A receptor reagent and uses thereof

Abstract

The present application relates to a kind of receptor reagent and its application.The receptor reagent provided by the present application includes buffer solution and the receptor particles suspended therein, the receptor particles can be reacted with active oxygen to produce chemiluminescence, characterized in that, the ZETA potential of the receptor particles is not higher than-10mV, and not lower than-50mV;The sugar content of each milligram of receptor particles is not higher than 25 micrograms.The receptor reagent provided by the present application can be mass-produced, low in cost, qualified in quality and stable in performance, can meet the sensitivity requirement, can also meet the linear range requirement;The kit comprising the receptor reagent has the advantages of strong sample anti-interference ability and good testing performance.

Description

Technical Field

[0001] This invention relates to the field of chemiluminescence detection, and more specifically, to a receptor reagent and its application. Background Technology

[0002] Immunoassay has evolved over half a century, resulting in numerous types. Based on whether the analyte is separated from the reaction system during the assay, it can be divided into heterogeneous and homogeneous immunoassays. Heterogeneous immunoassays involve introducing a probe for labeling, where various reagents are mixed and reacted, requiring separation to separate the analyte from the reaction system before detection. This is currently the mainstream method in immunoassays. Examples include the well-known enzyme-linked immunosorbent assay (ELISA) and magnetic particle chemiluminescence immunoassay. Homogeneous immunoassays, on the other hand, involve directly measuring the analyte after mixing and reacting with the relevant reagents in the reaction system, without any additional separation or washing steps. Currently, various sensitive detection methods are applied to homogeneous immunoassays, such as optical and electrochemical detection methods.

[0003] For example, Light-Initiated Chemiluminescence Assay (LiCA) is a typical homogeneous immunoassay method. It is based on the formation of an immune complex between two microspheres coated with antigens or antibodies in a liquid phase, bringing the two microspheres closer together. Under laser excitation, singlet oxygen transfer occurs between the microspheres, generating high-energy red light. The photon count is converted into target molecule concentration using a single-photon counter and mathematical fitting. When the sample does not contain target molecules, an immune complex cannot form between the two microspheres, and the distance between the microspheres exceeds the singlet oxygen propagation range. The singlet oxygen is rapidly quenched in the liquid phase, and no high-energy red light signal is generated during detection. It is characterized by its speed, homogeneity (no rinsing required), high sensitivity, and simple operation. Light-initiated chemiluminescence technology has been applied to many detection methods.

[0004] According to traditional optical detection theory, the more uniform the particle size of the microspheres used in homogeneous chemiluminescence detection, the better the performance of the chemiluminescence detection using those microspheres. Therefore, those skilled in the art tend to strive to obtain monodisperse microsphere systems with more uniform particle size. 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.

[0005] Therefore, there is an urgent need to develop a receptor reagent that can be mass-produced, is low in cost, has qualified quality, and stable performance, and can meet both sensitivity and linear range requirements. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a receptor reagent that addresses the shortcomings of the prior art. This receptor reagent can be mass-produced, is low in cost, has qualified quality and stable performance, and the kit containing this receptor reagent has the advantages of strong sample anti-interference ability and good testing performance.

[0007] To this end, the first aspect of the present invention provides a receptor reagent comprising a buffer solution and receptor particles suspended therein, the receptor particles being capable of reacting with reactive oxygen species to produce chemiluminescence, characterized in that the zeta potential of the receptor particles is not higher than -10mV and not lower than -50mV; and the sugar content per milligram of receptor particles is not higher than 25 micrograms.

[0008] In some preferred embodiments of the present invention, the ZETA potential of the receptor particles in the receptor reagent is not higher than -15mV and not lower than -40mV.

[0009] In some specific embodiments of the present invention, the ZETA potential of the receptor particles in the receptor reagent is selected from -10mV, -20mV, -30mV, -40mV, and -50mV.

[0010] In some embodiments of the present invention, the sugar content in each milligram of the receptor particle is not higher than 15 micrograms.

[0011] In some specific embodiments of the present invention, the sugar content in each milligram of the receptor particle can be 3 micrograms, 6 micrograms, 9 micrograms, 12 micrograms, 15 micrograms, 18 micrograms, 21 micrograms, or 24 micrograms.

[0012] In some embodiments of the present invention, the receptor particle includes a carrier, the interior of which is filled with a luminescent composition, and the surface of which is bonded with biomolecules.

[0013] In some embodiments of the present invention, the surface of the carrier has bonding functional groups that are bonded to biomolecules.

[0014] In some embodiments of the present invention, the bonding functional group is selected from at least one of amino, amide, hydroxyl, aldehyde, carboxyl, maleimide and thiol, preferably selected from aldehyde and / or carboxyl.

[0015] In some embodiments of the present invention, the surface of the carrier has aldehyde groups. The aldehyde groups react with amino groups on the biomolecules to form Schiff base bonds, thereby bonding the biomolecules to the surface of the carrier.

[0016] In some embodiments of the invention, the surface of the carrier has carboxyl groups. These carboxyl groups react with amino groups on biomolecules to form amide bonds, thereby attaching avidin molecules to the surface of the carrier.

[0017] In some embodiments of the present invention, the surface of the carrier is not coated with sugar molecules, but directly bonded with biomolecules.

[0018] In some embodiments of the present invention, the biomolecule is capable of specifically binding to the analyte molecule.

[0019] 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 not less than 5% and not more than 20%.

[0020] In some preferred embodiments of the present invention, the coefficient of variation (CV) of the particle size distribution of the receptor particles in the receptor reagent is not higher than 15%.

[0021] In some preferred embodiments of the present invention, the coefficient of variation (CV) of the particle size distribution of the receptor particles in the receptor reagent is not less than 8%.

[0022] In some specific embodiments of the present invention, the coefficient of variation (CV) of the particle size distribution of the receptor particles in reagent 1 can be selected from 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, and 25%.

[0023] In other embodiments of the invention, the receptor particles exhibit polydispersity in the size distribution of the receptor reagent.

[0024] In some embodiments of the present invention, the particle size of the carrier is selected from 100 to 400 nm, preferably 150 to 350 nm, and more preferably 180 to 220 nm.

[0025] In some embodiments of the present invention, the concentration of the receptor particles in the receptor reagent is from 10 μg / ml to 1 mg / ml, preferably from 20 μg / ml to 500 μg / ml, and more preferably from 50 μg / ml to 200 μg / ml.

[0026] In some embodiments of the present invention, the receptor reagent further includes a buffer solution with a pH of 7.0 to 9.0, in which the receptor particles are suspended.

[0027] In some embodiments of the present invention, the buffer solution contains a polysaccharide selected from carbohydrates containing three or more unmodified or modified monosaccharide units, preferably selected from dextran, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxyglucan and aminoglucan; more preferably selected from dextran, starch, glycogen and polyribose.

[0028] In some embodiments of the present invention, the molecular weight distribution Mw of the polysaccharide (e.g., dextran) is selected from 10,000 to 1,000,000 Da, preferably from 100,000 to 800,000 Da, and more preferably from 300,000 to 700,000 Da.

[0029] In some embodiments of the present invention, the sugar content in each liter of the buffer solution is not less than 0.2g and not more than 2g.

[0030] In some embodiments of the present invention, the sugar content in each liter of the buffer solution is not less than 0.5g and not more than 1.5g.

[0031] In other embodiments of the invention, the surface of the carrier is coated with a layer of at least two consecutive polysaccharide layers.

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

[0033] In some embodiments of the invention, each of the continuous polysaccharide layers is spontaneously associated with each of the preceding polysaccharide layers.

[0034] In some embodiments of the present invention, the polysaccharide has side group functional groups, and the functional groups of the continuous polysaccharide layers carry opposite charges to the functional groups of the preceding polysaccharide layer.

[0035] In some embodiments of the present invention, the polysaccharide has side group functional groups, and the continuous polysaccharide layer is covalently linked to the previous polysaccharide layer through a reaction between the functional groups and the functional groups of the previous polysaccharide layer.

[0036] In some embodiments of the invention, the functional groups of the continuous polysaccharide layer alternate between amine functional groups and amine reactive functional groups.

[0037] In some embodiments of the present invention, the amine reactive functional group is an aldehyde group or a carboxyl group.

[0038] In some embodiments of the present invention, the first polysaccharide layer is spontaneously associated with the carrier.

[0039] In some embodiments of the present invention, the outermost polysaccharide layer of the coating has at least one side functional group.

[0040] In some embodiments of the present invention, the side group functional groups of the outermost polysaccharide layer of the coating are selected from at least one of aldehyde, carboxyl, mercapto, amino, hydroxyl and maleamine groups; preferably selected from aldehyde and / or carboxyl groups.

[0041] In some embodiments of the present invention, the side group functional groups of the outermost polysaccharide layer of the coating are directly or indirectly chemically bonded to one of the paired members.

[0042] In some embodiments of the present invention, the polysaccharide is selected from carbohydrates containing three or more unmodified or modified monosaccharide units; preferably selected from dextran, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxyglucan and aminoglucan; more preferably selected from dextran, starch, glycogen and polyribose.

[0043] In some embodiments of the present invention, the method for preparing the receptor particles includes the following steps:

[0044] Step S1 involves reacting receptor microspheres containing bonded functional groups on the surface of the carrier with biomolecules in the presence of an activator to obtain an intermediate product.

[0045] Step S2: Add a sealing agent to seal the intermediate product obtained in step S1.

[0046] Step S3: The intermediate product after the sealing treatment in step S2 is washed to obtain receptor particles with biomolecules bonded to their surface.

[0047] In some embodiments of the present invention, the density of bonded functional groups on the surface of the support in step S1 is not less than 10 nmol / mg.

[0048] In some other embodiments of the present invention, the density of bonded functional groups on the surface of the carrier in step S1 is not less than 30 nmol / mg.

[0049] In some embodiments of the present invention, the mass ratio of the receptor microspheres to the biomolecules in step S1 is 10:(0.3 to 0.9).

[0050] In some other embodiments of the present invention, the mass ratio of the receptor microspheres to the biomolecules in step S1 is 10:(0.6 to 0.8).

[0051] In some embodiments of the present invention, the final concentration of the sealing agent after adding the sealing agent in step S2 is not less than 5 wt%.

[0052] In some other embodiments of the present invention, the final concentration of the sealing agent after adding the sealing agent in step S2 is not less than 10 wt%.

[0053] A second aspect of the present invention provides a chemiluminescence detection kit comprising the receptor reagent as described in the first aspect of the present invention.

[0054] In some embodiments of the present invention, the reagent kit has at least one reagent strip, the reagent strip having a plurality of reagent wells for holding reagents, wherein at least one reagent well is used to hold the receptor reagent.

[0055] In some embodiments of the present invention, the chemical yellowing detection kit, in addition to the receptor reagent, also includes other reagents as needed, depending on the target or detection method, such as donor reagents, biotin-coated secondary antibodies, diluents, etc. In the field of in vitro diagnostics, especially in immunoassay, manufacturers typically label or abbreviate the components in different vials of the kit as Reagent 1 or R1, Reagent 2 or R2, Reagent 3 or R3, and so on, to simplify the naming of different components in commercial kits. This facilitates customer identification, assembly, and use, and also serves the purpose of technical confidentiality. Therefore, kits from different in vitro diagnostic manufacturers may all contain Reagent 1, Reagent 2, Reagent 3, etc., but the specific components of each reagent differ between manufacturers.

[0056] A third aspect of the present invention provides the application of the receptor reagent according to the first aspect of the present invention or the kit according to the second aspect of the present invention in a chemiluminescence analyzer.

[0057] A fourth aspect of the present invention provides the application of the receptor reagent according to the first aspect of the present invention or the kit according to the second aspect of the present invention in a POCT instrument. POCT refers to on-site rapid testing or clinical testing performed at the patient's side.

[0058] The present invention also provides the application of the receptor reagent according to the first aspect of the present invention or the kit according to the second aspect of the present invention in in vitro diagnosis of diseases or non-diseases.

[0059] The beneficial effects of this invention are as follows:

[0060] The receptor reagent provided by this invention can be mass-produced, is low in cost, has qualified quality and stable performance, and can meet both sensitivity requirements and linear range requirements; the kit containing this receptor reagent has the advantages of strong sample anti-interference ability and good test performance. Attached Figure Description

[0061] Figure 1 This is a ZETA potential distribution diagram of experimental group 5 according to Embodiment 2 of the present invention. Detailed Implementation

[0062] To facilitate understanding of the present invention, it will be described in detail below. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terminology used herein is for describing specific embodiments only and is not intended to be restrictive.

[0063] Where numerical ranges are provided, it should be understood that every intermediate value between the upper and lower limits of the range and any other specified or intermediate value within the specified range is covered by this invention. The upper and lower limits of these smaller ranges may be independently included in the smaller range and are also covered by this invention, subject to any explicitly excluded limits within the specified range. Where a specified range includes one or two limits, the range excluding any or both of those included limits is also included by this invention.

[0064] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of this invention, preferred methods and materials are now described.

[0065] I. Terminology

[0066] The term "reactive oxygen species" as used in this invention refers to a general term for substances composed of oxygen in the body or natural environment that contain oxygen and are reactive in nature. It is mainly an excited-state oxygen molecule, including the one-electron reduction product of oxygen, superoxide anion (O2·-), the two-electron reduction product, hydrogen peroxide (H2O2), the three-electron reduction product, hydroxyl radical (·OH), as well as nitric oxide and singlet oxygen (1O2), etc.

[0067] As used in this invention, "receptor particle" refers to a compound particle containing a compound capable of reacting with reactive oxygen species to generate a detectable signal. The receptor particle is induced and activated by energy or an active compound, releasing high-energy reactive oxygen species. These high-energy reactive oxygen species are captured by nearby receptor particles, thereby transferring energy to activate the receptor particle. In one specific embodiment, the receptor particle comprises a luminescent composition and a carrier, wherein the luminescent composition is filled in the carrier and / or coated on the surface of the carrier.

[0068] The "carrier" described in this invention is selected from tapes, sheets, rods, tubes, pores, microtiter plates, beads, particles, and microspheres. It can be any microsphere or microparticle known to those skilled in the art, can be of any size, can be organic or inorganic, can be expandable or non-expandable, can be porous or non-porous, and can have any density, but preferably a density close to that of water. It is preferably buoyant in water and is composed of transparent, partially transparent, or opaque materials. The carrier may or may not have a charge; when charged, it is preferably negatively charged. The carrier can be latex particles or other particles containing organic or inorganic polymers, lipid bilayers such as liposomes, phospholipid vesicles, small oil droplets, silica particles, metal sols, cells, and microcrystalline dyes.

[0069] In this invention, the "luminescent composition" is a compound referred to as a marker that can undergo a chemical reaction to induce luminescence, for example, by being converted into another compound in an electronically excited state. The excited state can be a singlet state or a triplet excited state. The excited state can relax to the ground state and emit light directly, or it can recover to the ground state by transferring the excitation energy to the energy acceptor. In this process, the energy acceptor particle will transition to an excited state and emit light.

[0070] As used in this invention, "donor particle" refers to a particle containing a sensitizer that, upon activation by energy or an active compound, can generate an active intermediate, such as reactive oxygen species, that reacts with the acceptor particle. The donor particle can be photoactivated (e.g., dyes and aromatic compounds) or chemically activated (e.g., enzymes, metal salts, etc.). In some specific embodiments of this invention, the donor particle is a polymeric microsphere filled with a photosensitizer. The photosensitizer can be a photosensitizer known in the art, preferably a relatively photostable compound that does not react effectively with singlet oxygen. Non-limiting examples include, for example, compounds such as methylene blue, rose red, porphyrin, phthalocyanine, and chlorophyll disclosed in US Patent 5709994 (which is incorporated herein by reference in its entirety), and derivatives of these compounds having 1-50 substituents, which are used to make these compounds more lipophilic or more hydrophilic, and / or as linking groups to specifically binding pairing members. Other examples of photosensitizers known to those skilled in the art may also be used in this invention, such as those described in U.S. Patent US6406913, which is incorporated herein by reference.

[0071] In this invention, the "coefficient of variation (CV) of particle size distribution" refers to the coefficient of variation of particle size within a Gaussian distribution in the detection results of a nanoparticle size analyzer. The formula for calculating the coefficient of variation is: Coefficient of variation (CV) = (Standard deviation (SD) / Mean) × 100%. Standard deviation (SD), also known as standard deviation, describes the average distance of each data point from the mean (deviation from the mean). It is the square root of the average of the squared deviations, denoted by σ. Standard deviation is the arithmetic square root of variance. Standard deviation reflects the dispersion of a dataset; the smaller the standard deviation, the less these values ​​deviate from the mean, and vice versa. Standard deviation σ is the distance between the inflection point (0.607 times the peak height) on the normal distribution curve and the perpendicular line from the peak height to the time axis, i.e., half the distance between two inflection points on the normal distribution curve. Half-peak width (Wh / 2) refers to the peak width at half the peak height, Wh / 2 = 2.355σ. The intercept of the tangent line drawn from the inflection points on both sides of the normal distribution curve on the baseline is called the peak width or baseline width, W = 4σ or W = 1.699Wh / 2.

[0072] The term "sample to be tested" as used in this invention refers to a mixture containing or suspected of containing the target molecule. Samples to be tested that can be used in this invention include bodily fluids, such as blood (which may be anticoagulated blood commonly seen in collected blood samples), plasma, serum, urine, semen, saliva, cell cultures, tissue extracts, etc. Other types of samples to be tested include solvents, seawater, industrial water samples, food samples, environmental samples such as soil or water, plant material, eukaryotic cells, bacteria, plasmids, viruses, fungi, and cells derived from prokaryotes. Samples to be tested can be diluted with a diluent as needed before use. For example, to avoid the hook effect, the sample can be diluted with a diluent before being tested on the instrument.

[0073] The term "antibody" as used in this invention is used in the broadest sense, including any isotype of antibody, antibody fragments that retain specific binding to antigens, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, bispecific antibodies, and fusion proteins comprising the antigen-binding portion of an antibody and a non-antibody protein. Where desired, antibodies may be further conjugated to other portions, such as a member of a specific binding pair, for example, biotin or avidin (a member of the biotin-avidin specific binding pair).

[0074] The term "antigen" as used in this invention refers to a substance capable of stimulating an immune response in the body and binding to antibodies and sensitized lymphocytes, the products of the immune response, both in vivo and in vitro, to produce an immune effect. The antigen may be a fusion antigen, and where necessary, the antigen may be further conjugated to other parts, such as specific binding pair members, for example, biotin or avidin (one of the biotin-avidin specific binding pair members).

[0075] The terms “bonding” or “combination” used in this invention refer to the connection between two molecules caused by interactions such as covalent, electrostatic, hydrophobic, ionic and / or hydrogen bonding, including but not limited to physical or chemical interactions such as salt bridges and water bridges.

[0076] In this invention, "direct or indirect connection" means that the specified substance can be chemically or physically bonded to another substance (directly); or the specified substance can be chemically or physically bonded to another substance through an intermediate substance (compound, polymer, polysaccharide) (indirectly).

[0077] The term "specific binding" as used in this invention refers to a mutually distinguishable and selective binding reaction between two substances, which, from a stereostructural perspective, means the conformational correspondence between the corresponding reactants. Under the technical concept disclosed in this invention, the detection methods for specific binding reactions include, but are not limited to: double-antibody sandwich method, competitive method, neutralization competitive method, indirect method, or capture method.

[0078] The term "specifically binding pair" as used in this invention refers to a pair of molecules that can specifically bind to each other, such as enzyme-substrate, antigen-antibody, or ligand-receptor pairs. A specific example of a specific binding pair is the biotin-streptavidin system, where "biotin" is widely found in animal and plant tissues and has two ring structures: an imidazoline ring and a thiophene ring. The imidazoline ring is the primary site of binding to streptavidin. Activated biotin can couple with almost all known biomolecules, including proteins, nucleic acids, polysaccharides, and lipids, mediated by protein cross-linking agents. The avidin is selected from egg avidin, streptavidin, yolk avidin, neutral avidin, and avidin-like molecules, preferably neutral avidin and / or streptavidin. Avidin is a glycoprotein extracted from egg white, with a molecular weight of 60 kDa. Each molecule consists of four subunits, thus it can bind closely to four biotin molecules and plays an important role in the immune mechanism. Avidins mainly include ovalbumin, streptavidin, yolk avidin, and neutral avidin. Streptavidin is a protein secreted by Streptomyces. The streptavidin molecule consists of four identical peptide chains, each capable of binding one biotin. Therefore, each antigen or antibody can simultaneously couple multiple biotin molecules, creating a "tentacle effect" that enhances analytical sensitivity. Where necessary, any reagent used in this invention, including antigens, antibodies, receptor particles, or receptor granules, can be conjugated with any member of the biotin-streptavidin specific binding pair, as required.

[0079] The ZETA potential value described in this invention refers to the potential value of acceptor particles in a dispersion system with a pH of 6-9. The ZETA potential of a particle refers to the potential at the shear plane; that is, the potential difference between the continuous phase and the fluid-stabilized layer attached to the microspheres. Because the dispersed particles carry a charge on their surface, they attract surrounding anti-charge ions. These anti-charge ions are distributed in a diffuse state at the interface between the two phases, forming a diffuse electric double layer. According to the Stern double layer theory, the double layer can be divided into two parts: the Stern layer and the diffuse layer. The Stern layer is defined as a planar layer composed of a layer of ionic (IHP or OHP) charge centers adsorbed on the electrode surface. The potential of this planar layer relative to a point in the fluid far from the interface is called the Stern potential. The interface between the stationary layer (including the stern layer and the portion of the diffusion layer within the slipping plane) and the dispersion medium within the diffusion layer, where relative movement occurs, is called the slipping plane. The potential at this point relative to a point in the fluid far from the interface is called the Zeta potential or zeta potential. In other words, the Zeta potential is the potential difference between the continuous phase and the fluid stationary layer attached to the dispersed particles. It can be directly measured through electrokinetic phenomena. Currently, the main methods for measuring the Zeta potential include electrophoresis, electroosmosis, flow potential analysis, and ultrasound, with electrophoresis being the most widely used.

[0080] II. Specific Implementation Plan

[0081] The invention will now be described in more detail.

[0082] In vitro diagnostics (IVD) technology generally refers to products and services that obtain relevant clinical diagnostic information by testing samples from the body, including blood, body fluids, and tissues, outside the human body, thereby helping to determine diseases or bodily functions. Nanomaterials possess unique size-dependent physical or chemical properties. At the nanoscale, their optical, magnetic, electrical, thermal, and biological properties can be modulated by changing their size, shape, chemical composition, and surface functional groups. In particular, nanomaterials, with a specific surface area far exceeding that of macroscopic materials, provide ample space for modifying their surfaces with different molecules, making them crucial for applications such as bioanalysis and biosensors. These surface-modified nanomaterials can selectively detect small molecules, nucleic acids, proteins, and microorganisms. Clearly, the integration of nanomaterials with IVD technology promises lower detection limits, higher sensitivity, and stronger selectivity. Furthermore, the combination of nanomaterials and clinical diagnostic analysis techniques will propel the field of clinical IVD towards new growth points.

[0083] In the field of photo-induced chemiluminescence (PET), donor and acceptor particles together form a "double-sphere" system. The two types of particles communicate via antigen-antibody binding, enabling the transfer of singlet oxygen and inducing a photo-induced chemiluminescence process, thus achieving homogeneous immunoassay without separation. Both "spheres" are nanospheres that complement, interact with, cooperate with, and influence each other in the PET system; neither can be dispensed with. Both types of nanospheres exhibit excellent suspension properties in the liquid phase, and their interaction with antigens or antibodies in the liquid phase perfectly conforms to liquid dynamics. Under 680 nm laser irradiation, the photosensitizer in the donor particle excites oxygen in the surrounding environment into singlet oxygen molecules. When these singlet oxygen molecules diffuse to the acceptor particle, they react with the chemiluminescent agent in the acceptor particle to produce a series of chemiluminescent reactions, generating an emission wavelength light signal in the range of 610 nm to 620 nm.

[0084] The technical principle of photoluminescence immunoassay is as follows: A sensitizer, under laser irradiation, excites oxygen molecules in the surrounding environment into singlet oxygen molecules. These singlet oxygen molecules react with a luminescent composition approximately 200 nm away, generating a light signal of a specific wavelength. When the sample contains the antigen or antibody to be tested, the immunoreaction of this antigen and antibody allows receptor particles containing the sensitizer to bind to receptor particles containing the luminescent composition, thereby generating a light signal of a specific wavelength. Detecting this light signal allows for the detection of the content of the antigen or antibody to be tested. In the photoluminescence immunoassay described above, the diameter, material, and surface properties of the receptor particles significantly affect the efficiency of the sensitizer in exciting singlet oxygen molecules and the energy transfer efficiency of the singlet oxygen molecules. They also affect the non-specific binding of receptor particles, leading to errors in the detection results. Therefore, the diameter range of the receptor particles, the uniformity of particle size, the material of the particles, and the surface chemical properties are key areas for research and improvement in photoluminescence immunoassay technology, and are not simply common knowledge or industry practice.

[0085] Acceptor reagents are an indispensable component of photoluminescence systems, containing luminescent substances in the acceptor particles that react with singlet oxygen to generate a detection signal. The acceptor particle preparation process, particle size distribution, selection of luminescent substances, and surface treatment of the acceptor particles all directly affect the final detection results. Through extensive research, the inventors have discovered that by strictly controlling the sugar content within a suitable range in the acceptor particles and by regulating the ZETA value of the acceptor particles, the problem of producing low-cost, high-quality, and stable acceptor reagents for mass production can be effectively solved. This invention is based on the aforementioned methods.

[0086] The first aspect of this invention relates to a receptor reagent comprising a buffer solution and receptor particles suspended therein, the receptor particles being capable of reacting with singlet oxygen to produce chemiluminescence, characterized in that: the zeta potential of the receptor particles is not higher than -10mV and not lower than -50mV; and the sugar content per milligram of receptor particles is not higher than 25 micrograms.

[0087] In some preferred embodiments of the present invention, the ZETA potential of the receptor particles in the receptor reagent is not higher than -15mV and not lower than -40mV.

[0088] In some specific embodiments of the present invention, the ZETA potential of the receptor particles in the receptor reagent is selected from -10mV, -20mV, -30mV, -40mV, and -50mV. The inventors of this patent have discovered that precisely controlling the ZETA potential of the receptor particles in the receptor reagent within a suitable range can enable the reagent kit to have the advantages of strong anti-interference ability and excellent testing performance.

[0089] In some specific embodiments of the present invention, the sugar content in each milligram of the receptor particle is no more than 15 micrograms.

[0090] In some specific embodiments of the present invention, the sugar content in each milligram of the receptor particle can be 3 micrograms, 6 micrograms, 9 micrograms, 12 micrograms, 15 micrograms, 18 micrograms, 21 micrograms, or 24 micrograms.

[0091] In some embodiments of the present invention, the receptor particle includes a carrier, the interior of which is filled with a luminescent composition, and the surface of which is bonded with biomolecules.

[0092] In some embodiments of the present invention, the surface of the carrier has bonding functional groups that are bonded to biomolecules.

[0093] In some embodiments of the present invention, the bonding functional group is selected from at least one of amino, amide, hydroxyl, aldehyde, carboxyl, maleimide and thiol, preferably selected from aldehyde and / or carboxyl.

[0094] In some embodiments of the present invention, the surface of the carrier has aldehyde groups. The aldehyde groups react with amino groups on the biomolecules to form Schiff base bonds, thereby bonding the biomolecules to the surface of the carrier.

[0095] In some embodiments of the invention, the surface of the carrier has carboxyl groups. These carboxyl groups react with amino groups on biomolecules to form amide bonds, thereby attaching avidin molecules to the surface of the carrier.

[0096] In some embodiments of the present invention, the surface of the carrier is not coated with sugar molecules, but directly bonded with biomolecules.

[0097] In some embodiments of the present invention, the biomolecule is capable of specifically binding to the analyte molecule.

[0098] 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 not less than 5% and not more than 20%.

[0099] In some preferred embodiments of the present invention, the coefficient of variation (CV) of the particle size distribution of the receptor particles in the receptor reagent is not higher than 15%.

[0100] In some preferred embodiments of the present invention, the coefficient of variation (CV) of the particle size distribution of the receptor particles in the receptor reagent is not less than 8%.

[0101] In some specific embodiments of the present invention, the coefficient of variation (CV) of the particle size distribution of the receptor particles in reagent 1 can be selected from 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, and 25%.

[0102] In other embodiments of the invention, the receptor particles exhibit polydispersity in the size distribution of the receptor reagent.

[0103] In some embodiments of the present invention, the particle size of the carrier is selected from 100 to 400 nm, preferably 150 to 350 nm, and more preferably 180 to 220 nm.

[0104] In some embodiments of the present invention, the concentration of the receptor particles in the receptor reagent is from 10 μg / ml to 1 mg / ml, preferably from 20 μg / ml to 500 μg / ml, and more preferably from 50 μg / ml to 200 μg / ml.

[0105] In some embodiments of the present invention, the receptor reagent further includes a buffer solution with a pH of 7.0 to 9.0, in which the receptor particles are suspended.

[0106] In some embodiments of the present invention, the buffer solution contains a polysaccharide selected from carbohydrates containing three or more unmodified or modified monosaccharide units, preferably selected from dextran, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxyglucan and aminoglucan; more preferably selected from dextran, starch, glycogen and polyribose.

[0107] In some embodiments of the present invention, the molecular weight distribution Mw of the polysaccharide (e.g., dextran) is selected from 10,000 to 1,000,000 Da, preferably from 100,000 to 800,000 Da, and more preferably from 300,000 to 700,000 Da.

[0108] In some embodiments of the present invention, the sugar content in each liter of the buffer solution is not less than 0.2g and not more than 2g.

[0109] In some embodiments of the present invention, the sugar content in each liter of the buffer solution is not less than 0.5g and not more than 1.5g.

[0110] The sugar content in the receptor particles of the present invention can come from the polysaccharides coating the surface of the receptor particles, or from the polysaccharide components carried in the structure of the antigen, antibody, or specific binding pair member itself.

[0111] In some embodiments of the present invention, the polysaccharide is selected from carbohydrates containing three or more unmodified or modified monosaccharide units; preferably selected from at least one of dextran, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxyglucan, and aminoglucan; more preferably selected from at least one of dextran, starch, glycogen, and polyribose; and most preferably dextran and / or dextran derivatives. Polysaccharides, especially dextran and dextran derivatives, can increase the hydrophilicity of the carrier surface and provide conjugated sites for the connection between antibody molecules and the carrier surface. Coating the receptor microparticle surface with polysaccharides can increase the hydrophilicity of the microspheres and avoid non-specific adsorption, which greatly affects the optical signal of subsequent photochemiluminescence detection. The inventors of this patent have discovered that precisely controlling the sugar content on the surface of the microspheres within a suitable range can effectively solve some technical problems existing in the application of photochemiluminescence technology in the field of in vitro diagnostics.

[0112] In this invention, sugar concentration or sugar content can be determined using the anthrone method. The anthrone method for determining polysaccharides is known to those skilled in the art. Sugars dehydrate upon contact with concentrated sulfuric acid to produce furfural or its derivatives. Furfural or hydroxymethylfurfural further condenses with anthrone reagent to produce a blue-green substance with maximum absorption in the visible light region of 620 nm to 630 nm. Furthermore, its absorbance value is directly proportional to the sugar content within a certain range. This method can be used to determine the content of monosaccharides, oligosaccharides, and polysaccharides, and has advantages such as high sensitivity, simplicity, speed, and suitability for the determination of trace samples.

[0113] Coating receptor particles with polysaccharides can reduce non-specific adsorption. However, polysaccharides also bring a series of other problems, such as higher cost, more complex processing, and reduced detection signal. Especially in the field of in vitro diagnostics, due to the complex composition of human body fluids containing many unknown components, we have found that polysaccharide-coated receptor reagents often have a significant impact on detection signals when used in in vitro diagnostics. To comprehensively address these issues, the inventors discovered that strictly controlling the sugar content on receptor particles, reducing excessive sugar coating on receptor microspheres, or even eliminating polysaccharide coating altogether, yields better results.

[0114] In other embodiments of the invention, the surface of the carrier is coated with a layer of at least two consecutive polysaccharide layers.

[0115] In some embodiments of the present invention, the first polysaccharide layer of the coating is spontaneously associated with the second polysaccharide layer.

[0116] In some embodiments of the invention, each of the continuous polysaccharide layers is spontaneously associated with each of the preceding polysaccharide layers.

[0117] In some embodiments of the present invention, the polysaccharide has side group functional groups, and the functional groups of the continuous polysaccharide layers carry opposite charges to the functional groups of the preceding polysaccharide layer.

[0118] In some embodiments of the present invention, the polysaccharide has side group functional groups, and the continuous polysaccharide layer is covalently linked to the previous polysaccharide layer through a reaction between the functional groups and the functional groups of the previous polysaccharide layer.

[0119] In some embodiments of the invention, the functional groups of the continuous polysaccharide layer alternate between amine functional groups and amine reactive functional groups.

[0120] In some embodiments of the present invention, the amine reactive functional group is an aldehyde group or a carboxyl group.

[0121] In some embodiments of the present invention, the first polysaccharide layer is spontaneously associated with the carrier.

[0122] In some embodiments of the present invention, the outermost polysaccharide layer of the coating has at least one side functional group.

[0123] In some embodiments of the present invention, the side group functional groups of the outermost polysaccharide layer of the coating are selected from at least one of aldehyde, carboxyl, mercapto, amino, hydroxyl and maleamine groups; preferably selected from aldehyde and / or carboxyl groups.

[0124] In some embodiments of the present invention, the side group functional groups of the outermost polysaccharide layer of the coating are directly or indirectly chemically bonded to one of the paired members.

[0125] In some embodiments of the present invention, the polysaccharide is selected from carbohydrates containing three or more unmodified or modified monosaccharide units; preferably selected from dextran, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxyglucan and aminoglucan; more preferably selected from dextran, starch, glycogen and polyribose.

[0126] 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.

[0127] In some specific embodiments of the present invention, the chemiluminescent compound is selected from olefin compounds, preferably from dimethylthiophene, dibutyl dione compounds, dioxane, enol ethers, enamines, 9-alkylene oxalane, 9-alkylene-N-9,10 dihydroacrylidine, aryl ethyl ether olefins, aryl imidazoles and glossin and their derivatives, more preferably from dimethylthiophene and its derivatives.

[0128] In some other embodiments of the present invention, the metal chelate is a rare earth metal or a Group VIII metal, preferably selected from europium, terbium, dysprosium, samarium, osmium, and ruthenium, and more preferably europium.

[0129] In some specific embodiments of the present invention, the metal chelate comprises a chelating agent selected from the following: 4'-(10-methyl-9-anthrayl)-2,2':6'2”-tripyridine-6,6”-dimethylamine]tetraacetic acid (MTTA), 2-

[0130] (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) The following are included: 1,1,1-trifluoroacetone (TTA), 3-naphthyl-1,1,1-trifluoroacetone (NPPTA), naphthyltrifluorobutylene dione (NTA), trioctylphosphine oxide (TOPO), triphenylphosphine oxide (TPPO), 3-benzoyl-1,1,1-trifluoroacetone (BFTA), 2,2-dimethyl-4-perfluorobutyryl-3-butanone (fod), 2,2'-bipyridine (bpy), bipyridylcarboxylic acid, azacrown ethers, azacavitary ligands, and trioctylphosphine oxide, and their derivatives. In some embodiments of the present invention, the method for preparing the receptor particles includes the following steps:

[0131] Step S1 involves reacting receptor microspheres containing bonded functional groups on the surface of the carrier with biomolecules in the presence of an activator to obtain an intermediate product.

[0132] Step S2: Add a sealing agent to seal the intermediate product obtained in step S1.

[0133] Step S3: The intermediate product after the sealing treatment in step S2 is washed to obtain receptor particles with biomolecules bonded to their surface.

[0134] In some embodiments of the present invention, the density of bonded functional groups on the surface of the support in step S1 is not less than 10 nmol / mg.

[0135] In some other embodiments of the present invention, the density of bonded functional groups on the surface of the carrier in step S1 is not less than 30 nmol / mg.

[0136] In some embodiments of the present invention, the mass ratio of the receptor microspheres to the biomolecules in step S1 is 10:(0.3 to 0.9).

[0137] In some other embodiments of the present invention, the mass ratio of the receptor microspheres to the biomolecules in step S1 is 10:(0.6 to 0.8).

[0138] In some embodiments of the present invention, the final concentration of the sealing agent after adding the sealing agent in step S2 is not less than 5 wt%.

[0139] In some other embodiments of the present invention, the final concentration of the sealing agent after adding the sealing agent in step S2 is not less than 10 wt%.

[0140] A second aspect of the present invention relates to a chemiluminescence detection kit comprising the receptor reagent as described in the first aspect of the present invention.

[0141] In some embodiments of the present invention, the reagent kit has at least one reagent strip, the reagent strip having a plurality of reagent wells for holding reagents, wherein at least one reagent well is used to hold the receptor reagent.

[0142] In some embodiments of the present invention, the chemical yellowing detection kit, in addition to the receptor reagent, also includes other reagents as needed, depending on the target or detection method, such as donor reagents, biotin-coated secondary antibodies, diluents, etc. In the field of in vitro diagnostics, especially in immunoassay, manufacturers typically label or abbreviate the components in different vials of the kit as Reagent 1 or R1, Reagent 2 or R2, Reagent 3 or R3, and so on, to simplify the naming of different components in commercial kits. This facilitates customer identification, assembly, and use, and also serves the purpose of technical confidentiality. Therefore, kits from different in vitro diagnostic manufacturers may all contain Reagent 1, Reagent 2, Reagent 3, etc., but the specific components of each reagent differ between manufacturers.

[0143] A third aspect of the invention relates to the use of a receptor reagent according to the first aspect of the invention or a kit according to the second aspect of the invention in a chemiluminescence analyzer.

[0144] A fourth aspect of the invention relates to the use of a receptor reagent according to the first aspect of the invention or a kit according to the second aspect of the invention in a point-of-care testing (POCT) instrument. POCT refers to on-site rapid testing or clinical testing performed at the patient's side.

[0145] The present invention also relates to the use of the receptor reagent according to the first aspect of the present invention or the kit according to the second aspect of the present invention in in vitro diagnosis of diseases or non-diseases.

[0146] The inventors of this application have discovered that the ZETA potential of receptor particles directly affects the detection results of photochemiluminescence. To achieve the commercial application of photochemiluminescence systems in clinical immunodiagnostics, it is necessary to mass-produce low-cost, high-quality, and stable receptor reagents. Therefore, it is essential to strictly control the ZETA potential of the receptor particles in the receptor reagents within an appropriate range.

[0147] III. Specific Implementation Examples

[0148] To make the present invention easier to understand, the present invention will be further described in detail below with reference to embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of application of the present invention. Unless otherwise specified, the raw materials or components used in the present invention can be obtained commercially or by conventional methods.

[0149] Example 1: Preparation of Receptor Reagent A

[0150] 1.1 Synthesis and Characterization of Carboxylated Polystyrene Latex Microspheres

[0151] 1) Prepare a 100ml three-necked flask, add 40mmol styrene, 3mmol methacrylic acid, and 10ml water, stir for 10min, and then purge with N2 for 30min;

[0152] 2) Weigh 0.11g of ammonium persulfate and 0.2g of sodium chloride, dissolve them in 40ml of water to prepare an aqueous solution. Add this aqueous solution to the reaction system in step 1), and continue to purge with N2 for 30min;

[0153] 3) Heat the reaction system to 70℃ and react for 15 hours;

[0154] 4) Cool the emulsion after the reaction is complete to room temperature and filter it with a suitable filter cloth. Wash the resulting emulsion by centrifugation with deionized water multiple times until the conductivity of the supernatant after centrifugation is close to that of deionized water. Then dilute it with water and store it in emulsion form.

[0155] 5) The carboxyl content of the latex microspheres was determined to be 70 nmol / mg by conductivity titration.

[0156] 1.2. Landfilling process and characterization of the luminescent composition

[0157] 1) Prepare a 25ml round-bottom flask, add 0.1g of dimethylthiophene derivative and 0.1g of europium(III) complex (MTTA-EU) 3+ Add 10 ml of 95% ethanol, stir magnetically, and heat in a water bath to 70°C to obtain the complex solution;

[0158] 2) Prepare a 100ml three-necked flask, add 10ml of 95% ethanol, 10ml of water and 10ml of carboxylated polystyrene latex microspheres with a concentration of 10% obtained in step 1.1, stir magnetically, and heat in a water bath to 70℃.

[0159] 3) Slowly add the complex solution from step 1) to the three-necked flask from step 2), react at 70°C for 2 hours, then stop stirring and allow to cool naturally.

[0160] 4) Centrifuge the above emulsion for 1 hour at 30,000 g. After centrifugation, discard the supernatant to obtain carboxylated polystyrene microspheres embedded with the luminescent composition. Adjust the volume with 20 μm HEPES buffer to a final concentration of 20 mg / ml.

[0161] 5) The average particle size of the microspheres at this time, as measured by a nanoparticle size analyzer, is 204.9 nm, with a coefficient of variation (CV) of 5.03%.

[0162] 1.3 Coating of carboxylated luminescent microspheres with HIV antigen

[0163] 1) The HIV antigen was dialyzed into 50mM MES buffer with a pH of 5.0, and the concentration was measured to be 1mg / ml.

[0164] 2) Add 0.5 mL of carboxylated luminescent microspheres and 0.5 mL of dialyzed HIV antigen to a 2 mL centrifuge tube, mix well, and then add 100 μl of 10 mg / mL EDAC solution (50 mM MES buffer). React at 2-8 °C for 4 hours.

[0165] 3) After the reaction is complete, add 0.5 ml of 100 mg / ml BSA solution (50 mM MES buffer) and react at 2-8℃ for 2 hours.

[0166] 4) After the reaction is complete, centrifuge for 30 min at 20000g. Discard the supernatant after centrifugation and resuspend the sample in 50mM MES buffer. Repeat the centrifugation and washing four times, and dilute to a final concentration of 100μg / ml to obtain the HIV antigen-conjugated receptor particle solution.

[0167] Example 2: Determination of the ZETA potential of receptor particles in receptor reagents

[0168] This invention employs a method for detecting the Zeta potential: NICOMP 380Z3000 uses Doppler electrophoresis (ELS) to determine the Zeta potential value. This is primarily achieved by measuring the electrophoretic migration rate of charged particles in a suspension, thereby obtaining the Zeta potential value. The stability of the colloid is determined by measuring the Zeta potential of the microspheres. The main influencing factor on the Zeta potential of particles is the surface charge of the particles.

[0169] The method for measuring the ZETA potential in this invention is as follows:

[0170] 2.1. Different receptor reagents were prepared according to the method described in Example 1, wherein the sugar content in each receptor reagent was not higher than 25 micrograms per milligram of the receptor particle as determined by the anthrone method, as shown in Table 1.

[0171] 2.2. Prepare the sample by diluting the different receptor reagents prepared in step 2.1 into deionized water to a concentration of 10 μg / mL.

[0172] 2.3. The NICOMP 380Z3000 instrument was calibrated with standards before the Zeta potential was measured. The results are shown in Table 1. The Zeta potential of experimental group 5 is shown in Table 1. Figure 1 .

[0173] Table 1

[0174]

[0175] Results Analysis: Results from experimental groups 1-3 show that, with other conditions remaining constant, the absolute value of the zeta potential decreases when the activator content increases. Results from experimental groups 4-6 show that, with other conditions remaining constant, the absolute value of the zeta potential decreases when the coating ratio increases. Results from experimental groups 5, 7, and 8 show that, with other conditions remaining constant, the absolute value of the zeta potential decreases when the final concentration of the blocking agent increases. Results from experimental groups 5 and 9 show that, with other conditions remaining constant, the absolute value of the zeta potential increases when the carboxyl group density increases.

[0176] Example 3: Evaluation of the anti-interference properties of the receptor reagent

[0177] HIV antibody detection kits were used for evaluation. The chemiluminescence detection process was completed and the results were output on a fully automated photocatalytic chemiluminescence analysis system (LiCA HT) developed by Boyang Biotechnology (Shanghai) Co., Ltd. The specific experimental steps are as follows:

[0178] 1) The HIV antigen-coated carboxyl luminescent microspheres (1-9 in Example 2) prepared under different conditions were diluted to the same working concentration with luminescent reagent buffer.

[0179] 2) Perform the test according to the instructions for the HIV antibody test kit.

[0180] 3) Manually add the sample, HIV antigen-coated carboxyl luminescent microsphere receptor reagent, and reagent two from the kit according to the reaction mode for the first stage of incubation.

[0181] 4) Add photosensitive bead solution.

[0182] 5) Conduct the second stage of incubation.

[0183] 6) Place the LiCA HT sample in the container and take readings. The sample values ​​for different receptor reagents are shown in Table 2, and the sample discrimination of different receptor reagents is shown in Table 3.

[0184] In this embodiment, the anti-interference performance of the reagent was tested using negative serum and plasma tube samples to evaluate the reagent's ability to resist plasma tube interference. When the ratio of the plasma tube sample signal measurement value to the mean of the negative serum sample signal measurement value was greater than 1, plasma tube interference was present; when the ratio was significantly greater than 1, plasma tube interference was severe; and when the ratio was less than 1, no plasma tube interference was present.

[0185] Table 2

[0186]

[0187]

[0188] Table 3

[0189] 1 2 3 4 5 6 7 8 9 Plasma tube 01 96.20 6.63 0.80 0.97 0.67 2.24 3.01 4.79 1.79 Plasma tube 02 219.99 7.03 0.62 0.85 0.58 2.85 3.25 6.23 1.64 Plasma tube 03 1346.51 9.00 0.95 0.69 0.51 4.39 3.14 5.73 1.44 Plasma tube 04 545.17 6.48 0.78 0.64 0.84 3.65 3.17 4.39 1.11

[0190] Results Analysis: Table 3 shows that plasma tube interference was most severe in experimental group 1, while no interference was observed in experimental groups 3-5. Interference was relatively weak in the other groups. Therefore, plasma tube interference was weakest in experimental groups 3-5, indicating good reagent anti-interference ability.

[0191] Based on the results of Examples 2 and 3, it can be concluded that when the zeta potential of the receptor particles of HIV antigen-conjugated carboxyl microspheres is between -5 and -50 mV, the reagent has a strong ability to resist interference from plasma tubes; when the zeta potential is between -15 and -50 mV, the reagent has the best ability to resist interference from plasma tubes.

[0192] It should be noted that the embodiments described above are only for explaining the present invention and do not constitute any limitation on the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from the scope and spirit of the present invention. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention can be extended to all other methods and applications with the same function.

Claims

1. A receptor reagent comprising a buffer solution and receptor particles suspended therein, said receptor particles being capable of reacting with reactive oxygen species to produce chemiluminescence, characterized in that, The receptor particle includes a carrier, the interior of which is filled with a luminescent composition, and the surface of the carrier is not coated with sugar molecules but directly bonded with biomolecules. The ZETA potential of the receptor particles is not higher than -15mV and not lower than -40mV; the sugar content per milligram of receptor particles is not higher than 15 micrograms; the coefficient of variation (CV) of the particle size distribution of the receptor particles in the receptor reagent is not lower than 5% and not higher than 20%, and the particle size distribution of the receptor particles in the receptor reagent exhibits polydispersity.

2. The receptor reagent according to claim 1, characterized in that, The surface of the carrier has bonding functional groups that bond with biomolecules.

3. The receptor reagent according to claim 2, characterized in that, The bonding functional group is selected from at least one of amino, amide, hydroxyl, aldehyde, carboxyl, maleimide and thiol groups.

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

5. The receptor reagent according to claim 4, characterized in that, The surface of the carrier has carboxyl groups, which react with amino groups on the biomolecule to bond the biomolecule to the surface of the carrier.

6. The receptor reagent according to any one of claims 1-5, characterized in that, The method for preparing the receptor particles includes the following steps: Step S1: The receptor microspheres with bonded functional groups on the surface of the carrier are reacted with biomolecules in the presence of an activator to obtain an intermediate product. Step S2: Add a sealing agent to seal the intermediate product obtained in step S1. Step S3: The intermediate product after the sealing treatment in step S2 is washed to obtain receptor particles with biomolecules bonded to their surface.

7. The receptor reagent according to claim 6, characterized in that, The density of bonded functional groups on the surface of the support in step S1 is not less than 10 nmol / mg.

8. The receptor reagent according to claim 7, characterized in that, The density of bonded functional groups on the surface of the support in step S1 is not less than 30 nmol / mg.

9. The receptor reagent according to claim 6, characterized in that, In step S1, the mass ratio of the receptor microspheres to the biomolecules is 10:(0.3 to 0.9).

10. The receptor reagent according to claim 6, characterized in that, The amount of activator added in step S1 shall not be less than 30 nmol / mg.

11. The receptor reagent according to claim 6, characterized in that, The final concentration of the blocking agent after adding the blocking agent in step S2 shall not be less than 5 wt%.

12. A chemiluminescence detection kit comprising the receptor reagent as described in any one of claims 1 to 11.

13. The use of a receptor reagent according to any one of claims 1 to 11 or a kit according to claim 12 in a chemiluminescence analyzer.

14. The use of a receptor reagent according to any one of claims 1 to 11 or a kit according to claim 12 in a POCT instrument.

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