Serum autoantibodies for identifying lymph node metastasis of cardiac adenocarcinoma and application thereof

Abstract

The application belongs to the field of medicine and biotechnology, and specifically discloses serum autoantibodies for identifying lymph node metastasis of cardiac gland cancer and application thereof. The biomarker for diagnosing cardiac gland cancer provided by the application is at least one of LIMCH1 autoantibody, RSPH1 autoantibody, ENAH autoantibody, COTL1 autoantibody and ASS1 autoantibody, and the expression level of the biomarker in cardiac gland cancer lymph node metastasis positive serum is higher than that in cardiac gland cancer lymph node metastasis negative serum, and the difference is statistically significant. The application further provides a kit for identifying lymph node metastasis of cardiac gland cancer, and the kit contains reagents for detecting the above biomarkers. By detecting the expression level of the above biomarkers in human serum, the application can effectively distinguish cardiac gland cancer lymph node metastasis positive and negative, and can be used for auxiliary diagnosis of lymph node metastasis of cardiac gland cancer.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical biotechnology, and specifically discloses serum autoantibodies used to identify lymph node metastasis of gastric cardia adenocarcinoma and their applications. Background Technology

[0002] Gastric cardiac adenocarcinoma (GCA), often referred to as esophagogastric junction adenocarcinoma in Western countries, refers to adenocarcinoma with its center located 1 cm above and 2 cm below the esophagogastric junction. GCA has an insidious onset, with few or no early symptoms, and most patients are diagnosed at an advanced stage, resulting in a very poor prognosis. Lymph node metastasis is a core prognostic indicator in the international TNM staging system and a key basis for guiding decisions on the extent of surgical procedures and adjuvant therapy. However, currently, accurately determining whether lymph node metastasis exists in gastric cardia adenocarcinoma mainly relies on postoperative pathological examination. This means that preoperatively, especially in the early stages, there is a lack of highly sensitive and specific non-invasive detection methods to reliably assess lymph node status. Therefore, it is particularly important to find a biomarker that can predict lymph node metastasis non-invasively and accurately before surgery. Finding one or a group of serum autoantibodies that can identify lymph node metastasis in gastric cardia adenocarcinoma is especially important. This could provide a blood test method to non-invasively assess the risk of lymph node metastasis before surgery or treatment, guide individualized treatment decisions, and improve the survival rate of patients with gastric cardia adenocarcinoma.

[0003] A growing body of research indicates that the body produces autoantibodies against tumor-associated antigens very early in the development and progression of tumors. These autoantibodies are present in serum and can be obtained through non-invasive blood tests, making them ideal research subjects. Importantly, their appearance may precede clinical symptoms or imaging findings of the tumor, enabling early detection and intervention. Therefore, systematically screening serum autoantibody combinations closely related to the specific malignant biological behavior of lymph node metastasis in gastric cardia adenocarcinoma holds promise not only for developing a novel preoperative predictive tool but also for revealing the underlying molecular mechanisms driving metastasis at the level of immune response, providing clues for the development of new therapeutic targets.

[0004] Currently, there are relatively few studies on serum autoantibodies for identifying lymph node metastasis in gastric cardia adenocarcinoma. We used high-throughput protein microarray and other technologies to systematically screen the serum autoantibody profile of patients with gastric cardia adenocarcinoma. By comparing the differences in antibody expression between patients with and without lymph node metastasis, we screened and validated a serum autoantibody biomarker that can accurately predict lymph node metastasis. This exploration will provide important theoretical basis and translational application prospects for precise preoperative staging, prognostic assessment, and the development of individualized treatment strategies for gastric cardia adenocarcinoma. Summary of the Invention

[0005] In view of the problems and deficiencies in the existing technology, the present invention provides serum autoantibodies for identifying lymph node metastasis of gastric cardia adenocarcinoma and their application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of the present invention provides the use of a reagent for detecting a biomarker in the preparation of a product for identifying lymph node metastasis of gastric cardia adenocarcinoma, wherein the biomarker is at least one of LIMCH1 autoantibody, RSPH1 autoantibody, ENAH autoantibody, COTL1 autoantibody, and ASS1 autoantibody.

[0007] According to the above application, preferably, the reagent is a reagent for detecting the biomarker in the sample by enzyme-linked immunosorbent assay (ELISA), protein chip, immunoblotting, or microfluidic immunoassay.

[0008] According to the above application, preferably, the sample is serum, plasma, interstitial fluid or urine.

[0009] According to the above application, preferably, the reagent is an antigen or antibody for detecting the biomarker. More preferably, the detection reagent is an antigen for detecting the biomarker, and the antigen is at least one of LIMCH1 protein, RSPH1 protein, ENAH protein, COTL1 protein, and ASS1 protein.

[0010] Based on the above application, preferably, when the biomarker is a combination of LIMCH1 autoantibody and RSPH1 autoantibody, the probability calculation formula for the product to identify lymph node metastasis of gastric cardia adenocarcinoma is as follows: ; Where P represents the prediction probability, LIMCH1 represents the expression level of LIMCH1 autoantibody, and RSPH1 represents the expression level of RSPH1 autoantibody. When the biomarker is a combination of LIMCH1 autoantibody, RSPH1 autoantibody, and ENAH autoantibody, the probability calculation formula for the product to identify lymph node metastasis of gastric cardia adenocarcinoma is as follows: ; Where P represents the prediction probability, LIMCH1 represents the expression level of LIMCH1 autoantibody, RSPH1 represents the expression level of RSPH1 autoantibody, and ENAH represents the expression level of ENAH autoantibody. When the biomarker is a combination of LIMCH1 autoantibody, RSPH1 autoantibody, ENAH autoantibody, and COTL1 autoantibody, the probability calculation formula for the product to identify lymph node metastasis in gastric cardia adenocarcinoma is as follows: ; Where P represents the prediction probability, LIMCH1 represents the expression level of LIMCH1 autoantibody, RSPH1 represents the expression level of RSPH1 autoantibody, ENAH represents the expression level of ENAH autoantibody, and COTL1 represents the expression level of COTL1 autoantibody. When the biomarker is a combination of LIMCH1 autoantibody, RSPH1 autoantibody, ENAH autoantibody, COTL1 autoantibody, and ASS1 autoantibody, the probability calculation formula for the product to identify lymph node metastasis of gastric cardia adenocarcinoma is as follows: , ; Where P represents the prediction probability, LIMCH1 represents the expression level of LIMCH1 autoantibody, RSPH1 represents the expression level of RSPH1 autoantibody, ENAH represents the expression level of ENAH autoantibody, COTL1 represents the expression level of COTL1 autoantibody, and ASS1 represents the expression level of ASS1 autoantibody.

[0011] According to the above applications, preferably, the product is a protein chip, a reagent kit, or a formulation.

[0012] A second aspect of the present invention provides a kit for identifying lymph node metastasis in gastric cardia adenocarcinoma, the kit comprising reagents for detecting biomarkers; the biomarkers being at least one of LIMCH1 autoantibody, RSPH1 autoantibody, ENAH autoantibody, COTL1 autoantibody, and ASS1 autoantibody.

[0013] According to the above-described kit, preferably, the kit detects the biomarkers in the sample by enzyme-linked immunosorbent assay (ELISA), protein chip, immunoblotting, or microfluidic immunoassay.

[0014] According to the above-mentioned kit, preferably, when the biomarker is a combination of LIMCH1 autoantibody and RSPH1 autoantibody, the probability calculation formula for the kit to identify lymph node metastasis of gastric cardia adenocarcinoma is as follows: ; Where P represents the prediction probability, LIMCH1 represents the expression level of LIMCH1 autoantibody, and RSPH1 represents the expression level of RSPH1 autoantibody. When the biomarker is a combination of LIMCH1 autoantibody, RSPH1 autoantibody, and ENAH autoantibody, the probability calculation formula for the kit to identify lymph node metastasis in gastric cardia adenocarcinoma is as follows: ; Where P represents the prediction probability, LIMCH1 represents the expression level of LIMCH1 autoantibody, RSPH1 represents the expression level of RSPH1 autoantibody, and ENAH represents the expression level of ENAH autoantibody. When the biomarkers are a combination of LIMCH1 autoantibody, RSPH1 autoantibody, ENAH autoantibody, and COTL1 autoantibody, the probability calculation formula for the kit to identify lymph node metastasis in gastric cardia adenocarcinoma is as follows: ; Where P represents the prediction probability, LIMCH1 represents the expression level of LIMCH1 autoantibody, RSPH1 represents the expression level of RSPH1 autoantibody, ENAH represents the expression level of ENAH autoantibody, and COTL1 represents the expression level of COTL1 autoantibody. When the biomarker is a combination of LIMCH1 autoantibody, RSPH1 autoantibody, ENAH autoantibody, COTL1 autoantibody, and ASS1 autoantibody, the probability calculation formula for the kit to identify lymph node metastasis in gastric cardia adenocarcinoma is as follows: , ; Where P represents the prediction probability, LIMCH1 represents the expression level of LIMCH1 autoantibody, RSPH1 represents the expression level of RSPH1 autoantibody, ENAH represents the expression level of ENAH autoantibody, COTL1 represents the expression level of COTL1 autoantibody, and ASS1 represents the expression level of ASS1 autoantibody.

[0015] According to the above-described kit, preferably, the kit is an ELISA detection kit. More preferably, the ELISA detection kit includes a solid-phase carrier and an antigen coated on the solid-phase carrier; the antigen is at least one selected from LIMCH1 protein, RSPH1 protein, ENAH protein, COTL1 protein, and ASS1 protein.

[0016] According to the kit described above, preferably, the sample is serum, plasma, interstitial fluid, or urine.

[0017] According to the kit described above, preferably, the ELISA detection kit further includes a sample diluent, a second antibody, an antibody diluent, a washing buffer, a chromogenic solution, and a stop solution.

[0018] The basic information of LIMCH1 protein, RSPH1 protein, ENAH protein, COTL1 protein, and ASS1 protein in this invention is as follows: LIMCH1 protein: Enables myosin II head-neck binding activity, involving multiple processes, including actin-based cytoplasmic contraction during cell movement; positive regulation of stress fiber assembly; and regulation of local adhesion assembly. It is located in the myosin II complex and stress fibers. Serial number: NP-001317601.1.

[0019] RSPH1 protein: This gene encodes an acidic protein associated with chromosomes during metaphase of male meiosis. It is expressed in tissues with motile cilia or flagella, including the trachea, lungs, airway brushes, and testes. Serial number: NP-543136.1.

[0020] ENAH protein: Located on chromosome 3, this gene encodes a member of an activating / vasodilatory stimulating phosphoprotein. Members of this gene family are involved in actin-based movement. This protein is involved in regulating actin filament assembly, modulating cell adhesion and movement. Alternative splicing variations of this gene are associated with tumor invasiveness in certain tissues, and these variations may serve as prognostic markers. Serial number: NP-060682.2.

[0021] COTL1 protein: Located on chromosome 16, this gene encodes one of many actin-binding proteins that regulate the actin cytoskeleton. This protein binds to F-actin and interacts with 5-lipoxygenase, the first identified enzyme in leukotriene biosynthesis. Sequence number: NP-066972.1.

[0022] ASS1 protein: Located on chromosome 9, this gene encodes a protein that catalyzes the penultimate step of the arginine biosynthesis pathway. Mutations in the chromosome 9 copy of this gene result in citrullinemia. Serial number: NP-466464.1.

[0023] Compared with the prior art, the positive and beneficial effects achieved by the present invention are as follows: (1) This invention is the first to discover that the expression levels of LIMCH1, RSPH1, ENAH, COTL1, and ASS1 autoantibodies in the serum of patients with positive lymph node metastasis of gastric cardia adenocarcinoma are significantly higher than those in negative patients, and the differences are statistically significant. By detecting the expression levels of LIMCH1, RSPH1, ENAH, COTL1, and ASS1 autoantibodies in human serum, lymph node metastasis of gastric cardia adenocarcinoma can be effectively detected. Verification shows that when any one of the LIMCH1, RSPH1, ENAH, COTL1, and ASS1 autoantibodies is used alone for the diagnosis of lymph node metastasis of gastric cardia adenocarcinoma, the AUC value of the ROC curve is above 0.514; when multiple markers are used in combination, the differentiation and diagnostic effects are higher than those of using any single marker, resulting in better diagnostic efficacy. Therefore, the markers used in this invention for differentiating lymph node metastasis of gastric cardia adenocarcinoma can be used for the auxiliary diagnosis of lymph node metastasis in gastric cardia adenocarcinoma.

[0024] (2) When the five biomarkers—LIMCH1 autoantibody, RSPH1 autoantibody, ENAH autoantibody, COTL1 autoantibody, and ASS1 autoantibody—are combined for the diagnosis and detection of gastric cardia adenocarcinoma, the AUC of the ROC curve reaches 0.638; the sensitivity and specificity are 31.6% and 92.4%, respectively. The biomarkers of this invention effectively fill the gap in the lack of preoperative biomarkers for diagnosing lymph node metastasis in gastric cardia adenocarcinoma. Compared with traditional biomarkers, they greatly improve the sensitivity and specificity of lymph node metastasis diagnosis and have broad market application prospects. This method is simple, rapid, economical, practical, and easy to implement in clinical practice. Attached Figure Description

[0025] Figure 1 Flowchart for screening and identifying autoantibody markers for lymph node metastasis in gastric cardia adenocarcinoma; Figure 2 The expression levels of five autoantibodies in the positive and negative groups of lymph node metastasis in gastric cardia adenocarcinoma; Figure 3 The efficiency of five individual autoantibody indicators in the differential diagnosis of lymph node metastasis in gastric cardia adenocarcinoma; Figure 4 The efficiency of different combinations of five autoantibodies in the differential diagnosis of lymph node metastasis in gastric cardia adenocarcinoma. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below through embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0027] Example 1: Screening for biomarkers to identify lymph node metastasis in gastric cardia adenocarcinoma The procedure for screening autoantibody markers for identifying lymph node metastasis in gastric cardia adenocarcinoma is as follows: Figure 1 As shown, the specific process is as follows.

[0028] 1. Experimental Samples: Serum samples were collected from 120 patients newly diagnosed with gastric cardia adenocarcinoma at Linzhou Cancer Hospital. Among them, 60 cases were positive for lymph node metastasis, and 60 cases were negative for lymph node metastasis. Inclusion criteria: ① Underwent radical surgical treatment for gastric cardia cancer between 2020 and 2022, with postoperative pathological diagnosis of gastric cardia adenocarcinoma. ② Complete postoperative pathology and clear record of lymph node metastasis status. Exclusion criteria: Concomitant other types of malignant tumors.

[0029] Serum Collection: 5 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 tubes were labeled with sample numbers on the top and side and stored at -80°C. The collection date and storage location were recorded. Before use, the serum was thawed at 4°C and aliquoted to avoid repeated freeze-thaw cycles.

[0030] 2. Protein chip detection: Each serum sample is tested using one HuProt™ protein chip. Specific antibodies in the serum (including IgG, IgM or other types of antibodies) bind to proteins immobilized on the chip. Unbound antibodies and other proteins are washed away, and then the sample is detected by fluorescently labeled secondary antibodies Anti-human IgM (cy5 labeled, appearing red) and Anti-human IgG fluorescent secondary antibodies (cy3 labeled, appearing green). The signal is digitized using a fluorescence scanner.

[0031] (1) Experimental methods The specific steps are as follows: 1) Blocking: Using a chip incubation box, add 10 mL of blocking solution (1% bovine serum albumin (BSA) + 0.1% Tween-20 TBST), remove the chip from -80 ℃ and place it face up in the incubation box; side-shake at 50-60 rpm, room temperature, 1 hr; 2) Serum sample incubation: After blocking, pour out the blocking solution, and then quickly add the prepared serum incubation solution (the serum sample is first frozen and thawed in a chromatography cabinet at 4°C, and then the serum sample is diluted at a ratio of 1:200 using blocking solution (1% bovine serum albumin (BSA) + 0.1% Tween-20 TBST)). Incubate overnight at 4°C on a side-shaking incubator at 20 rpm. 3) Cleaning: After incubation, remove the chip and place it in a chip cleaning box containing cleaning solution (PBS + 1× Roti-Block + 0.1% Tween 20). Use a horizontal shaker at room temperature and 80 rpm to clean 3 times for 10 min each time. 4) Secondary antibody incubation: Transfer the chip to an incubator containing 3 mL of commercially available secondary antibody incubation solution (the secondary antibody is a fluorescently labeled anti-human IgM or IgG antibody; the secondary antibody is diluted with the incubation solution at a ratio of 1:1000 to obtain the secondary antibody incubation solution, the composition of which is: 1 mL of 10% BSA and 9 mL of 1×PBST solution). Shake the chip on a side at 40 rpm, protect it from light, and incubate at room temperature for 1 hour. 5) Cleaning: Remove the chip (be careful not to touch or scratch the top surface of the chip), place it in a chip cleaning box containing cleaning solution, place it on a horizontal shaker, 80 rpm, clean 3 times, 10 min each time, after completion, clean with ddH2O 2 times, 10 min each time; 6) Drying: After cleaning, place the chip in a chip dryer for centrifugal drying; 7) Scanning: Perform a standardized fluorescence scan on the dried chip according to the scanner's operating specifications and user manual, and record the fluorescence signal; (2) Data Analysis: After data normalization, statistical analysis was performed on 60 samples with positive lymph node metastasis and 60 samples with negative lymph node metastasis to screen for specific high-response protein biomarkers that distinguish the positive lymph node metastasis group from the negative group. The analysis logic is as follows: 1) For any protein, assume the samples to be compared come from two completely identical populations, and use the F-test to determine whether the variances of the two groups to be compared are homogeneous. Then, select the corresponding t-test (two-tailed) based on the F-test results, and characterize the t-test results as p-values. Define that when p-value < 0.05, reject the null hypothesis, i.e., there is a significant difference between the two groups; 2) For any protein, calculate the fold change between the lymph node metastasis positive group and the negative group, i.e., fold change = mean of the lymph node metastasis positive group / mean of the negative group, which is used to indicate the degree to which the lymph node metastasis positive group is higher than the negative group; by definition, fold change ≥ 1.2 is considered to be a potential difference, and it is generally believed that the larger the fold change, the more obvious the difference between the two groups.

[0032] 3) To avoid comparisons between negative proteins in different sample groups, before sample alignment, a positive protein judgment threshold was set based on the distribution of protein spot signal SNR values ​​on the normalized chip. Proteins were defined as positive when IgG-SNR > 4 and IgM-SNR > 5. Based on this, a cutoff threshold (cutoff-IgG ≥ 4, cutoff-IgM ≥ 5) was set using the SNR of the negative group on this protein as the calculation object. The positive rates (sensitivity) of the lymph node metastasis-positive group and the positive rates (1-specificity) of the negative group were calculated separately.

[0033] 4) Based on the above, the highest positive rate for negative lymph node metastasis is defined as 10%, that is, under the premise that the specificity of the protein is ≥90%, the protein has the greatest discrimination (discrimination = (specificity + sensitivity) / 2) when distinguishing between the positive and negative lymph node metastasis groups, and the discrimination ≥60% is the screening criterion.

[0034] (3) Experimental results: Based on the threshold settings above, a total of 5 differential autoantibodies associated with lymph node metastasis were screened out, namely LIMCH1 autoantibody, RSPH1 autoantibody, ENAH autoantibody, COTL1 autoantibody, and ASS1 autoantibody.

[0035] Example 2: ELISA detection of serum expression levels of LIMCH1 autoantibody, RSPH1 autoantibody, ENAH autoantibody, COTL1 autoantibody, and ASS1 autoantibody. The expression levels of five anti-tumor-associated antigen autoantibodies screened out were further detected in the serum of patients with positive and negative lymph node metastases of gastric cardia adenocarcinoma using an indirect enzyme-linked immunosorbent assay (ELISA) in a large sample population.

[0036] 1. Experimental Samples Serum samples were collected from another group of 308 patients with gastric cardia adenocarcinoma from the specimen bank of the State Key Laboratory for Esophageal Cancer Prevention and Control (co-constructed by the Ministry of Education and Henan Province). Among them, 190 cases were positive for lymph node metastasis, and 118 cases were negative for lymph node metastasis. These patients were pathologically diagnosed and had not received any treatment. This study was approved by the Ethics Committee of Zhengzhou University, and all participants signed informed consent forms.

[0037] The specific procedures for serum collection are the same as in Example 1.

[0038] 2. Experimental materials and reagents: (1) Five tumor-associated antigen proteins: recombinant LIMCH1 protein, recombinant RSPH1 protein, recombinant ENAH protein, recombinant COTL1 protein, and recombinant ASS1 protein were purchased from Abcam. (2) 96-well microplate (8 rows × 12 columns); (3) Coating solution: an aqueous solution containing 0.15% sodium carbonate (Na2CO3) and 0.29% sodium bicarbonate (NaHCO3); (4) Blocking solution: PBST buffer containing 2% (v / v) bovine serum albumin (BSA) in 0.2% (v / v) Tween 20; (5) Serum sample diluent: PBST buffer containing 1% (w / v) BSA; (6) Enzyme-labeled second antibody: Horseradish peroxidase (HRP) labeled mouse anti-human immunoglobulin antibody (hereinafter referred to as HRP-labeled mouse anti-human IgG antibody); (7) Antibody dilution buffer: PBST buffer containing 1% (w / v) BSA; (8) Washing buffer: PBST buffer containing 0.2% (v / v) Tween 20; (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 of 3.7% Na2HPO4•12H2O, 0.92% citric acid, and 0.75% hydrourea peroxide. When using, mix colorimetric solution A and colorimetric solution B in equal volumes at a ratio of 1:1. Prepare fresh solution immediately before use. (10) Termination solution: 10% sulfuric acid.

[0039] 3. Experimental methods: (1) Preparation of ELISA plates coated with five tumor-associated antigens: Five types of ELISA plates were prepared using the same method: LIMCH1-coated, RSPH1-coated, ENAH-coated, COTL1-coated, and ASS1-coated.

[0040] Taking the preparation of an ELISA plate coated with the tumor antigen LIMCH1 as an example, the specific operating steps are as follows: 1) Preparation of tumor antigen LIMCH1 protein solution: The LIMCH1 protein was prepared into a LIMCH1 protein solution with a concentration of 0.125 μg / mL using a coating solution.

[0041] 2) Coating the microplate: Add the LIMCH1 protein solution prepared in step 1) to each well of the 96-well microplate at a volume of 50 μL / well. Coat overnight at 4°C, then shake off the remaining coating solution and pat dry.

[0042] 3) Blocking: Add blocking solution to the reaction wells 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 with washing solution (300 μL / well) 3 times, and pat dry to obtain the ELISA plate coated with tumor-associated antigen LIMCH1.

[0043] 4) The preparation steps for the ELISA plates coated with the other 10 tumor-associated antigens are basically the same as those for the ELISA plates coated with the tumor-associated antigen LIMCH1, and the coating concentrations are also the same. The difference lies in the tumor-associated antigens used in step 1).

[0044] (2) Detection of autoantibody expression levels of five antitumor-related antigens in serum samples: Using the same serum sample, the expression levels of LIMCH1 autoantibody, RSPH1 autoantibody, ENAH autoantibody, COTL1 autoantibody, and ASS1 autoantibody in serum samples from patients with positive and negative lymph node metastasis of gastric adenocarcinoma were detected by ELISA using enzyme-linked immunosorbent assay (ELISA) plates coated with the five tumor antigens prepared above.

[0045] Taking the detection of LIMCH1 autoantibody expression levels as an example, the specific operating steps are as follows: 1) Serum sample incubation: Dilute the serum sample to be tested with serum sample diluent at a volume ratio of 1:100. Add the diluted serum sample to the reaction wells of the 96-well microplate coated with LIMCH1 protein prepared in step (1) above, column 1 to 11, with a sample volume of 50 μL / well; add quality control serum diluted 1:100 to reaction wells 1 to 5 of column 12 of the 96-well microplate coated with LIMCH1 protein, with a sample volume of 50 μL / well. The quality control serum is used as a quality control for standardization between different microplates; add serum-free antibody diluent (sample volume of 50 μL / well) to reaction wells 6 to 8 of column 12 of the 96-well microplate coated with LIMCH1 protein as a blank control; then incubate the 96-well microplate in a 37°C water bath for 1 h, then discard the liquid in the reaction wells, wash 5 times with washing buffer (sample volume of 300 μL / well) and pat dry.

[0046] 2) Secondary antibody incubation: Dilute the HRP-labeled mouse anti-human IgG antibody with antibody dilution buffer at a ratio of 1:5000 (v / v), then add the diluted HRP-labeled mouse anti-human IgG antibody to the corresponding reaction wells of the 96-well microplate at a volume of 50 μL / well, incubate at 37°C for 1 h, then discard the liquid in the reaction wells, wash with washing buffer (volume of 300 μL / well) 5 times, and pat dry.

[0047] 3) 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 a 96-well microplate at a volume of 50 μL / well. Incubate at room temperature in the dark for 5-15 min. Then, add 25 μL of stop solution to each reaction well to terminate the color development reaction. Use a microplate reader to read the absorbance at wavelengths of 450 nm and 620 nm, OD450 and OD620, respectively. The absorbance at 620 nm wavelength, OD620, is the background value. The difference between OD450 and OD620 is used as the final absorbance value.

[0048] The specific steps for detecting the expression levels of the remaining four autoantibodies in serum samples are basically the same as those for detecting LIMCH1 autoantibody, except that in step 1), the enzyme-labeled plates used for detection are coated with RSPH1, ENAH, COTL1, and ASS1 proteins, respectively.

[0049] 4. Data Processing: The Kolmogorov-Smirnova test was performed on the absorbance values ​​of serum samples from the positive and negative groups of lymph node metastasis in gastric cardia adenocarcinoma. The results showed that the expression levels of five anti-tumor-related antigen autoantibodies in the serum samples of the subjects did not conform to a normal distribution (P<0.05). Therefore, the nonparametric test (Wilcoxon) was used to compare whether there was a difference in the expression levels of autoantibodies between the positive and negative groups of lymph node metastasis in gastric cardia adenocarcinoma.

[0050] 5. Experimental Results: The distribution of expression levels of five anti-tumor-associated antigen autoantibodies in serum samples from the positive and negative groups of gastric cardia adenocarcinoma with lymph node metastasis is shown in the figure below. Figure 2 As shown, orange represents the Positive group and blue represents the Negative group. Figure 2 It was found that the five autoantibodies showed significant differences between the positive and negative groups of lymph node metastasis in gastric cardia adenocarcinoma (P<0.05). This indicates that autoantibodies against these five tumor antigens have an early warning effect on lymph node metastasis in gastric cardia adenocarcinoma. It also suggests that autoantibodies against these five anti-tumor-related antigens can be used as an adjunct to the diagnosis of lymph node metastasis in gastric cardia adenocarcinoma.

[0051] Example 3: Assessment of the ability of five autoantibodies to diagnose lymph node metastasis in gastric cardia adenocarcinoma According to Example 2, the expression levels of LIMCH1 autoantibody, RSPH1 autoantibody, ENAH autoantibody, COTL1 autoantibody, and ASS1 autoantibody in serum samples from 190 patients with positive and 118 negative lymph node metastases of gastric cardia adenocarcinoma were detected by ELISA. ROC curves for different combinations of single and multiple autoantibodies were plotted using SPSS 21 to differentiate between positive and negative lymph node metastases of gastric cardia adenocarcinoma, and the value of the five autoantibodies in the diagnosis of lymph node metastases of gastric cardia adenocarcinoma was analyzed.

[0052] 1. The ability of a single autoantibody to identify lymph node metastasis in gastric cardia adenocarcinoma: Based on the expression levels of LIMCH1, RSPH1, ENAH, COTL1, and ASS1 autoantibodies in serum samples from 190 patients with positive and 118 patients with negative lymph node metastases from gastric cardia adenocarcinoma, ROC curves were plotted for each autoantibody. The ROC curves were used to evaluate the ability of each autoantibody to independently diagnose and differentiate between patients with positive and negative lymph node metastases from gastric cardia adenocarcinoma.

[0053] ROC curves for diagnosing and differentiating patients with positive and negative lymph node metastases from LIMCH1, RSPH1, ENAH, COTL1, and ASS1 autoantibodies in gastric cardia adenocarcinoma are shown below. Figure 3 As shown. Based on the ROC curve, the OD value with the largest Youden index is used as the cutoff value, and the corresponding AUC, 95% confidence interval, sensitivity, and specificity are calculated.

[0054] Depend on Figure 3 It was found that when the five antitumor-associated antigen autoantibodies were used alone to differentiate between patients with positive and negative lymph node metastases in gastric cardia adenocarcinoma, the AUC of their ROC curves all reached above 0.514. Among them, the LIMCH1 autoantibody had the largest AUC (0.570), with a sensitivity of 73.7% and a specificity of 39.8%. This indicates that all five antitumor-associated antigen autoantibodies can be used for the auxiliary diagnosis of positive and negative lymph node metastases in gastric cardia adenocarcinoma.

[0055] 2. The ability of two autoantibodies to differentiate lymph node metastasis in gastric cardia adenocarcinoma: Using the expression levels of LIMCH1 and RSPH1 autoantibodies in serum samples from 190 patients with positive and 118 negative lymph node metastases of gastric adenocarcinoma as independent variables, and the presence or absence of positive lymph node metastasis events as the dependent variable, logistic regression analysis was performed on the expression levels of LIMCH1 and RSPH1 autoantibodies in serum samples from the positive and negative lymph node metastasis groups of gastric adenocarcinoma to construct a diagnostic model for differentiating lymph node metastasis in gastric adenocarcinoma. The diagnostic model is as follows: ,in In this diagnostic model: P represents the predicted probability, and LIMCH1 and RSPH1 represent the expression levels of these two autoantibodies in the subject's serum (expression levels are measured by the absorbance values ​​detected by the ELISA method described in Example 2). Substituting the expression levels of LIMCH1 and RSPH1 autoantibodies in each serum sample into the diagnostic model yields the predicted probability (i.e., the P-value) for each serum sample. An ROC curve is then plotted based on the predicted probabilities, as shown in the figure. Figure 4 As shown in Figure A. Further calculations were made based on the coordinates of the ROC curve using the Youden index (Youden index = sensitivity + specificity - 1). The P-value corresponding to the maximum Youden index was the optimal cutoff value for identifying lymph node metastasis in gastric cardia adenocarcinoma. Sensitivity and specificity were then calculated.

[0056] Depend on Figure 4 As shown in Figure A, the combination of LIMCH1 and RSPH1 autoantibodies can differentiate between patients with positive and negative lymph node metastases in gastric cardia adenocarcinoma, achieving an AUC of 0.590 on the ROC curve; the sensitivity and specificity are 34.7% and 83.1%, respectively. This indicates that the combination of LIMCH1 and RSPH1 autoantibodies can be used for the auxiliary diagnosis of positive and negative lymph node metastases in gastric cardia adenocarcinoma.

[0057] 3. The ability of three autoantibodies to differentiate lymph node metastasis in gastric cardia adenocarcinoma: Using the expression levels of LIMCH1, RSPH1, and ENAH autoantibodies in serum samples from 190 patients with positive and 118 negative lymph node metastases of gastric adenocarcinoma as independent variables, and the presence or absence of positive lymph node metastasis events in gastric adenocarcinoma as the dependent variable, logistic regression analysis was performed on the expression levels of LIMCH1, RSPH1, and ENAH autoantibodies in serum samples from the positive and negative groups of gastric adenocarcinoma lymph node metastasis to construct a diagnostic model for differentiating lymph node metastasis in gastric adenocarcinoma. The diagnostic model is as follows: ,in In this diagnostic model: P represents the predicted probability, and LIMCH1, RSPH1, and ENAH represent the expression levels of these three autoantibodies in the subject's serum (expression levels are measured by the absorbance values ​​detected by the ELISA method described in Example 2). Substituting the expression levels of LIMCH1, RSPH1, and ENAH autoantibodies in each serum sample into the diagnostic model yields the predicted probability (i.e., the P-value) for each serum sample. ROC curves are then plotted based on the predicted probabilities, as shown in the figure. Figure 4 As shown in B. Further calculations were made based on the coordinates of the ROC curve using the Youden index (Youden index = sensitivity + specificity - 1). The P-value corresponding to the maximum Youden index was the optimal cutoff value for identifying lymph node metastasis in gastric cardia adenocarcinoma. Sensitivity and specificity were then calculated.

[0058] Depend on Figure 4 As shown in Figure B, the combination of three autoantibodies—LIMCH1, RSPH1, and ENAH—achieved an AUC of 0.604 when differentiating between patients with positive and negative lymph node metastases in gastric cardia adenocarcinoma; the sensitivity and specificity were 41.1% and 78.8%, respectively. This indicates that the combination of LIMCH1, RSPH1, and ENAH autoantibodies can be used for the auxiliary diagnosis of positive and negative lymph node metastases in gastric cardia adenocarcinoma.

[0059] 4. The ability of four autoantibodies to differentiate lymph node metastasis in gastric cardia adenocarcinoma: Using the expression levels of LIMCH1, RSPH1, ENAH, and COTL1 autoantibodies in serum samples from 190 patients with positive and 118 negative lymph node metastases of gastric adenocarcinoma as independent variables and the presence or absence of lymph node metastasis as the dependent variable, logistic regression analysis was performed on the expression levels of LIMCH1, RSPH1, ENAH, and COTL1 autoantibodies in serum samples from both positive and negative groups of gastric adenocarcinoma lymph node metastasis to construct a diagnostic model for differentiating lymph node metastasis in gastric adenocarcinoma. The diagnostic model is as follows: ,in In this diagnostic model: P represents the predicted probability, and LIMCH1, RSPH1, ENAH, and COTL1 represent the expression levels of these four autoantibodies in the subject's serum (expression levels are measured by the absorbance values ​​detected by the ELISA method described in Example 2). Substituting the expression levels of LIMCH1, RSPH1, ENAH, and COTL1 autoantibodies in each serum sample into the diagnostic model yields the predicted probability (i.e., the P-value) for each serum sample. ROC curves are then plotted based on the predicted probabilities, as shown in the figure. Figure 4As shown in C. Further, the Youden index (Youden index = sensitivity + specificity - 1) is calculated based on the coordinates of the ROC curve. The P-value corresponding to the maximum Youden index is the optimal cutoff value for identifying lymph node metastasis in gastric cardia adenocarcinoma. Sensitivity and specificity are then calculated.

[0060] Depend on Figure 4 As shown in Figure C, the combination of four autoantibodies—LIMCH1, RSPH1, ENAH, and COTL1—achieved an AUC of 0.616 when differentiating between patients with positive and negative lymph node metastases in gastric cardia adenocarcinoma; the sensitivity and specificity were 53.7% and 65.3%, respectively. This indicates that the combination of these four autoantibodies can be used for the auxiliary diagnosis of positive and negative lymph node metastases in gastric cardia adenocarcinoma.

[0061] 5. The ability of five autoantibodies to differentiate lymph node metastasis in gastric cardia adenocarcinoma: Using the expression levels of LIMCH1, RSPH1, ENAH, COTL1, and ASS1 autoantibodies in serum samples from 190 patients with positive and 118 negative lymph node metastases of gastric cardia adenocarcinoma as independent variables and the presence or absence of positive lymph node metastasis events as the dependent variable, logistic regression analysis was performed on the expression levels of LIMCH1, RSPH1, ENAH, COTL1, and ASS1 autoantibodies in serum samples from both positive and negative lymph node metastasis groups of gastric cardia adenocarcinoma to construct a diagnostic model for differentiating lymph node metastasis in gastric cardia adenocarcinoma. The diagnostic model is as follows: ,in In this diagnostic model: P represents the predicted probability, and LIMCH1, RSPH1, ENAH, COTL1, and ASS1 represent the expression levels of these five autoantibodies in the subject's serum (expression levels are measured by the absorbance values ​​detected by the ELISA method described in Example 2). Substituting the expression levels of LIMCH1, RSPH1, ENAH, COTL1, and ASS1 autoantibodies in each serum sample into the diagnostic model yields the predicted probability (i.e., the P-value) for each serum sample. ROC curves are then plotted based on the predicted probabilities, as shown in the figure. Figure 4 As shown in D. Further, the Youden index (Youden index = sensitivity + specificity - 1) is calculated based on the coordinates of the ROC curve. The P-value corresponding to the maximum Youden index is the optimal cutoff value for identifying lymph node metastasis in gastric cardia adenocarcinoma. Sensitivity and specificity are then calculated.

[0062] Depend on Figure 4As shown in D, when the combination of five autoantibodies—LIMCH1, RSPH1, ENAH, COTL1, and ASS1—distinguished between patients with positive and negative lymph node metastases in gastric adenocarcinoma, the AUC of its ROC curve reached 0.638; the sensitivity and specificity were 31.6% and 92.4%, respectively.

[0063] For ease of comparison, the ROC curves, AUC, sensitivity, specificity, and Youden index of the above single or multiple autoantibody combinations for identifying lymph node metastasis in gastric cardia adenocarcinoma were statistically analyzed, and the results are shown in Table 1.

[0064] Table 1. Evaluation results of five autoantibodies in differentiating lymph node metastasis in gastric cardia adenocarcinoma. As shown in Table 1, compared with single autoantibodies, the AUC of the ROC curves for the identification of lymph node metastasis in gastric cardia adenocarcinoma using combinations of two, three, four, and five autoantibodies all reached above 0.590, significantly higher than that of single autoantibodies. When five autoantibodies were used to identify lymph node metastasis in gastric cardia adenocarcinoma, the AUC of the ROC curve reached the maximum of 0.638. Moreover, the sensitivity and specificity of the five autoantibodies in identifying lymph node metastasis in gastric cardia adenocarcinoma were both good, at 31.6% and 92.4%, respectively. This indicates that the combined use of five autoantibodies—LIMCH1, RSPH1, ENAH, COTL1, and ASS1—provides the greatest advantage as a biomarker for identifying lymph node metastasis in gastric cardia adenocarcinoma.

Claims

1. The use of reagents for detecting biomarkers in the preparation of products for identifying lymph node metastasis of gastric cardia adenocarcinoma, wherein the biomarker is at least one of LIMCH1 autoantibody, RSPH1 autoantibody, ENAH autoantibody, COTL1 autoantibody, and ASS1 autoantibody.

2. The application according to claim 1, characterized in that, The reagent is used to detect the biomarkers in the sample by enzyme-linked immunosorbent assay (ELISA), protein chip, immunoblotting, or microfluidic immunoassay.

3. The application according to claim 2, characterized in that, The sample may be serum, plasma, interstitial fluid, or urine.

4. The application according to claim 2, characterized in that, The reagent is an antigen or antibody used to detect the biomarker.

5. The application according to claim 1, characterized in that, When the biomarker is a combination of LIMCH1 autoantibody and RSPH1 autoantibody, the probability calculation formula for the product to identify lymph node metastasis of gastric cardia adenocarcinoma is as follows: ； Where P represents the prediction probability, LIMCH1 represents the expression level of LIMCH1 autoantibody, and RSPH1 represents the expression level of RSPH1 autoantibody. When the biomarker is a combination of LIMCH1 autoantibody, RSPH1 autoantibody, and ENAH autoantibody, the probability calculation formula for the product to identify lymph node metastasis of gastric cardia adenocarcinoma is as follows: ； Where P represents the prediction probability, LIMCH1 represents the expression level of LIMCH1 autoantibody, RSPH1 represents the expression level of RSPH1 autoantibody, and ENAH represents the expression level of ENAH autoantibody. When the biomarker is a combination of LIMCH1 autoantibody, RSPH1 autoantibody, ENAH autoantibody, and COTL1 autoantibody, the probability calculation formula for the product to identify lymph node metastasis in gastric cardia adenocarcinoma is as follows: ； Where P represents the prediction probability, LIMCH1 represents the expression level of LIMCH1 autoantibody, RSPH1 represents the expression level of RSPH1 autoantibody, ENAH represents the expression level of ENAH autoantibody, and COTL1 represents the expression level of COTL1 autoantibody. When the biomarker is a combination of LIMCH1 autoantibody, RSPH1 autoantibody, ENAH autoantibody, COTL1 autoantibody, and ASS1 autoantibody, the probability calculation formula for the product to identify lymph node metastasis of gastric cardia adenocarcinoma is as follows: ， ； Where P represents the prediction probability, LIMCH1 represents the expression level of LIMCH1 autoantibody, RSPH1 represents the expression level of RSPH1 autoantibody, ENAH represents the expression level of ENAH autoantibody, COTL1 represents the expression level of COTL1 autoantibody, and ASS1 represents the expression level of ASS1 autoantibody.

6. The application according to any one of claims 1-5, characterized in that, The product is a protein chip, reagent kit, or formulation.

7. A kit for identifying lymph node metastasis in gastric cardia adenocarcinoma, characterized in that, The kit contains reagents for detecting biomarkers; the biomarkers are at least one of LIMCH1 autoantibody, RSPH1 autoantibody, ENAH autoantibody, COTL1 autoantibody, and ASS1 autoantibody.

8. The reagent kit according to claim 7, characterized in that, The kit detects the biomarkers in a sample by enzyme-linked immunosorbent assay (ELISA), protein chip, immunoblotting, or microfluidic immunoassay.

9. The reagent kit according to claim 8, characterized in that, When the biomarker is a combination of LIMCH1 autoantibody and RSPH1 autoantibody, the probability calculation formula for the kit to identify lymph node metastasis in gastric cardia adenocarcinoma is as follows: ； Where P represents the prediction probability, LIMCH1 represents the expression level of LIMCH1 autoantibody, and RSPH1 represents the expression level of RSPH1 autoantibody. When the biomarker is a combination of LIMCH1 autoantibody, RSPH1 autoantibody, and ENAH autoantibody, the probability calculation formula for the kit to identify lymph node metastasis in gastric cardia adenocarcinoma is as follows: ； Where P represents the prediction probability, LIMCH1 represents the expression level of LIMCH1 autoantibody, RSPH1 represents the expression level of RSPH1 autoantibody, and ENAH represents the expression level of ENAH autoantibody. When the biomarkers are a combination of LIMCH1 autoantibody, RSPH1 autoantibody, ENAH autoantibody, and COTL1 autoantibody, the probability calculation formula for the kit to identify lymph node metastasis in gastric cardia adenocarcinoma is as follows: ； Where P represents the prediction probability, LIMCH1 represents the expression level of LIMCH1 autoantibody, RSPH1 represents the expression level of RSPH1 autoantibody, ENAH represents the expression level of ENAH autoantibody, and COTL1 represents the expression level of COTL1 autoantibody. When the biomarker is a combination of LIMCH1 autoantibody, RSPH1 autoantibody, ENAH autoantibody, COTL1 autoantibody, and ASS1 autoantibody, the probability calculation formula for the kit to identify lymph node metastasis in gastric cardia adenocarcinoma is as follows: ， ； Where P represents the prediction probability, LIMCH1 represents the expression level of LIMCH1 autoantibody, RSPH1 represents the expression level of RSPH1 autoantibody, ENAH represents the expression level of ENAH autoantibody, COTL1 represents the expression level of COTL1 autoantibody, and ASS1 represents the expression level of ASS1 autoantibody.

10. The reagent kit according to claim 8, characterized in that, The kit is an ELISA detection kit, which includes a solid-phase carrier and an antigen coated on the solid-phase carrier; the antigen is at least one of LIMCH1 protein, RSPH1 protein, ENAH protein, COTL1 protein, and ASS1 protein.

Images