Detection reagent and detection method for activity of isocitrate dehydrogenase

Abstract

The invention discloses a reagent for detecting the activity of isocitrate dehydrogenase. The reagent consists of a first reagent and a second reagent, the first reagent comprises an electron carrier, isocitric acid or isocitrate, divalent metal ions, a first reagent buffer solution, a first reagent additive, a first reagent preservative and a first reagent surfactant; the divalent metal ions comprise metal ions; the second reagent comprises a dye which comprises tetrazolium salt and has an oxidation state and a reduction state, a cofactor, a second reagent buffer solution, a second reagent additive, a coenzyme stabilizer, a second reagent surfactant and a second reagent preservative; the pH is buffered to 7-9.5 and 5-9 by the first buffer solution and the second buffer solution respectively; the first reagent additive and the second reagent additive comprise divalent metal salt, sugar, sugar alcohol, protein, chelating agent and basic amino acid; the first and second reagent surfactants comprise nonionic and anionic surfactants; the first reagent preservative and the second reagent preservative comprise sodium azide or isothiazolinones. The invention further discloses a corresponding detection method.

Description

Technical Field

[0001] The invention relates to the technical field of isocitrate dehydrogenase activity detection, and in particular to a detection reagent and a detection method for isocitrate dehydrogenase activity. Background Art

[0002] Isocitrate dehydrogenase (ICD or ICDH) is an important oxidoreductase with decarboxylation function in the body. It is divided into two categories in the human body. One category uses NAD+ as a coenzyme and is mainly involved in the tricarboxylic acid cycle and has nothing to do with clinical diagnosis; the other category uses NADP+ as a coenzyme and is mainly present in the liver, heart, skeletal muscle, kidney, adrenal gland, platelets and red blood cells; it is most abundant in the liver and exists in the mitochondria and cytoplasm of hepatocytes. Therefore, the detection of ICD activity can more accurately reflect the degree of hepatocyte lesions, especially necrotic changes, and provide an accurate basis for judging hepatocyte lesions and estimating the prognosis of patients with liver disease.

[0003] At present, the determination and detection methods include manual colorimetry, fixed time colorimetry, continuous monitoring colorimetry, spectrophotometry, etc. The detection principles can be divided into two types. One method is that isocitrate dehydrogenase catalyzes the conversion of isocitrate into α-ketoglutarate with the help of nicotinamide adenine dinucleotide phosphate (NADP) and Mn2+, while releasing carbon dioxide. The activity of isocitrate dehydrogenase can be determined by the amount of reduced nicotinamide adenine dinucleotide phosphate (NADPH) formed per unit time at a wavelength in the ultraviolet range (340 or 366nm). Another method is to couple α-ketoglutarate with phenylhydrazine to obtain a color complex that can be measured photometrically.

[0004] The disadvantage of the first method is that there is no visible color change of the reactant, and the enzyme activity cannot be judged from the color change. Its measurement requires an optical device that can measure the change in absorbance per unit time in the ultraviolet range. Moreover, the change in NADH in biological samples is usually small, especially when the activity of isocitrate dehydrogenase in the sample is small, it is easy to have reverse reactions and other reactions to produce negative changes. The disadvantage of the second method is that the reaction cannot be tracked kinetically, and the test operation is cumbersome, requiring multiple steps of reaction, and taking a lot of time and labor. Summary of the invention

[0005] The purpose of the present invention is to overcome the defects of the prior art and to address the problems in the related art. A detection reagent and a detection method for isocitrate dehydrogenase activity are provided, which convert a kinetic reaction that can only be measured in the ultraviolet range into a kinetic reaction that can be measured in the visible light range, and the isocitrate dehydrogenase activity can be accurately and conveniently determined. At the same time, the detection method catalyzes isocitrate into α-ketoglutarate and reduced coenzyme with the help of oxidized coenzyme and cofactor by isocitrate dehydrogenase (hereinafter referred to as ICD). Subsequently, the second enzyme in the detection method, such as diaphorase, reoxidizes the reduced coenzyme into oxidized coenzyme and reduces the tetrazolium salt into colored formazan. Since these reactions are carried out quantitatively and specifically, the formazan generated per unit time can be measured by colorimetry to achieve accurate measurement of ICD activity.

[0006] The first aspect of the present invention is to provide a detection reagent for isocitrate dehydrogenase activity, which is used for determining the isocitrate dehydrogenase activity in a biological sample and consists of a first reagent and a second reagent;

[0007] The first reagent comprises the following components: an electron carrier, isocitrate or isocitrate, a divalent metal ion, a first reagent buffer, a first reagent additive, a first reagent preservative and a first reagent surfactant; wherein:

[0008] The electron carrier includes diaphorase, phenazine methyl sulfate (PMS), 1-methoxyphenazine methyl sulfate (1-methoxy-PMS), 9-dimethylaminobenzo-a-phenazoline chloride and 2,6-dichlorophenol indophenol (DPIP); the divalent metal ions include metal ions, and the metal ions include magnesium ions, manganese ions, iron ions, nickel ions, zinc ions or cobalt ions; wherein the manganese ions and magnesium ions are natural ligands; the first reagent buffer is an alkaline buffer, including a buffer capable of buffering to a pH range of 7 to 9.5; the first reagent additive includes a divalent metal salt, a sugar, a sugar alcohol, a protein, a chelating agent and a basic amino acid; the first reagent preservative includes sodium azide or isothiazolinones; the first reagent surfactant includes a nonionic surfactant and an anionic surfactant;

[0009] The second reagent comprises the following components: a dye having an oxidized state and a reduced state, a cofactor, a second reagent buffer, a second reagent additive, a coenzyme stabilizer, a second reagent surfactant, and a second reagent preservative, wherein:

[0010] The dye having an oxidized state and a reduced state includes a tetrazolium salt, the type of the tetrazolium salt must be sufficient to be reduced to a colored formazan, and the amount of the tetrazolium salt must be sufficient to correlate the concentration of the formazan with the concentration of the analyte in the fluid sample; the cofactor includes β-nicotinamide adenine dinucleotide (NAD+, oxidized coenzyme I), β-nicotinamide adenine dinucleoside phosphate (NADP+, oxidized coenzyme II) or any salt thereof, any salt of which includes β-nicotinamide adenine dinucleotide disodium salt, β-nicotinamide adenine dinucleotide sodium salt or β-nicotinamide adenine dinucleotide lithium salt; the second buffer includes a buffer capable of buffering pH to a range of 5-9; the first reagent additive includes a divalent metal salt, a sugar, a sugar alcohol, a protein, a chelating agent and a basic amino acid; the second reagent surfactant includes a nonionic surfactant and an anionic surfactant; the second reagent preservative includes sodium azide or isothiazolinones.

[0011] Preferably, the divalent metal salt includes sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, sodium acetate or potassium acetate; the sugar includes sucrose and / or lactose; the sugar alcohol includes sorbitol and / or mannitol; the protein includes bovine serum albumin, human serum albumin and / or silk protein; the chelating agent includes EDTA and / or EGTA; the basic amino acid includes lysine, arginine and / or histidine.

[0012] Preferably, the tetrazolium salt includes 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT), iodonitrotetrazolium violet (INT), nitro blue tetrazolium chloride (NBT), tetranitro blue tetrazolium chloride (TNBT), tetrazolium blue (TB), tetrazolium violet (TV), thiocarbamoyl nitro blue tetrazolium chloride, triphenyl tetrazolium chloride, 2-(2'-benzothiazolyl)-5-phenylvinyl-3-(4'-phthaloylhydrazide) tetrazolium (BSPT), 2-benzothiazolyl-(2)-3,5-diphenyl tetrazolium (BTDP), 2,3-bis(4-nitrophenyl) tetrazolium (DNP), 2,5-diphenyl-3-(4-phenylvinylphenyl) tetrazolium (DPSP), distyryl nitro blue tetrazolium (DS-NBT), 2-phenyl-3-(4-carboxyphenyl)-5-methyl tetrazolium (PCPM), thiocarbamoyl nitro blue tetrazolium (TCNBT), water-soluble tetrazolium salts, such as 2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-phenyl-3H-tetrazolium-2',4'-disulfonic acid sodium salt (IPB), 2-benzothiazolyl-3-(4-carboxy-2-methoxyphenyl)-5-[4-(2-sulfoethylcarbamoyl)phenyl]-2H-tetrazolium (GLT004), 2,2'-dibenzothiazolyl-5,5'-bis[4-di(2-sulfoethyl)carbamoylphenyl]-3,3'-(3,3'-dimethoxy-4,4'-biphenylene)tetrazolyl disodium salt (WST-5) or 2-(2-methoxy-4-nitrobenzene)-3-(4-nitrobenzene)-5-(2,4-disulfonylphenyl)-2H-tetrazolyl monosodium salt (WST-8);

[0013] Preferably, the first reagent buffer and the second reagent buffer include a combination of one or more of tris(hydroxymethyl)aminomethane, tris(hydroxymethyl)methylglycine, 4-hydroxyethylpiperazinepropanesulfonic acid, 4-hydroxyethylpiperazineethanesulfonic acid, tris(hydroxymethyl)aminomethaneethanesulfonic acid, N,N-di(2-hydroxyethyl)-2-aminoethanesulfonic acid, 3-morpholinepropanesulfonic acid or 1,4-piperazinediethanesulfonic acid and bis(2-hydroxyethyl)amino(trihydroxymethyl)methane.

[0014] Preferably, the first reagent additive and the second reagent additive both include sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, sodium acetate or potassium acetate, sugar, sugar alcohol, protein, chelating agent and basic amino acid; the sugar includes sucrose and / or lactose; the sugar alcohol includes sorbitol and / or mannose; the protein includes bovine serum albumin, human serum albumin and / or silk protein; the chelating agent includes EDTA and / or EGTA; the basic amino acid includes lysine, arginine and / or histidine.

[0015] Preferably, the coenzyme stabilizer includes one or a combination of more of sericin, carboxylated chitosan, substrate stabilizer CCD, substrate stabilizer PPCD, stabilizer protector Phos-E, complex enzyme stabilizer AES, complex enzyme stabilizer DPD, complex enzyme stabilizer AFP, complex enzyme protector HP or protein protector TY.

[0016] Preferably, the nonionic surfactant includes one or more of polyethylene glycol monooctylphenyl ether (TritonX-100), sorbitan monolaurate (Span20), sorbitan monopalmitate (Span40), polyoxyethylene lauryl ether (Brij35), polyoxyethylene cetyl ester (Brij58), polyoxyethylene sorbitan monolaurate (Brij58), polyoxyethylene sorbitan monolaurate (Span40), Tween 20, Tween 40, Tween 80 and Tween 60; the anionic surfactant includes one or more of sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, sodium cholate, sodium deoxycholate and sodium lauryl sarcosine.

[0017] Preferably, the preservatives of the first reagent and the second reagent include ProClin150, ProClin200, ProClin300 or ProClin5000.

[0018] A second aspect of the present invention is to provide a method for detecting isocitrate dehydrogenase activity, which is implemented based on a detection reagent for isocitrate dehydrogenase activity and is used to perform an assay of an analyte from a fluid sample, comprising:

[0019] The detection reagent is provided in the form of a liquid reagent formed by adding an aqueous solution or in the form of a reagent film formed by adding a film-forming agent to contain the detection reagent in a film;

[0020] The detection method comprises: adding a fluid sample after appropriate treatment to the detection reagent provided in the form of a liquid reagent to react to form a solution medium; measuring the absorbance change of the formazan indicating the color in the solution medium by spectrophotometry; wherein the formazan is produced by the reduction of a tetrazolium salt under the assay conditions; or

[0021] After the fluid sample is properly treated, it is added to a reaction device combined with a reagent film for reaction; the reflectivity or transmittance of the formazan indicating color is measured in the film medium by spectrophotometry; the reaction device includes a test tube, a colorimetric dish, a colorimetric cup, an ELISA plate, a colorimetric card and any reaction device component suitable for the attachment of the reagent film.

[0022] Preferably, the step of appropriately treating the fluid sample comprises: diluting the fluid sample with a diluent;

[0023] The step of adding the fluid sample after appropriate treatment to a reaction device combined with a reagent membrane to perform a reaction comprises:

[0024] Adding a certain amount of the diluted fluid sample into the colorimetric well of the reaction device, and heating the reaction device to an appropriately selected temperature for reaction, wherein the appropriately selected temperature is 25° C.-37° C.;

[0025] The method of measuring the absorbance change of the color-indicating formazan in a solution medium by spectrophotometry comprises:

[0026] After the diluted fluid sample fully reacts with the detection reagent, a spectrophotometer or an automatic analysis instrument commonly used in clinical practice is selected to test the rate of increase of the absorbance value of the solution in the colorimetric well; the time for the full reaction is 5-10 minutes; the wavelength of the light source generated by the spectrophotometer is 530-760nm.

[0027] Beneficial effects of the detection reagent and detection method of the present invention:

[0028] By providing accurate determination of isocitrate dehydrogenase activity in the visible light range without the use of expensive optical auxiliary tools, the application field of ICD activity determination can be significantly broadened, especially in the medical field, and is suitable for clinical promotion and use; the detection instrument required for this method is relatively simple in principle and low in cost, and the present invention is suitable for disease screening tests in epidemiological field surveys and mobile and fixed medical diagnosis; the testing process of the present invention is simple and fast, and patients can determine their plasma isocitrate dehydrogenase activity more quickly, which is essential for the diagnosis of hepatitis and other dangerous diseases; the present invention can also provide an important supplement for the dynamic and continuous monitoring of many diseases and medical diagnosis in small laboratories. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the related technologies, the drawings required for use in the specific embodiments or the related technical descriptions will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 This is a schematic diagram of the determination capability of the detection method provided according to an embodiment of the present invention for isocitrate dehydrogenase, wherein the ordinate is the change in absorbance at 605 nm per minute, and the abscissa is the concentration of isocitrate dehydrogenase;

[0031] Figure 2This is a schematic diagram of the determination capability of the detection method provided according to an embodiment of the present invention for isocitrate dehydrogenase, wherein the ordinate is the change in absorbance per minute at 450 nm, and the abscissa is the concentration of isocitrate dehydrogenase;

[0032] Figure 3 The schematic diagram of the clinical correlation analysis results of the isocitrate dehydrogenase assay method proposed by the present invention is shown in FIG. 1 , with the values ​​measured by the reference method provided by the embodiment of the present invention as the ordinate and the values ​​measured by the detection method proposed by the present invention as the abscissa. DETAILED DESCRIPTION

[0033] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", and "third" are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance.

[0035] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0036] This embodiment provides a detection reagent for isocitrate dehydrogenase activity, which is used for determining the isocitrate dehydrogenase activity in a biological sample, and is composed of a first reagent and a second reagent;

[0037] The first reagent comprises the following components: an electron carrier, isocitrate or isocitrate, a divalent metal ion, a buffer, an additive, a preservative and a surfactant; wherein,

[0038] The electron carriers include diaphorase, phenazine methosulfate (PMS), 1-methoxyphenazine methosulfate (1-methoxy-PMS), 9-dimethylaminobenzo-a-phenazoline chloride, and 2,6-dichlorophenol indophenol (DPIP);

[0039] The divalent metal ions include magnesium ions, manganese ions, iron ions, nickel ions, zinc ions, cobalt ions and other metal ions. The detection of isocitrate dehydrogenase activity requires the participation of divalent metal ions. The ion radius determines the reaction efficiency. Manganese ions and magnesium ions are natural ligands. The most preferred embodiment of the present invention is to use magnesium ions as divalent metal ions.

[0040] The buffer is an alkaline buffer, including a buffer capable of buffering to a pH in the range of 7 to 9.5, such as tris(hydroxymethyl)aminomethane, tris(hydroxymethyl)methylglycine, 4-hydroxyethylpiperazinepropanesulfonic acid, 4-hydroxyethylpiperazineethanesulfonic acid, tris(hydroxymethyl)aminomethaneethanesulfonic acid, N,N-di(2-hydroxyethyl)-2-aminoethanesulfonic acid, 3-morpholinepropanesulfonic acid or 1,4-piperazinediethanesulfonic acid and bis(2-hydroxyethyl)amino(tris(hydroxymethyl)methane);

[0041] The additives include divalent metal salts such as chlorides, sulfates and acetates of sodium and potassium, sugars such as sucrose and lactose, sugar alcohols such as sorbitol and mannitol, proteins such as bovine serum albumin, human serum albumin, silk protein, chelating agents such as EDTA and EGTA, and basic amino acids such as lysine, arginine, and histidine;

[0042] The preservatives include sodium azide or isothiazolinones, such as ProClin150, ProClin200, ProClin300 or ProClin5000;

[0043] The surfactant includes a nonionic surfactant and an anionic surfactant; the nonionic surfactant includes polyethylene glycol monooctylphenyl ether (TritonX-100), sorbitan monolaurate (Span20), sorbitan monopalmitate (Span40), polyoxyethylene lauryl ether (Brij35), polyoxyethylene cetyl ester (Brij58), polyoxyethylene sorbitan monolaurate (Brij58), polyoxyethylene sorbitan monolaurate (Span40), Tween 20, Tween 40, Tween 80 or Tween 60; the anionic surfactant includes sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, sodium cholate, sodium deoxycholate or sodium lauryl sarcosine.

[0044] Application Example: In a preferred embodiment of the present invention, the reagent contains 1-1000U / ml, preferably 10-200U / ml diaphorase; 0.1-200mmol / L, preferably 1-100mmol / L DL isocitrate trisodium salt; preferably 0.5-100mmol / L divalent magnesium ion; 0.0001 to 10wt%, preferably 0.001 to 1wt% surfactant; 0.1-30wt% additive; 0.0001 to 10wt%, preferably 0.001 to 1wt% preservative; 1-100mmol / L buffer to adjust pH to 7.0-10.0. Particularly preferably, the buffer concentration is 10-500mmol / L, and the pH is adjusted to the range of 7.5-9.5.

[0045] The second reagent comprises the following components: a dye having an oxidized state and a reduced state, a cofactor, a buffer, an additive, a coenzyme stabilizer, a surfactant, and a preservative; wherein,

[0046] The dye having an oxidized state and a reduced state includes a tetrazolium salt, the type of the tetrazolium salt must be sufficient to be reduced to a colored formazan, and the amount of the tetrazolium salt must be sufficient to correlate the concentration of the formazan with the concentration of the analyte in the fluid sample; the tetrazolium salt includes 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT), p-iodonitrotetrazolium violet (INT), nitro tetrazolium blue chloride (NBT), tetranitro blue tetrazolium chloride (TNBT), tetrazolium Blue (TB), tetrazolium violet (TV), thiocarbamoyl nitro blue tetrazolium chloride, triphenyl tetrazolium chloride, 2-(2'-benzothiazolyl)-5-phenylvinyl-3-(4'-phthaloyl) tetrazolium (BSPT), 2-benzothiazolyl-(2)-3,5-diphenyl tetrazolium (BTDP), 2,3-di(4-nitrophenyl) tetrazolium (DNP), 2,5-diphenyl-3-(4-phenylvinyl) tetrazolium (DPSP), diphenylethylene 2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-phenyl-3H-tetrazolium-2',4'-disulfonic acid sodium salt (IPB), 2-benzothiazolyl-3-(4-carboxy-2-methoxyphenyl)-5-[4-(2-sulfonic acid)]-tetrazolyl blue tetrazolium (DS-NBT), 2-phenyl-3-(4-carboxyphenyl)-5-methyltetrazolium (PCPM), thiocarbamoyl nitro blue tetrazolium (TCNBT), water-soluble tetrazolium salts, such as ...

[0045] , 2,2'-dibenzothiazolyl-5,5'-bis[4-di(2-sulfoethyl)carbamoylphenyl]-3,3'-(3,3'-dimethoxy-4,4'-biphenylene)tetrazolyl disodium salt (WST-5) or 2-(2-methoxy-4-nitrobenzene)-3-(4-nitrobenzene)-5-(2,4-disulfonylphenyl)-2H-tetrazolium monosodium salt (WST-8).

[0047] The cofactor includes β-nicotinamide adenine dinucleotide (NAD+, oxidized coenzyme I), β-nicotinamide adenine dinucleoside phosphate (NADP+, oxidized coenzyme II) or any salt thereof, any salt including β-nicotinamide adenine dinucleotide phosphate disodium salt, β-nicotinamide adenine dinucleotide sodium salt or β-nicotinamide adenine dinucleotide lithium salt.

[0048] The buffer includes a buffer capable of buffering pH to a range of 5-9, and the buffer includes tris(hydroxymethyl)aminomethane, tris(hydroxymethyl)methylglycine, 4-hydroxyethylpiperazinepropanesulfonic acid, 4-hydroxyethylpiperazineethanesulfonic acid, tris(hydroxymethyl)aminomethaneethanesulfonic acid, N,N-di(2-hydroxyethyl)-2-aminoethanesulfonic acid, 3-morpholinepropanesulfonic acid, 2-morpholineethanesulfonic acid, 1,4-piperazinediethanesulfonic acid or bis(2-hydroxyethyl)amino(tris(hydroxymethyl)methane.

[0049] The additives include sodium chloride, potassium chloride, divalent metal salts of sulfate and acetate, sugar, sugar alcohol, protein, chelating agent and basic amino acid; the sugar includes sucrose or lactose; the sugar alcohol includes sorbitol or mannitol; the protein includes bovine serum albumin, human serum albumin or silk protein; the chelating agent includes EDTA or EGTA; the basic amino acid includes lysine, arginine or histidine;

[0050] The coenzyme stabilizer includes one or more of sericin, carboxylated chitosan, substrate stabilizer CCD, substrate stabilizer PPCD, stabilizer protector Phos-E, complex enzyme stabilizer AES, complex enzyme stabilizer DPD, complex enzyme stabilizer AFP, complex enzyme protector HP or protein protector TY;

[0051] The surfactant includes a nonionic surfactant and an anionic surfactant; the nonionic surfactant includes a combination of one or more of polyethylene glycol monooctylphenyl ether (TritonX-100), sorbitol monolaurate (Span20), sorbitan monopalmitate (Span40), polyoxyethylene lauryl ether (Brij35), polyoxyethylene cetyl ester (Brij58), polyoxyethylene sorbitol monolaurate (Brij58), polyoxyethylene sorbitol monolaurate (Span40), Tween 20, Tween 40, Tween 80 and Tween 60; the anionic surfactant includes a combination of one or more of sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, sodium cholate, sodium deoxycholate and sodium lauryl sarcosine. These surfactants can prevent the deposition and contamination of the generated formazan compounds, and can achieve the improvement of color development stability and the enhancement of coloring.

[0052] The preservatives include sodium azide or isothiazolinones, such as ProClin 150, ProClin 200, ProClin 300 or ProClin 5000.

[0053] Application example: In a preferred embodiment of the present invention, the reagent contains 0.01-20mmol / L, preferably 0.1-5mmol / L of tetrazolium salt; preferably 0.5-200mmol / L of β-phosphonicotinamide adenine dinucleotide disodium salt; 0.001 to 30wt%, preferably 0.01 to 10wt% of coenzyme stabilizer; 0.0001 to 10wt%, preferably 0.001 to 1wt% of surfactant; 0.1-30wt% of additive; 0.0001 to 10wt%, preferably 0.001 to 1wt% of preservative; 1-100mmol / L buffer to adjust pH to 5.0-10.0. It is particularly preferred that the buffer concentration is 10-500mmol / L, and the pH is adjusted to the range of 6.0-8.0.

[0054] A second aspect of the present invention is to provide a method for detecting isocitrate dehydrogenase activity, which is implemented based on a detection reagent for isocitrate dehydrogenase activity and is used to perform an assay of an analyte from a fluid sample, comprising:

[0055] The detection reagent is provided in the form of a liquid reagent formed by adding an aqueous solution or in the form of a reagent film formed by adding a film-forming agent to contain the detection reagent in a film;

[0056] The detection method comprises: adding a fluid sample after appropriate treatment to the detection reagent provided in the form of a liquid reagent to react to form a solution medium; measuring the absorbance change of the formazan indicating the color in the solution medium by spectrophotometry; wherein the formazan is produced by the reduction of a tetrazolium salt under the assay conditions; or

[0057] After the fluid sample is properly treated, it is added to a reaction device combined with a reagent film for reaction; the reflectivity or transmittance of the formazan indicating color is measured in the film medium by spectrophotometry; the reaction device includes a test tube, a colorimetric dish, a colorimetric cup, an ELISA plate, a colorimetric card and any reaction device component suitable for the attachment of the reagent film.

[0058] As a preferred embodiment, the step of appropriately processing the fluid sample comprises: diluting the fluid sample with a diluent.

[0059] As a preferred embodiment, the process of adding the fluid sample after appropriate treatment to a reaction device combined with a reagent membrane for reaction comprises:

[0060] A certain amount of the diluted fluid sample is added to the colorimetric well of the reaction device, and the reaction device is heated to an appropriately selected temperature for reaction, wherein the appropriately selected temperature is 25° C.-37° C.

[0061] As a preferred embodiment, the measuring of the absorbance change of the color-indicating formazan in a solution medium by spectrophotometry comprises:

[0062] After the diluted fluid sample fully reacts with the detection reagent, a spectrophotometer or an automatic analysis instrument commonly used in clinical practice is selected to test the rate of increase of the absorbance value of the solution in the colorimetric well; the time for the full reaction is 5-10 minutes, and the wavelength of the light source generated by the spectrophotometer is 530-760nm, preferably 605nm.

[0063] Application examples and verification:

[0064] Embodiment 1:

[0065] The isocitrate dehydrogenase activity assay reagent of this example includes the following components, and the assay reagent 1 (reagent 1 formula in Table 1) and reagent 2 (reagent 2 formula in Table 1) having the following compositions are prepared.

[0066] Table 1

[0067]

[0068]

[0069] Preparation of standard products:

[0070] Prepare multiple isocitrate dehydrogenase standards with different concentration gradients for constructing a standard curve. For example, more than 5 isocitrate dehydrogenase standards with a concentration range of 1U / L-100U / L can be prepared. In one example, the lyophilized isocitrate dehydrogenase standard [concentration: (100.0±20.0)U / L] is dissolved in double distilled water and then diluted to 100, 50, 25, 12.5, 6.25, 3.13, 1.57, and 0.79U / L with double distilled water to construct a quantitative standard curve. Commercial quality control substances can be selected for isocitrate dehydrogenase standards.

[0071] Measurement method: 10 μl of a standard sample with known isocitrate dehydrogenase activity was added to 60 μl of reagent 1, and the mixture was preheated to 37° C. Then, 30 μl of reagent 2 was added, and the absorbance change at 605 nm was measured per minute at 37° C.

[0072] Test results: Figure 1 As shown in the figure, the relationship between the isocitrate dehydrogenase activity and the absorbance change rate is as shown in the figure. When the isocitrate dehydrogenase activity is in the range of 0.8-100 U / L, the absorbance change per minute is linearly related to the isocitrate dehydrogenase activity.

[0073] Embodiment 2:

[0074] To demonstrate the applicability of WST-8 as a chromogenic substrate, isocitrate dehydrogenase activity level assay was performed in the same manner as in Example 1, except that the following reagent solutions were used.

[0075] The differences between this embodiment and embodiment 1 are shown in Table 2 below:

[0076] Table 2

[0077]

[0078]

[0079] The wavelength of the light source generated by the spectrophotometer used in the test is 450nm, and the results obtained are as follows Figure 2 As shown,

[0080] The relationship between the isocitrate dehydrogenase concentration and the absorbance change rate is shown in the figure. When the isocitrate dehydrogenase concentration is in the range of 0.8U / L-100U / L, the absorbance change per minute is linearly related to the isocitrate dehydrogenase concentration.

[0081] Example 3

[0082] Precision test: The test method provided in Example 1 of the present invention is used for sample detection, and a lyophilized reconstituted solution containing 5.0-10.0U / L isocitrate dehydrogenase is used as a low-concentration quality control; a lyophilized reconstituted solution containing 80.0-120.0U / L isocitrate dehydrogenase is used as a high-concentration quality control, and the low-concentration quality control with a known isocitrate dehydrogenase concentration of 6.8U / L and the high-concentration quality control with a concentration of 98.5U / L are tested 20 times each. Commercial quality control substances can be selected for isocitrate dehydrogenase quality control products.

[0083]

[0084] Where Xi is the concentration value measured for the i-th time, and n is the number of tests.

[0085] According to the above formula, the low-concentration quality control product with isocitrate dehydrogenase concentration of 6.8U / L and the high-concentration quality control product with isocitrate dehydrogenase concentration of 98.5U / L were tested 35 times, and the average value of the measured value was The results show that the isocitrate dehydrogenase activity determination method provided by the present invention has sufficient precision.

[0086] Example 4

[0087] Clinical relevance analysis:

[0088] The isocitrate dehydrogenase activity determination method provided in Example 1 of the present invention was used as the experimental group for sample detection, and the isocitrate dehydrogenase content of 30 serum samples of normal people and people with abnormal liver function was determined. In addition, the isocitrate dehydrogenase content of the same 30 serum samples was simultaneously determined using the existing clinical isocitrate dehydrogenase determination kit (NAFP+oxidoreductase method) as a reference method. The corresponding isocitrate dehydrogenase content (unit U / L) is as follows Figure 3 As shown, the ordinate value is the isocitrate dehydrogenase content in the serum sample measured by the reference method, and the abscissa value is the isocitrate dehydrogenase content in the serum sample measured by the method provided in Example 1 of the present invention.

[0089] The linear regression equation between the two groups of test results of the isocitrate dehydrogenase activity determination method provided by the embodiment of the present invention and the reference method is as follows:

[0090] y=1.0025x-0.4855;

[0091] Correlation coefficient R 2 =0.9972, the closer the correlation coefficient is to 1, the stronger the correlation between the two sets of data. Therefore, the isocitrate dehydrogenase activity determination method provided by the embodiment of the present invention has a strong correlation with the test results of the reference method.

[0092] Through the description of the above implementation methods, those skilled in the art can clearly understand that the above embodiments can be implemented by software, or by means of software plus necessary general hardware platforms. Based on such understanding, the technical solutions of the above embodiments can be embodied in the form of software products, which can be stored in a non-volatile storage medium (which can be a CD-ROM, a USB flash drive, a mobile hard disk, etc.), including several instructions for enabling a computer device (which can be a personal computer, a server, or a network device, etc.) to execute the methods described in the various embodiments of the present invention.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A detection reagent for isocitrate dehydrogenase activity, characterized in that: Used for determining the activity of isocitrate dehydrogenase in biological samples, comprising a first reagent and a second reagent; The first reagent comprises the following components: an electron carrier, isocitrate or isocitrate, a divalent metal ion, a first reagent buffer, a first reagent additive, a first reagent preservative and a first reagent surfactant; wherein: The electron carrier includes diaphorase, phenazine methyl sulfate (PMS), 1-methoxyphenazine methyl sulfate (1-methoxy-PMS), 9-dimethylaminobenzo-a-phenazoline chloride and 2,6-dichlorophenol indophenol (DPIP); the divalent metal ions include metal ions, and the metal ions include magnesium ions, manganese ions, iron ions, nickel ions, zinc ions or cobalt ions; wherein the manganese ions and magnesium ions are natural ligands; the first reagent buffer is an alkaline buffer, including a buffer capable of buffering pH to a range of 7-9.5; the first reagent additive includes a divalent metal salt, a sugar, a sugar alcohol, a protein, a chelating agent and a basic amino acid; the first reagent preservative includes sodium azide or isothiazolinones; the first reagent surfactant includes a nonionic surfactant and an anionic surfactant; The second reagent comprises the following components: a dye having an oxidized state and a reduced state, a cofactor, a second reagent buffer, a second reagent additive, a coenzyme stabilizer, a second reagent surfactant, and a second reagent preservative, wherein: The dye having an oxidized state and a reduced state includes a tetrazolium salt, the type of the tetrazolium salt must be sufficient to be reduced to a colored formazan, and the amount of the tetrazolium salt must be sufficient to correlate the concentration of the formazan with the concentration of the analyte in the fluid sample; the cofactor includes β-nicotinamide adenine dinucleotide (NAD+, oxidized coenzyme I), β-nicotinamide adenine dinucleoside phosphate (NADP+, oxidized coenzyme II) or any salt thereof, any salt of which includes β-nicotinamide adenine dinucleotide disodium salt, β-nicotinamide adenine dinucleotide sodium salt or β-nicotinamide adenine dinucleotide lithium salt; the second buffer includes a buffer capable of buffering pH to a range of 5-9; the first reagent additive includes a divalent metal salt, a sugar, a sugar alcohol, a protein, a chelating agent and a basic amino acid; the second reagent surfactant includes a nonionic surfactant and an anionic surfactant; the second reagent preservative includes sodium azide or isothiazolinones.

2. A detection reagent for isocitrate dehydrogenase activity according to claim 1, characterized in that: The divalent metal salt includes sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, sodium acetate or potassium acetate; the sugar includes sucrose and / or lactose; the sugar alcohol includes sorbitol and / or mannitol; the protein includes bovine serum albumin, human serum albumin and / or silk protein; the chelating agent includes EDTA and / or EGTA; the basic amino acid includes lysine, arginine and / or histidine.

3. A detection reagent for isocitrate dehydrogenase activity according to claim 2, characterized in that, The tetrazolium salts include 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT), iodonitrotetrazolium violet (INT), nitro blue tetrazolium chloride (NBT), tetranitro blue tetrazolium chloride (TNBT), tetrazolium blue (TB), tetrazolium violet (TV), thiocarbamoyl nitro blue tetrazolium chloride, triphenyl tetrazolium chloride, 2-(2'-benzothiazolyl)-5-phenylvinyl-3-(4'-phthaloylhydrazide) tetrazolium (BSPT), 2-benzothiazolyl-(2)-3,5-diphenyl tetrazolium (BTDP), 2,3-bis(4-nitrophenyl) tetrazolium (DNP), 2,5-diphenyl-3-(4-phenylvinylphenyl) tetrazolium (DPSP), distyryl nitro blue tetrazolium (DS-NBT), 2-phenyl-3-(4-carboxyphenyl)-5-methyl tetrazolium (PCPM), thiocarbamoyl nitro blue tetrazolium (TCNBT), water-soluble tetrazolium salts, such as 2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-phenyl-3H-tetrazolium-2',4'-disulfonic acid sodium salt (IPB), 2-benzothiazolyl-3-(4-carboxy-2-methoxyphenyl)-5-[4-(2-sulfoethylcarbamoyl)phenyl]-2H-tetrazolium ( GLT004), 2,2'-dibenzothiazolyl-5,5'-bis[4-di(2-sulfoethyl)carbamoylphenyl]-3,3'-(3,3'-dimethoxy-4,4'-biphenylene)tetrazolyl disodium salt (WST-5) or 2-(2-methoxy-4-nitrobenzene)-3-(4-nitrobenzene)-5-(2,4-disulfonylphenyl)-2H-tetrazolium monosodium salt (WST-8).

4. A detection reagent for isocitrate dehydrogenase activity according to claim 3, characterized in that: The first reagent buffer and the second reagent buffer include a combination of one or more of tris(hydroxymethyl)aminomethane, tris(hydroxymethyl)methylglycine, 4-hydroxyethylpiperazinepropanesulfonic acid, 4-hydroxyethylpiperazineethanesulfonic acid, tris(hydroxymethyl)aminomethaneethanesulfonic acid, N,N-di(2-hydroxyethyl)-2-aminoethanesulfonic acid, 3-morpholinepropanesulfonic acid or 1,4-piperazinediethanesulfonic acid and bis(2-hydroxyethyl)amino(tris(hydroxymethyl)methane).

5. A detection reagent for isocitrate dehydrogenase activity according to claim 4, characterized in that: The first reagent additive and the second reagent additive both include sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, sodium acetate or potassium acetate, sugar, sugar alcohol, protein, chelating agent and basic amino acid; the sugar includes sucrose and / or lactose; the sugar alcohol includes sorbitol and / or mannose; the protein includes bovine serum albumin, human serum albumin and / or silk protein; the chelating agent includes EDTA and / or EGTA; the basic amino acid includes lysine, arginine and / or histidine.

6. A detection reagent for isocitrate dehydrogenase activity according to claim 5, characterized in that: The coenzyme stabilizer includes one or a combination of sericin, carboxylated chitosan, substrate stabilizer CCD, substrate stabilizer PPCD, stabilizer protector Phos-E, complex enzyme stabilizer AES, complex enzyme stabilizer DPD, complex enzyme stabilizer AFP, complex enzyme protector HP or protein protector TY.

7. A detection reagent for isocitrate dehydrogenase activity according to claim 6, characterized in that: The nonionic surfactant includes one or more combinations of polyethylene glycol monooctylphenyl ether (TritonX-100), sorbitan monolaurate (Span20), sorbitan monopalmitate (Span40), polyoxyethylene lauryl ether (Brij35), polyoxyethylene cetyl ester (Brij58), polyoxyethylene sorbitan monolaurate (Brij58), polyoxyethylene sorbitan monolaurate (Span40), Tween 20, Tween 40, Tween 80 and Tween 60; the anionic surfactant includes one or more combinations of sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, sodium cholate, sodium deoxycholate and sodium lauryl sarcosine.

8. A detection reagent for isocitrate dehydrogenase activity according to claim 7, characterized in that: The preservatives of the first agent and the second agent include ProClin150, ProClin200, ProClin300 or ProClin5000.

9. A method for detecting isocitrate dehydrogenase activity, implemented based on the detection reagent for isocitrate dehydrogenase activity according to any one of claims 1 to 8, for performing an assay of an analyte from a fluid sample, characterized in that: include: The detection reagent is provided in the form of a liquid reagent formed by adding an aqueous solution or in the form of a reagent film formed by adding a film-forming agent to contain the detection reagent in a film; The detection method comprises: adding a fluid sample after appropriate treatment to the detection reagent provided in the form of a liquid reagent to react to form a solution medium; measuring the absorbance change of the formazan indicating the color in the solution medium by spectrophotometry; wherein the formazan is produced by the reduction of a tetrazolium salt under the assay conditions; or After the fluid sample is properly treated, it is added to a reaction device combined with a reagent film for reaction; the reflectivity or transmittance of the formazan indicating color is measured in the film medium by spectrophotometry; the reaction device includes a test tube, a colorimetric dish, a colorimetric cup, an ELISA plate, a colorimetric card and any reaction device component suitable for the attachment of the reagent film.

10. A method for detecting isocitrate dehydrogenase activity according to claim 9, characterized in that: The subjecting the fluid sample to appropriate treatment includes: diluting the fluid sample with a diluent; The step of adding the fluid sample after appropriate treatment to a reaction device combined with a reagent membrane to perform a reaction comprises: Adding a certain amount of the diluted fluid sample into the colorimetric well of the reaction device, and heating the reaction device to an appropriately selected temperature for reaction, wherein the appropriately selected temperature is 25° C.-37° C.; The method of measuring the absorbance change of the color-indicating formazan in a solution medium by spectrophotometry comprises: After the diluted fluid sample fully reacts with the detection reagent, a spectrophotometer or an automatic analysis instrument commonly used in clinical practice is selected to test the rate of increase of the absorbance value of the solution in the colorimetric well; the time for the full reaction is 5-10 minutes; the wavelength of the light source generated by the spectrophotometer is 530-760nm.

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