A double-staining immunohistochemistry kit and its application

Abstract

The present invention provides a double-staining immunohistochemistry kit and its application, which relates to the field of immunohistochemistry. Among them, the double-staining immunohistochemistry kit includes: a fixative, a membrane-breaking agent, a primary antibody reagent, a secondary antibody reagent, and a staining agent. Among them, the primary antibody reagent includes a LEF1 antibody and a CD20 antibody. The secondary antibody reagent includes a secondary antibody polymer labeled with alkaline phosphatase AP and a secondary antibody polymer labeled with horseradish peroxidase HRP. The kit can obtain stained sample cells with complete cell morphology, has a simple process, can effectively improve the recognition rate of cell morphology, and improve the diagnostic accuracy rate.

Description

Technical Field

[0001] The present invention relates to the field of immunohistochemistry, and particularly to a double-staining immunohistochemistry kit and its application. Background Art

[0002] Chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL) is one of the common lymphoproliferative diseases in adult patient populations, manifested as an increase in monoclonal B lymphocytes in peripheral blood or bone marrow. The diagnosis of CLL mainly relies on immunophenotyping and immunohistochemistry. Most CLL patients' cells express CD5 and B-cell antigens CD19, CD20, and CD23. The typical CLL immunophenotype is CD5+, CD23+, CD43+ / -, CD10-, CD19+, CD20 dim (weak expression of CD20), sIg dim (weak expression of sIg). Some CLL patients may show sIg bright (strong expression of sIg), CD23- / dim, and weakly positive for FMC7.

[0003] Currently, the clonality of B cells is mainly confirmed by staining bone marrow biopsy specimens. However, it has been found that mantle cell lymphoma (MCL) has an immunophenotype of CD5+ and CD20 expression. Sometimes, MCL patients may also show an immunophenotype similar to that of CLL. Therefore, new diagnostic markers are needed to achieve the differential diagnosis between CLL / SLL and MCL.

[0004] In addition, during the diagnosis of CLL / SLL, bone marrow biopsy specimens need to be stained. Since there is a large amount of bone in bone marrow biopsy specimens, they cannot be directly sectioned. Decalcification treatment needs to be carried out using weak or strong acids, and these solvents can cause certain damage to antigens, easily resulting in false positives and false negatives, leading to inaccurate results. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a double-staining immunohistochemistry kit and its application.

[0006] The present invention solves its technical problems by adopting the following technical solutions.

[0007] In a first aspect of the present invention, a double-staining immunohistochemistry kit is provided, including: a fixing solution, a membrane-breaking agent, a primary antibody reagent, a secondary antibody reagent, and a staining agent. Among them, the primary antibody reagent includes a LEF1 antibody and a CD20 antibody; the secondary antibody reagent includes a secondary antibody polymer labeled with alkaline phosphatase AP and a secondary antibody polymer labeled with horseradish peroxidase HRP.

[0008] In an exemplary embodiment of the present invention, the LEF1 antibody and the CD20 antibody are derived from two different species of animals.

[0009] In an exemplary embodiment of the present invention, the alkaline phosphatase (AP)-labeled secondary antibody polymer is used to bind to a primary antibody derived from an animal of a first species, and the horseradish peroxidase (HRP)-labeled secondary antibody polymer is used to bind to a primary antibody derived from an animal of a second species, wherein the first species is different from the second species.

[0010] In an exemplary embodiment of the present invention, the first species and the second species are respectively consistent with the animal species of the LEF1 antibody and the CD20 antibody.

[0011] In an exemplary embodiment of the present invention, the stain includes a peroxidase substrate staining solution, an alkaline phosphatase substrate staining solution, and a counterstain staining solution, and the color development of the counterstain staining solution does not mask the color development of the peroxidase substrate staining solution and the alkaline phosphatase substrate staining solution.

[0012] In an exemplary embodiment of the present invention, the fixing solution is a mixed solution of methanol and formaldehyde.

[0013] In an exemplary embodiment of the present invention, the double-staining immunohistochemistry kit further includes a protein blocker, which is selected from one or more of bovine serum albumin buffer solution, goat serum buffer solution, horse serum buffer solution, and casein buffer solution.

[0014] In an exemplary embodiment of the present invention, the permeabilizing agent is selected from one or more of saponin, Triton-X, Tween-20, aldehydes, alcohols, ketones, sodium chloride, calcium chloride, sodium benzoate, disodium ethylenediaminetetraacetate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium benzoate, nonylphenol polyoxyethylene ether, Triton X-100, Triton-X, and Tween-20, and is a PBS solution mixed therewith.

[0015] In an exemplary embodiment of the present invention, the double-staining immunohistochemistry kit is used for staining peripheral blood smears, bone marrow smears, and cell smears.

[0016] The second aspect of the present invention provides a staining method based on double staining of LEF1 / CD20, which uses the double-staining immunohistochemistry kit described in any one of the above.

[0017] In an exemplary embodiment of the present invention, the staining method includes:

[0018] Step S1: Drop the fixing solution on the smear for fixation to obtain a sample;

[0019] Step S2: Drop the protein blocker on the sample and incubate to obtain a blocked sample;

[0020] Step S3: Drop a permeabilizing agent onto the blocking sample and incubate for a period of time;

[0021] Step S4: Add the primary antibody reagent, incubate, and then wash;

[0022] Step S5: Add the secondary antibody reagent, incubate, and then wash;

[0023] Step S6: Drop peroxidase substrate staining solution, alkaline phosphatase substrate staining solution, and hematoxylin staining solution for staining treatment to obtain a stained sample.

[0024] In an exemplary embodiment of the present invention, in Step S6, the steps of the staining treatment include: first drop horseradish peroxidase substrate staining solution, incubate and wash, then drop alkaline phosphatase substrate staining solution, incubate and wash, and finally drop counterstain staining solution, incubate and wash to obtain a stained sample; or,

[0025] First drop alkaline phosphatase substrate staining solution, incubate and wash, then drop horseradish peroxidase substrate staining solution, incubate and wash, and finally drop counterstain staining solution, incubate and wash to obtain a stained sample.

[0026] The third aspect of the present invention provides the application of the double-staining immunohistochemistry kit as described in any one of the above in the diagnosis of chronic lymphocytic leukemia.

[0027] Compared with the prior art, the beneficial effects of the double-staining immunohistochemistry kit, staining method, and application of the present invention are:

[0028] The double-staining immunohistochemistry kit provided by the present invention can be applied to the auxiliary diagnosis of chronic lymphocytic leukemia, effectively improving the diagnostic accuracy. For CLL, both LFE1 (lymphocyte enhancer factor 1) and CD20 are expressed on monoclonal B lymphocytes. For MCL, since LEF1 is mainly expressed on CD3-positive T lymphocytes and rarely appears positive on CD20-positive monoclonal B lymphocytes. By performing double staining of LEF1 / CD20 in the present invention, the expression of LEF1 is stained by nuclear staining, and the expression of CD20 is stained by cytoplasmic staining. Using two different colors for positive double staining to identify the positions of the two types of cells can more clearly identify abnormal expression of B lymphocytes, exclude the interference of T cells, and achieve differential diagnosis of CLL and MCL. The double-staining immunohistochemistry kit uses double staining with LEF1 antibody and CD20 antibody, has strong specificity, and the staining method is simple and easy to implement, and can realize the whole staining process through an automated staining device. The present invention obtains clear staining results and the cell morphology is easy to distinguish.

[0029] In addition, the staining method of this embodiment can be applied to peripheral blood smears, with a wide range of specimen applicability. There is no need for pretreatment processes such as decalcification, effectively avoiding antigen damage and reducing the occurrence of false positives and false negatives. The sample is fixed with a fixative, and the cell morphology has a high degree of integrity. Brief Description of the Drawings

[0030] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0031] Figure 1 It is a flowchart of the staining method provided by the embodiment of the present invention;

[0032] Figure 2 It is a diagram of the staining result of sample A provided by Embodiment 1 of the present invention;

[0033] Figure 3 It is a diagram of the staining result of sample B provided by Embodiment 1 of the present invention;

[0034] Figure 4 It is a diagram of the staining result of sample C provided by Embodiment 1 of the present invention. Detailed Description of the Embodiments

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase. It should be noted that unless otherwise defined, all scientific or technical professional terms in this disclosure are in line with the general understanding of most ordinary people in the field. The following are the general definitions of most professional terms in the field in the literature, and the professional terms used in this patent are consistent with the descriptions of these professional terms in the above literature.

[0036] The double-staining immunohistochemistry kit, staining method, and application of the embodiments of the present disclosure will be specifically described below.

[0037] The double-staining immunohistochemistry kit provided by the embodiments of the present disclosure includes: a fixative, a membrane-breaking agent, a primary antibody reagent, a secondary antibody reagent, and a staining agent.

[0038] In the embodiments of the present invention, the double-staining immunohistochemistry kit further includes a protein blocker. The protein blocker can be selected, for example, from bovine serum albumin (BSA), etc. During the immunohistochemical staining process, there may be some interfering substances in the sample, such as endogenous antibodies, other binding proteins, autoantibodies, etc. The interfering substances can interfere with the reaction between the antibody and the analyte through non-specific binding, resulting in false positives or false negatives. The protein blocker can effectively block the interference that appears in the test and improve the test accuracy. In one embodiment, the protein blocker can further include a stabilizer, a surfactant, etc. The stabilizer can be, for example, sodium chloride, and the surfactant can be, for example, Tween 20, etc.

[0039] Further preferably, in other embodiments, the protein blocker can be selected from one or more of bovine serum albumin buffer solution, goat serum buffer solution, horse serum buffer solution, and casein buffer solution. Specifically, the protein blocker can be, for example, a bovine serum albumin buffer solution or a mixture of a bovine serum albumin buffer solution and a casein buffer solution. The protein blocker can also be a commercially available product, and the present disclosure does not make specific limitations.

[0040] In a preferred embodiment of the present disclosure, the fixing solution is a mixed solution of methanol and formaldehyde. Specifically, the fixing solution is a mixed solution of methanol and formaldehyde with a mass ratio of 1:0.5 to 2. More preferably, the fixing solution is a mixed solution of methanol and formaldehyde with a mass ratio of 1:0.9 to 1.1. For example, in a preferred embodiment of the present disclosure, the fixing solution is methanol and formaldehyde with a mass ratio of 1:1. This fixing solution can effectively fix the cells on the premise of ensuring the antigen activity, improve the integrity of the cell morphology, and is beneficial to the subsequent staining process and the recognition of the cell morphology after staining.

[0041] In a preferred embodiment of the present invention, the permeabilizing agent is selected from one or more of saponin, Triton-X, and Tween-20, aldehydes, alcohols, ketones, sodium chloride, calcium chloride, sodium benzoate, disodium ethylenediaminetetraacetate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium benzoate, nonylphenol polyoxyethylene ether, Triton X-100, Triton-X, and Tween-20 in a mixed PBS solution. By applying the permeabilizing agent, a perforation operation is performed on the cell membrane, so that the subsequent antibody can enter the cell to bind to the antigen.

[0042] Further preferably, the permeabilizing agent is preferably a BD permeabilizing agent. For example, a commercially available BD Phosflow TM Perm Buffer II permeabilizing agent (product number: 558052) or BD Phosflow TM Perm Buffer II permeabilizing agent (product number: 558050), etc.

[0043] In the embodiments of the present invention, the primary antibody reagent includes a LEF1 antibody and a CD20 antibody. Specifically, the LEF1 antibody and the CD20 antibody are derived from animals of two different species. For example, in one embodiment, the LEF1 antibody is a rabbit anti-human LEF1 monoclonal antibody (clone number BP6113, Tulin (Hangzhou) Biotechnology Co., Ltd.). In one embodiment, the CD20 antibody is a mouse monoclonal antibody (CD20 mouse Monoclonal Antibody, clone number L26) (Beyotime Biotechnology).

[0044] It should be noted that in other embodiments, it may also be that LFE1 is a mouse monoclonal antibody and CD20 is a rabbit monoclonal antibody. The LEF1 antibody and the CD20 antibody in this embodiment can also be other commercial products, as long as one of the LEF1 antibody and the CD20 antibody is rabbit anti and the other is mouse anti. The present invention does not make specific restrictions.

[0045] In the embodiments of the present invention, the secondary antibody reagent includes a secondary antibody polymer labeled with alkaline phosphatase AP and a secondary antibody polymer labeled with horseradish peroxidase HRP. Specifically, the secondary antibody polymer labeled with alkaline phosphatase AP is used to bind to the primary antibody derived from the first species of animal, and the secondary antibody polymer labeled with horseradish peroxidase HRP is used to bind to the primary antibody derived from the second species of animal, and the first species of animal is different from the second species of animal.

[0046] For example, in one embodiment, commercial products such as an alkaline phosphatase AP-labeled affinity-purified goat anti-rabbit IgG (H+L) secondary antibody and an HRP-labeled goat anti-mouse IgG F(ab')2 secondary antibody produced by AAT Bioquest can be selected.

[0047] It should be noted that the secondary antibody polymer labeled with alkaline phosphatase AP and the secondary antibody polymer labeled with horseradish peroxidase HRP can also be other commercial products, for example, one of them is an anti-rabbit polymer and the other is an anti-mouse polymer.

[0048] In the embodiments of the present invention, the secondary antibody polymer labeled with alkaline phosphatase AP and the secondary antibody polymer labeled with horseradish peroxidase HRP are respectively consistent with the animal species of the LEF1 antibody and the CD20 antibody. For example, if LEF1 is mouse anti and the CD20 antibody is rabbit anti, the secondary antibody polymer labeled with alkaline phosphatase AP is an anti-mouse polymer, and the secondary antibody polymer labeled with horseradish peroxidase HRP is an anti-rabbit polymer.

[0049] During the immunohistochemical detection process, the primary antibody reagent specifically binds to the target protein (antigen), and the secondary antibody reagent recognizes the primary antibody to provide a signal amplification effect for the detection.

[0050] In a preferred embodiment of the present invention, the staining agent includes a peroxidase substrate staining solution, an alkaline phosphatase substrate staining solution and a counterstaining solution. Preferably, in the given embodiment, the peroxidase substrate staining solution can be selected from DAB staining solution, and the alkaline phosphatase substrate staining solution can be selected from Fast Red staining solution. The color development of the counterstaining solution does not mask the color development of the peroxidase substrate staining solution and the alkaline phosphatase substrate staining solution.

[0051] In one embodiment, the DAB staining solution can be obtained through commercial products, and its components include DAB (3,3-diaminobenzidine tetrahydrochloride) solution and H2O2. The secondary antibody polymer labeled with horseradish peroxidase HRP can catalyze the decomposition of hydrogen peroxide in the DAB staining solution into highly active hydroxyl radical molecules, oxidize diaminobenzidine, and form a brown-yellow precipitate, thereby achieving positioning.

[0052] In one embodiment, the fast red staining solution can be obtained through commercial products, and its components include alkaline phosphatase substrate solution and fast red solution. Specifically, the alkaline phosphatase substrate solution is fast red (fast red), and the fast red solution includes naphthol phosphate and trishydroxymethylaminomethane. During the staining process, the secondary antibody polymer labeled with alkaline phosphatase (AP) can catalyze the hydrolysis of the phosphate group of the alkaline phosphatase substrate to form phosphate-free naphthol, which reacts chemically with the fast red solution to generate an insoluble red product precipitate at the location of the alkaline phosphatase, thereby achieving positioning.

[0053] In one embodiment, the counterstaining solution is a hematoxylin staining solution or a methyl green staining solution. The hematoxylin staining solution and the methyl green staining solution can be obtained through commercial products. The cell nucleus is stained blue-purple by the hematoxylin staining solution, or the cell nucleus is stained green by the methyl green staining solution. The cytoplasm, fibers, etc. are not stained, which has the effect of setting off the overall cell distribution and tissue morphology, making it easier to observe and identify.

[0054] In an embodiment of the present invention, the above-mentioned double staining immunohistochemistry kit can be used to stain peripheral blood smears, bone marrow smears, and cell smears, and the staining results are clear and have a wide range of applications. More preferably, the double staining immunohistochemistry kit is applied to bone marrow peripheral blood smears, and bone marrow peripheral blood smears do not need to undergo a complex pre-processing process, which greatly saves pre-processing time and avoids damage to cells caused by treatments such as decalcification.

[0055] like Figure 1 As shown, an embodiment of the present invention further provides a staining method based on LEF1 / CD20 double staining, using the double staining immunohistochemistry kit as described above. Specifically, in an embodiment of the present invention, the staining method includes:

[0056] Step S1, adding a fixing solution dropwise onto the smear for fixing to obtain a sample;

[0057] Step S2: Drop the protein blocker onto the sample, and obtain a blocked sample after incubation.

[0058] Step S3: Drop the membrane-breaking agent onto the blocked sample and incubate for a period of time.

[0059] Step S4: Add the primary antibody reagent, incubate and then wash.

[0060] Step S5: Add the secondary antibody reagent, incubate and then wash.

[0061] Step S6: Drop the peroxidase substrate staining solution, alkaline phosphatase substrate staining solution and hematoxylin staining solution for staining treatment to obtain a stained sample.

[0062] Specifically, in Step S1, the fixative is preferably methanol and formaldehyde with a mass ratio of 1:1. The fixation time is 1 - 30 min. Preferably, in one embodiment, the fixation time is 1 min. This fixation step can effectively improve the integrity of cell morphology while ensuring the antigen activity.

[0063] Step S2: Drop the protein blocker onto the sample, and obtain a blocked sample after incubation.

[0064] Specifically, in this step, the protein blocker is one or more of a primary antibody diluent, a secondary antibody diluent, a bovine serum albumin solution, a goat serum solution, a horse serum solution and a casein solution. Particularly, when applied to bone marrow peripheral blood smears, since there is a large amount of dry stain in the sample, adding a protein blocker can effectively improve the detection accuracy.

[0065] Further, in this step, the volume of the protein blocker dropped can be 50 - 200 μL, and the incubation time can be 1 - 60 min. Further preferably, the incubation time is 10 min, and the incubation temperature is 15 - 35°C. After incubation, drain off the liquid by centrifugation and no washing is required.

[0066] Step S3: Drop 50 - 200 μL of the membrane-breaking agent solution onto the blocked sample. The concentration of the membrane-breaking agent solution is 5% - 35%, and incubate for a period of time. Specifically, in one embodiment, the membrane-breaking agent solution contains sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, nonylphenol polyoxyethylene ether, Triton X - 100, paraformaldehyde.

[0067] More preferably, the membrane-breaking agent is the flow cytometry membrane-breaking agent produced by BD Company. After adding the membrane-breaking agent, the incubation time is 1 - 60 min. More preferably, the incubation time is 10 min, and the incubation temperature is 15 - 35°C. After this step, pores can be formed on the cells to facilitate the binding of the primary antibody reagent.

[0068] Step S4: Add the primary antibody reagent to the sample obtained in step S3. After incubation, wash with PBS buffer. Specifically, in this step, the primary antibody reagent is dropped. The specific dosage of the primary antibody reagent can be selected according to the instructions of the specifically purchased primary antibody reagent, and the present disclosure does not make specific limitations. After adding the primary antibody reagent, it can be incubated at 4 - 60 °C for 3 - 800 min, and then the smear is washed with PBS buffer. The step of washing with PBS buffer solution can be washing for 3 min each time and washing repeatedly for 3 times. It can be understood that the incubation time after adding the primary antibody reagent can be adjusted according to different incubation temperatures. For example, in a preferred embodiment, it is incubated at 30 - 40 °C for 20 - 40 min. Further, in this step, the incubation temperature is 35 °C and the incubation time is 30 min. Under this condition, the incubation time is shorter and the incubation effect is better.

[0069] It should be noted that the present disclosure uses CD20 antibody and LEF1 antibody as the primary antibody reagent. In one embodiment, CD20 antibody and LEF1 antibody can be added simultaneously for incubation. In other embodiments, it can also be to add CD20 antibody for incubation first and then add LEF1 antibody for incubation; or add LEF1 antibody for incubation first and then add CD20 antibody for incubation.

[0070] Step S5: Add the secondary antibody reagent and incubate at 4 - 60 °C for 5 - 800 min, and wash multiple times with PBS buffer. The dosage of the secondary antibody reagent can be determined according to the specifically purchased manufacturer. For example, 135 u of the secondary antibody reagent purchased from Xiamen Tongling Biotechnology Co., Ltd. can be added. It can be understood that the incubation time after adding the primary antibody reagent can be adjusted according to different incubation temperatures. For example, in a preferred embodiment, after adding the secondary antibody reagent, it is incubated at 20 - 45 °C for 10 - 40 min. Under this condition, the incubation time is shorter and the incubation effect is better.

[0071] It should be noted that in this step, in one embodiment, the secondary antibody polymer labeled with alkaline phosphatase AP and the secondary antibody polymer labeled with horseradish peroxidase HRP can be added simultaneously for incubation. In other embodiments, it can also be to add the secondary antibody polymer labeled with alkaline phosphatase AP and the secondary antibody polymer labeled with horseradish peroxidase HRP separately for incubation. It can be to add the secondary antibody polymer labeled with alkaline phosphatase AP for incubation first, or it can be to add the secondary antibody polymer labeled with horseradish peroxidase HRP for incubation first.

[0072] Step S6: Drop the peroxidase substrate staining solution, alkaline phosphatase substrate staining solution and hematoxylin staining solution for staining treatment to obtain a stained sample.

[0073] In one embodiment, step S6 specifically includes: first adding a horseradish peroxidase substrate staining solution, incubating, and washing, then adding an alkaline phosphatase substrate staining solution, incubating, and washing, and finally adding a hematoxylin staining solution. After incubation and washing, a stained sample is obtained. Specifically, in one embodiment, first add a DAB staining solution, incubate at 5 - 50 °C for 3 - 120 min, then wash twice with pure water for 2 min each time. Then add a fast red staining solution, incubate at 5 - 50 °C for 3 - 120 min, and wash twice with pure water for 2 min each time. Then smear and add 135 μl of hematoxylin staining solution, incubate at 5 - 50 °C for 3 - 120 min, and wash twice with pure water for 2 min each time.

[0074] In another embodiment, step S6 specifically includes: first adding an alkaline phosphatase substrate staining solution, incubating, and washing, then adding a horseradish peroxidase substrate staining solution, incubating, and washing, and finally adding a hematoxylin staining solution. After incubation and washing, a stained sample is obtained. Specifically, in one embodiment, first add a fast red staining solution, incubate at 5 - 50 °C for 3 - 120 min, then wash twice with pure water for 2 min each time. Then add a DAB staining solution, incubate at 5 - 50 °C for 3 - 120 min, and wash twice with pure water for 2 min each time. Then smear and add 135 μl of hematoxylin staining solution, incubate at 5 - 50 °C for 3 - 120 min, and wash twice with pure water for 2 min each time.

[0075] The embodiment of the present invention also provides the application of the double - staining immunohistochemistry kit as described above in the diagnosis of chronic lymphocytic leukemia.

[0076] The features and properties of the present disclosure are further described in detail below in conjunction with embodiments.

[0077] Example 1

[0078] Reagents:

[0079]

[0080]

[0081] The following samples A, B, and C are subjected to staining treatment:

[0082] Sample A: A peripheral blood smear from a patient diagnosed with CLL in a certain hospital;

[0083] Sample B: Negative control: A smear of normal T lymphocytes;

[0084] Sample C: A peripheral blood smear from a patient diagnosed with MCL in a certain hospital.

[0085] The staining process is as follows:

[0086] (1) Fixation: Add the fixing solution dropwise to the sample for fixation for 1 min;

[0087] (2) Blocking: Add 40 μl of protein blocking agent, incubate at room temperature for 10 min, and spin dry (no washing);

[0088] (3) Permeabilization: Add 150 μl of permeabilization agent solvent, incubate at room temperature for 10 min, and cover the blood film;

[0089] (4) Primary antibody incubation: Add 135 μl of primary antibody reagent, incubate at 35 °C for 30 min, then wash the section with PBS buffer 3 times, 3 min each time.

[0090] (5) Secondary antibody incubation: Add 135 μl of secondary antibody reagent, incubate at 35 °C for 18 min, then wash the section with PBS buffer 3 times, 3 min each time.

[0091] (6) Chromogenic reaction: Add 180 μl of DAB staining solution dropwise to the smear, incubate at 35 °C for 6 min, then wash with pure water 2 times, 2 min each time. Add 150 μl of fast red staining solution and incubate for 20 min.

[0092] (7) Counterstaining: Add 135 μl of hematoxylin staining solution dropwise to the smear, incubate at 35 °C for 4 min, then wash with pure water 2 times, 2 min each time.

[0093] (8) Microscopic examination: After acidification and bluing, examine under the microscope.

[0094] As Figure 2 shown is the staining result diagram of sample A, Figure 3 is the staining result diagram of sample B, Figure 4 is the staining result diagram of sample C.

[0095] It can be seen from the staining results that Figure 2 in, brown cell membrane indicates CD20 positive, red cell nucleus indicates LEF1 positive, and CD20 and LEF1 co-positive, identified as CLL cells. Figure 3 in, no brown cell membrane detected indicates CD20 negative, red cell nucleus indicates LEF1 positive, identified as normal T lymphocytes. Figure 4 in, brown cell membrane indicates CD20 positive, no red cell nucleus detected indicates LEF1 negative, identified as MCL cells. It can be seen that the staining method of the present disclosure can effectively distinguish CLL and MCL. It can be seen from the staining diagram that the morphology of the stained cells is clear, the colors of the cell nucleus, cell membrane and cytoplasm are distinct, the positive localization is accurate, the signal expression is clear, and it can be applied to the auxiliary diagnosis of chronic lymphocytic leukemia.

[0096] The embodiments described above are some, but not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention. 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 scope of protection of the present invention.

Claims

1. Use of a reagent in the preparation of a double-staining immunohistochemistry kit for the auxiliary diagnosis of chronic lymphocytic leukemia, characterized in that, The kit includes the following reagents: a fixing solution, a membrane-breaking agent, a primary antibody reagent, a secondary antibody reagent, a protein blocker, and a staining agent. Among them, the primary antibody reagent includes a LEF1 antibody and a CD20 antibody; the secondary antibody reagent includes a secondary antibody polymer labeled with alkaline phosphatase AP and a secondary antibody polymer labeled with horseradish peroxidase HRP; the fixing solution is a mixed solution of methanol and formaldehyde with a mass ratio of 1:1; the protein blocker is a mixture of bovine serum albumin buffer solution and casein buffer solution; the double-staining immunohistochemistry kit is used for staining peripheral blood smears.

2. Use of the reagent according to claim 1 in the preparation of a dual-staining immunohistochemistry kit for the auxiliary diagnosis of chronic lymphocytic leukemia, characterized in that, The LEF1 antibody and the CD20 antibody are derived from animals of two different species.

3. Use of the reagent according to claim 1 in the preparation of a double-staining immunohistochemistry kit for the auxiliary diagnosis of chronic lymphocytic leukemia, characterized in that, The secondary antibody polymer labeled with alkaline phosphatase AP is used to bind to the primary antibody derived from animals of the first species, and the secondary antibody polymer labeled with horseradish peroxidase HRP is used to bind to the primary antibody derived from animals of the second species, and the first species and the second species are different.

4. Use of the reagent according to claim 3 in the preparation of a dual-staining immunohistochemistry kit for the auxiliary diagnosis of chronic lymphocytic leukemia, characterized in that, The first species and the second species are respectively consistent with the animal species of the LEF1 antibody and the CD20 antibody.

5. Use of the reagent according to claim 1 in the preparation of a double-staining immunohistochemistry kit for the auxiliary diagnosis of chronic lymphocytic leukemia, characterized in that, The staining agent includes a peroxidase substrate staining solution, an alkaline phosphatase substrate staining solution, and a counterstaining solution, and the color development of the counterstaining solution does not cover the color development of the peroxidase substrate staining solution and the alkaline phosphatase substrate staining solution.

6. Use of the reagent according to claim 1 in the preparation of a double-staining immunohistochemistry kit for the auxiliary diagnosis of chronic lymphocytic leukemia, characterized in that, The membrane-breaking agent is selected from one or more of saponins, aldehydes, alcohols, ketones, sodium chloride, calcium chloride, sodium benzoate, disodium ethylenediaminetetraacetate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium benzoate, nonylphenol polyoxyethylene ether, Triton X-100, Triton-X, and Tween-20.

Images