A method for detecting immune status in thymic hyperplasia tissue under Epstein-Barr virus infection

Abstract

The present invention relates to a method for detecting the immune status in thymic hyperplasia tissue under Epstein-Barr virus infection. A kit comprising quantum dot-coupled CD19 antibody and quantum dot-coupled CD86 antibody is constructed. After staining a thymic hyperplasia tissue slice sample, a conjugate of quantum dots and specific antibodies is specifically bound to an antigen on a B cell in the tissue slice sample, thereby realizing the marking of B cell surface co-stimulatory molecules by quantum dots; and judging the immune status of thymic hyperplasia tissue according to the marking result. In the present invention, the quantum dot-coupled antibody can directly bind to the antigen on the tissue slice, realizing direct marking of the tissue slice by the quantum dot-coupled antibody, thereby quantifying and locating the B cell surface co-stimulatory molecules in the thymic hyperplasia tissue microenvironment, clarifying its relationship with thymic hyperplasia-related autoimmune diseases, evaluating the immune status of the thymic hyperplasia tissue microenvironment, and predicting autoimmune diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a method for detecting the immune state in thymic hyperplasia tissue under the state of EB virus infection. Background Art

[0002] Thymic hyperplasia usually refers to the enlargement of the thymus gland. In children, thymic hyperplasia is the most common benign lesion in the anterior mediastinum; in adults, it often occurs in people aged 40 to 50. According to pathological staining observations, thymic hyperplasia can be divided into two morphological types: true thymic hyperplasia and lymphofollicular thymic hyperplasia. True hyperplasia is an increase in the size and weight of the thymus due to an increase in the number of thymic epithelial cells, and the normal tissue morphology of the thymus is preserved; lymphofollicular thymic hyperplasia is caused by hyperplastic lymphoid follicles in the thymus. Lymphoid follicles are composed of GCs containing immune cells such as B lymphocytes, plasma cells, follicular dendritic cells, and macrophages containing cell fragments, which are distributed in the medulla and interlobular spaces. True hyperplasia is usually not highly correlated with autoimmune diseases, while follicular hyperplasia is closely related to them, such as myasthenia gravis (MG) and Graves' Disease (GD).

[0003] EBV is a γ-type herpes virus, belonging to the B-lymphotropic virus, and its biological characteristics are closely related to mature B cells. According to statistics, more than 90% of the world's population has been infected with EBV. The body's immune function keeps a state of balance with the virus, so that it cannot play a pathogenic role and maintains a lifelong latent infection. EBV is related to the occurrence of autoimmune diseases, such as multiple sclerosis (MS), systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), but the correlation between EBV and autoimmune diseases associated with thymic hyperplasia is not very clear at present.

[0004] Clinically, the classic method for evaluating the immune status of patients with thymic hyperplasia mainly relies on the patient's clinical manifestations, serum antibody level detection and other tests. Due to the limitations of sensitivity and specificity, the accuracy of immune status assessment for various early autoimmune diseases including myasthenia gravis is poor, and there is a lack of effective assessment methods at the pathogenesis level. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for detecting the immune status in thymic hyperplasia tissue under EB disease infection.

[0006] The technical solution adopted by the present invention is: a detection kit for the immune status in thymic hyperplasia tissue under EB virus infection, comprising quantum dot-coupled CD19 antibody and quantum dot-coupled CD86 antibody.

[0007] Preferably, the quantum dots coupled to the CD19 antibody are different from the quantum dots coupled to the CD86 antibody.

[0008] Preferably, it comprises quantum dot-antibody conjugates QDs-525-CD19, QDs-625-CD86, as well as antigen repair solution and nucleic acid staining solution.

[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 the immune status in thymic hyperplasia tissue under Epstein-Barr virus infection for non-disease diagnosis and treatment purposes, preparing thymic hyperplasia tissue slices, preparing thymic hyperplasia tissue slices, and staining the tissue slices using a kit;

[0011] First, antigen repair was performed using antigen repair solution, and then the tissue sections were stained with antibodies labeled with different quantum dots. Then, the cells were stained, and the fluorescence information of the tissue sections was collected. The immune status in the thymic hyperplasia tissue was obtained based on the fluorescence expression level and spatial position distribution information.

[0012] Preferably, quantum dots of different colors label CD19 and CD86 in the same tissue section.

[0013] Preferably, the co-localization expression trends of CD19 and CD86 under different EB virus infection states are compared.

[0014] Preferably, in thymic hyperplasia tissue infected with EB virus, the co-localization mean fluorescence intensity of B cell specific antibody CD19 and CD86 is greater than 501.818, indicating that the immune state is relatively active and the possibility of combined autoimmune disease is high;

[0015] Alternatively, in EB virus-infected thymic hyperplasia tissue, the co-localization mean fluorescence intensity of B cell-specific antibodies CD19 and CD86 is less than 339.179, indicating that the immune status is weak and the possibility of combined autoimmune diseases is low.

[0016] The advantages and positive effects of the present invention are: the existing multi-color immunofluorescence technology is improved, the method of using quantum dots to carry antibodies is used, the secondary antibody staining step is eliminated, the direct labeling method is adopted, and the advantages of quantum dots themselves are brought into play, the emission spectrum covers the entire visible light region, and it is not easy to have spectral overlap when multiple quantum dots are used at the same time, which saves experimental time, improves efficiency and has good accuracy and sensitivity;

[0017] By replacing fluorescent dyes with quantum dots, tissue sections can be directly labeled, B cells can be locally localized in the microenvironment of thymic hyperplasia tissue, and the number of co-stimulatory molecules on the surface of B cells can be detected, and the immune status can be evaluated after analysis and comparison. By in vitro detection and comparative analysis of the co-localized expression of co-stimulatory molecules on the surface of B cells in the tissue microenvironment of patients with thymic hyperplasia, the immune status of patients can be evaluated at the pathogenesis level, overcoming the disadvantages of previous limited evaluation methods and having good sensitivity and accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Expression of costimulatory molecules CD19 and CD86 on the surface of B cells in thymic hyperplasia tissues with myasthenia gravis without EB virus infection;

[0019] Figure 2 Expression of co-stimulatory molecules CD19 and CD86 on the surface of B cells in thymic hyperplasia tissues of Epstein-Barr virus-infected patients without myasthenia gravis. DETAILED DESCRIPTION

[0020] The embodiments of the present invention are described below with reference to the accompanying drawings.

[0021] The present invention relates to a method for detecting the immune status in thymic hyperplasia tissue under Epstein-Barr virus infection, comprising a detection kit, the kit comprising a quantum dot-labeled CD19 antibody and a quantum dot-labeled CD86 antibody. During the detection, the thymic hyperplasia tissue slice sample is first stained, and the antigen-specific connection on the B cells in the slice sample cells is sequentially labeled by multicolor quantum dot-antibody conjugates, so as to achieve the quantification of the costimulatory molecules on the surface of the B cells by the quantum dots.

[0022] EBV mainly infects B lymphocytes in the body, followed by epithelial cells. It usually replicates in two ways: infecting B cells and then proliferating or lysing the virus body for amplification. LMP1 is the main oncogene of EBV. It is expressed in the latent period of EBV infection. It can simulate CD40 and activate the NF-κB signaling pathway, which promotes EBV infection of B cells and cell transformation. LMP2A, the main proto-oncogene in LMP2, can work together with LMP1 to activate the B cell receptor and CD40 signaling pathways, respectively, thereby reducing B cell apoptosis in GC; LMP2A can also activate the B cell-related Ras pathway, activate the PI3k / Akt signaling pathway, promote B cell survival, and antagonize apoptosis. In the first few days of EBV infection, immediate early genes and early lytic genes are expressed. The immediate early gene BZLF1 is the key to the transformation from the latent phase to the lytic phase. One of its expression products, Zta, is a member of the basic leucine zipper transcription factor family, which can initiate and maintain EBV lytic infection. BARF1 is an EBV early lytic gene induced by Zta, which can enable immune escape and cell survival by causing epithelial cell immortalization, thereby causing carcinogenesis.

[0023] In the thymus, B cells play an important role in the induction of central tolerance by negatively selecting self-reactive T cells, a function consistent with their location in the thymic medulla, the expression of the key co-stimulatory molecule CD86 and high levels of MHC class II molecules. CD86 (B7-2) are members of the immunoglobulin (Ig) superfamily and are expressed on the surface of many types of cells, including T cells, B cells, macrophages and dendritic cells. CD86 expression on resting naive B cells is low or even absent. Upregulation of CD86 expression on B cells can be achieved by triggering the B-cell receptor (BCR). CD86-mediated signaling pathways may affect B cell responses and immunoglobulin production, and CD86 enhances B cell activity. The rapid and high-level expression of CD86 in early immune responses is considered to be an essential activation and signal transmitted during immune responses.

[0024] In MG patients, B cells play an important role in the pathogenesis of AChR+ MG by producing anti-AChR antibodies. However, B cells are also able to induce antigen-specific T cell responses through antigen presentation and co-stimulation. Molecules such as MHC-II class molecules, CD80, CD86, and CD40 play a role in BT cell interactions. The thymus of AChR+ MG may contain activated CD86-expressing B cells with stronger antigen presentation properties. One possible reason for the increased expression of co-stimulatory molecules in B cells of AChR+ MG patients is the difference in exposure to signaling molecules. Signals from Toll-like receptor (TLR) agonists or T cell-dependent cytokines (such as IL-21) can upregulate the expression of co-stimulatory molecules in B cells. EBV infection can activate B lymphocytes and transform them into lymphoblastoid cells, which are immortalized. Studies have also shown that CD86 is highly expressed in the thymus during infancy, but gradually decreases with age.

[0025] According to the above characteristics, the present invention targets the specific antibody proteins CD19 and CD86 of the co-stimulatory molecules on the surface of B cells, labels each target protein with multicolor quantum dots, and judges the immune status in the thymic hyperplasia tissue under the EB virus infection state from the content and position information. In certain embodiments of the present invention, the kit includes QDs-525-CD19, QDs-625-CD86, as well as antigen repair solution and nucleic acid staining solution.

[0026] Multicolor immunofluorescence techniques all use traditional fluorescent dyes, which have certain defects. Traditional dyes have limited emission spectrum wavelengths; poor photostability; spectral overlaps are easy to occur between different fluorescent dyes; poor biocompatibility, making it difficult to perform specific labeling; short fluorescence lifetimes, making it difficult to store for a long time, and strong background interference. Quantum dots are quasi-zero-dimensional nanomaterials and a type of semiconductor nanoparticles composed of a small number of atoms. Using the same excitation light source can achieve synchronous detection of quantum dots of different particle sizes, which can be used for multicolor labeling. In addition, quantum dots have narrow and symmetrical fluorescence emission peaks, so spectral overlaps are not easy to occur when multiple quantum dots are used simultaneously. Using quantum dots instead of fluorescent dyes can avoid some of the problems of traditional fluorescence technology as much as possible.

[0027] The coupling of quantum dots and antibodies includes the following steps:

[0028] Step 1: QDs, EDC, and NHS are 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 is adjusted to 8-9 with PBS to obtain an activated quantum dot solution;

[0029] Step 2: Take an appropriate amount of antibody stock solution, dilute it with PBS buffer, and mix it thoroughly; add antibody to the activated quantum dot solution, the molar ratio of quantum dots to antibody protein is 1:20-80 (1:20, 1:40, 1:80 can be used for labeling respectively), shake at 4°C in the dark, and obtain QDs-Ab conjugate after reaction;

[0030] Step 3: 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. The purified product is stored in a refrigerator at 4°C away from light for later use.

[0031] By changing the different feed ratios of quantum dots, required cross-linkers 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 different colors of quantum dots and different antibodies can be combined according to the steps in the instructions to achieve personalized customization.

[0032] An inappropriate ratio of QDs to Ab will result in poor stability of the obtained complex, easy aggregation, significantly affecting its biocompatibility and making it impossible to use it in biological applications, while an excessive amount of cross-linking agent will cause cross-linking between Abs and aggregation of QDs. It is necessary to precisely control the ratio of QDs, Abs 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.

[0033] In addition, the purification of the bioconjugate after the coupling of quantum dots and antibodies is currently commonly carried out 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.

[0034] The above kit is used to detect B cell surface co-stimulatory molecules in tissue sections, and the quantitative information of B cell surface co-stimulatory molecules expression is detected and analyzed to evaluate tissue microenvironment and body immune status; the specific method is as follows:

[0035] Step 1: Take a paraffin specimen of thymic hyperplasia tissue to make tissue sections, bake the tissue sections and dewax and dehydrate them;

[0036] Step 2: Use a microwave oven to perform antigen repair on tissue sections to facilitate the binding of specific antibodies;

[0037] Step 3: staining the tissue section with quantum dots and antibody conjugates. When there are multiple quantum dots and antibody conjugates, staining is performed in the reverse order of protein expression abundance. If the protein expression abundance is high, staining is performed first, and if the protein expression abundance is low, staining is performed later. Each quantum dot staining step includes staining, incubation and washing processes. After fully removing the unbound quantum dots and antibody conjugates, the next staining step is performed.

[0038] Step 4: After all quantum dots are stained, the cell nuclei are stained, the slides are sealed and photographed, and the location and number of target cells are analyzed based on the photo information.

[0039] Combining quantum dots with the required antibodies makes up for the shortcomings of traditional fluorescent dyes. At the same time, the special binding 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 of co-stimulatory molecules on the surface of B cells; the expression level of its fluorescent signal can be calculated with high accuracy, and the immune status can be judged without combining other markers, especially for early and latent autoimmune diseases. The immune status is evaluated by in vitro detection of the expression level of B cell surface co-stimulatory molecules in the microenvironment of the patient's thymic hyperplasia tissue. By quantifying the co-stimulatory molecules on the surface of B cells, the immune status of the microenvironment of thymic hyperplasia tissue can be evaluated, and its relationship with thymic hyperplasia-related autoimmune diseases can be clarified.

[0040] The mean fluorescence intensity of co-localization of B cell-specific antibodies CD19 and CD86 was statistically analyzed by ImageJ software using the following formula;

[0041] Mean fluorescence intensity Mean = total fluorescence intensity of the region InDen / area of ​​the region Area;

[0042] Through observation under a fluorescence microscope and statistics using ImageJ software, the average fluorescence intensity of co-localization of B cell-specific antibodies CD19 and CD86 in thymic hyperplasia tissue infected with EB virus was greater than 501.818, indicating that the immune state was relatively active and the possibility of combined autoimmune diseases was greater; the average fluorescence intensity of co-localization of B cell-specific antibodies CD19 and CD86 in thymic hyperplasia tissue infected with EB virus was less than 339.179, indicating that the immune state was weak and the possibility of combined autoimmune diseases was less.

[0043] 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.

[0044] Example 1: Preparation of QDs-Ab conjugates

[0045] The quantum dots used in this embodiment are QDs-525 and QDs-625.

[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 (pH 9.18) to obtain quantum dot activation solution. 100ul of quantum dot activation solution was added to the reaction container. An appropriate amount of antibody stock solution was taken, diluted to 0.01mg / ml with PBS buffer, mixed thoroughly, and added to the above reaction container according to different ratios (reference quantum dots: antibody protein molar ratio of 1:20, 1:40, 1:80 for labeling), and reacted on a shaker at 4℃ in the dark for 4 hours.

[0047] After the reaction was completed, centrifuge at 8000 rpm for 3 min to remove possible agglomerated precipitates and retain the supernatant.

[0048] 1% agarose gel electrophoresis was used to detect and analyze, and the best conditions were screened. The supernatant obtained under the best coupling conditions was purified using a PD10 column, and the purified product was stored in a refrigerator at 4°C away from light for later use.

[0049] Example 2: Tissue section preparation and detection

[0050] 30 tissues from patients with thymic hyperplasia with myasthenia gravis known to be infected by EB virus and 30 specimens from patients with thymic hyperplasia without myasthenia gravis infected by EB virus were obtained. The above specimens were obtained from surgical resection or puncture (EBV infection samples were positive for in situ hybridization of EBER; patients were clinically diagnosed with thymic hyperplasia with myasthenia gravis), and soaked in 4% formalin for more than 48 hours; wax blocks were made from thymic hyperplasia tissue specimens from different sources and placed on a microtome to make 3-4μm tissue sections. The source information of the sections, staining index, date, antibody ratio, etc. 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] Quantum dot-coupled antibodies QDs-525-CD19 and QDs-625-CD86 were prepared according to the method of Example 1. Each tissue section treated in Example 2 was stained with QDs-525-CD19 and QDs-625-CD86 in turn, and the detailed steps are as follows:

[0054] First round of dyeing:

[0055] Blocking: Remove 1× TBST solution, slowly add 10% BSA, and incubate at room temperature for 10 minutes.

[0056] Prepare antibodies: dilute the antibodies with 1×PBS to prepare QDs-525-CD19 quantum dot labeled antibody solution.

[0057] Incubation with the first antibody: remove excess serum, add QDs-525-CD19 quantum dot labeled antibody, place the tissue sections flat in an immunohistochemistry humidified box, and then place in a 4°C refrigerator overnight.

[0058] Rinse: Rinse three times with a wash bottle containing 1×TBST, 2 minutes each time, to remove unbound QDs-525-CD19 quantum dot-labeled antibodies.

[0059] After the first round of staining, the second round of staining was performed;

[0060] Blocking: Remove 1× TBST solution, slowly add 10% BSA, and incubate at room temperature for 10 minutes.

[0061] Prepare antibodies: dilute the antibodies with 1×PBS to prepare QDs-625-CD86 quantum dot-coupled antibody solution.

[0062] Secondary antibody incubation: remove excess serum, add QDs-625-CD86 quantum dot-coupled antibody, place tissue sections flat in an immunohistochemistry humidified box, and then place in a 4°C refrigerator overnight.

[0063] Rinse: Rinse three times with a wash bottle containing 1×TBST, 2 minutes each time, to remove unbound QDs-625-CD86 quantum dot-coupled antibodies.

[0064] After the staining, the tissue sections were stained with nuclei, and 1×DAPI solution was prepared with ddH20 1:100; 1×DAPI solution was 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 was added, and then the coverslip was covered. After the tissue sections were stable, the results were observed and photographed using a fluorescence microscope equipped with a 488nm laser. Among them, the green light signal represents CD19 and the red light signal represents CD86.

[0065] The test results of 30 tissue samples of Epstein-Barr virus-infected thymic hyperplasia patients with myasthenia gravis and 30 specimens of Epstein-Barr virus-infected thymic hyperplasia patients without myasthenia gravis were statistically analyzed, and the average fluorescence intensity of each quantum dot was analyzed by ImageJ software, corresponding to the expression level 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, and the average fluorescence intensity Mean = the sum of the fluorescence intensity of the region InDen / the area of ​​the region Area. The average values ​​of the average fluorescence intensity of the corresponding antibodies corresponding to multiple samples are shown in Table 1.

[0066] Table 1

[0067]

[0068] Note: Data are shown as Mean±SD, t-test was used for statistical analysis between the two groups.

[0069] As shown in Table 1, the mean fluorescence intensity of CD19CD86 co-expression in samples of the same group is similar. + CD86 + The expression of CD19 in 30 tissues of patients with thymic hyperplasia and myasthenia gravis infected by EB virus was detected. + CD86 + The minimum mean fluorescence intensity was 501.818; in 30 samples of EB virus-infected patients with thymic hyperplasia without myasthenia gravis, CD19 + CD86 + The maximum average fluorescence intensity is 339.179.

[0070] It can be seen that in the thymic hyperplasia tissue infected by EB virus, the co-localization average fluorescence intensity of B cell specific antibody CD19 and CD86 is greater than 501.818, indicating that the immune state is relatively active and the possibility of combined autoimmune diseases is greater; while in the thymic hyperplasia tissue infected by EB virus, the co-localization average fluorescence intensity of B cell specific antibody CD19 and CD86 is less than 339.179, indicating that the immune state is weak and the possibility of combined autoimmune diseases is small.

[0071] Example 3: Detection of CD19 and CD86 on the surface of B cells in tissues of patients with thymic hyperplasia infected by EB virus and associated with myasthenia gravis

[0072] The thymic hyperplasia tissue sections to be tested for EB virus infection were stained and photographed according to the method of Example 2. The results are as follows: Figure 1 As shown. Among them, the average fluorescence intensity of the co-localization of the co-stimulatory molecules CD19 and CD86 is 503.923, which is consistent with the average fluorescence intensity of the co-localization of the B cell specific antibody CD19 and CD86 being greater than 501.818, indicating that the immune state is relatively active, and the possibility of combined autoimmune diseases is high, and it is a thymic hyperplasia sample with viral infection and myasthenia gravis; it can be seen that by using this kit to detect the tissue of thymic hyperplasia patients infected with EB virus, it can be predicted that the possibility of thymic hyperplasia patients infected with EB virus have autoimmune diseases.

[0073] Example 4: Detection of CD19 and CD86 on the surface of B cells in tissues of patients with thymic hyperplasia but without myasthenia gravis infected by EB virus

[0074] The thymic hyperplasia tissue sections infected with EB virus to be detected were stained and photographed according to the method of Example 2. The results are as follows Figure 2 Among them, the average fluorescence intensity of co-localization of B cell specific antibody CD19 and CD86 is 338.137, which is consistent with the average fluorescence intensity of co-localization of B cell specific antibody CD19 and CD86 being less than 339.179, indicating that the immune status is weak and the possibility of combined autoimmune disease is small. It is a thymic hyperplasia sample without myasthenia gravis infected by EB virus.

[0075] 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 detecting the immune status in thymic hyperplasia tissue under Epstein-Barr virus infection, characterized in that: Including quantum dot-coupled CD19 antibody and quantum dot-coupled CD86 antibody; 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.

2. The kit for detecting the immune status in thymic hyperplasia tissue under EB virus infection according to claim 1, characterized in that: The quantum dots coupled to the CD19 antibody are different from the quantum dots coupled to the CD86 antibody.

3. The detection kit for the immune status in thymic hyperplasia tissue under EB virus infection according to claim 1, characterized in that: Including quantum dot-antibody conjugates QDs-525-CD19, QDs-625-CD86, as well as antigen repair solution and nucleic acid staining solution.

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