Formula of testosterone immunodetection reagent sample releasing agent

Abstract

The invention belongs to the technical field of immunoassay, and particularly relates to a testosterone immunodetection reagent sample releasing agent formula. The invention discloses a testosterone immunodetection reagent sample releasing agent formula, and the testosterone immunodetection reagent sample releasing agent formula comprises the following components: ethisterone (CAS: 434-03-7) which is dissolved in a buffer solution.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and in particular to a testosterone sample release agent formula. Background Art

[0002] Testosterone, also known as testosterone, testosterone or testosterone, is a steroid estrogen secreted by the male testicles or female ovaries. The adrenal glands also secrete a small amount of testosterone. Testosterone can promote the formation of epididymis, vas deferens and seminal vesicles, promote the growth and development of external reproductive organs and secondary sexual characteristics, and affect the production of sperm. It has an important impact on health for both men and women. For men, increased testosterone levels are common in adrenal cortical hyperplasia, adrenal cortical tumors, testicular tumors, male precocious puberty and other diseases. Reduced testosterone levels are common in primary male dysfunction such as cryptorchidism, anorchia, interstitial cell dysgenesis, congenital seminiferous tubule dysgenesis, as well as hyperprolactinemia, hypercortisolism and other diseases. For women, abnormal testosterone levels are common in diseases such as idiopathic hirsutism, polycystic ovary syndrome, pineal tumors, and ovarian masculinization tumors. Accurate detection of testosterone levels in the body can help assist in the diagnosis and treatment of these diseases.

[0003] In serum, testosterone is mainly bound to sex hormone binding protein, albumin and cortisol binding protein, and only 2% of testosterone is in a free state. The immunoassay of total serum testosterone requires the release of testosterone from sex hormone binding protein, albumin and cortisol binding protein to facilitate antibody recognition. Therefore, the formula composition of the testosterone release reagent in the detection reagent is related to the release effect of testosterone, which directly affects the sensitivity and specificity of the serum total testosterone immunoassay reagent. At present, the commercial TESTO reagent already has a corresponding testosterone releaser, but we have discovered a more effective testosterone releaser formula in our research that can better improve the sensitivity and specificity of the reagent. Summary of the invention

[0004] The object of the present invention is to provide a more effective testosterone releasing agent formula.

[0005] In order to achieve the above technical objectives, the technical solution adopted by the present invention is as follows:

[0006] A testosterone immunoassay reagent sample releaser formula, the key component of which is: ethisterone (CAS: 434-03-7), dissolved in a buffer. The buffer can be any one of PBS buffer, 2-morpholineethanesulfonic acid buffer (MES), TRIS-HCL buffer, citrate buffer, 3-(N-morpholinyl)-2-hydroxypropanesulfonic acid (MOPSO) buffer, glycine-hydrochloric acid buffer, acetic acid-sodium acetate buffer, and 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer.

[0007] As a preferred embodiment of the present invention, the concentrations of the components in the testosterone releasing reagent are as follows: the concentration of ethinyl progesterone (CAS: 434-03-7) is 0.05-0.25 mg / mL.

[0008] The preparation method of the testosterone releasing reagent is:

[0009] S1: Take a compound having the function of releasing testosterone according to the composition ratio, dissolve it with DMSO or DMF or pure water to prepare a mother solution with a concentration of 500 mg / mL.

[0010] S2: diluting the solution to a corresponding concentration using a buffer solution to prepare a testosterone releasing reagent.

[0011] By adopting the technical scheme of the present invention, compared with the prior art, the release agent in the detection reagent of the present invention, as a component of the testosterone enzymatic chemiluminescent immunoassay reagent, can more effectively dissociate testosterone from its binding protein, so that the sensitivity and specificity of the testosterone immunoassay reagent can be significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The present invention can be further illustrated by the non-limiting examples given in the accompanying drawings;

[0013] Figure 1 This is a test result diagram of a reference example without a testosterone-releasing reagent;

[0014] Figure 2 This is a test result diagram of progesterone as a reference example of a testosterone-releasing reagent;

[0015] Figure 3 This is a test result diagram of a reference example of using dihydrotestosterone as a testosterone-releasing reagent;

[0016] Figure 4 This is a test result diagram of dihydrotestosterone, heparin sodium, and danazol as a reference example of testosterone releasing reagents;

[0017] Figure 5 Example 1 test result diagram;

[0018] Figure 6 Example 2 test result diagram;

[0019] Figure 7 Example 3 test result diagram. DETAILED DESCRIPTION

[0020] In order to enable those skilled in the art to better understand the present invention, the technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0021] Take the magnetic particle enzymatic chemiluminescent immunoassay reagent of testosterone as an example, its components include: superparamagnetic particles coated with testosterone antigen, alkaline phosphatase labeled testosterone antibody, testosterone release reagent, alkaline phosphatase luminescent substrate and magnetic bead cleaning solution;

[0022] The testosterone releaser is characterized by ethisterone (CAS: 434-03-7) dissolved in a buffer solution.

[0023] Example 1: Testosterone sample releaser formulation 1, its composition: 50mM PBS buffer containing 0.1mg / mL ethinyl progesterone, pH 7.0;

[0024] Example 2: Testosterone sample releaser formulation 2, comprising: 50 mM MES buffer containing 0.1 mg / mL ethinyl progesterone and 5 mg / mL heparin sodium, pH 6.0;

[0025] Example 3: Testosterone sample releaser formulation 3, its composition: 50mM MES buffer containing 0.1mg / mL ethinyl progesterone, 5mg / mL heparin sodium, and 0.02mg / mL danazol, pH 6.0.

[0026] Specificity: 40 clinical samples were collected and tested with the testosterone assay kits with different sample releaser formulations in Examples 1-3. The test results were compared with the clinical test results. Figure 1-Figure 7 .

[0027] Figure 1 This is a test result diagram of a reference example without a testosterone-releasing reagent. Figure 2 This is the test result of the steroidal heterocyclic compound progesterone as a testosterone-releasing reagent. Figure 3 This is a test result diagram of dihydrotestosterone as a reference example of a testosterone releaser. Figure 4 This is a test result diagram of a reference example in which dihydrotestosterone, heparin sodium, and danazol are simultaneously used as testosterone-releasing reagents; Figure 5-Figure 7 They are the test result diagrams of Example 1 to Example 3 respectively.

[0028] Figure 1 The experimental results show that the equation of the straight line is y = 0.8722x + 1.4482, and the correlation coefficient is R 2 =0.5271;

[0029] Figure 2 The experimental results show that the equation of the straight line is y = 0.8864x + 1.6093, and the correlation coefficient is R 2 =0.6451;

[0030] Figure 3The experimental results show that the equation of the straight line is y = 1.0022x + 0.1944, and the correlation coefficient is R 2 =0.9729;

[0031] Figure 4 The experimental results show that the equation of the straight line is y = 1.0227x-0.0606, and the correlation coefficient is R 2 =0.9865;

[0032] Figure 5 The experimental results show that the equation of the straight line is y = 1.0223x + 0.0423, and the correlation coefficient is R 2 =0.9868;

[0033] Figure 6 The experimental results show that the equation of the straight line is y = 0.9731x + 0.0773, and the correlation coefficient is R 2 =0.9876;

[0034] Figure 7 The experimental results show that the equation of the straight line is y = 0.9999x + 0.0331, and the correlation coefficient is R 2 =0.9945.

[0035] pass Figure 1 , Figure 2 , Figure 3 , indicating that the steroidal heterocyclic compound progesterone has no effect on the release of testosterone molecules, while dihydrotestosterone can fully promote the release of testosterone molecules. Dihydrotestosterone can competitively bind to sex hormone binding protein, thereby releasing testosterone molecules bound to sex hormone binding protein, but a part of testosterone molecules are still bound to albumin and cortisol binding protein. Heparin sodium and danazol can release testosterone molecules from albumin and cortisol binding protein respectively. Figure 3 and Figure 4 The comparison shows the effect of heparin sodium and danazol. Figure 5 This indicates that the release of testosterone molecules in the presence of only ethinyl progesterone is better than that in the presence of dihydrotestosterone, sodium heparin and danazol at the same time.

[0036] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the technology may modify or change the above embodiments without violating the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A testosterone immunoassay reagent sample release agent formula, characterized in that: Its composition contains ethisterone (CAS: 434-03-7). After being dissolved in DMSO or DMF, it is added into PBS buffer or 2-morpholineethanesulfonic acid buffer (MES) or Tris-HCL buffer or citrate buffer or 3-(N-morpholinyl)-2-hydroxypropanesulfonic acid (MOPSO) buffer or glycine-hydrochloric acid buffer or acetic acid-sodium acetate buffer or 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer to prepare a testosterone releaser formula.

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