Use of a receptor reagent in diagnosing myocardial injury in a subject

Abstract

The present application relates to a kind of acceptor reagent in the preparation for the use of kit for the method for diagnosing whether subject is suffered from myocardial injury in vitro, wherein the method comprises: the body fluid from subject is contacted with acceptor reagent and donor reagent, and generates the mixture to be measured after reaction;The intensity of the chemiluminescence signal generated thereby is detected by exciting the mixture to be measured at least once with excitation light;The concentration of at least one myocardial marker in the body fluid is quantitatively calculated according to the intensity of the chemiluminescence signal, to determine whether subject is suffered from myocardial injury;Wherein, the acceptor reagent comprises acceptor particles capable of generating chemiluminescence by reacting with active oxygen, and the coefficient of variation C.V of the particle size distribution of the acceptor particles in acceptor reagent is controlled to be greater than or equal to 5%; The donor reagent comprises donor particles capable of generating active oxygen in the excited state.The method has both ultra-high sensitivity and very wide detection range.

Description

Technical Field

[0001] The present invention belongs to the field of chemiluminescence detection, and in particular relates to the use of a receptor reagent in diagnosing myocardial damage in a subject. Background Art

[0002] Cardiovascular disease is a serious threat to human health and life, and has become the leading cause of death in the 21st century. Among these, acute myocardial infarction is the most common and dangerous. Changes in personal behavior and lifestyle have caused the risk of cardiovascular disease in China to rapidly surpass that of developed countries like the United States.

[0003] Cardiovascular testing is a bottleneck in the cardiovascular field. Only by accurately diagnosing diseases in the shortest possible time can early detection and treatment be achieved, minimizing disability and mortality, and improving patient outcomes and quality of life. In recent years, research on cardiovascular biomarkers has deepened, accumulating extensive clinical experience and evidence, gradually clarifying their clinical indications and new research areas, and promoting their clinical application. Biomarker testing can directly impact the clinical diagnosis, risk stratification, treatment selection, and prognosis of patients with cardiovascular disease. Cardiac biomarkers, also known as early markers of cardiac injury, are markers whose blood levels increase within six hours of cardiac injury. They are crucial indicators in the clinical diagnosis of heart diseases such as myocardial infarction, myocardial ischemia, and heart failure. These markers include cardiac troponin I (cTnI), myoglobin (MYO), creatine kinase isoenzyme (CK-MB), and N-terminal pro-brain natriuretic peptide (NT-proBNP). With the continuous advancement of technology, the detection of heart failure markers in the blood is gradually being used in clinical practice, providing a method for the early diagnosis and prognosis of cardiovascular disease. For example, NT-proBNP is the inactive N-terminal fragment of the BNP prohormone. Compared to BNP, it has a longer half-life and is more stable. Its concentration reflects the transient release of newly synthesized rather than stored BNP, thus better reflecting BNP pathway activation. Plasma NT-proBNP levels increase with increasing severity of heart failure. NT-proBNP plays a crucial role in the diagnosis of acute heart failure.

[0004] Currently, the above markers have been widely accepted by hospitals. A variety of kits are available for detecting these indicators individually. However, the mainstream methods currently used are heterogeneous assays (such as ELISA and magnetic microparticle chemiluminescence). Although these markers can be measured, they suffer from disadvantages 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 deficiencies of the existing technology and provide a receptor reagent for use in preparing a kit for use in in vitro diagnosis of whether a subject has myocardial damage. When using the receptor reagent for detection, it has both ultra-high sensitivity and a wide detection range.

[0006] To this end, the first aspect of the present invention provides a use of a receptor reagent in preparing a kit for use in a method for in vitro diagnosis of whether a subject suffers from myocardial damage, wherein the method comprises: contacting a body fluid from a subject with a receptor reagent and a donor reagent to generate a test mixture after reaction; exciting the test mixture at least once with excitation light, and detecting the intensity of the chemiluminescence signal generated thereby; 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 suffers from myocardial damage;

[0007] The receptor reagent comprises receptor particles capable of reacting with active oxygen to produce chemiluminescence, and the particle size distribution coefficient of variation CV value of the receptor particles in the receptor reagent is controlled to be ≥5%; the donor reagent comprises donor particles capable of generating active oxygen in an excited state.

[0008] The myocardial marker is selected from one or more of troponin T, troponin I, troponin kinase isoenzyme, myoglobin, interleukin 6 and lactate dehydrogenase.

[0009] In some embodiments of the present invention, the coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent is controlled to be ≥8%; preferably, the coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent is controlled to be ≥10%.

[0010] In some preferred embodiments of the present invention, the coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent is controlled to be ≤40%; more preferably, the coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent is controlled to be ≤20%.

[0011] In some embodiments of the present invention, the particle size distribution of the receptor particles in the receptor reagent is polydisperse.

[0012] In some specific embodiments of the present invention, the coefficient of variation CV value of the particle size distribution is calculated by Gaussian distribution.

[0013] In other specific embodiments of the present invention, using Gaussian distribution analysis, the Gaussian distribution curve of the receptor particles in the receptor reagent presents two or more peaks.

[0014] In some preferred embodiments of the present invention, the receptor reagent comprises receptor particles with at least two average particle size distributions.

[0015] In some embodiments of the present invention, the acceptor particle comprises a luminescent composition and a carrier, and the luminescent composition is filled in the carrier and / or attached to the carrier.

[0016] In some embodiments of the present invention, the luminescent composition is capable of reacting with reactive oxygen species to generate a detectable chemiluminescent signal, and comprises a chemiluminescent compound and a metal chelate.

[0017] In other specific embodiments of the present invention, the chemiluminescent compound is selected from olefin compounds, preferably selected from dimethylthiophene, dibutanedione compounds, dioxine, enol ether, enamine, 9-alkylene xanthane, 9-alkylene-N-9,10 dihydroacridine, aryl ether ene, aryl imidazole and lucigenin and their derivatives, more preferably selected from dimethylthiophene and its derivatives.

[0018] In some specific embodiments of the present invention, the metal of the metal chelate is a rare earth metal or a Group VIII metal, preferably selected from europium, terbium, dysprosium, samarium, osmium and ruthenium, more preferably europium.

[0019] In other specific embodiments of the present invention, the metal chelate comprises a chelating agent selected from the group consisting of NHA, BHHT, BHHCT, DPP, TTA, NPPTA, NTA, TOPO, TPPO, BFTA, 2,2-dimethyl-4-perfluorobutyryl-3-butanone (FOD), 2,2'-bipyridine (BPY), bipyridyl carboxylic acid, azacrown ethers, azacryptands and trioctylphosphine oxide and derivatives thereof.

[0020] In some embodiments of the present invention, the carrier is selected from the group consisting of a strip, a sheet, a rod, a tube, a well, a microtiter plate, a bead, a particle, and a microsphere; preferably a bead and a microsphere.

[0021] In other embodiments of the present invention, the carrier is a magnetic or non-magnetic particle.

[0022] In some embodiments of the present invention, the support material is selected from natural, synthetic or modified naturally occurring polymers, including but not limited to: agarose, cellulose, nitrocellulose, cellulose acetate, polyvinyl chloride, polystyrene, polyethylene, polypropylene, poly(4-methylbutene), polyacrylamide, polymethacrylate, polyethylene terephthalate, nylon, polyvinyl butyrate or polyacrylate.

[0023] In other embodiments of the present invention, the carrier is formaldehyde-modified latex particles.

[0024] In some preferred embodiments of the present invention, the average particle size of the carrier is in the range of 50 nm to 1 μm; preferably 100 nm to 500 nm; more preferably 150 nm to 400 nm; most preferably 190 nm to 300 nm.

[0025] 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 a first polysaccharide layer is spontaneously associated with a second polysaccharide layer.

[0026] In other embodiments of the present invention, each of the successive polysaccharide layers is spontaneously associated with each of the previous polysaccharide layers.

[0027] In some embodiments of the present invention, the polysaccharide has pendant functional groups, and the functional groups of the successive polysaccharide layers have charges opposite to those of the functional groups of the previous polysaccharide layer.

[0028] In other embodiments of the present invention, the polysaccharide has pendant functional groups, and the successive layers of the polysaccharide are covalently linked to the previous polysaccharide layer by reaction between the functional groups of the successive layers and the functional groups of the previous layer.

[0029] In some embodiments of the invention, the functional groups of the continuous polysaccharide layer alternate between amine functional groups and amine-reactive functional groups.

[0030] In other specific embodiments of the present invention, the amine-reactive functional group is an aldehyde group or a carboxyl group.

[0031] In some embodiments of the invention, the first polysaccharide layer is spontaneously associated with the carrier.

[0032] In other embodiments of the present invention, the outermost polysaccharide layer of the coating has at least one pendant functional group.

[0033] In some embodiments of the present invention, the side functional groups of the outermost polysaccharide layer of the coating are selected from at least one of aldehyde, carboxyl, thiol, amino, hydroxyl and malein; preferably selected from aldehyde and / or carboxyl.

[0034] In other specific embodiments of the present invention, the side functional groups of the outermost polysaccharide layer of the coating are directly or indirectly connected to reporter molecules, and the reporter molecules can specifically bind to the target molecules to be detected.

[0035] In some embodiments of the present invention, the pendant functional groups of the outermost polysaccharide layer of the coating are directly or indirectly bound to a member of the specific binding pair.

[0036] In other specific embodiments of the present invention, the specific binding pair member is selected from a pair of substances consisting of an antibody, an antibody fragment, a ligand, an oligonucleotide, an oligonucleotide binding protein, a lectin, a hapten, an antigen, an immunoglobulin binding protein, avidin, avidin or biotin; preferably, the specific binding pair member is biotin-avidin.

[0037] In some specific embodiments of the present invention, the polysaccharide is selected from carbohydrates containing three or more unmodified or modified monosaccharide units; preferably selected from glucan, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxyglucan and aminoglucan; more preferably selected from glucan, starch, glycogen and polyribose.

[0038] It should be noted that the coefficient of variation (CV) of the particle size distribution of the receptor particles directly or indirectly linked to the reporter molecule or a member of the specific binding pair in the receptor reagent is still ≥5%. In some embodiments of the present invention, the coefficient of variation (CV) of the particle size distribution of the receptor particles directly or indirectly linked to the reporter molecule or a member of the specific binding pair in the receptor reagent is ≥8%; preferably, the coefficient of variation (CV) of the particle size distribution is ≥10%; further preferably, the coefficient of variation (CV) of the particle size distribution is ≤40%; and even more preferably, the coefficient of variation (CV) of the particle size distribution is ≤20%. In some preferred embodiments of the present invention, the particle size distribution of the receptor particles directly or indirectly linked to the reporter molecule or a member of the specific binding pair exhibits polydispersity.

[0039] In some embodiments of the invention, the donor particle is directly or indirectly linked to a reporter molecule or bound to a member of a specific binding pair.

[0040] In other embodiments of the present invention, the body fluid from the subject is diluted with a diluent before contacting with the receptor reagent and the donor reagent.

[0041] In some embodiments of the present invention, the detection wavelength of the chemiluminescence is 520-620 nm.

[0042] In other embodiments of the present invention, red excitation light of 600 to 700 nm is used for laser irradiation.

[0043] In some embodiments of the present invention, the concentration of the receptor particles in the receptor reagent is 1 ug / mL-1000 ug / mL; preferably 10 ug / mL-500 ug / mL; more preferably 20 ug / mL-200 ug / mL.

[0044] In other embodiments of the present invention, the reactive oxygen species is singlet oxygen.

[0045] In some embodiments of the present invention, the body fluid is whole blood, serum, plasma, sputum, lymph, semen, vaginal mucus, feces, urine, tears or spinal fluid from a subject.

[0046] The beneficial effects of the present invention are as follows: the present invention provides a use of a receptor reagent in preparing a kit for use in a method for in vitro diagnosis of whether a subject suffers from myocardial damage. The method adds a receptor reagent containing receptor particles to body fluids from the subject, wherein the coefficient of variation CV value of the receptor particle size distribution is ≥5%, so that the detection performance of the method of the present invention for myocardial damage markers is greatly improved compared with the existing technology, with both ultra-high sensitivity and a wide detection range. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The present invention will be further described below with reference to the accompanying drawings.

[0048] Figure 1 This is a Gaussian distribution curve of the formaldehyde polystyrene latex microspheres prepared in Example 1.

[0049] Figure 2 Gaussian distribution diagram of the dextran-coated aldehyde-based polystyrene latex microspheres filled with the luminescent composition prepared in Example 1

[0050] Figure 3 This is the Gaussian distribution diagram of the receptor particles prepared in Example 1 with an average particle size of about 250 nm.

[0051] Figure 4 This is the Gaussian distribution diagram of the receptor particles with a particle size of about 110 nm prepared in Example 1.

[0052] Figure 5 This is the Nicomp distribution diagram of the receptor particles with a particle size of about 110 nm prepared in Example 1.

[0053] Figure 6 This is the Gaussian distribution diagram of the receptor particles with a particle size of about 350 nm prepared in Example 1.

[0054] Figure 7 This is the Nicomp distribution diagram of the receptor particles with a particle size of about 350 nm prepared in Example 1.

[0055] Figure 8 This is the Gaussian distribution diagram of the particle size distribution of the mixed receptor particles in Example 2.

[0056] Figure 9This is the Nicomp distribution diagram of the particle size distribution of the mixed receptor particles in Example 2. DETAILED DESCRIPTION

[0057] To facilitate understanding of the present invention, the present invention 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 intended solely to describe specific embodiments and is not intended to be limiting. The practice of the present invention is not limited to the following examples, and any modifications and / or variations made to the present invention fall within the scope of the present invention.

[0058] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of the range and any other specified or intervening values ​​in the specified range is encompassed within the present invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the present invention, subject to any express exclusions in the specified range. Where a specified range includes one or both limits, ranges excluding either or both of those included limits are also encompassed within the present invention.

[0059] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, preferred methods and materials are now described.

[0060] I. Terminology

[0061] The term "active oxygen species" as used in the present invention refers to a general term for substances composed of oxygen, containing oxygen and having active properties in the body or the natural environment, mainly an excited state of oxygen molecules, including the one-electron reduction product of oxygen, the superoxide anion (O2·-), the two-electron reduction product of oxygen, the three-electron reduction product of oxygen, the hydroxyl radical (·OH), as well as nitric oxide and singlet oxygen (1O2).

[0062] The term "donor particles" as used herein refers to particles containing a sensitizer that can generate active intermediates such as reactive oxygen species that react with receptor particles after being activated by energy or an active compound. The donor particles can be photoactivated (such as dyes and aromatic compounds) or chemically activated (such as enzymes, metal salts, etc.). In some specific embodiments of the present invention, the donor particles are polymer microspheres filled with a photosensitizer, which can be a photosensitizer known in the art, preferably a compound that is relatively stable to light and does not effectively react with singlet oxygen. Non-limiting examples include compounds such as methylene blue, rose bengal, porphyrin, phthalocyanine, and chlorophyll disclosed in U.S. Pat. No. 5,709,994 (which is incorporated herein by reference in its entirety), as well as derivatives of these compounds having 1-50 atom substituents, the substituents being used to make these compounds more lipophilic or more hydrophilic, and / or as linking groups for attachment to specific binding pair members. Examples of other photosensitizers known to those skilled in the art can also be used in the present invention, such as those described in U.S. Pat. No. 6,406,913, which is incorporated herein by reference.

[0063] The term "acceptor particle" as used herein refers to a particle containing a compound capable of reacting with reactive oxygen species to produce a detectable signal. Donor particles are activated by energy or reactive compounds and release high-energy reactive oxygen species, which are captured by nearby acceptor particles, thereby transferring energy to activate the acceptor particles. In some embodiments of the present invention, the acceptor particles comprise a luminescent composition and a carrier, wherein the luminescent composition is filled in the carrier and / or coated on the surface of the carrier. The "carrier" herein is selected from a strip, sheet, rod, tube, well, microtiter plate, bead, particle, and microsphere, and may be a microsphere or microparticle known to those skilled in the art, and may be of any size, organic or inorganic, expandable or non-expandable, porous or non-porous, and of any density, but preferably has a density close to that of water, preferably floats in water, and is composed of a transparent, partially transparent, or opaque material. The carrier may or may not be charged; when charged, it is preferably negatively charged. The carrier can be a latex particle or other particle containing an organic or inorganic polymer, a lipid bilayer such as a liposome, a phospholipid vesicle, an oil droplet, a silica particle, a metal sol, a cell, and a microcrystalline dye.

[0064] In the present invention, the "chemiluminescent compound" is a compound, also known as a label, that can undergo a chemical reaction to induce luminescence, such as by being converted into another compound in an electronically excited state. The excited state can be a singlet or triplet excited state. The excited state can relax to the ground state directly, emitting light, or it can transfer the excitation energy to an emitting energy acceptor, thereby returning to the ground state. During this process, the energy acceptor particle will transition to an excited state, emitting light.

[0065] In the present application, the "directly or indirectly capable of binding" means that the specified entity is capable of specifically binding to the entity (directly), or the specified entity is capable of specifically binding to the specific binding partner (indirectly).

[0066] In the present application, the "specific binding partner" means a pair of substances capable of specifically binding to each other.

[0067] In the present application, the "coefficient of variation C.V value of particle size distribution" means the coefficient of variation in Gaussian distribution of particle size in the detection results of the nanometer particle size analyzer. The calculation formula of the coefficient of variation is: C.V value = (standard deviation SD / average value Mean) x 100%.

[0068] In the present application, the term "Nicomp distribution" refers to an algorithm distribution in the American PSS Nanoparticle Size Analyzer NICOMP. Compared with the Gaussian single-peak algorithm, the Nicomp multi-peak algorithm has unique advantages in the analysis of multi-component, non-uniform particle size distribution liquid dispersion systems and the stability analysis of colloidal systems.

[0069] In the present application, the term "body fluid from a subject" refers to a mixture containing or suspected to contain a cardiac marker to be detected. The body fluid from the subject is whole blood, serum, plasma, sputum, lymph, semen, vaginal mucus, feces, urine, tears or spinal fluid. The body fluid from the subject can be diluted as needed before use with a diluent. For example, in order to avoid the hook effect, the body fluid from the subject can be diluted with a diluent before detection on the detection instrument.

[0070] In the present application, the term "antibody" is used in the broadest sense, including antibodies of any isotype, antibody fragments that retain specific binding to an antigen, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, bispecific antibodies, and fusion proteins containing antigen-binding portions of antibodies and non-antibody proteins. Wherever necessary, the antibody can be further conjugated with other moieties, such as one of the specific binding partners, for example, biotin or avidin (one of the biotin-avidin specific binding partners), etc.

[0071] In the present application, the term "antigen" refers to a substance capable of stimulating the body to produce an immune response, and capable of binding to the immune response products, antibodies and sensitized lymphocytes in vivo and in vitro, and causing immune effects.

[0072] In the present application, the term "binding" refers to the direct combination between two molecules due to interactions such as covalent, electrostatic, hydrophobic, ionic and / or hydrogen bonds, including but not limited to interactions such as salt bridges and water bridges.

[0073] The term "specific binding" as used herein refers to the mutual recognition and selective binding reaction between two substances. From a stereostructural perspective, this refers to the conformational correspondence between the reactants. Within the technical concepts disclosed herein, methods for detecting specific binding reactions include, but are not limited to, double antibody sandwich assays, competitive assays, neutralization competition assays, indirect assays, and capture assays.

[0074] II. Implementation Method

[0075] The present invention will be described in more detail below with reference to the embodiments.

[0076] The existing common sense is that the more uniform the particle size of the microspheres, the better the performance of the homogeneous chemiluminescence detection using the microspheres. Therefore, the current research on microspheres used in homogeneous chemiluminescence tends to obtain microspheres with more uniform particle sizes. After research, the inventors of this application found that when using microspheres with uniform particle size for homogeneous chemiluminescence detection, the sensitivity and detection range of the detection results cannot be guaranteed at the same time. However, by using microspheres with appropriate particle size uniformity (such as the coefficient of variation of the microsphere particle size distribution is greater than 5%), the sensitivity of the photoinduced chemiluminescence detection can be guaranteed and the detection range can be widened.

[0077] The inventors of the present invention control the particle size distribution of the receptor particles in the receptor reagent, thereby controlling the amount of reporter molecules (e.g., antibodies / antigens) on the surface of each receptor particle (small-diameter microspheres have a large specific surface area, resulting in a high amount of reporter molecules on the surface per unit mass of the microspheres, while large-diameter microspheres have a small specific surface area, resulting in a low amount of reporter molecules on the surface per unit mass of the microspheres). A larger coefficient of variation in the particle size distribution of the receptor particles in the receptor reagent indicates a higher degree of heterogeneity, equivalent to the presence of receptor particles of various sizes in the system. This allows the method of the present invention to have both high sensitivity and a wide detection range.

[0078] Therefore, the first aspect of the present invention relates to the use of a receptor reagent in preparing a kit for use in a method for in vitro diagnosis of whether a subject suffers from myocardial damage, wherein the method comprises: contacting a body fluid from a subject with a receptor reagent and a donor reagent to generate a test mixture after reaction; exciting the test mixture at least once with excitation light, and detecting the intensity of the chemiluminescence signal generated thereby; 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 suffers from myocardial damage;

[0079] The receptor reagent comprises receptor particles capable of reacting with active oxygen to produce chemiluminescence, and the coefficient of variation (CV) of the particle size distribution of the receptor particles in the receptor reagent is controlled to be ≥5%; the donor reagent comprises donor particles capable of generating active oxygen in an excited state;

[0080] The myocardial marker is selected from one or more of troponin T (troponin T), troponin I (troponin I), troponin kinase isoenzyme (CK-MB), myoglobin (MYO), interleukin 6 and lactate dehydrogenase.

[0081] In some embodiments of the present invention, the coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent is controlled to be ≥8%; preferably, the coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent is controlled to be ≥10%.

[0082] In some preferred embodiments of the present invention, the coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent is controlled to be ≤40%; more preferably, the coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent is controlled to be ≤20%.

[0083] It is worth noting that the coefficient of variation CV value of the particle size distribution of the receptor particles described in the present invention refers to the coefficient of variation CV value of the particle size distribution after the receptor particles are coated with the required substance.

[0084] In some specific embodiments of the present invention, the coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent is controlled to be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35% and 40%, etc.

[0085] In some embodiments of the present invention, the particle size distribution of the receptor particles in the receptor reagent is polydisperse.

[0086] In some specific embodiments of the present invention, the coefficient of variation CV value of the particle size distribution is calculated by Gaussian distribution.

[0087] In other specific embodiments of the present invention, using Gaussian distribution analysis, the Gaussian distribution curve of the receptor particles in the receptor reagent presents two or more peaks.

[0088] In some preferred embodiments of the present invention, the receptor reagent comprises receptor particles with at least two average particle size distributions.

[0089] In some embodiments of the present invention, the acceptor particle comprises a luminescent composition and a carrier, and the luminescent composition is filled in the carrier and / or attached to the carrier.

[0090] In some embodiments of the present invention, the luminescent composition is capable of reacting with reactive oxygen species to generate a detectable chemiluminescent signal, and comprises a chemiluminescent compound and a metal chelate.

[0091] In other specific embodiments of the present invention, the chemiluminescent compound is selected from olefin compounds, preferably selected from dimethylthiophene, dibutanedione compounds, dioxine, enol ether, enamine, 9-alkylene xanthane, 9-alkylene-N-9,10 dihydroacridine, aryl ether ene, aryl imidazole and lucigenin and their derivatives, more preferably selected from dimethylthiophene and its derivatives.

[0092] In some specific embodiments of the present invention, the metal of the metal chelate is a rare earth metal or a Group VIII metal, preferably selected from europium, terbium, dysprosium, samarium, osmium and ruthenium, more preferably europium.

[0093] In other embodiments of the present invention, the metal chelate comprises a chelating agent selected from the group consisting of: 4'-(10-methyl-9-anthryl)-2,2':6'2"-terpyridine-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"-heptafluoro-4",6"-hexanedione- 6"-yl)-chlorosulfonyl-o-terphenyl (BHHCT), 4,7-biphenyl-1,10-phenanthroline (DPP), 1,1,1-trifluoroacetone (TTA), 3-naphthoyl-1,1,1-trifluoroacetone (NPPTA), naphthyltrifluorobutanedione (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, azacryptands and trioctylphosphine oxide and their derivatives.

[0094] In some embodiments of the present invention, the carrier is selected from the group consisting of a strip, a sheet, a rod, a tube, a well, a microtiter plate, a bead, a particle, and a microsphere; preferably a bead and a microsphere.

[0095] In other embodiments of the present invention, the carrier is a magnetic or non-magnetic particle.

[0096] In some embodiments of the present invention, the support material is selected from natural, synthetic or modified naturally occurring polymers, including but not limited to: agarose, cellulose, nitrocellulose, cellulose acetate, polyvinyl chloride, polystyrene, polyethylene, polypropylene, poly(4-methylbutene), polyacrylamide, polymethacrylate, polyethylene terephthalate, nylon, polyvinyl butyrate or polyacrylate.

[0097] In other embodiments of the present invention, the carrier is formaldehyde-modified latex particles.

[0098] In some preferred embodiments of the present invention, the average particle size of the carrier is in the range of 50 nm to 1 μm; preferably 100 nm to 500 nm; more preferably 150 nm to 400 nm; most preferably 190 nm to 300 nm.

[0099] 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 a first polysaccharide layer is spontaneously associated with a second polysaccharide layer.

[0100] In other embodiments of the present invention, each of the successive polysaccharide layers is spontaneously associated with each of the previous polysaccharide layers.

[0101] In some embodiments of the present invention, the polysaccharide has pendant functional groups, and the functional groups of the successive polysaccharide layers have charges opposite to those of the functional groups of the previous polysaccharide layer.

[0102] In other embodiments of the present invention, the polysaccharide has pendant functional groups, and the successive layers of the polysaccharide are covalently linked to the previous polysaccharide layer by reaction between the functional groups of the successive layers and the functional groups of the previous layer.

[0103] In some embodiments of the invention, the functional groups of the continuous polysaccharide layer alternate between amine functional groups and amine-reactive functional groups.

[0104] In other specific embodiments of the present invention, the amine-reactive functional group is an aldehyde group or a carboxyl group.

[0105] In some embodiments of the invention, the first polysaccharide layer is spontaneously associated with the carrier.

[0106] In other embodiments of the present invention, the outermost polysaccharide layer of the coating has at least one pendant functional group.

[0107] In some embodiments of the present invention, the side functional groups of the outermost polysaccharide layer of the coating are selected from at least one of aldehyde, carboxyl, thiol, amino, hydroxyl and malein; preferably selected from aldehyde and / or carboxyl.

[0108] In other specific embodiments of the present invention, the side functional groups of the outermost polysaccharide layer of the coating are directly or indirectly connected to reporter molecules, and the reporter molecules can specifically bind to the target molecules to be detected.

[0109] In some embodiments of the present invention, the pendant functional groups of the outermost polysaccharide layer of the coating are directly or indirectly bound to a member of the specific binding pair.

[0110] In other embodiments of the present invention, the specific binding pair member is selected from a pair of substances consisting of an antibody, an antibody fragment, a ligand, an oligonucleotide, an oligonucleotide binding protein, a lectin, a hapten, an antigen, an immunoglobulin binding protein, avidin, avidin, or biotin; preferably, the specific binding pair member is biotin-avidin. The avidin is selected from avidin, streptavidin, vitellogenin, neutravidin, and avidin-like substances, preferably neutravidin and / or streptavidin.

[0111] In some specific embodiments of the present invention, the polysaccharide is selected from carbohydrates containing three or more unmodified or modified monosaccharide units; preferably selected from glucan, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxyglucan and aminoglucan; more preferably selected from glucan, starch, glycogen and polyribose.

[0112] It should be noted that the coefficient of variation (CV) of the particle size distribution of the receptor particles directly or indirectly linked to the reporter molecule or a member of the specific binding pair in the receptor reagent is ≥5%. In some embodiments of the present invention, the coefficient of variation (CV) of the particle size distribution of the receptor particles directly or indirectly linked to the reporter molecule or a member of the specific binding pair in the receptor reagent is ≥8%; preferably, the coefficient of variation (CV) of the particle size distribution is ≥10%; further preferably, the coefficient of variation (CV) of the particle size distribution is ≤40%; even more preferably, the coefficient of variation (CV) of the particle size distribution is ≤20%. In some preferred embodiments of the present invention, the particle size distribution of the receptor particles directly or indirectly linked to the reporter molecule or a member of the specific binding pair is polydisperse.

[0113] In some preferred embodiments of the present invention, the body fluid from the subject is first contacted with the receptor reagent and then with the donor reagent.

[0114] In some embodiments of the invention, the donor particle is directly or indirectly linked to a reporter molecule or bound to a member of a specific binding pair.

[0115] To further improve the accuracy of the final test results and the stability of the test sample, in other embodiments of the present invention, the body fluid from the subject is diluted with a diluent before contacting with the receptor reagent and the donor reagent.

[0116] In some embodiments of the present invention, the chemiluminescence detection wavelength is 520-620 nm, preferably 610-620 nm, and more preferably 615 nm.

[0117] In other embodiments of the present invention, red excitation light of 600-700 nm is used for laser irradiation; preferably, red excitation light of 640-680 nm is used for laser irradiation; and more preferably, red excitation light of 660 nm is used for laser irradiation.

[0118] In some embodiments of the present invention, the concentration of the receptor particles in the receptor reagent is 1 ug / mL-1000 ug / mL; preferably 10 ug / mL-500 ug / mL; more preferably 20 ug / mL-200 ug / mL.

[0119] In other embodiments of the present invention, the reactive oxygen species is singlet oxygen.

[0120] In some embodiments of the present invention, the body fluid is whole blood, serum, plasma, sputum, lymph, semen, vaginal mucus, feces, urine, tears or spinal fluid from a subject.

[0121] III. Examples

[0122] Example 1: Preparation of Receptor Particle Solution Conjugated with Antibody I (cTnI Antibody)

[0123] (1) Preparation of antibody-coupled receptor particles with an average particle size of approximately 250 nm

[0124] 1.1 Preparation and characterization of formaldehyde-based polystyrene latex microspheres

[0125] 1) Prepare a 100ml three-necked flask, add 40mmol of styrene, 5mmol of acrolein, and 10ml of water, stir for 10min, and then pass N2 for 30min;

[0126] 2) Weigh 0.11g of ammonium persulfate and 0.2g of sodium chloride and 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 flow N2 for 30 minutes.

[0127] 3) The reaction system was heated to 70°C and reacted for 15 hours;

[0128] 4) Cool the emulsion after the reaction to room temperature and filter it through a suitable filter cloth. Wash the resulting emulsion with deionized water by centrifugation until the conductivity of the supernatant is close to that of deionized water. Then dilute it with water and store it as an emulsion.

[0129] 5) The Gaussian distribution average particle size of the latex microspheres measured by the nanoparticle size analyzer is 204.7 nm, the coefficient of variation (CV) is 5.01%, and the Gaussian distribution curve is as follows: Figure 1 The aldehyde content of the latex microspheres was determined to be 268 nmol / mg by conductometric titration.

[0130] 1.2 Filling process and characterization of luminescent composition

[0131] 1) Prepare a 25 ml round bottom flask, add 0.1 g of dimethylthiophene derivative and 0.1 g of europium (III) complex (MTTA-EU 3+ ), 10 ml of 95% ethanol, magnetic stirring, and heating in a water bath to 70°C to obtain a complex solution;

[0132] 2) Prepare a 100 ml three-necked flask and add 10 ml of 95% ethanol, 10 ml of water, and 10 ml of the 10% formaldehyde-based polystyrene latex microspheres obtained in step 1.1. Stir magnetically and heat in a water bath to 70°C.

[0133] 3) slowly adding the complex solution in step 1) dropwise to the three-necked flask in step 2), reacting at 70° C. for 2 hours, then stopping stirring and cooling naturally;

[0134] 4) Centrifuging the emulsion for 1 hour at 30,000 G, discarding the supernatant to obtain formaldehyde-based polystyrene microspheres filled with the luminescent composition.

[0135] 1.3 Surface coating of receptor particles with dextran

[0136] 1) Place 50 mg of aminodextran solid in a 20 mL round-bottom flask, add 5 mL of 50 mM / pH 10 carbonate buffer, and dissolve by stirring at 30°C in the dark.

[0137] 2) taking 100 mg of the prepared aldehyde-based polystyrene microspheres filled with the luminescent composition, adding them to the aminodextran solution and stirring for 2 hours;

[0138] 3) Dissolve 10 mg of sodium borohydride in 0.5 mL of 50 mM / pH 10 carbonate buffer and add dropwise to the above reaction solution. React at 30°C in the dark overnight.

[0139] 4) Centrifuge the reaction mixture at 30,000g, discard the supernatant, and add 50mM / pH 10 carbonate buffer for ultrasonic dispersion. Repeat the centrifugation and washing three times, then adjust the volume with 50mM / pH 10 carbonate buffer to a final concentration of 20mg / ml.

[0140] 5) Place 100 mg of aldehyde dextran solid in a 20 mL round-bottom flask, add 5 mL of 50 mM / pH 10 carbonate buffer, and dissolve by stirring at 30°C in the dark.

[0141] 6) Add the above microspheres to the aldehyde dextran solution and stir for 2 hours;

[0142] 7) Dissolve 15 mg of sodium borohydride in 0.5 mL of 50 mM / pH 10 carbonate buffer and add dropwise to the above reaction solution. React at 30°C in the dark overnight.

[0143] 8) The reaction mixture was centrifuged at 30,000 G, the supernatant discarded, and 50 mM / pH 10 carbonate buffer was added for ultrasonic dispersion. Repeat the centrifugation and washing three times, and then the volume was adjusted to 20 mg / ml with 50 mM / pH 10 carbonate buffer.

[0144] 9) The Gaussian distribution average particle size of the microspheres measured by the nanoparticle size analyzer is 241.7 nm, and the coefficient of variation (CV) is 18.40% (as shown in FIG. Figure 2 shown).

[0145] 1.4 Antibody Conjugation Process

[0146] 1) The paired antibody I was dialyzed into 50 mM CB buffer at pH 10, and the concentration was measured to be 1 mg / ml.

[0147] 2) Add 0.5 ml of the receptor particles 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, add 100 μl of 10 mg / ml NaBH4 solution (50 mM CB buffer), and react at 2-8°C for 4 hours.

[0148] 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°C for 2 hours.

[0149] 4) After the reaction is complete, centrifuge at 30,000g for 45 minutes. Discard the supernatant and resuspend in 50mM MES buffer. Repeat the centrifugation and washing process four times. Dilute to a final concentration of 100 μg / ml to obtain a solution of receptor particles conjugated to Antibody I.

[0150] 5) The Gaussian distribution average particle size of the microspheres measured by the nanoparticle size analyzer is 249.9 nm, and the coefficient of variation (CV value) is 11.60% (as shown in FIG. Figure 3 shown).

[0151] (II) Preparation of antibody-coupled receptor particles with an average particle size of approximately 110 nm

[0152] The preparation method is the same as the preparation process of the receptor particles with an average particle size of about 250nm in the above (1), and the Gaussian distribution of the receptor particle size is measured by a nanoparticle size analyzer (such as Figure 4 The average particle size is 108.2 nm, and the coefficient of variation (CV) is 7.5%. The Nicomp distribution is unimodal (as shown in FIG. Figure 5 shown).

[0153] (III) Preparation of antibody-coupled receptor particles with an average particle size of approximately 350 nm

[0154] The preparation method is the same as the preparation process of the receptor particles with an average particle size of about 250nm in the above (1), and the Gaussian distribution of the receptor particle size is measured by a nanoparticle size analyzer (such as Figure 6 The average particle size is 345.4 nm, the coefficient of variation (CV) is 4.0%, and the Nicomp distribution is unimodal (as shown in FIG. Figure 7 shown).

[0155] Example 2: Sensitivity and upper limit of detection of cTnI marker using the receptor reagent of the present invention

[0156] The sensitivity point is defined as the point where the signal at concentration Cx is twice that at concentration C0, i.e., RLU(Cx) > 2RLU(C0). The corresponding sensitivity of the assay is Cx. The upper limit of detection is defined as the upper limit of the range determined using the method in the National Committee for Clinical Laboratory Standards (NCCLS) Evaluation Protocol (EP) Series 6 document.

[0157] (1) The cTnI antigen was diluted to a concentration series of 1 pg / ml, 2 pg / ml, 5 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 100 pg / ml, 1000 pg / ml, 5000 pg / ml, 10000 pg / ml, 50000 pg / ml, and 1000 ng / ml. The receptor reagents (concentration of 100 μg / ml) prepared in Example 1, each containing receptor particles of different average particle sizes (110 nm and 350 nm) coupled to cTnI antibody I, were used to detect the cTnI antigen in the above concentration series together with the same biotin-labeled cTnI monoclonal antibody 2 (diluted to 2 μg / ml) and universal solution (reagent containing donor particles). The detection sensitivity and upper limit of detection of the photochemiluminescence analysis system developed by Boyang Biotechnology (Shanghai) Co., Ltd. are shown in Table 1.

[0158] Table 1

[0159]

[0160] As can be seen from Table 1, the upper detection limit of the receptor particles with an average particle size of 110 nm is high, but the sensitivity is poor; while the receptor particles with an average particle size of 350 nm have the best sensitivity, but the upper detection limit is low.

[0161] (2) A solution of receptor particles with an average particle size of 110 nm coupled to cTnI antibody I was mixed with a solution of receptor particles with an average particle size of 350 nm coupled to cTnI antibody I to obtain a new receptor reagent. The results of the measurement of the receptor particle size in the new receptor reagent are as follows:

[0162] The average particle size of the Gaussian distribution is 318.1 nm, and the coefficient of variation (CV value) of the particle size distribution is 37.5% (e.g. Figure 8 shown);

[0163] Nicomp distribution is bimodal: #1: average particle size 102.9nm, coefficient of variation (CV value) = 121%; #2: average particle size 328.4nm, coefficient of variation (CV value) of particle size distribution = 13.0% (e.g. Figure 9 shown).

[0164] The above-mentioned new receptor reagent was used with biotin-labeled cTnI monoclonal antibody 2 (diluted to 2 μg / ml) and universal solution (reagent containing donor particles) to detect the above-mentioned concentration series of cTnI antigen. The detection sensitivity and upper limit of detection of the photochemiluminescence analysis system developed by Boyang Biotechnology (Shanghai) Co., Ltd. are shown in Table 2.

[0165] Table 2

[0166]

[0167] As can be seen from Table 2, by increasing the coefficient of variation of the particle size distribution of the receptor particles in the receptor reagent, that is, appropriately increasing the heterogeneity of the particle size of the receptor particles, the detection performance of the method is significantly improved.

[0168] Example 3: Preparation of a Series of Antibody I-Conjugated Receptor Particle Solutions with an Average Particle Size of approximately 250 nm and Different Coefficients of Variation in Particle Size Distribution

[0169] According to the method described in Example 1 (I), receptor particle solutions of conjugated antibody I (cTnI antibody) with different coefficients of variation of particle size distribution were obtained.

[0170] Specifically:

[0171] Receptor particles 1: Gaussian distribution average particle size is 250.2 nm, particle size distribution coefficient of variation CV value = 4.5%; Nicomp distribution is unimodal.

[0172] Receptor particles 2: Gaussian distribution average particle size is 251.3 nm, particle size distribution coefficient of variation CV value = 5.0%; Nicomp distribution is unimodal.

[0173] Receptor particles 3: Gaussian distribution average particle size is 249.8 nm, particle size distribution coefficient of variation CV value = 6.0%; Nicomp distribution is unimodal.

[0174] Receptor particles 4: Gaussian distribution average particle size is 250.6 nm, particle size distribution coefficient of variation CV value = 11.0%; Nicomp distribution is unimodal.

[0175] Receptor particles 5: Gaussian distribution average particle size is 253.2 nm, particle size distribution coefficient of variation CV value = 15.8%; Nicomp distribution is unimodal.

[0176] Receptor particles 6: Gaussian distribution average particle size is 251.7 nm, particle size distribution coefficient of variation CV value = 36.8%; Nicomp distribution is bimodal.

[0177] Example 4: Sensitivity and upper limit of detection of cTnI marker using the receptor reagent of the present invention

[0178] The sensitivity point is defined as when the signal at concentration Cx is higher than twice the signal at concentration C0, that is, RLU(Cx)>2RLU(C0), and the corresponding detection reagent sensitivity is Cx. The upper detection limit is defined as the upper limit of the range determined using the method in the NCCLS EP-6 document.

[0179] The cTnI antigen was diluted to a concentration series of 1 pg / ml, 2 pg / ml, 5 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 100 pg / ml, 1000 pg / ml, 5000 pg / ml, 10000 pg / ml, 50000 pg / ml, and 1000 ng / ml. The cTnI antigen in the above concentration series was detected using the receptor reagent comprising receptor particles coupled to cTnI antibody I prepared in Example 3 (concentration of 100 μg / ml), the same biotin-labeled cTnI monoclonal antibody 2 (diluted to 2 μg / ml), and the universal solution (reagent containing donor particles). The detection sensitivity and upper limit of detection of the photochemiluminescence analysis system developed by Boyang Biotechnology (Shanghai) Co., Ltd. are shown in Table 3.

[0180] Table 3

[0181]

[0182] As can be seen from Table 3, when the coefficient of variation of the receptor particle size distribution is greater than or equal to 5%, the method of adding a receptor reagent containing the above receptor particles has both relatively suitable sensitivity and a wide detection range.

[0183] Example 5: Detection of cTnI marker standards

[0184] cTnI antigen was diluted to a concentration series of 1pg / ml, 2pg / ml, 5pg / ml, 10pg / ml, 20pg / ml, 30pg / ml, 40pg / ml, 50pg / ml, 100pg / ml, 1000pg / ml, 5000pg / ml, 10000pg / ml, 50000pg / ml, and 1000ng / ml, and different particle sizes (50nm, 80nm, 110nm, 140nm, 170nm, The receptor reagent (concentration of 100 μg / ml, CV values ​​of approximately 10%) of cTnI monoclonal antibody 1 coated with acceptor particles (200 nm, 250 nm, 300 nm, 350 nm, and 400 nm) was used to detect the cTnI antigen in the above concentration series, together with the same biotinylated cTnI monoclonal antibody 2 (diluted to 2 μg / ml) and universal solution (donor particle solution). The detection sensitivity and upper limit of detection of the photochemiluminescence analysis system developed by Boyang Biotechnology (Shanghai) Co., Ltd. are shown in Table 4.

[0185] Table 4

[0186]

[0187]

[0188] The cTnI test results in Table 4 show that receptor particles with a particle size of 50 nm and 80 nm have a high upper limit of detection but poor sensitivity, while receptor particles with a particle size of 300 nm have the best sensitivity but a low upper limit of detection. Receptor particles with a particle size of 50 nm and 80 nm were mixed with receptor particles with a particle size of 300 nm to form receptor reagents. The sensitivity and upper limit of detection of the method containing the corresponding receptor reagents were tested. The results are shown in Table 5.

[0189] Table 5

[0190]

[0191] From Table 5, it can be seen that the addition of the receptor reagent formed by combining the acceptor particles with a small average particle size and the acceptor particles with a large average particle size has both high sensitivity and a high detection upper limit (wide detection range), exhibiting the advantages of large and small particle size acceptor particles, and the performance of the receptor reagent containing two or more average particle sizes of acceptor particles is greatly improved compared to the single average particle size distribution of acceptor particles.

[0192] Example 6: Clinical detection of cTnl of normal people and patients suspected of having myocardial injury

[0193] This example detects 40 clinical samples (13 negative samples and 27 positive samples), and the cTnl quantitative detection reagent kit (light-induced chemiluminescence method) used is composed of reagent 1 (R1) containing first anti-cTnl monoclonal antibody coated acceptor particles, reagent 2 (R2) containing biotin-labeled second anti-cTnl monoclonal antibody, and in addition, a universal liquid (R3) containing donor particles. Among them, R1 is a receptor reagent (concentration of 100 ug / ml) prepared by using the acceptor particles 4 (particle size distribution coefficient of variation C.V value = 11%) in Example 3.

[0194] The detection process is completed on the fully automatic light-induced chemiluminescence analysis system developed by Boyang Biotech (Shanghai) Co., Ltd. and the detection results are output, and the specific detection steps include:

[0195] a. Add the clinical sample to the reaction well;

[0196] b. Add R1 and R2 to the reaction well in turn;

[0197] c. Incubate;

[0198] d. Add R3 to the reaction well;

[0199] e. Incubate;

[0200] f. Laser irradiate the reaction well and calculate the amount of light photons per well;

[0201] g. Calculate the concentration of cTnl in the sample to be tested.

[0202] When the cTnI marker is present in a clinical sample, cTnI simultaneously binds specifically to receptor particles coated with a first anti-cTnI monoclonal antibody and a second biotin-labeled anti-cTnI monoclonal antibody, forming a double-antibody sandwich complex on the receptor particle surface. At this point, if streptavidin-modified donor particles are added, the biotin binds to the streptavidin, bringing the two particles into close proximity. Under the excitation of an excitation light source, the donor particles release singlet oxygen, which, upon encountering the receptor particles in solution, produces chemiluminescence, further stimulating the fluorescent groups on the same particles to generate a cascade amplification reaction, producing fluorescence. At this point, the greater the amount of cTnI marker present, the stronger the fluorescence intensity. The intensity of the luminescence is used to quantitatively measure the amount of cTnI in the patient's serum. The specific test results are shown in Table 6 below:

[0203] Table 6

[0204]

[0205]

[0206] Data comparison revealed a correlation of 0.9973 between the Abbott values ​​and those measured in Example 6, with a slope of 1.0495. Samples 1-13 were from healthy patients undergoing physical examinations, with a distribution range of 1.77 pg / ml to 25.3 pg / ml, and a median of 6.77 pg / ml. Samples 14-40 were from patients identified as having myocardial damage, with a distribution range of 30.94 pg / ml to 29,896.88 pg / ml, and a median of 450.54 pg / ml.

[0207] Cardiac troponin I (cTnI) concentrations are low in the serum or plasma of healthy individuals. Within 4-8 hours after the onset of chest pain, necrotic myocardial cells release large amounts of cTnI into the bloodstream, reaching a peak within 12-48 hours. In patients with severe myocardial infarction, cTnI levels remain elevated for several days, making it an optimal marker for diagnosing myocardial injury and myocardial infarction. Data from Example 6 of the present invention demonstrate the feasibility of using the receptor reagent described herein in a kit for in vitro diagnosis of myocardial injury. Quantitative determination of cTnI in a subject's body fluids using the receptor reagent and corresponding method described herein can be used to diagnose myocardial injury and myocardial infarction.

[0208] Example 7: Clinical Detection of CKMB in Normal Subjects and Patients Suspected of Myocardial Injury

[0209] The present example detects 40 clinical samples, the CKMB quantitative detection reagent kit (light-activated chemiluminescence) used is composed of reagent 1 (R1') containing first anti-CKMB antibody coated receptor microparticles, reagent 2 (R2') containing biotin labeled second anti-CKMB antibody, and further including universal liquid (R3') containing donor particles. Among them, R1 is a receptor reagent (concentration of 200 ug / ml) prepared by using the receptor particles 4 (particle size distribution variation coefficient C.V value = 11%) in Example 3.

[0210] The specific experimental steps are as follows:

[0211] 1. Select 40 clinical samples, balance to room temperature, mix well;

[0212] 2. Add mixed samples, prepared R1' and R2' to 8x12 white plates respectively;

[0213] 3. Put the sample plate into LiCA HT instrument for reaction, the reaction mode used is as follows:

[0214] (1) Mix 40ul sample, 15ul R1' and 15ul R2';

[0215] (2) Incubate at 37℃ for 8min;

[0216] (3) Add 160ul universal liquid (R3');

[0217] (4) Incubate at 37℃ for 2min;

[0218] (5) Excite reading, the specific detection results are shown in Table 7.

[0219] Table 7

[0220]

[0221]

[0222] After comparison, the correlation between Roche value and Boyang value is 0.9877, and the slope is 0.9192. Samples 1-13 are normal physical examination patients, the distribution range is 0.23 ng / ml-3.88 ng / ml, and the median is 1.27 ng / ml; samples 14-40 are patients identified with myocardial injury, the distribution range is 6.53 ng / ml-150.90 ng / ml, and the median is 41.88 ng / ml.

[0223] Creatine kinase isoenzyme (CK-MB) is one of the three criteria recommended by the WHO for determining myocardial infarction. CK-MB is one of the three dimeric isoenzymes of creatine kinase, with a high proportion in the myocardium. It enters the blood circulation when myocardial cells necrotize. The concentration of patients with myocardial infarction increases 3-4 hours after onset, reaches a peak 18-24 hours later, and returns to normal within 72 hours. According to the data of Example 7 of the present invention, it can be shown that the use of the receptor reagent of the present invention in the preparation of a kit for use in a method for in vitro diagnosis of whether a subject has myocardial damage is feasible. The quantitative results of the CK-MB marker in the subject's body fluids using the receptor reagent of the invention and the corresponding method can be used to diagnose myocardial damage and myocardial infarction.

[0224] Example 8: Clinical Detection of MYO in Normal Subjects and Patients Suspected of Myocardial Injury

[0225] This example tested 40 clinical samples using a MYO quantitative assay kit (photochemiluminescence) consisting of Reagent 1 (R1') containing acceptor microparticles coated with a primary anti-MYO antibody, Reagent 2 (R2') containing a biotin-labeled secondary anti-MYO antibody, and a universal solution (R3') containing donor particles. R1 was a 200 μg / ml receptor reagent prepared using acceptor particles 4 (particle size distribution coefficient of variation CV = 11%) described in Example 3.

[0226] The specific experimental steps are as follows:

[0227] 1. Select 40 clinical samples, equilibrate to room temperature, and mix thoroughly;

[0228] 2. Add the mixed sample, prepared R1' and R2' to an 8×12 white plate;

[0229] 3. Place the sample-loaded white plate into the LiCA HT instrument for reaction. The reaction mode used is as follows:

[0230] (1) Mix 40ul sample, 15ul R1' and 15ul R2';

[0231] (2) Incubate at 37°C for 8 min;

[0232] (3) Add 160 μl of universal solution (R3');

[0233] (4) Incubate at 37°C for 2 min;

[0234] (5) Excitation readings. The specific test results are shown in Table 8 below.

[0235] Table 8

[0236]

[0237]

[0238] Comparison showed that the Roche and Boyang values ​​had a correlation of 0.994 and a slope of 0.954. Samples 1-13 were from healthy patients undergoing physical examinations, with a range of 23.36 ng / ml to 84.08 ng / ml and a median of 38.93 ng / ml. Samples 14-40 were from patients diagnosed with myocardial injury, with a range of 81.3 ng / ml to 1495.59 ng / ml and a median of 382.48 ng / ml.

[0239] Myoglobin (MYO) is present in myocardium and skeletal muscle. It is small in size and can rapidly diffuse into the bloodstream when myocardial cells necrotize. Its concentration can rise within 1-2 hours, making it an early indicator of acute myocardial infarction. Myoglobin concentrations can return to normal 24 hours after the onset of myocardial infarction, so MYO can be used to diagnose recurrent myocardial infarction. The data from Example 8 of the present invention demonstrate the feasibility of using the receptor reagent described herein in a kit for preparing an in vitro method for diagnosing whether a subject has myocardial injury. The quantitative results of measuring MYO markers in a subject's body fluids using the receptor reagent described herein and the corresponding method can be used to diagnose myocardial injury and myocardial infarction.

[0240] It should be noted that the embodiments described above are only used to explain the present invention and do not constitute any limitation of 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 words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein. On the contrary, the present invention can be extended to all other methods and applications with the same function.

Claims

1. Use of a receptor reagent in preparing a kit for use in a method for in vitro diagnosis of whether a subject has myocardial damage, wherein the method comprises: contacting a body fluid from a subject with a receptor reagent and a donor reagent to react to form a test mixture; Exciting the test mixture at least once with excitation light, and detecting the intensity of the chemiluminescence signal generated thereby; 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 suffers from myocardial damage; The receptor reagent comprises receptor particles capable of reacting with reactive oxygen species to produce chemiluminescence, the receptor particles comprising a luminescent composition and a carrier, the luminescent composition being filled in and / or attached to the carrier; the receptor reagent comprises receptor particles having at least two average particle size distributions; the coefficient of variation (CV) value of the particle size distribution of the receptor particles in the receptor reagent is controlled to be ≥11% and ≤15.8%; the donor reagent comprises donor particles capable of generating reactive oxygen species in an excited state; The myocardial marker is 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 particle size distribution of the receptor particles in the receptor reagent is polydisperse.

3. The use according to claim 1 or 2, characterized in that The coefficient of variation CV value of the particle size distribution is calculated through Gaussian distribution.

4. The use according to claim 1 or 2, characterized in that Using the Gaussian distribution analysis method, the Gaussian distribution curve of the receptor particles in the receptor reagent presents two or more peaks.

5. The use according to claim 1, characterized in that The luminescent composition can react with active oxygen to generate a detectable chemiluminescent signal, and comprises a chemiluminescent compound and a metal chelate.

6. The use according to claim 5, characterized in that The chemiluminescent compound is selected from olefin compounds.

7. The use according to claim 5, characterized in that The chemiluminescent compound is selected from dimethylthiophene, dibutanedione compounds, dioxine, enol ether, enamine, 9-alkylene xanthane, 9-alkylene-N-9,10 dihydroacridine, aryl ether ene, aryl imidazole and lucigenin and their derivatives.

8. The use according to claim 7, characterized in that The chemiluminescent compound is selected from dimethylthiophene and its derivatives.

9. The use according to any one of claims 5 to 8, characterized in that The metal of the metal chelate is a rare earth metal or a Group VIII metal.

10. The use according to claim 9, characterized in that The metal of the metal chelate is selected from the group consisting of europium, terbium, dysprosium, samarium, osmium and ruthenium.

11. The use according to claim 10, characterized in that The metal of the metal chelate is europium.

12. The use according to any one of claims 5 to 8, characterized in that The metal chelate comprises a chelating agent selected from the group consisting of NHA, BHHT, BHHCT, DPP, TTA, NPPTA, NTA, TOPO, TPPO, BFTA, 2,2-dimethyl-4-perfluorobutyryl-3-butanone (fod), 2,2'-bipyridine (bpy), bipyridyl carboxylic acid, azacrown ethers, azacryptands, trioctylphosphine oxide, and derivatives thereof.

13. The use according to claim 1, characterized in that The carrier is selected from beads.

14. The use according to claim 1, characterized in that The carrier is selected from microspheres.

15. The use according to claim 1, characterized in that The carrier is a magnetic or non-magnetic particle.

16. The use according to claim 1, characterized in that The support material is selected from natural, synthetic or modified naturally occurring polymers.

17. The use according to claim 16, characterized in that The carrier material includes: agarose, cellulose, nitrocellulose, cellulose acetate, polyvinyl chloride, polystyrene, polyethylene, polypropylene, poly(4-methylbutene), polyacrylamide, polymethacrylate, polyethylene terephthalate, nylon, polyvinyl butyrate or polyacrylate.

18. The use according to claim 16, characterized in that The carrier is aldehyde-modified latex particles.

19. The use according to claim 1, characterized in that The average particle size of the carrier is in the range of 50 nm to 1 μm.

20. The use according to claim 19, characterized in that The average particle size of the carrier is in the range of 100 nm to 500 nm.

21. The use according to claim 20, characterized in that The average particle size of the carrier is in the range of 150 nm to 400 nm.

22. The use according to claim 21, characterized in that The average particle size of the carrier is in the range of 190 nm to 300 nm.

23. The use according to claim 1, characterized in that The surface of the carrier is coated with a coating of at least two consecutive polysaccharide layers, wherein a first polysaccharide layer is spontaneously associated with a second polysaccharide layer.

24. The use according to claim 23, characterized in that Each of the successive polysaccharide layers is spontaneously associated with each of the previous polysaccharide layers.

25. The use according to claim 23 or 24, characterized in that The polysaccharide has pendant functional groups, and the functional groups of the successive polysaccharide layers are opposite in charge to the functional groups of the previous polysaccharide layer.

26. The use according to claim 23 or 24, characterized in that The polysaccharide has pendant functional groups, and the successive polysaccharide layer is covalently linked to the preceding polysaccharide layer by reaction between the functional groups of the successive polysaccharide layer and the functional groups of the preceding layer.

27. The use according to claim 26, characterized in that The functional groups of the continuous polysaccharide layer alternate between amine functional groups and amine-reactive functional groups.

28. The use according to claim 27, characterized in that The amine-reactive functional group is an aldehyde group or a carboxyl group.

29. The use according to claim 23 or 24, characterized in that The first polysaccharide layer is spontaneously associated with the carrier.

30. The use according to claim 23 or 24, characterized in that The outermost polysaccharide layer of the coating has at least one pendant functional group.

31. The use according to claim 23 or 24, characterized in that The side functional groups of the outermost polysaccharide layer of the coating are selected from at least one of aldehyde, carboxyl, thiol, amino, hydroxyl and malein.

32. The use according to claim 31, characterized in that The side functional groups of the outermost polysaccharide layer of the coating are selected from aldehyde groups and / or carboxyl groups.

33. The use according to claim 30, characterized in that The side functional groups of the outermost polysaccharide layer of the coating are directly or indirectly connected to the reporter molecules, and the reporter molecules can specifically bind to the target molecules to be detected.

34. The use according to claim 30, characterized in that The side functional groups of the outermost polysaccharide layer of the coating are directly or indirectly bound to one of the specific binding pair members.

35. The use according to claim 34, characterized in that The specific binding pair members are selected from a pair of substances consisting of antibodies, antibody fragments, ligands, oligonucleotides, oligonucleotide binding proteins, lectins, haptens, antigens, immunoglobulin binding proteins, avidin or biotin.

36. The use according to claim 35, characterized in that The specific binding pair member is biotin-avidin.

37. The use according to claim 23 or 24, characterized in that The polysaccharide is selected from carbohydrates containing three or more unmodified or modified monosaccharide units.

38. The use according to claim 37, characterized in that The polysaccharide is selected from the group consisting of dextran, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxydextran and aminodextran.

39. The use according to claim 38, characterized in that The polysaccharide is selected from the group consisting of dextran, starch, glycogen and polyribose.

40. The use according to claim 1, characterized in that The donor particle is directly or indirectly linked to a reporter molecule or bound to a member of a specific binding pair.

41. The use according to claim 1, characterized in that The body fluid from the subject is diluted with a diluent and then contacted with the receptor reagent and the donor reagent.

42. The use according to claim 1, characterized in that The detection wavelength of the chemiluminescence is 520-620 nm.

43. The use according to claim 1, characterized in that Red excitation light of 600-700 nm was used for laser irradiation.

44. The use according to claim 1, characterized in that The concentration of the receptor particles in the receptor reagent is 1 ug / mL-1000 ug / mL.

45. The use according to claim 44, characterized in that The concentration of the receptor particles in the receptor reagent is 10ug / mL-500ug / mL.

46. ​​The use according to claim 45, characterized in that The concentration of the receptor particles in the receptor reagent is 20ug / mL-200ug / mL.

47. The use according to claim 1, characterized in that The active oxygen is singlet oxygen.

48. The use according to claim 1, characterized in that The body fluid is whole blood, serum, plasma, sputum, lymph, semen, vaginal mucus, feces, urine, tears or spinal fluid from a subject.

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