A stimulating thyroid stimulating hormone receptor antibody subtype detection kit and its preparation method and application

Abstract

The present invention provides a stimulatory thyroid stimulating hormone receptor antibody subtype detection kit and its preparation method and application, which belongs to in vitro diagnostic reagents. The kit provided by the present invention includes a solid phase reagent, a labeling reagent, a calibration solution, and a quality control product, wherein the sequence of the mutant thyroid stimulating hormone receptor in the labeling reagent is shown in SEQ ID No. 1. The kit provided by the present invention only needs to test the sample to directly obtain the concentration value of the sample; the relevant test has been fully automated, and the addition of reagents and samples is completed by instruments, which is more convenient to operate and reduces human errors.

Description

Technical Field

[0001] The present invention relates to the field of in vitro diagnostic reagents, and in particular to a stimulating thyroid stimulating hormone receptor antibody subtype detection kit and a preparation method and application thereof. Background Art

[0002] Thyroid stimulating hormone receptor (TSHR) is a macromolecular glycoprotein located on the membrane of thyroid follicular epithelial cells. It consists of a single polypeptide chain containing 764 amino acids, with a molecular weight of approximately 84 kDa. The extracellular domain has approximately 414 to 418 amino acids, including six potential N-linked glycosylation sites, while the remaining sequence forms a transmembrane domain and an intracellular carboxyl terminus. The presence of the transmembrane region and extracellular domain of TSHR is necessary for high-affinity ligand binding, and the stability of the transmembrane region and intracellular structure plays an important role in maintaining the stability of TSHR. A large number of literatures have shown that TSHR itself has poor stability. Under physiological conditions, thyroid stimulating hormone binds to its receptor TSHR, and a cascade reaction is generated through G protein activation to achieve biological effects. Under pathological conditions, when TSHR becomes antigenic, it causes an autoimmune reaction and produces a large number of thyroid stimulating hormone receptor antibodies (TRAb). TRAb is a group of heterogeneous antibodies, which can be divided into the following three categories according to their functions: 1. Thyroid stimulating antibodies (TSAb), also known as thyroid stimulating immunoglobulin (TSI), bind to the TSH receptors on the thyroid follicle membrane, stimulate thyroid enlargement, enhance its functional activity, and are the main cause of Graves' disease; 2. Thyroid function inhibitory antibodies (TFIAb): also known as thyroid function inhibitory immunoglobulin (TFII), after binding to the TSH receptor, it can inhibit thyroid function and cause hypothyroidism; 3. Thyroid growth stimulating immunoglobulin (TGI): can stimulate thyroid enlargement but does not affect its function.

[0003] At present, the methods for detecting thyroid stimulating hormone receptor antibodies (TRAb) mainly include: 1. Enzyme-linked immunosorbent assay (ELISA): This method is widely used, but it has shortcomings such as cumbersome operation, multiple types of reagent bottles, cross-contamination risks, and manual operation errors; 2. Radioimmunoassay: This method has disadvantages such as radioactive contamination, complex operation, and unstable results; 3. Bioanalysis: This method can distinguish between stimulatory antibodies and blocking antibodies, but it is time-consuming, cumbersome to operate, and costly. Summary of the invention

[0004] The object of the present invention is to provide a method for detecting thyroid stimulating receptor antibodies which has simple operation, strong anti-interference ability, high accuracy and high sensitivity.

[0005] To achieve the above object, the present invention provides a first aspect of a stimulatory thyroid stimulating hormone receptor antibody subtype detection kit, the kit comprising:

[0006] A solid phase reagent, the solid phase reagent comprising magnetic particles coated with stimulating thyroid stimulating hormone receptor antibodies and a solid phase reagent diluent, the solid phase reagent diluent comprising a biological buffer, an inorganic salt, a protein protective agent, a surfactant, a chemical stabilizer, a preservative and a blocking agent;

[0007] A labeling reagent, the labeling reagent comprising a mutant human thyroid stimulating hormone receptor labeled with a marker and a labeling reagent diluent, the labeling reagent diluent comprising a biological buffer, an inorganic salt, a protein protective agent, a surfactant, a chemical stabilizer, a preservative and a blocking agent;

[0008] A calibration solution, wherein the calibration solution is a 0-40 IU / L thyroid stimulating hormone receptor antibody solution, and the solvent of the calibration solution is a calibration quality control diluent;

[0009] Quality control products, the quality control products comprising two thyroid stimulating hormone receptor antibody solutions of different concentrations, the solvent of the quality control products being a calibration quality control diluent;

[0010] The calibration quality control diluent comprises a biological buffer, an inorganic salt, a protein protectant, a surfactant, a chemical stabilizer, a preservative and a blocking agent.

[0011] The kit provided by the present invention adopts a fully automatic chemiluminescence method to detect the concentration of thyroid stimulating hormone receptor antibodies. The principle is that during the detection, after the detection sample, the solid phase reagent of magnetic particles coated with stimulating thyroid stimulating hormone receptor antibodies, and the labeling reagent of mutant human thyroid stimulating hormone receptors labeled by a labeling substance are mixed, TRAb in the sample and the stimulating thyroid stimulating hormone receptor antibodies on the magnetic particles compete with the mutant human thyroid stimulating hormone receptors. After a washing cycle, by adding a triggering reagent corresponding to the corresponding labeling substance, the reaction system will generate chemiluminescence, and the chemiluminescence intensity is detected by an instrument and compared with a standard curve to obtain the concentration of TRAb in the sample.

[0012] Preferably, the stimulating thyroid stimulating hormone receptor antibody is a humanized stimulating thyroid stimulating hormone receptor monoclonal antibody as shown in PDB: 3G04.

[0013] Preferably, the protein sequence of the mutant human thyroid stimulating hormone receptor is shown in SEQ ID No. 1, which is obtained by subjecting the extracellular segment of the protein shown in NCBI: P16473 to amino acid site-directed mutagenesis, specifically: the 275th amino acid of the protein shown in NCBI: P16473 is replaced by alanine to valine, the 361st amino acid is mutated from aspartic acid to glutamate, the 381st amino acid is mutated from phenylalanine to tyrosine, the 386th amino acid is mutated from aspartic acid to glutamate, and the 397th amino acid is mutated from valine to isoleucine.

[0014] The mutant human thyroid stimulating hormone receptor provided by the present invention is based on the existing ones, and site-directed mutation is performed on the non-antigenic determinant cluster position of the extracellular segment of the human thyroid stimulating hormone receptor, so that the human thyroid stimulating hormone receptor forms a more stable three-dimensional structure, which can remain stable under different environmental conditions and does not affect the binding with the stimulating thyroid stimulating hormone receptor antibody.

[0015] Preferably, the biological buffer is selected from one of Dulbecco's phosphate buffer, citrate buffer, HEPES buffer, Hank's buffer, EBSS buffer, and Tris buffer;

[0016] The inorganic salt is selected from one or more of sodium chloride, sodium bicarbonate, disodium hydrogen phosphate, and sodium dihydrogen phosphate;

[0017] The protein protective agent is a conventional protein protective agent;

[0018] The surfactant is selected from any one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant, a zwitterionic surfactant, and a polymer surfactant;

[0019] The chemical stabilizer is Tween-20;

[0020] The preservative is selected from any one or more of sodium azide, thimerosal, antibiotics, and cinnamaldehyde;

[0021] The blocking agent is IGG and / or IgM;

[0022] The label is any one of a ruthenium compound, an acridine compound, a peroxidase, an alkaline phosphatase and a horseradish peroxidase;

[0023] The magnetic particles are selected from any one of streptavidin magnetic particles, carboxyl magnetic particles, tosyl magnetic particles, amino magnetic particles and epoxy magnetic particles.

[0024] Further preferably, the inorganic salt is sodium chloride, and the mass fraction of sodium chloride in the solid phase reagent diluent is preferably 0.9%, the mass fraction in the labeling reagent diluent is preferably 0.9%, and the mass fraction in the calibration quality control diluent is preferably 0.9%.

[0025] Further preferably, the preservative is sodium azide, and the mass fraction of sodium azide in the solid phase reagent diluent is preferably 0.1%, the mass fraction in the labeling reagent diluent is preferably 0.1%, and the mass fraction in the calibration quality control diluent is preferably 0.1%.

[0026] More preferably, the label is an acridinium ester.

[0027] More preferably, the magnetic particles are carboxyl magnetic particles with a concentration of 0.1 mg / mL.

[0028] The second aspect of the present invention provides a method for preparing the kit according to the first aspect, comprising the following steps:

[0029] S1: prepare solid phase reagent diluent and solid phase reagent;

[0030] S2: prepare labeling reagent diluent and labeling reagent;

[0031] S3: Prepare calibration quality control dilutions and prepare calibrators and quality controls.

[0032] Preferably, the step S1 comprises the following steps:

[0033] S11: dissolving the biological buffer, inorganic salt, protein protective agent, surfactant, chemical stabilizer, preservative, and blocking agent in purified water in sequence, adjusting the pH to 6.0-7.0, fixing the volume, and sterilizing to obtain a solid phase reagent dilution solution;

[0034] S12: Take the magnetic particles and suspend them, perform magnetic separation and remove the supernatant, resuspend them with MES buffer at pH 5.0, activate the magnetic particles, add stimulating thyroid stimulating hormone receptor antibody, suspend them at room temperature for 2 hours, remove the supernatant after magnetic separation, resuspend them with 0.1M Tris buffer at pH 8.0 containing 2% BSA to obtain a high-concentration magnetic particle intermediate coated with stimulating thyroid stimulating hormone receptor antibody, and store them at 4°C for future use;

[0035] S13: Use a solid phase reagent diluent to dilute the high-concentration magnetic particle intermediate coated with the stimulating thyroid stimulating hormone receptor antibody to obtain a solid phase reagent.

[0036] Preferably, step S2 comprises the following steps:

[0037] S21: dissolving the biological buffer, inorganic salt, protein protective agent, surfactant, chemical stabilizer, preservative, and blocking agent in purified water in sequence, adjusting the pH to 7.0-7.4, fixing the volume, and sterilizing to obtain a labeling reagent dilution solution;

[0038] S22: Dissolve the marker with an organic solvent of anhydrous dimethyl sulfoxide or anhydrous N,N-dimethylformamide, and store at -20°C in a dark and dry condition; Take the mutant human thyroid stimulating hormone receptor, dilute it with PB to a concentration in the range of 0.5-2 mg / mL, add the marker and mix well, seal and let stand in the dark to react; After the reaction is completed, purify to obtain a high-concentration labeling reagent intermediate for use;

[0039] S23: dilute the high concentration labeling reagent intermediate using a labeling reagent diluent to obtain a labeling reagent.

[0040] Preferably, the step S3 comprises the following steps:

[0041] S31: Dissolve the biological buffer, inorganic salt, protein protectant, surfactant, chemical stabilizer, preservative, and blocking agent in purified water in sequence, adjust the pH to 6.5-7.5, make up to volume, and sterilize to obtain the calibration quality control dilution solution;

[0042] S32: Dissolve the thyroid stimulating hormone receptor antibody using the calibration quality control diluent to prepare several thyroid stimulating hormone receptor antibody solutions with different concentrations, which are the calibration solutions;

[0043] S33: Use the calibration quality control diluent to dissolve the thyroid stimulating hormone receptor antibody and prepare two thyroid stimulating hormone receptor antibody solutions with different concentrations, which are the quality control products.

[0044] The third aspect of the present invention provides an application of the kit described in the first aspect, wherein the application includes using the kit for quantitative detection of stimulatory thyroid stimulating hormone receptor antibody subtypes for non-disease diagnosis purposes.

[0045] Compared with the prior art, the present invention has the following beneficial effects: the stimulatory thyroid stimulating hormone receptor antibody subtype detection kit provided by the present invention selects the stimulatory thyroid stimulating hormone receptor antibody subtype to be coated on the magnetic microparticles, which can avoid the interference of the inhibitory thyroid stimulating hormone receptor antibody subtype and the thyroid growth stimulating immunoglobulin subtype on the detection system; acridinium ester is selected as the labeling material and applied to the chemiluminescence immunoassay system, which is not interfered by biotin or sodium fluorescein; the selected acridinium ester chemiluminescence immunoassay system has high repeatability, and the intra-batch and inter-batch differences are both within 5%; the kit provided by the present invention has a built-in standard curve in the test software, and the concentration value of the sample can be directly obtained by testing the sample; the relevant test has been fully automated, and the addition of reagents and samples is completed by the instrument, the operation is simpler, and the human error is reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a standard curve of the stimulating thyroid stimulating hormone receptor antibody subtypes of the kit in a specific embodiment of the present invention. DETAILED DESCRIPTION

[0047] In order to make the above-mentioned purposes, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation method and typical parameters of the present invention, and are not used to limit the parameter range described in the present invention. Reasonable changes derived therefrom are still within the scope of protection of the claims of the present invention.

[0048] It should be noted that the endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this article.

[0049] Unless otherwise defined, all terms, symbols and other scientific terms used herein are intended to have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. In some cases, terms with conventionally understood meanings are defined herein for the purpose of clarification or ease of reference, and such definitions herein should not be construed as indicating significant differences from conventional understandings in the art. The technical methods described or cited herein are generally well understood by those skilled in the art and are adopted by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents and instruments is carried out in accordance with the protocols and parameters given by the manufacturer.

[0050] In order to solve the technical difficulties in the background technology, the specific embodiment of the present invention provides a stimulatory thyroid stimulating hormone receptor antibody subtype detection kit, the kit comprising:

[0051] A solid phase reagent, the solid phase reagent comprising magnetic particles coated with stimulating thyroid stimulating hormone receptor antibodies and a solid phase reagent diluent, the solid phase reagent diluent comprising a biological buffer, an inorganic salt, a protein protective agent, a surfactant, a chemical stabilizer, a preservative and a blocking agent;

[0052] A labeling reagent, the labeling reagent comprising a mutant human thyroid stimulating hormone receptor labeled with a marker and a labeling reagent diluent, the labeling reagent diluent comprising a biological buffer, an inorganic salt, a protein protective agent, a surfactant, a chemical stabilizer, a preservative and a blocking agent;

[0053] A calibration solution, wherein the calibration solution is a 0-40 IU / L thyroid stimulating hormone receptor antibody solution, and the solvent of the calibration solution is a calibration quality control diluent;

[0054] Quality control products, the quality control products comprising two thyroid stimulating hormone receptor antibody solutions of different concentrations, the solvent of the quality control products being a calibration quality control diluent;

[0055] The calibration quality control diluent comprises a biological buffer, an inorganic salt, a protein protectant, a surfactant, a chemical stabilizer, a preservative and a blocking agent.

[0056] The kit provided by the present invention adopts a fully automatic chemiluminescence method to detect the concentration of thyroid stimulating hormone receptor antibodies. The principle is that during the detection, after the detection sample, the solid phase reagent of magnetic particles coated with stimulating thyroid stimulating hormone receptor antibodies, and the labeling reagent of mutant human thyroid stimulating hormone receptors labeled with a marker are mixed, the TRAb in the sample and the stimulating thyroid stimulating hormone receptor antibodies on the magnetic particles compete with the mutant human thyroid stimulating hormone receptors. After a washing cycle, by adding a triggering reagent corresponding to the corresponding marker, the reaction system will produce chemiluminescence, and the chemiluminescence intensity is detected by an instrument and compared with the standard curve to obtain the concentration of TRAb in the sample. In the above embodiment, the stimulating thyroid stimulating hormone receptor antibody is a humanized stimulating thyroid stimulating hormone receptor monoclonal antibody shown in PDB: 3G04.

[0057] In the above embodiment, the mutant human thyroid stimulating hormone receptor protein sequence is shown in SEQ ID No. 1, which is obtained by subjecting the extracellular segment of the protein shown in NCBI: P16473 to amino acid site-directed mutagenesis, specifically: the 275th amino acid of the protein shown in NCBI: P16473 is replaced by alanine (Ala) to valine (Val), the 361st amino acid is mutated from aspartic acid (Asp) to glutamic acid (Glu), the 381st amino acid is mutated from phenylalanine (Phe) to tyrosine (Tyr), the 386th amino acid is mutated from aspartic acid (Asp) to glutamic acid (Glu), and the 397th amino acid is mutated from valine (Val) to isoleucine (Ile).

[0058] The mutant human thyroid stimulating hormone receptor provided by the present invention is based on the existing ones, and site-directed mutation is performed on the non-antigenic determinant cluster position of the extracellular segment of the human thyroid stimulating hormone receptor, so that the human thyroid stimulating hormone receptor forms a more stable three-dimensional structure, which can remain stable under different environmental conditions and does not affect the binding with the stimulating thyroid stimulating hormone receptor antibody.

[0059] Specifically, the amino acid sequence of the mutant human thyroid stimulating hormone receptor is shown in SEQ ID No.1.

[0060] In the above embodiment, the biological buffer is selected from one of Dulbecco's phosphate buffer, citrate buffer, HEPES buffer, Hank's buffer, EBSS buffer, and Tris buffer;

[0061] The inorganic salt is selected from one or more of sodium chloride, sodium bicarbonate, disodium hydrogen phosphate, and sodium dihydrogen phosphate, preferably sodium chloride, wherein the mass fraction of sodium chloride in the solid phase reagent diluent is preferably 0.9%, the mass fraction in the labeling reagent diluent is preferably 0.9%, and the mass fraction in the calibration quality control diluent is preferably 0.9%;

[0062] The protein protective agent is a conventional protein protective agent;

[0063] The surfactant is selected from any one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant, a zwitterionic surfactant, and a polymer surfactant;

[0064] The chemical stabilizer is Tween-20;

[0065] The preservative is selected from any one or more of sodium azide, thimerosal, antibiotics, and cinnamaldehyde, preferably sodium azide, wherein the mass fraction of sodium azide in the solid phase reagent diluent is preferably 0.1%, the mass fraction in the labeling reagent diluent is preferably 0.1%, and the mass fraction in the calibration quality control diluent is preferably 0.1%;

[0066] The blocking agents are IgG and / or IgM;

[0067] The label is any one of ruthenium compounds, acridine compounds, peroxidase, alkaline phosphatase and horseradish peroxidase;

[0068] The magnetic particles are selected from any one of streptavidin magnetic particles, carboxyl magnetic particles, tosyl magnetic particles, amino magnetic particles and epoxy magnetic particles.

[0069] The specific embodiment of the present invention also provides a method for preparing the aforementioned kit, which specifically comprises the following steps:

[0070] S11: dissolving the biological buffer, inorganic salt, protein protective agent, surfactant, chemical stabilizer, preservative, and blocking agent in purified water in sequence, adjusting the pH to 6.0-7.0, fixing the volume, and sterilizing to obtain a solid phase reagent dilution solution;

[0071] S12: Take the magnetic particles and suspend them, perform magnetic separation and remove the supernatant, resuspend them with MES buffer at pH 5.0, activate the magnetic particles, add stimulating thyroid stimulating hormone receptor antibody, suspend them at room temperature, remove the supernatant after magnetic separation, resuspend them with 0.1M Tris buffer at pH 8.0 containing 2% BSA to obtain a high-concentration magnetic particle intermediate coated with stimulating thyroid stimulating hormone receptor antibody, and store them at 4°C for future use;

[0072] S13: diluting the high-concentration magnetic particle intermediate coated with the stimulating thyroid stimulating hormone receptor antibody using a solid phase reagent diluent to obtain a solid phase reagent;

[0073] S21: dissolving the biological buffer, inorganic salt, protein protective agent, surfactant, chemical stabilizer, preservative, and blocking agent in purified water in sequence, adjusting the pH to 7.0-7.4, fixing the volume, and sterilizing to obtain a labeling reagent dilution solution;

[0074] S22: Dissolve the marker with an organic solvent of anhydrous dimethyl sulfoxide or anhydrous N,N-dimethylformamide, and store at -20°C in a dark and dry condition; Take the mutant human thyroid stimulating hormone receptor, dilute it with PB to a concentration in the range of 0.5-2 mg / mL, add the marker and mix well, seal and let stand in the dark to react; After the reaction is completed, purify to obtain a high-concentration labeling reagent intermediate for use;

[0075] S23: diluting the high-concentration labeling reagent intermediate using a labeling reagent diluent to obtain a labeling reagent;

[0076] S31: Dissolve the biological buffer, inorganic salt, protein protectant, surfactant, chemical stabilizer, preservative, and blocking agent in purified water in sequence, adjust the pH to 6.5-7.5, make up to volume, and sterilize to obtain the calibration quality control dilution solution;

[0077] S32: Dissolve the thyroid stimulating hormone receptor antibody using the calibration quality control diluent to prepare several thyroid stimulating hormone receptor antibody solutions with different concentrations, which are the calibration solutions;

[0078] S33: Use the calibration quality control diluent to dissolve the thyroid stimulating hormone receptor antibody and prepare any two concentrations of thyroid stimulating hormone receptor antibody solutions, which are the quality control products.

[0079] The technical solution of the present invention is further described below through specific embodiments.

[0080] Example 1

[0081] Kit preparation

[0082] S11: Dissolve Dulbecco's phosphate buffer, sodium chloride, BSA, SDS, Tween 20, sodium azide, and IgG in purified water in sequence, adjust the pH to 6.0, make up to volume, and filter and sterilize to obtain a solid phase reagent dilution solution;

[0083] S12: Take 50 mg of carboxyl magnetic microparticles (particle size 0.8 μm) suspension, remove the supernatant by magnetic separation, resuspend with 0.02 M, pH 5.0 MES buffer, add 0.5 mL of freshly prepared 20 mg / mL EDC aqueous solution to activate the carboxyl groups on the surface of the magnetic microparticles, add 3 mg of stimulatory thyroid stimulating hormone receptor antibody, suspend at room temperature for 2 hours, remove the supernatant after magnetic separation, resuspend to 10 mg / mL with 0.1 M pH 8.0 Tris buffer containing 2% BSA, and obtain a high-concentration magnetic particle intermediate coated with stimulatory thyroid stimulating hormone receptor antibody, which is stored at 4°C for use;

[0084] S13: diluting the high-concentration magnetic particle intermediate coated with the stimulating thyroid stimulating hormone receptor antibody to 0.5 mg / mL with a solid phase reagent diluent to obtain a solid phase reagent;

[0085] S21: Dissolve Dulbecco's phosphate buffer, sodium chloride, casein, Tween-80, Tween-20, thimerosal, and IgM in purified water in sequence, adjust the pH to 7.0, make up to volume, and sterilize to obtain a labeling reagent dilution solution;

[0086] S22: Dissolve the acridinium ester in anhydrous dimethyl sulfoxide and store in a dry place at -20°C away from light; take the mutant human thyroid stimulating hormone receptor, dilute it with PB to a concentration of 0.5 mg / mL, add the acridinium ester solution and mix well, seal and let stand in the dark to react; after the reaction is completed, purify to obtain a high-concentration labeling reagent intermediate for use;

[0087] S23: diluting the high-concentration labeling reagent intermediate using a labeling reagent diluent to obtain a labeling reagent;

[0088] S31: Dissolve citric acid buffer, sodium chloride, casein, SDS, Tween-20, sodium azide, and IgG in purified water in sequence, adjust the pH to 6.5, make up to volume, and sterilize to obtain calibration quality control dilution solution;

[0089] S32: Use the calibration quality control diluent to dissolve the thyroid stimulating hormone receptor antibody to prepare several thyroid stimulating hormone receptor antibody solutions of different concentrations, namely the calibration solution. The concentrations of the thyroid stimulating hormone receptor antibody in the calibration solution are: 0 IU / L, 0.3 IU / L, 1.75 IU / L, 5.00 IU / L, 10.50 IU / L, 15.00 IU / L, 20.00 IU / L, 25.30 IU / L, 30.40 IU / L, 40.00 IU / L;

[0090] S33: Use the calibration quality control diluent to dissolve the thyroid stimulating hormone receptor antibody and prepare any two concentrations of thyroid stimulating hormone receptor antibody solutions, which are the quality control products. The concentrations of the thyroid stimulating hormone receptor antibodies in the quality control products are 1.50 IU / L and 12.00 IU / L.

[0091] Example 2

[0092] Kit preparation

[0093] S11: Dissolve HEPES buffer, sodium bicarbonate, gelatin, MES, Tween-20, cinnamaldehyde, and IgG in purified water in sequence, adjust the pH to 7.0, make up to volume, and filter and sterilize to obtain a solid phase reagent dilution solution;

[0094] S12: Take 50 mg of carboxyl magnetic microparticles (particle size 0.8 μm) suspension, perform magnetic separation and remove the supernatant, resuspend with 0.02 M MES buffer with a pH of 5.0, add 0.5 mL of a freshly prepared 20 mg / mL EDC aqueous solution to activate the magnetic microparticles, add stimulating thyroid stimulating hormone receptor antibody, suspend at room temperature, remove the supernatant after magnetic separation, resuspend with 0.1 M Tris buffer with a pH of 8.0 containing 2% BSA, and obtain a high-concentration magnetic particle intermediate coated with stimulating thyroid stimulating hormone receptor antibody, which is stored at 4°C for future use;

[0095] S13: diluting the high-concentration magnetic particle intermediate coated with the stimulating thyroid stimulating hormone receptor antibody using a solid phase reagent diluent to obtain a solid phase reagent;

[0096] S21: Dissolve Tris buffer, disodium hydrogen phosphate, whey protein, glyceryl monostearate, Tween-20, gentamicin, and IgG in purified water in sequence, adjust the pH to 7.4, make up to volume, and filter and sterilize to obtain a labeling reagent diluent;

[0097] S22: Dissolve the acridinium ester in anhydrous N,N-dimethylformamide and store at -20°C in a dark and dry condition; take the mutant human thyroid stimulating hormone receptor, dilute it with PB to a concentration of 2 mg / mL, add the acridinium ester solution and mix well, seal and let stand in the dark to react; after the reaction is completed, purify to obtain a high-concentration labeling reagent intermediate for use;

[0098] S23: diluting the high-concentration labeling reagent intermediate using a labeling reagent diluent to obtain a labeling reagent;

[0099] S31: Dissolve EBSS buffer, sodium dihydrogen phosphate, mannitol, AES, Tween-20, sodium azide, and IgG in purified water in sequence, adjust the pH to 7.5, make up to volume, and filter and sterilize to obtain calibration quality control dilution solution;

[0100] S32: Use the calibration quality control diluent to dissolve the thyroid stimulating hormone receptor antibody to prepare several thyroid stimulating hormone receptor antibody solutions with different concentrations, namely the calibration solution. The concentrations of the thyroid stimulating hormone receptor antibody in the calibration solution are: 0 IU / L, 0.3 IU / L, 1.75 IU / L, 5.00 IU / L, 10.50 IU / L, 15.00 IU / L, 20.00 IU / L, 25.30 IU / L, 30.40 IU / L, 40.00 IU / L;

[0101] S33: Use the calibration quality control diluent to dissolve the thyroid stimulating hormone receptor antibody and prepare two thyroid stimulating hormone receptor antibody solutions with different concentrations, namely the quality control products. The concentrations of the thyroid stimulating hormone receptor antibodies in the quality control products are 1.50 IU / L and 12.00 IU / L.

[0102] Example 3

[0103] Performance Evaluation

[0104] The kit prepared in Example 1 was subjected to standard curve determination using a fully automatic chemiluminescence immunoassay analyzer. The test results are shown in FIG. Figure 1 shown.

[0105] To measure the sensitivity:

[0106] The blank limit LOB refers to the experimental method and calculation formula of CLSI standard EP17-A.

[0107] Preparation of LOB samples: Prepare 5 portions of commercial hormone-free serum matrix with the analyte content close to 0. Use three batches of reagents. Repeat the test for 3 times for each sample for 4 consecutive days. Count the data results of 60 tests and calculate LOB according to the formula of EP17-A.

[0108] The experimental results are shown in Table 1 below:

[0109] Table 1

[0110]

[0111] As can be seen from Table 1, the kit prepared in Example 1 has higher sensitivity and better stability.

[0112] Determination of linearity:

[0113] Linear analysis was performed on standard samples with concentrations of 0, 0.28 IU / L, 1.74 IU / L, 3.41 IU / L, 11.31 IU / L, 23.79 IU / L, and 40.05 IU / L, and the linear correlation coefficient was calculated, r=0.9987. In addition, the linear range of the kit for the determination of thyroid stimulating hormone receptor antibody samples is 0.30 IU / L-40.00 IU / L.

[0114] Intra-batch and inter-batch differences:

[0115] Three stimulating thyroid stimulating hormone receptor antibody samples with concentrations of 1.51 IU / L, 7.53 IU / L and 33.09 IU / L were taken, and 3 parallels were made for each sample and each concentration. The three batches of test kits were used for determination, and the intra-batch and inter-batch differences of the test kits were calculated. The results showed that the intra-batch and inter-batch differences of the test kits were less than 5%.

[0116] Interference experiment:

[0117] The mixed serum was added with interfering substances including vitamin C, hemoglobin, conjugated bilirubin, unconjugated bilirubin, triglycerides, RF, and biotin to interfere with the measurement of the mixed serum and the mixed serum after adding various interfering substances, and the deviation between the two was calculated, with ±10% as the acceptable range. The results showed that the interference reached the relevant standard documents of NCCLS and can be used for accurate evaluation of the content of stimulating thyroid stimulating hormone receptor antibodies.

[0118] It can be seen from the above embodiments that the specific embodiments of the present invention select specific solid phase reagents, labeling reagents, and transform human thyroid stimulating hormone receptors, and the final stimulating thyroid stimulating hormone receptor antibody subtype detection kit has extremely high stability, sensitivity and anti-interference strength.

[0119] Although the disclosure is disclosed as above, the protection scope of the disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the disclosure, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. A stimulatory thyroid stimulating hormone receptor antibody subtype detection kit, characterized in that: The kit comprises: A solid phase reagent, the solid phase reagent comprising magnetic particles coated with stimulating thyroid stimulating hormone receptor antibodies and a solid phase reagent diluent, the solid phase reagent diluent comprising a biological buffer, an inorganic salt, a protein protective agent, a surfactant, a chemical stabilizer, a preservative and a blocking agent; A labeling reagent, the labeling reagent comprising a mutant human thyroid stimulating hormone receptor labeled with a label and a labeling reagent diluent, the protein sequence of the mutant human thyroid stimulating hormone receptor is shown in SEQ ID No. 1, which is obtained by site-directed mutation of the extracellular segment of the protein shown in NCBI: P16473, specifically: the 275th amino acid of the protein shown in NCBI: P16473 is replaced by alanine to valine, the 361st amino acid is mutated from aspartic acid to glutamic acid, the 381st amino acid is mutated from phenylalanine to tyrosine, the 386th amino acid is mutated from aspartic acid to glutamic acid, and the 397th amino acid is mutated from valine to isoleucine, and the labeling reagent diluent comprises a biological buffer, an inorganic salt, a protein protective agent, a surfactant, a chemical stabilizer, a preservative and a blocking agent; A calibration solution, wherein the calibration solution is a 0-40 IU / L thyroid stimulating hormone receptor antibody solution, and the solvent of the calibration solution is a calibration quality control diluent; Quality control products, the quality control products comprising two thyroid stimulating hormone receptor antibody solutions of different concentrations, the solvent of the quality control products being a calibration quality control diluent; The calibration quality control diluent comprises a biological buffer, an inorganic salt, a protein protectant, a surfactant, a chemical stabilizer, a preservative and a blocking agent.

2. The kit according to claim 1, characterized in that The stimulating thyroid stimulating hormone receptor antibody is a humanized stimulating thyroid stimulating hormone receptor monoclonal antibody shown in PDB: 3G04.

3. The kit according to claim 1, characterized in that The biological buffer is selected from one of Dulbecco's phosphate buffer, citrate buffer, HEPES buffer, Hank's buffer, EBSS buffer, and Tris buffer; The inorganic salt is selected from any one or more of sodium chloride, sodium bicarbonate, disodium hydrogen phosphate, and sodium dihydrogen phosphate; The protein protective agent is a conventional protein protective agent; The surfactant is selected from any one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant, a zwitterionic surfactant, and a polymer surfactant; The chemical stabilizer is Tween-20; The preservative is selected from any one or more of sodium azide, thimerosal, antibiotics, and cinnamaldehyde; The blocking agent is IgG and / or IgM; The label is any one of a ruthenium compound, an acridine compound, a peroxidase, an alkaline phosphatase and a horseradish peroxidase; The magnetic particles are selected from any one of streptavidin magnetic particles, carboxyl magnetic particles, tosyl magnetic particles, amino magnetic particles and epoxy magnetic particles.

4. A method for preparing the kit according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1: prepare solid phase reagent diluent and solid phase reagent; S2: prepare labeling reagent diluent and labeling reagent; S3: Prepare calibration quality control dilutions and prepare calibrators and quality controls.

5. The preparation method according to claim 4, characterized in that: The step S1 comprises the following steps: S11: dissolving the biological buffer, inorganic salt, protein protective agent, surfactant, chemical stabilizer, preservative, and blocking agent in purified water in sequence, adjusting the pH to 6.0-7.0, fixing the volume, and sterilizing to obtain a solid phase reagent dilution solution; S12: Take the magnetic particles and suspend them, perform magnetic separation and remove the supernatant, resuspend them with MES buffer at pH 5.0, activate the magnetic particles, add stimulating thyroid stimulating hormone receptor antibody, suspend them at room temperature, remove the supernatant after magnetic separation, resuspend them with 0.1M Tris buffer at pH 8.0 containing 2% BSA, and obtain a high-concentration magnetic particle intermediate coated with stimulating thyroid stimulating hormone receptor antibody, and store them at 4°C for future use; S13: Use a solid phase reagent diluent to dilute the high-concentration magnetic particle intermediate coated with the stimulating thyroid stimulating hormone receptor antibody to obtain a solid phase reagent.

6. The preparation method according to claim 4, characterized in that: The step S2 comprises the following steps: S21: dissolving the biological buffer, inorganic salt, protein protective agent, surfactant, chemical stabilizer, preservative, and blocking agent in purified water in sequence, adjusting the pH to 7.0-7.4, fixing the volume, and sterilizing to obtain a labeling reagent dilution solution; S22: Dissolve the marker with an organic solvent of anhydrous dimethyl sulfoxide or anhydrous N,N-dimethylformamide, and store at -20°C in a dark and dry condition; Take the mutant human thyroid stimulating hormone receptor, dilute it with PB to a concentration in the range of 0.5-2 mg / mL, add the marker and mix well, seal and let stand in the dark to react; After the reaction is completed, purify to obtain a high-concentration labeling reagent intermediate for use; S23: dilute the high concentration labeling reagent intermediate using a labeling reagent diluent to obtain a labeling reagent.

7. The preparation method according to claim 4, characterized in that: The step S3 comprises the following steps: S31: Dissolve the biological buffer, inorganic salt, protein protectant, surfactant, chemical stabilizer, preservative, and blocking agent in purified water in sequence, adjust the pH to 6.5-7.5, make up to volume, and sterilize to obtain the calibration quality control dilution solution; S32: Dissolve the thyroid stimulating hormone receptor antibody using the calibration quality control diluent to prepare several thyroid stimulating hormone receptor antibody solutions with different concentrations, which are the calibration solutions; S33: Use the calibration quality control diluent to dissolve the thyroid stimulating hormone receptor antibody and prepare two thyroid stimulating hormone receptor antibody solutions with different concentrations, which are the quality control products.

8. A use of the kit according to any one of claims 1 to 3, characterized in that: The application includes using the kit for the quantitative detection of stimulatory thyroid stimulating hormone receptor antibody subtypes for non-disease diagnosis purposes.

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