CN113125700A - Homogeneous detection kit for creatine kinase isoenzyme and application thereof - Google Patents
Homogeneous detection kit for creatine kinase isoenzyme and application thereof Download PDFInfo
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- CN113125700A CN113125700A CN201911411233.XA CN201911411233A CN113125700A CN 113125700 A CN113125700 A CN 113125700A CN 201911411233 A CN201911411233 A CN 201911411233A CN 113125700 A CN113125700 A CN 113125700A
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- creatine kinase
- kinase isoenzyme
- antibody
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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Abstract
The invention relates to a homogeneous detection kit of creatine kinase isoenzyme and application thereof, the kit comprises a reagent 1 and a reagent 3, wherein the reagent 1 comprises a first buffer solution and acceptor particles suspended in the first buffer solution, the reagent 3 comprises a second buffer solution and donor particles suspended in the second buffer solution, the acceptor particles comprise a first carrier, the first carrier is filled with a luminescent 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 comprises a second carrier, the inside of which is filled with a sensitizer, the donor particle having a sugar content of not more than 40mg per mg of the donor particle in reagent 3. The kit can be used for clinically auxiliary diagnosis of myocardial infarction, and has relatively 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 isoenzyme, and a preparation method and application thereof.
Background
Creatine Kinase (CK) is a dimeric enzyme that has four different forms: mitochondrial isozyme, cytoplasmic isozyme CK-MM (muscular type), creatine kinase brain isozyme CK-BB (brain type), creatine kinase isozyme (CK-MB), wherein the creatine kinase isozyme is mainly located in cardiac muscle.
The creatine kinase isozyme is 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 detected together with myoglobin and troponin to judge whether the myocardium is damaged, and is an index for researching and diagnosing the Acute Myocardial Infarction (AMI). The method is mainly used for auxiliary diagnosis of myocardial infarction clinically.
Common CK-MB detection methods include gold-labeled qualitative test, fluorescence immunoassay, combined immunosorbent assay (ELISA), magnetic particle chemiluminescence assay (CMIA) and biochemical detection. The basic principle of light-activated chemiluminescence is a homogeneous immune response. It is based on two kinds of antigens or antibodies coated on the surfaces of particles, and immune complexes are formed in a liquid phase to draw the two kinds of particles close. Under the excitation of laser, the transfer of singlet oxygen between particles occurs, and then high-level red light is generated, and the number of photons is converted into the concentration of an analyte through a single photon counter and mathematical fitting. When the sample does not contain the 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 level red light is generated during detection. Compared with the existing detection kit, the kit based on the light-activated chemiluminescence method has the advantages of convenience in operation, rapidness in detection, high sensitivity, good accuracy and the like; the application of the compound in monitoring myocardial damage can improve the accuracy of acute myocardial infarction diagnosis.
CN106093411A discloses a one-step homogeneous CK-MB detection kit for acute myocardial infarction diagnosis and application thereof in detecting CK-MB content, wherein a light-activated chemiluminescence method is adopted, and although the kit has the advantages of convenience in operation, rapidness in detection, strong specificity and the like compared with the prior art, the kit still has low sensitivity, and the kit cannot give consideration to high sensitivity and wide detection range.
Disclosure of Invention
The invention aims to solve the technical problem of providing a homogeneous detection kit of creatine kinase isoenzyme aiming at the defects of the prior art, and the quantitative result of the CK-MB marker in the body fluid of a main body can be used for clinically auxiliary diagnosis of myocardial infarction, so that the kit has proper sensitivity and wide detection range.
To this end, the present invention provides, in a first aspect, a homogeneous assay kit for creatine kinase isoenzyme, comprising a reagent 1 and a reagent 3, the reagent 1 comprising a first buffer solution and, suspended therein, receptor particles capable of reacting with reactive oxygen species to produce chemiluminescence, which is bound to an antibody for creatine kinase isoenzyme capable of specifically binding to creatine kinase isoenzyme; reagent 3 comprising a second buffered solution and, suspended therein, donor particles that bind to one of the specific pair members, characterized in that:
the acceptor 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 a creatine kinase isoenzyme antibody, 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 comprises a second support having a sensitizer filled therein, the second support having one of the specific pair members bonded to a surface thereof; the donor particle has a sugar content of no more than 40mg per mg of the donor particle in reagent 3.
In some embodiments of the invention, the acceptor particles have a coefficient of variation of particle size distribution C.V value in reagent 1 of no greater than 15%.
In some embodiments of the invention, the recipient particle may have a coefficient of variation of particle size distribution C.V value in reagent 1 selected from the group consisting of 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 bonding to a polysaccharide molecule.
In some embodiments of the invention, the surface of the first support is coated with at least two continuous polysaccharide layers.
In some embodiments of the invention, the first polysaccharide layer of the coating is spontaneously associated with the second polysaccharide layer.
In some embodiments of the invention, each polysaccharide layer in the coating has a functional group that is oppositely charged to the functional group of the previous polysaccharide layer.
In other embodiments of the present invention, each polysaccharide layer in the coating has functional groups, and each polysaccharide layer is covalently linked to a previous polysaccharide layer by a reaction between its functional groups and functional groups 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 binds to an antibody to creatine kinase isoenzyme.
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, carboxyl, thiol, amino, hydroxyl, and maleic groups.
In some preferred embodiments of the present invention, the pendant functional groups of the outermost polysaccharide layer of the coating are selected from aldehyde and/or carboxyl groups.
In some embodiments of the invention, the sugar content per liter of said first buffer solution is between 0.01 and 1 g.
In some preferred embodiments of the invention, the sugar content per liter of said first buffer solution is between 0.02 and 0.2 g.
In some embodiments of the invention, the acceptor particle comprises no less than 20 micrograms of sugar per milligram. In some embodiments of the invention, the sugar content per mg of the acceptor particle may be selected from 20.2 mg, 40mg, 45.1 mg, 55.8 mg, 60.3 mg.
In some preferred embodiments of the present invention, the sugar content per liter of 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, fructan, 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 coefficient of variation of particle size distribution C.V value in reagent 3 of no less than 5% and no more than 25%. In some embodiments of the invention, the donor particle has a coefficient of variation in size distribution C.V value in reagent 3 selected from the group consisting of 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 of known creatine kinase isoenzyme concentration, the concentration of creatine kinase isoenzyme in the series of calibrator solutions being 0-510 ng/mL.
In some embodiments of the invention, the concentration of creatine kinase isoenzyme in the series of calibrator solutions comprises 0, 4, 10, 50, 200, and 510 ng/mL.
In other embodiments of the invention, the kit further comprises reagent 2, said reagent 2 comprising a creatine kinase isoenzyme antibody linked to one of the specific binding pair members, said creatine kinase isoenzyme antibody being capable of specifically binding to 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 wells for containing reagents, wherein at least 3 reagent wells are respectively used for containing the reagent 1, the reagent 2, and the reagent 3.
A second aspect of 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 tested, a calibrator and/or a quality control product into the liquid containing device;
step S2, adding reagent 1, reagent 2 and reagent 3 into the liquid containing device;
and step S3, putting the liquid containing device on a chemiluminescence analyzer for reaction and detection.
The inventor of the present invention 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, and the quality thereof is easily affected by various factors, so that it is necessary to obtain the optimal control technical characteristics according to multiple detection experiments, such as C.V values of the variation coefficient of the particle size distribution of the acceptor particles and the donor particles, since the volume of the acceptor particles and the donor particles is internally fused with the luminescent composition or sensitizer and externally coated with antibody antigen or streptavidin, in order to ensure the uniformity of the particles and reduce the error caused by the uneven size of the particles, one skilled in the art would generally consider to control the value of the variation coefficient of the particle size distribution C.V to be in a smaller range, even lower, better. However, the inventors of the present invention have found that if the C.V value of the fine particles is small, the requirement for the production process is too high, which greatly increases the production cost of the reagent, and the reagent cannot be industrially produced, so that the reagent cannot be used in large quantities in clinical diagnosis. Particularly, after the polysaccharide is coated, the change of the variation coefficient C.V value of the particle size distribution of the nano-microspheres is more obvious and more unstable, and the requirements of medical appliance product registration and clinical application reagents are difficult to meet. However, if the value of C.V is too large, the effect of light-activated chemiluminescence detection is not good. Therefore, the inventors of the present invention surprisingly found that if the value of the variation coefficient C.V of the particle size distribution of the acceptor particle and the donor particle is controlled within a proper range, an optimal detection result of the photoluminescence can be obtained, and a wide detection range with proper sensitivity can be realized.
The invention has the beneficial effects that:
the kit provided by the invention has feasibility in-vitro diagnosis of whether a main body suffers from myocardial damage, and the quantitative result of the CK-MB marker in the body fluid of the main body determined by using the kit provided by the invention and the corresponding method can be used for auxiliary diagnosis of myocardial infarction. The kit of the invention not only has relatively proper sensitivity, but also has wide detection range.
Detailed Description
In order that the invention may be readily understood, a detailed description of the invention is provided below. However, before the 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, to the extent that there is no stated or intervening value in that stated range, to the extent that there is no such intervening value, 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 a specified range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. 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. Term(s) for
The term "homogeneous" as used herein is defined in english as "homogeneous" and means that the bound antigen-antibody complex and the remaining free antigen or antibody are detected without separation.
The term "sample to be tested" as used herein refers to a mixture that may contain an analyte. Typical test samples that may be used in the disclosed methods include body fluids such as blood, plasma, serum, urine, semen, saliva, and the like.
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. In any case desired, the antibody may be further conjugated to other moieties, such as a specific binding pair member, e.g., biotin or avidin (a member of a biotin-avidin specific binding pair member) or the like.
The term "antigen" as used herein refers to a substance that stimulates the body to produce an immune response and that binds to the immune response product antibodies and sensitized lymphocytes in vitro and in vivo to produce an immune effect. The antigen may be a fusion antigen and, in any case desired, the antigen may be further conjugated with other moieties such as a specific binding pair member, e.g. biotin or avidin (a member of the biotin-avidin specific binding pair member) or the like.
The term "bind" or "bonding" as used herein refers to the association between 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 discrimination and selective binding reaction between two substances, and is the conformational correspondence between the corresponding reactants from the perspective of the steric structure.
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 the molecule, an imidazolone ring and a thiophene ring, respectively, in which the imidazolone ring is the main site for binding to avidin. Activated biotin can be conjugated to almost any biological macromolecule known, including proteins, nucleic acids, polysaccharides, lipids, and the like, mediated by a protein cross-linking agent; while "avidin" is a protein secreted by Streptomyces and has a molecular weight of 65 kD. The "avidin" molecule consists of 4 identical peptide chains, each of which is capable of binding a biotin. Thus, each antigen or antibody can be conjugated to multiple biotin molecules simultaneously, thereby creating a "tentacle effect" that increases assay sensitivity. In the present invention, the avidin is selected from the group consisting of ovalbumin, streptavidin, vitellin, neutravidin and avidin, preferably selected from the group consisting of neutravidin and/or streptavidin.
The term "active oxygen" as used herein refers to a general term for a substance which is composed of oxygen, contains oxygen, and is active in nature, and is mainly an excited oxygen molecule, including superoxide anion (O) which is an electron reduction product of oxygen2(-) and the two-electron reduction product hydrogen peroxide (H)2O2) The three-electron reduction product hydroxyl radical (. OH) and nitric oxide and active oxygen (A)1O2) And the like.
The term "donor particle" as used herein refers to a sensitizer capable of generating a reactive intermediate, such as a reactive oxygen species, upon activation by energy or a reactive compound, which reacts with the acceptor particle. The donor particles may be light activated (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 the substrate through a functional group to form polymer microparticles filled with a photosensitive compound, which can generate active oxygen under light excitation, in this case, 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 photosensitizer may be one known in the art, preferably a compound that is relatively photostable and does not react efficiently with reactive oxygen species, non-limiting examples of which include compounds such as methylene blue, rose bengal, porphyrins, and phthalocyanines, as well as derivatives of these compounds having 1-50 atom substituents that serve to render these compounds more lipophilic or more hydrophilic, and/or as a linker for attachment to a member of a specific binding pair. 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. Other examples of donors include: 1, 4-dicarboxyethyl-1, 4-naphthalene endoperoxide, 9, 10-diphenylanthracene-9, 10-endoperoxide, etc., which are heated or which absorb light directly to release active oxygen, e.g., active oxygen.
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 by energy or an active compound to activate and release reactive oxygen species in a high energy state that are captured by the acceptor particles in close proximity, thereby transferring energy to activate the acceptor particles. In some embodiments of the present invention, the acceptor particles are filled in the carrier through the functional groups to form polymer microparticles filled with the luminescent composition, and the luminescent composition comprises a chemiluminescent compound capable of reacting with active oxygen. In some embodiments of the invention, the chemiluminescent compound undergoes a chemical reaction with reactive oxygen species to form an unstable metastable intermediate that can decompose with or subsequently luminesce. Typical examples of such substances include, but are not limited to: enol ethers, enamines, 9-alkylidene xanthan gums, 9-alkylidene-N-alkyl acridines, aryl ethylether alkenes, diepoxides, dimethylthiophenes, aryl imidazoles, or lucigenins.
In the present invention, the "luminescent composition", i.e. a compound referred to as a label, may undergo a chemical reaction in order to cause luminescence, for example 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 may return to the ground state itself by transferring excitation energy to an emission energy acceptor. In this process, the energy acceptor particle will be transitioned to an excited state to emit light.
The "carrier" according to the invention, which may be of any size, organic or inorganic, which may be swellable or non-swellable, which may be porous or non-porous, which has any density, but preferably has a density close to that of water, preferably is capable of floating in water, and is composed of a transparent, partially transparent or opaque material. The carrier may or may not have a charge, and when charged, is preferably a negative charge. The carrier may be a solid (e.g., polymers, metals, glasses, organic and inorganic materials 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 a latex particle or other particle 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 recipient 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, hydroxy, mercapto, and the like. One non-limiting example of a carrier suitable for use in the present invention is polystyrene latex microspheres.
The "C.V value of variation coefficient of particle size distribution" described in the present invention refers to the variation coefficient of particle size in Gaussian distribution in the detection result of the nanometer particle size analyzer. C.V is a statistic for measuring the variation of the particle size of each particle in the standard substance. The coefficient of variation of the particle size distribution of the standard substance is used to indicate 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 mean particle size of the standard substance, which is also referred to as the degree of dispersion. The coefficient of variation is calculated as: the coefficient of variation C.V value (standard deviation SD/Mean) x 100%. The Standard Deviation (SD), also known as the Standard Deviation, describes the mean of the distances of the data from the mean (mean Deviation), which is the square of the Deviation and the root of the mean, expressed as a. 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 mean, and vice versa. The standard deviation σ is a distance between an inflection point (0.607 times the peak height) on the normal distribution curve and a vertical line between the peak height and the time axis, that is, a distance between two inflection points on the normal distribution curve is half. The peak width at half height (Wh/2) is the width of the peak at half the peak height, Wh/2 ═ 2.355 σ. The tangent is drawn by the inflection points on both sides of the normal distribution curve, the intercept at the base line is called the peak width or base line width, and W is 4 sigma or 1.699 Wh/2.
Detailed description of the preferred embodiments
The present invention will be described in more detail below.
The technical principle of the light-activated 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 luminescent composition with a 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 or antibody can combine the donor particle containing the sensitizer with the acceptor particle containing the luminescent composition, so as to generate an optical signal with a specific wavelength, and the content of the antigen or antibody to be detected can be detected by detecting the optical signal. In the above-described photo-excited chemiluminescent immune reaction, the diameters, materials, surface properties, etc. of the acceptor particle and the donor particle significantly affect the efficiency of the sensitizer to excite singlet oxygen molecules and the energy transfer efficiency of singlet oxygen molecules; and non-specific binding between the donor particles and the acceptor particles is also affected, so that errors occur in the detection result, and therefore, the diameter range, the uniformity of the particle size, the material and the surface chemical properties of the particles, and the like of the donor particles and the acceptor particles are the key directions for developing and improving the light-activated chemiluminescence analysis technology, and are not the common knowledge or the industry convention in the field.
When the CK-MB marker exists in a clinical sample, CK-MB specifically binds to a receptor particle coated with CK-MB antibody I (monoclonal antibody) and biotin-labeled CK-MB antibody II (monoclonal antibody) at the same time, and forms a double-antibody sandwich complex on the surface of the receptor particle; at this time, if a donor particle modified by streptavidin is added, biotin and streptavidin are combined to enable the two particles to approach each other, under the excitation of an excitation light source, the donor particle releases singlet oxygen, and chemiluminescence is generated after the donor particle touches an acceptor particle in a solution, so that a fluorophore on the same particle is further excited to generate a cascade amplification reaction to generate fluorescence. At this time, the more CK-MB marker is present, the stronger the fluorescence intensity. The present invention is based on the above-mentioned method.
To this end, the present invention relates, in a first aspect, to a homogeneous assay kit for creatine kinase isoenzyme, comprising a reagent 1 and a reagent 3, the reagent 1 comprising a first buffer solution and, suspended therein, receptor particles capable of reacting with reactive oxygen species to produce chemiluminescence which is bound to antibodies to creatine kinase isoenzyme capable of specifically binding to creatine kinase isoenzyme; reagent 3 comprising a second buffered solution and, suspended therein, donor particles that bind to one of the specific pair members, characterized in that:
the acceptor 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 a creatine kinase isoenzyme antibody, 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 comprises a second support having a sensitizer filled therein, the second support having one of the specific pair members bonded to a surface thereof; the donor particle has a sugar content of no more than 40mg per mg of the donor particle in reagent 3.
In some embodiments of the invention, the acceptor particles have a coefficient of variation of particle size distribution C.V value in reagent 1 of no greater than 15%.
In some embodiments of the invention, the recipient particle has a coefficient of variation of particle size distribution C.V value in reagent 1 selected from the group consisting of 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 bonding to a polysaccharide molecule.
In some embodiments of the invention, the surface of the first support is coated with at least two continuous 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 two polysaccharide layers.
In some embodiments of the invention, the first polysaccharide layer of the coating is spontaneously associated with the second polysaccharide layer. "spontaneous" as used herein refers to the manner in which multiple 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 polysaccharide layer in the coating has a functional group that is oppositely charged to the functional group of the previous polysaccharide layer.
In other embodiments of the present invention, each polysaccharide layer in the coating has functional groups, and each polysaccharide layer is covalently linked to a previous polysaccharide layer by a reaction between its functional groups and functional groups 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 binds to an antibody to creatine kinase isoenzyme.
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, carboxyl, thiol, amino, hydroxyl, and maleic groups.
In some preferred embodiments of the present invention, the pendant functional groups of the outermost polysaccharide layer of the coating are selected from aldehyde and/or carboxyl groups.
In some embodiments of the invention, the sugar content per liter of said first buffer solution is between 0.01 and 1 g.
In some preferred embodiments of the invention, the sugar content per liter of said first buffer solution is between 0.02 and 0.2 g.
In some embodiments of the invention, the acceptor particle comprises no less than 20 micrograms of sugar per milligram. The saccharide content of the receptor particles of the present invention may be derived from polysaccharides coated on the surface of the receptor particles, or from polysaccharide components carried in the structure of the antigen-antibody or specific binding pair member itself.
In some embodiments of the invention, the acceptor particle comprises 20.2 mg, 40mg, 45.1 mg, 55.8 mg, 60.3 mg sugar per mg.
In some preferred embodiments of the present invention, the sugar content per liter of 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, fructan, 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, which can increase the hydrophilicity of the carrier surface and provide conjugate sites for the attachment of antibody molecules to the carrier surface. The polysaccharide coated on the surface of the receptor particles can increase the hydrophilicity of the microspheres, avoid the occurrence of non-specific adsorption phenomenon and greatly influence the optical signal of the later period light-activated chemiluminescence detection. 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 light-activated chemiluminescence technology in the field of in vitro diagnosis.
In some embodiments of the invention, the donor particles have a coefficient of variation of particle size distribution C.V value in reagent 3 of no less than 5% and no more than 25%. In some embodiments of the invention, the donor particle has a coefficient of variation of particle size distribution 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 of known creatine kinase isoenzyme concentration, the concentration of creatine kinase isoenzyme in the series of calibrator solutions being 0-510 ng/mL.
In some embodiments of the invention, the concentration of creatine kinase isoenzyme in the series of calibrator solutions comprises 0, 4, 10, 50, 200, and 510 ng/mL.
In other embodiments of the invention, the kit further comprises reagent 2, said reagent 2 comprising a creatine kinase isoenzyme antibody linked to one of the specific binding pair members, said creatine kinase isoenzyme antibody being capable of specifically binding to 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 wells for containing reagents, wherein at least 3 reagent wells 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 polysaccharide is measured by an anthrone method, which is known by the technical personnel in the field, saccharide is dehydrated in the presence of concentrated sulfuric acid to generate furfural or a derivative thereof, the furfural or hydroxymethyl furfural is further condensed with an anthrone reagent to generate a blue-green substance, the blue-green substance has the maximum absorption at the wavelength of 620 nm-630 nm in a visible light region, and the light absorption value of the blue-green substance is in a direct proportion relation with the content of sugar in a certain range. The method can be used for measuring the contents of monosaccharide, oligosaccharide and polysaccharide, and has the advantages of high sensitivity, simplicity, convenience, quickness, 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 application comprises the steps of:
step S1, respectively adding a sample to be tested, a calibrator and/or a quality control product into the liquid containing device;
step S2, adding reagent 1, reagent 2 and reagent 3 into the liquid containing device;
and step S3, putting the liquid containing device on a chemiluminescence analyzer for reaction and detection.
Detailed description of the preferred embodiments
In order that the present invention may be more readily understood, the following detailed description will proceed with reference being made to examples, which are intended to be illustrative only and are not intended to limit the scope of the invention. The starting materials or components used in the present invention may be commercially or conventionally prepared unless otherwise specified.
EXAMPLE 1 preparation of reagent 1
1.1 preparation and characterization procedure of the first support
1) Preparing a 100ml three-neck flask, adding 40mmol styrene, 5mmol methacrylic acid and 10ml water, stirring for 10min, introducing N2 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 in the step 1, and continuously introducing N2 30min；
3) Heating the reaction system to 70 ℃ and reacting for 15 hours;
4) the emulsion after completion of the reaction was cooled to room temperature and filtered through a suitable filter cloth. Centrifuging, settling and cleaning the obtained emulsion for multiple times by using deionized water until the conductivity of the supernatant at the beginning of centrifugation is close to that of the deionized water, diluting the obtained emulsion by using water, and storing the diluted emulsion in an emulsion form;
5) the Gaussian of the particle size of the carboxyl polystyrene latex microspheres at the moment is measured by a nanometer particle size analyzer
The distribution average particle diameter was 202.5nm, and the coefficient of variation (C.V) was 5.6%.
1.2 landfill Process of luminescent composition
1) A25 ml round-bottom flask was prepared, and 0.1g of a dimethylthiophene derivative and 0.1g of europium (III) complex (MTTA-EU) were added3+) 10ml of 95% ethanol, magnetically stirring, heating in a water bath to 70 ℃ to obtain a complex solution;
2) preparing a 100ml three-neck flask, adding 10ml of 95% ethanol, 10ml of water and 10ml of 10% carboxyl polystyrene latex microspheres obtained in the step 1.1, magnetically stirring, and heating in a water bath to 70 ℃;
3) slowly dripping the complex solution in the step 1) into the three-neck flask in the step 2), reacting for 2 hours at 70 ℃, stopping stirring, and naturally cooling;
4) and centrifuging the emulsion for 1 hour at 30000G, and removing supernatant after centrifugation to obtain the carboxyl polystyrene latex microspheres with the luminous composition embedded inside.
1.3 surface coating of receptor particles with dextran
1) Taking 50mg of aminodextran solid, putting the aminodextran solid into a 20mL round-bottom flask, adding 5mL of 50mM/pH 6 phosphate buffer solution, and stirring and dissolving the aminodextran solid at 30 ℃ in the dark;
2) adding 100mg of prepared carboxyl polystyrene latex microspheres with the luminous composition embedded inside into the aminodextran solution and stirring for 2 hours;
3) 10mg of EDC & HCl was dissolved in 0.5mL of 50mM/pH 6 phosphate buffer, and then added dropwise to the reaction mixture, followed by overnight reaction at 30 ℃ in the absence of light;
4) the reacted mixture was centrifuged at 30000G for 45min, and the supernatant was discarded, followed by ultrasonic dispersion in 50mM/pH 10 carbonate buffer. After repeated centrifugal washing for three times, the solution is fixed by 50mM/pH 10 carbonate buffer solution to a final concentration of 20 mg/ml;
5) putting 100mg aldehyde dextran solid into a 20mL round-bottom flask, adding 5mL 50mM/pH 10 carbonate buffer, and stirring and dissolving at 30 ℃ in the dark 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) dissolving 15mg of sodium borohydride in 0.5mL of 50mM/pH 10 carbonate buffer solution, dropwise adding the solution into the reaction solution, and reacting overnight at 30 ℃ in a dark place;
8) the reacted mixture was centrifuged at 30000G for 45min, and the supernatant was discarded, followed by ultrasonic dispersion in 50mM/pH 10 carbonate buffer. After repeated centrifugal washing three times, the volume is determined by 50mM/pH 10 carbonate buffer solution to a final concentration of 20mg/ml, and a semi-finished microsphere solution is obtained.
9) The average particle size of the semi-finished product microspheres in Gaussian distribution measured by a nanometer particle size analyzer is 241.6nm, and the coefficient of variation (C.V) is 11.50%.
1.4 conjugation of CK-MB antibody I
1) CK-MB antibody I was dialyzed into 50mM CB buffer at PH 10, and the measured concentration was 1.4 mg/ml.
2) 0.5ml (1.3) of the acceptor particles obtained in step 1) and 0.36ml of CK-MB antibody I obtained in step 1) were added to a 2ml centrifuge tube, mixed well, and reacted at 2 to 8 ℃ for 4 hours with 100. mu.l of a 10mg/ml NaBH4 solution (50mM CB buffer).
3) After completion of the reaction, 0.5ml of 100mg/ml BSA solution (50mM CB buffer) was added thereto, and the reaction was carried out at 2 to 8 ℃ for 2 hours.
4) After completion of the reaction, the reaction mixture was centrifuged at 30000G for 45min, and the supernatant was discarded after centrifugation and resuspended in 50mM MES buffer. The centrifugal washing was repeated four times, and diluted to a final concentration of 100. mu.g/ml to obtain CK-MB antibody I-coupled reagent 1.
5) The average particle size of the Gaussian distribution of the particle size of the microspheres measured by a nanometer particle size analyzer was 248.9nm, and the coefficient of variation (C.V value) was 9.90%.
EXAMPLE 2 preparation of reagent 2
2.1 CK-MB antibody II was dialyzed to 0.1M NaHCO3Buffer, measured at a concentration of 1.1 mg/ml.
2.2 taking CK-MB antibody II0.5mg in the step 1), adjusting the concentration to 1mg/ml, adding 2.7 mu l of biotin with the concentration of 16.8mg/ml, mixing evenly, and reacting for 14-18 hours at the temperature of 2-8 ℃.
2.3 reaction completion dialysis in 20mM Tris buffer.
EXAMPLE 3 preparation of reagent 3
3.1 preparation of the second support
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 10min, N was introduced thereinto2 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 continuingGeneral formula (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. And the obtained emulsion is centrifugally settled and washed by deionized water for multiple times until the conductivity of the supernatant at the beginning of centrifugation is close to that of the deionized water, and then the obtained emulsion is diluted by water and stored in the form of emulsion.
e) The average particle size of the aldehyde polystyrene latex microspheres in a Gaussian distribution manner is 201.3nm measured by a nanometer particle size analyzer, and the variation coefficient (C.V) is 8.0%.
3.2 filling of sensitizers
a) A25 ml round bottom flask was prepared, 0.11g of copper phthalocyanine and 10ml of N, N-dimethylformamide were added, magnetic stirring was carried out, and the temperature in the water bath was raised to 75 ℃ to obtain a photosensitizer solution.
b) Preparing a 100ml three-neck flask, adding 10ml 95% ethanol, 10ml water and 10ml aldehyde polystyrene latex microspheres obtained in 3.1 with the concentration of 10%, magnetically stirring, and heating in a water bath to 70 ℃.
c) Slowly dropwise adding the solution obtained in the step a) into the three-neck flask obtained in the step b), reacting at 70 ℃ for 2 hours, stopping stirring, and naturally cooling to obtain an emulsion.
d) The emulsion was centrifuged for 1 hour at 30000G, the supernatant discarded after centrifugation and resuspended in 50% ethanol. After repeated centrifugation washing three times, the suspension was resuspended in 50mM CB buffer at pH 10 to a final concentration of 20 mg/ml.
3.3 preparation of reagent 3
a) Treating microsphere suspension: and (3) sucking a certain amount of the microspheres prepared in the step (3.2) to centrifuge in a high-speed refrigerated centrifuge, removing the supernatant, adding a certain amount of MES buffer solution, performing ultrasonic treatment on an ultrasonic cell disruption instrument until the particles are resuspended, and adding the MES buffer solution to adjust the concentration of the microspheres to 100 mg/ml.
b) Preparing an avidin solution: a certain amount of streptavidin was weighed and dissolved in MES buffer to 8 mg/ml.
c) Mixing: mixing the treated microsphere suspension, 8mg/ml avidin and MES buffer solution in a volume ratio of 2:5:1, and quickly mixing to obtain a reaction solution.
d) Reaction: preparing 25mg/ml NaBH by MES buffer solution3Adding CN solution according to the volume ratio of 1:25 to the reaction solution, and quickly and uniformly mixing. The reaction was rotated at 37 ℃ for 48 hours.
e) And (3) sealing: MES buffer solution is prepared into 75mg/ml Gly solution and 25mg/ml NaBH3Adding CN solution into the solution according to the volume ratio of 2:1:10 of the reaction solution, mixing uniformly, and carrying out rotary reaction for 2 hours at 37 ℃. Then, 200mg/ml BSA solution (MES buffer) was added thereto at a volume ratio of 5:8, and the mixture was rapidly mixed and subjected to a rotary reaction at 37 ℃ for 16 hours.
f) Cleaning: adding MES buffer solution into the reacted solution, centrifuging by a high-speed refrigerated centrifuge, removing supernatant, adding fresh MES buffer solution, resuspending by an ultrasonic method, centrifuging again, washing for 3 times, finally suspending by using a small amount of buffer solution, measuring solid content, and adjusting the concentration to 150 mu g/ml by using the buffer solution to obtain a reagent 3 containing donor particles.
g) The donor particles a in reagent 3 had a gaussian distribution mean particle diameter of 227.7nm as measured by a nanometer particle sizer, and a coefficient of variation C.V value of 6.5%.
Example 4: detection of sugar content by anthrone method
4.1 microsphere sample pretreatment:
the test sample was prepared by taking 1mg of acceptor particle-containing reagent 1 in example 1 and 1mg of donor particle-containing reagent 3 in example 3, respectively, centrifuging at 20000g for 40min, pouring out the supernatant, ultrasonically dispersing with purified water, repeating the centrifugation and dispersion three times, and then diluting to 1mg/mL with purified water.
4.2 preparation of glucose standard solution:
a1 mg/mL glucose stock solution was prepared as a standard solution curve at 0mg/mL, 0.025mg/mL, 0.05mg/mL, 0.075mg/mL, 0.10mg/mL, 0.15mg/mL with purified water.
4.3 preparation of anthrone solution: the solution was made up to 2mg/mL with 80% sulfuric acid solution.
4.4 adding 0.1mL of glucose standard solution and sample to be detected with each concentration into the centrifuge tube, and adding 1mL of anthrone solution into each tube.
Incubate at 4.585 deg.C for 30 min.
4.6 centrifuge the sample reaction tube at 15000G for 40min, and the pipette tip sucks the clarified liquid from the bottom of the tube to measure the absorbance, avoiding sucking the suspended matter on the upper part.
4.7 Return to room temperature and measure the absorbance at 620 nm.
4.8 taking the concentration of the standard substance as the X value and the absorbance as the Y value, carrying out linear regression to obtain the absorbance value of the standard curve shown in the table 1, and detecting the sugar content of the sample to be detected.
The sugar content of the acceptor particles in example 1 and the donor particles in example 3 are shown in table 2 below.
TABLE 1
TABLE 2
Example 5 creatine kinase isoenzyme assay kit (light-activated chemiluminescence method)
The kit comprises a reagent 1 (containing CK-MB antibody I coated acceptor particles) prepared in example 1, a reagent 2 (containing biotin labeled CK-MB antibody II) prepared in example 2, creatine kinase isoenzyme series calibration products, creatine kinase isoenzyme quality control products and a reagent 3 (containing avidin coated donor particles) prepared in example 3, and the concentration of creatine kinase isoenzyme (CK-MB) in human serum and plasma samples is quantitatively detected by a double-antibody sandwich immuno-photoluminescence method under homogeneous conditions.
The following table 3 shows the main components of the creatine kinase isoenzyme detection kit (photoluminescence method) prepared in this example.
TABLE 3
EXAMPLE 6 determination of kit Properties
The performance of the kit of this example was tested using an LiCA500 automated light activated chemiluminescence detector developed by Boyang Biotechnology (Shanghai) Inc. The specific process steps of detecting the creatine kinase isozyme by the LiCA500 automatic light-activated chemiluminescence detector are as follows:
1. respectively adding 50 mu L of sample or calibrator and quality control material into the reaction hole;
2. adding 15 mu L of reagent 1 and 15 mu L of reagent 2 into the reaction hole in sequence;
3. shaking and incubating at 37 ℃ for 10 minutes;
4. automatically adding 150 mu L of reagent 3 (light-activated chemiluminescence analysis system general solution, LiCA general solution);
5. shaking and incubating at 37 ℃ for 2 minutes;
6. irradiating the micropores by laser and calculating the quantity of light photons emitted by each hole;
7. and calculating the concentration of the sample according to the calibration curve.
The method for judging the validity of the detection result comprises the following steps: the quality control product is detected and measured in a single hole in each batch of test, and the detection result is within the required quality control range, if the detection result exceeds the range, the test result is not credible, and the test is required to be re-detected and re-calibrated if necessary.
The kit detects 400 plasma samples of healthy people, wherein 200 women and 200 men respectively have corresponding 97.5 percent of human measured values: female: 3.5ng/mL, male: 4.8 ng/mL.
The results of evaluating the detection performance of the kit of this example are as follows:
blank limit: less than or equal to 0.1 ng/mL;
linearity: the linear range is 0.1-500 ng/mL, and the value of the linear correlation coefficient r is more than or equal to 0.9900;
accuracy: the recovery rate is in the range of 85-115 percent;
repeatability: the coefficient of variation (C.V) is less than or equal to 10 percent;
inter-batch difference: the inter-batch variation coefficient (C.V) is less than or equal to 15 percent.
EXAMPLE 7 determination of the sensitivity and the upper detection limit of the kit
The sensitivity point is defined as when the signal at concentration Cx is higher than the signal at twice the concentration of C0, i.e., RLU (Cx) >2RLU (C0), then the corresponding detection reagent sensitivity is Cx. The detection upper limit point is defined as the upper limit of the range determined using the method in the NCCLS EP-6 document.
(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, and 1500ng/ml, and the above concentration series of CK-MB antigen was detected using reagent 1 (comprising CK-MB antibody I-coupled acceptor particles at a concentration of 100. mu.g/ml) prepared in example 1, then reagent 2 (comprising the same biotin-labeled CK-MB antibody II diluted to 2ug/ml) prepared in example 2 and reagent 3 (comprising donor particles) prepared in example 3, respectively, and the detection sensitivity and detection upper limit were determined using a photo-excitation optical analytical system developed by Boyang Biotech (Shanghai) Limited As shown.
TABLE 4
As can be seen from Table 4, when the variation coefficient of the particle size distribution of the acceptor particles is not less than 5% and not more than 25%, the kit containing the acceptor particles has both appropriate sensitivity and 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 described above, and the sugar content in the acceptor particles was measured using the anthrone method given in example 4.
With reference 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 performed on an LiCA HT automated photo-activated chemiluminescence assay system developed by Boyang Biotechnology (Shanghai) Inc. and the assay results were outputted.
TABLE 5
As can be seen from Table 5, when the sugar content of the acceptor particle in the reagent 1 is not less than 20mg/g, the photo-activated 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 tested using the kit (photo-activated chemiluminescence) prepared in example 5. The CK-MB quantitative determination detection kit (light-activated chemiluminescence method) comprises the following components: a reagent 1 comprising a CK-MB antibody I (monoclonal antibody) -coated acceptor particle, a reagent 2 comprising a biotin-labeled CK-MB antibody II (monoclonal antibody), and a reagent 3 comprising a donor particle (photo-activated chemiluminescence analysis system-use solution). Wherein the concentration of the donor particles in the reagent 3 was 150ug/ml, the variation coefficient of particle size distribution C.V of the donor particles in the reagent R3 was 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, according to the anthrone assay given in example 4.
The detection process is completed on a full-automatic light-activated chemiluminescence analysis system (LiCA HT) developed by Boyang Biotechnology (Shanghai) Inc., and a detection result is output, and the specific detection steps comprise:
a. adding a clinical sample into the reaction hole;
b. sequentially adding a reagent 1 and a reagent 2 into the reaction hole;
c. incubation;
d. adding a reagent 3 into the reaction hole;
e. incubation;
f. irradiating the reaction holes with laser twice and calculating the quantity of light photons emitted by each hole;
g. and calculating the concentration of CK-MB in the sample to be detected.
When a CK-MB marker exists in a clinical sample, CK-MB specifically binds to a receptor particle coated with a CK-MB antibody I (monoclonal antibody) and a biotin-labeled CK-MB antibody II (monoclonal antibody) at the same time, and forms a double-antibody sandwich complex on the surface of the receptor particle; at this time, if a donor particle modified by streptavidin is added, biotin and streptavidin are combined to enable the two particles to approach each other, under the excitation of an excitation light source, the donor particle releases active oxygen, and chemiluminescence is generated after the donor particle touches an acceptor particle in a solution, so that 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 is present, the stronger the fluorescence intensity is, and the amount of CK-MB in the serum of the patient is quantitatively detected according to the intensity of luminescence, and the specific detection results are shown in the following Table 6:
TABLE 6
By comparison, the correlation between the roche measurement and the measurement of this example 8 is 0.9963, and the slope is 0.9632. Sample No. 1-23 is a patient for normal physical examination, the distribution range is 0.3ng/ml-4.13ng/ml, and the median value is 1.09 ng/ml; samples No. 24-40 identified patients with myocardial injury, with a distribution range of 4.55ng/ml to 260.48ng/ml, and a median of 20.06 g/ml.
Creatine Kinase (CK) is a dimeric enzyme that has four different forms: mitochondrial isozyme, cytoplasmic isozyme CK-MM (muscular type), creatine kinase brain isozyme CK-BB (brain type), creatine kinase isozyme (CK-MB), wherein the creatine kinase isozyme (CK-MB) is mainly located in cardiac muscle. The creatine kinase isozyme is 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 detected together with myoglobin and troponin to judge whether the myocardium is damaged, and is an index for researching and diagnosing the Acute Myocardial Infarction (AMI). The method is mainly used for auxiliary diagnosis of myocardial infarction clinically. The data according to embodiment 7 of the present invention can indicate that the use of the donor reagent of 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 the quantitative determination of the CKMB marker in the body fluid of the subject using the donor reagent of the present invention and the corresponding method can be used for the auxiliary diagnosis of myocardial infarction.
It should be noted that the above-mentioned embodiments are only for explaining the present invention, and do not constitute any limitation to the present invention. The present invention has been described with reference to exemplary embodiments, but the words which have been used herein are words of description and illustration, rather than words of limitation. The invention can be modified, as prescribed, within the scope of the claims and without departing from the scope and spirit of the invention. Although the invention has been described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, but rather extends to all other methods and applications having the same functionality.
Claims (18)
1. A homogeneous assay kit for creatine kinase isoenzyme, comprising a reagent 1 and a reagent 3, the reagent 1 comprising a first buffer solution and, suspended therein, receptor particles capable of reacting with reactive oxygen species to produce chemiluminescence which is bound to an antibody for creatine kinase isoenzyme capable of specifically binding to creatine kinase isoenzyme; reagent 3 comprising a second buffered solution and, suspended therein, donor particles that bind to one of the specific pair members, characterized in that:
the acceptor 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 a creatine kinase isoenzyme antibody, 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 comprises a second support having a sensitizer filled therein, the second support having one of the specific pair members bonded to a surface thereof; the donor particle has a sugar content of no more than 40mg per mg of the donor particle in reagent 3.
2. The kit of claim 1, wherein the receptor particles have a coefficient of variation of particle size distribution C.V value of no greater than 15% in reagent 1.
3. The kit according to claim 1 or 2, wherein the first carrier is coated with a polysaccharide, and the creatine kinase isoenzyme antibody is bound to the first carrier by bonding to a polysaccharide molecule.
4. A kit according to any one of claims 1 to 3, characterized in that the surface of the first support is coated with at least two successive 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 polysaccharide layer in the coating has a functional group, and each polysaccharide layer is 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 any one of claims 4-6, wherein the outermost polysaccharide layer of the coating has at least one pendant functional group that binds to an antibody to creatine kinase isoenzyme.
8. The kit according to any one of claims 4 to 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, thiol groups, amino groups, hydroxyl groups, and maleic groups; preferably selected from aldehyde groups and/or carboxyl groups.
9. Kit according to any one of claims 1 to 8, characterized in that the sugar content per litre of said first buffer solution is between 0.01 and 1g, preferably between 0.02 and 0.2 g.
10. Kit according to any one of claims 1 to 9, characterized in that the sugar content per milligram of said acceptor particles is not lower than 20 microgram, preferably not lower than 40 microgram.
11. Kit according to any one of claims 1 to 10, characterized in that the polysaccharide is selected from the group consisting of carbohydrates containing three or more unmodified or modified monosaccharide units; preferably at least one selected from the group consisting of dextran, starch, glycogen, inulin, fructan, 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.
12. The kit of any one of claims 1 to 11, wherein the donor particles have a variation coefficient of size distribution C.V value in reagent 3 of not less than 5% and not more than 25%.
13. The kit of any one of claims 1-12, wherein the kit further comprises 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 0-510 ng/mL.
14. The kit of claim 13, wherein the concentration of creatine kinase isoenzyme in the series of calibrator solutions comprises 0, 4, 10, 50, 200, and 510 ng/mL.
15. The kit of any one of claims 1-14, further comprising reagent 2, wherein reagent 2 comprises an antibody to creatine kinase isoenzyme linked to one of the members of the specific binding pair, wherein the antibody to creatine kinase isoenzyme is capable of specifically binding to creatine kinase isoenzyme.
16. The kit according to any one of claims 1 to 15, wherein the kit comprises at least 1 reagent strip, the reagent strip is provided with a plurality of reagent holes for containing reagents, and at least 3 reagent holes are respectively used for containing the reagent 1, the reagent 2 and the reagent 3.
17. Use of a kit according to any one of claims 1 to 16 in a chemiluminescent analyzer.
18. The application according to claim 17, characterized in that it comprises the following steps:
step S1, respectively adding a sample to be tested, a calibrator and/or a quality control product into the liquid containing device;
step S2, adding reagent 1, reagent 2 and reagent 3 into the liquid containing device;
and step S3, putting the liquid containing device on a chemiluminescence analyzer for reaction and detection.
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| CN113125700B (en) | 2023-08-08 |
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