner of indirect labeling comprises passage of fluorescein isothiocyanate with an anti-isothiocyanate fluorescein antibody system or streptavidin
  • the component and the component B are indirectly labeled with the ubiquitin and biotin system.
  • the component A or the component B is coated directly or indirectly with a magnetic sphere, and the indirect coating comprises a method of fluorescein isothiocyanate and a fluorescein isothiocyanate antibody system or streptavidin.
  • the bismuth and biotin systems indirectly coat the magnetic sphere.
  • an A I detection kit comprising a component A1 and a component B1, wherein the component A1 is an A I antigen or a linker of an A I antigen and a protein carrier, Component B1 is an anti-A I antibody, one of the components A1 and B1 is labeled with a tracer marker and the other is coated with a magnetic sphere.
  • the A I detection kit provided by the present invention is a chemiluminescent immunoassay kit.
  • the anti-A I antibody may be one or more anti-A I monoclonal antibodies and/or anti-A I polyclonal antibodies. In fact, all of the antibodies mentioned in the present invention may be monoclonal antibodies and/or polyclonal antibodies.
  • the protein carrier to which the present invention is applicable may be selected from at least one of protein carriers commonly used in the art.
  • the protein carrier is selected from the group consisting of bovine serum albumin (BSA), human serum albumin (HSA), rabbit serum albumin (RSA), hemocyanin (KLH), bovine IgG, human IgG, ovalbumin (OVA). At least one of myoglobin and thyroglobulin.
  • the tracer label may be selected from tracer markers commonly used in the art to label antigens or antibodies, for example selected from adamantane, luminol and its derivatives, isoluminol and its derivatives.
  • At least one of acridinium ester, alkaline phosphatase, and horseradish peroxidase is preferably N-(4-aminobutyl)-N-ethylisoluminol (ABEI).
  • the tracer marker directly labels or indirectly labels the A I antigen (or its linker to a protein carrier) or an anti-A I antibody.
  • indirect labeling includes, but is not limited to, indirect labeling by fluorescein isothiocyanate (FITC) and anti-FITC antibody system or streptavidin (SA) and biotin (Biotin) system.
  • FITC fluorescein isothiocyanate
  • SA streptavidin
  • Biotin biotin
  • the adaptor or an anti-A I antibody, including but not limited to FITC and anti-FITC antibody systems or streptavidin and biotin systems.
  • the inventors have found that indirect labeling is advantageous for attenuating spatial effects, facilitating amplification of signals, and making detection more sensitive.
  • the A I antigen (or its conjugate with a protein carrier) or the anti-A I antibody directly coats the magnetic sphere, or indirectly coated by the FITC and anti-FITC antibody system or streptavidin and the biotin system. ball.
  • Said “direct coating” Means that the magnetic sphere is directly coated with an A I antigen (or a linker thereof with a protein carrier) or an anti-A I antibody; the “indirect coating” refers to an A I antigen via an intermediate vector linking system (or The magnetic sphere is coated with a linker to the protein carrier or an anti-A I antibody, including but not limited to FITC and anti-FITC antibody systems or streptavidin and biotin systems.
  • the advantage of indirect coating is that it helps to reduce the spatial effect, facilitates the amplification of the signal, and makes the detection more sensitive.
  • Magnetic spheres suitable for use in the present invention are also referred to as magnetic beads and may be magnetic microspheres commonly used in the art.
  • the magnetic ball used in the present invention is a composite of nano-scale Fe 2 O 3 or Fe 3 O 4 magnetic particles and an organic polymer material to form a micron-scale having superparamagnetic and extremely large protein adsorption capacity.
  • the solid phase microspheres have the property of being magnetized rapidly under the action of an external magnetic field and having zero remanence after the magnetic field is withdrawn.
  • the type of the organic polymer material is not particularly limited and may be selected as needed.
  • the magnetic microspheres used in the present invention should be capable of satisfying a diameter of 0.1 to 5 ⁇ m, and the magnetic microspheres may also have various active functional groups by surface modification, including but not limited to -OH, -COOH, -NH 2 .
  • the magnetic ball is a composite of Fe 2 O 3 or Fe 3 O 4 magnetic nanoparticles and an organic polymer material, and has a particle diameter of 0.1-5 ⁇ m; and the magnetic ball is optional.
  • the surface is modified with one or more active functional groups by surface modification.
  • the kit comprises a component selected from any one of component O1 and component O2, and is selected from any one of component P1, component P2 and component P3.
  • a component wherein component O1 is an AI antigen directly labeled by ABEI (or a linker thereof to a protein carrier); component O2 is a streptavidin-labeled ABEI and a biotinylated A I antigen (or a linker to the protein carrier); component P1 is a magnetic sphere directly coated with an anti-A I antibody; component P2 is a biotinylated anti-A I antibody and a streptavidin-coated magnetic sphere; P3 is a magnetic ball coated with anti-A I antibody-labeled FITC and anti-FITC antibody.
  • the kit comprises a component selected from any one of component C1 and component C2, and is selected from any one of component D1, component D2 and component D3.
  • a component wherein, component C1 is an ABI directly labeled anti-A I antibody; component C2 is streptavidin-labeled ABEI and biotinylated anti-A I antibody; component D1 is A I antigen ( Or a magnetic particle directly coated with the protein carrier; component D2 is a biotinylated A I antigen (or a linker thereof with a protein carrier) and a streptavidin-coated magnetic sphere; Component D3 is a magnetic ball coated with FITC and anti-FITC antibody labeled with an A I antigen (or its linker to a protein carrier).
  • the kit may further comprise a low point calibrator and a high point calibrator of the A I antigen (or its conjugate to a protein carrier), and optionally a buffer.
  • the low-point calibrator and the high-point calibrator of the present invention are relative to each other, wherein the "low-point calibrator” refers to the A I antigen (or its conjugate with a protein carrier) using 50% bovine serum preparation. Dilution to a calibrator at a concentration of 0.2-2 ng/ml; and "high-point calibrator” means diluting the A I antigen (or its conjugate with a protein carrier) with a 50% bovine serum preparation to a concentration of 8-24 ng /ml Get the calibrator.
  • the concentration of each component contained is preferably as follows: AI antigen or AI and protein carrier conjugate is 0.002-0.01 mg/ml; anti-AI antibody is 0.05-1 mg/ml; magnetic sphere is 0.05- 1 mg/ml; FITC is 0.002-0.01 mg/ml; anti-FITC antibody is 0.05-1 mg/ml; streptavidin is 0.05-1 mg/ml; biotin is 0.002-0.01 mg/ml; tracer marker is 0.2-1 mg/l; and if a protein carrier is used, its concentration is 2-10 mg/l.
  • concentrations of each of the above ingredients are based on the amount of the individual kit components comprising the ingredients.
  • ABEI is labeled on the A I antigen (or its conjugate to a protein carrier) and the magnetic sphere is coated with an anti-A I antibody.
  • the kit comprises an ABEI labeled with an A I antigen (or a linker thereof to a protein carrier), an anti-A I antibody coated magnetic sphere, a low point calibrator, and High point calibrator.
  • the kit comprises an ABEI, a biotinylated anti-A I antibody, a streptavidin package labeled with an A I antigen (or a linker thereof to a protein carrier) Magnetic ball, low point calibrator and high Point the calibrator.
  • the kit comprises an ABEI labeled with an A I antigen (or a conjugate thereof to a protein carrier), an anti-A I antibody-labeled FITC, an anti-FITC polyclonal antibody coating Magnetic ball, low point calibrator and high point calibrator.
  • the kit comprises a biotinylated A I antigen (or a linker thereof to a protein carrier), a streptavidin-labeled ABEI, an anti-A I antibody package Magnetic ball, low point calibrator and high point calibrator.
  • ABEI is labeled on an anti-A I antibody and the magnetic sphere is coated with an A I antigen (or a linker to the protein carrier).
  • the kit comprises a magnetic sphere coated with an ABEI, an A antigen (or a linker to a protein carrier) labeled with an anti-A I antibody, a low-point calibrator, and High point calibrator.
  • the kit comprises an ABEI labeled with an anti-A I antibody, a FITC-labeled A I antigen (or a conjugate thereof with a protein carrier), an anti-FITC polyclonal antibody coating Magnetic ball, low point calibrator and high point calibrator.
  • the kit comprises a biotinylated anti-A I antibody, an ABEI labeled with streptavidin, an A I antigen (or a linker thereof to a protein carrier), Magnetic ball, low point calibrator and high point calibrator.
  • the kit comprises an ABEI labeled with an anti-A I antibody, a biotinylated A I antigen (or a linker thereof to a protein carrier), a streptavidin package Magnetic ball, low point calibrator and high point calibrator.
  • the present invention also provides a method for preparing a kit as described above, the method comprising: directly or indirectly labeling one of the component A1 and the component B1 with a tracer marker, and the other directly Or indirectly coated with a magnetic ball.
  • the indirect labeling comprises labeling the tracer label with fluorescein isothiocyanate and an anti-isothiocyanate fluorescein antibody system or streptavidin and a biotin system to label the component A1 Or component B1.
  • the indirect coating comprises passing the component A1 or the component B1 through a fluorescein isothiocyanate and an anti-isothiocyanate fluorescein antibody system or streptavidin and a biotin system. Indirectly coated with magnetic balls.
  • the method for preparing the kit comprises the steps of: i) an A I antigen (or a linker to a protein carrier) labeling step, or directly or indirectly labeling the A I antigen using ABEI (or Its linker to the protein carrier); and ii) the anti-A I antibody coated magnetic sphere step, the anti-A I antibody is coated directly or indirectly with the magnetic sphere.
  • the method for preparing the kit comprises the steps of: i') an anti-A I antibody labeling step, direct or indirect labeling of an anti-A I antibody using ABEI; and ii') A I antigen (or its linker to a protein carrier) is coated with a magnetic sphere step, and the A I antigen (or its linker to the protein carrier) is coated directly or indirectly with a magnetic sphere.
  • the ABEI in step i), is indirectly labeled with the A I antigen (or a linker thereof with a protein carrier) by FITC and an anti-FITC antibody system or streptavidin and a biotin system; and/or
  • the anti-A I antibody is indirectly coated with a magnetic sphere by FITC and an anti-FITC antibody system or streptavidin and a biotin system.
  • the ABEI in step i'), is indirectly labeled with an anti-AIC antibody by FITC and an anti-FITC antibody system or streptavidin and a biotin system; and/or in step ii'), A The I antigen (or its conjugate to a protein carrier) indirectly coats the magnetic sphere by FITC with an anti-FITC antibody system or streptavidin and a biotin system.
  • the kit preparation method according to the present invention may further include a configuration of an A I low point calibrator and a high point calibrator, and may further include assembly of the kit.
  • a method of detecting the concentration of A I which comprises detecting the concentration of A I in a sample to be tested by chemiluminescence immunoassay using a kit as described above.
  • the above A I concentration detecting method comprises the component A1 and the component B1 of the kit. Mixing with the sample to be tested, determining the light signal intensity of the sample, and calculating the angiotensin I concentration of the sample to be tested by comparing with the light signal intensity of the angiotensin I calibrator; wherein, in the kit, angiotensin The concentration of the I antigen or the angiotensin I antigen and the protein carrier is 0.002-0.01 mg/ml; the concentration of the anti-angiotensin I antibody is 0.05-1 mg/ml; and the concentration of the magnetic sphere is 0.05-1 mg/ml; The concentration of the tracer marker is 0.2-1 mg/l. The concentrations of each of the above ingredients are based on the amount of the individual kit components comprising the ingredients.
  • the method of detecting the AI concentration comprises detecting the AI concentration by a chemiluminescence immunoassay analyzer using a kit as described above.
  • the method is carried out fully automatically.
  • the chemiluminescence immunoassay analyzer is preferably a Maglumi series chemiluminescence immunoassay analyzer (manufactured by Shenzhen New Industry Biomedical Engineering Co., Ltd.).
  • the detecting step of performing chemiluminescence detection may include: 1) obtaining a sample to be tested.
  • the sample to be tested may be directly obtained serum, plasma and whole blood, or may be obtained by extracting a blood sample of a human body, or a sample treated by an enzyme inhibitor; 2) using an A I antigen labeled with ABEI (or The protein carrier linker and the magnetic ball with anti-A I antibody are mixed with the sample to be tested and then incubated to obtain a reaction product; 3) after washing, the chemiluminescence signal is detected on the machine to obtain corresponding optical signal data; 4) The corresponding optical signal data was analyzed to obtain the A I antigen content.
  • the detecting step of performing the chemiluminescence detection may include: 1) obtaining the sample to be tested.
  • the sample to be tested may be directly obtained serum, plasma and whole blood, or may be obtained by extracting a human blood sample or being subjected to an enzyme inhibitor treatment; 2) using an ABEI-labeled anti-A I antibody and The magnetic ball of the A I antigen (or its linkage with the protein carrier) is mixed with the sample to be tested and then incubated to obtain a reaction product; 3) after washing, the chemiluminescence signal is detected on the machine to obtain corresponding optical signal data; 4) The corresponding light signal data is analyzed to obtain the A I content, and the content of renin activity can be further calculated.
  • the present invention still further provides a method for measuring renin activity, which is used to measure the A I concentration using the test kit provided by the present invention, thereby obtaining a renin activity value.
  • an A II detection kit comprising a component A2 and a component B2, wherein the component A2 is an A II antigen or a linker of an A II antigen and a protein carrier Component B2 is an anti-A II antibody, one of the components A2 and B2 is labeled with a tracer marker and the other is coated with a magnetic sphere.
  • the A II detection kit provided by the present invention is a chemiluminescent immunoassay kit.
  • the anti-A II antibody may be one or more anti-A II monoclonal antibodies and/or anti-A II polyclonal antibodies. In fact, all of the antibodies mentioned in the present invention may be monoclonal antibodies and/or polyclonal antibodies.
  • the tracer marker directly labels or indirectly labels the A II antigen (or its protein linker) or the anti-A II antibody.
  • indirect labeling includes, but is not limited to, indirect labeling of the A II antigen (or its conjugate with a protein carrier) or an anti-A II antibody by FITC in combination with an anti-FITC antibody system or streptavidin and a biotin system.
  • the "direct labeling” is to indicate that the trace label is directly labeled with an A II antigen (or a linker thereof with a protein carrier) or an antibody to the antigen to be tested; the "indirect label” refers to an intermediate vector linkage system.
  • the tracer marker is labeled with an A II antigen (or a conjugate thereof with a protein carrier) or an anti-A II antibody, including but not limited to FITC and anti-FITC antibody systems or streptavidin and biotin system.
  • a II antigen or a conjugate thereof with a protein carrier
  • an anti-A II antibody including but not limited to FITC and anti-FITC antibody systems or streptavidin and biotin system.
  • the A II antigen (or its conjugate with a protein carrier) or the anti-A II antibody directly coats the magnetic sphere, or indirectly coated by the FITC and anti-FITC antibody system or streptavidin and the biotin system. ball.
  • the "direct coating” refers to the direct coating of a magnetic sphere by using an A II antigen (or a linker thereof with a protein carrier) or an anti-A II antibody; the “indirect coating” refers to an intermediate medium linking system.
  • the magnetic sphere is coated with an A II antigen (or a linker thereof with a protein carrier) or an anti-A II antibody, including but not limited to FITC and an anti-FITC antibody system or chain Mycomycin and biotin systems.
  • the advantage of indirect coating is that it helps to reduce the spatial effect, facilitates the amplification of the signal, and makes the detection more sensitive.
  • the magnetic sphere is a composite of Fe 2 O 3 or Fe 3 O 4 magnetic nanoparticles and an organic polymer material, and has a particle diameter of 0.1 to 5 ⁇ m;
  • the magnetic spheres are optionally provided with a plurality of reactive functional groups by surface modification.
  • the kit comprises a component selected from any one of component E1 and component E2, and is selected from any one of component F1, component F2 and component F3.
  • a component wherein component E1 is an AII antigen directly labeled by ABEI (or a linker thereof to a protein carrier); component E2 is a streptavidin-labeled ABEI and a biotinylated A II antigen (or a linker thereof to a protein carrier; a component F1 is a magnetic ball directly coated with an anti-A II antibody; a component F2 is a biotinylated anti-A II antibody and a streptavidin-coated magnetic sphere; Fraction F3 is a magnetic sphere coated with anti-A II antibody-labeled FITC and anti-FITC antibody.
  • the kit comprises a component selected from any one of component G1 and component G2, and is selected from any one of component H1, component H2 and component H3.
  • a component wherein, component G1 is an ABEI directly labeled anti-A II antibody; component G2 is streptavidin-labeled ABEI and biotinylated anti-A II antibody; component H1 is A II antigen ( Or a magnetic particle directly coated with the protein carrier; the component H2 is a biotinylated A II antigen (or a linker thereof with a protein carrier) and a streptavidin-coated magnetic sphere; Component H3 is a magnetic ball coated with FITC and anti-FITC antibody labeled with A II antigen (or its linker to a protein carrier).
  • the kit may further comprise a low point calibrator and a high point calibrator of the A II antigen, and optionally a buffer.
  • the low-point calibrator and the high-point calibrator of the present invention are relative to each other, wherein the "low-point calibrator” refers to diluting the A II antigen with about 50% of the bovine serum product to a concentration of 10-60 pg/ The calibrator obtained in ml; and the "high-point calibrator” refers to a calibrator obtained by diluting A II antigen with about 50% bovine serum preparation to a concentration of 400-800 pg/ml.
  • the concentration of each component contained is preferably as follows: A II antigen or a linker of A II antigen and protein carrier is 0.002-0.0 lmg/ml; anti-A II antibody is 0.05-1 mg/ml; magnetic The ball is 0.05-1 mg/ml; the FITC is 0.002-0.01 mg/ml; the anti-FITC antibody is 0.05-1 mg/ml; the streptavidin is 0.05-1 mg/ml; the biotin is 0.002-0.01 mg/ml; The trace marker is 0.2-1 mg/l; and if a protein carrier is used, the concentration is 2-10 mg/l.
  • the concentrations of each of the above ingredients are based on the amount of the individual kit components comprising the ingredients.
  • ABEI is labeled on the A II antigen (or its conjugate to a protein carrier) and the magnetic sphere is coated with an anti-A II antibody.
  • the kit comprises an AII antigen (or a linker to a protein carrier) labeled ABEI, an anti-A II antibody coated magnetic sphere, a low point calibrator, and High point calibrator.
  • the kit comprises an AII antigen (or a linker to a protein carrier) labeled ABEI, a biotinylated anti-A II antibody, a streptavidin package Magnetic ball, low point calibrator and high point calibrator.
  • the kit comprises an AII antigen (or a linker to a protein carrier) labeled ABEI, an anti-A II antibody-labeled FITC, an anti-FITC polyclonal antibody coating. Magnetic ball, low point calibrator and high point calibrator.
  • the kit comprises a biotinylated A II antigen (or a linker thereof to a protein carrier), a streptavidin-labeled ABEI, an anti-A II antibody package Magnetic ball, low point calibrator and high point calibrator.
  • kits of the invention in the kit of the invention, ABEI is labeled on an anti-A II antibody and the magnetic sphere is coated with an A II antigen (or a linker to the protein carrier).
  • the kit comprises a magnetic ball coated with an anti-A II antibody-labeled ABEI, A II antigen (or a linker thereof with a protein carrier), a low-point calibrator, and High point calibrator.
  • the kit comprises an anti-A II antibody-labeled ABEI, A II antigen (or its conjugate to a protein carrier) labeled FITC, anti-FITC polyclonal antibody coating Magnetic ball, low point calibrator and high point calibrator.
  • the kit comprises a biotinylated anti-A II antibody, streptavidin-labeled ABEI, A II antigen (or a linker thereof to a protein carrier) package Magnetic ball, low point calibrator and high point calibrator.
  • the kit comprises an anti-A II antibody-labeled ABEI, a biotinylated A II antigen (or a linker thereof to a protein carrier), a streptavidin package Magnetic ball, low point calibrator and high point calibrator.
  • the present invention also provides a method for the preparation of the A II detection kit as described above, comprising: component A2 (A II antigen or a conjugate thereof with a protein carrier) and component B2 (anti-A II antibody) One of them directly or indirectly marks the tracer marker and the other directly or indirectly coats the magnetic sphere.
  • the indirect labeling comprises passing the tracer label to fluorescein isothiocyanate and an anti-isothiocyanate fluorescein antibody system or streptavidin and a biotin system.
  • the A II antigen (or its linker to a protein carrier) or an anti-A II antibody is labeled.
  • the indirect coating comprises transposing the A II antigen (or a conjugate thereof with a protein carrier) or an anti-A II antibody with fluorescein isothiocyanate.
  • the fluorescein thiocyanate antibody system or streptavidin and the biotin system indirectly coat the magnetic sphere.
  • the method for preparing the A II detection kit comprises the steps of: i) an A II antigen labeling step, direct or indirect labeling of the A II antigen with a tracer label (or protein carrier thereof) And ii) the anti-A II antibody coated magnetic sphere step, the anti-A II antibody directly or indirectly coated with the magnetic sphere.
  • the method for preparing the A II detection kit comprises the steps of: i') an anti-A II antibody labeling step, direct or indirect labeling of an anti-A II antibody using a tracer label; Ii') The A II antigen (or its linker to the protein carrier) is coated with a magnetic sphere step, and the A II antigen (or its linkage to the protein carrier) is coated directly or indirectly with a magnetic sphere.
  • the tracer label is indirectly labeled with the A II antigen (or a linker thereof to the protein carrier) by FITC in combination with an anti-FITC antibody system or streptavidin and a biotin system; / or in step ii), the anti-A II antibody is indirectly coated with magnetic beads by FITC and anti-FITC antibody system or streptavidin and biotin system.
  • the tracer marker in step i'), is indirectly labeled with an anti-FIT antibody system or streptavidin and a biotin system by an anti-AII antibody; and/or in step ii')
  • the A II antigen (or its conjugate to a protein carrier) is indirectly coated with a magnetic sphere by FITC and an anti-FITC antibody system or streptavidin and a biotin system.
  • the A II detection kit preparation method according to the present invention may further include preparation of a low point calibrator and a high point calibrator, and may further include assembly of the kit.
  • a method for detecting A II concentration which comprises detecting a concentration of A II in a sample to be tested by a chemiluminescence immunoassay using an A II detection kit as described above.
  • the component A1 and the component B1 of the kit are mixed with the sample to be tested, and the optical signal intensity of the sample is measured, and the optical signal is passed through the calibration product with the A II.
  • Intensity control calculating the concentration of A II of the sample to be tested; wherein, in the kit, the concentration of the linker of the A II antigen or the A II antigen and the protein carrier is 0.002-0.01 mg/ml; the concentration of the anti-A II antibody It is 0.05-1 mg/ml; the concentration of the magnetic sphere is 0.05-1 mg/ml; and the concentration of the tracer marker is 0.2-1 mg/l.
  • the concentrations of each of the above ingredients are based on the amount of the individual kit components comprising the ingredients.
  • the AII concentration detection method comprises detecting the A II concentration by a chemiluminescence immunoassay analyzer using an A II detection kit as described above.
  • the method is fully automated get on.
  • the chemiluminescence immunoassay analyzer is preferably a Maglumi series chemiluminescence immunoassay analyzer (manufactured by Shenzhen New Industry Biomedical Engineering Co., Ltd.).
  • the detection step of performing chemiluminescence detection may include: 1) obtaining Test the sample.
  • the sample to be tested may be directly obtained serum, plasma and whole blood, or may be obtained by extracting a human blood sample or being subjected to an enzyme inhibitor treatment; 2) using an A II antigen labeled with a tracer label ( Or a protein-supporting linker thereof and a magnetic ball coated with an anti-A II antibody are mixed with the sample to be tested, and then incubated to obtain a reaction product; 3) after washing, the chemiluminescence signal is detected on the machine to obtain a corresponding optical signal. Data; 4) Analysis of the corresponding light signal data to obtain the A II antigen content.
  • the step of detecting the chemiluminescence detection may include: 1) obtaining the sample to be tested.
  • the sample to be tested may be directly obtained serum, plasma and whole blood, or may be obtained by extracting a human blood sample or being subjected to an enzyme inhibitor treatment; 2) an anti-A II antibody labeled with a tracer label And the magnetic ball coated with the A II antigen (or its linkage with the protein carrier) is mixed with the sample to be tested, and then incubated to obtain a reaction product; 3) after washing, the chemiluminescence signal is detected on the machine to obtain a corresponding optical signal. Data; 4) Analysis of the corresponding optical signal data to obtain A II content.
  • an aldosterone detecting kit comprising an aldosterone antigen and an anti-aldosterone antibody, one of the aldosterone antigen and the anti-aldosterone antibody, and a marker tracer, and the other The magnetic ball is coated.
  • the aldosterone detection kit provided by the present invention may be a chemiluminescent immunodetection kit.
  • the anti-aldosterone antibody may be one or more anti-aldosterone monoclonal antibodies and/or anti-aldosterone polyclonal antibodies.
  • all of the antibodies mentioned in the present invention may be monoclonal antibodies and/or polyclonal antibodies.
  • the aldosterone antigen refers to a aldosterone antigen alone or a conjugate of a aldosterone antigen alone to a protein carrier, unless otherwise specified.
  • the protein carrier may be selected from at least one of protein carriers commonly used in the art.
  • the protein carrier is selected from the group consisting of bovine serum albumin (BSA), human serum albumin (HSA), rabbit serum albumin (RSA), hemocyanin (KLH), bovine IgG, human IgG, ovalbumin (OVA). At least one of myoglobin and thyroglobulin.
  • the tracer label directly or indirectly labels an ALD antigen or an anti-ALD antibody.
  • indirect labeling includes, but is not limited to, indirect labeling of ALD antigen or anti-ALD antibody by FITC and anti-FITC antibody system or streptavidin and biotin system.
  • the "direct labeling" is a label indicating that the trace label is directly linked to an ALD antigen or an antibody against the antigen to be tested;
  • the "indirect label” means that the trace label is labeled with an ALD antigen or an anti-ALD by an intermediate vector linking system.
  • Antibodies, said intermediate vector linkage systems include, but are not limited to, FITC and anti-FITC antibody systems or streptavidin and biotin systems. The inventors have found that indirect labeling is advantageous for attenuating spatial effects, facilitating amplification of signals, and making detection more sensitive.
  • the ALD antigen or the anti-ALD antibody directly coats the magnetic sphere, or indirectly coats the magnetic sphere by FITC and the anti-FITC antibody system or streptavidin and the biotin system.
  • the "direct coating” refers to directly coating a magnetic ball with an ALD antigen or an anti-ALD antibody; the “indirect coating” refers to an ALD antigen or an anti-ALD antibody on a magnetic ball through an intermediate medium linking system.
  • the intermediate vector linkage system includes, but is not limited to, FITC and anti-FITC antibody systems or streptavidin and biotin systems.
  • the advantage of indirect coating is that it helps to reduce the spatial effect, facilitates the amplification of the signal, and makes the detection more sensitive.
  • the magnetic sphere is a composite of Fe 2 O 3 or Fe 3 O 4 magnetic nanoparticles and an organic polymer material, and has a particle diameter of 0.1 to 5 ⁇ m; Also, the magnetic ball is optionally provided with a plurality of reactive functional groups by surface modification.
  • the ALD detection kit comprises a component selected from any one of component S1 and component S2, and any one selected from the group consisting of component T1, component T2, and component T3. a component; wherein, component S1 ALD antigen directly labeled for ABEI; component S2 is streptavidin-labeled ABEI and biotinylated ALD antigen; component T1 is a magnetic sphere directly coated with an anti-ALD antibody; component T2 is biotinylated Anti-ALD antibody and streptavidin coated magnetic spheres; and component T3 is an anti-ALD antibody labeled FITC and anti-FITC antibody coated magnetic sphere.
  • the ALD detection kit comprises a component selected from any one of component U1 and component U2, and is selected from the group consisting of component V1, component V2 and component V3. a component of any one; wherein component U1 is an ABEI directly labeled anti-ALD antibody; component U2 is streptavidin-labeled ABEI and biotinylated anti-ALD antibody; component V1 is an ALD antigen direct package The magnetic sphere is; the component V2 is a biotinylated ALD antigen and a streptavidin-coated magnetic sphere; and the component V3 is an ALD antigen-labeled FITC and anti-FITC antibody-coated magnetic sphere.
  • the ALD detection kit can also include a low point calibrator of ALD and a high point calibrator, and optionally a buffer.
  • the low-point calibrator and the high-point calibrator of the present invention are relative to each other, wherein the "low-point calibrator” refers to the ALD antigen diluted with 50% bovine serum preparation to a concentration of 20-120 pg/ml.
  • Calibrator; and "high-point calibrator” refers to a calibrator obtained by diluting ALD antigen with 50% bovine serum preparation to a concentration of 800-1600 pg/ml.
  • the concentration of each component contained is preferably as follows: ALD antigen is 0.002-0.01 mg/ml; anti-ALD antibody is 0.05-1 mg/ml; magnetic sphere is 0.05-1 mg/ml; FITC is 0.002-0.01 mg/ml; anti-FITC antibody is 0.05-1 mg/ml; streptavidin is 0.05-1 mg/ml; biotin is 0.002-0.01 mg/ml; tracer marker is 0.2-1 mg/l; And if a protein carrier is used, its concentration is 2-10 mg/l.
  • concentrations of each of the above ingredients are based on the amount of the individual kit components comprising the ingredients.
  • ABEI is labeled on the ALD antigen and the magnetic sphere is coated with an anti-ALD antibody.
  • the ALD detection kit comprises an ALD antigen labeled ABEI, an anti-ALD antibody coated magnetic sphere, a low point calibrator, and a high point calibrator.
  • the ALD detection kit comprises an ALD antigen-labeled ABEI, a biotinylated anti-ALD antibody, a streptavidin-coated magnetic sphere, a low-point calibrator, and High point calibrator.
  • the ALD detection kit comprises an ALD antigen-labeled ABEI, an anti-ALD antibody-labeled FITC, an anti-FITC polyclonal antibody-coated magnetic sphere, a low-point calibrator, and a high Point the calibrator.
  • the ALD detection kit comprises a biotinylated ALD antigen, a streptavidin-labeled ABEI, an anti-ALD antibody-coated magnetic sphere, a low-point calibrator, and High point calibrator.
  • ABEI is labeled on an anti-ALD antibody and the magnetic sphere is coated with an ALD antigen.
  • the ALD detection kit comprises an anti-ALD antibody labeled ABEI, an ALD antigen coated magnetic sphere, a low point calibrator, and a high point calibrator.
  • the ALD detection kit comprises an anti-ALD antibody-labeled ABEI, an ALD antigen-labeled FITC, an anti-FITC polyclonal antibody-coated magnetic sphere, a low-point calibrator, and a high Point calibrator
  • the ALD detection kit comprises a biotinylated anti-ALD antibody, a streptavidin-labeled ABEI, an ALD antigen-coated magnetic sphere, a low-point calibrator, and High point calibrator;
  • the ALD detection kit comprises an anti-ALD antibody labeled ABEI, a biotinylated ALD antigen, a streptavidin coated magnetic sphere, a low point calibrator, and High point calibrator.
  • the present invention also provides a method for preparing an ALD detection kit as described above, comprising: labeling a tracer marker directly or indirectly with one of an aldosterone antigen and an aldosterone antibody, and directly or indirectly The magnetic ball is coated.
  • the indirect labeling comprises labeling the tracer label with fluorescein isothiocyanate and an anti-isothiocyanate fluorescein antibody system or streptavidin and a biotin system. Aldosterone resistance Pro- or anti-aldosterone antibody.
  • the indirect coating comprises passing the aldosterone antigen or an anti-aldosterone antibody to a fluorescein isothiocyanate antibody system or streptavidin.
  • the magnetic sphere is indirectly coated with the biotin system.
  • the method for preparing the ALD detection kit comprises the steps of: i) ALD antigen labeling step, labeling ALD antigen directly or indirectly with ABEI; and ii) anti-ALD antibody coating magnetic sphere step The anti-ALD antibody is coated directly or indirectly with a magnetic ball.
  • the method for preparing the ALD detection kit comprises the steps of: i') an anti-ALD antibody labeling step, direct or indirect labeling of an anti-ALD antibody using ABEI; and ii') an ALD antigen package
  • the ALD antigen is coated directly or indirectly with the magnetic ball by the magnetic ball step.
  • step i) ABEI is indirectly labeled with ALD antigen by FITC and anti-FITC antibody system or streptavidin and biotin system; and/or in step ii), anti-ALD antibody is passed through FITC
  • the magnetic sphere is indirectly coated with an anti-FITC antibody system or streptavidin and a biotin system.
  • step i' ABEI is indirectly labeled with an anti-ALDC antibody via FITC and an anti-FITC antibody system or streptavidin and a biotin system; and/or in step ii'), the ALD antigen is The magnetic sphere is indirectly coated by FITC and anti-FITC antibody system or streptavidin and biotin system.
  • the ALD detection kit preparation method according to the present invention may further include preparation of an ALD low point calibrator and a high point calibrator, and may further include assembly of the kit.
  • a method of detecting an ALD concentration comprising detecting an ALD concentration in a sample to be tested by a chemiluminescence immunoassay using an ALD detection kit as described above.
  • the ALD concentration detection method comprises mixing a kit component comprising an aldosterone antigen and an aldosterone antibody with a sample to be tested, determining the optical signal intensity of the sample, and passing the light with the aldosterone calibrator The signal intensity comparison is performed to calculate the aldosterone concentration of the sample to be tested; wherein, in the kit, the aldosterone antigen concentration is 0.002-0.01 mg/ml, the anti-aldosterone antibody concentration is 0.05-1 mg/ml, and the magnetic ball concentration is 0.05-1 mg. /ml, the tracer marker concentration is 0.2-1 mg/l.
  • the concentrations of each of the above ingredients are based on the amount of the individual kit components comprising the ingredients.
  • the method of detecting an ALD concentration comprises detecting an ALD concentration by a chemiluminescence immunoassay analyzer using an ALD detection kit as described above.
  • the method is carried out fully automatically.
  • the chemiluminescence immunoassay analyzer is preferably a Maglumi series chemiluminescence immunoassay analyzer (manufactured by Shenzhen New Industry Biomedical Engineering Co., Ltd.).
  • the detecting step of performing the chemiluminescence detection may include: 1) acquiring the sample to be tested.
  • the sample to be tested may be directly obtained serum, plasma and whole blood, or may be obtained by extracting a human blood sample or being subjected to an enzyme inhibitor treatment; 2) using an ABEI-labeled ALD antigen and having an anti-ALD
  • the magnetic ball of the antibody is mixed with the sample to be tested and then incubated to obtain a reaction product; 3) after washing, the chemiluminescence signal is detected on the machine to obtain the corresponding optical signal data; 4) the corresponding optical signal data is analyzed to obtain the ALD antigen content.
  • the detecting step of performing the chemiluminescence detection may include: 1) acquiring the sample to be tested.
  • the sample to be tested may be directly obtained serum, plasma and whole blood, or may be obtained by extracting a human blood sample; 2) mixing the anti-ALD antibody with ABEI label and the magnetic ball with ALD antigen with the sample to be tested The incubation is carried out to obtain the reaction product; 3) after washing, the chemiluminescence signal is detected on the machine to obtain the corresponding optical signal data; 4) the corresponding optical signal data is analyzed to obtain the ALD content.
  • kit of the invention can be combined with a chemiluminescence immunoassay analyzer (especially The Maglumi series of chemiluminescence immunoassay analyzers are used together to achieve full automation in the sample measurement process, so that the detection of aldosteronol factor concentration can be carried out simply, conveniently, quickly and in batches, while ensuring a small systematic error in detection.
  • chemiluminescence immunoassay analyzer especially The Maglumi series of chemiluminescence immunoassay analyzers are used together to achieve full automation in the sample measurement process, so that the detection of aldosteronol factor concentration can be carried out simply, conveniently, quickly and in batches, while ensuring a small systematic error in detection.
  • the present invention detects aldosteronism factors by chemiluminescence immunoassay, avoids the use of radioactive markers that pollute the environment and endanger human health, is safer and environmentally friendly; at the same time, the markers of the present invention are not only safe but also relatively stable. The problem of short half-life of the label present in prior art methods such as radioimmunoassay is overcome.
  • a I antigen purchased from Sigma;
  • Anti-A I antibody purchased from Biogenesis;
  • a II antigen purchased from Sigma;
  • Anti-A II antibody purchased from Biogenesis;
  • ALD antigen purchased from Sigma;
  • Anti-ALD antibody purchased from Meridian;
  • Sheep anti-FITC polyclonal antibody purchased from Jackson, USA;
  • the magnetic microspheres are produced by Shenzhen New Industry Biomedical Engineering Co., Ltd., with 80% particle size distribution of 1-5 ⁇ m, sedimentation time of 10-15 seconds when magnetization is 4000 Gauss, and protein adsorption concentration of 0.8mg when BSA is 30mg. -1.2mg;
  • Biotin, streptavidin all purchased from Biosources, USA;
  • ABEI provided by Shenzhen New Industry Biomedical Engineering Co., Ltd.;
  • the Maglumi 2000 chemiluminescence analyzer was supplied by Shenzhen New Industry Biomedical Engineering Co., Ltd.
  • a I antigen 100 ⁇ g was adjusted to 1 ml with 0.1 mol/L pH 9.5 carbonate buffer (F solution). The dialyzate was placed, and the mixture was dialyzed for 2 hours at room temperature, and the dialyzed solution was added to 300 ⁇ g of ABEI-activated ester, and reacted at 37 ° C for 2 hours.
  • the ligation product of the A I antigen and ABEI was purified by a G-25 gel column.
  • D2 solution 200 ml of 0.5 M phosphate buffer (P001 solution), 20 g of BSA, 8 g of NaN3, 2 g of MgCl2 ⁇ 6H2O, and 600 ml of glycerin were added to a 2000 ml beaker, diluted with purified water to 2000 ml, and filtered.
  • P001 solution 0.5 M phosphate buffer
  • BSA BSA
  • MgCl2 ⁇ 6H2O 600 ml of glycerin
  • the purified ligation product was double-diluted with a D 2 solution to obtain an A I antigen labeled with ABEI.
  • Preparation of solution A Weigh 2.55g of sodium acetate trihydrate into a 5000ml beaker, measure 4500ml of purified water into a beaker by using a measuring cylinder, add 14ml of acetic acid after mixing, and then add purified water to 5000ml (pH is 3.6).
  • the magnetic beads having a particle diameter of 1 ⁇ m were added to an equal volume of pH 3.6 acetate buffer to make the suspension concentration of the magnetic beads 20 mg/ml, and then 1-cyclohexyl-2-morpholine ethylcarbazone was added. Amine p-toluenesulfonate (CMC) was added to a concentration of 10 mg/ml and purified anti-A I antibody was added.
  • CMC Amine p-toluenesulfonate
  • the magnetic bead suspension was placed in a constant temperature shaking water bath for 24 hours at 37 ° C (shaking water bath shaking speed: 260 rpm).
  • solution C 160 g of MC (methylcellulose) was weighed and poured into a 5000 ml beaker, and purified water was added to 4000 ml, and the mixture was heated and stirred in a water bath at 90 ° C for 2 hours. Another 4000 ml of 0.5 M phosphate buffer was added, 80 g of NaN 3 (analytical grade), 80 ml of Tween-20 (analytical grade) were added, mixed, and filtered. After the two solutions were thoroughly mixed, 200 g of BSA was added, and water was added to 40,000 ml.
  • MC methylcellulose
  • the washed magnetic beads were suspended in a C solution at a suspension concentration of 20 mg/ml to obtain an anti-A I antibody-coated magnetic sphere, and the volume of the suspension was the coating volume described in the examples.
  • the above magnetic sphere suspension was further diluted to a suspension of 0.1 mg/ml in terms of a magnetic sphere, and was used.
  • the A I antigen was diluted with 50% bovine serum preparations into two high and low calibrator calibration points at concentrations of 14.386 ng/ml and 0.667 ng/ml, respectively.
  • a I antigen 100 ⁇ g was adjusted to 1 ml with 0.1 mol/L pH 9.5 carbonate buffer (F solution). The dialyzate was placed, and the mixture was dialyzed for 2 hours at room temperature, and the dialyzed solution was added to 300 ⁇ g of ABEI-activated ester, and reacted at 37 ° C for 2 hours.
  • the ligation product of the A I antigen and ABEI was purified by a G-25 gel column.
  • D 2 solution 200 ml of 0.5 M phosphate buffer, 20 g of BSA, 8 g of NaN 3 , 2 g of MgCl 2 ⁇ 6H 2 O, 600 ml of glycerol were added to a 2000 ml beaker, diluted with purified water to 2000 ml, and filtered.
  • the purified ligation product was diluted with a D 2 solution.
  • the solution A was prepared as in Example A-1.
  • the magnetic beads were added to a volume equivalent of pH 3.6 acetate buffer to make the magnetic beads have a suspension concentration of 20 mg/ml, and then CMC (concentration: 10 mg/ml) was added, and purified goat anti-FITC polyclonal antibody was added.
  • the magnetic bead suspension was placed in a constant temperature shaking water bath for 24 hours at 37 ° C (shaking water bath shaking speed: 260 rpm).
  • the magnetic beads after washing are suspended in the C solution at a concentration of 20 mg/ml, that is, a magnetic ball suspension coated with a goat anti-FITC polyclonal antibody is obtained, and the volume of the suspension is coated as described in the present embodiment. volume.
  • the above magnetic sphere suspension was further diluted to a suspension of 0.5 mg/ml in terms of a magnetic sphere, and was used.
  • the ligation product of the anti-A I antibody and FITC was purified by G-25 gel column.
  • the purified ligation product was diluted with a C 2 solution.
  • the A I antigen was diluted with 50% bovine serum preparation into two high and low calibrator calibration points at a concentration of 15.50 ng/ml and 0.85 ng/ml.
  • a suitable dialysis bag (common molecular weight 14000) was used to measure the size of the F solution. After wetting, the end was tightened and the purified water was leaked 3 times (no leakage required).
  • the ligation product of the anti-A I antibody and ABEI was purified by G-25 gel column.
  • the D 2 solution was prepared as in Example A-1.
  • the purified ligation product may be diluted with a D 2 solution.
  • the solution A was prepared as in Example A-1.
  • the magnetic beads were placed in a constant temperature shaking water bath for 24 hours at 37 ° C (shaking water bath shaking speed: 260 rpm).
  • the C solution was prepared in accordance with the procedure of Example A-1.
  • the washed magnetic beads were suspended in a C solution at a suspension concentration of 20 mg/ml to obtain an anti-A I antibody-coated magnetic sphere suspension, and the volume of the suspension was the coating volume described in the examples.
  • the above magnetic sphere suspension was further diluted to a suspension of 1 mg/ml in terms of a magnetic sphere, and was used.
  • the A I antigen was diluted with 50% bovine serum preparations into two high and low calibrator calibration points at concentrations of 14.718 ng/ml and 0.780 ng/ml, respectively.
  • the ligation product of the anti-A I antibody and ABEI was purified by G-25 gel column.
  • a D 2 solution was prepared in accordance with the procedure of Example A-1.
  • the purified ligation product was diluted with a D 2 solution.
  • the solution A was prepared as in Example A-1.
  • the magnetic beads were added to a volume equal volume of pH 3.6 acetate buffer to make the magnetic beads suspension concentration of 20 mg/ml, and then CMC (concentration: 10 mg/ml) was added, and the purified goat anti-FITC polyclonal was added at a certain ratio. antibody.
  • the magnetic beads were placed in a constant temperature shaking water bath for 24 hours at 37 ° C (shaking water bath shaking speed: 260 rpm).
  • the magnetic beads after the washing is suspended in the C solution at a suspension concentration of 20 mg/ml, that is, a magnetic ball suspension coated with a goat anti-FITC polyclonal antibody is obtained, and the volume of the suspension is the coating volume described in this embodiment. .
  • the above magnetic sphere suspension was further diluted to a suspension of 0.5 mg/ml in terms of a magnetic sphere, and was used.
  • a I antigen 100 ⁇ g of A I antigen was adjusted to 1 ml with 0.1 mol/L pH 9.5 carbonate buffer (F solution). The dialyzate was placed in the dialysate, and the mixture was dialyzed for 2 hours at room temperature. The dialyzed solution was added to 300 ⁇ g of FITC, and the mixture was shaken at room temperature for 24 hours.
  • the ligation product of the A I antigen and FITC was purified by a G-25 gel column.
  • the purified ligation product is diluted with a C 2 solution.
  • the A I antigen was diluted with 50% bovine serum preparations into two high and low calibrator calibration points of 16.480 ng/ml and 2.213 ng/ml, respectively.
  • the ligation product of the anti-A I antibody and ABEI was purified by a G-25 gel column.
  • the D 2 solution was prepared according to the procedure of Example A-1, and the purified ligation product was diluted with D 2 solution.
  • the purified ligation product may be diluted with a D 2 solution.
  • the solution A was prepared in the same manner as in Example A-1.
  • the magnetic beads were added to an equal volume of pH 3.6 acetate buffer to make the magnetic beads have a suspension concentration of 20 mg/ml, and then CMC (concentration: 10 mg/ml) was added, and purified SA was added.
  • the magnetic bead suspension was placed in a constant temperature shaking water bath for 24 hours at 37 ° C (shaking water bath shaking speed: 260 rpm).
  • the cleaned magnetic beads were suspended in a C solution at a suspension concentration of 20 mg/ml to obtain an SA-coated magnetic sphere suspension, and the volume of the suspension was the coating volume described in this example.
  • the above magnetic sphere suspension was further diluted to a suspension of 0.1 mg/ml in terms of a magnetic sphere, and was used.
  • the A I antigen was diluted with 50% bovine serum preparations into two high and low calibrator calibration points at a concentration of 16.668 ng/ml and 1.667 ng/ml.
  • D2 solution is prepared, and the purified ligation product is diluted with D 2 solution.
  • the purified ligation product can be diluted with D 2 solution.
  • the solution A was prepared in accordance with the method of Example A-1.
  • the magnetic bead suspension was placed in a constant temperature shaking water bath for 24 hours at 37 ° C (shaking water bath shaking speed: 260 rpm).
  • the C solution was prepared in accordance with the method of Example A-1.
  • the washed magnetic beads were suspended in a C solution at a suspension concentration of 20 mg/ml to obtain an anti-A I antibody-coated magnetic sphere suspension, and the volume of the suspension was the coating volume described in this example.
  • the above magnetic sphere suspension was further diluted to a suspension of 0.1 mg/ml in terms of a magnetic sphere, and was used.
  • the A I antigen was diluted with 50% bovine serum preparations into two high and low calibrator calibration points at concentrations of 15.386 ng/ml and 1.367 ng/ml.
  • a I antigen-BSA protein conjugate 100 ⁇ g was adjusted to 1 ml with 0.1 mol/L pH 9.5 carbonate buffer (F solution). The dialyzate was placed, and the mixture was dialyzed for 2 hours at room temperature, and the dialyzed solution was added to 300 ⁇ g of ABEI-activated ester, and reacted at 37 ° C for 2 hours.
  • the ligation product of the A I antigen-BSA protein linker with ABEI was purified by G-25 gel column.
  • D 2 solution 200 ml of 0.5 M phosphate buffer (P001 solution), 20 g of BSA, 8 g of NaN 3 , 2 g of MgCl 2 ⁇ 6H 2 O, 600 ml of glycerol were added to a 2000 ml beaker, diluted with purified water to 2000 ml, and filtered.
  • P001 solution 0.5 M phosphate buffer
  • BSA 8 g of NaN 3
  • MgCl 2 ⁇ 6H 2 O 600 ml of glycerol
  • the purified ligation product was double-diluted with a D 2 solution to obtain an A I antigen-BSA protein linker labeled with ABEI.
  • Preparation of solution A Weigh 2.55g of sodium acetate trihydrate into a 5000ml beaker, measure 4500ml of purified water into a beaker by using a measuring cylinder, add 14ml of acetic acid after mixing, and then add purified water to 5000ml (pH is 3.6).
  • the magnetic beads having a particle diameter of 1 ⁇ m were added to a buffering solution of pH 3.6 in the same volume as the coating volume, the suspension concentration of the magnetic beads was 20 mg/ml, and then CMC (concentration: 10 mg/ml) was added, and the purified anti-A was added. I antibody.
  • the magnetic bead suspension was placed in a constant temperature shaking water bath for 24 hours at 37 ° C (shaking water bath shaking speed: 260 rpm).
  • PBS pH 7.4 phosphate buffer solution
  • the washed magnetic beads were suspended in a C solution at a suspension concentration of 20 mg/ml to obtain an anti-A I antibody-coated magnetic sphere, and the volume of the suspension was the coating volume described in the examples.
  • the above magnetic sphere suspension was further diluted to a suspension of 0.1 mg/ml in terms of a magnetic sphere, and was used.
  • the A I antigen was diluted with 50% bovine serum preparations into two high and low calibrator calibration points at concentrations of 14.386 ng/ml and 0.667 ng/ml, respectively.
  • the ligation product of the anti-A I antibody and ABEI was purified by G-25 gel column.
  • a D 2 solution was prepared in accordance with the procedure of Example A-1.
  • the purified ligation product was diluted with a D 2 solution.
  • the solution A was prepared as in Example A-1.
  • the magnetic beads were added to a volume equal volume of pH 3.6 acetate buffer to make the magnetic beads suspension concentration of 20 mg/ml, and then CMC (concentration: 10 mg/ml) was added, and the purified goat anti-FITC polyclonal was added at a certain ratio. antibody.
  • the magnetic beads were placed in a constant temperature shaking water bath for 24 hours at 37 ° C (shaking water bath shaking speed: 260 rpm).
  • the magnetic beads after the washing is suspended in the C solution at a suspension concentration of 20 mg/ml, that is, a magnetic ball suspension coated with a goat anti-FITC polyclonal antibody is obtained, and the volume of the suspension is the coating volume described in this embodiment. .
  • the above magnetic sphere suspension was further diluted to a suspension of 0.1 mg/ml in terms of a magnetic sphere, and was used.
  • a I antigen-BSA protein conjugate 100 ⁇ g was adjusted to 1 ml with 0.1 mol/L pH 9.5 carbonate buffer (F solution). The dialyzate was placed in the dialysate, and the mixture was dialyzed for 2 hours at room temperature. The dialyzed solution was added to 300 ⁇ g of FITC, and the mixture was shaken at room temperature for 24 hours.
  • the ligation product of the A I antigen-BSA protein linker and FITC was purified by G-25 gel column.
  • the purified ligation product is diluted with a C 2 solution.
  • the A I antigen was diluted with 50% bovine serum preparations into two high and low calibrator calibration points of 16.480 ng/ml and 2.213 ng/ml, respectively.
  • the A I concentration of the sample was detected by a chemiluminescence immunocompetence assay, and the sample to be tested was 160 clinical samples.
  • the A I concentration is proportional to the relative light intensity (RLU), and the analyzer can be used to automatically fit the calculated A I concentration.
  • the sample to be tested is a sample treated by an enzyme inhibitor.
  • the sample is divided into two parts, one is placed at 2-8 ° C, and one is placed at 37 ° C. After accurately counting for one hour, the samples of the two treatment methods are placed. In the sample holder.
  • the sample to be tested is a sample treated by an enzyme inhibitor.
  • the sample is divided into two parts, one is placed at 2-8 ° C, and one is placed at 37 ° C. After accurately counting for one hour, the samples of the two treatment methods are placed. In the sample holder.
  • the sample to be tested is a sample treated by an enzyme inhibitor.
  • the sample is divided into two parts, one is placed at 2-8 ° C, and one is placed at 37 ° C. After accurately counting for one hour, the samples of the two treatment methods are placed. In the sample holder.
  • Example A-9 120 clinical samples of Example A-9 were tested using a commercially available commercial radioimmunoassay kit available on the market. The results are shown in Table A-1.
  • the A I detection kit and the detection method provided by the present invention have higher accuracy.
  • the renin level in the vertical position is generally low.
  • the samples obtained by using the kits prepared in Example A-1, Example A-4 and Example A-8 were close to the lower limit of the confidence interval, indicating that the level of renin activity in the vertical position was extremely low, and the clinical diagnosis of the sample.
  • the results of the vertical and horizontal position detection using the radioimmunoassay kit are close to the upper limit of the confidence interval, which is obviously inconsistent with the actual clinical situation. Therefore, the detection effect of the A I detection kit and the detection method thereof provided by the invention is better than that of the comparison radioimmunization kit, and the clinical situation can be more accurately and truly reflected.
  • the A I test kit prepared in the above other examples has been clinically tested, and the effects are consistent with those of the embodiment A-1, the embodiment A-4 and the embodiment A-7, and are not considered here for the sake of space saving. List the inspection data.
  • the measured value of the A I test kit provided according to the present invention is more consistent with the actual value, and the clinical coincidence rate is higher, indicating that the kit has a stronger diagnostic ability.
  • the A I detection kit provided by the present invention has higher stability, safety in use, and environmental friendliness than the radioimmunoassay kit.
  • a II antigen 100 ⁇ g was adjusted to 1 ml with 0.1 mol/L pH 9.5 carbonate buffer (F solution). The dialyzate was placed, and the mixture was dialyzed for 2 hours at room temperature, and the dialyzed solution was added to 300 ⁇ g of ABEI-activated ester, and reacted at 37 ° C for 2 hours.
  • the ligation product of the A II antigen and ABEI was purified by a G-25 gel column.
  • D 2 solution 200 ml of 0.5 M phosphate buffer (P001 solution), 20 g of BSA, 8 g of NaN 3 , 2 g of MgCl 2 ⁇ 6H 2 O, 600 ml of glycerol were added to a 2000 ml beaker, diluted with purified water to 2000 ml, and filtered.
  • P001 solution 0.5 M phosphate buffer
  • BSA 8 g of NaN 3
  • MgCl 2 ⁇ 6H 2 O 600 ml of glycerol
  • the purified ligation product was double-diluted with a D 2 solution to obtain an A II antigen labeled with ABEI.
  • Preparation of solution A Weigh 2.55g of sodium acetate trihydrate into a 5000ml beaker, measure 4500ml of purified water into a beaker by using a measuring cylinder, add 14ml of acetic acid after mixing, and then add purified water to 5000ml (pH is 3.6).
  • a magnetic particle having a particle diameter of 1 ⁇ m was added to a buffering solution of pH 3.6 in an equal volume of the coated solution to make the magnetic sphere have a suspension concentration of 20 mg/ml, and then CMC (concentration: 10 mg/ml) was added, and purified anti-A was added.
  • the magnetic ball suspension was placed in a constant temperature shaking water bath for 24 hours at 37 ° C (shaking water bath shaking speed: 260 rpm).
  • solution C 160 g of MC (methylcellulose) was weighed and poured into a 5000 ml beaker, and purified water was added to 4000 ml, and the mixture was heated and stirred in a water bath at 90 ° C for 2 hours. Another 4000 ml of 0.5 M phosphate buffer was added, 80 g of NaN 3 (analytical grade), 80 ml of Tween-20 (analytical grade) were added, mixed, and filtered. After the two solutions were thoroughly mixed, 200 g of BSA was added, and water was added to 40,000 ml.
  • MC methylcellulose
  • the magnetic sphere after washing is suspended in the C solution at a suspension concentration of 20 mg/ml, that is, an anti-A II antibody-coated magnetic sphere is obtained, and the volume of the suspension is the coating volume described in the step.
  • the above magnetic sphere suspension was further diluted to a suspension of 0.1 mg/ml in terms of a magnetic sphere, and was used.
  • the A II antigen was diluted with 50% bovine serum preparations into two high and low calibrator calibration points at concentrations of 613.238 pg/ml and 32.614 pg/ml, respectively.
  • a II antigen 100 ⁇ g was adjusted to 1 ml with 0.1 mol/L pH 9.5 carbonate buffer (F solution). The dialyzate was placed, and the mixture was dialyzed for 2 hours at room temperature, and the dialyzed solution was added to 300 ⁇ g of ABEI-activated ester, and reacted at 37 ° C for 2 hours.
  • the ligation product of the A II antigen and ABEI was purified by a G-25 gel column.
  • D 2 solution 200 ml of 0.5 M phosphate buffer, 20 g of BSA, 8 g of NaN 3 , 2 g of MgCl 2 ⁇ 6H 2 O, 600 ml of glycerol were added to a 2000 ml beaker, diluted with purified water to 2000 ml, and filtered.
  • the purified ligation product was diluted with a D 2 solution.
  • the solution A was prepared as in Example B-1.
  • the magnetic spheres were added to a volumetric equivalent of pH 3.6 acetate buffer to make the magnetic spheres have a suspension concentration of 20 mg/ml, and then CMC (concentration: 10 mg/ml) was added, and purified goat anti-FITC polyclonal antibody was added.
  • the magnetic ball suspension was placed in a constant temperature shaking water bath for 24 hours at 37 ° C (shaking water bath shaking speed: 260 rpm).
  • the C solution was prepared in the same manner as in Example B-1.
  • the magnetic ball after washing is suspended in the C solution at a concentration of 20 mg/ml, that is, a magnetic ball coated with a goat anti-FITC polyclonal antibody is obtained, and the volume of the suspension is the coating volume described in the step.
  • the above magnetic sphere suspension was further diluted to a suspension of 0.5 mg/ml in terms of a magnetic sphere, and was used.
  • the ligation product of the anti-A II antibody and FITC was purified by G-25 gel column.
  • C 2 solution 200 ml of 0.5 M phosphate buffer, 20 g of BSA, 8 g of NaN 3 8 g, 2 g of MgCl 2 ⁇ 6H 2 O, and purified water were added to 2000 ml (filtered). The C 2 solution was prepared, and the purified ligation product was diluted with a C 2 solution.
  • the A II antigen was diluted with 50% bovine serum preparations into two high and low calibrator calibration points at concentrations of 591.578 pg/ml and 42.260 pg/ml.
  • a suitable dialysis bag (common molecular weight 14000) was used to measure the size of the F solution. After wetting, the end was tightened and the purified water was leaked 3 times (no leakage required).
  • the ligation product of the anti-A II antibody and ABEI was purified by G-25 gel column.
  • the D 2 solution was prepared as in Example B-1.
  • the purified ligation product was diluted with a D 2 solution.
  • the solution A was prepared as in Example B-1.
  • the magnetic sphere was added to the volume equivalent of pH 3.6 acetate buffer to make the suspension concentration of the magnetic sphere 20 mg/ml, and then 1-cyclohexyl-2-morpholine ethylcarbodiimide p-toluenesulfonic acid was added.
  • Salt (CMC) to a concentration of 10 mg/ml, adding purified A II antigen at a certain ratio
  • the magnetic ball was placed in a constant temperature shaking water bath for 24 hours at 37 ° C (shaking water bath shaking speed: 260 rpm).
  • the C solution was prepared in the same manner as in Example B-1.
  • the magnetic sphere after washing is suspended in the C solution at a suspension concentration of 20 mg/ml, that is, an anti-A II antibody-coated magnetic sphere is obtained, and the volume of the suspension is the coating volume described in the step.
  • the above magnetic sphere suspension was further diluted to a suspension of 1 mg/ml in terms of a magnetic sphere, and was used.
  • the A II antigen was diluted with 50% bovine serum preparations into two high and low calibrator calibration points at concentrations of 562.34 pg/ml and 17.78 pg/ml, respectively.
  • the ligation product of the anti-A II antibody and ABEI was purified by G-25 gel column.
  • a D 2 solution was prepared in accordance with the procedure of Example B-1.
  • the purified ligation product was diluted with a D 2 solution.
  • the solution A was prepared as in Example B-1.
  • the magnetic ball was placed in a constant temperature shaking water bath for 24 hours at 37 ° C (shaking water bath shaking speed: 260 rpm).
  • the C solution was prepared in the same manner as in Example B-1.
  • the magnetic ball coated with the goat anti-FITC polyclonal antibody after washing is suspended in the C solution at a suspension concentration of 20 mg/ml to obtain a magnetic ball coated with the goat anti-FITC polyclonal antibody, and the volume of the suspension is The step covers the volume.
  • the above magnetic sphere suspension was further diluted to a suspension of 0.05 mg/ml in terms of magnetic spheres, and was used.
  • a II antigen 100 ⁇ g was adjusted to 1 ml with 0.1 mol/L pH 9.5 carbonate buffer (F solution).
  • the dialyzate was placed in the dialysate, and the mixture was dialyzed for 2 hours at room temperature.
  • the dialyzed solution was added to 300 ⁇ g of FITC, and the mixture was shaken at room temperature for 24 hours.
  • the ligation product of A II antigen and FITC was purified by G-25 gel column.
  • the purified ligation product is diluted with a C 2 solution.
  • the A II antigen was diluted to a concentration of 645.12 pg/ml and 46.50 with 50% bovine serum preparations. Pg/ml two high and low calibrator calibration points.
  • the ligation product of the anti-A II antibody and ABEI was purified by a G-25 gel column.
  • the D 2 solution was prepared according to the procedure of Example B-1, and the purified ligation product was diluted with D 2 solution.
  • the purified ligation product was diluted with a D 2 solution.
  • the solution A was prepared in the same manner as in Example B-1.
  • the magnetic sphere was added to an equal volume of pH 3.6 acetate buffer to make a magnetic sphere suspension concentration of 20 mg/ml, and then CMC (concentration: 10 mg/ml) was added, and purified SA was added.
  • the magnetic ball suspension was placed in a constant temperature shaking water bath for 24 hours at 37 ° C (shaking water bath shaking speed: 260 rpm).
  • the C solution was prepared in the same manner as in Example B-1.
  • the SA coated magnetic sphere after the cleaning is suspended in the C solution at a suspension concentration of 20 mg/ml, that is, the SA coated magnetic sphere is obtained, and the volume of the suspension is the coating volume described in the step.
  • the above magnetic sphere suspension was further diluted to a suspension of 0.5 mg/ml in terms of a magnetic sphere, and was used.
  • the A II antigen was diluted with 50% bovine serum preparations into two high and low calibrator calibration points at a concentration of 563.245 pg/ml and 15.337 pg/ml.
  • the purified ligation product can be diluted with D 2 solution.
  • the solution A was prepared in accordance with the method of Example B-1.
  • the magnetic sphere was added to a volume equal volume of pH 3.6 acetate buffer to make the magnetic sphere suspension concentration 20 mg/ml, and then CMC (concentration: 10 mg/ml) was added, and the purified A II antigen was added.
  • the magnetic ball suspension was placed in a constant temperature shaking water bath for 24 hours at 37 ° C (shaking water bath shaking speed: 260 rpm).
  • the magnetic ball after the washing was suspended in the C solution at a suspension concentration of 20 mg/ml to obtain a magnetic ball coated with the anti-A II antibody.
  • the above magnetic sphere suspension was further diluted to a suspension of 0.5 mg/ml in terms of a magnetic sphere, and was used.
  • the A II antigen was diluted with 50% bovine serum preparation into two high and low calibrator calibration points at a concentration of 515.73 pg/ml and 9.70 pg/ml.
  • a II antigen-BSA protein linker 100 ⁇ g was adjusted to 1 ml with 0.1 mol/L pH 9.5 carbonate buffer (F solution). The dialyzate was placed, and the mixture was dialyzed for 2 hours at room temperature, and the dialyzed solution was added to 300 ⁇ g of ABEI-activated ester, and reacted at 37 ° C for 2 hours.
  • the ligation product of the A II antigen-BSA protein linker to ABEI was purified by G-25 gel column.
  • D 2 solution 200 ml of 0.5 M phosphate buffer, 20 g of BSA, 8 g of NaN 3 , 2 g of MgCl 2 ⁇ 6H 2 O, 600 ml of glycerol were added to a 2000 ml beaker, diluted with purified water to 2000 ml, and filtered.
  • the purified ligation product was diluted with a D 2 solution.
  • the solution A was prepared as in Example B-1.
  • the magnetic spheres were added to a volumetric equivalent of pH 3.6 acetate buffer to make the magnetic spheres have a suspension concentration of 20 mg/ml, and then CMC (concentration: 10 mg/ml) was added, and purified goat anti-FITC polyclonal antibody was added.
  • the magnetic ball suspension was placed in a constant temperature shaking water bath for 24 hours at 37 ° C (shaking water bath shaking speed: 260 rpm).
  • the C solution was prepared in the same manner as in Example B-1.
  • the magnetic ball after washing is suspended in the C solution at a concentration of 20 mg/ml, that is, a magnetic ball coated with a goat anti-FITC polyclonal antibody is obtained, and the volume of the suspension is the coating volume described in the step.
  • the above magnetic sphere suspension was further diluted to a suspension of 0.5 mg/ml in terms of a magnetic sphere, and was used.
  • the ligation product of the anti-A II antibody and FITC was purified by G-25 gel column.
  • C 2 solution 200 ml of 0.5 M phosphate buffer, 20 g of BSA, 8 g of NaN 3 , 2 g of MgCl 2 ⁇ 6H 2 O, and purified water were added to 2000 ml (filtered). The C 2 solution was prepared, and the purified ligation product was diluted with a C 2 solution.
  • the A II antigen was diluted with 50% bovine serum preparations into two high and low calibrator calibration points at concentrations of 591.578 pg/ml and 42.260 pg/ml.
  • Example B-8 Chemiluminescence Detection Using a Detection Kit A II
  • the A II concentration of the sample was detected by a chemiluminescence immunocompetence assay, and the sample to be tested was 160 clinical samples.
  • the A II concentration is proportional to the relative light intensity (RLU), and the analyzer can be used to automatically fit the calculated A II concentration.
  • the sample to be tested is the sample treated by the enzyme inhibitor.
  • the specific loading step is: add 100 ⁇ l to the calibrator or sample to be tested, and then add the tracer labeled A II antigen solution. 50 ⁇ l, add anti-A II antibody coated magnetic microsphere suspension 20 ⁇ l, mix, incubate at 37 °C for 15 minutes, the instrument automatically cleans twice and directly into the measurement room to obtain the light intensity signal of each sample, through the ten point curve Two-point calibration automatically fits the A II antigen concentration value of the sample to be tested. The test results are shown in Table B-1.
  • the sample to be tested is the sample treated by the enzyme inhibitor.
  • the sample to be tested is the sample treated by the enzyme inhibitor.
  • Example B-8 The 160 clinical samples in Example B-8 were tested using the mainstream commercial radioimmunoassay kit available on the market. The test results are shown in Table B-1.
  • Sample No. 148 is a sample taken from patients with primary aldosteronism, and the level of A II in the supine position is generally low.
  • the results obtained by using the A II test kit prepared in Example B-1, Example B-4 and Example B-7 were close to the lower limit of the confidence interval, indicating that the level of the A II in the vertical position was extremely low, and the sample was The clinical diagnosis is consistent.
  • the results of the vertical and horizontal position detection using the radioimmunoassay kit are close to the upper limit of the confidence interval, which is obviously inconsistent with the actual clinical situation. Therefore, the detection effect of the A II detection kit and the detection method thereof provided by the invention is better than that of the comparison radioimmunization kit, and the clinical situation can be more accurately and truly reflected.
  • the A II test kit prepared in the above other examples has been clinically tested, and the effects are consistent with those of the embodiment B-1, the embodiment B-4 and the embodiment B-7, and are not considered here for the sake of space saving. List the inspection data.
  • the measured value of the A II test kit provided according to the present invention is more in conformity with the actual value, and the clinical coincidence rate is higher, indicating that the kit has a higher diagnostic ability.
  • the A II detection kit provided by the present invention has higher stability, safety of use and environmental friendliness compared to the radioimmunoassay kit.
  • ALD antigen 100 ⁇ g of ALD antigen was adjusted to 1 ml with 0.1 mol/L pH 9.5 carbonate buffer (F solution). The dialyzate was placed, and the mixture was dialyzed for 2 hours at room temperature, and the dialyzed solution was added to 300 ⁇ g of ABEI-activated ester, and reacted at 37 ° C for 2 hours.
  • the ligation product of the ALD antigen and ABEI was purified by a G-25 gel column.
  • D 2 solution 200 ml of 0.5 M phosphate buffer (P001 solution), 20 g of BSA, 8 g of NaN 3 , 2 g of MgCl 2 ⁇ 6H 2 O, 600 ml of glycerol were added to a 2000 ml beaker, diluted with purified water to 2000 ml, and filtered.
  • P001 solution 0.5 M phosphate buffer
  • BSA 8 g of NaN 3
  • MgCl 2 ⁇ 6H 2 O 600 ml of glycerol
  • the purified ligation product was double-diluted with a D 2 solution to obtain an ALD antigen labeled with ABEI.
  • Preparation of solution A Weigh 2.55g of sodium acetate trihydrate into a 5000ml beaker, measure 4500ml of purified water into a beaker by using a measuring cylinder, add 14ml of acetic acid after mixing, and then add purified water to 5000ml (pH is 3.6).
  • a magnetic particle having a particle diameter of 1 ⁇ m was added to an equal volume of pH 3.6 acetate buffer to make a suspension concentration of the magnetic sphere of 20 mg/ml, and then 1-cyclohexyl-2-morpholine ethylcarbazone was added.
  • the amine p-toluenesulfonate (CMC) was added to a concentration of 10 mg/ml and a purified anti-ALD antibody was added.
  • the magnetic ball suspension was placed in a constant temperature shaking water bath for 24 hours at 37 ° C (shaking water bath shaking speed: 260 rpm).
  • solution C 160 g of MC (methylcellulose) was weighed and poured into a 5000 ml beaker, and purified water was added to 4000 ml, and the mixture was heated and stirred in a water bath at 90 ° C for 2 hours. Another 4000 ml of 0.5 M phosphate buffer was added, 80 g of NaN 3 (analytical grade), 80 ml of Tween-20 (analytical grade) were added, mixed, and filtered. After the two solutions were thoroughly mixed, 200 g of BSA was added, and water was added to 40,000 ml.
  • MC methylcellulose
  • the magnetic ball of the anti-ALD antibody after washing is suspended in the C solution at a suspension concentration of 20 mg/ml to obtain an anti-ALD antibody-coated magnetic sphere suspension, and the volume of the suspension is the coating volume of the step. .
  • the above magnetic sphere suspension was further diluted to a suspension of 0.1 mg/ml in terms of a magnetic sphere, and was used.
  • the ALD antigen was diluted with 50% bovine serum preparations into two high and low calibrator calibration points at concentrations of 1226.476 pg/ml and 65.228 pg/ml, respectively.
  • ALD antigen 100 ⁇ g of ALD antigen was adjusted to 1 ml with 0.1 mol/L pH 9.5 carbonate buffer (F solution). The dialyzate was placed, and the mixture was dialyzed for 2 hours at room temperature, and the dialyzed solution was added to 300 ⁇ g of ABEI-activated ester, and reacted at 37 ° C for 2 hours.
  • the ligation product of the ALD antigen and ABEI was purified by a G-25 gel column.
  • D 2 solution 200 ml of 0.5 M phosphate buffer, 20 g of BSA, 8 g of NaN 3 , 2 g of MgCl 2 ⁇ 6H 2 O, 600 ml of glycerol were added to a 2000 ml beaker, diluted with purified water to 2000 ml, and filtered.
  • the purified ligation product was diluted with a D 2 solution.
  • the solution A was prepared as in Example C-1.
  • the magnetic ball suspension was placed in a constant temperature shaking water bath for 24 hours at 37 ° C (shaking water bath shaking speed: 260 rpm).
  • the C solution was prepared in the same manner as in Example C-1.
  • the washed anti-FITC polyclonal antibody-coated magnetic sphere was suspended in a C solution at a concentration of 20 mg/ml to obtain a magnetic ball suspension coated with a goat anti-FITC polyclonal antibody, and the volume of the suspension was That is, the volume of the coating described in this step.
  • the above magnetic sphere suspension was further diluted to a suspension of 0.5 mg/ml in terms of a magnetic sphere, and was used.
  • 1 mg of the anti-ALD antibody was adjusted to 1 ml with 0.1 mol/L pH 9.5 carbonate buffer (F solution).
  • the dialyzate was placed in the dialysate, and the mixture was dialyzed for 2 hours at room temperature.
  • the dialyzed solution was added to 300 ⁇ g of FITC, and the mixture was shaken at room temperature for 24 hours.
  • the ligation product of the anti-ALD antibody and FITC was purified by G-25 gel column.
  • the purified ligation product was diluted with a C 2 solution.
  • the ALD antigen was diluted with 50% bovine serum preparations into two high and low calibrator calibration points at a concentration of 1183.156 pg/ml and 84.520 pg/ml.
  • a suitable dialysis bag (common molecular weight 14000) was used to measure the size of the F solution. After wetting, the end was tightened and the purified water was leaked 3 times (no leakage required).
  • the ligation product of the anti-ALD antibody to ABEI was purified by G-25 gel column.
  • the D 2 solution was prepared as in Example C-1.
  • the purified ligation product was diluted with a D 2 solution.
  • the solution A was prepared as in Example C-1.
  • the magnetic ball was placed in a constant temperature shaking water bath for 24 hours at 37 ° C (shaking water bath shaking speed: 260 rpm).
  • the C solution was prepared in the same manner as in Example C-1.
  • the magnetic ball coated with the anti-ALD antibody after washing is suspended in the C solution at a suspension concentration of 20 mg/ml to obtain an anti-ALD antibody-coated magnetic sphere suspension, and the volume of the suspension is the package described in this step. Being volume.
  • the above magnetic sphere suspension was further diluted to a suspension of 1 mg/ml in terms of a magnetic sphere, and was used.
  • the ALD antigen was diluted with 50% bovine serum preparations into two high and low calibrator calibration points at concentrations of 1124.686 pg/ml and 35.566 pg/ml, respectively.
  • the ligation product of the anti-ALD antibody to ABEI was purified by G-25 gel column.
  • a D 2 solution was prepared in accordance with the procedure of Example C-1.
  • the purified ligation product was diluted with a D 2 solution.
  • the solution A was prepared as in Example C-1.
  • the magnetic ball was placed in a constant temperature shaking water bath for 24 hours at 37 ° C (shaking water bath shaking speed: 260 rpm).
  • the C solution was prepared in the same manner as in Example C-1.
  • the magnetic ball of the goat anti-FITC polyclonal antibody after washing is suspended in the C solution at a suspension concentration of 20 mg/ml to obtain a magnetic ball suspension coated with the goat anti-FITC polyclonal antibody, and the volume of the suspension is The step covers the volume.
  • the above magnetic sphere suspension was further diluted to a suspension of 0.05 mg/ml in terms of magnetic spheres, and was used.
  • ALD antigen 100 ⁇ g of ALD antigen was adjusted to 1 ml with 0.1 mol/L pH 9.5 carbonate buffer (F solution). The dialyzate was placed in the dialysate, and the mixture was dialyzed for 2 hours at room temperature. The dialyzed solution was added to 300 ⁇ g of FITC, and the mixture was shaken at room temperature for 24 hours.
  • the ligation product of ALD antigen and FITC was purified by G-25 gel column.
  • the purified ligation product is diluted with a C 2 solution.
  • the ALD antigen was diluted with 50% bovine serum preparations into two high and low calibrator calibration points at concentrations of 1290.242 pg/ml and 93.214 pg/ml, respectively.
  • the ligation product of the anti-ALD antibody and ABEI was purified by G-25 gel column.
  • the D 2 solution was prepared according to the method of Example C-1, and the purified ligation product was diluted with D 2 solution.
  • the purified ligation product was diluted with a D 2 solution.
  • the solution A was prepared in the same manner as in Example C-1.
  • the magnetic sphere was added to an equal volume of pH 3.6 acetate buffer to make a magnetic sphere suspension concentration of 20 mg/ml, and then CMC (concentration of 10 mg/ml) was added, and purified SA was added.
  • the magnetic ball suspension was placed in a constant temperature shaking water bath for 24 hours at 37 ° C (shaking water bath shaking speed: 260 rpm).
  • the C solution was prepared in the same manner as in Example C-1.
  • the SA-coated magnetic spheres after the washing is suspended in the C solution at a suspension concentration of 20 mg/ml, that is, a SA-coated magnetic sphere suspension is obtained, and the volume of the suspension is the coating volume described in the step.
  • the above magnetic sphere suspension was further diluted to a suspension of 0.5 mg/ml in terms of a magnetic sphere, and was used.
  • the ALD antigen was diluted with 50% bovine serum preparations into two high and low calibrator calibration points at a concentration of 1512.344 pg/ml and 76.559 pg/ml.
  • the purified ligation product can be diluted with D 2 solution.
  • the solution A was prepared in the same manner as in Example C-1.
  • the magnetic sphere was added to a volume equivalent of pH 3.6 acetate buffer to make the magnetic sphere suspension concentration 20 mg/ml, and then CMC (concentration of 10 mg/ml) was added, and the purified ALD antigen was added.
  • the magnetic ball suspension was placed in a constant temperature shaking water bath for 24 hours at 37 ° C (shaking water bath shaking speed: 260 rpm).
  • the C solution was prepared in the same manner as in Example C-1.
  • the anti-ALD antibody-coated magnetic sphere after washing is suspended in a C solution at a suspension concentration of 20 mg/ml to obtain an anti-ALD antibody-coated magnetic sphere suspension, and the volume of the suspension is the package of the present step. Being volume.
  • the above magnetic sphere suspension was further diluted to a suspension of 0.5 mg/ml in terms of a magnetic sphere, and was used.
  • the ALD antigen was diluted with 50% bovine serum preparations into two high and low calibrator calibration points at a concentration of 1031.466 pg/ml and 19..554 pg/ml.
  • ALD antigen-BSA protein conjugate 100 ⁇ g was adjusted to 1 ml with 0.1 mol/L pH 9.5 carbonate buffer (F solution). The dialyzate was placed, and the mixture was dialyzed for 2 hours at room temperature, and the dialyzed solution was added to 300 ⁇ g of ABEI-activated ester, and reacted at 37 ° C for 2 hours.
  • the ligation product of the ALD antigen-BSA protein linker to ABEI was purified by G-25 gel column.
  • D 2 solution 200 ml of 0.5 M phosphate buffer, 20 g of BSA, 8 g of NaN 3 , 2 g of MgCl 2 ⁇ 6H 2 O, 600 ml of glycerol were added to a 2000 ml beaker, diluted with purified water to 2000 ml, and filtered.
  • the purified ligation product was diluted with a D 2 solution.
  • the solution A was prepared as in Example C-1.
  • the magnetic spheres were added to a volumetric equivalent of pH 3.6 acetate buffer to make the magnetic spheres have a suspension concentration of 20 mg/ml, and then CMC (concentration: 10 mg/ml) was added, and purified goat anti-FITC polyclonal antibody was added.
  • the magnetic ball suspension was placed in a constant temperature shaking water bath for 24 hours at 37 ° C (shaking water bath shaking speed: 260 rpm).
  • the C solution was prepared in the same manner as in Example C-1.
  • the washed anti-FITC polyclonal antibody-coated magnetic sphere was suspended in a C solution at a concentration of 20 mg/ml to obtain a magnetic ball suspension coated with a goat anti-FITC polyclonal antibody, and the volume of the suspension was That is, the volume of the coating described in this step.
  • the above magnetic sphere suspension was further diluted to a suspension of 0.5 mg/ml in terms of a magnetic sphere, and was used.
  • 1 mg of the anti-ALD antibody was adjusted to 1 ml with 0.1 mol/L pH 9.5 carbonate buffer (F solution).
  • the dialyzate was placed in the dialysate, and the mixture was dialyzed for 2 hours at room temperature.
  • the dialyzed solution was added to 300 ⁇ g of FITC, and the mixture was shaken at room temperature for 24 hours.
  • the ligation product of the anti-ALD antibody and FITC was purified by G-25 gel column.
  • the C 2 solution was prepared, and the purified ligation product was diluted with a C 2 solution.
  • the ALD antigen was diluted with 50% bovine serum preparations into two high and low calibrator calibration points at a concentration of 1183.156 pg/ml and 84.520 pg/ml.
  • Example C-8 Chemiluminescence Detection ALD Using a Detection Kit
  • the ALD concentration of the sample was detected by a chemiluminescence immunocompetence assay, and the sample to be tested was 160 clinical samples.
  • the ALD concentration is proportional to the Relative Light Unit (RLU), and the analyzer can be used to automatically fit the calculated ALD concentration.
  • the sample to be tested is the sample treated by the enzyme inhibitor.
  • the sample to be tested is the sample treated by the enzyme inhibitor.
  • the sample to be tested is the sample treated by the enzyme inhibitor.
  • Example C-8 A total of 160 clinical samples of Example C-8 were tested using a commercially available commercial radioimmunoassay kit available on the market. The results are shown in Table C-1.
  • the aldosterone of healthy individuals was detected by the kit of the present invention, and the 95% confidence interval was standing position: 30-160 pg/ml, and lying position: 70-300 pg/ml. That is, the normal person's standing position and lying position detection values should fall within the above reference interval.
  • Sample 148 is a sample taken from patients with primary aldosteronism.
  • the aldosterone level in the supine position should be high and there should be no significant change in the level of the supine position.
  • the results of the samples prepared by using the kits prepared in Example C-1, Example C-4 and Example C-7 were above the confidence interval, indicating that the aldosterone level in the vertical position was extremely high, and the level of the vertical position did not change significantly. , in line with the nature of this sample as a patient sample.
  • the results of the vertical and horizontal position detection using the radioimmunoassay kit are quite different, which is obviously inconsistent with the actual clinical situation. Therefore, the detection effect of the ALD detection kit provided by the invention is better than that of the comparison radioimmunization kit, and the clinical situation can be more accurately and truly reflected.
  • the ALD detection kit prepared in the above other examples has been clinically tested, and the effects are consistent with those of the embodiment C-1, the embodiment C-4 and the embodiment C-7, and are not listed here for the sake of space saving. Test data.
  • the measured value of the ALD detection kit provided according to the present invention is more consistent with the actual value, and the clinical coincidence rate is higher, indicating that the diagnostic capability of the kit is stronger.
  • the ALD detection kit provided by the present invention has higher stability, safety in use, and environmental friendliness than the radioimmunoassay kit.

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Abstract

L'invention concerne un kit de réactif de détection de facteur d'hyperaldostéronisme, comprenant le composant A et le composant B, le composant A étant un antigène du facteur de l'hyperaldostéronisme ou un conjugué de l'antigène du facteur de l'hyperaldostéronisme et d'un support de protéine, le composant B étant un anticorps du facteur d'hyperaldostéronisme, l'un parmi le composant A et le composant B étant marqué avec un marqueur de suivi, l'autre étant revêtu sur des billes magnétiques. Le facteur d'hyperaldostéronisme est choisi parmi l'angiotensine I et l'angiotensine II. L'invention concerne également un procédé utilisant le kit de réactif pour détecter une concentration du facteur d'hyperaldostéronisme.
PCT/CN2015/072692 2015-02-10 2015-02-10 Kit de réactif de détection de facteur d'hyperaldostéronisme, et procédé de préparation et son application WO2016127322A1 (fr)

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