Glycated albumin detection kit, preparation method and application method thereof

Abstract

The invention discloses a saccharified albumin detection kit, a preparation method and an application method thereof. The saccharified albumin detection kit includes R1 reagent, R2 reagent and R3 reagent, R1 detection reagent includes a first buffer, fructose amino acid oxidase, catalase, vitamin C, a preservative, a stabilizer, R2 detection reagent includes a second buffer, sodium carbonate, 3-methylbenzothiazole sulfone hydrazone, calcium chloride, EDTA, and R3 detection reagent includes a third buffer, alkaline protease, proteinase K, fructose amino acid oxidase, peroxidase, polyethylene glycol PEG-400, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt. The present invention increases the accuracy of the measurement result by arranging a reagent that eliminates the influence of endogenous saccharified amino acids in advance, adds polyethylene glycol to the measurement reagent, increases the affinity between the generated hydrogen peroxide and the developer, increases the reaction rate, and reduces the technical problem that the measurement result is low due to the decomposition of hydrogen peroxide.

Description

Technical Field

[0001] The present invention relates to the technical field of clinical medical parameter detection, and in particular to a glycated albumin detection kit, a preparation method and an application method thereof. Background Art

[0002] Serum albumin is the product of non-enzymatic glycosylation reaction between glucose in the blood and the N-terminus of serum albumin. It is an important indicator that can reflect the average blood sugar level in the past 2-3 weeks. It has more outstanding advantages in confirming the therapeutic effect and adjusting the clinical dosage. Therefore, the accuracy of its measurement results is of great significance.

[0003] The detection methods of glycated albumin mainly include affinity chromatography, high performance liquid chromatography, immunoassay, enzyme method, etc. Among them, chromatography and high performance liquid chromatography require complex pretreatment of the samples to be tested. At the same time, the instruments are expensive, time-consuming, and costly, and are not suitable for routine clinical analysis. The conventional method for clinical detection of glycated albumin is mainly the enzyme method, namely the fructose amino acid oxidase method, also known as the ketoamine oxidase method.

[0004] The main process of the ketoaminoxidase method is: adding protease to the serum or plasma to be tested to decompose glycated albumin into amino acids and glycated amino acids; the glycated amino acids obtained by the decomposition of glycated albumin are decomposed into glucose ketoaldehyde, amino acids and hydrogen peroxide under the action of ketoaminoxidase; the generated hydrogen peroxide reacts with the color developer to generate a substance of specific color, the absorbance is measured, and the concentration of glycated albumin is calculated.

[0005] At present, a variety of kits for the detection of glycated albumin have been developed, such as the technical solutions disclosed in Chinese patents with application numbers 201410401260.X, 201811120574.7 and 201911287909.9. These kits basically include two reagents. First, glycated albumin is decomposed to obtain a solution containing glycated amino acids. Then, hydrogen peroxide is generated under the action of an enzyme. The hydrogen peroxide reacts with a color developer to produce a pigment corresponding to the concentration of glycated albumin. The concentration of glycated albumin is obtained based on the absorbance.

[0006] However, the existing kit for measuring glycated albumin has the following problems: the protease activity is not high, the influence of endogenous glycated amino acids cannot be eliminated, the reaction between hydrogen peroxide and pigment lags behind the generation process of hydrogen peroxide, resulting in partial decomposition of hydrogen peroxide, making the measurement result low, and the pigment generated by hydrogen peroxide and the developer is not stable enough, resulting in inaccurate measurement results of glycated albumin. Therefore, it is necessary to develop a glycated albumin detection kit, preparation method and application method thereof that can solve the above problems. Summary of the invention

[0007] In view of this, the present invention provides a glycated albumin detection kit, a preparation method and an application method thereof, which increases the accuracy of the measurement results by setting a reagent for first eliminating the influence of endogenous glycated amino acids, and adds polyethylene glycol to the measurement reagent to increase the affinity between the generated hydrogen peroxide and the color developer, thereby increasing the reaction rate and reducing the technical problem of low measurement results caused by the decomposition of hydrogen peroxide.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a glycated albumin detection kit, the glycated albumin detection kit comprising R1 reagent, R2 reagent and R3 reagent;

[0010] The components and concentrations of the R1 reagent are as follows:

[0011]

[0012] The components and concentrations of the R2 reagent are as follows:

[0013]

[0014] The components and concentrations of the R3 reagent are as follows:

[0015]

[0016]

[0017] Preferably, the first buffer comprises a tris buffer at pH 8, the second buffer comprises a tris buffer at pH 10, and the third buffer comprises a tris buffer at pH 7.

[0018] Preferably, the preservative is proclin 300 or sodium azide.

[0019] Preferably, the stabilizer is trehalose or sucrose.

[0020] In a second aspect, the present invention provides a method for preparing a glycated albumin detection kit, the method for preparing the glycated albumin detection kit comprising the following steps:

[0021] Prepare the volume of R1 reagent as needed, calculate and weigh the ingredients according to the following ratio: first buffer 50-150mmol / L, fructose amino acid oxidase 5-100KU / L, catalase 2-10KU / L, vitamin C 10-100mmol / L, preservative 1-5mL / L, stabilizer 1-5g / L, dissolve in the corresponding volume of deionized water to obtain R1 reagent;

[0022] Prepare the volume of R2 reagent as needed, calculate and weigh the ingredients according to the following ratio: 60-120 mmol / L of second buffer, 10-100 mmol / L of sodium carbonate, 5-20 mmol / L of 3-methylbenzothiazole sulfone hydrazone, 0.5-1 g / L of calcium chloride, 1-2 g / L of EDTA, 1-5 mL / L of preservative, 1-5 g / L of stabilizer, and dissolve them in the corresponding volume of deionized water to obtain R2 reagent;

[0023] The volume of R3 reagent is prepared as needed, and the ingredients are calculated and weighed according to the following ratio: third buffer 50-100 mmol / L, alkaline protease 1-5 KU / L, proteinase K 5-10 KU / L, fructose amino acid oxidase 50-200 KU / L, peroxidase 10-30 KU / L, polyethylene glycol PEG-400 1-8 g / L, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt 2-20 mmol / L, preservative 1-5 mL / L, stabilizer 1-5 g / L, and dissolved in the corresponding volume of deionized water to obtain R3 reagent.

[0024] Preferably, the components of the R1 reagent are added in the order of first buffer, stabilizer, preservative, fructose amino acid oxidase, catalase, and vitamin C.

[0025] Preferably, the components of the R2 reagent are added in the order of the second buffer, EDTA, calcium chloride, 3-methylbenzothiazole sulfone hydrazone, stabilizer, preservative, and sodium carbonate.

[0026] Preferably, the components of the R3 reagent are added in the following order: third buffer, stabilizer, preservative, polyethylene glycol PEG-400, fructose amino acid oxidase, peroxidase, alkaline protease, proteinase K, and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt.

[0027] In a third aspect, the present invention provides an application method of a glycated albumin detection kit, the application method comprising the following steps:

[0028] Mixing the sample to be tested with the R1 reagent;

[0029] After a certain period of time, add the R2 reagent, shake and mix, and measure the absorbance;

[0030] After a certain period of time, add the R3 reagent, shake and mix, and measure the absorbance;

[0031] The concentration of glycated albumin in the sample to be tested is calculated according to the absorbance.

[0032] Compared with the prior art, the present invention has the following beneficial technical effects: ① it can eliminate the influence of endogenous glycated amino acids in the blood sample to be tested to the greatest extent, thereby improving the accuracy of the measurement; ② it can improve the activity of the protease and accelerate the enzymatic hydrolysis rate of glycated albumin; ③ it can coordinate the generation rate and consumption rate of hydrogen peroxide, reduce the decomposition of hydrogen peroxide, and improve the accuracy of the measurement; ④ it can increase the stability of the pigment generated by hydrogen peroxide and the color developer, thereby improving the accuracy of the measurement. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is described in detail below in conjunction with embodiments.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] The present invention provides a glycated albumin detection kit, a preparation method and an application method thereof, which increases the accuracy of the measurement result by setting a reagent for first eliminating the influence of endogenous glycated amino acids, and adds polyethylene glycol to the measurement reagent to increase the affinity between the generated hydrogen peroxide and the color developer, thereby increasing the reaction rate and reducing the technical problem of low measurement results caused by the decomposition of hydrogen peroxide.

[0038] The present invention provides a glycated albumin detection kit, which includes an R1 reagent, an R2 reagent and an R3 reagent;

[0039] The components and concentrations of R1 reagent are as follows:

[0040]

[0041] The components and concentrations of R2 reagent are as follows:

[0042]

[0043] The components and concentrations of R3 reagent are as follows:

[0044]

[0045] Preferably, the first buffer comprises a tris buffer at pH 8, the second buffer comprises a tris buffer at pH 10, and the third buffer comprises a tris buffer at pH 7.

[0046] Preferably, the preservative is proclin 300 or sodium azide.

[0047] Preferably, the stabilizer is trehalose or sucrose.

[0048] It should be noted that the main function of the R1 reagent is to decompose the endogenous glycated amino acids in the sample to be tested into glucose ketoaldehyde, amino acids and hydrogen peroxide, and consume the hydrogen peroxide corresponding to the endogenous glycated amino acids, thereby eliminating the presence of endogenous glycated amino acids in the determination of glycated albumin concentration.

[0049] The function of fructose amino acid oxidase in R1 reagent is to decompose endogenous glycated amino acids.

[0050] Vitamin C and catalase in the R1 reagent can completely consume the hydrogen peroxide produced by the decomposition of endogenous glycated amino acids. Vitamin C has reducing properties and can react with oxidative hydrogen peroxide under certain pH conditions; catalase can also promote the decomposition of hydrogen peroxide into oxygen and water, thereby eliminating the influence of endogenous glycated amino acids.

[0051] It should be noted that the sodium carbonate in the R2 reagent not only further stabilizes the pH value of the measurement system in the alkaline range of 10-11, but also reacts with the excess vitamin C in the R1 reagent, causing the vitamin C to react with oxygen in the air in an alkaline environment and lose its reducing property, which will not affect the accuracy of the hydrogen peroxide measurement in the next step. Sodium carbonate also does not react with hydrogen peroxide, so it does not affect the measurement results.

[0052] It should be noted that 3-methylbenzothiazole sulfone hydrazone (MBTH) is added to the R2 reagent as a color developer, and the pigment formed by it, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt (TOOS) and hydrogen peroxide is more stable.

[0053] It should be noted that the addition of polyethylene glycol PEG-400 to the R3 reagent has the following effects: it enables the generation of hydrogen peroxide to be coupled with the reaction to prevent the decomposition or overflow of hydrogen peroxide, which would lead to a low measurement result; it also has the effect of stabilizing the purple or blue dye formed by the oxidative coupling reaction of TOOS and 3-methylbenzothiazole sulfone hydrazone (MBTH), hydrogen peroxide, and peroxidase.

[0054] It should be noted that there is a synergistic interaction between sodium carbonate, EDTA, vitamin C and calcium chloride. When the reagents are prepared and stored, EDTA forms a chelate structure with calcium, and the calcium ions do not form precipitation with carbonate ions. During the determination process, after the carbonate ions in the R2 reagent react with the vitamin C in the R1 reagent, the pH of the determination system decreases, causing EDTA to release the chelated calcium and generate free calcium ions, which is beneficial to improving enzyme activity and maintaining stability.

[0055] In some embodiments, the concentration of the first buffer in the R1 reagent can be any value between 50-150 mmol / L, for example, 50 mmol / L, 70 mmol / L, 90 mmol / L, 110 mmol / L, 130 mmol / L, or 150 mmol / L.

[0056] In some embodiments, the concentration of fructose amino acid oxidase in the R1 reagent can be any value between 5-100 KU / L, for example, 5 KU / L, 10 KU / L, 20 KU / L, 30 KU / L, 40 KU / L, 50 KU / L, 60 KU / L, 70 KU / L, 80 KU / L, 90 KU / L, 100 KU / L.

[0057] In some embodiments, the concentration of catalase in the R1 reagent may be any value between 2-10 KU / L, for example, 2 KU / L, 4 KU / L, 6 KU / L, 8 KU / L, or 10 KU / L.

[0058] In some embodiments, the concentration of vitamin C in the R1 reagent can be any value between 10-100 mmol / L, for example, 10 mmol / L, 20 mmol / L, 30 mmol / L, 40 mmol / L, 50 mmol / L, 60 mmol / L, 70 mmol / L, 80 mmol / L, 90 mmol / L, 100 mmol / L.

[0059] In some embodiments, the concentration of the preservative in the R1 reagent may be any value between 1-5 mL / L, for example, 1 mL / L, 2 mL / L, 3 mL / L, 4 mL / L, or 5 mL / L.

[0060] In some embodiments, the concentration of the stabilizer in the R1 reagent may be any value between 1-5 g / L, for example, 1 g / L, 2 g / L, 3 g / L, 4 g / L, or 5 g / L.

[0061] In some embodiments, the concentration of the second buffer in the R2 reagent can be any value between 60-120 mmol / L, for example, 60 mmol / L, 70 mmol / L, 80 mmol / L, 90 mmol / L, 100 mmol / L, 110 mmol / L, or 120 mmol / L.

[0062] In some embodiments, the concentration of sodium carbonate in the R2 reagent can be any value between 10-100 mmol / L, for example, 10 mmol / L, 20 mmol / L, 30 mmol / L, 40 mmol / L, 50 mmol / L, 60 mmol / L, 70 mmol / L, 80 mmol / L, 90 mmol / L, 100 mmol / L.

[0063] In some embodiments, the concentration of 3-methylbenzothiazole sulfone hydrazone in the R2 reagent can be any value between 5-20 mmol / L, for example, 5 mmol / L, 7 mmol / L, 10 mmol / L, 12 mmol / L, 14 mmol / L, 16 mmol / L, 18 mmol / L, or 20 mmol / L.

[0064] In some embodiments, the concentration of calcium chloride in the R2 reagent can be any value between 0.5-1 g / L, for example, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, or 1 g / L.

[0065] In some embodiments, the concentration of EDTA in the R2 reagent can be any value between 1-2 g / L, for example, 1 g / L, 1.2 g / L, 1.4 g / L, 1.6 g / L, 1.8 g / L, or 1 g / L.

[0066] In some embodiments, the concentration of the third buffer in the R3 reagent can be any value between 50-100 mmol / L, for example, 50 mmol / L, 60 mmol / L, 70 mmol / L, 80 mmol / L, 90 mmol / L, or 100 mmol / L.

[0067] In some embodiments, the concentration of alkaline protease in the R3 reagent can be any value between 1-5 KU / L, for example, 1 KU / L, 2 KU / L, 3 KU / L, 4 KU / L, or 5 KU / L.

[0068] In some embodiments, the concentration of proteinase K in the R3 reagent can be any value between 5-10 KU / L, for example, 5 KU / L, 6 KU / L, 7 KU / L, 8 KU / L, or 10 KU / L.

[0069] In some embodiments, the concentration of fructose amino acid oxidase in the R3 reagent can be any value between 50-200 KU / L, for example, 50 KU / L, 60 KU / L, 70 KU / L, 80 KU / L, 100 KU / L, 120 KU / L, 140 KU / L, 160 KU / L, 180 KU / L, 200 KU / L.

[0070] In some embodiments, the concentration of peroxidase in the R3 reagent can be any value between 10-30 KU / L, for example, 10 KU / L, 12 KU / L, 14 KU / L, 16 KU / L, 18 KU / L, 20 KU / L, 22 KU / L, 24 KU / L, 26 KU / L, 30 KU / L.

[0071] In some embodiments, the concentration of polyethylene glycol PEG-400 in the R3 reagent can be any value between 1-8 g / L, for example, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L.

[0072] In some embodiments, the concentration of N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt in the R3 reagent can be any value between 2-20 mmol / L, for example, 2 mmol / L, 4 mmol / L, 6 mmol / L, 7 mmol / L, 9 mmol / L, 11 mmol / L, 12 mmol / L, 14 mmol / L, 15 mmol / L, 18 mmol / L, 20 mmol / L.

[0073] The present invention also provides a method for preparing a glycated albumin detection kit, and the method for preparing the glycated albumin detection kit comprises the following steps:

[0074] Prepare the volume of R1 reagent as needed, calculate and weigh the ingredients according to the following ratio: first buffer 50-150mmol / L, fructose amino acid oxidase 5-100KU / L, catalase 2-20KU / L, vitamin C 10-100mmol / L, preservative 1-5mL / L, stabilizer 1-5g / L, dissolve in the corresponding volume of deionized water to obtain R1 reagent;

[0075] Prepare the volume of R2 reagent as needed, calculate and weigh the ingredients according to the following ratio: 60-120 mmol / L of second buffer, 10-100 mmol / L of sodium carbonate, 5-20 mmol / L of 3-methylbenzothiazole sulfone hydrazone, 0.5-1 g / L of calcium chloride, 1-2 g / L of EDTA, 1-5 mL / L of preservative, 1-5 g / L of stabilizer, and dissolve them in the corresponding volume of deionized water to obtain R2 reagent;

[0076] The volume of R3 reagent is prepared as needed, and the ingredients are calculated and weighed according to the following ratio: third buffer 50-100 mmol / L, alkaline protease 1-5 KU / L, proteinase K 5-10 KU / L, fructose amino acid oxidase 50-200 KU / L, peroxidase 10-30 KU / L, polyethylene glycol PEG-400 1-8 g / L, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt 2-20 mmol / L, preservative 1-5 mL / L, stabilizer 1-5 g / L, and dissolved in the corresponding volume of deionized water to obtain R3 reagent.

[0077] Preferably, the components of the R1 reagent are added in the order of first buffer, stabilizer, preservative, fructose amino acid oxidase, catalase, and vitamin C.

[0078] Preferably, the components of the R2 reagent are added in the order of the second buffer, EDTA, calcium chloride, 3-methylbenzothiazole sulfone hydrazone, stabilizer, preservative, and sodium carbonate.

[0079] Preferably, the components of the R3 reagent are added in the following order: third buffer, stabilizer, preservative, polyethylene glycol PEG-400, fructose amino acid oxidase, peroxidase, alkaline protease, proteinase K, and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt.

[0080] In one specific embodiment, the specific preparation process of the glycated albumin detection kit is as follows:

[0081] Take the preparation of 1.0L R1 reagent as an example: first, measure 0.8L water, add the first buffer according to the set ratio, and after it is completely dissolved, add the stabilizer and preservative in the set ratio, then add fructose amino acid oxidase and catalase, and finally add vitamin C. After it is completely dissolved, use water to make the volume to 1.0L, and seal it for storage to reduce oxygen entering the R1 reagent and consuming vitamin C;

[0082] Take the preparation of 1.0L of R2 reagent as an example: first, measure 0.8L of water, add the second buffer according to the set ratio, add EDTA and calcium chloride after complete dissolution, stir at room temperature for 30-60min, then add 3-methylbenzothiazole sulfone hydrazone, stabilizer and preservative in sequence, dissolve sodium carbonate in 0.1L of water, dissolve sodium carbonate to obtain sodium carbonate solution, then add the sodium carbonate solution dropwise to R2 reagent, control the dropping speed between 5-10 drops / min to avoid the formation of calcium carbonate precipitation, and finally dilute to 1.0L with water to obtain R2 reagent;

[0083] Take the preparation of R3 reagent with a volume of 1.0L as an example: first measure 0.7L of water, add the third buffer according to the set ratio, and after it is completely dissolved, add the stabilizer and the preservative, dissolve polyethylene glycol PEG-400 in 0.2L of water to obtain polyethylene glycol PEG-400 solution, slowly add the polyethylene glycol PEG-400 solution to the solution, and then add fructose amino acid oxidase, peroxidase, alkaline protease and proteinase K, and then add N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt to the solution, stir well, and wait for complete dissolution to obtain R3 reagent.

[0084] Based on the same inventive concept, the present invention also provides an application method of a glycated albumin detection kit, the application method comprising the following steps:

[0085] Mixing the sample to be tested with the R1 reagent;

[0086] After a certain period of time, add the R2 reagent, shake and mix, and measure the absorbance;

[0087] After a certain period of time, add the R3 reagent, shake and mix, and measure the absorbance;

[0088] The concentration of glycated albumin in the sample to be tested is calculated according to the absorbance.

[0089] In a specific embodiment, the measurement is performed using a fully automatic biochemical analyzer, and the specific use process of the glycated albumin detection kit is as follows:

[0090] Add 3-6 μL of the sample to be tested to 20-40 μL of R1 reagent, shake and mix at 37°C, incubate for 4-7 minutes, and keep shaking during the incubation process;

[0091] Add 140-180 μL of R2 reagent, shake and mix at 37°C, incubate for 5-10 min, and measure the absorbance A1. The main wavelength is 546 nm and the secondary wavelength is 700 nm.

[0092] Add 70-90 μL of R3 reagent, shake and mix at 37°C for 4-6 minutes, and measure the absorbance A2. The main wavelength is 546 nm and the secondary wavelength is 700 nm.

[0093] ΔA=A2-A1 is calculated, and then the concentration of glycated albumin in the sample to be tested is calculated based on the obtained standard curve.

[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] Examples 1-6

[0096] The R1, R2 and R3 reagents of the glycated albumin detection kit of the present invention were prepared according to the following steps and conditions.

[0097] Prepare the volume of R1 reagent as needed, calculate and weigh the ingredients of the following components according to the specific components and ratios shown in Table 1, and prepare according to the following method: first measure 0.8 times the volume of water to be prepared R1 reagent, add the first buffer according to the set ratio, and after it is completely dissolved, add the stabilizer and preservative in the set ratio, then add fructose amino acid oxidase and catalase, and finally add vitamin C. After it is completely dissolved, use water to make up the volume, seal and store, and obtain the R1 reagent of the corresponding embodiment;

[0098] Table 1 Preparation of the R1 reagent of the present invention, each group distribution ratio

[0099]

[0100] Prepare the volume of R2 reagent as needed, calculate and weigh the ingredients of the following components according to the specific components and ratios shown in Table 2, and prepare according to the following method: first measure 0.8 times the volume of R1 reagent to be prepared, add the second buffer according to the set ratio, and after complete dissolution, add EDTA and calcium chloride, stir at room temperature for 30-60 minutes, then add 3-methylbenzothiazole sulfone hydrazone, stabilizer and preservative in sequence, dissolve sodium carbonate in water with 0.1 times the volume of R1 reagent to be prepared, dissolve sodium carbonate to obtain sodium carbonate solution, and then add the sodium carbonate solution dropwise to R2 reagent, the dropping speed is controlled between 5-10 drops / min to avoid the formation of calcium carbonate precipitation, and finally dilute with water to obtain R2 reagent;

[0101] Table 2 Preparation of the R2 reagent of the present invention, the distribution ratio and preparation conditions of each group

[0102]

[0103] Prepare the volume of R3 reagent as needed, calculate and weigh the ingredients of the following components according to the specific components and ratios shown in Table 3, and prepare according to the following method: first measure 0.7 times the volume of water to be prepared R1 reagent, add the third buffer according to the set ratio, and after complete dissolution, add stabilizers and preservatives, dissolve polyethylene glycol PEG-400 in 0.2 times the volume of water to be prepared R1 reagent to obtain polyethylene glycol PEG-400 solution, slowly add the polyethylene glycol PEG-400 solution to the solution, then add fructose amino acid oxidase, peroxidase, alkaline protease and proteinase K, and then add N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt to the solution, stir well, and wait for complete dissolution to obtain R3 reagent;

[0104] Table 3 Preparation of the R3 reagent of the present invention, each group distribution ratio

[0105]

[0106]

[0107] The glycated albumin detection kit obtained in Examples 1 to 6 was tested using the following detection method:

[0108] Add 5 μL of the sample to be tested to 30 μL of R1 reagent, shake and mix at 37°C, and incubate for 6 min. Keep shaking during the incubation process.

[0109] Add 160 μL of R2 reagent, shake and mix at 37°C, incubate for 8 min, and measure the absorbance A1. The main wavelength is 546 nm and the secondary wavelength is 700 nm.

[0110] Add 90 μL of R3 reagent, shake and mix at 37°C for 5 min, and measure the absorbance A2. The main wavelength is 546 nm and the secondary wavelength is 700 nm.

[0111] ΔA=A2-A1 is calculated, and then the concentration of glycated albumin in the sample to be tested is calculated based on the obtained standard curve.

[0112] The detection reagent corresponding to each embodiment was used to perform multiple tests on a batch of samples to be tested, and the calculated detection limit and precision results are shown in Table 4. The detection limit and precision calculation method of the test results were carried out using the existing method, and the concentration of glycated albumin in the sample to be tested was determined using the existing high-performance liquid chromatography method as a benchmark for testing the accuracy of the test kit of the present invention. The parameter for measuring the accuracy was the deviation rate of the test result (the absolute value of the difference between the test result of the kit and the test result of the high-performance liquid chromatography method as a percentage of the test result of the high-performance liquid chromatography method)

[0113] Table 4 Test results of the detection kit of the present invention

[0114]

[0115] Comparative Example 1

[0116] The various components and proportions of the kit were prepared according to Example 3, but R1 reagent was not prepared, sodium carbonate, EDTA, and calcium chloride were not added to R2 reagent, and polyethylene glycol PEG-400 was not added to R3 reagent. The same determination method as in the example was used to determine the same sample to be tested. The test results of the detection reagent obtained in this comparative example 1 are as follows: detection limit 3.0 U / L, precision 7.5%, and accuracy 12.1%.

Claims

1. A glycated albumin detection kit, characterized in that: The glycated albumin detection kit includes R1 reagent, R2 reagent and R3 reagent; The components and concentrations of the R1 reagent are as follows: The first buffer 50-150mmol / L Fructose amino acid oxidase 5-100KU / L Catalase 2-10KU / L Vitamin C 10-100mmol / L Preservative 1-5mL / L Stabilizer 1-5g / L; The components and concentrations of the R2 reagent are as follows: Second buffer 60-120mmol / L Sodium carbonate 10-100mmol / L 3-Methylbenzothiazole sulfone hydrazone 5-20mmol / L Calcium chloride 0.5-1g / L EDTA 1-2g / L Preservative 1-5mL / L Stabilizer 1-5g / L; The components and concentrations of the R3 reagent are as follows: The third buffer 50-100mmol / L Alkaline protease 1-5KU / L Proteinase K 5-10KU / L Fructose amino acid oxidase 50-200KU / L Peroxidase 10-30KU / L Polyethylene glycol PEG-400 1-8g / L N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt 2-20mmol / L Preservative 1-5mL / L Stabilizer 1-5g / L; The pH value of the assay system composed of the R1 reagent, the R2 reagent and the R3 reagent is 10-11.

2. The glycated albumin detection kit according to claim 1, characterized in that: The first buffer comprises a tris buffer at pH 8, the second buffer comprises a tris buffer at pH 10, and the third buffer comprises a tris buffer at pH 7.

3. The glycated albumin detection kit according to claim 1, characterized in that: The preservative is proclin 300 or sodium azide.

4. The glycated albumin detection kit according to claim 1, characterized in that: The stabilizer is trehalose or sucrose.

5. A method for preparing a glycated albumin detection kit, characterized in that: The preparation method of the glycated albumin detection kit comprises the following steps: Prepare the volume of R1 reagent as needed, calculate and weigh the ingredients according to the following ratio: first buffer 50-150mmol / L, fructose amino acid oxidase 5-100KU / L, catalase 2-10KU / L, vitamin C 10-100mmol / L, preservative 1-5mL / L, stabilizer 1-5g / L, dissolve in the corresponding volume of deionized water to obtain R1 reagent; Prepare the volume of R2 reagent as needed, calculate and weigh the ingredients according to the following ratio: 60-120 mmol / L of second buffer, 10-100 mmol / L of sodium carbonate, 5-20 mmol / L of 3-methylbenzothiazole sulfone hydrazone, 0.5-1 g / L of calcium chloride, 1-2 g / L of EDTA, 1-5 mL / L of preservative, 1-5 g / L of stabilizer, and dissolve them in the corresponding volume of deionized water to obtain R2 reagent; The volume of R3 reagent is prepared as needed, and the ingredients are calculated and weighed according to the following ratio: third buffer 50-100 mmol / L, alkaline protease 1-5 KU / L, proteinase K 5-10 KU / L, fructose amino acid oxidase 50-200 KU / L, peroxidase 10-30 KU / L, polyethylene glycol PEG-400 1-8 g / L, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt 2-20 mmol / L, preservative 1-5 mL / L, stabilizer 1-5 g / L, and dissolved in the corresponding volume of deionized water to obtain R3 reagent.

6. The method for preparing the glycated albumin detection kit according to claim 5, characterized in that: The components of the R1 reagent are added in the following order: first buffer, stabilizer, preservative, fructose amino acid oxidase, catalase, and vitamin C.

7. The method for preparing the glycated albumin detection kit according to claim 5, characterized in that: The components of the R2 reagent are added in the following order: second buffer, EDTA, calcium chloride, 3-methylbenzothiazole sulfone hydrazone, stabilizer, preservative, and sodium carbonate.

8. The method for preparing the glycated albumin detection kit according to claim 5, characterized in that: The components of the R3 reagent are added in the following order: third buffer, stabilizer, preservative, polyethylene glycol PEG-400, fructose amino acid oxidase, peroxidase, alkaline protease, proteinase K, and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt.

9. An application method of the glycated albumin detection kit according to claim 1, characterized in that: The application method comprises the following steps: Mixing the sample to be tested with the R1 reagent; After a certain period of time, add the R2 reagent, shake and mix, and measure the absorbance; After a certain period of time, add the R3 reagent, shake and mix, and measure the absorbance; The concentration of glycated albumin in the sample to be tested is calculated according to the absorbance.

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