Preparation method and application of detection kit constructed based on biotin and avidin

Abstract

The invention discloses a preparation method and application of a detection kit constructed based on biotin and avidin, and relates to the field of kit preparation, and the detection kit comprises a sample to be detected, a biotinylated ST2 monoclonal antibody, an acridinium ester labeled ST2 monoclonal antibody, streptavidin coated magnetic particles, a chemiluminescence excitation liquid 1 and a chemiluminescence excitation liquid 2. According to the preparation method and application of the detection kit constructed on the basis of biotin and avidin, the kit has the advantages of simplicity and convenience in operation, high specificity, high sensitivity, wide linear range, high precision and the like in use; compared with a colloidal gold method and an immunofluorescence chromatography method, an accurate clinical judgment basis is provided; compared with a chemiluminescence detection kit constructed based on a biotin and avidin system, the kit provided by the invention eliminates or reduces the influence of free biotin in a measurement sample on an ST2 detection result; and a rapid, simple and accurate detection reagent with strong anti-interference capability is provided for clinical diagnosis.

Description

Technical Field

[0001] The invention relates to the field of preparation of a test kit, and in particular to a preparation method and application of a detection kit constructed based on biotin and avidin. Background Art

[0002] Heart failure refers to a group of syndromes in which ventricular filling or ejection capacity is impaired due to various cardiac structural or functional diseases, resulting in cardiac output unable to meet the body's metabolic needs, leading to insufficient blood perfusion of organs and tissues, often manifested as dyspnea, decreased exercise tolerance, circulatory congestion and peripheral edema. Heart failure is the terminal stage of various cardiovascular diseases. With the development of medical technology, the survival period of cardiovascular diseases has gradually prolonged, and the age-dependent mortality rate has significantly decreased, leading to an increase in the prevalence of heart failure. Timely diagnosis, standardized treatment, accurate risk stratification and prognosis assessment are crucial. However, the diagnostic sensitivity and specificity of symptoms and signs are relatively low, which often mislead doctors in diagnosis and treatment. Heart failure biomarkers can fill the gap, which mainly rely on the intrinsic mechanisms of heart failure, namely fibrosis, inflammation, oxidative stress, myocardial damage, neurohormonal disorders, and myocardial and matrix remodeling.

[0003] As a marker of heart failure, it needs to meet the following conditions: convenient and accurate measurement; reflect some important pathophysiological processes in the occurrence and progression of heart failure; cannot simply reflect the clinical information provided by existing markers; help to quickly establish a diagnosis or exclude a diagnosis, or help to accurately assess the prognosis and provide information for treatment. Serum brain sodium titanate and N-terminal B-type natriuretic peptide precursor, which have been widely used clinically, lack specificity and are both cleared by the kidneys. High blood volume and increased blood pressure in renal insufficiency increase the secretion of BNP and NT-proBNP. With age, the glomerular filtration rate decreases, accompanied by an increase in the concentration of BNP and NT-proBNP. Body mass index also has a certain effect on the concentration of the two. As BMI increases, the concentration of BNP and NT-proBNP decreases. In addition, the pathophysiological mechanism of heart failure is relatively complex, and BNP and NT-proBNP only reflect one aspect of it. The specific analysis still requires multi-factor analysis. Therefore, new biomarkers have come into being.

[0004] Growth-stimulated gene expression 2 protein is a member of the interleukin-1 receptor family, with soluble and transmembrane forms. IL-33 is a functional ligand of sST2 and ST2L. When cardiomyocytes and cardiac fibroblasts are stimulated by mechanical pressure, both ST2L and sST2 increase. The binding of IL-33 to ST2L can inhibit cardiomyocyte hypertrophy and fibrosis, thereby protecting the heart. However, sST2 acts as a decoy receptor and competitively binds to IL-33 with ST2L, inhibiting the binding of ST2L to IL-33, thereby preventing the protective effect of IL-33 on the heart and promoting myocardial remodeling and ventricular dysfunction. sST2 is also considered to be a new generation of cardiac markers that is more timely, accurate and stable than NT-proBNP. A large number of studies have shown that sST2 is related to myocardial fibrosis and myocardial remodeling, and has a high application value in the diagnosis and evaluation of heart failure, and has been written into multiple guidelines. Soluble ST2 can predict the hospitalization and death of patients with heart failure, and can assist NT-proBNP, providing a deeper diagnostic value for the diagnosis of heart failure.

[0005] With the advent of various biotin derivatives, BAS was soon widely used in various fields of medicine. In recent years, a large number of studies have confirmed that the biotin and avidin system can be combined with almost all the markers that have been successfully studied. The strong binding of biotin and avidin with high affinity and the multi-stage amplification effect make BAS immunolabeling and related tracer analysis more sensitive. It has become a new technology widely used in qualitative and quantitative detection of trace antigens and antibodies and location observation research. Biotin is water-soluble and rapidly excreted, however, if high-dose biotin supplementation is used, there may be relatively high levels of biotin in the circulation, and circulating biotin will also be present in samples used to measure analytes for in vitro analysis, that is, in samples such as serum or plasma, biotin contained in the sample, if present at high levels, will bind to streptavidin-coated magnetic particles, resulting in the antigen-antibody complex being unable to bind to the magnetic particles and being washed away, resulting in false positive or false negative assay results, thereby leading to clinical misdiagnosis. Summary of the invention

[0006] The main purpose of the present invention is to provide a preparation method and application of a detection kit based on biotin and avidin, which can effectively solve the problems in the background technology.

[0007] To achieve the above object, the technical solution adopted by the present invention is: The preparation method of the detection kit based on biotin and avidin includes the following steps: S1: prepare the materials for preparation, including the sample to be tested, biotinylated ST2 monoclonal antibody, acridinium ester-labeled ST2 monoclonal antibody, streptavidin-coated magnetic particles, chemiluminescent excitation solution 1, and chemiluminescent excitation solution 2; S2: 20 μL of the sample to be tested, 100 μL of biotinylated ST2 monoclonal antibody, and 100 μL of acridinium ester-labeled ST2 monoclonal antibody were reacted at 37°C for 10 minutes to form an antibody-antigen-antibody complex; S3: Add 20 μL of streptavidin-coated magnetic microparticles and react with the antibody-antigen-antibody complex of step S2 for 10 minutes. The antibody-antigen-antibody complex is linked to the magnetic microparticles through the action of biotinylated ST2 monoclonal antibody and streptavidin to form a magnetic microparticle-antibody-antigen-antibody complex, which is recorded as a magnetic complex suspension; S4: placing the magnetic complex suspension in step S3 in a magnetic field, and washing the magnetic complex contained in the magnetic complex suspension; S5: inject 275 μL of chemiluminescent excitation solution 1 into the magnetic complex washed in step S4, and after 1.5 seconds, inject 275 μL of chemiluminescent excitation solution 2 to detect the chemiluminescent photon intensity; S6: The instrument converts the signal value into a concentration value output, thereby completing the preparation of the detection kit based on biotin and avidin.

[0008] Preferably, the concentration of the biotinylated ST2 monoclonal antibody is 500 ng / mL, the concentration of the acridinium ester-labeled ST2 monoclonal antibody is 500 ng / mL, and the concentration of the streptavidin-coated magnetic microparticles is 0.9 mg / mL.

[0009] Preferably, the biotinylated ST2 monoclonal antibody is modified with a polymer, comprising the following steps: A: Synthesize initiators, including single-head initiators and double-head initiators, wherein the initiators are halogenated hydrocarbon compounds with maleimide protecting groups, and the initiators are modified onto biotinylated ST2 monoclonal antibodies; B: The biotinylated ST2 monoclonal antibody was subjected to atom transfer radical polymerization, the monomer, initiator, ligand, copper chloride and sodium ascorbate in the atom transfer radical polymerization were mixed, and the polymerization reaction was carried out in an aqueous solution, and a nitrogen atmosphere was introduced during the polymerization reaction to protect the reaction; C: After the reaction is completed, the obtained polymer is dialyzed in an aqueous solution, and then freeze-dried to complete the modification.

[0010] Preferably, in the atom transfer radical polymerization in step B, the molar ratio of monomer, initiator, ligand, copper chloride and sodium ascorbate is 50-500:1:12:10:1.

[0011] Preferably, in the atom transfer radical polymerization in step B, the molar ratio of monomer, initiator, ligand, copper chloride and sodium ascorbate is 50-200:1:12:10:1.

[0012] Preferably, the polymerization time is 30 min-24 h, preferably, the polymerization time is 30 min-480 min; more preferably, the polymerization time is 30 min-60 min.

[0013] Preferably, the reaction temperature is 0-30°C, more preferably, the reaction temperature is 20-30°C.

[0014] The application of the detection kit constructed based on biotin and avidin is applied to the above-mentioned method for preparing the detection kit constructed based on biotin and avidin. The detection kit constructed based on biotin and avidin is applied to qualitative and quantitative detection and positioning observation research of trace antigens and antibodies.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The sandwich method is used to detect ST2, including a streptavidin-coated magnetic particle suspension solution, a biotin-labeled ST2 protein capture antibody, and an acridinium ester-labeled ST2 protein detection antibody; wherein a self-developed modified streptavidin protein is added to the acridinium ester-labeled ST2 protein detection antibody solution, and the protein can bind to free biotin in the sample but not to biotin labeled on the antibody, thereby eliminating or reducing the influence of biotin in the measured sample on the ST2 detection result while using the biotin-avidin system to improve the reagent sensitivity. At present, as long as the reagent adopts the biotin and avidin system to amplify the reagent signal, the interference of biotin in the sample cannot be avoided, and this kit, because the reagent contains the self-developed modified streptavidin protein, can eliminate or reduce the influence of biotin in the measured sample on the ST2 detection result. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a structural diagram of the single-head initiator of the present invention; Figure 2 is a structural diagram of the double-head initiator of the present invention; Figure 3 is a structural diagram of a monomer initiator of the present invention; Figure 4 FIG. 4 is a structural diagram of another monomer initiator of the present invention.

[0017] In the figure: R includes but is not limited to ethyl, propyl and other groups. DETAILED DESCRIPTION

[0018] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] The preparation method of the detection kit based on biotin and avidin includes the following steps: S1: prepare the materials for preparation, including the sample to be tested, biotinylated ST2 monoclonal antibody, acridinium ester-labeled ST2 monoclonal antibody, streptavidin-coated magnetic microparticles, chemiluminescent excitation solution 1, and chemiluminescent excitation solution 2. The concentration of biotinylated ST2 monoclonal antibody is 500 ng / mL, the concentration of acridinium ester-labeled ST2 monoclonal antibody is 500 ng / mL, and the concentration of streptavidin-coated magnetic microparticles is 0.9 mg / mL. The biotinylated ST2 monoclonal antibody is modified with a polymer, including the following steps: A: Synthesize initiators, including single-head initiators and double-head initiators. The initiators are halogenated hydrocarbon compounds with maleimide protective groups. The initiators are modified onto biotinylated ST2 monoclonal antibodies. The structural formula is as follows: Figure 1-4 As shown; B: The biotinylated ST2 monoclonal antibody was subjected to atom transfer radical polymerization, the monomer, initiator, ligand, copper chloride and sodium ascorbate in the atom transfer radical polymerization were mixed, and the polymerization reaction was carried out in an aqueous solution, and a nitrogen atmosphere was introduced during the polymerization reaction to protect the reaction; C: After the reaction is completed, the obtained polymer is dialyzed in an aqueous solution, and then freeze-dried to complete the modification.

[0020] The molar ratio of monomer, initiator, ligand, copper chloride and sodium ascorbate in the atom transfer radical polymerization is 50-500:1:12:10:1.

[0021] The molar ratio of monomer, initiator, ligand, copper chloride and sodium ascorbate in the atom transfer radical polymerization is 50-200:1:12:10:1.

[0022] The polymerization time is 30 min-24 h, preferably, the polymerization time is 30 min-480 min; more preferably, the polymerization time is 30 min-60 min.

[0023] The reaction temperature is 0-30°C, more preferably, the reaction temperature is 20-30°C; S2: 20 μL of the sample to be tested, 100 μL of biotinylated ST2 monoclonal antibody, and 100 μL of acridinium ester-labeled ST2 monoclonal antibody were reacted at 37°C for 10 minutes to form an antibody-antigen-antibody complex; S3: Add 20 μL of streptavidin-coated magnetic microparticles and react with the antibody-antigen-antibody complex of step S2 for 10 minutes. The antibody-antigen-antibody complex is linked to the magnetic microparticles through the action of biotinylated ST2 monoclonal antibody and streptavidin to form a magnetic microparticle-antibody-antigen-antibody complex, which is recorded as a magnetic complex suspension; S4: placing the magnetic complex suspension in step S3 in a magnetic field, and washing the magnetic complex contained in the magnetic complex suspension; S5: inject 275 μL of chemiluminescent excitation solution 1 into the magnetic complex washed in step S4, and after 1.5 seconds, inject 275 μL of chemiluminescent excitation solution 2 to detect the chemiluminescent photon intensity; S6: The instrument converts the signal value into a concentration value output, thereby completing the preparation of the detection kit based on biotin and avidin.

[0024] The application of the detection kit constructed based on biotin and avidin is applied to the above-mentioned method for preparing the detection kit constructed based on biotin and avidin. The detection kit constructed based on biotin and avidin is applied to qualitative and quantitative detection and positioning observation research of trace antigens and antibodies. Specific embodiment one:

[0025] Samples containing different concentrations of free biotin were tested. The following is the free biotin content in 10ng / mL, as shown in Table 1: Table 1

[0027] The following is the free biotin content in 500ng / mL, as shown in Table 2: Table 2 Specific embodiment 2:

[0030] The soluble growth stimulating expression gene 2 protein chemiluminescence detection kit constructed using the biotin and avidin system, the detection results of the sample with free biotin content in 10ng / mL are shown in Table 3: Table 3

[0031] The soluble growth stimulating expression gene 2 protein chemiluminescence detection kit constructed using the biotin and avidin system, the detection results of the sample with free biotin content in 500ng / mL are shown in Table 4: Table 4

[0032] The present invention utilizes the biotin and avidin system to improve the sensitivity of the reagent. Since the reagent contains the modified streptavidin protein developed by the main developer, the influence of free biotin in the measurement sample on the ST2 detection result is eliminated or reduced. The method utilizes the ST2 antibody adsorbed on the magnetic particles by the biotin and avidin system to capture the ST2 protein in the test sample, utilizes the ST2 antibody labeled with acridinium ester to detect the ST2 protein in the test sample, and forms an antibody-antigen-antibody complex. When the luminescent marker acridinium ester is added with H2O2 in an alkaline environment, it emits light by itself, and the luminescence value (RLU) of the solution is detected by an instrument. The standard curve quantitatively calculates the concentration of ST2 protein in the test sample. Compared with the enzyme-linked immunosorbent assay kit, the kit of the present invention has the advantages of simple operation, strong specificity, high sensitivity, wide linear range, high precision, etc. in use due to the use of chemiluminescent immunoassay; compared with the colloidal gold method and immunofluorescence chromatography, it provides an accurate clinical judgment basis; compared with the chemiluminescent detection kit constructed based on the biotin and avidin system, it eliminates or reduces the influence of free biotin in the measurement sample on the ST2 detection result; and provides a rapid, simple, accurate and anti-interference detection reagent for clinical diagnosis.

[0033] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a detection kit based on biotin and avidin, characterized in that: The steps include: S1: prepare the materials for preparation, including the sample to be tested, biotinylated ST2 monoclonal antibody, acridinium ester-labeled ST2 monoclonal antibody, streptavidin-coated magnetic particles, chemiluminescent excitation solution 1, and chemiluminescent excitation solution 2; S2: 20 μL of the sample to be tested, 100 μL of biotinylated ST2 monoclonal antibody, and 100 μL of acridinium ester-labeled ST2 monoclonal antibody were reacted at 37°C for 10 minutes to form an antibody-antigen-antibody complex; S3: Add 20 μL of streptavidin-coated magnetic microparticles and react with the antibody-antigen-antibody complex of step S2 for 10 minutes. The antibody-antigen-antibody complex is linked to the magnetic microparticles through the action of biotinylated ST2 monoclonal antibody and streptavidin to form a magnetic microparticle-antibody-antigen-antibody complex, which is recorded as a magnetic complex suspension; S4: placing the magnetic complex suspension in step S3 in a magnetic field, and washing the magnetic complex contained in the magnetic complex suspension; S5: inject 275 μL of chemiluminescent excitation solution 1 into the magnetic complex washed in step S4, and after 1.5 seconds, inject 275 μL of chemiluminescent excitation solution 2 to detect the chemiluminescent photon intensity; S6: The instrument converts the signal value into a concentration value output, thereby completing the preparation of the detection kit based on biotin and avidin.

2. The method for preparing a detection kit based on biotin and avidin according to claim 1, characterized in that: The concentration of the biotinylated ST2 monoclonal antibody was 500 ng / mL, the concentration of the acridinium ester-labeled ST2 monoclonal antibody was 500 ng / mL, and the concentration of the streptavidin-coated magnetic particles was 0.9 mg / mL.

3. The method for preparing a detection kit based on biotin and avidin according to claim 1, characterized in that: The biotinylated ST2 monoclonal antibody is modified with a polymer, comprising the following steps: A: Synthesize initiators, including single-head initiators and double-head initiators, wherein the initiators are halogenated hydrocarbon compounds with maleimide protecting groups, and the initiators are modified onto biotinylated ST2 monoclonal antibodies; B: The biotinylated ST2 monoclonal antibody was subjected to atom transfer radical polymerization, the monomer, initiator, ligand, copper chloride and sodium ascorbate in the atom transfer radical polymerization were mixed, and the polymerization reaction was carried out in an aqueous solution, and a nitrogen atmosphere was introduced during the polymerization reaction to protect the reaction; C: After the reaction is completed, the obtained polymer is dialyzed in an aqueous solution, and then freeze-dried to complete the modification.

4. The method for preparing a detection kit based on biotin and avidin according to claim 3, characterized in that: In the atom transfer radical polymerization in step B, the molar ratio of monomer, initiator, ligand, copper chloride and sodium ascorbate is 50-500:1:12:10:

1.

5. The method for preparing a detection kit based on biotin and avidin according to claim 3, characterized in that: In the atom transfer radical polymerization in step B, the molar ratio of monomer, initiator, ligand, copper chloride and sodium ascorbate is 50-200:1:12:10:

1.

6. The method for preparing a detection kit based on biotin and avidin according to claim 3, characterized in that: The polymerization time is 30 min-24 h.

7. The method for preparing a detection kit based on biotin and avidin according to claim 3, characterized in that: The reaction temperature is 0-30°C.

8. Application of a detection kit based on biotin and avidin, applied to the above-mentioned method for preparing a detection kit based on biotin and avidin, characterized in that: The detection kit constructed based on biotin and avidin is applied to qualitative and quantitative detection and positioning observation research of trace antigens and antibodies.

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