WO2023103827A1 - Thyroid stimulating hormone receptor antigen reagent and thyroid stimulating hormone receptor antibody quantitative test kit - Google Patents
Thyroid stimulating hormone receptor antigen reagent and thyroid stimulating hormone receptor antibody quantitative test kit Download PDFInfo
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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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Classifications
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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
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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
- G01N33/53—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
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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
- G01N33/53—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
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- 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
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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.
Abstract
A human thyroid stimulating hormone receptor (TSHR) antibody quantitative test kit and a TSHR antigen reagent. The kit comprises: a solid-phase support coated with an anti-TSHR monoclonal antibody A; the antigen reagent which is an immune complex of an anti-TSHR monoclonal antibody B and a TSHR; and a human TSHR antibody marked by a marker. No competitive binding is caused between the anti-TSHR monoclonal antibody A and the anti-TSHR monoclonal antibody B; the concentration of the TSHR antibody in serum or plasma is measured by using a competitive method reaction principle. The TSHR antigen reagent comprises the immune complex of the anti-TSHR monoclonal antibody B and the TSHR. The kit is stable and can avoid interference of biotin or fluorescein sodium, the preparation process is simpler and more stable, and the repeatability is good.
Description
本申请要求于2021年12月7日提交中国专利局、申请号为202111487759.3、发明名称为“一种促甲状腺激素受体抗原试剂及促甲状腺激素受体抗体定量检测试剂盒”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。This application requires submission of a Chinese patent application to the China Patent Office on December 7, 2021, with the application number 202111487759.3 and the title of the invention "a thyroid-stimulating hormone receptor antigen reagent and thyroid-stimulating hormone receptor antibody quantitative detection kit". priority, the entire contents of which are incorporated by reference into this application.
本发明涉及体外诊断技术领域，具体是一种促甲状腺激素受体抗原试剂及促甲状腺激素受体抗体定量检测试剂盒。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.
促甲状腺激素受体(TSHR)是位于甲状腺滤泡上皮细胞膜上的一种大分子糖蛋白，属于G蛋白偶联受体家族成员。生理条件下，促甲状腺激素(TSH)与其受体TSHR结合，经G蛋白激活产生级联反应以实现生物学效应。在遗传和环境作用下，TSHR结构发生变异，产生了抗原性，引起自身免疫反应，产生大量的促甲状腺激素受体抗体(TRAb)。TRAb是一组异质性抗体，根据其功能不同可分为甲状腺刺激抗体(TSAb)、甲状腺刺激阻断抗体(TSBAb)和中性甲状腺激素受体抗体。TSAb可与TSH竞争性结合在TSHR上，通过产生cAMP，刺激甲状腺滤泡细胞分泌合成甲状腺素，继而可引起毒性弥漫性甲状腺肿(Graves病)。TSBAb与TSHR结合，可直接干扰TSH与其受体的结合，从而减弱TSH的生物学效应。另外，TSBAb还可抑制腺苷酸环化酶的活化引起甲减。中性抗体与TSHR结合后既不刺激也不抑制甲状腺功能。Thyroid-stimulating hormone receptor (TSHR) 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. In addition, 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由含764个氨基酸的单一多肽链组成，分子量约为84kDa，胞外域大约有414～418个氨基酸，包含六个潜在的N连接糖基化位点，而剩余的序列形成跨膜结构域和细胞内羧基末端。正确的折叠、糖基化和存在TSHR的跨膜区和胞外域是高亲和力配体结合所必需的，同时，跨膜区和细胞内结构的稳定对于维持TSHR的稳定有重要作用。有大量文献表明TSHR本身稳定性很 差(参考文献1：Furmaniak J,Sanders J,Sanders P,Miller-Gallacher J,Ryder MM,Rees Smith B.Practical applications of studies on the TSH receptor and TSH receptor autoantibodies.Endocrine.2020 May；68(2):261-264.doi:10.1007/s12020-019-02180-9.Epub 2020May 29.PMID:32472423；PMCID:PMC7266850.参考文献2：Iida Y,Amir SM,Ingbar SH.Stabilization,partial purification,and characterization of thyrotropin receptors in solubilized guinea pig fat cell membranes.Endocrinology.1987 Nov；121(5):1627-36.doi:10.1210/endo-121-5-1627.PMID:3665836.)。在体外诊断检测技术领域中，检测试剂在液体状态下长期保存是其能在自动化检测仪器中正常使用的必要条件，而TSHR稳定性差，易失去活性，很难在2-8℃条件下长期保存，因而限制了其在自动化监测仪器上的应用。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. There is a large amount of literature showing that TSHR itself is very poorly stable (reference 1: Furmaniak J, Sanders J, Sanders P, Miller-Gallacher J, Ryder MM, Rees Smith B. Practical applications of studies on the TSH receptor and TSH receptor autoantibodies. Endocrine .2020 May;68(2):261-264.doi:10.1007/s12020-019-02180-9.Epub 2020May 29.PMID:32472423;PMCID:PMC7266850.Reference 2:Iida Y,Amir SM,Ingbar SH. Stabilization, partial purification, and characterization of thyrotropin receptors in solubilized guinea pig fat cell membranes. Endocrinology. 1987 Nov; 121(5):1627-36.doi:10.1210/endo-121-5-1627.PMID:3 665836.). In the field of in vitro diagnostic testing technology, long-term storage of testing reagents in a liquid state is a necessary condition for their normal use in automated testing instruments, while TSHR has poor stability and is prone to lose activity, making it difficult to store for a long time at 2-8 °C , thus limiting its application in automated monitoring instruments.
TRAb是Graves患者血清中存在对TSHR的特异性自身抗体。血清TRAb的测定可以作为Graves病诊断、治疗及预后判断的良好指标，对妊娠期甲状腺功能亢进症的治疗以及新生儿甲状腺功能异常的风险评估也具有重要价值。目前TRAb的检测方法主要有生物分析法和免疫分析法。生物分析法能区分刺激性抗体和阻断性抗体，但因耗时、操作繁琐，成本高而不能应用于临床实验室的检测。常见的免疫分析法为放射免疫法、酶联免疫法、化学发光法，目前临床应用最广泛的为全自动磁微粒化学发光法。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. At present, 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.
现有技术已经公开了多种用于测定TRAb的方法，例如，申请号为CN201910875687.6的中国专利公开了一种刺激性促甲状腺激素受体抗体浓度的检测试剂盒，包括：包被有刺激性促甲状腺激素受体抗体的磁微粒，含有标记物标记的刺激性促甲状腺激素受体，刺激性促甲状腺激素受体抗体校准品，其固相包被为刺激性促甲状腺激素受体抗体，其解决的问题是避免了抑制性促甲状腺激素受体抗体对测量系统的影响。申请号为CN201611018792.0的中国专利公开了一种促甲状腺激素受体抗体化学发光免疫测定试剂盒包括：促甲状腺激素受体包被的磁微粒、鼠抗人IgG抗体包被的吖啶酯、促甲状腺激素受体抗体定标品，其固相包被为促甲状腺激素受体，其解决的问题是克服了现有测定方法测定成本高、测定灵敏度低、测定线性范围窄、重现性低、不能定量、操作复杂的缺点。申请号为CN201710115161.9的中国文献公开了一种促甲状 腺激素受体抗体的检测方法，其试剂盒组分主要包括FITC标记的羊抗人球蛋白等试剂及生物薄片，生物薄片为取自大鼠甲状腺细胞制成，包被在载片的反应区内；测定方法为：在反应区内加待测血清，经孵育、漂洗、甩干后，加入FITC标记的羊抗人球蛋白，经孵育、漂洗、甩干后，在荧光显微镜下，甲状腺细胞膜上形成FITC标记的羊抗人球蛋白荧光复合物，产生黄绿色荧光；其解决的问题是克服了促甲状腺激素受体抗体的检测方法中的漏检问题。申请号为CN201810506744.9的中国专利公开了一种能同时检测促甲状腺激素受体分型抗体的酶联免疫试剂盒及检测方法，该试剂盒主要包括：包被抗原、预包被酶标板、漂洗液、促甲状腺激素受体抗体标准品、酶标液，该试剂盒解决的主要问题是可用于定量检测促甲状腺激素受体分型抗体的浓度。文献《Evaluation of the application of TSH receptor stimulating autoantibodies and the optimization of detection strategy in Graves'disease》(Tong M,Ding J,Huang B,Chen J,Wei X,Li Z,Shu J,Hu Z,Jiang X,Sheng H.Clin Chim Acta.2021Oct；521:34-39.doi:10.1016/j.cca.2021.06.017.Epub 2021Jun 16.PMID:34144042)公开了某国外厂家的促甲状腺激素受体刺激性抗体检测试剂盒(化学发光法)是由一对重组的人TSHR构建，其检测原理为夹心法，并且只检测刺激性抗体。市面上另有其他采用竞争法原理检测TRAb的商品化试剂盒，其检测系统可大致分为两种：生物素-亲和素系统或FITC-抗FITC系统，这两种系统均有一定的弊端：生物素-亲和素系统：当标本中含有一定量的生物素会干扰检测结果(参考文献3：张国峰,郭锐,关海霞.重视化验单之外的信息——由生物素干扰检验而被误诊为Graves病甲状腺功能亢进症的实例谈诊断甲状腺疾病的要素[J].中华内分泌代谢杂志,2017,33(09):723-725.)；FITC-抗FITC系统：一些历经视网膜荧光血管造影术的患者血清中残留的荧光素也会对检测结果有一定影响(参考文献4：Bloom JN,Herman DC,Elin RJ,Sliva CA,Ruddel ME,Nussenblatt RB,Palestine AG.Intravenous fluorescein interference with clinical laboratory tests.Am J Ophthalmol.1989 Oct 15；108(4):375-9.doi:10.1016/s0002-9394(14)73304-5.PMID:2801858.)。上述两种系统均涉及复杂的标记工艺，同时会引入相应的干扰因素。Various methods for determining TRAb have been disclosed in the prior art. For example, 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. The main problem solved by this kit is that it can be used to quantitatively detect the concentration of thyroid-stimulating hormone receptor antibody. Document "Evaluation of the application of TSH receptor stimulating autoantibodies and the optimization of detection strategy in Graves' disease" (Tong M, Ding J, Huang B, Chen J, Wei X, Li Z, Shu J, Hu Z, Jiang X, Sheng H.Clin Chim Acta.2021Oct;521:34-39.doi:10.1016/j.cca.2021.06.017.Epub 2021Jun 16.PMID:34144042) disclosed the detection of thyroid stimulating hormone receptor stimulating antibody by a foreign manufacturer The kit (chemiluminescent method) is constructed from a pair of recombinant human TSHR, and its detection principle is the sandwich method, and only the stimulating antibody is detected. There are other commercial kits on the market that use the principle of the competition method to detect TRAb, and their detection systems can be roughly divided into two types: biotin-avidin system or FITC-anti-FITC system, both of which have certain drawbacks : 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]. Chinese Journal of Endocrinology and Metabolism, 2017,33(09):723-725.); FITC-anti-FITC system: Some undergoing fluorescein angiography of the retina Intravenous fluorescein interference with clinical laboratory tests .Am J Ophthalmol.1989 Oct 15;108(4):375-9.doi:10.1016/s0002-9394(14)73304-5.PMID:2801858.). Both of the above-mentioned systems involve a complex marking process and introduce corresponding interference factors.
发明内容Contents of the invention
有鉴于此，本发明所要解决的技术问题在于提供一种人促甲状腺激素受体抗原试剂及促甲状腺激素受体抗体定量检测试剂盒，本发明提供的试剂盒稳定性较好，且不受生物素、荧光素等干扰。In view of this, 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.
本发明提供了一种人促甲状腺激素受体抗体定量检测试剂盒，包括：包被有抗TSHR单克隆抗体A的固相载体；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;
抗原试剂，所述抗原试剂为抗TSHR单克隆抗体B和TSHR的免疫复合物；Antigen reagent, the antigen reagent is the immune complex of anti-TSHR monoclonal antibody B and TSHR;
含有标记物标记的人刺激性促甲状腺激素受体抗体；Contains marker-labeled human stimulating thyrotropin receptor antibody;
所述抗TSHR单克隆抗体A与抗TSHR单克隆抗体B不产生竞争性结合。The anti-TSHR monoclonal antibody A does not produce competitive binding with the anti-TSHR monoclonal antibody B.
本发明提供的试剂盒用于检测血清或血浆中促甲状腺激素受体抗体的浓度，其采用竞争法反应原理进行检测，原理如下：标记物标记的人刺激性促甲状腺激素受体抗体与人血浆或血清中TRAb竞争结合抗TSHR单克隆抗体B-TSHR免疫复合物，再与包被有抗TSHR单克隆抗体A的固相载体进行反应，通过免疫反应形成固相抗体-抗原-标记抗体复合物，该复合物催化发光底物液发光，发光强度与TRAb的浓度成反比，通过检测发光强度，从而获得待测血浆或血清中TRAb的浓度。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. Or 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. By detecting the luminous intensity, the concentration of TRAb in the plasma or serum to be tested can be obtained.
本发明提供的试剂盒包括包被有抗TSHR单克隆抗体A的固相载体，其中，抗TSHR单克隆抗体A的识别位点位于TSHR跨膜区和细胞内羧基末端，具体为TSHR氨基酸序列的414～764位。所述抗TSHR单克隆抗体A包括但不限于鼠源性单克隆抗体、兔单克隆抗体、羊单克隆抗体、人源性单克隆抗体和嵌合型单克隆抗体，优选为鼠源性单克隆抗体。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.
在一个实施例中，所述固相载体选自氨基、羧基、醛基、苯肼基或磺酸基表面修饰的磁性微球，进一步地，所述磁性微球的内核为四氧化三铁或三氧化二铁。In one embodiment, 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.
在一个实施例中，抗TSHR单克隆抗体A和磁性微球的比例为5～25μg/mg的磁性微球，优选为15μg/mg的磁性微球。In one embodiment, the ratio of the anti-TSHR monoclonal antibody A to the magnetic microspheres is 5-25 μg/mg magnetic microspheres, preferably 15 μg/mg magnetic microspheres.
在一个实施例中，所述包被有抗TSHR单克隆抗体A的羧基磁性微球可 以按照以下方法制备：In one embodiment, the carboxyl magnetic microspheres coated with anti-TSHR monoclonal antibody A can be prepared according to the following method:
将羧基磁性微球用EDC和NHS在酸性条件下进行活化，活化缓冲液为0.1M的MES(2-(N-吗啉代)乙磺酸)缓冲液，活化完成后，在磁场的作用下使羧基磁性微球与液体分开，弃去上清，加入适量的抗人TSHR鼠单克隆抗体A，将所述包被有抗TSHR单克隆抗体A的羧基磁性微球用含有1％牛血清白蛋白的0.01M的PBS缓冲液进行封闭，封闭结束后，加入封闭液以保存包被有抗TSHR单克隆抗体A的羧基磁性微球，其磁性微球的最终浓度为1mg/ml，将该混悬液置于2～8℃保存以备使用。Activate the carboxyl magnetic microspheres with EDC and NHS under acidic conditions. The activation buffer is 0.1M MES (2-(N-morpholino)ethanesulfonic acid) buffer. After the activation is completed, under the action of a magnetic field Separate the carboxyl magnetic microspheres from the liquid, discard the supernatant, add an appropriate amount of anti-human TSHR mouse monoclonal antibody A, and coat the carboxyl magnetic microspheres coated with anti-TSHR monoclonal antibody A with 1% bovine serum white 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.
本发明提供的试剂盒还包括抗原试剂，所述抗原试剂为抗TSHR单克隆抗体B和TSHR的免疫复合物。其中，抗TSHR单克隆抗体B的识别位点位于TSHR跨膜区和细胞内羧基末端，具体为TSHR氨基酸序列的661～764位，进一步的，抗TSHR单克隆抗体B的识别位点位于TSHR氨基酸序列的665～698位。所述抗TSHR单克隆抗体B包括但不限于鼠源性单克隆抗体、兔单克隆抗体、羊单克隆抗体、人源性单克隆抗体和嵌合型单克隆抗体，优选为鼠源性单克隆抗体。The kit provided by the invention also includes an antigen reagent, which is an immune complex of anti-TSHR monoclonal antibody B and TSHR. Among them, 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.
在一个实施例中，所述TSHR包括但不限于天然人源TSHR、天然猪源TSHR、重组表达的人源TSHR或重组表达的猪源TSHR。In one embodiment, the TSHR includes, but is not limited to, natural human TSHR, natural porcine TSHR, recombinantly expressed human TSHR or recombinantly expressed porcine TSHR.
在一个实施例中，所述抗原试剂由抗TSHR单克隆抗体B和TSHR在冻干液中反应得到或者由抗TSHR单克隆抗体B和TSHR在冻干液中反应、冻干后得到，或者由TSHR单克隆抗体B和TSHR在冻干液中反应、冻干、复溶后得到。In one embodiment, 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.
在一个实施例中，所述抗原试剂按照以下方法制备：In one embodiment, the antigen reagent is prepared according to the following method:
将抗TSHR单克隆抗体B和TSHR在抗原冻干液中反应，冻干得到冻干粉；Reacting the anti-TSHR monoclonal antibody B and TSHR in the antigen lyophilized liquid, and lyophilized to obtain a lyophilized powder;
将所述冻干粉在复溶液中进行复溶，得到抗原试剂。The lyophilized powder is reconstituted in a reconstitution solution to obtain an antigen reagent.
将抗TSHR单克隆抗体B和TSHR在抗原冻干液中进行反应，所述反应的温度优选为20～25℃，时间为1～5h。抗TSHR单克隆抗体B和TSHR复合，冻干再复溶后使得TSHR的稳定性得到极大提高。实验结果表明，复溶后的含有抗TSHR单克隆抗体B和TSHR免疫复合物的抗原可以在2～8℃下稳定28 天。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.
在一个实施例中，抗TSHR单克隆抗体B的浓度为1～10ug/mL，TSHR稀释的比例为1:10～1:100。In one embodiment, the concentration of anti-TSHR monoclonal antibody B is 1-10 ug/mL, and the dilution ratio of TSHR is 1:10-1:100.
在一个实施例中，所述抗原冻干液包括：缓冲液、1wt％-10wt％的甘露醇、1wt％-10wt％的蔗糖、0.1wt％-1wt％的血清白蛋白和0.1wt％-1wt％的聚乙二醇。其中，缓冲液可以为Bis-Tris缓冲液，血清白蛋白可以为牛血清白蛋白，聚乙二醇为聚乙二醇5000。In one embodiment, the antigen lyophilized solution comprises: buffer, 1wt%-10wt% mannitol, 1wt%-10wt% sucrose, 0.1wt%-1wt% serum albumin and 0.1wt%-1wt% % polyethylene glycol. Wherein, the buffer may be Bis-Tris buffer, the serum albumin may be bovine serum albumin, and the polyethylene glycol may be polyethylene glycol 5000.
反应完毕后，将得到的产物进行冻干，得到冻干粉，本发明对所述冻干没有特殊限制，本领域技术人员熟知的冻干工艺即可。After the reaction is completed, 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.
得到冻干粉后，将其在复溶液中复溶。在一个实施例中，所述复溶液包括：缓冲液和1wt％-5wt％的丙三醇。其中，缓冲液可以为Bis-Tris。After obtaining the lyophilized powder, it was reconstituted in the reconstitution solution. In one embodiment, the reconstitution solution includes: buffer and 1wt%-5wt% glycerol. Wherein, the buffer can be Bis-Tris.
在一个实施例中，所述抗原试剂按照以下方法制备：In one embodiment, the antigen reagent is prepared according to the following method:
将抗TSHR单克隆抗体B和TSHR在抗原冻干液中混合反应，形成抗TSHR单克隆抗体B和TSHR免疫复合物。The 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.
抗TSHR单克隆抗体B和TSHR免疫复合物在20～25℃抗原冻干液中能够稳定128h，在2-8℃抗原冻干液中能够稳定168h。Anti-TSHR monoclonal antibody B and TSHR immune complex can be stable for 128 hours in 20-25 ℃ antigen lyophilized solution, and can be stable for 168 hours in 2-8 ℃ antigen lyophilized solution.
本发明提供的试剂盒还含有标记物标记的人刺激性促甲状腺激素受体抗体，所述标记物选自钌化合物、吖啶化合物、过氧化物酶或碱性磷酸酶，其标记方式可以是直接标记或间接标记。在一个实施例中，所述含有标记物标记的人刺激性促甲状腺激素受体抗体在包含血清白蛋白、防腐剂和缓冲液的溶液中保存待用。在一个实施例中，所述缓冲液为Bis-Tris缓冲液，血清白蛋白为牛血清白蛋白，含量为3wt～8wt％，优选为5wt％；防腐剂为PC300，含量为0.1wt～5wt％，优选为0.5wt～4wt％。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. In one embodiment, the human-stimulating thyrotropin receptor antibody containing the marker is preserved in a solution comprising serum albumin, preservative and buffer until use. In one embodiment, 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%.
本发明提供的试剂盒还包括样本稀释液和/或刺激性促甲状腺激素受体抗体系列校准品。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.
在一个实施例中，所述样本稀释液包括血清白蛋白、防腐剂和缓冲液。在一个实施例中，所述缓冲液为Bis-Tris缓冲液，血清白蛋白为牛血清白蛋白，含量为1wt～3wt％；防腐剂为PC300，含量为0.1wt～0.3wt％。In one embodiment, the sample diluent includes serum albumin, a preservative, and a buffer. In one embodiment, the buffer is Bis-Tris buffer, the serum albumin is bovine serum albumin, the content is 1wt-3wt%, and the preservative is PC300, the content is 0.1wt-0.3wt%.
在一个实施例中，所述刺激性促甲状腺激素受体抗体系列校准品为浓度 0～100IU/L的系列校准品。在一个实施例中，所述校准品浓度分别是为0IU/L、2IU/L、5IU/L、10IU/L、20IU/L、50IU/L。In one embodiment, 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.
本发明提供的试剂盒采用竞争法实现对血清或血浆中促甲状腺激素受体抗体的浓度，原理如下：标记物标记的人刺激性促甲状腺激素受体抗体与人血浆或血清中TRAb竞争结合抗TSHR单克隆抗体B-TSHR免疫复合物，再与包被有抗TSHR单克隆抗体A的固相载体进行反应，通过免疫反应形成固相抗体-抗原-标记抗体复合物，该复合物催化发光底物液发光，发光强度与TRAb的浓度成反比；采用系列刺激性促甲状腺激素受体抗体系列校准品建立定标曲线，再检测待测品的发光强度，根据待测品的发光强度和定标曲线获得待测血浆或血清中TRAb的浓度。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.
本发明提供的试剂盒使用方法如下：The method for using the kit provided by the invention is as follows:
将待测品或校准品、抗原试剂和样本稀释液混合后、温育，温育温度为37℃，时间为17min；Mix the test product or calibrator, antigen reagent and sample diluent, and incubate at 37°C for 17 minutes;
然后加入包被有抗TSHR单克隆抗体A的固相载体和含有标记物标记的人刺激性促甲状腺激素受体抗体，混合均匀后，继续温育，温育温度为37℃，时间为15min；Then add the solid phase carrier coated with anti-TSHR monoclonal antibody A and the human stimulating thyrotropin receptor antibody containing the marker, mix well, and continue to incubate at 37°C for 15min;
加入发光底物，检测光信号强度；Add a luminescent substrate to detect the intensity of the light signal;
通过校准品反应曲线，根据样本检测光强度计算出待测样本中TRAb的浓度。Calculate the concentration of TRAb in the sample to be tested according to the response curve of the calibrator and the light intensity of the sample.
本发明以一组抗TSHR单克隆抗体A和抗TSHR单克隆抗体B构建定量检测试剂盒，其中，抗TSHR单克隆抗体B与TSHR形成免疫复合物，能够提高TSHR的稳定性，从而使得到的试剂盒具有良好的稳定性，实验结果表明，本发明提供的试剂盒，抗TSHR单克隆抗体B与TSHR免疫复合物在20～25℃的冻干液中稳定128h，在2-8℃的冻干液中稳定168h，抗原试剂复溶后能够在2～8℃条件下稳定28天。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.
本发明提供的人促甲状腺激素受体抗体定量检测试剂盒包括：包被有抗TSHR单克隆抗体A的固相载体；抗原试剂，所述抗原试剂为抗TSHR单克隆抗体B和TSHR的免疫复合物；含有标记物标记的人刺激性促甲状腺激素受体抗体。本发明提供的试剂盒采用竞争法反应原理检测血清或血浆中促甲状腺 激素受体抗体的浓度，与采用固相包被亲和素、生物素标记抗TSHR抗体(生物素-亲和素系统)或固相包被抗异硫氰酸荧光素(FITC)抗体、FITC标记抗TSHR抗体(FITC-抗FITC系统)的商品化试剂盒相比，本发明不仅减少了标记步骤，还能够避免生物素用药患者血浆或血清标本中生物素对生物素-亲和素系统的干扰或经荧光素眼底血管造影术的患者血浆或血清标本中荧光素钠对FITC-抗FITC系统的干扰，制备工艺更简单稳定，重复性良好，在提高产品性能的同时，大大降低了成本。The human thyroid-stimulating hormone receptor antibody quantitative detection kit provided by the present invention 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 The interference of biotin in the plasma or serum specimens of patients with medication on the biotin-avidin system or the interference of fluorescein sodium in the plasma or serum specimens of patients undergoing fluorescein angiography on the FITC-anti-FITC system, the preparation process is simpler It is stable and has good repeatability. While improving product performance, it greatly reduces costs.
图1是本发明实施例1提供的定标曲线；Fig. 1 is the calibration curve that the embodiment of the present invention 1 provides;
图2是本发明实施例及比较例提供的抗原试剂在冻干液中20～25℃下的稳定性评价结果；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;
图3是本发明实施例及比较例提供的抗原试剂在冻干液中2～8℃下的稳定性评价结果；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;
图4是本发明实施例及比较例提供的抗原试剂在复溶后在2～8℃下的稳定性评价结果；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;
图5是本发明实施例提供的试剂盒与其他试剂盒检测结果的相关性曲线图。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.
以下结合实施例对本发明提供的促甲状腺激素受体抗原试剂及促甲状腺激素受体抗体定量检测试剂盒进行进一步说明。The 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.
如无特殊说明，本发明所用的试剂和仪器均为市售产品，本发明采用的试验方法为本领域的常规方法。Unless otherwise specified, 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.
本发明的试剂盒中未详细提及的试剂组分(例如清洗液等一些必要的缓冲液等)、试剂盒外包装以及各试剂组分的独立包装容器等均可以按照所属领域的常规操作进行，符合相关行业规定即可。本发明试剂盒的方法中未详细提及的操作步骤也可参照所属领域的常规操作进行。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.
以下各实施例中，抗TSHR单克隆抗体B按照以下方法制备：In each of the following examples, anti-TSHR monoclonal antibody B was prepared according to the following method:
(1)化学合成：将TSHR氨基酸序列的665-698位序列片段PGDKDTKIAKRMAVLIFTDFICMAPISFYALSAI作为抗原进行化学合成，通过巯基偶联到KLH(Keyhole Limpet Hemocyanin)上作为免疫原，通过巯基偶联到BSA上作为筛选原。(1) Chemical synthesis: PGDKDTKIAKRMAVLIFTDFICMAPISFYALSAI, the 665-698-position sequence fragment of the TSHR amino acid sequence, was chemically synthesized as an antigen, coupled to KLH (Keyhole Limpet Hemocyanin) through sulfhydryl as an immunogen, and coupled to BSA through sulfhydryl as a screening agent .
(2)免疫：将纯化好的蛋白稀释到适当浓度，与弗氏完全佐剂1:1混合，经乳化后通过皮下注射给6周龄大小的BALB/c雌性小鼠。两周后，将等量的蛋白与弗氏不完全佐剂进行等比混合，乳化后进行第二次小鼠免疫。自此之后每隔两周，重复免疫一次。直至细胞融合前3-4天，进行一次加强免疫。(2) Immunization: The purified protein was diluted to an appropriate concentration, mixed with Freund's complete adjuvant 1:1, emulsified and subcutaneously injected into 6-week-old BALB/c female mice. Two weeks later, the same amount of protein was mixed with Freund's incomplete adjuvant in equal proportions, emulsified, and the mice were immunized for the second time. Every two weeks thereafter, the immunization was repeated. A booster immunization was performed until 3-4 days before cell fusion.
(3)细胞融合、亚克隆和腹水制备：此后，对小鼠进行颈托安乐死之后无菌取出脾脏与小鼠骨髓瘤系NS-1细胞进行细胞融合，将融合后的细胞经有限稀释法进行多次亚克隆培养后，经ELISA筛选出抗体水平较高的细胞株进行后续的腹水制备工作。(3) Cell fusion, subcloning and ascites preparation: Afterwards, the mice were euthanized with a neck collar and the spleen was aseptically removed to fuse with NS-1 cells of the mouse myeloma line, and the fused cells were performed by limiting dilution method. After multiple subcloning cultures, cell lines with higher antibody levels were screened by ELISA for subsequent ascites preparation.
(4)抗体纯化：将抗体进行SPA亲和层析纯化。(4) Antibody purification: the antibody was purified by SPA affinity chromatography.
实施例1Example 1
一种人促甲状腺激素受体抗体定量检测试剂盒，包括：包被有抗人TSHR鼠单克隆抗体A的磁微粒、抗原试剂(抗人TSHR鼠单克隆抗体B-重组人TSHR复合物)、样本稀释液、辣根过氧化物酶标记的人刺激性促甲状腺激素受体抗体和刺激性促甲状腺素受体抗体系列校准品，其制备过程如下：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:
(1)包被抗人TSHR鼠单克隆抗体A的磁微粒混悬液的制备：(1) Preparation of magnetic particle suspension coated with anti-human TSHR mouse monoclonal antibody A:
将羧基磁性微球用EDC和NHS在酸性条件下进行活化，活化缓冲液为0.1M的MES(2-(N-吗啉代)乙磺酸)缓冲液，活化时间为30min，活化完成后，在磁场的作用下使羧基磁性微球与液体分开，弃去上清，加入适量的抗人TSHR鼠单克隆抗体A，将所述包被有抗人TSHR单克隆抗体A的羧基磁性微球用含有1％牛血清白蛋白的0.01M的PBS缓冲液进行封闭，封闭结束后，加入封闭液以保存包被有抗TSHR单克隆抗体A的羧基磁性微球，其磁性微球的最终浓度为1mg/ml，将该混悬液置于2～8℃保存以备使用。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. 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, and store the suspension at 2-8°C for use.
(2)抗原试剂的配制：将5μg/mL的抗人TSHR鼠单克隆抗体B与1/20稀释比例的重组人TSHR在特定的抗原冻干液中于20～25℃条件下搅拌反应3h，制成冻干品，用特定复溶液复溶；取0.05mol/L的Bis-Tris缓冲液、5wt％ 甘露醇、5wt％蔗糖、0.5wt％牛血清白蛋白、0.2wt％PEG5000配制冻干液；取0.05mol/L的Bis-Tris缓冲液、含有2wt％的丙三醇配制特定复溶液复溶；(2) Preparation of antigen reagent: 5 μg/mL anti-human TSHR mouse monoclonal antibody B and recombinant human TSHR at a dilution ratio of 1/20 were stirred and reacted for 3 hours at 20-25°C in a specific antigen lyophilized solution, Make lyophilized product, redissolve with specific reconstitution solution; take 0.05mol/L Bis-Tris buffer, 5wt% mannitol, 5wt% sucrose, 0.5wt% bovine serum albumin, 0.2wt% PEG5000 to prepare lyophilized solution ; Take 0.05mol/L of Bis-Tris buffer and contain 2wt% glycerol to prepare a specific reconstitution solution for redissolution;
(3)样本稀释液的制备：Bis-Tris缓冲液中包含有3％牛血清白蛋白和0.2％PC300，然后混合均匀，2～8℃保存待用；(3) Preparation of sample diluent: Bis-Tris buffer contains 3% bovine serum albumin and 0.2% PC300, then mix well, store at 2-8°C until use;
(4)辣根过氧化物酶标记的人刺激性促甲状腺激素受体抗体的制备方法为：将辣根过氧化物酶标记的人刺激性促甲状腺激素受体抗体加入到含有5％牛血清白蛋白和3％PC300的Bis-Tris缓冲液，然后混合均匀；其辣根过氧化物酶标记的人刺激性促甲状腺激素受体抗体的稀释比例为1/1000～1/5000；2～8℃保存待用；(4) 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 ℃ for later use;
(5)刺激性促甲状腺素受体抗体系列校准品为浓度0～100IU/L的系列校准品，所述校准品的制备方法为：向Bis-Tris缓冲液中添加5％的牛血清白蛋白，混合均匀后得校准品稀释液，然后用该校准品稀释液将刺激性促甲状腺素受体抗体稀释成六个浓度点S0～S5，浓度分别是为0IU/L、2IU/L、5IU/L、10IU/L、20IU/L、50IU/L。(5) 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.
该试剂盒在全自动化学发光仪A2000Plus上的检测步骤：The detection steps of the kit on the automatic chemiluminescence instrument A2000Plus:
(1)准备测试样本(校准品或待测样本)并正确放置后，点击启动按钮进行定标程序或样本检测程序，仪器将执行以下操作；(1) After preparing the 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;
(2)将样本架传送到吸样位，将反应容器加载到加样位；(2) Transfer the sample rack to the sample suction position, and load the reaction container to the sample addition position;
(3)执行定标程序时，完成100μL校准品、20μL抗原试剂、50μL样本稀释液的分注；(3) When performing the calibration procedure, complete the dispensing of 100 μL calibrator, 20 μL antigen reagent, and 50 μL sample diluent;
(4)执行样本检测程序时，完成100μL样本、20μL抗原试剂、50μL样本稀释液的分注；(4) When performing the sample detection procedure, complete the dispensing of 100 μL sample, 20 μL antigen reagent, and 50 μL sample diluent;
(5)对反应液进行混匀、温育，温育温度为37℃，温育时间为17分钟；(5) Mix and incubate the reaction solution, the incubation temperature is 37°C, and the incubation time is 17 minutes;
(6)完成20μL包被抗人TSHR鼠单克隆抗体A的磁微粒混悬液、50μL辣根过氧化物酶标记的人刺激性促甲状腺激素受体抗体的分注；(6) Complete the dispensing of 20 μL magnetic particle suspension coated with anti-human TSHR mouse monoclonal antibody A and 50 μL horseradish peroxidase-labeled human stimulating thyrotropin receptor antibody;
(7)对反应液进行混匀、温育，温育温度为37℃，温育时间为15分钟；(7) Mix and incubate the reaction solution, the incubation temperature is 37°C, and the incubation time is 15 minutes;
(8)温育完成后，使用清洗液对反应液进行清洗分离；(8) After the incubation is completed, use a cleaning solution to clean and separate the reaction solution;
(9)完成50μL底物A液和50μL底物B液的分注；(9) Complete the dispensing of 50 μL of substrate A solution and 50 μL of substrate B solution;
(10)对反应液进行混匀，检测发光强度；(10) Mix the reaction solution and detect the luminous intensity;
以校准品浓度值为X轴，以校准品发光强度对数值为Y轴，建立定标曲线，根据待测样品的发光强度值回算相应的浓度值，其中，定标曲线见图1，图1是本发明实施例提供的定标曲线。Take the concentration value of the calibrator as the X-axis, and the logarithmic value of the luminous intensity of the calibrator as the Y-axis, establish a calibration curve, and calculate the corresponding concentration value according to the luminous intensity value of the sample to be tested. The calibration curve is shown in Figure 1, Fig. 1 is the calibration curve provided by the embodiment of the present invention.
对比例1Comparative example 1
与实施例1相比，区别在于，抗原试剂未采用抗人TSHR鼠单克隆抗体B复合，直接将重组人TSHR冻干后复溶。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.
实施例2稳定性评估Embodiment 2 stability evaluation
稳定性评估方法：分别采用对比例1和实施例1提供的试剂盒平行检测含有一系列用校准品稀释液稀释得到的刺激性促甲状腺素受体抗体浓度0IU/L、2IU/L、5IU/L、10IU/L、20IU/L、50IU/L的校准品，在全自动化学发光仪A2000Plus系统上按照检测步骤进行测定，得到各组的发光值，以信号值降幅≤10％视为稳定性较好，结果参见表1、图2、图3和图4，表1为本发明实施例及比较例提供的试剂盒的稳定性评价结果，图2是本发明实施例及比较例提供的抗原试剂在冻干液中20～25℃下的稳定性评价结果，其中曲线a是实施例1提供的抗原试剂的稳定性评价结果，曲线b是对比例1提供的抗原试剂的稳定性评价结果，图3是本发明实施例及比较例提供的抗原试剂在冻干液中2～8℃下的稳定性评价结果，其中曲线a是实施例1提供的抗原试剂的稳定性评价结果，曲线b是对比例1提供的抗原试剂的稳定性评价结果，图4是本发明实施例及比较例提供的抗原试剂在复溶后在2～8℃下的稳定性评价结果，其中曲线a是实施例1提供的抗原试剂的稳定性评价结果，曲线b是对比例1提供的抗原试剂的稳定性评价结果。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. 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 ℃, 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.
表1 本发明实施例及比较例提供的试剂盒的稳定性评价结果Table 1 The stability evaluation results of the kits provided by the embodiments of the present invention and comparative examples
注：/表示无。Note: / means none.
由表1、图2、图3和图4可知，本发明提供的试剂盒中，抗TSHR鼠单克隆抗体B-人TSHR免疫复合物在20～25℃的冻干液中稳定128h，在2～8℃ 的冻干液中稳定168h，同时抗原试剂复溶后稳定性满足2～8℃28天。It can be seen from Table 1, Figure 2, Figure 3 and Figure 4 that in the kit provided by the present invention, 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.
实施例3试剂盒比对Embodiment 3 kit comparison
按照实施例1构建的人促甲状腺激素受体抗体定量检测试剂盒与试剂盒C：促甲状腺激素受体抗体定量检测试剂盒(电化学发光法)、试剂盒D：人促甲状腺激素受体抗体检测试剂盒(酶促化学发光法)进行临床样本的检测比对，同时对这三种检测定量试剂盒的重复性和干扰进行分析和比较。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) is used for detection and comparison of clinical samples, and the repeatability and interference of these three detection and quantification kits are analyzed and compared at the same time.
试剂盒C的检测原理为：竞争法，用链霉亲和素包被磁粒，通过可溶性猪TSHR和生物素化抗猪TSHR C-端鼠单克隆抗体免疫复合体以及钌复合物标记的人单克隆自身抗体M22进行测定；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;
试剂盒D的的检测原理为：竞争法，用FITC抗体包被磁微粒，FITC标记的抗人TSHR鼠单克隆抗体-人TSHR免疫复合物制备抗原试剂，辣根过氧化物酶标记的TRAb与样本中的TRAb共同竞争抗原试剂中的人TSHR免疫复合物；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;
检测方法：采用本发明试剂盒、试剂盒C和试剂盒D平行考核梯度血清样本193例，其中阴性样本103份，浓度范围为0.8～40IU/L，检测结果进行临床相关性分析，相关性如图5所示，图5是本发明实施例提供的试剂盒与其他试剂盒检测结果的相关性曲线图，其中图5(A)是与试剂盒C的相关性曲线图，图5(B)是与试剂盒D的相关性曲线图。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.
由图5可知，将本发明试剂盒与试剂盒C、试剂盒D同时对193份临床血清样本进行测定，本发明试剂盒与二者测定结果的相关性较好，相关系数R
2为0.958-0.997之间。
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.
实施例4重复性考核Embodiment 4 repeatability assessment
考核方式：选取低、高(1.83IU/L；24.37IU/L)两个浓度水平的患者样本，采用实施例1提供的试剂盒和方法以及试剂盒D及其方法进行检测，每个样本连续重复测定20次，计算出均值(AV)，标准差(SD)和变异系数(CV)，结果参见表2，表2为本发明实施例1提供的试剂盒的重复性考核结果：Assessment method: select patient samples with two concentration levels of low and high (1.83IU/L; 24.37IU/L), and use the kit and method provided in Example 1, kit D and its method for detection, and each sample is continuously Repeat the measurement 20 times, calculate the mean value (AV), standard deviation (SD) and coefficient of variation (CV), the results are shown in Table 2, and Table 2 is the repeatability assessment result of the kit provided by Example 1 of the present invention:
表2 本发明实施例1提供的试剂盒的重复性考核结果Table 2 The repeatability assessment results of the kit provided in Example 1 of the present invention
结果表明，与同类产品相比，本发明试剂盒的重复性较好。The result shows that, compared with similar products, the repeatability of the kit of the present invention is better.
实施例5干扰考核： Embodiment 5 interference assessment:
选取低、高(3.58IU/L；20.13IU/L)两个浓度水平的患者样本，向其中加入10ng/mL的生物素，分别采用试剂盒C和实施例1提供的试剂盒进行检测；向其中加入0.5μg/mL的荧光素钠，分别采用试剂盒D和实施例1提供的试剂盒进行检测，结果参见表3，表3为本发明实施例和比较例提供的试剂盒的干扰试验考核结果。Select low and high (3.58IU/L; 20.13IU/L) patient samples at two concentration levels, add 10ng/mL biotin therein, adopt kit C and the kit that embodiment 1 provides to detect respectively; Add 0.5 μg/mL of sodium fluorescein, and use kit D and the kit provided in Example 1 to detect, the results are shown in Table 3, and Table 3 is the interference test assessment of the kits provided in the examples and comparative examples of the present invention result.
表3 本发明实施例和比较例提供的试剂盒的干扰试验考核结果Table 3 The results of the interference test assessment of the kits provided by the embodiments of the present invention and comparative examples
试剂盒C的TRAb检测试剂盒说明书中声称，当标本中生物素浓度＞41nmol/L或10ng/mL时，测定结果会偏高；试剂盒D的TRAb检测试剂盒说明书中声称，当样本中荧光素浓度超过0.5ug/mL时，可导致检测结果变化超过10％；而用本发明试剂盒检测添加10ng/mL的生物素或0.5ug/mL的荧光素钠的样本时，检测结果不受影响(结果变化在±10％以内)，上述结果表明本发明可避免生物素-亲和素系统或FITC-抗FITC系统引入的相关干扰，提升了试剂盒的抗干扰性能。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.
实施例6Example 6
一种人促甲状腺激素受体抗体定量检测试剂盒，包括包被有抗猪TSHR鼠单克隆抗体A的磁微粒、抗原试剂(抗猪TSHR鼠单克隆抗体B-重组猪TSHR)、碱性磷酸酶标记的人刺激性促甲状腺激素受体抗体和刺激性促甲状腺素受体抗体系列校准品，其制备过程如下：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:
(1)包被抗猪TSHR鼠单克隆抗体A的磁微粒混悬液的制备：将羧基磁性微球用EDC和NHS在酸性条件下进行活化，活化缓冲液为0.1M的MES(2-(N-吗啉代)乙磺酸)缓冲液，活化时间为30min，活化完成后，在磁场的作用下使羧基磁性微球与液体分开，弃去上清，加入适量的抗猪TSHR鼠单 克隆抗体A，将所述包被有抗人TSHR单克隆抗体A的羧基磁性微球用含有1％牛血清白蛋白的0.01M的PBS缓冲液进行封闭，封闭结束后，加入封闭液以保存包被有抗TSHR单克隆抗体A的羧基磁性微球，其磁性微球的最终浓度为1mg/ml，将该混悬液置于2～8℃保存以备使用。(1) Preparation of magnetic particle suspension coated with anti-pig TSHR mouse monoclonal antibody A: Carboxylated magnetic microspheres were activated with EDC and NHS under acidic conditions, and the activation buffer was 0.1M MES (2-( N-morpholino) ethanesulfonic acid) buffer solution, the activation time is 30min, after the activation is completed, the carboxyl magnetic microspheres are separated from the liquid under the action of a magnetic field, the supernatant is discarded, and an appropriate amount of anti-pig TSHR mouse monoclonal Antibody A, the carboxyl magnetic microspheres coated with anti-human TSHR monoclonal antibody A are blocked with 0.01M PBS buffer containing 1% bovine serum albumin, after the blocking is completed, add blocking solution to preserve the coating Carboxyl magnetic microspheres with anti-TSHR monoclonal antibody A, the final concentration of the magnetic microspheres is 1mg/ml, and the suspension is stored at 2-8°C for use.
(3)抗原试剂的配制：将5ug/mL的抗猪TSHR鼠单克隆抗体B与1/30稀释比例的重组猪TSHR在特定的抗原冻干液中于20～25℃条件下搅拌反应3h，制成冻干品，用特定复溶液复溶；取0.05mol/L的Bis-Tris缓冲液、5wt％甘露醇、5wt％蔗糖、0.5wt％牛血清白蛋白、0.2wt％PEG5000配制冻干液；取0.05mol/L的Bis-Tris缓冲液、含有2wt％的丙三醇配制特定复溶液复溶；(3) Preparation of antigen reagent: 5ug/mL anti-pig TSHR mouse monoclonal antibody B and recombinant porcine TSHR at a dilution ratio of 1/30 were stirred and reacted for 3 hours at 20-25°C in a specific antigen freeze-dried liquid, Make lyophilized product, redissolve with specific reconstitution solution; take 0.05mol/L Bis-Tris buffer, 5wt% mannitol, 5wt% sucrose, 0.5wt% bovine serum albumin, 0.2wt% PEG5000 to prepare lyophilized solution ; Take 0.05mol/L of Bis-Tris buffer and contain 2wt% glycerol to prepare a specific reconstitution solution for redissolution;
(3)样本稀释液的制备：Bis-Tris缓冲液中包含有1-3％牛血清白蛋白和和0.1-0.3％PC300，然后混合均匀，2～8℃保存待用；(3) Preparation of sample diluent: 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;
(4)碱性磷酸酶标记的人刺激性促甲状腺激素受体抗体的制备方法为：将碱性酶标记的人刺激性促甲状腺激素受体抗体加入到含有5％牛血清白蛋白和0.1-5％PC300的Bis-Tris缓冲液，然后混合均匀，2～8℃保存待用；(4) The preparation method of 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;
(5)刺激性促甲状腺素受体抗体系列校准品为浓度0～100IU/L的系列校准品，所述校准品的制备方法为：向Bis-Tris缓冲液中添加5％的牛血清白蛋白，混合均匀后得校准品稀释液，然后用该校准品稀释液将刺激性促甲状腺素受体抗体稀释成六个浓度点S0-S5，浓度分别是为0IU/L、2IU/L、5IU/L、10IU/L、20IU/L、50IU/L；(5) 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;
稳定性考核：Stability assessment:
稳定性评估方法：采用实施例6提供的试剂盒平行检测含有一系列用校准品稀释液稀释得到的刺激性促甲状腺素受体抗体浓度0IU/L、2IU/L、5IU/L、10IU/L、20IU/L、50IU/L的校准品，在全自动化学发光仪A2000Plus系统上按照检测步骤进行测定，得到各组的发光值。以信号值降幅≤10％视为稳定性较好，结果参见表4，表4为本发明实施例6提供的试剂盒的稳定性评价结果。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.
表4本发明实施例6提供的试剂盒的稳定性评价结果Table 4 The stability evaluation result of the kit provided by Example 6 of the present invention
注：/表示无。Note: / means none.
由表4可知，实施例6提供的试剂盒较为稳定。As can be seen from Table 4, the kit provided in Example 6 is relatively stable.
干扰考核：Interference assessment:
选取低、高(3.58IU/L；20.13IU/L)两个浓度水平的患者样本，向其中加入10ng/mL的生物素，分别采用试剂盒C和实施例6提供的试剂盒进行检测；向其中加入0.5μg/mL的荧光素钠，分别采用试剂盒D和实施例6提供的试剂盒进行检测，结果参见表5，表5为本发明实施例6和比较例提供的试剂盒的干扰试验考核结果。Select low and high (3.58IU/L; 20.13IU/L) patient samples at two concentration levels, add 10ng/mL biotin thereinto, respectively adopt kit C and the kit that embodiment 6 provides to detect; Add 0.5 μg/mL of sodium fluorescein, and use kit D and the kit provided in Example 6 to detect, the results are shown in Table 5, and Table 5 shows the interference test of the kit provided in Example 6 and Comparative Example of the present invention Assessment results.
表5 本发明实施例6和比较例提供的试剂盒的干扰试验考核结果Table 5 The interference test assessment result of the kit provided by Example 6 of the present invention and comparative example
由表5可知，本发明试剂盒检测添加10ng/mL的生物素或0.5ug/mL的荧光素钠的样本时，检测结果不受影响(结果变化在±10％以内)，上述结果表明本发明可避免生物素-亲和素系统或FITC-抗FITC系统引入的相关干扰，提升了试剂盒的抗干扰性能。As can be seen from Table 5, when 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.
实施例7Example 7
一种人促甲状腺激素受体抗体定量检测试剂盒，包括包被有抗猪TSHR鼠单克隆抗体A的磁微粒、抗原试剂(抗猪TSHR鼠单克隆抗体B-天然猪TSHR)、样本稀释液、吖啶酯标记的人刺激性促甲状腺激素受体抗体和刺激性促甲状腺素受体抗体系列校准品，其制备过程如下：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:
(1)包被抗猪TSHR鼠单克隆抗体A的磁微粒混悬液的制备：将羧基磁性微球用EDC和NHS在酸性条件下进行活化，活化缓冲液为0.1M的MES(2-(N-吗啉代)乙磺酸)缓冲液，活化时间为30min，活化完成后，在磁场的作用下使羧基磁性微球与液体分开，弃去上清，加入适量的抗猪TSHR鼠单克隆抗体A，将所述包被有抗人TSHR单克隆抗体A的羧基磁性微球用含有1％牛血清白蛋白的0.01M的PBS缓冲液进行封闭，封闭结束后，加入封闭液以保存包被有抗TSHR单克隆抗体A的羧基磁性微球，其磁性微球的最终浓度为1mg/ml，将该混悬液置于2～8℃保存以备使用。(1) Preparation of magnetic particle suspension coated with anti-pig TSHR mouse monoclonal antibody A: Carboxylated magnetic microspheres were activated with EDC and NHS under acidic conditions, and the activation buffer was 0.1M MES (2-( N-morpholino) ethanesulfonic acid) buffer solution, the activation time is 30min, after the activation is completed, the carboxyl magnetic microspheres are separated from the liquid under the action of a magnetic field, the supernatant is discarded, and an appropriate amount of anti-pig TSHR mouse monoclonal Antibody A, the carboxyl magnetic microspheres coated with anti-human TSHR monoclonal antibody A are blocked with 0.01M PBS buffer containing 1% bovine serum albumin, after the blocking is completed, add blocking solution to preserve the coating Carboxyl magnetic microspheres with anti-TSHR monoclonal antibody A, the final concentration of the magnetic microspheres is 1mg/ml, and the suspension is stored at 2-8°C for use.
(2)抗原试剂的配制：将5ug/mL的抗猪TSHR鼠单克隆抗体B与1/10稀释比例的天然猪TSHR在特定的抗原冻干液中于20-25℃条件下搅拌反应3h，制成冻干品，用特定复溶液复溶；取0.05mol/L的Bis-Tris缓冲液、5wt％甘露醇、5wt％蔗糖、0.5wt％牛血清白蛋白、0.2wt％PEG5000配制冻干液；取0.05mol/L的Bis-Tris缓冲液、含有2wt％的丙三醇配制特定复溶液复溶；(2) Preparation of antigen reagent: 5ug/mL anti-pig TSHR mouse monoclonal antibody B and 1/10 dilution ratio of natural porcine TSHR were stirred and reacted at 20-25°C for 3 hours in a specific antigen lyophilized solution, Make lyophilized product, redissolve with specific reconstitution solution; take 0.05mol/L Bis-Tris buffer, 5wt% mannitol, 5wt% sucrose, 0.5wt% bovine serum albumin, 0.2wt% PEG5000 to prepare lyophilized solution ; Take 0.05mol/L of Bis-Tris buffer and contain 2wt% glycerol to prepare a specific reconstitution solution for redissolution;
(3)样本稀释液的制备：Bis-Tris缓冲液中包含有1-3％牛血清白蛋白和和0.1-0.3％PC300，然后混合均匀，2～8℃保存待用；(3) Preparation of sample diluent: 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;
(4)标记物标记的人刺激性促甲状腺激素受体抗体的制备方法为：将吖啶酯标记的人刺激性促甲状腺激素受体抗体加入到含有5％牛血清白蛋白和0.1-5％PC300的Bis-Tris缓冲液，然后混合均匀，2～8℃保存待用；(4) 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;
(5)刺激性促甲状腺素受体抗体系列校准品为浓度0～100IU/L的系列校准品，所述校准品的制备方法为：向Bis-Tris缓冲液中添加5％的牛血清白蛋白，混合均匀后得校准品稀释液，然后用该校准品稀释液将刺激性促甲状腺素受体抗体稀释成六个浓度点S0-S5，浓度分别是为0IU/L、2IU/L、5IU/L、10IU/L、20IU/L、50IU/L。(5) 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.
稳定性考核：Stability assessment:
稳定性评估方法：利用实施例7制备的试剂盒平行检测含有一系列用校准品稀释液稀释得到的刺激性促甲状腺素受体抗体浓度0IU/L、2IU/L、5IU/L、10IU/L、20IU/L、50IU/L的校准品，在全自动化学发光仪A2000Plus系统上按照检测步骤进行测定，得到各组的发光值。以信号值降幅≤10％视为稳定性较好，结果参见表6，表6为本发明实施例7提供的试剂盒的稳定性评价结果。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.
表6 本发明实施例7提供的试剂盒的稳定性评价结果Table 6 The stability evaluation result of the kit provided by the embodiment of the present invention 7
注：/表示无。Note: / means none.
由表6可知，实施例7提供的试剂盒较为稳定。As can be seen from Table 6, the kit provided in Example 7 is relatively stable.
干扰考核：Interference assessment:
选取低、高(3.58IU/L；20.13IU/L)两个浓度水平的患者样本，向其中加 入10ng/mL的生物素，分别采用试剂盒C和实施例7提供的试剂盒进行检测；向其中加入0.5μg/mL的荧光素钠，分别采用试剂盒D和实施例7提供的试剂盒进行检测，结果参见表7，表7为本发明实施例7和比较例提供的试剂盒的干扰试验考核结果。Select low and high (3.58IU/L; 20.13IU/L) patient samples at two concentration levels, add 10ng/mL biotin therein, and adopt kit C and the kit that embodiment 7 provides to detect respectively; Add 0.5 μg/mL of sodium fluorescein, and use kit D and the kit provided in Example 7 to detect, the results are shown in Table 7, Table 7 shows the interference test of the kit provided in Example 7 and Comparative Example of the present invention Assessment results.
表7 本发明实施例7和比较例提供的试剂盒的干扰试验考核结果Table 7 The interference test assessment result of the kit provided by Example 7 of the present invention and comparative example
由表7可知，用本发明试剂盒检测添加10ng/mL的生物素或0.5ug/mL的荧光素钠的样本时，检测结果不受影响(结果变化在±10％以内)，上述结果表明本发明可避免生物素-亲和素系统或FITC-抗FITC系统引入的相关干扰，提升了试剂盒的抗干扰性能。As can be seen from Table 7, when the kit of the present invention is used to detect samples added with 10ng/mL of biotin or 0.5ug/mL of fluorescein sodium, the test results are not affected (the results vary within ± 10%), the above results show that this The 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.
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，根据本发明的技术方案及其发明构思加以等同替换或改变，都应涵盖在本发明的保护范围之内。The above is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto, any person familiar with the technical field within the technical scope disclosed in the present invention, according to the technical solution of the present invention Any equivalent replacement or change of the inventive concepts thereof shall fall within the protection scope of the present invention.
Claims (10)
- 一种人促甲状腺激素受体抗体定量检测试剂盒，包括：包被有抗TSHR单克隆抗体A的固相载体；A human thyroid-stimulating hormone receptor antibody quantitative detection kit, comprising: a solid-phase carrier coated with anti-TSHR monoclonal antibody A;抗原试剂，所述抗原试剂为抗TSHR单克隆抗体B和TSHR的免疫复合物；Antigen reagent, the antigen reagent is the immune complex of anti-TSHR monoclonal antibody B and TSHR;含有标记物标记的人刺激性促甲状腺激素受体抗体；Contains marker-labeled human stimulating thyrotropin receptor antibody;所述抗TSHR单克隆抗体A与抗TSHR单克隆抗体B不产生竞争性结合。The anti-TSHR monoclonal antibody A does not produce competitive binding with the anti-TSHR monoclonal antibody B.
- 根据权利要求1所述的试剂盒，其特征在于，还包括样本稀释液和/或刺激性促甲状腺激素受体抗体系列校准品。The kit according to claim 1, further comprising a sample diluent and/or a serial calibrator of stimulating thyroid stimulating hormone receptor antibody.
- 根据权利要求1或2所述的试剂盒，其特征在于，所述抗TSHR单克隆抗体A的识别位点位于TSHR氨基酸序列的414～764位；The kit according to claim 1 or 2, wherein the recognition site of the anti-TSHR monoclonal antibody A is located at positions 414-764 of the TSHR amino acid sequence;所述单克隆抗体B的识别位点位于TSHR氨基酸序列的661～764位。The recognition site of the monoclonal antibody B is located at positions 661-764 of the TSHR amino acid sequence.
- 根据权利要求3所述的试剂盒，其特征在于，所述单克隆抗体B的识别位点位于TSHR氨基酸序列的665～698位。The kit according to claim 3, wherein the recognition site of the monoclonal antibody B is located at positions 665-698 of the TSHR amino acid sequence.
- 根据权利要求1或2所述的试剂盒，其特征在于，所述抗TSHR单克隆抗体A和抗TSHR单克隆抗体B独立地选自鼠源性单克隆抗体、兔源性单克隆抗体、羊源性单克隆抗体、人源性单克隆抗体和嵌合型单克隆抗体。The kit according to claim 1 or 2, wherein said anti-TSHR monoclonal antibody A and anti-TSHR monoclonal antibody B are independently selected from mouse-derived monoclonal antibodies, rabbit-derived monoclonal antibodies, sheep Derived monoclonal antibodies, humanized monoclonal antibodies and chimeric monoclonal antibodies.
- 根据权利要求1或2所述的试剂盒，其特征在于，所述抗原试剂由抗TSHR单克隆抗体B和TSHR在冻干液中反应得到或者由抗TSHR单克隆抗体B和TSHR在冻干液中反应、冻干复溶后得到。The kit according to claim 1 or 2, wherein the antigen 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 It was obtained after medium reaction, freeze-drying and reconstitution.
- 根据权利要求1或2所述的试剂盒，其特征在于，所述固相载体为氨基、羧基、醛基、苯肼基或磺酸基表面修饰的磁性微球。The kit according to claim 1 or 2, characterized in that the solid phase carrier is a magnetic microsphere surface-modified by amino, carboxyl, aldehyde, phenylhydrazine or sulfonic acid groups.
- 根据权利要求1或2所述的试剂盒，其特征在于，所述含有标记物标记的人刺激性促甲状腺激素受体抗体中，所述标记物选自钌化合物、吖啶化合物、过氧化物酶或碱性磷酸酶。The kit according to claim 1 or 2, characterized in that, in the human stimulating thyrotropin receptor antibody labeled with a marker, the marker is selected from the group consisting of ruthenium compounds, acridine compounds, peroxide enzyme or alkaline phosphatase.
- 一种促甲状腺激素受体抗原试剂，包括：抗TSHR单克隆抗体B和TSHR的免疫复合物。A thyroid-stimulating hormone receptor antigen reagent, comprising: anti-TSHR monoclonal antibody B and TSHR immune complex.
- 根据权利要求1所述的抗原试剂，其特征在于，由抗TSHR单克隆抗 体B和TSHR在冻干液中反应得到或者由抗TSHR单克隆抗体B和TSHR在冻干液中反应、冻干复溶后得到。The antigen reagent according to claim 1, characterized in that, it is obtained by reacting anti-TSHR monoclonal antibody B and TSHR in lyophilized liquid or reacted by anti-TSHR monoclonal antibody B and TSHR in lyophilized liquid, and freeze-dried obtained after dissolution.
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| CN114113637A (en) | 2022-03-01 |
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