Detection kit for epstein-barr virus and plasma cells in lymphoid tissue microenvironment, and preparation method and application thereof

Abstract

The present application relates to a kind of detection kits for EB virus and plasma cell in lymphoid tissue microenvironment and its preparation method and application, kit includes colloidal quantum well labeled probe EBER, colloidal quantum well labeled antibody CD138 and colloidal quantum well labeled antibody MUM-1;By colloidal quantum well and specific antibody conjugate to thymoma paraffin section sample dyeing, conjugate can be expressed with EBER and related antigen specific connection in section, so as to realize colloidal quantum well to lymphoid tissue, EB virus and the quantity and spatial position detection of plasma cell.Colloidal quantum well replaces fluorescent dye, can realize the direct marking of tissue section, realizes the local positioning of EB virus and plasma cell in lymphoid tissue microenvironment, both can realize the detection of quantity also can realize the detection of position, it is convenient to evaluate the situation of plasma cell in EB virus infection tissue microenvironment.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of detection, and particularly relates to a detection kit for Epstein-Barr virus and plasma cells in a lymphoid tissue microenvironment and a preparation method and application thereof. BACKGROUND

[0002] Lymphoid tissues are important components of the immune system that recognize and respond to antigens, protecting the body from infection. According to the complexity of the organizational structure and the difference in function, lymphoid tissues can be divided into primary lymphoid tissues, secondary lymphoid tissues and tertiary lymphoid tissues.

[0003] Primary lymphoid tissues are mainly the sites of immune cell generation and initial differentiation, such as bone marrow and thymus. Secondary lymphoid tissues are the sites where immune cells meet antigens, responsible for the initiation and regulation of immune responses, including lymph nodes, spleen, MALT, etc. Tertiary lymphoid tissues are temporary lymphoid structures formed in local immune responses, usually appearing in inflammatory, infectious or tumor microenvironments, and have the function of local immune response. Lymphoid tissues work synergistically through different levels of structure and function, playing a crucial role in the body's immune defense, ensuring that the body can effectively recognize and eliminate foreign antigens and pathogens.

[0004] Tertiary lymphoid tissues refer to temporary immune tissue structures that appear in certain tissues or organs in the absence of persistent immune surveillance during local inflammation or immune response. Tertiary lymphoid tissues are generally formed in response to pathogens or tumors. Function: Tertiary lymphoid tissues usually appear during local immune responses and can form temporary lymphoid tissue structures in inflammatory areas. These structures contain active immune cells such as T cells, B cells, and dendritic cells, which can initiate immune responses at sites of infection or tumor occurrence. These tissues do not exist permanently but only appear under pathological conditions. Tertiary lymphoid tissues usually appear in areas of local immune response activation such as inflammation, infection, and tumors, and exhibit structures similar to lymph nodes, such as lymphoid follicles or lymphatic vessels, within the tissues. These structures help initiate local immune responses and are often found in places such as the lungs, liver, kidneys, and tumor tissues, especially in autoimmune diseases, chronic inflammation, or tumor microenvironments. The formation of tertiary lymphoid tissues indicates the immune system's response to local lesions and may be a marker of chronic inflammation or malignancy. In some cases, the appearance of tertiary lymphoid tissues may help the body fight pathogens or tumor cells, but it may also promote inappropriate immune responses (such as autoimmune responses or tumor escape from immune surveillance).

[0005] The main components of the thymic microenvironment include thymic epithelial cells, cortex TEC, dendritic cells (DCs), macrophages, thymic stromal cells, etc. The blood vessels and lymphatic vessels in the thymus play an important role in transportation, helping to transport immune cells, antigens, and cytokines into the thymic microenvironment. In addition, vascular endothelial cells are also involved in the selective migration of immune cells in the thymus.

[0006] Epstein-Barr virus (EBV) is a virus belonging to the herpes virus family and is one of the most common viruses in humans. It can infect human B cells and cause a series of clinical diseases. EB virus is associated with many malignant tumors, immune diseases, infectious mononucleosis, etc. It is transmitted through saliva and can remain latent in the body for a long time and may reactivate when the immune system is suppressed.

[0007] The lymphoid tissue environment infected by EB virus is filled with a complex immune microenvironment, including the infiltration and interaction of various immune cells, however, currently, there is no solution and development of techniques for detecting the relationship between the virus and the plasma cells in the above complex environment, and exploring the relationship and mechanism between the action of the virus and the plasma cells. SUMMARY

[0008] To solve the above technical problems, the present application provides a detection kit for EB virus and plasma cells in the lymphoid tissue microenvironment and its preparation method and application.

[0009] The technical solution adopted by the present application is: a detection kit for EB virus and plasma cells in the lymphoid tissue microenvironment, which comprises a colloidal quantum well-probe EBER conjugate, a colloidal quantum well-antibody CD138 conjugate and a colloidal quantum well-antibody MUM-1 conjugate.

[0010] Preferably, the colloidal quantum wells coupled with the probe EBER, the antibody CD138 and the antibody MUM-1 have different light emission wavelengths.

[0011] Preferably, the colloidal quantum well includes multiple types of CQW-525, CQW-585 and CQW-625.

[0012] Preferably, it further comprises an antigen repair solution and a DNA fluorescent dye solution.

[0013] The method for preparing the detection kit for EB virus and plasma cells in the microenvironment of lymphoid tissue mixes the colloidal quantum well with EDC and NHS at 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 the coupling reaction, colloidal quantum well-probe conjugates or colloidal quantum well-antibody conjugates are formed.

[0014] The molar ratio of CQW to nucleic acid probes is 1:1.5-3, or the molar ratio of CQW to antibodies is 1:10-20.

[0015] Preferably, the colloidal quantum well is one or more of CdSe, CdSe / CdS, CdSe / CdZnS, or CdZnSe / ZnS.

[0016] The method for using the detection kit for EB virus and plasma cells in the microenvironment of lymphoid tissue processes tissue sections of lymphoid tissue and uses antigen retrieval solution for antigen retrieval; colloidal quantum well probes or antibodies coupled to different targets are added for staining treatment; then cell nucleus staining is performed; finally, multi-channel fluorescence image information of the tissue section is collected, and the luminescence information is detected to quantify the luminescence intensity and spatial position information.

[0017] Preferably, the colloidal quantum well-probe EBER conjugate is used to determine whether the tissue section is EBER positive or negative;

[0018] The luminescence wavelength formed by the combination of the colloidal quantum well-antibody CD138 conjugate and the colloidal quantum well-antibody MUM-1 conjugate is collected, and the plasma cell information is determined by the co-expression information of antibody CD138 and antibody MUM-1.

[0019] The detection kit for EB virus and plasma cells in the microenvironment of lymphoid tissue or the method for using the detection kit for EB virus and plasma cells in the microenvironment of lymphoid tissue is applied to the detection of EBV-infected lymphoid tissue.

[0020] Preferably, if the average fluorescence intensity of EBER is greater than or equal to 613.208 and the spatial position distribution is dense, it is determined to be EBER positive; if the average fluorescence intensity of EBER is less than 271.006 and the spatial position distribution is sparse, it is determined to be EBER negative.

[0021] In the EBER-positive lymphoid tissue, the average fluorescence intensity of the co-expression of colloidal quantum well-labeled antibody CD138 and colloidal quantum well-labeled antibody MUM-1 is greater than 503.307, indicating that the degree of plasma cell infiltration in the EB virus-infected lymphoid tissue is high.

[0022] The present application has the advantages and positive effects that: by replacing fluorescent dyes with colloidal quantum wells, direct labeling of tissue sections can be achieved, local positioning of EB virus and plasma cells in lymphoid tissue microenvironment can be achieved, synchronous detection of EB virus infection related signals, plasma cell infiltration and spatial distribution in tissues can be achieved, effective support for quantitative evaluation and spatial positioning analysis of immune microenvironment changes caused by EBV infection is provided, and a more refined, multi-dimensional technical support platform is provided for pathological research, diagnosis development and clinical pathological mechanism research. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Expression of EBER, CD138 and MUM-1 in lymphoid tissues with positive EBER;

[0024] Figure 2 Expression of EBER, CD138 and MUM-1 in lymphoid tissues with negative EBER. DETAILED DESCRIPTION

[0025] The embodiments of the present application will be described below with reference to the accompanying drawings.

[0026] The present application relates to a detection kit for EB virus and plasma cells in lymphoid tissue microenvironment, and a preparation method and application thereof, the kit comprising colloidal quantum well labeled probe EBER, colloidal quantum well labeled antibody CD138 and colloidal quantum well labeled antibody MUM-1. When used, by sequentially staining the same tissue sample, qualitative or quantitative detection of EB virus and plasma cell indicators is achieved, and the plasma cell infiltration in the lymphoid microenvironment infected by EB virus is reacted.

[0027] EBER is one of the RNA molecules encoded by EB virus, which belongs to non-translational RNA. EBER molecules play an important role in cells infected by EB virus, which can help the virus maintain a long-term latent state in host cells and have immune escape function.

[0028] Plasma cells are immune cells differentiated from B cells, and their main function is to produce antibodies to resist external pathogens. They are generated in the bone marrow and reach the peripheral tissues through the blood. When encountering antigens, plasma cells will secrete a large amount of antibodies. The characteristics of plasma cells are large cell volume, nuclear deviation, and rich endoplasmic reticulum for synthesizing and secreting antibodies.

[0029] CD138 is a cell surface marker, also known as Syndecan-1, which is usually expressed on plasma cells. It plays an important role in the interaction between cells and the extracellular matrix, participating in cell adhesion and signal transduction. In clinical practice, the detection of CD138 is often used to identify plasma cells, especially in the diagnosis of multiple myeloma and other plasma cell-related diseases. MUM-1 (Multiple Myeloma Oncogene 1) is a transcription factor involved in the regulation of immune response. MUM-1 expression is common in plasma cells, B cells and some types of lymphoma cells. In some immune diseases and tumors (such as Hodgkin's lymphoma, non-Hodgkin's lymphoma, etc.), overexpression of MUM-1 may be related to the progression of tumors. In clinical diagnosis, MUM-1 is often used as a marker to help identify some types of lymphoma.

[0030] EB virus is closely related to immunity; plasma cells protect the body from pathogens by producing antibodies, and CD138 and MUM-1, as markers of plasma cells, play an important role in the detection of plasma cells. By detecting EB virus nucleic acid and related protein expression, combined with plasma cell marker localization analysis, changes in plasma cell infiltration caused by EB virus infection can be revealed, and the role of functional changes in autoimmune function can be evaluated. On the other hand, by comparing the lymphoid tissue microenvironment in EB virus infection and non-infection states, the number and morphological changes of plasma cells in EB virus infection state and the spatial relationship between virus and plasma cells are observed to speculate whether EB virus promotes the occurrence or development of autoimmune diseases by affecting the functional changes of plasma cells.

[0031] By coupling colloidal quantum wells with probes EBER, antibodies CD138 and antibodies MUM-1, reagents for labeling EB virus and plasma cells in tissue sections are obtained. The conjugate can specifically connect with EB virus and plasma cell-related antigens expressed in the section, so as to realize the qualitative or quantitative detection of EB virus and plasma cell indicators by colloidal quantum wells.

[0032] Coupling of colloidal quantum wells with nucleic acids includes the following steps:

[0033] Step 1: activate colloidal quantum wells; prepare carboxyl-modified water-soluble colloidal quantum wells with different emission wavelengths, the concentration is 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 under room temperature and light-free conditions for 30 minutes to activate the carboxyl groups on the surface of the colloidal quantum wells. The intermediate generated facilitates subsequent covalent connection with probes;

[0034] Step 2: Preparation of CQW-probe conjugate; after the activation is completed, the reaction solution is quickly passed through ultrafiltration (10 kDa filter membrane) to remove free EDC / NHS, and is resuspended to the original volume with PBS (pH 7.4) buffer. Then, the NH2 modified probe is added, and the probe to quantum dot molar ratio can be set to 1.5-3:1. The mixed system is shaken at room temperature for 2 hours or shaken at 4°C overnight to complete the coupling reaction;

[0035] Step 3: After the reaction is completed, the colloidal quantum well-probe complex is obtained, and is purified again using an ultrafiltration tube to remove uncoupled probes, and is 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.

[0036] The colloidal quantum well-EBER probe complex can be prepared by the above method, and the colloidal quantum well-EBER probe complex obtained can be used for EBER in situ hybridization experiments, realizes specific visualization of EBV infected cells, and has high light stability and signal strength.

[0037] Coupling of colloidal quantum well and antibody includes the following steps:

[0038] Step 1: CQW, EDC, and NHS are mixed at a molar ratio of 1:1-5:1.5-7.5, diluted with PBS, mixed uniformly on a shaker at room temperature for 30 min, and then the pH is adjusted to 8-9 with PBS (PH 9.18) to obtain a colloidal quantum well activation solution;

[0039] Step 2: 100 ul of the colloidal quantum well activation solution is added to a reaction container, and an appropriate amount of antibody stock solution is diluted to 0.01 mg / ml with PBS buffer and mixed thoroughly, wherein the molar ratio of colloidal quantum well to antibody is 1:10-20, and the above reaction container is added, and the reaction is carried out at 4°C on a shaker in the dark for 4 h;

[0040] Step 3: After the reaction is completed, the reaction solution is centrifuged at 8000 rpm for 3 min to remove possible agglomeration precipitates, and the supernatant is retained; the supernatant contains colloidal quantum well-antibody conjugates.

[0041] The molar ratio of colloidal quantum well and specific antibody binding is the result of a large number of verifications in EB virus infected lymphoid tissues. Molar ratios that do not meet this interval will result in unstable colloidal quantum well and antibody binding, which is manifested as follows: if the molar ratio of colloidal quantum well and antibody binding is high, such as 1:5, a large number of false positive results will occur when the average fluorescence intensity of co-expression is detected by ImageJ; if the molar ratio of colloidal quantum well and antibody binding is low, such as 1:30, the fluorescence of colloidal quantum well and antibody binding to antigen cannot be detected when the average fluorescence intensity of co-expression is detected by ImageJ. When the molar ratio of colloidal quantum well and antibody binding is 1:10-20, the expression and spatial position of the antigen can be accurately and clearly displayed.

[0042] In order to be able to realize the labeling of different target objects in the same slice respectively, colloidal quantum wells with different light-emitting wavelengths (colors) are used to couple with probes EBER or antibodies CD138h and MUM-1 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 experimental requirements. Among them, the CQW is a CdSe, CdSe / CdS, CdSe / CdZnS or CdZnSe / ZnS structure, and the emission wavelength is any set wavelength band within 450-650 nm. In some embodiments of the present application, one or more of CQW-525, CQW-585 and CQW-625 are selected as colloidal quantum wells, which are coupled with EBER, CD138h and MUM-1 respectively to obtain CQWs-525-EBER, CQWs-585-CD138 and CQWs-625-MUM-1. The EBER nucleic acid is coupled with the colloidal quantum well CQWs-525, and the emission light is displayed as green; the antibody CD138 is coupled with the colloidal quantum well CQWs-585, and the emission light is displayed as yellow; the antibody MUM-1 is coupled with the colloidal quantum well CQWs-625, and the emission 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.

[0043] Colloidal quantum wells (CQWs) are used as fluorescent probes for multi-immunolabeling to achieve high-throughput and multi-parameter precise detection of specific targets (e.g., 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 than traditional fluorescent probes (such as quantum dots and organic fluorescent dyes). In some embodiments of the present application, the CQWs are of CdSe, CdSe / CdS, CdSe / CdZnS, or CdZnSe / ZnS structure, and the emission wavelength is any set wavelength within 450-650 nm.

[0044] CQWs have several key advantages in terms of light emission performance. First, their emission peak has an extremely narrow full width at half maximum (FWHM), usually less than 35 nm, and can even be less than 10 nm under optimized structure, which is much better than the emission width of traditional QDs, which is usually in the range of 20-40 nm. This spectral concentration significantly reduces the spectral crosstalk between multiple channels, making it particularly suitable for multi-fluorescent 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). Their two-dimensional structure results in a larger optical cross-section, meaning that under the same concentration and excitation conditions, stronger fluorescence signal output can be obtained. This has significant advantages for the detection of low-expression targets, 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 particle, enabling the coupling of a larger number of antibody molecules. Compared to 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 to enhance the discrimination of background signals in time-resolved fluorescence imaging (such as FLIM); anti-flashing performance ensures signal stability during long-time or dynamic imaging, avoiding the "instant light-out" phenomenon of quantum dots in some applications, and improving experimental repeatability and data consistency.

[0045] 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.

[0046] The use of colloidal quantum wells to construct multi-label fluorescent antibody probes is 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 improves the performance of immunolabeling imaging in complex tissue environments.

[0047] The configuration of the detection kit for EB virus and plasma cells in the lymphoid tissue microenvironment includes colloidal quantum wells and probe EBER conjugates, colloidal quantum well labeled antibody CD138, and colloidal quantum well labeled antibody MUM-1, in addition to antigen retrieval solution and nucleic acid staining solution. EBER is a key indicator of EB virus, and the expression of nucleic acid EBER labeled with colloidal quantum wells detects the expression amount and spatial position of EB virus in lymphoid tissue, and calculates the average fluorescence intensity of the corresponding cells to reflect the EB virus infection in the lymphoid tissue microenvironment; the co-expression detection of colloidal quantum well labeled antibody CD138 and colloidal quantum well labeled antibody MUM-1 reflects the expression amount and spatial position of plasma cells in the lymphoid tissue, and calculates the average fluorescence intensity of the corresponding cells to reflect the plasma cell infiltration in the lymphoid microenvironment.

[0048] In use, after sequentially staining the same tissue section with the colloidal quantum well conjugate with probe EBER, the colloidal quantum well-labeled antibody CD138, and the colloidal quantum well-labeled antibody MUM-1, the EB virus infection and the expression of plasma cell-related indicators can be observed by color comparison, thereby judging the plasma cell infiltration in EB virus-infected lymphoid tissue.

[0049] When applying the above-mentioned detection kit to detect EB virus and plasma cells in the lymphoid tissue microenvironment, the specific steps may include the following:

[0050] Step 1: Take paraffin-embedded specimens from thymoma patients and prepare tissue sections (the tissue is derived from surgical puncture or excision). Bake the tissue sections and dewax and dehydrate them.

[0051] Step 2: Use a microwave oven to perform antigen retrieval on tissue sections;

[0052] Step 3: Stain tissue sections using colloidal quantum traps and nucleic acid conjugates; stain tissue sections using colloidal quantum traps and antibody conjugates. When multiple colloidal quantum traps and antibody conjugates are used, staining is performed sequentially according to the order of specific proteins in the tissue from strong to weak. In some embodiments of the present invention, EBER is stained first, then CD138 is stained, and finally MUM-1 is stained.

[0053] Step 4: After the colloidal quantum trap staining is completed, the cell nuclei are stained, the slides are mounted and photographed, and the quantity and location information of viruses, epithelium and stroma are detected and analyzed. The degree of EMT is evaluated by the expression of EB virus and plasma cell markers in lymphoid tissue.

[0054] EBER is a key indicator of Epstein-Barr virus (EBV). The expression level and spatial location of EBV in lymphoid tissue were detected using colloidal quantum well-labeled nucleic acid EBER. The average fluorescence intensity of the corresponding cells was calculated to reflect the EBV infection status in the lymphoid tissue microenvironment. The expression level and spatial location of plasma cells in lymphoid tissue were detected using colloidal quantum well-labeled antibodies CD138 and MUM-1. The average fluorescence intensity of the corresponding cells was calculated to reflect the plasma cell infiltration status in the lymphoid microenvironment.

[0055] CD138 marks the surface characteristics of plasma cells, which can effectively distinguish plasma cells from other types of immune cells; MUM-1 reveals the intrinsic molecular markers of plasma cells and their functional state; by analyzing the co-expression information of CD138 and MUM-1 to measure the situation of plasma cells, by co-localizing CD138 and MUM-1, plasma cells and other types of immune cells can be accurately identified and distinguished. CD138 and MUM-1 are surface and internal markers of plasma cells, respectively, and have high specificity in fluorescence detection. Co-localization of the two makes the identification of plasma cells more accurate, reduces the misidentification of other cells, and improves the reliability of experimental results. In addition, the combination of CD138 and MUM-1 can not only confirm whether the cells are plasma cells, but also provide information about the activity, maturity and functional state of these cells. This provides more data support for the study of the biological function of plasma cells, immune regulation mechanisms and related diseases (such as myasthenia gravis).

[0056] The average fluorescence intensity of EBER, CD138 and MUM-1 was statistically analyzed by ImageJ software, and the following formula was used:

[0057] The average fluorescence intensity Mean = the total fluorescence intensity InDen of the region / the area Area of the region.

[0058] In lymphoid tissues, the average fluorescence intensity of EBER is greater than or equal to 613.208, and the spatial position distribution is dense, then it is determined that there is meaningful EB virus infection in the tissue. In EBER-positive lymphoid tissues, the average fluorescence intensity of the co-expression position of colloidal quantum well-labeled antibody CD138 and colloidal quantum well-labeled antibody MUM-1 is greater than 503.307, indicating that the degree of plasma cell infiltration in EB virus-infected lymphoid tissues is high.

[0059] Through fluorescence microscopy observation and ImageJ software statistics, in lymphoid tissues, the average fluorescence intensity of EBER is less than 271.006, then it is determined to be negative; EBER-negative means that the expression degree of EBER fluorescence nucleus in lymphoid tissue sections is low or no fluorescence expression, and the spatial position is relatively dispersed. In the verified experiment, the expression amount of CD138 and MUM-1 in EBER-negative lymphoid tissue sections is also low, and the co-expression position is less, indicating that the degree of plasma cell infiltration in EB virus-uninfected lymphoid tissues is low.

[0060] By locating and quantifying CD138 and MUM-1 in tissue sections, the location and activity of plasma cells can be obtained, which can provide information on the differentiation position of plasma cells and the differentiation degree of plasma cells. By analyzing the co-expression of CD138 and MUM-1 in the same section, plasma cells can be more accurately identified, and by comparing with the staining information of CD138 and MUM-1 alone, the differentiation trend of plasma cells can be judged. Using CD138 and MUM-1 as fluorescent markers for plasma cell detection can not only improve the specificity and sensitivity of detection, but also provide detailed information about the activity, function and differentiation state of plasma cells. The advantage of this co-localization technology is that it can help accurately identify plasma cells and provide reliable data support for immunology research and treatment monitoring.

[0061] The impact of EBV infection on tissue sections is multifaceted, including changes in cells, immune and tissue structure, and often has a complex microenvironment, making it difficult to accurately identify cell changes in EBV-infected tissue sections using traditional labeling methods. By replacing fluorescent dyes with colloidal quantum wells, the degree of plasma cell infiltration in EB virus-infected lymphoid tissue can be detected, and the method is simple and fast, making it easy to determine the relationship between EB virus and plasma cells. By replacing fluorescent dyes with colloidal quantum wells, direct labeling of tissue sections can be achieved, enabling the localization of EB virus and plasma cells in the lymphoid tissue microenvironment, both in terms of quantity and location, facilitating the evaluation of the impact of EB virus on plasma cells. By coupling colloidal quantum wells with CD138 and MUM-1 antibodies, the high light stability, easy-to-identify color, and high sensitivity of colloidal quantum wells make them more suitable for the complex effects of EB virus infection on tissue section environments. By co-expressing CD138 and MUM-1, plasma cells can be accurately identified, and by analyzing the differences in CD138 and MUM-1 expression locations, the differentiation process of plasma cells can be analyzed, and the relationship between EB virus infection and plasma cell differentiation can be dynamically analyzed.

[0062] The EB virus and plasma cell detection kit in the lymphoid tissue microenvironment can be used to evaluate the degree of plasma cell infiltration in EBV-infected lymphoid tissue, and thus predict the immune status. By observing the expression levels and spatial distribution of key indicators of EB virus EBER and plasma cell-related indicators, important reference directions can be provided for studying how viruses affect the function of plasma cells in complex environments. EB virus plays a role by infecting B cells and other immune cells, and plasma cells are key cells in immune responses. Studying their role in lymphoid tissue can help deepen the understanding of the pathogenic mechanism of EB virus, immune escape mechanism, and interaction with the immune system. By detecting changes in plasma cells in lymphoid tissue, the degree of viral infection and immune system response can be evaluated, which can help develop personalized treatment plans.

[0063] The method can realize the synchronous detection of EB virus infection related signals, plasma cell infiltration and spatial distribution in tissues, effectively support the quantitative evaluation and spatial positioning analysis of the immune microenvironment changes caused by EBV infection, and provide a more refined and multi-dimensional technical support platform for pathological research, diagnosis development and clinical pathological mechanism research.

[0064] The present application will be described below in conjunction with the drawings, wherein the experimental methods not specifically described in the operation steps are carried out according to the corresponding product instructions. The instruments, reagents and consumables used in the examples can be purchased from commercial companies unless otherwise specified.

[0065] Example 1: Preparation of CQWs-EBER conjugate

[0066] Prepare the colloidal quantum well CQWs-525 with 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 under room temperature and light-free conditions for 30 minutes to activate the carboxyl groups on the surface of the colloidal quantum well, generating an intermediate for subsequent covalent connection with the probe. After activation, remove the free EDC / NHS by ultrafiltration (10 kDa filter membrane) and resuspend to the original volume with PBS (pH 7.4) buffer. Then add the NH2 modified EBER probe, with a probe to quantum dot molar ratio of 1.5-3:1. Shake the mixed system at room temperature for 2 hours or at 4°C overnight to complete the coupling reaction and obtain CQWs-525-EBER.

[0067] After the reaction is completed, the un-coupled probe is 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 realize specific visualization of EBV infected cells, and has high light stability and signal strength.

[0068] Example 2: Preparation of CQWs-antibody conjugate

[0069] CQWs, EDC, NHS were mixed in a molar ratio of 1:1-5:1.5-7.5, diluted with PBS, mixed on a shaker at room temperature for 30 min, and then the pH was adjusted to 8-9 with PBS (pH 9.18) to obtain a colloidal quantum well activation solution. Take 100 ul of colloidal quantum well activation solution and add it to the reaction vessel. Take the appropriate amount of antibody stock solution, dilute it with PBS buffer to 0.01 mg / ml, mix thoroughly, and add it to the reaction vessel in different proportions. Incubate at 4°C for 4 hours in the dark.

[0070] After the reaction, centrifuge at 8000 rpm for 3 min to remove the possible agglomeration precipitate, and keep the supernatant, which contains the CQWs-antibody conjugate.

[0071] The best conditions were screened and analyzed by 1% agarose gel electrophoresis. The supernatant obtained under the best coupling conditions was purified using a PD10 column, and the purified product was stored at 4°C in the dark for future use.

[0072] CQWs-585 and CQWs-625 were prepared by the above method.

[0073] Example 3: Detection of EBER, CD138 and MUM-1 in lymphoid tissue

[0074] CQWs-525-EBER prepared in Example 1 and CQWs-585 and CQWs-625 prepared in Example 2 were used for the detection of lymphoid tissue.

[0075] Sixty lymphoid tissue samples (the samples were obtained by puncture or surgical resection) were collected and soaked in 4% formalin for more than 48 hours. The lymphoid tissue samples were made into wax blocks and placed on a microtome to make 3-4 μm tissue sections. The information about the source of the sections, the staining index, the date, and the antibody ratio was recorded before staining.

[0076] 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, each for 5 min.

[0077] Antigen retrieval was performed on dewaxed and dehydrated tissue sections. During antigen retrieval, either an acidic or basic antigen retrieval solution was chosen according to the corresponding antibody instructions; the antigen retrieval solution was added to a container, which was placed in a microwave oven and heated to boiling, then the tissue sections were added and heated for 10 min at low heat, then removed. After the tissue sections were naturally cooled to room temperature, they were transferred to an immunohistochemistry rinse box and rinsed with sterile water for 1 min and 1x TBST for 2 min. The tissue was enclosed using an immunohistochemistry pen, and the entire tissue block on the section was located inside the enclosure.

[0078] First round of nucleic acid staining:

[0079] Blocking: The 1x TBST solution was removed, and 10% BSA was slowly added and incubated at room temperature for 10 min.

[0080] Antibody preparation: CQWs-525-EBER colloidal quantum well-labeled EBER solution was prepared.

[0081] Incubation: Excess serum was removed, and CQWs-525-EBER was added, and the tissue sections were placed flat in an immunohistochemistry wet box, then placed in a 4°C refrigerator overnight.

[0082] Rinsing: The wash bottle containing 1x TBST was used to rinse 3 times, each time for 2 min, to wash away unbound CQWs-525-EBER.

[0083] After the first round of staining, the second round of staining was performed;

[0084] Blocking: The 1x TBST solution was removed, and 10% BSA was slowly added and incubated at room temperature for 10 min.

[0085] Antibody preparation: The antibody was diluted with 1x PBS, and CQWs-585-CD138 colloidal quantum well-coupled antibody solution was prepared.

[0086] Second antibody incubation: Excess serum was removed, and CQWs-585-CD138 was added, and the tissue sections were placed flat in an immunohistochemistry wet box, then placed in a 4°C refrigerator overnight.

[0087] Rinsing: The wash bottle containing 1x TBST was used to rinse 3 times, each time for 2 min, to wash away unbound CQWs-585-CD138.

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

[0089] Blocking: The 1x TBST solution was removed, and 10% BSA was slowly added and incubated at room temperature for 10 min.

[0090] Antibody preparation: The antibody was diluted with 1x PBS, and CQWs-625-MUM-1 colloidal quantum well-coupled antibody solution was prepared.

[0091] Third antibody incubation: Excess serum was removed, CQWs-625-MUM-1 was added, the tissue section was placed in the immunohistochemical wet box, and then placed in a 4°C refrigerator overnight.

[0092] Rinse: The washing bottle filled with 1xTBST was used to rinse 3 times, 2 min each time, to remove unbound CQWs-625-MUM-1.

[0093] The stained tissue section was dyed with a 1:100 ddH20 solution of 1xDAPI solution; 1xDAPI solution was added to the section tissue, incubated at room temperature for 5 min in the dark, rinsed with 1xTBST for 2 min, and rinsed with ddH2O for 1 min. After removing the excess liquid on the section, 30 μl of anti-quenching mounting medium was added, then a cover glass was covered, and after the tissue section was stable, the results were observed and photographed using a fluorescence microscope with a 488 nm laser. Among them, EBER nucleic acid is coupled with colloidal quantum well CQWs-525, and the emitted light shows green; CD138 antibody is coupled with colloidal quantum well CQWs-585, and the emitted light shows yellow; MUM-1 antibody is coupled with colloidal quantum well CQWs-625, and the emitted light shows red; CD138 and MUM-1 co-expression position emits light shows pink.

[0094] The average fluorescence intensity of EBER, CD138, and MUM-1 was statistically analyzed by ImageJ software, using the following formula:

[0095] Mean fluorescence intensity Mean = total fluorescence intensity of the region InDen / area of the region Area;

[0096] Through detection, it is found that in 60 thymoma detection samples, the average fluorescence intensity of EBER is greater than 613.208, and the spatial position distribution is dense, then it is determined that there is meaningful EB virus infection in the tissue. Further analysis of the distribution of plasma cells and the activation of plasma cells in the EB virus infected sample can be carried out by observing the position information and fluorescence intensity information of CD138, MUM-1 and CD138-MUM-1 co-expression; if the average fluorescence intensity of the colloidal quantum well labeled antibody CD138 and the colloidal quantum well labeled antibody MUM-1 co-expression (pink fluorescence) in the EBER positive lymphoid tissue is greater than 503.307, it indicates that the degree of plasma cell infiltration in the EB virus infected lymphoid tissue is high.

[0097] Through detection of 60 thymoma detection samples, it is known that in lymphoid tissue, the average fluorescence intensity of EBER is less than 271.006, and it is determined to be negative; EBER negative means that the expression degree of EBER fluorescence nucleus in lymphoid tissue section is low or no fluorescence expression, and the spatial position is relatively dispersed. In order to further compare the growth of plasma cells in EBER negative and EBER positive tissue sections, the position information and fluorescence intensity information of CD138, MUM-1 and CD138-MUM-1 co-expression are analyzed; it is found that in EBER negative tissue section, the expression of CD138 and MUM-1 is also relatively low.

[0098] EBER negative section in thymoma detection sample is analyzed, and it is found that the average fluorescence intensity of co-expression position of colloidal quantum well labeled antibody CD138 and colloidal quantum well labeled antibody MUM-1 is generally less than 135.228.

[0099] As shown in Figure 1 , the average fluorescence intensity of EBER is 615.119, which indicates that the tissue section is EBER positive, and the average fluorescence intensity of co-expression of CD138 and MUM-1 representing plasma cells is 505.612, Figure 1 , which is a sample infected with EB virus and high degree of plasma cell infiltration. As shown in Figure 2 , the average fluorescence intensity of EBER is 265.106, which indicates that the tissue section is EBER negative, and the average fluorescence intensity of co-expression position of CD138 and MUM-1 representing plasma cells is 134.003.

[0100] From the experimental results, in the lymphoid tissue infected with EB virus, by detecting and comparing the expression amount and expression position of EBER, CD138 and MUM-1 in the same tissue section, the average fluorescence intensity can clearly determine the plasma cell infiltration of the section, and further explore the relationship between EB virus infection and plasma cell differentiation.

[0101] 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 detection kit for EB virus and plasma cells in the lymphoid tissue microenvironment, characterized in that: These include colloidal quantum well-probe EBER conjugates, colloidal quantum well-antibody CD138 conjugates, and colloidal quantum well-antibody MUM-1 conjugates; the colloidal quantum wells conjugated with probe EBER, antibody CD138, and antibody MUM-1 have different emission wavelengths.

2. The detection kit for EB virus and plasma cells in the lymphoid tissue microenvironment according to claim 1, characterized in that: Colloidal quantum wells include several types of CQW-525, CQW-585, and CQW-625.

3. The detection kit for EB virus and plasma cells in the lymphoid tissue microenvironment according to claim 1 or 2, characterized in that: It also includes antigen retrieval solution and DNA fluorescent staining solution.

4. The method for preparing the detection kit for EB virus and plasma cells in the lymphoid tissue microenvironment according to any one of claims 1-3, 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.

5. The method for preparing the detection kit for EB virus and plasma cells in the lymphoid tissue microenvironment according to claim 4, characterized in that: The colloidal quantum well is one or more of CdSe, CdSe / CdS, CdSe / CdZnS, or CdZnSe / ZnS.

6. The method of using the detection kit for EB virus and plasma cells in the lymphoid tissue microenvironment according to any one of claims 1-3, characterized in that: The lymphoid tissue was sectioned and antigen retrieval was performed using antigen retrieval solution; then, colloidal quantum trap probes or antibodies conjugated to different targets were added for staining. Cell nucleus staining was then performed; finally, multi-channel fluorescence images of tissue sections were acquired, and the luminescence intensity and spatial location information were quantified by detecting the luminescence information.

7. The method of using the detection kit for EB virus and plasma cells in the lymphoid tissue microenvironment according to claim 6, characterized in that: The emission wavelength of the color formed after the colloidal quantum trap-antibody CD138 conjugate and the colloidal quantum trap-antibody MUM-1 conjugate are collected, and plasma cell information is judged by the co-expression information of antibody CD138 and antibody MUM-1.

8. The method of using the detection kit for EBV and plasma cells in the lymphoid tissue microenvironment as described in any one of claims 1-3 or the detection kit for EBV and plasma cells in the lymphoid tissue microenvironment as described in claim 6 in detecting EBV-infected lymphoid tissue.

9. The application according to claim 8, characterized in that: If the average fluorescence intensity of EBER is greater than or equal to 613.208 and the spatial distribution is dense, it is judged as EBER positive; if the average fluorescence intensity of EBER is less than 271.006 and the spatial distribution is dispersed, it is judged as EBER negative. In EBER-positive lymphoid tissue, the average fluorescence intensity of the co-expressed colloidal quantum well-labeled antibody CD138 and colloidal quantum well-labeled antibody MUM-1 was greater than 503.307, indicating a high degree of plasma cell infiltration in EBV-infected lymphoid tissue.

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