An Epstein-Barr virus infection lymphatic tissue epithelial mesenchymal transformation detection kit and a preparation method and application thereof

Abstract

The present application relates to a kind of EB virus infection lymphatic epithelial-mesenchymal transition detection kit and its preparation method and application, including colloidal quantum well labeled probe EBER, colloidal quantum well labeled antibody CLDN4, colloidal quantum well labeled antibody N-cadherin;By colloidal quantum well-probe / antibody conjugate in turn to the staining of lymphatic tissue section, the EMT-related antigen expressed in section is marked, so as to realize colloidal quantum well to the qualitative or quantitative of lymphatic tissue cell that EMT occurs, can realize the comprehensive evaluation to virus infection state, epithelial-mesenchymal transition process and its immune function influence.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of detection, and particularly relates to an Epstein-Barr virus (EBV) infection lymphatic epithelial-mesenchymal transition detection kit as well as a preparation method and application thereof. BACKGROUND

[0002] Thymic epithelial tumor (TET) originates from thymic epithelial cells and is the most common tumor in the anterior mediastinum of adults. The pathological morphological structure of thymoma is complex, and different types are divided according to the proportion of epithelial cells and lymphocytes. The mechanism of thymoma is not clear at present, which includes epigenetic changes, abnormal immune microenvironment and other factors. It has been reported that viral infection exists in thymoma patients, which shows that the thymus can be infected by viruses, and the viruses may participate in the malignant transformation of normal thymus. Through the method of double immunofluorescence staining, researchers identified functional EBV receptors in immature human thymocyte subsets, and found that stable viral infection can be established in thymocytes after co-culture with EBV for 6 weeks. In addition, it has been reported that nearly half of the lymphoepithelioma-like carcinoma (LELC) cases are EBV positive. Since the thymus is a very important central lymphatic organ in the human body, the thymic epithelium has certain lymphoepithelial characteristics, so it is speculated that thymic epithelial tumors and EBV infection are related.

[0003] Epstein-Barr virus (EBV), also known as human herpesvirus 4 (HHV-4), is a member of the gamma herpesvirus family, which is a lymphotropic double-stranded DNA virus with an infection rate of about 95% in the global population. Due to its lymphocyte tropism, early studies have found that EBV mainly infects human B cells and replicates and latently. In recent years, more and more studies have proved that EBV can also infect various epithelial cells and cause lesions. The latent period of EBV refers to the state of hiding and latency after EBV infects epithelial cells, which is used to escape the monitoring and removal of the immune system, and this stage is beneficial to the long-term survival of the virus in the lymphatic tissue environment.

[0004] Epithelial-mesenchymal transition (EMT) is a biological process in which epithelial cells change in morphology and function to mesenchymal cells, characterized by reduced cell adhesion, enhanced cell migration and invasion. EMT occurs during embryonic development in normal physiological conditions, specifically involved in the formation of primitive gut and the process of neural crest cell layering. In the process of EMT, epithelial cells transdifferentiate into mesenchymal phenotype, accompanied by reduced intercellular adhesion, removal of cell polarity and remodeling of actin cytoskeleton. At the same time, through the change of cell and extracellular matrix (ECM) interaction, the cells undergoing EMT show increased expression of mesenchymal markers and enhanced migration ability.

[0005] The lymphoid tissue microenvironment infected by Epstein-Barr virus is a complex environment, including the interaction and mutual influence of various immune cells, epithelial cells and interstitial cell tissues, however, at present, the technology for detecting the relationship between the virus and epithelial-mesenchymal transition in the above complex environment, and exploring the relationship and mechanism between the action of the virus and epithelial-mesenchymal transition has not been solved and developed. SUMMARY

[0006] To solve the above technical problems, the present application provides an EB virus infection lymphoid tissue epithelial-mesenchymal transition detection kit and its preparation method and application.

[0007] The technical scheme adopted by the present application is: an EB virus infection lymphoid tissue epithelial-mesenchymal transition detection kit, comprising a colloidal quantum well-probe EBER conjugate, a colloidal quantum well-antibody CLDN4 conjugate, and a colloidal quantum well-antibody N-cadherin conjugate.

[0008] Preferably, it further comprises a colloidal quantum well-protein LMP2A conjugate.

[0009] Preferably, the colloidal quantum wells coupled with the probe EBER, the protein LMP2A, the antibody CLDN4 and the antibody N-cadherin have different light emitting wavelengths.

[0010] Preferably, the colloidal quantum well comprises one or more of CQW-525, CQW-565, CQW-585 and CQW-625.

[0011] Preferably, it further comprises an antigen repair solution and a DNA fluorescent dye solution.

[0012] The method for preparing the EB virus infection lymphatic tissue epithelial mesenchymal transformation detection kit, the colloidal quantum well is mixed with EDC and NHS at a molar ratio of 1:1-5:1.5-7.5 to activate the carboxyl group; the nucleic acid probe or the antibody is added, and the colloidal quantum well-probe conjugate or the colloidal quantum well-antibody conjugate is formed after the coupling reaction;

[0013] The molar ratio of CQW to nucleic acid probe is 1:1.5-3, or the molar ratio of CQW to antibody is 1:10-20.

[0014] Preferably, the colloidal quantum well is one or more of CdSe, CdSe / CdS, CdSe / CdZnS or CdZnSe / ZnS.

[0015] The method for using the EB virus infection lymphatic tissue epithelial mesenchymal transformation detection kit, the lymphatic tissue section is prepared, after antigen repair, the different colloidal quantum well-probe / antibody conjugates are used in sequence to carry out antigen antibody binding reaction on the tissue section, the image is collected under the fluorescence microscope after staining, and the luminescence information is detected to quantify the luminescence intensity and spatial position information.

[0016] Preferably, the staining sequence is colloidal quantum well-probe EBER conjugate, colloidal quantum well-protein LMP2A conjugate, colloidal quantum well-antibody CLDN4 conjugate, and colloidal quantum well-antibody N-cadherin conjugate.

[0017] Preferably, the expression of nucleic acid EBER labeled with colloidal quantum well is used to detect the expression amount and spatial position of EB virus in the lymphatic tissue, and the average fluorescence intensity of the corresponding cells is calculated to reflect the EB virus infection in the thymus microenvironment;

[0018] And / or the expression of antibody LMP2A labeled with colloidal quantum well is used to detect the expression amount and spatial position of the virus-acting protein in the lymphatic tissue, and the average fluorescence intensity of the corresponding protein is calculated to reflect the virus action in the thymus microenvironment;

[0019] And / or the expression of antibody CLDN4 labeled with colloidal quantum well is used to detect the expression amount and spatial position of epithelial cells in the lymphatic tissue, and the average fluorescence intensity of the corresponding cells is calculated to reflect the epithelial expression in the thymus microenvironment;

[0020] And / or the expression of antibody N-cadherin labeled with colloidal quantum well is used to detect the expression amount and spatial position of mesenchymal cells in the lymphatic tissue, and the average fluorescence intensity of the corresponding cells is calculated to reflect the mesenchymal expression in the thymus microenvironment;

[0021] The EB virus infection lymphatic tissue epithelial mesenchymal transition detection kit or the use method of the EB virus infection lymphatic tissue epithelial mesenchymal transition detection kit is applied to the detection of the EB virus infection lymphatic tissue epithelial mesenchymal transition.

[0022] Preferably, in the lymphatic tissue tissue that is EBER positive and LMP2A positive, the average fluorescence intensity of the marker antibody N-cadherin is greater than the average fluorescence intensity of the marker antibody CLDN4, indicating that the degree of epithelial mesenchymal transition in the EB virus infected lymphatic tissue tissue is high;

[0023] In the lymphatic tissue tissue that is EBER negative and LMP2A negative, the average fluorescence intensity of the marker antibody N-cadherin is less than the average fluorescence intensity of the marker antibody CLDN4, indicating that the degree of epithelial mesenchymal transition in the lymphatic tissue tissue is low or no epithelial mesenchymal transition occurs.

[0024] Preferably, if EBER is positive and the fluorescence intensity of N-cadherin is higher than that of CLDN4, it is judged that the degree of EMT is high and the antigen presentation ability is strong.

[0025] If EBER is negative and the fluorescence intensity of N-cadherin is lower than that of CLDN4, it is judged that the degree of EMT is low or no EMT occurs, and the antigen presentation ability is weak.

[0026] The application has the advantages and positive effects that the combination of the quantum well coupled probe EBER and the colloidal quantum well coupled antibodies LMP2A, CLDN4 and N-cadherin can realize the labeling of different target objects in the same tissue section, can realize the synchronous detection of the EB virus infection related signals, epithelial / interstitial structure changes and immune cell distribution in the tissue, and effectively support the quantitative evaluation and spatial positioning analysis of the immune microenvironment changes caused by EBV infection.

[0027] The detection method is simple and fast, and the colloidal quantum well replaces the fluorescent dye, and has outstanding performance in the control of the light emission spectrum, the light signal intensity, the stability and the antibody loading capacity, and can realize both quantitative detection and position detection. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 The fluorescence detection results of one parotid tissue detection sample in Example 5;

[0029] Figure 2 The expression of CLDN4 and N-cadherin in the EB infection positive parotid tissue in Example 6;

[0030] Figure 3 The expression of CLDN4 and N-cadherin in the EB infection negative parotid tissue in Example 6. DETAILED DESCRIPTION

[0031] Embodiments of the present application will be described below with reference to the accompanying drawings.

[0032] The present application relates to an Epstein-Barr virus infection lymphatic epithelial-mesenchymal transition detection kit, its preparation method and application, including colloidal quantum well coupled probe EBER, and colloidal quantum well coupled antibodies CLDN4 and N-cadherin. By detecting Epstein-Barr virus nucleic acid and related protein expression, combined with epithelial and mesenchymal marker positioning analysis, it can be used to reveal the degree of epithelial-mesenchymal transition caused by Epstein-Barr virus infection, and assist in evaluating the role of the transformation process in antigen presentation function. After being used for staining of lymphatic tissue sections, it can realize synchronous detection of Epstein-Barr virus infection related signals, epithelial / mesenchymal structural changes, and immune cell distribution in the tissue, effectively supporting quantitative evaluation and spatial positioning analysis of immune microenvironment changes caused by EBV infection, and providing a more refined, multi-dimensional technical support platform for pathological research, diagnosis development and clinical pathological mechanism research.

[0033] EBER (Epstein-Barr Virus-Encoded RNA) is a non-coding RNA molecule encoded by Epstein-Barr Virus (EBV), which belongs to small RNA encoded by viruses. EBER plays an important role in the process of EBV infection, and EBV can inhibit the immune response of host cells by producing EBER, especially the antiviral response of the host. EBER also plays a key role in the latent phase of the virus, and it is a signature molecule of EBV latent infection. After EBV infection, the presence of EBER helps the virus maintain a latent state in the host cell. EBER may also affect the gene expression of host cells by interacting with certain RNA or proteins of the host. It interacts with various regulatory factors of the host cell, which may promote cell survival and proliferation, which is beneficial for EBV to maintain its latent infection state.

[0034] The role of CLDN4 and N-cadherin proteins in epithelial-mesenchymal transition (EMT) is very important, as they play key roles in cell morphology, cell adhesion, migration, and other aspects. CLDN4 is one of the tight junction proteins, mainly located in the tight junction region of epithelial cells, maintaining the barrier function between cells. During epithelial-mesenchymal transition, the expression of CLDN4 often decreases or changes, leading to the destruction of tight junctions, which provides conditions for cell migration. In the detection of EMT, the change of CLDN4 is an important indicator for evaluating the change of cell barrier function and cell morphology. Tight junctions are structural connections between epithelial cells and endothelial cells, which play a key role in maintaining cell barrier function and regulating the permeability of substances through intercellular gaps. CLDN4 can affect how dendritic cells recognize and present antigens. When the expression of CLDN4 decreases, the stability and density of tight junctions may be affected, and the barrier function between cells becomes more relaxed. In this way, antigens and pathogens are more easily able to pass through the epithelial barrier into the immune system, which can lead to more antigens being taken up by immune cells (such as dendritic cells). In this case, the efficiency of antigen presentation may increase, thereby activating more immune responses. However, excessive immune responses can lead to abnormal reactions of the immune system, such as autoimmune reactions.

[0035] N-cadherin is a classic calcium-dependent adhesion molecule that plays an important role in cell adhesion between cells. N-cadherin is usually expressed in the process of EMT, and its increase helps cell migration and invasion, promoting the process of epithelial cells transforming into mesenchymal cells. N-cadherin can be used to track cell migration and invasion as a marker of cell adhesion during EMT. Its upregulation is usually accompanied by changes in cell morphology and is a key marker of the transition from epithelial to mesenchymal state.

[0036] The use of CLDN4 and N-cadherin in combination can more comprehensively evaluate the process of EMT, and the combination of the two can reveal changes in cell adhesion and loss of cell polarity between cells.

[0037] Epstein-Barr virus (EBV) is associated with epithelial-mesenchymal transition (EMT). During the latent phase of EB virus, key proteins such as LMP1, LMP2A, and other key proteins may play a role in activating host cells, causing host cells to change their original morphology or transcriptional expression of genes in the nucleus.

[0038] During EMT, epithelial cells lose intercellular connections and acquire mesenchymal-like migratory and invasive properties. LMP1 (Latent Membrane Protein 1) and LMP2A (Latent Membrane Protein 2A), as important latent membrane proteins of EBV, play a key role in EBV-induced cell transformation. Epstein-Barr virus latent membrane protein 1 (LMP 1) is a central factor encoded by EBV that plays a regulatory role in malignant processes, and it has been considered as a core effector protein of EBV; LMP2A is an important latent membrane protein encoded by Epstein-Barr virus (EBV) with six transmembrane regions. Compared with LMP1, LMP2A has a relatively simple structure, but it still plays a crucial role in the latent state of the virus and host immune escape. Despite their different structural characteristics and functions, LMP1 and LMP2A have complementary roles in the life cycle of EBV, especially in maintaining latent infection, immune escape, and promoting tumorigenesis. LMP2A promotes cell proliferation and anti-apoptosis by activating NF-κB, JAK / STAT signaling pathways, and to some extent, promotes the occurrence of EMT. LMP2A can promote the invasion and metastasis of tumor cells by up-regulating EMT-related genes such as Twist, Snail, and ZEB1. In addition, LMP2A further supports the transformation of epithelial cells to mesenchymal cells and enhances the invasiveness of tumors by regulating the remodeling of extracellular matrix (ECM) and cell-matrix interaction. The mechanism of action of LMP2A is different from LMP2A, but it can also promote the occurrence of EBV-related tumors and the EMT process. LMP2A inhibits the maturation and antigen presentation function of B cells by regulating the B cell receptor signaling pathway, and may promote the mesenchymalization of epithelial cells by down-regulating N-cadherin expression. LMP2A is involved in the changes of tumor microenvironment through its effects on cell adhesion and cell migration.

[0039] Based on the above, in some embodiments of the present application, the EB virus infection lymphatic tissue epithelial-mesenchymal transformation detection kit also includes a colloidal quantum well coupled antibody LMP2A. The expression amount and expression position of the probes EBER, LMP2A, CLDN4 and N-cadherin are detected at the same time, which can reveal the degree of epithelial-mesenchymal transformation caused by EB virus infection, assist in evaluating the role of the transformation process in antigen presentation function, and further explore whether EB virus plays a role through LMP2A protein. By staining the lymphatic tissue paraffin section sample with colloidal quantum well and probe EBER conjugate, and colloidal quantum well and CLDN4 and N-cadherin antibody conjugate, the expression of EB virus and EMT related antigens in the section can be specifically recognized, so as to realize the qualitative or quantitative detection of EB virus and EMT indicators. EBER is a key indicator of EB virus, and colloidal quantum well labeled EBER is used to evaluate the EB virus infection state in the lymphatic tissue microenvironment; CLDN4 is an epithelial marker, and colloidal quantum well labeled CLDN4 is used to evaluate the epithelial state in the lymphatic tissue microenvironment; N-cadherin is an interstitial marker, and colloidal quantum well labeled N-cadherin is used to evaluate the interstitial state in the lymphatic tissue microenvironment; CLDN4 is also an antigen presentation related molecule, and colloidal quantum well labeled CLDN4 is used to evaluate the antigen presentation in the lymphatic tissue microenvironment; by detecting the expression and position of EBER, CLDN4 and N-cadherin, the influence of EB virus on epithelial-mesenchymal transformation in EB virus infected lymphatic tissue can be evaluated.

[0040] Alternatively, by staining the lymphatic tissue paraffin section sample with colloidal quantum well and probe EBER conjugate, and colloidal quantum well and LMP2A, CLDN4 and N-cadherin antibody conjugate, the expression of EB virus, EB virus infection related pathways and EMT related antigens in the section can be specifically recognized, so as to realize the qualitative or quantitative detection of EB virus and EMT indicators. LMP2A is used to evaluate the mechanism of EB virus in the lymphatic tissue microenvironment.

[0041] In addition, when EBV is positive, the expression of CLDN4 protein is reduced and the expression of N-Cadherin protein is increased, indicating that epithelial-mesenchymal transformation occurs, suggesting that the cell migration and invasion ability in the tissue environment is enhanced, and thus predicting a poor prognosis of the patient; in addition, the decrease of CLDN4 protein further increases the antigen presentation efficiency, leading to enhanced immune response, thereby predicting the occurrence and development of autoimmune diseases. By detecting the expression and position of EBER, CLDN4 and N-cadherin in the same tissue section, the epithelial-mesenchymal transformation can be judged, and a more accurate prognosis can also be made.

[0042] The coupling of colloidal quantum wells with nucleic acids comprises the following steps:

[0043] Step 1: Activation of colloidal quantum wells; prepare carboxyl-modified water-soluble colloidal quantum wells with different emission wavelengths, respectively, at a concentration of about 8 μM, then add EDC (final concentration 0.2 mg / mL) and NHS (final concentration 0.5 mg / mL) to the colloidal quantum well solution, and gently shake the reaction at room temperature in the dark for 30 minutes to activate the carboxyl groups on the surface of the colloidal quantum wells, and the generated intermediates facilitate subsequent covalent connection with probes;

[0044] Step 2: Preparation of CQW-probe conjugates; after activation, the reaction solution is quickly filtered by ultrafiltration (10 kDa filter membrane) to remove free EDC / NHS, and then resuspended in PBS (pH 7.4) buffer to the original volume. Then add NH2-modified probes, and the molar ratio of probes to quantum dots can be set to 1.5-3:1. Shake the mixed system at room temperature for 2 hours or at 4°C overnight to complete the coupling reaction;

[0045] Step 3: After the reaction is completed, the colloidal quantum well-probe complex is obtained, which is purified again using an ultrafiltration tube to remove uncoupled probes, and then replaced into the final storage buffer (PBS, pH 7.4) with the addition of a final concentration of 0.1% BSA or 1 U / μl RNase inhibitor.

[0046] The colloidal quantum well-EBER probe complex can be prepared by the above method, and the obtained colloidal quantum well-EBER probe complex can be used for EBER in situ hybridization experiments to achieve specific visualization of EBV infected cells, and has high light stability and signal strength.

[0047] The coupling of colloidal quantum wells with antibodies comprises the following steps:

[0048] Step 1: Mix CQW, EDC, and NHS at a molar ratio of 1:1-5:1.5-7.5, dilute with PBS, mix uniformly on a shaker at room temperature for 30 min, then adjust the pH to 8-9 with PBS (PH 9.18) to obtain colloidal quantum well activation solution;

[0049] Step 2: Take 100 ul of colloidal quantum well activation solution and add it to the reaction container, take an appropriate amount of antibody stock solution, dilute it to 0.01 mg / ml with PBS buffer, mix thoroughly, and then add it to the above reaction container, and react for 4 h on a shaker at 4°C in the dark;

[0050] Step 3: After the reaction is completed, centrifuge at 8000 rpm for 3 min to remove possible agglomerated precipitates, and retain the supernatant; the supernatant contains colloidal quantum well-antibody conjugates.

[0051] The molar ratio of the colloidal quantum well and the specific antibody binding is the result of a large number of verifications in EB virus infected lymphoid tissues. The molar ratio not within the interval will cause the colloidal quantum well to be unstable in binding with the antibody, which is specifically manifested as follows: if the molar ratio of the colloidal quantum well and the antibody binding is high, such as 1:5, a large number of false positive results will appear when the average fluorescence intensity of co-expression is detected by ImageJ; if the molar ratio of the colloidal quantum well and the antibody binding is low, such as 1:30, the fluorescence of the colloidal quantum well binding with the antibody to the antigen cannot be detected when the average fluorescence intensity of co-expression is detected by ImageJ. When the molar ratio of the colloidal quantum well and the antibody binding is preferably 1:10-20, the expression and spatial position of the antigen can be accurately and clearly displayed.

[0052] In order to be able to realize the labeling of different target objects in the same slice respectively, the colloidal quantum wells with different light-emitting wavelengths (colors) are used to couple with the probes EBER or antibodies LMP2A, CLDN4 and N-cadherin respectively; the emission wavelength of the colloidal quantum well is located in the range of about 450-650 nm, and the specific wavelength selection can be adjusted according to the experimental requirements. In some embodiments of the present application, the colloidal quantum wells CQW-525, CQW-565, CQW-585 and CQW-625 are selected to couple with EBER, LMP2A, CLDN4 and N-cadherin respectively to obtain CQW-525-EBER, CQW-565-LMP2A, CQW-585-CLDN4 and CQW-625-N-cadherin. The EBER nucleic acid is coupled with the colloidal quantum well CQW-525, and the emitted light is displayed as green; the LMP2A antibody is coupled with the colloidal quantum well CQW-565, and the emitted light is displayed as yellow; the CLDN4 antibody is coupled with the colloidal quantum well CQW-585, and the emitted light is displayed as orange; and the N-cadherin antibody is coupled with the colloidal quantum well CQW-625, and the emitted light is displayed as red. In some other embodiments of the present application, the colloidal quantum wells coupled with different antibodies or probes can be interchanged to produce other color matching schemes.

[0053] In some embodiments of the present application, the EB virus infected lymphoid tissue epithelial mesenchymal transformation detection kit comprises colloidal quantum well and probe EBER coupling agent, colloidal quantum well labeled antibody CLDN4 and colloidal quantum well labeled antibody N-cadherin, antigen repair solution and nucleic acid staining solution.

[0054] In some other embodiments of the present application, the EB virus infected lymphoid tissue epithelial mesenchymal transformation detection kit comprises colloidal quantum well and probe EBER coupling agent, colloidal quantum well labeled antibody LMP2A, colloidal quantum well labeled antibody CLDN4 and colloidal quantum well labeled antibody N-cadherin, antigen repair solution and nucleic acid staining solution.

[0055] When the lymph tissue section is stained by the lymph tissue epithelial-mesenchymal transition detection kit for B virus infection, different colloidal quantum well-probe / antibody conjugates can be used to stain the same tissue section in turn, and the multi-channel fluorescence image information of the tissue section is collected. By combining the spatial positioning and intensity distribution of different channel fluorescence signals, the expression of EB virus infection and epithelial and mesenchymal related indicators can be observed by color contrast difference, and the EB virus infection state, the expression level of epithelial and mesenchymal cells and their mutual relationship can be judged, so as to judge the epithelial-mesenchymal transition in the EB virus infected lymph tissue. In some preferred embodiments of the present application, the staining order is EBER first, then CLDN4, and finally N-cadherin. This order can ensure accurate detection of EBV infection and EMT state; or the staining order is EBER first, then LMP2A, then CLDN4, and finally N-cadherin.

[0056] The specific use steps are as follows:

[0057] Step 1: Take the lymph tissue patient paraffin specimen to make tissue sections (the tissue is derived from surgical puncture or cutting sample), and bake and deparaffinize and dehydrate the tissue sections;

[0058] Step 2: Microwave the tissue sections for antigen repair;

[0059] Step 3: Stain the tissue sections by colloidal quantum well and nucleic acid conjugate; stain the tissue sections by colloidal quantum well and antibody conjugate, when the colloidal quantum well and antibody conjugate include multiple, the staining is carried out in turn according to the order of the specific protein in the tissue from strong to weak;

[0060] Step 4: Stain the cell nucleus, mount the section and take pictures, detect and analyze the quantity information and position information of virus, epithelium and mesenchyme, and evaluate the EMT degree by the expression of EB virus, epithelial indicators and mesenchymal indicators in the lymph tissue.

[0061] EBER is a key indicator of EB virus, colloidal quantum well labeled EBER is used to evaluate the infection status of EB virus in lymphoid tissue microenvironment, and colloidal quantum well-nucleic acid conjugates (such as CQW-525-EBER) can be used to locate and detect EBER; colloidal quantum well labeled LMP2A is used to evaluate the mechanism of EB virus in lymphoid tissue microenvironment; CLDN4 is an epithelial marker, colloidal quantum well labeled CLDN4 is used to evaluate the epithelial state in lymphoid tissue microenvironment; N-cadherin is a stromal marker, colloidal quantum well labeled N-cadherin is used to evaluate the stromal state in lymphoid tissue microenvironment; by detecting the expression and location of EBER, CLDN4 and N-cadherin, the expression and distribution information of virus, epithelial and stromal markers in the tissue can be obtained, which can evaluate the influence of EB virus on epithelial-mesenchymal transition in EB virus infected lymphoid tissue; evaluate the degree of epithelial-mesenchymal transition in EBV infected lymphoid tissue, and then predict the degree of thymocyte migration.

[0062] After staining the lymphoid tissue sections with different types of colloidal quantum well-nucleic acid conjugates and colloidal quantum well-antibody conjugates in turn, take photos by fluorescence microscope, analyze the multi-channel fluorescence images collected by ImageJ software, and calculate the average fluorescence intensity of EBER, LMP2A, CLDN4 and N-cadherin, using the following formula:

[0063] Average fluorescence intensity Mean = total fluorescence intensity InDen in the area / area of the area;

[0064] In the lymphoid tissue, if the average fluorescence intensity of EBER is greater than 503.267, it is determined that EBER is positive; at the same time, EBER positive means that the expression degree of EBER fluorescence nucleus in the lymphoid tissue section is high, and the spatial position is relatively dense. In the EBER positive tissue, the average fluorescence intensity of CLDN4 is less than 153.011 and the average fluorescence intensity of N-cadherin is greater than 501.629, indicating that the degree of epithelial-mesenchymal transition in EB virus infected lymphoid tissue is high. When the same lymphoid tissue section is also stained with colloidal quantum well-antibody LMP2A, if the average fluorescence intensity of LMP2A is greater than 403.309, it is determined that LMP2A is positive, which proves that the EB infection in the tissue section is related to the LMP2A related pathway.

[0065] In the lymphoid tissue, if the average fluorescence intensity of EBER is less than 503.267, it is determined that EBER is negative; at the same time, EBER negative means that the expression degree of EBER fluorescence nucleus in the lymphoid tissue section is low or no fluorescence expression, and the spatial position is more dispersed. In order to further compare the epithelial mesenchymal transition in the tissue section under the condition of EBER positive (EB virus infection) and EBER negative (EB virus non-infection), the expression of CLDN4 and N-cadherin in EBER negative tissue can be further evaluated; if the average fluorescence intensity of CLDN4 is greater than 500.026 and the average fluorescence intensity of N-cadherin is less than 148.277, it is indicated that the average fluorescence intensity of epithelial index CLDN4 is greater than that of mesenchymal index N-cadherin when EB virus is not infected, which indicates that the epithelial mesenchymal transition in the lymphoid tissue is low or does not occur.

[0066] Colloidal quantum wells (CQWs) are used as fluorescent probes for multiple immunolabeling to realize high-throughput and multi-parameter precise detection of specific targets (such as Tfh cells, dendritic cells, EB virus infection markers, etc.) in tissue sections. CQWs are a kind of semiconductor nanomaterials with a clear two-dimensional structure. Their nanoscale thickness and adjustable lateral size endow them with unique optical properties and surface chemical behavior, making them a new type of labeling material with more comprehensive performance advantages after traditional fluorescent probes (such as quantum dots and organic fluorescent dyes). In some embodiments of the present application, the CQW is of CdSe, CdSe / CdS, CdSe / CdZnS or CdZnSe / ZnS structure, and the emission wavelength is any set wavelength within 450-650 nm.

[0067] CQWs have several key advantages in luminescence performance. First, the emission peak has an extremely narrow full width at half maximum (FWHM), usually less than 35 nm, and can even reach below 10 nm under optimized structure, which is much better than the emission width of traditional QDs usually in the range of 20-40 nm. This spectral concentration significantly reduces the spectral crosstalk between multiple channels, especially suitable for multiple fluorescence labeling experiments that require high signal-to-noise ratio and high resolution. Second, CQWs have a higher optical absorption cross section, especially under high-energy excitation (such as short-wave ultraviolet or multi-photon excitation). The two-dimensional structure determines a larger optical cross-sectional area, which means that under the same concentration and excitation conditions, a stronger fluorescence signal output can be obtained. This has a significant advantage for the detection of low-expression target points, deep tissue imaging, and thick section imaging. More importantly, the flat two-dimensional structure of CQWs allows more surface functional groups to be exposed per unit particle, allowing more antibodies to be coupled. Compared with spherical or isotropic QDs, the antibody loading capacity of a single CQW is significantly enhanced, which helps to improve the binding efficiency of target antigens and the spatial coverage of imaging signals, and improves the overall labeling efficiency and biological recognition sensitivity. In addition, CQWs have a short fluorescence lifetime and excellent anti-flashing properties. Short fluorescence lifetime helps time-resolved fluorescence imaging (such as FLIM), enhancing the discrimination of background signals; Anti-flashing performance ensures signal stability during long-time or dynamic imaging, avoiding the "instant light-out" phenomenon of quantum dots in some applications, improving experimental repeatability and data consistency.

[0068] Compared with organic fluorescent dyes (such as FITC, Cy5, Alexa series, etc.), CQWs also have significant advantages in stability, light intensity and multiplicity. First, CQWs have higher light stability and are not prone to photobleaching. Traditional fluorescent dyes are prone to rapid decay under excitation light irradiation, while CQWs can maintain stable light emission after long-term exposure, repeated imaging, or even multiple experiments, which helps to improve the reliability and traceability of experimental data. Second, CQWs have higher fluorescence intensity and quantum efficiency. Under the same concentration and excitation intensity, their signal brightness is much higher than that of traditional dyes, and their advantages are particularly obvious in weak expression, deep tissue and low background imaging scenarios. At the same time, their emission wavelength can be precisely controlled, supporting a wide spectral range from ultraviolet to near-infrared, meeting the complex wavelength requirements of multi-label combination experiments. Third, CQWs have better surface functionalization ability and larger antibody coupling capacity. Traditional dyes usually rely on indirect labeling or single-point chemical bond connection, with poor coupling efficiency and stability; while CQWs can form stable antibody connection structures after surface shell or functional polymer modification, improving the consistency and biocompatibility of labeled probes. In addition, CQWs can directly replace traditional dyes to realize in situ multi-target immunolabeling of tissue sections, and further integrate EBER in situ hybridization steps in the present application to detect EB virus signals and epithelial / mesenchymal transition markers using CQW labeled probes, simplifying the process, saving time and reagents, and overall improving detection efficiency and reducing error risk.

[0069] Using colloidal quantum wells to construct multi-label fluorescent antibody probes, they are superior to existing QDs and traditional dye technology systems in terms of emission spectrum control, light signal intensity, stability, and antibody loading capacity, and overall improve the performance of immunolabeling imaging in complex tissue environments.

[0070] By replacing fluorescent dyes with colloidal quantum wells, it is possible to detect the epithelial-mesenchymal transition of lymphoid tissue cells infected with EB virus, and the detection method is simple and fast, which is convenient for clarifying the relationship between EB virus and lymphoid tissue. By replacing fluorescent dyes with colloidal quantum wells, it is possible to directly label tissue sections, realize the local positioning of EB virus and epithelial cells and mesenchymal tissue in lymphoid tissue, and both quantitative detection and positional detection are possible, which is convenient for evaluating the influence of EB virus on EMT.

[0071] EBV infection-related signals, epithelial / interstitial structure changes, and immune cell distribution in tissues can be simultaneously detected by the EBV infection lymphatic epithelial mesenchymal transition detection kit, which effectively supports quantitative evaluation and spatial positioning analysis of immune microenvironment changes caused by EBV infection, and provides a more refined and multi-dimensional technical support platform for pathological research, diagnosis and development, and clinical pathological mechanism research. The effect of antitumor drugs can be monitored in an in vitro lymphatic tissue animal model, and the effect of the drugs can be evaluated by comparing the EMT state before and after the drug action.

[0072] By observing the expression levels and spatial distribution of EBV key indicators EBER and LMP2A and epithelial mesenchymal transition-related indicators, important reference directions can be provided for studying how viruses affect epithelial mesenchymal transition in a complex environment; or the influence of EBV on EMT to cause angiogenesis in lymphatic tissue can be observed; or the antigen presentation and immune response state in the lymphatic tissue microenvironment can be evaluated. The thymus is a key place for T cell differentiation and development, and the occurrence of lymphatic tissue can lead to the generation and output of abnormal T cells. By evaluating the epithelial mesenchymal transition (EMT) process of EBV infection, important support and basis can be provided for the generation and output of abnormal T cells, and further exploration of how EBV infection affects the immune cell function of the thymus.

[0073] The experimental methods of the operation steps are not specifically described below, and are performed according to the corresponding product instructions. The instruments, reagents, and consumables used in the examples can be purchased from commercial companies unless otherwise specified.

[0074] Example 1: Preparation of CQW-EBER conjugate

[0075] Carboxyl-modified water-soluble colloidal quantum well CQW-525 with a concentration of about 8 μM was prepared, and EDC (final concentration 0.2 mg / mL) and NHS (final concentration 0.5 mg / mL) were added to the colloidal quantum well solution. The carboxyl groups on the surface of the colloidal quantum well were activated by slight shaking under room temperature and light-free conditions for 30 minutes to generate an intermediate for subsequent covalent connection with the probe. After activation, the reaction solution was quickly filtered by ultrafiltration (10 kDa filter membrane) to remove free EDC / NHS, and then resuspended with PBS (pH 7.4) buffer to the original volume.

[0076] NH2-modified EBER probe was added, and the molar ratio of the probe to CQW-525 was 1.5-3:1. The mixed system was reacted at 4°C overnight to complete the conjugation reaction to obtain CQW-525-EBER.

[0077] After the reaction is completed, the un-coupled probes are removed by ultrafiltration tube and replaced with the final storage buffer (PBS, pH 7.4) with a final concentration of 0.1% BSA or 1 U / μl RNase inhibitor. The obtained colloidal quantum well-EBER probe complex can be used for EBER in situ hybridization experiments to achieve specific visualization of EBV infected cells with high light stability and signal intensity.

[0078] Example 2: Preparation of CQW-Ab conjugates

[0079] Carboxyl-modified water-soluble colloidal quantum wells CQW-565, CQW-585 and CQW-625 were prepared and coupled with LMP2A, CLDN4 and N-cadherin, respectively.

[0080] CQW, EDC and NHS were mixed at a molar ratio of 1:1-5:1.5-7.5, diluted with PBS, and mixed uniformly on a shaker at room temperature for 30 min. Then the pH was adjusted to 8-9 with PBS (pH 9.18) to obtain colloidal quantum well activation solution. 100 ul of colloidal quantum well activation solution was added to the reaction vessel. An appropriate amount of antibody stock solution was diluted with PBS buffer to 0.01 mg / ml, mixed thoroughly, and added to the reaction vessel at a colloidal quantum well to antibody ratio of 1:10-20. The reaction was carried out at 4°C on a shaker in the dark for 4 h. After the reaction was completed, the possible agglomerated precipitates were removed by centrifugation at 8000 rpm for 3 min, and the supernatant was retained. CQW-565-LMP2A, CQW-585-CLDN4 and CQW-625-N-cadherin were prepared, respectively.

[0081] The optimal conditions were screened and compared by 1% agarose gel electrophoresis detection analysis. The supernatant obtained under the optimal coupling conditions was purified by PD10 column, and the purified product was stored at 4°C in the dark for standby use.

[0082] Example 3: EB virus infection lymphatic epithelial mesenchymal transformation detection kit

[0083] The kit includes CQW-525-EBER prepared in Example 1, CQW-565-LMP2A, CQW-585-CLDN4, CQW-625-N-cadherin prepared in Example 2, antigen repair solution and nucleic acid staining solution.

[0084] Example 4: EB virus infection lymphatic epithelial mesenchymal transformation detection kit

[0085] CQW-525-EBER prepared in Example 1, CQW-585-CLDN4, CQW-625-N-cadherin prepared in Example 2, antigen retrieval solution, and nucleic acid staining solution.

[0086] Example 5: Application of the Epstein-Barr virus infection lymphatic epithelial mesenchymal transformation detection kit

[0087] Sixty parotid gland samples of patients with Sjogren's syndrome were collected (the samples were obtained from puncture or surgical resection), and were immersed in 4% formalin for more than 48 hours; the parotid gland samples were made into wax blocks and placed on a microtome to make 3-4 μm tissue sections. Before staining, the information of the section source, staining index, date, and antibody ratio was recorded.

[0088] The tissue sections were placed in a 60°C oven for 30 min for baking. Then, the sections were transferred to No. 1 100% xylene for 15 min, No. 2 100% xylene for 15 min, No. 3 100% ethanol for 3 min, No. 4 100% ethanol for 3 min, No. 5 95% ethanol for 3 min, No. 6 75% ethanol for 3 min, No. 7 50% ethanol for 3 min, and PBS for 3 times of 5 min each time for deparaffinization and dehydration.

[0089] Antigen repair was performed on the deparaffinized and dehydrated tissue sections. During antigen repair, acid or alkaline antigen repair solution was selected according to the corresponding antibody instruction; the antigen repair solution was added to a container, heated and boiled in a microwave oven, and then the tissue sections were placed in the container; after heating at low heat for 10 min, the container was taken out; after the tissue sections were naturally cooled to room temperature, the container was transferred to an immunohistochemical washing box, washed with sterilized water for 1 min, and washed with 1×TBST for 2 min. An immunohistochemical pen was used to enclose the tissue, and the entire tissue block on the section was located inside the enclosure.

[0090] First round of nucleic acid staining:

[0091] Blocking: The 1×TBST solution was removed, and 10% BSA was slowly added, and incubated at room temperature for 10 min.

[0092] Antibody preparation: CQW-525-EBER colloidal quantum well labeled EBER solution was prepared.

[0093] Incubation: The excess serum was removed, and CQW-525-EBER colloidal quantum well labeled EBER was added; the tissue sections were placed flat in an immunohistochemical wet box, and then placed in a 4°C refrigerator overnight.

[0094] Washing: The washing bottle containing 1×TBST was used to wash 3 times, each time for 2 min, to remove the unbound CQW-525-EBER colloidal quantum well labeled nucleic acid.

[0095] Second round of antibody staining;

[0096] Blocking: Remove 1x TBST solution, slowly add 10% BSA, incubate at room temperature for 10 min.

[0097] Antibody configuration: Dilute the antibody with 1x PBS, configure the CQW-585-CLDN4 colloidal quantum well coupled antibody solution.

[0098] Second antibody incubation: Remove excess serum, add CQW-585-CLDN4 colloidal quantum well coupled antibody, place the tissue section in the immunohistochemical wet box, and then place it in the 4°C refrigerator overnight.

[0099] Rinse: Use a wash bottle filled with 1x TBST to rinse 3 times, 2 min each time, to remove unbound CQW-585-CLDN4 colloidal quantum well coupled antibody.

[0100] Third round of antibody staining;

[0101] Blocking: Remove 1x TBST solution, slowly add 10% BSA, incubate at room temperature for 10 min.

[0102] Antibody configuration: Dilute the antibody with 1x PBS, configure the CQW-625-N-cadherin colloidal quantum well coupled antibody solution.

[0103] Second antibody incubation: Remove excess serum, add CQW-625-N-cadherin colloidal quantum well coupled antibody, place the tissue section in the immunohistochemical wet box, and then place it in the 4°C refrigerator overnight.

[0104] Rinse: Use a wash bottle filled with 1x TBST to rinse 3 times, 2 min each time, to remove unbound CQW-625-N-cadherin colloidal quantum well coupled antibody.

[0105] The stained tissue section is stained with DAPI, and 1x DAPI solution is configured with ddH20 1:100; Add 1x DAPI solution to the tissue section, incubate at room temperature in the dark for 5 min, rinse with 1x TBST for 2 min, and rinse with ddH2O for 1 min. After removing the excess liquid on the section, add 30μl of anti-quenching mounting medium, then cover with a cover glass. After the tissue section is stable, observe the results and take pictures using a fluorescence microscope configured with a 488nm laser. Among them, EBER nucleic acid is coupled with colloidal quantum well CQW-525, and the emitted light shows green; CLDN4 antibody is coupled with colloidal quantum well CQW-585, and the emitted light shows orange; N-cadherin antibody is coupled with colloidal quantum well CQW-625, and the emitted light shows red.

[0106] Statistical findings, EBER positive tissue sections of EB virus infection, the average fluorescence intensity is greater than 503.267, and the spatial position is more intensive; while the average fluorescence intensity of CLDN4 is less than 153.011 and the average fluorescence intensity of N-cadherin is greater than 501.629, the degree of epithelial mesenchymal transition in EB virus infected lymphoid tissue is high. The average fluorescence intensity of EBER in EB virus infection negative tissue sections is less than 503.267, and the spatial position is more dispersed.

[0107] In order to further compare the epithelial mesenchymal transition in EBER positive (EB virus infection) and EBER negative (EB virus infection) tissue sections, the expression of CLDN4 and N-cadherin in EBER negative tissue can be further evaluated; the average fluorescence intensity of CLDN4 is greater than 500.026 and the average fluorescence intensity of N-cadherin is less than 148.277, which indicates that when EB virus is not infected, the average fluorescence intensity of epithelial index CLDN4 is greater than that of mesenchymal index N-cadherin, indicating that the degree of epithelial mesenchymal transition in lymphoid tissue is low or no epithelial mesenchymal transition occurs.

[0108] As shown in Figure 1 , it is a detection result of a parotid gland tissue detection sample, in which the average fluorescence intensity of EBER is 525.109, and the parotid gland tissue infected with EB virus is determined; the average fluorescence intensity of CLDN4 is 131.371, and the average fluorescence intensity of N-cadherin is 560.277; it can be seen that the degree of epithelial mesenchymal transition in the EB virus infected parotid gland tissue is high.

[0109] Example 6: Application of EB virus infected lymphoid tissue epithelial mesenchymal transition detection kit

[0110] The parotid gland specimens of patients with Sjogren's syndrome were taken as the detection object, and the parotid gland specimens known to be EBV positive and EBV negative were taken respectively, and the kit was used for detection.

[0111] Soak in 4% formalin for more than 48 hours; make paraffin blocks of parotid gland specimens and place them on a microtome to make 3-4 μm tissue sections. Record the information of the section source, staining index, date and antibody ratio, etc. before starting staining.

[0112] Tissue sections were baked in a 60°C oven for 30 minutes. Then, they were dewaxed and dehydrated, and transferred to 100% xylene solution (No. 1) for 15 minutes, 100% xylene solution (No. 2) for 15 minutes, 100% ethanol solution (No. 3) for 3 minutes, 100% ethanol solution (No. 4) for 3 minutes, 95% ethanol solution (No. 5) for 3 minutes, 75% ethanol solution (No. 6) for 3 minutes, and 50% ethanol solution (No. 7) for 3 minutes. The sections were then rinsed three times with PBS, each time for 5 minutes.

[0113] Antigen retrieval was performed on dewaxed and dehydrated tissue sections. For antigen retrieval, an acidic or alkaline antigen retrieval solution was selected according to the corresponding antibody's instructions. The antigen retrieval solution was added to a container, heated to boiling in a microwave oven, and then the tissue sections were placed inside. The sections were heated on medium-low heat for 10 minutes and then removed. After the tissue sections cooled naturally to room temperature, they were transferred to an immunohistochemistry washing box and rinsed with sterile water for 1 minute, followed by rinsing with 1×TBST for 2 minutes. The tissue was then enclosed using an immunohistochemical pen, ensuring the entire tissue block on the section was within the enclosing circle.

[0114] First round of antibody staining;

[0115] Blocking: Remove 1×TBST solution, slowly add 10% BSA, and incubate at room temperature for 10 min.

[0116] Antibody preparation: Dilute the antibody with 1×PBS to prepare CQW-585-CLDN4 colloidal quantum trap conjugated antibody solution.

[0117] Antibody incubation: Remove excess serum, add CQW-585-CLDN4 colloidal quantum trap conjugated antibody, place the tissue slices flat in the immunohistochemistry humidifier, and then place in a 4°C refrigerator overnight.

[0118] Rinsing: Rinse 3 times with a wash bottle containing 1×TBST for 2 minutes each time to remove unbound CQW-585-CLDN4 colloidal quantum trap conjugate antibody.

[0119] The second round of staining is performed after the first round of staining is completed;

[0120] Blocking: Remove 1×TBST solution, slowly add 10% BSA, and incubate at room temperature for 10 min.

[0121] Antibody preparation: Dilute the antibody with 1×PBS to prepare CQW-625-N-cadherin colloidal quantum trap conjugated antibody solution.

[0122] Antibody incubation: Remove excess serum, add CQW-625-N-cadherin colloidal quantum trap conjugated antibody, place the tissue sections flat in the immunohistochemistry humidifier, and then place in a 4°C refrigerator overnight.

[0123] Rinse: Wash the slides with 1x TBST for 3 times, 2 min each time, to remove unbound CQW-625-N-cadherin colloidal quantum well conjugated antibody.

[0124] After the staining of the tissue sections, the nuclei were dyed by using 1x DAPI solution prepared with ddH20 at 1:100. The 1x DAPI solution was added to the tissue sections, which were incubated at room temperature in the dark for 5 min, rinsed with 1x TBST for 2 min, and rinsed with ddH20 for 1 min. After the excess liquid on the tissue sections was removed, 30 μl of anti-quenching mounting medium was added, and then a cover glass was covered. After the tissue sections were stabilized, the results were observed and photographed by using a fluorescence microscope equipped with a 488 nm laser. The CLDN4 antibody was coupled with colloidal quantum well CQW-585, and the emitted light was displayed as orange. The N-cadherin antibody was coupled with colloidal quantum well CQW-625, and the emitted light was displayed as red.

[0125] Image format: Preferably use 32-bit grayscale image (to preserve original intensity information); if color image: Image > Type > 32-bit; Scale calibration: Analyze > Set Scale: nm / pixel according to experimental equipment (e.g. confocal microscope 20x objective ≈ 0.45 μm / pixel). The steps for fluorescence intensity analysis are as follows: (1) Background subtraction method: Process > Subtract Background, parameters: Rolling Ball Radius: 50-100 pixels (adjust according to image size), check Light background (fluorescence signal is light background); (2) Region of interest (ROI): automatic selection (suitable for multiple quantum wells): Process > Find Maxima (noise tolerance set to background + 3x SD); (3) Intensity measurement: Analyze > Measure (or shortcut Ctrl+M). Key output parameters: Mean Gray Value (average fluorescence intensity); Integrated Density (integrated fluorescence intensity, suitable for non-uniform light emission); Area (selected area for normalization).

[0126] The average fluorescence intensity of CLDN4 and N-cadherin was statistically analyzed by using ImageJ software, and the following formula was used:

[0127] Average fluorescence intensity Mean = total fluorescence intensity of the region InDen / area of the region Area.

[0128] For example, Figure 2Figure 3 As shown in the figure, the detection results of 1 EBV infection positive parotid gland tissue detection sample and 1 EBV infection negative parotid gland tissue detection sample are shown respectively. Figure 2 As shown in the figure, in the EBV infection positive parotid gland tissue detection sample, the average fluorescence intensity of CLDN4 is 150.701, and the average fluorescence intensity of N-cadherin is 501.806; it can be seen that the degree of epithelial mesenchymal transition in the EB virus infected parotid gland tissue is high. Figure 3 As shown in the figure, in the EBV infection negative parotid gland tissue detection sample, the average fluorescence intensity of CLDN4 is 536.411, and the average fluorescence intensity of N-cadherin is 129.073, which indicates that the degree of epithelial mesenchymal transition in the EB virus uninfected parotid gland tissue is low or no epithelial mesenchymal transition occurs.

[0129] From the above experimental results, in the EB virus infected lymphoid tissue, by comparing the contents of the same tissue section, the average fluorescence intensity can clearly determine the epithelial mesenchymal transition of the section.

[0130] The above embodiments of the present application are described in detail, but the content is only the preferred embodiment of the present application, and cannot be considered to limit the scope of the present application. Any equivalent changes and improvements made according to the scope of the present application should still belong to the scope of the present application.

Claims

1. A kit for detecting epithelial-mesenchymal transition in lymphoid tissue infected with Epstein-Barr virus (EBV), characterized in that: These include colloidal quantum well-probe EBER conjugates, colloidal quantum well-antibody CLDN4 conjugates, and colloidal quantum well-antibody N-cadherin conjugates; the colloidal quantum wells conjugated with probe EBER, antibody CLDN4, and antibody N-cadherin have different emission wavelengths.

2. The EB virus infection lymphoid tissue epithelial-mesenchymal transition detection kit according to claim 1, characterized in that: It also includes colloidal quantum trap-protein LMP2A conjugates; the colloidal quantum traps conjugated with probe EBER, protein LMP2A, antibody CLDN4 and antibody N-cadherin have different emission wavelengths.

3. The EB virus infection lymphoid tissue epithelial-mesenchymal transition detection kit according to claim 1 or 2, characterized in that: Colloidal quantum wells include one or more of CQW-525, CQW-565, CQW-585, and CQW-625.

4. The EB virus infection lymphoid tissue epithelial-mesenchymal transition detection kit according to claim 1 or 2, characterized in that: It also includes antigen retrieval solution and DNA fluorescent staining solution.

5. A method for preparing the EB virus-infected lymphoid tissue epithelial-mesenchymal transition detection kit according to any one of claims 1-4, characterized in that: The colloidal quantum trap is mixed with EDC and NHS in a molar ratio of 1:1–5:1.5–7.5 to activate the carboxyl group; nucleic acid probes or antibodies are added, and after coupling reaction, colloidal quantum trap-probe conjugates or colloidal quantum trap-antibody conjugates are formed. The molar ratio of CQW to nucleic acid probe is 1:1.5–3, or the molar ratio of CQW to antibody is 1:10–20.

6. The method of using the EB virus infection lymphoid tissue epithelial-mesenchymal transition detection kit according to any one of claims 1-4, characterized in that: Lymphoid tissue sections were prepared, and after antigen retrieval, different colloidal quantum trap probes / antibody conjugates were used to perform antigen-antibody binding reactions on the tissue sections. After staining, images were acquired under a fluorescence microscope, and the luminescence intensity and spatial location information were quantified by detecting the luminescence information.

7. The method of using the EB virus-infected lymphoid tissue epithelial-mesenchymal transition detection kit according to claim 6, characterized in that: The staining order was: EBER first, then CLDN4, and finally N-cadherin. Alternatively, the staining order can be as follows: colloidal quantum trap-probe EBER conjugate, colloidal quantum trap-protein LMP2A conjugate, colloidal quantum trap-antibody CLDN4 conjugate, and colloidal quantum trap-antibody N-cadherin conjugate.

8. The method of using the EB virus-infected lymphoid tissue epithelial-mesenchymal transition detection kit according to any one of claims 1-4 or the EB virus-infected lymphoid tissue epithelial-mesenchymal transition detection kit according to claim 6 or 7 in the detection of EB virus-infected lymphoid tissue epithelial-mesenchymal transition.

9. The application according to claim 8, characterized in that: If EBER is positive and the N-cadherin fluorescence intensity is higher than CLDN4, it is judged as a high degree of EMT and strong antigen presentation ability; If EBER is negative and the N-cadherin fluorescence intensity is lower than CLDN4, it is judged that the degree of EMT is low or has not occurred, and the antigen presentation ability is weak.

Images