Detection kit for diagnosing thymoma

Abstract

The invention relates to the field of biological medicine, discloses application of a reagent for detecting IL8 expression in total T cells in preparation of a thymoma diagnosis product, and discloses a thymoma diagnosis and detection kit. The detection process provided by the invention is suitable for large-scale screening and diagnosis of thymoma. Experiment operation is simplified through ELISA, IL-8 expression in peripheral blood total T cells is detected, a reliable basis is provided for early diagnosis of thymoma, and the kit has popularization value.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and more specifically, to a detection kit for diagnosing thymoma. Background Art

[0002] There are many types of anterior mediastinal tumors, mainly thymoma, thymic cyst, thymic carcinoma, germ cell tumor, lymphoma and other mesenchymal tissue-derived tumors. Among them, thymoma is the most common tumor in the anterior mediastinum, accounting for about 50% of anterior mediastinal tumors and 90% of thymic tumors. Thymoma can cause many harms to the human body. Thymoma spreads and grows in the anterior mediastinum, and can cause extensive implantation of the pericardium and pleura; thymoma invades surrounding tissue structures and can cause diaphragmatic paralysis or superior vena cava syndrome; thymoma often leads to abnormal development of T cells, causing many immune-related diseases, such as myasthenia gravis, low immunity, etc. Therefore, thymoma needs to be surgically removed once it is discovered.

[0003] Although traditional imaging (such as CT and MRI) has made great progress, there are still many misdiagnoses in the diagnosis of mediastinal tumors, which often lead to unnecessary biopsies, surgeries, and chemoradiotherapy. According to existing literature reports, the misdiagnosis rate of CT is as high as 22%-68%, and thymic cysts and lymphomas are often misdiagnosed as thymomas, resulting in many unnecessary surgeries; in addition, traditional imaging also causes a missed diagnosis rate of about 7%, which is common in missed diagnosis of thymomas without clinical symptoms. These missed diagnoses delay the treatment opportunity. Therefore, in order to accurately treat thymic tumors, the existing diagnostic methods of thymic tumors are in urgent need of further optimization, and a detection kit for diagnosing thymoma is urgently needed. Summary of the invention

[0004] The invention purpose of this application is to develop an efficient and convenient diagnostic kit, which is designed for the rapid and accurate identification and diagnosis of thymic tumors, in order to improve the early detection rate and diagnostic efficiency of thymoma, provide a reliable basis for the clinical diagnosis of thymoma, and optimize the existing detection process to improve the sensitivity and specificity of diagnosis.

[0005] In order to achieve the above-mentioned invention purpose, the applicant previously found through flow cytometry exploratory research that IL-8 positive primary T cells (CD4 + CD45RA + CCR7 + CD31 + IL-8 + T cells or CD8 + CD45RA + CCR7 + CD31 + IL-8 +The level of IL-8 positive initial T cells in the peripheral blood of patients with anterior mediastinal tumors was significantly increased, while the level of IL-8 positive initial T cells in the peripheral blood of patients with other types of anterior mediastinal tumors was lower; the level of IL-8 positive initial T cells in patients with thymoma decreased significantly after surgical resection, and increased again when thymoma recurred; ROC curve analysis showed that the level of IL-8 positive initial T cells could well distinguish thymoma from other types of anterior mediastinal tumors, with a sensitivity and specificity of nearly 95%

[0006] To date, there is no effective hematological biomarker for thymoma, and IL-8-positive naive T cells have shown good diagnostic value as a hematological biomarker in our previous studies. However, the flow cytometry detection of IL-8-positive naive T cells is cumbersome and time-consuming, taking about 12 hours.

[0007] In order to better utilize the clinical value of IL8 as a molecular marker for the diagnosis of thymoma, we intend to further optimize the detection process of IL-8. The IL8 level of thymoma patients after initial T cell stimulation is significantly higher than that of unstimulated patients, while the IL8 level of non-thymoma patients is not high even after stimulation. Based on this, we consider using enzyme-linked immunosorbent assay (ELISA) for detection, which can provide valuable basis for the clinical diagnosis of thymoma.

[0008] Based on the above, this application provides the following technical solutions:

[0009] In a first aspect, the present application provides a reagent for detecting IL8 expression in total T cells and its use in preparing a thymoma diagnostic product.

[0010] Furthermore, the test sample of the diagnostic product is a peripheral blood sample of the subject.

[0011] Furthermore, the diagnostic product includes a detection kit.

[0012] Furthermore, the detection kit is an ELISA kit.

[0013] In a second aspect, the present application provides a thymoma diagnostic detection kit, comprising a reagent for detecting IL8 expression in total T cells.

[0014] In summary, compared with the prior art, the present application has the following beneficial effects:

[0015] Simplified process: Compared with flow cytometry, the present invention has a shorter detection time and is suitable for clinical settings.

[0016] Low equipment requirements: The experiment only requires basic ELISA detection equipment, reducing dependence on professional equipment.

[0017] High diagnostic accuracy: Through optimized detection methods, the diagnostic accuracy is comparable to that of flow cytometry, which is suitable for large-scale screening and clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 :The level of IL-8 positive naive T cells is significantly increased in the peripheral blood of thymoma patients;

[0019] Figure 2 : The level of IL-8 positive initial T cells increases again when thymoma relapses after surgery;

[0020] Figure 3 : Detection IL8 expression in T cells (CD4 / CD8 cells) and total T cells was similar;

[0021] Figure 4 : Total T cell IL8 expression can better distinguish thymomas, thymic cysts and other lesions (n=41);

[0022] Figure 5 :T cells were isolated, incubated, stimulated, and then lysed, and IL8 in the supernatant was detected by ELISA;

[0023] Figure 6 : Effect of separating T cells using 96-well plates coated with CD3 antibodies;

[0024] Figure 7 : The expression level of IL8 in peripheral blood total T cells of thymoma patients was significantly increased after stimulation (n=41). DETAILED DESCRIPTION

[0025] The technical scheme and effects of the present application are further described in detail below in conjunction with the embodiments and drawings. It is to be understood that the specific embodiments described herein are only used to explain the invention, rather than to limit the invention. Unless otherwise specified, the methods used in the following embodiments are all existing methods, and the materials used are all commercially available.

[0026] Example 1

[0027] Peripheral blood samples were extracted from healthy volunteers, patients with thymic cysts, thymoma patients, thymic cancer patients, lymphoma patients and teratoma patients. Through flow cytometry, an exploratory study found that IL-8 positive naive T cells (CD4 + CD45RA + CCR7 + CD31 + IL-8 + T cells or CD8 + CD45RA + CCR7+ CD31 + IL-8 + T cells) were significantly increased, while the levels of IL-8 positive initial T cells in the peripheral blood of patients with other types of anterior mediastinal tumors were low (such as Figure 1 IL-8 positive initial T cell levels were significantly reduced after surgical resection of thymoma patients, and increased again when thymoma relapsed (as shown in Figure 2 ROC curve analysis showed that the level of IL-8 positive initial T cells could well distinguish thymoma from other types of anterior mediastinal tumors, with a sensitivity and specificity close to 95%.

[0028] Up to now, there is no effective hematological biomarker for thymoma, and IL-8 positive initial T cells have shown good diagnostic value as hematological biomarkers in the applicant's preliminary study (Example 1). However, the flow cytometry detection of IL-8 positive initial T cells is cumbersome and time-consuming. First, peripheral blood lymphocytes need to be obtained, and then phorbol ester (PMA) and ionomycin (Ionomycin) need to be cumulatively stimulated for 6 hours under cell culture grade conditions, and Brefeldin A (BFA) needs to be added after stimulating for 4 hours to process for 2 hours. After the stimulation, the cells are collected and used for flow cytometry analysis. Since IL8 is an intracellular cytokine, it is necessary to fix the cells after surface marker staining and break the membrane to stain the intracellular IL8. It takes about 12 hours to obtain the final result from obtaining peripheral blood to machine detection.

[0029] In order to better exert the clinical value of IL8 as a molecular marker for diagnosing thymoma, the applicant intends to further optimize the detection process of IL-8. The IL8 level of thymoma patients after initial T cell stimulation is significantly higher than that of unstimulated patients, while the IL8 level of non-thymoma patients is not high even after stimulation. Based on this, we considered using enzyme-linked immunosorbent assay (ELISA) for detection. Despite this, it is still necessary to obtain the target initial T cells for further experiments. The applicant compared the expression of IL8 in initial T cells and total T cells in the flow cytometry test results and found that the expression level of IL8 in total T cells and CD4 + CD45RA + CCR7 + CD31 + IL-8 + T cells or CD8 + CD45RA + CCR7 + CD31 + IL-8 +T cells are close to each other, and their diagnostic accuracy for thymoma is highly consistent (such as Figure 3 The analysis of the peripheral blood test results of 41 patients (Example 2 below) also showed that the expression level of IL8 in total T cells can better distinguish thymoma from thymic cysts and other non-thymic lesions (such as Figure 4 This indicates that detecting IL8 expression in total T cells can improve the diagnostic accuracy of thymoma.

[0030] Therefore, the applicant isolated the total peripheral blood T cells and detected the IL8 expression by ELISA. The experimental results were in line with the applicant's expectations. The expression of IL8 in peripheral blood T cells of thymoma patients was significantly higher than that of non-thymoma patients. This method can provide valuable basis for the clinical diagnosis of thymoma.

[0031] The specific steps of the above-mentioned embodiment 2 are implemented as follows:

[0032] Example 2

[0033] This example discloses a rough process for the separation of total T cells and IL8 detection in human peripheral blood lymphocytes. Figure 5 The total T cells in the peripheral blood of 41 patients who were simultaneously tested by flow cytometry were tested by ELISA for IL8. The specific steps were as follows:

[0034] Step 1: Prepare pre-coated 96-well plates:

[0035] Prepare and commercialize 96-well plates coated with CD3 capture antibodies and IL8 capture antibodies for direct use, and the preparation work is handed over to the biological company (the 96-well plates used in the preliminary test were purchased from Corning, USA, and the CD3 and IL8 antibodies used were purchased from CST, USA, and the coating work was completed by ourselves). The coating method is: dilute CD3 or IL-8 antibodies in an appropriate buffer, coat at a volume of 100 μl / well, incubate overnight at 4°C, wash and block the blank site, and store at 4°C for use.

[0036] Step 2: Plating and capturing T cells:

[0037] Plating: Separate peripheral blood lymphocytes (PBMCs, about 2-5x10 per well) ^5 cells) were evenly spread into a 96-well plate coated with CD3 capture antibody. The cells were evenly spread in 6 wells, 3 wells for the control group and 3 wells for the experimental group. Incubation: Place the 96-well plate in a cell culture incubator (37°C, 5-10% CO2) and incubate for 2 hours to ensure that the T cells bind to the CD3 antibody. Washing: After the incubation, wash the 96-well plate 3 times (200 μl / well each time) with PBS containing 0.05% Tween-20 to remove unbound non-T cells. After washing, tap the well plate to remove residual liquid and ensure that the T cells remain in the well plate. The test process found that the T cell separation effect was good, and the purity was close to 90% ( Figure 6 ).

[0038] Step 3, T cell stimulation:

[0039] A control group and an experimental group were set up, where the control group: 100 μl of T cell complete medium was added to the 3 control group wells, and no other treatment was done; the experimental group: 100 μl of T cell complete medium was added to the 3 experimental group wells, and phorbol ester (PMA, working concentration 1: 10000) and ionomycin (Ionomycin, working concentration 1: 1000) were added to stimulate T cell activation. Incubation: The 96-well plate was placed in a cell culture incubator (37°C, 5-10% CO2) for 4 hours, and then Brefeldin A (BFA, working concentration 1: 500) was added and incubated for another 2 hours.

[0040] Step 4: T cell lysis and collection of supernatant:

[0041] Lysis: After the incubation, remove the culture medium, add 100ul PBS to each well to wash and aspirate. Add 100μl of cell lysis solution to each well, gently pipette to mix, ensure sufficient lysis, and let stand for 30 minutes to complete the lysis. Centrifugation: Transfer the lysis solution in each well to a 200μl centrifuge tube, centrifuge at 12000rpm for 10 minutes at 4℃, and separate the supernatant. Collection: Collect the supernatants of the control group and the experimental group separately and store them on ice for later use, waiting for ELISA detection.

[0042] Step 5, ELISA test:

[0043] Sample loading: Dilute the supernatants of the control and experimental groups, as well as the standards of different concentration gradients, in the sample diluent. Add 100 μl of diluted supernatant or standard to each well of the 96-well plate coated with IL-8 capture antibody. Incubation: Incubate at 37°C for 2 hours to ensure that the IL-8 in the sample binds to the coated antibody. Washing: Wash the 96-well plate 3 to 5 times with 200 μl of washing buffer (PBS containing 0.05% Tween-20) to remove unbound antigen.

[0044] Step 6: Add detection antibody:

[0045] Add detection antibody: dilute the HRP-labeled IL-8 detection antibody in buffer and add 100 μl detection antibody to each well. Incubate: incubate at 37°C for 1 hour to form a sandwich structure of IL-8 antigen. Wash: wash again with wash buffer 3 to 5 times to remove unbound detection antibody.

[0046] Step 7, color reaction:

[0047] Add substrate: Add 100 μl TMB substrate to each well and incubate at room temperature in the dark for 10-30 minutes until a visible color change (blue) occurs. Stop reaction: Add 50 μl stop solution (2M H2SO4 or 1M HCl) to each well until the color changes to yellow.

[0048] Step 8, OD value determination and data analysis:

[0049] OD value determination: Use a microplate reader to measure the absorbance value (OD value) of each well at a wavelength of 450 nm. Draw a standard curve: Draw a standard curve based on the OD value of the standard, and use the curve to calculate the concentration of IL-8 in the control group and experimental group samples.

[0050] Analysis results: Compare the IL-8 concentrations in the experimental group and the control group. If the IL-8 concentration in the experimental group is significantly higher than that in the control group, it indicates that T cells secrete a large amount of IL-8 after stimulation, which may indicate that the patient is a thymoma patient. Figure 7 The statistical analysis results showed that the IL8 expression level of total T cells in the peripheral blood of thymoma patients increased significantly after stimulation, with an average increase of 5.87 times, while the average increase of thymic cyst patients after stimulation was only 1.38 times.

[0051] In summary, the detection process of the present application is suitable for large-scale screening and diagnosis of thymoma. By simplifying the experimental operation through ELISA, the expression of IL-8 in peripheral blood total T cells is detected, which provides a reliable basis for the early diagnosis of thymoma and has promotion value.

[0052] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. Application of reagents for detecting IL8 expression in total T cells in the preparation of thymoma diagnostic products.

2. The use according to claim 1, characterized in that: The test sample of the diagnostic product is a peripheral blood sample of a subject.

3. The use according to claim 1, characterized in that: The diagnostic products include test kits.

4. The use according to claim 3, characterized in that: The detection kit is an ELISA kit.

5. A thymoma diagnostic test kit, characterized in that: Includes reagents for detecting IL8 expression in total T cells.

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