A kit for simultaneously detecting angiogenesis and plasma cell infiltration in lymphoid tissues, and a preparation method and application thereof

Abstract

The present application relates to a kind of kit for simultaneously detecting angiogenesis and plasma cell infiltration in lymphoid tissue and its preparation method and application, kit includes quantum well-antibody conjugate CQWs-525-VEGF, CQWs-525-ACTA2 and CQWs-585-CD138, CQWs-625-Mum1, can be used to lymphoid tissue section staining;By marking the expression of VEGF, ACTA2, CD138 and Mum1, evaluate the degree of angiogenesis and plasma cell infiltration in lymphoid tissue.Quantum sheet has the characteristics of wide absorption spectrum, narrow light wave, high brightness, not easy to quench, after coupling with antibody, mark the blood vessel and plasma cell in tissue, can realize quantitative analysis simultaneously, and observe spatial position distribution, so as to more accurate and effective detection of tissue immune microenvironment, and then evaluate the immune state and immune function of organism.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological detection, and particularly relates to a kit for simultaneously detecting angiogenesis and plasma cell infiltration in lymphoid tissues as well as a preparation method and application thereof. BACKGROUND

[0002] The immune microenvironment in lymphoid tissues is closely related to the immune function of the body. The NH tissue of the thymus is an important lymphoid organ of the human body. Angiogenesis and the aggregation and distribution of plasma cells in the thymus tissue reflect the immune microenvironment of the thymus tissue. The number of angiogenesis in the thymus tissue, the number of plasma cells, and the spatial distribution of the blood vessels and the plasma cells can to some extent evaluate whether the immune function of the body is normal or not, and have important value for studying and evaluating the immune function of the human body. Epithelial-mesenchymal transition (EMT) is a process in which epithelial cells are transformed into mesenchymal cells through a series of molecular and morphological changes. The occurrence of EMT usually promotes angiogenesis. After EMT, cells secrete a large amount of pro-angiogenic factors to stimulate vascular endothelial cells and drive the generation of new blood vessels. Or the cells that have undergone EMT may acquire endothelial cell characteristics, such as expressing CD31, and form a vascular-like pipe structure, i.e., vasculogenic mimicry.

[0003] Specific antigens in lymphoid tissues can be labeled by biological staining technology. Traditional staining technology has defects. The traditional dye has a limited emission spectrum wavelength. The light stability is poor, and the attenuation quenching is easy to occur. There is a problem of light leakage and color mixing between different fluorescent dyes. The biological compatibility is poor, and it is not easy to perform specific labeling. The fluorescent lifetime is short, and it is difficult to be stored for a long time. The background interference is strong. SUMMARY

[0004] To solve the above technical problems, the application provides a kit for simultaneously detecting angiogenesis and plasma cell infiltration in lymphoid tissues as well as a preparation method and application thereof.

[0005] The technical scheme adopted by the application is as follows: a kit for simultaneously detecting angiogenesis and plasma cell infiltration in lymphoid tissues, comprising quantum well-antibody conjugates, the antibodies comprising CD138 and Mum1, and further comprising VEGF and / or ACTA2.

[0006] Preferably, the quantum well nanosheets coupled with VEGF and / or ACTA2 have different light emission wavelengths from the quantum well nanosheets coupled with CD138 and Mum1.

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

[0008] Preferably, the antigen repair solution and the DNA fluorescent dye solution are further included.

[0009] The method for preparing the kit for simultaneously detecting angiogenesis and plasma cell infiltration in lymphoid tissues comprises coupling quantum well nanosheets with antibodies by an EDC / NHS or SMCC method to prepare quantum well-antibody conjugates.

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

[0011] Preferably, the CQW, EDC and NHS are mixed at a molar ratio of 1:1-5:1.5-7.5, and the colloidal quantum well activation solution is obtained by incubation, and then the antibodies are added, and the molar ratio of CQW to antibodies is 1:10-20, and the quantum well-antibody conjugates CQWs-Ab are prepared by thoroughly mixing and reacting in the dark.

[0012] The method for using the kit for simultaneously detecting angiogenesis and plasma cell infiltration in lymphoid tissues comprises preparing a lymphoid tissue section, using quantum well-antibody conjugates with different light emission wavelengths in sequence for antigen-antibody binding reaction on the tissue section after antigen repair, collecting images under a fluorescence microscope after staining, and detecting light emission information and quantifying light emission intensity and spatial position information.

[0013] Preferably, the angiogenesis is evaluated by detecting VEGF and / or ACTA2; VEGF is determined to be positive when the average fluorescence intensity is greater than 72.75, ACTA2 is determined to be positive when the average fluorescence intensity is greater than 70.33, and angiogenesis is considered to occur when VEGF and / or ACTA2 are both positive; and the plasma cells are evaluated by detecting CD138 and Mum1; CD138 is determined to be positive when the average fluorescence intensity is greater than 45.63, Mum1 is determined to be positive when the average fluorescence intensity is greater than 47.51, and plasma cell infiltration is considered to occur when CD138 and Mum1 are both positive.

[0014] Whether the plasma cells are concentrated and distributed, and whether the plasma cells and the newly formed blood vessels are distributed in overlap is observed.

[0015] The kit for simultaneously detecting angiogenesis and plasma cell infiltration in lymphoid tissues or the method for using the kit for simultaneously detecting angiogenesis and plasma cell infiltration in lymphoid tissues is applied in evaluating lymphoid tissues.

[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 at 52-56℃, and immediately put the tissue after paraffin 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. Choose the appropriate position, and then 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, then flatten the wax sheet in an environment of 40-45℃, paste the label and make a 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 into a 60℃ oven for 0.5-2h. Then put the tissue section into 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, choose acidic or alkaline antigen repair solution; commonly used antigen repair solutions are 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 into 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 entire 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 Stain:

[0064] A. Block: Remove 1 x TBST and slowly add 10% BSA. Incubate at room temperature for 10 minutes.

[0065] B. Prepare Antibody: Dilute antibody with 1 x PBS. Prepare CQWs-585-CD138.

[0066] C. First Antibody Incubation: Remove excess serum and place the tissue section flat in the immunohistochemistry humidified chamber with the CQWs-585-CD138 quantum dot labeled antibody. Place in the 4°C refrigerator overnight.

[0067] D. Rinse: Rinse 3 times for 2 minutes each with wash bottle filled with 1 x TBST.

[0068] Second Stain:

[0069] A. Block: Remove 1 x TBST and slowly add 10% BSA. Incubate at room temperature for 10 minutes.

[0070] B. Prepare Antibody: Dilute antibody with 1 x PBS. Prepare CQWs-625-Mum1.

[0071] C. Second Antibody Incubation: Remove excess serum and place the tissue section flat in the immunohistochemistry humidified chamber with the CQWs-625-Mum1 quantum dot labeled antibody. Place in the 4°C refrigerator overnight.

[0072] D. Rinse: Rinse 3 times for 2 minutes each with wash bottle filled with 1 x TBST.

[0073] Third Stain:

[0074] A. Block: Remove 1 x TBST and slowly add 10% BSA. Incubate at room temperature for 10 minutes.

[0075] B. Prepare Antibody: Dilute antibody with 1 x PBS. Prepare CQWs-525-VEGF.

[0076] C. First Antibody Incubation: Remove excess serum and place the tissue section flat in the immunohistochemistry humidified chamber with the CQWs-525-VEGF quantum dot labeled antibody. Place in the 4°C refrigerator overnight.

[0077] D. Rinse: Rinse 3 times for 2 minutes each with wash bottle filled with 1 x TBST.

[0078] The nucleus DNA was dyed again. 1X DAPI solution was prepared by using ddH20 at 1:100; 1X DAPI solution was added to the section tissue, incubated at room temperature for 5 min in the dark, washed with 1X TBST for 2 min, and washed with ddH2O for 1 min; 30 μl of anti-quenching mounting medium was added, covered with a cover glass, and the periphery was sealed with a special sealing agent. After the tissue section was stable, the results were observed and photographed using a fluorescence microscope with a 488 nm laser. The obtained pictures were analyzed using imageJ, the picture format was set to 8-bit format; the background was set, the Threshold was adjusted, the threshold value was manually selected to ensure that the dyed area was selected; the measurement parameters were set, the average gray value was calculated; the data was obtained, analyzed, and the immune status was evaluated.

[0079] The detection results of 30 cases of thymoma combined with autoimmune disease tumor tissue section samples were statistically analyzed, and the average fluorescence intensity of quantum dots corresponding to the antibody was calculated by ImageJ software: the obtained immunofluorescence pictures were converted to 8-Bit format using ImageJ, the "Threshold" area was adjusted, the default threshold was used, the "Dark Background" was checked, and the average fluorescence intensity Mean was calculated using the default algorithm. The average fluorescence intensity of each group was calculated.

[0080] As shown in Figures 1-5 is the color development of a thymoma and a thymoma patient with autoimmune disease tumor section under different fluorescence channels. Figure 1 The medium blue light is the cell nucleus group; Figure 2 The medium orange fluorescence shows CD138 expressed on the surface of plasma cells; Figure 3 The medium red fluorescence shows Mum1 expressed in the nucleus of plasma cells; Figure 4 The medium green fluorescence shows VEGF expressed in the newly formed blood vessels; Figure 5 is the synthesis of CQWs-585-CD138, CQWs-625-Mum1, CQWs-525-VEGF and dapi four dyes in thymoma and thymoma patient with autoimmune disease tumor section. Through the fluorescence intensity and position of each antibody, the expression intensity and spatial position relationship of CD138, Mum1 and VEGF can be clearly shown.

[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 patient thymoma tumor tissue sections in a 60°C oven for 0.5-2h. Subsequently, place tissue sections 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 rinse with ddH2O for 3 times for 2min each. Select acid or base antigen retrieval solution according to antibody instructions; commonly used antigen retrieval solutions are 1x Tris-EDTA antigen retrieval solution and 1x citrate antigen retrieval solution. When antigen retrieval, add antigen retrieval solution to the immunohistochemistry antigen retrieval container, and place in a microwave oven on high heat until boiling; then place tissue sections in the antigen retrieval solution, and set the microwave oven to low heat for 10min, then remove the antigen retrieval container and cool to room temperature naturally.

[0085] Remove the tissue sections and rinse with ddH2O for 1min, and 1x TBST for 1 time for 2min. Use an immunohistochemistry pen to circle the tissue, so that the entire tissue block on the section is inside the circle.

[0086] In addition, take the above processed tissue sections and sequentially perform staining with different quantum dot-antibody conjugates.

[0087] First round of staining:

[0088] A, blocking: remove 1x TBST solution, slowly add 10% BSA, and incubate at room temperature for 10min.

[0089] B, antibody preparation: dilute the antibody with 1x PBS, and prepare CQWs-585-CD138.

[0090] C, first antibody incubation: remove excess serum, and place the tissue sections flat in an immunohistochemistry wet box after adding CQWs-585-CD138 quantum dot-labeled antibody, then place in a 4°C refrigerator overnight.

[0091] D, rinse: rinse with a wash bottle containing 1x TBST for 3 times, each for 2min.

[0092] Second round of staining:

[0093] A, blocking: remove 1x TBST solution, slowly add 10% BSA, and incubate at room temperature for 10min.

[0094] B, antibody preparation: dilute the antibody with 1x PBS, and prepare CQWs-625-Mum1.

[0095] C. Second antibody incubation: Remove excess serum, add CQWs-625-Mum1 quantum dot labeled antibody, then place the tissue section in the immunohistochemistry wet box, and then place in the 4°C refrigerator overnight.

[0096] D. Rinse: Apply a wash bottle filled with 1 x TBST to rinse 3 times, 2 min each time.

[0097] Third round of staining:

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

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

[0100] C. First antibody incubation: Remove excess serum, add CQWs-525-ACTA2 quantum dot labeled antibody, then place the tissue section in the immunohistochemistry wet box, and then place in the 4°C refrigerator overnight.

[0101] D. Rinse: Apply a wash bottle filled with 1 x TBST to rinse 3 times, 2 min each time.

[0102] Nuclear DNA staining: Dilute 1 x DAPI solution with ddH20 at a ratio of 1:100; add 1 x DAPI solution to the sectioned tissue, incubate at room temperature in the dark for 5 min, rinse with 1 x TBST for 2 min, and rinse with ddH2O for 1 min; add 30 μl of anti-quenching mounting medium, cover with a cover glass, and seal the periphery with a special sealing agent; after the tissue section is stable, observe the results and take pictures using a fluorescence microscope equipped with a 488 nm laser. Analyze the obtained pictures using imageJ, set the picture format to 8-bit format; set the background, adjust the Threshold, manually select the threshold value to ensure that the stained area is selected; set the measurement parameters, calculate the average gray value; obtain the data, analyze, and evaluate the immune status.

[0103] Statistical analysis of the detection results of 30 cases of thymoma combined with autoimmune disease tumor tissue section samples, and calculation of the average fluorescence intensity of quantum dots corresponding to the antibody by ImageJ software: convert the obtained immunofluorescence picture to 8-Bit format using ImageJ, adjust the "Threshold" area, use the default threshold, check "Dark Background", and use the default algorithm to calculate the average fluorescence intensity Mean, wherein the ImageJ software statistical formula is average fluorescence intensity Mean = total fluorescence intensity of the area InDen / area Area. Calculate the average fluorescence intensity average value of each group.

[0104] As Figures 6-10The color development of a thymoma and a thymoma patient with autoimmune disease tumor section under different fluorescence channels is shown. Figure 6 The medium blue light emission is the cell nucleus group; Figure 7 The medium orange fluorescence shows CD138 expressed on the surface of plasma cells; Figure 8 The medium red fluorescence shows Mum1 expressed in the nucleus of plasma cells; Figure 9 The medium green fluorescence shows ACTA2 expressed in the smooth muscle layer of blood vessels; Figure 10 The synthesis of CQWs-585-CD138, CQWs-625-Mum1, CQWs-525-ACTA2 and dapi four dyes in the thymoma and thymoma patient with autoimmune disease tumor section is shown. Through the fluorescence intensity and position of each antibody, the expression intensity and spatial position relationship of CD138, Mum1 and ACTA2 are clearly shown.

[0105] It can be calculated 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; the average fluorescence intensity of ACTA2 expression is 75.85±5.990, and the expression of CD138, Mum1 and ACTA2 in the figure is positive; CD138 and Mum1 are concentrated in the region, accounting for more than 50% in the lymphoid tissue, and overlapping with ACTA2 distribution, 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 and CQWs-585-CD138, CQWs-625-Mum1, Tris-EDTA and DNA fluorescent dye were constructed respectively.

[0108] According to the method of Example 3, antigen-repaired thymoma tumor tissue sections were prepared, and the closed tissue was applied to the immunohistochemical pen, so that the entire tissue block on the section was located inside the sealing ring. According to the method of Example 3, CQWs-585-CD138, CQWs-625-Mum1, CQWs-525-VEGF and CQWs-525-ACTA2 were sequentially applied, and then nuclear staining was performed. The color development of the pictures under different excitation wavelengths was observed by fluorescence microscopy, the distribution of plasma cells was determined by the co-labeling results of CQWs-585-CD138 and CQWs-625-Mum1, the angiogenesis was determined by the labeling results of CQWs-525-VEGF and CQWs-525-ACTA2, and the 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: Includes quantum trap antibody conjugates, the antibodies of which include CD138 and Mum1, and also include VEGF and / or ACTA2; the quantum trap antibody conjugates are CQWs-525-VEGF, CQWs-525-ACTA2, CQWs-585-CD138 or CQWs-625-Mum1.

2. The kit for simultaneously detecting angiogenesis and plasma cell infiltration in lymphoid tissue according to claim 1, characterized in that: It also includes antigen retrieval solution and DNA fluorescent staining solution.

3. A method for preparing the kit for simultaneously detecting angiogenesis and plasma cell infiltration in lymphoid tissue as described in claim 1 or 2, characterized in that: Quantum well-antibody conjugates were prepared by coupling quantum well nanosheets with antibodies via EDC / NHS or SMCC methods.

4. The method for preparing the reagent kit according to claim 3, characterized in that: The quantum well nanosheets are one or more of CdSe, CdSe / CdS, CdZnSe / ZnS, and CdSe / CdZnS.

5. The method for preparing the reagent kit according to claim 3, characterized in that: 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. Then, an antibody was added, with a CQW to antibody molar ratio of 1:10–20. After thorough mixing, the mixture was reacted in the dark to prepare the quantum well and antibody conjugate CQWs-Ab.

6. The method of using the kit for simultaneous detection of angiogenesis and plasma cell infiltration in lymphoid tissue as described in claim 1 or 2, characterized in that: Lymphoid tissue sections were prepared, and after antigen retrieval, the tissue sections were subjected to antigen-antibody binding reactions using quantum trap-antibody conjugates with different emission wavelengths. After staining, images were acquired under a fluorescence microscope, and the emission intensity and spatial location information were quantified by detecting the emission information.

7. The method of use according to claim 6, characterized in that: Angiogenesis is assessed by detecting VEGF and / or ACTA2; a mean fluorescence intensity of VEGF greater than 72.75 indicates VEGF positivity, a mean fluorescence intensity of ACTA2 greater than 70.33 indicates ACTA2 positivity, and positivity for both VEGF and / or ACTA2 indicates angiogenesis. Plasma cell status is assessed by detecting CD138 and Mum1; a mean fluorescence intensity of CD138 greater than 45.63 indicates CD138 positivity, a mean fluorescence intensity of Mum1 greater than 47.51 indicates Mum1 positivity, and positivity for both CD138 and Mum1 indicates plasma cell infiltration. Observe whether plasma cells are concentrated in one area and whether their distribution overlaps with that of new blood vessels.

8. The method of using the kit for simultaneous detection of angiogenesis and plasma cell infiltration in lymphoid tissue as described in claim 1 or 2, or the kit for simultaneous detection of angiogenesis and plasma cell infiltration in lymphoid tissue as described in claim 6 or 7, in the evaluation of lymphoid tissue.

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