Application of anti-ATP6V0D1 autoantibody in auxiliary diagnosis of lung adenocarcinoma

Abstract

The invention discloses application of an anti-ATP6V0D1 autoantibody in auxiliary diagnosis of lung adenocarcinoma, the expression level of the anti-ATP6V0D1 autoantibody in plasma of a patient with lung adenocarcinoma is obviously higher than that of a healthy control, and the difference has statistical significance. The novel anti-ATP6V0D1 autoantibody and a tumor marker CEA are combined to construct a lung adenocarcinoma differential diagnosis model, and the model has good differential diagnosis efficiency on a lung adenocarcinoma patient and has important significance on management and identification of the lung adenocarcinoma patient.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of anti-ATP6V0D1 autoantibodies in auxiliary diagnosis of lung adenocarcinoma. Background Art

[0002] This section merely provides background information related to the present disclosure and is not necessarily prior art.

[0003] Primary bronchial lung cancer, also known as lung cancer (LC), is one of the malignant tumors with the highest incidence and mortality rates in the world. Early lung cancer often has no obvious symptoms. Most patients are already in the late stage when they present symptoms and seek medical treatment. The overall 5-year survival rate of patients with advanced lung cancer is about 20%. Non-small cell lung carcinoma (NSCLC) accounts for about 85% of all lung cancer cases, of which lung adenocarcinoma (LUAD) accounts for about 70% of NSCLC. Lung adenocarcinoma is the pathological type with the highest incidence rate in lung cancer. Clinical studies have shown that the five-year survival rate of diagnosed early lung adenocarcinoma can be as high as 90% with surgery combined with radiotherapy and chemotherapy. However, the survival rate of lung adenocarcinoma that has metastasized is greatly reduced because it cannot be treated surgically. Due to the characteristics of early metastasis of adenocarcinoma, more than half of lung adenocarcinoma patients show local or distant metastasis at the time of initial diagnosis, and lose the best treatment opportunity. Therefore, improving the early diagnosis strategy of lung adenocarcinoma is the key to reducing the mortality rate of lung cancer. Screening out lung adenocarcinoma tumor markers with high tissue specificity and sensitivity is of great significance for the early diagnosis, prognosis and precise treatment of lung adenocarcinoma.

[0004] For many years, both at home and abroad, efforts have been made to achieve early diagnosis and treatment of lung cancer through screening, thereby reducing the mortality rate associated with lung cancer. The results of a randomized controlled study of the National Lung Cancer Screening Trial in the United States in 2011 showed that, compared with X-ray examinations, screening of high-risk lung cancer populations with low-dose computed tomography (LDCT) can reduce lung cancer mortality by 20%. The lung cancer screening guidelines of many medical organizations in Europe and the United States recommend the use of LDCT for lung cancer screening in high-risk populations. Although the mortality rate of lung cancer has been significantly reduced, non-high-risk populations are still likely to suffer from lung cancer. About 50% of new lung cancer cases do not meet the screening conditions; and the vast majority of patients with lung nodules cannot obtain important information from routine examinations, that is, they cannot effectively distinguish between benign and malignant nodules. Among patients diagnosed with LC by LDCT, the false positive rate is as high as 21.8%-26.6%. Overdiagnosis causes unnecessary harm to patients both physically and financially. Therefore, further research, development and verification of non-invasive biological markers that can be effectively used for the diagnosis of lung adenocarcinoma are still needed.

[0005] Blood tumor-related marker detection is the most easily accepted diagnostic method by patients. Protein markers are relatively stable and the detection method is simple. In the patient's serum, the increase of CEA, SCCA and CYFRA21-1 levels is helpful for the diagnosis of NSCLC. CEA is most significantly increased in lung adenocarcinoma and non-neuroendocrine large cell lung cancer, and has a high sensitivity. However, it should be noted that increased CEA can also be seen in digestive tract tumors and pulmonary interstitial fibrosis. Combined detection of CYFRA21-1 and CEA can improve the sensitivity and specificity of the diagnosis of lung adenocarcinoma. The CEA level of long-term smokers may be slightly higher than that of healthy people. CYFRA21-1 is also one of the sensitive indicators of NSCLC. Attention should be paid to trauma and saliva contamination, as well as the possibility of false increase of CYFRA21-1 in patients with renal failure. SCCA has a high specificity for squamous epithelial tumors such as squamous cell carcinoma of the lung, which can assist in histological diagnosis. However, a single marker cannot distinguish SCLC from NSCLC. About 10% of NSCLCs have immune responses to at least one of the neuroendocrine markers. If NSE, ProGRP, CYFRA21-1, CEA and SCCA are tested together, the accuracy of identification can be improved. However, traditional tumor markers are generally not elevated in patients with early lung cancer, so the sensitivity of detection is low. Therefore, it is necessary to find new minimally invasive blood molecular markers and lung cancer diagnosis-related biomarker detection, which can improve lung cancer risk assessment and help early detection of lung cancer patients.

[0006] Many studies have shown that tumors can stimulate humoral immunity in the host in the early stages of their occurrence. Autoantibodies against tumor-associated antigens (TAAbs) can be detected in the serum of patients with various tumors, including lung cancer. Tumor antigens refer to the general term for new antigens and overexpressed antigens that appear during cell carcinogenesis. Studies have reported that TAAbs can be detected in the blood 17-47 months before the diagnosis of cancer. Therefore, TAAbs are biomarkers with great development potential. This study aims to screen and identify IgG and IgM humoral immunity lung cancer antigens through plasma samples from patients with lung adenocarcinoma and healthy people. Through further verification, plasma protein markers or their combinations with good differential diagnostic value will be used as potential tools for the management and differential diagnosis of patients with lung adenocarcinoma in clinical practice. Summary of the invention

[0007] Purpose of the invention: The technical problem to be solved by the present invention is to provide an application of anti-ATP6V0D1 autoantibody in the auxiliary diagnosis of lung adenocarcinoma in view of the deficiencies in the prior art.

[0008] In order to solve the above technical problems, the present invention discloses the following technical solutions:

[0009] In a first aspect, the present invention discloses a product for detecting lung adenocarcinoma biomarkers.

[0010] Wherein, the biomarkers include anti-ATP6V0D1 autoantibodies, and the autoantibodies are IgG and IgM autoantibodies in peripheral blood.

[0011] Among them, the product also includes the traditional tumor marker CEA.

[0012] Among them, the products include kits and reagents.

[0013] Wherein, the kit is an ELISA detection kit; further, the kit includes ATP6V0D1 protein.

[0014] Wherein, the detection of lung adenocarcinoma includes distinguishing lung adenocarcinoma from healthy people. In some embodiments, lung adenocarcinoma and healthy people are distinguished by detecting autoantibodies or the expression of autoantibodies and traditional tumor markers CEA. In some embodiments, lung adenocarcinoma and healthy people are distinguished by detecting the expression of autoantibodies and traditional tumor markers CEA by ELISA.

[0015] In a second aspect, the present invention discloses the use of a biomarker containing anti-ATP6V0D1 autoantibody or a reagent for detecting a biomarker containing anti-ATP6V0D1 autoantibody in the preparation of a product for predicting the risk of lung adenocarcinoma, screening or prognosis assessment, or in the preparation of a product for detecting the concentration of autoantibodies and / or tumor antigens in a subject sample.

[0016] Wherein, the autoantibody is an anti-ATP6V0D1 autoantibody, and the autoantibody is an IgG or IgM autoantibody in peripheral blood.

[0017] Among them, the product also includes the traditional tumor marker CEA.

[0018] Among them, the products include kits and reagents.

[0019] Wherein, the kit is an ELISA detection kit; further, the kit includes ATP6V0D1 protein.

[0020] Wherein, the sample is serum of the subject.

[0021] The basic information of the tumor-associated antigen ATP6V0D1 in the present invention is: ATPase H+transportingV0subunit D1 (ATP6V0D1), located on human chromosome 16, contains 8 exons. The protein is a major subunit of vacuolar ATPase (V-ATPase) in lysosomes, and its related pathways include insulin receptor recycling and folded protein response (UPR). V-ATPase is a heteromultimeric enzyme composed of two complexes: ATP hydrolysis V1 complex and proton translocation V0 complex. V-ATPase is a multi-subunit enzyme that mediates acidification of organelles in eukaryotic cells. V-ATPase-dependent organelle acidification is necessary for intracellular processes such as protein sorting, zymogen activation, receptor-mediated endocytosis, and synaptic vesicle proton gradient generation.

[0022] The present invention first discovered that the expression levels of IgG and IgM autoantibodies against tumor-associated antigen ATP6V0D1 in the plasma of patients with lung adenocarcinoma were significantly higher than those of normal people, and there was a significant difference. Using Logistic regression to jointly detect IgG and IgM autoantibodies against ATP6V0D1 and traditional tumor marker CEA, it was found that the diagnostic efficacy of the combination for lung adenocarcinoma was better than that of CEA alone. CEA is the CEA content in the patient's serum obtained by clinical electrochemiluminescence detection, and the unit is ng / mL.

[0023] Beneficial effects:

[0024] The present invention screens and identifies a novel plasma protein marker (anti-ATP6V0D1 autoantibody) that can be used for differential diagnosis of lung adenocarcinoma patients and healthy people through enzyme-linked immunosorbent assay. Anti-ATP6V0D1 autoantibody and tumor marker CEA are combined to construct a differential diagnosis model for lung adenocarcinoma patients and healthy people. The model has good differential diagnosis efficacy for lung adenocarcinoma patients and healthy people, and provides help for better clinical management of lung adenocarcinoma patients. And the detection sample of the kit of the present invention is plasma, which can avoid invasive diagnosis. The risk of lung adenocarcinoma can be obtained in the early stage by taking plasma for detection in a minimally invasive manner, requiring less blood, less pain for the detected person, and high compliance; moreover, the operation is simple, the time to detect the result is short, and there is a broad market prospect and social benefit. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0026] Figure 1 The SBI scatter plot and ROC curve of anti-ATP6V0D1 IgG and IgM autoantibodies in lung adenocarcinoma and healthy controls in the training set samples.

[0027] Figure 2 This is a scatter plot of SBI of IgG and IgM autoantibodies against ATP6V0D1 in the validation set samples in lung adenocarcinoma and healthy controls.

[0028] Figure 3 Figure 3 is the ROC curve of anti-ATP6V0D1 IgG and IgM autoantibodies in the validation set for diagnosing lung adenocarcinoma and healthy controls.

[0029] Figure 4 This is the ROC curve of anti-ATP6V0D1 IgG, IgM autoantibodies combined with traditional tumor marker CEA in the diagnosis of lung adenocarcinoma and healthy controls. DETAILED DESCRIPTION

[0030] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0031] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0032] The screening method for identifying plasma protein markers in lung adenocarcinoma patients and healthy controls in the present invention specifically comprises the following steps:

[0033] (1) Blood samples were collected from lung adenocarcinoma patients and healthy people, and plasma samples were obtained after separation and processing;

[0034] (2) Screen and identify plasma protein markers in patients with lung adenocarcinoma and healthy people.

[0035] Furthermore, ELISA validation of differential plasma proteins between lung adenocarcinoma patients and healthy subjects was performed; the differential plasma protein ELISA validation included: optimal protein coating concentration of ATP6V0D1 and optimal dilution concentration of secondary antibody; ELISA testing of plasma samples according to the kit instructions; and statistical analysis of the obtained data.

[0036] The w / v described in the following examples are all mg / mL unless otherwise specified.

[0037] Example: ELISA detection of plasma expression levels of autoantibodies against tumor-associated antigen ATP6V0D1

[0038] 1. Experimental samples

[0039] This study included 166 training set samples, including 83 patients with lung adenocarcinoma and 83 healthy controls, and 440 validation set samples, including 220 patients with lung adenocarcinoma and 220 healthy people. All samples were collected from a tertiary hospital in Henan Province from November 2019 to May 2022. The basic information, pathological stage, and clinical traditional tumor marker levels of all subjects were summarized and organized through the hospital's internal system with the consent of the patients and the approval of the institutional review board and the hospital ethics committee. The basic information of the specific research subjects is shown in Tables 1 and 2.

[0040] Table 1 Basic characteristics of training set samples

[0041]

[0042]

[0043] Table 2 Basic characteristics of validation set samples

[0044]

[0045]

[0046] (1) Patient sample and information collection process

[0047] 1) For patients who were hospitalized for suspicious lung nodules detected by CT scans, blood samples were collected on the first day or the second day of hospitalization without receiving any treatment (blood samples were collected from some lung cancer patients after treatment), and the samples were numbered, packaged, and their basic information was registered before being included in the blood sample library.

[0048] 2) After the patient was diagnosed, the hospitalization number, gender, age and other information of the research subjects were consulted in the hospital patient management system and relevant clinical information was collected, including the smoking history, drinking history, nodule diameter, traditional tumor markers (CEA, CYFRA211 detection values ​​and cutoff values) of lung adenocarcinoma patients, TNM staging of lung cancer patients, lymph node metastasis and distant metastasis, etc., and a patient clinical information database was established.

[0049] (2) Healthy control samples and information collection process

[0050] The data were collected from people who had physical examinations in a tertiary hospital in Henan Province. The physical examination number, gender, age and other information of the research subjects were consulted in the hospital patient management system and relevant clinical information was collected. Their blood samples were collected, numbered, packaged, and registered in the blood sample data information database before being included in the blood sample library.

[0051] (3) Inclusion and exclusion criteria of research subjects

[0052] The inclusion criteria for patients with lung adenocarcinoma included: 1) no chemotherapy, radiotherapy, surgery, or other interventions; 2) clear histopathological results, confirmed as lung adenocarcinoma according to the primary lung cancer diagnosis and treatment guidelines, and pathological histological types (including adenocarcinoma, carcinoma, adenocarcinoma, large cell carcinoma, sarcomatoid carcinoma, and mixed carcinoma); 3) no history of other malignant tumors; 4) complete medical history; 5) no autoimmune diseases; 6) all were new cases; 7) patients were >18 years old.

[0053] The inclusion criteria for healthy controls included: 1) no history of malignant tumors in the lung or other parts of the body; 2) no autoimmune diseases; 3) age >18 years old.

[0054] Exclusion criteria: subjects who do not meet the above inclusion criteria.

[0055] (4) Plasma collection:

[0056] 5 ml of peripheral blood was collected from the subjects in a fasting state and placed in a blood collection tube without anticoagulant. After standing at room temperature for 1 hour, it was placed in a centrifuge set at 4°C and centrifuged at 3000 rpm for 10 minutes. The serum on the upper layer of the blood collection tube was then aspirated and dispensed into 1.5 ml EP tubes. The sample number was marked on the top and side of the EP tubes and placed in a -80°C refrigerator for frozen storage. The blood collection date and storage location were recorded. Before use, the serum was taken out and placed in a 4°C refrigerator to thaw and aliquot to avoid repeated freezing and thawing of the serum.

[0057] 2. Experimental Materials and Reagents

[0058] (1) ATP6V0D1 protein was purchased from Wuhan Huamei Bioengineering Co., Ltd.

[0059] (2) 96-well ELISA plate (8 rows × 12 columns).

[0060] (3) Coating solution: an aqueous solution containing 0.15% sodium carbonate (Na2CO3) and 0.29% sodium bicarbonate (NaHCO3).

[0061] (4) Blocking solution: PBST buffer containing 2% (v / v) bovine serum albumin (BSA) and 0.2% (v / v) Tween 20.

[0062] (5) Plasma sample diluent: PBST buffer containing 1% (w / v) BSA.

[0063] (6) Enzyme-labeled secondary antibody: IgG, IgM labeled with horseradish peroxidase (HRP).

[0064] (7) Antibody diluent: PBST buffer containing 1% (w / v) BSA.

[0065] (8) Washing solution: PBST buffer containing 0.2% (v / v) Tween 20.

[0066] (9) Color developing solution: The color developing solution consists of color developing solution A and color developing solution B, wherein color developing solution A is an aqueous solution of 20% tetramethylbenzidine dihydrochloride, and color developing solution B is an aqueous solution containing 3.7% Na2HPO4·12H2O, 0.92% citric acid and 0.75% (v / v) urea hydrogen peroxide; when using, color developing solution A and color developing solution B are mixed evenly in equal volumes of 1:1 and prepared before use.

[0067] (10) Quality control plasma: 100 normal control blood samples were mixed in equal amounts to prepare quality control blood samples for quality control (QC), which was used to eliminate the errors between the various ELISA plates and represent the general level of the population. If the CV of the quality control samples between all ELISA plates prepared on the same day was less than 20%, the data could be used for subsequent analysis.

[0068] (11) Stop solution: 10% sulfuric acid.

[0069] 3. Experimental methods

[0070] (1) Protein pretreatment and determination of protein concentration: Use the protein diluent recommended in the protein manual to dilute the protein to an appropriate concentration and then perform SDS-PAGE electrophoresis to confirm the protein concentration.

[0071] (2) Preliminary experiments explored the optimal protein coating concentration of ATP6V0D1 and the dilution concentrations of secondary antibodies IgG and IgM: Plasma from four patients with lung adenocarcinoma and four healthy controls was randomly selected for the preliminary experiment. Two concentration gradients were set for the protein coating concentration and the secondary antibody dilution concentration. After the experiment, the optimal multiple was selected based on the sample OD value and the standard OD value to ensure that the color development time of the sample and the standard was consistent and the sample OD value was between the highest and lowest OD values ​​of the standard.

[0072] (3) Plate layout: According to the layout of the 96-well ELISA plate, calculate the number of samples, sample volume, protein volume, sample layout, etc. in advance. Calculate the total amount of reagents required for subsequent use based on the sample volume and prepare them in advance. The blank well is 1% BSA, and the sample volume is 50 μL / well. The sample layout table is shown in Table 3.

[0073] Table 3 Sample layout table (taking the first board as an example)

[0074]

[0075]

[0076] (4) Sample subpackaging and sample dilution: After selecting the required experimental samples from the sample library, slowly thaw them in a 4°C refrigerator the day before the experiment. The next day, the samples are divided into 96-well pointed-bottom PCR plates according to the previous layout and the required sample volume. Then, according to the sample dilution ratio required for the experiment, the samples are added to the antibody diluent in the corresponding layout of the 96-well deep-well plate, and the surface is covered with a film for sealing, and then placed in a 4°C refrigerator for future use.

[0077] (5) Protein coating: After using the coating solution to dilute the protein to the optimal coating concentration (0.125 μg / mL) determined in the preliminary experiment, the prepared ATP6V0D1 protein solution was added to the reaction wells in columns 1 to 12 of the 96-well ELISA plate after shaking, with a loading volume of 50 μL / well. The plates were sealed with plastic wrap to prevent evaporation and coated overnight at 4°C.

[0078] (6) Blocking: After removing the coating solution, add blocking solution to each reaction well of the 96-well ELISA plate at a volume of 100 μL / well, block in a 37°C water bath for 2 h, then remove the blocking solution, wash three times with washing solution and pat dry to obtain an ELISA plate coated with the tumor-associated antigen ATP6V0D1.

[0079] (7) Primary antibody (plasma) reaction: The plasma sample to be tested was diluted with plasma sample diluent at a volume ratio of 1:100, and then the diluted plasma sample was added to the ELISA plate at a specification of 50 μL / well according to the layout of the plate, and the sample volume was 50 μL / well; 50 μL / well of antibody diluent (without any plasma sample) was also added to the blank wells. Then the 96-well ELISA plate was placed in a 37°C water bath for incubation for 1 hour, and then the liquid in the reaction wells was discarded, and the wells were washed 5 times with washing solution (the sample volume was 300 μL / well) and patted dry.

[0080] (8) Enzyme-labeled secondary antibody incubation: HRP-labeled IgG and IgM were diluted with antibody diluent according to the optimal dilution concentration determined in the preliminary experiment. The optimal concentration of IgG is 1:5000 (v / v) and the optimal concentration of IgM is 1:2500 (v / v). Then, the diluted HRP-labeled IgG and IgM were added to each reaction well of the 96-well ELISA plate at a volume of 50 μl / well, and incubated in a 37°C water bath for 1 h. Then, the liquid in the reaction wells was discarded, and the wells were washed with washing solution (the volume was 300 μl / well) for 5 times and patted dry.

[0081] (9) Color development and termination reaction: Color development solution A and color development solution B were mixed in a 1:1 ratio of equal volumes, and then the mixed color development solution was quickly added to the reaction wells of a 96-well ELISA plate at a volume of 50 μl / well. The mixture was kept at room temperature and protected from light. The color development was observed at all times during the color development process. After sufficient color development, 25 μl of stop solution was added to each reaction well to terminate the color development reaction. The absorbance (optical density, OD) at 450 nm and 620 nm was then read using an ELISA reader, where the absorbance at 620 nm was the background value. The difference between the absorbance at 450 nm and 620 nm was used as the absorbance value for subsequent analysis, and the blank control well was used to adjust the color to zero.

[0082] 4. Data processing

[0083] The statistical analysis method of the present invention is to use SPSS26.0, GraphPad Prism 8.0 software to perform statistical analysis on experimental data and visualize the results. The difference in the expression level of anti-ATP6V0D1 autoantibodies between different groups was analyzed by Mann-Whitney U test, and the difference in positive rate was analyzed by chi-square test; the SBI value corresponding to the maximum Youden's Index (YI) was taken as the cutoff value, and the diagnostic value indicators such as AUC, specificity, sensitivity and 95% confidence interval (95% CI) of anti-ATP6V0D1 autoantibodies were calculated by ROC curve. All statistical analyses were performed using two-sided tests, and when P<0.05, the difference was considered to be statistically significant.

[0084] 5. Results Analysis

[0085] (1) ELISA pre-experiment to explore the optimal protein coating concentration of ATP6V0D1 and the optimal dilution concentrations of secondary antibodies IgG and IgM: A double antibody sandwich ELISA experiment was used to conduct a pre-experimental analysis on a small number of samples (plasma from 4 patients with lung adenocarcinoma and 4 patients with benign lung nodules) to explore the optimal protein coating concentration of ATP6V0D1 and the optimal dilution concentrations of secondary antibodies IgG and IgM in this experiment. The optimal protein coating concentration of ATP6V0D1 and the optimal dilution multiples of secondary antibodies IgG and IgM are shown in Table 3.

[0086] Table 3 ELISA preliminary experiment to explore the optimal protein coating concentration of ATP6V0D1 and the optimal dilution multiples of secondary antibodies IgG and IgM

[0087]

[0088] (2) ELISA experiments were used to preliminarily verify the differential diagnostic efficacy of IgG and IgM autoantibodies against the tumor-associated antigen ATP6V0D1

[0089] In the training set samples, the expression levels of anti-ATP6V0D1 IgG and IgM autoantibodies in the plasma samples of the lung adenocarcinoma group and the healthy control group were as follows: Figure 1 As shown in the figure, the expression levels of IgM and IgG autoantibodies against ATP6V0D1 in the plasma samples of the lung adenocarcinoma group were significantly higher than those in the healthy control group (P<0.0001). According to the receiver operating characteristic (ROC) curve analysis, when anti-ATP6V0D1 IgG autoantibodies were used to diagnose and distinguish between the lung adenocarcinoma group and the healthy control group, the AUC of the ROC curve was 0.7151 (95% CI: 0.6367-0.7936), and when the cutoff value was 0.3855, the sensitivity of using anti-ATP6V0D1 IgG autoantibodies to diagnose and distinguish between lung adenocarcinoma and the healthy control group reached 65.06%, and the specificity reached 73.49%; when anti-ATP6V0D1 IgM autoantibodies were used to diagnose and distinguish between the lung adenocarcinoma group and the healthy control group, the AUC of the ROC curve was 0.8393 (95% CI: 0.7793-0.8993), and when the cutoff value was 0.5542, the sensitivity of using anti-ATP6V0D1 IgG autoantibodies to diagnose and distinguish between lung adenocarcinoma and the healthy control group reached 67.47%, and the specificity reached 87.95%.

[0090] In the validation set samples, the expression levels of IgM and IgG autoantibodies against ATP6V0D1 in the plasma samples of the lung adenocarcinoma group and the healthy control group were as follows: Figure 2 As shown in the figure, the expression level of anti-ATP6V0D1 autoantibodies in the plasma samples of the lung adenocarcinoma group was significantly higher than that in the healthy control group (P < 0.0001). Figure 3It can be seen that through the receiver operating characteristic (ROC) curve analysis, when the IgG autoantibody against ATP6V0D1 was used to diagnose and distinguish the lung adenocarcinoma group and the healthy control group, the AUC of the ROC curve was 0.6314 (95% CI: 0.5798-0.6829), and when the cutoff value was 0.2091, the sensitivity of the IgG autoantibody against ATP6V0D1 to diagnose and distinguish the lung adenocarcinoma group and the healthy control group reached 75%, and the specificity reached 45.91%; the AUC of the ROC curve for the diagnosis and differentiation of the lung adenocarcinoma group and the healthy control group using the IgM autoantibody against the tumor-associated antigen ATP6V0D1 was 0.6842 (95% CI: 0.6350-0.7334), and when the cutoff value was 0.3045, the sensitivity reached 66.36%, and the specificity reached 64.09%. Logistic regression was used to jointly detect IgG and IgM autoantibodies against the tumor-associated antigen ATP6V0D1 to diagnose and distinguish the lung adenocarcinoma group from the healthy control group. The AUC of the ROC curve for distinguishing the lung adenocarcinoma group from the healthy control group was 0.7175 (95% CI: 0.6679-0.7623), which was higher than the two groups diagnosed separately. This shows that both IgG and IgM autoantibodies against ATP6V0D1 can distinguish the lung adenocarcinoma group from the healthy control group. This also proves that autoantibodies against ATP6V0D1 can be used for auxiliary diagnosis of lung adenocarcinoma. Among them, there were 440 cases in the lung adenocarcinoma group (LUAD) and the healthy control group (NC).

[0091] Figure 4 Logistic regression was used to detect anti-ATP6V0D1 IgG, IgM autoantibodies and traditional tumor marker CEA. The AUC of the ROC curve for distinguishing lung adenocarcinoma group from healthy control group was 0.7276 (95% CI: 0.5038-0.7078), which was higher than the area under the curve of CEA alone for distinguishing lung adenocarcinoma group from healthy control group, which was 0.5128 (95% CI: 0.4234-0.6022); and higher than the area under the curve of anti-IgG combined with anti-IgM for distinguishing lung adenocarcinoma group from healthy control group, which was 0.7012 (0.6919-0.7806). Among them, there were 222 cases of lung adenocarcinoma group (LUAD) and healthy control group (NC) in the samples with CEA information.

[0092] In summary, the present invention provides a plasma biomarker for auxiliary diagnosis of lung adenocarcinoma, wherein the marker is an autoantibody against the tumor-associated antigen ATP6V0D1. The present invention first discovered that the expression levels of IgG and IgM autoantibodies against the tumor-associated antigen ATP6V0D1 in the plasma of patients with lung adenocarcinoma were significantly higher than those in healthy controls and, with significant differences. Logistic regression was used to jointly detect IgG and IgM autoantibodies of ATP6V0D1 and traditional tumor marker CEA, and it was found that the diagnostic efficacy of the combination for lung adenocarcinoma was better than that of CEA alone. CEA is the CEA content in the patient's serum obtained by clinical electrochemiluminescence detection, and the unit is ng / mL.

[0093] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A product for detecting lung adenocarcinoma biomarkers, characterized in that: The biomarkers include anti-ATP6V0D1 autoantibodies.

2. The product according to claim 1, characterized in that The autoantibodies are IgG and IgM autoantibodies in peripheral blood.

3. The product according to claim 1, characterized in that The product also includes the traditional tumor marker CEA.

4. The product according to any one of claims 1 to 3, characterized in that: The products include kits and reagents.

5. The product according to claim 4, characterized in that The kit is an ELISA detection kit; preferably, the kit comprises ATP6V0D1 protein.

6. The product according to claim 1 or 2, characterized in that: The detection of lung adenocarcinoma includes distinguishing lung adenocarcinoma from healthy people; preferably, lung adenocarcinoma and healthy people are distinguished by detecting autoantibodies or the expression levels of autoantibodies and traditional tumor marker CEA, and further preferably, lung adenocarcinoma and healthy people are distinguished by detecting the expression levels of autoantibodies and traditional tumor marker CEA by ELISA.

7. Use of a biomarker containing anti-ATP6V0D1 autoantibody or a reagent for detecting a biomarker containing anti-ATP6V0D1 autoantibody in the preparation of a product for predicting the risk of lung adenocarcinoma, screening or prognosis assessment, or in the preparation of a product for detecting the concentration of autoantibodies and / or tumor antigens in a subject's sample.

8. The use according to claim 7, characterized in that The autoantibodies are IgG and IgM autoantibodies against ATP6V0D1, preferably IgG and IgM autoantibodies against ATP6V0D1 in peripheral blood; preferably, the biomarkers also include the traditional tumor marker CEA.

9. The use according to claim 7, characterized in that: The product includes a kit and a preparation; preferably, the kit is an ELISA detection kit.

10. The use according to claim 7, characterized in that The sample is serum from a subject.

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