Use of a donor reagent in diagnosing myocardial injury

Abstract

This invention relates to the use of a donor reagent in the preparation of a kit for an in vitro diagnostic method for determining whether a subject has myocardial injury. The method includes: contacting a bodily fluid from a subject with a recipient reagent and a donor reagent, reacting to generate a test mixture; exciting the test mixture at least once with excitation light, and detecting the intensity of the resulting chemiluminescence signal; quantitatively calculating the concentration of at least one myocardial marker in the bodily fluid based on the intensity of the chemiluminescence signal, thereby determining whether the subject has myocardial injury; wherein the donor reagent comprises donor particles capable of generating reactive oxygen species in an excited state, the donor particles comprising a first carrier internally filled with a sensitizer, and the surface of the first carrier specifically binding one of the paired members via surface chemical bonding. The method exhibits both extremely high sensitivity and a wide detection range.

Description

Technical Field

[0001] This invention belongs to the field of chemiluminescence analysis, specifically relating to the use of a donor reagent in diagnosing myocardial injury. Background Technology

[0002] Cardiovascular diseases are serious threats to human health and life, and have become the number one killer of human health in the 21st century. Among them, acute myocardial infarction is the most common and the most dangerous.

[0003] Cardiovascular testing is a "bottleneck" discipline within the entire cardiovascular field because only by accurately diagnosing diseases in the shortest possible time can early detection and treatment be achieved, minimizing the risk of disability and death, and improving patient prognosis and quality of life. In recent years, research on cardiovascular biomarkers has deepened, accumulating a wealth of clinical experience and evidence, gradually clarifying their clinical indications and new research areas, and promoting their clinical application. Biomarker detection directly impacts the clinical diagnosis, risk stratification, treatment selection, and prognosis of cardiovascular disease patients. Myocardial biomarkers, also known as early markers of cardiac injury, refer to markers whose blood levels rise within 6 hours of cardiac injury. They are important indicators for diagnosing myocardial infarction, myocardial ischemia, heart failure, and other cardiac diseases. These mainly include cardiac troponin I (cTnI), myoglobin (MYO), creatine kinase isoenzyme (CK-MB), and N-terminal pro-brain natriuretic peptide (NT-proBNP). With continuous technological advancements, the detection of heart failure biomarkers in the blood is gradually being applied clinically, providing methods for the early diagnosis and prognosis of cardiovascular diseases. For example, NT-proBNP is the inactive N-terminal fragment of BNP progenitor after cleavage. Compared to BNP, it has a longer half-life and is more stable. Its concentration reflects the release of newly synthesized rather than stored BNP over a short period, thus better reflecting the activation of the BNP pathway. Plasma NT-proBNP levels increase with the severity of heart failure. NT-proBNP plays an important role in the diagnosis of acute heart failure.

[0004] Currently, the above biomarkers are widely accepted by hospitals. Various kits for individually detecting these indicators are also available on the market. However, the mainstream methods currently used are heterogeneous assays (such as ELISA and magnetic particle chemiluminescence). While these methods can detect the above biomarkers, they suffer from drawbacks such as slow detection speed, low sensitivity, and high cost. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing a donor reagent for the diagnosis of myocardial injury. When the donor particles are used for detection, they have both extremely high sensitivity and a wide detection range.

[0006] Therefore, a first aspect of the present invention provides the use of a donor reagent in a kit for preparing a method for in vitro diagnosis of whether a subject has myocardial injury, wherein the method includes: contacting a body fluid from a subject with a recipient reagent and a donor reagent, reacting to generate a test mixture; exciting the test mixture at least once with excitation light, detecting the intensity of the chemiluminescence signal generated therefrom; and quantitatively calculating the concentration of at least one myocardial marker in the body fluid based on the intensity of the chemiluminescence signal, thereby determining whether the subject has myocardial injury. The donor reagent comprises donor particles capable of generating reactive oxygen species in an excited state. The donor particles include a first carrier, the interior of which is filled with a sensitizer. The surface chemical bonds of the first carrier specifically bind to one of the paired members. The cardiac markers are selected from one or more of troponin T, troponin I, troponin kinase isoenzyme, myoglobin, interleukin-6, and lactate dehydrogenase.

[0007] In some embodiments of the present invention, the surface of the first carrier is not coated or connected to a polysaccharide substance that directly chemically binds to one of the paired members.

[0008] In other embodiments of the invention, the surface of the first carrier has bonding functional groups for chemically bonding one of the specific binding pair members to the surface of the first carrier.

[0009] In some embodiments of the present invention, the bonding functional group is selected from amino, amide, hydroxyl, aldehyde, carboxyl, maleimide and thiol groups; preferably selected from aldehyde and / or carboxyl groups.

[0010] In some embodiments of the present invention, the content of bonded functional groups on the surface of the first carrier is 100~500 nmol / mg, preferably 200~400 nmol / mg.

[0011] 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, wherein the first polysaccharide layer and the second polysaccharide layer are spontaneously associated.

[0012] In other embodiments of the invention, each of the continuous polysaccharide layers is spontaneously associated with each of the preceding polysaccharide layers.

[0013] In some embodiments of the present invention, the polysaccharide has side group functional groups, and the functional groups of the continuous polysaccharide layers carry opposite charges to the functional groups of the preceding polysaccharide layer.

[0014] In other embodiments of the invention, the polysaccharide has side functional groups, and the continuous polysaccharide layer is covalently linked to the preceding polysaccharide layer through a reaction between the functional groups and the functional groups of the preceding polysaccharide layer.

[0015] In some embodiments of the invention, the functional groups of the continuous polysaccharide layer alternate between amine functional groups and amine reactive functional groups.

[0016] In other embodiments of the invention, the amine reactive functional group is an aldehyde group or a carboxyl group.

[0017] In some embodiments of the present invention, the first polysaccharide layer is spontaneously associated with the first carrier.

[0018] In other embodiments of the invention, the outermost polysaccharide layer of the coating has at least one side functional group.

[0019] 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, mercapto, amino, hydroxyl and maleamine groups; preferably selected from aldehyde and / or carboxyl groups.

[0020] In other embodiments of the invention, the side group functional groups of the outermost polysaccharide layer of the coating are directly or indirectly chemically bonded to one of the paired members.

[0021] 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 dextran, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxyglucan and aminoglucan; more preferably selected from dextran, starch, glycogen and polyribose.

[0022] In some embodiments of the present invention, the particle size of the first carrier is selected from 100~400nm, preferably 150~350nm, and more preferably 180~220nm.

[0023] In other embodiments of the present invention, the first carrier is magnetic or non-magnetic, preferably non-magnetic.

[0024] In some embodiments of the present invention, the shape of the first carrier is selected from tape, sheet, rod, tube, hole, microtiter plate, bead, particle and microsphere; preferably microsphere.

[0025] In other embodiments of the present invention, the material of the first carrier is selected from natural, synthetic or modified naturally occurring polymers; preferably synthetic polymers.

[0026] In some specific embodiments of the present invention, the material of the first carrier is selected from agarose, cellulose, nitrocellulose, cellulose acetate, polyvinyl chloride, polystyrene, polyethylene, polypropylene, poly(4-methylbutene), polyacrylamide, polymethyl methacrylate, polyethylene terephthalate, nylon, polyvinyl butyrate, or polyacrylate; preferably selected from polystyrene, polypropylene, poly(4-methylbutene), polyacrylamide, polymethyl methacrylate, polyethylene terephthalate, or polyacrylate.

[0027] In some embodiments of the present invention, the first carrier is polystyrene latex microspheres.

[0028] In other embodiments of the present invention, the sensitizer is a photoactivated photosensitizer and / or a chemically activated initiator, preferably a photoactivated photosensitizer.

[0029] In some embodiments of the present invention, the sensitizer is selected from methylene blue, rose red, porphyrin, phthalocyanine and chlorophyll.

[0030] In other embodiments of the present invention, the specific binding pair members are selected from a pair of substances capable of specifically binding to each other, consisting of an antibody, antibody fragment, ligand, oligonucleotide, oligonucleotide-binding protein, lectin, hapten, antigen, immunoglobulin-binding protein, avidin, or biotin.

[0031] In some embodiments of the present invention, the specific binding pairing member is avidin-biotin.

[0032] In other embodiments of the invention, the avidin is selected from egg avidin, streptavidin, egg yolk avidin, neutral avidin, and avidin-like substances, preferably neutral avidin and / or streptavidin.

[0033] In some embodiments of the present invention, the avidin is chemically bonded to the surface of the first carrier by reacting an amino group with an aldehyde group on the surface of the first carrier to form a Schiff base.

[0034] In some embodiments of the present invention, the coefficient of variation (CV) of the particle size distribution of the donor particles in the donor reagent is controlled to be ≥5%.

[0035] In other embodiments of the present invention, the coefficient of variation (CV) of the particle size distribution of the donor particles in the donor reagent is controlled to be ≥8%; preferably, the coefficient of variation (CV) of the particle size distribution of the donor particles in the donor reagent is controlled to be ≥10%.

[0036] In some embodiments of the present invention, the coefficient of variation (CV) of the particle size distribution of the donor particles in the donor reagent is controlled to be ≤40%; more preferably, the coefficient of variation (CV) of the particle size distribution of the donor particles in the donor reagent is controlled to be ≤20%.

[0037] In other embodiments of the invention, the donor particles exhibit polydispersity in the particle size distribution of the donor reagent.

[0038] In some embodiments of the present invention, the concentration of the donor particles in the donor reagent is 10 μg / ml to 1 mg / ml, preferably 20 μg / ml to 500 μg / ml, and more preferably 50 μg / ml to 200 μg / ml.

[0039] In some other embodiments of the present invention, the donor reagent further includes a buffer solution with a pH value of 7.0 to 9.0, and the donor particles are suspended in the buffer solution.

[0040] In some embodiments of the present invention, the buffer solution contains a polysaccharide selected from carbohydrates containing three or more unmodified or modified monosaccharide units, preferably selected from dextran, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxyglucan and aminoglucan; more preferably selected from dextran, starch, glycogen and polyribose.

[0041] In other embodiments of the present invention, the molecular weight distribution Mw of the dextran is selected from 10,000 to 1,000,000 kDa, preferably from 100,000 to 800,000 kDa, and more preferably from 300,000 to 700,000 kDa.

[0042] In some embodiments of the present invention, the content of dextran in the buffer solution is 0.01~1wt%, preferably 0.05~0.5wt%.

[0043] In other embodiments of the present invention, the receptor particles in the receptor reagent include a second carrier, the interior of which is filled with a luminescent composition, and the surface of the second carrier is coated with at least one polysaccharide layer, the surface of which is connected to a reporter molecule that is capable of specifically binding to the target molecule to be tested.

[0044] In some embodiments of the present invention, the luminescent composition comprises a chemiluminescent compound and a metal chelate.

[0045] In other embodiments of the present invention, the chemiluminescent compound is selected from olefin compounds, preferably from dimethylthiophene, dibutyl dione compounds, dioxane, enol ethers, enamines, 9-alkylene oxalane, 9-alkylene-N-9,10 dihydroacrylidine, aryl ethyl ether olefins, aryl imidazoles and glossin and their derivatives, more preferably from dimethylthiophene and its derivatives.

[0046] In some embodiments of the present invention, the metal chelate is a rare earth metal or a Group VIII metal, preferably selected from europium, terbium, dysprosium, samarium, osmium, and ruthenium, and more preferably europium.

[0047] In other embodiments of the invention, 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.

[0048] In some specific embodiments of the present invention, the bodily fluids from the host are diluted with a diluent before being contacted with the recipient reagent and the donor reagent.

[0049] In some embodiments of the present invention, the detection wavelength of the chemiluminescence is 520~620nm.

[0050] In some other embodiments of the present invention, laser irradiation is performed using red excitation light of 600-700 nm.

[0051] In some embodiments of the present invention, the concentration of the receptor particles in the receptor reagent is 1 ug / mL to 1000 ug / mL; preferably 10 ug / mL to 500 ug / mL; more preferably 20 ug / mL to 200 ug / mL.

[0052] In other embodiments of the present invention, the active oxygen is singlet oxygen.

[0053] In other embodiments of the invention, the bodily fluids from the subject include, but are not limited to: blood, serum, plasma, sputum, lymph, semen, vaginal mucus, feces, urine, or cerebrospinal fluid.

[0054] The beneficial effects of this invention are as follows: This invention provides the use of a donor reagent in the preparation of a kit for in vitro diagnostic methods to determine whether a subject has myocardial injury. The donor particles in the donor reagent generate reactive oxygen species with high efficiency. These reactive oxygen species are more easily transferred to acceptor particles in a homogeneous system and are less susceptible to interference from other substances. Furthermore, the donor particles themselves have high stability and can exist stably in the donor reagent without easily becoming inactivated. When using the donor reagent for detection, it offers both extremely high sensitivity and a wide detection range. In addition, the production cost of the donor particles in the donor reagent is low, and it is convenient to use, making it applicable to various detection projects. Attached Figure Description

[0055] The invention will now be further described with reference to the accompanying drawings.

[0056] Figure 1 This is a Gaussian distribution diagram of the aldehyde-based polystyrene latex microspheres prepared in Example 1.

[0057] Figure 2 The image shows the Nicomp distribution of the aldehyde-based polystyrene latex microspheres prepared in Example 1.

[0058] Figure 3 This is a Gaussian distribution diagram of the donor particles prepared in Example 1.

[0059] Figure 4 Gaussian distribution map of the dextran-coated microspheres prepared in Example 2. Figure 5 The image shows the Gaussian distribution of the donor particles prepared in Example 2.

[0060] Figure 6 The image shows the Gaussian distribution curve of the aldehyde-based polystyrene latex microspheres prepared in Example 3.

[0061] Figure 7 The Gaussian distribution curve of the aldehyde-based polystyrene latex microspheres filled with the luminescent composition prepared in Example 3 is shown.

[0062] Figure 8 Gaussian distribution diagram of aldehyde polystyrene latex microspheres filled with luminescent composition and coated with dextran prepared in Example 3.

[0063] Figure 9 The Gaussian distribution diagram of the receptor particles with an average particle size of about 250 nm prepared in Example 3. Detailed Implementation

[0064] 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. The implementation of the present invention is not limited to the embodiments below, and any modifications and / or alterations made to the present invention will fall within the protection scope of the present invention.

[0065] 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.

[0066] 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 be used in the practice or testing of this invention, preferred methods and materials are now described.

[0067] I. Terminology The term "reactive oxygen species" as used in this invention refers to a general term for substances composed of oxygen in the body or natural environment that contain oxygen and are reactive in nature. It is mainly an excited-state oxygen molecule, including the one-electron reduction product of oxygen, superoxide anion (O2·-), the two-electron reduction product, hydrogen peroxide (H2O2), the three-electron reduction product, hydroxyl radical (·OH), as well as nitric oxide and singlet oxygen (1O2), etc.

[0068] As used in this invention, "donor particle" refers to a particle containing 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. The donor particle can be photoactivated (e.g., dyes and aromatic compounds) or chemically activated (e.g., enzymes, metal salts, etc.). In some specific embodiments of this invention, the donor particle is a polymeric microsphere 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 singlet oxygen. Non-limiting examples include, for example, compounds such as methylene blue, rose red, porphyrin, phthalocyanine, and chlorophyll disclosed in US Patent 5709994 (which is incorporated herein by reference in its entirety), and 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. Other examples of photosensitizers known to those skilled in the art may also be used in this invention, such as those described in U.S. Patent US6406913, which is incorporated herein by reference.

[0069] As used in this invention, "receptor particle" refers to a particle containing a compound capable of reacting with reactive oxygen species to generate a detectable signal. The donor particle is induced and activated by energy or an active compound, releasing high-energy reactive oxygen species. These high-energy reactive oxygen species are captured by a nearby receptor particle, thereby transferring energy to activate the receptor particle. In some specific embodiments of this invention, the receptor particle comprises a luminescent composition and a carrier, wherein the luminescent composition is filled in the carrier and / or coated on the surface of the carrier.

[0070] The "carrier" described in this invention is selected from tapes, sheets, rods, tubes, pores, microtiter plates, beads, particles, and microspheres. It can be microspheres or microparticles known to those skilled in the art, and can be of any size. It can be organic or inorganic, expandable or non-expandable, porous or non-porous, magnetic or non-magnetic, and has any density, but preferably has a density close to that of water. It is preferably able to float in water and is made of transparent, partially transparent, or opaque materials.

[0071] In this invention, the "chemiluminescent compound" 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.

[0072] The "specific binding pair" mentioned in this invention refers to a pair of substances that can specifically bind to each other.

[0073] The "coefficient of variation (CV) of particle size distribution" mentioned in this invention refers to the coefficient of variation of particle size in the Gaussian distribution in the detection results of a nanoparticle size analyzer. The formula for calculating the coefficient of variation is: CV = (standard deviation SD / mean Mean) × 100%.

[0074] The term "Nicomp distribution" used in this invention refers to an algorithmic distribution in the NICOMP nanoparticle size analyzer from the American PSS. Compared to the Gaussian single-peak algorithm, the Nicomp multi-peak algorithm has unique advantages for the analysis of multi-component, non-uniformly sized liquid dispersion systems and the stability analysis of colloidal systems.

[0075] The term "body fluid from the subject" as used in this invention refers to a mixture containing or suspected of containing cardiac markers. The body fluid is derived from the subject's whole blood, serum, plasma, sputum, lymph, semen, vaginal mucus, feces, urine, tears, or cerebrospinal fluid. The body fluid from the subject can be diluted with a diluent as needed before use. For example, to avoid the hook effect, the body fluid from the subject can be diluted with a diluent before testing on the instrument.

[0076] 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 a non-antibody protein. Where desired, antibodies may be further conjugated to other portions, such as a member of a specific binding pair, for example, biotin or avidin (a member of the biotin-avidin specific binding pair).

[0077] The term "antigen" as used in this invention refers to a substance that can stimulate the body to produce an immune response and can bind to immune response products, antibodies and sensitized lymphocytes in vivo and in vitro to produce an immune effect.

[0078] The term "bonding" as used in this invention refers to the direct union between two molecules caused by interactions such as covalent, electrostatic, hydrophobic, ionic and / or hydrogen bonding, including but not limited to interactions such as salt bridges and water bridges.

[0079] The term "specific binding" as used in this invention refers to a mutually distinguishable and selective binding reaction between two substances, which, from a stereostructural perspective, means the conformational correspondence between the corresponding reactants. Under the technical concept disclosed in this invention, the detection methods for specific binding reactions include, but are not limited to: double-antibody sandwich method, competitive method, neutralization competitive method, indirect method, or capture method.

[0080] II. Specific Implementation Plan The present invention will now be described in more detail with reference to the embodiments.

[0081] Those skilled in the art generally believe that the more uniform the particle size of microspheres, the better the performance of homogeneous chemiluminescence detection using those microspheres. Therefore, current research on microspheres used in homogeneous chemiluminescence tends to focus on obtaining microspheres with more uniform particle sizes. The inventors of this application, through research, discovered that when using microspheres with uniform particle sizes for homogeneous chemiluminescence detection, it is difficult to simultaneously guarantee the sensitivity and detection range of the detection results. However, by using microspheres with suitable particle size uniformity (e.g., a coefficient of variation of the microsphere particle size distribution > 5%), it is possible to both guarantee the sensitivity of photo-induced chemiluminescence detection and broaden the detection range.

[0082] Therefore, the use of the donor reagent involved in this invention in a kit for preparing a method for in vitro diagnosis of whether a subject has myocardial injury, wherein the method includes: contacting a body fluid from a subject with a recipient reagent and a donor reagent, reacting to generate a test mixture; exciting the test mixture at least once with excitation light, detecting the intensity of the chemiluminescence signal generated therefrom; and quantitatively calculating the concentration of at least one myocardial marker in the body fluid based on the intensity of the chemiluminescence signal, thereby determining whether the subject has myocardial injury. The donor reagent comprises donor particles capable of generating reactive oxygen species in an excited state. The donor particles include a first carrier, the interior of which is filled with a sensitizer. The surface chemical bonds of the first carrier specifically bind to one of the paired members. The cardiac markers are selected from one or more of troponin T, troponin I, troponin kinase isoenzyme, myoglobin, interleukin-6, and lactate dehydrogenase.

[0083] In some embodiments of the present invention, the surface of the first carrier is not coated or connected to a polysaccharide substance that directly chemically binds to one of the paired members.

[0084] In other embodiments of the invention, the surface of the first carrier has bonding functional groups for chemically bonding one of the specific binding pair members to the surface of the first carrier.

[0085] In some embodiments of the present invention, the bonding functional group is selected from amino, amide, hydroxyl, aldehyde, carboxyl, maleimide and thiol groups; preferably selected from aldehyde and / or carboxyl groups.

[0086] In some embodiments of the present invention, the content of bonded functional groups on the surface of the first carrier is 100~500 nmol / mg, preferably 200~400 nmol / mg.

[0087] 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, wherein the first polysaccharide layer and the second polysaccharide layer are spontaneously associated.

[0088] In other embodiments of the invention, each of the continuous polysaccharide layers is spontaneously associated with each of the preceding polysaccharide layers.

[0089] In some embodiments of the present invention, the polysaccharide has side group functional groups, and the functional groups of the continuous polysaccharide layers carry opposite charges to the functional groups of the preceding polysaccharide layer.

[0090] In other embodiments of the invention, the polysaccharide has side functional groups, and the continuous polysaccharide layer is covalently linked to the preceding polysaccharide layer through a reaction between the functional groups and the functional groups of the preceding polysaccharide layer.

[0091] In some embodiments of the invention, the functional groups of the continuous polysaccharide layer alternate between amine functional groups and amine reactive functional groups.

[0092] In other embodiments of the invention, the amine reactive functional group is an aldehyde group or a carboxyl group.

[0093] In some embodiments of the present invention, the first polysaccharide layer is spontaneously associated with the first carrier.

[0094] In other embodiments of the invention, the outermost polysaccharide layer of the coating has at least one side functional group.

[0095] 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, mercapto, amino, hydroxyl and maleamine groups; preferably selected from aldehyde and / or carboxyl groups.

[0096] In other embodiments of the invention, the side group functional groups of the outermost polysaccharide layer of the coating are directly or indirectly chemically bonded to one of the paired members.

[0097] 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 dextran, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxyglucan and aminoglucan; more preferably selected from dextran, starch, glycogen and polyribose.

[0098] In some embodiments of the present invention, the particle size of the first carrier is selected from 100~400nm, preferably 150~350nm, and more preferably 180~220nm.

[0099] In other embodiments of the present invention, the first carrier is magnetic or non-magnetic, preferably non-magnetic.

[0100] In some embodiments of the present invention, the shape of the first carrier is selected from tape, sheet, rod, tube, hole, microtiter plate, bead, particle and microsphere; preferably microsphere.

[0101] In other embodiments of the present invention, the material of the first carrier is selected from natural, synthetic or modified naturally occurring polymers; preferably synthetic polymers.

[0102] In some specific embodiments of the present invention, the material of the first carrier is selected from agarose, cellulose, nitrocellulose, cellulose acetate, polyvinyl chloride, polystyrene, polyethylene, polypropylene, poly(4-methylbutene), polyacrylamide, polymethyl methacrylate, polyethylene terephthalate, nylon, polyvinyl butyrate, or polyacrylate; preferably selected from polystyrene, polypropylene, poly(4-methylbutene), polyacrylamide, polymethyl methacrylate, polyethylene terephthalate, or polyacrylate.

[0103] In some embodiments of the present invention, the first carrier is polystyrene latex microspheres.

[0104] In other embodiments of the present invention, the sensitizer is a photoactivated photosensitizer and / or a chemically activated initiator, preferably a photoactivated photosensitizer.

[0105] In some embodiments of the present invention, the sensitizer is selected from methylene blue, rose red, porphyrin, phthalocyanine and chlorophyll.

[0106] In other embodiments of the present invention, the specific binding pair members are selected from a pair of substances capable of specifically binding to each other, consisting of an antibody, antibody fragment, ligand, oligonucleotide, oligonucleotide-binding protein, lectin, hapten, antigen, immunoglobulin-binding protein, avidin, or biotin.

[0107] In some embodiments of the present invention, the specific binding pairing member is avidin-biotin.

[0108] In other embodiments of the invention, the avidin is selected from egg avidin, streptavidin, egg yolk avidin, neutral avidin, and avidin-like substances, preferably neutral avidin and / or streptavidin.

[0109] In some embodiments of the present invention, the avidin is chemically bonded to the surface of the first carrier by reacting an amino group with an aldehyde group on the surface of the first carrier to form a Schiff base.

[0110] In some embodiments of the present invention, the coefficient of variation (CV) of the particle size distribution of the donor particles in the donor reagent is controlled to be ≥5%.

[0111] In other embodiments of the present invention, the coefficient of variation (CV) of the particle size distribution of the donor particles in the donor reagent is controlled to be ≥8%; preferably, the coefficient of variation (CV) of the particle size distribution of the donor particles in the donor reagent is controlled to be ≥10%.

[0112] In some embodiments of the present invention, the coefficient of variation (CV) of the particle size distribution of the donor particles in the donor reagent is controlled to be ≤40%; more preferably, the coefficient of variation (CV) of the particle size distribution of the donor particles in the donor reagent is controlled to be ≤20%.

[0113] It is worth noting that the coefficient of variation (CV) of the donor particle size distribution mentioned in this invention refers to the coefficient of variation (CV) of the particle size distribution after the donor particle is coated with the desired material.

[0114] In some specific embodiments of the present invention, the coefficient of variation (CV) of the donor particle size distribution in the acceptor reagent can be 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 25%, 30%, 35%, or 40%, etc.

[0115] In other embodiments of the invention, the donor particles exhibit polydispersity in the particle size distribution of the donor reagent.

[0116] In some embodiments of the present invention, the concentration of the donor particles in the donor reagent is 10 μg / ml to 1 mg / ml, preferably 20 μg / ml to 500 μg / ml, and more preferably 50 μg / ml to 200 μg / ml.

[0117] In some other embodiments of the present invention, the donor reagent further includes a buffer solution with a pH value of 7.0 to 9.0, and the donor particles are suspended in the buffer solution.

[0118] In some embodiments of the present invention, the buffer solution contains a polysaccharide selected from carbohydrates containing three or more unmodified or modified monosaccharide units, preferably selected from dextran, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxyglucan and aminoglucan; more preferably selected from dextran, starch, glycogen and polyribose.

[0119] In other embodiments of the present invention, the molecular weight distribution Mw of the dextran is selected from 10,000 to 1,000,000 kDa, preferably from 100,000 to 800,000 kDa, and more preferably from 300,000 to 700,000 kDa.

[0120] In some embodiments of the present invention, the content of dextran in the buffer solution is 0.01~1wt%, preferably 0.05~0.5wt%.

[0121] In some embodiments of the present invention, the coefficient of variation (CV) of the particle size distribution of the receptor particles in the receptor reagent is ≥5%.

[0122] In some embodiments of the present invention, the coefficient of variation (CV) of the particle size distribution of the receptor particles in the receptor reagent is ≥8%; preferably, the coefficient of variation (CV) of the particle size distribution of the receptor particles in the receptor reagent is ≥10%.

[0123] In other embodiments of the present invention, the coefficient of variation (CV) of the particle size distribution of the receptor particles in the receptor reagent is ≤40%; more preferably, the coefficient of variation (CV) of the particle size distribution of the receptor particles in the receptor reagent is ≤20%.

[0124] It is worth noting that the coefficient of variation (CV) of the receptor particle size distribution mentioned in this invention refers to the coefficient of variation (CV) of the particle size distribution after the receptor particle is coated with the desired substance.

[0125] In some specific embodiments of the present invention, the coefficient of variation (CV) of the particle size distribution of the receptor particles in the receptor reagent can be 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 25%, 30%, 35%, or 40%, etc.

[0126] In some embodiments of the present invention, the particle size distribution of the receptor particles in the receptor reagent exhibits polydispersity.

[0127] In some specific embodiments of the present invention, the particle size distribution variation coefficient (CV) value is calculated using the Gaussian distribution.

[0128] In some other embodiments of the present invention, the Gaussian distribution analysis method is used, and the Gaussian distribution curve of the receptor particles in the receptor reagent shows two or more peaks.

[0129] In some embodiments of the present invention, the receptor reagent comprises receptor particles with at least two average particle size distributions.

[0130] In other embodiments of the present invention, the receptor particles in the receptor reagent include a second carrier, the interior of which is filled with a luminescent composition, and the surface of the second carrier is coated with at least one polysaccharide layer, the surface of which is connected to a reporter molecule that is capable of specifically binding to the target molecule to be tested.

[0131] In some embodiments of the present invention, the surface of the carrier is coated with a coating of at least two consecutive polysaccharide layers, wherein the first polysaccharide layer and the second polysaccharide layer are spontaneously associated.

[0132] In other embodiments of the invention, each of the continuous polysaccharide layers is spontaneously associated with each of the preceding polysaccharide layers.

[0133] In some specific embodiments of the present invention, the polysaccharide has side group functional groups, and the functional groups of the continuous polysaccharide layer carry opposite charges to the functional groups of the preceding polysaccharide layer.

[0134] In some other embodiments of the invention, the polysaccharide has side functional groups, and the continuous polysaccharide layer is covalently linked to the preceding polysaccharide layer through a reaction between the functional groups and the functional groups of the preceding polysaccharide layer.

[0135] In some embodiments of the invention, the functional groups of the continuous polysaccharide layer alternate between amine functional groups and amine reactive functional groups.

[0136] In other embodiments of the invention, the amine reactive functional group is an aldehyde group or a carboxyl group.

[0137] In some embodiments of the present invention, the first polysaccharide layer is spontaneously associated with the carrier.

[0138] In other embodiments of the invention, the outermost polysaccharide layer of the coating has at least one side functional group.

[0139] 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, mercapto, amino, hydroxyl and maleamine groups; preferably selected from aldehyde and / or carboxyl groups.

[0140] In other embodiments of the invention, the side functional groups of the outermost polysaccharide layer of the coating are directly or indirectly connected to a reporter molecule, which is capable of specifically binding to the target molecule to be tested.

[0141] In some embodiments of the present invention, the side group functional groups of the outermost polysaccharide layer of the coating bind directly or indirectly to one of the specific binding pair members.

[0142] In some embodiments of the present invention, the luminescent composition comprises a chemiluminescent compound and a metal chelate.

[0143] In other embodiments of the present invention, the chemiluminescent compound is selected from olefin compounds, preferably from dimethylthiophene, dibutyl dione compounds, dioxane, enol ethers, enamines, 9-alkylene oxalane, 9-alkylene-N-9,10 dihydroacrylidine, aryl ethyl ether olefins, aryl imidazoles and glossin and their derivatives, more preferably from dimethylthiophene and its derivatives.

[0144] In some embodiments of the present invention, the metal chelate is a rare earth metal or a Group VIII metal, preferably selected from europium, terbium, dysprosium, samarium, osmium, and ruthenium, and more preferably europium.

[0145] In other embodiments of the invention, the metal chelate comprises a chelating agent selected from the following: 4'-(10-methyl-9-anthrayl)-2,2':6'2”-bitripyridine-6,6”-dimethylamine]tetraacetic acid (MTTA), 2-(1',1',2',2',3',3'-heptafluoro-4',6'-hexanedione-6'-yl)-naphthalene (NHA), 4,4'-bis(2”,3”,3”-heptafluoro-4”,6”-hexanedione-6”-yl)-o-terphenyl (BHHT), 4,4'-bis(1”,1”,1”,2”,2”,3”,3”,3”)-o-terphenyl (BHHT), 4,4'-bis(1”,1”,1”,2”,2”,3”,3”)-terphenyl (BHHT). -Hepheptafluoro-4”,6”-hexanedione-6”-yl)-chlorosulfonyl-o-terphenyl (BHHCT), 4,7-biphenyl-1,10-phenanthroline (DPP), 1,1,1-trifluoroacetone (TTA), 3-naphthyl-1,1,1-trifluoroacetone (NPPTA), naphthyltrifluorobutylene dione (NTA), trioctylphosphine oxide (TOPO), triphenylphosphine oxide (TPPO), 3-benzoyl-1,1,1-trifluoroacetone (BFTA), 2,2-dimethyl-4-perfluorobutyryl-3-butanone (fod), 2,2'-bipyridine (bpy), bipyridylcarboxylic acid, azacrown ethers, azacavitary ligands and trioctylphosphine oxide and their derivatives.

[0146] To further improve the accuracy of the final detection results and the stability of the sample to be tested, in some specific embodiments of the present invention, the body fluid from the host is diluted with a diluent before being brought into contact with the recipient reagent and the donor reagent.

[0147] In some embodiments of the present invention, the detection wavelength of the chemiluminescence is 520~620nm; preferably 610~620nm, more preferably 615nm.

[0148] In some other embodiments of the present invention, laser irradiation is performed using red excitation light of 600-700 nm; preferably, laser irradiation is performed using red excitation light of 640-680 nm; more preferably, laser irradiation is performed using red excitation light of 660 nm.

[0149] In some embodiments of the present invention, the concentration of the receptor particles in the receptor reagent is 1 ug / mL to 1000 ug / mL; preferably 10 ug / mL to 500 ug / mL; more preferably 20 ug / mL to 200 ug / mL.

[0150] In other embodiments of the present invention, the active oxygen is singlet oxygen.

[0151] In other embodiments of the invention, the bodily fluids from the subject include, but are not limited to: blood, serum, plasma, sputum, lymph, semen, vaginal mucus, feces, urine, or cerebrospinal fluid.

[0152] III. Examples Example 1: Preparation of donor particles and donor reagents with uncoated or polysaccharide-linked surfaces. (I) Preparation of aldehyde-based polystyrene latex microspheres 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.

[0153] 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.

[0154] c) Heat the reaction system to 70°C and react for 15 hours.

[0155] d) Cool the emulsion after the reaction is complete to room temperature and filter it with a suitable filter cloth. Wash the resulting emulsion with deionized water by centrifugation 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.

[0156] e) The Gaussian average particle size of the latex microspheres, measured by a nanoparticle size analyzer, is 201.3 nm, with a coefficient of variation (CV) of 8.0%. The Gaussian distribution diagram is shown below. Figure 1 As shown, the Nicomp distribution is multi-peaked (e.g. Figure 2 (As shown). The aldehyde content of the latex microspheres was determined to be 260 nmol / mg by conductivity titration.

[0157] (ii) Filling of sensitizers 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.

[0158] b) Prepare a 100ml three-necked flask, add 10ml of 95% ethanol, 10ml of water and 10ml of aldehyde-based polystyrene latex microspheres with a concentration of 10% (i), stir magnetically, and heat in a water bath to 70℃.

[0159] 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.

[0160] 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.

[0161] (III) Modification of microspheres with avidin to prepare donor reagents a) Microsphere suspension treatment: Take a certain amount of the microspheres prepared in step (II) 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.

[0162] b) Preparation of streptavidin solution: Weigh a certain amount of streptavidin and dissolve it in MES buffer to 8 mg / ml.

[0163] 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.

[0164] 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.

[0165] e) Blocking: Prepare 75 mg / ml Gly solution and 25 mg / ml NaBH3CN solution using MES buffer. Add these solutions to the reaction solution at a volume ratio of 2:1:10, 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 rapidly, and incubate at 37°C with rotation for 16 hours.

[0166] 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 repeat this washing process 3 times. Finally, suspend the solution with a small amount of donor particle buffer, determine the solid content, and adjust the concentration to 150 μg / ml with donor particle buffer to obtain the donor reagent containing donor particles.

[0167] g) The average Gaussian distribution particle size of the donor particles, measured by a nanoparticle size analyzer, was 227.7 nm, with a coefficient of variation (CV) of 6.5%. (Details are as follows...) Figure 3 As shown.

[0168] Example 2: Preparation of polysaccharide-coated donor particles and donor reagents The preparation of aldehyde-based polystyrene latex microspheres and the filling process of the sensitizer are the same as the preparation steps in (a) and (b) of Example 1.

[0169] (I) Preparation of aminoglucan a) Place a 500mL four-necked flask in an oil bath, attach the condenser, and purge with nitrogen.

[0170] b) Add 10g of dextran with an average molecular weight distribution of 500,000 kDa, 100ml of deionized water, 2g of NaOH, and 10g of N-(2,3-epoxypropyl)phthalimide in sequence, and stir mechanically.

[0171] c) After 2 hours in a 90℃ oil bath, turn off the heating and continue stirring while allowing the mixture to cool naturally.

[0172] d) The main mixture precipitates out in 2L of methanol from the reaction mixture, the solid is collected and dried.

[0173] e) Place a 200mL four-necked flask in an oil bath, attach the condenser, and purge with nitrogen.

[0174] f) Add the dried solid, 100 mL of deionized water, 1.8 g of sodium acetate, and 5 mL of 50% hydrazine hydrate in sequence, then adjust the pH to 4 and stir mechanically.

[0175] g) After 1 hour in an oil bath at 85℃, turn off the heating and allow the mixture to cool naturally while stirring.

[0176] h) After adjusting the pH of the reaction solution to neutral, filter it and collect the filtrate.

[0177] i) Place the filtrate in a dialysis bag and dialyze with deionized water at 4°C for 2 days, changing the water 3-4 times a day.

[0178] j) After dialysis, freeze-dry to obtain 9.0 g of aminoglucan solid.

[0179] k) The amino content was determined to be 0.83 mmol / g by the TNBSA method.

[0180] (II) Preparation of aldehyde dextran a) Weigh 10g of dextran with an average molecular weight distribution of 500,000 kDa and place it in a 250°C beaker. Add 100mL of 0.1M / pH=6.0 phosphate buffer and stir to dissolve at room temperature.

[0181] b) Weigh 1.8g of sodium periodate into a 50mL beaker, add 10mL of 0.1M / pH=6.0 phosphate buffer, and stir to dissolve at room temperature.

[0182] c) Slowly add sodium periodate solution to dextran solution, and continue stirring for 1 hour after the reaction stops and no more bubbles are generated.

[0183] d) Place the reaction mixture in a dialysis bag and dialyze with deionized water at 4°C for 2 days, changing the water 3-4 times a day.

[0184] e) After dialysis, freeze-dry to obtain 9.6g of aldehyde dextran solid.

[0185] f) The aldehyde content was measured to be 0.94 mmol / g using the BCA Kit.

[0186] (III) Microspheres coated with dextran a) Take 50 mg of aminoglucan solid into a 20 mL round-bottom flask, add 5 mL of 50 mM / pH=10 carbonate buffer, and stir to dissolve at 30 °C in the dark.

[0187] b) Take 100 mg of donor particles and add them to the aminoglucan solution and stir for 2 hours.

[0188] c) Dissolve 10 mg of sodium borohydride in 0.5 mL of 50 mM / pH=10 carbonate buffer and add it dropwise to the above reaction solution. React overnight at 30°C in the dark.

[0189] d) After centrifuging the reaction mixture at 30,000 g, discard the supernatant and add 50 mM / pH=10 carbonate buffer for ultrasonic dispersion. Repeat centrifugation and washing three times, then bring the volume up to 20 mg / ml with 50 mM / pH=10 carbonate buffer.

[0190] e) Take 100 mg of aldehyde dextran solid into a 20 mL round-bottom flask, add 5 mL of 50 mM / pH=10 carbonate buffer solution, and stir to dissolve at 30 °C in the dark.

[0191] f) Add the above particles to the aldehyde dextran solution and stir for 2 hours.

[0192] g) Dissolve 15 mg of sodium borohydride in 0.5 mL of 50 mM / pH=10 carbonate buffer and add it dropwise to the above reaction solution. React overnight at 30°C in the dark.

[0193] h) After centrifuging the reaction mixture at 30,000 g, discard the supernatant and add 50 mM / pH=10 carbonate buffer for ultrasonic dispersion. Repeat centrifugation and washing three times, then bring the volume up to 20 mg / ml with 50 mM / pH=10 carbonate buffer.

[0194] i) The Gaussian average particle size of the microspheres, measured by a nanoparticle size analyzer, is 235.6 nm, with a coefficient of variation (CV) of 8.1%. (Details are as follows...) Figure 4 As shown.

[0195] (iv) Modification of microspheres with avidin to prepare donor reagents h) Microsphere suspension treatment: Take a certain amount of the microspheres prepared in step (iii) and centrifuge them in a high-speed refrigerated centrifuge. Discard the supernatant, add a certain amount of MES buffer, and sonicate the microspheres on an ultrasonic cell disruptor until they are resuspended. Add MES buffer to adjust the donor particle concentration to 100 mg / ml.

[0196] i) Preparation of avidin solution: Weigh a certain amount of neutral avidin and dissolve it in MES buffer to 8 mg / ml.

[0197] j) 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.

[0198] k) 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 rapidly. Incubate at 37°C with the reaction mixture for 48 hours.

[0199] 1) Blocking: Prepare 75 mg / ml Gly solution and 25 mg / ml NaBH3CN solution using MES buffer. Add these solutions to the reaction solution at a volume ratio of 2:1:10, mix well, and incubate at 37°C for 2 hours. Then add 200 mg / ml BSA solution (MES buffer) at a volume ratio of 5:8, mix quickly, and incubate at 37°C for 16 hours.

[0200] m) 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 repeat this washing process 3 times. Finally, suspend the solution with a small amount of donor particle buffer, determine the solid content, and adjust the concentration to 150 μg / ml with donor particle buffer to obtain the donor reagent containing donor particles.

[0201] n) The average Gaussian distribution particle size of the donor particles, measured by a nanoparticle size analyzer, was 249.9 nm, with a coefficient of variation (CV) of 11.6%. (Details are as follows...) Figure 5 As shown.

[0202] Example 3: Preparation of receptor particles 1. Preparation and characterization process of aldehyde-based polystyrene latex microspheres 1) Prepare a 100ml three-necked flask, add 40mmol styrene, 5mmol acrolein and 10ml water, stir for 10min and then purge with N2 for 30min. 2) Weigh 0.11g of ammonium 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; 3) Heat the reaction system to 70℃ and react for 15 hours; 4) Cool the emulsion after the reaction is complete to room temperature and filter it with a suitable filter cloth. Wash the resulting emulsion with deionized water by centrifugation 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. 5) The Gaussian average particle size of the latex microspheres, measured by a nanoparticle size analyzer, was 202.2 nm, with a coefficient of variation (CV) of 4.60%. The Gaussian distribution curve is shown below. Figure 6 As shown, the aldehyde content of the latex microspheres was determined to be 280 nmol / mg by conductivity titration.

[0203] 2. Process and characterization of embedding luminescent compositions inside microspheres 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; 2) Prepare a 100ml three-necked flask, add 10ml of 95% ethanol, 10ml of water and 10ml of 10% aldehyde polystyrene latex microspheres obtained in step 1, stir magnetically and heat in a water bath to 70℃. 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; 4) Centrifuge the above emulsion for 1 hour at 30,000 g. After centrifugation, discard the supernatant to obtain aldehyde polystyrene microspheres filled with the luminescent composition.

[0204] 5) The Gaussian average particle size of the microspheres, as measured by a nanoparticle size analyzer, is 204.9 nm, with a coefficient of variation (CV) of 5.00%. Figure 7 (As shown) 3. Process and characterization of coating polysaccharide onto microsphere surface 1) Take 50 mg of aminoglucan solid into a 20 mL round bottom flask, add 5 mL of 50 mM / pH=10 carbonate buffer, and stir to dissolve at 30 °C in the dark; 2) Take 100 mg of the aldehyde polystyrene microspheres filled with the luminescent composition prepared in step 2, add them to the aminodextran solution and stir for 2 hours; 3) Dissolve 10 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. 4) After centrifuging the reaction mixture at 30,000g, 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. 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, and stir to dissolve at 30 °C in the dark. 6) Add the above microspheres to the aldehyde dextran solution and stir for 2 hours; 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. 8) After centrifuging the reaction mixture at 30,000 g, discard the supernatant and add 50 mM / pH=10 carbonate buffer for ultrasonic dispersion. Repeat centrifugation and washing three times, then bring the volume up to 20 mg / ml with 50 mM / pH=10 carbonate buffer.

[0205] 9) The Gaussian average particle size of the microspheres, as measured by a nanoparticle size analyzer, is 241.6 nm, with a coefficient of variation (CV) of 12.90%. Figure 8 (As shown).

[0206] 4. The conjugation process of troponin I antibody 1) Dialyze the paired troponin I antibody to 50mM CB buffer at pH=10 and measure the concentration as 1mg / ml.

[0207] 2) Add 0.5 ml of the microspheres obtained in step 3 and 0.5 ml of the paired antibody I obtained in step 1) to a 2 ml centrifuge tube, mix well, and then add 100 μl of 10 mg / ml NaBH4 solution (50 mM CB buffer). Incubate at 2-8 °C for 4 hours.

[0208] 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.

[0209] 4) After the reaction is complete, centrifuge for 45 min at 30000G. After centrifugation, discard the supernatant and resuspend the sample in 50 mM MES buffer. Repeat the centrifugation and washing four times, and dilute with buffer to a final concentration of 50 μg / ml to obtain the receptor particle solution of conjugated antibody I.

[0210] 5) The Gaussian average particle size distribution of the acceptor particles, measured by a nanoparticle size analyzer, was 253.1 nm, with a coefficient of variation (CV) of 9.54% (e.g., ...). Figure 9 (As shown).

[0211] Example 4: Preparation of a donor reagent comprising the following series of donor particles using the method described in Example 1. Donor reagent 1: The average particle size of the donor particles in the Gaussian distribution curve is 226.5 nm, and the coefficient of variation (CV) for particle size distribution is 3.8; the Nicomp distribution is unimodal.

[0212] Donor reagent 2: The average particle size of the donor particles in the Gaussian distribution curve is 225.3 nm, and the coefficient of variation (CV) for particle size distribution is 4.6; the Nicomp distribution is unimodal.

[0213] Donor reagent 3: The average particle size of the donor particles in the Gaussian distribution curve is 225.2 nm, and the particle size distribution variation coefficient CV value is 5.0; the Nicomp distribution is unimodal.

[0214] Donor reagent 4: The average particle size of the donor particles in the Gaussian distribution curve is 226.7 nm, and the particle size distribution variation coefficient CV value is 8.1; the Nicomp distribution is unimodal.

[0215] Donor reagent 5: The average particle size of the donor particles in the Gaussian distribution curve is 227.8 nm, and the coefficient of variation (CV) for particle size distribution is 15.6; the Nicomp distribution is unimodal.

[0216] Donor reagent 6: The average particle size of the donor particles in the Gaussian distribution curve is 225.9 nm, and the coefficient of variation (CV) for particle size distribution is 26.1; the Nicomp distribution is unimodal.

[0217] Donor reagent 7: The average particle size of the donor particles in the Gaussian distribution curve is 225.1 nm, and the particle size distribution variation coefficient CV value is 32.4; the Nicomp distribution is unimodal.

[0218] Example 5: A photo-induced chemiluminescence immunoassay analyzer The principle of the photo-induced chemiluminescence immunoassay analyzer described in this embodiment is as follows: The target molecule in the sample suspected of containing the cTnI marker reacts with donor and acceptor particles to form an immune complex. This interaction brings the donor and acceptor particles closer together. Under laser irradiation (wavelength 680 nm), the sensitizer in the donor particle converts oxygen in the surrounding environment into more reactive singlet oxygen. This singlet oxygen diffuses to the acceptor particle and reacts with the chemiluminescent agent in the acceptor particle, further activating the luminescent group also on the acceptor particle, causing it to emit light at a wavelength of 520-620 nm. The half-life of singlet oxygen is 4 μSec, and its diffusion distance in solution is approximately 200 nm. If there is no interaction between biomolecules, singlet oxygen cannot diffuse to the acceptor particle, and no light signal will be generated. Therefore, by measuring the light intensity emitted by the mixture, the concentration of the target molecule in the sample can be calculated. The donor particle includes a first carrier, the interior of which is filled with a sensitizer, and the surface of the first carrier is chemically bonded to specifically bind one of the paired members.

[0219] A preferred structure of the photo-induced chemiluminescence immunoassay analyzer described in this embodiment includes the following components: A reagent addition module is used to add the test sample and / or acceptor reagent and donor reagent to the reaction vessel; wherein the donor reagent includes donor particles, and the coefficient of variation (CV) of the particle size distribution of the donor particles in the donor reagent is ≥5%; An incubation module is used to provide a suitable temperature environment for the homogeneous chemiluminescence reaction in the reaction vessel; the incubation module can be a metal bath, water bath, or oil bath, etc. The detection module includes a laser exciter and a photon detector (PMT) for detecting the chemiluminescence signal generated by the homogeneous chemiluminescence reaction.

[0220] The reagent dispensing module, incubation module, and detection module are all electrically connected to the circuit control module. Under the control of the circuit control module, the incubation module is used to adjust the temperature of the immune reaction substance, the reagent dispensing module is used to transfer the substance in the reaction vessel, and the detection module is used to emit a laser and measure the light intensity emitted by the sample to be tested.

[0221] Example 6: Detection Results and Analysis (Detection Substance: Troponin I) (1) Using the analyzer in Example 5, the donor reagents from Examples 1 and 2 were simultaneously tested with the receptor reagent from Example 3 to detect troponin I. The results are shown in Table 1. The cTnI quantitative assay kit (photocatalytic chemiluminescence method) used in this example consists of reagent 1 (R1') containing receptor particles coated with a first anti-troponin I antibody, reagent 2 (R2') containing a biotin-labeled second anti-troponin I antibody, and a universal solution (R3') containing donor particles. R1' is the receptor reagent prepared using the receptor particles from Example 3; R3' is the donor reagent prepared using the donor particles from Examples 1 and 2.

[0222] Table 1

[0223] As shown in Table 1, the analytical method provided in this application exhibits excellent sensitivity and detection limit. Furthermore, the analytical method using the donor reagent in Example 1 demonstrates superior sensitivity and detection limit compared to the method using the donor reagent in Example 2. This indicates that the use of donor particles without polysaccharide coating results in superior performance.

[0224] (2) The results of simultaneous instrumental testing of the donor reagent in Example 4 and the recipient reagent in Example 3. The troponin I quantitative assay kit (photocatalytic chemiluminescence assay) used in this example consists of reagent 1 (R1') containing receptor particles coated with a first anti-cTnI antibody, reagent 2 (R2') containing a biotin-labeled second anti-cTnI antibody, and a universal solution (R3') containing donor particles. R1' is the receptor reagent prepared using the receptor particles from Example 3; R3' is a series of donor reagents prepared using the reagents from Example 4.

[0225] The detection process was completed and the results were output using a fully automated photocatalytic chemiluminescence analysis system (LiCA HT) developed by Boyang Biotechnology (Shanghai) Co., Ltd. The specific experimental steps are as follows: 1. Add the well-mixed sample, the prepared R1' and R2' to an 8×12 white plate respectively; 2. Place the prepared white plate into the LiCA HT instrument for reaction, using the following reaction mode; (1) Mix 40 μL of sample, 15 μL of R1' and 15 μL of R2'. (2) Incubate at 37℃ for 8 minutes; (3) Add 160 μL of R3'; (4) Incubate at 37℃ for 2 minutes; (5) Excitation reading, the specific test results are shown in Table 2 below.

[0226] Table 2

[0227] As shown in Table 2, when the coefficient of variation of the particle size distribution of the donor particles is greater than or equal to 5%, the detection using the donor reagent containing the donor particles has both suitable sensitivity and a wide detection range.

[0228] Example 7: Clinical detection of cTnI in normal individuals and patients suspected of having myocardial injury This embodiment tested 40 clinical samples (13 negative and 27 positive). The cTnI quantitative assay kit (photochemiluminescence method) used included: reagent 1 (R1) containing receptor particles coated with a primary anti-cTnI monoclonal antibody, reagent 2 (R2) containing a biotin-labeled secondary anti-cTnI monoclonal antibody, and a universal solution (R3) containing donor particles for the photochemiluminescence analysis system. The concentration of donor particles in reagent R3 was 100 μg / ml, and the coefficient of variation (CV) of the donor particle size distribution in reagent R3 was 11%.

[0229] 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: a. Add the clinical sample to the reaction well; b. Add R1 and R2 sequentially into the reaction well; c. Warmth and nurturing; d. Add R3 to the reaction well; e. nurturing; f. Irradiate the reaction hole twice with lasers and calculate the amount of photons emitted from each hole; g. Calculate the cTnI concentration in the sample to be tested.

[0230] When cTnI markers are present in clinical samples, cTnI specifically binds to both receptor particles coated with a primary anti-cTnI monoclonal antibody and a biotin-labeled secondary anti-cTnI monoclonal antibody, forming a double-antibody sandwich complex on the receptor particle surface. 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 reacts with the receptor particles in solution to produce chemiluminescence. This further excites the fluorophores on the same particle, resulting in a cascade amplification reaction and fluorescence. The higher the concentration of cTnI markers, the stronger the fluorescence intensity. The amount of cTnI in the patient's serum is quantitatively detected based on the intensity of the fluorescence. Specific detection results are shown in Table 3 below. Table 3

[0231] Data comparison showed that the Abbott test value had a correlation of 0.9973 with the test value mentioned in Example 7, with a slope of 1.0495. Samples 1-13 were normal physical examination patients, with a range of 1.77 pg / ml to 25.3 pg / ml and a median of 6.77 pg / ml; samples 14-40 were patients identified with myocardial injury, with a range of 30.94 pg / ml to 29896.88 pg / ml and a median of 450.54 pg / ml.

[0232] Cardiac troponin I (cTnI) concentrations are low in the serum or plasma of healthy individuals. After a patient experiences chest pain, necrotic cardiomyocytes release large amounts of cTnI into the bloodstream, reaching peak levels 12-48 hours later. In patients with severe myocardial infarction, cTnI levels remain high even several days later, making it the optimal biomarker for diagnosing myocardial injury and 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 kits for in vitro diagnostic methods for determining whether a subject has myocardial injury. The quantitative results of measuring cTnI biomarkers in the body fluids of a subject using the donor reagent and corresponding methods described in this invention can be used to diagnose myocardial injury and myocardial infarction.

[0233] Example 8: Clinical detection of CKMB in normal individuals and patients suspected of having myocardial injury This embodiment tested 40 clinical samples. The CKMB quantitative assay kit (photocatalytic chemiluminescence method) used included: reagent 1 (R1') containing receptor microparticles coated with a primary anti-CKMB antibody, reagent 2 (R2') containing a biotin-labeled secondary anti-CKMB antibody, and a universal solution (R3') containing donor particles. The concentration of donor particles in reagent R3 was 100 μg / ml, and the coefficient of variation (CV) of the donor particle size distribution in reagent R3 was 6.5%.

[0234] The specific experimental steps are as follows: 1. Select 40 clinical samples, equilibrate to room temperature, and mix thoroughly; 2. Add the mixed sample, the prepared R1' and R2' to an 8×12 white plate respectively; 3. Place the white plate with the added sample into the LiCA HT instrument for reaction, using the following reaction mode; (1) Mix 40 μL of sample, 15 μL of R1' and 15 μL of R2'. (2) Incubate at 37℃ for 8 minutes; (3) Add 160ul of general-purpose liquid (R3'); (4) Incubate at 37℃ for 2 minutes; (5) Multiple excitation readings were performed, and the specific test results are shown in Table 4 below.

[0235] Table 4

[0236] The correlation between Roche's and Boyang's measurements was 0.9877, with a slope of 0.9192. Samples 1-13 were normal physical examination patients, ranging from 0.23 ng / ml to 3.88 ng / ml, with a median of 1.27 ng / ml; samples 14-40 were patients identified with myocardial injury, ranging from 6.53 ng / ml to 150.90 ng / ml, with a median of 41.88 ng / ml.

[0237] Creatine kinase isoenzyme (CK-MB) is one of the three criteria recommended by the WHO for diagnosing myocardial infarction. CK-MB is one of the three dimeric isoenzymes of creatine kinase, and it is present in a high proportion in the myocardium. It enters the bloodstream when myocardial cells die. In patients with myocardial infarction, its concentration increases 3-4 hours after the onset of the disease, peaks at 18-24 hours, and returns to normal within 72 hours. Data from Embodiment 8 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 the CK-MB marker in the body fluids of a subject using the donor reagent and corresponding method described in this invention can be used to diagnose myocardial injury and myocardial infarction.

[0238] Example 9: Clinical detection of MYO in normal individuals and patients suspected of having myocardial injury This embodiment tested 40 clinical samples. The MYO quantitative assay kit (photocatalytic chemiluminescence method) used included: reagent 1 (R1') containing receptor microparticles coated with a first anti-MYO antibody, reagent 2 (R2') containing a biotin-labeled second anti-MYO antibody, and a universal solution (R3') containing donor particles. The concentration of donor particles in reagent R3 was 120 μg / ml, and the coefficient of variation (CV) of the donor particle size distribution in reagent R3 was 13.5%.

[0239] The specific experimental steps are as follows: 1. Select 40 clinical samples, equilibrate to room temperature, and mix thoroughly; 2. Add the mixed sample, the prepared R1' and R2' to an 8×12 white plate respectively; 3. Place the white plate with the added sample into the LiCA HT instrument for reaction, using the following reaction mode; (1) Mix 40 μL of sample, 15 μL of R1' and 15 μL of R2'. (2) Incubate at 37℃ for 8 minutes; (3) Add 160ul of general-purpose liquid (R3'); (4) Incubate at 37℃ for 2 minutes; (5) Excitation reading, the specific test results are shown in Table 5 below.

[0240] Table 5

[0241] The correlation between Roche's and Boyang's measurements was 0.994, with a slope of 0.954. Samples 1-13 were normal physical examination patients, ranging from 23.36 ng / ml to 84.08 ng / ml, with a median of 38.93 ng / ml; samples 14-40 were patients identified with myocardial injury, ranging from 81.3 ng / ml to 1495.59 ng / ml, with a median of 382.48 ng / ml.

[0242] Myoglobin (MYO) is present in cardiac and skeletal muscle. Due to its small size, it rapidly diffuses into the bloodstream when cardiac cells die, and its concentration can increase within 1-2 hours, making it an early indicator of acute myocardial infarction. In patients with myocardial infarction, MYO concentration can return to normal within 24 hours after the onset of the disease; therefore, MYO can be used to diagnose recurrent myocardial infarction. Data from Embodiment 9 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 the MYO marker in the body fluids of a subject using the donor reagent and corresponding method described in this invention can be used to diagnose myocardial injury and myocardial infarction.

[0243] 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. Use of a donor reagent in a kit for preparing a method for in vitro diagnostic testing of whether a subject has myocardial injury, wherein the method comprises: The body fluid from the host is brought into contact with the receptor reagent and the donor reagent, and the reaction produces the test mixture; The test mixture is excited at least once with excitation light, and the intensity of the chemiluminescence signal generated therefrom is detected; the concentration of at least one myocardial marker in the body fluid is quantitatively calculated based on the intensity of the chemiluminescence signal, thereby determining whether the subject has myocardial injury. The donor reagent comprises donor particles capable of generating reactive oxygen species in an excited state, the donor particles being suspended in a buffer solution containing 0.01-1 wt% dextran. The donor particles include a first carrier, which is a polystyrene latex microsphere with a particle size selected from 100 to 400 nm. The first carrier is filled with a sensitizer, and its surface is not coated or connected to a polysaccharide substance, which directly chemically binds to one of the paired members. The coefficient of variation (CV) of the particle size distribution of the donor particles in the donor reagent is controlled to be ≥5%, and the coefficient of variation (CV) of the particle size distribution of the donor particles in the donor reagent is controlled to be ≤40%. The cardiac markers are selected from one or more of troponin T, troponin I, troponin kinase isoenzyme, myoglobin, interleukin-6, and lactate dehydrogenase.

2. The use according to claim 1, characterized in that, The surface of the first carrier has bonding functional groups, which are used to chemically bond one of the specific binding pair members to the surface of the first carrier.

3. The use according to claim 2, characterized in that, The bonding functional groups are selected from amino, amide, hydroxy, aldehyde, carboxyl, maleimide and thiol groups.

4. The use according to claim 3, characterized in that, The bonding functional groups are selected from aldehyde and / or carboxyl groups.

5. The use according to claim 2, characterized in that, The content of bonded functional groups on the surface of the first carrier is 100~500 nmol / mg.

6. The use according to claim 5, characterized in that, The content of bonded functional groups on the surface of the first carrier is 200~400 nmol / mg.

7. The use according to any one of claims 1 to 6, characterized in that, The particle size of the first carrier is 150~350nm.

8. The use according to claim 7, characterized in that, The particle size of the first carrier is 180~220nm.

9. The use according to any one of claims 1 to 6, characterized in that, The coefficient of variation (CV) of the particle size distribution of the donor particles in the donor reagent is controlled to be ≥8%.

10. The use according to claim 9, characterized in that, The coefficient of variation (CV) of the particle size distribution of the donor particles in the donor reagent is controlled to be ≥10%.

11. The use according to any one of claims 1 to 6, characterized in that, The coefficient of variation (CV) of the particle size distribution of the donor particles in the donor reagent is controlled to be ≤20%.

12. The use according to any one of claims 1 to 6, characterized in that, In the donor reagent, the pH value of the buffer solution is 7.0~9.

0.

13. The use according to any one of claims 1 to 6, characterized in that, The molecular weight distribution Mw of the dextran is selected from 10,000 to 1,000,000 kDa.

14. The use according to claim 13, characterized in that, The molecular weight distribution Mw of the dextran is selected from 100,000 to 800,000 kDa.

15. The use according to claim 14, characterized in that, The molecular weight distribution Mw of the dextran is selected from 300,000 to 700,000 kDa.

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