A neutral dissociation agent for detecting the content of 25-hydroxy vitamin D in human blood sample and a preparation method thereof

Abstract

This invention discloses a neutral dissociation agent and its preparation method for detecting the content of 25-hydroxyvitamin D in human blood samples, belonging to the field of biomedical detection technology. The dissociation agent is composed of disodium EDTA, sodium dodecyl sulfonate, dithiothreitol, hexadecyltrimethylammonium bromide, and hydroxypropyl-β-cyclodextrin dissolved in 50 mM tris(hydroxymethyl)aminomethane buffer, with the mass-volume ratios of each component being 0.5%-1%, 0.5%-1%, 0.2%-0.5%, 0.2%-0.5%, and 0.2%-0.5%, respectively. During preparation, each component is dissolved according to the steps, and the pH is adjusted to 7.4. This dissociation agent exhibits mild dissociation conditions, does not damage protein activity, has high dissociation efficiency, and is non-corrosive and non-toxic. The detection results show high specificity and sensitivity, with a coefficient of variation ≤6%. It can be directly integrated into automated detection processes and is suitable for large-scale clinical and research applications.

Description

Technical Field

[0003] This invention relates to the field of biomedical detection technology, specifically to a neutral dissociating agent for detecting the content of 25-hydroxyvitamin D in human blood samples and its preparation method, and also to the application of the dissociating agent in the detection of 25-hydroxyvitamin D. Background Technology

[0005] Vitamin D is an essential fat-soluble steroid derivative for the human body, mainly existing in the forms of D2 and D3. It is hydroxylated in the liver to produce 25-hydroxyvitamin D, and its blood concentration is the best indicator for assessing a person's vitamin D status. Detection of 25-hydroxyvitamin D levels is of significant clinical importance for determining vitamin D deficiency / excess, guiding individualized supplementation, and preventing bone diseases, autoimmune diseases, and other related conditions.

[0006] Currently, the vitamin D dissociation agents used for pretreatment in the detection of 25-hydroxyvitamin D have many technical shortcomings:

[0007] (1) Acid-base method: Vitamin D is physically released by denaturing and precipitating vitamin D-binding proteins through extreme pH (strong acid or strong base). Although this method is simple and fast, it is highly destructive and may damage the structure of vitamin D. Moreover, the extreme pH of the treated sample will destroy antibody activity. It must be subjected to complex neutralization / dilution before it can be used for subsequent immune responses, and the release efficiency is difficult to balance with the pH intensity.

[0008] (2) Reduction method: The disulfide bonds of VDBP are broken by a strong reducing agent (such as dithiothreitol DTT), causing it to denature or undergo conformational changes and release vitamin D. Although this method can also release vitamin D, the strong reducing agent will also break the disulfide bonds of the antibody protein, inactivating it. The concentration of the reducing agent used needs to be strictly determined, and residual reducing agent may interfere with subsequent immune responses.

[0009] (3) Enzymatic hydrolysis method: Add a specific protease (such as proteinase K) to hydrolyze VDBP and gently release vitamin D. This method takes too long and does not meet the requirements for rapid detection. In addition, the enzyme preparation is expensive and has high requirements for reagent storage stability.

[0010] (4) Organic solvent extraction method: Proteins are precipitated using organic solvents (acetonitrile, methanol, etc.), while vitamin D is competitively dissociated and extracted to the supernatant based on the principle of "like dissolves like". This method has the highest release and extraction efficiency and is considered the "gold standard" pretreatment, which can effectively remove most protein interferences. However, it involves multiple steps such as adding liquid, vortexing, centrifugation, and taking the supernatant, and is not easy to integrate into a fully automated immunoassay analyzer.

[0011] (5) Displacement method: Adding a high concentration of vitamin D structural analogues or strong binding agents to competitively bind to VDBP with endogenous vitamin D, thereby displacing it. Currently, the most efficient displacement agent (such as perfluorooctanoic acid PFOA) is a persistent organic pollutant, which is environmentally unfriendly and poses safety and regulatory risks.

[0012] Therefore, developing a 25-hydroxyvitamin D dissociator that has mild dissociation conditions, high dissociation efficiency, environmental friendliness, and can be adapted to automated detection processes has become an urgent need in the field of biomedical detection. Summary of the Invention

[0014] This invention aims to overcome the technical deficiencies of existing 25-hydroxyvitamin D dissociation agents and provide a neutral dissociation agent that features mild conditions, high dissociation efficiency, no corrosive toxicity, and accurate and reliable detection results. It also provides a method for preparing this dissociation agent that is simple to operate, uses readily available raw materials, and is suitable for large-scale preparation. Furthermore, this invention discloses the application method of this dissociation agent, which can be directly integrated into automated detection processes to meet the needs of high-throughput clinical testing.

[0015] Technical solution:

[0016] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0017] A neutral dissociating agent for detecting the content of 25-hydroxyvitamin D in human blood samples comprises disodium EDTA, sodium dodecyl sulfonate, dithiothreitol, hexadecyltrimethylammonium bromide, hydroxypropyl-β-cyclodextrin, and a buffer solution; wherein the buffer solution is a 50 mM tris(hydroxymethyl)aminomethane buffer with a pH of 7.4; the mass-to-volume ratios of disodium EDTA, sodium dodecyl sulfonate, dithiothreitol, hexadecyltrimethylammonium bromide, and hydroxypropyl-β-cyclodextrin dissolved in the buffer solution are 0.5%-1%, 0.5%-1%, 0.2%-0.5%, 0.2%-0.5%, and 0.2%-0.5%, respectively.

[0018] Preferably, the mass-volume ratio of each component is: 0.5% disodium EDTA, 0.5% sodium dodecyl sulfonate, 0.2% dithiothreitol, 0.2% hexadecyltrimethylammonium bromide, and 0.2% hydroxypropyl-β-cyclodextrin. This ratio is the optimal ratio, with the best dissociation effect and the highest correlation between the test results and the gold standard.

[0019] The present invention also provides a method for preparing the above-mentioned neutral dissociation agent, comprising the following steps:

[0020] S1: Prepare a 50mM tris(hydroxymethyl)aminomethane buffer solution, adjust the pH to 7.4 using an acid-base adjuster, and set aside.

[0021] S2: Weigh out EDTA disodium, sodium dodecyl sulfonate and dithiothreitol according to the above proportions, add them to the buffer solution prepared in step S1, stir thoroughly to dissolve, and adjust the pH to 7.4.

[0022] S3: Weigh out hexadecyltrimethylammonium bromide according to the above proportion, add it to the solution in step S2, stir thoroughly to dissolve, and then adjust the pH to 7.4;

[0023] S4: Weigh out hydroxypropyl-β-cyclodextrin according to the above proportion, add it to the solution in step S3, stir thoroughly to dissolve, and adjust the pH to 7.4 to obtain the neutral dissociation agent.

[0024] Furthermore, in step S2, the mass-volume ratio of disodium EDTA and sodium dodecyl sulfonate is 0.5%, and the mass-volume ratio of dithiothreitol is 0.2%; in step S3, the mass-volume ratio of hexadecyltrimethylammonium bromide is 0.2%; and in step S4, the mass-volume ratio of hydroxypropyl-β-cyclodextrin is 0.2%.

[0025] The present invention also provides the application of the above-mentioned neutral dissociating agent in the detection of 25-hydroxyvitamin D content in human blood samples. The serum to be tested is mixed with the dissociating agent at a volume ratio of 1:1, and after reacting at 37°C for 5 minutes, subsequent detection can be performed directly using chemiluminescent immunoassay without additional pretreatment steps.

[0026] Compared with the prior art, the beneficial effects of the present invention are reflected in:

[0027] 1. Mild dissociation conditions: This invention is a neutral dissociation agent with a stable pH of 7.4. During the dissociation process, it does not damage the activity of 25-hydroxyvitamin D binding protein and antibody protein, ensuring the integrity of the 25-hydroxyvitamin D molecule conformation, thereby ensuring the specificity and sensitivity of the detection and obtaining true and reliable detection results.

[0028] 2. Highly efficient dissociation and adaptable to automation: It can quickly and fully release free 25-hydroxyvitamin D in blood samples without complicated pretreatment steps. It can be directly integrated into standardized and automated detection processes to achieve high-throughput and automated operation while ensuring the accuracy and repeatability of test results.

[0029] 3. Environmentally friendly and highly safe: This dissociation agent is non-toxic and non-corrosive, poses no harm to the health of operators, will not cause corrosive damage to testing instruments, and will not pollute the environment, thus solving the environmental safety problems of existing displacement agents.

[0030] 4. Simple preparation and low cost: The components of the dissociation agent are simple, all raw materials are commercially available products, the preparation steps are simple, no special equipment is required, it is easy to carry out large-scale production, and the cost of clinical testing is reduced.

[0031] 5. High accuracy of test results: After the sample is treated with the dissociation agent of this invention, the deviation of the test results from the Roche gold standard is between -13% and 12%, the coefficient of variation (CV) of repeated tests is no greater than 6%, and the deviation is basically no more than 4%, which is far superior to the detection effect of existing dissociation agents. Attached Figure Description

[0033] Figure 1 This is a correlation analysis chart between the test results of control group 1 and the results of Roche's assay.

[0034] Figure 2 This is a correlation analysis chart between the test results of control group 2 and the results of Roche's assay.

[0035] Figure 3 This is a correlation analysis chart between the test results of control group 3 and the Roche measurement results.

[0036] Figure 4 This is a correlation analysis graph between the test results of Example 1 and the Roche measurement results. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to specific embodiments. The scope of protection of the present invention is not limited to the following embodiments, and all technical solutions based on the present invention are within the scope of protection of the present invention.

[0039] All raw materials used in this invention are commercially available conventional reagents, and the fully automated chemiluminescence immunoassay analyzer used is the VIT900 fully automated chemiluminescence immunoassay analyzer developed by Shenzhen Tailede Medical Co., Ltd.

[0040] Example 1: Preparation scheme of the dissociation agent of the present invention

[0041] S1: Prepare a 50mM tris(hydroxymethyl)aminomethane buffer solution and adjust the pH to 7.4 with hydrochloric acid;

[0042] S2: Weigh 0.5g of disodium EDTA, 0.5g of sodium dodecyl sulfonate, and 0.2g of dithiothreitol, add them to 100mL of the buffer solution from step S1, stir thoroughly to dissolve, and then adjust the pH to 7.4.

[0043] S3: Weigh 0.2g of hexadecyltrimethylammonium bromide and add it to the solution in step S2. After stirring and dissolving thoroughly, adjust the pH to 7.4.

[0044] S4: Weigh 0.2g of hydroxypropyl-β-cyclodextrin and add it to the solution in step S3. After stirring and dissolving thoroughly, adjust the pH to 7.4 to obtain the neutral dissociation agent of the present invention.

[0045] Example 2: Dissociation effect detection

[0046] 2.1 Preparation scheme of dissociation agent for control group

[0047] Control group 1: Dissolve 0.5% disodium EDTA, 0.5% sodium dodecyl sulfonate, and 0.2% dithiothreitol in 50mM tris(hydroxymethyl)aminomethane buffer (pH 7.4) to obtain the dissociation agent of control group 1;

[0048] Control group 2: Dissolve 0.5% disodium EDTA, 0.5% sodium dodecyl sulfonate, 0.2% dithiothreitol, and 0.2% hexadecyltrimethylammonium bromide in 50mM tris(hydroxymethyl)aminomethane buffer (pH 7.4) to obtain the dissociation agent of control group 2;

[0049] Control group 3: Dissociation agent of control group 3 was prepared by dissolving 0.5% disodium EDTA, 0.5% sodium dodecyl sulfonate, 0.2% dithiothreitol and 0.2% hydroxypropyl-β-cyclodextrin in 50mM tris(hydroxymethyl)aminomethane buffer (pH 7.4);

[0050] 2.2 Sample processing and testing steps

[0051] Twenty human blood serum samples were selected and processed using four methods: Example 1 and Control Groups 1-3. The 25-hydroxyvitamin D content was detected using a VIT900 fully automated chemiluminescence immunoassay analyzer. At the same time, the Roche 25-hydroxyvitamin D assay kit was used as the gold standard for detection, and the detection deviation and correlation coefficient of each method were compared.

[0052] The testing steps are as follows:

[0053] 1. Mix the serum to be tested with the dissociation agent at a volume ratio of 1:1 and react at 37°C for 5 min;

[0054] 2. Add alkaline phosphatase-labeled 25-hydroxyvitamin D antibody to the mixture of treated serum and dissociation agent, and react at 37°C for 7.5 min;

[0055] 3. Add biotin-labeled 25-hydroxyvitamin D and streptavidin-labeled magnetic beads to the system with good reaction, and react at 37°C for 7.5 min;

[0056] 4. Clean the sample and add chemiluminescent solution. The machine measures the luminescence signal and reports the concentration of 25-hydroxyvitamin D in the serum.

[0057] 2.3 Analysis of Test Results

[0058] As shown in Table 1 and Figure 1As shown, the test results of control group 1 deviated from the Roche measured values ​​by -52% to 24%, with a K value of 0.54 and an R-squared value of 0.80. The correlation with the Roche measured values ​​was poor. The dissociation agent scheme of control group 1 had a certain dissociation effect, but the dissociation was not sufficient.

[0059] As shown in Table 2 and Figure 2 As shown, the deviation between the test results of control group 2 and the Roche measured values ​​was between -20% and 22%, with a K value of 0.85 and an R-squared value of 0.93. Compared with control group 1, the correlation with the Roche measured values ​​was better.

[0060] As shown in Table 3 and Figure 3 As shown, the deviation between the test results of control group 3 and the Roche measured values ​​was between -20% and 17%, with a K value of 0.90 and an R-squared value of 0.92. Compared with control groups 1 and 2, the correlation with Roche measured values ​​was better.

[0061] As shown in Table 4 and Figure 4 As shown, the deviation between the test results of Example 1 and the Roche measured values ​​was between -13% and 12%, with a K value of 0.95 and an R-squared value of 0.98. Compared with control groups 1, 2, and 3, it showed the best correlation with the Roche measured values.

[0062] Table 1. Results of dissociation effect measurement in control group 1

[0063]

[0064] Table 2. Results of dissociation effect measurement in control group 2

[0065]

[0066] Table 3. Results of dissociation effect measurement in control group 3

[0067]

[0068] Table 4 Results of dissociation effect measurement in Example 1

[0069]

[0070] Example 3 Repeatability Test

[0071] Six Roche-calibrated serum samples were selected and treated with the dissociation agent described in Example 1 of this invention. Each sample was tested 10 times, and the coefficient of variation (CV) and deviation from the target value were calculated.

[0072] Test results: The coefficients of variation (CV) of the test results of the 6 samples were 4%, 5%, 4%, 5%, 6%, and 5%, respectively, all ≤6%; the deviations from the target values ​​were -1%, -4%, -2%, -2%, -1%, and -2%, respectively, basically not exceeding 4%, indicating that the test results of the samples treated with the dissociation agent in Example 1 of this invention have good repeatability and high accuracy.

[0073] Table 5. Coefficient of variation and bias detection results (unit: ng / mL)

[0074]

[0075] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A neutral dissociating agent for detecting the content of 25-hydroxyvitamin D in human blood samples, characterized in that, It is composed of disodium EDTA, sodium dodecyl sulfonate, dithiothreitol, hexadecyltrimethylammonium bromide, hydroxypropyl-β-cyclodextrin, and a buffer solution; the buffer solution is a 50 mM tris(hydroxymethyl)aminomethane buffer solution with a pH of 7.4; the mass-to-volume ratios of disodium EDTA, sodium dodecyl sulfonate, dithiothreitol, hexadecyltrimethylammonium bromide, and hydroxypropyl-β-cyclodextrin dissolved in the buffer solution are 0.5%-1%, 0.5%-1%, 0.2%-0.5%, 0.2%-0.5%, and 0.2%-0.5%, respectively.

2. The neutral dissociation agent according to claim 1, characterized in that, The mass-to-volume ratio of disodium EDTA and sodium dodecyl sulfonate dissolved in the tris(hydroxymethyl)aminomethane buffer was 0.5%, and the mass-to-volume ratio of dithiothreitol, hexadecyltrimethylammonium bromide, and hydroxypropyl-β-cyclodextrin was 0.2%.

3. A method for preparing the neutral dissociation agent according to claim 1 or 2, characterized in that, Includes the following steps: S1: Prepare a 50 mM tris(hydroxymethyl)aminomethane buffer solution and adjust the pH to 7.4; S2: Weigh out disodium EDTA, sodium dodecyl sulfonate, and dithiothreitol according to the specified ratio, dissolve them in the buffer solution of step S1, and adjust the pH to 7.4; S3: Weigh out hexadecyltrimethylammonium bromide according to the specified ratio, dissolve it in the solution of step S2, and adjust the pH to 7.4; S4: Weigh out hydroxypropyl-β-cyclodextrin according to the specified ratio, dissolve it in the solution of step S3, and adjust the pH to 7.4 to obtain the neutral dissociating agent.

4. The preparation method according to claim 3, characterized in that, In step S2, the mass-volume ratio of disodium EDTA and sodium dodecyl sulfonate is 0.5%, and the mass-volume ratio of dithiothreitol is 0.2%.

5. The preparation method according to claim 3, characterized in that, In step S3, the mass-volume ratio of hexadecyltrimethylammonium bromide is 0.2%; in step S4, the mass-volume ratio of hydroxypropyl-β-cyclodextrin is 0.2%.

6. The application of the neutral dissociating agent according to claim 1 or 2 in the detection of 25-hydroxyvitamin D content in human blood samples, characterized in that, The serum to be tested is mixed with the dissociation agent at a volume ratio of 1:1, and after reacting at 37°C for 5 minutes, it can be directly detected by chemiluminescent immunoassay.

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