WO2016127322A1

WO2016127322A1 - Hyperaldosteronism factor detection reagent kit, and preparation method and application therefor

Info

Publication number: WO2016127322A1
Application number: PCT/CN2015/072692
Authority: WO
Inventors: 饶微; 高莉; 杜凯; 李钦; 徐红; 罗凯; 李婷华; 袁锦云
Original assignee: 深圳市新产业生物医学工程股份有限公司
Priority date: 2015-02-10
Filing date: 2015-02-10
Publication date: 2016-08-18

Abstract

A hyperaldosteronism factor detection reagent kit, comprising component A and component B, component A being a hyperaldosteronism factor antigen or a conjugate of the hyperaldosteronism factor antigen and a protein carrier, component B being a hyperaldosteronism factor antibody, either one of component A and component B being marked with a trace marker, the other one being coated on magnetic balls. The hyperaldosteronism factor is selected from either one of angiotensin I and angiotensin II. Also disclosed is a method using the reagent kit to detect a hyperaldosteronism factor concentration.

Description

Aldosterone factor detection kit and preparation method and application thereof

Technical field

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.

technical background

Primary aldosteronism (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).

The renin-angiotensin-aldosterone system (RAAS) is a hormonal system. When a large amount of blood loss or blood pressure drops, 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. 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.

At present, the methods for measuring A I clinically mainly include radioactive immunoassay, enzyme-linked immunosorbent assay and the like. For example, 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.

However, radioimmunoassay and enzyme-linked immunoassay have many shortcomings. For example, 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.

The renin-angiotensin-aldosterone system (RAAS) is a hormonal system. When a large amount of blood loss or blood pressure drops, 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.

At present, the methods for clinically determining A II mainly include radioactive immunoassay, enzyme-linked immunosorbent assay, and the like. For example, 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. After fully 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 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.

However, radioimmunoassay and enzyme-linked immunoassay have many shortcomings. For example, 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. When a large amount of blood loss or blood pressure drops, the kidney secretes renin, which regulates the hydrolysis of angiotensinogen to produce angiotensin I. (A I), 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.

In addition, the levels of sodium and potassium in the body, adrenocorticotropic hormone, adrenergic and dopaminergic systems also regulate the secretion of ALD. In vivo, 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. The urinary uncontaminated ALD accounts for about 6% of the secreted amount and has hormonal activity.

At present, the methods for clinically determining ALD mainly include radioactive immunoassay, enzyme-linked immunosorbent assay, and the like. For example, 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. Place overnight (16-24 hours), add anti-rabbit immunosuppressant, shake well, leave it 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 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.

However, radioimmunoassay and enzyme-linked immunoassay have many shortcomings. For example, 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. A test kit for detecting ALD.

Summary of the invention

In order to solve the above problems in the prior art, it is an object of the present invention to provide a kit 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.

In addition, 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.

According to the present invention, there is provided 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.

According to the invention, 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.

According to the invention, 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.

According to an aspect of the present invention, there is provided 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. More specifically, the A I detection kit provided by the present invention is a chemiluminescent immunoassay kit.

It is to be understood that in the above kit provided by the present invention, 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. For example, 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.

According to the invention, 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).

According to the invention, 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. Among them, 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. The "direct labeling" means that the ABEI is directly labeled with the antigen to be tested or the antibody to the antigen to be tested; the "indirect labeling" refers to the ABEI labeling of the A I antigen (or its protein carrier) via an intermediate vector linking system. 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.

According to the present invention, 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. Similarly, 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. Preferably, 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 μm, and the magnetic microspheres may also have various active functional groups by surface modification, including but not limited to -OH, -COOH, -NH ₂ .

In a specific embodiment, 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 μm; and the magnetic ball is optional. The surface is modified with one or more active functional groups by surface modification.

According to some embodiments of the present invention, 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.

According to further embodiments of the present invention, 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).

According to the invention, 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.

According to the kit provided by the present invention, 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. The concentrations of each of the above ingredients are based on the amount of the individual kit components comprising the ingredients.

In one embodiment of the invention, in the kit of the invention, 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.

For example, in a specific embodiment of the invention, 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.

For example, in a specific embodiment of the invention, 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.

For example, in a specific embodiment of the invention, 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.

For example, in a specific embodiment of the invention, 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.

In another embodiment of the invention, in the kit of the invention, 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).

For example, in a specific embodiment of the invention, 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.

For example, in a specific embodiment of the invention, 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.

For example, in a specific embodiment of the invention, 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.

For example, in a specific embodiment of the invention, 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.

According to the method provided by the present invention, 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.

According to the method provided by the present invention, 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.

In some embodiments, 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.

In other specific embodiments, 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.

In some embodiments, in step i), the ABEI 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 In step ii), 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.

In some embodiments, in step i'), the ABEI 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.

According to the present invention, there is also provided 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.

In a specific embodiment, 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.

In one embodiment, the method of detecting the AI concentration comprises detecting the AI concentration by a chemiluminescence immunoassay analyzer using a kit as described above. In a preferred embodiment of the invention, the method is carried out fully automatically. According to the present invention, the chemiluminescence immunoassay analyzer is preferably a Maglumi series chemiluminescence immunoassay analyzer (manufactured by Shenzhen New Industry Biomedical Engineering Co., Ltd.).

Specifically, in the case of labeling ABEI on the A I antigen (or its conjugate with a protein carrier) and coating the magnetic sphere with the anti-A I antibody, 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.

For the case where the ABI is labeled on the anti-A I antibody and the magnetic sphere is coated with the A I antigen (or a linker thereof with the protein carrier), 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.

Therefore, 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.

According to another aspect of the present invention, there is provided 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. More specifically, the A II detection kit provided by the present invention is a chemiluminescent immunoassay kit.

It is to be understood that in the above kit provided by the present invention, 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.

According to the invention, the tracer marker directly labels or indirectly labels the A II antigen (or its protein linker) or the anti-A II antibody. Among them, 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. The inventors have found that indirect labeling is advantageous for attenuating spatial effects, facilitating amplification of signals, and making detection more sensitive.

According to the present invention, 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. Similarly, 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.

According to the A II detection kit of the present invention, preferably, 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 μm; The magnetic spheres are optionally provided with a plurality of reactive functional groups by surface modification.

According to some embodiments of the present invention, 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.

According to further embodiments of the present invention, 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).

According to the invention, 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.

According to the kit provided by the present invention, 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.

In one embodiment of the invention, in the kit of the invention, 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.

For example, in a specific embodiment of the invention, 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.

For example, in a specific embodiment of the invention, 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.

For example, in a specific embodiment of the invention, 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.

For example, in a specific embodiment of the invention, 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.

In another embodiment 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).

For example, in a specific embodiment of the invention, 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.

For example, in a specific embodiment of the invention, 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.

For example, in a specific embodiment of the invention, 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.

For example, in a specific embodiment of the invention, 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.

According to the method for preparing an A II detection kit provided by the present invention, 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.

According to the method for preparing an A II detection kit provided by the present invention, 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.

In some embodiments, 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.

In other specific embodiments, 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.

In some embodiments, in step i), 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.

In some embodiments, in step i'), the tracer marker 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.

According to the present invention, there is also provided 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.

In some embodiments, in the A II concentration detection method, 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.

In one embodiment, 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. In a preferred embodiment of the invention, the method is fully automated get on. According to the present invention, the chemiluminescence immunoassay analyzer is preferably a Maglumi series chemiluminescence immunoassay analyzer (manufactured by Shenzhen New Industry Biomedical Engineering Co., Ltd.).

Specifically, in the case where a tracer marker is labeled on an A II antigen (or a linker thereof with a protein carrier) and a magnetic sphere is coated with an anti-A II antibody, 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.

In the case where the magnetic marker is coated with the A II antigen (or a linker thereof with the protein carrier) against the labeling of the tracer label on the anti-A II antibody, 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.

According to still another aspect of the present invention, there is provided 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. More specifically, the aldosterone detection kit provided by the present invention may be a chemiluminescent immunodetection kit.

It is to be understood that in the above kit provided by the present invention, the anti-aldosterone antibody may be one or more anti-aldosterone monoclonal antibodies and/or anti-aldosterone polyclonal antibodies. In fact, all of the antibodies mentioned in the present invention may be monoclonal antibodies and/or polyclonal antibodies.

It is to be noted that, in the present invention, the aldosterone antigen refers to a aldosterone antigen alone or a conjugate of a aldosterone antigen alone to a protein carrier, unless otherwise specified. Wherein, the protein carrier may be selected from at least one of protein carriers commonly used in the art. For example, 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.

According to the invention, the tracer label directly or indirectly labels an ALD antigen or an anti-ALD antibody. Among them, 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.

According to the present invention, 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. For coating, the intermediate vector linkage system includes, but is not limited to, FITC and anti-FITC antibody systems or streptavidin and biotin systems. Similarly, 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.

In the ALD detection kit according to the present invention, preferably, 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 μm; Also, the magnetic ball is optionally provided with a plurality of reactive functional groups by surface modification.

According to some embodiments of the present invention, 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.

According to further embodiments of the present invention, 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.

In accordance with the present invention, 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.

According to the ALD detection kit provided by the present invention, 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. The concentrations of each of the above ingredients are based on the amount of the individual kit components comprising the ingredients.

In one embodiment of the invention, in the ALD detection kit of the invention, ABEI is labeled on the ALD antigen and the magnetic sphere is coated with an anti-ALD antibody.

For example, in a specific embodiment of the invention, 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.

For example, in a specific embodiment of the invention, 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.

For example, in a specific embodiment of the invention, 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.

For example, in a specific embodiment of the invention, 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.

In another embodiment of the invention, in the ALD detection kit of the invention, ABEI is labeled on an anti-ALD antibody and the magnetic sphere is coated with an ALD antigen.

For example, in a specific embodiment of the invention, 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.

For example, in a specific embodiment of the invention, 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

For example, in a specific embodiment of the invention, 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;

For example, in a specific embodiment of the invention, 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.

According to the method for preparing an ALD detection kit provided by the present invention, 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.

According to the method for preparing an ALD detection kit provided by the present invention, 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.

In some embodiments, 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.

In other specific embodiments, 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.

In some embodiments, in 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.

In some embodiments, in 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.

According to the present invention, there is also provided a method of detecting an ALD concentration, the method comprising detecting an ALD concentration in a sample to be tested by a chemiluminescence immunoassay using an ALD detection kit as described above.

In some embodiments, the ALD concentration detection method according to the present invention 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.

In one embodiment, 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. In a preferred embodiment of the invention, the method is carried out fully automatically. According to the present invention, the chemiluminescence immunoassay analyzer is preferably a Maglumi series chemiluminescence immunoassay analyzer (manufactured by Shenzhen New Industry Biomedical Engineering Co., Ltd.).

Specifically, in the case of labeling ABEI on the ALD antigen and coating the magnetic sphere with the anti-ALD antibody, 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.

For the case where the ABEI is labeled on the anti-ALD antibody and the magnetic ball is coated by the ALD antigen, 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.

The beneficial effects of the invention are:

1. High specificity, good sensitivity and wide detection range.

2. The operation is more simple and easy, and the 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.

3. 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.

detailed description

The invention is further explained and illustrated in conjunction with the non-limiting examples. However, it should be noted that the following specific embodiments of the invention are not intended to limit the invention in any way. It should be understood by those skilled in the art that the details and the details of the present invention may be modified or substituted without departing from the spirit and scope of the invention.

In the following examples:

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;

FITC: purchased from Shanghai Ji Ning Industrial Co., Ltd.;

The magnetic microspheres are produced by Shenzhen New Industry Biomedical Engineering Co., Ltd., with 80% particle size distribution of 1-5μm, 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.

Example A-1

(1) Marking of A I antigen, the specific steps are as follows:

Preparation of dialysate (F solution): 14.31 g of Na ₂ CO ₃ and 26.46 g of NaHCO ₃ were added to a 5000 ml vessel, diluted with purified water to 4500 ml, and the prepared F solution was placed on a magnetic stirrer for use.

Select a suitable dialysis bag (common molecular weight 14000) dialysis bag, measure the size enough to accommodate the F solution, tighten the end after wetting, and test the purified water for 3 times (no leakage is required).

100 μg 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, and the mixture was dialyzed for 2 hours at room temperature, and the dialyzed solution was added to 300 μg 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.

Preparation of 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.

The purified ligation product was double-diluted with a D ₂ solution to obtain an A I antigen labeled with ABEI.

(2) Anti-A I antibody coated magnetic sphere, the specific steps are as follows:

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).

Add 5 times the coating volume of the A solution to the small white bottle, put it into the ultrasonic apparatus and stir it for 2-3 minutes while stirring, then place it on the magnet. After the supernatant is clear, pour out the supernatant. This step is repeated three times.

The magnetic beads having a particle diameter of 1 μm 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.

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).

Prepare 500 ml of pH 7.4 phosphate buffer (PBS) with P001: purified water = 1:9 by volume, add 2.5 g of BSA to dissolve, and mix, which is a magnetic bead cleaning solution.

Pour the magnetic bath suspension into a beaker, then deposit on the magnet, pour off the supernatant, add 5 times the volume of the magnetic bead cleaning solution, stir and then place on the magnet. After the supernatant is clear, The supernatant was discarded and the washing step was repeated four times.

Preparation of 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.

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.

(3) Preparation of A I low point calibrator and high point calibrator

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.

(4) Assembly

The above reagent components were dispensed and assembled into a kit, and stored at 2 to 8 °C.

Example A-2

(1) Marking of A I antigen, the specific steps are as follows:

Configuration of dialysate (F solution): 14.31 g of Na ₂ CO _{3 and} 26.46 g of NaHCO ₃ were added to a 5000 ml vessel, and diluted with purified water to 4500 ml. The prepared F solution was placed on a magnetic stirrer for use.

Preparation of 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.

(2) The goat anti-FITC polyclonal antibody is coated with a magnetic ball. The specific steps are as follows:

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.

500 ml of a pH 7.4 PBS buffer was prepared at a ratio of P001:purified water = 1:9, and 2.5 g of BSA was added to dissolve and dissolve, which was a magnetic bead cleaning solution.

The C solution was prepared in accordance with the procedure of Example A-1.

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.

(3) Anti-A I antibody labeling, the specific steps are as follows:

1 mg of anti-A I 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 μg of FITC, and the mixture was shaken at room temperature for 24 hours.

The ligation product of the anti-A I antibody and FITC was purified by G-25 gel column.

Preparation of 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).

After the C ₂ solution was prepared, the purified ligation product was diluted with a C ₂ solution.

(4) Preparation of A I low point calibrator and high point calibrator

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.

(5) Assembly

Example A-3

(1) Labeling of anti-A I antibody, the specific steps are as follows:

Configuration of dialysate (F solution): 14.31 g of Na ₂ CO _{3 and} 26.46 g of NaHCO ₃ were added to a 5000 ml beaker, diluted with purified water to 4500 ml, and the prepared F solution was placed on a magnetic stirrer for use.

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).

1 mg of anti-A I antibody 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 μg of ABEI-activated ester, and reacted at 37 ° C for 2 hours.

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.

(2) A I antigen coated magnetic ball, the specific steps are as follows:

The solution A was prepared as in Example A-1.

Add the magnetic beads to the coating volume of pH 3.6 acetate buffer to make the suspension concentration of the magnetic beads 20mg/ml, then add CMC (concentration is 10mg/ml), and add the purified A I antigen at a certain ratio.

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).

500 ml of pH 7.4 PBS buffer was prepared at a volume ratio of P001: purified water = 1:9, and 2.5 g of BSA was added to dissolve and dissolve, which was a magnetic bead cleaning solution.

Pour the magnetic bath suspension into a beaker, then deposit on the magnet, then pour off the supernatant and add 5 times the volume. The magnetic bead cleaning solution is stirred and washed, and then placed on the magnet. After the supernatant is clear, the supernatant is decanted, and the washing step is repeated four times.

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.

(3) A I low point calibrator, high point calibrator

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.

(4) Assembly

Example A-4

(1) Labeling of anti-A I antibody, the specific steps are as follows:

Configuration of dialysate (F solution): Add 14.31 g of Na ₂ CO _{3 ,} 26.46 g of NaHCO ₃ in a 5000 ml beaker, and dilute to 4500 ml with water. The prepared F solution was placed on a magnetic stirrer for use.

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.

Prepare 500 ml of pH 7.4 PBS buffer with P001: purified water = 1:9 volume ratio, add 2.5 g of BSA, mix and dissolve to dissolve, which is the magnetic bead cleaning solution.

The C solution was prepared in accordance with the procedure of Example A-1.

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. .

(3) A I antigen labeling, the specific steps are as follows:

For the configuration of the dialysate (F solution), 14.31 g of Na ₂ CO _{3 and} 26.46 g of NaHCO ₃ were added to a 5000 ml beaker, and the solution was adjusted to 4500 ml with water, and the prepared F solution was placed on a magnetic stirrer for use.

Use a suitable dialysis bag (usually 14000) to measure the appropriate size, tighten one end after wetting, and purify the water. Test leak 3 times (no leakage required).

100 μg 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 μg 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.

Preparation of C ₂ solution: 200 ml of P001 solution, 20 g of BSA, 8 g of NaN ₃ , 2 g of MgCi ₂ ·6H ₂ O, and a volume of 2000 ml (filtered) were added.

The purified ligation product is diluted with a C ₂ solution.

(4) Preparation of A I low point calibrator and high point calibrator

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.

(5) Assembly

Example A-5

(1) Labeling of anti-A I antibody, the specific steps are as follows:

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.

(2) A I antigen biotinylation

100 μg of biotin and 100 μg of A I antigen were 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 reacted at 37 ° C for 2 hours.

Purified by G-25 gel column.

The purified ligation product may be diluted with a D ₂ solution.

(3) SA package is magnetic ball, the specific steps are as follows:

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 C solution was prepared in accordance with the procedure of Example A-1.

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.

(4) Preparation of A I low point calibrator and high point calibrator

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.

(5) Assembly

Example A-6

(1) Biotinylation of anti-A I antibody, the specific steps are as follows:

100 μg of biotin and 1 mg of anti-A I antibody were 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 reacted at 37 ° C for 2 hours.

Purified by G-25 gel column.

D2 solution is prepared, and the purified ligation product is diluted with D ₂ solution.

(2) Mark of SA

100 μg of SA 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 μg of ABEI-activated ester, and reacted at 37 ° C for 2 hours.

G-25 gel column purification.

The purified ligation product can be diluted with D ₂ solution.

(3) A I antigen coated magnetic ball, the specific steps are as follows:

The solution A was prepared in accordance with the method of Example A-1.

Add the magnetic beads to the coating volume of pH 3.6 acetate buffer to make the suspension concentration of the magnetic beads 20mg/ml, then add CMC (concentration 10mg/ml), add the purified A I antigen.

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.

(4) A I low point calibrator, high point calibrator

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.

(5) Assembly

Example A-7

(1) A I antigen-BSA labeling, the specific steps are as follows:

100 μg of the A I antigen-BSA protein conjugate 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 μg 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.

Preparation of 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.

The purified ligation product was double-diluted with a D ₂ solution to obtain an A I antigen-BSA protein linker labeled with ABEI.

The magnetic beads having a particle diameter of 1 μm 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.

500 ml of pH 7.4 phosphate buffer solution (PBS) was prepared in a P001:purified water ratio of 1:9 by volume, dissolved in 2.5 g of BSA, and mixed to obtain a magnetic bead cleaning solution.

The C solution was prepared in accordance with the procedure of Example A-1.

(3) Preparation of A I low point calibrator and high point calibrator

(4) Assembly

Example A-8

(1) Labeling of anti-A I antibody, the specific steps are as follows:

1 mg of anti-A I antibody was adjusted to 1 ml with 0.1 mol/L pH 9.5 carbonate buffer (F solution). The dialyzate was placed in the dialyzate, and the mixture was dialyzed for 2 hours at room temperature, and the dialyzed solution was added to 300 μg of ABEI-activated ester, and reacted at 37 ° C for 2 hours.

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 C solution was prepared in accordance with the procedure of Example A-1.

(3) A I antigen-BSA protein linker labeling, the specific steps are as follows:

Use a suitable dialysis bag (usually 14000) to measure the appropriate size, tighten one end after wetting, and test the purified water for 3 times (no leakage is required).

100 μg of the A I antigen-BSA protein conjugate 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 μg 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.

Preparation of C ₂ solution: 200 ml of P001 solution, 20 g of BSA, 8 g of NaN ₃ 8 g, 2 g of MgCl ₂ ·6H ₂ O, and a volume of 2000 ml (filtered) were added.

The purified ligation product is diluted with a C ₂ solution.

(4) Preparation of A I low point calibrator and high point calibrator

(5) Assembly

Example A-9: Chemiluminescence Detection Using a Detection Kit A I

Using the A I detection kit prepared in the above Examples A-1 to A-8 and the Maglumi 2000 chemiluminescence analyzer, 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 specific steps for the chemiluminescence detection using the kit obtained in Example A-1 are as follows:

1. Load the calibrator or sample to be tested in sequence on 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.

2. Insert the sample holder into the sample chamber of the Maglumi 2000 chemiluminescence analyzer, edit the sample number and start the test. The specific sample loading procedure is: add 100 μl to the calibrator or sample to be tested, and then add the tracer labeled A I antigen solution. 50μl, add anti-A I antibody coated magnetic microsphere suspension 20μl, mix, incubate at 37 ° C for 15 minutes, the instrument automatically cleans twice Directly enter the measurement room to obtain the light intensity signal of each sample, and automatically fit the A I antigen concentration value of the sample to be tested by a ten-point curve and two-point calibration. Thus, the same sample obtained different concentration values under the two temperature treatments, and the sample concentration value processed at 37 ° C was subtracted from the sample concentration value treated at 2-8 ° C to obtain renin activity (PRA). The test results are shown in Table A-1.

The specific steps for the chemiluminescence detection using the kit obtained in Example A-4 are as follows:

2. Insert the sample holder into the sample chamber of the Maglumi series of chemiluminescence analyzers, edit the sample number and start the test. The specific loading steps are: add 100 μl to the calibrator or sample to be tested, and then add anti-A I labeled with the tracer label. 50 μl of antibody solution, add 50 μl of A I antigen-labeled FITC, add 20 μl of anti-FITC antibody-coated magnetic microsphere suspension, mix, incubate at 37 ° C for 15 minutes, automatically clean the instrument twice and directly into the measurement room to obtain each sample. The light intensity signal is automatically fitted to the A I antigen concentration value of the sample to be tested by a ten-point curve and two-point calibration. Thus, the same sample obtained different concentration values under the two temperature treatments, and the sample concentration value processed at 37 ° C was subtracted from the sample concentration value treated at 2-8 ° C to obtain renin activity (PRA). The test results are shown in Table A-1.

The specific steps for the chemiluminescence detection using the kit obtained in Example A-7 are as follows:

2. Insert the sample holder into the sample chamber of the Maglumi series of chemiluminescence analyzers, edit the sample number and start the test. The specific loading steps are: add 100 μl to the calibrator or sample to be tested, and then add anti-A I labeled with the tracer label. 50 μl of antibody solution, add 50 μl of FITC labeled with A I antigen-BSA protein conjugate, add 20 μl of anti-FITC antibody-coated magnetic microsphere suspension, mix well, incubate at 37 ° C for 15 minutes, and automatically clean the instrument twice after entering The measurement room obtains the light intensity signal of each sample, and automatically fits the A I antigen concentration value of the sample to be tested by a ten-point curve and two-point calibration. Thus, the same sample obtained different concentration values under the two temperature treatments, and the sample concentration value processed at 37 ° C was subtracted from the sample concentration value treated at 2-8 ° C to obtain renin activity (PRA). The test results are shown in Table A-1.

Comparative example A-1

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.

Table A-1

It can be seen from the above Examples A-1 to A-9, and Comparative Example A-1:

Of the 160 clinical samples selected in this clinical comparison trial, 16 samples from 145-160 were diagnosed with primary aldosteronism, and the remaining samples were normal samples. Patients with primary aldosteronism not only have low plasma renin activity under basal conditions (ie, under supine conditions), but also use a force method that stimulates renin release, such as the use of standing, low-sodium diet or diuretics, plasma. Renin activity did not increase or slightly increased. The plasma renin activity samples of healthy individuals were detected by the kit of the present invention, and the 95% confidence interval was 0.1-6.56 ng/ml/hr, and the lying position was 0-2.33 ng/ml/hr. According to clinical statistics, plasma renin activity in patients with primary aldosteronism should be less than 1.5 ng / ml / hr.

As shown in Table A-1, only sample No. 148 shows the A I test kit and radioactive exemption of the examples of the present invention. The results of the ELISA kit were inconsistent; except for the test results of No. 148, the test results of the samples of Example A-1, Example A-4 and Example A-7 were compared with those of the remaining samples by the radioimmunoassay kit. The test results were straight-line fitted. For the linear equation of Example A-1, y=1.004x+0.031, the correlation coefficient R=0.9989; for the linear equation of Example A-4, y=0.999x+0.040, the correlation coefficient R=0.9989 For the linear equation of Example A-7, y=0.997x+0.034 correlation coefficient R=0.9985. It can be seen that the A I detection kit provided by the present invention has substantially good consistency with the detection results of the mainstream commercial radiopheresis kits circulating on the market.

However, in combination with the measurement results of the sample No. 148, the A I detection kit and the detection method provided by the present invention have higher accuracy. For the 148 sample, patients with primary aldosteronism, 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. Compatible. However, 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.

In summary, compared with a commercially available radioimmunoassay kit, 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. . In addition, the A I detection kit provided by the present invention has higher stability, safety in use, and environmental friendliness than the radioimmunoassay kit.

Example B-1

(1) Labeling of A II antigen, the specific steps are as follows:

100 μg of A II 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 μg 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.

The purified ligation product was double-diluted with a D ₂ solution to obtain an A II antigen labeled with ABEI.

(2) Anti-A II antibody coated magnetic sphere, the specific steps are as follows:

A magnetic particle having a particle diameter of 1 μm 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. II antibody

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).

Prepare pH 7.4 phosphate buffer (PBS) 500ml with P001: purified water = 1:9 volume ratio and add 2.5g BSA to mix and dissolve, which is the magnetic ball cleaning solution.

Pour the magnetic bath suspension into a beaker, then deposit on the magnet, pour off the supernatant, add 5 times the volume of the magnetic ball cleaning solution, stir and then place on the magnet. After the supernatant is clear, Pour off the supernatant and repeat the cleaning step Four times.

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.

(3) Preparation of A II low-point calibrator and high-point calibrator

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.

(4) Assembly

Example B-2

(1) Labeling of A II antigen, the specific steps are as follows:

Preparation of dialysate (F solution): 14.31 g of Na ₂ CO _{3 and} 26.46 g of NaHCO ₃ were added to a 5000 ml vessel, and diluted with purified water to 4500 ml. The prepared F solution was placed on a magnetic stirrer for use.

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.

500 ml of a pH 7.4 PBS buffer was prepared at a ratio of P001:purified water = 1:9, and 2.5 g of BSA was added to dissolve and dissolve, which was a magnetic ball washing solution.

Pour the magnetic bath suspension into a beaker, then deposit on the magnet, pour off the supernatant, add 5 times the volume of the magnetic ball cleaning solution, stir and then place on the magnet. After the supernatant is clear, The supernatant was discarded and the washing step was repeated four times.

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.

(3) Anti-A II antibody labeling, the specific steps are as follows:

1 mg of anti-A II 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 μg of FITC, and the mixture was shaken at room temperature for 24 hours.

The ligation product of the anti-A II antibody and FITC was purified by G-25 gel column.

Preparation of 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.

(4) Preparation of A II low-point calibrator and high-point calibrator

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.

(5) Assembly

Example B-3

(1) Labeling of anti-A II antibodies, the specific steps are as follows:

Preparation of dialysate (F solution): 14.31 g of Na ₂ CO _{3 and} 26.46 g of NaHCO ₃ were added to a 5000 ml beaker, diluted with purified water to 4500 ml, and the prepared F solution was placed on a magnetic stirrer for use.

1 mg of anti-A II antibody 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 μg of ABEI-activated ester, and reacted at 37 ° C for 2 hours.

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.

(2) A II antigen coated magnetic ball, the specific steps are as follows:

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).

500 ml of pH 7.4 PBS buffer was prepared at a volume ratio of P001:purified water=9, and 2.5 g of BSA was added and mixed to dissolve, which was a magnetic ball washing solution.

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.

(3) A II low point calibrator, high point calibrator

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.

(4) Assembly

Example B-4

(1) Labeling of anti-A II antibodies, the specific steps are as follows:

Preparation of dialysate (F solution): Add 14.31 g of Na ₂ CO _{3 ,} 26.46 g of NaHCO ₃ in a 5000 ml beaker, and dilute to 4500 ml with water. The prepared F solution was placed on a magnetic stirrer for use.

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.

Add the magnetic sphere to the volume of the equal volume of pH 3.6 acetate buffer to make the magnetic sphere suspension concentration 20mg/ml, then add CMC (to the concentration of 10mg/ml), and add the purified goat anti-FITC according to a certain ratio. Polyclonal antibody.

500 ml of pH 7.4 PBS buffer was prepared at a ratio of P001:purified water = 1:9, and 2.5 g of BSA was added to dissolve and dissolve, which was a magnetic ball washing solution.

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.

(3) A II antigen labeling, the specific steps are as follows:

To prepare the dialysate (F solution), add 14.31 g of Na ₂ CO _{3 ,} 26.46 g of NaHCO ₃ in a 5000 ml beaker, dilute to 4500 ml with water, and place the prepared F solution on a magnetic stirrer for use.

100 μg of A II 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 μg 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.

Preparation of C ₂ solution: 200 ml of POO1 solution, 20 g of BSA, 8 g of NaN ₃ , 2 g of MgCl ₂ ·6H ₂ O, and a volume of 2000 ml (filtered) were added.

The purified ligation product is diluted with a C ₂ solution.

(4) Preparation of A II low-point calibrator and high-point calibrator

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.

(5) Assembly

Example B-5

(1) Labeling of anti-A II antibodies, the specific steps are as follows:

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.

(2) A II antigen biotinylation

100 μg of Biotin and 100 μg of A II antigen were 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 reacted at 37 ° C for 2 hours.

Purified by G-25 gel column.

The purified ligation product was diluted with a D ₂ solution.

(3) SA package is magnetic ball, the specific steps are as follows:

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 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.

(4) Preparation of A II low-point calibrator and high-point calibrator

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.

(5) Assembly

Example B-6

(1) Biotinylation of anti-A II antibody, the specific steps are as follows:

100 μg of Biotin and 1 mg of anti-A II antibody were 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 reacted at 37 ° C for 2 hours.

Purified by G-25 gel column.

D2 was prepared, purified product was a good connection with D _2-fold dilution solution.

(2) Mark of SA

G-25 gel column purification.

The purified ligation product can be diluted with D ₂ solution.

(3) A II antigen coated magnetic ball, the specific steps are as follows:

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.

Prepare 500 ml of pH 7.4 PBS buffer with P001: purified water = 1:9 by volume. Add 2.5 g of BSA and mix and dissolve to dissolve, which is the magnetic ball cleaning solution.

The C solution was prepared in accordance with the procedure of Example B-1.

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.

(4) A II low point calibrator, high point calibrator

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.

(5) Assembly

Example B-7:

(1) Labeling of the A II antigen-BSA protein linker, the specific steps are as follows:

100 μg of the A II antigen-BSA protein linker 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 μg 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.

The purified ligation product was diluted with a D ₂ solution.

The solution A was prepared as in Example B-1.

Add 5 times the volume of the solution A to the small white bottle, put it into the ultrasonic instrument and stir it while ultrasonicizing. For 2-3 minutes, then place on the magnet. After the supernatant is clear, pour off the supernatant. This step is repeated three times.

The C solution was prepared in the same manner as in Example B-1.

(3) Anti-A II antibody labeling, the specific steps are as follows:

Preparation of 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.

(4) Preparation of A II low-point calibrator and high-point calibrator

Example B-8: Chemiluminescence Detection Using a Detection Kit A II

Using the A II detection kit prepared in the above Examples B-1 to B-7 and the Maglumi 2000 chemiluminescence analyzer, 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 specific steps for the chemiluminescence detection using the kit obtained in Example B-1 are as follows:

1. Load the calibrator or the sample to be tested sequentially on the sample rack. The sample to be tested is the sample treated by the enzyme inhibitor.

2. Insert the sample holder into the sample chamber of the Maglumi 2000 chemiluminescence analyzer and edit the sample number to start the test. The specific loading step is: add 100 μl to the calibrator or sample to be tested, and then add the tracer labeled A II antigen solution. 50μl, add anti-A II antibody coated magnetic microsphere suspension 20μl, 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 specific steps for the chemiluminescence detection using the kit obtained in Example B-4 are as follows:

2. Insert the sample holder into the sample chamber of the Maglumi 2000 chemiluminescence analyzer, edit the sample number and start the test. The specific loading steps are: add 100 μl to the calibrator or sample to be tested, and then add anti-A II labeled with the tracer. 50 μl of antibody solution, add 50 μl of A II antigen-labeled FITC solution, add 20 μl of magnetic microsphere suspension coated with goat anti-FITC polyclonal antibody, mix well, incubate at 37 ° C for 15 minutes, and automatically clean the instrument twice before entering the measurement. The light intensity signal of each sample is obtained, and the A II antigen concentration value of the sample to be tested is automatically fitted by a ten-point curve and two-point calibration. Test results are shown in the table. B-1.

The specific steps for the chemiluminescence detection using the kit obtained in Example B-7 are as follows:

2. Insert the sample holder into the sample chamber of the Maglumi 2000 chemiluminescence analyzer, edit the sample number and start the test. The specific loading steps are: add 100 μl to the calibrator or sample to be tested, and then add anti-A II labeled with the tracer. 50 μl of antibody solution, add 50 μl of A II antigen-labeled FITC solution, add 20 μl of magnetic microsphere suspension coated with goat anti-FITC polyclonal antibody, mix well, incubate at 37 ° C for 15 minutes, and automatically clean the instrument twice before entering the measurement. The light intensity signal of each sample is obtained, and the A II antigen concentration value of the sample to be tested is automatically fitted by a ten-point curve and two-point calibration. The test results are shown in Table B-1.

Comparative Example B-1

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.

Table B-1

It can be seen from the above Examples B-1 to B-8, Comparative Example B-1, and Table B-1:

Of the 160 clinical samples selected in this clinical comparison trial, 16 samples from 145-160 were diagnosed with primary aldosteronism, and the remaining samples were normal samples. Patients with primary aldosteronism are not only under low A II in the basal state, but also use a force method that stimulates renin release, such as the use of standing, low-sodium diet or diuretics, A II does not increase or slightly increase. The healthy individual A II is detected by the kit of the embodiment of the present invention, and the 95% confidence interval is the standing position: 25-60 pg/ml, and the lying position: 50-120 pg/ml, that is, the normal person's standing position and the lying position detection value should fall separately. Enter the above reference interval.

As shown in Table B-1, only sample No. 148 shows that the A II test kit of the embodiment of the present invention is inconsistent with the test result of the radioimmunoassay kit; except for the test result No. 148, Example B-1, Example B -4 and the test samples of Example B-7 were used to directly fit the detection results of the remaining samples with the detection results of the remaining samples by the radioimmunoassay kit, and the linear equation for the example B-1 was y=0.998x-0.284, Correlation coefficient R = 0.9955; for the example B-4 linear equation is y = 0.998x + 0.316, correlation coefficient R = 0.9960; for the example B-7 linear equation is y = 0.996x + 0.499, correlation coefficient R = 0.9950. It can be seen that the detection kit provided by the invention has better consistency with the existing radioimmunization kit.

However, in combination with the measurement results of the No. 148 sample, the A II detection kit and the detection method provided by the present invention have higher accuracy. 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. However, 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.

In summary, compared with a commercially available radioimmunoassay kit, 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. . In addition, the A II detection kit provided by the present invention has higher stability, safety of use and environmental friendliness compared to the radioimmunoassay kit.

Example C-1

(1) Marking of ALD antigen, the specific steps are as follows:

100 μg 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 μg 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.

The purified ligation product was double-diluted with a D ₂ solution to obtain an ALD antigen labeled with ABEI.

(2) Anti-ALD antibody coated magnetic ball, the specific steps are as follows:

A magnetic particle having a particle diameter of 1 μm 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 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. .

(3) Preparation of ALD low-point calibrators and high-point calibrators

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.

(4) Assembly

Example C-2

(1) Marking of ALD antigen, the specific steps are as follows:

The purified ligation product was diluted with a D ₂ solution.

The solution A was prepared as in Example C-1.

Add the magnetic sphere to the volume of the equal volume of pH 3.6 acetate buffer to make the suspension concentration of the magnetic sphere 20mg/ml, then add CMC (to the concentration of 10mg/ml), and add the purified goat anti-FITC polyclonal. antibody.

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.

(3) Anti-ALD antibody labeling, the specific steps are as follows:

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 μg 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.

(4) Preparation of ALD low-point calibrator and high-point calibrator

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.

(5) Assembly

Example C-3

(1) Marking of anti-ALD antibodies, the specific steps are as follows:

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, and the mixture was dialyzed for 2 hours at room temperature, and the dialyzed solution was added to 300 μg of ABEI-activated ester, and reacted at 37 ° C for 2 hours.

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.

(2) ALD antigen coated magnetic ball, the specific steps are as follows:

The solution A was prepared as in Example C-1.

Add the magnetic sphere to the volume of the equal volume of pH 3.6 acetate buffer to make the suspension concentration of the magnetic sphere 20mg/ml, then add CMC (to the concentration of 10mg/ml), and add the purified ALD antigen at a certain ratio.

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.

(3) ALD low point calibrator, high point calibrator

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.

(4) Assembly

Example C-4

(1) Marking of anti-ALD antibodies, the specific steps are as follows:

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 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.

(3) ALD antigen labeling, the specific steps are as follows:

100 μg 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 μg 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.

(4) Preparation of ALD low-point calibrator and high-point calibrator

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.

(5) Assembly

Example C-5

(1) Marking of anti-ALD antibodies, the specific steps are as follows:

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.

(2) ALD antigen biotinylation

100 μg of Biotin and 100 μg of ALD antigen were 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 reacted at 37 ° C for 2 hours.

Purified by G-25 gel column.

The purified ligation product was diluted with a D ₂ solution.

(3) SA package is magnetic ball, the specific steps are as follows:

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.

500 ml of PBS buffer of pH 7.4 was prepared at a volume ratio of P001:purified water = 1:9, and 2.5 g of BSA was added to dissolve and dissolve, which was a magnetic ball washing solution.

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.

(4) Preparation of ALD low-point calibrator and high-point calibrator

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.

(5) Assembly

Example C-6

(1) Biotinylation of anti-ALD antibodies, the specific steps are as follows:

100 μg of Biotin and 1 mg of anti-ALD antibody were 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 reacted at 37 ° C for 2 hours.

Purified by G-25 gel column.

(2) Mark of SA

G-25 gel column purification.

The purified ligation product can be diluted with D ₂ solution.

(3) ALD antigen coated magnetic ball, the specific steps are as follows:

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 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.

(4) ALD low point calibrator, high point calibrator

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.

(5) Assembly

Example C-7:

(1) Labeling of ALD antigen-BSA protein linker, the specific steps are as follows:

100 μg of ALD antigen-BSA protein conjugate 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 μg 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.

The purified ligation product was diluted with a D ₂ solution.

The solution A was prepared as in Example C-1.

The C solution was prepared in the same manner as in Example C-1.

(3) Anti-ALD antibody labeling, the specific steps are as follows:

The C ₂ solution was prepared, and the purified ligation product was diluted with a C ₂ solution.

(4) Preparation of ALD low-point calibrator and high-point calibrator

(5) Assembly

Example C-8: Chemiluminescence Detection ALD Using a Detection Kit

Using the ALD detection kit prepared in the above Examples C-1 to C-7 and the Maglumi 2000 chemiluminescence analyzer, 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 specific steps for the chemiluminescence detection using the kit obtained in Example C-1 are as follows:

2. Insert the sample holder into the sample chamber of the Maglumi 2000 chemiluminescence analyzer, edit the sample number and start the test. The specific loading steps are: calibrator or sample to be tested plus 100μl, then add anti-ALD antibody coated magnetic microsphere suspension 20 μl of liquid, mix, incubate at 37 ° C for 10 minutes, add 40 μl of ALD antigen solution labeled with tracer marker, the instrument automatically cleans twice and directly enters the measurement room to obtain the light intensity signal of each sample, passing the ten point curve and two points The calibration automatically fits the ALD antigen concentration value of the sample to be tested. The test results are shown in Table C-1.

The specific steps for the chemiluminescence detection using the kit obtained in Example C-4 are as follows:

2. Insert the sample holder into the sample chamber of the Maglumi 2000 chemiluminescence analyzer, edit the sample number and start the test. The specific loading steps are: add 100 μl to the calibrator or the sample to be tested, and then add the anti-ALD antibody labeled with the tracer label. 50μl, add 50μl of ALD antigen-labeled FITC solution, add 20μl of magnetic microsphere suspension coated with goat anti-FITC polyclonal antibody, mix well, incubate for 10 minutes at 37°C, automatically clean the instrument twice and directly enter the measurement room to get each The light intensity signal of the sample is automatically fitted to the ALD antigen concentration value of the sample to be tested by a ten-point curve and two-point calibration. The test results are shown in Table C-1.

The specific steps for the chemiluminescence detection using the kit obtained in Example C-7 are described as follows:

Comparative example C-1

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.

Table C-1

It can be seen from Examples C-1 to C-8, Comparative Example C-1, and Table C-1:

Of the 160 clinical samples selected in this clinical comparison trial, 16 samples from 145-160 were diagnosed with primary aldosteronism, and the remaining samples were normal samples. Primary aldosteronism, regardless of the cause or type, is caused by excessive secretion of aldosterone. This type of patient not only has a high level of aldosterone in the basal state, but also uses a force method that stimulates the release of renin, such as the use of a standing position, a low-sodium diet or a diuretic, and there is no significant change in aldosterone. 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.

As shown in Table C-1, only sample No. 148 showed that the ALD detection kit of the present invention was inconsistent with the detection result of the radioimmunoassay kit; except for the detection result No. 148, Example C-1 and Example C were excluded. The detection results of the remaining samples of the -4 kit were directly fitted to the detection results of the remaining samples by the radioimmunoassay kit, and the linear equation for the example C-1 was y=0.999x-0.163, and the correlation coefficient R=0.9999; The linear equation for Example C-4 is y = 0.999x + 0.374, the correlation coefficient R = 0.9999; for the embodiment C-7, the linear equation is y = 0.999x - 0.004, and the correlation coefficient R = 0.9999. It can be seen that the ALD detection kit provided by the present invention has substantially good consistency with the measurement results of the radioimmunization kit.

However, in combination with the measurement results of the sample No. 148, the ALD detection kit and the detection method provided by the present invention have higher accuracy. 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. However, 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.

In summary, compared with a commercially available radioimmunoassay kit, 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. In addition, the ALD detection kit provided by the present invention has higher stability, safety in use, and environmental friendliness than the radioimmunoassay kit.

Modifications within the spirit and scope of the invention will be apparent to those skilled in the art. In addition, it should be understood that various aspects of the invention, various parts of the various embodiments, and the various features listed may be combined or in whole or in part. In the various embodiments described above, those embodiments with reference to another embodiment may be combined with other embodiments as appropriate, as will be understood by those skilled in the art. In addition, those skilled in the art will understand that the foregoing description is by way of example only and is not intended to limit the invention.

Claims

An aldosteronism factor detecting kit, the kit comprising a component A and a component B, wherein the component A is an aldosteronism factor antigen or a aldosteronism factor antigen and a protein carrier linker, component B An aldosteronism factor antibody, any one of the component A and the component B is labeled with a tracer marker, and the other is coated with a magnetic sphere; wherein the aldosteronism factor is selected from the group consisting of angiotensin I Any one of angiotensin II and aldosterone.
The kit according to claim 1, wherein

The tracer label directly or indirectly labels component A or component B, and the method of indirect labeling comprises passage of fluorescein isothiocyanate with an anti-isothiocyanate fluorescein antibody system or streptavidin and a living being The element system indirectly labels component A or component B;

Component A or component B directly or indirectly coated with magnetic spheres, including by fluorescein isothiocyanate and fluorescein isothiocyanate antibody system or streptavidin and biotin system Indirectly coated with magnetic balls.
An angiotensin I detecting kit comprising a component A1 and a component B1, wherein the component A1 is an angiotensin I antigen or a linker of an angiotensin I antigen and a protein carrier, the group The fraction B1 is an anti-angiotensin I antibody, one of which is labeled with a tracer marker and the other is coated with a magnetic sphere.
The kit according to claim 3, wherein the protein carrier is selected from the group consisting of bovine serum albumin, human serum albumin, rabbit serum albumin, hemocyanin, bovine IgG, human IgG, ovalbumin, muscle red. At least one of a protein and thyroglobulin.
The kit according to claim 3, wherein said tracer marker is selected from the group consisting of adamantane, luminol and its derivatives, isoluminol and its derivatives, acridinium ester, alkaline phosphatase, and At least one of horseradish peroxidase.
The kit according to claim 5, wherein the tracer marker is N-(4-aminobutyl)-N-ethylisoluminol.
The kit according to claim 3, wherein 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 μm; The magnetic sphere is optionally provided with one or more reactive functional groups by surface modification.
The kit according to claim 3, wherein in the kit, the concentration of the angiotensin I antigen or the angiotensin I antigen and the protein carrier is 0.002-0.01 mg/ml; The concentration of the angiotensin I antibody 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 kit according to claim 3, wherein

The tracer label directly or indirectly labels component A1 or component B1, and the method of indirect labeling comprises passage of fluorescein isothiocyanate with an anti-isothiocyanate fluorescein antibody system or streptavidin and a living being The element system indirectly labels component A1 or component B1;

Component A1 or component B1 directly or indirectly coated with magnetic spheres, including by fluorescein isothiocyanate and fluorescein isothiocyanate antibody system or streptavidin and biotin system Indirectly coated with magnetic balls.
The kit according to claim 9, wherein the kit comprises a component selected from any one of component O1 and component O2, and is selected from component P1, component P2 and component a component of any one of P3; wherein

Component O1 is a linker of angiotensin I antigen or angiotensin I antigen directly labeled with N-(4-aminobutyl)-N-ethylisoluminol and a protein carrier;

Component O2 is a link between streptavidin-labeled N-(4-aminobutyl)-N-ethylisoluminol and biotinylated angiotensin I antigen or angiotensin I antigen and protein carrier Object

Component P1 is a magnetic sphere directly coated with an anti-angiotensin I antibody;

Component P2 is a biotinylated anti-angiotensin I antibody and a streptavidin coated magnetic sphere;

Component P3 is coated with anti-angiotensin I antibody-labeled fluorescein isothiocyanate and anti-isothiocyanate antibody Magnetic ball.
The kit according to claim 9, wherein the kit comprises a component selected from any one of component C1 and component C2, and is selected from component D1, component D2 and component a component of any of D3; wherein

Component C1 is an anti-angiotensin I antibody directly labeled with N-(4-aminobutyl)-N-ethylisoluminol;

Component C2 is streptavidin-labeled N-(4-aminobutyl)-N-ethylisoluminol and biotinylated anti-angiotensin I antibody;

Component D1 is an angiotensin I antigen or a magnetic ball directly coated with an angiotensin I antigen and a protein carrier;

Component D2 is a biotinylated angiotensin I antigen or an angiotensin I antigen-protein carrier conjugate and a streptavidin-coated magnetic sphere;

Component D3 is an angiotensin I antigen or an angiotensin I antigen and a protein carrier conjugated labeled fluorescein isothiocyanate and an anti-isothiocyanate antibody-coated magnetic sphere.
The kit according to any one of claims 1 to 11, wherein the kit further comprises a low point calibrator and a high point calibrator of angiotensin I antigen, and optionally a buffer.
A method for preparing an angiotensin I detecting kit, the method comprising: marking one of component A1 and component B1 directly or indirectly with a tracer marker, and coating another one directly or indirectly with a magnetic sphere The component A1 is an angiotensin I antigen or a linker of an angiotensin I antigen to a protein carrier, and the component B1 is an anti-angiotensin I antibody.
The method of claim 13 wherein:

The indirect labeling comprises labeling the tracer label with the fluorescein isothiocyanate and the anti-isothiocyanate fluorescein antibody system or streptavidin and the biotin system to label the component A1 or component B1;

The indirect coating comprises indirectly coating the magnetic component with the component A1 or the component B1 by fluorescein isothiocyanate and the anti-isothiocyanate fluorescein antibody system or streptavidin and the biotin system.
The method of claim 13 wherein said tracer label is N-(4-aminobutyl)-N-ethylisoluminol.
The method according to claim 13, wherein 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 to 5 μm.
The method according to claim 13, wherein in the kit, the concentration of the angiotensin I antigen or the angiotensin I antigen and the protein carrier is 0.002-0.01 mg/ml; anti-vascular tension The concentration of the prime I antibody 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.
A method for detecting angiotensin I concentration, comprising: detecting an angiotensin I concentration in a sample to be tested by a chemiluminescence immunoassay using an angiotensin I detecting kit; wherein The kit comprises a component A1 and a component B1, which is an angiotensin I antigen or a linker of an angiotensin I antigen to a protein carrier, said component B1 being an anti-angiotensin I antibody, said One of component A1 and component B1 is labeled with a tracer marker and the other is coated with a magnetic sphere.
The method according to claim 18, wherein said tracer label is N-(4-aminobutyl)-N-ethylisoluminol; said magnetic sphere is Fe 2 O 3 or Fe 3 A composite of O 4 magnetic nanoparticles and an organic polymer material, and having a particle diameter of 0.1 to 5 μm.
The method according to claim 18, wherein in the method, component A1 and component B1 of the kit are mixed with the sample to be tested, and the optical signal intensity of the sample is measured, and is calibrated by angiotensin I. Calculating the angiotensin I concentration of the sample to be tested, wherein the concentration of the angiotensin I antigen or the angiotensin I antigen and the protein carrier is 0.002-0.01 in the kit The concentration of the anti-angiotensin I antibody 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 method of claim 18, wherein

The tracer label directly or indirectly labels component A1 or component B1, and the method of indirect labeling comprises passage of fluorescein isothiocyanate with an anti-isothiocyanate fluorescein antibody system or streptavidin and a living being The element system indirectly labels component A1 or component B1;

Component A1 or component B1 directly or indirectly coated with magnetic spheres, including by fluorescein isothiocyanate and fluorescein isothiocyanate antibody system or streptavidin and biotin system Indirectly coated with magnetic balls.
The method according to claim 18, wherein the method comprises detecting an angiotensin I concentration by a chemiluminescence immunoassay analyzer using the kit.
An angiotensin II detecting kit comprising a component A2 and a component B2, wherein the component A2 is an angiotensin II antigen or an angiotensin II antigen and a protein carrier, component B2 For an anti-angiotensin 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 kit according to claim 23, wherein said protein carrier is selected from the group consisting of bovine serum albumin, human serum albumin, rabbit serum albumin, hemocyanin, bovine IgG, human IgG, ovalbumin, and muscle red. At least one of a protein and thyroglobulin.
The kit according to claim 23, wherein said tracer marker is selected from the group consisting of adamantane, luminol and its derivatives, isoluminol and its derivatives, acridinium ester, alkaline phosphatase, and At least one of horseradish peroxidase.
The kit according to claim 23, wherein said tracer marker is N-(4-aminobutyl)-N-ethylisoluminol.
The kit according to claim 23, wherein 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 μm; The magnetic sphere is optionally provided with a plurality of reactive functional groups by surface modification.
The kit according to claim 23, wherein in the kit, the concentration of the angiotensin II antigen or the angiotensin II antigen and the protein carrier is 0.002-0.01 mg/ml; The concentration of the angiotensin II antibody 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 kit according to claim 23, wherein

The tracer label directly or indirectly labels component A2 or component B2, including by fluorescein isothiocyanate and fluorescein isothiocyanate antibody system or streptavidin and organism Indirect labeling of component A2 or component B2;

Component A2 or component B2 directly or indirectly coated with magnetic spheres, including by fluorescein isothiocyanate and fluorescein isothiocyanate antibody system or streptavidin and biotin system Indirectly coated with magnetic balls.
The kit according to claim 29, wherein said kit comprises a component selected from any one of component E1 and component E2, and is selected from component F1, component F2 and component a component of any of F3;

Component E1 is an angiotensin II antigen directly labeled with N-(4-aminobutyl)-N-ethylisoluminol;

Component E2 is streptavidin-labeled N-(4-aminobutyl)-N-ethylisoluminol and biotinylated angiotensin II antigen;

Component F1 is a magnetic sphere directly coated with an anti-angiotensin II antibody;

Component F2 is a biotinylated anti-angiotensin II antibody and streptavidin coated magnetic sphere;

Component F3 is a magnetic sphere coated with anti-angiotensin II antibody-labeled fluorescein isothiocyanate and goat anti-isothiocyanate antibody.
The kit according to claim 29, wherein said kit comprises a component selected from any one of component G1 and component G2, and is selected from component H1, component H2 and component a component of any one of H3;

Component G1 is an N-(4-aminobutyl)-N-ethylisoluminol directly labeled anti-angiotensin II antibody;

Component G2 is streptavidin-labeled N-(4-aminobutyl)-N-ethylisoluminol and biotinylated anti-angiotensin II antibody;

Component H1 is a magnetic sphere directly coated with angiotensin II antigen;

Component H2 is a magnetic ball coated with biotinylated angiotensin II antigen and streptavidin;

Component H3 is an angiotensin II antigen-labeled fluorescein isothiocyanate and a goat anti-isothiocyanate antibody-coated magnetic sphere.
The kit according to any one of claims 23 to 31, further comprising a low point calibrator and a high point calibrator of angiotensin II antigen, and optionally a buffer.
A method for preparing an angiotensin II detecting kit, the method comprising: labeling one of component A2 or component B2 directly or indirectly with a tracer marker, and coating another one directly or indirectly with a magnetic sphere Wherein component A2 is an angiotensin II antigen or a linker of an angiotensin II antigen to a protein carrier, said component B2 being an anti-angiotensin II antibody.
The method of claim 33, wherein

The indirect labeling comprises labeling the tracer label with the fluorescein isothiocyanate and the anti-isothiocyanate fluorescein antibody system or streptavidin and the biotin system to label the component A2 or component B2;

The indirect coating comprises indirectly coating the magnetic component by component A2 or component B2 by fluorescein isothiocyanate with an anti-isothiocyanate fluorescein antibody system or streptavidin and a biotin system.
The method of claim 33 wherein said tracer label is N-(4-aminobutyl)-N-ethylisoluminol.
The method according to claim 33, wherein 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 to 5 μm.
The method according to claim 33, wherein in the prepared kit, the concentration of the angiotensin II antigen or the angiotensin II antigen and the protein carrier is 0.002-0.01 mg/ml; The angiotensin II antibody concentration is 0.05-1 mg/ml; the magnetic sphere concentration is 0.05-1 mg/ml; and the tracer marker concentration is 0.2-1 mg/l.
A method for detecting angiotensin II concentration, comprising: detecting an angiotensin II concentration in a sample to be tested by a chemiluminescence immunoassay using an angiotensin II detecting kit; Including component A2 and component B2, which is an angiotensin II antigen or an angiotensin II antigen and a protein carrier, component B2 is an anti-angiotensin II antibody, the component A2 and One of the components B2 marks the tracer marker and the other is coated with a magnetic sphere.
The method according to claim 38, wherein said tracer label is N-(4-aminobutyl)-N-ethylisoluminol, and said magnetic sphere is Fe 2 O 3 or Fe 3 A composite of O 4 magnetic nanoparticles and an organic polymer material, and having a particle diameter of 0.1 to 5 μm.
The method according to claim 38, wherein in the method, component A2 and component B2 of the kit are mixed with the sample to be tested, and the optical signal intensity of the sample is measured, and the angiotensin II calibrator is passed. An optical angiotensin II concentration of the test sample, wherein the concentration of the angiotensin II antigen or the angiotensin II antigen and the protein carrier is 0.002-0.01 mg in the kit. /ml; anti-angiotensin II antibody concentration is 0.05-1 mg / ml; magnetic sphere concentration is 0.05-1 mg / ml; tracer marker concentration is 0.2-1 mg / l.
The method of claim 38, wherein

The tracer label directly or indirectly labels component A2 or component B2, including by fluorescein isothiocyanate and fluorescein isothiocyanate antibody system or streptavidin and organism Indirect labeling of component A2 or component B2;

Component A2 or component B2 directly or indirectly coated with magnetic spheres, including by fluorescein isothiocyanate and fluorescein isothiocyanate antibody system or streptavidin and biotin system Indirectly coated with magnetic balls.
An aldosterone detection kit comprising an aldosterone antigen and an anti-aldosterone antibody, one of the aldosterone antigen and the anti-aldosterone antibody is labeled with a tracer marker, and the other is coated with a magnetic sphere.
The kit according to claim 42, wherein said tracer marker is selected from the group consisting of adamantane, luminol and its derivatives, isoluminol and its derivatives, acridinium ester, alkaline phosphatase, and At least one of horseradish peroxidase.
The kit according to claim 42, wherein said tracer marker is N-(4-aminobutyl)-N-ethylisoluminol.
The kit according to claim 42, wherein 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 μm; The magnetic sphere is optionally provided with a plurality of reactive functional groups by surface modification.
The kit according to claim 42, 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 sphere concentration is 0.05-1 mg. /ml; tracer marker concentration is 0.2-1 mg / l.
The kit according to claim 42, wherein

The tracer marker directly or indirectly labels an aldosterone antigen or an anti-aldosterone antibody, and the indirect label comprises a fluorescein isothiocyanate antibody system or streptavidin and a biotin system by fluorescein isothiocyanate Indirect labeling of aldosterone antigen or anti-aldosterone antibody;

An aldosterone antigen or an anti-aldosterone antibody directly or indirectly coated with a magnetic sphere, and the indirect coating comprises indirect coating with a fluorescein isothiocyanate antibody system or a streptavidin antibody system and a biotin system Be magnetized.
The kit according to claim 47, wherein said kit comprises a component selected from any one of component S1 and component S2, and is selected from the group consisting of component T1, component T2 and component a component of any of T3;

Component S1 is an N-(4-aminobutyl)-N-ethylisoluminol directly labeled aldosterone antigen;

Component S2 is streptavidin-labeled N-(4-aminobutyl)-N-ethylisoluminol and a biotinylated aldosterone antigen;

Component T1 is a magnetic ball directly coated with an anti-aldosterone antibody;

Component T2 is a biotinylated anti-aldosterone antibody and streptavidin coated magnetic sphere;

Component T3 is an anti-aldosterone antibody-labeled fluorescein isothiocyanate and an anti-isothiocyanate antibody-coated magnetic sphere.
The kit according to claim 47, wherein said kit comprises a component selected from any one of component U1 and component U2, and is selected from component V1, component V2 and component a component of any of V3;

Component U1 is an N-(4-aminobutyl)-N-ethylisoluminol directly labeled anti-aldosterone antibody;

Component U2 is streptavidin-labeled N-(4-aminobutyl)-N-ethylisoluminol and biotinylated anti-aldosterone antibody;

Component V1 is a magnetic sphere directly coated with an aldosterone antigen;

Component V2 is a biotinylated aldosterone antigen and a streptavidin coated magnetic sphere;

Component V3 is an aldosterone antigen-labeled fluorescein isothiocyanate and an anti-isothiocyanate antibody-coated magnetic sphere.
The kit of any of claims 42-49, further comprising a low point calibrator of aldosterone and a high point calibrator, and optionally a buffer.
A method of preparing an aldosterone detection kit, the method comprising: directly or indirectly labeling a tracer marker with one of an aldosterone antigen and an aldosterone antibody, and coating the magnetic sphere directly or indirectly.
The method of claim 51 wherein:

The indirect labeling comprises labeling the tracer label with the aldosterone antigen or the anti-aldosterone antibody by a fluorescein isothiocyanate antibody system or streptavidin antibody system or streptavidin and a biotin system;

The indirect coating comprises indirectly coating the magnetic ball with the aldosterone antigen or the anti-aldosterone antibody by fluorescein isothiocyanate and the anti-isothiocyanate fluorescein antibody system or streptavidin and the biotin system.
The method according to claim 51, wherein said tracer marker is N-(4-aminobutyl)-N-B Kiwi luminol.
The method according to claim 51, wherein 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 to 5 μm.
The method according to claim 51, wherein in the prepared 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 sphere concentration is 0.05- 1 mg/ml; tracer marker concentration is 0.2-1 mg/l.
A method for detecting an aldosterone concentration, which comprises detecting an aldosterone concentration in a sample to be tested by a chemiluminescence immunoassay using an aldosterone detection kit; the kit comprising an aldosterone antigen and an anti-aldosterone antibody, One of the aldosterone antigens and the anti-aldosterone antibodies is labeled with a tracer marker, and the other is coated with a magnetic sphere.
The method of claim 56, wherein

The tracer marker directly or indirectly labels an aldosterone antigen or an anti-aldosterone antibody, and the indirect label comprises a fluorescein isothiocyanate antibody system or streptavidin and a biotin system by fluorescein isothiocyanate Indirect labeling of aldosterone antigen or anti-aldosterone antibody;

An aldosterone antigen or an anti-aldosterone antibody directly or indirectly coated with a magnetic sphere, and the indirect coating comprises indirect coating with a fluorescein isothiocyanate antibody system or a streptavidin antibody system and a biotin system Be magnetized.
The method according to claim 56, wherein said tracer marker is N-(4-aminobutyl)-N-ethylisoluminol, and said magnetic sphere is Fe 2 O 3 or Fe. A composite of 3 O 4 magnetic nanoparticles and an organic polymer material, and having a particle diameter of 0.1 to 5 μm.
The method according to claim 56, wherein in the method, a kit component comprising an aldosterone antigen and an anti-aldosterone antibody is mixed with the sample to be tested, and the optical signal intensity of the sample is determined by using an aldosterone calibrator. The light 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 sphere concentration is 0.05. -1 mg/ml, the tracer marker concentration is 0.2-1 mg/l.