A method for detecting EBV latent infection in epithelial cells and B cells in the microenvironment of thymus and salivary gland tissue

Abstract

The present invention relates to a thymus and salivary gland tissue microenvironment epithelial cells and B cells EBV latent infection detection kit and detection method, by coupling quantum dots and specific antibodies, staining in thymus and salivary gland tissue; quantum dots and specific antibody conjugates are combined with tissue sample specific antigens to achieve quantum dots to EBV latently infected epithelial cells and B cells marking. The present invention improves the existing multicolor immunofluorescence technology, and uses specific antibodies and quantum dots after coupling and purification, and directly combines with antigens on tissue sections to achieve quantum dot-coupled antibodies to directly mark tissue samples, thereby locating and quantifying EBV latently infected cells in the thymus and salivary gland tissue microenvironment, and evaluating the patient's viral infection from the pathogenesis level, overcoming the disadvantages of the limited evaluation methods in the past, and having good sensitivity and accuracy.

Description

Technical Field

[0001] The invention belongs to the field of detection technology, and in particular relates to a method for detecting EBV latent infection in epithelial cells and B cells in the microenvironment of thymus and salivary gland tissues. Background Art

[0002] The thymus is the main immune organ of the human body and is essential for maintaining a healthy immune system. Changes in the thymus structure can lead to damage to the body's immune system. Thymus organic and functional disorders caused by various thymic diseases increase the risk of infection, tumors and autoimmune diseases. The thymus of patients with myasthenia gravis is extensively hyperplastic and structurally disordered, and can form B cell follicles containing active germinal centers, thereby producing plasma cells and becoming a source of autoantibodies. In addition, thymic hyperplasia is also associated with many other autoimmune diseases, involving multiple systems such as the nervous, cardiovascular, gastrointestinal, blood, skin, and endocrine systems, such as Sjögren's syndrome, systemic lupus erythematosus, and rheumatoid arthritis, but the specific pathogenic mechanism of thymic hyperplasia in these diseases is still unclear.

[0003] Among the environmental influences that are considered to be likely to trigger the onset of autoimmune diseases, pathogen infection, especially bacterial and viral infection, is a major factor. Viral infection can trigger the connection between autoimmune responses through mechanisms such as molecular mimicry, bystander activation and epitope spreading. Thymic virus infection disrupts T cell maturation and selection, which may lead to autoimmune dysregulation. Thymic infection with human and mouse roseophore viruses, which belong to the herpesvirus family, leads to a transient decrease in CD4+ single-positive and CD4+CD8+ double-positive thymocytes. In addition, infection of neonatal mice with roseophore viruses has been associated with the development of autoimmune gastritis. At 12 weeks after infection, these mice developed autoimmune features, including gastritis characterized by the production of autoantibodies and autoreactive CD4+ T cells. Coxsackievirus is one of the viral environmental risk factors for the development of type 1 diabetes and is involved in the development of the disease by affecting the inflammatory environment and inducing bystander activation of CD8+ T cells and possible molecular mimicry mechanisms. Therefore, thymic virus infection may lead to severe thymic structural and immune dysregulation, which is associated with the development of autoimmune diseases.

[0004] EBV is a double-stranded DNA herpes virus that occupies a prominent position among environmental factors and is widespread worldwide, infecting more than 90% of the population, triggering the development of autoimmune diseases by causing epithelial damage and stimulating the innate and adaptive immune systems. Persistent EBV infection may cause damage to the salivary glands, leading to sicca symptoms. EBV can exist in the human organism in a latent form, allowing the virus to hide in host cells and reactivate under favorable conditions. In order to survive unnoticed, the virus uses various methods to hide its marks, allowing it to escape the control of the host immune system. EBV not only promotes the development of autoimmunity, but may also lead to the development of malignancies, especially lymphomas.

[0005] A deeper understanding of the pathogenic mechanism of EBV will not only help us understand the occurrence and development of autoimmune diseases, but also help us explore the role of EBV in autoimmune diseases related to thymic lesions, so as to deepen our understanding of thymic diseases leading to autoimmune diseases. At the same time, finding effective protection against EBV infection and its reactivation is particularly important for inhibiting the development of certain EBV virus-related cancers or enhancing their treatment, as well as preventing post-transplant lymphoproliferative diseases.

[0006] The classic clinical method for evaluating EBV infection mainly relies on the detection of serum antibody levels in patients. Due to the limitations of sensitivity and specificity, the accuracy of the assessment of various early infection states is poor, and there is a lack of effective assessment methods at the pathogenesis level. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a method for detecting EBV latent infection in epithelial cells and B cells in the microenvironment of thymus and salivary gland tissues.

[0008] The technical solution adopted by the present invention is: a kit for detecting EBV latent infection of epithelial cells and B cells in the microenvironment of thymus and salivary gland tissues, comprising quantum dot-labeled LMP1 antibody, quantum dot-labeled CK19 antibody, and quantum dot-labeled CD19 antibody.

[0009] Preferably, the quantum dots include QDs-525, QDs-585 and QDs-625.

[0010] Preferably, it specifically includes quantum dot-antibody conjugates QDs-525-LMP, QDs-585-CK19, QDs-625-CD19, as well as antigen repair solution and nucleic acid staining solution.

[0011] 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.

[0012] The kit is not intended for disease diagnosis or treatment. It is a method for detecting EBV latent infection in epithelial cells and B cells in the microenvironment of thymus and salivary gland tissues. Thymus and salivary gland tissue sections are prepared, antigen repair is performed using antigen repair solution, the tissue sections are stained with antibodies labeled with different quantum dots, the cells are stained, fluorescence information of the tissue sections is collected, and the EBV infection status is obtained based on the fluorescence expression information.

[0013] Preferably, the quantum dot-labeled LMP1 antibody specifically labels EBV, the quantum dot-labeled CK19 antibody specifically labels epithelial cells, and the quantum dot-labeled CD19 antibody specifically labels B cells.

[0014] Preferably, the co-labeling site of quantum dot-labeled LMP1 antibody and quantum dot-labeled CK19 antibody represents cells latently infected with EBV in epithelial cells; and the co-labeling site of quantum dot-labeled LMP1 antibody and quantum dot-labeled CD19 antibody represents cells latently infected with EBV in B cells.

[0015] Preferably, the EBV latently infected epithelial cells and / or B cells in the tissue are quantified to determine the EBV latent infection status in the thymus and salivary gland tissues. The higher the proportion, the greater the risk of infection.

[0016] Preferably, the degree of EBV infection is quantitatively evaluated by the ratio of the average fluorescence intensity of epithelial cells latently infected with EBV in the thymus and salivary gland tissue microenvironment to all epithelial cells in the thymus and salivary gland tissue microenvironment and / or the ratio of the average fluorescence intensity of B cells latently infected with EBV in the thymus and salivary gland tissue microenvironment to all B cells in the thymus and salivary gland tissue microenvironment.

[0017] Preferably, in thymus tissue, if the LMP1+CK19+ / CK19+DAPI+ ratio is greater than 62.42%, it is judged that the latent infection of EBV virus in epithelial cells is more serious; in salivary gland tissue, if the LMP1+CK19+ / CK19+DAPI+ ratio is greater than 66.67%, it is judged that the latent infection of EBV virus in epithelial cells is more serious;

[0018] In thymus tissue, if the LMP1+CD19+ / CD19+DAPI+ ratio is greater than 86.76%, it is judged that the latent infection of EBV virus in B lymphocytes is more severe; in salivary gland tissue, if the LMP1+CD19+ / CD19+DAPI+ ratio is greater than 84.87%, it is judged that the latent infection of EBV virus in B lymphocytes is more severe.

[0019] The advantages and positive effects of the present invention are: improving the existing multi-color immunofluorescence technology, eliminating the secondary antibody staining step by coupling the quantum dots to antibodies, and giving full play to the advantages of the quantum dots themselves by direct labeling. The emission spectrum covers the entire visible light region, and when multiple quantum dots are used simultaneously, it is not easy to have spectral overlap, saving experimental time, improving efficiency, and having good accuracy and sensitivity;

[0020] By replacing fluorescent dyes with quantum dots, tissue samples can be directly labeled, and EBV-infected latent epithelial cells and B cells in the thymus and salivary gland tissue microenvironment can be quantified, which can achieve quantitative detection and facilitate the evaluation of viral infection. By in vitro detection of the distribution and number of epithelial cells and B cells of EBV-infected latent cells in the patient's thymus and salivary gland tissue microenvironment, the patient's viral infection status is evaluated at the pathogenesis level, overcoming the disadvantages of previous limited evaluation methods and having good sensitivity and accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The distribution and expression of LMP1+CK19+ cells in the thymic tissue microenvironment;

[0022] Figure 2 The distribution and expression of LMP1+CK19+ cells in the salivary gland tissue microenvironment;

[0023] Figure 3 The distribution and expression of LMP1+CD19+ cells in the thymic tissue microenvironment;

[0024] Figure 4 Distribution and expression of LMP1+CD19+ cells in the salivary gland tissue microenvironment. 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 method for detecting EBV latent infection in epithelial cells and B cells in the microenvironment of thymus and salivary gland tissues, wherein quantum dots are coupled with specific antibodies, and thymus and salivary gland tissue sections are stained by the quantum dots and specific antibody conjugates, and the conjugates can be connected with specific antigens in the sectioned tissues to achieve the quantification of epithelial cells and B cells latently infected with EBV by quantum dots. By analyzing the number of epithelial cells and B cells in thymus and salivary gland tissues, the EBV infection of patients can be evaluated from the pathogenesis level, overcoming the disadvantages of the limited evaluation methods in the past, and having good accuracy and sensitivity.

[0027] Thymus and salivary tissue sections are used as test objects to detect EBV latently infected cells. The specific antibodies used to detect EBV latently infected cells are one or more of LMP1, CK19 and CD19. LMP1 is a hallmark antibody for EBV latency, and significant induction of full-length LMP1 is often observed during viral reactivation into the lytic cycle. LMP1 is essential for EBV-infected cell activation, adhesion and survival. CK19 and CD19 are common markers used to mark epithelial cells and B cells, respectively.

[0028] By coupling quantum dots with antibodies, thymus and salivary gland tissue sections are stained to achieve the localization and quantification of EBV latently infected epithelial cells and B cells by quantum dots. Multicolor immunofluorescence technology uses traditional fluorescent dyes, which have limited emission spectrum wavelengths; poor photostability; spectral overlap between different fluorescent dyes; poor biocompatibility, and difficulty in specific labeling; short fluorescence lifetime, difficult to preserve 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. The same excitation light source can be used to 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 overlap is not easy to occur when multiple quantum dots are used simultaneously. Using quantum dots instead of fluorescent dyes can avoid some problems of traditional fluorescence technology as much as possible.

[0029] The coupling of quantum dots and antibodies includes the following steps:

[0030] 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;

[0031] 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;

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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. In certain embodiments of the present invention, the kit may include quantum dot-antibody conjugates QDs-525-LMP, QDs-585-CK19, and QDs-625-CD19. When used, the quantum dot-antibody conjugates can be used in sequence to stain the same tissue section, and the staining results can be analyzed by different colors; in other embodiments of the present invention, the kit may include quantum dot-antibody conjugates QDs-525-LMP, QDs-625-CK19, and QDs-625-CD19. When used, QDs-525-LMP and QDs-625-CK19 are used to stain tissue sections, respectively, and then QDs-525-LMP and QDs-625-CD19 are used to stain homologous tissue sections to explore the co-expression of LMP1 and CK19, as well as the co-expression of LMP1 and CD19.

[0036] The specific steps of the method for detecting EBV latently infected epithelial cells and B cells are as follows:

[0037] Step 1: Take paraffin specimens of thymus or salivary gland tissue to make tissue sections, bake, dewax and hydrate the tissue sections;

[0038] Step 2: Use a microwave oven to perform antigen retrieval on tissue sections to expose antigens for binding to specific antibodies;

[0039] Step 3: Staining tissue sections with quantum dots and antibody conjugates. Multiple quantum dots and antibody conjugates are stained in the reverse order of protein expression abundance. Each quantum dot staining step includes staining, incubation and washing processes. After fully removing unbound quantum dots and antibody conjugates, proceed to the next staining step.

[0040] Step 4: After all quantum dots are stained, stain the cell nucleus, seal the slide and take a photo. Analyze the distribution and number of target cells based on the photo information and perform analysis.

[0041] Quantum dots coupled to specific antibodies can make up for the shortcomings of traditional fluorescent dyes, and the special binding method allows the quantum dots and antibodies to be stably coupled, achieving complementary advantages; quantum dot labeling technology can effectively observe the expression level and spatial distribution of EBV latently infected epithelial cells and B cells, with high accuracy, and can judge the viral infection status of autoimmune diseases without combining other markers. The expression level of EBV latently infected epithelial cells and B cells in the microenvironment of the patient's thymus and salivary gland tissues can be detected in vitro to evaluate the viral infection status.

[0042] 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. By detecting the latent infection of EBV in epithelial cells and B cells, the degree and risk of EBV infection can be further evaluated. By analyzing the proportion of EBV latently infected cells in the microenvironment of thymus and salivary gland tissues, the association between EBV and the activity of autoimmune diseases combined with thymic diseases, especially Sjögren's syndrome, is evaluated; by the spatial distribution of EBV latently infected epithelial cells and B cells in tissues, the latent infection status of EBV in thymus and salivary gland tissues is judged.

[0043] The proportion of co-expression of LMP1 and CK19 in the total CK19+DAPI+ epithelial cells is expressed as LMP1+CK19+ / CK19+ DAPI+%, and the proportion of co-expression of LMP1 and CD19 in the total CD19+DAPI+ B lymphocytes is expressed as LMP1+CD19+ / CD19+ DAPI+%. The degree of EBV infection can be predicted by the ratio respectively; the larger the ratio, the more serious the EBV latent degree and the increased risk of EBV reactivation. In thymus tissue, if the LMP1+CK19+ / CK19+DAPI+ ratio is greater than 62.42%, it is judged that the latent infection of EBV virus in epithelial cells is more serious; in salivary gland tissue, if the LMP1+CK19+ / CK19+DAPI+ ratio is greater than 66.67%, it is judged that the latent infection of EBV virus in epithelial cells is more serious. In thymus tissue, if the LMP1+CD19+ / CD19+DAPI+ ratio is greater than 86.76%, it is judged that the latent infection of EBV virus in B lymphocytes is more severe; in salivary gland tissue, if the LMP1+CD19+ / CD19+DAPI+ ratio is greater than 84.87%, it is judged that the latent infection of EBV virus in B lymphocytes is more severe.

[0044] In addition, in certain embodiments of the present invention, the quantity information and location information of EBV latently infected epithelial cells and B cells can be detected and analyzed simultaneously, and the combination of information from the two places can better achieve the purpose of early detection. The "LMP1+CK19+ / CK19+DAPI+%" or "LMP1+CD19+ / CD19+DAPI+%" value is detected in the microenvironment of thymus and salivary gland tissues to quantitatively evaluate the EBV infection in Sjögren's syndrome combined with thymic diseases. Based on the spatial distribution of EBV latently infected epithelial cells and B cells in the tissues, the EBV latent infection status in the thymus and salivary gland tissues is judged, indicating the detection of EBV.

[0045] 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.

[0046] Example 1: Preparation of QDs-Ab conjugates

[0047] The quantum dots used in this embodiment are QDs-525, QDs-585, and QDs-625.

[0048] 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.

[0049] After the reaction is completed, centrifuge at 8000 rpm for 3 minutes to remove possible agglomerated precipitates and keep the supernatant.

[0050] 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.

[0051] Example 2: Detection of EBV latently infected epithelial cells and B cells in the microenvironment of thymus and salivary gland tissues

[0052] According to the method of Example 1, quantum dot-coupled antibodies QDs-525-LMP1, QDs-585-CK19 and QDs-625-CD19 were prepared.

[0053] Thirty thymic surgical or puncture tissue specimens were collected (the above 30 samples were EBV virus-infected samples; EBER in situ hybridization test results were positive and judged to be EBV virus-infected); 30 salivary gland surgical or puncture tissue specimens were collected (the above 30 samples were EBV virus-infected samples; EBER in situ hybridization test results were positive and judged to be EBV virus-infected). Use 4% formalin to fix for more than 48 hours; make wax blocks from thymic and salivary gland tissue samples and place them on a microtome to make 4μm tissue sections. Record the section information, staining index, date, antibody dilution ratio and other information before staining.

[0054] The tissue sections were placed in a 60°C oven for 30 minutes for baking, followed by dewaxing and hydration, and the sections were placed in xylene (I) for 15 minutes - xylene (II) for 15 minutes - anhydrous ethanol (I) for 3 minutes - anhydrous ethanol (II) for 3 minutes - 95% ethanol for 3 minutes - 75% ethanol for 3 minutes - 50% ethanol for 3 minutes - 1× PBS buffer (I) for 5 minutes - 1× PBS buffer (II) for 5 minutes - 1× PBS buffer (III) for 5 minutes.

[0055] Antigen repair was performed on tissue sections that had been dewaxed to water. 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 the low heat was maintained for 10 minutes before being taken out; after the tissue sections naturally cooled to room temperature, they were moved to a wet box, rinsed with sterile water for 1 minute, and rinsed with 1×TBST for 2 minutes. The tissue was circled with a water blocking pen so that the entire tissue block on the section was located inside the sealing circle.

[0056] First round of dyeing:

[0057] Blocking: Remove 1× TBST solution, slowly add 10% BSA, and incubate at room temperature for 10 minutes.

[0058] Preparation of antibodies: dilute the antibodies with 1×PBS to prepare QDs-525-LMP1 quantum dot labeled antibody solution.

[0059] Incubation with the first antibody: Remove excess BSA, add QDs-525-LMP1 quantum dot-labeled antibody, place the tissue sections flat in a humidified box, and then place in a 4°C refrigerator overnight.

[0060] Rinse: Rinse three times with a wash bottle containing 1×TBST, 2 minutes each time, to remove unbound QDs-525-LMP1 quantum dot-labeled antibodies.

[0061] After the first round of staining, the second round of staining was performed;

[0062] Blocking: Remove 1× TBST solution, slowly add 10% BSA, and incubate at room temperature for 10 minutes.

[0063] Preparation of antibodies: dilute the antibodies with 1×PBS to prepare QDs-585-CK19 quantum dot-coupled antibody solution.

[0064] Secondary antibody incubation: remove excess BSA, add QDs-585-CK19 quantum dot-coupled antibody, place tissue sections flat in a humidified box, and then place in a 4°C refrigerator overnight.

[0065] Rinse: Rinse three times with a wash bottle containing 1×TBST, 2 minutes each time, to remove unbound QDs-585-CK19 quantum dot-coupled antibodies.

[0066] After the second round of staining, the third round of staining was performed;

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

[0068] Preparation of antibodies: dilute the antibodies with 1×PBS to prepare QDs-625-CD19 quantum dot-coupled antibody solution.

[0069] Secondary antibody incubation: remove excess BSA, add QDs-625-CD19 quantum dot-coupled antibody, place tissue sections flat in a 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-625-CD19 quantum dot-coupled antibodies.

[0071] After the tissue sections are stained, the nuclei are stained. 1×DAPI solution is prepared with ddH20 1:100; 1×DAPI solution is added to the tissue sections, incubated at room temperature in the dark for 5 minutes, rinsed with 1×TBST for 2 minutes, and rinsed with ddH2O for 1 minute. After removing the excess liquid on the sections, 30μl of anti-fade mounting medium is added, and then the coverslip is covered. After the tissue sections are stable, the results are observed and photographed using a fluorescence microscope equipped with a 488nm laser. The green light signal represents LMP1, the orange light signal represents CK19, and the red light signal represents CD19. The co-expression of LMP1+CK19+ is yellow, and the CK19+DAPI+ is purple; the co-expression of LMP1+CD19+ is brown, and the CD19+DAPI+ is dark brown.

[0072] The detection images were analyzed using ImageJ under default conditions, and the average fluorescence intensity of LMP1+CK19+, CK19+ DAPI+ and LMP1+CD19+, CD19+ DAPI+ was statistically analyzed by ImageJ software, using the following formula;

[0073] Mean fluorescence intensity Mean = total fluorescence intensity of the region InDen / area of ​​the region Area;

[0074] Through testing, it can be seen that in 30 EBV virus-infected thymus tissue specimens, the minimum value of LMP1+CK19+ / CK19+ DAPI+ is 62.42%; in 30 EBV virus-infected salivary gland tissue specimens, the minimum value of LMP1+CK19+ / CK19+DAPI+ is 66.67%. In 30 EBV virus-infected thymus tissue specimens, the minimum value of LMP1+CD19+ / CD19+DAPI+ is 86.76%, and in 30 EBV virus-infected salivary gland tissue specimens, the minimum value of LMP1+CD19+ / CD19+ DAPI+ is 84.87%. The following are the average fluorescence intensity and related proportions of related indicators.

[0075] Table 1 Expression levels of EBV latent infection-related indicators in epithelial cells

[0076]

[0077] Table 2 Expression levels of EBV latent infection-related indicators in B cells

[0078]

[0079] It can be seen from the above table that in thymus tissue, if the LMP1+CK19+ / CK19+DAPI+ ratio is greater than 62.42%, it is judged that the latent infection of EBV virus in epithelial cells is more severe; in salivary gland tissue, if the LMP1+CK19+ / CK19+DAPI+ ratio is greater than 66.67%, it is judged that the latent infection of EBV virus in epithelial cells is more severe.

[0080] In thymus tissue, if the LMP1+CD19+ / CD19+DAPI+ ratio is greater than 86.76%, it is judged that the latent infection of EBV virus in B lymphocytes is more severe; in salivary gland tissue, if the LMP1+CD19+ / CD19+DAPI+ ratio is greater than 84.87%, it is judged that the latent infection of EBV virus in B lymphocytes is more severe.

[0081] Example 3: Detection of EBV latently infected epithelial cells in the tissue microenvironment of thymic hyperplasia combined with Sjögren's syndrome

[0082] Quantum dot-coupled antibodies QDs-525-LMP1 and QDs-625-CK19 were prepared according to the method of Example 1. Thymus tissue specimens and salivary gland tissue specimens infected with EBV virus were taken, and the tissue sections were pretreated according to the method of Example 3, and then stained with quantum dot-coupled antibodies, and the specific steps were as follows;

[0083] First round of dyeing:

[0084] Blocking: Remove 1× TBST solution, slowly add 10% BSA, and incubate at room temperature for 10 minutes.

[0085] Preparation of antibodies: dilute the antibodies with 1×PBS to prepare QDs-525-LMP1 quantum dot labeled antibody solution.

[0086] Incubation with the first antibody: Remove excess BSA, add QDs-525-LMP1 quantum dot-labeled antibody, place the tissue sections flat in a humidified box, and then place in a 4°C refrigerator overnight.

[0087] Rinse: Rinse three times with a wash bottle containing 1×TBST, 2 minutes each time, to remove unbound QDs-525-LMP1 quantum dot-labeled antibodies.

[0088] After the first round of staining, the second round of staining was performed;

[0089] Blocking: Remove 1× TBST solution, slowly add 10% BSA, and incubate at room temperature for 10 minutes.

[0090] Preparation of antibodies: dilute the antibodies with 1×PBS to prepare QDs-625-CK19 quantum dot-coupled antibody solution.

[0091] Secondary antibody incubation: Remove excess BSA, add QDs-625-CK19 quantum dot-coupled antibody, place tissue sections flat in a humidified box, and then place in a 4°C refrigerator overnight.

[0092] Rinse: Rinse three times with a wash bottle containing 1×TBST, 2 minutes each time, to remove unbound QDs-625-CK19 quantum dot-coupled antibodies.

[0093] After the tissue sections have been stained, the nuclei are stained. 1×DAPI solution is prepared with ddH20 1:100; 1×DAPI solution is added to the tissue sections, incubated at room temperature in the dark for 5 minutes, rinsed with 1×TBST for 2 minutes, and rinsed with ddH2O for 1 minute. After removing the excess liquid on the sections, 30μl of anti-fade mounting medium is added, and then the coverslip is covered. After the tissue sections are stable, the results are observed and photographed using a fluorescence microscope equipped with a 488nm laser. The green light signal represents LMP1, and the red light signal represents CK19; the co-expression of LMP1+CK19+ is yellow, and the CK19+DAPI+ is purple; Figure 1 Figure 2 shown.

[0094] LMP1 and CK19 are two indicators marked in the microenvironment of thymus and salivary gland tissues. LMP1 positivity represents EBV latent infection, and CK19 positivity represents epithelial cells. Figure 1 and Figure 2 It means analyzing the proportion of EBV-infected epithelial cells in the thymus and salivary gland tissues in the latent infection period to all epithelial cells in the thymus and salivary gland tissues to evaluate the status and degree of EBV latent infection in epithelial cells, and then comparing LMP1+CK19+ / CK19+DAPI+ to evaluate the status and degree of EBV latent infection in epithelial cells. The higher the ratio, the more severe the latent infection. The proportion of LMP1+CK19+ / CK19+DAPI+ was calculated. In the thymus tissue sample, the ratio of LMP1+CK19+ / CK19+DAPI+ was 62.57%, which judged that the thymus tissue EBV virus latent infection was more severe in the epithelial cells; in the salivary gland tissue, the ratio of LMP1+CK19+ / CK19+DAPI+ was 68.03%, which judged that the salivary gland tissue EBV virus latent infection was more severe in the epithelial cells.

[0095] Example 4: Detection of EBV latently infected B cells in the tissue microenvironment of thymic hyperplasia combined with Sjögren's syndrome

[0096] Quantum dot-coupled antibodies QDs-525-LMP1 and QDs-625-CD19 were prepared according to the method of Example 1. Thymus tissue specimens infected with EBV virus and salivary gland tissue specimens were pretreated according to the method of Example 3, and then stained with quantum dot-coupled antibodies, and the specific steps were as follows;

[0097] First round of dyeing:

[0098] Blocking: Remove 1× TBST solution, slowly add 10% BSA, and incubate at room temperature for 10 minutes.

[0099] Preparation of antibodies: dilute the antibodies with 1×PBS to prepare QDs-525-LMP1 quantum dot labeled antibody solution.

[0100] Incubation with the first antibody: Remove excess BSA, add QDs-525-LMP1 quantum dot-labeled antibody, place the tissue sections flat in a humidified box, and then place in a 4°C refrigerator overnight.

[0101] Rinse: Rinse three times with a wash bottle containing 1×TBST, 2 minutes each time, to remove unbound QDs-525-LMP1 quantum dot-labeled antibodies.

[0102] After the first round of staining, the second round of staining was performed;

[0103] Blocking: Remove 1× TBST solution, slowly add 10% BSA, and incubate at room temperature for 10 minutes.

[0104] Preparation of antibodies: dilute the antibodies with 1×PBS to prepare QDs-625-CD19 quantum dot-coupled antibody solution.

[0105] Secondary antibody incubation: remove excess BSA, add QDs-625-CD19 quantum dot-coupled antibody, place tissue sections flat in a humidified box, and then place in a 4°C refrigerator overnight.

[0106] Rinse: Rinse three times with a wash bottle containing 1×TBST, 2 minutes each time, to remove unbound QDs-625-CD19 quantum dot-coupled antibodies.

[0107] After the tissue sections are stained, the nuclei are stained. 1×DAPI solution is prepared with ddH20 1:100; 1×DAPI solution is added to the tissue sections, incubated at room temperature in the dark for 5 minutes, rinsed with 1×TBST for 2 minutes, and rinsed with ddH2O for 1 minute. After removing the excess liquid on the sections, 30μl of anti-fade mounting medium is added, and then the coverslip is covered. After the tissue sections are stable, the results are observed and photographed using a fluorescence microscope equipped with a 488nm laser. The green light signal represents LMP1, and the red light signal represents CD19. Among them, the co-expression of LMP1+CD19+ is yellow light, and the CD19+DAPI+ is purple light. The results are as follows: Figure 3 Figure 4 shown.

[0108] LMP1 and CD19 are two indicators marked in the microenvironment of thymus and salivary gland tissues. LMP1 positivity represents EBV latent infection, and CD19 positivity represents B cells. Figure 3 and Figure 4 It indicates the analysis of the proportion of EBV-infected B cells in the latent infection period in the thymus and salivary gland tissues to all B cells in the thymus and salivary gland tissues. Among them, CD19+DAPI+ indicates the distribution of B cells in the thymus or salivary gland tissues, and LMP1+CD19+ indicates the latent infection of EBV in the thymus or salivary gland tissues. LMP1+CD19+ / CD19+DAPI+ is used to evaluate the state and degree of EBV latent infection in B cells. The higher the ratio, the more severe the latent infection, and the risk of EBV activation, proliferative diseases and tumors is greatly increased. Calculate the LMP1+CD19+ / CD19+DAPI+ ratio. In the thymus tissue, the LMP1+CD19+ / CD19+DAPI+ ratio was 87.21%, which indicated that the latent infection of EBV virus in the thymus tissue was more severe in B lymphocytes; in the salivary gland tissue, the LMP1+CD19+ / CD19+DAPI+ ratio was 84.90%, which indicated that the latent infection of EBV virus in the salivary gland tissue was more severe in B lymphocytes.

[0109] 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 latent infection of EBV in epithelial cells and B cells in the microenvironment of thymus and salivary gland tissues, characterized in that: Including quantum dot-labeled LMP1 antibody, quantum dot-labeled CK19 antibody, and quantum dot-labeled CD19 antibody; quantum dots include QDs-525, QDs-585 and QDs-625; quantum dots of different colors are combined with different antibodies; 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 EBV latent infection detection kit for epithelial cells and B cells in the thymus and salivary gland tissue microenvironment according to claim 1, characterized in that: Specifically include quantum dot-antibody conjugates QDs-525-LMP, QDs-585-CK19, QDs-625-CD19, as well as antigen repair solution and nucleic acid staining solution.

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