A ctni detection kit and application thereof

Abstract

The present application relates to a cTnI detection kit and application thereof. The cTnI detection kit comprises: reagent 1 containing first anti-cTnI antibody coated receptor particles, and donor reagent containing donor particles, the donor reagent comprising a buffer solution and donor particles suspended therein, the donor particles being capable of generating active oxygen after being excited in a liquid phase; the coefficient of variation C.V. of the particle size distribution of the donor particles in the donor reagent is not less than 5%, and the sugar content in the donor particles per gram of mass is not higher than 25 mg.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of chemiluminescence detection, and more particularly, the present technical solution relates to a cTnI detection kit and application thereof. BACKGROUND

[0002] In vitro diagnosis (IVD) technology generally refers to obtaining relevant clinical diagnostic information by detecting samples such as blood, body fluid and tissue outside the human body, so as to help judge diseases or body functions. Nanomaterials have unique size-dependent physical or chemical properties. In the nanometer scale, their optical, magnetic, electrical, thermal and biological properties can be adjusted by changing their size, shape, chemical composition and surface functional groups. In particular, nanomaterials have a specific surface area much higher than that of macroscopic materials, which can provide a large amount of space for surface modification of different molecules, making them play an important role in biological analysis and biosensors. Nanomaterials modified with different molecules can selectively detect small molecules, nucleic acids, proteins and microorganisms. Obviously, the combination of nanomaterials and in vitro diagnostic technology is expected to have lower detection limit, higher sensitivity and stronger selectivity. The combination of nanomaterials and clinical diagnostic analysis technology will also push the field of clinical in vitro diagnostics to a new growth point.

[0003] Immunoassay has developed for more than half a century, and many detection types have appeared. According to whether the measured substance needs to be separated from the reaction system during the determination process, it can be divided into heterogeneous immunoassay and homogeneous immunoassay. Heterogeneous immunoassay refers to the operation process in which the probe is labeled. After mixing and reaction of various related reagents, separation is needed, and the measured substance is separated from the reaction system before detection. It is the mainstream method in current immunoassay. For example, the widely known enzyme-linked immunosorbent assay (ELISA method) and magnetic particle chemiluminescence method. Homogeneous immunoassay refers to the direct determination of the measured substance mixed with the related reagents in the reaction system after mixing and reaction, without the separation or washing steps. So far, various sensitive detection methods have been applied to homogeneous immunoassay, such as chemiluminescence detection method, electrochemical detection method, etc.

[0004] Light Initiated Chemiluminescent Assay (LiCA) is a typical homogeneous immunoassay method. It is based on the reaction of an antigen coated on a nanoball and an antibody coated on another nanoball in a liquid phase to pull the two nanoballs close to form a "double ball" immune complex. The donor particle and the acceptor particle together form a pair of "double ball" system through the binding between the antigen and the antibody. Both of the "double ball" are nanoballs, which complement each other, interact with each other, cooperate with each other, and influence each other in the light-activated chemiluminescent system. The two nanoballs have good suspension properties in the liquid phase, and the nanoballs meet the liquid dynamics characteristics when meeting the antigen or the antibody in the liquid phase. Under the irradiation of 680 nm laser, the photosensitizer of the donor particle is responsible for exciting the oxygen in the surrounding environment into singlet oxygen molecules. When the singlet oxygen molecules diffuse to the acceptor particle in the "double ball" system, a series of chemiluminescence reactions occur between the singlet oxygen molecules and the chemiluminescent agent in the acceptor particle, thereby generating a light signal with an emission wavelength of 610 nm to 620 nm. Through photon counting and mathematical fitting, the number of photons is converted into the concentration of the target molecule, thereby realizing the "separation-free" homogeneous immunoassay. When the target molecule is not contained in the sample to be measured, the immune complex cannot be formed between the two nanoballs, and the distance between the two nanoballs exceeds the propagation range of the singlet oxygen within 200 nm. Therefore, the singlet oxygen is rapidly quenched in the liquid phase, and no high-energy red light signal is generated during detection. The method has the characteristics of rapidity, homogeneity (no washing), high sensitivity, and simple operation.

[0005] However, the "double ball" in the prior art has poor homogeneity in the liquid phase, poor repeatability when used for immunoassay, unstable light emission effect, and difficulty in balancing high sensitivity and wide detection range. SUMMARY

[0006] Based on the defects in the prior art, one of the embodiments of the present application provides a donor reagent, which comprises a buffer solution and donor particles suspended therein. The donor particles can generate active oxygen after being excited in the liquid phase. The coefficient of variation C.V of the particle size distribution of the donor particles in the donor reagent is not less than 5%, and the sugar content in the donor particles per gram of mass is not more than 25 mg.

[0007] In one specific embodiment, the donor particles comprise a carrier, the inside of the carrier is filled with a sensitizer, and the surface of the carrier is connected with one of the specific pairing members.

[0008] In one specific embodiment, the coefficient of variation C.V of the particle size distribution of the donor particles in the donor reagent is not more than 20%.

[0009] In one embodiment, the coefficient of variation (C.V.) of the particle size distribution of the donor particles in the donor reagent is not more than 15%.

[0010] In one embodiment, the coefficient of variation (C.V.) of the particle size distribution of the donor particles in the donor reagent is not less than 8%.

[0011] In one embodiment, the sugar content in the donor particles per gram of mass is not more than 15 mg.

[0012] In one embodiment, the sugar content in the buffer solution per liter of volume is not less than 0.2 g and not more than 2 g.

[0013] In one embodiment, the sugar content in the buffer solution per liter of volume is not less than 0.5 g and not more than 1.5 g.

[0014] In one embodiment, the sugar content is detected by anthrone method.

[0015] In one embodiment, the surface of the carrier is provided with a bonding functional group for chemically bonding one of the specific binding pair members on the surface of the carrier.

[0016] In one embodiment, the bonding functional group is selected from at least one of amine group, amide group, hydroxyl group, aldehyde group, carboxyl group, maleimide group and thiol group; preferably, the bonding functional group is selected from aldehyde group and / or carboxyl group.

[0017] In one embodiment, the particle size distribution of the donor particles in the donor reagent exhibits polydispersity.

[0018] In one embodiment, the donor reagent comprises at least two kinds of donor particles with different average particle size distribution.

[0019] In one embodiment, the specific binding pair members are avidin-biotin system, and the avidin is selected from egg white avidin, egg yolk avidin, streptavidin, neutral avidin and avidin-like molecules, preferably streptavidin.

[0020] In one embodiment, the avidin is selected from streptavidin.

[0021] The second aspect of the present application provides a chemiluminescent detection kit comprising the donor reagent as described in any one of the first aspect of the present application.

[0022] In one embodiment, the kit comprises a plurality of reagent strips, each of which is provided with a plurality of reagent wells for containing reagents, one of which is for containing the donor reagent.

[0023] The fourth aspect of the present application provides the use of the donor reagent of any one of the first aspect of the present application or the kit of any one of the second aspect of the present application in a POCT instrument.

[0024] The fourth aspect of the present application provides the use of the donor reagent of any one of the first aspect of the present application or the kit of any one of the second aspect of the present application in a POCT instrument.

[0025] The donor reagent of the present application functions in 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 distance of 200 nm from the acceptor particles, and finally a chemiluminescence signal is generated to realize the detection of unknown substances.

[0026] The term "active oxygen" in the present application refers to a general term for substances composed of oxygen, containing oxygen and having active properties in the body or natural environment, mainly an excited state oxygen molecule, including one-electron reduction product of oxygen, superoxide anion (O2·-), two-electron reduction product of oxygen, hydrogen peroxide (H2O2), three-electron reduction product of oxygen, hydroxyl radical (·OH), and nitric oxide and singlet oxygen (1O2) and the like.

[0027] In the present application, the term "directly or indirectly connected" refers to that a specified substance can be chemically or physically bonded to another substance (directly connected); or the specified substance is chemically or physically bonded to another substance through an intermediate substance (compound, polymer, polysaccharide) (indirectly connected).

[0028] The term "acceptor particle" in the present application refers to a compound particle containing a compound capable of reacting with active oxygen to generate a detectable signal. The donor particle is induced and activated by energy or active compound and releases high-energy state active oxygen, and the high-energy state active oxygen is captured by the nearby acceptor particle to transfer energy to activate the acceptor particle.

[0029] 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" of the present application is selected from the group consisting of strips, sheets, rods, tubes, wells, microtiter plates, beads, particles and microspheres, which can be microspheres or microparticles known to those skilled in the art, which can be of any size, which can be organic or inorganic, which can be swellable or non-swellable, which can be porous or non-porous, which can have any density, but preferably have a density close to that of water, preferably capable of floating in water, and which are composed of transparent, partially transparent or opaque materials. The carrier can be charged or uncharged, and when charged, is preferably negatively charged. The carrier can be a latex particle or other particle containing an organic or inorganic polymer, a lipid bilayer such as a liposome, a phospholipid vesicle, a small oil droplet, a silica particle, a metal sol, a cell and a microcrystalline dye.

[0030] In the present application, the "luminescent agent" is a compound known as a label, which undergoes a chemical reaction to cause luminescence, such as by being converted to another compound formed in an electronically excited state. The excited state can be a singlet or a triplet excited state. The excited state can relax to the ground state directly with luminescence, or by transferring the excitation energy to an energy acceptor, thereby recovering to the ground state itself. In this process, the energy acceptor particle will be transitioned to an excited state and luminesce.

[0031] The term "donor particle" of the present application refers to a particle containing a sensitizer which, upon activation by energy or an active compound, is capable of generating an active intermediate such as a reactive oxygen species which reacts with the acceptor particle. The donor particle can be photoactivated (such as dyes and aromatic compounds) or chemically activated (such as enzymes, metal salts, etc.). In one embodiment, the donor particle is a polymeric microsphere filled with a photosensitizer, which can be a photosensitizer known in the art, preferably a compound which is relatively photostable and does not react efficiently with singlet oxygen, non-limiting examples of which include compounds such as methylene blue, rose Bengal, porphyrins, phthalocyanines and chlorophylls disclosed in U.S. Patent No. 5,709,994 (the entire contents of which are incorporated herein by reference), and derivatives of these compounds having 1-50 atom substituents which serve to make the compounds more lipophilic or more hydrophilic, and / or as linking groups to specific binding partners. Examples of other photosensitizers known to those skilled in the art can also be used in the present application, such as those described in U.S. Patent No. 6,406,913, which is incorporated herein by reference.

[0032] In one embodiment, the surface of the carrier is coated with at least two successive layers of polysaccharides, wherein the polysaccharide layer is spontaneously associated with the second polysaccharide layer.

[0033] In one embodiment, each layer of the successive layers of polysaccharides is spontaneously associated with each layer of the preceding layer of polysaccharides.

[0034] In one embodiment, the polysaccharides have pendant functional groups, the pendant functional groups of the successive layers of polysaccharides bear opposite charges to the pendant functional groups of the preceding layer of polysaccharides.

[0035] In one embodiment, the polysaccharides have pendant functional groups, and the successive layers of polysaccharides are covalently linked to the preceding layer of polysaccharides through reactions between the pendant functional groups of the successive layers of polysaccharides and the pendant functional groups of the preceding layer of polysaccharides.

[0036] In one embodiment, in the successive layers of polysaccharides, the pendant functional groups of two adjacent layers of polysaccharides alternate between amine functional groups and amine-reactive functional groups. That is, where the pendant functional groups of one layer are amine functional groups, the pendant functional groups of the other layer are amine-reactive functional groups.

[0037] In one embodiment, the amine-reactive functional groups are aldehyde groups or carboxyl groups.

[0038] In one embodiment, the first layer of polysaccharides (i.e. the innermost layer) is spontaneously associated with the carrier.

[0039] In one embodiment, the polysaccharides in the outermost layer of polysaccharides of the coating have at least one pendant functional group.

[0040] In one embodiment, the pendant functional groups of the polysaccharides in the outermost layer of polysaccharides of the coating are selected from at least one of aldehyde groups, carboxyl groups, thiol groups, amino groups, hydroxyl groups and maleimide groups; preferably from aldehyde groups and / or carboxyl groups.

[0041] In one embodiment, the pendant functional groups of the polysaccharides in the outermost layer of polysaccharides of the coating bind directly or indirectly, via chemical bonds, to one of the specific binding partners.

[0042] In one embodiment, the polysaccharides are selected from carbohydrates comprising three or more unmodified or modified monosaccharide units; preferably from dextran, starch, glycogen, inulin, levan, mannan, agarose, galactan, carboxydextran and aminodextran; more preferably from dextran, starch, glycogen and polyribose.

[0043] In one embodiment, the particle size of the carrier is selected from 100 to 400 nm, preferably 150 to 350 nm, more preferably 180 to 220 nm.

[0044] In one embodiment, 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, more preferably from 50 μg / ml to 200 μg / ml.

[0045] In one embodiment, the donor reagent further comprises a buffer solution having a pH value of 7.0 to 9.0, and the donor particles are suspended in the buffer solution.

[0046] In one 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.

[0047] In one embodiment, the polysaccharide (e.g. dextran) has a molecular weight distribution Mw selected from the group consisting of 10000 to 1000000 Da, preferably from 100000 to 800000 Da, more preferably from 300000 to 700000 Da.

[0048] In one embodiment, the sugar content in the buffer solution per liter of volume is not less than 0.2 g and not more than 2 g.

[0049] In one embodiment, the sugar content in the buffer solution per liter of volume is not less than 0.5 g and not more than 1.5 g.

[0050] In the present application, the "coefficient of variation C.V value of particle size distribution" refers to the coefficient of variation in the Gaussian distribution of particle size in the detection results of the nanosizer. The calculation formula of the coefficient of variation is: coefficient of variation C.V value = (standard deviation SD / average value Mean) x 100%. The standard deviation (Standard Deviation, SD) is also called standard deviation, which describes the average of the distance (deviation from the mean) of each data from the mean. It is the square root of the average of the sum of deviations, represented by σ. The standard deviation is the arithmetic square root of the variance. The standard deviation can reflect the dispersion degree of a data set. The smaller the standard deviation, the less the values deviate from the mean, and vice versa. The standard deviation σ is the distance between the inflection point (0.607 times the peak height) on the normal distribution curve and the vertical line of the peak height and time axis, that is, 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 = 2.355σ. The intercept on the baseline by the tangent line drawn through the inflection points on both sides of the normal distribution curve is called the peak width or baseline width, W = 4σ or W = 1.699 Wh / 2.

[0051] The inventors have found through extensive research that the coefficient of variation (C.V) of the particle size distribution of the particles in the liquid phase affects the detection signal of the photochemical chemiluminescence. If the photochemical chemiluminescence system is to be applied in the commercialization of clinical immunodiagnosis, a large number of donor particles with qualified production quality and stable performance are needed, and therefore the C.V of the particle size distribution of the donor particles in the donor reagent must be strictly controlled within a suitable range. It is worth noting that, unlike the particle size distribution of the traditional blank polystyrene microspheres (i.e. the carrier described in the present application, which has no filling functional substance inside and no modified avidin molecules or polysaccharides on the surface), the C.V described in the present application refers to the coefficient of variation (C.V) of the particle size distribution of the whole donor particles in the donor reagent. Since the C.V of the particle size distribution of the donor particles is continuously changing from the carrier to the preparation process of the donor particles, especially after being coated with polysaccharides or avidin, the C.V of the particle size distribution of the nanoscale microspheres changes more significantly and is more unstable, which is difficult to meet the requirements of medical device product registration and clinical application of reagents. Therefore, the applicant has gradually explored and found the range and method of strictly controlling the C.V of the particle size distribution of the donor particles in the liquid phase through extensive experimental research, and finally realized the commercial application of photochemical chemiluminescence technology in clinical testing.

[0052] The term "sample to be tested" as used herein refers to a mixture containing or suspected of containing a target molecule to be tested. The sample to be tested that can be used in the present application includes body fluids such as blood (which can be anticoagulated blood commonly seen in collected blood samples), plasma, serum, urine, semen, saliva, cell culture, tissue extract, etc. Other types of samples to be tested include solvents, seawater, industrial water samples, food samples, environmental samples such as soil or water, plant materials, eukaryotic cells, bacteria, plasmids, viruses, fungi, and cells from prokaryotes. The sample to be tested can be diluted with a diluent as needed before use. For example, in order to avoid the HOOK effect, the sample to be tested can be diluted with a diluent before detection on the detection instrument.

[0053] The term "target molecule" as used herein refers to a substance in a sample to be detected. One or more substances having specific binding affinity for the target molecule are used to detect the target molecule. The target molecule can be a protein, a peptide, an antibody, or a hapten that can bind to an antibody. The target molecule can be a nucleic acid or an oligonucleotide that binds to a complementary nucleic acid or oligonucleotide. The target molecule can be any other substance that can form a specific binding pair member. Other examples of typical target molecules include: drugs, such as steroids, hormones, proteins, glycoproteins, mucoproteins, nucleoproteins, phosphoproteins, drugs of abuse, vitamins, antibacterial agents, antifungal agents, antiviral agents, purines, antineoplastic agents, amphetamines, heterocyclic compounds, nucleic acids, and prostaglandins, and metabolites of any of these drugs; pesticides and metabolites thereof; and receptors. Analytes also include cells, viruses, bacteria, and fungi.

[0054] The term "antibody" as used herein is used in the broadest sense, and includes antibodies of any isotype, antibody fragments that retain specific binding to an antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, bispecific antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. Where desired, the antibody can be further conjugated to other moieties, such as one member of a specific binding pair, e.g., biotin or avidin (one member of a biotin-avidin specific binding pair), and the like.

[0055] The term "antigen" as used herein refers to a substance that is capable of stimulating the body to make an immune response, and to which an immune response product, such as an antibody or a sensitized lymphocyte, can bind in vivo or in vitro to produce an immunological effect.

[0056] 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 bridges and water bridges.

[0057] The term "specific binding" as used herein refers to the mutual recognition and selective binding reaction between two substances, and from the perspective of steric structure, it refers to the conformational correspondence between the corresponding reactants. Under the technical concept disclosed in the present application, the detection methods of specific binding reaction include, but are not limited to, double antibody sandwich method, competition method, neutralization competition method, indirect method, or capture method.

[0058] The term "specific binding pair members" as used herein refers to a pair of molecules that specifically bind to each other, such as enzyme-substrate, antigen-antibody, ligand-receptor. An example of a specific binding pair member is the biotin-streptavidin system, wherein "biotin" is widely present in animal and plant tissues, and has two ring structures on the molecule, namely imidazolone ring and thiophene ring, wherein the imidazolone ring is the main part of the binding with streptavidin. Activated biotin can be coupled with almost all known biological macromolecules under the mediation of protein cross-linking agents, including proteins, nucleic acids, polysaccharides and lipids, etc.; and the said avidin is selected from egg white avidin, streptavidin, egg yolk avidin, neutral avidin and similar avidin, preferably neutral avidin and / or streptavidin. Avidin is a glycoprotein that can be extracted from egg white, with a molecular weight of 60 kD, and each molecule is composed of 4 subunits, so it can bind 4 biotin molecules closely, and plays an important role in the immune mechanism. Avidin mainly includes egg white avidin, streptavidin, egg yolk avidin and neutral avidin. Streptavidin is a protein secreted by streptomyces, and the "streptavidin" molecule is composed of 4 identical peptide chains, each of which can bind one biotin. Therefore, each antigen or antibody can be coupled with multiple biotin molecules at the same time, thereby generating a "tentacle effect" to improve the analysis sensitivity. In any case, any reagent used in the present application, including antigens, antibodies, receptor particles or donor particles, can be conjugated with any one of the biotin-streptavidin specific binding pair members as needed.

[0059] In the photochemical chemiluminescence system, in addition to the donor reagent, other reagents are also included according to the needs of the detection object or detection method, such as: acceptor reagent, biotin-coated secondary antibody, diluent, etc. In the field of in vitro diagnosis, especially in the field of immunoassay, in order to simplify the naming of different components in commercial reagent kits, manufacturers usually mark or simply refer to the components in different bottles of the kit as reagent 1 or R1, reagent 2 or R2, reagent 3 or R3, and so on, which is convenient for customers to identify, assemble and use, and also for the purpose of technical secrecy. Therefore, the reagent kit products of different in vitro diagnostic manufacturers may all contain reagent 1, reagent 2, reagent 3, …, but the corresponding reagent components of different manufacturers are different.

[0060] The beneficial effects of the present application are:

[0061] The liquid homogeneous phase mode makes the consistency between tests better, and the product has better repeatability and consistency. It is free of washing, avoids the introduction of unnecessary interference substances and other uncertain factors in the reaction, and makes the detection result more stable. It can introduce unique chemiluminescence technology in the field of rapid diagnosis, and make the overall detection performance better. BRIEF DESCRIPTION OF DRAWINGS

[0062] Figure 1 Gaussian distribution of donor particles a prepared in Example 1 is shown.

[0063] Figure 2 Gaussian distribution of donor particles b prepared in Example 2 is shown.

[0064] Figure 3 The relationship between glucose concentration and corresponding absorbance is shown. DETAILED DESCRIPTION

[0065] The application will be further described in conjunction with the following examples, which are merely exemplary and in no way limit the application.

[0066] The reagents used in the following examples, unless otherwise specified, are commercially available.

[0067] Preparation of donor particles a and donor reagent A containing the donor particles a of Example 1

[0068] 1.1 Preparation of carrier

[0069] a) Prepare a 100ml three-necked flask, add 40mmol of styrene, 5mmol of acrolein, 10ml of water, stir for 10min, and pass N2 for 30min.

[0070] b) Weigh 0.11g of ammonium persulfate and 0.2g of sodium chloride, dissolve in 40ml of water to prepare an aqueous solution. Add the aqueous solution to the reaction system of step a), and continue to pass N2 for 30min.

[0071] c) Raise the reaction system to 70°C, and react for 15 hours to obtain an emulsion.

[0072] d) Cool the emulsion after the reaction to room temperature, filter with a suitable filter cloth, and wash the obtained filtered emulsion with deionized water by repeated centrifugal sedimentation until the conductivity of the supernatant after centrifugation approaches that of deionized water, then dilute with water to obtain the final emulsion of aldehyde group-containing polystyrene microspheres.

[0073] e) The size of the carrier is measured by a nanoparticle size analyzer to be in Gaussian distribution, with an average particle size of 201.3nm and a coefficient of variation (C.V.) of 8.0%.

[0074] 1.2 Sensitizer-filled carrier

[0075] a) Prepare a 25ml round-bottom flask, add 0.11g of copper phthalocyanine, 10ml of N,N-dimethylformamide, and magnetically stir, then raise the temperature to 75°C in a water bath to obtain a photosensitizer solution.

[0076] b) Prepare a 100ml three-necked flask, add 10ml 95% ethanol, 10ml water, and 10ml of the carrier prepared in step 1.1 with a concentration of 10%, magnetically stir, and heat to 70°C in a water bath.

[0077] c) Slowly add the photosensitizer solution in step a) to the three-necked flask in step b), and stir at 70°C for 2 hours to fill the aldehyde polystyrene microspheres with the sensitizer, then stop stirring and cool naturally to obtain a filled emulsion.

[0078] d) Centrifuge the filled emulsion at 30000g for 1 hour, discard the supernatant after centrifugation, and resuspend with 50% ethanol. After repeating the centrifugation and washing three times, resuspend with 50mM CB buffer with pH = 10 to obtain a microsphere suspension with a final concentration of 20mg / ml of the aldehyde polystyrene microspheres filled with the sensitizer.

[0079] 1.3 Preparation of donor reagent A

[0080] a) Microsphere suspension treatment: centrifuge the microsphere suspension prepared in step (ii) in a high-speed refrigerated centrifuge, discard the supernatant, add MES buffer, and sonicate on an ultrasonic cell disruptor until the particles are resuspended, add MES buffer to adjust the mass concentration of the microspheres to 100mg / ml, and obtain a treated microsphere suspension.

[0081] b) Affinity solution preparation: weigh a certain amount of streptavidin, dissolve in MES buffer to 8mg / ml.

[0082] c) Mixing: mix the treated microsphere suspension, 8mg / ml of streptavidin, and MES buffer in a volume ratio of 2:5:1, mix quickly, and obtain a reaction solution.

[0083] d) Reaction: prepare a 25mg / ml NaBH3CN solution in MES buffer, add according to a volume ratio of 1:25 of the NaBH3CN solution to the reaction solution, mix quickly, and rotate at 37°C for 48 hours.

[0084] e) Blocking: prepare 75mg / ml glycine (Gly) solution and 25mg / ml NaBH3CN solution in MES buffer respectively, add to the solution in step d) according to a volume ratio of 2:1:10 of the glycine solution, NaBH3CN solution, and reaction solution, mix, rotate at 37°C for 2 hours, and then add 200mg / ml BSA solution (prepared in MES buffer), with a volume ratio of 5:8 of the BSA solution to the reaction solution, mix quickly, and rotate at 37°C for 16 hours.

[0085] f) Washing: MES buffer was added to the solution after step e) reaction, centrifuged by high speed refrigerated centrifuge, the supernatant was discarded, then fresh MES buffer was added again, after ultrasonic suspension, centrifuged again, washed for 3 times, finally suspended with a small amount of buffer, measured the solid content, and adjusted the solid content to 150 μg / ml concentration with the buffer, obtained the donor reagent A containing donor particles a, the composition of the buffer is as follows: 0.1 mol Tris-HCl, 0.3 mol NaCl, 25 mmol EDTA, 0.1% dextran, 0.01% gentamicin and 15 ppm of ProClin-300, pH 8.00.

[0086] g) The size of donor particles a in the donor reagent A was measured by a nanoparticle size analyzer, which showed a Gaussian distribution, the average particle size was 227.7 nm, and the coefficient of variation C.V. value was 6.5%, as shown in Figure 1

[0087] Example 2

[0088] Preparation of donor particles b and donor reagent B containing the same

[0089] The preparation of the carrier used in this example and the filling process of the sensitizing agent are the same as steps 1.1 and 1.2 in Example 1.

[0090] 2.1 Coating dextran on the surface of the carrier

[0091] a) 50 mg of aminodextran solid was taken in a 20 mL round-bottom flask, 5 mL of 50 mM / pH=10 carbonate buffer was added, and it was dissolved by stirring at 30°C in the dark.

[0092] b) 100 mg of donor particles a was taken and added to the aminodextran solution and stirred for 2 hours to obtain a reaction solution.

[0093] c) 10 mg of sodium borohydride was dissolved in 0.5 mL of 50 mM / pH=10 carbonate buffer and added dropwise to the above reaction solution, which was reacted overnight at 30°C in the dark to obtain a mixture of aminodextran-coated carriers.

[0094] d) The mixture of step c) was centrifuged at 30000 g, and the supernatant was discarded, and 50 mM / pH=10 carbonate buffer was added and ultrasonically dispersed. After repeating the centrifugal washing three times, the aminodextran-coated carrier was made to a final concentration of 20 mg / ml with 50 mM / pH=10 carbonate buffer, and an aminodextran carrier solution was obtained.

[0095] e) 100 mg of aldehyde dextran solid was taken in a 20 mL round-bottom flask, 5 mL of 50 mM / pH=10 carbonate buffer was added, and it was dissolved by stirring at 30°C in the dark. ​

[0096] f) Add the amino-dextran carrier solution to the aldehyde-dextran solution and stir for 2 hours.

[0097] g) Dissolve 15 mg of sodium borohydride in 0.5 mL of 50 mM / pH=10 carbonate buffer and add it dropwise to the reaction solution. React overnight at 30°C in the dark to obtain a mixture of the carrier coated with aldehyde-dextran.

[0098] h) Centrifuge the mixture of step g) at 30000 g and discard the supernatant. Ultrasonically disperse the carrier coated with aldehyde-dextran in 50 mM / pH=10 carbonate buffer. Repeat the centrifugation and washing three times and then make up to volume with 50 mM / pH=10 carbonate buffer to obtain a carrier coated with aldehyde-dextran at a final concentration of 20 mg / mL.

[0099] i) The size of the carrier coated with dextran is Gaussian distributed with an average particle size of 235.6 nm and a coefficient of variation (C.V.) of 8.1% as measured by a nanoparticle size analyzer.

[0100] 2.2 Preparation of donor reagent B

[0101] a) Treatment of the carrier coated with dextran: centrifuge the carrier coated with dextran obtained in step h) of Example 1.1 in a high-speed refrigerated centrifuge, discard the supernatant, add MES buffer and ultrasonically disperse the carrier coated with dextran in an ultrasonic cell disruptor until the microspheres are resuspended. Add MES buffer to adjust the mass concentration of the carrier to 100 mg / mL.

[0102] b) Preparation of the avidin solution: weigh a certain amount of neutral avidin and dissolve it in MES buffer to obtain a solution of 8 mg / mL.

[0103] c) Mixing: mix the treated microsphere suspension, the 8 mg / mL avidin solution and the MES buffer in a volume ratio of 2:5:1, mix rapidly and obtain a reaction solution.

[0104] d) Reaction: prepare a 25 mg / mL NaBH3CN solution in MES buffer and add it to the reaction solution in a volume ratio of 1:25, mix rapidly and react at 37°C for 48 hours.

[0105] e) Blocking: prepare a 75 mg / mL Gly solution and a 25 mg / mL NaBH3CN solution in MES buffer and add them to the solution of step d) in a volume ratio of 2:1:10, mix and react at 37°C for 2 hours. Then add a 200 mg / mL BSA solution (prepared in MES buffer) in a volume ratio of 5:8, mix rapidly and react at 37°C for 16 hours.

[0106] f) Washing: To the solution after step e) reaction, MES buffer was added, centrifuged by high speed refrigerated centrifuge, the supernatant was discarded, then fresh MES buffer was added again, after ultrasonic suspension, centrifuged again, so washing 3 times, finally suspended with a small amount of buffer, measured the solid content, and adjusted the solid content to 150 μg / ml with the buffer, obtained the donor reagent B containing donor particles b, the composition of the buffer is as follows: 0.1 mol Tris-HCl, 0.3 mol NaCl, 25 mmol EDTA, 0.1% dextran, 0.01% gentamicin and 15 ppm of ProClin-300, pH 8.00.

[0107] g) The size of donor particles b in the donor reagent B was measured by a nanoparticle size analyzer, which showed a Gaussian distribution, with an average particle size of 249.9 nm and a coefficient of variation C.V value of 11.6%, as shown in Figure 2 .

[0108] Example 3 Determination of sugar content in donor particles

[0109] 3.1 Determining the relationship between glucose concentration and corresponding absorbance value by anthrone method

[0110] a) Preparation of glucose standard solution: 1 mg / mL glucose stock solution was prepared into 0 mg / mL, 0.025 mg / mL, 0.05 mg / mL, 0.075 mg / mL, 0.10 mg / mL, 0.15 mg / mL standard solution curve with purified water.

[0111] b) Preparation of anthrone solution: 2 mg / mL (this solution is stable at room temperature within 24 h, prepare it fresh) with 80% sulfuric acid solution.

[0112] c) Add 0.1 mL of each concentration of glucose standard solution to each centrifuge tube, and add 1 mL of anthrone test solution to each tube.

[0113] d) Incubate at 85 degrees for 30 min, then centrifuge at 15000 g for 40 min, use a pipette gun to suck the clear liquid from the bottom of the tube, avoiding sucking the upper suspension.

[0114] e) After the clear liquid is sucked out and returned to room temperature, measure its absorbance at 620 nm (the measurement is preferably performed within 2 h), repeat twice, and take the average of the two repeats as the final absorbance value, the results are shown in Table 1.

[0115] f) Linear regression with standard concentration as X value and average absorbance as Y value, the results are shown in Figure 3 , to determine the sugar concentration of the sample to be tested.

[0116] Table 1

[0117] Serial 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

[0118] 3.2 Determination of sugar content in donor particles

[0119] a) Take 1 mg of donor particles in a donor reagent, centrifuge at 20000 g for 40 min, after discarding the supernatant, ultrasonically disperse with purified water, repeat the centrifugation and dispersion three times, then dilute with purified water to 1 mg / mL to prepare the sample to be tested.

[0120] b) Preparation of anthrone solution: prepare 2 mg / mL with 80% sulfuric acid solution (this solution is stable at room temperature within 24 h, prepare it fresh).

[0121] c) Add 0.1 mL of the sample to be tested and 1 mL of the anthrone reagent solution to the centrifuge tube in turn.

[0122] d) Incubate at 85 degrees for 30 min; centrifuge at 15000 g for 40 min, use a pipette gun to suck the clear liquid from the bottom of the tube, avoiding sucking the upper suspension.

[0123] e) After the clear liquid sucked out is restored to room temperature, measure its absorbance at 620 nm (the measurement is preferably performed within 2 h), repeat twice, take the average of the two repeats as the final absorbance value, then calculate the sugar content in the donor particles according to the Figure 3 obtained curve equation, the results are shown in Table 2.

[0124] Table 2

[0125] Serial number Absorbance A Absorbance B Absorbance mean Sugar concentration mg / g Donor particle a 0.0572 0.0564 0.0568 11.5 Donor particle b 0.172 0.165 0.1685 53.6

[0126] Example 5

[0127] Use the preparation method of the donor particles and donor reagent in Example 1 above to prepare donor reagents containing a series of donor particles as follows:

[0128] Donor reagent 1: the average particle size of the donor particles in the Gaussian distribution curve is 226.5 nm, the particle size distribution coefficient of variation C.V value = 3.8; the Nicomp distribution is unimodal.

[0129] Donor reagent 2: the average particle size of the donor particles in the Gaussian distribution curve is 225.3 nm, the particle size distribution coefficient of variation C.V value = 4.6; the Nicomp distribution is unimodal.

[0130] Donor reagent 3: the average particle size of the donor particles in the Gaussian distribution curve is 225.2 nm, the particle size distribution coefficient of variation C.V value = 5.0; the Nicomp distribution is unimodal.

[0131] Donor Reagent 4: The average particle size of the donor particles in the Gaussian distribution curve was 226.7 nm, and the particle size distribution coefficient of variation C.V value = 8.1; the Nicomp distribution was unimodal.

[0132] Donor Reagent 5: The average particle size of the donor particles in the Gaussian distribution curve was 227.8 nm, and the particle size distribution coefficient of variation C.V value = 15.6; the Nicomp distribution was unimodal.

[0133] Donor Reagent 6: The average particle size of the donor particles in the Gaussian distribution curve was 225.9 nm, and the particle size distribution coefficient of variation C.V value = 26.1; the Nicomp distribution was unimodal.

[0134] Troponin I quantitative detection reagent kits 1 to 6 were prepared using the above donor reagents 1 to 6.

[0135] The kits of this embodiment each consist of reagent 1 (R1') containing first anti-cTnI antibody-coated receptor particles, reagent 2 (R2') containing biotin-labeled second anti-cTnI antibody, and additionally including any one of donor reagents 1 to 6 (R3'). Obviously, R1' is a receptor reagent; R3' is a donor reagent.

[0136] The chemiluminescence detection process was completed on the LiCA HT automatic light-activated chemiluminescence analysis system developed by Boyang Biotech (Shanghai) Co., Ltd. and the detection results were output, and the specific experimental steps were as follows:

[0137] (1) 40 μl of the sample to be tested containing a known concentration of cTnI marker, 15 μl of R1' and 15 μl of R2' were added to an 8x12 96-well plate, and mixed well;

[0138] (2) Incubate at 37°C for 8 min;

[0139] (3) Add 160 μl of universal liquid (R3');

[0140] (4) Incubate at 37°C for 2 min;

[0141] (5) Put into LiCA HT instrument to excite reading, and the specific detection results are shown in Table 3. Then analyze the sensitivity and detection upper limit of kits 1 to 6 according to the detection results.

[0142] Table 3

[0143]

[0144] From Table 3, when the coefficient of variation of the particle size distribution of the donor particles is greater than or equal to 5%, the method has appropriate sensitivity and a relatively appropriate detection range, and the production cost of the reagent raw materials and the reagent kit is relatively low, which can meet the needs of clinical in vitro diagnosis applications.

[0145] Example 6

[0146] The donor reagents containing a series of donor particles were prepared by using the preparation methods of the donor particles and the donor reagents in Examples 1 and 2 above, and the sugar content in the donor particles was detected by using the anthrone method in Examples 4 and 5.

[0147] According to the method given in Example 5 above, Troponin I quantitative detection test kits A to E containing donor reagents A to E were prepared, and the chemiluminescence detection process was completed on the LiCA HT automatic light-activated chemiluminescence analysis system developed by Boyang Biotech (Shanghai) Co., Ltd. and the detection results were output. The specific experimental steps can be referred to the steps given in Example 5, and the experimental results are shown in Table 4.

[0148] Table 4

[0149] Donor reagent name Sugar content of donor particle mg / g Photoluminescence chemiluminescence 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

[0150] As can be seen from Table 4, when the sugar content of the donor particles in the donor reagent is less than 25 mg / g, the detection effect of light-activated chemiluminescence is better.

[0151] Example 7

[0152] In this example, the CRP was detected by using the homogeneous chemiluminescence POCT detection device disclosed in CN208568604U.

[0153] The kit prepared in this example includes a plurality of reagent cup strips, and the reagent cup strip is provided with a plurality of hole positions for containing reagents, including but not limited to a sample hole position, a first reagent hole position and a second reagent hole position. The sample hole position is used to contain a sample containing a target molecule to be detected. The first reagent hole position is used to contain a donor reagent containing donor particles, and the donor particles can generate singlet oxygen in an excited state. The second reagent hole position is used to contain a receptor reagent containing receptor particles, and the receptor particles can react with singlet oxygen to generate a chemiluminescence signal, and the particle size of the donor particles is larger than the particle size of the receptor particles. The hole positions can be selected and functionally expanded according to actual needs.

[0154] The donor reagent in the first reagent well of the embodiment is the donor reagent A prepared in Example 1, wherein the coefficient of variation C.V of the particle size distribution of the donor particles is 6.5%, and the sugar content is 11.5 mg / g. In one well of the reagent cup strip, 50 μL of the CRP clinical sample (containing serum and whole blood) of different concentrations, 50 μL of the biotinylated anti-CRP antibody, 50 μL of the receptor reagent containing the CRP receptor particles, and 50 μL of the donor reagent A prepared in Example 1 were added, and the parallel tubes in each well were averaged. The reaction was carried out at 37°C for 7.5 min, and then 50 μL of the donor reagent A prepared in Example 1 was added, and the reaction was carried out at 37°C for 5 min. The light excitation detection was carried out. The experimental results are shown in Table 5. As shown in Table 5, the correlation coefficient between the serum and the whole blood reaches 0.9988. The experimental results show that the donor particles of the application greatly reduce the non-specific adsorption in the sample, and the measurement results for the serum and the whole blood have very good correlation. The adaptability of the donor reagent to the clinical sample is greatly enhanced, and the donor reagent can be directly used for the detection of the clinical whole blood sample.

[0155] Table 5

[0156] Serum test results (unit: mg / L) Whole blood test results (unit: mg / L) 4.037 4.035 4.785 4.559 5.499 5.233 5.513 5.160 6.449 6.035 7.111 7.008 7.179 7.062 8.435 8.215 10.290 10.443 12.899 12.465 12.929 12.336 17.455 16.980 16.900 17.378 17.500 18.645 18.989 20.918 20.651 20.555 21.677 21.440 23.401 24.450 27.998 27.334 32.865 32.590 43.870 44.239 65.111 65.211 72.962 74.728 75.883 75.882 81.595 81.829 85.240 85.375 109.991 109.860 111.332 112.640 115.230 105.985 132.241 133.660 145.030 144.459 200.325 199.768

[0157] Example 8

[0158] In this embodiment, 40 clinical samples are detected, including 13 negative samples (Nos. 1 to 13) and 27 positive samples (Nos. 14 to 40). The cTnI quantitative detection reagent kit (light excitation chemiluminescence method) used includes reagent 1 (R1) containing first anti-cTnI monoclonal antibody coated receptor particles, reagent 2 (R2) containing biotin-labeled second anti-cTnI monoclonal antibody, and light excitation chemiluminescence analysis system universal liquid (R3) containing donor particles. The concentration of the donor particles in the reagent R3 is 100 μg / ml, the coefficient of variation C.V of the particle size distribution of the donor particles in the reagent R3 is 11%, and the sugar content of the donor particles is 14.5 mg / g.

[0159] The detection process is completed on the full-automatic light excitation chemiluminescence analysis system (LiCA HT) developed by Boyang Biotech (Shanghai) Co., Ltd., and the detection results are output. The specific detection steps are described in Example 5.

[0160] When the cTnI marker exists in the clinical sample, the cTnI marker is specifically bound to both the receptor particles coated with the first anti-cTnI monoclonal antibody and the biotin-labeled second anti-cTnI monoclonal antibody, and a double antibody sandwich complex is formed on the surface of the receptor particles; at this time, if the streptavidin-modified donor particles are added, the biotin binds to the streptavidin to make the two particles close to each other, under the excitation of the excitation light source, the donor particles release singlet oxygen, and after colliding with the receptor particles in the solution, chemical luminescence is generated, thereby further exciting the fluorescent groups on the same particle to produce a cascade amplification reaction to generate fluorescence. At this time, the more the content of the existing cTnI marker is, the stronger the fluorescence intensity is, and the amount of the cTnI marker in the serum of the patient is quantitatively detected according to the strength of the luminescence, and the specific cTnI marker content detection result is shown in Table 6.

[0161] Table 6

[0162]

[0163]

[0164] According to the data comparison, the correlation between the Abbott value and the Boyang value is 0.9973, and the slope is 1.0495. Samples 1-13 are normal physical examination patients, with a distribution range of 1.77 pg / ml to 25.3 pg / ml, and a median of 6.77 pg / ml; samples 14-40 are patients identified as having myocardial injury, with a distribution range of 30.94 pg / ml to 29896.88 pg / ml, and a median of 450.54 pg / ml.

[0165] The concentration of cardiac troponin I (cTnI) in the serum or plasma of healthy people is relatively low, and after the onset of chest pain in patients, a large number of necrotic myocardial cells release cTNI into the blood circulation system, and the peak value is reached in 12-48 hours, and the cTnI of severe heart attack patients still maintains a high level for several days, which is the optimal marker for diagnosing myocardial injury and myocardial infarction. According to the data of the embodiment 7 of the present application, it can be shown that the use of the donor reagent described in the present application in the preparation of a kit used in a method for diagnosing whether a subject has myocardial injury in vitro is feasible, and the quantitative results of the cTnI marker in the body fluid of the subject measured by using the donor reagent and the corresponding method described in the present application can be used for diagnosing myocardial injury and myocardial infarction.

Claims

1. A cTnI detection kit, characterized in that, include: The reagent 1 contains receptor particles coated with a first anti-cTnI antibody, and a donor reagent contains donor particles, wherein the donor reagent comprises a buffer solution and donor particles suspended therein, the donor particles being able to generate reactive oxygen species upon excitation in the liquid phase; the coefficient of variation (C·V) of the particle size distribution of the donor particles in the donor reagent is not less than 5%, and each gram of the donor particles includes a carrier, the interior of which is filled with a sensitizer, and the content of polysaccharides coated or linked on the surface of the carrier is not higher than 25 mg.

2. The reagent kit according to claim 1, characterized in that, It also includes reagent 2, which contains a biotin-labeled second anti-cTnI antibody.

3. The reagent kit according to claim 1, characterized in that, The coefficient of variation (C·V) of the particle size distribution of the donor particles in the donor reagent is not less than 8% and not more than 20%.

4. The reagent kit according to claim 1, characterized in that, The concentration of the donor particles in the donor reagent is from 10 μg / ml to 1 mg / ml.

5. The reagent kit according to claim 1, characterized in that, The concentration of the donor particles in the donor reagent is from 20 μg / ml to 500 μg / ml.

6. The reagent kit according to claim 1, characterized in that, The concentration of the donor particles in the donor reagent is from 50 μg / ml to 200 μg / ml.

7. The kit according to claim 1, characterized in that, The particle size of the carrier is selected from 100 nm to 400 nm.

8. The reagent kit according to claim 1, characterized in that, The particle size of the carrier is 150 nm to 350 nm.

9. The reagent kit according to claim 1, characterized in that, The particle size of the carrier is 180 nm to 220 nm.

10. The reagent kit according to claim 1, 7, 8 or 9, characterized in that, The surface of the carrier is coated with a coating of at least two consecutive polysaccharide layers, wherein the polysaccharide layers are spontaneously associated with the second polysaccharide layer; the molecular weight distribution Mw of the polysaccharide is selected from 10,000 Da to 1,000,000 Da.

11. The reagent kit according to claim 10, characterized in that, The molecular weight distribution Mw of the polysaccharide is selected from 100,000 Da to 800,000 Da.

12. The reagent kit according to claim 10, characterized in that, The molecular weight distribution Mw of the polysaccharide is selected from 300,000 Da to 700,000 Da.

13. The reagent kit according to claim 10, characterized in that, The polysaccharide is selected from carbohydrates containing three or more unmodified or modified monosaccharide units.

14. The kit according to claim 13, characterized in that, The polysaccharide is selected from glucan, starch, glycogen, inulin, fructan, mannan, agarose, galactan, carboxyglucan, and aminoglucan.

15. The reagent kit according to claim 13, characterized in that, The polysaccharide is selected from dextran, starch, glycogen, and polyribose.

16. The reagent kit according to claim 1, characterized in that, The donor reagent also includes a buffer solution with a pH value of 7.0 to 9.0, in which the donor particles are suspended.

17. The reagent kit according to any one of claims 1 to 9, 11 to 16, characterized in that, The content of the polysaccharide was determined by the anthrone method.

18. The application of the cTnI detection kit according to any one of claims 1 to 17 in a POCT instrument.

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