Application of CTHRC1 protein as a marker for aortic aneurysm and / or aortic dissection

Abstract

This invention discloses the application of CTHRC1 protein as a marker for aortic aneurysm and / or aortic dissection, belonging to the field of gene diagnosis and treatment technology in molecular biology. It aims to address the high mortality risk and poor long-term efficacy and prognosis associated with surgical treatment of aortic aneurysm and / or aortic dissection. This invention uses recombinant human CTHRC1 protein as an immunogen to obtain a murine anti-CTHRC1 monoclonal antibody that specifically binds to the human CTHRC1 antigen. This monoclonal antibody can neutralize and effectively block the specific biological activity of the CTHRC1 immunogen through high binding affinity, and can be applied as a novel therapeutic target for aortic aneurysm and / or aortic dissection, reducing disease mortality and improving patient prognosis.

Description

Technical Field

[0001] This invention belongs to the field of gene diagnosis and treatment technology in molecular biology, and particularly relates to the application of CTHRC1 protein as a marker for aortic aneurysm and / or aortic dissection. Background Technology

[0002] Aortic aneurysm and / or aortic dissection are serious cardiovascular diseases that severely endanger patients' lives. They have a rapid onset and are extremely dangerous, potentially causing aortic rupture and death within a short period. Due to the high risk of death associated with surgical treatment for aortic lesions, the long-term efficacy and prognosis for patients are poor. Although some medications are now effective in treating aortic dissection, such as antibiotics like doxycycline and roxithromycin, as well as statins, beta-blockers, and angiotensin-converting enzyme inhibitors, these drugs are not widely used due to significant side effects or limited clinical efficacy.

[0003] Aortic aneurysm is the result of pathological dilation of the aorta, which can exceed 50% of the normal aortic diameter. Aortic dissection occurs when the aortic intima tears, allowing blood to flow through the tear into the media under arterial pressure, forming a dissecting hematoma that extends and peels away along the aortic wall, creating two lumens, one true and one false. Untreated aortic dissection is a fatal emergency, with an initial mortality rate of approximately 40%, and this rate increases by 1% per hour, potentially reaching as high as 90% annually. Epidemiological studies indicate that the incidence of aortic dissection is approximately 3-5 per 100,000 cases per year, and is showing an increasing trend. Furthermore, there are currently no effective and safe drug treatments for aortic aneurysms and / or aortic dissections; the main approaches focus on surgical treatment and endovascular interventional repair. It is worth noting that due to the high risk of death associated with surgical treatment of aortic aneurysms and / or aortic dissections, the long-term efficacy and prognosis for patients are poor.

[0004] Recent studies have found that adventitia fibroblasts in the aortic wall can be activated into myofibroblasts under pathological conditions such as mechanical stress or injury. Simultaneously, adventitia fibroblasts can participate in pathological remodeling through autocrine pathways or complex paracrine interactions with vascular smooth muscle cells, providing a theoretical basis for novel diagnostic and therapeutic strategies targeting fibroblast populations for aortic aneurysms and / or aortic dissections.

[0005] Collagen Triple Helix Repeat Containing 1 (CTHRC1) was first identified by Pyagay et al. from differential gene expression analysis in a rat arterial injury model, where it was highly expressed. Subsequently, Tang et al. examined CTHRC1 expression in 19 solid tumors, finding increased expression in 16 of them. Currently, most research focuses on the role of CTHRC1 in the development and progression of various tumors, but studies on its application in aortic aneurysms and / or aortic dissections are scarce, and its biological functions and specific mechanisms of action involving adventitia fibroblast populations remain unclear. Summary of the Invention

[0006] To address the aforementioned issues, this application provides an application of CTHRC1 protein as a marker for aortic aneurysm and / or aortic dissection, including a diagnostic kit for accurately diagnosing selected aortic aneurysms and / or aortic dissection, the use of a diagnostic kit for the CTHRC1 gene, and a monoclonal antibody targeting the biological function of CTHRC1. The application of the aforementioned monoclonal antibody is also provided. This monoclonal antibody can bind with high affinity and neutralize to block the specific biological functional activity of the CTHRC1 immunogen, and can be applied to a new diagnostic and therapeutic target for aortic aneurysm and / or aortic dissection diseases.

[0007] In a first aspect, the present invention provides the application of CTHRC1 protein in the preparation of diagnostic reagents for aortic aneurysm and / or aortic dissection.

[0008] The amino acid sequence of the CTHRC1 protein is as follows:

[0009] SEIPKGKQKAQLRQREVVDLYNGMCLQGPAGVPGRDGSPGANGIPGTPGIPGRDGFKG

[0010] EKGECLRESFEESWTPNYKQCSWSSLNYGIDLGKIAECTFTKMRSNSALRVLFSGSLRLKCR

[0011] NACCQRWYFTFNGAECSGPLPIEAIIYLDQGSPEMNSTINIHRTSSVEGLCEGIGAGLVDVAIW

[0012] VGTCSDYPKGDASTGWNSVSRIIIEELPK;

[0013] In a preferred embodiment of this, the diagnostic reagent for aortic aneurysm and / or aortic dissection is a CTHRC1 protein monoclonal antibody.

[0014] CTHRC1 protein monoclonal antibody, as follows:

[0015] Light chain variable region amino acid sequence

[0016] QLILTQSSSASFSLGASAKLTCTLSSQHSTYTIEWYQQQPLKPPKYVMEVKKDGHSTGDGIP DRFSGSSSGADRYLSISNIQPEDEAIYICGVGDTIKEQFVYVFGGGTRVTVL;

[0017] Heavy chain variable region amino acid sequence

[0018] EVQLVETGGGLVQPKGSMKLSCAASGFTFNINAMNWVRQAPGKGLEWVARIRSKSNNYAT YYADSVKDRFTISRDDSQRMLYLQMNNLKTEDTAKYYCVSNWDWYFDVWGAGTTVTVSS;

[0019] Secondly, the present invention provides the application of CTHRC1 protein in the preparation of diagnostic kits for aortic aneurysm and / or aortic dissection.

[0020] An enzyme-linked immunosorbent assay (ELISA) kit for detecting the CTHRC1 protein content in a sample is provided in this regard, comprising a CTHRC1 protein monoclonal antibody.

[0021] Thirdly, the present invention provides the application of CTHRC1 protein as a drug target in screening drugs for the treatment of aneurysms and / or aortic dissection, and provides a monoclonal antibody that targets the biological function of anti-CTHRC1 against the adventitia fibroblast population, which can bind with high affinity and neutralize and block the specific biological functional activity of CTHRC1 immunogen.

[0022] In a preferred embodiment of this aspect, the drug comprises a monoclonal antibody against CTHRC1, which can bind with high affinity and neutralize and block the specific biological functional activity of the CTHRC1 immunogen, and can be applied to a new targeted therapeutic target for aortic aneurysm and / or aortic dissection.

[0023] Fourthly, the present invention provides the use of an inhibitor of CTHRC1 protein in the preparation of a medicament for treating aortic aneurysm and / or aortic dissection, wherein the inhibitor of CTHRC1 protein is a monoclonal antibody against CTHRC1 protein.

[0024] In a preferred embodiment of this aspect, the medicament comprises a pharmaceutically acceptable carrier and an effective amount of an active ingredient, wherein the active ingredient is an inhibitor of the CTHRC1 protein.

[0025] Fifthly, the present invention provides a medicament for treating aortic aneurysm and / or aortic dissection, comprising a pharmaceutically acceptable carrier and an effective amount of the following active ingredient, wherein the active ingredient is a monoclonal antibody against CTHRC1 protein.

[0026] Compared with the prior art, the present invention has the following advantages:

[0027] This invention uses recombinant human CTHRC1 protein as an immunogen to obtain a murine anti-CTHRC1 monoclonal antibody that specifically binds to the human CTHRC1 antigen. This monoclonal antibody can neutralize and effectively block the specific biological activity of the CTHRC1 immunogen through high binding affinity. It can be applied to a new therapeutic target for aortic aneurysm and / or aortic dissection, reduce disease mortality, and improve patient prognosis.

[0028] In this invention, vascular smooth muscle cells that interact with adventitia fibroblasts exhibit CTHRC1 protein stimulation in vitro, demonstrating biological activities including direct inhibition of vascular smooth muscle cell phenotypic transformation, such as suppressing the ERK signaling pathway. By sequencing the amino acid sequence of the antibody with the most ideal biological activity and humanizing it, a therapeutic anti-CTHRC1 monoclonal antibody is prepared. Using an anti-CTHRC1 monoclonal antibody targeting the adventitia fibroblast population offers advantages such as high efficiency, minimal invasiveness, and high safety, effectively reducing the risk of disease-related death from aortic aneurysm and / or aortic dissection and improving patient prognosis. Attached Figure Description

[0029] Figure 1 This is a structural diagram of the CTHRC1 protein.

[0030] Figure 2 The image shows the amino acid sequence of the variable region of a CTHRC1 protein monoclonal antibody; (a) light chain; (b) heavy chain.

[0031] Figure 3 The results are from SDS-PAGE analysis of CTHRC1 protein.

[0032] Figure 4 The CTHRC1 content in the serum of patients with aortic dissection.

[0033] Figure 5 To detect the expression level of CTHRC1 in vascular tissues of patients with human aortic dissection using immunohistochemistry.

[0034] Figure 6 The level of CTHRC1 in the serum of model mice was detected by ELISA.

[0035] Figure 7To detect the expression level of CTHRC1 in the aortic tissue of model mice using the Western blot method.

[0036] Figure 8 The expression level of CTHRC1 in the aortic tissue of model mice was detected by immunohistochemistry.

[0037] Figure 9 Gross images of aortic lesions in wild-type mice and CTHRC1 knockout mice.

[0038] Figure 10 The incidence of aortic dissection in wild-type mice and CTHRC1 knockout mice.

[0039] Figure 11 The mortality rate of aortic dissection in wild-type mice and CTHRC1 knockout mice.

[0040] Figure 12 Gross image of aortic lesions in wild-type mice after treatment with anti-CTHRC1 neutralizing antibody.

[0041] Figure 13 Incidence of aortic lesions in wild-type mice after treatment with anti-CTHRC1 neutralizing antibodies.

[0042] Figure 14 Mortality rate of aortic dissection in wild-type mice after treatment with anti-CTHRC1 neutralizing antibody.

[0043] Figure 15 The results show the in vitro functional blockade of the CHTRC1 neutralizing antibody. Detailed Implementation

[0044] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0045] Example 1: Preparation of CTHRC1 protein monoclonal antibody

[0046] 1. Preparation of CTHRC1 immunogenic antigen for sensitized mice

[0047] Using whole-genome synthesis to protein expression and purification (31-243 aa), eukaryotic protein expression of the His-containing CTHRC1 protein was performed, and SDS-PAGE analysis showed... Figure 3 .

[0048] The amino acid sequence of CTHRC1 protein:

[0049] SEIPKGKQKAQLRQREVVDLYNGMCLQGPAGVPGRDGSPGANGIPGTPGIPGRDGFKGEKGECLRESFEESWTPNYKQCSWSSLNYGIDLGKIAECTFTKMRSNSALRVLFS GSLRLKCRNACCQRWYFTFNGAECSGPLPIEAIIYLDQGSPEMNSTINIHRTSSVEGLCEGIGAGLVDVAIWVGTCSDYPKGDASTGWNSVSRIIIEELPK; CTHRC1 protein structure is as follows Figure 1 As shown.

[0050] 2. Evaluation of immunized mice and serum

[0051] Female Balb / c mice aged 7 to 11 weeks, weighing approximately 18-22g and aged 7-10 weeks, were selected as immunization animals. Healthy animals with glossy fur and free movement were chosen. The CTHRC1 antigen was removed from a -20℃ freezer and thawed at room temperature, avoiding repeated freeze-thaw cycles. Syringes were labeled with the project number and animal number. The antigen was thoroughly mixed. The initial immunization concentration was 0.5 mg / mL, with a dose of 0.1 mL per mouse. For the second to fourth immunizations, the antigen concentration was 0.5 mg / mL, with a dose of 0.1 mL per mouse. Immunization was primarily administered via subcutaneous or intraperitoneal injection at multiple sites. The immunization cycle was as follows: the second immunization was administered 21 days after the initial immunization, with a 14-day interval between the second and third immunizations, and between the third and fourth immunizations. Blood samples were collected from the orbital sinus 7 days after the fourth immunization. Finally, the serum titer of the mice was determined. The antigen was diluted to 1 μg / mL with coating buffer, and 50 μL / well was added to the microplate. Coating was performed overnight at 4°C. The liquid in the wells was then removed. 1% BSA was added to the microplate at 100 μL / well, and the plate was incubated at 37°C for 1 hour. The liquid in the wells was then removed again. Immunized mouse serum was serially diluted with PBS at dilutions of 1:300, 1:900, 1:2700, 1:8100, 1:24300, 1:72900, and 1:218700. 50 μL / well was added to the first to seventh wells in the same column of the microplate according to the increasing dilution. BSA was added to the eighth well as a negative control. Each dilution gradient was replicated. The plate was incubated at 37°C for 30 minutes. Washing buffer (1X TBST) was added to the microplate at 180 μL / well, and the plate was washed twice. Dilute GAM-HRP to the working concentration (1:5000) with 1% BSA, add 50 μL / well to the microplate, and incubate at 37°C for 30 min. For color development, termination, and reading: Discard the liquid in the wells, add washing buffer at 180 μL / well to the microplate, and wash the microplate three times; add 100 μL of freshly prepared TMB substrate to each well, and incubate at 37°C for 5 min; then add 90 μL / well of stop solution to terminate the reaction, and measure the OD value at 450 nm using a microplate reader. Specific measurement results are shown in Table 1.

[0052] Table 1: Serum titer results of immunized mice

[0053] Dilution factor YM246-10 YM246-12 negative mice 1：300 3.072 2.987 0.017 1：900 1.884 1.837 0.013 1：2700 0.79 0.712 0.002 1：8100 0.304 0.248 0.006 1：24300 0.117 0.089 0.007 1：72900 0.054 0.041 0.008 1：218700 0.028 0.023 0.006 PBS 0.016 0.015 0.01

[0054] 3. Mouse fusion rescreening

[0055] Take mice four days after booster immunization. Verify mouse identification numbers. Grasp the mouse by the tail, tighten the skin around its neck to prevent its head from turning freely, and make its eyes protrude. Use curved forceps to remove the protruding eyeballs and collect orbital blood. After the blood has drained, euthanize the mouse by cervical dislocation and place it in a beaker containing 75% alcohol for sterilization. Take the beaker, mouse, and alcohol into a sterile room. Use large forceps to remove the mouse from the alcohol, drain the alcohol, and place it in a kidney-shaped dish on a laminar flow hood. Use large forceps to lift the mouse's abdominal trichome, make an inverted triangular incision with sterile ophthalmic scissors, and then use hemostatic forceps to tear open the abdominal trichome to expose the abdominal cavity. Use sterile small forceps to lift the mouse's peritoneum, and use another sterile ophthalmic scissor to cut open the peritoneum until the entire abdominal cavity is exposed. Locate the spleen in the upper right quadrant of the abdominal cavity, carefully remove it, and place it in a glass petri dish containing preheated IMDM culture medium. Change the petri dish three times and wash three times. Using forceps, make a small hole at one end of the spleen. Then, use two forceps to squeeze out the spleen cells. Use a 1mL pipette to disperse the spleen cells and collect them in a 50mL centrifuge tube. Centrifuge at 1500rpm for 3 minutes. Discard the supernatant, break up the precipitate, and add 30mL of IMDM culture medium. Centrifuge at 1500rpm for 3 minutes. Simultaneously, detach well-growing SP2 / 0 cells and centrifuge at 1500rpm for 3 minutes. Discard the supernatant and resuspend the cells in 30mL of preheated IMDM culture medium. Centrifuge again at 1500rpm for 3 minutes. Discard the supernatant and resuspend the cells in 30mL of preheated IMDM culture medium. Take appropriate amounts of mouse spleen cells and SP2 / 0 cells at a ratio of 10:1, mix them thoroughly in an imported 50mL centrifuge tube, and centrifuge at 1500rpm for 3 minutes. Use a suction pump to remove the supernatant. Slowly add 1mL of preheated PEG (1500) along the bottom wall of the centrifuge tube, completing the addition within 60 seconds. Incubate at 37°C for 1 minute. Slowly add 5 mL of preheated IMDM culture medium dropwise along the tube wall, then gradually increase the speed by adding 15 mL of preheated IMDM culture medium, finally adding to a total of 40 mL. Centrifuge the fused cell suspension at 1200 rpm for 5 minutes. Discard the supernatant, add 25 mL of preheated SP2 / 0 myeloma cell culture medium, and carefully resuspend the cells using a 10 mL pipette. Spread 100 μL / well of the fused hybridoma cell suspension onto a 96-well cell culture plate. After 4–6 hours, add 100 μL / well of 2×HAT culture medium.

[0056] 4. Monoclonalization of positive hybridomas

[0057] Seven days after fusion, cells were tested for positivity. Positive cells were picked, and 200 cells were seeded from each well into a 96-well plate for sub-selection to identify monoclonal antibodies. After 8-10 days of growth, the supernatant was collected for testing, and positive cell lines were selected. Once monoclonal cell lines were selected, they were expanded, and the cell supernatant was collected and cryopreserved. The antigen was diluted to 1 μg / mL with coating buffer, and 50 μL was added to each 96-well ELISA plate. The plate was incubated overnight at 4°C. The next day, the solution in the wells was discarded, and the cells were washed once with 180 μL of 1xTBST washing buffer and blotted dry. 100 μL of 1% BSA was added to each well for blocking, and the plate was incubated at 37°C for 1 hour. The blocking solution was then discarded. 50 μL of cell supernatant was added to the previously blocked wells. Positive control wells (1:500 dilution of immunized mouse serum) and negative control wells (containing 1% BSA) were also prepared. Incubate at 37°C for 30 min, blot dry, and wash twice with 1xTBST wash buffer (180 μL per well). Blot dry. Add diluted goat anti-mouse secondary antibody-HRP (1:5000 dilution, diluted with 1% BSA) at 50 μL / well to the microplate wells, incubate at 37°C for 30 min, then discard the solution. Wash three times with 1xTBST wash buffer (180 μL per well), and blot dry. Add 100 μL of the prepared TMB substrate solution (prepared 1:1 with solution A and solution B, freshly prepared) to each reaction well, and incubate at 37°C for 5 min. Add 50 μL of 2M sulfuric acid to each reaction well to stop the reaction. Measure the OD value at 450 nm using a microplate reader. Specific measurement results are shown in Table 2.

[0058] Table 2: Results of titer detection in supernatant from positive hybridomas

[0059]

[0060] Count the positive hybridoma cells. Add 6.5 mL of sub-medium medium to the sample well, then add positive hybridoma cells to the medium to achieve a cell concentration of 125 cells / 200 μL. Add cells to a 96-well cell culture plate at a rate of 200 μL / well, in 3 columns (24 wells total). Then add 5 mL of fresh sub-medium medium, and seed the plate in the same manner, in 3 columns (24 wells total). Repeat this process sequentially. This will create four cell gradients: 125 cells / well, 25 cells / well, 5 cells / well, and 1 cell / well. Incubate the cell culture plate at 37°C and 5% CO2. Dilute the antigen to 1 μg / mL with coating buffer, add 50 μL to each 96-well ELISA plate, and incubate overnight at 4°C. The next day, discard the solution in the wells, wash once with 1xTBST washing buffer at 180 μL per well, and blot dry. Add 50 μL of cell supernatant to the previously sealed reaction wells. Incubate at 37°C for 30 min, blot dry, and wash twice with 1xTBST wash buffer (180 μL per well). For typing secondary antibody incubation: Add various typing secondary antibodies (1:1000 dilution, diluted with 1% BSA) at 50 μL / well to each well of the microplate, incubate at 37°C for 30 min, then discard the solution. Wash three times with 1xTBST wash buffer (180 μL per well), and blot dry. Add 100 μL of the prepared TMB substrate solution (prepared 1:1 with solutions A and B, freshly prepared) to each well, and incubate at 37°C for 5 min. Stop the reaction by adding 100 μL of 2M sulfuric acid to each well. Measure the OD value at 450 nm using a microplate reader. Specific measurement results are shown in Table 3.

[0061] Table 3: Titer Detection of Monoclonal Cell Supernatant

[0062]

[0063] 5. Preparation of purified antibodies

[0064] One week prior to the procedure, mice used for ascites preparation were sensitized by intraperitoneal injection of 0.5 mL of liquid paraffin. Ascites typically formed in mice within 7-10 days. A drainage needle was inserted into the mouse's peritoneal cavity, and the needle was carefully rotated to allow the ascites to flow out slowly. After collection, the ascites was centrifuged at 10,000 rpm for 5 minutes, and the supernatant was collected, its potency determined, and the solution purified. Semi-finished ascites was retrieved from the raw material storage and thawed at room temperature or 2-8°C. The sample was then observed. Normal ascites under natural light appears as follows: a light yellow, relatively clear liquid with some oil and tissue material. 10 mL of mouse ascites was added to a clean glass beaker, and the sample number and volume were recorded. 20 mL of acetate buffer (pH 4.0) was measured using a graduated cylinder and added to the beaker, which was then stirred on a magnetic stirrer. 330 μL of octanoic acid was added at a rate of 2-3 drops per 3.33% of the ascites volume, and the timer was started for 30 minutes after the addition was complete. After stirring, transfer the sample to a round-bottom centrifuge tube and centrifuge at 10,000 rpm for 30 min at 4°C. After centrifugation, filter the sample using 8 layers of gauze and a funnel, and transfer the supernatant to a clean beaker, discarding the precipitate. Dialyze the filtered supernatant to 1×PBS with a dialysate volume ratio of at least 30 times. During the day, keep the solution at room temperature with stirring, changing the solution every 3 hours. At night, place the solution in a 4°C display case and dialyze overnight with stirring, changing the solution a total of 3 times. Record the supernatant obtained in the previous step as a number and measure its volume as 20 mL. Set the peristaltic pump flow rate to 10 rpm and add 20 mL of saturated ammonium sulfate solution to the beaker using the peristaltic pump. Stir continuously while adding the solution, and stir thoroughly on a stirrer for 30 min. After stirring, centrifuge at 10,000 rpm for 10 min at 4°C, discard the supernatant, and dissolve the antibody precipitate with an appropriate amount of 1×PBS. Dialyze the dissolved antibody from the previous step to 1×PBS with a dialysate volume ratio greater than 50 times. During the day, the sample was stirred at room temperature, with the medium changed every 3 hours. At night, it was placed in a 4°C display case and dialyzed overnight with stirring, for a total of 4 medium changes. After dialysis, the antibody was centrifuged at 10,000 rpm for 10 min at 4°C and filtered through a 0.22 μm filter. The dialyzed sample was diluted appropriately, and the OD value was measured at 280 nm using a nucleic acid and protein detector, with an OD range of 0.2-0.8.

[0065] The following are monoclonal antibodies against the CTHRC1 protein:

[0066] Light chain variable region amino acid sequence

[0067] QLILTQSSSASFSLGASAKLTCTLSSQHSTYTIEWYQQQPLKPPKYVMEVKKDGHSTG DGIPDRFSGSSSGADRYLSISNIQPEDEAIYICGVGDTIKEQFVYVFGGGTRVTVL;

[0068] Where CDR1: TLSSQHSTYTIE; CDR2: VKKDGSHSTGD; CDR3: GVGDTIKEQFVYV, as Figure 2 As shown in (a).

[0069] Heavy chain variable region amino acid sequence

[0070] EVQLVETGGGLVQPKGSMKLSCAASGFTFNINAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQRMLYLQMNNLKTEDTAKYYCVSNWDWYFDVWGAGTTVTVSS;

[0071] Where CDR1: INAMN; CDR2: RIRSKSNNYATYYADSVKD; CDR3: NWDWYFDV, as Figure 2 As shown in (b).

[0072] Example 2: CTHRC1 content in the serum of patients with aortic aneurysm and / or aortic dissection

[0073] 1. Main reagents

[0074] Human CTHRC1 ELSIA kit. Serum samples from patients with aortic aneurysm and / or aortic dissection and healthy controls.

[0075] 2. Experimental Procedure

[0076] 2.1 Collect serum samples from patients with aortic aneurysm and / or aortic dissection and healthy controls. Collect whole blood into test tubes without anticoagulants, place at room temperature for 1 hour, and allow the whole blood to coagulate naturally and precipitate serum. Centrifuge at 1500g for 10 minutes at 4°C, and collect the yellow supernatant to obtain serum.

[0077] 2.2 Dilute the sample five times and add 100 μL / well to the corresponding well as the sample group, standard group, and blank group, and incubate at 37 degrees for 2 hours.

[0078] 2.3 Add 300-400 μL of washing solution to each well and wash the plate three times, and pat it dry on thick absorbent paper for the last wash.

[0079] 2.4 Add 100 μL of biotin-conjugated anti-human CTHRC1 detection antibody to all wells, cover with sealing film, and incubate at 37 degrees Celsius in the dark for 1 hour.

[0080] 2.5 Add 300-400 μL of washing solution to each well and wash the plate five times, and pat it dry on thick absorbent paper on the last wash.

[0081] 2.6 Add 100 μL of diluted streptavidin-HRP to all wells, cover with sealing film, and incubate at room temperature in the dark for 30 minutes.

[0082] 2.7 Add 100 μL of TMB colorimetric solution to all wells. Cover with sealing film and incubate at room temperature in the dark for 15 minutes.

[0083] 2.8 Add 50 μL of stop solution to all wells, mix well, and immediately measure the A450 value.

[0084] 3. ELISA results as follows Figure 4 As shown, CTHRC1 levels were significantly elevated in the serum of patients with aortic aneurysm and / or aortic dissection.

[0085] Example 3: CTHRC1 expression level in vascular tissue of patients with aortic aneurysm and / or aortic dissection

[0086] 1. Materials

[0087] CTHRC1 protein monoclonal antibody; vascular tissue from patients with aortic aneurysm and / or aortic dissection and healthy controls.

[0088] 2. Experimental Procedure

[0089] 2.1 Dewaxing paraffin sections to water: Vascular sections from patients with aortic aneurysm and / or aortic dissection and vascular sections from healthy controls were sequentially immersed in xylene I for 15 minutes, xylene II for 15 minutes, xylene III for 15 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 85% alcohol for 5 minutes, 75% alcohol for 5 minutes, and then washed with distilled water.

[0090] 2.2 Antigen Retrieval: Tissue slides were placed in a retrieval container filled with EDTA antigen retrieval buffer (pH 9.0) and microwaved for antigen retrieval. The microwave was heated on medium heat for 10 minutes until boiling, then turned off and kept warm for 10 minutes before being heated on medium-low heat for another 7 minutes. During this process, excessive evaporation of the buffer should be prevented, and the slides should not be allowed to dry. After natural cooling, the slides were placed in PBS (pH 7.4) and washed three times on a destaining shaker for 5 minutes each time.

[0091] 2.3 Blocking endogenous peroxidase: The slides were placed in 3% hydrogen peroxide solution (hydrogen peroxide: pure water = 1:9) and incubated at room temperature in the dark for 25 minutes. The slides were then placed in PBS (pH 7.4) and washed three times on a decolorizing shaker for 5 minutes each time.

[0092] 2.4 Treat with Triton-X100 (0.3%) for 10 minutes, then wash with PBS 3 times for 5 minutes each time.

[0093] 2.5 Serum blocking: Add 10% donkey serum to the histochemistry zone and evenly cover the tissue. Block at room temperature for 30 minutes.

[0094] 2.6 Add primary antibody: Gently shake off the blocking solution, add CTHRC1 monoclonal antibody prepared in PBS at a certain ratio to the slide, and incubate the slide flat in a humidified chamber at 4°C overnight (add a small amount of water to the humidified chamber to prevent antibody evaporation).

[0095] 2.7 Adding secondary antibody: Place the slide in PBS (pH 7.4) and wash three times on a decolorizing shaker for 5 minutes each time. After slightly drying the sections, add the corresponding species' secondary antibody (HRP-labeled) to the tissue in the circle and incubate at room temperature for 50 minutes.

[0096] 2.8 DAB staining: Place the slide in PBS (pH 7.4) and wash three times on a destaining shaker for 5 minutes each time. After slightly drying the slide, add freshly prepared DAB staining solution to the circle. Control the staining time under a microscope. A positive result is brownish-yellow. Rinse the slide with tap water to stop the staining process.

[0097] 2.9 Counterstaining cell nuclei: Harris hematoxylin counterstaining for about 3 minutes, rinse with tap water, differentiate with 1% hydrochloric acid alcohol for a few seconds, rinse with tap water, return to blue with ammonia water, and rinse with running water.

[0098] 2.10 Dehydration and mounting: Immerse the sections in 75% alcohol for 5 minutes, 85% alcohol for 5 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, and xylene I for 5 minutes to dehydrate and clear them. Remove the sections from the xylene and let them air dry slightly before mounting them with neutral resin.

[0099] 2.11 Microscopic examination, image acquisition and analysis.

[0100] 3. Immunohistochemical results as follows Figure 5 As shown, CTHRC1 expression levels were elevated in the vascular tissues of patients with human aortic aneurysms and / or aortic dissections.

[0101] Example 4: Mouse model of aortic aneurysm and / or aortic dissection induced by combination therapy of angiotensin II (Ang II) and β-aminopropionitrile (BAPN).

[0102] 1. Experimental study on the induction of aortic aneurysm and / or aortic dissection models in animals using a combination of Ang II and BAPN.

[0103] SPF-grade C57BL / 6 mice and CTHRC1 knockout mice were purchased from Cyagen Biosciences. Aortic aneurysm and / or aortic dissection mouse models were induced by the combined administration of Ang II and BAPN: Six-week-old male C57BL / 6 wild-type and CTHRC1- / - mice were administered subcutaneously via micro-infusion pumps containing Ang II and BAPN at rates of 2500 ng / (kg·min) and 150 mg / (kg·day), respectively, for 14 days. The survival of the four groups of mice was recorded during the 14-day experimental period, and all mice were sacrificed after 2 weeks. Aortic tissue and serum were collected for further experiments.

[0104] Animal experiments using CTHRC1 neutralizing antibodies to treat aortic structural disorders and phenotypic transformation of medial vascular smooth muscle: The induction and modeling experiments were the same as above. Six-week-old male C57BL / 6 wild-type mice were selected and divided into a CTHRC1 neutralizing antibody treatment group and an IgG treatment group. Both groups were administered 200ug / mouse / dose of neutralizing antibody or IgG intraperitoneally three times a week for two weeks. Forty-eight hours after the last injection, all mice were sacrificed, and the aorta and serum were collected for subsequent experiments.

[0105] 2. Experimental steps:

[0106] 2.1 ELISA method for detecting CTHRC1 levels in mouse serum

[0107] 2.1.1 Collect serum samples from mice in the model group and control group. Collect whole blood into test tubes without anticoagulant and place at room temperature for 1 hour. After the whole blood coagulates naturally and serum is separated, centrifuge at 1500g at 4℃ for 10 minutes and take the yellow supernatant to obtain serum.

[0108] 2.1.2 Dilute the sample five times and add 100 μL / well to the corresponding well as the sample group, standard group, and blank group, and incubate at 37 degrees for 2 hours.

[0109] 2.1.3 Add 300-400 μL of washing solution to each well and wash the plate three times, and pat it dry on thick absorbent paper for the last wash.

[0110] 2.1.4 Add 100 μL of biotin-conjugated anti-human CTHRC1 detection antibody to all wells, cover with sealing film, and incubate at 37 degrees Celsius in the dark for 1 hour.

[0111] 2.1.5 Add 300-400 μL of washing solution to each well and wash the plate five times, and pat it dry on thick absorbent paper on the last wash.

[0112] 2.1.6 Add 100 μL of diluted streptavidin-HRP to all wells, cover with sealing film, and incubate at room temperature in the dark for 30 minutes.

[0113] 2.1.7 Add 100 μL TMB of colorimetric solution to all wells. Cover with sealing film and incubate at room temperature in the dark for 15 minutes.

[0114] 2.1.8 Add 50 μL of stop solution to all wells, mix well, and immediately measure the A450 value.

[0115] 2.2 Immunohistochemical methods were used to detect the expression level of CTHRC1 in the vascular tissues of the model group and the control group mice.

[0116] 2.2.1 Dewaxing paraffin sections to water: The blood vessel sections of the model group and the control group mice were sequentially immersed in xylene I for 15 minutes, xylene II for 15 minutes, xylene III for 15 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 85% alcohol for 5 minutes, 75% alcohol for 5 minutes, and then washed with distilled water.

[0117] 2.2.2 Antigen Retrieval: Tissue slides were placed in a retrieval container filled with EDTA antigen retrieval buffer (pH 9.0) and microwaved for antigen retrieval. The microwave was heated on medium heat for 10 minutes until boiling, then turned off and kept warm for 10 minutes before being heated on medium-low heat for another 7 minutes. During this process, excessive evaporation of the buffer should be prevented, and the slides should not be allowed to dry. After natural cooling, the slides were placed in PBS (pH 7.4) and washed three times on a destaining shaker for 5 minutes each time.

[0118] 2.2.3 Blocking endogenous peroxidase: The slides were placed in 3% hydrogen peroxide solution (hydrogen peroxide: pure water = 1:9) and incubated at room temperature in the dark for 25 minutes. The slides were then placed in PBS (pH 7.4) and washed three times on a decolorizing shaker for 5 minutes each time.

[0119] 2.2.4 Treat with Triton-X100 (0.3%) for 10 minutes, then wash with PBS 3 times for 5 minutes each time.

[0120] 2.2.5 Serum blocking: Add 10% donkey serum evenly to the histochemistry zone and block at room temperature for 30 minutes.

[0121] 2.2.6 Add primary antibody: Gently shake off the blocking solution, add CTHRC1 monoclonal antibody prepared in PBS at a certain ratio to the slide, and incubate the slide flat in a humidified chamber at 4°C overnight (add a small amount of water to the humidified chamber to prevent antibody evaporation).

[0122] 2.2.7 Adding secondary antibody: Place the slide in PBS (pH 7.4) and wash three times on a decolorizing shaker for 5 minutes each time. After slightly drying the sections, add the corresponding species' secondary antibody (HRP-labeled) to the tissue in the circle and incubate at room temperature for 50 minutes.

[0123] 2.2.8 DAB staining: Place the slide in PBS (pH 7.4) and wash three times on a destaining shaker for 5 minutes each time. After slightly drying the slide, add freshly prepared DAB staining solution to the circle. Control the staining time under a microscope. A positive result is brownish-yellow. Rinse the slide with tap water to stop the staining process.

[0124] 2.2.9 Counterstaining cell nuclei: Harris hematoxylin counterstaining for about 3 minutes, rinse with tap water, differentiate with 1% hydrochloric acid alcohol for a few seconds, rinse with tap water, return to blue with ammonia water, and rinse with running water.

[0125] 2.2.10 Dehydration and mounting: Immerse the sections in 75% alcohol for 5 minutes, 85% alcohol for 5 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, and xylene I for 5 minutes to dehydrate and clear them. Remove the sections from the xylene and let them air dry slightly before mounting them with neutral resin.

[0126] 2.2.11 Microscopic examination, image acquisition and analysis.

[0127] 2.3 Immunoblotting method was used to detect the expression level of CTHRC1 in vascular tissues of model group and control group mice.

[0128] 2.3.1 Use pre-cooled tools to separate the vascular tissues of corresponding parts of the model group and control group mice, and place them on ice as much as possible to prevent protease hydrolysis;

[0129] 2.3.2 Place the tissue block in a round-bottomed microcentrifuge tube or EP tube, add liquid nitrogen to freeze the tissue, and homogenize it on ice. Add about 200 μl of pre-cooled lysis buffer (add protease and phosphatase inhibitors before use) for every 10 mg of tissue. After homogenizing on ice, place it at 4°C and shake for 2 h.

[0130] 2.3.3 Centrifuge at 12,000 rpm for 20 min at 4℃. Gently aspirate the supernatant and transfer it to a new, pre-cooled microcentrifuge tube. Place the tube on ice. This is the protein sample. Discard the precipitate.

[0131] 2.3.4 Protein Quantification: Based on the number of samples, prepare an appropriate amount of BCA working solution by adding 1 volume of BCA reagent B to 50 volumes of BCA reagent A (50:1), and mix thoroughly. Place the ELISA plate on a shaker and shake for 30 seconds, incubate at 37°C for 30 minutes, and then measure the absorbance at 562 nm. The corresponding protein concentration can be found on the standard curve based on the absorbance value of the measured sample.

[0132] 2.3.5 Denatured and reduced protein samples: Protein denaturation is generally performed using a loading buffer containing a cationic denaturing detergent such as SDS, and boiling at 95-100℃ for 5 minutes.

[0133] 2.3.6 Electrophoresis: The loading volume of vascular tissue protein antigen is 30 μg. Take the processed sample solution, use a microsyringe to draw an appropriate amount of sample solution, and slowly add the sample solution to the notch area (sample entry point) in the gel plate. During electrophoresis, use low-voltage constant-voltage electrophoresis for the upper gel, turn on the power and adjust the voltage to 80V (generally for about 15 minutes). When bromophenol blue enters the lower gel, use high-voltage constant-voltage electrophoresis, adjust the voltage to 120V until the bromophenol blue reaches near the bottom of the gel, then stop the electrophoresis.

[0134] 2.3.7 Transfer: After SDS-PAGE electrophoresis, gently pry open the two glass plates of the gel plate with a blade or thin plate, allowing the gel to lie flat on one of the glass plates. Using a blade, cut the separating gel along the boundary between the separating and stacking gels, and cut off a small corner from the upper left to mark the sample order. Then carefully transfer the gel into the transfer buffer. Cut a 0.45µm PVDF membrane of the same size as the separating gel and soak it in methanol for 5 seconds. Cut six pieces of filter paper of the same size and equilibrate them with the PVDF membrane and gel in transfer buffer for 15 minutes. Place the sponge pad, filter paper, gel, membrane, filter paper, and sponge pad (from bottom to top) on the transfer apparatus from negative (black background) to positive. Set the transfer current to a constant 200mA for approximately 40 minutes (the time varies depending on the protein size; generally, 1 kDa takes about 1 minute). After the electrophoresis is complete, remove the membrane and carefully cut off a corner at the same position as the gel to mark the direction of electrophoresis and the membrane surface with adsorbed proteins.

[0135] 2.3.8 Sealing: Shake slowly for 1-2 hours at room temperature or 37°C with 5% skim milk powder or BSA solution.

[0136] 2.3.9 Add primary antibody: Dilute CTHRC1 monoclonal antibody at a ratio of 1:1000 and incubate overnight at 4°C with gentle shaking.

[0137] 2.3.10 After incubation, take the primary antibody, add TBST to a square food storage box, place the membrane in the box, ensuring the TBST covers the PVDF membrane, and shake on a shaker at low speed for 10 minutes. Repeat the washing process 3 times.

[0138] 2.3.11 Dilute the secondary antibody of the corresponding species at a ratio of 1:10000 and incubate on a shaker at room temperature for 1 hour. After incubation, remove the secondary antibody, add TBST to a square food storage container, place the membrane in the container, ensuring that the TBST covers the PVDF membrane, and shake on a shaker at low speed for 10 minutes. Repeat the washing process 3-5 times.

[0139] 2.3.12 Chemiluminescence color development and band gray value analysis.

[0140] 3. ELISA, immunohistochemistry, and Western blotting revealed significantly upregulated CTHRC1 expression in the model group mice. Simultaneously, single-cell sequencing data from the diseased blood vessels showed that CTHRC1 was primarily highly expressed in a heterogeneous group of fibroblasts. The aortic lesions in the model group mice were more severe, and the medial smooth muscle cells exhibited a proliferative phenotype. Experimental results are as follows... Figure 6 , 7 As shown in Figure 8.

[0141] 4. Compared with CTHRC1 knockout mice, wild-type mice had a higher incidence of aortic aneurysm and / or aortic dissection, a higher mortality rate, and more severe lesions. Experimental results are as follows: Figure 9 , 10 As shown in Figure 11.

[0142] 5. In wild-type mice, CTHRC1 neutralizing antibodies reduced the incidence and mortality of aortic aneurysms and / or aortic dissections induced by the combined use of angiotensin II (Ang II) and β-aminopropionitrile (BPAN). They also alleviated the severity of aortic lesions, improved vascular structural disorder, and maintained normal function. Experimental results are as follows: Figure 12 , 13 As shown in Figure 14.

[0143] Example 5: Experiment on Phenotype Transformation of Vascular Smooth Muscle Cells

[0144] 1. Materials

[0145] CTHRC1 recombinant protein, CTHRC1 monoclonal antibody, vascular aortic smooth muscle cells, α-SMA, SM22α, p-ERK, and ERK primary antibody were purchased from Abcam.

[0146] 2. An expression plasmid for CTHRC1 was constructed and transfected into 293T cells. The recombinant CTHRC1 protein was purified. Immunoblotting experiments showed that the recombinant CTHRC1 protein promoted vascular smooth muscle cell migration and exacerbated the loss of the contractile phenotype in vascular smooth muscle cells by activating the ERK signaling pathway. This effect could be reversed using a CTHRC1 neutralizing antibody.

[0147] 3. Experimental Procedure: Detection of contractile marker protein levels in vascular smooth muscle cells using Western blotting.

[0148] 3.1 Wash the vascular smooth muscle cells in the six-well plate with pre-cooled PBS, aspirate the supernatant, and place on ice as much as possible to prevent protease hydrolysis;

[0149] 3.2 Add approximately 250 μl of pre-chilled lysis buffer (add protease and phosphatase inhibitors before use) to every 10^6 cells, scrape off the cells with a cell scraper, and incubate at 4°C for 15 minutes for lysis.

[0150] 3.3 Centrifuge at 12000 rpm for 20 min at 4℃, gently aspirate the supernatant, transfer it to a new pre-cooled microcentrifuge tube and place it on ice. This is the protein sample. Discard the precipitate.

[0151] 3.4 Protein Quantification: Based on the number of samples, prepare an appropriate amount of BCA working solution by adding 1 volume of BCA reagent B to 50 volumes of BCA reagent A (50:1), and mix thoroughly. Place the ELISA plate on a shaker and shake for 30 seconds, incubate at 37°C for 30 minutes, and then measure the absorbance at 562 nm. The corresponding protein concentration can be found on the standard curve based on the absorbance value of the measured sample.

[0152] 3.5 Denatured and reduced protein samples: Protein denaturation is generally performed using a loading buffer containing cationic denaturing detergents such as SDS, and boiling at 95-100℃ for 5 minutes.

[0153] 3.6 Electrophoresis: The loading volume of vascular tissue protein antigen is 30 μg. Take the processed sample solution, use a microsyringe to draw an appropriate amount of sample solution, and slowly add the sample solution to the notch area (sample entry point) in the gel plate. During electrophoresis, use low-voltage constant-voltage electrophoresis for the upper gel, turn on the power and adjust the voltage to 80V (generally for about 15 minutes). When bromophenol blue enters the lower gel, use high-voltage constant-voltage electrophoresis, adjust the voltage to 120V until the bromophenol blue reaches near the bottom of the gel, and then stop electrophoresis.

[0154] 3.7 Transfer: After SDS-PAGE electrophoresis, gently pry open the two glass plates of the gel plate with a blade or thin plate, allowing the gel to tilt onto one of the glass plates. Using a blade, cut the separating gel along the boundary between the separating and stacking gels, and cut off a small corner from the upper left to mark the sample order. Then carefully transfer the gel into the transfer buffer. Cut a 0.45µm PVDF membrane of the same size as the separating gel and soak it in methanol for 5 seconds. Cut six pieces of filter paper of the same size and equilibrate them with the PVDF membrane and gel in transfer buffer for 15 minutes. Place the sponge pad, filter paper, gel, membrane, filter paper, and sponge pad (from bottom to top) on the transfer apparatus from negative (black background) to positive. Set the transfer current to a constant 200mA for approximately 40 minutes (the time varies depending on the protein size; generally, 1 kDa takes about 1 minute). After the electrophoresis is complete, remove the membrane and carefully cut off a corner at the same position as the gel to mark the direction of electrophoresis and the membrane surface with adsorbed proteins.

[0155] 3.8 Sealing: Shake gently for 1-2 hours at room temperature or 37°C with 5% skim milk powder or BSA solution.

[0156] 3.9 Add primary antibody: Dilute α-SMA, SM22α, p-ERK1 / 2, and ERK1 / 2 at a ratio of 1:1000 and incubate overnight at 4°C with gentle shaking.

[0157] 3.10 After incubation, take the primary antibody, add TBST to a square food storage box, place the membrane in it, ensuring that the TBST covers the PVDF membrane, shake on a shaker at low speed for 10 minutes, and repeat the washing process 3 times.

[0158] 3.11 Dilute the secondary antibody of the corresponding species at a ratio of 1:10000 and incubate on a shaker at room temperature for 1 hour. After incubation, remove the secondary antibody, add TBST to a square food storage container, place the membrane in the container, ensuring that the TBST covers the PVDF membrane, and shake on a shaker at low speed for 10 minutes. Repeat the washing process 3-5 times.

[0159] 3.12 Chemiluminescence color development and band gray value analysis.

[0160] 4. Experimental results show that recombinant CTHRC1 protein can exacerbate the loss of the contractile phenotype in vascular smooth muscle cells by activating the ERK signaling pathway, while the CTHRC1 neutralizing antibody can reverse this effect, as shown in the results. Figure 15 As shown.

[0161] The above experiments confirm that CTHRC1 is involved in the occurrence and development of aortic aneurysms and / or aortic dissections, exacerbating the disease progression. In vivo, CTHRC1 can accelerate the progression of aortic aneurysms and / or aortic dissections, promote adventitia structural disorder, and exacerbate the loss of contractile phenotype in vascular media smooth muscle cells. In vitro, CTHRC1 can accelerate vascular smooth muscle cell migration and accelerate the loss of its contractile phenotype. Its effects can be blocked using CTHRC1 neutralizing antibodies. In vivo, the use of CTHRC1 monoclonal antibodies with high affinity to neutralize and block the disease can significantly reduce the incidence and mortality of aortic aneurysms and / or aortic dissections, and slow disease progression. These results demonstrate that this gene can be accurately used in diagnostic kits for aortic aneurysms and / or aortic dissections, providing the application of CTHRC1 gene diagnostic kits and a new target for targeted therapy of aortic aneurysms and / or aortic dissections.

[0162] The above description is merely a few embodiments of this application and is not intended to limit this application in any way. Although this application discloses preferred embodiments as described above, it is not intended to limit this application. Any changes or modifications made by those skilled in the art without departing from the scope of the technical solution of this application using the disclosed technical content are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. The use of a monoclonal antibody against CTHRC1 protein in the preparation of a kit for diagnosing aortic aneurysm and / or aortic dissection, characterized in that, The monoclonal antibody can specifically bind to human CTHRC1 protein and is used to detect the expression level of the protein in the serum of the subject, wherein the elevated expression level of CTHRC1 protein is positively correlated with the occurrence of aortic aneurysm and / or aortic dissection. The three CDR amino acid sequences of the light chain variable region of the monoclonal antibody are: CDR1: TLSSQHSTYTIE; CDR2: VKKDGSHSTGD; CDR3: GVGDTIKEQFVYV; The three CDR amino acid sequences of the heavy chain variable region of the monoclonal antibody are: CDR1: INAMN; CDR2: RIRSKSNNYATYYADSVKD; CDR3: NWDWYFDV.

2. A drug for treating aortic aneurysm and / or aortic dissection, characterized in that, The active ingredient is a monoclonal antibody targeting the adventitia fibroblast population and targeting the CTHRC1 protein, along with a pharmaceutically acceptable carrier. The three CDR amino acid sequences of the light chain variable region of the monoclonal antibody are: CDR1: TLSSQHSTYTIE; CDR2: VKKDGSHSTGD; CDR3: GVGDTIKEQFVYV; The three CDR amino acid sequences of the heavy chain variable region of the monoclonal antibody are: CDR1: INAMN; CDR2: RIRSKSNNYATYYADSVKD; CDR3: NWDWYFDV.

3. The use of a monoclonal antibody against CTHRC1 protein in the preparation of a medicament for treating aortic aneurysm and / or aortic dissection, characterized in that, The monoclonal antibody targets the adventitia fibroblast population, and by binding to and neutralizing the CTHRC1 protein with high affinity, it inhibits the activation of the ERK signaling pathway, thereby blocking the phenotypic transformation and migration of vascular smooth muscle cells. The three CDR amino acid sequences of the light chain variable region of the monoclonal antibody are: CDR1: TLSSQHSTYTIE; CDR2: VKKDGSHSTGD; CDR3: GVGDTIKEQFVYV; The three CDR amino acid sequences of the heavy chain variable region of the monoclonal antibody are: CDR1: INAMN; CDR2: RIRSKSNNYATYYADSVKD; CDR3: NWDWYFDV.

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