A homogeneous detection kit for creatine kinase isoenzyme and application thereof

Abstract

The present application relates to a kind of creatine kinase isozyme homogeneous detection kit and its application, the kit includes reagent 1 and reagent 3, the reagent 1 includes first buffer solution and the acceptor particle suspended in it, the reagent 3 includes second buffer solution and the donor particle suspended in it, the acceptor particle includes first carrier, the inside of the first carrier is filled with luminous composition, the particle size distribution variation coefficient C.V value of the acceptor particle in reagent 1 is not less than 5% and not more than 25%; The donor particle includes second carrier, the inside of the second carrier is filled with sensitizing agent, the sugar content in the donor particle in reagent 3 per gram of the donor particle is not more than 40 mg.The kit of the present application can be used for the auxiliary diagnosis of myocardial infarction in clinic, both have more suitable sensitivity, and have very wide detection range.

Description

Technical Field

[0001] This invention belongs to the field of homogeneous detection technology, specifically relating to a homogeneous detection kit for creatine kinase isoenzymes, its preparation method, and its application. Background Technology

[0002] Creatine kinase (CK) is a dimer enzyme that exists in four different forms: mitochondrial isoenzyme, cytoplasmic isoenzyme CK-MM (muscle type), creatine kinase brain type isoenzyme CK-BB (brain type), and creatine kinase isoenzyme (CK-MB). Creatine kinase isoenzyme is mainly located in the myocardium.

[0003] Creatine kinase isoenzymes are released into the peripheral blood during acute myocardial infarction (AMI) and myocardial necrosis. They can be detected 3-8 hours after the onset of clinical symptoms. Combined detection with myoglobin and troponin can determine whether the myocardium is damaged, making them indicators for the research and diagnosis of acute myocardial infarction (AMI). Clinically, they are mainly used as an adjunct to the diagnosis of myocardial infarction.

[0004] Commonly used CK-MB detection methods include gold nanoparticle qualitative assay, fluorescence immunoassay, ELISA, magnetic microparticle chemiluminescence immunoassay (CMIA), and biochemical analyzer detection. The basic principle of photo-induced chemiluminescence is a homogeneous immunoreaction. It is based on the formation of immune complexes between two microparticles coated with antigens or antibodies in a liquid phase, drawing the two microparticles closer together. Under laser excitation, singlet oxygen transfer occurs between the microparticles, generating high-energy red light. The photon count is converted into analyte concentration using a single-photon counter and mathematical fitting. When the sample does not contain analyte, immune complexes cannot form between the two microparticles, and the distance between them exceeds the singlet oxygen propagation range. The singlet oxygen is rapidly quenched in the liquid phase, and no high-energy red light is generated during detection. Kits based on photo-induced chemiluminescence have advantages over existing kits, including ease of operation, rapid detection, high sensitivity, and good accuracy. Applying it to the monitoring of myocardial injury can improve the accuracy of diagnosing acute myocardial infarction.

[0005] CN106093411A discloses a one-step homogeneous CK-MB detection kit for the diagnosis of acute myocardial infarction and its application in the detection of CK-MB content. It adopts photo-induced chemiluminescence method. Although it has the advantages of convenient operation, rapid detection and strong specificity compared with the prior art, the sensitivity is still not high. The kit cannot achieve both high sensitivity and wide detection range. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a homogeneous detection kit for creatine kinase isoenzymes to address the shortcomings of the prior art. The quantitative results of the CK-MB marker in the host body fluid can be used for the auxiliary diagnosis of myocardial infarction in clinical practice. It has both suitable sensitivity and a wide detection range.

[0007] Therefore, in a first aspect, the present invention provides a homogeneous detection kit for creatine kinase isoenzymes, comprising reagent 1 and reagent 3, wherein reagent 1 comprises a first buffer solution and receptor particles suspended therein, the receptor particles being capable of generating chemiluminescence by reacting with reactive oxygen species, and binding to a creatine kinase isoenzyme antibody, the creatine kinase isoenzyme antibody being capable of specifically binding to the creatine kinase isoenzyme; reagent 3 comprises a second buffer solution and donor particles suspended therein, the donor particles binding to one of the specifically paired members, characterized in that:

[0008] The receptor particle includes a first carrier, the interior of which is filled with a luminescent composition, and the surface of the first carrier is bonded with a creatine kinase isoenzyme antibody. The particle size distribution coefficient of variation (CV) of the receptor particle in reagent 1 is not less than 5% and not more than 25%.

[0009] The donor particle includes a second carrier, the interior of which is filled with a sensitizer, and the surface of the second carrier is bonded with one of the specific pairing members; the sugar content of the donor particle in reagent 3 is not higher than 40 mg per gram of the donor particle.

[0010] In some embodiments of the present invention, the coefficient of variation (CV) of the particle size distribution of the receptor particles in reagent 1 is not higher than 15%.

[0011] In some specific embodiments of the present invention, the coefficient of variation (CV) of the particle size distribution of the receptor particles in reagent 1 can be selected from 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, and 25%.

[0012] In some embodiments of the present invention, the surface of the first carrier is coated with polysaccharides, and the creatine kinase isoenzyme antibody binds to the first carrier by binding to the polysaccharide molecules.

[0013] In some embodiments of the present invention, the surface of the first carrier is coated with a coating of at least two consecutive polysaccharide layers.

[0014] In some embodiments of the present invention, the first polysaccharide layer of the coating is spontaneously associated with the second polysaccharide layer.

[0015] In some embodiments of the present invention, each polysaccharide layer in the coating has functional groups, the functional groups of the polysaccharide layer carrying opposite charges to the functional groups of the preceding polysaccharide layer.

[0016] In other embodiments of the invention, each polysaccharide layer in the coating has functional groups, and each polysaccharide layer is covalently linked to the previous polysaccharide layer by a reaction between its functional groups and the functional groups on the previous polysaccharide layer.

[0017] In some embodiments of the present invention, the outermost polysaccharide layer of the coating has at least one side functional group that is bonded to a creatine kinase isoenzyme antibody.

[0018] In some embodiments of the present invention, the side group functional groups of the outermost polysaccharide layer of the coating are selected from at least one of aldehyde, carboxyl, thiol, amino, hydroxyl and maleamine groups.

[0019] In some preferred embodiments of the present invention, the side group functional groups of the outermost polysaccharide layer of the coating are selected from aldehyde and / or carboxyl groups.

[0020] In some embodiments of the present invention, the sugar content in each liter of the first buffer solution is 0.01-1g.

[0021] In some preferred embodiments of the present invention, the sugar content in each liter of the first buffer solution is 0.02-0.2g.

[0022] In some embodiments of the present invention, the sugar content per gram of the receptor particle is not less than 20 mg. In some specific embodiments of the present invention, the sugar content per gram of the receptor particle may be selected from 20.2 mg, 40 mg, 45.1 mg, 55.8 mg, and 60.3 mg.

[0023] In some preferred embodiments of the present invention, the sugar content in each liter of the first buffer solution is not less than 40 micrograms.

[0024] In some embodiments of the present invention, the polysaccharide is selected from carbohydrates containing three or more unmodified or modified monosaccharide units; preferably selected from at least one of dextran, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxyglucan and aminoglucan; more preferably selected from at least one of dextran, starch, glycogen and polyribose, and most preferably dextran and / or dextran derivatives.

[0025] In some specific embodiments of the present invention, the polysaccharide is a dextran.

[0026] In some embodiments of the present invention, the coefficient of variation (CV) of the donor particles in reagent 3 is not less than 5% and not more than 25%. In some specific embodiments of the present invention, the CV of the donor particles in reagent 3 is selected from 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, and 25%. In some embodiments of the present invention, the kit further includes a series of calibrator solutions with known creatine kinase isoenzyme concentrations, wherein the concentration of the creatine kinase isoenzyme in the series of calibrator solutions is 0-510 ng / mL.

[0027] In some embodiments of the present invention, the concentrations of creatine kinase isoenzymes in the series of calibrator solutions include 0, 4, 10, 50, 200, and 510 ng / mL.

[0028] In some other embodiments of the invention, the kit further includes reagent 2, which contains a creatine kinase isoenzyme antibody linked to one of the specific binding pairing members, the creatine kinase isoenzyme antibody being capable of specifically binding to the creatine kinase isoenzyme.

[0029] In some embodiments of the present invention, the reagent kit contains at least one reagent strip, the reagent strip having a plurality of reagent wells for holding reagents, wherein at least three reagent wells are respectively used to hold reagent 1, reagent 2 and reagent 3.

[0030] A second aspect of the present invention provides an application of the reagent kit as described in the first aspect of the present invention in a chemiluminescence analyzer.

[0031] In some embodiments of the present invention, the application includes the following steps:

[0032] Step S1: Add the sample to be tested, calibrator and / or quality control sample to the liquid holding device respectively;

[0033] Step S2: Add reagent 1, reagent 2 and reagent 3 to the liquid holding device;

[0034] Step S3: Place the liquid container into the chemiluminescence analyzer for reaction and detection.

[0035] The inventors of this patent discovered that the small size of receptor and donor particles, their low concentration in the reagent kit, and the complex preparation process make their quality susceptible to various factors. Therefore, multiple testing experiments are needed to obtain optimal control characteristics, such as the coefficient of variation (CV) of the particle size distribution of the receptor and donor particles. Since the receptor and donor particles incorporate a luminescent composition or sensitizer internally and are coated with antibody antigens or streptavidin externally, to ensure particle uniformity and reduce errors caused by particle size inconsistencies, those skilled in the art typically consider controlling the CV value to a small range, or even as low as possible. However, the inventors of this patent found that achieving a very low CV value places excessively high demands on the production process, significantly increasing reagent production costs and hindering industrialization, thus preventing the reagent's widespread use in clinical diagnostics. Especially after polysaccharide coating, the CV value of the nanospheres changes more significantly and is more unstable, making it difficult to meet the reagent requirements for medical device registration and clinical application. However, if the CV value is too high, the photochemiluminescence detection effect is poor. Therefore, the inventors of this patent were surprised to find that if the coefficient of variation (CV) of the particle size distribution of the acceptor particles and the donor particles is controlled within a suitable range, the best photochemiluminescence detection results can be obtained, achieving both suitable sensitivity and a wide detection range.

[0036] The beneficial effects of this invention are as follows:

[0037] The kit provided by this invention is feasible for in vitro diagnosis of myocardial injury in a subject. The quantitative results of CK-MB marker determination in the subject's body fluids using the kit and corresponding methods provided by this invention can be used to assist in the diagnosis of myocardial infarction. The kit of this invention has both suitable sensitivity and a wide detection range. Detailed Implementation

[0038] 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.

[0039] 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.

[0040] 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.

[0041] I. Terminology

[0042] The English definition of "homogeneous" used in this invention is "homogeneous", which means that detection can be performed without separating the bound antigen-antibody complex from the remaining free antigen or antibody.

[0043] The term "sample to be tested" as used in this invention 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 bodily fluids such as blood, plasma, serum, urine, semen, saliva, etc.

[0044] The term "antibody" as used in this invention is used in the broadest sense, including any isotype of antibody, 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 specific binding pairing members, for example, biotin or avidin (one of the biotin-avidin specific binding pairing members).

[0045] As used in this invention, "antigen" refers to a substance capable of stimulating the body to produce an immune response and binding with immune response products, antibodies and sensitized lymphocytes, in vivo and in vitro to produce an immune effect. The antigen may be a fusion antigen, and where necessary, the antigen may be further conjugated with other parts, such as specific binding pair members, for example, biotin or avidin (one of the biotin-avidin specific binding pair members).

[0046] The terms “bonding” or “combination” used in this invention refer to the connection between two molecules caused by interactions such as covalent, electrostatic, hydrophobic, ionic and / or hydrogen bonding, including but not limited to physical or chemical interactions such as salt bridges and water bridges.

[0047] The term "specific binding" used in this invention refers to the mutual recognition and selective binding reaction between two substances, which, from a stereostructural perspective, means the conformational correspondence between the corresponding reactants.

[0048] The term "specifically binding pair" as used in this invention refers to a pair of molecules that can specifically bind to each other, such as enzyme-substrate, antigen-antibody, or ligand-receptor pairs. A specific example of a specific binding pair is the biotin-avidin system. 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. Avidin, on the other hand, 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 to multiple biotin molecules, creating a "tentacle effect" that enhances analytical sensitivity. The avidin described in this invention is selected from avidin, streptavidin, yolk avidin, neutral avidin, and avidin-like substances, preferably from neutral avidin and / or streptavidin.

[0049] The term "reactive oxygen species" as used in this invention refers to a general term for oxygen-containing and reactive substances in the body or natural environment. It mainly refers to excited-state oxygen molecules, including the one-electron reduction product of oxygen (superoxide anion (O2·-), the two-electron reduction product of oxygen (hydrogen peroxide (H2O2), the three-electron reduction product of oxygen (hydroxyl radical (·OH),) as well as nitric oxide and reactive oxygen species (NOS). 1 O2), etc.

[0050] The term "donor particle" as used in this invention refers to a sensitizer that, upon activation by energy or an active compound, can generate an active intermediate, such as reactive oxygen species, that reacts with the acceptor particle. Donor particles can be photoactivated (e.g., dyes and aromatic compounds) or chemically activated (e.g., enzymes, metal salts, etc.). In some embodiments of this invention, the donor particles are coated onto a matrix with functional groups to form high-molecular-weight microparticles filled with a photosensitive compound, capable of generating reactive oxygen species upon photoexcitation. In this case, the photosensitive microspheres can also be called oxygen-donating microspheres or photosensitive microspheres. The surface of the donor particles may have hydrophilic aldehyde dextran, and the interior is filled with a photosensitizer. The photosensitizer can be a photosensitizer known in the art, preferably a relatively photostable compound that does not react effectively with reactive oxygen species. Non-limiting examples include compounds such as methylene blue, rose red, porphyrin, and phthalocyanine, as well as derivatives of these compounds having 1-50 substituents, which are used to make these compounds more lipophilic or more hydrophilic, and / or as linking groups to specifically binding pairing members. The surface of the donor particles may also be filled with other sensitizers, non-limiting examples of which are certain compounds that catalyze the conversion of hydrogen peroxide into reactive oxygen species and water. Other examples of donors include 1,4-dicarboxyethyl-1,4-naphthalene endoperoxide, 9,10-diphenylanthracene-9,10-endoperoxide, etc. Heating these compounds or direct light absorption by these compounds releases reactive oxygen species, such as reactive oxygen species.

[0051] As used in this invention, "receptor particle" refers to a compound capable of reacting with reactive oxygen species (ROS) to generate a detectable signal. Donor particles are induced and activated by energy or an active compound, releasing high-energy ROS. This high-energy ROS is captured by a nearby receptor particle, thereby transferring energy to activate the receptor particle. In some embodiments of this invention, the receptor particles are filled into a carrier via functional groups to form polymeric microparticles filled with a luminescent composition comprising a chemiluminescent compound capable of reacting with ROS. In some specific embodiments of this invention, the chemiluminescent compound undergoes a chemical reaction with ROS to form an unstable metastable intermediate that can decompose, simultaneously or subsequently emitting light. Typical examples of such substances include, but are not limited to: enol ethers, enamines, 9-alkylidene xanthan gum, 9-alkylidene-N-alkylacridinium, arylethyl ethers, diethylene glycol, dimethylthiophene, arylimidazolium, or glossin.

[0052] In this invention, the "luminescent composition" is a compound referred to as a marker that can undergo a chemical reaction to induce luminescence, for example, by being converted into another compound 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.

[0053] The "carrier" described in this invention can be of any size, organic or inorganic, expandable or non-expandable, porous or non-porous, and has any density, but preferably close to that of water. It is preferably buoyant in water and is made of transparent, partially transparent, or opaque materials. The carrier may or may not be charged, and when charged, it is preferably negatively charged. The carrier can be a solid (such as polymers, metals, glass, organic and inorganic materials such as minerals, salts, and diatoms), small oil droplets (such as hydrocarbons, fluorocarbons, and silica fluids), or vesicles (such as synthetic phospholipids, or natural materials such as cells and organelles). The carrier can be latex particles or other particles containing organic or inorganic polymers, lipid bilayers such as liposomes, phospholipid vesicles, small oil droplets, silica particles, metal sols, cells, and microcrystalline dyes. The carrier is generally multifunctional or can bind to a donor or acceptor through specific or non-specific covalent or non-covalent interactions. Many functional groups are available or incorporated. Typical functional groups include carboxylic acids, acetaldehyde, amino groups, cyano groups, vinyl groups, hydroxyl groups, and mercapto groups. A non-limiting example of a carrier suitable for this invention is polystyrene latex microspheres.

[0054] The "coefficient of variation (CV) value" described in this invention refers to the coefficient of variation of particle size distribution within a Gaussian distribution in the detection results of a nanoparticle size analyzer. CV is a statistical measure of the degree of variation in particle size among standard materials. The coefficient of variation of particle size distribution in standard materials is used to represent the degree of particle size dispersion of the standard material, and is commonly expressed as a percentage of the ratio of the standard deviation to the average particle size of the standard material; the latter is also called dispersion. The formula for calculating the coefficient of variation is: Coefficient of variation (CV) value = (Standard deviation (SD) / Mean) × 100%. Standard deviation (SD), also known as standard deviation, describes the average distance of each data point from the mean (deviation from the mean). It is the square root of the average of the squared deviations, denoted by σ. Standard deviation is the arithmetic square root of variance. Standard deviation reflects the dispersion of a dataset; the smaller the standard deviation, the less these values ​​deviate from the mean, and vice versa. The standard deviation σ is the distance from the inflection point (0.607 times the peak height) on the normal distribution curve to the perpendicular line from the peak height to the time axis; that is, half the distance between two inflection points on the normal distribution curve. The half-peak width (Wh / 2) refers to the peak width at half the peak height, Wh / 2 = 2.355σ. The intercept of the tangent lines drawn through the inflection points on both sides of the normal distribution curve on the baseline is called the peak width or baseline width, W = 4σ or W = 1.699Wh / 2.

[0055] II. Specific Implementation Plan

[0056] The invention will now be described in more detail.

[0057] The technical principle of photoluminescence immunoassay is as follows: A sensitizer, under laser irradiation, excites oxygen molecules in the surrounding environment into singlet oxygen molecules. These singlet oxygen molecules react with a luminescent composition approximately 200 nm away, generating a light signal of a specific wavelength. When the sample contains the antigen or antibody to be tested, the immunoreaction of this antigen and antibody allows the donor particle containing the sensitizer to bind to the acceptor particle containing the luminescent composition, thereby generating a light signal of a specific wavelength. Detecting this light signal allows for the detection of the content of the antigen or antibody to be tested. In the photoluminescence immunoassay described above, the diameter, material, and surface properties of the acceptor and donor particles significantly affect the efficiency of the sensitizer in exciting singlet oxygen molecules and the energy transfer efficiency of the singlet oxygen molecules. They also affect the non-specific binding of the donor and acceptor particles, leading to errors in the detection results. Therefore, the diameter range of the donor and acceptor particles, the uniformity of particle size, the material of the particles, and the surface chemical properties are key areas for research and improvement in photoluminescence immunoassay technology, and are not simply common knowledge or industry practice.

[0058] When CK-MB biomarkers are present in clinical samples, CK-MB specifically binds to both receptor particles coated with CK-MB antibody I (monoclonal antibody) and biotin-labeled CK-MB antibody II (monoclonal antibody), forming a double-antibody sandwich complex on the surface of the receptor particles. If streptavidin-modified donor particles are added, biotin binds to streptavidin, causing the two particles to approach each other. Under excitation by a light source, the donor particles release singlet oxygen, which, upon encountering the receptor particles in solution, produces chemiluminescence. This further excites the fluorophores on the same particle, resulting in a cascade amplification reaction and fluorescence. The higher the concentration of CK-MB biomarkers, the stronger the fluorescence intensity. This invention is based on the above method.

[0059] Therefore, a first aspect of the present invention relates to a homogeneous detection kit for creatine kinase isoenzymes, comprising reagent 1 and reagent 3, wherein reagent 1 comprises a first buffer solution and receptor particles suspended therein, the receptor particles being capable of generating chemiluminescence by reacting with reactive oxygen species, and binding to a creatine kinase isoenzyme antibody, the creatine kinase isoenzyme antibody being capable of specifically binding to the creatine kinase isoenzyme; reagent 3 comprises a second buffer solution and donor particles suspended therein, the donor particles binding to one of the specifically paired members, characterized in that:

[0060] The receptor particle includes a first carrier, the interior of which is filled with a luminescent composition, and the surface of the first carrier is bonded with a creatine kinase isoenzyme antibody. The particle size distribution coefficient of variation (CV) of the receptor particle in reagent 1 is not less than 5% and not more than 25%.

[0061] The donor particle includes a second carrier, the interior of which is filled with a sensitizer, and the surface of the second carrier is bonded with one of the specific pairing members; the sugar content of the donor particle in reagent 3 is not higher than 40 mg per gram of the donor particle.

[0062] In some embodiments of the present invention, the coefficient of variation (CV) of the particle size distribution of the receptor particles in reagent 1 is not higher than 15%.

[0063] In some specific embodiments of the present invention, the coefficient of variation (CV) of the particle size distribution of the receptor particles in reagent 1 is selected from 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, and 25%.

[0064] In some embodiments of the present invention, the surface of the first carrier is coated with polysaccharides, and the creatine kinase isoenzyme antibody binds to the first carrier by binding to the polysaccharide molecules.

[0065] In some embodiments of the present invention, the surface of the first carrier is coated with a coating of at least two consecutive polysaccharide layers. The term "consecutive polysaccharide layers" as used in the present invention refers to multiple polysaccharide layers directly bonded together, with no other non-polysaccharide layers between the two polysaccharide layers.

[0066] In some embodiments of the present invention, the first polysaccharide layer and the second polysaccharide layer of the coating spontaneously associate. The term "spontaneous" as used in the present invention refers to the spontaneous formation of an ordered structure among multiple polysaccharide layers, for example, through charge interactions or molecular self-assembly.

[0067] In some embodiments of the present invention, each polysaccharide layer in the coating has functional groups, the functional groups of the polysaccharide layer carrying opposite charges to the functional groups of the preceding polysaccharide layer.

[0068] In other embodiments of the invention, each polysaccharide layer in the coating has functional groups, and each polysaccharide layer is covalently linked to the previous polysaccharide layer by a reaction between its functional groups and the functional groups on the previous polysaccharide layer.

[0069] In some embodiments of the present invention, the outermost polysaccharide layer of the coating has at least one side functional group that is bonded to a creatine kinase isoenzyme antibody.

[0070] In some embodiments of the present invention, the side group functional groups of the outermost polysaccharide layer of the coating are selected from at least one of aldehyde, carboxyl, thiol, amino, hydroxyl and maleamine groups.

[0071] In some preferred embodiments of the present invention, the side group functional groups of the outermost polysaccharide layer of the coating are selected from aldehyde and / or carboxyl groups.

[0072] In some embodiments of the present invention, the sugar content in each liter of the first buffer solution is 0.01-1g.

[0073] In some preferred embodiments of the present invention, the sugar content in each liter of the first buffer solution is 0.02-0.2g.

[0074] In some embodiments of the present invention, the sugar content per gram of the receptor particle is not less than 20 milligrams. The sugar content in the receptor particle of the present invention may originate from the polysaccharide coating on the surface of the receptor particle, or from the polysaccharide component carried in the structure of the antigen-antibody or specific binding pair itself.

[0075] In some specific embodiments of the present invention, the sugar content per gram of the receptor particle is 20.2 mg, 40 mg, 45.1 mg, 55.8 mg, and 60.3 mg.

[0076] In some preferred embodiments of the present invention, the sugar content in each liter of the first buffer solution is not less than 40 micrograms.

[0077] In some embodiments of the present invention, the polysaccharide is selected from carbohydrates containing three or more unmodified or modified monosaccharide units; preferably selected from at least one of dextran, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxyglucan, and aminoglucan; more preferably selected from at least one of dextran, starch, glycogen, and polyribose; and most preferably dextran and / or dextran derivatives. Polysaccharides, especially dextran and dextran derivatives, can increase the hydrophilicity of the carrier surface and provide conjugated sites for the connection between antibody molecules and the carrier surface. Coating the receptor microparticle surface with polysaccharides can increase the hydrophilicity of the microspheres and avoid non-specific adsorption, which greatly affects the optical signal of subsequent photochemiluminescence detection. The inventors of this patent have discovered that precisely controlling the sugar content on the surface of the microspheres within a suitable range can effectively solve some technical problems existing in the application of photochemiluminescence technology in the field of in vitro diagnostics.

[0078] In some embodiments of the present invention, the coefficient of variation (CV) of the donor particles in reagent 3 is not less than 5% and not more than 25%. In some specific embodiments of the present invention, the CV value of the donor particles in reagent 3 is selected from 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, and 25%.

[0079] In some embodiments of the present invention, the kit further includes a series of calibrator solutions with known concentrations of creatine kinase isoenzymes, wherein the concentration of the creatine kinase isoenzyme in the series of calibrator solutions is 0-510 ng / mL.

[0080] In some embodiments of the present invention, the concentrations of creatine kinase isoenzymes in the series of calibrator solutions include 0, 4, 10, 50, 200, and 510 ng / mL.

[0081] In some other embodiments of the invention, the kit further includes reagent 2, which contains a creatine kinase isoenzyme antibody linked to one of the specific binding pairing members, the creatine kinase isoenzyme antibody being capable of specifically binding to the creatine kinase isoenzyme.

[0082] In some embodiments of the present invention, the reagent kit contains at least one reagent strip, the reagent strip having a plurality of reagent wells for holding reagents, wherein at least three reagent wells are respectively used to hold reagent 1, reagent 2 and reagent 3.

[0083] In this invention, sugar concentration or sugar content can be determined using the anthrone method. The anthrone method for determining polysaccharides is known to those skilled in the art. Sugars dehydrate upon contact with concentrated sulfuric acid to produce furfural or its derivatives. Furfural or hydroxymethylfurfural further condenses with anthrone reagent to produce a blue-green substance with maximum absorption in the visible light region of 620 nm to 630 nm. Furthermore, its absorbance value is directly proportional to the sugar content within a certain range. This method can be used to determine the content of monosaccharides, oligosaccharides, and polysaccharides, and has advantages such as high sensitivity, simplicity, speed, and suitability for the determination of trace samples.

[0084] The second aspect relates to the application of a reagent kit as described in the first aspect in a chemiluminescence analyzer.

[0085] In other embodiments of the present invention, the application includes the following steps:

[0086] Step S1: Add the sample to be tested, calibrator and / or quality control sample to the liquid holding device respectively;

[0087] Step S2: Add reagent 1, reagent 2 and reagent 3 to the liquid holding device;

[0088] Step S3: Place the liquid container into the chemiluminescence analyzer for reaction and detection.

[0089] III. Specific Implementation Examples

[0090] To make the present invention easier to understand, the present invention will be further described in detail below with reference to embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of application of the present invention. Unless otherwise specified, the raw materials or components used in the present invention can be obtained commercially or by conventional methods.

[0091] Example 1 Preparation of Reagent 1

[0092] 1.1 Preparation and characterization process of the first carrier

[0093] 1) Prepare a 100ml three-necked flask, add 40mmol styrene, 5mmol methacrylic acid, and 10ml water, stir for 10min, and then purge with N2 for 30min;

[0094] 2) Weigh 0.12g of potassium persulfate and 0.2g of sodium chloride, dissolve them in 40ml of water to prepare an aqueous solution. Add this aqueous solution to the reaction system in step 1, and continue to purge with N2 for 30min;

[0095] 3) Heat the reaction system to 70℃ and react for 15 hours;

[0096] 4) Cool the emulsion after the reaction is complete to room temperature and filter it with a suitable filter cloth. Wash the resulting emulsion by centrifugation with deionized water multiple times until the conductivity of the supernatant after centrifugation is close to that of deionized water. Then dilute it with water and store it in emulsion form.

[0097] 5) The Gaussian particle size distribution of the carboxylated polystyrene latex microspheres at this point was measured using a nanoparticle size analyzer.

[0098] The average particle size was 202.5 nm, and the coefficient of variation (CV) was 5.6%.

[0099] 1.2 Landfilling process of luminescent compositions

[0100] 1) Prepare a 25ml round-bottom flask, add 0.1g of dimethylthiophene derivative and 0.1g of europium(III) complex (MTTA-EU) 3+ Add 10 ml of 95% ethanol, stir magnetically, and heat in a water bath to 70°C to obtain the complex solution;

[0101] 2) Prepare a 100ml three-necked flask, add 10ml of 95% ethanol, 10ml of water and 10ml of carboxylated polystyrene latex microspheres with a concentration of 10% obtained in step 1.1, stir magnetically, and heat in a water bath to 70℃.

[0102] 3) Slowly add the complex solution from step 1) to the three-necked flask from step 2), react at 70°C for 2 hours, then stop stirring and allow to cool naturally.

[0103] 4) Centrifuge the above emulsion for 1 hour at 30,000 g. After centrifugation, discard the supernatant to obtain carboxylated polystyrene latex microspheres with luminescent composition embedded inside.

[0104] 1.3 Surface coating of receptor particles with dextran

[0105] 1) Take 50 mg of aminoglucan solid into a 20 mL round bottom flask, add 5 mL of 50 mM / pH=6 phosphate buffer, and stir to dissolve at 30 °C in the dark;

[0106] 2) Take 100 mg of the prepared carboxylated polystyrene latex microspheres with the luminescent composition inside and add them to the aminodextran solution and stir for 2 hours;

[0107] 3) Dissolve 10 mg EDC·HCl in 0.5 mL of 50 mM / pH=6 phosphate buffer and add it dropwise to the above reaction solution. React overnight at 30°C in the dark.

[0108] 4) Centrifuge the reaction mixture at 30,000 g for 45 min, discard the supernatant, add 50 mM / pH=10 carbonate buffer, and sonicate to disperse. Repeat centrifugation and washing three times, then adjust the volume with 50 mM / pH=10 carbonate buffer to a final concentration of 20 mg / ml.

[0109] 5) Take 100 mg of aldehyde dextran solid into a 20 mL round bottom flask, add 5 mL of 50 mM / pH=10 carbonate buffer, stir and dissolve at 30 °C in the dark to obtain aldehyde dextran solution;

[0110] 6) Add the microsphere solution obtained in step 4) above to the aldehyde dextran solution and stir for 2 hours;

[0111] 7) Dissolve 15 mg of sodium borohydride in 0.5 mL of 50 mM / pH=10 carbonate buffer solution and add it dropwise to the above reaction solution. React overnight at 30°C in the dark.

[0112] 8) After centrifuging the reaction mixture at 30000G for 45 min, discard the supernatant and add 50mM / pH=10 carbonate buffer for ultrasonic dispersion. Repeat centrifugation and washing three times, then adjust the volume with 50mM / pH=10 carbonate buffer to a final concentration of 20mg / ml to obtain a semi-finished microsphere solution.

[0113] 9) The Gaussian average particle size of the semi-finished microspheres, as measured by a nanoparticle size analyzer, was 241.6 nm, with a coefficient of variation (CV) of 11.50%.

[0114] 1.4 Conjugation of CK-MB antibody I

[0115] 1) CK-MB antibody I was dialyzed to 50mM CB buffer at pH=10, and the concentration was measured to be 1.4mg / ml.

[0116] 2) Add 0.5 ml of the receptor particles obtained in (1.3) and 0.36 ml of the CK-MB antibody I obtained in step 1) to a 2 ml centrifuge tube, mix well, add 100 μl of 10 mg / ml NaBH4 solution (50 mM CB buffer), and react at 2-8 °C for 4 hours.

[0117] 3) After the reaction is complete, add 0.5 ml of 100 mg / ml BSA solution (50 mM CB buffer) and react at 2-8℃ for 2 hours.

[0118] 4) After the reaction is complete, centrifuge for 45 min at 30000G. After centrifugation, discard the supernatant and resuspend in 50 mM MES buffer. Repeat the centrifugation and washing four times, and dilute to a final concentration of 100 μg / ml to obtain reagent 1 conjugated with CK-MB antibody I.

[0119] 5) The average particle size of the microspheres at this time, as measured by a nanoparticle size analyzer, was 248.9 nm, with a coefficient of variation (CV) of 9.90%.

[0120] Example 2 Preparation of Reagent 2

[0121] 2.1 The CK-MB antibody II was dialyzed into 0.1M NaHCO3 buffer and the concentration was measured to be 1.1 mg / ml.

[0122] 2.2 Take 0.5 mg of CK-MB antibody II from step 1), adjust the concentration to 1 mg / ml, add 2.7 μl of biotin with a concentration of 16.8 mg / ml, mix well, and react at 2-8℃ for 14-18 hours.

[0123] 2.3 The reaction was completed by dialyzing in 20 mM Tris buffer.

[0124] Example 3 Preparation of Reagent 3

[0125] 3.1 Preparation of the second carrier

[0126] a) Prepare a 100ml three-necked flask, add 40mmol styrene, 5mmol acrolein and 10ml water, stir for 10min and then purge with N2 for 30min.

[0127] b) Weigh 0.11 g of ammonium persulfate and 0.2 g of sodium chloride, dissolve them in 40 ml of water to prepare an aqueous solution. Add this aqueous solution to the reaction system in step a), and continue to purge with N2 for 30 min.

[0128] c) Heat the reaction system to 70°C and react for 15 hours.

[0129] d) Cool the emulsion after the reaction is complete to room temperature and filter it with a suitable filter cloth. Wash the resulting emulsion by centrifugation with deionized water multiple times until the conductivity of the supernatant after centrifugation is close to that of deionized water. Then dilute it with water and store it in emulsion form.

[0130] e) The Gaussian average particle size of the aldehyde-based polystyrene latex microspheres, as measured by a nanoparticle size analyzer, was 201.3 nm, with a coefficient of variation (CV) of 8.0%.

[0131] 3.2 Filling of sensitizer

[0132] a) Prepare a 25ml round-bottom flask, add 0.11g copper phthalocyanine and 10ml N,N-dimethylformamide, stir magnetically, and heat in a water bath to 75℃ to obtain a photosensitizer solution.

[0133] b) Prepare a 100ml three-necked flask, add 10ml of 95% ethanol, 10ml of water and 10ml of aldehyde-based polystyrene latex microspheres obtained in 3.1 with a concentration of 10%, stir magnetically, and heat in a water bath to 70℃.

[0134] c) Slowly add the solution from step a) to the three-necked flask from step b), react at 70°C for 2 hours, then stop stirring and allow to cool naturally to obtain the emulsion.

[0135] d) Centrifuge the above emulsion for 1 hour at 30,000 g. After centrifugation, discard the supernatant and resuspend in 50% ethanol. Repeat the centrifugation and washing three times, then resuspend in 50 mM CB buffer at pH 10 to achieve a final concentration of 20 mg / ml.

[0136] 3.3 Preparation of Reagent 3

[0137] a) Microsphere suspension treatment: Take a certain amount of the microspheres prepared in step 3.2 and centrifuge them in a high-speed refrigerated centrifuge. Discard the supernatant, add a certain amount of MES buffer, and sonicate on an ultrasonic cell disruptor until the particles are resuspended. Add MES buffer to adjust the microsphere concentration to 100 mg / ml.

[0138] b) Preparation of streptavidin solution: Weigh a certain amount of streptavidin and dissolve it in MES buffer to 8 mg / ml.

[0139] c) Mixing: Mix the prepared microsphere suspension, 8 mg / ml avidin and MES buffer at a volume ratio of 2:5:1 and mix quickly to obtain the reaction solution.

[0140] d) Reaction: Prepare a 25 mg / ml NaBH3CN solution using MES buffer and add it to the reaction solution at a volume ratio of 1:25, then mix quickly. Incubate at 37°C with a rotating incubator for 48 hours.

[0141] e) Blocking: Prepare 75 mg / ml Gly solution and 25 mg / ml NaBH3CN solution in MES buffer. Add these solutions to the above solutions at a volume ratio of 2:1:10 to the reaction solution, mix well, and incubate at 37°C with rotation for 2 hours. Then add 200 mg / ml BSA solution (MES buffer) at a volume ratio of 5:8 to the reaction solution, mix quickly, and incubate at 37°C with rotation for 16 hours.

[0142] f) Washing: Add MES buffer to the reacted solution, centrifuge with a high-speed refrigerated centrifuge, discard the supernatant, add fresh MES buffer and resuspend by sonication, centrifuge again, and wash in this way 3 times. Finally, suspend with a small amount of buffer, determine the solid content, and adjust the concentration to 150 μg / ml with buffer to obtain reagent 3 containing donor particles.

[0143] g) The average Gaussian distribution particle size of donor particle a in reagent 3, measured by a nanoparticle size analyzer, was 227.7 nm, with a coefficient of variation (CV) of 6.5%.

[0144] Example 4: Determination of sugar content using the anthrone method

[0145] 4.1 Microsphere Sample Pretreatment:

[0146] Take reagent 1 containing 1 mg of receptor particles from Example 1 and reagent 3 containing 1 mg of donor particles from Example 3, respectively, centrifuge at 20000g for 40 min, discard the supernatant, and then disperse with purified water by ultrasonication. Repeat the centrifugation and dispersion three times, and then make up to 1 mg / mL with purified water to serve as the test samples.

[0147] 4.2 Preparation of glucose standard solution:

[0148] The 1 mg / mL glucose stock solution was prepared into standard solution curves with concentrations of 0 mg / mL, 0.025 mg / mL, 0.05 mg / mL, 0.075 mg / mL, 0.10 mg / mL, and 0.15 mg / mL using purified water.

[0149] 4.3 Preparation of anthrone solution: Prepare a solution of 2 mg / mL using 80% sulfuric acid solution.

[0150] 4.4 Add 0.1 mL of glucose standard solution of various concentrations and the sample to be tested to centrifuge tubes respectively, and add 1 mL of anthrone solution to each tube.

[0151] 4.5 Incubate at 85 degrees Celsius for 30 minutes.

[0152] 4.6 Centrifuge the sample reaction tube at 15000G for 40 min. Use a pipette tip to aspirate the clear liquid from the bottom of the tube for absorbance measurement, avoiding aspirating the suspended matter at the top.

[0153] 4.7 Return to room temperature and measure absorbance at 620 nm.

[0154] 4.8 Using the concentration of the standard as the X value and the absorbance as the Y value, a linear regression was performed to obtain the absorbance values ​​of the standard curve shown in Table 1, and the sugar content of the sample to be tested was determined based on these values.

[0155] The sugar content of the acceptor particles in Example 1 and the donor particles in Example 3 is shown in Table 2 below.

[0156] Table 1

[0157]

[0158]

[0159] Table 2

[0160]

[0161] Example 5: Creatine kinase isoenzyme detection kit (photocatalytic chemiluminescence method)

[0162] This kit contains Reagent 1 (containing receptor microparticles coated with CK-MB antibody I) prepared in Example 1, Reagent 2 (containing biotin-labeled CK-MB antibody II) prepared in Example 2, creatine kinase isoenzyme series calibrators, creatine kinase isoenzyme quality control samples, and Reagent 3 (containing donor particles coated with avidin) prepared in Example 3. Under homogeneous conditions, the concentration of creatine kinase isoenzyme (CK-MB) in human serum and plasma samples is quantitatively detected using a double antibody sandwich immunophotochemiluminescence assay.

[0163] Table 3 below shows the main components of the creatine kinase isoenzyme detection kit (photocatalytic chemiluminescence method) prepared in this embodiment.

[0164] Table 3

[0165]

[0166]

[0167] Example 6: Determination of reagent kit performance

[0168] The performance of the kit in this example was detected using the LiCA500 automated photo-induced chemiluminescence immunoassay analyzer developed by Boyang Biotechnology (Shanghai) Co., Ltd. The specific steps for detecting creatine kinase isoenzymes using the LiCA500 automated photo-induced chemiluminescence immunoassay analyzer are as follows:

[0169] 1. Add 50 μL of sample or calibrator / quality control to each reaction well;

[0170] 2. Add 15 μL of reagent 1 and 15 μL of reagent 2 to the reaction wells in sequence;

[0171] 3. Vibrate, incubate at 37℃ for 10 minutes;

[0172] 4. Automatically add 150 μL of reagent 3 (universal solution for photochemiluminescence analysis system, universal solution for LiCA);

[0173] 5. Vibrate, incubate at 37°C for 2 minutes;

[0174] 6. Irradiate the micro-holes with a laser and calculate the amount of photons emitted from each hole;

[0175] 7. Calculate the sample concentration based on the calibration curve.

[0176] The method for determining the validity of the test results in this embodiment is as follows: Quality control samples should be tested for each batch of tests, with single-well determination. The test results should be within the required quality control range. If they are outside the range, the test results are unreliable and must be retested. If necessary, recalibration should be performed.

[0177] This kit was used to test 400 plasma samples from healthy individuals, including 200 women and 200 men. The corresponding 97.5% of the population measured the following levels: women: 3.5 ng / mL, men: 4.8 ng / mL.

[0178] The detection performance evaluation results of the kit in this embodiment are as follows:

[0179] Blank limit: ≤0.1 ng / mL;

[0180] Linearity: Within the linear range of 0.1–500 ng / mL, the linear correlation coefficient r value is ≥0.9900;

[0181] Accuracy: Recovery rate is within the range of 85% to 115%;

[0182] Repeatability: Coefficient of variation (CV) ≤ 10%;

[0183] Inter-batch variation: Inter-batch coefficient of variation (CV) ≤ 15%.

[0184] Example 7: Determination of reagent kit sensitivity and upper limit of detection

[0185] The sensitivity point is defined as the point where the signal at concentration Cx is higher than twice the signal at concentration C0, i.e., RLU(Cx) > 2RLU(C0), and the sensitivity of the corresponding detection reagent is Cx. The upper limit of detection is defined as the upper limit of the range determined using the method in the NCCLS EP-6 document.

[0186] (1) CK-MB antigen was diluted to a series of concentrations of 0.1 ng / ml, 0.3 ng / ml, 0.5 ng / ml, 0.7 ng / ml, 0.9 ng / ml, 3 ng / ml, 5 ng / ml, 100 ng / ml, 300 ng / ml, 500 ng / ml, 700 ng / ml, 900 ng / ml, 1100 ng / ml, 1300 ng / ml, and 1500 ng / ml. The above-mentioned concentration series of CK-MB antigens were detected using reagent 1 (containing receptor particles conjugated with CK-MB antibody I, with a concentration of 100 μg / ml) prepared in Example 1, reagent 2 (containing the same biotin-labeled CK-MB antibody II, diluted to 2 μg / ml) prepared in Example 2, and reagent 3 (containing donor particles) prepared in Example 3. The detection sensitivity and detection limit of the photo-induced chemiluminescence analysis system developed by Boyang Biotechnology (Shanghai) Co., Ltd. are shown in Table 4.

[0187] Table 4

[0188]

[0189] As shown in Table 4, when the coefficient of variation of the particle size distribution of the receptor particles is not less than 5% and not more than 25%, the kit containing the receptor particles has both suitable sensitivity and a wide detection range.

[0190] Example 8: Effect of sugar content of receptor particles in reagent 1 on reagent kit performance

[0191] Reagent 1 containing the following series of receptor particles was prepared using the preparation method of reagent 1 in Example 1 above, and the sugar content in the receptor particles was detected using the anthrone method given in Example 4.

[0192] Referring to the method given in Example 5 above, a creatine kinase isoenzyme detection kit (photocatalytic chemiluminescence method) including reagent 1 with different sugar contents was prepared. The photocatalytic chemiluminescence detection process was completed and the detection results were output on the LiCA HT automated photocatalytic chemiluminescence analysis system developed by Boyang Biotechnology (Shanghai) Co., Ltd.

[0193] Table 5

[0194]

[0195] As shown in Table 5, when the sugar content of the receptor particles in reagent 1 is not less than 20 mg / g, the photo-induced chemiluminescence detection signal of the kit containing the receptor particles is relatively high.

[0196] Example 9: Detection of Clinical Samples

[0197] This embodiment tested 40 clinical samples using the kit (photochemiluminescence immunoassay) prepared in Example 5. The CK-MB quantitative assay kit (photochemiluminescence immunoassay) included: reagent 1 containing receptor particles coated with CK-MB antibody I (monoclonal antibody), reagent 2 containing biotin-labeled CK-MB antibody II (monoclonal antibody), and reagent 3 (a universal solution for photochemiluminescence analysis systems) containing donor particles. The concentration of donor particles in reagent 3 was 150 μg / ml, and the coefficient of variation (CV) of the donor particle size distribution in reagent R3 was 6.5%. The sugar content of the donor particles was determined to be 11.5 μg / mg using the anthrone method given in Example 4, and the sugar content of the receptor particles was 59.60 μg / mg.

[0198] The detection process was completed and the results were output on a fully automated photocatalytic chemiluminescence analysis system (LiCA HT) developed by Boyang Biotechnology (Shanghai) Co., Ltd. The specific detection steps include:

[0199] a. Add the clinical sample to the reaction well;

[0200] b. Add reagent 1 and reagent 2 to the reaction wells in sequence;

[0201] c. Warming;

[0202] d. Add reagent 3 to the reaction well;

[0203] e. Warming;

[0204] f. Irradiate the reaction hole twice with lasers and calculate the amount of photons emitted from each hole;

[0205] g. Calculate the CK-MB concentration in the sample to be tested.

[0206] When CK-MB biomarkers are present in clinical samples, CK-MB specifically binds to both receptor particles coated with CK-MB antibody I (monoclonal antibody) and biotin-labeled CK-MB antibody II (monoclonal antibody), forming a double-antibody sandwich complex on the surface of the receptor particles. If streptavidin-modified donor particles are added, biotin binds to streptavidin, causing the two types of particles to approach each other. Under excitation by a light source, the donor particles release reactive oxygen species, which, upon encountering the receptor particles in solution, produce chemiluminescence. This further excites the fluorophores on the same particle, resulting in a cascade amplification reaction and fluorescence. The higher the concentration of CK-MB biomarkers, the stronger the fluorescence intensity. The amount of CK-MB in the patient's serum is quantitatively detected based on the intensity of the fluorescence. Specific detection results are shown in Table 6 below.

[0207] Table 6

[0208]

[0209]

[0210] Data comparison showed that the correlation between the Roche measurement and the measurement in Example 8 was 0.9963, with a slope of 0.9632. Samples 1-23 were normal physical examination patients, with a range of 0.3 ng / ml-4.13 ng / ml and a median of 1.09 ng / ml; samples 24-40 were patients identified with myocardial injury, with a range of 4.55 ng / ml-260.48 ng / ml and a median of 20.06 g / ml.

[0211] Creatine kinase (CK) is a dimeric enzyme with four different forms: mitochondrial isoenzyme, cytoplasmic isoenzyme CK-MM (muscle type), creatine kinase brain isoenzyme CK-BB (brain type), and creatine kinase isoenzyme (CK-MB). CK-MB is primarily located in the myocardium. During acute myocardial infarction (AMI) and myocardial necrosis, creatine kinase is released into the peripheral blood and can be detected 3–8 hours after the onset of clinical symptoms. Combined detection with myoglobin and troponin can determine whether the myocardium is damaged, serving as an indicator for the research and diagnosis of AMI. Clinically, it is mainly used as an auxiliary diagnostic tool for myocardial infarction. Data from Embodiment 7 of the present invention demonstrate the feasibility of using the donor reagent described in this invention in the preparation of a kit for in vitro diagnosis of myocardial injury in a subject. The quantitative results of measuring CKMB markers in the body fluids of a subject using the donor reagent and corresponding method described in this invention can be used as an auxiliary diagnostic tool for myocardial infarction.

[0212] 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 homogeneous detection kit for creatine kinase isoenzymes, comprising reagent 1 and reagent 3, wherein reagent 1 contains a first buffer solution and receptor particles suspended therein, the receptor particles being capable of generating chemiluminescence by reacting with reactive oxygen species, the receptor particles comprising a first carrier, the interior of the first carrier being filled with a luminescent composition, the surface of the first carrier being coated with a polysaccharide, and a creatine kinase isoenzyme antibody binding to the first carrier by binding to the polysaccharide molecule, the creatine kinase isoenzyme antibody being capable of specifically binding to creatine kinase isoenzymes; reagent 3 comprises a second buffer solution and donor particles suspended therein, the donor particles comprising a second carrier, the interior of the second carrier being filled with a sensitizer, and the surface of the second carrier being bonded with one of the specific pairing members, characterized in that: The coefficient of variation (CV) of the recipient particles in reagent 1 is not less than 5% and not more than 25%, and the sugar content per gram of the recipient particles in reagent 1 is not less than 20 mg; the coefficient of variation (CV) of the donor particles in reagent 3 is not less than 5% and not more than 25%, and the sugar content per gram of the donor particles in reagent 3 is not more than 40 mg; the coefficient of variation (CV) is the coefficient of variation of particle size in a Gaussian distribution in the detection results of a nanoparticle size analyzer.

2. The reagent kit according to claim 1, characterized in that, The coefficient of variation (CV) of the size distribution of the receptor particles in reagent 1 is no higher than 15%.

3. The reagent kit according to claim 1, characterized in that, The sugar content in each liter of the first buffer solution is 0.01-1g.

4. The reagent kit according to claim 3, characterized in that, The sugar content in each liter of the first buffer solution is 0.02-0.2g.

5. The reagent kit according to claim 1, characterized in that, Each gram of the receptor particle in reagent 1 contains no less than 40 milligrams of sugar.

6. The reagent kit according to claim 1, characterized in that, The polysaccharide is selected from carbohydrates containing three or more unmodified or modified monosaccharide units.

7. The reagent kit according to claim 6, characterized in that, The polysaccharide is selected from at least one of glucan, fructan, mannan, and galactan.

8. The reagent kit according to claim 6, characterized in that, The polysaccharide is selected from at least one of starch, glycogen, inulin, agarose, carboxyglucan, and aminoglucan.

9. The reagent kit according to claim 6, characterized in that, The polysaccharide is a polyribose.

10. The reagent kit according to claim 6, characterized in that, The polysaccharide is a dextran and / or a dextran derivative.

11. The reagent kit according to claim 1, characterized in that, The kit also includes reagent 2, which contains a creatine kinase isoenzyme antibody linked to one of the specific binding pair members, the creatine kinase isoenzyme antibody being able to specifically bind to the creatine kinase isoenzyme.

12. The application of a kit as described in any one of claims 1-11 in a chemiluminescence analyzer.

13. The application according to claim 12, characterized in that, The application includes the following steps: Step S1: Add the sample to be tested, calibrator and / or quality control sample to the liquid holding device respectively; Step S2: Add reagent 1, reagent 2 and reagent 3 to the liquid holding device; Step S3: Place the liquid container into the chemiluminescence analyzer for reaction and detection.

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