An antibody recognizing vasp and uses thereof

Abstract

This invention relates to the field of biomedicine, specifically to antibody compositions for human VASP and their applications. The invention provides antibodies and compositions thereof capable of specifically binding to human VASP. These antibody compositions exhibit high specificity and affinity for human VASP, enabling specific and sensitive detection of human VASP. Human VASP detection kits developed based on these antibody compositions possess high specificity, sensitivity, and accuracy, a broad linear range, and high stability, meeting detection requirements and showing promising application prospects for human VASP detection in practice.

Description

Technical Field

[0001] This invention relates to the field of biological detection, and more particularly to an antibody that identifies VASP and its applications. Background Technology

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

[0003] VASPs participate in multiple signaling pathways, including the Rho family GTPase signaling pathway. They regulate cytoskeleton remodeling by interacting with these GTPases. Abnormal VASP function is associated with a variety of diseases, including certain types of cancer, cardiovascular disease, and neurodegenerative diseases. For example, altered VASP expression levels and activity may be associated with tumor invasiveness and metastasis.

[0004] In summary, while the direct link between VASP and some diseases requires further investigation, existing research has shown that it plays a role in the pathogenesis of thrombotic diseases and may serve as a potential biomarker for diagnosing thrombotic diseases. Future research may reveal the specific mechanisms of action of VASP in thrombotic diseases and how it, along with other biomarkers, can aid in the diagnosis and treatment of depression.

[0005] Immunoassays based on the specific reaction between antigens and antibodies are low-cost, fast, accurate, and sensitive, with specific antibodies being their core component. Currently, there are no diagnostic reagents or raw materials in the industry that have high specificity for VASPs.

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

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

[0008] This invention provides, in one aspect, an anti-VASP antibody or antigen-binding fragment, comprising:

[0009] Selected from at least one of the following CDR sequences or amino acid sequences that have at least 80% identity with them:

[0010] Light chain variable region CDR sequences: SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5;

[0011] Heavy chain variable region CDR sequences: SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8,

[0012] or

[0013] Light chain variable region CDR sequences: SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15;

[0014] Heavy chain variable region CDR sequences: SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18;

[0015] This invention provides an antibody or antigen-binding fragment against VASP, and an enzyme immunoassay kit prepared using the antibody or antigen-binding fragment has high sensitivity and specificity.

[0016] According to some embodiments of the present invention, the antibody or antigen-binding fragment comprises: 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, the heavy chain variable region CDR3 sequence shown in SEQ ID NO:8, or the light chain variable region CDR1 sequence shown in SEQ ID NO:13, the light chain variable region CDR2 sequence shown in SEQ ID NO:14, the light chain variable region CDR3 sequence shown in SEQ ID NO:15, the heavy chain variable region CDR1 sequence shown in SEQ ID NO:16, the heavy chain variable region CDR2 sequence shown in SEQ ID NO:17, and the heavy chain variable region CDR3 sequence shown in SEQ ID NO:18.

[0017] According to some embodiments of the present invention, the antibody or antigen-binding fragment comprises at least one of the following:

[0018] (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;

[0019] (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;

[0020] Compared with (a) or (b), the sequence identity is at least 80% of the amino acid sequence.

[0021] (c) The CDR1, CDR2 and CDR3 sequences having the light chain variable region shown in SEQ ID NO:1, and the CDR1, CDR2 and CDR3 sequences having the heavy chain variable region shown in SEQ ID NO:2;

[0022] (d) The CDR1, CDR2 and CDR3 sequences having the light chain variable region shown in SEQ ID NO:11, and the CDR1, CDR2 and CDR3 sequences having the heavy chain variable region shown in SEQ ID NO:12;

[0023] Or, compared to (c) or (d), it has an amino acid sequence with more than one conserved amino acid substitution.

[0024] According to some embodiments of the present invention, the antibody or antigen-binding fragment further includes at least one of a heavy chain constant region and a light chain constant region, at least a portion of the heavy chain constant region and the light chain constant region being derived from a mammalian antibody.

[0025] 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 mouse antibodies, rabbit antibodies, primate antibodies or mutants thereof.

[0026] According to some embodiments of the present invention, the heavy chain constant region and the light chain constant region are derived from mouse antibodies or mutants thereof.

[0027] According to some embodiments of the present invention, the antibody or antigen-binding fragment comprises a monoclonal antibody or a polyclonal antibody;

[0028] According to some embodiments of the present invention, the monoclonal antibody includes at least one of full-length antibody, Fv, single-chain antibody, Fab, single-domain antibody, and minimum recognition unit.

[0029] In another aspect, the present invention provides an antibody analog comprising the variable region or CDR region of the variable region of the antibody or antigen-binding fragment described above.

[0030] In another aspect, the present invention provides an isolated polynucleotide encoding the aforementioned antibody or antigen-binding fragment.

[0031] According to some embodiments of the present invention, the polynucleotide comprises:

[0032] (e) A nucleic acid sequence having the encoding of the light chain variable region as shown in SEQ ID NO:9,

[0033] Compared to (e), nucleic acid sequences with at least 80% sequence identity; or

[0034] (f) A nucleic acid sequence having the heavy chain variable region encoded as shown in SEQ ID NO:10,

[0035] Compared to (f), nucleic acid sequences with at least 80% sequence identity; or

[0036] (g) A nucleic acid sequence having the encoding of the light chain variable region shown in SEQ ID NO:19,

[0037] Compared to (g), nucleic acid sequences with at least 80% sequence identity; or

[0038] (h) A nucleic acid sequence having the heavy chain variable region encoded as shown in SEQ ID NO:20,

[0039] Compared to (h), the nucleic acid sequence has at least 80% sequence identity.

[0040] In another aspect, the present invention provides an expression vector comprising the above-mentioned polynucleotides.

[0041] In another aspect, the present invention provides a hybridoma cell that can produce the above-mentioned anti-VASP antibody or antigen-binding fragment.

[0042] In another aspect, the present invention provides a method for preparing anti-VASP antibodies or antigen fragments, comprising culturing the above-mentioned recombinant cells or hybridoma cells.

[0043] In another aspect, the present invention provides the use of the above-mentioned antibody or antigen-binding fragment and the above-mentioned antibody analog in the preparation of a kit for detecting VASP.

[0044] Another aspect of the present invention provides a kit comprising at least one of the above-described antibody or antigen-binding fragment, antibody analog, vector, recombinant cell, and hybridoma cell.

[0045] Another aspect of the present invention provides a composition comprising the above-described antibody or antigen-binding fragment, antibody analog, polynucleotide, vector, recombinant cell, or hybridoma cell.

[0046] 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 cells or hybridoma cells.

[0047] According to some embodiments of the present invention, the VASP-mediated diseases include platelet dysfunction, cardiovascular diseases, etc.

[0048] This invention provides a monoclonal antibody against VASP, which exhibits high affinity and specificity, and also provides an in vitro diagnostic detection kit based on this antibody. This invention addresses the current market shortage of antibody raw materials and kits for VASP detection.

[0049] Additional aspects and advantages of this disclosure will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this disclosure. Attached Figure Description

[0050] Figure 1 This is a diagram of the pCDNA3.1-VASP plasmid expressing the recombinant VASP protein.

[0051] Figure 2 SDS-PAGE was used to identify the expression of the target protein VASP.

[0052] Figure 3 This is a standard curve for an enzyme immunoassay kit prepared using antibodies HC8 and HC10. Detailed Implementation

[0053] The embodiments of this disclosure are described in detail below. The embodiments described below are exemplary and are only used to explain this disclosure, and should not be construed as limiting this disclosure.

[0054] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this disclosure, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0055] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0056] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0057] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0058] In this invention, the term "antibody" refers to a protein encoded by an animal's antibody gene and biosynthesized.

[0059] In this invention, the term "antibody analogue" refers to a derivative obtained by biological or chemical methods, based on an antibody structure, through the deletion, addition, or modification of chemical groups (such as amino acids). These derivatives still contain structures similar to the antibody variable region (or the CDR region within the antibody variable region) and can undergo reactions similar to antigen-antibody binding through these structures.

[0060] In this invention, the term "antibody variable region" refers to a domain within the heavy and light chains of an antibody. In nature, antibody variable regions are encoded by the V, D (only applicable to the heavy chain), and J segments from immunoglobulin (heavy and light chains) genes through gene recombination. The amino acid sequences of the variable regions differ significantly between different antibodies (while the amino acid sequences of other regions of the antibody are relatively highly similar), and they are responsible for recognizing and binding to specific antigenic determinants. The antibody variable region can be further subdivided into a backbone region and a CDR (completional determination region). A typical antibody variable region has three backbone regions and three CDR regions (interleaved with each other). The backbone region primarily functions to form the protein domain framework, while the CDR regions primarily function to specifically recognize and bind antigens to antibodies.

[0061] In this invention, the term "anti-VASP antibody (or antibody analog)" refers to a single (or monoclonal) form containing a single structure (such as a single amino acid sequence).

[0062] In this invention, the term "identity" is used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, 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 procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Institute)). Foundation, Washington, DC). Numerous algorithms exist 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, including but not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul...). See, Meth.Enzym., 266:460-480 (1996); or GAP, BESTFIT, BLAST Altschul, etc., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0063] In this invention, the term "antigen-binding fragment" is also known as "antibody fragment". Antibody fragments generally refer to antigen-binding antibody fragments, which may include a part of a complete antibody. They are generally antigen-binding regions or variable regions. Examples of antibody fragments include Fab, Fab', F(ab')2, Fv or scFv, biantibodies, linear antibodies, single-chain antibody molecules, etc.

[0064] Without substantially affecting antibody activity (retaining at least 95% 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 in the sequences of this invention to obtain variants of the antibody or its functional fragments. These are all considered to be included within the scope of protection of this invention. For example, amino acids with similar properties can be substituted in the variable region. The variant sequences of this invention can have at least 80% identity (or homology) with the reference sequence. Sequence identity described in this invention can be measured using sequence analysis software, such as the computer program BLAST using default parameters, especially BLASTP or TBLASTN. The amino acid sequences described in this invention are shown from the N-terminus to the C-terminus.

[0065] Anti-VASP antibody or antigen-binding fragment

[0066] This invention provides an anti-VASP antibody or antigen-binding fragment selected from (1) sequences having the CDR1, CDR2, and CDR3 sequences having the light chain variable region shown in SEQ ID NO:1, and sequences having the CDR1, CDR2, and CDR3 sequences having the light chain variable region shown in SEQ ID NO:2; or sequences having at least one conserved amino acid substitution compared to (1). The conserved amino acid substitution may be one, two, or three amino acid substitutions.

[0067] The present invention also provides an anti-VASP antibody or antigen-binding fragment selected from (1) 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; (2) having light chain CDR sequences shown in SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15, and 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 with (1) or (2).

[0068] In some embodiments, the present invention provides an antibody or antigen-binding fragment having 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, or having 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.

[0069] In other embodiments, the antibody or antigen-binding fragment provided by this invention has one or more conserved amino acid substitutions compared to the above sequence. "Antigen-binding fragment" refers to an antibody fragment that maintains its ability to specifically bind to antigens. "Conserved amino acid substitution" refers to the substitution of an amino acid by another amino acid with a biologically, chemically, or structurally similar residue. Biological similarity means that the substitution does not impair the biological activity of the VASP antibody or the VASP antigen. Structural similarity 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 both hydrophilic or hydrophobic. For example, hydrophobic residues such as isoleucine, valine, leucine, or methionine can be substituted for each other. Alternatively, polar amino acids can be used, such as arginine replacing lysine, glutamic acid replacing aspartic acid, glutamine replacing asparagine, serine replacing threonine, etc.

[0070] The antibodies described in this invention can also be directly synthesized from the variable region amino acid sequence provided by this invention, and can be further chemically modified to derive different antibody analogs.

[0071] In some specific embodiments, the present invention provides an antibody or antigen-binding fragment comprising at least one of the following:

[0072] (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;

[0073] (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;

[0074] Compared to (a) or (b), the sequence identity is at least 80% of the amino acid sequence.

[0075] In some embodiments, the present invention provides an antibody or antigen-binding fragment having a light chain variable region sequence as shown in SEQ ID NO:1 and a heavy chain variable region sequence as shown in SEQ ID NO:2, or having a light chain variable region sequence as shown in SEQ ID NO:11 and a heavy chain variable region sequence as 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 conserved amino acid substitutions compared to the amino acid sequences 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 conserved amino acid substitutions compared to the amino acid sequences shown in SEQ ID NO:2 or SEQ ID NO:12. Of course, these conserved amino acid substitutions do not alter the biological function of the antibody or antigen-binding fragment. In some specific embodiments, these conserved amino acid substitutions may occur on amino acids in the heavy chain variable region and light chain variable region other than the CDR region.

[0076] According to specific embodiments of the present invention, the antibody or antigen-binding fragment is a single-chain antibody, a multimeric antibody, a CDR transplantation antibody, or a small molecule antibody. For example, the antibody is a single-chain antibody. For example, the small molecule antibodies mentioned include Fab antibodies, Fv antibodies, single-chain antibodies, and at least one of the smallest recognition units.

[0077] According to a specific embodiment of the present invention, the light chain variable region sequence of the provided HC8 antibody is as follows:

[0078] DIVLTQSPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPWTFGGGTKLEIK

[0079] (SEQ ID NO:1)

[0080] According to a specific embodiment of the present invention, the heavy chain variable region sequence of the provided HC8 antibody is as follows:

[0081] LVKLEESGGGLVKPGGSLKLSCAVSGFAFSSYDMSWVRQTPEKRLEWVAYIDNGGGYTYYPDTVKGRFTISRDDNAKNTLYLQMSSLKSEDTAMYYCVRLGRDYWGQGTTLTVSS (SEQ ID NO: 2)

[0082] According to a specific embodiment of the present invention, the light chain variable region sequence of the provided HC10 antibody is as follows:

[0083] DIVLTQSPATLSVTPGYRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYVSQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGTKLELK (SEQ ID NO: 11)

[0084] According to a specific embodiment of the present invention, the heavy chain variable region sequence of the provided HC10 antibody is as follows:

[0085] DFKLQESGGGLVQPGGSRKLSCEASGFTISSFGMHWVRQAPEKGLEWVAYISSGSNTIYYADTVKGRFTISRDIPKNTLFLQMTSLRSEDTAMYYCARRLAYWGQGTLVTVSA

[0086] (SEQ ID NO:12)

[0087] Polynucleotides, expression vectors, recombinant cells, hybridoma cells

[0088] In the process of preparing or obtaining these antibodies, the polynucleotides expressing these antibodies can be linked to different vectors and then expressed in different cells to obtain the corresponding antibodies.

[0089] Therefore, the present invention also provides an isolated polynucleotide encoding the aforementioned antibody or antigen-binding fragment. The provided nucleotide sequence can be obtained using techniques known to those skilled in the art. Furthermore, it can be species-optimized for easier expression in mammalian cells.

[0090] Based on the variable region (or CDR region) sequences of the antibody heavy and light chains 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 means of chemical synthesis and / or expression of antibody genes in heterologous cells (i.e., recombinant protein expression).

[0091] This invention also provides an expression vector comprising the isolated polynucleotides described above. When ligating the isolated polynucleotides to the vector, the polynucleotides can be directly or indirectly linked to control elements on the vector, as long as these control elements can control the translation and expression of the polynucleotides. These control elements can be directly derived from the vector itself or are exogenous, i.e., not derived from the vector itself. Of course, the polynucleotides and control elements need to be operatively linked. In this context, "operatively linked" means ligating a foreign gene to the vector so that the control elements within the vector, such as transcriptional control sequences and translational control sequences, can perform their intended functions of regulating the transcription and translation of the foreign gene. The polynucleotides used to encode the antibody heavy and light chains can be inserted independently into different vectors, but commonly they are inserted into the same vector. Commonly used vectors include plasmids, phages, etc., such as the pCDNA 3.1 plasmid.

[0092] This invention also provides a recombinant cell containing the expression vector. The expression vector can be introduced into mammalian cells to construct recombinant cells, which can then be used to express the antibody or antigen-binding fragment provided by this invention. The corresponding antibody can be obtained by culturing these recombinant cells. These usable mammalian cells can be, for example, CHO cells.

[0093] The present invention also provides a hybridoma cell that can be generated by the classical method for preparing monoclonal antibodies.

[0094] In classic monoclonal antibody preparation methods, a suitable antigen is first required, which is then used to immunize animals. To prepare anti-VASP antibodies (or antibody analogs), the suitable antigen must contain VASP. This antigen can be obtained through isolation and purification from natural human tissue or blood, chemical synthesis, artificial recombinant expression, or a combination of these methods. The antigen is then prepared for use in animal immunization and antibody screening.

[0095] In classic monoclonal antibody preparation methods, animals are first immunized with artificially recombinant VASP antigen, and blood is collected at intervals to verify whether the animals have developed an antibody response to the artificially recombinant VASP antigen. Then, B cells are isolated from the spleens of animals with antibody responses and fused in vitro with immortalized myeloma cells to obtain hybridoma cells. These hybridoma cells are then extremely diluted in culture plates and regrow (monoclonal hybridoma cell lines), and the culture supernatant of these hybridoma cell lines is collected to detect whether they contain specific antibodies against the antigen. Based on antibody yield, quality, and cell line growth characteristics, the optimal monoclonal antibody production cell line can be selected for subsequent monoclonal antibody production.

[0096] Other methods can also be used to obtain monoclonal antibodies. For example, spleen cells from the aforementioned animals can be isolated and incubated with labeled antigens (such as fluorescein-labeled VASP). Since antibody-producing B cells typically have antibody molecules present on their cell membranes, these cells bind to the labeled antigens (staining) and can then be sorted using a fluorescence flow cytometry sorter. The mRNA from these sorted B cells can be isolated, and a library of antibody variable region cDNA can be obtained through in vitro reverse transcription and specific PCR. 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 that plasmid. These host cells (or phages) can be isolated and purified (cloned) using various methods (such as the previously described cell limiting dilution culture method, or phage plating method). These cell lines or phage clones can be used to produce antibodies and to analyze and identify the antibodies.

[0097] The antibody or antigen-binding fragment, antibody analog, polynucleotide sequence, expression vector, recombinant cell, hybridoma cell provided by the present invention play a role in the auxiliary diagnosis of VASP deficiency and related diseases, and the enzyme immunoassay kit based on HC8 and HC10 antibodies greatly improves the accuracy and convenience of diagnosis.

[0098] According to a preferred embodiment of the present invention, animal immunization is performed by multiple subcutaneous injections at multiple sites. This increases the titer of the final animal serum and improves the positive clone rate.

[0099] According to a preferred embodiment of the present invention, the addition of the HAT screening reagent is delayed from the day of fusion to the second day, which significantly improves the cell positivity rate.

[0100] According to a preferred embodiment of the present invention, the VASP-positive samples used in the present invention are all VASP-positive serums detected by liquid chromatography-mass spectrometry, to ensure the authenticity of the subsequent kit detection.

[0101] 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 the 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.).

[0102] This invention provides monoclonal antibodies HC8 and HC10 against VASP, which exhibit high affinity and specificity. It also provides in vitro diagnostic kits based on these antibodies. This invention addresses the current market shortage of antibody raw materials and kits for VASP detection, and can serve as an auxiliary diagnostic tool for VASP syndrome and related neurological diseases, filling a gap in the domestic market.

[0103] The nucleic acids encoding the heavy and / or light chains of the antibodies of this invention are within the scope of this invention. Based on the amino acid sequences of the heavy and / or light chains, 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 are defined according to the Kabat CDR encoding rules. Those skilled in the art should understand that CDR sequences obtained from different databases may differ, but these variations should all be included within the scope of protection of this invention.

[0104] Table 1

[0105]

[0106]

[0107]

[0108]

[0109]

[0110] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0111] Example 1: Preparation of recombinant VASP protein

[0112] 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 nucleotide sequence for recombinant expression 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 the sequence was then synthesized by a gene company. The synthesized gene and pGEX-6P-1 were double-digested with restriction endonucleases BamHI and XhoI, respectively. The gene fragments recovered from the gel were ligated overnight at 16°C using 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 diagram is shown in [link to plasmid diagram]. Figure 1 .

[0113] Pour into a container containing 15 ml of SOB medium (each liter of medium contains 20 g of trypsin and 5 g of yeast extract).

[0114] Add 30 μL of ampicillin solution (final concentration 100 μg / mL) to a test tube containing 0.5 g NaCl (pH 7.0). Then add approximately 1-2 μL of glycerol bacteria picked from a sterile pipette tip to the culture medium. Place the test tube on a shaker (Jiangsu Peiying, #DHZ-DA) for fixation and incubate overnight at 37℃ and 200 rpm. Take 4 ml of the overnight culture (1% bacterial solution added) and add it to an Erlenmeyer flask. Incubate at 37℃ and 200 rpm for 4 hours. When the bacterial density (OD600) reaches 0.8-1.0, add 1 MIPTG solution (final concentration 1 mM) to the Erlenmeyer flask and induce incubation at 37℃ and 200 rpm for 4 hours. After induction, add 200 ml of the solution to a centrifuge tube, centrifuge at 10000×g for 20 minutes, and discard the supernatant. The bacterial cells were homogenized using a high-pressure homogenizer (Zhejiang Xinzhi, SCIENTZ-150) at 800 Bar, centrifuged at 10000×g for 20 minutes, and the supernatant was collected after homogenization.

[0115] Soluble proteins in the supernatant were purified using a 5 mL gravity column packed with GST affinity packing material (YEASON, 20507ES10). After bacterial lysis, the supernatant was loaded intact at 1 mL / min. Washing: The affinity column was washed at 1 mL / min with equilibration buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 8.0), 20 column volumes (CV). Elution: Elution was performed with elution buffer containing 10 mM glutathione (reduced form). Elution was performed at 1 mL / min, and the eluent was collected. SDS-PAGE was used to identify the expression of the target protein VASP. Figure 2 ),like Figure 2 The eluent shown (lanes 8, 9, 10) contains recombinant VASP protein at 67.7 kDa. The eluent was mixed and dialyzed overnight into equilibration buffer. The recombinant VASP protein (GST Tag) was concentrated to 1.1 mg / mL using an ultrafiltration tube (Amicon, #UFC5003).

[0116] Example 2: Immunization of BALB / c mice with VASP protein

[0117] BALB / c mice aged 6-8 weeks were immunized according to the following procedure: For the initial immunization, 25 µg of recombinant VASP protein (His Tag, abcam, #ab105601) was mixed with an equal volume of Freund's complete adjuvant, emulsified, and injected subcutaneously at multiple sites. Fourteen days after the initial immunization, a booster immunization was performed using 12.5 µg of recombinant VASP protein mixed with an equal volume of Freund's incomplete adjuvant, emulsified, and then administered. Fourteen days after the second immunization, a booster immunization was performed using 12.5 µg of recombinant VASP protein (His Tag, abcam, #ab105601) mixed with an equal volume of Freund's incomplete adjuvant, emulsified, and then administered. Fourteen days after the third immunization, blood was collected and serum was separated. An ELISA plate was coated with 1 µg / mL of recombinant VASP protein (GSTTag), and an indirect ELISA assay was performed to determine the serum titer. The results showed that the prepared mouse antiserum titer was 1:72900. Typical serum test results from immunized mice are shown in Table 2.

[0118] Table 2

[0119]

[0120] Example 3: Cell Fusion

[0121] In Example 3, cell fusion was performed 3 days after booster immunization of mice. After blood was collected from the mice's eyes, they were euthanized by dislocation, placed in a bottle of 70% alcohol for 2 minutes, and then fixed to a foam board in a biosafety cabinet. The abdominal skin was opened to locate the spleen, which was removed with forceps and gently crushed in a 200-mesh stainless steel filter. The cells were gently washed with DMEM medium (Thermo, 11965092), and then centrifuged at 200 g for 10 minutes at room temperature. The supernatant was discarded and the cells were used for further processing. For feeder cell preparation, mice were euthanized by dislocation, placed in a bottle of 70% alcohol for 2 minutes, and then fixed to a foam board in a biosafety cabinet. The abdominal skin was opened, and PBS was gently injected subperitoneally using a syringe. The feeder cell-containing liquid was washed out from the other side, and then centrifuged at 200 g for 10 minutes at room temperature. The supernatant was discarded and the cells were used for further processing. 0.3 × 10⁻⁶ cells were then used. 7 One FO myeloma cell and 1.1 × 10 8 Mix the spleen cells thoroughly, centrifuge at 200 g for 10 minutes, discard the supernatant, gently vortex to mix, and in a 37°C water bath, add 1 mL of 50% PEG-1450 (Merk, P1458) aqueous solution over 90 seconds. Then add 20 mL of DMEM medium, centrifuge at 200 g for 10 minutes, discard the supernatant, repeat the washing once, centrifuge at 200 g for 10 minutes, discard the supernatant, and obtain hybridoma cells. Seed the cells into 10 96-well culture plates, 150 μL per well. Add 10,000 feeder cells / well to 10 wells of the above-mentioned 96-well cell culture plates, 100 μL per well. After labeling the culture plates, incubate them in a cell culture incubator at 37°C with 5% CO2. On the second day, add HAT selection medium (Merk, H0262) and continue HAT selection culture for 1-2 days. 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. Maintain the HAT selection culture medium for 7-10 days, then switch to HT culture medium (Merk, H0137) and maintain for another 2 weeks. Then, continue culturing in DMEM medium with 20% FBS (ExCell, FSP500). During the above selection culture, when the hybridoma cells cover 1 / 10 of the bottom area of ​​the well, you can start detecting specific antibodies and screening for the desired hybridoma cell lines. During the selection culture, generally change half of the culture medium every 2-3 days.

[0122] Example 4: Screening and subclonal culture of positive hybridoma cell lines

[0123] First, the optimal coating amount of recombinant VASP protein (GST Tag) was determined using a square titration method. 0.5, 1.0, 2.0, and 4.0 µg of recombinant VASP protein (GST Tag) were coated onto 96-well plates, with each concentration represented by 6 wells (3 positive and 3 negative). Square titration was performed using positive serum from immunized mice at different dilutions, with negative serum from unimmunized mice serving as a negative control. 0.5 μg of purified recombinant VASP protein (GST Tag) was coated onto each well of a 96-well ELISA plate and incubated overnight at 4°C. The plates were washed twice with PBST. 200 µL of 1% BSA in PBS was added to each well, and the plates were blocked at room temperature for 2 hours, then patted dry on folded paper. Sample loading: 0.1 mL of the test sample was added to each well, and the plates were incubated at 37°C for 1 hour, followed by washing. Blank wells (without sample), negative control wells, and positive control wells were also prepared. 0.1 mL of freshly diluted antibody was added to each well, and the plates were incubated at 37°C for 1 hour, followed by washing three times. Add enzyme-labeled secondary antibody: Add 0.1 mL of freshly diluted enzyme-labeled antibody to each reaction well. Incubate at 37 ℃ for 1 hour, washing 3 times. Add substrate solution for color development: Add 0.1 mL of TMB substrate solution to each reaction well and incubate at room temperature for 10 minutes. Add 0.1 mL of 1M H2SO4 to each reaction well. Measure OD value to determine the result: Measure the absorbance at 450 nm (A450) using a microplate reader. A positive result is defined as OD value greater than 2.1 times that of the negative control (calculated after zeroing the blank control well). Select single clones of hybridoma cells that are anti-VASP.

[0124] Following the above method, the selected positive hybridoma cells were subcloned. The original wells were diluted with HAT selective medium using a limiting dilution method and then re-distributed into 96-well plates. Cell morphology and quantity were then observed. The cell density was adjusted to 3-5 cells / mL. 100 µL of diluted cells was added to each well of a cell culture plate containing a feeder cell layer prepared the previous day. The plates were incubated statically at 37 ℃ in a 5% CO2 incubator. The medium was changed on day 7, and thereafter every 2-3 days. Cell clone formation was observed on days 8-9, and antibody activity was promptly assessed. Cells from the positive wells were transferred to 24-well plates for further culture. Each clone was cryopreserved as soon as possible, and ultimately 10 hybridoma cell lines were selected for antibody production.

[0125] Example 5: Large-scale preparation of monoclonal antibodies and determination of antibody titer

[0126] (1) Large-scale preparation of monoclonal antibodies

[0127] Eight-week-old BALB / c mice were injected intraperitoneally with 0.5 mL of Freund's incomplete adjuvant, followed by intraperitoneal injection of 1×10⁻⁶ mL two weeks later. 6Hybridoma cells from different cell lines can produce ascites 7-10 days after inoculation. Closely observe the animals' health and signs of ascites. When ascites is abundant but the mice are close to death, euthanize them and aspirate the ascites into test tubes using a dropper. 5-10 ml of ascites can be obtained from one mouse. Alternatively, ascites can be extracted using a syringe, and collection can be repeated several times. Centrifuge the obtained ascites at 3000 g for 10 minutes, discarding the upper layer of oil and the bottom precipitate. Collect the supernatant and aliquot it at -20℃. After thawing and equilibrating the ascites supernatant to room temperature, add 1 / 10 volume of 1 M Tris–HCl (pH 8.0) to adjust the sample pH to 8.0. Equilibrate the protein G affinity column with 20 column volumes of 100 mM Tris-HCl at pH 8.0. Load the ascites supernatant (adjusted to pH 8.0) onto the column, then wash with 20 column volumes of 100 mM Tris-HCl at pH 8.0. Finally, elute the antibody with 100 mM Glycine-HCl at pH 2.5. Add the antibody eluent to a concentration tube (Millipore, UFC801008, 10K) and centrifuge at 3000 g for 20 minutes at room temperature using a centrifuge (Xiangyi, L550). Centrifuge in batches to a final volume of 1 ml per concentration tube (2 tubes). Add 4 mL of 10 mM PBS pH 7.4 buffer and centrifuge again at 3000 × g for 20 minutes at room temperature. Repeat centrifugation three times to bring the antibody buffer to 10 mM PBS pH 7.4, then add 10 mM PBS pH 7.4 to a final volume of 10 mL. Finally, aliquot the concentrated antibody solution into centrifuge tubes at 2 mL / tube and store at -80°C. The antibody concentration was determined using a BCA kit (Solepro, PC0020) to measure the concentration of the purified monoclonal antibody.

[0128] (2) Antibody titer determination

[0129] The titers of antibodies against different VASP cell lines were detected using an indirect ELISA method. Recombinant VASP protein (GST Tag) was diluted with PBS and coated into 96-well microplates at a concentration of 0.2 µg / mL (100 µL / well). After incubation at 4°C overnight, the plates were washed twice with PBST solution (300 µL / well each time) and dried. The plates were then blocked with 200 µL / well of 1% BSA in PBS and incubated at room temperature for 2 hours. After blocking, the plates were dried. Anti-VASP cell line antibodies of different cell lines were serially diluted with PTB to 1020.6–0.5 ng / mL and incubated at 37°C for 1 hour. The plates were then washed twice with PBST solution (300 µL / well each time) and dried. Finally, HRP-labeled goat anti-mouse antibody diluted 5000-fold with PTB was added and incubated at 37°C for 1 hour. The plates were then washed twice with PBST solution (300 µL / well each time). After washing, pat dry. Add 100 µL of TMB substrate solution per well and react at room temperature for 10 minutes. Stop the reaction by adding 100 µL of 1M H₂SO₄ per well. Measure the absorbance at 450 nm (A450) using a microplate reader. Plot a saturation curve for each monoclonal antibody to determine the optimal antibody concentration for antigen saturation and the half-maximal saturation concentration (IC50). Results are shown in Table 3.

[0130] Table 3

[0131]

[0132] Example 6: Screening of anti-VASP monoclonal antibody pairs

[0133] (1) Using epitope superposition experiments to preliminarily screen antibody pairs

[0134] Epitopes of antibodies against different VASP cell lines were detected using an indirect ELISA epitope superposition assay. Recombinant VASP protein (GST) was diluted with PBS and coated into 96-well microplates at a concentration of 0.2 µg / mL (100 µL / well). After incubation at 4°C overnight, the plates were washed twice with PBST solution (300 µL / well each time) and then dried. The plates were then blocked with 200 µL / well of 1% BSA in PBS solution at room temperature for 2 hours and then dried. Following the half-maximal saturation concentration (IC50) for different cell lines in "Example 7", PTB-diluted antibodies against different VASP cell lines (one or a mixture of two antibodies) were added and reacted at 37°C for 1 hour. The plates were then washed twice with PBST solution (300 µL / well each time) and then dried. Add PTB to dilute HRP-labeled goat anti-mouse antibody 5000-fold, and incubate at 37°C for 1 hour. Wash the microplate twice with PBST solution, 300 µL / well each time. Pat dry after washing. Add TMB substrate solution, 100 µL / well, and incubate at room temperature for 10 minutes. Stop the reaction by adding 100 µL / well of 1M H2SO4. Measure the absorbance at 450 nm (A450) using a microplate reader.

[0135] The first and second saturated concentrations of monoclonal antibodies, as well as a mixture of both saturated concentrations, were bound to the antigen, and the corresponding A450 values ​​were measured, namely A1A2 and A1+2, respectively. The additive index (AI) was obtained from the A value, calculated using the formula AI (%) = [2 × A1+2 / (A1+A2) - 1] × 100%. Results are generally judged as AI > or < 50%. If the monoclonal antibody concentration can saturate the antigen, and two monoclonal antibodies target the same or similar epitopes, AI ≤ 50%; if the two antibodies target different epitopes, AI ≥ 50%. The results are shown in Table 4. The results indicate that 5 monoclonal antibodies recognized the same epitope, while the other 2 recognized other epitopes. The additive index AI (%) was obtained by detecting antibodies from different clone numbers. Paired antibodies with AI (%) > 90 (23A3, 30A3, 8D5, 18H1, 2H3, HC10, 5C12, HC8, 8E6) were selected for labeling and pairing experiments. The results are shown in Table 4.

[0136] Table 4

[0137]

[0138] (2) Antibody labeling

[0139] Take 0.5 mL of 20 nmol (3 mg) of antibody (5C12, HC10, 8E6, 23A3, HC8, HC10) and add it to a dialysis bag (10 kDa, 1 cm wide). 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 in 1 L of 10 mM carbonate buffer (pH 9.5) and dialyze at room temperature with stirring for 2 hours, preparing for coupling with activated HRP. At the same time, accurately weigh 1 mg of HRP using an analytical balance and dissolve it in 0.2 mL of ultrapure water to make the HRP concentration 5 mg / mL. Add 40 µl of 0.1 M NaIO4 to the above HRP solution, place 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, 1 cm wide) 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. Mix the antibody and HRP (for the coupling reaction) and incubate on a horizontal shaker at room temperature in the dark for 4 hours. After the coupling reaction is complete, add 10 μL of freshly prepared NaBH4 (using pre-cooled ultrapure water) and incubate overnight at 4°C in the dark to terminate the reaction. Transfer the antibody solution after the coupling reaction is terminated to a dialysis bag (10 kDa, 1 cm wide) and dialyze at room temperature with stirring for 2 hours. Finally, transfer the solution to a brown centrifuge tube and store at 4°C.

[0140] (3) Identification of optimal antibody pairing

[0141] The pairing of antibodies against different VASP cell lines was compared using an ELISA sandwich assay to select the optimal paired antibody. Antibodies from different cell lines were diluted with PBS and coated into 96-well microplates at 1 µg / mL (100 µL / well). After incubation at 4°C overnight, the plates were washed twice with PBST solution (300 µL / well each time) and dried. The plates were then blocked with 200 µL / well of 1% BSA-containing PBS solution at room temperature for 2 hours and dried. Different concentrations of recombinant VASP protein diluted with PTB were added, and the plates were incubated at 37°C for 1 hour. The plates were then washed twice with PBST solution (300 µL / well each time) and dried. Finally, HRP-labeled anti-VASP antibodies of different cell lines diluted with PTB were added, and the plates were incubated at 37°C for 1 hour. The plates were then washed twice with PBST solution (300 µL / well each time) and dried. Add 100 µL of TMB substrate solution to each well and react at room temperature for 10 minutes. Terminate the reaction by adding 100 µL of 1 M H₂SO₄ to each well. Measure the absorbance at 450 nm (A450) using a microplate reader. Compare the detection ranges of different antibodies for detecting recombinant VASP protein (see Table 5). The optimal antibody pair is HC8-coated antibody followed by HC10-labeled antibody.

[0142] Table 5

[0143]

[0144] Example 7: Sequence Analysis of Monoclonal Antibodies

[0145] (1) Identification of monoclonal antibody subtypes

[0146] Hybridoma cell lines HC8 and HC10 were cultured in DMEM medium (GIBCO, #C11995500BT) supplemented with 10% serum in 10 cm diameter cell culture dishes (37℃, 5% CO2). After 7 days of culture, the cells were transferred to 15 ml centrifuge tubes, counted using a hemocytometer, and 5 × 10⁶ cells were collected. 6 Centrifuge the cells at 200 g for 5 minutes, discard the supernatant, and invert the centrifuge tube to drain the liquid. Use the QIAGEN reverse transcription kit (Qiagen, 74134) to synthesize cDNA from the cells.

[0147] Antibody subtypes were determined by PCR using subtype-specific primers. The synthesized cDNA was used as the PCR template. The PCR reaction solution consisted of: 0.25 μL TAKARA Ex Taq (5 U / µL, TAKARA, RR001B); 5 µL 10×Ex Taq Buffer; 4 µL dNTP mixture (2.5 mM each); 1 µL template cDNA; 1 µL upstream primer (100 µM); 1 µL downstream primer (100 µM); and double-distilled water to a total volume of 50 µL. The PCR temperature program was: 94℃ for 5 minutes of pre-denaturation, followed by 30 cycles of 94℃ for 1 minute, 57℃ for 1 minute, and 72℃ for 1 minute, and then extension at 72℃ for 10 minutes. After the reaction, 10 µL of each PCR product was loaded onto a 1% agarose gel for electrophoresis. The antibody subtype was deduced from the PCR product results (Table 6). The monoclonal antibody HC8 obtained in this 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.

[0148] Table 6

[0149]

[0150] where S = C or G, M = A or C, R= A or G, and W = A or T

[0151] (2) Sequencing of the variable region (V region) of the hybridoma cell lines HC8 and HC10 antibodies

[0152] The V region fragments of the antibodies from cell lines HC8 and HC10, obtained after PCR amplification, were cut from agarose gels and extracted using a DNA extraction kit (Qiagen, 74134). The extracted DNA fragments were ligated into the pEASY-T1 cloning vector and transformed into Trans1-T1 competent cells (Transgen, CT101-1). Transformed bacterial colonies were picked and cultured overnight in LB medium for DNA sequencing. The light chain V region nucleic acid sequence of the anti-VASP antibody (HC8) provided by this invention is shown in SEQ ID NO. 17, and the heavy chain V region nucleic acid sequence is shown in SEQ ID NO. 18. The light chain V region nucleic acid sequence of the antibody (HC10) is shown in SEQ ID NO. 19, and the heavy chain V region nucleic acid sequence is shown in SEQ ID NO. 20.

[0153] Example 8: Application of an enzyme immunoassay kit prepared using antibodies HC8 and HC10 in the detection of VASP in human serum.

[0154] (1) Horseradish peroxidase (HRP) labeling of antibody HC10 and identification of the labeled product

[0155] Add 0.5 mL of antibody HC10 (20 nmol, 3 mg) to a dialysis bag (10 kDa, 1 cm wide) and 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 at room temperature for 2 hours with stirring, in preparation for coupling with activated HRP.

[0156] Meanwhile, accurately weigh 1 mg of HRP using an analytical balance and dissolve it in 0.2 mL of ultrapure water to achieve an HRP concentration of 5 mg / mL. Add 40 µl of 0.1 M NaIO4 to the above HRP solution and place it on a horizontal shaker to react (activate) at room temperature in the dark for 20 minutes. Add the activated HRP solution to a dialysis bag (10 kDa, 1 cm wide) 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.

[0157] Mix the above antibodies with HRP (for conjugation), place on a horizontal shaker, and incubate at room temperature in the dark for 4 hours. After the conjugation reaction is complete, add 10 μL of freshly prepared NaBH4 (using pre-cooled ultrapure water) and incubate overnight at 4°C in the dark to terminate the reaction. Transfer the antibody solution after the conjugation reaction is terminated to a dialysis bag (10 kDa, 1 cm wide) and dialyze at room temperature with stirring for 2 hours. Finally, transfer the solution to brown centrifuge tubes and store at 4°C.

[0158] (2) Determination of the standard curve of the enzyme immunoassay kit prepared using antibodies HC8 and HC10

[0159] An ELISA sandwich assay kit was prepared using antibody HC8 diluted in PBS and coated into 96-well microplates at a concentration of 1 µg / mL (100 µL / well). After incubation at 4°C overnight, the plates were washed twice with PBST solution (300 µL / well each time) and then blotted dry. The plates were then blocked with 200 µL / well of 1% BSA in PBS and incubated at room temperature for 2 hours, followed by blotting dry. Different concentrations of VASP recombinant protein diluted with PTB were added and incubated at 37°C for 1 hour. The plates were then washed twice with PBST solution (300 µL / well each time) and blotted dry. Finally, HRP-labeled HC10 antibody diluted with PTB was added and incubated at 37°C for 1 hour. The plates were then washed twice with PBST solution (300 µL / well each time) and blotted dry. Add 100 µL of TMB substrate solution to each well and react at room temperature for 10 minutes. Terminate the reaction by adding 100 µL of 1 M H₂SO₄ to each well. Measure the absorbance at 450 nm using a microplate reader (A450). The linear relationship between sample concentration and absorbance is shown in the figure. Figure 3 As shown, the results indicate that curve R 2 =0.994, the standard curve range of this method is 0-960ng / ml.

[0160] (3) Enzyme immunoassay kits prepared using antibodies HC8 and HC10 were used to detect VASP in human serum.

[0161] Antibody HC8 was diluted with PBS and coated into 96-well microplates at a concentration of 1 µg / mL (100 µL / well). After incubation at 4°C overnight, the plates were washed twice with PBST solution (300 µL / well each time) and dried. The plates were then blocked with 200 µL / well of 1% BSA in PBS and incubated at room temperature for 2 hours, followed by drying. Different concentrations of VASP recombinant protein diluted with PTB were added and incubated at 37°C for 1 hour. The plates were then washed twice with PBST solution (300 µL / well each time) and dried. HRP-labeled HC10 antibody diluted with PTB was added and incubated at 37°C for 1 hour. The plates were then washed twice with PBST solution (300 µL / well each time) and dried. TMB substrate solution was added at 100 µL / well and incubated at room temperature for 10 minutes. The reaction was terminated by adding 100 µL / well of 1M H2SO4. The absorbance was measured at 450 nm using a microplate reader (A450). The enzyme immunoassay kit of this invention can be detected via OD... 450 Substitute the numerical values ​​into the standard curve ( Figure 3 The results of the quantitative detection of VASP in human serum, as shown in Table 7, indicate that this kit can preliminarily determine positive and negative serum samples.

[0162] Table 7

[0163]

[0164] (4) Identification of the specificity of antibodies against HC8 and HC10 in detecting natural VASP

[0165] The specificity of the antibodies against HC8 and HC10 for detecting native VASP was evaluated using a sandwich ELISA. In this experiment, antibody HC8 was diluted with PBS and coated into 96-well microplates at a concentration of 1 µg / mL, with a volume of 100 µL / well. After overnight coating at 4°C, the microplates were washed twice with PBST solution, 300 µL / well each time. After washing and drying, the microplates were blocked with PBS solution containing 1% BSA, 200 µL / well, at room temperature for 2 hours. After drying, different concentrations of cell lysis buffer (HeLa) and recombinant VASP protein were diluted with PTB. The lysis buffer from the VASP knockout HeLa cell line (abcam, #ab265892) was used as a negative control because it does not express VASP protein. The microplates were incubated on a shaker at room temperature for 30 minutes, then added to the blocked microplates at 100 µL / well and incubated at 37°C for 1 hour. Pat dry, wash the plate three times with PBST (300 µL / well each time). Pat dry, add 1000-fold diluted HRP-labeled HC10 antibody to the plate with PTB, and incubate at 37°C for 1 hour. Wash the plate three times with PBST (300 µL / well). Pat dry, add 100 µL of TMB substrate solution to the plate, incubate at room temperature for 10 minutes, then add 100 µL of 1M H2SO4 to stop the reaction. Measure the absorbance at 450 nm (A450) using a microplate reader. The experimental results are shown in Table 8, indicating that the antibody has good specificity for detecting VASP in natural cells using HC8 and HC10.

[0166] Table 8

[0167]

[0168] This invention provides a monoclonal antibody against VASP, which exhibits high affinity and specificity, and also provides an in vitro diagnostic detection kit based on this antibody. This invention addresses the current market shortage of antibody raw materials and kits for VASP detection.

[0169] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled 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 its antigen-binding fragment for detecting VASP, characterized in that, The amino acid sequences of the complementary determinant regions LCDR1, LCDR2, and LCDR3 of the light chain variable region 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 complementary determinant regions HCDR1, HCDR2, and HCDR3 of the heavy chain variable region are shown in SEQ ID NO.6, 7, and 8, respectively.

2. The antibody or its antigen-binding fragment as described in claim 1, characterized in that, 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 its antigen-binding fragment as described in claim 1 or 2, 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.9, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.

10.

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

5. The antibody or its antigen-binding fragment as described in claim 4, characterized in that, 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 its antigen-binding fragment as described in 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, Fv, or a single-chain antibody.

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

9. A biomaterial, characterized in that, The biomaterial contains the nucleic acid molecule as described in claim 8; the biomaterial is an expression cassette, a vector, or a host cell.

10. An antibody conjugate, characterized in that, The antibody-drug conjugate is obtained by conjugating the antibody or its antigen-binding fragment according to any one of claims 1-6 with a label, wherein the label is 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 for VASP, characterized in that, The antibody composition comprises a first antibody and a second antibody, wherein the first antibody is selected from the antibodies described in any one of claims 1-3, and the second antibody is selected from the antibodies described in any one of claims 4-6.

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

13. The use of the antibody or antigen-binding fragment thereof 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, said product being a product for detecting platelet dysfunction or a product for detecting thrombotic diseases.

14. A reagent kit, characterized in that, It comprises the antibody or antigen-binding fragment thereof 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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