CN113125700B - Homogeneous detection kit for creatine kinase isozymes and application thereof - Google Patents
Homogeneous detection kit for creatine kinase isozymes and application thereof Download PDFInfo
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- CN113125700B CN113125700B CN201911411233.XA CN201911411233A CN113125700B CN 113125700 B CN113125700 B CN 113125700B CN 201911411233 A CN201911411233 A CN 201911411233A CN 113125700 B CN113125700 B CN 113125700B
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- G—PHYSICS
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Abstract
The invention relates to a homogeneous detection kit of creatine kinase isozymes and application thereof, wherein the kit comprises a reagent 1 and a reagent 3, the reagent 1 comprises a first buffer solution and acceptor particles suspended therein, the reagent 3 comprises a second buffer solution and donor particles suspended therein, the acceptor particles comprise a first carrier, the interior of the first carrier is filled with a luminous composition, and the variation coefficient C.V value of the particle size distribution of the acceptor particles in the reagent 1 is not lower than 5% and not higher than 25%; the donor particle includes a second carrier, the interior of which is filled with a sensitizer, and the sugar content of the donor particle in the reagent 3 is not higher than 40mg per mg of the donor particle. The kit can be used for clinically assisting in diagnosis of myocardial infarction, and has proper sensitivity and wide detection range.
Description
Technical Field
The invention belongs to the technical field of homogeneous detection, and particularly relates to a homogeneous detection kit for creatine kinase isozymes, and a preparation method and application thereof.
Background
Creatine Kinase (CK) is a dimeric enzyme, which has four different forms: mitochondrial isozymes, cytoplasmic isozymes CK-MM (myotype), creatine kinase brain isozymes CK-BB (brain type), creatine kinase isozymes (CK-MB), wherein the creatine kinase isozymes are mainly located in the heart muscle.
Creatine kinase isozymes are released into peripheral blood when Acute Myocardial Infarction (AMI) and myocardial necrosis occur, can be detected after clinical symptoms appear for 3-8 hours, can be combined with myoglobin and troponin for detection, can judge whether the cardiac muscle is damaged, and is an index for researching and diagnosing the Acute Myocardial Infarction (AMI). The method is mainly used for clinically assisting diagnosis of myocardial infarction.
Common CK-MB detection methods include gold calibration assays, fluorescent immunoassays, combined immunoadsorption assays (ELISA), magnetic particle Chemiluminescence (CMIA), and biochemical instrumentation. The basic principle of photoexcitation is a homogeneous immune response. It is based on two particles surface coated antigens or antibodies, forming immune complexes in the liquid phase, drawing the two particles closer together. Under the excitation of laser, the transfer of singlet oxygen between particles occurs, and then high-level red light is generated, and the photon number is converted into the concentration of the analyte through a single photon counter and mathematical fitting. When the sample does not contain an analyte, immune complexes cannot be formed between the two particles, the distance between the two particles exceeds the propagation range of singlet oxygen, the singlet oxygen is rapidly quenched in a liquid phase, and no high-energy red light is generated during detection. Compared with the existing detection kit, the kit based on the light excitation chemiluminescence method has the advantages of convenience in operation, rapidness in detection, high sensitivity, good accuracy and the like; the method is applied to the monitoring of myocardial damage, and can improve the accuracy of diagnosis of acute myocardial infarction.
CN106093411a discloses a one-step homogeneous CK-MB detection kit for acute myocardial infarction diagnosis and application thereof in detecting CK-MB content, which adopts a photoexcitation luminescence method, and although the kit has the advantages of convenient operation, rapid detection, strong specificity and the like compared with the prior art, the sensitivity is still not high, and the kit cannot consider both high sensitivity and wide detection range.
Disclosure of Invention
The invention aims to solve the technical problem of providing a homogeneous detection kit for creatine kinase isozymes, which aims at overcoming the defects of the prior art, and the quantitative result of CK-MB markers in body fluid of a measured subject can be used for clinically assisting diagnosis of myocardial infarction, and has proper sensitivity and wide detection range.
To this end, a first aspect of the present invention provides a homogeneous assay kit for a creatine kinase isoenzyme comprising reagent 1 and reagent 3, the reagent 1 comprising a first buffer solution and, suspended therein, a receptor particle capable of producing chemiluminescence upon action of reactive oxygen species, which binds to a creatine kinase isoenzyme antibody capable of specifically binding to a creatine kinase isoenzyme; the reagent 3 comprises a second buffer solution and donor particles suspended therein, the donor particles being bound to one of the specific pairing members, characterized in that:
The receptor particles comprise a first carrier, the interior of the first carrier is filled with a luminous composition, the surface of the first carrier is bonded with creatine kinase isoenzyme antibodies, and the variation coefficient C.V value of the particle size distribution of the receptor particles in the reagent 1 is not lower than 5% and not higher than 25%;
the donor particle comprises a second carrier, the interior of the second carrier is filled with a sensitizer, and one member of a specific pairing member is bonded to the surface of the second carrier; the sugar content of the donor particle in reagent 3 per mg of the donor particle is not higher than 40mg.
In some embodiments of the invention, the acceptor particle has a particle size distribution coefficient of variation C.V value in reagent 1 of no more than 15%.
In some embodiments of the invention, the receptor particle may have a particle size distribution coefficient of variation C.V value in reagent 1 selected from 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, and 25%.
In some embodiments of the invention, the surface of the first carrier is coated with a polysaccharide, and the creatine kinase isoenzyme antibody is bound to the first carrier by a linkage to a polysaccharide molecule.
In some embodiments of the invention, the surface of the first carrier is coated with a coating of at least two successive polysaccharide layers.
In some embodiments of the invention, the first polysaccharide layer and the second polysaccharide layer of the coating spontaneously associate.
In some embodiments of the invention, each layer of polysaccharide in the coating has functional groups that are oppositely charged to the functional groups of the previous polysaccharide layer.
In other embodiments of the invention, each layer of polysaccharide in the coating has a functional group, and each polysaccharide layer is covalently linked to the previous polysaccharide layer by a reaction between its functional group and a functional group on the previous polysaccharide layer.
In some embodiments of the invention, the outermost polysaccharide layer of the coating has at least one pendant functional group that is bound to an creatine kinase isozyme antibody.
In some embodiments of the invention, the pendant functional groups of the outermost polysaccharide layer of the coating are selected from at least one of aldehyde groups, carboxyl groups, thiol groups, amino groups, hydroxyl groups, and maleamine groups.
In some preferred embodiments of the invention, the pendant functional groups of the outermost polysaccharide layer of the coating are selected from aldehyde groups and/or carboxyl groups.
In some embodiments of the invention, the sugar content per liter of the first buffer solution is 0.01-1g.
In some preferred embodiments of the invention, the sugar content per liter of the first buffer solution is between 0.02 and 0.2g.
In some embodiments of the invention, the sugar content per mg of the acceptor particle is not less than 20 micrograms. In some embodiments of the invention, the sugar content per mg of the acceptor particle may be selected from 20.2 mg, 40 mg, 45.1 mg, 55.8 mg, 60.3 mg.
In some preferred embodiments of the present invention, the sugar content in the first buffer solution is not less than 40 micrograms per liter.
In some embodiments of the invention, the polysaccharide is selected from carbohydrates containing three or more unmodified or modified monosaccharide units; preferably at least one selected from the group consisting of dextran, starch, glycogen, inulin, levan, mannan, agarose, galactan, carboxydextran and aminodextran; more preferably at least one selected from the group consisting of dextran, starch, glycogen and polyribose, most preferably dextran and/or dextran derivatives.
In some embodiments of the invention, the polysaccharide is dextran.
In some embodiments of the invention, the donor particles have a particle size distribution coefficient of variation C.V in reagent 3 of no less than 5% and no more than 25%. In some embodiments of the invention, the donor particle has a particle size distribution coefficient of variation C.V value in reagent 3 selected from 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, and 25%. In some embodiments of the invention, the kit further comprises a series of calibrator solutions having known concentrations of creatine kinase isozymes, the concentration of creatine kinase isozymes in the series of calibrator solutions being between 0 and 510ng/mL.
In some embodiments of the invention, the concentration of creatine kinase isozymes in the series of calibrator solutions comprises 0, 4, 10, 50, 200, and 510ng/mL.
In other embodiments of the invention, the kit further comprises reagent 2, the reagent 2 comprising an creatine kinase isoenzyme antibody linked to one of the specific binding pair members, the creatine kinase isoenzyme antibody being capable of specifically binding to the creatine kinase isoenzyme.
In some embodiments of the present invention, the kit contains at least 1 reagent strip, and the reagent strip is provided with a plurality of reagent holes for containing reagents, wherein at least 3 reagent holes are respectively used for containing the reagent 1, the reagent 2 and the reagent 3.
In a second aspect the invention provides the use of a kit according to the first aspect of the invention in a chemiluminescent analyzer.
In some embodiments of the invention, the application comprises the steps of:
step S1, respectively adding a sample to be detected, a calibrator and/or a quality control product into a liquid containing device;
step S2, adding a reagent 1, a reagent 2 and a reagent 3 into the liquid containing device;
and S3, placing the liquid containing device on a chemiluminescence analyzer for reaction and detection.
The inventors of the present patent studied and found that the volume of the acceptor particles and the donor particles is small, the concentration in the kit is low, the preparation process is very complicated, the quality is easily affected by various factors, and therefore, it is necessary to obtain the optimal control technical characteristics, such as the value of the variation coefficient C.V of the particle size distribution of the acceptor particles and the donor particles, according to multiple detection experiments, since the light-emitting composition or the sensitizer is to be incorporated into the volume of the acceptor particles and the donor particles, the antibody antigen or the streptavidin is to be coated on the outside, in order to ensure the uniformity of the particles, the error caused by the variation of the particle size is reduced, and the person skilled in the art will generally consider to control the value of the variation coefficient C.V of the particle size distribution to be in a smaller numerical range, even lower and better. However, the inventors of the present patent found that if the C.V value of the microparticles is too small, the requirement for the production process is too high, greatly increasing the production cost of the reagent, failing to form industrialization, and making the reagent unusable in clinical diagnosis in large amounts. Especially after the polysaccharide is coated, the change of the C.V value of the particle size distribution coefficient of variation of the nano-microsphere is more remarkable and unstable, and the reagent requirements of medical instrument product registration and clinical application are difficult to meet. However, if the C.V value is too large, the effect of the photo-excitation chemiluminescence detection is not good. Therefore, the inventors of the present patent have surprisingly found that if the values of the variation coefficients C.V of the particle size distribution of the acceptor particles and the donor particles are controlled within a suitable range, the optimal photoexcitation chemiluminescence detection result can be obtained, and thus, the detection device has a relatively suitable sensitivity and a very wide detection range.
The beneficial effects of the invention are as follows:
the kit provided by the invention has feasibility in-vitro diagnosis of whether a subject suffers from myocardial damage, and the quantitative result of the CK-MB marker in body fluid of the subject can be used for assisting in diagnosis of myocardial infarction by using the kit and a corresponding method. The kit provided by the invention has the advantages of proper sensitivity and wide detection range.
Detailed Description
In order that the invention may be readily understood, the invention will be described in detail. Before the present invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Unless defined otherwise, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.
I. Terminology
The term "homogeneous" as used herein is defined as "homogeneous" and refers to a method that allows detection without the need to separate the bound antigen-antibody complex from the remaining free antigen or antibody.
The term "sample to be tested" as used herein refers to a mixture that may contain an analyte. Typical samples to be tested that can be used in the methods disclosed herein include body fluids such as blood, plasma, serum, urine, semen, saliva, and the like.
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 specific binding pair members, e.g., biotin or avidin (a member of the biotin-avidin specific binding pair), 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 antigen may be a fusion antigen, and in any desired case, the antigen may be further conjugated to other moieties, such as a specific binding pair member, e.g., biotin or avidin (one of the biotin-avidin specific binding pair members), or the like.
The term "binding" or "bonding" as used herein refers to the association of two molecules due to interactions such as covalent, electrostatic, hydrophobic, ionic and/or hydrogen bonding, including but not limited to physical or chemical 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.
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 pair is the biotin-avidin system, in which "biotin" is widely present in animal and plant tissues and has two cyclic structures on its molecule, an imidazolone ring and a thiophene ring, respectively, in which the imidazolone ring is the primary site of binding to avidin. Activated biotin can be coupled to almost all known biomacromolecules, including proteins, nucleic acids, polysaccharides, lipids, etc., mediated by protein cross-linking agents; while "avidin" is a protein secreted by Streptomyces and has a molecular weight of 65kD. The "avidin" molecule consists of 4 identical peptide chains, each of which is capable of binding one biotin. Thus, each antigen or antibody can be conjugated to multiple biotin molecules simultaneously, thereby producing a "tentacle effect" that enhances assay sensitivity. The avidin in the present invention is selected from the group consisting of avidin, streptavidin, vitellin, neutravidin and avidin-like, preferably selected from the group consisting of neutravidin and/or streptavidin.
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 active oxygen 1 O 2 ) Etc.
The term "donor particle" as used herein refers to 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 some embodiments of the present invention, the donor particles are coated on a substrate by a functional group to form polymer microparticles filled with a photosensitive compound, which can generate active oxygen under light excitation, and the photosensitive microspheres may also be referred to as oxygen supplying microspheres or photosensitive microspheres. The surface of the donor particle can be provided with hydrophilic aldehyde dextran, and the inside of the donor particle is filled with a photosensitizer. The photosensitizers may be photosensitizers known in the art, preferably compounds that are relatively light stable and do not react effectively with active oxygen, non-limiting examples of which include methylene blue, rose bengal, porphyrin, and phthalocyanine compounds, and derivatives of these compounds having 1-50 atom substituents that are used to render these compounds more lipophilic or hydrophilic, and/or as linking groups to specific binding pair members. The donor particle surface may also be filled with other sensitizers, non-limiting examples of which are certain compounds that catalyze the conversion of hydrogen peroxide to active oxygen and water. Examples of other donors include: 1, 4-dicarboxyethyl-1, 4-naphthalene endoperoxide, 9, 10-diphenylanthracene-9, 10-endoperoxide, and the like, and active oxygen, such as active oxygen, may be released by heating these compounds or by direct absorption of light by these compounds.
The term "acceptor particle" as used herein refers to a compound that is capable of reacting with reactive oxygen species to produce a detectable signal. The donor particles are induced to activate by energy or an active compound and release active oxygen in a high energy state which is captured by the acceptor particles in close proximity, thereby transferring energy to activate the acceptor particles. In some embodiments of the invention, the acceptor particles are filled with functional groups in a carrier to form polymeric microparticles filled with a luminescent composition comprising a chemiluminescent compound capable of reacting with reactive oxygen species. In some embodiments of the invention, the chemiluminescent compound undergoes a chemical reaction with reactive oxygen species to form an unstable metastable intermediate that may decompose with or subsequent to luminescence. Typical examples of such substances include, but are not limited to: enol ethers, enamines, 9-alkylidene xanthan gums, 9-alkylidene-N-alkyl acridines, aryl ether olefins, bisoxyethylene, dimethylthiophene, aryl imidazoles or gloss concentrates.
In the present invention, the "light-emitting composition", i.e., a compound called a label, may undergo a chemical reaction to cause light emission, 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 "carrier" according to the invention, which may be of any size, may be organic or inorganic, may be expandable or non-expandable, may be porous or non-porous, has any density, but preferably has a density close to that of water, is preferably floatable in water, and is composed of a transparent, partially transparent or opaque material. The carrier may or may not be charged and when charged is preferably negatively charged. The carrier may be a solid (e.g., polymers, metals, glass, organic and inorganic substances such as minerals, salts, and diatoms), oil droplets (e.g., hydrocarbons, fluorocarbons, siliceous fluids), vesicles (e.g., synthetic such as phospholipids, or natural such as cells, and organelles). 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. The carrier is generally multifunctional or capable of binding to a donor or acceptor by specific or non-specific covalent or non-covalent interactions. Many functional groups are available or incorporated. Typical functional groups include carboxylic acid, acetaldehyde, amino, cyano, vinyl, hydroxyl, mercapto, and the like. One non-limiting example of a carrier suitable for use in the present invention is polystyrene latex microspheres.
The term "C.V value of the particle size distribution coefficient of variation" as used herein refers to the coefficient of variation of the particle size in gaussian distribution in the result of the detection by the nanoparticle analyzer. C.V is a statistic that measures the degree of variation in particle size of individual particles in a standard substance. The standard substance particle size distribution variation coefficient is used to represent the degree of dispersion of the particle size of the standard substance, and is usually expressed as a percentage of the ratio of the standard deviation to the average particle size of the standard substance, which is also called the degree of dispersion. 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.
II. Detailed description of the preferred embodiments
The present invention will be described in more detail below.
The technical principle of the photoexcitation chemiluminescence analysis technology is that a sensitizer can excite oxygen molecules in the surrounding environment into singlet oxygen molecules under the irradiation of laser, and the singlet oxygen molecules can react with a luminous composition with the distance of about 200nm to generate a light signal with a certain wavelength; when the sample contains the antigen or antibody to be detected, the immune reaction of the antigen and the antibody can enable the donor particles containing the sensitizer to be combined with the acceptor particles containing the luminous composition, so that a light signal with a specific wavelength is generated, and the content of the antigen or antibody to be detected can be detected by detecting the light signal. In the above-described photoexcitation chemiluminescent immune reaction, the diameters, materials, surface properties and the like of the acceptor particles and the donor particles may significantly affect the efficiency of exciting singlet oxygen molecules by the sensitizer and the energy transfer efficiency of the singlet oxygen molecules; non-specific binding of donor particles and acceptor particles is also affected, and thus errors occur in detection results, so that the diameter ranges of the donor particles and the acceptor particles, uniformity of particle sizes, materials of particles, surface chemical properties and the like are important directions for developing and improving the photo-activated chemiluminescence analysis technology, and are not common in the art or common in industry practice.
When CK-MB markers exist in the clinical sample, the CK-MB is combined with the receptor particles coated with the CK-MB antibody I (monoclonal antibody) and the biotin-labeled CK-MB antibody II (monoclonal antibody) in a specific way, and a double-antibody sandwich complex is formed on the surfaces of the receptor particles; 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 the CK-MB marker content is present, the stronger the fluorescence intensity. The present invention has been made based on the above-described method.
To this end, a first aspect of the invention relates to a homogeneous assay kit for a creatine kinase isoenzyme comprising a reagent 1 and a reagent 3, the reagent 1 comprising a first buffer solution and, suspended therein, a receptor particle capable of generating chemiluminescence upon action of reactive oxygen species, which binds to a creatine kinase isoenzyme antibody capable of specifically binding to a creatine kinase isoenzyme; the reagent 3 comprises a second buffer solution and donor particles suspended therein, the donor particles being bound to one of the specific pairing members, characterized in that:
The receptor particles comprise a first carrier, the interior of the first carrier is filled with a luminous composition, the surface of the first carrier is bonded with creatine kinase isoenzyme antibodies, and the variation coefficient C.V value of the particle size distribution of the receptor particles in the reagent 1 is not lower than 5% and not higher than 25%;
the donor particle comprises a second carrier, the interior of the second carrier is filled with a sensitizer, and one member of a specific pairing member is bonded to the surface of the second carrier; the sugar content of the donor particle in reagent 3 per mg of the donor particle is not higher than 40mg.
In some embodiments of the invention, the acceptor particle has a particle size distribution coefficient of variation C.V value in reagent 1 of no more than 15%.
In some embodiments of the invention, the receptor particle has a particle size distribution coefficient of variation C.V value in reagent 1 selected from 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, and 25%.
In some embodiments of the invention, the surface of the first carrier is coated with a polysaccharide, and the creatine kinase isoenzyme antibody is bound to the first carrier by a linkage to a polysaccharide molecule.
In some embodiments of the invention, the surface of the first carrier is coated with a coating of at least two successive polysaccharide layers. The term "continuous polysaccharide layer" as used herein means that a plurality of polysaccharide layers are directly bonded to each other, and no other non-polysaccharide layer is present between the two polysaccharide layers.
In some embodiments of the invention, the first polysaccharide layer and the second polysaccharide layer of the coating spontaneously associate. By "spontaneous" as used herein is meant that the polysaccharide layers spontaneously form ordered structures with respect to each other, e.g., charge interactions or molecular self-assembly.
In some embodiments of the invention, each layer of polysaccharide in the coating has functional groups that are oppositely charged to the functional groups of the previous polysaccharide layer.
In other embodiments of the invention, each layer of polysaccharide in the coating has a functional group, and each polysaccharide layer is covalently linked to the previous polysaccharide layer by a reaction between its functional group and a functional group on the previous polysaccharide layer.
In some embodiments of the invention, the outermost polysaccharide layer of the coating has at least one pendant functional group that is bound to an creatine kinase isozyme antibody.
In some embodiments of the invention, the pendant functional groups of the outermost polysaccharide layer of the coating are selected from at least one of aldehyde groups, carboxyl groups, thiol groups, amino groups, hydroxyl groups, and maleamine groups.
In some preferred embodiments of the invention, the pendant functional groups of the outermost polysaccharide layer of the coating are selected from aldehyde groups and/or carboxyl groups.
In some embodiments of the invention, the sugar content per liter of the first buffer solution is 0.01-1g.
In some preferred embodiments of the invention, the sugar content per liter of the first buffer solution is between 0.02 and 0.2g.
In some embodiments of the invention, the sugar content per mg of the acceptor particle is not less than 20 micrograms. The sugar content in the receptor particles of the present invention may be derived from polysaccharides coated on the surface of the receptor particles, or may be derived from polysaccharide components carried in the structure of the antigen-antibody or specific binding pair member itself.
In some embodiments of the invention, the sugar content per mg of the acceptor particle is 20.2 mg, 40 mg, 45.1 mg, 55.8 mg, 60.3 mg.
In some preferred embodiments of the present invention, the sugar content in the first buffer solution is not less than 40 micrograms per liter.
In some embodiments of the invention, the polysaccharide is selected from carbohydrates containing three or more unmodified or modified monosaccharide units; preferably at least one selected from the group consisting of dextran, starch, glycogen, inulin, levan, mannan, agarose, galactan, carboxydextran and aminodextran; more preferably at least one selected from the group consisting of dextran, starch, glycogen and polyribose, most preferably dextran and/or dextran derivatives. Polysaccharides, particularly dextran and dextran derivatives, can increase the hydrophilicity of the support surface and provide conjugation sites for the attachment of antibody molecules to the support surface. The surface of the receptor particles is coated with polysaccharide, so that the hydrophilicity of the microspheres can be increased, the occurrence of non-specific adsorption phenomenon is avoided, and the optical signal of the later photo-excitation chemiluminescence detection is greatly influenced. The inventor of the patent finds that the sugar content on the surface of the microsphere is accurately controlled within a proper range, and can well solve certain technical problems existing in the application of the photo-excitation chemiluminescence technology in the field of in-vitro diagnosis.
In some embodiments of the invention, the donor particles have a particle size distribution coefficient of variation C.V in reagent 3 of no less than 5% and no more than 25%. In some embodiments of the invention, the donor particle has a particle size distribution coefficient of variation C.V in reagent 3 of a value selected from 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, and 25%.
In some embodiments of the invention, the kit further comprises a series of calibrator solutions having known concentrations of creatine kinase isozymes, the concentration of creatine kinase isozymes in the series of calibrator solutions being between 0 and 510ng/mL.
In some embodiments of the invention, the concentration of creatine kinase isozymes in the series of calibrator solutions comprises 0, 4, 10, 50, 200, and 510ng/mL.
In other embodiments of the invention, the kit further comprises reagent 2, the reagent 2 comprising an creatine kinase isoenzyme antibody linked to one of the specific binding pair members, the creatine kinase isoenzyme antibody being capable of specifically binding to the creatine kinase isoenzyme.
In some embodiments of the present invention, the kit contains at least 1 reagent strip, and the reagent strip is provided with a plurality of reagent holes for containing reagents, wherein at least 3 reagent holes are respectively used for containing the reagent 1, the reagent 2 and the reagent 3.
In the present invention, the sugar concentration or sugar content can be determined by the anthrone method. The method for measuring polysaccharide by using anthrone method is known to a person skilled in the art, the polysaccharide is dehydrated by using concentrated sulfuric acid to generate furfural or derivatives thereof, the furfural or hydroxymethylfurfural is further condensed with an anthrone reagent to generate blue-green substances, the blue-green substances have maximum absorption at the wavelength of 620 nm-630 nm in a visible light region, and the light absorption value of the blue-green substances is in direct proportion to the content of the sugar within a certain range. The method can be used for measuring the content of monosaccharide, oligosaccharide and polysaccharide, and has the advantages of high sensitivity, simplicity, convenience, rapidness, suitability for measuring trace samples and the like.
A second aspect relates to the use of a kit according to the first aspect in a chemiluminescent analyzer.
In other embodiments of the invention, the use comprises the steps of:
step S1, respectively adding a sample to be detected, a calibrator and/or a quality control product into a liquid containing device;
step S2, adding a reagent 1, a reagent 2 and a reagent 3 into the liquid containing device;
and S3, placing the liquid containing device on a chemiluminescence analyzer for reaction and detection.
III. Detailed description of the invention
In order that the invention may be more readily understood, the invention will be further described in detail with reference to the following examples, which are given by way of illustration only and are not limiting in scope of application. The starting materials or components used in the present invention may be prepared by commercial or conventional methods unless specifically indicated.
Example 1 preparation of reagent 1
1.1 preparation and characterization of the first Carrier
1) A100 ml three-necked flask was prepared, 40mmol of styrene, 5mmol of methacrylic acid and 10ml of water were added thereto, and after stirring for 10 minutes, N was introduced 2 30min；
2) 0.12g of potassium 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 1, and continuing to introduce N 2 30min；
3) Heating the reaction system to 70 ℃ for reaction for 15 hours;
4) The emulsion after completion of the reaction was cooled to room temperature and filtered through a suitable filter cloth. Washing the obtained emulsion with deionized water for multiple times through centrifugal sedimentation until the conductivity of the supernatant fluid at the beginning of centrifugation is close to that of the deionized water, diluting with water, and preserving in an emulsion form;
5) The particle size of the carboxyl polystyrene latex microsphere is measured by a nanometer particle size analyzer
The average particle size was 202.5nm, and the coefficient of variation (C.V) =5.6%.
1.2 landfill Process of luminescent compositions
1) A25 ml round-bottomed flask was prepared and charged with 0.1g of a dimethylthiophene derivative and 0.1g of europium (III) complex (MTTA-EU) 3+ ) 10ml of 95% ethanol is magnetically stirred, and the temperature of the water bath is raised to 70 ℃ to obtain a complex solution;
2) Preparing a 100ml three-neck flask, adding 10ml 95% ethanol, 10ml water and 10ml carboxyl polystyrene latex microspheres with concentration of 10% obtained in the step 1.1, magnetically stirring, and heating to 70 ℃ in a water bath;
3) Slowly dripping the complex solution in the step 1) into the three-neck flask in the step 2), stopping stirring after reacting for 2 hours at 70 ℃, and naturally cooling;
4) Centrifuging the emulsion for 1 hour, 30000G, and discarding supernatant after centrifuging to obtain carboxyl polystyrene latex microsphere with luminous composition embedded therein.
1.3 surface coating of receptor particles with dextran
1) 50mg of aminodextran solid was taken in a 20mL round bottom flask, 5mL of 50 mm/ph=6 phosphate buffer was added, and the mixture was stirred and dissolved at 30 ℃ in the absence of light;
2) Taking 100mg of prepared carboxyl polystyrene latex microspheres with the luminous composition embedded inside, adding the carboxyl polystyrene latex microspheres into an aminodextran solution, and stirring for 2 hours;
3) 10mg of EDC/HCl is dissolved in 0.5ml of 50 mM/pH=6 phosphate buffer solution and then added dropwise to the reaction solution, and the reaction is carried out overnight at 30 ℃ in a dark place;
4) After the reaction mixture was centrifuged at 30000G for 45min, the supernatant was discarded, and 50 mM/ph=10 carbonate buffer was added for ultrasonic dispersion. After repeating the centrifugal washing three times, the volume is fixed by 50 mM/pH=10 carbonate buffer solution to make the final concentration of the solution be 20mg/ml;
5) Taking 100mg of aldehyde dextran solid into a 20mL round bottom flask, adding 5mL of 50 mM/pH=10 carbonate buffer solution, and stirring and dissolving at 30 ℃ in a dark place to obtain aldehyde dextran solution;
6) Adding the microsphere solution obtained in the step 4) into an aldehyde dextran solution, and stirring for 2 hours;
7) 15mg of sodium borohydride was dissolved in 0.5ml of 50 mM/pH=10 carbonate buffer, and then added dropwise to the above reaction solution, followed by reaction at 30℃overnight in the absence of light;
8) After the reaction mixture was centrifuged at 30000G for 45min, the supernatant was discarded, and 50 mM/ph=10 carbonate buffer was added for ultrasonic dispersion. After repeating the centrifugal washing three times, the volume is fixed by 50 mM/pH=10 carbonate buffer solution to make the final concentration of the solution be 20mg/ml, and a semi-finished microsphere solution is obtained.
9) The average particle size of the Gaussian distribution of the particle size of the semi-finished microspheres at this time was 241.6nm as measured by a nanoparticle sizer, and the coefficient of variation (C.V) =11.50%.
1.4 coupling of CK-MB antibody I
1) CK-MB antibody I was dialyzed to 50mM CB buffer at ph=10, and the concentration was measured to be 1.4mg/ml.
2) Into a 2ml centrifuge tube, 0.5ml (1.3) of the receptor particles obtained in step 1) and 0.36ml of the CK-MB antibody I obtained in step 1 were added, and after mixing, 100. Mu.l of a 10mg/ml NaBH4 solution (50 mM CB buffer) was added, and reacted at 2-8℃for 4 hours.
3) After completion of the reaction, 0.5ml of 100mg/ml BSA solution (50 mM CB buffer) was added, and the reaction was carried out at 2-8℃for 2 hours.
4) After the reaction, the mixture was centrifuged for 45min at 30000G, the supernatant was discarded after centrifugation, and resuspended in 50mM MES buffer. The centrifugation washing was repeated four times and diluted to a final concentration of 100. Mu.g/ml to obtain reagent 1 conjugated with CK-MB antibody I.
5) The average particle diameter of the Gaussian distribution of the particle diameters of the microspheres at this time was 248.9nm as measured by a nanoparticle analyzer, and the coefficient of variation (C.V value) =9.90%.
Example 2 preparation of reagent 2
2.1 dialysis of CK-MB antibody II against 0.1M NaHCO 3 Buffer, measured at a concentration of 1.1mg/ml.
2.2 taking 0.5mg of the CK-MB antibody II0.5mg of the CK-MB antibody in the step 1), adjusting the concentration to 1mg/ml, adding 2.7 mu l of biotin with the concentration of 16.8mg/ml, uniformly mixing, and reacting for 14-18 hours at the temperature of 2-8 ℃.
2.3 reaction completion was dialyzed against 20mM Tris buffer.
Example 3 preparation of reagent 3
3.1 preparation of the second Carrier
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.
d) The emulsion after completion of the reaction was cooled to room temperature and filtered through a suitable filter cloth. The obtained emulsion is subjected to centrifugal sedimentation and cleaning for a plurality of times by using deionized water until the conductivity of supernatant fluid at the beginning of centrifugation is close to that of the deionized water, and then the emulsion is diluted by using water and is stored in an emulsion form.
e) The average gaussian distribution particle diameter of the aldehyde-based polystyrene latex microsphere is 201.3nm, and the variation coefficient (C.V) =8.0% is measured by a nano-particle sizer.
3.2 filling of sensitizer
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 aldehyde-based polystyrene latex microspheres obtained in concentration of 10% and 3.1 were added, and the mixture was magnetically stirred, and the temperature was raised to 70℃in a water bath.
c) Slowly dripping the solution in the step a) into the three-neck flask in the step b), stopping stirring after reacting for 2 hours at 70 ℃, and naturally cooling to obtain emulsion.
d) The emulsion was centrifuged for 1 hour, 30000G, after which the supernatant was discarded and resuspended in 50% ethanol. After repeating the centrifugation washing three times, the mixture was resuspended in 50mM CB buffer having a pH value of 10 to a final concentration of 20mg/ml.
3.3 preparation of reagent 3
a) Microsphere suspension treatment: sucking a certain amount of microspheres prepared in the step 3.2, centrifuging in a high-speed refrigerated centrifuge, discarding the supernatant, adding a certain amount of MES buffer, performing ultrasonic treatment on an ultrasonic cell disruption instrument until particles are resuspended, and adding the MES buffer to adjust the concentration of the microspheres to 100mg/ml.
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 The CN solution was added in a volume ratio of 1:25 to the reaction solution, and the mixture was rapidly and uniformly mixed. The reaction was rotated at 37℃for 48 hours.
e) Closing: preparing 75mg/ml Gly solution and 25mg/ml NaBH in MES buffer 3 CN solution according to the reactionAdding the liquid 2:1:10 by volume ratio into the solution, uniformly mixing, and carrying out rotary reaction for 2 hours at 37 ℃. Then 200mg/ml BSA solution (MES buffer) was added thereto in a volume ratio of 5:8, and the mixture was swiftly mixed and reacted at 37℃for 16 hours.
f) Cleaning: adding MES buffer solution into the reacted solution, centrifuging by a high-speed refrigerated centrifuge, discarding the supernatant, adding fresh MES buffer solution, suspending again by an ultrasonic method, centrifuging again, washing for 3 times, suspending with a small amount of buffer solution, measuring the solid content, and regulating the concentration to 150 mug/ml by the buffer solution to obtain the reagent 3 containing donor particles.
g) The average particle diameter of the Gaussian distribution of the donor particles a in the reagent 3 was 227.7nm, and the coefficient of variation C.V was 6.5% as measured by a nanoparticle analyzer.
Example 4: sugar content determination by anthrone method
4.1 pretreatment of microsphere samples:
reagent 1 containing 1mg of acceptor particles in example 1 and reagent 3 containing 1mg of donor particles in example 3 were taken respectively, centrifuged at 20000g for 40min, the supernatant was removed and then dispersed ultrasonically with purified water, and after repeated centrifugation and dispersion three times, the volumes were each fixed to 1mg/mL with purified water as samples to be examined.
4.2 preparation of 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.
4.3 preparation of anthrone solution: 2mg/mL was prepared using 80% sulfuric acid solution.
4.4 adding 0.1mL of glucose standard solution with each concentration and the sample to be tested into a centrifuge tube, and adding 1mL of anthrone solution into each tube.
4.5 Incubation was performed at 85 degrees celsius for 30min.
4.6 centrifuging the sample reaction tube 15000G for 40min, and sucking clear liquid from the bottom of the tube by the pipette tip to measure absorbance, so as to avoid sucking out suspended matters on the upper part.
4.7 was returned to room temperature and absorbance at 620nm was measured.
4.8, carrying out linear regression once by taking the concentration of the standard substance as an X value and the absorbance as a Y value to obtain the absorbance value of a standard curve shown in table 1, and detecting the sugar content of the sample to be detected.
The sugar content of the acceptor particle in example 1 and the donor particle in example 3 is shown in table 2 below.
TABLE 1
TABLE 2
Example 5 creatine kinase isozyme detection kit (light excitation chemiluminescence method)
The kit contains a reagent 1 (containing CK-MB antibody I coated receptor particles) prepared in the example 1, a reagent 2 (containing biotin-labeled CK-MB antibody II) prepared in the example 2, a creatine kinase isoenzyme series calibrator, a creatine kinase isoenzyme quality control product and a reagent 3 (containing avidin coated donor particles) prepared in the example 3, and the concentration of creatine kinase isoenzyme (CK-MB) in human serum and plasma samples is quantitatively detected by adopting a double-antibody sandwich immunofluorescence excitation chemiluminescence method under a homogeneous condition.
Table 3 below shows the main components of the creatine kinase isoenzyme assay kit (photoexcitation chemiluminescence method) prepared in this example.
TABLE 3 Table 3
Example 6 determination of the Performance of the kit
The performance of the kit in this example was measured using an automatic photo-activated chemiluminescent detector of LiCA500 developed by Boyang biosciences (Shanghai). The specific flow steps of the LiCA500 automatic photo-excitation chemiluminescence detector for detecting creatine kinase isozymes are as follows:
1. respectively adding 50 mu L of sample or calibrator and quality control material into the reaction holes;
2. sequentially adding 15 mu L of the reagent 1 and 15 mu L of the reagent 2 into the reaction well;
3. shaking and incubating for 10 minutes at 37 ℃;
4. automatically adding 150. Mu.L of reagent 3 (photo-activated chemiluminescent assay system universal solution, liCA universal solution);
5. shaking and incubating for 2 minutes at 37 ℃;
6. irradiating the micropores by laser and calculating the luminous photon quantity of each hole;
7. the sample concentration was calculated from the calibration curve.
The method for judging the validity of the detection result in the embodiment comprises the following steps: and (3) detecting quality control products in each batch of tests, wherein the detection results are required to be in a quality control range by single-hole measurement, if the detection results exceed the required quality control range, the test results are unreliable, the detection must be repeated, and if necessary, the calibration is repeated.
The kit is used for detecting 400 plasma samples of healthy people, wherein 200 women and 200 men have the corresponding 97.5% of measured values of the people respectively: female: 3.5ng/mL, male: 4.8ng/mL.
The detection performance evaluation results of the kit of this example were as follows:
blank limit: less than or equal to 0.1ng/mL;
linearity: the linear correlation coefficient r value is more than or equal to 0.9900 within the linear range of 0.1-500 ng/mL;
accuracy: the recovery rate is in the range of (85% -115%);
repeatability: the variation coefficient (C.V) is less than or equal to 10 percent;
batch-to-batch difference: the variation coefficient (C.V) between batches is less than or equal to 15 percent.
Example 7 determination of the sensitivity and upper detection limit of the kit
The sensitivity point is defined as when the signal of concentration Cx is higher than the signal of double concentration C0, i.e. RLU (Cx) >2RLU (C0), the corresponding detection reagent sensitivity is Cx. The upper detection limit point is defined as the upper range limit determined using the method in NCCLS EP-6 file.
(1) CK-MB antigen was diluted to a series of concentrations of 0.1ng/ml, 0.3ng/ml, 0.5ng/ml, 0.7ng/ml, 0.9ng/ml, 3ng/ml, 5ng/ml, 100ng/ml, 300ng/ml, 500ng/ml, 700ng/ml, 900ng/ml, 1100ng/ml, 1300ng/ml, 1500ng/ml, respectively, using reagent 1 prepared in example 1 (acceptor particle containing conjugated CK-MB antibody I at a concentration of 100. Mu.g/ml), and then reagent 2 prepared in example 2 (CK-MB antibody II containing the same biotin label, diluted to 2 ug/ml) and reagent 3 prepared in example 3 were assayed for the above-mentioned series of CK-MB antigen, and the detection sensitivity and upper limit of the assay were as shown in Table 4 using a photo-excitation chemiluminescence assay system developed by Bosun biotechnology (Shanghai).
TABLE 4 Table 4
As can be seen from Table 4, when the coefficient of variation of the particle size distribution of the receptor particles is not less than 5% and not more than 25%, the kit comprising the receptor particles has both a relatively good sensitivity and a wide detection range.
Example 8 Effect of sugar content of acceptor particles in reagent 1 on kit Performance
Reagent 1 comprising the following series of acceptor particles was prepared using the preparation method of reagent 1 in example 1 above, and the sugar content in the acceptor particles was detected using the anthrone method given in example 4.
Referring to the method given in example 5 above, a creatine kinase isoenzyme assay kit (photo-activated chemiluminescence method) including reagents 1 of different sugar contents was prepared, and the photo-activated chemiluminescence assay process was completed on a LiCA HT automated photo-activated chemiluminescence assay system developed by bosa biotechnology (shanghai) limited and the assay result was outputted.
TABLE 5
As is clear from Table 5, when the sugar content of the acceptor particle in the reagent 1 is not less than 20mg/g, the photo-excitation chemiluminescent detection signal amount of the kit comprising the acceptor particle is high.
Example 9 detection of clinical samples
In this example, 40 clinical specimens were tested, and the test was performed using the kit (photoexcitation chemiluminescence method) prepared in example 5. The CK-MB quantitative determination detection kit (a light excitation chemiluminescence method) comprises: reagent 1 comprising a CK-MB antibody I (monoclonal antibody) coated acceptor particle, reagent 2 comprising a biotin-labeled CK-MB antibody II (monoclonal antibody), and reagent 3 comprising a donor particle (photo-activated chemiluminescent assay system universal solution). Wherein the concentration of donor particles in reagent 3 was 150ug/ml, the variation coefficient C.V value of the particle size distribution of the donor particles in reagent R3=6.5%, the sugar content of the donor particles was 11.5ug/mg, and the sugar content of the acceptor particles was 59.60ug/mg, as measured by the anthrone method given in example 4.
The detection process is completed on a fully automatic light-activated chemiluminescence analysis system (LiCA HT) developed by Boyang biotechnology (Shanghai) limited company, and the detection result is output, and the specific detection steps comprise:
a. adding a clinical sample into the reaction well;
b. sequentially adding a reagent 1 and a reagent 2 into the reaction hole;
c. incubating;
d. adding a reagent 3 into the reaction hole;
e. incubating;
f. irradiating the reaction holes twice by laser and calculating the luminous photon quantity of each hole;
g. and calculating the concentration of CK-MB in the sample to be tested.
When CK-MB markers exist in the clinical sample, the CK-MB is combined with receptor particles coated with CK-MB antibody I (monoclonal antibody) and biotin-labeled CK-MB antibody II (monoclonal antibody) in a specific way, and a double-antibody sandwich complex is formed on the surface of the receptor particles; 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, active oxygen is released by the donor particle 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 the content of the CK-MB marker is, the stronger the fluorescence intensity, and the amount of CK-MB in the serum of the patient is quantitatively detected based on the intensity of the luminescence, and the specific detection results are shown in the following Table 6:
TABLE 6
By data comparison, the rogowski assay correlated with the assay described above for example 8 was 0.9963 with a slope of 0.9632. Sample numbers 1-23 are normal physical examination patients, the distribution range is 0.3ng/ml-4.13ng/ml, and the median value is 1.09ng/ml; samples 24-40 were identified as patients with myocardial damage, ranging from 4.55ng/ml to 260.48ng/ml with a median of 20.06g/ml.
Creatine Kinase (CK) is a dimeric enzyme, which has four different forms: mitochondrial isozymes, cytoplasmic isozymes CK-MM (myotype), creatine kinase brain isozymes CK-BB (brain type), creatine kinase isozymes (CK-MB), wherein the creatine kinase isozymes (CK-MB) are mainly located in the heart muscle. Creatine kinase isozymes are released into peripheral blood when Acute Myocardial Infarction (AMI) and myocardial necrosis occur, can be detected after clinical symptoms appear for 3-8 hours, can be combined with myoglobin and troponin for detection, can judge whether the cardiac muscle is damaged, and is an index for researching and diagnosing the Acute Myocardial Infarction (AMI). The method is mainly used for clinically assisting diagnosis of 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 myocardial damage in a subject is feasible, and that the quantitative determination of CKMB markers in body fluids of a subject using the donor reagent according to the present invention and the corresponding method can be used to aid in the diagnosis of myocardial infarction.
It should be noted that the above-described embodiments are only for explaining the present invention and do not constitute any limitation of the present invention. The invention has been described with reference to exemplary embodiments, but it is understood that the words which have been used are words of description and illustration, rather than words of limitation. Modifications may be made to the invention as defined in the appended claims, and the invention may be modified without departing from the scope and spirit of the invention. Although the invention is described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, as the invention extends to all other means and applications which perform the same function.
Claims (22)
1. A homogeneous assay kit for a creatine kinase isozyme comprising reagent 1 and reagent 3, the reagent 1 comprising a first buffer solution and acceptor particles suspended therein, the acceptor particles being capable of producing chemiluminescence upon reactive oxygen species, which bind to a creatine kinase isozyme antibody capable of specifically binding to a creatine kinase isozyme; the reagent 3 comprises a second buffer solution and donor particles suspended therein, the donor particles being bound to one of the specific pairing members, characterized in that:
The receptor particles comprise a first carrier, the interior of the first carrier is filled with a luminous composition, the surface of the first carrier is bonded with creatine kinase isoenzyme antibodies, and the variation coefficient C.V value of the particle size distribution of the receptor particles in the reagent 1 is not lower than 5% and not higher than 25%; a sugar content of not less than 20 micrograms per milligram of the acceptor particle;
the donor particle comprises a second carrier, the interior of the second carrier is filled with a sensitizer, and one member of a specific pairing member is bonded to the surface of the second carrier; the sugar content of the donor particle in reagent 3 per gram of the donor particle is not higher than 40mg.
2. The kit according to claim 1, wherein the receptor particles have a coefficient of variation C.V of the particle size distribution in reagent 1 of not more than 15%.
3. The kit according to claim 1 or 2, wherein the surface of the first carrier is coated with a polysaccharide, and the creatine kinase isoenzyme antibody is bound to the first carrier by binding to a polysaccharide molecule.
4. A kit according to claim 3, wherein the surface of the first carrier is coated with a coating of at least two consecutive polysaccharide layers.
5. The kit of claim 4, wherein the first polysaccharide layer of the coating spontaneously associates with the second polysaccharide layer.
6. The kit of claim 4 or 5, wherein each layer of polysaccharide in the coating has a functional group, each polysaccharide layer being covalently linked to a previous polysaccharide layer by a reaction between its functional group and a functional group on the previous polysaccharide layer.
7. The kit of claim 6, wherein the outermost polysaccharide layer of the coating has at least one pendant functional group that is bound to an creatine kinase isoenzyme antibody.
8. The kit of claim 7, wherein the pendant functional groups of 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.
9. The kit of claim 8, wherein the pendant functional groups of the outermost polysaccharide layer of the coating are selected from aldehyde groups and/or carboxyl groups.
10. The kit according to claim 1, wherein the sugar content per liter of the first buffer solution is 0.01-1g.
11. The kit according to claim 10, wherein the sugar content per liter of the first buffer solution is 0.02-0.2g.
12. The kit of claim 1, wherein the sugar content per mg of the acceptor particle is not less than 40 micrograms.
13. A kit according to claim 3, wherein the polysaccharide is selected from carbohydrates containing three or more unmodified or modified monosaccharide units.
14. The kit of claim 13, wherein the polysaccharide is selected from at least one of dextran, starch, glycogen, inulin, levan, mannan, agarose, galactan, polyribose, dextran derivatives.
15. The kit of claim 14, wherein the dextran is selected from the group consisting of carboxydextran and aminodextran.
16. The kit according to claim 1, wherein the donor particles have a coefficient of variation C.V of the particle size distribution in reagent 3 of not less than 5% and not more than 25%.
17. The kit of claim 1 or 2, further comprising a series of calibrator solutions of known creatine kinase isoenzyme concentration, wherein the concentration of creatine kinase isoenzyme in the series of calibrator solutions is 0-510ng/mL.
18. The kit of claim 17, wherein the concentration of creatine kinase isozymes in the series of calibrator solutions comprises 0, 4, 10, 50, 200, and 510ng/mL.
19. The kit of claim 1 or 2, further comprising a reagent 2, the reagent 2 comprising a creatine kinase isoenzyme antibody linked to one of the specific binding pair members, the creatine kinase isoenzyme antibody capable of specifically binding to the creatine kinase isoenzyme.
20. The kit according to claim 1 or 2, wherein the kit comprises at least 1 reagent strip, and a plurality of reagent holes for containing reagents are formed in the reagent strip, wherein at least 3 reagent holes are respectively used for containing the reagents 1, 2 and 3.
21. Use of a kit according to any one of claims 1 to 20 in a chemiluminescent analyzer.
22. The application according to claim 21, characterized in that it comprises the steps of:
step S1, respectively adding a sample to be detected, a calibrator and/or a quality control product into a liquid containing device;
step S2, adding a reagent 1, a reagent 2 and a reagent 3 into the liquid containing device;
and S3, placing the liquid containing device on a chemiluminescence analyzer for reaction and detection.
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| CN116859041A (en) | 2023-10-10 |
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