A monoclonal antibody against von Willebrand factor-cleaving protease (ADAMTS13), a preparation method thereof, and applications thereof

Abstract

The present invention relates to the field of monoclonal antibodies. Specifically, the present invention relates to a monoclonal antibody against von Willebrand factor-cleaving protease (ADAMTS13), a preparation method thereof, and an application thereof. The ADAMTS13 monoclonal antibody provided by the present invention has high sensitivity and strong affinity, and can effectively inhibit the enzyme activity of ADAMTS13. In addition, the present invention also develops an in vitro diagnostic detection kit using this antibody. The kit has the advantages of a wide detection range, few interfering substances, a short detection time, good stability and repeatability, providing new ideas for the treatment and research of diseases.

Description

Technical Field

[0001] The present invention relates to the field of monoclonal antibodies. Specifically, the present invention relates to a monoclonal antibody against von Willebrand factor-cleaving protease (ADAMTS13), a preparation method thereof, and an application thereof. Background Art

[0002] ADAMTS13 is a metalloprotease containing 1,427 amino acid residues, which is mainly secreted by interstitial hepatic stellate cells and is a cleaving protease for the multimeric protein von Willebrand factor (vWF). vWF is a large multimeric glycoprotein released by endothelial cells in the form of ultra-large multimers (UL-vWF) of different sizes, with a molecular weight of up to 20,000 kDa. ADAMTS13 cleaves the A2 domain of UL-vWF to produce smaller vWF multimers, and this proteolysis is crucial for maintaining normal hemostasis and regulating the inflammatory process. The inability to cleave ultra-large vWF leads to the accumulation of highly adhesive vWF on the surface of activated or damaged endothelium, resulting in excessive microvascular thrombosis, end-organ damage, and death. Thrombotic thrombocytopenic purpura (TTP) is closely related to a severe deficiency of von Willebrand factor-cleaving protease (ADAMTS13) in plasma. TTP is a rare blood disease with an incidence of 4 to 13 cases per million residents, and its clinical manifestations include thrombocytopenia, hemolytic anemia, neurological abnormalities, renal insufficiency, and fever, etc. von Willebrand disease (VWD) is a bleeding disorder caused by a defect in the vWF gene. In some patients with type 2A VWD, there is a change in the amino acid conformation in the A2 region of the vWF factor, making the cleavage site of ADAMTS13 easily exposed, increasing the affinity for binding to ADAMT13, and enhancing the ability to shear vWF multimers into inactive vWF monomers.

[0003] Therefore, the development of monoclonal antibodies against ADAMTS13 is of great significance for the treatment and research of VWD: type 2A disease. In addition, evaluating the ADAMTS13 biomarker is not only crucial for establishing a preliminary diagnosis, but also can be applied to risk stratification and early detection of disease recurrence. Summary of the Invention

[0004] In order to fill the gaps in the prior art, the present invention provides a monoclonal antibody against ADAMTS13 protein with high sensitivity, strong affinity, and the ability to inhibit the enzyme activity of ADAMTS13, providing new ideas for the treatment and research of diseases. The in vitro diagnostic detection kit using this antibody has the advantages of a wide detection range, few interfering substances, a short detection time, good stability, and repeatability. Specifically, the present invention provides the following technical solutions:

[0005] First, in a first aspect, the present invention provides a monoclonal antibody Z08 against ADAMTS13 protein, and the antibody comprises a light chain CDR1 amino acid sequence as shown in SEQ ID NO:3, a light chain CDR2 amino acid sequence as shown in SEQ ID NO:4, a light chain CDR3 amino acid sequence as shown in SEQ ID NO:5, a heavy chain CDR1 amino acid sequence as shown in SEQ ID NO:6, a heavy chain CDR2 amino acid sequence as shown in SEQ ID NO:7, and a heavy chain CDR3 amino acid sequence as shown in SEQ ID NO:8.

[0006] In one embodiment, the antibody comprises a light chain variable region amino acid sequence as shown in SEQ ID NO:1 and a heavy chain variable region amino acid sequence as shown in SEQ ID NO:2.

[0007] In a second aspect, the present invention provides a nucleic acid molecule encoding the monoclonal antibody Z08 against ADAMTS13 protein, which comprises a nucleic acid sequence encoding the light chain variable region as shown in SEQ ID NO:9 and a nucleic acid sequence encoding the heavy chain variable region as shown in SEQ ID NO:10.

[0008] In a third aspect, the present invention provides an expression vector containing the aforementioned nucleic acid molecule.

[0009] In a fourth aspect, the present invention provides a recombinant cell, characterized in that the cell contains the aforementioned nucleic acid molecule or expression vector.

[0010] In a fifth aspect, the present invention provides the use of the aforementioned monoclonal antibody and / or nucleic acid molecule in the preparation of a product for detecting ADAMTS13 protein.

[0011] In one embodiment, the product is a test strip or a kit.

[0012] In a sixth aspect, the present invention provides an enzyme immunoassay kit for detecting ADAMTS13 protease, and the kit contains the monoclonal antibody Z08.

[0013] In a seventh aspect, the present invention provides the use of the aforementioned monoclonal antibody, nucleic acid molecule, expression vector and / or recombinant cell in the preparation of an ADAMTS13 inhibitor, wherein the inhibitor can inhibit the enzyme activity of ADAMTS13.

[0014] In an eighth aspect, the present invention provides an ADAMTS13 inhibitor, and the inhibitor contains the aforementioned monoclonal antibody Z08. Description of the Drawings

[0015] The accompanying drawings are used to provide a further understanding of the present invention and form a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation to the present invention. In the accompanying drawings:

[0016] Figure 1 It is the SDS-PAGE of ADAMTS13 protein. Specific embodiments

[0017] The following describes the preferred embodiments of the present invention with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.

[0018] In the following embodiments:

[0019] The ADAMTS13 protein expression plasmid was constructed by General Biosystems Co., Ltd.; Expi293 cells were purchased from ThermoFisher, A14528; the cell culture medium used was Duoning Biologics Transpro CD 01 PLUS expression medium, M021-1L-02.

[0020] Example 1: Antigen preparation

[0021] 1.1 Expression and purification of ADAMTS13 protein

[0022] 1) One day before transfection, inoculate Expi293 at a density of 3.0×10 6 cells / mL into a 1L shake flask containing 200ml of medium, and transfect and feed the ADAMTS13 protein expression plasmid according to the usage guide of the Duoning Biologics transient transfection medium system. When the cell viability reaches 60%, stop culturing, centrifuge the culture broth at 8000 rpm for 20 minutes, and collect the supernatant for purification.

[0023] 2) Purify the supernatant sample using Ni affinity packing (cytiva, 2025-02), collect the flow-through, and wash the affinity column with the equilibration buffer (50 mM Tris, 150 mM Nacl, PH 7.5). Elute with 10 mM, 30 mM, 100 mM, 250 mM, 500 mM imidazole (prepared with 50 mM Tris, 150 mM Nacl, PH 8.0, and after adding 10 mM, 30 mM, 100 mM, 250 mM, 500 mM imidazole, adjust the PH to 7.5 with NaOH).

[0024] 1.2 Identification of ADAMTS13 protein

[0025] 1) Identify the expression of the target protein using SDS-PAGE. According to the sample loading order in Table 1, respectively take 16 μL of each of the above samples and add 4 μL of 5× loading buffer (Solarbio, P1040), boil the samples at 100 °C for 5 minutes, and centrifuge at 12,000 rpm for 1 minute.

[0026] Table 1 SDS-PAGE sample loading order of ADAMTS13 protein

[0027]

[0028] 2) Dilute 20× MOPS electrophoresis buffer (Invitrogen, #NP0001) to 1× MOPS electrophoresis buffer using precast Bis-Tris gel (Invitrogen, #NP0342BOX). After mixing, pour it into the inner tank of the electrophoresis tank. The liquid level in the outer tank is lower than that in the inner tank. Run electrophoresis at 200 V for 50 minutes. TM After completion of electrophoresis, take out the gel and place it in an incubation box. Add 50 mL of deionized water, heat it in a microwave oven at high power for 3 minutes and discard. Add 50 mL of staining solution (Solarbio, #P1300), stain it in a microwave oven at high power for 3 minutes, and continue staining on a horizontal shaker for 10 minutes, then discard. Add 50 mL of deionized water, decolorize it in a microwave oven at high power for 3 minutes, and repeat the operation until the bands are clearly visible. The band results are as

[0029] shown: Bands of the target protein (ADAMTS13) appear in the 100, 250, and 500 mM imidazole elution solutions. Collect the three-component liquid, mix and concentrate it, and take a sample for testing. The test results show that the protein has the activity of cleaving VWF. Figure 1 If these modifications and variations of Example 2: Animal Immunization and Titer Detection fall within the scope of the claims of the present invention and their equivalent technologies, then the present invention also intends to include these modifications and variations.

[0030] 2.1 Mouse Immunization

[0031] Use 6 - 8-week-old female BALB / c mice. For the primary immunization, mix 50 μg of the ADAMTS13 protein prepared in Example 1 with Freund's adjuvant (F5881 - 10ML) at a ratio of 1:1 and fully emulsify it. Immunize at multiple points on the back and intraperitoneally of the mice, with an immunization dose of 200 μL per mouse. Immunize once every two weeks thereafter, each time with 25 μg, mix it with Freund's incomplete adjuvant (F5506 - 10ML) at a ratio of 1:1 and fully emulsify it for booster immunization, with an immunization dose of 200 μL per mouse.

[0032] 2.2 Titer Detection

[0033]

[0034] 1) Coated antigen: Dilute the ADAMTS13 protein prepared in Example 1 to 1 μg / mL with 1×CB buffer (33.92 g of Na2CO3 and 57.13 g of NaHCO3 are added to 1 L of purified water, adjust the pH to 9.6, and dilute 20 times), coat it in an ELISA microplate, 100 μL / well, overnight at 4°C.

[0035] 2) Blocking: Wash the coated microplate 3 times with a 1× PBST (1×PBS, 0.5% Tween-20) washer, 300 ul / well. After patting dry, add the blocking solution (1×PBS, 1% BSA) and incubate at 37°C for 2 hours, 200 μL / well. Wash the microplate 3 times with a 1× PBST (1×PBS, 0.5% Tween-20) washer, and wait for measurement after patting dry.

[0036] 3) Primary antibody: Seven days after the fourth immunization, take blood from the mouse's eye socket, centrifuge at 4000 g for 20 minutes to separate the supernatant, and then dilute it 100 times with PTB diluent (1×PBS, 0.5% Tween-20, 1% BSA). Take out 150 μL of the serum diluent and add it to well 1# (the first well) of the antigen detection strip, and then make a serial dilution in a 1:3 ratio longitudinally (after gradient dilution in each well, the final volume is 100 ul / well), a total of 7 wells, the 8th well is blank, and incubate at 37°C for half an hour.

[0037] 4) Secondary antibody: Wash the microplate 3 times with a 1× PBST washer, 300 μL / well. After patting dry, add goat anti-mouse IgG-HRP diluted 5000 times (Solarbio, Goat anti-mouse IgG, HRP labeled, SE131-0.1ml), 100 μL / well, and incubate at 37°C for half an hour.

[0038] 5) Color development: Wash the microplate 3 times with a 1× PBST washer, 300 μL / well. Add 100 μL / well of the color developing solution (Solarbio, TMB two-component color developing solution, PR1210-2*500ml, mix solution A and solution B in a 1:1 ratio, prepare and use immediately), and develop color at 37°C for 10 minutes.

[0039] 6) Termination: Termination solution 1 M HCL, 50 μL / well.

[0040] 7) Data reading: Use an enzyme-linked immunosorbent assay analyzer (Rayto, RT-6100) to measure OD 450 , and the results are shown in Table 2.

[0041] Table 2

[0042]

[0043] As shown in Table 2, the antibody titer of the mouse exceeded 72,000, and subsequent hybridoma cell fusion could be carried out.

[0044] Example 3: Cell fusion and screening

[0045] 3.1 Cell fusion

[0046] 1) Resuscitate mouse myeloma cells (FO cells) one week before fusion. Take out the F0 cells from the liquid nitrogen tank, resuscitate them within 1 - 2 minutes in a 37°C water bath, and then add the cell suspension in the cryopreservation tube to a 10 cm culture dish containing 10 mL of complete DMEM medium (DMEM: Gibco, C11995500BT - 500ml; added with penicillin - streptomycin - amphotericin B: Solarbio, P7630 - 100ml; glutamine solution: Solarbio, G022; 20% FBS: EXCELL, #140306) in a biosafety cabinet. Transfer the cells in the CO2 incubator at 37°C for more than 3 passages in a timely manner. On the day of cell fusion, select FO cells with good cell state, centrifuge them at 200 g for 5 minutes, discard the supernatant, resuspend them in 40 mL of DMEM medium, take 10 μL of the cell suspension to count the total number of cells, and place them in a 37°C incubator for use in fusion within 30 minutes.

[0047] 2) Strengthen the immunization of the mouse 3 days in advance with 25 μg of ADAMTS13 protein (prepared in Example 1) and an immunization dose of 200 μL.

[0048] 3) One day before cell fusion, decapitate and sacrifice two 16 - week - old BALB / c mice (blank mice), soak them in 75% alcohol for 3 minutes, and then peel off the outer skin with ophthalmic scissors and forceps in a laminar flow hood. Use a 5 mL syringe to aspirate 5 mL of complete DMEM medium into each mouse, insert it into the peritoneal cavity of the mouse and aspirate repeatedly several times, then add it to a total of 100 mL of complete DMEM medium and mix well. Add 100 μL / well to a 96 - well cell culture plate, for a total of 10 plates. Culture in a CO2 incubator at 37°C with 5% CO2.

[0049] 4) B cell separation: On the day of fusion, use ophthalmic forceps to unilaterally remove the eyeball of the ADAMTS13 - immunized BALB / c mouse to bleed (about 1 mL), decapitate and sacrifice it, and soak it in 75% ethanol for 5 minutes. Take out the spleen (avoid rupture) and remove excess tissue with ophthalmic scissors and forceps in a sterile workbench. Add an appropriate amount of DMEM and grind it to single cells with a cell sieve (Jet Biofil, CSS013070) and a cell grinding rod (Jet Biofil, CSP001001), add it to a 15 ml centrifuge tube, centrifuge at 300 g for 5 minutes, discard the supernatant, resuspend and wash it once with 10 mL of DMEM medium, centrifuge at 300 g for 5 minutes, and discard the supernatant. Add 10 mL of medium to count the total number of cells.

[0050] 5) Cell fusion: Mix F0 cells and B cells at a ratio of 1:4, and centrifuge at 250 g for 5 minutes after mixing. Discard the supernatant, and slowly add 1 mL of PEG (sigma, #P7306) solution to the centrifuge tube within 1 minute at a uniform speed. Let it stand for 90 seconds, and then slowly add 40 mL of DMEM medium to terminate the reaction. Centrifuge at 250 g for 5 minutes. Resuspend the fused cells in complete medium containing 20% FBS, and plate them in a 96-well plate pre-cultured with feeder cells the previous day, 100 μL / well, for a total of 10 plates. Incubate in a CO2 incubator at 37°C with 5% CO2.

[0051] 6) On the second day of culture, add complete medium containing 20% FBS with HAT to the cell plate, 50 μL / well, to make the final concentration 1×HAT. Incubate in a CO2 incubator at 37°C with 5% CO2. At this time, a large number of myeloma cells die.

[0052] 3.2 ELISA screening and labeling

[0053] Use the method in 2.2 to detect the antibody expression in the supernatant of the fusion plate cells. Add the supernatant of the fusion plate as the primary antibody to the coated and blocked ELISA plate, 100 μL / well. The results are shown in Table 3.

[0054] Table 3: ELISA OD of the fusion plate 450 Test results:

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065] Note: The last two wells of the 96-well ELISA plate are negative and positive control wells.

[0066] Pick out the OD in the experimental results 450A total of 19 positive cell lines with a value greater than 0.3 were subjected to subsequent scale-up culture.

[0067] 3.3 Cell scale-up culture and cryopreservation

[0068] The screened positive cell lines were re-plated in 96-well plates by the limited dilution method. Observe the cell number and status, and adjust to 0.5 - 1 cell / well. Incubate statically in a 37 °C, 5% CO2 incubator. When clone clusters form, detect the activity and subclones in a timely manner, gradually scale up from 96-well plates to 24-well plates and cryopreserve in a timely manner. Select the hybridoma cell line with the highest titer, clone number Z08, for antibody production.

[0069] Example 4: Large-scale preparation of monoclonal antibodies

[0070] Inject 0.5 mL of Freund's incomplete adjuvant intraperitoneally into 8-week-old BALB / C mice. After 1 - 2 weeks, inject the hybridoma cells into the abdominal cavity at a dose of 1×10 6 cells / mouse. One to two weeks after inoculation, the abdomen of the mice can be seen to be enlarged. At this time, ascites can be extracted. Generally, 5 - 10 mL of ascites can be obtained from one mouse. Centrifuge the obtained ascites at 3000 g for 10 minutes, discard the upper layer of grease and the bottom cell precipitate, and collect the supernatant and aliquot it at -20 °C.

[0071] Example 5: Purification and activity detection of monoclonal antibodies

[0072] 5.1 Purification of monoclonal antibody Z08

[0073] After thawing and equilibrating the ascites supernatant to room temperature, purify the antibody using a protein G affinity column (Huiyan Bio, HQ170827100M - 100 mL). Slowly add the supernatant sample to the purification column and collect the flow-through. Wash the miscellaneous proteins with 10×CV volume of loading buffer (1×PBS, pH 7.2 - 7.4) until no protein is detected. Elute the antibody using elution buffer (0.1 M glycine, 0.5 M NaCl, pH 2.7). After elution, quickly add the neutralization solution (1 M Tris, pH 9.0) in aliquots.

[0074] The collected antibody was immediately dialyzed (the dialysis bag size is 8 K - 14 K), and dialyzed at 4 °C for 16 hours using the loading buffer at a ratio of at least 1:100. After dialysis, collect the sample and detect the protein concentration of the eluate to be 0.904 mg / mL using the NanoDrop A280 method.

[0075] 5.2 Activity detection of monoclonal antibody Z08

[0076] 1) Coated antigen: Dilute the ADAMTS13 protein with 1×CB buffer (add 33.92 g of Na2CO3 and 57.13 g of NaHCO3 to 1 L of purified water, adjust the pH to 9.6, and dilute 20 times) to 1 μg / mL, coat it in an ELISA microplate, 100 μL / well, overnight at 4°C.

[0077] 2) Blocking: Wash the coated microplate 3 times with a 1×PBST (1×PBS, 0.5% Tween-20) washer, 300 ul / well. After patting dry, add the blocking solution (1×PBS, 1% BSA) and incubate at 37°C for 2 hours, 200 μL / well. Wash the microplate 3 times with a 1×PBST (1×PBS, 0.5% Tween-20) washer, and pat dry for further measurement.

[0078] 3) Primary antibody: Dilute the purified antibody with PTB diluent (1×PBS, 0.5% Tween-20, 1% BSA) to 1 μg / mL, take 150 μL and add it to well 1# (the first well) of the antigen detection strip, then make a 1:3 serial dilution longitudinally (after gradient dilution in each well, the final volume is 100 ul / well), a total of 7 wells, well 8 is blank, incubate at 37°C for half an hour.

[0079] 4) Secondary antibody: Wash the microplate 3 times with a 1×PBST washer, 300 μL / well. After patting dry, add goat anti-mouse IgG-HRP diluted 5000 times (Solarbio, Goat anti-mouse IgG, HRP labeled, SE131-0.1ml), 100 μL / well, incubate at 37°C for half an hour.

[0080] 5) Color development: Wash the microplate 3 times with a 1×PBST washer, 300 μL / well. Add 100 μL of color development solution per well (Solarbio, TMB two-component color development solution, PR1210-2*500ml, mix solution A and solution B in a 1:1 ratio, prepare and use immediately), incubate at 37°C for 10 minutes.

[0081] 6) Termination: Termination solution 1 M HCL, 50 μL / well.

[0082] 7) Data reading: Use an enzyme-linked immunosorbent assay analyzer (Rayto, RT-6100) to measure OD 450 , and the results are shown in Table 4, indicating that the antibody has antigen (i.e., ADAMTS13 protein) binding activity.

[0083] Table 4: OD 450 value

[0084]

[0085] Example 6: Sequencing of Monoclonal Antibody Against ADAMTS

[0086] Culture the hybridoma cell line with clone number Z08 to 5×10 6 cells, collect the cell precipitate and send it to Nanjing Zhongding Biotechnology Co., Ltd. for sequencing. The amino acid sequence of the light chain variable region of the ADAMTS13 monoclonal antibody provided by the present invention is as shown in SEQ ID NO:1, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:2, the amino acid sequence of the light chain CDR1 is as shown in SEQ ID NO:3, the amino acid sequence of the light chain CDR2 is as shown in SEQ ID NO:4, the amino acid sequence of the light chain CDR3 is as shown in SEQ ID NO:5, the amino acid sequence of the heavy chain CDR1 is as shown in SEQ ID NO:6, the amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID NO:7, the amino acid sequence of the heavy chain CDR3 is as shown in SEQ ID NO:8, the nucleotide sequence encoding the light chain variable region is as shown in SEQ ID NO:9, and the nucleotide sequence encoding the heavy chain variable region is as shown in SEQ ID NO:10.

[0087] Table 5: Sequence information of monoclonal antibody Z08

[0088]

[0089]

[0090] Example 7: Preparation and application of an enzyme immunoassay kit for ADAMTS13

[0091] The present invention uses the purified ADAMTS13 monoclonal antibody Z08 as the capture antibody and the detection antibody to prepare an enzyme immunoassay kit for detecting ADAMTS13 protein.

[0092] 7.1 Horseradish peroxidase (HRP) labeling of monoclonal antibody Z08

[0093] 1) Take 3 mg of antibody Z08 and add it to a dialysis bag, then place it in 1 L of 10 mM carbonate buffer (pH 9.5) and stir and dialyze at room temperature for 2 hours. Prepare a 5 mg / mL HRP solution, add 40 µL of 0.1 M NaIO4 to the above HRP solution, place it on a horizontal shaker, and react (activate) at room temperature in the dark for 20 minutes. Dialyze the activated HRP solution in 2 L of 1 mM sodium acetate buffer (pH 4.4) at 4°C overnight. Add 1 / 10 volume of 0.2 M carbonate buffer to raise the pH of the activated HRP solution to 9.0 - 9.5.

[0094] 2) Mix the above antibody and HRP, 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 uL of freshly prepared NaBH4, and terminate the reaction overnight in the dark at 4°C. Dialyze the antibody solution after terminating the conjugation reaction with stirring at room temperature for 2 hours. Finally, transfer the solution to a brown centrifuge tube and store it at 4°C.

[0095] 7.2 Detection with the ADAMTS13 ELISA Kit

[0096] 1) Coat the 96-well ELISA plate with the ADAMTS13 monoclonal antibody Z08 at a coating concentration of 2 µg / mL and a volume of 100 µL / well. After coating overnight at 4°C, wash the ELISA plate twice with PBST solution, 300 µL / well each time. After washing, pat dry. Block the ELISA plate with PBS solution containing 1% BSA, 200 µL / well, and pat dry at room temperature for 2 hours.

[0097] 2) Add different concentrations of diluted ADAMTS13 protein samples in PTB to each well, 100 μL / well, and let it stand and react at 37°C for 1 hour. Wash the plate 3 times with PBST, 300 µL / well each time. Pat dry. Add the HRP-labeled Z08 antibody to the ELISA plate and react in an incubator at 37°C for 1 hour. Wash the plate 3 times with PBST, 300 µL / well. Pat dry. Add the TMB substrate solution to the ELISA plate, 100 µL / well, and react at room temperature for 10 minutes, then add 100 µL / well of 1M H2SO4 to terminate the reaction. Measure the absorbance (A450) at 450 nm using an ELISA reader. The experimental results are shown in Table 6. The antibody Z08 can specifically bind to ADAMTS13. The standard curve range of this method is 0 - 60 ng / mL, and the minimum detection limit is 0.5 ng / mL. The results are shown in Table 6.

[0098] Table 6: ELISA Detection Results of ADAMTS13

[0099]

[0100] Example 8: Application of the ADAMTS13 Monoclonal Antibody in Inhibiting the Activity of ADAMTS13

[0101] Detect the activity of ADAMTS13 by detecting the residual substrate. Use a chemiluminescence analyzer to measure, and its luminescence intensity directly reflects the concentration of the residual substrate, which is inversely proportional to the activity of ADAMTS13. Mix normal human serum and the diluted Z08 antibody at a ratio of 1:1, and the final dilution concentration gradient is 0, 12.5, 25, 50 μg / mL. The experimental results are shown in Table 7:

[0102] Table 7: Detection Results of Z08 Inhibiting Enzyme Activity

[0103]

[0104] The results showed that the monoclonal antibody Z08 could inhibit the activity of ADAMTS13 enzyme, and the inhibition was enhanced with the increase of antibody concentration.

[0105] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A monoclonal antibody Z08 against ADAMTS13 protein, characterized in that: The antibody includes a light chain CDR1 amino acid sequence as shown in SEQ ID NO:3, a light chain CDR2 amino acid sequence as shown in SEQ ID NO:4, a light chain CDR3 amino acid sequence as shown in SEQ ID NO:5, a heavy chain CDR1 amino acid sequence as shown in SEQ ID NO:6, a heavy chain CDR2 amino acid sequence as shown in SEQ ID NO:7 and a heavy chain CDR3 amino acid sequence as shown in SEQ ID NO:

8.

2. The monoclonal antibody Z08 according to claim 1, characterized in that The antibody comprises a light chain variable region amino acid sequence as shown in SEQ ID NO:1 and a heavy chain variable region amino acid sequence as shown in SEQ ID NO:

2.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the monoclonal antibody Z08 against ADAMTS13 protein, which includes the nucleic acid sequence encoding the light chain variable region as shown in SEQ ID NO:9 and the nucleic acid sequence encoding the heavy chain variable region as shown in SEQ ID NO:

10.

4. An expression vector comprising the nucleic acid molecule according to claim 3.

5. A recombinant cell, characterized in that The cell comprises the nucleic acid molecule of claim 3 or the expression vector of claim 4.

6. Use of the monoclonal antibody according to any one of claims 1-2 and / or the nucleic acid molecule according to claim 3 in the preparation of a product for detecting ADAMTS13 protein.

7. The use according to claim 6, characterized in that The product is a test strip or a test kit.

8. A protease immune detection kit for detecting ADAMTS13, characterized in that: The kit comprises the monoclonal antibody according to claim 1 or 2.

9. Use of the monoclonal antibody according to any one of claims 1 to 2, the nucleic acid molecule according to claim 3, the expression vector according to claim 4 and / or the recombinant cell according to claim 5 in the preparation of an ADAMTS13 inhibitor, characterized in that: The inhibitor is capable of inhibiting ADAMTS13 enzyme activity.

10. An ADAMTS13 inhibitor, characterized in that The inhibitor comprises the monoclonal antibody according to any one of claims 1-2.

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