Application of LDHA function-based biomarker in tumor diagnosis

Abstract

The invention provides an application of a modified LDHA S319 phosphorylated LDHA as a biomarker in preparation of a tumor diagnosis reagent. The invention further provides a kit capable of quantitatively or semi-quantitatively detecting the protein, and the kit contains the phosphorylated antibody capable of being specifically combined with LDHA S319. According to the application disclosed by the invention, by detecting the expression level of LDHA S319 phosphorylation, the tumor condition of a subject can be more effectively and preliminarily evaluated and predicted. The application of the invention is helpful for improving the diagnosis accuracy of tumors, and provides an important basis for early treatment of patients. The invention also provides an application of the LDHA S319 phosphorylated antibody in preparation of a tumor diagnosis kit.

Description

Technical Field

[0001] The present invention relates to the application of a biomarker based on LDHA function in tumor diagnosis, and belongs to the field of tumor medicine. Background Art

[0002] The key to tumor treatment lies in whether it can be detected and diagnosed early so that appropriate treatment measures can be taken. At present, tumors can only be completely cured if they are treated early. If the tumor develops to the middle or late stages, the tumor tissue expands or has metastasized, it will be difficult to cure it even if various treatment methods are taken. On the contrary, if the tumor can be discovered in the early or relatively early stages and before metastasis occurs and is given timely and reasonable treatment, a considerable number of patients can achieve satisfactory treatment results, and even achieve a cure.

[0003] At present, the methods of tumor diagnosis are divided into five levels: ① Clinical diagnosis: a speculative diagnosis based on clinical symptoms, signs and imaging examinations, with reference to the law of disease development; ② Surgical diagnosis: a judgment made only on the tumor seen by the naked eye through surgery or various endoscopic examinations, without pathological confirmation; ③ Physical and chemical diagnosis: clinically consistent with cancer manifestations, and supported by positive results of physical and chemical examinations, such as X-ray, B-ultrasound, CT and MRI nuclear magnetic resonance examinations, or carcinoembryonic antigen, alpha-fetoprotein determination, etc.; ④ Cytopathological diagnosis: a diagnosis made based on various exfoliated cells and puncture cell examinations; ⑤ Histopathological diagnosis: a diagnosis made after pathological analysis of tissue obtained after coarse needle puncture. Among these five levels, the reliability of the diagnosis increases in sequence, with the fifth level being the most ideal. However, the above methods cannot achieve early detection and early diagnosis of tumors. More often than not, after the tumor develops to a certain extent, the patient may even develop serious symptoms and signs. At this time, the best treatment period is often missed, making the later treatment extremely passive. In addition, pathological diagnosis with better diagnostic effect requires puncture to obtain cells or tissue slices, which has great damage and negative effects on the patient's mental and physical health. It is crucial to find more effective biomarkers to achieve early diagnosis of tumors.

[0004] At present, there are still many deficiencies in the research on the mechanism of occurrence and development of liver cancer, which seriously hinders the development of new means of diagnosis and treatment for this type of tumor. Lactate dehydrogenase A (LDHA) is a key glycolytic enzyme that catalyzes the conversion of pyruvate to lactate in the last step of glycolysis. Lactate produced by glycolysis serves as a substrate for histone or non-histone lactylation, regulating gene expression and protein activity in immune cells and cancer cells in a glycolysis-dependent manner. Although the key metabolic function of LDHA has been found in glycolysis, its potential function in apoptosis anemia and tumor development during immunotherapy remains unclear. Ferroptosis is a different form of regulated cell death that depends on iron accumulation and is driven by an increase in lipid peroxides within the cell membrane. It plays a key role in cancer development and immunotherapy.

[0005] In view of the above background, in order to more accurately judge tumor development and more reasonably select treatment plans, thereby improving the effectiveness of tumor immunotherapy, it is necessary to find key markers related to tumor cell ferroptosis and immune escape mechanisms for early diagnosis of tumors. Summary of the invention

[0006] The technical problem or primary purpose to be solved by the present invention is to perform early diagnosis of tumors in subjects.

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

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

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

[0010] In the scheme of 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 the present invention, by using an immunoassay reagent for LDHA S319 phosphorylation to detect the expression level of LDHA S319 phosphorylation in a sample from a subject, early diagnosis of cancer in the subject can be performed. Furthermore, the sample from the subject is any one of the subject's venous peripheral blood, paracancerous tissue or tumor tissue.

[0012] In the application of the present invention, the tumor diagnostic reagent is an immunodetection reagent based on Elisa or Western Blot method.

[0013] In a second aspect, the present invention further provides an antibody, wherein the antibody is an antibody capable of specifically binding to phosphorylated LDHA S319.

[0014] In a third aspect, the present invention further provides a kit for quantitative or semi-quantitative detection of proteins, comprising the antibody described in the second aspect of the present invention.

[0015] In a preferred embodiment of the present invention, the kit capable of quantitatively or semi-quantitatively detecting proteins is a kit capable of detecting proteins by an enzyme-linked immunosorbent assay (ELISA) method or a Western Blot method; the kit contains a first antibody and a second antibody; the first antibody is an antibody capable of specifically binding to phosphorylated LDHA S319, and the second antibody is an enzyme-labeled antibody homologous to the first antibody.

[0016] In a fourth aspect, the present invention also provides use of an antibody that specifically binds to the phosphorylated LDHA S319 in the preparation of a tumor diagnosis kit.

[0017] In a further preferred embodiment of the present invention, the kit further contains 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 use the kit for testing, and how to use the test results to judge tumor development and select a treatment plan. The components of the kit may be packaged in the form of an aqueous medium or in a lyophilized form.

[0018] The tumor diagnostic kit of the present invention can be used for the early, middle or late diagnosis of any of the following tumor diseases: oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, genitourinary 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, Leigh-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteogenic sarcoma tumor, 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] The present invention proposes a new biomarker LDHA S319 phosphorylation for use in tumor diagnosis. By detecting the expression level of the biomarker, the tumor condition of the subject can be diagnosed in the early, middle or late stages.

[0020] The LDHA S319 phosphorylation described in the present invention is a specific modification form of LDHA, which is highly related to the ferroptosis and immune escape mechanism of 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 receives PD-1 immunotherapy, CD8 + IFNγ secreted by T cells can activate S6K, triggering activated S6K to phosphorylate the S319 site of LDHA. This phosphorylation of LDHA can inhibit liver cancer cell ferroptosis, thereby reducing the effect of PD-1 immunotherapy for liver cancer. In addition, in clinical specimens of liver cancer, the level of LDHA S319 phosphorylation was positively correlated with the expression of 4-HNE, a marker of ferroptosis, and was associated with the poor prognosis of liver cancer patients. These findings reveal the key mechanism by which IFNγ induces immune escape of liver cancer cells by inducing LDHA S319 phosphorylation to inhibit the non-metabolic function of liver cancer cell ferroptosis when liver cancer cells receive PD-1 immunotherapy, and emphasize that the combination therapy targeting LDHA S319 phosphorylation and anti-PD-1 antibodies is a promising HCC immunotherapy.

[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) Athymic nude mice were BALB / c athymic nude mice;

[0026] (3) Patient samples: Surgically resected, formalin-fixed, paraffin-embedded HCC tissue samples 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 pathological diagnosis of HCC who had not received surgical treatment were selected as an independent cohort. Clinical data were obtained by reviewing the patients' medical histories. Pathological staging was assessed according to the 8th edition of the American Joint Committee on Cancer / Consortium for International Cancer Control TNM classification system.

[0027] (4) The shRNA sequences used for gene knockdown are 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] By contacting the sample with an antibody that specifically binds to the phosphorylated polypeptide and determining the amount of bound antibody, for example by detecting or measuring the formation of a complex between the antibody and the polypeptide. The antibody may be labeled (radioactive, fluorescent, etc.) to facilitate detection of the complex.

[0033] The detection systems for the polypeptide levels of the present invention include: autoradiographic immunoassay RIA, immunofluorescence, gel electrophoresis, and Western immunoblotting.

[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), JAK2pY1007 / 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 LDHApS319 specific antibody was synthesized by abclonal.

[0035] Mouse tumor sample acquisition and analysis: 6-week-old male immunocompetent mice were housed under specific pathogen-free conditions with a 14-h / 10-h 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 5 ) were suspended in 20 μl of PBS and injected directly into the liver, or suspended in 50 μl of PBS and injected subcutaneously into 6-week-old male immunocompetent mice, with or without anti-PD-1 injection 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. Bioluminescence imaging of in situ cancer was performed using an IVIS system (AniView 100, Biolight Biotechnology, Guangzhou, China). Subsequently, the animals were euthanized, and the tumor of each mouse was dissected, fixed in 4% formaldehyde, and embedded in paraffin. Tumor formation and phenotype were evaluated by histological analysis of hematoxylin and eosin-stained sections. The formula V = 1 / 2a was used.2 b Calculation of tumor volume (where V is volume, a is the shortest diameter, and b is the longest diameter).

[0036] (6) Immunohistochemistry: Paraffin-embedded human liver cancer sample sections were stained with specific primary antibodies and nonspecific IgG as a negative control. The specific method was as follows: after deparaffinization, rehydration and antigen retrieval of paraffin sections, TMA slides were incubated with primary antibody rabbit anti-human phosphorylated LDHA S319 (dilution 1:200) or nonspecific IgG (as a negative control) at 4°C overnight. The slides were then incubated with anti-rabbit secondary antibodies (ready-to-use solution; Cell Signaling Technology; #8114), followed by dichromatic diaminobenzidine (DAB) staining (Cell Signaling Technology) and hematoxylin staining, and fixed in xylene. Tissue slides were quantitatively scored under a microscope according to the percentage of positive cells and staining intensity. The present invention assigns the following positive cell proportion 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, from 71% to 100% of cells are positive. The staining intensity is also rated on a scale of 0 to 3: 0, negative (i.e., no visible staining); 1, weak (very light color); 2, moderate (obvious color); 3, strong (very dark color). As previously described, the positive cell proportion score and the staining intensity grade are added to obtain a total score (range 0-8). Two pathologists who were unaware of the clinical information independently verified the reproducibility of the scoring system.

[0037] (7) Kaplan-Meier plots of patients' overall survival were used for data analysis using SPSS version 20.0 software (SPSS Inc., Chicago, IL, USA). The expression levels of biomarkers in tumors and normal tissues were compared using the independent sample t-test. One-way analysis of variance (ANOVA, posthoc Bonferroni test) was used to perform multiple comparisons and the least significant difference test for the correlation between the expression levels of biomarkers and the clinical pathological characteristics of patients. The Pearson correlation coefficient was used to analyze the correlation between the expression levels of biomarkers. Overall survival (OS) was defined as the duration from the date of diagnosis to the date of death or the last follow-up. K-means cluster analysis was used to classify the expression levels of relevant markers, the Kaplan-Meier method was used to draw survival curves, the log-rank test was used to compare survival rates, and the Cox regression model with a two-way Wald test was used to calculate the hazard ratio (HR) and 95% confidence intervals (CIs) for survival analysis. Censored data were used for patients who were alive at the last follow-up or were lost due to follow-up. Univariate analysis variables with a P value of less than 0.05 were included in the multivariate analysis. P < 0.05 was considered statistically significant. All statistical tests were two-sided.

[0038] 2 Results and analysis

[0039] (1) Figure 1 As shown, after Huh7 cells were treated with IFNγ for 12 hours, immunohistochemical analysis showed that IFNγ could induce the interaction between LDHA and S6K.

[0040] (2) Figure 2 As shown, Co-IP combined with mass spectrometry analysis found 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 detection found that the LDHA phosphorylation site S319A mutation and the enzyme-inactive site K155R mutation induced lipid peroxidation in cells.

[0043] (5) Figure 5 As shown, flow cytometry analysis revealed that LDHA phosphorylation site S319A mutation and enzyme-inactivating site K155R mutation induced cell death.

[0044] (6) Figure 6As shown, cell proliferation experiments found that LDHA phosphorylation site S319A mutation and enzyme inactivation site K155R mutation inhibited cell proliferation.

[0045] (7) The LDHA phosphorylation site S319A mutation can increase the sensitivity of transplanted tumors to anti-PD-1 treatment. Hep1-6-LUC cells were injected orally into 6-week-old male immunocompetent mice and treated with anti-PD-1 at the same time. The results showed that the expression of LDHA S319 phosphorylation site can reduce the sensitivity of transplanted tumors to anti-PD-1 treatment, while the LDHA S319A mutation can increase the sensitivity of transplanted tumors to anti-PD-1 treatment ( Fig. 7A , Figure 7B , Fig. 8A and Figure 8B ), immunohistochemical experiments showed that LDHA S319A mutation can promote ferroptosis and improve the effect of PD-1 immunotherapy ( Fig. 9 ).

[0046] (8) LDHA S319 phosphorylation is associated with the prognosis of patients with liver cancer. We used immunohistochemical staining of validated anti-LDHA-pS319 to analyze human primary liver cancer specimens and found that the levels of these protein markers in liver cancer tissues were significantly higher than those in adjacent tissues ( Fig.10 In addition, a negative correlation between LDHA-pS319 and the expression of ferroptosis marker 4-HNE was observed ( Fig.11 ). Furthermore, phosphorylation of LDHAS319 is positively correlated with poor survival outcomes in patients with liver cancer ( Fig.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. Samples can be collected from venous peripheral blood to complete the test, which greatly reduces the psychological burden and physical damage of the subjects. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 It reflects the results of immunoprecipitation and immunoblotting analysis after IFNγ treatment of cells;

[0049] Figure 2 It reflects the mass spectrometry identification of the post-translational modification of LDHA in Huh7 liver cancer cells;

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

[0051] Figure 4It reflects the effect of LDHA S319 phosphorylation on cellular lipid peroxidation detected by flow cytometry;

[0052] Figure 5 It reflects the effect of LDHA S319 phosphorylation on cell death detected by flow cytometry;

[0053] Figure 6 The cell proliferation experiment detected the effect of LDHA S319 phosphorylation on cell proliferation;

[0054] Fig. 7A , Figure 7B This study analyzed the effect of LDHA S319 mutation on anti-PD-1 immunotherapy in orthotopic xenografts in mice;

[0055] Fig. 8A , Figure 8B Figure 7 shows the photos and weight statistics of mouse transplanted tumors;

[0056] Fig. 9 It reflects the immunohistochemical results of mouse transplanted tumors in Figure 7;

[0057] Fig.10 It reflects the immunohistochemical results of human liver cancer and adjacent liver cancer samples;

[0058] Fig.11 It reflects the immunohistochemical results of different liver cancer patients;

[0059] Fig.12 The figure shows the survival curve of LDHA S319 phosphorylation level in tumor tissue of liver cancer patients and their prognosis. DETAILED DESCRIPTION

[0060] In order to make the purpose and technical solution of the present invention more clear, the present invention is further described in detail below in conjunction with the accompanying drawings. The experimental methods described in the following experiments are all conventional methods unless otherwise specified; if no specific technology or conditions are specified in the experiment, the technology or conditions described in the literature in the field or the product instructions are used; the reagents and materials described can be obtained from commercial channels unless otherwise specified.

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

[0062] The present invention also provides the application of LDHA S319 phosphorylation in the preparation of early tumor diagnosis reagents.

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

[0064] The reagents include a first antibody and a second antibody. The first antibody is an antibody LDHA pS319 that can specifically bind to phosphorylated LDHA S319. The second antibody is an antibody that is homologous to the first antibody and is labeled with horseradish peroxidase.

[0065] The present invention also provides a tumor early diagnosis kit based on Elisa or Western Blot method, the kit contains reagents for detecting the expression level of LDHA S319 phosphorylation, each kit includes a first antibody and a second antibody, the first antibody is LDHA S319 phosphorylated antibody LDHA pS319, and the second antibody is homologous to the first antibody and is labeled with horseradish peroxidase. The kit also includes a container, instructions for use, positive controls, negative controls, buffers, adjuvants and solvents. The instructions for use record how to use the kit for detection, and how to use the detection results to judge tumor development and select treatment plans. The components of the kit can be packaged in the form of an aqueous medium.

[0066] The kit can be used for the early, middle or late diagnosis 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 or hematopoietic system cancer, glioma, sarcoma, epithelial cancer, lymphoma, melanoma, fibroma, meningioma, brain cancer, kidney cancer, biliary system cancer, pheochromocytoma, islet cell carcinoma, Leigh-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteogenic sarcoma tumor, 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.

[0067] The method for early diagnosis of tumor using the kit of the present invention comprises:

[0068] 1) detecting the expression level of the biomarker in the sample;

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

[0070] Using Elisa or Western Blot to detect the phosphorylation expression level of LDHA S319 in the treated samples;

[0071] 2) Determine whether the subject suffers from a tumor based on the LDHA S319 phosphorylation expression level in the sample detected in 1).

[0072] The Western Blot method mentioned in the evaluation or prediction method of the present invention is a protein immunoblotting experiment, which is a technique commonly used to separate and identify proteins in research. It uses SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to separate various proteins contained in a specified sample, and then transfers the separated proteins to a nitrocellulose or PVDF membrane, and then incubates the membrane with a specific antibody for the target protein. During the membrane washing process, unbound antibodies are washed away, leaving only antibodies bound to the target protein, and finally the bound antibodies are detected by developing film or fluorescent scanning. Since the antibody only binds to the target protein, generally only a clear band can be seen, and the thickness of the band corresponds to the amount of protein. By analyzing the position and intensity of a specific reaction, the expression information of the target protein in a given cell or tissue homogenate can be obtained. Due to the high resolution of gel electrophoresis and the strong specificity and high sensitivity of immunity, Western blot analysis can detect target proteins as low as 1ng. This method is widely used in molecular biology, biochemistry, immunogenetics and other molecular biology fields.

[0073] Based on the description of the above specific implementation methods, the present invention specifically lists the following embodiments to further illustrate the technical solution and technical effects of the present invention in detail.

[0074] Example 1.

[0075] An ELISA detection kit comprises a pre-coated microplate, a standard substance, an enzyme-labeled secondary antibody, a substrate solution, a stop solution, a washing buffer solution, a sample diluent, a control sample, an instruction manual and a sealing film; the wells of the microplate are pre-fixed with a 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 WB detection kit containing the following reagents and items:

[0078] Primary antibody: LDHA pS319;

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

[0080] Substrate solution;

[0081] Blocking solution: milk powder blocking solution;

[0082] Wash buffer: TBS / T;

[0083] Transfer buffer;

[0084] PVDF membrane;

[0085] Electroporation fluid;

[0086] Membrane staining solution;

[0087] Sample buffer;

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

[0089] Instructions for use.

[0090] Example 3.

[0091] A method for early diagnosis of tumor in a subject, comprising the following steps:

[0092] 1. The WB detection kit described in Example 2 was used to detect the phosphorylation expression level of LDHA S319 in the patient's venous peripheral serum.

[0093] The specific detection steps are as follows:

[0094] 1. Sample Preparation

[0095] 1) Sample collection: Collect peripheral blood from the subject's vein, place it on ice for 15 minutes, and centrifuge it at 4°C, 3500 rpm for 15 minutes to obtain serum for testing.

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

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

[0098] 4) Centrifuge at 4°C, 12,000 g for 15 min and carefully collect the supernatant into another 1.5 ml tube.

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

[0100] 6) Add 5× Loading Buffer (2.5 ml Buffer / 10 ml protein) to the remaining samples and cook at 95°C for 10 minutes, shaking once during the cooking.

[0101] 7) Load the sample directly onto the gel or aliquot and store at -80℃ for long term storage.

[0102] 2. SDS-PAGE polyacrylamide gel electrophoresis

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

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

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

[0106] 4) Inject the concentrated gel into the upper end of the separation gel, taking care to avoid bubbles.

[0107] 5) Insert the comb and wait for the concentrated gel to solidify (there should be a clear boundary between the gel and the comb, and the solidification time of the separation gel should be greater than 2 hours), wash the wells with double distilled water to remove the gel fragments, and then absorb the water 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 5μl of prestained marker in the marker well and add an appropriate amount of 1× loading buffer to make the total volume the same as that of the sample well. The sample loading volume is generally 15-25μl. First, cook it in a heating block at 95℃ for 5-10 minutes, shake it once, then quickly centrifuge it and load it on the gel; add an equal volume of 1× loading buffer to the well without loaded sample.

[0110] 8) Electrophoresis: Start with a constant voltage of 60-80V. After running through the concentrated 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 runs to two-thirds of the separation gel.

[0111] 3. Membrane Transfer

[0112] 1) Cut the gel according to the marker and the position of the target band (make sure to mark the gel cutting angle), and immerse the eluted gel in transfer buffer for 15 minutes.

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

[0114] 3) Prepare the "sandwich": follow the following order: fiber pad - filter paper - PVDF membrane - gel - filter paper - fiber pad; Note: each item must be aligned to ensure there are no 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 transfer, wash the membrane with 10 ml of 1× TBS at room temperature for 10 minutes.

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

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

[0120] 4) Add 5 ml of primary antibody dilution buffer (antibodies are diluted according to the instructions), gently shake at room temperature for 2 hours or at 4°C overnight, recover the primary antibody, add sodium azide (which can inhibit bacterial growth) at 5 μl / ml primary antibody solution, and store at 4°C (uncommon antibodies can be stored at -20°C for a long time), which can be reused.

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

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

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

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

[0125] 1) Color development steps: first lay out plastic wrap, then lay out plastic wrap on absorbent paper; pour water, developer (cannot be used if the color becomes darker) 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 mixed solution, membrane, etc. into the darkroom. Close the door, lock it, and pull back the cloth. Pour the ECL mixed solution into a small box, slightly dry the membrane with absorbent paper, put it into the ECL mixed solution and shake it at room temperature for 5 minutes (so that the ECL is evenly spread on the membrane), dry it with absorbent paper and place it on the plastic wrap, wrap it face down and place it on the clip, cut the X-film (note that you can only hold the edge of the X-film with your hands) and place it on the membrane, and the cut corner side is the marker (note: when taking the film, adjust the light to the minimum, and do not face the light, but face away from the light). Adjust the exposure time according to the brightness of the strip. Generally, you can expose it for 2 minutes first, observe the depth of the strip, and then determine the best exposure time.

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

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

[0128] 2. Evaluate the patient's sensitivity to immunotherapy based on the protein expression results detected in step 1.

[0129] Diagnostic analysis of test results:

[0130] If the following protein bands of corresponding sizes appear on the PVDF membrane: LDHA S3191 phosphorylation, it indicates that the subject may have invasive cancer, is in the progressive stage of invasive cancer, or has a poor prognosis.

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

[0132] According to the method of this example, the above-mentioned subjects were diagnosed with liver cancer at an early stage. After the expression level of serum LDHA S319 phosphorylation modification of 46 cases was detected by the above-mentioned WB detection kit, the evaluation and prediction were performed based on the obtained protein bands. The results showed that 17 out of 20 liver cancer patients were positive and 3 out of 26 healthy people were positive, indicating that the sensitivity of this method was 85% and the specificity was 88.5%.

[0133] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. Application of LDHA S319 phosphorylation, a modified form of LDHA, as a biomarker in the preparation of tumor diagnostic reagents.

2. The use according to claim 1, characterized in that: The tumor diagnostic reagent shown is used to detect the expression level of LDHA S319 phosphorylation in a sample from a subject.

3. The use according to claim 2, characterized in that: Detection of the expression level of LDHA S319 phosphorylation in samples from subjects is achieved by Elisa or Western Blot method.

4. The use according to any one of claims 2 or 3, characterized in that: The detection of the expression level of LDHA S319 phosphorylation in the sample from the subject is to detect the expression level of LDHA S319 phosphorylation in the venous peripheral blood, paracancerous tissue or tumor tissue from the subject.

5. A kit for quantitative or semi-quantitative detection of proteins, comprising an antibody that can specifically bind to LDHA S319 phosphorylation.

6. The kit according to claim 5, characterized in that: The kit capable of quantitatively or semi-quantitatively detecting proteins is a kit capable of detecting proteins by an enzyme-linked immunosorbent assay (ELISA) method or a Western Blot method; the kit contains a first antibody and a second antibody; the first antibody is an antibody capable of specifically binding to phosphorylated LDHAS319, and the second antibody is an enzyme-labeled antibody homologous to the first antibody.

7. The kit according to any one of claims 5 to 6, characterized in that: The antibody that can specifically bind to phosphorylated LDHA S319 is a monoclonal antibody that is phosphorylated LDHA S319.

8. Application of LDHA S319 phosphorylated antibody in the preparation of tumor diagnostic kit.

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