A chemiluminescence method for protein detection using magnetic microparticles

Abstract

This invention discloses a magnetic microparticle chemiluminescence protein detection method, comprising the following steps: (1) combining surface-modified magnetic microparticles with chicken anti-mouse IgY antibody to form R1 complex; (2) combining chicken anti-mouse IgY antibody coated with magnetic beads with target mouse anti-human IgG antibody to form R2 complex; (3) combining streptavidinized Protein A with biotinylated alkaline phosphatase to form biotin-avidin complex to form R3 complex; (4) combining R3 complex with target mouse anti-human IgG antibody to form R4 complex, thus obtaining the final product. This method avoids the destruction of immune activity of the coated antibody and detection antibody during the coupling process, allowing the coated antibody and detection antibody to directly participate in the immune reaction. Furthermore, the introduction of the biotin-avidin system greatly improves detection sensitivity, making it suitable for in vitro detection of low-abundance disease markers such as early diagnosis of Alzheimer's disease or cytokine storms.

Description

Technical Field

[0001] This invention belongs to the technical field of medical immunology, and in particular relates to a magnetic microparticle chemiluminescence protein detection method. Background Technology

[0002] Chemiluminescence technology is currently the mainstream direction of immunodiagnostic technology after continuous updates and iterations. Reagent products have advantages such as high sensitivity, wide linear kinetic range, accurate quantitative detection, stable results with small errors, and simple operation, making them stand out in clinical applications and becoming the mainstream products in the field of quantitative immunoassay. Chemiluminescence immunoassay is a recognized advanced labeled immunoassay technology. The principle generally involves coating the surface of superparamagnetic microparticles with captured antigens or antibodies, and then adsorbing or covalently coupling the labeled antigens or antibodies onto a luminescent compound. Common luminescent compounds include alkaline phosphatase, luminol, luciferin, alkaline phosphatase, horseradish peroxidase, and ruthenium tripyridine.

[0003] In existing processes, chemical cross-linking agents are commonly used for protein modification. For example, in the process of coating proteins with carboxylated 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 alkaline phosphatase-labeled antigens or antibodies, two proteins are usually chemically modified. (N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC) is used to modify the protein into an amide bond intermediate, while 2-iminothione hydrochloride (Traut's) is used to treat the antigen or antibody, modifying it into an intermediate with a thiol group (-SH). After purification, the two are combined to form a bispecific activating group for protein coupling. The process of using cross-linking agents to activate antibodies or antigens is essentially a modification or disruption of the protein structure. This structural change leads to a certain degree of reduction in the protein's natural affinity or immune activity.

[0004] In the development of immunological in vitro diagnostic reagents, the immune response between antigens and antibodies is often affected by chemical modifications, which reduces the reaction efficiency, leading to decreased sensitivity or failure to meet specificity requirements. In particular, for the coupling between large protein molecules, the steric hindrance effect is quite significant during the formation of immune complexes, making it difficult to capture low-abundance markers in the blood, which greatly reduces the affinity of the antigen or antibody itself. Summary of the Invention

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

[0006] To solve the above-mentioned technical problems, the present invention is achieved through the following technical solution:

[0007] A magnetic particle chemiluminescence protein detection method includes the following steps:

[0008] (1) Formation of R1 component: Surface-modified magnetic microparticles and chicken anti-mouse IgY antibody are mixed at a mass ratio of 100:1 and the first diluent is added to prepare a 10 mg / mL magnetic microparticle reagent semi-finished product;

[0009] (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 a 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.

[0010] (3) Formation of R3 component: 5 μL of biotinylated alkaline phosphatase was added to 0.1 mg of streptavidinized protein A for non-specific binding. The mixture was slowly mixed at 37 °C in the dark and reacted for 1 h. The mixture was then purified by ultrafiltration. The purified solution was diluted with the third diluent to a final concentration of 0.4 mg / mL to obtain R3 component.

[0011] (4) Formation of R4 component: Unmodified mouse anti-human detection antibody was added to R2 component, reacted fully, and after magnetic separation and washing, it was added to R3 component at a mass ratio of 0.5:1. The mixture was slowly mixed at 37℃ for 30 min to prepare R4 component. R4 component was added to the fourth dilution solution at a mass ratio of 1:10000. In the final system, the concentration of detection antibody was 0.5 μg / mL.

[0012] (5) Add substrate solution to R4 component for photoexcitation. The luminescence value is linearly related to the target protein, thereby quantitatively detecting the target protein content in the sample.

[0013] Furthermore, the surface-modified magnetic microparticles corresponding to component R1 are carboxyl magnetic microparticles with surface-modifying groups of -COOH, -NH2, or -SH groups, and a particle size of 270nm~300nm.

[0014] Magnetic microparticles can be used in different detection fields depending on their surface chemical groups. The surrounding polymer materials can be activated by physical or chemical methods to generate groups such as amino (NH2-), carboxyl (COOH-), or epoxy (—CH(O)), which can be coupled with bioactive molecules such as proteins. Among them, carboxylated magnetic microparticles are the most widely used. Carboxylated magnetic microparticles are magnetic microparticles with carboxyl functional groups on their surface and superparamagnetic. They are a type of functional biomagnetic beads. They can quickly aggregate in a magnetic field and can be uniformly dispersed by magnetic separation after leaving the magnetic field.

[0015] Surface modification of carboxyl magnetic microparticles often involves using 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) chemical crosslinking agent to activate the carboxyl groups (COOH-) on the surface of the magnetic microparticles, forming an amine-reactive O-acylisourea intermediate. This intermediate can react rapidly with amino groups to form amide bonds, thereby covalently coupling the primary amino group of the target protein with the activated O-acylisourea intermediate. This coupling method is stable, does not detach, and is highly efficient, making it the most widely used coupling method currently.

[0016] Carboxyl magnetic microspheres are monodisperse and non-aggregate regular spherical shapes. They have the characteristics of superparamagnetism, rapid magnetic response, abundant carboxyl functional groups, monodispersity and submicron scale particle size. They can covalently couple biological ligands such as peptides, proteins, antibodies, and oligonucleotides to the surface of microspheres under the action of special chemical reagents (such as EDC), making them an important carrier tool in medical and molecular biology research.

[0017] Furthermore, the first dilution solution corresponding to component R1 includes Na2HPO4 2H2O 7.21g / L, NaH2PO4 12H2O 1.31g / L, NaCl 9.00g / L, ProClin 950 1.00g / L, sucrose 50.00g / L, Albumin Bovine V 5.00g / L, and Tween-80 0.50g / L.

[0018] Furthermore, the second diluent corresponding to component R2 includes 2% Albumin Bovine V, 1% ProClin950, and 0.5% Tween-80.

[0019] Furthermore, the third diluent in component R3 is a 1×PBS diluent, comprising 0.1 mol / L Tris base, 0.9% NaCl, 0.5% Albumin Bovine V, and 1% ProClin950.

[0020] Furthermore, the fourth diluent in component R4 includes 0.5% Albumin Bovine V, 1% sucrose, 5% glycerol, 0.1% Proclin 950, and 0.05% Tween-80.

[0021] The detection principle is a double-antibody sandwich method. Chicken anti-mouse IgY antibody coated with magnetic beads binds to target mouse anti-human IgG antibody to form the R1 complex. Streptavidin-modified protein A binds to biotinylated alkaline phosphatase to form a biotin-avidin complex. This complex binds to the target mouse anti-human IgG antibody to form the R2 complex. The clinical sample to be tested is incubated with the R1 and R2 complexes. During this process, the analyte in the sample forms a sandwich-structured complex with R1 and R2. After washing, unbound components are removed from the sample. Pre-activation and activation solutions are then added. Under the action of the activation solution, alkaline phosphatase emits light of a specific wavelength. 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, thus enabling quantitative detection of the analyte in the clinical sample.

[0022] The present invention has the following beneficial effects: The methodology provided by the present invention is a modeling system applicable to any immunodiagnostic reagent project. It avoids the destruction of the immune activity of the coating antibody and the detection antibody during the coupling process, and allows the coating antibody and the detection antibody to directly participate in the immune reaction. Furthermore, the introduction of the biotin-avidin system greatly improves the detection sensitivity and is suitable for the detection of low-abundance disease biomarkers such as Alzheimer's disease or cytokine storm.

[0023] The labeling process of chicken anti-mouse IgY antibody with magnetic microparticle conjugate is stable, as is the labeling process of streptavidin-modified Protein A with biotinylated alkaline phosphatase conjugate. The methodology of this invention simplifies the process of labeling process optimization and system optimization, ensuring process stability and production stability to the greatest extent, and saving time, manpower and financial costs in project development. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0025] Figure 1 : Schematic diagram of the technical principle of this invention.

[0026] Figure 2 The sensitivity of the pTau-217 reagent prepared by this method.

[0027] Figure 3 Linear range of the pTau-217 reagent prepared by this method.

[0028] Figure 4 Precision of the pTau-217 reagent prepared by this method.

[0029] Figure 5 The thermal stability of the pTau-217 reagent prepared by this method.

[0030] Figure 6 Clinical evaluation of the pTau-217 reagent prepared using this method. Detailed Implementation

[0031] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0032] This embodiment of a human phosphorylated Tau-217 protein detection kit (magnetic microparticle chemiluminescence method) includes kit components such as R1, R2, R3, and R4;

[0033] The preparation of R1 involves mixing carboxyl magnetic microparticles with chicken anti-mouse IgY antibody at a mass ratio of 100:1 to obtain a 10 mg / mL reagent semi-finished product. The selected carboxyl magnetic microparticles are JSR, Thomas Fisher, 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.

[0034] The preparation of R2 involves combining R1 with human phosphorylated Tau-217 mouse anti-human monoclonal antibody (Mouse AntiHuman p-Tau217) at a mass ratio of 1:0.04. After adding the human phosphorylated Tau-217 mouse anti-human monoclonal antibody to the magnetic microparticle reagent semi-finished product, the mixture is slowly mixed at 37°C for 30 minutes. The diluent comprises the following components and concentrations: 2% Albumin Bovine V, 1% ProClin950, and 0.5% Tween-80.

[0035] The preparation of R3 involved mycoagulant-modified Protein A (purchased from Thermo Fisher Scientific) and biotinylated alkaline phosphatase (purchased from BBI Solution). 5 μL of biotinylated alkaline phosphatase was added to 0.1 mg of streptavidin-modified Protein A, and the mixture was slowly mixed at 37°C in the dark for 1 h. The solution was then purified via ultrafiltration, and the purified solution was diluted with 1×PBS to a final concentration of 0.4 mg / mL. The diluent contained the following components and concentrations: 0.1 mol / L Tris base, 0.9% NaCl, 0.5% Albumin Bovine V, and 1% ProClin 950.

[0036] The preparation of R4 involves combining the aforementioned biotin-avidin complex with the target mouse anti-human IgG detection antibody at a mass ratio of 1:0.5. After adding the target mouse anti-human IgG detection antibody to the biotin-avidin complex, the mixture is slowly mixed at 37°C for 30 minutes to prepare the R4 complex. This complex is then added to the reagent buffer at a mass ratio of 1:10000 to obtain the R4 reagent. The final concentration of the detection antibody in the system is 0.5 μg / mL. The diluent comprises the following components and concentrations: 0.5% Albumin Bovine V, 1% sucrose, 5% glycerol, 0.1% Proclin 950, and 0.05% Tween-80.

[0037] These embodiments are selected and specifically described in this specification 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 non-diagnostic magnetic particle chemiluminescence protein detection method, characterized in that: It includes the following steps, (1) Formation of R1 component: Surface-modified magnetic microparticles and chicken anti-mouse IgY antibody are mixed at a mass ratio of 100:1 and the first diluent is added to prepare a 10 mg / mL magnetic microparticle 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 a 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: 5 μL of biotinylated alkaline phosphatase was added to 0.1 mg of streptavidinized protein A for non-specific binding. The mixture was slowly mixed at 37 °C in the dark and reacted for 1 h. The mixture was then purified by ultrafiltration. The purified solution was diluted with the third diluent to a final concentration of 0.4 mg / mL to obtain R3 component. (4) Formation of R4 component: Unmodified mouse anti-human detection antibody was added to R2 component, reacted fully, and after magnetic separation and washing, it was added to R3 component at a mass ratio of 0.5:

1. The mixture was slowly mixed at 37℃ for 30 min to prepare R4 component. R4 component was added to the fourth dilution solution at a mass ratio of 1:10000. In the final system, the concentration of detection antibody was 0.5 μg / mL. (5) Add substrate solution to R4 component for photoexcitation. The luminescence value is linearly related to the target protein, thereby quantitatively detecting the target protein content in the sample.

2. The non-diagnostic magnetic particle chemiluminescence protein detection method according to claim 1, characterized in that: The surface-modified magnetic microparticles corresponding to component R1 are carboxyl magnetic microparticles with surface-modifying groups of -COOH, -NH2, or -SH groups, and a particle size of 270nm~300nm.

3. The non-diagnostic magnetic particle chemiluminescence protein detection method according to claim 2, characterized in that: The modification of magnetic microparticles involves using 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) chemical crosslinking agent to activate the carboxyl groups (COOH-) on the surface of the magnetic microparticles, forming an amine-reactive O-acylisourea intermediate, which reacts rapidly with amino groups to form amide bonds, thereby covalently coupling the primary amino group of the target protein with the activated O-acylisourea intermediate.

4. The non-diagnostic magnetic particle chemiluminescence protein detection method according to claim 1, characterized in that: The first dilution solution corresponding to component R1 includes 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.

5. The non-diagnostic chemiluminescence protein detection method using magnetic microparticles according to claim 1, characterized in that: The second diluent for component R2 consists of 2% Albumin Bovine V, 1% ProClin950, and 0.5% Tween-80.

6. The non-diagnostic magnetic particle chemiluminescence protein detection method according to claim 1, characterized in that: The third diluent in component R3 is a 1×PBS diluent, which includes 0.1 mol / L Tris base, 0.9% NaCl, 0.5% Albumin Bovine V, and 1% ProClin 950.

7. The non-diagnostic magnetic particle chemiluminescence protein detection method according to claim 1, characterized in that: The fourth diluent in component R4 includes 0.5% Albumin Bovine V, 1% sucrose, 5% glycerol, 0.1% Proclin 950, and 0.05% Tween-80.

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