Combined double-staining immune cell chemical detection kit and application thereof

Abstract

The invention belongs to the field of immunodetection, and particularly relates to a combined double-staining immune cell chemical detection kit and application thereof, which can be used for screening and identifying HPV infection related and non-HPV infection related cervical cancer and precancerous lesions. Comprising a CLDN18.2 / Ki-67 primary antibody working mixed solution, a p16 / Ki-67 primary antibody working mixed solution, a goat anti-mouse / rabbit secondary antibody working mixed solution, a DAB working mixed solution, an RED working mixed solution and hematoxylin. According to the invention, liquid-based slide preparation is carried out on cervical exfoliated cells, vagina exfoliated cytology and the like, and CLDN18.2 / Ki-67 and p16 / Ki-67 are combined with immunochemical double staining, so that not only can cervical squamous carcinoma and precancerous lesions, but also cervical adenocarcinoma and precancerous lesions related to HPV infection be covered, and cervical adenocarcinoma and precancerous lesions related to non-HPV infection can be covered; and the kit has good specificity and sensitivity.

Description

Technical Field

[0001] The present invention relates to an immuno(cyto)chemical detection kit which can be used for screening and identifying HPV infection-related and non-HPV infection-related cervical cancer and precancerous lesions, belonging to the field of immunoassay. Background Art

[0002] Among cervical cancer and its precancerous lesions, cervical squamous cell carcinoma and its precancerous lesions are associated with HPV; cervical adenocarcinoma and its precancerous lesions are divided into two types, HPV-related and non-HPV-related, according to the 2019 IECC and 2020 WHO pathological diagnostic criteria. The existing screening and diagnostic strategy—HPV combined with cytology—has the following urgent issues that need to be addressed: ① The cytological diagnostic accuracy of cervical adenomatous lesions is low; ② There is a lack of specific diagnostic screening methods for non-HPV-related cervical gastric adenocarcinoma; ③ In particular, with the application of HPV vaccines, the proportion of non-HPV-related cervical adenocarcinomas is gradually increasing. Efficient diagnostic screening methods and kits will be an even more important need for the clinical diagnosis and prevention of cervical lesions. Preliminary research results of the project show that ① p16 / Ki-67 double-staining cytology combined with existing screening methods can improve the diagnostic accuracy of HPV-related cervical cancer and precancerous lesions. ②For cervical gastric adenocarcinoma, which accounts for approximately 95% of non-HPV-related cervical adenocarcinomas, the immunochemical diagnostic accuracy of the CLDN18.2 target is significantly higher than that of the immune markers MUC6 and HIK1083 suggested by existing guidelines and literature.

[0003] Immunochemical staining is a commonly used technical method in histopathology. It can use the principle of specific binding between antigens and antibodies to detect whether target antigens exist in cells or tissues and determine the protein expression in tumor cells or tissues. It is the gold standard for tumor pathology diagnosis. Immunochemical staining is highly specific, sensitive, simple, fast and low-cost, which is conducive to in-depth research in the field of pathology.

[0004] Immunochemical double staining is one of the most commonly used immuno(cyto)chemical staining methods. It is often used as an effective tool for identifying tumors, especially for screening and identifying cervical cancer and its precancerous lesions. It uses two primary antibodies of different species, two secondary antibodies with different reaction systems, and different color development systems. It can simultaneously detect the expression of two antigens in a single section to discover their localization, morphology, and functional relationships.

[0005] In summary, providing a highly accurate, easy-to-use, highly sensitive, and non-invasive product and method for detecting cervical cancer and precancerous lesions is of great significance for the early diagnosis and screening of cervical cancer. It holds promise for developing new screening strategies, methods, and kits suitable for the post-vaccine era, where the proportion of non-HPV-related cervical diseases is increasing significantly. Summary of the Invention

[0006] To address the practical needs of existing cervical cancer and precancerous lesion screening strategies, the present invention provides a combined double-staining immunocytochemical detection kit and its application. This method uses liquid-based preparation of cervical exfoliated cells and vaginal exfoliated cytology, combined with a combined immunochemical double-staining method for CLDN18.2 / Ki-67 and p16 / Ki-67, to screen for cervical cancer and precancerous lesions. This method can screen for cervical squamous cell carcinoma and precancerous lesions, HPV-related cervical adenocarcinoma and precancerous lesions, as well as non-HPV-related cervical adenocarcinoma and precancerous lesions, and can identify these. The detection is highly sensitive, specific, and accurate.

[0007] To achieve the above object, the present invention adopts the following technical solutions: One of the purposes of the present invention is to provide: a combined double-staining immunocytochemistry detection kit and its application, comprising reagents: p16 / Ki-67 primary antibody working mixture, goat anti-mouse / rabbit secondary antibody working mixture, CLDN18.2 / Ki-67 primary antibody working mixture, DAB working mixture, RED working mixture and hematoxylin.

[0008] Preferably, the p16 / Ki-67 primary antibody working mixture includes antibody diluent, p16 primary antibody concentrate and Ki-67 primary antibody concentrate; the CLDN18.2 / Ki-67 primary antibody working mixture includes antibody diluent, CLDN18.2 primary antibody concentrate and Ki-67 primary antibody concentrate; the DAB working mixture includes DAB staining substrate and DAB staining buffer; the RED working mixture includes AP-red color development buffer and AP-red chromogen concentrate.

[0009] A second object of the present invention is to provide a method for interpreting the above-mentioned detection kit. After cervical cell sampling, two cervical exfoliated cell slides, designated as slide 1 and slide 2, are prepared. Slide 1 is immunohistochemically stained for p16 / Ki-67 using a p16 / Ki-67 primary antibody working mixture, a goat anti-mouse / rabbit secondary antibody working mixture, a DAB working mixture, and hematoxylin. Slide 2 is immunohistochemically stained for CLDN18.2 / Ki-67 using a CLDN18.2 / Ki-67 primary antibody working mixture, a RED working mixture, a goat anti-mouse / rabbit secondary antibody working mixture, and hematoxylin. The result is determined by combining the staining results with the cell count. p16 / Ki-67 positivity is defined as cells expressing both p16 and Ki-67, independent of cell morphology, with the number of positive cells being ≥1. CLDN18.2 / Ki-67 positivity was defined as CLDN18.2 positivity and Ki-67 positivity or negativity, independent of cell morphology, with ≥1 positive cell.

[0010] Preferably, a p16 / Ki-67 primary antibody working mixture is prepared by first mixing the antibody diluent and the p16 primary antibody concentrate, and then adding the mixture to the Ki-67 primary antibody concentrate and mixing thoroughly.

[0011] Preferably, a CLDN18.2 / Ki-67 primary antibody working mixture is prepared by adding the antibody diluent and the CLDN18.2 primary antibody concentrate to the Ki-67 primary antibody concentrate and mixing.

[0012] Preferably, a goat anti-mouse / rabbit secondary antibody working mixture is prepared by adding the goat anti-mouse secondary antibody working solution to the goat anti-rabbit secondary antibody working solution and mixing.

[0013] Preferably, a DAB working mixture is prepared by mixing DAB staining substrate and DAB staining buffer.

[0014] Preferably, the RED working mixture is prepared by mixing AP-red color development buffer and AP-red chromogen concentrate.

[0015] Preferably, p16 / Ki-67 immunohistochemical staining is performed on the cell preparation 1: first, the p16 / Ki-67 primary antibody working mixture is added to the cell preparation 1; then, a goat anti-mouse / rabbit secondary antibody working mixture is added to the cell preparation 1, and finally, DAB working mixture, RED working mixture and hematoxylin are added in sequence for staining.

[0016] Preferably, CLDN18.2 / Ki-67 immunohistochemical staining is performed on cell preparation 2: first, the CLDN18.2 / Ki-67 primary antibody working mixture is added to cell preparation 2, then the goat anti-mouse / rabbit secondary antibody working mixture is added to the cell preparation 2, and finally, DAB working mixture, RED working mixture and hematoxylin are added in sequence for staining.

[0017] Preferably, the sample comprises any one of human cervical exfoliated cells, vaginal exfoliated cells or cervical tissue.

[0018] Compared with existing detection methods, the present invention has the following beneficial effects: The p16 / Ki-67 double staining screening method can improve the screening accuracy of HPV-related cervical cancer and precancerous lesions. For cervical gastric adenocarcinoma and precancerous lesions, which account for approximately 95% of non-HPV-related cervical adenocarcinomas, the immunochemical diagnostic accuracy of the CLDN18.2 target is significantly higher than that of existing immune markers - MUC6 and HIK1083. In the post-HPV vaccine era, cervical screening strategies need to be changed. In the past, screening strategies mainly prioritized HPV screening, but in the future, screening strategies will require that HPV screening be combined with screening for non-HPV-related cervical lesions. The present invention uses a combined immunochemical double staining method of CLDN18.2 / Ki-67 and p16 / Ki-67 to take into account the screening strategies for both HPV and non-HPV-related cervical lesions, and has good detection effects, high specificity and sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the immunochemical double staining image of p16 / Ki -67 double staining (+) in a patient with cervical squamous cell carcinoma; Figure 2 This is the immunochemical double staining image of HPV-related cervical adenocarcinoma p16 / Ki -67 double staining (+); Figure 3 This is an immunochemical double staining image of CLDN18.2 / Ki-67 double staining (+) in non-HPV-related cervical adenocarcinoma. DETAILED DESCRIPTION

[0020] A combined double-staining immunocytochemistry detection kit comprises a CLDN18.2 / Ki-67 primary antibody working mixture, a p16 / Ki-67 primary antibody working mixture, a goat anti-mouse / rabbit secondary antibody working mixture, a DAB working mixture, a RED working mixture and hematoxylin.

[0021] The p16 / Ki-67 primary antibody working mixture includes antibody diluent, p16 primary antibody concentrate, and Ki-67 primary antibody concentrate; CLDN18.2 / Ki-67 primary antibody working solution includes antibody diluent, CLDN18.2 primary antibody concentrate, and Ki-67 primary antibody concentrate; Goat anti-mouse / rabbit secondary antibody working mixture includes goat anti-mouse secondary antibody working solution and goat anti-rabbit secondary antibody working solution; The DAB working mixture includes DAB staining substrate and DAB staining buffer; RED working mixture includes AP-red color development buffer and AP-red chromogen concentrate.

[0022] Two cervical exfoliated cell slides, designated as slide 1 and slide 2, were prepared. Immunohistochemical staining for p16 / Ki-67 was performed on slide 1 using a p16 / Ki-67 primary antibody working mixture, a goat anti-mouse / rabbit secondary antibody working mixture, a DAB working mixture, a RED working mixture, and hematoxylin. Immunohistochemical staining for CLDN18.2 / Ki-67 was performed on slide 2 using a CLDN18.2 / Ki-67 primary antibody working mixture, a goat anti-mouse / rabbit secondary antibody working mixture, a RED working mixture, and hematoxylin. The staining results and cell number were combined to determine whether p16 / Ki-67 was positive, defined as cells expressing both p16 and Ki-67, independent of cell morphology, with a positive cell count of ≥1. Positive CLDN18.2 / Ki-67 was defined as cells expressing CLDN18.2 and either Ki-67 or CLDN18.2, independent of cell morphology, with a positive cell count of ≥1.

[0023] The technical concept of the present invention is to perform immunocytochemical double staining on cervical exfoliated cells after liquid-based preparation.

[0024] The specific operation of the method based on liquid-based preparation combined with immunochemical double staining is as follows: (1) Cell preservation: centrifuge the cervical exfoliated cells in the preservation solution (1000-2000 rpm, 5-10 min), slowly discard the supernatant, resuspend the cell sediment with PBS buffer, centrifuge again (2000 rpm, 5-10 min), slowly discard the supernatant, and add an appropriate amount of cervical exfoliated cell preservation solution to the washed cell sediment; (2) Specimen preparation: After the cervical exfoliated cells are placed in the preservation solution for more than 30 minutes, the specimen can be prepared by sedimentation preparation method. The sample sedimentation time is 10 to 15 minutes; (3) Fixation: Fix with 10% neutral buffered formalin for 5–20 min and wash twice with washing buffer; (4) Antigen repair: Discard the washing buffer and heat the antigen repair solution to 60-90°C using a heating device. Heat the specimen for 5-20 minutes. After cooling, discard the antigen repair solution and wash twice with the washing buffer. (5) Peroxidase blocking: Add peroxidase blocking agent, incubate at room temperature for 3-5 minutes, and then wash twice with washing buffer; (6) Primary antibody incubation: Add CLDN18.2 / Ki-67 or p16 / Ki-67 combination primary antibodies, incubate at room temperature for 20-60 min, and wash twice with washing buffer; (7) Secondary antibody incubation: discard the washing buffer, add the combined secondary antibody, incubate at room temperature for 10-40 min, and wash with the washing buffer; (8) DAB staining: discard the washing buffer, add DAB staining solution, incubate at room temperature for 3-8 minutes, and wash twice with washing buffer; (9) AP staining: discard the washing buffer, add basic fuchsin staining solution, incubate at room temperature for 3-8 minutes, and wash twice with pure water; (10) Restaining: discard the pure water, restain with hematoxylin solution for 30-60 seconds, and rinse twice with pure water; (11) Seal the slides with aqueous mounting medium.

Claims

1. A combined double-staining immunocytochemistry detection kit, characterized in that: Includes CLDN18.2 / Ki-67 primary antibody working mix, p16 / Ki-67 primary antibody working mix, goat anti-mouse / rabbit secondary antibody working mix, DAB working mix, RED working mix, and hematoxylin.

2. A combined double-staining immunocytochemistry detection kit according to claim 1, characterized in that: The p16 / Ki-67 primary antibody working mixture includes antibody diluent, p16 primary antibody concentrate, and Ki-67 primary antibody concentrate; CLDN18.2 / Ki-67 primary antibody working solution includes antibody diluent, CLDN18.2 primary antibody concentrate, and Ki-67 primary antibody concentrate; Goat anti-mouse / rabbit secondary antibody working mixture includes goat anti-mouse secondary antibody working solution and goat anti-rabbit secondary antibody working solution; The DAB working mixture includes DAB staining substrate and DAB staining buffer; RED working mixture includes AP-red color development buffer and AP-red chromogen concentrate.

3. Use of a combined double-staining immunocytochemistry detection kit according to claim 1 or 2, characterized in that: Interpretation method of the detection kit: Liquid-based preparation of cervical exfoliated cells was performed with a double-staining immunocytochemical detection kit, and then immunocytochemical double staining was performed to prepare two cervical exfoliated cell preparations, namely cell preparation 1 and cell preparation 2; p16 / Ki-67 immunohistochemical staining was performed on cell preparation 1 using a p16 / Ki-67 primary antibody working mixture, goat anti-mouse / rabbit secondary antibody working mixture, DAB working mixture, RED working mixture, and hematoxylin; CLDN18.2 / Ki-67 immunohistochemical staining was performed on cell preparation 1 using a CLDN18.2 / Ki-67 primary antibody working mixture, goat anti-mouse / rabbit secondary antibody working mixture, DAB working mixture, RED working mixture, and hematoxylin; Cell preparation 2 was immunohistochemically stained for CLDN18.2 / Ki-67 using a rabbit secondary antibody working mixture, a RED working mixture, and hematoxylin. The staining results and cell number were combined to determine whether p16 / Ki-67 was positive, defined as cells expressing both p16 and Ki-67, independent of cell morphology, with ≥1 positive cell; and CLDN18.2 / Ki-67 was defined as CLDN18.2-positive and either Ki-67-positive or negative, independent of cell morphology, with ≥1 positive cell.

4. The use of a combined double-staining immunocytochemistry detection kit according to claim 3, characterized in that: Prepare the p16 / Ki-67 primary antibody working mixture: first mix the antibody diluent and the p16 primary antibody concentrate, then add to the Ki-67 primary antibody concentrate and mix thoroughly.

5. The use of a combined double-staining immunocytochemistry detection kit according to claim 3, characterized in that: Prepare CLDN18.2 / Ki-67 primary antibody working solution: add antibody diluent and CLDN18.2 primary antibody concentrate to Ki-67 primary antibody concentrate and mix thoroughly.

6. Use of a combined double-staining immunocytochemistry detection kit according to claim 3, characterized in that: Prepare goat anti-mouse / rabbit secondary antibody working mixture: add goat anti-mouse secondary antibody working solution to goat anti-rabbit secondary antibody working solution and mix.

7. A combined double-staining immunocytochemistry detection kit and its use according to claim 3, characterized in that: Prepare DAB working mixture: mix DAB staining substrate and DAB staining buffer.

8. The use of a combined double-staining immunocytochemistry detection kit according to claim 3, characterized in that: Prepare RED working mixture: mix AP-red color development buffer and AP-red chromogen concentrate.

9. The use of a combined double-staining immunocytochemistry detection kit according to claim 3, characterized in that: Perform p16 / Ki-67 immunohistochemical staining on cell preparation 1: first, add the p16 / Ki-67 primary antibody working mixture to cell preparation 1; then, add the goat anti-mouse / rabbit secondary antibody working mixture to cell preparation 1, and finally, add DAB working mixture, RED working mixture, and hematoxylin in sequence for staining.

10. The use of a combined double-staining immunocytochemistry detection kit according to claim 3, characterized in that: Perform CLDN18.2 / Ki-67 immunohistochemical staining on cell preparation 2: First, add the CLDN18.2 / Ki-67 primary antibody working mixture to cell preparation 2, then add the goat anti-mouse / rabbit secondary antibody working mixture to cell preparation 2, and finally add DAB working mixture, RED working mixture, and hematoxylin in sequence for staining.

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