A kit for detecting plasma cells and related antibody levels in thymus microenvironment and its application

Abstract

The present invention relates to a kit for detecting plasma cells and related antibody levels in a thymic microenvironment and its application. The kit includes quantum dot-labeled antibody CD138, quantum dot-labeled antibody CD21, quantum dot-labeled AchR, Musk and / or LRP4 receptors, antigen repair solution and DNA fluorescent dye; the kit can detect one or more of plasma cells, follicular DC and related antibodies in a thymic (tumor) sample, so as to obtain the immune level of a tissue section sample. The method of using quantum dots to carry antibodies eliminates the secondary antibody staining link in the existing multi-color immunofluorescence technology and improves efficiency; by replacing fluorescent dyes with quantum dots, direct labeling of tissue sections can be achieved, and local positioning of plasma cells and follicular DC in the thymoma tumor microenvironment can be achieved, which can achieve both quantity detection and position detection.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a kit for detecting plasma cells and related antibody levels in a thymus microenvironment and its application. Background Art

[0002] The main biological function of the thymus is to produce a diverse pool of T cells, which constitute an important part of the host's adaptive immune response to foreign pathogens. The thymus also plays a key role in immune tolerance through negative selection and the production of Tregs (regulatory T cells). TECs (thymic epithelial cells) are the most important components in the thymic microenvironment, supporting the development of thymocytes and the establishment of self-tolerance. Since the thymus plays a key role in maintaining the balance between the host's immune response and tolerance, thymic dysfunction can lead to a variety of diseases in the human body, which are called thymus-related diseases, namely thymic diseases.

[0003] Pathological changes in the thymus, including thymoma and thymic hyperplasia, can lead to a variety of pathological changes in the body. Thymoma is associated with a variety of autoimmune diseases, among which myasthenia gravis is the most common and has the strongest association with thymoma. Other diseases, including pure red cell aplasia, hypogammaglobulinemia, systemic lupus erythematosus (SLE), rheumatoid arthritis, Sjögren's syndrome, and autoimmune thyroiditis, have a lower incidence than myasthenia gravis, but they are relatively recognized thymoma-related diseases. The specific mechanism of the association between thymoma and most of these diseases is still unclear. Immunohistochemistry helps to identify thymoma. Plasma cell and related antigen staining of different pathological types of thymomas will be more helpful in studying the relationship between different histological types of thymomas and related immune diseases.

[0004] Since the thymus plays such an important role in immunity and related immune diseases such as myasthenia gravis, the detection of specific cells or components in thymic tissue is helpful to explore the role of "thymic function modification" in related diseases from the source of the disease, and to improve the further understanding of thymic related diseases. The existing technology usually uses flow cytometry to detect peripheral blood T, B cells and their subsets, but only in extreme immune states will there be changes in CD20+B cells in peripheral blood. Although this can indicate the severity of the disease, it is not applicable to the early state of the disease. Although confocal microscopy or multi-labeled immunofluorescence staining technology can more accurately identify a certain type of multi-marker cell subsets, traditional staining technology has defects. The emission spectrum wavelength of traditional dyes is limited; the photostability is poor; spectral overlap is easy to occur between different fluorescent dyes; poor biocompatibility, it is not easy to carry out specific labeling; the fluorescence lifetime is short, it is difficult to preserve for a long time, and the background interference is strong. It is difficult to reveal the occurrence process of thymus-related autoimmune diseases. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a kit and application for detecting plasma cells and related antibody levels in the thymus microenvironment.

[0006] The technical solution adopted by the present invention is: a kit for detecting plasma cells and related antibody levels in the thymus microenvironment, including quantum dot-labeled antibody CD138, quantum dot-labeled antibody CD21, and quantum dot-labeled AchR, Musk and / or LRP4 receptors.

[0007] Preferably, it comprises antibody CD138 labeled with quantum dots QDs-525, antibody CD21 labeled with quantum dots QDs-585, one or more of AchR, MuSK and LRP4 receptors labeled with quantum dots QDs-625, antigen repair solution and DNA fluorescent dye.

[0008] Preferably, quantum dots, EDC, and NHS are mixed at a molar ratio of 1:2500-3500:5000-6000 to activate the quantum dots, and antibodies are added to the activated quantum dot solution, with a molar ratio of quantum dots to antibodies of 1:20-80, to obtain QDs-Ab conjugates after the reaction; the supernatant is taken as a quantum dot-labeled antibody solution that can be used for staining.

[0009] The kit for detecting the levels of plasma cells and related antibodies in the thymic microenvironment is used for detecting the levels of plasma cells and related antibodies in autoimmune disease samples for non-disease diagnosis and treatment purposes.

[0010] Preferably, the content and location of one or more of plasma cells, follicular DCs, anti-acetylcholine receptor AchR antibodies, anti-muscle specific receptor tyrosine kinase MuSK antibodies and anti-low-density lipoprotein receptor-related protein 4 LRP4 antibodies in thymic tissue sections or thymoma tissue sections are detected.

[0011] Preferably, the autoimmune disease is myasthenia gravis, rheumatoid arthritis, pure red cell aplasia, Sjögren's syndrome, autoimmune thyroiditis or systemic lupus erythematosus.

[0012] Preferably, thymic tissue sections or thymoma tissue sections are prepared first, and antigen repair is performed using an antigen repair solution. The tissue sections are then stained using antibodies labeled with different quantum dots, and then the cells are stained. The fluorescence information of the tissue sections is collected to obtain the immune level in the detected thymic microenvironment or thymoma microenvironment.

[0013] Preferably, the average fluorescence intensity of CD138 accounts for more than 55%, indicating that its immune infiltration is relatively rich; the fluorescence intensity is less than 15% and is judged to be a weak immune state in the tumor microenvironment;

[0014] and / or, the CD21 mean fluorescence intensity accounted for more than 43%, indicating that the immune infiltration was relatively abundant;

[0015] And / or, if the fluorescence intensity of one or more of AchR, Musk and LRP4 is less than 50%, it is judged as simple tumor tissue and tumor combined with mild myasthenia gravis; if the fluorescence intensity of one or more of AchR, Musk and LRP4 is greater than 50%, it is judged as tumor combined with severe myasthenia gravis.

[0016] The advantages and positive effects of the present invention are: the method of using quantum dots to carry antibodies eliminates the secondary antibody staining step in the existing multi-color immunofluorescence technology, adopts direct labeling, and gives full play to the advantages of quantum dots themselves, saving experimental time and improving efficiency; by replacing fluorescent dyes with quantum dots, direct labeling of tissue sections can be achieved, and local positioning of plasma cells and follicular DCs in the tumor microenvironment of thymoma can be achieved, which can realize both quantitative detection and position detection, making it easier to clarify their biological behavior;

[0017] In addition, the patient's autoimmune diseases can be predicted based on the expression levels of plasma cells, follicular DCs, and AchR, Musk and / or LRP4. The potential occurrence of autoimmune diseases can be determined without combining other markers for comprehensive judgment, which is more conducive to the early diagnosis of thymopathy-related autoimmune diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 HE staining of simple thymoma and thymoma combined with myasthenia gravis tumor tissue sections;

[0019] Figure 2 The expression of plasma cells in simple thymoma and thymoma combined with myasthenia gravis tumor tissues;

[0020] Figure 3 The expression of DC cells in simple thymoma and thymoma combined with myasthenia gravis tumor tissues;

[0021] Figure 4 The expression of AchR antibody in tumor tissues of simple thymoma (T), thymoma combined with mild myasthenia gravis (TMGL), and thymoma combined with severe myasthenia gravis (TMGH);

[0022] Figure 5 The expression of Musk antibody in tumor tissues of simple thymoma (T), thymoma combined with mild myasthenia gravis (TMGL), and thymoma combined with severe myasthenia gravis (TMGH);

[0023] Figure 6The expression of LRP4 antibody in tumor tissues of simple thymoma (T), thymoma combined with mild myasthenia gravis (TMGL), and thymoma combined with severe myasthenia gravis (TMGH). DETAILED DESCRIPTION

[0024] The embodiments of the present invention are described below with reference to the accompanying drawings.

[0025] The present invention relates to a kit for detecting plasma cells and related antibody levels in a thymic microenvironment and an application thereof. The kit for detecting plasma cells and related antibody levels in a thymic microenvironment comprises quantum dot-labeled antibody CD138, quantum dot-labeled antibody CD21, quantum dot-labeled AchR, Musk and / or LRP4 receptors, antigen repair solution and DNA fluorescent dye solution.

[0026] Related antibodies unique to the thymic (tumor) microenvironment, anti-acetylcholine receptor (AchR) antibodies, anti-muscle-specific receptor tyrosine kinase (MuSK) antibodies, and anti-low-density lipoprotein receptor-related protein 4 (LRP4) antibodies; AchR, Musk, and LRP4 antibodies are the most common autoantibodies in myasthenia gravis. Among them, AchR antibodies can be detected in more than 80% of myasthenia gravis patients. Myasthenia gravis (MG) is an autoimmune disease with acquired neuromuscular junction (NMJ) transmission disorders mediated by autoantibodies. Acetylcholine receptor antibodies (Anti-AchR Ab) are the most common pathogenic antibodies in myasthenia gravis; in addition, antibodies against other components of the postsynaptic membrane, including muscle-specific receptor tyrosine kinase (MuSK), low-density lipoprotein receptor-related protein 4 (LRP4) and ryanodine receptor (RyR), have been found to be involved in the pathogenesis of MG. These antibodies can interfere with AChR aggregation, affect AChR function and NMJ signaling. Ocular myasthenia gravis (OMG) symptoms are limited to the eyes. Adult-onset OMG is prone to transform into systemic type within 2 years of the onset of ocular muscle symptoms. Anti-AchR antibodies can be detected in half of OMG patients. AChR-generalized MG (GMG) has positive serum AChR antibodies and can be divided into early-onset myasthenia gravis (EOMG) and late-onset myasthenia gravis (LOMG) according to the age of onset. MuSK antibodies can be detected in the serum of about 1%-10% of MG patients. Most MuSK antibodies belong to the IgG4 subtype. The affected muscle groups of MuSK-MG are relatively limited, mainly the bulbar, neck and respiratory muscles, followed by the extraocular muscles and limb muscles, mainly manifested as bulbar palsy and facial and neck muscle weakness. Changes in anti-MuSK antibody titers usually indicate the activity of the patient's condition. LRP4 antibodies can be detected in 1%~5% MG and 7%~33% of AChR and MuSK antibody-negative MG patients. Antibody-negative MG has no detectable antibodies, and the thymus may be normal or hyperplastic. Thymoma-related MG accounts for about 10% to 15% of MG patients and is a paraneoplastic syndrome. AChR antibodies can be detected in most of them. In addition, titin antibodies and RyR antibodies are often present.The thymus of patients with myasthenia gravis often has germinal centers, and many of these germinal centers are AChR-specific in patients with myasthenia gravis; lymphocytes recovered from the thymus of patients with myasthenia gravis easily produce anti-AChR antibodies in vitro. The germinal centers in the MG thymus provide an environment for AChR-specific B cells to proliferate and increase the affinity of the antibodies they produce for AChR through a process of affinity maturation. The detection indicators of AchR, Musk, and LRP4 antibodies play a vital role in the progression of myasthenia gravis, and observing their expression can better understand the disease situation.

[0027] Plasma cells mainly refer to a type of B lymphocyte subset that can secrete antibodies and promote the progression of autoimmune diseases; follicular DCs are a rare and mysterious type of stromal cells in the B cell follicles of secondary lymphoid organs, which can store antigens and present antigens to B cells; the initiation of plasma cell differentiation is attacked by T cell-dependent antigens, and antigen-specific B cells and CD4+T cells interact homologously at the junction of T and B cells, and activated B cells migrate to the outer area of ​​the follicle. B cells proliferate in large numbers, forming extracellular aggregation areas and differentiating into plasma cells that secrete antibodies. Extrafollicular antibody responses play an important role as the first line of defense for acquired immunity by providing rapid protection against pathogens. CD21 is a type II complement receptor, encoding the gene CR2, located at 1q32, and is a C3d complement receptor and EBV receptor. It is strongly positively expressed in FDC (follicular dendritic cells) and positively located on the cell membrane. CD21 forms a CD21-CD19-CD81 complex with other membrane proteins CD19 and CD81 on the surface of mature B cells, called the B cell co-receptor complex. In the immunohistochemical staining of frozen tissues, CD21 is expressed on mature B cells (CD21L) and FDCs (CD21S), but is only expressed on FDCs in the immunohistochemical staining of paraffin-embedded tissues.

[0028] The kit for detecting the levels of plasma cells and related antibodies in the thymic microenvironment can examine the content and location of plasma cells, follicular DCs and related antibodies in thymic (tumor) samples. Antibody CD138 can specifically connect to plasma cells, and quantum dots labeled with antibody CD138 can specifically label plasma cells, and the content and location of plasma cells can be reflected by the corresponding fluorescent color; antibody CD21 can specifically connect to follicular DCs, and quantum dots labeled with antibody CD21 can specifically label follicular DCs, and the content and location of follicular DCs can be reflected by the corresponding fluorescent color. AchR, Musk and LRP4 receptors can specifically connect to AchR, Musk and LRP4 antibodies, respectively, and quantum dot-labeled AchR, Musk and LRP4 receptors can specifically label AchR, Musk and LRP4 antibodies in the thymic (tumor) microenvironment, and the quantum dot information obtained by the detection can reflect the content and location of the corresponding antibodies. Usually, a test sample will only include one of AchR, Musk and LRP4, and the quantum dots coupled with AchR, Musk and LRP4 antibodies in the kit can be the same color or different colors.

[0029] Compared with tumor tissue samples from patients with simple thymoma, there are more B lymphocyte infiltrations in the thymic (tumor) tissue microenvironment combined with autoimmune diseases, and the expression levels of follicular DCs, plasma cells and related antibodies are increased. Observing the B cell infiltration in the thymic tissue microenvironment, as well as the expression levels of plasma cells, follicular DCs and related antibodies, can better monitor the status of thymic disease-related autoimmune diseases.

[0030] The kit for detecting plasma cells and related antibody levels in the thymus microenvironment of the present invention can be used for the detection of key elements of various autoimmune diseases, including rheumatoid arthritis, pure red cell aplastic anemia, Sjögren's syndrome, autoimmune thyroiditis and systemic lupus erythematosus, in addition to myasthenia gravis. The test object of the kit is a thymus (tumor) tissue specimen, and the detection of immune cells in the thymus (tumor) tissue is achieved by staining it with one or more quantum dots and specific antibody conjugates.

[0031] In certain embodiments of the present invention, coupling quantum dots with specific antibodies comprises the following steps:

[0032] A1: Activation of quantum dots: QDs, EDC, and NHS were mixed at a molar ratio of 1: (2500-3500): (5000-6000), preferably 1: 3000: 5500, diluted with PBS, mixed on a shaker at room temperature, and then the pH was adjusted to 8-9 with PBS to obtain an activated quantum dot solution;

[0033] A2: Preparation of QDs-Ab conjugate: adding specific antibody to activated quantum dot solution, the molar ratio of quantum dots to specific antibody is 1:20-80; reacting on a shaker at 4°C in the dark, and obtaining QDs-Ab conjugate after reaction;

[0034] A3: After the reaction is completed, centrifuge at 8000 rpm to remove possible agglomerated precipitates, retain the supernatant, and purify the supernatant using a PD10 column to finally obtain specific antibodies coupled to quantum dots that can be used for staining.

[0035] By changing the different feed ratios of quantum dots, required cross-linking agents and antibodies, stable loading conditions were developed to achieve stable binding of quantum dots and antibodies. The obtained QDs-Ab can be used immediately, and quantum dots of different colors and different antibodies are combined according to the steps in the instructions to achieve personalized customization. The inappropriate ratio of QDs to Ab will result in poor stability of the obtained complex, easy polymerization, significantly affecting its biological compatibility, and unable to be used for biological applications, while excessive cross-linking agents will cause cross-linking between Ab and polymerization of QDs. It is necessary to accurately control the ratio between QDs, Ab and cross-linking agents to obtain a conjugate with high uniformity (i.e., a conjugate with only one Ab molecule bound to the surface of each nanoparticle) and stability. In addition, the purification of the biological conjugate after the coupling of quantum dots and antibodies is currently commonly used by gel filtration chromatography and membrane ultrafiltration, and the separation and purification rate is low. Some embodiments of the present invention use PD10 column purification, which effectively improves the purification rate and simplifies the purification steps.

[0036] The method for detecting plasma cells, follicular DCs and AchR, Musk, and LRP4 antibodies in thymic (tumor) tissues by using the above kit is as follows:

[0037] Step 1: Take thymus (tumor) tissue specimens to make tissue sections, bake the tissue sections and dewax and dehydrate them;

[0038] Step 2: Perform antigen repair on tissue sections using antigen repair solution to facilitate the binding of specific antibodies;

[0039] Step 3: staining the treated tissue sections with quantum dots and antibody protein conjugates. When there are multiple quantum dots and antibody protein conjugates, staining is performed in sequence. Each quantum dot staining step includes staining, incubation and washing processes. After fully removing the unbound quantum dots and antibody protein conjugates, the next staining step is performed.

[0040] Step 4: After all quantum dots are stained, the cell nuclei are stained, the slides are sealed and photographed, and the location and number of target cells are analyzed based on the photo information.

[0041] The kit can be used to mark B cells, Breg cells and high endothelial venules in the immune microenvironment of thymic (tumor) tissue. The specific antibody used to mark B cells is CD19; the specific antibody used to mark Breg cells is CD1d, and the specific antibodies used to mark high endothelial venules are CD31 and PNad. The quantum dots are coupled with the above antibodies, and the content of B cells, Breg cells and high endothelial venules can be reflected through the corresponding quantum dots. When used, first prepare thymic tissue sections, use antigen repair solution for antigen repair, then stain the tissue sections with antibodies labeled with different quantum dots, then stain the cells, collect the fluorescence information of the tissue sections, and obtain the immune infiltration status of the detected thymic tissue according to the fluorescence expression information, evaluate and predict the autoimmune reaction. By detecting the expression levels of plasma cells, follicular DCs and AchR, MuSK or LRP4 antibodies in the immune microenvironment of the thymus (tumor), the average fluorescence intensity percentage of the corresponding indicators can be calculated, and the immune infiltration status in the thymic immune microenvironment can be analyzed. At the same time, it provides an evaluation method for thymic tumors combined with myasthenia gravis or other autoimmune diseases.

[0042] Thymic (tumor) tissue sections were used as the detection object, and plasma cells were labeled with QDs-525-CD138. Under fluorescence microscopy, the average fluorescence intensity of CD138 accounted for more than 55%, indicating that its immune infiltration was relatively rich. Fluorescence intensity less than 15% was judged to be weak in the immune state of the tumor microenvironment. Follicular DCs were labeled with QDs-585-CD21. Under fluorescence microscopy, the average fluorescence intensity of CD21 accounted for more than 43%, indicating that its immune infiltration was relatively rich. The most common antibody in myasthenia gravis patients is AchR antibody positivity, and less common are Musk and LRP4 antibody positivity. AchR, MuSK and LRP4 antibodies were labeled with QDs-625-AchR receptor, QDs-625-Musk receptor or QDs-625-LRP4 receptor, respectively, and observed under a fluorescence microscope. If the average fluorescence intensity of any of AchR, Musk and LRP4 receptors is less than 50%, it is judged as simple tumor tissue and tumor combined with mild myasthenia gravis; if the fluorescence intensity is greater than 50%, it is judged as tumor combined with severe myasthenia gravis.

[0043] Combining quantum dots with the required antibodies makes up for the shortcomings of traditional fluorescent dyes. At the same time, the special combination method makes quantum dots and antibodies stably combined to achieve complementary advantages. Quantum dot multi-labeling immunofluorescence technology can effectively observe the expression level and spatial position structure of plasma cells, follicular DCs and related antibodies. The 95% confidence interval of the expression level of each cell subpopulation can be calculated with high accuracy. It can judge the status of thymic disease-related autoimmune diseases without combining other markers, especially for early and latent autoimmune diseases. The immune status can be evaluated by detecting the expression levels of plasma cells, follicular DCs and related antibodies in the patient's thymic tumor microenvironment in vitro.

[0044] The classic clinical diagnosis and evaluation methods for autoimmune diseases mainly rely on the patient's clinical manifestations, serum antibody level detection, etc. Due to the limitations of sensitivity and specificity, the accuracy of the disease assessment for various early autoimmune diseases including myasthenia gravis is poor, and there is a lack of effective evaluation methods at the pathogenesis level. Instead of the gene level or peripheral blood antibody detection method, the thymus (tumor) tissue sections are directly used as the detection object to detect plasma cells, follicular DC cells and related antibodies. By analyzing the expression levels of plasma cells, follicular DC and related antibodies in thymic tissue, the patient's immune status can be evaluated from the pathogenesis level, overcoming the disadvantages of the previous limited evaluation methods. The relevant cells and antibodies are counted by quantum dot multi-labeling immunofluorescence technology with better accuracy.

[0045] The scheme of the present invention is described below in conjunction with the accompanying drawings, wherein the experimental methods without specific operating steps are all carried out in accordance with the corresponding product instructions, and the instruments, reagents, and consumables used in the examples can all be purchased from commercial companies unless otherwise specified.

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

[0047] QDs, EDC, and NHS were mixed at a molar ratio of 1:3000:5500, diluted with PBS, and mixed on a shaker at room temperature for 30 minutes, and then the pH was adjusted to 8-9 with PBS to obtain a quantum dot activation solution. Take 100ul of the quantum dot activation solution and add it to the reaction container. Take an appropriate amount of antibody stock solution, dilute it to 0.01mg / ml with PBS buffer, mix it thoroughly, add it to the above reaction container according to different ratios, and react for 4 hours on a shaker at 4℃ in the dark. After the reaction is completed, centrifuge at 8000 rpm for 3 min to remove possible agglomerated precipitates, and keep the supernatant, which is the prepared QDs-Ab conjugate. The supernatant is purified with a PD10 column, and the purified product is stored in a refrigerator at 4℃ in the dark for later use. The supernatant can be detected and analyzed using 1% agarose gel electrophoresis.

[0048] In order to screen the optimal coupling conditions, samples with different mixing ratios of quantum dots and antibodies were prepared, and the molar ratios of quantum dots QDs and antibody proteins were 1:20, 1:40, and 1:80, respectively. The optimal coupling conditions can be screened by analyzing the results through 1% agarose gel electrophoresis.

[0049] According to the above method, quantum dots QDs-525, QDs-585, and QDs-625 were taken respectively, and connected with the specific antibody protein CD138 for plasma cells, the specific antibody CD21 for follicular DC cells, and the AchR, MuSK, and LRP4 receptors for detecting the related antibodies AchR, Musk, and LRP4, respectively, to form quantum dots and antibody protein conjugates, specifically QDs-525-CD138, QDs-585-CD21, QDs-625-AchR ab, QDs-625-Musk ab, and QDs-625-LRP4 ab.

[0050] Example 2: Preparation of test samples

[0051] Thymoma tumor tissue specimens from patients with simple thymoma (T), thymoma combined with mild myasthenia gravis (TMGL), and thymoma combined with severe myasthenia gravis (TMGH) were collected. The samples were from thymoma (tumor) specimens removed by surgery in the Department of Cardiothoracic Surgery, Tianjin Medical University General Hospital. They were soaked in 4% formalin for more than 48 hours. Wax blocks were made from thymoma tissue specimens and placed on a microtome to make 4μm tissue sections. The source information of the sections, staining index, date, antibody ratio, etc. were recorded before staining.

[0052] Place the tissue slices in a 60°C oven for 30 minutes for baking. Dewax and dehydrate, transfer the slices to No. 1 100% xylene for 15 minutes, No. 2 100% xylene for 15 minutes, No. 3 100% ethanol for 3 minutes, No. 4 100% ethanol for 3 minutes, No. 5 95% ethanol for 3 minutes, No. 6 75% ethanol for 3 minutes, No. 7 50% ethanol for 3 minutes, and rinse with PBS 3 times for 5 minutes each time.

[0053] Antigen repair was performed on the dewaxed and dehydrated tissue sections. During antigen repair, acidic or alkaline antigen repair solution was selected according to the instructions of the corresponding antibody; the antigen repair solution was added to the container, placed in a microwave oven to heat and boil, and then the tissue sections were placed in the container, and heated on medium-low heat for 10 minutes before being taken out; after the tissue sections naturally cooled to room temperature, they were transferred to the immunohistochemistry washing box, washed with sterile water for 1 minute, and washed with 1×TBST for 2 minutes. The tissue was enclosed with an immunohistochemistry pen so that the entire tissue block on the section was located inside the seal.

[0054] HE staining was performed on the prepared slice samples, and the results were as follows Figure 1 shown.

[0055] Example 3: Detection of plasma cells in the thymoma tumor microenvironment

[0056] The slice sample prepared in Example 2 was stained with QDs-525-CD138, as follows:

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

[0058] Prepare antibodies: dilute the antibodies with 1×PBS to prepare QDs-525-CD138 quantum dot labeled antibody solution.

[0059] Incubation with the first antibody: remove excess serum, add QDs-525-CD138 quantum dot-labeled antibody, place the tissue sections flat in an immunohistochemistry humidified box, and then place in a 4°C refrigerator overnight.

[0060] Rinse: Rinse three times with a wash bottle containing 1×TBST, 2 minutes each time, to remove unbound QDs-525-CD138 quantum dot-labeled antibodies.

[0061] After the tissue sections are stained, the nuclei are stained. 1×DAPI solution is prepared with ddH20 1:100; 1×DAPI solution is added to the tissue sections, incubated at room temperature in the dark for 5 minutes, rinsed with 1×TBST for 2 minutes, and rinsed with ddH2O for 1 minute. After removing the excess liquid on the sections, 30μl of anti-fade mounting medium is added, and then the coverslip is covered. After the tissue sections are stable, the results are observed and photographed using a fluorescence microscope equipped with a 488nm laser.

[0062] Thymoma tumor tissue specimens from multiple groups of patients with simple thymoma (T), thymoma combined with mild myasthenia gravis (TMGL), and thymoma combined with severe myasthenia gravis (TMGH) were tested, and the statistical results showed that the average fluorescence intensity of CD138 accounted for more than 55%, indicating that the immune infiltration was relatively rich. A fluorescence intensity of less than 15% was judged to be a weak immune state in the tumor microenvironment.

[0063] like Figure 2 The results are shown for a simple thymoma sample and a thymoma combined with myasthenia gravis tumor tissue sample. QDs-525 is displayed as a green fluorescent signal. The green light signal in the figure represents CD138, reflecting the content and location of plasma cells in the test sample. The left picture shows the expression of plasma cells in simple thymoma tissue, and the right picture shows the expression of plasma cells in thymoma combined with myasthenia gravis tumor tissue. The comparison shows that the content of plasma cells in thymoma combined with myasthenia gravis tumor tissue is significantly increased. Figure 2In the right figure, the average fluorescence intensity of CD138 accounts for more than 55%, indicating rich immune infiltration, which is consistent with the tumor tissue characteristics of thymoma combined with myasthenia gravis.

[0064] Example 4: Detection of follicular DCs in the thymoma tumor microenvironment

[0065] The slice samples prepared in Example 2 were stained with QDs-585-CD21 as follows:

[0066] Blocking: Remove 1× TBST solution, slowly add 10% BSA, and incubate at room temperature for 10 minutes.

[0067] Prepare antibodies: dilute the antibodies with 1×PBS to prepare QDs-585-CD21 quantum dot labeled antibody solution.

[0068] Incubation with the first antibody: remove excess serum, add QDs-585-CD21 quantum dot labeled antibody, place the tissue sections flat in an immunohistochemistry humidified box, and then place in a 4°C refrigerator overnight.

[0069] Rinse: Rinse three times with a wash bottle containing 1×TBST, 2 minutes each time, to remove unbound QDs-585-CD21 quantum dot-labeled antibodies.

[0070] Rinse: Rinse three times with a wash bottle containing 1×TBST, 2 minutes each time, to remove unbound QDs-585-CD21 quantum dot-coupled antibodies.

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

[0072] Thymoma tumor tissue specimens from multiple groups of patients with simple thymoma (T), thymoma combined with mild myasthenia gravis (TMGL), and thymoma combined with severe myasthenia gravis (TMGH) were tested. Statistics showed that the average fluorescence intensity of CD21 accounted for more than 43%, indicating that its immune infiltration was relatively rich.

[0073] like Figure 3The results are shown for a simple thymoma sample and a thymoma combined with myasthenia gravis tumor tissue sample. The orange light signal represents CD21, reflecting the content and location of follicular DC cells in the test sample. The left picture shows the expression of DC cells in simple thymoma tissue, and the right picture shows the expression of DC cells in thymoma combined with myasthenia gravis tumor tissue. The comparison shows that the plasma cell content in thymoma combined with myasthenia gravis tumor tissue is significantly higher. Figure 3 In the right figure, the average fluorescence intensity of CD21 accounts for more than 43%, indicating that its immune infiltration is relatively rich, which is consistent with the tumor tissue characteristics of thymoma combined with myasthenia gravis.

[0074] Example 5: Detection of AchR, MuSK, and LRP4 antibodies in the thymoma tumor microenvironment

[0075] The slice samples prepared in Example 2 were stained with QDs-625-AchR ab, QDs-625-Musk ab and QDs-625-LRP4 ab, respectively. The details are as follows:

[0076] Blocking: Remove 1× TBST solution, slowly add 10% BSA, and incubate at room temperature for 10 minutes.

[0077] Prepare antibodies: dilute the antibodies with 1×PBS to prepare QDs-625- AchR ab / Musk ab / LRP4 ab quantum dot labeled antibody solution.

[0078] Incubation with the first antibody: remove excess serum, add QDs-625-AchR ab / Musk ab / LRP4 ab quantum dot-labeled antibodies, place the tissue sections flat in an immunohistochemistry humidified box, and then place in a 4°C refrigerator overnight.

[0079] Washing: Wash three times with a washing bottle filled with 1×TBST, 2 min each time, to remove unbound QDs-625-AchR ab / Musk ab / LRP4 ab quantum dot-labeled antibodies.

[0080] Rinse: Rinse three times with a wash bottle filled with 1×TBST, 2 minutes each time, to remove unbound QDs-625-AchR ab / Musk ab / LRP4 ab quantum dot-coupled antibodies.

[0081] After the tissue sections are stained, the nuclei are stained. 1×DAPI solution is prepared with ddH20 1:100; 1×DAPI solution is added to the tissue sections, incubated at room temperature in the dark for 5 minutes, rinsed with 1×TBST for 2 minutes, and rinsed with ddH2O for 1 minute. After removing the excess liquid on the sections, 30μl of anti-fade mounting medium is added, and then the coverslip is covered. After the tissue sections are stable, the results are observed and photographed using a fluorescence microscope equipped with a 488nm laser.

[0082] Thymoma tumor tissue specimens from multiple groups of patients with simple thymoma (T), thymoma combined with mild myasthenia gravis (TMGL), and thymoma combined with severe myasthenia gravis (TMGH) were tested. Statistics showed that when the fluorescence intensity of the AchR / Musk / LRP4 receptor was less than 50%, it was judged as simple tumor tissue and tumor combined with mild myasthenia gravis; when the fluorescence intensity was greater than 50%, it was judged as tumor combined with severe myasthenia gravis.

[0083] like Figure 4-Figure 6 Shown are the test results of tumor tissues of a simple thymoma (T), a thymoma combined with mild myasthenia gravis (TMGL), and a thymoma combined with severe myasthenia gravis (TMGH). The red light signal represents the AchR / Musk / LRP4 antibody, which reflects the content and location of AchR, Musk or LRP4 in each test sample. Among them, T is the test result of a simple thymoma sample, TMGL is the test result of a thymoma combined with mild myasthenia gravis sample, and TMGH is the test result of a thymoma combined with severe myasthenia gravis sample; it can be seen from the figure that the expression of AchR, Musk or LRP4 in the tissue increases with the intensification of the disease, proving that the course of the disease can be learned by comparing the intensity and position information of the quantum dot fluorescence signal. Figure 4-Figure 6 The corresponding average fluorescence intensity of AchR, Musk or LRP4 in the TMGH group was greater than 50%, which was judged as tumor combined with severe myasthenia gravis, which was consistent with the sample classification.

[0084] Example 3-Example 5 can be used to perform quantum dot staining detection on slices of the same tissue, or the same tissue slice can be sequentially stained with plasma cell quantum dot staining, follicular DC quantum dot staining, and one of the related antibodies. Thymic hyperplasia is characterized by the aggregation of a large number of germinal centers (places where B cells develop and mature), in which B cells produce anti-AchR antibodies (Musk / LRP4 antibodies), mediating the loss of immune tolerance to AchR; thymic epithelial cells, muscle cells, and follicular dendritic cells are all involved in this immune response process. By detecting plasma cells, follicular DCs, and related antibodies at the same time, the immune development can be judged based on the contents of the three types of detection objects.

[0085] like Figure 1The figure shows HE staining of the slice samples, which reflects the HE staining of simple thymoma and thymoma combined with myasthenia gravis tumor tissue sections; Figure 2-Figure 6 The images separately reflect the quantum dot staining situation. By switching different channels, the single expression of light signals of different colors can be observed, so as to know the expression situations of plasma cells, follicular DC, anti-acetylcholine receptor AchR antibody, anti-muscle-specific receptor tyrosine kinase MuSK antibody and anti-low-density lipoprotein receptor-related protein 4 LRP4 antibody respectively. The correlation between the expression positions of plasma cells, follicular DC and anti-AchR / MuSK / LRP4 antibodies can be known through the overall image.

[0086] The embodiments of the present invention are described in detail above, but the contents described are only preferred embodiments of the present invention and cannot be considered to limit the scope of implementation of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A kit for detecting plasma cells and related antibody levels in the thymus microenvironment, characterized in that: Including quantum dot-labeled antibody CD138, quantum dot-labeled antibody CD21, quantum dot-labeled AchR, Musk and / or LRP4 receptors; The quantum dots, EDC and NHS are mixed at a molar ratio of 1:2500-3500:5000-6000 to activate the quantum dots, and the antibody is added to the activated quantum dot solution, the molar ratio of quantum dots to antibodies is 1:20-80, and QDs-Ab conjugates are obtained after the reaction; the supernatant is taken as a quantum dot-labeled antibody solution that can be used for staining.

2. The kit for detecting plasma cells and related antibody levels in the thymic microenvironment according to claim 1, characterized in that: It includes antibody CD138 labeled with quantum dots QDs-525, antibody CD21 labeled with quantum dots QDs-585, one or more of AchR, MuSK and LRP4 receptors labeled with quantum dots QDs-625, antigen repair solution and DNA fluorescent dye.

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