Antibody for recognizing VASP and application thereof

Abstract

The invention relates to the field of biological medicine, in particular to a VASP antibody composition and application thereof. The invention provides an antibody capable of specifically binding to human VASP and a composition thereof, and the antibody composition has high specificity and higher affinity to human VASP, and can realize specific and sensitive detection of human VASP. The human VASP detection kit developed on the basis of the antibody composition has relatively high specificity, sensitivity and accuracy, relatively wide linear range and relatively high stability, can meet detection requirements, can be used for human VASP detection in practice, and has a relatively good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and particularly to an antibody that recognizes VASP and its applications. Background Art

[0002] VASP (Vasodilator-Stimulated Phosphoprotein) is a protein that plays an important role in cell signal transduction. VASP is a small-molecular-weight protein that mainly functions in the dynamic reorganization and signal transduction of the cytoskeleton. It participates in a variety of cell processes, including cell migration, cell division, cell morphological changes, and vasodilation. The VASP protein contains multiple domains, including an N-terminal EVH1 domain, a middle Pro-rich domain, and a C-terminal Enigma domain. These domains enable VASP to interact with a variety of proteins.

[0003] VASP participates in multiple signal transduction pathways, including the Rho family GTPase signal pathway. It can regulate the reorganization of the cytoskeleton by interacting with these GTPases. And it participates in multiple signal transduction pathways, including the Rho family GTPase signal pathway. It can regulate the reorganization of the cytoskeleton by interacting with these GTPases. The abnormal function of VASP is related to a variety of diseases, including certain types of cancer, cardiovascular diseases, and neurodegenerative diseases. For example, changes in the expression level and activity of VASP may be related to the invasiveness and metastasis of tumors.

[0004] In summary, although the direct connection between VASP and some diseases still requires further research to clarify, existing research has shown that it plays a role in the pathogenesis of thrombotic diseases and may be a potential biomarker for the diagnosis of thrombotic diseases. Future research may reveal the specific mechanism of action of VASP in thrombotic diseases and how it, together with other biomarkers, can help in the diagnosis and treatment of depression.

[0005] Immunoassay methods based on the specific reaction between antigens and antibodies are low-cost, fast, highly accurate, and highly sensitive, and their core is specific antibodies. Currently, there are no diagnostic reagent raw materials with high specificity for VASP in the industry.

[0006] Currently, there is an urgent need to develop an anti-VASP antibody with high specificity and high sensitivity. Summary of the Invention

[0007] The present invention aims to solve at least one of the technical problems in the related art to some extent.

[0008] On the one hand, the present invention provides an anti-VASP antibody or antigen-binding fragment, comprising: A CDR sequence selected from at least one of the following or an amino acid sequence having at least 80% identity thereto: CDR sequences of the light chain variable region: SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5; CDR sequences of the heavy chain variable region: SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, Or CDR sequences of the light chain variable region: SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15; CDR sequences of the heavy chain variable region: SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18; The present invention provides an anti-VASP antibody or antigen-binding fragment. An enzyme immunoassay kit prepared using the antibody or antigen-binding fragment has high sensitivity and specificity.

[0009] According to some embodiments of the present invention, the antibody or antigen-binding fragment comprises: the CDR1 sequence of the light chain variable region shown in SEQ ID NO:3, the CDR2 sequence of the light chain variable region shown in SEQ ID NO:4, the CDR3 sequence of the light chain variable region shown in SEQ ID NO:5, the CDR1 sequence of the heavy chain variable region shown in SEQ ID NO:6, the CDR2 sequence of the heavy chain variable region shown in SEQ ID NO:7, the CDR3 sequence of the heavy chain variable region shown in SEQ ID NO:8, or the CDR1 sequence of the light chain variable region shown in SEQ ID NO:13, the CDR2 sequence of the light chain variable region shown in SEQ ID NO:14, the CDR3 sequence of the light chain variable region shown in SEQ ID NO:15, the CDR1 sequence of the heavy chain variable region shown in SEQ ID NO:16, the CDR2 sequence of the heavy chain variable region shown in SEQ ID NO:17, and the CDR3 sequence of the heavy chain variable region shown in SEQ ID NO:18.

[0010] According to some embodiments of the present invention, the antibody or antigen-binding fragment comprises at least one of the following: (a) having the light chain variable region shown in SEQ ID NO:1 and the heavy chain variable region shown in SEQ ID NO:2; (b) having the light chain variable region shown in SEQ ID NO:11 and the heavy chain variable region shown in SEQ ID NO:12; An amino acid sequence having at least 80% sequence identity compared to (a) or (b).

[0011] (c) the CDR1, CDR2 and CDR3 sequences of the light chain variable region shown in SEQ ID NO:1, and the CDR1, CDR2 and CDR3 sequences of the heavy chain variable region shown in SEQ ID NO:2; (d) the CDR1, CDR2 and CDR3 sequences of the light chain variable region shown in SEQ ID NO:11, and the CDR1, CDR2 and CDR3 sequences of the heavy chain variable region shown in SEQ ID NO:12; or an amino acid sequence having more than one conservative amino acid substitution compared to (c) or (d).

[0012] 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, and at least a part of at least one of the heavy chain constant region and the light chain constant region is derived from a mammalian antibody.

[0013] 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 a mutant thereof.

[0014] 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.

[0015] According to some embodiments of the present invention, the antibody or antigen-binding fragment comprises a monoclonal antibody or a polyclonal antibody; According to some embodiments of the present invention, the monoclonal antibody comprises at least one of a full-length antibody, Fv, single-chain antibody, Fab, single-domain antibody and minimum recognition unit.

[0016] Another aspect of the present invention provides an antibody analogue, which comprises the variable region or the CDR region in the variable region of the above-mentioned antibody or antigen-binding fragment.

[0017] Another aspect of the present invention provides an isolated polynucleotide, which encodes the above-mentioned antibody or antigen-binding fragment.

[0018] According to some embodiments of the present invention, the polynucleotide comprises: (e) a nucleic acid sequence encoding the light chain variable region shown in SEQ ID NO:9, a nucleic acid sequence having at least 80% sequence identity compared to (e); or (f) a nucleic acid sequence encoding the heavy chain variable region shown in SEQ ID NO:10, a nucleic acid sequence having at least 80% sequence identity compared to (f); or (g) A nucleic acid sequence encoding a light chain variable region as shown in SEQ ID NO: 19, A nucleic acid sequence having at least 80% sequence identity compared to (g); or (h) A nucleic acid sequence encoding a heavy chain variable region as shown in SEQ ID NO: 20, A nucleic acid sequence having at least 80% sequence identity compared to (h).

[0019] Another aspect of the present invention provides an expression vector comprising the above polynucleotide.

[0020] Another aspect of the present invention provides a hybridoma cell that can produce the above anti-VASP antibody or antigen-binding fragment.

[0021] Another aspect of the present invention provides a method for preparing an anti-VASP antibody or antigen fragment, comprising culturing the above recombinant cell or the above hybridoma cell.

[0022] Another aspect of the present invention provides the use of the above antibody or antigen-binding fragment and the above antibody analog in the preparation of a kit for detecting VASP.

[0023] Another aspect of the present invention provides a kit comprising at least one of the above antibody or antigen-binding fragment, antibody analog, vector, recombinant cell, hybridoma cell.

[0024] Another aspect of the present invention provides a composition comprising the above antibody or antigen-binding fragment, antibody analog, polynucleotide, vector, recombinant cell, hybridoma cell.

[0025] Another aspect of the present invention provides a method for preparing the above antibody or antigen-binding fragment, comprising culturing the above recombinant cell, hybridoma cell.

[0026] According to some embodiments of the present invention, the VASP-mediated related diseases include platelet dysfunction, cardiovascular diseases, etc.

[0027] The present invention provides a monoclonal antibody against VASP, which has the characteristics of high affinity and specificity, and also provides an in vitro diagnostic detection kit based on the above antibody. The present invention solves the problem of relative shortage of antibody raw materials and kits for VASP detection items in the current market.

[0028] Additional aspects and advantages of the present disclosure will be given in part in the following description, become apparent in part from the following description, or be learned through the practice of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1It is a plasmid map of pCDNA3.1-VASP expressing VASP recombinant protein.

[0030] Figure 2 It is to identify the expression of the target protein VASP by SDS-PAGE.

[0031] Figure 3 It is the standard curve of the enzyme immunoassay kit prepared using antibodies HC8 and HC10. Specific implementation manners

[0032] 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 a limitation to the present disclosure.

[0033] In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly indicating the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present disclosure, the meaning of "a plurality" is at least two, such as two, three, etc., unless otherwise specifically defined.

[0034] To make it easier to understand the present invention, 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 of ordinary skill in the art to which the present invention pertains.

[0035] In this article, the term "comprising" or "including" is an open expression, that is, it includes the content specified by the present invention, but does not exclude other aspects of the content.

[0036] In this article, the terms "optionally", "optional" or "option" generally mean that the subsequent event or condition may or may not occur, and this description includes the situation where the event or condition occurs and the situation where the event or condition does not occur.

[0037] In the present invention, the term "antibody" refers to a protein encoded by the antibody gene of an animal and biosynthesized.

[0038] In the present invention, the term "antibody analog" refers to a derivative obtained by biological or chemical methods, based on the antibody structure, through deletion, addition, or modification of chemical groups (such as amino acids). These derivatives still contain a structure similar to the variable region of the antibody (or the CDR region in the variable region of the antibody) and can undergo a reaction similar to antigen-antibody binding through these structures.

[0039] In the present invention, the term "antibody variable region" refers to a domain in the heavy and light chains of an antibody. In nature, the antibody variable region is encoded by the recombination of V, D (only applicable to heavy chains), and J segments in immunoglobulin (heavy and light chain) genes. The amino acid sequences of the variable regions among different antibodies are highly diverse (the amino acid sequences of other regions of the antibody are relatively highly identical), and are responsible for the recognition and binding to specific antigenic determinants. In the antibody variable region, it can be further subdivided into a framework region and a CDR region (complementarity determining region). 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 forming the framework of the protein domain, while the CDR region mainly plays a role in the specific recognition and binding of antigen-antibody.

[0040] In the present invention, the term "anti-VASP antibody (or antibody analog)" exists in a single (or monoclonal) form, and the single antibody (or antibody analog) contains a single structure (such as a single amino acid sequence).

[0041] In the present invention, as used herein, the term "identity", when describing an amino acid sequence or a nucleic acid sequence relative to a reference sequence, refers to the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences determined by conventional methods. See, for example, 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 Foundation, Washington, D.C.)). 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. 70:173-187 (1997)); and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs utilizing 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, 8th ed., Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0042] In the present invention, the term "antigen-binding fragment", also known as "antibody fragment", generally refers to an antigen-binding antibody fragment, which may include a part of a complete antibody, typically the antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab’, F(ab’)2, Fv or scFv, diabodies, linear antibodies, single-chain antibody molecules, etc.

[0043] Without substantially affecting the antibody activity (retaining at least 95% of the activity), those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids to obtain variants of the sequence of the antibody or its functional fragment. They are all considered to be included within the scope of protection of the present invention. For example, amino acids with similar properties are substituted in the variable region. The variant sequences of the present invention may have at least 80% identity (or homology) with the reference sequence. The sequence identity described in the present invention can be measured using sequence analysis software. For example, using the computer program BLAST with default parameters, especially BLASTP or TBLASTN. The amino acid sequences described in the present invention are shown in the N-terminal to C-terminal manner.

[0044] Anti-VASP antibody or antigen-binding fragment The present invention provides an anti-VASP antibody or antigen-binding fragment selected from (1) the CDR1, CDR2, and CDR3 sequences of the light chain variable region shown in SEQ ID NO:1, and the CDR1, CDR2, and CDR3 sequences of the light chain variable region shown in SEQ ID NO:2; or a sequence having at least one conservative amino acid substitution compared to (1). The mentioned conservative amino acid substitutions can be one, two, or three amino acid substitutions.

[0045] The present invention also provides an anti-VASP antibody or antigen-binding fragment selected from (1) the light chain CDR sequences shown in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, and the heavy chain CDR sequences shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8; (2) the light chain CDR sequences shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, and the heavy chain CDR sequences shown in SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18; or an amino acid sequence having at least 80% sequence identity compared to (1) or (2).

[0046] In some embodiments, the present invention provides an antibody or antigen-binding fragment thereof having light chain CDR sequences shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, and heavy chain CDR sequences shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, or having light chain CDR sequences shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, and having heavy chain CDR sequences shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.

[0047] In other embodiments, the antibody or antigen-binding fragment provided by the present invention has more than one conservative amino acid substitution compared with the above sequences. An "antigen-binding fragment" refers to an antibody fragment that retains the ability to specifically bind an antigen. A "conservative amino acid substitution" means that an amino acid is replaced by another residue that is biologically, chemically or structurally similar. Biologically similar means that the substitution does not disrupt the biological activity of the VASP antibody or the VASP antigen. Structurally similar means that the amino acids have side chains of similar length, such as alanine, glycine or serine, or side chains of similar size. Chemical similarity means that the amino acids have the same charge or are all hydrophilic or hydrophobic. For example, hydrophobic residues such as isoleucine, valine, leucine or methionine are substituted for each other. Or polar amino acids are used, such as lysine is replaced by arginine, aspartic acid is replaced by glutamic acid, asparagine is replaced by glutamine, and threonine is replaced by serine, and so on.

[0048] The antibody of the present invention can also be directly synthesized according to the amino acid sequence of the antibody variable region provided by the present invention, and can be derivatized into different antibody analogs through further chemical modification.

[0049] In some specific embodiments, the present invention provides an antibody or antigen-binding fragment thereof, including 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; (b) having a light chain variable region shown in SEQ ID NO: 11 and a heavy chain variable region shown in SEQ ID NO: 12; An amino acid sequence having at least 80% sequence identity compared with (a) or (b). An amino acid sequence having at least 80% identity.

[0050] In some embodiments, the present invention provides an antibody or antigen-binding fragment thereof having 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, or having a light chain variable region sequence shown in SEQ ID NO: 11 and a heavy chain variable region sequence shown in SEQ ID NO: 12. 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 or SEQ ID NO: 11. 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 or SEQ ID NO: 12. Of course, these conservative amino acid substitutions do not change the biological function of the antibody or antigen-binding fragment. In some specific ways, these conservative amino acid substitutions can occur on amino acids other than the CDR regions in the heavy chain variable region and the light chain variable region.

[0051] 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 antibodies mentioned include at least one of Fab antibody, Fv antibody, single-chain antibody, and the minimum recognition unit.

[0052] According to a specific embodiment of the present invention, the light chain variable region sequence of the provided HC8 antibody is as follows: DIVLTQSPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPWTFGGGTKLEIK (SEQ ID NO:1) According to a specific embodiment of the present invention, the heavy chain variable region sequence of the provided HC8 antibody is as follows: LVKLEESGGGLVKPGGSLKLSCAVSGFAFSSYDMSWVRQTPEKRLEWVAYIDNGGGYTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCVRLGRDYWGQGTTLTVSS(SEQ ID NO:2) According to a specific embodiment of the present invention, the light chain variable region sequence of the provided HC10 antibody is as follows: DIVLTQSPATLSVTPGYRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYVSQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGTKLELK (SEQ ID NO:11) According to the specific embodiments of the present invention, the heavy chain variable region sequence of the provided HC10 antibody is as follows: DFKLQESGGGLVQPGGSRKLSCEASGFTISSFGMHWVRQAPEKGLEWVAYISSGSNTIYYADTVKGRFTISRDIPKNTLFLQMTSLRSEDTAMYYCARRLAYWGQGTLVTVSA (SEQ ID NO:12) Polynucleotide, expression vector, recombinant cell, hybridoma cell In the process of preparing or obtaining these antibodies, polynucleotides expressing these antibodies can be ligated to different vectors and then expressed in different cells to obtain the corresponding antibodies.

[0053] For this purpose, 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 well known to those skilled in the art. Moreover, it can be optimized for species to be more easily expressed in mammalian cells.

[0054] Based on the variable region (or CDR region) sequences of the heavy and light chains of the antibody 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) identical 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).

[0055] The present invention also provides an expression vector, which contains the above-mentioned isolated polynucleotide. When ligating the above-mentioned isolated polynucleotide to a vector, the polynucleotide can be directly or indirectly linked to the control elements on the vector, as long as these control elements can control the translation and expression of the polynucleotide, etc. Of course, these control elements can directly come from the vector itself or be exogenous, that is, not from the vector itself. Of course, it is only necessary to operably link the polynucleotide to the control elements. As used herein, "operably linked" means ligating a foreign gene to a vector such that the control elements within the vector, such as transcriptional control sequences and translational control sequences, etc., can exert their expected functions of regulating the transcription and translation of the foreign gene. Of course, the polynucleotides encoding the heavy and light chains of the antibody can be independently inserted into different vectors, and commonly are inserted into the same vector. Commonly used vectors can be, for example, plasmids, phages, etc. For example, the pCDNA 3.1 plasmid.

[0056] The present invention also provides a recombinant cell, which contains the above-mentioned expression vector. The expression vector can be introduced into mammalian cells to construct the recombinant cell, and then these recombinant cells are used to express the antibody or antigen-binding fragment provided by the present invention. By culturing these recombinant cells, the corresponding antibody can be obtained. These available mammalian cells can be, for example, CHO cells, etc.

[0057] The present invention also provides a hybridoma cell, which can be produced by the classical method for preparing monoclonal antibodies.

[0058] In the classical method for preparing monoclonal antibodies, first, a suitable antigen is required and used to immunize an animal. To prepare an antibody (or antibody analog) against VASP, the suitable antigen must contain VASP. This antigen can be obtained by isolating and purifying from natural human tissues or blood, chemical synthesis, recombinant expression in vitro, or a combination of these methods for use in immunizing animals and screening and detecting antibodies.

[0059] In the classical method for preparing monoclonal antibodies, first, an animal is immunized with the recombinant expressed VASP antigen, and blood is collected at intervals to verify whether the animal has produced an antibody response to the recombinant expressed VASP antigen. Then, B cells isolated from the spleen of the animal with an antibody response are fused with immortalized myeloma cells in vitro to obtain hybridoma cells. Then, these hybridoma cells are serially diluted 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. According to the antibody yield, quality, and cell line growth characteristics, the best monoclonal antibody-producing cell line can be selected for subsequent monoclonal antibody production.

[0060] Other methods can also be used to obtain monoclonal antibodies. For example, the spleen cells of the above animals can be isolated and then incubated with a labeled antigen (such as fluorescein-labeled VASP). Since the B cells that produce antibodies usually have antibody molecules presented on the cell membrane surface, these cells will be bound (stained) by the labeled antigen and thus can be sorted out by a fluorescence-activated cell sorter. The mRNA of these sorted B cells can be isolated, and a cDNA library of the variable region of the antibody can be obtained through 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 host cells 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 or the phage plaque plating method described above). These cell lines or phage clones can be used to produce antibodies and analyze and identify the antibodies.

[0061] According to the antibody or antigen-binding fragment, antibody analog, polynucleotide sequence, expression vector, recombinant cell, hybridoma cell provided by the present invention, their roles in the auxiliary diagnosis of VASP deficiency and related diseases, and the enzyme immunoassay kit based on the HC8 and HC10 antibodies greatly improve the accuracy and convenience of diagnosis.

[0062] According to a preferred embodiment of the present invention, the animals in the present invention are immunized by multiple subcutaneous injections at multiple points. This improves the titer of the final animal serum and increases the positive cloning rate.

[0063] According to a preferred embodiment of the present invention, the addition of the HAT screening reagent in the present invention is postponed from the day of fusion to the second day, which significantly increases the positive rate of the cells.

[0064] According to a preferred embodiment of the present invention, the VASP-positive samples used in the present invention are all VASP-positive sera detected by liquid chromatography-mass spectrometry to ensure the authenticity of the later detection by the kit.

[0065] The anti-VASP antibody of the present invention can be used to detect VASP in human serum or body fluids, or substances containing VASP, as well as substances that can specifically bind to VASP (such as anti-VASP antibodies). The detection principle is mainly based on the specific recognition and binding of the VASP antibody of the present invention to VASP, and chemical labeling techniques (such as labeled antibodies or labeled specific secondary antibodies) or physical detection techniques (such as light scattering techniques, surface plasmon resonance techniques, etc.) are used for detection.

[0066] The present invention provides monoclonal antibodies HC8 and HC10 against VASP, which have the characteristics of high affinity and specificity. At the same time, it also provides an in vitro diagnostic detection kit based on the above antibodies. The present invention solves the problem of relative shortage of antibody raw materials and kits for VASP detection items in the current market, and can be used as an auxiliary diagnosis for VASP syndrome and related neurological diseases, filling the domestic related market gap.

[0067] Nucleic acids encoding the heavy chain and / or light chain of the antibodies of the present invention are within the scope of the present invention. According to the amino acid sequences of the heavy chain and / or light chain, those skilled in the art can easily obtain the corresponding nucleic acid sequences, as shown in Table 1. It should be noted that the CDR sequences listed in Table 1 below are defined according to the Kabat CDR encoding rules. Those skilled in the art should know that the CDR sequences analyzed by different databases may be different, but these variations should all be included within the protection scope of the present invention.

[0068] Table 1 The solution of the present invention will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the examples for specific techniques or conditions, they shall be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. Those reagents or instruments not indicated with the manufacturer can all be conventional products obtained through commercial purchase.

[0069] Example 1: Preparation of Recombinant Protein of VASP First, referring to the GenBank nucleotide sequence NM_005022.4 (Homo sapiens profilin 1 (PFN1), transcript variant 2, mRNA), a Kappa secretion signal peptide sequence was inserted upstream and a 6×His Tag was inserted downstream. The recombinant expression nucleotide sequence of VASP is shown in SEQ ID NO.21. BamHI and XhoI restriction enzyme sites were inserted at the 5' and 3' ends respectively, and then the gene was synthesized by a gene company. The synthesized gene and pGEX-6P-1 were double digested with the restriction enzymes BamHI and XhoI respectively, and the gene fragment recovered by gel extraction was ligated overnight at 16°C with T4 ligase. The ligation product was transformed into TOP10 competent cells, and the synthesized plasmid was extracted and named pGEX-6P-1-VASP. The expression plasmid map is shown in Figure 1 .

[0070] Add 30 μL of ampicillin solution (final concentration 100 μg / mL) to a test tube containing 15 ml of SOB medium (containing 20 g of tryptone, 5 g of yeast extract 0.5 g of NaCl, pH 7.0) and then add the glycerol bacteria (about 1 - 2 μL) picked with a sterile 200 μl pipette tip to the medium. Fix the test tube on a shaker (Jiangsu Peiying, #DHZ-DA), incubate at 37°C and 200 rpm overnight. Take 4 ml of the overnight culture (1% of the bacterial solution added) and add it to a conical flask, and culture at 37°C and 200 rpm for 4 hours. When the OD600 of the bacterial density is detected to be 0.8 - 1.0, add 1 M IPTG solution (final concentration 1 mM) to the conical flask and induce culture at 37°C and 200 rpm for 4 hours. After induction, add 200 ml / tube to centrifuge tubes, centrifuge at 10000×g for 20 minutes, and discard the supernatant. The bacteria were lysed using a high-pressure homogenizer (Zhejiang Xinzhi, SCIENTZ-150) at 800 Bar, and then centrifuged at 10000×g for 20 minutes to collect the supernatant after lysis.

[0071] Use a gravity column containing 5 mL of GST affinity packing material (YEASON, 20507ES10) to purify the soluble protein in the supernatant. First, load the supernatant after lysis onto the column at a rate of 1 ml / minute. Washing: Wash the affinity column with the equilibration buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 8.0) at a rate of 1 ml / minute for 20 CV (column volume). Elution: Elute with an elution buffer prepared with the equilibration buffer containing 10 mM glutathione (reduced form). Elute and collect the eluate at a rate of 1 mL / minute, and identify the expression of the target protein VASP by SDS-PAGE (Figure 2 ), such as Figure 2 The eluate (lanes 8, 9, 10) as shown contains the VASP recombinant protein at 67.7KDa. Mix the above eluates and dialyze overnight into the equilibrium buffer. Concentrate the recombinant protein of VASP (GST Tag) to 1.1 mg / mL using an ultrafiltration tube (Amicon, #UFC5003).

[0072] Example 2: Immunization of BALB / c mice with VASP protein Select 6 - 8 week - old BALB / c mice for the following immunization procedure: At the primary immunization, mix 25 µg of VASP recombinant protein (His Tag, abcam, #ab105601) with an equal volume of Freund's complete adjuvant, emulsify it, and inject subcutaneously at multiple points; 14 days after the first immunization, mix 12.5 µg of VASP recombinant protein with an equal volume of Freund's incomplete adjuvant, emulsify it, and boost the immunization. 14 days after the second immunization, mix 12.5 µg of VASP recombinant protein (His Tag, abcam, #ab105601) with an equal volume of Freund's incomplete adjuvant, emulsify it, and boost the immunization. Blood is collected 14 days after the third immunization and serum is separated. Coat an ELISA plate with 1 µg / mL of VASP recombinant protein (GSTTag) and perform an indirect ELISA test to determine the serum titer. The results show that the titer of the prepared mouse antiserum is 1:72900. A typical result of detecting the serum of immunized mice is shown in Table 2.

[0073] Table 2 Example 3: Cell fusion Three days after boosting the immunization of mice in Example 3, cell fusion is performed. After collecting blood by removing the mouse's eyeballs, the mouse is sacrificed by dislocation of the cervical vertebrae and placed in a 70% alcohol bottle for 2 minutes. Then, the mouse is fixed on a foam board in a biosafety cabinet. The abdominal skin is cut open to find the spleen, which is removed with forceps and placed in a 200 - mesh stainless - steel filter membrane and gently ground. The cells are gently rinsed with DMEM medium (Thermo, 11965092), and then centrifuged at 200 g for 10 minutes in a centrifuge at room temperature. The supernatant is discarded and reserved for later use; when preparing feeder cells, the mouse is sacrificed by dislocation of the cervical vertebrae and placed in a 70% alcohol bottle for 2 minutes. Then, the mouse is fixed on a foam board in a biosafety cabinet. The abdominal skin is cut open, and PBS is aspirated with a syringe and gently injected subcutaneously. The liquid containing feeder cells is washed out from the other side, and then centrifuged at 200 g for 10 minutes in a centrifuge at room temperature. The supernatant is discarded and reserved for later use. Mix 0.3×10 7 cells of FO myeloma cells with 1.1×10 8Mix the spleen cells, centrifuge them at 200 g for 10 minutes in a centrifuge, discard the supernatant, gently shake to mix evenly, in a 37 °C water bath, add 1 mL of an aqueous solution of PEG-1450 (Merk, P1458) with a volume concentration of 50% dropwise within 90 seconds, then add 20 mL of DMEM medium, centrifuge at 200 g for 10 minutes in a centrifuge, discard the supernatant, repeat the washing once, centrifuge at 200 g for 10 minutes in a centrifuge, discard the supernatant to obtain hybridoma cells, and seed the cells into 10 96-well culture plates, 150 μL per well. Add 10,000 feeder cells per well to the above 10 96-well cell culture plates, 100 μL per well. After labeling the culture plates, place them in a cell culture incubator at 37 °C containing 5% CO2 for culture. On the second day, add HAT selection medium (Merk, H0262). During the 1 - 2 days of HAT selection culture, a large number of tumor cells will die, and after 3 - 4 days, the tumor cells will disappear, and hybrid cells will form small colonies. After maintaining the HAT selection culture medium for 7 - 10 days, it should be replaced with HT medium (Merk, H0137), and then maintained for another 2 weeks, and then switched to DMEM medium containing 20% FBS (ExCell, FSP500) for continued culture. During the above selection culture period, when the hybridoma cells cover 1 / 10 of the bottom area of the well, the detection of specific antibodies can be started to screen out the required hybridoma cell line. During the selection culture period, generally, half of the culture medium is changed every 2 - 3 days.

[0074] Example 4: Screening and subcloning culture of positive hybridoma cell lines First, determine the optimal coating amount of VASP recombinant protein (GST Tag) as an antigen through Fangzheng experiment. Coat a 96-well plate with 0.5, 1.0, 2.0, 4.0 μg of VASP recombinant protein (GST Tag), with 6 wells for each concentration, and set 3 wells as positive and 3 wells as negative respectively. Perform Fangzheng titration with immune mouse positive sera at different dilution multiples, and use non-immune mouse negative sera as a negative control. Coat a 96-well ELISA plate with 0.5 μg of purified VASP recombinant protein (GST Tag) per well and incubate overnight at 4 °C; wash twice with PBST; add 200 μL of 1% BSA in PBS to each well, block at room temperature for 2 hours, and then pat dry on filter paper; add samples: add 0.1 mL of the sample to be tested to the reaction wells, incubate at 37 °C for 1 hour, then wash, and at the same time, set up blank wells (without adding samples), negative control wells, and positive control wells. Add 0.1 mL of the 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 the 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 judge the result, and use an enzyme-linked immunosorbent assay reader to measure the absorbance value (A450) at 450 nm. A value more than 2.1 times the OD value of the negative control is considered positive (calculated after zeroing with the blank control well). Select monoclonal hybridoma cells against VASP.

[0075] According to the above method, perform subcloning on the selected positive hybridoma cells. Dilute the original wells with HAT selection medium by the limited dilution method and then re-distribute them into a 96-well culture plate, and then observe the cell morphology and quantity. Adjust the cells to 3 - 5 cells / mL. Take the cell culture plate of the feeder cell layer prepared the previous day, and add 100 μL of the diluted cells to each well. Incubate statically in a 37 °C, 5% CO2 incubator. Change the medium on the 7th day, and then change the medium once every 2 - 3 days. Cell clones can be seen on the 8th - 9th day, and detect the antibody activity in a timely manner. Transfer the cells in the positive wells to a 24-well plate for expansion culture. Each clone should be frozen as soon as possible, and finally select 10 hybridoma cells for antibody production.

[0076] Example 5: Large-scale preparation of monoclonal antibody and determination of antibody titer (1) Large-scale preparation of monoclonal antibody Inject 0.5 mL of Freund's incomplete adjuvant intraperitoneally into 8-week-old BALB / C mice. Two weeks later, inject 1×10 6Hybridoma cells of different cell lines can produce ascites 7 to 10 days after inoculation of cells. Closely observe the health status of the animals and ascites signs. Before the ascites is as much as possible and the mice are on the verge of death, sacrifice the mice. Use a dropper to suck the ascites into a test tube. One mouse can obtain 5 to 10 ml of ascites. Ascites can also be extracted with a syringe and collected repeatedly several times. Centrifuge the obtained ascites at 3000 g for 10 minutes, discard the upper layer of grease and the bottom precipitate, collect the supernatant and aliquot it at -20°C. After the ascites supernatant is thawed and equilibrated to room temperature, add 1 / 10 volume of 1 M Tris–HCl PH8.0 to adjust the sample PH value to 8.0. Equilibrate the protein G affinity column with 20 column volumes of 100 mM PH8.0 Tris–HCl. Load the ascites supernatant with adjusted PH to 8.0 onto the column, then wash it with 20 column volumes of 100 mM PH 8.0 Tris–HCl, and finally elute the antibody with 100 mM Glycine–HCL PH 2.5. Add the antibody eluate to a concentrator tube (Millipore, UFC801008, 10K) and centrifuge it at 3000 g for 20 minutes at room temperature in a centrifuge (Xiangyi, L550). Centrifuge in batches until the solution volume reaches 1 ml / concentrator tube (2 tubes), add 4 mL of 10 mM PBS PH 7.4 buffer, and continue to centrifuge at 3000×g for 20 minutes at room temperature. Repeat centrifugation 3 times to make the buffer of the antibody 10 mM PBS PH7.4, and add 10 mM PBS PH 7.4 to a total volume of 10 mL. Finally, aliquot the concentrated antibody solution at 2 mL / tube into centrifuge tubes and store it at -80°C. Use a BCA kit (Solarbio, PC0020) to measure the antibody concentration and measure the concentration of the purified monoclonal antibody.

[0077] (2)Antibody titer determination The titer of antibodies against VASP in different cell lines was detected by the indirect ELISA method. The recombinant VASP protein (GST Tag) was diluted with PBS and coated in a 96-well ELISA plate at 0.2 μg / mL, 100 μL / well. After incubation overnight at 4°C, the ELISA plate was washed twice with PBST solution, 300 μL / well each time, and patted dry after washing. The ELISA plate was blocked with PBS solution containing 1% BSA, 200 μL / well, at room temperature for 2 hours, and patted dry after blocking. Antibodies against VASP in different cell lines diluted with PTB to 1020.6 - 0.5 ng / mL were added and reacted in an incubator at 37°C for 1 hour. The ELISA plate was washed twice with PBST solution, 300 μL / well each time. After washing, it was patted dry. HRP-labeled goat anti-mouse antibody diluted 5000-fold with PTB was added and reacted in an incubator at 37°C for 1 hour. The ELISA plate was washed twice with PBST solution, 300 μL / well each time. After washing, it was patted dry. TMB substrate solution was added, 100 μL / well, and reacted at room temperature for 10 minutes. Then, 100 μL / well of 1M H2SO4 was added to terminate the reaction. The absorbance value (A450) was measured at 450 nm using an ELISA reader. A saturation curve was made for each monoclonal antibody to determine the appropriate antibody concentration that could saturate the antigen, and the half-saturation concentration (IC50 concentration) of the antibody was determined. The results are shown in Table 3.

[0078] Table 3 Example 6: Screening of anti-VASP monoclonal antibody pairs (1) Preliminary screening of antibody pairs using epitope overlay experiment The epitopes of antibodies against VASP in different cell lines were detected by the epitope overlay experiment of the indirect ELISA method. The recombinant VASP protein (GST) was diluted with PBS and coated in a 96-well ELISA plate at 0.2 μg / mL, 100 μL / well. After incubation overnight at 4°C, the ELISA plate was washed twice with PBST solution, 300 μL / well each time, and patted dry after washing. The ELISA plate was blocked with PBS solution containing 1% BSA, 200 μL / well, at room temperature for 2 hours, and patted dry after blocking. Referring to the half-saturation concentration (IC50) of different cell lines in "Example 7", antibodies against VASP in different cell lines diluted with PTB (one or two antibodies mixed) were added and reacted in an incubator at 37°C for 1 hour. The ELISA plate was washed twice with PBST solution, 300 μL / well each time. After washing, it was patted dry. HRP-labeled goat anti-mouse antibody diluted 5000-fold with PTB was added and reacted in an incubator at 37°C for 1 hour. The ELISA plate was washed twice with PBST solution, 300 μL / well each time. After washing, it was patted dry. TMB substrate solution was added, 100 μL / well, and reacted at room temperature for 10 minutes.

[0079] The first and second saturation concentrations and the monoclonal mixture of the two saturation concentrations were respectively combined with the antigen, and the corresponding A450 values, namely A1, A2, and A1+2, were measured. The addition index (AI) was obtained from the A values, and the formula was AI (%) = [2×A1+2 / (A1 + A2) - 1] × 100%. The results were generally judged based on whether AI was > or < 50%. If the monoclonal antibody concentration could saturate the antigen, when the two monoclonal antibodies targeted the same or similar epitopes, AI ≤ 50%, and when the epitopes of the two antibodies were different, AI would ≥ 50%. The results are shown in Table 4, which indicates that 5 monoclonal antibodies recognize the same epitope, and the other 2 recognize other epitopes. The addition index AI (%) obtained from the A values was compared for antibodies with different clone numbers, and paired antibodies with AI (%) > 90 (23A3, 30A3, 8D5, 18H1, 2H3, HC10, 5C12, HC8, 8E6) were selected for the labeling pairing experiment. The results are shown in Table 4.

[0080] Table 4 (2)Labeling of antibodies 0.5 mL of 20 nmol (3 mg) of each antibody (5C12, HC10, 8E6, 23A3, HC8, HC10) was separately added into a dialysis bag (10 KDa, width 1 cm) and dialyzed overnight at 4 °C in 2 L of 10 mM PBS solution (pH 7.4). 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 with stirring at room temperature for 2 hours to prepare for coupling with the activated HRP. Meanwhile, 1 mg of HRP was accurately weighed using an analytical balance, dissolved in 0.2 mL of ultrapure water to make the HRP concentration 5 mg / mL. 40 μL of 0.1 M NaIO4 was added to the above HRP solution, placed on a horizontal shaker, and reacted (activated) at room temperature in the dark for 20 minutes. The activated HRP solution was added into a dialysis bag (10 KDa, width 1 cm) and dialyzed overnight at 4 °C in 2 L of 1 mM sodium acetate buffer (pH 4.4). The dialyzed HRP solution was carefully aspirated and transferred into a new 1.5 mL centrifuge tube, and 1 / 10 volume of 0.2 M carbonate buffer was added to raise the pH of the activated HRP solution to 9.0 - 9.5. The above antibody and HRP were mixed (for the coupling reaction), placed on a horizontal shaker, and reacted at room temperature in the dark for 4 hours. After the coupling reaction was completed, 10 μL of freshly prepared NaBH4 (using pre-cooled ultrapure water) was added to terminate the reaction at 4 °C in the dark overnight. The antibody solution after terminating the coupling reaction was transferred into a dialysis bag (10 KDa, width 1 cm) and dialyzed with stirring at room temperature for 2 hours. Finally, the solution was transferred into a brown centrifuge tube and stored at 4 °C.

[0081] (3)Identification of the best antibody pair The sandwich ELISA was used to compare the pairing of antibodies against VASP in different cell lines and select the optimal paired antibodies. The antibodies from different cell lines were diluted with PBS and coated on a 96-well ELISA plate at a concentration of 1 µg / mL and a volume of 100 µL / well. After incubation overnight at 4°C, the ELISA plate was washed twice with PBST solution at a volume of 300 µL / well each time, and then patted dry after washing. The ELISA plate was blocked with PBS solution containing 1% BSA at a volume of 200 µL / well for 2 hours at room temperature, and then patted dry after blocking. Different concentrations of recombinant VASP protein diluted with PTB were added and reacted in an incubator at 37°C for 1 hour. The ELISA plate was washed twice with PBST solution at a volume of 300 µL / well each time, and then patted dry after washing. Antibodies against VASP from different cell lines labeled with HRP diluted with PTB were added and reacted in an incubator at 37°C for 1 hour. The ELISA plate was washed twice with PBST solution at a volume of 300 µL / well each time, and then patted dry after washing. TMB substrate solution was added at a volume of 100 µL / well and reacted at room temperature for 10 minutes, and then the reaction was terminated by adding 100 µL / well of 1M H2SO4. The absorbance (A450) was measured at 450 nm using an ELISA reader. The detection ranges of different antibody pairs for detecting recombinant VASP protein were compared. See Table 5. The optimal antibody pair was the coated HC8 antibody and the labeled HC10 antibody.

[0082] Table 5 Example 7: Sequence analysis of monoclonal antibodies (1)Identification of monoclonal antibody subtypes The hybridoma cell lines HC8 and HC10 were cultured in a 10-cm diameter cell culture dish (37°C, 5% CO2) with DMEM medium (GIBCO, #C11995500BT) supplemented with 10% serum. After 7 days of culture, the cells were transferred to a 15-ml centrifuge tube. After counting with a hemocytometer, 5×10 6 cells were taken out, centrifuged at 200 g for 5 minutes, the supernatant was discarded, and the centrifuge tube was inverted to drain the liquid inside. The cells in the tube were used to synthesize cDNA using the reverse transcription kit (Qiagen, 74134) from QIAGEN.

[0083] The antibody subtypes were determined by performing PCR using subtype-specific primers for antibodies. The synthesized cDNA described above was used as the template for the PCR reaction. PCR reaction solution system: TAKARA Ex Taq (5 U / µL, TAKARA, RR001B), 0.25 μL; 10×Ex Taq Buffer, 5 µL; dNTP mixture (each 2.5 mM), 4 µL; template cDNA, 1 µL; upstream primer (100 µM), 1 µL; downstream primer (100 µM), 1 µL; double-distilled water was added to a total volume of 50 µL. PCR reaction temperature program: pre-denaturation at 94°C for 5 minutes, 30 temperature cycles (94°C for 1 minute, 57°C for 1 minute, 72°C for 1 minute), extension at 72°C for 10 minutes. After the reaction, 10 µL of each PCR product was loaded onto a 1% agarose gel for electrophoresis, and the antibody subtype could be inferred based on the PCR product results (Table 6). The monoclonal antibody HC8 obtained in the present invention has a heavy chain of IgG2A and a light chain of kappa. The monoclonal antibody HC10 has a heavy chain of IgG1 and a light chain of kappa.

[0084] Table 6 Where S = C or G, M = A or C, R = A or G, and W = A or T (2)Sequencing of the variable regions (V regions) of the antibodies of hybridoma cell lines HC8 and HC10 The fragments obtained after PCR amplification of the V regions of the antibodies of cell lines HC8 and HC10 were cut from the agarose gel and extracted using a DNA extraction kit (Qiagen, 74134). The extracted DNA fragments were ligated to the pEASY-T1 cloning vector and transformed into Trans1-T1 competent cells (Transgen, CT101-1). Transformed bacterial colonies were picked into LB medium and subjected to DNA sequencing after overnight culture. The nucleic acid sequence of the light chain V region of the anti-VASP antibody (HC8) provided by the present invention is shown in SEQ ID NO.17, and the nucleic acid sequence of the heavy chain V region is shown in SEQ ID NO.18. The nucleic acid sequence of the light chain V region of the antibody (HC10) is shown in SEQ ID NO.19, and the nucleic acid sequence of the heavy chain V region is shown in SEQ ID NO.20.

[0085] Example 8: Application of the enzyme immunoassay kits prepared using antibodies HC8 and HC10 in detecting VASP in human serum (1)Labeling of antibody HC10 with horseradish peroxidase (HRP) and identification of the labeled product Take 0.5 mL of antibody HC10 (20 nmol, 3 mg) and add it to a dialysis bag (10 KDa, width 1 cm). Dialyze overnight at 4 °C in 2 L of 10 mM PBS solution (pH 7.4). The next day, place the dialysis bag containing the antibody solution into 1 L of 10 mM carbonate buffer (pH 9.5) and dialyze with stirring at room temperature for 2 hours to prepare for coupling with the activated HRP.

[0086] Meanwhile, accurately weigh 1 mg of HRP using an analytical balance, add 0.2 mL of ultrapure water to dissolve it, so that the concentration of HRP is 5 mg / mL. Add 40 μl of 0.1 M 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 (10 KDa, width 1 cm), and dialyze overnight at 4 °C in 2 L of 1 mM sodium acetate buffer (pH 4.4). Carefully aspirate the dialyzed HRP solution and transfer it to a new 1.5 mL centrifuge tube. Add 1 / 10 volume of 0.2 M carbonate buffer to raise the pH of the activated HRP solution to 9.0 - 9.5.

[0087] Mix the above antibody and HRP (for the coupling reaction), place it on a horizontal shaker, and react at room temperature in the dark for 4 hours. After the coupling reaction is completed, add 10 μl of freshly prepared NaBH4 (using pre-cooled ultrapure water) and terminate the reaction at 4 °C in the dark overnight. Transfer the antibody solution after terminating the coupling reaction to a dialysis bag (10 KDa, width 1 cm) and dialyze with stirring at room temperature for 2 hours. Finally, transfer the solution to a brown centrifuge tube and store it at 4 °C.

[0088] (2)Determination of the standard curve of the enzyme immunoassay kit prepared using antibodies HC8 and HC10 An enzyme immunoassay kit prepared by the ELISA sandwich method. The antibody HC8 was diluted with PBS and coated on a 96-well enzyme-linked immunosorbent assay (ELISA) plate at a concentration of 1 μg / mL and a volume of 100 μL / well. After incubation overnight at 4°C, the ELISA plate was washed twice with PBST solution at a volume of 300 μL / well each time, and then patted dry after washing. The ELISA plate was blocked with PBS solution containing 1% BSA at a volume of 200 μL / well for 2 hours at room temperature, and then patted dry after blocking. Different concentrations of recombinant VASP protein diluted with PTB were added and reacted in an incubator at 37°C for 1 hour. The ELISA plate was washed twice with PBST solution at a volume of 300 μL / well each time, and then patted dry after washing. HRP-labeled HC10 antibody diluted with PTB was added and reacted in an incubator at 37°C for 1 hour. The ELISA plate was washed twice with PBST solution at a volume of 300 μL / well each time, and then patted dry after washing. TMB substrate solution was added at a volume of 100 μL / well and reacted at room temperature for 10 minutes, and then 100 μL / well of 1M H2SO4 was added to terminate the reaction. The absorbance value (A450) was measured at 450 nm using an ELISA reader. The linear relationship between the sample concentration and the absorbance value is as Figure 3 shown. The results show that the curve R 2 = 0.994, and the standard curve range of this method is 0 - 960 ng / ml.

[0089] (3) The enzyme immunoassay kit prepared using antibodies HC8 and HC10 can detect VASP in human serum. The antibody HC8 was diluted with PBS and coated on a 96-well ELISA plate at a concentration of 1 μg / mL and a volume of 100 μL / well. After incubation overnight at 4°C, the ELISA plate was washed twice with PBST solution at a volume of 300 μL / well each time, and then patted dry after washing. The ELISA plate was blocked with PBS solution containing 1% BSA at a volume of 200 μL / well for 2 hours at room temperature, and then patted dry after blocking. Different concentrations of recombinant VASP protein diluted with PTB were added and reacted in an incubator at 37°C for 1 hour. The ELISA plate was washed twice with PBST solution at a volume of 300 μL / well each time, and then patted dry after washing. HRP-labeled HC10 antibody diluted with PTB was added and reacted in an incubator at 37°C for 1 hour. The ELISA plate was washed twice with PBST solution at a volume of 300 μL / well each time, and then patted dry after washing. TMB substrate solution was added at a volume of 100 μL / well and reacted at room temperature for 10 minutes, and then 100 μL / well of 1M H2SO4 was added to terminate the reaction. The absorbance value (A450) was measured at 450 nm using an ELISA reader. The enzyme immunoassay kit of the present invention can quantify VASP in human serum by substituting the OD 450 value into the standard curve ( Figure 3 ). By comparing the results in Table 7, it shows that this kit can preliminarily determine positive and negative sera.

[0090] Table 7 (4)Identification of the Specificity of Antibodies HC8 and HC10 for Detecting Native VASP The ability of antibodies HC8 and HC10 to detect the specificity of native VASP was evaluated by sandwich ELISA. In this experiment, antibody HC8 was diluted with PBS as the coating antibody and coated on a 96-well ELISA plate at a coating concentration of 1 μg / mL and a volume of 100 μL / well. After overnight coating at 4 °C, the ELISA plate was washed twice with PBST solution, 300 μL / well each time. After washing and patting dry, the ELISA plate was blocked with PBS solution containing 1% BSA, 200 μL / well, for 2 hours at room temperature. After patting dry, cell lysates of Hela and VASP recombinant proteins at different concentrations were diluted with PTB respectively. Among them, the VASP knockout Hela cell line (abcam, #ab265892) was used as a negative control because it does not express VASP protein. After reacting on a shaker at room temperature for 30 minutes, they were added to the above-blocked ELISA plate, 100 μL / well, and reacted in an incubator at 37 °C for 1 hour. After patting dry, the ELISA plate was washed 3 times with PBST, 300 μL / well each time. After patting dry, HRP-labeled antibody HC10 diluted 1000 times with PTB was added to the ELISA plate and reacted in an incubator at 37 °C for 1 hour. The ELISA plate was washed 3 times with PBST, 300 μL / well. After patting dry, TMB substrate solution was added to the ELISA plate, 100 μL / well, for 10 minutes at room temperature, and then 100 μL / well of 1 M H2SO4 was added to terminate the reaction. The absorbance (A450) was measured at 450 nm using an ELISA reader. The experimental results are shown in Table 8, indicating that antibodies HC8 and HC10 have good specificity for detecting VASP in native cells.

[0091] Table 8 The present invention provides monoclonal antibodies against VASP, which have the characteristics of high affinity and specificity. At the same time, an in vitro diagnostic detection kit based on the above antibodies is also provided. The present invention solves the problem of the relative shortage of antibody raw materials and kits for VASP detection items in the current market.

[0092] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof for detecting VASP, characterized in that, The amino acid sequences of the light chain variable region complementarity determining regions LCDR1, LCDR2, and LCDR3 of the antibody or its antigen-binding fragment are shown in SEQ ID NO.3, 4, and 5 respectively, and the amino acid sequences of the heavy chain variable region complementarity determining regions HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO.6, 7, and 8 respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

2.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein The nucleotide sequence of the light chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO.9, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.

10.

4. An antibody or antigen-binding fragment thereof for detecting VASP, characterized in that, The amino acid sequences of the light chain variable region complementarity determining regions LCDR1, LCDR2, and LCDR3 of the antibody or its antigen-binding fragment are shown in SEQ ID NO.13, 14, and 15 respectively, and the amino acid sequences of the heavy chain variable region complementarity determining regions HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO.16, 17, and 18 respectively.

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein The amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO.11, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

12.

6. The antibody or antigen-binding fragment thereof according to claim 4 or 5, characterized in that, The nucleotide sequence of the light chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO.19, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.

20.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1-6, characterized in that, The antibody or its antigen-binding fragment is a monoclonal antibody, Fab, Fab', F(ab')2, Fd, Fv, or single-chain antibody.

8. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody or its antigen-binding fragment according to any one of claims 1-6.

9. A biological material, characterized in that, The biological material contains the nucleic acid molecule according to claim 8; the biological material is an expression cassette, vector, or host cell.

10. An antibody conjugate, characterized in that, The antibody conjugate is obtained by conjugating the antibody or its antigen-binding fragment according to any one of claims 1-6 with a label selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, colloidal gold labeling, and radioactive labeling.

11. An antibody composition against VASP, characterized in that, The antibody composition consists of a first antibody and a second antibody, wherein the first antibody is selected from the antibodies according to any one of claims 1-3, and the second antibody is selected from the antibodies according to any one of claims 4-6.

12. The antibody composition according to claim 11, wherein The first antibody is a coating antibody, and the second antibody is a labeled antibody.

13. Use of the antibody or its antigen-binding fragment according to any one of claims 1 to 7, or the nucleic acid molecule according to claim 8, or the biological material according to claim 9, or the antibody conjugate according to claim 10, or the antibody composition according to claim 11 or 12 in the preparation of a product for detecting the presence or level of VASP in a sample.

14. The application according to claim 13, wherein The product includes: (1) A product for detecting platelet dysfunction; (2) A product for detecting Wiskott-Aldrich syndrome (WAS); (3) Products for detecting thrombotic diseases; (4) Products for detecting diabetes; (5) Products for detecting cardiovascular diseases; (6) Products for detecting neurodegenerative diseases; (7) Products for detecting immune diseases; (8) Products for detecting and / or prognostic evaluation of malignant tumors.

15. A kit, characterized in that, It comprises the antibody or its antigen-binding fragment as described in any one of claims 1 to 7, or the nucleic acid molecule as described in claim 8, or the biological material as described in claim 9, or the antibody conjugate as described in claim 10, or the antibody composition as described in claim 11 or 12.

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