A kit and method for simultaneously detecting IL-6, IL-21 and T cell CD4 surface molecules in lymphoid tissue

Abstract

The present invention relates to a kit and method for simultaneously detecting IL-6, IL-21 and T cell CD4 surface molecules in lymphoid tissue, belonging to the field of biomedicine; the kit comprises quantum dots for labeling IL-6 antibodies, quantum dots for labeling IL-21 antibodies, and quantum dots for labeling CD4 antibodies; the antibodies labeled by quantum dots specifically bind to antigens in lymphoid tissue respectively; CD4T cells have the ability to secrete IL-6 and IL-21, and the relationship between them and IL-6 and IL-21 reflects the tissue immune activity. The present invention provides a kit for simultaneously detecting IL-6, IL-21 and T cell surface molecules in lymphoid tissue, locating the spatial relationship between two cytokines and T cells, calculating and analyzing the corresponding fluorescence intensity, and evaluating the immune situation; the kit has the advantages of high fluorescence intensity, accurate detection results, wide spectrum selection, wide applicability, stable fluorescence structure, long storage time, high sensitivity, good specificity, etc.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a kit and a method for simultaneously detecting IL-6, IL-21 and T cell CD4 surface molecules in lymphoid tissue. Background Art

[0002] As a multipotent cytokine, IL-6 was first discovered to have the function of promoting T cell proliferation and activation, B cell differentiation and participating in the acute phase of inflammation. The classic regulatory function of IL-6 is that IL-6 binds to its specific receptor IL-6R, leading to the dimerization of IL-6ST and activating the JAK / STAT signaling pathway, thereby exerting various physiological effects. In T cells, the effects of IL-6 mainly include the following aspects: (1) Promoting T cell proliferation: IL-6 can stimulate T cell proliferation and participate in the activation and differentiation of T cells; (2) Affecting T cell polarization: IL-6 can affect T cell differentiation, promote the generation of Th17 cells, and inhibit the development of Treg cells.

[0003] IL-21 is a cytokine that is mainly produced by activated CD4+ helper T cells (Th cells). IL-21 can affect multiple types of T cells, including CD4+ T cells, CD8+ T cells, and Treg cells. It can promote the generation and function of memory T cells and enhance the effector function of CD8+ T cells. In addition, IL-21 is involved in regulating the proliferation and maturation of B cells. IL-21 can induce T cell-dependent and T cell-independent B cell proliferation. In vitro mature B cells also rely on IL-21 and co-stimulatory signals for proliferation and survival. IL-21 can also promote the differentiation of B cells into plasma cells, playing an important role in the regulation of immune responses.

[0004] IL-6 and IL-21 also have certain interactions in T cell immunity. For example, IL-21 can promote the differentiation of Th17 cells, and cytokines such as IL-17 produced by Th17 cells can induce the surrounding tissues to produce IL-6, and IL-6 can promote the production of T cells into Th cells, forming a positive feedback regulation loop. In addition, IL-6 and IL-21 can also interact and regulate the function of T cells through shared signaling pathways, such as the STAT3 signaling pathway.

[0005] However, the effects of IL-6 and IL-21 on T cells may differ in different immune environments and disease states, and the specific mechanism of action requires further research to gain a deeper understanding. To solve the above problems, the present invention provides a kit and method for in vitro detection of the relationship between IL-6, IL-21 and T cells in the thymic tumor microenvironment, quantitatively studying IL-6, IL-21 and CD4+T cells through quantum dot staining, and observing the spatial relationship between the three in lymphoid tissue, thereby evaluating the immune environment and immune status of lymphoid tissue. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a kit and method for simultaneously detecting IL-6, IL-21 and T cell CD4 surface molecules in lymphoid tissue.

[0007] The technical solution adopted by the present invention is: a kit for simultaneously detecting IL-6, IL-21 and T cell CD4 surface molecules in lymphoid tissue, including quantum dot-labeled IL-6 monoclonal antibody, quantum dot-labeled IL-21 monoclonal antibody, and quantum dot-labeled T cell surface molecule CD4 monoclonal antibody.

[0008] Preferably, the quantum dots are QDs-525, QDs-585, and QDs-625, respectively.

[0009] Preferably, it comprises IL-6 antibody labeled with quantum dots QDs-525, IL-21 antibody labeled with quantum dots QDs-585, T cell surface molecule CD4 antibody labeled with quantum dots QDs-625, antigen repair solution and DNA fluorescent dye.

[0010] 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 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.

[0011] Preferably, the antigen retrieval solution is Tris-EDTA, and the DNA fluorescent stain is DAPI stain.

[0012] The method for detecting lymphoid tissue using a kit for simultaneously detecting IL-6, IL-21 and T cell CD4 surface molecules in lymphoid tissue comprises: first preparing lymphoid tissue sections from the test samples, performing antigen repair using an antigen repair solution, then staining the tissue sections with antibodies labeled with different quantum dots, then staining the cells, and collecting fluorescence information of the tissue sections to obtain the immune environment and immune status of the detected lymphoid tissue.

[0013] Preferably, when the quantum dots are stained, IL-6 is stained first, then IL-21, and finally CD4.

[0014] Preferably, the quantitative status and spatial localization status of IL-6, IL-21 and T cells are reflected by the fluorescence information of quantum dots of different colors.

[0015] Preferably, when IL-6 is scattered and the mean fluorescence intensity is enhanced; IL-21 is scattered and the mean fluorescence intensity is enhanced; CD4 T cells increase and the mean fluorescence intensity is enhanced; and the spatial position relationship of the three is that IL-6 and IL-21 are evenly distributed on CD4 T cells, it is considered that the immune state is hyperactive.

[0016] Preferably, when the average fluorescence intensity of CD4 is greater than 23.206, the average fluorescence intensity of IL-6 is less than 32.608, the average fluorescence intensity of IL-21 is less than 50.645, and the fluorescence overlap rate of CD4, IL-6 and IL-21 is greater than 60%, the test sample is judged to have hyperimmune function.

[0017] The beneficial effects of the kit provided by the present invention are as follows: quantum dots are used for fluorescent labeling of samples, so that the kit has the advantages of high fluorescence intensity, accurate detection results, wide spectrum selection, wide applicability, stable fluorescence structure, long storage time, high sensitivity, good specificity, etc.; the kit is simple to use, the labeling process is stable and effective, and it is easy to use;

[0018] Through the coordination of three-color quantum dots, IL-6, IL-21 and T cells can be quantified and located simultaneously, which can not only realize the quantitative detection of the three components at the same time, but also observe the spatial position relationship of the three, so as to more effectively and accurately evaluate the body's immune status and immune function;

[0019] The present invention provides a kit for simultaneously detecting IL-6, IL-21 and T cell surface molecule CD4 in lymphoid tissue in vitro. The kit integrates a primary antibody for labeling immune cells and all reagents for quantum dot staining, which is convenient and fast and can provide more accurate services for scientific researchers. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 HE staining of tumor tissue sections of patients with simple thymoma and thymoma combined with autoimmune disease;

[0021] Figure 2 The expression of CD4+T lymphocytes in the tumor microenvironment of patients with simple thymoma and thymoma combined with autoimmune disease;

[0022] Figure 3The expression of IL-6 cytokine in tumor tissues of simple thymoma and thymoma combined with autoimmune disease;

[0023] Figure 4 The expression of IL-21 cytokine in tumor tissues of simple thymoma and thymoma combined with autoimmune disease;

[0024] Figure 5 The spatial distribution of CD4+T lymphocytes, IL-6 cytokines, and IL-21 cytokines in tumor tissues of simple thymoma and thymoma combined with autoimmune disease. DETAILED DESCRIPTION

[0025] The embodiments of the present invention are described below with reference to the accompanying drawings.

[0026] The present invention relates to a kit and method for simultaneously detecting IL-6, IL-21 and T cell CD4 surface molecules in lymphoid tissue. The kit comprises antibodies respectively labeled with different quantum dots, which can specifically label IL-6, IL-21 and T cell CD4 surface molecules in lymphoid tissue respectively; the selected quantum dots are QDs-525, QDs-585 and QDs-625, which are green, orange and red respectively; the antibodies labeled with quantum dots respectively label IL-6, IL-21 and T cell CD4 in tissue samples, and different factors or proteins in the same sample can be detected respectively, and IL-6, IL-21 and CD4+T cells can be quantitatively and positionally analyzed simultaneously, so that the immune environment and immune status of the tissue sample can be evaluated.

[0027] The kit for in vitro detection of IL-6, IL-21 and T cell surface molecule CD4 in lymphoid tissues includes quantum dot-labeled IL-6 monoclonal antibody, quantum dot-labeled IL-21 monoclonal antibody, quantum dot-labeled T cell surface molecule CD4 monoclonal antibody, antigen repair solution, and DNA fluorescent dye. The quantum dot-labeled IL-6, IL-21, and CD4 monoclonal antibodies specifically bind to the corresponding antigens in the lymphoid tissue samples, respectively, so as to quantitatively analyze and spatially locate IL-6, IL-21 and T cells. Among them, the IL-6 antibody is coupled with quantum dots QDs-525, and the emission light is displayed as green; the IL-21 antibody is coupled with quantum dots QDs-585, and the emission light is displayed as orange; the T cell surface molecule CD4 antibody is coupled with quantum dots QDs-625, and the emission light is displayed as red. In view of the differences in the expression levels of surface receptors in lymphoid tissues, the order of staining IL-6 first, then IL-21, and finally CD4 was selected; in certain embodiments of the present invention, the antigen repair solution is Tris-EDTA, and the DNA fluorescent stain is DAPI stain.

[0028] The coupling of quantum dots to specific antibodies includes the following steps:

[0029] 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;

[0030] 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

[0031] 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.

[0032] When the kit for simultaneously detecting IL-6, IL-21 and T cell surface molecule CD4 in lymphoid tissue in vitro is used to detect lymphoid tissue samples, IL-6 is stained first, then IL-21, and finally CD4. Each quantum dot staining step includes staining, incubation and washing processes. The specific operation process is as follows:

[0033] B1: Preparation of lymphatic tissue pathological sections, gradient ethanol dehydration, immersion and transparency, paraffin permeation, paraffin embedding, sectioning, mounting, baking, and hydration;

[0034] B2: Use antigen retrieval solution to perform antigen retrieval on the hydrated pathological tissue specimens;

[0035] B3: Use quantum dot-labeled antibody reagents to stain pathological tissue specimens; each quantum dot staining step includes staining, incubation and rinsing processes; first seal the treated sample, then mix and incubate the prepared antibody solution with the sample slices, and then rinse after staining to complete a round of staining; according to the above steps, stain IL-6 first, then IL-21, and finally CD4;

[0036] B4: Use DNA fluorescent dye to stain the cell nucleus;

[0037] B5: Dehydrate the stained lymphoid tissue specimens, seal the slides, observe and draw conclusions.

[0038] When IL-6 is scattered, the average fluorescence intensity increases; when IL-21 is scattered, the average fluorescence intensity increases; when the number of CD4 T cells increases, the average fluorescence intensity increases; the spatial position relationship of the three is: IL-6 and IL-21 are evenly distributed on CD4 T cells, which can be considered as an immune hyperactivity.

[0039] A kit and method for simultaneously detecting IL-6, IL-21 and T cell surface molecule CD4 in lymphoid tissue in vitro, by coupling quantum dots with specific antibodies IL-6, IL-21 and T cell surface molecule CD4, so that the kit has the advantages of high fluorescence intensity, accurate detection results, wide spectrum selection, wide applicability, stable fluorescence structure, long storage time, high sensitivity, good specificity, etc. Specific antibodies labeled with quantum dots specifically bind to IL-6, IL-21 and T cell surface molecule CD4 in lymphoid tissue, and the expression of IL-6, IL-21 and T cell surface molecule CD4 in lymphoid tissue can be effectively observed through fluorescence intensity, and the spatial distribution of IL-6, IL-21 and T cell surface molecule CD4 can be displayed through different colors of emitted light, so as to effectively evaluate the number and function of T cells in lymphoid tissue and the immune status of lymphoid tissue in related areas.

[0040] Through calculation, the fluorescence overlap rate of CD4, IL-6 and IL-21 in lymphoid tissue with normal immune function is less than 35%. In lymphoid tissue with hyperimmune function, when the average fluorescence intensity of CD4 is greater than 23.206, the average fluorescence intensity of IL-6 is less than 32.608, the average fluorescence intensity of IL-21 is less than 50.645, and the fluorescence overlap rate of CD4, IL-6 and IL-21 is greater than 60%, the test sample is judged to have hyperimmune function.

[0041] The present invention uses quantum dot staining technology to perform in vitro detection of tumor tissue specimens (or lymph node tissue with tumor metastasis or adjacent tumor tissue) of patients including but not limited to thymoma, locates the tumor microenvironment through immune molecule markers CD4+ T lymphocytes, cytokines IL6 and IL21, and then analyzes the spatial localization relationship and number of immune cells therein, and evaluates the immune infiltration in the tumor microenvironment in vitro, which has guiding significance for thymoma research.

[0042] 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.

[0043] Example 1: Preparation of QDs-Ab conjugates

[0044] 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.

[0045] 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.

[0046] Embodiment 2:

[0047] According to the method of Example 1, quantum dots QDs-525 were used to label IL-6, QDs-58 to label IL-21, and QDs-625 to label CD4. Among them, the IL-6 monoclonal antibody was abcam's catalog number ab9324, the IL-21 monoclonal antibody was abcam's catalog number ab154767, and the T cell surface molecule CD4 monoclonal antibody was abcam's catalog number ab133616.

[0048] Collect 30 simple thymoma and 30 thymoma combined with autoimmune disease tumor tissue specimens, fix them in 4% formalin for more than 48 hours, take out the tissues and put them in embedding boxes, number and mark them, and then dehydrate them with gradient ethanol, soaking them in 70% ethanol for 30 minutes, 80% ethanol for 30 minutes, 90% ethanol for 30 minutes, 90% ethanol for 30 minutes, 100% ethanol for 30 minutes, and 100% ethanol for 30 minutes. Mix equal amounts of pure alcohol and xylene for 15 minutes, and xylene for 0.5 hours. If the tissue is not transparent enough, it should be soaked in xylene until the slices are transparent. Put the sliced ​​sample into a mixture of half xylene and half paraffin for 15 minutes, and then put it into paraffin I and paraffin II for 20-30 minutes each; melt the paraffin at 52-56℃, and immediately put the paraffined tissue and the melted paraffin into cold water to solidify it into a wax block; fix the fixed and repaired paraffin block on the clamping table of the slicer, adjust the blade angle, and stick the blade to the wax block. Select a suitable position and slice the wax block continuously with a thickness of 5um; take a clean glass slide, drop a drop of sticky tablet in the center, put the cut wax tape on the water surface with tweezers, and then stick it on the glass slide, pay attention to the placement, and then flatten the wax sheet at 40-45℃, stick a label and mark it, and dry it in a 37℃ oven. Record the slice information, staining index, date, antibody ratio and other information.

[0049] Place the patient's thymoma tumor tissue sections in a 60°C oven for 0.5-2h. Then place the tissue sections in No. 1 100% xylene for 5min, No. 2 100% xylene for 5min, No. 3 100% ethanol for 5min, No. 4 100% ethanol for 5min, No. 5 95% ethanol for 5min, No. 6 75% ethanol for 5min, No. 7 50% ethanol for 5min, and finally rinse with ddH2O 3 times for 2min each time. Choose an acidic or alkaline antigen repair solution according to the antibody instructions; commonly used antigen repair solutions are 1×Tris-EDTA antigen repair solution and 1×citric acid antigen repair solution. During antigen repair, add the antigen repair solution to the immunohistochemistry antigen repair container and heat it in a microwave oven at high heat until boiling; then place the tissue sections in the antigen repair solution, adjust the microwave oven to low heat for 10min, take out the antigen repair container, and cool it naturally to room temperature.

[0050] The tissue sections were removed and rinsed with ddH2O for 1 min and 1×TBST for 2 min. The tissue was enclosed with an immunohistochemical pen so that the entire tissue block on the section was within the enclosed circle.

[0051] The above-treated tissue sections were stained with HE. The results were as follows: Figure 1 As shown, Figure 1 The left is the HE staining result of simple thymoma tissue section. Figure 1 The right side shows the HE staining results of tumor tissue sections of thymoma combined with autoimmune disease.

[0052] In addition, the above-treated tissue sections were sequentially stained with different quantum dot-antibody conjugates.

[0053] First round of dyeing:

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

[0055] B. Prepare antibodies: dilute the antibodies with 1×PBS to prepare QDs-525 quantum dot-labeled IL-6 antibody solution.

[0056] C. Primary antibody incubation: Remove excess serum, add QDs-525-IL-6 quantum dot labeled antibody, place tissue sections flat in an immunohistochemistry humidified box, and then place in a 4°C refrigerator overnight.

[0057] D. Rinse: Rinse three times with a wash bottle containing 1×TBST, each time for 2 minutes.

[0058] Second round of dyeing:

[0059] A. Blocking: Remove 1× TBST solution, slowly add 10% BSA, and incubate at room temperature for 10 minutes.

[0060] B. Prepare antibodies: dilute the antibodies with 1×PBS to prepare QDs-585 quantum dot-labeled IL-21 antibody solution.

[0061] C. Secondary antibody incubation: Remove excess serum, add QDs-585-IL-21 quantum dot labeled antibody, place tissue sections flat in an immunohistochemistry humidified box, and then place in a 4°C refrigerator overnight.

[0062] D. Rinse: Rinse three times with a wash bottle containing 1×TBST, each time for 2 minutes.

[0063] The third round of dyeing:

[0064] A. Blocking: Remove 1× TBST solution, slowly add 10% BSA, and incubate at room temperature for 10 minutes.

[0065] B. Prepare antibodies: dilute the antibodies with 1×PBS and prepare CD4 antibody solution labeled with QDs-625 quantum dots.

[0066] C. Primary antibody incubation: Remove excess serum, add QDs-625-CD4 quantum dot labeled antibody, place tissue sections flat in an immunohistochemistry humidified box, and then place in a 4°C refrigerator overnight.

[0067] D. Rinse: Rinse three times with a wash bottle containing 1×TBST, each time for 2 minutes.

[0068] Nuclear DNA staining: 1×DAPI solution was prepared with ddH20 at a ratio of 1:100; 1×DAPI solution was added to the sliced ​​tissue, incubated at room temperature in the dark for 5 min, rinsed with 1×TBST for 2 min, and rinsed with ddH2O for 1 min; 30 μl of anti-fade mounting agent was added and the coverslip was covered, and the periphery was sealed with a special edge sealing agent; after the tissue slices were stabilized, a fluorescence microscope equipped with a 488nm laser was used to observe the results and take pictures, where the green light signal represents IL-6, the orange light signal represents IL-21, and the red light signal represents CD4. ImageJ was used to analyze the obtained images, and the image format was set to 8-bit format; the background was set, the Threshold was adjusted, and the threshold was manually selected to ensure that all stained areas were selected; the measurement parameters were set, and the average gray value was calculated; the data was obtained, analyzed, and the immune status was evaluated.

[0069] The test results of tumor tissue sections of 30 cases of simple thymoma and 30 cases of thymoma combined with autoimmune diseases were statistically analyzed. The mean fluorescence intensity of the quantum dot corresponding antibody was calculated by ImageJ software. The obtained immunofluorescence images were converted to 8-Bit format using ImageJ, and the "Threshold" area was adjusted to use the default threshold, "DarkBackground" was checked, and the default algorithm was used to calculate the mean fluorescence intensity Mean; the statistical formula of ImageJ software is Mean fluorescence intensity Mean = the sum of the fluorescence intensity of the region InDen / the area of ​​the region Area. The average value of the mean fluorescence intensity of each group was calculated, and the details are shown in Table 1.

[0070] Table 1

[0071]

[0072] From the data in the table, it can be seen that the expression of IL-6, IL-21 or CD4 in the same group is similar. The immune function status can be determined by the expression of IL-6, IL-21 or CD4. Through the analysis of 60 groups of data, the average fluorescence intensity of IL-6 in lymphoid tissues with normal immune function is 44.543±5.584, while that in lymphoid tissues with hyperimmunity is: 28.432±4.176; the average fluorescence intensity of IL-21 in lymphoid tissues with normal immune function is 66.953±6.038, while that in lymphoid tissues with hyperimmunity is: 45.657±4.988; the average fluorescence intensity of CD4 in lymphoid tissues with normal immune function is: 7.867±0.909; while that in lymphoid tissues with hyperimmunity is 26.318±3.112. When the average fluorescence intensity of CD4 is greater than 23.206, the average fluorescence intensity of IL-6 is less than 32.608, and the average fluorescence intensity of IL-21 is less than 50.645, the subject is judged to have hyperimmunity; the fluorescence overlap rate of CD4 and IL-6, IL-21 in lymphoid tissue with normal immune function is less than 35%, while it is greater than 60% in lymphoid tissue with hyperimmunity.

[0073] The results are as follows Figure 2-Figure 5 As shown, the left picture is a simple thymoma tissue section sample, and the right picture is a tumor tissue section sample of thymoma combined with autoimmune disease; Figure 2 The red light signal distribution represents the expression content and spatial position of CD4. The average fluorescence intensity of CD4 in simple thymoma tissue is 7.927, and the average fluorescence intensity of CD4 in tumor tissue of thymoma combined with autoimmune disease is 28.577; Figure 3 The distribution of green light signals represents the expression content and spatial location of IL-6. The average fluorescence intensity of IL-6 in simple thymoma tissue is 50.182, and the average fluorescence intensity of IL-6 in tumor tissue of thymoma combined with autoimmune disease is 26.489; Figure 4 The orange light signal distribution represents the expression content and spatial location of IL-21. The average fluorescence intensity of IL-21 in simple thymoma tissue is 64.042, and the average fluorescence intensity of IL-21 in tumor tissue of thymoma combined with autoimmune disease is 43.898; Figure 5 for Figure 2-Figure 4 The image formed after mixing can determine the positional relationship between IL-6, IL-21 and CD4 from the various color signals and positional relationships in the image. The fluorescence overlap rate of CD4 with IL-6, IL-21 in simple thymoma tissue is 15.326%, and the fluorescence overlap rate of CD4 with IL-6, IL-21 in tumor tissue of thymoma combined with autoimmune diseases is 77.86%.

[0074] 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 kit for simultaneously detecting IL-6, IL-21 and T cell CD4 surface molecules in lymphoid tissue, characterized in that: Used to detect lymphatic tissue sections, the kit includes quantum dot-labeled IL-6 monoclonal antibody, quantum dot-labeled IL-21 monoclonal antibody, and quantum dot-labeled T cell surface molecule CD4 monoclonal antibody; the quantum dots are QDs-525, QDs-585, and QDs-625 respectively; When in use, different quantum dot-labeled antibodies are used to stain lymph tissue sections, and the quantitative and spatial localization of IL-6, IL-21 and T cells are reflected through the fluorescence information of quantum dots of different colors. When IL-6 is scattered, the average fluorescence intensity is enhanced; IL-21 is scattered, the average fluorescence intensity is enhanced; the number of CD4 T cells is increased, and the average fluorescence intensity is enhanced; the spatial position relationship of the three is that IL-6 and IL-21 are evenly distributed on CD4 T cells, then it is considered that the immune state is hyperactive.

2. The kit for simultaneously detecting IL-6, IL-21 and T cell CD4 surface molecules in lymphoid tissue according to claim 1, characterized in that: It includes IL-6 antibody labeled with quantum dots QDs-525, IL-21 antibody labeled with quantum dots QDs-585, T cell surface molecule CD4 antibody labeled with quantum dots QDs-625, antigen repair solution and DNA fluorescent dye.

3. The kit for simultaneously detecting IL-6, IL-21 and T cell CD4 surface molecules in lymphoid tissue according to claim 1 or 2, 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 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.

4. A kit for simultaneously detecting IL-6, IL-21 and T cell CD4 surface molecules in lymphoid tissue according to any one of claims 1 to 3, characterized in that: The antigen retrieval solution was Tris-EDTA, and the DNA fluorescent stain was DAPI stain.

5. A kit for simultaneously detecting IL-6, IL-21 and T cell CD4 surface molecules in lymphoid tissue according to any one of claims 1 to 3, characterized in that: First, the test sample is used to prepare lymphatic tissue sections, and antigen repair is performed using antigen repair solution. The tissue sections are then stained with antibodies labeled with different quantum dots, and then the cells are stained. The fluorescence information of the tissue sections is collected to obtain the immune environment and immune status of the tested lymphatic tissue.

6. The kit for simultaneously detecting IL-6, IL-21 and T cell CD4 surface molecules in lymphoid tissue according to claim 5, characterized in that: When staining with quantum dots, IL-6 was stained first, then IL-21, and finally CD4.

Images