A light-activated chemiluminescence detection kit for influenza b virus antigen and an analysis method thereof

Abstract

The application provides a light-activated chemiluminescence detection kit for influenza B virus antigen and an analysis method thereof. The light-activated chemiluminescence detection kit for influenza B virus antigen provided by the application comprises a light-emitting latex microsphere solution coated with anti-influenza B virus antibody and other reagents, and the light-emitting latex microsphere contains optical reaction substances; the light-emitting latex microsphere solution and / or other reagents also contain blank latex microspheres which do not contain any optical reaction substances and have no biological active substances connected to the surface. The kit provided by the application can eliminate the interference of the sample to be detected in the light-activated chemiluminescence method, and improve the accuracy of detection.

Description

Technical Field

[0001] This invention belongs to the field of photo-induced chemiluminescence analysis, specifically relating to a photo-induced chemiluminescence detection kit for influenza B virus antigen and its analytical method. Background Technology

[0002] Influenza B virus (B virus) is a type of influenza virus belonging to the genus *Betainfluenzavirus* in the family Orthomyxoviridae. It causes influenza. B virus only infects humans and pinnipeds (such as seals), therefore it has not caused the various influenza pandemics caused by influenza A virus. Although both viruses undergo gene rearrangement and antigenic drift, the evolution rate of B virus in the human population is lower than that of influenza A virus (2-3 times lower, but higher than that of influenza C virus). Nevertheless, B virus can still cause significant casualties worldwide, especially among adolescents and children. The genetic sequence of B virus contains 14,548 nucleotides, divided into eight single-stranded RNA segments. Based on the antigenic characteristics of its surface glycoprotein hemagglutinin, there are two main branches of circulating B virus: B / Yamagata / 16 / 88-virus and B / Victoria / 2 / 87-virus.

[0003] Oropharyngeal or nasopharyngeal swab samples are typically collected from the oropharynx or nasopharynx using a relatively long cotton swab. These secretions are then placed in a pathogen extract solution. This type of sample is used clinically to test for pathogens, commonly used to detect respiratory pathogens such as influenza virus and coronavirus. Saliva is a digestive fluid, mostly water, but also contains mucin and salivary amylase. Nasal aspirate is secretion extracted from the nasal cavity using a special device. These samples contain components such as mucin secreted by epithelial cells. When using photoluminescence immunoassay (PLIA) to detect these sample types, mucin can strongly interfere with the detection system. This is because mucin carries a charge, while the microspheres used in PLATIC carry the opposite charge. Within the reaction system, the mucin attracts the two types of microspheres, directly linking them together to generate a signal. Therefore, it is necessary to address sample interference when using PLATIC to detect these types of samples. Summary of the Invention

[0004] The technical problem this invention aims to solve is to provide a photocatalytic chemiluminescence analysis method for detecting influenza B virus antigens, addressing the shortcomings of existing technologies and resolving the issue of non-characteristic signals generated when using photocatalytic chemiluminescence analysis on samples such as throat swabs. This invention also provides a photocatalytic chemiluminescence detection kit for influenza B virus antigens and its usage method.

[0005] Therefore, a first aspect of the present invention provides a photoluminescence detection kit for influenza B virus antigen, comprising a solution of luminescent latex microspheres coated with anti-influenza B virus antibodies and other reagents, wherein the luminescent latex microspheres contain an optically reactive substance; the luminescent latex microsphere solution and / or other reagents further contain blank latex microspheres, wherein the blank latex microspheres do not contain any optically reactive substance and have no bioactive substances attached to their surface.

[0006] In some embodiments of the present invention, the other reagents include a biotin-labeled anti-influenza B virus antibody solution; preferably, the other reagents also include a sample diluent.

[0007] According to some embodiments of the present invention, the surface of the blank latex microspheres is modified with chemical groups, wherein the chemical groups are selected from at least one of epoxy, chloromethyl, mercapto, amino, hydroxy, maleamine, sulfonic acid, carboxyl and aldehyde groups.

[0008] According to some embodiments of the present invention, the chemical group is selected from at least one of sulfonic acid group, carboxyl group and aldehyde group.

[0009] According to some embodiments of the present invention, the chemical group is a carboxyl group.

[0010] According to some embodiments of the present invention, the blank latex microspheres are prepared by an emulsion polymerization method without emulsifiers.

[0011] In some embodiments of the present invention, the method for preparing the blank latex microspheres includes: reacting deionized water, styrene, hydroxyethyl methacrylate, and a small amount of divinylbenzene in the presence of potassium persulfate initiator, and obtaining microspheres with a clean surface and hydroxyl groups, with a diameter of approximately 60-1000 nm. Preferably, the reaction conditions include: polymerization reaction at 60-90°C, for example, 70°C, under a nitrogen atmosphere for 5-6 hours.

[0012] According to some embodiments of the present invention, the blank latex microspheres are polystyrene microspheres.

[0013] According to some embodiments of the present invention, both the luminescent latex microspheres and the blank latex microspheres are polystyrene microspheres.

[0014] According to some embodiments of the present invention, the average particle size of the blank latex microspheres is 60-1000 nm, for example, it can be 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, or 900 nm.

[0015] According to a preferred embodiment of the present invention, the average particle size of the blank latex microspheres is 100-300 nm.

[0016] According to some embodiments of the present invention, the particle size of the blank latex microspheres is smaller than that of the luminescent latex microspheres.

[0017] According to some embodiments of the present invention, the surface of the blank latex microspheres carries a negative charge.

[0018] According to some embodiments of the present invention, the zeta potential of the blank latex microspheres is -10 to -50 mV, for example, it can be -15 mV, -20 mV, -25 mV, -30 mV, -35 mV, -40 mV, -45 mV, or -50 mV.

[0019] According to some embodiments of the present invention, the zeta potential of the blank latex microspheres is -15 to -30 mV.

[0020] According to some embodiments of the present invention, the blank latex microspheres and the luminescent latex microspheres carry the same type of charge.

[0021] According to some embodiments of the present invention, the concentration of the blank latex microspheres in the first mixture is higher than 31.25 μg / mL.

[0022] According to a preferred embodiment of the present invention, in the first mixture, the concentration of the blank latex microspheres is 40 μg / mL-15 mg / mL, preferably 62.5 μg / mL-12.5 mg / mL.

[0023] A second aspect of the present invention provides a photo-induced chemiluminescence analysis method for detecting influenza B virus antigens, comprising the following steps:

[0024] Step S1: Mix the detection reagents in the kit with the sample to be tested, so that the influenza B virus antigen in the sample to be tested comes into contact with blank latex microspheres, luminescent latex microspheres containing labeled anti-influenza B virus antibodies, and biotin-labeled anti-influenza B virus antibodies to obtain a first mixture.

[0025] Step S2: Mix the first mixture with a solution of photosensitive latex microspheres labeled with streptavidin to obtain a second mixture;

[0026] Step S3: Irradiate the second mixture with a laser and detect the chemiluminescence signal.

[0027] According to some embodiments of the present invention, in step S1, the contact temperature is 30-45°C and the contact time is 10-30 minutes.

[0028] According to a preferred embodiment of the present invention, in step S2, the mixing temperature is 30-45°C and the time is 5-30 minutes.

[0029] According to some embodiments of the present invention, in step S3, the excitation wavelength of the laser irradiation is 650-700nm, and the detection wavelength is 550-650nm.

[0030] According to a preferred embodiment of the present invention, in step S3, the excitation wavelength of the laser irradiation is 675-685nm, and the detection wavelength is 610-620nm.

[0031] A third aspect of the invention provides the use of the kit according to the first aspect or the method according to the second aspect in detecting influenza B virus in oropharyngeal swabs, saliva or nasal aspirate samples.

[0032] In this invention, when detecting influenza B virus antigen using photoluminescence chemiluminescence, a certain amount of blank latex microspheres are added to the reaction system. These blank latex microspheres carry the same charge as the microspheres used in photoluminescence chemiluminescence. By adsorbing interfering substances such as mucins through the blank latex microspheres, interference from samples such as oropharyngeal swabs, nasopharyngeal swabs, saliva, and nasal aspirates is eliminated in the photoluminescence chemiluminescence method. This solves the problem of non-specific signals generated by such samples when using photoluminescence analysis technology, thereby improving the accuracy of detection. Detailed Implementation

[0033] To facilitate understanding of the present invention, it will be described in detail below. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terminology used herein is for describing specific embodiments only and is not intended to be restrictive.

[0034] Where numerical ranges are provided, it should be understood that every intermediate value between the upper and lower limits of the range and any other specified or intermediate value within the specified range is covered by this invention. The upper and lower limits of these smaller ranges may be independently included in the smaller range and are also covered by this invention, subject to any explicitly excluded limits within the specified range. Where a specified range includes one or two limits, the range excluding any or both of those included limits is also included by this invention.

[0035] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of this invention, preferred methods and materials are now described.

[0036] The technical principle of photo-induced chemiluminescence analysis is as follows: Under laser irradiation, the sensitizer can excite oxygen molecules in the surrounding environment into singlet oxygen molecules. The singlet oxygen molecules can react with optical reactive substances about 200 nm away to generate a light signal of a certain wavelength. When the sample contains the antigen or antibody to be tested, the immune reaction of the antigen and antibody can cause the photosensitive latex microspheres containing the sensitizer to bind with the luminescent latex microspheres containing the optical reactive substances, thereby generating a light signal of a specific wavelength. The content of the antigen or antibody to be tested can be detected by detecting this light signal.

[0037] An "antigen" is a substance that can stimulate the body to produce an immune response and bind to the immune response products, antibodies and sensitized lymphocytes, in vivo and in vitro to produce an immune effect. The antigen can be a fusion antigen, and when necessary, it can be further conjugated with other components, such as specific binding pairs, like biotin or avidin (one of the biotin-avidin specific binding pairs). When influenza B virus antigen is present in the sample to be tested, the influenza B virus antigen specifically binds to both luminescent latex microspheres coated with anti-influenza B virus antibodies and biotin-labeled anti-influenza B virus antibodies, forming a double-antibody sandwich complex on the surface of the luminescent latex microspheres. If streptavidin-modified photosensitive latex microspheres are added, the biotin binds to the streptavidin, causing the two microspheres to approach each other. Under excitation by a light source, the photosensitive latex microspheres release reactive oxygen species, which, upon encountering the luminescent latex microspheres in solution, produce chemiluminescence, further exciting the fluorescent groups on the same microsphere to produce a cascade amplification reaction and generate fluorescence. At this time, the higher the content of influenza B virus antigen markers, the stronger the fluorescence intensity.

[0038] "Sample to be tested" refers to a mixture that may contain the analyte. Typical samples to be tested that can be used in the methods disclosed in this invention include, but are not limited to, oropharyngeal swabs, nasopharyngeal swabs, saliva, nasal aspirate, serum, plasma, ascites, and pleural effusion. Samples to be tested typically contain mucin and / or other protein components with the same type of charge as mucin. These components are charged, while the microspheres used in the photo-induced chemiluminescence method carry the opposite charge. In the reaction system, mucin can directly bind to the microspheres through the attraction between positive and negative charges, thereby achieving the function of generating a signal through photo-induced chemiluminescence, which can cause strong interference with the detection system. Therefore, when using photoluminescence to detect samples that may contain influenza B virus antigens, this invention adds a certain amount of blank latex microspheres to the reaction system. These blank latex microspheres carry the same charge as the microspheres used in photoluminescence. By adsorbing interfering substances such as mucins through the blank latex microspheres, the interference caused by samples such as oropharyngeal swabs, nasopharyngeal swabs, saliva, and nasal aspirate in the photoluminescence method is eliminated. This solves the problem of non-specific signals generated by such samples when using photoluminescence analysis technology, and improves the accuracy of detection.

[0039] Therefore, in a first aspect, the present invention provides a photoluminescence detection kit for influenza B virus antigen, comprising a solution of luminescent latex microspheres coated with anti-influenza B virus antibodies and other reagents, wherein the luminescent latex microspheres contain an optically reactive substance; the luminescent latex microsphere solution and / or other reagents further contain blank latex microspheres, wherein the blank latex microspheres do not contain any optically reactive substance and have no bioactive substances attached to their surface.

[0040] The term "antibody" as used in this invention is used in the broadest sense, including any isotype of antibody or immunoglobulin, antibody fragments that retain specific binding to antigens, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, bispecific antibodies, and fusion proteins comprising the antigen-binding portion of an antibody and non-antibody proteins. Where desired, antibodies may be further conjugated to other portions, such as specifically binding pairing members, for example, biotin or streptavidin.

[0041] As used in this invention, the term "luminescent latex microspheres" refers to a type of microsphere, known as a marker, that can undergo a chemical reaction to induce luminescence, for example, by being transformed into another type of microsphere in an electronically excited state. The excited state can be a singlet state or a triplet excited state. The excited state can relax to the ground state and emit light directly, or it can recover to the ground state by transferring the excitation energy to the energy acceptor. In this process, the energy acceptor particle will transition to an excited state and emit light.

[0042] The term "bioactive substance" as used in this invention refers to a biologically active compound, typically a member of a biologically active "specific binding pair." A "specific binding pair" is a pair of molecules that can specifically bind to each other, such as an enzyme-substrate pair, an antigen-antibody pair, or a ligand-receptor pair. A specific example of a specific binding pair is the biotin-avidin system, where biotin, widely found in plant and animal tissues, has two ring structures: an imidazoline ring and a thiophene ring, with the imidazoline ring being the primary binding site for avidin. Activated biotin can couple to almost all known biomolecules, including proteins, nucleic acids, polysaccharides, and lipids, mediated by protein cross-linking agents; while avidin is a protein secreted by Streptomyces with a molecular weight of 65 kDa. The avidin molecule consists of four identical peptide chains, each capable of binding one biotin. Therefore, each antigen or antibody can simultaneously couple multiple biotin molecules, creating a "tentacle effect" that enhances analytical sensitivity.

[0043] As used in this invention, the term "optically reactive substance" refers to a substance capable of participating in optical reactions, including substances capable of absorbing or emitting light of a specific wavelength. According to some embodiments of the invention, the "optically reactive substance" comprises a chemiluminescent compound and a metal chelate. Further, the chemiluminescent compound is selected from olefin compounds, preferably from dimethylthiophene, dibutyl ketone compounds, dioxane, enol ethers, enamines, 9-alkylene-xanthane, 9-alkylene-N-9,10-dihydroacridine, arylethyl ethers, aryl imidazoles, and luster compounds, and their derivatives, more preferably from dimethylthiophene and its derivatives. The metal in the metal chelate is a rare earth metal or a Group VIII metal, preferably selected from europium, terbium, dysprosium, samarium, osmium, and ruthenium, more preferably europium. The metal chelate comprises a chelating agent selected from the following: NHA, BHHT, BHHCT, DPP, TTA, NPPTA, NTA, TOPO, TPPO, BFTA, 2,2-dimethyl-4-perfluorobutyryl-3-butanone, 2,2'-bipyridine, bipyridylcarboxylic acid, azirocrown ethers, azirocavitary ligands, and trioctylphosphine oxide and their derivatives.

[0044] In some embodiments of the present invention, the other reagents include a biotin-labeled anti-influenza B virus antibody solution; preferably, the other reagents also include a sample diluent.

[0045] The blank latex microspheres described in this invention can be present in any or several reagents of the kit. According to some embodiments of the invention, the blank latex microspheres are in a luminescent latex microsphere solution, a biotin-labeled antigen or antibody solution, or a sample diluent; in other embodiments, the blank latex microspheres are in any two of the above solutions; in still other embodiments, the blank latex microspheres are present in all three of the above solutions.

[0046] The "biotin" described in this invention is widely found in animal and plant tissues. Its molecule has two ring structures: an imidazoline ring and a thiophene ring, with the imidazoline ring being the primary site for binding to avidin. Activated biotin can be coupled to almost all known biomolecules, including proteins, nucleic acids, polysaccharides, and lipids, mediated by protein cross-linking agents.

[0047] According to some embodiments of the present invention, the surface of the blank latex microspheres is modified with chemical groups, wherein the chemical groups are selected from at least one of epoxy, chloromethyl, mercapto, amino, hydroxy, maleamine, sulfonic acid, carboxyl, and aldehyde groups. Experiments have shown that blank latex microspheres modified with different chemical groups in the same kit can all achieve the experimental objectives. The chemical groups used can be selected based on the properties of other reagents in the kit, and the kit performance can be optimized through reaction system optimization. In some embodiments of the present invention, the chemical groups are selected from at least one of sulfonic acid, carboxyl, and aldehyde groups, or a combination of multiple groups can be used. In other embodiments of the present invention, the chemical group is a carboxyl group.

[0048] According to some embodiments of the present invention, the blank latex microspheres are prepared by an emulsion polymerization method without emulsifiers.

[0049] In some embodiments of the present invention, the method for preparing the blank latex microspheres includes: reacting deionized water, styrene, hydroxyethyl methacrylate, and a small amount of divinylbenzene in the presence of potassium persulfate initiator, and obtaining microspheres with a clean surface and hydroxyl groups, with a diameter of approximately 60-1000 nm. Preferably, the reaction conditions include: polymerization reaction at 60-90°C, for example, 70°C, under a nitrogen atmosphere for 5-6 hours.

[0050] The latex microspheres of the present invention may be made of, but are not limited to, agarose, cellulose, nitrocellulose, cellulose acetate, polyvinyl chloride, polystyrene, polyethylene, polypropylene, poly(4-methylbutene), polyacrylamide, polymethacrylate, polyethylene terephthalate, nylon, polyvinyl butyrate, or polyacrylate; preferably selected from polystyrene, polypropylene, poly(4-methylbutene), polyacrylamide, polymethacrylate, polyethylene terephthalate, or polyacrylate.

[0051] According to some embodiments of the present invention, the blank latex microspheres are polystyrene microspheres. According to some embodiments of the present invention, both the luminescent latex microspheres and the blank latex microspheres are polystyrene microspheres. That is, the blank latex microspheres and luminescent latex microspheres of the present invention are made of the same material, the main difference being the substances they contain and the substances used for surface modification, giving them different functions. The luminescent latex microspheres contain optically reactive substances and have bioactive substances attached to their surface, while the blank latex microspheres do not contain either of these substances.

[0052] According to some embodiments of the present invention, the average particle size of the blank latex microspheres is 60-1000 nm, for example, it can be 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, or 900 nm. According to a preferred embodiment of the present invention, the average particle size of the blank latex microspheres is 100-300 nm.

[0053] According to some embodiments of the present invention, the particle size of the blank latex microspheres is smaller than that of the luminescent latex microspheres.

[0054] According to some embodiments of the present invention, the surface of the blank latex microspheres carries a negative charge.

[0055] According to some embodiments of the present invention, the zeta potential of the blank latex microspheres is -10 to -50 mV, for example, it can be -15 mV, -20 mV, -25 mV, -30 mV, -35 mV, -40 mV, -45 mV, or -50 mV.

[0056] According to some embodiments of the present invention, the zeta potential of the blank latex microspheres is -15 to -30 mV. The zeta potential value described in this invention refers to the potential value of the acceptor particles in a dispersion system with a pH of 6 to 9. The zeta potential of the particles refers to the potential of the particles at the shear plane; that is, the potential difference between the continuous phase and the fluid-stabilized layer attached to the microspheres. Because the dispersed particles carry a charge on their surface, they attract surrounding anti-charge ions. These anti-charge ions are distributed in a diffuse state at the interface between the two phases, forming a diffused electric double layer. According to the Stern double layer theory, the electric double layer can be divided into two parts: the Stern layer and the diffused layer. The Stern layer is defined as a planar layer composed of a layer of ionic (IHP or OHP) charge centers adsorbed on the electrode surface. The potential of this planar layer relative to a point in the fluid far from the interface is called the Stern potential. The interface between the stationary layer (including the stern layer and the portion of the diffusion layer within the slipping plane) and the dispersion medium within the diffusion layer, where relative movement occurs, is called the slipping plane. The potential at this point relative to a point in the fluid far from the interface is called the ZETA potential or zeta potential, i.e., the ZETA potential is the potential difference between the continuous phase and the fluid stationary layer attached to the dispersed particles. It can be directly measured through electrokinetic phenomena. Currently, the main methods for measuring the ZETA potential include electrophoresis, electroosmosis, flow potential analysis, and ultrasound, with electrophoresis being the most widely used. According to some embodiments of the present invention, the blank latex microspheres and the luminescent latex microspheres carry the same type of charge.

[0057] The antibodies described in this invention can be monoclonal antibodies, polyclonal antibodies, or antibody fragments; including but not limited to animal-derived antibodies, human-derived antibodies, chimeric antibodies, or genetically engineered antibodies. According to some embodiments of this invention, the anti-influenza B virus antibody is selected from murine monoclonal antibodies.

[0058] According to some embodiments of the present invention, the kit further includes at least one of a positive control, a negative control, and a critical value control. In the test, the critical value control is added to the reaction microwell outside the test sample according to the detection mode of the sample to be tested, and the sample is detected simultaneously. Its luminescence signal intensity is used as the critical value for a positive result. When the luminescence signal intensity of the test sample is ≥ the critical value (i.e., S / CO value ≥ 1), the sample is determined to be positive for the target; otherwise, it is determined to be negative for the target.

[0059] According to the present invention, the method of using the kit is photo-induced chemiluminescence detection.

[0060] In some embodiments of the present invention, the method of using the kit includes the following steps:

[0061] S1: The sample to be tested is mixed with reagent R1 (a solution of luminescent latex microspheres coated with anti-influenza B virus antibody 1), reagent R2 (a solution of biotin-labeled anti-influenza B virus antibody 2) and reagent R3 (sample diluent) to obtain the first mixture;

[0062] S2: Mix the first mixture with the photosensitive latex microsphere solution to obtain the second mixture;

[0063] S3: Detect the chemiluminescence signal of the second mixture.

[0064] In some embodiments of the present invention, the method of use includes the following steps:

[0065] a. Add 10-30 μL of the sample to be tested, 10-30 μL of reagent R1 (solution of luminescent latex microspheres coated with anti-influenza B virus antibody 1), 10-30 μL of reagent R2 (solution of biotin-labeled anti-influenza B virus antibody 2) and 10-30 μL of reagent R3 (sample diluent) to the reaction wells.

[0066] b. Incubate at 30-45℃ for 10-30 minutes;

[0067] c. Add 100-250 μL of photosensitive latex microsphere solution to the reaction well;

[0068] d. Incubate at 30-45℃ for 5-30 minutes;

[0069] e. Irradiate the reaction well with a laser and detect the chemiluminescence signal. The excitation wavelength is 650-700nm and the detection wavelength is 550-650nm.

[0070] In some specific embodiments of the present invention, the excitation wavelength of the laser irradiation is 680 nm and the detection wavelength is 615 nm.

[0071] In some embodiments of the present invention, the sample to be tested is selected from at least one of oropharyngeal swabs, nasopharyngeal swabs, saliva, nasal aspirate, serum, plasma, ascites, and pleural effusion.

[0072] In some embodiments of the present invention, the sample to be tested contains mucin and / or other protein components carrying the same type of charge as mucin.

[0073] In a second aspect, the present invention provides a photo-induced chemiluminescence analysis method for detecting influenza B virus antigens using the aforementioned kit, comprising the following steps:

[0074] Step S1: Mix the test reagent in the kit with the sample to be tested, so that the influenza B virus antigen in the sample to be tested comes into contact with blank latex microspheres, luminescent latex microspheres containing labeled anti-influenza B virus antibodies, and anti-influenza B virus antibodies containing biotin to obtain a first mixture;

[0075] Step S2: The first mixture is mixed with a solution of photosensitive latex microspheres labeled with streptavidin to obtain a second mixture;

[0076] Step S3: Irradiate the second mixture with a laser and detect the chemiluminescence signal.

[0077] According to some embodiments of the present invention, the blank latex microspheres and the luminescent latex microspheres carry the same type of charge.

[0078] According to some embodiments of the present invention, the concentration of the blank latex microspheres in the first mixture is higher than 31.25 μg / mL.

[0079] According to some embodiments of the present invention, the concentration of the blank latex microspheres in the first mixture is 40 μg / mL to 15 mg / mL. In some specific embodiments of the present invention, the concentration of the blank latex microspheres in the first mixture can be 40 μg / mL, 50 μg / mL, 62.5 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, 600 μg / mL, 800 μg / mL, 1 mg / mL, 3 mg / mL, 5 mg / mL, 8 mg / mL, 10 mg / mL, 12 mg / mL, 15 mg / mL, and any value between them. According to a preferred embodiment of the present invention, the concentration of the blank latex microspheres in the first mixture is 62.5 μg / mL to 12.5 mg / mL.

[0080] According to some embodiments of the present invention, the blank latex microspheres may be present in one or more reagents in the kit, and the amount of the blank latex microspheres is sufficient to satisfy the concentration in the first mixture being within the aforementioned range. If the kit contains multiple reagents containing blank latex microspheres, the concentrations of blank latex microspheres in different reagents may be the same or different, and the concentration of blank latex microspheres in the reagents may be adjusted according to the original component characteristics of the reagents. For example, if n reagents are required for the test, but only one reagent in the kit contains blank latex microspheres, when all reagents are used in equal amounts during the test, the concentration of blank latex microspheres in that reagent shall not be less than (40n) μg / mL and not more than (12.5n) mg / mL; if the kit contains two reagents containing blank latex microspheres of equal concentration, when all reagents are used in equal amounts during the test, the concentration of blank latex microspheres in both reagents shall not be less than (20n) μg / mL and not more than (6.25n) mg / mL.

[0081] According to a preferred embodiment of the present invention, the blank latex microspheres are present in one of the reagents of the kit, and preferably, the concentration of the blank latex microspheres in the reagent is not less than 0.125 mg / mL.

[0082] According to a preferred embodiment of the present invention, the concentration of the blank latex microspheres in the reagent is 0.25-50 mg / mL. In some specific embodiments of the present invention, the concentration of the blank latex microspheres in the reagent may be 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, or 50 mg / mL.

[0083] According to some embodiments of the present invention, in step S1, the contact temperature is 30-45°C and the contact time is 10-30 minutes.

[0084] According to a preferred embodiment of the present invention, in step S2, the mixing temperature is 30-45°C and the time is 5-30 minutes.

[0085] According to some embodiments of the present invention, in step S3, the excitation wavelength of the laser irradiation is 650-700nm, and the detection wavelength is 550-650nm.

[0086] According to a preferred embodiment of the present invention, in step S3, the excitation wavelength of the laser irradiation is 675-685nm, and the detection wavelength is 610-620nm.

[0087] In a third aspect, the present invention provides the use of the kit according to the first aspect or the method according to the second aspect in detecting influenza B virus in oropharyngeal swabs, saliva or nasal aspirate samples. Specific Implementation

[0089] To make the present invention easier to understand, specific embodiments of the influenza B virus antigen detection kit are provided below to further illustrate the invention. These embodiments are for illustrative purposes only and are not intended to limit the scope of application of the invention. Unless otherwise specified, all raw materials or components used in the present invention can be obtained commercially or by conventional methods.

[0090] Example 1

[0091] Preparation of blank latex microspheres:

[0092] The microspheres are prepared by emulsion polymerization without emulsifiers. Specifically, deionized water, styrene, hydroxyethyl methacrylate, and a small amount of divinylbenzene are mixed, and potassium persulfate is added to initiate the reaction. The polymerization reaction is carried out at 60-90℃, for example, 70℃, under a nitrogen atmosphere for 5-6 hours. After purification, microspheres with a clean surface and hydroxyl groups and a diameter of approximately 60-1000 nm are obtained. The diameter of the microspheres can be screened, preferably 100-300 nm. The blank latex microspheres obtained are made of polystyrene and can be modified with chemical groups. The chemical groups used for modification can be selected from epoxy, chloromethyl, mercapto, amino, hydroxyl, maleamine, sulfonic acid, carboxyl, and aldehyde groups.

[0093] Blank latex microspheres can be added to any one, two, or three of the solutions R1, R2, and R3, and their concentration can be adjusted as needed.

[0094] The zeta potential of blank latex microspheres was determined using Doppler electrophoresis (ELS) with a NICOMP 380Z3000, ranging from -10 to -50 mV.

[0095] Example 2

[0096] The influenza B antigen test kit includes the following reagents:

[0097] Reagent R1: Luminescent latex microspheres coated with 25 mg / mL anti-influenza B virus antibody, with a diameter of 200 ± 5 nm;

[0098] Reagent R2-1: Contains 0.5 μg / mL of biotin-labeled anti-influenza B virus antibody 2, and 5 mg / mL of carboxyl-labeled blank latex microspheres. The microspheres have a diameter of 150 ± 5 nm and a measured Zeta potential of -20 mV.

[0099] Reagent R2-2: Contains 0.5 μg / mL of biotin-labeled anti-influenza B virus antibody 2;

[0100] Reagent R3: Sample diluent.

[0101] Both the blank latex microspheres and the luminescent latex microspheres are polystyrene microspheres, and both have a negative charge on their surface.

[0102] Other required reagents: 40 μg / mL streptomycin affinity-labeled photosensitive latex microsphere solution.

[0103] The following is a detailed implementation method for testing 10 positive samples and 35 negative samples:

[0104] 1. Add 25 μL of reagent R2-1 or reagent R2-2, 25 μL of the sample to be tested, then add 25 μL of reagent R1 and 25 μL of reagent R3 to the detection wells and mix well. Incubate at 37°C for 15 minutes. Simultaneously, add the pre-calibrated critical value control to the reaction wells according to the detection mode of the first well and detect it at the same time. The intensity of its luminescence signal is used as the critical value for antigen-positive results.

[0105] 2. Continue to add 175 μL of photosensitive latex microsphere solution and incubate at 37°C for another 15 minutes.

[0106] 3. After the warm bath, the light signal intensity was detected using a photo-induced chemiluminescence analyzer (excitation wavelength 680nm, detection wavelength 615nm). If the light signal intensity of the sample was ≥ the critical value (i.e., S / CO value ≥ 1), the sample was determined to be positive for influenza B virus antigen.

[0107] The results of the clinical sample tests are shown in Table 1 below:

[0108] Table 1. Results of clinical samples detected by influenza B virus antigen reagent.

[0109]

[0110]

[0111]

[0112] As shown in Table 1, this method reduced the false positive rate of the influenza B virus antigen detection kit from 34.29% to 0%, demonstrating a significant effect. It also ensured that the reagent's test results were consistent with those of third-party kits.

[0113] Example 3

[0114] The influenza B antigen test kit includes the following reagents:

[0115] Reagent R1-1: Luminescent latex microspheres coated with 25 mg / mL anti-influenza B virus antibody, with a diameter of 200 ± 5 nm;

[0116] Reagent R1-2: Luminescent latex microspheres coated with 25 mg / mL anti-influenza B virus antibody, with a diameter of 200±5 nm; also containing 5 mg / mL carboxyl blank latex microspheres, with a diameter of 150±5 nm, and the measured Zeta potentials were -30 mV / -15 mV, respectively.

[0117] Reagent R2: Contains 0.5 μg / mL of biotin-labeled anti-influenza B virus antibody 2;

[0118] Reagent R3: Sample diluent.

[0119] Both the blank latex microspheres and the luminescent latex microspheres are polystyrene microspheres, and both have a negative charge on their surface.

[0120] Other required reagents: 40 μg / mL streptomycin affinity-labeled photosensitive latex microsphere solution.

[0121] The specific implementation method for testing 10 positive samples and 20 negative samples is as follows:

[0122] 1. Add 25 μL of reagent R1-1 or reagent R1-2, 25 μL of sample, then add 25 μL of reagent R2 and 25 μL of reagent R3 to the detection wells and mix well. Incubate at 37°C for 15 minutes. Simultaneously, add the pre-calibrated critical value control to the reaction wells according to the detection mode of the first well and detect it at the same time. The intensity of its luminescence signal is used as the critical value for antigen-positive results.

[0123] 2. Continue to add 175 μL of photosensitive latex microsphere solution and incubate at 37°C for another 15 minutes.

[0124] 3. After the warm bath, the light signal intensity was detected using a photo-induced chemiluminescence analyzer (excitation wavelength 680nm, detection wavelength 615nm). If the light signal intensity of the sample was ≥ the critical value (i.e., S / CO value ≥ 1), the sample was determined to be positive for influenza B virus antigen.

[0125] The results of the clinical sample tests are shown in Table 2 below:

[0126] Table 2 Results of clinical samples detected by blank latex microspheres with different charges

[0127]

[0128]

[0129]

[0130] As can be seen from Table 2, when using the influenza B virus antigen detection kit of the present invention, the zeta potential of the blank latex microspheres in the sample dilution solution is different, ranging from -15 to -30 mV, and the detection results are consistent with the nucleic acid detection results.

[0131] Example 4

[0132] The influenza B antigen test kit includes the following reagents:

[0133] Reagent R1: Luminescent latex microspheres coated with 25 mg / mL anti-influenza B virus antibody, with a diameter of 200 ± 5 nm;

[0134] Reagent R2: Contains 0.5 μg / mL of biotin-labeled anti-influenza B virus antibody 2;

[0135] Reagent R3-1: Sample diluent;

[0136] Reagent R3-2: Sample diluent containing 0.125 mg / mL / 0.25 mg / mL / 5 mg / mL / 50 mg / mL carboxyl-containing blank latex microspheres with a diameter of 150 ± 5 nm and a measured Zeta potential of -25 mV.

[0137] Both the blank latex microspheres and the luminescent latex microspheres are polystyrene microspheres, and both have a negative charge on their surface.

[0138] Other required reagents: 40 μg / mL streptavidin-labeled photosensitive latex microsphere solution.

[0139] The specific implementation method for testing 10 positive samples and 20 negative samples is as follows:

[0140] 1. Add 25 μL of reagent R3-1 or reagent R3-2, 25 μL of reagent R2, and 25 μL of sample sequentially to the detection wells. Then add 25 μL of reagent R1 and mix well to obtain the first mixture. Incubate at 37°C for 15 minutes. Simultaneously, add the pre-calibrated critical value control to the reaction wells according to the detection mode of the first well and detect it at the same time. The intensity of its luminescence signal is used as the critical value for antigen-positive results.

[0141] 2. Continue to add 175 μL of photosensitive latex microsphere solution to obtain the second mixture, and incubate at 37°C for another 15 minutes.

[0142] 3. After the warm bath, the light signal intensity was detected using a photo-induced chemiluminescence analyzer (excitation wavelength 680nm, detection wavelength 615nm). If the light signal intensity of the sample was ≥ the critical value (i.e., S / CO value ≥ 1), the sample was determined to be positive for influenza B virus antigen.

[0143] The results of clinical sample testing are shown in Table 3 below:

[0144] Table 3. Results of clinical samples detected with blank latex microspheres at different concentrations.

[0145]

[0146]

[0147] As can be seen from Table 3, when using the influenza B virus antigen detection kit of the present invention, the expected purpose was not achieved when 0.125 mg / mL blank latex microspheres (i.e., 31.25 μg / mL blank latex microspheres were added to the reagent (i.e., the first mixture contained 31.25 μg / mL blank latex microspheres); however, when 0.25 mg / mL, 5 mg / mL, and 50 mg / mL blank latex microspheres (i.e., 62.5 μg / mL, 1.25 mg / mL, and 12.5 mg / mL blank latex microspheres were added, the results of the test samples were consistent with the nucleic acid test results.

[0148] It should be noted that the embodiments described above are only for explaining the present invention and do not constitute any limitation on the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from the scope and spirit of the present invention. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention can be extended to all other methods and applications with the same function.

Claims

1. A photoluminescence detection kit for influenza B virus antigen, comprising a solution of luminescent latex microspheres coated with anti-influenza B virus antibodies and other reagents, wherein the luminescent latex microspheres contain an optically reactive substance; the luminescent latex microsphere solution and / or other reagents further contain blank latex microspheres, wherein the blank latex microspheres do not contain any optically reactive substance and have no bioactive substances attached to their surface; the blank latex microspheres and the luminescent latex microspheres carry the same type of charge.

2. The reagent kit according to claim 1, characterized in that, The surface of the blank latex microspheres carries a negative charge.

3. The reagent kit according to claim 2, characterized in that, The zeta potential of the blank latex microspheres is -10 to -50 mV.

4. The reagent kit according to claim 2, characterized in that, The zeta potential of the blank latex microspheres is -15 to -30 mV.

5. The reagent kit according to claim 1, characterized in that, The other reagents include a biotin-labeled solution of antibodies against influenza B virus.

6. The reagent kit according to claim 1, characterized in that, Other reagents include sample diluents.

7. The kit according to any one of claims 1-6, characterized in that, The surface of the blank latex microspheres is modified with chemical groups, which are selected from at least one of epoxy, chloromethyl, mercapto, amino, hydroxy, maleamine, sulfonic acid, carboxyl, and aldehyde groups.

8. The kit according to any one of claims 1-6, characterized in that, The surface of the blank latex microspheres is modified with chemical groups, which are selected from at least one of sulfonic acid groups, carboxyl groups, and aldehyde groups.

9. The kit according to any one of claims 1-6, characterized in that, The surface of the blank latex microspheres is modified with chemical groups, namely carboxyl groups.

10. The kit according to any one of claims 1-6, characterized in that, The particle size of the blank latex microspheres is 60nm-1000nm.

11. The kit according to claim 7, characterized in that, The particle size of the blank latex microspheres is 100nm-300nm.

12. The reagent kit according to any one of claims 1-6 and 11, characterized in that, The particle size of the blank latex microspheres is smaller than that of the luminescent latex microspheres.

13. The reagent kit according to any one of claims 1-6 and 11, characterized in that, The blank latex microspheres are polystyrene microspheres.

14. The kit according to claim 13, characterized in that, Both the luminescent latex microspheres and the blank latex microspheres are polystyrene microspheres.

15. The kit according to any one of claims 1-6, 11, and 14, characterized in that, The blank latex microspheres were prepared by emulsion polymerization without emulsifiers.

16. A photo-induced chemiluminescence assay method for detecting influenza B virus antigen using the kit according to any one of claims 1-15, wherein the method is for non-diagnostic purposes and comprises the following steps: Step S1: Mix the test reagent in the kit with the sample to be tested, so that the influenza B virus antigen in the sample to be tested comes into contact with blank latex microspheres, luminescent latex microspheres containing labeled anti-influenza B virus antibodies, and anti-influenza B virus antibodies containing biotin to obtain a first mixture; Step S2: Mix the first mixture with a solution of photosensitive latex microspheres labeled with streptavidin to obtain a second mixture; Step S3: Irradiate the second mixture with a laser and detect the chemiluminescence signal.

17. The method according to claim 16, characterized in that, In the first mixture, the concentration of the blank latex microspheres is higher than 31.25 μg / mL.

18. The method according to claim 16, characterized in that, In the first mixture, the concentration of the blank latex microspheres is 40 μg / mL to 15 mg / mL.

19. The method according to claim 16, characterized in that, In the first mixture, the concentration of the blank latex microspheres is 62.5 μg / mL to 12.5 mg / mL.

20. The use of a kit according to any one of claims 1-15 or a method according to any one of claims 16-19 for non-diagnostic purposes in detecting influenza B virus in oropharyngeal swabs, nasopharyngeal swabs, saliva or nasal aspirate samples.

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