A 25-hydroxyvitamin D dissociating agent, its preparation method and application

Abstract

The present invention discloses a 25-hydroxyvitamin D dissociating agent, which is characterized by comprising the following components: 10-500 mM PBS, 0.005-0.05 g / mL n-octyl-β-D-maltoside, 0.01-0.1 g / mL 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate, 0.001-0.01 g / mL sodium dodecyl sulfate, 0.001-0.01 g / mL BSA, 0.001-0.01 g / mL sodium chloride, 0.01-1% of PC300, and pH 6.0-8.0. The present invention has no toxic effect on the human body and no direct or indirect toxic hazards and risks to users and producers; most of the components of the present invention are novel and highly efficient detergents, and have an obvious effect on protein dissociation.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a 25-hydroxyvitamin D dissociating agent, a preparation method thereof, and an application thereof. Background Art

[0002] The detection of 25-hydroxyvitamin D can reflect the vitamin D status of the human body, and has important significance in the diagnosis and monitoring of rickets, osteoporosis, etc.; 25-hydroxyvitamin D in the blood is often tightly bound to binding proteins, and the bound complex needs to be fully dissociated before accurate quantification.

[0003] Currently, the commonly used dissociating agents include several categories such as organic solvents (DMSO, etc.), strong acids and bases, and fluoride-containing substances. They all have a certain dissociating effect, but at the same time they all have a common fatal defect - containing toxic, harmful or even highly toxic components, posing risks to biosafety or environmental hazards. Specifically, organic substances such as DMSO are highly toxic substances; strong acids and bases have extremely strong corrosiveness; fluoride-containing substances are highly toxic and are difficult to degrade in the natural environment, having potential bioaccumulation and environmental persistence. As a result, the European Union has added a restriction clause in Annex XVII, Item 68 of the REACH Regulation regarding perfluorooctanoic acid (PFOA), officially including PFOA, its salts, and related substances in the REACH Regulation restriction list. Summary of the Invention

[0004] Object of the Invention: The technical problem to be solved by the present invention is to provide an environmentally friendly and safe 25-hydroxyvitamin D dissociating agent to solve the technical problem that the 25-hydroxyvitamin D dissociating agent in the prior art contains toxic and harmful components.

[0005] The present invention also needs to solve the technical problem of providing a preparation method of the above-mentioned 25-hydroxyvitamin D dissociating agent.

[0006] The last technical problem to be solved by the present invention is to provide an application of the above-mentioned 25-hydroxyvitamin D dissociating agent in detecting the content of 25-hydroxyvitamin D in blood.

[0007] Technical Solution: To solve the above technical problems, the present invention provides the following technical solutions:

[0008] A 25-hydroxyvitamin D dissociating agent, characterized in that it comprises the following components:

[0009] 10 - 500 mM PBS, 0.005 - 0.05 g / mL n - octyl - β - D - maltoside, 0.01 - 0.1 g / mL 3 - [(3 - cholamidopropyl)dimethylammonio]-2 - hydroxy - 1 - propanesulfonate, 0.001 - 0.01 g / mL sodium dodecyl sulfate, 0.001 - 0.01 g / mL BSA, 0.001 - 0.01 g / mL sodium chloride, 0.01 - 1% PC300, pH 6.0 - 8.0.

[0010] Further, the 25 - hydroxyvitamin D dissociating agent preferably has the following formulation:

[0011] 20 mM PBS, 0.01 g / mL n - octyl - β - D - maltoside, 0.02 g / mL 3 - [(3 - cholamidopropyl)dimethylammonio]-2 - hydroxy - 1 - propanesulfonate, 0.005 g / mL sodium dodecyl sulfate, 0.005 g / mL BSA, 0.01 g / mL sodium chloride, 0.05% PC300, pH 7.5.

[0012] The preparation method of the above - mentioned 25 - hydroxyvitamin D dissociating agent includes the following steps:

[0013] Weigh disodium hydrogen phosphate and sodium dihydrogen phosphate to prepare a 20 mM PBS solution. While continuously stirring with a stir bar, successively weigh n - octyl - β - D - maltoside, 3 - [(3 - cholamidopropyl)dimethylammonio]-2 - hydroxy - 1 - propanesulfonate, sodium dodecyl sulfate, and sodium chloride, and successively add them to the PBS solution. Add another component after one component is fully dissolved to obtain a stable system. Then, use 5 M sodium hydroxide to adjust the pH to 7.5. Then add BSA and PC300, mix well, and filter through a 0.45 μm filter membrane to obtain the 25 - hydroxyvitamin D dissociating agent.

[0014] The application of the above - mentioned 25 - hydroxyvitamin D dissociating agent in detecting the content of 25 - hydroxyvitamin D in blood is within the protection scope of the present invention.

[0015] Beneficial effects: The present invention has the following outstanding technical effects:

[0016] (1) The present invention has no toxic effect on the human body and has no direct or indirect toxic hazards and risks to users and producers;

[0017] (2) The components of the present invention are biodegradable components and will not accumulate in nature or the environment, causing no pollution to the environment;

[0018] (3) Most of the components of the present invention are novel and highly efficient detergents and surfactants, and have an obvious effect on protein dissociation;

[0019] (4) The components of the present invention are stable, with loose storage conditions and stable and uniform dissociation effects. Description of the Drawings

[0020] Figure 1 The figure showing the results of a comparative experiment with a well-known commercially available reagent of the present invention.

[0021] Figure 2 Correlation between Roche results and Disassociant 1.

[0022] Figure 3 Correlation between Roche results and Disassociant 2.

[0023] Figure 4 Correlation between Roche results and Disassociant 3.

[0024] Figure 5 Correlation between Roche results and Disassociant 4.

[0025] Figure 6 Correlation between Roche results and Disassociant 5.

[0026] Figure 7 Correlation between Roche results and Disassociant 6.

[0027] Specific Implementation Steps

[0028] According to the following embodiments, the present invention can be better understood. However, those skilled in the art can easily understand that the content described in the embodiments is only used to illustrate the present invention and should not and will not limit the present invention described in detail in the claims.

[0029] Example 1: This example provides the preparation process of the disassociant of the present invention.

[0030] The contents of the components in the disassociant described in the present invention are: 20 mM PBS, 0.01 g / mL n-octyl-β-D-maltoside, 0.02 g / mL 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate, 0.005 g / mL sodium dodecyl sulfate, 0.005 g / mL BSA, 0.01 g / mL sodium chloride, 0.05% PC300, pH 7.5.

[0031] Preparation method: Weigh disodium hydrogen phosphate, sodium dihydrogen phosphate, etc. according to the above ratio to prepare a 20 mM PBS solution. While continuously stirring with a stir bar, weigh n-octyl-β-D-maltoside, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate, sodium dodecyl sulfate, and sodium chloride in sequence, and add them to the PBS solution one by one. After one component is fully dissolved, add another component to obtain a stable system. Then add 5 M sodium hydroxide to adjust the pH to 7.5. Then add BSA and PC300, mix well, and filter through a 0.45 μm filter to obtain the dissociating agent.

[0032] Example 2: Detection test of the correlation of this dissociating agent

[0033] Take 25 clinical samples, conduct an experimental comparison using the dissociating agent of the present invention and Roche reagent, and the correlation of the detection results is shown in Table 1 and Figure 1 。

[0034] Table 1. Roche results VS data of the present invention

[0035]

[0036]

[0037] The experimental results show that the correlation between the detection results of the present invention and those of Roche reagent is better, and the correlation coefficient r is greater than 0.99, indicating that the dissociating effect of the dissociating agent of the present invention is good, very close to the performance of the currently market-recognized products, can meet the current clinical detection requirements for dissociating agents, and has a very large application space.

[0038] Examples of comparative examples

[0039] Comparative dissociating agent 1: Only contains 0.005 g / mL sodium dodecyl sulfate;

[0040] Comparative dissociating agent 2: Only contains 0.02 g / mL 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate, 0.005 g / mL sodium dodecyl sulfate,

[0041] Comparative dissociating agent 3: Only contains 0.01 g / mL n-octyl-β-D-maltoside, 0.005 g / mL sodium dodecyl sulfate;

[0042] According to the above components, compare dissociating agents 1-3, detect the 25-hydroxyvitamin D level in 25 clinical samples, with Roche reagent as the comparison, and the detection result data are shown in Tables 2-4, and the correlation analysis is shown in Figures 2 - 4 。

[0043] Table 2. Roche results VS data of dissociating agent 1

[0044]

[0045]

[0046] Table 3. Roche Results VS Dissociator 2 Data

[0047]

[0048]

[0049] Table 4. Roche Results VS Dissociator 3 Data

[0050]

[0051]

[0052] The results showed that the correlation coefficient r between the test results of dissociator 1 and the detection results of Roche reagent was 0.3968; the correlation coefficient r between the test results of dissociator 2 and the detection results of Roche reagent was 0.4882; the correlation coefficient r between the test results of dissociator 3 and the detection results of Roche reagent was 0.8018. In summary, the consistency of the above three dissociators compared with the detection results of Roche reagent was poor and could not meet the requirements of clinical detection.

[0053] Example 3: Verification of the addition amount of this dissociator.

[0054] Take 25 clinical samples, compare the dissociation effects of the dissociator of commercially available Company B and different usage amounts of the present invention, and detect using the chemiluminescence detection reagent of our company. The detection results are shown in Table 5.

[0055] Table 5. Comparison of the dissociation effects of the dissociator of commercially available Company B and different usage amounts of the present invention

[0056]

[0057]

[0058] Analyzing from the usage amount of the dissociator, comparing the usage amount of the dissociator of commercially available Company B, it can be fully dissociated in the range of about 100 - 150 μL. From the data results of this example, when the usage amount of the present invention is 50 μL, the effect of full dissociation can be achieved. Although a small part does not reach the effect of Company B, the effect is basically at the same level and there is no obvious difference. Thus, it can be seen that the dissociator of the present invention has obvious advantages in terms of usage amount. Compared with conventional products on the market, the usage amount is reduced by 1 / 2.

[0059] Example 4: Verification of the dissociation effect of this dissociator after long-term storage

[0060] The dissociation agents of the present invention were stored at room temperature and at 2 - 8°C for 0 month, 3 months, 6 months, 9 months, and 12 months respectively. Here, 0 month refers to the time at the start of the verification test. 25 clinical samples were taken to verify the dissociation effects under different storage environments and at different time points, and detected using the chemiluminescence detection reagent of our company. The detection results are shown in Table 6 - 7.

[0061] Table 6. Comparison of dissociation effects of the present invention stored at room temperature for different times

[0062]

[0063] Table 7. Comparison of dissociation effects of the present invention stored at 2 - 8°C for different times

[0064]

[0065]

[0066] The dissociation effects showed that when stored at room temperature and at 2 - 8°C for 0 month, 3 months, 6 months, 9 months, and 12 months, there were no obvious changes in the dissociation effects of the dissociation agents of the present invention. The storage conditions were loose and the dissociation effects were stable; the dissociation effects were not affected by the storage environment. In specific clinical and scientific research applications, appropriate storage methods can be selected according to the specific laboratory. Within the validity period of 12 months, the dissociation effects are not affected by the storage environment.

[0067] Example 5: Verification of dissociation effects when different concentrations are added to the components of this dissociation agent

[0068] Verification of the dissociation effects of different added concentrations of the main components of the dissociation agent of the present invention. 25 clinical samples were taken to verify the dissociation effects under different storage environments and at different time points, and detected using the chemiluminescence detection reagent of our company. The detection results are shown in Table 8, and the analysis results are shown in Figures 2 to 4 .

[0069] Table 8. Comparison of dissociation effects of different concentrations of the main components of the dissociation agent of the present invention

[0070]

[0071]

[0072] Comparative dissociation agent 4: 10 PBS, 0.005 g / mL n - octyl - β - D - maltoside, 0.01 g / mL 3 - [(3 - cholesterylaminopropyl)dimethylamino] - 2 - hydroxy - 1 - propanesulfonic acid, 0.001 g / mL sodium dodecyl sulfate, 0.001 g / mL BSA, 0.001 g / mL sodium chloride, 0.01% PC300, pH 6.0 - 8.0.

[0073] Comparison dissociating agent 5: 200 mM PBS, 0.02 g / mL n-octyl-β-D-maltoside, 0.05 g / mL 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate, 0.005 g / mL sodium dodecyl sulfate, 0.005 g / mL BSA, 0.005 g / mL sodium chloride, 0.5% PC300, pH 6.0 - 8.0.

[0074] Comparison dissociating agent 6: 500 mM PBS, 0.05 g / mL n-octyl-β-D-maltoside, 0.1 g / mL 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate, 0.01 g / mL sodium dodecyl sulfate, 0.01 g / mL BSA, 0.01 g / mL sodium chloride, 1% PC300, pH 6.0 - 8.0.

[0075] The dissociation effect shows that within the main component concentration range stated in the present invention, the dissociation effects of the high, medium, and low concentrations of the dissociating agent of the present invention have no obvious change and can all achieve an ideal dissociation effect. In specific clinical and scientific research applications, the appropriate main component concentration can be selected according to the specific laboratory.

Claims

1. A 25-hydroxyvitamin D dissociating agent, characterized in that, it consists of the following components: 10 - 500 mM PBS, 0.005 - 0.05 g / mL n-octyl-β-D-maltoside, 0.01 - 0.1 g / mL 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate, 0.001 - 0.01 g / mL sodium dodecyl sulfate, 0.001 - 0.01 g / mL BSA, 0.001 - 0.01 g / mL sodium chloride, 0.01 - 1% PC300, pH 6.0 - 8.

0.

2. The 25-hydroxyvitamin D dissociating agent according to claim 1, characterized in that, it consists of the following components: 20 mM PBS, 0.01 g / mL n-octyl-β-D-maltoside, 0.02 g / mL 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate, 0.005 g / mL sodium dodecyl sulfate, 0.005 g / mL BSA, 0.01 g / mL sodium chloride, 0.05% PC300, pH 7.

5.

3. The preparation method of the 25-hydroxyvitamin D dissociating agent according to claim 1 or 2, characterized in that, it includes the following steps: Weigh disodium hydrogen phosphate and sodium dihydrogen phosphate to prepare a PBS solution. While continuously stirring with a stir bar, weigh n-octyl-β-D-maltoside, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate, sodium dodecyl sulfate, and sodium chloride in sequence, and add them to the PBS solution in sequence. After one component is fully dissolved, then add another component to obtain a stable system. Then adjust the pH with 5M sodium hydroxide, add BSA and PC300, mix well, and filter through a 0.45 μm filter membrane to obtain the 25-hydroxyvitamin D dissociating agent.

4. The application of the 25-hydroxyvitamin D dissociating agent according to claim 1 or 2 in detecting the content of 25-hydroxyvitamin D in blood for non-diagnostic purposes.

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