Immunohistochemical kit for diagnosing main subtypes of lung cancer, application and detection method

Abstract

The invention belongs to the field of biological medicine, and particularly relates to an immunohistochemical kit for diagnosis of main subtypes of lung cancer, application and a detection method. The detection method comprises the following steps: (1) preparing a paraffin tissue sample; (2) sequentially slicing, baking and dewaxing and hydrating the paraffin tissue sample; (3) eliminating endogenous peroxidase in the tissue slice; (4) sealing the tissue slice, and incubating an antibody on the tissue slice; (5) sequentially dyeing and dehydrating the tissue slice; (6) carrying out sealing treatment on the tissue slice, and then carrying out microscopic imaging; (7) quantifying the expression level of SOX30 in cell nucleus and cytoplasm, performing ROC curve analysis, and finally making a judgment; wherein the main subtypes of the lung cancer are lung adenocarcinoma and lung squamous cell carcinoma; the judgment rule in the step (7) is that the SOX30 protein is only expressed in cytoplasm, and the sample is judged to be lung adenocarcinoma when the expression specificity in a cell nucleus is deleted; the SOX30 protein is expressed in both the cell nucleus and the cytoplasm, and when the total expression quantity of the SOX30 protein in the cell nucleus and the cytoplasm is obviously higher than that of normal tissues, the sample is judged to be lung squamous cell carcinoma. The examination method can accurately distinguish lung adenocarcinoma, lung squamous carcinoma and normal tissues, is simple, can improve the early diagnosis rate and treatment progress, and improves the prognosis of patients.

Description

Technical Field

[0001] This application belongs to the field of biomedicine, and specifically relates to an immunohistochemistry kit for diagnosing the main subtypes of lung cancer, its uses, and detection methods. Background Art

[0002] Lung cancer is one of the malignant tumors with the highest incidence and mortality rates globally, seriously threatening human health. In 2022, there were 2.48 million new cases of lung cancer globally, accounting for 12.4% of the total number of new cancer cases, and the mortality rate accounted for 18.7% of the total global cancer mortality rate, far exceeding the incidence and mortality rates of other cancers. Due to the large population base in China, the incidence rate of lung cancer in 2022 was 106.06 / 4.824 million (21.99%), and the mortality rate was 73.33 / 2.5442 million (28.49%), higher than the global data, making it the leading cause of cancer incidence and death in China. It is predicted that the incidence rate of lung cancer in China will remain high in the next few decades, becoming a major public health challenge in China. Despite the significant progress made in the diagnosis and treatment of lung cancer in recent years, the treatment and prognosis of patients are still not ideal. Late detection and delayed diagnosis are the main reasons for the extremely high mortality rate of lung cancer.

[0003] Clinically, lung cancer is divided into non-small cell lung cancer (NSCLC) and small cell lung cancer. Non-small cell lung cancer accounts for 85% - 90% of the total number of lung cancer patients, with a wide coverage, and is the main focus of current and future lung cancer treatment. Among non-small cell lung cancer, lung adenocarcinoma (ADC) and lung squamous cell carcinoma (SCC) are common histological subtypes, accounting for approximately 90%. Different pathological classifications have obvious differences in tissue location, pathological characteristics, treatment regimens, and prognosis. Currently, the main molecular characteristics and mechanisms of ADC and SCC are still unclear, and there is no rapid and accurate diagnostic method. At present, the diagnosis of clinical lung cancer subtypes still mainly relies on the judgment of pathological characteristics, which takes a long time and requires high professionalism, seriously delaying the implementation of effective treatment measures.

[0004] Therefore, there is an urgent need to find new reliable, rapid, popular, and accurate identification methods for more effective precise diagnosis and treatment of lung cancer. Summary of the Invention

[0005] Technical Problem

[0006] This application provides a kit for rapidly, popularly, and accurately detecting ADC and SCC, which can help clinical lung cancer patients quickly identify the lung cancer subtype, thereby formulating precise treatment plans, improving the early diagnosis rate and treatment progress, and improving the prognosis of patients.

[0007] Technical Solution

[0008] The first aspect of the present invention provides a method for qualitatively detecting the main subtypes of lung cancer using an immunohistochemistry kit for non-diagnostic purposes. The steps of the detection method are as follows: (1) Prepare a paraffin tissue sample; (2) Successively perform sectioning, baking, dewaxing and hydration treatment, and antigen repair on the paraffin tissue sample; (3) Eliminate endogenous peroxidase in the tissue section; (4) Block the tissue section, and then incubate the antibody on the tissue section; (5) Successively perform staining and dehydration treatment on the tissue section; (6) Mount the tissue section, and then perform microscopic imaging; (7) Quantify the expression levels of SOX30 in the cell nucleus and cytoplasm, and perform ROC curve analysis, and finally make a judgment; wherein, the main subtypes of lung cancer are ADC and SCC; the judgment rule in step (7) is: when SOX30 protein is only expressed in the cytoplasm and specifically lacks expression in the cell nucleus, the sample is judged to be ADC; when SOX30 protein is expressed in both the cell nucleus and cytoplasm, and the total expression level in the cell nucleus and cytoplasm is more than 1.3 times that of normal tissue, the sample is judged to be SCC.

[0009] The SOX gene is a class of nuclear transcription factors, a gene superfamily composed of SRY (sex determination region of Y chromosome)-related genes. All SOX members contain an SRY-related HMG box DNA-binding domain, which is widely involved in the regulation of embryonic development and cell fate determination and plays an important role in the formation of various organs and tumorigenesis. The transcription factor SOX30 (SRY box containing gene 30) is the only member of the H subgroup of the SOX gene family. It contains a conserved HMG box DNA domain and has been proven to be an essential gene for spermatogenesis, participates in gonadal development, and plays a role as a tumor suppressor gene in the progression and prognosis of ovarian cancer, lung cancer, and acute myeloid leukemia.

[0010] In some embodiments, when SOX30 protein is expressed in both the cell nucleus and cytoplasm, and the value of the total expression level in the cell nucleus and cytoplasm is greater than 4, the sample is judged to be SCC.

[0011] In some embodiments, the immunohistochemistry kit includes the following reagents: Reagent A: Monoclonal antibody against SOX30 protein, Reagent B: HRP-labeled secondary antibody working solution, Reagent C: Peroxidase blocker, Reagent D: Blocking agent, Reagent E: Antibody diluent, Reagent F: Chromogenic agent, Reagent G: Counterstain.

[0012] In some embodiments, Reagent A is a mouse anti-human monoclonal antibody against SOX30 protein; Reagent B is HRP-labeled rabbit anti-mouse IgG.

[0013] In some embodiments, the peroxidase blocker is 3% H2O2, and the blocking agent is bovine serum albumin (BSA).

[0014] The antigen retrieval means is heat retrieval.

[0015] In some embodiments, the antibody diluent is one of PBS and the primary antibody diluent for immunostaining.

[0016] In some embodiments, the chromogenic agent is DAB chromogenic solution, and the counterstain is hematoxylin counterstain solution.

[0017] In some embodiments, the dewaxing and hydration treatment is specifically as follows: soak in 100% ethanol for 5 - 8 min twice, soak in 95% ethanol for 3 - 5 min once, soak in 90% ethanol for 3 - 5 min once, soak in 80% ethanol for 3 - 5 min once, soak in 70% ethanol for 3 - 5 min once, soak in 60% ethanol for 3 - 5 min once, wash with distilled water for 3 - 5 min once, and wash with PBS for 3 - 5 min twice.

[0018] In some embodiments, the antibody incubation is specifically as follows: drop the monoclonal antibody of SOX30 protein on the tissue section and incubate overnight at 4°C, wash with PBS for 3 - 5 min three times; drop the working solution of the secondary antibody labeled with HRP on the tissue section, incubate at room temperature for 50 - 60 min, and wash with PBS buffer for 3 - 5 min three times.

[0019] The second aspect of the present invention provides an immunohistochemistry kit, and the immunohistochemistry kit uses the detection method described in any one of the above to perform qualitative detection of the main subtypes of lung cancer.

[0020] The third aspect of the present invention provides the use of the above immunohistochemistry kit for identifying the main subtypes of lung cancer for non - disease diagnosis and treatment purposes, and the main subtypes of lung cancer are ADC and SCC.

[0021] Technical effects

[0022] Our research findings indicate that subcellular expression localization plays an important guiding role in the rapid diagnosis and effective clinical intervention of lung cancer subtypes. Specifically, at the protein level, the overall expression level of SOX30 protein is significantly elevated in lung cancer tissues. There is no difference in its expression in adenocarcinoma of the lung (ADC), but it is significantly elevated in squamous cell carcinoma of the lung (SCC). Therefore, the overall expression level of SOX30 in tissues can be used to rapidly diagnose lung cancer and SCC subtypes. The subcellular localization of SOX30 protein shows that SOX30 is specifically absent in the nuclei of ADC, indicating that ADC can be rapidly diagnosed by detecting the subcellular localization of SOX30 protein. Further, by detecting the expression levels of SOX30 in the nucleus and cytoplasm, lung cancer, ADC, and SCC lung cancer subtypes can also be distinguished and diagnosed: compared with the nuclei of normal tissues, the level of SOX30 in the nuclei of ADC tissues is significantly decreased, and the level of SOX30 in the cytoplasm is significantly increased; the levels of SOX30 in both the nuclei and cytoplasm of SCC tissues are significantly increased. In tumor tissues, ADC and SCC can be rapidly distinguished by detecting the overall expression level and the expression level in the nucleus of SOX30: compared with ADC, the overall level of SOX30 and the level of SOX30 in the nucleus in SCC tissues are both significantly increased. In summary, the present invention provides a new method for detecting and judging to rapidly distinguish lung cancer and its subtypes, that is, to rapidly and accurately distinguish lung cancer, ADC, and SCC subtypes by the subcellular localization and expression level of SOX30 protein; this method is simple and easy to operate, with a high accuracy rate, can improve the diagnosis rate of early lung cancer, enable patients to receive targeted treatment in a timely manner, thereby improving the prognosis of patients, and has high diagnostic value. Description of the Drawings

[0023] Figure 1 Microscopic imaging diagrams of immunohistochemistry (IHC) for adjacent normal tissues, adenocarcinoma of the lung (ADC), and squamous cell carcinoma of the lung (SCC) tissues, with a scale bar of 20 μm;

[0024] Figure 2A Results of the analysis of the expression differences of SOX30 protein levels in non-small cell lung cancer tissues (NSCLC, including ADC and SCC samples) and normal tissues and the corresponding ROC curves;

[0025] Figure 2B Results of the analysis of the expression differences of SOX30 protein levels in ADC and normal tissues and the corresponding ROC curves;

[0026] Figure 2C Results of the analysis of the expression differences of SOX30 protein levels in SCC and normal tissues and the corresponding ROC curves;

[0027] Figure 2D Results of the analysis of the expression differences of SOX30 protein levels in ADC and SCC tissues and the corresponding ROC curves;

[0028] Figure 3A The expression difference of SOX30 protein level in the nucleus of ADC cells and the nucleus of normal tissues and the corresponding ROC curve analysis;

[0029] Figure 3B The expression difference of SOX30 protein level in the cytoplasm of ADC cells and the cytoplasm of normal tissues and the corresponding ROC curve analysis;

[0030] Figure 4A The expression difference of SOX30 protein level in the nucleus of SCC and normal tissues and the ROC curve analysis;

[0031] Figure 4B The expression difference of SOX30 protein level in the cytoplasm of SCC and normal tissues and the ROC curve analysis;

[0032] Figure 5A The expression difference and ROC curve analysis of SOX30 protein level in normal tissues and the nucleus of ADC cells;

[0033] Figure 5B The expression difference and ROC curve analysis of SOX30 protein level in normal tissues and the cytoplasm of ADC cells;

[0034] Figure 6A The expression difference and ROC curve analysis of SOX30 protein level in the nucleus of ADC and SCC cells;

[0035] Figure 6B The expression difference and ROC curve analysis of SOX30 protein level in the cytoplasm of ADC and SCC cells. Detailed implementation manners

[0036] For the convenience of describing the technical solutions of the application, the following first gives a general description and definition of the terms and expressions involved in this application.

[0037] The term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, such that a process, method, article or device comprising a series of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article or device. Without further limitation, an element defined by the phrase "comprising a..." does not exclude the presence of additional identical elements in the process, method, article or device comprising the element.

[0038] All lung cancer tissue samples had not received treatments such as radiotherapy and chemotherapy before surgery, and the tissue specimens were diagnosed and identified by at least 2 pathological experts. Informed consent forms from the patients were obtained for all samples used in the study, and the study was approved by the ethics committee of the relevant institution.

[0039] In the present invention, subcellular localization can localize a certain protein or expression product to a specific location in the cell, such as in the nucleus, various organelles, and the plasma membrane, thereby providing a research direction for understanding the mechanism of action of genes.

[0040] In each group of comparative experiments provided in this application, unless otherwise specified, except for the differences pointed out in each group, other experimental conditions, materials, etc. are kept consistent for comparability.

[0041] The reagents, instruments, and equipment used in the embodiments of the present invention can be purchased from the market without detailed description.

[0042] The following further describes an immunohistochemistry kit, its use, and detection method for diagnosing the main subtypes of lung cancer provided in this application.

[0043] Example 1 Section Preparation

[0044] The tissue obtained by surgery was fixed in formalin, dehydrated with gradient alcohol, embedded, and sectioned. The sections were baked in an oven at 65 °C for 2 h to remove moisture. The sections were dewaxed by soaking in xylene 3 times, 8 min each time; soaked in absolute ethanol 2 times, 5 min each time; soaked in gradient alcohol (95%-90%-80%-70%-60%) once each, 3 min each time. Washed once with distilled water; washed 2 times with PBS buffer solution, 3 min each time. Antigen retrieval was performed with sodium citrate buffer solution (boiled for 5 min), and washed 2 times with PBS buffer solution, 3 min each time; treated with 3% H2O2 at room temperature in the dark for 10 min to eliminate endogenous peroxidase, and then washed 2 times with PBS buffer solution, 3 min each time. Finally, blocked by incubating with bovine serum albumin (BSA) at room temperature for 15 min.

[0045] Example 2 Incubation and Color Development of SOX30 Antibody

[0046] Add the mouse anti-human monoclonal antibody of diluted SOX30 protein (purchased from Santa Cruz Biotechnology, diluted with PBS at a dilution ratio of 1:100) dropwise to the section that has been blocked, place it in a refrigerator at 4°C and incubate overnight, then wash it 3 times with PBS buffer for 3 minutes each time. Add the diluted secondary antibody solution (i.e., rabbit anti-mouse IgG, diluted with PBS at a dilution ratio of 1:500), incubate at room temperature for 50 minutes, then wash it 3 times with PBS buffer for 3 minutes each time. Add the horseradish peroxidase-labeled solution with the working concentration, incubate at room temperature for 15 minutes, then wash it 3 times with PBS buffer for 3 minutes each time. Add the chromogenic working solution DAB, observe the staining and terminate it, then rinse with tap water for 1 hour. Counterstain with hematoxylin solution for 30 seconds, and rinse with tap water again for 30 minutes. Differentiate and clear the section with 1% hydrochloric acid-alcohol, rinse with tap water for bluing, air dry, then mount the section with neutral resin, let it stand for a while, and then examine it under a microscope and take pictures for analysis.

[0047] Example 3 ROC curve (Receiver Operating Characteristic curve) analysis

[0048] Import the quantitative data of SOX30 expression in the nuclei and cytoplasm of ADC and SCC into the graphing and analysis software GraphPad Prism 8.0. In the software toolbar, select Analysis → Column analyses → ROC Curve. The control values are the normal lung tissue group, and the patient values are the lung cancer tissue group. Statistical analysis is performed using the Wilson method with a 95% confidence interval, and the area under the curve (AUC) is calculated.

[0049] We analyzed the expression of SOX30 in ADC and SCC tissues by IHC (as Figure 1 shown), and performed quantitative analysis and ROC curve analysis through staining intensity and positive rate.

[0050] The evaluation criteria for staining intensity and positive rate are as follows:

[0051] The expression level of SOX30 was quantified by a cell staining intensity and positive rate scoring system. When the IHC staining immunoreactivity was ≥10%, SOX30 was evaluated as positive. The positive rate of cell staining was divided into 5 grades: 0 points (immunoreactivity <10%), 1 point (immunoreactivity 10% - 25%), 2 points (immunoreactivity 26% - 50%), 3 points (immunoreactivity 51% - 75%), 4 points (immunoreactivity ≥76%). The cell staining intensity was divided into 4 grades: negative (0 points), weakly positive (1 point), moderately positive (2 points), strongly positive (3 points). Protein expression was quantified by multiplying the cell staining intensity score by the cell staining positive rate score, and the range of the protein quantitative expression value was 0 - 12 points.

[0052] The criteria for ROC curve analysis were as follows:

[0053] The ROC curve used the TPR (sensitivity, also known as the true positive rate) as the Y-axis. The higher this index, the higher the diagnostic accuracy. The FPR (1 - Specificity, also known as the false positive rate) was used as the X-axis to classify different predicted values. Different TPRs and FPRs corresponded to each point on the ROC curve. Therefore, the ROC curve reflected the dynamic relationship curve between the FPR and the TPR. The faster the TPR increased, that is, the higher the sensitivity and the lower the misjudgment rate. The diagnostic value of the SOX30 expression level in NSCLC, ADC, and SCC was detected by the AUC and P values. Among them, the AUC value was between 0.5 and 1.0. A larger AUC value represented a better classification effect; when 0.9 < AUC < 1.0, it indicated a very good diagnostic value; when 0.7 < AUC < 0.9, it indicated a good diagnostic value; when 0.5 < AUC < 0.7, it indicated a diagnostic value; when AUC < 0.5, it indicated no diagnostic value.

[0054] According to the above evaluation criteria, the results of the present invention were as follows:

[0055] a) IHC staining showed that the protein localization of SOX30 was different in normal tissues, lung adenocarcinoma, and lung squamous cell carcinoma; the statistical results showed that SOX30 was expressed in the cell nucleus and cytoplasm of adjacent normal tissues and squamous cell carcinoma tissues, while it was only expressed in the cytoplasm of ADC tissues (as shown in Table 1). These results suggested that the expression localization of SOX30 protein could be used to quickly diagnose lung adenocarcinoma.

[0056] Table 1 Statistical results of the expression localization of SOX30 in adjacent normal tissues, lung adenocarcinoma, and lung squamous cell carcinoma tissues of lung cancer

[0057]

[0058] Statistical analysis of the expression and localization of SOX30 in adjacent tissues of lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma tissues (n = 148; among them, n = 75 for adenocarcinoma and n = 73 for squamous cell carcinoma)

[0059] b) The average expression levels in the cell nucleus and cytoplasm were used as the overall expression level of SOX30 in tumor cells / normal cells. The results showed that the overall expression level of SOX30 protein was significantly higher in non-small cell lung cancer tissues than in normal tissues (as Figure 2A shown), indicating that normal tissues and non-small cell lung cancer tissues could be distinguished by the expression level of SOX30 protein.

[0060] c) ROC curve analysis was performed on the expression level of SOX30 protein. The results showed that the expression level of SOX30 could diagnose normal tissues and non-small cell lung cancer tissues (as Figure 2A shown: AUC = 0.7145, P < 0.0001, sensitivity was 77.33%, and specificity was 75.33%).

[0061] d) Quantitative staining of the IHC samples of the above-mentioned ADC and SCC found that there was no significant difference in the overall level of SOX30 protein between ADC and adjacent normal tissues (as Figure 2B shown), but it was significantly increased in SCC (as Figure 2C shown); furthermore, the overall level of SOX30 protein in ADC was significantly lower than that in SCC (as Figure 2D shown).

[0062] e) ROC curve analysis showed that the overall level of SOX30 protein could not distinguish ADC from normal tissues (as shown in 2B: AUC = 0.5328, P < 0.4880, sensitivity was 73.33%, and specificity was 60%), but it could distinguish SCC from normal tissues well (as shown in 2C: AUC = 0.8385, P < 0.0001, sensitivity was 84%, and specificity was 90.67%); moreover, the expression of SOX30 could diagnose SCC from ADC well (as shown in 2D: AUC = 0.7802, P < 0.0001, sensitivity was 78.67%, and specificity was 88%).

[0063] f) The expression levels of SOX30 in the cell nucleus and cytoplasm of tissues were separately statistically analyzed. The results showed that, compared with normal tissues, the expression level of SOX30 in the cell nucleus of non-small cell lung cancer tissues was significantly decreased, and tumor tissues could be distinguished from normal tissues (as Figure 3A shown: AUC = 0.6716, P < 0.001, sensitivity was 70.67%, and specificity was 83.33%). The expression of SOX30 in the cytoplasm of non-small cell lung cancer tissues was significantly higher than that in normal tissues and had good diagnostic value (asFigure 3B As shown: AUC = 0.8297, P < 0.0001, sensitivity was 84.67%, and specificity was 99.33%). These results indicate that normal tissues and non-small cell lung cancer tissues can be distinguished and diagnosed by the expression level of SOX30 in subcellular compartments.

[0064] g) Combining the different localizations of SOX30 protein in ADC, SCC, and normal tissues, the IHC staining quantitative results also showed that: the expression level of SOX30 protein in the nucleus of ADC was significantly lower than that in the nucleus of normal tissues (as Figure 4A shown); while the expression level of SOX30 protein in the cytoplasm of ADC cells was significantly higher than that in the cytoplasm of normal lung tissues (as Figure 4B shown). Compared with normal lung tissues, higher levels of SOX30 protein expression were observed in the nuclei and cytoplasm of SCC (as Figures 2A - 2D well as shown in Figure 5).

[0065] h) ROC curve analysis showed that the expression level of SOX30 in the nucleus of ADC could diagnose ADC very well (as Figure 4A shown: AUC = 0.9933, P < 0.0001, sensitivity was 100%, and specificity was 98.67%); the expression level of SOX30 in the cytoplasm of ADC could also diagnose ADC well (as Figure 4B shown: AUC = 0.8303, P < 0.0001, sensitivity was 85.33%, and specificity was 98.67%); in SCC, the expression level of SOX30 in the nucleus of SCC could diagnose SCC well (as Figure 5A shown: AUC = 0.7684, P < 0.0001, sensitivity was 73.33%, and specificity was 76%), and the expression level of SOX30 in the cytoplasm could also diagnose SCC well (as Figure 5B shown: AUC = 0.8290, P < 0.0001, sensitivity was 84%, and specificity was 100%).

[0066] i) Further, the expression level of SOX30 in the nucleus of ADC was significantly lower than that in SCC, while there was no significant difference in the expression level of SOX30 in the cytoplasm of ADC and that in the cytoplasm of SCC; ROC curve analysis showed that the expression of SOX30 in the nucleus could well distinguish SCC from ADC (as Figure 6A shown: AUC = 0.9667, P < 0.0001, sensitivity was 93.33%, and specificity was 100%). The diagnostic value of the expression of SOX30 in the cytoplasm was poor in ADC and SCC (as Figure 6BShown: AUC = 0.5031, P = 0.9476, sensitivity was 73.33%, and specificity was 26.67%). These results indicate that the expression levels of SOX30 in the cytoplasm and nucleus can distinguish and diagnose ADC and SCC subtypes.

[0067] These results indicate that the expression of SOX30 is significantly different between non-small cell lung cancer tissues and normal tissues; and there are obvious differences in the expression of SOX30 in non-small cell lung cancer subtypes ADC tissues and SCC tissues. The expression of SOX30 is generally up-regulated in non-small cell lung cancer tissues, and is very significantly up-regulated in the SCC subtype. Compared with ADC tissues, the expression of SOX30 in SCC is also significantly increased, indicating that there are obvious differences in the expression levels of SOX30 in ADC and SCC. Subcellular localization shows that the expression of SOX30 is specifically absent in the nucleus of ADC, indicating that ADC can be quickly diagnosed by the subcellular localization of SOX30 protein. Further, the expression levels of SOX30 in the cytoplasm and nucleus can distinguish normal and non-small cell lung cancer tissues: compared with normal tissues, the overall expression level of SOX30 in the nucleus of non-small cell lung cancer tissues decreases, while the expression level in the cytoplasm increases significantly. For these non-small cell lung cancer tissues, according to the expression levels of SOX30 protein in the cytoplasm / nucleus, ADC and SCC subtypes can be further distinguished and diagnosed: compared with normal tissues, SOX30 is lowly expressed in the nucleus of ADC and highly expressed in the cytoplasm; while SOX30 is highly expressed in both the nucleus and cytoplasm of SCC; and compared with ADC, the expression of SOX30 in the nucleus of SCC is significantly higher. In summary, according to the subcellular localization, overall expression level and subcellular expression level of SOX30 protein, non-small cell lung cancer patients can be distinguished and diagnosed, and ADC and SCC subtypes can be further distinguished and diagnosed.

[0068] The above specific embodiments further elaborate on the purpose, technical solutions and beneficial effects of the present application. It should be understood that the above are only specific embodiments of the present application and are not used to limit the protection scope of the present application. Any modifications, equivalent replacements, improvements, etc. made on the basis of the technical solutions of the present application should be included in the protection scope of the present application.

Claims

1. An immunohistochemical kit for qualitative detection of major subtypes of lung cancer for non-diagnostic purposes, characterized in that: The steps of the detection method are: (1) Preparation of paraffin tissue samples; (2) The paraffin tissue samples are sliced, baked, dewaxed and hydrated in sequence; (3) Eliminate endogenous peroxidase in tissue sections; (4) blocking the tissue sections and then incubating the antibodies on the tissue sections; (5) staining and dehydrating the tissue sections in sequence; (6) Seal the tissue sections and then perform microscopic imaging; (7) Quantify the expression levels of SOX30 in the nucleus and cytoplasm and perform ROC curve analysis to make a judgment; Among them, the main subtypes of lung cancer are lung adenocarcinoma and lung squamous cell carcinoma; The judgment rule described in step (7) is: when the SOX30 protein is only expressed in the cytoplasm and specifically lacks expression in the nucleus, the sample is judged to be lung adenocarcinoma; when the SOX30 protein is expressed in both the nucleus and cytoplasm, and the total expression level in the nucleus and cytoplasm is more than 1.3 times that of normal tissue, the sample is judged to be lung squamous cell carcinoma.

2. The detection method according to claim 1, characterized in that: The immunohistochemistry kit includes the following reagents: Reagent A: Monoclonal antibody against SOX30 protein, Reagent B: HRP-labeled secondary antibody working solution, Reagent C: Peroxidase Blocker, Reagent D: blocking agent, Reagent E: Antibody diluent, Actual F: color developer, Reagent G: Counterstain.

3. The detection method according to claim 2, characterized in that: The reagent A is a mouse anti-human monoclonal antibody against SOX30 protein; the reagent B is a rabbit anti-mouse IgG labeled with HRP.

4. The method according to claim 2, characterized in that: The peroxidase blocking agent is 3% H2O2, and the sealing agent is bovine serum albumin.

5. The detection method according to claim 2, characterized in that: The antibody diluent was PBS.

6. The detection method according to claim 2, characterized in that: The color developer is DAB color developer, and the counterstain is hematoxylin counterstain.

7. The detection method according to claim 1, characterized in that: The dewaxing and hydration treatment is specifically as follows: soaking in 100% ethanol for 5 to 8 minutes twice, soaking in 95% ethanol for 3 to 5 minutes once, soaking in 90% ethanol for 3 to 5 minutes once, soaking in 80% ethanol for 3 to 5 minutes once, soaking in 70% ethanol for 3 to 5 minutes once, soaking in 60% ethanol for 3 to 5 minutes once, washing with distilled water for 3 to 5 minutes once, and washing with PBS for 3 to 5 minutes twice.

8. The detection method according to claim 1, characterized in that: The incubation antibody is specifically as follows: adding monoclonal antibody of SOX30 protein to the tissue section, incubating at 4°C overnight, and washing with PBS for 3 to 5 minutes 3 times; adding HRP-labeled secondary antibody working solution to the tissue section, incubating at room temperature for 50 to 60 minutes, and washing with PBS buffer for 3 to 5 minutes 3 times.

9. An immunohistochemistry kit, characterized in that: The immunohistochemistry kit uses the detection method described in any one of claims 1 to 8 to perform qualitative detection of major subtypes of lung cancer.

10. Use of the immunohistochemical kit according to claim 9 for identifying major subtypes of lung cancer for purposes other than disease diagnosis and treatment, characterized in that: The main subtypes of lung cancer are lung adenocarcinoma and lung squamous cell carcinoma.

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