Detection method for full-automatic multiplex fluorescent staining of EBER, CD3 and CD20 and application of detection method

Abstract

The invention discloses a full-automatic multiplex fluorescent staining detection method for EBER, CD3 and CD20 and application of the full-automatic multiplex fluorescent staining detection method, and belongs to the technical field of biomarker detection. The invention relates to an EBER, CD3 and CD20 detection method, which comprises: carrying out digestion treatment on a sample to be detected, adding an EBER probe, and sequentially adding a primary antibody, a secondary antibody and a fluorescent dye A so as to complete color development; sequentially adding a CD3 antibody, an HRP polymer and a fluorescent dye B into the to-be-detected sample to finish CD3 dyeing; sequentially adding a CD20 antibody, an HRP polymer, a fluorescent dye C and a DAPI dye into a sample to be detected to finish CD20 dyeing; and observing or counting the dyeing result of the to-be-detected sample through fluorescence scanning equipment.

Description

Technical Field

[0001] The present invention relates to the technical field of biomarker detection, and specifically relates to a detection method and application for full-automatic multiplex fluorescence staining of EBER, CD3, and CD20. Background Art

[0002] EBER is a small nuclear RNA encoded by Epstein-Barr Virus (EBV), which is widely present in EBV-infected lymphocytes. EBV is closely related to the occurrence of Hodgkin lymphoma and certain non-Hodgkin lymphomas. Therefore, the detection of EBER can help identify these lymphoma types and provide diagnostic support.

[0003] CD3 is a T cell surface marker, which is widely used to identify the presence of T cells. T cell lymphoma is an important type of lymphoma. The expression of CD3 helps to determine the cell origin of the tumor and provides a basis for the classification of lymphoma.

[0004] CD20 is a specific marker, which is mainly expressed in B cells and their related lymphomas. The detection of CD20 not only helps in the diagnosis of B cell lymphoma, but also can guide targeted therapy using anti-CD20 monoclonal antibodies (such as rituximab). Therefore, the detection of CD20 has important application value in clinical practice.

[0005] The detection of the above-mentioned markers has the following technical problems:

[0006] 1. When using traditional techniques to detect CD3, CD20, and EBER, only one or a few antigens can be detected in one experiment. To detect all three markers, multiple independent experiments are required, which is cumbersome and may lead to poor result comparability due to differences in experimental conditions. Moreover, traditional techniques have a certain role in the localization and observation of single or a few antigens, but they are limited in demonstrating the spatial relationship between multiple antigens and cell-cell interactions, and it is difficult to comprehensively present the complex tissue microenvironment information.

[0007] 2. The sensitivity of traditional detection methods is relatively limited, and it may be difficult to accurately detect antigens with low expression or trace expression, and false negative results are likely to occur. When expressed at a low level in early lymphoma cells, it may be missed due to weak signals.

[0008] 3. The results of traditional detection methods are usually observed through an optical microscope and semi-quantitatively analyzed based on the color depth and the proportion of positive cells, etc. The subjectivity is strong, and there may be differences in judgment between different observers, and it is difficult to perform precise quantitative analysis. When judging the proliferation index of tumor cells, there may be deviations in the counting of positive cells and the evaluation of staining intensity by different pathologists. Summary of the Invention

[0009] In view of the deficiencies of the prior art, the present invention provides a detection method and application for full-automatic multiplex fluorescence staining of EBER, CD3, and CD20.

[0010] The object of the present invention can be achieved by the following technical solutions:

[0011] In a first aspect of the present invention, there is provided a detection method for EBER, CD3, and CD20, comprising the following steps:

[0012] After digestion and treatment of the sample to be tested, an EBER probe is added, followed by the sequential addition of a primary antibody, a secondary antibody, and fluorescent dye A to complete color development.

[0013] A CD3 antibody, an HRP polymer, and fluorescent dye B are sequentially added to the sample to be tested to complete CD3 staining.

[0014] A CD20 antibody, an HRP polymer, fluorescent dye C, and DAPI dye are sequentially added to the sample to be tested to complete CD20 staining.

[0015] The staining results of the sample to be tested are observed or counted by a fluorescence scanning device.

[0016] Among them, the fluorescent dye A, fluorescent dye B, and fluorescent dye C have different colors.

[0017] Optionally, the primary antibody and the secondary antibody are an anti-digoxin antibody and an enzyme-labeled goat anti-mouse IgG polymer, respectively.

[0018] Optionally, the EBER probe is:

[0019] Optionally, the CD3 antibody and the CD20 antibody are:

[0020] Optionally, after the addition of the EBER probe, the primary antibody, and / or the secondary antibody, soaking and rinsing are carried out in PBS; after the addition of the CD3 antibody, the CD20 antibody, the HRP polymer, fluorescent dye B, and / or fluorescent dye C, soaking and rinsing are carried out in PBS.

[0021] In a second aspect of the present invention, there is provided a detection system for detecting EBER, CD3, and CD20, comprising:

[0022] An EBER color development reagent, comprising: an EBER probe, a primary antibody, a secondary antibody, and fluorescent dye A;

[0023] A CD3 staining reagent, comprising: a CD3 antibody, an HRP polymer, and fluorescent dye B;

[0024] A CD20 staining reagent, comprising: a CD20 antibody, an HRP polymer, fluorescent dye C, and DAPI dye;

[0025] and a fluorescence scanning device for observing or counting the staining results;

[0026] Among them, the fluorescent dyes A, B, and C have different colors.

[0027] Optionally, the primary antibody and the secondary antibody are respectively an anti-digoxin antibody and an enzyme-labeled sheep anti-mouse IgG polymer.

[0028] Optionally, the EBER probe is:

[0029] The CD3 antibody and the CD20 antibody are respectively:

[0030] Optionally, the fluorescence scanning device is a multiplex immunofluorescence digital pathology scanner.

[0031] The third aspect of the present invention relates to the application of the above detection method and the above detection system in the preparation of a detection product for detecting lymphoma.

[0032] In the present invention, the detection methods for EBER, CD3, and CD20 can also be used for non-diagnostic purposes. For example, for scientific research purposes, to identify mouse or other animal models, and to evaluate the status and classification of lymphoma in patients in clinical trials, etc.

[0033] Advantages of the present invention:

[0034] Compared with the prior art, the present invention can simultaneously detect the markers EBER, CD3, and CD20. This method can provide more comprehensive lymphoma cell characteristics, help pathologists more accurately classify lymphoma types, and thus formulate personalized treatment plans. Specifically:

[0035] 1. By performing multiplex fluorescence staining on the same section, the spatial relative position information of the markers can be displayed, providing a unique diagnostic advantage for the diagnosis of lymphoma.

[0036] 2. Quantitative analysis can be performed more accurately. In the diagnosis of lymphoma, simultaneously detecting the markers CD3, CD20, and EBER of tumor cells, and combining the expression levels and distribution of the three, provides a more accurate basis for tumor grading, staging, and the selection of treatment plans. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The present invention will be further described below with reference to the accompanying drawings.

[0038] Figure 1 It is a schematic diagram of the multiplex immunofluorescence scanning result in Example 1 of this application;

[0039] Figure 2 It is a schematic diagram of the multiplex immunofluorescence scanning result in Example 2 of this application. Detailed implementation manners

[0040] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0041] Embodiment 1:

[0042] Manual operation.

[0043] 1) Sample preparation: Select a paraffin block of lymphoma tissue with clear positive expression of CD20, CD3, and EBER, then section and dewax and hydrate.

[0044] 2) Tissue pretreatment

[0045] a. EDTA repair: Boil for 5 - 20 min and cool naturally.

[0046] b. Cool the repair solution and sections to room temperature with running water and rinse with purified water.

[0047] c. Remove excess liquid, add digestive enzyme (about 100 uL), incubate at room temperature for 5 - 10 min, and rinse with purified water.

[0048] d. Remove excess liquid, add EBER probe (product number: DC004 from Xiamen Longjin Biotechnology Co., Ltd.), and cover with a coverslip.

[0049] e. Place the sections in a hybridization instrument or incubation box and incubate at 37 °C for 30 - 50 min.

[0050] f. Soak the sections in PBS at room temperature for 3 - 5 min and carefully remove the coverslip.

[0051] g. Soak and rinse the sections in fresh PBS at 48 °C for 1 - 5 min.

[0052] 3) Color development

[0053] ① Remove excess liquid, add mouse anti - digoxin antibody, and incubate in a humid box at 37 °C for 10 - 15 min.

[0054] ② Soak and rinse in PBS at room temperature for 2 min × 3 times.

[0055] ③ Remove excess liquid, add enzyme - labeled goat anti - mouse IgG polymer, and incubate in a humid box at room temperature for 3 - 5 min.

[0056] ④ Soak and rinse in PBS at room temperature for 2 min × 3 times.

[0057] ⑤ Remove the excess liquid, add the fluorescent dye dropwise, incubate in a humid box at room temperature for 1 - 5 min, and rinse thoroughly with PBS;

[0058] 4) CD3 Staining

[0059] ① Repair with EDTA by boiling gently for 10 - 15 min, and let the repair solution and the sections cool to room temperature with running water;

[0060] ② Remove the excess liquid, add block dropwise, incubate at room temperature for 10 min, and soak and rinse with PBS for 2 min × 3 times;

[0061] ③ Remove the excess liquid, add the CD3 antibody reagent (product number: R50264 from Chengdu Zhengneng Biotechnology Co., Ltd.), incubate at room temperature for 10 - 15 min, and soak and rinse with PBS for 2 min × 3 times;

[0062] ④ Remove the excess liquid, add the HRP polymer, incubate at room temperature for 10 min, and soak and rinse with PBS for 2 min × 3 times;

[0063] ⑤ Remove the excess liquid, add the fluorescent dye dropwise, incubate at room temperature for 1 - 5 min, and soak and rinse with PBS for 2 min × 3 times;

[0064] 5) CD20 Staining

[0065] ① Repair with EDTA by boiling gently for 10 - 15 min, and let the repair solution and the sections cool to room temperature with running water;

[0066] ② Remove the excess liquid, add block dropwise, incubate at room temperature for 10 min, and soak and rinse with PBS for 2 min × 3 times;

[0067] ③ Remove the excess liquid, add the CD20 antibody reagent (product number: BX50134 from Bailing Biotechnology Co., Ltd.), incubate at room temperature for 10 - 15 min, and soak and rinse with PBS for 2 min × 3 times;

[0068] ④ Remove the excess liquid, add the HRP polymer, incubate at room temperature for 10 min, and soak and rinse with PBS for 2 min × 3 times;

[0069] ⑤ Remove the excess liquid, add the fluorescent dye dropwise, incubate at room temperature for 1 - 5 min, and soak and rinse with PBS for 2 min × 3 times;

[0070] ⑥ Remove the excess liquid, add DAPI staining solution for nuclear counterstaining for observation and comparison, incubate at room temperature for 1 - 3 min, soak and rinse with PBS for 2 min × 3 times, then soak with purified water for 2 min, and mount the slides;

[0071] 6) Imaging and Result Interpretation and Analysis:

[0072] When selecting a multi-immunofluorescence scanning device, it is necessary to use the digital pathology scanner (KR-HT5, Jiangsu KuoRan Biopharmaceutical Technology Co., Ltd., Jiangsu Medical Device Registration No. 20232221657) produced by Jiangsu KuoRan Biopharmaceutical Technology Co., Ltd. to image the stained tissue sections under fluorescence and interpret and analyze the imaging pictures. The results are as Figure 1 shown.

[0073] CD3( Figure 1 showing red fluorescence signal in it): The positive expression level is 78.5%;

[0074] CD20( Figure 1 showing green fluorescence signal in it): The positive expression level is 21.9%;

[0075] EBER( Figure 1 showing yellow fluorescence signal in it): The positive expression level is 68.7%.

[0076] Example 2:

[0077] In this example, the fully automatic immunohistochemical staining machine Krast 300 (Jiangsu Medical Device Preparation No. 20230100) or Krast600 (Jiangsu Medical Device Preparation No. 20230101) produced by Jiangsu KuoRan Biopharmaceutical Technology Co., Ltd. is mainly used for staining and imaging. The specific steps are as follows:

[0078] Prepare before loading the machine. Select a lymphoma tissue wax block with clear positive expression of CD20, CD3, and EBER, and then section it.

[0079] 1) Turn on and connect the fully automatic immunohistochemical staining machine

[0080] Open the main power switch on the side of the instrument host, turn on the computer host, monitor, and label printer. Enter the host login interface, input the username and password to log in, and enter the main interface.

[0081] 2) Check before operation

[0082] a) Check all reagents in large quantities to confirm that the installed reagents are correct and sufficient, and the system does not prompt insufficient reagents;

[0083] b) Check the high-concentration waste liquid bottle to confirm that there is enough space for the experiment and the system does not prompt the need to process the waste liquid;

[0084] c) Check the low-concentration waste liquid bucket to confirm that there is enough space for the experiment and the system does not prompt the need to process the waste liquid.

[0085] 3) Enter reagent information

[0086] a) When used for the first time, click on Reagent Management - Settings - Add in sequence, edit and add the reagent information to be used. Among them, the antibody reagent type and EBER probe reagent type are "Open primary antibody", the fluorescent dye type is "Dye", and the blocking diluent, secondary antibody, and DAPI fluorescent dye types are "Auxiliary reagents" (multiplex detection system). After entering the reagent information, click OK, and the reagent will appear in the reagent list. Among them, the EBER probe used in this example is from Xiamen Longjin Biotechnology Co., Ltd. (product number: DC004), the CD3 antibody reagent is from Chengdu Zhengneng Biotechnology Co., Ltd. (product number: R50264), and the CD20 antibody reagent is from Bailing Biotechnology Co., Ltd. (product number: BX50134);

[0087] b) Click on Input and Query, scan the barcode on the bottle body of the new open reagent bottle, select the reagent name, and then click "OK" to complete the input;

[0088] c) Enter all the required reagent bottles in the above way in sequence;

[0089] d) If not used for the first time, use the reagent bottles with the already entered information.

[0090] 4) Case and Slide Settings

[0091] a) Click on Slide Settings - Add Case, enter the case details (Case ID is a required item), and the default wax removal is set for the preparation procedure. You can select baking and wax removal according to your needs;

[0092] b) Select the corresponding case, and then click "Add Slide" to add slide annotations. Select the staining method as "Multiplex", click "+" to increase the number of experimental rounds, select the markers, select the dyes, select the corresponding experimental procedures (you can use the default staining procedure or customize the staining procedure). After each round of settings is completed, click "Add Slide". Repeat this operation to add more slides;

[0093] 5) Upload Slides and Reagents

[0094] a) Click on "Print Label" in the lower right corner of the page, print the label, and stick the label on the upper end of the slide;

[0095] b) Place the slide on the slide rack, and place the liquid cover film on the slide (the slide and the liquid cover film need to be aligned with the card slots);

[0096] c) Push the slide rack horizontally into the machine;

[0097] d) Press the "Reaction Chamber" button to confirm that all slides have been recognized;

[0098] e) Load all the required reagents (in reagent bottles) onto the reagent carrier, and then load the reagent carrier onto the instrument. Confirm that all reagents have been recognized.

[0099] 6) Start the staining process and monitor the operation status

[0100] a) Click the button to start the staining process;

[0101] b) Delayed start: If the experimenter wishes to start the experiment at a certain time in the future, after getting ready, select the slide in the corresponding reaction chamber, right-click the mouse and select delayed start;

[0102] c) Monitor the staining status and the staining process until the staining is completed;

[0103] d) When the green indicator light of the instrument button flashes, press the button to unload the slide.

[0104] 7) Cleaning: After the program runs to completion, take out the slide and place it in the staining tank. Wash it 3 times with purified water for 1 minute each time.

[0105] 8) Sealing the slide: Gently shake off the water on the slide, add a drop of anti-quenching mounting medium at the tissue area, gently touch one side of the coverslip and then put it down. Wait until it fully unfolds and spreads without bubbles.

[0106] 3. Imaging and result analysis:

[0107] Select a multi-immunofluorescence scanning device to image the stained tissue section under fluorescence, and interpret and analyze the imaging pictures. The results are as Figure 2 shown.

[0108] CD3 ( Figure 2 shows a red fluorescence signal in it): The positive expression level is 83.2%;

[0109] CD20 ( Figure 2 shows a green fluorescence signal in it): The positive expression level is 45.0%;

[0110] EBER ( Figure 2 shows a yellow fluorescence signal in it): The positive expression level is 59.4%.

[0111] In the description of this specification, the descriptions referring to terms such as "one embodiment", "example", "specific example", etc. mean that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0112] The basic principles, main features and advantages of the present invention have been shown and described above. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements fall within the scope of the present invention claimed.

Claims

1. A detection method for EBER, CD3 and CD20, characterized in that Comprising the following steps: After the test sample is digested and processed, add the EBER probe, then sequentially add the primary antibody, the secondary antibody, and the fluorescent dye A to complete the color development; Sequentially add the CD3 antibody, the HRP polymer, and the fluorescent dye B to the test sample to complete the CD3 staining; Sequentially add the CD20 antibody, the HRP polymer, the fluorescent dye C, and the DAPI dye to the test sample to complete the CD20 staining; Observe or count the staining results of the test sample through a fluorescence scanning device; Wherein, the fluorescent dye A, the fluorescent dye B, and the fluorescent dye C have different colors.

2. The detection method of EBER, CD3 and CD20 according to claim 1, characterized in that The primary antibody and the secondary antibody are respectively an anti-digoxin antibody and an enzyme-labeled goat anti-mouse IgG polymer.

3. The detection method of EBER, CD3 and CD20 according to claim 1, characterized in that The EBER probe is: the probe with the product number DC004 of Xiamen Longjin Biotechnology Co., Ltd.

4. The detection method of EBER, CD3 and CD20 according to claim 1, wherein The CD3 antibody and the CD20 antibody are respectively: the antibody with the product number R50264 of Chengdu Zhengneng Biotechnology Co., Ltd. and the antibody with the product number BX50134 of Bailing Biotechnology Co., Ltd.

5. The detection method of EBER, CD3 and CD20 according to claim 1, characterized in that, After adding the EBER probe, the primary antibody and / or the secondary antibody, soak and rinse in PBS; after adding the CD3 antibody, the CD20 antibody, the HRP polymer, the fluorescent dye B and / or the fluorescent dye C, soak and rinse in PBS.

6. A detection system for detecting EBER, CD3 and CD20, characterized in that, Including: The EBER color development reagent, including: the EBER probe, the primary antibody, the secondary antibody, and the fluorescent dye A; The CD3 staining reagent, including: the CD3 antibody, the HRP polymer, and the fluorescent dye B; The CD20 staining reagent, including: the CD20 antibody, the HRP polymer, the fluorescent dye C, and the DAPI dye; And, a fluorescence scanning device for observing or counting the staining results; Wherein, the fluorescent dye A, the fluorescent dye B, and the fluorescent dye C have different colors.

7. The detection system for detecting EBER, CD3 and CD20 according to claim 6, wherein The primary antibody and the secondary antibody are respectively an anti-digoxin antibody and an enzyme-labeled goat anti-mouse IgG polymer.

8. The detection system for detecting EBER, CD3 and CD20 according to claim 6, wherein The EBER probe is: the probe with the product number DC004 of Xiamen Longjin Biotechnology Co., Ltd.; The CD3 antibody and the CD20 antibody are respectively: the antibody with the product number R50264 of Chengdu Zhengneng Biotechnology Co., Ltd. and the antibody with the product number BX50134 of Bailing Biotechnology Co., Ltd.

9. The detection system for detecting EBER, CD3 and CD20 according to claim 6, characterized in that, The fluorescence scanning device is a multi-immunofluorescence digital pathology scanner.

10. Use of the detection method according to any one of claims 1 to 5 and the detection system according to any one of claims 6 to 9 in the preparation of a detection product for detecting lymphoma.

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