Application of Anti-ATP6V0D1 Autoantibody in the Auxiliary Diagnosis of Lung Adenocarcinoma

Abstract

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

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of anti-ATP6V0D1 autoantibodies in the auxiliary diagnosis of lung adenocarcinoma. Background Technology

[0002] This section provides only background information relevant to this disclosure and is not necessarily prior art.

[0003] Primary bronchogenic carcinoma, or lung cancer (LC) for short, is one of the malignant tumors with high incidence and mortality rates worldwide. Early-stage lung cancer often presents with no obvious symptoms, and most patients are already in advanced stages when they seek medical attention. The overall 5-year survival rate for patients with advanced lung cancer is approximately 20%. Non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancer cases, with lung adenocarcinoma (LUAD) accounting for about 70% of NSCLC. Lung adenocarcinoma is the most common pathological type of lung cancer. Clinical studies show that early-stage lung adenocarcinoma treated with surgery combined with radiotherapy and chemotherapy can achieve a 5-year survival rate as high as 90%. However, the survival rate of lung adenocarcinoma that has metastasized is significantly lower because surgery is not an option. Due to the characteristic of early metastasis in adenocarcinoma, more than half of lung adenocarcinoma patients show local or distant metastases at the time of initial diagnosis, missing the optimal treatment window. Therefore, improving early diagnosis strategies for lung adenocarcinoma is key to reducing lung cancer mortality. Screening for lung adenocarcinoma tumor markers with high tissue specificity and sensitivity is of great significance for early diagnosis, prognosis, and precise treatment of lung adenocarcinoma.

[0004] For many years, both domestic and international efforts have focused on early diagnosis and treatment of lung cancer through screening, thereby reducing lung cancer-related mortality. The 2011 US National Lung Cancer Screening Trial, a randomized controlled study, showed that compared to X-ray examination, screening high-risk groups for lung cancer using low-dose computed tomography (LDCT) reduced lung cancer mortality by 20%. Multiple medical organizations in Europe and the US recommend LDCT for lung cancer screening in high-risk populations. While significantly reducing lung cancer mortality, non-high-risk individuals still have the potential to develop lung cancer; approximately 50% of newly diagnosed lung cancer cases do not meet screening criteria. Furthermore, most patients with pulmonary nodules cannot obtain crucial information from routine examinations, making it difficult to effectively differentiate 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' physical and financial well-being. Therefore, further research, development, and validation of non-invasive biomarkers that can effectively diagnose lung adenocarcinoma are still needed.

[0005] Blood-based tumor marker testing is the most readily accepted diagnostic method by patients. Protein markers are relatively stable and the testing methods are simple. Elevated levels of CEA, SCCA, and CYFRA21-1 in a patient's serum are helpful in diagnosing NSCLC. CEA shows the most significant elevation in lung adenocarcinoma and non-neuroendocrine large cell lung cancer, and has high sensitivity. However, it should be noted that elevated CEA can also be seen in gastrointestinal tumors and pulmonary interstitial fibrosis. Combined detection of CYFRA21-1 and CEA can improve the sensitivity and specificity of lung adenocarcinoma diagnosis. CEA levels in long-term smokers may be slightly higher than in healthy individuals. CYFRA21-1 is also a sensitive marker for NSCLC; however, it should be noted that trauma, saliva contamination, and patients with renal failure may cause false elevations in CYFRA21-1. SCCA has high specificity for squamous epithelial tumors such as lung squamous cell carcinoma and can assist in histological diagnosis. However, a single marker cannot differentiate between SCLC and NSCLC. Approximately 10% of NSCLC patients exhibit an immune response to at least one of the neuroendocrine biomarkers. Combined detection of NSE, ProGRP, CYFRA21-1, CEA, and SCCA can improve diagnostic accuracy. However, traditional tumor markers generally do not elevate in early-stage lung cancer patients, resulting in low detection sensitivity. Therefore, it is necessary to explore new minimally invasive blood molecular markers and biomarkers relevant to lung cancer diagnosis to improve lung cancer risk assessment and facilitate early detection of lung cancer patients.

[0006] Numerous studies have shown that tumors can trigger humoral immunity in the host at an early stage of development. 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 collective term for neoantigens and overexpressed antigenic substances that emerge during cell carcinogenesis. Studies have reported that TAAbs can be detected in the blood 17-47 months before cancer diagnosis. Therefore, TAAbs are highly promising biomarkers. This study aimed to screen and identify IgG and IgM humoral immune lung cancer antigens using serum samples from lung adenocarcinoma patients and healthy individuals. Further validation will allow serum protein biomarkers or combinations thereof with good diagnostic value to serve as potential tools for the clinical management and differential diagnosis of lung adenocarcinoma patients. Summary of the Invention

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

[0008] To solve the above-mentioned technical problems, the present invention discloses the following technical solution:

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

[0010] The biomarkers include anti-ATP6V0D1 autoantibodies, which are IgG and IgM type autoantibodies in peripheral blood.

[0011] The product also includes the traditional tumor marker CEA.

[0012] The products mentioned include reagent kits and reagents.

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

[0014] The detection of lung adenocarcinoma includes differentiating between lung adenocarcinoma and healthy individuals. In some embodiments, this is achieved by detecting autoantibodies or the expression levels of autoantibodies compared to the traditional tumor marker CEA. In other embodiments, it is achieved by using ELISA to detect the expression levels of autoantibodies and the traditional tumor marker CEA.

[0015] Secondly, the present invention discloses the use of biomarkers containing anti-ATP6V0D1 autoantibodies or reagents for detecting biomarkers containing anti-ATP6V0D1 autoantibodies in the preparation of products for risk prediction, screening or prognostic assessment of lung adenocarcinoma, or in the preparation of products for detecting the concentration of autoantibodies and / or tumor antigens in subject samples.

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

[0017] The product also includes the traditional tumor marker CEA.

[0018] The products mentioned include reagent kits and reagents.

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

[0020] The sample is the subject's serum.

[0021] The basic information of the tumor-associated antigen ATP6V0D1 in this invention is as follows: ATPase H+ transporting V0subunit D1 (ATP6V0D1), located on human chromosome 16, contains 8 exons. This protein is a major subunit of lysosomal vacuolar ATPase (V-ATPase), and its related pathways include insulin receptor recycling and the folding protein response (UPR). V-ATPase is a heteromultimeric enzyme composed of two complexes: the ATP hydrolysis V1 complex and the proton translocation V0 complex. V-ATPase is a multi-subunit enzyme that mediates organelle acidification in eukaryotic cells. V-ATPase-dependent organelle acidification is essential for intracellular processes such as protein sorting, zymogen activation, receptor-mediated endocytosis, and synaptic vesicle proton gradient generation.

[0022] This invention is the first to discover that the expression levels of IgG and IgM autoantibodies against the tumor-associated antigen ATP6V0D1 in the serum of patients with lung adenocarcinoma are significantly higher than those in healthy individuals, and the differences are statistically significant. Using logistic regression combined with the detection of IgG and IgM autoantibodies against ATP6V0D1 and the traditional tumor marker CEA, it was found that the combined method showed superior diagnostic efficacy for lung adenocarcinoma compared to CEA alone. CEA is the serum CEA content obtained clinically using electrochemiluminescence immunoassay, measured in ng / mL.

[0023] Beneficial effects:

[0024] This invention screens and identifies a novel serum protein biomarker (anti-ATP6V0D1 autoantibody) for the differential diagnosis of lung adenocarcinoma patients and healthy individuals using enzyme-linked immunosorbent assay (ELISA). The anti-ATP6V0D1 autoantibody is combined with the tumor marker CEA to construct a differential diagnostic model for lung adenocarcinoma patients and healthy individuals. This model demonstrates good diagnostic efficacy for both types of lung adenocarcinoma, providing assistance for better clinical management of lung adenocarcinoma patients. Furthermore, the kit of this invention uses serum as the test sample, avoiding invasive diagnosis. Early detection of lung adenocarcinoma risk can be achieved through minimally invasive serum collection and testing, requiring a small blood volume, causing minimal discomfort and high compliance among test subjects. Moreover, the operation is simple, and the results are available quickly, indicating broad market prospects and social benefits. Attached Figure Description

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

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

[0027] Figure 2 To validate the SBI scatter plot of anti-ATP6V0D1 IgG and IgM autoantibodies in lung adenocarcinoma and healthy controls.

[0028] Figure 3 ROC curves were used to validate the diagnosis of lung adenocarcinoma by IgG and IgM autoantibodies against ATP6V0D1 in the validation set samples compared with those of healthy controls.

[0029] Figure 4 ROC curves of combined IgG and IgM autoantibodies against ATP6V0D1 and traditional tumor marker CEA for the diagnosis of lung adenocarcinoma compared with healthy controls. Detailed Implementation

[0030] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

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

[0032] The method for screening serum protein biomarkers to distinguish between lung adenocarcinoma patients and healthy controls in this invention specifically includes the following steps:

[0033] (1) Collect blood samples from patients with lung adenocarcinoma and healthy individuals, and obtain serum samples after separation and processing;

[0034] (2) Screening serum protein markers to distinguish between patients with lung adenocarcinoma and healthy individuals.

[0035] Furthermore, differential serum protein ELISA validation was performed between lung adenocarcinoma patients and healthy individuals. This differential serum protein ELISA validation included determining the optimal protein coating concentration of ATP6V0D1 and the optimal dilution concentration of the secondary antibody; performing ELISA detection on serum samples according to the kit instructions; and statistically analyzing the obtained data.

[0036] Unless otherwise specified, all w / v values ​​mentioned in the following examples are in mg / mL.

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

[0038] 1. Experimental Samples

[0039] This study included 166 training set participants (83 patients with lung adenocarcinoma and 83 healthy controls) and 440 validation set participants (220 patients with lung adenocarcinoma and 220 healthy controls). All samples were collected from November 2019 to May 2022 at a tertiary hospital in Henan Province. Basic information, pathological stage, and levels of traditional clinical tumor markers for all participants were compiled and organized through the hospital's internal system with patient consent and approval from the institutional review committee and hospital ethics committee. Specific basic information of the study participants is shown in Tables 1 and 2.

[0040] Table 1 Basic characteristics of training set samples

[0041]

[0042] Table 2 Basic characteristics of the validation set samples

[0043]

[0044] (1) Patient sample and information collection process

[0045] 1) For patients who were hospitalized for this cause after CT scans detected suspicious lung nodules, blood samples were collected on the first day or the day after hospitalization when no treatment was received (for some lung cancer patients, blood samples were collected after treatment). The samples were numbered, packaged, and their basic information was registered before being entered into the blood sample bank.

[0046] 2) After the patient was diagnosed, the relevant clinical information was retrieved and collected in the hospital patient management system using the patient's hospital number, gender, age, etc., including smoking history, drinking history, nodule diameter, traditional tumor markers (detection values ​​and cutoff values ​​of CEA and CYFRA21-1) for lung adenocarcinoma patients, TNM stage, lymph node metastasis and distant metastasis for lung cancer patients, and a patient clinical information database was constructed.

[0047] (2) Healthy control sample and information collection process

[0048] The study collected data from individuals undergoing physical examinations at a tertiary hospital in Henan Province. Information such as the subjects' examination numbers, genders, and ages was retrieved from the hospital's patient management system, and relevant clinical information was collected. Blood samples were collected, numbered, packaged, and registered in the blood sample database.

[0049] (3) Inclusion and exclusion criteria for study subjects

[0050] The inclusion criteria for patients with lung adenocarcinoma include: 1) not having received chemotherapy, radiotherapy, surgery, or other interventions; 2) having a clear histopathological result, diagnosed with lung adenocarcinoma according to the guidelines for the diagnosis and treatment of primary lung cancer, and the pathological histological type (including adenocarcinoma, carcinoma, large cell carcinoma, sarcomatoid carcinoma, and mixed carcinoma); 3) no history of other malignant tumors; 4) having a complete medical history; 5) no autoimmune diseases; 6) all being newly diagnosed cases; and 7) patients aged >18 years.

[0051] The inclusion criteria for healthy controls included: 1) no history of malignant tumors in the lungs or other parts of the body; 2) no autoimmune diseases; and 3) age > 18 years.

[0052] Exclusion criteria: Research subjects that do not meet the above inclusion criteria.

[0053] (4) Serum collection:

[0054] Five ml of peripheral blood was collected from the subjects while they were fasting and placed in a blood collection tube without anticoagulant. After standing at room temperature for 1 hour, the tube was centrifuged at 3000 rpm for 10 minutes at 4°C. The serum from the top of the blood collection tube was then aspirated and aliquoted into 1.5 ml EP tubes. The sample numbers were marked on the top and side of the EP tubes, and the tubes were frozen at -80°C. The blood collection date and storage location were recorded. Before use, the serum was thawed at 4°C and aliquoted to avoid repeated freeze-thaw cycles.

[0055] 2. Experimental materials and reagents

[0056] (1) ATP6V0D1 protein, purchased from Wuhan Huamei Biotechnology Co., Ltd.

[0057] (2) 96-well microplate (8 rows × 12 columns).

[0058] (3) Coating solution: an aqueous solution containing 0.15% sodium carbonate (Na2CO3) and 0.29% sodium bicarbonate (NaHCO3).

[0059] (4) Blocking solution: PBST buffer containing 2% (v / v) bovine serum albumin (BSA) in 0.2% (v / v) Tween 20.

[0060] (5) Serum sample diluent: PBST buffer containing 1% (w / v) BSA.

[0061] (6) Enzyme-labeled second antibody: horseradish peroxidase (HRP) labeled IgG and IgM.

[0062] (7) Antibody diluent: PBST buffer containing 1% (w / v) BSA.

[0063] (8) Washing solution: PBST buffer containing 0.2% (v / v) Tween 20.

[0064] (9) Colorimetric solution: The colorimetric solution consists of colorimetric solution A and colorimetric solution B. Colorimetric solution A is an aqueous solution of 20% tetramethylbenzidine dihydrochloric acid, and colorimetric solution B is an aqueous solution containing 3.7% Na2HPO4•12H2O, 0.92% citric acid and 0.75% (v / v) hydrourea peroxide. When using, colorimetric solution A and colorimetric solution B are mixed evenly in equal volumes at a ratio of 1:1 and prepared fresh for each use.

[0065] (10) Quality control serum: 100 blood samples from normal controls were thoroughly and equally mixed to prepare a quality control serum for quality control (QC) to eliminate errors between different ELISA plates and to represent the general level of the population. If the CV of the quality control samples among all ELISA plates processed on the same day is <20%, the data are considered usable for subsequent analysis.

[0066] (11) Termination solution: 10% sulfuric acid.

[0067] 3. Experimental Methods

[0068] (1) Protein pretreatment and protein concentration determination: Dilute the protein to a suitable concentration using the protein dilution buffer recommended in the protein instruction manual and then perform SDS-PAGE electrophoresis to confirm the protein concentration.

[0069] (2) Preliminary experiment to explore the optimal protein coating concentration of ATP6V0D1 and the dilution concentration of secondary antibody IgG and IgM: Serum from 4 lung adenocarcinoma patients and 4 healthy controls were randomly selected for preliminary experiment. Two concentration gradients were set for protein coating concentration and secondary antibody dilution concentration. After the experiment, the optimal multiple was selected by combining the OD value of the sample and the OD value of the standard to ensure that the color development time of the sample and the standard was consistent and that the OD value of the sample was between the highest and lowest values ​​of the OD value of the standard.

[0070] (3) Plate preparation: Calculate the number of samples, sample volume, protein volume, and sample layout in advance according to the layout of a 96-well microplate. Calculate the total amount of reagents required for subsequent processing based on the sample volume and prepare them in advance. The blank wells contain 1% BSA, with a sample volume of 50 μL / well. The sample layout is shown in Table 3.

[0071] Table 3 Sample Layout Table (Taking the first board as an example)

[0072]

[0073] (4) Sample aliquoting and dilution: After selecting the required experimental samples from the sample library, thaw them slowly in a 4°C refrigerator the day before the experiment. The next day, aliquot the samples into 96-well pointed-bottom PCR plates according to the previously arranged plate layout and the required sample volume. Then, according to the required sample dilution ratio, add the antibody diluent to the corresponding layout in the 96-well deep-well plates, cover the surface with a membrane for sealing, and place in a 4°C refrigerator for later use.

[0074] (5) Protein coating: After diluting the protein to the optimal coating concentration (0.125 μg / mL) determined in the pre-experiment using coating buffer, the prepared ATP6V0D1 protein solution was added to the reaction wells in columns 1-12 of a 96-well microplate after shaking. The sample volume was 50 μL / well. The plate was sealed with plastic wrap to prevent evaporation and coated overnight at 4°C.

[0075] (6) Blocking: Add blocking solution to each reaction well of the 96-well microplate after removing the coating solution. The sample volume is 100 μL / well. Block in a 37℃ water bath for 2 h. Then remove the blocking solution, wash 3 times with washing solution and pat dry to obtain the microplate coated with tumor-associated antigen ATP6V0D1.

[0076] (7) Primary antibody (serum) reaction: Dilute the serum sample to be tested with serum sample diluent at a volume ratio of 1:100. Then, add the diluted serum sample to the microplate at a density of 50 μL / well according to the plate layout. The sample volume is 50 μL / well. Add 50 μL of antibody diluent (without any serum sample) to the blank wells. Then, incubate the 96-well microplate at 37°C for 1 hour. Then, discard the liquid in the reaction wells, wash 5 times with washing buffer (sample volume of 300 μL / well), and pat dry.

[0077] (8) Enzyme-labeled secondary antibody incubation: HRP-labeled IgG and IgM were diluted with antibody diluent according to the optimal dilution ratio determined in the pre-experiment. The optimal concentration of IgG was 1:5000 (v / v), and the optimal concentration of IgM was 1:2500 (v / v). Then, the diluted HRP-labeled IgG and IgM were added to each well of the 96-well microplate, with a sample volume of 50 μl / well. The wells were incubated in a 37°C water bath for 1 h. The liquid in the wells was then discarded, and the wells were washed 5 times with washing buffer (sample volume of 300 μl / well) and patted dry.

[0078] (9) Color development and termination of reaction: Mix color development solution A and color development solution B in equal volumes at a ratio of 1:1. Then, quickly add the mixed color development solution to the reaction wells of the 96-well microplate at a volume of 50 μl / well. Keep at room temperature and away from light. Observe the color development during the color development process. After sufficient color development, add 25 μl of termination solution to each reaction well to terminate the color development reaction. Then, use a microplate reader to read the absorbance (optical density, OD) at wavelengths of 450 nm and 620 nm. The absorbance value at wavelength 620 nm is the background value. The difference between the absorbance at wavelengths of 450 nm and 620 nm is used as the absorbance value for subsequent analysis. Zero the plate with a blank control well.

[0079] 4. Data Processing

[0080] The statistical analysis method of this invention utilizes SPSS 26.0 and GraphPad Prism 8.0 software for statistical analysis and visualization of experimental data. Differences in anti-ATP6V0D1 autoantibody expression levels among different groups were analyzed using the Mann-Whitney U test, and differences in positive rates were analyzed using the chi-square test. The SBI value corresponding to the maximum Youden's Index (YI) was used as the cutoff value. Diagnostic value indicators such as AUC, specificity, sensitivity, and 95% confidence interval (95% CI) of anti-ATP6V0D1 autoantibody were calculated using ROC curves. All statistical analyses employed two-tailed tests, and a p-value < 0.05 was considered statistically significant.

[0081] 5. Results Analysis

[0082] (1) Preliminary ELISA experiment to explore the optimal protein coating concentration of ATP6V0D1 and the optimal dilution concentrations of secondary antibodies IgG and IgM: A preliminary ELISA experiment was conducted on a small number of samples (serum from 4 patients with lung adenocarcinoma and 4 patients with benign pulmonary 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 factors of secondary antibodies IgG and IgM are shown in Table 4.

[0083] Table 4. Preliminary ELISA experiments to explore the optimal protein coating concentration of ATP6V0D1 and the optimal dilution ratios of secondary antibodies IgG and IgM.

[0084]

[0085] (2) The ELISA assay was used to preliminarily verify the differential diagnostic efficacy of IgG and IgM type autoantibodies against tumor-associated antigen ATP6V0D1.

[0086] In the training set samples, the expression levels of anti-ATP6V0D1 IgG and IgM autoantibodies in serum samples from the lung adenocarcinoma group and the healthy control group are as follows: Figure 1 As shown, the expression levels of IgM and IgG autoantibodies against ATP6V0D1 in serum samples of the lung adenocarcinoma group were significantly higher than those in the healthy control group (P<0.0001). Receiver operating characteristic (ROC) curve analysis showed that when using anti-ATP6V0D1 IgG autoantibodies to differentiate 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). Furthermore, when the cutoff value was 0.3855, the sensitivity and specificity of using anti-ATP6V0D1 IgG autoantibodies to differentiate between lung adenocarcinoma and the healthy control group reached 65.06% and 73.49%, respectively. When using anti-ATP6V0D1 IgM autoantibodies to differentiate 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). Furthermore, when the cutoff value was 0.5542, the sensitivity and specificity of using anti-ATP6V0D1 IgG autoantibodies to differentiate between lung adenocarcinoma and the healthy control group reached 67.47% and 87.95%, respectively.

[0087] In the validation set samples, the expression levels of IgM and IgG autoantibodies against ATP6V0D1 in serum samples from the lung adenocarcinoma group and the healthy control group were as follows: Figure 2 As shown, the expression level of anti-ATP6V0D1 autoantibodies in serum samples from the lung adenocarcinoma group was significantly higher than that in the healthy control group (P < 0.0001). Figure 3The receiver operating characteristic (ROC) curve analysis showed that when using the IgG autoantibody against ATP6V0D1 to differentiate between the lung adenocarcinoma group and the healthy control group, the AUC of the ROC curve was 0.6314 (95% CI: 0.5798-0.6829). Furthermore, when the cutoff value was 0.2091, the sensitivity of using the IgG autoantibody against ATP6V0D1 to differentiate between the lung adenocarcinoma group and the healthy control group reached 75%, and the specificity reached 45.91%. When using the IgM autoantibody against the tumor-associated antigen ATP6V0D1 to differentiate between the lung adenocarcinoma group and the healthy control group, the AUC of the ROC curve was 0.6842 (95% CI: 0.6350-0.7334). Furthermore, when the cutoff value was 0.3045, the sensitivity reached 66.36%, and the specificity reached 64.09%. Logistic regression combined with the detection of IgG and IgM autoantibodies against the tumor-associated antigen ATP6V0D1 was used to differentiate between the lung adenocarcinoma (LUAD) group and the healthy control group. The combined ROC curve AUC for differentiating the LUAD group and the healthy control group was 0.7175 (95% CI: 0.6679–0.7623), higher than that of the two groups diagnosed individually. This indicates that both IgG and IgM autoantibodies against ATP6V0D1 can distinguish the LUAD group from the healthy control group. This also proves that anti-ATP6V0D1 autoantibodies can be used for the auxiliary diagnosis of LUAD. The LUAD group and the healthy control group (NC) comprised a total of 440 cases.

[0088] Figure 4 To investigate the use of logistic regression to detect IgG and IgM autoantibodies against ATP6V0D1 in combination with the traditional tumor marker CEA, the AUC of the ROC curve for distinguishing between the lung adenocarcinoma (LUAD) group and the healthy control group was 0.7276 (95% CI: 0.6491-0.8086). This was higher than the area under the curve (AUC) of 0.5128 (95% CI: 0.4234-0.6022) for CEA alone in distinguishing between the LUAD group and the healthy control group, and higher than the AUC of 0.7012 (0.6919-0.7806) for anti-IgG combined with anti-IgM in distinguishing between the LUAD group and the healthy control group. Among the samples containing CEA information, there were a total of 222 cases in the LUAD group and the healthy control group (NC).

[0089] In summary, this invention provides a serum biomarker for the auxiliary diagnosis of lung adenocarcinoma, wherein the biomarker is an autoantibody against the tumor-associated antigen ATP6V0D1. This invention is the first to discover that the expression levels of both IgG and IgM autoantibodies against the tumor-associated antigen ATP6V0D1 in the serum of lung adenocarcinoma patients are significantly higher than those in healthy controls, and the differences are statistically significant. Using logistic regression combined with the detection of IgG and IgM autoantibodies against ATP6V0D1 and the traditional tumor marker CEA, it was found that the combined detection is superior to CEA alone in terms of diagnostic efficacy for lung adenocarcinoma. CEA is the serum CEA content obtained clinically using electrochemiluminescence immunoassay, measured in ng / mL.

[0090] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. The use of a reagent for detecting biomarkers containing anti-ATP6V0D1 autoantibodies in the preparation of products for risk prediction and screening of lung adenocarcinoma; wherein the autoantibody is an IgG or IgM type autoantibody against ATP6V0D1 in peripheral blood.

2. The use according to claim 1, characterized in that, The biomarkers also include the traditional tumor marker CEA.

3. The use according to claim 1, characterized in that, The products include reagent kits and formulations.

4. The use according to claim 3, characterized in that, The kit is an ELISA detection kit.

Images