CN113125419B - Donor reagent and application thereof - Google Patents
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- Publication number
- CN113125419B CN113125419B CN201911420699.6A CN201911420699A CN113125419B CN 113125419 B CN113125419 B CN 113125419B CN 201911420699 A CN201911420699 A CN 201911420699A CN 113125419 B CN113125419 B CN 113125419B
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- donor
- reagent
- particles
- agent
- avidin
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Images
Classifications
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/5436—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand physically entrapped within the solid phase
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to a donor reagent and application thereof. The donor agent comprises a buffer solution and donor particles suspended therein, the donor particles comprising a carrier, the carrier having a sensitizer filled therein, the carrier having a surface to which one member of a specific pair of binding members is directly or indirectly attached, characterized in that the donor particles have a coefficient of variation C.V in particle size distribution in the donor agent of not less than 5% and a sugar content in the donor particles of not more than 25mg per gram mass.
Description
Technical Field
The invention relates to the field of chemiluminescent detection, in particular to a donor reagent and application thereof.
Background
In vitro diagnostic (In Vitro diagnosis, IVD) techniques generally refer to products and services that assist in determining disease or body function by detecting samples of the body, including blood, body fluids, and tissues, outside the body to obtain relevant clinical diagnostic information. Nanomaterials have unique size-dependent physical or chemical properties, and in the nanoscale, their optical, magnetic, electrical, thermal and biological properties can be regulated by changing their size, shape, chemical composition, surface functional groups, etc., and in particular nanomaterials can provide a large amount of space to modify different molecules on their surfaces due to their much higher specific surface area than macroscopic materials, making them important in applications such as bioanalysis and biosensors. The nanometer material with the surface modified with different molecules can selectively detect small molecules, nucleic acids, proteins, microorganisms and the like. Obviously, the nano material is hopeful to have the characteristics of lower detection limit, higher sensitivity, stronger selectivity and the like after being fused with the in-vitro diagnosis technology, and the combination of the nano material and the clinical diagnosis analysis technology also pushes the clinical in-vitro diagnosis discipline to a new development growth point.
Immunoassays have evolved over half a century to give rise to a number of detection categories. The separation of the substance to be measured from the reaction system in the measurement process may be classified into heterogeneous immunoassay and homogeneous immunoassay. Heterogeneous immunoassay refers to the main method in the prior immunoassay, wherein various related reagents are required to be separated after mixing reaction in the operation process of marking a probe, and the detection is performed after separating an object to be detected from a reaction system. Such as enzyme-linked immunosorbent assay (ELISA) and magnetic particle chemiluminescence, which are widely known. Homogeneous immunoassay refers to a method in which an analyte is mixed with a reagent in a reaction system and then directly measured in a measurement process without redundant separation or washing steps. Up to now, various sensitive detection methods are applied to homogeneous immunoassays, such as chemiluminescent detection methods, electrochemical detection methods, and the like.
A typical homogeneous immunoassay is known as photo-activated chemiluminescence (Light Initiated Chemiluminescent Assay, liCA). The method is based on the reaction of antigen coated on the surface of one nanometer microsphere and antibody coated on the surface of the other nanometer microsphere in liquid phase to pull the two nanometer microspheres together, thereby forming a 'double-sphere' immune complex. That is, the donor particle and the acceptor particle together form a pair of "double sphere" systems by means of antigen-antibody binding. The double spheres are nano microspheres which complement each other, interact, cooperate and influence each other in a light-activated chemiluminescence system, and are indispensable. The two nano-microspheres have good suspension characteristics in a liquid phase, and the microspheres meet antigen or antibody in the liquid phase to completely meet the liquid dynamic characteristics. Under 680nm laser irradiation, the photosensitizer of the donor particle is responsible for exciting oxygen in the surrounding environment into singlet oxygen molecules. When singlet oxygen molecules diffuse into receptor particles in a 'double-sphere' system, a series of chemiluminescent reactions are generated with chemiluminescent agents in the receptor particles, so that emission wavelength optical signals of 610nm to 620nm are generated, and photon numbers are converted into target molecule concentrations through photon counting and mathematical fitting, so that 'separation-free' homogeneous immunoassay is realized. When the sample to be detected does not contain target molecules, immune complexes cannot be formed between the two nano-microspheres, and the distance between the two nano-microspheres exceeds the propagation range of singlet oxygen within 200nm, so that the singlet oxygen is rapidly quenched in a liquid phase, and no high-energy level red light signal is generated during detection. The method has the characteristics of rapidness, homogeneous phase (no flushing), high sensitivity and simple operation.
However, the "double spheres" in the prior art have the defects of poor homogeneity in a liquid phase, poor repeatability and unstable luminous effect when being used for immunoassay, difficulty in combining high sensitivity, wide detection range and the like.
Disclosure of Invention
Based on the drawbacks of the prior art, one of the present invention provides a donor reagent comprising a buffer solution and donor particles suspended therein, said donor particles being capable of generating active oxygen after being excited in a liquid phase, said donor particles having a coefficient of variation C.V of the particle size distribution in the donor reagent of not less than 5% and a sugar content in said donor particles of not more than 25mg per gram of mass.
In a specific embodiment, the donor particle comprises a carrier, the interior of which is filled with a sensitizer, the surface of which is attached to one of the specific counterpart binding members.
In a specific embodiment, the donor particle has a particle size distribution coefficient of variation C.V in the donor agent of not more than 20%.
In a specific embodiment, the donor particle has a particle size distribution coefficient of variation C.V in the donor agent of no more than 15%.
In a specific embodiment, the donor particle has a coefficient of variation C.V of the particle size distribution in the donor agent of not less than 8%.
In one embodiment, the sugar content in the donor particle is no higher than 15mg per gram mass.
In a specific embodiment, the sugar content in the buffer solution per liter of volume is not less than 0.2g and not more than 2g.
In a specific embodiment, the sugar content in the buffer solution per liter of volume is not less than 0.5g and not more than 1.5g.
In a specific embodiment, the sugar content is detected by an anthrone method.
In a specific embodiment, the surface of the support carries a binding functionality for chemically binding one member of a specific binding pair member to the surface of the support.
In a specific embodiment, the bonding functional group is selected from at least one of an amine group, an amide group, a hydroxyl group, an aldehyde group, a carboxyl group, a maleimide group, and a thiol group; preferably, the bonding functional group is selected from aldehyde groups and/or carboxyl groups.
In a specific embodiment, the particle size distribution of the donor particles in the donor agent exhibits polydispersity.
In a specific embodiment, the donor agent comprises at least two donor particles having an average particle size distribution.
In a specific embodiment, the specific pair binding member is an avidin-biotin system, and the avidin is selected from the group consisting of avidin, vitellin, streptavidin, neutravidin and avidin-like molecules, preferably streptavidin.
In a specific embodiment, the avidin is selected from streptavidin.
The second aspect of the present invention provides a chemiluminescent assay kit comprising a donor reagent according to any one of the present invention.
In one specific embodiment, the kit comprises a plurality of reagent strips, each reagent strip is provided with a plurality of reagent holes for containing the reagent, and one of the reagent holes is used for containing the donor reagent.
The third invention provides the use of the donor reagent according to any one of the first invention or the kit according to any one of the second invention in a chemiluminescent analyzer.
The fourth invention provides the use of the donor reagent according to any one of the inventions or the kit according to any one of the two inventions in the use of POCT instruments.
The donor reagent provided by the invention has the effects that the donor particles in the reagent can generate singlet oxygen after being excited by external excitation light, the singlet oxygen transmits energy to the donor particles within 200nm from the acceptor particles, and finally, chemiluminescent signals can be generated, so that the detection of unknown substances is realized.
The term "active oxygen" as used herein refers to a substance which is composed of oxygen in the body or in the natural environment, contains oxygen and is active in nature, and is mainly an excited oxygen molecule, including an electron reduction product of oxygen, superoxide anion (O 2 Hydrogen peroxide (H), a two-electron reduction product 2 O 2 ) Hydroxyl radical (OH) of three-electron reduction product, nitric oxide and singlet oxygen (1O) 2 ) Etc.
In the present invention, the term "directly or indirectly attached" means that a given substance is capable of being chemically or physically bonded to another substance (direct attachment); or the specified substance is chemically or physically re-bonded to another substance (indirectly linked) via an intermediate substance (compound, polymer, polysaccharide).
The term "acceptor particle" as used herein refers to particles comprising a compound capable of reacting with reactive oxygen species to produce a detectable signal. The donor particles are induced to activate by energy or active compounds and release active oxygen in a high energy state which is captured by the closely spaced acceptor particles, thereby transferring energy to activate the acceptor particles.
In one embodiment, the acceptor 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. The "carrier" according to the invention is selected from the group consisting of tapes, sheets, rods, tubes, wells, microtiter plates, beads, particles and microspheres, which may be microspheres or microparticles well known to the person skilled in the art, which may be of any size, which may be organic or inorganic, which may be expandable or non-expandable, which may be porous or non-porous, which has any density, but preferably has a density close to that of water, preferably floats in water, and is composed of transparent, partially transparent or opaque materials. The carrier may or may not be charged and when charged is preferably negatively charged. The carrier may be latex particles or other particles containing organic or inorganic polymers, lipid bilayers such as liposomes, phospholipid vesicles, oil droplets, silica particles, metal sols, cells and microcrystalline dyes.
In the present invention, the "luminescent agent", a compound called a label, may undergo a chemical reaction to cause luminescence, such as by being converted into another compound formed in an electronically excited state. The excited state may be a singlet state or a triplet excited state. The excited state may relax to the ground state to emit light directly or by transferring excitation energy to an emission energy acceptor, thereby restoring itself to the ground state. In this process, the energy acceptor particles will be transitioned to an excited state to emit light.
The term "donor particle" as used herein refers to particles containing a sensitizer which upon activation of energy or an active compound is capable of generating an active intermediate such as active oxygen which reacts with the acceptor particle. The donor particles may be photoactivated (e.g., dyes and aromatic compounds) or chemically activated (e.g., enzymes, metal salts, etc.). In a specific embodiment, the donor particle is a polymeric microsphere filled with a photosensitizer, which may be a photosensitizer known in the art, preferably a compound that is relatively light stable and does not react efficiently with singlet oxygen, non-limiting examples of which include compounds such as methylene blue, rose bengal, porphyrin, phthalocyanine and chlorophyll as well as derivatives of these compounds having 1-50 atom substituents for making these compounds more lipophilic or hydrophilic, and/or as linking groups to specific binding partners, as disclosed in U.S. patent No. 5709994 (which is incorporated herein by reference in its entirety). Examples of other photosensitizers known to those skilled in the art may also be used in the present invention, such as described in U.S. patent No. 6406913, which is incorporated herein by reference.
In a specific embodiment, the surface of the carrier is coated with a coating of at least two successive polysaccharide layers, wherein the polysaccharide layer is spontaneously associated with the second polysaccharide layer.
In a specific embodiment, each of the continuous polysaccharide layers is spontaneously associated with each of the previous polysaccharide layers.
In a specific embodiment, the polysaccharide has pendant functional groups, the pendant functional groups in the continuous polysaccharide layer being oppositely charged to the pendant functional groups in the preceding polysaccharide layer.
In a specific embodiment, the polysaccharide has pendant functional groups and the continuous polysaccharide layer is covalently linked to the previous polysaccharide layer by a reaction between the pendant functional groups and the pendant functional groups of the previous polysaccharide layer.
In a specific embodiment, in the continuous polysaccharide layer, the pendant functional groups in adjacent two polysaccharide layers alternate between amine functional groups and amine reactive functional groups. I.e. wherein the pendant functional groups in one layer are amine functional groups, then the pendant functional groups in the other layer are amine reactive functional groups.
In a specific embodiment, the amine-reactive functional group is an aldehyde group or a carboxyl group.
In a specific embodiment, the first polysaccharide layer (i.e., the innermost layer) is spontaneously associated with the carrier.
In a specific embodiment, the polysaccharide in the outermost polysaccharide layer of the coating has at least one pendant functional group.
In a specific embodiment, the pendant functional groups of the polysaccharide in the outermost polysaccharide layer of the coating are selected from at least one of aldehyde groups, carboxyl groups, sulfhydryl groups, amino groups, hydroxyl groups, and maleamine groups; preferably selected from aldehyde groups and/or carboxyl groups.
In a specific embodiment, the pendant functional groups of the polysaccharide in the outermost polysaccharide layer of the coating are bound directly or indirectly by a member of a specific binding pair.
In a specific embodiment, the polysaccharide is selected from the group consisting of carbohydrates containing three or more unmodified or modified monosaccharide units; preferably selected from the group consisting of dextran, starch, glycogen, inulin, levan, mannan, agarose, galactan, carboxydextran and aminodextran; more preferably selected from the group consisting of dextran, starch, glycogen and polyribose.
In a specific embodiment, the particle size of the support is selected from 100 to 400nm, preferably 150 to 350nm, more preferably 180 to 220nm.
In a specific embodiment, the concentration of the donor particles in the donor agent is from 10 μg/ml to 1mg/ml, preferably from 20 μg/ml to 500 μg/ml, more preferably from 50 μg/ml to 200 μg/ml.
In a specific embodiment, the donor agent further comprises a buffer solution having a PH of 7.0 to 9.0, and the donor particles are suspended in the buffer solution.
In a specific embodiment, the buffer solution contains a polysaccharide selected from the group consisting of carbohydrates containing three or more unmodified or modified monosaccharide units, preferably selected from the group consisting of dextran, starch, glycogen, inulin, levan, mannan, agarose, galactan, carboxydextran and aminodextran; more preferably selected from the group consisting of dextran, starch, glycogen and polyribose.
In a specific embodiment, the molecular weight distribution Mw of the polysaccharide (e.g. dextran) is selected from 10000 to 1000000Da, preferably from 100000 to 800000Da, more preferably from 300000 to 700000Da.
In a specific embodiment, the sugar content in the buffer solution per liter of volume is not less than 0.2g and not more than 2g.
In a specific embodiment, the sugar content in the buffer solution per liter of volume is not less than 0.5g and not more than 1.5g.
In the present invention, the "coefficient of variation C.V of particle size distribution" refers to the coefficient of variation of particle size in gaussian distribution in the result of detection by a nanoparticle analyzer. The calculation formula of the variation coefficient is as follows: coefficient of variation C.V value= (standard deviation SD/Mean) ×100%. The standard deviation (Standard Deviation, SD), also called standard deviation, describes the average of the distances (from mean deviation) of the individual data from the average, which is the square root after the sum of the squares of the deviations, denoted sigma. The standard deviation is the arithmetic square root of the variance. The standard deviation reflects the degree of dispersion of a data set, and the smaller the standard deviation, the less the values deviate from the average and vice versa. The standard deviation sigma is the distance from the inflection point (0.607 times the peak height) on the normal distribution curve to the perpendicular line of the peak height and the time axis, i.e., half the distance between the two inflection points on the normal distribution curve. The half height peak width (Wh/2) refers to the peak width at half the peak height, wh/2=2.355 σ. The intercept at the base line is called the peak width or base line width, w=4σ or w=1.699 Wh/2, by making tangents to the inflection points on both sides of the normal distribution curve.
The inventors have found through a great deal of research that the value of the variation coefficient C.V of the particle size distribution of particles in a liquid phase affects the photo-excitation chemiluminescence detection signal. If commercial application of the photoexcitation luminescence system in clinical immunodiagnosis is desired, a large quantity of qualified donor particles with stable performance are required to be produced, and the C.V value of the particle size distribution of the donor particles in the donor reagent must be strictly controlled within a proper range. It should be noted that, unlike the conventional blank polystyrene microsphere (i.e., the carrier of the present invention, which is not filled with a functional material on the inside and modified with avidin molecules or polysaccharides on the surface), the C.V value of the present invention refers to the value of C.V of the variation coefficient of the particle size distribution of the whole donor particle in the donor reagent. Since the change of the C.V value of the particle size distribution of the donor particles is more remarkable and unstable after the coating of the polysaccharide or the avidin, the change of the C.V value of the particle size distribution of the nano-microsphere is more remarkable and unstable in the preparation process of the donor particles, and the reagent requirements of medical instrument product registration and clinical application are difficult to meet. Therefore, through a great deal of experimental study, the applicant gradually searches and discovers a range and a method for strictly controlling the variation coefficient C.V value of the particle size distribution of the donor particles in the liquid phase, and finally realizes the commercial application of the photo-excitation chemiluminescence technology in clinical examination.
The term "test sample" as used herein refers to a mixture to be tested that contains or is suspected of containing a target molecule to be tested. Samples to be tested that may be used in the present invention include body fluids such as blood (which may be anticoagulated blood as is commonly found in collected blood samples), plasma, serum, urine, semen, saliva, cell cultures, tissue extracts, and the like. 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 from prokaryotes. The sample to be measured can be diluted with a diluent as required before use. For example, in order to avoid the HOOK effect, the sample to be tested may be diluted with a diluent before on-machine testing and then tested on a testing instrument.
The term "target molecule to be detected" as used herein refers to a substance in a sample to be detected during detection. One or more substances having a specific binding affinity for the target molecule to be detected may be used to detect the target molecule. The target molecule to be tested may be a protein, peptide, antibody or hapten which can be conjugated to an antibody. The target molecule to be detected may be a nucleic acid or oligonucleotide that binds to a complementary nucleic acid or oligonucleotide. The target molecule to be tested may be any other substance that can form a specific binding pair member. Examples of other typical target molecules to be measured include: drugs such as steroids, hormones, proteins, glycoproteins, mucins, nucleoproteins, phosphoproteins, drugs of abuse, vitamins, antibacterial agents, antifungal agents, antiviral agents, purines, antitumor agents, amphetamines, heteronitrogen compounds, nucleic acids and prostaglandins, and metabolites of any of these drugs; pesticides and metabolites thereof; and a recipient. Analytes also include cells, viruses, bacteria, and fungi.
The term "antibody" as used herein is used in its broadest sense and includes antibodies of any isotype, antibody fragments that retain specific binding to an antigen, including but not limited to Fab, fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies, bispecific antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. In any desired case, the antibody may be further conjugated to other moieties, such as a member of a specific binding pair member, e.g., biotin or avidin (a member of a biotin-avidin specific binding pair member), and the like.
The term "antigen" as used herein refers to a substance that stimulates the body to produce an immune response and binds to antibodies and sensitized lymphocytes, which are the products of the immune response, in vivo and in vitro, resulting in an immune effect.
The term "binding" as used herein refers to the direct association between two molecules due to interactions such as covalent, electrostatic, hydrophobic, ionic and/or hydrogen bonding, including but not limited to interactions such as salt and water bridges.
The term "specific binding" as used herein refers to the mutual recognition and selective binding reaction between two substances, and from a steric perspective, corresponds to the conformational correspondence between the corresponding reactants. Under the technical ideas disclosed in the present invention, the detection method of the specific binding reaction includes, but is not limited to: a diabody sandwich method, a competition method, a neutralization competition method, an indirect method or a capture method.
The term "specific binding pair member" as used herein refers to a pair of molecules that are capable of specifically binding to each other, e.g., enzyme-substrate, antigen-antibody, ligand-receptor. An example of a specific binding pair member is the biotin-streptavidin system, where "biotin" is widely found in animal and plant tissues and has two cyclic structures on the molecule, an imidazolone ring and a thiophene ring, respectively, where the imidazolone ring is the primary site of binding to streptavidin. Activated biotin can be coupled to almost all known biomacromolecules, including proteins, nucleic acids, polysaccharides, lipids, etc., mediated by protein cross-linking agents; and the avidin is selected from the group consisting of ovalbumin, streptavidin, vitellin, neutravidin and avidins, preferably neutravidin and/or streptavidin. Avidin is a glycoprotein which can be extracted from egg white and has a molecular weight of 60kD, and each molecule consists of 4 subunits, so that the avidin can be closely combined with 4 biotin molecules, and plays an important role in an immune mechanism. The avidin mainly comprises ovalbumin, streptavidin, yolk avidin, neutravidin and the like. Streptavidin is a protein secreted by streptomyces, and the "streptavidin" molecule consists of 4 identical peptide chains, each of which is capable of binding to a biotin. Thus, each antigen or antibody can be conjugated to multiple biotin molecules simultaneously, thereby producing a "tentacle effect" that enhances assay sensitivity. In any case where desired, any agent used in the present invention, including antigen, antibody, acceptor particle or donor particle, may be conjugated to any member of the biotin-streptavidin specific binding pair member as desired.
In the photoexcitation chemiluminescent system, in addition to the donor reagent, other reagents are included according to the requirement of the detection object or detection method, such as: receptor reagents, biotin-coated secondary antibodies, dilutions, and the like. In the field of in vitro diagnosis, especially in the field of immunoassay, in order to simplify the naming of different components in a commercial kit, each manufacturer usually marks or simply refers to the components of different bottles in the kit as reagent 1 or R1, reagent 2 or R2, reagent 3 or R3, … …, and so on, so that the kit is convenient for customer identification, assembly and use, and also for technical security purposes. Therefore, the kit products of different in vitro diagnostic manufacturers may contain the reagent 1, the reagent 2, the reagent 3 and the reagent … …, but the corresponding reagent components of different manufacturers are different.
The invention has the beneficial effects that:
the liquid homogeneous mode ensures that the consistency among the tests is better, and the product has better repeatability and consistency. And no flushing is adopted, so that unnecessary interferents and other uncertain factors are prevented from being introduced in the reaction, and the detection result is more stable. Unique chemiluminescence technology can be introduced in the field of rapid diagnosis, so that the overall detection performance is better.
Drawings
Fig. 1 shows the gaussian distribution of donor particles a prepared in example 1.
Fig. 2 shows the gaussian distribution of donor particles b prepared in example 2.
FIG. 3 shows the relationship between glucose concentration and the corresponding absorbance.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The invention is further illustrated below with reference to the examples, which are merely illustrative of the invention and do not constitute a limitation of the invention in any way.
The reagents used in the following examples were commercially available unless otherwise specified.
Example 1 preparation of donor particles a and donor agent A containing such donor particles a
1.1 preparation of the vector
a) A100 ml three-necked flask was prepared, 40mmol of styrene, 5mmol of acrolein and 10ml of water were added thereto, and after stirring for 10 minutes, N was introduced 2 30min。
b) 0.11g of ammonium persulfate and 0.2g of sodium chloride were weighed and dissolved in 40ml of water to prepare an aqueous solution. Adding the aqueous solution into the reaction system of the step a), and continuing to introduce N 2 30min。
c) The reaction system was warmed to 70℃and reacted for 15 hours to obtain an emulsion.
d) And cooling the emulsion after the reaction to room temperature, filtering the emulsion by using a proper filter cloth, centrifugally settling and cleaning the obtained filtered emulsion for a plurality of times by using deionized water until the conductivity of the supernatant obtained after centrifugation is close to that of the deionized water, and then diluting the supernatant by using water to obtain the final emulsion aldehyde polystyrene microsphere liquid.
e) The size of the support was measured by a nanoparticle analyzer and was gaussian, the average particle diameter was 201.3nm, and the coefficient of variation (c.v.) was=8.0%.
1.2 sensitizer filled Carrier
a) A25 ml round bottom flask was prepared, 0.11g copper phthalocyanine, 10ml N, N-dimethylformamide was added thereto, and magnetically stirred, and the temperature was raised to 75℃in a water bath to obtain a photosensitizer solution.
b) A100 ml three-necked flask was prepared, 10ml of 95% ethanol, 10ml of water, and 10ml of the carrier prepared in step 1.1 having a concentration of 10% were added, and magnetically stirred, and the temperature was raised to 70℃in a water bath.
c) Slowly dripping the photosensitizer solution in the step a) into the three-neck flask in the step b), stirring and reacting for 2 hours at 70 ℃ to enable the sensitizer to be filled into the aldehyde-based polystyrene microspheres, stopping stirring, and naturally cooling to obtain a filled emulsion.
d) The filled emulsion was centrifuged for 1 hour at 30000g, after which the supernatant was discarded and resuspended in 50% ethanol. After repeating the centrifugal washing three times, the suspension was resuspended in 50mM CB buffer having a pH value of=10 so that the final concentration of the aldehyde-based polystyrene microspheres filled with the sensitizer was 20mg/ml, to obtain a microsphere suspension.
1.3 preparation of Donor reagent A
a) Microsphere suspension treatment: centrifuging the microsphere suspension prepared in the step (II) in a high-speed refrigerated centrifuge, discarding the supernatant, adding MES buffer, performing ultrasonic treatment on an ultrasonic cell disruption instrument until particles are resuspended, and adding the MES buffer to adjust the mass concentration of the microspheres to 100mg/ml to obtain the microsphere suspension after treatment.
b) Avidin solution preparation: a quantity of streptavidin was weighed and dissolved to 8mg/ml in MES buffer.
c) Mixing: mixing the treated microsphere suspension, avidin of 8mg/ml and MES buffer in the volume ratio of 2:5:1, and rapidly and uniformly mixing to obtain a reaction solution.
d) The reaction: 25mg/ml NaBH is prepared by MES buffer solution 3 CN solution according to NaBH 3 The CN solution and the reaction solution are added in a volume ratio of 1:25 and are rapidly and uniformly mixed. The reaction was rotated at 37℃for 48 hours.
e) Closing: 75mg/ml glycine (Gly) solution and 25mg/ml NaBH were prepared separately using MES buffer 3 CN solution, according to glycine solution and NaBH 3 Adding the CN solution and the reaction solution in the volume ratio of 2:1:10 into the solution in the step d), uniformly mixing, and carrying out rotary reaction for 2 hours at 37 ℃. Then 200mg/ml BSA solution (prepared by MES buffer solution) was added, the volume ratio of BSA solution to reaction solution was 5:8, and the mixture was rapidly and uniformly mixed and reacted at 37℃for 16 hours.
f) Cleaning: adding MES buffer solution into the solution after the reaction in the step e), centrifuging by a high-speed refrigerated centrifuge, discarding the supernatant, adding fresh MES buffer solution again, carrying out ultrasonic suspension, centrifuging again, washing 3 times in this way, finally suspending by using a small amount of buffer solution, measuring the solid content, and regulating the solid content to the concentration of 150 mug/ml by using the buffer solution to obtain a donor reagent A containing donor particles a, wherein the buffer solution comprises the following components: 0.1mol Tris-HCl, 0.3mol NaCl, 25mmol EDTA, 0.1% dextran, 0.01% gentamicin and 15ppm ProClin-300, pH 8.00.
g) The size of donor particles a in donor reagent a was gaussian, the average particle diameter was 227.7nm, and the coefficient of variation c.v. value was 6.5% as measured by a nanoparticle analyzer, as shown in fig. 1.
Example 2 preparation of donor particles B and Donor reagent B containing such donor particles B
The procedure for preparing the support used in this example and for filling the sensitizer is the same as in steps 1.1 and 1.2 of example 1.
2.1 coating of the surface of the Carrier with dextran
a) 50mg of aminodextran solid was taken in a 20mL round bottom flask, 5mL of 50 mm/ph=10 carbonate buffer was added and dissolved by stirring away from light at 30 ℃.
b) 100mg of donor particles a were added to the aminodextran solution and stirred for 2 hours to obtain a reaction solution.
c) 10mg of sodium borohydride was dissolved in 0.5ml of 50 mM/pH=10 carbonate buffer, and then added dropwise to the reaction solution, followed by reaction at 30℃overnight in the absence of light, to obtain a mixed solution of the carrier coated with aminodextran.
d) Centrifuging 30000g of the mixture obtained in the step c), discarding the supernatant, and adding 50 mM/pH=10 carbonate buffer for ultrasonic dispersion. After repeating the centrifugation washing three times, the volume was fixed with 50 mM/pH=10 carbonate buffer so that the final concentration of the carrier coated with aminodextran was 20mg/ml, to obtain aminodextran carrier fluid.
e) 100mg of aldehyde dextran solid was taken in a 20mL round bottom flask, 5mL of 50 mm/ph=10 carbonate buffer was added and dissolved by stirring away from light at 30 ℃.
f) The aminodextran carrier fluid was added to the aldehyde dextran solution and stirred for 2 hours.
g) 15mg of sodium borohydride was dissolved in 0.5ml of 50 mM/pH=10 carbonate buffer, and then added dropwise to the reaction solution, followed by reaction at 30℃overnight in the absence of light, to obtain a mixture of carriers coated with aldehyde dextran.
h) Centrifuging 30000g of the mixture obtained in the step g), discarding the supernatant, and adding 50 mM/pH=10 carbonate buffer for ultrasonic dispersion. After repeating the centrifugation washing three times, the volume was fixed with 50 mM/pH=10 carbonate buffer so that the final concentration of the carrier coated with the aldehyde dextran was 20mg/ml, to obtain a dextran-coated carrier fluid.
i) The size of the dextran-coated carrier was measured by a nanoparticle sizer as a gaussian distribution with an average particle size of 235.6nm and a coefficient of variation (c.v.) of 8.1%.
2.2 preparation of donor reagent B
a) Treatment of dextran-coated carrier liquid: centrifuging the glucan coated carrier liquid prepared in the step h) in the step 2.1 of the embodiment in a high-speed refrigerated centrifuge, discarding the supernatant, adding an MES buffer solution, performing ultrasonic treatment on an ultrasonic cytoclasis instrument until the microspheres are resuspended, and then adding the MES buffer solution to adjust the mass concentration of the carrier to 100mg/ml.
b) Avidin solution preparation: a certain amount of neutravidin was weighed and dissolved in MES buffer to 8mg/ml.
c) Mixing: mixing the treated microsphere suspension, avidin of 8mg/ml and MES buffer in the volume ratio of 2:5:1, and rapidly and uniformly mixing to obtain a reaction solution.
d) The reaction: 25mg/ml NaBH is prepared by MES buffer solution 3 CN solution according to NaBH 3 The CN solution and the reaction solution are added in a volume ratio of 1:25 and are rapidly and uniformly mixed. The reaction was rotated at 37℃for 48 hours.
e) Closing: preparation of 75mg/ml Gly solution and 25mg/ml NaBH with MES buffer distribution 3 CN solution, according to Gly solution and NaBH 3 Adding the CN solution and the reaction solution in the volume ratio of 2:1:10 into the solution in the step d), uniformly mixing, and carrying out rotary reaction for 2 hours at 37 ℃. Further 200mg/ml BSA solution (prepared with MES buffer) was added, BSThe volume ratio of the solution A to the reaction solution is 5:8, and the solution A and the reaction solution are quickly and evenly mixed and are subjected to rotary reaction at 37 ℃ for 16 hours.
f) Cleaning: adding MES buffer solution into the solution after the reaction in the step e), centrifuging by a high-speed refrigerated centrifuge, discarding the supernatant, adding fresh MES buffer solution again, carrying out ultrasonic suspension, centrifuging again, washing 3 times in this way, finally suspending by using a small amount of buffer solution, measuring the solid content, and regulating the solid content to 150 mug/ml by using the buffer solution to obtain a donor reagent B containing donor particles B, wherein the buffer solution comprises the following components: 0.1mol Tris-HCl, 0.3mol NaCl, 25mmol EDTA, 0.1% dextran, 0.01% gentamicin and 15ppm ProClin-300, pH 8.00.
g) The size of donor particles B in donor reagent B was gaussian, the average particle diameter was 249.9nm, and the coefficient of variation C.V was 11.6% as measured by a nanoparticle analyzer, as shown in fig. 2.
Example 3 determination of sugar content in donor particles
3.1 determination of the relationship between the glucose concentration and the corresponding absorbance Using the anthrone method
a) Preparing a glucose standard solution: the glucose stock solution of 1mg/mL was formulated with purified water into standard solution curves of 0mg/mL, 0.025mg/mL, 0.05mg/mL, 0.075mg/mL, 0.10mg/mL, 0.15 mg/mL.
b) Configuration of anthrone solution: 2mg/mL of the solution (stable at room temperature for 24h, ready-to-use) was prepared with 80% sulfuric acid solution.
c) To each centrifuge tube, 0.1mL of glucose standard solution at each concentration was added, and 1mL of anthrone test solution was added to each tube.
d) Incubating at 85 ℃ for 30min; then, 15000g was centrifuged for 40min, and clarified liquid was aspirated from the bottom of the tube with a pipette tip to avoid aspiration of the upper suspension.
e) After the aspirated clear liquid was returned to room temperature, its absorbance at 620nm was measured (measurement was preferably performed within 2 hours), repeated twice, and the average of the two repetitions was taken as the final absorbance, and the results are shown in table 1.
f) And (3) carrying out primary linear regression by taking the concentration of the standard substance as an X value and the average absorbance as a Y value, wherein the result is shown in figure 3, so that the sugar concentration of the sample to be detected is obtained.
TABLE 1
| Sequence number | Concentration mg/mL | Absorbance A | Absorbance B | Absorbance mean |
| 1 | 0.15 | 0.415 | 0.411 | 0.4130 |
| 2 | 0.1 | 0.293 | 0.302 | 0.2975 |
| 3 | 0.075 | 0.214 | 0.227 | 0.2205 |
| 4 | 0.05 | 0.146 | 0.153 | 0.1495 |
| 5 | 0.025 | 0.101 | 0.098 | 0.0995 |
| 6 | 0 | 0.032 | 0.031 | 0.0315 |
3.2 determination of sugar content in donor particles
a) Taking a donor reagent containing 1mg of donor particles, centrifuging for 40min at 20000g, pouring out supernatant, performing ultrasonic dispersion by using purified water, repeating the centrifugal dispersion for three times, and then performing constant volume to 1mg/mL by using the purified water to prepare a sample to be tested.
b) Configuration of anthrone solution: 2mg/mL of the solution (stable at room temperature for 24h, ready-to-use) was prepared with 80% sulfuric acid solution.
c) To the centrifuge tube, 0.1mL of the sample to be tested and 1mL of the anthrone test solution were added sequentially.
d) Incubating at 85 ℃ for 30min; the solution was centrifuged at 15000g for 40min and clarified liquid was aspirated from the bottom of the tube with a pipette tip to avoid aspiration of the upper suspension.
e) After the aspirated clarified liquid was returned to room temperature, its absorbance at 620nm (preferably within 2 h) was measured, repeated twice, the average of the two repetitions was taken as the final absorbance value, and then the sugar content in the donor particles was calculated according to the curve equation obtained in fig. 3, and the results are shown in table 2.
TABLE 2
| Sequence number | Absorbance A | Absorbance B | Absorbance mean | Sugar concentration mg/g |
| Donor particles a | 0.0572 | 0.0564 | 0.0568 | 11.5 |
| Donor particles b | 0.172 | 0.165 | 0.1685 | 53.6 |
Example 4
The donor agent comprising the following series of donor particles was prepared using the method of preparation of donor particles in example 1 above:
Donor reagent 1: the average particle diameter of the donor particles in the gaussian distribution curve is 226.5nm, and the variation coefficient C.V value of the particle diameter distribution=3.8; nicomp distribution is unimodal.
Donor reagent 2: the average particle diameter of the donor particles in the gaussian distribution curve is 225.3nm, and the variation coefficient C.V value of the particle diameter distribution=4.6; nicomp distribution is unimodal.
Donor reagent 3: the average particle diameter of the donor particles in the gaussian distribution curve is 225.2nm, and the variation coefficient C.V value of the particle diameter distribution=5.0; nicomp distribution is unimodal.
Donor reagent 4: the average particle diameter of the donor particles in the gaussian distribution curve is 226.7nm, and the variation coefficient C.V value of the particle diameter distribution=8.1; nicomp distribution is unimodal.
Donor reagent 5: the average particle diameter of the donor particles in the gaussian distribution curve is 227.8nm, and the variation coefficient C.V value of the particle diameter distribution=15.6; nicomp distribution is unimodal.
Donor reagent 6: the average particle diameter of the donor particles in the gaussian distribution curve is 225.9nm, and the particle diameter distribution variation coefficient C.V value=26.1; nicomp distribution is unimodal.
Troponin I quantitative assay kits 1 to 6 were prepared using the above donor reagents 1 to 6.
The kit of this example consists of reagent 1 (R1 ') comprising a first anti-cTnI antibody-coated acceptor particle, reagent 2 (R2 ') comprising a biotin-labeled second anti-cTnI antibody, and further comprises any one of donor reagents 1 to 6 (R3 '). Obviously, R1' is the acceptor agent; r3' is a donor agent.
The chemiluminescent detection process is completed on a fully automatic light-activated chemiluminescent analysis system (LiCA HT) developed by Boyang biotechnology (Shanghai) limited company, and the detection result is output, and the specific experimental steps are as follows:
(1) Adding 40 μl of a sample to be tested containing cTnI markers with known concentration, 15 μ l R 'and 15 μ l R2' into a 96-well plate of 8×12, and mixing;
(2) Incubation at 37℃for 8min;
(3) 160 μl of universal solution (R3') was added;
(4) Incubation at 37℃for 2min;
(5) Excitation readings were carried out in a LiCA HT instrument and specific detection results are shown in Table 3 below. The sensitivity and upper limit of detection of the kits 1 to 6 are then analyzed based on the detection results.
TABLE 3 Table 3
As can be seen from Table 3, when the variation coefficient of the particle size distribution of the donor particles is greater than or equal to 5%, the method has the advantages of proper sensitivity, proper detection range, relatively low production cost of the reagent raw materials and the kit, and capability of meeting the requirements of clinical in-vitro diagnosis application.
Example 5
Donor agents comprising the following series of donor particles were prepared using the donor particles and donor agent preparation methods described above in examples 1 and 2, and sugar content in the donor particles was detected using the anthrone method in example 3.
Referring to the method given in example 4 above, troponin I quantitative assay test kits a to E containing donor reagents a to E were prepared, and the chemiluminescent assay process was performed on a fully automated light activated chemiluminescent assay system (LiCA HT) developed by bosa biotechnology (shanghai) limited and the assay results were output, and specific experimental procedures were taken in the procedure given in example 4, and the experimental results are shown in table 4.
TABLE 4 Table 4
| Donor reagent name | Sugar content of donor particles mg/g | Photo-activated chemiluminescent detection signal |
| Donor reagent a in example 1 | 11.5 | 1117775 |
| Donor reagent B in example 2 | 53.6 | 42500 |
| Donor reagent C | 23.8 | 1079654 |
| Donor reagent D | 45.3 | 32642 |
| Donor reagent E | 60.3 | 25056 |
As can be seen from Table 4, the detection effect of photoexcitation is better when the sugar content of the donor particles in the donor reagent is lower than 25 mg/g.
Example 6
This example utilizes the homogeneous chemiluminescent POCT detection device disclosed in CN208568604U to detect CRP.
The kit prepared by the embodiment comprises a plurality of reagent cup strips, wherein a plurality of hole sites for containing reagents are arranged on the reagent cup strips, and the hole sites comprise, but are not limited to, a hole site of a sample to be tested, a hole site of a first reagent and a hole site of a second reagent, wherein the hole site of the sample to be tested is used for containing a sample to be tested containing target molecules to be tested. The first reagent well is for holding a donor reagent comprising donor particles capable of generating singlet oxygen in an excited state. The second reagent pore site is configured to hold an acceptor reagent comprising an acceptor particle capable of reacting with singlet oxygen to generate a chemiluminescent signal, and the donor particle has a particle size greater than the particle size of the acceptor particle. The hole site can be selected and the function can be expanded according to actual needs.
The donor reagent contained in the first reagent well of this example was donor reagent A prepared in example 1, in which the donor particles had a coefficient of variation C.V in the particle size distribution of 6.5% and a sugar content of 11.5mg/g. 50. Mu.L of clinical samples (containing serum and whole blood) of different concentrations of CRP were added to one well site of the reagent cup strip, the average value was taken from each well of parallel tubes, 50. Mu.L of biotinylated anti-CRP antibody, 50. Mu.L of acceptor reagent containing coupled CRP acceptor particles was reacted at 37℃for 7.5min, 50. Mu.L of donor reagent A prepared in example 1 was further added, and reacted at 37℃for 5min, and the light excitation detection was performed, and the experimental results were shown in Table 5. Table 5 shows that the correlation coefficient between serum and whole blood reached 0.9988. The experimental result shows that the donor particles greatly reduce the non-specific adsorption in the sample, so that the measurement results of serum and whole blood have very good correlation, the adaptability of the donor reagent to clinical samples is greatly enhanced, and the donor reagent can be directly used for detecting the clinical whole blood samples.
TABLE 5
Example 7
The present example detects 40 clinical specimens, including 13 negative specimens (numbered 1 to 13) and 27 positive specimens (numbered 14 to 40), and the cTnI quantitative assay kit (photoexcitation chemiluminescence method) used includes: reagent 1 (R1) comprising a first anti-cTnI monoclonal antibody coated acceptor particle, reagent 2 (R2) comprising a biotin-labeled second anti-cTnI monoclonal antibody, and photostimulated chemiluminescent assay system universal solution (R3) comprising donor particles. Wherein the concentration of donor particles in the reagent R3 is 100 μg/ml, the variation coefficient C.V value of the particle size distribution of the donor particles in the reagent R3=11%, and the sugar content of the donor particles is 14.5mg/g.
The detection process was performed on a fully automatic light-activated chemiluminescence analysis system (LiCA HT) developed by bosa biotechnology (Shanghai) limited and the detection result was outputted, and the specific detection procedure is described in example 4.
When a cTnI marker exists in a clinical sample, the cTnI marker is simultaneously specifically combined with a receptor particle coated with a first anti-cTnI monoclonal antibody and a biotin-labeled second anti-cTnI monoclonal antibody, and a double-antibody sandwich complex is formed on the surface of the receptor particle; at this time, if the streptavidin-modified donor particle is added, biotin and streptavidin are combined to enable the two particles to be close to each other, the donor particle releases singlet oxygen under the excitation of the excitation light source, chemiluminescence is generated after the donor particle is hit against the acceptor particle in the solution, and therefore fluorescent groups on the same particle are further excited to generate cascade amplification reaction to generate fluorescence. At this time, the more cTnI marker content is present, the stronger the fluorescence intensity, the amount of cTnI marker in the serum of the patient is quantitatively detected according to the intensity of the luminescence, and the specific cTnI marker content detection result is shown in table 6.
TABLE 6
The correlation between Abbott measurement and Boyang measurement is 0.9973 and the slope is 1.0495. Sample nos. 1-13 were normal physical examination patients with a distribution range of 1.77pg/ml to 25.3pg/ml, with a median value of 6.77pg/ml; samples 14-40 were identified as having myocardial damage and were distributed over a range of 30.94pg/ml to 29896.88pg/ml with a median value of 450.54pg/ml. Cardiac troponin I (cTnI) is low in serum or plasma of healthy people, necrotic cardiac myocytes release a large amount of cTNI into the blood circulation system 4-8 hours after chest pain of patients occurs, peak value is reached 12-48 hours, and cTnI is still maintained at a high level after a few days of severe myocardial infarction patients, so that the cardiac troponin I is an optimal marker for diagnosing myocardial injury and myocardial infarction. The data of embodiment 7 according to the present invention show that the use of the donor reagent according to the present invention in the preparation of a kit for use in a method for in vitro diagnosis of whether a subject has myocardial damage is feasible, and that the quantitative determination of cTnI markers in body fluids of a subject using the donor reagent according to the present invention and the corresponding method can be used for diagnosis of myocardial damage and myocardial infarction.
Claims (20)
1. A donor reagent comprising a buffer solution and donor particles suspended therein, the donor particles being capable of generating active oxygen upon excitation in a liquid phase, the donor particles comprising a carrier, the interior of which is filled with a sensitizer, the surface of which is linked to one of the specific counterpart binding members; the specific pairing-binding member is an avidin-biotin system, and the avidin is selected from avidin, vitelline avidin, streptavidin, neutravidin and avidin-like molecules; characterized in that the donor particles have a coefficient of variation C.V in the particle size distribution in the donor agent of not less than 5% and a sugar content in the donor particles per gram mass of not more than 25mg.
2. The donor agent of claim 1, wherein the donor particles have a coefficient of variation C.V of the particle size distribution in the donor agent of not more than 20%.
3. The donor agent of claim 2, wherein the donor particles have a coefficient of variation C.V of the particle size distribution in the donor agent of not less than 15%.
4. The donor agent of claim 1, wherein the donor particles have a coefficient of variation C.V of the particle size distribution in the donor agent of not less than 8%.
5. The donor agent of claim 1, wherein the sugar content in the donor particle per gram mass is not higher than 15mg.
6. The donor reagent according to claim 1, wherein the sugar content in the buffer solution per liter of volume is not lower than 0.2g and not higher than 2g.
7. The donor reagent of claim 6 wherein the sugar content in the buffer solution per liter of volume is not less than 0.5g and not more than 1.5g.
8. The donor reagent of claim 1 wherein the sugar content is detected by an anthrone method.
9. The donor agent of claim 1, wherein the surface of the support bears a binding functionality for chemically binding one of the specific binding pair members to the surface of the support.
10. The donor agent of claim 9 wherein the surface of the support is not coated with a polysaccharide and one of the specific binding pair members is directly chemically bonded to a bonding functional group on the surface of the support.
11. The donor reagent of claim 9 wherein the bonding functional group is selected from at least one of an amine group, an amide group, a hydroxyl group, an aldehyde group, a carboxyl group, a maleimide group, and a thiol group.
12. The donor agent of claim 11, wherein the bonding functional groups are selected from aldehyde groups and/or carboxyl groups.
13. The donor agent of any of claims 1 to 12, wherein the particle size distribution of the donor particles in the donor agent exhibits polydispersity.
14. The donor agent according to any of claims 1 to 12, wherein the donor agent comprises at least two donor particles having an average particle size distribution.
15. The donor agent of any one of claims 1 to 12 wherein the surface of the carrier is directly bonded to one of the members of the specific mating binding pair.
16. The donor agent of any one of claims 1 to 12, wherein the avidin is streptavidin.
17. A chemiluminescent detection kit comprising the donor reagent of any one of claims 1 to 16.
18. The kit of claim 17, wherein the kit comprises a plurality of reagent strips, each reagent strip having a plurality of reagent wells for holding a reagent, wherein one of the reagent wells is for holding the donor reagent.
19. Use of a donor reagent according to any one of claims 1 to 16 or a kit according to claim 17 or 18 in a chemiluminescent analyzer.
20. Use of the donor reagent of any one of claims 1 to 16 or the kit of claim 17 or 18 in a POCT instrument.
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| CN116449001A (en) | 2023-07-18 |
| CN116400074A (en) | 2023-07-07 |
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