Antibody for detecting thyroid stimulating hormone, kit, detection method and sample analyzer

Abstract

The invention relates to an antibody for detecting thyroid stimulating hormone, a kit, a detection method and a sample analyzer, in particular to a monoclonal antibody specifically binding to thyroid stimulating hormone or an antigen binding antibody fragment thereof, the antibody can recognize TSH variant R55G, and the TSH variant R55G can recognize TSH variant R55G or TSH variant R55G or TSH variant R55G or TSH variant R55G. Moreover, the TSH detection kit is not subjected to cross reaction with alpha-subunits shared by human luteinizing hormone (LH), follicle stimulating hormone (FSH), human chorionic gonadotropin (hCG) and free glycoprotein hormone, and is particularly suitable for developing the TSH detection kit with functional sensitivity less than or equal to 0.0015 mu IU / ml. The invention also relates to a thyroid stimulating hormone detection kit, a detection method and a sample analyzer.

Description

Technical Field

[0001] The present invention belongs to the field of molecular immunology, and in particular relates to a thyroid stimulating hormone (TSH) specific antibody and a kit, a detection method and a sample analyzer for detecting TSH. Background Art

[0002] Thyroid dysfunction is common in adults, mainly hypothyroidism and hyperthyroidism. Thyroid stimulating hormone (TSH) in the blood is an effective marker for thyroid function. The normal range of TSH in serum is 0.58-4.1μIU / ml. The TSH concentration in the blood of patients with hypothyroidism is increased, while the TSH concentration in the blood of patients with hyperthyroidism is decreased.

[0003] The concentration of TSH in human serum samples is usually determined by sandwich immunoassay. Currently, most third-generation TSH assays are used, with a functional sensitivity of 0.01-0.02 μIU / mL. These tests accurately distinguish patients with hyperthyroidism from those with normal thyroid function. However, in some patients, serum TSH concentrations are below the detection limit of the third-generation TSH assay. The fourth-generation TSH analysis provides 10 times the functional sensitivity (0.001-0.002 μIU / mL), so TSH concentrations in the serum of these patients can be quantified. The functional sensitivity is determined by the affinity of the antibody used and the sensitivity of the detection system. Therefore, the fourth-generation TSH assay requires high-affinity antibodies that can provide high sensitivity.

[0004] TSH is a member of the glycoprotein hormone family, which consists of thyroid stimulating hormone, luteinizing hormone (LH), follicle stimulating hormone (FSH) and human chorionic gonadotropin (hCG). TSH is a heterodimer composed of α- and β-subunits. The α-subunit of TSH is similar to the α-subunits of LH, FSH and hCG, and the β-subunit is specific for TSH. However, there is a high degree of homology between the β-subunit sequences of human TSH, LH, FSH and CG. In order to accurately determine the concentration of TSH in serum samples, it is required that the antibodies used in the test do not recognize other glycoprotein hormones. In particular, in the one-step TSH sandwich immunoassay, the capture and detection antibodies are incubated simultaneously with the serum sample, and it is required that the two antibodies used should not cross-react with LH, FSH and hCG. Therefore, antibodies that are specific to TSH dimers or TSH β-subunits and do not cross-react with other glycoprotein hormones are required.

[0005] In order to accurately measure the concentration of TSH in serum, it is required to be able to measure all forms of TSH in the blood. In 2014, a new point mutation in the thyrotropin β subunit was reported, which resulted in the mutation of arginine at position 55 to glycine (R55G) (Drees JC et al. Falsely undetectable TSH in a cohort of South Asianeuthyroid patients. J Clin Endocrinol Metab. 2014Apr;99(4):1171-9.) This mutation changes an epitope on TSH, preventing the binding of antibodies against the epitope. In patients homozygous for the R55G mutation, TSH is undetectable by four widely used U.S. Food and Drug Administration (FDA)-approved TSH immunoassays. In this case, patients carrying the TSH R55G variant cannot be distinguished from patients with subclinical hyperthyroidism. Patients with undetectable TSH levels are mistakenly diagnosed with hyperthyroidism and treated. There is still a need to develop a kit that can accurately measure TSH in patients with the R55G mutation. Summary of the invention

[0006] One object of the present invention is to provide a novel monoclonal antibody or an antigen-binding antibody fragment thereof, which can specifically bind to TSH dimer or TSH β-subunit, and does not cross-react with the common α-subunit of human LH, FSH, CG and free glycoprotein hormones. It is suitable for developing a fourth-generation functional sensitivity TSH determination method. Another object of the present invention is to provide a monoclonal antibody or an antigen-binding antibody fragment thereof that recognizes TSH variant R55G.

[0007] Antibodies, nucleic acid molecules, expression vectors, host cells

[0008] Specifically, in one aspect, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to TSH or a mutant thereof, comprising:

[0009] 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, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 2, 8, 14, 20, 26, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98 and variants thereof, and / or a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99 and variants thereof; and / or,

[0010] 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, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101 and variants thereof, and / or a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102 and variants thereof.

[0011] In some embodiments, 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). In some embodiments, the substitution is a conservative substitution.

[0012] In some embodiments, 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.

[0013] In some embodiments, 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, Chothia, or IMGT numbering systems.

[0014] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0015] (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;

[0016] (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;

[0017] (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;

[0018] (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;

[0019] (1e) a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 25 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 26 or a variant thereof, and CDR-H3 having 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 having an amino acid sequence of SEQ ID NO: 28 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 29 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 30 or a variant thereof;

[0020] (1f) a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 31 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 32 or a variant thereof, and CDR-H3 having 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 having an amino acid sequence of SEQ ID NO: 34 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 35 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 36 or a variant thereof;

[0021] (1g) a heavy chain variable region comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 37 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 38 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 39 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: 40 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 41 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 42 or a variant thereof;

[0022] (1h) a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 43 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 44 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 45 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: 46 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 47 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 48 or a variant thereof;

[0023] (1i) a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 49 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 50 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 51 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: 52 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 53 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 54 or a variant thereof;

[0024] (1j) a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 55 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 56 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 57 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: 58 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 59 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 60 or a variant thereof;

[0025] (1k) a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 61 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 62 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 63 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: 64 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 65 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 66 or a variant thereof;

[0026] (11) a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 67 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 68 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 69 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: 70 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 71 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 72 or a variant thereof;

[0027] (1m) a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 73 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 74 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 75 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: 76 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 77 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 78 or a variant thereof;

[0028] (1n) a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 79 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 80 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 81 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: 82 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 83 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 84 or a variant thereof;

[0029] (1o) a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 85 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 86 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 87 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: 88 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 89 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 90 or a variant thereof;

[0030] (1p) a heavy chain variable region comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 91 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 92 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 93 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: 94 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 95 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 96 or a variant thereof; or

[0031] (1q) a heavy chain variable region comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 97 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 98 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 99 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: 100 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 101 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 102 or a variant thereof.

[0032] 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 are mainly based on a β-folded conformation, and three complementary determining regions are connected in the form of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein the CDR region is mainly based on a loop conformation, and forms a part of a β-folded structure in some cases. The complementary determining regions in each chain are closely together through FR, and together with the complementary determining regions from another chain, 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 each independently derived from the heavy chain framework region and the light chain framework region of a rabbit or sheep immunoglobulin. In some embodiments, the heavy chain framework region and the light chain framework region are each independently derived from the heavy chain framework region and the light chain framework region of a human or mouse immunoglobulin. In some embodiments, the heavy chain framework region and the light chain framework region each independently comprise a sequence derived from the heavy chain framework region and the light chain framework region of a mouse immunoglobulin, the heavy chain framework region and the 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 a sequence derived from the heavy chain framework region and the light chain framework region of a rabbit or sheep immunoglobulin, the heavy chain framework region and the 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 a sequence derived from the heavy chain framework region and the light chain framework region of a human germline antibody.

[0033] In some embodiments, the antibody or its antigen-binding fragment 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 rabbit or sheep heavy chain constant region and a rabbit or sheep 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. 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).

[0034] 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 rabbit or sheep antibody, a chimeric antibody or a humanized antibody.

[0035] In some embodiments, the antibody or antigen-binding fragment thereof is further detectably labeled.

[0036] 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 32 P), 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.

[0037] In some embodiments, the 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.

[0038] In some embodiments, the antibody or antigen-binding fragment thereof specifically recognizes TSH dimer or TSH β subunit. In some embodiments, the antibody or antigen-binding fragment thereof can also recognize TSH R55G mutant.

[0039] 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.

[0040] In another aspect, the present invention provides an expression vector comprising the isolated nucleic acid molecule described in any one of the above items.

[0041] In some embodiments, the vector is a plasmid, a virus, a phage, a bacterium, or a viroid.

[0042] In another aspect, the present invention provides a host cell comprising the isolated nucleic acid molecule or expression vector described in any one of the above.

[0043] In some embodiments, the host cell is a eukaryotic cell, preferably a mammalian cell.

[0044] In some embodiments, the host cell is a prokaryotic cell, preferably Escherichia coli.

[0045] In another aspect, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to TSH or a mutant thereof, wherein the antibody or antigen-binding fragment thereof:

[0046] Escherichia coli Rosetta, deposited at the Russian National Collection of Industrial Microorganisms (VKPM) with the deposit number B-14884 TM (DE3)pLysS rTS01 VL plasmid, and Escherichia coli Rosetta with the deposit number B-14885 TM Plasmid generation in (DE3)pLysS rTS01 VH;

[0047] Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14886 TM (DE3)pLysShTS02 VL plasmid, and Escherichia coli Rosetta with the deposit number B-14887 TMPlasmid production in (DE3)pLysShTS02 VH;

[0048] Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14888 TM (DE3)pLysShTS03 VL plasmid, and Escherichia coli Rosetta with the deposit number B-14889 TM Plasmid production in (DE3)pLysShTS03 VH;

[0049] Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14890 TM (DE3)pLysShTS04 VL plasmid, and Escherichia coli Rosetta with the deposit number B-14891 TM Plasmid production in (DE3)pLysShTS04 VH;

[0050] Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14892 TM (DE3)pLysShTS05 VL plasmid, and Escherichia coli Rosetta with the deposit number B-14893 TM Plasmid generation in (DE3)pLysShTS05 VH;

[0051] Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14894 TM (DE3)pLysShTS06 VL plasmid, and Escherichia coli Rosetta with the accession number B-14895 TM Plasmid production in (DE3)pLysShTS06 VH;

[0052] Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14896 TM (DE3)pLysShTS07 VL plasmid, and Escherichia coli Rosetta with the deposit number B-14897 TM Plasmid production in (DE3)pLysShTS07 VH;

[0053] Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14898 TM (DE3)pLysShTS08 VL plasmid, and Escherichia coli Rosetta with the deposit number B-14899 TM Plasmid production in (DE3)pLysShTS08 VH;

[0054] Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14900 TM (DE3)pLysShTS09 VL plasmid, and Escherichia coli Rosetta with the deposit number B-14901 TM Plasmid production in (DE3)pLysShTS09 VH;

[0055] Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14902 TM (DE3)pLysShTS10 VL plasmid, and Escherichia coli Rosetta with the accession number B-14903 TM Plasmid production in (DE3)pLysShTS10 VH;

[0056] Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14904 TM (DE3)pLysShTS11 VL plasmid, and Escherichia coli Rosetta with the accession number B-14905 TM Plasmid production in (DE3)pLysShTS11 VH;

[0057] Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14906 TM (DE3)pLysShTS12 VL plasmid, and Escherichia coli Rosetta with the deposit number B-14907 TM Plasmid production in (DE3)pLysShTS12 VH;

[0058] Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14908 TM(DE3)pLysShTS13 VL plasmid, and Escherichia coli Rosetta with the deposit number B-14909 TM Plasmid generation of (DE3)pLysShTS13 VH;

[0059] Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14910 TM (DE3)pLysShTS14 VL plasmid, and Escherichia coli Rosetta with the accession number B-14911 TM Plasmid production in (DE3)pLysShTS14 VH;

[0060] Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14912 TM (DE3)pLysShTS15 VL plasmid, and Escherichia coli Rosetta with the accession number B-14913 TM Plasmid generation in (DE3)pLysShTS15 VH;

[0061] Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14914 TM (DE3)pLysShTS16 VL plasmid, and Escherichia coli Rosetta with the accession number B-14915 TM Plasmid production in (DE3)pLysShTS16 VH; or

[0062] Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14916 TM (DE3)pLysShTS17 VL plasmid, and Escherichia coli Rosetta with accession number B-14917 TM Plasmid generation in (DE3)pLysShTS17 VH.

[0063] Reagent test kit

[0064] At present, the mainstream TSH test is still based on the third-generation test reagents. The functional sensitivity of TSH is 0.01-0.02 μIU / mL, which still has obvious shortcomings in some special clinical scenarios:

[0065] 1. For patients with severe hyperthyroidism, accurate TSH measurement is particularly important in doctors' initial judgment and treatment monitoring of patients. However, the TSH content in the circulation of these patients is often lower than 0.01 μIU / mL, so the sensitivity of existing detection reagents may not meet this specific clinical need;

[0066] 2. For patients with thyroid cancer, after their thyroid gland is completely removed, they need to take levothyroxine tablets to inhibit the synthesis and secretion of TSH in the body. In order to better inhibit the recurrence of thyroid cancer, the optimization of this inhibition therapy is to suppress TSH to below 0.01μIU / mL. Therefore, the sensitivity of existing detection reagents cannot meet this specific clinical need.

[0067] The antibody of the present invention has excellent specificity and affinity for TSH, and on the one hand does not cross-react with the α-subunit shared by LH, FSH, CG and free glycoprotein hormones, and on the other hand can recognize the TSH variant R55G. The kit developed based on the antibody of the present invention can achieve a fourth-generation functional sensitivity of <0.002μIU / mL.

[0068] Therefore, in one aspect, the present invention provides a TSH detection kit for detecting the presence or level of TSH or a mutant thereof in a sample.

[0069] In some embodiments, the detection sensitivity of the kit to TSH is ≤0.0015 μIU / mL, preferably ≤0.001 μIU / mL.

[0070] In some embodiments, the TSH or a mutant thereof is selected from a TSH dimer, a β subunit of TSH, and / or an R55G mutant of TSH.

[0071] In some embodiments, the kit includes a first reagent and a second reagent, wherein the first reagent contains magnetic beads coated with a first antibody that can recognize TSH or a mutant thereof; the second reagent contains a second antibody bound to a chemiluminescent marker, wherein after the sample and the first reagent and the second reagent are mixed, a sandwich complex of the first antibody-antigen-second antibody can be formed, and the antigen is TSH or a mutant thereof.

[0072] The marker is well known to those skilled in the art, and may be, for example, enzymes such as oxidase, microperoxidase, horseradish peroxidase, alkaline phosphatase (ALP), β-galactosidase, glucose oxidase, and glucose 6-phosphate dehydrogenase; fluorescent substances such as fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, fluorescein, rhodamine, europium, and green fluorescent protein; chemiluminescent substances such as luminol, isoluminol, phenanthridinium, and acridinium ester.

[0073] In some embodiments, the first antibody and / or the second antibody is selected from an antibody or an antigen-binding fragment thereof that specifically binds to TSH or a mutant thereof, and comprises:

[0074] 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, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 2, 8, 14, 20, 26, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98 and variants thereof, and / or a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99 and variants thereof; and / or,

[0075] 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, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101 and variants thereof, and / or a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102 and variants thereof.

[0076] In some embodiments, the first antibody and / or the second antibody:

[0077] 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;

[0078] 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;

[0079] 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;

[0080] 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;

[0081] A heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 25 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 26 or a variant thereof, and CDR-H3 having 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 having an amino acid sequence of SEQ ID NO: 28 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 29 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 30 or a variant thereof;

[0082] A heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 31 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 32 or a variant thereof, and CDR-H3 having 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 having an amino acid sequence of SEQ ID NO: 34 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 35 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 36 or a variant thereof;

[0083] A heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 37 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 38 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 39 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: 40 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 41 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 42 or a variant thereof;

[0084] A heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 43 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 44 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 45 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: 46 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 47 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 48 or a variant thereof;

[0085] A heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 49 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 50 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 51 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: 52 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 53 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 54 or a variant thereof;

[0086] A heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 55 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 56 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 57 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: 58 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 59 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 60 or a variant thereof;

[0087] A heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 61 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 62 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 63 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: 64 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 65 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 66 or a variant thereof;

[0088] A heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 67 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 68 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 69 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: 70 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 71 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 72 or a variant thereof;

[0089] A heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 73 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 74 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 75 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: 76 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 77 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 78 or a variant thereof;

[0090] A heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 79 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 80 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 81 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: 82 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 83 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 84 or a variant thereof;

[0091] A heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 85 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 86 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 87 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: 88 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 89 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 90 or a variant thereof;

[0092] A heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 91 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 92 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 93 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: 94 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 95 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 96 or a variant thereof; or

[0093] A heavy chain variable region comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 97 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 98 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 99 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: 100 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 101 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 102 or a variant thereof.

[0094] In some embodiments, the first antibody is an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 31 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 32 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 33 and variants thereof; and, a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 34 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 35 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 36 and variants thereof; and,

[0095] The second antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence selected from SEQ ID NO: 49 and variants thereof, CDR-H2 having an amino acid sequence selected from SEQ ID NO: 50 and variants thereof, and CDR-H3 having an amino acid sequence selected from SEQ ID NO: 51 and variants thereof; and, a light chain variable region comprising the following three CDRs: CDR-L1 having an amino acid sequence selected from SEQ ID NO: 52 and variants thereof, CDR-L2 having an amino acid sequence selected from SEQ ID NO: 53 and variants thereof, and CDR-L3 having an amino acid sequence selected from SEQ ID NO: 54 and variants thereof.

[0096] In some embodiments, the first antibody is an antibody or antigen-binding fragment thereof comprising a heavy chain variable region of the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 37 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 38 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 39 and variants thereof; and, a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 40 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 41 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 42 and variants thereof; and,

[0097] The second antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence selected from SEQ ID NO: 43 and variants thereof, CDR-H2 having an amino acid sequence selected from SEQ ID NO: 44 and variants thereof, and CDR-H3 having an amino acid sequence selected from SEQ ID NO: 45 and variants thereof; and, a light chain variable region comprising the following three CDRs: CDR-L1 having an amino acid sequence selected from SEQ ID NO: 46 and variants thereof, CDR-L2 having an amino acid sequence selected from SEQ ID NO: 47 and variants thereof, and CDR-L3 having an amino acid sequence selected from SEQ ID NO: 48 and variants thereof.

[0098] In some embodiments, the first antibody is an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence selected from SEQ ID NO: 25 and variants thereof, CDR-H2 having an amino acid sequence selected from SEQ ID NO: 26 and variants thereof, and CDR-H3 having an amino acid sequence selected from SEQ ID NO: 27 and variants thereof; and, a light chain variable region comprising the following three CDRs: CDR-L1 having an amino acid sequence selected from SEQ ID NO: 28 and variants thereof, CDR-L2 having an amino acid sequence selected from SEQ ID NO: 29 and variants thereof, and CDR-L3 having an amino acid sequence selected from SEQ ID NO: 30 and variants thereof; and,

[0099] The second antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence selected from SEQ ID NO: 49 and variants thereof, CDR-H2 having an amino acid sequence selected from SEQ ID NO: 50 and variants thereof, and CDR-H3 having an amino acid sequence selected from SEQ ID NO: 51 and variants thereof; and, a light chain variable region comprising the following three CDRs: CDR-L1 having an amino acid sequence selected from SEQ ID NO: 52 and variants thereof, CDR-L2 having an amino acid sequence selected from SEQ ID NO: 53 and variants thereof, and CDR-L3 having an amino acid sequence selected from SEQ ID NO: 54 and variants thereof.

[0100] In some embodiments, the first antibody is an antibody or antigen-binding fragment thereof comprising a heavy chain variable region of the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 19 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 20 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 21 and variants thereof; and, a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 22 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 23 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 24 and variants thereof; and,

[0101] The second antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region of the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 61 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 62 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 63 and variants thereof; and, a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 64 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 65 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 66 and variants thereof.

[0102] In some embodiments, the first antibody is an antibody or antigen-binding fragment thereof comprising a heavy chain variable region of the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 19 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 20 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 21 and variants thereof; and, a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 22 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 23 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 24 and variants thereof; and,

[0103] The second antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence selected from SEQ ID NO: 43 and variants thereof, CDR-H2 having an amino acid sequence selected from SEQ ID NO: 44 and variants thereof, and CDR-H3 having an amino acid sequence selected from SEQ ID NO: 45 and variants thereof; and, a light chain variable region comprising the following three CDRs: CDR-L1 having an amino acid sequence selected from SEQ ID NO: 46 and variants thereof, CDR-L2 having an amino acid sequence selected from SEQ ID NO: 47 and variants thereof, and CDR-L3 having an amino acid sequence selected from SEQ ID NO: 48 and variants thereof.

[0104] In another aspect, the present invention provides a composition or a TSH detection kit, which contains the antibody or antigen-binding fragment thereof described in any one of the above items.

[0105] In some embodiments, the composition or TSH detection kit contains:

[0106] A first antibody, which is selected from at least one of the antibodies or antigen-binding fragments thereof described in any one of the preceding items, and

[0107] A second antibody, which is selected from at least one of the antibodies or antigen-binding fragments thereof described in any one of the preceding items;

[0108] The first antibody and the second antibody are respectively directed against different epitopes of TSH or its mutants.

[0109] In some embodiments, the composition or TSH detection kit, wherein:

[0110] I) the first antibody is the antibody or antigen-binding fragment thereof according to the first aspect (1a), or a combination thereof with any one or more of (1b)-(1q) defined in the first aspect;

[0111] The second antibody is selected from one or more of (1b)-(1q) as defined in the first aspect, preferably (1c) or (1f);

[0112] II) the first antibody is the antibody or antigen-binding fragment thereof according to the first aspect (1b), or a combination thereof with any one or more of (1a), (1c)-(1q) as defined in the first aspect;

[0113] The second antibody is selected from one or more of (1a), (1c)-(1q) as defined in the first aspect, preferably (1i) or (1j);

[0114] III) the first antibody is the antibody or antigen-binding fragment thereof according to the first aspect (1c), or a combination thereof with any one or more of (1a), (1b), (1d)-(1q) as defined in the first aspect;

[0115] The second antibody is selected from one or more of (1a), (1b), (1d)-(1q) as defined in the first aspect, preferably (1j);

[0116] IV) the first antibody is the antibody or antigen-binding fragment thereof according to the first aspect (1d), or a combination thereof with any one or more of (1a)-(1c), (1e)-(1q) as defined in the first aspect;

[0117] The second antibody is selected from one or more of (1a)-(1c), (1e)-(1q) as defined in the first aspect, preferably (1h), (1k) or (1o);

[0118] V) the first antibody is the antibody or antigen-binding fragment thereof according to the first aspect (1e), or a combination thereof with any one or more of (1a)-(1d), (1f)-(1q) as defined in the first aspect;

[0119] The second antibody is selected from one or more of (1a)-(1d), (1f)-(1q) as defined in the first aspect, preferably (1m) or (1o);

[0120] VI) the first antibody is the antibody or antigen-binding fragment thereof according to the first aspect (1f), or a combination thereof with any one or more of (1a)-(1e), (1g)-(1q) as defined in the first aspect;

[0121] The second antibody is selected from one or more of (1a)-(1e), (1g)-(1q) as defined in the first aspect, preferably (1i);

[0122] VII) the first antibody is the antibody or antigen-binding fragment thereof according to the first aspect (1g), or a combination thereof with any one or more of (1a)-(1f), (1h)-(1q) as defined in the first aspect;

[0123] The second antibody is selected from one or more of (1a)-(1f), (1h)-(1q) as defined in the first aspect, preferably (1h) or (1j);

[0124] VIII) the first antibody is the antibody or antigen-binding fragment thereof according to the first aspect (1h), or a combination thereof with any one or more of (1a)-(1g), (1i)-(1q) as defined in the first aspect;

[0125] The second antibody is selected from one or more of (1a)-(1g), (1i)-(1q) as defined in the first aspect, preferably (1f);

[0126] IX) the first antibody is the antibody or antigen-binding fragment thereof according to the first aspect (1i), or a combination thereof with any one or more of (1a)-(1h), (1j)-(1q) as defined in the first aspect;

[0127] The second antibody is selected from one or more of (1a)-(1h), (1j)-(1q) as defined in the first aspect, preferably (1f);

[0128] X) the first antibody is the antibody or antigen-binding fragment thereof according to the first aspect (1j), or a combination thereof with any one or more of (1a)-(1i), (1k)-(1q) as defined in the first aspect;

[0129] The second antibody is selected from one or more of (1a)-(1i), (1k)-(1q) as defined in the first aspect;

[0130] XI) the first antibody is the antibody or antigen-binding fragment thereof according to the first aspect (1k), or a combination thereof with any one or more of (1a)-(1j), (1l)-(1q) as defined in the first aspect;

[0131] The second antibody is selected from one or more of (1a)-(1j), (1l)-(1q) as defined in the first aspect;

[0132] XII) the first antibody is the antibody or antigen-binding fragment thereof according to the first aspect (11), or a combination thereof with any one or more of (1a)-(1k), (1m)-(1q) as defined in the first aspect;

[0133] The second antibody is selected from one or more of (1a)-(1k), (1m)-(1q) as defined in the first aspect, preferably (1f);

[0134] XIII) the first antibody is the antibody or antigen-binding fragment thereof according to the first aspect (1m), or a combination thereof with any one or more of (1a)-(1l), (1n)-(1q) as defined in the first aspect;

[0135] The second antibody is selected from one or more of (1a)-(1l), (1n)-(1q) as defined in the first aspect;

[0136] XIV) the first antibody is the antibody or antigen-binding fragment thereof according to the first aspect (1n), or a combination thereof with any one or more of (1a)-(1m), (1o)-(1q) as defined in the first aspect;

[0137] The second antibody is selected from one or more of (1a)-(1m), (1o)-(1q) as defined in the first aspect, preferably (1f); or

[0138] XV) the first antibody is the antibody or antigen-binding fragment thereof according to the first aspect (1o), or a combination thereof with any one or more of (1a)-(1n), (1p)-(1q) as defined in the first aspect;

[0139] The second antibody is selected from one or more of (1a)-(1n), (1p)-(1q) as defined in the first aspect;

[0140] XVI) the first antibody is the antibody or antigen-binding fragment thereof according to the first aspect (1p), or a combination thereof with any one or more of (1a)-(1o) and (1q) as defined in the first aspect;

[0141] The second antibody is selected from one or more of (1a)-(1o), (1q), preferably (1c) or (1f) as defined in the first aspect; or

[0142] XVII) the first antibody is the antibody or antigen-binding fragment thereof according to the first aspect (1q), or a combination thereof with any one or more of (1a)-(1p) as defined in the first aspect;

[0143] The second antibody is selected from one or more of (1a)-(1p) defined in the first aspect.

[0144] In some embodiments, the composition or TSH detection kit, wherein:

[0145] The first antibody is the antibody or antigen-binding fragment thereof according to any one of (1b) to (1g) of the first aspect;

[0146] The second antibody is the antibody or antigen-binding fragment thereof according to any one of (1a), (1h) to (1q) of the first aspect.

[0147] In some embodiments, the composition or TSH detection kit contains:

[0148] The first antibody of the first aspect (1a), and the second antibody of the first aspect (1c);

[0149] The first antibody of the first aspect (1a), and the second antibody of the first aspect (1f);

[0150] The first antibody of the first aspect (1h), and the second antibody of the first aspect (1f);

[0151] The first antibody of the first aspect (1i), and the second antibody of the first aspect (1f);

[0152] The first antibody of the first aspect (1b), and the second antibody of the first aspect (1i);

[0153] The first antibody of the first aspect (1b), and the second antibody of the first aspect (1j);

[0154] The first antibody of the first aspect (1c), and the second antibody of the first aspect (1j);

[0155] The first antibody of the first aspect (1d), and the second antibody of the first aspect (1o);

[0156] The first antibody of the first aspect (11), and the second antibody of the first aspect (1f);

[0157] The first antibody of the first aspect (1n), and the second antibody of the first aspect (1f)

[0158] The first antibody of the first aspect (1e), and the second antibody of the first aspect (1m);

[0159] The first antibody of the first aspect (1e), and the second antibody of the first aspect (1o);

[0160] The first antibody of the first aspect (1g), and the second antibody of the first aspect (1j);

[0161] The first antibody of the first aspect (1p), and the second antibody of the first aspect (1c);

[0162] The first antibody of the first aspect (1p), and the second antibody of the first aspect (1f);

[0163] The first antibody of the first aspect (1f), and the second antibody of the first aspect (1i);

[0164] The first antibody of the first aspect (1d), and the second antibody of the first aspect (1h);

[0165] The first antibody of the first aspect (1d), and the second antibody of the first aspect (1k);

[0166] The first antibody of the first aspect (1g), and the second antibody of the first aspect (1h); or

[0167] The first antibody of the first aspect (1e), and the second antibody of the first aspect (1i).

[0168] In some embodiments, the composition or TSH detection kit contains:

[0169] The first antibody of the first aspect (1b), and the second antibody of the first aspect (1i);

[0170] The first antibody of the first aspect (1b), and the second antibody of the first aspect (1j);

[0171] The first antibody of the first aspect (1c), and the second antibody of the first aspect (1j);

[0172] The first antibody of the first aspect (1d), and the second antibody of the first aspect (1o);

[0173] The first antibody of the first aspect (1e), and the second antibody of the first aspect (1m);

[0174] The first antibody of the first aspect (1e), and the second antibody of the first aspect (1o);

[0175] The first antibody of the first aspect (1g), and the second antibody of the first aspect (1j);

[0176] The first antibody of the first aspect (1f), and the second antibody of the first aspect (1i);

[0177] The first antibody of the first aspect (1d), and the second antibody of the first aspect (1h);

[0178] The first antibody of the first aspect (1d), and the second antibody of the first aspect (1k);

[0179] The first antibody of the first aspect (1g), and the second antibody of the first aspect (1h); or

[0180] The first antibody of the first aspect (1e), and the second antibody of the first aspect (1i).

[0181] In some embodiments, the first antibody is a capture antibody and the second antibody is a detection antibody.

[0182] In some embodiments, the immunoassay kit further comprises a solid phase carrier.

[0183] In some embodiments, the solid support is selected from magnetic particles or microtiter plates (eg, microtiter plates or ELISA plates).

[0184] In some embodiments, the first antibody is coated on the surface of the solid phase carrier, preferably fixed on the surface of magnetic particles.

[0185] In some embodiments, the second antibody is detectably labeled.

[0186] In some embodiments, the 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.

[0187] In some embodiments, the second antibody is labeled with alkaline phosphatase.

[0188] In some embodiments, the kit further comprises a primary antibody diluent and a secondary antibody diluent, wherein:

[0189] The first antibody diluent contains one or more of the following ingredients: a buffer solution, an inorganic salt, a preservative, a surfactant and a stabilizer; and / or

[0190] 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.

[0191] In some embodiments, the first antibody diluent and the first antibody are placed in the same or different formulation units.

[0192] In some embodiments, the second antibody diluent and the second antibody are placed in the same or different formulation units.

[0193] In some embodiments, the buffer solution is selected from Tris, Hepes and PBS buffer solutions.

[0194] In some embodiments, the inorganic salt is selected from one or more of NaCl, KCl, CaCl2, MgCl2 and ZnCl2.

[0195] In some embodiments, the preservative is selected from Proclin 300, PC-300, BND (eg, BND-10 or BND-99), and BIT (eg, BIT-10).

[0196] In some embodiments, the surfactant is selected from Tween-20, TritonX-100 and S9.

[0197] In some embodiments, the stabilizer is selected from calf serum, bovine serum albumin, and gelatin.

[0198] In some embodiments, the immunoassay kit further comprises one or more of the following: a TSH calibrator, a substrate for the detection marker, a washing solution, a stop solution, and instructions for use.

[0199] In some embodiments, the immunoassay kit is used to detect the presence or level of TSH or its mutants in a sample.

[0200] In some embodiments, the TSH or a mutant thereof is selected from a TSH dimer, a β subunit of TSH, or an R55G mutant of TSH.

[0201] In some embodiments, the sample is from a human, such as a human body fluid (eg, blood, serum, or plasma).

[0202] In some embodiments, the detection sensitivity of the immunoassay kit to TSH or its mutants is ≤0.0015 μIU / mL, preferably ≤0.001 μIU / mL.

[0203] Detection Methods

[0204] Based on the aforementioned antibodies and kits, the present invention further provides a method for detecting the presence or level of TSH or its mutants in a sample, which comprises the step of using any of the above-mentioned antibodies or antigen-binding fragments thereof or immunoassay kits for detection.

[0205] In some embodiments, the method comprises the steps of:

[0206] contacting the sample with two or more of the antibodies or antigen-binding fragments thereof, and

[0207] Qualitatively or quantitatively detecting the binding of the two or more antibodies or antigen-binding fragments thereof to TSH or a mutant thereof;

[0208] The binding indicates the presence or concentration of TSH or a mutant thereof in the sample, wherein

[0209] The two or more antibodies or antigen-binding fragments thereof are directed against two or more epitopes of TSH or its mutants.

[0210] In some embodiments, at least one of the two or more antibodies or antigen-binding fragments thereof is a capture antibody and at least another one is a detection antibody.

[0211] In some embodiments, the capture antibody is a primary antibody as defined in any of the preceding items.

[0212] In some embodiments, the detection antibody is a secondary antibody as defined in any of the preceding items.

[0213] In some embodiments, the method comprises the following steps:

[0214] (1) contacting the sample with the capture antibody to form an antibody-antigen complex;

[0215] (2) contacting the antibody-antigen complex with the detection antibody to form an antibody-antigen-antibody complex; and

[0216] (3) Determining the amount of the antibody-antigen-antibody complex.

[0217] The antibodies or antigen-binding antibody fragments thereof of the present invention do not have cross-reactions with other glycoprotein hormones and can be used in a single-step sandwich immunoassay, in which the capture and detection antibodies are incubated with human blood samples simultaneously. Therefore, in some embodiments, step (1) and step (2) can be performed separately or simultaneously.

[0218] In some embodiments, the method is ELISA immunoassay, such as double antibody sandwich ELISA immunoassay, preferably a one-step double antibody sandwich ELISA immunoassay.

[0219] application

[0220] In another aspect, the present invention provides the use of the antibody or antigen-binding fragment thereof in preparing a kit or the use of any of the above immunoassay kits, wherein the kit is used for:

[0221] (1) detecting the presence or level of TSH or its mutants in a sample;

[0222] (2) Diagnosis or auxiliary diagnosis of thyroid dysfunction (such as hypothyroidism or hyperthyroidism);

[0223] (3) Diagnosis or auxiliary diagnosis of thyroid-related diseases; and / or

[0224] (4) Evaluation or auxiliary evaluation of the treatment effect of thyroid-related diseases.

[0225] In another aspect, the present invention provides a 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 one of the above, or detecting a sample from a subject using the method described in any one of the above, and quantifying TSH or a mutant thereof in the sample, wherein the method is used for:

[0226] (1) Diagnosis or auxiliary diagnosis of thyroid dysfunction (such as hypothyroidism or hyperthyroidism);

[0227] (2) diagnosis or auxiliary diagnosis of thyroid-related diseases; and / or

[0228] (3) Evaluation or auxiliary evaluation of the treatment effect of thyroid-related diseases.

[0229] In some embodiments, the thyroid-related disease is hyperthyroidism or thyroid cancer.

[0230] In some embodiments, the sample is from a human, such as a human body fluid (eg, blood, serum, or plasma).

[0231] Detection device

[0232] As we all know, the basic principle of the photomultiplier tube is that a photon outputs a beam of electrons through the photomultiplier tube, which corresponds to an electrical pulse signal. However, since the electrical pulse signals caused by photons entering the photomultiplier tube are randomly distributed, as the intensity of the light signal to be measured increases, the electrical pulse signals caused by photons entering the photomultiplier tube become more and more, resulting in the phenomenon of pulse accumulation in which two or more electrical pulses are connected together. Fig.14 and Fig.15 As shown, Fig.14 shows the electrical pulse signal under normal conditions, while Fig.15 The electrical pulse signal under pulse accumulation is shown, wherein when the peak value of the identified pulse is greater than the threshold, it is considered that a photon is identified. Fig.14 The number of photons can be accurately detected in the case of Fig.15 In some cases, multiple accumulated electrical pulses will be identified as one electrical pulse, resulting in low counting results in the higher light intensity segment, and the upper limit of the linear range of the photometric device is insufficient. For example, the upper limit of the linear range of existing photometric devices is only 30 million photons per second. When the measured luminescence value of the sample (such as a high-value sample of TSH) is greater than 30 million photons per second, the test result is unreliable, and the sample usually needs to be diluted and retested, but this seriously affects the measurement speed, resulting in a longer reporting time for the sample.

[0233] The immunological kit of the present invention can realize ultrasensitive detection of samples. However, due to the linear range limitation of the measurement signal of the device, one sample measurement cannot simultaneously meet the ultrasensitive detection of low-value samples and the detection of high-value samples.

[0234] Based on the aforementioned antibodies and kits, the present invention further provides a sample analyzer with a high linear range, which can detect optical signals in a high linear range and can be adapted to the kit of the present invention, so that the sample can be detected in one time with ultra-sensitivity and accurate results.

[0235] Specifically, the sample analyzer includes the antibody or antigen-binding fragment thereof or the immunoassay kit described in any one of the above.

[0236] In some embodiments, the sample analyzer comprises:

[0237] A loading device, configured to load the sample to be tested and the reagents required for the chemiluminescent reaction into the reaction container, wherein the reagents include the antibody or antigen-binding fragment thereof, or the immunoassay kit;

[0238] A reaction incubation device, configured to provide a reaction and incubation place for a reaction container containing a sample to be tested and a reagent required for a chemiluminescent reaction, so that the sample to be tested and the reagent required for a chemiluminescent reaction in the reaction container form a sample liquid to be tested; and

[0239] A light measuring device, configured to measure light on the sample liquid to be tested; wherein the light measuring device comprises:

[0240] A receiving component configured to receive a light signal emitted by the sample to be tested through a chemiluminescent reaction and convert the light signal into a corresponding electrical signal; and

[0241] A processing component is configured to be electrically connected to a receiving component and receive the electrical signal from the receiving component, the processing component includes a first photon counting module and a second photon counting module, the first photon counting module is configured to detect the number of pulses of the electrical signal using a pulse recognition method to obtain a first photon counting result, the second photon counting module is configured to process the electrical signal to obtain a parameter characterizing the number of photons, and calculate a second photon counting result based on the parameter characterizing the number of photons and a preset calibration function, wherein the calibration function represents a mapping relationship between the parameter characterizing the number of photons and the photon counting result, and the processing component is further configured to output a final photon counting result of the sample to be tested based on the first photon counting result and the second photon counting result.

[0242] In some embodiments, the processing component is further configured to: when the first photon counting result is lower than a first threshold, output the first photon counting result as the final photon counting result; when the first photon counting result is higher than a second threshold, output the second photon counting result as the final photon counting result, wherein the second threshold is greater than or equal to the first threshold.

[0243] In some embodiments, the second photon counting module includes an integration circuit and a second counting circuit electrically connected to each other, the integration circuit is configured to integrate the electrical signal within a predetermined time period to obtain a DC component signal, the parameters characterizing the number of photons include parameters related to the DC component signal, and the second counting circuit is configured to calculate the second photon counting result based on the parameters related to the DC component signal and a preset calibration function.

[0244] In some embodiments, the sample analyzer comprises:

[0245] A loading device, configured to load the sample to be tested and the reagents required for the chemiluminescent reaction into the reaction container, wherein the reagents include the antibody or antigen-binding fragment thereof, or the immunoassay kit;

[0246] A reaction incubation device, configured to provide a reaction and incubation place for a reaction container containing a sample to be tested and a reagent required for a chemiluminescent reaction, so that the sample to be tested and the reagent required for a chemiluminescent reaction in the reaction container form a sample liquid to be tested; and

[0247] A photometric device, the photometric device is configured to measure the photometry of a sample liquid to be tested in a reaction container that has completed reaction and incubation, the linear detection range of the photometric device is [A1, A2] photons / second, the linear range represents the range of the output photon number of the photometric device, within the linear detection range, the intensity of the luminescent signal generated in the chemiluminescent reaction is linear with the output photon number of the detection device, wherein A1 is less than or equal to 2000 and A2 is greater than or equal to 10 8 .

[0248] like Fig.16 As shown, an embodiment of the present invention further provides a sample analyzer 1, which includes a loading device 200, a reaction incubation device 300 and a light measuring device 100 according to any of the above embodiments.

[0249] The loading device 200 is configured to load the sample to be tested, such as a blood sample, and the reagents required for the chemiluminescent reaction into the reaction container. The reaction incubation device 300 is configured to provide a place for reaction and incubation for the reaction container containing the sample to be tested and the reagents required for the chemiluminescent reaction, so that the sample to be tested and the reagents required for the chemiluminescent reaction in the reaction container form a sample liquid to be tested. The photometric device 100 is configured to perform photometric measurement on the sample liquid to be tested in the reaction container that has completed the reaction and incubation.

[0250] In a specific embodiment, Fig.16As shown, the sample analyzer 1 is constructed as a chemiluminescent immunoassay analyzer for analyzing samples, such as blood samples. The loading device 200 includes a sample supply unit 210 and a reagent supply unit 220, the sample supply unit 210 is configured to absorb the sample to be tested and supply it to the reaction container, and the reagent supply unit 220 is configured to absorb the immune reagent (such as a labeled antigen or antibody) and the magnetic bead reagent and supply them to the reaction container. The chemiluminescent immunoassay analyzer also includes a magnetic separation device 400, a substrate supply device (not shown) and a transport device 500, the magnetic separation device 400 is configured to perform a magnetic separation operation on the reaction container containing the sample to be tested, the immune reagent and the magnetic bead reagent, the substrate supply device is configured to supply the luminescent substrate to the reaction container that has completed the magnetic separation operation, and the transport device 500 is configured to transport the reaction container that has completed the magnetic separation operation from the magnetic separation device 500 to the reaction incubation device 300. The incubated reaction container is transported to the photometric device 100 for photometry.

[0251] In some embodiments, the reagent supply unit 220 includes a reagent tray 221 and a reagent needle 222. The reagent tray 221 is used to place reagent bottles containing immunoreagents and magnetic bead reagents and has the function of refrigerating the reagent bottles. The reagent needle 222 is used to absorb the reagent from the reagent bottle at the reagent absorption position and discharge the absorbed reagent into the reaction container at the sample addition position.

[0252] In some embodiments, the chemiluminescent immunoassay analyzer further includes a mixer 700 disposed between the reaction disk and the magnetic separation disk, the mixer being configured to perform a mixing operation on the reaction container to which the reagent and the sample have been added. The mixer can be configured, for example, as a vortex mixer for non-contact mixing, which can effectively avoid cross contamination.

[0253] In some embodiments, the chemiluminescent immunoassay analyzer also includes a first manipulator 600 for transporting a reaction vessel, the first manipulator being configured to move in three dimensions and to clamp the reaction vessel. The first manipulator is configured to load a new reaction vessel into the sample loading position so that the sample needle can inject the sample. The first manipulator is also configured to discard an empty reaction cup. The first manipulator is further configured to transport the reaction vessel between the sample loading position, the reaction incubation device 300, and the dilution position.

[0254] In some embodiments, the upper limit of the linear range of the light measuring device 100 to detect the light signal is greater than or equal to 10 8 (i.e. 100 million) photons / second; the upper limit of the linear range of the photometric device 100 is greater than or equal to 10 8photons / second, the upper limit of the detection range of the aforementioned test kit can reach 100μIU / mL, which can achieve accurate results for high-value samples in one measurement; preferably, the linear range of the light signal detected by the photometric device 100 is greater than or equal to 120 million photons / second; further preferably, the linear range of the light signal detected by the photometric device 100 is greater than or equal to 150 million photons / second; more preferably, the linear range of the light signal detected by the photometric device 100 is greater than or equal to 200 million photons / second, and the linear detection range refers to the linear detection range of a single detection, that is, the linear detection range that can be achieved in one detection of a sample.

[0255] According to some embodiments of the light measuring device 100 of the present invention, Fig.17 As shown, the light measuring device 100 includes a receiving component 10 and a processing component 20 .

[0256] The receiving component 10 is configured to receive the optical signal emitted by the sample to be tested, such as the blood sample to be tested, after the chemiluminescent reaction and convert the optical signal into a corresponding electrical signal. The receiving component 10 can be configured as a photomultiplier tube that converts weak optical signals into electrical signals.

[0257] The processing component 20 is configured to be electrically connected to the receiving component 10 so as to receive an electrical signal from the receiving component 10 , and to process the received electrical signal so as to obtain the number of photons.

[0258] According to the present invention, the processing component 20 includes a first photon counting module 21 and a second photon counting module 22, wherein the first photon counting module 21 is configured to process the electrical signal using a first photon counting method to obtain a first photon counting result, and the second photon counting module 22 is configured to process the electrical signal using a second photon counting method different from the first photon counting method to obtain a second photon counting result. The processing component 20 is also configured to output a final photon counting result of the sample to be tested based on the first photon counting result and the second photon counting result. The final photon counting result can then be used to calculate the content of the substance to be tested in the sample to be tested.

[0259] In the embodiment of the present invention, two photon counting modules are used to count photons simultaneously, wherein the first photon counting module uses a first counting method for a weak light segment to obtain a first photon counting result, and the second photon counting module uses a second counting method for a strong light segment to obtain a second photon counting result, and finally the first photon counting result and the second photon counting result are integrated and the final counting result is output. This can greatly expand the linear photometric range of the photometric device. Fig.18As shown, curve S1 represents the photometric linear characteristics of the photometric device in the prior art, and curve S2 represents the photometric linear characteristics of the photometric device according to the present invention. The present invention can achieve the expansion of the linear photometric range of the photometric device from the original linear photometric range of 0 to 30 million photons / second (i.e., 0 to 30 million photons per second) to 0 to 200 million photons / second (i.e., 0 to 200 million photons per second).

[0260] After obtaining the first photon counting result and the second photon counting result, the processing component 20 is further configured to output a final photon counting result according to one of the following embodiments.

[0261] In some embodiments, the processing component 20 may be configured to: when the first photon counting result is lower than a first threshold, output the first photon counting result as the final photon counting result; when the first photon counting result is higher than a second threshold, output the second photon counting result as the final photon counting result, wherein the second threshold is greater than or equal to the first threshold. For example, when the first photon counting result is lower than 30 million photons / second, the first photon counting result is output; and when the first photon counting result is not lower than 30 million photons / second, the second photon counting result is output.

[0262] Further, the processing component may be further configured to: when the first photon counting result is between a first threshold and a second threshold, calculate the final photon counting result according to the first photon counting result and the second photon counting result by a preset fusion function, wherein the second threshold is greater than the first threshold. The preset fusion function may be, for example, a function for finding an average value or a function for finding a weighted average value.

[0263] In other embodiments, multiple threshold intervals may be set for the first photon counting module. In this case, the processing component 20 may also be configured to: when the first photon counting result is in different threshold intervals, use different fusion functions to calculate the first photon counting result and the second photon counting result, and output the calculation result as the final photon counting result. Here, the fusion function may be, for example, a function for obtaining a weighted average, and the fusion functions for different threshold intervals have different weights for the first photon counting result and the second photon counting result.

[0264] As the photometric device 10 ages and the usage environment changes, the first photon counting result and the second photon counting result will show different variations, and there will be deviations between the two near the point where the first threshold is located. Therefore, the setting of at least two thresholds can enable the photometer to meet the linearity or precision requirements in long-term use.

[0265] Next, some embodiments of the first photon counting module 21 and the second photon counting module 22 according to the present invention are described.

[0266] In some embodiments, the first counting method may be a pulse recognition method for calculating the number of photons by identifying the electrical pulses caused by photons entering the receiving component, while the second counting method does not calculate the number of photons by identifying the electrical pulses caused by photons entering the receiving component, but estimates the number of photons by processing the electrical signal into parameters that can characterize the number of photons (i.e., the number of electrical pulses caused by photons entering the receiving component).

[0267] Here, those skilled in the art can understand that, in an embodiment of the present invention, the pulse recognition method can be understood as a method of calculating the number of photons by identifying pulses in an electrical signal, that is, when the peak value of the pulse identified from the electrical signal is greater than a threshold, it is considered that a photon is identified.

[0268] In some embodiments, the first photon counting module 21 can be configured to detect the number of pulses of the electrical signal using a pulse recognition method to obtain a first photon counting result, and the second photon counting module 22 can be configured to process the electrical signal to obtain parameters characterizing the number of photons, and calculate the second photon counting result based on the parameters characterizing the number of photons and a preset calibration function, wherein the calibration function represents the mapping relationship between the parameters characterizing the number of photons and the photon counting result.

[0269] Combine the following Figures 19 to 20 Some embodiments of the first photon counting module 21 according to the present invention are described.

[0270] In some embodiments, Fig.19 As shown, the first photon counting module 21 may include a level identification circuit 211, a shaping and frequency division circuit 212, and a first counting circuit 213. The level identification circuit 211 is configured to convert an electrical signal into a square wave signal, the shaping and frequency division circuit 212 is configured to perform frequency division and shaping on the square wave signal, and the first counting circuit 213 is configured to count the signal output by the shaping and frequency division circuit 212, that is, to identify the number of pulses, to obtain a first photon counting result. The first photon counting module 21 according to this example can achieve a linear photometric range of 0 to 30 million photons / second.

[0271] In other alternative embodiments, such as Fig. 20As shown, the first photon counting module 21 may include at least one first AD conversion circuit 214 and a first counting circuit 213. The first AD conversion circuit 214 is configured to collect electrical signals at a sampling frequency greater than 1 GHz / s and convert the collected electrical signals into digital signals and output them to the first counting circuit 213. The first counting circuit 213 is configured to analyze the collected digital signals to identify the number of pulses, thereby obtaining a first photon counting result. Thus, by high-speed sampling at a sampling frequency of 1 GHz / s, the first counting circuit 213 can more accurately identify the number of pulses in the electrical signal, thereby obtaining a more accurate first photon counting result.

[0272] Furthermore, the first counting circuit 213 may also be configured to correct the first photon counting result by using a Poisson distribution compensation algorithm, thereby obtaining a more accurate photon counting result.

[0273] In some embodiments, the first counting circuit 213 may include an FPGA chip and its surrounding circuits.

[0274] Combine the following Figure 21 to Figure 24 Some embodiments of the second photon counting module 22 according to the present invention are described.

[0275] In an embodiment of the present invention, the preset calibration function is a conversion function that converts the value of the parameter characterizing the number of photons into a photon counting value, which represents the mapping relationship between the parameter characterizing the number of photons and the photon counting result (or counting value).

[0276] In a specific example, a calibration function used by the second photon counting module of the photometric device is calibrated using a reference light source, such as an LED lamp, and the first photon counting module of the photometric device, that is, the calibration method of the conversion function of the parameter characterizing the number of photons and the photon counting result is as follows:

[0277] Controlling the input value of the reference light source, using the photometric device to be calibrated to test the photon counting result of the reference light source from the weak light segment to the strong light segment and detecting the value of the parameter characterizing the number of photons, wherein the weak light segment is a light signal with a photon count not higher than a predetermined threshold value per second, and the strong light segment is a light signal with a photon count higher than the predetermined threshold value per second, and the predetermined threshold value is, for example, any value selected from 20 million to 30 million photons / second, for example, 30 million photons / second;

[0278] According to the linear function relationship between the weak light segment input value of the reference light source and the weak light segment photon counting result measured by the first photon counting module of the light measuring device to be calibrated, the theoretical photon counting result corresponding to the strong light segment input value of the reference light source is calculated; and under the premise that the input value of the reference light source is fixed, the corresponding value of the parameter characterizing the number of photons and the theoretical photon counting result are one-to-one corresponding, and a set of parameter values ​​characterizing the number of photons in the strong light segment and the theoretical photon counting result are fitted, such as n-order polynomial weighted least squares fitting, to finally obtain the conversion parameters of the parameters characterizing the number of photons and the photon counting result, that is, the calibration function.

[0279] In another alternative example, a reference light source and a reference photometric device can also be used to calibrate the conversion function of the parameters characterizing the number of photons and the photon counting result, in the following manner: the input value of the reference light source is controlled, and for the period from the weak light section to the strong light section of the reference light source, the value of the parameter characterizing the number of photon pulses is tested using the photometric device to be calibrated according to the present invention, and the counting result of the reference light source from the weak light section to the strong light section is tested using the reference photometric device; based on the linear function relationship between the input value of the reference light source and the counting result of the reference photometric device, the theoretical photon counting result corresponding to the input value of the reference light source is deduced; and under the premise that the input value of the reference light source is fixed, the corresponding value of the parameter characterizing the number of photons and the theoretical photon counting result are one-to-one corresponding, and a set of values ​​of the parameters characterizing the number of photons in the strong light section and the theoretical photon counting result are fitted, such as n-order polynomial weighted least squares fitting, to finally obtain the conversion parameters of the parameters characterizing the number of photons and the photon counting result, that is, the calibration function.

[0280] It can be understood that the calibration function can be pre-stored in the light measuring device, especially in the second photon counting module 22 .

[0281] In the embodiment of the present invention, the second photon counting module 22 which uses the parameter characterizing the number of photons and the predetermined calibration function to obtain the photon counting result can achieve a linear photometric range of 10 million to 200 million photons / second, such as Fig.21 As shown, S3 represents the Fig.19 The first photon counting module of the embodiment shown in FIG. 1 is a photometric linear feature, and S4 is a photometric linear feature of the second photon counting module according to the embodiment of the present invention. Thus, the combination of the first photon counting module 21 and the second photon counting module 22 can achieve a linear photometric range of at least 0 to 200 million photons / second.

[0282] In some embodiments, the second photon counting module 22 may be configured to process the electrical signal, for example, by integration processing to obtain a DC component signal, and the parameter characterizing the number of photons includes a parameter related to the DC component signal, for example, the DC component signal itself per unit time. The second photon counting module 22 is further configured to calculate a second photon counting result based on the parameter related to the DC component signal and a preset calibration function.

[0283] Alternatively or additionally, the second photon counting module 22 is configured to integrate the electrical signal within a predetermined time period to obtain an integration result, such as a DC component signal, and the parameter characterizing the number of photons includes a parameter related to the integration result, such as an integration result per unit time. The second photon counting module 22 is further configured to calculate a second photon counting result based on the parameter related to the integration result and a preset calibration function.

[0284] In a specific example, Fig. 22 and Fig.23 As shown, the second photon counting module 22 includes an integration circuit 221 and a second counting circuit 222 electrically connected to each other. The integration circuit 221 is configured to integrate the electrical signal within a predetermined time period to obtain a DC component signal, and the parameter characterizing the number of photons includes a parameter related to the DC component signal. The second counting circuit 222 is configured to calculate the second photon counting result according to the parameter related to the DC component signal and a preset calibration function.

[0285] Furthermore, if Fig.24 As shown, an AD conversion circuit 224 is further provided between the integration circuit 221 and the second counting circuit 222, and the AD conversion circuit 224 is configured to convert the DC component signal output by the integration circuit 221 into a digital signal. The sampling frequency of the AD conversion circuit 224 can be set to less than 1 MHz / s, for example, hundreds of kHz / s.

[0286] For example, the second counting circuit 222 may be configured to perform Fourier transform on the digital signal to obtain a DC component signal.

[0287] Alternatively or additionally, the second counting circuit 222 may be configured to integrate or sum the digital signal within a predetermined time period to obtain a parameter related to the integration result or the summation result as the parameter characterizing the number of photons.

[0288] Furthermore, in some embodiments, Fig.25As shown, the processing component 20 may further include a preamplifier circuit 23 electrically connected to the receiving component 10, the first photon counting module 21, and the second photon counting module 22. The preamplifier circuit 23 is configured to amplify the electrical signal output by the receiving component 10 and transmit it to the first photon counting module 21 and the second photon counting module 22. For example, the preamplifier circuit 23 may amplify the electrical signal output by the receiving component 10 by 60 to 90 times, and in particular by 70 to 80 times.

[0289] Of course, in other embodiments, the processing component 20 may include a first pre-amplifier circuit electrically connected to the receiving component 10 and the first photon counting module 21 and / or a second pre-amplifier circuit electrically connected to the receiving component 10 and the second photon counting module 22. The first pre-amplifier circuit is configured to amplify the electrical signal output by the receiving component 10 and transmit it to the first photon counting module 22, and the second pre-amplifier circuit is configured to amplify the electrical signal output by the receiving component 10 and transmit it to the second photon counting module 22.

[0290] Furthermore, if Fig.26 As shown, the processing component 20 may include a resistor 24 disposed between the receiving component 10 and the pre-amplifier circuit 23, and the resistor is used to convert the electrical signal in the form of a current signal output by the receiving component 10 into a voltage signal so that the pre-amplifier circuit 23 amplifies the voltage signal.

[0291] According to some embodiments of the light measuring device 100 of the present invention, the light measuring device 100 includes a receiving component 10 and a switchable light attenuation component (not shown) arranged in front of the receiving component, the light attenuation component is, for example, a filter; at the beginning of measurement, the light signal that has not been attenuated by the light attenuation component is received by the receiving component 10, and a weak light signal can be measured at this time; then the light attenuation component is switched so that the receiving component 10 receives the light signal that has been attenuated by the light attenuation component, even if it is a strong light signal at this time, the strong light signal becomes weak light after light attenuation and is detected by the receiving component 10. The linear range of the light measuring device can also be extended by the switchable light attenuation component.

[0292] Definition of terms

[0293] 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.

[0294] 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). D The 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.

[0295] 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).

[0296] 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 LIt 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.

[0297] 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).

[0298] In the present invention, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention can be determined 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 preferably determined by the Kabat, Chothia or IMGT numbering systems.

[0299] 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.

[0300] 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.

[0301] 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.

[0302] 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).

[0303] 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.

[0304] 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.

[0305] 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)).

[0306] 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.

[0307] 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.

[0308] 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.

[0309] 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.

[0310] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of the 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 the other 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 the binding activity to the target antigen (USP4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:68516855 (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).

[0311] 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 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.).

[0312] 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.

[0313] 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.

[0314] 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).

[0315] 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.

[0316] 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.

[0317] 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.

[0318] 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).

[0319] 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.

[0320] 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.

[0321] As used herein, the term "subject" includes, but is not limited to, various animals, particularly mammals, such as humans.

[0322] Herein, unless otherwise indicated, "%" refers to mass percentage. BRIEF DESCRIPTION OF THE DRAWINGS

[0323] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0324] Figure 1 The antibody was tested for cross-reactivity with the human glycoprotein hormones TSH, FSH, LH and CG. Antibodies specific for the alpha subunit common to the glycoprotein hormones were used as positive controls for FSH, LH and CG immunoreactivity.

[0325] Figure 2 The antibody is shown to be tested for cross reactivity with the human glycoprotein hormones FSH (10 IU / ml), LH (10 IU / ml) and CG (1000 IU / ml), using TSH at a concentration of 0.137 μIU / ml as a control.

[0326] Figure 3 Antibodies shown were tested for cross-reactivity with human recombinant wild-type TSH and the TSH variant R55G.

[0327] Figure 4 The functional sensitivity test results of the kit in Example 7 are shown.

[0328] Figure 5 The functional sensitivity test results of the kit in Example 8 are shown.

[0329] Figure 6 The functional sensitivity test results of the kit in Example 9 are shown.

[0330] Figure 7 The functional sensitivity test results of the kit in Example 10 are shown.

[0331] Figure 8 The functional sensitivity test results of the kit in Example 11 are shown.

[0332] Fig. 9 The methodological comparison results of the kit of Example 7 and the commercial TSH kit are shown.

[0333] Fig.10 The methodological comparison results of the kit of Example 8 and the commercial TSH kit are shown.

[0334] Fig.11 The methodological comparison results of the kit of Example 9 and the commercial TSH kit are shown.

[0335] Fig.12The methodological comparison results of the kit of Example 10 and the commercial TSH kit are shown.

[0336] Fig.13 The methodological comparison results of the kit of Example 11 and the commercial TSH kit are shown.

[0337] Fig.14 is a schematic graph of a pulse signal with normal pulses.

[0338] Fig.15 is a schematic graph of a pulse signal with pile-up pulses.

[0339] Fig.16 FIG. 4 is a schematic structural diagram of an embodiment of a chemical immunoassay analyzer according to the present invention.

[0340] Fig.17 The following are light-metering linear characteristic curves of a light-metering device in the prior art and a light-metering device according to the present invention.

[0341] Fig.18 FIG. 4 is a schematic block diagram of a first embodiment of a light measuring device according to the present invention.

[0342] Fig.19 FIG. 4 is a schematic block diagram of a second embodiment of a light measuring device according to the present invention.

[0343] Fig. 20 Graph showing the photometric linear characteristics of the first photon counting module and the second photon counting module according to the present invention.

[0344] Fig.21 FIG. 4 is a schematic block diagram of a third embodiment of a light measuring device according to the present invention.

[0345] Fig. 22 FIG. 4 is a schematic block diagram of a fourth embodiment of a light measuring device according to the present invention.

[0346] Fig.23 FIG. 5 is a schematic block diagram of a fifth embodiment of a light measuring device according to the present invention.

[0347] Fig.24 FIG. 4 is a schematic block diagram of a sixth embodiment of a light measuring device according to the present invention.

[0348] Fig.25 FIG. 4 is a schematic block diagram of a seventh embodiment of a light measuring device according to the present invention.

[0349] Fig.26 FIG. 8 is a schematic block diagram of an eighth embodiment of a light measuring device according to the present invention. DETAILED DESCRIPTION

[0350] 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.

[0351] Example 1 Preparation of monoclonal antibodies

[0352] 1. Immunization of rabbits and sheep with human TSH

[0353] Recombinant human thyroid stimulating hormone (TSH) (the protein does not contain any tags and its sequence is the same as that of natural TSH (WHO International Standard NIBSC code 81 / 565)) is used to immunize rabbits and sheep. The immunized hybridoma cells are then screened to obtain hybridoma cells that produce human thyroid stimulating hormone (TSH).

[0354] 2. Screening for antibodies that do not cross-react with other glycoprotein hormones

[0355] Hybridoma culture supernatants were tested by ELISA for the presence of antibodies specific to free common α subunits of human glycoprotein hormones, human luteinizing hormone (LH), human follicle stimulating hormone (FSH), and human chorionic gonadotropin (hCG). Hybridomas that produced only weakly cross-reactive antibodies to free α subunits, LH, FSH, or CG were excluded. Rabbit-mouse hybridomas and sheep-mouse hybridomas that secreted antibodies that did not cross-react with other glycoprotein hormones were further selected.

[0356] 3. Selection of sheep antibodies that recognize TSH with the R55G mutation in the TSHβ subunit

[0357] To select antibodies that recognize TSH with the R55G mutation in the β subunit (TSH R55G mutant), we developed recombinant human TSH containing this mutation. The sequences encoding the α- and β-subunits are provided in Table 1. The β-subunit contains glycine instead of arginine at position 55 (R55G mutation).

[0358] Table 1 Amino acid sequence of TSH mutant R55G

[0359]

[0360] Culture supernatants from sheep-mouse hybridomas were tested by ELISA with wild-type TSH and TSH mutant R55G. Hybridomas that recognized both wild-type TSH and TSH variant R55G were selected. In the later stages of antibody development, the interaction of rabbit antibodies with the TSH R55G variant was studied on purified antibody preparations.

[0361] 4. Screening of antibodies that recognize TSH with high sensitivity

[0362] The hybridoma with the highest signal-to-noise ratio in the chemiluminescent sandwich immunoassay was selected.

[0363] TSH-specific high-affinity polyclonal antibodies were isolated from the blood of immunized rabbits and sheep for sandwich immunoassays. Among them, rabbit polyclonal antibodies were used as capture antibodies in the screening test for sheep antibodies. Sheep polyclonal antibodies were used as capture antibodies in the screening test for rabbit antibodies. Hybridomas were graded based on the signal-to-noise ratio. Rabbit-mouse hybridomas and sheep-mouse hybridomas showing the highest signal-to-noise ratio were selected for the development of recombinant forms. Some antibody sequencing results are shown in "5. Antibody Sequencing".

[0364] 5. Antibody Sequencing

[0365] Total RNA was extracted from 50-500 hybridoma cells using the Norgen Single Cell Purification Kit (Norgen Biotek) and sequenced according to conventional procedures in the art. The nucleotide sequences and deduced amino acid sequences of all three CDRs of the VH, VK and VL domains are given in Tables 2 and 3. Among them, TS01 is a rabbit antibody and the rest are goat antibodies.

[0366] Table 2 Partial sequencing results of heavy chain (nucleotide + amino acid sequence)

[0367]

[0368]

[0369]

[0370]

[0371] Table 3 Partial sequencing results of light chain (nucleotide + amino acid sequence)

[0372]

[0373]

[0374]

[0375]

[0376] 6. Antibody Production and Affinity Purification

[0377] The antibodies were obtained in recombinant form. The design of appropriate molecular genetic constructs was performed. To develop molecular genetic constructs for rabbit antibodies, variable domains of antibodies and rabbit immunoglobulin constant domains of heavy chain IgG isotype and light chain λ isotype were used. To develop molecular genetic constructs for sheep antibodies, variable domains of antibodies and constant domains of human immunoglobulins were used, wherein IgG1 isotype was used for heavy chain and λ isotype was used for light chain. The molecular genetic constructs of recombinant antibodies were obtained and purified in preparation amounts in bacterial cells. Expi293F cells were transfected with molecular genetic constructs of recombinant antibodies.

[0378] 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 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.

[0379] The antibodies were purified from the conditioned medium using protein A affinity chromatography (Mab Select SuRe, Cytiva). The supernatant was loaded onto a column containing Mab Select SuRe equilibrated with PBS. The column was washed with PBS and the antibodies were eluted with 0.1 M citric acid (pH 3.0). The eluate was then neutralized and dialyzed with PBS containing 0.09% sodium azide. The antibody solution was concentrated using an Amicon Ultra-15 centrifugal filter 50 kDa MWCO (Millipore). Recombinant rabbit monoclonal antibody rTS01 and recombinant chimeric antibody hTS02-hTS17 were obtained. The antibody purity was analyzed by SDS-PAGE under reducing conditions and size exclusion chromatography using a Superdex200Increase5 / 150 column (Cytiva). The purity of the antibody was >90%.

[0380] Collection information:

[0381] rTS01: Escherichia coliRosetta deposited at VKPM on July 10, 2024, with the deposit number B-14884 TM (DE3)pLysS rTS01 VL, the plasmid contained in the cell produces the rTS01 light chain; Escherichia coli RosettaTM (DE3)pLysS rTS01 VH, deposited with VKPM on July 10, 2024, with the deposit number B-14885, the plasmid contained in the cell produces the rTS01 heavy chain;

[0382] hTS02: Escherichia coli RosettaTM (DE3) pLysS hTS02 VL, deposited with VKPM on July 10, 2024, with the deposit number B-14886, the plasmid contained in the cell produces the hTS02 light chain; Escherichia coli RosettaTM (DE3) pLysS hTS02 VH, deposited with VKPM on July 10, 2024, with the deposit number B-14887, the plasmid contained in the cell produces the hTS02 heavy chain;

[0383] hTS03: Escherichia coli RosettaTM (DE3) pLysS hTS03 VL, deposited with VKPM on July 10, 2024, with the deposit number B-14888, the plasmid contained in the cell produces the hTS03 light chain; Escherichia coli RosettaTM (DE3) pLysS hTS03 VH, deposited with VKPM on July 10, 2024, with the deposit number B-14889, the plasmid contained in the cell produces the hTS03 heavy chain;

[0384] hTS04: Escherichia coli RosettaTM (DE3) pLysS hTS04 VL, deposited with VKPM on July 10, 2024, with the deposit number B-14890, the plasmid contained in the cell produces the hTS04 light chain; Escherichia coli RosettaTM (DE3) pLysS hTS04 VH, deposited with VKPM on July 10, 2024, with the deposit number B-14891, the plasmid contained in the cell produces the hTS04 heavy chain;

[0385] hTS05: Escherichia coli RosettaTM (DE3) pLysS hTS05 VL, deposited with VKPM on July 10, 2024, with the deposit number B-14892, the plasmid contained in the cell produces the hTS05 light chain; Escherichia coli RosettaTM (DE3) pLysS hTS05 VH, deposited with VKPM on July 10, 2024, with the deposit number B-14893, the plasmid contained in the cell produces the hTS05 heavy chain;

[0386] hTS06: Escherichia coli RosettaTM (DE3) pLysS hTS06 VL, deposited with VKPM on July 10, 2024, with the deposit number B-14894, the plasmid contained in the cell produces the hTS06 light chain; Escherichia coli RosettaTM (DE3) pLysS hTS06 VH, deposited with VKPM on July 10, 2024, with the deposit number B-14895, the plasmid contained in the cell produces the hTS06 heavy chain;

[0387] hTS07: Escherichia coli RosettaTM (DE3) pLysS hTS07 VL, deposited with VKPM on July 10, 2024, with the deposit number B-14896, the plasmid contained in the cell produces the hTS07 light chain; Escherichia coli RosettaTM (DE3) pLysS hTS07 VH, deposited with VKPM on July 10, 2024, with the deposit number B-14897, the plasmid contained in the cell produces the hTS07 heavy chain;

[0388] hTS08: Escherichia coli RosettaTM (DE3) pLysS hTS08 VL, deposited with VKPM on July 10, 2024, with the deposit number B-14898, the plasmid contained in the cell produces the hTS08 light chain; Escherichia coli RosettaTM (DE3) pLysS hTS08 VH, deposited with VKPM on July 10, 2024, with the deposit number B-14899, the plasmid contained in the cell produces the hTS08 heavy chain;

[0389] hTS09: Escherichia coli RosettaTM (DE3) pLysS hTS09 VL, deposited with VKPM on July 10, 2024, with the deposit number B-14900, the plasmid contained in the cell produces the hTS09 light chain; Escherichia coli RosettaTM (DE3) pLysS hTS09 VH, deposited with VKPM on July 10, 2024, with the deposit number B-14901, the plasmid contained in the cell produces the hTS09 heavy chain;

[0390] hTS10: Escherichia coli RosettaTM (DE3) pLysS hTS10 VL, deposited with VKPM on July 10, 2024, with the deposit number B-14902, the plasmid contained in the cell produces the hTS10 light chain; Escherichia coli RosettaTM (DE3) pLysS hTS10 VH, deposited with VKPM on July 10, 2024, with the deposit number B-14903, the plasmid contained in the cell produces the hTS10 heavy chain;

[0391] hTS11: Escherichia coli RosettaTM (DE3) pLysS hTS11 VL, deposited with VKPM on July 10, 2024, with the deposit number B-14904, the plasmid contained in the cell produces the hTS11 light chain; Escherichia coli RosettaTM (DE3) pLysS hTS11 VH, deposited with VKPM on July 10, 2024, with the deposit number B-14905, the plasmid contained in the cell produces the hTS11 heavy chain;

[0392] hTS12: Escherichia coli RosettaTM (DE3) pLysS hTS12 VL, deposited with VKPM on July 10, 2024, with the deposit number B-14906, the plasmid contained in the cell produces the hTS12 light chain; Escherichia coli RosettaTM (DE3) pLysS hTS12 VH, deposited with VKPM on July 10, 2024, with the deposit number B-14907, the plasmid contained in the cell produces the hTS12 heavy chain;

[0393] hTS13: Escherichia coli RosettaTM (DE3) pLysS hTS13 VL, deposited with VKPM on July 10, 2024, with the deposit number B-14908, the plasmid contained in the cell produces the hTS13 light chain; Escherichia coli RosettaTM (DE3) pLysS hTS13 VH, deposited with VKPM on July 10, 2024, with the deposit number B-14909, the plasmid contained in the cell produces the hTS13 heavy chain;

[0394] hTS14: Escherichia coli RosettaTM (DE3) pLysS hTS14 VL, deposited with VKPM on July 10, 2024, with the deposit number B-14910, the plasmid contained in the cell produces the hTS14 light chain; Escherichia coli RosettaTM (DE3) pLysS hTS14 VH, deposited with VKPM on July 10, 2024, with the deposit number B-14911, the plasmid contained in the cell produces the hTS14 heavy chain;

[0395] hTS15: Escherichia coli RosettaTM (DE3) pLysS hTS15 VL, deposited with VKPM on July 10, 2024, with the deposit number B-14912, the plasmid contained in the cell produces the hTS15 light chain; Escherichia coli RosettaTM (DE3) pLysS hTS15 VH, deposited with VKPM on July 10, 2024, with the deposit number B-14913, the plasmid contained in the cell produces the hTS15 heavy chain;

[0396] hTS16: Escherichia coli RosettaTM (DE3) pLysS hTS16 VL, deposited with VKPM on July 10, 2024, with the deposit number B-14914, the plasmid contained in the cell produces the hTS16 light chain; Escherichia coli RosettaTM (DE3) pLysS hTS16 VH, deposited with VKPM on July 10, 2024, with the deposit number B-14915, the plasmid contained in the cell produces the hTS16 heavy chain;

[0397] hTS17: Escherichia coli RosettaTM (DE3) pLysS hTS17 VL, deposited in VKPM on July 10, 2024, with the deposit number B-14916. The plasmid contained in this cell produces the hTS17 light chain; Escherichia coli RosettaTM (DE3) pLysS hTS17 VH, deposited in VKPM on July 10, 2024, with the deposit number B-14917. The plasmid contained in this cell produces the hTS17 heavy chain.

[0398] Example 2 Testing of Monoclonal Antibody Specificity

[0399] The specificity of the monoclonal antibodies was determined by ELISA using TSH free β-subunit, free glycoprotein hormone common α-subunit, TSH dimer, FSH, LH and hCG. All proteins used for cross-reactivity studies were human, recombinant and highly purified. The specific method is as follows.

[0400] Add 30ng / 0.05ml of PBS solution of any protein to each well and incubate for 30 minutes. Wash the plate twice with PBST. Then incubate the plate with the antibody solution (10μg / ml) in PBST for 30 minutes and wash it twice with PBST. Incubate with 0.05ml of HRP-labeled anti-human or anti-rabbit antibody for 30 minutes to show antibodies that bind to TSH. After incubation with the secondary antibody, wash the plate six times with PBST and add 0.05ml of o-phenylenediamine dihydrochloride (OPD) HRP substrate. After incubation for 10 minutes, stop the reaction by adding 0.025ml 0.5M sulfuric acid and measure the absorbance in the well at 490nm.

[0401] Figure 1 Results of antibody testing with glycoprotein hormones are shown, with none of the antibodies recognizing free α-subunits, FSH, LH, or hCG.

[0402] Based on the detection results of TSH dimers and free subunits, antibodies can be divided into two groups. The first group of antibodies recognizes TSH dimers and free TSH β subunits (including rTS01, hTS08~hTS17). We believe that the epitopes of these antibodies are located on the β subunit. The second group of antibodies only interact with TSH dimers and do not interact with free α- and β-subunits (including hTS02~hTS07). We believe that these antibodies are specific for TSH dimers. Table 4 summarizes the specificity of the antibodies.

[0403] Table 4 Anti-TSH antibody specificity test results

[0404]

[0405]

[0406] Note: “+” indicates recognition of the corresponding antigen.

[0407] Example 3 Establishment of Sandwich Immunoassay Method

[0408] Using a plate-based sandwich immunoassay, rTS01 and hTS02-hTS17 were tested in paired combinations to find the combination with the best properties. For the chemiluminescent sandwich immunoassay, the antibody was labeled with alkaline phosphatase (ALP). The specific assay method is as follows.

[0409] Incubate 50 ng / 0.05 ml of capture antibody in PBS per well in a 96-well plate (Greiner) for 30 minutes. Wash the plate twice with Tris buffer containing 0.05% Tween-20 (TBST) and 10 ng / 0.025 ml of ALP-labeled detection antibody. At the same time, 0.025 ml of human TSH (containing 1% bovine serum albumin (assay buffer)) diluted with TBST to a concentration of 0.05 μIU / ml was added to the well. The same volume of detection buffer was used as a blank. After incubation for 30 minutes, the plate was washed 6 times with TBST and 0.05 ml of AMPPD chemiluminescent substrate was added. The plate was incubated at 37°C for 10 minutes and the signal was quantified using a plate reader capable of luminescent detection. The antibody pairs that exhibited the highest signal-to-noise ratio were selected for further evaluation.

[0410] Example 4 Establishment of fully automated microparticle chemiluminescent immunoassay (sandwich method)

[0411] The antibody pairs selected in Example 3 were retested in the automated CLIA. The antibody labeled with ALP was used as the detection antibody. The capture antibody was biotinylated and bound to the magnetic beads coated with streptavidin. The magnetic beads immobilized with the capture antibody were used for determination at a concentration of 0.25 mg / ml; the detection antibody was used for detection at a concentration of 0.5 μg / ml. Human natural TSH (WHO International Standard NIBSC code 81 / 565) was used as an antigen. TSH serial dilutions were prepared with assay buffer (0.05, 0.016, 0.0055 μIU / ml). The assay buffer was used as a blank. 50 μL of TSH solution was mixed with 50 μL of magnetic bead suspension and 50 μL of detection antibody solution and incubated at 37°C for 16 minutes. After the reaction, the magnetic beads were washed with TBST. AMPPD chemiluminescent substrate was used to detect chemiluminescence. Finally, the 20 antibody pairs that showed the highest signal-to-noise ratio in this experiment were selected (Table 5).

[0412] Table 5 Optimal antibody pairs obtained by fully automated microparticle chemiluminescent immunoassay (sandwich method)

[0413]

[0414]

[0415] Example 5 Fully automated microparticle chemiluminescent immunoassay (sandwich method) characterization of selected antibody pairs

[0416] 1. Limit of Detection (LoD) Determination

[0417] We used human native TSH (WHO International Standard NIBSC code 81 / 565) diluted to concentrations of 0.0024, 0.0018, 0.0014, 0.001, 0.00077 and 0.00059 μIU / ml in assay buffer to determine the LoD of the selected antibody pairs. Twenty replicates of assay buffer and TSH dilutions were tested per experiment in the corresponding chemiluminescent sandwich immunoassay. The limit of blank (LoB) and limit of detection (LoD) were calculated using the following formula:

[0418] LoB = Mean 空白 +1.645(SD 空白 )

[0419] LoD = LoB + 1.645 (SD 低浓度样品 )

[0420] The LoD of TSH diluted in sustained release was the same for all antibody pairs at 0.00077 μIU / ml. The coefficient of variation CV was <2% for all TSH dilutions tested. This data suggests that the selected antibody pairs exhibit sensitivity equivalent to that of the 4th generation TSH assay.

[0421] 2. Cross-reactivity test with LH, FSH, and CG

[0422] Recombinant human LH, FSH, and CG were diluted in assay buffer to a concentration of 10 IU / ml (LH, FSH) or 1000 IU / ml (CG) and tested in the corresponding chemiluminescent sandwich immunoassays. TSH (WHO International Standard NIBSC code 81 / 565) at a concentration of 0.137 μIU / ml was used as a control. For all antibody pairs, no signal was detected in samples containing LH, FSH, or CG that was significantly higher than that in blank controls ( Figure 2 ). Thus, we confirmed that neither the individual antibodies nor the antibody pairs consisting of these antibodies cross-reacted with other human glycoprotein hormones.

[0423] 3. Cross-reactivity test of TSH variant R55G

[0424] Recombinant human wild-type TSH and TSH variant R55G were serially diluted in assay buffer to concentrations of 1, 0.33, 0.11, 0.037, 0.012 ng / ml and tested in the corresponding chemiluminescent sandwich immunoassays. The selected antibody pairs recognized wild-type TSH and TSH variant R55G equally. Figure 3 ).

[0425] Example 6 Antibody Affinity Detection

[0426] To determine the equilibrium dissociation constant, the analysis was performed using the Octet Biolayer Interferometry platform. The biotinylated antibody was immobilized on a high-precision streptavidin biosensor (SAX). Human TSH was serially diluted to concentrations of 15, 10, 5, and 2.5 nmol in PBST containing 0.1% bovine serum albumin and 10 μg / ml biocytin. A reference biosensor without immobilized antibody was incubated with the highest TSH concentration (15 nmol). The analysis temperature was 30°C. The experimental protocol was as follows:

[0427] 1. Hydration of Sensor with PBS

[0428] 2. Binding of sensor to antibody (4 μg / ml in PBS, 10 min)

[0429] 3. Clean the sensor (PBS)

[0430] 4. Sensor blocking (PBST containing 0.1% bovine serum albumin and 10 μg / ml biocytin)

[0431] 5. Binding (5 minutes for each test antibody concentration)

[0432] 6. Dissociation (PBS, 25 minutes)

[0433] Octet Analysis Studio software was used to calculate the binding constant (Ka), equilibrium dissociation constant (KD), and dissociation constant (Kd) (results are shown in Table 6). Global fitting and a 1:1 binding model were used to analyze the data.

[0434] Table 6 Binding constants of antibodies of the present invention

[0435]

[0436] Example 7 Preparation and Detection of TSH Kit and Calibrator

[0437] Step 1: Preparation of R1 reagent (including capture antibody)

[0438] The specific TSH antibody and superparamagnetic particles 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 particles coated with the TSH antibody are retained. The TBS buffer is repeated 3 times, and the superparamagnetic particles coated with the TSH antibody are suspended in R1 diluent to prepare R1 reagent, in which the superparamagnetic particle content is 0.07%, and the TSH antibody content is 0.0007%. Refrigerate at 2-8°C for use.

[0439] Step 2: Preparation of R2 reagent (including detection antibody)

[0440] Another specific TSH antibody is coupled to alkaline phosphatase, wherein the molar ratio of alkaline phosphatase to antibody is 15:1, and then the conjugate is dissolved with R2 diluent to prepare R2 reagent, wherein the concentration of the conjugate is 0.5 μg / mL, and refrigerated at 2-8°C for use; (R1 reagent and R2 reagent are combined together to form a TSH kit).

[0441] Step 3: Preparation of TSH Calibrator

[0442] The TSH antigen was diluted to concentration points of 0 μIU / mL, 0.05 μIU / mL, 0.5 μIU / mL, 1 μIU / mL, 2 μIU / mL, 4 μIU / mL, 10 μIU / mL, 20 μIU / mL, 40 μIU / mL, 60 μIU / mL, 80 μIU / mL, 100 μIU / mL, and 120 μIU / mL to prepare TSH calibrators.

[0443] Step 4: TSH kit measurement procedure

[0444] The TSH test 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.

[0445] Use the matching TSH calibrator and perform the calibration test according to the operating steps in the instrument manual. Based on the calibration data, the system software uses a weighted four-parameter logarithmic curve (4PLC) mathematical fitting model to fit the luminescence signal and concentration. The final result is given in the form of μIU / mL concentration.

[0446] TSH kits 1-5 (antibody and antigen composition see Table 7 below) were obtained according to the above method, and their performances were analyzed.

[0447] Table 7 TSH kit

[0448] TSH Kit Capture Antibody Detection antibodies Kit 1 hT06 hTS09 Kit 2 hT07 hT08 Kit 3 hTS05 hTS09 Kit 4 hTS04 hTS11 Kit 5 hTS04 hT08

[0449] 1. Functional sensitivity test results:

[0450] A series of low-concentration samples were prepared by gradient dilution, and these samples were tested multiple times for 10 consecutive days. The test was repeated twice a day to obtain 20 measurement results. The average value of each concentration was calculated, and the daily CV value of each concentration was calculated. A dot plot of the average value of each concentration and its daily CV was made. The system software used a weighted five-parameter logarithmic curve (5PLC) mathematical fitting model to fit the daily coefficient of variation CV% and the sample concentration value. The concentration value corresponding to the 20% CV value was calculated as the functional sensitivity of TSH. Results are shown in Figure 4-Figure 8 .

[0451] The functional sensitivity of current TSH detection kits can reach <0.0015μIU / mL.

[0452] 2. TSH kit analog cross-reaction test results:

[0453] TSH analog samples reaching the target concentration were prepared by adding three analog antigens to the TSH zero value calibrator, and the constructed TSH kit was used for detection, and the cross-reaction rate was calculated. The results are shown in Table 8.

[0454] The cross-reaction rates of the current TSH detection kits and the three analogs FSH, LH, and hCG are

[0455] <0.001%.

[0456] Table 8 TSH detection sensitivity and cross-reaction test results of the kit of the present invention

[0457]

[0458]

[0459] 3. TSH kit methodology comparison data

[0460] like Figure 9-13 As shown in the figure, the current TSH test kit is compared with the commercial TSH test kit (Thyroid Stimulating Hormone Test Kit (Electrochemiluminescence), Roche, (240)

[0461] 08429324190) have good methodological consistency, and the correlation coefficient R 2 All of them were greater than 0.95, which showed good clinical applicability.

[0462] Example 8 TSH kit can simultaneously detect wild-type TSH and mutant R55G TSH antigen

[0463] Three concentrations of wild-type TSH recombinant antigen and mutant R55G TSH antigen were prepared respectively, and the two types of samples were tested on Mindray's chemiluminescent immunoassay analyzer CL8000i using the kits 1-3 in the aforementioned Example 7. At the same time, a commercial TSH detection kit was used to test on the corresponding instrument. The results are shown in the following table, indicating that the reagents of the present application can detect wild-type TSH antigen and mutant R55G TSH antigen, while the commercial kit did not detect the mutant R55G TSH antigen.

[0464]

[0465]

[0466] The information of the amino acid sequences involved in this application is described in the following table:

[0467]

[0468]

[0469]

[0470]

[0471]

[0472]

[0473]

[0474]

[0475]

[0476]

[0477] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and substitutions may be made to those details based on all the teachings disclosed, and these changes are within the scope of protection of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to TSH (thyroid stimulating hormone) or a mutant thereof, 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, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 2, 8, 14, 20, 26, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98 and variants thereof, and / or a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99 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 the group consisting of SEQ ID NO: 4, 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100 and variants thereof, a CDR-L2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101 and variants thereof, and / or a CDR-L3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102 and variants thereof; wherein each of the variants 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; Preferably, each such 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.

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 having an amino acid sequence of SEQ ID NO: 25 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 26 or a variant thereof, and CDR-H3 having 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 having an amino acid sequence of SEQ ID NO: 28 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 29 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 30 or a variant thereof; (1f) a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 31 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 32 or a variant thereof, and CDR-H3 having 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 having an amino acid sequence of SEQ ID NO: 34 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 35 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 36 or a variant thereof; (1g) a heavy chain variable region comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 37 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 38 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 39 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: 40 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 41 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 42 or a variant thereof; (1h) a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 43 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 44 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 45 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: 46 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 47 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 48 or a variant thereof; (1i) a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 49 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 50 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 51 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: 52 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 53 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 54 or a variant thereof; (1j) a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 55 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 56 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 57 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: 58 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 59 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 60 or a variant thereof; (1k) a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 61 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 62 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 63 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: 64 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 65 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 66 or a variant thereof; (11) a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 67 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 68 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 69 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: 70 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 71 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 72 or a variant thereof; (1m) a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 73 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 74 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 75 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: 76 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 77 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 78 or a variant thereof; (1n) a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 79 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 80 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 81 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: 82 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 83 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 84 or a variant thereof; (1o) a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 85 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 86 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 87 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: 88 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 89 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 90 or a variant thereof; (1p) a heavy chain variable region comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 91 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 92 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 93 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: 94 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 95 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 96 or a variant thereof; or (1q) a heavy chain variable region comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 97 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 98 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 99 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: 100 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 101 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 102 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 rabbit or sheep heavy chain constant region, and the light chain constant region is a rabbit or sheep 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 rabbit or sheep antibody, a chimeric antibody or a humanized antibody.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which is detectably labeled; 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.

6. An antibody or antigen-binding fragment thereof that specifically binds to TSH or a mutant thereof, wherein the antibody or antigen-binding fragment thereof: Escherichiacoli Rosetta, deposited at the Russian National Collection of Industrial Microorganisms (VKPM) with the deposit number B-14884 TM (DE3)pLysS rTS01 VL plasmid, and Escherichiacoli Rosetta with the deposit number B-14885 TM Plasmid generation in (DE3)pLysS rTS01 VH; Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14886 TM (DE3)pLysShTS02 VL plasmid, and Escherichia coli Rosetta with the deposit number B-14887 TM Plasmid production in (DE3)pLysShTS02 VH; Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14888 TM (DE3)pLysShTS03 VL plasmid, and Escherichia coli Rosetta with the deposit number B-14889 TM Plasmid production in (DE3)pLysShTS03 VH; Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14890 TM (DE3)pLysShTS04 VL plasmid, and Escherichia coli Rosetta with the accession number B-14891 TM Plasmid production in (DE3)pLysShTS04 VH; Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14892 TM (DE3)pLysShTS05 VL plasmid, and Escherichia coli Rosetta with the deposit number B-14893 TM Plasmid generation in (DE3)pLysShTS05 VH; Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14894 TM (DE3)pLysShTS06 VL plasmid, and Escherichia coli Rosetta with the accession number B-14895 TM Plasmid production in (DE3)pLysShTS06 VH; Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14896 TM (DE3)pLysShTS07 VL plasmid, and Escherichia coli Rosetta with the deposit number B-14897 TM Plasmid production in (DE3)pLysShTS07 VH; Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14898 TM (DE3)pLysShTS08 VL plasmid, and Escherichia coli Rosetta with the deposit number B-14899 TM Plasmid production in (DE3)pLysShTS08 VH; Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14900 TM (DE3)pLysShTS09 VL plasmid, and Escherichia coli Rosetta with the accession number B-14901 TM Plasmid production in (DE3)pLysShTS09 VH; Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14902 TM (DE3)pLysShTS10 VL plasmid, and Escherichia coli Rosetta with the accession number B-14903 TM Plasmid production in (DE3)pLysShTS10 VH; Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14904 TM (DE3)pLysShTS11 VL plasmid, and Escherichia coli Rosetta with the deposit number B-14905 TM Plasmid production in (DE3)pLysShTS11 VH; Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14906 TM (DE3)pLysShTS12 VL plasmid, and Escherichia coli Rosetta with the deposit number B-14907 TM (DE3)pLysShTS12 VH plasmid generation; based on Escherichia coli Rosetta deposited at VKPM with accession number B-14908 TM (DE3)pLysS hTS13 VL plasmid, and Escherichia coli Rosetta with the accession number B-14909 TM Plasmid generation of (DE3)pLysS hTS13VH; Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14910 TM (DE3)pLysShTS14 VL plasmid, and Escherichia coli Rosetta with the accession number B-14911 TM Plasmid production in (DE3)pLysShTS14 VH; Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14912 TM (DE3)pLysShTS15 VL plasmid, and Escherichia coli Rosetta with the deposit number B-14913 TM Plasmid generation in (DE3)pLysShTS15 VH; Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14914 TM (DE3)pLysShTS16 VL plasmid, and Escherichia coli Rosetta with the deposit number B-14915 TM Plasmid production in (DE3)pLysShTS16 VH; or Based on Escherichia coli Rosetta deposited at VKPM with accession number B-14916 TM (DE3)pLysShTS17 VL plasmid, and Escherichia coli Rosetta with accession number B-14917 TM Plasmid generation in (DE3)pLysShTS17 VH.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, which specifically recognizes TSH dimer or β subunit of TSH, and optionally R55G mutant of TSH.

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. A TSH detection kit for detecting the presence or level of TSH or its mutants in a sample; Preferably, the detection function sensitivity of the detection kit for TSH or its mutants is ≤0.0015 μIU / mL, preferably ≤0.001 μIU / mL; Preferably, the TSH or a mutant thereof is selected from TSH dimer, β subunit of TSH and / or R55G mutant of TSH.

12. The detection kit according to claim 11, comprising a first reagent and a second reagent, wherein the first reagent comprises magnetic beads coated with a first antibody capable of recognizing TSH or a mutant thereof; and the second reagent comprises a second antibody combined with a chemiluminescent marker, wherein after the sample and the first reagent and the second reagent are mixed, a sandwich complex of the first antibody-antigen-second antibody can be formed, and the antigen is TSH or a mutant thereof.

13. The detection kit according to claim 12, wherein the first antibody and / or the second antibody is selected from an antibody or an antigen-binding fragment thereof that specifically binds to TSH or a mutant thereof, and the antibody or the antigen-binding fragment thereof 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, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 2, 8, 14, 20, 26, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98 and variants thereof, and / or a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99 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 the group consisting of SEQ ID NO: 4, 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100 and variants thereof, a CDR-L2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101 and variants thereof, and / or a CDR-L3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102 and variants thereof; Preferably, the first antibody and / or the second antibody: 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; 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; 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; 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; A heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 25 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 26 or a variant thereof, and CDR-H3 having 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 having an amino acid sequence of SEQ ID NO: 28 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 29 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 30 or a variant thereof; A heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 31 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 32 or a variant thereof, and CDR-H3 having 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 having an amino acid sequence of SEQ ID NO: 34 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 35 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 36 or a variant thereof; A heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 37 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 38 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 39 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: 40 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 41 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 42 or a variant thereof; A heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 43 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 44 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 45 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: 46 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 47 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 48 or a variant thereof; A heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 49 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 50 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 51 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: 52 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 53 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 54 or a variant thereof; A heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 55 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 56 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 57 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: 58 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 59 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 60 or a variant thereof; A heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 61 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 62 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 63 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: 64 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 65 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 66 or a variant thereof; A heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 67 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 68 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 69 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: 70 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 71 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 72 or a variant thereof; A heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 73 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 74 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 75 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: 76 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 77 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 78 or a variant thereof; A heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 79 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 80 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 81 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: 82 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 83 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 84 or a variant thereof; A heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 85 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 86 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 87 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: 88 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 89 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 90 or a variant thereof; A heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 91 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 92 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 93 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: 94 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 95 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 96 or a variant thereof; or A heavy chain variable region comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 97 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 98 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 99 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: 100 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 101 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 102 or a variant thereof.

14. The detection kit according to claim 13, wherein: i) the first antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 31 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 32 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 33 and variants thereof; and, a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 34 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 35 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 36 and variants thereof; and, The second antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region of the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 49 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 50 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 51 and variants thereof; and, a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 52 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 53 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 54 and variants thereof; ii) the first antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 37 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 38 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 39 and variants thereof; and, a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 40 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 41 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 42 and variants thereof; and, The second antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region of the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 43 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 44 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 45 and variants thereof; and, a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 46 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 47 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 48 and variants thereof; iii) the first antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 25 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 26 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 27 and variants thereof; and, a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 28 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 29 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 30 and variants thereof; and, The second antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region of the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 49 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 50 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 51 and variants thereof; and, a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 52 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 53 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 54 and variants thereof; iv) the first antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 19 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 20 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 21 and variants thereof; and, a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 22 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 23 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 24 and variants thereof; and, The second antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region of the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 61 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 62 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 63 and variants thereof; and, a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 64 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 65 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 66 and variants thereof; Alternatively, v) the first antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 19 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 20 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 21 and variants thereof; and, a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 22 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 23 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 24 and variants thereof; and, The second antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence selected from SEQ ID NO: 43 and variants thereof, CDR-H2 having an amino acid sequence selected from SEQ ID NO: 44 and variants thereof, and CDR-H3 having an amino acid sequence selected from SEQ ID NO: 45 and variants thereof; and, a light chain variable region comprising the following three CDRs: CDR-L1 having an amino acid sequence selected from SEQ ID NO: 46 and variants thereof, CDR-L2 having an amino acid sequence selected from SEQ ID NO: 47 and variants thereof, and CDR-L3 having an amino acid sequence selected from SEQ ID NO: 48 and variants thereof.

15. A composition or TSH detection kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

16. A composition or TSH detection kit comprising: The first antibody is an antibody or antigen-binding fragment thereof comprising a heavy chain variable region of the following three CDRs: a CDR-H1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97 and variants thereof, a CDR-H2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 14, 20, 26, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98 and variants thereof, and / or a CDR-H3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99 and variants thereof; and / or a light chain variable region comprising the following three CDRs: an amino acid sequence selected from the group consisting of SEQ ID NOs: NO:4, 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO:5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101 and variants thereof, and / or a CDR-L3 having an amino acid sequence selected from SEQ ID NO:6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102 and variants thereof, and, The second antibody is an antibody or antigen-binding fragment thereof comprising a heavy chain variable region of the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 1, 7, 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 2, 8, 14, 20, 26, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98 and variants thereof, and / or a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99 and variants thereof; and / or a light chain variable region comprising the following three CDRs: an amino acid sequence selected from SEQ ID NO: NO:4, 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100 and variants thereof, a CDR-L2 having an amino acid sequence selected from the group consisting of SEQ ID NO:5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101 and variants thereof, and / or a CDR-L3 having an amino acid sequence selected from the group consisting of SEQ ID NO:6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102 and variants thereof; in, The first antibody and the second antibody are directed against different epitopes of TSH or its mutants respectively.

17. The composition or immunoassay kit of claim 16, wherein: The first antibody is an antibody or antigen-binding fragment thereof comprising a heavy chain variable region of the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 7, 13, 19, 25, 31, 37 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 8, 14, 20, 26, 32, 38 and variants thereof, and / or a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 9, 15, 21, 27, 33, 39 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: 10, 16, 22, 28, 34, 40 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 11, 17, 23, 29, 35, 41 and variants thereof, and / or a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 12, 18, 24, 30, 36, 42 and variants thereof, and, The second antibody is an antibody or antigen-binding fragment thereof comprising a heavy chain variable region of the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 1, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 2, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98 and variants thereof, and / or a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 3, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99 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, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100 and variants thereof, and an amino acid sequence selected from SEQ ID NO: 5 NO:5, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101 and variants thereof, and / or a CDR-L3 having an amino acid sequence selected from SEQ ID NO:6, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102 and variants thereof.

18. The composition or TSH detection kit according to claim 15 or 16, comprising: i) a first antibody, which is an antibody or an antigen-binding fragment thereof, 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 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 2 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 3 and variants thereof; and a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 4 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 5 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 6 and variants thereof, and, The second antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 13 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 14 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 15 and variants thereof; and a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 16 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 17 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 18 and variants thereof; ii) a first antibody, which is an antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region of the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 1 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 2 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 3 and variants thereof; and, a light chain variable region of the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 4 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 5 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 6 and variants thereof, and, The second antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 31 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 32 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 33 and variants thereof; and a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 34 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 35 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 36 and variants thereof; iii) a first antibody, which is an antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 43 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 44 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 45 and variants thereof; and, a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 46 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 47 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 48 and variants thereof; and, The second antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 31 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 32 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 33 and variants thereof; and a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 34 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 35 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 36 and variants thereof; iv) a first antibody, which is an antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 49 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 50 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 51 and variants thereof; and a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 52 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 53 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 54 and variants thereof, and, The second antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 31 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 32 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 33 and variants thereof; and a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 34 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 35 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 36 and variants thereof; v) a first antibody, which is an antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 7 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 8 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 9 and variants thereof; and a light chain variable region comprising three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 10 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 11 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 12 and variants thereof, and, The second antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 55 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 56 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 57 and variants thereof; and a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 58 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 59 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 60 and variants thereof; vi) a first antibody, which is an antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region of three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 7 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 8 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 9 and variants thereof; and a light chain variable region of three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 10 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 11 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 12 and variants thereof, and, The second antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 49 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 50 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 51 and variants thereof; and a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 52 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 53 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 54 and variants thereof; vii) a first antibody, which is an antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising three CDRs: a CDR-H1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 13 and variants thereof, a CDR-H2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 14 and variants thereof, and a CDR-H3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 15 and variants thereof; and, a light chain variable region comprising three CDRs: a CDR-L1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 16 and variants thereof, a CDR-L2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 17 and variants thereof, and a CDR-L3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 18 and variants thereof; and, The second antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 55 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 56 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 57 and variants thereof; and a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 58 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 59 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 60 and variants thereof; viii) a first antibody, which is an antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 19 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 20 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 21 and variants thereof; and, a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 22 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 23 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 24 and variants thereof; and, The second antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 85 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 86 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 87 and variants thereof; and a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 88 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 89 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 90 and variants thereof; ix) a first antibody, which is an antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 67 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 68 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 69 and variants thereof; and, a light chain variable region comprising three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 70 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 71 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 72 and variants thereof; and, The second antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 31 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 32 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 33 and variants thereof; and a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 34 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 35 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 36 and variants thereof; x) a first antibody, which is an antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 79 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 80 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 81 and variants thereof; and, a light chain variable region comprising three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 82 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 83 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 84 and variants thereof; and, The second antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 31 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 32 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 33 and variants thereof; and a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 34 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 35 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 36 and variants thereof; xi) a first antibody, which is an antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 25 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 26 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 27 and variants thereof; and, a light chain variable region comprising three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 28 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 29 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 30 and variants thereof; and, The second antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 73 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 74 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 75 and variants thereof; and a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 76 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 77 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 78 and variants thereof; xii) a first antibody, which is an antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 25 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 26 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 27 and variants thereof; and, a light chain variable region comprising three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 28 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 29 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 30 and variants thereof; and, The second antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 85 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 86 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 87 and variants thereof; and a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 88 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 89 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 90 and variants thereof; xiii) a first antibody, which is an antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 37 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 38 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 39 and variants thereof; and, a light chain variable region comprising three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 40 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 41 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 42 and variants thereof; and, The second antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 55 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 56 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 57 and variants thereof; and a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 58 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 59 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 60 and variants thereof; xiv) a first antibody, which is an antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 91 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 92 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 93 and variants thereof; and a light chain variable region comprising three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 94 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 95 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 96 and variants thereof, and, The second antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 13 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 14 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 15 and variants thereof; and a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 16 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 17 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 18 and variants thereof; Alternatively, xv) the first antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence selected from SEQ ID NO: 91 and variants thereof, CDR-H2 having an amino acid sequence selected from SEQ ID NO: 92 and variants thereof, and CDR-H3 having an amino acid sequence selected from SEQ ID NO: 93 and variants thereof; and, a light chain variable region comprising the following three CDRs: CDR-L1 having an amino acid sequence selected from SEQ ID NO: 94 and variants thereof, CDR-L2 having an amino acid sequence selected from SEQ ID NO: 95 and variants thereof, and CDR-L3 having an amino acid sequence selected from SEQ ID NO: 96 and variants thereof, and, The second antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence selected from SEQ ID NO: 31 and variants thereof, CDR-H2 having an amino acid sequence selected from SEQ ID NO: 32 and variants thereof, and CDR-H3 having an amino acid sequence selected from SEQ ID NO: 33 and variants thereof; and, a light chain variable region comprising the following three CDRs: CDR-L1 having an amino acid sequence selected from SEQ ID NO: 34 and variants thereof, CDR-L2 having an amino acid sequence selected from SEQ ID NO: 35 and variants thereof, and CDR-L3 having an amino acid sequence selected from SEQ ID NO: 36 and variants thereof.

19. The composition or TSH detection kit according to claim 15 or 16, comprising: i) a first antibody, which is an antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region of three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 31 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 32 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 33 and variants thereof; and a light chain variable region of three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 34 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 35 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 36 and variants thereof; and The second antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 49 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 50 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 51 and variants thereof; and a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 52 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 53 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 54 and variants thereof; ii) a first antibody, which is an antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region of three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 37 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 38 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 39 and variants thereof; and, a light chain variable region of three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 40 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 41 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 42 and variants thereof; and, The second antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 43 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 44 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 45 and variants thereof; and a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 46 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 47 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 48 and variants thereof; iii) a first antibody, which is an antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region of three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 25 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 26 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 27 and variants thereof; and, a light chain variable region of three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 28 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 29 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 30 and variants thereof; and, The second antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 49 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 50 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 51 and variants thereof; and a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 52 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 53 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 54 and variants thereof; iv) a first antibody, which is an antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region of three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 19 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 20 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 21 and variants thereof; and, a light chain variable region of three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 22 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 23 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 24 and variants thereof; and, The second antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 61 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 62 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 63 and variants thereof; and a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 64 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 65 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 66 and variants thereof; Alternatively, v) the first antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the following three CDRs: a CDR-H1 having an amino acid sequence selected from SEQ ID NO: 19 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 20 and variants thereof, and a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 21 and variants thereof; and, a light chain variable region comprising the following three CDRs: a CDR-L1 having an amino acid sequence selected from SEQ ID NO: 22 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO: 23 and variants thereof, and a CDR-L3 having an amino acid sequence selected from SEQ ID NO: 24 and variants thereof; and, The second antibody is an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence selected from SEQ ID NO: 43 and variants thereof, CDR-H2 having an amino acid sequence selected from SEQ ID NO: 44 and variants thereof, and CDR-H3 having an amino acid sequence selected from SEQ ID NO: 45 and variants thereof; and, a light chain variable region comprising the following three CDRs: CDR-L1 having an amino acid sequence selected from SEQ ID NO: 46 and variants thereof, CDR-L2 having an amino acid sequence selected from SEQ ID NO: 47 and variants thereof, and CDR-L3 having an amino acid sequence selected from SEQ ID NO: 48 and variants thereof.

20. The composition or TSH detection kit according to any one of claims 16 to 19, wherein the first antibody is a capture antibody, and the second antibody is a detection antibody.

21. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 in preparing a kit or use of the immunoassay kit according to any one of claims 11 to 20, wherein the kit is used for: (1) detecting the presence or level of TSH or its mutants in a sample; (2) Diagnosis or auxiliary diagnosis of thyroid dysfunction (such as hypothyroidism or hyperthyroidism); (3) Diagnosis or auxiliary diagnosis of thyroid-related diseases; and / or (4) Evaluation or auxiliary evaluation of the treatment effect of thyroid-related diseases; Preferably, the thyroid-related disease is hyperthyroidism or thyroid cancer.

22. A sample analyzer, comprising: A loading device, configured to load the sample to be tested and the reagents required for the chemiluminescent reaction into the reaction container, wherein the reagents include an antibody or an antigen-binding fragment thereof that specifically binds to TSH or a mutant thereof; A reaction incubation device, configured to provide a reaction and incubation place for a reaction container containing a sample to be tested and a reagent required for a chemiluminescent reaction, so that the sample to be tested and the reagent required for a chemiluminescent reaction in the reaction container form a sample liquid to be tested; and A light measuring device is configured to measure light on the sample liquid to be tested; wherein, The antibody or antigen-binding fragment thereof 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, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 2, 8, 14, 20, 26, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98 and variants thereof, and / or a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99 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 the group consisting of SEQ ID NO: 4, 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100 and variants thereof, a CDR-L2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101 and variants thereof, and / or a CDR-L3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102 and variants thereof; The light measuring device comprises: A receiving component configured to receive a light signal emitted by the sample to be tested through a chemiluminescent reaction and convert the light signal into a corresponding electrical signal; and A processing component is configured to be electrically connected to a receiving component and receive the electrical signal from the receiving component, the processing component includes a first photon counting module and a second photon counting module, the first photon counting module is configured to detect the number of pulses of the electrical signal using a pulse recognition method to obtain a first photon counting result, the second photon counting module is configured to process the electrical signal to obtain a parameter characterizing the number of photons, and calculate a second photon counting result based on the parameter characterizing the number of photons and a preset calibration function, wherein the calibration function represents a mapping relationship between the parameter characterizing the number of photons and the photon counting result, and the processing component is further configured to output a final photon counting result of the sample to be tested based on the first photon counting result and the second photon counting result.

23. The sample analyzer according to claim 22, characterized in that: The processing component is also configured to: when the first photon counting result is lower than a first threshold, output the first photon counting result as the final photon counting result; when the first photon counting result is higher than a second threshold, output the second photon counting result as the final photon counting result, wherein the second threshold is greater than or equal to the first threshold.

24. The sample analyzer according to claim 22 or 23, characterized in that: The second photon counting module includes an integration circuit and a second counting circuit electrically connected to each other, the integration circuit is configured to integrate the electrical signal within a predetermined time period to obtain a DC component signal, the parameters characterizing the number of photons include parameters related to the DC component signal, and the second counting circuit is configured to calculate the second photon counting result based on the parameters related to the DC component signal and a preset calibration function.

25. A sample analyzer, comprising: A loading device, configured to load a sample to be tested and a reagent required for a chemiluminescent reaction into a reaction container, wherein the reagent comprises an antibody or an antigen-binding fragment thereof; A reaction incubation device, configured to provide a reaction and incubation place for a reaction container containing a sample to be tested and a reagent required for a chemiluminescent reaction, so that the sample to be tested and the reagent required for a chemiluminescent reaction in the reaction container form a sample liquid to be tested; as well as A photometric device is configured to measure the photometry of the sample liquid to be tested, wherein the linear detection range of the photometric device is [A1, A2] photons / second, wherein the linear range represents the range of the output photon number of the photometric device, within which the intensity of the luminescent signal generated in the chemiluminescent reaction is linearly proportional to the output photon number of the detection device, wherein A1 is less than or equal to 2000 and A2 is greater than or equal to 10 8 ; The antibody or antigen-binding fragment thereof 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, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97 and variants thereof, a CDR-H2 having an amino acid sequence selected from SEQ ID NO: 2, 8, 14, 20, 26, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98 and variants thereof, and / or a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99 and variants thereof; and / or a light chain variable region comprising the following three CDRs: an amino acid sequence selected from SEQ ID NO: NO:4, 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100 and variants thereof, a CDR-L2 having an amino acid sequence selected from SEQ ID NO:5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101 and variants thereof, and / or a CDR-L3 having an amino acid sequence selected from SEQ ID NO:6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102 and variants thereof.

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