A kind of aspartate aminotransferase mitochondrial isozyme detection kit and preparation method thereof

Abstract

The present invention discloses a kit for detecting mitochondrial isozymes of aspartate aminotransferase and a preparation method thereof. The detection kit comprises a cytoplasmic aspartate aminotransferase-specific adsorption membrane, a first reagent, a second reagent and a third reagent. The structure of the cytoplasmic aspartate aminotransferase-specific adsorption membrane is that acrylic gel-type anion exchange resin particles are sandwiched in the middle region of two layers of cellulose acetate membranes. The first reagent comprises an alkaline reagent. The present invention removes c-AST in a sample to be tested by adding a cytoplasmic aspartate aminotransferase-specific adsorption membrane to the kit, eliminates interference in determination, and simultaneously removes L-aspartic acid and α-ketoglutaric acid originally present in the sample to be tested, thereby reducing background value and improving detection accuracy.

Description

Technical Field

[0001] The present invention relates to the technical field of clinical medical parameter detection, and in particular to a kit for detecting mitochondrial isozymes of aspartate aminotransferase and a preparation method thereof. Background Art

[0002] Aspartate aminotransferase (AST) is widely present in the heart, liver, lung and other tissue cells. It has two isoenzymes, one is present in the cell cytoplasm, called cytoplasmic aspartate aminotransferase (c-AST), and the other is present in the mitochondria, the mitochondrial isoenzyme of aspartate aminotransferase (m-AST). The change of AST concentration has important clinical diagnostic significance, among which m-AST can objectively reflect the severity of some diseases and can serve as an important reference for guiding treatment and prognosis of some diseases.

[0003] At present, the main methods for detecting m-AST include electrophoresis, ion exchange, enzyme immunoassay, radioimmunoassay, etc. Electrophoresis requires a complex staining process and is not suitable for clinical testing of a large number of samples; ion exchange requires the use of chromatography columns and cannot achieve the purpose of rapid detection; enzyme immunoassay has a low degree of automation and consumes a lot of manpower; radioimmunoassay is radioactive and is not environmentally friendly.

[0004] The enzyme kinetics method of m-AST is to infer the enzyme activity or concentration of m-AST based on the speed of the chemical reaction catalyzed by m-AST. It has the advantages of automated operation, large sample throughput, and short detection time. The basic principle of detection is: m-AST catalyzes L-aspartate and α-ketoglutarate to produce oxaloacetate and L-glutamate, and oxaloacetate is reduced to L-malate by malate dehydrogenase (MDH). At the same time, reduced coenzyme I (NADH) is oxidized to oxidized coenzyme I (NAD+), thereby reducing the absorbance at 340nm. By monitoring the rate of decrease in absorbance at 340nm, the activity of m-AST in the sample can be calculated. However, it is easily interfered by c-AST, resulting in a large deviation in the detection results. Therefore, it is necessary to develop a detection kit and its preparation method that can eliminate c-AST interference and accurately determine the concentration of m-AST. Summary of the invention

[0005] In view of this, the present invention provides a kit for detecting mitochondrial isozymes of aspartate aminotransferase and a preparation method thereof, wherein a cytoplasmic aspartate aminotransferase-specific adsorption membrane is added to the kit to remove c-AST in the sample to be tested, thereby eliminating measurement interference, and simultaneously removing L-aspartic acid and α-ketoglutaric acid originally present in the sample to be tested, thereby reducing the background value and improving the detection accuracy.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a kit for detecting mitochondrial isozymes of aspartate aminotransferase, the kit comprising a cytoplasmic aspartate aminotransferase-specific adsorption membrane, a first reagent, a second reagent and a third reagent;

[0008] The structure of the cytoplasmic aspartate aminotransferase specific adsorption membrane is as follows: acrylic gel-type anion exchange resin particles are sandwiched between two layers of cellulose acetate membranes;

[0009] The middle area of ​​the two layers of cellulose acetate film refers to the middle area of ​​the film except the area 2-5 mm away from the edge of the film;

[0010] The cellulose acetate membrane is a cellulose acetate membrane loaded with a cytoplasmic aspartate aminotransferase-specific monoclonal antibody;

[0011] The cytoplasmic aspartate aminotransferase-specific monoclonal antibody is composed of a first specific monoclonal antibody and a second monoclonal antibody in a molar ratio of 1:2-2:1, the antigenic determinant amino acid sequence corresponding to the first specific monoclonal antibody is LAN HLE IYG LLE FAS NAM RLA, and the antigenic determinant amino acid sequence corresponding to the second specific monoclonal antibody is KEI ASN MKH RQL FPF FGS;

[0012] The first reagent includes a first buffer solution 30-110 mmol / L, potassium hydroxide 0.5-0.6 mmol / L, glycerol 0.3-1.2 g / L, polyethylene glycol 0.1-0.9 g / L, ethanolamine 0.1-2 mmol / L, reduced coenzyme I NADH 200-270 mmol / L, a first antibacterial agent 30 mg / L-69 mg / L, and an antioxidant 140-310 mg / L;

[0013] The second reagent includes a second buffer solution 60-90 mmol / L, malate dehydrogenase 3-6 kU / L, a surfactant 20-67 mmol / L, a second antibacterial agent 50 mg / L-90 mg / L, and a protein stabilizer 40-70 mmol / L;

[0014] The third reagent includes a third buffer solution 50-100 mmol / L, L-aspartic acid 30-50 mmol / L, α-ketoglutaric acid 30-50 mmol / L, and a third antibacterial agent 40 mg / L-80 mg / L.

[0015] Preferably, the pore size of the cellulose acetate membrane is 0.45 micrometers.

[0016] Preferably, the acrylic gel-type anion exchange resin includes a weakly basic anion exchange resin.

[0017] Preferably, the number of layers of the acrylic acid gel-type anion exchange resin particles is 1-3, and the particle size of the acrylic acid gel-type anion exchange resin particles is 0.9-1.3 μm.

[0018] Preferably, the first antibacterial agent and the third antibacterial agent include gentamicin, the second antibacterial agent includes sodium azide, the first buffer and the third buffer include phosphate buffer, the second buffer is Tris buffer, the surfactant includes at least one of lauroyl diethanolamine, Tween 60, and Tween 80, the antioxidant includes vitamin C, and the protein stabilizer includes at least one of alkyl glycoside and oligohalogen trehalose.

[0019] Preferably, the aspartate aminotransferase mitochondrial isozyme detection kit also includes a membrane washing reagent, which includes a first buffer solution 30-110 mmol / L, potassium hydroxide 0.06-0.09 mmol / L, glycerol 0.3-1.2 g / L, polyethylene glycol 0.1-0.9 g / L, a first antibacterial agent 30 mg / L-69 mg / L, and an antioxidant 140-310 mg / L.

[0020] In a second aspect, the present invention further provides a method for preparing the aspartate aminotransferase mitochondrial isozyme detection kit as described above, the preparation method comprising the following steps:

[0021] Preparation of cytoplasmic aspartate aminotransferase-specific monoclonal antibody: the first specific monoclonal antibody and the second specific monoclonal antibody are prepared according to conventional methods, wherein the first specific monoclonal antibody and the second specific monoclonal antibody are goat anti-human monoclonal antibodies; the amino acid sequence of the antigenic determinant used to prepare the first specific monoclonal antibody is LAN HLE IYG LLE FAS NAM RLA, and the amino acid sequence of the antigenic determinant used to prepare the second specific monoclonal antibody is KEI ASN MKH RQL FPF FGS;

[0022] Preparation of a cellulose acetate membrane loaded with a cytoplasmic aspartate aminotransferase-specific monoclonal antibody: immersing the cellulose acetate membrane in a phosphate buffer, adding an activator NHS-EDC, stirring evenly, adding a certain amount of morpholine and o-hydroxyphenylacetic acid, adding the first specific monoclonal antibody and the second specific monoclonal antibody in a molar ratio of 1:2-2:1, stirring evenly, and incubating for a certain period of time to obtain the cellulose acetate membrane loaded with a cytoplasmic aspartate aminotransferase-specific monoclonal antibody;

[0023] Preparation of acrylic acid gel type anion exchange resin particles: using a pulverizer to crush the acrylic acid gel type anion exchange resin particles from which surface water has been removed to obtain acrylic acid gel type anion exchange resin small particles, and then using a hydraulic sieving method to sieve the acrylic acid gel type anion exchange resin small particles to obtain resin small particles with a particle size of 0.9-1.3 microns;

[0024] Preparation of cytoplasmic aspartate aminotransferase specific adsorption membrane: The acetic acid solution of mussel mucin is evenly sprayed on the center of one side of the cellulose acetate membrane loaded with cytoplasmic aspartate aminotransferase specific monoclonal antibody, and a 2-5 mm area is reserved at the edge without spraying. The spraying amount is 10-30 mL / m 2 , drying at 28-36° C. for 30-70 min, wherein the mass concentration of the acetic acid solution is 0.1%-0.3%, and the mass concentration of the mussel mucin in the acetic acid solution is 0.5%-1.3%, to obtain a cellulose acetate membrane loaded with cytoplasmic aspartate aminotransferase-specific monoclonal antibodies and covered with mussel mucin on one side; the resin small particles with a particle size of 0.9-1.3 μm are evenly coated on the cellulose acetate membrane loaded with cytoplasmic aspartate aminotransferase-specific monoclonal antibodies and covered with mussel mucin on one side, and sterile air is introduced into the side not covered with mussel mucin to remove the unadhered resin particles. Blow off, and at the same time form a gas-liquid channel in the mussel mucin layer; spray glue on the reserved unsprayed area on the edge of the cellulose acetate membrane loaded with cytoplasmic aspartate aminotransferase-specific monoclonal antibodies and covered with mussel mucin on one side, and press the reserved edge area of ​​the two cellulose acetate membranes loaded with cytoplasmic aspartate aminotransferase-specific monoclonal antibodies and covered with mussel mucin on one side, with the surfaces coated with resin particles facing each other, to form a structure in which the two cellulose acetate membranes loaded with cytoplasmic aspartate aminotransferase-specific monoclonal antibodies wrap the resin particles, and obtain the cytoplasmic aspartate aminotransferase-specific adsorption membrane;

[0025] Prepare the first reagent: prepare the volume of the first reagent as needed, calculate and weigh the ingredients according to the following ratio: first buffer 30-110 mmol / L, potassium hydroxide 0.5-0.6 mmol / L, glycerol 0.3-1.2 g / L, polyethylene glycol 0.1-0.9 g / L, ethanolamine 0.1-2 mmol / L, reduced coenzyme I NADH 200-270 mmol / L, first antibacterial agent 30 mg / L-69 mg / L, antioxidant 140-310 mg / L, to obtain the first reagent;

[0026] Prepare the second reagent: prepare the volume of the second reagent as needed, calculate and weigh the ingredients according to the following ratio: 60-90 mmol / L of the second buffer, 3-6 kU / L of malate dehydrogenase, 20-67 mmol / L of a surfactant, 50 mg / L-90 mg / L of a second antibacterial agent, and 40-70 mmol / L of a protein stabilizer to obtain the second reagent;

[0027] Prepare the third reagent: prepare the volume of the third reagent as needed, calculate and weigh the ingredients according to the following ratio: 50-100 mmol / L of the third buffer, 30-50 mmol / L of L-aspartic acid, 30-50 mmol / L of α-ketoglutaric acid, and 40 mg / L-80 mg / L of the third antibacterial agent to obtain the third reagent.

[0028] Preferably, the method further comprises the step of immersing the acrylic gel-type anion exchange resin particles in an alkaline solution for transformation, wherein the alkaline solution comprises sodium hydroxide, potassium hydroxide, and ammonia water, and the concentration of the alkaline solution is 1.3-2.0 mol / L.

[0029] Preferably, the mussel mucin refers to a protein with adhesive function separated from the byssus tissue of mussel.

[0030] Preferably, the method further includes a step of preparing a membrane washing reagent. The volume of the membrane washing reagent is prepared as needed, and the ingredients are calculated and weighed according to the following ratio: 30-110 mmol / L of a first buffer solution, 0.06-0.09 mmol / L of potassium hydroxide, 0.3-1.2 g / L of glycerol, 0.1-0.9 g / L of polyethylene glycol, 30 mg / L-69 mg / L of a first antibacterial agent, and 140-310 mg / L of an antioxidant to obtain the membrane washing reagent.

[0031] Compared with the prior art, the present invention has the following beneficial technical effects: ① a cytoplasmic aspartate aminotransferase-specific adsorption membrane is added to the test kit to remove c-AST in the sample to be tested and eliminate measurement interference; ② by adding anion exchange resin, L-aspartic acid and α-ketoglutaric acid originally present in the sample to be tested are removed, the background value is reduced, and the detection accuracy is improved. DETAILED DESCRIPTION

[0032] The technical solution of the present invention is described in detail below in conjunction with embodiments.

[0033] It should be noted that the following embodiments and features in the embodiments may be combined with each other in the absence of conflict; and, based on the embodiments in the present disclosure, all other embodiments obtained by ordinary technicians in the field without making any creative work are within the scope of protection of the present disclosure.

[0034] It should be noted that various aspects of the embodiments within the scope of the appended claims are described below. It should be apparent that the aspects described herein can be embodied in a wide variety of forms, and any specific structure and / or function described herein is merely illustrative. Based on this disclosure, it should be understood by those skilled in the art that an aspect described herein can be implemented independently of any other aspect, and two or more of these aspects can be combined in various ways. For example, any number of aspects described herein can be used to implement the device and / or practice the method.

[0035] It should be noted that the serial numbers in the application documents, such as "first", "second", "third", etc., only distinguish the objects described and do not have any order or technical meaning.

[0036] The aspartate aminotransferase mitochondrial isozyme detection kit and preparation method thereof provided by the present invention remove c-AST in the sample to be tested by adding a cytoplasmic aspartate aminotransferase-specific adsorption membrane in the kit, thereby eliminating measurement interference and simultaneously removing L-aspartic acid and α-ketoglutaric acid originally present in the sample to be tested, thereby reducing background values ​​and improving detection accuracy.

[0037] The present invention provides a kit for detecting mitochondrial isozymes of aspartate aminotransferase, which comprises a cytoplasmic aspartate aminotransferase specific adsorption membrane, a first reagent, a second reagent and a third reagent;

[0038] The structure of the cytoplasmic aspartate aminotransferase specific adsorption membrane is as follows: acrylic gel-type anion exchange resin particles are sandwiched between two layers of cellulose acetate membranes;

[0039] The middle area of ​​two layers of cellulose acetate membrane refers to the middle area of ​​the membrane except the area 2-5 mm away from the edge of the membrane;

[0040] The cellulose acetate membrane is a cellulose acetate membrane loaded with a monoclonal antibody specific for cytoplasmic aspartate aminotransferase;

[0041] The cytoplasmic aspartate aminotransferase-specific monoclonal antibody is composed of a first specific monoclonal antibody and a second monoclonal antibody in a molar ratio of 1:2-2:1, the antigenic determinant amino acid sequence corresponding to the first specific monoclonal antibody is LAN HLE IYG LLE FAS NAM RLA, and the antigenic determinant amino acid sequence corresponding to the second specific monoclonal antibody is KEI ASN MKH RQL FPF FGS;

[0042] The first reagent includes a first buffer solution 30-110 mmol / L, potassium hydroxide 0.5-0.6 mmol / L, glycerol 0.3-1.2 g / L, polyethylene glycol 0.1-0.9 g / L, ethanolamine 0.1-2 mmol / L, reduced coenzyme I NADH 200-270 mmol / L, a first antibacterial agent 30 mg / L-69 mg / L, and an antioxidant 140-310 mg / L;

[0043] The second reagent includes a second buffer solution 60-90 mmol / L, malate dehydrogenase 3-6 kU / L, a surfactant 20-67 mmol / L, a second antibacterial agent 50 mg / L-90 mg / L, and a protein stabilizer 40-70 mmol / L;

[0044] The third reagent includes a third buffer solution 50-100 mmol / L, L-aspartic acid 30-50 mmol / L, α-ketoglutaric acid 30-50 mmol / L, and a third antibacterial agent 40 mg / L-80 mg / L.

[0045] It should be noted that the reason for choosing cellulose acetate membrane is that it has high hydrophilicity and has little nonspecific adsorption of m-AST in the test samples, which can minimize the deviation of the test results.

[0046] Preferably, the pore size of the cellulose acetate membrane is 0.45 micrometers.

[0047] Further preferably, the loading density of the first specific monoclonal antibody and the second monoclonal antibody on the cellulose acetate membrane is 1-10 IU / cm 2 .

[0048] Preferably, the acrylic gel-type anion exchange resin includes a weakly basic anion exchange resin.

[0049] More preferably, the acrylic gel-type anion exchange resin is in the hydroxide form, which can exchange and remove substances such as c-AST to the greatest extent.

[0050] Further preferably, the acrylic gel-type weakly basic anion exchange resin includes DuPont's HPR6700 weakly basic anion exchange resin.

[0051] It should be noted that the acrylic acid gel-type weak base anion exchange resin has the following two functions: first, the functional groups on the surface of the resin particles can exchange and remove c-AST, L-aspartic acid and α-ketoglutaric acid with a certain negative charge; second, the functional groups inside the resin particles can exchange and remove L-aspartic acid and α-ketoglutaric acid. c-AST and m-AST with larger molecular weight cannot enter the interior of the resin particles, reducing the loss of m-AST caused by entering the interior of the resin particles. In addition, acrylic acid resins have good hydrophilicity, low non-specific adsorption of protein enzymes, and can also reduce non-specific adsorption of m-AST.

[0052] Preferably, the number of layers of the acrylic acid series gel type anion exchange resin particles is 1-3 layers, and the particle size of the acrylic acid series gel type anion exchange resin particles is 0.9-1.3 microns.

[0053] It should be noted that the particle size of the acrylic gel-type anion exchange resin particles plays a key role in achieving the technical effect of the present invention. In order to avoid unnecessary loss of m-AST, it cannot be allowed to enter the interior of the resin particles, nor can the sample to be tested pass through an overly thick resin layer, but it is also necessary to ensure that c-AST, which is close to the molecular weight of m-AST, is completely adsorbed, so it is necessary to provide enough sites that can adsorb c-AST; it is also necessary to remove L-aspartic acid and α-ketoglutaric acid in the sample to be tested. These two acids with smaller molecular weights can enter the interior of the resin particles, but the time they pass through the resin layer is limited, so a faster diffusion speed in the particles is required, so a smaller resin particle size is required. It is also necessary to ensure that the resin particles are completely intercepted and do not enter the liquid system to be tested, so as not to affect the spectrophotometric determination results.

[0054] Preferably, the first antibacterial agent and the third antibacterial agent include gentamicin, the second antibacterial agent includes sodium azide, the first buffer and the third buffer include phosphate buffer, the second buffer is Tris buffer, the surfactant includes at least one of lauroyl diethanolamine, Tween 60, and Tween 80, the antioxidant includes vitamin C, and the protein stabilizer includes at least one of alkyl glycoside and oligohalogen trehalose.

[0055] More preferably, the alkyl glycoside includes alkyl glucoside, which is produced by fatty alcohol and glucose in the presence of a catalyst; and the oligosaccharide includes oligosaccharides formed by polymerization of 5-8 trehalose monomers.

[0056] Preferably, the aspartate aminotransferase mitochondrial isozyme detection kit also includes a membrane washing reagent, which includes a first buffer solution 30-110 mmol / L, potassium hydroxide 0.06-0.09 mmol / L, glycerol 0.3-1.2 g / L, polyethylene glycol 0.1-0.9 g / L, a first antibacterial agent 30 mg / L-69 mg / L, and an antioxidant 140-310 mg / L.

[0057] It should be noted that the role of the membrane washing reagent is to wash out the sample to be tested that is present in the cytoplasmic aspartate aminotransferase-specific adsorption membrane, and to recover the m-AST in the sample to be tested into the system to be tested as much as possible. The present invention sets the pH of the membrane washing reagent to a higher pH value than the system to be tested, in order to increase the negative charge of c-AST, L-aspartic acid and α-ketoglutaric acid that have been exchanged onto the cellulose acetate membrane and the resin particles, so that they are more firmly adsorbed, and prevent them from falling off during the membrane washing process and entering the system to be tested, causing an increase in the degree of interference and weakening the technical effect of the present application.

[0058] It should be noted that coupling the cytoplasmic aspartate aminotransferase-specific monoclonal antibody to the cellulose acetate membrane solves the technical problems of insufficient stability, easy inactivation and poor shielding effect of this type of monoclonal antibody.

[0059] It should be noted that L-aspartic acid and α-ketoglutaric acid are in excess in the third reagent of the present application, and malate dehydrogenase is also in excess in the second reagent to avoid low test results due to insufficient amount of auxiliary reactants; NADH is appropriate, and the test results should not be low due to insufficient amount, nor should it be excessive, resulting in excessive zero-point absorbance, small change value, and reduced measurement accuracy.

[0060] The present invention also provides a method for preparing the above-mentioned aspartate aminotransferase mitochondrial isozyme detection kit, and the preparation method comprises the following steps:

[0061] Preparation of cytoplasmic aspartate aminotransferase-specific monoclonal antibody: a first specific monoclonal antibody and a second specific monoclonal antibody are prepared according to a conventional method, wherein the first specific monoclonal antibody and the second specific monoclonal antibody are goat anti-human monoclonal antibodies; the amino acid sequence of the antigenic determinant used to prepare the first specific monoclonal antibody is LANHLE IYG LLE FAS NAM RLA, and the amino acid sequence of the antigenic determinant used to prepare the second specific monoclonal antibody is KEI ASN MKH RQL FPF FGS;

[0062] Preparation of a cellulose acetate membrane loaded with a cytoplasmic aspartate aminotransferase-specific monoclonal antibody: immersing the cellulose acetate membrane in a phosphate buffer, adding an activator NHS-EDC, stirring evenly, adding a certain amount of morpholine and o-hydroxyphenylacetic acid, adding a first specific monoclonal antibody and a second specific monoclonal antibody at a molar ratio of 1:2-2:1, stirring evenly, and incubating for a certain period of time to obtain a cellulose acetate membrane loaded with a cytoplasmic aspartate aminotransferase-specific monoclonal antibody;

[0063] Preparation of acrylic acid gel type anion exchange resin particles: using a pulverizer to crush the acrylic acid gel type anion exchange resin particles from which surface water has been removed to obtain acrylic acid gel type anion exchange resin small particles, and then using a hydraulic sieving method to sieve the acrylic acid gel type anion exchange resin small particles to obtain resin small particles with a particle size of 0.9-1.3 microns;

[0064] Preparation of cytoplasmic aspartate aminotransferase specific adsorption membrane: The acetic acid solution of mussel mucin is evenly sprayed on the center of one side of the cellulose acetate membrane loaded with cytoplasmic aspartate aminotransferase specific monoclonal antibody, leaving a 2-5mm area on the edge without spraying, and the spraying amount is 10-30mL / m 2 , drying at 28-36°C for 30-70min, the mass concentration of the acetic acid solution is 0.1%-0.3%, the mass concentration of the mussel mucin in the acetic acid solution is 0.5%-1.3%, and a cellulose acetate membrane loaded with a cytoplasmic aspartate aminotransferase-specific monoclonal antibody and covered with mussel mucin on one side is obtained; small resin particles with a particle size of 0.9-1.3 microns are evenly coated on the cellulose acetate membrane loaded with a cytoplasmic aspartate aminotransferase-specific monoclonal antibody and covered with mussel mucin on one side, sterile air is introduced into the side not covered with mussel mucin, and the unadhered resin particles are removed. Blow off, and at the same time form a gas-liquid channel in the mussel mucin layer; spray glue on the unsprayed area reserved at the edge of the cellulose acetate membrane loaded with cytoplasmic aspartate aminotransferase-specific monoclonal antibodies and covered with mussel mucin on one side, and press the reserved edge areas of two cellulose acetate membranes loaded with cytoplasmic aspartate aminotransferase-specific monoclonal antibodies and covered with mussel mucin on one side, with the surfaces coated with resin particles facing each other, to form a structure in which two cellulose acetate membranes loaded with cytoplasmic aspartate aminotransferase-specific monoclonal antibodies wrapped with resin particles, and obtain a cytoplasmic aspartate aminotransferase-specific adsorption membrane;

[0065] Prepare the first reagent: prepare the volume of the first reagent as needed, calculate and weigh the ingredients according to the following ratio: first buffer 30-110 mmol / L, potassium hydroxide 0.5-0.6 mmol / L, glycerol 0.3-1.2 g / L, polyethylene glycol 0.1-0.9 g / L, ethanolamine 0.1-2 mmol / L, reduced coenzyme I NADH 200-270 mmol / L, first antibacterial agent 30 mg / L-69 mg / L, antioxidant 140-310 mg / L, to obtain the first reagent;

[0066] Prepare the second reagent: prepare the volume of the second reagent as needed, calculate and weigh the ingredients according to the following ratio: 60-90 mmol / L of the second buffer, 3-6 kU / L of malate dehydrogenase, 20-67 mmol / L of a surfactant, 50 mg / L-90 mg / L of a second antibacterial agent, and 40-70 mmol / L of a protein stabilizer to obtain the second reagent;

[0067] Prepare the third reagent: prepare the volume of the third reagent as needed, calculate and weigh the ingredients according to the following ratio: 50-100mmol / L of the third buffer solution, 30-50mmol / L of L-aspartic acid, 30-50mmol / L of α-ketoglutaric acid, and 40mg / L-80mg / L of the third antibacterial agent to obtain the third reagent.

[0068] Further preferably, the concentration of NHS-EDC in the buffer for loading the monoclonal antibody onto the cellulose acetate membrane is 3.5-4.8 g / L, the concentration of morpholine is 0.9-2.6 g / L, and the concentration of o-hydroxyphenylacetic acid is 1.1-1.4 g / L.

[0069] Further preferably, the reaction temperature for loading the monoclonal antibody onto the cellulose acetate membrane is 38-43°C.

[0070] More preferably, the reaction incubation time for loading the monoclonal antibody onto the cellulose acetate membrane is 8-11 h.

[0071] Further preferably, the glue for bonding the edges of the cellulose acetate film comprises epoxy resin glue.

[0072] It should be noted that the reason for using hydraulic sieving to crush the resin is that hydraulic sieving can reduce the proportion of hydrophobic resin particles and reduce non-specific adsorption of proteins in serum.

[0073] Preferably, the method further comprises the step of immersing the acrylic gel-type anion exchange resin particles in an alkaline solution for transformation, wherein the alkaline solution comprises sodium hydroxide, potassium hydroxide, and ammonia water, and the concentration of the alkaline solution is 1.3-2.0 mol / L.

[0074] Preferably, the mussel mucin refers to a protein having an adhesive effect separated from the byssus tissue of mussel.

[0075] It should be noted that mussel mucin is a commercially available product.

[0076] Further preferably, the method for controlling the number of resin particle layers is as follows: first, a 0.1-0.3 micron thick hydrophilic glue is coated on the middle area of ​​the cellulose acetate membrane, and then after an appropriate interval, dry resin particles are dipped and reversely ventilated, in order to form a channel in the glue layer to prevent the membrane from being blocked; a single layer of dipping can control the number of resin particle layers to 1-2 layers, and the upper and lower membranes are both coated with glue and dipped in resin particles, then the number of resin particle layers can be controlled to 2-3 layers; after the edges of the upper and lower membranes are bonded and pressed tightly, a specific adsorption membrane can be obtained.

[0077] Preferably, the method further includes a step of preparing a membrane washing reagent. The volume of the membrane washing reagent is prepared as needed, and the ingredients are calculated and weighed according to the following ratio: 30-110 mmol / L of a first buffer solution, 0.06-0.09 mmol / L of potassium hydroxide, 0.3-1.2 g / L of glycerol, 0.1-0.9 g / L of polyethylene glycol, 30 mg / L-69 mg / L of a first antibacterial agent, and 140-310 mg / L of an antioxidant to obtain a membrane washing reagent.

[0078] In one embodiment, the application process of the aspartate aminotransferase mitochondrial isozyme detection kit is as follows:

[0079] (1) taking fresh blood and preparing a sample to be tested;

[0080] (2) mixing the sample to be tested with the first reagent, wherein the volume ratio of the sample to be tested to the first reagent is 10:1-16:1, and after mixing, the pH value is 8-9, to obtain a first mixed solution;

[0081] (3) subjecting the first mixed solution to a cytoplasmic aspartate aminotransferase-specific adsorption membrane to obtain a filtrate;

[0082] (4) adding a second reagent to the filtrate and mixing evenly to obtain a second mixed solution;

[0083] (5) adding the third reagent to the second mixed solution and mixing evenly to obtain a third mixed solution;

[0084] (6) Using an analyzer to measure the rate of change of absorbance during the reaction (wavelength 340 nm);

[0085] (7) Calculate the concentration of m-AST in the sample based on the absorbance change.

[0086] In another specific embodiment, the application process of the aspartate aminotransferase mitochondrial isozyme detection kit is as follows:

[0087] (1) taking fresh blood and preparing a sample to be tested;

[0088] (2) mixing the sample to be tested with the first reagent, wherein the volume ratio of the sample to be tested to the first reagent is 10:1-16:1, and after mixing, the pH value is 8-9, to obtain a first mixed solution;

[0089] (3) passing the first mixed solution through a cytoplasmic aspartate aminotransferase-specific adsorption membrane to obtain a filtrate, and washing the filter membrane with a membrane washing reagent having a volume of 0.1 to 0.2 times that of the first mixed solution, and combining the filtrate with the membrane washing reagent;

[0090] (4) adding a second reagent to the filtrate and mixing evenly to obtain a second mixed solution;

[0091] (5) adding the third reagent to the second mixed solution and mixing evenly to obtain a third mixed solution;

[0092] (6) Using an analyzer to measure the rate of change of absorbance during the reaction (wavelength 340 nm);

[0093] (7) Calculate the concentration of m-AST in the sample based on the absorbance change.

[0094] In order to further understand the present invention, the technical solution of the present invention is described below in conjunction with embodiments, but it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the present invention.

[0095] Example 1

[0096] The aspartate aminotransferase mitochondrial isozyme detection kit of the present invention is prepared according to the following steps and conditions:

[0097] Preparation of cytoplasmic aspartate aminotransferase-specific monoclonal antibody: prepare and extract a first specific monoclonal antibody and a second specific monoclonal antibody according to a conventional method, wherein the first specific monoclonal antibody and the second specific monoclonal antibody are goat anti-human monoclonal antibodies; the amino acid sequence of the antigenic determinant used to prepare the first specific monoclonal antibody is LAN HLE IYG LLE FAS NAM RLA, and the amino acid sequence of the antigenic determinant used to prepare the second specific monoclonal antibody is KEI ASN MKH RQL FPF FGS;

[0098] Preparation of a cellulose acetate membrane loaded with a cytoplasmic aspartate aminotransferase-specific monoclonal antibody: immersing a cellulose acetate membrane with a pore size of 0.45 μm in a phosphate buffer, adding an activator NHS-EDC to a concentration of 4.1 g / L in the phosphate buffer, stirring evenly, adding morpholine at a concentration of 0.9 g / L, and also adding o-hydroxyphenylacetic acid at a concentration of 1.1 g / L, adding a first specific monoclonal antibody and a second specific monoclonal antibody at a molar ratio of 1:2, stirring evenly, and reacting for 11 hours at a reaction temperature of 38° C. to obtain a cellulose acetate membrane loaded with a cytoplasmic aspartate aminotransferase-specific monoclonal antibody; the total loading density of the first specific monoclonal antibody and the second monoclonal antibody on the cellulose acetate membrane is 5 IU / cm 2 ;

[0099] Preparation of acrylic acid gel anion exchange resin particles: using a grinder to crush the hydroxyl-type DuPont HPR6700 weakly alkaline anion exchange resin from which surface water has been removed to obtain acrylic acid gel anion exchange resin small particles, and then using a hydraulic sieving method to sieve the acrylic acid gel anion exchange resin small particles to obtain resin small particles with a particle size of 1.3 microns;

[0100] Preparation of cytoplasmic aspartate aminotransferase specific adsorption membrane: The acetic acid solution of mussel mucin was evenly sprayed on the center of one side of the cellulose acetate membrane loaded with cytoplasmic aspartate aminotransferase specific monoclonal antibody, leaving a 3.5 mm area on the edge without spraying, and the spraying amount was 10 mL / m 2 , dried at 36°C for 70 minutes, the mass concentration of the acetic acid solution is 0.2%, the mass concentration of mussel mucin in the acetic acid solution is 0.5%, and a cellulose acetate membrane loaded with cytoplasmic aspartate aminotransferase-specific monoclonal antibody and covered with mussel mucin on one side is obtained; small resin particles are evenly coated on the cellulose acetate membrane loaded with cytoplasmic aspartate aminotransferase-specific monoclonal antibody and covered with mussel mucin on one side, the number of layers of resin particles is 1, sterile air is introduced into the side not covered with mussel mucin, the non-adherent resin particles are blown off, and at the same time, the mussel mucin is coated with small resin particles. The method comprises the following steps: forming a gas-liquid channel by layer; spraying epoxy resin glue on the reserved unsprayed area on the edge of the cellulose acetate membrane loaded with cytoplasmic aspartate aminotransferase-specific monoclonal antibodies and covered with mussel mucin on one side, pressing the reserved edge areas of two cellulose acetate membranes loaded with cytoplasmic aspartate aminotransferase-specific monoclonal antibodies and covered with mussel mucin on one side, with the surfaces coated with resin particles facing each other, to form a structure in which two cellulose acetate membranes loaded with cytoplasmic aspartate aminotransferase-specific monoclonal antibodies wrapped the resin particles, and obtaining a cytoplasmic aspartate aminotransferase-specific adsorption membrane;

[0101] Prepare the first reagent: prepare the volume of the first reagent as needed, calculate and weigh the ingredients according to the following ratio: phosphate buffer 110 mmol / L, potassium hydroxide 0.5 mmol / L, glycerol 0.7 g / L, polyethylene glycol 0.9 g / L, ethanolamine 0.1 mmol / L, reduced coenzyme I NADH 200 mmol / L, gentamicin 69 mg / L, vitamin C 280 mg / L, to obtain the first reagent.

[0102] Prepare the second reagent: prepare the volume of the second reagent as needed, calculate and weigh the ingredients according to the following ratio: Tris buffer 75 mmol / L, malate dehydrogenase 6 kU / L, lauroyl diethanolamine 44 mmol / L, sodium azide 90 mg / L, alkyl glucoside 55 mmol / L, to obtain the second reagent.

[0103] Prepare the third reagent: prepare the volume of the third reagent as needed, calculate and weigh the ingredients according to the following ratio: 50 mmol / L phosphate buffer, 50 mmol / L L-aspartic acid, 50 mmol / L α-ketoglutaric acid, and 40 mg / L gentamicin to obtain the third reagent.

[0104] A quality control sample with a known m-AST concentration was used as a control, and a mixed sample with a certain concentration of c-AST and a known m-AST concentration added was used as a test sample. Multiple measurements were performed to investigate the anti-c-AST interference performance of the aspartate aminotransferase mitochondrial isozyme detection kit prepared above. The results showed that the results obtained for the two samples were very close, the standard deviation of the measurement results from the total average was less than 3.1%, and the standard deviation from the nominal m-AST concentration value of the quality control sample was 3.5%, indicating that the aspartate aminotransferase mitochondrial isozyme detection kit of the present application has a strong anti-c-AST ability.

[0105] Example 2

[0106] The aspartate aminotransferase mitochondrial isozyme detection kit of the present invention is prepared according to the following steps and conditions:

[0107] Preparation of cytoplasmic aspartate aminotransferase-specific monoclonal antibody: prepare and extract a first specific monoclonal antibody and a second specific monoclonal antibody according to a conventional method, wherein the first specific monoclonal antibody and the second specific monoclonal antibody are goat anti-human monoclonal antibodies; the amino acid sequence of the antigenic determinant used to prepare the first specific monoclonal antibody is LAN HLE IYG LLE FAS NAM RLA, and the amino acid sequence of the antigenic determinant used to prepare the second specific monoclonal antibody is KEI ASN MKH RQL FPF FGS;

[0108] Preparation of a cellulose acetate membrane loaded with a cytoplasmic aspartate aminotransferase-specific monoclonal antibody: immersing a cellulose acetate membrane with a pore size of 0.45 μm in a phosphate buffer, adding an activator NHS-EDC to a concentration of 3.5 g / L in the phosphate buffer, stirring evenly, adding morpholine at a concentration of 2.6 g / L, and also adding o-hydroxyphenylacetic acid at a concentration of 1.25 g / L, adding a first specific monoclonal antibody and a second specific monoclonal antibody at a molar ratio of 2:1, stirring evenly, reacting for 9.5 hours at a reaction temperature of 43°C, and obtaining a cellulose acetate membrane loaded with a cytoplasmic aspartate aminotransferase-specific monoclonal antibody; the total loading density of the first specific monoclonal antibody and the second monoclonal antibody on the cellulose acetate membrane is 1 IU / cm 2 ;

[0109] Preparation of acrylic acid gel anion exchange resin particles: using a grinder to crush the hydroxide-type DuPont HPR6700 weakly alkaline anion exchange resin from which surface water has been removed to obtain acrylic acid gel anion exchange resin small particles, and then using a hydraulic sieving method to sieve the acrylic acid gel anion exchange resin small particles to obtain resin small particles with a particle size of 0.9 microns;

[0110] Preparation of cytoplasmic aspartate aminotransferase specific adsorption membrane: The acetic acid solution of mussel mucin was evenly sprayed on the center of one side of the cellulose acetate membrane loaded with cytoplasmic aspartate aminotransferase specific monoclonal antibody, leaving a 5mm area on the edge without spraying, and the spraying amount was 16mL / m 2 , dried at 32°C for 50 minutes, the mass concentration of the acetic acid solution is 0.1%, the mass concentration of mussel mucin in the acetic acid solution is 1.3%, and a cellulose acetate membrane loaded with cytoplasmic aspartate aminotransferase-specific monoclonal antibodies and covered with mussel mucin on one side is obtained; small resin particles are evenly coated on the cellulose acetate membrane loaded with cytoplasmic aspartate aminotransferase-specific monoclonal antibodies and covered with mussel mucin on one side, the number of layers of resin particles is 2, sterile air is introduced into the side not covered with mussel mucin, the non-adherent resin particles are blown off, and at the same time, the mussel mucin is coated with small resin particles. The method comprises the following steps: forming a gas-liquid channel by layer; spraying epoxy resin glue on the reserved unsprayed area on the edge of the cellulose acetate membrane loaded with cytoplasmic aspartate aminotransferase-specific monoclonal antibodies and covered with mussel mucin on one side, pressing the reserved edge areas of two cellulose acetate membranes loaded with cytoplasmic aspartate aminotransferase-specific monoclonal antibodies and covered with mussel mucin on one side, with the surfaces coated with resin particles facing each other, to form a structure in which two cellulose acetate membranes loaded with cytoplasmic aspartate aminotransferase-specific monoclonal antibodies wrapped the resin particles, and obtaining a cytoplasmic aspartate aminotransferase-specific adsorption membrane;

[0111] Prepare the first reagent: prepare the volume of the first reagent as needed, calculate and weigh the ingredients according to the following ratio: phosphate buffer 70 mmol / L, potassium hydroxide 0.55 mmol / L, glycerol 0.3 g / L, polyethylene glycol 0.5 g / L, ethanolamine 0.9 mmol / L, reduced coenzyme I NADH 240 mmol / L, gentamicin 48 mg / L, vitamin C 140 mg / L, to obtain the first reagent.

[0112] Prepare the second reagent: prepare the volume of the second reagent as needed, calculate and weigh the ingredients according to the following ratio: 60 mmol / L Tris buffer, 4.5 kU / L malate dehydrogenase, 44 mmol / L Tween 60, 70 mg / L sodium azide, 55 mmol / L 8 trehalose monomer oligosaccharide, to obtain the second reagent.

[0113] Prepare the third reagent: prepare the volume of the third reagent as needed, calculate and weigh the ingredients according to the following ratio: phosphate buffer 75 mmol / L, L-aspartic acid 40 mmol / L, α-ketoglutaric acid 40 mmol / L, gentamicin 60 mg / L, to obtain the third reagent.

[0114] A quality control sample with a known m-AST concentration was used as a control, and a mixed sample with a certain concentration of c-AST and a known m-AST concentration added was used as a test sample. Multiple measurements were performed to investigate the anti-c-AST interference performance of the aspartate aminotransferase mitochondrial isozyme detection kit prepared above. The results showed that the results obtained for the two samples were very close, the standard deviation of the measurement results from the total average was less than 3.9%, and the standard deviation from the nominal m-AST concentration value of the quality control sample was 3.76%, indicating that the aspartate aminotransferase mitochondrial isozyme detection kit of the present application has a strong ability to resist c-AST.

[0115] Example 3

[0116] The aspartate aminotransferase mitochondrial isozyme detection kit of the present invention is prepared according to the following steps and conditions:

[0117] Preparation of cytoplasmic aspartate aminotransferase-specific monoclonal antibody: prepare and extract a first specific monoclonal antibody and a second specific monoclonal antibody according to a conventional method, wherein the first specific monoclonal antibody and the second specific monoclonal antibody are goat anti-human monoclonal antibodies; the amino acid sequence of the antigenic determinant used to prepare the first specific monoclonal antibody is LAN HLE IYG LLE FAS NAM RLA, and the amino acid sequence of the antigenic determinant used to prepare the second specific monoclonal antibody is KEI ASN MKH RQL FPF FGS;

[0118] Preparation of a cellulose acetate membrane loaded with a cytoplasmic aspartate aminotransferase-specific monoclonal antibody: immersing a cellulose acetate membrane with a pore size of 0.45 μm in a phosphate buffer, adding an activator NHS-EDC to a concentration of 4.8 g / L in the phosphate buffer, stirring evenly, adding morpholine at a concentration of 1.8 g / L, and also adding o-hydroxyphenylacetic acid at a concentration of 1.4 g / L, adding a first specific monoclonal antibody and a second specific monoclonal antibody at a molar ratio of 1:1, stirring evenly, and reacting for 8 hours at a reaction temperature of 40°C to obtain a cellulose acetate membrane loaded with a cytoplasmic aspartate aminotransferase-specific monoclonal antibody; the total loading density of the first specific monoclonal antibody and the second monoclonal antibody on the cellulose acetate membrane is 10 IU / cm 2 ;

[0119] Preparation of acrylic acid gel-type anion exchange resin particles: using a grinder to crush the hydroxide-type DuPont HPR6700 weakly alkaline anion exchange resin from which surface water has been removed to obtain acrylic acid gel-type anion exchange resin small particles, and then using a hydraulic sieving method to sieve the acrylic acid gel-type anion exchange resin small particles to obtain resin small particles with a particle size of 1.1 microns;

[0120] Preparation of cytoplasmic aspartate aminotransferase specific adsorption membrane: The acetic acid solution of mussel mucin was evenly sprayed on the center of one side of the cellulose acetate membrane loaded with cytoplasmic aspartate aminotransferase specific monoclonal antibody, leaving a 2mm area on the edge without spraying, and the spraying amount was 30mL / m 2 , dried at 28°C for 30 minutes, the mass concentration of the acetic acid solution is 0.3%, the mass concentration of mussel mucin in the acetic acid solution is 0.9%, and a cellulose acetate membrane loaded with cytoplasmic aspartate aminotransferase-specific monoclonal antibody and covered with mussel mucin on one side is obtained; small resin particles are evenly coated on the cellulose acetate membrane loaded with cytoplasmic aspartate aminotransferase-specific monoclonal antibody and covered with mussel mucin on one side, the number of layers of resin particles is 3, sterile air is introduced into the side not covered with mussel mucin, the non-adherent resin particles are blown off, and at the same time, the mussel mucin is coated with small resin particles. The method comprises the following steps: forming a gas-liquid channel by layer; spraying epoxy resin glue on the reserved unsprayed area on the edge of the cellulose acetate membrane loaded with cytoplasmic aspartate aminotransferase-specific monoclonal antibodies and covered with mussel mucin on one side, pressing the reserved edge areas of two cellulose acetate membranes loaded with cytoplasmic aspartate aminotransferase-specific monoclonal antibodies and covered with mussel mucin on one side, with the surfaces coated with resin particles facing each other, to form a structure in which two cellulose acetate membranes loaded with cytoplasmic aspartate aminotransferase-specific monoclonal antibodies wrapped the resin particles, and obtaining a cytoplasmic aspartate aminotransferase-specific adsorption membrane;

[0121] Prepare the first reagent: prepare the volume of the first reagent as needed, calculate and weigh the ingredients according to the following ratio: phosphate buffer 30mmol / L, potassium hydroxide 6mmol / L, glycerol 1.2g / L, polyethylene glycol 0.1g / L, ethanolamine 2mmol / L, reduced coenzyme I NADH 270mmol / L, gentamicin 30mg / L, vitamin C 310mg / L, to obtain the first reagent.

[0122] Prepare the second reagent: prepare the volume of the second reagent as needed, calculate and weigh the ingredients according to the following ratio: Tris buffer 90 mmol / L, malate dehydrogenase 3 kU / L, Tween 80 44 mmol / L, sodium azide 50 mg / L, 5 trehalose monomer oligosaccharide 55 mmol / L, to obtain the second reagent.

[0123] Prepare the third reagent: prepare the volume of the third reagent as needed, calculate and weigh the ingredients according to the following ratio: phosphate buffer 100 mmol / L, L-aspartic acid 30 mmol / L, α-ketoglutaric acid 30 mmol / L, gentamicin 80 mg / L, to obtain the third reagent.

[0124] A quality control sample with a known m-AST concentration was used as a control, and a mixed sample with a certain concentration of c-AST and a known m-AST concentration added was used as a test sample. Multiple measurements were performed to investigate the anti-c-AST interference performance of the aspartate aminotransferase mitochondrial isozyme detection kit prepared above. The results showed that the results obtained for the two samples were very close, the standard deviation of the measurement results from the total average value was less than 3.3%, and the standard deviation from the nominal m-AST concentration value of the quality control sample was 3.6%, indicating that the aspartate aminotransferase mitochondrial isozyme detection kit of the present application has a strong ability to resist c-AST.

[0125] Example 4

[0126] The contents not mentioned in the following statements are the same as those in Embodiment 1.

[0127] The method also includes a preparation step of a membrane washing reagent, wherein the volume of the membrane washing reagent is prepared as required, and the ingredients are calculated and weighed according to the following ratio: 110 mmol / L phosphate buffer, 0.07 mmol / L potassium hydroxide, 0.7 g / L glycerol, 0.9 g / L polyethylene glycol, 69 mg / L gentamicin, and 280 mg / L vitamin C, to obtain the membrane washing reagent.

[0128] A quality control sample with a known m-AST concentration was used as a control, and a mixed sample with a certain concentration of c-AST and a known m-AST concentration was used as a test sample. Multiple measurements were performed to investigate the anti-c-AST interference performance of the aspartate aminotransferase mitochondrial isozyme detection kit prepared above.

[0129] The difference from the detection process of Example 1 is that after the mixed solution is treated with the cytoplasmic aspartate aminotransferase-specific adsorption membrane, a filtrate is obtained, and then a membrane washing reagent with a volume of 0.15 times that of the mixed solution is used to top-wash the filter membrane, which is then combined with the filtrate for subsequent detection operations.

[0130] The results showed that the standard deviation of the measured results from the total mean value was less than 1.56%, and the standard deviation from the nominal m-AST concentration value of the quality control sample was 1.93%, indicating that the operation of top-washing the filter membrane can further improve the detection accuracy.

[0131] Comparative Example 1 (cytoplasmic aspartate aminotransferase specific adsorption membrane and first reagent not provided)

[0132] This comparative example is the same as Example 1 except that the cytoplasmic aspartate aminotransferase-specific adsorption membrane and the first reagent are not included.

[0133] A quality control sample with a known m-AST concentration was used as a control, and a mixed sample with a certain concentration of c-AST and a known m-AST concentration was used as a test sample. After the test sample processed according to the conventional method was mixed with the second reagent and the third reagent, the two samples were tested and measured multiple times. The results showed that the standard deviation of the measurement results from the total average value was higher than 8.35%, and the standard deviation from the nominal m-AST concentration value of the quality control sample was 9.51%, indicating that the measurement results were seriously interfered by c-AST.

Claims

1. A kit for detecting mitochondrial isozymes of aspartate aminotransferase, characterized in that: The detection kit comprises a cytoplasmic aspartate aminotransferase specific adsorption membrane, a first reagent, a second reagent and a third reagent; The structure of the cytoplasmic aspartate aminotransferase specific adsorption membrane is as follows: acrylic gel-type anion exchange resin particles are sandwiched between two layers of cellulose acetate membranes; The particle size of the acrylic gel-type anion exchange resin particles is 0.9-1.3 microns; The acrylic gel-type anion exchange resin is a hydroxide-type DuPont HPR6700 weakly basic anion exchange resin; The middle area of ​​the two layers of cellulose acetate film refers to the middle area of ​​the film except the area 2-5 mm away from the edge of the film; The cellulose acetate membrane is a cellulose acetate membrane loaded with a cytoplasmic aspartate aminotransferase-specific monoclonal antibody; The cytoplasmic aspartate aminotransferase-specific monoclonal antibody is composed of a first specific monoclonal antibody and a second monoclonal antibody in a molar ratio of 1:2-2:1, the antigenic determinant amino acid sequence corresponding to the first specific monoclonal antibody is LAN HLE IYG LLE FAS NAM RLA, and the antigenic determinant amino acid sequence corresponding to the second specific monoclonal antibody is KEI ASN MKH RQL FPF FGS; The first reagent includes a first buffer solution 30-110 mmol / L, potassium hydroxide 0.5-0.6 mmol / L, glycerol 0.3-1.2 g / L, polyethylene glycol 0.1-0.9 g / L, ethanolamine 0.1-2 mmol / L, reduced coenzyme I NADH 200-270 mmol / L, a first antibacterial agent 30 mg / L-69 mg / L, and an antioxidant 140-310 mg / L; The second reagent includes a second buffer solution of 60-90 mmol / L, malate dehydrogenase of 3-6 kU / L, a surfactant of 20-67 mmol / L, a second antibacterial agent of 50 mg / L-90 mg / L, and a protein stabilizer of 40-70 mmol / L; The third reagent includes a third buffer solution 50-100 mmol / L, L-aspartic acid 30-50 mmol / L, α-ketoglutaric acid 30-50 mmol / L, and a third antibacterial agent 40 mg / L-80 mg / L; The first antibacterial agent and the third antibacterial agent include gentamicin, the second antibacterial agent includes sodium azide, the first buffer and the third buffer include phosphate buffer, the second buffer is Tris buffer, the surfactant includes at least one of lauroyl diethanolamine, Tween 60, and Tween 80, the antioxidant includes vitamin C, and the protein stabilizer includes at least one of alkyl glycoside and oligohalogen trehalose; The alkyl glycoside is an alkyl glucoside; The oligosaccharide is an oligosaccharide formed by polymerization of 5 or 8 trehalose monomers.

2. The aspartate aminotransferase mitochondrial isozyme detection kit according to claim 1, characterized in that: The pore size of the cellulose acetate membrane is 0.45 μm.

3. The aspartate aminotransferase mitochondrial isozyme detection kit according to claim 1, characterized in that: The number of layers of the acrylic gel-type anion exchange resin particles is 1-3.

4. The aspartate aminotransferase mitochondrial isozyme detection kit according to claim 1, characterized in that: The aspartate aminotransferase mitochondrial isozyme detection kit also includes a membrane washing reagent, which is prepared from 110 mmol / L phosphate buffer, 0.07 mmol / L potassium hydroxide, 0.7 g / L glycerol, 0.9 g / L polyethylene glycol, 69 mg / L gentamicin, and 280 mg / L vitamin C.

5. A method for preparing the aspartate aminotransferase mitochondrial isozyme detection kit according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: Preparation of cytoplasmic aspartate aminotransferase-specific monoclonal antibody: the first specific monoclonal antibody and the second specific monoclonal antibody are prepared according to conventional methods, wherein the first specific monoclonal antibody and the second specific monoclonal antibody are goat anti-human monoclonal antibodies; the amino acid sequence of the antigenic determinant used to prepare the first specific monoclonal antibody is LAN HLE IYG LLE FAS NAM RLA, and the amino acid sequence of the antigenic determinant used to prepare the second specific monoclonal antibody is KEI ASN MKH RQL FPF FGS; Preparation of a cellulose acetate membrane loaded with a cytoplasmic aspartate aminotransferase-specific monoclonal antibody: immersing the cellulose acetate membrane in a phosphate buffer, adding an activator NHS-EDC, stirring evenly, adding a certain amount of morpholine and o-hydroxyphenylacetic acid, adding the first specific monoclonal antibody and the second specific monoclonal antibody in a molar ratio of 1:2-2:1, stirring evenly, and incubating for a certain period of time to obtain the cellulose acetate membrane loaded with a cytoplasmic aspartate aminotransferase-specific monoclonal antibody; Preparation of acrylic acid gel type anion exchange resin particles: using a pulverizer to crush the acrylic acid gel type anion exchange resin particles from which surface water has been removed to obtain acrylic acid gel type anion exchange resin small particles, and then using a hydraulic sieving method to sieve the acrylic acid gel type anion exchange resin small particles to obtain resin small particles with a particle size of 0.9-1.3 microns; Preparation of cytoplasmic aspartate aminotransferase specific adsorption membrane: The acetic acid solution of mussel mucin is evenly sprayed on the center of one side of the cellulose acetate membrane loaded with cytoplasmic aspartate aminotransferase specific monoclonal antibody, leaving a 2-5 mm area on the edge without spraying, and the spraying amount is 10-30 mL / m 2 , drying at 28-36°C for 30-70 min, the mass concentration of the acetic acid solution is 0.1%-0.3%, the mass concentration of the mussel mucin in the acetic acid solution is 0.5%-1.3%, and a cellulose acetate membrane loaded with cytoplasmic aspartate aminotransferase-specific monoclonal antibodies and covered with mussel mucin on one side is obtained; the resin small particles with a particle size of 0.9-1.3 microns are evenly coated on the cellulose acetate membrane loaded with cytoplasmic aspartate aminotransferase-specific monoclonal antibodies and covered with mussel mucin on one side, sterile air is introduced into the side not covered with mussel mucin to blow off the unadhered resin particles, and at the same time, the resin particles are removed from the mussel mucin on the other side. The mucin layer forms a gas-liquid channel; glue is sprayed on the reserved unsprayed area on the edge of the cellulose acetate membrane loaded with cytoplasmic aspartate aminotransferase-specific monoclonal antibodies and covered with mussel mucin on one side, and the reserved edge area of ​​two pieces of cellulose acetate membranes loaded with cytoplasmic aspartate aminotransferase-specific monoclonal antibodies and covered with mussel mucin on one side are pressed together, with the surfaces coated with resin particles facing each other, to form a structure in which the two pieces of cellulose acetate membranes loaded with cytoplasmic aspartate aminotransferase-specific monoclonal antibodies wrap the resin particles, thereby obtaining the cytoplasmic aspartate aminotransferase-specific adsorption membrane; The acrylic gel-type anion exchange resin is a hydroxide-type DuPont HPR6700 weakly basic anion exchange resin; Prepare the first reagent: prepare the volume of the first reagent as needed, calculate and weigh the ingredients according to the following ratio: first buffer 30-110 mmol / L, potassium hydroxide 0.5-0.6 mmol / L, glycerol 0.3-1.2 g / L, polyethylene glycol 0.1-0.9 g / L, ethanolamine 0.1-2 mmol / L, reduced coenzyme I NADH 200-270 mmol / L, first antibacterial agent 30 mg / L-69 mg / L, antioxidant 140-310 mg / L, to obtain the first reagent; Prepare the second reagent: prepare the volume of the second reagent as needed, calculate and weigh the ingredients according to the following ratio: 60-90 mmol / L of second buffer, 3-6 kU / L of malate dehydrogenase, 20-67 mmol / L of surfactant, 50 mg / L-90 mg / L of second antibacterial agent, and 40-70 mmol / L of protein stabilizer to obtain the second reagent; Prepare the third reagent: prepare the volume of the third reagent as needed, calculate and weigh the ingredients according to the following ratio: 50-100 mmol / L of the third buffer, 30-50 mmol / L of L-aspartic acid, 30-50 mmol / L of α-ketoglutaric acid, and 40 mg / L-80 mg / L of the third antibacterial agent to obtain the third reagent; The first antibacterial agent and the third antibacterial agent include gentamicin, the second antibacterial agent includes sodium azide, the first buffer and the third buffer include phosphate buffer, the second buffer is Tris buffer, the surfactant includes at least one of lauroyl diethanolamine, Tween 60, and Tween 80, the antioxidant includes vitamin C, and the protein stabilizer includes at least one of alkyl glycoside and oligohalogen trehalose; The alkyl glycoside is an alkyl glucoside; The oligosaccharide is an oligosaccharide formed by polymerization of 5 or 8 trehalose monomers.

6. The method for preparing the aspartate aminotransferase mitochondrial isozyme detection kit according to claim 5, characterized in that: The method also includes the step of immersing the acrylic gel-type anion exchange resin particles in an alkaline solution for transformation. The alkaline solution includes sodium hydroxide, potassium hydroxide, and ammonia water. The concentration of the alkaline solution is 1.3-2.0 mol / L.

7. The method for preparing the aspartate aminotransferase mitochondrial isozyme detection kit according to claim 5, characterized in that: The mussel mucin refers to a protein with adhesive function separated from the byssus tissue of mussel.

8. The method for preparing the aspartate aminotransferase mitochondrial isozyme detection kit according to claim 5, characterized in that: The method also includes a preparation step of a membrane washing reagent, wherein the volume of the membrane washing reagent is prepared as required, and the ingredients are calculated and weighed according to the following ratio: 110 mmol / L phosphate buffer, 0.07 mmol / L potassium hydroxide, 0.7 g / L glycerol, 0.9 g / L polyethylene glycol, 69 mg / L gentamicin, and 280 mg / L vitamin C, to obtain the membrane washing reagent.