A thymic tumor microenvironment antigen detection kit and its application in myasthenia gravis evaluation

Abstract

The present invention relates to a thymic tumor microenvironment antigen detection kit and its application in myasthenia gravis evaluation. The thymic tumor microenvironment antigen detection kit includes quantum dot-coupled antibody AchR, quantum dot-coupled antibody Titin, quantum dot-coupled antibody MuSK, quantum dot-coupled antibody LRP4 and quantum dot-coupled antibody RyR; quantum dot staining is performed on thymic tumor tissue sections by the kit, and different antigen staining can be performed on the same tissue section at the same time. There are differences in the expression levels of AchR, Titin, MuSK, LRP4 and RyR antigens in patients with simple thymoma, simple myasthenia gravis and thymoma combined with myasthenia gravis, and this difference can be used to distinguish the cause, course and prognosis of myasthenia gravis. The method of using quantum dots to directly carry specific antibodies eliminates the complex staining link of secondary antibodies and improves work efficiency; and observes its expression level and spatial position.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a thymus tumor microenvironment antigen detection kit and its application in myasthenia evaluation. Background Art

[0002] The thymus is an important lymphoid organ in the body. Its function is closely related to immunity. It secretes thymic hormones and hormone-like substances and is an organ with endocrine function. It is located in the anterior mediastinum of the thoracic cavity. The thymus is an immune organ of the human body that can store and secrete immune cells and immune molecules, etc. It gradually shrinks with age. Thymic tumors are tumors that occur in central immune organs. The occurrence of tumors breaks this normal immune structure and causes a series of autoimmune diseases. The tumor microenvironment (TME) is mainly composed of tumor cells and their surrounding immune and inflammatory cells, tumor-associated fibroblasts and nearby interstitial tissues, microvessels, and various cytokines and chemokines. It is a complex integrated system that can be divided into an immune microenvironment dominated by immune cells and a non-immune microenvironment dominated by fibroblasts. There is infiltration of immune cells inside the tumor tissue. The lymphocytes infiltrating into the tumor are called tumor infiltration lymphocytes (TILs), including T lymphocytes, B lymphocytes, macrophages, natural killer (NK) cells, myeloid-derived suppressor cells (MDSC) and dendritic cells (DC) that play an antigen-presenting role. Tumor cells and tumor microenvironment complement each other. The heterogeneity of the microenvironment is closely related to the different response rates of tumor patients to immunotherapy. It is very important to explore the internal structure of the tumor microenvironment and the biological relationship of various immune cells for the precise treatment of tumors.

[0003] The occurrence of tumors is often caused by the disruption of the dynamic balance of immunity. The body cannot effectively eliminate the proliferation of tumor cells, thus inducing the occurrence of tumors. The bodies of patients with autoimmune diseases are mostly in a state of hyperimmunity, recognizing self-antigens, thereby inducing damage to target organs. When tumors appear in central immune organs, specific antigens will be produced, and then corresponding antibodies will appear. For patients who have both immune tolerance due to tumor factors and hyperimmunity due to autoimmune diseases, detecting and distinguishing specific antibodies has an effective prompting effect on controlling the direction of disease progression and mastering the treatment of the disease. The detection of its antigens can provide guidance for clinical practice one step ahead.

[0004] Myasthenia gravis is an autoimmune disease caused by multiple factors that result in an immune response to self-antigens, resulting in damage to the body's own tissues. At present, there are many ways to classify myasthenia gravis, and there is a lack of early and rapid diagnosis methods. Myasthenia gravis caused by different antibodies, myasthenia gravis related to paraneoplasm, myasthenia gravis caused by ion channel changes, etc., the prognosis of different classifications and different antibody phenotypes varies greatly. For the diagnosis of myasthenia gravis, there is still a low sensitivity and specificity. The diagnosis of the disease often requires multiple indicators to be tested simultaneously to support the diagnosis of the corresponding disease. For myasthenia gravis, long-term immune hyperstimulation induces damage to the body's own tissues, but currently most people have not paid attention to the immune status of the central immune organ-thymus, and its immune status is unknown; clinically, the current application is to detect related antibodies in the blood to assist in the identification of myasthenia gravis. Detecting the immune status in thymic tumors can better understand the degree of disease progression. At present, there is no analytical method for the association between the expression of antigens and antibodies in thymoma tissues and myasthenia gravis. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a thymic tumor microenvironment antigen detection kit and its application in myasthenia gravis evaluation. The application of this kit plays a vital role in early prevention and early diagnosis of the disease, timely improvement and relief of clinical symptoms, and delaying the disease and improving the quality of life of patients.

[0006] The technical solution adopted by the present invention is: a thymic tumor microenvironment antigen detection kit, including quantum dot-coupled antibody AchR, quantum dot-coupled antibody Titin, quantum dot-coupled antibody MuSK, quantum dot-coupled antibody LRP4 and quantum dot-coupled antibody RyR.

[0007] Preferably, the quantum dot-coupled antibodies include QDs-525-AchR, QDs-585-Titin, QDs-625-MuSK, QDs-585-LRP4, and QDs-625-RyR.

[0008] Preferably, antigen repair solution and nucleic acid staining solution are also included.

[0009] 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.

[0010] Application of thymic tumor microenvironment antigen detection kit in the evaluation of myasthenia gravis.

[0011] Preferably, 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, and the presence and location of various antigens in the detected thymoma tissue are determined based on the fluorescence expression level and spatial position distribution information.

[0012] Preferably, the tissue sections are stained in sequence using quantum dot-coupled antibodies AchR, quantum dot-coupled antibodies Titin and quantum dot-coupled antibodies MuSK, and the content and position information of each antibody in the tissue section are observed through quantum dot information;

[0013] And / or, the tissue sections are stained in sequence using quantum dot-coupled antibody AchR, quantum dot-coupled antibody LRP4 and quantum dot-coupled antibody RyR, and the content and position information of each antibody in the tissue section are observed through quantum dot information.

[0014] Preferably, the average fluorescence intensity of quantum dots is used to determine whether the corresponding antibody test is positive, and the critical value for positive definition is that the average fluorescence intensity of QDs-525-AchR accounts for more than 55%, the average fluorescence intensity of QDs-585-Titin accounts for more than 43%, the average fluorescence intensity of QDs-625-MuSK accounts for more than 47%, the average fluorescence intensity of QDs-585-LRP4 accounts for more than 40%, and the average fluorescence intensity of QDs-625-RyR accounts for more than 45%.

[0015] Preferably, the tissue sections are stained in the order of QDs-525-AchR, QDs-585-Titin and QDs-625-MuSK;

[0016] And / or, the tissue sections are stained in the order of QDs-525-AchR, QDs-585-LRP4 and QDs-625-RyR.

[0017] Preferably, the classification and prognosis of myasthenia gravis are determined based on the fluorescence information.

[0018] The advantages and positive effects of the present invention are: providing a kit for evaluating myasthenia gravis, capable of performing multi-type quantum dot staining on thymus tissue sections, and achieving classification and prognosis of myasthenia gravis by simultaneously labeling multiple target antigens;

[0019] Instead of multi-color immunofluorescence technology, the method of using quantum dots to directly carry specific antibodies eliminates the complex staining process of secondary antibodies, gives full play to the advantages of quantum dots themselves, saves experimental time, and improves work efficiency; by replacing fluorescent dyes with quantum dots, its expression level and spatial position structure can be effectively observed;

[0020] In addition, by calculating the 95% confidence interval of myasthenia gravis patients, a high degree of accuracy can be guaranteed, and the type of myasthenia gravis can be determined without combining other markers. It has a good suggestive effect on early and latent myasthenia gravis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 HE staining of tumor tissue sections of patients with simple thymoma and thymoma combined with autoimmune disease;

[0022] Figure 2 The expression of AchR, Titin and MuSK antigens and the mean fluorescence intensity scatter plot in the tumor tissues of simple thymoma and thymoma combined with myasthenia gravis; A is the simple thymoma group; B is the thymoma combined with MG group;

[0023] Figure 3 The expression of AchR, LRP4, and RyR antigens and the mean fluorescence intensity scatter plots in the tumor tissues of simple thymoma and thymoma combined with myasthenia gravis; A is the simple thymoma group, and B is the thymoma combined with MG group;

[0024] Figure 4 The expression of AchR antigen in tumor tissues of simple thymoma and thymoma combined with myasthenia gravis. DETAILED DESCRIPTION

[0025] The embodiments of the present invention are described below with reference to the accompanying drawings.

[0026] The present invention relates to a thymic tumor microenvironment antigen detection kit and application thereof in myasthenia evaluation. The thymic tumor microenvironment antigen detection kit comprises quantum dot-coupled antibody AchR, quantum dot-coupled antibody Titin, quantum dot-coupled antibody MuSK, quantum dot-coupled antibody LRP4 and quantum dot-coupled antibody RyR, antigen repair solution and nucleic acid staining solution.

[0027] The levels of Titin and MuSK antibodies in myasthenia gravis patients are related to the course of the disease. The level of AChR antibodies is related to the severity of the disease. In MG patients, the expression of AChR antibodies, Titin antibodies, and MuSK antibodies may show different trends depending on the time of onset, whether there is thymic abnormality, and the severity of the disease. LRP4 antibodies can be detected in 7% to 33% of MG patients with negative AChR and MuSK antibodies. AChR antibodies can be detected in the vast majority of thymoma-related MG. In addition, Titin antibodies and RyR antibodies are often combined. Thymoma-related MG is slightly more severe and requires a longer course of immunosuppressive treatment. In myasthenia gravis patients, the expression of AchR, Titin, MuSK, LRP4, and RyR antibodies may show different trends depending on the time of onset, whether there is thymic abnormality, and the severity of the disease. As the antigen corresponding to the antibody, it appears earlier than the antibody. The antibody is a change in the terminal stage of the disease. Early detection of the antigen can guide the evaluation and classification of myasthenia gravis earlier.

[0028] In certain embodiments of the present invention, coupling quantum dots with specific antibodies comprises the following steps:

[0029] 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;

[0030] A2: Preparation of QDs-Ab conjugate: Add specific antibody to activated quantum dot solution, with the molar ratio of quantum dots to specific antibody being 1:20-80; react on a shaker at 4°C in the dark, and obtain QDs-Ab conjugate after reaction

[0031] 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.

[0032] The thymic tumor microenvironment antigen detection kit can be used to evaluate myasthenia gravis. First, thymoma tissue sections are prepared, and antigen repair is performed using antigen repair solution. The tissue sections are stained with antibodies labeled with different quantum dots, and then the cells are stained. The fluorescence information of the tissue sections is collected, and the presence and location of various antigens in the detected thymoma tissue are obtained based on the fluorescence expression and spatial position distribution information. The thymic tumor microenvironment antigen detection kit can detect tissue sections from the perspective of antigens, which has a better effect on the early judgment of myasthenia gravis.

[0033] The kit includes quantum dot-coupled antibodies QDs-525-AchR, QDs-585-Titin, QDs-625-MuSK, QDs-585- LRP4, QDs-625-RyR, as well as antigen retrieval solution Tris-EDTA and nucleic acid staining solution DAPI stain.

[0034] In certain embodiments of the present invention, the five quantum dot-labeled antibodies can be divided into two combinations to detect the AchR, Titin, and MuSK antigens in the same tissue section, or to detect the AchR, LRP4, and RyR antigens in the same tissue section. Specifically, the tissue sections can be processed and stained according to the following steps.

[0035] 1. Take the paraffin specimen of thymoma tissue and cut 3-5um tissue sections, bake the tissue sections, dewax and hydrate them;

[0036] 2. Use a microwave oven to perform antigen repair on tissue sections, using Tris-EDTA as the repair solution;

[0037] 3. Staining tissue sections with quantum dots and specific antibody conjugates. When there are multiple quantum dots and specific antibody conjugates, staining is performed in sequence; for example, staining tissue sections in the order of QDs-525-AchR, QDs-585-Titin and QDs-625-MuSK; or staining tissue sections in the order of QDs-525-AchR, QDs-585-LRP4 and QDs-625-RyR;

[0038] 4 After staining, use DAPI to stain the cell nucleus, seal the slide and take photos.

[0039] The corresponding AchR, Titin, MuSK, LRP4, and RyR antigens were labeled by quantum dot staining technology, and the average fluorescence intensity of the corresponding cell expression level was calculated. Ten fields of view were selected for analysis of tumor tissue sections from the same patient. The semi-quantitative technology of quantum dot staining was used to indirectly evaluate the immune status of thymoma combined with autoimmune diseases.

[0040] The average fluorescence intensity of each quantum dot was detected, and whether the corresponding antibody was detected as positive was determined by the average fluorescence intensity of the quantum dots. The critical value for positive definition was: the average fluorescence intensity of QDs-525-AchR accounted for more than 55%, the average fluorescence intensity of QDs-585-Titin accounted for more than 43%, the average fluorescence intensity of QDs-625-MuSK accounted for more than 47%, the average fluorescence intensity of QDs-585-LRP4 accounted for more than 40%, and the average fluorescence intensity of QDs-625-RyR accounted for more than 45%; the above critical values ​​were used to determine the accuracy of positive judgment for existing samples to be more than 90%.

[0041] There are differences in the expression levels of AchR, Titin, MuSK, LRP4, and RyR antigens in patients with simple thymoma, simple myasthenia gravis, and thymoma combined with myasthenia gravis. This difference can be used to distinguish the cause, course, and prognosis of myasthenia gravis, so the classification and outcome of myasthenia gravis can be judged based on the fluorescence information. The detection of multiple antigens in the thymic tumor microenvironment requires tumor tissue sections of patients. The present invention improves the existing multicolor immunofluorescence technology, uses a method of directly carrying specific antibodies with quantum dots, eliminates the complex staining links of the second antibody, gives full play to the advantages of the quantum dots themselves, saves experimental time, and improves work efficiency. By replacing fluorescent dyes with quantum dots, its expression level and spatial position structure can be effectively observed. In addition, by calculating the 95% confidence interval of myasthenia gravis patients, a higher accuracy can be guaranteed, and the type of myasthenia gravis can be judged without combining other markers, which has a good prompting effect on early and latent myasthenia gravis. In addition, the tissue sections stained with quantum dots can be preserved for a long time, and by preserving and comparing sections of multiple periods, it is beneficial to study the study of myasthenia gravis.

[0042] 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.

[0043] Example 1: Tissue section preparation and processing

[0044] Thymoma tissue specimens were collected from 30 patients with simple thymoma (T), 30 patients with simple myasthenia gravis (MG), and 30 patients with thymoma combined with myasthenia gravis (TMG) during thymectomy.

[0045] Thymic tissue or thymoma tissue was obtained respectively, and the tissues were sliced ​​and pre-processed according to the following steps.

[0046] S1: Record the patient information, staining index, date and antibody ratio of the slice.

[0047] S2: Place the patient's thymoma tumor tissue slices in a 60°C oven for 30min-2h.

[0048] S3: Dewaxing and dehydrating: Soak the tissue sections in No. 1 100% xylene for 5 min, No. 2 100% xylene for 5 min, No. 3 100% ethanol for 5 min, No. 4 100% ethanol for 5 min, No. 5 95% ethanol for 5 min, No. 6 75% ethanol for 5 min, No. 7 50% ethanol for 5 min, and finally rinse with ddH2O for 3 times, each time for 2 min.

[0049] S4: Antigen repair: Choose acidic or alkaline antigen repair solution according to the antibody instructions; commonly used antigen repair solutions are 1×Tris-EDTA antigen repair solution and 1×citric acid antigen repair solution. When repairing antigens, add the antigen repair solution to the immunohistochemistry antigen repair container and heat it in a microwave oven at high heat until it boils; then put the tissue section into the antigen repair solution, adjust the microwave oven to low heat and maintain for 10 minutes, then take out the antigen repair container and cool it naturally to room temperature (this process must be cooled naturally, otherwise it will affect the repair effect).

[0050] S5: Remove the tissue sections and rinse them with ddH2O for 1 min and 1×TBST once for 2 min.

[0051] S6: Use an immunohistochemistry pen to enclose the tissue so that the entire tissue block on the slice is located inside the seal.

[0052] S7: Add 5% BSA to the inside of the sealing ring to completely cover the tissue specimen and incubate at room temperature for 10 minutes.

[0053] Example 2: Preparation of quantum dot-antibody conjugates

[0054] The coupling of quantum dots to antibody proteins includes the following steps:

[0055] Step 1: QDs, EDC, and NHS are 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 is adjusted to 8-9 with PBS to obtain an activated quantum dot solution;

[0056] Step 2: Take an appropriate amount of antibody stock solution, dilute it with PBS buffer, and mix it thoroughly; add antibody to the activated quantum dot solution, the molar ratio of quantum dots to antibody protein is 1:20-80 (1:20, 1:40, 1:80 can be used for labeling respectively), shake at 4°C in the dark, and obtain QDs-Ab conjugate after reaction;

[0057] Step 3: After the reaction is completed, centrifuge at 8000 rpm to remove possible agglomerated precipitates and retain the supernatant. Purify the supernatant using a PD10 column and store the purified product in a 4°C refrigerator away from light for later use.

[0058] By changing the different feed ratios of quantum dots, required cross-linkers 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 different colors of quantum dots and different antibodies can be combined according to the steps in the instructions to achieve personalized customization.

[0059] An inappropriate ratio of QDs to Ab will result in poor stability of the obtained complex, easy aggregation, significantly affecting its biocompatibility and making it impossible to use it in biological applications, while an excessive amount of cross-linking agent will cause cross-linking between Abs and aggregation of QDs. It is necessary to precisely control the ratio of QDs, Abs 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.

[0060] In addition, the purification of the bioconjugate after the coupling of quantum dots and antibodies is currently commonly performed by gel filtration chromatography and membrane ultrafiltration, and the separation and purification rate is low. In this embodiment, PD10 column purification is used to effectively improve the purification rate and simplify the purification steps.

[0061] Comparative example: HE staining of tumor tissue sections of patients with simple thymoma and thymoma combined with autoimmune disease

[0062] The tissue sections treated according to the method of Example 1 were stained with HE. The results are as follows: Figure 1 As shown, the left picture shows the HE staining of a simple thymoma (T) tissue section, and the right picture shows the HE staining of a thymoma (TMG) tissue section combined with autoimmune diseases. By comparing the two pictures, it can be seen that there are differences in the cell structure in the tumor tissues of patients with simple thymoma and thymoma combined with autoimmune diseases.

[0063] Example 3: Expression of AchR, Titin and MuSK antigens in tumor tissues of simple thymoma and thymoma combined with myasthenia gravis

[0064] According to the method of Example 2, quantum dot-antibody conjugates QDs-525-AchR, QDs-585-Titin and QDs-625-MuSK were prepared respectively, and the tissue sections obtained by the treatment of Example 1 were stained with QDs-525-AchR, QDs-585-Titin and QDs-625-MuSK in turn; the specific steps are as follows:

[0065] First round of dyeing:

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

[0067] B. Prepare antibodies: dilute the antibodies with 1×PBS and prepare AchR antibody solution labeled with QDs-525 quantum dots.

[0068] C. Primary antibody incubation: remove excess serum, add QDs-525-AchR quantum dot labeled antibody, place tissue sections flat in an immunohistochemistry humidified box, and then place in a 4°C refrigerator overnight.

[0069] D. Rinse: Rinse three times with a wash bottle containing 1×TBST, each time for 2 minutes.

[0070] Second round of dyeing:

[0071] A. Blocking: Remove 1× TBST solution, slowly add 10% BSA, and incubate at room temperature for 10 minutes.

[0072] B. Prepare antibodies: dilute the antibodies with 1×PBS and prepare QDs-585 quantum dot-labeled titin antibody solution.

[0073] C. Secondary antibody incubation: Remove excess serum, add QDs-585-Titin quantum dot labeled antibody, place tissue sections flat in an immunohistochemistry humidified box, and then place in a 4°C refrigerator overnight.

[0074] D. Rinse: Rinse three times with a wash bottle containing 1×TBST, each time for 2 minutes.

[0075] The third round of dyeing:

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

[0077] B. Prepare antibodies: dilute the antibodies with 1×PBS and prepare MuSK antibody solution labeled with QDs-625 quantum dots.

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

[0079] D. Rinse: Rinse three times with a wash bottle containing 1×TBST, each time for 2 minutes.

[0080] Nuclear DNA staining: 1×DAPI solution was prepared with ddH20 at a ratio of 1:100; 1×DAPI solution was added to the sliced ​​tissue, incubated at room temperature in the dark for 5 min, rinsed with 1×TBST for 2 min, and rinsed with ddH2O for 1 min; 30 μl of anti-fade sealing agent was added and the coverslip was covered, and the periphery was sealed with a special sealing agent; after the tissue slices were stabilized, a fluorescence microscope equipped with a 488nm laser was used to observe the results and take pictures, where the green light signal represents AchR, the orange light signal represents Titin, and the red light signal represents MuSK. Use imageJ to analyze the obtained pictures and set the picture format to 8-bit format; set the background, adjust the Threshold, and manually select the threshold to ensure that all stained areas are selected; set the measurement parameters and calculate the average gray value; obtain data, analyze it, and evaluate the immune status.

[0081] The results are as follows Figure 2 As shown in the figure, it can be seen that Achr, Titin, and Musk are distinguished by different colors when they are developed separately, and finally Merge is a combination of the three colors, which reflects the expression quantity and distribution of different antibodies in tissues. In simple thymoma tissue sections, Achr, Titin, and Musk are less in content, among which the average fluorescence intensity of Achr accounts for 59%, the average fluorescence intensity of Titin accounts for 47%, and the average fluorescence intensity of Musk accounts for 49%; in the sections of thymoma combined with MG group, it can be seen that Achr, Titin, and Musk are all expressed in large amounts, with the average fluorescence intensity of Achr accounting for 62%, the average fluorescence intensity of Titin accounting for 51%, and the average fluorescence intensity of Musk accounting for 55%; in simple thymoma tissue sections and thymoma combined with MG group sections, each antibody is positive, and the expression of each antibody in the sections of thymoma combined with MG group is higher. In the merged images of the two groups of sections, the relationship between the spatial positions of Achr, Titin, and Musk expression can also be observed according to different signal colors.

[0082] Example 4: Expression of AchR, LRP4 and RyR antigens in tumor tissues of simple thymoma and thymoma combined with myasthenia gravis

[0083] According to the method of Example 2, quantum dot-antibody conjugates QDs-525-AchR, QDs-585-LRP4 and QDs-625-RyR were prepared respectively, and the tissue sections obtained by the treatment of Example 1 were stained with QDs-525-AchR, QDs-585-LRP4 and QDs-625-RyR in turn; the specific steps are as follows:

[0084] First round of dyeing:

[0085] A. Blocking: Remove 1× TBST solution, slowly add 10% BSA, and incubate at room temperature for 10 minutes.

[0086] B. Prepare antibodies: dilute the antibodies with 1×PBS and prepare AchR antibody solution labeled with QDs-525 quantum dots.

[0087] C. Primary antibody incubation: remove excess serum, add QDs-525-AchR quantum dot labeled antibody, place tissue sections flat in an immunohistochemistry humidified box, and then place in a 4°C refrigerator overnight.

[0088] D. Rinse: Rinse three times with a wash bottle containing 1×TBST, each time for 2 minutes.

[0089] Second round of dyeing:

[0090] A. Blocking: Remove 1× TBST solution, slowly add 10% BSA, and incubate at room temperature for 10 minutes.

[0091] B. Prepare antibodies: dilute the antibodies with 1× PBS to prepare the LRP4 antibody solution labeled with QDs-585 quantum dots.

[0092] C. Secondary antibody incubation: Remove excess serum, add QDs-585-LRP4 quantum dot labeled antibody, place tissue sections flat in an immunohistochemistry humidified box, and then place in a 4°C refrigerator overnight.

[0093] D. Rinse: Rinse three times with a wash bottle containing 1×TBST, each time for 2 minutes.

[0094] The third round of dyeing:

[0095] A. Blocking: Remove 1× TBST solution, slowly add 10% BSA, and incubate at room temperature for 10 minutes.

[0096] B. Prepare antibodies: dilute the antibodies with 1× PBS and prepare the RyR antibody solution labeled with QDs-625 quantum dots.

[0097] C. Primary antibody incubation: Remove excess serum, add QDs-625-RyR quantum dot labeled antibody, place tissue sections flat in an immunohistochemistry humidified box, and then place in a 4°C refrigerator overnight.

[0098] D. Rinse: Rinse three times with a wash bottle containing 1×TBST, each time for 2 minutes.

[0099] Nuclear DNA staining: 1×DAPI solution was prepared with ddH20 at a ratio of 1:100; 1×DAPI solution was added to the sliced ​​tissue, incubated at room temperature in the dark for 5 min, rinsed with 1×TBST for 2 min, and rinsed with ddH2O for 1 min; 30 μl of anti-fade mounting agent was added and the coverslip was covered, and the periphery was sealed with a special edge sealing agent; after the tissue slices were stabilized, a fluorescence microscope equipped with a 488nm laser was used to observe the results and take pictures, where the green light signal represents AchR, the orange light signal represents LRP4, and the red light signal represents RyR. Use imageJ to analyze the obtained pictures and set the picture format to 8-bit format; set the background, adjust the Threshold, manually select the threshold, and ensure that all stained areas are selected; set the measurement parameters and calculate the average gray value; obtain data, analyze it, and evaluate the immune status.

[0100] Representative results are shown in Figure 3As shown in the figure, it can be seen that Achr, LRP4, and RyR are distinguished by different colors when they are colored separately, and finally Merge is a combination of the three colors, which reflects the expression quantity and distribution of different antibodies in tissues. In simple thymoma tissue sections, the contents of AchR, LRP4, and RyR are relatively low, with the average fluorescence intensity of AchR accounting for 59%, the average fluorescence intensity of LRP4 accounting for 43%, and the average fluorescence intensity of RyR accounting for 48%; in the sections of thymoma combined with MG group, it can be seen that AchR, LRP4, and RyR are all expressed in large amounts, with the average fluorescence intensity of AchR accounting for 62%, the average fluorescence intensity of LRP4 accounting for 47%, and the average fluorescence intensity of RyR accounting for 54%; in simple thymoma tissue sections and thymoma combined with MG group sections, each antibody is positive, and the expression of each antibody in the sections of thymoma combined with MG group is higher; in the merged images of the two groups of sections, the relationship between the spatial positions of AchR, LRP4, and RyR expression can also be observed according to different signal colors.

[0101] Example 5: AchR antigen expression in tumor tissues of simple thymoma and thymoma combined with myasthenia gravis

[0102] According to the method of Example 2, quantum dot-antibody conjugates QDs-525-AchR were prepared respectively, and the tissue sections obtained by the treatment of Example 1 were stained by QDs-525-AchR; the specific steps are as follows:

[0103] A. Blocking: Remove 1× TBST solution, slowly add 10% BSA, and incubate at room temperature for 10 minutes.

[0104] B. Prepare antibodies: dilute the antibodies with 1×PBS and prepare AchR antibody solution labeled with QDs-525 quantum dots.

[0105] C. Primary antibody incubation: remove excess serum, add QDs-525-AchR quantum dot labeled antibody, place tissue sections flat in an immunohistochemistry humidified box, and then place in a 4°C refrigerator overnight.

[0106] D. Rinse: Rinse three times with a wash bottle containing 1×TBST, each time for 2 minutes.

[0107] Nuclear DNA staining: 1×DAPI solution was prepared with ddH20 at a ratio of 1:100; 1×DAPI solution was added to the sliced ​​tissue, incubated at room temperature in the dark for 5 min, rinsed with 1×TBST for 2 min, and rinsed with ddH2O for 1 min; 30 μl of anti-fade mounting agent was added and the coverslip was covered, and the periphery was sealed with a special edge sealing agent; after the tissue slices were stabilized, a fluorescence microscope equipped with a 488nm laser was used to observe the results and take pictures, where the green light signal represents AchR. Use imageJ to analyze the obtained pictures, set the picture format to 8-bit format; set the background, adjust the Threshold, manually select the threshold, and ensure that all stained areas are selected; set the measurement parameters, calculate the average gray value; obtain data, analyze, and evaluate the immune status.

[0108] Representative results are shown in Figure 4 As shown, Figure 4 The left is the staining result of simple thymoma section. Figure 4 The right side shows the staining results of thymoma sections combined with autoimmune diseases. After QDs-525-AchR staining, the difference in AchR distribution between simple tumor sections and thymoma sections combined with autoimmune diseases can be clearly seen. The average fluorescence intensity of AchR in thymoma sections combined with autoimmune diseases is 62%.

[0109] 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 thymic tumor microenvironment antigen detection kit, characterized in that: Used for differential detection of simple thymoma, simple myasthenia gravis and thymoma combined with myasthenia gravis, including quantum dot-coupled antibody AchR, quantum dot-coupled antibody Titin, quantum dot-coupled antibody MuSK, quantum dot-coupled antibody LRP4 and quantum dot-coupled antibody RyR; When used, thymic tissue or thymoma tissue sections are prepared, and the tissue sections are stained in sequence with quantum dot-coupled antibodies AchR, quantum dot-coupled antibodies Titin, and quantum dot-coupled antibodies MuSK, and the distribution, content, and location information of the quantum dot-labeled antibodies in the tissue sections are observed by fluorescence microscopy; And / or, the tissue sections are stained in sequence using quantum dot-coupled antibody AchR, quantum dot-coupled antibody LRP4 and quantum dot-coupled antibody RyR, and the distribution, content and location information of the quantum dot-labeled antibodies in the tissue sections are observed by fluorescence microscopy.

2. The thymic tumor microenvironment antigen detection kit according to claim 1, characterized in that: Including quantum dot-conjugated antibodies QDs-525-AchR, QDs-585-Titin, QDs-625-MuSK, QDs-585-LRP4, and QDs-625-RyR.

3. The thymic tumor microenvironment antigen detection kit according to claim 1 or 2, characterized in that: Also included are antigen retrieval solution and nucleic acid staining solution.

4. The thymic tumor microenvironment antigen detection kit according to claim 1 or 2, characterized in that: 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 the quantum dot-labeled antibody solution that can be used for staining.

5. The thymic tumor microenvironment antigen detection kit according to claim 1, characterized in that: First, thymoma tissue sections are prepared, and antigen retrieval solution is used for antigen retrieval. The tissue sections are then stained in turn using antibodies labeled with different quantum dots. Then the cells are stained, and the fluorescence information of the tissue sections is collected. The presence and location of various antigens in the detected thymoma tissue are obtained based on the fluorescence expression level and spatial distribution information.

6. The thymic tumor microenvironment antigen detection kit according to claim 1, characterized in that: The positive result of the corresponding antibody test was determined by the average fluorescence intensity of quantum dots. The critical value of positive definition was that the average fluorescence intensity of QDs-525-AchR accounted for more than 55%, the average fluorescence intensity of QDs-585-Titin accounted for more than 43%, the average fluorescence intensity of QDs-625-MuSK accounted for more than 47%, the average fluorescence intensity of QDs-585-LRP4 accounted for more than 40%, and the average fluorescence intensity of QDs-625-RyR accounted for more than 45%.

7. The thymic tumor microenvironment antigen detection kit according to claim 1, characterized in that: Tissue sections were stained in the order of QDs-525-AchR, QDs-585-Titin, and QDs-625-MuSK; And / or, the tissue sections are stained in the order of QDs-525-AchR, QDs-585-LRP4 and QDs-625-RyR.

Images