Glycosylated protein fluid, preparation method and application thereof, and fructosamine detection kit

Abstract

The invention relates to the technical field of biological detection, and discloses a glycosylated protein fluid, a preparation method and application thereof and a fructosamine detection kit.The preparation method comprises the following steps that human serum albumin, bovine serum albumin and glucose with the mass ratio being (70-80): (20-30) are added into a buffer solution with the pH ranging from 7.0 to 8.0, incubation is conducted for 14 days to 28 days at the temperature of 36 DEG C to 38 DEG C in a dark place, an obtained reaction solution is purified, and the glycosylated protein fluid is obtained. A glycosylated protein solution is obtained; the ratio of the total protein mass to the glucose mass is 2: 1. The specific proportion of the human serum albumin and the bovine serum albumin can generate a synergistic effect, and an optimal balance is achieved between reduction of a heterologous matrix effect and improvement of stability, so that a calibrator prepared from the glycosylated protein liquid can simultaneously and remarkably improve the correction capability on reagent batch difference and instrument difference. Meanwhile, by controlling the pH value, the temperature and the time and adopting a purification process, the glycosylated protein fluid can have good stability.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to a glycosylated protein solution, its preparation method and application, and a fructoamine detection kit. Background Technology

[0002] Fructosamine (also known as glycated serum protein) is a product of the non-enzymatic glycation reaction between glucose and proteins (mainly albumin) in the blood. Its concentration can effectively reflect the average blood glucose level of diabetic patients over the past 2-3 weeks and is an important indicator for the diagnosis and monitoring of diabetes. In clinical testing, the accuracy and comparability of test results are core to ensuring the quality of medical care.

[0003] Currently, clinical fructosamine testing faces a prominent problem: consistency of results. Significant differences in test results arise between different batches of reagents (batch-to-batch variation) and between different instrument platforms (instrument-to-instrument variation), reducing the reliability and comparability of the results. Specifically, commercially available fructosamine test kits often use chemically synthesized fructosamine analogs, such as fructose morpholine as a calibrator. While these substances are chemically stable and highly pure, as small molecules, their reaction kinetics differ fundamentally from the large glycosylated proteins found in clinical samples. Specifically, in fructosamine detection based on the nitrotetrazole blue (NBT) reduction method, the Amadori rearrangement process and colorimetric reaction of glycosylated proteins in an alkaline environment are significantly affected by the protein matrix environment. Small-molecule synthetic calibrators cannot simulate this complex matrix effect, leading to inconsistencies in their reaction behavior compared to real serum samples. Therefore, existing calibrators have limited ability to correct for systematic errors introduced by reagent batch-to-batch and instrument-to-instrument variations, failing to achieve true standardization of testing.

[0004] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a glycosylated protein solution, its preparation method and application, and a fructosamine detection kit, in order to solve the problem that existing calibrators for fructosamine detection have limited ability to correct systematic errors introduced by reagent batch-to-batch differences and instrument-to-instrument differences.

[0006] The technical solution of the present invention is as follows: A first aspect of the present invention provides a method for preparing a glycosylated protein solution, comprising the following steps: Human serum albumin, bovine serum albumin, and glucose were added to a buffer solution with a pH of 7.0-8.0 and incubated at 36-38°C in the dark for 14-28 days to obtain the reaction solution. The mass ratio of human serum albumin to bovine serum albumin was (70-80):(20-30), and the ratio of the total mass of human serum albumin and bovine serum albumin to the mass of glucose was 2:1. After purifying the reaction solution, the glycosylated protein solution is obtained.

[0007] Optionally, the total concentration of human serum albumin and bovine serum albumin in the reaction solution is 150-250 g / L; The buffer solution includes at least one of phosphate buffer and HEPES buffer; The concentration of the buffer solution is 50~200 mM.

[0008] In a second aspect, the present invention provides a glycosylated protein solution, wherein the solution is prepared by the preparation method described above.

[0009] A third aspect of the present invention provides the application of the glycosylated protein solution described above in the preparation of fructosamine calibrators or fructosamine quality control products.

[0010] A fourth aspect of the present invention provides a method for preparing a fructosamine calibrator, comprising the following steps: The glycosylated protein solution of the present invention as described above is added to a stabilizer solution containing trehalose and histidine to obtain a mixture; in the stabilizer solution, the concentration of trehalose is 10-50 mM and the concentration of histidine is 5-25 mM. The mixture is the fructosamine calibrator; or, the mixture is freeze-dried to obtain the fructosamine calibrator.

[0011] In a fifth aspect, the present invention provides a fructoamine calibrator, wherein it is prepared by the preparation method of the present invention as described above.

[0012] In a sixth aspect, the present invention provides a method for preparing a fructose amine quality control product, comprising the following steps: The glycosylated protein solution of the present invention is added to a stabilizer solution containing trehalose and histidine to obtain a mixture; in the stabilizer solution, the concentration of trehalose is 10-50 mM and the concentration of histidine is 5-25 mM. The mixture is the fructose amine quality control product; or, the mixture is freeze-dried to obtain the fructose amine quality control product.

[0013] In a seventh aspect, the present invention provides a fructose amine quality control product, wherein it is prepared by the preparation method of the present invention as described above.

[0014] In an eighth aspect, the present invention provides a fructosamine detection kit, wherein the fructosamine detection kit includes fructosamine calibrators and / or fructosamine quality control products.

[0015] Optionally, the fructosamine detection kit further includes a fructosamine detection reagent, which includes reagent R1 and reagent R2; The reagent R1 comprises the following components: 300-500 mM first buffer and 1-10 KU / L PEGylated uricase; The pH of the first buffer solution is 9.0~11.0; The reagent R2 comprises the following components: A second buffer of 5–50 mM and a chromogenic substrate of 0.5–3.0 mM.

[0016] Beneficial Effects: This invention involves incubating human serum albumin and bovine serum albumin in a specific mass ratio of (70-80):(20-30) with glucose at a specific pH and temperature in the dark for a specific number of days, followed by purification to obtain a glycosylated protein solution. The specific ratio of human serum albumin to bovine serum albumin produces a synergistic effect, achieving an optimal balance between reducing heterogeneous matrix effects and improving stability. This allows calibrators prepared from this glycosylated protein solution to significantly improve their ability to correct for both batch-to-batch and instrument-to-institutional differences, thereby enhancing consistency between reagent batches and instruments. Furthermore, the control of pH, temperature, and time, along with the purification process, ensures that the prepared glycosylated protein solution exhibits good stability. Attached Figure Description

[0017] Figure 1 This is a comparison chart of clinical sample measurements using different formulations of fructosamine detection reagents and commercially available reagents.

[0018] Figure 2 This is a comparison of the interference rate of uric acid on the detection of fructosamine when different formulations of fructosamine detection reagents were placed at 37℃ for 0 days and 37℃ for 14 days to test the fructosamine concentration.

[0019] Figure 3 The graph shows the measured values ​​of calibrators with different HSA to BSA ratios and 1-deoxy-1-morpholine-D-fructose calibrator in accelerated stability tests.

[0020] Figure 4 This is a graph showing the comparison of the mean coefficient of variation (CV%) between batches of the same group of clinical samples after calibrating three batches of different fructosamine detection reagents using the calibrators in Example 3 and Comparative Examples 4-8.

[0021] Figure 5The regression analysis plots show the consistency of results for detecting the same group of clinical samples on different instrument platforms using the fructosamine detection reagent in Example 2 and the fructosamine calibrator in Example 3. (a) is Calendar 7180 and Dirui CS-2000, and (b) is Beckman AU5800 and Dirui CS-2000. Detailed Implementation

[0022] This invention provides a glycosylated protein solution, its preparation method and application, and a fructosamine detection kit. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0024] If the embodiments of the present invention involve descriptions such as "first" or "second", such descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated.

[0025] This invention provides a method for preparing a glycosylated protein solution, comprising the following steps: Human serum albumin, bovine serum albumin, and glucose were added to a buffer solution with a pH of 7.0–8.0 and incubated at 36–38°C (e.g., 36°C, 37°C, or 38°C) in the dark for 14–28 days (to carry out glycosylation reaction) to obtain the reaction solution; wherein the mass ratio of human serum albumin to bovine serum albumin was (70–80):(20–30), and the ratio of the total mass of human serum albumin and bovine serum albumin to the mass of glucose was 2:1; After purifying the reaction solution, the glycosylated protein solution is obtained (wherein, the glycosylated protein is fructosamine).

[0026] The inventors discovered through research that calibrators prepared using only human serum albumin exhibit poor long-term stability, while calibrators prepared using only bovine serum albumin suffer from batch-to-batch and instrument-to-instance consistency due to heterogeneity. This invention involves incubating human serum albumin and bovine serum albumin in a specific mass ratio of (70-80):(20-30) with glucose at a specific pH and temperature in the dark for a specific number of days, followed by purification to obtain a glycosylated protein solution. The specific ratio of human serum albumin to bovine serum albumin produces a synergistic effect, achieving an optimal balance between reducing heterogeneous matrix effects and improving stability. This allows calibrators prepared from this glycosylated protein solution to significantly improve their ability to correct for batch-to-batch and instrument-to-instance differences, thereby enhancing batch-to-batch and instrument-to-instance consistency. Furthermore, the control of pH, temperature, and time, along with the purification process, ensures that the prepared glycosylated protein solution possesses excellent stability.

[0027] In some embodiments, the total concentration of human serum albumin and bovine serum albumin in the reaction solution is 150-250 g / L, for example, it can be 150 g / L, 160 g / L, 180 g / L, 200 g / L, 210 g / L, 220 g / L or 250 g / L.

[0028] In some embodiments, the buffer solution includes at least one of phosphate buffer and HEPES (4-hydroxyethylpiperazine ethanesulfonic acid) buffer, but is not limited thereto.

[0029] In some embodiments, the concentration of the buffer solution is 50-200 mM (i.e., mmol / L), for example, it can be 50 mM, 60 mM, 80 mM, 100 mM, 120 mM, 150 mM, 180 mM or 200 mM.

[0030] In some embodiments, the purification is performed by ultrafiltration of the reaction solution using an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa to completely remove unreacted glucose.

[0031] This invention also provides a glycosylated protein solution, which is prepared using the preparation method described above.

[0032] This invention also provides an application of the glycosylated protein solution described above in the preparation of fructosamine calibrators or fructosamine quality control products.

[0033] This invention also provides a method for preparing a fructosamine calibrator, comprising the following steps: The glycosylated protein solution of the present invention as described above is added to a stabilizer solution containing trehalose and histidine to obtain a mixture; in the stabilizer solution, the concentration of trehalose is 10-50 mM and the concentration of histidine is 5-25 mM. The mixture is the fructosamine calibrator; or, the mixture is freeze-dried to obtain the fructosamine calibrator.

[0034] Trehalose and histidine are used as stabilizers to stabilize the prepared glycosylated protein.

[0035] In some specific embodiments, the molar ratio of trehalose to histidine in the fructosamine calibrator is 2:1. This invention also provides a fructosamine calibrator prepared using the method described above.

[0036] In this embodiment, the fructosamine calibrator exhibits high stability, significantly improving the consistency between batches and between instruments for fructosamine detection reagents. Specifically, the fructosamine calibrator can reduce the batch-to-batch coefficient of variation (CV%) to less than 3.5% and the inter-instrument correlation coefficient R0 to [missing value]. 2 It reaches 0.99 or higher.

[0037] This invention also provides a method for preparing a fructose amine quality control product, comprising the following steps: The glycosylated protein solution of the present invention as described above is added to a stabilizer solution containing trehalose and histidine to obtain a mixture; in the stabilizer solution, the concentration of trehalose is 10-50 mM and the concentration of histidine is 5-25 mM. The mixture is the fructose amine quality control product; or, the mixture is freeze-dried to obtain the fructose amine quality control product.

[0038] In some specific embodiments, the molar ratio of trehalose to histidine in the fructose amine quality control product is 2:1.

[0039] This invention also provides a fructose amine quality control product, which is prepared using the preparation method described above.

[0040] In this embodiment, the fructose amine quality control sample has good uniformity, and the fructose amine detection system has good accuracy and reliability for monitoring and evaluating it.

[0041] This invention also provides a fructosamine detection kit, wherein the fructosamine detection kit includes fructosamine calibrators and / or fructosamine quality control products.

[0042] Currently, the clinical detection of fructosamine still faces the long-standing problem of interference from endogenous uric acid. High concentrations of uric acid (UA) in serum nonspecifically reduce the chromogenic substrate in the nitrotetrazole blue (NBT) reduction method, leading to positive bias in the results and severely affecting accuracy. The conventional solution is to add uricase, but the strong alkalinity (pH 9.0~11.0) required for fructosamine detection destroys the structure of ordinary uricase, causing its activity to decrease during the reagent's shelf life and failing to provide continuous and stable anti-interference capabilities. This has become a long-standing technical problem in this field that has not been properly resolved.

[0043] The inventors have noted that in the pharmaceutical field, PEGylation is commonly used to modify therapeutic proteins (such as uricase) to improve their pharmacokinetic properties. The primary purpose of this modification is to prolong their half-life in vivo, enhance their stability, and reduce their immunogenicity. This technique has become a routine practice in the biopharmaceutical industry. However, those skilled in the art may consider PEGylation a technique specifically developed for in vivo drug therapy, its value primarily lying in handling complex physiological environments. For in vitro diagnostic reagents, which exist in relatively simple and controllable buffer systems and do not require immunogenicity of the enzyme, there is a general lack of motivation and technical inspiration in the art to apply PEGylated enzymes to in vitro diagnostic reagents. Furthermore, the alkaline environment (pH 9.0–11.0) required for fructosamine detection poses a significant challenge to the stability of any enzyme, and those skilled in the art cannot predict whether PEGylated uricase can remain stable under these conditions and effectively resist uric acid interference.

[0044] Therefore, in some embodiments of the present invention, the fructosamine detection kit (resistant to uric acid interference) includes fructosamine calibrators and / or fructosamine quality controls, and the fructosamine detection kit further includes fructosamine detection reagents, the fructosamine detection reagents including reagent R1 and reagent R2; The reagent R1 comprises the following components: 300-500 mM first buffer and 1-10 KU / L (thousand units per liter) of polyethylene glycol-modified uricase (obtained by covalently linking uricase to activated polyethylene glycol); The pH of the first buffer solution is 9.0~11.0 (e.g., it can be 9.0, 9.2, 9.5, 9.8, 10, 10.2, 10.5, 10.8 or 11, etc.). The reagent R2 comprises the following components: A second buffer of 5-50 mM and a chromogenic substrate of 0.5-3.0 mM (such as NBT, which can be reduced to formazan by fructosamine in an alkaline environment, and has a characteristic absorption peak near 550 nm).

[0045] For example, the concentration of the first buffer solution can be 300 mM, 350 mM, 400 mM, 450 mM or 500 mM, etc.

[0046] The concentration of the polyethylene glycol-modified uricase can be 1 KU / L, 2 KU / L, 3 KU / L, 4 KU / L, 5 KU / L, 6 KU / L, 7 KU / L, 8 KU / L, 9 KU / L, or 10 KU / L, etc.

[0047] The concentration of the second buffer solution is 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM, etc. The concentration of the chromogenic substrate is 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, or 3.0 mM, etc.

[0048] The inventors discovered that PEGylated uricase can withstand the high pH environment required for fructosamine detection using the NBT method while maintaining high anti-interference capabilities. It remains stable in this alkaline environment and continues to degrade uric acid. This invention applies PEGylated uricase, originally used primarily in drug development, to fructosamine in vitro diagnostic reagents. Under alkaline conditions of pH 9.0–11.0, its stable resistance to uric acid interference is significantly superior to that of ordinary uricase, solving a long-standing technical problem in the field and ensuring that the reagent effectively eliminates uric acid interference throughout its shelf life. Furthermore, the alkali-resistant anti-interference reagent R1 ensures reaction specificity, the highly stable and consistent fructosamine calibrator significantly reduces batch-to-batch and instrument-to-instrument variability, providing a reliable metrological traceability benchmark, and the well-homogenized fructosamine quality control ensures the accuracy and reliability of the detection. The synergistic effect of these three components enables accurate and stable detection of complex clinical samples, greatly improving the standardization level of fructosamine detection. This invention not only improves batch-to-batch and instrument-to-instrument consistency but also fundamentally solves the accuracy problem in fructosamine detection.

[0049] In this embodiment, the detection principle of the reagent kit is as follows: First, the test sample is mixed with reagent R1. In an alkaline environment with a pH of 9.0-11.0, uric acid in the test sample is efficiently decomposed by PEGylated uricase, thus eliminating its interference. Simultaneously, fructosamine (glycosylated protein) in the test sample undergoes an Amadori rearrangement, generating an enol structure. Reagent R2 is then added, where the substrate (such as NBT) is reduced by the enol structure, generating purple formazan. The concentration is detected using a biochemical analyzer at a wavelength of 550 nm.

[0050] In some embodiments, the volume ratio of reagent R1 to reagent R2 is 4:1.

[0051] In some embodiments, the first buffer includes at least one of carbonate buffer, glycine buffer, Tris-HCl buffer, borate buffer, CHES (2-cyclohexylaminoethanesulfonic acid) buffer, and CAPS (3-cyclohexylaminopropanesulfonic acid) buffer, but is not limited thereto; The pH of the second buffer is 6.5 to 7.8 (e.g., 6.5, 6.8, 7, 7.5, or 7.8, etc.), and the second buffer includes, but is not limited to, at least one of phosphate buffer, HEPES buffer, MOPS (3-morpholinopropanesulfonic acid) buffer, sodium barbital-hydrochloric acid buffer, Tris-hydrochloric acid buffer, disodium hydrogen phosphate-citrate buffer, and potassium dihydrogen phosphate-sodium hydroxide buffer.

[0052] The present invention will be further described below through specific embodiments.

[0053] Unless otherwise specified, the raw materials used in the following embodiments are all commercially available products.

[0054] The PEGylated uricase was purchased from Beijing Dacheng Biotechnology Co., Ltd., model number E016-P.

[0055] Example 1 This embodiment provides a fructosamine detection reagent, comprising reagent R1 and reagent R2 in a volume ratio of 4:1; Reagent R1 comprises the following components: 400 mM carbonate buffer (pH 10.2) and 1 KU / L PEGylated uricase.

[0056] The reagent R2 comprises the following components: 15 mM phosphate buffer (pH 7.0) and 2 mM NBT.

[0057] The preparation method of reagent R1 is as follows: Weigh an appropriate amount of sodium carbonate and sodium bicarbonate, dissolve them in purified water, and prepare a 400mM carbonate buffer solution with pH 10.2. Then add polyethylene glycol-modified uricase to make the final concentration 1 KU / L, mix well, filter for sterilization, and store at 2-8℃.

[0058] The preparation method of reagent R2 is as follows: Weigh an appropriate amount of sodium dihydrogen phosphate and disodium hydrogen phosphate, dissolve them in purified water to prepare a 15 mM phosphate buffer solution with pH 7.0, then add NBT to make the final concentration 2 mM, mix well, filter to remove bacteria, and store at 2-8℃.

[0059] Example 2 This embodiment provides a fructosamine detection reagent, comprising reagent R1 and reagent R2 in a volume ratio of 4:1. The only difference from Example 1 is that the concentration of polyethylene glycol uricase in reagent R1 is 5 KU / L.

[0060] Comparative Example 1 This comparative example provides a fructosamine detection reagent, comprising reagent R1 and reagent R2 in a volume ratio of 4:1. The only difference from Example 1 is that reagent R1 does not contain polyethylene glycol uricase.

[0061] Comparative Example 2 This comparative example provides a fructosamine detection reagent, comprising reagent R1 and reagent R2 in a volume ratio of 4:1. The only difference from Example 1 is that the 1 KU / L polyethylene glycol uricase in reagent R1 is replaced with 5 KU / L uricase.

[0062] Comparative Example 3 This comparative example provides a fructosamine detection reagent, comprising reagent R1 and reagent R2 in a volume ratio of 4:1. The only difference from Example 1 is that the concentration of polyethylene glycol uricase in reagent R1 is 0.5 KU / L.

[0063] The following tests were conducted: The fructosamine calibrator used in the following tests was prepared by the following method: (1) Preparation of protein mixture: Dissolve human serum albumin and bovine serum albumin in phosphate buffer (200 mM, pH 7.4) at a mass ratio of 75:25 to prepare a protein mixture with a total protein concentration of 200 g / L. (2) Glycosylation reaction: Add D-glucose to the above protein mixture until the concentration is 100 g / L, mix well and filter to remove bacteria, then place in a 37℃ constant temperature incubator and incubate in the dark for 21 days to obtain the reaction solution.

[0064] (3) Purification: The reaction solution was purified by ultrafiltration using an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa to completely remove unreacted glucose and obtain glycosylated protein solution.

[0065] (4) Stabilization and preparation: The purified glycosylated protein solution was diluted to 270 μM with a stabilizer solution containing 30 mM trehalose and 15 mM histidine (solvent is phosphate buffer, pH 7.4) to obtain fructoamine calibrator.

[0066] The preparation method of the fructose amine quality control sample is the same as that of the fructose amine calibrator mentioned above.

[0067] (1) Evaluation experiment on the difference of clinical samples in fructosamine detection reagent test A fully automated biochemical analyzer (Dirui CS-2000) was selected, and the reagents from Examples 1-2 and Comparative Examples 1-3 were used. Calibration was performed using calibrators, and quality control was conducted using quality control materials. Then, 15 human serum samples covering a clinical range were tested, and the deviations from clinical measurements using commercially available reagents were calculated. The results are shown in Table 1 and... Figure 1 As shown.

[0068] Table 1. Clinical measurement deviations between different formulations of fructosamine detection reagents and commercially available reagents.

[0069] Note: The commercially available reagent is the Roche fructosamine detection kit (colorimetric method), model number 05171962188.

[0070] From Table 1 and Figure 1 It can be seen that the clinical measurements of Comparative Examples 1-3 were all high, indicating that uric acid in the samples interfered with the fructosamine measurement. Although Comparative Example 2 contained uricase, the activity of ordinary uricase was inhibited to varying degrees under alkaline conditions at pH 10.2, resulting in poor results against uric acid interference. Comparative Example 3 contained polyethylene glycol-modified uricase, but the concentration was low, which also led to high clinical measurements. The clinical measurement deviation in Example 1 was basically acceptable (average deviation of 3.8%), and the concentration of polyethylene glycol-modified uricase used in this case was 1 KU / L. The clinical measurement deviation in Example 2 was the smallest (average deviation of 0.4%), indicating that the uric acid in the sample was completely hydrolyzed by polyethylene glycol-modified uricase, with the best effect. The concentration of polyethylene glycol-modified uricase used in this case was 5 KU / L.

[0071] (2) Evaluation of the ability of fructosamine detection reagent to resist uric acid interference Five samples with the same baseline fructosamine value but different uric acid concentrations (0, 100, 200, 400, and 800 μM, respectively) were prepared. All samples were incubated at 37°C for 0 days and 14 days with fructosamine calibrators and quality control samples, using the fructosamine detection reagents from Examples 1-2 and Comparative Examples 1-3, respectively. The fructosamine concentrations of the five samples were then measured, and the interference rate (B) of uric acid on fructosamine detection was calculated according to the following formula (1). The results are shown in Tables 2.1 and 2.2. Figure 2 As shown.

[0072] (1) Where: M - the test results of the interference samples (i.e., the measured values ​​of samples 2 to 5 with different concentrations of uric acid added); The test results of the T-blank control sample (i.e., the measured value of sample 1 with uric acid of 0 μM).

[0073] Table 2.1 Results of the test on the anti-uric acid interference rate of the fructosamine detection reagent in Comparative Examples 1-3

[0074] Table 2.2, Test results of the uric acid interference rate of the fructosamine detection reagent in Examples 1-2

[0075] Note: The normal concentration of uric acid in human serum is approximately 150~400 μM.

[0076] From Tables 2.1 and 2.2 and Figure 2 It was found that reagents without either of the two enzymes (Comparative Example 1), or those with only ordinary uricase (Comparative Example 2, where the activity of ordinary uricase is inhibited at pH 10.2), or those with a low concentration of PEGylated uricase (Comparative Example 3, where the amount of added PEGylated uricase was too low, resulting in incomplete hydrolysis of uric acid in the sample), all exhibited uric acid interference rates exceeding ±10% at 37°C for 0 days. Furthermore, the interference rate increased with increasing uric acid concentration. Reagents with 1 KU / L (Example 1) and 5 KU / L (Example 2) of PEGylated uricase both resisted interference from 800 μM uric acid at 37°C for 0 days and 37°C for 14 days, with the reagent in Example 2 showing the best performance.

[0077] Furthermore, the reagent with added ordinary uricase (Comparative Example 2) showed an increased uric acid interference rate and a significant decrease in anti-interference performance after incubation at 37°C for 14 days. In contrast, the reagent with added polyethylene glycol-modified uricase showed essentially the same anti-uric acid interference performance after incubation at 37°C for 14 days as it did after incubation at 37°C for 0 days, indicating that the anti-uric acid interference ability of the reagent with added polyethylene glycol-modified uricase is stable within its shelf life. Considering that increasing the dosage of polyethylene glycol-modified uricase would increase reagent costs, its usage range was determined to be 1~10 KU / L (preferably 5 KU / L).

[0078] Example 3 This embodiment provides a method for preparing a fructosamine calibrator, comprising the following steps: (1) Prepare protein mixture: Dissolve human serum albumin (HSA) and bovine serum albumin (BSA) in phosphate buffer (200 mM, pH 7.4) at a mass ratio of 75:25 to prepare a protein mixture with a total protein concentration of 200 g / L.

[0079] (2) Glycosylation reaction: Add D-glucose to the above protein mixture until the concentration is 100 g / L, mix well and filter to remove bacteria, then place in a 37°C constant temperature incubator and incubate in the dark for 21 days to obtain the reaction solution.

[0080] (3) Purification: The reaction solution was purified by ultrafiltration using an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa to completely remove unreacted glucose and obtain glycosylated protein solution.

[0081] (4) Stabilization and preparation: The purified glycosylated protein solution was diluted to 270 μmol / L with a stabilizer solution containing 30 mM trehalose and 15 mM histidine (solvent is phosphate buffer, pH 7.4).

[0082] Comparative Example 4 This comparative example provides a method for preparing a fructosamine calibrator, which differs from Example 3 only in that it uses only HSA and not BSA (i.e., the mass ratio of HSA to BSA is 100:0).

[0083] Comparative Example 5 This comparative example provides a method for preparing a fructosamine calibrator, which differs from Example 3 only in that only BSA is used, instead of HSA (i.e., the mass ratio of HSA to BSA is 0:100).

[0084] Comparative Example 6 This comparative example provides a method for preparing a fructosamine calibrator, which differs from Example 3 only in that the mass ratio of HSA to BSA is 90:10.

[0085] Comparative Example 7 This comparative example provides a method for preparing a fructosamine calibrator, which differs from Example 3 only in that the mass ratio of HSA to BSA is 60:40.

[0086] Comparative Example 8 This comparative example provides a method for preparing a fructoamine calibrator, which differs from Example 3 only in that steps (1) to (3) are omitted, and in step (4), 1-deoxy-1-morpholine-D-fructose is used to replace the purified glycosylated protein solution.

[0087] The following tests were performed on the fructosamine calibrators used in Example 3 and Comparative Examples 4-8: (1) Accelerated stability test The fructosamine calibrators from Example 4 and Comparative Examples 4-8 were placed in a 37°C incubator, and their fructosamine content was measured on day 0, day 7, and day 14. The rate of change of the measured values ​​for each period was calculated, and the results are shown in Table 3. Figure 3 As shown.

[0088] Table 3. Results of Accelerated Stability Test

[0089] From Table 3 and Figure 3It can be seen that the calibrators using 1-deoxy-1-morpholine-D-fructose as raw material, as well as the calibrators with HSA to BSA mass ratios of 75:25, 60:40, and 0:100, all showed a change rate of ±5% after 14 days of accelerated testing, which meets the stability requirements. The calibrator with the smallest change rate was the one with an HSA to BSA mass ratio of 75:25 (Example 3).

[0090] (2) Batch consistency test The fructosamine detection reagents (reagents R1 and R2) in Example 2 were prepared three times to obtain three batches of reagents from different production batches. These were calibrated using the fructosamine calibrators from Example 3 and Comparative Examples 4-8, respectively. Then, 15 fresh human serum samples were tested. The coefficient of variation (CV%) of the test results for each sample among the three batches of reagents was calculated according to formulas (2), (3), and (4). The average CV% of the 15 samples was then calculated. The results are shown in Table 4 and [Table data missing]. Figure 4 As shown.

[0091] (2) (3) (4) Where n represents the number of samples, x i This indicates the test result for each sample. This represents the average of the test results for 15 samples, and SD represents the standard deviation.

[0092] Table 4. Coefficients of variation of measured values ​​of three batches of reagents after calibration with different calibrators

[0093] As shown in Table 4, the fructosamine calibrators prepared with HSA to BSA mass ratios of 100:0, 90:10, and 75:25 showed small fluctuations in CV% and an average CV% of less than 3.5% when testing plasma samples after calibrating three batches of reagents, thus meeting the requirements for batch-to-batch variation of the reagents.

[0094] (3) Instrument difference test Three fully automated biochemical analyzers of different models (Dirui CS-2000, Hitachi 7180, and Beckman AU5800) were selected. Using the same batch of fructosamine detection reagents (reagents R1 and R2, obtained via the method in Example 2), each instrument was calibrated using zero-time fructosamine calibrators from Example 3 and Comparative Examples 4-8. Fifteen human serum samples covering a clinical range were then tested. The correlation coefficient (R²) between the results of the two instruments under each calibration group was calculated. 2 Finally, the average correlation coefficient for this calibration group was calculated, and the results are shown in Table 5. Figure 5 As shown.

[0095] Table 5. Correlation coefficients of measured values ​​of the same batch of fructosamine detection reagents after calibration with different fructosamine calibrators on three instruments.

[0096] As shown in Table 5, when the mass ratio of HSA to BSA is 100:0, 90:10, and 75:25, the prepared fructoamine calibrators significantly reduced inter-instrument variability, resulting in a lower inter-instrument correlation coefficient R0. 2 A value of 0.99 or higher meets the instrument's consistency requirements.

[0097] The above results show that the fructosamine calibrator provided by this invention can meet the requirement of a change rate of less than ±5% after 14 days of accelerated testing at 37 °C, and can significantly reduce the inter-batch and inter-instrumental differences in fructosamine detection, resulting in an inter-batch coefficient of variation (CV%) of less than 3.5% and an inter-instrumental correlation coefficient R0. 2 The value reached above 0.99. This solved the problem of balancing stability and consistency for single-protein source calibrators (the calibrators in the above comparative examples could not simultaneously achieve stability, as well as batch-to-batch and instrument-to-instrument consistency).

[0098] Example 4 This embodiment provides a method for preparing fructose amine quality control material, including the following steps: Following the method in Example 3, the prepared glycosylated protein solution was diluted with a stabilizer solution containing 30 mM trehalose and 15 mM histidine (solvent being phosphate buffer, pH 7.4) to achieve glycosylated protein concentrations of 380 μM and 245 μM, respectively, which were used as high-value and low-value quality control products.

[0099] Repeat the above method 5 times to obtain 5 bottles of high-value quality control products and 5 bottles of low-value quality control products respectively.

[0100] Comparative Example 9 This comparative example provides a method for preparing a fructose amine quality control sample, comprising the following steps: Using 1-deoxy-1-morpholine-D-fructose as a raw material, it was added to phosphate buffer (pH 7.4) to prepare high-value quality control products with a concentration of 380 μM and low-value quality control products with a concentration of 245 μM.

[0101] Repeat the above method 5 times to obtain 5 bottles of high-value quality control products and 5 bottles of low-value quality control products respectively.

[0102] Uniformity evaluation was performed on the quality control samples in Example 4 and Comparative Example 9: For 5 bottles of high-value quality control samples, each bottle was tested once, and the average of the 5 measurement results was calculated. ) and standard deviation SD1; in addition, one of the five high-value quality control samples was tested five times consecutively, and the average of the five measurement results was calculated ( ) and standard deviation SD2; calculate the CV (%) value of inter-bottle uniformity according to formula (5) and formula (6). The value represents the quality control product uniformity. When SD1 < SD2, let CV 瓶间 =0. The same method was used to test 5 bottles of low-value control samples. The results are shown in Table 6.

[0103] (5) (6) Table 6. Results of the uniformity test for quality control samples

[0104] As shown in Table 6, the uniformity of high and low values ​​of the quality control samples of this invention are 0.60% and 0.51%, respectively, which is less than 3.5%, meeting the uniformity requirements for quality control samples and significantly better than those using 1-deoxy-1-morpholine-D-fructose as a raw material. The quality control samples provided by this invention have a uniformity of less than 3.5%, demonstrating good performance and can be used to monitor and evaluate fructosamine detection systems (or kits), ensuring accuracy and reliability.

[0105] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A method for preparing a glycosylated protein solution, characterized by, The method comprises the following steps: adding human serum albumin, bovine serum albumin and glucose into a buffer solution with a pH of 7.0-8.0, and incubating the mixture at 36-38 °C in the dark for 14-28 days to obtain a reaction solution; wherein the mass ratio of human serum albumin to bovine serum albumin is (70-80):(20-30), and the mass ratio of the total mass of human serum albumin and bovine serum albumin to the mass of glucose is 2:1; purifying the reaction solution to obtain the glycosylated protein solution.

2. The production method according to claim 1, characterized by, In the reaction solution, the total concentration of human serum albumin and bovine serum albumin is 150-250 g / L. The buffer solution comprises at least one of a phosphate buffer solution and a HEPES buffer solution. The concentration of the buffer solution is 50-200 mM.

3. A glycosylated protein solution, characterized in that, The glycosylated protein solution is prepared by the method of any one of claims 1-2.

4. Use of the glycosylated protein solution of claim 3 in the preparation of a fructosamine calibrator or a fructosamine quality control.

5. A method of preparing a fructosamine calibrator, characterized by, The method comprises the following steps: adding the glycosylated protein solution of claim 3 into a stabilizer solution containing trehalose and histidine to obtain a mixed solution; in the stabilizer solution, the concentration of the trehalose is 10-50 mM, and the concentration of the histidine is 5-25 mM; the mixed solution is the fructosamine calibrator; or, the mixed solution is lyophilized to obtain the fructosamine calibrator.

6. A glucosamine calibrator characterized in that, The fructosamine calibrator is prepared by the method of claim 5.

7. A method for preparing a fructosamine quality control product, characterized by, The method comprises the following steps: adding the glycosylated protein solution of claim 3 into a stabilizer solution containing trehalose and histidine to obtain a mixed solution; in the stabilizer solution, the concentration of the trehalose is 10-50 mM, and the concentration of the histidine is 5-25 mM; the mixed solution is the fructosamine quality control; or, the mixed solution is lyophilized to obtain the fructosamine quality control.

8. A glucosamine quality control characterized in that, The fructosamine quality control is prepared by the method of claim 7.

9. A fructosamine test kit characterized in that, The fructosamine detection kit comprises a fructosamine calibrator and / or a fructosamine quality control.

10. The fructosamine test kit according to claim 9, characterized in that The fructosamine detection kit further comprises a fructosamine detection reagent, and the fructosamine detection reagent comprises reagent R1 and reagent R2; The reagent R1 comprises the following components: 300-500 mM of a first buffer solution and 1-10 KU / L of pegylated uricase; the pH of the first buffer solution is 9.0-11.0; The reagent R2 comprises the following components: 5-50 mM of a second buffer solution and 0.5-3.0 mM of a chromogenic substrate.

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