Double antibody sandwich elisa kit for detecting cthrc1 content in blood and its detection method and application

Abstract

The application discloses a double-antibody sandwich ELISA kit for detecting the content of CTHRC1 in blood and a detection method and application thereof, and belongs to the field of molecular biological techniques. In view of the problem that it is difficult to identify early acute aortic dissection at present, a pair of capture antibodies and detection antibodies capable of recognizing different epitopes of CTHRC1 protein antigens are used in the application to assemble a double-antibody sandwich ELISA kit for detecting the content of CTHRC1 in blood. The double-antibody sandwich ELISA kit has high specificity and sensitivity for CTHRC1 protein in serum, can quantitatively detect the CTHRC1 protein in serum, and thus can accurately identify early acute aortic dissection, thereby avoiding misdiagnosis.

Description

TECHNICAL FIELD

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

[0002] Acute aortic dissection (AAD) is an extremely critical cardiovascular disease, with an initial visit mortality rate of about 40%, and a subsequent mortality rate rising at a rate of 1% per hour. Epidemiological studies have shown that the incidence of AAD is about 3-5 per 100,000 cases per year, and is showing an increasing trend year by year. Its pathological manifestations are the separation of the layers of the aortic wall, the tearing of the intimal layer, and the progression of the dissection (proximal or retrograde), with blood entering the intima and media through the intimal tear, which can cause aortic rupture in a short period of time and cause patient death. However, due to its diverse clinical manifestations, the disease is often confused with myocardial infarction, pulmonary embolism and other diseases, making diagnosis more difficult.

[0003] On the one hand, the typical symptoms of acute aortic dissection are sudden and severe chest and back tearing pain, but some patients may only show abdominal pain, syncope, limb ischemia or nervous system symptoms (such as hemiplegia), which are easily confused with acute myocardial infarction, pulmonary embolism, acute abdomen or stroke. In addition, about 10%-15% of patients have asymmetric or disappearing limb pulses due to the compression of the true lumen by the false lumen caused by intimal tear, but this sign may not be obvious in the early stage, especially in obese or hypotensive patients, and is more likely to be ignored.

[0004] On the other hand, the diagnosis of acute aortic dissection disease requires CT angiography (CTA), transesophageal echocardiography (TEE) or magnetic resonance imaging (MRI), but it is worth noting that CTA, although highly sensitive and specific, is the preferred examination method in current emergency, but it relies on iodinated contrast agents and ionizing radiation, limiting its application in patients with renal dysfunction and contrast agent allergic populations. MRI has unique advantages due to its lack of radiation and excellent image quality, but it takes a long time to examine and requires high patient cooperation, which is not suitable for critically ill or unstable patients. TEE can be completed at the bedside and is suitable for patients who cannot tolerate transportation, but this technique is highly dependent on the experience of the operator, and there are certain limitations in the display of the distal ascending aorta and aortic arch. In addition, the clinical manifestations of aortic dissection are non-specific, and some patients may even have no obvious symptoms, making it more difficult to identify early.

[0005] According to clinical retrospective studies, the misdiagnosis rate of aortic dissection at the initial diagnosis can reach 30%-50%. Especially when the coronary artery is not involved or the aortic valve regurgitation does not occur, the electrocardiogram may have no specific changes, and it is easy to be misjudged as other cardiovascular diseases. The delay in diagnosis directly leads to an increase in mortality (1%-2% per hour), further exacerbating the pressure of clinical decision-making. Therefore, it is urgent to improve the diagnosis rate of acute aortic dissection, and the development of disease plasma marker detection kit may provide a new direction for early identification.

[0006] Collagen Triple Helix Repeat Containing 1 (CTHRC1) was first discovered in the differential expression analysis of normal rat arteries and globular injury arteries. It is a secreted protein molecule containing an N-terminal signal peptide, a 36-amino acid collagen triple helix, and a C-terminal globular domain. The expression of CTHRC1 is affected by the vascular regulatory factors TGF-β (Transforming growth factor-β) and BMP4 (Bone morphogenetic protein-4). In colorectal cancer cells, CTHRC1 can induce the expression of MMP2 and MMP9 in the Matrix metalloproteinases (MMPs) family by activating the ERK signal, enhancing the invasion ability of tumor cells. The plasma level of CTHRC1 in rheumatoid arthritis patients is significantly higher than that in healthy controls. However, the effect of CTHRC1 in aortic dissection has not been clearly defined. SUMMARY

[0007] In view of the above problems, the present application aims to provide a double antibody sandwich ELISA kit for detecting the content of CTHRC1 in blood, a detection method and application thereof. A pair of antibodies that can recognize different epitopes of CTHRC1 protein antigens are used to assemble a double antibody sandwich ELISA kit, which realizes quantitative detection of CTHRC1 protein. The double antibody sandwich ELISA kit has high specificity and sensitivity for the diagnosis of acute aortic dissection marker CTHRC1 protein, is simple to operate, and can be used for the detection of acute aortic dissection disease in scientific research and clinical practice, playing a role in auxiliary diagnosis, treatment guidance, and prognosis judgment.

[0008] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0009] The application provides a double antibody sandwich ELISA kit for detecting the content of CTHRC1 in blood, which comprises a CTHRC1 capture antibody coated on an enzyme-labeled plate, a CTHRC1 detection antibody labeled with horseradish peroxidase, a CTHRC1 protein standard, a washing solution, a blocking solution, a color developing agent and a termination solution.

[0010] The CTHRC1 capture antibody and the CTHRC1 detection antibody are a set of antibodies matched with each other and capable of recognizing different sites of a CTHRC1 protein antigen.

[0011] Based on the above scheme, the application uses a recombinant human CTHRC1 protein as an immunogen, screens to obtain a CTHRC1 capture antibody and a CTHRC1 detection antibody, and uses the pair of antibodies capable of recognizing different epitopes of a CTHRC1 protein antigen to assemble a double antibody sandwich ELISA kit, so that quantitative detection of a CTHRC1 protein in serum can be realized.

[0012] Preferably, the preparation method of the CTHRC1 detection antibody labeled with horseradish peroxidase comprises the following steps:

[0013] The horseradish peroxidase is activated and subjected to dialysis treatment to obtain dialyzed HRP;

[0014] The purified CTHRC1 detection antibody is subjected to dialysis treatment to obtain dialyzed detection antibody;

[0015] The dialyzed HRP, ethylene glycol and the dialyzed detection antibody are uniformly mixed and subjected to dialysis treatment to obtain an HRP-antibody mixed solution;

[0016] NaBH4 solution and saturated ammonium sulfate are added to the HRP-antibody mixed solution, and the mixture is uniformly stirred and subjected to centrifugal treatment; the supernatant is discarded, and the precipitate is 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 color developing agent is a TMB solution.

[0020] Preferably, the termination solution is H2SO4.

[0021] In another aspect, the application further provides a detection method of the double antibody sandwich ELISA kit as described above, which comprises the following steps:

[0022] The CTHRC1 capture antibody is coated with Na2CO3 and NaHCO3 with pH 9.6, added to a porous enzyme-labeled plate and tightly combined with the enzyme-labeled plate to form the CTHRC1 capture antibody coated on the enzyme-labeled plate;

[0023] The enzyme-labeled plate is washed with a washing solution, and then the blocking solution is added to each hole, and incubated at 37 DEG C for 3h;

[0024] After the blocking is completed, the protein extract solution to be detected and the CTHRC1 protein standard are added to the holes of the enzyme-labeled plate, and incubated at 37 DEG C for 1h; then the horseradish peroxidase-labeled CTHRC1 detection antibody is added, and incubated for 1h;

[0025] Finally, the color developing agent is added to the formed complex, and incubated at room temperature for 3min, and the color developing agent changes color; the reaction is terminated by adding a termination solution, and the presence and concentration of the CTHRC1 protein in the protein extract solution to be detected are determined according to the OD 450 value.

[0026] On the other hand, the application also provides the use of the double antibody sandwich ELISA kit as described above in the preparation of products for the diagnosis / treatment of acute aortic dissection.

[0027] Based on the above scheme, the double antibody sandwich ELISA kit in the application can detect the content of the CTHRC1 protein in the serum, and the content of the CTHRC1 protein in the patient with acute aortic dissection is significantly increased compared with the healthy control population and the patient with acute myocardial infarction, so that the patient with acute aortic dissection can be quickly and accurately diagnosed based on the detection result of the content of the CTHRC1 protein.

[0028] The application has the following beneficial effects:

[0029] The application uses a pair of capture antibodies and detection antibodies that can recognize different epitopes of the CTHRC1 protein antigen to assemble a double antibody sandwich ELISA kit, so that the quantitative detection of the CTHRC1 protein is realized. The test proves that the ELISA kit of the application has high specificity and sensitivity for the CTHRC1 protein which is a diagnostic marker for mouse and human acute aortic dissection, and the operation is simple, and can be used for the detection of acute aortic dissection disease in scientific research and clinical practice, so as to play a role in auxiliary diagnosis, treatment guidance and prognosis judgment, and optimize the current diagnosis strategy of acute aortic dissection disease, and further improve the early recognition rate and diagnosis efficiency of the disease for clinical medical workers. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The CTHRC1 standard curve of the double antibody sandwich ELISA kit in the application.

[0031] Figure 2The content of CTHRC1 in serum of mice in the acute aortic dissection model group in Example Three of the present application was detected.

[0032] Figure 3 The ROC curve of the content of CTHRC1 in serum of mice in the acute aortic dissection model group in Example Three of the present application was obtained.

[0033] Figure 4 The content of CTHRC1 in plasma of healthy control population and acute aortic dissection patients in Example Four of the present application was detected.

[0034] Figure 5 The content of CTHRC1 in plasma of acute myocardial infarction patients and acute aortic dissection patients in Example Four of the present application was detected. DETAILED DESCRIPTION

[0035] In order to enable a person of ordinary skill in the art to better understand the technical solutions of the present application, the technical solutions of the present application are further described below in combination with the accompanying drawings and examples.

[0036] Example One

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

[0038] The kit comprises: CTHRC1 capture antibody coated on an enzyme-labeled plate, CTHRC1 detection antibody labeled with horseradish peroxidase (HRP), CTHRC1 protein standard, washing solution, blocking solution, color developing agent and termination solution.

[0039] It should be noted that the CTHRC1 capture antibody and the CTHRC1 detection antibody are a set of antibodies that can recognize different sites of the CTHRC1 protein antigen and are matched with each other. The capture antibody and the detection antibody can be obtained by direct purchase. The washing solution is PBST, the blocking solution is 2% bovine serum albumin, the color developing agent is TMB solution, and the termination solution is H2SO4.

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

[0041] HRP (horseradish peroxidase, purchased from Sigma) was weighed at 6 mg and dissolved in 1 ml of double distilled water, then 0.30 ml of freshly prepared 0.1 M NaIO4 solution was slowly added drop by drop, and the HRP was activated by stirring at 4°C in the dark for 35 min. The color of the HRP solution changed from brown to green. The activated HRP solution was placed in a dialysis bag and dialyzed against 0.01 M sodium acetate buffer, pH 4.4, at 4°C overnight. The color of the solution changed to brown-green. The presence of precipitate was observed, and the precipitate was analyzed. The precipitate was removed by centrifugation at 10,000 r / min at 4°C for 10 min, and the dialyzed HRP was obtained.

[0042] The purified CTHRC1 detection antibody in Example 1 was dialyzed against 0.05 M carbonate buffer, pH 9.5, at 4°C overnight. The presence of precipitate was observed, and the precipitate was analyzed. The precipitate was removed by centrifugation at 10,000 r / min at 4°C for 10 min, and the dialyzed detection antibody was obtained.

[0043] The dialyzed HRP was added to 0.16 M ethylene glycol (0.1 ml of ethylene glycol per 1 mg of HRP), and stirred at 4°C in the dark for 1 h, then the dialyzed detection antibody was added. The mixture was dialyzed against 0.05 M carbonate buffer, pH 9.5, at 4°C overnight to obtain a HRP-antibody mixture.

[0044] To the HRP-antibody mixture, 0.1 ml of 5 mg / ml NaBH4 solution (0.1-0.2 mg of NaBH4 per 1 mg of HRP) was added, and stirred at 4°C in the dark for 3 h. An equal volume of saturated ammonium sulfate solution was added drop by drop, and stirred at 4°C in the dark for 2 h. The supernatant was discarded by centrifugation at 10,000 r / min at 4°C for 10 min. The precipitate was dissolved in PBS to obtain the CTHRC1 detection antibody labeled with horseradish peroxidase.

[0045] The detection method of the kit in this example includes the following steps:

[0046] CTHRC1 capture antibody (2 μg / ml) was coated with Na2CO3, NaHCO3, pH 9.6, and 100 ml was added to each well of a 96-well enzyme-labeled plate, and coated at 4°C for 12 h to form a CTHRC1 capture antibody coated on the enzyme-labeled plate.

[0047] After coating, the enzyme-labeled 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 the end of the blocking, the protein extract solution to be tested (dilution ratio of the sample to be tested to PBS buffer) and the CTHRC1 protein standard (5 μg / ml per well, diluted at a gradient of 1:5 to 1.56 ng / ml, three replicates for each sample) were added to the wells of the enzyme-labeled plate, 100 ml / well, and incubated at 37°C for 1 h. Then, the CTHRC1 detection antibody labeled with horseradish peroxidase (HRP) was added, 100 μl / well, and incubated at 37°C for 1 h.

[0049] Finally, the color developing agent 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 developing agent changed color; 2M stop solution H2SO4 was added, 50 μl / well, to stop the reaction. According to the OD 450 value, the presence and concentration of CTHRC1 protein in the protein extract solution to be tested were determined.

[0050] The concentration of the CTHRC1 protein standard was used as the vertical coordinate, and the OD 450 value was used as the horizontal coordinate to make a standard curve. The results are shown in the accompanying Figure 1 figure. The relationship between the concentration y of the CTHRC1 protein standard and the OD 450 value x was simulated as y = 7.9496x 2 - 0.6665x, R 2 = 0.9909. Therefore, the content of CTHRC1 in the protein extract solution to be tested can be calculated according to the detected OD 450 value.

[0051] Example 2:

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

[0053] Step 1: Preparation and purification of immunogen CTHRC1 protein;

[0054] Specifically,

[0055] (1) CTHRC1 expression plasmid synthesis

[0056] The base sequence of the recombinant protein encoding CTHRC1 (31-243 aa) with His tag was synthesized (this process was entrusted to GenScript).

[0057] (2) Plasmid transformation

[0058] The DH5α competent cells were thawed on ice and divided into 50 μL portions in sterile 1.5 mL centrifuge tubes.

[0059] Add 1 μL of the fully gene-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 then incubate on ice for 2 minutes. Add 400 μL of LB medium without antibiotics, and incubate at 220 rpm and 37°C for 1 hour. Take about 10 μL of the recovered liquid, and evenly spread it on LB medium containing Amp antibiotic until the liquid is dry. After sealing, place the plate upside down in a 37°C incubator, and incubate overnight. Inoculate the bacterial liquid into 100 mL of 100 μg / mL Amp LB medium, and incubate overnight.

[0060] (3) Plasmid extraction

[0061] Collect the bacterial liquid, centrifuge at 8000 rpm for 3 minutes, discard the culture medium, and invert on an absorbent paper to dry 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 for 8-15 times, and then incubate for 3-5 minutes. Add 5 mL of S3K, invert and mix for 15-20 times, and centrifuge at 8000 rpm for 10 minutes. Remove the plunger of the filter, and pour the supernatant from the previous step into the filter. Add 0.1 times the volume of Buffer ER2 to the filtrate, and invert for 10-15 times. Add 0.5 times the volume of anhydrous ethanol to the mixture, and invert and mix for 8-10 times (equilibrate the column). Place the HiPure DNA Maxi Column C in a 50 mL collection tube, transfer 2.5 mL of Buffer CL (equilibrium liquid) to the column, and stand for 2 minutes. Centrifuge at 8000 rpm for 3 minutes. Discard the filtrate, and place the column back in the collection tube. Repeat the above steps until all the mixture is transferred to the column and centrifuged.

[0062] Discard the filtrate, and place the column back in the collection tube. Add 5 mL of Buffer PW1 to the column, and centrifuge at 8000 rpm for 3 minutes. Discard the filtrate, and place the column back in the collection tube. Add 9 mL of Buffer PW2 to the column, and centrifuge at 8000 rpm for 10 minutes. Place the column in a collection tube C, add 0.7-1.5 mL of Elution Buffer or sterile water to the center of the column membrane, stand for 3 minutes, centrifuge at 8000 rpm for 3 minutes, remove the preparation column, and measure the concentration of the plasmid in the eluate using a microspectrophotometer. Send the extracted plasmid to GenScript for sequencing for quality control.

[0063] (4) Expression and purification of immunogenic proteins

[0064] ① Preparation for protein expression

[0065] Cell preparation: One day before transfection, count the cell viability using trypan blue, and the viability is 98%. On the day of transfection, dilute the cells to 1.0 x 106 / mL with fresh culture medium. Add 30 mL of cell suspension to a shake flask.

[0066] Transfection: Transfection reaction system was prepared, in which A solution included: 1.2 mL Opti-MEM, 42 μg Plasmid; B solution included 1.2 mL Opti-MEM, 120 μL PEI.

[0067] After mixing plasmid and Opti-MEM, PEI and Opti-MEM respectively, incubate at room temperature for 5 minutes. After mixing A solution and B solution, incubate at room temperature for 20 minutes. Add mixed plasmid and PEI drop by drop into cells, mix gently and uniformly. Incubate at 37°C, 5% CO2 for 5 days, collect the conditioned medium. Collect the conditioned medium in a 50 mL centrifuge tube, remove the cells after centrifugation at 8000 rpm for 5 min. The supernatant is used for antibody affinity purification.

[0068] ②Protein expression and purification

[0069] Prepare buffer: in which,

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

[0071] Washing 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, load ~ 8 mg / mL His-tag protein, flow rate about 1 mL / min, room temperature column; 15 mL deionized water wash column, wash once; 15 mL equilibrium buffer, equilibrium once; sample, repeat sample once; 15 mL washing buffer to wash the column; 15 mL elution buffer to 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 equilibration column and save; dialysis of qualified elution sample to 1xPBS, pH 7.4 solution, 4°C dialysis overnight; centrifuge at 4500 rpm, 4°C, concentrate the target protein to the appropriate concentration with ultrafiltration tube, get the purified immunogen CTHRC1 protein.

[0074] ③Amino acid sequence alignment

[0075] The amino acid sequence information of the purified immunogen CTHRC1 protein was queried by using the Uniprot database, and was compared with the amino acid sequence of mouse and human CTHRC1 protein. The results are shown in Table 1. In Table 1, due to the large length of the amino acid sequence, the amino acid sequence of the immunogen CTHRC1 protein is compared with the amino acid sequence of mouse and human CTHRC1 protein in three rows. As can be seen from Table 1, the amino acid sequence of the immunogen CTHRC1 protein has high homology with the amino acid sequence of mouse and human CTHRC1 protein.

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

[0077]

[0078] Step 2: Using the immunogen CTHRC1 protein to immunize animals, culture and screen hybridoma cells;

[0079] The 7- to 11-week-old Balb / c female mice were immunized with the purified immunogen CTHRC1 protein in step 1, and each mouse was injected subcutaneously and intraperitoneally. The immunization cycle was as follows: the second immunization was performed at 21 days after the first immunization, the second immunization to the third immunization, and the third immunization to the fourth immunization interval was 14 days. The mouse was taken 7 days after the booster immunization (fourth immunization), and the antiserum titer of the mouse serum against the immunogen was detected by indirect ELISA method (wavelength 450 nm).

[0080] The mouse with the highest titer was selected for spleen artery suspension preparation. The mouse abdominal membrane was lifted with a sterile small forceps, and the abdominal membrane was cut with another sterile ophthalmic scissors until the entire abdominal cavity was exposed. At the right upper part of the abdominal cavity, the spleen was found, which was carefully taken out and placed in a glass dish containing preheated IMDM culture solution, and three dishes were changed for washing three times. A small hole was first made at one end of the spleen with a forceps, and then the spleen cells were squeezed out with two forceps. The spleen cells were blown off with a 1 mL syringe and collected into a 50 mL centrifuge tube, and centrifuged at 1500 rpm for 3 min. The supernatant was discarded, and the precipitate was knocked off and added with 30 mL of IMDM culture solution, and centrifuged at 1500 rpm for 3 min.

[0081] Meanwhile, the SP2 / 0 cells in good growth state were blown down, and centrifuged at 1500 rpm for 3 min. The supernatant was discarded, and the cells were resuspended with 30 mL of preheated IMDM culture solution. Centrifuged at 1500 rpm for 3 min again. Discard the supernatant, resuspend the cells with 30 mL of preheated IMDM culture solution.

[0082] According to the ratio of mouse spleen cells and SP2 / 0 cells 10:1, the corresponding cell amount is taken, mixed uniformly in the imported 50 mL centrifuge tube, centrifuged at 1500 rpm for 3 min. The supernatant is pumped dry. Along the tube wall at the bottom of the centrifuge tube, 1 mL of preheated PEG (1500) is slowly added, which needs to be completed within 60 s, and then 37°C water bath is placed for 1 min. 5 mL of preheated IMDM culture solution is slowly added along the tube wall, and then the speed is gradually increased, 15 mL of preheated IMDM culture solution is added, and finally it is added to 40 mL. The cell suspension after the above fusion is centrifuged at 1200 rpm for 5 min. Discard the supernatant, add 25 mL of preheated SP2 / 0 myeloma cell culture solution, and resuspend the cells with a 10 mL pipette. The hybridoma cell suspension after fusion is 100 μL / well to 96-well cell culture plate. After 4-6 h, 2×HAT culture solution is added, 100 μL / well.

[0083] After the hybridoma cells after fusion grow for 7 days, positive detection is carried out, positive cells are picked, 200 cells of the positive cells are blown uniformly and placed in a 96-well plate for sub-screening. After the sub-plate cells grow for 8-10 days, the supernatant is detected, and a single clone cell strain is selected. After the single clone cell strain is expanded, the cell supernatant is finally collected, the hybridoma cells are screened, and the hybridoma cells are stored for standby use.

[0084] According to the principle of mutual pairing of capture antibodies and detection antibodies, a pair of hybridoma cells that can be paired with each other are screened, which are respectively marked as 1A and 2A.

[0085] Step 3: The hybridoma cells screened in step 2 are subjected to antibody variable region sequence determination to verify that the hybridoma cells screened are single clone cell strains.

[0086] Specifically, step 301: The pair of hybridoma cells screened in step 2 are expanded;

[0087] Step 302: The total RNA of the expanded hybridoma cells is extracted by the Rizol method;

[0088] Step 303: The total RNA is reversely transcribed 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 uniformly, 70°C for 5 min, and then 4°C for 5 min. Then add 5 μl of RT buffer (5×), 2.5 μl of DTT (0.1 M) and 1 μl of reverse transcriptase, 42°C for 1 hour. Incubate at 70°C for 15 min to terminate the reaction, and obtain cDNA.

[0090] Step 304: The cDNA is used as a template to perform variable region gene PCR amplification and sequencing;

[0091] The cDNA obtained in step 303 is used as a template, and forward and reverse primers (as shown in Table 2 below) are designed according to the design rules of the mouse monoclonal antibody primer sequence to perform PCR amplification. In a 50 L reaction system, 25 pmol of each primer is added. The remaining dNTPs and buffers are added according to the conventional method, and finally 1 μl of cDNA template and 1 U of hot-start Taq DNA polymerase are added. The PCR amplification program is set as 94°C for 40 seconds, 52°C for 40 seconds, and 72°C for 40 seconds, for 20 to 25 cycles, and finally 72°C for 3 minutes. The product can be placed at 4°C for standby or directly electrophoresed. 20 μl of the PCR product is electrophoresed and analyzed, separated on a 1.5% agarose gel, and the gel is recovered. The obtained heavy chain variable region and light chain variable region are respectively cloned into a pMD18T plasmid vector (TaKaRa) for sequencing.

[0092] Table 2 PCR amplification primers

[0093]

[0094] In the present application, the nucleotide sequence of the light chain variable region of the monoclonal antibody secreted by the hybridoma cell strain 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] CAACTTATACTCACTCAGTCATCTTCAGCCTCTTTCTCCCTGGGAGCCTCAGCAAAACTCACGTGCACCTTGAGTAGTCAGCACAGTACGTACACCATTGAATGGTATCAGCAACAGCCACTCAAGCCTCCTAAGTATGTGATGGAGGTTAAGAAAGATGGAAGCCACAGCACAGGTGATGGGATTCCTGATCGCTTCTCTGGATCCAGCTCTGGTGCTGATCGCTACCTTAGCATTTCCAACATCCAGCCTGAAGATGAAGCAATATACATCTGTGGTGTGGGTGATACAATTAAGGAACAATTTGTGTATGTTTTCGGCGGTGGAACCAGGGTCACTGTCCTA

[0097] SEQ ID NO: 2:

[0098] QLILTQSSSASFSLGASAKLTCTLSSQHSTYTIEWYQQQPLKPPKYVMEVKKDGSHSTGDGIPDRFSGSSSGADRYLSISNIQPEDEAIYICGVGDTIKEQFVYVFGGGTRVTVL

[0099] SEQ ID NO: 3:

[0100] GAGGTGCAGCTTGTTGAGACTGGTGGAGGATTGGTGCAGCCTAAAGGGTCAATGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATATCAATGCCATGAATTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAAAGTAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCACAAAGGATGCTCTATCTGCAAATGAACAACTTGAAAACTGAGGACACAGCCAAGTATTACTGTGTGTCTAACTGGGACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA

[0101] SEQ ID NO: 4:

[0102] EVQLVETGGGLVQPKGSMKLSCAASGFTFNINAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQRMLYLQMNNLKTEDTAKYYCVSNWDWYFDVWGAGTTVTVSS

[0103] In the present application, the light chain variable region nucleotide sequence of the monoclonal antibody secreted by the hybridoma cell strain 2A is shown as SEQ ID NO: 5, and the amino acid sequence is shown as SEQ ID NO: 6; the heavy chain variable region nucleotide sequence is shown as SEQ ID NO: 7, and the amino acid sequence is shown as 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 the RNA of hybridoma cells numbered 1A and 2A, reverse transcribing the cDNA, and analyzing the sequencing results after PCR amplification, it can be proved that the hybridoma cells screened in step 2 are indeed monoclonal cell strains, and the variable region sequences thereof can specifically recognize different epitopes of CTHRC1 antigen, thereby being used for the combination of paired antibodies and providing reliable basis for the preparation of capture antibodies and detection antibodies.

[0113] Step 4: CTHRC1 antibodies are prepared and purified using the hybridoma cells in step 2.

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

[0115] Ascites preparation: 0.5 mL liquid paraffin is injected into the abdominal cavity of a mouse for ascites preparation to sensitize the mouse. After one week, the hybridoma cells are injected into the abdominal cavity of the mouse, and the ascites of the mouse is formed after 7-10 days. A drainage needle is inserted into the abdominal cavity of the mouse, and the needle is carefully rotated to make the ascites slowly flow out. After the ascites is collected, it is centrifuged at 10,000 rpm for 5 min, and the supernatant layer is collected. The titer is determined and purified.

[0116] Purification of CTHRC1 antibodies: The collected ascites is centrifuged at 4°C and 4,000 rpm for 10 min. The middle ascites is carefully sucked out and collected in a centrifuge tube, which is stored at 4°C or -20°C. The CTHRC1 antibodies are purified from the ascites by HiTrapr Protein A FF (GE Company) affinity chromatography according to the instructions, and CTHRC1 capture antibodies and CTHRC1 detection antibodies are obtained, respectively. The purity of the CTHRC1 antibodies is identified by SDS-PAGE gel, and the concentration of the CTHRC1 antibodies is determined by the Bradford method. The purified CTHRC1 antibodies are stored at -20°C for standby.

[0117] Example Three:

[0118] In Example Three, the kit and detection method in Example Two are used to detect the content of CTHRC1 in the serum of mice with acute aortic dissection.

[0119] Collect blood of acute aortic dissection model mice (Ang-II / BAPN) and control mice (PBS). Collect 1 ml of mouse orbital blood into a blood collection tube, stand for 30 minutes, centrifuge at 3000 rpm for 15 minutes, and take the upper plasma into an EP tube.

[0120] Add 50 μl / well of the sample to be tested to the enzyme-labeled plate coated with the antibody in the kit. Incubate at 37°C for 30 min. Wash the enzyme-labeled plate twice with 180 μl / well of the washing solution (1X PBST). Dilute GAM-HRP to the working concentration (1:5000) with 1% BSA, and add 50 μl / well to the enzyme-labeled plate, and incubate at 37°C for 30 min. Discard the liquid in the wells again, wash the enzyme-labeled plate three times with 180 μl / well of the washing solution, add 100 μl of freshly prepared TMB color developing substrate to each reaction well, and incubate at 37°C for 5 min. Then, add 90 μl / well of the stop solution to stop the reaction, and measure the OD value at 450 nm on an enzyme-labeled instrument.

[0121] The results of the detection of the content of CTHRC1 in the serum of the acute aortic dissection model mice are shown in FIG. 2, and the ROC curve is shown in FIG. 3. As can be seen from FIGS. 2 and 3, the content of CTHRC1 in the serum of the acute aortic dissection model mice is significantly increased, and the ROC curve analysis shows that CTHRC1 can be used as a specific serum marker for the diagnosis of acute aortic dissection. Figure 2 Figure 3 Figure 2 Figure 3 The results of the detection of the content of CTHRC1 in the serum of the acute aortic dissection model mice are shown in FIG. 2, and the ROC curve is shown in FIG. 3. As can be seen from FIGS. 2 and 3, the content of CTHRC1 in the serum of the acute aortic dissection model mice is significantly increased, and the ROC curve analysis shows that CTHRC1 can be used as a specific serum marker for the diagnosis of acute aortic dissection.

[0122] Example Four:

[0123] Example Four utilizes the kit and detection method in Example Two to detect the content of CTHRC1 in the plasma samples of acute aortic dissection patients, acute myocardial infarction patients, and healthy people.

[0124] Collect peripheral venous blood of healthy control people, acute aortic dissection patients, and acute myocardial infarction patients with 5 ml of EDTA-containing anticoagulant tubes, centrifuge at 3000 rpm for 15 minutes, and take the upper plasma into 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 min. Add washing buffer (1X PBST) to the ELISA plate at a rate of 180 μl / well and wash the ELISA plate twice. Dilute GAM-HRP to the working concentration (1:5000) with 1% BSA, add 50 μl / well to the ELISA plate, and incubate at 37°C for 30 min. Discard the liquid in the wells again, add washing buffer to the ELISA plate at a rate of 180 μl / well, and wash the ELISA plate three times. Add 100 μl of freshly prepared TMB chromogenic substrate to each reaction 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.

[0126] The plasma CTHRC1 levels in healthy controls (CTRL) and patients with acute aortic dissection (AAD) are shown in the attached figure. Figure 4 The results of plasma CTHRC1 testing in patients with acute myocardial infarction (AMI) and acute aortic dissection (AAD) are shown in the attached figure. Figure 5 As shown. From the appendix Figure 4 and attached Figure 5 The results show that 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, the CTHRC1 levels (ng / ml) in patients with acute aortic dissection were significantly increased. This indicates that CTHRC1 can be a candidate biomarker for the diagnosis (positive or negative) of acute aortic dissection, monitoring of disease course and progression, and risk of recurrence.

[0127] In summary, the occurrence and development of acute aortic dissection are closely related to the level of CTHRC1 in plasma, making CTHRC1 a candidate biomarker for the diagnosis (positive or negative) of acute aortic dissection, monitoring of disease course and progression, and risk of recurrence.

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

Claims

1. A double antibody sandwich ELISA kit for detecting the content of CTHRC1 in blood for diagnosing acute aortic dissection, characterized in that: The kit comprises CTHRC1 capture antibodies coated on an enzyme-labeled plate, horseradish peroxidase-labeled CTHRC1 detection antibodies, CTHRC1 protein standards, washing solution, blocking solution, color developing agent and termination solution. ​ The CTHRC1 capture antibodies and the CTHRC1 detection antibodies are a set of antibodies that can recognize different sites of the CTHRC1 protein antigen and are paired with each other. The light chain variable region nucleotide sequence of the CTHRC1 capture antibodies is shown as SEQ ID NO: 1, and the amino acid sequence is shown as SEQ ID NO: 2; the heavy chain variable region nucleotide sequence is shown as SEQ ID NO: 3, and the amino acid sequence is shown as SEQ ID NO: 4; the light chain variable region nucleotide sequence of the CTHRC1 detection antibodies is shown as SEQ ID NO: 5, and the amino acid sequence is shown as SEQ ID NO: 6; the heavy chain variable region nucleotide sequence is shown as SEQ ID NO: 7, and the amino acid sequence is shown as SEQ ID NO:

8.

2. The double antibody sandwich ELISA kit according to claim 1, characterized in that: The preparation method of the horseradish peroxidase-labeled CTHRC1 detection antibodies comprises the following steps: The horseradish peroxidase is activated and dialyzed to obtain the dialyzed HRP; The purified CTHRC1 detection antibodies are dialyzed to obtain the dialyzed detection antibodies; The dialyzed HRP, ethylene glycol and the dialyzed detection antibodies are mixed uniformly and then dialyzed to obtain the HRP-antibody mixed solution; NaBH4 solution and saturated ammonium sulfate are added to the HRP-antibody mixed solution, and then the mixture is stirred uniformly and centrifuged; the supernatant is discarded, and the precipitate is dissolved in PBS to obtain the horseradish peroxidase-labeled CTHRC1 detection antibodies.

3. The double antibody sandwich ELISA kit according to claim 1, characterized in that: The washing solution is PBST.

4. The double antibody sandwich ELISA kit according to claim 1, characterized in that: The blocking solution is 2% bovine serum albumin.

5. The diabodies sandwich ELISA kit according to claim 1, characterized in that: The color developing agent is TMB solution.

6. The diabodies sandwich ELISA kit according to claim 1, characterized in that: The termination solution is H2SO4.

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

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