A homogeneous chemiluminescence detection method and its application

Abstract

The present invention relates to a homogeneous chemiluminescence detection method and its application in the field of chemiluminescence detection technology. The method comprises the following steps: step S1, contacting a sample to be tested with a receptor reagent including receptor particles and a donor reagent including donor particles, and generating a mixture to be tested after reaction; controlling the coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent to be ≥5%. Step S2, using energy or active compounds to excite the chemiluminescence of the mixture to be tested, analyzing the signal intensity of the chemiluminescence, and judging whether the sample to be tested contains the target molecule to be tested and / or the concentration of the target molecule to be tested in the sample to be tested. The detection performance of the method of the present invention is greatly improved compared with the existing technology, with both ultra-high sensitivity and a wide detection range.

Description

Technical Field

[0001] The present invention belongs to the field of chemiluminescence detection, and in particular relates to a homogeneous chemiluminescence detection method and application thereof. Background Art

[0002] After more than half a century of development, immunoassays have developed into many types. Depending on whether the analyte needs to be separated from the reaction system during the measurement process, it can be divided into heterogeneous immunoassays (Heterogenous) and homogeneous immunoassays (Homogeneous). Heterogeneous immunoassay refers to the process of introducing probes for labeling, in which various related reagents need to be separated after the mixed reaction. The analyte is separated from the reaction system before detection. It is the mainstream method in immunoassays today. Such as the widely known enzyme-linked immunosorbent assay (ELISA) and magnetic particle chemiluminescence method. Homogeneous immunoassay refers to the process of directly measuring the analyte after mixing the analyte with the relevant reagents in the reaction system during the measurement process, without any unnecessary separation or washing steps. To date, a variety of sensitive detection methods have been applied to homogeneous immunoassays, such as optical detection methods, electrochemical detection methods, etc.

[0003] For example, light initiated chemiluminescent assay (LiCA) is a typical homogeneous immunoassay method. It is based on two microspheres coated with antigens or antibodies, which form an immune complex in the liquid phase and bring the two microspheres closer together. Under the excitation of the laser, the transfer of singlet oxygen occurs between the microspheres, thereby generating high-energy red light. The number of photons is converted into the concentration of the target molecule through a single-photon counter and mathematical fitting. When the sample does not contain the target molecule, the immune complex cannot form between the two microspheres, and the distance between the two microspheres exceeds the propagation range of singlet oxygen. The singlet oxygen is quickly quenched in the liquid phase, and no high-energy red light signal is generated during detection. It has the characteristics of rapidity, homogeneity (no washing required), high sensitivity and simple operation. Photochemiluminescence technology has been applied to many detection projects.

[0004] Photochemiluminescence testing is based on a "double-sphere" system, which refers to a system composed of luminescent microspheres and photosensitive microspheres, with both types of microspheres exhibiting excellent suspension properties in the liquid phase. The interaction of the microspheres with antigens or antibodies in the liquid phase perfectly conforms to the dynamics of the liquid state. Luminescent microspheres are monodisperse polymer microspheres with luminescent substances attached to their surfaces or embedded within them. These can be directly or indirectly coupled to antigens or antibodies for immunoassays via active functional groups on their surfaces. Monodisperse polymer microspheres generally refer to particles with a uniform appearance, such as shape and size. Therefore, they are also called polymer microspheres of uniform size.

[0005] According to traditional optical detection theory, the more uniform the particle size of the microspheres used in homogeneous chemiluminescence detection, the better the chemiluminescence detection performance. Therefore, those skilled in the art tend to strive to obtain monodisperse microspheres with more uniform particle sizes. However, with the advancement of the detection industry, the demand for ultrasensitive reagents is increasing, requiring not only extremely high sensitivity but also a very wide detection range. Existing homogeneous chemiluminescence detection methods struggle to meet these requirements.

[0006] Therefore, there is an urgent need to develop a homogeneous chemiluminescence detection method that can meet both sensitivity requirements and linear range requirements. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a homogeneous chemiluminescence detection method in view of the deficiencies in the prior art, which has both ultra-high sensitivity and a wide detection range when used for detection.

[0008] To this end, the first aspect of the present invention provides a homogeneous chemiluminescence detection method, which comprises the following steps:

[0009] Step S1, contacting a sample to be tested with a receptor reagent including receptor particles and a donor reagent including donor particles to generate a test mixture after reaction; controlling the coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent to be ≥5%;

[0010] Step S2: Using energy or an active compound to stimulate chemiluminescence in the test mixture, analyzing the chemiluminescent signal intensity to determine whether the test sample contains the target molecule and / or the concentration of the target molecule in the test sample. 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 controlled to be ≥8%; preferably, the coefficient of variation (CV) of the particle size distribution of the receptor particles in the receptor reagent is controlled to be ≥10%.

[0011] 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%.

[0012] In some embodiments of the present invention, the particle size distribution of the receptor particles in the receptor reagent is polydisperse.

[0013] In some specific embodiments of the present invention, the coefficient of variation CV value of the particle size distribution is calculated by Gaussian distribution.

[0014] 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.

[0015] In some preferred embodiments of the present invention, the receptor reagent comprises receptor particles with at least two average particle size distributions.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] In other embodiments of the present invention, the carrier is a magnetic or non-magnetic particle.

[0023] 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.

[0024] In other embodiments of the present invention, the carrier is aldehyde-modified latex particles.

[0025] 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.

[0026] 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.

[0027] In other embodiments of the present invention, each of the successive polysaccharide layers is spontaneously associated with each of the previous polysaccharide layers.

[0028] 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.

[0029] 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.

[0030] In some embodiments of the invention, the functional groups of the continuous polysaccharide layer alternate between amine functional groups and amine-reactive functional groups.

[0031] In other specific embodiments of the present invention, the amine-reactive functional group is an aldehyde group or a carboxyl group.

[0032] In some embodiments of the invention, the first polysaccharide layer is spontaneously associated with the carrier.

[0033] In other embodiments of the present invention, the outermost polysaccharide layer of the coating has at least one pendant functional group.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] In other embodiments of the present invention, the sample to be tested is diluted with a diluent and then contacted with an acceptor reagent comprising acceptor particles and a donor reagent comprising donor particles.

[0042] In some embodiments of the present invention, the detection wavelength of the chemiluminescence is 520-620 nm.

[0043] In other embodiments of the present invention, red excitation light of 600 to 700 nm is used for laser irradiation.

[0044] 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.

[0045] In other embodiments of the present invention, the reactive oxygen species is singlet oxygen.

[0046] The second aspect of the present invention provides a use of the method according to the first aspect of the present invention in homogeneous chemiluminescence detection for non-diagnostic purposes.

[0047] The third aspect of the present invention provides a homogeneous chemiluminescence analyzer, which uses the method described in the first aspect of the present invention to detect the presence and / or concentration of a target molecule in a sample.

[0048] In some embodiments of the present invention, the homogeneous chemiluminescence detector includes the following components:

[0049] A sample adding mechanism, which is used to add the sample to be tested into the reaction container;

[0050] a reagent adding mechanism for adding an acceptor reagent and / or a donor reagent to the reaction container;

[0051] an incubation module, which is used to provide a suitable temperature for the homogeneous chemiluminescence reaction in the reaction container;

[0052] The detection module is used to detect the chemiluminescent signal generated by the reaction between the receptor particles and the active oxygen.

[0053] In some preferred embodiments of the present invention, the homogeneous chemiluminescence detector is a POCT analyzer.

[0054] A fourth aspect of the present invention provides a method for controlling the POCT analyzer to perform homogeneous chemiluminescence analysis, comprising the following steps:

[0055] Step Q1, contacting a sample to be tested with a receptor reagent comprising receptor particles and a donor reagent comprising donor particles to generate a test mixture after reaction; the donor particles are capable of generating reactive oxygen species when excited; the receptor particles are capable of reacting with the reactive oxygen species to produce a detectable chemiluminescent signal, and the coefficient of variation (CV) of the particle size distribution of the receptor particles in the receptor reagent is ≥5%;

[0056] Step Q2, using excitation light with a wavelength of 600 to 700 nm to excite the test mixture to perform chemiluminescence, and detecting the signal intensity of the chemiluminescence; the detection wavelength of the chemiluminescence is 520 to 620 nm;

[0057] Step Q3: Analyze the signal intensity of the chemiluminescence to determine whether the sample contains the target molecule and / or the concentration of the target molecule in the sample.

[0058] The present invention provides the following beneficial effects: The homogeneous chemiluminescence detection method of the present invention, by adding a receptor reagent containing receptor particles to the sample to be tested, wherein the receptor particle size distribution coefficient of variation (CV) value is ≥5%, significantly improves the detection performance of the method of the present invention compared to existing technologies, achieving both ultra-high sensitivity and a wide detection range. The receptor reagent of the present invention not only has low production costs but also enables high detection precision in homogeneous chemiluminescence detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The present invention will be further described below with reference to the accompanying drawings.

[0060] Figure 1 This is a Gaussian distribution curve of the formaldehyde polystyrene latex microspheres prepared in Example 1.

[0061] Figure 2 This is a Gaussian distribution curve of the formaldehyde polystyrene latex microspheres filled with the luminescent composition prepared in Example 1.

[0062] Figure 3 Gaussian distribution diagram of the dextran-coated aldehyde-based polystyrene latex microspheres filled with the luminescent composition prepared in Example 1

[0063] Figure 4 This is the Gaussian distribution diagram of the receptor particles prepared in Example 1 with an average particle size of about 250 nm.

[0064] Figure 5 This is the Gaussian distribution diagram of the receptor particles with a particle size of about 110 nm prepared in Example 1.

[0065] Figure 6This is the Nicomp distribution diagram of the receptor particles with a particle size of about 110 nm prepared in Example 1.

[0066] Figure 7 This is the Gaussian distribution diagram of the receptor particles with a particle size of about 350 nm prepared in Example 1.

[0067] Figure 8 This is the Nicomp distribution diagram of the receptor particles with a particle size of about 350 nm prepared in Example 1.

[0068] Figure 9 This is the Gaussian distribution diagram of the particle size distribution of the mixed receptor particles in Example 2.

[0069] Figure 10 This is the Nicomp distribution diagram of the particle size distribution of the mixed receptor particles in Example 2. DETAILED DESCRIPTION

[0070] 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.

[0071] 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.

[0072] 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.

[0073] I. Terminology

[0074] 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).

[0075] 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.

[0076] 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.

[0077] 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.

[0078] In the present invention, "capable of binding directly or indirectly" means that the specified entity can specifically bind to the entity (directly), or the specified entity can specifically bind to a specific binding pair member (indirectly).

[0079] The "specific binding pair members" of the present invention refer to a pair of substances that can specifically bind to each other.

[0080] The "coefficient of variation of particle size distribution CV value" mentioned in the present invention refers to the coefficient of variation of particle size in Gaussian distribution in the detection results of nanoparticle size analyzer. The formula for calculating the coefficient of variation is: CV value = (standard deviation SD / mean value) × 100%.

[0081] The term "Nicomp distribution" as used herein refers to an algorithmic distribution used in the American PSS nanoparticle size analyzer NICOMP. Compared to the Gaussian unimodal algorithm, the Nicomp multimodal algorithm offers unique advantages for analyzing multicomponent liquid dispersions with uneven particle size distributions, as well as for analyzing the stability of colloidal systems.

[0082] The term "test sample" as used herein refers to a mixture containing or suspected to contain target molecules to be tested. The test sample that can be used in the present invention includes body fluids, such as blood (which can be anticoagulant blood commonly seen in the blood sample collected), plasma, serum, urine, semen, saliva, cell culture, tissue extracts, etc. Other types of test samples include solvents, seawater, industrial water samples, food samples, environmental samples such as soil or water, plant materials, eukaryotic cells, bacteria, plasmids, viruses, fungi, and cells from prokaryotes. The test sample can be diluted with diluent as needed before use. For example, in order to avoid the HOOK effect, the test sample can be diluted with diluent before being tested on the machine and then tested on the detection instrument.

[0083] As used herein, the term "target molecule to be detected" refers to a substance in a sample to be detected. One or more substances having a specific binding affinity for the target molecule to be detected are used to detect the target molecule. The target molecule to be detected can be a protein, peptide, antibody, or a hapten that can bind to an antibody. The target molecule to be detected can be a nucleic acid or oligonucleotide that binds to a complementary nucleic acid or oligonucleotide. The target molecule to be detected can be any other substance that can form a specific binding pair member. Other typical examples of target molecules to be detected include: drugs such as steroids, hormones, proteins, glycoproteins, mucins, nucleoproteins, phosphoproteins, drugs of abuse, vitamins, antibacterial drugs, antifungal drugs, antiviral drugs, purines, antitumor agents, amphetamines, nitrogen compounds, nucleic acids, and prostaglandins, as well as metabolites of any of these drugs; pesticides and their metabolites; and receptors. Analytes also include cells, viruses, bacteria, and fungi.

[0084] The term "antibody" as used herein is used in the broadest sense and includes 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 comprising an antigen-binding portion of an antibody and a non-antibody protein. Where desired, the antibody may be further conjugated to other moieties, such as a member of a specific binding pair, e.g., biotin or avidin (a member of a biotin-avidin specific binding pair), etc.

[0085] The term "antigen" as used in the present invention refers to a substance that can stimulate the body to produce an immune response and can combine with the immune response products antibodies and sensitized lymphocytes in vivo or in vitro to produce an immune effect.

[0086] As used herein, the term "binding" refers to the direct association 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.

[0087] 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.

[0088] II. Implementation Method

[0089] The present invention will be described in more detail below with reference to the embodiments.

[0090] 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.

[0091] 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.

[0092] Therefore, the homogeneous chemiluminescence detection method involved in the first aspect of the present invention comprises the following steps:

[0093] Step S1, contacting a sample to be tested with a receptor reagent including receptor particles and a donor reagent including donor particles to generate a test mixture after reaction; controlling the coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent to be ≥5%;

[0094] Step S2: using energy or active compounds to excite the test mixture to produce chemiluminescence, analyzing the signal intensity of the chemiluminescence, and determining whether the test sample contains the target molecule and / or the concentration of the target molecule in the test sample.

[0095] 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%.

[0096] 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%.

[0097] 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.

[0098] 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.

[0099] In some embodiments of the present invention, the particle size distribution of the receptor particles in the receptor reagent is polydisperse.

[0100] In some specific embodiments of the present invention, the coefficient of variation CV value of the particle size distribution is calculated by Gaussian distribution.

[0101] 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.

[0102] In some preferred embodiments of the present invention, the receptor reagent comprises receptor particles with at least two average particle size distributions.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] In other embodiments of the present invention, the carrier is a magnetic or non-magnetic particle.

[0110] 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.

[0111] In other embodiments of the present invention, the carrier is aldehyde-modified latex particles.

[0112] 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.

[0113] 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.

[0114] In other embodiments of the present invention, each of the successive polysaccharide layers is spontaneously associated with each of the previous polysaccharide layers.

[0115] 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.

[0116] 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.

[0117] In some embodiments of the invention, the functional groups of the continuous polysaccharide layer alternate between amine functional groups and amine-reactive functional groups.

[0118] In other specific embodiments of the present invention, the amine-reactive functional group is an aldehyde group or a carboxyl group.

[0119] In some embodiments of the invention, the first polysaccharide layer is spontaneously associated with the carrier.

[0120] In other embodiments of the present invention, the outermost polysaccharide layer of the coating has at least one pendant functional group.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] In some preferred embodiments of the present invention, in step S1, the sample to be tested is first mixed with an acceptor reagent and then mixed with a donor reagent.

[0128] 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.

[0129] 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 test sample is diluted with a diluent and then contacted with an acceptor reagent including acceptor particles and a donor reagent including donor particles.

[0130] In some embodiments of the present invention, the chemiluminescence detection wavelength is 520-620 nm, preferably 610-620 nm, and more preferably 615 nm.

[0131] 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.

[0132] 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.

[0133] In other embodiments of the present invention, the reactive oxygen species is singlet oxygen.

[0134] It should be noted that the method of the present invention can be used for double antigen sandwich detection of antibodies or double antibody sandwich detection of antigens. In addition, the method can also be used for competitive detection of antigens or antibodies, indirect detection of antigens or antibodies, and capture detection of antigens or antibodies.

[0135] The second aspect of the present invention relates to the use of the method according to the first aspect of the present invention in homogeneous chemiluminescence detection for non-diagnostic purposes.

[0136] The third aspect of the present invention relates to a homogeneous chemiluminescence analyzer, which uses the method described in the first aspect of the present invention to detect the presence and / or concentration of a target molecule in a sample.

[0137] In some embodiments of the present invention, the homogeneous chemiluminescence detector includes the following components:

[0138] A sample adding mechanism, which is used to add the sample to be tested into the reaction container;

[0139] a reagent adding mechanism for adding an acceptor reagent and / or a donor reagent to the reaction container;

[0140] an incubation module, which is used to provide a suitable temperature for the homogeneous chemiluminescence reaction in the reaction container;

[0141] The detection module is used to detect the chemiluminescent signal generated by the homogeneous chemiluminescent reaction.

[0142] In some preferred embodiments of the present invention, the homogeneous chemiluminescence detector is a POCT analyzer. A POCT analyzer herein refers to a point-of-care testing instrument for clinical testing (bedside testing) performed at the patient's side. The principle of the homogeneous immunoassay POCT analyzer is as follows: the biomolecules to be tested in the test sample react with donor particles and acceptor particles to form an immune complex. This interaction draws the donor and acceptor particles closer together. Under irradiation with a laser (wavelength of 680 nm), the sensitizer in the donor particles converts oxygen in the surrounding environment into more active singlet oxygen. Singlet oxygen diffuses into the acceptor particles, reacts with the chemiluminescent agent in the acceptor particles, and further activates the luminescent groups also on the acceptor particles, causing them to emit light with a wavelength of 520-620 nm. The half-life of singlet oxygen is 4 μSec, and the diffusion distance in solution is approximately 200 nm. If the biomolecules do not interact, singlet oxygen cannot diffuse into the acceptor particles, and no light signal is generated. Therefore, by measuring the light intensity emitted by the mixture, the concentration of the target molecule in the sample can be calculated.

[0143] The POCT analyzer includes a sample loading mechanism, a reagent loading mechanism, an incubation module, a detection module, and a circuit control module; the sample loading mechanism, reagent loading mechanism, 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 reagent card and the substances within the reagent card, the reagent loading mechanism is used to transfer the substances within the reagent card, and the detection module is used to emit laser light and measure the light intensity emitted by the sample to be tested.

[0144] In other embodiments of the present invention, the sample to be tested is selected from materials suspected of containing the target molecule to be tested, including but not limited to: blood, serum, plasma, sputum, lymph, semen, vaginal mucus, feces, urine or cerebrospinal fluid.

[0145] A fourth aspect of the present invention relates to a method for controlling the POCT analyzer to perform homogeneous chemiluminescence analysis, comprising the following steps:

[0146] Step Q1, contacting a sample to be tested with a receptor reagent comprising receptor particles and a donor reagent comprising donor particles to generate a test mixture after reaction; the donor particles are capable of generating reactive oxygen species when excited; the receptor particles are capable of reacting with the reactive oxygen species to produce a detectable chemiluminescent signal, and the coefficient of variation (CV) of the particle size distribution of the receptor particles in the receptor reagent is ≥5%;

[0147] Step Q2, using excitation light with a wavelength of 600 to 700 nm to excite the test mixture to perform chemiluminescence, and detecting the signal intensity of the chemiluminescence; the detection wavelength of the chemiluminescence is 520 to 620 nm;

[0148] Step Q3: Analyze the signal intensity of the chemiluminescence to determine whether the sample contains the target molecule and / or the concentration of the target molecule in the sample.

[0149] III. Examples

[0150] Example 1: Preparation of Receptor Particle Solution Conjugated with Antibody I (PCT Antibody) (I) Preparation of Receptor Particles Conjugated with Antibody with an Average Particle Size of About 250 nm

[0151] 1.1 Preparation and characterization of formaldehyde-based polystyrene latex microspheres

[0152] 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;

[0153] 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.

[0154] 3) The reaction system was heated to 70°C and reacted for 15 hours;

[0155] 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.

[0156] 5) The Gaussian distribution average particle size of the latex microspheres measured by the nanoparticle size analyzer is 202.2 nm, the coefficient of variation (CV) is 4.60%, and the Gaussian distribution curve is as follows: Figure 1 The aldehyde content of the latex microspheres was determined to be 280 nmol / mg by conductometric titration.

[0157] 1.2 Filling process and characterization of luminescent composition

[0158] 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;

[0159] 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.

[0160] 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;

[0161] 4) Centrifuging the emulsion for 1 hour at 30,000 G, discarding the supernatant to obtain formaldehyde-based polystyrene microspheres embedded with the luminescent composition.

[0162] 5) The Gaussian distribution average particle size of the microspheres measured by the nanoparticle size analyzer is 204.9 nm, and the coefficient of variation (CV) is 5.00% (as shown in FIG. Figure 2 shown).

[0163] 1.3 Surface coating of receptor particles with dextran

[0164] 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.

[0165] 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;

[0166] 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.

[0167] 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.

[0168] 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.

[0169] 6) Add the above microspheres to the aldehyde dextran solution and stir for 2 hours;

[0170] 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.

[0171] 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.

[0172] 9) The Gaussian distribution average particle size of the microspheres measured by the nanoparticle size analyzer is 241.6 nm, and the coefficient of variation (CV) is 12.90% (as shown in FIG. Figure 3 shown).

[0173] 1.4 Antibody Conjugation Process

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] 5) The Gaussian distribution average particle size of the microspheres measured by the nanoparticle size analyzer is 253.5 nm, and the coefficient of variation (CV) is 9.60%. Figure 4 shown).

[0179] (II) Preparation of antibody-coupled receptor particles with an average particle size of approximately 110 nm

[0180] 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 5 The average particle size is 107.1 nm, and the coefficient of variation (CV) is 7.6%. The Nicomp distribution is unimodal (as shown in FIG. Figure 6 shown).

[0181] (III) Preparation of antibody-coupled receptor particles with an average particle size of approximately 350 nm

[0182] 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 7 The average particle size is 347.5 nm, the coefficient of variation (CV) is 3.9%, and the Nicomp distribution is unimodal (as shown in FIG. Figure 8 shown).

[0183] Example 2: Determination of sensitivity and upper limit of detection of the method of the present invention

[0184] 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.

[0185] (1) PCT antigen was diluted to a series of concentrations of 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 80 pg / ml, 160 pg / ml, 500 pg / ml, 1000 pg / ml, 5000 pg / ml, 20000 pg / ml, 50000 pg / ml, 100000 pg / ml and 200000 pg / ml. The receptor reagent (concentration of 100 μg / ml) containing receptor particles of different average particle sizes (110 nm and 350 nm) coupled to PCT antibody I prepared in Example 1 was used, and then the PCT antigen of the above concentration series was detected with the same biotin-labeled PCT 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.

[0186] Table 1

[0187]

[0188]

[0189] As can be seen from Table 1, the upper detection limit of the receptor particles with an average particle size of 110 nm is higher, but the sensitivity is lower, while the receptor particles with an average particle size of 350 nm have the best sensitivity, but the upper detection limit is lower.

[0190] (2) A solution of receptor particles with an average particle size of 110 nm coupled to PCT antibody I was mixed with a solution of receptor particles with an average particle size of 350 nm coupled to PCT 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:

[0191] The average particle size of the Gaussian distribution is 317.7 nm, and the coefficient of variation (CV value) of the particle size distribution is 37.2% (e.g. Figure 9 shown);

[0192] The Nicomp distribution is bimodal: #1: average particle size 103.1nm, coefficient of variation (CV value) = 11.8%; #2: average particle size 328.8nm, coefficient of variation (CV value) of particle size distribution = 13.0% (e.g. Figure 10 shown).

[0193] The above-mentioned new receptor reagent was used with biotin-labeled PCT monoclonal antibody 2 (diluted to 2ug / ml) and universal solution (reagent containing donor particles) to detect the above-mentioned concentration series of PCT antigen. The detection sensitivity and detection limit of the photochemiluminescence analysis system developed by Boyang Biotechnology (Shanghai) Co., Ltd. are shown in Table 2.

[0194] Table 2

[0195]

[0196] 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.

[0197] Example 3: Preparation of a series of receptor particle solutions of coupled antibody I (PCT antibody) with an average particle size of about 250 nm and different coefficients of variation of particle size distribution

[0198] According to the method described in Example 1 (I), receptor particle solutions of coupled antibody I (PCT antibody) with different coefficients of variation of particle size distribution were obtained.

[0199] Specifically:

[0200] Receptor particles 1: Gaussian distribution average particle size is 251.2 nm, particle size distribution coefficient of variation CV value = 3.7%; Nicomp distribution is unimodal.

[0201] Receptor particles 2: Gaussian distribution average particle size is 254.9 nm, particle size distribution coefficient of variation CV value = 5.0%; Nicomp distribution is unimodal.

[0202] Receptor particles 3: Gaussian distribution average particle size is 251.3 nm, particle size distribution coefficient of variation CV value = 8.0%; Nicomp distribution is unimodal.

[0203] Receptor particles 4: Gaussian distribution average particle size is 251.9 nm, particle size distribution coefficient of variation CV value = 10.5%; Nicomp distribution is unimodal.

[0204] Receptor particles 5: Gaussian distribution average particle size is 252.3 nm, particle size distribution coefficient of variation CV value = 16.8%; Nicomp distribution is unimodal.

[0205] Receptor particles 6: Gaussian distribution average particle size is 240.8 nm, particle size distribution coefficient of variation CV value = 34.5%; Nicomp distribution is bimodal.

[0206] Example 4: Determination of sensitivity and upper limit of detection of the method of the present invention

[0207] 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.

[0208] (1) The PCT antigen was diluted to a series of concentrations of 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 80 pg / ml, 160 pg / ml, 500 pg / ml, 1000 pg / ml, 5000 pg / ml, 20000 pg / ml, 50000 pg / ml, 100000 pg / ml and 200000 pg / ml. The receptor reagent (concentration of 100 μg / ml) containing receptor particles coupled to PCT antibody I prepared in Example 3 was used respectively. Then, the PCT antigen in the above concentration series was detected with the same biotin-labeled PCT 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 3.

[0209] Table 3

[0210]

[0211]

[0212] 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.

[0213] Example 5: Detection of cTnI and PCT marker standards

[0214] 5.1 Detection of cTnI Antigen Standards

[0215] cTnI antigen was diluted to a series of concentrations 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, 1000 ng / ml, and 1000 ng / ml. Different particle sizes (50 nm, 80 nm, 110 nm, 140 nm, 170 nm, 2 The receptor reagent (concentration of 100 μg / ml, CV values ​​of approximately 10%) containing cTnI monoclonal antibody 1 was coated with acceptor particles (00 nm, 250 nm, 300 nm, 350 nm, and 400 nm) and the same biotinylated cTnI monoclonal antibody 2 (diluted to 2 μg / ml) and universal solution (containing donor particle solution) was used to detect the cTnI antigen in the above concentration series. 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.

[0216] Table 4

[0217]

[0218] As shown in Table 4, the cTnI test results 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, and the results are shown in Table 5.

[0219] Table 5

[0220]

[0221] As can be seen from Table 5, by adding a receptor reagent formed by combining receptor particles with a smaller average particle size and receptor particles with a larger average particle size, this method has both high sensitivity and a high upper detection limit (wide detection range), showing the advantages of large-size receptor particles and small-size receptor particles. Compared with receptor particles with a single average particle size distribution, the performance of the receptor reagent containing receptor particles with two or more average particle sizes is greatly improved.

[0222] 5.2 Detection of PCT Antigen Standards

[0223] PCT antigen was diluted to a series of concentrations of 20pg / ml, 30pg / ml, 40pg / ml, 50pg / ml, 60pg / ml, 80pg / ml, 160pg / ml, 500pg / ml, 1000pg / ml, 5000pg / ml, 20000pg / ml, 100000pg / ml and 2000ng / ml, and different particle sizes (50nm, 80nm, 110nm, 140nm, 170nm, 20 The PCT antigen in the above concentration series was detected using a receptor reagent (concentration of 100 μg / ml, CV values ​​of about 10%) coated with PCT monoclonal antibody 1 (donor particles with diameters of 0 nm, 250 nm, 300 nm, 350 nm, and 400 nm) and the same biotinylated PCT 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 6.

[0224] Table 6

[0225]

[0226]

[0227] As shown in Table 6 for the PCT test results, the 110 nm receptor particles have a high upper limit of detection but poor sensitivity, while the 300 nm and 350 nm receptor particles have the best sensitivity but a lower upper limit of detection. The 110 nm receptor particles were mixed with the 300 nm and 350 nm receptor particles to form receptor reagents. The sensitivity and upper limit of detection of the method containing the corresponding receptor reagents were measured using a photochemiluminescence analysis system developed by Boyang Biotechnology (Shanghai) Co., Ltd. The results are shown in Table 7.

[0228] Table 7

[0229]

[0230] As can be seen from Table 7, by adding a receptor reagent formed by combining small-size receptor particles with similar CV value distribution and large-size receptor particles, this method has both high sensitivity and a high detection limit (wide detection range), showing the advantages of large-size receptor particles and small-size receptor particles. Compared with receptor particles of a single particle size, the performance of the receptor reagent containing receptor particles of two or more particle sizes is greatly improved.

[0231] Example 6: Detection of cTnI marker levels in samples from healthy individuals and patients suspected of having myocardial injury

[0232] In this example, 40 clinical samples (13 negative and 27 positive) were tested using a cTnI quantitative assay kit (photochemiluminescence) consisting of 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. R1 was a receptor reagent (at a concentration of 100 μg / ml) prepared using receptor particles with an average particle size of approximately 250 nm and a particle size distribution coefficient of variation (CV) of 11%.

[0233] The detection process is completed and the test results are output on a fully automatic photochemiluminescence analysis system developed by Boyang Biotechnology (Shanghai) Co., Ltd. The specific detection steps include:

[0234] a. Add clinical samples to the reaction wells;

[0235] b. Add R1 and R2 to the reaction wells in sequence;

[0236] c. Incubation;

[0237] d. Add R3 to the reaction well;

[0238] e. Incubation;

[0239] f. Irradiate the reaction wells with laser and calculate the amount of photons emitted from each well;

[0240] g. Calculate the cTnI concentration in the test sample.

[0241] 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 8 below:

[0242] Table 8

[0243]

[0244]

[0245] 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.

[0246] Example 7: Detection of PCT marker levels in samples from normal subjects and patients suspected of having inflammation

[0247] In this example, 40 clinical samples were tested using a PCT quantitative assay kit (photochemiluminescence) consisting of Reagent 1 (R1') containing acceptor microparticles coated with a primary anti-PCT antibody, Reagent 2 (R2') containing a biotin-labeled secondary anti-PCT antibody, and a universal solution (R3') containing donor particles. R1 was an acceptor reagent (at a concentration of 200 μg / ml) prepared using acceptor particles 4 (particle size distribution coefficient of variation CV = 10.5%) described in Example 3.

[0248] The specific experimental steps are as follows:

[0249] 1. Select 40 clinical samples, equilibrate to room temperature, and mix thoroughly;

[0250] 2. Add the mixed sample, prepared R1' and R2' to an 8×12 white plate;

[0251] 3. Place the sample-loaded white plate into the LiCA HT instrument for reaction. The reaction mode used is as follows:

[0252] (1) Mix 40ul sample, 15ul R1' and 15ul R2';

[0253] (2) Incubate at 37°C for 8 min;

[0254] (3) Add 160 μl of universal solution (R3');

[0255] (4) Incubate at 37°C for 2 min;

[0256] (5) Excitation readings. The specific test results are shown in Table 9 below.

[0257] Table 9

[0258]

[0259]

[0260] Data comparison revealed that the Roche values ​​correlated well with the values ​​measured in Example 7, with a slope of 0.9977 and a slope of 0.9984. Samples 1-11 were obtained from healthy patients undergoing physical examinations, with a distribution range of 30 pg / ml to 70 pg / ml, and a median of 50 pg / ml. Samples 12-40 were obtained from patients identified as having inflammation, with a distribution range of 120 pg / ml to 63.23 ng / ml, and a median of 580 pg / ml.

[0261] 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. A homogeneous chemiluminescence analysis method comprising the following steps: Step S1, contacting a sample to be tested with a receptor reagent comprising receptor particles and a donor reagent comprising donor particles to generate a test mixture after reaction; the receptor particles comprise a luminescent composition and a carrier, the luminescent composition being filled in the carrier and / or attached to the carrier; the receptor reagent comprises receptor particles having at least two average particle size distributions; and the coefficient of variation (CV) value of the particle size distribution of the receptor particles in the receptor reagent is controlled to be ≥5% and ≤16.8%. Step S2: using energy or active compounds to excite the test mixture to produce chemiluminescence, analyzing the signal intensity of the chemiluminescence, and determining whether the test sample contains the target molecule and / or the concentration of the target molecule in the test sample.

2. The method according to claim 1, characterized in that The coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent is controlled to be ≥8%.

3. The method according to claim 2, characterized in that The coefficient of variation CV value of the particle size distribution of the receptor particles in the receptor reagent is controlled to be ≥10%.

4. The method according to any one of claims 1 to 3, characterized in that The particle size distribution of the receptor particles in the receptor reagent is polydisperse.

5. The method according to any one of claims 1 to 3, characterized in that The coefficient of variation CV value of the particle size distribution is calculated through Gaussian distribution.

6. The method according to any one of claims 1 to 3, 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.

7. The method 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.

8. The method according to claim 7, characterized in that The chemiluminescent compound is selected from olefin compounds.

9. The method according to claim 7, 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.

10. The method according to claim 9, characterized in that The chemiluminescent compound is selected from dimethylthiophene and its derivatives.

11. The method according to any one of claims 7 to 10, characterized in that The metal of the metal chelate is a rare earth metal or a Group VIII metal.

12. The method according to claim 11, characterized in that The metal of the metal chelate is selected from the group consisting of europium, terbium, dysprosium, samarium, osmium and ruthenium.

13. The method according to claim 12, characterized in that The metal of the metal chelate is europium.

14. The method according to any one of claims 7 to 10, 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.

15. The method according to claim 1, wherein The carrier is selected from beads.

16. The method according to claim 1, wherein The carrier is selected from microspheres.

17. The method according to claim 1, wherein The carrier is a magnetic or non-magnetic particle.

18. The method according to claim 1, wherein The support material is selected from natural, synthetic or modified naturally occurring polymers.

19. The method according to claim 18, 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.

20. The method according to claim 18, wherein The carrier is aldehyde-modified latex particles.

21. The method according to claim 1, wherein The average particle size of the carrier is in the range of 50 nm to 1 μm.

22. The method according to claim 21, characterized in that The average particle size of the carrier is in the range of 100 nm to 500 nm.

23. The method according to claim 22, characterized in that The average particle size of the carrier is in the range of 150 nm to 400 nm.

24. The method according to claim 23, wherein The average particle size of the carrier is in the range of 190 nm to 300 nm.

25. The method according to claim 1, wherein 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.

26. The method according to claim 25, characterized in that Each of the successive polysaccharide layers is spontaneously associated with each of the previous polysaccharide layers.

27. The method according to claim 25 or 26, 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.

28. The method according to claim 25 or 26, 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.

29. The method according to claim 28, characterized in that The functional groups of the continuous polysaccharide layer alternate between amine functional groups and amine-reactive functional groups.

30. The method according to claim 29, wherein The amine-reactive functional group is an aldehyde group or a carboxyl group.

31. The method according to claim 25 or 26, characterized in that The first polysaccharide layer is spontaneously associated with the carrier.

32. The method according to any one of claims 25 or 26, wherein: The outermost polysaccharide layer of the coating has at least one pendant functional group.

33. The method according to any one of claims 25 or 26, 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.

34. The method according to claim 33, wherein The side functional groups of the outermost polysaccharide layer of the coating are selected from aldehyde groups and / or carboxyl groups.

35. The method according to claim 32, wherein 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.

36. The method according to claim 32, wherein 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.

37. The method according to claim 36, wherein 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.

38. The method according to claim 37, wherein The specific binding pair member is biotin-avidin.

39. The method according to claim 25 or 26, characterized in that The polysaccharide is selected from carbohydrates containing three or more unmodified or modified monosaccharide units.

40. The method according to claim 39, wherein The polysaccharide is selected from the group consisting of dextran, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxydextran and aminodextran.

41. The method according to claim 40, wherein The polysaccharide is selected from the group consisting of dextran, starch, glycogen and polyribose.

42. The method according to claim 1, wherein The donor particle is directly or indirectly linked to a reporter molecule or bound to a member of a specific binding pair.

43. The method according to claim 1, wherein The sample to be tested is diluted with a diluent and then brought into contact with a receptor reagent comprising receptor particles and a donor reagent comprising donor particles.

44. The method according to claim 1, wherein The detection wavelength of the chemiluminescence is 520-620 nm.

45. The method according to claim 1, wherein Red excitation light of 600~700nm was used for laser irradiation.

46. ​​The method according to claim 1, wherein The concentration of the receptor particles in the receptor reagent is 1 ug / mL-1000 ug / mL.

47. The method according to claim 46, wherein The concentration of the receptor particles in the receptor reagent is 10ug / mL-500ug / mL.

48. The method according to claim 47, wherein The concentration of the receptor particles in the receptor reagent is 20ug / mL-200ug / mL.

49. The method according to claim 1, wherein The active oxygen is singlet oxygen.

50. Use of the method according to any one of claims 1 to 49 in homogeneous chemiluminescence detection for non-diagnostic purposes.

51. A method for controlling a POCT analyzer to perform homogeneous chemiluminescence analysis, comprising the following steps: Step Q1: contacting a sample to be tested with a receptor reagent comprising receptor particles and a donor reagent comprising donor particles to generate a test mixture after reaction; the donor particles are capable of generating reactive oxygen species when excited; the receptor particles are capable of reacting with the reactive oxygen species to produce a detectable chemiluminescent signal; the receptor particles comprise 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; and the coefficient of variation of the particle size distribution of the receptor particles in the receptor reagent is 5% ≤ CV value ≤ 16.8%; Step Q2, using excitation light with a wavelength of 600-700 nm to excite the test mixture to perform chemiluminescence, and detecting the signal intensity of the chemiluminescence; the detection wavelength of the chemiluminescence is 520-620 nm; Step Q3: judging whether the sample to be tested contains the target molecule to be tested and / or the concentration of the target molecule to be tested in the sample to be tested based on the analysis of the chemiluminescence signal intensity.

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