CN115703837B

CN115703837B - Recombinant antibody for resisting growth stimulus expression gene 2 protein

Info

Publication number: CN115703837B
Application number: CN202110944249.8A
Authority: CN
Inventors: 孟媛; 钟冬梅; 黄玉玲
Original assignee: Dongguan Pengzhi Biotechnology Co Ltd
Current assignee: Dongguan Pengzhi Biotechnology Co Ltd
Priority date: 2021-08-17
Filing date: 2021-08-17
Publication date: 2023-10-03
Anticipated expiration: 2041-08-17
Also published as: CN115703837A

Abstract

The application relates to an isolated binding protein containing a growth stimulation expression gene 2 protein antigen binding domain, and researches on preparation, application and the like of the binding protein. The binding protein has strong activity and high affinity with the growth stimulation expression gene 2 protein, and can be widely applied to the field of detection of the growth stimulation expression gene 2 protein.

Description

Recombinant antibody for resisting growth stimulus expression gene 2 protein

Technical Field

The application relates to the technical field of immunity, in particular to a recombinant antibody for resisting growth stimulation expression gene 2.

Background

The growth stimulation expressed gene 2 protein (ST 2) is one of the interleukin 1 (IL-1) receptor superfamily members, and ST2 was first found in mouse fibroblasts by Tominga et al in 1987; richard Lee, a affiliated female hospital at the university of Harvard, 2002, reported that myocardial stress and myocardial injury caused expression of ST2; researchers such as Schmitz found that the ST2 gene was not only involved in the pathological processes of inflammatory reaction and immune diseases, but also found that the specific functional ligand IL-33 of the ST2 gene; the 2013 american ACC/AHA heart failure guideline and 2014 chinese heart failure diagnosis and treatment guideline introduced ST2 (soluble ST 2) into the recommendation of biomarkers of heart failure, which are cardiac fibrosis, based on ST2 clinical study data accumulation.

The growth stimulatory expressed gene 2 protein (ST 2), also known as the T1, IL1RL1 or Fit1 gene and located on chromosome 2q12 in humans, is about 40KD, the transcription product of the ST2 gene has 4 subtypes, two of which are most important due to the subtype regulated by the different promoters, the first being transmembrane ST2 (ST 2L), consisting of 3 extracellular immunoglobulin G domains, one transmembrane domain and one intracellular domain, being a membrane receptor member of the interleukin-1 receptor family; the other is soluble ST2 (sST 2), sST2 being free flowing in the blood due to the lack of transmembrane and intracellular domains, making it detectable in serum.

Studies of growth stimulation expressed gene 2 protein (ST 2) show that the protein is related to cardiac dysfunction, ST2L receptor has heart protection effect, and after the combination of specific ligand IL-33, the structure of fibrosis and hypertrophic burden of cardiac myocytes can be reduced, and the degree of myocardial injury, myocardial apoptosis, inflammatory myocardial activation, myocardial failure reconstruction and other pathological symptoms of myocardial infarction can be reduced. Kakkar et al found that mechanical stretching of living cells could enhance the release of IL-33 in cytoplasmic vesicles, could induce reconstitution of IL-33 by heart due to volume or pressure overload, that vasopressin II could cause damage to heart by generating various Reactive Oxygen Species (ROS), that IL-33 could exert anti-myocardial hypertrophy by blocking the action of vasopressin II or phenylephrine on the myocardium, and that ST2L and IL33 combined to activate nuclear transcription factor NF-kB and mitogen activated protein kinase MAPK pathway, resulting in release of Th 2-like cytokines IL-4, IL-5 by target cells, which could exert anti-myocardial hypertrophy myocardial fibrosis, thus initiating repair protection. It was also found that in vitro administration of IL-33 enhances the activation of ST2L and its own intracellular biochemical pathways, as well as reduces ROS production induced by angiotension II, thereby preventing or at least alleviating the deterioration of cardiac clinical and structural states;

sST2, which is a decoy receptor for IL-33, is a marker of cardiovascular diseases, and blocks the 1L-33/ST2L signal pathway after being combined with IL-33, so that the effectiveness of interaction between IL-33 and myomembrane receptors is reduced, sST2 is obviously induced in myocardial cells with heavy mechanical load, and therefore, heart failure, chronic heart failure state deterioration in the past, myocardial infarction induced injury lead to poor myocardial extension, and all the conditions can be detected by measuring blood sST2 level. Weinberg et al showed that myocardial infarction resulted in elevated levels of sST2 in plasma. Thus, elevated sST2 can reduce the protective effect of IL-33 on cardiomyocytes and can negatively impact the overall cardiovascular risk profile.

Cardiovascular disease (CVD) is the leading cause of death worldwide, brain Natriuretic Peptide (BNP) and brain natriuretic peptide precursor NT-proBNP are the most well known markers of Heart Failure (HF), troponin markers improve the diagnosis of acute and chronic coronary artery disease, however, single biomarkers can only reflect a unilateral pathology of heart failure, but also be affected by pulmonary arterial hypertension, etc. due to renal function, age, BMI, etc., reducing the accuracy of these biomarkers. With the increasing awareness of the effects of sST2 and ST2L on the cardiovascular system, one began to consider the assessment of plasma sST2 levels as a new marker of cardiovascular events, particularly the indices and clinical conditions associated with heart failure and ischemic heart disease, and sST2 was not affected by indices of kidney function, age, body weight, etc. sST2 is also considered a possible biomarker for patients with acute heart failure and associated asthma. Studies also prove that sST2 plays a role in predicting mortality, sST2 can be combined with other biomarkers to serve as a prognosis biomarker, sST2 can also be used for monitoring the pharmacological response of heart failure, has a correlation with the recommended therapeutic drugs for heart failure, and can provide a reference for clinical selection of the therapeutic drugs and the scheme.

The main methods currently used for detecting ST2 are enzyme-linked immunosorbent assay (ELISA), magnetic particle chemiluminescence assay, chromatography and the like; different detection methods all require specific monoclonal antibodies directed against ST 2. There are drawbacks to the sensitivity or specificity of the monoclonal antibodies currently used to detect ST 2.

In view of this, the present application has been made.

Disclosure of Invention

The present application relates to a novel isolated binding protein comprising an ST2 antigen binding domain, and studies have been made on the preparation, use, etc. of the binding protein.

Wherein the antigen binding domain comprises at least one complementarity determining region selected from the group consisting of:

a complementarity determining region CDR-VH1 comprising SEQ ID NO:1 or consists of the amino acid sequence shown in 1;

a complementarity determining region CDR-VH2 comprising SEQ ID NO:2 or consists of the amino acid sequence shown in the figure;

a complementarity determining region CDR-VH3 comprising SEQ ID NO:3 or consists of the amino acid sequence shown in 3;

a complementarity determining region CDR-VL1 comprising SEQ ID NO:4 or consists of the amino acid sequence shown in figure 4;

a complementarity determining region CDR-VL2 comprising SEQ ID NO:5 or consists of the amino acid sequence shown in figure 5;

a complementarity determining region CDR-VL3 comprising SEQ ID NO:6 or consists of the amino acid sequence shown in figure 6.

The application has the beneficial technical effects that the binding protein has strong activity and high affinity with ST2, and is suitable for the content detection of ST2, in particular for the content detection of human ST 2. Meanwhile, the binding protein obtained by the recombination method has small individual difference, small batch-to-batch difference and stable quality, and is more beneficial to quality control and detection stability.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present application, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is an electrophoresis chart of an anti-ST2 monoclonal antibody in example 1 of the present application.

Detailed Description

The application may be understood more readily by reference to the following detailed description of some embodiments of the application and the examples included therein.

Before the present application is further described, it is to be understood that this application is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present application will be limited only by the appended claims.

Unless defined otherwise herein, scientific and technical terms used in connection with the present application shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms should be clear, however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or extraneous definition. In the present application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "include" and other forms is not limiting.

Generally, the nomenclature used in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein and the techniques thereof are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present application are generally well known in the art and are performed according to conventional methods as described in various general and more specific references cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to manufacturer's instructions, as commonly accomplished in the art, or as described herein. Nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques therefor, are those well known and commonly employed in the art.

In order that the application may be more readily understood, selected terms are defined below.

The term "amino acid" refers to a naturally occurring or non-naturally occurring spindle alpha-amino acid. The term "amino acid" as used herein may include naturally occurring amino acids and non-naturally occurring amino acids. Naturally occurring amino acids include alanine (three letter code: A1a, one letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, c), glutamine (G1N, Q), glutamic acid (G1 u, E), glycine (G1Y, G), histidine (His, H), isoleucine (I1E, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Va 1, V). Non-naturally occurring amino acids include, but are not limited to, alpha-aminoadipic acid, aminobutyric acid, citrulline, homocysteine, homoleucine, homoarginine, hydroxyproline, norleucine, pyridylalanine, sarcosine, and the like.

The term "isolated binding protein" is a protein that, due to its origin or source of derivation, does not bind to a naturally bound component that accompanies it in its natural state; substantially free of other proteins from the same species; expressed by cells from different species; or not present in nature. Thus, a protein that is chemically synthesized or synthesized in a cellular system other than the cell from which it naturally originates will be "isolated" from the component with which it naturally binds. Protein purification techniques well known in the art can also be used by isolation so that the protein is substantially free of substantially bound components.

The term "isolated binding protein comprising an antigen binding domain" broadly refers to all proteins/protein fragments comprising CDR regions. The term "antibody" includes polyclonal and monoclonal antibodies as well as antigen compound binding fragments of such antibodies, including Fab, F (ab') ₂ Fd, fv, scFv, bispecific antibodies and antibody minimal recognition units, and single chain derivatives of these antibodies and fragments. The type of antibody may be selected from the group consisting of IgG1, igG2, igG3, igG4, and IgA, igM, igE, igD. Furthermore, the term "antibody" includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric (chimeric), bifunctional (bifunctional) and humanized (humanzed) antibodies, as well as related synthetic isomeric forms (isoforms). The term "antibody" is used interchangeably with "immunoglobulin".

"variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of a heavy chain may be referred to as "VH". The variable domain of the light chain may be referred to as "VL". These domains are typically the most variable parts of an antibody and contain antigen binding sites. The light or heavy chain variable region (VL or VH) is composed of framework regions interrupted by three hypervariable regions called "complementarity determining regions" or "CDRs". The framework regions and CDR ranges have been precisely defined, for example, in Kabat (see sequence of immunologically important proteins (Sequences of Proteins of Immunological Interest), E.Kabat et al, U.S. department of health and human services (U.S. device of Health and Human Services), (1983)) and Chothia. The framework regions of antibodies, i.e., the framework regions that make up the combination of the essential light and heavy chains, function to locate and align the CDRs, which are primarily responsible for binding to the antigen.

Although the 2 domains of the Fv fragment (VL and VH) are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made into a single protein chain, in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv), "single chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of an antibody, in some embodiments the Fv polypeptide additionally comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding.

"variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of a heavy chain may be referred to as "VH". The variable domain of the light chain may be referred to as "VL". These domains are typically the most variable parts of an antibody and contain antigen binding sites. The light or heavy chain variable region is composed of framework regions interrupted by three hypervariable regions called "complementarity determining regions" or "CDRs". The framework regions of antibodies, i.e., the framework regions that make up the combination of the essential light and heavy chains, function to locate and align the CDRs, which are primarily responsible for binding to the antigen.

As used herein, "framework," "framework," or "FR" regions mean regions of an antibody variable domain that are excluding those regions defined as CDRs. Each antibody variable domain framework can be further subdivided into contiguous regions (FR 1, FR2, FR3, and FR 4) separated by CDRs.

Typically, the variable regions VL/VH of the heavy and light chains are obtained by joining the CDRs numbered below with the FR in a combination arrangement as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

CDR labeling methods commonly used in the art include: the Kabat numbering scheme, the IMGT, chothia and Lesk numbering schemes, and the 1997 Lefranc et al, were introduced as a new standardized numbering system for all protein sequences of the immunoglobulin superfamily. 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 Kabat numbering schemes, which are generally considered as widely adopted criteria for numbering antibody residues. The application adopts Kabat annotation standard to mark CDR regions, but other methods to mark CDR regions also belong to the protection scope of the application.

As used herein, the term "purified" or "isolated" in connection with a polypeptide or nucleic acid means that the polypeptide or nucleic acid is not in its natural medium or in its natural form. Thus, the term "isolated" includes polypeptides or nucleic acids that are removed from their original environment, e.g., if it is naturally occurring. For example, an isolated polypeptide is generally free of at least some protein or other cellular component that is normally associated therewith or that is normally admixed therewith or in solution. Isolated polypeptides include naturally produced said polypeptides contained in cell lysates, purified or partially purified forms of said polypeptides, recombinant polypeptides, said polypeptides expressed or secreted by cells, and said polypeptides in heterologous host cells or cultures. In connection with a nucleic acid, the term isolated or purified indicates, for example, that the nucleic acid is not in its native genomic context (e.g., in a vector, as an expression cassette, linked to a promoter, or artificially introduced into a heterologous host cell).

The term "affinity" as used herein refers to the equilibrium constant of reversible binding of 2 agents and is expressed as KD. The affinity of the binding protein for the ligand, such as the affinity of the antibody for the epitope, may be, for example, about 100 nanomolar (nM) to about 0.1nM, about 100nM to about 1 picomolar (pM), or about 100nM to about 1 femtomole (fM). The term "affinity" as used herein means the resistance of a complex of 2 or more agents to dissociation after dilution. Apparent affinity can be determined by methods such as enzyme-linked immunosorbent assay (ELISA) or any other technique familiar to those skilled in the art.

The term "homology" or "identity" or "similarity" as used herein refers to sequence similarity between two peptides or between two nucleic acid molecules. Each of homology and identity may be determined by comparing the positions in each sequence that are aligned for comparison purposes. When an equivalent position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position; when an equivalent position is occupied by the same or a similar amino acid residue (e.g., similar in terms of steric and/or electronic properties), then the molecules may be said to be homologous (similar) at that position. The percent homology/similarity or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences. In comparing two sequences, the absence of residues (amino acids or nucleic acids) or the presence of additional residues also reduces identity and homology/similarity.

The present application relates to an isolated binding protein comprising an antigen binding domain, wherein the antigen binding domain comprises at least one complementarity determining region selected from the group consisting of:

In some embodiments, the binding protein comprises at least 3 CDRs (e.g., 3 light chain CDRs or 3 heavy chain CDRs).

In some embodiments, the binding protein comprises at least 6 CDRs.

In some embodiments, the binding protein is an intact antibody comprising a variable region and a constant region.

In some embodiments, the binding protein is an antibody, F (ab') ₂ One of Fab', fab, fv, scFv, bispecific antibodies and antibody minimal recognition units.

In some embodiments, the binding protein comprises the light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4 that are shown in sequence SEQ ID NO. 11-14 or have at least 90% homology thereto, and/or the heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4 that are shown in sequence SEQ ID NO. 7-10 or have at least 90% homology thereto.

In alternative embodiments, the binding protein comprises the light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4 that are shown in SEQ ID NO. 11-14 in sequence or have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and/or the heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4 that are shown in SEQ ID NO. 7-10 in sequence or have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

In some embodiments, the binding protein further comprises an antibody constant region sequence.

In some embodiments, the constant region sequence is selected from the group consisting of the sequence of any one of the constant regions of IgG1, igG2, igG3, igG4, igA, igM, igE, igD.

In some embodiments, the constant region is of a species origin of cow, horse, cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, or human.

In some embodiments, the constant region is derived from a mouse;

the light chain constant region sequence is shown in SEQ ID NO: shown at 16;

the heavy chain constant region sequence is shown in SEQ ID NO: 15.

In another aspect, the application also provides an isolated nucleic acid molecule, which is DNA or RNA, encoding a binding protein as described above.

In this context, a nucleic acid comprises variants of its conservative substitutions (e.g., substitutions of degenerate codons) and the complementary sequence. The terms "nucleic acid" and "polynucleotide" are synonymous and include genes, cDNA molecules, mRNA molecules and fragments thereof, e.g., oligonucleotides.

According to an aspect of the present application, there is also provided a vector comprising the nucleic acid molecule described above.

Wherein the nucleic acid sequence is operably linked to at least one regulatory sequence. "operably linked" refers to a coding sequence being linked to regulatory sequences in a manner that allows for the expression of the coding sequence. Regulatory sequences are selected to direct expression of the protein of interest in a suitable host cell, and include promoters, enhancers and other expression control elements.

Herein, a vector may refer to a molecule or agent comprising a nucleic acid of the application or a fragment thereof, which is capable of carrying genetic information and which may deliver the genetic information into a cell. Typical vectors include plasmids, viruses, phages, cosmids, and minichromosomes. The vector may be a cloning vector (i.e., a vector for transferring genetic information into a cell, the cell may be propagated and the cell may be selected for the presence or absence of the genetic information) or an expression vector (i.e., a vector comprising the necessary genetic elements to allow expression of the genetic information of the vector in a cell). Thus, a cloning vector may contain a selectable marker, and an origin of replication that matches the cell type specified by the cloning vector, while an expression vector contains regulatory elements necessary to effect expression in the specified target cell.

The nucleic acid of the application or a fragment thereof may be inserted into a suitable vector to form a cloning vector or an expression vector carrying the nucleic acid fragment of the application. Such novel vectors are also part of the present application. The vector may include a plasmid, phage, cosmid, minichromosome, or virus, as well as naked DNA that is transiently expressed only in a particular cell. The cloning vectors and expression vectors of the present application are capable of spontaneous replication and thus can provide high copy numbers for high level expression or high level replication purposes for subsequent cloning. The expression vector may include a promoter for driving expression of the nucleic acid fragment of the application, optionally a nucleic acid sequence encoding a signal peptide that causes secretion or integration of the peptide expression product onto a membrane, the nucleic acid fragment of the application, and optionally a nucleic acid sequence encoding a terminator. When the expression vector is manipulated in a producer strain or cell line, the vector may or may not be integrated into the host cell genome when introduced into the host cell. The vector typically carries a replication site, as well as a marker sequence capable of providing phenotypic selection in transformed cells.

In another aspect, the application also provides a host cell transformed with a vector as described above.

The expression vectors of the application are useful for transforming host cells. Such transformed cells are also part of the application and may be cultured cells or cell lines for propagation of the nucleic acid fragments and vectors of the application, or for recombinant production of the polypeptides of the application. Transformed cells of the application include microorganisms such as bacteria (e.g., E.coli, bacillus, etc.). Host cells also include cells derived from multicellular organisms such as fungi, insect cells, plant cells or mammalian cells, preferably mammalian derived cells, e.g., CHO cells. The transformed cell is capable of replicating the nucleic acid fragment of the application. When the peptide combinations of the application are recombinantly produced, the expression product may be exported into culture medium or carried on the surface of the transformed cells.

According to an aspect of the present application, there is also provided a method for producing the above binding protein, comprising the steps of:

the above-described host cells are cultured in a suitable culture condition, and the produced binding protein is recovered from the culture medium or from the cultured host cells.

The method may be, for example, transfecting a host cell with a nucleic acid vector encoding at least a portion of the binding protein, and culturing the host cell under suitable conditions to express the binding protein. The host cell may also be transfected with one or more expression vectors, which may comprise, alone or in combination, DNA encoding at least a portion of the binding protein. The binding proteins may be isolated from the culture medium or cell lysate using conventional techniques for purifying proteins and peptides, including ammonium sulfate precipitation, chromatography (e.g., ion exchange, gel filtration, affinity chromatography, etc.), and/or electrophoresis.

Construction of a suitable vector containing the coding and regulatory sequences of interest can be performed using standard ligation and restriction techniques well known in the art. The isolated plasmid, DNA sequence or synthetic oligonucleotide is cleaved, tailing and religated as desired. Mutations may be introduced into the coding sequence by any method to produce variants of the application, and these mutations may comprise deletions or insertions or substitutions, etc.

According to one aspect of the application, the application also provides the use of a binding protein as described above for the preparation of a diagnostic agent or kit for diagnosing heart failure and evaluating heart function.

According to an aspect of the present application, there is also provided a method of detecting ST2 in a test sample, comprising:

a) Contacting ST2 antigen in the test sample with a binding protein as described above under conditions sufficient for an antibody/antigen binding reaction to occur to form an immune complex; and

b) Detecting the presence of the immune complex.

In the above detection method, the presence of said complex in step b) is indicative of the presence of said ST2 in said test sample.

In some embodiments, in step a), a second antibody is also included in the immunocomplex, the second antibody binding to the binding protein.

In some embodiments, in step a), a second antibody is also included in the immune complex, the second antibody binding to the ST2;

in this embodiment, the binding protein forms a partner antibody in the form of a first antibody with the second antibody for binding to a different epitope of ST 2.

In some embodiments, the binding protein may be labeled with an indicator that shows signal strength to allow the complex to be readily detected.

In some embodiments, in step a), a second antibody is also included in the immune complex, the second antibody binding to the ST2 antigen;

in this embodiment, the binding protein serves as an antigen for the second antibody, which may be labeled with an indicator that shows signal strength, so that the complex is easily detected.

In some embodiments, the indicator that displays signal intensity comprises any one of a fluorescent substance, a quantum dot, a digoxin-labeled probe, biotin, a radioisotope, a radiocontrast agent, a paramagnetic ion fluorescent microsphere, an electron dense substance, a chemiluminescent label, an ultrasound contrast agent, a photosensitizer, colloidal gold, or an enzyme.

In some embodiments, the fluorescent material comprises Alexa 350, alexa 405, alexa 430, alexa488, alexa 555, alexa 647, AMCA, aminoacridine, BODIPY 630/650, BODIPY650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4 ',5' -dichloro-2 ',7' -dimethoxyfluorescein, 5-carboxy-2 ',4',5',7' -Tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxytetramethyl rhodamine, cascade Blue, cy2, cy3, cy5, cy7, 6-FAM, dansyl chloride, fluorescein, HEX, 6-JOE, NBD (7-nitrobenzo-2-oxa-1, 3-diazole), oregon Green 488, oregon Green 500, oregon Green514, pacific Blue, phthalic acid, terephthalic acid, isophthalic acid, cresol purple, light cresol Blue, para-aminobenzoic acid, erythrosin, phthalocyanine, cyanine, alternet-2-oxa-3-diazole azomethine, cyanine, xanthine, succinyl fluorescein, rare earth metal cryptate, tripyridyl diamine europium, europium cryptate or chelate, diamine, bis anthocyanin, la Jolla Blue dye, allophycocyanin b, phycocyanin C, phycocyanin R, thiamine, phycoerythrin R, REG, rhodamine Green, rhodamine isothiocyanate, rhodamine red, ROX, TAMRA, TET, TRIT (tetramethyl rhodamine isothiol), tetramethyl rhodamine and texas red.

In some embodiments, the radioisotope comprises ¹¹⁰ In、 ¹¹¹ In、 ¹⁷⁷ Lu、 ¹⁸ F、 ⁵² Fe、 ⁶² Cu、 ⁶⁴ Cu、 ⁶⁷ Cu、 ⁶⁷ Ga、 ⁶⁸ Ga、 ⁸⁶ Y、 ⁹⁰ Y、 ⁸⁹ Zr、 ⁹⁴ mTc、 ⁹⁴ Tc、 ⁹⁹ mTc、 ¹²⁰ I、 ¹²³ I、 ¹²⁴ I、 ¹²⁵ I、 ¹³¹ I、 ^154-158 Gd、 ³² P、 ¹¹ C、 ¹³ N、 ¹⁵ O、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ⁵¹ Mn、 ⁵² mMn、 ⁵⁵ Co、 ⁷² As、 ⁷⁵ Br、 ⁷⁶ Br、 ⁸² mRb and is provided with ⁸³ Sr.

In some embodiments, the enzyme comprises any one of horseradish peroxidase, alkaline phosphatase, and glucose oxidase.

In some embodiments, the fluorescent microsphere is: the polystyrene fluorescent microsphere is internally wrapped with rare earth fluorescent ion europium.

According to one aspect of the application, the application also relates to a detection reagent or kit comprising a binding protein as described above.

In some embodiments, the kit or kit further comprises one or more of a pharmaceutically acceptable excipient, buffer, stabilizer, diluent, or carrier.

Embodiments of the present application will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present application and should not be construed as limiting the scope of the present application. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer.

Example 1

This example provides an exemplary method of preparing a recombinant antibody against ST 2.

1. Construction of expression plasmids

Restriction enzyme, prime Star DNA polymerization in this exampleEnzymes were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO company. BD SMART ^TM RACE cDNA Amplification Kit kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kits were purchased from Tiangen. Primer synthesis and gene sequencing were accomplished by Invitrogen corporation.

1.1 preparation of Anti-ST2 antibody Gene

mRNA is extracted from hybridoma cell strains secreting Anti-ST2 monoclonal antibodies, DNA products are obtained through an RT-PCR method, rTaq DNA polymerase is used for carrying out an A adding reaction on the products, the products are inserted into a pMD-18T vector and are transformed into DH5 alpha competent cells, after colonies grow out, the Heavy Chain and Light Chain gene clones are respectively taken for 4 clones to send to a gene sequencing company for sequencing.

1.2 sequence analysis of variable region Gene of Anti-ST211F9 antibody

The gene sequence obtained by sequencing is placed in a Kabat antibody database for analysis, and VNTI11.5 software is utilized for analysis to determine that the amplified genes of the heavy Chain primer pair and the Light Chain primer pair are correct, wherein in the gene fragment amplified by the Light Chain, the VL gene sequence is 321bp, belongs to the VkII gene family, and the front part of the VL gene sequence is 57bp leader peptide sequence; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence is 345bp, belongs to the VH1 gene family, and a 57bp leader peptide sequence is arranged in front of the VH gene family.

1.3 construction of recombinant antibody expression plasmids

pcDNA ^TM 3.4vector is a constructed eukaryotic expression vector of the recombinant antibody, and the expression vector is introduced into a HindIII, bamHI, ecoRI polyclonal enzyme cutting site, named pcDNA3.4A expression vector and is hereinafter abbreviated as 3.4A expression vector; according to the result of the antibody variable region gene sequencing in pMD-18T, VL and VH gene specific primers of the Anti-ST211F9 antibody are designed, hindIII, ecoRI restriction sites and protective bases are respectively arranged at two ends, and a Light Chain gene fragment of 0.73KB and a Heavy Chain gene fragment of 1.4KB are amplified by a PCR amplification method.

The Heavy Chain gene fragment and the Light Chain gene fragment are respectively cut by HindIII/EcoRI double enzyme, the 3.4A vector is cut by HindIII/EcoRI double enzyme, the Heavy Chain gene and the Light Chain gene after the fragment and the vector are purified and recovered are respectively connected into the 3.4A expression vector, and recombinant expression plasmids of the Heavy Chain and the Light Chain are respectively obtained. 2. Stable cell line selection

2.1 transient transfection of recombinant antibody expression plasmids into CHO cells, determination of expression plasmid Activity

The plasmid was diluted to 40ug/100ul with ultrapure water and CHO cells were conditioned to 1.43X 10 ⁷ 100ul of plasmid is mixed with 700ul of cells in a centrifuge tube, transferred into an electrorotating cup, electrorotated, sampled and counted on days 3, 5 and 7, and collected and detected on day 7.

Coating liquid (main component NaHCO) ₃ ) Diluting sheep anti-mouse IgG 1ug/ml, coating a microplate, and performing 100 uL/well overnight at 4 ℃; the next day, the washing liquid (main component Na ₂ HPO ₄ +NaCl) for 2 times, and beating to dry; blocking solution (20% BSA+80% PBS) was added and dried at 37℃for 1h in 120uL per well; adding diluted cell supernatant at 100 uL/well, 37deg.C for 60min; removing liquid in the plate, beating, adding 20% mouse negative blood, sealing, and sealing at 37deg.C for 1 hr; removing liquid in the plate, beating, adding diluted ST2 antigen, 100uL per well, and 37 ℃ for 40min; washing with washing liquid for 5 times, and drying; adding HRP-labeled ST2 monoclonal antibody, 100uL per well at 37 ℃ for 30min; adding a developing solution A (50 uL/hole) and a developing solution B (50 uL/hole) for 10min; adding a stop solution, 50 uL/well; OD was read on the microplate reader at 450nm (reference 630 nm). The results showed that the reaction OD after 1000-fold dilution of the cell supernatant was still greater than 1.0, and that the reaction OD without cell supernatant was less than 0.1, indicating that the antibodies produced after transient transformation of the plasmid were active against ST2 antigen.

Remarks: liquid A (main component of citric acid, sodium acetate, acetanilide and carbamide peroxide); liquid B (main component citric acid+EDTA.2Na+TMB+concentrated HCl); stop solution (EDTA.2Na+ concentrated H) ₂ SO ₄ )

2.2 linearization of recombinant antibody expression plasmids

The following reagents were prepared: buffer 50ul, DNA 100 ug/tube, pvuI enzyme 10ul, sterile water to 500ul, water bath at 37 ℃ for enzyme digestion overnight; firstly, extracting with equal volume of phenol/chloroform/isoamyl alcohol (lower layer) 25:24:1, and then sequentially extracting with chloroform (water phase); precipitating 0.1 times volume (water phase) of 3M sodium acetate and 2 times volume of ethanol on ice, rinsing the precipitate with 70% ethanol, removing organic solvent, completely volatilizing ethanol, re-thawing with appropriate amount of sterilized water, and measuring concentration.

2.3 stable transfection of recombinant antibody expression plasmid, pressure screening of stable cell lines

The plasmid was diluted to 40ug/100ul with ultrapure water and CHO cells were conditioned to 1.43X 10 ⁷ Placing cells/ml in a centrifuge tube, mixing 100ul of plasmid with 700ul of cells, transferring into an electric rotating cup, carrying out electric rotation, and counting the next day; 25umol/L MSX 96-well pressure culture for about 25 days.

Observing the clone holes with the cells under a microscope, and recording the confluency; taking culture supernatant, and carrying out sample feeding detection; selecting cell strains with high antibody concentration and relative concentration, turning 24 holes, and turning 6 holes about 3 days; seed preservation and batch culture are carried out after 3 days, and cell density is regulated to be 0.5x10 ⁶ Batch culture was performed with cells/ml,2.2ml, and cell density was 0.3X10 ⁶ Performing seed preservation by using cells/ml and 2 ml; and (3) carrying out sample feeding detection on the culture supernatant of the 6-hole batch culture for 7 days, and selecting cell strains with smaller antibody concentration and smaller cell diameter to transfer TPP for seed preservation and passage.

3. Recombinant antibody production

3.1 cell expansion culture

After cell recovery, the cells were first cultured in 125ml shake flasks with an inoculation volume of 30ml and a medium of 100% dynamis and placed in a shaker at a speed of 120r/min at 37℃and with 8% carbon dioxide. Culturing for 72h, inoculating and expanding culture at 50 ten thousand cells/ml inoculating density, and calculating the expanding culture volume according to production requirements, wherein the culture medium is 100% Dynamis culture medium. After that, the culture was spread every 72 hours. When the cell quantity meets the production requirement, the inoculation density is strictly controlled to be about 50 ten thousand cells/ml for production.

3.2 shaking flask production and purification

Shake flask parameters: the rotating speed is 120r/min, the temperature is 37 ℃, and the carbon dioxide is 8%. Feeding: feeding was started every day until 72h of culture in shake flasks, hyCloneTM Cell BoostTM Feed a fed-batch was 3% of the initial culture volume every day, feed 7b fed-batch was one thousandth of the initial culture volume every day, and fed-batch was continued until day 12 (day 12 Feed). Glucose was fed at 3g/L on day six. Samples were collected on day 13. Affinity purification was performed using a proteona affinity column. 4. Mu.g of purified antibody was subjected to reducing SDS-PAGE, and 4. Mu.g of external control antibody was used as a control, and the electrophoretogram was shown in the figure. Two bands are shown after reducing SDS-PAGE, 1 Mr is 50KD (heavy chain), and the sequence is shown as SEQ ID NO. 18; the other Mr is 28KD (light chain), and the sequence is shown as SEQ ID NO. 20.

Example 2

Antibody affinity assay and Activity characterization

The resulting antibodies Anti-ST 2F 9 of example 1 (having the heavy and light chain variable regions shown in SEQ ID NO:17 and SEQ ID NO: 19) were analyzed for complementarity determining regions of the heavy chain:

CDR-VH1, SEQ ID NO:1, and a polypeptide sequence shown in the specification;

CDR-VH2, SEQ ID NO:2, and a polypeptide sequence represented by the following formula (2);

CDR-VH3, SEQ ID NO:3, an amino acid sequence shown in 3;

complementarity determining regions of the light chain:

CDR-VL1, SEQ ID NO:4, and a polypeptide sequence shown in the figure;

CDR-VL2, SEQ ID NO:5, and a polypeptide sequence shown in the figure;

CDR-VL3, SEQ ID NO:6, and a polypeptide having the amino acid sequence shown in FIG. 6.

The sequences of the light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4 are shown as SEQ ID NO. 11-14 in sequence;

the sequences of the heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4 are shown as SEQ ID NO. 7-10.

1. Affinity analysis

The purified antibody is diluted to 10ug/ml by PBST by using an AMC sensor, and ST2 antigen is subjected to gradient dilution by using PBST;

the operation flow is as follows: equilibration for 60s in buffer 1 (PBST), antibody 300s in antibody solution, incubation for 180s in buffer 2 (PBST), binding for 420s in antigen solution, dissociation for 1200s in buffer 2, sensor regeneration with 10mM pH 1.69GLY solution and buffer 3 (PBST), output data. (KD represents equilibrium dissociation constant, i.e., affinity; kon represents binding rate; kdis represents dissociation rate.) Table 1 shows the obtained affinity detection data.

Table 1 affinity assay data

Sample name	KD(M)	kon(1/Ms)	kdis(1/s)
				Control	7.30E-09	9.90E+05	7.23E-03
Anti-ST2 11F9	7.61E-10	9.79E+05	7.45E-04

2. Activity assay

Coating liquid (main component NaHCO) ₃ ) Diluting sheep anti-mouse IgG 1ug/ml, coating a microplate, and performing 100 uL/well overnight at 4 ℃; the next day, the washing liquid (main component Na ₂ HPO ₄ +NaCl) for 2 times, and beating to dry; blocking solution (20% BSA+80% PBS) was added and dried at 37℃for 1h in 120uL per well; adding diluted purified antibody at 100 uL/well, 37deg.C for 60min; removing liquid in the plate, beating, adding 20% mouse negative blood, sealing, and sealing 120ul,3 in each hole7 ℃ for 1h; removing liquid in the plate, beating, adding diluted ST2 antigen, 100uL per well, and 37 ℃ for 40min; washing with washing liquid for 5 times, and drying; the HRP-labeled ST2 monoclonal antibody (available from the Fipeng organism paired with purified antibody) was added at 100uL per well, 37℃for 30min; adding a developing solution A (50 uL/hole) and a developing solution B (50 uL/hole) for 10min; adding a stop solution, 50 uL/well; OD values read at 450nm (reference 630 nm) on a microplate reader, and the obtained antibody activity analysis data are shown in Table 2.

TABLE 2 data from antibody Activity analysis

Sample concentration (ng/ml)	31.250	15.625	7.813	3.906	1.953	0.000
							Control	1.509	0.665	0.370	0.260	0.163	0.046
Anti-ST2 11F9	1.883	1.495	0.984	0.489	0.205	0.061

3. Stability analysis

Bare stability assessment:

the self-produced antibody is placed at 4 ℃ (refrigerator), 80 ℃ (refrigerator) and 37 ℃ (incubator) for 21 days, 7 days, 14 days and 21 days are taken for state observation, and activity detection is carried out on the 21 days, so that the result shows that no obvious protein state change is seen for the antibody placed for 21 days under three examination conditions, the activity is not in a descending trend along with the increase of the examination temperature, and the stability of the self-produced antibody is indicated. Table 3 shows the results of the detection of OD after 21 days of enzyme-free activity.

TABLE 3 antibody stability test data

Sample concentration (ng/ml)	31.25	15.625	0
				4 ℃,21 days sample	1.829	1.358	0.094
Sample at-80℃for 21 days	1.835	1.331	0.083
				37 ℃ and 21 days of sample	1.833	1.345	0.067

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present application, and not for limiting the same; although the application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the application.

SEQUENCE LISTING

<110> Dongguan City, pengzhi biotechnology Co., ltd

<120> recombinant antibody against growth-stimulating expressed Gene 2 protein

<130> P2021030CN01

<160> 20

<170> PatentIn version 3.5

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Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

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Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Gln Thr Val

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Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

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Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro

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Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

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Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

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Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

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Val His Thr Ala Gln Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

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Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

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Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

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Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln

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Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

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Ser Leu Thr Cys Met Ile Thr Asn Phe Phe Pro Glu Asp Ile Thr Val

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Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln

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Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

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Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

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Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

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Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

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Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

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Phe Gln Ile Asn Asn Leu Arg Asn Glu Asp Thr Ala Thr Tyr Phe Cys

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Thr Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

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Ser Ile His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met

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Gly Trp Ile His Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe

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Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr

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Phe Gln Ile Asn Asn Leu Arg Asn Glu Asp Thr Ala Thr Tyr Phe Cys

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Ala Arg Asp Asp Gly Leu Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr

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Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro

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Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val

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Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser

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Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu

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Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser

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Gln Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val

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Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys

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Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys

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Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val

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Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp

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Asp Val Glu Val His Thr Ala Gln Thr Lys Pro Arg Glu Glu Gln Phe

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Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp

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Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe

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Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys

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Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys

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Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asn Phe Phe Pro Glu Asp

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Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys

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Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser

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Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr

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Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser

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Leu Ser His Ser Pro Gly

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Ser Arg Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Asp

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Tyr Asp Thr Asn Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly

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Ser Arg Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Asp

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Glu Asp Met Ala Thr Tyr Phe Cys Leu Gln His Ser Tyr Leu Pro Leu

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Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala

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Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly

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Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile

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Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu

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Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser

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Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr

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Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser

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Phe Asn Arg Asn Glu Cys

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Claims

1. An antibody comprising an ST2 antigen binding domain, wherein the antibody comprises a complementarity determining region of the amino acid sequence:

the amino acid sequence of the CDR-VH1 of the complementarity determining region is shown in SEQ ID NO:1 is shown in the specification;

the amino acid sequence of the complementarity determining region CDR-VH2 is shown in SEQ ID NO:2 is shown in the figure;

the amino acid sequence of the CDR-VH3 of the complementarity determining region is shown in SEQ ID NO:3 is shown in the figure;

the amino acid sequence of CDR-VL1 of the complementarity determining region is shown in SEQ ID NO:4 is shown in the figure;

the amino acid sequence of CDR-VL2 of the complementarity determining region is shown in SEQ ID NO:5 is shown in the figure; and

the amino acid sequence of CDR-VL3 of the complementarity determining region is shown in SEQ ID NO: shown at 6.

2. The antibody of claim 1, wherein the antibody is selected from any one of F (ab ') 2, fab', fab, fv, and scFv.

3. The antibody of claim 1, wherein the antibody comprises light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4 having a sequence as shown in SEQ ID NOs 11-14 or at least 90% homology thereto, and/or heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4 having a sequence as shown in SEQ ID NOs 7-10 or at least 90% homology thereto.

4. An antibody comprising an ST2 antigen binding domain, wherein said antibody has a heavy chain variable region of sequence set forth in SEQ ID No. 17 and a light chain variable region set forth in SEQ ID No. 19.

5. The antibody of any one of claims 1 to 4, further comprising an antibody constant region sequence.

6. The antibody of claim 5, wherein the constant region sequence is selected from the group consisting of the sequence of any one of the constant regions of IgG1, igG2, igG3, igG4, igA, igM, igE, igD.

7. The antibody of claim 5, wherein the constant region is of a species origin of bovine, equine, porcine, ovine, caprine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, or human.

8. The antibody of claim 7, wherein the cow is a dairy cow.

9. The antibody of claim 5, wherein the constant region is of mouse origin.

10. The antibody of claim 5, wherein the constant region comprises a heavy chain constant region and/or a light chain constant region,

the light chain constant region sequence is shown in SEQ ID NO: shown at 16;

the heavy chain constant region sequence is shown in SEQ ID NO: 15.

11. An isolated nucleic acid molecule, wherein the nucleic acid molecule is DNA or RNA encoding the antibody of any one of claims 1 to 10.

12. A vector comprising the nucleic acid molecule of claim 11.

13. A host cell transformed with the vector of claim 12.

14. The host cell of claim 13, wherein the host cell is a mammalian cell.

15. The host cell of claim 13, wherein the host cell is a Chinese Hamster Ovary (CHO) cell, a HeLa cell, a milbemouse kidney cell, an NS0 mouse myeloma cell.

16. A method of producing the antibody of any one of claims 1 to 10, comprising the steps of: culturing the host cell of any one of claims 13 to 15 under suitable culture conditions and recovering the produced antibody from the culture medium or from the cultured host cell.

17. Use of an antibody according to any one of claims 1 to 10 for the preparation of a diagnostic agent for diagnosing heart failure and evaluating heart function.

18. Use of an antibody according to any one of claims 1 to 10 in the preparation of a kit for detecting ST2 in a test sample comprising:

a) Contacting ST2 antigen in the test sample with the antibody of any one of claims 1-10 under conditions sufficient for an antibody/antigen binding reaction to occur to form an immune complex; and

b) Detecting the presence of said immune complex, the presence of said complex being indicative of the presence of said ST2 in said test sample.

19. The use according to claim 18, wherein in step a) the immune complex further comprises a second antibody, which second antibody binds to the antibody.

20. The use according to claim 18, wherein in step a) the immune complex further comprises a second antibody, which binds to ST 2.

21. A reagent or kit comprising the antibody of any one of claims 1-10.

22. The kit of claim 21, wherein the reagent or kit further comprises one or more of a pharmaceutically acceptable excipient, buffer, stabilizer, diluent or carrier.