Autoantibody reagent for screening early lung squamous carcinoma

Abstract

The invention discloses an autoantibody reagent for screening early-stage lung squamous cell carcinoma. The autoantibody comprises autoantibodies of tumor-associated antigens: RNF43; or, RNF43 and KLF4; or, RNF43, KLF4, and P53; or, RNF43, KLF4, P53 and NCOR1, or, RNF43, KLF4, P53 and NCOR1; or, RNF43, KLF4, P53, NCOR1, and CCND1 are selected from the group consisting of: RNF43, KLF4, P53, the reagent provided by the invention is used for detecting the expression level of the antibody in a human body, can effectively detect the lung squamous cell carcinoma, especially early lung squamous cell carcinoma, has high sensitivity and specificity, and can enable the sensitivity to be up to 62.4% and the specificity to be up to 82.3% when the combination of RNF43, KLF4, P53, NCOR1 and CCND1 is used for diagnosing the early lung squamous cell carcinoma. Meanwhile, compared with other combinations, the Youden index is closer to 1, which shows that the combination has higher diagnostic value and higher application value.

Description

Technical Field

[0001] The present invention relates to the fields of molecular biology and oncology, and in particular to an autoantibody reagent for early-stage lung squamous cell carcinoma screening. Background Art

[0002] Globally, the mortality rate of lung cancer ranks high among all cancers, among which squamous cell lung cancer accounts for about 25%-30% of lung cancer cases. The overall prognosis of patients with squamous cell lung cancer is not optimistic, and the 5-year survival rate is relatively low. At present, surgical resection, radiotherapy and chemotherapy are the main traditional means of treating squamous cell lung cancer, but these methods are only effective for some patients, especially for advanced patients, the treatment effect is often unsatisfactory.

[0003] In the early stages of squamous cell lung cancer, patients usually lack typical and obvious clinical symptoms, and most patients have already progressed to the middle and late stages when diagnosed. Middle and late stage squamous cell lung cancer not only has severe local invasion of the tumor, but may also metastasize to distant sites, which greatly increases the complexity and difficulty of treatment. Therefore, it is urgent to achieve early and accurate diagnosis of squamous cell lung cancer. Early diagnosis can provide patients with the best treatment opportunity and help significantly improve patients' survival rate and quality of life during survival.

[0004] Studies have shown that in the process of squamous cell lung cancer, tumor cells release some special protein substances into the blood circulation. These protein substances can trigger the response of the body's immune system and are defined as tumor-associated antigens. At the same time, the body's immune system will produce corresponding antibodies, namely tumor-associated autoantibodies, against these abnormally expressed tumor-associated antigens through a series of complex immune cascade reactions. These autoantibodies have unique advantages. They can exist in the serum of patients with squamous cell lung cancer before they have obvious clinical symptoms, and can be stable in the blood for a long time. In addition, methods for detecting such autoantibodies, such as protein chip technology and enzyme-linked immunosorbent assay, have high sensitivity, and the detection process causes minimal trauma to patients. Based on these characteristics, tumor-associated autoantibodies show great potential application value in the field of early immunological diagnosis of squamous cell lung cancer. In summary, in order to improve the accuracy and efficiency of early diagnosis of squamous cell lung cancer and improve the survival of patients with squamous cell lung cancer, it is urgent to systematically screen out serological autoantibody markers with strong specificity and high sensitivity for early and accurate diagnosis of squamous cell lung cancer. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide an autoantibody reagent for early screening of lung squamous cell carcinoma in view of the shortcomings of the prior art.

[0006] In order to solve the above technical problems, the present invention discloses the following technical solutions:

[0007] In a first aspect, the present invention discloses a reagent for detecting a biomarker.

[0008] Among them, the biomarker is used for screening squamous cell lung cancer; the biomarker is an autoantibody, and the autoantibody includes autoantibodies against the following tumor-associated antigens:

[0009] RNF43 (IgM); or,

[0010] RNF43 (IgM) and KLF4 (IgM); or

[0011] RNF43 (IgM), KLF4 (IgM) and P53 (IgG); or,

[0012] RNF43 (IgM), KLF4 (IgM), P53 (IgG) and, NCOR1 (IgG); or,

[0013] RNF43 (IgM), KLF4 (IgM), P53 (IgG), NCOR1 (IgG) and CCND1 (IgG).

[0014] Among them, the autoantibody is an IgM-type or IgG-type autoantibody in peripheral blood.

[0015] Among them, the squamous cell lung cancer is preferably early-stage squamous cell lung cancer.

[0016] Among them, the reagent is a reagent for enzyme-linked immunosorbent assay, protein / peptide chip detection, immunoblotting, bead immunoassay or microfluidic immunoassay; preferably, the reagent detects the biomarker by enzyme-linked immunosorbent assay.

[0017] In a second aspect, the present invention discloses the application of autoantibodies against tumor-associated antigens as biomarkers in the preparation of a reagent for screening squamous cell lung cancer.

[0018] Among them, the autoantibodies against tumor-associated antigens are:

[0019] RNF43 (IgM); or,

[0020] RNF43 (IgM) and KLF4 (IgM); or

[0021] RNF43 (IgM), KLF4 (IgM) and P53 (IgG); or,

[0022] RNF43 (IgM), KLF4 (IgM), P53 (IgG) and, NCOR1 (IgG); or,

[0023] RNF43 (IgM), KLF4 (IgM), P53 (IgG), NCOR1 (IgG) and CCND1 (IgG).

[0024] Among them, the protein sequence numbers of the tumor - related antigens in NCBI in this application are: RNF43: NP_001292473.1, KLF4: NP_001300981.1, P53: NP_001040616.1, NCOR1: NP_001177369.1, CCND1: NP_444284.1.

[0025] Among them, the autoantibodies are IgM - type or IgG - type autoantibodies in peripheral blood.

[0026] Among them, the squamous cell carcinoma of the lung is preferably early - stage squamous cell carcinoma of the lung.

[0027] Among them, the reagent is a reagent for enzyme - linked immunosorbent assay, protein / peptide chip detection, immunoblotting, microbead immunoassay or microfluidic immunoassay; preferably, the reagent detects the biomarker by enzyme - linked immunosorbent assay.

[0028] In a third aspect, the present invention discloses a kit.

[0029] Among them, the kit includes the reagent described in the first aspect above.

[0030] Among them, the kit is a kit for enzyme - linked immunosorbent assay, protein / peptide chip detection, immunoblotting, microbead immunoassay or microfluidic immunoassay; preferably, the kit is a kit for enzyme - linked immunosorbent assay detection.

[0031] In a fourth aspect, the present invention discloses the use of the reagent described in the first aspect above in the preparation of a product for screening squamous cell carcinoma of the lung.

[0032] Among them, the product is used to screen whether a subject has squamous cell carcinoma of the lung; the subject is preferably a mammal, more preferably a primate mammal, and most preferably a human.

[0033] Beneficial effects:

[0034] For the first time, the present invention combines RNF43, the combination of RNF43 and KLF4, the combination of RNF43, KLF4 and P53, the combination of RNF43, KLF4, P53 and NCOR1, and the combination of RNF43, KLF4, P53, NCOR1 and CCND1 to detect the antibody expression level in the human body, which can effectively detect squamous cell lung cancer, especially early squamous cell lung cancer, with high sensitivity and high specificity. Especially when using the combination of RNF43, KLF4, P53, NCOR1 and CCND1 for the diagnosis of early squamous cell lung cancer, the sensitivity can reach 62.4% while the specificity can reach 82.3%. At the same time, its Youden index is closer to 1 compared with other combinations, indicating that this combination has higher diagnostic value and application value. The present invention is beneficial to the early screening of asymptomatic squamous cell lung cancer, improves the detection specificity while greatly reducing the misdiagnosis rate, bringing great well-being to patients and their families. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The following further specifically describes the present invention in conjunction with the drawings and specific embodiments, and the above and / or other advantages of the present invention will become clearer.

[0036] Figure 1 It is the detection result of the expression levels of autoantibodies against 5 anti-tumor related antigens by a protein chip in squamous cell lung cancer serum and normal serum.

[0037] Figure 2 It is a scatter plot of the expression levels of autoantibodies against 5 related antigens in squamous cell lung cancer and healthy controls in an ELISA experiment.

[0038] Figure 3 It is the ROC curve of autoantibodies against 5 related antigens for differentiating squamous cell lung cancer from healthy controls.

[0039] Figure 4 It is the ROC curve graph after the step-by-step combination of autoantibodies against 5 related antigens; Note: Figure A is the ROC curve after Logistic regression of RNF43 (IgM) + KLF4 (IgM); Figure B is the ROC curve after Logistic regression of RNF43 (IgM) + KLF4 (IgM) + P53 (IgG); Figure C is the ROC curve after Logistic regression of RNF43 (IgM) + KLF4 (IgM) + P53 (IgG) + NCOR1 (IgG); Figure D is the ROC curve after Logistic regression of RNF43 (IgM) + KLF4 (IgM) + P53 (IgG) + NCOR1 (IgG) + CCND1 (IgG).

[0040] Figure 5 It is the nomogram for the combined application of autoantibodies against 5 related antigens. Detailed implementation manners

[0041] The present invention can be better understood according to the following embodiments. However, those skilled in the art can easily understand that the content described in the embodiments is only used to illustrate the present invention and should not and will not limit the present invention detailed in the claims.

[0042] In the following embodiments, the experimental methods are conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0043] Embodiment

[0044] 1. Experimental samples

[0045] As shown in Table 1, a total of 216 serum samples of patients with lung squamous cell carcinoma and 235 serum samples of healthy control subjects were included in this study, including 79 protein chip detection samples (30 cases of lung squamous cell carcinoma and 49 healthy controls) in the screening stage and 372 ELISA verification samples (186 cases of lung squamous cell carcinoma and 186 healthy controls each) in the verification stage.

[0046] The inclusion and exclusion criteria for the research subjects are as follows:

[0047] Lung squamous cell carcinoma: The inclusion criteria for lung squamous cell carcinoma samples are: (1) age > 18 years old; (2) classified as lung squamous cell carcinoma according to the "WHO Histological Classification of Lung Cancer 2015"; (3) having a complete medical history, no history of other malignant tumors, and no history of autoimmune diseases; (4) newly diagnosed cases without chemotherapy, radiotherapy, surgery, and other intervention measures. Exclusion criteria: (1) suffering from autoimmune diseases, taking immunosuppressive drugs for a long time, and taking tonics for a long time; (2) having coexisted with benign lung tumors, benign and malignant tumors in other parts, and lung tumors metastasized from tumors in other parts; (3) having coexisted with other serious diseases and being unable to tolerate the collection, surgery, and detection of pathological diagnosis specimens; (4) patients with hemolyzed or lipemic blood specimens.

[0048] Healthy control samples: The inclusion criteria are: (1) age > 18 years old; (2) no history of malignant tumors in the lungs and other parts, and no autoimmune diseases; (3) no obvious abnormalities were found in the lung CT, excluding benign and malignant lung tumors and other diseases; (4) no obvious abnormalities were found in B-ultrasound, electrocardiogram, and other imaging tests. Exclusion criteria: (1) autoimmune diseases, taking drugs that affect immune function for a long time, and using or taking foods or drugs that enhance immunity in the long term or in the past 2 months; (2) pregnant and lactating patients; (3) patients with a history of blood transfusion, surgery, and previous malignant tumors; (4) patients with hemolyzed or lipemic blood specimens.

[0049] 2. Collection of clinical information of blood samples

[0050] Collect 5 mL of peripheral blood from the population in a fasting state using a blood collection tube without anticoagulant. After standing at room temperature for 1 hour, place it in a centrifuge at a speed of 3000 rpm for 10 minutes. Then aspirate the serum on the upper layer of the blood collection tube and dispense it into 1.5 mL EP tubes. Mark the corresponding sample numbers on the EP tubes, and then store the serum samples in a -80°C refrigerator. Before the experiment, extract the required serum and thaw it in a 4°C refrigerator to avoid repeated freezing and thawing of the serum. By querying this and previous medical records, collect and record general clinical information of the subjects such as gender and age. This study has been approved by the Ethics Committee of our school, and all research subjects have signed informed consent forms.

[0051] Table 1 Basic Information Table of Samples

[0052]

[0053] 3. Protein Chip Experiment

[0054] (1) Detection Principle

[0055] The customized Focused Array protein chip contains a total of 154 human recombinant proteins, including 143 proteins encoded by cancer driver genes and 11 proteins (IMP1, IMP2, IMP3, CyclinB1, c-Myc, CIP2A / p90, RalA, YWHAZ, RBM39, and two fragments of Survivin) that showed good results in previous studies in our laboratory. These human recombinant proteins are all from the CDI laboratory in the United States; the recombinant proteins are fixed on the chip, and then the proteins bind to specific antibodies in the serum. Wash away the antibodies and other components that are not bound by the specific proteins, and then use anti-human IgM fluorescently labeled secondary antibody (cy5-labeled showing red) and anti-human IgG fluorescently labeled secondary antibody (cy3-labeled showing green) for detection. Obtain the fluorescence intensity of the chip through a fluorescence scanner, and there is a positive correlation between the intensity of the fluorescence signal and the affinity and quantity of the corresponding antibodies.

[0056] (2) Protein Chip Samples: Aspirate 30 μL of the sample serum collected in step 2 and place it in a 200 μL EP tube, and transport it to Guangzhou Bochong Biotechnology Co., Ltd. under cold chain.

[0057] (3) Experimental Procedures

[0058] 1) Rewarming: Take out the customized Focused Array protein chip from the -80°C freezer, place it in a 4°C refrigerator for 30 minutes, and then place it at room temperature for 15 minutes.

[0059] 2) Blocking: Fix 14 fences for each rewarmed chip, add blocking solution to each fence, then place the chip on a side-sway shaker, gently shake, and block at room temperature for 3 h.

[0060] 3) Primary antibody incubation: After blocking is completed, discard the blocking solution, and then quickly add the pre-diluted serum (the serum sample is first stored in a chromatography cabinet at 4 °C and diluted with 1×PBST containing 1% BSA at a volume ratio of 1:50). Each protein chip can incubate 14 serum samples (200 μL / fence), in a 4 °C cold storage, in a side-sway shaker, incubate overnight at a rotation speed of 20 rpm.

[0061] 4) Washing: Take out the chip and chip holder from the shaker, discard the serum sample, quickly add PBST (200 μL / fence), and repeat this operation several times. Use tweezers to take out the chip holder, place the chip in a box containing washing solution, on a horizontal shaker, at a rotation speed of 80 rpm, wash at room temperature 3 times, and wash for 10 min each time.

[0062] 5) Secondary antibody incubation: Place the chip in a box containing 3 mL of secondary antibody incubation solution (fluorescently labeled secondary antibodies against human IgM and IgG), then transfer to a side-sway shaker, at a rotation speed of 40 rpm, protected from light, incubate at room temperature for 60 min.

[0063] 6) Washing: The previous operation is the same as in step 4). After completion, wash 2 times with double-distilled water, 10 min each time (pay attention to protecting from light).

[0064] 7) Drying: Place the chip in a chip dryer for centrifugal drying.

[0065] 8) Scanning: Scan the chip according to the operation instructions of the scanning instrument in a standardized manner.

[0066] 9) Data extraction: Open the corresponding GAL format file, align each queue of the chip image with the whole GAL file, then click the auto-align button, extract the data and save it as GPR format.

[0067] (4) Data extraction and preprocessing

[0068] For individual samples with very high background values, they need to be excluded to avoid affecting subsequent analysis. First, calculate the mean and standard deviation based on the background values of 150 chip detection samples, and then set the cut-off value to Mean + 2SD. In data analysis, samples with background values greater than the threshold are excluded.

[0069] F532Median: The median value of the signal foreground value in the 532nm channel, which refers to the median intensity value of all pixel points corresponding to each protein signal point, representing the signal intensity; B532Median: The median value of the background value in the 532nm channel, which refers to the median intensity value of the pixel points within a certain range of the background around each protein signal point, representing the background value. After extracting the data of F532Median and B532Median, calculate the ratio of the foreground value to the background value of each protein, that is, F Median / BMedian, to eliminate the deviation caused by inconsistent background values among different samples. On this basis, define SNR (signal-to-noise ratio), that is, the mean value of F Median / B Median of two repeated proteins. Based on SNR, perform statistical analysis on the samples.

[0070] (5) Experimental results

[0071] Figure 1 Shows the detection results of the expression levels of autoantibodies against 5 anti-tumor related antigens in the sera of patients with squamous cell lung cancer and normal sera by protein chip (LC = 30, NC = 49). It can be Figure 1 seen that in the protein chip detection, the expressions of autoantibodies against 5 tumor related antigens, RNF43 (IgM), KLF4 (IgM), CCND1 (IgG), P53 (IgG), NCOR1 (IgG), in squamous cell lung cancer are all higher than those in healthy controls (P < 0.05).

[0072] 4. ELISA experiment

[0073] (1) Materials and reagents:

[0074] 1) P53 protein was purchased from Wuhan Sanying Biotechnology Co., Ltd., RNF43 protein was purchased from Wuhan Huamei Bio-Engineering Co., Ltd., and KLF4, CCND1, and NCOR1 proteins were purchased from Wuhan Yunke Long Biotechnology Co., Ltd.;

[0075] 2) 96-well enzyme-linked immunosorbent assay (ELISA) plate (8 rows × 12 columns);

[0076] 3) Coating solution: An aqueous solution containing 0.15% sodium carbonate (Na2CO3) and 0.29% sodium bicarbonate (NaHCO3);

[0077] 4) Blocking solution: PBST buffer containing 2% (v / v) bovine serum albumin (BSA) and 0.2% (v / v) Tween 20;

[0078] 5) Serum sample diluent: PBST buffer containing 1% (w / v) BSA;

[0079] 6) Enzyme-labeled secondary antibody: Mouse anti-human immunoglobulin G and M antibodies labeled with horseradish peroxidase (HRP) (hereinafter referred to as HRP-labeled mouse anti-human IgG and IgM antibodies);

[0080] 7) Antibody diluent: PBST buffer containing 1% (w / v) BSA;

[0081] 8) Washing solution: PBST buffer containing 0.2% (v / v) Tween 20;

[0082] 9) Chromogenic solution: The chromogenic solution consists of chromogenic solution A and chromogenic solution B. Among them, chromogenic solution A is an aqueous solution of 20% tetramethylbenzidine dihydrochloride; chromogenic solution B is an aqueous solution containing 3.7% NaHPO·12H₂O, 0.92% citric acid, and 0.75% (v / v) hydrogen peroxide urea; when in use, chromogenic solution A and chromogenic solution B are mixed evenly in equal volume according to 1:1, and prepared immediately before use;

[0083] 10) Stop solution: 10% sulfuric acid

[0084] (2) Experimental steps:

[0085] 1) Preparation of enzyme-labeled plate coated with tumor-associated antigen: According to the results of preliminary experiments, dilute RNF43, KLF4, CCND1, P53, and NCOR1 proteins with coating solution at a concentration of 0.125 μg / mL, add the diluted proteins to a 96-well enzyme-labeled plate, 50 μL per well. Place it in a 4°C refrigerator and coat overnight for 16 h;

[0086] 2) Serum dilution: Add serum and serum diluent to a 96-well deep-well plate at a ratio of 1:100, and mix well. Store it in a 4°C refrigerator;

[0087] 3) Blocking: Take out the coated enzyme-labeled plate, discard the coating solution in the enzyme-labeled plate, and then add the prepared blocking solution (2% BSA) to the enzyme-labeled plate with a 12-channel pipette for blocking, 100 μL per well. Then incubate in a 37°C constant temperature water bath for 2 h;

[0088] 4) Plate washing: Take out the blocked enzyme-labeled plate, discard the blocking solution in the enzyme-labeled plate, and then wash the enzyme-labeled plate with an automatic enzyme-labeled plate washer. Use the pre-prepared 1×PBST as the washing solution. After washing the plate 3 times, pat the enzyme-labeled plate dry on clean absorbent paper;

[0089] 5) Primary antibody (serum) incubation: Take out the pre-diluted serum, use a 12-channel pipette to extract 50 μL of sample from each well of the deep-well plate and add it to the corresponding ELISA plate, including the quality control serum sample. Add 50 μL of serum diluent (1% BSA) without serum sample to each well of the 2 blank control wells. After all wells are loaded, incubate in a 37°C constant temperature water bath for 1 hour;

[0090] 6) Washing: Take out the ELISA plate after the primary antibody incubation, shake out the liquid in the ELISA plate, and then wash the plate with an automatic ELISA plate washer. Use the 1×PBST prepared in advance as the washing solution. Wash the plate 5 times in total. After washing, pat the plate dry on clean absorbent paper. The absorbent paper needs to be changed when patting different plates to avoid cross contamination.

[0091] 7) Secondary antibody incubation: Dilute the horseradish peroxidase-labeled mouse anti-human IgG and IgM with secondary antibody diluent (1% BSA) according to the dilution ratio required for different recombinant proteins, and add 50 μL of the diluted secondary antibody to each well of the ELISA plate using a 12-channel pipette. Incubate in a 37°C constant temperature water bath for 1 hour;

[0092] 8) Washing: Take out the ELISA plate after the secondary antibody incubation, discard the liquid in the ELISA plate, and then wash the ELISA plate with an automatic ELISA plate washer. Use the 1× PBST prepared in advance as the washing solution. After washing the plate 5 times, pat the ELISA plate dry on a clean absorbent paper.

[0093] 8) Color development: Mix solution A and solution B in a ratio of 1:1, and add 50 μL of the mixed color development solution to each well of the ELISA plate using a 12-channel pipette. Cover the plate with tin foil and react at room temperature in the dark for 5-10 minutes.

[0094] 10) Stop the reaction: Use a 12-channel pipette to add 25 μL of stop solution to each well of the ELISA plate to stop the reaction;

[0095] 11) Scanning: Wipe the moisture on the bottom of the ELISA plate to avoid interference with the scanning and reading. Place the ELISA plate on the ELISA reader for scanning. The values read are the absorbance values (optical density, OD) at wavelengths of 450nm and 620nm. Save the experimental data.

[0096] 12) Data preprocessing: The OD required for the final analysis is the value read at two wavelengths of 450nm and 620nm minus the value of the blank well, that is, (OD450-OD620 absorbance value)-(average of the blank well OD values).

[0097] 13) Data quality control: The value of the blank well of each plate should be < 0.1, indicating that the plate is not contaminated; at the same time, the 6 quality control samples added between each plate are the same. According to the OD values of the quality control samples, the coefficient of variation (CV = standard deviation / mean) of the 6 quality control samples per plate can be calculated, and the calculated CV value should not be greater than 15%. Then, using the plate with the minimum CV value as the standard, the OD values of the remaining plates are standardized.

[0098] (3) Data processing:

[0099] Perform Kolmogrov - Smirnova test on the absorbance values of serum samples in the lung squamous cell carcinoma group and the normal control group. After the Kolmogrov - Smirnova test, it is found that the expression levels of autoantibodies against 5 anti - tumor - related antigens in the serum samples of the study subjects do not conform to the normal distribution (P < 0.05). Therefore, the 25th percentile (P25), median (P50), and 75th percentile (P75) are used to describe the distribution of the expression levels of autoantibodies against nine anti - tumor - related antigens; then, non - parametric test (Mann - Whitney U) is used to compare the expression levels of autoantibodies in the hepatocellular carcinoma group and the normal control group. Use GraphPad 8.0.2 software to draw the ROC curve of the autoantibodies against tumor - related antigens for differentiating lung squamous cell carcinoma and healthy controls. According to the area under the ROC curve (AUC), analyze the diagnostic value of the autoantibodies against 5 anti - tumor - related antigens for lung squamous cell carcinoma. All statistical analyses are performed using SPSS 21.0 software and GraphPad 8.0.2 software, and P < 0.05 is used as the statistical judgment criterion.

[0100] (4) Results:

[0101] Figure 2 It is a scatter plot obtained after processing the OD values measured for the autoantibodies against 5 related antigens and healthy controls in the ELISA experiment (LC = 186, NC = 186). (NC in the figure represents healthy controls, and LC represents lung squamous cell carcinoma samples).

[0102] Figure 3 It is the ROC curve of the autoantibodies against 5 related antigens compared with the healthy control group. Figure 4 It is the ROC curve graph after combining the autoantibodies against 5 related antigens.

[0103] Figure 3 and Figure 4 It can be seen that the AUC range of the 5 tumor - related autoantibodies before combination is 0.509 - 0.716, and the AUC value for diagnosing lung squamous cell carcinoma after combining using the Logistic regression model and performing ten - fold cross - validation is 0.788, indicating a great improvement in its diagnostic performance compared with before combination.

[0104] Figure 5 It is a nomogram for the combined application of 5 related antigen autoantibodies. It can be seen that the combined nomogram shows that 5 tumor-related antigen autoantibodies can help patients better predict and diagnose squamous cell lung cancer. This shows that the combined diagnosis of 5 tumor-related antigen autoantibodies, namely RNF43 (IgM), KLF4 (IgM), CCND1 (IgG), P53 (IgG), and NCOR1 (IgG), can be used for the auxiliary diagnosis of squamous cell lung cancer.

[0105] 5. Combined detection results of different combinations of tumor-related antigen autoantibodies

[0106] According to the ELISA detection results of the expression levels of five antibodies in 372 samples, the ROC curves for diagnosing and differentiating squamous cell lung cancer and normal controls were respectively plotted using GraphPad Prism 8.0 for single autoantibodies and combinations of multiple autoantibodies. The detection results of the sequential combination of 5 autoantibodies for the diagnosis of squamous cell lung cancer were analyzed by the stepwise Logistic regression method. The results are shown in Table 2. When using the combination of two related antigens, RNF43 and KLF4, and the combination of four antigens, RNF43, KLF4, P53, and NCOR1, for the early diagnosis of squamous cell lung cancer, although the sensitivity or specificity can be improved, both cannot be improved simultaneously. When using the combination of three related antigens, RNF43, KLF4, and P53, and the combination of five tumor-related antigens, RNF43, KLF4, P53, NCOR1, and CCND1, alone for the early diagnosis of squamous cell lung cancer, both the sensitivity and specificity can be relatively high. Especially when using the combination of five tumor-related antigens, RNF43, KLF4, P53, NCOR1, and CCND1, for the early diagnosis of squamous cell lung cancer, the sensitivity can reach 62.4% while the specificity can reach 82.3%. At the same time, its Youden index is closer to 1 compared to other combinations, indicating that this combination has a higher diagnostic value and application value.

[0107] Table 2 Combined detection results of different combinations of tumor-related antigen autoantibodies

[0108]

[0109] The above-described embodiments only represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent for the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be subject to the appended claims.

Claims

1. A reagent for detecting biomarkers, characterized in that, The biomarker is used for screening squamous cell lung cancer; the biomarker is an autoantibody, and the autoantibody includes autoantibodies against the following tumor-associated antigens: RNF43; or, RNF43 and KLF4; or, RNF43, KLF4 and P53; or, RNF43, KLF4, P53 and NCOR1; or, RNF43, KLF4, P53, NCOR1 and CCND1.

2. The reagent according to claim 1, wherein The autoantibody is an IgM-type or IgG-type autoantibody in peripheral blood; the squamous cell lung cancer is preferably early-stage squamous cell lung cancer.

3. The reagent according to claim 1, characterized in that, The reagent is a reagent for enzyme-linked immunosorbent assay, protein / peptide chip detection, immunoblotting, bead immunoassay or microfluidic immunoassay; preferably, the reagent detects the biomarker by enzyme-linked immunosorbent assay.

4. Use of autoantibodies against tumor-associated antigens as biomarkers in the preparation of a reagent for screening squamous cell lung cancer, characterized in that, The autoantibody against the tumor-associated antigen is: RNF43; or, RNF43 and KLF4; or RNF43, KLF4 and P53; or, RNF43, KLF4, P53 and, NCOR1; or, RNF43, KLF4, P53, NCOR1 and CCND1.

5. The application according to claim 4, wherein The autoantibody is an IgM-type or IgG-type autoantibody in peripheral blood; the squamous cell lung cancer is preferably early-stage squamous cell lung cancer.

6. The application according to claim 4, wherein The reagent is a reagent for enzyme-linked immunosorbent assay, protein / peptide chip detection, immunoblotting, bead immunoassay or microfluidic immunoassay; preferably, the reagent detects the biomarker by enzyme-linked immunosorbent assay.

7. A kit, characterized in that, including the reagent according to any one of claims 1-3.

8. The kit according to claim 7, wherein The kit is a kit for enzyme-linked immunosorbent assay, protein / peptide chip detection, immunoblotting, bead immunoassay or microfluidic immunoassay; preferably, the kit is a kit for enzyme-linked immunosorbent assay detection.

9. Use of the reagent according to any one of claims 1-3 in the preparation of a product for screening squamous cell lung cancer.

10. The use according to claim 9, characterized in that, The product is used for screening whether a subject has squamous cell lung cancer; the subject is preferably a mammal, preferably a primate mammal, preferably a human.

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