Intraoperative frozen tissue section staining kit and application thereof

Abstract

The invention discloses an intraoperative frozen tissue section staining kit and application thereof. The intraoperative frozen tissue section staining kit comprises the following components in percentage by volume: 15%-20% of a blocking agent, 15%-20% of a reinforcing agent, 15%-20% of a polymer enzyme-labeled antibody reagent, 20%-25% of an immune color developing agent and 15%-20% of a hematoxylin staining agent. The enhancer is introduced and can be combined with primary antibodies of mouse species and rabbit species, double antibody binding epitopes are exposed by utilizing the symmetry characteristic of the antibodies, the dyeing effect is remarkably enhanced, the dyeing time is shortened, the dyeing speed is higher, and rapid pathological diagnosis in an operation can be assisted.

Description

Technical Field

[0001] The invention belongs to the technical field of immunoassay, and particularly relates to a kit for staining frozen tissue sections during surgery and an application thereof. Background Art

[0002] Intraoperative frozen tissue section staining involves rapidly freezing the resected lesion during surgery, preparing sections, and then rapidly testing them using immunohistochemistry (IHC). This technique is primarily used to facilitate rapid intraoperative pathological diagnosis, enabling surgeons to adjust surgical plans immediately. For example, intraoperative frozen tissue section staining can help surgeons determine tumor boundaries and whether the tumor has been completely removed (e.g., for gliomas and breast cancer), minimizing postoperative tumor recurrence.

[0003] Simply put, the complete process of intraoperative frozen tissue section staining is as follows: first, the diseased tissue is removed during surgery and sent to the pathology department; then, the surgically removed tissue sample is rapidly frozen using liquid nitrogen or a cryogenic freezing agent; next, the frozen tissue sample is cut into thin slices (4-8μm thick) using a freezing microtome and attached to a glass slide; finally, an intraoperative frozen tissue section staining kit (e.g., shortening the incubation time) is used, and the entire process, from endogenous enzyme blocking to hematoxylin counterstaining, is typically completed within 30-60 minutes. Based on the expression of markers (such as CK, S-100, GFAP, etc.) on the frozen tissue section, the pathologist can provide a diagnosis and assist the surgeon in completing the operation.

[0004] Compared with conventional paraffin section staining, which takes 1-2 days, intraoperative frozen tissue section staining is faster. Therefore, its staining results can provide a basis for adjusting the scope of surgery immediately, and it has important application value in clinical practice. However, this staining technology still has several defects that need to be overcome. The first is that frozen sections are prone to produce ice crystals, which affect cell morphology and antigen preservation. Secondly, not all antibodies are suitable for rapid processes, and false negatives / positives may occur. Finally, the results of intraoperative frozen tissue section staining need to be verified by postoperative paraffin section staining. Therefore, in current clinical practice, intraoperative frozen tissue section staining requires an experienced pathology team to operate to avoid technical errors, resulting in certain restrictions on the promotion of this technology. Summary of the Invention

[0005] In order to address the deficiencies of the prior art, the purpose of the present invention is to provide an intraoperative frozen tissue section staining kit and its application, which introduces an enhancer that can be combined with primary antibodies of mouse and rabbit species, utilizes the symmetry of the antibodies to expose double the antibody binding epitopes, significantly enhances the staining effect, shortens the staining time, and stains faster, which can assist in rapid intraoperative pathological diagnosis.

[0006] In order to achieve the above objectives, the present invention adopts the following technical solutions: A kit for staining frozen tissue sections during surgery comprises the following components in percentage by volume: 15% to 20% of a blocking agent, 15% to 20% of an enhancing agent, 15% to 20% of a polymerase-labeled antibody reagent, 20% to 25% of an immunochromogen, and 15% to 20% of a hematoxylin stain. The polymerase-labeled antibody reagent is one or both of a goat anti-mouse IgG antibody or a goat anti-rabbit IgG antibody coupled with 8 to 12 alkaline phosphatases or horseradish peroxidases, and can further amplify the antibody staining signal.

[0007] Preferably, the aforementioned blocking agent is one or both of a 0.1-1.0% by mass levamisole hydrochloride-methanol solution or a 1.0-3.0% by volume hydrogen peroxide-methanol solution, which can effectively block endogenous alkaline phosphatase and peroxidase in tissue section samples.

[0008] Preferably, the aforementioned enhancer is one or both of rabbit anti-mouse IgG antibody and mouse anti-rabbit IgG antibody, which can be combined with the primary antibody of mouse species or rabbit species to enhance the antibody staining effect.

[0009] Preferably, the aforementioned immunochromogenic reagent is one or both of Fast Red colorimetric reagent and DAB colorimetric reagent, and is applicable to alkaline phosphatase (AP) colorimetric system and horseradish peroxidase (HRP) colorimetric system.

[0010] The application of the above-mentioned staining kit in intraoperative immune marker-assisted diagnosis.

[0011] The present invention is beneficial in that: (1) The staining kit of the present invention introduces an enhancer that can be combined with primary antibodies of mouse and rabbit species. By utilizing the symmetry of the antibody, the kit exposes double the antibody binding epitopes, significantly enhancing the staining effect, shortening the staining time, and increasing the staining speed. This can assist in rapid pathological diagnosis during surgery and provide information support for surgeons to adjust the surgical plan immediately during the operation. (2) The staining kit of the present invention can be fully automatically operated on the intraoperative frozen tissue staining instrument CATYS 12, and can stain 12 different intraoperative frozen tissue sections at the same time, greatly improving the staining efficiency and significantly reducing the error of manual liquid addition, which is very beneficial to the clinical standardization of this technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The figure is a comparison of the dyeing effects of the kits of Example 1 and Comparative Example 1; Figure 2 This is a color development diagram of the kit of Example 2 using the alkaline phosphatase system; Figure 3 This is a diagram showing the fully automatic dyeing effect of the kit of Example 3 on the instrument; Figure 4 This is a staining effect diagram of the kit of Comparative Example 2; Figure 5 This is a diagram of the dyeing effect of the kit of Comparative Example 3. DETAILED DESCRIPTION

[0013] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0014] The polymerase-labeled antibody reagent is a goat anti-mouse IgG antibody or goat anti-rabbit IgG antibody conjugated to 8-12 alkaline phosphatase or horseradish peroxidase residues, or both. The antibody and enzyme (alkaline phosphatase AP or horseradish peroxidase HRP) are conjugated using the heterobifunctional crosslinker SMCC (succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate) in a two-step process. The specific steps are as follows: S1. Activation of the amino group of the antibody Antibody solution (1 mg / mL goat anti-mouse IgG antibody or goat anti-rabbit IgG antibody, dissolved in 0.1 M PBS) and SMCC crosslinker (dissolved in anhydrous DMSO to a final concentration of 10 mM) were mixed at a molar ratio of 1:20 and reacted in the dark at 25°C for 2 hours. Unreacted SMCC was removed by ultrafiltration centrifugation (10 kDa molecular weight cutoff) and the activated antibody was resuspended in PBS buffer (pH 6.8).

[0015] S2. Sulfhydryl modification of enzymes Mix the enzyme solution (5 mg / mL alkaline phosphatase (AP) or horseradish peroxidase (HRP), dissolved in 0.1 M PBS) with Traut's reagent (2-iminothiolane hydrochloride) at a molar ratio of 1:15 (enzyme:Traut's reagent). Incubate at 25°C in the dark for 1 hour to generate the sulfhydryl enzyme. Use a desalting column (PD-10) to remove the unreacted Traut's reagent and collect the sulfhydryl enzyme fraction.

[0016] S3: Conjugation of Antibodies to Enzymes The activated antibody and thiol-linked enzyme were mixed at a molar ratio of 1:15 and reacted at 25°C in the dark for 12 hours. The reaction was terminated by adding 5 mM cysteine ​​and incubated at room temperature for 30 minutes. The coupled products were separated by size exclusion chromatography (Superdex 200 column) to remove uncoupled antibody and enzyme, and the high molecular weight peak (corresponding to the antibody-enzyme complex) was collected to determine the coupling efficiency.

[0017] S4. Enzyme activity assay after coupling AP activity: Using pNPP (p-nitrophenyl phosphate) as the substrate, the absorbance change at 405 nm was detected. After coupling, the enzyme activity retention was ≥80%.

[0018] HRP activity: Using TMB as substrate, the absorbance change at 450 nm was detected, and the activity retention should be ≥85%.

[0019] Storage stability: The coupled product can be stored stably in PBS (containing 1% BSA and 0.05% NaN3) at 4°C for 6 months with enzyme activity decreasing by <10%.

[0020] This method uses a heterobifunctional crosslinker to achieve efficient, directional conjugation of antibodies and enzymes, with each antibody molecule conjugated to an average of 8-12 enzymes, significantly enhancing staining signal intensity. The technology is comprehensive, highly reproducible, and meets patent disclosure requirements. It also demonstrates ingenuity through its activity retention and scalable purification design.

[0021] Example 1: TTF-1 mouse monoclonal antibody was selected and compared with the staining kit of the present invention. The tissue sample was lung adenocarcinoma.

[0022] The operation process is as follows: Step 1. Endogenous peroxidase blocking: Add 80-100 μL of 3% hydrogen peroxide blocking agent to the frozen lung adenocarcinoma tissue sample section, incubate at room temperature in the dark for 30 seconds, and then rinse with TBST buffer for 5 seconds; Step 2. Primary antibody binding: Add 80-100 μL of TTF-1 mouse monoclonal antibody reagent to the slice, incubate at room temperature for 2-4 minutes, and then rinse with TBST buffer for 10 seconds; Step 3, Signal Enhancement: Add 80-100 μL of rabbit anti-mouse IgG antibody reagent to the slice, incubate at room temperature for 1-3 minutes, and then rinse with TBST buffer for 10 seconds; Step 4. Polymerase-labeled antibody incubation: Add 80-100 μL of HRP enzyme-labeled goat anti-mouse / rabbit polymer antibody reagent to the slice, incubate at room temperature for 1-3 minutes, and then rinse with TBST buffer for 10 seconds; Step 5, DAB color development: Prepare DAB colorimetric working solution by mixing DAB chromogen and diluent in a ratio of 1:20 in advance. Then, add 80-100 μL of this solution to the sections, incubate at room temperature for 1-2 minutes, and rinse with distilled water for 10 seconds. Step 6, counterstaining - dehydration - mounting: add 80-100 μL of hematoxylin staining solution to the slices, incubate at room temperature for 10-30 seconds, rinse with distilled water for 10 seconds, use a hair dryer to dry the moisture on the tissue slices, and finally mount the slices with neutral gum; Step 7. Observation: Use an ordinary optical microscope to observe the protein staining. Positive staining is located in the cell nucleus.

[0023] Example 2: Cytokeratin CK pan rabbit monoclonal antibody was selected and the staining kit of the present invention was used for comparison. The tissue sample used in this experiment was breast cancer.

[0024] The operation process is as follows: Step 1. Endogenous alkaline phosphatase blocking: Add 80-100 μL of 1% levamisole hydrochloride blocking agent to the frozen breast cancer tissue sample section, incubate at room temperature in the dark for 30 seconds, and then rinse with TBST buffer for 5 seconds; Step 2. Primary antibody binding: Add 80-100 μL of CK Pan rabbit monoclonal antibody reagent to the slice, incubate at room temperature for 2-4 minutes, and then rinse with TBST buffer for 10 seconds; Step 3, signal enhancement: add 80-100 μL of mouse anti-rabbit IgG antibody reagent to the slice, incubate at room temperature for 1-3 minutes, and then rinse with TBST buffer for 10 seconds; Step 4. Polymerase-labeled antibody incubation: Add 80-100 μL of HRP enzyme-labeled goat anti-mouse / rabbit polymer antibody reagent to the slice, incubate at room temperature for 1-3 minutes, and then rinse with TBST buffer for 10 seconds; Step 5. Fast Red color development: Add 1 mL of the diluent to the Fast Red chromogen in advance to prepare the Fast Red color development working solution. Then, add 80-100 μL of this solution to the sections, incubate at room temperature for 3-5 minutes, and rinse with distilled water for 10 seconds. Step 6, counterstaining - dehydration - mounting: add 80-100 μL of hematoxylin staining solution to the slices, incubate at room temperature for 10-30 seconds, rinse with distilled water for 10 seconds, use a hair dryer to dry the moisture on the tissue slices, and finally mount the slices with neutral gum; Step 7. Observation: Observe protein staining under an optical microscope. Positive staining is localized in the cytoplasm.

[0025] Example 3: Dyeing effect of dyeing kit on machine CD3 mouse monoclonal antibody was selected and the staining kit of the present invention was used to perform fully automatic staining on an intraoperative freezing stainer CATYS 12. The tissue sample used in this experiment was the appendix.

[0026] The operation process is as follows: Step 1. Primary antibody selection: On the primary antibody reagent selection interface of the intraoperative cryo-staining instrument CATYS 12, search for and select CD3 mouse monoclonal antibody; Step 2. Sample selection: On the sample type selection interface of the intraoperative frozen stainer CATYS 12, search for the lymph node tissue sample and select it; Step 3. Method selection: On the staining method selection interface of the intraoperative cryo-stainer CATYS 12, search for mouse enhanced staining and select it. Step 4. Start the operation: On the instrument operation status interface of the intraoperative frozen stainer CATYS 12, click the Start button and wait for 10-15 minutes for automatic staining; Step 5. Seal the slides and observe the protein staining under an optical microscope. Positive staining is localized to the cell membrane.

[0027] Comparative Example 1: The operation steps of this comparative example are the same as those of Example 1, with the specific difference that: the intraoperative rapid immunohistochemistry kit provided by Henan Synotech Biotechnology Co., Ltd. (patent publication number: CN 111766385 A) was used.

[0028] Comparative Example 2: The operation steps of this comparative example are the same as those of Example 1, with the specific difference that: the signal enhancement step (Step 3) is not performed, that is, no enhancer is added to the kit.

[0029] Comparative Example 3: The operation steps of this comparative example are the same as those of Example 1, with the specific difference that: the polymerase-labeled antibody incubation step (Step 4) is not performed, that is, the polymerase-labeled antibody reagent is not added to the kit.

[0030] Performance test (1) Comparison of the dyeing effect diagrams of Example 1 and Comparative Example 1, Figure 1 (a) shows the staining effect of the kit of the present invention, Figure 1 (b) shows the staining effect of using a commercial kit. By comparison, Figure 1 The staining intensity of (a) is higher and the staining background is cleaner.

[0031] (2) The staining effect of the kit of Example 2 is as follows Figure 2 As shown, it can be seen that the color development intensity of the alkaline phosphatase system used in the kit of the present invention is very high.

[0032] (3) The fully automatic staining effect of the kit of Example 3 on the instrument is as follows Figure 3 As shown, it can be seen that the staining intensity is high and the staining background is clean.

[0033] (4) The dyeing effect diagrams of Example 1 and Comparative Example 2 are as follows: Figure 4 As shown, the dyeing effect of Comparative Example 2 is unclear and cannot be accurately judged. Therefore, it can be seen that adding an enhancer can improve the dyeing effect of the dyeing kit.

[0034] (5) The dyeing effect diagrams of Example 1 and Comparative Example 3 are as follows: Figure 5 As shown, the staining effect of Comparative Example 3 is poor and the background is messy. It can be seen that adding a polymerase-labeled antibody reagent can improve the staining effect of the staining kit.

[0035] In summary, the staining kit of the present invention, by adapting to the alkaline phosphatase system, addresses the limited applicability of antibodies used in intraoperative freezing techniques. Furthermore, by utilizing a fully automated staining instrument, it overcomes the manual staining errors associated with intraoperative freezing techniques. Finally, the inclusion of an enhancer further improves the staining intensity of intraoperative freezing techniques, removing obstacles to their clinical application and facilitating their rapid adoption.

[0036] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solutions obtained by equivalent replacement or equivalent transformation fall within the scope of protection of the present invention.

Claims

1. A kit for staining frozen tissue sections during surgery, characterized in that: The invention comprises the following components in volume percentage: 15% to 20% of a blocking agent, 15% to 20% of an enhancing agent, 15% to 20% of a polymerase-labeled antibody reagent, 20% to 25% of an immunochromogen and 15% to 20% of a hematoxylin stain. The polymerase-labeled antibody reagent is one or both of a goat anti-mouse IgG antibody or a goat anti-rabbit IgG antibody coupled with 8 to 12 alkaline phosphatases or horseradish peroxidases.

2. A kit for staining frozen tissue sections during surgery according to claim 1, characterized in that: The blocking agent is one or both of a 0.1-1.0% mass fraction levamisole hydrochloride-methanol solution or a 1.0-3.0% volume fraction hydrogen peroxide-methanol solution.

3. The intraoperative frozen tissue section staining kit according to claim 1, characterized in that: The enhancer is one or both of rabbit anti-mouse IgG antibody and mouse anti-rabbit IgG antibody.

4. The intraoperative frozen tissue section staining kit according to claim 1, characterized in that: The immunochromogenic reagent is one or both of Fast Red color developing reagent and DAB color developing reagent.

5. Use of the staining kit according to any one of claims 1 to 4 in intraoperative immunolabeling-assisted diagnosis.

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