Anti-tetraiodothyronine antibody or its functional fragment, reagent and kit for detecting tetraiodothyronine

Abstract

The present invention discloses an anti-tetraiodothyronine antibody or a functional fragment thereof, a reagent and a kit for detecting tetraiodothyronine, and relates to the field of antibodies. The anti-tetraiodothyronine antibody disclosed in the present invention comprises a heavy chain complementary determining region and a light chain complementary determining region, and the antibody provides an important source of raw materials for the detection of tetraiodothyronine, and has improved affinity and activity.

Description

Technical Field

[0001] The present invention relates to the field of antibodies, in particular to an anti-tetraiodothyronine antibody or a functional fragment thereof, and a reagent and a kit for detecting tetraiodothyronine. Background Art

[0002] Tetraiodothyronine (3,5,3′,5′-tetraiodo-L-thyronine, abbreviated as T4) is a hormone secreted from the thyroid follicular epithelium. After being released into the blood, most of T4 binds to TBG (thyroid binding globulin), and binds to specific receptor proteins in the cell nucleus, inducing the protein to produce an active conformation, so that its DNA binding site effectively binds to a specific site of DNA, thereby activating the DNA transcription process and achieving the biological function of regulating gene expression. About 0.03% of the total serum T4 is in a free state, which is in dynamic balance with the binding hormone to maintain normal physiological functions. A large number of biological and physiological experimental results have proved that thyroid hormones play an important role in regulating and controlling the normal development and differentiation of cells, the temperature regulation of animals, and the maintenance of various sugar, fat and protein metabolic balances.

[0003] If the concentration of T4 in serum is higher than the normal value, it may be due to: (1) hyperthyroidism; (2) drug effects: such as taking thyroxine, estrogen drugs and contraceptives, perphenazine, etc.; (3) other diseases: such as congenital hereditary hyperthyroidism, acute infectious hepatitis, intermittent porphyria, etc.; (4) physiological factors: such as pregnancy. If the concentration of T4 in serum is lower than the normal value, it may be due to: (1) thyroid disease: primary hypothyroidism, chronic lymphocytic thyroiditis; (2) drug effects: antithyroid drugs; (3) other diseases: severe liver and kidney failure, nephrotic syndrome, active acromegaly, hereditary hypothyroidism, etc. Therefore, the detection of serum T4 is of great significance for the diagnosis or differential diagnosis of hyperthyroidism, hypothyroidism and thyroid tumors, and for the development process, efficacy and prognosis evaluation of thyroid function-related diseases.

[0004] Immunological detection is a means of detection based on the specific reaction of antibodies and antigens. Since it can amplify and display the detected signal using isotopes, enzymes, chemiluminescent substances, etc., it is often used to detect trace bioactive substances such as proteins and hormones. At present, immunological detection methods have been widely used in clinical diagnosis and biochemistry research as an important detection method. Commonly used immunological detection methods include radioimmunoassay, enzyme-linked immunosorbent assay, chemiluminescence, etc. Different methods have their own advantages and disadvantages. For example, radioimmunoassay inevitably uses radioactive elements, which have a certain impact on people and the environment; enzyme-linked immunosorbent assay has a low sensitivity; chemiluminescence requires expensive instruments and reagents. However, when any of the above immunological detection methods is used to detect T4, a specific monoclonal antibody against T4 is required. Therefore, there is a strong demand in this field for antibodies that effectively and specifically bind to and detect T4 and detect it.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide an anti-tetraiodothyronine antibody or a functional fragment thereof, and a reagent and a kit for detecting tetraiodothyronine.

[0007] The present invention is achieved in that:

[0008] In a first aspect, an embodiment of the present invention provides an antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises any one of (a) to (d): (a) HCDR1 to 3 and LCDR1 to 3, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID Nos: 1, 2 and 4, or in sequence; the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID Nos: 5, 6, 7, or in sequence; (b) a heavy chain variable region with an amino acid sequence as shown in any one of SEQ ID NOs: 9 to 16 and a light chain variable region with an amino acid sequence as shown in any one of SEQ ID NOs: 26 to 29; (c) a heavy chain variable region and a light chain variable region with an amino acid sequence having more than 80% identity with the sequence shown in (b), and comprising HCDR1 to HCDR3 and LCDR1 to LCDR3 of the sequence shown in (a); (d) a heavy chain with an amino acid sequence as shown in any one of SEQ ID NOs: 18 to 25, and an amino acid sequence as shown in SEQ ID NOs: NO: The light chain shown in any one of items 31 to 34.

[0009] In a second aspect, an embodiment of the present invention provides an antibody conjugate, which includes the antibody or a functional fragment thereof as described in the above embodiments.

[0010] In a third aspect, an embodiment of the present invention provides a reagent or a kit, which includes the antibody or its functional fragment as described in the above embodiments or the antibody conjugate as described in the above embodiments.

[0011] In a fourth aspect, an embodiment of the present invention provides a method for detecting tetraiodothyronine, comprising: mixing the antibody or its functional fragment as described in the above embodiment with a sample to be detected, so that the antibody or its functional fragment contacts the tetraiodothyronine in the sample to be detected to form an immune complex.

[0012] In a fifth aspect, an embodiment of the present invention provides an isolated nucleic acid encoding the antibody or a functional fragment thereof described in the preceding embodiments.

[0013] In a sixth aspect, an embodiment of the present invention provides a vector comprising the isolated nucleic acid described in the preceding embodiment.

[0014] In a seventh aspect, an embodiment of the present invention provides a cell containing the isolated nucleic acid described in the preceding embodiment or the vector described in the preceding embodiment.

[0015] In an eighth aspect, an embodiment of the present invention provides a method for preparing the antibody or a functional fragment thereof described in the preceding embodiment, comprising: culturing the cells described in the preceding embodiment.

[0016] The present invention has the following beneficial effects:

[0017] The anti-tetraiodothyronine antibody disclosed in the present invention comprises the heavy chain complementary determining region and the light chain complementary determining region. The antibody provides an important source of raw materials for the detection of tetraiodothyronine and has improved affinity and activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0019] Figure 1 The results of reducing SDS-PAGE of Anti-T4 8F6 are shown. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0021] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0022] An embodiment of the present invention provides an antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises:

[0023] (a) The amino acid sequences of HCDR1-3 and LCDR1-3, HCDR1, HCDR2 and HCDR3 are shown in SEQ ID Nos: 1, 2 and 4, or 1, 3 and 4, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID Nos: 5, 6, 7, or 5, 6, 8, respectively.

[0024] In the present invention, the term "antibody" is used in the broadest sense and may include full-length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies, so long as they exhibit the desired biological activity.

[0025] In the present invention, the term "complementarity determining region", "CDR" or "CDRs" refers to the highly variable region of the heavy and light chains of immunoglobulins, and refers to the region containing one or more or even all of the major amino acid residues that play a role in the binding of an antibody or antigen-binding fragment to its recognized antigen or epitope. In a specific embodiment of the present invention, CDRs refers to the highly variable region of the heavy and light chains of the antibody.

[0026] In the present invention, the heavy chain complementary determining region is represented by HCDR, and the three CDRs contained in the heavy chain variable region include HCDR1, HCDR2 and HCDR3; the light chain complementary determining region is represented by LCDR, and the three CDRs contained in the light chain variable region include LCDR1, LCDR2 and LCDR3. Commonly used CDR marking methods in the art include: Kabat numbering scheme, IMGT numbering scheme, Chothia and Lesk numbering scheme, and the new standardized numbering system introduced by Lefranc et al. for all protein sequences of the immunoglobulin superfamily in 1997. Kabat et al. were the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences has led to the creation of the KABATMAN database, and the Kabat numbering scheme is generally considered to be a widely used standard for numbering antibody residues. The embodiments of the present invention use the Kabat annotation standard to mark the CDR region, but CDR regions marked by other methods also fall within the scope of protection of the present invention.

[0027] On the other hand, an embodiment of the present invention further provides an antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises:

[0028] (b) a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 9 to 16 and a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 26 to 29.

[0029] In the present invention, the "framework region" or "FR" region includes the heavy chain framework region and the light chain framework region, and refers to the region other than the CDR in the heavy chain variable region and the light chain variable region of the antibody; wherein the heavy chain framework region can be further subdivided into adjacent regions separated by CDR, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by CDR, including LFR1, LFR2, LFR3 and LFR4 framework regions.

[0030] In the present invention, the heavy chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0031] On the other hand, an embodiment of the present invention further provides an antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises:

[0032] (c) A heavy chain variable region and a light chain variable region whose amino acid sequence has more than 80% identity with the sequence shown in (b), and includes HCDR1 to HCDR3 and LCDR1 to LCDR3 of the sequence shown in (a). The above antibody has improved activity.

[0033] In an optional embodiment, the antibody or a functional fragment thereof comprises a heavy chain framework region in the heavy chain variable region shown in SEQ ID NOs: 9 to 16, and a light chain framework region in the light chain variable region shown in SEQ ID NOs: 26 to 29.

[0034] In an optional embodiment, the framework region amino acid sequence of the antibody or its functional fragment may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the above-mentioned framework region.

[0035] In an alternative embodiment, the antibody or a functional fragment thereof further comprises a constant region.

[0036] In an alternative embodiment, the constant region includes a heavy chain constant region and / or a light chain constant region.

[0037] In an optional embodiment, the heavy chain constant region is selected from the heavy chain constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; and the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0038] In an optional embodiment, the species origin of the constant region is any one of cattle, horses, pigs, sheep, rats, mice, dogs, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese and humans.

[0039] In an alternative embodiment, the species origin of the constant region is sheep.

[0040] In an alternative embodiment, the heavy chain constant region sequence is as shown in SEQ ID NO: 17 or has at least 80% identity thereto.

[0041] In an alternative embodiment, the light chain constant region sequence is as shown in SEQ ID NO: 30 or has at least 80% identity thereto.

[0042] Specifically, the constant region sequence can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the above-mentioned constant region (SEQ ID NO: 17 or 30).

[0043] In an optional embodiment, the functional fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

[0044] The functional fragments of the above antibodies usually have the same binding specificity as the antibodies from which they are derived. It is easy for a person skilled in the art to understand based on the contents described in the present invention that the functional fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction to split disulfide bonds. Based on the structure of the complete antibody disclosed in the present invention, a person skilled in the art can easily obtain the above functional fragments.

[0045] The functional fragments of the above antibodies can also be synthesized by recombinant genetic techniques known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0046] On the other hand, an embodiment of the present invention further provides an antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises:

[0047] (d) a heavy chain having an amino acid sequence as shown in any one of SEQ ID NOs: 18 to 25, and a light chain having an amino acid sequence as shown in any one of SEQ ID NOs: 31 to 34.

[0048] In an optional embodiment, the antibody or its functional fragment described in the above embodiment has a KD of less than 7.17×10 - 09 M binds tetraiodothyronine with high affinity.

[0049] In an optional embodiment, the antibody or its functional fragment described in the above embodiment has a KD≤10 -09 M, KD≤10 -10 M, KD≤10 -11 M or KD≤10 -12 M binds tetraiodothyronine with high affinity.

[0050] In an optional embodiment, the antibody or its functional fragment described in the above embodiment has a KD≤9.67×10 - 10 M, KD≤4.56×10-10 M, KD≤3.43×10 -10 M, KD≤3.18×10 -10 M, KD≤3.15×10 -10 M, KD≤2.44×10 -10 M, KD≤1.53×10 -10 M, KD≤1.49×10 -10 M or KD≤1.29×10 -10 M binds tetraiodothyronine with high affinity.

[0051] On the other hand, an embodiment of the present invention further provides an antibody conjugate, which includes the antibody or a functional fragment thereof as described in the above embodiment.

[0052] In an optional embodiment, the antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody or a functional fragment thereof.

[0053] In an optional embodiment, the antibody conjugate further comprises a label coupled to the antibody or a functional fragment thereof.

[0054] In an optional embodiment, the above-mentioned marker refers to a class of substances with properties that can be directly observed by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0055] In an optional embodiment, the marker is selected from at least one of a fluorescent dye, an enzyme, a radioactive isotope, a chemiluminescent agent and a nanoparticle marker.

[0056] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it belongs to the protection scope of the present invention.

[0057] In an optional embodiment, the fluorescent dye is not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy3.7, Cy3.8, Cy3.9, Cy3.10, Cy3.11, Cy3.12, Cy3.13, Cy3.14, Cy3.15, Cy3.16, Cy3.17, Cy3.18, Cy3.19, Cy3.20, Cy3.21, Cy3.22, Cy3.23, Cy3.24, Cy3.25, Cy3.26, Cy3.27, Cy3.28, Cy3.29, Cy3.30, Cy3.31, Cy3.32, Cy3.33, Cy3.34, Cy3.35, Cy3.36, Cy3.37, Cy3.38, Cy3.39, Cy3.40, Cy3.41, Cy3.42, Cy3.43, Cy3.44, Cy3.45, Cy3.46, Cy3.47, Cy3.48, Cy3.49, Cy3.50, Cy3.51, Cy3.52, Cy3.53, Cy3.54, Cy3.55, Cy3.56, Cy3.57, Cy3.58, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.5 y5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), pre-chlorophyll protein (preCP), etc.).

[0058] In alternative embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxygenase.

[0059] In an optional embodiment, the radioactive isotopes include but are not limited to 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu and 18F.

[0060] In an optional embodiment, the chemiluminescent reagent includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxyoxalates and their derivatives.

[0061] In an optional embodiment, the nanoparticle markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0062] In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, colloidal selenium, disperse dyes, dye-labeled microspheres, and latex.

[0063] In an optional embodiment, the colloidal metal includes but is not limited to colloidal gold or colloidal silver.

[0064] In an optional embodiment, the colloidal metal is colloidal gold.

[0065] In an optional embodiment, the antibody conjugate further comprises a solid phase carrier coupled to the antibody or its functional fragment. In the antibody conjugate, the antibody is coupled to the solid phase carrier.

[0066] In an alternative embodiment, the solid support is selected from microspheres, plates and membranes.

[0067] In an optional embodiment, the solid phase includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic microparticles, microplates, glass, capillaries, nylon, and nitrocellulose membranes.

[0068] In an optional embodiment, the solid phase carrier is a nitrocellulose membrane.

[0069] On the other hand, an embodiment of the present invention further provides a reagent or a kit, which includes the antibody or its functional fragment as described in any of the foregoing embodiments or the antibody conjugate as described in any of the foregoing embodiments.

[0070] On the other hand, an embodiment of the present invention further provides a method for detecting tetraiodothyronine, comprising:

[0071] The antibody or its functional fragment as described in any of the foregoing embodiments, the antibody conjugate as described in any of the foregoing embodiments, or the reagent or kit as described in any of the foregoing embodiments is mixed with the sample to be detected, so that the antibody or its functional fragment contacts the tetraiodothyronine in the sample to be detected to form an immune complex.

[0072] In a preferred embodiment, the immune complex further comprises a second antibody, which binds to the antibody or a functional fragment thereof.

[0073] In a preferred embodiment, the immune complex further comprises a second antibody, which binds to tetraiodothyronine.

[0074] On the other hand, an embodiment of the present invention further provides the use of the antibody or functional fragment described in any of the foregoing embodiments, or the antibody conjugate described in any of the foregoing embodiments, or the reagent or kit described in any of the foregoing embodiments in detecting tetraiodothyronine or preparing a product for detecting tetraiodothyronine.

[0075] On the other hand, an embodiment of the present invention further provides the use of the antibody or functional fragment described in any of the preceding embodiments, or the antibody conjugate described in any of the preceding embodiments, or the reagent or kit described in any of the preceding embodiments in the preparation of a product having at least one of the following uses, wherein the uses include: diagnosing or assisting in the diagnosis of diseases related to tetraiodothyronine metabolism, and predicting or assisting in the prediction of at least one of the prognosis and efficacy of diseases related to tetraiodothyronine metabolism.

[0076] In an optional embodiment, the tetraiodothyronine metabolism-related disease includes at least one of hyperthyroidism, hypothyroidism and thyroid tumor.

[0077] In an alternative embodiment, the product comprises a reagent or a kit.

[0078] On the other hand, an embodiment of the present invention further provides an isolated nucleic acid encoding the antibody or a functional fragment thereof described in any of the aforementioned embodiments.

[0079] On the other hand, an embodiment of the present invention further provides a vector comprising the isolated nucleic acid described in any of the above embodiments.

[0080] On the other hand, an embodiment of the present invention further provides a cell, which contains the isolated nucleic acid described in any of the foregoing embodiments or the vector described in any of the foregoing embodiments.

[0081] On the other hand, an embodiment of the present invention further provides a method for preparing the antibody or a functional fragment thereof as described in any of the foregoing embodiments, comprising: culturing the cell as described in any of the foregoing embodiments.

[0082] Based on the amino acid sequence of the antibody or its functional fragment disclosed in the present invention, those skilled in the art can easily think of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the antibody or its functional fragment, for example, separating and purifying the antibody or its functional fragment from the culture product of a recombinant cell that can recombinantly express the antibody or its functional fragment as described in any of the above items, which is easy to achieve for those skilled in the art. Based on this, no matter what technology is used to prepare the antibody or its functional fragment of the present invention, it belongs to the protection scope of the present invention.

[0083] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0084] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used for the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise stated, the techniques adopted or considered herein are standard methods. Materials, methods and examples are illustrative and non-restrictive only.

[0085] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0086] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0087] Example 1 Preparation of Anti-T4 8F6 Monoclonal Antibody

[0088] In this example, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART TM RACE cDNA Amplification Kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were completed by a gene sequencing company.

[0089] 1. Expression plasmid construction

[0090] 1.1. Anti-T4 8F6 antibody gene preparation

[0091] mRNA was extracted from the hybridoma cell line secreting Anti-T4 8F6 monoclonal antibody, and the DNA product was obtained by RT-PCR. It was inserted into the pMD-18T vector and transformed into DH5α competent cells. After the colonies grew, 4 positive clones of the Heavy Chain and Light Chain genes were taken and sent to a gene sequencing company for sequencing.

[0092] 1.2. Sequence analysis of the variable region gene of Anti-T4 8F6 antibody

[0093] The gene sequences obtained by the above sequencing were placed in the Kabat antibody database for analysis, and the VNTI11.5 software was used for analysis to determine that the genes amplified by the heavy chain and light chain primer pairs were correct. Among the gene fragments amplified by the Light Chain, the VL gene sequence was 318bp, preceded by a 57bp leader peptide sequence; among the gene fragments amplified by the Heavy Chain primer pair, the VH gene sequence was 348bp, belonging to the VH1 gene family, preceded by a 57bp leader peptide sequence.

[0094] 1.3. Construction of recombinant antibody expression plasmid

[0095] pcDNA TM 3.4 vector is a recombinant antibody eukaryotic expression vector constructed, which has been modified to introduce multiple cloning restriction sites and is subsequently referred to as 3.4A expression vector; based on the sequencing results of the antibody variable region genes in the above pMD-18T, the VL and VH gene-specific primers of the Anti-T4 8F6 antibody were designed, with restriction endonuclease cleavage sites and protective bases at both ends, respectively, and the 0.72kb Light Chain gene fragment and the 1.43kb Heavy Chain gene fragment were amplified by the PCR amplification method.

[0096] The Heavy Chain and Light Chain gene fragments were double-digested with restriction endonucleases, and the 3.4A vector was double-digested with restriction endonucleases. After the fragments and vectors were purified and recovered, the Heavy Chain gene and the Light Chain gene were respectively connected to the 3.4A expression vector to obtain the recombinant expression plasmids of the Heavy Chain and Light Chain, respectively.

[0097] 2. Screening of stable cell lines

[0098] 2.1 Transient transfection of recombinant antibody expression plasmid into CHO cells to determine the activity of the expression plasmid

[0099] The plasmid was diluted to 40 μg / 100 μL with ultrapure water and the CHO cells were adjusted to 1.43×10 7 cells / ml in a centrifuge tube, mix 100 μL of plasmid with 700 μL of cells, transfer to an electroporation cup, electroporate, take samples and count on the 3rd, 5th and 7th days, and collect samples for detection on the 7th day.

[0100] The coating solution (main component NaHCO3) was diluted with T4-BSA (purchased from biospacific, VI9100) to 2 μg / mL, 100 μL per well, and incubated at 4°C overnight; the next day, the cells were washed twice with washing solution (main component Na2HPO4+NaCl), and patted dry; blocking solution (20% BSA+80% PBS) was added, 120 μL per well, incubated at 37°C for 1 hour, and patted dry; the diluted cell supernatant was added, 100 μL per well, and the cells were incubated at 4°C overnight. μL / well, 37℃, 30min (partial supernatant 1h); wash 5 times with washing solution, pat dry; add mouse anti-sheep IgG-HRP, 100μL per well, 37℃, 30min; wash 5 times with washing solution, pat dry; add colorimetric solution A (50μL / well), add colorimetric solution B (50μL / well), 10min; add stop solution, 50μL / well; read OD value at 450nm (reference 630nm) on the microplate reader. The results showed that the reaction OD was still greater than 1.0 after the cell supernatant was diluted 1000 times, and the reaction OD of the well without cell supernatant was less than 0.1, indicating that the antibody produced after transient plasmid transfection was active against T4-BSA (purchased from biospacific, VI9100).

[0101] The reactivity of cell supernatant with BSA was detected by the same method as above. The results showed that the reaction OD was less than 0.1 after the cell supernatant was diluted 1000 times, and the reaction OD of the well without cell supernatant was less than 0.1, indicating that the antibody produced after transient transfection of plasmid was inactive to BSA, further proving that the cell supernatant was active to T4.

[0102] Note: Solution A (main components: citric acid + sodium acetate + acetanilide + urea peroxide); Solution B (main components: citric acid + EDTA·2Na + TMB + concentrated HCL); Stop solution (EDTA·2Na + concentrated H2SO4).

[0103] 2.2 Linearization of recombinant antibody expression plasmid

[0104] Prepare the following reagents: 50μL Buffer, 100ug DNA / tube, 10μl Pvu I enzyme, and make up to 500μl with sterile water. Digest in a 37℃ water bath overnight; first use an equal volume of phenol / chloroform / isoamyl alcohol (lower layer) 25:24:1, and then use chloroform (aqueous phase) to extract in sequence; 0.1 volume (aqueous phase) 3M sodium acetate and 2 volumes of ethanol are precipitated on ice, and the precipitate is rinsed with 70% ethanol to remove the organic solvent. After the ethanol is completely evaporated, it is reconstituted with an appropriate amount of sterile water, and finally the concentration is measured.

[0105] 2.3 Stable transfection of recombinant antibody expression plasmid and pressure screening of stable cell lines

[0106] The plasmid was diluted to 40 μg / 100 μL with ultrapure water and the CHO cells were adjusted to 1.43×10 7 cells / ml in a centrifuge tube, mix 100 μL of plasmid with 700 μL of cells, transfer to an electroporation cup, electrotransform, and count the next day; culture under pressure in 25 μmol / L MSX 96-well plates for about 25 days.

[0107] Observe the clone wells with cells under a microscope and record the confluence; take the culture supernatant and send it for testing; select cell lines with high antibody concentration and relative concentration and transfer them to 24 wells, and transfer them to 6 wells after about 3 days; after 3 days, maintain the seed batch culture and adjust the cell density to 0.5×10 6 cells / ml, 2.2 mL for batch culture, cell density 0.3×10 6 cells / mL, 2mL for seed preservation; 7-day 6-well batch culture supernatant samples were sent for testing, and cell lines with smaller antibody concentrations and cell diameters were selected for TPP seed preservation and subculture.

[0108] 3. Recombinant Antibody Production

[0109] 3.1 Cell expansion

[0110] After cell recovery, culture in a 125mL shake flask with an inoculation volume of 30mL and a culture medium of 100% Dynamis medium. Place the flask in a shaker at a speed of 120r / min, a temperature of 37°C, and a carbon dioxide content of 8%. Culture for 72 hours, inoculate and expand at an inoculation density of 500,000 cells / mL. The expansion volume is calculated according to production requirements, and the culture medium is 100% Dynamis medium. Expand the culture every 72 hours. When the cell volume meets the production requirements, strictly control the inoculation density to about 500,000 cells / mL for production.

[0111] 3.2 Shake flask production and purification

[0112] Shake flask parameters: rotation speed 120r / min, temperature 37℃, carbon dioxide 8%. Feeding: Start feeding every day when the culture reaches 72h in the shake flask. HyCloneTM Cell BoostTM Feed 7a is fed daily at 3% of the initial culture volume, and Feed 7b is fed daily at one thousandth of the initial culture volume until the 12th day (feeding on the 12th day). Glucose is added at 3g / L on the sixth day. Samples are collected on the 13th day. Affinity purification is performed using a proteinA affinity chromatography column. Take 6μg of the purified antibody for reducing SDS-PAGE, and the electrophoresis is shown as follows Figure 1 After reducing SDS-PAGE, two bands were shown, one with Mr of 50KD (heavy chain) and the other with Mr of 28KD (light chain).

[0113] Example 2 Affinity and activity optimization

[0114] Although the Anti-T4 8F6 monoclonal antibody obtained in Example 1 has the ability to bind tetraiodothyronine, its affinity and antibody activity are not ideal. Therefore, the applicant conducted directed mutations on the light chain CDR and heavy chain CDR of the antibody. That is, a computer was used to simulate the structure of the antibody variable region, the structure of the complex of the antigen and the antibody variable region, analyze the key amino acids of the antibody and design mutations. According to the mutation scheme, bidirectional primers covering the mutation site were designed and synthesized, primers at both ends of the target DNA were synthesized, a high-fidelity PCR reaction was performed, the PCR product was cloned into a vector, and then the mutant antibody was prepared according to the method described in Example 1. After screening, monoclonal antibodies with significantly improved affinity and antibody activity were obtained and named Anti-T4-8F6RMb1 to Anti-T4-8F6RMb9, and the heavy chain and light chain amino acid sequences are as follows:

[0115] Table 1 Antibody sequences

[0116] Sample name Heavy chain number Light chain number Anti-T4-8F6RMb1 SEQ ID NO:18 SEQ ID NO:31 Anti-T4-8F6RMb2 SEQ ID NO:23 SEQ ID NO:31 Anti-T4-8F6RMb3 SEQ ID NO:21 SEQ ID NO:31 Anti-T4-8F6RMb4 SEQ ID NO:19 SEQ ID NO:31 Anti-T4-8F6RMb5 SEQ ID NO:18 SEQ ID NO:34 Anti-T4-8F6RMb6 SEQ ID NO:18 SEQ ID NO:32 Anti-T4-8F6RMb7 SEQ ID NO:23 SEQ ID NO:32 Anti-T4-8F6RMb8 SEQ ID NO:21 SEQ ID NO:32 Anti-T4-8F6RMb9 SEQ ID NO:19 SEQ ID NO:32

[0117] Example 3 Affinity Analysis

[0118] Using the AMC sensor, the purified antibody was diluted to 10 μg / ml with PBST, and T4-BSA (purchased from biospacific, VI9100) was serially diluted with PBST.

[0119] Operation process: 60s of equilibration in buffer 1 (PBST), 300s of antibody fixation in antibody solution, 180s of incubation in buffer 2 (PBST), 420s of binding in antigen solution, 1200s of dissociation in buffer 2, sensor regeneration with 10mM pH 1.69GLY solution and buffer 3 (PBST), and data output. (K D represents the equilibrium dissociation constant or affinity; kon represents the binding rate; kdis represents the dissociation rate. The main components of PBST are Na2HPO4+NaCl+TW-20).

[0120] Table 2 Affinity analysis data

[0121] Sample name <![CDATA[K D (M)]]> kon(1 / Ms) kdis(1 / s) Control Antibodies 7.17E-09 1.23E+05 8.82E-04 Anti-T4-8F6RMb1 2.44E-10 2.30E+06 5.60E-04 Anti-T4-8F6RMb2 4.56E-10 2.59E+05 1.18E-04 Anti-T4-8F6RMb3 3.18E-10 3.37E+05 1.07E-04 Anti-T4-8F6RMb4 1.53E-10 6.81E+05 1.04E-04 Anti-T4-8F6RMb5 3.43E-10 3.24E+06 1.11E-03 Anti-T4-8F6RMb6 3.15E-10 3.32E+06 1.05E-03 Anti-T4-8F6RMb7 9.67E-10 4.13E+05 3.99E-04 Anti-T4-8F6RMb8 1.49E-10 2.13E+06 3.17E-04 Anti-T4-8F6RMb9 1.29E-10 2.67E+06 3.44E-04

[0122] Example 4 Activity Identification

[0123] The coating solution (main component NaHCO3) was used to dilute T4-BSA (purchased from biospacific, VI9100) to 3μg / ml, 100μL per well, and incubated at 4℃ overnight; the next day, the cells were washed twice with washing solution (main component Na2HPO4+NaCl) and patted dry; blocking solution (20% BSA+80% PBS) was added, 120μL per well, incubated at 37℃ for 1h, and patted dry; diluted purified antibody and control antibody were added, 100μL / well, 37℃, 30min; the cells were washed 5 times with washing solution and patted dry; goat anti-mouse IgG-HRP was added, 100μL per well, 37℃, 30min; the cells were washed 5 times with washing solution and patted dry; color developing solution A (50μL / well) and color developing solution B (50μL / well) were added for 10min; stop solution was added, 50μL / well; OD value was read at 450nm (reference 630nm) on the microplate reader.

[0124] Note: Solution A (main components: citric acid + sodium acetate + acetanilide + urea peroxide); Solution B (main components: citric acid + EDTA·2Na + TMB + concentrated HCL); Stop solution (EDTA·2Na + concentrated H2SO4).

[0125] Table 3 Activity data

[0126]

[0127]

[0128] Example 5 Antibody Stability Assessment

[0129] The above antibodies were placed at 4°C (refrigerator), -80°C (refrigerator), and 37°C (constant temperature box) for 21 days, and samples were taken for 7 days, 14 days, and 21 days for status observation, and the activity of the 21-day samples was tested. The results showed that there was no obvious protein state change in the antibodies under the three test conditions for 21 days, and the activity did not show a downward trend with the increase of the test temperature, indicating that the expressed antibodies were stable. The following table shows the OD results of the enzyme immunoassay activity test after 21 days of testing.

[0130] Table 4 Stability data

[0131] Sample concentration (ng / ml) 1000 250 0 4℃, 21-day samples 2.256 1.290 0.040 -80℃, 21 days sample 2.243 1.270 0.051 37℃, 21 days sample 2.277 1.278 0.054

[0132] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0133] The partial amino acid sequences involved in this application are as follows:

[0134]

Claims

1. An anti-tetraiodothyronine antibody or an antigen-binding fragment thereof, characterized in that: The antibody or its antigen-binding fragment comprises HCDR1-3 and LCDR1-3, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID Nos: 1, 2 and 4, or 1, 3 and 4, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID Nos: 5, 6, 7, or SEQ ID Nos: 5, 6, 8, respectively.

2. An anti-tetraiodothyronine antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, and the amino acid sequences of the heavy chain variable region and the light chain variable region are selected from any combination of the following: 。 3. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof has a heavy chain variable region and a light chain variable region that are 80% or more identical to the sequence shown in claim 2.

4. An anti-tetraiodothyronine antibody, characterized in that: The antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, and the amino acid sequences of the heavy chain and the light chain are selected from any combination of the following:

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 or the antibody according to claim 4, characterized in that: The antibody or antigen-binding fragment thereof is expressed as K D <7.17×10 -09 M binds tetraiodothyronine with high affinity.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that: The antibody or antigen-binding fragment thereof further comprises a constant region.

7. The antibody or antigen-binding fragment thereof according to claim 6, characterized in that: The constant region includes a heavy chain constant region and a light chain constant region.

8. The antibody or antigen-binding fragment thereof according to claim 7, characterized in that: The heavy chain constant region is selected from the heavy chain constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; the light chain constant region is selected from the κ type or λ type light chain constant region.

9. The antibody or antigen-binding fragment thereof according to claim 6, characterized in that: The species origin of the constant region is any one of cattle, horses, pigs, sheep, rats, mice, dogs, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese and humans.

10. The antibody or antigen-binding fragment thereof according to claim 6, characterized in that: The species origin of the constant region is sheep.

11. The antibody or antigen-binding fragment thereof according to claim 7, characterized in that: The heavy chain constant region sequence is as shown in SEQ ID NO: 17 or has at least 80% identity thereto; the light chain constant region sequence is as shown in SEQ ID NO: 30 or has at least 80% identity thereto.

12. The 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 any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

13. An antibody conjugate, characterized in that: The antibody conjugate consists of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 and 5 to 12 or the antibody according to claim 4 and biotin or a label conjugated thereto.

14. The antibody conjugate according to claim 13, characterized in that: The antibody conjugate further comprises a solid phase carrier coupled to the antibody or antigen-binding fragment thereof.

15. The antibody conjugate according to claim 13, characterized in that The marker is selected from at least one of fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents and nanoparticle markers.

16. The antibody conjugate according to claim 13, characterized in that: The label is colloidal gold.

17. A reagent or a kit, characterized in that: It includes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, 5 to 12, the antibody according to claim 4, or the antibody conjugate according to claims 10 to 13.

18. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, 5 to 12, or the antibody according to claim 4 in the preparation of a product for detecting tetraiodothyronine, characterized in that: It includes: The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, 5 to 12 or the antibody according to claim 4 is mixed with a sample to be detected, so that the antibody or antigen-binding fragment thereof contacts tetraiodothyronine in the sample to be detected to form an immune complex.

19. The use according to claim 18, characterized in that The immune complex also includes a second antibody that binds to the antibody or antigen-binding fragment thereof.

20. The use according to claim 18, characterized in that The immune complex also includes a second antibody that binds to tetraiodothyronine.

21. An isolated nucleic acid, characterized in that It encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, 5 to 12 or the antibody according to claim 4.

22. A carrier, characterized in that It contains the nucleic acid according to claim 21.

23. A recombinant cell, characterized in that It contains the nucleic acid according to claim 21 or the vector according to claim 22.

24. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, 5 to 12 or the antibody according to claim 4, characterized in that: It includes: Cultivate the recombinant cell of claim 23.

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