WO2023103827A1 - Réactif antigénique de récepteur d'hormones de stimulation de la thyroïde (tshr) et kit de test quantitatif d'anticorps de tshr - Google Patents

Réactif antigénique de récepteur d'hormones de stimulation de la thyroïde (tshr) et kit de test quantitatif d'anticorps de tshr Download PDF

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WO2023103827A1
WO2023103827A1 PCT/CN2022/134765 CN2022134765W WO2023103827A1 WO 2023103827 A1 WO2023103827 A1 WO 2023103827A1 CN 2022134765 W CN2022134765 W CN 2022134765W WO 2023103827 A1 WO2023103827 A1 WO 2023103827A1
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tshr
monoclonal antibody
antibody
kit
hormone receptor
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PCT/CN2022/134765
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English (en)
Chinese (zh)
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徐海娈
靳增明
赵艳芳
韩阳瑞
田晓平
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郑州安图生物工程股份有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)

Definitions

  • the invention relates to the technical field of in vitro diagnosis, in particular to a thyroid-stimulating hormone receptor antigen reagent and a thyroid-stimulating hormone receptor antibody quantitative detection kit.
  • Thyroid-stimulating hormone receptor is a macromolecular glycoprotein located on the membrane of thyroid follicular epithelial cells and belongs to the G protein-coupled receptor family. Under physiological conditions, thyroid-stimulating hormone (TSH) binds to its receptor TSHR, and is activated by G protein to generate a cascade reaction to achieve biological effects. Under the influence of heredity and environment, the structure of TSHR changes, resulting in antigenicity, causing autoimmune reactions, and producing a large number of thyroid-stimulating hormone receptor antibodies (TRAb).
  • TRAb is a group of heterogeneous antibodies, which can be divided into thyroid stimulating antibody (TSAb), thyroid stimulating blocking antibody (TSBAb) and neutral thyroid hormone receptor antibody according to their different functions.
  • TSAb can compete with TSH to bind to TSHR, and stimulate thyroid follicular cells to secrete and synthesize thyroxine by producing cAMP, which in turn can cause toxic diffuse goiter (Graves' disease).
  • the combination of TSBAb and TSHR can directly interfere with the combination of TSH and its receptor, thereby weakening the biological effects of TSH.
  • TSBAb can also inhibit the activation of adenylyl cyclase to cause hypothyroidism.
  • the binding of neutral antibodies to TSHR neither stimulates nor inhibits thyroid function.
  • TSHR consists of a single polypeptide chain containing 764 amino acids, with a molecular weight of about 84 kDa, and the extracellular domain is about 414-418 amino acids, including six potential N-linked glycosylation sites, while the remaining sequences form the transmembrane domain and Intracellular carboxyl terminus. Correct folding, glycosylation and the presence of the transmembrane region and extracellular domain of TSHR are necessary for high-affinity ligand binding. At the same time, the stability of the transmembrane region and intracellular structure plays an important role in maintaining the stability of TSHR.
  • TRAb is a specific autoantibody to TSHR in the serum of Graves patients.
  • the determination of serum TRAb can be used as a good indicator for the diagnosis, treatment and prognosis of Graves' disease, and it is also of great value in the treatment of hyperthyroidism during pregnancy and the risk assessment of neonatal thyroid dysfunction.
  • the detection methods of TRAb mainly include bioanalysis and immunoassay.
  • Bioanalysis can distinguish stimulatory antibodies from blocking antibodies, but due to time-consuming, cumbersome operation and high cost, it cannot be applied to the detection of clinical laboratories.
  • Common immunoassay methods include radioimmunoassay, enzyme-linked immunoassay, and chemiluminescence. At present, the most widely used clinical method is fully automatic magnetic particle chemiluminescence.
  • the Chinese patent application number CN201910875687.6 discloses a detection kit for the concentration of stimulatory thyroid-stimulating hormone receptor antibody, including: coated with stimulatory hormone Magnetic particles of TSH receptor antibody, containing marker-labeled TSH receptor, TSH receptor antibody calibrator, whose solid phase is coated with TSH receptor antibody, The problem it solves is to avoid the influence of inhibitory thyroid-stimulating hormone receptor antibody on the measurement system.
  • the Chinese patent with the application number CN201611018792.0 discloses a TSH receptor antibody chemiluminescence immunoassay kit including: TSH receptor coated magnetic particles, mouse anti-human IgG antibody coated acridinium ester, Thyroid-stimulating hormone receptor antibody calibrator, whose solid phase is coated with thyroid-stimulating hormone receptor, solves the problem of overcoming the high cost, low sensitivity, narrow linear range and low reproducibility of existing assay methods , can not be quantified, the shortcomings of complex operation.
  • the Chinese document with the application number CN201710115161.9 discloses a detection method for thyroid stimulating hormone receptor antibody.
  • the kit components mainly include reagents such as FITC-labeled goat antihuman globulin and biological thin slices.
  • the biological thin slices are obtained from large Made of rat thyroid cells, coated in the reaction area of the slide; the determination method is: add the serum to be tested in the reaction area, after incubation, rinsing, and drying, add FITC-labeled goat antihuman globulin, and incubate , after rinsing and drying, under a fluorescent microscope, a FITC-labeled goat anti-human globulin fluorescent complex is formed on the thyroid cell membrane, producing yellow-green fluorescence; the problem it solves is to overcome the thyroid-stimulating hormone receptor antibody detection method. missing detection problem.
  • the Chinese patent with the application number CN201810506744.9 discloses an enzyme-linked immunosorbent assay kit and detection method capable of simultaneously detecting thyroid stimulating hormone receptor typing antibodies.
  • the kit mainly includes: coated antigen, pre-coated ELISA plate , rinsing solution, thyroid-stimulating hormone receptor antibody standard, and enzyme-labeled solution.
  • coated antigen pre-coated ELISA plate
  • rinsing solution thyroid-stimulating hormone receptor antibody standard
  • enzyme-labeled solution enzyme-labeled solution.
  • Biotin-avidin system When a certain amount of biotin in the specimen will interfere with the test results (Reference 3: Zhang Guofeng, Guo Rui, Guan Haixia. Pay attention to information other than the test sheet——By biotin interference test An example of misdiagnosed as Graves' disease hyperthyroidism discusses the elements of diagnosing thyroid diseases[J].
  • the technical problem to be solved by the present invention is to provide a human thyroid-stimulating hormone receptor antigen reagent and a quantitative detection kit for thyroid-stimulating hormone receptor antibody.
  • the kit provided by the present invention has good stability and is not affected by biological interferences such as fluorescein and fluorescein.
  • the invention provides a human thyroid-stimulating hormone receptor antibody quantitative detection kit, comprising: a solid-phase carrier coated with anti-TSHR monoclonal antibody A;
  • Antigen reagent is the immune complex of anti-TSHR monoclonal antibody B and TSHR;
  • the anti-TSHR monoclonal antibody A does not produce competitive binding with the anti-TSHR monoclonal antibody B.
  • the kit provided by the present invention is used to detect the concentration of thyroid-stimulating hormone receptor antibody in serum or plasma, which adopts the reaction principle of competition method for detection.
  • TRAb in the serum competes for the binding of anti-TSHR monoclonal antibody B-TSHR immune complex, and then reacts with the solid-phase carrier coated with anti-TSHR monoclonal antibody A, and forms a solid-phase antibody-antigen-labeled antibody complex through immune reaction , the complex catalyzes the luminescent substrate solution to emit light, and the luminous intensity is inversely proportional to the concentration of TRAb.
  • the concentration of TRAb in the plasma or serum to be tested can be obtained.
  • the kit provided by the present invention includes a solid phase carrier coated with anti-TSHR monoclonal antibody A, wherein the recognition site of anti-TSHR monoclonal antibody A is located at the transmembrane region of TSHR and the intracellular carboxyl terminal, specifically the amino acid sequence of TSHR 414 to 764 bits.
  • the anti-TSHR monoclonal antibody A includes but not limited to murine monoclonal antibody, rabbit monoclonal antibody, goat monoclonal antibody, human monoclonal antibody and chimeric monoclonal antibody, preferably murine monoclonal antibody Antibody.
  • the solid phase carrier is selected from magnetic microspheres modified on the surface of amino, carboxyl, aldehyde, phenylhydrazine or sulfonic acid groups, further, the inner core of the magnetic microspheres is ferric iron tetroxide or Ferric oxide.
  • the ratio of the anti-TSHR monoclonal antibody A to the magnetic microspheres is 5-25 ⁇ g/mg magnetic microspheres, preferably 15 ⁇ g/mg magnetic microspheres.
  • the carboxyl magnetic microspheres coated with anti-TSHR monoclonal antibody A can be prepared according to the following method:
  • the activation buffer is 0.1M MES (2-(N-morpholino)ethanesulfonic acid) buffer.
  • MES 2-(N-morpholino)ethanesulfonic acid
  • the 0.01M PBS buffer solution of the protein was used for blocking. After the blocking was completed, the blocking solution was added to preserve the carboxyl magnetic microspheres coated with anti-TSHR monoclonal antibody A.
  • the final concentration of the magnetic microspheres was 1mg/ml. The suspension was stored at 2-8°C for use.
  • the kit provided by the invention also includes an antigen reagent, which is an immune complex of anti-TSHR monoclonal antibody B and TSHR.
  • an antigen reagent which is an immune complex of anti-TSHR monoclonal antibody B and TSHR.
  • the recognition site of the anti-TSHR monoclonal antibody B is located in the transmembrane region of TSHR and the carboxyl terminal of the cell, specifically the 661-764 position of the TSHR amino acid sequence, further, the recognition site of the anti-TSHR monoclonal antibody B is located in the TSHR amino acid 665-698 bits of the sequence.
  • the anti-TSHR monoclonal antibody B includes but not limited to murine monoclonal antibody, rabbit monoclonal antibody, goat monoclonal antibody, human monoclonal antibody and chimeric monoclonal antibody, preferably murine monoclonal antibody Antibody.
  • the TSHR includes, but is not limited to, natural human TSHR, natural porcine TSHR, recombinantly expressed human TSHR or recombinantly expressed porcine TSHR.
  • the antigenic reagent is obtained by reacting anti-TSHR monoclonal antibody B and TSHR in a lyophilized solution, or by reacting an anti-TSHR monoclonal antibody B and TSHR in a lyophilized solution and freeze-drying, or by TSHR monoclonal antibody B and TSHR are obtained after reacting in lyophilized liquid, lyophilized and reconstituted.
  • the antigen reagent is prepared according to the following method:
  • the lyophilized powder is reconstituted in a reconstitution solution to obtain an antigen reagent.
  • the anti-TSHR monoclonal antibody B is reacted with TSHR in the antigen freeze-dried liquid, the temperature of the reaction is preferably 20-25° C., and the time is 1-5 h.
  • the anti-TSHR monoclonal antibody B is compounded with TSHR, and the stability of TSHR is greatly improved after freeze-drying and reconstitution.
  • Experimental results show that the reconstituted antigen containing anti-TSHR monoclonal antibody B and TSHR immune complex can be stable for 28 days at 2-8°C.
  • the concentration of anti-TSHR monoclonal antibody B is 1-10 ug/mL, and the dilution ratio of TSHR is 1:10-1:100.
  • the antigen lyophilized solution comprises: buffer, 1wt%-10wt% mannitol, 1wt%-10wt% sucrose, 0.1wt%-1wt% serum albumin and 0.1wt%-1wt% % polyethylene glycol.
  • the buffer may be Bis-Tris buffer
  • the serum albumin may be bovine serum albumin
  • the polyethylene glycol may be polyethylene glycol 5000.
  • the obtained product is freeze-dried to obtain a freeze-dried powder.
  • the present invention has no special limitation on the freeze-drying, and the freeze-drying process well-known to those skilled in the art will suffice.
  • the reconstitution solution includes: buffer and 1wt%-5wt% glycerol.
  • the buffer can be Bis-Tris.
  • the antigen reagent is prepared according to the following method:
  • anti-TSHR monoclonal antibody B and TSHR are mixed and reacted in the antigen freeze-dried liquid to form anti-TSHR monoclonal antibody B and TSHR immune complex.
  • Anti-TSHR monoclonal antibody B and TSHR immune complex can be stable for 128 hours in 20-25 °C antigen lyophilized solution, and can be stable for 168 hours in 2-8 °C antigen lyophilized solution.
  • the kit provided by the present invention also contains human stimulating thyrotropin receptor antibody labeled with a marker, the marker is selected from ruthenium compound, acridine compound, peroxidase or alkaline phosphatase, and the labeling method can be Direct marking or indirect marking.
  • the human-stimulating thyrotropin receptor antibody containing the marker is preserved in a solution comprising serum albumin, preservative and buffer until use.
  • the buffer is Bis-Tris buffer
  • the serum albumin is bovine serum albumin
  • the content is 3wt-8wt%, preferably 5wt%
  • the preservative is PC300
  • the content is 0.1wt-5wt% , preferably 0.5wt-4wt%.
  • the kit provided by the present invention also includes a sample diluent and/or a serial calibrator of the stimulating thyroid-stimulating hormone receptor antibody.
  • the sample diluent includes serum albumin, a preservative, and a buffer.
  • the buffer is Bis-Tris buffer
  • the serum albumin is bovine serum albumin
  • the content is 1wt-3wt%
  • the preservative is PC300
  • the content is 0.1wt-0.3wt%.
  • the stimulatory thyroid stimulating hormone receptor antibody serial calibrator is a serial calibrator with a concentration of 0-100 IU/L. In one embodiment, the concentrations of the calibrator are 0IU/L, 2IU/L, 5IU/L, 10IU/L, 20IU/L, 50IU/L, respectively.
  • the kit provided by the present invention uses a competition method to achieve the concentration of thyroid stimulating hormone receptor antibody in serum or plasma.
  • the TSHR monoclonal antibody B-TSHR immune complex reacts with the solid-phase carrier coated with anti-TSHR monoclonal antibody A, and forms a solid-phase antibody-antigen-labeled antibody complex through an immune reaction, which catalyzes the luminescent substrate
  • the substance liquid emits light, and the luminous intensity is inversely proportional to the concentration of TRAb; a series of stimulatory thyroid-stimulating hormone receptor antibody series calibrator is used to establish a calibration curve, and then the luminous intensity of the test product is detected, according to the luminous intensity of the test product and the calibration Curve to obtain the concentration of TRAb in plasma or serum to be tested.
  • the method for using the kit provided by the invention is as follows:
  • test product or calibrator Mix the test product or calibrator, antigen reagent and sample diluent, and incubate at 37°C for 17 minutes;
  • the present invention uses a set of anti-TSHR monoclonal antibody A and anti-TSHR monoclonal antibody B to construct a quantitative detection kit, wherein the anti-TSHR monoclonal antibody B forms an immune complex with TSHR, which can improve the stability of TSHR, so that the obtained The kit has good stability.
  • the experimental results show that, in the kit provided by the present invention, the anti-TSHR monoclonal antibody B and the TSHR immune complex are stable in the freeze-dried liquid at 20-25°C for 128 hours, and stable in the freeze-dried solution at 2-8°C. It is stable in dry solution for 168 hours, and the antigen reagent can be stable for 28 days at 2-8°C after reconstitution.
  • the human thyroid-stimulating hormone receptor antibody quantitative detection kit comprises: a solid-phase carrier coated with anti-TSHR monoclonal antibody A; an antigen reagent, which is an immune complex of anti-TSHR monoclonal antibody B and TSHR substance; contains human stimulating thyroid-stimulating hormone receptor antibody labeled with a marker.
  • the kit provided by the invention adopts the reaction principle of competition method to detect the concentration of thyroid-stimulating hormone receptor antibody in serum or plasma, and adopts solid-phase coated avidin and biotin-labeled anti-TSHR antibody (biotin-avidin system) or solid-phase coated anti-fluorescein isothiocyanate (FITC) antibody, FITC-labeled anti-TSHR antibody (FITC-anti-FITC system) compared to commercial kits, the present invention not only reduces the labeling steps, but also avoids biotin
  • Fig. 1 is the calibration curve that the embodiment of the present invention 1 provides
  • Fig. 2 is the stability evaluation result of the antigen reagent provided by the embodiment of the present invention and the comparative example in the lyophilized solution at 20-25°C;
  • Fig. 3 is the stability evaluation result of the antigen reagent provided by the embodiment of the present invention and the comparative example in the lyophilized solution at 2-8°C;
  • Figure 4 is the stability evaluation results at 2-8°C after reconstitution of the antigen reagents provided by the examples and comparative examples of the present invention.
  • Fig. 5 is a correlation curve diagram of the detection results of the kit provided by the embodiment of the present invention and other kits.
  • thyroid-stimulating hormone receptor antigen reagent and the thyroid-stimulating hormone receptor antibody quantitative detection kit provided by the present invention will be further described below in conjunction with the examples.
  • the reagents and instruments used in the present invention are commercially available products, and the test methods used in the present invention are conventional methods in the art.
  • Reagent components not mentioned in detail in the kit of the present invention (such as some necessary buffers such as cleaning solution, etc.), the outer packaging of the kit and the independent packaging container of each reagent component, etc. can be carried out according to the conventional operations in the field , in line with relevant industry regulations. Operational steps not mentioned in detail in the method of the kit of the present invention can also be performed with reference to conventional operations in the field.
  • anti-TSHR monoclonal antibody B was prepared according to the following method:
  • Antibody purification the antibody was purified by SPA affinity chromatography.
  • a human thyroid-stimulating hormone receptor antibody quantitative detection kit comprising: magnetic particles coated with anti-human TSHR mouse monoclonal antibody A, antigen reagent (anti-human TSHR mouse monoclonal antibody B-recombinant human TSHR complex), Sample diluent, horseradish peroxidase-labeled human stimulating thyrotropin receptor antibody and stimulating thyrotropin receptor antibody serial calibrator, the preparation process is as follows:
  • the carboxyl magnetic microspheres were activated under acidic conditions with EDC and NHS, the activation buffer was 0.1M MES (2-(N-morpholino)ethanesulfonic acid) buffer, and the activation time was 30min. After the activation was completed, Under the action of a magnetic field, the carboxyl magnetic microspheres are separated from the liquid, the supernatant is discarded, and an appropriate amount of anti-human TSHR mouse monoclonal antibody A is added, and the carboxyl magnetic microspheres coated with anti-human TSHR monoclonal antibody A are used The 0.01M PBS buffer containing 1% bovine serum albumin was used for blocking.
  • the blocking solution was added to preserve the carboxyl magnetic microspheres coated with anti-TSHR monoclonal antibody A.
  • the final concentration of the magnetic microspheres was 1mg /ml, and store the suspension at 2-8°C for use.
  • Bis-Tris buffer contains 3% bovine serum albumin and 0.2% PC300, then mix well, store at 2-8°C until use;
  • the preparation method of the human stimulating thyrotropin receptor antibody labeled with horseradish peroxidase is as follows: the human stimulating thyrotropin receptor antibody labeled with horseradish peroxidase is added to a mixture containing 5% bovine serum Albumin and 3% PC300 Bis-Tris buffer, then mix well; the dilution ratio of horseradish peroxidase-labeled human stimulating thyrotropin receptor antibody is 1/1000 ⁇ 1/5000; 2 ⁇ 8 Store at °C for later use;
  • the stimulatory thyrotropin receptor antibody serial calibrator is a serial calibrator with a concentration of 0-100 IU/L, and the preparation method of the calibrator is: add 5% bovine serum albumin to the Bis-Tris buffer , and mix well to obtain the calibration product dilution, and then use the calibration product diluent to dilute the stimulating thyrotropin receptor antibody to six concentration points S0 ⁇ S5, the concentrations are 0IU/L, 2IU/L, 5IU/L L, 10IU/L, 20IU/L, 50IU/L.
  • test sample (calibrator or sample to be tested) and placing it correctly, click the start button to perform the calibration procedure or sample detection procedure, and the instrument will perform the following operations;
  • Fig. 1 is the calibration curve provided by the embodiment of the present invention.
  • Example 1 Compared with Example 1, the difference is that the antigen reagent is not complexed with anti-human TSHR mouse monoclonal antibody B, and the recombinant human TSHR is directly freeze-dried and reconstituted.
  • Stability evaluation method using the kits provided in Comparative Example 1 and Example 1 to detect in parallel a series of stimulating thyrotropin receptor antibody concentrations 0IU/L, 2IU/L, 5IU/ Calibrator of L, 10IU/L, 20IU/L, 50IU/L is measured on the automatic chemiluminescence instrument A2000Plus system according to the detection steps, and the luminescence value of each group is obtained, and the signal value drop ⁇ 10% is regarded as stability Better, see Table 1, Figure 2, Figure 3 and Figure 4 for the results, Table 1 is the stability evaluation results of the kits provided in the examples of the present invention and comparative examples, and Figure 2 is the antigens provided in the examples of the present invention and comparative examples The stability evaluation result of the reagent at 20-25°C in the lyophilized solution, wherein curve a is the stability evaluation result of the antigen reagent provided in Example 1, and curve b is the stability evaluation result of the antigen reagent provided in Comparative Example 1, Fig.
  • FIG. 3 is the stability evaluation result of the antigen reagent provided in the embodiment of the present invention and comparative example in lyophilized liquid at 2 ⁇ 8 °C, wherein curve a is the stability evaluation result of the antigen reagent provided in embodiment 1, and curve b is The stability evaluation results of the antigen reagents provided in Comparative Example 1,
  • Figure 4 is the stability evaluation results of the antigen reagents provided in the Examples of the present invention and Comparative Examples at 2-8°C after reconstitution, where curve a is the results of Example 1
  • the stability evaluation result of the antigen reagent provided, curve b is the stability evaluation result of the antigen reagent provided in Comparative Example 1.
  • the anti-TSHR mouse monoclonal antibody B-human TSHR immune complex is stable in the lyophilized solution at 20-25°C for 128 hours, and is stable at 2 It is stable in the freeze-dried solution at ⁇ 8°C for 168 hours, and the stability of the antigen reagent after reconstitution meets the requirement of 28 days at 2-8°C.
  • Quantitative detection kit for human thyroid stimulating hormone receptor antibody constructed according to Example 1 and kit C: quantitative detection kit for thyroid stimulating hormone receptor antibody (electrochemiluminescence method), kit D: human thyroid stimulating hormone receptor antibody
  • the detection kit enzyme-catalyzed chemiluminescence method
  • kit C The detection principle of kit C is: competition method, magnetic particles coated with streptavidin, human immunocomplexes labeled with soluble porcine TSHR and biotinylated anti-porcine TSHR C-terminal mouse monoclonal antibody and ruthenium complex Monoclonal autoantibody M22 was tested;
  • kit D The detection principle of kit D is: competition method, using FITC antibody to coat magnetic particles, FITC-labeled anti-human TSHR mouse monoclonal antibody-human TSHR immune complex to prepare antigen reagent, horseradish peroxidase-labeled TRAb and The TRAb in the sample competes with the human TSHR immune complex in the antigen reagent;
  • Detection method adopt the kit of the present invention, kit C and kit D to assess 193 cases of gradient serum samples in parallel, wherein 103 negative samples, the concentration range is 0.8 ⁇ 40IU/L, and the detection results are subjected to clinical correlation analysis, and the correlation is as follows: As shown in Fig. 5, Fig. 5 is a correlation curve diagram between the test kit provided by the embodiment of the present invention and other kit detection results, wherein Fig. 5 (A) is a correlation curve diagram with kit C, and Fig. 5 (B) is the correlation curve with Kit D.
  • kit of the present invention As can be seen from Figure 5, the kit of the present invention, kit C and kit D are used to measure 193 clinical serum samples at the same time, and the correlation between the kit of the present invention and the results of the two assays is better, and the correlation coefficient R is 0.958- between 0.997.
  • Embodiment 4 repeatability assessment
  • Embodiment 5 interference assessment :
  • the instruction of the TRAb detection kit of kit C states that when the biotin concentration in the sample is >41nmol/L or 10ng/mL, the measurement result will be high; the instruction of the TRAb detection kit of kit D states that when the biotin concentration in the sample When the concentration of biotin exceeds 0.5ug/mL, it can cause the detection result to change by more than 10%; and when the kit of the present invention is used to detect samples added with 10ng/mL biotin or 0.5ug/mL fluorescein sodium, the detection result will not be affected (The results vary within ⁇ 10%).
  • the above results show that the present invention can avoid the related interference introduced by the biotin-avidin system or the FITC-anti-FITC system, and improve the anti-interference performance of the kit.
  • a quantitative detection kit for human thyroid-stimulating hormone receptor antibody comprising magnetic particles coated with anti-pig TSHR mouse monoclonal antibody A, antigen reagent (anti-pig TSHR mouse monoclonal antibody B-recombinant pig TSHR), alkaline phosphoric acid Enzyme-labeled human stimulating thyrotropin receptor antibody and stimulating thyrotropin receptor antibody serial calibrator, the preparation process is as follows:
  • Bis-Tris buffer contains 1-3% bovine serum albumin and 0.1-0.3% PC300, then mix well, and store at 2-8°C for later use;
  • alkaline phosphatase-labeled human stimulating thyroid-stimulating hormone receptor antibody is as follows: adding alkaline enzyme-labeled human stimulating thyroid-stimulating hormone receptor antibody to a mixture containing 5% bovine serum albumin and 0.1- 5% PC300 Bis-Tris buffer, then mix well, store at 2-8°C until use;
  • the stimulatory thyrotropin receptor antibody serial calibrator is a serial calibrator with a concentration of 0-100 IU/L, and the preparation method of the calibrator is: add 5% bovine serum albumin to the Bis-Tris buffer , and mix well to get the calibration product dilution, and then use the calibration product dilution to dilute the stimulating thyrotropin receptor antibody into six concentration points S0-S5, the concentrations are 0IU/L, 2IU/L, 5IU/L L, 10IU/L, 20IU/L, 50IU/L;
  • Stability evaluation method use the kit provided in Example 6 to detect in parallel the concentration of stimulating thyrotropin receptor antibody obtained by diluting a series of calibrator diluents: 0IU/L, 2IU/L, 5IU/L, 10IU/L , 20IU/L, and 50IU/L calibration products were measured on the automatic chemiluminescence instrument A2000Plus system according to the detection steps, and the luminescence values of each group were obtained. The stability is considered to be good if the decrease in signal value is less than or equal to 10%. For the results, see Table 4. Table 4 shows the stability evaluation results of the kit provided in Example 6 of the present invention.
  • Example 6 As can be seen from Table 4, the kit provided in Example 6 is relatively stable.
  • the kit of the present invention detects samples added with 10ng/mL of biotin or 0.5ug/mL of fluorescein sodium, the detection results are not affected (the results vary within ⁇ 10%), the above results show that the present invention The related interference introduced by the biotin-avidin system or FITC-anti-FITC system can be avoided, and the anti-interference performance of the kit is improved.
  • a human thyroid-stimulating hormone receptor antibody quantitative detection kit including magnetic particles coated with anti-pig TSHR mouse monoclonal antibody A, antigen reagent (anti-pig TSHR mouse monoclonal antibody B-natural pig TSHR), sample diluent , acridinium ester-labeled human stimulating thyroid-stimulating hormone receptor antibody and stimulating thyroid-stimulating hormone receptor antibody serial calibrator, the preparation process of which is as follows:
  • Bis-Tris buffer contains 1-3% bovine serum albumin and 0.1-0.3% PC300, then mix well, and store at 2-8°C for later use;
  • the preparation method of marker-labeled human stimulating thyroid stimulating hormone receptor antibody is as follows: adding acridinium ester-labeled human stimulating thyroid stimulating hormone receptor antibody to a mixture containing 5% bovine serum albumin and 0.1-5% Bis-Tris buffer solution of PC300, then mix well, store at 2-8°C until use;
  • the stimulatory thyrotropin receptor antibody serial calibrator is a serial calibrator with a concentration of 0-100 IU/L, and the preparation method of the calibrator is: add 5% bovine serum albumin to the Bis-Tris buffer , and mix well to get the calibration product dilution, and then use the calibration product dilution to dilute the stimulating thyrotropin receptor antibody into six concentration points S0-S5, the concentrations are 0IU/L, 2IU/L, 5IU/L L, 10IU/L, 20IU/L, 50IU/L.
  • Stability evaluation method use the kit prepared in Example 7 to detect in parallel the concentrations of 0IU/L, 2IU/L, 5IU/L, and 10IU/L of the stimulating thyrotropin receptor antibody obtained by diluting a series of calibrator diluents , 20IU/L, and 50IU/L calibration products were measured on the automatic chemiluminescence instrument A2000Plus system according to the detection steps, and the luminescence values of each group were obtained. The stability is considered to be good if the decrease in signal value is less than or equal to 10%. See Table 6 for the results. Table 6 shows the stability evaluation results of the kit provided in Example 7 of the present invention.
  • Example 7 As can be seen from Table 6, the kit provided in Example 7 is relatively stable.

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Abstract

Kit de test quantitatif d'anticorps de Récepteur d'hormone de stimulation de la thyroïde (TSHR) humaine et réactif antigénique de TSHR. Le kit comprend : un support en phase solide, revêtu d'un anticorps monoclonal anti-TSHR A ; le réactif antigénique, c'est-à-dire un complexe immun d'un anticorps monoclonal anti-TSHR B et d'un TSHR ; et un anticorps humain de TSHR, marqué d'un marqueur. Aucune liaison concurrentielle n'a lieu entre l'anticorps monoclonal anti-TSHR A et l'anticorps monoclonal anti-TSHR B. La concentration de l'anticorps de TSHR dans le sérum ou dans le plasma se mesure selon un principe de réaction à méthodes concurrentielles. Le réactif antigénique de TSHR comprend le complexe immun de l'anticorps monoclonal anti-TSHR B et le TSHR. Le kit est stable et permet d'éviter l'interférence de biotine ou de fluorescéine sodique. Le processus de préparation est simplifié et stabilisé et la répétabilité est bonne.
PCT/CN2022/134765 2021-12-07 2022-11-28 Réactif antigénique de récepteur d'hormones de stimulation de la thyroïde (tshr) et kit de test quantitatif d'anticorps de tshr WO2023103827A1 (fr)

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WO2024046384A1 (fr) * 2022-08-31 2024-03-07 长春金赛药业有限责任公司 Anticorps se liant au récepteur de l'hormone stimulant la thyroïde et son utilisation

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