Sex hormone binding globulin calibrator diluent and application thereof

Abstract

The invention discloses a sex hormone binding globulin calibrator diluent and an application thereof. The calibrator diluent comprises the following components and contents: 50-100mM of a buffer solution; 0.01% to 0.1% of estradiol or an estradiol derivative; 0.5%-2% of a protein protective agent; 1%-10% of an anti-freezing agent; 0.1-1% of metal ions; 0.1%-0.5% of a preservative; 0.1%-2% of an ion regulator; 1%-10% of a saccharide protective agent; 0.1%-1% of a complex; and the balance of water. The in-vitro diagnostic reagent calibrator is prepared in a liquid form, and compared with freeze-dried calibrators adopted by well-known foreign manufacturers such as Rogowski and other domestic and overseas manufacturers in the market, the in-vitro diagnostic reagent calibrator has the advantages of convenience and rapidness in use; the problem of inaccurate calibration product concentration caused by operation errors possibly introduced when doctors in clinical laboratories in hospitals redissolve the calibration product is avoided, and under the synergistic effect of the components, the unstable dimer structure of the SHBG can not be degraded in a diluent for a long time, so that the ability of the calibration product to correct a reagent curve is effectively ensured.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostic reagent technology, specifically relating to a diluent for a sex hormone-binding globulin calibrator and its application. Background Technology

[0002] SHBG is a blood test. SHBG stands for sex hormone-binding globulin, also known as testosterone-estradiol-binding globulin, and is a carrier test for transporting sex hormones. SHBG is used to detect the binding of sex hormones such as estrogen, testosterone, androgens, follicle-stimulating hormone, and luteinizing hormone to proteins in the body. A deficiency in SHBG indicates downregulation or receptor insensitivity of sex hormones. 1. Decreased SHBG levels: Conditions such as polycystic ovary syndrome, obesity, hypothyroidism, hirsutism in women, and virilization can all lead to decreased SHBG levels and increased levels of free androgens. 2. Elevated SHBG levels: Commonly seen in men with hypogonadism, hyperthyroidism, liver disease, or precocious puberty. SHBG testing can serve not only as a diagnostic indicator but also as a basis for assessing treatment effectiveness.

[0003] In the blood, SHBG typically exists as a dimer, where two SHBG monomers are linked non-covalently to form a functional dimer. SHBG has two main hormone-binding sites, located at the N-terminal region of each monomer. Each binding site can specifically bind to one hormone, such as testosterone or estrogen. The structure of these binding sites allows SHBG to bind sex hormones with high affinity, preventing them from freely diffusing and acting uncontrollably on target cells. The molecular weight of SHBG is approximately 90 kDa, while due to its dimer structure, each monomer has a molecular weight of approximately 45 kDa. SHBG is primarily synthesized and secreted in the liver, but it can also be expressed in other tissues such as the testes and brain.

[0004] Currently, domestic and foreign manufacturers prepare calibrators using lyophilization and liquid forms. Lyophilized calibrators require reconstitution, and the use of pipettes to draw purified water can easily introduce errors. Furthermore, the operation steps are cumbersome and difficult for laboratory professionals to accept. Therefore, liquid calibrators are the preferred choice for calibrator development. SHBG protein itself has a dimer structure, which is less stable than many large protein molecules. Therefore, the physicochemical factors such as pH and temperature of the diluent can easily affect the protein structure. Thus, developing a diluent that can stabilize SHBG protein over a long period of time is an urgent problem to be solved in the field of in vitro diagnostics. Summary of the Invention

[0005] This invention discloses a diluent for sex hormone-binding globulin calibrators and its application, in order to solve the problem of poor stability of calibrators prepared by existing technologies.

[0006] In a first aspect, the present invention provides a diluent for a sex hormone-binding globulin calibrator, comprising the following components and amounts: buffer solution 50mM-100mM; estradiol or estradiol derivative 0.01%-0.1%; protein protectant 0.5%-2%; antifreeze 1%-10%; metal ions 0.1%-1%; preservative 0.1%-0.5%; ion regulator 0.1%-2%; carbohydrate protectant 1%-10%; complex 0.1%-1%; and the balance being water.

[0007] Optionally, it may also include the following components and amounts: surfactant 0.1-0.5%.

[0008] Optionally, the buffer solution is selected from at least one of Tris-HCl buffer, PB buffer, and MES buffer.

[0009] Optionally, the estradiol derivative is dibromoestradiol.

[0010] Optionally, the protein protectant is casein or BSA.

[0011] Optionally, the metal ion is calcium ion, the ion regulator is NaCl, the complex is EDTA-2Na, and the antifreeze is glycerol.

[0012] Optionally, the preservative is selected from at least one of Proclin 300 and sodium azide.

[0013] Optionally, the sugar protectant is selected from at least one of trehalose, mannitol, and sucrose.

[0014] Optionally, the surfactant is Tween 20 or Tween 80.

[0015] A second aspect of the present invention provides a kit for detecting sex hormone-binding globulin, comprising a sex hormone-binding globulin calibrator diluent provided in any implementation of the first aspect.

[0016] This invention prepares in vitro diagnostic reagent calibrators in liquid form. Compared with the lyophilized calibrators used by well-known foreign manufacturers such as Roche, this method is convenient and quick to use. It avoids the problem of inaccurate calibrator concentration caused by operational errors that may be introduced when hospital laboratory doctors reconstitute the calibrators. Under the synergistic effect of each component, the unstable dimer structure of SHBG can remain undegraded in the diluent for a long time, thus effectively ensuring the calibrator's ability to correct reagent curves.

[0017] Furthermore, the calibrator diluent formulation of this invention uses the estrogen derivative dibromoestradiol to simulate the binding of natural estradiol and SHBG. However, compared to natural estradiol, dibromoestradiol has a larger molecular weight and better stability. At the same time, dibromoestradiol has a stronger affinity for SHBG. As a result, after forming a more robust structure with SHBG, dibromoestradiol is less prone to degradation than estradiol. Therefore, the key binding sites of dibromoestradiol and SHBG will not be exposed for a longer period of time, thus ensuring the stability of the key intermediate structure of the SHBG dimer. Detailed Implementation

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] To address the instability of sex hormone-binding globulin antigen in diluents, this invention provides a diluent for sex hormone-binding globulin calibrators, comprising: 50mM-100mM buffer; 0.01%-0.1% estradiol or estradiol derivatives; 0.5%-2% protein protectant; 0.1%-1% complex; 1%-10% antifreeze; 0.1%-1% metal ions; 0.1%-0.5% preservative; 0.1%-2% ion modifier; 1%-10% glycoprotectant; and the balance being water. Optionally, it may also include the following components and amounts: 0.1-0.5% surfactant.

[0020] Furthermore, the buffer solution is selected from at least one of Tris-HCl buffer, PB phosphate buffer, and MES buffer.

[0021] Furthermore, the estradiol derivative is dibromoestradiol. Dibromoestradiol is a synthetic estrogen derivative whose specific chemical and biological properties are enhanced by introducing bromine atoms into the estradiol molecule. For example, the bromine atom can increase molecular stability and reduce its metabolic degradation in vivo. Dibromoestradiol may have a higher affinity for estrogen receptors, thereby enhancing its effect in target tissues. Through structural modification, dibromoestradiol may exhibit higher tissue specificity; for instance, it may have stronger estrogenic activity in some tissues and weaker activity in others, thereby reducing side effects. As a synthetic estrogen derivative, the introduction of bromine atoms exhibits unique advantages, making dibromoestradiol a promising candidate for applications in medicine and research. Therefore, dibromoestradiol may also see wider application in in vitro diagnostics due to its advantages.

[0022] Sex hormone-binding globulin (SHBG), also known as testosterone-estradiol-binding globulin, is a trace protein found in the plasma of many mammals. Its main function is to specifically bind and transport sex hormones. SHBG primarily binds to testosterone and estradiol. Human SHBG has a high affinity for testosterone and a lower affinity for estradiol. Each SHBG molecule can only bind one sex hormone molecule, and the sex hormone binding site is located between two monomers, forming a sandwich structure. The polymerization of SHBG monomers is necessary to form a single sex hormone binding site, and the binding with ligands forms chemical bonds to obtain energy, thus making the dimer structure more stable. Previous studies have shown that the efficiency of the sex hormone binding site is further enhanced in the presence of androgens and calcium ions, suggesting that the binding of divalent cations and sex hormones promotes the maintenance of the dimer structure. SHBG is a globulin, and divalent cations are essential for the activity of this type of protein, likely acting on the sulfhydryl groups of the globulin to exert their effect. For long-term storage, the addition of calcium ions could be considered to increase stability. Previous studies have found that the 205 amino acids at the N-terminus of human SHBG are related to its affinity, among which Met139 is essential for binding to the B-ring of sex hormones. Related studies have shown that in buffer solutions with added calcium ions, the accelerated 7-day decrease in human recombinant protein is less than 10%, while in buffer solutions without added calcium ions, the accelerated 7-day decrease is 47%.

[0023] This invention verified that calcium ions also help stabilize SHBG, but do not play a crucial role, possibly due to differences in the structure of recombinant SHBG proteins from different manufacturers. This invention demonstrates that the key component is the estrogen derivative dibromoestradiol; the combined use of dibromoestradiol and divalent cations can maintain the long-term stability of recombinant SHBG proteins.

[0024] Furthermore, the protein protectant is casein or BSA.

[0025] Furthermore, the preservative is selected from at least one of Proclin 300 and sodium azide.

[0026] This invention prepares in vitro diagnostic reagent calibrators in liquid form, which, compared to the lyophilized calibrators used by well-known international manufacturers such as Roche, offers advantages such as convenience and speed. It also avoids the problem of inaccurate calibrator concentrations caused by operational errors introduced during the reconstitution of calibrators by hospital laboratory physicians. Each component protects the SHBG protein in the calibrator, and the concentrations of each component are optimized. Through the synergistic effect of the components, the unstable dimer structure of SHBG remains undegraded in the diluent for a long period, thus effectively ensuring the calibrator's ability to correct reagent curves.

[0027] The calibrator diluent formulation of this invention uses the estrogen derivative dibromoestradiol to simulate the binding of natural estradiol and SHBG. However, compared to natural estradiol, dibromoestradiol has a larger molecular weight and better stability. At the same time, dibromoestradiol has a stronger affinity for SHBG. As a result, after forming a more robust structure with SHBG, dibromoestradiol is less prone to degradation than estradiol. Therefore, the key binding sites of dibromoestradiol and SHBG will not be exposed for a longer period of time, thus ensuring the stability of the key intermediate structure of the SHBG dimer.

[0028] This invention optimizes the dosage of dibromoestradiol. The preferred final dosage is 0.01% of dibromoestradiol dissolved in 5 mg / ml DMF added to the diluent. Excessive concentration can actually increase the stability of the SHBG calibrator by more than 10% after 7 days; therefore, careful attention must be paid to the appropriate dosage of dibromoestradiol. The addition of EDTA-2NA to the diluent prevents turbidity when CaCl2 is added, and also acts as a preservative to eliminate the influence of external environmental microorganisms on the antigen protein.

[0029] The SHBG recombinant protein antigen used in the calibrator formulation of this invention is sourced from Getein Biotech Co., Ltd., while the antigen from the comparison manufacturer is from Shanghai Nearshore Co., Ltd. Both antigens are recombinant antigens and are suitable for the diluent formulation of this invention. The calibrator reagents of this invention are developed and manufactured by Getein Biotech Co., Ltd., and the reagents use a double-antibody sandwich method to test the SHBG antigen in the sample or calibrator.

[0030] In this application, the stability test of the calibrators includes 30 days after opening, 7 days of accelerated freezing and thawing, 3 freeze-thaw cycles, and 1 year of storage at 4°C. The pass criterion is that the measured values ​​of the calibrators for accelerated freeze-thaw stability after opening are all within ±10% compared to the control. The calculation method for the stability deviation of the calibrators is as follows:

[0031]

[0032] The low and high values ​​of the calibrator must be within the acceptable range required by the registration certificate of the Base Egg Biological Reagent. The low value concentration requirement is 12-18 nmol / L, and the high value concentration requirement is 80-120 nmol / L. Calculate the amount of antigen to be used, add the antigen to the diluent, shake to mix, label clearly, and set aside for later use.

[0033] The testing process of this invention is as follows: acridinium ester-labeled monoclonal antibody, biotin-labeled monoclonal antibody, streptavidin-coated magnetic beads, and calibrators are added to a reaction vessel, incubated at 37°C for a period of time, washed, and then excitation solution is added. Finally, the luminescence value measured by the fully automated chemiluminescence analyzer is directly proportional to the concentration of the SHBG antigen calibrator, that is, the higher the antigen concentration, the higher the luminescence value.

[0034] The present application will be further described in detail below with reference to specific embodiments.

[0035] Example 1

[0036] Accurately dissolve Tris-HCl and casein in 800 mL of purified water. While stirring, adjust the pH of the solution to 12.0 ± 0.3 and stir overnight. Once the solution is clear and free of precipitates, accurately add calcium chloride, trehalose, EDTA-2NA, and sodium chloride to the solution and mix well. Then accurately add glycerol and Proclin 300, mix well, and adjust the pH to 7.4 ± 0.1 with 3 mol / L HCl (a small amount of precipitate may form during pH adjustment; dissolve and continue adjusting. If the pH exceeds the range, adjust to 7.40 ± 0.1 with 1 M HCl or 1 M NaOH). Add purified water to bring the final volume to 1000 mL (weight: 1055 g, density: 1.055 g / mL). Mix well for 4 hours. Then accurately pipette 5 mg / mL dibromoestradiol diluted with DMF and add it to the solution, mixing well. Label the contents for future reference.

[0037] Table 1. Components and content of the calibrator diluent in Example 1

[0038] Components content buffer solution 50mM Tris-HCl (pH=7.4) Hormone derivatives 0.01% 5 mg / ml dibromoestradiol solution protein protectants 1% casein antifreeze 4% glycerin metal ions <![CDATA[0.2%CaCl2]]> preservative 0.1% Proclin300 Complex 0.5% EDTA-2NA Salt ions 0.9% NaCl Sugar protectants 10% Trehalose

[0039] Example 2

[0040] In Example 1, dibromoestradiol was replaced with estradiol, and the other components were the same as in Example 1.

[0041] Example 3

[0042] The protein protectant in Example 1 was replaced with BSA, and the Tris-HCl (pH=7.4) buffer was replaced with MES (pH=7.4). All other components were the same as in Example 1.

[0043] Example 4

[0044] The concentration of dibromoestradiol in Example 1 was increased to 0.1%, while the other components remained the same as in Example 1.

[0045] Example 5

[0046] In Example 1, trehalose was replaced with sucrose, and the other components were the same as in Example 1.

[0047] Example 6

[0048] Compared to Example 1, EDTA-2NA and CaCl2 were replaced with 0.5% Tween 20 surfactant, while the other components were the same as in Example 1.

[0049] Example 7

[0050] Compared with Example 1, without the addition of metal ions and EDTA-2NA, the casein content was increased by 2%, while the other components were the same as in Example 1.

[0051] Example 8

[0052] Compared to Example 1, the buffer solution was 100 mM Tris-HCl, and the other components were the same as in Example 1.

[0053] Example 9

[0054] The content of dibromoestradiol solution in Example 1 was increased to 0.02%, while other components remained the same as in Example 1.

[0055] Example 10

[0056] The components and contents of Example 10 are shown in Table 2. The preparation method is the same as that of Example 1.

[0057] Table 2. Components and content of the calibrator diluent in Example 10

[0058] Components content buffer solution 50mM PB (pH=7.4) Hormone derivatives 0.01% 5 mg / ml dibromoestradiol solution protein protectants 0.5% casein antifreeze 1% glycerin surfactants 0.1% Tween 20 metal ions <![CDATA[0.1%CaCl2]]> preservative <![CDATA[0.1%Proclin300+0.1%NaN3]]> Complex 0.1% EDTA-2NA Salt ions 0.1% NaCl Sugar protectants 1% Trehalose

[0059] Example 11

[0060] The components and their contents in Example 11 are shown in Table 3. The preparation method is the same as that in Example 1.

[0061] Table 3. Components and content of the calibrator diluent in Example 11

[0062] Components content buffer solution 100mM PB (pH=7.4) Hormone derivatives 0.01% 5 mg / ml dibromoestradiol solution protein protectants 2% casein antifreeze 10% glycerin surfactants 0.5% Tween 80 metal ions <![CDATA[1%CaCl2]]> preservative <![CDATA[0.2%Proclin300+0.3%NaN3]]> Complex 1% EDTA-2NA Salt ions 2% NaCl Sugar protectants 10% Mannitol

[0063] Comparative Example 1

[0064] Compared to Example 1, dibromoestradiol was not added, but the other components were the same as in Example 1.

[0065] Comparative Example 2

[0066] Compared to Example 1, no casein was added, but the other components were the same as in Example 1.

[0067] Comparative Example 3

[0068] Compared to Example 1, CaCl2 was not added, but the other components were the same as in Example 1.

[0069] Comparative Example 4

[0070] Compared to Example 1, trehalose was not added, but the other components were the same as in Example 1.

[0071] Comparative Example 5

[0072] Compared to Example 1, glycerol was not added, but the other components were the same as in Example 1.

[0073] Comparative Example 6

[0074] Compared to Example 1, dibromoestradiol and casein were not added, but the other components were the same as in Example 1.

[0075] Comparative Example 7

[0076] Compared to Example 1, dibromoestradiol, casein, CaCl2, and sugar protectants were not added, while other components were the same as in Example 1.

[0077] Experiments were conducted on Examples 1-11 and Comparative Examples 1-7 respectively, and the experimental results are shown in Table 4.

[0078] Table 4 Experimental Results

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086] Based on the above experimental results, the analysis is as follows:

[0087] 1. The calibrators in Example 1 were stored at 4°C and 37°C. The maximum decrease in the low and high values ​​of the calibrators after 7 days of accelerated storage was -1.35% and -0.28%, respectively. According to the Arrhenius formula, 7 days of accelerated storage at 37°C simulates a one-year shelf life, indicating that the protective agents such as dibromoestradiol in this example can keep the SHBG antigen relatively stable.

[0088] 2. In Example 2, the calibrators L and H decreased by -10.55% and -9.87% respectively after 7 days of accelerated degradation at 37°C. The lower value exceeded the 10% pass threshold, indicating that the estradiol in Example 2 was not as effective as dibromoestradiol in protecting the stability of SHBG antigen.

[0089] 3. In Example 3, after replacing casein in the diluent with BSA, the maximum reduction in L and H of the calibrator over 7 days was -5.32% and -6.41%, respectively, indicating that BSA has a weaker protective effect against SHBG antigen compared to casein.

[0090] 4. In Example 4, increasing the concentration of dibromoestradiol from 0.01% to 0.1% in the diluents accelerated the increase in the measured value of the 7-day calibrator by 14.91% and 12.68%, respectively. This indicates that the dosage of dibromoestradiol needs to be controlled appropriately. The increase in measured value may be related to the fact that the addition of dibromoestradiol exposes more sites of SHBG antigen.

[0091] 5. In Example 5, the diluent was replaced with sucrose, which accelerated the reduction of the low and high values ​​of the calibrator by 5.17% and 5.44% respectively over 7 days. The values ​​remained stable at 4 degrees Celsius, indicating that trehalose can better protect the SHBG antigen structure than sucrose under high temperature conditions.

[0092] 6. In Example 6, replacing EDTA-2NA and Cacl2 with Tween 20 accelerated the reduction of the low and high values ​​of the 7-day calibrator to -7.37% and -6.86%, respectively, indicating that calcium ions can play a certain protective role, and Tween 20 as a surfactant does not improve the stability of SHBG.

[0093] 7. In Example 7, the casein content of the diluent was increased to 2%, without the addition of metal ions and EDTA-2NA. The reduction in the low and high values ​​of the 7-day calibrator was accelerated to -6.19% and -6.44%, respectively, indicating that increasing the casein content no longer improved the SHBG protection effect, and 1% casein content was sufficient.

[0094] 8. In Example 8, the buffer system Tris-HCl was increased from 50 mM to 100. The calibrator values ​​decreased by 1.11% and 0.22% over 7 days, respectively, indicating that the buffer system of 50 mM already has sufficient buffering capacity.

[0095] 9. In Example 9, the content of dibromoestradiol was increased from 0.01% to 0.02%, while the other components and concentrations remained the same as in Example 1. The calibrator showed an increasing trend in the low value after 7 days of acceleration. The changes in the low and high values ​​after 7 days of acceleration were 4.31% and -1.25%, respectively. Therefore, the optimal content of dibromoestradiol as a key stabilizer is set at around 0.01%. Adding too much dibromoestradiol would be detrimental to the accelerated protective effect.

[0096] 10. The experimental results of Examples 10 and 11 show that the decrease in both the low and high values ​​of the accelerated calibrator after 7 days is within 5%, indicating that Examples 10 and 11 have good stability when used as calibrator diluents.

[0097] 11. In Comparative Example 1 without dibromoestradiol, the accelerated 7-day decreases in the low and high values ​​of the calibrator were -12.34% and -9.44%, respectively, indicating that dibromoestradiol plays a key role in protecting the SHBG structure.

[0098] 12. In Comparative Example 2 without casein, the calibrator values ​​decreased by -18.41% and -13.30% respectively after 7 days of accelerated reduction, indicating the routine protective effect of casein on the SHBG antigen structure at 37 degrees Celsius.

[0099] 13. In Comparative Example 3 without CaCl2, the calibrator values ​​decreased by -6.48% and -5.66% respectively after 7 days of accelerated testing, indicating that CaCl2 can bind SHBG and thus stabilize the structure. However, compared with Example 1, the calibrator has poor accelerated stability, so dibromoestradiol needs to be added to make it more stable.

[0100] 14. In Comparative Example 4 without trehalose, the acceleration of 7 days resulted in a decrease of -5.16% and -5.79% in the low and high values ​​of the calibrator, respectively, indicating that trehalose has a certain protective effect on acceleration.

[0101] 15. In Comparative Example 5, without the addition of glycerin, the low and high values ​​of the calibrator decreased by -5.42% and -5.94% respectively after opening the bottle, and remained stable after 7 days of accelerated testing, indicating that glycerin has a certain protective effect on the stability after opening the bottle.

[0102] 16. Comparative Example 6, without the addition of dibromoestradiol and casein, showed accelerated reductions of -19.69% and -23.29% in the low and high values ​​of the calibrator after 7 days, respectively, indicating the synergistic protective effect of dibromoestradiol and casein on SHBG protein.

[0103] 17. Comparative Example 7 dilution without dibromoestradiol, casein, CaCl2, and sugar protectants showed accelerated 7-day calibrator values ​​decreasing by -32.21% and -29.33% at the low and high values, respectively. Simultaneously, the values ​​decreased by -14.97% and -18.63% after opening at 4 degrees Celsius. This indicates that these four substances provide better protection for SHBG than dibromoestradiol and casein. The protectants have different mechanisms of action, and the simultaneous addition of different protectants can better maintain the relative stability of the SHBG structure.

[0104] The above are merely embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention are included within the scope of the claims of the present invention pending approval.

Claims

1. A diluent for a sex hormone-binding globulin calibrator, characterized in that, Substances including the following components and amounts: Buffer solution 50mM - 100mM; 0.01%-0.1% estradiol or estradiol derivatives; Protein protectant 0.5%-2%; Antifreeze 1%-10%; Metal ions 0.1-1%; Preservative 0.1%-0.5%; Ion regulator 0.1%-2%; Carbohydrate protectant 1%-10%; Complex 0.1%-1%; The remainder is water.

2. The sex hormone-binding globulin calibrator diluent according to claim 1, characterized in that, It also includes the following components and amounts: surfactant 0.1-0.5%.

3. The diluent for a sex hormone-binding globulin calibrator according to claim 1, characterized in that, The buffer solution is selected from at least one of Tris-HCl buffer, PB buffer, and MES buffer.

4. The diluent for a sex hormone-binding globulin calibrator according to claim 1, characterized in that, The estradiol derivative is dibromoestradiol.

5. The diluent for a sex hormone-binding globulin calibrator according to claim 1, characterized in that, The protein protectant is casein or BSA.

6. The diluent for a sex hormone-binding globulin calibrator according to claim 1, characterized in that, The metal ion is calcium ion, the ion regulator is NaCl, the complex is EDTA-2Na, and the antifreeze is glycerol.

7. The diluent for a sex hormone-binding globulin calibrator according to claim 1, characterized in that, The preservative is selected from at least one of Proclin 300 and sodium azide.

8. The diluent for a sex hormone-binding globulin calibrator according to claim 1, characterized in that, The sugar protectant is selected from at least one of trehalose, mannitol, and sucrose.

9. The diluent for a sex hormone-binding globulin calibrator according to claim 2, characterized in that, The surfactant is Tween 20 or Tween 80.

10. A kit for detecting sex hormone-binding globulin, characterized in that, The diluent for a sex hormone-binding globulin calibrator, as described in any one of claims 1 to 9.

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