Double-antibody sandwich ELISA kit for detecting content of CTHRC1 in blood and detection method and application thereof

Abstract

The invention discloses a double-antibody sandwich ELISA kit for detecting the content of CTHRC1 in blood as well as a detection method and application of the double-antibody sandwich ELISA kit, and belongs to the technical field of molecular biology. Aiming at the problem of high difficulty in early recognition of acute aortic dissection at present, a pair of capture antibody and detection antibody capable of recognizing different epitopes of a CTHRC1 protein antigen is assembled to obtain the double-antibody sandwich ELISA kit for detecting the content of CTHRC1 in blood. The double-antibody sandwich ELISA kit has relatively high specificity and sensitivity to the CTHRC1 protein in the serum, and can be used for quantitatively detecting the CTHRC1 protein in the serum, so that the early acute aortic dissection is accurately recognized, and misdiagnosis is avoided.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology, and in particular to a double-antibody sandwich ELISA kit for detecting the CTHRC1 content in blood, and a detection method and application thereof. Background Art

[0002] Acute aortic dissection (AAD) is an extremely serious cardiovascular disease with an initial mortality rate of approximately 40%, and subsequent mortality rates increase at a rate of 1% per hour. Epidemiological studies have shown that the incidence of AAD is approximately 3-5 cases per 100,000 cases per year, with an increasing trend year by year. Its pathological manifestations are separation of the layers of the aortic wall and tearing of the intimal layer, leading to the progression of dissection (proximal or retrograde). Blood enters the space between the intima and media through the intimal tear, which can cause aortic rupture and death in the short term. However, due to its diverse clinical manifestations, this disease is often confused with other diseases such as myocardial infarction and pulmonary embolism, making diagnosis difficult.

[0003] On the one hand, the typical symptoms of acute aortic dissection are sudden, severe, tearing pain in the chest and back, but some patients may only present with abdominal pain, syncope, limb ischemia, or neurological symptoms (such as hemiplegia), which can easily be confused with acute myocardial infarction, pulmonary embolism, acute abdomen, or stroke. Furthermore, approximately 10%-15% of patients experience asymmetric or absent limb pulses due to intimal tearing, resulting in compression of the true lumen by the false lumen. However, this sign may not be obvious in the early stages and is particularly prone to being overlooked in obese or hypotensive patients.

[0004] On the other hand, the diagnosis of acute aortic dissection requires CT angiography (CTA), transesophageal ultrasound (TEE) or magnetic resonance imaging (MRI). However, it is worth noting that although CTA has high sensitivity and specificity and is currently the preferred examination method in emergency departments, it relies on iodine contrast agents and ionizing radiation, which limits its application in patients with renal insufficiency and those who are allergic to contrast agents. MRI has unique advantages due to its lack of radiation and excellent image quality, but its examination time is long and requires high patient cooperation, making it unsuitable for critically ill or unstable patients. TEE can be performed at the bedside and is suitable for patients who cannot tolerate transfer, but this technology is highly dependent on the operator's experience and has certain limitations in displaying the distal ascending aorta and the aortic arch. In addition, the clinical manifestations of aortic dissection are nonspecific, and some patients do not even have obvious symptoms, which increases the difficulty of early identification.

[0005] According to clinical retrospective studies, the initial misdiagnosis rate for aortic dissection can reach 30%-50%. In particular, when the coronary arteries are not involved or aortic regurgitation does not occur, the electrocardiogram may not show specific changes and can be easily misdiagnosed as other cardiovascular diseases. Delayed diagnosis directly leads to an increase in mortality (1%-2% increase per hour), further increasing the pressure on clinical decision-making. Therefore, there is an urgent need to improve the diagnosis rate of acute aortic dissection, and the development of plasma marker detection kits for the disease may provide a new direction for early identification.

[0006] Collagen Triple Helix Repeat Containing 1 (CTHRC1) was first discovered through differential expression analysis between normal rat arteries and arteries with bulbar injury. It is a secreted protein consisting of an N-terminal signal peptide, a 36-amino acid collagen triple helix, and a C-terminal globular domain. CTHRC1 expression is influenced by the vascular regulatory factors TGF-β (Transforming growth factor-β) and BMP4 (Bonemorphogenetic protein-4). In colorectal cancer cells, CTHRC1 can induce the expression of matrix metalloproteinases (MMPs) such as MMP2 and MMP9 by activating ERK signaling, thereby enhancing tumor cell invasion. Plasma CTHRC1 levels in patients with rheumatoid arthritis are significantly elevated compared with healthy controls. However, the role of CTHRC1 in aortic dissection remains unclear. Summary of the Invention

[0007] To address the above-mentioned problems, the present invention aims to develop a double-antibody sandwich ELISA kit for detecting the CTHRC1 content in blood, as well as its detection method and application. A double-antibody sandwich ELISA kit is assembled using a pair of antibodies that can recognize different epitopes of the CTHRC1 protein antigen to achieve quantitative detection of the CTHRC1 protein. The double-antibody sandwich ELISA kit has high specificity and sensitivity for the CTHRC1 protein, a diagnostic marker for human acute aortic dissection, and is simple to operate. It can be used for the detection of acute aortic dissection in scientific research and clinical practice, playing a role in assisting diagnosis, guiding treatment, and determining prognosis.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] On the one hand, the present invention provides a double-antibody sandwich ELISA kit for detecting the CTHRC1 content in blood, the kit comprising a CTHRC1 capture antibody coated on an ELISA plate, a CTHRC1 detection antibody labeled with horseradish peroxidase, a CTHRC1 protein standard, a washing solution, a blocking solution, a color developer, and a stop solution;

[0010] Among them, the CTHRC1 capture antibody and the CTHRC1 detection antibody are a group of paired antibodies that can recognize different sites of the CTHRC1 protein antigen.

[0011] Based on the above scheme, the present invention uses recombinant human CTHRC1 protein as an immunogen to screen and obtain CTHRC1 capture antibodies and CTHRC1 detection antibodies. Utilizing this pair of antibodies that can recognize different epitopes of the CTHRC1 protein antigen, a double-antibody sandwich ELISA kit is assembled to achieve quantitative detection of CTHRC1 protein in serum.

[0012] Preferably, the method for preparing a CTHRC1 detection antibody labeled with horseradish peroxidase comprises the following steps:

[0013] Activate and dialyze horseradish peroxidase to obtain dialyzed HRP;

[0014] dialyzing the purified CTHRC1 detection antibody to obtain a dialyzed detection antibody;

[0015] The dialyzed HRP, ethylene glycol, and the dialyzed detection antibody are mixed evenly and then dialyzed to obtain an HRP-antibody mixture;

[0016] NaBH4 solution and saturated ammonium sulfate were added to the HRP-antibody mixture, stirred evenly, and then centrifuged; the supernatant was discarded, and the precipitate was dissolved in PBS to obtain the CTHRC1 detection antibody labeled with horseradish peroxidase.

[0017] Preferably, the washing solution is PBST.

[0018] Preferably, the blocking solution is 2% bovine serum albumin.

[0019] Preferably, the developer is TMB solution.

[0020] Preferably, the stop solution is H2SO4.

[0021] On the other hand, the present invention also provides a detection method of the double antibody sandwich ELISA kit as described above, comprising the following steps:

[0022] The CTHRC1 capture antibody was coated with Na2CO3 and NaHCO3 at pH 9.6, added to a multi-well ELISA plate, and tightly bound to the ELISA plate to form a CTHRC1 capture antibody coated on the ELISA plate;

[0023] Wash the ELISA plate with washing solution, then add blocking solution to each well and incubate at 37°C for 3 h;

[0024] After blocking, the protein extract solution to be tested and the CTHRC1 protein standard were added to the wells of the ELISA plate and incubated at 37°C for 1 hour. Then, the CTHRC1 detection antibody labeled with horseradish peroxidase was added and incubated for another 1 hour.

[0025] Finally, a color developer was added to the formed complex and incubated at room temperature for 3 minutes until the color of the developer changed. The reaction was terminated by adding a stop solution. 450 The value is used to determine the presence and concentration of CTHRC1 protein in the protein extract solution to be tested.

[0026] On the other hand, the present invention also provides use of the aforementioned double-antibody sandwich ELISA kit in the preparation of a product for diagnosing / treating acute aortic dissection.

[0027] Based on the above scheme, the double-antibody sandwich ELISA kit of the present invention detects the content of CTHRC1 protein in the serum. The CTHRC1 content in patients with acute aortic dissection is significantly increased compared with healthy controls and patients with acute myocardial infarction. Therefore, acute aortic dissection patients can be quickly and accurately diagnosed based on the CTHRC1 protein content detection results.

[0028] The beneficial effects of the present invention are:

[0029] The present invention utilizes a pair of capture and detection antibodies that recognize different epitopes of the CTHRC1 protein antigen to assemble a double-antibody sandwich ELISA kit for quantitative detection of the CTHRC1 protein. Experiments have demonstrated that the ELISA kit of the present invention exhibits high specificity and sensitivity for the CTHRC1 protein, a diagnostic marker for acute aortic dissection in both mice and humans. It is simple to operate and can be used for the detection of acute aortic dissection in both scientific research and clinical settings, serving as an aid in diagnosis, guiding treatment, and assessing prognosis. This optimizes current diagnostic strategies for acute aortic dissection and can further enhance the early recognition rate and diagnostic efficiency of this disease among clinical practitioners. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the CTHRC1 standard curve of the double antibody sandwich ELISA kit of the present invention.

[0031] Figure 2This is the detection result of the CTHRC1 content in the serum of mice in the acute aortic dissection model group in Example 3 of the present invention.

[0032] Figure 3 This is the ROC curve of the CTHRC1 content in the serum of mice in the acute aortic dissection model group in Example 3 of the present invention.

[0033] Figure 4 These are the test results of plasma CTHRC1 levels in healthy controls and patients with acute aortic dissection in Example 4 of the present invention.

[0034] Figure 5 These are the test results of plasma CTHRC1 levels in patients with acute myocardial infarction and acute aortic dissection in Example 4 of the present invention. DETAILED DESCRIPTION

[0035] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0036] Example 1:

[0037] Example 1 provides a double-antibody sandwich ELISA kit for detecting the CTHRC1 content in blood and a detection method thereof.

[0038] The kit comprises: a CTHRC1 capture antibody coated on an ELISA plate, a CTHRC1 detection antibody labeled with horseradish peroxidase (HRP), a CTHRC1 protein standard, a washing solution, a blocking solution, a color developer and a stop solution.

[0039] It should be noted that the CTHRC1 capture antibody and CTHRC1 detection antibody are a pair of paired antibodies that recognize different sites on the CTHRC1 protein antigen. These antibodies can be purchased directly. The wash solution is PBST, the blocking solution is 2% bovine serum albumin, the colorimetric reagent is TMB solution, and the stop solution is H2SO4.

[0040] Furthermore, the preparation method of the CTHRC1 detection antibody labeled with horseradish peroxidase (HRP) specifically comprises the following steps:

[0041] Weigh 6 mg of HRP (horseradish peroxidase, purchased from Sigma) and dissolve it in 1 ml of double-distilled water. Then, slowly add 0.30 ml of freshly prepared 0.1 M NaIO4 solution dropwise. Stir at 4°C in the dark for 35 minutes to activate the HRP, causing the HRP solution to change color from brown to green. Pour the activated HRP solution into a dialysis bag and dialyze overnight at 4°C against 0.01 M sodium acetate buffer, pH 4.4, until the solution changes color to brown-green. Observe for any precipitation and analyze its properties. Centrifuge at 10,000 rpm, 4°C for 10 minutes to remove the precipitate and obtain dialyzed HRP.

[0042] The purified CTHRC1 detection antibody in Example 1 was dialyzed against 0.05 M carbonate buffer, pH 9.5, at 4°C overnight. Observe for the presence of a precipitate and analyze its properties. Centrifuge at 10,000 rpm, 4°C for 10 minutes to remove the precipitate and obtain the dialyzed detection antibody.

[0043] Add the dialyzed HRP to 0.16 M ethylene glycol (0.1 ml ethylene glycol per 1 mg HRP) and stir at 4°C in the dark for 1 hour. Then add the dialyzed detection antibody. Mix the two and dialyze overnight at 4°C against 0.05 M carbonate buffer, pH 9.5, to obtain an HRP-antibody mixture.

[0044] Add 0.1 ml of a 5 mg / ml NaBH4 solution (0.1-0.2 mg NaBH4 per 1 mg HRP) to the HRP-antibody mixture and stir at 4°C in the dark for 3 hours. Add an equal volume of saturated ammonium sulfate to the NaBH4 solution dropwise and stir at 4°C in the dark for 2 hours. Centrifuge at 10,000 rpm and 4°C for 10 minutes, and discard the supernatant. Dissolve the precipitate in PBS to obtain the horseradish peroxidase-labeled CTHRC1 detection antibody.

[0045] The detection method of the kit in this embodiment comprises the following steps:

[0046] CTHRC1 capture antibody (2 μg / ml) was coated with Na2CO3 and NaHCO3 at pH 9.6, and 100 ml was added to each well of a 96-well ELISA plate. The antibody was coated at 4°C for 12 hours to allow it to bind tightly to the ELISA plate, thereby forming a CTHRC1 capture antibody coated on the ELISA plate.

[0047] After coating, the ELISA plate was washed 6 times with washing solution (PBST), and 300 μl of 2% bovine serum albumin was added to each well as blocking solution, and incubated at 37° C. for 3 h.

[0048] After blocking, add the protein extract solution to be tested (the dilution factor is the volume ratio of the test sample to PBS buffer) and CTHRC1 protein standard (5 μg / ml per well, diluted 1:5 to 1.56 ng / ml, with three replicates for each sample) to the ELISA plate wells at 100 ml / well and incubate at 37°C for 1 hour. Then, add CTHRC1 detection antibody labeled with horseradish peroxidase (HRP) at 100 μl / well and incubate at 37°C for 1 hour.

[0049] Finally, TMB solution was added to the formed complex, 100 μl per well, and incubated at room temperature for 3 min. Under the action of horseradish peroxidase, the color of the developer changed. 2M stop solution H2SO4 was added, 50 μl / well to terminate the reaction. 450 The value is used to determine the presence and concentration of CTHRC1 protein in the protein extract solution to be tested.

[0050] The concentration of CTHRC1 protein standard is used as the vertical axis, OD 450 The standard curve is made with the value as the horizontal axis, and the results are shown in the attached Figure 1 As shown, the numerical simulation results show the concentration y and OD of CTHRC1 protein standard. 450 The relationship between the values ​​x is y = 7.9496x 2 -0.6665x, R 2 =0.9909. Therefore, according to the OD of the protein extract solution to be tested 450 The CTHRC1 content in the protein extract solution to be tested can be calculated based on the value.

[0051] Example 2:

[0052] Example 2 provides another method for obtaining CTHRC1 capture antibody and CTHRC1 detection antibody, which specifically includes the following steps:

[0053] Step 1: Prepare and purify the immunogen CTHRC1 protein;

[0054] Specifically,

[0055] (1) Synthesis of CTHRC1 expression plasmid

[0056] The base sequence encoding the recombinant protein of CTHRC1 (31-243aa) with a His tag was fully synthesized (this process was commissioned to Qingke Bio).

[0057] (2) Plasmid transformation

[0058] Thaw the DH5α competent cells on ice and dispense 50 μL aliquots into sterile 1.5 mL centrifuge tubes.

[0059] Gently add 1 μL of the fully synthesized plasmid from step (1) to the competent cells and incubate on ice for 25 minutes. Heat shock at 42°C for 45 seconds and incubate on ice for 2 minutes. Add 400 μL of resistance-free LB culture medium and incubate at 220 rpm at 37°C for 1 hour. Pipette about 10 μL of the recovery solution and evenly spread it on the LB culture medium containing Amp antibiotic until the liquid dries. After sealing, place the plate upside down in a 37°C incubator and culture overnight. Inoculate the bacterial liquid into 100 mL of 100 μg / mL Amp LB culture medium and culture overnight.

[0060] (3) Plasmid extraction

[0061] Collect the bacterial culture and centrifuge at 8,000 rpm for 3 minutes. Discard the culture medium and invert onto absorbent paper to drain the LB medium. Add 10 mL of Buffer P1 / RNase A mixture and vortex at high speed. Add 10 mL of Buffer P2, invert and mix 8-15 times, then incubate for 3-5 minutes. Add 5 mL of S3K, invert and mix 15-20 times, and centrifuge at 8,000 rpm for 10 minutes. Remove the filter piston and pour the entire supernatant from the previous step into the filter. Add 0.1 volume of Buffer ER2 to the filtrate and invert 10-15 times. Add 0.5 volume of anhydrous ethanol to the mixture and invert and mix 8-10 times (to equilibrate the column). Place the HiPure DNA Maxi Column C in a 50 mL collection tube, transfer 2.5 mL of Buffer CL (equilibration solution) to the column, and incubate for 2 minutes. Centrifuge at 8,000 rpm for 3 minutes. Discard the filtrate and place the column back into the collection tube. Repeat the above steps until the mixture is transferred to the column and centrifuged.

[0062] Discard the filtrate and return the column to the collection tube. Add 5 mL of Buffer PW1 to the column and centrifuge at 8,000 rpm for 3 minutes. Discard the filtrate and return the column to the collection tube. Add 9 mL of Buffer PW2 to the column and centrifuge at 8,000 rpm for 10 minutes. Place the column in collection tube C and add 0.7–1.5 mL of Elution Buffer or sterile water to the center of the column membrane. Let stand for 3 minutes. Centrifuge at 8,000 rpm for 3 minutes. Remove the preparative column and measure the plasmid concentration in the eluate using a microspectrophotometer. Send the extracted plasmid to Qingke Bio for sequencing and quality control.

[0063] (4) Immunogen protein expression and purification

[0064] ① Preparation for protein expression

[0065] Cell preparation: Count cells using trypan blue the day before transfection to ensure viability is 98%. Dilute cells to 1.0 × 10⁶ / mL in fresh culture medium on the day of transfection. Add 30 mL of cell suspension to a shake flask.

[0066] Transfection: Prepare a transfection reaction system, where Solution A includes: 1.2 mL Opti-MEM, 42 μg Plasmid; Solution B includes: 1.2 mL Opti-MEM, 120 μL PEI.

[0067] Mix the plasmid with Opti-MEM, PEI, and Opti-MEM, respectively, and incubate at room temperature for 5 minutes. Mix Solution A and Solution B, and incubate at room temperature for 20 minutes. Add the mixed plasmid and PEI to the cells dropwise and mix gently. Incubate at 37°C, 5% CO2 for 5 days, and collect the conditioned medium. Collect the conditioned medium in a 50 mL centrifuge tube and centrifuge at 8000 rpm for 5 minutes to remove the cells. The supernatant is used for antibody affinity purification.

[0068] ②Protein expression and purification

[0069] Prepare buffer solution:

[0070] Equilibration buffer: 20 mM PB, 0.5 M NaCl, pH 7.4.

[0071] Wash buffer: 20 mM PB, 0.5 M NaCl, 10 mM Imidazole, pH 7.4.

[0072] Elution buffer: 20 mM PB, 0.5 M NaCl, 100 mM Imidazole, pH 7.4.

[0073] Purification steps: 3.0 mL filler, loading capacity ~8 mg / mL His-tag protein, flow rate of about 1 mL / min, room temperature column; 15 mL deionized water wash column, wash once; 15 mL equilibration buffer, equilibrate once; load sample, repeat loading once; 15 mL wash buffer wash column; 15 mL elution buffer elute protein; 15 mL deionized water wash column, wash once; 15 mL 0.5 M NaOH wash column; 15 mL deionized water wash column, wash three times; 20% ethanol equilibrate column and store; the qualified eluted sample is dialyzed into 1xPBS, pH 7.4 solution, dialyzed overnight at 4°C; centrifuge at 4500 rpm, 4°C, and ultrafiltration tube is used to concentrate the target protein to an appropriate concentration to obtain the purified immunogen CTHRC1 protein.

[0074] ③Amino acid sequence alignment

[0075] The amino acid sequence of the purified immunogen CTHRC1 protein was queried using the Uniprot database and compared with the amino acid sequences of mouse and human CTHRC1 proteins. The results are shown in Table 1 below. In Table 1, due to the length of the amino acid sequence, the amino acid sequence of the immunogen CTHRC1 protein was separated into three rows for comparison. As can be seen from Table 1, the amino acid sequence of the immunogen CTHRC1 protein has a high degree of homology with the amino acid sequences of mouse and human CTHRC1 proteins.

[0076] Table 1 Comparison of the amino acid sequence of the immunogen CTHRC1 protein with the amino acid sequences of mouse and human CTHRC1 proteins

[0077]

[0078] Step 2: Immunize animals with the immunogen CTHRC1 protein, culture and screen hybridoma cells;

[0079] Immunize 7- to 11-week-old Balb / c female mice with the purified immunogen CTHRC1 protein from Step 1 via multiple subcutaneous and intraperitoneal injections. Immunizations were repeated 21 days after the first immunization, followed by a second immunization, followed by a third immunization, and then a fourth immunization, with 14 days between the second and third immunizations, and finally the fourth immunization. Seven days after the booster immunization (fourth immunization), the titer of polyclonal antibodies against the immunogen in mouse sera was determined by indirect ELISA (wavelength 450 nm).

[0080] Select the mouse with the highest titer for the preparation of splenic artery suspension. Use sterile tweezers to lift the mouse peritoneal membrane, and use another pair of sterile ophthalmic scissors to cut the peritoneal membrane until the entire abdominal cavity is exposed. Find the spleen in the upper right corner of the abdominal cavity, carefully remove it, and place it in a glass dish filled with preheated IMDM culture medium. Change three dishes and wash three times. Use tweezers to make a small hole at one end of the spleen, then use two tweezers to squeeze out the spleen cells, use a 1mL gun to blow the spleen cells away, collect them into a 50mL centrifuge tube, and centrifuge at 1500rpm for 3min. Discard the supernatant, knock out the precipitate, add 30mL IMDM culture medium, and centrifuge at 1500rpm for 3min.

[0081] Meanwhile, dissociate healthy SP2 / 0 cells and centrifuge at 1500 rpm for 3 minutes. Discard the supernatant and resuspend the cells in 30 mL of prewarmed IMDM medium. Centrifuge again at 1500 rpm for 3 minutes. Discard the supernatant and resuspend the cells in 30 mL of prewarmed IMDM medium.

[0082] Prepare the appropriate amount of mouse spleen cells and SP2 / 0 cells at a 10:1 ratio, mix thoroughly in a 50 mL centrifuge tube, and centrifuge at 1500 rpm for 3 minutes. Drain the supernatant using a pump. Slowly add 1 mL of preheated PEG (1500) along the bottom wall of the centrifuge tube within 60 seconds, then incubate in a 37°C water bath for 1 minute. Slowly add 5 mL of preheated IMDM medium dropwise along the tube wall, gradually increasing the speed to add 15 mL of preheated IMDM medium, bringing the total volume to 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. Plate 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 medium.

[0083] After 7 days of growth, the fused hybridoma cells were tested for positive results. Positive cells were picked, and 200 cells were spread onto a 96-well plate for sub-screening. After 8-10 days of growth, the supernatant was collected for testing. After a monoclonal cell line was selected, the culture was expanded, and the cell supernatant was collected to screen the hybridoma cells and freeze them for future use.

[0084] Based on the principle of pairing capture antibodies and detection antibodies, a pair of hybridoma cells that can pair with each other were screened out and labeled as 1A and 2A respectively.

[0085] Step 3: Sequencing the antibody variable regions of the hybridoma cells screened in step 2 to verify that the screened hybridoma cells are monoclonal cell lines;

[0086] Specifically, step 301: amplifying a pair of hybridoma cells screened in step 2;

[0087] Step 302: extracting total RNA from the amplified hybridoma cells using the Rizol method;

[0088] Step 303: reverse transcribing the total RNA into cDNA;

[0089] Take 9 μl of the total RNA obtained in step 302, add 2.5 μl of oligo(dT)12-18 primer (10 mM) and 5 μl of dNTPs, mix thoroughly, and incubate at 70°C for 5 minutes, then at 4°C for 5 minutes. Then, add 5 μl of 5× RT buffer, 2.5 μl of 0.1 M DTT, and 1 μl of reverse transcriptase, and incubate at 42°C for 1 hour. Terminate the reaction by incubating at 70°C for 15 minutes to obtain cDNA.

[0090] Step 304: Using the cDNA as a template, perform PCR amplification and sequencing of the variable region gene;

[0091] Using the cDNA obtained in step 303 above as a template, forward and reverse primers (as shown in Table 2 below) were designed according to the mouse monoclonal antibody primer sequence design rules for PCR amplification. 25 pmol of each primer was added to a 50 μL reaction system. The remaining dNTPs and buffer were added as usual. Finally, 1 μL of the cDNA template and 1 U of hot-start Taq DNA polymerase were added. The PCR amplification program was set to 94°C for 40 seconds, 52°C for 40 seconds, and 72°C for 40 seconds for 20 to 25 cycles, followed by a final extension at 72°C for 3 minutes. The product was stored at 4°C until ready for use or subjected to direct electrophoresis. 20 μL of the PCR product was analyzed by electrophoresis and separated on a 1.5% agarose gel. The resulting heavy and light chain variable regions were cloned into the pMD18T plasmid vector (TaKaRa) for sequencing.

[0092] Table 2 PCR amplification primers

[0093]

[0094] In the present invention, the nucleotide sequence of the light chain variable region of the monoclonal antibody secreted by hybridoma cell line 1A is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2; the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4.

[0095] SEQ ID NO: 1:

[0096] CAACTTATACTCACTCAGTCATCTTCAGCCTCTTTCCCCTGGGAGCCTCAGCAAAACTCACGTGCACCTTGAGTAGTCAGCACAGTACGTACACCATTGAATGGTATCAGCAACAGCCACTCAAGCCTCCTAAGTATGTGATGGAGGTTAAGAAAGATGGAAGCCACAGCA CAGGTGATGGGATTCCTGATCGCTTCTCTGGATCCAGCTCTGGTGCTGATCGCTACCTTAGCATTTCCAACATCCAGCCTGAAGATGAAGCAATATACATCTGTGGTGTGGGTGATACAATTAAGGAACAATTTGTGTATGTTTTCGGCGGTGGAACCAGGGTCACTGTCCTA

[0097] SEQ ID NO: 2:

[0098] QLILTQSSSASFSLGASAKLTCTLSSQHSTYTIEWYQQQPLKPPKYVMEVKKDGHSTGDGIPDRFSGSSSGADRYLSISNIQPEDEAIYICGVGDTIKEQFVYVFGGGTRVTVL

[0099] SEQ ID NO: 3:

[0100] GAGGTGCAGCTTGTTGAGACTGGTGGAGGATTGGTGCAGCCTAAAGGGTCAATGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATATCAATGCCATGAATTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAAAGTAATAATTATGCAA CATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCACAAAGGATGCTCTATCTGCAAATGAACAACTTGAAAACTGAGGACACAGCCAAGTATTACTGTGTGTCTAACTGGGACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA

[0101] SEQ ID NO: 4:

[0102] EVQLVETGGGLVQPKGSMKLSCAASGFTFNINAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQRMLYLQMNNLKTEDTAKYYCVSNWDWYFDVWGAGTTVTVSS

[0103] In the present invention, the nucleotide sequence of the light chain variable region of the monoclonal antibody secreted by hybridoma cell line 2A is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 6; the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO: 7, and the amino acid sequence is shown in SEQ ID NO: 8.

[0104] SEQ ID NO: 5:

[0105] GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCTTTGTGCATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

[0106] SEQ ID NO:6:

[0107] DVLMTQTPLSLPVSLGDQASISCRSSQSFVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGAGTKLELK

[0108] SEQ ID NO:7:

[0109] GAGGTTCAGCTGCAGCAGTCTGGGGCAGAGCTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAACTTCTGGCTTCAACATTAAAGACACCTATATACACTGGGTGAGGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGAATGGTAATACTAAATATGACCCGAAGTTCCAGGGCAAGGCCACTATAACAGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTTACATCTGAGGACACTGCCGTCTATCACTGTGCTAGAGAGGGTAACTACGAGGAGGCTATGGACTATTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

[0110] SEQ ID NO:8:

[0111] EVQLQQSGAELVKPGASVKLSCTTSGFNIKDTYIHWVRQRPEQGLEWIGRIDPANGNTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYHCAREGNYEEAMDYWGQGTSVTVSS

[0112] By extracting RNA from hybridoma cells No. 1A and 2A, reverse transcribing cDNA, and analyzing the sequencing results after PCR amplification, it can be confirmed that the hybridoma cells screened in step 2 are indeed monoclonal cell lines, and their variable region sequences can specifically recognize different epitopes of the CTHRC1 antigen, thereby being used for pairing antibody combinations and providing a reliable basis for the preparation of capture antibodies and detection antibodies.

[0113] Step 4: Use the hybridoma cells from step 2 to produce and purify CTHRC1 antibodies.

[0114] Antibodies were prepared using the hybridoma cells numbered 1A and 2A obtained in step 2, to obtain CTHRC1 capture antibodies and CTHRC1 detection antibodies, respectively. The specific steps are as follows:

[0115] Ascites Preparation: Inject 0.5 mL of liquid paraffin into the peritoneal cavity of mice to sensitize them. One week later, inject hybridoma cells into the peritoneal cavity of the mice. After 7-10 days, ascites will form. Insert a drainage needle into the mouse's peritoneal cavity and carefully rotate the needle to allow the ascites to drain slowly. After collecting the ascites, centrifuge at 10,000 rpm for 5 minutes, collect the supernatant, determine the titer, and purify the supernatant.

[0116] Purification of CTHRC1 Antibody: Centrifuge the collected ascites at 4000 rpm for 10 minutes at 4°C. Carefully aspirate the ascites fluid and collect it in a centrifuge tube. Store at 4°C or -20°C. Purify CTHRC1 antibody from the ascites using HiTraprProtein A FF (GE) affinity chromatography according to the manufacturer's instructions to obtain CTHRC1 capture antibody and CTHRC1 detection antibody, respectively. Assay the purity of the CTHRC1 antibody using SDS-PAGE gels, and determine the concentration of the CTHRC1 antibody using the Bradford assay. Store the purified CTHRC1 antibody at -20°C until use.

[0117] Example 3:

[0118] Example 3 The kit and detection method in Example 2 were used to detect the level of CTHRC1 in the serum of mice with acute aortic dissection.

[0119] Blood was collected from mice with acute aortic dissection (Ang-II / BAPN) and mice in the control group (PBS). 1 ml of blood was collected from the mouse's orbital cavity and placed into a blood collection tube. The tube was allowed to stand for 30 minutes and then centrifuged at 3000 rpm for 15 minutes. The upper layer of plasma was collected and placed into an EP tube.

[0120] Add 50 μl / well of the sample to be tested to the antibody-coated ELISA plate in the kit. Incubate at 37°C for 30 minutes. Add 180 μl / well of 1X PBST to the plate and wash twice. Dilute GAM-HRP to a working concentration of 1:5000 in 1% BSA and add 50 μl / well of PBS to the plate. Incubate at 37°C for 30 minutes. Discard the liquid from the wells again and add 180 μl / well of PBS to the plate and wash three times. Add 100 μl of freshly prepared TMB chromogenic substrate to each reaction well and incubate at 37°C for 5 minutes. Then, add 90 μl / well of stop solution to terminate the reaction and measure the OD value at 450 nm on a microplate reader.

[0121] The results of the detection of CTHRC1 content in the serum of mice in the acute aortic dissection model group are shown in the attached figure. Figure 2 As shown in the attached ROC curve Figure 3 As shown. Figure 2 and attached Figure 3 It can be seen that the CTHRC1 content in the serum of the mouse model of acute aortic dissection was significantly increased. At the same time, combined with ROC curve analysis, it showed that CTHRC1 can be used as a specific serum marker for the diagnosis of acute aortic dissection.

[0122] Example 4:

[0123] Example 4 The reagent kit and detection method in Example 2 were used to detect the CTHRC1 content in plasma samples from patients with acute aortic dissection, patients with acute myocardial infarction and healthy people.

[0124] Peripheral venous blood was collected from healthy controls, patients with acute aortic dissection, and patients with acute myocardial infarction. 5 ml of blood was collected using an EDTA anticoagulant tube and centrifuged at 3000 rpm for 15 minutes. The upper plasma layer was transferred to an EP tube.

[0125] Add 50 μl / well of the sample to be tested to the antibody-coated ELISA plate in the kit. Incubate at 37°C for 30 minutes. Add 180 μl / well of 1X PBST to the plate and wash twice. Dilute GAM-HRP to a working concentration of 1:5000 in 1% BSA and add 50 μl / well of PBS to the plate. Incubate at 37°C for 30 minutes. Discard the liquid from the wells again and add 180 μl / well of PBS to the plate and wash three times. Add 100 μl of freshly prepared TMB chromogenic substrate to each reaction well and incubate at 37°C for 5 minutes. Then, add 90 μl / well of stop solution to terminate the reaction and measure the OD value at 450 nm on a microplate reader.

[0126] The results of plasma CTHRC1 detection in healthy controls (CTRL) and patients with acute aortic dissection (AAD) are shown in the attached figure. Figure 4 The results of plasma CTHRC1 detection in patients with acute myocardial infarction (AMI) and acute aortic dissection (AAD) are shown in the attached figure. Figure 5 As shown. Figure 4 and attached Figure 5 As can be seen, the mean plasma CTHRC1 level in patients with acute aortic dissection was 42.5 ng / ml; the mean plasma CTHRC1 level in healthy controls was 4.95 ng / ml; and the mean plasma CTHRC1 level in patients with acute myocardial infarction was 12.96 ng / ml. Compared with the mean serum CTHRC1 levels (ng / ml) in healthy controls and patients with acute myocardial infarction, CTHRC1 levels (ng / ml) in patients with acute aortic dissection were significantly increased. This suggests that CTHRC1 could be a candidate biomarker for the diagnosis of acute aortic dissection (positive or negative), monitoring of disease course and progression, and recurrence risk.

[0127] In summary, the occurrence and development of acute aortic dissection is closely correlated with the plasma CTHRC1 content, which makes CTHRC1 a biomarker candidate for the diagnosis of acute aortic dissection (positive or negative diagnosis), monitoring of disease course and progression, and recurrence risk.

[0128] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A double-antibody sandwich ELISA kit for detecting CTHRC1 content in blood, characterized by: The kit includes a CTHRC1 capture antibody coated on an ELISA plate, a CTHRC1 detection antibody labeled with horseradish peroxidase, a CTHRC1 protein standard, a washing solution, a blocking solution, a color developer, and a stop solution; Among them, the CTHRC1 capture antibody and the CTHRC1 detection antibody are a group of paired antibodies that can recognize different sites of the CTHRC1 protein antigen.

2. The double-antibody sandwich ELISA kit for detecting the CTHRC1 content in blood according to claim 1, characterized in that: The preparation method of the CTHRC1 detection antibody labeled with horseradish peroxidase comprises the following steps: Activate and dialyze horseradish peroxidase to obtain dialyzed HRP; dialyzing the purified CTHRC1 detection antibody to obtain a dialyzed detection antibody; The dialyzed HRP, ethylene glycol, and the dialyzed detection antibody are mixed evenly and then dialyzed to obtain an HRP-antibody mixture; NaBH4 solution and saturated ammonium sulfate were added to the HRP-antibody mixture, stirred evenly, and then centrifuged; the supernatant was discarded, and the precipitate was dissolved in PBS to obtain the CTHRC1 detection antibody labeled with horseradish peroxidase.

3. The double-antibody sandwich ELISA kit for detecting the CTHRC1 content in blood according to claim 1, characterized in that: The washing solution is PBST. 4 . The double-antibody sandwich ELISA kit for detecting the CTHRC1 content in blood according to claim 1 , wherein the blocking solution is 2% bovine serum albumin. The double-antibody sandwich ELISA kit for detecting the CTHRC1 content in blood according to claim 1 , wherein the color developing agent is TMB solution. The double-antibody sandwich ELISA kit for detecting the CTHRC1 content in blood according to claim 1 , wherein the stop solution is H 2 SO 4 .

7. The detection method of the double antibody sandwich ELISA kit according to any one of claims 1 to 6, characterized in that: The following steps are involved: The CTHRC1 capture antibody was coated with Na2CO3 and NaHCO3 at pH 9.6, added to a multi-well ELISA plate, and tightly bound to the ELISA plate to form a CTHRC1 capture antibody coated on the ELISA plate; Wash the ELISA plate with washing solution, then add blocking solution to each well and incubate at 37°C for 3 h; After blocking, the protein extract solution to be tested and the CTHRC1 protein standard were added to the wells of the ELISA plate and incubated at 37°C for 1 hour. Then, the CTHRC1 detection antibody labeled with horseradish peroxidase was added and incubated for another 1 hour. Finally, a color developer was added to the formed complex and incubated at room temperature for 3 minutes until the color of the developer changed. The reaction was terminated by adding a stop solution. 450 The value is used to determine the presence and concentration of CTHRC1 protein in the protein extract solution to be tested.

8. Use of the double antibody sandwich ELISA kit according to any one of claims 1 to 6 in the preparation of a product for diagnosing / treating acute aortic dissection.

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