Protein detection method by magnetic particle chemiluminescence method

Abstract

The invention discloses a magnetic particle chemiluminescence protein detection method, which comprises the following steps: (1) combining surface modified magnetic particles with a chicken anti-mouse IgY antibody to prepare an R1 compound; (2) combining the chicken anti-mouse IgY antibody coated with the magnetic beads with a target mouse anti-human IgG antibody to prepare an R2 compound; (3) combining the Protein A subjected to streptavidin treatment with the alkaline phosphatase subjected to biotinylation to form a biotin-avidin compound, and preparing an R3 compound; and (4) combining the R3 compound with a target mouse anti-human IgG antibody to prepare an R4 compound. According to the present invention, the immunocompetence of the coating antibody and the detection antibody is prevented from being damaged during the coupling process, the coating antibody and the detection antibody directly participate in the immunoreaction, and the biotin-avidin system is introduced, such that the detection sensitivity is substantially improved, and the kit is suitable for the in vitro detection of the low-abundance disease markers such as the early diagnosis of the Alzheimer's disease or the cytokine storm.

Description

Technical Field

[0001] The invention belongs to the technical field of medical immunity, and particularly relates to a magnetic particle chemiluminescence protein detection method. Background Art

[0002] Chemiluminescence technology is the mainstream direction of immunodiagnostic technology after continuous updates and iterations. The reagent products have the advantages of high sensitivity, wide linear dynamic range, accurate quantitative detection, stable results with small errors, and simple operation. They stand out in clinical applications and become the mainstream products in the field of immunoquantitative analysis. Chemiluminescence immunoassay is a recognized advanced labeling immunoassay technology. The principle is generally to coat the capture antigen or antibody on the surface of superparamagnetic particles, and then adsorb or covalently couple the labeled antigen or antibody to the luminescent compound. Common luminescent compounds include alkaline phosphatase, luminol, lucigenin, alkaline phosphatase, horseradish peroxidase, terpyridine ruthenium, etc.

[0003] In the existing process technology, chemical crosslinkers are usually used for protein modification. For example, in the process of coating proteins with carboxyl magnetic beads, 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) is used to activate the carboxyl group. After activation, the amide residue forms a stable covalent bond with the ε-amino acid of the antigen or antibody. In the alkaline phosphatase labeling antigen or antibody, two proteins are usually chemically modified. (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester (SMCC) is used to modify it into an amide bond intermediate. At the same time, 2-iminothiolane hydrochloride (Traut's) is used to treat the antigen or antibody and modify it into an intermediate with a thiol group (-SH). After purification, the two are formed into a bispecific activation group for protein coupling. The process of using a crosslinker to activate the antibody or antigen is essentially a modification or destruction of the protein structure. The change in this structure leads to a decrease in the natural affinity or immune activity of the protein to a certain extent.

[0004] In the development process of immunological in vitro diagnostic reagents, the immune response of antigens and antibodies is often affected by chemical modifications, which reduces the reaction efficiency, resulting in decreased sensitivity or specificity that does not meet the requirements. In particular, for the coupling between large molecular proteins, the steric hindrance effect is more obvious during the formation of immune complexes, and low-abundance markers in the blood are often difficult to capture, which greatly reduces the affinity of the antigen or antibody itself. Summary of the invention

[0005] The purpose of the present invention is to provide a magnetic particle chemiluminescence protein detection method, which mainly solves the problem that the immune reaction of antigen and antibody is affected by chemical modification and the reaction efficiency is reduced.

[0006] In order to solve the above technical problems, the present invention is achieved through the following technical solutions: A magnetic particle chemiluminescence protein detection method comprises the following steps: (1) Formation of R1 component: The surface-modified magnetic particles and chicken anti-mouse IgY antibody were mixed at a mass ratio of 100:1, and the first diluent was added to prepare a 10 mg / mL magnetic particle reagent semi-finished product; (2) Formation of R2 component: The unmodified mouse anti-human capture antibody was fully reacted with the target protein in the sample, and then added to the R1 component at a mass ratio of 0.04:1 for non-specific binding. The mixture was slowly mixed at 37°C for 30 min, and the second diluent was added to make the concentration of the coated antibody in the final system 0.1-0.5 μg / mL to prepare the R2 component. (3) Formation of R3 component: Take 5 μL of biotinylated alkaline phosphatase and add it to 0.1 mg of streptavidin-ylated protein A for non-specific binding. Slowly mix at 37°C in the dark and react for 1 h. Purify by ultrafiltration tube. Take the purified solution and dilute it with the third dilution solution to a final concentration of 0.4 mg / mL to obtain R3 component. (4) Formation of R4 component: Add unchemically modified mouse anti-human detection antibody to R2 component, allow to react fully, and after magnetic separation and cleaning, add to R3 component at a mass ratio of 0.5:1. Slowly mix at 37°C for 30 min to prepare R4 component, which is added to the fourth dilution at a mass ratio of 1:10,000. In the final system, the concentration of the detection antibody is 0.5 μg / mL; (5) Substrate solution is added to the R4 component for light excitation. The luminescence value is linearly related to the target protein, thereby quantitatively detecting the target protein content in the sample.

[0007] Furthermore, the surface-modified magnetic particles corresponding to the R1 component are carboxyl magnetic particles, whose surface modification groups are -COOH, -NH2, or -SH groups, and whose particle sizes are between 270nm and 300nm.

[0008] Magnetic particles can be used in different detection fields according to the different surface chemical groups. The peripheral polymer materials can be activated by physical or chemical methods to produce amino (NH2-), carboxyl (COOH-) or epoxy (—CH(O)) groups, which can be coupled with biologically active molecules such as proteins. Among them, the most widely used are carboxylated magnetic particles. Carboxyl magnetic particles are magnetic particles with superparamagnetism modified with carboxyl functional groups on the surface. They are a kind of functional biological magnetic beads. They can quickly aggregate in the magnetic field and can be evenly dispersed after leaving the magnetic field to facilitate magnetic separation.

[0009] The surface modification of carboxyl magnetic particles often uses 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) chemical crosslinker to activate the carboxyl groups (COOH-) on the surface of the magnetic particles to form amine-reactive O-acylisourea intermediates, which can react rapidly with amino groups to form amide bonds, thereby covalently coupling the primary amino groups of the target protein to the activated O-acylisourea intermediates. This coupling method is stable, does not fall off, and has high efficiency, and is currently the most commonly used coupling method.

[0010] Carboxyl magnetic microspheres are monodisperse, non-agglomerated, regular spherical particles with the characteristics of superparamagnetism, rapid magnetic responsiveness, abundant carboxyl functional groups, monodispersity and submicron particle size. They can covalently couple biological ligands such as peptides, proteins, antibodies, oligonucleotides to the surface of microspheres under the action of special chemical reagents (such as EDC), and are important carrier tools in medical and molecular biology research.

[0011] Furthermore, the first dilution solution corresponding to the R1 component includes Na2HPO4 2H2O 7.21 g / L, NaH2PO4 12H2O1.31 g / L, NaCl 9.00 g / L, ProClin 950 1.00 g / L, sucrose 50.00 g / L, Albumin Bovine V5.00 g / L, and Tween-80 0.50 g / L.

[0012] Furthermore, the second dilution solution corresponding to the R2 component includes 2% Albumin Bovine V, 1% ProClin950, and 0.5% Tween-80.

[0013] Furthermore, the third diluent in the R3 component is a 1×PBS diluent, including 0.1 mol / L Tris base, 0.9% NaCl, 0.5% Albumin Bovine V, and 1% ProClin950.

[0014] Furthermore, the fourth dilution in the R4 component includes 0.5% Albumin Bovine V, 1% sucrose, 5% glycerol, 0.1% Proclin950, and 0.05% Tween-80.

[0015] The detection principle is the double antibody sandwich method. The chicken anti-mouse IgY antibody coated with magnetic beads is combined with the target mouse anti-human IgG antibody to form the R1 complex; the streptavidin-ylated protein A is combined with the biotinylated alkaline phosphatase to form a biotin-avidin complex, and the complex is combined with the target mouse anti-human IgG antibody to form the R2 complex; the clinical sample to be tested is incubated with the R1 complex and the R2 complex. During this process, the analyte in the sample forms a sandwich structure complex with R1 and R2. After washing, other unbound components in the sample are removed. Subsequently, the pre-excitation solution and the excitation solution are added. The alkaline phosphatase will emit light of a specific wavelength under the action of the excitation solution. The luminescence intensity is proportional to the concentration of the analyte in the sample. The content of the analyte in the sample can be obtained by processing the calibration curve, thereby quantitatively detecting the analyte in the clinical sample to be tested.

[0016] The present invention has the following beneficial effects: the method provided by the methodology of the present invention is a modeling system, which is applicable to any immunodiagnostic reagent project, avoids the destruction of the immune activity of the coating antibody and the detection antibody during the coupling process, directly involves the coating antibody and the detection antibody in the immune response, and introduces the biotin-avidin system, which greatly improves the detection sensitivity and is applicable to the detection of low-abundance disease markers such as Alzheimer's disease or cytokine storm.

[0017] The labeling process of chicken anti-mouse IgY antibody and magnetic microparticle conjugate is stable, and the labeling process of streptavidin-ylated Protein A and biotinylated alkaline phosphatase conjugate is stable. The methodology system of the present invention simplifies the process of labeling process optimization and system optimization, ensures process stability and production stability to the greatest extent, and saves time cost, manpower cost and financial cost of project development. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for describing the embodiments are briefly introduced below.

[0019] Figure 1 : Technical principle diagram of the present invention.

[0020] Figure 2 :The sensitivity of the pTau-217 reagent prepared by this method.

[0021] Figure 3 : The linear range of the pTau-217 project reagent prepared by this method.

[0022] Figure 4 : The precision of the pTau-217 reagent prepared by this method.

[0023] Figure 5 : Thermal stability of the pTau-217 project reagent prepared by this method.

[0024] Figure 6 : Clinical test evaluation of the pTau-217 project reagent prepared by this method. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0026] A human phosphorylated Tau-217 protein detection kit (magnetic particle chemiluminescence method) of this embodiment includes kit components such as R1, R2, R3, and R4; The preparation of R1 includes preparing a 10 mg / mL reagent semi-finished product by mixing carboxyl magnetic particles and chicken anti-mouse IgY antibodies in a mass ratio of 100:1. The selected carboxyl magnetic particles are JSR, Thomerfisher, GE, etc. with a particle size of 270 nm to 300 nm. The diluent includes the following components and contents: Na2HPO4 2H2O 7.21 g / L, NaH2PO4 12H2O 1.31 g / L, NaCl 9.00 g / L, ProClin 950 1.00 g / L, sucrose 50.00 g / L, Albumin Bovine V 5.00 g / L, and Tween-80 0.50 g / L.

[0027] The preparation of R2 includes combining the above R1 with the human phosphorylated Tau-217 mouse anti-human monoclonal antibody (Mouse AntiHuman p-Tau217) at a mass ratio of 1:0.04, adding the human phosphorylated Tau-217 mouse anti-human monoclonal coated antibody to the magnetic particle reagent semi-finished product, and slowly mixing at 37°C for 30 minutes. The components and contents of the diluent are: 2% Albumin Bovine V, 1% ProClin950, and 0.5% Tween-80.

[0028] The preparation of R3 includes streptavidin-protein A purchased from Thermo Fisher Scientific, biotinylated alkaline phosphatase purchased from BBI Solution, 5 μL of biotinylated alkaline phosphatase is added to 0.1 mg of streptavidin-protein A, slowly mixed at 37°C in the dark, and reacted for 1 hour. Purify through an ultrafiltration tube, take the purified solution, and dilute it with 1×PBS to a final concentration of 0.4 mg / mL. The components and contents of the diluent are: 0.1 mol / L Tris base, 0.9% NaCl, 0.5% Albumin Bovine V, and 1% ProClin950.

[0029] The preparation of R4 includes combining the above-mentioned biotin-avidin complex with the target mouse anti-human IgG detection antibody at a mass ratio of 1:0.5. After the target mouse anti-human IgG detection antibody is added to the biotin-avidin complex, it is slowly mixed at 37°C for 30 minutes to prepare the R4 complex. It is added to the reagent buffer at a mass ratio of 1:10000 with the R4 reagent buffer to prepare the R4 reagent. In the final system, the concentration of the detection antibody is 0.5μg / mL. The components and contents of the diluent are: 0.5% Albumin Bovine V, 1% sucrose, 5% glycerol, 0.1% Proclin950, and 0.05% Tween-80.

[0030] This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can better understand and utilize the present invention.

Claims

1. A magnetic particle chemiluminescence protein detection method, characterized in that: It includes the following steps: (1) Formation of R1 component: The surface-modified magnetic particles and chicken anti-mouse IgY antibody were mixed at a mass ratio of 100:1, and the first diluent was added to prepare a 10 mg / mL magnetic particle reagent semi-finished product; (2) Formation of R2 component: The unmodified mouse anti-human capture antibody was fully reacted with the target protein in the sample, and then added to the R1 component at a mass ratio of 0.04:1 for non-specific binding. The mixture was slowly mixed at 37°C for 30 min, and the second diluent was added to make the concentration of the coated antibody in the final system 0.1-0.5 μg / mL to prepare the R2 component. (3) Formation of R3 component: Take 5 μL of biotinylated alkaline phosphatase and add it to 0.1 mg of streptavidin-ylated protein A for non-specific binding. Slowly mix at 37°C in the dark for 1 h. Purify by ultrafiltration tube. Take the purified solution and dilute it with the third dilution solution to a final concentration of 0.4 mg / mL to obtain R3 component. (4) Formation of R4 component: Add unchemically modified mouse anti-human detection antibody to R2 component, allow to react fully, and after magnetic separation and cleaning, add to R3 component at a mass ratio of 0.5:

1. Slowly mix at 37°C for 30 min to prepare R4 component, which is added to the fourth dilution at a mass ratio of 1:10,000. In the final system, the concentration of the detection antibody is 0.5 μg / mL; (5) Substrate solution is added to the R4 component for light excitation. The luminescence value is linearly related to the target protein, thereby quantitatively detecting the target protein content in the sample.

2. The method for detecting protein by magnetic particle chemiluminescence according to claim 1, characterized in that: The surface-modified magnetic particles corresponding to the R1 component are carboxyl magnetic particles, whose surface modification groups are -COOH, -NH2, or -SH groups, and whose particle sizes are between 270nm and 300nm.

3. A magnetic particle chemiluminescence protein detection method according to claim 2, characterized in that: The modification of magnetic particles uses 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) chemical crosslinker to activate the carboxyl groups (COOH-) on the surface of the magnetic particles to form amine-reactive O-acylisourea intermediates, which are used to quickly react with amino groups to form amide bonds, so that the primary amino groups of the target protein are covalently coupled to the activated O-acylisourea intermediates.

4. The method for detecting protein by magnetic particle chemiluminescence according to claim 1, characterized in that: The first dilution solution corresponding to the R1 component includes Na2HPO4 2H2O 7.21 g / L, NaH2PO4 12H2O 1.31 g / L, NaCl 9.00 g / L, ProClin950 1.00 g / L, sucrose 50.00 g / L, Albumin Bovine V 5.00 g / L, and Tween-80 0.50 g / L.

5. The method for detecting protein by magnetic particle chemiluminescence according to claim 1, characterized in that: The second dilution corresponding to the R2 component includes 2% Albumin Bovine V, 1% ProClin950, and 0.5% Tween-80.

6. The method for detecting protein by magnetic particle chemiluminescence according to claim 1, characterized in that: The third diluent in the R3 component is a 1×PBS diluent, including 0.1 mol / L Tris base, 0.9% NaCl, 0.5% Albumin Bovine V, and 1% ProClin950.

7. The method for detecting protein by magnetic particle chemiluminescence according to claim 1, characterized in that: The fourth dilution in the R4 component includes 0.5% Albumin Bovine V, 1% sucrose, 5% glycerol, 0.1% Proclin950, and 0.05% Tween-80.

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