Glycated albumin detection kit and preparation method thereof

Abstract

The invention relates to the field of in vitro diagnostic reagents. The invention discloses a glycated albumin detection kit. The glycated albumin detection kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises the following components: a first buffer solution, a first pH adjusting solution, inorganic salt, a chromogen substrate, a first biological enzyme, a first surfactant, an anti-interference agent and a stabilizer; and the R2 reagent comprises the following components: a second buffer solution, a second pH adjusting solution, a Trinder reaction substrate, a second biological enzyme and a metal ion chelating agent. Based on the same inventive concept, the invention also provides a preparation method of the glycated albumin detection kit. According to the glycated albumin detection kit provided by the invention, the surfactant octadecyl trimethyl ammonium chloride is added to be compounded with cocamidopropyl betaine, so that the use concentration of the glycated albumin detection kit in the R1 reagent is determined, the use amount of protease is greatly reduced, the reagent cost is reduced, and meanwhile, the sensitivity is good, and the test accuracy is high.

Description

Technical Field

[0001] The present invention relates to the field of in vitro diagnostic reagents, and in particular to a glycated albumin detection kit and a preparation method thereof. Background Art

[0002] Glycated albumin (GA) is a product of the glycosylation of serum albumin and is defined as albumin containing lysine residues bound to glucose (glycated lysine).

[0003] Albumin is the most abundant protein in human blood, with a molecular mass of 67 kDa and a biological half-life of about 20 days. Human serum albumin contains 59 lysine residues (Lys). Four of these lysine residues (Lys) are the main sites for glycosylation. Human serum albumin (HSA) is the most abundant protein in human plasma or serum. This protein is usually present in serum at a concentration of 30 to 50 g / , accounting for about 60% of the total protein content in serum. The molecular weight of HSA is 66.7 kDa, and it consists of a polypeptide chain consisting of 585 amino acids and 17 disulfide chains. The crystal structure of HSA reveals a globular protein composed of approximately 67% α-helix, 23% extended chain, and 10% β-turn. The N-terminus and 59 lysine residues can serve as potential sites for the formation of early glycosylation products of this protein.

[0004] Glycated albumin is an indicator that reflects the average blood sugar level in the past 2-3 weeks. It has a shorter response period than the "gold standard" of blood sugar testing, glycosylated hemoglobin. Therefore, GA has advantages over glycosylated hemoglobin in confirming the treatment effect and adjusting the clinical dosage.

[0005] Common detection methods for GA detection include immunoassay, chromatography, colorimetry and enzyme methods. The above detection methods determine the content of glycated albumin by measuring different target molecules. Therefore, there will be significant differences in the measurement results. Among them, the immunoassay method mainly uses the chemical steric effect of antibodies as the main analysis basis; the chromatography method cannot be widely used due to the disadvantages of long test time and certain limitations in the test sample range; when the colorimetric method is tested, it may be affected by uric acid and lipemia factors, resulting in low accuracy of the test value. Compared with the above detection methods, the detection advantage of the enzyme method is more obvious. It can realize the centralized detection of a large number of samples with the help of a fully automated detector, and the sample is serum, which is the same as the test sample of other items. There is no need to collect samples multiple times, which provides great convenience for clinical examination. In addition, the advantages of this detection method are also reflected in the fact that it will not be affected and interfered by other factors of the patient's body, and the test results are relatively stable. It is one of the detection methods with more extensive clinical application at this stage.

[0006] The principle of enzyme reagent testing is that the specific protease in the reagent first acts on the glycated albumin in the sample to obtain small molecule products, glycated amino acids or glycated peptides. The small molecule product is used as the substrate for the second step reaction. Under the catalysis of ketoamine oxidase, the product hydrogen peroxide is generated. Under the action of peroxidase, hydrogen peroxide combines with 4-aminoantipyrine and chromogen to undergo Trinder reaction, deoxygenation condensation to generate colored substances. The amount of colored substances generated is proportional to the content of glycated albumin in the sample, so the content of glycated albumin in serum can be determined. In order to meet the inspection requirements of automatic biochemical analyzers, the entire reaction must be completed within 10 minutes. In order for subsequent reactions to proceed synchronously and stably, the generation rate of glycated amino acids often requires a high concentration of protease to ensure that the reagent cost is increased, and the risk of reagent storage stability is increased. Due to the non-specific recognition nature of proteases, high concentrations will cause the decomposition of other protein components in the reagent during the long-term storage of the reagent, resulting in reagent failure.

[0007] In existing research, Chinese patent CN107870170A provides a method for reducing the non-specific color development of the reagent Trinder component and causing the blank to increase. Chinese patent CN101297044A uses a quaternary ammonium or its salt albumin denaturant with a hydrocarbon group having more than 12 carbon atoms to improve the efficiency of protease decomposition of albumin in the sample; Chinese patent CN104614459A discloses a method for rapid enzymatic hydrolysis of saccharified albumin using ionic liquids; Chinese patent CN109212238A adds an enzyme activator composed of three components of MgCl2, glutathione, and EDTA to the R2 reagent, which effectively improves the analytical sensitivity of the kit; Chinese patent CN109837255A provides a method for chemically modifying ketoamine oxidase, and the ketoamine oxidase chemically modified by this method can maintain a high enzyme activity and is not easily degraded by protease. The research directions of each patent are to improve the stability of the reagent and the catalytic efficiency of the enzyme. However, there are still problems in the stability of the reagent, which need to be solved urgently. Summary of the invention

[0008] The purpose of the present invention is to provide a saccharified albumin detection kit and a preparation method thereof in view of the deficiencies in the prior art. The present invention uses enzymatic reaction as the test principle, further improves the catalytic efficiency of the enzyme and the storage stability of the reagent by compounding with a surfactant, starts from reducing the concentration of the protease, and after comparative studies, it is found that the use of octadecyl trimethyl ammonium chloride compounded with cocamidopropyl betaine in the R1 reagent can specifically decompose albumin, reduce the amount of protease, and has no significant effect on other components of the reagent, thereby improving the storage stability of the reagent.

[0009] To achieve the above-mentioned object of the invention, the technical solution of the present invention is implemented as follows: A glycated albumin detection kit, comprising R1 reagent and R2 reagent; wherein: The R1 reagent comprises the following components: a first buffer solution, a first pH adjusting solution, an inorganic salt, a chromogen substrate, a first biological enzyme, a first surfactant, an anti-interference agent, and a stabilizer; The R2 reagent includes the following components: a second buffer solution, a second pH regulating solution, a Trinder reaction substrate, a second biological enzyme, and a metal ion chelating agent.

[0010] In the above-mentioned glycated albumin detection kit, the anti-interference agent includes a first anti-interference agent, a second anti-interference agent and a third anti-interference agent, and the R1 reagent includes the following components in the following concentrations: First buffer 50-100 mM / L; pH 7.5-8.5; Inorganic salt 8-20g / L; Chromogen substrate 0.5-1g / L; First biological enzyme 50-400KU / L; The first surfactant 0-4%; The first anti-interference agent is 50-100KU / L; The second anti-interference agent 2-5g / L; The third anti-interference agent 0-10g / L; Stabilizer 10-30g / L; The R2 reagent includes the following components at the following concentrations: Second buffer 50-100 mM / L; pH 6.5-8.0; Trinder reaction substrate 0.5-2g / L; The second biological enzyme is 50-100KU / L; Metal ion chelator 0.5-2g / L.

[0011] Preferably, in the above-mentioned glycated albumin detection kit, the anti-interference agent includes a first anti-interference agent, a second anti-interference agent and a third anti-interference agent, and the R1 reagent includes the following components in the following concentrations: First buffer 100 mM / L; pH 7.8-8.0; Inorganic salt 9-14.5g / L; Chromogen substrate 0.6 g / L; First biological enzyme 100-300KU / L; First surfactant 3.5%; The first anti-interference agent is 50KU / L; The second anti-interference agent 2.5g / L; The third anti-interference agent 0-10g / L; Stabilizer 20g / L; The R2 reagent includes the following components at the following concentrations: Second buffer 100 mM / L; pH 7.0; Trinder reaction substrate 0.5 g / L; The second biological enzyme 80KU / L; Metal ion chelator 0.5g / L.

[0012] Most preferably, in the above-mentioned glycated albumin detection kit, the anti-interference agent includes a first anti-interference agent, a second anti-interference agent and a third anti-interference agent, and the R1 reagent includes the following components in concentration: TRIS buffer 100 mM / L; pH 8.0; Sodium chloride 9g / L; Calcium chloride 5.5 g / L; TOOS0.6g / L; Proteinase K 100 KU / L; Octadecyltrimethylammonium chloride 1.5%; Cocamidopropyl Betaine 2%; Ascorbate oxidase 50KU / L; Potassium ferrocyanide 2.5g / L; PEG600010g / L; Mannitol 20g / L; The R2 reagent includes the following components at the following concentrations: TRIS buffer solution 100 mM / L; pH 7.4-7.8; 4-aminoantipyrine 0.5 g / L; Peroxidase 80KU / L; Glycosylated amino acid oxidase 10KU / L; EDTA-Na20.5g / L.

[0013] In the glycated albumin detection kit as described above, the first surfactant is at least one of cocamidopropyl betaine, octadecyltrimethylammonium chloride, laurylamidopropyl betaine, palmitamidopropyl betaine, hexadecyltrimethylammonium chloride, dodecyltrimethylammonium chloride, octadecyldimethylbenzylammonium chloride, and hexadecyldimethylbenzylammonium chloride, or a mixture of more thereof.

[0014] As described above, the first surfactant is a mixture of cocamidopropyl betaine and octadecyl trimethyl ammonium chloride. Under this condition, the surfactant cocamidopropyl betaine added in the present invention is compounded with octadecyl trimethyl ammonium chloride, which acts on the substrate albumin, improves the enzymolysis efficiency, reduces the use concentration of the protease, and maintains the high sensitivity and wide linear range of the reagent. Based on the single denaturation means of albumin in the previous similar methodology, the present invention provides a rapid denaturation method for combining albumin surface charge and internal hydrophobic structure by a surfactant, so as to reduce the reagent cost and improve the reagent stability.

[0015] In the above-mentioned glycated albumin detection kit, the mass ratio of cocamidopropyl betaine to octadecyl trimethyl ammonium chloride is (1:4) to (4:1). Preferably, the mass ratio of cocamidopropyl betaine to octadecyl trimethyl ammonium chloride is (3:4) to (4:3). Under this condition, the kit has good linear relationship, high analytical sensitivity, good repeatability and high accuracy. Most preferably, the mass ratio of octadecyl trimethyl ammonium chloride to cocamidopropyl betaine is 3:4.

[0016] In the above-mentioned glycated albumin detection kit, the inorganic salt is any one of sodium chloride, calcium chloride, and potassium chloride, or a mixture of several of them. Preferably, the inorganic salt is any one of sodium chloride and calcium chloride, or a mixture of two of them. Optionally, the inorganic salt is sodium chloride; Optionally, the inorganic salt is a mixture of sodium chloride and calcium chloride, and the mass ratio of sodium chloride to calcium chloride is 18:11.

[0017] In the above-mentioned glycated albumin detection kit, the first biological enzyme is proteinase K; the concentration of proteinase K is 100-300 KU / L.

[0018] In the above-mentioned glycated albumin detection kit, the first anti-interference agent is ascorbic acid oxidase, the second anti-interference agent is potassium ferrocyanide, and the third anti-interference agent is PEG6000. Preferably, the concentration of the ascorbic acid oxidase is 50KU / L, the concentration of the potassium ferrocyanide is 2.5g / L, and the concentration of the PEG6000 is 10g / L.

[0019] In the above-mentioned glycated albumin detection kit, the first buffer and the second buffer are any one of TRIS buffer, phosphate buffer, HEPES buffer, and citric acid buffer, or a mixture of several thereof. Preferably, the first buffer and the second buffer are both TRIS buffer or both HEPES buffer.

[0020] Based on the same inventive concept, the present invention provides a method for preparing a glycated albumin detection kit, comprising the following steps: (1) Preparation of R1 reagent According to the component content of the R1 reagent, a first buffer solution is prepared, and the pH is adjusted to 7.5-8.5 using a first pH adjusting solution, and then an inorganic salt, a chromogen substrate, a first biological enzyme, a first surfactant, an anti-interference agent, and a stabilizer are added in sequence, and stirred evenly to obtain the R1 reagent; (2) Preparation of R2 reagent According to the component content of R2 reagent, prepare the second buffer solution, adjust the pH to 6.5-8.0 with the second pH adjusting solution, then add the Trinder reaction substrate, the second biological enzyme, and the metal ion chelator in sequence, stir evenly, and obtain R2 reagent.

[0021] Compared with the existing technology, the effects and advantages of the present invention are: 1. The glycated albumin detection kit provided by the present invention determines the use concentration in the R1 reagent by adding the surfactant octadecyltrimethylammonium chloride to compound cocamidopropyl betaine, greatly reduces the amount of protease used, reduces the reagent cost, and has good sensitivity and high test accuracy. It has high consistency with the measured values ​​of commercially available reagents, and the correlation coefficient is greater than 0.99.

[0022] 2. The glycated albumin detection kit provided by the present invention uses ascorbic acid oxidase and potassium ferrocyanide to reduce the consumption of the main reaction products by reducing substances (ascorbic acid, uric acid, bilirubin, glucose) in the sample, so that the Trinder reaction is more complete, the false negative of the test result is reduced, and the accuracy of the reagent is further improved.

[0023] 3. The glycated albumin detection kit provided by the present invention uses PEG6000 to eliminate the interference of globulin. PEG6000 can specifically bind to globulin and avoid the binding reaction between protease and globulin.

[0024] 4. The preparation method of the glycated albumin detection kit provided by the present invention has a simple preparation process and low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a linear curve graph of experimental group 5 of the present invention; Figure 2 This is a correlation diagram between the experimental group 5 of the present invention and the measured values ​​of commercially available reagents. DETAILED DESCRIPTION

[0026] The following will combine the contents in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in this specification includes any and all combinations of one or more of the related listed items.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0029] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0030] Embodiment 1: The present embodiment provides a detection kit for glycated albumin, comprising R1 reagent and R2 reagent, and the preparation method thereof comprises the following steps: (1) Preparation of R1 reagent According to the component content of the R1 reagent, a first buffer solution is prepared, and the pH is adjusted to 8.0 using a first pH adjusting solution, and then an inorganic salt, a chromogen substrate, a first biological enzyme, an anti-interference agent, and a stabilizer are added in sequence, and stirred evenly to obtain the R1 reagent; In this embodiment, the first buffer solution is a TRIS buffer solution prepared by dissolving TRIS in water, the first pH adjusting solution is a 6 mol / L hydrochloric acid solution adjusted to pH 8.0, the inorganic salt is sodium chloride, the chromogen substrate is N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt (hereinafter referred to as "TOOS"), the first biological enzyme is proteinase K, the first anti-interference agent is ascorbic acid oxidase, the second anti-interference agent is potassium ferrocyanide, and the stabilizer is mannitol. This embodiment does not contain the first surfactant.

[0031] After preparation, the final concentrations of the components of R1 are: TRIS buffer solution 100mM / L pH 8.0 Sodium chloride 9g / L TOOS0.6g / L Proteinase K300KU / L Ascorbate oxidase 50KU / L Potassium ferrocyanide 2.5g / L Mannitol 20g / L (2) Preparation of R2 reagent According to the component content of the R2 reagent, a second buffer solution is prepared, and the pH is adjusted to 7.6 using a second pH adjusting solution, and then the Trinder reaction substrate, the second biological enzyme, and the metal ion chelator are added in sequence, and stirred evenly to obtain the R2 reagent; In this embodiment, the second buffer solution is a HEPES buffer solution prepared by dissolving HEPES in water, the second pH adjusting solution is a 6 mol / L hydrochloric acid solution, the Trinder reaction substrate is 4-aminoantipyrine, the second biological enzyme is peroxidase and glycosylated amino acid oxidase, and the metal ion chelator is EDTA-Na2.

[0032] After preparation, the final concentrations of the components of R2 are: HEPES buffer solution 100mM / L pH 7.6 4-aminoantipyrine 0.5g / L Peroxidase 80KU / L Glycosylated amino acid oxidase 10KU / L EDTA-Na20.5g / L Embodiment 2: Referring to the preparation method of the glycated albumin detection kit of Example 1, the kits of the following multiple experimental groups were prepared according to the following table, wherein implementation group 1 is the technical solution of Example 1.

[0033]

[0034] Among them, the kits of experimental groups 1 to 5 were subjected to subsequent kit linearity, analytical sensitivity, repeatability, and accuracy experiments together; the kits of experimental group 1, experimental group 5, and Example 5 were subjected to subsequent anti-interference experiments together; the kits of Example 1, Example 3, Example 4, and Example 5 were subjected to subsequent kit stability experiments together.

[0035] Embodiment 3: The present embodiment provides a detection kit for glycated albumin, comprising R1 reagent and R2 reagent, and the preparation method thereof comprises the following steps: (1) Preparation of R1 reagent According to the component content of the R1 reagent, a first buffer solution is prepared, and the pH is adjusted to 7.8 using a first pH adjusting solution, and then an inorganic salt, a chromogen substrate, a first biological enzyme, an anti-interference agent, a first surfactant, and a stabilizer are added in sequence, and stirred evenly to obtain the R1 reagent; In the present embodiment, the first buffer solution is a HEPES buffer solution prepared by dissolving HEPES in water, the first pH adjusting solution is a 6 mol / L hydrochloric acid solution, the inorganic salt is sodium chloride, the chromogenic substrate is TOOS, the first biological enzyme is proteinase K, the first anti-interference agent is ascorbic acid oxidase, the second anti-interference agent is potassium ferrocyanide, the first surfactant is 1.5% by mass of octadecyltrimethylammonium chloride and 2% of cocamidopropyl betaine, and the stabilizer is mannitol.

[0036] After preparation, the final concentrations of the components of R1 are: HEPES buffer solution 100mM / L pH 7.8 Sodium chloride 9g / L TOOS0.6g / L Proteinase K100KU / L Ascorbate oxidase 50KU / L Potassium ferrocyanide 2.5g / L Octadecyltrimethylammonium chloride 1.5% Cocamidopropyl Betaine 2% Mannitol 20g / L (2) Preparation of R2 reagent According to the component content of the R2 reagent, a second buffer solution is prepared, and the pH is adjusted to 7.0 using a second pH adjusting solution, and then the Trinder reaction substrate, the second biological enzyme, and the metal ion chelating agent are added in sequence, and stirred evenly to obtain the R2 reagent; In this embodiment, the second buffer solution is a TRIS buffer solution prepared by dissolving TRIS in water, the second pH adjusting solution is a 6 mol / L hydrochloric acid solution, the Trinder reaction substrate is 4-aminoantipyrine, the second biological enzyme is peroxidase and glycosylated amino acid oxidase, and the metal ion chelator is EDTA-Na2.

[0037] After preparation, the final concentrations of the components of R2 are: TRIS buffer solution 100mM / L pH 7.0 4-aminoantipyrine 0.5g / L Peroxidase 80KU / L Glycosylated amino acid oxidase 10KU / L EDTA-Na20.5g / L Embodiment 4: The present embodiment provides a detection kit for glycated albumin, comprising R1 reagent and R2 reagent, and the preparation method thereof comprises the following steps: (1) Preparation of R1 reagent According to the component content of the R1 reagent, a first buffer solution is prepared, and the pH is adjusted to 8.0 using a first pH adjusting solution, and then an inorganic salt, a chromogen substrate, a first biological enzyme, an anti-interference agent, a first surfactant, and a stabilizer are added in sequence, and stirred evenly to obtain the R1 reagent; In this embodiment, the first buffer solution is a TRIS buffer solution prepared by dissolving TRIS in water, the first pH adjusting solution is a 6 mol / L hydrochloric acid solution, the inorganic salts are sodium chloride and calcium chloride, the chromogen substrate is TOOS, the first biological enzyme is proteinase K, the first anti-interference agent is ascorbic acid oxidase, the second anti-interference agent is potassium ferrocyanide, the first surfactant is 1.5% by mass of octadecyltrimethylammonium chloride and 2% of cocamidopropyl betaine, and the stabilizer is mannitol.

[0038] After preparation, the final concentrations of the components of R1 are: TRIS buffer solution 100mM / L pH 8.0 Sodium chloride 9g / L Calcium chloride 5.5g / L TOOS0.6g / L Proteinase K100KU / L Ascorbate oxidase 50KU / L Potassium ferrocyanide 2.5g / L Octadecyltrimethylammonium chloride 1.5% Cocamidopropyl Betaine 2% Mannitol 20g / L (2) Preparation of R2 reagent According to the component content of the R2 reagent, a second buffer solution is prepared, and the pH is adjusted to 7.0 using a second pH adjusting solution, and then the Trinder reaction substrate, the second biological enzyme, and the metal ion chelating agent are added in sequence, and stirred evenly to obtain the R2 reagent; In this embodiment, the second buffer solution is a TRIS buffer solution prepared by dissolving TRIS in water, the second pH adjusting solution is a 6 mol / L hydrochloric acid solution, the Trinder reaction substrate is 4-aminoantipyrine, the second biological enzyme is peroxidase and glycosylated amino acid oxidase, and the metal ion chelator is EDTA-Na2.

[0039] After preparation, the final concentrations of the components of R2 are: TRIS buffer solution 100mM / L pH 7.0 4-aminoantipyrine 0.5g / L Peroxidase 80KU / L Glycosylated amino acid oxidase 10KU / L EDTA-Na20.5g / L Embodiment 5: The present embodiment provides a detection kit for glycated albumin, comprising R1 reagent and R2 reagent, and the preparation method thereof comprises the following steps: (1) Preparation of R1 reagent According to the component content of the R1 reagent, a first buffer solution is prepared, and the pH is adjusted to 8.0 using a first pH adjusting solution, and then an inorganic salt, a chromogen substrate, a first biological enzyme, an anti-interference agent, a first surfactant, and a stabilizer are added in sequence, and stirred evenly to obtain the R1 reagent; In this embodiment, the first buffer solution is a TRIS buffer solution prepared by dissolving TRIS in water, the first pH adjusting solution is a 6 mol / L hydrochloric acid solution, the inorganic salts are sodium chloride and calcium chloride, the chromogen substrate is TOOS, the first biological enzyme is proteinase K, the first anti-interference agent is ascorbic acid oxidase, the second anti-interference agent is potassium ferrocyanide, the third anti-interference agent is PEG6000, the first surfactant is 1.5% by mass of octadecyltrimethylammonium chloride and 2% of cocamidopropyl betaine, and the stabilizer is mannitol.

[0040] After preparation, the final concentrations of the components of R1 are: TRIS buffer solution 100mM / L pH 8.0 Sodium chloride 9g / L Calcium chloride 5.5g / L TOOS0.6g / L Proteinase K100KU / L Ascorbate oxidase 50KU / L Potassium ferrocyanide 2.5g / L PEG600010g / L Octadecyltrimethylammonium chloride 1.5% Cocamidopropyl Betaine 2% Mannitol 20g / L (2) Preparation of R2 reagent According to the component content of the R2 reagent, a second buffer solution is prepared, and the pH is adjusted to 7.0 using a second pH adjusting solution, and then the Trinder reaction substrate, the second biological enzyme, and the metal ion chelating agent are added in sequence, and stirred evenly to obtain the R2 reagent; In this embodiment, the second buffer solution is a TRIS buffer solution prepared by dissolving TRIS in water, the second pH adjusting solution is a 6 mol / L hydrochloric acid solution, the Trinder reaction substrate is 4-aminoantipyrine, the second biological enzyme is peroxidase and glycosylated amino acid oxidase, and the metal ion chelator is EDTA-Na2.

[0041] After preparation, the final concentrations of the components of R2 are: TRIS buffer solution 100mM / L pH 7.0 4-aminoantipyrine 0.5g / L Peroxidase 80KU / L Glycosylated amino acid oxidase 10KU / L EDTA-Na20.5g / L Example 6 Application of Glycated Albumin Detection Kit A detection method of the above-mentioned glycated albumin detection kit in this embodiment comprises the following steps: Take Hitachi 7600 operation as an example: the measurement wavelength is 600nm, the calibration volume is (4uL), add R1 reagent (160uL), mix, incubate at 37℃ for 5 minutes, read the absorbance value A1, add R2 reagent (40uL) and mix, incubate at 37℃ for 5 minutes, read the absorbance value A2, calculate the absorbance difference △A=A2-A1, repeat the measurement twice for each tube, take the average value of the absorbance difference △A measured twice for each calibration tube as the ordinate, and the corresponding calibration concentration as the abscissa, and draw the "concentration-absorbance difference" calibration curve. Take the serum or plasma sample to be tested, determine the absorbance difference of the sample in the same way, substitute it into the calibration curve, and the content of glycated albumin (GA) in the sample to be tested can be calculated. If the concentration of glycated albumin in the sample exceeds the range of the calibration curve, the sample needs to be diluted and then tested to ensure the accuracy of the test result. This test kit is not only suitable for Hitachi 7600, but also for other brands and models of semi-automatic and fully automatic biochemical analyzers. The specific parameters can be adjusted according to the instrument.

[0042] Example 7 Quality Evaluation of Glycated Albumin Detection Kit (1) Linear range A. Experimental Methods The linear range is 3.8-28.8g / L. Use a concentrated sample close to the high value of the linearity (28.42g / L) and a sample close to the low value of the linearity (3.62g / L), and mix them in a ratio of 1:0; 5:1; 1:1; 1:5; 0:1 for the high value: low value to obtain a batch of mixed samples. Use the above detection procedure to determine the concentration. Each sample is measured 2-3 times, and the average value is taken. A linear graph is drawn based on the average value and the theoretical value, and the correlation coefficient r and linear deviation are calculated. Correlation coefficient |r| ≥ 0.9900; in the range of [3.8, 14.4] g / L, the absolute linear deviation should not exceed ±1.4g / L; in the range of (14.4, 28.8] g / L, the relative linear deviation should not exceed ±10%.

[0043] The linearity test results of the kit are shown in Table 1.

[0044] Table 1 Linearity test results

[0045] As shown in the results of Table 1, the linear correlation coefficients and deviations of experimental groups 1-5 are within the acceptable range, and the linearity meets the requirements. The linear curve of experimental group 5 is shown in Figure 1 The results of experimental group 5 were the best.

[0046] (2) Analytical sensitivity A. Experimental Methods When the concentration of the tested glycated albumin sample was 15 g / L, its absorbance changed between 0.02 and 0.20.

[0047] B. Experimental Results and Analysis Table 2 Analytical sensitivity test results

[0048] As shown in the results of Table 2, the analytical sensitivities of experimental groups 1-5 all met the requirements, among which the sensitivity of experimental group 5 was closest to the control group (experimental group 1).

[0049] (3) Repeatability testing A. Experimental Methods The samples with a glycated albumin concentration of 7.0 g / L ± 1.0 g / L were tested 10 times, and the mean (Mean) and standard deviation (SD) were calculated, and the coefficient of variation (CV) was calculated. The repeatability of the kits in experimental groups 1-5 was investigated, and the acceptable range of CV was ± 8%.

[0050] B. Experimental Results and Analysis Table 3 Repeatability test results

[0051] As shown in the results of Table 3, the coefficients of variation CV of experimental groups 1-5 are all within an acceptable range, indicating that the components and their concentration ranges involved in the present invention can have good in-bottle uniformity, and the CV result of the kit of experimental group 5 is the best.

[0052] (4) Accuracy test A. Experimental Methods Using commercially available reagents as comparison reagents, 40 human samples of different concentrations within the detection concentration range were tested, and each sample was tested separately according to the above operation method and comparison method. The correlation coefficient (r) of the results and the relative deviation or absolute deviation of each concentration point were calculated using the linear regression method. In the range of 3.8-28.8g / L, the correlation coefficient r≥0.9900.

[0053] B. Experimental Results and Analysis Table 4 Accuracy test results

[0054] The correlation between experimental group 5 and the measured values ​​of commercially available reagents is as follows Figure 2 As shown, the measured values ​​have good linear correlation within the test interval.

[0055] As shown in the results of Table 4, comparing the linear correlation coefficients of experimental groups 1-5 with the measured values ​​of commercially available reagent samples, the results of experimental group 5 are closest to the control group (experimental group 1).

[0056] (5) Anti-interference performance test A. Experimental Methods Take healthy serum samples, accurately measure high concentration interfering substances and mix them with samples (the final volume of the interfering substances is 5%, and a solvent control is prepared) to prepare mixed samples with different interfering substance concentrations. Each sample is measured 3 times and the average value is taken. The interfering substances used in this experiment are 10g / L ascorbic acid, 50g / L hemoglobin, 6000μmol / L bilirubin, 10g / L glucose, and 10g / L uric acid. The preparation process of the reagents used is Experimental Group 1, Experimental Group 5, and Example 5 in Example 2.

[0057] The calculation method of interference degree is: Interference degree = (mean value of samples with interference substance added - mean value of control samples without interference substance added solvent) / mean value of control samples without interference substance added solvent × 100% The acceptable interference range is ±10%.

[0058] B. Experimental Results and Analysis Table 5 Test results of kit interference (%)

[0059] As shown in Table 5, the interference degree of the samples added with interference substances in Experimental Group 5 and Example 5 can be controlled within ±10% compared with the control (Experimental Group 1), indicating that the reagent has a certain anti-interference ability. The data show that the anti-interference ability of Example 5 is better, which should be because the anti-interference agent reacts with the reducing substance in the sample to avoid its consumption of the reaction product H2O2, thereby achieving the purpose of anti-interference. When a certain concentration of interfering substances such as bilirubin, hemoglobin, ascorbic acid, glucose and uric acid exist in the test sample, the measurement results of the kit of the present invention are less affected.

[0060] (6) Accelerated stability test A. Accelerated stability test method At the same time, the glycated albumin detection kits of Example 1, Example 3, Example 4, and Example 5 were placed in a 37° C. water bath, and were taken out on 3 days, 5 days, and 7 days respectively for calibration and testing together with the normally stored reagents. The quality control products were tested 3 times each, and the relative deviation was calculated.

[0061] B. Experimental Results and Analysis Table 6 Accelerated stability test results

[0062] As shown in the results of Table 6, the relative deviations of the measured values ​​were calculated by comparing the reagents stored at 37°C for 3, 5, and 7 days with those of the reagents stored normally. The deviations of Example 5 were all within an acceptable range and were superior to other solutions.

[0063] (7) Opening stability test A. Opening stability test method At the same time, the glycated albumin detection kits of Example 1, Example 3, Example 4, and Example 5 were placed in an automatic biochemical analyzer and stored with the lid opened for 30 days. Two levels of quality control products were calibrated and tested on the day of opening. The quality control products were tested 3 times each on the 5th, 8th, 11th, 15th, 20th, 22nd, 24th, 26th, 28th, and 30th day after opening together with the normally stored reagents, and the relative deviation was calculated.

[0064] B. Experimental Results and Analysis Table 7 Opening stability test results

[0065] As shown in the results of Table 7, the relative deviations of the measured values ​​were calculated by comparing the reagents on the day of opening with those on days 5, 11, 15, 20, 26 and 30 after opening, and the deviations of Example 5 were all within an acceptable range. The result of Example 5 was the best, indicating that the buffer system and components used in Example 5 were stable during the opening process and were not disturbed by air and moisture. This may be due to the formation of a micellar structure on the liquid surface with the appropriate surfactant concentration and the buffer system, which increases the surface tension of the liquid and avoids the interaction of moisture and air components with the main components of the reagent.

[0066] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A glycated albumin detection kit, characterized in that: Comprising R1 reagent and R2 reagent; wherein: The R1 reagent comprises the following components: a first buffer solution, a first pH adjusting solution, an inorganic salt, a chromogen substrate, a first biological enzyme, a first surfactant, an anti-interference agent, and a stabilizer; The R2 reagent includes the following components: a second buffer solution, a second pH regulating solution, a Trinder reaction substrate, a second biological enzyme, and a metal ion chelating agent.

2. The glycated albumin detection kit according to claim 1, characterized in that: The anti-interference agent includes a first anti-interference agent, a second anti-interference agent and a third anti-interference agent, and the R1 reagent includes the following components: First buffer 50-100 mM / L; pH 7.5-8.5; Inorganic salt 8-20g / L; Chromogen substrate 0.5-1g / L; First biological enzyme 50-400KU / L; The first surfactant 0-4%; The first anti-interference agent is 50-100KU / L; The second anti-interference agent 2-5g / L; The third anti-interference agent 0-10g / L; Stabilizer 10-30g / L; The R2 reagent includes the following components in the following concentrations: Second buffer 50-100 mM / L; pH 6.5-8.0; Trinder reaction substrate 0.5-2g / L; The second biological enzyme is 50-100KU / L; Metal ion chelator 0.5-2g / L.

3. The glycated albumin detection kit according to claim 2, characterized in that: The first surfactant is at least one of cocamidopropyl betaine, octadecyltrimethylammonium chloride, laurylamidopropyl betaine, palmitamidopropyl betaine, hexadecyltrimethylammonium chloride, dodecyltrimethylammonium chloride, octadecyldimethylbenzylammonium chloride, and hexadecyldimethylbenzylammonium chloride, or a mixture of more thereof.

4. The glycated albumin detection kit according to claim 3, characterized in that: The first surfactant is a mixture of cocamidopropyl betaine and octadecyl trimethyl ammonium chloride.

5. The glycated albumin detection kit according to claim 4, characterized in that: The mass ratio of cocamidopropyl betaine to octadecyltrimethylammonium chloride is (1:4) to (4:1).

6. The glycated albumin detection kit according to claim 2, characterized in that: The inorganic salt is any one of sodium chloride, calcium chloride and potassium chloride or a mixture of several of them.

7. The glycated albumin detection kit according to claim 2, characterized in that: The first biological enzyme is proteinase K; the concentration of proteinase K is 100-300 KU / L.

8. The glycated albumin detection kit according to claim 2, characterized in that: The first anti-interference agent is ascorbic acid oxidase, the second anti-interference agent is potassium ferrocyanide, and the third anti-interference agent is PEG6000.

9. The glycated albumin detection kit according to claim 2, characterized in that: The first buffer and the second buffer are any one of TRIS buffer, phosphate buffer, HEPES buffer, and citrate buffer, or a mixture of several of them.

10. The method for preparing the glycated albumin detection kit according to any one of claims 1 to 9, characterized in that: The following steps are involved: (1) Preparation of R1 reagent According to the component content of the R1 reagent, a first buffer solution is prepared, and the pH is adjusted to 7.5-8.5 using a first pH adjusting solution, and then an inorganic salt, a chromogen substrate, a first biological enzyme, a first surfactant, an anti-interference agent, and a stabilizer are added in sequence, and stirred evenly to obtain the R1 reagent; (2) Preparation of R2 reagent According to the component content of R2 reagent, prepare the second buffer solution, adjust the pH to 6.5-8.0 with the second pH adjusting solution, then add the Trinder reaction substrate, the second biological enzyme, and the metal ion chelator in sequence, stir evenly, and obtain R2 reagent.

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