An antibody against phosphorylated VASP-Ser 239 protein and its application

Abstract

This invention relates to an antibody or antigen-binding fragment of phosphorylated VASP-Ser 239 protein and its applications. The antibody comprises specific light and heavy chain variable region complementarity-determining region (CDR) sequences or amino acid sequences having at least 80% sequence identity with them. The invention also provides isolated polynucleotides encoding the antibody, expression vectors, recombinant cells, and methods for preparing the antibody. The antibody and its derivatives of this invention can efficiently and specifically detect phosphorylated VASP-Ser 239 protein in human whole blood or platelet cells, and are suitable for monitoring the inhibitory effect of platelet ADP receptor (P2Y12) antagonists (such as clopidogrel) on platelet activation, clarifying clopidogrel resistance, and guiding rational clinical drug use. This invention solves the current shortage of antibody raw materials and kits for related detection projects on the market.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a phosphorylated VASP-Ser 239 protein antibody and its applications. Background Technology

[0002] Angiotensin-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 filamentous actin formation and may play a broad role in cell adhesion and motility. VASP may also be involved in regulating intracellular signaling pathways that regulate integrin-extracellular matrix interactions. VASP is regulated by cyclic nucleotide-dependent kinases PKA and PKG.

[0003] VASP proteins are actin-associated proteins involved in a series of processes dependent on cytoskeleton remodeling and cell polarity, such as axonal guidance, platelet and filamentous dynamics, platelet activation, and cell migration. VASP promotes actin filament elongation. It protects the barbed ends of growing actin filaments from capping and increases the rate of actin polymerization in the presence of capping proteins. VASP stimulates actin filament elongation by promoting the transfer of actin monomers bound to fasciculin to the barbed ends of growing actin filaments. It plays a role in the actin-based migration of Listeria monocytogenes into host cells. It also 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. Phosphorylation is regulated by the cAMP cascade, which is activated by prostacyclin E1 (PGE1). VASP phosphorylation is directly related to the level of P2Y12 receptor inhibition. Therefore, the effects of antiplatelet drugs such as clopidogrel can be evaluated by measuring phosphorylated VASP, and thus it can also be used to assess population variability in antiplatelet drug efficacy. The method for measuring phosphorylated VASP has high sensitivity and specificity, good parallelism with platelet aggregation rate measurements, requires a small blood sample, and can use whole blood.

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

[0006] To address the current market shortage of antibody raw materials and kits for detecting phosphorylated VASP-Ser 239 protein, this invention provides a monoclonal antibody for phosphorylated VASP-Ser 239 protein, which exhibits high affinity and specificity. The invention also provides products prepared based on this antibody and their applications.

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

[0008] This invention provides a phosphorylated VASP-Ser 239 protein antibody or antigen-binding fragment. An enzyme immunoassay kit prepared using this antibody or antigen-binding fragment exhibits high sensitivity and specificity. According to some embodiments of the 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 invention, the antibody or antigen-binding fragment comprises at least one of the following:

[0009] (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;

[0010] Compared to (a), the sequence identity is at least 80% of the amino acid sequence.

[0011] According to some embodiments of the present invention, the antibody or antigen-binding fragment comprises at least one of the following:

[0012] (b) The CDR1, CDR2 and CDR3 sequences having the light chain variable region shown in SEQ ID NO:1, and the CDR1, CDR2 and CDR3 sequences having the heavy chain variable region shown in SEQ ID NO:2;

[0013] Or, compared to (b), it has an amino acid sequence with more than one conserved amino acid substitution.

[0014] According to some embodiments of the present invention, the antibody or antigen-binding fragment further includes at least one of a heavy chain constant region and a light chain constant region, at least a portion of the heavy chain constant region and the light chain constant region being derived from a mammalian antibody.

[0015] According to some embodiments of the present invention, the heavy chain constant region and the light chain constant region are derived from at least one of mouse antibodies, rabbit antibodies, primate antibodies or mutants thereof.

[0016] According to some embodiments of the present invention, the heavy chain constant region and the light chain constant region are derived from mouse antibodies or mutants thereof.

[0017] According to some embodiments of the present invention, the N end of the heavy chain constant region is connected to the C end of the heavy chain variable region; and / or the N end of the light chain constant region is connected to the C end of the light chain variable region.

[0018] According to some embodiments of the present invention, the heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; or the light chain constant region includes a light chain constant region selected from κ-type or λ-type.

[0019] According to some embodiments of the present invention, the antibody or antigen-binding fragment comprises a monoclonal antibody or a polyclonal antibody;

[0020] According to some embodiments of the present invention, the monoclonal antibody includes at least one of full-length antibody, Fv, single-chain antibody, Fab, single-domain antibody, and minimum recognition unit.

[0021] In another aspect, the present invention provides an antibody analog comprising the variable region or CDR region of the variable region of the antibody or antigen-binding fragment described above.

[0022] Another aspect of the present invention provides a coupling comprising:

[0023] The aforementioned phosphorylated VASP-Ser 239 protein antibody or antigen-binding fragment; and

[0024] The coupling portion is linked to the phosphorylated VASP-Ser 239 protein antibody or antigen-binding fragment.

[0025] According to some embodiments of the present invention, the coupling portion includes at least one of a carrier, a drug, a toxin, a cytokine, a protein tag, a modifier, and a chemotherapeutic agent.

[0026] According to some embodiments of the present invention, the carrier comprises a fluorescent dye.

[0027] According to some embodiments of the present invention, the fluorescent dye includes any one or more of the following: FITC, PE, Cy5, PI, 7AAD, APC, AlexaFluor, eFluor, PECy7, APC Cy7, PerCP, PerCPcy5.5, PB, BV series, QDot series and BUV series dyes.

[0028] In another aspect, the present invention provides an isolated polynucleotide encoding the aforementioned antibody or antigen-binding fragment.

[0029] According to some embodiments of the present invention, the polynucleotide comprises:

[0030] (c) A nucleic acid sequence having the light chain variable region encoded as shown in SEQ ID NO:9

[0031] Compared to (c), nucleic acid sequences with at least 80% sequence identity; or

[0032] (d) A nucleic acid sequence having the heavy chain variable region encoded as shown in SEQ ID NO:10,

[0033] Compared to (d), the nucleic acid sequence has at least 80% sequence identity.

[0034] In another aspect, the present invention provides an expression vector comprising the above-mentioned polynucleotides.

[0035] In another aspect, the present invention provides a recombinant cell into which the above-mentioned expression vector has been transferred.

[0036] In another aspect, the present invention provides a cell that can produce the above-mentioned phosphorylated VASP-Ser 239 protein antibody or antigen-binding fragment.

[0037] In another aspect, the present invention provides a method for preparing phosphorylated VASP-Ser 239 protein antibodies or antigen fragments, comprising culturing the aforementioned recombinant cells.

[0038] In another aspect, this invention provides the use of the aforementioned antibodies or antigen-binding fragments and antibody analogs in the preparation of a kit for the in vitro quantitative detection of the phosphorylation level of vasodilator-stimulating phosphoprotein (VASP) in human whole blood. This kit is used to monitor the inhibitory effect and degree of platelet activation by specific platelet ADP receptor (P2Y12) antagonists (such as clopidogrel), to determine the presence of clopidogrel resistance, and to guide the rational adjustment of dual antibody drugs.

[0039] According to some embodiments of the present invention, the kit is used to quantitatively detect the phosphorylation level of vasodilator-stimulating phosphoprotein (VASP) in human whole blood in vitro, to monitor the inhibitory effect and degree of platelet activation by specific platelet ADP receptor (P2Y12) antagonists (such as clopidogrel), to determine whether clopidogrel resistance exists, and to guide the rational adjustment of dual antiplatelet drugs.

[0040] 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 vasodilatory stimulating phosphoprotein (VASP) in human whole blood. This is used to monitor the inhibitory effect and degree of platelet activation by specific platelet ADP receptor (P2Y12) antagonists (such as clopidogrel), to determine the presence of clopidogrel resistance, and to guide the rational adjustment of dual antiplatelet therapy.

[0041] Another aspect of the present invention provides a kit comprising at least one of the above-described antibody or antigen-binding fragment, conjugate, expression vector, and recombinant cells.

[0042] Another aspect of the present invention provides a composition comprising at least one of the above-described antibody or antigen-binding fragments, conjugates, isolated polynucleotides, expression vectors, recombinant cells, and hybridoma cells.

[0043] Another aspect of the present invention provides a method for preparing the above-mentioned antibody or antigen-binding fragment, comprising culturing the above-mentioned recombinant cells.

[0044] Another aspect of the present invention provides a method for detecting phosphorylated VASP-Ser 239 protein, comprising:

[0045] The phosphorylated VASP-Ser 239 protein antibody or antigen-binding fragment, conjugate or kit is contacted with the sample to be tested to form an immune complex; based on the signal of the immune complex, the phosphorylated VASP-Ser 239 protein in the sample to be tested is quantitatively and / or qualitatively analyzed.

[0046] According to some embodiments of the present invention, the signal of the immune complex is detected by at least one of ELISA, WB and FACS.

[0047] According to some embodiments of the present invention, the signal includes a fluorescence signal.

[0048] Another aspect of the present invention provides a drug comprising at least one of the above-described antibody or antigen-binding fragments, conjugates, isolated polynucleotides, expression vectors, recombinant cells, and compositions.

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

[0050] Figure 1 The kit of the present invention is for the quantitative detection of phosphorylated VSAP protein in human platelet cells. 450 Standard curve. Detailed Implementation

[0051] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0052] The nucleic acids encoding the heavy and / or light chains of the antibodies of this invention are within the scope of this invention. Based on the amino acid sequences of the heavy and / or light chains, those skilled in the art can easily obtain the corresponding nucleic acid sequences, as shown in Table 1. It should be noted that the CDR sequences listed in Table 1 below were obtained from the IMGT database. Those skilled in the art should understand that CDR sequences obtained from different databases may differ, but these variations should all be included within the scope of protection of this invention.

[0053] Table 1 Sequence Information

[0054]

[0055]

[0056] Example 1: Synthesis of non-phosphorylated VASP-Ser 239 protein peptide, phosphorylated VASP-Ser 239 protein peptide, and full-length phosphorylated VASP-Ser 239 protein antigen

[0057] The full-length amino acid sequence of the non-phosphorylated VASP protein is shown below:

[0058] MSETVICSSRATVMLYDDGNKRWLPAGTGPQAFSRVQIYHNPTANSFRVVGRKMQPDQQVVINCAIVRGVKYNQATPNFHQWRDARQVWGLNFGSKEDAAQFAAGMASALEALEGGGPPPPPALPTWSVPNGPSPEEVEQQKRQQPGPSEHIERRVSNAGGPPAPPAGGPPPPPGPPPPPGPPPPPGLPP SGVPAAAHGAGGGPPPAPPLPAAQGPGGGGAGAPGLAAAIAGAKLRKVSKQEEASGGPTAPKAESGRSGGGGLMEEMNAMLARRRKATQVGEKTPKDESANQEEPEARVPAQSESVRRPWEKNSTTLPRMKSSSSVTTSETQPCTPSSSDYSDLQRVKQELLEEVKKELQKVKEEIIEAFVQELRKRGSP (SEQ ID NO:11)

[0059] Using a portion of the sequence, phosphorylated and non-phosphorylated VASP-Ser 239 protein peptides were synthesized using conventional methods in the art. The specific peptide sequences are as follows:

[0060] Table 2. Peptide synthesis sequence information

[0061]

[0062] Note: In the table, "(p)" indicates that the preceding amino acid has a carboxylation modification. "BSA" indicates that the C-segment of the phosphorylated VASP-Ser 239 protein or the non-phosphorylated VASP-Ser 239 protein peptide is linked to bovine serum albumin, synthesized by Shanghai Botai Biotechnology Co., Ltd.; "KLH" indicates that the C-segment of the phosphorylated VASP-Ser 239 protein is linked to hemocyanin, synthesized by Shanghai Synthetic Botai Biotechnology Co., Ltd.

[0063] Example 2: Identification of phosphorylated VASP-Ser 239 protein

[0064] First, phosphorylated VASP-Ser 239 protein (VASP-BSA) was coated onto an ELISA plate at a concentration of 1 μg / ml. Purified phosphorylated VASP-Ser 239 protein antibody was used as the primary antibody in the indirect ELISA method, and HRP-labeled goat anti-rat antibody was used as the secondary antibody. TMB was used as the chromogenic buffer, and the reaction was terminated with 1M H₂SO₄. The OD of the ELISA plate was measured. 450The readings were compared with the antigenic activity of phosphorylated VASP-Ser 239 protein, and it can be seen that the antigenic activity of phosphorylated VASP-Ser 239 protein was detected to be high.

[0065] Table 3 Identification of phosphorylated VASP-Ser 239 protein

[0066]

[0067] Example 3: Immunizing BALB / c mice with phosphorylated VASP-Ser 239 protein

[0068] BALB / c mice aged 6–8 weeks were immunized according to the following procedure: For the initial immunization, 25 μg of phosphorylated VASP-Ser 239 protein pVASP239-KLH was mixed with an equal volume of Freund's complete adjuvant, emulsified, and injected subcutaneously at multiple sites. Fourteen days after the initial immunization, a booster immunization was performed using 12.5 μg of phosphorylated VASP-Ser 239 protein pVASP239-KLH mixed with Freund's incomplete adjuvant, emulsified, and then administered. Fourteen days after the second immunization, a booster immunization was performed using 12.5 μg of phosphorylated VASP-Ser 239 protein pVASP239-KLH mixed with Freund's incomplete adjuvant, emulsified, and then administered. Fourteen days after the third immunization, blood was collected and serum separated. An ELISA plate was coated with 1 μg / mL phosphorylated VASP-Ser 239 protein pVASP239-BSA, and an indirect ELISA assay was performed to determine the serum titer. The results showed that the titer of the prepared mouse antiserum was 1:72900, indicating successful immunization. Typical results of immunized mouse serum testing are shown in Table 4.

[0069] Table 4 Typical serum detection results from immunized mice

[0070]

[0071] Example 4: Cell Fusion

[0072] In Example 3, cell fusion was performed 3 days after booster immunization of mice. After blood was collected from the mice's eyes, they were euthanized by dislocation, placed in a bottle of 70% alcohol for 2 minutes, and then fixed to a foam board in a biosafety cabinet. The abdominal skin was opened to locate the spleen, which was removed with forceps and gently crushed in a 200-mesh stainless steel filter. The cells were gently washed with DMEM medium (Thermo, 11965092), and then centrifuged at 200g for 10 minutes at room temperature. The supernatant was discarded and the cells were used for further processing. For feeder cell preparation, mice were euthanized by dislocation, placed in a bottle of 70% alcohol for 2 minutes, and then fixed to a foam board in a biosafety cabinet. The abdominal skin was opened, and PBS was gently injected subperitoneally using a syringe. The feeder cell-containing liquid was washed out from the other side, and then centrifuged at 200g for 10 minutes at room temperature. The supernatant was discarded and the cells were used for further processing. 2.0 × 10⁻⁶ cells were then prepared. 7 One FO myeloma cell and 2.0 × 10 8 Mix the spleen cells thoroughly, centrifuge at 200g for 10 minutes, discard the supernatant, gently vortex to mix, and incubate at 37°C. Add 1 ml of 50% PEG-1450 (Merk, P1458) aqueous solution within 90 seconds, then add 20 mL of DMEM medium, centrifuge at 200g for 10 minutes, discard the supernatant, repeat the washing once, centrifuge at 200g for 10 minutes, discard the supernatant, and obtain hybridoma cells. Plate the cells into 10 96-well culture plates, 150 μL per well. Add 10,000 feeder cells / well to 10 wells of the above-mentioned 96-well cell culture plates, 100 μL per well. After labeling the culture plates, incubate them in a cell culture incubator at 37°C with 5% CO2. On the second day, add HAT selection medium (Merk, H0262) and continue HAT selection culture for 1-2 days. A large number of tumor cells will die, and after 3-4 days, the tumor cells will disappear, and hybrid cells will form small colonies. Maintain the HAT selection culture medium for 7-10 days, then switch to HT culture medium (Merk, H0137) and maintain for another 2 weeks. Then, continue culturing in DMEM medium containing 20% ​​FBS (ExCell, FSP500). During the above selection culture, when the hybridoma cells cover 1 / 10 of the well bottom area, you can start detecting specific antibodies and screening for the desired hybridoma cell lines. During the selection culture, generally change half of the culture medium every 2-3 days.

[0073] Example 5: Screening and subclonal culture of positive hybridoma cell lines

[0074] First, orthogonal experiments were used to determine the optimal coating amount of phosphorylated VASP-Ser 239 protein as the antigen. 0.5, 1.0, 2.0, and 4.0 μg of phosphorylated VASP-Ser 239 protein pVASP239-BSA were coated onto 96-well plates, with each concentration represented by 6 wells (3 positive and 3 negative). Square titration was performed using positive serum from mice immunized with phosphorylated VASP-Ser 239 protein pVASP239-KLH at different dilutions, while negative serum from unimmunized mice served 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 blot dry on folded paper. Add 0.1 mL of the test sample to each well, incubate at 37°C for 1 hour, then wash. Simultaneously prepare blank wells (without sample), negative control wells, and positive control wells. Add 0.1 mL of freshly diluted antibody to each well, incubate at 37°C for 1 hour, then wash three times. Add 0.1 mL of freshly diluted enzyme-labeled antibody to each well. Incubate at 37°C for 1 hour, then wash three times. Add substrate solution for color development: Add 0.1 mL of TMB substrate solution to each well and incubate at room temperature for 10 minutes. Add 0.1 mL of 1M H₂SO₄ to each well. The results were determined by measuring the OD value using an ELISA reader at 450 nm (A450). A positive result was defined as an OD value greater than 2.1 times that of the negative control (calculated after zeroing the blank control well). Hybridoma cell clones resistant to phosphorylated VASP-Ser 239 protein were selected.

[0075] Following the above method, the selected positive hybridoma cells were subcloned. The original wells were diluted with HAT selective medium using a limiting dilution method and then re-distributed into 96-well culture plates. Cell morphology and quantity were then observed. The cell density was adjusted to 3–10 cells / mL. 100 μL of diluted cells was added to each well of a cell culture plate containing a feeder cell layer prepared the previous day. The plates were then incubated statically at 37°C in a 5% CO2 incubator.

[0076] Change the medium on day 7, and then every 2-3 days thereafter. Cell clones will be observed on days 8-9; antibody activity should be monitored promptly. Transfer cells from positive wells to 24-well plates for further culture. Each clone should be cryopreserved as soon as possible, and the hybridoma cell line with clone number G085 will be selected for antibody production.

[0077] Example 6: Large-scale preparation of monoclonal antibodies and determination of antibody titer

[0078] (1) Large-scale preparation of monoclonal antibodies

[0079] Eight-week-old BALB / c mice were injected intraperitoneally with 0.5 mL of Freund's incomplete adjuvant, followed by an intraperitoneal injection of 1 × 10⁻⁶ mg / mL two weeks later. 6 Hybridoma cells G085, after 7-10 days of inoculation, will produce ascites. Closely observe the health status and signs of ascites in the animals. When ascites is as abundant as possible and the mice are close to death, sacrifice the mice and aspirate the ascites into test tubes using a dropper. 5-10 ml of ascites can be obtained from one mouse. Alternatively, ascites can be extracted using a syringe, and can be collected repeatedly. Centrifuge the obtained ascites at 3000g for 10 minutes, discard the upper layer of oil and the bottom precipitate, collect the supernatant, and aliquot it at -20℃. After thawing and equilibrating the ascites supernatant to room temperature, add 1 / 10 volume of 1M Tris-HCl pH 8.0 to adjust the sample pH to 8.0. Equilibrate a protein G affinity column with 20 column volumes of 100 mM Tris-HCl at pH 8.0. Load the ascites supernatant (adjusted to pH 8.0) onto the column, then wash with 20 column volumes of 100 mM Tris-HCl at pH 8.0. Finally, elute the antibody (i.e., phosphorylated VASP-Ser239 protein antibody G085, abbreviated as antibody G085) with 100 mM Glycine HCl at pH 2.5. Add the antibody eluent to a concentration tube (Millipore, UFC801008, 10K) and centrifuge at 3000×g for 20 minutes at room temperature using a centrifuge (Xiangyi, L550). Centrifuge in batches to a final volume of 1 ml / concentration tube (2 tubes), add 4 mL of 10 mM PBS (pH 7.4) buffer, and continue centrifuging at 3000×g for 20 minutes at room temperature. Repeat centrifugation three times to prepare the antibody buffer at 10 mM PBS (pH 7.4), then add 10 mM PBS (pH 7.4) to a total volume of 10 mL. Finally, aliquot the concentrated antibody solution into centrifuge tubes (2 mL / tube) and store at -80°C. The antibody concentration was determined using a BCA kit (Solepro, PC0020), and the purified monoclonal antibody concentration was found to be 3.8 mg / mL.

[0080] (2) Antibody titer determination

[0081] The titer of phosphorylated VASP-Ser 239 protein antibody G085 was detected using an indirect ELISA method. Phosphorylated VASP-Ser 239 protein pVASP239-BSA was diluted with PBS and coated into 96-well microplates at a concentration of 0.2 μg / mL (100 μL / well). After incubation at 4°C overnight, the plates were washed twice with PBST solution (300 μL / well each time) and dried. The plates were then blocked with 200 μL / well of 1% BSA in PBS and incubated at room temperature for 2 hours, followed by drying. PTB diluted to 20 ng / mL of phosphorylated VASP-Ser 239 protein antibody G085 was added and incubated at 37°C for 1 hour. The plates were then washed twice with PBST solution (300 μL / well each time) and dried. HRP-labeled goat anti-mouse antibody was diluted 5000-fold with PTB and reacted at 37°C for 1 hour. The plate was washed twice with PBST solution, 300 μL / well each time. After washing, the plate was patted dry. TMB substrate solution was added at 100 μL / well and reacted at room temperature for 10 minutes. The reaction was stopped by adding 100 μL / well of 1M H2SO4. The absorbance was measured at 450 nm (A450) using a microplate reader. The results are shown in Table 5, indicating that the purified antibody titer was 1:729000.

[0082] Table 5 Antibody titer

[0083]

[0084] Example 7: Sequence Analysis of Monoclonal Antibodies

[0085] (1) Identification of monoclonal antibody subtypes

[0086] Hybridoma cell line G085 was cultured in DMEM medium (GIBCO, #C11995500BT) supplemented with 10% serum in 10 cm diameter cell culture dishes (37℃, 5% CO2). After 7 days of culture, the cells were transferred to 15 ml centrifuge tubes, counted using a hemocytometer, and 4 × 10⁴ cells were collected. 6 Centrifuge the cells at 200g for 5 minutes, discard the supernatant, and invert the centrifuge tube to drain the liquid. Use the Qiagen reverse transcription kit (Qiagen, 74134) to synthesize cDNA from the cells.

[0087] Antibody subtypes were determined by PCR using subtype-specific primers. The synthesized cDNA was used as the PCR template. The PCR reaction solution consisted of: 0.25 μL TAKARA Ex Taq (5 U / μL, TAKARA, RR001B); 5 μL 10×Ex Taq Buffer; 4 μL dNTP mixture (2.5 mM each); 1 μL template cDNA; 1 μL upstream primer (100 μM); 1 μL downstream primer (100 μM); and double-distilled water to a total volume of 50 μL. The PCR temperature program was: 94℃ for 5 minutes of pre-denaturation, followed by 30 temperature cycles (94℃ for 1 minute, 57℃ for 1 minute, 72℃ for 1 minute), and 72℃ for 10 minutes of extension. After the reaction, 10 μL of each PCR product was loaded onto a 1% agarose gel for electrophoresis. The antibody subtype could be deduced from the PCR product results (Table 6). The monoclonal antibody G085 obtained in this invention has a heavy chain of IgG1 and a light chain of kappa.

[0088] Table 6 PCR Primer Information

[0089]

[0090] Where S=C or G, M=A or C, R=A or G, and W=A or T

[0091] (2) Sequencing of the variable region (V region) of the hybridoma cell line G085 antibody

[0092] The V region fragment of the antibody from cell line G085, obtained after PCR amplification (see above), was cut from an agarose gel and extracted using a DNA extraction kit (Qiagen, 74134). The extracted DNA fragment was ligated into the pEASY-T1 cloning vector and transformed into Trans1-T1 competent cells (Transgen, CT101-1). The transformed bacterial colonies were picked into LB medium, cultured overnight, and then subjected to DNA sequencing. The light chain V region nucleic acid sequence of the phosphorylated VASP-Ser 239 protein antibody (G085) provided by this invention is shown in SEQ ID NO. 9, and the heavy chain V region nucleic acid sequence is shown in SEQ ID NO. 10.

[0093] Example 8: Identification of the specificity of antibody G085 in detecting phosphorylated VASP-Ser 239 protein

[0094] The ability of antibody G085 to detect the specificity of phosphorylated VASP-Ser 239 protein was evaluated using ELISA. In this experiment, the non-phosphorylated VASP-Ser 239 protein peptide VASP239-BSA and the phosphorylated VASP-Ser 239 protein pVASP239-BSA were diluted with PBS and coated into 96-well microplates at a concentration of 1 μg / mL and a volume of 100 μL / well. After overnight coating at 4°C, the microplates were washed twice with PBST solution, 300 μL / well each time. After washing and drying, the microplates were blocked with PBS solution containing 1% BSA, 200 μL / well, at room temperature for 2 hours. After drying, antibody G085 (diluted with PTB to different concentrations) was added to the blocked microplates, 100 μL / well, and incubated at 37°C for 1 hour. After drying, the microplates were washed three times with PBST, 300 μL / well each time. After patting the plate dry, add HRP-labeled goat anti-mouse antibody diluted 5000-fold with PTB to the microplate and incubate at 37°C for 1 hour. Wash the plate three times with PBST, 300 μL / well. After patting the plate dry, add 100 μL / well of TMB substrate solution and incubate at room temperature for 10 minutes. Then add 100 μL / well of 1M H2SO4 to stop the reaction. Measure the absorbance at 450 nm (A450) using a microplate reader. The experimental results are shown in Table 7, indicating that antibody G085 has good specificity for detecting phosphorylated VASP-Ser 239 protein.

[0095] Table 7 Identification of the specificity of antibody G085

[0096]

[0097] Example 9: Application of an enzyme immunoassay kit prepared using antibody G085 in the detection of phosphorylated VASP-Ser 239 protein in human platelet cells.

[0098] (1) Horseradish peroxidase (HRP) labeling of antibody G085 and identification of the labeled product

[0099] Add 0.5 mL of antibody G085 (20 nmol, 3 mg) to a dialysis bag (10 kDa, 1 cm wide) and dialyze overnight at 4°C in 2 L of 10 mM PBS solution (pH 7.4). The next day, place the dialysis bag containing the antibody solution into 1 L of 10 mM carbonate buffer (pH 9.5) and dialyze at room temperature for 2 hours with stirring, preparing for coupling with activated HRP.

[0100] Meanwhile, accurately weigh 1 mg of HRP using an analytical balance and dissolve it in 0.2 mL of ultrapure water to achieve an HRP concentration of 5 mg / mL. Add 40 μL of 0.1 M NaIO4 to the above HRP solution and place it on a horizontal shaker to react (activate) at room temperature in the dark for 20 minutes. Add the activated HRP solution to a dialysis bag (10 kDa, 1 cm wide) and dialyze overnight at 4°C in 2 L of 1 mM sodium acetate buffer (pH 4.4). Carefully aspirate the dialyzed HRP solution and transfer it to a new 1.5 mL centrifuge tube. Add 1 / 10 volume of 0.2 M carbonate buffer to raise the pH of the activated HRP solution to 9.0-9.5.

[0101] Mix the above antibodies with HRP (for coupling reaction) and place on a horizontal shaker. Incubate at room temperature in the dark for 4 hours. After the coupling reaction is complete, add 10 μl of freshly prepared NaBH4 (using pre-cooled ultrapure water) and incubate overnight at 4°C in the dark to terminate the reaction. Transfer the antibody solution after the coupling reaction is terminated to a dialysis bag (10 kDa, 1 cm wide) and dialyze at room temperature with stirring for 2 hours. Finally, transfer the solution to brown centrifuge tubes and store at 4°C.

[0102] (2) Determination of the standard curve of the enzyme immunoassay kit prepared using antibody G085

[0103] In this experiment, phosphorylated VASP-Ser 239-BSA conjugate protein (pVASP239-BSA) was diluted with PBS and coated onto 96-well microplates at a concentration of 1 µg / mL and a volume of 100 µL / well. After overnight coating at 4°C, the microplates were washed twice with PBST solution, 300 µL / well each time. After washing, the plates were blotted dry and 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 incubated on a shaker at room temperature for 30 minutes, and then added to the blocked microplates, 100 µL / well, and incubated at 37°C for 1 hour. The plates were then washed three times with PBST, 300 µL / well each time. Pat dry, add HRP-labeled goat anti-mouse antibody diluted 5000-fold with PTB to the plate, and incubate at 37°C for 1 hour. Wash the plate 3 times with PBST, 300 µL / well. Pat dry, add TMB substrate solution, 100 µL / well, incubate at room temperature for 10 minutes, then add 100 µL / well of 1MH2SO4 to terminate the reaction. Measure the absorbance (OD) at 450 nm using a microplate reader. 450 The linear relationship between sample concentration and absorbance is shown in the figure. Figure 1 As shown, the results indicate that curve R 2=0.9976, the standard curve range of this method is 0-10 ng / ml.

[0104] (3) An enzyme immunoassay kit prepared using antibody G085 was used to detect phosphorylated VASP-Ser 239 protein in human platelet cells.

[0105] In this experiment, phosphorylated VASP-Ser 239 protein antibody was diluted to 1 μg / mL with PBS, and then 100 μL / well was coated into a 96-well microplate (4°C overnight). The plate was washed twice with 300 μL / well of PBST. After drying, the plate was blocked with 200 μL / well of PBS containing 1% BSA and incubated at room temperature for 2 hours. After drying, 25 μL / well of a 1000-fold diluted HRP-labeled antibody G085 solution (diluted with PTB) was added to each well, followed by 75 μL / well of human platelet sample (platelet cell protein extract from patients with clinically confirmed platelet-related diseases was used as the test sample, and platelet cell protein extract from healthy individuals was used as the negative sample), and the plate was incubated at 37°C for 45 minutes. The plate was washed three times with 300 μL / well of PBST each time. Pat dry, add 100 μL of TMB substrate solution per well, and incubate at room temperature for 10 minutes. Then, stop the reaction by adding 100 μL of 1M H2SO4 per well. Measure the absorbance (OD) at 450 nm using a microplate reader. 450 The results, shown in Table 8, demonstrate that the enzyme immunoassay kit prepared using the antibody G085 of this invention can efficiently detect phosphorylated VASP-Ser 239 protein in human platelets. The enzyme immunoassay kit of this invention can be detected via OD... 450 Substitute the numerical values ​​into the standard curve ( Figure 1 The kit was used to quantitatively detect phosphorylated VASP239 protein in human platelets. Comparison of the results in Table 8 shows that this kit can preliminarily determine positive or negative blood types.

[0106] Table 8 Platelet Samples from Patients

[0107]

[0108] This invention provides a monoclonal antibody against phosphorylated VASP-Ser 239 protein, which exhibits high affinity and specificity. It also provides an in vitro diagnostic detection kit based on this antibody. This invention addresses the current market shortage of antibody raw materials and kits for detecting phosphorylated VASP-Ser 239 protein.

[0109] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0110] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A phosphorylated VASP-Ser 239 protein antibody or antigen-binding fragment, characterized in that, include: The light chain variable region CDR1 sequence shown in SEQ ID NO:3, the light chain variable region CDR2 sequence shown in SEQ ID NO:4, the light chain variable region CDR3 sequence shown in SEQ ID NO:5, the heavy chain variable region CDR1 sequence shown in SEQ ID NO:6, the heavy chain variable region CDR2 sequence shown in SEQ ID NO:7, and the heavy chain variable region CDR3 sequence shown in SEQ ID NO:

8.

2. The antibody or antigen-binding fragment according to claim 1, characterized in that, It includes the light chain variable region shown in SEQ ID NO:1 and the heavy chain variable region shown in SEQ ID NO:

2.

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

2.

4. A coupling agent, characterized in that, It comprises: a phosphorylated VASP-Ser 239 protein antibody or antigen-binding fragment as described in any one of claims 1 to 3; and a coupling portion, wherein the coupling portion is linked to the phosphorylated VASP-Ser 239 protein antibody or antigen-binding fragment; wherein the coupling portion is a fluorescent dye or a protein tag.

5. An isolated polynucleotide, characterized in that, The polynucleotide encodes a phosphorylated VASP-Ser 239 protein antibody or antigen-binding fragment as described in any one of claims 1 to 3; the polynucleotide comprises the nucleic acid sequence encoding the light chain variable region shown in SEQ ID NO:9 and the nucleic acid sequence encoding the heavy chain variable region shown in SEQ ID NO:

10.

6. An expression carrier, characterized in that, It comprises the isolated polynucleotide as described in claim 5.

7. A recombinant cell, characterized in that, It includes the expression vector as described in claim 6.

8. A method for preparing phosphorylated VASP-Ser 239 protein antibody or antigen fragment, characterized in that, This includes culturing the recombinant cells as described in claim 7.

9. The use of the antibody or antigen-binding fragment of any one of claims 1 to 3 or the conjugate of claim 4 in the preparation of a kit, characterized in that, The kit is used to detect phosphorylated VASP-Ser 239 protein in samples.

10. The use as described in claim 9, characterized in that, The kit is used to detect the phosphorylation level of VASP in human whole blood in vitro.

11. A reagent 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 3, the conjugate according to claim 4, the isolated polynucleotide according to claim 5, the expression vector according to claim 6, and / or the recombinant cell according to claim 7.

12. 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 3, the conjugate according to claim 4, the isolated polynucleotide according to claim 5, the expression vector according to claim 6, and / or the recombinant cell according to claim 7.

13. A method for preparing phosphorylated VASP-Ser 239 protein antibody or antigen-binding fragment according to any one of claims 1 to 3, characterized in that, This includes culturing the recombinant cells as described in claim 7.

14. A method for detecting phosphorylated VASP-Ser 239 protein for non-diagnostic purposes, characterized in that, include: The phosphorylated VASP-Ser 239 protein antibody or antigen-binding fragment according to any one of claims 1 to 3, or the conjugate according to claim 4, is contacted with the sample to be tested to form an immune complex; based on the signal of the immune complex, the phosphorylated VASP-Ser 239 protein in the sample to be tested is quantitatively and / or qualitatively analyzed; the signal of the immune complex is detected by at least one of ELISA, WB, and FACS; the signal includes a fluorescence signal.

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