Monoclonal antibody of von Willebrand factor cleavage protease (ADAMTS13) as well as preparation method and application of monoclonal antibody

Abstract

The invention relates to the field of monoclonal antibodies, in particular to a von Willebrand factor cleavage protease (ADAMTS13) monoclonal antibody as well as a preparation method and application thereof. The ADAMTS13 monoclonal antibody provided by the invention is high in sensitivity and strong in affinity and can effectively inhibit ADAMTS13 enzyme activity, in addition, the invention also develops an in-vitro diagnosis detection kit applying the antibody, and the kit has the advantages of wide detection range, few interfering substances, short detection time and good stability and repeatability. And a new thought is provided for treatment and research of diseases.

Description

Technical Field

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

[0002] ADAMTS13 is a metalloproteinase containing 1,427 amino acid residues that is mainly secreted by interstitial hepatic stellate cells and is a cleavage 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 varying sizes, with a molecular weight of up to 20,000 kDa. ADAMTS13 cleaves the UL-vWF A2 domain to produce smaller vWF multimers, and this proteolysis is essential for maintaining normal hemostasis and regulating inflammatory processes. The inability to cleave the ultra-large vWF results in the accumulation of highly adherent vWF on the surface of activated or damaged endothelium, leading to excessive microvascular thrombosis, end-organ damage, and death. Thrombotic thrombocytopenic purpura (TTP) is closely related to the 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 inhabitants. Clinical manifestations include thrombocytopenia, hemolytic anemia, nervous system abnormalities, renal insufficiency and fever. Von Willebrand disease (VWD) is a bleeding disease caused by a defect in the vWF gene. Some patients with type 2A VWD have changes in the amino acid conformation of the A2 region of the vWF factor, which makes the ADAMTS13 cleavage site easy to expose, increases the affinity for binding to ADAMT13, and enhances the ability to cleave vWF polymers into inactive vWF monomers.

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

[0004] In order to fill the gap in the prior art, the present invention provides a monoclonal antibody to ADAMTS13 protein with high sensitivity, strong affinity, and the ability to inhibit the activity of ADAMTS13 enzyme, providing a new idea for the treatment and research of diseases. The in vitro diagnostic test kit using the antibody has the advantages of wide detection range, few interfering substances, short detection time, good stability and repeatability. Specifically, the present invention provides the following technical solutions: First, in the first aspect, the present invention provides a monoclonal antibody Z08 against ADAMTS13 protein, wherein 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.

[0005] 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.

[0006] In a second aspect, the present invention provides a nucleic acid molecule encoding the monoclonal antibody Z08 against ADAMTS13 protein, which includes a nucleic acid sequence encoding a light chain variable region as shown in SEQ ID NO:9 and a nucleic acid sequence encoding a heavy chain variable region as shown in SEQ ID NO:10.

[0007] In a third aspect, the present invention provides an expression vector comprising the aforementioned nucleic acid molecule.

[0008] In a fourth aspect, the present invention provides a recombinant cell, characterized in that the cell comprises the aforementioned nucleic acid molecule or expression vector.

[0009] 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.

[0010] In one embodiment, the product is a test strip or a kit.

[0011] In a sixth aspect, the present invention provides a protease immunoassay kit for detecting ADAMTS13, wherein the kit comprises the monoclonal antibody Z08.

[0012] In a seventh aspect, the present invention provides 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 is capable of inhibiting ADAMTS13 enzyme activity.

[0013] In an eighth aspect, the present invention provides an ADAMTS13 inhibitor, wherein the inhibitor comprises the aforementioned monoclonal antibody Z08. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The accompanying drawings are used to provide a further understanding of the present invention and constitute 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 of the present invention. In the accompanying drawings: Figure 1 This is the SDS-PAGE of ADAMTS13 protein. DETAILED DESCRIPTION

[0015] The preferred embodiments of the present invention are described below in conjunction with 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.

[0016] In the following examples: The ADAMTS13 protein expression plasmid was constructed by General Biotech Co., Ltd.; Expi293 cells were purchased from ThermoFisher, A14528; and the cell culture medium used was Transpro CD 01 PLUS expression medium from Donin Biotech, M021-1L-02.

[0017] Example 1: Antigen preparation 1.1 ADAMTS13 protein expression and purification 1) One day before transfection, add Expi293 at 3.0×10 6 The cells were inoculated at a density of 1 / mL in a 1L shake flask containing 200ml of culture medium, and the ADAMTS13 protein expression plasmid was transfected and fed according to the instructions for use of the transient culture medium system of Duoning Biotechnology. When the cell viability reached 60%, the culture was stopped, the culture solution was centrifuged at 8000rpm for 20 minutes, and the supernatant was collected for purification.

[0018] 2) The supernatant sample was purified using Ni affinity filler (cytiva, 2025-02), the flow-through was collected, and the affinity column was washed with equilibration buffer (50 mM Tris, 150 mM Nacl, PH 7.5). Elution was performed with 10 mM, 30 mM, 100 mM, 250 mM, and 500 mM imidazole (prepared with 50 mM Tris, 150 mM Nacl, PH 8.0, after adding 10 mM, 30 mM, 100 mM, 250 mM, and 500 mM imidazole, the pH was adjusted to 7.5 with NaOH).

[0019] 1.2 Identification of ADAMTS13 protein 1) Use SDS-PAGE to identify the expression of the target protein. According to the loading order in Table 1, take 16 μL of each sample and add 4 μL of 5× loading buffer (Solabo, P1040), boil the sample at 100°C for 5 minutes, and centrifuge at 12,000 rpm for 1 minute.

[0020] Table 1 SDS-PAGE loading order of ADAMTS13 protein

[0021] 2) Use precast gel Bis-Tris (Invitrogen, #NP0342BOX) to mix 20 × MOPS electrophoresis buffer (Invitrogen TM , #NP0001) was diluted to 1×MOPS electrophoresis buffer, mixed and poured into the inner tank of the electrophoresis tank. The liquid level in the outer tank was lower than that in the inner tank. The voltage was 200 V for 50 minutes.

[0022] 3) After electrophoresis, take the gel and put it in an incubation box. Add 50 mL of deionized water and heat it in a microwave oven at high temperature for 3 minutes and discard it. Add 50 mL of staining solution (solarbio, #P1300) and stain it in a microwave oven at high temperature for 3 minutes. Continue staining on a horizontal shaker for 10 minutes and discard it. Add 50 ml of deionized water and decolorize it in a microwave oven at high temperature for 3 minutes. Repeat the operation until the bands are clearly visible. The band results are as follows: Figure 1 As shown: The target protein (ADAMTS13) bands appeared in the 100, 250, and 500 mM imidazole eluates. The three components were collected, mixed, and concentrated. The results of sampling and testing showed that the protein had the activity of enzymatic cleavage of VWF.

[0023] Embodiment 2: These modifications and variations of animal immunization and titer detection fall within the scope of the claims of the present invention and their equivalent technologies, and the present invention is also intended to include these modifications and variations.

[0024] 2.1 Immunization of mice BALB / c female mice aged 6-8 weeks were taken for the first immunization with 50 μg of the ADAMTS13 protein prepared in Example 1, which was mixed with Freund's adjuvant (F5881-10ML) at a ratio of 1:1 and fully emulsified, and then immunized at multiple points on the back of the mice and in the abdominal cavity, with an immunization dose of 200 μL per mouse. Immunization was performed once every two weeks, with 25 μg per immunization, which was mixed with Freund's incomplete adjuvant (F5506-10ML) at a ratio of 1:1 and fully emulsified, and then boosted with 200 μL per mouse.

[0025] 2.2 Potency detection 1) Coating antigen: The ADAMTS13 protein prepared in Example 1 was diluted to 1 μg / mL with 1×CB buffer (1L purified water, 33.92g Na2CO3, 57.13g NaHCO3, adjusted to pH 9.6, diluted 20 times), and coated in an ELISA plate at 100 μL / well at 4°C overnight.

[0026] 2) Blocking: Wash the coated ELISA plate 3 times with 1× PBST (1×PBS, 0.5% Tween-20) plate washer, 300 ul / well. Pat dry and add blocking solution (1×PBS, 1% BSA) and incubate at 37℃ for 2 hours, 200μL / well. Wash the plate 3 times with 1× PBST (1×PBS, 0.5% Tween-20) plate washer, pat dry and wait for detection.

[0027] 3) Primary antibody: Seven days after the fourth immunization, blood was collected from the mice’s orbits, and the supernatant was separated by centrifugation at 4000 g for 20 minutes. The serum was diluted to 100 times using PTB diluent (1×PBS, 0.5% Tween-20, 1% BSA). 150 μL of serum diluent was taken out and added to well 1# (the first well) of the antigen detection plate, and then gradient dilution was made in the vertical direction at 1:3 (after gradient dilution in each well, the final volume was 100 ul / well), for a total of 7 wells, with the 8th well left blank. The cells were incubated at 37°C for half an hour.

[0028] 4) Secondary antibody: Wash the plate 3 times with 1× PBST plate washer, 300 μL / well. After patting dry, add 5000-fold diluted goat anti-mouse IgG-HRP (Solebo, Goat anti-mouse IgG, HRP labeled, SE131-0.1ml), 100 μL / well, incubate at 37°C for half an hour.

[0029] 5) Color development: Wash the plate 3 times with a 1× PBST plate washer, 300 μL / well. Add 100 μL / well of color development solution (Solebol, TMB two-component color development solution, PR1210-2*500ml, A solution and B solution are mixed in a 1:1 ratio, ready for use), 37°C, color development for 10 minutes.

[0030] 6) Stopping solution: 1 M HCL, 50 μL / well.

[0031] 7) Data reading: Use a microplate reader (Radoo, RT-6100) to measure OD 450 , the results are shown in Table 2.

[0032] Table 2

[0033] As shown in Table 2, the mouse antibody titer exceeds 7.2W, and subsequent hybridoma cell fusion can be performed.

[0034] Example 3: Cell fusion and screening 3.1 Cell fusion 1) One week before fusion, mouse myeloma cells (FO cells) were revived. F0 cells were taken out of the liquid nitrogen tank and revived in a 37°C water bath for 1-2 minutes. Then, the cell solution in the cryopreserved tube was added to a 10 cm culture dish containing 10 mL DMEM complete medium (DMEM: Gibco, C11995500BT-500ml; penicillin-streptomycin-amphotericin B: Solebol, P7630-100ml; glutamine solution: Solebol, G022; 20% FBS: EXCELL, #140306) in a safety cabinet and subcultured for more than 3 generations in a CO2 incubator at 37°C. On the day of cell fusion, FO cells with good cell status were selected and centrifuged at 200 g for 5 minutes, the supernatant was discarded, 40 mL DMEM medium was added for resuspension, 10 μL of cell solution was taken to count the total number of cells, and the cells were placed in a 37°C incubator and used for fusion within 30 minutes.

[0035] 2) The mice were boosted with immunization 3 days in advance, with 25 μg of ADAMTS13 protein (prepared in Example 1) and an immunization dose of 200 μL.

[0036] 3) One day before cell fusion, two 16-week-old BALB / c mice (blank mice) were killed by pulling their necks, soaked in 75% alcohol for 3 minutes, and then peeled off the skin with ophthalmic scissors in a clean bench. Each mouse was injected with 5 mL of DMEM complete medium using a 5 mL syringe, and the mouse peritoneum was pierced and aspirated several times. Then, 100 mL of DMEM complete medium was added to the mixture and mixed. 100 μL / well was added to 96-well cell culture plates for a total of 10 plates. The plates were cultured in a CO2 incubator at 37°C and 5% CO2.

[0037] 4) B cell separation: On the day of fusion, ADAMTS13 BALB / c mice were unilaterally removed from the eyeball with ophthalmic forceps to bleed (about 1 mL), and then killed by pulling the neck, and soaked in 75% ethanol for 5 minutes. The spleen was removed with ophthalmic scissors and forceps on a sterile workbench (to avoid rupture) and excess tissue was removed. An appropriate amount of DMEM was added and ground into single cells using a cell screen (Jet, CSS013070) and a cell mesh grinding rod (Jet, CSP001001). The cells were added to a 15 ml centrifuge tube, centrifuged at 300g for 5 minutes, and the supernatant was discarded. The cells were resuspended and rinsed once with 10 mL DMEM culture medium, centrifuged at 300g for 5 minutes, and the supernatant was discarded. 10 mL culture medium was added to count the total number of cells.

[0038] 5) Cell fusion: Mix F0 cells and B cells at a ratio of 1:4 and centrifuge at 250 g for 5 minutes. Discard the supernatant and add 1 mL of PEG (sigma, #P7306) solution to the centrifuge tube at a constant speed within 1 minute. Let stand for 90 seconds, then slowly add 40 mL of DMEM medium to terminate the reaction. Centrifuge at 250 g for 5 minutes. Resuspend the fused cells in 20% FBS complete medium and plate them in a 96-well plate with feeder cells cultured the day before, 100 μL / well, 10 plates in total, in a CO2 incubator at 37°C, 5% CO2.

[0039] 6) On the second day of culture, add 20% FBS complete medium containing HAT to the cell plate, 50 μL / well, to make the final concentration 1×HAT. Incubate in a CO2 incubator at 37°C and 5% CO2. At this time, a large number of myeloma cells die.

[0040] 3.2 ELISA screening and labeling The antibody expression in the supernatant of the fusion plate cells was detected using the method in 2.2. The supernatant of the fusion plate was added as the primary antibody to the coated and blocked ELISA plate at 100 μL / well. The results are shown in Table 3.

[0041] Table 3: Fusion Plate ELISA OD 450 Test results:

[0042]

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051] Note: 1. The last two wells of the 96-well ELISA plate are negative and positive control wells.

[0052] Pick the OD in the experimental results 450 A total of 19 positive cell lines with values ​​greater than 0.3 were subsequently expanded and cultured.

[0053] 3.3 Cell expansion and cryopreservation The screened positive cell lines were re-plated in 96-well plates by limiting dilution method, the cell number and state were observed, and the concentration was adjusted to 0.5-1 cells / well, and cultured statically in a 37°C, 5% CO2 incubator. When clones were formed, the activity and subclones were detected in time, and the clones were gradually expanded from 96-well plates to 24-well plates and frozen in time. The hybridoma cell line with the highest titer, clone number Z08, was selected for antibody production.

[0054] Example 4: Large-scale preparation of monoclonal antibodies 8-week-old BALB / C mice were intraperitoneally injected with 0.5 mL of Freund's incomplete adjuvant. Hybridoma cells were plated at 1×10 6 The amount of cells / mouse was injected into the abdominal cavity. One to two weeks after inoculation, the abdomen of the mouse was swollen and the ascites could be extracted. Generally, 5 to 10 mL of ascites could be obtained from one mouse. The ascites was centrifuged at 3000 g for 10 minutes, the upper layer of fat and the cell sediment at the bottom were discarded, and the supernatant was collected and aliquoted at -20℃.

[0055] Example 5: Monoclonal antibody purification and activity detection 5.1 Purification of monoclonal antibody Z08 After the ascites supernatant was thawed and equilibrated to room temperature, the antibody was purified using a protein G affinity column (Huiyan Bio, HQ170827100M-100mL). The supernatant sample was slowly added to the purification column and the flow-through was collected. The impurities were washed with 10×CV volume of loading buffer (1×PBS, pH7.2-7.4) until no protein was detected. The antibody was eluted with elution buffer (0.1 M glycine, 0.5 M NaCl, pH2.7). After elution, the neutralization solution (1 M Tris, pH9.0) was quickly added to the tubes.

[0056] The collected antibodies were immediately dialyzed (dialysis bag specifications were 8K-14K) using loading buffer at a ratio of at least 1:100 at 4°C for 16 hours. After dialysis, samples were collected and the protein concentration of the eluate was determined to be 0.904 mg / mL using the NanoDrop A280 method.

[0057] 5.2 Activity detection of monoclonal antibody Z08 1) Coating antigen: Dilute the ADAMTS13 protein to 1 μg / mL with 1×CB buffer (1L purified water, add 33.92g Na2CO3, 57.13g NaHCO3, adjust the pH to 9.6, and dilute 20 times), and coat the plate at 100 μL / well at 4°C overnight.

[0058] 2) Blocking: Wash the coated ELISA plate 3 times with 1× PBST (1×PBS, 0.5% Tween-20) plate washer, 300 ul / well. Pat dry and add blocking solution (1×PBS, 1% BSA) and incubate at 37℃ for 2 hours, 200μL / well. Wash the plate 3 times with 1× PBST (1×PBS, 0.5% Tween-20) plate washer, pat dry and wait for detection.

[0059] 3) Primary antibody: Dilute the purified antibody to 1 μg / mL using PTB diluent (1×PBS, 0.5% Tween-20, 1% BSA), take 150 μL and add it to well 1# (the first well) of the antigen detection plate, then make a gradient dilution of 1:3 vertically (after the gradient dilution of each well, the final volume is 100ul / well), a total of 7 wells, leave the 8th well blank, and incubate at 37°C for half an hour.

[0060] 4) Secondary antibody: Wash the plate 3 times with 1× PBST plate washer, 300 μL / well. After patting dry, add 5000-fold diluted goat anti-mouse IgG-HRP (Solebo, Goat anti-mouse IgG, HRP labeled, SE131-0.1ml), 100 μL / well, incubate at 37°C for half an hour.

[0061] 5) Color development: Wash the plate 3 times with a 1× PBST plate washer, 300 μL / well. Add 100 μL / well of color development solution (Solebol, TMB two-component color development solution, PR1210-2*500ml, A solution and B solution are mixed in a 1:1 ratio, ready for use), 37°C, color development for 10 minutes.

[0062] 6) Stopping solution: 1 M HCL, 50 μL / well.

[0063] 7) Data reading: Use a microplate reader (Radoo, RT-6100) to measure OD 450 The results are shown in Table 4, indicating that the antibody has antigen (ie, ADAMTS13 protein) binding activity.

[0064] Table 4: Antibody OD after purification 450 value

[0065] Example 6: Monoclonal antibody sequencing of ADAMTS The hybridoma cell line with clone number Z08 was cultured to 5×10 6cells, and the cell precipitate was collected and sent to Nanjing Zhongding Biotechnology Co., Ltd. for sequencing. The light chain variable region amino acid sequence of the ADAMTS13 monoclonal antibody provided by the present invention is shown in SEQ ID NO: 1, the heavy chain variable region amino acid sequence is shown in SEQ ID NO: 2, the light chain CDR1 amino acid sequence is shown in SEQ ID NO: 3, the light chain CDR2 amino acid sequence is shown in SEQ ID NO: 4, the light chain CDR3 amino acid sequence is shown in SEQ ID NO: 5, the heavy chain CDR1 amino acid sequence is shown in SEQ ID NO: 6, the heavy chain CDR2 amino acid sequence is shown in SEQ ID NO: 7, the heavy chain CDR3 amino acid sequence is shown in SEQ ID NO: 8, the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO: 9, and the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO: 10.

[0066] Table 5: Monoclonal antibody Z08 sequence information

[0067]

[0068] Example 7: Preparation and application of ADAMTS13 ELISA kit The present invention adopts the purified ADAMTS13 monoclonal antibody Z08 as a capture antibody and a detection antibody to prepare an enzyme-immune detection kit that can be used to detect ADAMTS13 protein.

[0069] 7.1 Horseradish peroxidase (HRP) labeling of monoclonal antibody Z08 1) Take 3 mg of antibody Z08 and add it to the dialysis bag. Place it in 1 L of 10 mM carbonate buffer (pH 9.5) and dialyze it at room temperature for 2 hours. Prepare 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.

[0070] 2) Mix the above antibodies and HRP, place on a horizontal shaker, and react at room temperature in the dark for 4 hours. After the coupling reaction is completed, add 10uL of freshly prepared NaBH4 and terminate the reaction at 4℃ in the dark overnight. After the coupling reaction is terminated, stir and dialyze the antibody solution at room temperature for 2 hours. Finally, transfer the solution to a brown centrifuge tube and store at 4℃.

[0071] 7.2ADAMTS13 ELISA Kit Detection 1) ADAMTS13 monoclonal antibody Z08 was coated in a 96-well ELISA plate at a coating concentration of 2 µg / mL and a volume of 100 µL / well. After overnight coating at 4°C, the ELISA plate was washed twice with PBST solution, 300 µL / well each time. After washing, the plate was patted dry and blocked with PBS solution containing 1% BSA, 200 µL / well, and patted dry at room temperature for 2 hours.

[0072] 2) Add different concentrations of ADAMTS13 protein samples diluted with PTB to each well, 100 μL / well, and react at 37℃ for 1 hour. Wash the plate 3 times with PBST, 300 μL / well each time. Pat dry. Add HRP-labeled Z08 antibody to the ELISA plate and react in a 37℃ incubator for 1 hour. Wash the plate 3 times with PBST, 300 μL / well. Pat dry, add TMB substrate solution to the ELISA plate, 100 μL / well, and incubate at room temperature for 10 minutes, then add 100 μL / well of 1M H2SO4 to terminate the reaction. Use an ELISA reader to measure the absorbance value (A450) at 450 nm. The experimental results are shown in Table 6. 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.

[0073] Table 6: ADAMTS13 ELISA results

[0074] Example 8: Application of ADAMTS13 monoclonal antibody to inhibit ADAMTS13 activity The activity of ADAMTS13 was detected by detecting the residual substrate. The luminescence intensity was directly reflected by the concentration of the residual substrate and was inversely proportional to the activity of ADAMTS13 using a chemiluminescence instrument. Normal human serum was mixed with the diluted Z08 antibody in a 1:1 ratio, and the final dilution concentration gradient was 0, 12.5, 25, and 50 μg / mL. The experimental results are shown in Table 7: Table 7: Z08 inhibition enzyme activity test results

[0075] The results showed that monoclonal antibody Z08 could inhibit the enzyme activity of ADAMTS13, and the inhibition increased with the increase of antibody concentration.

[0076] 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 equivalents, 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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