Phosphorylated VASP-Ser 239 protein antibody and application thereof

Abstract

The invention relates to a phosphorylated VASP-Ser 239 protein antibody or an antigen binding fragment and application thereof. The antibody comprises specific light chain and heavy chain variable region complementarity determining region (CDR) sequences or amino acid sequences with at least 80% sequence identity with the CDR sequences. The invention also provides isolated polynucleotide for coding the antibody, an expression vector, a recombinant cell and a method for preparing the antibody. The antibody and derivative products thereof can efficiently and specifically detect phosphorylated VASP-Ser 239 protein in human whole blood or platelet cells, and are suitable for monitoring the inhibition effect of a platelet ADP receptor (P2Y12) antagonist (such as clopidogrel) on platelet activation, determining the clopidogrel resistance situation and guiding clinical reasonable medication. The invention solves the problem of shortage of antibody raw materials and kits of related detection items in the current market.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to an antibody against phosphorylated VASP-Ser 239 protein and its application. Background Art

[0002] Vasodilator-stimulated phosphoprotein (VASP) is a member of the Ena-VASP protein family. Members of the Ena-VASP family contain an EHV1 N-terminal domain that binds proteins containing the E / DFPPPPXD / E motif. In the middle region of the protein, family members have a proline-rich domain that binds proteins containing SH3 and WW domains. Their C-terminal EVH2 domain mediates tetramerization and binds G- and F-actin. VASP is associated with the formation of filamentous actin and may play a wide role in cell adhesion and motility. VASP may also be involved in regulating intracellular signaling pathways that mediate integrin-extracellular matrix interactions. VASP is regulated by the cyclic nucleotide-dependent kinases PKA and PKG.

[0003] VASP protein is an actin-related protein that participates in a series of processes dependent on cytoskeletal remodeling and cell polarity, such as axon guidance, lamellipodial and filopodial dynamics, platelet activation, and cell migration. VASP promotes the elongation of actin filaments. It protects the barbed ends of growing actin filaments from capping and increases the actin polymerization rate in the presence of capping protein. VASP stimulates actin filament elongation by promoting the transfer of actin monomers bound to profilin to the barbed ends of growing actin filaments. It plays a role in the actin-based motility of Listeria monocytogenes in host cells. It regulates actin dynamics in platelets and plays an important role in regulating platelet aggregation.

[0004] VASP is a platelet protein that is in a non-phosphorylated state at baseline. The phosphorylation process is regulated by the cAMP cascade, and prostaglandin E1 (PGE1) can activate this cascade. The phosphorylation of VASP is directly related to the inhibition level of the P2Y12 receptor. Therefore, the effects of antiplatelet drugs such as clopidogrel can be evaluated by measuring phosphorylated VASP, and it can also be used to evaluate the population differences of antiplatelet drugs. The method for measuring phosphorylated VASP has high sensitivity and specificity, good parallelism with the measurement of platelet aggregation rate, requires a small blood sample, and can use whole blood.

[0005] Currently, there is an urgent need to develop an antibody against phosphorylated VASP-Ser 239 protein with high specificity and sensitivity. Summary of the Invention

[0006] To solve the problem of the relative shortage of antibody raw materials and kits for the detection of phosphorylated VASP-Ser 239 protein in the current market, the present invention provides a monoclonal antibody against phosphorylated VASP-Ser 239 protein, which has the characteristics of high affinity and specificity. At the same time, the present invention also provides products prepared based on the above antibody and their applications.

[0007] On the one hand, the present invention provides an antibody or antigen-binding fragment against phosphorylated VASP-Ser 239 protein, comprising: CDR sequences selected from at least one of the following or amino acid sequences having at least 80% identity therewith: light chain variable region CDR sequences: SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5; heavy chain variable region CDR sequences: SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.

[0008] The present invention provides an antibody or antigen-binding fragment against phosphorylated VASP-Ser 239 protein, and an enzyme immunoassay kit prepared using the antibody or antigen-binding fragment has high sensitivity and specificity. 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, and the heavy chain variable region CDR3 sequence shown in SEQ ID NO:8. According to some embodiments of the present invention, the antibody or antigen-binding fragment comprises at least one of the following: (a) having the light chain variable region shown in SEQ ID NO:1 and the heavy chain variable region shown in SEQ ID NO:2; An amino acid sequence having at least 80% sequence identity compared to (a).

[0009] According to some embodiments of the present invention, the antibody or antigen-binding fragment comprises at least one of the following: (b) having the CDR1, CDR2 and CDR3 sequences of the light chain variable region shown in SEQ ID NO:1, and the CDR1, CDR2 and CDR3 sequences of the heavy chain variable region shown in SEQ ID NO:2; Or an amino acid sequence having one or more conservative amino acid substitutions compared to (b).

[0010] In some embodiments of the present invention, the antibody or antigen-binding fragment further comprises at least one of a heavy-chain constant region and a light-chain constant region, and at least a part of at least one of the heavy-chain constant region and the light-chain constant region is derived from a mammalian antibody.

[0011] In some embodiments of the present invention, the heavy-chain constant region and the light-chain constant region are derived from at least one of a murine antibody, a rabbit antibody, a primate antibody or a mutant thereof.

[0012] In some embodiments of the present invention, the heavy-chain constant region and the light-chain constant region are derived from a murine antibody or a mutant thereof.

[0013] In some embodiments of the present invention, the N-terminus of the heavy-chain constant region is connected to the C-terminus of the heavy-chain variable region; and / or the N-terminus of the light-chain constant region is connected to the C-terminus of the light-chain variable region.

[0014] In some embodiments of the present invention, the heavy-chain constant region comprises a heavy-chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; or the light-chain constant region comprises a light-chain constant region selected from κ-type or λ-type.

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

[0016] In another aspect of the present invention, there is provided an antibody analogue, which comprises the variable region or the CDR region in the variable region of the above-mentioned antibody or antigen-binding fragment.

[0017] In another aspect of the present invention, there is provided a conjugate, which comprises: the above-mentioned phosphorylated VASP-Ser 239 protein antibody or antigen-binding fragment; and a conjugate part, which is connected to the phosphorylated VASP-Ser 239 protein antibody or antigen-binding fragment In some embodiments of the present invention, the conjugate part comprises at least one of a carrier, a drug, a toxin, a cytokine, a protein tag, a modifier and a chemotherapeutic agent.

[0018] In some embodiments of the present invention, the carrier comprises a fluorescent dye.

[0019] According to some embodiments of the present invention, the fluorescent dye includes any one or more of FITC, PE, Cy5, PI, 7AAD, APC, Alexa Fluor, eFluor, PECy7, APC Cy7, PerCP, PerCPcy5.5, PB, BV series, QDot series, and BUV series dyes.

[0020] In yet another aspect, the present invention provides an isolated polynucleotide encoding the above antibody or antigen-binding fragment.

[0021] According to some embodiments of the present invention, the polynucleotide comprises: (c) a nucleic acid sequence encoding the variable region of the light chain shown in SEQ ID NO: 9, a nucleic acid sequence having at least 80% sequence identity compared to (c); or (d) a nucleic acid sequence encoding the variable region of the heavy chain shown in SEQ ID NO: 10, a nucleic acid sequence having at least 80% sequence identity compared to (d).

[0022] In yet another aspect, the present invention provides an expression vector comprising the above polynucleotide.

[0023] In yet another aspect, the present invention provides a recombinant cell into which the above expression vector has been transfected.

[0024] In yet another aspect, the present invention provides a cell that can produce the above antibody or antigen-binding fragment of phosphorylated VASP-Ser 239 protein.

[0025] In yet another aspect, the present invention provides a method for preparing an antibody or antigen fragment of phosphorylated VASP-Ser 239 protein, comprising culturing the above recombinant cell.

[0026] In yet another aspect, the present invention provides the use of the above antibody or antigen-binding fragment and the above antibody analog in the preparation of a kit for in vitro quantitative detection of the phosphorylation level of vasodilator-stimulated phosphoprotein (VASP) in human whole blood. It is used to monitor the inhibitory effect and degree of inhibition of specific platelet ADP receptor (P2Y12) antagonists (such as drugs like clopidogrel) on platelet activation, clarify whether there is clopidogrel resistance, and guide the rational adjustment of dual anti-drugs.

[0027] According to some embodiments of the present invention, the kit is used for in vitro quantitative detection of the phosphorylation level of vasodilator-stimulated phosphoprotein (VASP) in human whole blood, for monitoring the inhibitory effect and degree of inhibition of specific platelet ADP receptor (P2Y12) antagonists (such as drugs like clopidogrel) on platelet activation, for determining whether there is clopidogrel resistance, and for guiding the rational adjustment of dual anti-drugs.

[0028] According to some embodiments of the present invention, the phosphorylated VASP-Ser 239 protein is used for in vitro quantitative detection of the phosphorylation level of vasodilator-stimulated phosphoprotein (VASP) in human whole blood. For monitoring the inhibitory effect and degree of inhibition of specific platelet ADP receptor (P2Y12) antagonists (such as drugs like clopidogrel) on platelet activation, for determining whether there is clopidogrel resistance, and for guiding the rational adjustment of dual anti-drugs.

[0029] On the other hand, the present invention provides a kit comprising at least one of the above-mentioned antibody or antigen-binding fragment, conjugate, expression vector, and recombinant cell.

[0030] On the other hand, the present invention provides a composition comprising at least one of the above-mentioned antibody or antigen-binding fragment, conjugate, isolated polynucleotide, expression vector, recombinant cell, and hybridoma cell.

[0031] On the other hand, the present invention provides a method for preparing the above-mentioned antibody or antigen-binding fragment, comprising culturing the above-mentioned recombinant cell.

[0032] On the other hand, the present invention provides a method for detecting phosphorylated VASP-Ser 239 protein, comprising: Contacting the phosphorylated VASP-Ser 239 protein antibody or antigen-binding fragment, conjugate, or kit described above with a sample to be detected to form an immune complex; based on the signal of the immune complex, quantitatively and / or qualitatively analyzing the phosphorylated VASP-Ser 239 protein in the sample to be detected.

[0033] According to some embodiments of the present invention, the signal of the immune complex is detected by at least one of the methods of ELISA, WB, and FACS.

[0034] According to some embodiments of the present invention, the signal includes a fluorescence signal.

[0035] On the other hand, the present invention provides a drug comprising at least one of the above-mentioned antibody or antigen-binding fragment, conjugate, isolated polynucleotide, expression vector, recombinant cell, and composition.

[0036] Additional aspects and advantages of the present disclosure will be given in part in the following description, become apparent in part from the following description, or be learned by practice of the present disclosure. Description of the Drawings

[0037] Figure 1 It is the OD for quantitatively detecting phosphorylated VSAP protein in human platelet cells by the kit of the present invention according to the present invention. 450 Standard curve. Detailed Embodiments

[0038] The solution of the present invention will be explained below in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. For those not specified in the embodiments regarding specific techniques or conditions, they shall be carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0039] Nucleic acids encoding the heavy chain and / or light chain of the antibody of the present invention are within the scope of the present invention. According to the amino acid sequences of the heavy chain and / or light chain, those skilled in the art can easily obtain the corresponding nucleic acid sequences, as shown in Table 1. It should be noted that the CDR sequences listed in Table 1 below are obtained according to the IMGT database. Those skilled in the art should know that the CDR sequences analyzed by different databases may be different, but these variations should all be included within the protection scope of the present invention.

[0040] Table 1 Sequence Information

[0041]

[0042] Example 1: Synthesis of non-phosphorylated VASP-Ser 239 protein peptide segment, phosphorylated VASP-Ser 239 protein peptide segment, and phosphorylated VASP-Ser 239 full-length protein antigen The full-length amino acid sequence of non-phosphorylated VASP protein is shown as follows: MSETVICSSRATVMLYDDGNKRWLPAGTGPQAFSRVQIYHNPTANSFRVVGRKMQPDQQVVINCAIVRGVKYNQATPNFHQWRDARQVWGLNFGSKEDAAQFAAGMASALEALEGGGPPPPPALPTWSVPNGPSPEEVEQQKRQQPGPSEHIERRVSNAGGPPAPPAGGPPPPPGPPPPPGPPPPPGLPPSGVPAAAHGAGGGPPPAPPLPAAQGPGGGGAGAPGLAAAIAGAKLRKVSKQEEASGGPTAPKAESGRSGGGGLMEEMNAMLARRRKATQVGEKTPKDESANQEEPEARVPAQSESVRRPWEKNSTTLPRMKSSSSVTTSETQPCTPSSSDYSDLQRVKQELLEEVKKELQKVKEEIIEAFVQELRKRGSP (SEQ ID NO:11) The phosphorylated VASP-Ser 239 protein and the non-phosphorylated VASP-Ser 239 protein peptide were synthesized by using a partial sequence thereof and conventional methods in the art. The specific peptide sequences are as follows: Table 2 Polypeptide Synthesis Sequence Information

[0043] Note: “(p)” in the table indicates that the amino acid in front of it has carboxylation modification. “BSA” indicates that bovine serum albumin is connected to the C-terminus of the phosphorylated VASP-Ser 239 protein or the non-phosphorylated VASP-Ser 239 protein peptide, and it was synthesized by Shanghai Botai Biotechnology Co., Ltd.; “KLH” indicates that hemocyanin is connected to the C-terminus of the phosphorylated VASP-Ser 239 protein, and it was synthesized by Shanghai Botai Biotechnology Co., Ltd.

[0044] Example 2: Identification of Phosphorylated VASP-Ser 239 Protein First, the phosphorylated VASP-Ser 239 protein 239VASP-BSA was coated on an ELISA plate at 1 μg / ml. The purified phosphorylated VASP-Ser 239 protein antibody was used as the primary antibody in the indirect ELISA method, and the HRP-labeled goat anti-rat antibody was used as the secondary antibody. TMB was used as the chromogenic solution, and then the reaction was terminated with 1M H2SO4. The OD 450 reading value was measured by an ELISA reader. By comparing the antigen activity of the phosphorylated VASP-Ser 239 protein, it can be seen that the antigen activity of the phosphorylated VASP-Ser 239 protein was detected to be relatively high.

[0045] Table 3 Identification of Phosphorylated VASP-Ser 239 Protein

[0046] Example 3: Immunizing BALB / c Mice with Phosphorylated VASP-Ser 239 Protein Select BALB / c mice aged 6 - 8 weeks for the following immunization procedure: At the primary immunization, mix 25 μg of phosphorylated VASP-Ser 239 protein pVASP239-KLH with an equal volume of Freund's complete adjuvant, emulsify it, and then inject it subcutaneously at multiple points; 14 days after the first immunization, mix 12.5 μg of phosphorylated VASP-Ser 239 protein pVASP239-KLH with Freund's incomplete adjuvant, emulsify it, and boost the immunization. 14 days after the second immunization, mix 12.5 μg of phosphorylated VASP-Ser 239 protein pVASP239-KLH with Freund's incomplete adjuvant, emulsify it, and boost the immunization. 14 days after the third immunization, collect blood and separate the serum. Coat an ELISA plate with 1 μg / mL of phosphorylated VASP-Ser239 protein pVASP239-BSA, and perform an indirect ELISA test to determine the serum titer. The results show that the titer of the prepared mouse antiserum is 1:72900, and the immunization is successful. See Table 4 for the typical serum test results of immunized mice.

[0047] Table 4 Typical Serum Test Results of Immunized Mice

[0048] Example 4: Cell Fusion Three days after boosting the immunization of the mice in Example 3, perform cell fusion. After collecting blood from the mouse eyeballs, dislocate the mouse's neck to sacrifice it, place it in a 70% alcohol bottle for 2 minutes, then fix the mouse on a foam board in a biosafety cabinet. Untie the abdominal skin to find the spleen, remove it with forceps, place it in a 200-mesh stainless steel filter membrane and gently grind it. Gently rinse the cells with DMEM medium (Thermo, 11965092), and then centrifuge at 200 g for 10 minutes in a centrifuge at room temperature. Discard the supernatant and reserve it; when preparing feeder cells, dislocate the mouse's neck to sacrifice it, place it in a 70% alcohol bottle for 2 minutes, then fix the mouse on a foam board in a biosafety cabinet. Untie the abdominal skin, draw PBS with a syringe and gently inject it subcutaneously, and wash out the liquid containing feeder cells from the other side. Then centrifuge at 200 g for 10 minutes in a centrifuge at room temperature. Discard the supernatant and reserve it. Mix 2.0×10 7 FO myeloma cells with 2.0×10 8Mix the splenocytes, centrifuge at 200 g for 10 minutes in a centrifuge, discard the supernatant, gently oscillate to mix, in a 37 °C water bath, add 1 ml of an aqueous solution of PEG-1450 (Merk, P1458) with a volume concentration of 50% dropwise within 90 seconds, then add 20 mL of DMEM medium, centrifuge at 200 g for 10 minutes in a centrifuge, discard the supernatant, repeat the washing once, centrifuge at 200 g for 10 minutes in a centrifuge, discard the supernatant to obtain hybridoma cells, and seed the cells into 10 96-well culture plates, 150 μL per well. Add 10,000 feeder cells per well to the above 10 96-well cell culture plates, 100 μL per well. After labeling the culture plates, place them in a cell culture incubator at 37 °C containing 5% CO2 for culture. On the second day, add HAT selection medium (Merk, H0262), and then use HAT selection culture. Within 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. After maintaining the HAT selection culture medium for 7 - 10 days, it should be replaced with HT medium (Merk, H0137), and then maintained for another 2 weeks, and then switched to DMEM medium containing 20% FBS (ExCell, FSP500) for continued culture. During the above selection culture period, when the hybridoma cells cover 1 / 10 of the bottom area of the well, the detection of specific antibodies can be started to screen out the required hybridoma cell line. During the selection culture period, generally, half of the culture medium is changed every 2 - 3 days.

[0049] Example 5: Screening and subcloning culture of positive hybridoma cell lines First, determine the optimal coating amount of phosphorylated VASP-Ser 239 protein as an antigen through orthogonal experiments. Coat a 96-well plate with 0.5, 1.0, 2.0, and 4.0 μg of phosphorylated VASP-Ser 239 protein pVASP239-BSA. Set 6 wells for each concentration, with 3 positive wells and 3 negative wells. Perform checkerboard titration with the positive serum of mice immunized with phosphorylated VASP-Ser 239 protein pVASP239-KLH at different dilution multiples, and use the negative serum of non-immunized mice as a negative control. Coat a 96-well ELISA plate with 0.5 μg of purified phosphorylated VASP-Ser 239 protein pVASP239-BSA per well and incubate overnight at 4°C; wash twice with PBST; add 200 μL of 1% BSA in PBS to each well, block at room temperature for 2 hours, and then pat dry on filter paper; add samples: add 0.1 mL of the sample to be tested to the reaction wells, incubate at 37°C for 1 hour, then wash, and at the same time, set up blank wells (without adding samples), negative control wells, and positive control wells. Add 0.1 mL of the newly diluted antibody to each reaction well, incubate at 37°C for 1 hour, and then wash 3 times. Add enzyme-labeled secondary antibody: add 0.1 mL of the newly diluted enzyme-labeled antibody to each reaction well. Incubate at 37°C for 1 hour and wash 3 times. Add substrate solution for color development: add 0.1 mL of TMB substrate solution to each reaction well and let stand at room temperature for 10 minutes. Add 0.1 mL of 1M H2SO4 to each reaction well. Measure the OD value to judge the result, and use an enzyme-linked immunosorbent assay reader to measure the absorbance value (A450) at 450 nm. A value more than 2.1 times the OD value of the negative control is considered positive (calculated after zeroing with the blank control well). Select the hybridoma cell monoclonal antibody against phosphorylated VASP-Ser 239 protein.

[0050] According to the above method, perform subcloning on the selected positive hybridoma cells. Dilute the original wells with HAT selection medium by the limiting dilution method and then re-distribute them into a 96-well culture plate, and then observe the cell morphology and quantity. Adjust the cells to 3 - 10 cells / mL. Take the cell culture plate of the feeder cell layer prepared the previous day, and add 100 μL of the diluted cells to each well. Incubate statically in a 37°C, 5% CO2 incubator.

[0051] Change the medium on the 7th day, and then change the medium once every 2 - 3 days. Cell clones can be seen on the 8th - 9th day, and detect the antibody activity in a timely manner. Transfer the cells in the positive wells to a 24-well plate for expansion culture. Each clone should be frozen as soon as possible, and finally select the hybridoma cell line with clone number G085 for antibody production.

[0052] Example 6: Large-scale preparation of monoclonal antibody and determination of antibody titer (1) Large-scale preparation of monoclonal antibody Inject 0.5 mL of Freund's incomplete adjuvant intraperitoneally into 8-week-old BALB / C mice. Two weeks later, inject 1×106 Hybridoma cell G085 can produce ascites 7 to 10 days after cell inoculation. Closely observe the health status of the animals and ascites signs. Before the ascites is as much as possible and the mice are on the verge of death, sacrifice the mice. Use a dropper to suck the ascites into a test tube. One mouse can obtain 5 to 10 ml of ascites. Ascites can also be extracted with a syringe and collected repeatedly several times. Centrifuge the obtained ascites at 3000 g for 10 minutes, discard the upper layer of grease and the bottom precipitate, collect the supernatant, and aliquot it at -20°C. After the ascites supernatant is thawed and equilibrated to room temperature, add 1 / 10 volume of 1M Tris-HCl pH 8.0 to adjust the pH of the sample to 8.0. Equilibrate the protein G affinity column with 20 column volumes of 100 mM Tris-HCl pH 8.0. Load the ascites supernatant with adjusted pH to 8.0 onto the column, then wash it with 20 column volumes of 100 mM Tris-HCl pH 8.0, and finally elute the antibody (i.e., phosphorylated VASP-Ser239 protein antibody G085, which can be abbreviated as antibody G085 or G085) with 100 mM GlycineHCl pH 2.5. Add the antibody eluate to a concentrator tube (Millipore, UFC801008, 10K), and centrifuge it at 3000×g at room temperature for 20 minutes in a centrifuge (Xiangyi, L550). Centrifuge in batches until the solution volume reaches 1 ml / concentrator tube (2 tubes), add 4 mL of 10 mM PBS (pH 7.4) buffer, and continue to centrifuge at 3000×g at room temperature for 20 minutes. Repeat centrifugation 3 times to make the buffer of the antibody 10 mM PBS (pH 7.4), and add 10 mM PBS (pH 7.4) to a total volume of 10 mL. Finally, aliquot the concentrated antibody solution at 2 mL / tube into centrifuge tubes and store it at -80°C. Use a BCA kit (Solarbio, PC0020) to measure the antibody concentration, and the concentration of the purified monoclonal antibody is measured to be 3.8 mg / mL.

[0053] (2) Antibody titer determination The titer of the phosphorylated VASP-Ser 239 protein antibody G085 was detected by the indirect ELISA method. The phosphorylated VASP-Ser 239 protein pVASP239-BSA was diluted with PBS and coated on a 96-well ELISA plate at 0.2 μg / mL, 100 μL / well. After overnight incubation at 4 °C, the ELISA plate was washed 2 times with PBST solution, 300 μL / well each time, and patted dry after washing. The ELISA plate was blocked with PBS solution containing 1% BSA, 200 μL / well, at room temperature for 2 hours, and patted dry after blocking; the phosphorylated VASP-Ser239 protein antibody G085 diluted to 20 ng / mL with PTB was added and reacted in an incubator at 37 °C for 1 hour. The ELISA plate was washed 2 times with PBST solution, 300 μL / well each time. After washing, it was patted dry. The HRP-labeled goat anti-mouse antibody diluted 5000-fold with PTB was added and reacted in an incubator at 37 °C for 1 hour. The ELISA plate was washed 2 times with PBST solution, 300 μL / well each time. After washing, it was patted dry. The TMB substrate solution was added, 100 μL / well, and reacted at room temperature for 10 minutes. Then 100 μL / well of 1 M H2SO4 was added to terminate the reaction. The absorbance value (A450) was measured at 450 nm using an ELISA reader. The results are shown in Table 5. It can be seen that the titer of the purified antibody is 1:729000.

[0054] Table 5 Antibody titer

[0055] Example 7: Sequence analysis of monoclonal antibody (1) Identification of monoclonal antibody subtype The hybridoma cell line G085 was cultured in a 10-cm diameter cell culture dish (37 °C, 5% CO2) with DMEM medium (GIBCO, #C11995500BT) supplemented with 10% serum. After 7 days of culture, the cells were transferred to a 15-ml centrifuge tube. After counting with a hemocytometer, 4×10 6 cells were taken out, centrifuged at 200 g for 5 minutes, the supernatant was discarded, and the centrifuge tube was inverted to drain the liquid in the tube. The cells in the tube were used to synthesize cDNA using the reverse transcription kit (Qiagen, 74134) from QIAGEN.

[0056] The antibody subtype was determined by performing PCR with subtype-specific primers for the antibody. The synthesized cDNA described above was used as the template for the PCR reaction. PCR reaction solution system: TAKARA Ex Taq (5 U / μL, TAKARA, RR001B), 0.25 μL; 10× Ex Taq Buffer, 5 μL; dNTP mixture (2.5 mM each), 4 μL; template cDNA, 1 μL; upstream primer (100 μM), 1 μL; downstream primer (100 μM), 1 μL; add double-distilled water to a total volume of 50 μL. PCR reaction temperature program: pre-denaturation at 94°C for 5 minutes, 30 temperature cycles (94°C for 1 minute, 57°C for 1 minute, 72°C for 1 minute), extension at 72°C for 10 minutes. After the reaction, 10 μL of each PCR product was loaded onto a 1% agarose gel for electrophoresis, and the antibody subtype could be inferred based on the PCR product results (Table 6). The monoclonal antibody G085 obtained in the present invention has a heavy chain of IgG1 and a light chain of kappa.

[0057] Table 6 PCR primer information

[0058] where S = C or G, M = A or C, R = A or G, and W = A or T (2) Sequencing of the variable region (V region) of the antibody of hybridoma cell line G085 The fragment obtained after PCR amplification of the V region of the antibody of cell line G085 (see the above item) was cut from the agarose gel and extracted using a DNA extraction kit (Qiagen, 74134). The extracted DNA fragment was ligated to the pEASY-T1 cloning vector and transformed into Trans1-T1 competent cells (Transgen, CT101-1). Transformed bacterial colonies were picked into LB medium and subjected to DNA sequencing after overnight culture. The nucleic acid sequence of the light chain V region of the phosphorylated VASP-Ser 239 protein antibody (G085) provided by the present invention is shown in SEQ ID NO.9, and the nucleic acid sequence of the heavy chain V region is shown in SEQ ID NO.10.

[0059] Example 8: Identification of the specificity of antibody G085 in detecting phosphorylated VASP-Ser 239 protein The ability of antibody G085 to detect phosphorylated VASP-Ser 239 protein was evaluated by ELISA. In this experiment, the non-phosphorylated VASP-Ser 239 protein peptide VASP239-BSA and the phosphorylated VASP-Ser 239 protein pVASP239-BSA were used as antigens and diluted with PBS, respectively, and then coated on a 96-well ELISA plate at a coating concentration of 1 μg / mL and a volume of 100 μL / well. After overnight coating at 4 °C, the ELISA plate was washed twice with PBST solution at 300 μL / well each time. After washing and blotting dry, the ELISA plate was blocked with PBS solution containing 1% BSA at 200 μL / well for 2 hours at room temperature. After blotting dry, antibody G085 (diluted to different concentrations with PTB) was added to the blocked ELISA plate at 100 μL / well and reacted in an incubator at 37 °C for 1 hour. After blotting dry, the plate was washed 3 times with PBST at 300 μL / well each time. After blotting dry, HRP-labeled goat anti-mouse antibody diluted 5000-fold with PTB was added to the ELISA plate and reacted in an incubator at 37 °C for 1 hour. The plate was washed 3 times with PBST at 300 μL / well. After blotting dry, TMB substrate solution was added to the ELISA plate at 100 μL / well and reacted at room temperature for 10 minutes, and then 100 μL / well of 1 M H2SO4 was added to terminate the reaction. The absorbance value (A450) was measured at 450 nm using an ELISA reader. The experimental results are shown in Table 7, indicating that antibody G085 has good specificity for detecting phosphorylated VASP-Ser 239 protein.

[0060] Table 7 Identification of the specificity of antibody G085

[0061] Example 9: Application of the ELISA kit prepared with antibody G085 in detecting phosphorylated VASP-Ser 239 protein in human platelet cells (1) Horseradish peroxidase (HRP) labeling of antibody G085 and identification of the labeled product Take 0.5 mL of antibody G085 (20 nmol, 3 mg) and add it to a dialysis bag (10 KDa, width 1 cm), and dialyze overnight at 4 °C in 2 L of 10 mM PBS solution (pH 7.4). The next day, put the dialysis bag containing the antibody solution into 1 L of 10 mM carbonate buffer (pH 9.5) and stir and dialyze at room temperature for 2 hours to prepare for coupling with the activated HRP.

[0062] Meanwhile, accurately weigh 1 mg of HRP using an analytical balance, add 0.2 mL of ultrapure water to dissolve it, and make the HRP concentration 5 mg / mL. Add 40 μl of 0.1 M NaIO4 to the above HRP solution, place it on a horizontal shaker, and react (activate) in the dark at room temperature for 20 minutes. Add the activated HRP solution into a dialysis bag (10KDa, width 1 cm), and dialyze overnight at 4°C in 2 L of 1 mM sodium acetate buffer (PH 4.4). Carefully aspirate the dialyzed HRP solution and transfer it into 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.

[0063] Mix the above antibody and HRP (for conjugation reaction), place it on a horizontal shaker, and react in the dark at room temperature for 4 hours. After the conjugation reaction is completed, add 10 μl (freshly prepared with pre-cooled ultrapure water) of NaBH4, and terminate the reaction overnight at 4°C in the dark. Transfer the antibody solution after terminating the conjugation reaction into a dialysis bag (10KDa, width 1 cm), and dialyze with stirring at room temperature for 2 hours. Finally, transfer the solution into a brown centrifuge tube and store it at 4°C.

[0064] (2)Determination of the standard curve of the enzyme immunoassay kit prepared with antibody G085 In this experiment, the phosphorylated VASP-Ser 239-BSA conjugate protein (pVASP239-BSA) was diluted with PBS as an antigen and coated on a 96-well enzyme-linked immunosorbent assay (ELISA) plate at a coating concentration of 1 μg / mL and a volume of 100 μL / well. After coating overnight at 4°C, the ELISA plate was washed 2 times with PBST solution, 300 μL / well each time. After washing and blotting dry, the ELISA plate was blocked with PBS solution containing 1% BSA, 200 μL / well, at room temperature for 2 hours. After blotting dry, different concentrations of phosphorylated VASP-Ser 239-BSA diluted with antibody G085 (diluted to 10 ng / mL with PTB) were reacted on a shaker at room temperature for 30 minutes, and then added to the above-blocked ELISA plate, 100 μL / well, and reacted in an incubator at 37°C for 1 hour. After blotting dry, the plate was washed 3 times with PBST, 300 μL / well each time. After blotting dry, add HRP-labeled goat anti-mouse antibody diluted 5000 times with PTB to the plate and react in an incubator at 37°C for 1 hour. Wash the plate 3 times with PBST, 300 μL / well. After blotting dry, add TMB substrate solution to the ELISA plate, 100 μL / well, at room temperature for 10 minutes, and then add 100 μL / well of 1 M H2SO4 to terminate the reaction. Measure the absorbance value (OD 450 ) using an enzyme-linked immunosorbent assay reader at 450 nm. The linear result of the sample concentration and absorbance value is as Figure 1 shown. The results show that the curve R 2 = 0.9976, and the standard curve range of this method is 0 - 10 ng / ml.

[0065] (3) The enzyme immunoassay kit prepared with antibody G085 for detecting phosphorylated VASP-Ser 239 protein in human platelet cells In this experiment, the phosphorylated VASP-Ser 239 protein antibody was diluted to 1 μg / mL with PBS and then coated in a 96-well enzyme-linked immunosorbent assay (ELISA) plate at 100 μL / well (overnight at 4°C). The plate was washed twice with PBST at 300 μL / well. After patting dry, the ELISA plate was blocked with PBS solution containing 1% BSA at 200 μL / well for 2 hours at room temperature. After patting dry, 25 μL / well of the HRP-labeled antibody G085 (diluted with PTB) solution diluted 1000-fold was added to the ELISA plate, and then 75 μL / well of human platelet samples (the protein extract of platelet cells from patients clinically diagnosed with platelet-related diseases was selected as the test sample, and the protein extract of platelet cells from healthy individuals was used as the negative sample), 75 μL / well, were added and reacted in an incubator at 37°C for 45 minutes. The plate was washed 3 times with PBST at 300 μL / well each time. After patting dry, 100 μL / well of TMB substrate solution was added and reacted at room temperature for 10 minutes. Then 100 μL / well of 1 M H2SO4 was added to terminate the reaction, and the absorbance value (OD 450 ) was measured at 450 nm using an ELISA reader. The results are shown in Table 8, indicating that the enzyme immunoassay kit prepared with the antibody G085 of the present invention can efficiently detect phosphorylated VASP-Ser 239 protein in human platelets. The enzyme immunoassay kit of the present invention can perform quantitative detection of phosphorylated VASP239 protein in human platelets by substituting the OD 450 value into the standard curve ( Figure 1 ). By comparing the results in Table 8, it is shown that this kit can preliminarily determine positive and negative blood samples Table 8 Patient platelet samples

[0066] The present invention provides a monoclonal antibody against phosphorylated VASP-Ser 239 protein, which has the characteristics of high affinity and specificity. At the same time, it also provides an in vitro diagnostic detection kit based on the above antibody. The present invention solves the problem of the relative shortage of antibody raw materials and kits for the detection item of phosphorylated VASP-Ser 239 protein in the current market

[0067] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0068] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A phosphorylated VASP-Ser 239 protein antibody or antigen-binding fragment, characterized in that, Comprising: A CDR sequence selected from at least one of the following or an amino acid sequence having at least 80% identity thereto: Light chain variable region CDR sequences: SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5; Heavy chain variable region CDR sequences: SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:

8.

2. The antibody or antigen-binding fragment according to claim 1, wherein Comprising: The light chain variable region CDR1 sequence shown in SEQ ID NO:3, the light chain variable region CDR2 sequence shown in SEQ ID NO:4, the light chain variable region CDR3 sequence shown in SEQ ID NO:5, the heavy chain variable region CDR1 sequence shown in SEQ ID NO:6, the heavy chain variable region CDR2 sequence shown in SEQ ID NO:7, and the heavy chain variable region CDR3 sequence shown in SEQ ID NO:

8.

3. The antibody or antigen-binding fragment according to claim 1, wherein Comprising at least one of the following: (a) Having a light chain variable region shown in SEQ ID NO:1 and a heavy chain variable region shown in SEQ ID NO:2; An amino acid sequence having at least 80% sequence identity compared to (a).

4. The antibody or antigen-binding fragment according to claim 1, wherein, Comprising at least one of the following: (b) Having the CDR1, CDR2, and CDR3 sequences of the light chain variable region shown in SEQ ID NO:1, and the CDR1, CDR2, and CDR3 sequences of the heavy chain variable region shown in SEQ ID NO:2; Or an amino acid sequence having more than one conservative amino acid substitution compared to (b).

5. A conjugate, characterized in that, Comprising: The phosphorylated VASP-Ser 239 protein antibody or antigen-binding fragment according to any one of claims 1 to 4; And a conjugate part, the conjugate part being linked to the phosphorylated VASP-Ser 239 protein antibody or antigen-binding fragment; Optionally, the conjugate part includes at least one of a carrier, a drug, a toxin, a cytokine, a protein tag, a modifier, and a chemotherapeutic agent; Optionally, the carrier includes a fluorescent dye; Optionally, the fluorescent dye includes any one or more of FITC, PE, Cy5, PI, 7AAD, APC, Alexa Fluor, eFluor, PECy7, APC Cy7, PerCP, PerCPcy5.5, PB, BV series, QDot series, and BUV series dyes.

6. An isolated polynucleotide, characterized in that, The polynucleotide encodes the phosphorylated VASP-Ser 239 protein antibody or antigen-binding fragment according to any one of claims 1 to 4; Optionally, the polynucleotide comprises: (c) A nucleic acid sequence encoding a light chain variable region shown in SEQ ID NO:9, a nucleic acid sequence having at least 80% sequence identity compared to (c); Or (d) A nucleic acid sequence encoding a heavy chain variable region shown in SEQ ID NO:10, a nucleic acid sequence having at least 80% sequence identity compared to (d).

7. An expression vector, characterized in that, Comprising the isolated polynucleotide according to claim 6.

8. A recombinant cell, characterized in that, Comprising the expression vector according to claim 7.

9. A method for preparing a phosphorylated VASP-Ser 239 protein antibody or antigen fragment, characterized in that, Including culturing the recombinant cell according to claim 8.

10. Use of the antibody or antigen-binding fragment according to any one of claims 1 to 4 or the conjugate according to claim 5 in the preparation of a kit, characterized in that, The kit is used for detecting phosphorylated VASP-Ser 239 protein in a sample; optionally, the kit detects the phosphorylation degree of VASP in human whole blood in vitro.

11. The use according to claim 10, characterized in that, The kit is used for monitoring the inhibitory effect and degree of inhibition of a specific platelet ADP receptor (P2Y12) antagonist on platelet activation, clarifying whether there is clopidogrel resistance, and guiding the reasonable adjustment of dual anti-drugs.

12. A kit, characterized in that, It includes the phosphorylated VASP-Ser 239 protein antibody or antigen-binding fragment according to any one of claims 1 to 4, the conjugate according to claim 5, the isolated polynucleotide according to claim 6, the expression vector according to claim 7, and / or the recombinant cell according to claim 8.

13. A composition, characterized in that, It includes the phosphorylated VASP-Ser 239 protein antibody or antigen-binding fragment according to any one of claims 1 to 4, the conjugate according to claim 5, the isolated polynucleotide according to claim 6, the expression vector according to claim 7, and / or the recombinant cell according to claim 8.

14. A method for preparing the phosphorylated VASP-Ser 239 protein antibody or antigen-binding fragment according to any one of claims 1 to 4, characterized in that, It includes culturing the recombinant cell according to claim 8.

15. A method for detecting phosphorylated VASP-Ser 239 protein, characterized in that, It includes: Contacting the phosphorylated VASP-Ser 239 protein antibody or antigen-binding fragment according to any one of claims 1 to 4, the conjugate according to claim 5, or the kit according to claim 12 with a sample to be detected to form an immune complex; based on the signal of the immune complex, quantitatively and / or qualitatively analyzing the phosphorylated VASP-Ser 239 protein in the sample to be detected; optionally, the signal of the immune complex is detected by at least one method among ELISA, WB, and FACS; optionally, the signal includes a fluorescence signal.

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