Kit for simultaneously detecting angiogenesis and plasma cell infiltration in lymphatic tissue as well as preparation method and application of kit

Abstract

The invention relates to a kit for simultaneously detecting angiogenesis and plasma cell infiltration in lymphatic tissue and a preparation method and application of the kit, the kit comprises quantum well-antibody conjugates CQWs-525-VEGF, CQWs-525-ACTA2 and CQWs-585-CD138 and CQWs-625-Mum1, and the kit can be used for staining a lymphatic tissue section; by marking expression conditions of VEGF, ACTA2, CD138 and Mum1, the angiogenesis and plasma cell infiltration degree in the lymphatic tissue is evaluated. The quantum sheet has the characteristics of wide absorption spectrum, narrow light-emitting spectrum, high brightness and difficulty in quenching, and is used for marking blood vessels and plasma cells in tissues after being coupled with an antibody, so that quantitative analysis can be realized and spatial position distribution can be observed at the same time, the immune microenvironment of the tissues can be more accurately and effectively detected, and the immune state and immune function of the organism can be further evaluated.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and in particular relates to a kit for simultaneously detecting angiogenesis and plasma cell infiltration in lymphatic tissue, and a preparation method and application thereof. Background Art

[0002] The immune microenvironment within lymphoid tissues is closely linked to immune function. Thymic NH tissue is a crucial lymphoid organ in the human body. Angiogenesis and the accumulation and distribution of plasma cells within the thymus reflect the immune microenvironment. The amount of angiogenesis, the number of plasma cells, and the spatial distribution of these cells can, to a certain extent, assess the normality of immune function and are therefore of great value in the study and assessment of human immune function. For example, epithelial-mesenchymal transition (EMT) is a process in which epithelial cells transform into mesenchymal cells through a series of molecular and morphological changes. EMT typically promotes angiogenesis. Following EMT, cells secrete a large number of pro-angiogenic factors, which stimulate endothelial cells and drive angiogenesis. Alternatively, cells undergoing EMT may acquire endothelial characteristics, such as CD31 expression, forming a vessel-like structure known as vascular mimicry.

[0003] Specific antigens in lymphatic tissue can be labeled through biological staining technology. However, traditional staining technology has defects. The emission spectrum wavelength of traditional dyes is limited; the photostability is poor and they are easily attenuated and quenched; spectral overlap is easy to occur between different fluorescent dyes, resulting in problems of light leakage and color mixing; and the biocompatibility is poor, making it difficult to perform specific labeling; the fluorescence lifetime is short, making it difficult to preserve for a long time, and the background interference is strong. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a kit for simultaneously detecting angiogenesis and plasma cell infiltration in lymphoid tissue, as well as a preparation method and application thereof.

[0005] The technical solution adopted by the present invention is: a kit for simultaneously detecting angiogenesis and plasma cell infiltration in lymphatic tissue, comprising a quantum well-antibody conjugate, wherein the antibodies include CD138 and Mum1, and also include VEGF and / or ACTA2.

[0006] Preferably, the quantum well nanosheet coupled with VEGF and / or ACTA2 emits light at a wavelength different from that of the quantum well nanosheet coupled with CD138 and Mum1.

[0007] Preferably, the quantum well-antibody conjugates include CQWs-525-VEGF, CQWs-525-ACTA2, CQWs-585-CD138, and CQWs-625-Mum1.

[0008] Preferably, it also includes antigen repair solution and DNA fluorescent staining solution.

[0009] A method for preparing a kit for simultaneously detecting angiogenesis and plasma cell infiltration in lymphatic tissue comprises coupling quantum well nanosheets with antibodies through an EDC / NHS or SMCC method to prepare a quantum well-antibody conjugate.

[0010] Preferably, the quantum well nanosheets are one or more of CdSe, CdSe / CdS, CdZnSe / ZnS and CdSe / CdZnS.

[0011] Preferably, CQW, EDC and NHS are mixed in a molar ratio of 1:1–5:1.5–7.5, incubated to obtain a colloidal quantum well activation solution, and then the antibody is added in a molar ratio of CQW to antibody of 1:10–20. After thorough mixing, the reaction is carried out in the dark to prepare a quantum well and antibody conjugate CQWs-Ab.

[0012] The method for using the kit for simultaneously detecting angiogenesis and plasma cell infiltration in lymphatic tissue comprises preparing lymphatic tissue sections, performing antigen-antibody binding reactions on the tissue sections using quantum well-antibody conjugates with different luminescence wavelengths after antigen repair, collecting images under a fluorescence microscope after staining, and quantifying the luminescence intensity and spatial position information by detecting the luminescence information.

[0013] Preferably, angiogenesis is assessed by detecting VEGF and / or ACTA2; when the mean fluorescence intensity of VEGF is greater than 72.75, it is considered VEGF-positive; when the mean fluorescence intensity of ACTA2 is greater than 70.33, it is considered ACTA2-positive; when both VEGF and / or ACTA2 are positive, it is considered vascular proliferation; the plasma cell situation is assessed by detecting CD138 and Mum1; when the mean fluorescence intensity of CD138 is greater than 45.63, it is considered CD138-positive; when the mean fluorescence intensity of Mum1 is greater than 47.51, it is considered Mum1-positive; when both CD138 and Mum1 are positive, it is considered plasma cell infiltration;

[0014] Observe whether plasma cells are concentrated and whether plasma cells overlap with new blood vessels.

[0015] Application of a kit for simultaneously detecting angiogenesis and plasma cell infiltration in lymphoid tissue or a method for using the kit for simultaneously detecting angiogenesis and plasma cell infiltration in lymphoid tissue in evaluating lymphoid tissue.

[0016] The application has the advantages and positive effects that the quantum well is coupled with the antibody, compared with the conventional immunofluorescence staining, the quantum well nanosheet has a wider absorption spectrum, more accurate light emission, higher brightness, better efficiency, and the characteristic of no quenching compared with the organic fluorescent material, and compared with the ordinary quantum dot, the characteristic of multiple light emission channels, which can better meet the needs of multiple receptor labeling in biology to more accurately determine the lymphocyte subpopulation.

[0017] Based on the combination of the multi-color quantum sheet coupled antibody, VEGF, ACTA2, CD138 and Mum1 multiple markers can be used for simultaneous quantitative and positioning detection of neovascularization and plasma cells, the blood vessel formation and plasma cell infiltration in the lymphoid tissue are comprehensively evaluated, and the immune state and immune function of the body are accurately evaluated. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 A dapi graph in a tumor slice of a thymoma and a thymoma patient with autoimmune disease;

[0019] Figure 2 A CQWs-585-CD138 staining graph in a tumor slice of a thymoma and a thymoma patient with autoimmune disease;

[0020] Figure 3 A CQWs-625-Mum1 staining graph in a tumor slice of a thymoma and a thymoma patient with autoimmune disease;

[0021] Figure 4 A CQWs-525-VEGF staining graph in a tumor slice of a thymoma and a thymoma patient with autoimmune disease;

[0022] Figure 5 A synthesis graph of four kinds of staining in a tumor slice of a thymoma and a thymoma patient with autoimmune disease.

[0023] Figure 6 A dapi graph in a tumor slice of a thymoma and a thymoma patient with autoimmune disease;

[0024] Figure 7 A CQWs-585-CD138 staining graph in a tumor slice of a thymoma and a thymoma patient with autoimmune disease;

[0025] Figure 8 A CQWs-625-Mum1 staining graph in a tumor slice of a thymoma and a thymoma patient with autoimmune disease;

[0026] Figure 9 A CQWs-525-ACTA2 staining graph in a tumor slice of a thymoma and a thymoma patient with autoimmune disease;

[0027] Figure 10 Synthetic image of four stains in tumor sections of thymoma and thymoma patients with autoimmune diseases. DETAILED DESCRIPTION

[0028] Embodiments of the application will be described below with reference to the accompanying drawings.

[0029] The present application relates to a kit for simultaneously detecting angiogenesis and plasma cell infiltration in lymphoid tissues, and a preparation method and application thereof, taking VEGF, ACTA2, CD138 and Mum1 as detection objects to evaluate the degree of angiogenesis and plasma cell infiltration in lymphoid tissues.

[0030] VEGF and ACTA2 are two key proteins in neovascularization. Among them, VEGF is a core factor for regulating angiogenesis, which activates the downstream signaling pathway by combining with the VEGF receptor on the surface of vascular endothelial cells, and induces microvascular generation. ACTA2, also known as alpha smooth muscle actin, is a key protein for smooth muscle cell contraction and vascular stability, and its abnormal function is usually closely related to the physiological and pathological conditions of blood vessels. In particular, in some pathological states, overexpression of ACTA2 can promote the differentiation of myofibroblasts and collagen deposition, exacerbate perivascular fibrosis, indirectly affect the function of vascular endothelium, and possibly enhance VEGF-driven abnormal angiogenesis.

[0031] In the immune microenvironment, a large amount of angiogenesis can be accompanied by the occurrence of plasma cell infiltration. CD138 (Syndecan-1) is a transmembrane proteoglycan widely expressed on the surface of plasma cells and is one of the most specific markers of plasma cells. It regulates the adhesion, migration and survival of plasma cells by binding to extracellular matrix (such as collagen, fibronectin). MUM1 is a transcription factor encoded by IRF4 gene, which is located in the nucleus and mainly regulates the process of B cell differentiation into plasma cells, and is involved in plasma cell maturation and immunoglobulin secretion. The double positive markers of CD138 located on the cell membrane and MUM1 located on the cell nucleus can improve the specificity of plasma cell markers and accurately locate the distribution of plasma cells.

[0032] By simultaneously detecting the specific markers of angiogenesis and plasma cells, the local lymphoid tissue immune microenvironment can be reflected, so as to evaluate the overall immune status and function of the body.

[0033] Semiconductor colloidal quantum dots (CQDs) have high brightness, narrow-band emission, excellent optical stability, and tunable wavelength, which can be applied in the field of biological labeling. Based on this, two-dimensional semiconductor colloidal quantum wells (CQWs) such as CdSe, CdSe / CdS, CdSe / CdZnS, and CdZnSe / ZnS have higher excitation cross-section, stronger multi-photon response, and better optical purity, and perform better in multiple fluorescence labeling and high spatial resolution imaging. In particular, the two-dimensional structure of semiconductor colloidal quantum wells gives them a larger surface area, which can significantly increase the antibody coupling sites, thus achieving efficient and stable antibody binding. This structural characteristic not only improves the labeling specificity, ensuring high-affinity binding of antibodies to target antigens, but also greatly improves the detection sensitivity. The unique optical properties of semiconductor colloidal quantum wells effectively reduce signal crosstalk in multi-color immunofluorescence and background interference in complex lymphoid tissue sections through narrow emission spectrum and optimized optical properties, enabling high-resolution spatial localization.

[0034] By coupling quantum well nanosheet material CQW with various antibodies to form CQWs-Ab conjugates, it can be used to label the corresponding antigens in lymphoid tissue sections, thereby realizing the quantification and localization of target antigens in lymphoid tissue sections. Quantum well nanosheet antibody luminescence technology is a new detection technology that combines nanomaterials with antibody targeting functions, with characteristics such as broad absorption spectrum, narrow luminescence wavelength, high brightness, and good efficiency. Since its principle is to achieve different color luminescence by changing the quantum size, it has the characteristic of not being quenched compared to organic fluorescent materials. In addition, compared to ordinary quantum dots, it has the characteristic of multiple luminescence channels, which can better meet the needs of multiple receptor labeling in biology to more accurately determine lymphocyte subgroups.

[0035] Two-dimensional colloidal quantum well nanosheets of CdSe, CdSe / CdZnS, CdSe / CdS, and CdZnSe / ZnS structures are prepared by hot injection method. During synthesis, the luminescence wavelength (e.g., 510-650 nm) is adjusted by controlling the core layer thickness and chemical composition, as well as the shell layer thickness and chemical composition, and the quantum yield is optimized to more than 60%. The quantum well nanosheets are activated to obtain water-soluble CQWs with carboxyl groups on the surface, which are then coupled with antibodies for use. Further, block polymer ligands such as -NHS, -PEG, and -COOH can be grafted onto the activated quantum well nanosheets to improve colloidal stability and reduce non-specific adsorption. The quantum wells are modified by functional surface ligands, including polyethylene glycol (PEG), small molecule ligands or polymers containing carboxyl groups / amino groups; the quantum well nanosheets can be coupled with antibodies through methods such as EDC / NHS or SMCC.

[0036] In some embodiments of the present application, oil phase is used to synthesize CdSe, CdSe / CdS, CdZnSe / ZnS, CdSe / CdZnS and other CQWs; ligand exchange strategy (such as using mercapto PEG or polyamine ligand) is used for water phase transfer and surface functionalization; EDC / NHS or SMCC coupling method is used to stably graft target antibody to the surface of quantum well; CQW-antibody probe is purified by ultrafiltration, chromatography, centrifugation and other methods to obtain stable water-soluble fluorescent markers. CQW, EDC and NHS are mixed and incubated to obtain nanosheet colloidal quantum well activation solution, and then appropriate nucleic acid probe or antibody is added, mixed thoroughly and reacted in the dark to prepare quantum well nanosheet and antibody conjugate CQWs-Ab. In the process, quantum well nanosheet material is mixed with EDC and NHS at a molar ratio of 1:(1-5):(1.5-7.5) for carboxyl activation; nucleic acid probe (CQW to probe molar ratio 1:1.5-3) or antibody (CQW to antibody molar ratio 1:10-20) is coupled to form CQW-probe or CQW-antibody conjugate.

[0037] In order to distinguish different antigens in the same tissue section picture, quantum well nanosheet and antibody conjugate with different light emitting wavelengths (colors) are used; or according to different identification strategies, antibodies are grouped, and different light emitting wavelengths (colors) of quantum well nanosheet are used for different groups of antibodies. In some embodiments of the present application, the quantum well nanosheet used is CQW-525, CQW-585 and CQW-625, CQW-525 is coupled with angiogenesis related antibodies VEGF, ACTA2, CQW-585 and CQW-625 are coupled with plasma cell related antibodies CD138, Mum1.

[0038] In some embodiments of the present application, reagents or kits based on different CQWs-Ab conjugates can be prepared, in addition to different quantum well-antibody conjugates, antigen repair solution and DNA fluorescent dye are also included. The antigen repair solution is Tris-EDTA, and the DNA fluorescent dye is DAPI staining agent.

[0039] When used, first, antigen repair is performed using antigen repair solution, then antigen antibody binding reaction is performed on the tissue section using quantum well-antibody conjugates with different light emitting, the tissue section is stained, and finally the image is obtained under a fluorescence microscope, the light emitting information is detected, the light emitting intensity and spatial position information are quantified, and the immune environment and immune state of the detected lymphoid tissue are known. When staining using quantum well-antibody conjugate, each quantum well-antibody conjugate staining process includes blocking, antibody preparation, incubation and washing steps, and then the next quantum well-antibody conjugate staining is performed after sufficient washing.

[0040] When staining with nanosheet-antibody conjugates, CD138 is expressed on the cell membrane surface and is heat repair sensitive, so it needs to be stained first. Mum1 is located in the nucleus, so it needs to be stained second. VEGF needs strong repair, while ACTA2 is resistant, so it can be placed at the end. Therefore, the staining order of the four antibodies is: CD138 first, Mum1 second, and VEGF and / or ACTA2 last.

[0041] By labeling different color quantum well nanosheets, the expression of VEGF, ACTA2, and CD138 and Mum1 representing plasma cells can be labeled in the same lymphoid tissue section. By measuring the fluorescence intensity and analyzing the spatial position ratio of the staining results of multiple antibodies, the immune status in the local tumor microenvironment can be obtained. When the expression intensity of VEGF and / or ACTA2 increases and is regionally distributed, it represents an increase in angiogenesis in the lymphoid tissue. When the expression intensity of CD138 and Mum1 increases and is scattered, it represents plasma cell infiltration in the lymphoid tissue. When angiogenesis and plasma cell infiltration occur simultaneously, it represents immune hyperfunction in the tissue immune microenvironment. When the fluorescence intensity of VEGF and / or ACTA2 increases, it is considered that there are more new blood vessels. When the fluorescence intensity of CD138 and Mum1 increases and is scattered, it is considered that there is plasma cell infiltration. When both occur simultaneously, it is considered that the immune function is hyperfunction.

[0042] Through a large number of experiments, it was found that the minimum average fluorescence intensity of CD138 expression was 45.63; the minimum average fluorescence intensity of Mum1 expression was 47.51; the minimum average fluorescence intensity of VEGF expression was 72.75; the minimum average fluorescence intensity of ACTA2 expression was 70.33; it can be determined that when the average fluorescence intensity of VEGF is greater than 72.75, it is considered to be VEGF positive, when the average fluorescence intensity of ACTA2 is greater than 70.33, it is considered to be ACTA2 positive, when VEGF and / or ACTA2 are both positive, it is considered to be angiogenesis; when the average fluorescence intensity of CD138 is greater than 45.63, it is considered to be CD138 positive, when the average fluorescence intensity of Mum1 is greater than 47.51, it is considered to be Mum1 positive, when CD138 and Mum1 are both positive, it is considered to be plasma cell infiltration.

[0043] In terms of spatial distribution, the plasma cell CD138 and Mum1 regionally distribute, accounting for more than 50% in the lymphoid tissue, and overlap with the distribution of new blood vessels VEGF or ACTA2, with a close spatial relationship, and are considered to be plasma cell infiltration, angiogenesis and plasma cell infiltration, and are considered to be immune hyperfunction.

[0044] By coupling antibodies with the combination of multicolor quantum plate, the markers such as VEGF, ACTA2, CD138, Mum1, etc. can be simultaneously quantified and positioned to detect neovascularization and plasma cells, and the immune state and immune function of the body can be accurately evaluated through the angiogenesis and plasma cell infiltration in lymphoid tissues. Compared with conventional immunofluorescence staining, the quantum plate technology is coupled with antibodies, and the nanosheet has a wider absorption spectrum, more accurate luminescence, higher brightness, better efficiency, and non-quenching characteristics compared with organic fluorescent materials. In addition, compared with ordinary quantum dots, the nanosheet has the characteristics of multiple luminescence channels, which can better meet the needs of biological multiple receptor labeling and more accurate lymphocyte subpopulation determination. The kit is simple to use, stable and effective in labeling process, and convenient to use. At the same time, the kit for detecting VEGF, ACTA2, CD138 and Mum1 in lymphoid tissues in vitro integrates the primary antibody for labeling immune cells and all reagents for quantum dot staining, which is convenient and fast, and can provide more accurate services for researchers.

[0045] The application will be described below in conjunction with the drawings, wherein the experimental methods of the operation steps are not specifically described, and are performed according to the corresponding product instructions. The instruments, reagents and consumables used in the examples can be purchased from commercial companies if not specifically stated.

[0046] Example 1: Preparation of CQWs-Ab conjugate

[0047] The quantum well nanosheet material used in this example is CQW-525, CQW-585 and CQW-625, which are respectively coupled with antibodies for detecting VEGF, ACTA2, CD138 and Mum1 in lymphoid tissues.

[0048] 1.1 Nanosheet preparation

[0049] Two-dimensional colloidal quantum well nanosheets of CdSe, CdSe / CdZnS, CdSe / CdS, CdZnSe / ZnS and the like structure are prepared by hot injection method. During the synthesis process, the luminescence wavelength (such as 510-650 nm) is adjusted by controlling the core layer thickness and chemical composition, as well as the shell layer thickness and chemical composition, and the quantum yield is optimized to more than 60%.

[0050] The quantum well nanosheet is activated. The oil phase CQWs is resuspended in chloroform or hexane, and a basic aqueous solution (pH 10-11) containing mercaptopropionic acid (MPA) or mercaptoacetic acid (TGA) is slowly added dropwise; shake or ultrasonic treatment at room temperature for 30-60 minutes, and observe the fluorescence transfer to the water phase; use ultrafiltration (10 kDa MWCO) or chromatography to remove free ligands, and obtain water-soluble CQWs with carboxyl on the surface for subsequent antibody coupling.

[0051] Further grafting of -NHS, -PEG, -COOH, etc. block polymer ligands to improve colloidal stability and reduce non-specific adsorption.

[0052] 1.2 Coupling of CQWs with antibodies

[0053] EDC / NHS-mediated amide bond coupling between carboxyl-modified CQWs and amino groups on antibody molecules. Mix CQW, EDC, and NHS at a molar ratio of 1:1-5:1.5-7.5, dilute with PBS, mix well on a shaker at room temperature for 30 min, then adjust the pH to 8-9 with PBS (pH 9.18) to obtain a nanosheet colloidal quantum well activation solution. Take 100 ul of the activation solution and add it to the reaction vessel. Add an appropriate amount of specific antibody stock solution, dilute with PBS buffer to 0.01 mg / ml, mix well, and add the nanosheet colloidal quantum well and antibody at a ratio of 1:10-20 according to different conditions. React at 4°C on a shaker in the dark for 4 h.

[0054] After the reaction is complete, centrifuge at 8000 rpm for 3 min to remove any agglomerated precipitates, and retain the supernatant. Use 1% agarose gel electrophoresis to detect and analyze the best conditions. Purify the supernatant obtained under the best coupling conditions using a PD10 column, and store the purified product at 4°C in the dark for future use.

[0055] Example 2: Configuration of the kit

[0056] According to the method of Example 1, CQWs-525-VEGF, CQWs-525-ACTA2, and CQWs-585-CD138, CQWs-625-Mum1, Tris-EDTA, and DNA fluorescent staining solution were constructed, respectively.

[0057] Example 3: Application of the kit for simultaneous detection of angiogenesis and plasma cell infiltration in lymphoid tissues

[0058] The kit of Example 2 was used to detect lymphoid tissue sections.

[0059] Collect 30 cases of simple thymoma, 30 cases of thymoma combined with autoimmune disease tumor tissue specimens, fixed in 4% formalin, fixed for 48h or more, remove the tissue into the embedding box, do well the number and label, then gradient ethanol dehydration, in turn 70% ethanol immersion 30min, 80% ethanol immersion 30min, 90% ethanol immersion 30min, 90% ethanol immersion 30min, 100% ethanol immersion 30min, 100% ethanol immersion 30min. The pure alcohol and xylene equal mixture liquid 15min, xylene 0.5h, if the tissue is not enough bright, should be in xylene immersion to slice transparent. The slice sample is put into the mixture of xylene and paraffin each half for 15min, and then put into paraffin I, paraffin II for 20-30min; melt the paraffin in an environment of 52-56℃, and immediately put the tissue after paraffinization into cold water to make it solidify into a wax block; fix the fixed and repaired wax block on the clamping table of the microtome, adjust the blade angle, and make the blade close to the wax block, select the appropriate position, and continuously slice the wax block with a thickness of 5um; take a clean glass slide, drop a drop of adhesive in the center, put the cut wax tape on the water surface with tweezers, and then paste it on the glass slide, pay attention to the placement position, then flatten the wax sheet in an environment of 40-45℃, paste the label and make the mark, and place it in a 37℃ oven for drying. Record the slice information, staining index, date and antibody ratio, etc.

[0060] Put the patient's thymoma tumor tissue section in a 60℃ oven for 0.5-2h. Then put the tissue section in 1# 100% xylene for 5min, 2# 100% xylene for 5min, 3# 100% ethanol for 5min, 4# 100% ethanol for 5min, 5# 95% ethanol for 5min, 6# 75% ethanol for 5min, 7# 50% ethanol for 5min, and finally wash with ddH2O for 3 times, each time for 2min. According to the antibody instruction, select the acidic or alkaline antigen repair solution; the commonly used antigen repair solution is 1x Tris-EDTA antigen repair solution and 1x citric acid antigen repair solution. When the antigen is repaired, add the antigen repair solution to the immunohistochemical antigen repair container, and put it in the microwave oven to boil; then put the tissue section into the antigen repair solution, adjust the microwave oven to low heat for 10min, then take out the antigen repair container and cool it to room temperature naturally.

[0061] Take out the tissue section and wash it with ddH2O for 1min and 1x TBST for 2min. Use the immunohistochemical pen to seal the tissue, so that the whole tissue block on the slice is inside the seal.

[0062] In addition, the above treated tissue sections are sequentially dyed with different quantum well-antibody conjugates.

[0063] First round of dyeing:

[0064] A. Blocking: Remove 1×TBST solution, slowly add 10% BSA, and incubate at room temperature for 10 minutes.

[0065] B. Antibody preparation: Dilute the antibody with 1× PBS and prepare CQWs-585-CD138.

[0066] C. Primary antibody incubation: Remove excess serum, add CQWs-585-CD138 quantum dot labeled antibody, and place the tissue sections flat in an immunohistochemical humidified box, then place in a 4°C refrigerator overnight.

[0067] D. Rinse: Rinse three times with a wash bottle containing 1×TBST, each time for 2 minutes.

[0068] Second round of dyeing:

[0069] A. Blocking: Remove 1×TBST solution, slowly add 10% BSA, and incubate at room temperature for 10 minutes.

[0070] B. Antibody preparation: Dilute the antibody with 1× PBS to prepare CQWs-625-Mum1.

[0071] C. Secondary antibody incubation: Remove excess serum, add CQWs-625-Mum1 quantum dot-labeled antibody, and place the tissue sections flat in an immunohistochemical humidified box, then place in a 4°C refrigerator overnight.

[0072] D. Rinse: Rinse three times with a wash bottle containing 1×TBST, each time for 2 minutes.

[0073] Third round of dyeing:

[0074] A. Blocking: Remove 1×TBST solution, slowly add 10% BSA, and incubate at room temperature for 10 minutes.

[0075] B. Antibody preparation: Dilute the antibody with 1× PBS and prepare CQWs-525-VEGF.

[0076] C. Primary antibody incubation: Remove excess serum, add CQWs-525-VEGF quantum dot labeled antibody, and place the tissue sections flat in an immunohistochemical humidified box, then place in a 4°C refrigerator overnight.

[0077] D. Rinse: Rinse three times with a wash bottle containing 1×TBST, each time for 2 minutes.

[0078] Nuclear DNA was then stained. A 1× DAPI solution was prepared at a 1:100 ratio using ddH₂O. The 1× DAPI solution was added to the tissue sections and incubated in the dark for 5 minutes at room temperature. The sections were then rinsed with 1× TBST for 2 minutes and then rinsed with ddH₂O for 1 minute. After adding 30 μl of antifade mounting medium, the sections were covered with a coverslip and sealed with a dedicated edge sealant. After the sections stabilized, fluorescence microscopy with a 488 nm laser was used to observe and photograph the sections. Images were analyzed using ImageJ, with the image format set to 8-bit. The background was set, the threshold was adjusted, and the threshold was manually selected to ensure that all stained areas were selected. Measurement parameters were set and the mean grayscale value was calculated. Data were acquired, analyzed, and the immune status assessed.

[0079] The test results of 30 thymoma tissue samples with concurrent autoimmune diseases were statistically analyzed, and the mean fluorescence intensity of the quantum dot-specific antibodies was calculated using ImageJ software. The immunofluorescence images were converted to 8-bit format using ImageJ. The "Threshold" field was adjusted, the default threshold was used, "Dark Background" was selected, and the default algorithm was used to calculate the mean fluorescence intensity (Mean). The ImageJ statistical formula is: Mean fluorescence intensity (Mean) = Total fluorescence intensity (InDen) / Area (Area). The mean fluorescence intensity for each group was calculated.

[0080] like Figure 1-Figure 5 Shown are the color development of tumor sections from a thymoma patient and a thymoma patient with autoimmune disease under different fluorescence channels. Figure 1 The medium blue light is the cell nucleus; Figure 2 The medium orange fluorescence shows CD138 expressed on the surface of plasma cells; Figure 3 Middle red fluorescence shows Mum1 expressed in the plasma cell nucleus; Figure 4 The green fluorescence shows VEGF expressed in new blood vessels; Figure 5 This is a composite image of four stainings, CQWs-585-CD138, CQWs-625-Mum1, CQWs-525-VEGF, and DAPI, in tumor sections from patients with thymoma and thymoma complicated by autoimmune diseases. The relationship between the expression intensity and spatial position of the CD138, Mum1, and VEGF antibodies can be clearly shown through the relationship between the fluorescence intensity and position.

[0081] The calculation shows that the average fluorescence intensity of CD138 expression is 51.61±3.417; the average fluorescence intensity of Mum1 expression is 51.38±2.653; the average fluorescence intensity of VEGF expression is 73.27±1.328, and the expressions of CD138, Mum1 and VEGF in the figure are all positive; CD138 and Mum1 are concentrated in the region, and the proportion in the lymphoid tissue is more than 50%, and the distribution overlaps with VEGF. The tissue section shows increased angiogenesis, plasma cell infiltration, and hyperimmunity.

[0082] Example 4: Application of the kit for simultaneously detecting angiogenesis and plasma cell infiltration in lymphoid tissue

[0083] Thirty tumor tissue specimens of thymoma combined with autoimmune diseases were collected, fixed in 4% formalin for more than 48 hours, taken out of the tissue and placed in an embedding box, numbered and labeled, then dehydrated with gradient ethanol, soaked in 70% ethanol for 30 minutes, 80% ethanol for 30 minutes, 90% ethanol for 30 minutes, 90% ethanol for 30 minutes, 100% ethanol for 30 minutes, and 100% ethanol for 30 minutes. Mix equal amounts of pure alcohol and xylene for 15 minutes, xylene for 0.5 hours, and if the tissue is not transparent enough, it should be soaked in xylene until the section is transparent. Put the section sample into a mixture of half xylene and half paraffin for 15 minutes, then into paraffin I and paraffin II for 20-30 minutes; melt the paraffin in a 52-56°C environment, and immediately pour the melted paraffin into cold water to solidify the tissue into a wax block; fix the fixed and repaired wax block on the clamping table of the microtome, adjust the blade angle, and place the blade close to the wax block, select the appropriate position, and continuously section the wax block with a thickness of 5um; take a clean glass slide, drop a drop of adhesive in the center, place the cut wax strip on the water surface with tweezers, and then place it on the glass slide, pay attention to the placement position, then flatten the wax sheet in a 40-45°C environment, label and mark, and place it in a 37°C oven for drying. Record the section information, staining index, date and antibody ratio, etc.

[0084] Place the patient's thymoma tumor tissue sections in a 60°C oven for 0.5-2 hours. Then, soak the sections in 100% xylene (#1) for 5 minutes, 100% xylene (#2) for 5 minutes, 100% ethanol (#3) for 5 minutes, 100% ethanol (#4) for 5 minutes, 95% ethanol (#5) for 5 minutes, 75% ethanol (#6) for 5 minutes, and 50% ethanol (#7) for 5 minutes. Finally, rinse with ddH2O three times for 2 minutes each. Choose an acidic or alkaline antigen retrieval solution according to the antibody instructions; commonly used antigen retrieval solutions include 1× Tris-EDTA and 1× Citric Acid. For antigen retrieval, add the antigen retrieval solution to an immunohistochemistry (IHC) antigen retrieval container and microwave on high until boiling. Then, place the tissue sections in the antigen retrieval solution and microwave on low for 10 minutes. Remove the container and allow to cool to room temperature.

[0085] Remove the tissue sections and rinse them with ddH2O for 1 minute and 1×TBST for 2 minutes. Use an immunohistochemistry pen to encircle the tissue so that the entire tissue block on the section is within the encircled area.

[0086] In addition, the above-treated tissue sections were sequentially stained with different quantum dot-antibody conjugates.

[0087] First round of dyeing:

[0088] A. Blocking: Remove 1×TBST solution, slowly add 10% BSA, and incubate at room temperature for 10 minutes.

[0089] B. Antibody preparation: Dilute the antibody with 1× PBS and prepare CQWs-585-CD138.

[0090] C. Primary antibody incubation: Remove excess serum, add CQWs-585-CD138 quantum dot labeled antibody, and place the tissue sections flat in an immunohistochemical humidified box, then place in a 4°C refrigerator overnight.

[0091] D. Rinse: Rinse three times with a wash bottle containing 1×TBST, each time for 2 minutes.

[0092] Second round of dyeing:

[0093] A. Blocking: Remove 1×TBST solution, slowly add 10% BSA, and incubate at room temperature for 10 minutes.

[0094] B. Antibody preparation: Dilute the antibody with 1× PBS to prepare CQWs-625-Mum1.

[0095] C. Secondary antibody incubation: Remove excess serum, add CQWs-625-Mum1 quantum dot-labeled antibody, and place the tissue sections flat in an immunohistochemical humidified box, then place in a 4°C refrigerator overnight.

[0096] D. Rinse: Rinse three times with a wash bottle containing 1×TBST, each time for 2 minutes.

[0097] Third round of dyeing:

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

[0099] B. Antibody preparation: Dilute the antibody with 1× PBS to prepare CQWs-525-ACTA2.

[0100] C. Primary antibody incubation: Remove excess serum, add CQWs-525-ACTA2 quantum dot-labeled antibody, and place the tissue sections flat in an immunohistochemical humidified chamber, then place in a 4°C refrigerator overnight.

[0101] D. Rinse: Rinse three times with a wash bottle containing 1×TBST, each time for 2 minutes.

[0102] Nuclear DNA staining: Prepare a 1× DAPI solution at a 1:100 ratio using ddH₂O. Add the 1× DAPI solution to the tissue sections, incubate at room temperature in the dark for 5 minutes, rinse with 1× TBST for 2 minutes, and then rinse with ddH₂O for 1 minute. Add 30 μl of antifade mounting medium, cover with a coverslip, and seal the perimeter with a dedicated edge sealant. Once the tissue sections are stable, observe and photograph using a fluorescence microscope equipped with a 488 nm laser. Analyze the images using ImageJ, setting the image format to 8-bit. Set the background, adjust the threshold, and manually select the threshold to ensure that all stained areas are selected. Set measurement parameters and calculate the mean grayscale value. Acquire data, analyze, and assess immune status.

[0103] The test results of 30 thymoma tissue samples with concurrent autoimmune diseases were statistically analyzed, and the mean fluorescence intensity of the quantum dot-specific antibodies was calculated using ImageJ software. The immunofluorescence images were converted to 8-bit format using ImageJ. The "Threshold" field was adjusted, the default threshold was used, "Dark Background" was selected, and the default algorithm was used to calculate the mean fluorescence intensity (Mean). The ImageJ statistical formula is: Mean fluorescence intensity (Mean) = Total fluorescence intensity (InDen) / Area (Area). The mean fluorescence intensity for each group was calculated.

[0104] like Figures 6-10Shown are the color development of tumor sections from a patient with thymoma and thymoma complicated with autoimmune disease under different fluorescence channels. Figure 6 The medium blue light is the cell nucleus; Figure 7 The medium orange fluorescence shows CD138 expressed on the surface of plasma cells; Figure 8 Middle red fluorescence shows Mum1 expressed in the plasma cell nucleus; Figure 9 The medium green fluorescence shows ACTA2 expressed in the basal layer of blood vessels; Figure 10 This is a composite image of four stainings, CQWs-585-CD138, CQWs-625-Mum1, CQWs-525-ACTA2, and DAPI, in tumor sections from patients with thymoma and thymoma complicated by autoimmune diseases. The relationship between the expression intensity and spatial position of the CD138, Mum1, and ACTA2 antibodies is clearly shown through the relationship between the fluorescence intensity and position.

[0105] Calculation shows that the average fluorescence intensity of CD138 expression is 49.24±0.866; the average fluorescence intensity of Mum1 expression is 53.98±3.398; and the average fluorescence intensity of ACTA2 expression is 75.85±5.990. The figure shows that CD138, Mum1 and ACTA2 are all positive; CD138 and Mum1 are concentrated in the lymphoid tissue, accounting for more than 50% and overlapping with the distribution of ACTA2. The tissue section shows plasma cell infiltration and hyperimmune function.

[0106] Example 5: Configuration and Application of a Kit for Simultaneously Detecting Angiogenesis and Plasma Cell Infiltration in Lymphoid Tissue

[0107] According to the method of Example 1, CQWs-525-VEGF, CQWs-525-ACTA2, CQWs-585-CD138, CQWs-625-Mum1, Tris-EDTA and DNA fluorescent dye were constructed and obtained respectively.

[0108] Antigen-retrieved thymoma tumor tissue sections were prepared according to the method of Example 3. The tissue was enclosed using an immunohistochemistry pen, so that the entire tissue block on the section was located within the enclosed circle. Staining for CQWs-585-CD138, CQWs-625-Mum1, CQWs-525-VEGF, and CQWs-525-ACTA2 was performed sequentially according to the method of Example 3. After nuclear staining, the images were observed under a fluorescence microscope for color development at different excitation wavelengths. Co-labeling with CQWs-585-CD138 and CQWs-625-Mum1 was used to determine plasma cell distribution, while labeling with CQWs-525-VEGF and CQWs-525-ACTA2 was used to determine angiogenesis. Immune function was further analyzed and evaluated.

[0109] The above detailed description of the embodiments of the present application is only preferred embodiments of the present application, and should not be considered as limiting 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 patent coverage of the present application.

Claims

1. A kit for simultaneously detecting angiogenesis and plasma cell infiltration in lymphoid tissue, characterized in that: It includes quantum well-antibody conjugates, wherein the antibodies include CD138 and Mum1, and also include VEGF and / or ACTA2.

2. The kit for simultaneous detection of angiogenesis and plasma cell infiltration in lymphoid tissue according to claim 1, characterized in that: The emission wavelength of the quantum well nanosheets coupled with VEGF and / or ACTA2 is different from that of the quantum well nanosheets coupled with CD138 and Mum1.

3. The kit for simultaneous detection of angiogenesis and plasma cell infiltration in lymphoid tissue according to claim 1, characterized in that: Quantum well-antibody conjugates include CQWs-525-VEGF, CQWs-525-ACTA2, CQWs-585-CD138, and CQWs-625-Mum1.

4. The kit for simultaneously detecting angiogenesis and plasma cell infiltration in lymphoid tissue according to any one of claims 1 to 3, characterized in that: Also included are antigen retrieval solution and DNA fluorescent stain.

5. A method for preparing a kit for simultaneously detecting angiogenesis and plasma cell infiltration in lymphoid tissue according to any one of claims 1 to 4, characterized in that: The quantum well nanosheets are coupled with antibodies through EDC / NHS or SMCC method to prepare quantum well-antibody conjugates.

6. The method for preparing the kit according to claim 5, wherein: The quantum well nanosheets are one or more of CdSe, CdSe / CdS, CdZnSe / ZnS and CdSe / CdZnS.

7. The method for preparing the kit according to claim 5, wherein: CQW, EDC, and NHS were mixed in a molar ratio of 1:1–5:1.5–7.5 and incubated to obtain a colloidal quantum well activation solution. Antibodies were then added with a CQW to antibody molar ratio of 1:10–20. After thorough mixing, the reaction was carried out in the dark to prepare a quantum well and antibody conjugate CQWs-Ab.

8. A method for using the kit for simultaneously detecting angiogenesis and plasma cell infiltration in lymphoid tissue according to any one of claims 1 to 4, characterized in that: Prepare lymphatic tissue sections, and after antigen retrieval, use quantum well-antibody conjugates with different luminescence wavelengths to perform antigen-antibody binding reactions on the tissue sections. After staining, collect images under a fluorescence microscope, and detect the luminescence information to quantify the luminescence intensity and spatial position information.

9. The method of use according to claim 8, characterized in that: Angiogenesis was assessed by detecting VEGF and / or ACTA2; VEGF-positive cells were identified when the mean fluorescence intensity of VEGF was greater than 72.75, and ACTA2-positive cells were identified when the mean fluorescence intensity of ACTA2 was greater than 70.

33. Vascular proliferation was considered when both VEGF and / or ACTA2 were positive. Plasma cell status was assessed by detecting CD138 and Mum1; CD138-positive cells were identified when the mean fluorescence intensity of CD138 was greater than 45.63, and Mum1-positive cells were identified when the mean fluorescence intensity of Mum1 was greater than 47.

51. Plasma cell infiltration was considered when both CD138 and Mum1 were positive. Observe whether plasma cells are concentrated and whether plasma cells overlap with new blood vessels.

10. Use of the kit for simultaneously detecting angiogenesis and plasma cell infiltration in lymphoid tissue according to any one of claims 1 to 4 or the method for using the kit for simultaneously detecting angiogenesis and plasma cell infiltration in lymphoid tissue according to claim 8 or 9 in evaluating lymphoid tissue.

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