A method for detecting B cell subsets in lymphoid tissue

Abstract

The present invention relates to a method for detecting B cell subsets in lymphoid tissue, wherein lymphoid tissue sections are stained by a kit including quantum dot-antibody CD19 conjugate, quantum dot-antibody CD138 conjugate and quantum dot-antibody CD1d conjugate, and different antibodies are marked by quantum dots of different colors to realize the confirmation of the content and position of B cells, plasma cells and regulatory B cells in tissue sections, so as to facilitate the clarification of their biological behaviors. The method of the present invention improves the existing multicolor immunofluorescence technology, uses a method of carrying antibodies with quantum dots, eliminates the secondary antibody staining link, adopts direct labeling, and gives full play to the advantages of quantum dots themselves, saves experimental time, and improves efficiency.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a method for detecting B cell subsets in lymphoid tissue. Background Art

[0002] Lymphoid organs are immune organs in the body that realize immune functions. They are usually classified into primary lymphoid organs, secondary lymphoid organs, and tertiary lymphoid organs according to the hierarchical structure. Primary lymphoid organs include the thymus and bone marrow, secondary lymphoid organs mainly include lymph nodes, spleen, tonsils, etc. Tertiary lymphoid organs are aggregates of lymphocytes formed after birth. These lymphocyte aggregates can appear in any chronic inflammatory sites of non-lymphoid tissues in the body, such as autoimmune diseases, to regulate the immune response of local tissues. During the immune response process, lymphoid tissues are directly exposed to the inflammatory environment and receive the effects of stimulating antigens and cytokines. While rapidly generating an immune response, excessive lymphocyte activation may also occur, leading to an "inflammatory factor storm", which may also be closely related to the onset of autoimmune system diseases.

[0003] Autoimmune diseases are immunological disorders caused by multiple factors, often accompanied by hyperfunction of B cells and inhibition of T cell function, which also cause the body to produce immune responses to self-antigens, secrete multiple antibodies to attack target organs and tissues, and thus cause diseases. The immune imbalance caused by autoimmune diseases is mainly due to the excessive participation of T cells in the differentiation and maturation of B cells into plasma cells, which causes plasma cells to produce a large number of autoantibodies. As an important source of antibody-producing cells, B cells play an important role in autoimmune diseases. They have certain differences in cell subsets both in lymphoid tissues and in the peripheral blood of patients, and different B cell subsets also play different functions in the disease. At present, relevant research on B cells in bone marrow, peripheral blood and peripheral lymph nodes has made certain progress, but the expression and mechanism of action of B cell subsets in lymphoid tissues are still unclear, which has caused great obstacles to the research on autoimmune diseases with central immunity as the core.

[0004] Existing technologies usually use confocal microscopy or multi-marker immunofluorescence staining technology to detect the classification of cell subpopulations in tissue microenvironment. Although it can accurately identify a certain type of multi-marker cell subpopulation, traditional staining technology has defects and poor stability, making it difficult to reveal the important role played by B cell subpopulations in lymphoid tissues during the occurrence of autoimmune diseases. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for detecting B cell subsets in lymphoid tissue.

[0006] The technical solution adopted by the present invention is: a kit for detecting B cell subpopulations in lymphoid tissue, comprising a quantum dot-antibody CD19 conjugate, a quantum dot-antibody CD138 conjugate and a quantum dot-antibody CD1d conjugate.

[0007] Preferably, the quantum dots are QDs-525, QDs-585, and QDs-625, respectively.

[0008] Preferably, it comprises quantum dot-coupled antibody QDs-585-CD19, quantum dot-coupled antibody QDs-525-CD138, quantum dot-coupled antibody QDs-525-CD1d, antigen repair solution and DNA fluorescent dye.

[0009] 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.

[0010] A method for detecting B cell subsets in lymphoid tissue uses a kit for detecting B cell subsets in lymphoid tissue; antibody CD19, antibody CD138 and antibody CD1d in lymphoid tissue sections are labeled in sequence with different quantum dot-antibody conjugates to achieve detection of B cell subsets in lymphoid tissue.

[0011] Preferably, the specific antibody used to mark B cells is CD19; the specific antibodies used to mark plasma cells are CD19 and CD138; and the specific antibodies used to mark regulatory B cells are CD19 and CD1d.

[0012] Preferably, lymphoid tissue sections are prepared first, and antigen repair is performed using an antigen repair solution. The tissue sections are then stained using antibodies labeled with different quantum dots, and then the cells are stained. The fluorescence information of the tissue sections is collected to obtain the composition of B cell subpopulations in the detected lymphoid tissue.

[0013] The application of detection methods of B cell subsets in lymphoid tissue in studying the state of autoimmune diseases for non-disease diagnosis and treatment purposes.

[0014] Preferably, the fluorescence intensity of quantum dots is counted and the average fluorescence intensity of the corresponding antibodies is calculated; when the average fluorescence intensity of CD19 is greater than 484.029, it indicates that the subject's autoimmune state is hyperactive; at the same time, the average fluorescence intensity of CD138 is greater than 416.87 and the average fluorescence intensity of CD1d is less than 85.614, indicating that the subject's autoimmune hyperactivity is severe.

[0015] The advantages and positive effects of the present invention are: by replacing fluorescent dyes with quantum dots, direct labeling of tissue sections can be achieved, local localization of total B cells, plasma cells and regulatory B cells in lymphoid tissues can be achieved, and both the quantity and position of cells can be detected, which facilitates the clarification of their biological behaviors;

[0016] By detecting the expression of total B cells, plasma cells and regulatory B cells in the patient's lymphoid tissue in vitro, the patient's immune status is evaluated from the pathogenesis level, which overcomes the disadvantages of the previous limited evaluation methods and is more conducive to the prediction of the onset of clinical lymphoid tissue-related autoimmune diseases;

[0017] The existing multi-color immunofluorescence technology has been improved by using quantum dot staining. The method of using quantum dots to carry antibodies eliminates the secondary antibody staining step, adopts direct labeling, and takes advantage of the quantum dots themselves to save experimental time and improve efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Expression of CD19 in lymphoid tissue sections of severe myasthenia gravis;

[0019] Figure 2 The expression and distribution of CD19 and CD138 in the tissue sections of lymphadenopathy in severe myasthenia gravis;

[0020] Figure 3 The expression and distribution of CD19 and CD1d in the tissue sections of lymphadenopathy in severe myasthenia gravis. DETAILED DESCRIPTION

[0021] The embodiments of the present invention are described below with reference to the accompanying drawings.

[0022] The present invention relates to a method for detecting B cell subsets in lymphatic tissue. After dyeing lymphatic tissue sections with a quantum dot detection kit, the content and position of specific antibodies are observed to judge the immune level of lymphatic tissue. The quantum dot detection kit includes a quantum dot-specific antibody conjugate, an antigen repair solution and a DNA fluorescent dye, and the quantum dot-specific antibody conjugate specifically includes a quantum dot-antibody CD19 conjugate, a quantum dot-antibody CD138 conjugate and a quantum dot-antibody CD1d conjugate. The quantum dots in the kit are used to mark B cells, plasma cells and regulatory B cells. After dyeing a lymphatic tissue section sample, a specific antibody is specifically connected to the antigen on the total B cells, plasma cells and regulatory B cells in the section sample cells, and the quantum dots are used to quantify or locate the total B cells, plasma cells and regulatory B cells. Instead of the gene level or antibody detection method, the detection of total B cells, plasma cells and regulatory B cells is directly achieved by taking lymphatic tissue sections as the detection object.

[0023] The specific antibody used to mark B cells is CD19; the specific antibodies used to mark plasma cells are CD19 and CD138; the specific antibodies used to mark regulatory B cells are CD19 and CD1d. Quantum dots of different colors are coupled to antibody proteins respectively, and then used to stain tissue sections, so as to achieve the positioning or quantification of total B cells, plasma cells and regulatory B cells. The distribution trend of various types of B cells can be judged by the position distribution of quantum dots of different colors, and the proportion of plasma cells and regulatory B cells in total B cells can also be judged by the ratio between different quantum dots. Multicolor immunofluorescence technology uses traditional fluorescent dyes, which have certain defects. The emission spectrum wavelength of traditional dyes is limited; the photostability is poor; the spectrum overlaps easily between different fluorescent dyes; the biocompatibility is poor, and it is not easy to carry out specific labeling; the fluorescence lifetime is short, it is difficult to preserve for a long time, and the background interference is strong. The use of quantum dots instead of fluorescent dyes can avoid the above problems as much as possible.

[0024] The coupling of quantum dots to specific antibodies includes the following steps:

[0025] 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;

[0026] A2: Preparation of QDs-Ab conjugate: adding specific antibody to activated quantum dot solution, the molar ratio of quantum dots to specific antibody is 1:20-80; reacting on a shaker at 4°C in the dark, and obtaining QDs-Ab conjugate after reaction;

[0027] 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.

[0028] By changing the different feed ratios of quantum dots, required cross-linking agents and antibodies, stable loading conditions were developed to achieve stable binding of quantum dots and antibodies. The obtained QDs-Ab can be used immediately, and quantum dots of different colors and different antibodies can be combined according to the steps in the instructions to achieve personalized customization. An inappropriate ratio of QDs to Ab will result in poor stability of the obtained complex, easy polymerization, significantly affecting its biological compatibility, and unable to be used for biological applications, while excessive cross-linking agents will cause cross-linking between Ab and polymerization of QDs. It is necessary to accurately control the ratio between QDs, Ab and cross-linking agents to obtain a conjugate with high uniformity (i.e., a conjugate with only one Ab molecule bound to the surface of each nanoparticle) and stability. In addition, the purification of the biological conjugate after the coupling of quantum dots and antibodies is currently commonly used by gel filtration chromatography and membrane ultrafiltration, and the separation and purification rate is low. The method of the present invention uses PD10 column purification, which effectively improves the purification rate and simplifies the purification steps.

[0029] The kit for detecting B cell subsets in lymphoid tissue includes quantum dot-coupled antibody QDs-585-CD19, quantum dot-coupled antibody QDs-525-CD138, quantum dot-coupled antibody QDs-525-CD1d, antigen retrieval solution Tris-EDTA and DNA fluorescent dye DAPI.

[0030] The specific steps of the method for detecting the composition of total B cells, plasma cells and regulatory B cells in lymphoid tissue by using the above different QDs-Ab conjugates are as follows:

[0031] Step 1: Take lymphatic tissue specimens to make tissue sections, bake the tissue sections and dewax and dehydrate them;

[0032] Step 2: Perform antigen repair on tissue sections to facilitate the binding of specific antibodies;

[0033] Step 3: staining the tissue section with the quantum dot and antibody protein conjugate. When there are multiple quantum dots and antibody protein conjugates, the staining is performed in sequence. Each quantum dot staining step includes staining, incubation and washing processes. After fully removing the unbound quantum dots and antibody protein conjugates, the next staining step is performed.

[0034] Step 4: After all quantum dots are stained, the cell nuclei are stained, the slides are sealed and photographed, the location and number of target cells are analyzed based on the photo information, and statistical analysis is performed.

[0035] Combining quantum dots with the required antibodies makes up for the shortcomings of traditional fluorescent dyes. At the same time, the special combination method allows quantum dots and antibodies to be stably combined to achieve complementary advantages. Quantum dot multi-labeling immunofluorescence technology can effectively observe the expression level and spatial position structure of total B cells, plasma cells and regulatory B cells; the average fluorescence intensity of the corresponding immune cell markers can be calculated with high accuracy, and the status of autoimmune diseases can be judged without combining other markers. It has good sensitivity and specificity for early and latent autoimmune diseases. The immune status is evaluated by detecting the expression levels of total B cells, plasma cells and regulatory B cells in the patient's lymphoid tissue in vitro.

[0036] The mean fluorescence intensity of plasma cells, regulatory B cells and total B cells in lymphoid tissue is quantitatively associated with the severity of the autoimmune disease. In addition, in certain embodiments of the present invention, the quantity information and position information of total B cells, plasma cells and regulatory B cells can be detected simultaneously, and the quantity information and position information of total B cells, plasma cells and regulatory B cells can be analyzed.

[0037] The fluorescence intensity of each quantum dot was counted by ImageJ software, and the average fluorescence intensity of the corresponding antibody was calculated to compare the expression of each antibody. The statistical formula of ImageJ software is average fluorescence intensity Mean = the sum of the fluorescence intensity of the region InDen / the area of ​​the region Area. The possibility and severity of autoimmune diseases were measured by calculating the average fluorescence intensity of CD19, CD138 and CD1d. When the average fluorescence intensity of CD19 is greater than 484.029, it indicates that the possibility of autoimmune disease is high; at the same time, the average fluorescence intensity of CD138 is greater than 416.87 and the average fluorescence intensity of CD1d is less than 85.614, indicating that the degree of autoimmune disease is severe.

[0038] The classic means of clinical diagnosis and evaluation of autoimmune diseases mainly rely on the patient's clinical manifestations, serum antibody level detection, etc. Due to the limitations of sensitivity and specificity, the accuracy of the disease assessment for various early autoimmune diseases is poor, and there is a lack of effective evaluation methods at the pathogenesis level. By analyzing the expression of total B cells, plasma cells and regulatory B cells in lymphoid tissue, the patient's immune status can be evaluated from the pathogenesis level, overcoming the disadvantages of the limited evaluation methods in the past. The total B cells, plasma cells and regulatory B cells are counted by quantum dot multi-labeled immunofluorescence technology, with better sensitivity and specificity, which is more conducive to obtaining accurate expression of total B cells, plasma cells and regulatory B cells in lymphoid tissue. In certain embodiments of the present invention, the B cell subset detection method can be used not only for lymphoid tissue, but also for thymoma, thymic hyperplasia, thymic carcinoma, thymic cyst and lymph node lesion tissue.

[0039] 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.

[0040] Example 1: Preparation of QDs-Ab conjugates

[0041] 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.

[0042] 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.

[0043] Example 2: Detection of total B cells in lymphoid tissue

[0044] The quantum dot-coupled antibody QDs-585-CD19 was prepared according to the method of Example 1. The antibody CD19 product number is ab134114.

[0045] Lymph node tissue specimens from 30 patients with mild myasthenia gravis, 30 patients with severe myasthenia gravis, and 30 normal lymph node tissue specimens (specimens were obtained from surgical resection or biopsy); soaked in 4% formalin for more than 48 hours; wax blocks were made from lymph node tissue specimens and placed on a microtome to make 4μm tissue sections. Information such as the source of the sections, staining index, date, and antibody ratio were recorded before staining began.

[0046] 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.

[0047] 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.

[0048] Staining of tissue sections with quantum dot-conjugated antibody QDs-585-CD19:

[0049] Blocking: Remove 1× TBST solution, slowly add 10% BSA, and incubate at room temperature for 10 minutes.

[0050] Prepare antibodies: dilute the antibodies with 1×PBS to prepare QDs-585-CD19 quantum dot labeled antibody solution.

[0051] 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.

[0052] 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.

[0053] After the tissue sections are stained, the nuclei are stained and 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. The orange light signal represents CD19, and the overall B cells can be located and quantified by the quantum dot light signal in the figure.

[0054] Each group of samples was stained and observed under a fluorescence microscope. The test images were analyzed using ImageJ under default conditions. The statistical results were obtained using ImageJ software to calculate the average fluorescence intensity, which was calculated as Mean = the total fluorescence intensity of the region InDen / the area of ​​the region Are. The average values ​​of the average fluorescence intensity of different groups are shown in Table 1.

[0055] Table 1

[0056]

[0057] The data were analyzed between groups using the Two-way ANOVA statistical method;

[0058] It can be seen that samples in the same group showed relatively similar mean fluorescence intensities. The total number of B cells in the lymph node tissue of patients with severe myasthenia gravis was greater. The minimum mean fluorescence intensity detected in the lymph node tissue of 30 patients with severe myasthenia gravis was 484.029, and the CD19 mean fluorescence intensity accounted for more than 484.029, indicating that they were more likely to have autoimmune diseases. By using this method to detect lymph node tissue of patients with autoimmune diseases, the possibility of autoimmune diseases can be predicted.

[0059] like Figure 1 The figure shows the test results of lymph node tissue of a patient with severe myasthenia gravis, in which the average fluorescence intensity of CD19 is 486.019, which is greater than the minimum average fluorescence intensity of 484.029, verifying that the tissue section belongs to the lymph node tissue of a patient with severe myasthenia gravis.

[0060] Example 3: Detection of plasma cells and regulatory B cells in lymphoid tissue

[0061] Quantum dot-coupled antibodies QDs-585-CD19, QDs-525-CD138, and QDs-525-CD1d were prepared according to the method of Example 1. The catalog number of CD19 is ab134114, the catalog number of CD138 is ab128936, and the catalog number of CD1d is ab238657.

[0062] Lymph node tissue specimens from 30 patients with mild myasthenia gravis, 30 patients with severe myasthenia gravis, and 30 normal lymph node tissue specimens (specimens were obtained from surgical resection or biopsy); soaked in 4% formalin for more than 48 hours; wax blocks were made from lymph node tissue specimens and placed on a microtome to make 4μm tissue sections. Information such as the source of the sections, staining index, date, and antibody ratio were recorded before staining began.

[0063] 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.

[0064] 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.

[0065] Quantum dot-antibody conjugate staining:

[0066] The first round of staining, QDs-585-CD19 staining;

[0067] Blocking: Remove 1× TBST solution, slowly add 10% BSA, and incubate at room temperature for 10 minutes.

[0068] Prepare antibodies: dilute the antibodies with 1×PBS to prepare QDs-585-CD19 quantum dot labeled antibody solution.

[0069] Incubation: Remove excess serum, add QDs-585-CD19 quantum dot labeled antibody, place tissue sections flat in an immunohistochemistry humidified box, and then place in a 4°C refrigerator overnight.

[0070] 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.

[0071] The tissue sections obtained from the first round of staining were subjected to a second round of staining with QDs-525-CD138;

[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-525-CD138 quantum dot-coupled antibody solution.

[0074] Incubation: Remove excess serum, add QDs-525-CD138 quantum dot-coupled antibody, place 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-525-CD138 quantum dot-coupled antibodies.

[0076] The tissue sections obtained from the second round of staining were subjected to the third round of staining, QDs-625-CD1d staining;

[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-625-CD1d quantum dot-coupled antibody solution.

[0079] Incubation: Remove excess serum, add QDs-625-CD1d 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] After the tissue sections are stained, the nuclei are stained, and 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. After the thymoma tissue sections are stained, the orange light signal represents CD19, the green light signal represents CD138, and the red light signal represents CD1d. The orange light signal marks the location and content of the total B cells, the green light signal indicates the location and content of the plasma cells, and the red light signal marks the location and content of the regulatory B cells. The content and location of plasma cells and regulatory B cells in B cells can be observed by the distribution of orange light, green light and red light; further, the immune status of the lymphoid tissue can be determined.

[0082] Each group of samples was stained and observed under a fluorescence microscope. The average fluorescence intensity was calculated using ImageJ software. The average fluorescence intensity of different groups is shown in Table 2.

[0083] Table 2

[0084]

[0085] Two-way ANOVA statistical method was used to analyze the data between groups.

[0086] The samples in the same group showed similar mean fluorescence intensity. The minimum mean fluorescence intensity of CD19 detected in lymph node tissues of 30 patients with severe myasthenia gravis was 484.029, the minimum mean fluorescence intensity of CD138 was 416.87, and the maximum fluorescence intensity of CD1d was 85.614.

[0087] It can be seen that when the average fluorescence intensity of CD19 is greater than 484.029, the average fluorescence intensity of CD138 is greater than 416.87 and the average fluorescence intensity of CD1d is less than 85.614, it indicates that the degree of the autoimmune disease is relatively severe; thus, it can be seen that by using this method to detect lymph node tissues of patients with autoimmune diseases, the severity of the autoimmune disease can be predicted.

[0088] like Figure 2 Figure 3 The results of lymph node tissue testing in a patient with severe myasthenia gravis are shown. Figure 2 The distribution of plasma cells in total B cells. Figure 3 The distribution of B cells in the total B cells was regulated, where the average fluorescence intensity of CD19 was 486.105, the average fluorescence intensity of CD138 was 421.059, and the average fluorescence intensity of CD1d was 81.614, verifying that the tissue section belongs to the MGH group. The immune status of the tissue section can be inferred by the average fluorescence intensity of each antibody.

[0089] 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 according to the scope of application of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. Use of a kit for detecting B cell subsets in lymphoid tissue in immune status evaluation for non-disease diagnosis and treatment purposes, characterized in that: A kit for detecting B cell subsets in lymphoid tissue is used, the kit includes quantum dot-antibody CD19 conjugate, quantum dot-antibody CD138 conjugate and quantum dot-antibody CD1d conjugate; the quantum dots are QDs-525, QDs-585 and QDs-625, respectively, and quantum dots of different colors are combined with different antibodies; the kit includes quantum dot-conjugated antibody QDs-585-CD19, quantum dot-conjugated antibody QDs-525-CD138, quantum dot-conjugated antibody QDs-625-CD1d, antigen repair solution and DNA fluorescent dye; By sequentially labeling CD19, CD138 and CD1d in lymphoid tissue sections with different quantum dot-antibody conjugates, the detection of lymphoid tissue B cell subsets can be achieved; the specific antibody used to label B cells is CD19; the specific antibodies used to label plasma cells are CD19 and CD138; the specific antibodies used to label regulatory B cells are CD19 and CD1d; the detection of total B cells, plasma cells and regulatory B cells can be achieved directly using lymphoid tissue sections as the detection object; The fluorescence information of tissue sections was collected, the fluorescence intensity of quantum dots was counted, and the average fluorescence intensity of the corresponding antibodies was calculated; when the average fluorescence intensity of CD19 was greater than 484.029, it indicated that the subject's autoimmune state was hyperactive; at the same time, the average fluorescence intensity of CD138 was greater than 416.87, and the average fluorescence intensity of CD1d was less than 85.614, indicating that the subject's autoimmune hyperactivity was severe.

2. The use according to claim 1, 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.

3. The use according to claim 1, characterized in that: First, prepare lymphatic tissue sections, use antigen repair solution to perform antigen repair, then use different quantum dot-labeled antibodies to stain the tissue sections, and then stain the cells, collect the fluorescence information of the tissue sections, and learn the composition of B cell subpopulations in the detected lymphatic tissue.

Images