Anti-B-type natriuretic peptide antibody or its functional fragment, reagent and kit for detecting B-type natriuretic peptide

Abstract

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

Description

Technical Field

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

[0002] B-type natriuretic peptide, also known as brain natriuretic peptide (BNP), is a biologically active natural hormone synthesized by myocardial cells. It is mainly expressed in the ventricles and also exists in brain tissue. When the left ventricle is dysfunctional, it is rapidly synthesized and released into the blood due to myocardial dilation, which helps regulate heart function. BNP secreted by myocardial cells first exists in the form of a precursor composed of 108 amino acids. When myocardial cells are stimulated, they are cleaved into an inactive linear polypeptide composed of 76 amino acids and an active cyclic polypeptide composed of 32 amino acids under the action of activating enzymes, and released into the blood circulation, which are called NT-proBNP and BNP respectively. BNP can increase the glomerular filtration rate, inhibit sodium reabsorption, and thus facilitate sodium excretion. BNP can also inhibit the activity of central and peripheral sympathetic nerves and the renin-angiotensin-aldosterone system, thereby relaxing vascular smooth muscle, dilating arteries and veins, and reducing blood pressure and ventricular preload.

[0003] BNP and NT-proBNP are both good clinical markers for diagnosing heart failure (HF). The increase of BNP or NT-proBNP in HF is related to the degree of HF and has unique predictive value for the mortality of left ventricular dysfunction or AMI. It can be used for condition estimation, efficacy monitoring, prognosis judgment or risk classification of HF patients.

[0004] Immunological detection methods are a means of detection based on the specific reaction of antibodies and antigens. Since they can use isotopes, fluorescence, colloidal metals, enzymes, chemiluminescent substances, etc. to amplify and display the detected signals, they are often used to detect trace bioactive substances such as proteins and hormones. At present, the commonly used immunological detection methods for BNP detection include fluorescent immunochromatography, colloidal gold immunochromatography and chemiluminescence. When any of the above immunological detection methods is used for BNP detection, antibodies against BNP are required.

[0005] Therefore, there is a strong need in the art for antibodies that effectively bind to and detect BNP.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to provide an anti-B-type natriuretic peptide antibody or a functional fragment thereof, and a reagent and a kit for detecting B-type natriuretic peptide.

[0008] The present invention is achieved in that:

[0009] In a first aspect, an embodiment of the present invention provides an antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises any one of (a) to (d): (a) HCDR1 to 3 and LCDR1 to 3, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID Nos: 1, 2 and 3, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in any of the following: SEQ ID Nos: 4, 6 and 8; SEQ ID Nos: 5, 6 and 8; SEQ ID Nos: 4, 6 and 7; SEQ ID Nos: 5, 6 and 7; (b) a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 9 or 10, and a light chain variable region with an amino acid sequence as shown in any one of SEQ ID NOs: 14 to 17; (c) a heavy chain variable region and a light chain variable region with an amino acid sequence having more than 80% identity with the sequence shown in (b), and comprising HCDR1 to HCDR3 and LCDR1 to LCDR3 of the sequence shown in (a); (d) an amino acid sequence as shown in SEQ ID The heavy chain is shown in NO:12 or 13, and the light chain has an amino acid sequence as shown in any one of SEQ ID NOs:19-22.

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

[0011] In a third aspect, an embodiment of the present invention provides a reagent or a kit, which includes the antibody or its functional fragment as described in the above embodiments or the antibody conjugate as described in the above embodiments.

[0012] In a fourth aspect, an embodiment of the present invention provides a method for detecting B-type natriuretic peptide, comprising: mixing the anti-B-type natriuretic peptide antibody as described in the previous embodiment with a sample to be detected, allowing the antibody or its functional fragment to contact the B-type natriuretic peptide in the sample to be detected to form an immune complex.

[0013] In a fifth aspect, an embodiment of the present invention provides an isolated nucleic acid encoding the antibody or a functional fragment thereof described in the preceding embodiments.

[0014] In a sixth aspect, an embodiment of the present invention provides a vector comprising the isolated nucleic acid described in the preceding embodiment.

[0015] In a seventh aspect, an embodiment of the present invention provides a cell containing the isolated nucleic acid described in the preceding embodiment or the vector described in the preceding embodiment.

[0016] In an eighth aspect, an embodiment of the present invention provides a method for preparing the antibody or a functional fragment thereof described in the preceding embodiment, comprising: culturing the cells described in the preceding embodiment.

[0017] The present invention has the following beneficial effects:

[0018] The anti-B-type natriuretic peptide antibody disclosed in the present invention comprises the heavy chain complementary determining region and the light chain complementary determining region. The antibody provides an important source of raw materials for the detection of B-type natriuretic peptide and has improved affinity and activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0020] Figure 1 The results of reducing SDS-PAGE of Anti-BNP 46E5 are shown in FIG. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

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

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

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

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

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

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

[0028] (b) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 9 or 10, and a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 14 to 17.

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

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

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

[0032] (c) A heavy chain variable region and a light chain variable region whose amino acid sequences are more than 80% identical to the sequences shown in (b), and include HCDR1 to HCDR3 and LCDR1 to LCDR3 of the sequences shown in (a).

[0033] In an optional embodiment, the antibody or a functional fragment thereof comprises a heavy chain framework region in the heavy chain variable region shown in SEQ ID NO: 9 or 10, and a light chain framework region in the light chain variable region shown in any one of SEQ ID NOs: 14 to 17.

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

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

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

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

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

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

[0040] In an alternative embodiment, the heavy chain constant region sequence is as shown in SEQ ID NO: 11 or has at least 80% identity thereto.

[0041] In an alternative embodiment, the light chain constant region sequence is as shown in SEQ ID NO: 18 or has at least 80% identity thereto.

[0042] Specifically, the constant region sequence can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the above-mentioned constant region (SEQ ID NO: 11 or 18).

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

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

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

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

[0047] (d) a heavy chain having an amino acid sequence as shown in SEQ ID NO: 12 or 13, and a light chain having an amino acid sequence as shown in any one of SEQ ID NOs: 19 to 22.

[0048] In an optional embodiment, the antibody or its functional fragment has a KD of less than 2.64×10 -07 Affinity binding of M to B-type natriuretic peptide.

[0049] In an optional embodiment, the antibody or its functional fragment has a KD≤10 -07 M, KD≤10 -08 M, KD≤10 -09 M, KD≤10 -10M, KD≤10 -11 M or KD≤10 -12 Affinity binding of M to B-type natriuretic peptide.

[0050] In an optional embodiment, the antibody or its functional fragment has a KD≤1.18×10 -08 M, KD≤6.33×10 -09 M, KD≤5.61×10 -09 M, KD≤3.65×10 -09 M binds to B-type natriuretic peptide with affinity.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0070] On the other hand, an embodiment of the present invention further provides a method for detecting B-type natriuretic peptide, comprising:

[0071] The anti-B-type natriuretic peptide antibody or its functional fragment as described in any of the foregoing embodiments, or the antibody conjugate as described in any of the foregoing embodiments, or the reagent or kit as described in any of the foregoing embodiments is mixed with the sample to be tested, so that the anti-B-type natriuretic peptide antibody or its functional fragment contacts the B-type natriuretic peptide in the sample to be tested to form an immune complex.

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

[0073] In a preferred embodiment, the immune complex further comprises a second antibody, which binds to B-type natriuretic peptide.

[0074] On the other hand, an embodiment of the present invention also provides the use of the antibody or functional fragment described in any of the preceding embodiments, or the antibody conjugate described in any of the preceding embodiments, or the reagent or kit described in any of the preceding embodiments in detecting B-type natriuretic peptide or preparing a product for detecting B-type natriuretic peptide.

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

[0076] In an optional embodiment, the B-type natriuretic peptide metabolism-related diseases include at least one of heart failure, acute myocardial infarction (AMI), acute coronary syndrome (ACS), transient myocardial ischemia, hypertension, left ventricular hypertrophy, valvular heart disease, atrial fibrillation, hypertrophic cardiomyopathy, restrictive cardiomyopathy, constrictive pericarditis, diabetic cardiomyopathy, cardiac amyloid deformation, COPD, cor pulmonale, pulmonary embolism, ARDS, acute pulmonary edema, renal insufficiency and atherosclerosis.

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

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

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

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

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

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

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

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

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

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

[0087] Example 1 Preparation of Anti-BNP 46E5 Monoclonal Antibody

[0088] In this example, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART 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 a gene sequencing company.

[0089] 1. Expression plasmid construction

[0090] 1.1. Anti-BNP 46E5 antibody gene preparation

[0091] mRNA was extracted from the hybridoma cell line secreting Anti-BNP 46E5 monoclonal antibody, and the DNA product was obtained by RT-PCR. It was inserted into the pMD-18T vector and transformed into DH5α competent cells. After the colonies grew, 4 positive clones of the Heavy Chain and Light Chain genes were taken and sent to a gene sequencing company for sequencing.

[0092] 1.2. Sequence analysis of the variable region gene of Anti-BNP 46E5 antibody

[0093] The gene sequences obtained by the above sequencing were placed in the Kabat antibody database for analysis, and the VNTI11.5 software was used for analysis to determine that the genes amplified by the heavy chain and light chain primer pairs were correct. In the gene fragment amplified by the Light Chain, the VL gene sequence was 333bp, and there was a 57bp leader peptide sequence in front of it; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence was 372bp, belonging to the VH1 gene family, and there was a 57bp leader peptide sequence in front of it.

[0094] 1.3. Construction of recombinant antibody expression plasmid

[0095] pcDNA TM 3.4 vector is a constructed recombinant antibody eukaryotic expression vector, which has been modified to introduce multiple cloning restriction sites and is subsequently referred to as 3.4A expression vector; based on the sequencing results of the antibody variable region genes in the above-mentioned pMD-18T, VL and VH gene-specific primers of the Anti-BNP 46E5 antibody were designed, with restriction endonuclease cleavage sites and protective bases at both ends, respectively, and 0.74kb Light Chain gene fragment and 1.45kb Heavy Chain gene fragment were amplified by PCR amplification method.

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

[0097] 2. Recombinant Antibody Sample Preparation

[0098] Resuscitate HEK293 cells in advance and subculture them to 200 ml system to make the cell density reach 3-5×10 6 cells / ml, cell viability>95%; wash the cells by centrifugation, re-dissolve with culture medium, and adjust the cell density to 2.9×10 6 cells / ml, as the cell diluent. Use culture medium to prepare plasmid DNA and transfection reagent diluents respectively. Add the transfection reagent diluent to the plasmid DNA diluent, mix well and let stand at room temperature for 15 minutes; slowly add the mixture to the cell diluent within 1 minute, mix well and take samples to count, record and observe the viability of the cells after transfection, and place them in a 35°C constant temperature incubator for culture, with a speed of 120rmp and a CO2 content of 8%. Centrifuge and collect samples after 13 days. The centrifuged supernatant was affinity purified using a protein A affinity chromatography column. Take 6μg of the purified antibody for reducing SDS-PAGE, and the electrophoresis pattern is shown as follows Figure 1 After reducing SDS-PAGE, two bands were shown, one with Mr of 50KD (heavy chain) and the other with Mr of 28KD (light chain).

[0099] 3. Affinity and activity optimization

[0100] Although the Anti-BNP 46E5 monoclonal antibody obtained in Example 1 has the ability to bind to B-type natriuretic peptide, its affinity and antibody activity are not ideal, so the applicant performs directed mutation on the light chain and heavy chain of the antibody. That is, the computer is used to simulate the structure of the antibody variable region, the structure of the antigen and antibody variable region complex, the key amino acid analysis of the antibody and the mutation design, and the bidirectional primers covering the mutation site are designed and synthesized according to the mutation scheme, the primers at both ends of the target DNA are synthesized, and a high-fidelity PCR reaction is performed. The PCR product is cloned into a vector, and then the mutant antibody is prepared according to the method described in Example 1. After screening, monoclonal antibodies with significantly improved affinity and antibody activity are obtained, and the heavy chain and light chain amino acid sequences of each antibody are as follows.

[0101] Table 1 Antibody sequences

[0102] Sample name Heavy chain number Light chain number Anti-BNP-46E5RMb1 SEQ ID NO:12 SEQ ID NO:19 Anti-BNP-46E5RMb2 SEQ ID NO:13 SEQ ID NO:19 Anti-BNP-46E5RMb3 SEQ ID NO:12 SEQ ID NO:20 Anti-BNP-46E5RMb4 SEQ ID NO:12 SEQ ID NO:21

[0103] Example 2 Affinity Analysis

[0104] Using the AMC sensor, the purified antibody (Example 1) was diluted to 10 μg / ml with PBST, and the proBNP antigen (obtained from Feipeng Bio) was graded diluted with PBST.

[0105] Operation process: 60s of equilibration in buffer 1 (PBST), 300s of antibody fixation in antibody solution, 180s of incubation in buffer 2 (PBST), 420s of binding in antigen solution, 1200s of dissociation in buffer 2, sensor regeneration with 10mM pH 1.69GLY solution and buffer 3 (PBST), and data output. (K D represents the equilibrium dissociation constant or affinity; kon represents the binding rate; kdis represents the dissociation rate. The main components of PBST are Na2HPO4+NaCl+TW-20).

[0106] Table 2 Affinity analysis data

[0107]

[0108] Example 3 Activity Identification

[0109] The coating solution (main component NaHCO3) was used to dilute the proBNP antigen (obtained from Feipeng Bio) to 1 μg / ml, 100 μL per well, and incubated at 4°C overnight; the next day, the cells were washed twice with the washing solution (main component Na2HPO4+NaCl) and patted dry; the blocking solution (20% BSA+80% PBS) was added, 120 μL per well, incubated at 37°C for 1 hour, and patted dry; the diluted purified antibody and control antibody were added, 100 μL / well, incubated at 37°C for 30 minutes; the cells were washed 5 times and patted dry; the goat anti-mouse IgG-HRP was added, 100 μL per well, incubated at 37°C for 30 minutes; the cells were washed 5 times and patted dry; the colorimetric solution A (50 μL / well) and the colorimetric solution B (50 μL / well) were added for 10 minutes; the stop solution was added, 50 μL / well; the OD value was read at 450 nm (reference 630 nm) on the microplate reader.

[0110] Note: Solution A (main components: citric acid + sodium acetate + acetanilide + urea peroxide); Solution B (main components: citric acid + EDTA·2Na + TMB + concentrated HCL); Stop solution (EDTA·2Na + concentrated H2SO4).

[0111] Table 3 Activity data

[0112] Concentration (ng / ml) 62.50 31.25 15.63 7.81 3.91 0.00 Control Antibodies 1.421 0.856 0.514 0.287 0.151 0.006 Anti-BNP-46E5RMb1 2.069 1.705 1.201 0.780 0.417 0.008 Anti-BNP-46E5RMb2 1.949 1.602 1.204 0.764 0.410 0.007 Anti-BNP-46E5RMb3 1.787 0.963 0.636 0.312 0.144 0.008 Anti-BNP-46E5RMb4 2.046 1.532 0.980 0.626 0.274 0.005

[0113] Example 4 Antibody Stability Assessment

[0114] The above antibodies were placed at 4°C (refrigerator), -80°C (refrigerator), and 37°C (constant temperature box) for 21 days, and samples were taken for 7 days, 14 days, and 21 days for status observation, and the activity of the 21-day samples was tested. The results showed that there was no obvious protein state change in the antibodies under the three test conditions for 21 days, and the activity did not show a downward trend with the increase of the test temperature, indicating that the expressed antibodies were stable. The following table shows the OD results of the enzyme immunoassay activity test after 21 days of testing.

[0115] Table 4 Stability data

[0116]

[0117] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0118] The partial amino acid sequences involved in this application are as follows:

[0119]

Claims

1. An anti-B-type natriuretic peptide antibody or an antigen-binding fragment thereof, characterized in that: The antibody or its antigen-binding fragment comprises: HCDR1~HCDR3 and LCDR1~LCDR3, the amino acid sequences of the HCDR1~HCDR3 are shown in SEQ ID NO: 1, 2 and 3 respectively; the amino acid sequences of the LCDR1~LCDR3 are shown in any of the following items: SEQ ID NO: 4, 6 and 8; SEQ ID No: 4, 6 and 7; SEQ ID No: 5, 6 and 7.

2. An anti-B-type natriuretic peptide antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof comprises: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:14; or, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 14; or, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:15; or, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

16.

3. An anti-B-type natriuretic peptide antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof comprises: a heavy chain variable region and a light chain variable region whose amino acid sequences are more than 80% identical to the heavy chain variable region and the light chain variable region described in claim 2, and comprises HCDR1~HCDR3 and LCDR1~LCDR3 described in claim 1.

4. An anti-B-type natriuretic peptide antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof comprises: A heavy chain having an amino acid sequence as shown in SEQ ID NO: 12, and a light chain having an amino acid sequence as shown in SEQ ID NO: 19; or, A heavy chain having an amino acid sequence as shown in SEQ ID NO: 13, and a light chain having an amino acid sequence as shown in SEQ ID NO: 19; or, A heavy chain having an amino acid sequence as shown in SEQ ID NO: 12, and a light chain having an amino acid sequence as shown in SEQ ID NO: 20; or, The heavy chain has an amino acid sequence as shown in SEQ ID NO:12, and the light chain has an amino acid sequence as shown in SEQ ID NO:

21.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that: The antibody or antigen-binding fragment thereof is expressed as K D <2.64×10 -07 M binds to B-type natriuretic peptide with high affinity.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that: The antibody or antigen-binding fragment thereof further comprises a constant region.

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

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

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

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

11. The antibody or antigen-binding fragment thereof according to claim 7, characterized in that: The heavy chain constant region sequence is shown in SEQ ID NO:

11.

12. The antibody or antigen-binding fragment thereof according to claim 7, characterized in that: The heavy chain constant region sequence has at least 80% identity with the sequence shown in SEQ ID NO:

11.

13. The antibody or antigen-binding fragment thereof according to claim 7, characterized in that: The light chain constant region sequence is shown in SEQ ID NO:

18.

14. The antibody or antigen-binding fragment thereof according to claim 7, characterized in that: The light chain constant region sequence has at least 80% identity with the sequence shown in SEQ ID NO:

18.

15. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 and 7 to 14, characterized in that: The antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

16. An antibody conjugate, characterized in that: It comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15.

17. The antibody conjugate according to claim 16, characterized in that The antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody or antigen-binding fragment thereof.

18. The antibody conjugate according to claim 16, characterized in that The antibody conjugate further comprises a solid phase carrier coupled to the antibody or antigen-binding fragment thereof.

19. The antibody conjugate according to claim 16, characterized in that: The antibody conjugate further comprises a label coupled to the antibody or antigen-binding fragment thereof.

20. The antibody conjugate according to claim 19, characterized in that The marker is selected from at least one of fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents and nanoparticle markers.

21. The antibody conjugate according to claim 19, characterized in that The label is colloidal gold.

22. A reagent or a kit, characterized in that: It comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15 or the antibody conjugate according to any one of claims 16 to 21.

23. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, or the antibody conjugate according to any one of claims 16 to 21, or the reagent or kit according to claim 22 in the preparation of a product for detecting B-type natriuretic peptide, characterized in that: The antibody or antigen-binding fragment thereof according to any one of claims 1 to 15 is mixed with a sample to be detected, so that the antibody or antigen-binding fragment thereof contacts the B-type natriuretic peptide in the sample to be detected to form an immune complex.

24. The use according to claim 23, characterized in that The immune complex also includes a second antibody that binds to the antibody or antigen-binding fragment thereof.

25. The use according to claim 23, characterized in that The immune complex also includes a second antibody that binds to B-type natriuretic peptide.

26. An isolated nucleic acid, characterized in that It encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15.

27. A carrier, characterized in that It contains the isolated nucleic acid according to claim 26.

28. A cell, characterized in that It contains the isolated nucleic acid according to claim 26 or the vector according to claim 27.

29. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, characterized in that: It includes: Cultivate the cell of claim 28.

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