Glycated albumin concentration detection reagent and detection method

Abstract

The glycated albumin concentration detection reagent provided by the invention comprises a first reagent and a second reagent, the volume ratio of the first reagent to the second reagent is 4: 1, and the first reagent and the second reagent are added with a special protective agent and reduce protease to participate in a reaction. The problem of poor reagent stability caused by high protease dosage and enzyme activity loss in the preservation process in the prior art is avoided. In addition, the invention also provides a preparation method of the glycated albumin concentration detection reagent.

Description

Technical Field

[0001] The present application relates to the field of medical detection technology, and in particular to a glycated albumin concentration detection reagent and detection method. Background Art

[0002] Glycated albumin (GA) is the product of non-enzymatic glycation reaction between glucose and plasma albumin, reflecting the average blood sugar level of diabetic patients in the past 2 to 3 weeks. In recent years, glycated albumin has gradually attracted the attention of researchers and clinicians. Compared with glycated serum protein, it is not affected by the protein concentration, bilirubin, chylomicron and low molecular weight substances in the blood, and is used to assist in the diagnosis of the true blood sugar level in patients with anemia, pregnancy and abnormal hemoglobin metabolism. Therefore, glycated albumin has gradually become an important indicator for diabetes monitoring in the future.

[0003] The detection methods of glycated albumin include colorimetry, chromatography, immunoassay, enzymatic method, etc. The colorimetric method has poor specificity and is easily interfered by uric acid, lipemia, etc.; the chromatography method is time-consuming and cannot meet the needs of a large number of clinical sample determinations; the immunoassay method needs to take into account the antibody specificity and the chemical steric effect of the antibody. Therefore, the enzyme method is currently widely used in clinical testing. The enzymatic method uses albumin-specific proteases to generate glycated amino acids from glycated albumin, which are converted into glucosone, amino acids and hydrogen peroxide under the action of specific fructose amino acid oxidase (FAOD); under the action of peroxidase, hydrogen peroxide reacts with 4AAP (4-aminoantipyrine) and TOOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt) to produce red quinone imine, and its absorbance change is measured to determine the concentration of glycated albumin in the sample. Secondly, the amount of albumin in serum or plasma is determined using bromocresol purple or bromocresol green reagents, and finally GA% is calculated by the formula

[0004] During the research, it was found that in the process of protease decomposing glycated albumin, the albumin in the blood combines with various substances such as bilirubin or fat. In order to obtain glycated amino acids that can be decomposed under the action of fructose amino acid oxidase, a large amount of protease is needed to increase the reaction rate. However, adding a large amount of protease to increase the reaction will not only increase the cost, but also the storage stability of the reagent will deteriorate, and it may also lead to the inactivation of other enzymes in the reagent itself, such as peroxidase. Summary of the invention

[0005] In view of this, it is necessary to provide a glycated albumin concentration detection reagent, detection method and kit that can improve the stability of the reagent in response to the shortcomings of the current detection reagent technology.

[0006] To solve the above problems, this application adopts the following technical embodiments:

[0007] One of the purposes of the present application is to provide a glycated albumin concentration detection reagent, comprising a first reagent and a second reagent, wherein the volume ratio of the first reagent to the second reagent is 4:1, wherein:

[0008] The first reagent includes substances with the following concentrations: 50-150 mmol / L buffer, 0.5-5 mmol / L EDTA, 6-10 mg / L potassium ferrocyanide, 0.5-1 g / L sodium azide, 10-250 KU / L protease, 0.1-10 g / L 4-aminoantipyrine and 1-20 g / L surfactant;

[0009] The first reagent includes substances with the following concentrations: 50-150 mmol / L buffer, 0.5-5 mmol / L EDTA, 6-10 mg / L potassium ferrocyanide, 0.5-1 g / L sodium azide, 20-100 KU / L fructose amino acid oxidase, 1-100 KU / L peroxidase and 0.1-10 g / L color developer.

[0010] In some embodiments, the buffer is one of TrisHCL buffer, phosphate buffer, Tricine buffer, HEPES buffer and MES buffer.

[0011] In some embodiments, the protease is selected from one of alkaline protease, papain, subtilisin and proteinase K.

[0012] In some embodiments, the surfactant includes polysorbate-20, dextran 40, sodium dodecyl sulfate SDS, polyoxyethylene derivative Emulgen B66, Triton X-100, Triton X-405, betaine and polyethylene glycol monoalkyl ether. One or more of X-080.

[0013] In some embodiments, the surfactant is polysorbate 20, and the added amount of polysorbate 20 is 0.1-0.5%.

[0014] In some embodiments, the protease concentration is 100-150 KU / L.

[0015] In some embodiments, the surfactant further comprises a polyoxyethylene derivative B66, and the added amount of the polyoxyethylene derivative B66 is 0.5-1.0%.

[0016] In some of the embodiments, the color developer is selected from one or two of N,N-bis(4-sulfobutyl)-3-methylaniline disodium salt (TODB), N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt (TOPS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt (DAOS), and 3-(N-ethyl-3-methylanilino)-2-hydroxypropanesulfonic acid sodium salt (TOOS).

[0017] The second object of the present application is to provide a method for detecting the glycated albumin concentration detection reagent, comprising the following steps:

[0018] Adding the first reagent to the glycated albumin sample to be tested and mixing to obtain a first mixed solution, and incubating the first mixed solution and then reading a first absorbance A1;

[0019] Adding the second reagent to the first mixed solution and mixing well to obtain a second mixed solution, and incubating the second mixed solution and then reading a second absorbance A2;

[0020] An absorbance change value ΔA is obtained based on the first absorbance and the second absorbance, where ΔA=[(A2-A1) calibration tube / sample tube]-[(A2-A1) blank tube].

[0021] In some embodiments, the incubation temperature is 37° C. and the incubation time is 5 minutes.

[0022] This application adopts the above technical embodiments, and its beneficial effects are as follows:

[0023] The glycated albumin concentration detection reagent and detection method provided in the present application adopt the addition of special protective agents and the reduction of protease participation in the reaction, thereby avoiding the problem of poor reagent stability caused by large protease dosage and enzyme activity loss during storage in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical embodiments of the embodiments of the present application, the drawings required for use in the embodiments of the present application or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 This is a schematic diagram of the verification experiment of different concentrations of the protective agent polysorbate-20 provided in Examples 1 to 5 of the present application.

[0026] Figure 2Schematic diagram of the verification experiment of protease addition concentration under the condition of a certain amount of polysorbate-20 added provided in Examples 6 to 10 of the present application.

[0027] Figure 3 Schematic diagrams of experiments on the effects of different surfactants on reagent effects provided in Examples 11 to 15 of the present application.

[0028] Figure 4 Schematic diagram of the compatibility experiment of polysorbate-20 and polyoxyethylene derivatives at different concentrations provided in Examples 16 to 20 of the present application.

[0029] Figure 5 Schematic diagram of the compatibility experiment of polysorbate-20 and polyoxyethylene derivatives at different concentrations provided in Examples 21 to 25 of the present application.

[0030] Figure 6 Schematic diagram of the deviation of the test results of the reagent provided in the embodiment of the present application at 37°C for 7 days.

[0031] Figure 7 A schematic diagram of the deviation in test results of long-term storage of the reagents provided in the embodiments of the present application. DETAILED DESCRIPTION

[0032] Embodiments of the present application are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present application, and should not be construed as limiting the present application.

[0033] In the description of the present application, it should be understood that the terms "upper", "lower", "horizontal", "inside", "outside", etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present application 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 should not be understood as a limitation on the present application.

[0034] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of this application, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0035] In order to make the objectives, technical embodiments and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0036] The glycated albumin concentration detection reagent provided in the embodiment of the present application includes a first reagent and a second reagent, wherein the volume ratio of the first reagent to the second reagent is 4:1, wherein:

[0037] The first reagent includes substances with the following concentrations: 50-150 mmol / L buffer, 0.5-5 mmol / L EDTA, 6-10 mg / L potassium ferrocyanide, 0.5-1 g / L sodium azide, 10-250 KU / L protease, 0.1-10 g / L 4-aminoantipyrine and 1-20 g / L surfactant;

[0038] The first reagent includes substances with the following concentrations: 50-150 mmol / L buffer, 0.5-5 mmol / L EDTA, 6-10 mg / L potassium ferrocyanide, 0.5-1 g / L sodium azide, 20-100 KU / L fructose amino acid oxidase, 1-100 KU / L peroxidase and 0.1-10 g / L color developer.

[0039] In some embodiments, the buffer is one of TrisHCL buffer, phosphate buffer, Tricine buffer, HEPES buffer and MES buffer.

[0040] In some embodiments, the protease is selected from one of alkaline protease, papain, subtilisin and proteinase K.

[0041] In some embodiments, the surfactant includes polysorbate-20 TWEEN-20, dextran 40 Dextran 40 sodium dodecyl sulfate SDS, etc., polyoxyethylene derivatives Emulgen B66, Triton X-100, Triton X-405, betaine and polyethylene glycol monoalkyl ether. One or more of X-080.

[0042] In some embodiments, the surfactant is polysorbate 20, and the added amount of polysorbate 20 is 0.1-0.5%.

[0043] In some embodiments, the protease concentration is 100-150 KU / L.

[0044] In some embodiments, the surfactant further comprises a polyoxyethylene derivative B66, and the added amount of the polyoxyethylene derivative B66 is 0.5-1.0%.

[0045] In some of the embodiments, the color developer is selected from one or two of N,N-bis(4-sulfobutyl)-3-methylaniline disodium salt (TODB), N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt (TOPS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt (DAOS), and 3-(N-ethyl-3-methylanilino)-2-hydroxypropanesulfonic acid sodium salt (TOOS).

[0046] The present application also provides a method for detecting the glycated albumin concentration detection reagent, comprising the following steps:

[0047] S110: adding the first reagent to the glycated albumin sample to be tested and mixing to obtain a first mixed solution, and incubating the first mixed solution and then reading a first absorbance Al;

[0048] S120: adding the second reagent to the first mixed solution and mixing to obtain a second mixed solution, and incubating the second mixed solution and then reading a second absorbance A2;

[0049] S130: Obtaining an absorbance change value ΔA based on the first absorbance and the second absorbance, ΔA=[(A2-A1) calibration tube / sample tube]-[(A2-A1) blank tube].

[0050] It can be understood that a calibrator tube is a substance with known concentration or properties, which is used to calibrate the test instrument or test method. By comparing with the calibrator, the test instrument can be adjusted to make its measurement results more accurate. Blank tubes are generally used as controls. They do not contain samples to be tested or quality control products, but only contain solvents, buffers and other non-test substance components used in the test process. Sample tubes are test tubes that store experimental samples.

[0051] It is understandable that during the detection process, there may be background interference signals from the experimental environment, reagents, etc. By measuring the blank tube, the extent of these background interferences can be determined, and this part of the interference can be deducted from the subsequent test results to obtain a more accurate test value.

[0052] Please refer to the table below. In the absorbance detection provided in this embodiment, a dual wavelength method using a main wavelength and a sub-wavelength is used, which has higher accuracy.

[0053]

[0054] The glycated albumin concentration detection reagent, detection method and kit provided in the present application adopt the addition of special protective agents and the reduction of protease participation in the reaction, thereby avoiding the problem of poor reagent stability caused by large protease dosage and enzyme activity loss during storage in the prior art.

[0055] The above technical embodiments of the present application are described in detail below in conjunction with specific embodiments.

[0056] Example 1

[0057] The first reagent includes: TrisHCL buffer 150mmol / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, sodium azide 0.5g / L, 4-aminoantipyrine 10g / L, pH adjusted to 8.0, protease 200KU / L, ascorbate oxidase 50KU / L;

[0058] The second reagent includes: HEPES buffer 150mmol / L, sodium azide 0.5g / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, pH adjusted to 7.5, fructose amino acid oxidase 100KU / L, peroxidase 50KU / L, color developer N, N-bis (4-sulfobutyl) -3-methylaniline disodium salt (TODB) 10g / L;

[0059] The surfactant is: 0.2% polysorbate-20 TWEEN-20.

[0060] Example 2

[0061] The first reagent includes: TrisHCL buffer 150mmol / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, sodium azide 0.5g / L, 4-aminoantipyrine 10g / L, pH adjusted to 8.0, protease 200KU / L, ascorbate oxidase 50KU / L;

[0062] The second reagent includes: HEPES buffer 150mmol / L, sodium azide 0.5g / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, pH adjusted to 7.5, fructose amino acid oxidase 100KU / L, peroxidase 50KU / L, color developer N, N-bis (4-sulfobutyl) -3-methylaniline disodium salt (TODB) 10g / L;

[0063] The surfactant is: 0.4% polysorbate-20 TWEEN-20.

[0064] Example 3

[0065] The first reagent includes: TrisHCL buffer 150mmol / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, sodium azide 0.5g / L, 4-aminoantipyrine 10g / L, pH adjusted to 8.0, protease 200KU / L, ascorbate oxidase 50KU / L;

[0066] The second reagent includes: HEPES buffer 150mmol / L, sodium azide 0.5g / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, pH adjusted to 7.5, fructose amino acid oxidase 100KU / L, peroxidase 50KU / L, color developer N, N-bis (4-sulfobutyl) -3-methylaniline disodium salt (TODB) 10g / L;

[0067] The surfactant is: 0.8% polysorbate-20 TWEEN-20.

[0068] Example 4

[0069] The first reagent includes: TrisHCL buffer 150mmol / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, sodium azide 0.5g / L, 4-aminoantipyrine 10g / L, pH adjusted to 8.0, protease 200KU / L, ascorbate oxidase 50KU / L;

[0070] The second reagent includes: HEPES buffer 150mmol / L, sodium azide 0.5g / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, pH adjusted to 7.5, fructose amino acid oxidase 100KU / L, peroxidase 50KU / L, color developer N, N-bis (4-sulfobutyl) -3-methylaniline disodium salt (TODB) 10g / L;

[0071] The surfactant is: 1.0% polysorbate-20 TWEEN-20.

[0072] Example 5

[0073] The first reagent includes: TrisHCL buffer 150mmol / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, sodium azide 0.5g / L, 4-aminoantipyrine 10g / L, pH adjusted to 8.0, protease 200KU / L, ascorbate oxidase 50KU / L;

[0074] The second reagent includes: HEPES buffer 150mmol / L, sodium azide 0.5g / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, pH adjusted to 7.5, fructose amino acid oxidase 100KU / L, peroxidase 50KU / L, color developer N, N-bis (4-sulfobutyl) -3-methylaniline disodium salt (TODB) 10g / L;

[0075] The surfactant is: 1.5% polysorbate-20 TWEEN-20.

[0076] Example 6

[0077] The first reagent includes: TrisHCL buffer 150mmol / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, sodium azide 0.5g / L, 4-aminoantipyrine 10g / L, pH adjusted to 8.0, protease 50KU / L, ascorbate oxidase 50KU / L;

[0078] The second reagent includes: HEPES buffer 150mmol / L, sodium azide 0.5g / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, pH adjusted to 7.5, fructose amino acid oxidase 100KU / L, peroxidase 50KU / L, color developer N, N-bis (4-sulfobutyl) -3-methylaniline disodium salt (TODB) 10g / L;

[0079] Surfactant: 0.2% polysorbate-20 TWEEN-20,

[0080] Example 7

[0081] The first reagent includes: TrisHCL buffer 150mmol / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, sodium azide 0.5g / L, 4-aminoantipyrine 10g / L, pH adjusted to 8.0, protease 100KU / L, ascorbate oxidase 50KU / L;

[0082] The second reagent includes: HEPES buffer 150mmol / L, sodium azide 0.5g / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, pH adjusted to 7.5, fructose amino acid oxidase 100KU / L, peroxidase 50KU / L, color developer N, N-bis (4-sulfobutyl) -3-methylaniline disodium salt (TODB) 10g / L;

[0083] The surfactant is: 0.2% polysorbate-20 TWEEN-20.

[0084] Embodiment 8:

[0085] The first reagent includes: TrisHCL buffer 150mmol / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, sodium azide 0.5g / L, 4-aminoantipyrine 10g / L, pH adjusted to 8.0, protease 150KU / L, ascorbate oxidase 50KU / L;

[0086] The second reagent includes: HEPES buffer 150mmol / L, sodium azide 0.5g / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, pH adjusted to 7.5, fructose amino acid oxidase 100KU / L, peroxidase 50KU / L, color developer N, N-bis (4-sulfobutyl) -3-methylaniline disodium salt (TODB) 10g / L;

[0087] The surfactant is: 0.2% polysorbate-20 TWEEN-20.

[0088] Example 9

[0089] The first reagent includes: TrisHCL buffer 150mmol / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, sodium azide 0.5g / L, 4-aminoantipyrine 10g / L, pH adjusted to 8.0, protease 200KU / L, ascorbate oxidase 50KU / L;

[0090] The second reagent includes: HEPES buffer 150mmol / L, sodium azide 0.5g / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, pH adjusted to 7.5, fructose amino acid oxidase 100KU / L, peroxidase 50KU / L, color developer N, N-bis (4-sulfobutyl) -3-methylaniline disodium salt (TODB) 10g / L;

[0091] The surfactant is: 0.2% polysorbate-20 TWEEN-20.

[0092] Example 10

[0093] The first reagent includes: TrisHCL buffer 150mmol / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, sodium azide 0.5g / L, 4-aminoantipyrine 10g / L, pH adjusted to 8.0, protease 250KU / L, ascorbate oxidase 50KU / L;

[0094] The second reagent includes: HEPES buffer 150mmol / L, sodium azide 0.5g / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, pH adjusted to 7.5, fructose amino acid oxidase 100KU / L, peroxidase 50KU / L, color developer N, N-bis (4-sulfobutyl) -3-methylaniline disodium salt (TODB) 10g / L;

[0095] The surfactant is: 0.2% polysorbate-20 TWEEN-20.

[0096] Embodiment 11

[0097] The first reagent includes: TrisHCL buffer 150mmol / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, sodium azide 0.5g / L, 4-aminoantipyrine 10g / L, pH adjusted to 8.0, protease 150KU / L, ascorbate oxidase 50KU / L;

[0098] The second reagent includes: HEPES buffer 150mmol / L, sodium azide 0.5g / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, pH adjusted to 7.5, fructose amino acid oxidase 100KU / L, peroxidase 50KU / L, color developer N, N-bis (4-sulfobutyl) -3-methylaniline disodium salt (TODB) 10g / L;

[0099] The surfactants are: 0.2% polysorbate-20 TWEEN-20, 1.0% TX-405.

[0100] Example 12

[0101] The first reagent includes: TrisHCL buffer 150mmol / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, sodium azide 0.5g / L, 4-aminoantipyrine 10g / L, pH adjusted to 8.0, protease 150KU / L, ascorbate oxidase 50KU / L;

[0102] The second reagent includes: HEPES buffer 150mmol / L, sodium azide 0.5g / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, pH adjusted to 7.5, fructose amino acid oxidase 100KU / L, peroxidase 50KU / L, color developer N, N-bis (4-sulfobutyl) -3-methylaniline disodium salt (TODB) 10g / L;

[0103] The surfactants are: 0.2% polysorbate-20 TWEEN-20, 1.0% dextran 40.

[0104] Example 13

[0105] The first reagent includes: TrisHCL buffer 150mmol / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, sodium azide 0.5g / L, 4-aminoantipyrine 10g / L, pH adjusted to 8.0, protease 150KU / L, ascorbate oxidase 50KU / L;

[0106] The second reagent includes: HEPES buffer 150mmol / L, sodium azide 0.5g / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, pH adjusted to 7.5, fructose amino acid oxidase 100KU / L, peroxidase 50KU / L, color developer N, N-bis (4-sulfobutyl) -3-methylaniline disodium salt (TODB) 10g / L;

[0107] The surfactants are: 0.2% polysorbate-20 TWEEN-20, 1.0% TX-100.

[0108] Embodiment 14

[0109] The first reagent includes: TrisHCL buffer 150mmol / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, sodium azide 0.5g / L, 4-aminoantipyrine 10g / L, pH adjusted to 8.0, protease 150KU / L, ascorbate oxidase 50KU / L;

[0110] The second reagent includes: HEPES buffer 150mmol / L, sodium azide 0.5g / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, pH adjusted to 7.5, fructose amino acid oxidase 100KU / L, peroxidase 50KU / L, color developer N, N-bis (4-sulfobutyl) -3-methylaniline disodium salt (TODB) 10g / L;

[0111] The surfactants are: 0.2% polysorbate-20 TWEEN-20, 1.0% X-80.

[0112] Embodiment 15

[0113] The first reagent includes: TrisHCL buffer 150mmol / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, sodium azide 0.5g / L, 4-aminoantipyrine 10g / L, pH adjusted to 8.0, protease 150KU / L, ascorbate oxidase 50KU / L;

[0114] The second reagent includes: HEPES buffer 150mmol / L, sodium azide 0.5g / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, pH adjusted to 7.5, fructose amino acid oxidase 100KU / L, peroxidase 50KU / L, color developer N, N-bis (4-sulfobutyl) -3-methylaniline disodium salt (TODB) 10g / L;

[0115] The surfactants are: 0.2% polysorbate-20 TWEEN-20, 1.0% polyoxyethylene derivative Emulgen B66.

[0116] Example 16

[0117] The first reagent includes: TrisHCL buffer 150mmol / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, sodium azide 0.5g / L, 4-aminoantipyrine 10g / L, pH adjusted to 8.0, protease 150KU / L, ascorbate oxidase 50KU / L;

[0118] The second reagent includes: HEPES buffer 150mmol / L, sodium azide 0.5g / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, pH adjusted to 7.5, fructose amino acid oxidase 100KU / L, peroxidase 50KU / L, color developer N, N-bis (4-sulfobutyl) -3-methylaniline disodium salt (TODB) 10g / L;

[0119] The surfactants are: 0.2% polysorbate-20 TWEEN-20, 0.2% polyoxyethylene derivative Emulgen B66.

[0120] Embodiment 17

[0121] The first reagent includes: TrisHCL buffer 150mmol / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, sodium azide 0.5g / L, 4-aminoantipyrine 10g / L, pH adjusted to 8.0, protease 150KU / L, ascorbate oxidase 50KU / L;

[0122] The second reagent includes: HEPES buffer 150mmol / L, sodium azide 0.5g / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, pH adjusted to 7.5, fructose amino acid oxidase 100KU / L, peroxidase 50KU / L, color developer N, N-bis (4-sulfobutyl) -3-methylaniline disodium salt (TODB) 10g / L;

[0123] The surfactants are: 0.2% polysorbate-20 TWEEN-20, 0.5% polyoxyethylene derivative Emulgen B66.

[0124] Embodiment 18

[0125] The first reagent includes: TrisHCL buffer 150mmol / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, sodium azide 0.5g / L, 4-aminoantipyrine 10g / L, pH adjusted to 8.0, protease 150KU / L, ascorbate oxidase 50KU / L;

[0126] The second reagent includes: HEPES buffer 150mmol / L, sodium azide 0.5g / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, pH adjusted to 7.5, fructose amino acid oxidase 100KU / L, peroxidase 50KU / L, color developer N, N-bis (4-sulfobutyl) -3-methylaniline disodium salt (TODB) 10g / L;

[0127] The surfactants are: 0.2% polysorbate-20 TWEEN-20, 0.8% polyoxyethylene derivative Emulgen B66.

[0128] Embodiment 19

[0129] The first reagent includes: TrisHCL buffer 150mmol / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, sodium azide 0.5g / L, 4-aminoantipyrine 10g / L, pH adjusted to 8.0, protease 150KU / L, ascorbate oxidase 50KU / L;

[0130] The second reagent includes: HEPES buffer 150mmol / L, sodium azide 0.5g / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, pH adjusted to 7.5, fructose amino acid oxidase 100KU / L, peroxidase 50KU / L, color developer N, N-bis (4-sulfobutyl) -3-methylaniline disodium salt (TODB) 10g / L;

[0131] The surfactants are: 0.2% polysorbate-20 TWEEN-20, 1.5% polyoxyethylene derivative Emulgen B66.

[0132] Embodiment 20

[0133] The first reagent includes: TrisHCL buffer 150mmol / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, sodium azide 0.5g / L, 4-aminoantipyrine 10g / L, pH adjusted to 8.0, protease 150KU / L, ascorbate oxidase 50KU / L;

[0134] The second reagent includes: HEPES buffer 150mmol / L, sodium azide 0.5g / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, pH adjusted to 7.5, fructose amino acid oxidase 100KU / L, peroxidase 50KU / L, color developer N, N-bis (4-sulfobutyl) -3-methylaniline disodium salt (TODB) 10g / L;

[0135] The surfactants are: 0.2% polysorbate-20 TWEEN-20, 2.0% polyoxyethylene derivative Emulgen B66.

[0136] Embodiment 21

[0137] The first reagent includes: TrisHCL buffer 150mmol / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, sodium azide 0.5g / L, 4-aminoantipyrine 10g / L, pH adjusted to 8.0, protease 100KU / L, ascorbate oxidase 50KU / L;

[0138] The second reagent includes: HEPES buffer 150mmol / L, sodium azide 0.5g / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, pH adjusted to 7.5, fructose amino acid oxidase 100KU / L, peroxidase 50KU / L, color developer N, N-bis (4-sulfobutyl) -3-methylaniline disodium salt (TODB) 10g / L;

[0139] The surfactants are: 0.1% polysorbate-20 TWEEN-20, 0.5% polyoxyethylene derivative Emulgen B66.

[0140] Embodiment 22

[0141] The first reagent includes: TrisHCL buffer 150mmol / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, sodium azide 0.5g / L, 4-aminoantipyrine 10g / L, pH adjusted to 8.0, protease 100KU / L, ascorbate oxidase 50KU / L;

[0142] The second reagent includes: HEPES buffer 150mmol / L, sodium azide 0.5g / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, pH adjusted to 7.5, fructose amino acid oxidase 100KU / L, peroxidase 50KU / L, color developer N, N-bis (4-sulfobutyl) -3-methylaniline disodium salt (TODB) 10g / L;

[0143] The surfactants are: 0.2% polysorbate-20 TWEEN-20, 0.50% polyoxyethylene derivative Emulgen B66.

[0144] Embodiment 23

[0145] The first reagent includes: TrisHCL buffer 150mmol / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, sodium azide 0.5g / L, 4-aminoantipyrine 10g / L, pH adjusted to 8.0, protease 150KU / L, ascorbate oxidase 50KU / L;

[0146] The second reagent includes: HEPES buffer 150mmol / L, sodium azide 0.5g / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, pH adjusted to 7.5, fructose amino acid oxidase 100KU / L, peroxidase 50KU / L, color developer N, N-bis (4-sulfobutyl) -3-methylaniline disodium salt (TODB) 10g / L;

[0147] The surfactants are: 0.2% polysorbate-20 TWEEN-20, 0.5% polyoxyethylene derivative Emulgen B66.

[0148] Embodiment 24

[0149] The first reagent includes: TrisHCL buffer 150mmol / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, sodium azide 0.5g / L, 4-aminoantipyrine 10g / L, pH adjusted to 8.0, protease 150KU / L, ascorbate oxidase 50KU / L;

[0150] The second reagent includes: HEPES buffer 150mmol / L, sodium azide 0.5g / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, pH adjusted to 7.5, fructose amino acid oxidase 100KU / L, peroxidase 50KU / L, color developer N, N-bis (4-sulfobutyl) -3-methylaniline disodium salt (TODB) 10g / L;

[0151] The surfactants are: 0.5% polysorbate-20 TWEEN-20, 0.5% polyoxyethylene derivative Emulgen B66.

[0152] Embodiment 25

[0153] The first reagent includes: TrisHCL buffer 150mmol / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, sodium azide 0.5g / L, 4-aminoantipyrine 10g / L, pH adjusted to 8.0, protease 150KU / L, ascorbate oxidase 50KU / L;

[0154] The second reagent includes: HEPES buffer 150mmol / L, sodium azide 0.5g / L, EDTA 0.5mmol / L, potassium ferrocyanide 6mg / L, pH adjusted to 7.5, fructose amino acid oxidase 100KU / L, peroxidase 50KU / L, color developer N, N-bis (4-sulfobutyl) -3-methylaniline disodium salt (TODB) 10g / L;

[0155] The surfactants are: 0.5% polysorbate-20 TWEEN-20, 1.0% polyoxyethylene derivative Emulgen B66.

[0156] The reagents of the present application adopt the addition of special protective agents and surfactants and the reduction of protease participation in the reaction to avoid the problems of poor reagent stability caused by large protease dosage and enzyme activity loss during storage in the prior art.

[0157] The following experiments were conducted according to the different reagents prepared according to the above embodiments and placed at 37°C for accelerated destruction for 7 days. The control reagents were from Asahi Denka Co., Ltd. of Japan, and the reagents used in the clinical samples tested were normally stored within the validity period. The reagents of different schemes were calibrated on the same biochemical analyzer for experiments. The results of the specific embodiments are as follows:

[0158] Table 1 is the experimental results of different reagents provided in Examples 1 to 5 calibrated on the same biochemical analyzer

[0159]

[0160]

[0161] See also Figure 1 , represents the protective agent polysorbate-20 different concentration verification experiments of Example 1 to Example 5, the horizontal axis X represents the test results of the control group reagent, the vertical axis Y represents the test results of the example reagent of this scheme, and "XY" represents the data set of two variables, which is used for correlation analysis, regression analysis, etc. By studying the relationship between X and Y, the internal connection and law between the variables can be revealed.

[0162] It can be seen that when 200KU / L protease was added, different concentrations of the protective agent polysorbate-20 were verified. It can be seen that when the addition amount of polysorbate-20 was 0.2%, the deviation between the calibration test results and the imported reagent test results was the smallest, and the correlation was the best.

[0163] Table 2 is the experimental results of different reagents provided in Examples 6 to 10 calibrated on the same biochemical analyzer

[0164]

[0165]

[0166] See also Figure 2 , respectively represent schematic diagrams of verification experiments on the concentration of protease added when the amount of polysorbate-20 added is constant, as provided in Examples 6 to 10.

[0167] It can be seen that the protease concentration was further verified when the amount of polysorbate-20 added was constant. It was obvious that when the protease concentration was 150KU / L, the test results had the best correlation with the imported reagent control, and the relative deviation was lower than that of the other groups.

[0168] Table 3 is the experimental results of different reagents provided in Examples 11 to 15 calibrated on the same biochemical analyzer

[0169]

[0170]

[0171] See also Figure 3 , which are schematic diagrams of experiments on the effects of different surfactants on reagent effects provided in Examples 11 to 15, respectively.

[0172] Table 4 shows the experimental results of different reagents provided in Examples 16 to 20 calibrated on the same biochemical analyzer.

[0173]

[0174]

[0175] See also Figure 4 , respectively represent schematic diagrams of compatibility experiments of polysorbate-20 and polyoxyethylene derivatives at different concentrations provided in Examples 16 to 20.

[0176] From the above, we can see that the performance of the reagent can be further optimized by adding different surfactants. From the above results, we can see that when appropriate polysorbate-20 and protease concentrations are selected, when 1.0% polyoxyethylene derivative B66 is added, the test result data is improved compared with the control group. The correlation R of 50 clinical samples tested can reach above 0.99.

[0177] Table 5 shows the experimental results of different reagents provided in Examples 21 to 25 calibrated on the same biochemical analyzer.

[0178]

[0179]

[0180] See also Figure 5 , which are schematic diagrams of the compatibility experiments of polysorbate-20 and polyoxyethylene derivatives at different concentrations provided in Examples 21 to 25, respectively.

[0181] It can be seen that the performance of the reagent can be optimized by combining the addition of different concentrations of protease and surfactant. From the above results, it can be seen that when 0.1-0.5% polysorbate-20 and 100-150KU / L protease concentrations are selected, and 0.5-1.0% polyoxyethylene derivative B66 is added, the test results data basically meet the requirements compared with the control group. The correlation R-square of 50 clinical samples tested can reach above 0.97.

[0182] Further, using Examples 1, 8, 15, 21, 22, 25 and comparative reagents, the effects of various components of the present application on reagent stability were investigated, and the high and low quality controls were compared with the test data on day 0 by calibration testing. The test results are shown in the following table:

[0183]

[0184]

[0185] As can be seen from the table, in Examples 1, 8, and 15, under the conditions of 37°C acceleration and 2-8°C, the deviation of the test result of Comparative Case 15 from the day 0 is greatly improved compared with the others, indicating that the stability of the reagent has improved after adding a protective agent or a surfactant.

[0186] See also Figure 6 and Figure 7 As can be seen from the figure, in Examples 1, 8, 15, 21, 22, and 25, under the conditions of 37°C acceleration and 2-8°C, the deviation of the test results of Comparative Cases 21, 22, and 25 from the day 0 is greatly improved compared with the others, among which the deviation of the test results of Example 15 is the smallest, indicating that the stability of the reagent is better after adding a protective agent or a surfactant.

[0187] It can be understood that the technical features of the above-described embodiments can be arbitrarily combined. In order to make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0188] The above are only preferred embodiments of the present application, and only specifically describe the technical principles of the present application. These descriptions are only for explaining the principles of the present application and cannot be interpreted as limiting the scope of protection of the present application in any way. Based on the explanation here, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application, and other specific implementation methods of the present application that can be associated with the technicians in this field without creative work, should be included in the scope of protection of the present application.

Claims

1. A glycated albumin concentration detection reagent, characterized in that: The method comprises a first reagent and a second reagent, wherein the volume ratio of the first reagent to the second reagent is 4:1, wherein: The first reagent includes substances with the following concentrations: 50-150 mmol / L buffer, 0.5-5 mmol / L EDTA, 6-10 mg / L potassium ferrocyanide, 0.5-1 g / L sodium azide, 10-250 KU / L protease, 0.1-10 g / L 4-aminoantipyrine and 1-20 g / L surfactant; The first reagent includes substances with the following concentrations: 50-150 mmol / L buffer, 0.5-5 mmol / L EDTA, 6-10 mg / L potassium ferrocyanide, 0.5-1 g / L sodium azide, 20-100 KU / L fructose amino acid oxidase, 1-100 KU / L peroxidase and 0.1-10 g / L color developer.

2. The glycated albumin concentration detection reagent according to claim 1, characterized in that: The buffer is one of TrisHCL buffer, phosphate buffer, Tricine buffer, HEPES buffer and MES buffer.

3. The glycated albumin concentration detection reagent according to claim 1, characterized in that: The protease is selected from one of alkaline protease, papain, subtilisin and proteinase K.

4. The glycated albumin concentration detection reagent according to claim 1, characterized in that: The surfactants include polysorbate-20, dextran 40, sodium dodecyl sulfate SDS, polyoxyethylene derivative Emulgen B66, Triton X-100, Triton X-405, betaine and polyethylene glycol monoalkyl ether One or more of X-080.

5. The glycated albumin concentration detection reagent according to claim 4, characterized in that: The surfactant is polysorbate-20, and the added amount of the polysorbate-20 is 0.1-0.5%.

6. The glycated albumin concentration detection reagent according to claim 5, characterized in that: The protease concentration is 100-150 KU / L.

7. The glycated albumin concentration detection reagent according to claim 6, characterized in that: The surfactant also includes a polyoxyethylene derivative B66, and the addition amount of the polyoxyethylene derivative B66 is 0.5-1.0%.

8. The glycated albumin concentration detection reagent according to claim 1, characterized in that: The color developer is selected from one or two of N,N-bis(4-sulfobutyl)-3-methylaniline disodium salt, N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt, and 3-(N-ethyl-3-methylanilino)-2-hydroxypropanesulfonic acid sodium salt.

9. A method for detecting the glycated albumin concentration detection reagent according to claim 1, characterized in that: The steps include: Adding the first reagent to the glycated albumin sample to be tested and mixing to obtain a first mixed solution, and incubating the first mixed solution and then reading a first absorbance A1; Adding the second reagent to the first mixed solution and mixing well to obtain a second mixed solution, and incubating the second mixed solution and then reading a second absorbance A2; An absorbance change value ΔA is obtained based on the first absorbance and the second absorbance, where ΔA=[(A2-A1) calibration tube / sample tube]-[(A2-A1) blank tube].

10. The method for detecting the glycated albumin concentration detection reagent according to claim 6, characterized in that: The incubation temperature is 37° C. and the incubation time is 5 minutes.

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