Application of anti-FUCA2 autoantibody in preparation of lung adenocarcinoma auxiliary diagnosis product

Abstract

The invention discloses an application of an anti-FUCA2 autoantibody in preparation of a lung adenocarcinoma auxiliary diagnosis product. The anti-FUCA2 autoantibody is an anti-FUCA2 IgG and IgM type autoantibody. By detecting the expression levels of the two biomarkers in human plasma, lung adenocarcinoma patients and healthy people as well as lung adenocarcinoma patients and benign pulmonary nodule patients can be effectively distinguished, and by combining the two autoantibodies with the tumor marker CEA, the differential diagnosis efficiency of the lung adenocarcinoma patients can be improved; the method is of great significance to management and identification of lung adenocarcinoma patients.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to use of anti-FUCA2 autoantibodies in preparing lung adenocarcinoma (LUAD) auxiliary diagnosis products. Background Art

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

[0003] At present, the monitoring of the therapeutic efficacy of advanced lung cancer relies heavily on the evaluation criteria of imaging. However, in daily clinical practice, it is often not possible to evaluate the progression of tumors simply through imaging tests. For example, imaging examinations often have a lag, making it difficult to detect changes in the disease such as lymphatic invasion and pleural lung cancer at an early stage. In addition, long-term repeated imaging examinations will bring greater radiation hazards and economic burdens to patients. Therefore, how to achieve early diagnosis and treatment of lung cancer and better evaluate the efficacy and adjust the treatment plan in time is crucial to improve the survival of lung cancer patients. At present, histopathological examination is still the "gold standard" for diagnosing lung cancer. However, tissue biopsy has the problems of large trauma, painful process and difficulty in accurately sampling small lesions. Common non-invasive examination methods in clinical practice have included low-dose computed tomography (LDCT), positron emission tomography / computed tomography (PET-CT), serological tumor markers, etc., but the false positive rate of LDCT is high, and other means are needed for auxiliary diagnosis, and repeated imaging examinations increase the risk of radiation exposure. PET-CT examination is mainly used to determine tumor metastasis, but has a high false negative rate for small nodules and is expensive, so it is not used as a routine screening method. 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.

[0004] In the early stages of a tumor, tumor cell remodeling causes changes in protein expression patterns and the tumor microenvironment. During tumorigenesis, extracellular vesicles (EVs) shed by tumor cells and intracellular proteins released by tumor cell death also affect the tumor microenvironment. The defense system may recognize them as external mediators and trigger humoral and cellular immune responses. These abnormal proteins that the body's immune system can recognize are tumor-associated antigens (TAAs), and the specific antibodies produced by the immune system against them are called tumor-associated autoantibodies (TAAbs). Related studies have shown that tumor-associated autoantibodies can be detected several years before the tumor is diagnosed. Some studies have shown that TAAbs have the characteristics of strong specificity, good stability, and long half-life, which are conducive to improving the accuracy and stability of the test results. Therefore, TAAbs are expected to become a good biomarker for lung cancer diagnosis and efficacy evaluation.

[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. A large number of studies have been conducted on biomarkers for biological macromolecules in the human body, including circulating tumor cells (CTCs), circulating tumor cell DNA (ctDNA), micRNA, etc. in tumor "liquid biopsy" and tumor-related proteins such as carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), neuron-specific enolase (NSE), etc. that have been routinely tested in clinical practice. Although the above-mentioned types of biomarkers have shown certain application prospects, most markers are difficult to enter clinical practice. The content of CTC or ctDNA in the blood circulation of early cancer patients is extremely small. RNA markers require tissue specimens or fresh blood specimens. Tissue samples are invasive and difficult to obtain. Long-term frozen blood specimens are not suitable for RNA detection. In addition, traditional tumor markers are generally not elevated in patients with early lung cancer, so the detection sensitivity is low. At the same time, CA125 and CEA can be elevated in a variety of tumors and lack tumor specificity. Therefore, it is necessary to find new minimally invasive blood molecular markers and biomarkers related to lung cancer diagnosis, which can improve lung cancer risk assessment and help in early detection of lung cancer patients.

[0006] Compared with self-antigens, autoantibodies against tumor-associated antigens (TAAbs) in the blood are more stable and easier to detect after being amplified by immune response. TAAbs can be stably present in the blood, with high expression and long half-life, and show high levels in the very early stage of cancer. Studies have reported that TAAbs can be detected in the blood 17-47 months before cancer is diagnosed. Therefore, TAAbs are biomarkers with great development potential. However, there are few studies on TAAbs, and existing studies have focused on the study of IgG antibodies, and there are few reports on the study of other types of antibodies. This study aims to screen and identify lung cancer antigens of IgG and IgM humoral immunity through plasma samples of lung adenocarcinoma patients and healthy people. Through further verification, plasma protein markers or their combinations with good differential diagnostic value will be used as potential tools for 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 provide the use of anti-FUCA2 autoantibodies in the preparation of products for diagnosing 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 diagnosing biomarkers of lung adenocarcinoma.

[0010] Among them, the biomarker for diagnosing lung adenocarcinoma includes anti-FUCA2 autoantibodies, and the anti-FUCA2 autoantibodies are anti-FUCA2 IgG and IgM type autoantibodies. Furthermore, the anti-FUCA2 autoantibodies are anti-FUCA2 IgG and IgM type autoantibodies in peripheral blood.

[0011] The present invention firstly finds that the expression levels of anti-FUCA2 IgG and IgM autoantibodies in the plasma of patients with lung adenocarcinoma are significantly higher than those of healthy controls and patients with benign lung nodules, and there is a significant difference.

[0012] Among them, the product also includes the traditional tumor marker CEA.

[0013] Among them, the products include kits and preparations.

[0014] In some embodiments, the kit is an ELISA detection kit; in some embodiments, the ELISA detection kit includes FUCA2 protein.

[0015] Among them, the diagnosis of lung adenocarcinoma includes distinguishing lung adenocarcinoma from healthy people, distinguishing lung adenocarcinoma from benign lung nodules, such as diagnosing lung adenocarcinoma by detecting autoantibodies or the expression of autoantibodies and traditional tumor marker CEA, such as diagnosing lung adenocarcinoma by detecting the expression of autoantibodies and traditional tumor marker CEA through ELISA.

[0016] In a second aspect, the present invention discloses the use of a biomarker containing anti-FUCA2 autoantibodies or a reagent for detecting a biomarker containing anti-FUCA2 autoantibodies in the preparation of a product for diagnosing lung adenocarcinoma, or in the preparation of a product for predicting, screening or evaluating the risk of adenocarcinoma, or in the preparation of a product for detecting the concentration of autoantibodies and / or tumor antigens in a sample from a subject.

[0017] Among them, the diagnosis of lung adenocarcinoma includes distinguishing lung adenocarcinoma from healthy people, distinguishing lung adenocarcinoma from benign lung nodules, such as diagnosing lung adenocarcinoma by detecting autoantibodies or the expression of autoantibodies and traditional tumor marker CEA, such as diagnosing lung adenocarcinoma by detecting the expression of autoantibodies and traditional tumor marker CEA through ELISA.

[0018] The anti-FUCA2 autoantibodies are anti-FUCA2 IgG and IgM autoantibodies, and further are anti-FUCA2 IgG and IgM autoantibodies in peripheral blood.

[0019] Wherein, the biomarkers also include the traditional tumor marker CEA.

[0020] Among them, the products include kits and preparations.

[0021] In some embodiments, the kit is an ELISA detection kit; in some embodiments, the ELISA detection kit includes FUCA2 protein.

[0022] Wherein, the sample is serum of the subject.

[0023] The basic information of the tumor-associated antigen FUCA2 in the present invention is: Plasma alpha-L-fucosidase (FUCA2) is the full name of plasma alpha-L-fucosidase. FUCA2 glycosidases and glycosyltransferases greatly affect the malignant phenotype of tumors through genetic and epigenetic mechanisms. As a member of the glycoside hydrolase (GH) family 29A, alpha-L-fucosidase (AFU) is involved in the hydrolysis of the terminal L-fucose residues at the reducing end of N-acetylglucosamine (GlcNAc) connected to the oligosaccharide chain through α-1,2, α-1,3, α-1,4 or α-1,6. The deglycosylation process mediated by AFU promotes the development of various diseases, such as chronic inflammatory diseases, immune diseases and autoimmune diseases, by reducing the interaction between fucosylated adhesion molecules that support leukocyte extravasation. AFU also impairs key cell-extracellular matrix (ECM) interactions and possible subsequent cell signaling pathways, leading to changes in tumor function and behavior. There are two isoforms of AFU in humans, α-L-fucosidase 1 (FUCA1) and α-L-fucosidase 2 (FUCA2). FUCA1 is a p53 target gene that hydrolyzes different fucosylation sites on the epidermal growth factor receptor (EGFR), thereby determining EGFR activation. FUCA2 mediates the adhesion of Helicobacter pylori to the gastric mucosa and is upregulated in 24 tumor types.

[0024] The biomarker for auxiliary diagnosis of lung adenocarcinoma provided by the present invention is an autoantibody against Galactosidase Beta1 (FUCA2). The expression level of the marker in the plasma of lung adenocarcinoma patients is higher than that in patients with benign lung nodules and healthy controls, and the difference is statistically significant.

[0025] The novel plasma anti-FUCA2 IgG and IgM provided by the present invention are combined with serum tumor markers such as carcinoembryonic antigen (CEA) to have good efficacy in the differential diagnosis of lung adenocarcinoma, and are of great significance for the management and identification of lung adenocarcinoma patients.

[0026] The diagnosis of lung adenocarcinoma in the present invention includes auxiliary diagnosis of lung adenocarcinoma.

[0027] Beneficial effects:

[0028] The present invention screens and identifies novel plasma biomarkers (anti-FUCA2 IgG, IgM autoantibodies) that can be used for differential diagnosis of lung adenocarcinoma patients, healthy people, and benign pulmonary nodules patients through enzyme-linked immunosorbent assay. Anti-FUCA2 IgG, IgM and tumor marker CEA are combined to construct a differential diagnosis model for lung adenocarcinoma patients, healthy people, and benign pulmonary nodules. The model has good differential diagnosis efficacy for lung adenocarcinoma patients, healthy people, and benign pulmonary nodules, and provides help for better clinical management of lung adenocarcinoma patients. The test 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 person being tested, and high compliance; moreover, the operation is simple, the time to detect the result is short, and it has broad market prospects and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] 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.

[0030] Figure 1 The SBI scatter plot and ROC curve of anti-FUCA2 IgG and IgM autoantibodies in the training sample set in lung adenocarcinoma and healthy controls.

[0031] Figure 2 To verify the SBI scatter plot and ROC curve of anti-FUCA2 IgG and IgM autoantibodies in the sample set for diagnosing lung adenocarcinoma, benign pulmonary nodules and healthy controls.

[0032] Figure 3 ROC curves of IgG and IgM autoantibodies against tumor-associated antigen FUCA2 in the validation set combined with traditional tumor marker CEA for the diagnosis of lung adenocarcinoma, benign lung nodules and healthy controls. DETAILED DESCRIPTION

[0033] 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.

[0034] 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.

[0035] The w / v described in the following examples, unless otherwise specified, is (g / mL).

[0036] The method for screening lung adenocarcinoma markers described in the present invention specifically comprises the following steps: (1) collecting blood samples from lung adenocarcinoma patients and healthy people, and obtaining plasma samples after separation and treatment; (2) screening and identifying plasma protein markers of lung adenocarcinoma patients and healthy people.

[0037] The method for verifying the lung adenocarcinoma markers described in the present invention is specifically to verify the differential plasma protein ELISA between lung adenocarcinoma patients and healthy people and patients with benign lung nodules.

[0038] The differential plasma protein ELISA verification includes: the optimal protein coating concentration of FUCA2 and the optimal dilution concentration of the secondary antibody; performing ELISA testing on the plasma samples according to the instructions of the kit; and performing statistical analysis on the obtained data.

[0039] Example: ELISA to detect the plasma expression level of anti-FUCA2 autoantibodies

[0040] 1. Experimental samples

[0041] This study included 166 training set samples, including 83 patients with lung adenocarcinoma and 83 healthy controls, and 660 validation set samples, including 220 patients with lung adenocarcinoma, 220 patients with benign pulmonary nodules, 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.

[0042] Table 1 Basic characteristics of the research subjects in the training set samples

[0043]

[0044]

[0045] Table 2 Basic characteristics of the research subjects in the validation set samples

[0046]

[0047]

[0048] (1) Patient sample and information collection process

[0049] 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.

[0050] 2) After the patient is diagnosed, the hospitalization number, gender, age and other information of the research subject are consulted in the hospital patient management system and relevant clinical information is collected, including the smoking history, drinking history, traditional tumor markers (detection values ​​and cutoff values ​​of CEA, etc.) of patients with lung adenocarcinoma and benign lung nodules, and a patient clinical information database is established.

[0051] (2) Healthy control samples and information collection process

[0052] 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.

[0053] (3) Inclusion and exclusion criteria of research subjects

[0054] 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.

[0055] The inclusion criteria for patients with benign pulmonary nodules included: 1) clear histopathological biopsy confirmed its benign nature and pathological histological type; 2) no history of malignant tumors in the lungs or other parts; 3) all were new cases without any treatment; 4) age >18 years old.

[0056] 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.

[0057] Exclusion criteria: subjects who do not meet the above inclusion criteria.

[0058] (4) Plasma collection:

[0059] 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.

[0060] 2. Experimental Materials and Reagents

[0061] (1) FUCA2 protein was purchased from Wuhan Cloud-Clone Technology Service Co., Ltd.

[0062] (2) 96-well ELISA plate (8 rows × 12 columns).

[0063] (3) Coating solution: an aqueous solution containing 0.15% sodium carbonate (Na2CO3) and 0.29% sodium bicarbonate (NaHCO3).

[0064] (4) Blocking solution: PBST buffer containing 2% (v / v) bovine serum albumin (BSA) and 0.2% (v / v) Tween 20.

[0065] (5) Plasma sample diluent: PBST buffer containing 1% (w / v) BSA.

[0066] (6) Enzyme-labeled secondary antibodies: HRP anti-human IgG monoclonal antibody, purchased from Wuhan Aokebot Biotechnology Co., Ltd., and anti-human IgM (μ-chain specific) goat antibody, purchased from Merck Biotechnology Co., Ltd.

[0067] (7) Antibody diluent: PBST buffer containing 1% (w / v) BSA.

[0068] (8) Washing solution: PBST buffer containing 0.2% (v / v) Tween 20.

[0069] (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.

[0070] (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.

[0071] (11) Stop solution: 10% sulfuric acid.

[0072] 3. Experimental methods

[0073] (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.

[0074] (2) Preliminary experiments explored the optimal protein coating concentration of FUCA2 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 a 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 that the sample OD value was between the highest and lowest OD values ​​of the standard.

[0075] (3) Training set sample experiment: After determining the optimal protein coating concentration of FUCA2 and the dilution concentration of the secondary antibody IgG, plasma from lung adenocarcinoma patients and healthy controls were randomly selected for the training set sample experiment, including 83 lung adenocarcinoma patients and 83 healthy controls.

[0076] (4) Validation set sample experiment: The plasma of patients with lung adenocarcinoma, benign lung nodules and healthy controls was randomly selected for validation set sample experiment, including 220 patients with lung adenocarcinoma, 220 healthy controls and 220 benign lung nodules.

[0077] (5) 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 2 and Table 3.

[0078] Table 3 Training set sample layout table (taking the first board as an example)

[0079]

[0080]

[0081] Table 4 Validation set sample layout table (taking the first board as an example)

[0082]

[0083] (6) 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.

[0084] (7) Protein coating: Use coating solution to dilute the protein to the optimal coating concentration (0.125 μg / mL) determined in the preliminary experiment. After mixing, add the prepared FUCA2 protein solution to the reaction wells in columns 1 to 12 of the 96-well ELISA plate at a volume of 50 μL / well. Cover with plastic wrap to prevent evaporation and coat overnight at 4°C.

[0085] (8) Blocking: After removing the coating solution, add blocking solution to each reaction well of the coated 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 FUCA2.

[0086] (9) Primary antibody (plasma) reaction: dilute the plasma sample to be tested with plasma sample diluent at a volume ratio of 1:100, then add the diluted plasma sample to the corresponding ELISA plate according to the plate layout, with a sample volume of 50 μL / well; 50 μL / well of antibody diluent (without any plasma sample) is also added to the blank well. Then place the 96-well ELISA plate in a 37°C water bath for 1 hour, then discard the liquid in the reaction wells, wash 5 times with washing solution (sample volume of 300 μL / well) and pat dry.

[0087] (10) Enzyme-labeled secondary antibody incubation: For the secondary antibody reaction, HRP-labeled IgG and IgM were diluted with antibody diluent according to the optimal dilution ratio determined in the preliminary experiment. The optimal concentration of IgG was 1:5000 (v / v) and the optimal concentration of IgM was 1:5000 (v / v). The diluted HRP-labeled IgG and IgM were then added to each reaction well of the 96-well ELISA plate at a volume of 50 μl / well. The plate was incubated in a 37°C water bath for 1 h, and then the liquid in the reaction wells was discarded. The plates were washed with washing solution (300 μl / well) for 5 times and patted dry.

[0088] (11) 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 for zero adjustment.

[0089] 4. Data processing

[0090] The statistical analysis method of the present invention is to use SPSS26.0, GraphPad Prism 8.0 software to perform statistical analysis on the experimental data and visualize the results. The difference in the expression level of anti-FUCA2 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-FUCA2 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.

[0091] 5. Results Analysis

[0092] (1) ELISA preliminary experiment to explore the optimal protein coating concentration of FUCA2 and the optimal dilution concentration of the secondary antibody IgG: A double antibody sandwich ELISA experiment was used to conduct a preliminary analysis of 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 FUCA2 and the optimal dilution concentration of the secondary antibody IgG in this experiment. The optimal protein coating concentration of FUCA2 and the optimal dilution multiple of the secondary antibody IgG are shown in Table 3.

[0093] Table 3 ELISA preliminary experiment to explore the optimal protein coating concentration of FUCA2 and the optimal dilution multiple of secondary antibody IgG

[0094]

[0095] (2) Preliminary verification of the differential diagnostic efficacy of ELISA experiments for anti-FUCA2 IgG and IgM autoantibodies

[0096] The expression levels of anti-FUCA2 IgG and IgM autoantibodies in the plasma samples of lung adenocarcinoma group and healthy control group are as follows Figure 1 As shown in the figure, the expression levels of anti-FUCA2 IgM and IgG autoantibodies in the plasma samples of the lung adenocarcinoma group were significantly higher than those in the healthy control group (P≤0.0001).

[0097] The expression levels of anti-FUCA2 IgM and IgG autoantibodies in plasma samples of lung adenocarcinoma group, healthy control group and benign pulmonary nodule group are shown in Figure 2 Figure 2 As shown in the figure, the expression level of anti-FUCA2 autoantibodies in the plasma samples of the lung adenocarcinoma group was significantly higher than that of the healthy control group (P < 0.0001) and the benign pulmonary nodule group (P < 0.0001). The expression levels of IgG and IgM autoantibodies against the tumor-associated antigen FUCA2 in the plasma samples of the lung adenocarcinoma group, the healthy control group and the benign pulmonary nodule group are shown in the figure. Figure 2 As shown. Figure 2 It can be seen that the expression level of autoantibodies against tumor-associated antigen FUCA2 in the plasma samples of the lung adenocarcinoma group was significantly higher than that of the healthy control group (P < 0.0001) and the benign lung disease group (P < 0.0001). According to the receiver operating characteristic (ROC) curve analysis, when the IgG autoantibodies against tumor-associated antigen FUCA2 were used to diagnose and distinguish the lung adenocarcinoma group from the healthy control group, the AUC of the ROC curve was 0.617 (95% CI: 0.565-0.669), and the area under the curve of the IgG autoantibodies against tumor-associated antigen FUCA2 to diagnose and distinguish the lung adenocarcinoma group from the benign lung nodule group was 0.622 (95% CI: 0.570-0.674). The AUC of the ROC curve for the diagnosis of lung adenocarcinoma group and healthy control group by IgM autoantibodies was 0.811 (95% CI: 0.772-0.851), and the area under the curve for the diagnosis of lung adenocarcinoma group and benign lung nodule group by IgM autoantibodies against tumor-associated antigen FUCA2 was 0.608 (95% CI: 0.555-0.660), which shows that both IgG and IgM autoantibodies against tumor-associated antigen FUCA2 can distinguish lung adenocarcinoma group from healthy control group and benign lung nodule group. This also proves that autoantibodies against tumor-associated antigen FUCA2 can be used for auxiliary diagnosis of lung adenocarcinoma.

[0098] Figure 3 Logistic regression was used to detect IgG and IgM autoantibodies of FUCA2 and traditional tumor marker CEA. The AUC of the ROC curve for distinguishing lung adenocarcinoma group from healthy control group was 0.847 (95% CI: 0.800-0.894), which was higher than the area under the curve of CEA alone for distinguishing lung adenocarcinoma group from healthy control group (0.558 (95% CI: 0.484-0.633); the AUC of the ROC curve for distinguishing lung adenocarcinoma group from benign pulmonary nodule group was 0.707 (95% CI: 0.621-0.794), which was also higher than the area under the curve of CEA alone for distinguishing lung adenocarcinoma group from benign pulmonary nodule group (0.509 (95% CI: 0.415-0.602). Among them, there were 273 cases in lung adenocarcinoma group (LUAD), healthy control group (NC) and benign pulmonary nodule group (BPN).

[0099] In summary, the present invention provides a plasma biomarker for assisting the diagnosis of lung adenocarcinoma, wherein the marker is an autoantibody against the tumor-associated antigen FUCA2. The present invention discovered for the first time that the expression levels of IgG and IgM autoantibodies against the tumor-associated antigen FUCA2 in the plasma of patients with lung adenocarcinoma were significantly higher than those in healthy controls and patients with benign lung nodules, and there were significant differences. Logistic regression was used to jointly detect IgG and IgM autoantibodies of FUCA2 and the 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.

[0100] 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 diagnosing lung adenocarcinoma biomarkers, characterized in that: The biomarkers include anti-FUCA2 autoantibodies.

2. The product according to claim 1, characterized in that The anti-FUCA2 autoantibodies are anti-FUCA2 IgG and IgM autoantibodies.

3. The product according to claim 1, characterized in that The anti-FUCA2 autoantibodies are IgG and IgM autoantibodies against FUCA2 in peripheral blood.

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

5. The product according to any one of claims 1 to 4, characterized in that: The product includes a kit and a preparation; preferably, the kit is an ELISA detection kit; preferably, the ELISA detection kit includes FUCA2 protein.

6. The product according to claim 1 or 2, characterized in that: Therefore, the diagnosis of lung adenocarcinoma includes differentiating lung adenocarcinoma from healthy people, and differentiating lung adenocarcinoma from benign lung nodules; preferably, lung adenocarcinoma is diagnosed by detecting autoantibodies or the expression of autoantibodies and the traditional tumor marker CEA, preferably, lung adenocarcinoma is diagnosed by detecting the expression of autoantibodies and the traditional tumor marker CEA by ELISA.

7. Use of a biomarker containing anti-FUCA2 autoantibodies or a reagent for detecting a biomarker containing anti-FUCA2 autoantibodies in the preparation of a product for diagnosing lung adenocarcinoma, or in the preparation of a product for predicting, screening or evaluating the risk of adenocarcinoma, or in the preparation of a product for detecting the concentration of autoantibodies and / or tumor antigens in a sample from a subject.

8. The use according to claim 7, characterized in that The anti-FUCA2 autoantibodies are anti-FUCA2 IgG and IgM autoantibodies, preferably anti-FUCA2 IgG and IgM autoantibodies 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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