Donor reagent and application thereof

Abstract

The invention relates to a donor reagent and application thereof. The donor reagent provided by the invention comprises a buffer solution and donor particles suspended in the buffer solution, the donor particles can generate active oxygen after being excited, and the donor reagent is characterized in that the ZETA potential of the donor particles in the donor reagent is not higher than-10 mV and not lower than-60 mV. The kit containing the donor reagent can be used for determining the content of the marker in the body fluid of a main body, and has the advantages of high sample discrimination and good test performance.

Description

Technical Field

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

[0002] 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.

[0003] Immunoassay has evolved over half a century, resulting in numerous detection methods. Based on whether the analyte is separated from the reaction system during the assay, it can be categorized into heterogeneous and homogeneous immunoassays. Heterogeneous immunoassay, on the other hand, involves separating the analyte from the reaction system after the reagents are mixed and reacted during the probe labeling process. This is the mainstream method in current immunoassays. Examples include the well-known enzyme-linked immunosorbent assay (ELISA) and magnetic particle chemiluminescence immunoassay. Homogeneous immunoassay, on the other hand, involves directly measuring the analyte after it has been mixed and reacted with the reagents in the reaction system, without any additional separation or washing steps. Currently, many sensitive detection methods are applied to homogeneous immunoassay, such as chemiluminescence and electrochemical detection methods.

[0004] Light-induced chemiluminescence (LiCA) is a typical homogeneous immunoassay method. It is based on the reaction of an antigen coated on one type of nanosphere with an antibody coated on another in a liquid phase, drawing the two nanospheres closer together to form a "double-sphere" immunocomplex. LiCA is characterized by its "double-sphere" structure, where the system consists of a microsphere that generates singlet oxygen and a microsphere that accepts singlet oxygen, and both microspheres require specific suspension properties in the liquid phase. The antigen-linked microsphere and the antibody-linked microsphere form a "double-sphere" through an antigen-antibody reaction in the liquid phase. However, existing "double-sphere" techniques suffer from poor homogeneity in the liquid phase, resulting in poor reproducibility and unstable luminescence effects in immunoassays.

[0005] 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.

[0006] Therefore, there is an urgent need to develop a donor reagent that can be mass-produced, is low in cost, has qualified quality, and has stable performance, and can meet both sensitivity and linear range requirements. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a donor reagent to address the shortcomings of the prior art. The kit containing the donor reagent can be used to determine the content of markers in body fluids. The kit has the advantages of high sample discrimination and good testing performance.

[0008] To this end, the first aspect of the present invention provides a donor reagent comprising a buffer solution and donor particles suspended therein, wherein the donor particles are capable of generating reactive oxygen species upon excitation, characterized in that the zeta potential of the donor particles in the donor reagent is not higher than -10 mV and not lower than -60 mV.

[0009] In some preferred embodiments of the present invention, the ZETA potential of the donor particles in the donor reagent is not higher than -20 mV and not lower than -40 mV.

[0010] In some specific embodiments of the present invention, the ZETA potential of the donor particles in the donor reagent is selected from -10mV, -20mV, -30mV, -40mV, -50mV, and -60mV.

[0011] In some embodiments of the present invention, the sugar content in each milligram of the donor particle is not higher than 40 μg.

[0012] In some embodiments of the present invention, the sugar content in each milligram of the donor particle is not higher than 20 μg.

[0013] In some embodiments of the present invention, the donor particle includes a carrier, the interior of which is filled with a sensitizer, and the surface of which is bonded with a member of a specific binding pair.

[0014] In some embodiments of the present invention, the surface of the carrier has bonding functional groups that are bonded to one of the specific binding pairing members.

[0015] 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 groups.

[0016] In some embodiments of the present invention, the surface of the carrier is modified with an aldehyde group, which is directly bonded to one of the specific pairing binding members to bind one of the specific pairing binding members to the surface of the carrier.

[0017] In some embodiments of the invention, the surface of the carrier has aldehyde groups. These aldehyde groups react with amino groups on the avidin molecule to form Schiff base bonds, thereby attaching the avidin molecule to the surface of the carrier.

[0018] In some embodiments of the invention, the surface of the carrier has carboxyl groups. These carboxyl groups react with amino groups on the avidin molecule to form amide bonds, thereby attaching the avidin molecule to the surface of the carrier.

[0019] In some embodiments of the present invention, the surface of the carrier is modified with carboxyl groups, which are directly bonded to one of the specific pairing binding members to bind one of the specific pairing binding members to the surface of the carrier.

[0020] In some embodiments of the present invention, the surface of the carrier is not coated with sugar molecules but directly binds to one of the paired members.

[0021] In some embodiments of the present invention, the specific binding pairing member is an avidin-biotin system, preferably, the avidin is selected from at least one of egg avidin, egg vitellin, streptavidin, neutral avidin and avidin-like molecules.

[0022] In some preferred embodiments of the present invention, the avidin is streptavidin.

[0023] In some embodiments of the present invention, the coefficient of variation (CV) of the particle size distribution of the donor particles in the donor reagent is not less than 5% and not more than 20%.

[0024] In some preferred embodiments of the present invention, the coefficient of variation (CV) of the particle size distribution of the donor particles in the donor reagent is not higher than 15%.

[0025] In some preferred embodiments of the present invention, the coefficient of variation (CV) of the particle size distribution of the donor particles in the donor reagent is not less than 8%.

[0026] 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%.

[0027] In other embodiments of the invention, the donor particles exhibit polydispersity in the particle size distribution of the donor reagent.

[0028] 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.

[0029] In some embodiments of the present invention, the concentration of the donor particles in the donor 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.

[0030] In some embodiments of the present invention, the donor reagent further includes a buffer solution with a pH value of 7.0 to 9.0, and the donor particles are suspended in the buffer solution.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] In other embodiments of the invention, the surface of the carrier is coated with a layer of at least two consecutive polysaccharide layers.

[0036] In some embodiments of the present invention, the first polysaccharide layer of the coating is spontaneously associated with the second polysaccharide layer.

[0037] In some embodiments of the invention, each of the continuous polysaccharide layers is spontaneously associated with each of the preceding polysaccharide layers.

[0038] 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.

[0039] 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.

[0040] In some embodiments of the invention, the functional groups of the continuous polysaccharide layer alternate between amine functional groups and amine reactive functional groups.

[0041] In some embodiments of the present invention, the amine reactive functional group is an aldehyde group or a carboxyl group.

[0042] In some embodiments of the present invention, the first polysaccharide layer is spontaneously associated with the carrier.

[0043] In some embodiments of the present invention, the outermost polysaccharide layer of the coating has at least one side functional group.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] In some embodiments of the present invention, the method for preparing the donor particles includes the following steps: Step S1 involves reacting donor microspheres containing bonded functional groups on the surface of the support with one of the specifically bound pairing members in the presence of an activator to obtain an intermediate product.

[0048] 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 cleaned to obtain donor particles with one of the specific binding members bonded to the surface.

[0049] 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.

[0050] 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 50 nmol / mg.

[0051] In some embodiments of the present invention, the mass ratio of the donor microsphere to one of the specific binding pair members in step S1 is 10:(0.3~2).

[0052] In some other embodiments of the present invention, the mass ratio of the donor microsphere to one of the specific binding pair members in step S1 is 10:(0.6~1.2).

[0053] 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%.

[0054] 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%.

[0055] A second aspect of the present invention provides a chemiluminescence detection kit comprising donor reagents as described in the first aspect of the present invention.

[0056] 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 donor reagent.

[0057] In some embodiments of the present invention, the chemical yellowing test kit, in addition to the donor reagent, also includes other reagents as needed, depending on the target or detection method, such as receptor 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.

[0058] A third aspect of the present invention provides the application of the donor 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.

[0059] A fourth aspect of the present invention provides the application of the donor 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.

[0060] The present invention also provides the application of the donor 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.

[0061] The role of the donor reagent described in this invention is that the donor particles in the reagent can generate singlet oxygen after being excited by external excitation light. The singlet oxygen transfers energy to the donor particles within a range of 200 nm from the acceptor particles, which can finally generate a chemiluminescent signal, thereby realizing the detection of unknown substances.

[0062] The beneficial effects of this invention are as follows: The donor reagent described in this invention functions by generating singlet oxygen upon excitation by external excitation light. This singlet oxygen transfers energy to the donor particle within a 200 nm range of the acceptor particle, ultimately generating a chemiluminescent signal, thus enabling the detection of unknown substances. The kit containing the donor reagent of this invention can be used to determine the content of markers in body fluids, and this kit has the advantages of high sample discrimination and excellent testing performance. Attached Figure Description

[0063] Figure 1 This is a ZETA potential distribution diagram of experimental group 7 according to Embodiment 2 of the present invention. Detailed Implementation

[0064] To facilitate understanding of the present invention, it will be described in detail below. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terminology used herein is for describing specific embodiments only and is not intended to be restrictive.

[0065] Where numerical ranges are provided, it should be understood that every intermediate value between the upper and lower limits of the range and any other specified or intermediate value within the specified range is covered by this invention. The upper and lower limits of these smaller ranges may be independently included in the smaller range and are also covered by this invention, subject to any explicitly excluded limits within the specified range. Where a specified range includes one or two limits, the range excluding any or both of those included limits is also included by this invention.

[0066] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of this invention, preferred methods and materials are now described.

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

[0068] As used in this invention, "receptor particle" refers to a compound particle containing a compound capable of reacting with reactive oxygen species to generate a detectable signal. The donor particle is induced and activated by energy or an active compound, releasing high-energy reactive oxygen species. These high-energy reactive oxygen species are captured by a nearby receptor particle, thereby transferring energy to activate the receptor particle. In one specific embodiment, the receptor particle comprises a luminescent agent and a carrier, the luminescent agent being filled in the carrier and / or coated on the surface of the carrier.

[0069] 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.

[0070] 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.

[0071] 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 one specific embodiment, the donor particle is a polymeric microsphere filled with a photosensitizer, which 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, said substituents being 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.

[0072] 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 from the inflection point (0.607 times the peak height) on the normal distribution curve to the perpendicular line from the peak height to the time axis, 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.

[0073] 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.

[0074] The term "antibody" as used in this invention is used in the broadest sense, including any isotype of antibody, antibody fragments that retain specific binding to antigens, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, bispecific antibodies, and fusion proteins comprising the antigen-binding portion of an antibody and non-antibody proteins. Where desired, antibodies may be further conjugated to other portions, such as one of the specific binding pairing members, for example, biotin or avidin (a biotin-avidin specific binding pairing member).

[0075] 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).

[0076] 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.

[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 donor particles, 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 The invention will now be described in more detail.

[0081] The technical principle of photoluminescence immunoassay is as follows: A sensitizer, under laser irradiation, excites oxygen molecules in the surrounding environment into singlet oxygen molecules. These singlet oxygen molecules react with a luminescent composition approximately 200 nm away, generating a light signal of a specific wavelength. When the sample contains the antigen or antibody to be tested, the immunoreaction of this antigen and antibody allows the donor particle containing the sensitizer to bind to the acceptor particle containing the luminescent composition, thereby generating a light signal of a specific wavelength. Detecting this light signal allows for the detection of the content of the antigen or antibody to be tested. In the photoluminescence immunoassay described above, the diameter, material, and surface properties of the acceptor and donor particles significantly affect the efficiency of the sensitizer in exciting singlet oxygen molecules and the energy transfer efficiency of the singlet oxygen molecules. They also affect the non-specific binding of the donor and acceptor particles, leading to errors in the detection results. Therefore, the diameter range of the donor and acceptor particles, the uniformity of particle size, the material of the particles, and the surface chemical properties are key areas for research and improvement in photoluminescence immunoassay technology, and are not simply common knowledge or industry practice.

[0082] The donor reagent described in this invention functions by generating singlet oxygen upon excitation by external excitation light. This singlet oxygen transfers energy to the donor particles within a 200 nm range of the acceptor particles, ultimately generating a chemiluminescent signal and thus enabling the detection of unknown substances. This invention is based on the aforementioned method.

[0083] The first aspect of the present invention provides a donor reagent comprising a buffer solution and donor particles suspended therein, wherein the donor particles are capable of generating reactive oxygen species upon excitation, characterized in that the zeta potential of the donor particles in the donor reagent is not higher than -10 mV and not lower than -60 mV.

[0084] In some preferred embodiments of the present invention, the ZETA potential of the donor particles in the donor reagent is not higher than -20 mV and not lower than -40 mV.

[0085] In some specific embodiments of the present invention, the ZETA potential of the donor particles in the donor reagent is selected from -10mV, -20mV, -30mV, -40mV, -50mV, and -60mV. The inventors of this patent have discovered that precisely controlling the ZETA potential of the donor particles in the donor reagent within a suitable range enables the reagent kit to have the advantages of high sample discrimination and excellent testing performance.

[0086] In some embodiments of the present invention, the sugar content in each milligram of the donor particle is not higher than 40 μg.

[0087] In some embodiments of the present invention, the sugar content in each milligram of the donor particle is not higher than 20 μg.

[0088] In some embodiments of the present invention, the donor particle includes a carrier, the interior of which is filled with a sensitizer, and the surface of which is bonded with a member of a specific binding pair.

[0089] In some embodiments of the present invention, the surface of the carrier has bonding functional groups that are bonded to one of the specific binding pairing members.

[0090] 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 groups.

[0091] In some embodiments of the present invention, the carrier surface has an aldehyde group that is bonded to one of the specific pairing binding members, thereby binding one of the specific pairing binding members to the surface of the carrier.

[0092] In some embodiments of the present invention, the carrier surface has a carboxyl group, which is bonded to one of the specific pairing binding members, thereby binding one of the specific pairing binding members to the surface of the carrier.

[0093] In some embodiments of the invention, the surface of the carrier has aldehyde groups. These aldehyde groups react with amino groups on the avidin molecule to form Schiff base bonds, thereby attaching the avidin molecule to the surface of the carrier.

[0094] In some embodiments of the invention, the surface of the carrier has carboxyl groups. These carboxyl groups react with amino groups on the avidin molecule to form amide bonds, thereby attaching the avidin molecule to the surface of the carrier.

[0095] In some embodiments of the present invention, the surface of the carrier is not coated with sugar molecules but directly binds to one of the paired members.

[0096] In some embodiments of the present invention, the specific binding pairing member is an avidin-biotin system, preferably, the avidin is selected from at least one of egg avidin, egg vitellin, streptavidin, neutral avidin and avidin-like molecules.

[0097] In some preferred embodiments of the present invention, the avidin is streptavidin.

[0098] In some embodiments of the present invention, the coefficient of variation (CV) of the particle size distribution of the donor particles in the donor reagent is 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 donor particles in the donor 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 donor particles in the donor 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 donor particles exhibit polydispersity in the particle size distribution of the donor 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 donor particles in the donor 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 donor reagent further includes a buffer solution with a pH value of 7.0 to 9.0, and the donor particles are suspended in the buffer solution.

[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 donor particles of the present invention can come from the polysaccharides coated on the surface of the donor particles, or from the polysaccharide components carried in the structure of the antigen antibody or the 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 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 620nm-630nm. 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 method for preparing the donor particles includes the following steps: Step S1 involves reacting donor microspheres containing bonded functional groups on the surface of the support with one of the specifically bound pairing members in the presence of an activator to obtain an intermediate product.

[0127] 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 cleaned to obtain donor particles with one of the specific binding members bonded to the surface.

[0128] 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.

[0129] 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 50 nmol / mg.

[0130] In some embodiments of the present invention, the mass ratio of the donor microsphere to one of the specific binding pair members in step S1 is 10:(0.3~2).

[0131] In some other embodiments of the present invention, the mass ratio of the donor microsphere to one of the specific binding pair members in step S1 is 10:(0.6~1.2).

[0132] 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%.

[0133] 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%.

[0134] A second aspect of the present invention provides a chemiluminescence detection kit comprising donor reagents as described in the first aspect of the present invention.

[0135] 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 donor reagent.

[0136] In some embodiments of the present invention, the chemical yellowing test kit, in addition to the donor reagent, also includes other reagents as needed, depending on the target or detection method, such as receptor 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.

[0137] A third aspect of the present invention provides the application of the donor 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.

[0138] A fourth aspect of the present invention provides the application of the donor 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.

[0139] The present invention also provides the application of the donor 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.

[0140] The donor reagent described in this invention functions by generating singlet oxygen upon excitation by external excitation light. This singlet oxygen transfers energy to the donor particle within a 200 nm range of the acceptor particle, ultimately generating a chemiluminescence signal and enabling the detection of unknown substances. The inventors of this application have discovered that the ZETA potential of the donor particle directly affects the detection results of photo-induced chemiluminescence. To achieve the commercial application of photo-induced chemiluminescence systems in clinical immunodiagnostics, large-scale production of low-cost, high-quality, and stable donor reagents is necessary. Therefore, it is crucial to strictly control the ZETA potential of the donor particles within a suitable range.

[0141] III. Specific Implementation Examples 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.

[0142] Example 1: Preparation of Donor Reagent A 1.1 Preparation of the carrier a) Prepare a 100ml three-necked flask, add 40mmol styrene, 5mmol acrolein and 10ml water, stir for 10min and then purge with N2 for 30min.

[0143] b) Weigh 0.11 g of ammonium persulfate and 0.2 g of sodium chloride, dissolve them in 40 ml of water to prepare an aqueous solution. Add this aqueous solution to the reaction system in step a), and continue to purge with N2 for 30 min.

[0144] c) Heat the reaction system to 70°C and react for 15 hours to obtain an emulsion.

[0145] d) Cool the emulsion after the reaction 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 with water to obtain aldehyde-based polystyrene microspheres.

[0146] e) The size of the aldehyde-based polystyrene microspheres (carrier), as measured by a nanoparticle size analyzer, exhibits a Gaussian distribution with an average particle size of 201.3 nm and a coefficient of variation (CV) of 8.0%. The aldehyde content in the aldehyde-based polystyrene microspheres, as determined by conductivity titration, is 90 nmol / mg.

[0147] 1.2 Sensitizer Filler Carrier a) Prepare a 25ml round-bottom flask, add 0.11g copper phthalocyanine and 10ml N,N-dimethylformamide, stir magnetically, and heat in a water bath to 75℃ to obtain a photosensitizer solution.

[0148] b) Prepare a 100ml three-necked flask, add 10ml of 95% ethanol, 10ml of water and 10ml of aldehyde-based polystyrene latex microspheres with a concentration of 10% obtained in 3.1, stir magnetically, and heat in a water bath to 70℃.

[0149] c) Slowly add the solution from step a) to the three-necked flask from step b), stir at 70°C for 2 hours to allow the sensitizer to fill the aldehyde polystyrene microspheres, then stop stirring and allow to cool naturally to obtain a filled emulsion.

[0150] d) Centrifuge the emulsion at 30,000g for 1 hour. Discard the supernatant after centrifugation and resuspend in 50% ethanol. Repeat the centrifugation and washing three times, and then resuspend in 50mM CB buffer at pH=10 to achieve a final concentration of 20mg / ml of sensitized aldehyde polystyrene microspheres, thus obtaining a microsphere suspension.

[0151] 1.3 Preparation of Donor Reagent A a) Microsphere suspension treatment: The microsphere suspension prepared in step 3.2 was centrifuged at 20000G for 0.5 hours in a high-speed refrigerated centrifuge, the supernatant was discarded, MES buffer was added, and the microspheres were sonicated on an ultrasonic cell disruptor until they were resuspended. MES buffer was added to adjust the mass concentration of the microspheres to 100mg / ml to obtain the treated microsphere suspension.

[0152] b) Preparation of streptavidin solution: Weigh a certain amount of streptavidin and dissolve it in MES buffer to 8 mg / ml.

[0153] c) Mixing: Mix the prepared microsphere suspension, 8 mg / ml avidin and MES buffer at a volume ratio of 2:5:1 and mix quickly to obtain the reaction solution.

[0154] d) Reaction: Prepare a 25 mg / ml NaBH3CN solution using MES buffer, add it to the reaction solution at a volume ratio of 1:25, and mix quickly. Incubate at 37°C for 48 hours using a rotary osmosis method.

[0155] e) Blocking: Prepare 75 mg / ml glycine (Gly) solution and 25 mg / ml NaBH3CN solution using MES buffer. Add these solutions to the solution from step d) above at a volume ratio of 2:1:10 (glycine solution, NaBH3CN solution to reaction solution), mix well, and incubate at 37°C for 2 hours by rotation. Then add 200 mg / ml BSA solution (prepared with MES buffer), with a BSA solution to reaction solution volume ratio of 5:8, mix rapidly, and incubate at 37°C for 16 hours by rotation.

[0156] f) Washing: Add MES buffer to the solution after reaction in step e), centrifuge at 20000G for 0.5 hours using a high-speed refrigerated centrifuge, discard the supernatant, then add fresh MES buffer, sonicate to suspend, and centrifuge again. Repeat this washing process 3 times. Finally, suspend the solution with a small amount of buffer, determine the solid content, and adjust the solid content to a concentration of 150 μg / ml with buffer to obtain donor reagent A containing donor particles a.

[0157] g) The size of donor particles a in donor reagent A, as measured by a nanoparticle size analyzer, exhibits a Gaussian distribution with an average particle size of 227.7 nm and a coefficient of variation (CV) of 6.5%.

[0158] Example 2: Determination of the ZETA potential of donor particles in the donor reagent This invention employs a method for detecting the Zeta potential: NICOMP 380 Z3000 uses Doppler electrophoresis (ELS) to determine the Zeta potential value. The Zeta potential value is primarily obtained by measuring the electrophoretic migration rate of charged particles in a suspension. 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.

[0159] The method for measuring the ZETA potential of the present invention is as follows: 2.1. Different donor reagents were prepared according to the method described in Example 1, wherein the sugar content in each donor reagent was not higher than 40 μg per milligram of the donor particle as determined by the anthrone method, as shown in Table 1.

[0160] 2.2. Prepare the sample by diluting the different donor reagents prepared in step 2.1 into deionized water to a concentration of 10 μg / mL.

[0161] 2.3. The NICOMP 380 Z3000 instrument was calibrated with standards before the Zeta potential was measured. The results are shown in Table 1. The Zeta potential of experimental group 7 is shown in Table 1. Figure 1 .

[0162] Table 1

[0163] Results Analysis: Results from experimental groups 1-4 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 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 2 and 5 show that, with other conditions remaining constant, the absolute value of the zeta potential decreases when the aldehyde group density increases.

[0164] The above analysis shows that the zeta potential of the donor particles is affected by the aldehyde density, coating ratio, and blocking agent concentration. Under single factors, the absolute value of the zeta potential decreases when the coating ratio, blocking agent concentration, or aldehyde density increases.

[0165] Example 3 Performance Evaluation of Donor Reagents The CA19-9 carbohydrate antigen detection kit was used for detection and evaluation. The chemiluminescence detection process was completed and the results were output on the fully automated photo-induced chemiluminescence analysis system (LiCA HT) developed by Boyang Biotechnology (Shanghai) Co., Ltd.

[0166] The kits in this embodiment all consist of reagent 1 (R1) containing receptor particles coated with a first anti-CA199 antibody, reagent 2 (R2) containing a biotin-labeled second anti-CA199 antibody, and any one of the donor reagents 1 to 7 prepared in Example 2 (R3). Clearly, R1 is the receptor reagent; R3 is the donor reagent. The specific experimental steps are as follows: 1) Perform the test according to the instructions of the CA19-9 carbohydrate antigen detection kit.

[0167] 2) Manually add the sample and receptor reagent R1 and reagent 2 from the kit according to the reaction mode for the first stage of incubation.

[0168] 3) Add donor reagents 1-7 prepared under different conditions in Example 2.

[0169] 4) Conduct the second stage of incubation.

[0170] 5) Place the LiCA HT and take the readings. The calibrator values ​​for different donor reagents in the CA19-9 test are shown in Table 2, and the calibrator discrimination for different donor reagents in the CA19-9 test is shown in Table 3.

[0171] Table 2

[0172] Table 3

[0173] Results Analysis: Table 3 shows that the calibrators in experimental groups 1 and 7 had the lowest discrimination, while the calibrators in experimental groups 3-5 had higher discrimination. Therefore, the donor reagents in experimental groups 3-5 showed the best detection performance for the CA19-9 test.

[0174] Based on the results of Examples 2 and 3, it can be concluded that the reagent performance is better when the ZETA potential of the donor reagent is between -10 and -60 mV, and the performance is best when the ZETA potential is between -20 and -40 mV.

[0175] 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 donor reagent comprising a buffer solution and donor particles suspended therein, said donor particles being capable of generating reactive oxygen species upon excitation, characterized in that, The ZETA potential of the donor particles in the donor reagent is not higher than -10 mV and not lower than -60 mV; the sugar content in each milligram of the donor particles is not higher than 40 μg, and the sugar content in each liter of the buffer solution is not lower than 0.2 g and not higher than 2 g; The donor particles comprise a carrier, the inside of the carrier is filled with a sensitizing agent, and the surface of the carrier is bonded with one of the specific binding pair members; the surface of the carrier has an aldehyde group bonded with one of the specific binding pair members, thereby binding one of the specific binding pair members to the surface of the carrier.

2. The donor reagent of claim 1, wherein, The surface of the carrier is directly bonded with one of the specific binding pair members without coating sugar molecules.

3. The donor reagent of claim 1, wherein, The density of the bonding functional groups on the surface of the carrier is not lower than 10 nmol / mg.

4. The donor reagent of claim 1, wherein, The mass ratio of the donor particles to one of the specific binding pair members is 10:(0.3-2).

5. The donor reagent of claim 1, wherein, The size of the donor particles in the donor reagent is in Gaussian distribution, and the particle size distribution of the donor particles in the donor reagent shows polydispersity.

6. The donor reagent of claim 1, wherein, The coefficient of variation (C.V.) of the particle size distribution of the donor particles in the donor reagent is not lower than 5% and not higher than 20%.

7. The donor reagent according to any one of claims 1 to 6, wherein The preparation method of the donor particles comprises the following steps: Step S1, reacting the donor microspheres with aldehyde groups on the surface of the carrier with one of the specific binding pair members in the presence of an activating agent to obtain an intermediate product; Step S2, adding a blocking agent to block the intermediate product obtained in step S1; Step S3, washing the intermediate product after the blocking treatment in step S2 to obtain the donor particles with one of the specific binding pair members bonded on the surface.

8. The donor reagent of claim 7, wherein, The final concentration of the blocking agent after adding the blocking agent in step S2 is not lower than 5 wt%.

9. A chemiluminescence detection kit comprising the donor reagent according to any one of claims 1 to 8.

10. Use of the donor reagent according to any one of claims 1 to 8 or the kit of claim 9 on a chemiluminescence analyzer.

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