A non-carboxylated matrix Gla protein antibody and its application

Abstract

The present invention relates to the field of biomedicine, and provides an antibody of non-carboxylated matrix Gla protein and its application. The non-carboxylated matrix Gla protein antibody or antigen binding fragment comprises at least one selected from the following: (1) having a light chain CDR sequence shown in SEQ ID NO: 3, 4 and 5, and a heavy chain CDR sequence shown in SEQ ID NO: 6, 7 and 8; or an amino acid sequence having at least 80% sequence identity compared with (1). In addition, the present invention also discloses a kit containing the antibody. The non-carboxylated matrix Gla protein antibody or antigen binding fragment or kit of the present invention can realize the detection of non-carboxylated matrix Gla protein, and has the advantages of high accuracy, high sensitivity and high specificity, and high application value.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a non-carboxylated matrix Gla protein antibody and application thereof. Background Art

[0002] Matrix Gla protein (MGP) is a 10.4kD small molecule matrix protein, a vitamin K-dependent protein, containing 84 amino acids, including 5 γ-carboxylated glutamic acid residues and a disulfide bond. MGP is mainly synthesized by chondrocytes in cartilage and smooth muscle cells in arterial walls. It is almost exclusively expressed by smooth muscle cells in the tunica media, while the intima is mainly expressed by smooth muscle cells, macrophages and endothelial cells. MGP is associated with many diseases, such as osteoporosis, cardiovascular disease, ectopic calcification, tumors, etc.

[0003] MGP includes the inactive form, i.e., uncarboxylated MGP (ucMGP), and the biologically active carboxylated MGP (cMGP). The precursor of MGP after transcription must undergo vitamin K-dependent carboxylation to be biologically active. Vitamin K deficiency leads to the inactive, non-carboxylated form of MGP. When blood vessels calcify, smooth muscle cells continuously produce MGP to inhibit the progression of calcification, but only cMGP has an inhibitory effect. When vitamin K is deficient, as MGP continues to increase, ucMGP continues to accumulate. Therefore, the higher the ucMGP content in the initial stage of vascular calcification, the closer it is to vascular calcification. The ucMGP concentration can also reflect the deficiency of vitamin K. ucMGP accumulates in large quantities in atherosclerotic and calcified blood vessels, and cMGP is almost undetectable in these blood vessels. Therefore, ucMGP can be used as a biomarker for vascular calcification.

[0004] There are two forms of matrix Gla protein, namely carboxylated matrix Gla protein and non-carboxylated matrix Gla protein. It is difficult to screen antibodies with good specificity that can distinguish the two forms, which makes it difficult to identify positive serum for clinical non-carboxylated matrix Gla protein.

[0005] Currently, there is an urgent need to develop a non-carboxylated matrix Gla protein antibody with strong specificity and high sensitivity. Summary of the invention

[0006] The present invention aims to solve one of the technical problems in the related art at least to a certain extent.

[0007] In one aspect, the present invention provides a non-carboxylated matrix Gla protein antibody or antigen-binding fragment, comprising:

[0008] A CDR sequence selected from at least one of the following or an amino acid sequence having at least 80% identity thereto:

[0009] Light chain variable region CDR sequences: SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5;

[0010] Heavy chain variable region CDR sequences: SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.

[0011] The invention provides a non-carboxylated matrix Gla protein antibody or antigen binding fragment. An enzyme immunoassay kit prepared by using the antibody or antigen binding fragment has high sensitivity and specificity.

[0012] According to some embodiments of the present invention, the antibody or antigen-binding fragment comprises: a light chain variable region CDR1 sequence shown in SEQ ID NO:3, a light chain variable region CDR2 sequence shown in SEQ ID NO:4, a light chain variable region CDR3 sequence shown in SEQ ID NO:5, a heavy chain variable region CDR1 sequence shown in SEQ ID NO:6, a heavy chain variable region CDR2 sequence shown in SEQ ID NO:7, and a heavy chain variable region CDR3 sequence shown in SEQ ID NO:8.

[0013] According to some embodiments of the present invention, the antibody or antigen-binding fragment comprises at least one of the following:

[0014] (a) having a light chain variable region shown in SEQ ID NO: 1 and a heavy chain variable region shown in SEQ ID NO: 2;

[0015] An amino acid sequence with at least 80% sequence identity compared to (a).

[0016] According to some embodiments of the present invention, the antibody or antigen-binding fragment comprises at least one of the following:

[0017] (b) having the CDR1, CDR2 and CDR3 sequences of the light chain variable region shown in SEQ ID NO: 1, and having the CDR1, CDR2 and CDR3 sequences of the heavy chain variable region shown in SEQ ID NO: 2;

[0018] Or an amino acid sequence having one or more conservative amino acid substitutions compared to (b).

[0019] According to some embodiments of the present invention, the antibody or antigen-binding fragment further comprises at least one of a heavy chain constant region and a light chain constant region, at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from a mammalian antibody.

[0020] According to some embodiments of the present invention, the heavy chain constant region and the light chain constant region are derived from at least one of a murine antibody, a rabbit antibody, a primate antibody or mutants thereof.

[0021] According to some embodiments of the present invention, the heavy chain constant region and the light chain constant region are derived from a murine antibody or a mutant thereof.

[0022] According to some embodiments of the present invention, the N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region; and / or the N-terminus of the light chain constant region is connected to the C-terminus of the light chain variable region.

[0023] According to some embodiments of the present invention, the heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; or the light chain constant region includes a light chain constant region selected from κ or λ.

[0024] According to some embodiments of the present invention, the heavy chain constant region comprises: an amino acid sequence as shown in SEQ ID NO:22 or an amino acid sequence that has at least 80% identity thereto; the light chain constant region comprises: an amino acid sequence as shown in SEQ ID NO:23 or an amino acid sequence that has at least 80% identity thereto.

[0025] According to some embodiments of the present invention, the antibody or antigen-binding fragment comprises a monoclonal antibody or a polyclonal antibody;

[0026] According to some embodiments of the present invention, the monoclonal antibody includes at least one of a full-length antibody, Fv, a single-chain antibody, Fab, a single-domain antibody and a minimum recognition unit.

[0027] According to some embodiments of the present invention, the antibody or antigen-binding fragment comprises: a heavy chain having an amino acid sequence as shown in SEQ ID NO:24 or an amino acid sequence having at least 90% homology thereto; and / or a light chain having an amino acid sequence as shown in SEQ ID NO:25 or an amino acid sequence having at least 90% homology thereto.

[0028] Another aspect of the present invention provides an antibody analog, which comprises the variable region or the CDR region in the variable region of the above-mentioned antibody or antigen-binding fragment.

[0029] Another aspect of the present invention provides a conjugate, comprising:

[0030] The above-mentioned non-carboxylated matrix Gla protein antibody or antigen-binding fragment; and

[0031] A coupling portion, the coupling portion is connected to the non-carboxylated matrix Gla protein antibody or antigen binding fragment

[0032] According to some embodiments of the invention, the coupling moiety comprises at least one of a carrier, a drug, a toxin, a cytokine, a protein tag, a modifier and a chemotherapeutic agent.

[0033] According to some embodiments of the invention, the carrier comprises a fluorescent dye.

[0034] According to some embodiments of the present invention, the fluorescent dye includes any one or more of FITC, PE, Cy5, PI, 7 AAD, APC, AlexaFluor, eFluor, PECy7, APC Cy7, PerCP, PerCPcy5.5, PB, BV series, QDot series and BUV series dyes.

[0035] In another aspect, the present invention provides an isolated polynucleotide encoding the above-mentioned antibody or antigen-binding fragment.

[0036] According to some embodiments of the invention, the polynucleotide comprises:

[0037] (c) having a nucleic acid sequence encoding a light chain variable region as shown in SEQ ID NO:9,

[0038] A nucleic acid sequence having at least 80% sequence identity to (c); or

[0039] (d) having a nucleic acid sequence encoding a heavy chain variable region as shown in SEQ ID NO: 10,

[0040] A nucleic acid sequence having at least 80% sequence identity compared to (d).

[0041] In another aspect, the present invention provides an expression vector comprising the above-mentioned polynucleotide.

[0042] In another aspect, the present invention provides a recombinant cell into which the above expression vector is transferred.

[0043] In another aspect, the present invention provides a cell, which can produce the above-mentioned non-carboxylated matrix Gla protein antibody or antigen-binding fragment.

[0044] In yet another aspect, the present invention provides a method for preparing a non-carboxylated matrix Gla protein antibody or antigen fragment, comprising culturing the above-mentioned recombinant cell.

[0045] In yet another aspect, the present invention provides use of the above-mentioned antibody or antigen-binding fragment and the above-mentioned antibody analog in preparing a kit for detecting non-carboxylated matrix Gla protein in a sample.

[0046] According to some embodiments of the present invention, the kit is used for diagnosing related diseases mediated by non-carboxylated matrix Gla protein.

[0047] According to some embodiments of the present invention, the non-carboxylated matrix Gla protein-mediated related diseases include vascular calcification, cartilage calcification, kidney calcification, kidney stones, severe atherosclerosis, and ectopic calcification of the nasal septum.

[0048] Another aspect of the present invention provides a kit, comprising at least one of the above-mentioned antibody or antigen-binding fragment, conjugate, expression vector, and recombinant cell.

[0049] Another aspect of the present invention provides a composition comprising at least one of the above-mentioned antibodies or antigen-binding fragments, conjugates, isolated polynucleotides, expression vectors, recombinant cells and hybridoma cells.

[0050] Another aspect of the present invention provides a method for preparing the above-mentioned antibody or antigen-binding fragment, comprising culturing the above-mentioned recombinant cell.

[0051] Another aspect of the present invention provides a method for detecting non-carboxylated matrix Gla protein, comprising:

[0052] The non-carboxylated matrix Gla protein antibody or antigen-binding fragment, conjugate or kit is contacted with the sample to be tested to form an immune complex;

[0053] Based on the signal of the immune complex, the non-carboxylated matrix Gla protein in the sample to be detected is quantitatively and / or qualitatively analyzed.

[0054] The above method for detecting non-carboxylated matrix Gla protein can be used for diagnosis purposes, such as diagnosing non-carboxylated matrix Gla protein-related diseases; or for non-diagnosis purposes, such as achieving qualitative and quantitative analysis of non-carboxylated matrix Gla protein.

[0055] According to some embodiments of the present invention, the signal of the immune complex is detected by at least one of ELISA, WB and FACS.

[0056] According to some embodiments of the invention, the signal comprises a fluorescent signal.

[0057] Another aspect of the present invention provides a drug comprising at least one of the above-mentioned antibodies or antigen-binding fragments, conjugates, isolated polynucleotides, expression vectors, recombinant cells and compositions.

[0058] Another aspect of the present invention provides a use of the above-mentioned antibody or antigen-binding fragment, conjugate, isolated polynucleotide, expression vector, recombinant cell or composition in the preparation of a drug, wherein the drug is used to prevent or treat related diseases mediated by non-carboxylated matrix Gla protein;

[0059] According to some embodiments of the present invention, the non-carboxylated matrix Gla protein-mediated related diseases include vascular calcification, cartilage calcification, kidney calcification, kidney stones, severe atherosclerosis, and ectopic calcification of the nasal septum.

[0060] The present invention provides a monoclonal antibody of non-carboxylated matrix Gla protein, which has the characteristics of high affinity and specificity, and also provides a detection kit based on the antibody. The present invention solves the problem of relative shortage of antibody raw materials and kits for non-carboxylated matrix Gla protein detection projects in the current market.

[0061] Additional aspects and advantages of the present disclosure will be given in part in the following description and in part will be obvious from the following description or will be learned through practice of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 It is a gel map of subtype identification of the antibody expressed by the cell line G071 and cloning of the antibody variable region according to the embodiment of the present invention. The lane sequence is shown in Table 5. DETAILED DESCRIPTION OF THE INVENTION

[0063] The embodiments of the present disclosure are described in detail below. The embodiments described below are exemplary and are only used to explain the present disclosure, and should not be construed as limiting the present disclosure.

[0064] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of the present disclosure, "plurality" means at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0065] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0066] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0067] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0068] In the present invention, the term "antibody" refers to a protein encoded by an antibody gene of an animal and biosynthesized.

[0069] In the present invention, the term "antibody analogs" refers to derivatives produced by biological or chemical methods, which are obtained by deleting, adding, or modifying chemical groups (such as amino acids) based on the antibody structure. These derivatives still contain structures similar to the antibody variable region (or the CDR region in the antibody variable region), and can produce reactions similar to antigen-antibody binding through these structures.

[0070] In the present invention, the term "antibody variable region" refers to a domain in the heavy chain and light chain of an antibody. In nature, the antibody variable region is encoded by V, D (only heavy chain is applicable), and J fragments in the immunoglobulin (heavy chain and light chain) gene through gene recombination splicing. The amino acid sequence of the variable region has a high degree of diversity between different antibodies (the amino acid sequences in other regions of the antibody are relatively highly identical), and is responsible for recognizing and binding with specific antigenic determinants. In the antibody variable region, the framework region and CDR region (comlementarity determining region) can be further subdivided. A typical antibody variable region has 3 framework regions and 3 CDR regions (which are interspersed with each other). The framework region mainly plays a role in the formation of the protein domain framework, while the CDR region mainly plays a role in the specific recognition and binding of antigen-antibody.

[0071] In this document, the amino acid sequences described in the present invention are all shown from N-terminus to C-terminus.

[0072] The antibodies and antigen-binding fragments described in the present invention are usually prepared by biosynthetic methods. According to the nucleotide sequence described in the present invention, those skilled in the art can easily prepare the encoding nucleic acid of the present invention by various known methods. These methods are, for example, but not limited to: PCR, DNA artificial synthesis, etc., and specific methods can be found in J. Sambrook, "Molecular Cloning Laboratory Guide". As an embodiment of the present invention, the encoding nucleic acid sequence of the present invention can be constructed by synthesizing nucleotide sequences in segments and then performing overlap extension PCR. Among them, the antibodies and antigen fragments in the present invention are numbered and defined using the IMGT numbering system. However, those of ordinary skill in the art are fully capable of converting the sequence IMGT numbering of the sequence table into "HCDRs" and / or "LCDRs" under other numbering systems (including but not limited to AbM, Kabat, Chothia), and the CDRs determined based on the heavy chain variable region and light chain variable region disclosed in this application using other rules disclosed in the art also belong to the scope of protection of the present disclosure.

[0073] As used herein, the "IMGT definition" refers to the new standardized numbering system introduced by Lefranc et al. for all protein sequences of the immunoglobulin superfamily, see Lefranc, et al., The Immunologist, 7, 132-136 (1999). The "Kabat definition" refers to the definition system described by Kabat et al., U. S. Pat. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). The "Chothia definition" refers to Chothia et al., J Mol Biol 196:901-917 (1987).

[0074] In the present invention, the term "non-carboxylated matrix Gla protein antibody" exists in a single (or monoclonal) form, and a single antibody contains a single structure (such as a single amino acid sequence).

[0075] In the present invention, when the term "identity" is used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 48:443); the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:24 ... .70:173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs that utilize these algorithms are also available, and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0076] In the present invention, the term "antigen-binding fragment" also refers to "antibody fragment". Antibody fragments generally refer to antigen-binding antibody fragments and may include a portion of an intact antibody, generally an antigen-binding region or a variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, Fv or scFv, diabodies, linear antibodies, single-chain antibody molecules, etc.

[0077] Under the premise of not substantially affecting the activity of the antibody (retaining at least 95% of the activity), those skilled in the art can replace, add and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids in the sequence of the present invention to obtain variants of the sequence of the antibody or its functional fragment. They are all considered to be included in the scope of protection of the present invention. For example, amino acids with conservative modifications are replaced in the variable region. The variant sequence of the present invention may have at least 80% identity (or homology) with the reference sequence. The sequence identity of the present invention can be measured using sequence analysis software. For example, the computer program BLAST with default parameters is used, especially BLASTP or TBLASTN. The amino acid sequences described in the present invention are all shown in a manner from N-terminus to C-terminus.

[0078] Non-carboxylated matrix Gla protein antibody or antigen binding fragment

[0079] The present invention provides a non-carboxylated matrix Gla protein antibody or antigen-binding fragment, selected from (1) having CDR1, CDR2 and CDR3 sequences of the light chain variable region shown in SEQ ID NO: 1, and having CDR1, CDR2 and CDR3 sequences of the heavy chain variable region shown in SEQ ID NO: 2; or a sequence having at least one conservative amino acid substitution compared to (1). The conservative amino acid substitution mentioned can be one, two or three amino acid substitutions.

[0080] The present invention also provides a non-carboxylated matrix Gla protein antibody or antigen-binding fragment, selected from (1) having a light chain CDR sequence shown in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, and a heavy chain CDR sequence shown in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8; or an amino acid sequence having at least 80% sequence identity compared to (1).

[0081] In some embodiments, the present invention provides an antibody or antigen-binding fragment having a light chain CDR sequence shown in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, and a light chain CDR sequence shown in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.

[0082] In other embodiments, the antibodies or antigen-binding fragments provided by the present invention have more than one conservative amino acid substitution compared to the above sequences. "Antigen-binding fragment" refers to an antibody fragment that retains the ability to specifically bind to an antigen. "Conservative amino acid substitution" refers to the substitution of an amino acid with another amino acid residue that is biologically, chemically or structurally similar. Biologically similar means that the substitution does not destroy the biological activity of the non-carboxylated matrix Gla protein antibody or the non-carboxylated matrix Gla protein antigen. Structurally similar means that the amino acids have side chains of similar length, such as alanine, glycine or serine, or have side chains of similar size. Chemical similarity means that the amino acids have the same charge or are hydrophilic or hydrophobic. For example, the hydrophobic residues isoleucine, valine, leucine or methionine replace each other. Or with polar amino acids, such as arginine replacing lysine, glutamic acid replacing aspartic acid, glutamine replacing asparagine, serine replacing threonine, etc.

[0083] The antibodies of the present invention can also be directly synthesized based on the antibody variable region amino acid sequences provided by the present invention, and can be derived into different antibody analogs through further chemical modification.

[0084] In some specific embodiments, the present invention provides an antibody or antigen-binding fragment, comprising at least one of the following: (a) having a light chain variable region shown in SEQ ID NO:1 and a heavy chain variable region shown in SEQ ID NO:2; an amino acid sequence with at least 80% identity compared to (a).

[0085] In some embodiments, the present invention provides an antibody or antigen-binding fragment, which has a light chain variable region sequence shown in SEQ ID NO: 1 and a heavy chain variable region sequence shown in SEQ ID NO: 2. In other embodiments, the heavy chain variable region sequence of the antibody or antigen-binding fragment has one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the light chain variable region sequence of the antibody or antigen-binding fragment has one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 2. Of course, these conservative amino acid substitutions will not change the biological function of the antibody or antigen-binding fragment. In some specific embodiments, these conservative amino acid substitutions may occur in amino acids other than the CDR region in the heavy chain variable region and the light chain variable region.

[0086] According to a specific embodiment of the present invention, the antibody or antigen-binding fragment is a single-chain antibody, a multimeric antibody, a CDR-grafted antibody or a small molecule antibody. For example, the antibody is a single-chain antibody. For example, the small molecule antibody mentioned includes at least one of a Fab antibody, a Fv antibody, a single-chain antibody and a minimum recognition unit.

[0087] According to a specific embodiment of the present invention, the light chain variable region sequence provided is as follows:

[0088] DIQMTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWKSN(SEQ ID NO:1)

[0089] According to a specific embodiment of the present invention, the heavy chain variable region sequence provided is as follows:

[0090] EVQLQESGGGLVQPGESLKLSYESNEYEFPFHDISWVRKTPEMRLELVAAINSDGGNTYYPDTMERRFIISRDNTKKTLYLQMSSLRSEDTALFYCGRHKYSYRYGVCYGLLGSRNLSHRLL(SEQ ID NO:2)

[0091] In another aspect of the present invention, the present invention proposes a conjugate. According to an embodiment of the present invention, the conjugate comprises: the non-carboxylated matrix Gla protein antibody or antigen binding fragment described above; and a coupling portion, wherein the coupling portion is connected to the antibody or antigen binding fragment or recombinant protein. The present invention proposes that the conjugate can specifically bind to the non-carboxylated matrix Gla protein, and the generated signal intensity is high, which helps to accurately detect the non-carboxylated matrix Gla protein with high accuracy, and has important application value for the diagnosis, prevention or treatment of diseases related to the non-carboxylated matrix Gla protein. According to an embodiment of the present invention, the coupling portion includes at least one selected from a carrier, a drug, a toxin, a cytokine, a protein tag, a modifier and a chemotherapeutic agent. In this article, the carrier can be a substance that can be suspended or dispersed in a liquid phase (for example, solid phase carriers such as particles and magnetic beads), or a solid phase that can contain or carry a liquid phase (for example, a support such as a plate, a membrane, a test tube, and a container such as a well plate, a microfluidic channel, a glass capillary, a nanocolumn, a monolithic column, etc.); it can also be a labeling carrier for labeling an antibody or antigen-binding fragment or a recombinant protein, such as an enzyme (for example, peroxidase, alkaline phosphatase, luciferin, β-galactosidase), Affinity substances (e.g., one of streptavidin and biotin, one of the complementary positive and antisense nucleic acids), fluorescent substances (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substances (e.g., luciferin, aequorin, acridinium ester, tris(2,2'bipyridine)ruthenium, luminol), radioactive isotopes (e.g., 3H, 14C, 32P, 35S, 125I) and gold colloids, etc. According to an embodiment of the present invention, the drug is a small molecule drug that can be bound to an antibody or antigen-binding fragment or a recombinant protein. According to an embodiment of the present invention, the protein tag includes but is not limited to a His tag, a Flag tag, a GST tag, an MBP tag, a SUMO tag, and a C-Myc tag, etc. According to an embodiment of the present invention, the modifier should be understood in a broad sense and may refer to a substance used to modify a protein. Exemplarily, it may be polyethylene glycol or a derivative thereof. According to an embodiment of the present invention, the chemotherapeutic agents include, but are not limited to, albumin paclitaxel, cyclophosphamide, ifosfamide, phenylalanine mustard, methotrexate, fluorouracil, actinomycin D, and vincristine. It should be noted that the binding method of the coupling part and the antibody or antigen-binding fragment or recombinant protein can use methods known in the art. For example, physical adsorption, covalent binding, methods using affinity substances (e.g., biotin, streptavidin), and ion binding methods can be cited.

[0092] Polynucleotides, expression vectors, recombinant cells, hybridoma cells

[0093] In the process of preparing or obtaining these antibodies, the polynucleotides expressing these antibodies can be connected to different vectors and then expressed in different cells to obtain the corresponding antibodies.

[0094] To this end, the present invention also provides an isolated polynucleotide encoding the above-mentioned antibody or antigen-binding fragment. The provided nucleotide sequence can be obtained by techniques known to those skilled in the art. It can also be species-optimized to be more easily expressed in mammalian cells.

[0095] Based on the variable region (or CDR region) sequences of the antibody heavy chain and light chain provided by the present invention, or the isolated polynucleotide sequences provided by the present invention, those skilled in the art can prepare antibodies (or antibody analogs) that are the same as or similar to those of the present invention by chemical synthesis and / or expression of antibody genes in heterologous cells (i.e., recombinant protein expression).

[0096] The present invention also provides an expression vector, which comprises the above-mentioned isolated polynucleotide. When the above-mentioned isolated polynucleotide is connected to the vector, the polynucleotide can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the polynucleotide. Of course, these control elements can come directly from the vector itself, or they can be exogenous, that is, they are not from the vector itself. Of course, the polynucleotide can be operably connected to the control element. Herein, "operably connected" means connecting the exogenous gene to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their expected functions of regulating the transcription and translation of the exogenous gene. Of course, the polynucleotides used to encode the heavy chain and light chain of the antibody can be inserted into different vectors separately and independently, and it is common to insert them into the same vector. Commonly used vectors can be, for example, plasmids, bacteriophages, etc. For example, pcDNA plasmids.

[0097] The present invention also provides a recombinant cell, which contains the expression vector. The expression vector can be introduced into mammalian cells to construct recombinant cells, and then these recombinant cells are used to express the antibody or antigen-binding fragment provided by the present invention. The corresponding antibody can be obtained by culturing the recombinant cells. These available mammalian cells can be, for example, 293F cells, CHO cells, etc.

[0098] The present invention also provides a hybridoma cell, which can be produced by a classical method for preparing monoclonal antibodies.

[0099] In the classic monoclonal antibody preparation method, first of all, a suitable antigen is required and animals are immunized with it. In order to prepare antibodies (or antibody analogs) against non-carboxylated matrix Gla protein, the suitable antigen must contain non-carboxylated matrix Gla protein. This antigen can be obtained by separation and purification from natural human tissue or blood, chemical synthesis, or a combination of these methods, and is used for animal immunization and antibody screening detection.

[0100] In the classic monoclonal antibody preparation method, animals are first immunized with non-carboxylated matrix Gla protein antigens, and blood is collected at intervals to verify whether the animals have produced antibody responses to the non-carboxylated matrix Gla protein antigens. B cells are then isolated from the spleen of animals with antibody responses and fused with immortal myeloma cells in vitro to obtain hybridoma cells. These hybridoma cells are then diluted to the limit in a culture plate and regrown (monoclonal hybridoma cell lines), and the culture supernatant of these hybridoma cell lines is taken to detect whether it contains specific antibodies against the antigen. Based on the antibody yield, quality and cell line growth characteristics, the best monoclonal antibody production cell line can be selected for subsequent monoclonal antibody production.

[0101] Other methods can also be used to obtain monoclonal antibodies. For example, spleen cells from the above animals can be isolated and then incubated with labeled antigens (such as fluorescein-labeled non-carboxylated matrix Gla protein). Since antibody-producing B cells usually have antibody molecules presented on the cell membrane surface, these cells will be bound (stained) by the labeled antigen and can be sorted by a fluorescent flow cytometer. The mRNA of these sorted B cells can be isolated, and a library of antibody variable region cDNA can be obtained by in vitro reverse transcription and specific PCR reactions. This cDNA library can be inserted into an expression plasmid (such as an antibody expression plasmid suitable for expression in mammalian cells, or a phage expression plasmid suitable for expression in bacterial cells) and expressed in a host cell suitable for this expression plasmid. These host cells (or phages) can be isolated and purified (cloned) by different methods (such as the cell limiting dilution culture method described above, or the phage plaque plating method). These cell lines or phage clones can be used to produce antibodies and analyze and identify the antibodies.

[0102] The antibodies or antigen-binding fragments, antibody analogs, polynucleotide sequences, expression vectors, recombinant cells, hybridoma cells provided by the present invention, their role in auxiliary diagnosis of related diseases, and the enzyme-immunoassay kit prepared based on the G071 antibody greatly improve the accuracy and convenience of diagnosis.

[0103] According to a preferred embodiment of the present invention, the animal immunization in the present invention adopts multiple subcutaneous injections at multiple points, which improves the titer of the final animal serum and increases the positive clone rate.

[0104] According to a preferred embodiment of the present invention, the addition of HAT screening reagent is postponed from the day of fusion to the second day, so that the cell positive rate is significantly improved.

[0105] According to a preferred embodiment of the present invention, the present invention selects a variety of non-carboxylated matrix Gla protein structural analogs to compare antibody specificity detection. After multiple rounds of screening, the G071 antibody is obtained as an antibody with good specificity for non-carboxylated matrix Gla protein.

[0106] The non-carboxylated matrix Gla protein antibody of the present invention can be used to detect non-carboxylated matrix Gla protein in human serum or body fluids, or substances containing non-carboxylated matrix Gla protein, and substances that can specifically bind to non-carboxylated matrix Gla protein (such as non-carboxylated matrix Gla protein antibody). The principle of detection is mainly based on the specific recognition and binding of the non-carboxylated matrix Gla protein antibody of the present invention to non-carboxylated matrix Gla protein, and detection is performed using chemical labeling technology (such as labeled antibodies, or labeled specific secondary antibodies) or physical detection technology (such as light scattering technology, plasma resonance technology, etc.).

[0107] The present invention provides a monoclonal antibody G071 against non-carboxylated matrix Gla protein antibody, which has the characteristics of high affinity and specificity, and also provides an in vitro diagnostic test kit based on the above antibody. The present invention solves the problem of relative shortage of antibody raw materials and test kits for non-carboxylated matrix Gla protein detection projects in the current market, and can be used as an auxiliary diagnosis for related diseases, filling the gap in the domestic related market.

[0108] The nucleic acid encoding the heavy chain and / or light chain of the antibody of the present invention is within the scope of the present invention. According to the amino acid sequence of the heavy chain and / or light chain, those skilled in the art can easily obtain the corresponding nucleic acid sequence, as shown in Table 1. It should be noted that the CDR sequences listed in Table 1 below are obtained based on the IMGT database. Those skilled in the art should know that the CDR sequences analyzed from different databases may be different, but these changes should be included in the scope of protection of the present invention.

[0109] Table 1 Sequence information

[0110]

[0111]

[0112]

[0113] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0114] Example 1: Synthesis of non-carboxylated matrix Gla protein peptides, carboxylated matrix Gla protein peptides and matrix Gla protein full-length antigens

[0115] The full-length amino acid sequence of the non-carboxylated matrix Gla protein is shown below:

[0116] MKSLILLAILAALAVVTLCYESHESMESYELNPFINRRNANTFISPQQRWRAKVQERIRERSKPVHELNR EACDDYRLCERYAMVYGYNAAYNRYFRKRRGTK(SEQ ID NO:11)

[0117] Part of the sequence was used to synthesize carboxylated matrix Gla protein and non-carboxylated matrix Gla protein peptides using conventional methods in the art. The specific peptide sequences are as follows:

[0118]

[0119] Note: "(c)" in the table indicates that the preceding amino acid has carboxyl modification. "BSA" indicates that the C segment of carboxylated matrix Gla protein or non-carboxylated matrix Gla protein peptide is linked to bovine serum albumin, synthesized by Shanghai Botai Biotechnology Co., Ltd.; "KLH" indicates that the C segment of non-carboxylated matrix Gla protein is linked to hemocyanin, synthesized by Shanghai Synthetic Botai Biotechnology Co., Ltd.

[0120] Example 2: Identification of non-carboxylated matrix Gla protein

[0121] First, the non-carboxylated matrix Gla protein MGP-BSA was coated in an ELISA plate at 1 μg / ml, and the purified non-carboxylated matrix Gla protein antibody was used as the primary antibody of the indirect ELISA method, and the HRP-labeled goat anti-rat antibody was used as the secondary antibody. TMB was used as the color developing solution, and then 1M H2SO4 was used to terminate the reaction. The OD value of the microplate reader was 450 By comparing the data with the antigenic activity of the non-carboxylated matrix Gla protein, it can be seen that the antigenic activity of the non-carboxylated matrix Gla protein is detected to be higher.

[0122]

[0123] Example 3: Immunization of BALB / c mice with non-carboxylated matrix Gla protein

[0124] BALB / c mice aged 6 to 8 weeks were selected for the following immunization procedure: for the first immunization, 25 μg of non-carboxylated matrix Gla protein MGP-KLH was mixed with an equal amount of Freund's complete adjuvant and injected subcutaneously at multiple points after emulsification; 14 days after the first immunization, 12.5 μg of non-carboxylated matrix Gla protein MGP-KLH was mixed with Freund's incomplete adjuvant and emulsified for booster immunization. 14 days after the second immunization, 12.5 μg of non-carboxylated matrix Gla protein MGP-KLH was mixed with Freund's incomplete adjuvant and emulsified for booster immunization. 14 days after the third immunization, blood was collected and serum was separated. ELISA plates were coated with 1 μg / mL non-carboxylated matrix Gla protein MGP-BSA for indirect ELISA test to determine the serum titer. The results showed that the titer of the prepared mouse antiserum was 1:72900, and the immunization was successful. The typical immunized mouse serum test results are shown in Table 2.

[0125] Table 2 Typical results of serum testing in immune mice

[0126] Serum dilution multiple <![CDATA[OD 450 ]]> 100 2.482 300 2.759 900 2.324 2700 1.486 8100 0.969 24300 0.598 72900 0.239 Background 0.048

[0127] Example 4: Cell fusion

[0128] In Example 3, cell fusion was performed 3 days after the booster immunization of mice. After removing the eyeballs of the mice to collect blood, the mice were killed by dislocation, placed in a 70% alcohol bottle for 2 minutes, and then fixed on a foam board in a biosafety cabinet. The abdominal skin was untied to find the spleen, which was removed with forceps and gently ground into a 200-mesh stainless steel filter. The cells were gently rinsed with DMEM culture medium (Thermo, 11965092), and then centrifuged at 200g for 10 minutes at room temperature. The supernatant was discarded and set aside. When preparing feeder cells, the mice were killed by dislocation, placed in a 70% alcohol bottle for 2 minutes, and then fixed on a foam board in a biosafety cabinet. The abdominal skin was untied, and PBS was sucked up with a syringe and gently injected into the peritoneum. The liquid containing the feeder cells was washed out from the other side, and then centrifuged at 200g for 10 minutes at room temperature. The supernatant was discarded and set aside. 2.0×10 7 FO myeloma cells and 2.0×10 8The spleen cells were mixed, centrifuged at 200g for 10 minutes, the supernatant was discarded, and the mixture was gently shaken to mix. In a 37°C water bath, 1 ml of a 50% PEG-1450 (Merk, P1458) aqueous solution was added dropwise within 90 seconds, followed by 20 mL of DMEM culture medium. The mixture was centrifuged at 200g for 10 minutes, the supernatant was discarded, and the washing was repeated. The mixture was centrifuged at 200g for 10 minutes, the supernatant was discarded, and the hybridoma cells were obtained. The cells were plated into 10 96-well culture plates with 150 μL per well. 10,000 cells / well of feeder layer cells were added to the above 10 96-well cell culture plates, 100 μL per well, and the culture plates were marked and placed in a cell culture incubator at 37°C containing 5% CO2 for culture. On the second day, HAT screening medium (Merk, H0262) was added, and then HAT selection culture was used for 1-2 days. A large number of tumor cells died, and after 3-4 days, the tumor cells disappeared, and hybrid cells formed small colonies. After 7-10 days of HAT screening culture medium, HT culture medium (Merk, H0137) should be used, and then maintained for another 2 weeks, and then 20% FBS (ExCell, FSP500) DMEM culture medium was used for continued culture. During the above selection culture period, when the hybridoma cells covered 1 / 10 of the bottom area of ​​the well, specific antibodies could be detected to screen out the desired hybridoma cell lines. During the selection culture period, half of the culture medium was generally replaced every 2-3 days.

[0129] Example 5: Screening and subcloning of positive hybridoma cell lines

[0130] First, the optimal coating amount of non-carboxylated matrix Gla protein as antigen was determined by orthogonal experiment. 0.5, 1.0, 2.0, 4.0 μg of non-carboxylated matrix Gla protein MGP-BSA was coated on 96-well plate, and 6 wells of each concentration were set as 3 positive and 3 negative. Founder titration was performed with positive serum of mice immunized with non-carboxylated matrix Gla protein MGP-KLH at different dilution multiples, and negative serum of non-immunized mice was used as negative control. 96-well ELISA plate was coated with 0.5 μg purified non-carboxylated matrix Gla protein MGP-BSA per well, incubated at 4℃ overnight; washed twice with PBST; 200 μL of 1% BSA in PBS was added to each well, blocked at room temperature for 2 hours, and then placed on trifold paper and patted dry; sample addition: 0.1 mL of the sample to be tested was added to the reaction well, incubated at 37℃ for 1 hour, then washed, and blank wells (no sample added), negative control wells and positive control wells were made at the same time. Add 0.1 mL of newly diluted antibody to each reaction well, incubate at 37°C for 1 hour, and then wash 3 times. Add enzyme-labeled secondary antibody: Add 0.1 mL of newly diluted enzyme-labeled antibody to each reaction well. Incubate at 37°C for 1 hour, and wash 3 times. Add substrate solution for color development: Add 0.1 mL of TMB substrate solution to each reaction well and let stand at room temperature for 10 minutes. Add 0.1 mL of 1M H2SO4 to each reaction well. Measure the OD value to determine the result, and use an enzyme reader to measure the absorbance value (A450) at 450 nm. A value that is 2.1 times or more of the negative control OD value is considered positive (calculated after adjusting the blank control well to zero). Select hybridoma cell monoclones that are resistant to non-carboxylated matrix Gla protein.

[0131] According to the above method, the positive hybridoma cells obtained by screening are subcloned, and the original wells are diluted with HAT selection medium by limiting dilution method and then redistributed into 96-well culture plates, and then the morphology and number of cells are observed. The cells are adjusted to 3-10 cells / mL. Take the cell culture plate with the feeder cell layer prepared the day before, and add 100 μL of diluted cells to each well. Culture statically in a 37°C, 5% CO2 incubator. Change the medium on the 7th day, and change the medium once every 2-3 days thereafter. Cell clones can be seen on the 8th to 9th day, and the antibody activity is detected in time. The cells in the positive wells are transferred to a 24-well plate for expansion and culture. Each clone should be frozen as soon as possible, and the hybridoma cell line with clone number G071 is finally selected for antibody production.

[0132] Example 6: Preparation of monoclonal antibodies in large quantities and determination of antibody titer

[0133] (1) Large-scale preparation of monoclonal antibodies

[0134] 8-week-old BALB / C mice were intraperitoneally injected with 0.5 mL of Freund's incomplete adjuvant. Two weeks later, 1 × 10 6Hybridoma G071 cells can produce ascites 7 to 10 days after inoculation. Closely observe the health status of the animals and the signs of ascites. When the ascites is as much as possible and the mice are about to die, kill the mice and use a dropper to suck the ascites into a test tube. One mouse can obtain 5 to 10 ml of ascites. The ascites can also be extracted with a syringe and can be collected repeatedly several times. The ascites obtained is centrifuged at 3000g for 10 minutes, the upper layer of fat and the bottom sediment are discarded, the supernatant is collected and distributed at -20℃, the ascites supernatant is thawed and equilibrated to room temperature, and 1 / 10 volume of 1M Tris-HCl PH8.0 is added to adjust the sample pH value to 8.0. The protein G affinity column was balanced with 20 column volumes of 100mM Tris-HCl at pH 8.0, and the ascites supernatant after adjusting the pH to 8.0 was loaded onto the column, and then washed with 20 column volumes of 100mM Tris-HCl at pH 8.0, and finally eluted with 100mM Glycine-HCL pH 2.5 (i.e., non-carboxylated matrix Gla protein antibody G071, which can be referred to as antibody G071 or G071). The antibody eluate was added to a concentrator tube (Millipore, UFC801008, 10K) and centrifuged at room temperature at 3000×g for 20 minutes in a centrifuge (Xiangyi, L550). The solution was centrifuged in batches until the volume of the solution reached 1ml / concentrator tube (2 tubes), 4mL of 10mM PBS (PH 7.4) buffer was added, and centrifugation at room temperature at 3000×g was continued for 20 minutes. Repeat centrifugation 3 times to make the antibody buffer 10mM PBS (PH 7.4), add 10mM PBS (PH 7.4) to a total volume of 10mL. Finally, 2mL / tube of concentrated antibody solution was dispensed into centrifuge tubes and stored at -80°C. The antibody concentration was determined using a BCA kit (Solebo, PC0020), and the concentration of the purified monoclonal antibody was 4.5mg / mL.

[0135] (2) Antibody titer determination

[0136] The titer of the non-carboxylated matrix Gla protein antibody G071 was detected by the indirect ELISA method. The non-carboxylated matrix Gla protein MGP-BSA was diluted with PBS and coated in a 96-well ELISA plate at 0.2μg / mL and 100μL / well. After overnight at 4℃, the ELISA plate was washed twice with PBST solution, 300μL / well each time, and patted dry after washing. The ELISA plate was blocked with 1% BSA in PBS solution, 200μL / well, at room temperature for 2 hours, and patted dry after blocking; the non-carboxylated matrix Gla protein antibody G071 diluted to 20ng / mL with PTB was added, and reacted in a 37℃ incubator for 1 hour. The ELISA plate was washed twice with PBST solution, 300μL / well each time. Patted dry after washing. HRP-labeled goat anti-mouse antibody diluted 5000 times with PTB was added, and reacted in a 37℃ incubator for 1 hour. The ELISA plate was washed twice with PBST solution, 300μL / well each time. After washing, pat dry. Add TMB substrate solution, 100 μL / well, react at room temperature for 10 minutes, add 100 μL / well 1M H2SO4 to terminate the reaction. Use an ELISA reader to measure the absorbance value (A450) at 450nm. The results are shown in Table 3, and it can be seen that the titer of the purified antibody is 1:729000.

[0137] Table 3 Antibody titer

[0138]

[0139]

[0140] Example 7: Sequence analysis of monoclonal antibodies

[0141] (1) Monoclonal antibody subtype identification

[0142] The hybridoma cell line G071 was cultured in a 10 cm diameter cell culture dish (37°C, 5% CO2) using DMEM medium (GIBCO, #C11995500BT) supplemented with 10% serum. After 7 days of culture, the cells were transferred to a 15 ml centrifuge tube and 4 × 10 6 After centrifugation at 200g for 5 minutes, the supernatant was discarded, and the centrifuge tube was inverted to drain the liquid in the tube. The cells in the tube were synthesized into cDNA using the reverse transcription kit (Qiagen, 74134) of QIAGEN.

[0143] The antibody subtype is determined by PCR using antibody subtype-specific primers. The above-synthesized cDNA is used as a PCR reaction template. PCR reaction solution system: TAKARA Ex Taq (5U / μL, TAKARA, RR001B), 0.25μL; 10×Ex Taq Buffer, 5μL; dNTP mixture (2.5mM each), 4μL; template cDNA, 1μL; upstream primer (100μM), 1μL; downstream primer (100μM), 1μL; add double distilled water to a total volume of 50μL. PCR reaction temperature program: 94°C for 5 minutes for pre-denaturation, temperature cycle 30 times (94°C for 1 minute, 57°C for 1 minute, 72°C for 1 minute), and 72°C for 10 minutes for extension. After the reaction, 10μL of each PCR product was loaded on a 1% agarose gel for electrophoresis. The electrophoresis pattern is shown as follows. Figure 1 The order of samples added to each lane is shown in Table 5. The subtype of the antibody can be inferred from the PCR product results (Table 4). The monoclonal antibody G071 obtained in the present invention has a heavy chain of IgG1 and a light chain of kappa.

[0144] Table 4 PCR primer information

[0145]

[0146] Where S=C or G, M=A or C, R=A or G, and W=A or T

[0147] Table 5 Antibody information

[0148] Lanes PCR primers Antibody subtype specificity 1 DNA molecular weight standard (full gold, DNA MarkerII, #BM411) N / A 2 PCR products of primers F-VH and R-VH 1 IgG1 heavy chain 3 PCR products of primers F-VH and R-VH 2A IgG2A heavy chain 4 PCR products of primers F-VH and R-VH 2B IgG2B heavy chain 5 PCR products of primers F-VH and R-VH 3 IgG3 heavy chain 6 Blank N / A 7 PCR products of primers F-VH and R-VK IgKappa light chain 8 PCR products of primers F-VL and R-VL1 IgLambda1 light chain 9 PCR products of primers F-VL and R-VL2 IgLambda2 light chain

[0149] (2) Sequencing of the variable region (V region) of the hybridoma cell line G071

[0150] The fragments obtained after PCR amplification of the V region of the antibody of cell line G071 (see above) were cut from agarose gel and extracted using a DNA extraction kit (Qiagen, 74134). The extracted DNA fragments were linked to the pEASY-T1 cloning vector and transformed into Trans1-T1 competent cells (Transgen, CT101-1). The transformed bacterial colonies were picked into LB culture medium and DNA sequencing was performed after overnight culture. The light chain V region nucleic acid sequence of the non-carboxylated matrix Gla protein antibody (G071) provided by the present invention is shown in SEQ ID NO.9, and the nucleic acid sequence of the heavy chain V region is shown in SEQ ID NO.10.

[0151] Example 8: Identification of the specificity of antibody G071 in detecting non-carboxylated matrix Gla protein

[0152] The ability of antibody G071 to specifically detect free non-carboxylated matrix Gla protein was evaluated by ELISA. In this experiment, non-carboxylated matrix Gla protein peptide fragment MGP-BSA and carboxylated matrix Gla protein peptide fragment cMGP-BSA were used as antigens and diluted in PBS, respectively, and coated in 96-well ELISA plates at a coating concentration of 1μg / mL and 0.5μg / ml, with a volume of 100μL / well. After overnight coating at 4°C, the ELISA plate was washed twice with PBST solution, 300μL / well each time. After washing, pat dry, the ELISA plate was blocked with PBS solution containing 1% BSA, 200μL / well, at room temperature for 2 hours. Pat dry, antibody G071 (diluted to 10ng / mL with PTB) was added to the blocked ELISA plate, 100μL / well, and reacted in a 37°C incubator for 1 hour. Pat dry, and wash the plate 3 times with PBST, 300μL / well each time. Pat dry, add HRP-labeled goat anti-mouse antibody diluted 5000 times with PTB to the ELISA plate, and react in a 37°C incubator for 1 hour. Wash the plate 3 times with PBST, 300 μL / well. Pat dry, add TMB substrate solution to the ELISA plate, 100 μL / well, at room temperature for 10 minutes, then add 100 μL / well of 1M H2SO4 to terminate the reaction. Use an ELISA reader to measure the absorbance (A450) at 450nm. The experimental results are shown in Table 6, indicating that antibody G071 has good specificity for detecting non-carboxylated matrix Gla protein.

[0153] Table 6 Absorbance values

[0154]

[0155] Example 9: Application of the ELISA kit prepared using antibody G071 in detecting non-carboxylated matrix Gla protein in human serum

[0156] (1) Horseradish peroxidase (HRP) labeling of antibody G071 and identification of the labeled product

[0157] 0.5 mL of antibody G071 (20 nmol, 3 mg) was added to a dialysis bag (10 KDa, 1 cm width) and dialyzed in 2 L of 10 mM PBS solution (PH 7.4) at 4 ° C overnight. The next day, the dialysis bag containing the antibody solution was placed in 1 L of 10 mM carbonate buffer (PH 9.5) and dialyzed for 2 hours at room temperature to prepare for coupling with activated HRP.

[0158] At the same time, use an analytical balance to accurately weigh 1 mg of HRP, add 0.2 mL of ultrapure water to dissolve, and make the HRP concentration 5 mg / mL. Add 40 μl of 0.1M NaIO4 to the above HRP solution, place it on a horizontal shaker, and react (activate) at room temperature in the dark for 20 minutes. Add the activated HRP solution to a dialysis bag (10KDa, width 1 cm) and dialyze it in 2L 1mM sodium acetate buffer (PH4.4) at 4°C overnight. Carefully aspirate the dialyzed HRP solution and transfer it to a new 1.5 mL centrifuge tube, add 1 / 10 volume of 0.2M carbonate buffer, and raise the pH of the activated HRP solution to 9.0-9.5.

[0159] Mix the above antibodies and HRP (for coupling reaction), place on a horizontal shaker, and react at room temperature in the dark for 4 hours. After the coupling reaction is completed, add 10μl (pre-cooled ultrapure water) freshly prepared NaBH4, and terminate the reaction at 4℃ in the dark overnight. Move the antibody solution after the coupling reaction is terminated to a dialysis bag (10KDa, width 1cm), stir and dialyze at room temperature for 2 hours. Finally, transfer the solution to a brown centrifuge tube and store at 4℃.

[0160] (2) ELISA kit prepared using antibody G071 for the detection of non-carboxylated matrix Gla protein in human serum

[0161] In this experiment, matrix Gla protein antibody (Abcam, ab273657) was diluted to 1 μg / mL with PBS, and then 100 μL / well was coated in a 96-well ELISA plate (overnight at 4°C). The plate was washed twice with 300 μL / well PBST. Pat dry, block the ELISA plate with PBS solution containing 1% BSA, 200 μL / well, at room temperature for 2 hours. Pat dry, add 1000-fold diluted HRP-labeled antibody G071 (diluted with PTB) solution 25 μl / well to the ELISA plate, and then add different concentrations of human serum samples (serum of patients with clinically confirmed bone-related diseases was selected as the sample to be tested, and serum of healthy people was used as the negative sample), 75 μL / well, and reacted in a 37°C incubator for 45 minutes. Wash the plate 3 times with PBST, 300 μL / well each time. Pat dry, add TMB substrate solution, 100 μL / well, and react at room temperature for 10 minutes. Then, 100 μL / well of 1M H2SO4 was added to terminate the reaction, and the absorbance (A450) was measured at 450 nm using an ELISA reader. The results are shown in Table 7, indicating that the ELISA kit prepared using the antibody G071 of the present invention can efficiently detect non-carboxylated matrix Gla protein in human serum.

[0162] Table 7

[0163] Patient serum samples <![CDATA[OD 450 ]]> <![CDATA[OD 450(空白) ]]> Patient 1 0.764 0.023 Patient 2 0.963 0.056 Patient 3 0.765 0.073 Patient 4 0.579 0.054 Patient 5 0.769 0.085

[0164] The present invention provides a monoclonal antibody of non-carboxylated matrix Gla protein, which has the characteristics of high affinity and specificity, and also provides an in vitro diagnostic test kit based on the antibody. The present invention solves the problem of relative shortage of antibody raw materials and test kits for non-carboxylated matrix Gla protein detection projects in the current market.

[0165] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0166] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A non-carboxylated matrix Gla protein antibody or antigen-binding fragment, characterized in that include: The light chain variable region CDR1 sequence shown in SEQ ID NO:3, the light chain variable region CDR2 sequence shown in SEQ ID NO:4, the light chain variable region CDR3 sequence shown in SEQ ID NO:5, the heavy chain variable region CDR1 sequence shown in SEQ ID NO:6, the heavy chain variable region CDR2 sequence shown in SEQ ID NO:7, and the heavy chain variable region CDR3 sequence shown in SEQ ID NO:

8.

2. The antibody or antigen-binding fragment according to claim 1, characterized in that include: (a) has a light chain variable region shown in SEQ ID NO: 1 and a heavy chain variable region shown in SEQ ID NO:

2.

3. The antibody or antigen-binding fragment according to claim 1, characterized in that include: (b) having the CDR1, CDR2 and CDR3 sequences of the light chain variable region shown in SEQ ID NO:1, and having the CDR1, CDR2 and CDR3 sequences of the heavy chain variable region shown in SEQ ID NO:

2.

4. The antibody or antigen-binding fragment according to any one of claims 1 to 3, characterized in that It further comprises at least one of a heavy chain constant region and a light chain constant region, wherein at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from a mammalian-derived antibody.

5. The antibody or antigen-binding fragment according to claim 4, characterized in that: The heavy chain constant region and the light chain constant region are derived from at least one of a mouse antibody, a rabbit antibody, and a primate antibody.

6. The antibody or antigen-binding fragment according to claim 4, characterized in that The heavy chain constant region and the light chain constant region are derived from a murine antibody.

7. The antibody or antigen-binding fragment according to claim 4, characterized in that The N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region; and / or The N-terminus of the light chain constant region is connected to the C-terminus of the light chain variable region.

8. The antibody or antigen-binding fragment according to claim 4, characterized in that The heavy chain constant region comprises a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; or The light chain constant region comprises a light chain constant region selected from a kappa-type or a lambda-type.

9. The antibody or antigen-binding fragment according to claim 4, characterized in that The heavy chain constant region comprises: an amino acid sequence as shown in SEQ ID NO: 22; The light chain constant region includes: the amino acid sequence shown in SEQ ID NO:

23.

10. The antibody or antigen-binding fragment according to claim 1, characterized in that The antibody or antigen-binding fragment includes a monoclonal antibody or a polyclonal antibody.

11. The antibody or antigen-binding fragment according to claim 10, characterized in that: The monoclonal antibody comprises at least one of a full-length antibody, Fv, a single-chain antibody, and a Fab; The antibody or antigen-binding fragment comprises: The heavy chain of the amino acid sequence shown in SEQ ID NO: 24; and / or The light chain has the amino acid sequence shown in SEQ ID NO:

25.

12. A conjugate, characterized in that: Include: The non-carboxylated matrix Gla protein antibody or antigen-binding fragment according to any one of claims 1 to 11; and A coupling portion is connected to the non-carboxylated matrix Gla protein antibody or antigen binding fragment.

13. The conjugate according to claim 12, characterized in that The coupling part includes at least one of a carrier, a drug, a toxin, a cytokine, a protein tag, a modifier and the like.

14. The conjugate according to claim 12, characterized in that The conjugated moiety includes a chemotherapeutic agent.

15. The conjugate according to claim 13, characterized in that The carrier includes a fluorescent dye.

16. The conjugate according to claim 15, characterized in that The fluorescent dye includes any one or more of FITC, PE, Cy5, PI, 7-AAD, APC, AlexaFluor, eFluor, PE-Cy7, APC-Cy7, PerCP, PerCPcy5.5, PB, BV series, QDot series and BUV series dyes.

17. An isolated polynucleotide, characterized in that The polynucleotide encodes the non-carboxylated matrix Gla protein antibody or antigen-binding fragment according to any one of claims 1 to 11.

18. The isolated polynucleotide according to claim 17, characterized in that The polynucleotide comprises: (c) having a nucleic acid sequence encoding a light chain variable region as shown in SEQ ID NO:9; or (d) having a nucleic acid sequence encoding a heavy chain variable region as shown in SEQ ID NO:

10.

19. An expression vector, characterized in that: Comprising the isolated polynucleotide of claim 17 or 18.

20. A recombinant cell, characterized in that Comprising the expression vector of claim 19.

21. A method for preparing a non-carboxylated matrix Gla protein antibody or antigen fragment, characterized in that: Comprising culturing the recombinant cell of claim 20.

22. Use of the antibody or antigen-binding fragment according to any one of claims 1 to 11 or the conjugate according to any one of claims 12 to 16 in preparing a kit, characterized in that: The kit is used for detecting non-carboxylated matrix Gla protein in a sample.

23. A kit, characterized in that: The invention comprises at least one of the non-carboxylated matrix Gla protein antibody or antigen-binding fragment according to any one of claims 1 to 11, the conjugate according to any one of claims 12 to 16, the expression vector according to claim 19, and the recombinant cell according to claim 20.

24. A composition, characterized in that The invention comprises at least one of the non-carboxylated matrix Gla protein antibody or antigen-binding fragment according to any one of claims 1 to 11, the conjugate according to any one of claims 12 to 16, the isolated polynucleotide according to claim 17 or 18, the expression vector according to claim 19, and the recombinant cell according to claim 20.

25. A method for preparing the non-carboxylated matrix Gla protein antibody or antigen-binding fragment according to any one of claims 1 to 11, characterized in that: Comprising culturing the recombinant cell of claim 20.

26. A method for detecting non-carboxylated matrix Gla protein, characterized in that: include: contacting the non-carboxylated matrix Gla protein antibody or antigen-binding fragment according to any one of claims 1 to 11, the conjugate according to any one of claims 12 to 16, or the kit according to claim 23 with a sample to be detected to form an immune complex; Based on the signal of the immune complex, the non-carboxylated matrix Gla protein in the sample to be detected is quantitatively and / or qualitatively analyzed.

27. The method according to claim 26, characterized in that The signal of the immune complex is detected by at least one of ELISA, Western blot and FACS.

28. The method according to claim 26, characterized in that The signal comprises a fluorescent signal.

29. A drug, characterized in that The invention comprises at least one of the non-carboxylated matrix Gla protein antibody or antigen-binding fragment according to any one of claims 1 to 11, the conjugate according to any one of claims 12 to 16, the isolated polynucleotide according to claim 17 or 18, the expression vector according to claim 19, the recombinant cell according to claim 20 and the composition according to claim 24.

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