An antibody that binds to a T4-T4 antibody complex and its application

Abstract

This invention discloses an antibody that binds to a T4-T4 antibody complex and its applications, relating to the field of immunodiagnostics. The antibody that binds to the T4-T4 antibody complex disclosed in this invention includes a heavy chain variable region and a light chain variable region. This antibody exhibits good activity, detection sensitivity, and clinical relevance, providing an important source of raw materials for the detection of T4.

Description

[0001] Cross-referencing: This application claims priority to Chinese Patent Application No. 202411997436.2, filed on December 31, 2024, entitled "An antibody binding a T4-T4 antibody complex and its application thereof", the entire contents of which are incorporated herein by reference.

[0002] This application claims priority to Chinese Patent Application No. 202511257651.3, filed on September 3, 2025, entitled "An antibody that binds to a T4-T4 antibody complex and its application thereto", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention belongs to the field of immunodiagnostic technology, and more specifically, relates to an antibody that binds to a T4-T4 antibody complex and its application. Background Technology

[0004] Thyroxine, also known as tetraiodothyronine (3,5,3',5'-Tetraiodothyronine, T4), has a molar mass of 776.87 g·mol⁻¹ and is the main hormone secreted by the thyroid gland. All T4 in the blood originates from thyroid secretion. Thyroxine affects almost all physiological processes, including growth and development, metabolism, body temperature, and heart rate. The vast majority of T4 released into serum is in a bound state: approximately 60% is bound to thyroid-binding protein TBG, 30% to thyroid-binding prealbumin TBPA, 0.97% to albumin, and only about 0.03% exists in a free, biologically active form (FT4). A dynamic balance exists between free T4 (FT4) and bound T4 (TT4) to maintain normal physiological function. Since T4 is an important component of the hypothalamic-pituitary-thyroid regulatory system, TT4 and FT4 are important indicators for routine clinical assessment of thyroid function. The measurement results can be used as a basis for differential diagnosis of thyroid diseases, such as for diagnosing hyperthyroidism, primary and secondary hypothyroidism, and for monitoring thyroid-stimulating hormone suppression therapy.

[0005] Currently, methods for detecting thyroxine in human serum include radioimmunoassay, chromatography, and immunoassay. Radioimmunoassay has safety concerns due to radioactive isotopes, while chromatography is time-consuming and expensive, limiting its widespread use. Competitive immunoassays such as chemiluminescence and immunochromatography are limited by inherent limitations of competitive methods, such as insufficient low-end sensitivity, lack of precision, inaccuracy, susceptibility to interference, and narrow linear range. Since T4 is a small molecule, and most small molecules have only one antigenic determinant, they cannot support sandwich detection with two different antibodies. With the development of detection technologies, small-molecule sandwich methods using antibody complexes can better achieve quantitative detection of T4. The specific detection principle is as follows: one antibody binding to T4 (primary antibody) forms an immune complex with the target antigen T4, and another antibody against the complex (secondary antibody) binds to the aforementioned immune complex to form an immune sandwich complex. The recognition site is a new epitope formed after the primary antibody and T4 molecule bind, and the complex antibody must not bind to the free primary antibody or T4 molecule, or bind weakly. This method can greatly improve the sensitivity and accuracy of small molecule antigen detection. Its key technology lies in the development of antibodies that specifically recognize and bind to thyroxine and its antibody complex. Therefore, there is a strong demand in this field for antibody raw materials that can effectively bind to the T4-T4 complex. Summary of the Invention

[0006] This application provides an antibody that binds to a T4-T4 antibody complex, which binds only to T4 and the complex formed by T4 antibody, but not to T4 and T4 antibody. It can simultaneously detect total T4 and free T4 in a sample, providing an important source of raw materials for T4 detection. It also has good activity and sensitivity, good linearity, and high clinical relevance.

[0007] To achieve the above objectives, in a first aspect, the present invention provides an antibody that binds to a T4-T4 antibody complex, the antibody comprising three complementary determining regions having a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NO:19, 20, 21, 41 and three complementary determining regions having a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NO:22, 23, 24, 43.

[0008] Secondly, the present invention provides an antibody that binds to a T4-T4 antibody complex, comprising a complementarity-determining region, wherein the complementarity-determining region comprises any one of (a) to (c): (a) HCDR1 with amino acid sequences as shown in SEQ ID NO:1 (SYGVG), HCDR2 with amino acid sequences as shown in SEQ ID NO:2 (GVEAGGYRGYNPTLKS), HCDR3 with amino acid sequences as shown in SEQ ID NO:3 (VPLGYGHGYGRLDY), and LCDR1 with amino acid sequences as shown in SEQ ID NO:4 (SGSSSNVGYGDYVG), LCDR2 with amino acid sequences as shown in SEQ ID NO:5 (DTTSRAA), and LCDR3 with amino acid sequences as shown in SEQ ID NO:6 (ASYDDDSYGV); (b) HCDR1 with amino acid sequences as shown in SEQ ID NO:7 (SYTVG), HCDR2 with amino acid sequences as shown in SEQ ID NO:8 (NIESGGYTVYNPALKS), HCDR3 with amino acid sequences as shown in SEQ ID NO:9 (ENAVGGGDYDYDYYIDY), and LCDR1 with amino acid sequences as shown in SEQ ID NO:10 (SGSSSNVGYGNYVS), LCDR2 with amino acid sequences as shown in SEQ ID NO:11 (GATSRAS), and LCDR3 with amino acid sequences as shown in SEQ ID NO:12 (ASYDSNSNI); (c) HCDR1 with amino acid sequences as shown in SEQ ID NO:13 (SHGVG), HCDR2 with amino acid sequences as shown in SEQ ID NO:14 (GRENGGYAYYNSALKS), HCDR3 with amino acid sequences as shown in SEQ ID NO:15 (NSDGGIFLNNIDY), and LCDR1 with amino acid sequences as shown in SEQ ID NO:16 (TGTSTDIGAWDGVG), LCDR2 with amino acid sequences as shown in SEQ ID NO:17 (NVDKRPS), and LCDR3 with amino acid sequences as shown in SEQ ID NO:18 (GSPKSDYTIV); and (d) HCDR1 with amino acid sequences as shown in SEQ ID NO:35 (SNTID), HCDR2 with amino acid sequences as shown in SEQ ID NO:36 (GIDWAGDTGYNPALKS), HCDR3 with amino acid sequences as shown in SEQ ID NO:37 (GPRYYGRDSYGYSYLDH), and LCDR1 with amino acid sequences as shown in SEQ ID NO:38 (SGSSSNVGYGDYVG), LCDR2 with amino acid sequences as shown in SEQ ID NO:39 (DATSRAS), and LCDR3 with amino acid sequences as shown in SEQ ID NO:40 (ASYDSNFYRV).

[0009] Thirdly, the present invention provides an antibody that binds to a T4-T4 antibody complex, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the aforementioned heavy chain variable region is shown in any one of SEQ ID NO: 19, 20, 21, 41; and the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NO: 22, 23, 24, 43.

[0010] Fourthly, the present invention provides an antibody that binds to a T4-T4 antibody complex, comprising a heavy chain and a light chain, wherein the amino acid sequence of the aforementioned heavy chain is as shown in any one of SEQ ID NO:25, 26, 27, 42; and the amino acid sequence of the light chain is as shown in any one of SEQ ID NO:28, 29, 30, 44.

[0011] Fifthly, the present invention provides an antibody conjugate comprising the antibodies described in the first to fourth aspects.

[0012] In a sixth aspect, the present invention provides a reagent or kit comprising the antibody described in one to the fourth aspect or the antibody conjugate described in the fifth aspect.

[0013] In a seventh aspect, the present invention provides the use of the antibodies described in the first to fourth aspects, or the antibody conjugates described in the fifth aspect, or the reagents or kits described in the sixth aspect in the preparation of products for detecting T4 or products for detecting T4-T4 antibody complexes.

[0014] The eighth aspect provides a method for diagnosing thyroid disease, including: a) Under conditions sufficient to induce antibody / antigen binding, T4 in the test sample is contacted with T4 antibody to form immune complex 1; and b) Contacting the antibody of any one of claims 1-6, or the antibody conjugate of claim 7, or the reagent or kit of claim 8 with the immune complex 1 in a) to form immune complex 2; and c) Detect the presence of immune complex 2, the presence of complex 2 indicating the presence of T4 in the sample to be tested.

[0015] In a ninth aspect, the present invention provides a nucleic acid molecule, a vector, a cell, and a method for preparing antibodies according to the first to fourth aspects.

[0016] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 Graph showing the purity results of antibody anti-T4-T4-9H15 as determined by HPLC-SEC. Figure 2 Graph showing the purity results of antibody anti-T4-T4-10D9 as determined by HPLC-SEC. Figure 3 Graph showing the purity results of antibody anti-T4-T4-6J10 as determined by HPLC-SEC. Figure 4 Clinical correlation standard curve of anti-T4-T4-9H15 antibody detection on chemiluminescent platform with Roche-defined TT4 values ​​in samples. Figure 5 Clinical correlation standard curve of anti-T4-T4-9H15 antibody detection on chemiluminescent platform with Roche-defined FT4 values ​​in samples. Figure 6 Clinical correlation standard curve of anti-T4-T4-10D9 antibody detection on chemiluminescent platform with Roche-defined TT4 values ​​in samples. Figure 7 Clinical correlation standard curve of anti-T4-T4-10D9 antibody detection on chemiluminescent platform with Roche-defined FT4 values ​​in samples. Figure 8 Clinical correlation standard curve of anti-T4-T4-6J10 antibody detection on Roche-defined TT4 samples. Figure 9 Clinical correlation standard curve of anti-T4-T4-6J10 antibody detection on Roche-defined FT4 samples. Figure 10 Graph showing the purity results of antibody anti-T4-T4-9K13 as determined by HPLC-SEC. Figure 11 Antibody anti-T4-T4-9K13 detection Roche-defined FT4 sample clinical correlation standard curve Detailed Implementation To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below.

[0019] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0020] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless explicitly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. Abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0021] In this invention, the terms "comprising" and "including" are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0022] In this invention, the terms “optional,” “optional,” “alternatively,” “optional,” and “optional” generally refer to events or conditions described below that may but may not occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0023] In this invention, the term "antibody" is used in the broadest sense, and can include full-length monoclonal antibodies, bispecific, multispecific antibodies, chimeric antibodies, or antigen-binding fragments of antibodies, with no specific structure limitations, as long as they exhibit the desired antigen-binding activity.

[0024] In this article, the terms “full-length antibody”, “full-length monoclonal antibody” or “full-length monoclonal antibody” refer to antibodies composed of at least two identical light chains and at least two identical heavy chains linked by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), or immunoglobulin E (IgE).

[0025] In this document, the terms "polyclonal antibody" and "multispecific antibody" are synonymous, both referring to antibodies that can recognize multiple antigenic epitopes. For example, antibodies that recognize two antigenic epitopes (bispecific antibodies, or simply biantibodies), three antigenic epitopes, or four antigenic epitopes are used in a broad sense, and their specific structures are not limited, as long as they can recognize multiple antigenic epitopes. In this invention, at least one of the multiple antigenic epitopes is derived from T4, T4 antibody, or T4-T4 antibody complex.

[0026] In this document, the term "antigen-binding fragment" refers to a fragment containing part or all of an antibody that lacks at least some of the amino acids present in the full-length chain but still possesses the performance activity of specifically binding to an antigen. For example, the fragment may contain part or all of the antibody's CDR. Such fragments bind to antigens and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. Such fragments include, but are not limited to, Fab, Fab', F(ab)2, F(ab')2, Fv, scFv, scFv-Fc fusion proteins, scFv-Fv fusion proteins, single-domain antibodies, or minimal recognition units. Such fragments can be generated using recombinant nucleic acid technology or through enzymatic or chemical cleavage of antigen-binding molecules (including intact antibodies).

[0027] In this article, the term "Fab antibody" or "Fab fragment" generally refers to an antibody or fragment containing only Fab molecules, which consists of the VH and CH1 of the heavy chain and the complete light chain, linked by a disulfide bond.

[0028] In this paper, the term “F(ab')2 antibody” or “F(ab')2 fragment” refers to two antigen-binding Fab molecules linked together by disulfide bonds.

[0029] In this article, the term "Fv antibody" or "Fv fragment" generally refers to an antibody or fragment consisting only of a light chain variable region (VL) and a heavy chain variable region (VH) linked by non-covalent bonds. It is the smallest functional fragment of an antibody that retains the complete antigen-binding site.

[0030] In this paper, the terms "single-chain antibody" and "scFv fragment" refer to antibodies or fragments formed by linking the variable regions of the antibody heavy chain and light chain through short peptides.

[0031] In this article, the terms "minimum recognition unit" and "MRU" both refer to antibodies or fragments consisting of only one CDR, with a very small molecular weight, accounting for only about 1% of a complete antibody.

[0032] Antigen-binding fragments of antibodies typically possess the same binding specificity as the antibody from which they originate. Those skilled in the art will readily understand, based on the description of this invention, that these antigen-binding fragments can be obtained, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds. Given the complete antibody structure disclosed in this invention, those skilled in the art can readily obtain the aforementioned antigen-binding fragments.

[0033] Antigen-binding fragments can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesizing, for example, automated peptide synthesizers sold by Applied BioSystems.

[0034] In this paper, the CH1 region, hinge region (optional), CH2 region, CH3 region, CH4 region (optional), and tail peptide (optional) are all components of the heavy chain constant region, which is located at the C-terminus of the heavy chain of the antibody molecule. Each heavy chain constant region, from the N-terminus to the C-terminus, includes the CH1 region, hinge region (optional), CH2 region, CH3 region, CH4 region (optional), and tail peptide (optional). Different types of antibodies (such as IgG, IgA, IgM, etc.) have different amino acid sequences and structures in their heavy chain constant regions, but they all have relatively conserved structural features. These conserved structures enable the heavy chain constant region to perform its biological function. The heavy chain constant region, CH1 region, hinge region (optional), CH2 region, CH3 region, CH4 region (optional), and tail peptide (optional) of different species and subclasses are well known in the art, and their amino acid sequences can be determined based on bioinformatics databases, such as the IMGT database (https: / / www.imgt.org / IMGTrepertoire / Proteins / ). It should be understood that different bioinformatics databases or software may not have completely consistent results in the division and sequence identification of constant regions. However, those skilled in the art have a general and unified understanding of the concept, division and sequence identification of constant regions and their segments. Therefore, the constant regions that those skilled in the art can identify and divide using common knowledge and ordinary methods are all within the scope of protection of this invention.

[0035] For example, the amino acid sequence of the corresponding segment (such as the IgM CH2 region) divided by the IMGT database can be used as the reference sequence. The start or end position of the reference sequence can be moved forward by several amino acid residues (i.e., moved to the IgM CH1 region) or backward by several amino acid residues (i.e. moved to the IgM CH3 region) to obtain the corresponding segment sequence which is longer or shorter than the reference sequence.

[0036] In this document, the term "hinge region" refers to a polypeptide that links the CH1 and CH2 domains within the constant region of the heavy chain of an antibody. This region is rich in proline, thus allowing for stretching and bending, and typically contains at least one proline (P). Hinge regions are usually dimers, consisting of two polypeptides with the same amino acid sequence. Specific amino acid sequences are not limited and are all within the scope of protection of this application. Hinge regions of different species and subclasses are well known.

[0037] In this paper, the term "tail peptide" refers to a short peptide sequence of about a dozen amino acid residues at the end of the CH3 or CH4 region of an antibody. Tail peptides from different species are well known.

[0038] In this document, the term "IgM tail peptide" refers to a short peptide sequence at the end of the CH4 region of an IgM antibody, located at the C-terminus of the CH4 region. The specific amino acid sequence is not limited and is within the scope of protection of this application. The IgM tail peptide contains cysteine ​​residues that participate in polymer formation and can also bind to the J chain to further stabilize the multimeric structure.

[0039] In this invention, the term "T4" refers to thyroxine T4, which exists in both bound and free forms. The T4 detected in the sample includes, but is not limited to, total T4 (TT4) and free T4 (FT4). Sample types include, but are not limited to, blood, thyroid gland, tissue fluid, tissue cells, and cerebrospinal fluid.

[0040] In this invention, the term "T4-T4 antibody complex" refers to a complex formed by the binding of T4 and an anti-T4 antibody (which can specifically bind to T4), i.e., an antigen-antibody complex. In this invention, the term "antibody that binds to T4-T4 antibody complex" refers to an antibody that binds to the aforementioned antigen-antibody complex, and has the following characteristics: (1) it binds to T4-T4 antibody complex, but does not bind to T4 antigen or anti-T4 antibody; and / or (2) it binds strongly to T4-T4 antibody complex, and binds weakly or not to anti-T4 antibody or T4 antigen; the binding strength can be at least 1, 1.5, 2, 2.5, 5, 10, 15, 20, 30, 50, 80, or 100 times the difference in binding activity.

[0041] In this paper, the terms “complementarity-determining region”, “CDR”, or “CDRs” refer to highly variable regions of the heavy and light chains of immunoglobulins, specifically regions containing one or more or all of the major amino acid residues that contribute to the binding affinity of antibodies to the antigens or epitopes they recognize.

[0042] In this paper, the heavy chain complementarity determination regions (heavy chain variable regions CDRs) are referred to as "HCDRs" or "HCDRs", which include HCDR1 (also known as CDR-H1), HCDR2 (also known as CDR-H2), and HCDR3 (also known as CDR-H3); the light chain complementarity determination regions (light chain variable regions CDRs) are referred to as "LCDRs" or "LCDRs", which include LCDR1 (also known as CDR-L1), LCDR2 (also known as CDR-L2), and LCDR3 (also known as CDR-L3).

[0043] Commonly used CDR definition systems in this field include the Kabat definition, the Chothia definition, the IMGT definition, the Contact definition, and the AbM definition. As described herein, the “Kabat definition” refers to the system described in Kabat et al., USDept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983). The “Chothia definition” is found in Chothia et al., J Mol Biol 196:901-917 (1987). Other CDR definition methods may not strictly follow any of the above schemes, but will still overlap at least partially with the CDR region defined by Kabat, although they may be shortened or lengthened based on predictions or experimental results of specific residues or residue groups. Exemplary CDR definitions are listed in Table 1 below. Definitions vary slightly in different literatures, and given the variable region amino acid sequence of an antibody, those skilled in the art can routinely determine which residues contain a specific CDR. It should be noted that the CDR defined in this invention includes, but is not limited to, the CDRs defined by the methods in Table 1. CDRs defined by other methods disclosed in the art based on the heavy chain variable region and light chain variable region disclosed in this application are also within the scope of protection of this disclosure.

[0044] Table 1: CDR Definition 1

[0045] 1 In Table 1, except for the Contact definition system where CDRs are based on the Chothia numbering system, the CDRs for other definition systems are based on the Kabat numbering system (see below). The amino acid numbers on the heavy chain are represented by "H + number", and the amino acid numbers on the light chain are represented by "L + number".

[0046] 2 As used in Table 1, “AbM” with a lowercase “b” refers to the CDR defined by the “AbM” antibody modeling software of Oxford Molecular.

[0047] 3 If neither H35A nor H35B exists, then HCDR1 ends at bit 35; if only H35A exists, then HCDR1 ends at bit 35A; if both H35A and H35B exist, then HCDR1 ends at bit 35B.

[0048] 4If neither H35A nor H35B exists, then HCDR1 ends at bit 32; if only H35A exists, then HCDR1 ends at bit 33; if both H35A and H35B exist, then HCDR1 ends at bit 34.

[0049] 5 If neither H35A nor H35B exists, then HCDR1 ends at bit 33; if only H35A exists, then HCDR1 ends at bit 34; if both H35A and H35B exist, then HCDR1 ends at bit 35.

[0050] 6 The CDR numbering shown in Table 1 under the Contact definition system is based on the Chothia numbering system, and the Contact definition system is only applicable to the Chothia or Martin numbering system.

[0051] Kabat et al. also defined a numbering system applicable to the variable region sequence of any antibody. Those skilled in the art can readily map this Kabat numbering system to any variable region sequence without relying on any experimental data outside the sequence itself. As used herein, “Kabat numbering” refers to numbering using the numbering system described in Kabat et al., USDept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983). “Chothia numbering” as used herein refers to the numbering system described in Al-Lazikaniet al., (1997) JMB273, 927-948.

[0052] It should be noted that the polypeptide sequences of this invention are not numbered according to the Kabat numbering system. However, those skilled in the art are fully capable of converting the sequence numbers in the sequence listing to Kabat numbers.

[0053] In this application, the term "backbone region," "framework region," or "FR" region includes heavy chain frame regions and light chain frame regions, referring to the regions in the antibody heavy chain variable region (which may be represented as VH) and light chain variable region (which may be represented as VL) excluding the CDR. Given the amino acid sequence of the variable region of the antibody, those skilled in the art can routinely determine which residues contain a specific CDR and also determine the amino acid sequence of the frame region. The heavy chain frame region is represented by "HFR" and can be further subdivided into adjacent regions separated by CDRs, including HFR1, HFR2, HFR3, and HFR4 frame regions; the light chain frame region is represented by "LFR" and can be further subdivided into adjacent regions separated by CDRs, including LFR1, LFR2, LFR3, and LFR4 frame regions.

[0054] In this application, the heavy chain variable region is obtained by connecting the following CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by connecting the following CDRs and FRs in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0055] In this application, the terms “identity,” “homology,” or “similarity” are used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined by conventional methods, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Institute)). Foundation, Washington, DC). Numerous algorithms exist for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48: 443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2: 482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70: 173-187 (1997); and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215: 403-410). Computer programs utilizing these algorithms are also available, including but not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul...). See, Meth.Enzym., 266:460-480 (1996); or GAP, BESTFIT, BLAST Altschul, etc., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0056] In this application, the term "at least 80% identity" means at least 80% identity with each reference sequence, which may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0057] In this application, the term "vector" refers to a delivery vehicle that can operatively insert a genetic element (such as the aforementioned nucleic acid molecule) therein and enable the expression of that genetic element, for example, to produce a protein, RNA, or DNA encoded by the genetic element, or to replicate the genetic element. Vectors can be used to transform, transduce, or transfect host cells, enabling the expression of the genetic element they carry within the host cells. The vectors may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The vectors also include vectors having a variety of the aforementioned functions. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable cell or host. Typically, by culturing a suitable cell or host containing the vector, the vector can produce the desired expression product.

[0058] In this document, the term "cell" generally refers to a cell whose genetic material has been modified or recombined using genetic engineering or cell fusion techniques to obtain a unique trait with stable inheritance. Specifically, the term "cell" refers to prokaryotic or eukaryotic cells into which recombinant vectors can be introduced. The terms "transformed" or "transfected" as used herein refer to the introduction of nucleic acids (e.g., vectors) into cells using various techniques known in the art, followed by expression of the antibodies provided by this invention in these cells, and subsequent culturing of these cells to obtain the corresponding antibodies.

[0059] This invention proposes a method for preparing antibodies, antibody-drug conjugates, reagents or kits that combine T4-T4 antibody complexes and their uses, as well as nucleic acid molecules, vectors, cells, and antibodies, which are described in detail below.

[0060] Antibody In a first aspect, embodiments of the present invention provide an antibody that binds to a T4-T4 antibody complex, wherein the antibody comprises three complementary determining regions of a heavy chain variable region having an amino acid sequence as shown in any of SEQ ID NO:19, 20, 21, 41, and three complementary determining regions of a light chain variable region having an amino acid sequence as shown in any of SEQ ID NO:22, 23, 24, 43.

[0061] In an optional embodiment, the complementary determination region of the variable region is defined by any one or a combination of systems such as Kabat, Chothia, IMGT, AbM, or Contact.

[0062] In an optional embodiment, the complementary determination region of the aforementioned variable region is defined by the Kabat system.

[0063] In an alternative embodiment, the complementary determination region of the aforementioned variable region is defined by the Chothia system.

[0064] In an optional embodiment, the complementary determination region of the aforementioned variable region is defined by the IMGT system.

[0065] In an optional embodiment, the complementary determination region of the aforementioned variable region is defined by the AbM system.

[0066] In an optional embodiment, the complementary determination region of the aforementioned variable region is defined by the Contact system.

[0067] Secondly, embodiments of the present invention provide an antibody that binds to a T4-T4 antibody complex, including a complementarity-determining region, wherein the complementarity-determining region is selected from any one of (a) to (d): (a) HCDR1 with amino acid sequences as shown in SEQ ID NO:1 (SYGVG), HCDR2 with amino acid sequences as shown in SEQ ID NO:2 (GVEAGGYRGYNPTLKS), HCDR3 with amino acid sequences as shown in SEQ ID NO:3 (VPLGYGHGYGRLDY), and LCDR1 with amino acid sequences as shown in SEQ ID NO:4 (SGSSSNVGYGDYVG), LCDR2 with amino acid sequences as shown in SEQ ID NO:5 (DTTSRAA), and LCDR3 with amino acid sequences as shown in SEQ ID NO:6 (ASYDDDSYGV); (b) HCDR1 with amino acid sequences as shown in SEQ ID NO:7 (SYTVG), HCDR2 with amino acid sequences as shown in SEQ ID NO:8 (NIESGGYTVYNPALKS), HCDR3 with amino acid sequences as shown in SEQ ID NO:9 (ENAVGGGDYDYDYYIDY), and LCDR1 with amino acid sequences as shown in SEQ ID NO:10 (SGSSSNVGYGNYVS), LCDR2 with amino acid sequences as shown in SEQ ID NO:11 (GATSRAS), and LCDR3 with amino acid sequences as shown in SEQ ID NO:12 (ASYDSNSNI); and (c) HCDR1 with amino acid sequences as shown in SEQ ID NO:13 (SHGVG), HCDR2 with amino acid sequences as shown in SEQ ID NO:14 (GRENGGYAYYNSALKS), HCDR3 with amino acid sequences as shown in SEQ ID NO:15 (NSDGGIFLNNIDY), and LCDR1 with amino acid sequences as shown in SEQ ID NO:16 (TGTSTDIGAWDGVG), LCDR2 with amino acid sequences as shown in SEQ ID NO:17 (NVDKRPS), and LCDR3 with amino acid sequences as shown in SEQ ID NO:18 (GSPKSDYTIV); and (d) HCDR1 with amino acid sequences as shown in SEQ ID NO:35 (SNTID), HCDR2 with amino acid sequences as shown in SEQ ID NO:36 (GIDWAGDTGYNPALKS), HCDR3 with amino acid sequences as shown in SEQ ID NO:37 (GPRYYGRDSYGYSYLDH), and LCDR1 with amino acid sequences as shown in SEQ ID NO:38 (SGSSSNVGYGDYVG), LCDR2 with amino acid sequences as shown in SEQ ID NO:39 (DATSRAS), and LCDR3 with amino acid sequences as shown in SEQ ID NO:40 (ASYDSNFYRV).

[0068] In an optional embodiment, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Kabat system.

[0069] In optional embodiments, the antibodies described in the first and second aspects further include a framework region.

[0070] In optional embodiments, the antibodies described in the first and second aspects further have the framework regions shown by HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4.

[0071] Thirdly, embodiments of the present invention provide an antibody that binds to a T4-T4 antibody complex, comprising a heavy chain variable region and a light chain variable region, wherein the aforementioned heavy chain variable region and light chain variable region are selected from any one of (a') to (d'): (a') A heavy chain variable region having at least 80% identity with SEQ ID NO:19, a light chain variable region having at least 80% identity with SEQ ID NO:22, and including the complementarity-determining region shown in claim 2(a); (b') A heavy chain variable region having at least 80% identity with SEQ ID NO:20, a light chain variable region having at least 80% identity with SEQ ID NO:23, and including the complementarity-determining region shown in claim 2 (b); (c') A heavy chain variable region having at least 80% identity with SEQ ID NO:21, a light chain variable region having at least 80% identity with SEQ ID NO:24, and including the complementarity-determining region shown in claim 2 (c); and (d') The heavy chain variable region having at least 80% identity with the amino acid sequence of SEQ ID NO:41, the light chain variable region having at least 80% identity with the amino acid sequence of SEQ ID NO:43, and including the complementarity determining region shown in (d) of claim 2.

[0072] Fourthly, embodiments of the present invention provide an antibody that binds to a T4-T4 antibody complex, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID NO: 19, 20, 21, 41; and the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NO: 22, 23, 24, 43; In an optional embodiment, the combination of the heavy chain variable region and the light chain variable region is selected from any of the following combinations:

[0073] In optional embodiments, the antibodies described in the first, second, third, or fourth aspects above further include a constant region.

[0074] In an optional embodiment, the aforementioned constant region includes a heavy chain constant region and / or a light chain constant region.

[0075] In an optional embodiment, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments.

[0076] In an optional embodiment, the heavy chain constant region includes CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM, CH4 of IgM and / or the tail peptide of IgM.

[0077] In an optional embodiment, the light chain constant region includes a light chain constant region selected from κ-type or λ-type.

[0078] In an optional embodiment, the species source of the constant region is any one of cattle, horses, pigs, sheep, goats, rats, mice, dogs, camels, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese, or humans.

[0079] In an optional embodiment, the species source of the constant region is sheep.

[0080] In an optional embodiment, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO:31 or has at least 80% identity with it.

[0081] In an optional embodiment, the amino acid sequence of the light chain constant region is as shown in SEQ ID NO:32 or has at least 80% identity with it.

[0082] According to an embodiment of the present invention, the N end of the heavy chain constant region is connected to the C end of the heavy chain variable region, and the N end of the light chain constant region is connected to the C end of the light chain variable region.

[0083] In this application, the division of the variable and constant regions of the sequence refers to the IMGT partitioning method, see Lefranc, M.-P. IMGT®, the international ImMunoGeneTics database. Nucl. Acids Res., 29(1):207-209 (2001). DOI:10.1093 / nar / 29.1.207. PMID:11125093. and Martinez-Jean C. and Bosc N. or François Ehrenmann, Patrice Duroux, Chantal Ginestoux, Genetable: house mouse (Mus musculus) IGHC, IMGT Repertoire. IMGT®, the international ImMunoGenetics information system® http: / / www.imgt.org. Created: 16 / 03 / 2011. Version: 17 / 01 / 20 ... Ginestoux, Gene table: house mouse (Mus musculus) IGLC, IMGTRepertoire. IMGT®, the international ImMunoGenetics information system® http: / / www.imgt.org. Created: 16 / 03 / 2011. Version: 17 / 01 / 2020. The variable regions delineated by different methods may differ in some amino acids from the C-terminus or N-terminus of the variable regions delineated by IMGT. Variable regions or constant regions delineated by other methods known in the art are also within the scope of protection of this application.

[0084] Fifthly, embodiments of the present invention provide an antibody that binds to a T4-T4 antibody complex, comprising a heavy chain and a light chain, wherein the amino acid sequence of the aforementioned heavy chain is as shown in any one of SEQ ID NO: 25, 26, 27, 42; and the amino acid sequence of the aforementioned light chain is as shown in any one of SEQ ID NO: 28, 29, 30, 44.

[0085] In an optional embodiment, the amino acid sequence combination of the heavy chain and the light chain is selected from any of the following combinations:

[0086] Those skilled in the art will understand that the features and advantages described above with respect to the first aspect of the antibody also apply to the antibodies described in the second to fifth aspects, and will not be repeated here.

[0087] Antibody conjugates, reagents or kits and their uses In a sixth aspect, the present invention provides an antibody conjugate comprising: the antibody described in the first aspect, the second aspect, the third aspect, the fourth aspect, or the fifth aspect, and a conjugated portion thereof.

[0088] The term "coupler" generally refers to any substance formed by the binding of separate parts together. In a coupler, the separate parts may be interconnected at one or more active sites. Furthermore, the separate parts may covalently or non-covalently associate or connect with each other.

[0089] In an optional embodiment, the conjugation of the antibody with the conjugate does not affect the reaction characteristics of the antibody-conjugate with the T4-T4 complex as described in the sixth aspect, nor does it change the activity of the conjugate itself.

[0090] In optional embodiments, the above conjugate can specifically bind to the T4-T4 antibody complex and can be used for qualitative or quantitative detection of T4, or to indicate T4-related diseases.

[0091] In optional embodiments, the above-mentioned coupling portion includes any one or more of purification tags, affinity substances, solid-phase carriers, or markers.

[0092] In optional embodiments, the purification label includes any one or more of the following: His label, Flag label, GST label, MBP label, SUMO label, or C-Myc label.

[0093] In optional embodiments, the aforementioned affinity substance includes any one or more of biotin, biotin derivatives, or streptavidin.

[0094] In this application, the term "solid support" can refer to a substance that can be suspended or dispersed in a liquid phase (e.g., solid supports such as particles or magnetic beads), or a solid phase that can contain or carry a liquid phase (e.g., supports such as plates, membranes, or test tubes, as well as containers such as perforated plates, microfluidic paths, glass capillaries, nanopillars, or monolithic columns).

[0095] In an optional embodiment, the solid support is selected from any one of microspheres, plates, or membranes.

[0096] In optional embodiments, the solid support described above includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic microparticles, microporous plates, glass, capillaries, nylon, and nitrocellulose membranes.

[0097] In this application, the term "marker" refers to a class of substances that have properties such as luminescence, color development, and radioactivity that can be directly observed by the naked eye or detected or probed by instruments, thereby enabling qualitative or quantitative detection of the corresponding target.

[0098] In optional embodiments, the markers include any one or more of fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, or nanoparticle markers.

[0099] In practical use, those skilled in the art can select appropriate markers according to the detection conditions or actual needs. Regardless of the marker used, it falls within the protection scope of this application.

[0100] In optional embodiments, the fluorescent dyes mentioned above include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C...). y5, Cy5.5, Cy3 and other similar dyes, Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750 and other similar dyes) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP) and other similar dyes).

[0101] In optional embodiments, the enzymes mentioned above include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0102] In optional embodiments, the aforementioned radioactive isotopes include, but are not limited to, those mentioned above. 212 Bi、 131 I, 111 In、 90 Y、 186 Re、 211 At、 125 I, 188 Re、 153 Sm、 213 Bi、 32 P, 94 mTc,99 mTc, 203 Pb, 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn, 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb, 166 Ho、 105 Rh、 177 Lu、 172 Lu and 18 F.

[0103] In optional embodiments, the chemiluminescent reagents mentioned above include, but are not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0104] In optional embodiments, the above-mentioned nanoparticle markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0105] In optional embodiments, the colloids mentioned above include, but are not limited to, colloidal metals, colloidal carbon, dispersed dyes, dye-labeled microspheres, and latexes.

[0106] In optional embodiments, the colloidal metals mentioned above include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0107] In a seventh aspect, embodiments of the present invention provide a reagent or kit, the aforementioned reagent or kit comprising the antibody described in the first aspect, second aspect, third aspect, fourth aspect, or fifth aspect, or the antibody conjugate described in the sixth aspect.

[0108] As previously described, the antibodies in some embodiments of the present invention can effectively bind to the T4-T4 antibody complex. Therefore, reagents or kits containing the antibodies that bind to the T4-T4 antibody complex can effectively perform qualitative or quantitative detection of T4. The reagents or kits provided by the present invention can be used, for example, for detections involving the specific binding properties of the T4-T4 antibody complex and its antibodies, such as immunoblotting and immunoprecipitation. As previously described, the antibodies in some embodiments of the present invention have higher binding activity and specificity to the T4-T4 antibody complex; therefore, reagents or kits containing the aforementioned antibodies have higher detection sensitivity or specificity.

[0109] The above kit may contain any one or more of the following: processing solution, antibody binding to T4-T4 antibody complex, T4 quality control, anti-IgG antibody, instructions for use, or literature. Antibodies binding to T4-T4 antibody complex can be used for different types of diagnostic tests, such as detecting the presence of various diseases, drugs, or other proteins in vitro or in vivo. For example, they can be used to test for related diseases by detecting the serum or blood of the subject.

[0110] Eighthly, embodiments of the present invention provide the use of an antibody as described in any one of the first to fifth aspects, or a conjugate as described in the sixth aspect, or a reagent or kit as described in the seventh aspect, in detecting T4, detecting T4-T4 antibody complexes, diagnosing T4-related diseases, preparing products for detecting T4, or preparing products for diagnosing T4-related diseases.

[0111] According to embodiments of this aspect, the aforementioned T4-related diseases include, but are not limited to, any one or more of hyperthyroidism, primary and secondary hypothyroidism.

[0112] According to embodiments of this aspect, the above-mentioned products include, but are not limited to, reagents, kits, test strips, or test plates.

[0113] In a ninth aspect, the present invention provides a method for detecting T4-T4 antibody complexes, comprising: a) contacting T4 in a test sample with a T4 antibody under conditions sufficient to cause an antibody / antigen binding reaction to form an immune complex 1; and b) contacting an antibody described in any one of the first to fifth aspects, or an antibody-drug conjugate described in the sixth aspect, or a reagent or kit described in the seventh aspect with the immune complex 1 in a) to form an immune complex 2; and c) detecting the presence of the aforementioned immune complex 2, the presence of the aforementioned complex 2 indicating the presence of the aforementioned T4 in the test sample.

[0114] As used herein, the term "subject" refers to a vertebrate, preferably a mammal, and most preferably a human. Mammals include, but are not limited to, rodents, apes, humans, livestock, racing animals, and pets. Tissues, cells, and their progeny from biological entities obtained in vivo or cultured in vitro are also included.

[0115] Nucleic acid molecules, vectors, cells, and methods for preparing antibodies In the process of preparing antibodies according to any one of the first to fifth aspects, nucleic acid molecules expressing these antibodies can be linked to different vectors and then expressed in different cells to obtain the corresponding antibodies.

[0116] In a tenth aspect of the present invention, an embodiment of the present invention provides a nucleic acid molecule that encodes an antibody as described in any one of the first to fifth aspects.

[0117] The sequence of a nucleic acid molecule can be derived from the antibody's amino acid sequence using conventional methods such as codon coding rules. The full-length sequence of a nucleic acid molecule or a fragment thereof can usually be obtained using PCR amplification, recombination, or artificial synthesis.

[0118] In an eleventh aspect of the present invention, an embodiment provides a vector comprising the nucleic acid molecule described in the tenth aspect. When the nucleic acid molecule is ligated to the vector, it can be directly or indirectly linked to control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. The vector may contain various elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may contain a replication initiation site. The vector may also include components that facilitate its entry into cells, including but not limited to viral particles, liposomes, or protein coats. The vector can be an expression vector or a cloning vector. The expression vector can be directly derived from the vector itself or it can be exogenous, i.e., not derived from the vector itself. Of course, the aforementioned nucleic acid molecule and control elements need to be operably linked. In this document, "operably linked" means ligating a foreign gene to the vector so that the control elements within the vector, such as transcription control sequences and translation control sequences, can perform their intended function of regulating the transcription and translation of the foreign gene.

[0119] In an optional embodiment, the aforementioned expression vector is a plasmid expression vector.

[0120] In a twelfth aspect of the invention, an embodiment of the invention provides a cell comprising the nucleic acid molecule described in the tenth aspect and the vector described in the eleventh aspect; or expressing an antibody described in any one of the first to fifth aspects. Using this cell, under suitable conditions, the aforementioned antibody can be effectively expressed intracellularly.

[0121] It should be noted that the cells used in this invention are not particularly limited and can be prokaryotic cells, eukaryotic cells, or bacteriophages. The aforementioned prokaryotic cells include, but are not limited to, *Escherichia coli*, *Bacillus subtilis*, *Streptomyces*, or *Proteus mirabilis*. The aforementioned eukaryotic cells include fungi such as *Pichia pastoris*, *Saccharomyces cerevisiae*, *Schizosaccharomyces cerevisiae*, and *Trichoderma*; insect cells such as *Ardisia crenata*; plant cells such as tobacco; mammalian cells such as BHK cells, CHO cells, COS cells, NSO cells, 293 series cells, HepG2, HEK293 cell lines, Huh7 cells, and myeloma cells, but do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0122] It should be noted that the "suitable conditions" mentioned above in this invention refer to conditions suitable for the antibody expression described above. Those skilled in the art will readily understand that suitable conditions for the antibody expression include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy cell state, suitable cell density, suitable cell culture environment, and suitable cell culture time. The term "suitable conditions" is not particularly limited, and those skilled in the art can optimize the optimal conditions for the antibody expression based on the specific environment of their laboratory.

[0123] In a thirteenth aspect of the invention, the invention provides a method for preparing antibodies according to any one of the first to fifth aspects, wherein the aforementioned preparation method includes culturing the cells described in the twelfth aspect.

[0124] Based on the amino acid sequence of the antibody binding the T4-T4 antibody complex disclosed in this invention, those skilled in the art will readily conceive of preparing the antibody binding the T4-T4 antibody complex using genetic engineering or other techniques (chemical synthesis, recombinant expression). For example, the antibody can be isolated and purified from the culture product of recombinant cells capable of recombinantly expressing the antibody as described above. This is easily achievable by those skilled in the art. Therefore, regardless of the technique used to prepare the antibody binding the T4-T4 antibody complex of this invention, it falls within the protection scope of this invention.

[0125] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0126] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Unless otherwise stated, the techniques employed or considered herein are standard methods. Materials, methods, and examples are illustrative and not limiting in nature.

[0127] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. "Reaction" (Mullis et al., eds., 1994); and "Current Protocols in Immunology" (JEColigan et al., eds., 1991), each of which is explicitly incorporated herein by reference.

[0128] Based on long-term, inventive research on T4, the inventors discovered an antibody that meets the requirements for T4 detection. The preparation, activity identification, and performance testing of the antibody are illustrated in the examples.

[0129] The features and performance of the present invention will be further described in detail below with reference to the embodiments. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0130] Example 1: Antibody discovery of immune complexes formed by thyroxine and anti-thyroxine antibodies. 1. Sheep's Immunity An immune complex formed by thyroxine and anti-thyroxine antibody (exemplarily shown is anti-thyroxine antibody anti-T4-8C2, the heavy chain amino acid sequence of which includes but is not limited to SEQ ID NO:33, and the light chain amino acid sequence of which includes but is not limited to SEQ ID NO:34; antibodies that bind to T4 antigen can be used in this invention, and immune complexes formed by T4 antigen and anti-T4 antibody can be used as immunogens) is used as an immunogen. An emulsion preparation prepared with incomplete Freund's adjuvant is injected subcutaneously into sheep to stimulate an immune response. Serum is collected before and after immunization on days 0, 14, 28, 42, and 69, respectively. After serum titer testing, sheep with titers meeting the requirements are selected. Whole blood is collected from sheep, and PBMC cells are prepared using a lymphocyte separation kit.

[0131] 2. Construction of phage libraries RNA was extracted from sheep PBMCs and reverse transcribed into cDNA. Using specifically designed sheep antibody gene amplification primers, the heavy chain variable region (VH) and light chain variable region (VL) fragments were amplified using cDNA as a template. Then, the VH and VL gene fragments were sequentially inserted into the phage vector VO2 using enzyme digestion and ligation. Finally, the ligated phage plasmid was electroporated into TG1 competent cells. The next day, single colonies were selected for PCR identification and antibody gene sequencing to assess the quality of the phage library. Once qualified, the phage library was screened.

[0132] 3. Screening of phage libraries The TG1 phage library was inoculated into a shake flask and cultured to a suitable bacterial concentration (OD600 of 0.8-1.0). Helper phages were then added for infection for 1 hour, followed by overnight incubation. The next day, the bacterial culture was collected, centrifuged, and the supernatant was purified by salting out to obtain the displayed phage library.

[0133] The phage library was panned 3-4 times using magnetic beads. Then, monoclonal phage-infected colonies were selected for antibody expression in the supernatant. Next, the monoclonal phage antibody expression supernatant was screened and identified using ELISA. Finally, three dominant monoclonal phage antibodies were selected. These antibodies only bind to T4 and the complex formed by the antibody, and do not bind to T4 or T4 antibody. The antibodies that bind to the T4-T4 complex were named anti-anti-T4-T4-9H15, anti-T4-T4-10D9, and anti-T4-T4-6J10.

[0134] The experiment was repeated following the methods described above for sheep immunization, phage library construction, and phage library construction. One dominant monoclonal antibody was obtained through screening. This monoclonal antibody only binds to the complex formed by T4 and its antibody, and does not bind to T4 or T4 antibody. The antibody that binds to the T4-T4 complex was named anti-T4-T4-9K13.

[0135] 4. Phage antibody gene sequencing Antibody gene sequencing was performed on antibodies anti-T4-T4-9H15, anti-T4-T4-10D9, and anti-T4-T4-6J10. Through sequence analysis, duplicate and invalid sequences were removed to obtain a unique sheep monoclonal antibody sequence.

[0136] The heavy chain (H) and light chain (L) sequences of the above antibodies are shown in Table 2: Table 2: Antibody Sequences

[0137] The antibody anti-T4-T4-9K13 was subjected to antibody gene sequencing. Through sequence analysis, duplicate and invalid sequences were removed to obtain a unique sheep monoclonal antibody sequence. The heavy chain amino acid sequence of the antibody is shown in SEQ ID NO:42, and the light chain amino acid sequence is shown in SEQ ID NO:44.

[0138] Example 2 Preparation of recombinant antibodies binding to T4-T4 antibody complex The antibody that binds to the T4-T4 antibody complex obtained in Example 1 was recombinantly expressed.

[0139] 1. Expression plasmid construction The pcDNA™ 3.4 TOPO® vector is a recombinant antibody eukaryotic expression vector that has been modified to introduce polyclonal restriction enzyme sites, and will be referred to as the 3.4A expression vector. Based on the variable region gene, VL and VH gene-specific primers were designed, with restriction endonuclease sites and protective bases at both ends, respectively. The heavy chain gene fragment and the light chain gene fragment were amplified by PCR.

[0140] The heavy chain gene fragment and the light chain gene fragment were double-digested with restriction endonucleases, and the 3.4A vector was double-digested with restriction endonucleases. After purification and recovery of the gene fragments and the vector, the heavy chain gene and the light chain gene were respectively ligated into the 3.4A expression vector to obtain recombinant expression plasmids of the heavy chain and the light chain, respectively.

[0141] 2. Preparation of recombinant cells HEK293 cells were revived early and passaged to a 200ml volume to achieve a cell density of 3-5 × 10⁻⁶ cells / mL. 6Cells / ml, cell viability >95%; centrifuged to wash cells, reconstituted with culture medium, and adjusted cell density to 2.9 × 10⁶ cells / ml. 6 Cells / ml were used as cell dilution buffers. Plasmid DNA and transfection reagent dilution buffers from step 1 of Example 2 were prepared separately using culture medium. The transfection reagent dilution buffer was added to the three plasmid DNA dilution buffers, mixed well, and incubated at room temperature for 15 min. This mixture was then slowly added to the cell dilution buffer over 1 min, mixed well, and samples were taken for counting. Cell viability after transfection was recorded and observed, and the cells were incubated in a 35°C incubator at 120 rpm and 8% CO2 concentration.

[0142] 3. Specific detection of recombinant antibody supernatant 3.1 Binding assay of supernatant antibodies with thyroxine and anti-thyroxine antibody complexes (detection mode 1) (1) ELISA plate coating ELISA plates were coated with streptavidin at a concentration of 3 μg / mL, 100 μl / well, and incubated overnight at 4°C; washed twice with 1×PBST; blocked with 1% casein, 120 μl / well, at 37°C for 1 h, patted dry, and ready for use.

[0143] (2) Preparation of thyroxine and anti-thyroxine antibody complex 1 μg / mL of biotin-conjugated anti-thyroxine antibody (anti-T4-8C2) and 0.1 μg / mL of free thyroxine (from Phytobio) were first incubated in centrifuge tubes at 37°C with gentle shaking for 1.5 h to form a thyroxine and anti-thyroxine antibody complex, hereinafter referred to as the T4-T4 antibody complex.

[0144] Add the incubated T4-T4 antibody complex to the ELISA detection plate in step 3.1-(1), 100 μL / well, and incubate at 37°C for 1 h; discard the supernatant, do not tap or wash the plate, and set aside for use.

[0145] (3) Specific detection of antibody supernatant Take the antibody expression supernatant from the recombinant cells and dilute it with 1% casein by a certain factor.

[0146] Add the prepared antibody expression supernatant and blank control (1% casein) to the ELISA plate in step 3.1-(1), 100 μL / well, 2 wells in each case. Gently mix and incubate at 37°C for 45 min; wash 3 times with 1×PBST; add rabbit anti-goat FC-HRP (1% casein diluted 15000 times), 100 μL / well, incubate at 37°C for 45 min; wash 3 times with 1×PBST; add 50 μL of each of solutions A and B, display for 10 min, add 50 μL of stop solution / well, and read the values. The results are shown in Table 3.

[0147] 3.2 Binding assay of supernatant antibody and anti-thyroxine antibody (detection mode 2) The same experimental steps as detection mode 1 were used, except that the antibody complex in step 3.1-(2) was replaced with 1 μg / mL anti-thyroid antibody anti-T4-8C2 conjugated with biotinylate, and the supernatant antibody and anti-thyroid antibody binding experiment were performed. The experimental results are shown in Table 3.

[0148] 3.3 Binding assay of supernatant antibody with free thyroxine (detection mode 3) The same experimental steps as detection mode 1 were used, except that the preparation of antibody complex in step 3.1-(2) was replaced with 1 μg / mL free thyroxine. The supernatant antibody and free thyroxine binding experiment was carried out, and the experimental results are shown in Table 3.

[0149] The results showed that anti-T4-T4-9H15, anti-T4-T4-10D9, and anti-T4-T4-6J10 only bind to the T4-T4 antibody complex and do not bind to the T4 antibody or T4.

[0150] Table 3: Antibody Binding Experiment Data in Supernatant

[0151] The antibody expression supernatant of anti-T4-T4-9K13 was detected using the method described above, and the experimental results are shown in Table 3'.

[0152] Table 3': Anti-T4-T4-9K13 supernatant antibody binding experimental data

[0153] 3.5. Purification of recombinant antibodies Thirteen days later, the supernatant from the recombinant cells was collected by centrifugation. The supernatant was then purified using a Protein A affinity chromatography column to obtain purified antibodies anti-T4-T4-9H15, anti-T4-T4-10D9, and anti-T4-T4-6J10. The purity of the purified antibodies was tested by HPLC-SEC, and the results are as follows. Figures 1 to 3 As shown, the purity of antibodies anti-T4-T4-9H15, anti-T4-T4-10D9, and anti-T4-T4-6J10 is all greater than 97%.

[0154] Similarly, after 13 days, the supernatant from the recombinant cells was collected by centrifugation. The supernatant was then purified using a protein A affinity chromatography column to obtain the purified antibody anti-T4-T4-9K13. The purity of the purified antibody was tested using HPLC-SEC, and the results are as follows. Figure 10As shown, the purity of the anti-T4-T4-9K13 antibody is greater than 99%. Example 3: Antibody Performance Detection 1. Performance in chemiluminescence detection of magnetic particles 1.1 Coating process of thyroxine antibodies Wash 10 mg / mL carboxyl magnetic beads three times with MES buffer. Resuspend the beads in MES buffer, add EDC to a final concentration of 1 mg / mL, mix on a shaker at 25°C, and react for 30 min. Resuspend the beads in MES buffer again, add anti-thyroxine antibody anti-T4-8C2 to a final concentration of 0.2 mg / mL, mix on a shaker at 25°C, and react for 120 min. Store in Tris buffer at 2–8°C.

[0155] 1.2 Antibody labeling process Antibodies anti-T4-T4-9H15 and anti-T4-T4-10D9 were replaced with PBS (100 mM PB, 50 mM sodium chloride, pH 8.0) using a Zeba desalting column (10K MWCO). Acridinium ester was prepared into a 4 mM solution using DMSO. 10 mM acridinium ester was added to each of the anti-T4-T4-9H15 and anti-T4-T4-10D9 antibody solutions, and the reaction was carried out at 25°C for 2 hours. After desalting to remove excess reagents, the acridinium ester-modified anti-T4-T4-9H15 and anti-T4-T4-10D9 antibodies were stored at 4°C for later use.

[0156] 1.3 Testing Process The anti-T4-8C2 thyroxine antibody coated with magnetic beads was diluted to prepare a magnetic microparticle working solution using diluent. The acridinium ester-modified anti-T4-T4-9H15 and anti-T4-T4-10D9 antibodies were diluted to prepare an acridinium ester working solution. Detection was performed on a Shinei 2910 fully automated chemiluminescence immunoassay analyzer using a double-antibody sandwich method. Specifically, 30 μL of sample (containing different concentrations of TT4 or FT4 calibrators and different concentrations of Roche TT4 or FT4 baseline samples) and 70 μL of magnetic microparticle working solution were added sequentially to the instrument, mixed, incubated for 10 minutes, washed, and then 100 μL of acridinium ester working solution was added, mixed, and incubated for 10 minutes. After incubation, the reaction mixture was rinsed, pre-excitation and excitation solutions were added, and the relative luminescence intensity (RLU) was measured.

[0157] 1.4 Test Results Detection results of anti-T4-T4-9H15: 1.4.1 Results of total thyroxine (TT4) detection using anti-T4-T4-9H15 antibody (1) Detection results of the luminescence platform of the calibrator Table 4 shows the detection data of total thyroxine (TT4) calibrators using the antibody anti-T4-T4-9H15 on the chemiluminescence platform. The results show that the detection linearity of the antibody anti-T4-T4-9H15 on the chemiluminescence platform is very good in the range of 2-23.09 μg / dL.

[0158] Table 4: Test Results of Luminescence Platform Calibrators

[0159] (2) Correlation with Roche TT4 results Using anti-T4-T4-9H15 as the labeling antibody and anti-T4-8C2 as the coating antibody, the results of the chemiluminescence platform detection of samples for Roche TT4 determination are shown in Table 5. The clinical correlation standard curve is shown in... Figure 4 As shown in the figure. The results indicate that the antibody anti-T4-T4-9H15 has high sensitivity and clinical relevance on the chemiluminescence platform.

[0160] Table 5: Detection Results of Roche Fixed Value Samples on the Luminescence Platform

[0161] 1.4.2 Results of Anti-T4-T4-9H15 Detection of Free Thyroxine (FT4) (1) Testing working calibrators Table 6 shows the detection data of free thyroxine (FT4) calibrators using the antibody anti-T4-T4-9H15 on the chemiluminescence platform. The results show that the detection linearity of the antibody anti-T4-T4-9H15 on the chemiluminescence platform is very good in the range of 0-6.19 ng / dL.

[0162] Table 6: Test Results of Luminescence Platform Calibrators

[0163] (2) Correlation with Roche FT4 results Using anti-T4-T4-9H15 as the labeling antibody and anti-T4-8C2 as the coating antibody, the results of the Roche FT4 assay on the chemiluminescence platform are shown in Table 7. The clinical correlation standard curve is shown in... Figure 5 As shown in the figure. The results indicate that the antibody anti-T4-T4-9H15 has high sensitivity and clinical relevance on the chemiluminescence platform.

[0164] Table 7: Detection Results of Roche Fixed Value Samples on the Luminescence Platform

[0165] Detection results of anti-T4-T4-10D9: 1.4.3 Antibody anti-T4-T4-10D9 and total thyroxine (TT4) test results (1) Testing working calibrators Table 8 shows the detection data of total thyroxine (TT4) calibrators using the antibody anti-T4-T4-10D9 on the chemiluminescence platform. The results show that the detection linearity of the antibody anti-T4-T4-10D9 on the chemiluminescence platform is very good in the range of 2-23.09 μg / dL.

[0166] Table 8: Test Results of Luminescence Platform Calibrators

[0167] (2) Correlation with Roche TT4 results Using anti-T4-T4-10D9 as the labeling antibody and anti-T4-8C2 as the coating antibody, the results of the chemiluminescence platform assay for samples used to detect Roche TT4 values ​​are shown in Table 9. The clinical correlation standard curve is shown in... Figure 6 As shown in the figure. The results indicate that the use of the antibody anti-T4-T4-10D9 on the chemiluminescence platform has high sensitivity and clinical relevance.

[0168] Table 9: Detection Results of Roche Fixed Value Samples on the Luminescence Platform

[0169] 1.4.4 Anti-T4-T4-10D9 Free Thyroxine (FT4) Detection Results (1) Testing working calibrators Table 10 shows the detection data of free thyroxine (FT4) calibrators using the antibody anti-T4-T4-10D9 on the chemiluminescence platform. The results show that the detection linearity of the antibody anti-T4-T4-10D9 on the chemiluminescence platform is very good in the range of 0-6.19 ng / dL.

[0170] Table 10: Test Results of Luminescence Platform Calibrators

[0171] (2) Correlation with Roche FT4 results Using anti-T4-T4-10D9 as the labeling antibody and anti-T4-8C2 as the coating antibody, the results of the Roche FT4 assay on the chemiluminescence platform are shown in Table 11. The clinical correlation standard curve is shown in... Figure 7As shown in the figure. The results indicate that the use of the antibody anti-T4-T4-10D9 on the chemiluminescence platform has high sensitivity and clinical relevance.

[0172] Table 11: Detection Results of Roche Fixed Value Samples on the Luminescence Platform

[0173] 2. Performance testing of the fluorescence immunochromatography platform 2.1 Antibody labeling T-labeled microspheres: Add 100 μL of 1% solids fluorescent microspheres to 900 μL of activation buffer and mix well. After centrifugation and removal of supernatant, add 1 mL of activation buffer and sonicate to mix well. Then add activator, mix well in the dark by shaking for 20 min, centrifuge to remove supernatant, add a coupling buffer (MES buffer recommended, pH=6.0) of equal volume to the microspheres, mix well by sonication, add 0.1-0.2 mg of anti-thyroxine antibody anti-T4-8C2, mix well in the dark by shaking for 3 h, and finally add blocking buffer for blocking. After mixing well in the dark by shaking for 45 min, stop labeling, centrifuge to remove supernatant, reconstitute the microspheres with microsphere preservation solution, mix well by sonication, and store at 4℃ for use.

[0174] C-labeled material: Add 100 μL of 1% solids fluorescent microspheres to 900 μL of activation buffer and mix well. After centrifugation and removal of supernatant, add 1 mL of activation buffer and sonicate to mix well. Then add activator, and mix well in the dark by shaking for 20 min. After centrifugation and removal of supernatant, add an equal volume of coupling buffer (MES buffer, pH=6.5) to the microspheres and sonicate to mix well. Then add 0.4-0.8 mg of goat anti-chicken IgY labeled antibody, and mix well in the dark by shaking for 3 h. Finally, add blocking buffer for blocking, and stop labeling by shaking for 45 min in the dark. Centrifuge to remove supernatant, reconstitute the microspheres with microsphere preservation solution, sonicate to mix well, and store at 4℃ for use.

[0175] 2.2 Preparation of microsphere working solution The anti-thyroxine antibody T marker was diluted to 10-20% using microsphere diluent, and the goat anti-chicken IgY marker was diluted to 1-2%. After mixing them together, the markers were sprayed onto glass fiber using a spray pad device.

[0176] 2.3 Preparation of dried microsphere pads: Place the sprayed microsphere pads in a 50℃ oven and dry for more than 2 hours.

[0177] 2.4 Sample pad preparation: Dilute the blocking agent to 0.4 mg / ml using sample pad diluent, spread it on glass fiber, and dry it in a 50°C oven overnight.

[0178] 2.5 NC membrane coating T-line: Anti-T4-T4-6J10 and anti-T4-T4-9K13 were diluted to 0.5 mg / ml using coating dilution buffer before coating. Line C: Chicken IgY-coated antibodies were diluted to 1.0 mg / ml using coating dilution buffer before coating; After completing the T and C line scribing, place the film in a 50℃ oven to dry overnight.

[0179] 2.6 Preparation of Fluorescence Chromatography Strips Use a strip cutter to cut the fluorescence chromatography strips to the required width, assemble them, add samples, and perform detection.

[0180] 2.7 Testing Process Add samples containing different concentrations of thyroxine (different concentrations of TT4 / FT4 calibrators and different concentrations of TT4 / FT4 Roche-assigned samples) to the sample diluent, mix thoroughly, and then load the sample: load 75 μL onto a 3.5 mm wide strip, add the sample to the test card and react for 15 min, then immediately insert the test card into the instrument to read the value and calculate the T / C.

[0181] The test results for anti-T4-T4-6J10 are as follows: 2.7.1 Results of anti-T4-T4-6J10 detection of total thyroxine (TT4) (1) Testing working calibrators Table 12 shows the detection data of total thyroxine (TT4) calibrators using the antibody anti-T4-T4-6J10 on the fluorescence chromatography platform. The results show that the detection linearity of the antibody anti-T4-T4-6J10 on the fluorescence chromatography platform is very good in the range of 2-23.09 μg / dL.

[0182] Table 12: Detection Results of Calibrators for Fluorescence Chromatography Platform

[0183] (2) Correlation with Roche TT4 results Table 13 shows the clinical test results of samples with Roche TT4 values ​​when anti-T4-T4-6J10 was used as the coating antibody, and the clinical relevance is shown in the table. Figure 8 As shown in the figure. The results indicate that the antibody anti-T4-T4-6J10 has high sensitivity and clinical relevance on the fluorescence immunochromatographic platform.

[0184] Table 13: Results of Roche-calibrated sample detection on the fluorescence chromatography platform

[0185] 2.7.2 Results of Free Thyroxine (FT4) Detection (1) Testing working calibrators Table 14 shows the detection data of free thyroxine (FT4) calibrators using the antibody anti-T4-T4-6J10 on the fluorescence chromatography platform. The results show that the detection linearity of the antibody anti-T4-T4-6J10 on the fluorescence chromatography platform is very good in the range of 0-6.19 ng / dL.

[0186] Table 14: Detection Results of Fluorescence Chromatography Platform Calibrators

[0187] (2) Correlation with Roche FT4 results The clinical test results of samples with Roche FT4 values ​​when anti-T4-T4-6J10 was used as the coating antibody are shown in Table 15. The clinical relevance is as follows: Figure 9 As shown in the figure. The results indicate that the antibody anti-T4-T4-6J10 has high sensitivity and clinical relevance on the fluorescence immunochromatographic platform.

[0188] Table 15: Results of Roche Value Detection on Fluorescence Chromatography Platform

[0189] The test results for anti-T4-T4-9K13 are as follows: 2.7.3 Results of Free Thyroxine (FT4) Detection (1) Testing working calibrators Table 16 shows the detection data of free thyroxine (FT4) calibrators using the antibody anti-T4-T4-9K13 on the fluorescence chromatography platform. The results show that the detection linearity of the antibody anti-T4-T4-9K13 on the fluorescence chromatography platform is very good in the range of 0-8 ng / dL.

[0190] Table 16: Detection Results of Fluorescence Chromatography Platform Calibrators

[0191] (2) Correlation with Roche FT4 results The clinical test results of samples with Roche FT4 values ​​when anti-T4-T4-9K13 was used as the coating antibody are shown in Table 17. The clinical relevance is as follows: Figure 11 As shown in the figure. The results indicate that the antibody anti-T4-T4-9K13 has high sensitivity and clinical relevance on the fluorescence immunochromatographic platform.

[0192] Table 17: Results of Roche Value Detection on Fluorescence Chromatography Platform Sample number Roche FT4 value (ng / dL) Anti-T4-T4-9K13 measurement (ng / dL) 1 0.46 0.391 2 0.54 0.505 3 0.55 0.484 4 0.58 0.452 5 0.66 0.494 6 0.66 0.877 7 0.7 0.56 8 0.71 0.634 9 0.71 0.543 10 0.76 0.617 11 0.77 0.663 12 0.78 0.704 13 0.79 0.762 14 0.8 0.684 15 0.82 0.779 16 0.82 0.803 17 0.82 0.718 18 0.83 0.788 19 0.85 0.539 20 0.85 0.734 21 0.86 0.828 22 0.86 0.857 23 0.86 0.761 24 0.86 0.812 25 0.91 0.558 26 0.93 0.917 27 1.02 1.194 28 1.03 1.106 29 1.04 1.041 30 1.04 0.88 31 1.07 1.105 32 1.11 1.05 33 1.25 0.956 34 1.26 1.276 35 1.3 1.282 36 1.35 1.149 37 1.39 1.25 38 1.39 1.152 39 1.4 1.344 40 1.4 1.273 41 1.43 1.119 42 1.49 1.427 43 1.5 1.092 44 1.7 1.231 45 1.72 1.538 46 1.72 1.641 47 1.82 1.514 48 1.85 2.095 49 1.86 2.15 50 1.88 1.144 51 2.03 1.618 52 2.04 1.823 53 2.07 2.257 54 2.08 1.464 55 2.08 1.943 56 2.11 2.045 57 2.14 2.015 58 2.14 2.158 59 2.16 2.118 60 2.17 1.734 61 2.17 2.194 62 2.19 1.499 63 2.27 2.836 64 2.29 2.389 65 2.33 2.21 66 2.33 2.181 67 2.39 2.018 68 2.45 2.157 69 2.45 2.764 70 2.64 2.957 71 3.04 3.602 72 3.22 3.397 73 3.23 3.373 74 3.25 3.508 75 3.35 4.131 76 4.09 3.794 77 4.11 3.802 78 4.13 3.872 79 4.15 3.587 80 4.2 4.313 81 4.2 3.645 82 4.41 3.82 83 4.41 4.231 84 4.47 4.377 85 4.49 3.947 86 4.55 5.545 87 4.62 4.705 88 4.68 5.204 89 4.76 4.597 90 4.79 5.046 91 4.8 4.882 92 4.95 4.679 93 5.19 4.692 94 5.23 6.038 95 5.26 5.449 96 5.27 5.026 97 5.29 5.358 98 5.3 5.293 99 5.32 5.942 100 5.32 5.292 101 5.36 5.632 102 5.39 5.487 103 5.48 4.591 104 5.5 5.503 105 5.52 4.536 106 5.66 5.362 107 5.74 5.126 108 5.9 4.89 109 5.99 5.455 110 6.11 5.188 111 6.11 5.103 112 6.11 4.784 113 6.14 5.14 114 6.26 5.341 115 6.35 5.464 116 6.37 5.718 117 6.39 6.124 118 6.64 5.585 119 6.65 5.907 120 6.67 6.078 121 6.69 6.032 The partial amino acid sequences involved in this application are shown in Table 18: Table 18: Amino Acid Sequence List

[0194] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An antibody that binds to a T4-T4 antibody complex, characterized in that, The antibody comprises three complementary determinant regions of the heavy chain variable region as shown in any of SEQ ID NO: 19, 20, 21, 41 and three complementary determinant regions of the light chain variable region as shown in any of SEQ ID NO: 22, 23, 24, 43. Optionally, the complementary determination region of the variable region is defined by any one or a combination of systems such as Kabat, Chothia, IMGT, AbM, or Contact.

2. An antibody that binds to a T4-T4 antibody complex, comprising a complementarity-determining region, characterized in that, The complementary determination region includes any one selected from (a) to (d): (a) HCDR1 with amino acid sequences as shown in SEQ ID NO:1 (SYGVG), HCDR2 with amino acid sequences as shown in SEQ ID NO:2 (GVEAGGYRGYNPTLKS), HCDR3 with amino acid sequences as shown in SEQ ID NO:3 (VPLGYGHGYGRLDY), and LCDR1 with amino acid sequences as shown in SEQ ID NO:4 (SGSSSNVGYGDYVG), LCDR2 with amino acid sequences as shown in SEQ ID NO:5 (DTTSRAA), and LCDR3 with amino acid sequences as shown in SEQ ID NO:6 (ASYDDDSYGV); (b) HCDR1 with amino acid sequences as shown in SEQ ID NO:7 (SYTVG), HCDR2 with amino acid sequences as shown in SEQ ID NO:8 (NIESGGYTVYNPALKS), HCDR3 with amino acid sequences as shown in SEQ ID NO:9 (ENAVGGGDYDYDYYIDY), and LCDR1 with amino acid sequences as shown in SEQ ID NO:10 (SGSSSNVGYGNYVS), LCDR2 with amino acid sequences as shown in SEQ ID NO:11 (GATSRAS), and LCDR3 with amino acid sequences as shown in SEQ ID NO:12 (ASYDSNSNI); (c) HCDR1 with amino acid sequences as shown in SEQ ID NO:13 (SHGVG), HCDR2 with amino acid sequences as shown in SEQ ID NO:14 (GRENGGYAYYNSALKS), HCDR3 with amino acid sequences as shown in SEQ ID NO:15 (NSDGGIFLNNIDY), and LCDR1 with amino acid sequences as shown in SEQ ID NO:16 (TGTSTDIGAWDGVG), LCDR2 with amino acid sequences as shown in SEQ ID NO:17 (NVDKRPS), and LCDR3 with amino acid sequences as shown in SEQ ID NO:18 (GSPKSDYTIV); and (d) HCDR1 with amino acid sequences as shown in SEQ ID NO:35 (SNTID), HCDR2 with amino acid sequences as shown in SEQ ID NO:36 (GIDWAGDTGYNPALKS), HCDR3 with amino acid sequences as shown in SEQ ID NO:37 (GPRYYGRDSYGYSYLDH), and LCDR1 with amino acid sequences as shown in SEQ ID NO:38 (SGSSSNVGYGDYVG), LCDR2 with amino acid sequences as shown in SEQ ID NO:39 (DATSRAS), and LCDR3 with amino acid sequences as shown in SEQ ID NO:40 (ASYDSNFYRV).

3. An antibody that binds to a T4-T4 antibody complex, comprising a heavy chain variable region and a light chain variable region, characterized in that, The heavy chain variable region and light chain variable region of the antibody are selected from any one of (a') to (d'): (a') A heavy chain variable region having at least 80% identity with SEQ ID NO:19, a light chain variable region having at least 80% identity with SEQ ID NO:22, and including the complementarity-determining region shown in claim 2(a); (b') A heavy chain variable region having at least 80% identity with SEQ ID NO:20, a light chain variable region having at least 80% identity with SEQ ID NO:23, and including the complementarity-determining region shown in claim 2 (b); (c') A heavy chain variable region having at least 80% identity with SEQ ID NO:21, a light chain variable region having at least 80% identity with SEQ ID NO:24, and including the complementarity-determining region shown in claim 2 (c); and (d') The heavy chain variable region having at least 80% identity with the amino acid sequence of SEQ ID NO:41, the light chain variable region having at least 80% identity with the amino acid sequence of SEQ ID NO:43, and including the complementarity determining region shown in (d) of claim 2.

4. An antibody that binds to a T4-T4 antibody complex, comprising a heavy chain variable region and a light chain variable region, characterized in that, The amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NO: 19, 20, 21, and 41; The amino acid sequences of the light chain variable region are as shown in any one of SEQ ID NO: 22, 23, 24, and 43; Optionally, the combination of the heavy chain variable region and the light chain variable region is selected from any of the following combinations: 。 5. The antibody according to any one of claims 1-4, characterized in that, The antibody also includes a constant region; Optionally, the constant region includes a heavy chain constant region and / or a light chain constant region; Optionally, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments; Optionally, the heavy chain constant region includes CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM, CH4 of IgM and / or the tail peptide of IgM. Optionally, the light chain constant region includes a light chain constant region selected from κ-type or λ-type; Optionally, the species source of the constant region is any one of cattle, horses, pigs, sheep, goats, rats, mice, dogs, camels, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese, or humans; Optionally, the species source of the constant region is sheep; Optionally, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO:31 or has at least 80% identity with it; Optionally, the amino acid sequence of the light chain constant region is as shown in SEQ ID NO:32 or has at least 80% identity with it.

6. An antibody that binds to a T4-T4 antibody complex, comprising a heavy chain and a light chain, characterized in that, The amino acid sequence of the heavy chain is shown in any one of SEQ ID NO: 25, 26, 27, 42; the amino acid sequence of the light chain is shown in any one of SEQ ID NO: 28, 29, 30, 44.

7. An antibody conjugate, characterized in that, It includes the antibody as described in any one of claims 1-6 and the conjugated portion thereof; Optionally, the coupling portion includes at least one of a purification tag, an affinity substance, a solid-phase carrier, or a marker; Optionally, the purification tag includes at least one selected from His tag, Flag tag, GST tag, MBP tag, SUMO tag or C-Myc tag; Optionally, the affinity substance includes at least one selected from biotin or streptavidin; Optionally, the solid support comprises at least one selected from microspheres, plates, or membranes; Optionally, the marker includes at least one selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, or nanoparticle markers.

8. A reagent or kit, characterized in that, The reagent or kit comprises the antibody as described in any one of claims 1-6 or the antibody conjugate as described in claim 7.

9. The use of the antibody as described in any one of claims 1-6, or the antibody conjugate as described in claim 7, or the reagent or kit as described in claim 8 in the preparation of a product for detecting T4 or a product for detecting T4-T4 antibody complex; Optionally, the application includes: a) Under conditions sufficient to induce antibody / antigen binding, T4 in the test sample is brought into contact with T4 antibody to form immune complex 1; and b) Contacting the antibody of any one of claims 1-6, or the antibody conjugate of claim 7, or the reagent or kit of claim 8 with the immune complex 1 in a) to form immune complex 2; and c) Detect the presence of immune complex 2, the presence of complex 2 indicating the presence of T4 in the test sample.

10. A nucleic acid molecule, a vector, a cell, or a method for preparing an antibody according to any one of claims 1-6, wherein the nucleic acid molecule encodes an antibody according to any one of claims 1-6; the vector contains a nucleic acid molecule encoding an antibody according to any one of claims 1-6; the cell contains the aforementioned nucleic acid molecule or vector; and the method comprises culturing the aforementioned cell.

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