Anti-thyroglobulin antibody, kit and application

Abstract

The invention relates to an anti-thyroglobulin antibody, a kit and application. Specifically, the invention provides an anti-thyroglobulin specific antibody, a kit containing the antibody, and an application of the antibody in detecting the existence or the level of thyroglobulin and / or a thyroglobulin autoantibody in a sample.

Description

Technical Field

[0001] The invention belongs to the field of molecular immunology, and in particular relates to an anti-thyroglobulin specific antibody, a kit comprising the antibody and uses thereof. Background Art

[0002] Thyroglobulin (Tg) is a 660 kDa protein homodimer with a large number (approximately 60) disulfide bonds per monomer and is heavily glycosylated at 17 glycosylation sites. Tg is produced by thyroid follicular cells (thyrocytes) and stored in the follicular colloid of the gland, constituting 80% of the total thyroid protein. Tg functions as a prohormone for the synthesis of the iodinated tyrosine hormones thyroxine (T4) and triiodothyronine (T3) within the thyroid gland. Nearly 30 of the 66 tyrosine residues of Tg are iodinated; proteolysis of the polypeptide chain in lysosomes results in the release of T3 and T4 into the cytoplasm of thyrocytes. T3 and T4 are subsequently secreted into the blood.

[0003] Thyroglobulin antibodies (TgAbs) are usually associated with thyroid autoimmune diseases (AITD): such as Hashimoto's thyroiditis (HT) or Graves' disease (GD), but they also exist in one in ten healthy individuals. In addition, TgAbs are also found in differentiated thyroid cancer (DTC): TgAbs-positive sera are more than twice as common in DTC patients as in the general population (25% vs. 10%). This makes TgAbs a prognostic indicator for the risk assessment of DTC development.

[0004] Since Tg and TgAbs are often present simultaneously in patient serum, these two analytes can interfere with each other in immunoassays. Tg is widely used as a biomarker in the follow-up of patients with DTC after thyroidectomy: Tg is a thyroid-specific protein and an increase in Tg concentration during follow-up predicts tumor recurrence or metastasis. In this setting, the presence of Tg antibodies in a sample may result in a falsely low Tg value. It is common practice to measure TgAbs in all samples undergoing Tg evaluation.

[0005] The epitopes recognized by human antibodies on macromolecular Tg are usually conformational and cannot be determined using synthetic peptides. Serological studies have shown that there are many antigenic epitopes on Tg, and there are at least 40 such epitopes on human Tg. It was also found that TgAbs antibodies from AITD patients have a different binding pattern from natural TgAbs antibodies. The specificity of TgAbs in DTC patients may also be different from that in AITD patients or healthy individuals. The fact that various TgAbs exist in the blood of a single individual is also extremely relevant to clinical significance.

[0006] Currently, several immunochemical methods are used for TgAbs measurement, such as indirect immunoassay, double antigen sandwich immunoassay or competitive immunoassay. In competitive immunoassay, a labeled antibody that specifically binds to Tg is used as a tracer; TgAbs in the sample compete with the tracer for binding sites on the Tg molecule. The quantitative method for TgAbs in human serum developed by BRAHMS Diagnostica GmbH (Bergmann, A, US / 5,919,632) is based on human TgAbs purified from human serum as tracers.

[0007] The main problems with the use of polyclonal antibodies in immunoassays are suboptimal binding properties, limited amounts of material, and their irreproducibility, especially in the case of human-derived preparations. The reproducibility and stability of the assay can also become a complicating issue. In TgAbs measurements, the epitope specificity and affinity of the antibodies used as tracers are crucial for the assay performance. Therefore, there is a need to develop immunoassays for TgAbs assessment that are based on antibodies with a combination of binding characteristics of specificity and affinity that enable them to competitively bind to Tg with all major TgAbs present in the human population. Summary of the invention

[0008] The present invention describes a monoclonal antibody specific for human Tg, in particular, the binding characteristics (epitope specificity and affinity) of the antibody are comparable to those of the major TgAbs autoantibodies present in the human population, and the antibody can compete with TgAbs in a sample for binding to Tg, thereby being applied to the measurement of TgAbs in a sample.

[0009] Antibodies, nucleic acid molecules, expression vectors, host cells

[0010] In one aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to Tg (thyroglobulin), comprising:

[0011] A heavy chain variable region comprising the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 1, 7, 13, 19, 25, 31 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 2, 8, 14, 20, 26, 32 and variants thereof, and / or a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 3, 9, 15, 21, 27, 33 and variants thereof; and / or,

[0012] A light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 4, 10, 16, 22, 28, 34 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 5, 11, 17, 23, 29, 35 and variants thereof, and / or a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 6, 12, 18, 24, 30, 36 and variants thereof;

[0013] wherein the variant contains an amino acid mutation compared to the amino acid sequence from which it is derived, and the amino acid mutation is a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids); preferably, the substitution is a conservative substitution;

[0014] Preferably, the variant is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence from which it is derived;

[0015] Preferably, the three CDRs contained in the heavy chain variable region, and / or the three CDRs contained in the light chain variable region, are defined by the Kabat numbering system.

[0016] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0017] (1a) a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 1 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 2 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 3 or a variant thereof; and, a light chain variable region comprising the following three CDRs: CDR-L1 having an amino acid sequence of SEQ ID NO: 4 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 5 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 6 or a variant thereof;

[0018] (1b) a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 7 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 8 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 9 or a variant thereof; and, a light chain variable region comprising the following three CDRs: CDR-L1 having an amino acid sequence of SEQ ID NO: 10 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 11 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 12 or a variant thereof;

[0019] (1c) a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 13 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 14 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 15 or a variant thereof; and, a light chain variable region comprising the following three CDRs: CDR-L1 having an amino acid sequence of SEQ ID NO: 16 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 17 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 18 or a variant thereof;

[0020] (1d) a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 19 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 20 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 21 or a variant thereof; and, a light chain variable region comprising the following three CDRs: CDR-L1 having an amino acid sequence of SEQ ID NO: 22 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 24 or a variant thereof;

[0021] (1e) a heavy chain variable region comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 25 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 26 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 27 or a variant thereof; and, a light chain variable region comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 28 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 29 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 30 or a variant thereof; or

[0022] (1f) a heavy chain variable region comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 31 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 32 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 33 or a variant thereof; and a light chain variable region comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 34 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 35 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 36 or a variant thereof.

[0023] In some embodiments, the heavy chain variable region also includes a heavy chain framework region (FR). In some embodiments, the light chain variable region also includes a light chain framework region (FR). The framework region is highly conserved, and it provides a scaffold for the six CDRs in three-dimensional space to form an antigen binding surface. The variable domains of naturally occurring heavy and light chains each include four FR regions (FR1, FR2, FR3, and FR4), and these four FR regions mainly use a β-folded configuration, connected by three hypervariable regions, which form a loop connecting the β-folded structure, and in some cases form a part of the β-folded structure. The hypervariable regions in each chain are closely together through FR, and together with the hypervariable regions from another chain, they contribute to the formation of the antigen binding side end (see Kabat et al., cited above). The heavy chain framework region and / or the light chain framework region can be independently derived from the framework region of any species immunoglobulin. In some embodiments, the heavy chain framework region and the light chain framework region are independently derived from the heavy chain framework region and the light chain framework region of human or mouse immunoglobulin. In some embodiments, the heavy chain framework region and the light chain framework region each independently comprise an amino acid sequence derived from a heavy chain framework region and a light chain framework region of a mouse immunoglobulin, a heavy chain framework region and a light chain framework region of a human immunoglobulin, or a combination thereof. In some embodiments, the heavy chain framework region and the light chain framework region each independently comprise an amino acid sequence derived from a heavy chain framework region and a light chain framework region of a human germline antibody.

[0024] In some embodiments, the antibody or its antigen-binding fragment further comprises a heavy chain constant region (CH) and a light chain constant region (CL). In some embodiments, the antibody or its antigen-binding fragment comprises a mouse heavy chain constant region and a mouse light chain constant region. In some embodiments, the antibody or its antigen-binding fragment comprises a rat heavy chain constant region and a rat light chain constant region. In some embodiments, the antibody or its antigen-binding fragment comprises a human heavy chain constant region and a human light chain constant region. In some embodiments, the antibody or its antigen-binding fragment is an IgG, IgM, IgE, IgD or IgA antibody. In some embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, and for example a mouse IgG1, IgG2a, IgG2b or IgG3 constant region. In some embodiments, the light chain constant region is a κ or λ light chain constant region (eg, a human λ light chain constant region).

[0025] In some embodiments, the antigen-binding fragment is selected from scFv, Fab, Fab', (Fab')2, Fd, Fv, CDR fragment, nanobody, disulfide-linked Fv (dsFv), diabody, bispecific antibody and multispecific antibody; and / or, the antibody is a murine antibody, a chimeric antibody or a humanized antibody.

[0026] In some embodiments, the antibody or antigen-binding fragment thereof is further detectably labeled.

[0027] In this context, the detectable label may be any substance detectable by fluorescent, spectral, photochemical, biochemical, immunological, electrical, optical or chemical means. It is particularly preferred that such labels are suitable for use in immunological assays (e.g., enzyme-linked immunosorbent assays, radioimmunoassays, fluorescent immunoassays, chemiluminescent immunoassays, etc.). Such labels are well known in the art and include, but are not limited to, enzymes (e.g., oxidases, microperoxidases, horseradish peroxidases, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3 H. 125 I. 35 S. 14 C or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750), europium and green fluorescent protein, etc.), chemiluminescent substances (e.g., luminol, isoluminol, phenanthridinium, acridinium ester compounds, etc.), and biotin for binding to avidin modified with the above-mentioned markers (e.g., streptavidin). The markers encompassed in the present invention can be detected by methods known in the art. For example, radioactive labels can be detected using photographic film or a scintillation counter, and fluorescent markers can be detected using a photodetector to detect the emitted light. Enzyme markers are generally detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate. Chemiluminescent substances (e.g., acridinium ester compounds) are generally detected by providing an excitation liquid and / or a catalyst to the luminescent substance to detect the emitted light. Biotin is generally detected by providing biotin with avidin (e.g., streptavidin) modified with the above-mentioned label and detecting the label carried by the avidin connected to biotin. In certain embodiments, the detectable label as described above can be connected to the antibody or antigen-binding fragment thereof of the present invention through a linker of different lengths to reduce potential steric hindrance.

[0028] In some embodiments, the detectable label is selected from fluorescein, chemiluminescent substances (eg, acridinium ester compounds), enzymes (eg, horseradish peroxidase, alkaline phosphatase), radioactive isotopes, biotin, colloidal gold, and magnetic particles.

[0029] In some embodiments, the antibody or antigen-binding fragment thereof competes with TgAbs (thyroglobulin antibodies) for binding to Tg.

[0030] In another aspect, the present invention provides an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof as described in any one of the preceding items.

[0031] In another aspect, the present invention provides an expression vector comprising the isolated nucleic acid molecule described in any of the above. In some embodiments, the vector is a plasmid, a virus, a phage, a bacterium or a viroid.

[0032] In another aspect, the present invention provides a host cell comprising the isolated nucleic acid molecule or expression vector described in any of the foregoing. In some embodiments, the host cell is a eukaryotic cell, preferably a mammalian cell. In some embodiments, the host cell is a prokaryotic cell, preferably an Escherichia coli.

[0033] In another aspect, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to Tg, wherein the antibody or antigen-binding fragment thereof:

[0034] Produced by the hybridoma cell line The hybridoma of TG01 deposited in the Russian National Collection of Industrial Microorganisms (VKPM), with a deposit number of H-223 and a deposit date of June 4, 2024;

[0035] Escherichiacoli Rosetta deposited with VKPM on June 26, 2024, under the number B-14843 TM (DE3)pLysS hTG02 VH plasmid and Escherichia coli Rosetta with accession number B-14844 TM (DE3) plasmid production in pLysS hTG02 VL;

[0036] Escherichiacoli Rosetta deposited with VKPM on June 26, 2024, under the number B-14845 TM (DE3)pLysS hTG03 VH plasmid and Escherichia coli Rosetta with the accession number B-14846 TM Plasmid production in (DE3)pLysS hTG03 VL;

[0037] Escherichiacoli Rosetta deposited with VKPM on June 26, 2024, under the number B-14847 TM (DE3)pLysS hTG04 ​​VH plasmid and Escherichia coli Rosetta with accession number B-14848 TM (DE3) plasmid production in pLysS hTG04 ​​VL;

[0038] Escherichiacoli Rosetta deposited with VKPM on June 26, 2024, under the number B-14849 TM (DE3)pLysS hTG05 VH plasmid and Escherichia coli Rosetta with accession number B-14850 TM (DE3)pLysS hTG05 VL plasmid production; or

[0039] Escherichiacoli Rosetta deposited with VKPM on June 26, 2024, under the number B-14851 TM(DE3) Plasmid generation in pLysS hTG06.

[0040] Reagent test kit

[0041] The consistency between the current mainstream TgAbs detection methods and clinical diagnosis needs to be improved, and there are obvious differences in the methodological comparisons between different commercial reagents. For patients diagnosed with autoimmune thyroid diseases (Hashimoto's thyroiditis, Graves' disease, etc.), the positive rate of TgAbs varies between different types of diseases. The positive rate of patients diagnosed with Hashimoto's thyroiditis is about 50%-80%, and the positive rate of patients diagnosed with Graves is about 30%-50%. Even if the judgment can be assisted by adding the detection of Anti-TPO from the same manufacturer, the positive rate of Anti-TPO varies significantly among different types of patients. Therefore, clinical diagnosis and medication guidance often need to be based on the results of puncture or imaging, but these testing methods take a long time to process and will cause more pain and burden to patients.

[0042] The binding characteristics (epitope specificity and affinity) of the antibody specifically binding to human Tg of the present invention are comparable to those of the main TgAbs autoantibodies present in the human population, and can compete with TgAbs in the sample for binding to Tg, and can be used for the measurement of TgAbs. On this basis, the present invention provides a kit with a higher clinical compliance rate for in vitro detection of autoimmune thyroid diseases (Hashimoto's thyroiditis, Graves' disease, etc.).

[0043] Specifically, an object of the present invention is to provide an immunoassay kit, which contains at least one antibody or antigen-binding fragment thereof as described above.

[0044] An object of the present invention is an immunoassay kit comprising:

[0045] A first reagent, which contains Tg antigen;

[0046] The second reagent contains at least one antibody or antigen-binding fragment thereof as described in any one of the preceding items.

[0047] In some embodiments, the Tg antigen is a natural or recombinant Tg antigen.

[0048] In some embodiments, one of the Tg antigen and the antibody or its antigen-binding fragment is detectably labeled, and the other is coated on the surface of a solid carrier.

[0049] In some embodiments, the solid support is selected from magnetic particles or microtiter plates (eg, microtiter plates or ELISA plates).

[0050] In some embodiments, the detectable label is selected from fluorescein, a chemiluminescent label (eg, acridinium ester compounds), an enzyme (eg, horseradish peroxidase, alkaline phosphatase), a radioactive isotope, biotin, and colloidal gold.

[0051] In some embodiments, the first reagent further contains one or more of the following ingredients: a buffer solution, an inorganic salt, a preservative, a surfactant, and a stabilizer; and / or

[0052] The second reagent further contains one or more of the following components: a buffer solution, an inorganic salt, a preservative, a surfactant and a stabilizer.

[0053] In some embodiments, the components in the first reagent and the Tg antigen are placed in the same or different formulation units.

[0054] In some embodiments, the components of the second reagent and the antibody or antigen-binding fragment thereof are placed in the same or different formulation units.

[0055] In some embodiments, the buffer solution is selected from Tris, Hepes and PBS buffer solutions.

[0056] In some embodiments, the inorganic salt is selected from one or more of NaCl, KCl, CaCl2, MgCl2 and ZnCl2.

[0057] In some embodiments, the preservative is selected from Proclin 300, PC-300, BND (eg, BND-10 or BND-99), and BIT (eg, BIT-10).

[0058] In some embodiments, the surfactant is selected from Tween-20, TritonX-100 and S9.

[0059] In some embodiments, the stabilizer is selected from calf serum, bovine serum albumin, and gelatin.

[0060] In some embodiments, the Tg antigen is coated on the surface of the solid phase carrier, and the antibody or antigen-binding fragment thereof carries a detectable label.

[0061] In some embodiments, the Tg antigen is coated on superparamagnetic microparticles. In some embodiments, the content of the superparamagnetic microparticles in the first reagent is 0.01%-0.1%, such as 0.03%-0.1%, and further such as 0.05%-0.1%.

[0062] In some embodiments, the working concentration of the Tg antigen (referring to the concentration of the Tg antigen in the first reagent when used for measurement) is 0.01%-0.20%, such as 0.01%-0.15% or 0.05%-0.15%.

[0063] In some embodiments, the working concentration of the antibody or antigen-binding fragment thereof (referring to the concentration of the antibody or antigen-binding fragment thereof in the second reagent when used for measurement) is 0.05-10 μg / mL, for example 1-5 μg / mL, specifically 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL or 5 μg / mL.

[0064] In some embodiments, the kit may further include a reagent for detecting the corresponding detectable label (a substrate for the detectable label described above). For example, when the detectable label is an enzyme, the kit may also include a chromogenic substrate for the corresponding enzyme, such as o-phenylenediamine (OPD), tetramethylbenzidine (TMB), ABTS or luminol compounds for horseradish peroxidase, or p-nitrophenyl phosphate (p-NPP) or AMPPD for alkaline phosphatase. For example, when the detectable label is a chemiluminescent reagent (e.g., an acridinium ester compound), the kit may also include a pre-excitation solution and / or an excitation solution for chemiluminescence.

[0065] In some embodiments, the kit includes one or more of the following: a Tg calibrator, a detectably labeled substrate, a wash solution, a stop solution, and instructions for use.

[0066] The binding properties of the antibody of the present invention are comparable to the main TgAbs autoantibodies present in the human population, and can compete with the TgAbs in the sample for binding to the Tg antigen, and the TgAbs content in the sample is determined by quantitatively testing the enzyme-labeled antibodies in the test group and the control group (without adding the sample). Based on the measured TgAbs content, further diagnosis can be made on the individual providing the sample.

[0067] Therefore, the immunoassay kit of the present invention can be used to detect Tg and / or TgAbs, in particular the presence or level of Tg and / or TgAbs in a sample.

[0068] In some embodiments, the sample is from a human, such as a human body fluid (eg, blood, serum, or plasma).

[0069] In some embodiments, the immunoassay kit has a positive diagnostic rate of no less than 80% for thyroiditis (eg, Hashimoto's thyroiditis), and / or a positive diagnostic rate of no less than 60% for goiter (eg, Graves' disease).

[0070] In another aspect, the present invention provides a kit for competitive ELISA, comprising a Tg antigen and a Tg antibody, wherein:

[0071] For patients with clinically confirmed thyroiditis, the consistency rate between the diagnosis result using the kit and the clinical diagnosis result is not less than 80%, and / or,

[0072] For patients with clinically confirmed goiter, the consistency rate between the diagnosis result using the kit and the clinical diagnosis result is not less than 60%.

[0073] In some embodiments, the Tg antibody is selected from any of the antibodies or antigen-binding fragments thereof described above.

[0074] Detection Methods

[0075] Based on the antibody and / or kit of the present invention, the present invention further provides a method for detecting the presence or level of Tg and / or TgAbs in a sample, which comprises the step of using the antibody or antigen-binding fragment thereof or immunoassay kit described in any one of the preceding items for detection.

[0076] In some embodiments, the method comprises the following steps:

[0077] The sample is reacted with the first reagent and the second reagent, and quantitative analysis is performed to obtain the presence or concentration of TgAbs in the sample. In some embodiments, the reaction and quantitative analysis are performed in a chemiluminescent immunoassay analyzer.

[0078] In some embodiments, the method further comprises a step of calibrating with a Tg antigen calibrator. In some embodiments, the Tg antigen calibrator is a natural or recombinant Tg antigen.

[0079] In some embodiments, the limit of detection (LOD) of TgAbs in a sample is <10 IU / mL, preferably <5 IU / mL, more preferably <1 IU / mL.

[0080] In some embodiments, the detection range of TgAbs in the sample is 0.1-5000 IU / mL, such as below 4500 IU / mL, below 4000 IU / mL, or below 3500 IU / mL.

[0081] In some embodiments, the method is an ELISA immunoassay, such as a competitive ELISA immunoassay.

[0082] In some embodiments, the sample is from a human, such as a human body fluid (eg, blood, serum, or plasma).

[0083] application

[0084] Thyroid autoimmune diseases can be diagnosed based on the quantitative detection of TgAbs.

[0085] Specifically, the present invention provides a use of the antibody or antigen-binding fragment thereof or the kit described in any one of the above in the preparation of a kit, wherein the kit is used for:

[0086] (1) detecting the presence or level of Tg and / or TgAbs in a sample;

[0087] (2) Diagnosis of thyroid autoimmune disease (eg, thyroiditis or goiter, preferably Hashimoto's thyroiditis or Graves' disease) or thyroid cancer (eg, differentiated thyroid cancer).

[0088] In another aspect, the present invention provides a diagnostic method, comprising the steps of detecting a sample from a subject using the antibody or antigen-binding fragment thereof or the kit described in any of the foregoing items, or detecting a sample from a subject using the method described in any of the foregoing items, and quantifying the TgAbs in the sample, wherein the method is used to diagnose thyroid autoimmune diseases (e.g., thyroiditis or goiter, preferably Hashimoto's thyroiditis or Graves' disease) or thyroid cancer (e.g., differentiated thyroid cancer).

[0089] In some embodiments, the sample is from a human, such as a human body fluid (eg, blood, serum, or plasma).

[0090] Definition of terms

[0091] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the virology, biochemistry, and immunology laboratory operation steps used herein are conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0092] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules (i.e., a binding molecule and a target molecule), such as the reaction between an antibody and its antigen. The binding affinity between two molecules can be expressed as K. D Value description. K D The value refers to the dissociation constant obtained by the ratio of kd (dissociation rate of a specific binding molecule-target molecule interaction; also known as koff) to ka (association rate of a specific binding molecule-target molecule interaction; also known as kon), or kd / ka expressed as a molar concentration (M). DThe smaller the value, the tighter the two molecules bind and the higher the affinity. In certain embodiments, an antibody that specifically binds to an antigen (or has specificity for an antigen) refers to an antibody that binds to an antigen with a specificity of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or smaller K D Bind to the antigen. D The value can be determined by methods well known in the art, for example using surface plasmon resonance (SPR) in a BIACORE instrument.

[0093] As used herein, the term "immunological detection" refers to a determination using the specific interaction / binding affinity between antigen and antibody, which can generally be used to detect the presence or level of a specific antigen or antibody in a sample. Such immunological detection is well known to those skilled in the art, and includes, but is not limited to, enzyme immunoassay (EIA), chemiluminescent immunoassay (CLIA), radioimmunoassay (RIA), fluorescent immunoassay (FIA), Western blotting, immunoturbidimetry, surface plasmon resonance, etc. "Enzyme-linked immunosorbent assay" (i.e., ELISA) is to bind an antigen or antibody to the surface of a solid phase carrier, and cross-link an antigen or antibody-related substance with an enzyme to form an enzyme conjugate, which, on the one hand, maintains the immune characteristics of binding to the corresponding antigen or antibody, and on the other hand, has enzyme activity. When the enzyme conjugate is combined with the corresponding antigen or antibody, an enzyme-labeled antigen-antibody complex is formed. When the enzyme on the complex encounters the corresponding substrate, it can catalyze the product hydrolysis, oxidation or reduction, thereby producing a colored substance. According to the presence or absence of colored substances and their concentration, it is possible to indirectly infer whether the corresponding antigen or antibody exists in the sample and how much it is, thereby achieving the purpose of qualitative and quantitative determination. Among them, there are multiple modes for competitive ELISA to measure antibodies. The sample and enzyme-labeled antibody can be competitively bound to the solid phase antigen, or the sample and antigen are added to the solid phase antibody for competitive binding, and then the enzyme-labeled antibody is added after washing to react with the antigen bound to the solid phase. For a detailed description of immunological detection, see, for example, Fundamental Immunology, Ch.7 Paul, W., ed., 2nd edition, Raven Press, NY (1989).

[0094] As used herein, the terms "antibody" and "monoclonal antibody" are used interchangeably and refer to immunoglobulin molecules that are generally composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA and IgE, respectively. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant domain is not directly involved in the binding of the antibody to the antigen, but exhibits a variety of effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). H and V L It consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form antigen binding sites, respectively. The distribution of amino acids in each region or domain can follow the definition of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883.

[0095] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. There are three CDRs in each of the variable regions of the heavy and light chains, designated as CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883) or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). For a given antibody, a person skilled in the art will easily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art (eg, see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0096] In the present invention, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention can be identified according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention are identified by the Kabat numbering system.

[0097] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

[0098] The term "antibody" is not limited to any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibody can be an antibody of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibody.

[0099] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion." See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, complementarity determining region (CDR) fragments, scFv, diabodies, single domain antibodies, chimeric antibodies, linear antibodies, and polypeptides that contain at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0100] As used herein, the term "Fd" means an antibody fragment consisting of VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab' fragment" means a fragment obtained after reducing the disulfide bonds linking two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light chain and the Fd fragment of the heavy chain (consisting of VH and CH1 domains).

[0101] As used herein, the term "Fv" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen binding site. It is generally believed that the six CDRs confer antigen binding specificity to an antibody. However, even a variable region (e.g., a Fd fragment, which contains only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of a complete binding site.

[0102] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, ed., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also be present between the VH and VL of the scFv.

[0103] As used herein, the term "diabody" means that its VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0104] As used herein, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art, and refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen bound by a full-length antibody. Single-domain antibodies are also called nanobodies.

[0105] Each of the above antibody fragments retains the ability to specifically bind to the same antigen as the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0106] Antibody antigen-binding fragments (e.g., the antibody fragments described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antibody antigen-binding fragments can be screened for specificity in the same manner as for intact antibodies.

[0107] Herein, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only intact antibodies but also antigen-binding fragments of antibodies.

[0108] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of its light chain or / and heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of the light chain or / and heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains binding activity to the target antigen (USP 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)). For example, the term "chimeric antibody" may include antibodies (e.g., human-mouse chimeric antibodies) in which the heavy chain and light chain variable regions of the antibody are derived from a first antibody (e.g., a murine antibody), and the heavy chain and light chain variable regions of the antibody are derived from a second antibody (e.g., a human antibody).

[0109] As used herein, the term "humanized antibody" refers to a non-human antibody that has been genetically engineered, and its amino acid sequence has been modified to increase the homology with the amino acid sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, etc. The donor antibody can be a mouse, rat, rabbit or non-human primate (e.g., cynomolgus monkey) antibody with expected properties (e.g., antigen specificity, affinity, reactivity, etc.).

[0110] Chimeric antibodies or humanized antibodies of the present invention can be prepared based on the sequence of the mouse monoclonal antibody prepared above. DNA encoding heavy and light chains can be obtained from the target mouse hybridoma and engineered to contain non-mouse (eg, human) immunoglobulin sequences using standard molecular biology techniques.

[0111] To prepare chimeric antibodies, methods known in the art can be used to connect mouse immunoglobulin variable regions to human immunoglobulin constant regions. For example, the DNA encoding VH is operably connected to another DNA molecule encoding the heavy chain constant region to obtain a full-length heavy chain gene. The sequence of the human heavy chain constant region gene is known in the art (see, for example, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No.91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but is generally preferably IgG1 or IgG4 constant region. For example, the DNA encoding VL is operably connected to another DNA molecule encoding the light chain constant region CL to obtain a full-length light chain gene (and Fab light chain gene). The sequences of human light chain constant region genes are known in the art (see, e.g., Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments comprising these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region, but is generally preferably a kappa constant region.

[0112] To prepare humanized antibodies, the murine CDR regions can be inserted into human framework sequences using methods known in the art (see U.S. Pat. No. 5,225,539 to Winter; U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.; and Lo, Benny, KC, editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004).

[0113] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0114] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0115] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match). Typically, the comparison is made when the two sequences are aligned to produce maximum identity. Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol. Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0116] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0117] The compilation of the twenty conventional amino acids referred to herein follows conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.

[0118] In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0119] As used herein, the term "subject" includes, but is not limited to, various animals, particularly mammals, such as humans.

[0120] Herein, unless otherwise indicated, "%" refers to mass percentage. BRIEF DESCRIPTION OF THE DRAWINGS

[0121] Figure 1 Binding curves of TG01, hTG02-hTG06 monoclonal antibodies are shown. DETAILED DESCRIPTION

[0122] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0123] Example 1 Preparation of human thyroglobulin-specific monoclonal antibody

[0124] Thyroglobulin (HyTest Cat#8TG52, SDS-PAGE analysis purity>90%) of human thyroid gland was used to immunize mice and rats. Six highly sensitive antibodies TG01-TG06 against thyroglobulin autoantibodies were obtained by hybridoma screening (see Example 2 for the sensitivity test of antibodies against thyroglobulin autoantibodies), of which TG01 is a mouse antibody and TG02-TG06 are rat antibodies. These six antibodies were sequenced, and the results are shown in Tables 1 and 2.

[0125] Table 1 Antibody heavy chain sequencing results

[0126]

[0127]

[0128]

[0129] Table 2 Antibody light chain sequencing results

[0130]

[0131]

[0132] Note: The CDRs of the heavy chain variable region and light chain variable region are defined by the Kabat numbering system.

[0133] To develop the expression vector, the variable domains of TG02, TG03, TG04, TG05 and TG06 rat antibodies and the constant domains of human immunoglobulins of heavy chain IgG1 isotype and light chain λ isotype were used. The recombinant antibody expression vector was obtained in a prepared amount in bacterial cells and purified. The mammalian cell line Expi293F was transfected with the expression vector of the recombinant antibody.

[0134] For example, the light chain and heavy chain gene sequences are transferred to the open reading frame (ORF) of the expression vector through homologous recombination or restriction enzyme cutting, and finally the "promoter-antibody light chain gene-terminator" and "promoter-antibody heavy chain gene-terminator" structures are formed on the two expression vectors respectively. Then, the two expression vectors (antibody light chain expression vector and heavy chain expression vector) are simultaneously transferred into the mammalian cell line Expi293F for expression through biological, physical and chemical methods. Finally, the two light chains and two heavy chains are reassembled into a complete antibody in the cell and secreted into the culture supernatant. Alternatively, the expression vector is modified so that one expression vector contains two open reading frames, and then the light chain and heavy chain gene sequences are respectively inserted into the two open reading frames (ORF, Open Reading Frame) of the expression vector through homologous recombination or restriction enzyme cutting, forming a "promoter-antibody light chain gene-terminator-vector sequence-promoter-antibody heavy chain gene-terminator" or "promoter-antibody heavy chain gene-terminator-vector sequence-promoter-antibody light chain gene-terminator" structure, and then the expression vector containing both the antibody light chain and heavy chain gene sequences is transferred into the mammalian cell line Expi293F for expression through biological, physical and chemical methods, and finally the two light chains and the two heavy chains are reassembled into a complete antibody in the cell and secreted into the culture supernatant.

[0135] The antibodies were purified from the conditioned medium by protein A affinity chromatography, the resin was from GE health care Life Sciences (Piscataway, NJ), and the purification was performed according to the manufacturer's instructions. The above-mentioned recombinant chimeric antibodies hTG02-hTG06 were obtained. The purified monoclonal antibodies were stored in 50% ammonium sulfate as a suspension at 4°C.

[0136] Collection information:

[0137] TG01: TG01 hybridoma cell line The hybridoma of TG01 was deposited in VKPM on June 4, 2024, with the deposit number H-223;

[0138] hTG02: Escherichia coliRosetta deposited at VKPM on June 26, 2024, with the deposit number B-14843 TM (DE3)pLysS hTG02 VH, the plasmid contained in the cell produces hTG02 heavy chain; deposited in VKPM on June 26, 2024, with the deposit number B-14844 in Escherichia coli Rosetta TM (DE3)pLysS hTG02 VL, the plasmid contained in these cells produces the hTG02 light chain;

[0139] hTG03: Escherichia coliRosetta deposited at VKPM on June 26, 2024, with the deposit number B-14845 TM (DE3)pLysS hTG03 VH, the plasmid contained in the cell produces hTG03 heavy chain; deposited in VKPM on June 26, 2024, with the deposit number B-14846 in Escherichia coli Rosetta TM (DE3)pLysS hTG03 VL, the plasmid contained in these cells produces the hTG03 light chain;

[0140] hTG04: Escherichia coliRosetta deposited at VKPM on June 26, 2024, with the deposit number B-14847 TM (DE3)pLysS hTG04 ​​VH, the plasmid contained in the cells produces hTG04 ​​heavy chain; deposited in VKPM on June 26, 2024, with the deposit number B-14848 in Escherichia coli Rosetta TM (DE3)pLysS hTG04 ​​VL, the plasmid contained in these cells produces the hTG04 ​​light chain;

[0141] hTG05: Escherichia coliRosetta deposited at VKPM on June 26, 2024, with the deposit number B-14849 TM (DE3)pLysS hTG05 VH, the plasmid contained in the cell produces hTG05 heavy chain; deposited in VKPM on June 26, 2024, with the deposit number B-14850 in Escherichia coli Rosetta TM (DE3)pLysS hTG05 VL, the plasmid contained in these cells produces the hTG05 light chain;

[0142] hTG06: Escherichia coliRosetta deposited at VKPM on June 26, 2024, with the deposit number B-14851 TM (DE3)pLysS hTG06, the plasmid contained in this cell produces hTG06.

[0143] Example 2: Detection of Antibody Sensitivity to Anti-Thyroglobulin Autoantibodies

[0144] The sensitivity of the developed monoclonal antibody to thyroglobulin autoantibodies was determined by a competitive immunoassay method using Eu-labeled thyroglobulin and mixed serum samples containing high titers of anti-thyroglobulin autoantibodies. First, the PBS solution of captured anti-mouse or anti-rat IgG was incubated at 50 μL / well (2 μg / ml) in a 96-well plate with continuous shaking at room temperature for 30 minutes. The culture plate was washed with 10 mM Tris-HCl (pH 7.8) buffer supplemented with 0.15 M NaCl, 0.025% Tween-20 and 0.5 g / L NaN3 (washing solution). The antibody solution to be tested was then added to the culture plate and incubated under the same conditions, followed by washing with washing solution. Next, the plate was incubated with a stable Eu 3+ Chelate-labeled native thyroglobulin was pre-incubated with buffer (50 mM Tris-HCl buffer, pH 7.8, 0.9% NaCl, 0.01% Tween-40, 0.5% BSA and 0.05% NaN3) or pooled serum with a final concentration of 30 ng / mL and a final autoantibody level of 250 IU / mL for 15 minutes at room temperature and then added to the culture plate. The plate was incubated at room temperature with continuous shaking for 30 minutes. After washing with washing buffer, 0.1 ml of Enhancement solution (Perkin Elmer, Finland) was added and incubated at room temperature with gentle shaking for 10 min. Eu was measured on a Victor 1420 multilabel counter (Wallac-Perkin Elmer, Finland). 3+ The fluorescence is expressed in counts per second (CPS). The selection of monoclonal antibodies with high sensitivity to anti-thyroglobulin autoantibodies is based on the percentage of signal quenching, calculated as follows:

[0145] [Tg-Eu+ Assay Buffer] cps / [Tg-Eu+ Master Mix] cps*100%.

[0146] According to the results of sensitivity analysis of anti-thyroglobulin autoantibodies, TG01 and hTG02-hTG06 all showed the highest sensitivity to anti-thyroglobulin autoantibodies.

[0147] Example 3 Development of Competitive Chemiluminescent Immunoassay

[0148] For the continuous competitive chemiluminescent particle-based immunoassay, the detection monoclonal antibody was labeled with alkaline phosphatase (Cat. No. 03137031103, Roche Custom Biotech) and the capture antigen (recombinant thyroglobulin, HyTest Cat. No. 8RTG4) was labeled with biotin (Cat. No. PG82075, Thermo). The capture antigen at a concentration of 0.2 ug / mL was incubated with streptavidin-labeled particles (MyOne T1 Dynabeads Streptavidin PMP Hydrophobic Particles 100 um, Cat# 35604D, Thermo) and the resulting antigen-labeled particles were prepared at a concentration of 0.25 mg / ml in a buffer solution containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2% BSA, 0.05% γ-globulin (bovine), 0.2% Tween-20, 0.05% ProClin 300, 0.09% NaN3. The detection monoclonal antibody was prepared at a concentration of 0.15 μg / ml in a buffer solution containing 50 mM MES, pH 6, 0.9% NaCl, 1% BSA, 0.05% γ-globulin (bovine), 0.05% Tween-20, 0.048% ProClin 300, 2 mM MgCl2, 0.1 mM ZnCl2. A mixed serum sample with high levels of anti-thyroglobulin autoantibodies was serially diluted in a buffer solution containing 50 mM MES, pH 6, 0.9% NaCl, 1% BSA, 0.05% Tween-20, 0.048% ProClin 300, 2 mM MgCl2, 0.1 mM ZnCl2 for the generation of a calibration curve. A continuous competitive immunoassay was performed using an automatic analyzer Mindray CL-6000i according to the manufacturer's instructions (first incubation with biotinylated Tg and sample, second incubation with labeled antibody). Signals are expressed in relative light units (RLU).

[0149] Limit of Detection (LOD) Determination

[0150] 60 replicates of zero analyte (50 mM MES, pH 6, 0.9% NaCl, 1% BSA, 0.05% Tween-20, 0.048% ProClin300, 2 mM MgCl2, 0.1 mM ZnCl2) were analyzed in the corresponding competitive chemiluminescent immunoassay according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI) (Testability Evaluation of Clinical Laboratory Measurement Procedures; Approved Guidelines—Second Edition; EP17-A Vol. 24 No. 34). The general formula is:

[0151] LOB=1,645*mean;

[0152] LOD=LOB+1.645*SD

[0153] Table 3 CLIA final LOD of 6 antibodies

[0154]

[0155] Example 4 Affinity detection of antibodies TG01, hTG02, hTG03, hTG04, hTG05, and hTG06

[0156] Perform affinity biolayer interferometry according to the following protocol

[0157] 10 μg / mL of antibody buffer (50 mM Tris-HCl buffer, pH 7.8, 0.9% NaCl, 0.01% Tween-40, 0.5% BSA and 0.05% NaN3) was applied to the sensor; the sensor was blocked; the antigen (recombinant thyroglobulin) was applied at a concentration of 20 nM; the complete experimental protocol includes the following steps

[0158] (1) Sensor hydration;

[0159] (2) Antibody binding to sensor;

[0160] (3) Clean the sensor;

[0161] (4) Sensor closure;

[0162] (5) Binding (separately for each concentration of the tested antibody);

[0163] (6) dissociation;

[0164] (7) Regeneration of the sensor.

[0165] The results are as follows Figure 1 and Table 4.

[0166] Table 4 Affinity determination results of hTG06, hTG03, hTG04, hTG02, hTG05 and TG01

[0167] Antibody Ka(1 / Ms) Kd(1 / s) KD(M) HtTG06 1.13E+05 5.41E-05 4.77E-10 hTG03 1.29E+05 7.59E-05 5.89E-10 hTG04 1.35E+05 9.17E-07 6.79E-12 hTG02 1.36E+05 9.55E-05 7.02E-10 hTG05 1.33E+05 2.84E-05 2.13E-10 TG01 7.15E+05 1.78E-05 2.49E-11

[0168] Example 5 Preparation of TgAbs Detection Kit and Calibrators and Detection of TgAbs

[0169] Step 1: Preparation of R1 reagent (containing Tg antigen)

[0170] The Tg antigen obtained by natural extraction or recombinant transformation and the superparamagnetic microparticles are mixed in TBS buffer. After sufficient reaction, they are placed on a magnetic separator until the supernatant is free of turbidity. The supernatant is removed and the superparamagnetic microparticles coated with the Tg antigen are retained. The TBS buffer is then used repeatedly for 2-3 times. The superparamagnetic microparticles coated with the Tg antigen are dissolved in R1 reagent buffer to prepare R1 reagent, in which the content of superparamagnetic microparticles is 0.05%, and the content of Tg antigen is 0.05%. The reagent is refrigerated at 2-8°C for use.

[0171] Step 2: Preparation of R2 reagent (including detection antibody)

[0172] The specific Tg antibody is coupled to alkaline phosphatase, and then the conjugate is dissolved with R2 reagent buffer, wherein the molar ratio of alkaline phosphatase to antibody is 10:1, and then the conjugate is dissolved with R2 diluent to prepare R2 reagent, wherein the concentration of the conjugate is 5μg / mL, and refrigerated at 2-8℃ for use. (R1 reagent and R2 reagent are combined together to form TgAbs kit)

[0173] Step 3: Preparation of TgAbs Calibrators

[0174] TgAbs were diluted to concentration points of 0 IU / mL, 10 IU / mL, 20 IU / mL, 50 IU / mL, 100 IU / mL, 300 IU / mL, 500 IU / mL, 1000 IU / mL, 2000 IU / mL, 3000 IU / mL, 4200 IU / mL, etc. to prepare TgAbs calibrators.

[0175] Step 4: TgAbs kit measurement procedure

[0176] The TgAbs kit (including R1 reagent and R2 reagent) was loaded into the Mindray CL series fully automatic chemiluminescence immunoassay analyzer, and the measurement was performed according to the operating steps in the instrument manual.

[0177] The calibration test was performed using the matching TgAbs calibrator. Based on the calibration data, the system software used a weighted four-parameter logarithmic curve (4PLC) mathematical method to fit the luminescence signal to the concentration. The final result was given in the form of IU / mL concentration.

[0178] TgAbs kits 1-5 (antibody and antigen composition see Table 5 below) were obtained according to the above method and tested on samples from patients with clinically confirmed thyroid inflammation and goiter.

[0179] Table 5 TgAbs kit

[0180]

[0181]

[0182] Test items and results:

[0183] 1. Compliance with clinical diagnosis

[0184] The samples of patients with thyroid inflammation (Hashimoto's thyroiditis) and goiter (Graves' disease) with a clear clinical diagnosis were tested using the kit of the above embodiment, and the results were judged to be positive or negative based on the concentration value of TgAbs, where >115 IU / mL was judged as positive (the number was recorded as n), and ≤115 IU / mL was judged as negative (the number was recorded as m). The positive rate of the kit of each embodiment was counted, and the positive rate % = n / (n+m).

[0185] The results showed that the test results of the current test kit were highly consistent with the clinical diagnosis, with a positive rate of ≥80% in samples from patients diagnosed with Hashimoto's thyroiditis and a positive rate of ≥60% in samples from patients diagnosed with goiter.

[0186] Table 6 Statistics of positive rate of samples of patients diagnosed with Hashimoto's thyroiditis

[0187]

[0188]

[0189] Note: * Thyroglobulin antibody detection kit (electrochemiluminescence method), manufacturer: Roche, Germany.

[0190] Table 7 Statistics of positive rate of samples of patients diagnosed with goiter

[0191]

[0192]

[0193] Note: * Thyroglobulin antibody detection kit (electrochemiluminescence method), manufacturer: Roche, Germany.

[0194] Example 6 Anti-interference effect of TgAbs detection kit

[0195] The anti-interference effect of the kit was evaluated according to the following scheme

[0196] (1) Endogenous interference assessment (triglycerides, biotin, bilirubin, total protein):

[0197] A high-concentration triglyceride stock solution was prepared using physiological saline (0.9% NaCl solution), and samples of high and low concentration levels were taken and divided into two groups. Triglyceride with a final concentration of 1500 mg / dL was added to one group of samples to prepare an interference sample; the same volume of physiological saline was added to the other group of samples as a control sample.

[0198] Use physiological saline (0.9% NaCl solution) to prepare high-concentration biotin stock solution, take samples of high and low concentration levels, and divide them into two groups. Add biotin with a final concentration of 3600ng / mL to one group of samples to make interference samples; add the same volume of physiological saline to the other group of samples as control samples.

[0199] Use 0.1mol / L NaOH solution to prepare high-concentration bilirubin stock solution. Take samples with high and low concentration levels and divide them into two groups. Add bilirubin stock solution to one group of samples to prepare an interference sample containing 80mg / dL bilirubin; add the same volume of 0.1mol / L NaOH solution to the other group of samples as a control sample.

[0200] Samples at high and low concentration levels were taken and divided into two groups. BSA (the volume was negligible) was added to one group of samples to prepare interference samples with 15 g / dL total protein. The other group was used as control samples.

[0201] Repeat the test twice for each interference sample and control sample. The mean test result of the interference sample is recorded as M, and the mean test result of the control sample is recorded as T. The relative deviation B1 is calculated according to the following formula, and the deviation should be within the range of ±10%.

[0202] B1=(MT) / T×100%

[0203] Where:

[0204] B1—relative deviation;

[0205] M—mean value of interfering sample concentration;

[0206] T—mean value of control sample concentration.

[0207] (2) ANA, RF interference: Normal human serum samples were used as base samples, and RF, ANA positive serum was used as RF, ANA positive samples. High concentration analyte samples were added to the base samples and RF, ANA positive samples, respectively, and the added volume was controlled within 1 / 20 of the final volume to obtain base added samples and RF, ANA positive added samples. The test mean result of the base sample was recorded as X1, the test mean result of the base added sample was recorded as X2, the test mean result of the autoantibody ANA, RF positive samples was recorded as Y1, and the test mean result of the autoantibody ANA, RF positive added samples was recorded as Y2. The recovery deviation B2 of the measured concentration was calculated according to formula (2).

[0208] Formula (2): B2=((Y2-Y1) / (X2-X1)-1)×100%;

[0209] The results showed that the specific deviation of the current kit against endogenous interference and against ANA and RF interference were within the acceptance standard range (±10%), indicating that the kit of the present invention has strong ability to resist endogenous interference and against ANA and RF interference.

[0210] The evaluation results are shown in Tables 8 and 9.

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to Tg (thyroglobulin), comprising: A heavy chain variable region comprising the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 1, 7, 13, 19, 25, 31 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 2, 8, 14, 20, 26, 32 and variants thereof, and / or a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 3, 9, 15, 21, 27, 33 and variants thereof; and / or, A light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 4, 10, 16, 22, 28, 34 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 5, 11, 17, 23, 29, 35 and variants thereof, and / or a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 6, 12, 18, 24, 30, 36 and variants thereof; Wherein the variant contains an amino acid mutation compared to the amino acid sequence from which it is derived, and the amino acid mutation is a substitution, deletion or addition of one or several amino acids (for example, a substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids); preferably, the substitution is a conservative substitution; preferably, the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence from which it is derived.

2. The antibody or antigen-binding fragment thereof according to claim 1, comprising: (1a) a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 1 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 2 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 3 or a variant thereof; and, a light chain variable region comprising the following three CDRs: CDR-L1 having an amino acid sequence of SEQ ID NO: 4 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 5 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 6 or a variant thereof; (1b) a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 7 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 8 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 9 or a variant thereof; and, a light chain variable region comprising the following three CDRs: CDR-L1 having an amino acid sequence of SEQ ID NO: 10 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 11 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 12 or a variant thereof; (1c) a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 13 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 14 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 15 or a variant thereof; and, a light chain variable region comprising the following three CDRs: CDR-L1 having an amino acid sequence of SEQ ID NO: 16 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 17 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 18 or a variant thereof; (1d) a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 19 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 20 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 21 or a variant thereof; and, a light chain variable region comprising the following three CDRs: CDR-L1 having an amino acid sequence of SEQ ID NO: 22 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 24 or a variant thereof; (1e) a heavy chain variable region comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 25 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 26 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 27 or a variant thereof; and, a light chain variable region comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 28 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 29 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 30 or a variant thereof; or (1f) a heavy chain variable region comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 31 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 32 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 33 or a variant thereof; and a light chain variable region comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 34 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 35 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 36 or a variant thereof.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, further comprising a heavy chain constant region (CH) and a light chain constant region (CL); Preferably, the heavy chain constant region is a mouse heavy chain constant region, and the light chain constant region is a mouse light chain constant region; Preferably, the heavy chain constant region is a rat heavy chain constant region, and the light chain constant region is a rat light chain constant region; Preferably, the heavy chain constant region is a human heavy chain constant region, and the light chain constant region is a human light chain constant region; Preferably, the antibody or antigen-binding fragment thereof is an IgG, IgM, IgE, IgD or IgA antibody; Preferably, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region; Preferably, the light chain constant region is a kappa or lambda light chain constant region (eg, a human lambda light chain constant region).

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein The antigen-binding fragment is selected from scFv, Fab, Fab', (Fab')2, Fd, Fv, CDR fragment, nanobody, disulfide-linked Fv (dsFv), diabody, bispecific antibody and multispecific antibody; and / or, the antibody is a murine antibody, a chimeric antibody or a humanized antibody.

5. An antibody or antigen-binding fragment thereof that specifically binds to Tg, wherein the antibody or antigen-binding fragment thereof: Produced by the hybridoma cell line The hybridoma of TG01, which is deposited in the Russian National Collection of Industrial Microorganisms (VKPM) and has the deposit number H-223; Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14843 TM (DE3)pLysShTG02 VH plasmid and Escherichia coli Rosetta with accession number B-14844 TM (DE3) plasmid production in pLysShTG02 VL; Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14845 TM (DE3)pLysShTG03 VH plasmid and Escherichia coli Rosetta with the accession number B-14846 TM (DE3) plasmid production in pLysShTG03 VL; Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14847 TM Plasmids in (DE3)pLysShTG04 ​​VH and Escherichia coli Rosetta with accession number B-14848 TM (DE3) plasmid production in pLysShTG04 ​​VL; Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14849 TM (DE3)pLysShTG05 VH plasmid and Escherichia coli Rosetta with accession number B-14850 TM (DE3) plasmid generation in pLysS hTG05VL; Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14851 TM (DE3) Plasmid generation in pLysShTG06.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, further comprising a detectable label; Preferably, the label is selected from fluorescein, chemiluminescent substances (such as acridinium ester compounds), enzymes (such as horseradish peroxidase, alkaline phosphatase), radioactive isotopes, biotin, colloidal gold and magnetic particles.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, which competitively binds to Tg with TgAbs (thyroglobulin antibodies).

8. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

9. An expression vector comprising the isolated nucleic acid molecule of claim 8; Preferably, the vector is a plasmid, a virus, a phage, a bacterium or a viroid.

10. A host cell comprising the isolated nucleic acid molecule of claim 8 or the expression vector of claim 9; Preferably, the host cell is a eukaryotic cell, preferably a mammalian cell; Preferably, the host cell is a prokaryotic cell, preferably Escherichia coli.

11. An immunoassay kit comprising at least one antibody according to any one of claims 1 to 7.

12. An immunoassay kit comprising: A first reagent, comprising a Tg antigen, preferably, the Tg antigen is a natural or recombinant Tg antigen; The second reagent, which contains at least one antibody or antigen-binding fragment thereof that specifically binds to Tg, comprises: A heavy chain variable region comprising the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 1, 7, 13, 19, 25, 31 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 2, 8, 14, 20, 26, 32 and variants thereof, and / or a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 3, 9, 15, 21, 27, 33 and variants thereof; and / or, A light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 4, 10, 16, 22, 28, 34 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 5, 11, 17, 23, 29, 35 and variants thereof, and / or a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 6, 12, 18, 24, 30, 36 and variants thereof.

13. The immunoassay kit of claim 12, wherein the second reagent comprises: (1a) a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 1 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 2 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 3 or a variant thereof; and, a light chain variable region comprising the following three CDRs: CDR-L1 having an amino acid sequence of SEQ ID NO: 4 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 5 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 6 or a variant thereof; (1b) a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 7 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 8 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 9 or a variant thereof; and, a light chain variable region comprising the following three CDRs: CDR-L1 having an amino acid sequence of SEQ ID NO: 10 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 11 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 12 or a variant thereof; (1c) a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 13 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 14 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 15 or a variant thereof; and, a light chain variable region comprising the following three CDRs: CDR-L1 having an amino acid sequence of SEQ ID NO: 16 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 17 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 18 or a variant thereof; (1d) a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 19 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 20 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 21 or a variant thereof; and, a light chain variable region comprising the following three CDRs: CDR-L1 having an amino acid sequence of SEQ ID NO: 22 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 24 or a variant thereof; (1e) a heavy chain variable region comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 25 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 26 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 27 or a variant thereof; and, a light chain variable region comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 28 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 29 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 30 or a variant thereof; or (1f) a heavy chain variable region comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 31 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 32 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 33 or a variant thereof; and a light chain variable region comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 34 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 35 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 36 or a variant thereof.

14. A kit for competitive ELISA, comprising a Tg antigen and a Tg antibody, wherein: For patients with clinically confirmed thyroiditis, the consistency rate between the diagnosis result using the kit and the clinical diagnosis result is not less than 80%, and / or, For patients with clinically confirmed goiter, the consistency rate between the diagnosis result using the kit and the clinical diagnosis result is not less than 60%.

15. The kit of claim 14, wherein the Tg antibody is selected from the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

16. A method for detecting the presence or level of Tg and / or TgAbs in a sample, comprising the step of using the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 or the kit according to any one of claims 11 to 15 for detection.

17. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 in the preparation of a kit or use of the kit according to any one of claims 11 to 15, wherein the kit is used for: (1) detecting the presence or level of Tg and / or TgAbs in a sample; or (2) Diagnosis of thyroid autoimmune disease (eg, thyroiditis or goiter, preferably Hashimoto's thyroiditis or Graves' disease) or thyroid cancer (eg, differentiated thyroid cancer).

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