A diagnostic kit for constrictive pericarditis and its application

Abstract

The present invention discloses a diagnostic kit for constrictive pericarditis and its application, and belongs to the field of molecular biology and clinical detection technology. The kit includes: a specific antibody for detecting a specific protein marker, an enzyme-labeled secondary antibody, and a chromogenic substrate for binding and color development. The present invention adopts specific antibodies such as VEGF antibody, IFN-γ antibody, MMP-9 antibody, and then uses them in combination with a secondary antibody, which can quickly and accurately detect the levels of protein markers such as VEGF, IFN-γ, MMP-9, MCP-1, TIMP-1, etc., has high sensitivity and specificity, is easy to operate, is suitable for routine clinical detection, and can help clinicians evaluate the progression of the patient's disease and the treatment effect, thereby formulating a more accurate treatment plan.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology and clinical detection technology, and in particular to the use of protein markers in products for judging constrictive pericarditis diagnosis and a diagnostic kit for judging constrictive pericarditis through protein markers. Background Art

[0002] Constrictive pericarditis (CP) is a serious heart disease caused by a variety of pathogens such as mycobacteria. The thickening and stiffness of the pericardium affect the normal systolic and diastolic function of the heart, eventually inducing severe heart failure or cardiac complications, leading to disability or death. At present, the pathogenesis of the disease is still unclear. In addition to conventional anti-cancer and surgical treatment strategies, there are no special drugs or cell therapy methods.

[0003] During the development of constrictive pericarditis, there are dynamic changes in pericardial cell subsets, among which the changes in lymphocytes are the most obvious. In addition, pericardial lymphocytes are often accompanied by many genetic changes during the onset of the disease, including fibrosis-related cytokine VEGF, inflammatory cytokine IFN-γ, matrix remodeling-related proteins MMP-9, MCP-1 and TIMP-1.

[0004] Publication No. CN102175873A discloses a combined parallel detection method for protein markers of cardiovascular and cerebrovascular diseases and a diagnostic kit thereof. The technical solution is to form a triple complex through "protein marker-capture antibody-microspheres", in which the protein markers are vascular endothelial growth factor (VEGF), monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinase-9 (MMP-9), human matrix metalloproteinase inhibitor-1 (TIMP-1), and interferon-γ (IFN-γ). Publication No. CN115561464 A discloses a joint detection kit, method and application for detecting cardiovascular and cerebrovascular diseases, which includes a new protein marker and a conventional protein marker for diagnosing cardiovascular and cerebrovascular diseases. Both of the above technical solutions achieve the purpose of detection through specific protein markers, and the diseases they target are mainly cardiovascular and cerebrovascular diseases, and no detection indication is given for constrictive pericarditis.

[0005] In view of this, there is an urgent need in this field to develop protein markers or marker combinations that can be used to diagnose and judge constrictive pericarditis, so as to predict the chance of recurrence and the best or worst benefit before clinical treatment. Summary of the invention

[0006] In order to solve the above problems, the present invention proposes a diagnostic kit for constrictive pericarditis and its application. The kit can quickly detect by detecting the level of specific protein markers, thereby providing a basis for the diagnosis of constrictive pericarditis.

[0007] In order to achieve the above object, the present invention is implemented by the following technical solutions: A diagnostic kit for constrictive pericarditis, comprising a. Primary antibody: a specific antibody used to detect protein markers; b. Enzyme-labeled secondary antibody; c. a chromogenic substrate for binding and color development; The specific antibody is selected from at least one of human antibodies, chimeric antibodies, recombinant antibodies, humanized antibodies, monoclonal antibodies, antigen-binding fragments of monoclonal antibodies and polyclonal antibodies.

[0008] Furthermore, the specific antibody includes any one or more of VEGF antibody, IFN-γ antibody, MMP-9 antibody, MCP-1 antibody and TIMP-1 antibody. Furthermore, the enzyme-labeled secondary antibody is a horseradish peroxidase (HRP)-labeled secondary antibody (HRP-conjugated Goat Anti-Rabbit Ig G (H+L).

[0009] Furthermore, the color developing substrate is tetramethylbenzidine (TMB).

[0010] The present invention also protects a method for using a diagnostic kit for constrictive pericarditis, which is not for diagnostic purposes and comprises the following steps: (1) Obtaining biological samples to be tested; (2) Add the biological sample to the detection well containing the specific antibody in the kit and incubate for 1-2 hours; wash the detection well with PBS buffer to remove unbound substances; (3) Add enzyme-labeled secondary antibody to the detection well and incubate for 20-40 minutes; wash the detection well again with PBS buffer to remove unbound enzyme-labeled secondary antibody; (4) Add a chromogenic substrate to the detection well and develop color through an enzyme reaction; determine the concentration of the protein marker based on the color intensity and evaluate the test results.

[0011] Furthermore, the protein markers in step (4) include any one or more of VEGF, IFN-γ, MMP-9, TIMP-1, and MCP-1.

[0012] The protein marker VEGF is the fibrosis-related cytokine VEGF. VEGF plays a key role in the process of tissue fibrosis, promoting the production of extracellular matrix and inhibiting its degradation, leading to hardening and thickening of tissues.

[0013] The protein marker IFN-γ is the inflammatory cytokine IFN-γ: it plays an important role in the immune response and enhances the host's disease resistance by activating macrophages and other immune cells.

[0014] Protein marker MMP-9, also known as matrix remodeling-related protein MMP-9, participates in the degradation and remodeling of the extracellular matrix and affects the tissue repair process.

[0015] Protein markers MCP-1 and TIMP-1: They play important roles in the adaptive immune process of constrictive pericarditis.

[0016] Furthermore, the step (4) of determining the concentration of the protein marker according to the color intensity is as follows: after the color development reaction is completed, an enzyme-linked immunosorbent assay (ELISA) plate reader is used to read the absorbance of each detection well, and a standard curve is generated to obtain the slope of the standard curve, and the concentration of the protein marker is calculated; The calculation formula is: concentration = absorbance / slope of standard curve.

[0017] The present invention discloses a diagnostic kit for constrictive pericarditis and application thereof, and has the beneficial effects that: the present invention adopts specific antibodies such as VEGF antibody, IFN-γ antibody, MMP-9 antibody, MCP-1 antibody, TIMP-1 antibody, etc., and then uses them in combination with a secondary antibody, so as to be able to quickly and accurately detect the levels of protein markers such as VEGF, IFN-γ, MMP-9, MCP-1, TIMP-1, etc., has high sensitivity and specificity, is easy to operate, is suitable for routine clinical detection, and can help clinicians evaluate the progression of the patient's disease and the treatment effect, thereby formulating a more accurate treatment plan. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a standard curve diagram of protein marker concentration, in which the vertical axis is absorbance and the horizontal axis is protein marker concentration; Figure 2 The present invention is a schematic diagram of the application process of the diagnostic kit for constrictive pericarditis.

[0019] Figure 3 This is a diagram for verifying the IHC staining in Example 3 of the present invention. DETAILED DESCRIPTION

[0020] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] Example 1

[0022] A diagnostic kit for constrictive pericarditis, comprising a. Primary antibody (10 µg / mL): a variety of specific antibodies used to detect specific protein markers, mainly including VEGF antibody, IFN-γ antibody, MMP-9 antibody, MCP-1 antibody and TIMP-1 antibody; b. Horseradish peroxidase-labeled secondary antibody HRP-conjugated Goat Anti-Rabbit IgG (H+L) (5 µg / mL); c. Chromogenic substrate tetramethylbenzidine (100 µL) for binding and color development.

[0023] In the diagnostic kit for constrictive pericarditis in this embodiment, the primary antibody is fixed on a microplate with detection wells, and the secondary antibody and the color development substrate are separately assembled.

[0024] Example 2

[0025] An application of a diagnostic kit for constrictive pericarditis, comprising the following steps (process as shown in Figure 2 shown): (1) Obtaining biological samples to be tested; Plasma (serum) samples: Plasma (serum) samples were collected from patients with constrictive pericarditis, centrifuged at 1000 g for 15 min, and the supernatant was aspirated for subsequent testing; Pericardial effusion samples: Pericardial effusion samples from patients with constrictive pericarditis were collected after surgical opening of the pericardium, centrifuged at 1000 g for 15 min, and the supernatant was aspirated for subsequent testing; Pericardial tissue samples: Patients with different degrees of constrictive pericarditis were selected (200 patients with different degrees were selected). The pericardial tissue was sent to the laboratory within 1 hour after pericardectomy for constrictive pericarditis. First, an appropriate amount of tissue sample (about 50-100 mg) was cut and placed in a pre-cooled microcentrifuge tube, and then the sample was quickly frozen in liquid nitrogen to make it completely frozen. Then, a pre-cooled mortar and pestle was used to quickly grind the tissue in liquid nitrogen until the sample was completely crushed into fine powder. The ground sample powder was transferred to a new microcentrifuge tube, and an appropriate amount of pre-cooled lysis buffer (about 500 µL for every 50 mg of tissue) was added. After gently mixing, the sample was incubated on ice for 30 minutes, and the sample was gently shaken every 5-10 minutes to promote protein release. Centrifuge at 12,000 g for 15-20 minutes at 4°C to separate the supernatant, which is the pericardial tissue sample (i.e., the extracted total protein). The pericardial tissue samples are transferred to new microcentrifuge tubes for later use. The sample numbers are: S01, S02, S03, S04, and S05; (2) Add the biological sample to the detection well containing specific antibodies (MCP-1 antibody and TIMP-1 antibody) in the kit and incubate for 1-2 hours; wash the detection well with PBS buffer to remove unbound substances. The specific method is: add 100 μL of standard or plasma (serum) sample, pericardial effusion sample and pericardial tissue sample to the detection wells of different kits, incubate at 37°C for 2 hours, then discard the liquid in the well, wash the detection well with PBS buffer to remove unbound substances.

[0026] (3) Add 100 μL HRP-biotin and incubate at 37°C for 1 hour. Wash the plate five times with washing buffer for 2 minutes each time.

[0027] (4) Add 90 μL of TMB substrate, incubate at 37°C for 15-30 minutes, and then add 50 μL of stop solution. Read the sample at 450 nm using an ELISA reader. Determine the concentration of the protein marker based on the color intensity and evaluate the test results.

[0028] The specific operation of step (4) is as follows: after the color development reaction is completed, the concentration of the protein marker is obtained by using an enzyme-linked immunosorbent assay (ELISA). Specifically, the absorbance of each detection well is read at 450 nm using a plate reader, and a standard curve is generated (eg Figure 1 As shown), obtain the slope of the standard curve and calculate the concentration of the protein marker; The calculation formula is: concentration = absorbance / slope of the standard curve. The protein concentration is calculated by the absorbance and the slope of the standard curve. The higher the concentration, the more serious the disease.

[0029] The results are shown in Table 1: Table 1 Kit test results Sample No. VEGF (OD450) MMP-9 (OD450) <![CDATA[IF N-γ (OD450)]]> TIMP-1 (OD450) MCP-1(OD450) Calculated concentration (μg / mL) Grouping S01 0.500 0.450 0.470 0.540 0.490 11.2 Mild S02 0.450 0.390 0.410 0.520 0.443 9.4 Mild S03 0.700 0.620 0.670 0.750 0.685 15.5 Moderate S04 1.200 1.050 1.150 1.300 1.175 26.7 Severe S05 0.800 0.650 0.720 0.950 0.78 18.0 Severe .

[0030] Example 3

[0031] Validation method: Immunohistochemistry (IHC), specifically (1) Obtaining biological samples to be tested; The specific biological sample preparation method is as follows: the surgical tissues obtained from different patients (200) selected in Example 2 after constrictive pericardiectomy are fixed in neutral formalin fixative overnight, rinsed with running water, dehydrated, transparent and waxed to prepare tissue wax blocks. Three pericardial tissues are selected from each patient, made into paraffin wax blocks and 4 μm sections are prepared; (2) Place the slice obtained in step (1) in a 65°C oven with the front side facing the wind for 40 minutes to make the biological sample adhere better, immerse it in xylene wash tanks I, II, and III, place it in a 25°C constant temperature water bath for 10 minutes, transfer it to 100%, 85%, and 75% ethanol for 3 minutes each for gradient hydration, then transfer it to PBS buffer I, II, and III for 5 minutes each time, and place it in a 3% hydrogen peroxide wash tank for 15 minutes to block endogenous peroxidase. Immerse it in heated sodium citrate buffer and microwave it on low heat for 20 minutes. Immerse it in PBS buffer I, II, and III for 3 minutes each time. Use a PAP Pen immunohistochemical paraffin pen to draw the range along the edge of the slice tissue, use a 200μL micropipette to spread the diluted primary antibody solution or antibody working solution on the tissue surface within the drawn range and place it in a wet box, incubate it in a 37°C constant temperature incubator for 2 hours or in a 4°C refrigerator overnight. Remove excess primary antibody on a paper towel, and immerse the tissue chip in PBS buffer I, II, and III in turn for 3 minutes each time. Add reagent II in the two-step immunohistochemistry detection kit, ensure that the tissue surface within the drawn range is covered and placed in a wet box, and incubate in a 37°C constant temperature incubator for 15 minutes. Remove excess reagent on a paper towel, and immerse the tissue chip in PBS buffer I, II, and III in turn for 3 minutes each time. Add reagent III (Polyperoxidase-anti-mouse / rabbit Ig G ), make sure to cover the tissue surface within the drawn range and place it in a wet box, incubate in a 37℃ constant temperature incubator for 30 minutes. Remove excess reagents on a paper towel, and immerse the tissue chip in PBS buffers I, II, and III in turn, for 3 minutes each time. Take an appropriate amount of reagents from the DAB kit, and mix them in a ratio of 1mL DAB diluent to 50μLDB concentrate; use a 200μL micropipette to cover the diluted DAB solution on the tissue surface within the drawn range for color development, and keep the color development time consistent for each slice; observe the color development effect under the microscope until a significant contrast is formed within the same slice and between different slices, then remove excess DAB solution on a paper towel and place it in distilled water; the color development time is 60s, and the color development time varies between different antibodies. Use a 1000μL micropipette to quickly cover the surface of the tissue array within the drawn range for staining, and rinse it immediately with slow running water after 10s; place the stained slices in 1% ammonia water. Then, immerse the slices in 75%, 85%, and 100% ethanol for 3 minutes each for gradient dehydration. Immerse in a xylene washing tank for 10 minutes to remove the marks drawn by the PAP Pen immunohistochemical wax pen, place the slices in a ventilated place to dry, and seal them with a quick-drying sealing agent.

[0032] (3) Positive judgment criteria: Count 20 high-power fields of immunohistochemically stained cells (eg Figure 3 As shown in the figure, the staining intensity score is: no staining 0 points, weak staining 1 point, moderate staining 2 points, strong staining 3 points; the staining area score is: staining range ≤10% is 0 points, >10%-25% is 1 point, >25%-50% is 2 points, >50%-75% is 3 points, >75% is 4 points. If the sum of the two scores is ≥3 points, it is positive, and if it is less than 3 points, it is negative.

[0033] The 200 patients in Example 2 and Example 3 were divided into three groups: mild, moderate and severe (mild: pericardial thickening>0.5cm; moderate: pericardial thickening>1cm, accompanied by pericardial effusion; severe: pericardial thickening>1.2cm, accompanied by pericardial effusion and color Doppler ultrasound indicating that the heart contractile function is affected). The results obtained by the kit detection of the present invention and the immunohistochemistry experiment (IHC) verification are shown in Table 2; Table 2 Test results of the kit Group Test kit testing (number of people) IHC test, number of people Patient coincidence degree, % Mild 40 38 95 Moderate 93 89 96 Severe 67 64 95.5 As can be seen from Table 2, the patient overlap between the test kit of the present invention and the immunohistochemistry experiment (IHC) verification is above 95%, indicating that the test kit of the present invention is fast and accurate.

[0034] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present invention. The appearance of the phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0035] Finally, it should be noted that the embodiments disclosed in the present invention are only preferred embodiments of the present invention, which are only used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A diagnostic kit for constrictive pericarditis, characterized in that: include a. Primary antibody: a specific antibody used to detect protein markers, the specific antibody including any one or more of VEGF antibody, IFN-γ antibody, MMP-9 antibody, MCP-1 antibody and TIMP-1 antibody; b. Enzyme-labeled secondary antibody; c. a chromogenic substrate for binding and color development; The specific antibody is selected from at least one of human antibodies, chimeric antibodies, recombinant antibodies, humanized antibodies, monoclonal antibodies, antigen-binding fragments of monoclonal antibodies and polyclonal antibodies.

2. The diagnostic kit for constrictive pericarditis according to claim 1, characterized in that: The enzyme-labeled secondary antibody is a horseradish peroxidase-labeled secondary antibody.

3. The diagnostic kit for constrictive pericarditis according to claim 2, characterized in that: The color developing substrate is tetramethylbenzidine.

4. A method for using the diagnostic kit for constrictive pericarditis according to any one of claims 1 to 3 for non-diagnostic purposes, characterized in that: The following steps are involved: (1) Obtaining biological samples to be tested; (2) Add the biological sample to the detection well containing the specific antibody in the kit and incubate for 1-2 hours; Wash the detection wells with PBS buffer to remove unbound substances; (3) Add enzyme-labeled secondary antibody to the detection well and incubate for 20-40 minutes; wash the detection well again with PBS buffer to remove unbound enzyme-labeled secondary antibody; (4) Add a chromogenic substrate to the detection well and develop color through an enzyme reaction; determine the concentration of the protein marker based on the color intensity and evaluate the test results.

5. The method for using the diagnostic kit for constrictive pericarditis according to claim 4, characterized in that: The protein markers in step (4) include any one or more of VEGF, IFN-γ, MMP-9, TIMP-1, and MCP-1.

6. The method for using the diagnostic kit for constrictive pericarditis according to claim 4, characterized in that: Determining the concentration of the protein marker according to the color intensity in step (4) is as follows: after the color development reaction is completed, using an enzyme-linked immunosorbent assay plate reader to read the absorbance of each detection well, and generating a standard curve, obtaining the slope of the standard curve, and calculating the concentration of the protein marker; The calculation formula is: concentration = absorbance / slope of standard curve.

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