A monoclonal antibody specific for a vascular expansion-stimulated phosphoprotein and uses thereof

Abstract

The application discloses a vascular expansion stimulating phosphoprotein specific monoclonal antibody and application. In the monoclonal antibody, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 5. The monoclonal antibody only recognizes VASP, and does not exist cross reaction with EVL and Mena in the same family, and the antibody can recognize all isomers of VASP, and can recognize phosphorylated and non-phosphorylated VASP.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a vasodilatory stimulating phosphoprotein-specific monoclonal antibody and its applications. Background Technology

[0002] The enabled / vasodilator-stimulated phosphoprotein (Ena / VASP) family is a group of multifunctional proteins related to actin regulation. Its members include vasodilator-stimulated phosphoprotein (VASP), Ena / VASP-like protein (EVL), mammalian enabled (Mena), Ena (Drosophila Enabled), C. elegans Unc-34 (Caenorhabditis eleganshomologe of enabled), and DdVASP (Dictyostelium discoideum VASP). VASP, EVL, and Mena are found in vertebrates, Ena and C. elegans Unc-34 are found in invertebrates, and DdVASP is found in the genus Dictyostelium.

[0003] VASP is the founding member of this family, initially isolated from platelets and identified as a target of cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG). Subsequently, based on sequence similarity, two other mammalian family members, EVL and Mena, were identified. Ena / VASP family proteins are widely distributed in the body, abundant in the lungs, stomach, large intestine, and smooth muscle, and also present in the brain, heart, kidneys, and small intestine. Ena / VASP family proteins are mainly located at the edges of cell sheet-like pseudopodia and in areas of high actin turnover such as focal adhesion and adhesion junctions. By promoting actin nucleation and fibrous elongation, they regulate cytoskeleton remodeling, directly affecting cell motility and pseudopodia formation. Ena / VASP family members are upregulated in various cancers, including breast cancer, cervical cancer, colorectal cancer, malignant melanoma, pancreatic cancer, and lung cancer, and are closely related to biological processes such as tumor cell migration, invasion, endothelial pathway finding, cell-cell adhesion, and communication. In addition, these proteins also have many important physiological functions in cardiovascular cells, including regulating platelet activation, endothelial barrier function, cardiomyocyte contraction, conduction vasodilation, and smooth muscle relaxation. VASP is specifically expressed in platelets and participates in regulating platelet morphological changes and activation processes. Its phosphorylation state is regulated by the P2Y12 receptor signaling pathway, directly affecting platelet aggregation and contraction. P2Y12 receptor antagonists such as clopidogrel inhibit platelet activation by blocking VASP phosphorylation through the ADP-P2Y12 signaling pathway. Therefore, VASP phosphorylation level can serve as a specific indicator for assessing the antiplatelet effect of clopidogrel. By detecting the degree of VASP phosphorylation, the inhibitory effect of P2Y12 receptor can be quantified, guiding personalized medication.

[0004] Members of the Ena / VASP family share 60-70% structural homology, all containing the same domains, including the EVH1 (Ena / VASP homology 1) domain at the amino terminus, the proline-rich region PRR (Proline-Rich Region) in the middle, and the EVH2 (Ena / VASP homology 2) domain at the carboxyl terminus (see appendix). Figure 1 From the following reference: Benz, PM; T.; Laban, H.; Zink, J.; Ulrich, L.; Groneberg, D.; Boon, RA; Polly, P.; Renne, T.; de Wit, C. et al., Cardiovascular Functions of Ena / VASP Proteins: Past, Present and Beyond, Cells 2023, 12(13):1740. doi:10.3390 / cells12131740.PMID:37443774; PMCID:PMC10340426). The EVH1 domain contains approximately 115 amino acid residues and is present in a large number of multi-domain proteins involved in regulating the actin cytoskeleton or signal transduction. Four main families of proteins contain the EVH1 domain: Ena / VASP family proteins, Wiscottt-Aldrich syndrome (WASP) proteins, Homer / Vessel synaptic scaffold proteins, and Sprouty-associated proteins (SPRED) with the EVH1 domain. A distinctive feature of the EVH1 domain is the highly conserved aromatic side chains Y16, W23, and F79 (amino acid sequence numbering based on the human VASP isoform 1, accession number ALQ33839.1). The PRR region is composed of glycine (G) and multiple proline (P) motifs, and this region can bind to SH3 fragments and another important actin regulator, profilin. Mena has the longest PRR region, spanning 64 amino acids, followed by VASP's PRR region with 50 amino acids, and EVL's PRR region is the shortest with only 25 amino acids. VASP's PRR region contains three GP5 motifs, Mena's contains GP6 and GP9 motifs, and EVL's contains one GP8 motif. The EVH2 domain of Ena / VASP family proteins is located at the carboxyl terminus of the protein and consists of 160-190 amino acids, which can be further divided into three parts: A (225-245), B (259-278), and C (343-377) (the numbers are based on the human VASP amino acid sequence number). Region A contains the phosphorylation site serine 239 (S239); Region B is the region where Ena / VASP family proteins bind to actin filaments, and this region contains the phosphorylation site threonine 278 (T278); Region C is essential for the formation of the Ena / VASP tetramer and is a hallmark structure of the Ena / VASP family.

[0005] Ena / VASP family proteins are recognized targets of serine, threonine, and tyrosine protein kinase pathways. Phosphorylation can control the subcellular targeting of Ena / VASP family proteins and their ability to regulate actin dynamics. Notably, the phosphorylation status of VASPs is frequently used to assess the dependence of PKA / PKG in cardiovascular cells and platelet reactivity. The most important tyrosine phosphorylation sites in human VASPs are Y16 and Y39 within the EVH1 domain, which are conserved in both EVL and Mena. Serine phosphorylation sites include S157 in the PRR region, corresponding to EVL S160 and Mena S265; S239 in the EVH2 domain adjacent to the GAB motif, corresponding to Mena S405, which is not conserved in EVL; and S322 in the EVH2 domain located between the FAB and tetrameric motifs, corresponding to EVL S362 and Mena S512. The most important threonine phosphorylation site in VASP is T278 of the EVH2 domain, but it is not conserved in EVL or Mena.

[0006] The sequence analysis above shows that all members of the Ena / VASP family contain the same domains. The EVH1 and EVH2 domains are highly conserved and exhibit high sequence homology among Ena / VASP family members. Furthermore, each member of the Ena / VASP family possesses multiple different transcriptional splicing variants; for example, VASP has 7 isoforms, EVL has 5 distinct isoforms, and the Mena gene has the most complex structure, exhibiting abundant alternative splicing. Ena / VASP family proteins perform overlapping biological functions in the same or different tissues and organs. To accurately study their individual functions and develop corresponding therapeutic drugs, highly specific antibodies capable of accurately distinguishing different members of the Ena / VASP family, as well as specific antibodies targeting different phosphorylation sites, are needed.

[0007] Currently, commercially available monoclonal antibodies target VSAP and its different phosphorylation sites exist. However, the immunogens used in antibodies targeting total VASP (including phosphorylated and non-phosphorylated VASP) are all long amino acid segments (251-363aa, 271-360aa, 276-355aa, 353-385aa), all located within the EVH2 domain. The specific antigenic epitopes recognized by these antibodies are not clearly defined. Furthermore, the EVH2 domain is highly homologous among different members of the Ena / VASP family, and its long amino acid segments may contain cross-reactive antigenic epitopes, leading to unclear specificity of existing antibodies. This may be one reason for some inconsistent or contradictory results in existing studies. Therefore, the purpose of this invention is to prepare a monoclonal antibody that can specifically recognize total VASP (including phosphorylated and non-phosphorylated VASP) but does not cross-react with EVL and Mena from the same family or other proteins with similar domains. Summary of the Invention

[0008] Therefore, the present invention aims to provide a monoclonal antibody specific to vasodilatory stimulating phosphoprotein prepared using a fused hybridoma cell line, wherein the monoclonal antibody recognizes only VASP and does not cross-react with EVL and Mena of the same family, and the antibody can recognize all VASP isoforms and can recognize phosphorylated and non-phosphorylated VASP.

[0009] Therefore, one aspect of the present invention relates to a vasodilatory stimulating phosphoprotein-specific monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, and the light chain variable region comprises light chain CDR1, light chain CDR2, and light chain CDR3, wherein,

[0010] The amino acid sequence of the heavy chain CDR1 is the sequence shown in SEQ ID NO.2 or an amino acid sequence with one conserved amino acid substitution compared to the sequence shown in SEQ ID NO.2;

[0011] The amino acid sequence of the heavy chain CDR2 is the sequence shown in SEQ ID NO.3 or an amino acid sequence with one conserved amino acid substitution compared to the sequence shown in SEQ ID NO.3;

[0012] The amino acid sequence of the heavy chain CDR3 is the sequence shown in SEQ ID NO.4 or an amino acid sequence with one conserved amino acid substitution compared to the sequence shown in SEQ ID NO.4;

[0013] The amino acid sequence of the light chain CDR1 is the sequence shown in SEQ ID NO.6 or an amino acid sequence with one conserved amino acid substitution compared to the sequence shown in SEQ ID NO.6;

[0014] The amino acid sequence of the light chain CDR2 is LVS or an amino acid sequence with one conserved amino acid substitution compared to LVS.

[0015] The amino acid sequence of the light chain CDR3 is the sequence shown in SEQ ID NO.7 or an amino acid sequence with one conserved amino acid substitution compared to the sequence shown in SEQ ID NO.7.

[0016] In a further aspect, the present invention also relates to a vasodilatory stimulating phosphoprotein-specific monoclonal antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.1 and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO.5.

[0017] The present invention also relates to the above-mentioned monoclonal antibody or its antigen-binding fragment, wherein the antibody or antigen-binding fragment is a Fab fragment, a Fab' fragment, an F(ab')2 fragment, a single-chain antibody or a humanized antibody, which, because they retain the variable regions of the light chain and the heavy chain, or only retain the variable region of the heavy chain, are able to recognize and bind to vasodilatory stimulating phosphoproteins.

[0018] Furthermore, the present invention relates to a nucleic acid molecule comprising a nucleic acid encoding the aforementioned antibody or its antigen-binding fragment, and an expression vector comprising the aforementioned nucleic acid molecule, the expression vector being capable of expressing the aforementioned antibody or its antigen-binding fragment. The present invention also relates to a recombinant comprising the aforementioned nucleic acid molecule or the aforementioned expression vector, which can produce the aforementioned antibody or its antigen-binding fragment. On the other hand, the present invention relates to a hybridoma cell line that produces a vasodilatory stimulating phosphoprotein-specific monoclonal antibody, the monoclonal antibody hybridoma cell line secreting the aforementioned monoclonal antibody. Further, the present invention relates to a hybridoma cell line that produces a vasodilatory stimulating phosphoprotein-specific monoclonal antibody, the monoclonal antibody hybridoma cell line being mouse hybridoma cell line 9E06, with accession number CGMCC No. 46354.

[0019] Furthermore, this invention relates to the application of the aforementioned monoclonal antibody or its antigen-binding fragment in the preparation of products for detecting vasodilatory stimulating phosphoproteins. Further, this invention relates to a method for detecting vasodilatory stimulating phosphoproteins, the method comprising using the aforementioned monoclonal antibody or its antigen-binding fragment via an immunobinding method, preferably via immunohistochemistry, Western blotting, or enzyme-linked immunosorbent assay (ELISA). Further, this invention relates to a kit for detecting vasodilatory stimulating phosphoproteins, comprising the aforementioned monoclonal antibody or its antigen-binding fragment, wherein the monoclonal antibody or its antigen-binding fragment binds to vasodilatory stimulating phosphoproteins via an immunobinding method, preferably via immunohistochemistry, Western blotting, or ELISA.

[0020] Furthermore, this invention relates to the use of the aforementioned monoclonal antibody or its antigen-binding fragment in the preparation of products for evaluating the efficacy of clinically used antiplatelet drugs. More specifically, this invention relates to the use of the aforementioned monoclonal antibody or its antigen-binding fragment in the preparation of products for evaluating the efficacy of clinically used antiplatelet drugs, wherein the antiplatelet drug is a P2Y12 receptor blocker.

[0021] Instructions for the Preservation of Biological Materials

[0022] The monoclonal antibody hybridoma cell line of this invention, mouse hybridoma cell line 9E06, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46354 and deposit date of April 25, 2025. The address of the China General Microbiological Culture Collection Center is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. Attached Figure Description

[0023] Figure 1 These are the domains and phosphorylation sites of Ena / VASP family proteins.

[0024] Figure 2 Amino acid sequence alignment analysis of different VASP isomers.

[0025] Figure 3 The recognition specificity identification results of the anti-VASP high-specific monoclonal antibody 9E06, wherein 1 is the anti-VASP high-specific monoclonal antibody 9E06 prepared in this invention, 2 and 3 are commercial anti-VASP antibodies, 4 is a commercial anti-EVL antibody, and 5 is a commercial anti-Mena antibody.

[0026] Figure 4 Immunohistochemical detection of breast cancer tissues with different degrees of differentiation using the highly specific anti-VASP monoclonal antibody 9E06. Detailed Implementation

[0027] The purpose of this invention is to provide a monoclonal antibody specific to vasodilatory stimulating phosphoproteins, prepared using a fused hybridoma cell line. This monoclonal antibody recognizes only VASPs and exhibits no cross-reactivity with EVL and Mena cells of the same family. Furthermore, the antibody can recognize all VASP isoforms, including both phosphorylated and non-phosphorylated VASPs. The specific implementation is as follows: First, highly conserved sequences among different VASP isoforms are analyzed and determined. Second, the distribution of antigenic epitopes in the highly conserved VASP sequences is analyzed to identify high-scoring conserved antigenic epitopes. Third, the high-scoring conserved VASP antigenic epitopes are compared with the amino acid sequences of different isoforms of Mena and EVL cells of the same family, and BLAST analysis is performed in the GenBank database to identify VASP-specific antigenic epitopes. Fourth, a synthetic peptide containing VASP-specific antigenic epitopes is used as an immunogen to immunize mice to prepare the monoclonal antibody, and the specific epitopes recognized by the monoclonal antibody are identified. Fifth, the specificity of the highly specific VASP monoclonal antibody is determined, and its application value in medical detection is evaluated. Finally, the subtype and sequence characterization of the highly specific VASP monoclonal antibody prepared according to this invention are performed. The advantages of the highly specific VASP monoclonal antibody prepared in this invention are as follows: First, the epitope recognized by the highly specific VASP monoclonal antibody of this invention is not in a highly conserved domain among different members of the Ena / VASP family, but in the linker region between the EVH1 domain and the PRR region, which is a highly specific epitope sequence for VASP. Therefore, it only recognizes VASP and does not cross-react with EVL and Mena from the same family. Second, the epitope recognized by the highly specific VASP monoclonal antibody of this invention is a highly conserved sequence among different VASP isoforms, thus enabling the recognition of all VASP isoforms. Third, the epitope recognized by the highly specific VASP monoclonal antibody of this invention does not contain a phosphorylation site, enabling the simultaneous recognition of non-phosphorylated VASP and VASP phosphorylated at different sites.

[0028] On April 25, 2025, the inventors deposited cell line 9E06 at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 46354.

[0029] Subsequently, the inventors sequenced and analyzed the immunoglobulin domain sequence of the monoclonal antibody secreted by the mouse hybridoma cell line CGMCC No. 46354. They found that the amino acid sequence of its heavy chain variable region was 113 amino acids, and its sequence was: QVQLEQSGAELAKPGASVKMSCKASGHTFTNFWMHWIKQRPGQGLDWIGNITPSTG YTDYNQNFKDKATLTADTSSSTAYMQLSSLTSVDSAVYVTTKALGLLGSRNHSHRLL (SEQ ID NO. 1). Among them, the heavy chain CDR1 is located at 26-33 aa, and the amino acid sequence is GHTFTNFW (SEQ ID NO. 2); the heavy chain CDR2 is located at 51-58 aa, and the amino acid sequence is ITPSTGYT (SEQ ID NO. 3); the heavy chain CDR3 is located at 97-102 aa, and the amino acid sequence is TKALGL (SEQ ID NO. 4). The amino acid sequence of the light chain variable region consists of 109 amino acids, and its sequence is: DIVLTQSPASLAVSLGERATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLE SGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGAPSWKS (SEQ ID NO. 5), wherein light chain CDR1 is located at 27-36 aa, and the amino acid sequence is KSVSTSGYSY (SEQ ID NO. 6); light chain CDR2 is located at 54-56 aa, and the amino acid sequence is LVS; light chain CDR3 is located at 93-100 aa, and the amino acid sequence is QHIRELTR (SEQ ID NO. 7).

[0030] The inventors used immunohistochemistry and the breast cancer cell line MDA-MB-231, which simultaneously expresses three members of the Ena / VASP family—VASP, EVL, and Mena—to determine the specificity of the aforementioned monoclonal antibody. They found that the highly specific anti-VASP monoclonal antibody 9E06 prepared in this invention can specifically recognize VASP, including non-phosphorylated VASP and phosphorylated VASP (VASP-P), without any non-specific bands. Furthermore, it specifically recognizes only VASP and does not cross-react with EVL and Mena proteins from the same family.

[0031] As is well known in the art, the CDR regions of the antibody heavy chain and light chain are important amino acid sequence regions for recognizing and binding to corresponding antigens. Furthermore, a single conserved amino acid substitution in the amino acid sequence of these CDR regions generally does not alter the protein structure; therefore, a single conserved amino acid substitution within these regions may still possess the ability to bind to the corresponding antigen. Thus, monoclonal antibodies or their antigen-binding fragments obtained by making a single conserved amino acid substitution to heavy chain CDR1 and / or heavy chain CDR2 and / or heavy chain CDR3 and / or light chain CDR1 and / or light chain CDR2 and / or light chain CDR3 can still recognize and bind to vasodilatory stimulating phosphoproteins. In this patent application, conservative amino acid substitution refers to the replacement of one amino acid in a protein with another chemically similar amino acid. Examples include the substitution between aromatic amino acids Phe, Trp, and Tyr; the substitution between aliphatic amino acids Ala, Gly, Leu, Ile, and Val; the substitution between polar amino acids Gln and Asn; the substitution between basic amino acids Lys, Arg, and His; the substitution between acidic amino acids Asp and Glu; and the substitution between hydroxy amino acids Ser and Thr.

[0032] Those skilled in the art can also use existing techniques to prepare various antibody fragments, i.e., antigen-binding fragments, from the monoclonal antibodies of this invention capable of binding to vasodilatory stimulating phosphoproteins, such as, but not limited to, Fab, Fab', and F(ab')2. The Fab fragment is a region in the antibody structure that can bind to the antigen, consisting of a complete light chain and a variable region VH and a constant region CH1 domain (Fd segment) of the heavy chain. Both the light and heavy chains have a constant region and a variable region, and disulfide bonds link the light and heavy chains. The antigen-binding fragment can be prepared as follows: for example, after enzymatic digestion with papain, antibody IgG is degraded into two Fab fragments and one Fc fragment. Under the action of pepsin, antibody IgG is degraded into an F(ab')2 fragment and a pFc' fragment, and the F(ab')2 fragment is further reduced to form two Fab' fragments. Because the above antigen-binding fragments can still bind to the corresponding antigens, they can be used to prepare products for detecting vasodilatory stimulating phosphoproteins and products for evaluating the efficacy of clinically used antiplatelet drugs, preferably P2Y12 receptor blockers. Antiplatelet drugs are well known in this field.

[0033] Those skilled in the art can also prepare single-chain antibodies (scFv) from the monoclonal antibodies of the present invention using existing techniques. A single-chain antibody is an antibody composed of a heavy chain variable region and a light chain variable region linked by a short peptide linker of several amino acids; it has only one chain and is a synthetically produced antibody. A single-chain antibody may also contain only the heavy chain variable region. The length and amino acid composition of the short peptide linker are well known in the art, and usable short peptide linkers for the monoclonal antibodies of the present invention can be determined through simple, repeatable experiments. The single-chain antibody can be expressed, for example, in *E. coli* using genetic engineering techniques. The single-chain antibody of the present invention prepared in this way has the property of binding to vasodilatory stimulating phosphoproteins and can be used to prepare products for detecting vasodilatory stimulating phosphoproteins and products for evaluating the efficacy of clinically used antiplatelet drugs, preferably P2Y12 receptor blockers.

[0034] Those skilled in the art can design and synthesize nucleic acid molecules encoding the variable region of the aforementioned vasodilator-stimulating phosphoprotein-specific monoclonal antibody. They can also insert the synthesized nucleic acid molecules into a nucleic acid vector to construct an expression vector capable of expressing the vasodilator-stimulating phosphoprotein-specific monoclonal antibody or its antigen-binding fragment. Furthermore, those skilled in the art can introduce the synthesized nucleic acid molecules or constructed expression vectors into organisms such as cells, bacteria, and yeast to obtain recombinant bodies, and express the antibodies or their antigen-binding fragments of the present invention through these recombinant bodies. The expressed antibodies or their antigen-binding fragments can bind to and recognize vasodilator-stimulating phosphoproteins. Therefore, the aforementioned nucleic acid molecules, expression vectors, and recombinant bodies are within the scope of protection of the claims of this invention. Moreover, the above-mentioned techniques are all well-known in the art and can be carried out by those skilled in the art without inventive effort.

[0035] As described above, the antibody or its antigen-binding fragment of the present invention can specifically recognize and bind to vasodilatory stimulating phosphoprotein, and therefore can be used in methods for detecting vasodilatory stimulating phosphoprotein. These methods include any reaction utilizing the antibody or its antigen-binding fragment of the present invention to bind with vasodilatory stimulating phosphoprotein, including but not limited to detection of vasodilatory stimulating phosphoprotein by immunohistochemistry, Western blotting, enzyme-linked immunosorbent assay (ELISA), colloidal gold immunochromatography, fluorescence immunochromatography, chemiluminescence, etc. Furthermore, the antibody or its antigen-binding fragment of the present invention can be used to prepare a kit for detecting vasodilatory stimulating phosphoprotein based on the above-described methods. The kit contains the antibody or its antigen-binding fragment of the present invention, which utilizes the reaction utilizing the antibody or its antigen-binding fragment of the present invention to bind with vasodilatory stimulating phosphoprotein, including but not limited to detection of vasodilatory stimulating phosphoprotein by immunohistochemistry, Western blotting, enzyme-linked immunosorbent assay (ELISA), colloidal gold immunochromatography, fluorescence immunochromatography, chemiluminescence, etc.

[0036] Furthermore, VASP is specifically expressed within platelets, existing in both phosphorylated (VASP-P) and unphosphorylated (VASP) protein forms. Prostaglandin E1 (PGE1) can induce VASP phosphorylation, while adenosine diphosphate (ADP) can activate the P2Y12 receptor, causing VASP dephosphorylation. When ADP and PGE1 are added simultaneously to whole blood, ADP plays a major role in inducing VASP dephosphorylation; if the P2Y12 receptor is blocked by a corresponding receptor blocker, this dephosphorylation cannot occur effectively, resulting in a phosphorylated state of VASP. Therefore, detecting VASP phosphorylation levels using immunological methods can evaluate the clinical use of antiplatelet drugs, prioritizing the efficacy of P2Y12 receptor blockers. P2Y12 receptor blockers are well-known in the field and include, but are not limited to, clopidogrel, prasugrel, ticagrelor, cangrelor, and ticlopidine. Therefore, the monoclonal antibody or its antigen-binding fragment of the present invention can be used in the preparation of products for evaluating the efficacy of antiplatelet drugs used clinically. Furthermore, the above-mentioned monoclonal antibody or its antigen-binding fragment of the present invention can be used in the preparation of products for evaluating the efficacy of antiplatelet drugs used clinically, wherein the antiplatelet drug is a P2Y12 receptor blocker.

[0037] To explain in detail the technical content, objectives, and effects of the technical solution, the following description is provided in conjunction with specific embodiments.

[0038] Example 1: Searching for Ena / VASP family member isomers and amino acid sequences

[0039] We searched the GenBank database (https: / / www.ncbi.nlm.nih.gov / ) of the National Center for Biotechnology Information (NCBI) for all isomers of the Ena / VASP family members VASP, EVL, and Mena, and their amino acid sequences. The amino acid sequences found are summarized in Table 1. A total of 7 VASP isomers, 5 EVL isomers, and 9 Mena isomers with accession numbers and sequences were found.

[0040] Table 1. Isomers and amino acid sequence accession numbers of Ena / VASP family members

[0041] Note: The length in parentheses is the amino acid length.

[0042] Table 2. Amino acid sequences corresponding to the accession numbers in Table 1.

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049] Example 2: Determination of highly conserved sequences in different VASP isomers

[0050] The amino acid sequences of seven VASP isoforms were compared and analyzed using the bioinformatics analysis software DNAMAN 6.0. The results are as follows: Figure 2 As shown, the amino acid sequence homology of the seven isoform proteins is 90.38%, among which the three amino acid segments 1-168aa, 261-291aa and 293-380aa (amino acid sequence numbering based on human VASP isoform 1) have 100% sequence homology, which are highly conserved sequences among different VASP isoforms.

[0051] Example 3: Screening and identification of highly conserved antigenic epitopes specific to VASP

[0052] The distribution of B cell epitopes of VASP protein (isoform 1) was analyzed using the bioinformatics analysis software BIOSUN. First, its full-length amino acid sequence was input, and then the B cell epitopes were analyzed. A total of 18 antigenic epitopes were found in highly conserved sequences of different isoforms, and their epitope sequences are shown in Table 3. Epitopes were screened according to the following principles: First, to obtain highly active immunogens, epitopes with a score of 3.0 or higher were selected, namely epitopes 9, 12, 13, 14, 15, 16, and 17; Second, to recognize both phosphorylated and non-phosphorylated VASPs, epitopes containing phosphorylation sites Y16, Y39, S157, S239, S322, and T278 were avoided, therefore epitope 12 was excluded, leaving epitopes 9, 13, 14, 15, 16, and 17; Third, to specifically recognize VASPs, the remaining epitope sequences were compared with the sequences of all isoforms of EVL and Mena, and the Protein BLAST function on the NCBI website was also used for comparison, excluding epitopes 13, 16, and 17 which have 6 consecutive amino acid sequences completely identical to non-VASP proteins. Finally, the highly conserved VASP-specific epitopes were determined to be epitopes 9, 14, and 15 (in bold in the table below).

[0053] Table 3. Epitope sequences and scores within highly conserved sequences of different VASP isomers.

[0054]

[0055]

[0056] Example 4: Preparation of synthetic peptides containing VASP-specific, highly conserved antigenic epitopes

[0057] To specifically detect VASP proteins in the Ena / VASP family, synthetic peptides were prepared using highly conserved VASP-specific epitopes identified in Example 3 as immunogens for monoclonal antibody preparation. First, highly conserved VASP-specific epitopes 9, 14, and 15 were synthesized into a single synthetic peptide. To ensure epitope integrity, each epitope was preceded and followed by an additional amino acid. Furthermore, to facilitate conjugation, a cysteine ​​residue (C) was added to the N-terminus of the synthetic peptide, resulting in the sequence CVEQQKRQQPRPWEKNSTTSSSVTTSET (SEQ ID NO. 47). The immunogen synthetic peptide was conjugated to hemocyanin KLH to enhance immunogenicity. To screen and identify the epitopes recognized by monoclonal antibodies, synthetic peptides for epitopes 9, 14, and 15 were synthesized. To facilitate conjugation, a cysteine ​​residue (C) was added to the N-terminus of each synthetic peptide. The resulting sequences are: Epitope 9: CVEQQKRQQP (SEQ ID NO. 48); Epitope 14: CRPWEKNSTT (SEQ ID NO. 49); and Epitope 15: CSSSVTTSET (SEQ ID NO. 50). The synthetic peptides were conjugated with bovine serum albumin (BSA) for ELISA plate coating. Peptide synthesis and KLH or BSA conjugation were performed by Shanghai Dechi Biotechnology Co., Ltd., following standard techniques in the field.

[0058] Example 5: Preparation of monoclonal antibodies specifically recognizing VASP

[0059] Using the VASP-specific, highly conserved epitope-specific peptide KLH conjugate synthesized in Example 4 as the immunogen, 6-8 week old female BALB / c mice were immunized with 100 μg of antigen per mouse plus an equal volume of Freund's complete adjuvant. After thorough emulsification with a stirrer, the mice were immunized subcutaneously in the back and intraperitoneally, with 3 mice immunized. A second immunization was performed 4 weeks later, and a third immunization was performed 8 weeks later. For each third immunization, 50 μg of antigen per mouse plus incomplete Freund's adjuvant was emulsified thoroughly with a stirrer and then injected subcutaneously in the back and intraperitoneally. One week after the third immunization, blood was collected from the tail vein of the mice to detect the immune serum titer. Mice with the highest titer were selected for a booster immunization via intraperitoneal injection (50 μg per mouse). Spleen cells were harvested 3 days later for fusion. SP20 myeloma cells were resuscitated and cultured until they entered the logarithmic growth phase. Spleen cells were prepared from the spleens of immunized BALB / c mice. The spleen cells and myeloma cells were mixed in serum-free DMEM medium at a ratio of 9:1, centrifuged at 1500 rpm for 5 minutes, the supernatant was aspirated, and the cells were gently shaken to disperse them. The cells were then fused in a 37°C water bath. 1 mL of pre-warmed 50% PEG fusion cells was added within 1 minute while gently shaking. After the addition was complete, the cells were allowed to stand for 90 seconds. Serum-free DMEM medium was added to terminate the fusion. The cells were incubated at 37°C for 10 minutes, centrifuged at 1500 rpm for 5 minutes, and the pellet was resuspended in HAT medium. The pellet was aliquoted into 96-well cell culture plates containing feeder cells and cultured in a 37°C, 5% CO2 cell culture incubator for 5 days. The medium was changed once with HAT medium. The medium was changed again on day 10. When the fusion cells covered about 60% of the bottom of the wells, the cell culture supernatant was collected and serially diluted with PBS at dilutions of 400, 800, 1600, 3200, and 6400. Positive clones were screened using an indirect ELISA method. The specific method was as follows: BSA conjugates of the synthetic peptides of epitopes 9, 14, and 15 were diluted with carbonate coating buffer to a concentration of 2.5 μg / ml, with 100 μl per well, and incubated overnight at 4°C. The plate was washed twice with washing buffer. 150 μl / well of blocking buffer was added, and the plate was blocked at room temperature for 6 hours. The plate was washed 5 times with washing buffer. After adding 100 μl of sample dilution buffer to each well, 10 μl of cell culture supernatant in different serial dilutions was added, and the plate was incubated at room temperature for 30 min, then discarded. The plate was washed 5 times, and the washed ELISA plate was inverted on absorbent paper to dry. 100 μl / well of HRP-labeled goat anti-mouse IgG antibody was added, and the plate was incubated at room temperature for 30 min. The plate was washed 5 times. 50 μl each of TMB chromogenic solutions A and B were added to each well, and the plate was incubated at room temperature in the dark for 15 min. 50 μl of 2M H₂SO₄ stop solution was added to each well to stop the reaction. The microplate reader was set to a detection wavelength of 450 nm, and the OD value of each well was measured. Readings were taken within 10 minutes after the reading was stopped. A total of 9 positive clones were obtained, all recognizing epitope 9. No positive clones recognizing epitopes 14 and 15 were obtained. Therefore, one clone, 9E06, with a titer of 1:6400, was selected from the positive clones recognizing epitope 9 for subsequent experiments.

[0060] Hybridoma cell lines were collected and cultured in 1640 medium containing 10% fetal bovine serum. Each male BALB / c mouse was intraperitoneally injected with 0.5 mL of liquid paraffin. After 10 days, cells were collected and resuspended in 10 mL of physiological saline at a cell density of 1 × 10⁻⁶ cells / mL. 7 0.5 mL of antibody was administered intraperitoneally to each mouse. Two weeks later, ascites fluid was collected. One mL of ascites fluid was taken, and 1 mL of physiological saline and 2 mL of acetate buffer (pH 4.8) were added. Then, 25 μL / mL of caprylic acid was added, and the mixture was stirred for 30 minutes. The mixture was centrifuged at 12,000 rpm for 30 minutes. The supernatant was collected and mixed with an equal volume of physiological saline. Ammonium sulfate (0.23 g / mL) was added, and the mixture was stirred for 30 minutes. The mixture was then incubated overnight at 4°C. After centrifugation at 12,000 rpm for 30 minutes, the supernatant was discarded. The precipitate was dissolved in PBS buffer, and the mixture was then dialyzed against PBS at 4°C for 2 days with a volume greater than 20 times the volume, changing the PBS several times. The purified antibody was aliquoted and stored at -20°C.

[0061] Example 6: Identification of the specificity of the VASP-highly specific monoclonal antibody 9E06

[0062] MDA-MB-231, a breast cancer cell line simultaneously expressing three members of the Ena / VASP family—VASP, EVL, and Mena—was selected and cultured statically in RPMI 1640 medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. When the cells reached the logarithmic growth phase, they were washed twice with pre-chilled PBS solution. Cells were scraped off on ice, and 1 ml of PBS solution was added. The resulting cell suspension was transferred to an EP tube and placed on ice. The tube was centrifuged at 3000 rpm for 5 min at 4°C, removing as much liquid as possible. 100 μl of cell lysis buffer was added to the cell pellet, mixed thoroughly with a pipette tip, and the cells were lysed. The lysate was collected by centrifugation at 12000 rpm for 10 min at 4°C. Protein content was determined using the BCA method. SDS-PAGE gel electrophoresis was performed. The lysate of the MDA-MB-231 breast cancer cell line was loaded into five wells at a rate of 60 μg per well, with pre-stained protein standards added as molecular weight controls. After electrophoresis, the protein was transferred to a PVDF membrane via electrotransfer. The membrane was stained with Ponceau S, and the five wells were cut open. The membrane was then washed with TBST to remove the stain. The membrane was blocked with TBST containing 5% skim milk powder at room temperature for 1.5 hours to remove non-specific binding. The following antibodies were added to the five wells of the PVDF membrane: the highly specific anti-VASP monoclonal antibody 9E06 prepared according to this invention; a commercial anti-VASP antibody (ab229624, purchased from Abcam); a commercial anti-VASP C-terminal antibody (ab205952, purchased from Abcam); an anti-EVL antibody (ab204835, purchased from Abcam); and a Mena antibody (ab176820, purchased from Abcam). All antibodies were diluted 1:2000, and the membrane was incubated overnight at 4°C. The membrane was washed three times with TBST for 5 minutes each time. HRP-labeled secondary antibodies (1:2000) were added, and the membrane was incubated at room temperature for 2 hours. The membrane was then washed three times with TBST for 5 minutes each time. Develop with ECL for 2 minutes, then take a picture under a Bio-Rad developer.

[0063] The results are as follows Figure 3 As shown, the highly specific anti-VASP monoclonal antibody 9E06 prepared in this invention can specifically recognize VASPs, including non-phosphorylated VASPs and phosphorylated VASPs (VASP-P), with a clean background and no other non-specific bands. In contrast, the commercially available control antibody either exhibits non-specific bands or fails to recognize phosphorylated VASPs. Furthermore, the highly specific anti-VASP monoclonal antibody 9E06 prepared in this invention specifically recognizes only VASPs and does not cross-react with EVL and Mena proteins from the same family.

[0064] Example 7: Immunohistochemical detection of VASP expression level in breast cancer tissue samples by the highly specific monoclonal antibody 9E06.

[0065] Paraffin sections were prepared from cancer tissue and adjacent normal tissue samples of different differentiation levels from breast cancer patients. After drying, the sections were sequentially immersed in xylene I, xylene II, anhydrous ethanol, 95% ethanol, and 70% ethanol, each for 5-8 minutes. The sections were washed once with TBS for 5 minutes each time. After removing the TBS, 3% H2O2 solution was added and the sections were humidified at room temperature for 15 minutes. The sections were then washed three times with TBS for 3 minutes each time. After removing the TBS, blocking buffer was added and the sections were blocked at 37°C for 30 minutes. After removing the blocking buffer, VASP high-specificity monoclonal antibody 9E06 incubation solution was added to cover the entire specimen, and the sections were incubated overnight at 4°C in a humidified chamber. The sections were washed three times with TBS for 3 minutes each time. After cleaning and drying the slides, endogenous peroxidase inhibitor (reagent 1) (product number PV-6000, purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) was added and the slides were incubated at 37°C for 20 minutes in a humidified chamber. The sections were washed three times with TBS for 3 minutes each time. Add HRP-labeled goat anti-mouse IgG polymer working solution (Reagent 2) (product number PV-6000, purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.), and incubate at 37°C for 20 minutes in a humidified chamber. Wash three times with TBS, 3 minutes each time. After removing TBS from the slide, add DAB chromogenic solution and incubate at room temperature in the dark for 10 minutes. Stop the reaction by adding water and then soak and wash for 5 minutes. After drying the slide, immerse it in hematoxylin for 3 minutes and rinse for 20 seconds. Then immerse the slide in 1% HCl for 3 seconds and wash once with water. Flavour with 1% ammonia for about 3 minutes and wash once with water. Dehydrate with a gradient of 80%, 95%, 100%, and 100% alcohols for 2 minutes each, and treat with xylene for 7 minutes. Mount with neutral resin. Observe and photograph under a microscope.

[0066] The results are as follows Figure 4 As shown, the highly specific anti-VASP monoclonal antibody 9E06 prepared in this invention can specifically recognize VASP protein in cancer tissues with different degrees of differentiation. High, medium and low differentiation breast cancer tissues all show a positive reaction for VASP, while they show a negative reaction in adjacent normal tissues. Therefore, the highly specific anti-VASP monoclonal antibody 9E06 prepared in this invention can be used for immunohistochemical detection of VASP expression levels to assist in tumor diagnosis.

[0067] Example 8: Application of VASP-specific monoclonal antibody 9E06 in the preparation of products for evaluating the efficacy of clinically used antiplatelet drugs

[0068] VASP is specifically expressed in platelets, existing in both phosphorylated (VASP-P) and unphosphorylated (VASP) protein forms. Prostaglandin E1 (PGE1) can induce VASP phosphorylation, while adenosine diphosphate (ADP) can activate the P2Y12 receptor, causing VASP dephosphorylation. When both ADP and PGE1 are added to whole blood, ADP plays a major role in inducing VASP dephosphorylation; if the P2Y12 receptor is blocked by a corresponding receptor blocker, this dephosphorylation cannot occur effectively, resulting in a phosphorylated state of VASP. Therefore, detecting VASP phosphorylation levels using enzyme-linked immunosorbent assay (ELISA) can evaluate the efficacy of clinically used antiplatelet drugs (P2Y12 receptor blockers).

[0069] First, the highly specific anti-VASP monoclonal antibody 9E06 prepared in this invention was diluted to a concentration of 2.0 μg / mL with carbonate buffer (0.05 M, pH 9.6). 100 μL of the antibody dilution was added to each well of the ELISA plate, and the plate was coated overnight at 4°C. The plate was washed twice with washing buffer. 120 μL / well of blocking buffer containing BSA was added, and the plate was blocked at room temperature for 6 hours. The liquid in the wells was discarded, and the plate was vacuum dried. For each experiment, one blank control well was set up, and one well each of stimulant I and stimulant II was added to each sample. No liquid was added to the blank control well. For the sample stimulant I well, 40 μL of stimulant I (PGE1, 1-5 μM) was added; for the sample stimulant II well, 40 μL of stimulant II (PGE1, 1-5 μM; ADP, 5-20 μM) was added. Then, 40 μL of the same whole blood sample was added to each stimulant well, and the sample was mixed by pipetting 8-10 times. For the assay, seal the ELISA plate coated with the highly specific anti-VASP monoclonal antibody 9E06 with a sealing film and incubate at 18-26°C for 15 minutes. Add 100 μL of lysis buffer to each well (except the blank control well), pipette 8-10 times to mix, seal the plate again, and incubate at 18-26°C for 30 minutes. Discard the reaction solution. Add 300 μL of diluted wash buffer to each well, incubate at room temperature for 30 seconds, and discard the wash buffer. Wash the plate three times consecutively, and finally pat dry on absorbent paper. Add 100 μL of biotin-labeled mouse anti-human VASP-P (S239) monoclonal antibody (ab218619, purchased from Abcam) to each well, followed by 100 μL of horseradish peroxidase-labeled streptavidin, pipette 8-10 times to mix (excluding the blank control well). Cover the ELISA plate with a sealing film and incubate at 18-26℃ for 30 minutes. Wash the plate three times consecutively, following the same procedure. Add 100 μL each of TMB chromogenic solution A and B to each well sequentially, and pipette 8-10 times to mix (including the blank control well). Cover the ELISA plate with a sealing film and incubate at 18-26℃ for 20 minutes. Add 100 μL of stop solution to each well (including the blank control well) to terminate the reaction. Measure the absorbance of each well using a single wavelength of 450 nm on a microplate reader. The absorbance values ​​for stimulant I, stimulant II, and blank control are expressed as OD[stimulant I], OD[stimulant II], and OD[blank control], respectively. Each experimental result is used independently, and the result is determined by the platelet reaction index (PRI).

[0070] Platelet Reactivity Index (PRI) Calculation:

[0071] PRI (%) = (OD[stimulant I] - OD[stimulant II]) / (OD[stimulant I] - OD[blank control]) × 100% Interpretation of test results:

[0072] 1. A positive result is defined as PRI ≤ 65% in the tested sample, indicating a good response to antiplatelet drugs (P2Y12 receptor blockers).

[0073] 2. A negative PRI of >65% in the test sample indicates poor response to antiplatelet drugs (P2Y12 receptor blockers) or a healthy person.

[0074] Sixty-seven fresh clinical whole blood samples were tested in parallel using the method of this invention and the Stago CY-QUANTVASP / P2Y12 specific platelet ADP receptor antagonist detection kit (enzyme-linked immunosorbent assay). The concordance rate between the method and the Stago VASP detection kit was calculated, and the results are shown in Table 4. The positive concordance rate between the method established using the VASP high-specific monoclonal antibody 9E06 of this invention and the Stago VASP detection kit was 100% (39 / 39), the negative concordance rate was 96.43% (27 / 28), and the overall concordance rate was 98.51% (66 / 67). These results demonstrate that the method of this invention has a high concordance rate with the Stago VASP detection kit. The anti-VASP high-specific monoclonal antibody 9E06 prepared in this invention can be used to detect VASP phosphorylation levels and evaluate the efficacy of clinically used antiplatelet drugs (P2Y12 receptor blockers).

[0075] Table 4. Results of VASP detection for serine 239 phosphorylation in whole blood platelets

[0076]

[0077] Example 9: Determination of VASP High-Specific Monoclonal Antibody 9E06 Subtype

[0078] The heavy and light chain isotypes of mouse antibodies were identified using the rapid mouse antibody subtype detection card (catalog number: THJ-ISO-M8a-10 / 20) from Antaiji (Beijing) Biotechnology Co., Ltd. First, the antibody was diluted to 1 μg / mL with PBS. Then, 100 μl of the diluted antibody was added to each well, and the results were observed and recorded after standing for 5-10 min. The results showed that the highly specific anti-VASP monoclonal antibody 9E06 prepared in this invention was of the mouse IgG1 subtype, and the antibody light chain was of the Igκ subtype.

[0079] Example 10: Sequencing of the variable region of VASP-specific monoclonal antibody 9E06

[0080] Mouse hybridoma cell line 9E06 was cultured, and total RNA was extracted from the hybridoma cells using the Trizol method. After reverse transcription of cDNA, PCR amplification was performed using primers for the Fab fragment of mouse monoclonal antibodies synthesized by Beijing Qingke Biotechnology Co., Ltd. The cells were preheated at 95℃ for 2 min, followed by 30 cycles of 95℃ for 30 seconds, 58℃ for 30 seconds, and 72℃ for 30 seconds, with a final extension at 72℃ for 5 min. The resulting cells were ligated into the pMD18-T vector and transformed into *E. coli* JM109. Positive clones were selected for sequencing. The sequenced data were compared with the mouse-derived monoclonal antibody CDR region sequence using IgBLAST (https: / / www.ncbi.nlm.nih.gov / igblast / ) in the NCBI website's BLAST module.

[0081] Sequence analysis revealed that the heavy chain variable region contains 113 amino acids, with the following sequence: QVQLEQSGAELAKPGASVKMSCKASGHTFTNFWMHWIKQRPGQGLDWIGNITPSTG YTDYNQNFKDKATLTADTSSSTAYMQLSSLTSVDSAVYVTTKALGLLGSRNHSHRLL (SEQ ID NO.1). Among these, heavy chain CDR1 is located at 26-33 amino acids, with the amino acid sequence GHTFTNFW (SEQ ID NO.2); heavy chain CDR2 is located at 51-58 amino acids, with the amino acid sequence ITPSTGYT (SEQ ID NO.3); and heavy chain CDR3 is located at 97-102 amino acids, with the amino acid sequence TKALGL (SEQ ID NO.4). The light chain variable region has an amino acid sequence of 109 amino acids, as follows: DIVLTQSPASLAVSLGERATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLE SGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGAPSWKS (SEQ ID NO.5), where light chain CDR1 is located at 27-36 aa and has the amino acid sequence KSVSTSGYSY (SEQ ID NO.6); light chain CDR2 is located at 54-56 aa and has the amino acid sequence LVS; and light chain CDR3 is located at 93-100 aa and has the amino acid sequence QHIRELTR (SEQ ID NO.7).

Claims

1. A vasodilatory stimulating phosphoprotein-specific monoclonal antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, and the light chain variable region comprises light chain CDR1, light chain CDR2, and light chain CDR3, characterized in that, The amino acid sequence of the heavy chain CDR1 is the sequence shown in SEQ ID NO.2; The amino acid sequence of the heavy chain CDR2 is the sequence shown in SEQ ID NO.3; The amino acid sequence of the heavy chain CDR3 is the sequence shown in SEQ ID NO.4; The amino acid sequence of the light chain CDR1 is the sequence shown in SEQ ID NO.6; The amino acid sequence of the light chain CDR2 is LVS; The amino acid sequence of the light chain CDR3 is the sequence shown in SEQ ID NO.

7.

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

5.

3. The monoclonal antibody according to claim 2, characterized in that, It is secreted by mouse hybridoma cell line 9E06 with accession number CGMCC No.46354.

4. The monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that, The antibody or antigen-binding fragment is a Fab fragment, Fab' fragment, F(ab')2 fragment, single-chain antibody, or humanized antibody.

5. A nucleic acid molecule, characterized in that, It is a nucleic acid encoding the antibody or its antigen-binding fragment as described in any one of claims 1 to 4.

6. An expression vector, characterized by, It comprises the nucleic acid molecule as described in claim 5.

7. A cell recombination body, characterized in that, It comprises the nucleic acid molecule of claim 5 or the expression vector of claim 6.

8. The cellular recombinant of claim 7, characterized in that, It is a bacterial recombinant or a yeast recombinant.

9. A hybridoma cell line secreting a monoclonal antibody specific for the vasodilator-stimulated phosphoprotein, characterized in that, It is the mouse hybridoma cell line 9E06 with accession number CGMCC No. 46354.

10. The use of the vasodilator-stimulating phosphoprotein-specific monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 4 in the preparation of a product for detecting vasodilator-stimulating phosphoprotein.

11. A kit for detecting a VASP protein, characterized by, It comprises the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 4.

12. The kit of claim 11, wherein The monoclonal antibody or its antigen-binding fragment binds to vasodilatory phosphoprotein by immunohistochemistry, Western blotting, or enzyme-linked immunosorbent assay.

13. The use of the vasodilatory stimulating phosphoprotein-specific monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 4 in the preparation of products for evaluating the efficacy of clinically used P2Y12 receptor blocker drugs.

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