CN114113637B - Thyroid stimulating hormone receptor antigen reagent and thyroid stimulating hormone receptor antibody quantitative detection kit - Google Patents
Thyroid stimulating hormone receptor antigen reagent and thyroid stimulating hormone receptor antibody quantitative detection kit Download PDFInfo
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- CN114113637B CN114113637B CN202111487759.3A CN202111487759A CN114113637B CN 114113637 B CN114113637 B CN 114113637B CN 202111487759 A CN202111487759 A CN 202111487759A CN 114113637 B CN114113637 B CN 114113637B
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- 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
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- 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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- 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)
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Abstract
The invention provides a quantitative detection kit for a human thyroid stimulating hormone receptor antibody, which comprises the following components: a solid phase carrier coated with an anti-TSHR monoclonal antibody a; an antigenic agent which is an immune complex of anti-TSHR monoclonal antibody B and TSHR; a human stimulatory thyroid stimulating hormone receptor antibody comprising a label; the anti-TSHR monoclonal antibody a does not produce competitive binding with the anti-TSHR monoclonal antibody B. The kit provided by the invention adopts the competition reaction principle to detect the concentration of the thyroid stimulating hormone receptor antibody in serum or plasma, is stable, can avoid the interference of biotin or sodium fluorescein, has simpler and more stable preparation process and good repeatability, and greatly reduces the cost while improving the product performance.
Description
Technical Field
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.
Background
Thyroid Stimulating Hormone Receptor (TSHR) is a macromolecular glycoprotein located on the membrane of the thyroid follicle epithelium and belongs to the G protein-coupled receptor family members. Under physiological conditions, thyroid Stimulating Hormone (TSH) binds to its receptor TSHR, and is activated by G protein to produce a cascade of reactions to achieve a biological effect. Under genetic and environmental effects, the TSHR structure is mutated, antigenic, and causes an autoimmune response, producing a large number of thyroid stimulating hormone receptor antibodies (trabs). Trabs are a group of heterogeneous antibodies, which can be classified according to their functions into thyroid stimulating antibodies (tsabs), thyroid stimulating blocking antibodies (tsbabs) and neutral thyroid hormone receptor antibodies. Tsabs can bind competitively to TSHs on TSHRs, stimulating thyroid follicular cells to secrete thyroxine by producing cAMP, which in turn can cause toxic diffuse goiter (Graves disease). TSBAb binds to TSHR and can interfere directly with TSH binding to its receptor, thereby attenuating the biological effects of TSH. In addition, TSBAb can also inhibit adenylate cyclase activation leading to hypothyroidism. Neutral antibodies neither stimulate nor inhibit thyroid function upon binding to TSHR.
TSHR consists of a single polypeptide chain of 764 amino acids, of molecular weight about 84kDa, with an extracellular domain of about 414 to 418 amino acids, containing six potential N-linked glycosylation sites, with the remaining sequence forming the transmembrane domain and intracellular carboxy-terminal end. Proper folding, glycosylation and the presence of the transmembrane and extracellular domains of the TSHR are essential for high affinity ligand binding, whilst stability of the transmembrane and intracellular structure plays an important role in maintaining stability of the TSHR. There are a number of references indicating that TSHR itself is very poorly stable (ref 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 auto ibodies.endocrine.2020May;68 (2): 261-264.Doi: 10.1007/s12020-019-02180-9.Epub 2020May 29.PMID:32472423;PMCID:PMC7266850. Ref 2:Iida Y,Amir SM,Ingbar SH.Stabilization,partial purification,and characterization of thyrotropin receptors in solubilized guinea pig fat cell membranes.Endocrinology.1987Nov;121 (5): 1627-36.Doi: 10.1210/endo-121-5-1627.PMID: 3665836.). In the technical field of in vitro diagnostic detection, long-term storage of a detection reagent in a liquid state is a necessary condition for normal use in an automatic detection instrument, while TSHR has poor stability, is easy to lose activity, and is difficult to store for a long time at 2-8 ℃, so that the application of the detection reagent in the automatic detection instrument is limited.
Trabs are autoantibodies specific for TSHR present in serum from Graves patients. The determination of serum TRAb can be used as a good index for Graves disease diagnosis, treatment and prognosis judgment, and has important value for treating gestational hyperthyroidism and risk assessment of neonatal thyroid dysfunction. Currently, the detection methods of trabs mainly comprise bioassays and immunoassays. The bioassay method can distinguish between stimulatory antibodies and blocking antibodies, but is time-consuming, cumbersome to operate, and costly to use in clinical laboratory testing. The common immunoassay method is a radioimmunoassay, an enzyme-linked immunoassay and a chemiluminescent method, and the most widely used clinical method at present is a full-automatic magnetic particle chemiluminescent method.
Various methods for determining TRAb have been disclosed in the prior art, for example, chinese patent application No. CN201910875687.6 discloses a kit for detecting the concentration of an antibody to the stimulatory thyroid stimulating hormone receptor, comprising: the magnetic particles coated with the thyroid stimulating hormone receptor antibody comprise a labeled thyroid stimulating hormone receptor and a thyroid stimulating hormone receptor antibody calibrator, wherein the solid phase of the thyroid stimulating hormone receptor antibody calibrator is coated with the thyroid stimulating hormone receptor antibody, and the problem is solved by avoiding the influence of the inhibitory thyroid stimulating hormone receptor antibody on a measurement system. Chinese patent application No. CN201611018792.0 discloses a thyroid stimulating hormone receptor antibody chemiluminescent immunoassay kit comprising: the solid phase of the thyroid stimulating hormone receptor coated magnetic particles, the acridinium ester coated by the mouse anti-human IgG antibody and the thyroid stimulating hormone receptor antibody standard substance is coated with the thyroid stimulating hormone receptor, and the problems of high measurement cost, low measurement sensitivity, narrow measurement linear range, low reproducibility, incapacity of quantification and complex operation of the existing measurement method are solved. The Chinese literature with the application number of CN201710115161.9 discloses a detection method of thyroid stimulating hormone receptor antibody, wherein the kit mainly comprises reagents such as FITC labeled goat anti-human globulin and a biological sheet, wherein the biological sheet is prepared from rat thyroid cells and is coated in a reaction area of a slide glass; the measuring method comprises the following steps: adding serum to be detected into a reaction area, incubating, rinsing and spin-drying, adding FITC-labeled goat anti-human globulin, incubating, rinsing and spin-drying, and forming FITC-labeled goat anti-human globulin fluorescent complex on thyroid cell membranes under a fluorescent microscope to generate yellowish green fluorescence; the problem of the detection method of the thyroid stimulating hormone receptor antibody is solved. The Chinese patent with the application number of CN201810506744.9 discloses an ELISA kit and a detection method for simultaneously detecting thyroid stimulating hormone receptor typing antibodies, wherein the kit mainly comprises: the kit solves the main problems of being capable of being used for quantitatively detecting the concentration of thyroid stimulating hormone receptor typing antibodies. 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) discloses that a thyroid stimulating hormone receptor stimulating antibody detection kit (chemiluminescent method) of a foreign manufacturer is constructed from a pair of recombinant human TSHR's, the detection principle of which is a sandwich method, and only stimulating antibodies are detected. Other commercial kits for detecting trabs by adopting the competition principle exist in the market, and the detection systems can be roughly divided into two types: biotin-avidin or FITC-anti-FITC systems, both of which have certain drawbacks: biotin-avidin system: when a certain amount of biotin is contained in a specimen, the detection result is disturbed (reference 3: zhang Guofeng, guo Rui, guan Haixia. Importance is attached to information other than the laboratory sheet-an example of hyperthyroidism caused by the biotin disturbance test, which is misdiagnosed as Graves' disease, talks about an element for diagnosing thyroid disease [ J ]. Journal of endocrine metabolism, 2017,33 (09): 723-725.); FITC-anti-FITC System: residual fluorescein in the serum of some patients subjected to retinal fluorescence angiography also has some effect on the detection results (ref 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.1989Oct 15;108 (4): 375-9.Doi:10.1016/s0002-9394 (14) 73304-5.PMID: 2801858.). Both of the above systems involve complex marking processes, while introducing corresponding interference factors.
Disclosure of Invention
In view of the above, the technical problem to be solved by the invention is to provide a human thyroid stimulating hormone receptor antigen reagent and a thyroid stimulating hormone receptor antibody quantitative detection kit, which have better stability and are not interfered by biotin, fluorescein and the like.
The invention provides a quantitative detection kit for a human thyroid stimulating hormone receptor antibody, which comprises the following components: a solid phase carrier coated with an anti-TSHR monoclonal antibody a;
an antigenic agent which is an immune complex of anti-TSHR monoclonal antibody B and TSHR;
a human stimulatory thyroid stimulating hormone receptor antibody comprising a label;
the anti-TSHR monoclonal antibody a does not produce competitive binding with the anti-TSHR monoclonal antibody B.
The kit provided by the invention is used for detecting the concentration of thyroid stimulating hormone receptor antibodies in serum or plasma, and adopts the reaction principle of a competition method for detection, and the principle is as follows: the labeled human stimulated thyroid stimulating hormone receptor antibody competes with TRAb in human blood plasma or serum for binding with an anti-TSHR monoclonal antibody B-TSHR immune complex, then reacts with a solid-phase carrier coated with an anti-TSHR monoclonal antibody A, forms a solid-phase antibody-antigen-labeled antibody complex through immune reaction, catalyzes luminescent substrate liquid to emit light, the luminous intensity is inversely proportional to the concentration of the TRAb, and the concentration of the TRAb in the blood plasma or serum to be detected is obtained through detecting the luminous intensity.
The kit provided by the invention comprises a solid phase carrier coated with an anti-TSHR monoclonal antibody A, wherein the recognition site of the anti-TSHR monoclonal antibody A is positioned at the TSHR transmembrane region and the intracellular carboxyl terminal, in particular to the 414-764 positions of the TSHR amino acid sequence. The anti-TSHR monoclonal antibodies a include, but are not limited to, murine monoclonal antibodies, rabbit monoclonal antibodies, sheep monoclonal antibodies, humanized monoclonal antibodies and chimeric monoclonal antibodies, preferably murine monoclonal antibodies.
In one embodiment, the solid support is selected from amino, carboxyl, aldehyde, phenylhydrazine or sulfonic acid surface modified magnetic microspheres, further wherein the inner core of the magnetic microsphere is ferric oxide or ferric oxide.
In one embodiment, the proportion of anti-TSHR monoclonal antibody A to magnetic microspheres is in the range 5 to 25. Mu.g/mg magnetic microspheres, preferably 15. Mu.g/mg magnetic microspheres.
In one example, the carboxyl magnetic microsphere coated with anti-TSHR monoclonal antibody a can be prepared as follows:
activating carboxyl magnetic microspheres with EDC and NHS under acidic condition, wherein the activating buffer is 0.1M MES (2- (N-morpholino) ethanesulfonic acid) buffer, separating the carboxyl magnetic microspheres from the liquid under the action of a magnetic field after activation is completed, discarding the supernatant, adding a proper amount of anti-human TSHR mouse monoclonal antibody A, blocking the carboxyl magnetic microspheres coated with the anti-TSHR monoclonal antibody A with 0.01M PBS buffer containing 1% bovine serum albumin, adding a blocking solution after blocking is completed to preserve the carboxyl magnetic microspheres coated with the anti-TSHR monoclonal antibody A, wherein the final concentration of the magnetic microspheres is 1mg/ml, and preserving the suspension at 2-8 ℃ for use.
The kit provided by the invention also comprises an antigen reagent which is an immune complex of the anti-TSHR monoclonal antibody B and the TSHR. Wherein the recognition site of the anti-TSHR monoclonal antibody B is positioned at the transmembrane region of the TSHR and the carboxyl terminal end in the cell, specifically 661-764 of the amino acid sequence of the TSHR, and further the recognition site of the anti-TSHR monoclonal antibody B is positioned at 665-698 of the amino acid sequence of the TSHR. The anti-TSHR monoclonal antibodies B include, but are not limited to, murine monoclonal antibodies, rabbit monoclonal antibodies, sheep monoclonal antibodies, humanized monoclonal antibodies and chimeric monoclonal antibodies, preferably murine monoclonal antibodies.
In one embodiment, the TSHR includes, but is not limited to, a natural human TSHR, a natural porcine TSHR, a recombinantly expressed human TSHR or a recombinantly expressed porcine TSHR.
In one embodiment the antigenic agent is obtained by reacting the anti-TSHR monoclonal antibody B and the TSHR in a lyophilizate or by reacting the anti-TSHR monoclonal antibody B and the TSHR in a lyophilizate, after lyophilization or by reacting the TSHR monoclonal antibody B and the TSHR in a lyophilizate, after lyophilization, after reconstitution.
In one embodiment, the antigenic agent is prepared according to the following method:
reacting the anti-TSHR monoclonal antibody B with TSHR in an antigen freeze-drying liquid, and freeze-drying to obtain freeze-dried powder;
and re-dissolving the freeze-dried powder in a re-solution to obtain the antigen reagent.
The anti-TSHR monoclonal antibody B and TSHR are reacted in an antigen lyophilizate, preferably at a temperature of 20 to 25 ℃ for a period of 1 to 5 hours. The anti-TSHR monoclonal antibody B is compounded with the TSHR, and the stability of the TSHR is greatly improved after freeze-drying and re-dissolving. Experimental results show that the reconstituted antigen containing anti-TSHR monoclonal antibody B and TSHR immune complex can be stabilized at 2-8deg.C for 28 days.
In one embodiment, the concentration of anti-TSHR monoclonal antibody B is 1-10 ug/mL and the ratio of TSHR dilution is 1:10-1:100.
In one embodiment, the antigen lyophilization fluid comprises: buffer solution, mannitol 1wt% -10wt%, sucrose 1wt% -10wt%, serum albumin 0.1wt% -1wt% and polyethylene glycol 0.1wt% -1 wt%. Wherein the buffer solution can be Bis-Tris buffer solution, the serum albumin can be bovine serum albumin, and the polyethylene glycol is polyethylene glycol 5000.
After the reaction is finished, the obtained product is freeze-dried to obtain freeze-dried powder, and the freeze-drying process is not particularly limited, and is well known to those skilled in the art.
After the freeze-dried powder is obtained, the freeze-dried powder is redissolved in a redissolution. In one embodiment, the complex solution comprises: buffer and 1wt% to 5wt% glycerol. Wherein the buffer may be Bis-Tris.
In one embodiment, the antigenic agent is prepared according to the following method:
anti-TSHR monoclonal antibody B and TSHR are mixed in an antigen lyophilizate to react to form an anti-TSHR monoclonal antibody B and TSHR immune complex.
The anti-TSHR monoclonal antibody B and the TSHR immune complex can be stabilized for 128 hours in antigen lyophilized liquid at 20-25 ℃ and can be stabilized for 168 hours in antigen lyophilized liquid at 2-8 ℃.
The kit provided by the invention further comprises a human stimulatory thyroid stimulating hormone receptor antibody labeled with a label selected from ruthenium compounds, acridine compounds, peroxidases or alkaline phosphatases, wherein the label can be directly labeled or indirectly labeled. In one embodiment, the human stimulatory thyroid stimulating hormone receptor antibody containing a marker label is maintained in a solution comprising serum albumin, a preservative and a buffer for use. In one embodiment, the buffer is Bis-Tris buffer, the serum albumin is bovine serum albumin, and the content is 3wt% to 8wt%, preferably 5wt%; the preservative is PC300, and the content is 0.1-5 wt%, preferably 0.5-4 wt%.
The kit provided by the invention further comprises a sample diluent and/or a series of stimulatory thyroid stimulating hormone receptor antibody calibrators.
In one embodiment, the sample diluent comprises serum albumin, a preservative, and a buffer. In one embodiment, the buffer solution is Bis-Tris buffer solution, the serum albumin is bovine serum albumin, and the content is 1-3 wt%; the preservative is PC300, and the content is 0.1-0.3 wt%.
In one embodiment, the series of stimulatory thyroid stimulating hormone receptor antibodies is a series of calibrators at a concentration of 0-100 IU/L. In one embodiment, the calibrator concentration is 0IU/L, 2IU/L, 5IU/L, 10IU/L, 20IU/L, 50IU/L, respectively.
The kit provided by the invention adopts a competition method to realize the concentration of thyroid stimulating hormone receptor antibody in serum or plasma, and the principle is as follows: the labeled human stimulatory thyroid stimulating hormone receptor antibody competes with trabs in human blood plasma or serum for binding with an anti-TSHR monoclonal antibody B-TSHR immune complex, then reacts with a solid-phase carrier coated with an anti-TSHR monoclonal antibody A, and forms a solid-phase antibody-antigen-labeled antibody complex through immune reaction, wherein the complex catalyzes luminescent substrate liquid to emit light, and the luminous intensity is inversely proportional to the concentration of the trabs; a calibration curve is established by adopting a series of stimulatory thyroid stimulating hormone receptor antibody series calibrator, then the luminous intensity of the to-be-detected sample is detected, and the TRAb concentration in the blood plasma or serum to be detected is obtained according to the luminous intensity of the to-be-detected sample and the calibration curve.
The application method of the kit provided by the invention comprises the following steps:
mixing the sample to be tested or the calibrator, the antigen reagent and the sample diluent, and then incubating at 37 ℃ for 17min;
then adding a solid phase carrier coated with an anti-TSHR monoclonal antibody A and a human stimulatory thyroid stimulating hormone receptor antibody containing a marker label, uniformly mixing, and continuously incubating at 37 ℃ for 15min;
adding a luminescent substrate, and detecting the intensity of a light signal;
and calculating the TRAb concentration in the sample to be detected according to the sample detection light intensity through the calibrator reaction curve.
The invention constructs a quantitative detection kit by a group of anti-TSHR monoclonal antibody A and anti-TSHR monoclonal antibody B, wherein the anti-TSHR monoclonal antibody B and the TSHR form an immune complex, so that the stability of the TSHR can be improved, the obtained kit has good stability, and experimental results show that the kit provided by the invention has the advantages that the anti-TSHR monoclonal antibody B and the TSHR immune complex are stable in a freeze-dried liquid at 20-25 ℃ for 128 hours, the anti-TSHR monoclonal antibody B and the TSHR immune complex are stable in a freeze-dried liquid at 2-8 ℃ for 168 hours, and antigen reagents can be stable at 2-8 ℃ for 28 days after being re-dissolved.
The quantitative detection kit for the human thyroid stimulating hormone receptor antibody provided by the invention comprises the following components: a solid phase carrier coated with an anti-TSHR monoclonal antibody a; an antigenic agent which is an immune complex of anti-TSHR monoclonal antibody B and TSHR; a human stimulatory thyroid stimulating hormone receptor antibody comprising a label. Compared with the commercial kit adopting solid-phase coated avidin, biotin-marked anti-TSHR antibody (biotin-avidin system) or solid-phase coated anti-Fluorescein Isothiocyanate (FITC) antibody and FITC-marked anti-TSHR antibody (FITC-anti-FITC system), the kit provided by the invention not only reduces the marking step, but also can avoid the interference of biotin in the plasma or serum sample of a patient with biotin on the biotin-avidin system or the interference of sodium fluorescein in the plasma or serum sample of the patient with fluorescein fundus angiography on the FITC-anti-FITC system, has simpler and more stable preparation process and good repeatability, and greatly reduces the cost while improving the product performance.
Drawings
FIG. 1 is a calibration curve provided in example 1 of the present invention;
FIG. 2 shows the results of evaluation of the stability of the antigen reagent provided in the examples of the present invention and the comparative examples at 20 to 25℃in a lyophilized liquid;
FIG. 3 is a graph showing the results of evaluation of the stability of an antigen reagent provided in examples and comparative examples of the present invention at 2 to 8℃in a lyophilized liquid;
FIG. 4 is a graph showing the results of evaluation of the stability of the antigen reagent provided in the examples of the present invention and comparative examples at 2 to 8℃after reconstitution;
FIG. 5 is a graph showing the correlation between the detection results of the kit provided by the embodiment of the invention and other kits.
Detailed Description
The thyroid stimulating hormone receptor antigen reagent and thyroid stimulating hormone receptor antibody quantitative detection kit provided by the invention are further described in the following combination examples.
Unless otherwise specified, the reagents and apparatus used in the present invention are commercially available products, and the test methods employed in the present invention are conventional in the art.
The reagent components (such as a cleaning solution and other necessary buffers) not mentioned in detail in the kit, the external package of the kit, and the independent packaging containers of the reagent components can all be carried out according to the conventional operation in the field, and the kit meets the relevant industry regulations. The procedure not mentioned in detail in the method of the kit of the invention can also be carried out with reference to the routine procedures of the art.
In the following examples, anti-TSHR monoclonal antibody B was prepared as follows:
(1) Chemical synthesis: the 665-698 sequence fragment PGDKDTKIAKRMAVLIFTDFICMAPISFYALSAI of the TSHR amino acid sequence was synthesized chemically as an antigen, coupled to KLH (Keyhole Limpet Hemocyanin) via a thiol group as an immunogen, and coupled to BSA via a thiol group as a screening agent.
(2) Immunization: the purified protein was diluted to an appropriate concentration, mixed with Freund's complete adjuvant 1:1, emulsified and injected subcutaneously into 6-week-old BALB/c female mice. Two weeks later, equal amounts of protein were mixed with Freund's incomplete adjuvant in equal proportions, emulsified and a second mouse immunization was performed. Immunization was repeated every two weeks since then. A booster immunization was performed until 3-4 days before cell fusion.
(3) Cell fusion, subcloning and ascites preparation: after neck support euthanasia is carried out on the mice, the spleen and the mouse myeloma line NS-1 cells are taken out in a sterile mode to carry out cell fusion, the fused cells are subjected to multiple subcloning culture by a limiting dilution method, and then cell strains with higher antibody levels are screened by ELISA (enzyme-Linked immuno sorbent assay) to carry out subsequent ascites preparation work.
(4) Antibody purification: the antibodies were purified by SPA affinity chromatography.
Example 1
A kit for quantitatively detecting a human thyroid stimulating hormone receptor antibody, comprising: the preparation method comprises the following steps of coating magnetic particles of an anti-human TSHR mouse monoclonal antibody A, an antigen reagent (an anti-human TSHR mouse monoclonal antibody B-recombinant human TSHR complex), a sample diluent, a horseradish peroxidase-labeled human thyroid stimulating hormone receptor antibody and a series of thyrotropin receptor antibody calibrator:
(1) Preparation of a magnetic particle suspension coated with anti-human TSHR murine monoclonal antibody a:
activating carboxyl magnetic microspheres with EDC and NHS under acidic condition, wherein the activating buffer is 0.1M MES (2- (N-morpholino) ethanesulfonic acid) buffer, the activating time is 30min, separating the carboxyl magnetic microspheres from the liquid under the action of a magnetic field after the activating is completed, discarding the supernatant, adding a proper amount of anti-human TSHR mouse monoclonal antibody A, blocking the carboxyl magnetic microspheres coated with the anti-human TSHR monoclonal antibody A with 0.01M PBS buffer containing 1% bovine serum albumin, adding a blocking solution after the blocking is completed to preserve the carboxyl magnetic microspheres coated with the anti-TSHR monoclonal antibody A, wherein the final concentration of the magnetic microspheres is 1mg/ml, and preserving the suspension at 2-8 ℃ for use.
(2) Preparation of antigen reagent: mixing 5 mug/mL of anti-human TSHR mouse monoclonal antibody B and 1/20 dilution ratio recombinant human TSHR in specific antigen freeze-drying liquid at 20-25 ℃ for 3h, preparing freeze-drying product, and re-dissolving with specific re-dissolving solution; preparing a freeze-dried solution from Bis-Tris buffer solution with the concentration of 0.05mol/L, mannitol with the concentration of 5wt%, sucrose with the concentration of 5wt%, bovine serum albumin with the concentration of 0.5wt% and PEG5000 with the concentration of 0.2 wt%; preparing a specific compound solution from 0.05mol/L Bis-Tris buffer solution and glycerol containing 2 wt%;
(3) Preparation of sample dilutions: the Bis-Tris buffer solution contains 3% bovine serum albumin and 0.2% PC300, and then is uniformly mixed and stored at the temperature of 2-8 ℃ for standby;
(4) The preparation method of the horseradish peroxidase-labeled human stimulatory thyroid stimulating hormone receptor antibody comprises the following steps: adding horseradish peroxidase-labeled human stimulatory thyroid stimulating hormone receptor antibody into Bis-Tris buffer solution containing 5% bovine serum albumin and 3% PC300, and uniformly mixing; the dilution ratio of the horseradish peroxidase marked human stimulating thyroid stimulating hormone receptor antibody is 1/1000-1/5000; preserving at 2-8 ℃ for standby;
(5) The series calibrator of the stimulating thyrotropin receptor antibody is a series calibrator with the concentration of 0-100 IU/L, and the preparation method of the calibrator comprises the following steps: adding 5% bovine serum albumin into Bis-Tris buffer solution, uniformly mixing to obtain a calibrator diluent, and diluting the stimulatory thyrotropin receptor antibody into six concentration points S0-S5 with the calibrator diluent, wherein the concentrations are 0IU/L, 2IU/L, 5IU/L, 10IU/L, 20IU/L and 50IU/L respectively.
The detection steps of the kit on a full-automatic chemiluminescence instrument A2000Plus are as follows:
(1) After preparing a test sample (a calibrator or a sample to be tested) and placing the test sample correctly, clicking a start button to perform a calibration procedure or a sample detection procedure, and performing the following operations by the instrument;
(2) Transferring the sample rack to a sample sucking position, and loading the reaction container to a sample adding position;
(3) When the calibration procedure was performed, 100 μl of calibrator, 20 μl of antigen reagent, and 50 μl of sample diluent were dispensed;
(4) When the sample detection program is executed, 100 mu L of sample, 20 mu L of antigen reagent and 50 mu L of sample diluent are dispensed;
(5) Uniformly mixing and incubating the reaction solution, wherein the temperature of the incubation is 37 ℃ and the time of the incubation is 17 minutes;
(6) The 20. Mu.L of the magnetic particle suspension coated with the anti-human TSHR murine monoclonal antibody A and 50. Mu.L of horseradish peroxidase-labeled human stimulatory thyroid stimulating hormone receptor antibody were dispensed;
(7) Uniformly mixing and incubating the reaction solution, wherein the temperature of the incubation is 37 ℃ and the time of the incubation is 15 minutes;
(8) After the incubation is completed, cleaning and separating the reaction liquid by using a cleaning liquid;
(9) Dispensing 50. Mu.L of substrate A and 50. Mu.L of substrate B was completed;
(10) Uniformly mixing the reaction liquid, and detecting the luminous intensity;
and establishing a calibration curve by taking the concentration value of the calibrator as an X axis and the logarithmic value of the luminous intensity of the calibrator as a Y axis, and recalculating the corresponding concentration value according to the luminous intensity value of the sample to be measured, wherein the calibration curve is shown in FIG. 1, and FIG. 1 is the calibration curve provided by the embodiment of the invention.
Comparative example 1
In contrast to example 1, the difference is that the antigen reagent was reconstituted without the use of anti-human TSHR murine monoclonal antibody B, directly after lyophilization of the recombinant human TSHR.
Example 2 stability assessment
Stability evaluation method: the test kits provided in comparative example 1 and example 1 were used to detect in parallel calibrator containing a series of concentrations of 0IU/L, 2IU/L, 5IU/L, 10IU/L, 20IU/L and 50IU/L of the stimulatory thyrotropin receptor antibody diluted with the calibrator dilution, respectively, the luminescence values of each group were measured according to the detection procedure on a full-automatic luminescence apparatus A2000Plus system, the decrease of the signal value by less than or equal to 10% was regarded as good stability, and as a result, see Table 1, FIG. 2, FIG. 3 and FIG. 4, table 1 is the stability evaluation results of the test kits provided in examples and comparative examples of the present invention, FIG. 2 is the stability evaluation results of the antigen reagents provided in examples and comparative examples of the present invention at 20 to 25℃in the lyophilized solution, wherein curve a is the stability evaluation result of the antigen reagent provided in example 1, 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 examples and comparative examples according to the present invention at 2 to 8℃in a lyophilized liquid, wherein curve a is the stability evaluation result of the antigen reagent provided in example 1, curve b is the stability evaluation result of the antigen reagent provided in comparative example 1, FIG. 4 is the stability evaluation result of the antigen reagent provided in examples and comparative examples according to the present invention at 2 to 8℃after reconstitution, 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.
TABLE 1 stability evaluation results of the kits provided in examples and comparative examples of the present invention
Note that: and/indicates none.
As can be seen from Table 1, FIG. 2, FIG. 3 and FIG. 4, in the kit provided by the invention, the anti-TSHR mouse monoclonal antibody B-human TSHR immune complex is stabilized in the freeze-dried liquid at 20-25 ℃ for 128h, is stabilized in the freeze-dried liquid at 2-8 ℃ for 168h, and meanwhile, the stability of the antigen reagent after redissolution meets 2-8 ℃ for 28 days.
Example 3 alignment of kits
Human thyroid stimulating hormone receptor antibody quantitative detection kit and kit C constructed according to example 1: kit for quantitative detection of thyroid stimulating hormone receptor antibody (electrochemiluminescence method), kit D: the detection and comparison of clinical samples are carried out by the human thyroid stimulating hormone receptor antibody detection kit (enzymatic chemiluminescence method), and the repeatability and the interference of the three detection and quantification kits are analyzed and compared.
The detection principle of the kit C is as follows: the competition method, which uses streptavidin to coat magnetic particles, uses soluble pig TSHR and biotinylation anti-pig TSHR C-end mouse monoclonal antibody immune complex and ruthenium complex marked human monoclonal autoantibody M22 to determine;
the detection principle of the kit D is as follows: the competition method comprises the steps of coating magnetic particles with FITC antibodies, preparing an antigen reagent by using FITC-labeled anti-human TSHR mouse monoclonal antibody-human TSHR immune complex, and jointly competing human TSHR immune complex in the antigen reagent by horseradish peroxidase-labeled TRAb and TRAb in a sample;
the detection method comprises the following steps: 193 cases of gradient serum samples are examined in parallel by adopting the kit, the kit C and the kit D, wherein 103 cases of negative samples are subjected to clinical correlation analysis, the correlation of detection results is shown in FIG. 5, FIG. 5 is a correlation curve chart of detection results of the kit and other kits provided by the embodiment of the invention, FIG. 5 (A) is a correlation curve chart of the kit C, and FIG. 5 (B) is a correlation curve chart of the kit D.
As can be seen from FIG. 5, 193 clinical serum samples are measured simultaneously by the kit of the invention, the kit C and the kit D, the correlation between the kit of the invention and the measurement results of the kit is good, and the correlation coefficient R is good 2 Between 0.958 and 0.997.
Example 4 repeatability assessment
The examination mode is as follows: patient samples with low concentration level and high concentration level (1.83 IU/L;24.37 IU/L) are selected, detection is carried out by adopting the kit and the method provided in the embodiment 1, the kit D and the method thereof, each sample is continuously and repeatedly measured for 20 times, an Average Value (AV), a Standard Deviation (SD) and a variation Coefficient (CV) are calculated, the results are shown in the table 2, and the table 2 is the repeated check result of the kit provided in the embodiment 1:
TABLE 2 repeatability test results of the kit provided in example 1 of the present invention
The result shows that compared with the similar products, the kit has better repeatability.
Example 5 interference assessment:
patient samples with low concentration level and high concentration level (3.58 IU/L;20.13 IU/L) are selected, 10ng/mL of biotin is added into the patient samples, and the detection is carried out by adopting the kit C and the kit provided in the example 1 respectively; to this, 0.5. Mu.g/mL of sodium fluorescein was added, and the test was performed using the kit D and the kit provided in example 1, respectively, with the results shown in Table 3, and the results of the interference test for the kits provided in examples of the present invention and comparative examples are shown in Table 3.
TABLE 3 interference test assessment results for the kits provided in examples and comparative examples of the present invention
The TRAb detection kit specification of kit C states that the assay results are high when the biotin concentration in the sample is > 41nmol/L or 10 ng/mL; the TRAb detection kit specification of kit D states that when the concentration of fluorescein in the sample exceeds 0.5ug/mL, it can result in a detection result that varies by more than 10%; when the kit is used for detecting samples added with 10ng/mL of biotin or 0.5ug/mL of sodium fluorescein, the detection result is not affected (the change of the result is within +/-10 percent), and the result shows that the kit can avoid related interference introduced by a biotin-avidin system or a FITC-anti-FITC system, and improves the anti-interference performance of the kit.
Example 6
The quantitative detection kit for the human thyrotropic hormone receptor antibody comprises magnetic particles coated with an anti-porcine TSHR mouse monoclonal antibody A, an antigen reagent (anti-porcine TSHR mouse monoclonal antibody B-recombinant porcine TSHR), an alkaline phosphatase-labeled human thyrotropic hormone receptor antibody and a series of thyrotropic hormone receptor antibody calibrator, and the preparation process comprises the following steps:
(1) Preparation of a magnetic particle suspension coated with anti-porcine TSHR murine monoclonal antibody a: activating carboxyl magnetic microspheres with EDC and NHS under acidic condition, wherein the activating buffer is 0.1M MES (2- (N-morpholino) ethanesulfonic acid) buffer, the activating time is 30min, separating the carboxyl magnetic microspheres from the liquid under the action of a magnetic field after the activating is completed, discarding the supernatant, adding a proper amount of anti-pig TSHR mouse monoclonal antibody A, blocking the carboxyl magnetic microspheres coated with the anti-human TSHR monoclonal antibody A with 0.01M PBS buffer containing 1% bovine serum albumin, adding a blocking solution after the blocking is completed to preserve the carboxyl magnetic microspheres coated with the anti-TSHR monoclonal antibody A, wherein the final concentration of the magnetic microspheres is 1mg/ml, and preserving the suspension at 2-8 ℃ for use.
(3) Preparation of antigen reagent: mixing 5ug/mL of anti-pig TSHR mouse monoclonal antibody B and 1/30 dilution ratio recombinant pig TSHR in specific antigen freeze-drying liquid at 20-25 ℃ for reaction for 3h, preparing a freeze-dried product, and re-dissolving by using specific re-dissolving solution; preparing a freeze-dried solution from Bis-Tris buffer solution with the concentration of 0.05mol/L, mannitol with the concentration of 5wt%, sucrose with the concentration of 5wt%, bovine serum albumin with the concentration of 0.5wt% and PEG5000 with the concentration of 0.2 wt%; preparing a specific compound solution from 0.05mol/L Bis-Tris buffer solution and glycerol containing 2 wt%;
(3) Preparation of sample dilutions: the Bis-Tris buffer solution contains 1-3% of bovine serum albumin and 0.1-0.3% of PC300, and then the mixture is uniformly mixed and stored at the temperature of 2-8 ℃ for standby;
(4) The preparation method of the alkaline phosphatase-labeled human stimulatory thyroid stimulating hormone receptor antibody comprises the following steps: adding alkaline enzyme marked human stimulatory thyroid stimulating hormone receptor antibody into Bis-Tris buffer solution containing 5% bovine serum albumin and 0.1-5% PC300, then uniformly mixing, and preserving at 2-8 ℃ for later use;
(5) The series calibrator of the stimulating thyrotropin receptor antibody is a series calibrator with the concentration of 0-100 IU/L, and the preparation method of the calibrator comprises the following steps: adding 5% bovine serum albumin into Bis-Tris buffer solution, uniformly mixing to obtain a calibrator diluent, and diluting the stimulatory thyrotropin receptor antibody into six concentration points S0-S5 with the calibrator diluent, wherein the concentrations are 0IU/L, 2IU/L, 5IU/L, 10IU/L, 20IU/L and 50IU/L respectively;
stability assessment:
stability evaluation method: the kit provided in example 6 was used to detect in parallel a calibrator containing a series of concentrations of 0IU/L, 2IU/L, 5IU/L, 10IU/L, 20IU/L, and 50IU/L of the stimulatory thyrotropin receptor antibodies diluted with the calibrator dilution, and the luminescence values of each group were obtained by performing the detection steps on an A2000Plus system of a full-automatic luminescence apparatus. The stability is better when the signal value is reduced by less than or equal to 10%, the result is shown in table 4, and table 4 is the stability evaluation result of the kit provided in the embodiment 6 of the invention.
TABLE 4 evaluation results of stability of the kit provided in example 6 of the present invention
/>
Note that: and/indicates none.
As can be seen from Table 4, the kit provided in example 6 was relatively stable.
Interference assessment:
patient samples of low and high (3.58 IU/L;20.13 IU/L) concentration levels were selected, 10ng/mL biotin was added thereto, and detection was performed using kit C and the kit provided in example 6, respectively; to this, 0.5. Mu.g/mL sodium fluorescein was added, and the test was performed using the kit D and the kit provided in example 6, respectively, with the results shown in Table 5, and Table 5 shows the results of the interference test for the kits provided in example 6 and comparative examples of the present invention.
TABLE 5 interference test assessment results for the kits provided in example 6 and comparative example of the present invention
As can be seen from Table 5, when the kit of the invention detects a sample added with 10ng/mL of biotin or 0.5ug/mL of sodium fluorescein, the detection result is not affected (the result changes within + -10%), and the result shows that the kit of the invention can avoid related interference introduced by a biotin-avidin system or a FITC-anti-FITC system, thereby improving the anti-interference performance of the kit.
Example 7
The quantitative detection kit for the human thyroid stimulating hormone receptor antibody comprises magnetic particles coated with an anti-pig TSHR mouse monoclonal antibody A, an antigen reagent (anti-pig TSHR mouse monoclonal antibody B-natural pig TSHR), a sample diluent, an acridine ester labeled human thyroid stimulating hormone receptor antibody and a series of calibrator for the thyroid stimulating hormone receptor antibody, and the preparation process is as follows:
(1) Preparation of a magnetic particle suspension coated with anti-porcine TSHR murine monoclonal antibody a: activating carboxyl magnetic microspheres with EDC and NHS under acidic condition, wherein the activating buffer is 0.1M MES (2- (N-morpholino) ethanesulfonic acid) buffer, the activating time is 30min, separating the carboxyl magnetic microspheres from the liquid under the action of a magnetic field after the activating is completed, discarding the supernatant, adding a proper amount of anti-pig TSHR mouse monoclonal antibody A, blocking the carboxyl magnetic microspheres coated with the anti-human TSHR monoclonal antibody A with 0.01M PBS buffer containing 1% bovine serum albumin, adding a blocking solution after the blocking is completed to preserve the carboxyl magnetic microspheres coated with the anti-TSHR monoclonal antibody A, wherein the final concentration of the magnetic microspheres is 1mg/ml, and preserving the suspension at 2-8 ℃ for use.
(2) Preparation of antigen reagent: mixing 5ug/mL of anti-pig TSHR mouse monoclonal antibody B with 1/10 dilution ratio of natural pig TSHR in specific antigen freeze-drying liquid at 20-25 ℃ for 3h, preparing freeze-drying product, and re-dissolving with specific re-dissolving solution; preparing a freeze-dried solution from Bis-Tris buffer solution with the concentration of 0.05mol/L, mannitol with the concentration of 5wt%, sucrose with the concentration of 5wt%, bovine serum albumin with the concentration of 0.5wt% and PEG5000 with the concentration of 0.2 wt%; preparing a specific compound solution from 0.05mol/L Bis-Tris buffer solution and glycerol containing 2 wt%;
(3) Preparation of sample dilutions: the Bis-Tris buffer solution contains 1-3% of bovine serum albumin and 0.1-0.3% of PC300, and then the mixture is uniformly mixed and stored at the temperature of 2-8 ℃ for standby;
(4) The preparation method of the human stimulatory thyroid stimulating hormone receptor antibody marked by the marker comprises the following steps: adding acridinium ester marked human stimulated thyroid stimulating hormone receptor antibody into Bis-Tris buffer solution containing 5% bovine serum albumin and 0.1-5% PC300, then uniformly mixing, and preserving at 2-8 ℃ for later use;
(5) The series calibrator of the stimulating thyrotropin receptor antibody is a series calibrator with the concentration of 0-100 IU/L, and the preparation method of the calibrator comprises the following steps: adding 5% bovine serum albumin into Bis-Tris buffer solution, uniformly mixing to obtain a calibrator diluent, and diluting the stimulatory thyrotropin receptor antibody into six concentration points S0-S5 with the calibrator diluent, wherein the concentrations are 0IU/L, 2IU/L, 5IU/L, 10IU/L, 20IU/L and 50IU/L respectively.
Stability assessment:
stability evaluation method: the kit prepared in example 7 was used to detect a calibrator containing a series of concentrations of 0IU/L, 2IU/L, 5IU/L, 10IU/L, 20IU/L and 50IU/L of the stimulatory thyrotropin receptor antibody diluted with the calibrator dilution in parallel, and the luminescence values of each group were obtained by performing the detection steps on an full-automatic luminescence apparatus A2000Plus system. The stability is better when the signal value is reduced by less than or equal to 10%, the result is shown in table 6, and table 6 is the stability evaluation result of the kit provided in the embodiment 7 of the invention.
TABLE 6 stability evaluation results of the kit provided in example 7 of the present invention
/>
Note that: and/indicates none.
As can be seen from Table 6, the kit provided in example 7 was relatively stable.
Interference assessment:
patient samples of low and high (3.58 IU/L;20.13 IU/L) concentration levels were selected, 10ng/mL biotin was added thereto, and detection was performed using kit C and the kit provided in example 7, respectively; to this, 0.5. Mu.g/mL sodium fluorescein was added, and the test was performed using the kit D and the kit provided in example 7, respectively, with the results shown in Table 7, and Table 7 shows the results of the interference test for the kits provided in example 7 and comparative example of the present invention.
TABLE 7 interference test assessment results for the kits provided in example 7 and comparative example of the present invention
As can be seen from Table 7, when the kit of the present invention is used for detecting samples added with 10ng/mL of biotin or 0.5ug/mL of sodium fluorescein, the detection result is not affected (the result changes within + -10%), and the above results indicate that the present invention can avoid the relevant interference introduced by biotin-avidin system or FITC-anti-FITC system, and improve the anti-interference performance of the kit.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.
Claims (9)
1. A kit for quantitatively detecting a human thyroid stimulating hormone receptor antibody, comprising: a solid phase carrier coated with an anti-TSHR monoclonal antibody a; the recognition site of the anti-TSHR monoclonal antibody A is positioned at 414-764 positions of the TSHR amino acid sequence;
an antigenic agent which is an immune complex of anti-TSHR monoclonal antibody B and TSHR; the recognition site of the monoclonal antibody B is positioned at 661-764 positions of the TSHR amino acid sequence;
a human stimulatory thyroid stimulating hormone receptor antibody comprising a label;
the anti-TSHR monoclonal antibody a does not produce competitive binding with the anti-TSHR monoclonal antibody B.
2. The kit of claim 1, further comprising a sample diluent and/or a series of stimulatory thyroid stimulating hormone receptor antibodies calibrator.
3. A kit according to claim 1, wherein the recognition site for monoclonal antibody B is located at positions 665-698 of the TSHR amino acid sequence.
4. A kit according to claim 1 or 2, wherein the anti-TSHR monoclonal antibody a and anti-TSHR monoclonal antibody B are independently selected from murine monoclonal antibodies, rabbit monoclonal antibodies, sheep monoclonal antibodies, human monoclonal antibodies and chimeric monoclonal antibodies.
5. A kit according to claim 1 or claim 2 wherein the antigenic agent is obtained by reacting anti-TSHR monoclonal antibody B and TSHR in a lyophilizate or by reacting anti-TSHR monoclonal antibody B and TSHR in a lyophilizate, freeze-drying and reconstitution.
6. The kit according to claim 1 or 2, wherein the solid support is a magnetic microsphere surface modified with amino, carboxyl, aldehyde, phenylhydrazine or sulfonic acid groups.
7. The kit according to claim 1 or 2, wherein the human stimulatory thyroid stimulating hormone receptor antibody containing a label selected from the group consisting of ruthenium compounds, acridine compounds, peroxidases and alkaline phosphatases.
8. A thyroid stimulating hormone receptor antigen reagent comprising: immune complexes against TSHR monoclonal antibodies B and TSHR;
the recognition site of monoclonal antibody B is located at positions 661-764 of the amino acid sequence of the TSHR.
9. An antigenic agent as claimed in claim 8 wherein the anti-TSHR monoclonal antibody B and TSHR are reacted in a lyophilizate or the anti-TSHR monoclonal antibody B and TSHR are reacted in a lyophilizate, lyophilized and reconstituted.
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