Glycated hemoglobin dissociation reagent and application thereof

Abstract

The application provides a glycated hemoglobin dissociation reagent and application thereof, and belongs to the technical field of biological detection. The dissociation reagent comprises 10-50 mM Tris buffer, 0.2-0.5 M ammonium chloride, 0.5%-1.0% (v / v) triton X-100, 0.2 wt%-1.0 wt% BSA and 0.02-0.2 M sodium chloride. When the dissociation reagent is used to dissociate a whole blood sample, the hemoglobin can be dissociated by mixing the dissociation reagent with the whole blood sample for 20-60 s, and a clear and transparent solution is obtained. The dissociation reagent can be used to prepare a kit for dissociating or / and detecting glycated hemoglobin. The glycated hemoglobin obtained by using the dissociation reagent can be directly used for fluorescence immunoassay, the interference of the color of hemoglobin itself on immunochromatographic detection is avoided, and the sensitivity is high and the repeatability is good.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, and particularly relates to the processing of glycated hemoglobin, specifically to a glycated hemoglobin dissociation reagent and its application. Background Technology

[0002] Glycated hemoglobin (GHb) is a product of the binding of hemoglobin in red blood cells with glucose. It can be classified into glucose-glycosylated hemoglobin, fructose-glycosylated hemoglobin, and lactose-glycosylated hemoglobin, among others. The amount of free glucose in the human body is much higher than that of other carbohydrates, and the valine residue at the N-terminus of the β-chain of hemoglobin has the highest probability of glycation. Therefore, most GHb in the human body is a combination of the β-chain N-terminal valine residue and glucose (hemoglobin A1c, HbA1c), accounting for 60% of GHb. The formation of GHb is an irreversible reaction, directly proportional to blood glucose concentration, and remains stable for approximately 120 days. Therefore, blood glucose concentrations from 120 days ago can be detected, and it is unaffected by the time of blood draw, whether fasting is required, whether insulin is used, or other factors that can cause temporary fluctuations in blood glucose levels. The 2010 American Diabetes Association guidelines included HbA1c ≥ 6.5% as one of the diagnostic criteria for diabetes, and in 2011 the World Health Organization also recommended that countries and regions with the necessary conditions adopt this standard for diagnosing diabetes. Glycated hemoglobin (HbA1c) testing is highly specific, reproducible, and sensitive, and has been internationally recognized as the "gold standard" for diabetes monitoring.

[0003] Currently, there are many methods for HbA1c detection used in clinical and laboratory settings. These can be broadly categorized into two types based on their principles: (1) methods based on the difference in charge between HbA1c and non-glycated hemoglobin, primarily using ion exchange and electrophoresis; and (2) methods based on the difference in structure between HbA1c and non-glycated hemoglobin, mainly due to glycosylation sites, primarily using immunoassay and affinity chromatography. Immunochromatography is an immunoassay technique based on antigen-antibody specific reactions on a chromatographic membrane. It is widely used in pathogen detection, food safety measurement, and environmental monitoring due to its advantages of speed, simplicity, low cost, and high sensitivity. When using immunochromatography to detect glycated hemoglobin, whole blood cannot be detected using chromatographic reagents due to the inherent color of red blood cells. Therefore, before using immunochromatography to detect glycated hemoglobin, red blood cells need to be hemolyzed and broken. However, hemoglobin and glycated hemoglobin are themselves analytes, and their color cannot be filtered out to eliminate interference from the detection.

[0004] Chinese patent application CN114778242A discloses a method and pretreatment agent for pretreatment of glycated hemoglobin. This pretreatment agent includes a hemolysin and a decolorizing agent. In practice, the whole blood sample is first hemolyzed with the hemolysin, followed by decolorization with the decolorizing agent. This process requires 10 minutes for hemolysis and 5-30 minutes for decolorization; the entire procedure involves two steps, hemolysis and decolorization, making it cumbersome and time-consuming. CN114878839A discloses a glycated hemoglobin dissociation solution, comprising phosphate buffer, ethylphenyl polyethylene glycol, sodium dodecyl sulfate, and sodium casein. Compared to the pretreatment agent in CN114778242A and the dissociation solution in a kit sold by Badita Biotechnology Co., Ltd., this dissociation solution significantly reduces the dissociation time, but still requires 5 minutes. Summary of the Invention

[0005] This invention provides a glycated hemoglobin dissociation reagent. Using this dissociation reagent, glycated hemoglobin can be rapidly and efficiently dissociated, and the resulting solution is clear and transparent. It is simple to operate, safe to use, and can be directly used in immunochromatographic reactions. It can reduce non-specific adsorption, improve sensitivity, and has good repeatability.

[0006] Specifically, the present invention adopts the following technical solution:

[0007] A glycated hemoglobin dissociation reagent comprising 10–50 mmol / L Tris buffer, 0.2–0.5 mol / L ammonium chloride, 0.5%–1.0% (v / v) Triton X-100, 0.2 wt%–1.0 wt% bovine serum albumin and 0.02–0.2 mol / L sodium chloride.

[0008] Tris buffer solution controls the pH of the dissociation reagent at 7.2±0.5, allowing the dissociated glycated hemoglobin to directly bind to glycated hemoglobin antibodies in an immunoassay. Ammonium chloride hydrolyzes into ammonium ions in solution, increasing intracellular osmotic pressure and causing erythrocytes to swell. Triton X-100 increases cell membrane permeability to small molecules, facilitating erythrocyte swelling and rupture, releasing glycated hemoglobin, thus improving chromatography speed and reducing non-specific adsorption. Bovine serum albumin acts as a stabilizer, protecting hemoglobin and reducing non-specific adsorption, resulting in a more specific immunoassay between dissociated glycated hemoglobin and glycated hemoglobin antibodies, and lowering detection costs. Sodium chloride regulates the acid-base balance in solution, maintaining a certain ionic strength and osmotic pressure, which is beneficial for the immunoassay between glycated hemoglobin and glycated hemoglobin antibodies, and also reduces non-specific adsorption, improving repeatability. The introduction of bovine serum albumin and sodium chloride further enhances the sensitivity and repeatability of the fluorescence immunoassay.

[0009] Preferably, the concentration of the Tris buffer solution is 10–40 mmol / L.

[0010] More preferably, the concentration of the Tris buffer solution is 20–40 mmol / L.

[0011] Preferably, the concentration of ammonium chloride is 0.3 to 0.5 mol / L.

[0012] More preferably, the concentration of the ammonium chloride is 0.4–0.5 mol / L.

[0013] Preferably, the concentration of Triton X-100 is 0.5% to 0.6% (v / v).

[0014] Preferably, the concentration of bovine serum albumin is 0.5 wt% to 1.0 wt%.

[0015] More preferably, the concentration of bovine serum albumin is 0.5 wt% to 0.7 wt%.

[0016] Preferably, the concentration of sodium chloride is 0.03 to 0.1 mol / L.

[0017] More preferably, the concentration of sodium chloride is 0.05–0.1 mol / L.

[0018] The present invention also provides a method for using the glycated hemoglobin dissociation reagent to dissociate glycated hemoglobin, comprising the following steps: taking a whole blood sample, adding (50-150) times the volume of the glycated hemoglobin dissociation reagent to the whole blood sample, mixing for 20-60 seconds, and obtaining a clear and transparent solution.

[0019] The glycated hemoglobin dissociation reagent of the present invention can also be used to prepare a kit for separating and / or detecting glycated hemoglobin.

[0020] The beneficial effects of this invention are:

[0021] The glycated hemoglobin dissociation reagent provided by this invention can efficiently and completely dissociate glycated hemoglobin in whole blood samples. The dissociated solution is clear and transparent, avoiding interference from the hemoglobin's own color on immunochromatographic detection. The obtained glycated hemoglobin can directly bind to glycated hemoglobin antibodies in an immunoassay. Furthermore, the dissociation time is a maximum of 60 seconds, compared to the shortest time of 5 minutes in existing technologies, reducing the reaction time by 80%. The resulting solution can be directly used in fluorescence immunoassay, improving the sensitivity of the method and exhibiting good repeatability. Attached Figure Description

[0022] Figure 1The graph shows the linear correlation between the glycated hemoglobin results measured after dissociating whole blood samples using the glycated hemoglobin dissociation reagent prepared in Example 1 and the results measured by the MQ6000 glycated hemoglobin analyzer of Shanghai Huizhong Biotechnology.

[0023] Figure 2 The graph shows the linear correlation between the glycated hemoglobin results measured after dissociating whole blood samples using the glycated hemoglobin dissociation reagent prepared in Example 2 and the results measured by the MQ6000 glycated hemoglobin analyzer of Shanghai Huizhong Biotechnology.

[0024] Figure 3 The graph shows the linear correlation between the glycated hemoglobin results measured after dissociating whole blood samples using the glycated hemoglobin dissociation reagent prepared in Example 3 and the results measured by the MQ6000 glycated hemoglobin analyzer of Shanghai Huizhong Biotechnology.

[0025] Figure 4 The graph shows the linear correlation between the glycated hemoglobin results measured after dissociating whole blood samples using the glycated hemoglobin dissociation reagent prepared in Example 4 and the results measured by the MQ6000 glycated hemoglobin analyzer of Shanghai Huizhong Biotechnology.

[0026] Figure 5 The graph shows the linear correlation between the glycated hemoglobin results measured after dissociating whole blood samples using the glycated hemoglobin dissociation reagent prepared in Example 5 and the results measured by the MQ6000 glycated hemoglobin analyzer of Shanghai Huizhong Biotechnology.

[0027] Figure 6 The graph shows the linear correlation between the glycated hemoglobin results measured after dissociating whole blood samples using the glycated hemoglobin dissociation reagent prepared in Comparative Example 1 and the results measured by the MQ6000 glycated hemoglobin analyzer of Shanghai Huizhong Biotechnology.

[0028] Figure 7 The graph shows the linear correlation between the glycated hemoglobin results measured after dissociating whole blood samples using the glycated hemoglobin dissociation reagent prepared in Comparative Example 2 and the results measured by the MQ6000 glycated hemoglobin analyzer of Shanghai Huizhong Biotechnology.

[0029] Figure 8 The graph shows the linear correlation between the glycated hemoglobin results measured after dissociating whole blood samples using the glycated hemoglobin dissociation reagent prepared in Comparative Example 3 and the results measured by the MQ6000 glycated hemoglobin analyzer of Shanghai Huizhong Biotechnology.

[0030] Figure 9 The graph shows the linear correlation between the glycated hemoglobin results measured after dissociating whole blood samples using the glycated hemoglobin dissociation reagent prepared in Comparative Example 4 and the results measured by the MQ6000 glycated hemoglobin analyzer of Shanghai Huizhong Biotechnology.

[0031] Figure 10 The graph shows the linear correlation between the glycated hemoglobin (HbA1c) results measured after dissociating whole blood samples using the HbA1c dissociation reagent prepared in Comparative Example 5 and the results measured by the MQ6000 HbA1c analyzer from Shanghai Huizhong Biotechnology. Detailed Implementation

[0032] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] The main components of the glycated hemoglobin dissociation reagents in Examples 1-5 and Comparative Examples 1-2 are shown in Table 1.

[0034] Table 1. Main components of the glycated hemoglobin dissociation reagents in Examples 1-5 and Comparative Examples 1-2

[0035]

[0036] The main components of the glycated hemoglobin dissociation reagents in Comparative Examples 3–5 are shown in Table 2.

[0037] Table 2. Main components of the glycated hemoglobin dissociation reagents in Comparative Examples 3-5

[0038] Comparative Example 3 Comparative Example 4 Comparative Example 5 Tris buffer (mmol / L) 25 25 / PBS buffer (mol / L) / / 25 Ammonium chloride (mol / L) 0.4 0.4 0.4 Triton X-100 (%, v / v) 0.5 0.5 0.5 Bovine serum albumin (wt%) / / 0.5 Sodium caseinate (wt%) 0.5 / / PEG20000 (wt%) / 0.5 / Sodium chloride (mol / L) 0.1 0.1 0.1 pH 7.2±0.5 7.2±0.5 7.2±0.5

[0039] The method of using the glycated hemoglobin dissociation reagents in Examples 1, 4, 5 and Comparative Examples 1-2 includes the following steps:

[0040] S1. Dissolve each component in Table 1 in purified water to prepare glycated hemoglobin dissociation reagents containing the corresponding amounts of each component.

[0041] S2. Take 10 μL of whole blood sample into a centrifuge tube, add 800 μL of the glycated hemoglobin dissociation reagent, vortex and mix for 40 s to obtain a clear and transparent solution.

[0042] The method of using the glycated hemoglobin dissociation reagent in Example 2 is basically the same as that in Example 1, except that the volume of the glycated hemoglobin dissociation reagent in step S2 is 1500 μL and the vortex mixing time is 20 s.

[0043] The method of using the glycated hemoglobin dissociation reagent in Example 3 is basically the same as that in Example 1, except that the volume of the glycated hemoglobin dissociation reagent in step S2 is 500 μL and the vortex mixing time is 1 min.

[0044] The method of using the glycated hemoglobin dissociation reagents in Comparative Examples 3-5 is basically the same as that in Example 1, except that the glycated hemoglobin dissociation reagents in step S1 are prepared according to the components and amounts in Table 2.

[0045] 100 μL of the solutions obtained from the dissociation in each of the examples and comparative examples were added to glycated hemoglobin fluorescence immunochromatographic reagent (manufactured by Wuhan Youen Biotechnology Co., Ltd.) and reacted for 5 min. The results were then read using a fluorescence immunoassay analyzer. The results obtained from each example and comparative example were linearly fitted to the values ​​measured by the MQ6000 glycated hemoglobin analyzer of Shanghai Huizhong Biotechnology Co., Ltd., and the resulting curves are shown below. Figures 1-10 As shown, the linear equations are as follows:

[0046] Example 1: y1 = 0.9595x1 + 0.249(R) 2 =0.986);

[0047] Example 2: y2 = 0.979x2 + 0.0398(R) 2 =0.9952);

[0048] Example 3: y3 = 0.9767x3 + 0.2819(R) 2 =0.9837);

[0049] Example 4: y4 = 0.9739x4 + 0.0278(R) 2 =0.9918);

[0050] Example 5: y5 = 0.9568x5 + 0.4544(R) 2 =0.9806);

[0051] Comparative Example 1: y6 = 0.9102x6 + 0.5485(R) 2 =0.9582);

[0052] Comparative Example 2: y7 = 1.0187x7 - 0.0384(R) 2 =0.9496);

[0053] Comparative Example 3: y8 = 0.9121x8 + 0.5389(R) 2 =0.9607);

[0054] Comparative Example 4: y9 = 0.8923x9 + 0.6956(R) 2 =0.9517);

[0055] Comparative Example 5: y 10 =0.988x 10 -0.3116(R2 =0.9531).

[0056] from Figures 1-5 As can be seen from the examples 1-5, R 2 All values ​​were above 0.98, indicating good linear correlation, with the degree of linear correlation increasing sequentially from Example 5, Example 3, Example 1, Example 4, to Example 2. Based on the degree of linear correlation, it can be seen from Table 1 that when the Tris buffer concentration was 10–50 mmol / L, R... 2 It can reach above 0.98; at concentrations of 10–40 mmol / L, R 2 It can reach above 0.985; at concentrations of 20–40 mmol / L, R 2 It can reach above 0.99. When the ammonium chloride concentration is 0.1–0.5 mol / L, R 2 It can reach above 0.98; when the concentration is 0.3–0.5 mol / L, R 2 It can reach above 0.985; when the concentration is 0.4–0.5 mol / L, R 2 It can reach above 0.99. When the concentration of Triton X-100 is 0.5% to 1.0%, R 2 It can reach above 0.98; when the concentration is 0.5% to 0.6%, R 2 It can reach above 0.99. When the bovine serum albumin concentration is 0.2%–1.0%, R 2 It can reach above 0.98; when the concentration is 0.5% to 1.0%, R 2 It can reach above 0.985; when the concentration is 0.5% to 0.7%, R 2 It can reach above 0.99. When the sodium chloride concentration is 0.02–0.2 mol / L, R 2 It can reach above 0.98; when the concentration is 0.03–0.1 mol / L, R 2 It can reach above 0.985; when the concentration is 0.05–0.1 mol / L, R 2 It can reach above 0.99.

[0057] from Figures 6-10 As can be seen from the comparison examples 1 to 5, R 2All values ​​were below 0.98, indicating a significantly lower linear correlation compared to the examples. This is because the concentrations of Tris buffer and sodium chloride in the glycated hemoglobin dissociation reagent of Comparative Example 1 were outside the specified range; similarly, the concentrations of Tris buffer, ammonium chloride, and Triton X-100 in the glycated hemoglobin dissociation reagent of Comparative Example 2 were also outside the specified range. This is because the sodium chloride concentration was too high, resulting in an excessively high concentration of salt ions in the dissociation solution, which affected the antigen-antibody reaction; while the concentrations of ammonium chloride and Triton X-100 were too low, affecting the dissociation of glycated hemoglobin and thus impacting the accuracy of the test results. In Comparative Example 3, bovine serum albumin was replaced with sodium caseinate, and in Comparative Example 4, bovine serum albumin was replaced with PEG20000, leading to a significantly lower linear correlation in the test results compared to the examples. This may be because the blocking effect of sodium caseinate or PEG20000 in the entire dissociation reagent system is not as effective as that of BSA in the dissociation reagent. In Comparative Example 5, replacing the Tris buffer with phosphate buffer significantly worsened the linear correlation of the detection results. This could be because the system of buffer and other reagents is not conducive to lysis, or because the Tris buffer system is more conducive to the binding of glycated hemoglobin and glycated hemoglobin antibodies. Therefore, the components of the glycated hemoglobin dissociation reagent of this invention cannot be replaced with other similar reagents through conventional methods, and the component concentrations must not exceed the specified range; otherwise, the detection results may be inaccurate.

[0058] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Various modifications and variations can be made to the present invention by any person skilled in the art. Any simple equivalent changes and modifications made based on the scope of protection of the present invention and the content of the specification should be included within the scope of protection of the present invention.

Claims

1. A glycated hemoglobin dissociation reagent, characterized in that, The glycosylated hemoglobin dissociation reagent is composed of 10-40 mmol / L Tris buffer, 0.3-0.5 mol / L ammonium chloride, 0.5-1.0 v / v% Triton X-100, 0.5-1.0 wt% bovine serum albumin and 0.03-0.1 mol / L sodium chloride; the method for dissociating glycosylated hemoglobin using the glycosylated hemoglobin dissociation reagent comprises the following steps: taking a whole blood sample, adding 50-150 times the volume of the glycosylated hemoglobin dissociation reagent to the whole blood sample, mixing for 20-60 s to obtain a clear and transparent solution.

2. The glycated hemoglobin dissociation reagent according to claim 1, wherein, The concentration of the Tris buffer is 20-40 mmol / L.

3. The glycated hemoglobin dissociation reagent according to claim 1, wherein The concentration of the ammonium chloride is 0.4-0.5 mol / L.

4. The glycated hemoglobin dissociation reagent according to claim 1, wherein, The concentration of the Triton X-100 is 0.5-0.6 v / v%.

5. The glycated hemoglobin dissociation reagent according to claim 1, wherein, The concentration of the bovine serum albumin is 0.5-0.7 wt%.

6. The glycated hemoglobin dissociation reagent according to claim 1, wherein, The concentration of the sodium chloride is 0.05-0.1 mol / L.

7. A kit characterized in that, The kit comprises the glycosylated hemoglobin dissociation reagent according to any one of claims 1-6.

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