An Antibody Against NT-proBNP and Its Application

Abstract

This invention discloses an anti-NT-proBNP antibody and its applications, relating to the field of antibodies. The anti-NT-proBNP antibody disclosed in this invention includes a heavy chain complementarity-determining region and a light chain complementarity-determining region. This antibody provides an important source of raw materials for the detection of NT-proBNP and exhibits good affinity or activity.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibody technology, in particular, to an antibody against NT-proBNP and application thereof. BACKGROUND

[0002] In 1988, Japanese scholar Sudoh first isolated a polypeptide with strong diuresis, vasodilation and antihypertensive effect from pig brain, which was named brain natriuretic peptide (BNP). BNP is distributed most in the heart, but the myocardial cells first synthesize proBNP (BNP precursor) containing 108 amino acids. When the myocardial cells are stimulated, proBNP is cleaved into N-terminal B-type natriuretic peptide (NT-proBNP) containing 76 amino acids and having no biological activity and B-type natriuretic peptide (BNP) containing 32 amino acids and having activity by the action of endonuclease. The two are of the same source and are released into the blood circulation in equimolar amounts.

[0003] When the cardiac volume load increases or the cardiac function is impaired, the index concentration of N-terminal brain natriuretic peptide precursor (NT-proBNP) and BNP abnormally increases. NT-proBNP has better biological stability than BNP, a longer half-life (120 min), a relatively stable concentration, a long effective detection time, and a relatively higher content in blood than BNP by about 16-20 times. Therefore, the detection is relatively easy, and the stability of the plasma sample in vitro is long (>48 h). NT-proBNP is the best myocardial marker for diagnosing heart failure and evaluating cardiac function.

[0004] The content of NT-proBNP in normal human blood is generally less than 0.3 ng / mL. When the cardiac function is impaired and the myocardium is dilated, NT-proBNP is rapidly synthesized and secreted into the human body in large amounts. When some related early symptoms are found, accurate, sensitive, efficient and stable determination of the amount of NT-proBNP in blood can provide a rapid and accurate early diagnosis basis for early heart failure, heart failure, dyspnea, cardiac and non-cardiac heart failure treatment and prognosis monitoring, and classification of acute coronary syndrome.

[0005] At present, the methods for detecting NT-proBNP mainly include gold standard qualitative test, fluorescence immunoassay, enzyme-linked immunosorbent assay (ELISA) and magnetic microparticle chemiluminescence method (CMIA). However, these detection methods need antibodies against NT-proBNP. Therefore, there is a strong demand in the art for NT-proBNP antibodies with good performance. SUMMARY

[0006] The present application provides an antibody against NT-proBNP, which provides an important raw material source for the detection of NT-proBNP and has good activity or affinity.

[0007] To achieve the above-mentioned purpose, according to one aspect of the present application, an antibody against NT-proBNP is provided, which comprises three complementarity determining regions of a heavy chain variable region having an amino acid sequence shown in SEQ ID NO: 17 and three complementarity determining regions of a light chain variable region having an amino acid sequence shown in SEQ ID NO: 19.

[0008] To achieve the above-mentioned purpose, according to a second aspect of the present application, an antibody against NT-proBNP is provided, which comprises the following complementarity determining regions:

[0009] HCDR1 comprising or consisting of an amino acid sequence shown in SEQ ID NO: 1;

[0010] HCDR2 comprising or consisting of an amino acid sequence shown in SEQ ID NO: 2;

[0011] HCDR3 comprising or consisting of an amino acid sequence shown in SEQ ID NO: 3;

[0012] LCDR1 comprising or consisting of an amino acid sequence shown in SEQ ID NO: 4;

[0013] LCDR2 comprising or consisting of an amino acid sequence shown in SEQ ID NO: 5; and

[0014] LCDR3 comprising or consisting of an amino acid sequence shown in SEQ ID NO: 6.

[0015] To achieve the above-mentioned purpose, according to a third aspect of the present application, an antibody against NT-proBNP is provided, which comprises a heavy chain variable region and / or a light chain variable region, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 17; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 19.

[0016] To achieve the above-mentioned purpose, according to a fourth aspect of the present application, an antibody against NT-proBNP is provided, which comprises a heavy chain and / or a light chain, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 18; the amino acid sequence of the light chain is shown in SEQ ID NO: 20.

[0017] To achieve the above-mentioned purpose, according to a fifth aspect of the present application, an antibody conjugate is provided, which comprises the above-mentioned antibody.

[0018] To achieve the above object, according to a sixth aspect of the present application, there is provided a reagent or a kit comprising the above-mentioned antibody or the above-mentioned antibody conjugate.

[0019] To achieve the above object, according to an eighth aspect of the present application, there is provided a use of the above-mentioned antibody, antibody conjugate, reagent or kit in the manufacture of a product for detecting NT-proBNP.

[0020] To achieve the above object, the present application further provides a nucleic acid, a vector, a cell and a method for preparing the above-mentioned antibody. BRIEF DESCRIPTION OF DRAWINGS

[0021] To more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments will be briefly introduced as follows. It should be understood that the following drawings only show some of the embodiments of the present application, and therefore should not be considered as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0022] Figure 1 Results of the reducing SDS-PAGE of Anti-NT-proBNP 3F12 Rmb1. DETAILED DESCRIPTION

[0023] In a first aspect, the embodiments of the present application provide an antibody against NT-proBNP, which comprises three complementarity determining regions of a heavy chain variable region having an amino acid sequence shown in SEQ ID NO: 17 and three complementarity determining regions of a light chain variable region having an amino acid sequence shown in SEQ ID NO: 19.

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

[0025] The above-mentioned antigen-binding fragments generally have the same binding specificity as the antibody from which they are derived. Those skilled in the art can easily understand from the content disclosed in the present application that the above-mentioned antigen-binding fragments can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by methods of splitting disulfide bonds by chemical reduction. Those skilled in the art can easily obtain the above-mentioned antigen-binding fragments on the basis of the structural basis of the complete antibody disclosed in the present application.

[0026] The above antigen-binding fragments can also be obtained by recombinant genetics techniques, also known to those skilled in the art, or by synthesis, for example, by means of an automatic peptide synthesizer, such as the automatic peptide synthesizer sold by Applied BioSystems.

[0027] In the present application, the term "complementarity determining region", "CDR" or "CDRs" refers to the highly variable regions of the heavy and light chains of immunoglobulins, referring to the regions comprising one or more, or even all, of the main amino acid residues that play a role in the binding of the antibody or antigen-binding fragment to the antigen or epitope it recognizes. In the detailed description of the application, CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

[0028] In the present application, the heavy chain complementarity determining region is denoted as HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determining region is denoted as LCDR, which includes LCDR1, LCDR2 and LCDR3.

[0029] The definition method of CDR is well known in the art, and the CDR definition method includes: Kabat definition, Chothia definition, IMGT definition, Contact definition and AbM definition. As described herein, the "Kabat definition" refers to the definition system described in Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). The "Chothia definition" is described in Chothia et al., J Mol Biol 196:901-917 (1987). There can be other CDR definition methods that do not strictly follow one of the above schemes, but still overlap at least part of the CDR regions defined by Kabat, although they can be shortened or lengthened according to the prediction or experimental results of specific residues or groups of residues. Exemplary defined CDRs are listed in Table 1 below, with slight differences in the definitions in different literatures. Given the variable region amino acid sequence of a given antibody, one skilled in the art can routinely determine which residues comprise a particular CDR. It should be noted that CDRs defined by other methods in addition to Table 1 are also within the protection scope of the present disclosure.

[0030] Table 1: CDR definition 1

[0031] CDR Kabat AbM2 IMGT Chothia HCDR1 [H31-H35 3 ]]> [H26-H35 3 ]] [H26~H33..5 5 ]]> 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

[0032] 1The numbering of all CDR definitions in Table 1 is according to the Kabat numbering system (see below), with "H+ numbers" for amino acid positions on the heavy chain and "L+ numbers" for amino acid positions on the light chain. One of ordinary skill in the art can unambiguously correlate this Kabat numbering system to any variable region sequence without reliance on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0033] 2 "AbM" as used in Table 1 with a lower case "b" refers to CDRs defined by the "AbM" antibody modeling software of Oxford Molecular.

[0034] 3 If neither H35A nor H35B is present, then CDR-H1 ends at position 35; if only H35A is present, then CDR-H1 ends at position 35A; if both H35A and H35B are present, then CDR-H1 ends at position 35B.

[0035] 4 If neither H35A nor H35B is present, then CDR-H1 ends at position 32; if only H35A is present, then CDR-H1 ends at position 33; if both H35A and H35B are present, then CDR-H1 ends at position 34.

[0036] 5 If neither H35A nor H35B is present, then CDR-H1 ends at position 33; if only H35A is present, then CDR-H1 ends at position 34; if both H35A and H35B are present, then CDR-H1 ends at position 35.

[0037] According to embodiments of the application, 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.

[0038] In some alternative embodiments of the application, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Kabat system.

[0039] In some alternative embodiments of the present application, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Chothia system.

[0040] In some alternative embodiments of the present application, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT system.

[0041] In some alternative embodiments of the present application, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the AbM system.

[0042] In some alternative embodiments of the present application, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Contact system.

[0043] In some alternative embodiments of the present application, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a combination of the Kabat, Chothia, IMGT, AbM or Contact system.

[0044] According to embodiments of the present application, the Kabat, Chothia, AbM or IMGT system defined HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 amino acid sequence corresponding Kabat numbering position is as follows:

[0045] CDR Kabat AbM IMGT Chothia HCDR1 H31~H35 H26~H35 H26~H33 H26~H32 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

[0046] According to embodiments of the present application, the antibody comprises the following complementarity determining regions:

[0047] HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 1;

[0048] HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 2;

[0049] HCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 3;

[0050] LCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 4;

[0051] LCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 5; and

[0052] LCDR3, which comprises or consists of the amino acid sequence set forth in SEQ ID NO: 6.

[0053] In a second aspect, embodiments of the present application provide an antibody against NT-proBNP, which comprises the following complementarity determining regions:

[0054] HCDR1, which comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1;

[0055] HCDR2, which comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2;

[0056] HCDR3, which comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3;

[0057] LCDR1, which comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4;

[0058] LCDR2, which comprises or consists of the amino acid sequence set forth in SEQ ID NO: 5; and

[0059] LCDR3, which comprises or consists of the amino acid sequence set forth in SEQ ID NO: 6.

[0060] According to embodiments of the present application, the HCDRs and LCDRs are defined by the Kabat system.

[0061] In the present application, the "framework region" or "FR" region, which includes the heavy chain framework region and the light chain framework region, refers to the region in the antibody heavy chain variable region and the light chain variable region other than the CDR; wherein the heavy chain framework region can be further subdivided into adjacent regions separated by CDRs, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by CDRs, including LFR1, LFR2, LFR3 and LFR4 framework regions.

[0062] In the present application, the heavy chain variable region is obtained by connecting the CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by connecting the CDRs and FRs in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0063] In an optional embodiment, the antibody of the first aspect or the second aspect further has HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4.

[0064] In optional embodiments, the HFR1 comprises / is an amino acid sequence of SEQ ID NO: 7 or at least 80% identical thereto;

[0065] the HFR2 comprises / is an amino acid sequence of SEQ ID NO: 8 or at least 80% identical thereto;

[0066] the HFR3 comprises / is an amino acid sequence of SEQ ID NO: 9 or at least 80% identical thereto;

[0067] the HFR4 comprises / is an amino acid sequence of SEQ ID NO: 10 or at least 80% identical thereto;

[0068] the LFR1 comprises / is an amino acid sequence of SEQ ID NO: 11 or at least 80% identical thereto;

[0069] the LFR2 comprises / is an amino acid sequence of SEQ ID NO: 12 or at least 80% identical thereto;

[0070] the LFR3 comprises / is an amino acid sequence of SEQ ID NO: 13 or at least 80% identical thereto; and

[0071] the LFR4 comprises / is an amino acid sequence of SEQ ID NO: 14 or at least 80% identical thereto.

[0072] It is to be noted that in other embodiments, each of the framework regions of the antibody against NT-proBNP provided by the present application can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the corresponding framework region (SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, or 14) described above.

[0073] In optional embodiments, the antibody binds NT-proBNP with an affinity of KD< 2.75 x 10 -9 M.

[0074] In optional embodiments, the antibody binds NT-proBNP with an affinity of KD≤ 10 -8 M, KD≤ 10 -9 M, KD≤ 10 -10 M, KD≤ 10 -11 M, or KD≤ 10 -12 M.

[0075] In an alternative embodiment, the antibody binds NT-proBNP with an affinity of KD≤ 2.56 x 10 -10 M with an affinity of KD≤ 2.56 x 10

[0076] There are many methods for determining the affinity (KD) of an antibody, which can be classified into thermodynamic detection methods, kinetic detection methods and dynamic equilibrium detection methods according to the detection principle. Among them, the thermodynamic detection method is commonly known as isothermal titration calorimetry (ITC); the kinetic detection method is commonly known as surface plasmon resonance (SPR) and bio-layer interferometry (BLI); and the dynamic equilibrium detection method is commonly known as enzyme-linked immunosorbent assay (ELISA) and the like.

[0077] In an alternative embodiment, the determination of KD is performed using a kinetic detection method; alternatively, surface plasmon resonance, for example by using a system such as Biacore®. Biacore® system.

[0078] In an alternative embodiment, the antibody comprises a heavy chain variable region and / or a light chain variable region, the heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 17, and the light chain variable region having an amino acid sequence as shown in SEQ ID NO: 19.

[0079] In a third aspect, an embodiment of the present application provides an antibody against NT-proBNP, comprising a heavy chain variable region and / or a light chain variable region, the heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 17, and the light chain variable region having an amino acid sequence as shown in SEQ ID NO: 19.

[0080] In an alternative embodiment, the antibody of the first aspect, the second aspect or the third aspect further comprises a constant region.

[0081] In an alternative embodiment, the constant region comprises a heavy chain constant region and / or a light chain constant region.

[0082] In an alternative embodiment, the heavy chain constant region is selected from any one of IgG, IgA, IgM, IgE, IgD heavy chain constant region or a combination of multiple constant region segments.

[0083] In an alternative embodiment, the heavy chain constant region comprises CH1 of IgG, hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM.

[0084] In an alternative embodiment, the IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0085] In an alternative embodiment, the light chain constant region is selected from kappa type or lambda type light chain constant region.

[0086] In an alternative embodiment, the species origin of the constant region is bovine, equine, bovine, porcine, ovine, rat, mouse, dog, camel, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, cock or human.

[0087] In an alternative embodiment, the species origin of the constant region is mouse.

[0088] In the present text, the division of variable and constant region sequences refers to the IMGT division, see Lefranc, M.-P., Pommie, C, Ruino, C, Giudicelli, V., Jabado- the international ImMunoGeneTics database. Nucl. Acids Res., 29(1): 207-209 (2001). DOI: 10.1093 / nar / 29.1.207. PMID: 11125093. and Martinez-Jean C. and Bosc N. or Ehrenmann, Patrice Duroux, Chantal Ginestoux, Gene table: house mouse (Mus musculus) IGHG, IMGT Repertoire. the international ImMunoGeneTics 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. the international ImMunoGeneTics information http: / / www.imgt.org .Created: 16 / 03 / 2011. Version: 17 / 01 / 2020. Different methods of division of the variable region can have differences in the C-terminal end of the variable region or in the N-terminal end of the constant region compared to the IMGT division. Other methods of division of the variable region or the constant region known in the art are also within the scope of the present application.

[0089] In an alternative embodiment, the heavy chain constant region sequence (CH) is as shown in SEQ ID NO: 15 and the light chain constant region (CL) sequence is as shown in SEQ ID NO: 16.

[0090] It should be noted that in other embodiments, the constant region sequence can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the constant region sequence (SEQ ID NO: 15 or 16).

[0091] In optional embodiments, the antibody includes any one of F(ab)2, F(ab')2, Fab', Fab, Fv, and scFv.

[0092] In optional embodiments, the antibody includes a heavy chain and / or a light chain, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain is shown in SEQ ID NO: 20.

[0093] In a fourth aspect, the present application provides an antibody against NT-proBNP, including a heavy chain and / or a light chain, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain is shown in SEQ ID NO: 20.

[0094] In a fifth aspect, the present application provides an antibody conjugate, including the antibody described above.

[0095] In optional embodiments, the antibody conjugate described above further includes biotin or a biotin derivative coupled to the antibody.

[0096] In optional embodiments, the antibody conjugate further includes a label coupled to the antibody.

[0097] In optional embodiments, the label refers to a kind of substance having a property that can be directly observed by the naked eye or detected or probed by an instrument, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0098] In optional embodiments, the label includes but is not limited to fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

[0099] In actual use, a person skilled in the art can select a suitable label according to the detection conditions or actual needs, and no matter what label is used, it falls within the protection scope of the present application.

[0100] In optional embodiments, the fluorescent dye includes, but is not limited to, fluorescein-based dyes and their derivatives (such as, but not limited to, fluorescein isothiocyanate (FITC), hydroxycoumarin (FAM), tetra-chloro-fluorescein (TET), and the like or analogs thereof), rhodamine-based dyes and their derivatives (such as, but not limited to, red rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), and the like or analogs thereof), Cy series dyes and their derivatives (such as, but not limited to, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy3, and the like or analogs thereof), Alexa series dyes and their derivatives (such as, but not limited to, Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, and the like or analogs thereof), and protein-based dyes and their derivatives (such as, but not limited to, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), and the like).

[0101] In optional embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphogluconate dehydrogenase.

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

[0103] In optional embodiments, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, crustacyanin, ruthenium bis-2,2'-bipyridine and its derivatives, acridinium ester and its derivatives, dioxetane and its derivatives, luminol and its derivatives, and peroxyoxalate and its derivatives.

[0104] In optional embodiments, the nanoparticle-based label includes, but is not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0105] In optional embodiments, the colloid includes, but is not limited to, colloidal metals, colloidal carbon, dispersed dyes, dye-labeled microspheres, and latex.

[0106] In optional embodiments, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver and colloidal selenium.

[0107] In optional embodiments, the colloidal metal is colloidal gold.

[0108] In optional embodiments, the antibody conjugate further comprises a solid support coupled to the antibody.

[0109] In optional embodiments, the solid support is selected from the group consisting of microspheres, plates and membranes.

[0110] In optional embodiments, the solid support includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic microparticles, microplates, glass, capillary tubes, nylon and nitrocellulose membranes.

[0111] In a sixth aspect, the present application provides a reagent or a kit, which comprises the antibody or the antibody conjugate as described above.

[0112] As described above, the antibody in some embodiments or examples of the present application can effectively bind to NT-proBNP, and thus the reagent or the kit comprising the NT-proBNP antibody can effectively detect NT-proBNP qualitatively or quantitatively. The reagent or the kit provided by the present application can be used in, for example, immunoblotting, immunoprecipitation and other detections involving the specific binding of NT-proBNP and its antibody. As described above, the antibody in some embodiments or examples of the present application has higher binding activity or affinity to NT-proBNP, and thus the reagent or the kit comprising the antibody has higher detection sensitivity or specificity.

[0113] In a seventh aspect, the present application provides a method for detecting NT-proBNP, comprising: a) contacting the antibody, the antibody conjugate, the reagent or the kit as described above with NT-proBNP in a sample to be detected under conditions sufficient for an antibody / antigen binding reaction to occur to form an immunocomplex; and b) detecting the presence of the immunocomplex, wherein the presence of the complex indicates the presence of the antigen in the test sample.

[0114] In optional embodiments, the immunocomplex further comprises a second antibody, which binds to the antibody.

[0115] In optional embodiments, the immunocomplex further comprises a second antibody, which binds to NT-proBNP.

[0116] In an eighth aspect, the present application provides use of the above-mentioned anti-NT-proBNP antibody, antibody conjugate or the above-mentioned reagent or kit in the manufacture of a product for detecting NT-proBNP.

[0117] In a ninth aspect, the present application provides a nucleic acid molecule encoding the above-mentioned antibody.

[0118] In a tenth aspect, the present application provides a vector containing the above-mentioned nucleic acid molecule.

[0119] In an eleventh aspect, the present application provides a cell containing the above-mentioned vector.

[0120] In a twelfth aspect, the present application provides a method for producing an anti-NT-proBNP antibody, comprising culturing the above-mentioned cell.

[0121] In a thirteenth aspect, the present application provides use of the above-mentioned antibody, antibody conjugate or the above-mentioned reagent or kit in the detection of NT-proBNP or in the indication of NT-proBNP related disease.

[0122] In a fourteenth aspect, the present application provides a method for indicating NT-proBNP related disease in a subject, comprising:

[0123] a) contacting the above-mentioned antibody, antibody conjugate or the above-mentioned reagent or kit with NT-proBNP in a sample from the subject under conditions sufficient for an antibody / antigen binding reaction to occur to form an immunocomplex; and

[0124] b) detecting the presence of the immunocomplex, the presence of the complex indicating the presence or status of NT-proBNP related disease in the subject.

[0125] In an optional embodiment, the immunocomplex further comprises a second antibody, which binds to the antibody.

[0126] In an optional embodiment, the immunocomplex further comprises a second antibody, which binds to NT-proBNP.

[0127] In an optional embodiment, the NT-proBNP related disease in the thirteenth aspect or the fourteenth aspect is selected from the group consisting of cardiac dysfunction, heart failure, acute coronary syndrome, etc.

[0128] On the basis of the amino acid sequences of the anti-NT-proBNP antibody disclosed in the present application, it is easy for those skilled in the art to prepare the anti-NT-proBNP antibody by using genetic engineering technology or other technologies (chemical synthesis, recombinant expression), for example, to isolate and purify the antibody from the culture product of recombinant cells capable of recombinantly expressing the antibody as described in any one of the above, which is easy for those skilled in the art to achieve. Therefore, no matter what technology is used to prepare the anti-NT-proBNP antibody of the present application, it all falls within the protection scope of the present application.

[0129] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not indicated in the embodiments, the conventional conditions or the conditions suggested by the manufacturers are adopted. If the manufacturers of the reagents or instruments are not indicated, they are all the conventional products that can be purchased in the market.

[0130] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the preparations or unit doses herein, some methods and materials are now described. The techniques employed or contemplated herein are standard methodologies unless otherwise indicated. The materials, methods, and examples are illustrative only and not limiting.

[0131] Laboratory Manual, 2nd Ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Current Protocols in Immunology (J. E. Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0132] The features and nature of the present application will be further described in connection with the following examples.

[0133] Example 1 Preparation of Anti-NT-proBNP 3F12 Monoclonal Antibody

[0134] In this example, restriction enzymes and Prime Star DNA polymerase were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART TMRACE cDNA Amplification Kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were completed by Invitrogen. Hybridoma cell line secreting Anti-NT-proBNP 3F12 monoclonal antibody was prepared in the laboratory.

[0135] (1) Antibody gene preparation

[0136] The mRNA was extracted from the hybridoma cell line secreting Anti-NT-proBNP 3F12 monoclonal antibody, and the DNA product was obtained by RT-PCR method. The product was inserted into the pMD-18T vector after A reaction with rTaq DNA polymerase, and transformed into DH5a competent cells. After the colonies were grown, the Heavy Chain and Light Chain gene clones were taken respectively, and each 4 clones were sent to the gene sequencing company for sequencing.

[0137] (2) Sequence analysis of Anti-NT-proBNP 3F12 antibody variable region gene

[0138] The gene sequence obtained by the above sequencing was placed in the kabat antibody database for analysis, and the VNTI11.5 software was used for analysis to determine that the amplified genes by the heavy chain and light chain primer pairs were correct. Among them, the VL gene sequence in the Light Chain amplified gene fragment was 339 bp, and there was a 57 bp leader peptide sequence in front of it; the Vh gene sequence in the Heavy Chain amplified gene fragment was 354 bp, belonging to the VH1 gene family, and there was a 57 bp leader peptide sequence in front of it.

[0139] (3) Construction of recombinant antibody expression plasmid

[0140] pcDNA TM 3.4 vector is the constructed recombinant antibody eukaryotic expression vector. The expression vector has introduced HindIII, BamHI, EcoRI and other multiple enzyme digestion sites, and is named pcDNA3.4A expression vector, which is referred to as 3.4A expression vector hereinafter; according to the antibody variable region gene sequencing results in the above pMD-18T, the VL and VH gene specific primers of the antibody were designed, which have HindIII, EcoRI enzyme digestion sites and protection bases at both ends. The 0.71 kb Light Chain gene fragment and the 1.38 kb Heavy Chain gene fragment were amplified by PCR amplification method.

[0141] The Heavy Chain and Light Chain gene fragments were digested with HindIII / EcoRI, and the 3.4A vector was also digested with HindIII / EcoRI. After purification and recovery of the fragments and vector, the Heavy Chain gene and Light Chain gene were ligated into the 3.4A expression vector to obtain recombinant expression plasmids of Heavy Chain and Light Chain, respectively.

[0142] 2. Recombinant antibody production

[0143] HEK293 cells were revived early and passaged to a 200ml volume to achieve a cell density of 3–5 × 10⁻⁶ cells / mL. 6 Cell density reached the required antibody concentration and cell viability >95%; cells were washed by centrifugation, reconstituted with culture medium, and the cell density was adjusted to 2.9 × 10⁻⁶ cells / ml. 6 Cells were washed at a concentration of cells / ml and reconstituted with culture medium, which was used as a cell dilution buffer. Plasmid DNA and transfection reagent dilution buffers were prepared separately using culture medium. The transfection reagent dilution buffer was added to the plasmid DNA dilution buffer, mixed well, and incubated at room temperature for 15 min. This mixture was then slowly added to the cell dilution buffer over 1 min, mixed well, and samples were taken for cell counting. Cell viability after transfection was recorded and observed. The cells were then incubated at 35°C with a rotation speed of 120 rpm and a CO2 concentration of 8%. After 13 days, the samples were centrifuged and collected. The supernatant was purified using a protein A affinity chromatography column. 6 μg of the purified antibody was subjected to reducing SDS-PAGE, and the electrophoresis result is shown in the figure. The reducing SDS-PAGE showed two bands: one with a Mr of 50 kDa (heavy chain) and the other with a Mr of 28 kDa (light chain).

[0144] The resulting antibody was named Anti-NT-proBNP 3F12Rmb1. The heavy chain amino acid sequence of the antibody Anti-NT-proBNP 3F12Rmb1 is shown in SEQ ID NO:18, and the light chain amino acid sequence is shown in SEQ ID NO:20.

[0145] Example 2: Antibody Performance Detection

[0146] 1. Affinity Analysis

[0147] Purified antibody was diluted in advance, and NT-proBNP antigen (from Fupeng Biotech) was gradient diluted; the dissociation curve of antigen-antibody binding was tested on a Biacore 8K+ device using a CM5 chip pre-coupled with sheep anti-mouse IgG, and the instrument automatically fitted to obtain the affinity constant, binding rate, and dissociation rate. (KD represents the equilibrium dissociation constant, i.e., the affinity constant; ka represents the binding rate; and kd represents the dissociation rate)

[0148] Table 2 Affinity data

[0149] Sample name KD ka kd Control 2.75E-09 5.67E+05 1.56E-03 Anti-NT-proBNP 3F12Rmb1 2.56E-10 2.59E+06 6.64E-04

[0150] 2. Activity identification

[0151] Coating solution (main component NaHCO3) diluted NT-PROBNP antigen (from Fupeng Biotech) to 1 ug / ml, 100 uL per well, 4°C overnight; the next day, wash solution (main components Na2HPO4+Nacl) was washed twice, and then dried; blocking solution (20% BSA+80% PBS) was added, 120 uL per well, 37°C, 1h, and then dried; diluted purified antibody and control antibody were added, 100 uL per well, 37°C, 30min; wash solution was washed 5 times, and then dried; sheep anti-mouse IgG-HRP was added, 100 uL per well, 37°C, 30min; wash solution was washed 5 times, and then dried; color developing solution A (50 uL per well) was added, color developing solution B (50 uL per well) was added, 10min; stop solution was added, 50 uL per well; and OD value was read on a microplate reader at 450nm (reference 630nm).

[0152] Note: A solution (main components citric acid+ sodium acetate+acetanilide+urea peroxide); B solution (main components citric acid+EDTA·2Na+TMB+concentrated HCL); stop solution (EDTA·2Na+concentrated H2SO4)

[0153] Table 3 Activity data

[0154]

[0155] 3. Stability evaluation

[0156] The above antibody was placed at 4°C (refrigerator), -80°C (refrigerator), and 37°C (incubator) for 21 days, and samples taken at 7 days, 14 days, and 21 days were observed for state, and the 21-day sample was detected for activity. The results showed that under the three evaluation conditions, the antibody placed for 21 days did not show obvious changes in protein state, and the activity did not show a downward trend with the increase of the evaluation temperature, indicating that the above antibody was stable. Table 4 below shows the OD results of enzyme immunoactivity detection of the antibody Anti-NT-proBNP 3F12Rmb1 evaluated for 21 days.

[0157] Table 4 Stability data

[0158] Sample concentration (ng / ml) 3.91 1.95 0.00 4°C, 21 days sample 1.455 0.908 0.031 -80°C, 21 days sample 1.462 0.921 0.033 37°C, 21 days sample 1.448 0.913 0.032

[0159] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

[0160] Part of the amino acid sequences involved in the present application are shown in Table 5:

[0161]

Claims

1. An antibody against NT-proBNP, characterized in that, the antibody comprises three complementarity determining regions of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 17 and three complementarity determining regions of a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 19; the complementarity determining regions of the variable regions are defined by any one of the Kabat, Chothia, IMGT, AbM, or Contact systems.

2. An antibody against NT-proBNP, characterized in that the antibody comprises the following complementarity determining regions: HCDR1 having the amino acid sequence set forth in SEQ ID NO: 1 ; HCDR2 having the amino acid sequence set forth in SEQ ID NO: 2; HCDR3 having the amino acid sequence set forth in SEQ ID NO: 3; LCDR1 having the amino acid sequence set forth in SEQ ID NO: 4; LCDR2 having the amino acid sequence set forth in SEQ ID NO: 5; and LCDR3 having the amino acid sequence set forth in SEQ ID NO:

6.

3. The antibody according to claim 1 or 2, characterized in that the antibody further has HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4.

4. The antibody of claim 3, wherein the HFR1 comprises SEQ ID NO: 7 or an amino acid sequence at least 80% identical thereto; the HFR2 comprises SEQ ID NO: 8 or an amino acid sequence at least 80% identical thereto; the HFR3 comprises SEQ ID NO: 9 or an amino acid sequence at least 80% identical thereto; the HFR4 comprises SEQ ID NO: 10 or an amino acid sequence at least 80% identical thereto; the LFR1 comprises SEQ ID NO: 11 or an amino acid sequence at least 80% identical thereto; the LFR2 comprises SEQ ID NO: 12 or an amino acid sequence at least 80% identical thereto; the LFR3 comprises SEQ ID NO: 13 or an amino acid sequence at least 80% identical thereto; and the LFR4 comprises SEQ ID NO: 14 or an amino acid sequence at least 80% identical thereto.

5. The antibody of any one of claims 1, 2, 4, characterized in that, The antibody binds NT-proBNP with a KD < 2.75 x 10 -9 M binds NT-proBNP with high affinity.

6. An antibody against NT-proBNP comprising a heavy chain variable region and a light chain variable region, characterized in that, the heavy chain variable region amino acid sequence is set forth in SEQ ID NO: 17; and the light chain variable region amino acid sequence is set forth in SEQ ID NO:

19.

7. The antibody of any one of claims 1, 2, 4, 6, wherein, the antibody further comprises a constant region.

8. The antibody of claim 7, wherein the constant region comprises a heavy chain constant region and / or a light chain constant region.

9. The antibody of claim 8, wherein the heavy chain constant region is selected from the group consisting of a heavy chain constant region of any one of IgG, IgA, IgM, IgE, IgD, or a combination of constant region segments.

10. The antibody of claim 8, wherein the heavy chain constant region comprises CH1 of IgG, a hinge region of IgG, CH2 of IgM, CH3 of IgM, and / or CH4 of IgM.

11. The antibody of claim 7, wherein the species of origin of the constant region is bovine, equine, porcine, ovine, caprine, rat, mouse, dog, camel, cat, rabbit, donkey, deer, mink, chicken, duck, goose, or human.

12. The antibody of claim 7, wherein the species of origin of the constant region is mouse.

13. The antibody of claim 8, wherein the heavy chain constant region sequence is set forth in SEQ ID NO: 15 or is at least 80% identical thereto.

14. The antibody of claim 8, wherein the light chain constant region sequence is set forth in SEQ ID NO: 16 or is at least 80% identical thereto.

15. The antibody of any one of claims 1, 2, 4, 6, wherein, The antibody comprises any one of F(ab')2, Fab', Fab, Fv and scFv.

16. An antibody against NT-proBNP comprising a heavy chain and a light chain, characterized in that, The amino acid sequence of the heavy chain is shown as SEQ ID NO: 18; and the amino acid sequence of the light chain is shown as SEQ ID NO:

20.

17. An antibody conjugate, characterized in that, The antibody conjugate consists of the antibody of any one of claims 1 to 16 and a label or a solid phase carrier conjugated to the antibody.

18. The antibody conjugate of claim 17, wherein, The label is selected from the group consisting of biotin, a fluorescent dye, an enzyme, a radioisotope, a chemiluminescent reagent and a nanoparticle-based label.

19. A reagent or kit characterized in that, The reagent or kit comprises the antibody of any one of claims 1 to 16 or the antibody conjugate of any one of claims 17 to 18.

20. Use of the antibody of any one of claims 1 to 16, the antibody conjugate of claim 17 or 18, or the reagent or kit of claim 19 in the manufacture of a product for detecting NT-proBNP.

21. Use according to claim 20, characterized in that, The use comprises: a) contacting the antibody of any one of claims 1 to 16, the antibody conjugate of claim 17 or 18, or the reagent or kit of claim 19 with NT-proBNP in a sample to be tested under conditions sufficient for an antibody / antigen binding reaction to occur to form an immunocomplex; and b) detecting the presence of the immunocomplex, the presence of the complex indicating the presence of the antigen in the sample to be tested.

22. Use according to claim 21, characterized in that, The immunocomplex further comprises a second antibody which binds to the antibody.

23. The use according to claim 21, characterized in that, The immunocomplex further comprises a second antibody which binds to NT-proBNP.

24. A nucleic acid, comprising: which encodes the antibody of any one of claims 1 to 16.

25. A vector, comprising: which contains the nucleic acid of claim 24.

26. A cell, comprising: which contains the nucleic acid of claim 24 or the vector of claim 25.

27. A method of producing an antibody according to any one of claims 1 to 16, characterized in that, which comprises: culturing the cell of claim 26.

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