Preparation method and application of releasing agent

Abstract

The invention relates to a preparation method and application of a releasing agent, and belongs to the technical field of steroid hormone releasing agents. The releasing agent is prepared from the following components in percentage by mass: 0.2 to 1 percent of an amide compound, 0.02 to 0.06 percent of a metal chelate, 0.05 to 0.15 percent of a preservative, 0.2 to 0.5 percent of a surfactant, 0.015 to 0.045 percent of a steroid heterocyclic compound and the balance of deionized water, wherein the amide compound is a composition of sodium alpha-mercaptoethanesulfonate and N-methylacetamide, and the amide compound is a compound of sodium alpha-mercaptoethanesulfonate and N-methylacetamide; the steroid heterocyclic compound is a composition of methyltestosterone, danazol and stanolone. According to the universal releasing agent, the four hormones in a sample can be effectively released from respective binding proteins to be in a free state, and clinical test data of the sample can be obtained by measuring the content of the free hormones, so that the measured test result is more accurate.

Description

Technical Field

[0001] The invention relates to the technical field of steroid hormone releasers, and in particular to a preparation method of a releaser and application thereof. Background Art

[0002] Testosterone, estradiol, progesterone, and cortisol are important components of clinical hormone testing as steroid hormone types. The difficulty in developing this project lies in how to release the bound steroid hormone molecules from their respective binding proteins, thereby becoming free molecules to facilitate the detection of related reagents and improve clinical accuracy. Therefore, the research and development of this type of release agent is of great significance to the development of steroid hormone type detection reagents, which can greatly improve the accuracy of the reagents and avoid risks.

[0003] Among them, 98% of testosterone molecules in human blood are bound to sex hormone binding globulin (SHBG), almost all estradiol molecules exist in the form of binding proteins, progesterone is mainly bound to albumin, followed by sex hormone binding globulin (SHBG), and most of the cortisol in the blood is bound to binding proteins. About 1% of cortisol is excreted in the urine in its original form. This particularity has brought difficulties and challenges to the research and development of steroid hormone quantitative detection reagents. In clinical diagnosis, only the detection of free states of these four steroid hormones is clinically significant. How to release these four steroid hormones in the sample serum from the bound state to become free molecules has become a difficult problem in experimental research and development. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a universal release agent that can release steroid hormone (including testosterone, estradiol, progesterone, cortisol) molecules in serum from their respective binding proteins, and also relates to a preparation method and use of the universal release agent.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] In the first aspect, the present invention provides a release agent, comprising the following components in mass percentage: 0.2-1% amide compound, 0.02-0.06% metal chelate, 0.05-0.15% preservative, 0.2-0.5% surfactant, 0.015-0.045% steroidal heterocyclic compound, and the balance is deionized water; wherein the amide compound is a combination of sodium α-mercaptoethylsulfonate and N-methylacetamide; the steroidal heterocyclic compound is a combination of methylhydrotestosterone, danazol and androsol; the pH of the release agent system is 6.0-6.5.

[0007] The amide composition of sodium α-mercaptoethylsulfonate and N-methylacetamide in the releaser of the present invention may react with hormone binding protein to change its conformation, thereby making the hormone molecule more easily released. The steroidal heterocyclic compound composed of methylhydrotestosterone, danazol and androsol has a similar structure to steroid hormones and may displace the hormone molecule from the binding protein by competing for binding sites. The surfactant can reduce the surface tension of the solution, enhance the permeability of the solution to the hormone binding protein, and help release the hormone molecule. Controlling the pH of the releaser system to 6.0-6.5 can help maintain the stability and activity of the releaser.

[0008] The release agent of the present invention can effectively release hormone molecules from binding proteins by targeting the interaction between steroid hormones and binding proteins in serum through a carefully designed combination of components, thereby facilitating detection accuracy.

[0009] Preferably, the release agent comprises the following components in percentage by mass: 0.2-1% amide compound, 0.05% metal chelate, 0.1% preservative, 0.4% surfactant, 0.015-0.045% steroidal heterocyclic compound, and the remainder is deionized water.

[0010] More preferably, the release agent comprises the following components in percentage by weight: 0.5% amide compound, 0.05% metal chelate, 0.1% preservative, 0.4% surfactant, 0.03% steroidal heterocyclic compound, and the remainder is deionized water.

[0011] Preferably, the mass ratio of methylhydrotestosterone, danazol and androsan is 1:1:1.

[0012] Preferably, the mass ratio of sodium α-mercaptoethylsulfonate to N-methylacetamide is (1-5): (1-5).

[0013] Preferably, the mass ratio of sodium α-mercaptoethylsulfonate to N-methylacetamide is 1:1.

[0014] Preferably, the metal chelate is DTPA and / or EDTA, more preferably EDTA.

[0015] Preferably, the preservative is at least one of Proclin 300, sodium azide and thimerosal, more preferably sodium azide.

[0016] Preferably, the surfactant is at least one of Tween-20, Tween-40, Triton X-100 and sodium dodecyl sulfate.

[0017] Preferably, the pH of the release agent system is 6.4.

[0018] In a second aspect, the present invention provides a method for preparing the release agent described in the first aspect, comprising the following steps: adding all the components in the formula amount into deionized water and mixing, then adjusting the pH to 6.0-6.5 to obtain the release agent, and then storing it in an environment of 2-8°C away from light.

[0019] Preferably, the reagent for adjusting pH is 1 M hydrochloric acid solution.

[0020] In a third aspect, the present invention provides use of the release agent described in the first aspect in preparing a reagent for releasing steroid hormones in serum.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention overcomes a difficulty in the research and development of steroid hormone projects. Through the universal releaser, the four hormones in the sample can be effectively released from their respective binding proteins into a free state. The sample clinical trial data is obtained by measuring the content of the free hormone, making the measured experimental results more accurate, which is of great significance for the experimental research and development of the entire project and is convenient for subsequent research and development and optimization of the project. Moreover, the present invention is a releaser that is universal for these four steroid hormones, which greatly simplifies the experimental process, becomes more convenient, greatly reduces the workload of the experimenters, and improves efficiency.

[0023] The universal release agent prepared by the present invention is used as a sample pretreatment liquid in a steroid hormone project detection reagent, and is experimentally verified by using a chemiluminescent immunoassay (CLIA). By comparing with a conventional project treatment liquid, the experimental results show that the release agent of the present invention has a good release effect when used as a sample pretreatment liquid, and the sample result measurement value is accurate. DETAILED DESCRIPTION

[0024] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0025] Unless otherwise specified, other materials, reagents, etc. used in the examples and comparative examples can be obtained from commercial sources.

[0026] Examples 1-5 and Comparative Examples 1-3

[0027] The components of the release agents of Examples 1-5 and Comparative Examples 1-3 are shown in Table 1.

[0028] The preparation method of the release agent of Examples 1-5 and Comparative Examples 1-3 comprises the following steps:

[0029] All the components in the formula were added into deionized water and mixed, and then a 1M hydrochloric acid solution was added to adjust the pH to 6.4 to obtain the release agent, which was then stored at 5° C. and away from light.

[0030] Table 1 Component composition (mass percentage) of Examples 1-5 and Comparative Examples 1-3

[0031]

[0032]

[0033] Performance Testing

[0034] 15 specimens of each of the four hormone items measured by the imported test kit were collected (including low, medium and high values), a total of 60 specimens (testosterone specimens numbered 1 to 15, estradiol specimens numbered 16 to 30, progesterone specimens numbered 31 to 45, and cortisol specimens numbered 46 to 60). Each specimen was taken with a pipette gun and 50 μL of the specimen was put into 9 sample reaction cups, and 100 μL of the release agent of Examples 1-5 and Comparative Examples 1-3 and the conventional item treatment liquid were added to each sample reaction cup, mixed and left to stand at room temperature for 15 minutes, and then the chemiluminescent immunoassay (CLIA) was used for machine experimental verification, and the measured results were compared and analyzed with the results of the imported test kit. The following is the detection comparison results obtained by the specific embodiment of the present invention, specifically shown in Table 2-5. Among them, the conventional item treatment liquid is an aqueous solution containing 5% protein precipitant, 2% buffer and 1% stabilizer, the protein precipitant is acetonitrile, the buffer is Tris buffer, and the stabilizer is cyclodextrin.

[0035] Table 2 Testosterone comparison test results

[0036]

[0037]

[0038] Table 3 Estradiol comparison test results

[0039]

[0040]

[0041] Table 4 Progesterone comparison test results

[0042]

[0043] Table 5 Cortisol comparison test results

[0044]

[0045]

[0046] Combining Comparative Examples 1-3 and Example 2 in Tables 2-5, it can be seen that the amide compounds in the release agent play a leading role in the release effect of the hormone, and sodium α-mercaptoethylsulfonate and N-methylacetamide in the amide compounds have a synergistic effect, which may interact with the hormone binding protein and change its conformation, thereby making the hormone molecules easier to be released.

[0047] As can be seen from Tables 2-5, the measured values ​​after the specimens were treated with conventional project treatment fluids were generally much lower, so it can be inferred that the hormone molecules in the specimens were not released and did not have the effect of releasing them from the binding protein, while the concentration obtained after the dissociation agent prepared in the specific example diluted the specimen was very close to the concentration measured by the imported test kit. It can be inferred that the universal release agent prepared in Examples 1-5 is very sufficient for the release of the four hormones in the specimens. According to the exploration of the mass ratio of each component in the release agent, the release effect of Example 2 is the most obvious, which is basically consistent with the results measured by the imported reagents, with the highest correlation, reaching more than 0.99, and the highest release efficiency.

[0048] Therefore, the present invention provides a universal releaser capable of releasing steroid hormones (including testosterone, estradiol, progesterone, and cortisol) molecules in serum from their respective binding proteins. Pretreating clinical serum specimens with the releaser can release steroid hormones in serum from the binding proteins, thereby greatly simplifying the experimental process, making it more convenient, greatly reducing the workload of experimenters, and improving efficiency.

[0049] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A releasing agent, characterized in that The invention comprises the following components in percentage by mass: 0.2-1% of amide compounds, 0.02-0.06% of metal chelates, 0.05-0.15% of preservatives, 0.2-0.5% of surfactants, 0.015-0.045% of steroidal heterocyclic compounds, and the balance is deionized water; wherein the amide compounds are a combination of sodium α-mercaptoethylsulfonate and N-methylacetamide; the steroidal heterocyclic compounds are a combination of methylhydrotestosterone, danazol and androsol; and the pH of the release agent system is 6.0-6.

5.

2. The release agent according to claim 1, characterized in that The release agent comprises the following components in percentage by mass: 0.2-1% of amide compounds, 0.05% of metal chelates, 0.1% of preservatives, 0.4% of surfactants, 0.015-0.045% of steroid heterocyclic compounds, and the balance is deionized water.

3. The release agent according to claim 1, characterized in that The release agent comprises the following components in percentage by mass: 0.5% amide compound, 0.05% metal chelate, 0.1% preservative, 0.4% surfactant, 0.03% steroid heterocyclic compound, and the balance is deionized water.

4. The release agent according to claim 1 or 3, characterized in that The mass ratio of methylhydrotestosterone, danazol and androsan is 1:1:

1.

5. The release agent according to claim 1 or 2, characterized in that The mass ratio of sodium α-mercaptoethylsulfonate and N-methylacetamide is (1-5): (1-5).

6. The release agent according to claim 1 or 3, characterized in that The mass ratio of sodium α-mercaptoethylsulfonate and N-methylacetamide is 1:

1.

7. The release agent according to claim 1, characterized in that The component of the release agent is selected from at least one of the following: (I) the metal chelate is DTPA and / or EDTA; (II) the preservative is at least one of Proclin 300, sodium azide and thimerosal; (III) The surfactant is at least one of Tween-20, Tween-40, Triton X-100 and sodium dodecyl sulfate.

8. The release agent according to claim 1, characterized in that The pH of the release agent system is 6.

4.

9. The method for preparing the release agent according to any one of claims 1 to 8, characterized in that: The method comprises the following steps: adding all the components in the formula amount into deionized water for mixing, then adjusting the pH value to 6.0-6.5 to obtain the release agent, and then storing the release agent in an environment of 2-8°C and away from light.

10. Use of the release agent according to any one of claims 1 to 8 in the preparation of a reagent for releasing steroid hormones in serum.

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