Anti-thyroglobulin antibodies, kits and uses

Abstract

The present invention relates to anti-thyroglobulin antibodies, kits and uses. In particular, the present invention provides anti-thyroglobulin specific antibodies, as well as kits containing said antibodies, and their use in detecting the presence or level of thyroglobulin and / or thyroglobulin autoantibodies in a sample.

Description

Technical Field

[0001] This invention belongs to the field of molecular immunology, specifically relating to anti-thyroglobulin specific antibodies, kits containing such antibodies, and their uses. Background Technology

[0002] Thyroglobulin (Tg) is a 660 kDa homodimer protein, each monomer containing numerous (approximately 60) disulfide bonds and highly glycosylated at 17 glycosylation sites. Tg is produced by thyroid follicular cells (thyroid cells) and stored in the follicular colloid of the gland, accounting for 80% of the total protein in the thyroid gland. Tg functions as a pro-hormone in the thyroid gland, synthesizing the iodinated tyrosine hormones thyroxine (T4) and triiodothyronine (T3). Nearly 30 of Tg's 66 tyrosine residues are iodinated; proteolytic hydrolysis of the polypeptide chain in lysosomes leads to the release of T3 and T4 into the cytoplasm of thyroid cells. T3 and T4 are subsequently secreted into the bloodstream.

[0003] Thyroglobulin antibodies (TgAbs) are commonly associated with autoimmune thyroid diseases (AITDs), such as Hashimoto's thyroiditis (HT) or Graves' disease (GD), but they are also present in about one in ten healthy individuals. Furthermore, TgAbs have also been found in differentiated thyroid cancer (DTC): TgAb-positive serology is more than twice as common in DTC patients as in the general population (25% vs. 10%). This makes TgAbs a prognostic indicator for assessing the risk of DTC development.

[0004] Because Tg and TgAbs are often present in patient serum, these two analytes can interfere with each other in immunoassays. Tg is widely used as a biomarker for follow-up of patients with DTC after thyroidectomy: Tg is a thyroid-specific protein, and elevated Tg concentrations during follow-up predict tumor recurrence or metastasis. In this case, the presence of Tg antibodies in the sample may lead to erroneous low Tg values. The usual practice is to measure TgAbs in all samples undergoing Tg assessment.

[0005] The epitopes recognized by human antibodies on the macromolecular Tg are usually conformational and cannot be determined by synthetic peptides. Serological studies have shown that there are numerous antigenic epitopes on Tg, and at least 40 such epitopes on human Tg. TgAbs antibodies from AITD patients have also been found to have different binding patterns than naturally occurring TgAbs antibodies. The specificity of TgAbs in DTC patients may also differ from that in AITD patients or healthy individuals. The fact that a variety of TgAbs are present in the blood of individual individuals is also of great 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 specifically binds to Tg and is used as a tracer; TgAbs in the sample compete with the tracer for binding sites on the Tg molecule. A method for quantifying TgAbs in human serum, developed by BRAHMS Diagnostica GmbH (Bergmann, A, US / 5,919,632), is based on using purified human TgAbs from human serum as a tracer.

[0007] The main problems with using polyclonal antibodies in immunoassays are imperfect binding properties, limited material availability, and reproducibility, especially with human preparations. Reproducibility and stability of the assay can also be complex issues. In TgAbs measurements, the epitope specificity and affinity of the antibody used as a tracer are crucial for assay performance. Therefore, there is a need to develop immunoassays for TgAbs evaluation based on antibodies with a combination of specific and affinity binding characteristics, enabling them to competitively bind to Tg with all major TgAbs present in the human population. Summary of the Invention

[0008] This invention describes a monoclonal antibody specific to human Tg, in particular, whose binding characteristics (epitope specificity and affinity) are comparable to those of autoantibodies against the major TgAbs present in the human population, enabling it to compete with TgAbs in a sample for binding to Tg, and thus be used for 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] The heavy chain variable region contains the following three CDRs: CDR-H1 with amino acid sequences selected from SEQ ID NO:1, 7, 13, 19, 25, 31 and their variants; CDR-H2 with amino acid sequences selected from SEQ ID NO:2, 8, 14, 20, 26, 32 and their variants; and / or CDR-H3 with amino acid sequences selected from SEQ ID NO:3, 9, 15, 21, 27, 33 and their variants; and / or,

[0012] The light chain variable region contains the following three CDRs: CDR-L1 with amino acid sequences selected from SEQ ID NO:4, 10, 16, 22, 28, 34 and their variants; CDR-L2 with amino acid sequences selected from SEQ ID NO:5, 11, 17, 23, 29, 35 and their variants; and / or CDR-L3 with amino acid sequences selected from SEQ ID NO:6, 12, 18, 24, 30, 36 and their variants.

[0013] The variant contains an amino acid mutation compared to the amino acid sequence from which it originates, the amino acid mutation being a substitution, deletion, or addition of one or more 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 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;

[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 its antigen-binding fragment comprises:

[0017] (1a) A heavy chain variable region comprising the following three CDRs: CDR-H1 with the amino acid sequence of SEQ ID NO:1 or a variant thereof, CDR-H2 with the amino acid sequence of SEQ ID NO:2 or a variant thereof, and CDR-H3 with the 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 with the amino acid sequence of SEQ ID NO:4 or a variant thereof, CDR-L2 with the amino acid sequence of SEQ ID NO:5 or a variant thereof, and CDR-L3 with the 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 with the amino acid sequence of SEQ ID NO:7 or a variant thereof, CDR-H2 with the amino acid sequence of SEQ ID NO:8 or a variant thereof, and CDR-H3 with the 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 with the amino acid sequence of SEQ ID NO:10 or a variant thereof, CDR-L2 with the amino acid sequence of SEQ ID NO:11 or a variant thereof, and CDR-L3 with the 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 with the amino acid sequence of SEQ ID NO:13 or a variant thereof, CDR-H2 with the amino acid sequence of SEQ ID NO:14 or a variant thereof, and CDR-H3 with the 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 with the amino acid sequence of SEQ ID NO:16 or a variant thereof, CDR-L2 with the amino acid sequence of SEQ ID NO:17 or a variant thereof, and CDR-L3 with the 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 with the amino acid sequence of SEQ ID NO:19 or a variant thereof, CDR-H2 with the amino acid sequence of SEQ ID NO:20 or a variant thereof, and CDR-H3 with the 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 with the amino acid sequence of SEQ ID NO:22 or a variant thereof, CDR-L2 with the amino acid sequence of SEQ ID NO:23 or a variant thereof, and CDR-L3 with the 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 the amino acid sequence SEQ ID NO:25 or a variant thereof, CDR-H2 with the amino acid sequence SEQ ID NO:26 or a variant thereof, and CDR-H3 with the amino acid sequence SEQ ID NO:27 or a variant thereof; and a light chain variable region comprising the following three CDRs: CDR-L1 with the amino acid sequence SEQ ID NO:28 or a variant thereof, CDR-L2 with the amino acid sequence SEQ ID NO:29 or a variant thereof, and CDR-L3 with the amino acid sequence SEQ ID NO:30 or a variant thereof; or

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

[0023] In some embodiments, the heavy chain variable region further comprises a heavy chain framework region (FR). In some embodiments, the light chain variable region further comprises a light chain framework region (FR). The framework regions are highly conserved and provide a scaffold for the six CDRs in three-dimensional space to form an antigen-binding surface. The naturally occurring variable domains of the heavy and light chains each contain four FR regions (FR1, FR2, FR3, and FR4), which primarily utilize a β-sheet configuration and are connected by three hypervariable regions that form a loop connecting the β-sheet structure and, in some cases, form part of the β-sheet structure. The hypervariable regions in each chain are closely adjacent to each other via FRs and, together with hypervariable regions from the other chain, contribute to the formation of antigen-binding side ends (see Kabat et al., cited above). The heavy chain framework region and / or light chain framework region can each be independently derived from the framework region of any species immunoglobulin. In some embodiments, the heavy chain framework region and light chain framework region are each independently derived from the heavy chain framework region and 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 amino acid sequences derived from mouse immunoglobulin, human immunoglobulin, or combinations thereof. In some embodiments, the heavy chain framework region and the light chain framework region each independently comprise amino acid sequences derived from human germline antibodies.

[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 the IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, or, for example, the 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 (e.g., the 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 its antigen-binding fragment is further labeled with a detectable tag.

[0027] In this document, the detectable label can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, or chemical means. Particularly preferred are those labels suitable for immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). Such labels are well known in the art and include, but are not limited to, enzymes (e.g., oxidases, microperoxidases, horseradish peroxidases, alkaline phosphatases, β-galactosidases, ureases, glucose oxidases, etc.) and 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, phenanthrene, acridine esters, etc.), and biotin for binding avidin (e.g., streptavidin) modified with the above-mentioned markers. The markers covered in this invention can be detected by methods known in the art. For example, radioactive markers can be detected using photographic film or a scintillator, and fluorescent markers can be detected using a photodetector to detect emitted light. Enzyme markers are generally detected by providing a substrate to the enzyme and detecting the reaction product produced by the enzyme's action on the substrate. Chemiluminescent substances (such as acridine esters) are generally detected by providing an excitation solution and / or a catalyst to the luminescent substance to detect emitted light. Biotin is generally detected by providing biotin with an avidin modified with the aforementioned label (e.g., streptavidin) and detecting the label carried by the avidin linked to biotin. In some embodiments, the detectable label described above can be linked to the antibody or its antigen-binding fragment of the present invention via connectors of varying lengths to reduce potential steric hindrance.

[0028] In some embodiments, the detectable label is selected from fluorescein, chemiluminescent substances (e.g., acridine esters), enzymes (e.g., horseradish peroxidase, alkaline phosphatase), radioisotopes, biotin, colloidal gold, and magnetic particles.

[0029] In some embodiments, the antibody or its antigen-binding fragment competitively binds to Tg with TgAbs (thyroglobulin antibody).

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

[0031] In another aspect, the present invention provides an expression vector containing the isolated nucleic acid molecule described in any of the preceding embodiments. In some embodiments, the vector is a plasmid, virus, bacteriophage, bacterium, or 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 preceding embodiments. 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 *Escherichia coli*.

[0033] In another aspect, this 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 of TG01, deposited at the Russian State Industrial Microbial Collection Center (VKPM) with accession number H-223;

[0035] Based on Escherichia coli Rosetta, deposited at VKPM, accession number B-14843 TM The plasmid in (DE3)pLysShTG02 VH and Escherichia coli Rosetta with accession number B-14844 TM Plasmid generation in (DE3)pLysShTG02 VL;

[0036] Based on Escherichia coli Rosetta, deposited at VKPM, accession number B-14845 TM The plasmid in (DE3)pLysShTG03 VH and Escherichia coli Rosetta with accession number B-14846 TM Plasmid generation in (DE3)pLysShTG03 VL;

[0037] Based on Escherichia coli Rosetta, deposited at VKPM, accession number B-14847 TM The plasmid in (DE3)pLysShTG04 ​​VH and Escherichia coli Rosetta with accession number B-14848 TM Plasmid generation in (DE3)pLysShTG04 ​​VL;

[0038] Based on Escherichia coli Rosetta, deposited at VKPM, accession number B-14849 TM The plasmid in (DE3)pLysShTG05 VH and Escherichia coli Rosetta with accession number B-14850 TM Plasmid generation in (DE3)pLysShTG05 VL; or

[0039] Based on Escherichia coli Rosetta, deposited at VKPM, accession number B-14851 TM Plasmid generation from (DE3)pLysShTG06.

[0040] Reagent test kit

[0041] Currently, the consistency between mainstream TgAb detection methods and clinical diagnosis needs improvement, and significant differences exist in 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 among different disease types. The positive rate is approximately 50%-80% for patients diagnosed with Hashimoto's thyroiditis and approximately 30%-50% for patients diagnosed with Graves' disease. Even with the addition of Anti-TPO testing from the same manufacturer to aid in diagnosis, the positive rate of Anti-TPO varies significantly among different patient types. Therefore, clinical diagnosis and medication guidance often rely on biopsy or imaging results, but these testing methods are time-consuming and cause more pain and burden to patients.

[0042] The binding characteristics (epitope specificity and affinity) of the antibody that specifically binds to human Tg described in this invention are comparable to those of the major TgAbs autoantibodies present in the human population. This allows it to compete with TgAbs in the sample for Tg binding and to be used for TgAb measurement. Based on this, this invention provides a kit with higher clinical concordance rates for the in vitro detection of autoimmune thyroid diseases (Hashimoto's thyroiditis, Graves' disease, etc.).

[0043] Specifically, one object of the present invention is to provide an immunoassay kit containing at least one of the antibodies or antigen-binding fragments described above.

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

[0045] The first reagent contains Tg antigen;

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

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

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

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

[0050] In some embodiments, the detectable label is selected from fluorescein, chemiluminescent labels (e.g., acridine esters), enzymes (e.g., horseradish peroxidase, alkaline phosphatase), radioisotopes, biotin, and colloidal gold.

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

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

[0053] In some embodiments, the components of 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 its antigen-binding fragment 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 (e.g., BND-10 or BND-99) and BIT (e.g., BIT-10).

[0058] In some embodiments, the surfactant is selected from Tween-20, Triton X-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 support, and the antibody or its antigen-binding fragment is labeled with a detectable tag.

[0061] In some embodiments, the Tg antigen is coated with superparamagnetic microparticles. In some embodiments, the content of the superparamagnetic microparticles in the first reagent is 0.01%-0.1%, for example 0.03%-0.1%, or even 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%, for example 0.01%-0.15% or 0.05%-0.15%.

[0063] In some embodiments, the working concentration of the antibody or its antigen-binding fragment (referring to the concentration of the antibody or its antigen-binding fragment 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 comprise reagents for detecting the corresponding detectable label (the substrate of the detectable label described above). For example, when the detectable label is an enzyme, the kit may also comprise 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., acrid ester compounds), the kit may also comprise 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 substrate capable of detecting the label, a washing solution, a stop solution, and an instruction manual.

[0066] The antibody described in this invention has binding characteristics comparable to the major TgAbs autoantibodies present in the human population. It can competitively bind to Tg antigens with TgAbs in the sample. The TgAb content in the sample is determined by quantitatively testing the enzyme-labeled antibody in both the test group and the control group (without sample). Based on the measured TgAb content, further diagnosis can be performed on the individuals providing the sample.

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

[0068] In some implementations, the sample is derived from a human being, such as human bodily fluids (e.g., blood, serum, or plasma).

[0069] In some embodiments, the immunoassay kit has a positive rate of not less than 80% for diagnosing thyroiditis (e.g., Hashimoto's thyroiditis) and / or a positive rate of not less than 60% for diagnosing goiter (e.g., Graves' disease).

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

[0071] For patients with clinically diagnosed thyroiditis, the consistency rate between the diagnostic results obtained using the aforementioned kit and the clinical diagnosis is no less than 80%, and / or,

[0072] For patients with clinically diagnosed goiter, the consistency rate between the diagnostic results obtained using the aforementioned kit and the clinical diagnostic results is no less than 60%.

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

[0074] Detection methods

[0075] Based on the antibodies and / or kits described in this invention, this invention further provides a method for detecting the presence or level of Tg and / or TgAbs in a sample, comprising the step of using the antibodies or antigen-binding fragments thereof or immunoassay kits described in any of the preceding claims for detection.

[0076] In some implementations, the method includes the following steps:

[0077] The sample is reacted with the first and second reagents, 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 includes a calibration operation using a Tg antigen calibrator. In some embodiments, the Tg antigen calibrator is a natural or recombinant Tg antigen.

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

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

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

[0082] In some implementations, the sample is derived from a human being, such as human bodily fluids (e.g., blood, serum, or plasma).

[0083] application

[0084] Quantitative detection of TgAbs can be used to diagnose thyroid autoimmune diseases.

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

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

[0087] (2) Diagnose thyroid autoimmune diseases (e.g., thyroiditis or goiter, preferably Hashimoto's thyroiditis or Graves' disease) or thyroid cancer (e.g., differentiated thyroid cancer).

[0088] In another aspect, the present invention provides a diagnostic method comprising the steps of detecting a sample from a subject with an antibody or antigen-binding fragment thereof or a kit as described in any of the preceding claims, or detecting a sample from a subject with the method described in any of the preceding claims, and quantifying TgAbs in the sample, said method being 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 implementations, the sample is derived from a human being, such as human bodily fluids (e.g., blood, serum, or plasma).

[0090] Terminology Definition

[0091] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the virological, biochemical, and immunological laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of 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 target antigen. The binding affinity between two molecules can be measured using Kx. D Value description. K D The value refers to the dissociation constant obtained by the ratio of kd (the dissociation rate of a specific binding molecule-target molecule interaction; also known as koff) to ka (the association rate of a specific binding molecule-target molecule interaction; also known as kon), or kd / ka expressed as molar concentration (M). D The smaller the value, the tighter the binding between the two molecules, and the higher the affinity. In some embodiments, an antibody that specifically binds to a certain antigen (or an antibody that is specific to a certain antigen) refers to an antibody with a binding affinity of less than approximately 10. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10-8 M, 10 -9 M or 10 -10 M or smaller K D Bind to the antigen. K D The value can be determined by methods well known in the art, such as using surface plasmon resonance (SPR) in a BIACORE instrument.

[0093] As used herein, the term "immunological assay" refers to a determination that utilizes the specific interaction / binding affinity between an antigen and antibody, and is generally used to detect the presence or level of a specific antigen or antibody in a sample. Such immunological assays are well known to those skilled in the art and include, but are not limited to, enzyme immunoassay (EIA), chemiluminescent immunoassay (CLIA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), Western blotting, immunoturbidimetry, and surface plasmon resonance assays. "Enzyme-linked immunosorbent assay" (ELISA) involves binding antigens or antibodies to the surface of a solid-phase carrier, cross-linking the antigen or antibody-related substances with an enzyme to form an enzyme conjugate. This conjugate retains the immunogenic properties of binding to the corresponding antigen or antibody while also possessing enzymatic activity. When the enzyme conjugate binds to 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 hydrolysis, oxidation, or reduction of the product, thereby producing a colored substance. Based on the presence and concentration of colored substances, the presence and quantity of corresponding antigens or antibodies in the tested sample can be indirectly inferred, thus achieving qualitative and quantitative determination. Among these, competitive ELISA for antibody detection has several modes. One mode involves the sample and enzyme-labeled antibody competing for binding with the solid-phase antigen, or the sample and antigen being added together to the solid-phase antibody for competitive binding, followed by washing and then adding the enzyme-labeled antibody 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 typically composed of two pairs of polypeptide chains (each pair consisting of 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 antibody isotypes are 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 a single domain, CL. The constant domain does not directly participate in antibody-antigen binding but exhibits various 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 highly variable regions (called complementarity-determining regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). Each V H and V L It consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antigen-binding sites. The allocation of amino acids in each region or domain can follow the definitions in 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 residue in the antibody variable region responsible for antigen binding. Each of the heavy and light chain variable regions contains three CDRs, designated 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, those skilled in the art will readily identify the CDR as defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0096] In this invention, the CDR contained in the antibody or antigen-binding fragment thereof can be determined according to various numbering systems known in the art. In some embodiments, the CDR contained in the antibody or antigen-binding fragment thereof is determined using the Kabat numbering system.

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

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

[0099] As used herein, the term “antigen-binding fragment” of an antibody refers to a polypeptide containing a fragment of the 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; it is also referred to as the “antigen-binding moiety”. See Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated 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, diabody, single-domain antibody, chimeric antibody, linear antibody, and peptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the peptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

[0100] As used herein, the term “Fd” refers to an antibody fragment consisting of VH and CH1 domains; the term “dAb fragment” refers to an antibody fragment consisting of VH domains (Ward et al., Nature 341:544 546 (1989)); the term “Fab fragment” refers to an antibody fragment consisting of VL, VH, CL and CH1 domains; the term “F(ab')2 fragment” refers to an antibody fragment containing two Fab fragments connected by disulfide bridges on the hinge region; the term “Fab' fragment” refers to the fragment obtained by reducing the disulfide bonds connecting the two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light chain and heavy chain Fd fragment (consisting of VH and CH1 domains).

[0101] As used herein, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as an Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to the 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 containing VL and VH domains linked by a linker (see, for example, 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, edited by Roseburg and Moore, 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 a repeating GGGGS amino acid sequence or a variant 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 this 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 exist between VH and VL of scFv.

[0103] As used herein, the term “biantibody” means that its VH and VL domains are expressed on a single polypeptide chain, but the linker is too short to allow pairing between the two domains on the same chain, thus forcing the domain to pair with the complementary domain of another chain and creating two antigen-binding sites (see, for example, 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 as an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that maintains the ability to specifically bind to the same antigen bound by a full-length antibody. Single-domain antibodies are also known as nanobodies.

[0105] Each of the above antibody fragments 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.

[0106] Antigen-binding fragments (e.g., the antibody fragments described above) of a given antibody (e.g., the antibody provided in this invention) can be obtained using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be specifically screened in the same manner as those used for intact antibodies.

[0107] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0108] As used herein, the term "chimeric antibody" refers to an antibody whose light chain and / or heavy chain portion is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and whose light chain and / or heavy chain portion 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 which retains its binding activity to the target antigen in any case (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 variable regions of the heavy and light chains of the antibody are derived from a first antibody (e.g., a murine antibody), while the variable regions of the heavy and light chains 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 genetically engineered non-human antibody whose amino acid sequence has been modified to increase homology with that of a human antibody. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). Humanized antibodies generally retain the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, and reactivity. Donor antibodies can be mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibodies with the intended properties (e.g., antigen specificity, affinity, reactivity).

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

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

[0112] To prepare humanized antibodies, mouse CDR regions can be inserted into human frame sequences using methods known in the art (see Winter’s U.S. Patent No. 5,225,539; Queen et al.’s U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762 and 6,180,370; 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 polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may 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" refers to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are identical at a position when the same base or amino acid monomeric subunit occupies the same location (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage 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 × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods readily available, for example, computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage 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)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoIBiol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights 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 alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using 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 that are 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 or hydrogen bonds). 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 and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, 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 twenty common amino acids discussed in this article were written in accordance with standard usage. See, for example, Immunology-ASynthesis (2nd Edition, ESGolub and DRGren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.

[0118] In this invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Furthermore, in this 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] In this article, unless otherwise taught, "%" refers to mass percentage. Attached Figure Description

[0121] Figure 1 The binding curves of the TG01, hTG02-hTG06 monoclonal antibodies are shown. Detailed Implementation

[0122] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

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

[0124] Mice and rats were immunized with thyroglobulin from the human thyroid gland (HyTest Cat#8TG52, SDS-PAGE purity >90%). Six highly sensitive anti-thyroglobulin autoantibodies, TG01-TG06, were obtained through hybridoma screening (see Example 2 for antibody sensitivity testing against anti-thyroglobulin autoantibodies), where 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 for the heavy chain variable region and the light chain variable region are defined by the Kabat numbering system.

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

[0134] For example, light chain and heavy chain gene sequences are transferred into the open reading frame (ORF) of an expression vector through homologous recombination or restriction enzyme digestion, respectively. This results in the formation of "promoter-antibody light chain gene-terminator" and "promoter-antibody heavy chain gene-terminator" structures on the two expression vectors, respectively. Then, using biological, physical, and chemical methods, the two expression vectors (antibody light chain expression vector and heavy chain expression vector) are simultaneously transformed into the mammalian cell line Expi293F for expression. 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 can be modified to contain two open reading frames (ORFs). Then, through homologous recombination or restriction enzyme digestion, the light chain and heavy chain gene sequences are inserted into the two ORFs of the expression vector, forming a structure of "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". This expression vector containing both antibody light chain and heavy chain gene sequences is then transformed into the mammalian cell line Expi293F for expression using biological, physical, and chemical methods. Finally, the two light chains and two heavy chains reassemble into a complete antibody in the cell and are secreted into the culture supernatant.

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

[0136] Preservation information:

[0137] TG01: The hybridoma cell line TG01 was deposited at VKPM on June 4, 2024, with accession number H-223;

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

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

[0140] hTG04: Escherichia coli Rosetta deposited at VKPM on June 26, 2024, accession number B-14847. TM (DE3)pLysS hTG04 ​​VH, the plasmid contained in this cell produces the hTG04 ​​heavy chain; deposited on June 26, 2024 at VKPM, accession number B-14848, in *Escherichia coli* Rosetta. TM (DE3)pLysS hTG04 ​​VL, the plasmid contained in this cell produces the hTG04 ​​light chain;

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

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

[0143] Example 2: Sensitivity of Antibody Against Thyroglobulin Autoantibodies

[0144] The sensitivity of the developed monoclonal antibody to thyroglobulin autoantibodies was determined using a competitive immunoassay method with Eu-labeled thyroglobulin and a mixed serum sample containing a high titer of anti-thyroglobulin autoantibody. First, 50 μL / well (2 μg / ml) of PBS solution capturing anti-mouse or anti-rat IgG was added to each well of a 96-well plate and incubated at room temperature with shaking for 30 minutes. The plate was then 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 (wash buffer). The antibody solution to be tested was then added to the plate and incubated under the same conditions, followed by washing with wash buffer. Next, the plate was stabilized with Eu... 3+ Chelated native thyroglobulin was pre-incubated for 15 minutes at room temperature with either a buffer (50 mM Tris-HCl buffer, pH 7.8, 0.9% NaCl, 0.01% Tween-40, 0.5% BSA, and 0.05% NaN3) or a mixed serum solution with an autoantibody final level of 250 IU / mL and a final concentration of 30 ng / mL, and then added to a culture plate. The plate was incubated with shaking at room temperature for 30 minutes. After washing with washing buffer, 0.1 ml of the solution was added to each well. Enhanced solution (Perkin Elmer, Finland), incubated with gentle shaking for 10 minutes at room temperature. Eu was measured on a Victor 1420 multi-label counter (Wallac-Perkin Elmer, Finland). 3+ Fluorescence. Fluorescence is expressed in counts per second (CPS). The selection of monoclonal antibodies with high sensitivity against thyroglobulin autoantibodies is based on the percentage of signal quenching, calculated as follows:

[0145] [Tg-Eu+ analysis buffer] cps / [Tg-Eu+ master mixture] cps * 100%.

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

[0147] Example 3: Development of a Competitive Chemiluminescence Immunoassay Method

[0148] To perform continuous immunoassays based on competitive chemiluminescent particles, the detection monoclonal antibody was labeled with alkaline phosphatase (catalog number 03137031103, Roche Custom Biotech), and the capture antigen (recombinant thyroglobulin, HyTest catalog number 8RTG4) was labeled with biotin (catalog number PG82075, Thermo). The capture antigen at a concentration of 0.2 μg / mL was incubated with streptavidin-labeled particles (MyOne T1 Dynabeads streptavidin PMP hydrophobic particles 100 μm, Cat#35604D, Thermo). 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, and 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, and 0.1 mM ZnCl2. Serial dilutions of a mixed serum sample with high levels of anti-thyroglobulin autoantibodies were performed 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, and 0.1 mM ZnCl2 to generate a calibration curve. Continuous competitive immunoassays were performed using a Mindray CL-6000i automated analyzer according to the manufacturer's instructions (first incubation with biotinylated Tg and sample, second incubation with labeled antibody). The signal is expressed in relative light units (RLU).

[0149] Limit of Detection (LOD) Determination

[0150] Following the recommendations of the Clinical and Laboratory Standards Institute (CLSI) (Clinical Laboratory Measurement Procedures: Assessment of Detection Capabilities; Approved Guideline—Second Edition; EP17-A, Vol. 24, No. 34), 60 replicates of the zero analyte (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) were analyzed in the corresponding competitive chemiluminescent immunoassay. The general formula is:

[0151] LOB = 1,645 * mean;

[0152] LOD = LOB + 1.645 * SD

[0153] Table 3. LOD of the 6 antibodies in CLIA

[0154]

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

[0156] Affinity layer interferometry was performed according to the following scheme.

[0157] Spread 10 μg / mL antibody buffer (50 mM Tris-HCl buffer, pH 7.8, 0.9% NaCl, 0.01% Tween-40, 0.5% BSA, and 0.05% NaN3) onto the sensor; block the sensor; administer 20 nM antigen (recombinant thyroglobulin); the complete experimental protocol includes the following steps.

[0158] (1) Sensor hydration;

[0159] (2) Antibody binds to sensor;

[0160] (3) Clean the sensor;

[0161] (4) The sensor is sealed;

[0162] (5) Combining (for each concentration of the test antibody, respectively);

[0163] (6) Dissociation;

[0164] (7) Sensor regeneration.

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

[0166] Table 4. Affinity test results for hTG06, hTG03, hTG04, hTG02, hTG05, and TG01.

[0167] Antibody Ka(1 / Ms) Kd(1 / s) KD(M) hTG06 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] Tg antigen obtained from natural extraction or recombinant transformation is mixed with superparamagnetic microparticles in TBS buffer. After sufficient reaction, the mixture is placed on a magnetic separator until the supernatant is clear. The supernatant is removed, and the superparamagnetic microparticles coated with Tg antigen are collected. The mixture is then repeatedly mixed with TBS buffer 2-3 times. The superparamagnetic microparticles coated with 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 stored at 2-8℃ for later use.

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

[0172] The specific Tg antibody was conjugated with alkaline phosphatase, and then the conjugate was dissolved in R2 reagent buffer (alkaline phosphatase to antibody molar ratio of 10:1). The conjugate was then dissolved in R2 dilution buffer to prepare R2 reagent, with a concentration of 5 μg / mL of the conjugate. The reagent was stored at 2–8°C for later use. (R1 and R2 reagents together constitute the TgAbs kit.)

[0173] Step 3: Preparation of TgAbs calibrators

[0174] TgAbs were diluted to concentrations 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, and 4200 IU / mL to prepare TgAbs calibrators.

[0175] Step 4: Measurement Procedure for the TgAbs Kit

[0176] Load the TgAbs kit (including reagents R1 and R2) into the Mindray CL series fully automated chemiluminescence immunoassay analyzer and perform measurements according to the operating procedures 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 concentration in IU / mL.

[0178] TgAbs kits 1-5 were obtained according to the above method (antibody and antigen composition is shown in Table 5 below), and samples from patients clinically diagnosed with thyroiditis and goiter were tested.

[0179] Table 5 TgAbs Reagent Kit

[0180]

[0181]

[0182] Test items and results:

[0183] 1. Concordance rate with clinical diagnosis

[0184] For patient samples with a clear clinical diagnosis of thyroiditis (Hashimoto's thyroiditis) and goiter (Graves' disease), the kits described above were used for testing. The results were determined to be positive or negative based on the concentration of TgAbs. Samples >115 IU / mL were considered positive (number denoted as n), and samples ≤115 IU / mL were considered negative (number denoted as m). The positive rate of the kits in each embodiment was calculated as follows: positive rate % = n / (n+m).

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

[0186] Table 6. Positive rate statistics of patient samples diagnosed with Hashimoto's thyroiditis

[0187]

[0188]

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

[0190] Table 7. Positive rate statistics of patient samples diagnosed with goiter

[0191]

[0192]

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

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

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

[0196] (1) Assessment of endogenous interference (triglycerides, biotin, bilirubin, total protein):

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

[0198] A high-concentration biotin stock solution was prepared using physiological saline (0.9% NaCl solution). Samples at two concentration levels (high and low) were divided into two groups. An interference sample was prepared by adding biotin to one group at a final concentration of 3600 ng / mL; the other group was treated with the same volume of physiological saline as a control sample.

[0199] A high-concentration bilirubin stock solution was prepared using a 0.1 mol / L NaOH solution. Samples at two concentration levels (high and low) were divided into two groups. The bilirubin stock solution was added to one group of samples to prepare an interference sample containing 80 mg / dL bilirubin; the same volume of 0.1 mol / L NaOH solution was added to the other group of samples as a control sample.

[0200] Two samples with high and low concentrations were taken and divided into two groups. BSA (volume negligible) was added to one group of samples to prepare an interference sample with 15 g / dL total protein. The other group served as the control sample.

[0201] The interference sample and the control sample were each tested twice. The mean test result of the interference sample was denoted as M, and the mean test result of the control sample was denoted as T. The relative deviation B1 was calculated according to the following formula, and the deviation should be within ±10%.

[0202] B1 = (MT) / T × 100%

[0203] In the formula:

[0204] B1—Relative deviation;

[0205] M—mean concentration of interfering samples;

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

[0207] (2) ANA and RF interference: Normal human serum samples were used as the baseline samples, and RF and ANA positive serum samples were used as RF and ANA positive samples. High concentrations of analyte were added to the baseline samples and RF and ANA positive samples respectively, with the added volume controlled within 1 / 20 of the final volume, to obtain the baseline supplemented samples and RF and ANA positive supplemented samples. The mean test result of the baseline samples was recorded as X1, the mean test result of the baseline supplemented samples was recorded as X2, the mean test result of the autoantibody ANA and RF positive samples was recorded as Y1, and the mean test result of the autoantibody ANA and RF positive supplemented 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 show that the specificity deviations of the current kit against endogenous interference and against ANA and RF interference are both within the acceptable range (±10%), indicating that the kit of the present invention has strong resistance to endogenous interference and resistance to 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 that specifically binds to thyroglobulin (Tg) or an antigen-binding fragment thereof, wherein the antibody: Based on Escherichia coli Rosetta, deposited at VKPM, accession number B-14845 TM The plasmid in (DE3)pLysShTG03 VH and Escherichia coli Rosetta with accession number B-14846 TM Plasmid generation in (DE3)pLysShTG03 VL; or Based on Escherichia coli Rosetta, deposited at VKPM, accession number B-14851 TM Plasmid generation from (DE3)pLysShTG06.

2. The antibody or antigen-binding fragment thereof according to claim 1, further comprising a detectable marker.

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein, The detectable markers are selected from fluorescein, chemiluminescent substances, enzymes, radioisotopes, biotin, colloidal gold, and magnetic particles.

4. The antibody or antigen-binding fragment thereof according to claim 2, wherein, The detectable markers are selected from acridine esters, horseradish peroxidase, and alkaline phosphatase.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, which competitively binds to Tg with TgAbs (thyroglobulin antibody).

6. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof as described in any one of claims 1-5.

7. An expression vector containing the isolated nucleic acid molecule as described in claim 6.

8. The expression vector of claim 7, wherein the expression vector is a plasmid, virus, bacteriophage, or viroid.

9. A host cell comprising the isolated nucleic acid molecule of claim 6 or the expression vector of claim 7 or 8.

10. The host cell of claim 9, wherein, The host cell is a eukaryotic cell or a prokaryotic cell.

11. The host cell of claim 9, wherein, The host cell can be a mammalian cell or a bacterium.

12. The host cell of claim 9, wherein, The host cell is Escherichia coli.

13. An immunoassay kit comprising at least one antibody as described in any one of claims 1-5.

14. An immunoassay kit, comprising: The first reagent contains Tg antigen; The second reagent contains at least one antibody or antigen-binding fragment thereof that specifically binds to Tg, wherein the antibody or antigen-binding fragment thereof is selected from the antibodies or antigen-binding fragments thereof described in any one of claims 1-5.

15. The immunoassay kit of claim 14, wherein, The Tg antigen is either natural or recombinant Tg antigen.

16. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-5 or the immunoassay kit according to any one of claims 13-15 in the preparation of the kit, wherein the kit is used for: (1) Detect the presence or level of Tg or TgAbs in the sample; or (2) Diagnose thyroid autoimmune diseases or thyroid cancer.

17. The use of claim 16, wherein the kit is used for diagnosing thyroiditis, goiter, or differentiated thyroid cancer.

18. The use of claim 16, wherein the kit is used to diagnose Hashimoto's thyroiditis or Graves' disease.

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