Thyroid stimulating hormone receptor complex, kit, preparation method and use

Abstract

The present invention belongs to the field of medicine and detection, and relates to a thyroid stimulating hormone receptor complex, a kit, a preparation method and use. Specifically, it relates to a thyroid stimulating hormone receptor complex, comprising: a thyroid stimulating hormone receptor, a thyroid stimulating hormone receptor C-terminal monoclonal antibody and magnetic particles, wherein the thyroid stimulating hormone receptor is connected to the magnetic particles through the thyroid stimulating hormone receptor C-terminal monoclonal antibody.

Description

Technical Field

[0001] The present invention belongs to the field of medicine and detection, and relates to a thyroid stimulating hormone receptor complex, a kit, a preparation method and use thereof. Specifically, it relates to a magnetic particle-thyrotropin receptor C-terminal monoclonal antibody-thyrotropin receptor complex, a kit containing the complex, and a preparation method and use thereof. Background Art

[0002] Hyperthyroidism (abbreviated as hyperthyroidism) is a clinical syndrome in which the thyroid gland synthesizes and secretes too much thyroid hormone, leading to a series of symptoms in the body. The main clinical manifestations of patients are hypermetabolism and increased excitability of the nervous, circulatory, digestive and other systems, such as fatigue, heat intolerance, sweating, irritability, insomnia, anxiety, inattention, infiltrative exophthalmos, etc. If not treated in time or treated improperly, patients may experience high fever, heart failure, pulmonary edema, and even shock, coma and other life-threatening symptoms. According to an epidemiological survey of thyroid diseases in 10 cities in my country in 2010, the prevalence of hyperthyroidism was 0.89%. With the pressure of work and life and changes in eating habits, the prevalence of hyperthyroidism has increased year by year. The incidence rate in women is higher than that in men, and the peak age is between 30 and 60 years old. According to the cause of the disease, hyperthyroidism includes autoimmune hyperthyroidism, namely toxic diffuse goiter (Graves' disease, abbreviated as GD), inflammatory hyperthyroidism, drug-induced hyperthyroidism, hCG-related hyperthyroidism and pituitary TSH tumor. Different causes of the disease correspond to different treatment methods. Therefore, it is very necessary to distinguish the types of hyperthyroidism.

[0003] Clinically, 80% of hyperthyroidism is caused by Graves' disease. Graves' disease is an autoimmune disease caused by antibodies produced by the thyroid stimulating hormone receptor (TSHR) on the membrane of thyroid follicular cells. This type of antibody is the thyroid stimulating hormone receptor antibody (TRAb), which is a group of polyclonal antibodies. According to their functions, they are divided into stimulating antibodies (TSAb), blocking antibodies (TBAb) and neutral antibodies. TSAb binds to TSHR to produce cAMP, stimulating thyroid follicular cells to secrete synthetic thyroxine, thereby causing Graves' disease; TBAb can block the binding of TSH to the receptor, leading to the occurrence of hypothyroidism. Based on the biological effects of TRAb, TRAb is currently considered to be the most important marker for Graves' disease (Note: Strictly speaking, the "TRAb" here should theoretically be TSAb, but the current relevant diagnosis and treatment guidelines and most of the literature refer to it as TRAb, and there is currently no detection reagent on the market that can absolutely distinguish between TSAb, TBAb and neutral antibodies). It has the following three main applications in clinical practice: First, the diagnosis and exclusion of autoimmune hyperthyroidism; second, monitoring the treatment and prognosis of patients with Graves' disease; third, TRAb is a type of IgG antibody that can be transmitted to the fetus through the mother. Monitoring TRAb in patients with thyroid disease during pregnancy can not only assess the risk of miscarriage, but also predict the risk of thyroid disease in newborns. It is a routine mandatory indicator in obstetrics and gynecology.

[0004] At present, the main in vitro detection methods of TRAb are: (1) Bioanalysis, which uses the different biological effects caused by TRAb binding to TSHR receptors to detect TRAb. The most classic one is cAMP bioanalysis, which uses the stimulation of TRAb on living cells cultured in vitro to quantify the cAMP production. This method is cumbersome to operate, has high technical requirements, and the detection process is time-consuming and costly, making it difficult to apply to routine clinical testing; (2) Receptor analysis, which uses the immunological activity principle of TRAb specific binding to TSHR to design an analysis method. Compared with bioanalysis, this method has the advantages of simple operation, relatively accurate results, and low economic cost. It has been widely used in clinical departments. After a long period of technical iteration and development, the detection principles of this analysis method in clinical practice are mainly competitive and sandwich. The sandwich method mainly includes Siemens and Snibe, which use one or two TSHR antigens to detect the TRAb level in patient serum. At present, this type of detection method has not been widely used in clinical practice. The competitive method is the main detection method currently used in clinical practice, mainly by Roche, which measures TRAb concentration by competing between TRAb in patient serum and labeled recombinant human thyroid stimulating monoclonal antibody (M22) for binding to soluble porcine TSHR immune complex (connecting porcine TSHR with biotinylated porcine TSHR antibody to form porcine TSHR-biotinylated porcine TSHR antibody). Regardless of which of the above detection methods is used, the preparation of TSHR is involved.

[0005] TSHR is a member of the G protein-coupled receptor family. Its polypeptide chain consists of three main parts: the extracellular N-terminal part (including 5 regions and 6 N-glycosylation sites), the transmembrane part (including 7 hydrophobic transmembrane α helices, 3 extracellular loop structures and 3 intracellular loop structures), and the intracytoplasmic carboxyl part (forming the fourth intracellular loop structure through disulfide bonds). The transmembrane part is highly homologous in the G protein-coupled receptor family. In addition to the above three parts, it also contains a protein kinase C phosphorylation signal fragment as a key site for the decoupling of phosphorylated receptors from G proteins. TSHR is not a simple polypeptide chain monomer, but a heterodimer in which the extracellular α subunit and the transmembrane β subunit are connected by disulfide bonds. The epitope that has a biological effect with TRAb is an intermittent fragment in the polypeptide chain in the natural state, which preferentially recognizes glycosylated TSHR, while complex glycosylation requires the correct folding of TSHR and its localization in thyroid follicular cells. Changes in the spatial conformation of TSHR directly affect its biological activity, so it is very necessary to maintain the spatial conformation of TSHR during the establishment of the detection method.

[0006] At present, there are two forms of TSHR in TRAb detection kits: one is to directly coat the TSHR antigen on magnetic particles, mainly from Xinye and Siemens, and react the TRAb in the patient's serum with the TSHR antigen on the magnetic particles, and then add the labeled antigen to form an immune complex. The kit prepared in this form has not been widely used in clinical practice, and its sensitivity and specificity for clinical guidance are still under discussion; the other is the form of TSHR in the widely used kits in clinical practice, where the porcine TSHR is combined with the biotinylated porcine thyrotropin receptor antibody to form a complex, and the complex formed is placed in the kit in the form of a lyophilized product. Customers need to re-prepare the complex when using it. The specific method is to carefully add a certain amount of pretreatment buffer to dissolve the lyophilized complex, place it in a sealed container for 60 minutes, and continue to gently stir it with a rotator to obtain a completely dissolved liquid. The re-prepared TSHR complex can only be stored for up to 56 hours at room temperature (20-25°C), and can only be stored for 3 weeks when used alternately at 2-8°C and at room temperature (20-25°C) on the analytical instrument (see product instructions).

[0007] The preparation of the TSHR complex in the detection kits currently widely used in the market is cumbersome and time-consuming, and has low stability after reconstitution. If the customer cannot use it up within 3 weeks after opening, it will inevitably cause waste, which is not only not conducive to customer use, but also increases the production cost of the manufacturer and reduces the economic benefits. Summary of the invention

[0008] After in-depth research and creative work, the inventors found that although TSHR directly coupled to magnetic particles has certain activity, its stability is poor, and the constructed TRAb detection reagent has low sensitivity and specificity when detecting clinical samples; in addition, TSHR is connected to the thyrotropin receptor antibody biotin to form the entire complex, and the complex can only reach 3 weeks at 2-8°C after freeze-drying and re-dissolving. The inventors creatively coupled the monoclonal antibody at the C-terminus of the thyrotropin receptor to the magnetic particles, and the coated magnetic particles are preferably connected to TSHR in the environment of trimethylamine N-oxide, thereby improving the sensitivity and specificity of the reagent. The inventors also surprisingly found that the TSHR complex coupled to the magnetic particles can be stably stored at 2-8°C in liquid for at least 12 weeks and at room temperature for at least 3 weeks. The present invention realizes that the TRAb detection reagent has a longer shelf life, is easy to use, and has a lower cost under the premise of high sensitivity and specificity. The following invention is provided:

[0009] One aspect of the present invention relates to a thyroid stimulating hormone receptor complex, comprising: a thyroid stimulating hormone receptor (TSHR), a thyroid stimulating hormone receptor C-terminal monoclonal antibody and magnetic particles.

[0010] Wherein, the thyroid stimulating hormone receptor is connected to the magnetic particle through the thyroid stimulating hormone receptor C-terminal monoclonal antibody.

[0011] In some embodiments of the present invention, the thyroid stimulating hormone receptor complex, wherein the thyroid stimulating hormone receptor is a mammalian thyroid stimulating hormone receptor, such as human thyroid stimulating hormone receptor (hTSHR) or porcine thyroid stimulating hormone receptor.

[0012] In the present invention, the source of TSHR or hTSHR is not particularly limited, and can be obtained, for example, by extraction and separation from tissues, or by expression and purification of proteins via expression vectors.

[0013] In some embodiments of the present invention, the thyroid stimulating hormone receptor complex, wherein the region where the thyroid stimulating hormone receptor C-terminal monoclonal antibody binds to the thyroid stimulating hormone receptor is located at the 261st to 764th amino acids from the N-terminal of the thyroid stimulating hormone receptor;

[0014] Preferably, the thyrotropin receptor C-terminal monoclonal antibody is the TSHR C-terminal mouse antibody 10F8 produced by Xiamen Inbomai Biotechnology Co., Ltd., or an antibody having the same heavy chain variable region and light chain variable region as 10F8.

[0015] Those skilled in the art will appreciate that, in addition to the TSHR C-terminal mouse antibody 10F8 used herein, other thyrotropin receptor C-terminal monoclonal antibodies can also achieve the same or similar technical effects.

[0016] In some embodiments of the present invention, in the thyroid stimulating hormone receptor complex, the average particle size of the magnetic particles is 100nm-5000nm, preferably 100-500nm, and more preferably 100nm-300nm.

[0017] In some embodiments of the present invention, the thyroid stimulating hormone receptor complex, wherein the average particle size of the magnetic particles is 100nm, 150nm, 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm.

[0018] In some embodiments of the present invention, the thyroid stimulating hormone receptor complex, wherein the mass ratio of the thyroid stimulating hormone receptor C-terminal monoclonal antibody to the magnetic particles is (1-5) μg:1 mg or (2-4) μg:1 mg; preferably (2-3) μg:1 mg or 2.5 μg:1 mg.

[0019] In some embodiments of the present invention, the thyroid stimulating hormone receptor complex, wherein the mass ratio of the thyroid hormone receptor to the magnetic particles is (1-12) μg:1 mg; preferably (5-10) μg:1 mg, for example 5 μg:1 mg, 6 μg:1 mg, 7 μg:1 mg, 8 μg:1 mg, 9 μg:1 mg or 10 μg:1 mg; more preferably 6.25 μg:1 mg.

[0020] In some embodiments of the present invention, the thyroid stimulating hormone receptor complex, wherein the thyroid stimulating hormone receptor C-terminal monoclonal antibody is directly coupled to the magnetic particles or coupled via a cross-linking agent;

[0021] Preferably, the cross-linking agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide.

[0022] In some embodiments of the present invention, the thyrotropin receptor complex is used to detect the presence or level of thyrotropin receptor antibodies.

[0023] Another aspect of the present invention relates to a method for preparing the thyroid stimulating hormone receptor complex described in any one of the present invention, comprising the following steps:

[0024] (1) connecting the monoclonal antibody of the C-terminal end of the thyrotropin receptor to the magnetic particles to obtain a magnetic particle-thyrotropin receptor C-terminal monoclonal antibody conjugate;

[0025] (2) in the presence or absence of trimethylamine N-oxide, allowing the thyroid stimulating hormone receptor to bind to the thyroid stimulating hormone receptor C-terminal monoclonal antibody in the magnetic particle-thyroid stimulating hormone receptor C-terminal monoclonal antibody conjugate to obtain a thyroid stimulating hormone receptor complex;

[0026] Preferably, the method further comprises the following steps:

[0027] (3) Lyophilizing the product of step (2).

[0028] In some embodiments of the present invention, in the preparation method, wherein in step (1), the mass ratio of the magnetic particles to the thyrotropin receptor C-terminal monoclonal antibody is 1 mg: (1-5) μg or 1 mg: (2-4) μg; preferably 1 mg: (2-3) μg or 1 mg: 2.5 μg.

[0029] In some embodiments of the present invention, in the preparation method, wherein in step (2), the mass ratio of magnetic particles to thyroid hormone receptor is 1 mg:(1-12) μg; preferably 1 mg:(5-10) μg, for example 1 mg:5 μg, 1 mg:6 μg, 1 mg:7 μg, 1 mg:8 μg, 1 mg:9 μg or 1 mg:10 μg; more preferably 1 mg:6.25 μg.

[0030] In some embodiments of the present invention, in the preparation method, in step (2), the mass of trimethylamine N-oxide is 0.1‰-5‰ of the mass of the magnetic particle-thyrotropin hormone receptor C-terminal monoclonal antibody conjugate, preferably 0.5‰-2‰, 0.5‰-1.5‰ or 0.8‰-1.2‰, and more preferably 1‰.

[0031] In some embodiments of the present invention, in the preparation method, in step (3), the freeze-drying procedure is as follows:

[0032] Refrigeration control at -40℃ for 30 minutes → Cool the water trap at -45℃ for 5 minutes → Pre-vacuum at 0.0670mbar for 60 minutes → Based on -40℃, gradually increase the temperature to 0℃ for a drying process. The whole process takes 170 minutes → Perform analytical drying at 30℃ for 31 minutes → Slowly increase the pressure to normal.

[0033] Another aspect of the present invention relates to a kit comprising the thyroid stimulating hormone receptor complex described in any one of the present invention;

[0034] Preferably, the kit further comprises a TRAb analogue with a label, such as an acridinium ester-labeled hsTRAb;

[0035] Preferably, the kit further comprises a buffer solution, preferably a Tris-hydrochloric acid buffer solution, more preferably a 20 mM / L Tris-hydrochloric acid buffer solution.

[0036] Another aspect of the present invention relates to use of the thyrotropin receptor complex described in any one of the present invention in the preparation of a reagent for detecting thyrotropin receptor antibodies.

[0037] Another aspect of the present invention relates to use of any one of the thyroid stimulating hormone receptor complexes of the present invention in the preparation of a drug for diagnosis and prognosis monitoring of hyperthyroidism or TRAb monitoring in patients with thyroid diseases during pregnancy;

[0038] Preferably, the hyperthyroidism is Graves' disease.

[0039] In the present invention, the term "magnetic particles" is composed of a core-shell covering a magnetic layer and a polymer layer, and has a uniform particle size. The long chain end of the polyethylene glycol connected to the surface of the magnetic particles can be connected to an oligoamine group, the long chain can reduce nonspecific adsorption, and the oligoamine end can form a strong covalent bond between the antibody and the solid surface.

[0040] In some embodiments of the present invention, magnetic particles with Carboxyl groups on the surface (such as magnetic particles M-270Carboxyl), magnetic particles with Tosylactivated groups on the surface, and magnetic particles with NH2 groups on the surface can all achieve the coupling of the antibody.

[0041] In the present invention, the principle of coupling the antibody to the magnetic particles is mainly that the functional groups (carboxyl, amino or toluenesulfonyl etc.) on the surface of the magnetic particles react chemically with the amino, carboxyl and thiol groups on the antibody.

[0042] The commonly used particle size range of magnetic particles is 100nm-5000nm, wherein the preferred particle size range is 100nm-500nm, and more preferably 100nm-300nm.

[0043] Advantageous Effects of the Invention

[0044] The present invention achieves one or more of the following technical effects (1)-(4):

[0045] (1) The thyroid stimulating hormone receptor complex or kit of the present invention has high sensitivity and specificity for thyroid stimulating hormone receptor antibody detection.

[0046] (2) The thyroid stimulating hormone receptor complex or kit of the present invention has good stability and a long shelf life. After opening, the liquid reagent can be stably stored at 2-8°C for at least 3 months and at room temperature for at least 3 weeks. The shelf life of the kit after opening is significantly extended.

[0047] (3) The operation is simple and convenient, and can meet the needs of clinical testing.

[0048] (4) In the preparation method of the present invention, the addition of trimethylamine N-oxide can further improve the stability of the thyroid stimulating hormone receptor complex or the kit and extend the shelf life. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 : TRAb detection kit 1 linear fitting curve.

[0050] Figure 2 : TRAb detection kit 2 linear fitting curve.

[0051] Figure 3 : Comparison results between TRAb detection kit 1 and Roche kit.

[0052] Figure 4 : Comparison results between TRAb detection kit 2 and Roche kit. DETAILED DESCRIPTION

[0053] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0054] Some abbreviations involved in the present invention:

[0055] TSHR---Thyroid stimulating hormone receptor

[0056] TRAb-Thyrotropin receptor antibodies

[0057] GD-Graves Disease Toxic Diffuse Goiter

[0058] cAMP---cyclic adenosine monophosphate

[0059] Main reagents and equipment:

[0060] 1. Main reagents:

[0061] 10F8 (TSHR C-terminal mouse antibody, catalog number M3441) and hTSHR were purchased from Xiamen Inboma Biotechnology Co., Ltd.

[0062] Magnetic particles M270-Carboxyl were obtained from Life Technologies AS.

[0063] Acridinium ester comes from Suzhou Zhongsheng Chemical Co., Ltd.

[0064] Trimethylamine N-oxide was purchased from J&K Scientific.

[0065] 2. Main instruments:

[0066] The fully automatic chemiluminescence analyzer Caris200 was purchased from Xiamen Youmaike Medical Instrument Co., Ltd.

[0067] The magnetic rack was purchased from Wuxi Biomag Biotechnology Co., Ltd.

[0068] The rotary mixer was purchased from Kylin-Bell Lab Instruments.

[0069] Example 1: Preparation of magnetic microparticle-thyrotropin hormone receptor C-terminal monoclonal antibody conjugate

[0070] 1. Balance the magnetic particles M-270 Carboxyl and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) to room temperature, take the magnetic particles (colloidal state) with a concentration of 4 mg / ml, add 0.033 mg of EDC per mg of magnetic particles, and activate at room temperature for 30 minutes.

[0071] 2. Take the activated magnetic particles, collect the magnetism using a magnetic stand (using the magnetic layer Fe3O4 of the magnetic particles to combine with the magnet), remove the supernatant, and add MES buffer solution to resuspend.

[0072] 3. Add 2.5 μg of thyrotropin hormone receptor C-terminal monoclonal antibody 10F8 per mg of magnetic particles, mix well and react at room temperature for 3 hours.

[0073] 4. After collecting the magnet using a magnetic rack, remove the supernatant and add an equal volume of phosphate buffer containing 0.1% bovine serum albumin (for blocking) and react at room temperature for 3 hours.

[0074] 5. After collecting the magnet using a magnetic rack, remove the supernatant, add an equal volume of cleaning solution (phosphate buffer solution containing 0.05% Tween-20) and repeat the washing 4 times.

[0075] 6. The washed magnetic particles coated with the monoclonal antibody against the C-terminus of the thyrotropin receptor are stored in a tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution containing 0.75% (v / v) polyethylene glycol octylphenyl ether and stored at 2-8°C.

[0076] Thus, the magnetic particle-thyrotropin hormone receptor C-terminal monoclonal antibody 10F8 conjugate was prepared and used in the following Examples 2 and 3.

[0077] Example 2: Preparation of magnetic microparticle-thyrotropin receptor C-terminal monoclonal antibody-TSHR complex (1)

[0078] 1. Mix the magnetic particles-thyrotropin hormone receptor C-terminal monoclonal antibody 10F8 conjugate prepared in Example 1, add 12.5 μL of 0.5 mg / mL thyrotropin receptor per mg of magnetic particles, and rotate and mix at room temperature for 30 minutes.

[0079] 2. Remove the supernatant using a magnetic rack, add an equal volume of washing solution (phosphate buffer solution containing 0.05% Tween-20) and repeat washing 4 times to remove unreacted thyrotropin receptors.

[0080] 3. The washed magnetic microparticle product is stored in a tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution containing 0.75% (v / v) polyethylene glycol octylphenyl ether and stored at 2-8°C.

[0081] Thus, magnetic particle-thyrotropin receptor C-terminal monoclonal antibody-TSHR complex sample 1 was prepared.

[0082] Example 3: Preparation of magnetic microparticle-thyrotropin receptor C-terminal monoclonal antibody-TSHR complex (2)

[0083] 1. Add trimethylamine N-oxide to the magnetic particle-thyrotropin hormone receptor C-terminal monoclonal antibody 10F8 conjugate prepared in Example 1 at a mass ratio of 1:1000 and mix well.

[0084] 2. Add 12.5 μL of 0.5 mg / mL thyrotropin receptor per mg of magnetic particles and rotate to mix at room temperature for 30 minutes.

[0085] 3. Remove the supernatant using a magnetic rack, add an equal volume of washing solution (phosphate buffer solution containing 0.05% Tween-20) and repeat washing 4 times to remove unreacted thyrotropin receptors.

[0086] 4. The washed magnetic microparticle product is stored in a tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution containing 0.75% (v / v) polyethylene glycol octylphenyl ether and stored at 2-8°C.

[0087] Thus, magnetic particle-thyrotropin receptor C-terminal monoclonal antibody-TSHR complex sample 2 was prepared.

[0088] Example 4: Lyophilized magnetic microparticles-thyrotropin receptor C-terminal monoclonal antibody-TSHR complex sample

[0089] 1. The magnetic particle-thyrotropin receptor C-terminal monoclonal antibody-TSHR complex sample 1 (numbered M1) and the magnetic particle-thyrotropin receptor C-terminal monoclonal antibody-TSHR complex sample 2 (numbered M2) prepared in Example 2 and Example 3 were respectively packaged into vials at 1 mL / bottle, covered with rubber stoppers, and marked M1 and M2.

[0090] 2. Place the vials to be freeze-dried neatly on a stainless steel tray, and spread the vials in the tray evenly on the plate layer.

[0091] 3. Start the freeze drying equipment according to the standard operating procedures of the freeze dryer, and start the freezing program to run the freeze drying. The specific process is as follows: Refrigerate the vial containing the composite sample at -40℃ for 30 minutes → Refrigerate the water trap at -45℃ for 5 minutes → Pre-vacuum at 0.0670mbar for 60 minutes → Based on -40℃, gradually increase the temperature to 0℃ for a drying process, the whole process lasts 170 minutes → Perform analytical drying at 30℃ for 31 minutes → Slowly increase the pressure to normal.

[0092] 4. After the automatic freezing program is completed, press the stopper into the vial, take out the vial with the cap pressed, and store it at 2-8℃.

[0093] The freeze-dried magnetic microparticle-thyrotropin receptor C-terminal monoclonal antibody-TSHR complex sample 1 was named magnetic microparticle freeze-dried product M1, and the freeze-dried magnetic microparticle-thyrotropin receptor C-terminal monoclonal antibody-TSHR complex sample 2 was named magnetic microparticle freeze-dried product M2.

[0094] Example 5: Reconstitution stability of lyophilized product

[0095] 1. Add 1 mL of phosphate buffer solution containing 0.5% bovine serum albumin to the magnetic particle freeze-dried product M1 and magnetic particle freeze-dried product M2 respectively, let stand for 15 minutes, and then gently mix to obtain the reconstituted liquids (named as reagent 1-M1 and reagent 1-M2 respectively);

[0096] 2. Transfer the two reconstituted liquids to reagent bottles respectively, mix gently, and respectively construct TRAb detection kit 1 and TRAb detection kit 2 with acridinium ester-labeled hsTRAb (named reagent 2) and buffer solution (named reagent 3). Wherein:

[0097] hsTRAb is the abbreviation of human stimulatory thyrotropin receptor antibody, which is an analog of TRAb and competes with TRAb in the sample to be tested in the detection system. 50μg of hsTRAb is connected with 5μL acridinium ester (5mM / L), diluted 1000 times with 20mM phosphate buffer and separately dispensed into another reagent bottle as reagent 2. 50μL is used for each test;

[0098] Reagent 3 contains 20 mM / L Tris-HCl buffer solution.

[0099] Kit 1 and Kit 2 are respectively installed on a fully automatic chemiluminescence instrument.

[0100] 3. TRAb kit 1 and kit 2 installed on the fully automatic chemiluminometer respectively detect 5 samples containing the marker TRAb to be tested at different concentrations (sera collected from clinical TRAb detection projects or recombinant human TRAb can be used). The luminometer performs the following specific operations to detect samples: move the sample conveyor to the sampling point → load the reaction cup into the reaction plate → aspirate the sample and transfer the sample to the reaction cup → move the reaction cup forward one position, and add reagent 1-M1 or reagent 1-M2 and reagent 3 to the reaction cup → mix, incubate at 37°C and rinse the reaction mixture → add reagent 2 to the reaction cup → mix, incubate at 37°C and rinse the reaction mixture → add pre-excitation solution and excitation solution → detect relative luminescence value (RLU) → aspirate the contents of the reaction cup into the waste liquid, and put the reaction cup into the solid waste bucket to display the results. The first test result is used as a control, and the reagents are placed at 2-8°C and room temperature, respectively, and placed on the luminometer for detection once a week to evaluate the stability of the magnetic particle freeze-dried product M1 and the magnetic particle freeze-dried product M2 after reconstitution.

[0101] The results are shown in Tables 1 to 8 below.

[0102] Table 1: Stability results of lyophilized magnetic microparticles (M1) at 2-8°C after reconstitution (0-4 weeks)

[0103]

[0104] Table 2: Stability results of lyophilized magnetic microparticles (M1) at 2-8°C after reconstitution (5-8 weeks)

[0105]

[0106] Table 3: Stability results of lyophilized magnetic microparticles (M1) at 2-8°C after reconstitution (9-12 weeks)

[0107]

[0108] Table 4: Room temperature stability results of the reconstituted magnetic microparticle freeze-dried product (M1) (0-3 weeks)

[0109]

[0110] Table 5: Stability results of lyophilized magnetic microparticles (M2) at 2-8°C after reconstitution (0-4 weeks)

[0111]

[0112] Table 6: Stability results of lyophilized magnetic microparticles (M2) at 2-8°C after reconstitution (5-8 weeks)

[0113]

[0114] Table 7: Stability results of lyophilized magnetic microparticles (M2) at 2-8°C after reconstitution (9-13 weeks)

[0115]

[0116] Table 8: Room temperature stability results of the reconstituted magnetic microparticle freeze-dried product (M2) (0-3 weeks)

[0117]

[0118] Note: In Tables 1 to 8, S1-S3 are samples of the same batch at three different concentration levels. S1 in each table is the same sample, that is, according to the time set for the stability test, each time is to test the same batch of samples at three different concentration levels. The purpose of testing three different concentration levels is to fully demonstrate that the test results of the reconstituted reagent at different concentration levels are stable, considering the rigor of the experimental design.

[0119] The results in Tables 1 to 4 show that the lyophilized magnetic microparticles M1 prepared by linking magnetic microparticles-10F8 to TSHR without the addition of trimethylamine N-oxide can be stable for at least 12 weeks at 2-8°C and at least 3 weeks at room temperature after reconstitution;

[0120] The results in Tables 5 to 8 show that the lyophilized magnetic particle M2 prepared by connecting magnetic particles-10F8 to TSHR under the condition of adding trimethylamine N-oxide can be stable for at least 13 weeks at 2-8°C and at least 3 weeks at room temperature, indicating that the stability period of the preparation formed by connecting magnetic particles-10F8 to TSHR is significantly extended.

[0121] The results also showed that the magnetic microparticle freeze-dried product had a longer stability period and higher activity at 2-8°C after reconstitution compared to room temperature.

[0122] Example 6: Clinical application of lyophilized magnetic beads

[0123] 1. Install the TRAb detection kit 1 and kit 2 prepared by re-dissolving according to Example 5 on a fully automatic chemiluminescence instrument.

[0124] 2. Place 60 clinical samples (serum samples collected by the testing agency) with test results of the Roche test kit and 8 gradient diluted samples (formed by diluting TRAb with a concentration of 40 IU / L in a 2-fold gradient with a diluent not containing TRAb) on the luminometer, and use Kit 1 and Kit 2 to detect the above samples respectively. The luminometer performs the following specific operations to detect the samples: move the sample conveyor to the sampling point → load the reaction cup into the reaction disk → aspirate the sample and transfer the sample to the reaction cup → move the reaction cup forward one position, and add reagent 1-M1 or reagent 1-M2 and reagent 3 to the reaction cup → mix, incubate at 37°C and rinse the reaction mixture → add reagent 2 to the reaction cup → mix, incubate at 37°C and rinse the reaction mixture → add pre-excitation solution and excitation solution → detect the relative luminescence value (RLU) → aspirate the contents of the reaction cup into the waste liquid, and put the reaction cup into the solid waste bin.

[0125] 3. The relative luminescence values ​​(RLU) and corresponding concentrations of 8 gradient dilution samples were fitted using the Log-logit method. The fitting curve is as follows: Figure 1 and Figure 2 As shown, Figure 1 and Figure 2 The results showed that the linear fitting correlation coefficients r in the range of 0.6-40 IU / L were all greater than 0.9900, indicating that the reagent had good linearity in the range of 0.6-40 IU / L.

[0126] 4. The results of 60 clinical samples tested by Kit 1 and Kit 2 were compared with the test results of Roche's TSH receptor antibody assay kit (electrochemiluminescence method, National Medical Device Injection No. 20162404030). The results of the Roche kit were used as the horizontal axis, and the results of Kit 1 and Kit 2 were used as the vertical axis. A scatter plot was drawn, and the correlation coefficient was fitted and calculated. The results are as follows: Figure 3 and Figure 4 As shown, Figure 3 and Figure 4 The results showed that the correlation coefficient R between the TRAb detection kit prepared by magnetic microparticle-10F8 linked to TSHR and the Roche TRAb detection kit was 2 Greater than 0.95, the correlation is high.

[0127] According to the stability data of the kit of the present invention, the lyophilized magnetic microparticles of the present invention can be reconstituted for at least 12 weeks at 2-8°C and at least 3 weeks at room temperature, which is longer than the reconstituted stability of Roche's TSHR complex.

[0128] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art. According to all the teachings disclosed, various modifications and replacements can be made to those details, and these changes are all within the protection scope of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. A thyroid stimulating hormone receptor complex, comprising: TSH receptor, TSH receptor C-terminal monoclonal antibody and magnetic particles, in, The thyroid stimulating hormone receptor is connected to the magnetic particle via a monoclonal antibody at the C-terminus of the thyroid stimulating hormone receptor; The region where the thyrotropin receptor C-terminal monoclonal antibody binds to the thyrotropin receptor is located at the 261st to 764th amino acids from the N-terminal of the thyrotropin receptor; and The thyrotropin receptor C-terminal monoclonal antibody is the TSHR C-terminal mouse antibody 10F8 produced by Xiamen Inbomai Biotechnology Co., Ltd. Wherein, the thyroid stimulating hormone receptor is a human thyroid stimulating hormone receptor.

2. The thyroid stimulating hormone receptor complex according to claim 1, wherein The average particle size of the magnetic particles is 100nm-5000nm.

3. The thyroid stimulating hormone receptor complex according to claim 1, wherein The average particle size of the magnetic particles is 100-500 nm.

4. The thyroid stimulating hormone receptor complex according to claim 1, wherein The average particle size of the magnetic particles is 100nm-300nm.

5. The thyroid stimulating hormone receptor complex according to claim 1, wherein The mass ratio of the thyrotropin receptor C-terminal monoclonal antibody to the magnetic particles is (1-5) μg:1 mg.

6. The thyroid stimulating hormone receptor complex according to claim 1, wherein The mass ratio of the thyrotropin receptor C-terminal monoclonal antibody to the magnetic particles is (2-4) μg:1 mg.

7. The thyroid stimulating hormone receptor complex according to claim 1, wherein The mass ratio of the thyrotropin receptor C-terminal monoclonal antibody to the magnetic particles is 2.5 μg:1 mg.

8. The thyroid stimulating hormone receptor complex according to claim 1, wherein The mass ratio of thyroid stimulating hormone receptor to magnetic particles is (1-12) μg:1 mg.

9. The thyroid stimulating hormone receptor complex according to claim 1, wherein The mass ratio of thyroid stimulating hormone receptor to magnetic particles is (5-10) μg:1 mg.

10. The thyroid stimulating hormone receptor complex according to claim 1, wherein The mass ratio of thyroid stimulating hormone receptor to magnetic particles is 6.25 μg:1 mg.

11. The thyroid stimulating hormone receptor complex according to claim 1, wherein The monoclonal antibody against the C-terminus of the thyrotropin receptor is coupled to the magnetic particles directly or through a cross-linking agent.

12. The thyroid stimulating hormone receptor complex according to claim 11, wherein The cross-linking agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide.

13. The thyrotropin receptor complex according to claim 1, which is used to detect the presence or level of thyrotropin receptor antibodies.

14. A method for preparing the thyroid stimulating hormone receptor complex according to any one of claims 1 to 13, comprising the following steps: (1) connecting the monoclonal antibody of the C-terminal end of the thyrotropin receptor to the magnetic particles to obtain a magnetic particle-thyrotropin receptor C-terminal monoclonal antibody conjugate; (2) In the presence or absence of trimethylamine N-oxide, the thyroid stimulating hormone receptor is combined with the thyroid stimulating hormone receptor C-terminal monoclonal antibody in the magnetic particle-thyroid stimulating hormone receptor C-terminal monoclonal antibody conjugate to obtain a thyroid stimulating hormone receptor complex.

15. The preparation method according to claim 14, further comprising the steps of: (3) Lyophilizing the product of step (2).

16. The preparation method according to any one of claims 14 to 15, wherein: In step (1), the mass ratio of the magnetic particles to the thyrotropin receptor C-terminal monoclonal antibody is 1 mg: (1-5) μg.

17. The preparation method according to any one of claims 14 to 15, wherein: In step (1), the mass ratio of the magnetic particles to the monoclonal antibody against the C-terminus of the thyrotropin receptor is 1 mg: (2-4) μg.

18. The preparation method according to any one of claims 14 to 15, wherein: In step (1), the mass ratio of the magnetic particles to the monoclonal antibody against the C-terminus of the thyrotropin receptor is 1 mg:2.5 μg.

19. The preparation method according to any one of claims 14 to 15, wherein: In step (2), the mass ratio of magnetic particles to thyroid hormone receptor is 1 mg: (1-12) μg.

20. The preparation method according to any one of claims 14 to 15, wherein: In step (2), the mass ratio of magnetic particles to thyroid hormone receptor is 1 mg: (5-10) μg.

21. The preparation method according to any one of claims 14 to 15, wherein: In step (2), the mass ratio of magnetic particles to thyroid hormone receptor is 1 mg:6.25 μg.

22. The preparation method according to any one of claims 14 to 15, wherein: In step (2), the mass of trimethylamine N-oxide is 0.1‰-5‰ of the mass of the magnetic particle-thyrotropin hormone receptor C-terminal monoclonal antibody conjugate.

23. The preparation method according to any one of claims 14 to 15, wherein: In step (2), the mass of trimethylamine N-oxide is 0.5‰-2‰ of the mass of the magnetic particle-thyrotropin hormone receptor C-terminal monoclonal antibody conjugate.

24. The preparation method according to any one of claims 14 to 15, wherein: In step (2), the mass of trimethylamine N-oxide is 1‰ of the mass of the magnetic particle-thyrotropin hormone receptor C-terminal monoclonal antibody conjugate.

25. The preparation method according to claim 15, wherein: In step (3), the freeze-drying procedure is as follows: Refrigeration control at -40℃ for 30 minutes → Cool the water trap at -45℃ for 5 minutes → Pre-vacuum at 0.0670mbar for 60 minutes → Based on -40℃, gradually increase the temperature to 0℃ for a drying process. The whole process takes 170 minutes → Perform analytical drying at 30℃ for 31 minutes → Slowly increase the pressure to normal.

26. A kit comprising the thyroid stimulating hormone receptor complex according to any one of claims 1 to 13.

27. The kit according to claim 26, wherein The kit also contains a TRAb analog with a label.

28. The kit according to claim 27, wherein The labeled TRAb analog is hsTRAb labeled with an acridinium ester.

29. The kit according to any one of claims 26 to 28, wherein The kit also includes a buffer solution.

30. The kit according to claim 29, wherein The buffer solution is tris(hydroxymethylaminomethane)-hydrochloric acid buffer solution.

31. The kit according to claim 29, wherein The buffer solution is 20 mM / L Tris-hydrochloric acid buffer solution.

32. Use of the thyroid stimulating hormone receptor complex according to any one of claims 1 to 13 in the preparation of a reagent for detecting thyroid stimulating hormone receptor antibodies.

33. Use of the thyroid stimulating hormone receptor complex according to any one of claims 1 to 13 in the preparation of a drug for diagnosis and prognosis monitoring of hyperthyroidism or TRAb monitoring in patients with thyroid diseases during pregnancy.

34. The use according to claim 33, wherein The hyperthyroidism is Graves' disease.

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