A liquid reagent for stabilizing thyrotropin receptor and TRAb detection kit

Abstract

The present invention discloses a liquid reagent for stabilizing thyrotropin receptor and a TRAb detection kit, wherein the liquid reagent comprises: a preservative, a surfactant, a metal ion chelator, an amino acid and a protein; the concentration of the metal ion chelator is 0.1-10 mM, the concentration of the amino acid is 0.1-2.0%, and the concentration of the protein is 0.1-10%, thereby extending the validity period of the kit and reducing the preparation cost.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro diagnosis, and in particular to a liquid reagent for stabilizing thyrotropin receptors and a TRAb detection kit. Background Art

[0002] Graves' disease (GD) is one of the most common thyroid diseases in clinical practice, manifested by endocrine organ dysfunction. The concentration level of thyroid stimulating hormone receptor antibody (TRAb) plays an auxiliary diagnostic role in the disease.

[0003] Thyrotropin receptor (TSH receptor, TSHR) is the main target antigen of TRAb and plays an important role in the pathogenesis of toxic diffuse goiter. THSR is a transmembrane glycoprotein present on the membrane of human thyroid follicular epithelial cells (TEC), which is involved in regulating the growth, differentiation, synthesis and release of thyroid hormones of TEC. TSHR is one of the main autoantigens of toxic diffuse goiter and stimulates the body to produce TRAb.

[0004] In the field of in vitro diagnosis, TSHR as a raw material is a key component of the TRAb detection kit and has important value for the differential diagnosis of Graves' disease. Since TSHR is greatly affected by the storage temperature in the liquid matrix, lyophilization is often used to extend the shelf life of TSHR, and the stability of the freeze-dried powder in the machine is short after reconstitution, such as Roche Diagnostics Registration Certificate No.: Guoxie Zhujin 20162404030; Shenzhen Mindray Bio-Medical Electronics Co., Ltd. Registration No.: Yuexie Zhuzhun 20192400121; Zhengzhou Antu Biotechnology Co., Ltd. Patent No.: CN 111208309 A shows that the TSHR component is a lyophilized powder. Among them, according to recent reports, the stability of the TRAb detection kit reagent developed by Xiamen Wantai Biological is only 3 weeks in the machine.

[0005] It can be seen that the instability of TSHR in liquid matrix is ​​a common problem faced by various reagent manufacturers. This will seriously shorten the shelf life of the test kit, increase the cost of lyophilization, and the early reconstitution of the lyophilized powder is not conducive to the use of end customers.

[0006] Therefore, there is an urgent need for a liquid dilution reagent that can improve the stability of the thyroid stimulating hormone receptor liquid reagent. Summary of the invention

[0007] In order to solve one or more problems existing in the prior art, the present invention provides a liquid reagent for stabilizing thyrotropin receptors. The technical solution adopted by the present invention to solve the above problems is: the liquid reagent includes: a preservative, a surfactant, a metal ion chelator, an amino acid and a protein; the concentration of the metal ion chelator is 0.1-10 mM, the concentration of the amino acid is 0.1-2.0%, and the concentration of the protein is 0.1-10%.

[0008] In some embodiments, the metal ion chelator is selected from any one or more of EDTA-2Na, EDTA-2K, EDTA-4Na and hydrates thereof, wherein the metal ion chelator is preferably EDTA-2Na hydrate.

[0009] In some embodiments, the amino acid is selected from any one or more of glycine, arginine, and lysine, wherein preferably the amino acid is glycine.

[0010] In some embodiments, the protein is selected from any one or more of bovine serum albumin, human serum albumin, goat serum albumin, casein, and skimmed milk powder, wherein the protein is preferably bovine serum albumin.

[0011] And a TRAb detection kit, the kit comprises: a first reagent, the first reagent is prepared by diluting the thyrotropin receptor with the liquid reagent mentioned above.

[0012] The technical effect achieved by the present invention is: the thyrotropin receptor is diluted by the above-mentioned liquid reagent to prepare the corresponding liquid detection reagent, and the above-mentioned liquid reagent can effectively prolong the storage time of the liquid unopened reagent containing the thyrotropin receptor (12 months), and prolong the in-machine storage time of the opened reagent in an environment of 2-8° (1 month), thereby achieving the purpose of extending the validity period of the test kit and reducing the preparation cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Prepare a component table for the liquid reagents of the control group and Examples 1-3;

[0014] Figure 2 This is a titer evaluation table for the control group and Examples 1-3;

[0015] Figure 3 2-8° Storage Stability Evaluation Table for the Control Group and Examples 1-3;

[0016] Figure 4 This is the on-machine stability evaluation table after opening of the control group and Examples 1-3;

[0017] Figure 5This is the 37°C±1°C accelerated stability evaluation table for the control group and Examples 1-3. DETAILED DESCRIPTION

[0018] In order to make the above-mentioned objects, features and advantages of the present invention more understandable, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from this description, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below.

[0019] The present invention discloses a liquid reagent for stabilizing thyrotropin receptors, the liquid reagent comprising: a preservative, a surfactant, a metal ion chelating agent, an amino acid and a protein; the concentration of the metal ion chelating agent is 0.1-10 mM, the concentration of the amino acid is 0.1-2.0%, and the concentration of the protein is 0.1-10%;

[0020] Among them, the metal ion chelator can be selected from any one or more of EDTA-2Na, EDTA-2K, EDTA-4Na and their hydrates, preferably EDTA-2Na hydrate; the amino acid can be selected from any one or more of glycine, arginine, and lysine, preferably glycine; the protein can be selected from any one or more of bovine serum albumin, human serum albumin, sheep serum albumin, casein, and skimmed milk powder, preferably bovine serum albumin.

[0021] Preferably, the contents of the various components in the liquid reagent are: 20 mM buffer, 1 mM metal ion chelator, 0.3% amino acid, 2.0% protein, 0.1% preservative, and 0.1% surfactant, wherein the buffer is 20 mM Tris with a pH of 7.5, the preservative is PC-300, and the surfactant is Tween-20.

[0022] And a corresponding TRAb detection kit, the kit comprises: a first reagent, the first reagent is prepared by diluting the thyrotropin receptor with the liquid reagent.

[0023] It should be noted that the following reactions may occur in the solution during the preparation of the TRAb detection reagent: reaction of metal ions with sulfhydryl groups of proteins, reaction of metal ions with disulfide bonds, reaction of metal ions with tryptophan and histidine;

[0024] For the reaction of metal ions with sulfhydryl groups of proteins: Metal ions can react with sulfhydryl groups in proteins (such as cysteine ​​sulfhydryl groups) to form thiolates, resulting in protein inactivation. For example, some metal ions (such as Mn²⁺, Fe²⁺, Ni²⁺, Co²⁺, and Cu²⁺) will react with sulfhydryl groups of proteins in Tris buffer;

[0025] For metal ion reaction with disulfide bonds: Metal ions can react with disulfide bonds in proteins, causing protein aggregation or inactivation. This aggregation can be reversible or irreversible, depending on the conditions of the aggregation;

[0026] For the reaction of metal ions with tryptophan and histidine: Metal ions can react with tryptophan and histidine to form stable coordination compounds, thereby affecting the activity of proteins. For example, the coordination of Zn²⁺ and the imidazole group of histidine plays a key role in the functional regulation of insulin;

[0027] Effects of metal coordination on protein structure: Metal ions bind to proteins through coordination compounds, which can change the secondary and tertiary structures of proteins and thus affect their activity. For example, zinc ions in zinc finger structural proteins coordinate with cysteine ​​to drive protein folding to form functionally active finger-like structures;

[0028] Metal ion-induced aggregation: Certain metal ions (such as cobalt and chromium) can induce protein aggregation. This aggregation may lead to changes in protein structure, thereby affecting its activity. For example, cobalt ions (Co²⁺) can significantly induce aggregation of bovine serum albumin;

[0029] Therefore, adding an appropriate dose of metal chelators can effectively prevent metal ions from coordinating with proteins, thereby protecting the activity of proteins.

[0030] Amino acids are the basic building blocks of proteins, the most important of which are α-amino acids. They are composed of an amino group (-NH2), a carboxyl group (-COOH), a hydrogen atom (-H) and an R group (-R) linked to an α carbon atom. Since amino acid ions have both acidic and alkaline properties, they can inhibit the pH change of the solution during the low-temperature storage of biological products, thereby achieving the purpose of protecting the active components. Amino acids with small side chains (such as glycine) do not occupy too much space in the protein structure because the side chain is only a hydrogen atom, thereby reducing the spatial conflict between amino acids and facilitating the flexibility and mobility of proteins.

[0031] The principle of bovine serum albumin (BSA) as a protective protein is mainly reflected in the following aspects: 1. Providing a stable environment: BSA provides a stable environment in the solution to prevent protein denaturation; 2. Increasing the concentration of protein in the solution: By increasing the concentration of protein in the solution, this can reduce protein denaturation and nonspecific adsorption, thereby improving the stability of the enzyme; 3. Anti-oxidation and anti-denaturation: BSA contains cysteine ​​residues, which gives it a certain antioxidant ability. It can bind to free Cu²⁺ and prevent the generation of free radicals; 4. Maintaining plasma osmotic pressure and fluid balance: BSA is abundant in plasma, accounting for 60% of the total plasma protein. It has the function of maintaining plasma osmotic pressure and fluid balance, which helps to maintain the stability of the internal and external environment of the protein; 5. Structural protection and spatial stability: There are 17 pairs of disulfide bonds in the BSA molecule, which makes it have good stability. The spatial structure of BSA can change with temperature, pH, and salt concentration. It is a flexible molecule that can enter the gap between surface antibodies and close redundant sites.

[0032] Therefore, bovine serum albumin (BSA) effectively protects proteins from the adverse effects of the external environment by providing a stable environment, increasing protein concentration, anti-oxidation and anti-denaturation, maintaining osmotic pressure and fluid balance, and structural protection and spatial stabilization.

[0033] The following further describes the effect of the liquid reagent of the present invention on the thyrotropin receptor in combination with the control group and Examples 1-3.

[0034] Combination Figure 1 As shown, the control group and Example 1-3 were diluted with thyroid stimulating hormone receptor (TSHR) to prepare the corresponding liquid detection reagent to form a TRAb detection kit, and the potency, 2-8°C storage stability, on-machine stability after opening, and 37°C±1°C accelerated stability of the reagents in this kit were evaluated. In particular, " / " in the figure means none. A series of serum samples with TRAb labeled concentrations of 1.8 IU / L, 10.6 IU / L, and 38.5 IU / L were tested in parallel on the Yingkai fully automatic chemiluminescence instrument Shine i2910 system. Each test of each group was repeated 3 times, and the average value was taken. The results are detailed in Figure 2-5 .

[0035] Among them, the potency evaluation method: the TRAb detection kit is constructed by the above method, the sample signal value is detected, and the relative luminescence value of each group is obtained. The potency of Examples 1-3 is measured by the control group, and the relative deviation between Examples 1-3 and the control group is ≤±10% and is considered to be stable. The results are detailed in Figure 2 .

[0036] Evaluation method for storage stability at 2-8℃: The TRAb detection kit was constructed using the above method and stored at 2-8℃. The sample signal values ​​were tested at 0, 3, 6, 9, 11, 12, and 13 months, respectively. The sample signal value tested at 0 month was used as the control. The relative deviation of the signal value at other monitoring time points was ≤±10%, which was considered to be stable. For details, see Figure 3 .

[0037] Evaluation method for on-machine stability after opening: The TRAb detection kit was constructed using the above method. After opening, it was placed on the test instrument. The sample signal values ​​were tested on days 0, 7, 14, 21, 28, 30, and 32. The sample signal value on day 0 was used as the control. The relative deviation of the signal value at other monitoring time points was ≤±10%, which was considered to be stable. For details, see Figure 4 .

[0038] 37℃±1℃ accelerated stability assessment method: The TRAb detection kit was constructed using the above method and stored at 37℃±1℃. The sample signal values ​​were detected on day 0, day 1, day 4, day 7, and day 8, respectively. The sample signal value detected on day 0 was used as the control. The relative deviation of the signal value at other monitoring time points was ≤±10%, which was considered to be stable. For details, see Figure 5 .

[0039] like Figure 2-Figure 5 The results shown in the control group showed that the relative deviation of the signal value of the thyroid stimulating hormone receptor (TSHR) reagent stored at 2-8°C for 13 months exceeded -70%, the relative deviation of the signal value after opening and in the machine for 32 days exceeded -15%, and the relative deviation of the signal value after 8 days of acceleration at 37°C±1°C exceeded -60%. The stability of TSHR was extremely poor.

[0040] In Example 1, the relative deviation of the thyroid stimulating hormone receptor (TSHR) reagent potency and the control group was ≤±10%, the relative deviation of the signal value after storage at 2-8°C for 13 months exceeded -50%, the relative deviation of the signal value after opening and in the machine for 32 days was less than -10%, and the relative deviation of the signal value after accelerated storage at 37°C±1°C for 8 days exceeded -40%. The stability of TSHR was improved compared with the control group, indicating that 1mM EDTA-2Na hydrate has a certain help on the stability of the thyroid stimulating hormone receptor (TSHR) reagent.

[0041] In Example 2, the relative deviation of the thyroid stimulating hormone receptor (TSHR) reagent potency and the control group is ≤±10%, the relative deviation of the signal value after 13 months of storage at 2-8°C exceeds -15%, the relative deviation of the signal value after 32 days in the machine after opening is less than -10%, and the relative deviation of the signal value after 8 days of acceleration at 37°C±1°C exceeds -10%, and the stability of TSHR is significantly improved compared with the control group, indicating that the 1mM EDTA-2Na hydrate and 0.3% glycine composition plays a key role in the stability of the thyroid stimulating hormone receptor (TSHR) reagent;

[0042] In Example 3, the relative deviation of the thyroid stimulating hormone receptor (TSHR) reagent titer and the control group was ≤±10%, and the relative deviations of the signal values ​​after storage at 2-8°C for 13 months, in the machine for 32 days after opening, and accelerated at 37°C±1°C for 8 days were all less than -10%, indicating that the combination formula of 1mM EDTA-2Na hydrate, 0.3% glycine and 2.0% bovine serum albumin can stabilize TSHR.

[0043] In summary, the above-mentioned liquid reagent can effectively prolong the storage time of the unopened liquid reagent containing thyrotropin receptor (12 months), and prolong the in-machine storage time of the opened reagent in an environment of 2-8° (1 month), thereby achieving the purpose of extending the shelf life of the test kit and reducing the preparation cost.

[0044] The above-described embodiments only express one or more implementation modes of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the patent of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. A liquid reagent for stabilizing thyrotropin receptors, characterized in that: The liquid reagent consists of 0.1% PC-300, 0.1% Tween-20, 1 mM EDTA-2Na hydrate, 0.3% glycine, 2.0% bovine serum albumin and 20 mM Tris buffer.

2. A TRAb detection kit, characterized in that: The kit comprises: a first reagent, wherein the first reagent is prepared by diluting the thyroid stimulating hormone receptor using the liquid reagent according to claim 1.

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