A detection kit for B cells and high endothelial venules in the immune microenvironment of thymus tissue

Abstract

The present invention relates to a detection kit for B cells and high endothelial venules in the immune microenvironment of thymus tissue, comprising quantum dot-labeled antibody CD19, quantum dot-labeled antibody CD1d, quantum dot-labeled CD31 and quantum dot-labeled PNad. After the specific antibody coupled with the quantum dot is specifically combined with the antigen on the B cell and high endothelial venule on the tissue section sample, the quantum dot can be used to quantify or locate the B cell and high endothelial venule. Instead of the traditional multicolor immunofluorescence technology, the method of directly carrying specific antibodies with quantum dots is used to save experimental time and improve work efficiency. The expression of B cells and Breg cells in the thymus immune microenvironment and the expression of high endothelial venules can be detected, and at the same time, the relative spatial positions of the above cells and blood vessels can be observed, which directly reflects the state of immune infiltration and autoimmune reaction in the thymus immune microenvironment, and has broad application prospects in the thymus-related immunity and tumor fields.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a detection kit for thymus tissue immune microenvironment B cells and high endothelial venules. Background Art

[0002] The thymus is the central immune organ of the human immune system. Abnormalities in the thymus can lead to autoimmune diseases, and the relationship between the two has become a research hotspot. Autoimmune diseases are diseases caused by immunological dysfunction caused by multiple factors, which lead to the body's immune response to its own antigens and damage to its own tissues. At present, there are many types of autoimmune diseases with complex causes, and most diseases lack specific prediction methods. Autoimmune disease detection kits play a vital role in the early prediction and evaluation of diseases.

[0003] Breg cells are a subpopulation of B lymphocytes with immunosuppressive function, which can play a negative immunoregulatory role by secreting IL-10, TGF-β and IL-35. In autoimmune diseases, the expression level of Breg cells is reduced and immune tolerance is lost, thus promoting the progression of autoimmune diseases. In thymoma, the infiltration of Breg cells can promote tumor progression by inhibiting tumor immunity.

[0004] For patients with both thymus abnormalities and autoimmune diseases, B lymphocytes and Breg cells in the target organs are detected, and the ratio of Breg cells to B lymphocytes is further calculated. The larger the ratio, the milder the degree of autoimmune disease. The number of Breg cells is related to autoimmune diseases, and the severity of autoimmunity is negatively correlated with the number of Breg cells.

[0005] At present, it is difficult to further separate and purify B lymphocytes in the thymus, resulting in unclear classification of B lymphocytes in thymic tissue and a lack of research on the mechanism of action of B lymphocytes and Breg cells in thymic diseases and combined autoimmune diseases. Among them, Breg cells, as protective cells for autoimmune diseases, have not been reported on their expression level in the immune microenvironment of thymic tissue.

[0006] Due to the difficulties in research, the current research on B lymphocytes and Breg cells in thymic tissue is limited to blood. There is a lack of detection of B lymphocytes and Breg cells in tissues of thymic diseases and thymic diseases combined with autoimmune diseases, and it is impossible to judge the disease status through tissue staining technology. At present, the detection of angiogenesis in the immune microenvironment of thymic tissue mainly relies on tissue morphological observation, and mainly focuses on HE staining and immunohistochemical staining, which requires high experience of pathologists, is highly subjective, and cannot form a quantitative evaluation system.

[0007] The tumor microenvironment (TME) is the internal environment where tumor cells are produced and survive. It is mainly composed of tumor cells and their surrounding immune and inflammatory cells, tumor-associated fibroblasts and nearby interstitial tissue, microvessels, and various cytokines and chemokines. It is a complex integrated system. Tumor cells and the tumor microenvironment complement each other. The heterogeneity of the microenvironment is closely related to the different response rates of tumor patients to immunotherapy. Exploring the internal structure of the tumor microenvironment and the biological relationship of various cells is very important for the precise treatment of tumors.

[0008] High endothelial venules (HEV) are another important marker of angiogenesis in the tumor microenvironment. They belong to the tertiary lymphoid structure and can assist immune responses in non-lymphoid tissues, especially in response to tumors or chronic inflammation, and can promote anti-tumor immune responses. B cells from the blood can enter tumor tissues through HEVs, so HEVs in the tumor microenvironment can reflect the immune infiltration in tumor tissues and further evaluate tumor growth status or treatment effects. Peripheral lymph node addressin (PNAd) is strongly expressed in HEV endothelial cells and can therefore be used as a specific staining indicator for HEVs. Summary of the invention

[0009] In order to solve the above technical problems, the present invention provides a detection kit for B cells and high endothelial venules in the immune microenvironment of thymus tissue.

[0010] The technical solution adopted by the present invention is: a detection kit for thymus tissue immune microenvironment B cells and high endothelial venules, including quantum dot-labeled antibody CD19, quantum dot-labeled antibody CD1d, quantum dot-labeled CD31 and quantum dot-labeled PNad.

[0011] Preferably, the quantum dots include QDs-585, QDs-625, QDs-525 and QDs-565.

[0012] Preferably, quantum dot-antibody conjugates include QDs-585-CD19, QDs-625-CD1d, QDs-525-CD31 and QDs-565-PNAd.

[0013] Preferably, antigen repair solution and nucleic acid staining solution are also included.

[0014] Preferably, quantum dots, EDC, and NHS are mixed at a molar ratio of 1:2500-3500:5000-6000 to activate the quantum dots, and antibodies are added to the activated quantum dot solution, with a molar ratio of quantum dots to specific antibodies of 1:20-80, to obtain QDs-Ab conjugates after the reaction; the supernatant is taken as a quantum dot-labeled antibody solution that can be used for staining.

[0015] To use the kit, first prepare thymus tissue sections of the test sample (the thymus tissue comes from the thymus specimen removed during surgery), use antigen repair solution to perform antigen repair, then use different quantum dot-labeled antibodies to stain the tissue sections, then stain the cells, collect the fluorescence information of the tissue sections, and based on the fluorescence expression level and spatial position distribution information, obtain the immune infiltration status of the tested thymus tissue, evaluate and predict the autoimmune response.

[0016] Preferably, the quantum dot-labeled antibody CD19 can label B cells, the quantum dot-labeled antibody CD1d can label Breg cells, and the quantum dot-labeled CD31 and quantum dot-labeled PNad can label high endothelial venules.

[0017] Preferably, quantum dot-labeled antibody CD19, quantum dot-labeled CD31, and quantum dot-labeled PNad are used to stain tissue sections in sequence to detect the expression and spatial location of B cells and high endothelial venules, reflecting the degree of immune infiltration in the thymic microenvironment;

[0018] Alternatively, the tissue sections are stained with quantum dot-labeled antibody CD19 and quantum dot-labeled antibody CD1d in sequence to detect the expression of B cells and their subpopulation Breg cells to evaluate and predict autoimmune diseases;

[0019] Alternatively, the tissue sections were stained with quantum dot-labeled antibodies CD1d, quantum dot-labeled CD31, and quantum dot-labeled PNad in sequence to detect the expression of Breg cells and high endothelial venules and analyze the immune status of the thymic tissue immune microenvironment.

[0020] Preferably, thymus tissue sections are used as the detection object, QDs-585-CD19 is used to mark B cells, and QDs-525-CD31 and QDs-565-PNAd are used to mark high endothelial venules. Fluorescence microscopy observation shows that the perivascular space (PVS) area is accompanied by B cell aggregation, and the expression of high endothelial venules increases with the increase of immune infiltration, indicating that its immune state is relatively active;

[0021] Specifically, when the mean fluorescence intensity of CD19 is greater than 426.721, at the same time, the mean fluorescence intensity of CD31 is greater than 544.131, and the mean fluorescence intensity of PNAd is greater than 395.623, it indicates that the test sample has rich immune infiltration;

[0022] Alternatively, thymus tissue sections were used as the detection object, B cells were labeled with QDs-585-CD19 and Breg cells were labeled with QDs-525-CD1d, and the samples were observed under a fluorescence microscope; when the average fluorescence intensity of CD19 was less than 87.125 and the average fluorescence intensity of CD1d was greater than 365.113, the detection sample was judged to be simple tumor tissue; when the average fluorescence intensity of CD19 was less than 245.527 and the average fluorescence intensity of CD1d was greater than 102.006, the detection sample was judged to be tumor combined with mild autoimmune disease tissue; when the average fluorescence intensity of CD19 was greater than 426.721 and the average fluorescence intensity of CD1d was less than 58.826, the detection sample was judged to be tumor combined with severe autoimmune disease tissue;

[0023] Alternatively, using thymus tissue sections as the detection object, using QDs-525-CD1d to label Breg cells, QDs-525-CD31 and QDs-565-PNAd to label high endothelial venules, and observing under a fluorescence microscope, the expression of high endothelial venules increased and became more densely distributed, while the expression of Breg cells gradually decreased and became more discretely distributed, judging that the detection samples were gradually weakened in the immune status in the tumor microenvironment;

[0024] Specifically, the average fluorescence intensity of CD1d was less than 58.826, while the average fluorescence intensity of CD31 was greater than 544.131, and the average fluorescence intensity of PNAd was greater than 395.623, indicating that the immune status of the detected sample was relatively active in the tumor microenvironment.

[0025] The advantages and positive effects of the present invention are: the method of using quantum dots to directly carry specific antibodies saves experimental time and improves work efficiency; quantum dots are used instead of fluorescent dyes to detect the expression of B cells, Breg cells and high endothelial venules in the thymic immune microenvironment, and at the same time, the relative spatial positions of the above cells and blood vessels can be observed, and the state of immune infiltration and autoimmune reaction in the thymic immune microenvironment can be reflected by calculating the average fluorescence intensity of the corresponding cells, which has very broad application prospects in the fields of thymus-related immunity and tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Expression of CD31, CD19 and PNAd in tissue sections of simple thymoma, thymoma combined with mild myasthenia gravis and thymoma combined with severe myasthenia gravis;

[0027] Figure 2Expression of CD19 and CD1d in tissue sections of simple thymoma, thymoma combined with mild myasthenia gravis, and thymoma combined with severe myasthenia gravis;

[0028] Figure 3 Expression of CD31, CD1d and PNAd in tissue sections of simple thymoma, thymoma combined with mild myasthenia gravis and thymoma combined with severe myasthenia gravis. DETAILED DESCRIPTION

[0029] The embodiments of the present invention are described below with reference to the accompanying drawings.

[0030] The present invention relates to a detection kit for B cells and high endothelial venules in the immune microenvironment of thymus tissue. The kit can be used to detect the expression of B cells, Breg cells and high endothelial venules in the immune microenvironment of thymus tissue. The kit can mark specific antibodies with quantum dot-antibody conjugates to achieve the marking of B cells, Breg cells and high endothelial venules in the immune microenvironment of thymus tissue. The kit can intuitively obtain the expression amounts of B cells, Breg cells and high endothelial venules through the effect of a fluorescent microscope, and can more accurately know the state of immune infiltration and autoimmune reaction in the immune microenvironment of thymus tissue.

[0031] The detection kit for B cells and high endothelial venules in the immune microenvironment of thymus tissue includes quantum dot-labeled antibody CD19, quantum dot-labeled antibody CD1d, quantum dot-labeled CD31, quantum dot-labeled PNad, antigen repair solution and nucleic acid staining solution; wherein the quantum dots can be selected from QDs-585, QDs-625, QDs-525 and QDs-565, respectively connected to each specific antibody. In certain embodiments of the present invention, the kit may include quantum dot-antibody conjugates QDs-585-CD19, QDs-625-CD1d, QDs-525-CD31, QDs-565-PNAd, antigen repair solution and nucleic acid staining solution. The antigen repair solution is Tris-EDTA, and the DNA fluorescent stain is DAPI stain.

[0032] In certain embodiments of the present invention, coupling quantum dots with specific antibodies comprises the following steps:

[0033] A1: Activation of quantum dots: QDs, EDC, and NHS were mixed at a molar ratio of 1: (2500-3500): (5000-6000), preferably 1: 3000: 5500, diluted with PBS, mixed on a shaker at room temperature, and then the pH was adjusted to 8-9 with PBS to obtain an activated quantum dot solution;

[0034] A2: Preparation of QDs-Ab conjugate: Add specific antibody to activated quantum dot solution, with the molar ratio of quantum dots to specific antibody being 1:20-80; react on a shaker at 4°C in the dark, and obtain QDs-Ab conjugate after reaction;

[0035] A3: After the reaction is completed, centrifuge at 8000 rpm to remove possible agglomerated precipitates, retain the supernatant, and purify the supernatant using a PD10 column to finally obtain specific antibodies coupled to quantum dots that can be used for staining.

[0036] The kit can be used to mark B cells, Breg cells and high endothelial venules in the immune microenvironment of thymic tissue. The specific antibody used to mark B cells is CD19; the specific antibody used to mark Breg cells is CD1d, and the specific antibodies used to mark high endothelial venules are CD31 and PNad. The quantum dots are coupled with the above antibodies, and the content of B cells, Breg cells and high endothelial venules can be reflected through the corresponding quantum dots. When used, first prepare thymic tissue sections, use antigen repair solution for antigen repair, then use different quantum dot-labeled antibodies to stain the tissue sections, then stain the cells, collect the fluorescence information of the tissue sections, and obtain the immune infiltration status of the detected thymic tissue according to the fluorescence expression information, evaluate and predict the autoimmune reaction. By calculating the average fluorescence intensity of the corresponding indicators, an analysis method for the immune infiltration status in the thymic immune microenvironment is provided. By detecting the expression levels of B cells and Breg cells in the thymic immune microenvironment and calculating the average fluorescence intensity of the corresponding indicators, a method for evaluating tumors combined with early and latent autoimmune diseases is provided.

[0037] The images taken with the fluorescence microscope were analyzed by ImageJ software, and the average fluorescence intensity Mean = the total fluorescence intensity InDen of the area / the area of ​​the area Area.

[0038] In certain embodiments of the present invention, B cell marker antibodies and high endothelial venule marker antibodies are coupled to corresponding quantum dots to detect the expression of B cells and newly formed high endothelial venules in the immune microenvironment of thymic tissue, and thymic tissue sections are used as the detection object, and QDs-585-CD19 is used to mark B cells and QDs-525-CD31 and QDs-565-PNAd are used to mark high endothelial venules, and fluorescence microscopy is used for observation. When the average fluorescence intensity of CD19 is greater than 426.721, at the same time, the average fluorescence intensity of CD31 accounts for more than 544.131, and the average fluorescence intensity of PNad accounts for more than 395.623, it indicates that the sample has rich immune infiltration.

[0039] In certain embodiments of the present invention, Breg cell marker antibodies and B cell marker antibodies coupled with corresponding quantum dots are used to detect and observe the expression of B cells and their subpopulation Breg cells in the thymic immune microenvironment, and the average fluorescence intensity of the corresponding indicators is calculated to evaluate and predict autoimmune diseases; specifically, thymus tissue sections are used as detection objects, QDs-585-CD19 is used to mark B cells and QDs-585-CD19 and QDs-525-CD1d are used to mark Breg cells, and fluorescence microscope observation is performed. When the average fluorescence intensity of CD19 is less than 87.125, and the average fluorescence intensity of CD1d is greater than 365.113, the detection sample is judged to be a simple tumor tissue; when the average fluorescence intensity of CD19 is less than 245.527 and the average fluorescence intensity of CD1d is greater than 102.006, the detection sample is judged to be a tumor combined with a mild autoimmune disease tissue; when the average fluorescence intensity of CD19 is greater than 426.721 and the average fluorescence intensity of CD1d is less than 58.826, it is judged to be a tumor combined with a severe autoimmune disease tissue.

[0040] In certain embodiments of the present invention, Breg cell marker antibodies and high endothelial venule marker antibodies are coupled to corresponding quantum dots to detect the expression levels and relative spatial positions of the two in the thymic immune microenvironment, and the immune status of the thymic tissue immune microenvironment is analyzed by calculating the average fluorescence intensity of the corresponding indicators; specifically, thymic tissue sections are used as detection objects, QDs-525-CD1d is used to mark Breg cells, QDs-525-CD31 and QDs-565-PNAd are used to mark high endothelial venules; under fluorescence microscopy, the average fluorescence intensity of CD1d is less than 58.826, the average fluorescence intensity of CD31 is greater than 544.131, and the average fluorescence intensity of PNad is greater than 395.623, which indicates that the immune status in the tumor microenvironment is relatively active.

[0041] The immune infiltration status reflects the immune status of the immune microenvironment of the thymus tissue and further evaluates and predicts autoimmune diseases. In certain embodiments of the present invention, the above-mentioned detection process can be integrated to achieve mutual verification of the test results and improve the accuracy of the detection.

[0042] Based on quantum dots coupled to specific antibodies, thymic tissue specimens (tumor tissue or lymph node tissue with tumor metastasis or adjacent tissue to the tumor or other thymic disease tissue) are tested in vitro. The antigen-specific binding of quantum dots coupled to specific antibodies to label B cells, Breg cells, and high endothelial venules is used to achieve the localization observation and quantitative detection of B cells, Breg cells, and high endothelial venule structures in the thymic immune microenvironment, and to evaluate the immune infiltration status in the thymic immune microenvironment in vitro, further predict autoimmune diseases, and evaluate their severity. The detection kit for B cells and high endothelial venules in the thymic tissue immune microenvironment provides a detection and analysis method for detecting B cells, Breg cells, and high endothelial venules in the thymic immune microenvironment at the tissue level using quantum dots coupled to specific antibodies, which clarifies the concentration of quantum dots and specific antibodies and improves the accuracy and stability of the detection.

[0043] On the other hand, through quantum dot labeling, the content and location of B cells, Breg cells, and high endothelial venules can be clearly identified in the same tissue section. By studying tissue sections with known immune conditions, the role of B cells and Breg cells in autoimmune diseases can be further studied.

[0044] The scheme of the present invention is described below in conjunction with the accompanying drawings, wherein the experimental methods without specific operating steps are all carried out in accordance with the corresponding product instructions, and the instruments, reagents, and consumables used in the examples can all be purchased from commercial companies unless otherwise specified.

[0045] Example 1: Preparation of quantum dot-antibody conjugates

[0046] QDs, EDC, and NHS were mixed at a molar ratio of 1:3000:5500, diluted with PBS, and mixed on a shaker at room temperature for 30 minutes, and then the pH was adjusted to 8-9 with PBS to obtain a quantum dot activation solution. Take 100ul of the quantum dot activation solution and add it to the reaction container. Take an appropriate amount of antibody stock solution, dilute it to 0.01mg / ml with PBS buffer, mix it thoroughly, add it to the above reaction container according to different ratios, and react for 4 hours on a shaker at 4℃ in the dark. After the reaction is completed, centrifuge at 8000 rpm for 3 min to remove possible agglomerated precipitates, and keep the supernatant, which is the prepared QDs-Ab conjugate. The supernatant is purified with a PD10 column, and the purified product is stored in a refrigerator at 4℃ in the dark for later use. The supernatant can be detected and analyzed using 1% agarose gel electrophoresis.

[0047] In order to screen the optimal coupling conditions, samples with different mixing ratios of quantum dots and antibodies were prepared, and the molar ratios of quantum dots QDs and antibody proteins were 1:20, 1:40, and 1:80, respectively. The optimal coupling conditions can be screened by analyzing the results through 1% agarose gel electrophoresis.

[0048] QDs-585-CD19, QDs-525-CD31, QDs-565-PNAd and QDs-625-CD1d were prepared respectively according to the above method.

[0049] Example 2: Detection of thymus tissue

[0050] Thirty cases of simple thymoma (T), thymoma combined with mild myasthenia gravis (TMG L ) 30 cases, thymoma combined with severe myasthenia gravis (TMG H ) Thymoma tissue specimens from 30 patients were soaked in 4% formalin for more than 48 hours; wax blocks were made from thymoma tissue specimens and placed on a microtome to make 3-4μm tissue sections. Information such as the source of the sections, staining index, date, and antibody ratio were recorded before staining began.

[0051] Place the tissue slices in a 60°C oven for 30 minutes for baking. Dewax and dehydrate, transfer the slices to No. 1 100% xylene for 15 minutes, No. 2 100% xylene for 15 minutes, No. 3 100% ethanol for 3 minutes, No. 4 100% ethanol for 3 minutes, No. 5 95% ethanol for 3 minutes, No. 6 75% ethanol for 3 minutes, No. 7 50% ethanol for 3 minutes, and rinse with PBS 3 times for 5 minutes each time.

[0052] Antigen repair was performed on the dewaxed and dehydrated tissue sections. During antigen repair, acidic or alkaline antigen repair solution was selected according to the instructions of the corresponding antibody; the antigen repair solution was added to the container, placed in a microwave oven to heat and boil, and then the tissue sections were placed in the container, and heated on medium-low heat for 10 minutes before being taken out; after the tissue sections naturally cooled to room temperature, they were transferred to the immunohistochemistry washing box, washed with sterile water for 1 minute, and washed with 1×TBST for 2 minutes. The tissue was enclosed with an immunohistochemistry pen so that the entire tissue block on the section was located inside the seal.

[0053] The treated tissue sections were stained using QDs-585-CD19, QDs-525-CD31, QDs-565-PNAd and QDs-625-CD1d prepared in Example 1. The staining steps are as follows:

[0054] Blocking: Remove 1× TBST solution, slowly add 10% BSA, and incubate at room temperature for 10 minutes.

[0055] Prepare antibodies: dilute antibodies with 1× PBS to prepare quantum dot-labeled antibody solution.

[0056] Primary antibody incubation: Remove excess serum, add quantum dot-labeled antibodies, place tissue sections flat in an immunohistochemistry humidified box, and then place in a 4°C refrigerator overnight.

[0057] Rinse: Rinse three times with a wash bottle containing 1×TBST, 2 minutes each time, to remove unbound quantum dot-labeled antibodies.

[0058] When multiple quantum dot-labeled antibodies are needed for staining, the second quantum dot-labeled antibody can be stained after washing, and the staining steps are the same as above.

[0059] After the tissue sections are stained, the nuclei are stained. 1×DAPI solution is prepared with ddH20 1:100; 1×DAPI solution is added to the tissue sections, incubated at room temperature in the dark for 5 minutes, rinsed with 1×TBST for 2 minutes, and rinsed with ddH2O for 1 minute. After removing the excess liquid on the sections, 30μl of anti-fade mounting medium is added, and then the coverslip is covered. After the tissue sections are stable, the results are observed and photographed using a fluorescence microscope equipped with a 488nm laser. Among them, the green light signal represents CD31, the orange light signal represents CD19, the yellow light signal represents PNAd, and the red light signal represents CD1d.

[0060] 30 cases of simple thymoma (T), thymoma combined with mild myasthenia gravis (TMG L ) 30 cases, thymoma combined with severe myasthenia gravis (TMG H ) The test results of 30 patient samples were statistically analyzed, and the average fluorescence intensity of each quantum dot was analyzed by ImageJ software, which corresponds to the expression of the relevant antibody; the test images were analyzed by ImageJ under default conditions, and the images taken by the fluorescence microscope were analyzed by ImageJ software. The average fluorescence intensity Mean = the total fluorescence intensity of the region InDen / the area of ​​the region Area. The average fluorescence intensity of the corresponding antibodies corresponding to multiple samples is shown in Table 1.

[0061] Table 1

[0062]

[0063] Note: Data are shown as Mean±SD One-way ANOVA was used to detect the differences among the three groups, and comparisons were made between the two groups.

[0064] From the data in Table 1, it can be seen that thymoma tumor tissue samples in the same group will present relatively similar data results. The expression of CD19, CD31, PNAd or CD1d can be determined by the test results, thereby knowing the differentiation of B cells, Breg cells and high endothelial venules, and thus judging the immune status in the tumor microenvironment.

[0065] Further investigation was performed on 30 cases of simple thymoma (T), thymoma combined with mild myasthenia gravis (TMGL ) 30 cases, thymoma combined with severe myasthenia gravis (TMG H ) The detection data of 30 thymoma tumor tissue samples were analyzed to obtain standard data for determining the immune status in the tumor microenvironment.

[0066] In the TMGH group, the minimum mean fluorescence intensity of CD19 was 426.721, while the minimum mean fluorescence intensity of CD31 was 544.131, and the minimum mean fluorescence intensity of PNAd was 395.623. In the thymus tissue, the mean fluorescence intensity of CD19 was greater than 426.721, while the mean fluorescence intensity of CD31 was greater than 544.131, and the mean fluorescence intensity of PNAd was greater than 395.623, indicating that the sample was rich in immune infiltration.

[0067] In the T group, the maximum value of the CD19 mean fluorescence intensity was 87.125, and the minimum value of the CD1d mean fluorescence intensity was 365.113; in the TMGL group, the maximum value of the CD19 mean fluorescence intensity was 245.527, and the minimum value of the CD1d mean fluorescence intensity was 102.006; in the TMGH group, the minimum value of the CD19 mean fluorescence intensity was 426.721, and the maximum value of the CD1d mean fluorescence intensity was 58.826. When the CD19 mean fluorescence intensity was less than 87.125, and the CD1d mean fluorescence intensity was greater than 365.113, the test sample was judged to be pure tumor tissue; when the CD19 mean fluorescence intensity was less than 245.527 and the CD1d mean fluorescence intensity was greater than 102.006, the test sample was judged to be tumor combined with mild autoimmune disease tissue; when the CD19 mean fluorescence intensity was greater than 426.721 and the CD1d mean fluorescence intensity was less than 58.826, the test sample was judged to be tumor combined with severe autoimmune disease tissue.

[0068] In the TMGH group, the maximum value of the mean fluorescence intensity of CD1d was 58.826, the minimum value of the mean fluorescence intensity of CD31 was 544.131, and the minimum value of the mean fluorescence intensity of PNAd was 395.623. The mean fluorescence intensity of CD1d was less than 58.826, while the mean fluorescence intensity of CD31 was greater than 544.131, and the mean fluorescence intensity of PNAd was greater than 395.623, which was judged to be a relatively active immune state in the tumor microenvironment.

[0069] Example 3: Detection of B cells and high endothelial venules

[0070] Quantum dot-coupled antibodies QDs-585-CD19, QDs-525-CD31, and QDs-565-PNAd were prepared according to the method of Example 1. The tissue sections to be tested were processed according to the method of Example 2, and stained with QDs-585-CD19, QDs-525-CD31, and QDs-565-PNAd in turn. The detailed steps are as follows:

[0071] First round of dyeing:

[0072] Blocking: Remove 1× TBST solution, slowly add 10% BSA, and incubate at room temperature for 10 minutes.

[0073] Prepare antibodies: dilute the antibodies with 1×PBS to prepare QDs-585-CD19 quantum dot labeled antibody solution.

[0074] Incubation with the first antibody: remove excess serum, add QDs-585-CD19 quantum dot labeled antibody, place the tissue sections flat in an immunohistochemistry humidified box, and then place in a 4°C refrigerator overnight.

[0075] Rinse: Rinse three times with a wash bottle containing 1×TBST, 2 minutes each time, to remove unbound QDs-585-CD19 quantum dot-labeled antibodies.

[0076] After the first round of staining, the second round of staining was performed;

[0077] Blocking: Remove 1× TBST solution, slowly add 10% BSA, and incubate at room temperature for 10 minutes.

[0078] Prepare antibodies: dilute the antibodies with 1×PBS to prepare QDs-525-CD31 quantum dot-coupled antibody solution.

[0079] Secondary antibody incubation: remove excess serum, add QDs-525-CD31 quantum dot-coupled antibody, place tissue sections flat in an immunohistochemistry humidified box, and then place in a 4°C refrigerator overnight.

[0080] Rinse: Rinse three times with a wash bottle containing 1×TBST, 2 minutes each time, to remove unbound QDs-625-CD1d quantum dot-coupled antibodies.

[0081] Then carry out the third round of dyeing;

[0082] Blocking: Remove 1× TBST solution, slowly add 10% BSA, and incubate at room temperature for 10 minutes.

[0083] Prepare antibodies: dilute the antibodies with 1×PBS to prepare QDs-565-PNAd quantum dot-coupled antibody solution.

[0084] Incubation with the third antibody: remove excess serum, add QDs-565-PNAd quantum dot-coupled antibody, place the tissue sections flat in an immunohistochemistry humidified box, and then place in a 4°C refrigerator overnight.

[0085] Rinse: Rinse three times with a wash bottle containing 1×TBST, 2 minutes each time, to remove unbound QDs-525-CD31 quantum dot-coupled antibodies.

[0086] After the tissue sections are stained, the nuclei are stained. 1×DAPI solution is prepared with ddH20 1:100; 1×DAPI solution is added to the tissue sections, incubated at room temperature in the dark for 5 minutes, rinsed with 1×TBST for 2 minutes, and rinsed with ddH2O for 1 minute. After removing the excess liquid on the sections, 30μl of anti-fade mounting medium is added, and then the coverslip is covered. After the tissue sections are stable, the results are observed and photographed using a fluorescence microscope equipped with a 488nm laser. Among them, the green light signal represents CD31, the orange light signal represents CD19, and the yellow light signal represents PNAd.

[0087] The result is shown in Figure 1, where T is a simple thymoma tissue sample, TMGL is a thymoma combined with mild myasthenia gravis tissue sample, and TMGH is a thymoma combined with severe myasthenia gravis tissue sample; for the same tissue section, the single expression of different color light signals can be observed by switching different channels, so that the expression of CD31, CD19 and PNAd can be known respectively, and the correlation of CD31, CD19 and PNAd expression positions can be known through the overall image. The average fluorescence intensity of each quantum dot in the figure is shown in Table 2.

[0088] Table 2

[0089]

[0090] From the data in the table, we can know that in the tissues of patients with thymoma combined with severe myasthenia gravis, the average fluorescence intensity of CD19 is greater than 426.721, the average fluorescence intensity of CD31 is greater than 544.131, and the average fluorescence intensity of PNAd is greater than 395.623, indicating that the tissue sections provide rich immune infiltration; the average fluorescence intensity of the corresponding antibodies in the tissue samples of simple thymoma and thymoma combined with mild myasthenia gravis does not exceed the above standard value. The degree of immune infiltration of tissue samples can be judged based on the average fluorescence intensity of CD31, CD19 and PNAd obtained by detection.

[0091] Example 4: Detection of B cells and Breg cells

[0092] Quantum dot-coupled antibodies QDs-585-CD19 and QDs-625-CD1d were prepared according to the method of Example 1. The tissue sections of the sample to be tested were processed according to the method of Example 2 or 3, and stained with quantum dot-coupled antibodies in turn. The stained tissue sections were stained nuclear, and the results were observed and photographed using a fluorescence microscope equipped with a 488nm laser. Among them, the orange light signal represents CD19, and the red light signal represents CD1d.

[0093] The results are as follows Figure 2 As shown, T is a simple thymoma tissue sample, TMGL is a thymoma combined with mild myasthenia gravis tissue sample, and TMGH is a thymoma combined with severe myasthenia gravis tissue sample; for the same tissue section, the single expression of different color light signals can be observed by switching different channels, so that the expression of CD19 and CD1d can be known respectively, and the correlation between the expression positions of CD19 and CD1d can be known through the overall image. The average fluorescence intensity of each quantum dot in the figure is shown in Table 3.

[0094] Table 3

[0095]

[0096] From the data, we can see that the average fluorescence intensity of CD19 in the T group is less than 87.125, and the average fluorescence intensity of CD1d is greater than 365.113, judging that the test sample is pure tumor tissue; the average fluorescence intensity of CD19 in the TMGL group is less than 245.527 and the average fluorescence intensity of CD1d is greater than 102.006, judging that the test sample is tumor combined with mild autoimmune disease tissue; the average fluorescence intensity of CD19 in the TMGH group is greater than 426.721 and the average fluorescence intensity of CD1d is less than 58.826, judging that the test sample is tumor combined with severe autoimmune disease tissue. The judgment results obtained based on the data are consistent with the sample classification.

[0097] As myasthenia gravis worsens, CD19-marked B cells gradually increase, while CD1d-marked Breg cells gradually decrease.

[0098] Example 5: Detection of Breg cells and high endothelial venules

[0099] Quantum dot-coupled antibodies QDs-625-CD1d, QDs-525-CD31, and QDs-565-PNAd were prepared according to the method of Example 1. The tissue sections of the sample to be tested were processed according to the method of Example 2 or 3, and stained with quantum dot-coupled antibodies in turn. The stained tissue sections were stained nuclear, and the results were observed and photographed using a fluorescence microscope equipped with a 488nm laser. Among them, the green light signal represents CD31, the red light signal represents CD1d, and the yellow light signal represents PNAd.

[0100] The results are as follows Figure 3 As shown, T is a simple thymoma tissue sample, TMGL is a thymoma combined with mild myasthenia gravis tissue sample, and TMGH is a thymoma combined with severe myasthenia gravis tissue sample; for the same tissue section, the single expression of different color light signals can be observed by switching different channels, so that the expression of CD31, CD1d and PNAd can be known respectively. The average fluorescence intensity of each quantum dot in the figure is shown in Table 4.

[0101] Table 4

[0102]

[0103] From the data, we can see that the average fluorescence intensity of CD1d in the TMGH group is less than 58.826, while the average fluorescence intensity of CD31 is greater than 544.131, and the average fluorescence intensity of PNAd is greater than 395.623, which indicates that the immune state in the tumor microenvironment is relatively active. Breg cells marked by CD1d are expressed at a low level in thymoma combined with severe myasthenia gravis, while high endothelial venules marked by CD31 and PNAd are expressed at a high level. It can be concluded that as the autoimmune disease worsens, the immune state in the thymoma microenvironment gradually increases.

[0104] The embodiments of the present invention are described in detail above, but the contents described are only preferred embodiments of the present invention and cannot be considered to limit the scope of implementation of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A detection kit for B cells, Breg cells and high endothelial venules in the immune microenvironment of thymus tissue, characterized in that: It includes quantum dot-labeled antibody CD19, quantum dot-labeled antibody CD1d, quantum dot-labeled CD31 and quantum dot-labeled PNad.

2. The detection kit for thymus tissue immune microenvironment B cells, Breg cells and high endothelial venules according to claim 1, characterized in that: Quantum dots include QDs-585, QDs-625, QDs-525 and QDs-565.

3. The detection kit for thymus tissue immune microenvironment B cells, Breg cells and high endothelial venules according to claim 1, characterized in that: Including quantum dot-antibody conjugates QDs-585-CD19, QDs-625-CD1d, QDs-525-CD31, QDs-565-PNAd, as well as antigen repair solution and nucleic acid staining solution.

4. The detection kit for thymus tissue immune microenvironment B cells, Breg cells and high endothelial venules according to any one of claims 1 to 3, characterized in that: The quantum dots, EDC and NHS are mixed at a molar ratio of 1:2500-3500:5000-6000 to activate the quantum dots, and the antibody is added to the activated quantum dot solution. The molar ratio of quantum dots to specific antibodies is 1:20-80, and QDs-Ab conjugates are obtained after the reaction; the supernatant is taken as a quantum dot-labeled antibody solution that can be used for staining.

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