Anti-beta1-ar-ecii antibody and use thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of beta1-AR-ECII or its autoantibody as a marker or target point in diagnosis and / or treatment of cardiovascular diseases, and particularly provides an anti-beta1-AR-ECII antibody or antigen binding fragment thereof and application thereof. Firstly, two anti-beta1-AR-ECII antibodies or antigen binding fragments thereof are newly screened in the application, the two antibodies combine with N-terminal peptide and C-terminal peptide of beta1-AR-ECII respectively, beta1-AR-ECII detection is realized, cost is low, and it is fast and effective.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an anti-β1-AR-ECII antibody, its preparation method, and its application. Background Technology

[0002] β1-adrenoceptor autoantibody (β1-AA), a product of immune system disorders, is an autoantibody against the β1-adrenoceptor (β1-AR). It is present in various cardiovascular diseases such as cardiomyopathy (e.g., hypertensive cardiomyopathy, dilated cardiomyopathy, or rheumatic cardiomyopathy), arrhythmia, and heart failure, and is related to the pathogenesis of these diseases.

[0003] Low levels of autoantibodies are normal in human blood and do not cause disease. However, if the titer of autoantibodies exceeds a certain level, it may damage the body and eventually induce disease. High positive rates of β1-AA are also found in the serum of patients with heart failure. The long-term presence of these high positive rates of β1-AA can lead to changes in cardiac morphology and function. Studies have confirmed that the long-term presence of β1-AA can lead to heart failure, and the production of β1-AA can also be induced during the process of myocardial remodeling leading to heart failure. The two are mutually causal and mutually reinforcing (see non-patent literature: Li Xiao, Du Yunhui, Zuo Qian, Liu Huirong, et al. β1-adrenergic receptor autoantibodies and their role in the development of heart failure [J]. Progress in Physiological Sciences, 2013(05):67-71.). Further research has shown that the imbalance between β1-AAs and β2-AAs in patients with heart failure is a mechanism that exacerbates HF, and an elevated β1-AAs / β2-AAs ratio should be considered a clinical assessment factor for the deterioration of cardiac function in patients with heart failure (see reference: Ning Cao, β2-adrenergic receptor autoantibodies alleviated myocardial damage induced by β1-adrenergic receptor autoantibodies in heart failure, [J]., 2018: 114(11)). However, the mechanism of β1-AA production is still unclear at this stage. Summary of the Invention

[0004] To fill the gaps in existing technologies, this application investigates the formation mechanism of β1-AA using β1-AR-ECII. Unexpectedly, it was discovered that β1-AR-ECII is directly related to cardiovascular diseases (especially acute myocardial infarction) and can serve as a biomarker or target for the diagnosis and treatment of cardiovascular diseases. Furthermore, two novel anti-β1-AR-ECII antibodies or their antigen-binding fragments were screened. These two antibodies bind to the N-terminal and C-terminal peptides of β1-AR-ECII, respectively, enabling accurate, rapid, and effective detection of β1-AR-ECII. The specific protocol is as follows:

[0005] A first aspect of the present invention provides the use of β1-AR-ECII or its autoantibody as a marker or target in the preparation of products for the diagnosis and / or treatment of cardiovascular diseases.

[0006] A second aspect of the invention provides the use of reagents for detecting β1-AR-ECII or its autoantibodies in the preparation of products for diagnosing and / or treating cardiovascular diseases.

[0007] The β1-AR-ECII or its autoantibody mentioned is β1-AR-ECII or its autoantibody in body fluids or cells.

[0008] Preferably, the β1-AR-ECII or its autoantibody is β1-AR-ECII or its autoantibody in blood, serum, plasma or cardiomyocytes.

[0009] The products include reagents for detecting β1-AR-ECII.

[0010] Preferably, the reagent for detecting β1-AR-ECII includes an anti-β1-AR-ECII antibody or its antigen-binding fragment.

[0011] The product described can be a test kit.

[0012] Preferably, the diagnosis of cardiovascular disease includes detecting the presence or content of β1-AR-ECII.

[0013] Preferably, the treatment of cardiovascular disease includes administering a β2-AR / Gi pathway agonist, such as ICI118551, to a subject in need.

[0014] Preferably, the cardiovascular diseases include acute myocardial infarction, cardiomyopathy (such as hypertensive cardiomyopathy, dilated cardiomyopathy or rheumatic cardiomyopathy), arrhythmia or heart failure.

[0015] Preferably, the diagnosis and / or treatment of cardiovascular diseases can be for diagnosing and / or treating cardiovascular diseases in humans or non-human animals.

[0016] A third aspect of the present invention provides an antibody against the second extracellular loop (β1-Adrenergic Receptor Second Extracellular Loop, β1-AR-ECII) or an antigen-binding fragment thereof, wherein the anti-β1-AR-ECII antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein,

[0017] I) The heavy chain variable region contains: an amino acid sequence containing or as shown in SEQ ID NO: 1 VHCDR1; an amino acid sequence containing or as shown in SEQ ID NO: 2 VHCDR2; an amino acid sequence containing or as shown in SEQ ID NO: 3 VHCDR3; and the light chain variable region contains: an amino acid sequence containing or as shown in SEQ ID NO: 4 VLCDR1; an amino acid sequence containing or as shown in SEQ ID NO: 5 VLCDR2; an amino acid sequence containing or as shown in SEQ ID NO: 6 VLCDR3;

[0018] or,

[0019] II) The heavy chain variable region contains: an amino acid sequence containing or as shown in SEQ ID NO: 7 VHCDR1; an amino acid sequence containing or as shown in SEQ ID NO: 8 VHCDR2; an amino acid sequence containing or as shown in SEQ ID NO: 9 VHCDR3; and the light chain variable region contains: an amino acid sequence containing or as shown in SEQ ID NO: 10 VLCDR1; an amino acid sequence containing or as shown in SEQ ID NO: 11 VLCDR2; an amino acid sequence containing or as shown in SEQ ID NO: 12 VLCDR3.

[0020] Preferably, the anti-β1-AR-ECII antibody or its antigen-binding fragment further comprises a FR region. More preferably, the FR region is derived from the FR region of a human or a non-human animal (e.g., a non-human mammal), and more preferably from the FR region of a human, rodent, or primate.

[0021] Preferably, the anti-β1-AR-ECII antibody or its antigen-binding fragment includes VHFR1 (e.g., SEQ ID NO: 19), VHFR2 (SEQ ID NO: 20), VHFR3 (SEQ ID NO: 21), and VHFR4 (SEQ ID NO: 22) in the heavy chain variable region, and VLFR1 (SEQ ID NO: 23), VLFR2 (SEQ ID NO: 24), VLFR3 (SEQ ID NO: 25), and VLFR4 (SEQ ID NO: 26) in the light chain variable region.

[0022] In one specific embodiment of the present invention, the heavy chain variable region includes VHFR1, VHCDR1, VHFR2, VHCDR2, VHFR3, VHCDR3, and VHFR4 sequentially from the N end to the C end.

[0023] In one specific embodiment of the present invention, the light chain variable region includes VLFR1, VLCDR1, VLFR2, VLCDR2, VLFR3, VLCDR3, and VLFR4 sequentially from the N end to the C end.

[0024] Preferably, the amino acid sequence of the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 13, or includes an amino acid sequence having more than 90%, more than 95%, or more than 99% homology with the amino acid sequence shown in SEQ ID NO: 13, or includes an amino acid sequence having at least one, two, or more than three, or at most ten, nine, eight, seven, six, five, four, three, or two substituted, deleted, or mutated amino acids on SEQ ID NO: 13, and having the same or similar activity as SEQ ID NO: 13 (e.g., binding activity with β1-AR-ECII).

[0025] Preferably, the amino acid sequence of the light chain variable region includes the amino acid sequence shown in SEQ ID NO:14, or includes an amino acid sequence having more than 90%, more than 95%, or more than 99% homology with the amino acid sequence shown in SEQ ID NO:14, or includes an amino acid sequence having at least one, two, or more than three, or at most ten, nine, eight, seven, six, five, four, three, or two substituted, deleted, or mutated amino acids on SEQ ID NO:14, and having the same or similar activity as SEQ ID NO:14 (e.g., binding activity with β1-AR-ECII).

[0026] Preferably, the amino acid sequence of the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 15, or includes an amino acid sequence having more than 90%, more than 95%, or more than 99% homology with the amino acid sequence shown in SEQ ID NO: 15, or includes an amino acid sequence having at least one, two, or more than three, or at most ten, nine, eight, seven, six, five, four, three, or two substituted, deleted, or mutated amino acids on SEQ ID NO: 15, and having the same or similar activity as SEQ ID NO: 15 (e.g., binding activity with β1-AR-ECII).

[0027] Preferably, the amino acid sequence of the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 16, or includes an amino acid sequence having more than 90%, more than 95%, or more than 99% homology with the amino acid sequence shown in SEQ ID NO: 16, or includes an amino acid sequence having at least one, two, or more than three, or at most ten, nine, eight, seven, six, five, four, three, or two substituted, deleted, or mutated amino acids on SEQ ID NO: 16, and having the same or similar activity as SEQ ID NO: 16 (e.g., binding activity with β1-AR-ECII).

[0028] In one specific embodiment of the present invention, the amino acid sequence of the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 13; the amino acid sequence of the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 14.

[0029] In one specific embodiment of the present invention, the amino acid sequence of the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 15; the amino acid sequence of the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 16.

[0030] In one specific embodiment of the present invention, the anti-β1-AR-ECII antibody or its antigen-binding fragment is an IgG type antibody.

[0031] Preferably, the anti-β1-AR-ECII antibody or its antigen-binding fragment is a murine antibody.

[0032] Preferably, the anti-β1-AR-ECII antibody or its antigen-binding fragment includes Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2 or Fd.

[0033] The anti-β1-AR-ECII antibody or its antigen-binding fragment specifically binds to the β1-AR-ECII protein or its fragment in humans or non-human animals.

[0034] In a fourth aspect, the present invention provides an antigen receptor comprising the above-described anti-β1-AR-ECII antibody or an antigen-binding fragment thereof.

[0035] Preferably, the antigen receptor can be a chimeric antigen receptor (CAR) or a synthetic T-cell receptor antigen receptor (STAR).

[0036] In a fifth aspect, the present invention provides an antibody-drug conjugate comprising the anti-β1-AR-ECII antibody or its antigen-binding fragment as described in the third aspect, and a cytotoxic agent.

[0037] In a sixth aspect, the present invention provides a nucleic acid encoding the anti-β1-AR-ECII antibody or its antigen-binding fragment as described in the third aspect, or the antigen receptor as described in the fourth aspect, or the antibody-drug conjugate (ADC) as described in the fifth aspect.

[0038] In a seventh aspect, the invention provides a cell expressing the anti-β1-AR-ECII antibody or its antigen-binding fragment as described in the third aspect, or expressing the antigen receptor as described in the fourth aspect, or containing the nucleic acid as described in the sixth aspect.

[0039] The cells mentioned can be eukaryotic cells or prokaryotic cells.

[0040] Eukaryotic cells include animal and plant cells, such as T cells, yeast cells, HEK293 cells, or CHO cells, etc.

[0041] Prokaryotic cells, such as Escherichia coli.

[0042] Preferably, the cells can be hybridoma cells.

[0043] An eighth aspect of the present invention provides an anti-β1-AR-ECII antibody or an antigen-binding fragment thereof as described in the third aspect, or a method for preparing an antigen receptor as described in the fourth aspect, wherein the preparation method comprises introducing the nucleic acid as described in the sixth aspect into a host cell and then inducing its expression.

[0044] A ninth aspect of the present invention provides a method for detecting β1-AR-ECII in a sample, the method comprising using the anti-β1-AR-ECII antibody or its antigen-binding fragment as described in the third aspect, the nucleic acid as described in the sixth aspect, the cell as described in the seventh aspect, and the anti-β1-AR-ECII antibody or its antigen-binding fragment obtained by the preparation method described in the eighth aspect.

[0045] Preferably, the method includes contacting an anti-β1-AR-ECII antibody or its antigen-binding fragment with a sample and detecting the complex formed with β1-AR-ECII.

[0046] More preferably, the method includes:

[0047] 1) Link the anti-β1-AR-ECII antibody or its antigen-binding fragment 1 to a solid-phase carrier;

[0048] 2) Add the sample to be tested;

[0049] 3) Further add anti-β1-AR-ECII antibody or its antigen-binding fragment 2;

[0050] 4) Add labeled secondary antibody;

[0051] 5) Develop color and detect OD450 value.

[0052] Preferably, the sample comprises bodily fluids or cells. More preferably, the bodily fluids comprise blood, plasma, or serum. More preferably, the cells comprise cardiomyocytes.

[0053] Preferably, the sample is serum, and the volume added is 1-1000 μl, such as 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, 20 μl, 30 μl, 40 μl, 50 μl, 60 μl, 70 μl, 80 μl, 90 μl, 100 μl, 200 μl, 500 μl, or 1000 μl.

[0054] Preferably, if the sample is cells, the volume of cell culture supernatant added is 10-1000 μl, such as 10 μl, 15 μl, 20 μl, 25 μl, 30 μl, 35 μl, 40 μl, 45 μl, 50 μl, 60 μl, 70 μl, 80 μl, 90 μl, 100 μl, 200 μl, 500 μl, or 1000 μl.

[0055] Preferably, the sample can be bodily fluids or cells of a human or non-human animal.

[0056] Preferably, the final concentration of the anti-β1-AR-ECII antibody or its antigen-binding fragment 1 in the reaction system is 1 μg / ml to 100 μg / ml, such as 1 μg / ml, 2 μg / ml, 3 μg / ml, 4 μg / ml, 5 μg / ml, 6 μg / ml, 7 μg / ml, 8 μg / ml, 9 μg / ml, 10 μg / ml, 20 μg / ml, 40 μg / ml, 50 μg / ml, 80 μg / ml, or 100 μg / ml.

[0057] Preferably, the final concentration of the anti-β1-AR-ECII antibody or its antigen-binding fragment 2 in the reaction system is 1 μg / ml to 100 μg / ml, such as 1 μg / ml, 2 μg / ml, 3 μg / ml, 4 μg / ml, 5 μg / ml, 6 μg / ml, 7 μg / ml, 8 μg / ml, 9 μg / ml, 10 μg / ml, 20 μg / ml, 40 μg / ml, 50 μg / ml, 80 μg / ml, or 100 μg / ml.

[0058] The anti-β1-AR-ECII antibody or its antigen-binding fragment 1 binds to amino acids 14-26 of β1-AR-ECII (SEQ ID NO: 17).

[0059] Anti-β1-AR-ECII antibody or its antigen-binding fragment 2 binds to amino acids 1-13 of β1-AR-ECII (SEQ ID NO: 18); or

[0060] Anti-β1-AR-ECII antibody or its antigen-binding fragment 1 binds to amino acids 1-13 of β1-AR-ECII (SEQ ID NO: 18).

[0061] Anti-β1-AR-ECII antibody or its antigen-binding fragment 2 binds to amino acids 14-26 of β1-AR-ECII (SEQ ID NO: 17).

[0062] The β1-AR-ECII in the detection sample refers to the presence or content of β1-AR-ECII in the detection sample. "Presence" indicates whether the sample is present or absent, and "content" can refer to expression level or protein concentration, etc.

[0063] In a tenth aspect of the present invention, a detection kit for β1-AR-ECII is provided, the detection kit comprising an anti-β1-AR-ECII antibody or an antigen-binding fragment thereof as described in the third aspect.

[0064] Preferably, the detection kit further includes a solid support, buffer solution, blocking solution, diluent, enzyme-labeled secondary antibody, and chromogenic reagent. The buffer solution includes, but is not limited to, substrate buffer and washing buffer.

[0065] Preferably, the test kit also includes standards.

[0066] The eleventh aspect of the present invention provides an application of the anti-β1-AR-ECII antibody or its antigen-binding fragment described in the third aspect in the study of the generation mechanism of β1-adrenoceptor autoantibody (β1-AA).

[0067] A twelfth aspect of the present invention provides the use of the anti-β1-AR-ECII antibody or its antigen-binding fragment as described in the third aspect in the preparation of products for diagnosing and / or treating cardiovascular diseases.

[0068] Preferably, the cardiovascular diseases include acute myocardial infarction, cardiomyopathy (such as hypertensive cardiomyopathy, dilated cardiomyopathy or rheumatic cardiomyopathy), arrhythmia or heart failure.

[0069] In a thirteenth aspect of the present invention, a method for diagnosing cardiovascular disease is provided, the method comprising detecting the presence or amount of β1-AR-ECII or its autoantibody in a subject sample.

[0070] The subjects can be humans or non-human animals.

[0071] Preferably, the sample comprises bodily fluids or cells. More preferably, the bodily fluids comprise blood, plasma, or serum. More preferably, the cells comprise cardiomyocytes.

[0072] In one specific embodiment of the present invention, the sample is cell culture supernatant or serum.

[0073] Preferably, the sample can be derived from humans or non-human animals.

[0074] The "antigen-binding fragment" described in this invention is a portion of an antibody that retains the specific binding activity of the antibody; that is, any part of the antibody can specifically bind to an epitope on the antibody's target molecule. It includes, for example, Fab, Fab', F(ab')2, Fv, scFv, Fd, Fab'-SH, and variants of these fragments. For example, the heavy and / or light chains of the antibody, the variable regions of the heavy and / or light chains of the antibody, or one or more CDRs (Complementarity Determining Regions) from the heavy or light chains of the antibody. Specifically, Fab is a monovalent fragment consisting of VL, VH, CL, and CH1 domains. F(ab')2 is a divalent fragment comprising two Fab fragments connected by disulfide bonds in a hinge region. Fd is an Fd fragment consisting of VH and CH1 domains. Fv is an Fv fragment consisting of the VL and VH domains of a single arm of the antibody. Fab' is a Fab fragment having one or more cysteine ​​residues at the C-terminus of the CH1 domain. Fab'-SH refers to Fab' whose constant domain has at least one free thiol group attached to its cysteine ​​residues. scFv, or single-chain antibody fragment, consists of a heavy chain variable region and a light chain variable region, which can be linked by a short peptide (linker) typically 15-20 amino acids in length. Here, VH represents the heavy chain variable region, VL represents the light chain variable region, and CL represents the light chain.

[0075] The “CDR” as used in this invention refers to the complementarity-determining region within the variable sequence of an antibody. For each variable region, there are three CDRs in each variable region of the heavy and light chains, referred to as CDR1, CDR2, and CDR3. The exact boundaries of these CDRs are defined differently depending on the system. The system described by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides a definitive residue numbering system applicable to antibody variable regions but also provides residue boundaries defining the three CDRs. These CDRs can be referred to as Kabat CDRs. Each complementarity-determining region can contain amino acid residues from what is defined as a “complementarity-determining region” as by Kabat. Chothia et al. (Chothia & Lesk., J. Mol. Biol., 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (-1989)) found that Kabat… Certain sub-regions within a CDR adopt nearly identical peptide skeletons, despite significant diversity at the amino acid sequence level. These sub-regions are referred to as L1, L2, and L3, or H1, H2, and H3, where "L" and "H" denote the light and heavy chain regions, respectively. These regions may be termed Chothia CDRs, with boundaries overlapping with the Kabat CDR. Other CDR boundary definitions may not strictly adhere to one of the aforementioned systems but will still overlap with the Kabat CDR. The methods used herein can utilize CDRs defined according to any of these systems, although the preferred embodiment uses CDRs defined by Kabat or Chothia.

[0076] The term "comprising" or "including" as used in this invention is an open-ended expression. When used to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the same or similar activity or function as the original sequence.

[0077] The "homology" referred to in this invention means that, in the use of protein or nucleotide sequences, those skilled in the art can adjust the sequence according to actual work needs, so that the sequence used has (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 4% homology compared to sequences obtained by existing technologies. 1%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% of the sameness, and retain the same or similar activity or function.

[0078] The term "non-human animals" as used in this invention includes non-human mammals, including but not limited to wild animals, zoo animals, economically owned animals, pets, laboratory animals, etc. Preferably, the non-human mammals include but are not limited to pigs, cattle, sheep, horses, donkeys, foxes, raccoon dogs, minks, camels, dogs, cats, rabbits, rats (e.g., rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels) or monkeys, etc.

[0079] The method described in this invention can be used for the purpose of diagnosing or treating diseases, or for the purpose of diagnosing or treating non-diseases.

[0080] The present invention has the following beneficial effects:

[0081] (1) This application has screened two new anti-β1-AR-ECII antibodies or their antigen-binding fragments. These two antibodies bind to the N-terminal peptide and C-terminal peptide of β1-AR-ECII respectively, realizing rapid, effective, economical and batch detection of β1-AR-ECII.

[0082] (2) The newly screened anti-β1-AR-ECII antibody or its antigen-binding fragment in this application is used to detect β1-AR-ECII. When detecting β1-AR-ECII in serum, the detection range is 1 ng / ml-1400 ng / ml, which covers most of the short peptides present in serum. It is especially suitable for detecting the content of β1-AR-ECII in serum of patients with myocardial infarction, serum of actively immunized mice or rats, and cardiomyocytes of primary milk mice cultured in ischemia and hypoxia.

[0083] (3) By detecting β1-AR-ECII, this application can further explore the generation and intervention mechanism of β1-adrenoceptor autoantibody (β1-AA). Attached Figure Description

[0084] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0085] Figure 1 The image shows the Coomassie Brilliant Blue purity identification of β1-AR-mAb1 and β1-AR-mAb2 antibodies.

[0086] Figure 2 The image shows the mass spectrum of CDR1 in the heavy chain variable region of β1-AR-mAb1;

[0087] Figure 3 The image shows the mass spectrum of CDR2 in the heavy chain variable region of β1-AR-mAb1;

[0088] Figure 4 The image shows the mass spectrum of CDR3 in the variable region of the β1-AR-mAb1 heavy chain.

[0089] Figure 5 The image shows the mass spectrum of CDR1 in the variable region of the β1-AR-mAb1 light chain;

[0090] Figure 6 The image shows the mass spectrum of CDR2 in the variable region of the β1-AR-mAb1 light chain;

[0091] Figure 7 The image shows the mass spectrum of CDR3 in the variable region of the β1-AR-mAb1 light chain;

[0092] Figure 8 The image shows the mass spectrum of CDR1 in the heavy chain variable region of β1-AR-mAb2;

[0093] Figure 9 The image shows the mass spectrum of CDR2 in the heavy chain variable region of β1-AR-mAb2;

[0094] Figure 10The image shows the mass spectrum of CDR3 in the heavy chain variable region of β1-AR-mAb2;

[0095] Figure 11 The image shows the mass spectrum of CDR1 in the variable region of the β1-AR-mAb2 light chain;

[0096] Figure 12 The image shows the mass spectrum of CDR2 in the variable region of the β1-AR-mAb2 light chain;

[0097] Figure 13 The image shows the mass spectrum of CDR3 in the variable region of the β1-AR-mAb2 light chain;

[0098] Figure 14 The figures shown are the EC50 and R of β1-AR-mAb1 (Figure a) and β1-AR-mAb2 (Figure b). 2 result;

[0099] Figure 15 The diagram shown illustrates the principle of the double-antibody sandwich ELISA method for detecting β1-AR-ECII.

[0100] Figure 16 The figure shows the concentration and OD of the β1-AR-ECII standard. 450 A standard curve plotted using the values;

[0101] Figure 17 The figures show elevated serum β1-AA levels in patients with ST-segment elevation myocardial infarction (STEMI), which are negatively correlated with cardiac function. Figure a represents the OD values ​​of serum β1-AA in non-coronary artery disease patients (NCHD, n=50) and STEMI patients (n=160) with negative coronary angiography detected by ELISA. Data are presented as mean ± standard error; unpaired t-test, *** indicates P<0.001. Figure b represents the positive rate of serum β1-AA in NCHD and STEMI patients, chi-square test, *** indicates P<0.001. Figures cd represent the correlation between serum β1-AA OD values ​​in STEMI patients and left ventricular stroke volume (LVSV) and left ventricular ejection fraction (LVEF) as shown by echocardiography. In Pearson correlation analysis, "r" represents the correlation coefficient, and P<0.05 indicates statistical significance.

[0102] Figure 18The images show the presence of β1-AR-ECII in the serum of patients with primary STEMI and the culture supernatant of ischemic and hypoxic rat cardiomyocytes. Figure a shows the experimental flowchart, which first uses mouse monoclonal β1-AA to purify peptides from the serum of patients with primary STEMI and the culture supernatant of ischemic and hypoxic rat cardiomyocytes. Then, time-of-flight mass spectrometry was used to identify whether the purified peptide sequences contained the β1-AR-ECII sequence. Figure b shows that mass spectrometry detection revealed the presence of the β1-AR-ECII sequence in the serum of the three selected patients with primary STEMI (n=3). Figure c shows that mass spectrometry detection revealed the presence of the β1-AR-ECII sequence in the culture supernatant of serum-free and hypoxic rat primary cardiomyocytes (NRCMs) (n=3 or 1).

[0103] Figure 19 The diagram illustrates the generation of β1-AA due to abnormal exposure to β1-AR-ECII caused by acute myocardial infarction. Figure a represents the experimental flowchart for the production of β1-AA by actively immunizing mice with the supernatant of ischemic and hypoxic cultured cardiomyocytes. 1×10 6 The culture supernatant of NRCMs cultured for 24 hours under ischemic hypoxia was mixed 1:1 with Freund's complete adjuvant to prepare an emulsified antigen, which was injected intraperitoneally once a week for 2 weeks. Figure b shows the removal of β1-AR-ECII from the ischemic hypoxia culture supernatant using immunoprecipitation with β1-AA monoclonal antibody. The serum β1-AA level of mice at week 2 of active immunization was detected by ELISA. n=5. Data are presented as mean ± standard error. One-way ANOVA combined with Tukey's post-test was used. *** represents P<0.001, NS (No significance), indicating no statistical significance. Figure c shows the collection of venous serum from STEMI patients at initial onset and readmission two months later. The β1-AA level was detected by ELISA. n=30. Data are presented as mean ± standard error, paired t-test was used. *** P<0.001;

[0104] Figure 20 The results show that activation of the β2-AR / Gi pathway reduces the level of β1-AR-ECII in the supernatant of NRCMs cultured under ischemic and hypoxic conditions. NRCMs were pretreated with 1 μM of the β2-AR / Gi pathway-biased agonist ICI118551 for 30 minutes, followed by serum-free and anaerobic culture for 24 hours. The level of β1-AR-ECII in the cell supernatant was detected by ELISA. Data are presented as mean ± standard error. One-way ANOVA combined with Tukey post-test; *** represents P < 0.001, ** represents P < 0.01.

[0105] Figure 21The image shows cardiac injury directly induced by β1-AR-ECII in mice. C57BL / 6 mice were injected with 5 mg / kg / day of β1-AR-ECII or β1-AR-ECI / ECIII (50% each) for 3 consecutive days. One week later, echocardiography was used to assess systolic and diastolic function (a). Mmode represents M-mode echocardiography, PWD represents pulsed Doppler echocardiography, and TD represents tissue Doppler echocardiography. Sirius red staining was used to show collagen deposition area to determine the degree of cardiac fibrosis in mice (b). LVEF (left ventricular ejection fraction), E / E' (early diastolic flow velocity at the mitral valve tip / early diastolic myocardial motion velocity at the mitral annulus), and Collagen area (N=5) were used. Data are presented as mean ± standard error. Scale bar: 50 μm. One-way ANOVA combined with Tukey post-test was used. *** represents P<0.001; ** represents P<0.01; * represents P<0.05. Detailed Implementation

[0106] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0107] It should be noted that, unless otherwise specified, the methods used in this invention are conventional methods, and the reagents used in this invention are commercially available products unless otherwise specified.

[0108] Animals used in the examples: BALB / c mice: SPF (Specific Pathogen Free) grade BALB / c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., female, aged 56-64 days.

[0109] The main reagents used in the examples are shown in Table 1.

[0110] Table 1

[0111]

[0112]

[0113] Some of the experimental materials used in Examples 3 and 4:

[0114] High-affinity ELISA plate (FST015-200pcs, Beyotime); substrate buffer (R21957, ThermoFisher); antibody dilution buffer (Zhongshan Jinqiao, ZLI-9028); ELISA blocking buffer (C04-01002, Bioss); washing buffer 0.5% PBST (Pulley, B1019); goat anti-mouse IgG / HRP secondary antibody (ZB-2305, Zhongshan Jinqiao); ABTS (A1888-2G, Sigma); 0.5% hydrogen peroxide solution (AE4828A / ES, Chuangshi).

[0115] Accuracy calculation formula: (Average concentration by mass spectrometry / Average concentration by the detection method in this application) * 100%

[0116] STEMI standard for sizing:

[0117] Inclusion criteria were: ① STEMI patients meeting the diagnostic criteria of the "Guidelines for the Diagnosis and Treatment of Acute ST-Segment Elevation Myocardial Infarction (2015)"; ② STEMI patients undergoing emergency PCI must have undergone the procedure within 12 hours of symptom onset at Beijing Friendship Hospital affiliated with Capital Medical University; other included patients underwent coronary angiography during their hospitalization at Beijing Friendship Hospital, and PCI was performed as necessary (for patients with chest pain and negative angiography, this means the coronary angiography showed no abnormalities); ③ Enrolled patients were able to complete echocardiography; ④ Patients agreed to be enrolled in the trial and signed an informed consent form. Exclusion criteria were: ① Previous myocardial infarction or revascularization; ② Congestive heart failure; ③ Atrial fibrillation; ④ Renal insufficiency; ⑤ Acute infectious disease within the past 3 months; ⑥ Rheumatic immune system disease; ⑦ Malignant tumor; ⑧ Claudication; ⑨ Patients who did not agree to be enrolled in the trial.

[0118] Example 1: Detection of mouse immunity and mouse polyclonal antibody serum

[0119] 1. Preparation of immunogens

[0120] β1-AR-ECII-1 peptide (HWWRAESDEARRC (SEQ ID NO:18)) and β1-AR-ECII-2 peptide (YNDPKCCDFVTNR (SEQ ID NO:17)) were provided by Jier Biochemical (Shanghai) Co., Ltd. They were dissolved in DMSO to a final concentration of 5 mg / mL. 2 mg of each β1-AR-ECII-1 and β1-AR-ECII-2 peptide were conjugated to KLH carrier protein using the EDC method to obtain β1-AR-ECII-1-KLH and β1-AR-ECII-2-KLH conjugate proteins.

[0121] Immunogen 1 and immunogen 2 were prepared by β1-AR-ECII-1-KLH and β1-AR-ECII-2-KLH conjugate proteins, respectively. The conjugate proteins were mixed with Freund's complete adjuvant in equal volume and then emulsified by ultrasonication to prepare immunogen 1 and immunogen 2.

[0122] 2. Immunizing mice

[0123] One group of mice (n=3) was immunized with β1-AR-ECII-1-KLH, numbered 1-3#. Another group of mice (n=3) was immunized with β1-AR-ECII-2-KLH, numbered 4-6#. The two groups of mice were immunized with prepared immunogens 1 and 2 respectively. The immunization procedure for the mice was as follows: for the initial immunization, the mice were immunized with immunogen 1 and immunogen 2 respectively; once a day for a total of 4 times, the immunization sites were the subcutaneous tissue on the back and the muscle of the leg.

[0124] 3. Serum testing

[0125] Tail blood was collected from mice on day 14 post-immunization, and serum antibody titers were detected using an indirect ELISA method. The steps are as follows:

[0126] (1) Coating: Add 100 μL of 1 μg / mL β1-AR-ECII-1 or β1-AR-ECII-2 to each well for coating and react overnight at 4℃; (2) Washing: Wash the plate 3 times with PBS solution; (3) Blocking: Block with 5% milk-PBS at room temperature for 1 h and then wash the plate once with PBS solution; (4) Adding primary antibody: Add serially diluted mouse tail blood at 1 / 500, 1 / 1000, 1 / 5000, 1 / 10000, and 1 / 50000 to the ELISA plate for reaction and react at room temperature for 1 h; (5) Washing: Wash the plate 3 times with PBS solution and pat dry; (6) Adding secondary antibody: Add HRP-labeled goat anti-mouse IgG (Fc) secondary antibody diluted 1:2000 and react at room temperature for 1 h; (7) Washing: Wash the plate 5 times with PBS solution and pat dry; (8) Color development: Add TMB for color development and react for 20 min in the dark at room temperature. Then add 50 μL of stop solution, mix well, and read the OD value using a microplate reader. 450 Values. Tail blood was evaluated based on the test results. The test results are shown in Table 2 (coated antigen β1-AR-ECII-1, NC is the pre-immune negative serum control) and Table 3 (coated antigen β1-AR-ECII-2, NC is the pre-immune negative serum control).

[0127] Table 2: ELISA results of tail blood from mice in the β1-AR-ECII-1 immunized group on day 14.

[0128] No. 1 / 500 1 / 1,000 1 / 5,000 1 / 10,000 1 / 50,000 NC 1# 1.267 0.523 0.136 0.056 0.011 0.006 2# 1.880 1.013 0.303 0.094 0.036 0.012 3# 1.443 0.660 0.165 0.070 0.016 0.007

[0129] Table 3: ELISA results of tail blood from mice in the β1-AR-ECII-2 immunized group on day 14.

[0130] No. 1 / 500 1 / 1,000 1 / 5,000 1 / 10,000 1 / 50,000 NC 4# 0.790 0.520 0.290 0.126 0.019 0.006 5# 0.321 0.175 0.031 0.015 0.007 0.008 6# 0.165 0.051 0.009 0.007 0.007 0.007

[0131] As can be seen from the tail blood evaluation results in Table 2-3, the antibody titers of the D14 tail blood from the β1-AR-ECII-1 and β1-AR-ECII-2 immunization groups were relatively high, reaching 1 / 50,000. After further booster immunization, the tail blood was evaluated again.

[0132] Example 2: Cell fusion and hybridoma screening confirmation

[0133] Based on the ELISA evaluation results of tail blood in Example 1, mice #1 and #4 were selected. Spleen cells from these mice were fused with myeloma cells SP2 / 0. The specific steps for fusion were as follows: SP2 / 0 cells in good growth condition were pipetted off and centrifuged at 1500 rpm for 3 min; the supernatant was discarded, and the cells were resuspended in 30 mL of preheated DMEM culture medium; after centrifugation at 1500 rpm for 3 min, the supernatant was discarded, and an appropriate amount of preheated DMEM culture medium was added to resuspend the cells and perform cell counting; appropriate amounts of mouse spleen cells and SP2 / 0 cells were taken at a ratio of 10:1. Cells were mixed thoroughly in imported 50mL centrifuge tubes and centrifuged at 1500rpm for 3min, discarding the supernatant. Then, 1mL of preheated PEG was slowly added, and the mixture was incubated at 37℃ for 25min. 5mL of preheated DMEM medium containing 10% fetal bovine serum was slowly added dropwise along the tube wall, gradually increasing the speed by adding 15mL of preheated DMEM medium containing 10% fetal bovine serum, finally reaching a volume of 40mL. The mixture was centrifuged at 1200rpm for 5min, the supernatant was discarded, and the contents were added to 25mL of recovery medium. After culturing for 48 hours, the cells were transferred to selective semi-solid medium. After 10 days of culture, the cells were transferred to 96-well plates for further culture. After 7 days of culture in the 96-well plates, the supernatant was collected for analysis, and positive cell lines were selected. The cell culture supernatant (after 1:1 dilution) in the 96-well plate was evaluated by indirect ELISA (the steps are the same as in Example 1) to screen monoclonal cell lines that can secrete monoclonal antibodies that recognize β1-AR-ECII-1 and β1-AR-ECII-2 antigens.

[0134] Positive hybridoma cells were transferred and cultured in 48-well plates. After culturing for another 2 days, the cell supernatant (diluted 1:1) was collected for further screening and verification using the indirect ELISA method. The results are shown in Table 4 below (NC is the negative control, diluted with 5% milk-PBS; PC is the positive control, diluted 1:500 by bloodletting of mice at the end).

[0135] Table 4: Validation of Positive Clones Through Rescreening

[0136]

[0137] From the results in Table 4, three positive clones with stable antigen recognition were obtained from the seven positive clones. After expansion culture, the three monoclonal antibodies were subjected to competitive confirmation experiments and ELISA detection. For the normal group, the cell supernatant was serially diluted 1:1, 1:10, 1:100, 1:1000, and 1:10000. For the competitive group, 3 μg / mL of β1-AR-ECII-1 or β1-AR-ECII-2 was added to the cell supernatant. The results are shown in Tables 5 and 6 below (NC is the negative control, diluted with 5% milk-PBS; PC is the positive control, diluted 1:500 by bloodletting of mice at the end).

[0138] Table 5: Positive clone competition experiment (β1-AR-ECII-1)

[0139]

[0140]

[0141] Table 6: Positive clone competition experiment (β1-AR-ECII-2)

[0142]

[0143] As can be seen from the results in Tables 5-6, all three monoclonal antibodies showed significant competitive effects. The second strain targeting β1-AR-ECII-1 and the third strain targeting β1-AR-ECII-2 were selected for subsequent antibody extraction experiments.

[0144] Example 3: Antibody Purification and Validation

[0145] 1. Purification

[0146] Using the reagent kit (Melon) TMAfter separating and purifying β1-AR-mAb1 and β1-AR-mAb2 using the Gel Monoclonal IgG Purification Kit (Thermo Fisher, #45214), SDS-PAGE analysis (4 μg sample) was performed. Following Coomassie Brilliant Blue staining, compared with two commercially available β1-AR antibodies (Commercial antibody-1: CST#12271, Commercial antibody-2: Abcam#ab85037) and a rabbit IgG control (Rabbit IgGs control: Abcam#ab313801), the gray values ​​of the light and heavy chains of β1-AR-mAb1 IgG accounted for 95.7% of the total lane gray values ​​(i.e., purity), and the gray values ​​of the light and heavy chains of β1-AR-mAb2 IgG accounted for 97.4% of the total lane gray values ​​(Image J, v1.8.0.345). This was significantly higher than that of Commercial antibody-1 (97.1%) and Commercial antibody-2 (97.1%). antibody-2 (96.4%), Rabbit IgGs control (98.3%), see details. Figure 1 The above results demonstrate that the β1-AR-mAb1 and β1-AR-mAb2 isolated and purified in this embodiment achieved the purity of commercially available antibodies.

[0147] 2. Sequencing

[0148] The variable region sequences of the two antibodies were analyzed by Biotech Corporation using time-of-flight mass spectrometry. The variable region sequences of β1-AR-mAb1 are shown in Table 7, and those of β1-AR-mAb2 are shown in Table 8. The mass spectrum of the CDR1-3 region of the heavy chain variable region of β1-AR-mAb1 is shown in Table 8. Figure 2-4 The mass spectrum of the CDR1-3 variable region of the light chain of β1-AR-mAb1 is shown below. Figure 5-7 The mass spectrum of the CDR1-3 region of the heavy chain variable region of β1-AR-mAb2 is shown below. Figure 8-10 The mass spectrum of the CDR1-3 variable region of the light chain of β1-AR-mAb2 is shown below. Figure 11-13 .

[0149] Table 7

[0150]

[0151]

[0152] Table 8

[0153]

[0154]

[0155] 3. Detection of binding activity with β1-AR-ECII

[0156] (1) Incubation of β1-AR-mAb1 or β1-AR-mAb2: Dilute β1-AR-mAb1 or β1-AR-mAb2 into substrate buffer at a final concentration of 1:1-1:10^6, add 100 μl / well to the microplate, and let stand overnight at 4°C.

[0157] (2) Washing the plate: Discard the liquid in the microplate. Add 200 μl of 0.5% PBST, let stand for 5 minutes, and repeat 3 times.

[0158] (3) Blocking: Add 5% ELISA blocking solution, incubate at 37°C for 1 hour, and seal the plate with sealing film to prevent evaporation.

[0159] (4) Wash the plate: Discard the liquid in the microplate. Add 200 μl of 0.5% PBST, let stand for 5 minutes, and repeat 3 times.

[0160] (5) Add antigen: Add 100 μl of β1-AR-ECII standard with a concentration of 100 ng / ml, incubate at 37℃ for 1.5 h, and seal the ELISA plate with sealing film to prevent evaporation.

[0161] (6) Wash the plate: Discard the liquid in the microplate. Add 200 μl of 0.5% PBST, let stand for 5 minutes, and repeat 3 times.

[0162] (7) Incubation of secondary antibody: Add 100 μl of HRP-labeled goat anti-mouse secondary antibody to the microplate at a dilution of 1:2000, incubate at 37°C for 1 h, and seal the microplate with sealing film to prevent evaporation.

[0163] (8) Wash the plate: Discard the liquid in the microplate. Add 200 μl of 0.5% PBST, let stand for 5 minutes, and repeat 3 times.

[0164] (9) Color development: Dissolve 1.1 mg / ml ABTS in 100 ml of 0.5% hydrogen peroxide solution, add 100 μl of color development solution to each well of the microplate, and incubate at 37°C for 0.5 h.

[0165] (10) Read the OD value. Read the OD value of the microplate in the microplate reader. 450 Numerical values, calculating EC50 and R 2 .

[0166] The results are as follows Figure 14As shown, the EC50 of β1-AR-mAb1 is 3.482 and the EC50 of β1-AR-mAb2 is 3.560, indicating that the purified antibodies have a strong ability to bind to the antigen.

[0167] Example 4: Detection of β1-AR-ECII (see principle) Figure 15 )

[0168] 1. The antibodies and antigens used in this embodiment

[0169] Example 3 shows the purified β1-AR-mAb1 (variable region sequence shown in Table 7, 50 μg / ml), β1-AR-mAb2 (variable region sequence shown in Table 8, 50 μg / ml), and the full-length β1-AR-ECII peptide (HWWRAESDEARRCYNDPKCCDFVTNR (SEQ ID NO: 27), synthesized by Jier Biochemical).

[0170] 2. Experimental steps:

[0171] (1) Incubation of β1-AR-mAb1: Dilute β1-AR-mAb1 to substrate buffer at a final concentration of 1:100, add 100 μl / well to the microplate, and let stand overnight at 4°C.

[0172] (2) Washing the plate: Discard the liquid in the microplate. Add 200 μl of 0.5% PBST, let stand for 5 minutes, and repeat 3 times.

[0173] (3) Blocking: Add 5% ELISA blocking solution, incubate at 37°C for 1 hour, and seal the plate with sealing film to prevent evaporation.

[0174] (4) Wash the plate: Discard the liquid in the microplate. Add 200 μl of 0.5% PBST, let stand for 5 minutes, and repeat 3 times.

[0175] (5) Add samples: Add 100 μl of β1-AR-ECII standard at different concentrations (0, 1 ng / ml, 10 ng / ml, 50 ng / ml, 100 ng / ml, 500 ng / ml, 1000 ng / ml, 2000 ng / ml), or patient / rat / mouse serum diluted 1:100, or the original supernatant of ischemic and hypoxic cultured neonatal mouse cardiomyocytes; dilute the standard and serum with PBS, incubate at 37°C for 1.5 h, and seal the ELISA plate with sealing film to prevent evaporation.

[0176] (6) Wash the plate: Discard the liquid in the microplate. Add 200 μl of 0.5% PBST, let stand for 5 minutes, and repeat 3 times.

[0177] (7) Incubation of β1-AR-mAb2: Dilute β1-AR-mAb2 to antibody buffer at a final concentration of 1:1000, add 100 μl / well to the microplate, incubate at 37°C for 1.5 h, and seal the microplate with sealing film to prevent evaporation.

[0178] (8) Wash the plate: Discard the liquid in the microplate. Add 200 μl of 0.5% PBST, let stand for 5 minutes, and repeat 3 times.

[0179] (9) Incubation of secondary antibody: Add 100 μl of HRP-labeled goat anti-mouse secondary antibody to the microplate at a ratio of 1:2000, incubate at 37°C for 1 h, and seal the microplate with sealing film to prevent evaporation.

[0180] (10) Wash plate: Discard the liquid in the microplate. Add 200 μl of 0.5% PBST, let stand for 5 minutes, and repeat 3 times.

[0181] (11) Color development: Dissolve 1.1 mg / ml ABTS in 100 ml of 0.5% hydrogen peroxide solution, add 100 μl of color development solution to each well of the microplate, and incubate at 37°C for 0.5 h.

[0182] (12) Read the OD value. Read the OD value of the microplate in the microplate reader. 450 The concentration of β1-AR-ECII in serum or culture supernatant is obtained by using a standard curve derived from standards.

[0183] 3. Experimental Results

[0184] The determination results of β1-AR-ECII standards at different concentrations are shown in Table 9:

[0185] Table 9

[0186] Concentration (ng / ml) 2000 1000 500 100 50 10 1 0 <![CDATA[OD 450 (Average) 1.291 0.727 0.4215 0.2555 0.195 0.1845 0.1385 0.0955

[0187] A standard curve was plotted based on the test results of the standard samples. Figure 16 ).

[0188] The results of the human serum β1-AR-ECII assay are shown in Table 10.

[0189] Table 10

[0190]

[0191] Compared to mass spectrometry, which is considered the gold standard, this ELISA kit achieves an accuracy of 90.87% in detecting myocardial infarction.

[0192] The results of the determination of β1-AR-ECII in rat serum are shown in Table 11, where the concentration of immunogen applied in active immunizations 1, 2 and 3 was 500 ng / kg.

[0193] Table 11

[0194]

[0195]

[0196] Compared to mass spectrometry, which is the gold standard, this ELISA kit achieves an accuracy of 91.47% in detecting actively immunized rats.

[0197] The results of the determination of β1-AR-ECII in mouse serum are shown in Table 12, where the concentration of immunogen applied in active immunizations 1, 2 and 3 was 500 ng / kg.

[0198] Table 12

[0199]

[0200] Compared to mass spectrometry, which is the gold standard, this ELISA kit achieves an accuracy of 92.83% in detecting actively immunized mice.

[0201] Based on the above results, it can be seen that the detection method of this application can effectively detect the content of β1-AR-ECII in the serum of patients with myocardial infarction, the serum of mice actively immunized with β1-AR-ECII, and the serum of rats actively immunized with β1-AR-ECII.

[0202] Example 5: Serum β1-AA levels are elevated in patients with acute myocardial infarction and are associated with poorer cardiac function.

[0203] To evaluate the OD value and positive rate of β1-AA in patients with ST-segment elevation myocardial infarction (STEMI), this study selected 160 STEMI patients (regardless of whether it was a first-time case) and 50 patients with chest pain and negative coronary angiography (non-coronary heart disease, NCHD) according to inclusion and exclusion criteria. Patient information is shown in Table 13 (values ​​are expressed as mean ± SD, median (upper quartile, lower quartile), n (%); p-value was obtained by Student's test, Mann-Whitney U test, and chi-square test). Serum β1-AA levels were detected using ELISA, and patients with a p / n value > 2.1 were defined as positive. The results showed that the OD value of serum β1-AA in STEMI patients was significantly higher than that in NCHD patients. Figure 17a) and the β1-AA positivity rate in STEMI patients was 34.4%, which was also higher than the 6.0% in NCHD patients. Figure 17 b) The above results suggest that acute myocardial infarction can cause an increase in β1-AA production. This example also investigated the correlation between serum β1-AA levels and cardiac function in STEMI patients. The results showed that the OD value of serum β1-AA in STEMI patients was negatively correlated with left ventricular stroke volume (LVSV). Figure 17 c, r = -0.198, P = 0.014;) was negatively correlated with left ventricular ejection fraction (LVEF). Figure 17 d, r = -0.170, P = 0.032;) indicates that the increase in serum β1-AA caused by acute myocardial infarction is associated with poor cardiac function in patients.

[0204] Table 13

[0205]

[0206]

[0207] Among them, pNT-proBNP: peak value of N-terminal pro-B-type natriuretic peptide; pCK-MB: peak value of creatine kinase-MB; pTNI: peak value of troponin I; HsCRP: high-sensitivity C-reactive protein; WBC: white blood cells; FBG: fasting blood glucose; GLU: blood glucose level at admission; eGFR: estimated glomerular filtration rate; HbA1c: glycated hemoglobin; LDL-C: low-density lipoprotein cholesterol; HDL-C: high-density lipoprotein cholesterol.

[0208] Example 6: Abnormal exposure to β1-AR-ECII during acute myocardial infarction

[0209] To clarify the abnormal exposure of cardiomyocytes to β1-AR-ECII during myocardial infarction, this example used β1-AA affinity purification of serum from three β1-AA-positive newly diagnosed STEMI patients and the culture supernatant of primary cardiomyocytes from one SD neonatal rat cultured for 24 hours under ischemic and hypoxic conditions. The presence of β1-AR-ECII was then identified using time-of-flight mass spectrometry (experimental steps are described in [link to experimental procedure]). Figure 18 a). The results showed that amino acid fragments containing the β1-AR-ECII sequence were found in the serum of STEMI patients and in the supernatant of ischemic and hypoxic cultured cardiomyocytes. Figure 18bc), while no amino acid fragments of the β1-AR-ECII sequence were found in the supernatant of chest pain patients with negative contrast imaging and in the supernatant of normally cultured cardiomyocytes, suggesting that ischemic and hypoxic injury caused by myocardial infarction can lead to abnormal exposure of cardiomyocyte β1-AR-ECII to the blood.

[0210] Example 7: Acute myocardial infarction causes abnormal exposure of β1-AR-ECII in the blood, which can lead to the production of β1-AA.

[0211] To clarify that abnormal exposure to β1-AR-ECII in the blood can lead to the production of β1-AA, this study mixed and emulsified the supernatant of primary lactating rat cardiomyocytes cultured for 24 hours under ischemic hypoxia with Freund's complete adjuvant at a 1:1 ratio. The mixture was then administered via active immunization to 10-week-old male C57BL / 6N mice via intraperitoneal injection once weekly. The results showed that, two weeks after active immunization with the supernatant of ischemic hypoxia-cultured cells, the serum β1-AA level of mice significantly increased. However, after removing β1-AR-ECII using β1-AA, active immunization with the supernatant of ischemic hypoxia-cultured cardiomyocytes did not induce an increase in β1-AA. Figure 19 ab). This embodiment also examined the serum β1-AA levels of 30 newly diagnosed STEMI patients upon readmission two months later. The results showed that, compared with the initial myocardial infarction, the OD values ​​of serum β1-AA in these STEMI patients were significantly increased two months later. Figure 19 c). The above results suggest that abnormal exposure to β1-AR-ECII in the blood caused by acute myocardial infarction can lead to the production of β1-AA.

[0212] Example 8: Activation of the β2-AR / Gi pathway can reduce the abnormal exposure of β1-AR-ECII in ischemic and hypoxic cardiomyocytes.

[0213] In primary lactating rat cardiomyocytes cultured under ischemic and hypoxic conditions, pretreatment with 1 μM β2-AR / Gi pathway-biased agonist ICI118551 hydrochloride (Merrck, #72795-01-8) for 30 minutes was performed. The level of β1-AR-ECII in the culture supernatant was then detected using the method described in this application (principle explained in the appendix). Figure 15 The results showed that activation of the β2-AR / Gi pathway could partially reverse the increase in β1-AR-ECII levels in the cellular supernatant caused by ischemia and hypoxia. Figure 20 ).

[0214] Example 9: β1-AR-ECII directly induced cardiac damage in mice

[0215] To clarify whether β1-AR-ECII directly affects cardiac injury and decreased cardiac function in mice, this study administered intraperitoneal injections of β1-AR-ECII peptide or β1-AR-ECI / ECIII peptide (5 mg / kg / day, ip) to 10-week-old C57 mice for three consecutive days. Cardiac function was assessed using echocardiography one week later. Figure 21 a) Sirius red staining was used to detect the degree of cardiac fibrosis in mice. Figure 21 (b) At this stage, β1-AA has not yet formed (antibody generation requires 2 weeks). Results showed that β1-AR-ECII itself significantly impaired the systolic and diastolic functions of the mouse heart, increased cardiac collagen area, and caused cardiac fibrosis; while the use of other β1-AR extracellular cyclic peptides (β1-AR-ECI + β1-AR-ECIII) did not affect the cardiac function and structure of mice. These results suggest that β1-AR-ECII directly affects cardiac function and causes cardiac structural damage in mice.

Claims

1. An anti-β1-AR-ECII antibody or its antigen-binding fragment, characterized in that, The anti-β1-AR-ECII antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region, wherein, I) The heavy chain variable region contains: VHCDR1 with the amino acid sequence shown in SEQ ID NO: 1; VHCDR2 with the amino acid sequence shown in SEQ ID NO: 2; VHCDR3 with the amino acid sequence shown in SEQ ID NO: 3; and the light chain variable region contains: VLCDR1 with the amino acid sequence shown in SEQ ID NO: 4; VLCDR2 with the amino acid sequence shown in SEQ ID NO: 5; VLCDR3 with the amino acid sequence shown in SEQ ID NO: 6; or, II) The heavy chain variable region contains: VHCDR1 with the amino acid sequence shown in SEQ ID NO: 7; VHCDR2 with the amino acid sequence shown in SEQ ID NO: 8; VHCDR3 with the amino acid sequence shown in SEQ ID NO: 9; and the light chain variable region contains: VLCDR1 with the amino acid sequence shown in SEQ ID NO: 10; VLCDR2 with the amino acid sequence shown in SEQ ID NO: 11; and VLCDR3 with the amino acid sequence shown in SEQ ID NO:

12.

2. The anti-β1-AR-ECII antibody or its antigen-binding fragment according to claim 1, characterized in that, in: I) The amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 13, or an amino acid sequence having more than 90% homology with the amino acid sequence shown in SEQ ID NO: 13; the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO: 14, or an amino acid sequence having more than 90% homology with the amino acid sequence shown in SEQ ID NO:

14. or II) The amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 15, or an amino acid sequence having more than 90% homology with the amino acid sequence shown in SEQ ID NO: 15; the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO: 16, or an amino acid sequence having more than 90% homology with the amino acid sequence shown in SEQ ID NO:

16.

3. The anti-β1-AR-ECII antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The anti-β1-AR-ECII antibody or its antigen-binding fragment includes Fab, Fab', Fab'-SH, Fv, scFv or F(ab')2.

4. A nucleic acid, characterized in that, The nucleic acid described herein encodes the anti-β1-AR-ECII antibody or its antigen-binding fragment as described in any one of claims 1-3.

5. A cell comprising the nucleic acid of claim 4.

6. A method for detecting β1-AR-ECII in a sample, characterized in that, The method includes using the anti-β1-AR-ECII antibody or its antigen-binding fragment as described in any one of claims 1-3, and the method is not a diagnosis or treatment for the disease.

7. The method according to claim 6, characterized in that, The method includes: contacting an anti-β1-AR-ECII antibody or its antigen-binding fragment with a sample, and detecting the complex formed by the anti-β1-AR-ECII antibody or its antigen-binding fragment and β1-AR-ECII.

8. The method according to claim 6, characterized in that, The method includes: 1) An anti-β1-AR-ECII antibody or its antigen-binding fragment 1 is linked to a solid-phase carrier, wherein the anti-β1-AR-ECII antibody or its antigen-binding fragment 1 includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: VHCDR1 with the amino acid sequence shown in SEQ ID NO: 1; VHCDR2 with the amino acid sequence shown in SEQ ID NO: 2; and VHCDR3 with the amino acid sequence shown in SEQ ID NO: 3; and the light chain variable region comprises: VLCDR1 with the amino acid sequence shown in SEQ ID NO: 4; VLCDR2 with the amino acid sequence shown in SEQ ID NO: 5; and VLCDR3 with the amino acid sequence shown in SEQ ID NO: 6; 2) Add the sample to be tested; 3) Further addition of anti-β1-AR-ECII antibody or its antigen-binding fragment 2, wherein the anti-β1-AR-ECII antibody or its antigen-binding fragment 2 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: VHCDR1 with an amino acid sequence as shown in SEQ ID NO: 7; VHCDR2 with an amino acid sequence as shown in SEQ ID NO: 8; and VHCDR3 with an amino acid sequence as shown in SEQ ID NO: 9; and the light chain variable region comprises: VLCDR1 with an amino acid sequence as shown in SEQ ID NO: 10; VLCDR2 with an amino acid sequence as shown in SEQ ID NO: 11; and VLCDR3 with an amino acid sequence as shown in SEQ ID NO: 12; 4) Add labeled secondary antibody; 5) Develop color and detect OD 450 value.

9. The method according to claim 7, characterized in that, The samples mentioned include body fluids or cells.

10. The method according to claim 9, characterized in that, The bodily fluids mentioned include blood, plasma, or serum.

11. The method according to claim 9, characterized in that, The cells mentioned include cardiomyocytes.

12. The method according to claim 8, characterized in that, The final concentration of anti-β1-AR-ECII antibody or its antigen-binding fragment 1 in the reaction system is 1 μg / ml-10 μg / ml.

13. The method according to claim 8, characterized in that, The final concentration of anti-β1-AR-ECII antibody or its antigen-binding fragment 2 in the reaction system is 1 μg / ml-10 μg / ml.

14. The use of any one of the anti-β1-AR-ECII antibodies or antigen-binding fragments thereof as described in claims 1-3 in the preparation of products for the diagnosis and / or treatment of acute myocardial infarction.

15. A detection kit for β1-AR-ECII, characterized in that, The detection kit comprises the anti-β1-AR-ECII antibody or its antigen-binding fragment as described in any one of claims 1-3.

16. The detection kit according to claim 15, characterized in that, The detection kit also includes one or more of the following: solid support, buffer solution, blocking solution, diluent, enzyme-labeled secondary antibody, or colorimetric reagent.

Images