CN119306831A

CN119306831A - Anti-NT-proBNP antibodies, reagents and kits for detecting NT-proBNP

Info

Publication number: CN119306831A
Application number: CN202310851617.3A
Authority: CN
Inventors: 钟冬梅; 孟媛; 梁碧; 唐丽娜
Original assignee: Dongguan Pengzhi Biotechnology Co Ltd
Current assignee: Dongguan Pengzhi Biotechnology Co Ltd
Priority date: 2023-07-11
Filing date: 2023-07-11
Publication date: 2025-01-14

Abstract

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

Description

Anti-NT-proBNP antibody, reagent and kit for detecting NT-proBNP

Technical Field

The invention relates to the technical field of antibodies, in particular to an anti-NT-proBNP antibody, a reagent for detecting NT-proBNP and a kit.

Background

Japanese scholars Sudoh in 1988 isolated a polypeptide with potent diuretic, vasodilatory and antihypertensive effects from pig brain for the first time, named brain natriuretic peptide (Brain Natriuretic Peptide, BNP). BNP is distributed at the highest heart content, but proBNP (BNP precursor) containing 108 amino acids is first synthesized by cardiomyocytes, and when the cardiomyocytes are stimulated, the proBNP is cleaved under the action of endoenzymes to form N-terminal pro-B-natriuretic peptide (NT-proBNP) containing 76 amino acids and having no biological activity.

When the cardiac capacity load is increased or the cardiac function is impaired, the index concentration of the N-terminal brain natriuretic peptide precursor (NT-proBNP) is abnormally increased, the NT-proBNP has better biostability, longer half-life (120 min), relatively stable concentration and long effective detection time, so that the detection is relatively easy, and the stability of the plasma specimen in vitro is long, thus being the optimal cardiac marker for diagnosing heart failure and evaluating cardiac function.

Normal human blood typically has a NT-proBNP level of less than 0.3ng/mL. When the cardiac function is impaired and the myocardium dilates, NT-proBNP is rapidly synthesized and secreted in large quantities into the human blood. When some related early symptoms are found, the amount of NT-proBNP in blood can be accurately, sensitively, efficiently and stably measured, and the rapid and accurate early diagnosis reference can be provided for the aspects of cardiac and non-cardiac heart failure treatment, prognosis monitoring, grading of acute coronary syndrome and the like of early cardiac insufficiency, heart failure and dyspnea. Current methods for detecting NT-proBNP levels include gold-calibrated assays, fluorescent immunoassays, enzyme-linked immunosorbent assays (ELISA) and magnetic particle Chemiluminescence (CMIA), which all require monoclonal antibodies specific for NT-proBNP. Thus, there is a strong need for anti-NT-proBNP antibodies with good properties for the person skilled in the art.

Disclosure of Invention

The application provides an antibody or antigen binding fragment thereof, which provides an important raw material source for the detection of NT-proBNP and has good activity or affinity.

In order to achieve the above object, according to one aspect of the present invention, there is provided an antibody or an antigen-binding fragment thereof comprising three complementarity determining regions having any one of the heavy chain variable regions of amino acid sequences SEQ ID NO. 17, 18, 19, 20 and three complementarity determining regions having the light chain variable region of amino acid sequence SEQ ID NO. 21.

In order to achieve the above object, according to a second aspect of the present invention, there is provided an antibody or an antigen-binding fragment thereof comprising the following complementarity determining regions:

HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO. 1;

HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID No. 2;

HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID No. 3;

LCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO. 4;

LCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO. 5;

LCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO. 6.

In order to achieve the above object, according to a third aspect of the present invention, there is provided an antibody or antigen-binding fragment thereof comprising a heavy chain variable region having an amino acid sequence shown in SEQ ID NO. 17, 18, 19 or 20 and/or a light chain variable region having an amino acid sequence shown in SEQ ID NO. 21.

In order to achieve the above object, according to a fourth aspect of the present invention, there is provided an antibody or antigen-binding fragment thereof comprising a heavy chain having an amino acid sequence shown in SEQ ID NO. 22, 23, 24 or 25 and/or a light chain having an amino acid sequence shown in SEQ ID NO. 26.

In order to achieve the above object, according to a fifth aspect of the present invention, there is provided an antibody conjugate comprising the above antibody or antigen-binding fragment thereof.

In order to achieve the above object, according to a sixth aspect of the present invention, there is provided a reagent or kit comprising the above antibody or antigen-binding fragment thereof or the above antibody conjugate.

To achieve the above object, according to a seventh aspect of the present invention, there is provided a method for detecting NT-proBNP comprising a) contacting an antibody or antigen binding fragment thereof, antibody conjugate, or reagent or 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 immune complex, and b) detecting the presence of said immune complex, the presence of said complex being indicative of the presence of said antigen in said test sample.

In order to achieve the above object, according to an eighth aspect of the present invention, there is provided a nucleic acid encoding the above antibody or antigen-binding fragment thereof.

In order to achieve the above object, according to a ninth aspect of the present invention, there is provided a vector comprising the nucleic acid described above.

In order to achieve the above object, according to a tenth aspect of the present invention, there is provided a cell comprising the above nucleic acid, vector or antibody or antigen-binding fragment thereof expressed as described above.

In order to achieve the above object, according to an eleventh aspect of the present invention, there is provided a method for producing the above antibody or antigen-binding fragment thereof, the method comprising culturing the above cell.

In order to achieve the above object, according to a twelfth aspect of the present invention there is provided the use of an antibody or antigen binding fragment, antibody conjugate, reagent or kit as described above for the detection or preparation of a product for the detection of NT-proBNP.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the results of reducing SDS-PAGE of Anti-NT-proBNP 16F9 Rmb1 to Rmb 4.

Detailed Description

In a first aspect, the invention provides an antibody or antigen-binding fragment thereof comprising three complementarity determining regions having any one of the heavy chain variable regions of amino acid sequences SEQ ID NO. 17, 18, 19 or 20 and three complementarity determining regions having the light chain variable region of amino acid sequence SEQ ID NO. 21.

The HCDR1, HCDR2 and HCDR3 are amino acid sequences identical to the HCDR1, HCDR2 and HCDR3 of the same heavy chain variable region defined in the antibody or antigen-binding fragment thereof according to the first aspect, and the LCDR1, LCDR2 and LCDR3 are amino acid sequences identical to the LCDR1, LCDR2 and LCDR3 of the same light chain variable region defined in the antibody or antigen-binding fragment thereof according to the first aspect.

For example, the HCDR1, HCDR2 and HCDR3 are amino acid sequences identical to HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in SEQ ID NO. 17, and the LCDR1, LCDR2 and LCDR3 are amino acid sequences identical to LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in SEQ ID NO. 21.

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

In the present invention, the terms "complementarity determining regions", "CDRs" or "CDRs" refer to the highly variable regions of the heavy and light chains of immunoglobulins, and refer to regions comprising one or more or even all of the major amino acid residues responsible for the binding of an antibody or antigen-binding fragment to the antigen or epitope recognized by it. In a specific embodiment of the invention, CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

In the present invention, the heavy chain complementarity determining region is represented by HCDR and includes HCDR1, HCDR2 and HCDR3, and the light chain complementarity determining region is represented by LCDR and includes LCDR1, LCDR2 and LCDR3.

CDR definition methods are well known in the art and include Kabat definition, chothia definition, IMGT definition, contact definition and AbM definition. As used herein, "Kabat definition" refers to the definition system described by Kabat et al, U.S. Dept. Of HEALTH AND Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia definition" see Chothia et al, J Mol Biol 196:901-917 (1987). Still other CDR definition methods may not strictly follow one of the above schemes, but still overlap at least a portion of the Kabat-defined CDR regions, although they may be shortened or lengthened depending on the predicted or experimental outcome of a particular residue or group of residues. Exemplary defined CDRs are listed in table 1 below, with slightly different definitions in the different documents. Given the variable region amino acid sequence of an antibody, one of skill in the art can routinely determine which residues comprise a particular CDR. It should be noted that CDRs defined by other methods not limited to table 1 are also within the scope of the disclosure.

TABLE 1 CDR definition ¹

CDR	Kabat	AbM²	IMGT	Chothia
					HCDR1	H31~H35³	H26~H35³	H26~H33..5⁵	H26~H32..34⁴
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

¹ The numbering of all CDR definitions in Table 1 is according to the Kabat numbering system (see below), with the amino acid numbers on the heavy chain being indicated by "H+ numbers" and the amino acid numbers on the light chain being indicated by "L+ numbers". The Kabat numbering system can be specifically mapped to any variable region sequence by one of ordinary skill in the art without relying on any experimental data outside of the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al, U.S. Dept. Of HEALTH AND HumanServices, "Sequence of Proteins of Immunological Interest" (1983).

² The "AbM" as used in table 1 has a lower case "b" referring to CDRs defined by the "AbM" antibody modeling software of Oxford Molecular.

³ CDR-H1 ends at position 35 if both H35A and H35B are absent, CDR-H1 ends at position 35A if only H35A is present, and CDR-H1 ends at position 35B if both H35A and H35B are present.

⁴ CDR-H1 ends at bit 32 if both H35A and H35B are absent, at bit 33 if only H35A is present, and at bit 34 if both H35A and H35B are present.

⁵ CDR-H1 ends at position 33 if both H35A and H35B are absent, CDR-H1 ends at position 34 if only H35A is present, and CDR-H1 ends at position 35 if both H35A and H35B are present.

According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one system or combination of systems Kabat, chothia, IMGT, abM or contacts.

In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a Kabat system.

In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a Chothia system.

In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by an IMGT system.

In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by an AbM system.

In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a Contact system.

In some alternative embodiments of the invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by Kabat, chothia, IMGT, abM or Contact system combinations.

According to an embodiment of the present invention, the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 defined by the Kabat, chothia, abM or IMGT system correspond to the following Kabat numbering positions:

in a second aspect, the invention provides an antibody or antigen-binding fragment thereof comprising the complementarity determining regions:

In alternative embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

In the present invention, a "framework region" or "FR" region includes a heavy chain framework region and a light chain framework region, which refers to regions of an antibody heavy chain variable region and a light chain variable region other than CDRs, wherein the heavy chain framework region can be further subdivided into contiguous regions separated by CDRs, including HFR1, HFR2, HFR3, and HFR4 framework regions, and the light chain framework region can be further subdivided into contiguous regions separated by CDRs, including LFR1, LFR2, LFR3, and LFR4 framework regions.

In the present invention, the heavy chain variable region is obtained by ligating the CDRs numbered from HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 with the FRs in a combinatorial arrangement, and the light chain variable region is obtained by ligating the CDRs numbered from LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4 with the FRs in a combinatorial arrangement.

In an alternative embodiment, the antibody or antigen binding fragment thereof of the first or second aspect further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR 4;

the HFR1 comprises/has an amino acid sequence as shown in SEQ ID NO 7 or having at least 80% identity thereto;

the HFR2 comprises/has an amino acid sequence as shown in SEQ ID NO 8 or having at least 80% identity thereto;

the HFR3 comprises/has as SEQ ID NO 9 or an amino acid sequence having at least 80% identity thereto;

the HFR4 comprises/has an amino acid sequence as shown in SEQ ID NO 10 or having at least 80% identity thereto;

The LFR1 comprises/is as SEQ ID No. 11 or an amino acid sequence having at least 80% identity thereto;

The LFR2 comprises/is as SEQ ID No. 12 or an amino acid sequence having at least 80% identity thereto;

the LFR3 comprises/is as SEQ ID No. 13 or an amino acid sequence having at least 80% identity thereto;

the LFR4 comprises/is as set forth in SEQ ID No. 14 or an amino acid sequence having at least 80% identity thereto.

In an alternative embodiment, the HFR1 includes/is as shown in SEQ ID NO: 27.

In alternative embodiments, the LFR1 comprises/is shown as SEQ ID NO 28, 29 or 30.

In other embodiments, each of the framework region amino acid sequences of an antibody or antigen binding fragment thereof provided herein may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the corresponding framework region (SEQ ID NO:7, 8, 9, 10, 11, 12, 13 or 14) described above.

In an alternative embodiment, the antibody or antigen binding fragment thereof binds NT-proBNP with an affinity of KD <7.30 x 10 ^-9 M.

In an alternative embodiment, the antibody or antigen binding fragment thereof binds NT-proBNP with an affinity of KD.ltoreq.10 ^-9M、KD≤10^-10M、KD≤10^-11M、KD≤10^-12 M.

In an alternative embodiment, the antibody or antigen binding fragment thereof binds NT-proBNP with an affinity of KD.ltoreq.2.80X 10 ^-10 M.

Antibody affinity (KD) assays are widely varied and can be classified into thermodynamic, kinetic and dynamic equilibrium assays based on the principle of detection. Among them, thermodynamic detection methods are commonly known as Isothermal Titration Calorimetry (ITC), kinetic detection methods are commonly known as Surface Plasmon Resonance (SPR) and biological membrane light interferometry (BLI), and dynamic equilibrium detection methods are commonly known as enzyme-linked immunosorbent assay (ELISA).

In alternative embodiments, the KD is determined using kinetic detection methods, alternatively surface plasmon resonance, for example, by using techniques such asA biosensor system of the system.

In a third aspect, the invention provides an antibody or antigen binding fragment thereof comprising a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO. 17, 18, 19 or 20 and/or a light chain variable region having an amino acid sequence as shown in SEQ ID NO. 21.

In an alternative embodiment, the heavy chain variable region and the light chain variable region of the first aspect or the third aspect above are selected from any combination of:

Combination of two or more kinds of materials	Heavy chain variable region	Light chain variable region
			1	SEQ ID NO:17	SEQ ID NO:21
2	SEQ ID NO:18	SEQ ID NO:21
			3	SEQ ID NO:19	SEQ ID NO:21
4	SEQ ID NO:20	SEQ ID NO:21

。

In an alternative embodiment, the antibody or antigen binding fragment thereof of the first, second or third aspects above further comprises a constant region.

In alternative embodiments, the constant region comprises a heavy chain constant region and/or a light chain constant region.

In alternative embodiments, the heavy chain constant region is selected from the group consisting of a heavy chain constant region of any of IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD, or a combination of segments of heavy chain constant regions.

In alternative embodiments, the heavy chain constant region comprises a CH1 region of IgG, a hinge region of IgG, a CH2 region of IgM, a CH3 region of IgM, and/or a CH4 region of IgM.

In alternative embodiments, the light chain constant region is selected from kappa-type or lambda-type light chain constant regions.

In alternative embodiments, the constant region is of a species derived from bovine, equine, dairy cow, porcine, ovine, murine, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, chicken, or human.

In an alternative embodiment, the constant region is of murine species origin.

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

In other embodiments, the constant region sequence may 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 (SEQ ID NO:15 or 16) described above.

In alternative embodiments, the antigen binding fragment is selected from any one of F (ab ') 2, fab', fab, fv, and scFv of the antibody.

The antigen binding fragments of the above antibodies typically have the same binding specificity as the antibody from which they were derived. It will be readily appreciated by those skilled in the art from the teachings herein that antigen binding fragments of the above antibodies may be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by methods of chemical reduction cleavage of disulfide bonds. The antigen binding fragments described above are readily available to those skilled in the art based on the disclosure of the structure of the intact antibodies.

The antigen binding fragments of the above antibodies may also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, automated peptide synthesizers such as those sold by Applied BioSystems and the like.

In a fourth aspect, the invention provides an antibody or antigen binding fragment thereof comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO. 22, 23, 24 or 25 and/or a light chain having an amino acid sequence as shown in SEQ ID NO. 26.

In alternative embodiments, the antibody or antigen binding fragment thereof of the first, second, third, or fourth aspects above, comprises a heavy chain and a light chain in any one of the following combinations:

Combination of two or more kinds of materials	Heavy chain	Light chain
			1	SEQ ID NO:22	SEQ ID NO:26
2	SEQ ID NO:23	SEQ ID NO:26
			3	SEQ ID NO:24	SEQ ID NO:26
4	SEQ ID NO:25	SEQ ID NO:26

In a fifth aspect, the invention provides an antibody conjugate comprising an antibody or antigen binding fragment thereof as described above.

In an alternative embodiment, the above antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody or antigen binding fragment thereof.

In alternative embodiments, the antibody conjugate further comprises a label conjugated to the antibody or antigen binding fragment thereof.

In an alternative embodiment, the above-mentioned marker refers to a substance having a property such as luminescence, color development, radioactivity, etc., which can be directly observed by naked eyes or detected by an instrument, by which qualitative or quantitative detection of the corresponding target can be achieved.

In alternative embodiments, the labels include, but are not limited to, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

In the actual use process, a person skilled in the art can select a suitable marker according to the detection conditions or actual needs, and no matter what marker is used, the marker belongs to the protection scope of the invention.

In alternative embodiments, the fluorescent dyes include, but are not limited to, fluorescein-based dyes and derivatives thereof (including, but not limited to, fluorescein Isothiocyanate (FITC) hydroxy-light (FAM), tetrachlorolight (TET), and the like, or analogs thereof), rhodamine-based dyes and derivatives thereof (including, but not limited to, red Rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), and the like, or analogs thereof), cy-based dyes and derivatives thereof (including, but not limited to, cy2, cy3B, cy3.5, cy5, cy5.5, cy3, and the like, or analogs thereof), alexa-based dyes and derivatives thereof (including, but not limited to, alexa fluor350, 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 derivatives thereof (including, but not limited to, for example, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polyazosin (preCP), and the like).

In alternative embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose 6-phosphate deoxygenase.

In alternative 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.

In alternative embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and its derivatives, dioxane and its derivatives, lomustine and its derivatives, and peroxyoxalate and its derivatives.

In alternative embodiments, the nanoparticle-based labels include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

In alternative embodiments, the colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

In alternative embodiments, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

In an alternative embodiment, the colloidal metal is colloidal gold.

In an alternative embodiment, the above antibody conjugate further comprises a solid support coupled to the antibody or antigen binding fragment thereof.

In alternative embodiments, the solid support is selected from the group consisting of microspheres, plates, and membranes.

In alternative embodiments, the solid support includes, but is not limited to, magnetic microspheres, plastic microparticles, microplates, glass, capillaries, nylon, and nitrocellulose membranes.

In a sixth aspect, the invention provides a reagent or kit comprising an antibody or antigen binding fragment thereof as described above or an antibody conjugate as described above.

As previously mentioned, the antibodies or antigen-binding fragments thereof of some embodiments or examples of the invention are capable of efficiently binding to NT-proBNP, and thus, the reagents or kits comprising the NT-proBNP antibodies or antigen-binding fragments thereof are capable of efficiently performing a qualitative or quantitative detection of NT-proBNP. The reagent or the kit provided by the invention can be used for detection of specific binding properties of NT-proBNP and antibodies thereof, such as immunoblotting, immunoprecipitation and the like. As mentioned before, the antibodies or antigen binding fragments thereof in some embodiments or examples of the invention have a higher binding activity or affinity to NT-proBNP, and thus the reagents or kits comprising said antibodies or antigen binding fragments thereof have a higher detection sensitivity or specificity.

In a seventh aspect, the present invention provides a method for detecting NT-proBNP comprising a) contacting an antibody or antigen-binding fragment, antibody conjugate, reagent or kit 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 immune complex, and b) detecting the presence of said immune complex, the presence of said complex being indicative of the presence of said antigen in said test sample.

In an alternative embodiment, the immune complex further comprises a second antibody that binds to the antibody or antigen binding fragment thereof.

In an alternative embodiment, the immune complex further comprises a second antibody, which binds to NT-proBNP.

In an eighth aspect, the invention provides a nucleic acid molecule encoding an antibody or antigen binding fragment thereof as described above.

In a ninth aspect, the present invention provides a vector comprising the nucleic acid molecule described above.

In a tenth aspect, the present invention provides a cell comprising the vector described above.

In an eleventh aspect, the invention provides a method of producing an antibody or antigen-binding fragment thereof comprising culturing a cell as described above.

In a twelfth aspect, the present invention provides the use of an antibody as described above or an antigen binding fragment thereof, an antibody conjugate or a reagent or kit as described above for the detection of NT-proBNP or for the preparation of a product for the detection of NT-proBNP.

On the basis of the present disclosure of the amino acid sequence of an antibody or antigen-binding fragment thereof, it is readily apparent to those skilled in the art that the preparation of the antibody or antigen-binding fragment thereof by genetic engineering techniques or other techniques (chemical synthesis, recombinant expression), e.g., isolation and purification from a culture of recombinant cells capable of recombinantly expressing an antibody or antigen-binding fragment thereof as described in any of the above, is within the scope of the present disclosure, irrespective of the technique used to prepare the antibody or antigen-binding fragment thereof.

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Unless defined otherwise, 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 formulations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not intended to be limiting.

Unless otherwise indicated, practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. Such techniques are well explained in the literature, e.g., in the molecular cloning laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989), oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait et al, 1984), animal cell Culture (ANIMAL CELL Culture) (R.I. Freshney, 1987), enzymatic methods (Methods in Enzymology) (academic Press Co., ltd. (ACADEMIC PRESS, inc.), experimental immunology Manual (Handbook of Experimental Immunology) (D.M.Weir and C.Blackwell, inc.), mammalian cell gene transfer Vectors (GENE TRANSFER Vectors for MAMMALIAN CELLS) (J.M.Miller and M.P.Calos, 1987), contemporary molecular biology methods (F.M.Ausubel et al, 1987), polymerase chain reactions (28) (J.M.Weir. And C.Blackwell, inc.), PCR methods (J.34.J.37, J.J.37, J.F.37) and PCR methods (J.37, J.F.37, J.J.F.37, J.J.J.J.F.37, J.J.J.J.J.J.J.J.F.J.J.J.J.J.F.J.J.J.L).

The features and capabilities of the present invention are described in further detail below in connection with the examples.

EXAMPLE 1 preparation of Anti-NT-proBNP 16F9 monoclonal antibody

Restriction enzymes, PRIME STAR DNA polymerase in this example 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. The hybridoma cell strain secreting the Anti-NT-proBNP 16F9 monoclonal antibody is the hybridoma cell strain prepared in the laboratory and is recovered for later use.

(1) Antibody Gene production

MRNA is extracted from hybridoma cell strain secreting Anti-NT-proBNP 16F9 monoclonal antibody, DNA product is obtained by RT-PCR method, the product is inserted into pMD-18T vector after adding A reaction by rTaq DNA polymerase, and is transformed into DH5 alpha competent cells, HEAVY CHAIN and LIGHT CHAIN gene clones are respectively taken after colony growth, and 4 clones are sent to gene sequencing company for sequencing.

(2) Sequence analysis of the variable region Gene of the Anti-NT-proBNP 16F9 antibody

The gene sequence obtained by sequencing is placed in a kabat antibody database for analysis, and VNTI 11.5.5 software is used for analysis to determine that the amplified genes of the heavy chain and light chain primer pair are correct, wherein the VL gene sequence in the LIGHT CHAIN amplified gene fragment is 336bp, the front of the VL gene sequence is 57bp leader peptide sequence, and the VH gene sequence in the HEAVY CHAIN primer pair amplified gene fragment is 357bp, belongs to the VH1 gene family, and the front of the VL gene fragment is 57bp leader peptide sequence.

(3) Construction of recombinant antibody expression plasmids

pcDNA^TM3.4Vector is a constructed eukaryotic expression vector of recombinant antibody, which has been introduced with HindIII, bamHI, ecoRI and other polyclonal enzyme cutting sites and named pcDNA3.4A expression vector, 3.4A expression vector, VL and VH gene specific primers of the antibody are designed according to the result of gene sequencing of the antibody variable region in pMD-18T, and both ends have HindIII, ecoRI enzyme cutting sites and protective bases respectively, and a LIGHT CHAIN gene fragment of 0.71kb and a HEAVY CHAIN gene fragment of 1.40kb are amplified by a PCR amplification method.

The HEAVY CHAIN and LIGHT CHAIN gene fragments are respectively cut by HindIII/EcoRI double enzyme, the 3.4A vector is cut by HindIII/EcoRI double enzyme, the HEAVY CHAIN gene and LIGHT CHAIN gene after the fragments and the vector are purified and recovered are respectively connected with the 3.4A expression vector, and recombinant expression plasmids of HEAVY CHAIN and LIGHT CHAIN are respectively obtained.

2. Recombinant antibody production

Resuscitate HEK293 cells in advance, subculture to a 200ml system to enable the cell density to reach 3-5×10 ⁶ cells/ml, the cell density to reach the concentration of selected antibodies and cell viability to be more than 95%, centrifugally clean the cells, re-dissolve the cells with a culture medium, simultaneously adjust the cell density to 2.9×10 ⁶ cells/ml, re-dissolve the cells with the culture medium, and simultaneously serve as a cell dilution. The medium was used to prepare dilutions of plasmid DNA and transfection reagent, respectively. Adding transfection reagent diluent into plasmid DNA diluent, mixing uniformly, standing at room temperature for 15min, slowly adding the mixture into cell diluent within 1min, mixing uniformly, sampling, counting, recording and observing activity of transfected cells, culturing in a 35 ℃ constant temperature incubator at a rotating speed 120rmp and a CO2 content of 8%, and centrifuging and collecting samples after 13 days. The supernatant was affinity purified using a proteona affinity column. 6ug of the purified antibody was subjected to reducing SDS-PAGE, and the electrophoresed pattern was as shown. Two bands were shown after reducing SDS-PAGE, 1 Mr was 50KD (heavy chain) and the other Mr was 28KD (light chain).

The resulting antibody was designated as Anti-NT-proBNP 16F9Rmb1, and the sequences of the heavy (H) and light (L) chains of the antibody are shown in the following table:

TABLE 2 antibody sequences

Antibody name	Heavy chain	Light chain
			Anti-NT-proBNP 16F9Rmb1	SEQ ID NO:22	SEQ ID NO:26
Anti-NT-proBNP 16F9Rmb2	SEQ ID NO:23	SEQ ID NO:26
			Anti-NT-proBNP 16F9Rmb3	SEQ ID NO:24	SEQ ID NO:26
Anti-NT-proBNP 16F9Rmb4	SEQ ID NO:25	SEQ ID NO:26

Example 2 detection of Performance of antibodies

1. Affinity analysis

The antibody is diluted and purified in advance, meanwhile, NT-proBNP antigen (from Phpeng organism) is subjected to gradient dilution, the CM5 chip of the goat anti-mouse IgG which is coupled in advance is utilized to test the binding dissociation curve of the antigen-antibody on Biacore 8K+ equipment, and the affinity constant, the binding rate and the dissociation rate are obtained by automatic fitting of an instrument. (KD represents equilibrium dissociation constant, i.e., affinity constant; ka represents binding rate; KD represents dissociation rate)

TABLE 3 affinity data

Sample name	KD	ka	kd
				Control	7.30E-09	2.59E-05	1.89E-03
Anti-NT-proBNP 16F9Rmb1	2.73E-10	3.84E+06	1.05E-03
				Anti-NT-proBNP 16F9Rmb2	2.69E-10	3.87E+06	1.04E-03
Anti-NT-proBNP 16F9Rmb3	2.73E-10	3.59E+06	9.80E-04
				Anti-NT-proBNP 16F9Rmb4	2.80E-10	3.64E+06	1.02E-03

2. Activity assay

The coating solution (NaHCO 3 as main component) was diluted to 3ug/ml of NT-proBNP antigen (from the organism Phpeng) and washed at 100uL,37℃per well for 30min, 5 times per day, 50 uL/well, 10min, stop solution, 50 uL/well, OD read at 450nm (reference nm) on the microplate reader, wash solution (Na2HPO4+Nacl as main component) 2 times per well, 1h, 3 ug/well, 100 uL/well, 37℃for 30min, 5 times per well, 100 uL/well, 30min, 5 times per well, 50 uL/well, 10min, stop solution, 50 uL/well, and 450nm (reference nm).

Remarks are liquid A (main component citric acid+sodium acetate+acetanilide+carbamide peroxide), liquid B (main component citric acid+EDTA.2Na+TMB+concentrated HCL), and stop solution (EDTA.2Na+concentrated H2SO 4)

TABLE 4 Activity data

3. Stability assessment

Placing the antibody at 4 ℃, -80 ℃ (refrigerator) and 37 ℃ (incubator) for 21 days, taking 7 days, 14 days and 21 days for state observation, and detecting the activity of the 21 days, wherein the results show that no obvious protein state change is seen in the antibody placed for 21 days under three examination conditions, and the activity is not in a descending trend along with the increase of the examination temperature, thus indicating that the antibody is stable. Table 5 below shows the results of the detection of OD by the antibody Anti-NT-proBNP 16F9Rmb1 on a 21-day basis.

Table 5 stability data

Sample concentration (ng/ml)	9.766	4.883	0.000
				4 ℃ 21 Day sample	1.453	0.967	0.021
-80 ℃,21 Day sample	1.467	0.964	0.017
				37 ℃ 21 Day sample	1.464	0.956	0.033

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

The partial amino acid sequences involved in the present application are shown in Table 6:

TABLE 6 amino acid sequence

Claims

1. An antibody or antigen-binding fragment thereof comprising three complementarity determining regions having any one of the heavy chain variable regions of amino acid sequences SEQ ID NOs 17, 18, 19, 20 and three complementarity determining regions having the light chain variable region of amino acid sequence SEQ ID NO 21.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the complementarity determining region is defined by any one or a combination of systems Kabat, chothia, IMGT, abM or Contact.

3. An antibody or antigen-binding fragment thereof, comprising the following complementarity determining regions:

LCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO. 6;

optionally, the antibody or antigen binding fragment thereof further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR 4;

The HFR1 comprises the amino acid sequence of SEQ ID NO. 7 or at least 80% identity thereto;

The HFR2 comprises the amino acid sequence of SEQ ID NO 8 or at least 80% identity thereto;

The HFR3 comprises the amino acid sequence of SEQ ID NO 9 or at least 80% identity thereto;

The HFR4 comprises the amino acid sequence of SEQ ID NO 10 or at least 80% identity thereto;

The LFR1 comprises SEQ ID No. 11 or an amino acid sequence having at least 80% identity thereto;

The LFR2 comprises the amino acid sequence of SEQ ID No. 12 or at least 80% identity thereto;

the LFR3 comprises SEQ ID No. 13 or an amino acid sequence having at least 80% identity thereto;

the LFR4 comprises SEQ ID No. 14 or an amino acid sequence having at least 80% identity thereto;

Alternatively, the antibody or antigen binding fragment thereof binds NT-proBNP with an affinity of KD <7.30 x 10 ^-9 M.

4. An antibody or antigen binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, and is characterized in that the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 17, 18, 19 or 20, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 21.

5. The antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof further comprises a constant region;

optionally, the constant region comprises a heavy chain constant region and/or a light chain constant region;

alternatively, the heavy chain constant region is selected from the group consisting of a heavy chain constant region of any one of IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD or a combination of segments of multiple heavy chain constant regions;

Optionally, the heavy chain constant region comprises a CH1 region of IgG, a hinge region of IgG, a CH2 region of IgM, a CH3 region of IgM, and/or a CH4 region of IgM;

Alternatively, the constant region is of bovine, equine, porcine, ovine, caprine, murine, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, or human origin;

alternatively, the constant region is of murine species origin;

alternatively, the heavy chain constant region sequence is as shown in SEQ ID NO. 15 or at least 80% identical thereto;

alternatively, the light chain constant region sequence is as shown in SEQ ID NO. 16 or at least 80% identical thereto;

alternatively, the antigen binding fragment is selected from any one of F (ab ') 2, fab', fab, fv, and scFv of the antibody.

6. An antibody or antigen binding fragment thereof comprises a heavy chain and/or a light chain, and is characterized in that the amino acid sequence of the heavy chain is shown as SEQ ID NO. 22, 23, 24 or 25, and the amino acid sequence of the light chain is shown as SEQ ID NO. 26.

7. An antibody conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 6;

Optionally, the antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody or antigen binding fragment thereof;

optionally, the antibody conjugate further comprises a label conjugated to the antibody or antigen binding fragment thereof;

Optionally, the label is selected from the group consisting of fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels;

Optionally, the antibody conjugate further comprises a solid support coupled to the antibody or antigen binding fragment thereof;

alternatively, the solid support is selected from the group consisting of microspheres, plates, and membranes.

8. A reagent or kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 6 or the antibody conjugate of claim 7.

9. A method for detecting NT-proBNP, comprising:

a) Contacting an antibody or antigen binding fragment thereof according to any one of claims 1 to 6, an antibody conjugate according to claim 7, or a reagent or kit according to claim 8 with NT-proBNP in a sample to be tested under conditions sufficient for an antibody/antigen binding reaction to form an immune complex, and

B) Detecting the presence of the immune complex, the presence of the complex being indicative of the presence of the antigen in the test sample;

optionally, the immune complex further comprises a second antibody that binds to the antibody or antigen binding fragment thereof;

optionally, the immune complex further comprises a second antibody, which binds to NT-proBNP.

10. A nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 6.

11. A vector comprising the nucleic acid of claim 10.

12. A cell comprising the nucleic acid of claim 10 or the vector of claim 11.

13. A method for producing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, comprising culturing the cell according to claim 12.

14. Use of the antibody or antigen binding fragment thereof of any one of claims 1-6, the antibody conjugate of claim 7, or the reagent or kit of claim 8 for the preparation of a product for detecting NT-proBNP.