Use of biomarkers based on the function of ldha in the diagnosis of tumors

Abstract

The application provides application of a modified form of LDHA, LDHA S319 phosphorylation, as a biomarker in preparation of a tumor diagnosis reagent, and provides a kit for quantitatively or semi-quantitatively detecting a protein, which contains an antibody capable of specifically combining with LDHA S319 phosphorylation. The application can more effectively preliminarily evaluate and predict whether a subject has a tumor by detecting the expression level of LDHA S319 phosphorylation. The application can help improve the diagnosis accuracy of the tumor and provide an important basis for early treatment of the patient. The application also provides application of the antibody of LDHA S319 phosphorylation in preparation of a tumor diagnosis kit.

Description

Technical Field

[0001] This invention relates to the application of LDHA-based biomarkers in tumor diagnosis, belonging to the field of oncology medicine. Background Technology

[0002] The key to cancer treatment lies in early detection and diagnosis, so that appropriate treatment measures can be taken. Currently, only by striving for early treatment can cancer be completely cured. If the cancer develops to the middle or late stage, and the tumor tissue expands or has metastasized, it is difficult to cure even with various treatment methods. Conversely, if the cancer can be detected in the early or relatively early stage and before metastasis occurs, and timely and reasonable treatment is given, a considerable number of patients can achieve satisfactory treatment results, and even achieve a radical cure.

[0003] Currently, tumor diagnosis methods are classified into five levels: ① Clinical diagnosis: a presumptive diagnosis based on clinical symptoms, signs, and imaging examinations, referencing the disease progression pattern; ② Surgical diagnosis: a judgment made solely based on visual observation of the tumor after surgery or various endoscopic examinations, without pathological confirmation; ③ Physicochemical diagnosis: clinically consistent with cancer presentation, supported by positive physiochemical results, such as X-ray, ultrasound, CT, and MRI examinations, or carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) measurements; ④ Cytopathological diagnosis: a diagnosis based on various exfoliated cells and fine-needle aspiration cytology; ⑤ Histopathological diagnosis: a diagnosis obtained through pathological analysis of tissue obtained via core needle biopsy. The reliability of the diagnosis increases sequentially among these five levels, with level five being the most ideal. However, the methods mentioned above cannot achieve early detection and diagnosis of tumors. More often, the tumor has progressed to a certain stage, or even when the patient presents with severe symptoms and signs, at which point the optimal treatment period is often missed, leaving later treatment in a highly passive position. Furthermore, pathological diagnosis, which yields relatively good results, requires puncture to obtain cells or tissue sections, which can cause significant harm and negative impact on the patient's mental and physical well-being. Therefore, finding more effective biomarkers to achieve early diagnosis of tumors is crucial.

[0004] Current research on the mechanisms of liver cancer development and progression remains insufficient, severely hindering the development of new diagnostic and therapeutic approaches for this type of tumor. Lactate dehydrogenase A (LDHA) is a key glycolytic enzyme that catalyzes the conversion of pyruvate to lactate in the final step of glycolysis. Lactate produced by glycolysis serves as a substrate for histone or non-histone lactation, regulating gene expression and protein activity in immune and cancer cells in a glycolysis-dependent manner. Although key metabolic functions of LDHA have been identified in glycolysis, its potential role in iron deficiency anemia and tumor development during immunotherapy remains unclear. Ferroptosis, a different form of regulatory cell death, depends on iron accumulation and is driven by an increase in intracellular lipid peroxides. It plays a crucial role in cancer development and immunotherapy.

[0005] Given the above background, in order to more accurately determine tumor development, more rationally select treatment options, and thus improve the effectiveness of tumor immunotherapy, it is necessary to find key biomarkers related to tumor cell ferroptosis and immune escape mechanisms for early tumor diagnosis. Summary of the Invention

[0006] The technical problem or primary objective of this invention is to make an early diagnosis of tumors in subjects.

[0007] Another object of the present invention is to provide a product for the aforementioned early diagnosis.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In one aspect, the present invention uses the modified form of LDHA, LDHA S319 phosphorylation, as a biomarker for the preparation of tumor diagnostic reagents.

[0010] In the present invention, LDHA S319 phosphorylation refers to phosphorylation at position 319 of the amino acid sequence of LDHA; the NCBI sequence number of LDHA is NP_005557.1.

[0011] In the scheme described in this invention, early diagnosis of cancer in the subject can be achieved by detecting the expression level of LDHA S319 phosphorylation in a sample from the subject using an immunoassay reagent targeting LDHA S319 phosphorylation. Furthermore, the sample from the subject can be any one of the subject's peripheral venous blood, adjacent normal tissue, or tumor tissue.

[0012] In the applications described in this invention, the tumor diagnostic reagent is an immunoassay reagent based on ELISA or Western Blot methods.

[0013] Secondly, the present invention also provides an antibody that is capable of specifically binding to LDHA S319 phosphorylation.

[0014] Thirdly, the present invention also provides a kit for quantitative or semi-quantitative detection of proteins, containing the antibody described in the second aspect of the present invention.

[0015] In a preferred embodiment of the present invention, the reagent kit for quantitative or semi-quantitative detection of proteins is a reagent kit for detecting proteins by enzyme-linked immunosorbent assay (ELISA) or Western blotting; the reagent kit contains a first antibody and a second antibody; the first antibody is an antibody that can specifically bind to LDHA S319 phosphorylation, and the second antibody is an enzyme-labeled antibody homologous to the first antibody.

[0016] Fourthly, the present invention also provides the use of antibodies that specifically bind to phosphorylated LDHA S319 in the preparation of tumor diagnostic kits.

[0017] In a further preferred embodiment of the present invention, the kit further comprises one or more substances selected from the group consisting of: a container, instructions for use, a positive control, a negative control, a buffer, an adjuvant, or a solvent. The instructions for use describe how to perform the test using the kit, and how to use the test results to assess tumor development and select a treatment plan. The components of the kit may be packaged in an aqueous medium or in a lyophilized form.

[0018] The tumor diagnostic kit described in this invention can be used for the early, intermediate, or late-stage diagnosis of any of the following tumor diseases: oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory system cancer, genitourinary system cancer, gastrointestinal cancer, central or peripheral nervous system tissue cancer, endocrine or neuroendocrine system cancer, hematopoietic system cancer, glioma, sarcoma, epithelial cancer, lymphoma, melanoma, fibroma, meningioma, brain cancer, kidney cancer, biliary system cancer, pheochromocytoma, islet cell carcinoma, Levi-Flaumeni tumor, thyroid cancer, parathyroid cancer, pituitary adenoma, adrenal adenoma, bone-derived sarcoma, neuroendocrine system tumor, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer.

[0019] This invention proposes the application of a novel biomarker, LDHA S319 phosphorylation, in tumor diagnosis. By detecting the expression level of this biomarker, the condition of a subject with tumors can be diagnosed at an early, intermediate, or late stage.

[0020] The LDHA S319 phosphorylation described in this invention, as a specific modified form of LDHA, is highly correlated with ferroptosis and immune escape mechanisms in tumor cells, and induces immune escape of cancer cells through non-metabolic functions. Specifically, in previous studies, the inventors first discovered that when liver cancer patients received PD-1 immunotherapy, CD8... + IFNγ secreted by T cells can activate S6K, triggering activated S6K phosphorylation of LDHA at the S319 site. This phosphorylation of LDHA can inhibit ferroptosis in liver cancer cells, thereby reducing the efficacy of PD-1 immunotherapy in liver cancer. Furthermore, in clinical liver cancer specimens, the level of LDHA S319 phosphorylation is positively correlated with the expression of the ferroptosis marker 4-HNE, and is associated with poor prognosis in liver cancer patients. These findings reveal a key mechanism by which IFNγ induces immune escape from liver cancer cells during PD-1 immunotherapy by inducing LDHA S319 phosphorylation and inhibiting the non-metabolic function of ferroptosis. They also highlight that combination therapy targeting LDHA S319 phosphorylation and anti-PD-1 antibodies is a promising immunotherapy for HCC.

[0021] The above findings were obtained through the following research:

[0022] 1. Materials and Methods

[0023] 1.1 Materials Used

[0024] (1) Cell types: Huh7 cells (human liver cancer cell line Huh7 cells) and BEL-7405 cells (human liver cancer cell line Hep3B cells), from ATCC;

[0025] (2) The athymic nude mice were BALB / c athymic nude mice;

[0026] (3) Patient Samples: HCC tissue samples that had undergone surgical resection, formalin fixation, and paraffin embedding were retrospectively collected from the biobank of the Second Affiliated Hospital of Zhejiang University School of Medicine (Hangzhou, China). Tissue samples from 100 patients with pathologically diagnosed liver cancer who had not undergone surgical treatment were selected as an independent cohort. Clinical data were obtained by reviewing the patients' medical history. Pathological staging was assessed according to the 8th edition of the American Joint Committee on Cancer / International Consortium for the Classification of Cancer by TNM (TNM) system.

[0027] (4) The shRNA sequence used for gene knockdown is as follows:

[0028] LDHAshRNA:5′-GCAAACTCCAAGCTGGTCATT-3′;

[0029] S6KshRNA: 5′-CCCATGATCTCCAAACGGCCA-3′.

[0030] 1.2 Test Methods

[0031] (1) Detection of phosphorylated protein levels:

[0032] The amount of antibody bound is determined by contacting a sample with an antibody that specifically binds to a phosphorylated peptide, for example by detecting or measuring the path of the complex between the antibody and the peptide. The antibody can be labeled (radioactive, fluorescent, etc.) to facilitate the detection of the complex.

[0033] The peptide level detection system used in this invention includes: autoradiographic immunoassay (RIA), immunofluorescence, gel electrophoresis, and Western blotting.

[0034] Antibodies used (added): anti-FLAG (14793S) for IP), LDHA (3582S), S6K pT389 (9234S) (for WB), Ub (43124S), HA (3724S) (for IP), TYK2 pY1054 / 1055 (68790), TYK2 (14193), JAK2 pY1007 / 1008 (3771), JAK2 (3230), STAT3 pY705 (9145), STAT3 (9139), FLAG (F1804) (for WB), His (SAB1305538), 4-HNE (BS-6313R), GST (sc-138), TUBULIN (sc-8035) were purchased from CST, and the LDHA pS319 specific antibody was synthesized by Abclonal.

[0035] Mouse tumor sample acquisition and analysis: Six-week-old male immunocompetent mice were housed under specific pathogen-free conditions with a 14-hour / 10-hour light cycle, an ambient temperature of 21-23°C, and 50% humidity, with free access to water and food. Hep1-6 cells expressing luciferase (3 × 10⁻⁶ cells) were used for tumor sampling. 5 Anti-PD-1 was administered either by suspension in 20 μl of PBS and directly injected into the liver, or by suspension in 50 μl of PBS and subcutaneously injected into 6-week-old male immunocompetent mice, with or without anti-PD-1 administration starting from day 2. For anti-PD-1 blockade, each mouse received 200 μg of anti-PD-1 antibody or control IgG2a intraperitoneally every 3 days. Bioluminescent imaging of in situ carcinomas was performed using an IVIS system (AniView 100, Biolight Biotechnology, Guangzhou, China). Subsequently, the animals were euthanized, and the tumors of each mouse were dissected, fixed in 4% formaldehyde, and embedded in paraffin. Tumor formation and phenotype were assessed by histological analysis of sections stained with hematoxylin and eosin. The formula V = 1 / 2a was used.2 b calculates the tumor volume (where V is the volume, a is the shortest diameter, and b is the longest diameter).

[0036] (6) Immunohistochemistry: Paraffin-embedded human liver cancer samples were stained with specific primary antibodies and non-specific IgG as negative controls. The specific method was as follows: After dewaxing, rehydration, and antigen retrieval of the paraffin sections, TMA slides were incubated overnight at 4°C with primary antibody rabbit anti-human phosphorylated LDHA S319 (dilution 1:200) or non-specific IgG (as a negative control). The slides were then incubated with anti-rabbit secondary antibody (ready-to-use solution; CellSignaling Technology; #8114), followed by dichromatographic diaminobenzidine (DAB) staining (CellSignaling Technology) and hematoxylin staining, and fixed on xylene. The tissue slides were quantitatively scored under a microscope based on the percentage of positive cells and staining intensity. This invention assigns the following positive cell percentage scores: 0 points, 0% of cells are positive; 1 point, 0% to 1% of cells are positive; 2 points, 2% to 10% of cells are positive; 3 points, 11% to 30% of cells are positive; 4 points, 31% to 70% of cells are positive; and 5 points, 71% to 100% of cells are positive. Staining intensity is also graded on a scale of 0 to 3: 0, negative (i.e., no visible staining); 1, weak (very light color); 2, moderate (distinct color); 3, strong (very dark color). As previously described in the literature, the positive cell percentage score and staining intensity grade are summed to obtain a total score (range 0-8). The reproducibility of the scoring system was independently verified by two pathologists without clinical knowledge.

[0037] (7) Overall survival Kaplan-Meier plotting was performed using SPSS version 20.0 (SPSS Inc., Chicago, Illinois, USA). Independent samples t-tests were used to compare biomarker expression levels in tumor and normal tissues. One-way ANOVA (posthoc-Bonferroni test) was used for multiple comparisons and minimum significance tests to assess the correlation between biomarker expression levels and patient clinicopathological features. Pearson correlation coefficients were used to analyze the correlation between biomarker expression levels. Overall survival (OS) was defined as the duration from diagnosis to death or the last follow-up. K-means clustering analysis was used to classify the expression levels of relevant biomarkers, Kaplan-Meier survival curves were plotted, log-rank tests were used to compare survival rates, and Cox regression models with two-way Wald tests were used to calculate hazard ratios (HR) and 95% confidence intervals (CIs) for survival analysis. Data reviewed were for patients who were alive at the last follow-up or who were lost at follow-up. Variables with p-values ​​less than 0.05 were included in multivariate analyses. A p-value < 0.05 is considered statistically significant. All statistical tests are two-sided.

[0038] 2 Results and Analysis

[0039] (1) As Figure 1 As shown, after 12 hours of IFNγ treatment in Huh7 cells, immunoassay results indicated that IFNγ could induce the interaction between LDHA and S6K.

[0040] (2) Figure 2 As shown, Co-IP combined with mass spectrometry analysis revealed that the LDHA site in Huh7 cells treated with IFNγ could undergo phosphorylation modification.

[0041] (3) Figure 3 As shown, after the LDHA S319 site inactivation mutation, LDHA cannot be phosphorylated by S6K.

[0042] (4) Figure 4 As shown, flow cytometry analysis revealed that mutations at the S319A phosphorylation site and the K155R site of enzyme inactivation induced lipid peroxidation in cells.

[0043] (5) Figure 5 As shown, flow cytometry analysis revealed that mutations at the S319A phosphorylation site and the K155R site, which inhibit enzyme inactivation, induced cell death.

[0044] (6) Figure 6As shown, cell proliferation experiments revealed that mutations at the S319A phosphorylation site of LDHA and the K155R site of enzyme inactivation inhibited cell proliferation.

[0045] (7) LDHA phosphorylation site S319A mutation can increase the sensitivity of xenografts to anti-PD-1 therapy. Hep1-6-LUC cells were orally injected into 6-week-old male immunocompetent mice, and simultaneously treated with anti-PD-1. The results showed that LDHA S319 phosphorylation decreased the sensitivity of xenografts to anti-PD-1 therapy, while LDHA S319A mutation increased the sensitivity of xenografts to anti-PD-1 therapy. Figure 7A , Figure 7B , Figure 8A and Figure 8B Immunohistochemical experiments showed that LDHA S319A mutations can promote ferroptosis and improve the efficacy of PD-1 immunotherapy. Figure 9 ).

[0046] (8) LDHA S319 phosphorylation is associated with prognosis in patients with hepatocellular carcinoma. We analyzed human primary hepatocellular carcinoma specimens using validated anti-LDHA-pS319 immunohistochemical staining and found that the levels of these protein markers in hepatocellular carcinoma tissues were significantly higher than in adjacent normal tissues. Figure 10 Furthermore, a negative correlation was observed between LDHA-pS319 and the expression of the ferroptosis marker 4-HNE. Figure 11 Furthermore, phosphorylation of LDHAS319 is positively correlated with poor survival outcomes in liver cancer patients. Figure 12 ).

[0047] Compared with the prior art, the beneficial effects of the present invention are: 1. Based on the mechanism of tumor metabolism, the present invention has found new biomarkers for tumor metabolism, and early diagnosis of tumors can be achieved by detecting the expression level of biomarkers; 2. The test can be completed by collecting peripheral venous blood samples, which greatly reduces the psychological burden and physical damage to the subjects. Attached Figure Description

[0048] Figure 1 This reflects the results of immunoprecipitation and immunoblotting analysis performed after cell IFNγ treatment;

[0049] Figure 2 This demonstrates the post-translational modification of LDHA in Huh7 hepatocellular carcinoma cells identified by mass spectrometry.

[0050] Figure 3 This reflects the results of in vitro phosphorylation assays and S6K phosphorylation of LDHA;

[0051] Figure 4This demonstrates the effect of flow cytometry on LDHA S319 phosphorylation on cellular lipid peroxidation.

[0052] Figure 5 This demonstrates the effect of flow cytometry on LDHA S319 phosphorylation on cell death;

[0053] Figure 6 This study demonstrated the effect of LDHA S319 phosphorylation on cell proliferation as detected by cell proliferation assays.

[0054] Figure 7A , Figure 7B This study demonstrates the impact of LDHA S319 mutations on anti-PD-1 immunotherapy in mouse orthotopic xenografts.

[0055] Figure 8A , Figure 8B Figure 7 shows photographs of mouse xenografts and their weights.

[0056] Figure 9 This shows the immunohistochemical results of the mouse xenograft in Figure 7;

[0057] Figure 10 This reflects the immunohistochemical results of human liver cancer and adjacent normal samples;

[0058] Figure 11 This reflects the immunohistochemical results of different liver cancer patients;

[0059] Figure 12 This study reflects the relationship between LDHA S319 phosphorylation levels in liver cancer patients' tumor tissues and patient prognosis, as shown by the survival curve. Detailed Implementation

[0060] To make the objectives and technical solutions of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings. Unless otherwise specified, the experimental methods described in the following tests are conventional methods; for tests where specific techniques or conditions are not specified, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions; unless otherwise specified, the reagents and materials described are commercially available.

[0061] This invention provides the application of LDHA S319 phosphorylation, a modified form of LDHA, as a biomarker in the early diagnosis of tumors.

[0062] This invention also provides the application of LDHA S319 phosphorylation in the preparation of early tumor diagnostic reagents.

[0063] The early tumor diagnostic reagent includes a reagent for detecting the phosphorylation expression level of LDHA S319 in a sample. The sample is peripheral venous blood, adjacent normal tissue, or tumor tissue.

[0064] The reagent comprises a first antibody and a second antibody. The first antibody is an antibody that specifically binds to LDHA pS319 phosphorylated by LDHA. The second antibody is an antibody homologous to the first antibody and labeled with horseradish peroxidase.

[0065] This invention also provides early tumor diagnostic kits based on ELISA or Western Blot methods. The kits contain reagents for detecting the expression level of LDHA S319 phosphorylation. Each kit includes a first antibody and a second antibody. The first antibody is an antibody against LDHA S319 phosphorylation, LDHA pS319, and the second antibody is an antibody homologous to the first antibody and labeled with horseradish peroxidase. The kits also include containers, instructions for use, positive controls, negative controls, buffers, adjuvants, and solvents. The instructions for use describe how to use the kits for detection and how to use the detection results to assess tumor development and select treatment options. The kit components can be packaged in an aqueous medium.

[0066] This kit can be used for the diagnosis of early, intermediate, or late-stage tumors, including: oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancers, genitourinary cancers, gastrointestinal cancers, central or peripheral nervous system cancers, endocrine or neuroendocrine system cancers or hematopoietic system cancers, gliomas, sarcomas, epithelial cancers, lymphomas, melanomas, fibromas, meningiomas, brain cancers, kidney cancers, biliary tract cancers, pheochromocytomas, islet cell carcinomas, Levi-Flaumeni tumors, thyroid cancer, parathyroid cancer, pituitary adenoma, adrenal adenoma, osteosarcoma, neuroendocrine system tumors, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer.

[0067] A method for early tumor diagnosis using the kit described in this invention includes:

[0068] 1) Detect the expression levels of the biomarkers mentioned in the samples;

[0069] When using peripheral venous blood as a sample, the sample processing method includes: after collecting peripheral venous blood, it should be placed on ice for 15 minutes, and then centrifuged at 3500 rpm for 15 minutes to obtain serum or plasma, which can then be tested or stored at -80℃. Samples that cannot be tested immediately should be stored at -80℃ and thawed on ice before testing.

[0070] The phosphorylation expression level of LDHA S319 in the processed samples was detected by methods such as ELISA or Western Blot.

[0071] 2) Determine whether the subject has a tumor based on the phosphorylation expression level of LDHA S319 in the sample obtained in 1).

[0072] The Western blotting method mentioned in the evaluation or prediction method described in this invention, also known as protein immunoblotting, is a technique commonly used in research to isolate and identify proteins. It utilizes SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to separate various proteins contained in a specified sample. The separated proteins are then transferred to a nitrocellulose or PVDF membrane, which is subsequently incubated with a specific antibody against the target protein. During membrane washing, unbound antibodies are washed away, leaving only antibodies bound to the target protein. Finally, the bound antibodies are detected by developing film or fluorescence scanning. Because the antibody binds only to the target protein, typically only a single, clear band is observed, with the band thickness corresponding to the protein content. By analyzing the location and intensity of specific reactions, information about the expression of the target protein in a given cell or tissue homogenate can be obtained. Due to the high resolution of gel electrophoresis and the high specificity and sensitivity of immunoblotting, Western blotting analysis can detect target proteins as low as 1 ng. This method is widely used in molecular biology fields such as molecular biology, biochemistry, and immunogenetics.

[0073] Based on the above description of specific embodiments, the present invention provides the following examples to further illustrate the technical solution and its technical effects.

[0074] Example 1.

[0075] An ELISA detection kit contains a pre-coated microplate, standards, enzyme-labeled secondary antibody, substrate solution, stop solution, washing buffer, sample dilution solution, control sample, instructions, and sealing film; the wells of the microplate are pre-immobilized with the capture antibody LDHA pS319; the enzyme-labeled secondary antibody is an antibody homologous to the capture antibody LDHA pS319 and labeled with horseradish peroxidase.

[0076] Example 2.

[0077] A Western blot (WB) test kit containing the following reagents and items:

[0078] Primary antibody against LDHA pS319;

[0079] Secondary antibody (antibody homologous to LDHA pS319 and labeled with horseradish peroxidase);

[0080] Substrate solution;

[0081] Sealing solution: Milk powder sealing solution;

[0082] Washing buffer: TBS / T;

[0083] Transfer buffer;

[0084] PVDF membrane;

[0085] Electro-hydraulic transfer;

[0086] Membrane staining solution;

[0087] Sample buffer solution;

[0088] Standard protein or marker protein: used to estimate the molecular weight of the target protein;

[0089] Instruction manual.

[0090] Example 3.

[0091] A method for early diagnosis of tumors in subjects includes the following steps:

[0092] I. The WB detection kit described in Example 2 was used to detect the phosphorylation expression level of LDHA S319 in the peripheral serum of patients.

[0093] The specific testing steps are as follows:

[0094] 1. Prepare samples

[0095] 1) Sample collection: Collect peripheral blood from the subjects' veins, let it stand on ice for 15 min, centrifuge at 3500 rpm for 15 min at 4℃ to obtain serum for testing.

[0096] 2) Prepare ice packs; prepare cell lysis buffer: determine the required lysis buffer based on the number of cells: 50 μl / well of a six-well plate; lysis buffer formula: 100 μl cell lysis buffer + 0.1 μl phosphatase inhibitor mixture + 1 μl protease inhibitor mixture.

[0097] 3) Prepare the cells according to the experimental requirements: Remove the culture medium, wash 3 times with 1×PBS (to remove the serum from the culture medium), and add 50 μl of lysis buffer containing protease / phosphatase inhibitors to each well (6-well plate). Quickly scrape the cells off with a cell scraper and transfer them to a 1.5 ml centrifuge tube. Incubate on ice for 20 min to lyse, vortex to mix, and then incubate on ice for another 10 min.

[0098] 4) Centrifuge at 4℃, 12000g for 15 minutes, and carefully collect the supernatant into another 1.5ml tube.

[0099] 5) Take 2.5 μl of sample and dilute it with 22.5 μl of triple-distilled water for use in the BCA method to determine protein concentration.

[0100] 6) Add 5× Loading Buffer (at a ratio of 2.5ml Buffer / 10ml protein) to the remaining samples and boil at 95℃ for 10 minutes, shaking once during the process.

[0101] 7) Directly load the sample for gel running or dispense it for long-term storage at -80℃.

[0102] 2. SDS-PAGE polyacrylamide gel electrophoresis

[0103] 1) Prepare the separating gel (6 ml / gel).

[0104] 2) Carefully inject the separating gel, leaving about 2cm of space for the concentrating gel, cover the top with deionized water, and let stand for about 30 minutes.

[0105] 3) Prepare the concentrated gel (2 ml / gel).

[0106] 4) Pour the stacking gel into the top of the separating gel, being careful to avoid air bubbles.

[0107] 5) Insert the comb and wait for the concentrated gel to solidify (there is a clear boundary between the gel and the comb, and the solidification time of the separating gel should be greater than 2 hours). Clean the hole with double-distilled water to remove gel fragments, and then blot dry with filter paper.

[0108] 6) Place the gel into the electrophoresis tank, and add 1× electrophoresis buffer to both the upper and lower tanks (do not reuse more than 3 times).

[0109] 7) Sample loading: Take 5 μl of prestained marker into the marker well and add an appropriate amount of 1× loading buffer to make the total volume the same as the sample well. The sample loading volume is generally 15-25 μl. First, boil it in heating block at 95℃ for 5-10 minutes, shaking once during the process, then quickly centrifuge and load the sample for gel running; add an equal volume of 1× loading buffer to the wells without sample loading.

[0110] 8) Electrophoresis: Start with a constant voltage of 60-80V. After running through the stacking gel, increase the current to 100-120V. The electrophoresis time is determined according to the size of the target protein and the position of the marker. Generally, the target protein is ready when it reaches two-thirds of the separating gel.

[0111] 3. Membrane transfer

[0112] 1) Cut the gel according to the marker indication and the position of the target band (note to mark the cut corner of the gel). Immerse the eluted gel in the transfer buffer for 15 minutes.

[0113] 2) After marking the PVDF membrane, immerse it in methanol for 1 minute, then immerse it along with 4 sheets of 3mm filter paper and a sponge in transfer buffer for 15 minutes.

[0114] 3) Prepare the "sandwich": Follow the order below: fiber mat -- filter paper -- PVDF membrane -- gel -- filter paper -- fiber mat; Note: Align each item as it is added to ensure there are no air bubbles.

[0115] 4) Transfer: The transfer time is determined according to 1). One side of the PVDF membrane is connected to the positive electrode (red), and the other side of the gel is connected to the negative electrode (black).

[0116] 4. Membrane blocking and antibody incubation

[0117] 1) After the membrane transfer, wash the membrane with 10 ml of 1×TBS at room temperature for 10 minutes.

[0118] 2) Incubate with 5ml of 5% milk powder sealing solution at room temperature for 2 hours or at 4℃ with gentle shaking overnight. Since milk powder is relatively difficult to dissolve, it should be prepared at least 1 hour in advance.

[0119] 3) Wash the membrane three times with 10ml of TBS / T solution, each time for 5 minutes.

[0120] 4) Add 5 ml of primary antibody dilution buffer (divide the antibody according to the instructions), incubate at room temperature for 2 h or at 4 °C with gentle shaking overnight, recover the primary antibody, add sodium azide (which can inhibit bacterial growth) at 5 μl / ml of primary antibody solution, and store at 4 °C (less frequently used antibodies can be stored at -20 °C for a long time). It can be reused.

[0121] 5) Wash the membrane three times with 10ml of TBS / T solution, each time for 5 minutes.

[0122] 6) Add the secondary antibody (usually diluted 1:2000) and shake gently at room temperature for 1 hour.

[0123] 7) Wash the membrane three times with 10ml of TBS / T solution, each time for 5 minutes.

[0124] 5. Development and fixing (or direct fluorescence scanning after incubation with fluorescently labeled secondary antibody)

[0125] 1) Developing steps: First, lay down plastic wrap, then place another layer of plastic wrap on top of absorbent paper. Pour water, developer (if the color darkens, do not use), and fixer into their respective trays. Mix 2.5ml of ECL-A and 2.5ml of ECL-B, and keep away from light. Take the ECL mixture and film into the darkroom. Close and lock the door, and pull back the curtain. Pour the ECL mixture into a small box. Slightly pat the film dry with absorbent paper and place it in the ECL mixture, shaking at room temperature for 5 minutes (to ensure the ECL is evenly spread over the film). After patting dry with absorbent paper, place it on plastic wrap, film side down, and place it on a clip. Cut an X-film (only hold the edge of the X-film) and place it on the film, cutting one corner as a marker. (Note: When handling the film, turn the light to minimum and do not face the light; keep it away from the light.) Adjust the exposure time according to the brightness of the strips. Generally, you can expose for 2 minutes first, observe the depth of the strips, and then determine the optimal exposure time.

[0126] 2) Development and fixing: After taking out the X-film and placing it in the developer for a certain period of time (depending on the intensity of the target band and the background), wash it once with water and then place it in the fixer for at least 5 minutes.

[0127] Finally, the target protein band on the PVDF membrane is obtained. By comparing the size with the protein marker, the size and location of the target protein can be determined (LDHA protein size is 37KD), and the expression result of the target protein can be obtained.

[0128] II. Evaluate the sensitivity of patients to immunotherapy based on the protein expression results detected in step one.

[0129] Diagnostic analysis of test results:

[0130] If the following protein bands of the corresponding size appear on the PVDF membrane: LDHA S3191 phosphorylation, it suggests that the subject may have invasive cancer, be in the advanced stage of invasive cancer, or have a poor prognosis.

[0131] We collected serum samples from 46 subjects at the Second Affiliated Hospital of Zhejiang University, including 20 patients with liver cancer (diagnosed according to current clinical diagnostic standards) and 26 healthy individuals.

[0132] The method described in this embodiment was used to diagnose early-stage liver cancer in the aforementioned subjects. The expression level of serum LDHA S319 phosphorylation modification in 46 subjects was detected using the aforementioned Western blot (WB) detection kit, and the protein bands were used for evaluation and prediction. The results showed that 17 out of 20 liver cancer patients were positive, and 3 out of 26 healthy individuals were positive; indicating that the method had a sensitivity of 85% and a specificity of 88.5%.

[0133] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. Application of modified LDHA form LDHA S319 phosphorylated protein as a biomarker in the preparation of liver tumor diagnostic reagents.

2. The application as described in claim 1, characterized in that: The tumor diagnostic reagent shown is used to detect the expression level of LDHA S319 phosphorylated protein in samples from subjects.

3. The application as described in claim 2, characterized in that: The expression level of LDHA S319 phosphorylated protein in samples from subjects was detected by ELISA or Western Blot.

4. The application as described in any one of claims 2 or 3, characterized in that: The detection of the expression level of LDHA S319 phosphorylated protein in samples from the subject refers to the detection of the expression level of LDHA S319 phosphorylated protein in peripheral venous blood, adjacent normal tissue, or tumor tissue from the subject.

5. A kit for quantitative or semi-quantitative detection of proteins, containing an antibody that specifically binds to LDHA S319 phosphorylated protein.

6. The kit as described in claim 5, characterized in that: The aforementioned kit for quantitative or semi-quantitative protein detection is a kit that can detect proteins using enzyme-linked immunosorbent assay (ELISA) or Western blotting. The kit contains a first antibody and a second antibody. The first antibody is an antibody that specifically binds to LDHAS319 phosphorylated protein, and the second antibody is an enzyme-labeled antibody homologous to the first antibody.

7. The kit according to any one of claims 5-6, characterized in that: The antibody that specifically binds to LDHAS319 phosphorylated protein is a monoclonal antibody against LDHAS319 phosphorylated protein.

8. Application of antibodies against LDHA S319 phosphorylated protein in the preparation of liver tumor diagnostic kits.

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