Homocysteine hydrolase preserving fluid and detection kit containing same

Abstract

The invention discloses a homocysteine hydrolase preserving fluid and a detection kit containing the same, and belongs to the technical field of biological detection. The preservation solution comprises at least one buffer solution component of MES, Tris, monopotassium phosphate and disodium hydrogen phosphate, an S-adenosine homocysteine hydrolase, a prolin 300 preservative, a Tween20 / glycerol / SDS composite surfactant, 8wt% bovine serum albumin, PEG2000, S-inosyl-L-homocysteine, betaine, adenosine, creatinine and other small molecules, and all the components are compounded according to a specific concentration range. The natural conformation and the activity stability of the S-adenosyl homocysteine hydrolase are remarkably maintained, and meanwhile, the S-adenosyl homocysteine hydrolase has a strong anti-interference capability. A detection kit containing the preservation solution is divided into a component R1 and a component R2, the component R1 contains PBS, reduced coenzyme I, DTT and adenosine, the component R2 is the preservation solution, the detection result is accurate and reliable, the preservation solution can be stably preserved for more than 5 months at 2-8 DEG C, and the preservation solution is suitable for accurate and quantitative detection of homocysteine and has important application value.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, specifically to a homocysteine ​​hydrolase preservation solution and a homocysteine ​​detection kit containing the same, which is particularly suitable for improving reagent stability and anti-interference ability in the homocysteine ​​detection process. Background Technology

[0002] Homocysteine ​​(HCY) is a sulfur-containing amino acid produced by the demethylation of methionine within cells. During extracorporeal circulation, most homocysteine ​​exists in its oxidized form, bound to plasma proteins, specifically albumin via disulfide bonds to form protein-HCY. Only small amounts of reduced homocysteine ​​and homocysteine ​​disulfide (HCY-SS-HCY) are present in the blood. Total homocysteine ​​(tHCY) refers to all types of homocysteine ​​(free HCY and protein-bound HCY) in serum or plasma.

[0003] Homocysteine ​​is primarily metabolized through the transsulfurization and remethylation pathways. In the vitamin B6-dependent transsulfurization pathway, homocysteine ​​is irreversibly metabolized to cysteine. Most homocysteine ​​is then remethylated to methionine by folic acid and cobalamin-dependent methionine synthases. When these metabolic pathways are blocked, homocysteine ​​accumulates in the body. Elevated homocysteine ​​levels are closely related to the development and progression of various diseases, including cardiovascular and cerebrovascular diseases and neurological disorders. Therefore, accurate detection of total homocysteine ​​(tHCY) levels in serum or plasma has significant clinical value.

[0004] Currently, methods for detecting homocysteine ​​mainly include chemiluminescent immunoassay and cyclic enzymatic methods. The core principle of chemiluminescent immunoassay is that, under the action of a pretreatment solution, different forms of homocysteine ​​are reduced to a free state, and then converted to S-adenosyl-L-homocysteine ​​(SAH) by S-adenosyl homocysteine ​​hydrolase (SAHH) and adenosine. Quantitative analysis is then achieved through immunoassay and chemiluminescent signal detection. In this process, the activity and stability of S-adenosyl homocysteine ​​hydrolase directly affect the accuracy and reliability of the detection results.

[0005] However, existing homocysteine ​​hydrolase preservation systems have the following problems: First, the hydrolase is prone to conformational changes or degradation during preservation, leading to decreased activity and affecting the shelf life of the kit; second, the detection system has weak anti-interference ability, and substances such as S-adenosylmethionine, glutathione, and cystathionine in blood samples can easily interfere with the detection results, leading to quantitative deviations. Therefore, developing a preservation solution that can improve the stability of homocysteine ​​hydrolase and has strong anti-interference ability is of great significance for improving the accuracy of homocysteine ​​detection and the practicality of the kit. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a homocysteine ​​hydrolase preservation solution, which can significantly improve the stability of S-adenosine homocysteine ​​hydrolase and enhance the anti-interference ability of the detection system. Another purpose of this invention is to provide a homocysteine ​​detection kit containing the preservation solution.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A homocysteine ​​hydrolase preservation solution includes at least one buffer component selected from MES, Tris, potassium dihydrogen phosphate and disodium hydrogen phosphate, as well as S-adenosyl homocysteine ​​hydrolase, preservative, surfactant, bovine serum albumin and ligand small molecules.

[0009] The concentration ranges of each component are as follows: S-adenosyl homocysteine ​​hydrolase 1-100 U / L; buffer 2.5-10 g / L; preservative 0.4-0.6 g / L; surfactants Tween 20 and SDS 2.8-3.2 g / L, glycerol 1-10 g / L; bovine serum albumin 8 wt%; ligand small molecules 1-100 μmol / L.

[0010] Furthermore, the preservative is proclin300, which can effectively inhibit the growth and reproduction of microorganisms in the preservation solution, avoid the destruction of hydrolytic enzyme activity by microorganisms, and extend the shelf life of the preservation solution.

[0011] Furthermore, the surfactant is selected from at least two of Tween20, glycerol, and SDS, preferably Tween20 and glycerol. The surfactant can improve the dispersibility of the preservation solution, reduce the aggregation between hydrolytic enzyme molecules, and maintain the native conformation of the hydrolytic enzyme, thereby improving its stability.

[0012] Furthermore, the ligand small molecule is selected from any one of adenosine, creatinine, PEG2000, S-inosyl-L-homocysteine, and betaine, with adenosine being preferred. The ligand small molecule (adenosine, creatinine, PEG, betaine, etc.) can specifically bind to S-adenosine homocysteine ​​hydrolase through hydrogen bonds and intermolecular forces to form a stable complex. This stabilizes the stereostructure of S-adenosine homocysteine ​​hydrolase, increases its stability in the preservation solution, thereby inhibiting its degradation. In addition, it enhances the specificity of the detection reaction and reduces the influence of interfering substances.

[0013] Furthermore, the buffer solution is preferably MES.

[0014] The present invention also provides a homocysteine ​​detection kit, comprising component R1 and component R2, wherein component R2 contains the homocysteine ​​hydrolase preservation solution described in any one of the above claims;

[0015] The R1 component comprises: PBS 0.5-2 mmol / L, reduced coenzyme I 0.4-0.5 mmol / L, DTT 0.3-0.5 mmol / L, and adenosine 1-100 mg / L. DTT in the R1 component can reduce different forms of homocysteine ​​in the sample to their free state, preparing for subsequent reactions; reduced coenzyme I participates in the signal transduction process in the detection reaction.

[0016] Furthermore, the concentrations of each component in the R2 component conform to the concentration range of the above-mentioned preservation solution, ensuring the activity stability of the hydrolytic enzyme and the anti-interference ability of the detection system.

[0017] The beneficial effects of this invention are as follows:

[0018] 1. The preservation solution of this invention, through the rational combination of buffer solution, preservative, surfactant, and ligand small molecules, forms a stable hydrolytic enzyme preservation system. The ligand small molecules specifically bind to S-adenosine homocysteine ​​hydrolase, effectively maintaining the native conformation of the hydrolase, reducing its degradation and inactivation, significantly improving the stability of the hydrolase, and extending the shelf life of the kit.

[0019] 2. This preservation solution has strong anti-interference ability and can resist the influence of common interfering substances in the sample, such as S-adenosylmethionine, glutathione, cystathionine, adenosine, blood ammonia, triglycerides, ascorbic acid, etc., to ensure the accuracy and reliability of the test results.

[0020] 3. The homocysteine ​​detection kit containing this preservation solution has a simple structure, is easy to use, and has excellent detection performance. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the protein crystal structure of S-adenosine homocysteine ​​hydrolase (SAHH) and its ligand adenosine (the original crystal structure file is from the PDB website, PDB ID: 7R39).

[0022] Figure 2 This is a schematic diagram illustrating the surface charge analysis of S-adenosine homocysteine ​​hydrolase (SAHH) and its ligand adenosine.

[0023] Appendix Figure 1 and attached Figure 2 This indicates that the ligand adenosine can form a stable complex structure with S-adenosine homocysteine ​​hydrolase, thereby maintaining the conformational stability of the hydrolase. This is an important molecular mechanism by which the preservation solution of this invention can improve the stability of the hydrolase. Detailed Implementation

[0024] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0025] Example 1

[0026] A homocysteine ​​hydrolase preservation solution has the following composition: S-adenosine homocysteine ​​hydrolase 50 U / L; MES 6 g / L; proclin300 0.5 g / L; Tween20 3.0 g / L; glycerol 5 g / L; bovine serum albumin 8 wt%; adenosine 50 μmol / L.

[0027] A homocysteine ​​assay kit, comprising component R1 and component R2:

[0028] R1 components: PBS 1 mmol / L, reduced coenzyme I 0.45 mmol / L, DTT 0.4 mmol / L, adenosine 100 mg / L;

[0029] R2 component: the above-mentioned homocysteine ​​hydrolase preservation solution.

[0030] Example 2

[0031] A homocysteine ​​hydrolase preservation solution has the following composition: S-adenosine homocysteine ​​hydrolase 30 U / L; Tris 8 g / L; proclin300 0.45 g / L; Tween20 2.9 g / L; SDS 3.0 g / L; bovine serum albumin 8 wt%; PEG2000 60 μmol / L.

[0032] A homocysteine ​​assay kit, comprising component R1 and component R2:

[0033] R1 component: PBS 0.8 mmol / L, reduced coenzyme I 0.42 mmol / L, DTT 0.35 mmol / L, adenosine 80 mg / L;

[0034] R2 component: the above-mentioned homocysteine ​​hydrolase preservation solution.

[0035] Example 3

[0036] A homocysteine ​​hydrolase preservation solution has the following composition: S-adenosine homocysteine ​​hydrolase 100 U / L; potassium dihydrogen phosphate and disodium hydrogen phosphate (mass ratio 1:1) 7 g / L; proclin 300 0.55 g / L; Tween 20 3.1 g / L; glycerol 10 g / L; bovine serum albumin 8 wt%; creatinine 40 μmol / L.

[0037] A homocysteine ​​assay kit, comprising component R1 and component R2:

[0038] R1 components: PBS 2.0 mmol / L, reduced coenzyme I 0.5 mmol / L, DTT 0.5 mmol / L, adenosine 50 mg / L;

[0039] R2 component: the above-mentioned homocysteine ​​hydrolase preservation solution.

[0040] Example 4

[0041] A homocysteine ​​hydrolase preservation solution has the following composition: S-adenosine homocysteine ​​hydrolase 20 U / L; Tris 5 g / L; proclin300 0.48 g / L; glycerol 4.0 g / L; SDS 2.8 g / L; bovine serum albumin 8 wt%; betaine 70 μmol / L.

[0042] A homocysteine ​​assay kit, comprising component R1 and component R2:

[0043] R1 components: PBS 1.2 mmol / L, reduced coenzyme I 0.46 mmol / L, DTT 0.38 mmol / L, adenosine 30 mg / L;

[0044] R2 component: the above-mentioned homocysteine ​​hydrolase preservation solution.

[0045] Example 5

[0046] A homocysteine ​​hydrolase preservation solution has the following composition: S-adenosyl homocysteine ​​hydrolase 60 U / L; MES 10 g / L; proclin300 0.6 g / L; Tween20 3.2 g / L; glycerol 3 g / L; bovine serum albumin 8 wt%; S-inosyl-L-homocysteine ​​80 μmol / L.

[0047] A homocysteine ​​assay kit, comprising component R1 and component R2:

[0048] R1 component: PBS 0.5 mmol / L, reduced coenzyme I 0.4 mmol / L, DTT 0.3 mmol / L, adenosine 20 mg / L;

[0049] R2 component: the above-mentioned homocysteine ​​hydrolase preservation solution.

[0050] Experimental verification

[0051] I. Stability Experiment

[0052] The reagent kit prepared in Example 1 (experimental group) and 10 commercially available similar reagent kits (control group) were stored in a refrigerator at 2-8℃. After 1 month, 2 months, 3 months, 4 months and 5 months of storage, the homocysteine ​​standard with a concentration of 30 μmol / L was tested. The test was performed twice each month, and the average value of the two test results was recorded. The specific data are shown in Table 1.

[0053] Table 1. Stability test results (unit: μmol / L)

[0054]

[0055] As shown in Table 1, the control group kit showed a significant decrease in detection results after 5 months of storage compared to the initial values, while the experimental group kit maintained a detection result of around 29 μmol / L after 5 months of storage, with a small deviation from the standard concentration (30 μmol / L). This indicates that the preservation solution of the present invention can significantly improve the stability of the kit.

[0056] II. Interference Experiment

[0057] Take the kit prepared in Example 5, and add different concentrations of interfering substances to a homocysteine ​​standard with a concentration of 30 μmol / L. These substances include S-adenosylmethionine (35 μmol / L), glutathione (0.7 mmol / L), cystathionine (32 μmol / L), cystine (170 μmol / L), blood ammonia (65 μmol / L), triglycerides (2800 mg / dL), and ascorbic acid (25 mmol / L). Then, perform the detection, record the results, and compare them with the detection results of the standard without interfering substances. Calculate the relative deviation. The specific data are shown in Table 2.

[0058] Table 2. Results of anti-interference experiment (unit: μmol / L)

[0059]

[0060] As shown in Table 2, within the above-mentioned concentration range of interfering substances, the relative deviation of the detection results is less than 1%, indicating that the preservation solution of the present invention has strong anti-interference ability, can effectively resist the influence of common interfering substances in the sample, and ensure the accuracy of the detection results.

[0061] The homocysteine ​​hydrolase preservation solution of this invention significantly improves the stability of S-adenosyl homocysteine ​​hydrolase and extends the shelf life of the kit by scientifically combining buffer, preservative, surfactant, and small ligand molecules. Simultaneously, this preservation solution has strong anti-interference capabilities, effectively resisting the influence of various interfering substances in the sample, ensuring the accuracy and reliability of the test results. The homocysteine ​​detection kit containing this preservation solution has a simple structure, is easy to use, and has broad clinical application prospects.

[0062] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art to the above embodiments based on the technical essence of the present invention without departing from the scope of the technical solution of the present invention should be included within the protection scope of the present invention.

Claims

1. A preservation solution for homocysteine ​​hydrolase, characterized in that, The preservation solution includes at least one buffer component selected from MES, Tris, potassium dihydrogen phosphate and disodium hydrogen phosphate, as well as S-adenosyl homocysteine ​​hydrolase, preservative, surfactant, bovine serum albumin and ligand small molecules. The concentration ranges of each component are as follows: S-adenosyl homocysteine ​​hydrolase 1-100 U / L; buffer 2.5-10 g / L; preservative 0.4-0.6 g / L; surfactants Tween 20 and SDS 2.8-3.2 g / L, glycerol 1-10 g / L; bovine serum albumin 8 wt%; ligand small molecules 1-100 μmol / L.

2. The homocysteine ​​hydrolase preservation solution according to claim 1, characterized in that, The preservative is proclin300.

3. The homocysteine ​​hydrolase preservation solution according to claim 1, characterized in that, The surfactant is selected from at least two of Tween20, glycerol, and SDS, preferably Tween20 and glycerol.

4. The homocysteine ​​hydrolase preservation solution according to claim 1, characterized in that, The ligand molecule is selected from any one of adenosine, creatinine, PEG2000, S-inosyl-L-homocysteine, and betaine, with adenosine being preferred.

5. The homocysteine ​​hydrolase preservation solution according to claim 1, characterized in that, The buffer solution is preferably MES.

6. A homocysteine ​​detection kit, characterized in that, It includes component R1 and component R2, wherein component R2 contains the homocysteine ​​hydrolase preservation solution according to any one of claims 1-5; component R1 includes: PBS 0.5-2 mmol / L, reduced coenzyme I 0.4-0.5 mmol / L, DTT 0.3-0.5 mmol / L, and adenosine 1-100 mg / L.

7. The homocysteine ​​detection kit according to claim 6, characterized in that, The concentrations of each component in the R2 component conform to the concentration range defined in claim 1.

8. The use of the homocysteine ​​hydrolase preservation solution according to any one of claims 1-5 in improving the stability and anti-interference ability of homocysteine ​​detection.

9. The application according to claim 8, characterized in that, The anti-interference ability is specifically demonstrated by the fact that under the conditions of S-adenosylmethionine ≤35μmol / L, glutathione ≤0.7mmol / L, cystathionine ≤32μmol / L, cystine ≤170μmol / L, blood ammonia ≤65μmol / L, triglycerides ≤2800mg / dL, and ascorbic acid ≤25mmol / L, there is no significant interference with the detection results.

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