Monoclonal antibody against triiodothyronine, detection reagent based thereon and application thereof

Abstract

The present invention relates to a monoclonal antibody or an antigen-binding fragment thereof targeting triiodothyronine (T3), and related nucleic acid products, cell products, detection reagents or kits, and preparation methods and applications thereof. The monoclonal antibody or antigen-binding fragment thereof of the present invention can bind to T3 with high specificity and high affinity, thereby improving the detection accuracy and sensitivity of T3; and the present invention also develops a high-performance paired antibody suitable for double antibody sandwich immunoassay, thereby breaking through the limitations of low sensitivity and poor clinical relevance of traditional competitive detection reagents, and realizing double antibody sandwich immunoassay of T3, which is of great significance for the early diagnosis and treatment of thyroid diseases and the domestic substitution of core antibody raw materials.

Description

[0001] This divisional application is a divisional application based on the Chinese patent application with application number 2024101554569, application date February 2, 2024, and invention name “Monoclonal antibodies against triiodothyronine, detection reagents based thereon and their applications”. Technical Field

[0002] The present invention relates to the technical field of monoclonal antibody preparation and immunological detection, and in particular to a monoclonal antibody targeting triiodothyronine, a detection reagent based thereon and application thereof. Background Art

[0003] Triiodothyronine (T3) is a hormone secreted by the thyroid gland. In the human body, about 65% is produced directly by the thyroid gland, and 35% is formed by deiodination of thyroid hormone (T4) in peripheral tissues. Its molecular weight is about 650Da.

[0004] T3 is an important clinical indicator for evaluating thyroid function, and has important clinical significance for the diagnosis and efficacy testing of thyroid diseases such as hyperthyroidism and hypothyroidism. Part of the T3 in the body is synthesized from T4. The two differ in structure by only one iodine, so the specificity of the antibody is required to be high. At present, most T3 antibodies on the market have cross-reaction problems. Existing T3 immunoassay methods mainly include chemiluminescence and immunochromatography, and these immunoassay methods mainly use the principle of competition, which is lacking in detection specificity and sensitivity, and the linear range is narrow, resulting in a small degree of distinction between the low and high ends of clinical samples, thereby causing misjudgment of the test results.

[0005] In view of the above-mentioned problems existing in the existing technology, there is an urgent need to develop highly specific and high-affinity monoclonal antibodies against T3 to meet the needs of immunological detection, especially the development of high-performance paired antibodies to break through the limitations of traditional competitive methods and realize the double antibody sandwich method for detecting T3. Summary of the invention

[0006] Purpose of the Invention

[0007] In view of the problems or needs existing in the prior art, the purpose of the present invention is to provide a highly specific and high-affinity monoclonal antibody or its antigen-binding fragment against triiodothyronine (T3 in English), and to develop a high-performance paired antibody suitable for double antibody sandwich immunological detection.

[0008] Solution

[0009] In order to achieve the above objectives, the present invention has obtained, through a large number of screenings, highly specific and high affinity monoclonal antibodies or antigen-binding fragments thereof against triiodothyronine (T3 in English), their related nucleic acids and cell products, their preparation methods and applications, and immunoassay reagents based thereon, thereby providing a basis for highly specific and highly sensitive immunoassays of T3.

[0010] Specifically, the present invention provides the following technical solutions:

[0011] In a first aspect, the present invention provides a monoclonal antibody or an antigen-binding fragment thereof against triiodothyronine, characterized in that the monoclonal antibody or the antigen-binding fragment thereof comprises:

[0012] (1) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO:6, QAS and SEQ ID NO:7, respectively; or,

[0013] (2) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 15, LAS and SEQ ID NO: 16, respectively; or,

[0014] (3) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 24, LAS and SEQ ID NO: 25, respectively; or,

[0015] (4) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO:33, YAS and SEQ ID NO:34, respectively; or,

[0016] (5) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:37, SEQ ID NO:38 and SEQ ID NO:39, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO:42, SAS and SEQ ID NO:43, respectively; or,

[0017] (6) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:46, SEQ ID NO:47 and SEQ ID NO:48, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO:51, SAS and SEQ ID NO:52, respectively; or,

[0018] (7) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 as shown in the amino acid sequences of SEQ ID NO:55, SEQ ID NO:56 and SEQ ID NO:57, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 as shown in the amino acid sequences of SEQ ID NO:60, DAS and SEQ ID NO:61, respectively; or,

[0019] (8) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 as shown in the amino acid sequences of SEQ ID NO:64, SEQ ID NO:65 and SEQ ID NO:66, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 as shown in the amino acid sequences of SEQ ID NO:69, DAS and SEQ ID NO:70, respectively; or,

[0020] (9) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 as shown in the amino acid sequences of SEQ ID NO:73, SEQ ID NO:74 and SEQ ID NO:75, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 as shown in the amino acid sequences of SEQ ID NO:78, DAS and SEQ ID NO:79, respectively;

[0021] (10) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:82, SEQ ID NO:83 and SEQ ID NO:84, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO:87, DAS and SEQ ID NO:88, respectively.

[0022] Preferably, the monoclonal antibody or antigen-binding fragment thereof comprises:

[0023] (1) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:4; and a light chain variable region having the amino acid sequence shown in SEQ ID NO:8; or,

[0024] (2) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 13; and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 17; or,

[0025] (3) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 22; and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 26; or,

[0026] (4) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 31; and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 35; or,

[0027] (5) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:40; and a light chain variable region having the amino acid sequence shown in SEQ ID NO:44; or,

[0028] (6) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:49; and a light chain variable region having the amino acid sequence shown in SEQ ID NO:53; or,

[0029] (7) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 58; and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 62; or,

[0030] (8) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 67; and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 71; or,

[0031] (9) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 76; and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 80; or,

[0032] (10) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:85; and a light chain variable region having the amino acid sequence shown in SEQ ID NO:89.

[0033] In addition, the monoclonal antibody or antigen-binding fragment thereof further comprises a constant region; preferably, the constant region is any one selected from the following: a constant region of an IgG, IgA or IgM antibody.

[0034] Further preferably, the monoclonal antibody or antigen-binding fragment thereof comprises:

[0035] (1) a heavy chain having the amino acid sequence shown in SEQ ID NO:5; and a light chain having the amino acid sequence shown in SEQ ID NO:9; or,

[0036] (2) a heavy chain having the amino acid sequence shown in SEQ ID NO: 14; and a light chain having the amino acid sequence shown in SEQ ID NO: 18; or,

[0037] (3) a heavy chain having the amino acid sequence shown in SEQ ID NO: 23; and a light chain having the amino acid sequence shown in SEQ ID NO: 27; or,

[0038] (4) a heavy chain having the amino acid sequence shown in SEQ ID NO: 32; and a light chain having the amino acid sequence shown in SEQ ID NO: 36; or,

[0039] (5) a heavy chain having the amino acid sequence shown in SEQ ID NO:41; and a light chain having the amino acid sequence shown in SEQ ID NO:45; or,

[0040] (6) a heavy chain having the amino acid sequence shown in SEQ ID NO: 50; and a light chain having the amino acid sequence shown in SEQ ID NO: 54; or,

[0041] (7) a heavy chain having the amino acid sequence shown in SEQ ID NO: 59; and a light chain having the amino acid sequence shown in SEQ ID NO: 63; or,

[0042] (8) a heavy chain having the amino acid sequence shown in SEQ ID NO: 68; and a light chain having the amino acid sequence shown in SEQ ID NO: 72; or,

[0043] (9) a heavy chain having the amino acid sequence shown in SEQ ID NO: 77; and a light chain having the amino acid sequence shown in SEQ ID NO: 81; or,

[0044] (10) a heavy chain having the amino acid sequence shown in SEQ ID NO:86; and a light chain having the amino acid sequence shown in SEQ ID NO:90.

[0045] In the above first aspect, the monoclonal antibodies defined in items (1) to (10) are hereinafter respectively named as antibodies 1 to 10.

[0046] In some feasible embodiments, the antigen-binding fragment of the monoclonal antibody is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment, single chain antibody, human antibody, chimeric antibody or bispecific or multispecific antibody.

[0047] In a second aspect, the present invention provides a polynucleotide encoding the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect. The polynucleotide is not limited by the method of its production and can be obtained by genetic engineering recombination technology or chemical synthesis method.

[0048] In a third aspect, the present invention provides a nucleic acid construct comprising the polynucleotide as described in the second aspect above, and, optionally, at least one expression control element operably linked to the polynucleotide.

[0049] In a fourth aspect, the present invention provides a recombinant vector comprising the polynucleotide as described in the second aspect above, or the nucleic acid construct as described in the third aspect above.

[0050] The vector of the present invention may be a cloning vector or an expression vector, for example, it may be a plasmid, a cosmid, a phage, etc.

[0051] In some preferred embodiments, the recombinant vector is a recombinant expression vector, preferably a eukaryotic expression vector.

[0052] In a fifth aspect, the present invention provides a transformed host cell, wherein the polynucleotide as described in the second aspect, the nucleic acid construct as described in the third aspect, or the recombinant vector as described in the fourth aspect is transformed;

[0053] The host cell includes, but is not limited to, prokaryotic cells, such as Escherichia coli cells, eukaryotic cells, such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). The host cell can also be a cell line, such as a 293T cell line.

[0054] Preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell.

[0055] In a sixth aspect, the present invention provides use of the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect, the polynucleotide as described in the second aspect, the nucleic acid construct as described in the third aspect, the recombinant vector as described in the fourth aspect and / or the transformed host cell as described in the fifth aspect in the preparation of a detection reagent or kit for detecting triiodothyronine.

[0056] In a feasible embodiment, the sample is a biological sample of a subject, preferably a serum or plasma sample of a subject.

[0057] In a seventh aspect, the present invention provides the use of any two monoclonal antibodies or antigen-binding fragments thereof selected from the monoclonal antibodies or antigen-binding fragments thereof as described in the first aspect above in the preparation of a detection reagent or kit for detecting triiodothyronine.

[0058] Preferably, the two monoclonal antibodies or antigen-binding fragments thereof are selected from:

[0059] (I) Antibody 1 or an antigen-binding fragment thereof, and Antibody 2 or an antigen-binding fragment thereof;

[0060] (II) Antibody 3 or an antigen-binding fragment thereof, and Antibody 4 or an antigen-binding fragment thereof;

[0061] Wherein, antibody 1 or its antigen-binding fragment is defined by any item (1) of the above-mentioned first aspect, antibody 2 or its antigen-binding fragment is defined by any item (2) of the above-mentioned first aspect, antibody 3 or its antigen-binding fragment is defined by any item (3) of the above-mentioned first aspect, and antibody 4 or its antigen-binding fragment is defined by any item (4) of the above-mentioned first aspect.

[0062] In an eighth aspect, the present invention provides a detection reagent for detecting triiodothyronine, comprising the monoclonal antibody or its antigen-binding fragment as described in the first aspect above, the polynucleotide as described in the second aspect above, the nucleic acid construct as described in the third aspect above, the recombinant vector as described in the fourth aspect above and / or the transformed host cell as described in the fifth aspect above.

[0063] Preferably, the detection reagent is a double antibody sandwich immunoassay kit, which comprises any two monoclonal antibodies or antigen-binding fragments thereof selected from the monoclonal antibodies or antigen-binding fragments thereof described in the first aspect above, preferably comprises two monoclonal antibodies or antigen-binding fragments thereof selected from the following:

[0064] (I) Antibody 1 or an antigen-binding fragment thereof, and Antibody 2 or an antigen-binding fragment thereof;

[0065] (II) Antibody 3 or an antigen-binding fragment thereof, and Antibody 4 or an antigen-binding fragment thereof;

[0066] Wherein, antibody 1 or its antigen-binding fragment is defined by any item (1) of the above-mentioned first aspect, antibody 2 or its antigen-binding fragment is defined by any item (2) of the above-mentioned first aspect, antibody 3 or its antigen-binding fragment is defined by any item (3) of the above-mentioned first aspect, and antibody 4 or its antigen-binding fragment is defined by any item (4) of the above-mentioned first aspect.

[0067] In a feasible embodiment, the double antibody sandwich immunoassay kit comprises an immunochromatographic test strip, which is based on the principle of the double antibody sandwich method and uses any two monoclonal antibodies or antigen-binding fragments thereof selected from the monoclonal antibodies or antigen-binding fragments thereof described in the first aspect as capture antibodies and detection antibodies, respectively;

[0068] In some preferred embodiments, the immunochromatographic test strip uses any one of (1) antibody 1 or its antigen-binding fragment and (2) antibody 2 or its antigen-binding fragment as a capture antibody, and uses the other as a detection antibody; further preferably, the immunochromatographic test strip uses antibody 1 or its antigen-binding fragment as a detection antibody, and uses antibody 2 or its antigen-binding fragment as a capture antibody;

[0069] In other preferred embodiments, the immunochromatographic test strip uses any one of (1) antibody 3 or its antigen-binding fragment and (2) antibody 4 or its antigen-binding fragment as a capture antibody, and uses the other one as a detection antibody; further preferably, the immunochromatographic test strip uses antibody 3 or its antigen-binding fragment as a detection antibody, and uses antibody 4 or its antigen-binding fragment as a capture antibody;

[0070] Preferably, the monoclonal antibody or antigen-binding fragment thereof used as the detection antibody is labeled with a detectable marker; such detectable markers are well known to those skilled in the art, and include, but are not limited to, radioactive isotopes, fluorescent substances, luminescent substances, colored substances and enzymes (e.g., horseradish peroxidase), etc.

[0071] In a preferred embodiment, the detectable marker is fluorescent microspheres, latex microspheres or colloidal gold particles, preferably colloidal gold particles.

[0072] In a ninth aspect, the present invention provides a method for preparing the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect above, the method comprising: allowing the transformed host cell as described in the fifth aspect above to express the monoclonal antibody or antigen-binding fragment thereof under conditions suitable for the expression of the monoclonal antibody or antigen-binding fragment thereof, and recovering the expressed monoclonal antibody or antigen-binding fragment thereof from the culture of the host cell.

[0073] In the tenth aspect, the present invention provides a method for detecting the presence or level of triiodothyronine in a sample, the method comprising using the monoclonal antibody or its antigen-binding fragment as described in the first aspect above, the polynucleotide as described in the second aspect above, the nucleic acid construct as described in the third aspect above, the expression vector as described in the fourth aspect above, the transformed host cell as described in the fifth aspect above and / or the detection reagent as described in the eighth aspect above.

[0074] In a feasible embodiment, the sample includes but is not limited to serum, plasma, etc. from a subject.

[0075] The method can be used for diagnostic purposes (eg, the sample is a sample from a patient), or for non-diagnostic purposes (eg, the sample is a cell sample rather than a sample from a patient).

[0076] The general method of using monoclonal antibodies or their antigen binding fragments to detect the presence of target antigens in samples or their levels is well known to those skilled in the art. In certain preferred embodiments, the detection method can use enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay, chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography, competitive method and similar detection methods.

[0077] Beneficial Effects

[0078] The monoclonal antibody against triiodothyronine of the present invention can bind to triiodothyronine with high affinity and high specificity (the EC50 of its binding activity reaches the ng / ml level, and the KD is 10 -10 M~10 -11 M), achieving high-accuracy and high-sensitivity detection of triiodothyronine; and, the present invention also develops a high-performance paired antibody suitable for double-antibody sandwich immunological detection, thereby breaking through the limitations of low sensitivity and poor clinical relevance of traditional competitive detection reagents, and achieving double-antibody sandwich immunological detection of triiodothyronine. The detection method has higher specificity and sensitivity, and its clinical compliance rate is above 0.95. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] One or more embodiments are exemplarily described by the pictures in the accompanying drawings, and these exemplary descriptions do not constitute limitations on the embodiments. The special word "exemplary" here means "used as an example, embodiment or illustrative". Any embodiment described as "exemplary" here is not necessarily interpreted as being superior or better than other embodiments.

[0080] Figure 1 This is a gel image of SDS-PAGE detection of 10 anti-T3 monoclonal antibodies expressed and purified in Example 3 of the present invention; wherein M represents the electrophoresis band of the molecular weight marker, lane 1 shows the electrophoresis band of each monoclonal antibody under reducing conditions, and lane 2 shows the electrophoresis band of each monoclonal antibody under non-reducing conditions.

[0081] Figure 2 This is a graph showing the binding activity test results of the 10 anti-T3 monoclonal antibodies expressed and purified in Example 3 of the present invention and the T3 coupling protein, as tested in Example 4.

[0082] Figure 3 This is a structural diagram of the colloidal gold immunochromatographic test strip prepared in Example 5 of the present invention; wherein 1 is a PVC base plate; 2 is a sample pad; 3 is a nitrocellulose membrane; 4 is a gold line; 5 is a detection line; 6 is a quality control line; and 7 is a water absorbent pad.

[0083] Figure 4 The result diagram of the correlation between the two colloidal gold immunochromatographic test strips based on the two paired antibodies of the present invention and the clinical samples of the Roche detection reagent, as tested in Example 6; wherein, Figure 4 A is the result diagram of colloidal gold immunochromatographic test strip 1 based on T3mAb1+T3mAb2 paired antibodies, Figure 4 B is a graph showing the results of colloidal gold immunochromatographic test strip 2 based on the T3mAb3+T3mAb4 paired antibodies. DETAILED DESCRIPTION

[0084] Unless explicitly stated otherwise, throughout the specification and claims, the term “comprise” or variations such as “include” or “comprising”, etc., will be understood to include the stated elements or components but not to exclude other elements or components.

[0085] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

[0086] In order to make the present invention more easily understood, some technical terms are specifically defined as follows. Unless otherwise clearly defined in other parts of this document, the technical terms used herein have the meanings commonly understood by ordinary technicians in the field to which the present invention belongs.

[0087] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value, including but not limited to ±5%, ±2%, ±1%, and ±0.1%, as such variations are appropriate for performing the disclosed methods.

[0088] The term "and / or" should be understood to mean any one of the optional items or a combination of any two or more of the optional items.

[0089] As used herein, the term "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, that is, including at least one of the numbers or elements in the list, but also including more than one, and optionally, additional unlisted items. Only when terms are clearly indicated to the contrary, such as "only one" or "exactly one" or when "consisting of..." is used in the claims, it will refer to only one number listed or one element of the list.

[0090] The term "percent (%) amino acid sequence identity" or simply "identity" is defined as the percentage of amino acid residues in a candidate amino acid sequence that are identical to the amino acid residues in a reference amino acid sequence, after the amino acid sequences are aligned (and gaps introduced where necessary) to obtain the maximum percent sequence identity, and any conservative substitutions are not considered part of the sequence identity. Sequence alignments can be performed using various methods in the art to determine percent amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignments, including any algorithm required to achieve maximum alignment over the entire length of the compared sequences.

[0091] The term "antibody" refers to any form of antibody having the desired biological activity. Therefore, it is used in the broadest sense, specifically including but not limited to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies and camelized single domain antibodies.

[0092] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0093] "Antigen binding fragment" refers to an antigen binding fragment of an antibody and antibody analogs, which generally include at least a portion of the antigen binding region or variable region of the parent antibody, such as one or more CDRs. The antibody fragment retains at least some of the binding specificity of the parent antibody. Antigen binding fragments include those selected from Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, diabodies, peptides containing CDRs, etc.

[0094] The "Fab fragment" consists of a light chain and the CH1 and variable regions of a heavy chain.

[0095] The "Fc" region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic interaction of the CH3 domains.

[0096] A "Fab' fragment" contains a light chain and a portion of a heavy chain including a VH domain, a CH1 domain, and a portion of the constant region between the CH1 and CH2 domains, with an interchain disulfide bond formed between the two heavy chains of the two Fab' fragments to form a F(ab')2 molecule.

[0097] A "F(ab')2 fragment" contains two light chains and a portion of two heavy chains including a VH domain, a CH1 domain, and a portion of the constant region between the CH1 and CH2 domains, whereby an interchain disulfide bond is formed between the two heavy chains. Thus, a F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0098] The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[0099] "Single-chain Fv antibody (scFv antibody)" refers to an antigen-binding fragment comprising the VH and VL domains of an antibody, which domains are contained in a single polypeptide chain. Generally speaking, the scFv polypeptide comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0100] "Diabodies" are small antigen-binding fragments with two antigen-binding sites. The fragments contain a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are allowed to pair with the complementary domains of another chain and form two antigen-binding sites.

[0101] "Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can be generally represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art.

[0102] The term "high affinity" for IgG antibodies refers to a KD for the antigen of 1.0 × 10 -9 M or lower. For other antibody subtypes, "high affinity" binding may vary.

[0103] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in either single- or double-stranded form.

[0104] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0105] Example 1: Preparation of monoclonal antibodies against T3

[0106] (1) Animal immunization:

[0107] The coupled protein T3-BSA (purchased from Nanjing Mingyan Biotechnology Co., Ltd., product number MY-T3-003) was used as an antigen, and after being fully emulsified with an equal amount of Freund's complete adjuvant, it was subcutaneously injected to immunize rabbits (species: New Zealand white rabbits, female, 6-8 weeks old), and booster immunization was performed at intervals of two weeks, for a total of 3 immunizations. 5-8 days after the last booster immunization, blood was collected from the rabbit vein, and the serum titer was detected by ELISA. When the serum titer reached the standard of >128K, peripheral blood mononuclear cell (PBMC) suspension was prepared;

[0108] (2) PBMC preparation:

[0109] Add Ficoll, a lymphocyte separation solution, to the centrifuge tube, take anticoagulated peripheral blood (whole blood) and sterile PBS and mix thoroughly at a ratio of 1:1-1:5, use a pipette to slowly superimpose on the stratified liquid surface along the tube wall, centrifuge horizontally at 400g×30 minutes, remove part of the upper liquid after centrifugation, and about 1mL is left. Use a pipette to insert into the cloud layer to absorb the mononuclear cells. Place in another centrifuge tube, add more than 5 times the volume of PBS, centrifuge at 300g×10 minutes, and wash the cells twice. After the last centrifugation, discard the supernatant, add red blood cell lysis solution, incubate at room temperature for 2 minutes, lyse the red blood cells, add 10mL PBS, centrifuge at 300g×10 minutes, and wash the cells twice. Discard the supernatant, add RPMI1640 containing 10% FBS, resuspend the cells, and count;

[0110] (3) Obtaining single B cells:

[0111] The prepared PBMCs are used to label antigens and perform cell screening using a single-cell chip screening platform to obtain antigen-specific B cells.

[0112] Through the above steps, a total of 10 monoclonal antibodies against T3 were obtained, which were identified as IgG antibodies, respectively referred to as T3 mAb1 to T3 mAb10. In the following examples, the light and heavy chain gene sequences of these 10 monoclonal antibodies were detected, and expression, purification and binding activity detection were performed.

[0113] Example 2: Obtaining the light and heavy chain gene sequences of monoclonal antibodies against T3

[0114] cDNA preparation:

[0115] RNA was extracted from the 10 B cells obtained by sorting in Example 1, and reverse transcription was performed using RNA as a template to obtain cDNA, and the genes of the light and heavy chains of the antibody were amplified by PCR;

[0116] Light, heavy chain gene acquisition:

[0117] Amplification was performed using rabbit-specific primers, and the amplified DNA sequences were cloned into vectors, respectively, and then transformed and plated, clones were picked, plasmids were extracted and sequenced, and the light and heavy chain gene sequences of 10 antibodies were obtained; based on the measured light and heavy chain gene sequences of each antibody, the amino acid sequences of the light and heavy chains of each antibody were deduced; specifically, the heavy chain amino acid sequences of antibodies T3mAb1 to T3 mAb10 are shown in SEQ ID NOs: 5, 14, 23, 32, 41, 50, 59, 68, 77, and 86, respectively, and the light chain amino acid sequences are shown in SEQ ID NOs: 9, 18, 27, 36, 45, 54, 63, 72, 81, and 90, respectively;

[0118] Analysis of light and heavy chain variable region gene sequences:

[0119] VH and VL were analyzed according to the sequencing results, and finally the amino acid sequences of the light chain variable region and the heavy chain variable region were obtained.

[0120] The amino acid sequences of the heavy chain variable regions and light chain variable regions of the 10 monoclonal antibodies are shown below, wherein the CDR regions are underlined.

[0121] T3 mAb1:

[0122] VH (SEQ ID NO:4)

[0123] QSLEESGGDLVKPGASLTLTCTAS GFTLSSAW ICWVRQAPGKGLEWIAC IVGGSSGNT YDATWAKGRFTIPKTSSTTVTLQMTSLTAADTATYFC OUR FRIENDS WGPGTLVTVSS;

[0124] VL (SEQ ID NO: 8)

[0125] DVVMTQTPASVSEPVGGTVTIKCQAN QNIYSL LAWYQQKPGQPPKLLIY QAS KLASGVPSRFKGSGSGTEYTLTISDLECADAATYYC QNDYGISSYGHA FGGGTEVVVK; T3 mAb2:

[0126] VH (SEQ ID NO: 13)

[0127] QSLEESGGRLVTPGTPLTLTCTAS GFSISSYY MSWVRQAPGEGLEWIAM INIGGRA YYANWALGRFTISRTSTTVDLKMTSPTTEDTATYFC VRRVGDSGLYDL WGQGTLVTVSS;

[0128] VL (SEQ ID NO: 17)

[0129] DVVMTQTPASVEAAVGGTVTIKCQAS QNIYSW LSWYQQKPGQPPKLLIY LAS TLASGVPSRFKGSGSGTEYTLTISDLECADAATYYC SYMPTOMS FGGGTEVVVK;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0130] <h2 style=";text-align:left;direction:ltr"> T3 mAb3:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0131] <h2 style=";text-align:left;direction:ltr"> VH(SEQ ID NO:22)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0132] <h2 style=";text-align:left;direction:ltr"> QSVEESGGRLVTPGTPLTLTCTVS<h2 style=";text-align:left;direction:ltr"> GIDLSNYA <h2 style=";text-align:left;direction:ltr"> MGWVRQAPGKGLEYIGF<h2 style=";text-align:left;direction:ltr"> INTGGSA <h2 style=";text-align:left;direction:ltr"> YYANWAKGRFTISKTSSTTVDLKMTSLTTEDTATYFC<h2 style=";text-align:left;direction:ltr"> AKAYVGYVGDGYWVWTL <h2 style=";text-align:left;direction:ltr"> WGQGTLVTVSS;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0133] <h2 style=";text-align:left;direction:ltr"> VL(SEQ ID NO:26)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0134] <h2 style=";text-align:left;direction:ltr"> DIVMTQTPASVEAAVGGTVTIKCQAS<h2 style=";text-align:left;direction:ltr"> SHORT <h2 style=";text-align:left;direction:ltr"> LAWYQQKPGQPPKLLIY<h2 style=";text-align:left;direction:ltr"> LAS <h2 style=";text-align:left;direction:ltr"> TLASGVPSRFKGSGSGTQFTLTTISDLECADAATYYC<h2 style=";text-align:left;direction:ltr"> QSDYYGSGNTYAVA <h2 style=";text-align:left;direction:ltr"> FGGGTEVVVK;T3 mAb4:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0135] <h2 style=";text-align:left;direction:ltr"> VH(SEQ ID NO:31)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0136] <h2 style=";text-align:left;direction:ltr"> QSVEESGGRLVTPGTPLTLTCTAS<h2 style=";text-align:left;direction:ltr"> GFSLSSYW <h2 style=";text-align:left;direction:ltr"> MIWVRQAPGKGLEWIGC<h2 style=";text-align:left;direction:ltr"> IDAGGATT <h2 style=";text-align:left;direction:ltr"> YYATWAKGRFTISETSTTVDLKITSPATEDTATYFC<h2 style=";text-align:left;direction:ltr"> GRGYDTYDSNTFNI <h2 style=";text-align:left;direction:ltr"> WGPGTLVTVSS;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0137] <h2 style=";text-align:left;direction:ltr"> VL(SEQ ID NO:35)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0138] <h2 style=";text-align:left;direction:ltr"> DVVMTQTPASVEAAVGGTVTIKCQAS<h2 style=";text-align:left;direction:ltr"> QSISSW <h2 style=";text-align:left;direction:ltr"> LSWYQQKPGQPPKLLIY<h2 style=";text-align:left;direction:ltr"> AGE <h2 style=";text-align:left;direction:ltr"> TLASGVPSRFKGSGSGTEYTLTISDLECADAAIYYC<h2 style=";text-align:left;direction:ltr"> QTTYGISSYGAA <h2 style=";text-align:left;direction:ltr"> FGGGTEVVVK;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0139] <h2 style=";text-align:left;direction:ltr"> T3 mAb5:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0140] <h2 style=";text-align:left;direction:ltr"> VH(SEQ ID NO:40)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0141] <h2 style=";text-align:left;direction:ltr"> QSVEESGGRLVTPGTPLTLTCTVS<h2 style=";text-align:left;direction:ltr"> GFTISSYH <h2 style=";text-align:left;direction:ltr"> MSWVRQAPGKGLEWIGY<h2 style=";text-align:left;direction:ltr"> IWGGGNT<h2 style=";text-align:left;direction:ltr">DYASWAKGRFTIAKTSSTTVDLKITSPTTEDTATYFC<h2 style=";text-align:left;direction:ltr"> AKAYAGYAGDGYGFDL <h2 style=";text-align:left;direction:ltr"> WGQGTLVTVSS;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0142] <h2 style=";text-align:left;direction:ltr"> VL(SEQ ID NO:44)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0143] <h2 style=";text-align:left;direction:ltr"> DIVMTQTPASVEAAVGGTVTIKCQAS<h2 style=";text-align:left;direction:ltr"> QGIYSS <h2 style=";text-align:left;direction:ltr"> LAWYQQKPGQPPKLLIY<h2 style=";text-align:left;direction:ltr"> SAS <h2 style=";text-align:left;direction:ltr"> TLASGVPSRFKGSGSGTQFTLTTISDLECADAATYYC<h2 style=";text-align:left;direction:ltr"> QCDYYGSGNIYAGS <h2 style=";text-align:left;direction:ltr"> FGGGTEVVVK;T3 mAb6:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0144] <h2 style=";text-align:left;direction:ltr"> VH(SEQ ID NO:49)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0145] <h2 style=";text-align:left;direction:ltr"> EQLKESGGGLVTPGGTLTLTCTVS<h2 style=";text-align:left;direction:ltr"> GFTISTYH <h2 style=";text-align:left;direction:ltr"> MTWVRQAPGKGLEWIGY<h2 style=";text-align:left;direction:ltr"> IWGGDNT <h2 style=";text-align:left;direction:ltr"> DYASWAKGRFTIARTSSSTVDLKITSPTTEDTATYFC<h2 style=";text-align:left;direction:ltr"> AKAYAGYAGDGYGFDL <h2 style=";text-align:left;direction:ltr"> WGQGTLVTVSS;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0146] <h2 style=";text-align:left;direction:ltr"> VL(SEQ ID NO:53)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0147] <h2 style=";text-align:left;direction:ltr"> DIVMTQTPASVEAAVGDTVTIKCQAS<h2 style=";text-align:left;direction:ltr"> QSIYSN <h2 style=";text-align:left;direction:ltr"> LAWYQQKPGQPPKLLIY<h2 style=";text-align:left;direction:ltr"> SAS <h2 style=";text-align:left;direction:ltr"> TLASGVPSRFKGSGSGTQFTLTTISDLECADAATYYC<h2 style=";text-align:left;direction:ltr"> QCDDYGSGNTYNGS <h2 style=";text-align:left;direction:ltr"> FGGGTEVVVK;T3 mAb7:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0148] <h2 style=";text-align:left;direction:ltr"> VH(SEQ ID NO:58)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0149] <h2 style=";text-align:left;direction:ltr"> QSVEESGGRLVTPGTPLTLTCTAS<h2 style=";text-align:left;direction:ltr"> GASLSSYA <h2 style=";text-align:left;direction:ltr"> MTWVRQAPGKGLEYIGI<h2 style=";text-align:left;direction:ltr"> YESSSST <h2 style=";text-align:left;direction:ltr"> WYASWAKGRFTISKTSSTTVDLKITSPTTEDTATYFC<h2 style=";text-align:left;direction:ltr"> ARDIHTGSSYYSSTYFNL <h2 style=";text-align:left;direction:ltr"> WGPGTLVTVSS;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0150] <h2 style=";text-align:left;direction:ltr"> VL(SEQ ID NO:62)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0151] <h2 style=";text-align:left;direction:ltr"> AQVLTQTPSPVSAAVGGTVTIKCQAS<h2 style=";text-align:left;direction:ltr"> ACTUAL <h2 style=";text-align:left;direction:ltr"> LAWYQQKPGQPPKLLIY<h2 style=";text-align:left;direction:ltr"> DASDL AAGVPSRFSGSGSGTEYTLTISDLECDDAATYC QGGYYGSGDGA FGGGTEVVVK;T3 mAb8:

[0152] VH(SEQ ID NO:67)

[0153] QSVEESGGRLVTPGTPLTLTCTAS GFSLSSYA MSWVRQAPGKGLEWIGI IYGSGS T WYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFC ARDIHTGSSYYSSTYFNL WGP GTLVTVSS:

[0154] VL(SEQ ID NO:71)

[0155] AQVLTQTPSPVCAAVGGTVTICQAS ACTUAL LAWYQQKPGQPPKLLIY DAS DL ASGVPSRFSGSGSGTEYTLTISDLECDDAATYYC QGGYYGSGYGA FGGGTEVVVK;

[0156] T3 mAb9:

[0157] VH(SEQ ID NO:76)

[0158] QSVEESGGRLVTPGTPLTLTCTVS GFSLSDYA MSWVRQAPGEGLEWIGT I WANT T YYATWAKGRFTISKTSTTVDLKITRPTTEDTATYFC ARELNSYYSSAWGEGYFTL W GQGTLVTVSS;

[0159] VL(SEQ ID NO:80)

[0160] AAVLTQTPSPVSASVGGTVTISCQAS EDITION LSWYQQKPGQPPKLLIY DAS TLAS GVPSRFKGSGSGTQFTLTISDLECADAATYYC QSAVYSTSTSDAA FGGGTEVVVK:

[0161] T3 mAb10:

[0162] VH(SEQ ID NO:85)

[0163] QSVEESGGRLVTPGTPLTLTCTVS GFSLSDYA MSWVRQAPGKGLEWIGI ISTGGT T YYASWAKGRFTISRTSTTVDLKITSPTTEDTATYFC ARELNIYYSSWGEGYFSL WG PGTLVTVSS;

[0164] VL (SEQ ID NO: 89)

[0165] AAVLTQTPSPVSAAVGGTVTIKCQSS DIASY LSWYQQKPGQPPKQLIY DAS KLAS GVPSRFKGSGSGTQFTLTISGVQCDDAATYYC QSAVYSSSAVNA FGGGTEVVVK.

[0166] Example 3: Expression and purification of monoclonal antibodies against T3

[0167] (1) Cultivate N293 suspension cells until the density reaches (1-3)×10 6 When the cell count is 10 cells / ml and the survival rate is >80-90%, cell transfection is performed;

[0168] (2) The light and heavy chain genes of the 10 monoclonal antibodies T3 mAb1 to T3 mAb10 measured in Example 2 were synthesized, and the synthesized light and heavy chain genes were respectively constructed into the pcDNA3.4 plasmid; then, the recombinant plasmid containing the antibody light and heavy chain genes was transfected into N293 cells, and the transfection conditions were: the mass ratio of PEI / plasmid was 3:1, and the transfection ratio was 2 μg plasmid / 1 ml N293 cells;

[0169] (3) 5-7 days after transfection, the cell supernatant was collected and affinity purified using Protein A resin to obtain high-purity monoclonal antibodies. The elution buffer was replaced with PBS by dialysis, and the antibody concentration was measured using Nanodrop;

[0170] Next, the purity of the antibody was tested by SDS-PAGE and HPLC. The specific method is as follows:

[0171] SDS-PAGE detection:

[0172] Take 2 μg of the antibody to be tested, add an appropriate amount of SDS-PAGE protein loading buffer to make the total volume 20 μl; prepare reducing SDS-PAGE samples and non-reducing SDS-PAGE samples at the same time, and then load them; first, electrophoresis at 80V for 30 minutes, and then electrophoresis at 120V until the bands are clearly separated; remove the PAGE gel after electrophoresis, stain with Coomassie Brilliant Blue, remove the staining solution after 15 minutes, rinse with clean water, and then add decolorizing solution for decolorization until clear bands are seen.

[0173] The results are as follows Figure 1 As shown, Figure 1 The results showed that under reducing conditions, all 10 monoclonal antibodies had two electrophoretic bands, and the corresponding molecular weights of the two electrophoretic bands were consistent with the sizes of their respective light and heavy chains; under non-reducing conditions, all 10 monoclonal antibodies had a single band, and the corresponding molecular weights of the electrophoretic bands were consistent with the sizes of their respective complete antibodies; these results indicated that the expression of these 10 monoclonal antibodies was accurate.

[0174] In addition, all electrophoresis bands had clear edges and no foreign bands, indicating that the monoclonal antibodies obtained by the above expression and purification steps had high purity.

[0175] Example 4: Antigen Binding Activity and Affinity Detection of Monoclonal Antibodies Against T3

[0176] In this example, the antigen activity and affinity of the 10 monoclonal antibodies T3 mAb1 to T3mAb10 expressed and purified in Example 3 were tested respectively.

[0177] Binding activity and affinity assays

[0178] ① Antigen coating: dilute the antigen (i.e., the aforementioned coupling protein T3-BSA) to an appropriate concentration, and then add it to an empty ELISA plate at a volume of 100 μl / well; place the ELISA plate in a 4°C refrigerator and incubate overnight;

[0179] ② Blocking: Wash the plate 5 times with a plate washer, tap dry, add blocking solution to the ELISA plate, 150 μl per well, place in a 37°C incubator, block for 1 hour; wash the plate 5 times with a plate washer, tap dry;

[0180] ③ Incubate the primary antibody: dilute the antibody to be tested to a suitable concentration gradient with 1×PBS; add the diluted antibody to be tested to the ELISA plate, 100 μl per well, reaction time: 37℃ / 1h; wash the plate 5 times with a plate washer and dry;

[0181] ④ Incubate the secondary antibody: dilute the enzyme-labeled secondary antibody in an appropriate ratio, and then add it to the ELISA plate, 100 μl per well, reaction time: 37°C / 30 min; wash the plate 5 times with a plate washer and dry;

[0182] ⑤ Color development: add the color development solution to the ELISA plate, 100 μl per well, reaction time: 37 degrees / 15 minutes;

[0183] ⑥ Stop and read: add stop solution, volume 100μl / well; detection wavelength setting: detection wavelength 450nm, reference wavelength 620nm, export the detection results into Excel table.

[0184] ⑦Data analysis: Use software to analyze and plot data, fit EC50, and derive its KD.

[0185] The results of the binding activity and affinity test of these 10 monoclonal antibodies to antigens are as follows Figure 2 and as shown in Table 1 below.

[0186] Table 1

[0187] antibody no EC50(ng / ml) KD T3mAb1 22.33 <![CDATA[1.49*10 -10 M]]> T3mAb2 20.27 <![CDATA[1.35*10 -10 M]]> T3mAb3 11.76 <![CDATA[7.78*10 -11 M]]> T3mAb4 12.67 <![CDATA[8.45*10 -11 M <!-- 11 -->]]> T3mAb5 34.45 <![CDATA[2.3*10 -10 M]]> T3mAb6 20.62 <![CDATA[1.37*10 -10 M]]> T3mAb7 43.12 <![CDATA[2.87*10 -10 M]]> T3mAb8 34.08 <![CDATA[2.27*10 -10 M]]> T3mAb9 22.75 <![CDATA[1.52*10 -10 M]]> T3mAb10 23.55 <![CDATA[1.57*10 -10 M]]>

[0188] Figure 2 Table 1 shows that the EC50 of the 10 monoclonal antibodies binding to the antigen all reached the ng / ml level, and the KD values ​​were all above 10 -10 M~10 -11 M level.

[0189] As mentioned above, for IgG antibodies, it is generally believed in the art that when the KD value of binding to the antigen reaches 10 -9 M is considered to have high affinity, and the KD values ​​of the 10 monoclonal antibodies of the present invention for binding to the antigen all reached 10 -10 M~10 -11 M is 10-100 times the above-defined value, from which it can be concluded that the monoclonal antibody of the present invention has extremely strong antigen binding activity and affinity.

[0190] Example 5: Preparation of double antibody sandwich colloidal gold immunochromatographic test strip based on anti-T3 monoclonal antibody

[0191] In this example, T3 mAb1 and T3 mAb2, T3 mAb3 and T3mAb4 expressed and purified in Example 3 were used as paired antibodies to prepare two colloidal gold immunochromatographic test strips for detecting T3 using the double antibody sandwich method. The schematic diagram of the structure of the test strip is shown in FIG. Figure 3 As shown; the preparation method of the test strip is as follows:

[0192] (1) Preparation of Au-anti-T3 monoclonal antibody complex

[0193] ① Take 3 ml of colloidal gold particles (colloidal gold particle size: 20 nm, maximum absorption peak wavelength of 529 nm, absorbance of 2.0 A), add 18 μl of 0.1 M K2CO3, mix well, add 10 μg of anti-T3 antibody (i.e., detection antibody), and react at room temperature for 1 hour;

[0194] ② Add 600 μl 5% BSA blocking solution to the system in step ① and block for 30 min at room temperature;

[0195] ③ After blocking, centrifuge at 11000 rpm at 10°C for 15 min, discard the supernatant, and re-dissolve the precipitate with 50 μl of gold-labeled diluent (i.e., concentrated 60 times, 60×) to obtain the Au-anti-T3 monoclonal antibody complex; the gold-labeled diluent contains 100 mM Tris buffer at pH 7.5-8.5, 5% sucrose, 1% trehalose, and 5% BSA.

[0196] (2) Deposit

[0197] ① Draw a closed line: Take 5% BSA solution, set the instrument water output to 3μL / cm, and place the drawn bottom plate in a 37℃ oven to dry for 2 hours;

[0198] ② Dilute the Au-anti-T3 monoclonal antibody complex to a final concentration of 50×, set the water output to 2 μL / cm, draw a line at the closed line, and then place the bottom plate in a 37°C oven to dry for 2 hours. Then, place the marking pad in an aluminum foil bag pre-added with desiccant at a humidity not higher than 36% and seal it for storage;

[0199] (3) Coating quality control line C, detection line T

[0200] ① Preparation of coating solution for control line C and test line T: dilute goat anti-rabbit antibody with 10mM PBS buffer at pH 7.4 to a 1.0mg / mL antibody solution; dilute another anti-T3 monoclonal antibody (i.e., capture antibody) with 10mM PBS buffer at pH 7.4 to a 1.0mg / mL antibody solution;

[0201] ② Coating: Coat the control line C and the detection line T coating solution to the control line and the detection line of the nitrocellulose membrane respectively, with the interval between the C and T lines being 8 mm, and the coating volume being 1 μL / cm; place the coated PVC base plate in a 37°C oven for 16 hours, and then place the PVC base plate in an aluminum foil bag pre-added with desiccant at a humidity not higher than 36% and seal it for future use;

[0202] (4) Pretreatment of sample pad

[0203] Clean the drying net, soak the sample pad with the sample pad pretreatment solution, and then drain it until no water drops. Then, dry it in a 37°C oven for 4 hours, and then seal it in an aluminum foil bag with desiccant added in advance at a humidity not higher than 36% for storage;

[0204] The sample pad pretreatment solution contains 10 mM Tris buffer at pH 8.5, 0.1% PVP 10, 0.1% PEG 20000, 0.1% Tween 20 and 0.5% sodium caseinate;

[0205] (5) Assembly and cutting of membrane materials

[0206] At a humidity not higher than 36%, take out the absorbent pad and the treated sample pad, cut them into a size of 10 mm × 300 mm, take out the coated PVC base plate, and assemble them according to the following process: first stick the sample pad on the PVC base plate so that the sample pad part presses the nitrocellulose membrane for 2 mm, and finally stick the absorbent pad so that the absorbent pad presses the nitrocellulose membrane for 2 mm; cut the assembled PVC base plate into 4 mm, which is the colloidal gold immunochromatographic test strip for detecting T3.

[0207] The antibody pairing of test strip 1 is: T3 mAb1 (for preparing Au-antibody complex) + T3 mAb2 (for T-line coating); the antibody pairing of test strip 2 is: T3 mAb3 (for preparing Au-antibody complex) + T3mAb4 (for T-line coating).

[0208] Example 6: Detection performance of the double antibody sandwich colloidal gold immunochromatographic test strip based on anti-T3 monoclonal antibody

[0209] In this example, the detection performance of the two colloidal gold immunochromatographic test strips prepared in Example 5 for T3 in clinical serum samples was tested, and compared with the detection performance of the traditional Roche detection reagent.

[0210] Specifically, for the two colloidal gold immunochromatographic test strips prepared in Example 5, the clinical serum sample was diluted with a sample diluent (whose composition is: 10mMPBS+5% sodium dodecyl sulfate+5% disodium EDTA+0.5% guanidine isothiocyanate+0.1% proclin300, pH7.0), reacted at room temperature for 10min, then 75μl of the sample was added, and after 15 minutes, the colloidal gold immunoassay was used for detection, the detection value was read, and the data was processed to obtain the T3 concentration in the sample of each dilution; for the Roche detection reagent, the operation was carried out according to the flow of its instruction manual, the serum sample was diluted, and then detected to obtain the T3 concentration data. Then, the T3 concentration data detected by the two test strips were compared with the T3 concentration data detected by the Roche reagent (as a standard), and a clinical correlation (i.e., the coincidence rate with the Roche detection result) curve was prepared.

[0211] The results are as follows Figure 4 As shown; among them, Figure 4 A and 4B are the clinical relevance results of colloidal gold immunochromatographic test strip 1 and test strip 2, respectively, which show that the clinical relevance of the two test strips is R 2 >0.95 and R 2 >0.97.

[0212] The above results show that the T3 colloidal gold immunochromatographic test strip prepared based on the paired antibodies of the present invention has a high coincidence rate (R 2 above 0.95), thus having good clinical relevance.

[0213] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof against triiodothyronine, characterized in that: The monoclonal antibody or antigen-binding fragment thereof comprises: The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO:82, SEQ ID NO:83 and SEQ ID NO:84, respectively; and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO:87, DAS and SEQ ID NO:88, respectively.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The monoclonal antibody or antigen-binding fragment thereof comprises: The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:85; and the light chain variable region has an amino acid sequence as shown in SEQ ID NO:

89.

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 2, characterized in that: The monoclonal antibody or antigen-binding fragment thereof comprises: a heavy chain, whose amino acid sequence is shown in SEQ ID NO:86; and a light chain, whose amino acid sequence is shown in SEQ ID NO:

90.

4. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that: The antigen binding fragment is selected from Fab, Fab', F(ab')2, Fv, a complementarity determining region fragment, a single chain antibody, a human antibody, a chimeric antibody or a bispecific or multispecific antibody.

5. A polynucleotide encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

6. A nucleic acid construct comprising the polynucleotide of claim 5 and, optionally, at least one expression control element operably linked to the polynucleotide.

7. A recombinant vector comprising the polynucleotide according to claim 5, or the nucleic acid construct according to claim 6.

8. A transformed host cell comprising the polynucleotide according to claim 5, the nucleic acid construct according to claim 6 or the recombinant vector according to claim 7.

9. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the polynucleotide according to claim 5, the nucleic acid construct according to claim 6, the recombinant vector according to claim 7 and / or the transformed host cell according to claim 8 in the preparation of a detection reagent or kit for detecting triiodothyronine.

10. A detection reagent for detecting triiodothyronine, comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

11. A method for preparing the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the method comprising: Under conditions suitable for the expression of the monoclonal antibody or its antigen-binding fragment, the transformed host cell according to claim 8 is allowed to express the monoclonal antibody or its antigen-binding fragment, and the expressed monoclonal antibody or its antigen-binding fragment is recovered from the culture of the host cell.

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