Anti-NT-proBNP antibodies and uses thereof

Abstract

The invention discloses an anti-NT-proBNP antibody and application thereof, and relates to the field of antibodies. The anti-NT-proBNP antibody disclosed by the invention comprises a heavy chain complementarity determining region and a light chain complementarity determining region, provides an important raw material source for the detection of NT-proBNP, and has excellent affinity or activity.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present disclosure claims priority to a Chinese patent application with application number 202311395873.2 filed with the Chinese Patent Office on October 25, 2023, entitled “Anti-NT-proBNP Antibodies and Uses Thereof,” the entire contents of which are incorporated by reference into the present disclosure. Technical Field

[0003] The present invention belongs to the technical field of antibodies. Specifically, the present invention relates to an NT-proBNP antibody and uses thereof. More specifically, the present invention relates to an NT-proBNP antibody, a nucleic acid molecule, a vector, a cell or a host, a method for preparing the antibody, a conjugate, a reagent or a kit and uses thereof, a method for detecting NT-proBNP, a method for screening NT-proBNP antibodies and a mutation library. Background Art

[0004] In 1988, Japanese scholar Sudoh first isolated a polypeptide with strong diuretic, vasodilator and hypotensive effects from pig brain and named it brain natriuretic peptide (BNP). BNP is distributed in the heart with the highest content, but the first thing synthesized by myocardial cells is proBNP (BNP precursor) containing 108 amino acids. When myocardial cells are stimulated, proBNP is cleaved into N-terminal pro-B-type natriuretic peptide (NT-proBNP) containing 76 amino acids and no biological activity under the action of endonucleases.

[0005] When the cardiac volume load increases or the heart function is impaired, the index concentration of N-terminal pro-brain natriuretic peptide (NT-proBNP) will increase abnormally. NT-proBNP has good biological stability, a long half-life (120 minutes), a relatively stable concentration, and a long effective detection time. Therefore, it is relatively easy to detect. In addition, the plasma specimen has a long stability in vitro and is the best myocardial marker for diagnosing heart failure and evaluating cardiac function.

[0006] The NT-proBNP content in normal human blood is generally less than 0.3 ng / mL. When cardiac function is impaired and the myocardium is dilated, NT-proBNP will be rapidly synthesized and secreted and released into the human blood in large quantities. When some related early symptoms are discovered, accurate, sensitive, efficient and stable determination of the amount of NT-proBNP in the blood can provide a rapid and accurate early diagnostic reference for the treatment and prognosis monitoring of early heart failure, heart failure, dyspnea, cardiogenic and non-cardiogenic heart failure, and the classification of acute coronary syndrome. The methods currently used to detect the NT-proBNP content mainly include gold-labeled qualitative tests, fluorescent immunoassays, enzyme-linked immunosorbent assays (ELISA) and magnetic particle chemiluminescence assays (CMIA), and these measurement methods all require specific monoclonal antibodies against NT-proBNP. Therefore, those skilled in the art have a strong demand for anti-NT-proBNP antibodies with good performance. Summary of the invention

[0007] The present invention aims to provide an anti-NT-proBNP antibody, and a reagent or kit for detecting NT-proBNP.

[0008] In one aspect of the present invention, the present invention provides an anti-NT-proBNP antibody, comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, wherein the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 have the amino acid sequences shown below:

[0009] HCDR 1:DFAIH;

[0010] HCDR 2: WINTRNNKX1TYAX2DFKG, wherein X1 is S, A or Q, and X2 is D or A;

[0011] HCDR 3:SRHDWFAY;

[0012] LCDR 1:TLSX3QHSTYTIE, where X3 is S, A, or P;

[0013] LCDR 2: VEKDGSLX4X5GD, where X4 is S, L or V, and X5 is T or P;

[0014] LCDR 3:GVGDTIEEQFVYV.

[0015] In a second aspect of the present invention, the present invention provides an anti-NT-proBNP antibody, comprising a heavy chain variable region and / or a light chain variable region; the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 17 or a variant thereof, and compared with the amino acid sequence as shown in SEQ ID NO: 17, the variant of SEQ ID NO: 17 comprises a mutation in at least one of the following sites: S58A, S58Q, and D62A;

[0016] The light chain variable region includes an amino acid sequence as shown in SEQ ID NO: 18 or a variant thereof. Compared with the amino acid sequence shown in SEQ ID NO: 18, the variant of SEQ ID NO: 18 includes mutations in the following sites: at least one of S26A, S26P, S57L, S57V, and T58P.

[0017] In the third aspect of the present invention, the present invention proposes an anti-NT-proBNP antibody, comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, wherein the HCDR1, HCDR2, HCDR3 are amino acid sequences consistent with the HCDR1, HCDR2, HCDR3 of the heavy chain variable region defined by the antibody described in the second aspect; the LCDR1, LCDR2, LCDR3 are amino acid sequences consistent with the LCDR1, LCDR2, LCDR3 of the light chain variable region defined by the antibody described in the second aspect.

[0018] In the fourth aspect of the present invention, the present invention proposes a nucleic acid molecule, a vector, a cell or a host or a method for preparing the above-mentioned antibody, wherein the nucleic acid molecule encodes the antibody described in the first aspect, the second aspect or the third aspect, the vector comprises the aforementioned nucleic acid molecule, the cell or host comprises the aforementioned nucleic acid molecule or vector or expresses the antibody described in the first aspect, the second aspect or the third aspect, and the method comprises culturing the aforementioned cell or host.

[0019] In the fifth aspect of the present invention, the present invention provides a conjugate, which comprises: the antibody described in the first aspect, the second aspect or the third aspect and a conjugated part conjugated thereto.

[0020] In the sixth aspect of the present invention, the present invention provides a reagent or a kit, wherein the reagent or the kit comprises: the antibody described in the first aspect, the second aspect or the third aspect or the conjugate described in the fifth aspect.

[0021] In the seventh aspect of the present invention, the present invention proposes use of the antibody described in the first, second or third aspect, the conjugate described in the fifth aspect, or the reagent or kit described in the sixth aspect in detecting NT-proBNP, preparing a product for detecting NT-proBNP, or preparing a product for diagnosing NT-proBNP-related diseases.

[0022] In the seventh aspect of the present invention, a method for detecting NT-proBNP is proposed, comprising: contacting the antibody described in the first, second or third aspect, the conjugate described in the fifth aspect, or the reagent or kit described in the sixth aspect with a sample to be detected to form an immune complex.

[0023] In the eighth aspect of the present invention, a method for screening NT-proBNP antibodies is provided, the method comprising: a) designing primers for performing amino acid substitutions at sites X1, X2, X3, X4 and X5 defined in the antibody described in the first aspect, or at mutation sites defined in the antibody described in the second aspect; b) using the nucleic acid molecule, vector or cell described in the fourth aspect as a template and constructing a mutation library using the primers described in a); c) screening NT-proBNP antibodies from the mutation library.

[0024] In the ninth aspect of the present invention, the present invention provides a mutation library, which includes the antibody described in the first aspect, the second aspect or the third aspect.

[0025] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. DETAILED DESCRIPTION

[0026] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0027] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0028] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0029] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. The abbreviations for amino acid residues are standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0030] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0031] As used herein, the terms "optionally", "optional", "optionally", "optional" or "optional" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur.

[0032] Herein, the term "antibody" is used in the broadest sense and may include full-length monoclonal antibodies, multispecific antibodies, chimeric antibodies or antigen-binding fragments without limitation to the specific structure, as long as they exhibit the desired antigen-binding ability.

[0033] As used herein, the terms "full-length antibody", "full-length monoclonal antibody" or "full-length monoclonal antibody" are composed of at least two identical light chains and at least two identical heavy chains connected by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD) or immunoglobulin E (IgE).

[0034] In this article, the terms "polyantibody" and "multispecific antibody" are synonymous, and both refer to antibodies that can recognize multiple antigenic epitopes, such as antibodies that can recognize two antigenic epitopes (bispecific antibodies, referred to as bispecific antibodies), antibodies that recognize three antigenic epitopes, or antibodies that recognize four antigenic epitopes. They are understood in a broad sense, and the specific structure is not limited, as long as they can recognize multiple antigenic epitopes. In the present invention, at least one of the multiple antigenic epitopes is derived from NT-proBNP.

[0035] In this article, the term "antigen binding fragment" is a fragment comprising part or all of an antibody, which lacks at least some of the amino acids present in the full-length chain but is still capable of specifically binding to the antigen. For example, the fragment may comprise part or all of the antibody CDR. Such fragments bind to the antigen and can compete with other antigen binding molecules (including intact antibodies) for binding to a given epitope. Such fragments include, but are not limited to, Fab, Fab', F(ab)2, F(ab')2, Fv, scFv, scFv-Fc fusion protein, scFv-Fv fusion protein, single domain antibody, or minimum recognition unit. Such fragments can be produced by recombinant nucleic acid technology, or can be produced by enzymatic cleavage or chemical cleavage of antigen binding molecules (including intact antibodies).

[0036] As used herein, the term "Fab antibody" or "Fab fragment" generally refers to an antibody or fragment containing only the Fab molecule, which is composed of the VH and CH1 of the heavy chain and a complete light chain, with the light chain and the heavy chain connected by a disulfide bond.

[0037] As used herein, the term "F(ab')2 antibody" or "F(ab')2 fragment" has two antigen-binding F(ab') portions linked together by a disulfide bond.

[0038] As used herein, the term "Fv antibody" or "Fv fragment" generally refers to an antibody or fragment consisting only of a light chain variable region (VL) and a heavy chain variable region (VH) connected by non-covalent bonds, and is the smallest functional fragment of an antibody that retains a complete antigen binding site.

[0039] As used herein, the terms "single-chain antibody" and "scFv fragment" refer to antibodies or fragments formed by connecting the heavy chain variable region and the light chain variable region of an antibody via a short peptide.

[0040] In this article, the terms "minimum recognition unit" and "MRU" both refer to antibodies or fragments consisting of only one CDR, and their molecular weight is very small, accounting for only about 1% of the complete antibody.

[0041] As used herein, the terms "complementarity determining region", "CDR" or "CDRs" refer to the highly variable regions of the heavy and light chains of immunoglobulins, and refer to the regions containing one or more or even all of the major amino acid residues that contribute to the binding affinity of the antibody to its recognized antigen or epitope.

[0042] In this article, the heavy chain complementary determining region (heavy chain variable region CDR) is represented by "HCDRs" or "HCDR", which includes HCDR1 (also known as CDR-H1), HCDR2 (also known as CDR-H2) and HCDR3 (also known as CDR-H3); the light chain complementary determining region (light chain variable region CDR) is represented by "LCDRs" or "LCDR", which includes LCDR1 (also known as CDR-L1), LCDR2 (also known as CDR-L2) and LCDR3 (also known as CDR-L3). Commonly used CDR definition schemes in the art include: Kabat definition, Chothia definition, IMGT definition, Contact definition and AbM definition. As described herein, "Kabat definition" refers to the definition system described in Kabat et al., USDept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia definition" refers to Chothia et al., J Mol Biol 196: 901-917 (1987). Exemplary defined CDRs are listed in Table 1 below. The definitions in different documents are slightly different. Given the variable region amino acid sequence of an antibody, a person skilled in the art can routinely determine which residues comprise a specific CDR. It should be noted that the CDRs in the present invention include but are not limited to CDRs defined by other methods in Table 1. CDRs determined based on the heavy chain variable region and light chain variable region disclosed in this application using other rules disclosed in the art also fall within the scope of protection of the present disclosure.

[0043] Table 1: CDR Definition 1

[0044] CDR Kabat <![CDATA[AbM 2 ]]> IMGT Chothia HCDR1 <![CDATA[H31~H35 3 ]]> <![CDATA[H26~H35 3 ]]> <![CDATA[H26~H33..35 5 ]]> <![CDATA[H26~H32..34 4 ]]> HCDR2 H50~H65 H50~H58 H51~H57 H52~H56 HCDR3 H95~H102 H95~H102 H93~H102 H95~H102 LCDR1 L24~L34 L24~L34 L27~L32 L24~L34 LCDR2 L50~L56 L50~L56 L50~L51 L50~L56 LCDR3 L89~L97 L89~L97 L89~L97 L89~L97

[0045] 1 The numbering of all CDR definitions in Table 1 is based on the Kabat numbering system (see below), with amino acid numbers on the heavy chain represented by "H+numbers" and amino acid numbers on the light chain represented by "L+numbers".

[0046] 2 "AbM" as used in Table 1 with a lowercase "b" refers to CDRs defined by Oxford Molecular's "AbM" antibody modeling software.

[0047] 3 If both H35A and H35B are absent, HCDR1 ends at position 35; if only H35A is present, HCDR1 ends at position 35A; if both H35A and H35B are present, HCDR1 ends at position 35B.

[0048] 4 If both H35A and H35B are absent, HCDR1 ends at position 32; if only H35A is present, HCDR1 ends at position 33; if both H35A and H35B are present, HCDR1 ends at position 34.

[0049] 5 If both H35A and H35B are absent, HCDR1 ends at position 33; if only H35A is present, HCDR1 ends at position 34; if both H35A and H35B are present, HCDR1 ends at position 35.

[0050] Kabat et al. also defined a numbering system applicable to the variable region sequence of any antibody. A person of ordinary skill in the art can clearly correspond the Kabat numbering system to any variable region sequence without relying on any experimental data other than the sequence itself. As described herein, "Kabat numbering" refers to numbering using the numbering system described in "Kabat et al., U. S. Patent. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983)". The above numbering system is used to number the HCDRs and LCDRs of the antibodies of the present application, and the specific numbering results are shown in Table 1. It should be noted that the polypeptide sequences of the present invention are not numbered according to the Kabat numbering system. However, a person of ordinary skill in the art is fully capable of converting the sequence numbers of the sequence table into Kabat numbers.

[0051] As used herein, the term "framework region" or "FR" region includes a heavy chain framework region and a light chain framework region, and refers to the region other than CDR in the antibody heavy chain variable region (which may be represented as VH) and the light chain variable region (which may be represented as VL); wherein the heavy chain framework region is represented by "HFR" and can be further subdivided into adjacent regions separated by CDRs, comprising HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region is represented by "LFR" and can be further subdivided into adjacent regions separated by CDRs, comprising LFR1, LFR2, LFR3 and LFR4 framework regions.

[0052] In this article, the heavy chain variable region is obtained by arranging and connecting the CDRs and FRs with the following numbers in the following combinations: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by arranging and connecting the CDRs and FRs with the following numbers in the following combinations: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0053] As used herein, the terms "identity", "homology" or "similarity" are used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 48:443); the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:24 ... .70:173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs that utilize these algorithms are also available, and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0054] Under the premise of not substantially affecting the activity of the antibody (retaining at least 90% of the activity), those skilled in the art can replace, add and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids in the sequence of the present invention to obtain variants of the antibody sequence. They are all considered to be included in the scope of protection of the present invention. For example, amino acids with similar properties are replaced in the variable region. The variant sequence of the present invention can have at least 90%, 95%, 96%, 97%, 98% or 99% consistency (or homology) with the reference sequence. The sequence consistency of the present invention can be measured using sequence analysis software. For example, the computer program BLAST with default parameters is used, especially BLASTP or TBLASTN. The amino acid sequences described in the present invention are all shown in a manner from N-terminus to C-terminus.

[0055] It should be noted that the position or number of the mutation sites defined in the specification and claims of the present invention also needs to be adjusted according to the number and position of the added and / or deleted amino acids.

[0056] As used herein, the term "at least 80% homology" refers to at least 80%, and may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% homology to each reference sequence. The term "at least 90% homology" refers to at least 90%, and may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% homology to each reference sequence.

[0057] As used herein, the term "variant" or "mutant" may refer to any naturally occurring or engineered molecule comprising one or more nucleotide or amino acid mutations.

[0058] In this article, the term "vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and the inserted nucleic acid molecule is transferred to a cell or host and / or between cells or hosts. The vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used to express the transcription and / or translation of DNA or RNA. The vector also includes a vector with a variety of the above functions. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable cell or host. Typically, the vector can produce a desired expression product by cultivating a suitable cell or host that contains the vector.

[0059] In this article, the term "cell" generally refers to the use of genetic engineering technology or cell fusion technology to modify or reorganize the genetic material of the host cell to obtain a cell with a unique trait of stable inheritance. Among them, the term "host cell" refers to a prokaryotic cell or eukaryotic cell that can be introduced into a recombinant vector. The terms "transformed" or "transfected" used herein refer to the introduction of nucleic acids (such as vectors) into cells by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequence of the present invention, and can be used for the expression and / or secretion of target proteins. Examples of suitable host cells that can be used in the present invention include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid) and CoS cells.

[0060] The present invention provides an anti-NT-proBNP antibody, a nucleic acid molecule, a vector, a cell or a host, a method for preparing the antibody, a conjugate, a reagent or a kit and uses thereof, a method for detecting NT-proBNP, a method for screening NT-proBNP antibodies and a mutant library, which will be described in detail below.

[0061] Antibody

[0062] In the first aspect of the present invention, the present invention provides an anti-NT-proBNP antibody. According to an embodiment of the present invention, the antibody comprises HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, and the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 are the amino acid sequences shown below:

[0063] HCDR 1:DFAIH;

[0064] HCDR 2: WINTRNNKX1TYAX2DFKG, wherein X1 is S, A or Q, and X2 is D or A;

[0065] HCDR 3:SRHDWFAY;

[0066] LCDR 1:TLSX3QHSTYTIE, where X3 is S, A, or P;

[0067] LCDR 2: VEKDGSLX4X5GD, where X4 is S, L or V, and X5 is T or P;

[0068] LCDR 3:GVGDTIEEQFVYV.

[0069] According to an embodiment of the present invention, the HCDRs and LCDRs are defined by the Kabat system.

[0070] It can be seen from the aforementioned definition principles of CDR that different systems are used for definition, even if the same heavy chain variable region or the same light chain variable region, the obtained CDR is different. "HCDR 1: DFAIH" in this application means that the amino acid sequence of HCDR 1 includes or is DFAIH, which can also be adjusted according to different systems to obtain a HCDR 1 with a sequence longer than DFAIH.

[0071] In some optional embodiments of the present invention, X1 is S;

[0072] In some optional embodiments of the present invention, X1 is A;

[0073] In some optional embodiments of the present invention, X1 is Q;

[0074] In some optional embodiments of the present invention, X2 is D;

[0075] In some optional embodiments of the present invention, X2 is A;

[0076] In some optional embodiments of the present invention, X3 is S;

[0077] In some optional embodiments of the present invention, X3 is A;

[0078] In some optional embodiments of the present invention, X3 is P;

[0079] In some optional embodiments of the present invention, X4 is S;

[0080] In some optional embodiments of the present invention, X4 is L;

[0081] In some optional embodiments of the present invention, X4 is V;

[0082] In some optional embodiments of the present invention, X5 is T;

[0083] In some optional embodiments of the present invention, X5 is P;

[0084] In some optional embodiments of the present invention, X1 is S, X2 is D, X3 is S, X4 is S and X5 is T are not simultaneously true;

[0085] In some optional embodiments of the present invention, X1, X2, X3, X4 and X5 are selected from any one of the following combinations:

[0086] combination X1 X2 X3 X4 X5 1 S D A S T 2 S D P S T 3 S D S L T 4 S D S V T 5 S D S S P 6 A D S S T 7 Q D S S T 8 S A S S T 9 Q A S S T 10 Q A P S P 11 Q A A V P 12 Q D S S P 13 Q D P V P 14 Q D S L P .

[0087] The antibody described in the first aspect of the present invention has excellent activity, affinity, stability or specificity.

[0088] The antibody according to the first aspect of the present invention has improved activity, affinity, stability or specificity.

[0089] In the second aspect of the present invention, the present invention provides an anti-NT-proBNP antibody. According to an embodiment of the present invention, the antibody or functional fragment comprises: a heavy chain variable region and / or a light chain variable region; the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 17 or a variant thereof, and compared with the amino acid sequence as shown in SEQ ID NO: 17, the variant of SEQ ID NO: 17 comprises a mutation at the following sites: at least one of S58A, S58Q, and D62A; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 18 or a variant thereof, and compared with the amino acid sequence as shown in SEQ ID NO: 18, the variant of SEQ ID NO: 18 comprises a mutation at the following sites: at least one of S26A, S26P, S57L, S57V, and T58P;

[0090] It should be noted that the numbering of the heavy chain variable region sites is obtained by sequentially numbering the amino acid sequence shown in SEQ ID NO: 17 from the N-terminus to the C-terminus. For example, position 58 refers to the 58th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 17; the "S58A" means that the serine at position 58 of the amino acid sequence shown in SEQ ID NO: 17 is replaced by alanine.

[0091] The numbering of the above light chain variable region sites is obtained by sequentially numbering the amino acid sequence shown in SEQ ID NO: 18 from the N-terminus to the C-terminus. For example, position 26 refers to the 26th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 18; the "S26A" means that the serine at position 26 of the amino acid sequence shown in SEQ ID NO: 18 is replaced by alanine.

[0092] According to an embodiment of the present invention, the above antibody may further include at least one of the following additional technical features:

[0093] In some optional embodiments of the present invention, the mutation at position 58 is S58A;

[0094] In some optional embodiments of the present invention, the mutation at position 58 is S58Q;

[0095] In some optional embodiments of the present invention, the mutation at position 62 is D62A;

[0096] In some optional embodiments of the present invention, the mutation at position 26 is S26A;

[0097] In some optional embodiments of the present invention, the mutation at position 26 is S26P;

[0098] In some optional embodiments of the present invention, the mutation at position 57 is S57L;

[0099] In some optional embodiments of the present invention, the mutation at position 57 is S57V;

[0100] In some optional embodiments of the present invention, the mutation at position 58 is T58P;

[0101] In some optional embodiments of the present invention, the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 17, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 18, which are not true at the same time;

[0102] In some optional embodiments of the present invention, the heavy chain variable region and the light chain variable region are selected from any one of the following combinations:

[0103]

[0104] It should be noted that the “,” in the above table means “and”, that is, a combined mutation. For example, “S58Q, D62A” means a combined mutation of S58Q and D62A.

[0105] In the third aspect of the present invention, the present invention provides an anti-NT-proBNP antibody. According to an embodiment of the present invention, the antibody comprises HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, wherein the HCDR1, HCDR2, HCDR3 are amino acid sequences consistent with the HCDR1, HCDR2, HCDR3 of the heavy chain variable region defined by the antibody described in the second aspect; and the LCDR1, LCDR2, LCDR3 are amino acid sequences consistent with the LCDR1, LCDR2, LCDR3 of the light chain variable region defined by the antibody described in the second aspect.

[0106] It should be noted that the HCDR1, HCDR2 and HCDR3 in the antibody described in the third aspect are amino acid sequences consistent with the HCDR1, HCDR2 and HCDR3 of the same heavy chain variable region defined in the antibody described in the second aspect, and the LCDR1, LCDR2 and LCDR3 are amino acid sequences consistent with the LCDR1, LCDR2 and LCDR3 of the same light chain variable region defined in the antibody described in the second aspect.

[0107] For example, when the heavy chain variable region defined in the antibody described in the second aspect has no mutation compared to SEQ ID NO: 17, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region defined by Kabat are HCDR 1: DFAIH; HCDR 2: WINTRNNKSTYADDFKG; HCDR 3: SRHDWFAY; then the HCDR1, HCDR2, and HCDR3 contained in the antibody described in the third aspect are also HCDR 1: DFAIH; HCDR 2: WINTRNNKSTYADDFKG; HCDR 3: SRHDWFAY.

[0108] For example, when the light chain variable region defined in the antibody described in the second aspect only has a mutation at the S26A site compared to SEQ ID NO: 18, the LCDR1, LCDR2, and LCDR3 of the light chain variable region defined by Kabat are LCDR 1: TLSAQHSTYTIE; LCDR 2: VEKDGSLSTGD; LCDR 3: GVGDTIEEQFVYV, respectively; then the LCDR1, LCDR2, and LCDR3 contained in the antibody described in the third aspect are also LCDR 1: TLSAQHSTYTIE; LCDR 2: VEKDGSLSTGD; LCDR 3: GVGDTIEEQFVYV, respectively.

[0109] According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 is defined by any one system or a combination of multiple systems of Kabat, Chothia, IMGT, AbM or Contact.

[0110] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

[0111] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Chothia system.

[0112] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT system.

[0113] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the AbM system.

[0114] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a Contact system.

[0115] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a combination of Kabat, Chothia, IMGT, AbM or Contact systems.

[0116] According to an embodiment of the present invention, the Kabat numbering positions corresponding to the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 defined by the Kabat, Chothia, AbM or IMGT systems are as follows:

[0117]

[0118]

[0119] According to an embodiment of the present invention, the antibody described in the first aspect or the third aspect further comprises at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4.

[0120] In an optional embodiment of the present invention, at least a portion of at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4 is derived from at least one of mouse antibodies, human antibodies, primate antibodies, bovine antibodies, horse antibodies, dairy cow antibodies, porcine antibodies, sheep antibodies, goat antibodies, dog antibodies, cat antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting cock antibodies or their mutants.

[0121] According to an embodiment of the present invention, the HFR1 includes the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having at least 80% homology thereto; the HFR2 includes the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence having at least 80% homology thereto; the HFR3 includes the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having at least 80% homology thereto; the HFR4 includes the amino acid sequence shown in SEQ ID NO:10 or an amino acid sequence having at least 80% homology thereto; the LFR1 includes the amino acid sequence shown in SEQ ID NO:11 or an amino acid sequence having at least 80% homology thereto; the LFR2 includes the amino acid sequence shown in SEQ ID NO:12 or an amino acid sequence having at least 80% homology thereto; the LFR3 includes the amino acid sequence shown in SEQ ID NO:13 or an amino acid sequence having at least 80% homology thereto; the LFR4 includes the amino acid sequence shown in SEQ ID NO:14 or an amino acid sequence having at least 80% homology thereto.

[0122] According to an embodiment of the present invention, the antibody described in the first aspect, the second aspect or the third aspect may further include at least one of the following technical features:

[0123] According to an embodiment of the present invention, the affinity of the antibody is KD<10 -9 M.

[0124] According to an embodiment of the present invention, the affinity of the antibody is KD<10 -10 M.

[0125] According to an embodiment of the present invention, the affinity of the antibody is KD<10 -11 M.

[0126] According to an embodiment of the present invention, the affinity of the antibody is KD<10 -12 M.

[0127] According to an embodiment of the present invention, the antibody further comprises a constant region; wherein the constant region comprises at least one of a heavy chain constant region and a light chain constant region.

[0128] According to an embodiment of the present invention, at least a portion of at least one of the heavy chain constant region and the light chain constant region is from at least one of a mouse antibody, a human antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy cow antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting cock antibody or a mutant thereof.

[0129] According to an embodiment of the present invention, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments.

[0130] In an optional embodiment of the present invention, the heavy chain constant region includes CH1 of IgG, a hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM.

[0131] In an optional embodiment of the present invention, the IgG is selected from IgG1, IgG2, IgG3, and IgG4.

[0132] According to an embodiment of the present invention, the light chain constant region comprises a light chain constant region selected from a κ type or a λ type.

[0133] In an optional embodiment of the present invention, the heavy chain constant region includes or is a heavy chain constant region as shown in SEQ ID NO:15 or an amino acid sequence with at least 80% identity thereto; or the light chain constant region includes or is a light chain constant region as shown in SEQ ID NO:16 or an amino acid sequence with at least 80% identity thereto.

[0134] According to an embodiment of the present invention, the N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region, and the N-terminus of the light chain constant region is connected to the C-terminus of the light chain variable region.

[0135] In this article, the division of variable and constant region sequences refers to the IMGT division method, see Lefranc, and Martinez-Jean C. and Bosc N. or Ehrenmann,Patrice Duroux,Chantal Ginestoux,Gene table:housemouse(Mus musculus)IGHC,IMGT Repertoire. the internationalImMunoGenetics information http: / / www.imgt.org.Created:16 / 03 / 2011.Version:17 / 01 / 2020.or Ehrenmann,Patrice Duroux,Chantal Ginestoux,Gene table:house mouse(Mus musculus)IGLC,IMGT Repertoire. theinternational ImMunoGenetics information http: / / www.imgt.org.Created:16 / 03 / 2011.Version:17 / 01 / 2020. The variable regions divided by different methods may have some amino acid differences with the variable region C-terminus or constant region N-terminus divided by IMGT. The variable regions or constant regions divided by other methods known in the art are also within the protection scope of the present invention.

[0136] According to an embodiment of the present invention, the heavy chain and light chain of the antibody are selected from any one of the following combinations:

[0137] combination Heavy chain SEQ ID NO: Light chain SEQ ID NO: 1 19 25 2 19 26 3 19 27 4 19 28 5 19 29 6 21 20 7 22 20 8 23 20 9 24 20 10 24 30 11 24 31 12 22 29 13 22 32 14 22 33 .

[0138] Those skilled in the art will appreciate that the features and advantages described above for the antibody described in the first aspect are also applicable to the antibody described in the second or third aspect and will not be described in detail herein.

[0139] Nucleic acid molecule, vector, cell or host, and method for preparing antibody

[0140] In the process of preparing or obtaining the antibodies described in the first aspect, the second aspect or the third aspect, nucleic acid molecules expressing these antibodies can be connected to different vectors and then expressed in different cells to obtain the corresponding antibodies.

[0141] In a fourth aspect of the present invention, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the antibody described in the first aspect, the second aspect or the third aspect. The nucleic acid molecule according to an embodiment of the present invention can encode the above-mentioned antibody.

[0142] According to an embodiment of the present invention, the nucleic acid molecule includes DNA or RNA.

[0143] It should be noted that, for the nucleic acid molecules mentioned herein, those skilled in the art will understand that they actually include any one or two of the complementary double strands. For convenience, in this article, although only one strand is provided in most cases, the other strand complementary thereto is actually disclosed. In addition, the molecular sequence in the present invention includes a DNA form or an RNA form, and disclosing one of them means that the other is also disclosed.

[0144] In the fifth aspect of the present invention, the present invention proposes a vector. According to an embodiment of the present invention, the vector includes the nucleic acid molecule described in the fourth aspect. When the above-mentioned nucleic acid molecule is connected to the vector, the nucleic acid molecule can be directly or indirectly connected to the control element on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can come directly from the vector itself, or they can be exogenous, that is, they are not from the vector itself. Of course, the nucleic acid molecule can be operably connected to the control element. Herein, "operably connected" means connecting the exogenous gene to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their expected functions of regulating the transcription and translation of the exogenous gene. Commonly used vectors can be, for example, plasmids, bacteriophages, etc. After the vectors according to some specific embodiments of the present invention are introduced into suitable recipient cells, the expression of the aforementioned antibodies can be effectively achieved under the mediation of the regulatory system, thereby achieving a large amount of antibodies obtained in vitro.

[0145] In some specific embodiments of the present invention, the vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus or a bacteriophage.

[0146] In an optional embodiment of the present invention, the expression vector is a plasmid expression vector.

[0147] In the sixth aspect of the present invention, the present invention provides a cell or host. According to an embodiment of the present invention, the cell or host comprises: the nucleic acid molecule described in the fourth aspect or the vector described in the fifth aspect; or expresses the antibody described in the first aspect, the second aspect or the third aspect. The cell can effectively express the aforementioned antibody in the cell under suitable conditions.

[0148] According to an embodiment of the present invention, the cell is obtained by introducing the vector described in the fifth aspect into the cell.

[0149] It should be noted that the cells of the present invention are not particularly limited and can be prokaryotic cells, eukaryotic cells or bacteriophages. The prokaryotic cells can be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc. The eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, insect cells such as armyworm, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells.

[0150] In an optional embodiment of the present invention, the cell is a mammalian cell, including a BHK cell, a CHO cell, a NSO cell or a COS cell, and does not include an animal germ cell, a fertilized egg or an embryonic stem cell.

[0151] It should be noted that the "suitable conditions" described in the present invention refer to conditions suitable for the expression of the antibodies of the present invention. It is easy for those skilled in the art to understand that the conditions suitable for the expression of the antibodies include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy cell states, suitable cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the antibodies according to the specific environment of the laboratory.

[0152] In the seventh aspect of the present invention, the present invention provides a method for preparing the antibody of the first aspect, the second aspect or the third aspect. According to an embodiment of the present invention, the method comprises culturing the cell or host of the sixth aspect. The method according to some specific embodiments of the present invention can effectively obtain the antibody in large quantities.

[0153] Based on the amino acid sequence of the antibody disclosed herein, it is easy for those skilled in the art to conceive of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the antibody, for example, separating and purifying the antibody from the culture product of a recombinant cell that can recombinantly express the antibody 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 disclosed herein, it belongs to the protection scope of the present disclosure.

[0154] Those skilled in the art will appreciate that the features and advantages described above for the antibodies described in the first, second or third aspects are also applicable to the nucleic acid molecule, vector, cell or host, and method for preparing antibodies, and will not be described in detail here.

[0155] Conjugate, reagent or kit and use thereof

[0156] In the eighth aspect of the present invention, the present invention provides a conjugate. According to an embodiment of the present invention, the conjugate comprises: the antibody according to the first aspect, the second aspect or the third aspect and a conjugated part conjugated thereto. The conjugate according to an embodiment of the present invention can specifically bind to NT-proBNP, and can be used for qualitative or quantitative detection of NT-proBNP, or for diagnosing NT-proBNP-related diseases.

[0157] According to an embodiment of the present invention, the conjugate may further include at least one of the following additional technical features:

[0158] According to an embodiment of the present invention, the coupling part includes at least one of a purification tag, an affinity substance, a marker and a solid phase carrier.

[0159] According to an embodiment of the present invention, the purification tag includes at least one of a His tag, a Flag tag, a GST tag, an MBP tag, a SUMO tag and a C-Myc tag.

[0160] Herein, the affinity substance may be, for example, biotin, a biotin derivative or streptavidin, one of the sense and antisense strands of nucleic acids that are complementary to each other.

[0161] In some optional embodiments of the present invention, the affinity substance includes at least one selected from biotin, a biotin derivative or streptavidin.

[0162] In this article, "marker" refers to a class of substances that have properties that can be directly observed by the naked eye or detected or detected by instruments, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0163] According to an embodiment of the present invention, the marker includes at least one selected from fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent agents and nanoparticle markers.

[0164] 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.

[0165] According to an embodiment of the present invention, the fluorescent dyes include but are 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, Cy3.39, 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. 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.).

[0166] According to an embodiment of the present invention, the enzyme includes but is not limited to horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase and 6-phosphoglucose deoxidase.

[0167] According to an embodiment of the present invention, the radioactive isotopes include but are not limited to 212 Bi, 131 I. 111 In, 90 Y. 186 Re, 211 At 125 I. 188 Re, 153 Sm, 213 Bi, 32 P. 94 mTc, 99 mTc, 203 Pb, 67 Ga, 68 Ga, 43 Sc, 47 Sc, 110 mIn、 97 Such as 62 Cu, 64 Cu, 67 Cu, 68 Cu, 86 Y. 88 Y. 121 Sn, 161 Tb, 166 Ho, 105 Rh, 177 Lu, 172 Lu and 18 F.

[0168] According to an embodiment of the present invention, 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.

[0169] According to an embodiment of the present invention, the nanoparticle markers include but are not limited to nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles.

[0170] According to an embodiment of the present invention, the colloid includes but is not limited to colloidal metal, dispersed dye, dye-labeled microspheres and latex.

[0171] According to an embodiment of the present invention, the colloidal metal includes but is not limited to colloidal gold, colloidal silver and colloidal selenium.

[0172] In this article, the solid phase carrier can be a substance that can be suspended or dispersed in a liquid phase (for example, solid phase carriers such as particles and magnetic beads), or a solid phase that can contain or carry a liquid phase (for example, supports such as plates, membranes, test tubes, and containers such as well as well plates, microfluidics, glass capillaries, nanocolumns, monolithic columns, etc.).

[0173] According to an embodiment of the present invention, the solid phase carrier includes at least one selected from microspheres, plates and membranes.

[0174] In the ninth aspect of the present invention, the present invention provides a reagent or a kit. According to an embodiment of the present invention, the reagent or the kit comprises: the antibody described in the first aspect, the second aspect or the third aspect or the conjugate described in the eighth aspect. As mentioned above, the antibody in some specific embodiments or examples of the present invention can bind to NT-proBNP, and therefore, the reagent or the kit comprising the antibody can effectively detect NT-proBNP qualitatively or quantitatively. The reagent or the kit provided by the present invention can be used, for example, for detection involving the specific binding properties of NT-proBNP and its antibody, such as immunoblotting and immunoprecipitation. As mentioned above, the mutant antibody of the present invention has improved NT-proBNP binding activity, affinity, stability or specificity, and therefore the reagent or the kit comprising the antibody has improved detection sensitivity or specificity.

[0175] The above kit may include any one or more of the following: treatment solution, anti-NT-proBNP antibody, NT-proBNP quality control product, anti-IgG antibody, instructions or literature, etc. Anti-NT-proBNP antibody can be used for different types of diagnostic tests, for example, it can be used to detect the presence of various diseases or drugs, or other proteins in vitro or in vivo. For example, it can be used to test related diseases by testing the serum or blood of the subject.

[0176] In the tenth aspect of the present invention, the present invention proposes use of the antibody of the first, second or third aspect, the conjugate of the eighth aspect, or the reagent or kit of the ninth aspect in detecting NT-proBNP, diagnosing NT-proBNP-related diseases, preparing a product for detecting NT-proBNP, or preparing a product for diagnosing NT-proBNP-related diseases.

[0177] According to an embodiment of the present invention, the "NT-proBNP-related disease" refers to a disease that uses NT-proBNP, including its protein or encoding nucleic acid, as a marker. In particular, in one or more embodiments of the present disclosure, the NT-proBNP-related disease includes heart failure, heart failure or acute coronary syndrome.

[0178] In the eleventh aspect of the present invention, the present invention provides a method for detecting NT-proBNP. According to an embodiment of the present invention, the method comprises: contacting the antibody described in the first aspect, the second aspect or the third aspect, the conjugate described in the eighth aspect or the reagent or kit described in the ninth aspect with a sample to be detected to form an immune complex.

[0179] It should be noted that the above-mentioned "samples to be tested" can be samples to be tested from patients, such as serum samples; or they can be non-patient samples that may contain NT-proBNP. For example, in scientific research, the above-mentioned method is only used to detect the presence or content of NT-proBNP in the sample and does not involve disease diagnosis.

[0180] According to an embodiment of the present invention, whether the sample to be tested contains NT-proBNP or the content of NT-proBNP is determined based on the signal of the immune complex.

[0181] According to an embodiment of the present invention, the immune complex further includes a second antibody, which binds to the antibody.

[0182] According to an embodiment of the present invention, the immune complex further comprises a second antibody, and the second antibody binds to NT-proBNP.

[0183] According to an embodiment of the present invention, the signal comprises a fluorescent signal.

[0184] Methods, mutation library

[0185] In the twelfth aspect of the present invention, the present invention provides a method for screening NT-proBNP antibodies. According to an embodiment of the present invention, the method comprises: a) designing primers for amino acid substitution at 1, 2, 3, 4 or 5 sites among X1, X2, X3, X4 and X5 defined in the antibody of the first aspect, or the mutation sites defined in the antibody of the second aspect; b) using the nucleic acid molecule of the fourth aspect, the vector of the fifth aspect or the cell or host of the sixth aspect as a template, and constructing a mutation library using the primers of a);

[0186] c) screening NT-proBNP antibodies from the mutant library.

[0187] According to an embodiment of the present invention, the mutation library is a single-site saturation mutation library.

[0188] According to an embodiment of the present invention, the NT-proBNP antibody includes or is the antibody described in the first aspect, the second aspect or the third aspect.

[0189] In the thirteenth aspect of the present invention, the present invention provides a mutation library, which includes the antibody described in the first aspect, the second aspect or the third aspect.

[0190] According to an embodiment of the present invention, the mutant library is obtained by the method described in the twelfth aspect.

[0191] In a fourteenth aspect of the present invention, the present invention provides a method for diagnosing NT-proBNP-related diseases. According to an embodiment of the present invention, the method comprises: contacting the antibody described in the first aspect, the second aspect or the third aspect, the conjugate described in the eighth aspect or the reagent or kit described in the ninth aspect with a sample to be detected from a subject to form an immune complex.

[0192] According to an embodiment of the present invention, the presence or absence of a NT-proBNP-related disease in the subject is indicated based on the signal of the immune complex.

[0193] In a fifteenth aspect of the present invention, the present invention provides a method for evaluating the prognosis of NT-proBNP-related diseases. According to an embodiment of the present invention, the method comprises: contacting the antibody described in the first aspect, the second aspect or the third aspect, the conjugate described in the eighth aspect or the reagent or kit described in the ninth aspect with a sample to be detected from a subject to form an immune complex.

[0194] According to an embodiment of the present invention, the signal of the immune complex indicates the prognosis or treatment effect of the NT-proBNP-related disease in the subject.

[0195] According to an embodiment of the present invention, the method described in the fourteenth and fifteenth aspects may further include at least one of the following technical features:

[0196] According to an embodiment of the present invention, the immune complex further includes a second antibody, which binds to the antibody.

[0197] According to an embodiment of the present invention, the immune complex further comprises a second antibody, and the second antibody binds to NT-proBNP.

[0198] According to an embodiment of the present invention, the signal comprises a fluorescent signal.

[0199] As used herein, the term "subject" refers to a vertebrate, preferably a mammal, most preferably a human. Mammals include, but are not limited to, rodents, apes, humans, livestock, competitive animals, and pets. Tissues, cells, and progeny of biological entities obtained in vivo or cultured in vitro are also included.

[0200] The amino acid sequences involved in this article are shown in Table 2:

[0201] Table 2: Amino acid sequences

[0202]

[0203]

[0204]

[0205]

[0206] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0207] 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.

[0208] Practice of the present disclosure 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., 2011), each of which is expressly incorporated herein by reference.

[0209] In this example, restriction endonucleases, T4 DNA ligase, and DNA polymerase were purchased from New England Biolabs, Taq DNA polymerase was purchased from TaKaRa, gel recovery kits and plasmid extraction kits were commercially available, and primer synthesis and gene sequencing were completed by an outsourcing company. The sequence of NT-proBNP monoclonal antibody (hereinafter referred to as WT antibody) was derived from mouse hybridoma cell sequencing.

[0210] Example 1: Construction and screening of mutation library

[0211] 1. Construction of wild-type (WT) NT-proBNP antibody (abbreviated as WT antibody) template plasmid:

[0212] (1) WT antibody gene synthesis:

[0213] The nucleotide sequences of VH and VL of the WT antibody were optimized for E. coli codons, and then the antibody gene sequences were synthesized. The VH and VL amino acid sequences of the WT antibody are shown in SEQ ID NO: 17 and SEQ ID NO: 18, respectively, and the heavy chain and light chain amino acid sequences are shown in SEQ ID NO: 19 and SEQ ID NO: 20, respectively.

[0214] (2) WT antibody gene fragment amplification:

[0215] The nucleotide sequences of the antibody VH and VL synthesized in step (1) are amplified by PCR using DNA polymerase, and then the antibody bands are separated by agarose gel electrophoresis, followed by purification using a gel recovery kit to obtain the antibody gene fragments.

[0216] (3) Enzyme digestion and ligation of WT antibody gene fragments:

[0217] The antibody gene fragment and V01 vector plasmid (including the nucleotide sequence of the constant region) obtained in step (2) were double-digested with restriction endonucleases at the same time, and then purified with a gel recovery kit to obtain the antibody gene fragment and V01 vector with sticky ends. Then, the antibody gene fragment and V01 vector were connected with T4 DNA ligase at 22°C for 4 hours, and the connection reaction product was recovered and purified, and the DNA concentration was determined. Finally, 100 ng of the plasmid was transformed into 100 μl of TG1 Escherichia coli competent cells to obtain a bacterial solution. The bacterial solution was then spread on a plate containing ampicillin resistance and cultured at 37°C overnight.

[0218] (4) Extraction and sequencing verification of WT template plasmid

[0219] Select 10 monoclonal colonies cultured overnight in step (3), perform colony PCR and gel electrophoresis detection using Taq DNA polymerase, select bacteria with the antibody gene sequence correctly inserted for culture and amplification, use a plasmid extraction kit to obtain the WT template plasmid, and send it to a sequencing company for gene sequencing verification.

[0220] 2. Construction of single point mutation library

[0221] Single-point saturation mutations were performed on the entire CDR regions of VH and VL of the WT antibody obtained in step 1 to construct a single-point mutation library.

[0222] (1) Primer design and synthesis

[0223] Using degenerate base codons, upstream and downstream primer pairs for single-point saturation mutation of amino acid sites in the entire CDR region of VH and VL were designed and handed over to an outsourcing company for primer synthesis.

[0224] (2) Single-site saturation mutation plasmid PCR amplification

[0225] The primers obtained in step (1) were used to perform PCR amplification on the single-point saturation mutation plasmid using the PCR method. The reaction system was configured according to Table 3, and then the PCR reaction conditions in Table 4 were used to amplify and prepare the single-point saturation mutation library plasmid. Finally, the WT template plasmid obtained in step 1 was digested with a restriction endonuclease at 37° C. for 1 hour to obtain the plasmid of the mutation library of the amino acid sites in the entire CDR region.

[0226] Table 3: PCR amplification reaction system

[0227]

[0228]

[0229] Table 4: PCR reaction conditions

[0230] Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 temperature 95℃ 95℃ 55-60℃ 72℃ 72℃ 4℃ time 5min 30s 30s 2min 5min ∞

[0231] Note: Step 2 to Step 4 are repeated 22 times.

[0232] (3) Single-site saturation mutation plasmid transformation:

[0233] Take the plasmid of the mutation library of amino acid sites in the entire CDR region obtained in step (2), take 10 μl of the reaction product and transform it into 100 μl of TG1 Escherichia coli competent cells to obtain bacterial solution, then spread the entire bacterial solution on a plate containing ampicillin resistance and culture at 37°C overnight.

[0234] 3. Screening of single point mutation library

[0235] (1) Expression of mutant library antibodies

[0236] 500 μl of culture medium was added to a 96-well culture plate in advance. For each single-point mutation library, a monoclonal colony transformed with the single-point saturation mutation plasmid cultured overnight in step 2-(3) was selected, and WT, negative control (i.e., colonies without VH\VL genes inserted), and blank control (i.e., only culture medium without colonies) colonies were set. After culturing at 37°C for 5-6 hours, the bacterial solution was transferred to a new 96-well culture plate, and then cultured at 37°C for 1-2 hours. Finally, an induction medium was added, and the antibodies were expressed at 37°C overnight to obtain the antibody expression supernatant of the mutation library of the entire CDR region amino acid site.

[0237] (2) Mutation library screening and sequencing

[0238] The commercially available NT-proBNP protein was added to the ELISA plate at 0.004 μg / ml and 100 μl / well, and the plate was coated overnight at 4°C. The next day, the plate was blocked with 1-2% skim milk powder. The antibody expression supernatant of the mutation library of the amino acid sites of the entire CDR region obtained in step (1) was diluted and added to the ELISA plate at 100 μl / well. WT, negative control (i.e., colonies without VH\VL genes) and blank control (i.e., only culture medium without colonies) were set up and incubated at room temperature for 2 hours. The conventional ELISA detection method was used for subsequent plate washing, color development and reading. Finally, the data results were sorted and analyzed, and the clones with improved affinity were sent for sequencing. Finally, the sequencing results were analyzed, and the mutation sites of 8 unique mutation candidate clones (see Table 5) were selected for combinatorial mutation library construction. (The Ratio value in Table 5 represents the degree of affinity improvement. When the Ratio value is equal to 1, it means that the affinity of the mutant clone is the same as that of the WT).

[0239] Table 5: Screening results and mutation sites of candidate clones

[0240]

[0241]

[0242] Note: WT in this article represents no mutation relative to the wild-type sequence (same as Table 6 and Table 7); the positions of the mutant amino acids in this article are obtained by numbering the amino acid sequences of WT VH or VL in sequence from N-terminus to C-terminus.

[0243] 4. Construction of combinatorial mutation library

[0244] (1) Library primer design and synthesis:

[0245] Based on the mutation sites on VH and VL of the NT-proBNP antibody obtained in step 3-(2), primers for amplification of the combinatorial mutation library are designed and primer synthesis is performed.

[0246] (2) Fragment amplification and ligation

[0247] The antibody mutant fragments were amplified according to the PCR system in Table 3 and the PCR reaction conditions in Table 4, and then the antibody mutant fragments were recovered by gel. The antibody mutant fragments were spliced ​​into complete antibody fragments (heavy chain variable region or light chain variable region) by Overlap PCR.

[0248] Finally, the antibody fragment was inserted into the V01 vector by enzyme digestion and ligation to form a complete antibody expression plasmid (for specific steps, see step 1-(3) "WT antibody gene fragment digestion and ligation"). Take 100 ng of the plasmid and transform it into 100 μl TG1 Escherichia coli competent cells, spread the entire bacterial solution on a plate containing ampicillin resistance, and culture at 37°C overnight.

[0249] 5. Screening of combinatorial mutation libraries

[0250] Six combined mutant antibodies were randomly selected for supernatant expression, ELISA screening, and positive clone sequencing analysis. The mutation site information and Ratio value determination are shown in Table 6.

[0251] Table 6: Information of candidate clones for combined mutation

[0252]

[0253] Example 2: Expression of mutant NT-proBNP antibodies

[0254] In this example, the mutant NT-proBNP antibody obtained by screening in Example 1 was expressed, and the specific experimental operation was as follows:

[0255] 1. Construction of eukaryotic recombinant expression plasmid

[0256] pcDNA TM 3.4 vector is a constructed recombinant antibody eukaryotic expression vector, which has introduced multiple cloning restriction sites such as HindIII, BamHI, and EcoRI, and is named pcDNA3.4A expression vector, hereinafter referred to as 3.4A expression vector; according to the variable region gene sequence in the candidate clone screened in Example 1, VL and VH gene-specific amplification primers and constant region overlap primers of the corresponding antibody sequence are designed, and the primers at both ends have HindIII and EcoRI restriction sites and protective bases, respectively, and the light chain gene fragment and the heavy chain gene fragment are amplified by PCR amplification method.

[0257] The heavy chain and light chain gene fragments were double-digested with HindIII / EcoRI, and the 3.4A vector was double-digested with HindIII / EcoRI. The antibody light and heavy chain gene fragments and vectors after digestion were purified and recovered, and then the gene fragments encoding the antibody light and heavy chains were connected to the 3.4A expression vector and transformed into DH5α Escherichia coli competent cells. After the colonies grew, single colonies were picked for PCR identification of positive clones, and the positive clones were picked for sequencing to determine the correctness of the sequence. The clones with correct sequencing were selected for plasmid extraction and standby.

[0258] 2. Sample preparation of recombinant antibodies

[0259] Resuscitate HEK293 cells in advance and subculture them to 200 ml system to make the cell density reach (3-5)×10 6 cells / ml, cell viability>95%; wash the cells by centrifugation, re-dissolve with culture medium, and adjust the cell density to 3.3×10 6 cells / ml, as cell diluent. Prepare plasmid DNA and transfection reagent diluents with culture medium respectively. Add transfection reagent diluent to plasmid DNA diluent, mix well and let stand at room temperature for 15 minutes; slowly add the mixture to cell diluent within 1 minute, mix well and take samples to count, record and observe the viability of cells after transfection, and place them in a 35°C constant temperature incubator for culture, with a speed of 120rmp and a CO2 content of 8%. Centrifuge and collect samples after 13 days. Affinity purification was performed using protein A affinity chromatography column to obtain 14 antibodies, among which the amino acid sequences of the heavy and light chains of the 14 antibodies are shown in Table 7.

[0260] Table 7: Amino acid sequences of heavy and light chains of antibodies

[0261]

[0262] Example 3: Affinity Analysis

[0263] The wild-type antibody (WT) and the mutant antibody prepared in Example 2 were subjected to affinity detection analysis. The specific steps were: the binding and dissociation curves of NT-proBNP and NT-proBNP antibody were tested on the Biacore8K+ device, and the instrument automatically fitted to obtain the affinity constant, binding rate, and dissociation rate. In the affinity test results, KD represents the equilibrium dissociation constant, that is, the affinity constant. The smaller the KD value, the higher the affinity; ka represents the binding rate; kd represents the dissociation rate. The results show that the affinity of the mutant antibody to NT-proBNP is better than that of the wild-type antibody to NT-proBNP. The affinity test results of the exemplary display antibodies are shown in Table 8 below:

[0264] Table 8: Antibody affinity test results

[0265] Antibody Name ka(1 / Ms) kd(1 / s) KD(M) Antibody 1 1.77E+06 1.99E-04 1.13E-10 Antibody 2 1.81E+06 3.96E-04 2.19E-10 Antibody 3 1.91E+06 3.00E-04 1.57E-10 Antibody 4 2.01E+06 3.37E-04 1.67E-10 Antibody 5 1.90E+06 1.71E-04 9.03E-11 Antibody 6 2.04E+06 2.80E-04 1.37E-10 Antibody 7 1.86E+06 1.70E-04 9.17E-11 Antibody 8 2.16E+06 2.78E-04 1.29E-10 Antibody 9 2.00E+06 1.43E-04 7.14E-11 Antibody 10 1.33E+06 2.46E-04 1.85E-10 Antibody 11 2.18E+06 1.77E-04 8.12E-11 Antibody 12 1.88E+06 2.40E-04 1.27E-10 Antibody 13 2.25E+06 1.68E-04 7.45E-11 Antibody 14 1.83E+06 1.70E-04 9.27E-11 WT 1.18E+05 1.05E-04 8.89E-10

[0266] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0267] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. An anti-NT-proBNP antibody comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, characterized in that: The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 are the amino acid sequences shown below: HCDR 1:DFAIH; HCDR 2: WINTRNNKX1TYAX2DFKG, wherein X1 is S, A or Q, and X2 is D or A; HCDR 3:SRHDWFAY; LCDR 1:TLSX3QHSTYTIE, where X3 is S, A, or P; LCDR 2: VEKDGSLX4X5GD, where X4 is S, L or V, and X5 is T or P; LCDR 3:GVGDTIEEQFVYV; Optionally, X1 is S; Optionally, X1 is A; Optionally, X1 is Q; Optionally, X2 is D; Optionally, X2 is A; Optionally, X3 is S; Optionally, X3 is A; Optionally, X3 is P; Optionally, X4 is S; Optionally, X4 is L; Optionally, X4 is V; Optionally, X5 is T; Optionally, X5 is P; Optionally, X1 is S, X2 is D, X3 is S, X4 is S and X5 is T are not simultaneously true; Optionally, X1, X2, X3, X4 and X5 are selected from any one of the following combinations:

2. An anti-NT-proBNP antibody, characterized in that include: a heavy chain variable region and a light chain variable region; The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 17 or a variant thereof, wherein compared with the amino acid sequence shown in SEQ ID NO: 17, the variant of SEQ ID NO: 17 comprises a mutation in at least one of the following sites: S58A, S58Q, and D62A; The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 18 or a variant thereof, wherein compared with the amino acid sequence shown in SEQ ID NO: 18, the variant of SEQ ID NO: 18 comprises a mutation in at least one of the following sites: S26A, S26P, S57L, S57V, and T58P; Optionally, the mutation at position 58 is S58A; Optionally, the mutation at position 58 is S58Q; Optionally, the mutation at position 62 is D62A; Optionally, the mutation at position 26 is S26A; Optionally, the mutation at position 26 is S26P; Optionally, the mutation at position 57 is S57L; Optionally, the mutation at position 57 is S57V; Optionally, the mutation at position 58 is T58P; Optionally, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 18, but they are not true at the same time; Optionally, the heavy chain variable region and the light chain variable region are selected from any one of the following combinations:

3. An anti-NT-proBNP antibody comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, characterized in that: The HCDR1, HCDR2, and HCDR3 are amino acid sequences consistent with the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region defined by the antibody according to claim 2; the LCDR1, LCDR2, and LCDR3 are amino acid sequences consistent with the LCDR1, LCDR2, and LCDR3 of the light chain variable region defined by the antibody according to claim 2; Optionally, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one of the Kabat, Chothia, IMGT, AbM or Contact systems or a combination of multiple systems; Optionally, the antibody comprises at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4; Optionally, at least a portion of at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4 is derived from at least one of mouse antibody, human antibody, primate antibody, bovine antibody, horse antibody, dairy cow antibody, porcine antibody, sheep antibody, goat antibody, dog antibody, cat antibody, rabbit antibody, camel antibody, donkey antibody, deer antibody, mink antibody, chicken antibody, duck antibody, goose antibody, turkey antibody, fighting cock antibody or mutants thereof; Optionally, the HFR1 comprises an amino acid sequence as shown in SEQ ID NO: 7 or an amino acid sequence having at least 80% homology thereto; The HFR2 comprises an amino acid sequence as shown in SEQ ID NO: 8 or an amino acid sequence having at least 80% homology thereto; The HFR3 comprises an amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence having at least 80% homology thereto; The HFR4 comprises the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having at least 80% homology thereto; The LFR1 comprises an amino acid sequence as shown in SEQ ID NO: 11 or an amino acid sequence having at least 80% homology thereto; The LFR2 comprises an amino acid sequence as shown in SEQ ID NO: 12 or an amino acid sequence having at least 80% homology thereto; The LFR3 comprises an amino acid sequence as shown in SEQ ID NO: 13 or an amino acid sequence having at least 80% homology thereto; The LFR4 comprises the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 80% homology thereto.

4. The antibody according to any one of claims 1 to 3, characterized in that The antibody further comprises a constant region; Wherein, the constant region includes at least one of a heavy chain constant region and a light chain constant region; Optionally, at least a portion of at least one of the heavy chain constant region and the light chain constant region is from at least one of a mouse antibody, a human antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy cow antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting cock antibody, or a mutant thereof; Optionally, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments; Optionally, the heavy chain constant region includes CH1 of IgG, hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM; Optionally, the light chain constant region comprises a light chain constant region selected from a κ type or a λ type; Optionally, the heavy chain constant region comprises or is the heavy chain constant region shown in SEQ ID NO: 15 or an amino acid sequence having at least 80% identity thereto; or The light chain constant region comprises or is the light chain constant region shown in SEQ ID NO: 16 or an amino acid sequence having at least 80% identity thereto; Optionally, the heavy chain and light chain of the antibody are selected from any one of the following combinations: 。 5. A nucleic acid molecule, vector, cell or host or a method for preparing the antibody according to any one of claims 1 to 4, characterized in that: The nucleic acid molecule encodes the antibody according to any one of claims 1 to 4; the vector comprises the nucleic acid molecule; the cell or host comprises: the nucleic acid molecule or the vector or expresses the antibody according to any one of claims 1 to 4; the method comprises culturing the cell or host.

6. A conjugate, characterized in that: include: The antibody according to any one of claims 1 to 4 and the conjugated part thereof; Optionally, the coupling part includes at least one of a purification tag, an affinity substance, a marker and a solid phase carrier; Optionally, the purification tag includes at least one of a His tag, a Flag tag, a GST tag, an MBP tag, a SUMO tag, and a C-Myc tag; Optionally, the solid support comprises at least one selected from microspheres, plates and membranes; Optionally, the label comprises at least one selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent agents and nanoparticle labels; Optionally, the affinity substance includes at least one selected from biotin, a biotin derivative or streptavidin.

7. A reagent or a kit, characterized in that: include: The antibody according to any one of claims 1 to 4 or the conjugate according to claim 6.

8. Use of the antibody according to any one of claims 1 to 4, the conjugate according to claim 6, or the reagent or kit according to claim 7 in detecting NT-proBNP, preparing a product for detecting NT-proBNP, or preparing a product for diagnosing NT-proBNP-related diseases; Optionally, the NT-proBNP-related disease includes at least one of heart failure, heart failure or acute coronary syndrome.

9. A method for detecting NT-proBNP, characterized in that: include: The antibody according to any one of claims 1 to 4, the conjugate according to claim 6, or the reagent or kit according to claim 7 is contacted with a sample to be detected to form an immune complex; Optionally, based on the signal of the immune complex, determining whether the sample to be tested contains NT-proBNP or the content of the NT-proBNP; Optionally, the immune complex further comprises a second antibody, which binds to the antibody; Optionally, the immune complex further comprises a second antibody, which binds to NT-proBNP.

10. A method for screening NT-proBNP antibodies, characterized in that: include: a) designing primers for performing amino acid substitution at the sites of X1, X2, X3, X4 and X5 defined in the antibody of claim 1, or at the mutation sites defined in the antibody of claim 2; b) using the nucleic acid molecule, vector, cell or host of claim 5 as a template and the primers described in a) to construct a mutation library; c) screening NT-proBNP antibodies from the mutant library; Optionally, the mutation library is a single-site saturation mutation library; Optionally, the NT-proBNP antibody includes or is the antibody according to any one of claims 1 to 4.

11. A mutation library, characterized in that The mutant library comprises the antibody according to any one of claims 1 to 4; Optionally, the mutant library is obtained by the method of claim 10.

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