Serum protein markers for early screening and diagnosis of esophageal squamous cell carcinoma and application thereof

Abstract

The application discloses a serum protein marker for early screening and diagnosis of esophageal squamous cell carcinoma, which is an anti-tumor associated antigen MAGEA autoantibody. The application screens potential anti-tumor associated antigen MAGEA family protein autoantibodies which can be used for diagnosing esophageal squamous cell carcinoma, and then verifies the autoantibodies through an ELISA indirect method experiment, thereby screening a group of anti-tumor associated antigen MAGEA autoantibodies for early screening and diagnosis of esophageal squamous cell carcinoma, in particular, a combination of anti-tumor associated antigen MAGEA1-IgG antibody, anti-tumor associated antigen MAGEA4-IgG antibody, anti-tumor associated antigen MAGEA6-IgG antibody and anti-tumor associated antigen MAGEA12-IgG antibody, which is used for assisting clinical diagnosis of esophageal squamous cell carcinoma and has good reference value.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical biotechnology, specifically relating to serum protein markers and their applications for early screening and diagnosis of esophageal squamous cell carcinoma. Background Technology

[0002] Esophageal cancer is the sixth leading cause of cancer death worldwide. Global cancer epidemiological statistics show that in 2020, there were 604,000 new cases of esophageal cancer globally, and 544,000 people died from it, making it the sixth leading cause of death among malignant tumors. The main pathological types of esophageal cancer are squamous cell carcinoma and adenocarcinoma, with 90% of cases being esophageal squamous cell carcinoma (ESCC). Because early symptoms are nonspecific and easily overlooked, most ESCC patients are diagnosed at an advanced stage. Furthermore, current treatment options for ESCC are very limited, resulting in a poor prognosis; the overall 5-year survival rate is less than 20%. However, unlike patients with advanced ESCC, existing treatments for early-stage ESCC can achieve a 5-year survival rate of over 60%. Currently, the main diagnostic methods for ESCC include barium meal X-ray, CT / PET-CT scans, electronic endoscopy, and pathological tissue biopsy. However, their limited effectiveness in early diagnosis, high cost, or invasive procedures restrict their widespread use in screening. Therefore, exploring molecular markers for the early diagnosis of ESCC has significant potential clinical value. Currently, there are no ideal, non-invasive diagnostic markers for ESCC available for clinical use.

[0003] The melanoma-associated antigen A (MAGEA) family belongs to the class of cancer / testis antigens (CTAs). It is almost not expressed in normal tissues but is highly expressed to varying degrees in most tumor tissues. The MAGEA family promotes tumor growth, metastasis, and drug resistance, making it a marker of tumor development and progression. MAGEA family genes and their expression products have significant research value in cancer diagnosis. Tumor-associated autoantibodies (TAAbs) appear early in serum, are stable, and are easily measured; they have been proven to have diagnostic value for the early stages of various cancers. MAGEA family proteins have long been identified as tumor-associated antigens (TAAs) and can elicit a strong immune response, producing large amounts of autoantibodies. Anti-TAA family protein autoantibodies have significant potential for cancer diagnosis.

[0004] Currently, no studies have fully explored the diagnostic value of antitumor-associated antigen MAGEA family protein autoantibodies in esophageal squamous cell carcinoma, and further research is urgently needed on the diagnostic potential of antitumor-associated antigen MAGEA family protein autoantibodies in esophageal squamous cell carcinoma. Summary of the Invention

[0005] The purpose of this invention is to provide a serum protein biomarker for early screening and diagnosis of esophageal squamous cell carcinoma, and providing its application is another objective of this invention.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] Serum protein biomarkers for early screening and diagnosis of esophageal squamous cell carcinoma, wherein the serum protein biomarkers are anti-tumor-associated antigen MAGEA autoantibodies.

[0008] The serum protein markers are any one or a combination of two or more of the following: antitumor-associated antigen MAGEA1 autoantibody, antitumor-associated antigen MAGEA3 autoantibody, antitumor-associated antigen MAGEA4 autoantibody, antitumor-associated antigen MAGEA6 autoantibody, antitumor-associated antigen MAGEA10 autoantibody, and antitumor-associated antigen MAGEA12 autoantibody.

[0009] The antitumor-associated antigen MAGEA1 autoantibody is an antitumor-associated antigen MAGEA1-IgG antibody; the antitumor-associated antigen MAGEA3 autoantibody is one or both of antitumor-associated antigen MAGEA3-IgG antibody and antitumor-associated antigen MAGEA3-IgM antibody; the antitumor-associated antigen MAGEA4 autoantibody is an antitumor-associated antigen MAGEA4-IgG antibody; the antitumor-associated antigen MAGEA6 autoantibody is an antitumor-associated antigen MAGEA6-IgG antibody; the antitumor-associated antigen MAGEA10 autoantibody is an antitumor-associated antigen MAGEA10-IgG antibody; and the antitumor-associated antigen MAGEA12 autoantibody is one or both of antitumor-associated antigen MAGEA12-IgG antibody and antitumor-associated antigen MAGEA12-IgM antibody.

[0010] The serum protein markers are a combination of antitumor-associated antigen MAGEA1-IgG antibodies, antitumor-associated antigen MAGEA4-IgG antibodies, antitumor-associated antigen MAGEA6-IgG antibodies, and antitumor-associated antigen MAGEA12-IgG antibodies.

[0011] The serum protein markers are a combination of antitumor-associated antigen MAGEA1-IgG antibodies, antitumor-associated antigen MAGEA3-IgG antibodies, antitumor-associated antigen MAGEA3-IgM antibodies, antitumor-associated antigen MAGEA4-IgG antibodies, antitumor-associated antigen MAGEA6-IgG antibodies, antitumor-associated antigen MAGEA10-IgG antibodies, and antitumor-associated antigen MAGEA12-IgG antibodies.

[0012] Application of reagents for detecting serum protein markers in the preparation of products for early screening and diagnosis of esophageal squamous cell carcinoma.

[0013] The product used for early screening and diagnosis of esophageal squamous cell carcinoma is a kit.

[0014] The kit includes reagents for detecting serum protein markers; the reagents are antigens for detecting serum protein markers.

[0015] The antigen is any one or a combination of two or more of the following: tumor-associated antigen MAGEA1 protein, tumor-associated antigen MAGEA3 protein, tumor-associated antigen MAGEA4 protein, tumor-associated antigen MAGEA6 protein, tumor-associated antigen MAGEA10 protein, and tumor-associated antigen MAGEA12 protein; the antigen is coated on a solid-phase support.

[0016] The solid-phase support is made of polyvinyl chloride, polystyrene, polyacrylamide, or cellulose; the kit also includes any one or more combinations of positive control serum, negative control serum, blocking solution, sample diluent, secondary antibody, secondary antibody diluent, washing solution, chromogenic solution, or stop solution.

[0017] A method for detecting serum protein biomarkers used in the early screening and diagnosis of esophageal squamous cell carcinoma is characterized by comprising the following steps:

[0018] 1) Each serum protein marker was coated and blocked separately, then washed;

[0019] 2) Incubate with the diluted serum for testing with primary antibody, wash, then incubate with secondary antibody, and wash.

[0020] 3) After the colorimetric system has developed color, terminate the reaction and measure the absorbance value;

[0021] 4) Using OD450-OD620 as the relative OD value, and then subtracting the blank control, substitute the absorbance value into the following formula to calculate the predicted probability P value (taking the combination of antibodies against antitumor-related antigens MAGEA1-IgG, MAGEA4-IgG, MAGEA6-IgG, and MAGEA12-IgG as an example).

[0022] PRE (P=ESCC)=1 / (1+EXP(-(-2.438+8.255×OD MAGEA1-IgG + 3.718×OD MAGEA4-IgG +2.119 ×OD MAGEA6-IgG -4.411 ×OD MAGEA12-IgG )));

[0023] In the formula, OD MAGEA1-IgG OD MAGEA4-IgG OD MAGEA6-IgG OD MAGEA12-IgG The absorbance values ​​of each serum protein marker are calculated by subtracting the absorbance value of the blank control from the OD value.

[0024] When the P-value is ≥0.5, the sample is preliminarily identified as esophageal squamous cell carcinoma.

[0025] When the P value is < 0.5, it is preliminarily determined to be a normal sample.

[0026] It also includes step 5), which calculates the positive rate, sensitivity, specificity, Youden index, positive predictive value, negative predictive value, positive likelihood ratio, and negative likelihood ratio for each serum protein marker.

[0027] The basic information of tumor-associated antigens MAGEA1, MAGEA3, MAGEA4, MAGEA6, MAGEA10, and MAGEA12 in this invention is as follows:

[0028] The MAGEA family of genes comprises 12 members (MAGEA1–MAGEA12), among which MAGEA5 and MAGEA7 are pseudogenes with no transcripts. The promoters and first exons of MAGEA genes exhibit considerable variability, indicating that the existence of this gene family allows the same function to be expressed under different transcriptional controls. Most proteins in the MAGEA family possess a homology domain (MHD), showing 60–80% conservation across family members. Despite high sequence and structural similarity, individual members of the MAGEA family exhibit structural dynamics due to conformational changes, thus displaying different functions. MAGEA gene expression in normal somatic cells is suppressed by epigenetic mechanisms. In untransformed cells, MAGEA genes are silenced, while frequent epigenetic reprogramming in tumor cells leads to global DNA hypomethylation and MAGEA expression. In NCBI, the sequence numbers for MAGEA1, MAGEA3, MAGEA4, MAGEA6, MAGEA10, and MAGEA12 are: NP_001159859.1.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1) This invention is based on HuProt, which includes MAGEA1-MAGEA12 (excluding MAGEA5 and MAGEA7). TM The Human Proteome Chip V3.1 screened for potential MAGEA family protein autoantibodies that could be used to diagnose esophageal squamous cell carcinoma. These autoantibodies were then validated using an indirect ELISA method. A group of MAGEA autoantibodies for early screening and diagnosis of esophageal squamous cell carcinoma was identified, particularly the combination of MAGEA1-IgG, MAGEA4-IgG, MAGEA6-IgG, and MAGEA12-IgG antibodies. This combination is of good reference value for assisting in the clinical diagnosis of esophageal squamous cell carcinoma.

[0031] 2) The kit of the present invention is used to detect esophageal squamous cell carcinoma. It has the advantages of high sensitivity, strong specificity and low cost. It is also simple and quick to operate, providing a basis for the early diagnosis of esophageal squamous cell carcinoma. The test sample of the kit is serum, which can avoid invasive diagnosis. The risk of esophageal squamous cell carcinoma can be obtained by collecting serum in a minimally invasive manner. It requires a small amount of blood, causes little pain to the test subjects and has high compliance. It is simple to operate and has a short time to detect results. It has broad market prospects and social benefits. Attached Figure Description

[0032] To more clearly illustrate the technical solution of the present invention, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 As in Example 1, based on HuProt TM Schematic diagram of human protein chip detection principle;

[0034] Figure 2 In the figures, 2-1 and 2-2 are box plots of the expression levels of autoantibodies against candidate antitumor-associated antigens MAGEA family proteins, plotted based on the SNR values ​​after median normalization and z-score normalization of the protein chip in the experimental examples; where ESCC represents the esophageal squamous cell carcinoma group and NC represents the normal control group.

[0035] Figure 3 In the figure, 3-1 and 3-2 are scatter plots showing the OD values ​​of MAGEA family autoantibodies against tumor-associated antigens detected by ELISA in the training set and validation set, respectively, in the esophageal squamous cell carcinoma group and the normal control group.

[0036] Figure 4 To train a set of ROC curves for differentiating esophageal squamous cell carcinoma patients from healthy individuals using four tumor-associated antigen autoantibodies;

[0037] Figure 5 To validate the ROC curve plot of four tumor-associated antigen autoantibodies in distinguishing between esophageal squamous cell carcinoma patients and normal individuals;

[0038] Figure 6 To train a combination of autoantibodies against the tumor-associated antigen MAGEA to diagnose and differentiate between patients with esophageal squamous cell carcinoma and normal individuals;

[0039] Figure 7 To verify the effectiveness of the combined antitumor-associated antigen MAGEA autoantibody combination in diagnosing and differentiating esophageal squamous cell carcinoma patients from normal individuals, ROC curves were plotted. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention are described in detail below. However, the following embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0041] Unless otherwise specified, the experimental methods in the following examples all employ conventional techniques in this technical field or follow the conditions recommended by the manufacturer; reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased.

[0042] Example 1: Screening for MAGEA autoantibodies against esophageal squamous cell carcinoma-associated antitumor antigen using human whole-proteome microarray technology.

[0043] 1. Experimental Samples

[0044] Serum samples were collected from 30 patients with esophageal squamous cell carcinoma (ESCC) (ESCC group) and 24 healthy controls from the specimen bank of the Henan Provincial Key Laboratory of Tumor Epidemiology. The 30 ESCC patients were pathologically diagnosed and had not received any treatment. The 24 healthy controls were healthy subjects. Inclusion criteria for healthy subjects included: no history of cardiovascular, respiratory, liver, kidney, gastrointestinal, endocrine, hematological, mental, or neurological diseases; no acute or chronic diseases; no autoimmune diseases; no evidence of tumors; and no statistically significant gender differences between the 30 ESCC patients and the 24 healthy subjects. This study was approved by the Ethics Committee of Zhengzhou University, and all participants signed informed consent forms.

[0045] The serum from 30 patients with esophageal squamous cell carcinoma was mixed into one mixed esophageal squamous cell carcinoma serum sample, resulting in a total of 10 mixed esophageal squamous cell carcinoma serum samples; the serum from 21 healthy individuals was mixed into one mixed normal serum sample, resulting in a total of 7 mixed normal serum samples.

[0046] 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 EP 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.

[0047] 2. Human proteome microarray detection

[0048] Using HuProt TM Human proteome microarrays were used to detect the expression levels of autoantibodies in 10 serum samples from mixed esophageal squamous cell carcinoma patients, 7 mixed normal serum samples, and 3 normal human serum samples. Each microarray can simultaneously detect 14 serum samples. Proteins immobilized on the microarray interact with and bind to specific autoantibodies in the serum. See the experimental schematic diagram. Figure 1 .

[0049] (1) Experimental methods

[0050] 1) Rewarming: HuProt TM The human proteome chip was removed from the -80 °C freezer, placed in a 4 °C freezer for 30 min to rewarm, and then continued to rewarm to room temperature for 15 min.

[0051] 2) Blocking: Place the warmed chip face up in the chip incubation box, add 10 mL of blocking solution (3 mL 10% BSA, 7 mL 1 × PBS solution), and place it in a side-swinging shaker at 50-60 rpm for 1 h at room temperature.

[0052] 3) Serum sample incubation: After blocking, discard the blocking solution and quickly add the pre-diluted serum incubation solution (the serum sample is diluted at a ratio of 1:200 with diluent to obtain the diluted serum incubation solution, which is prepared by adding 1 ml of 10% BSA to 9 ml of 1 × PBST solution). Place the solution in a side-shaking shaker and incubate overnight at 20 rpm and 4 ℃.

[0053] 4) Cleaning: After incubation, remove the chip and place it in a chip cleaning box containing cleaning solution (1×PBST solution). Shake horizontally at 80 rpm at room temperature for 3 times, 10 min each time.

[0054] 5) Secondary antibody incubation: After washing, transfer the chip to the incubation box and add 3 mL of secondary antibody incubation solution diluted at a ratio of 1:1000 (the secondary antibody is a fluorescently labeled anti-human IgM and IgG antibody, and the diluent consists of 1 g BSA and 100 mL 1×PBST solution. The secondary antibody is diluted with the diluent at a ratio of 1:1000 to obtain the secondary antibody incubation solution). Place the box on a side-shaking incubator at 40 rpm, protect it from light, and incubate at room temperature for 1 h.

[0055] 6) Cleaning: Remove the chip (be careful not to touch or scratch the top surface of the chip), place it in a chip cleaning container, add chip cleaning solution (1 × PBST solution), place on a horizontal shaker, and clean 3 times at 80 rpm for 10 min each time. After completion, repeat the cleaning twice with ddH2O for 10 min each time;

[0056] 7) Drying: After cleaning, place the chip in a chip dryer for centrifugal drying;

[0057] 8) Scanning: Perform a standardized fluorescence scan on the dried chip according to the scanner's instructions and record the fluorescence signal (the intensity of the fluorescence signal is positively correlated with the affinity and quantity of the corresponding antibody).

[0058] 9) Data extraction: Open the corresponding GAL file, align each array on the GAL file with the chip image, click the automatic alignment button, extract the data and save it as GPR.

[0059] (2) Data processing

[0060] F532 Median refers to the median foreground value of the signal point in the 532 nm channel, and B532Median refers to the median background value of the signal point in the 532 nm channel. To eliminate signal inhomogeneity caused by inconsistent background values ​​among different protein points within the same chip, a background normalization method was used. The signal-to-noise ratio (SNR) was defined as F532 Median / B532 Median. SNR values ​​were calculated according to the formula for 10 mixed esophageal squamous cell carcinoma serum samples, 7 mixed normal serum samples, and 3 normal serum samples. Then, the SNR values ​​of the serum samples were linearly normalized to the median or standardized using z-score. Based on the Mann-Whitney U test, statistical tests were performed on various anti-tumor-related antigen MAGEA autoantibodies. The screening criterion was that the mean rank of the SNR value in the esophageal squamous cell carcinoma group was greater than the mean rank of the SNR value in the healthy control group. P <0.05.

[0061] (3) Experimental results

[0062] After screening, by Figure 2-1 It can be seen that, based on the SNR value linearly normalized to the median, five autoantibodies against the tumor-associated antigen MAGEA were finally screened. Figure 2-2Based on the z-score-normalized SNR value, six MAGEA autoantibodies were ultimately screened. The union of candidate MAGEA autoantibodies from the two standardization methods was used for further research, resulting in the inclusion of eight MAGEA autoantibodies: MAGEA1-IgG, MAGEA3-IgG, MAGEA3-IgM, MAGEA4-IgG, MAGEA6-IgG, MAGEA10-IgG, MAGEA12-IgG, and MAGEA12-IgM. The expression levels of these eight MAGEA autoantibodies in the serum of the esophageal squamous cell carcinoma group were significantly higher than those in the normal control group.

[0063] Example 2: ELISA detection of serum expression levels of anti-tumor-associated antigen MAGEA autoantibodies

[0064] The expression levels of the eight antitumor-associated antigen MAGEA autoantibodies screened in Example 1 in human serum were detected by enzyme-linked immunosorbent assay (ELISA).

[0065] 1. Experimental Samples

[0066] This study included serum samples from 423 patients with esophageal squamous cell carcinoma (ESCC) and 423 healthy controls (NCC), all sourced from the specimen bank of the Henan Provincial Key Laboratory of Tumor Epidemiology. The samples were divided into a training set and a validation set. The training set consisted of serum samples from 230 ESCC patients and 230 healthy controls. These samples were obtained from ESCC patients who visited a tertiary hospital in Henan Province between June and December 2022, and from healthy individuals undergoing physical examinations. The validation set consisted of serum samples from 193 ESCC patients, 193 healthy controls, and 173 patients with benign esophageal diseases. The cancer and benign esophageal disease serum samples in the validation set were obtained from patients who visited a tertiary hospital in Henan Province between August 2017 and April 2018, while the healthy controls were obtained from healthy individuals in Henan Province participating in the "Zhongyuan Gene Project" between January and September 2019. All patients with esophageal squamous cell carcinoma were pathologically diagnosed and had not received radiotherapy, chemotherapy, or surgery prior to sampling. All healthy controls and patients with benign diseases were free of autoimmune diseases and malignant tumors. This study was approved by the Ethics Committee of Zhengzhou University, and all participants were informed of the details and signed relevant informed consent forms before sampling.

[0067] Serum collection: Same as the serum collection method in Example 1.

[0068] Five ml of peripheral blood was collected from the subjects while they were fasting and placed in a blood collection tube without anticoagulant. After standing at room temperature for 1 hour, the tube was centrifuged at 3000 rpm for 10 minutes at 4°C. The serum from the top of the blood collection tube was then aspirated and aliquoted into 1.5 ml EP tubes. The sample numbers were marked on the top and side of the EP tubes, and the tubes were frozen at -80°C. The blood collection date and storage location were recorded. Before use, the serum was thawed at 4°C and aliquoted to avoid repeated freeze-thaw cycles.

[0069] 2. Reagents, Kit Materials, and Experimental Materials

[0070] (1) Six tumor-associated antigen MAGEA proteins: MAGEA1 recombinant protein, MAGEA4 recombinant protein, MAGEA6 recombinant protein, MAGEA10 recombinant protein and MAGEA12 recombinant protein were purchased from Wuhan Huamei Bioengineering Co., Ltd.; MAGEA3 recombinant protein was purchased from Wuhan Yunclone Technology Co., Ltd.

[0071] (2) 96-well microplate (8 rows × 12 columns);

[0072] (3) Coating solution: an aqueous solution containing 0.15% sodium carbonate (Na2CO3) and 0.29% sodium bicarbonate (NaHCO3);

[0073] (4) Blocking solution: PBST buffer containing 2% (v / v) bovine serum albumin (BSA) in 0.2% (v / v) Tween 20;

[0074] (5) Serum sample diluent: PBST buffer containing 1% (w / v) BSA;

[0075] (6) Enzyme-labeled second antibodies: horseradish peroxidase (HRP) labeled mouse anti-human immunoglobulin antibody G (hereinafter referred to as HRP-labeled mouse anti-human IgG antibody) and horseradish peroxidase (HRP) labeled mouse anti-human immunoglobulin antibody M (hereinafter referred to as HRP-labeled mouse anti-human IgM antibody);

[0076] (7) Antibody dilution buffer: PBST buffer containing 1% (w / v) BSA;

[0077] (8) Washing buffer: PBST buffer containing 0.2% (v / v) Tween 20;

[0078] (9) Colorimetric solution: The colorimetric solution consists of colorimetric solution A and colorimetric solution B. Colorimetric solution A is an aqueous solution of 20% tetramethylbenzidine dihydrochloric acid, and colorimetric solution B is an aqueous solution containing 3.7% Na2HPO4•12H2O, 0.92% citric acid and 0.75% hydrourea peroxide. When using, colorimetric solution A and colorimetric solution B are mixed evenly in equal volumes at a ratio of 1:1 and prepared fresh each time.

[0079] (10) Termination solution: 10% sulfuric acid.

[0080] 3. Experimental Methods

[0081] (1) Preparation of ELISA plates coated with MAGEA protein, a six-tumor-associated antigen:

[0082] Microplates coated with tumor-associated antigen MAGEA1 protein, MAGEA3 protein, MAGEA4 protein, MAGEA6 protein, MAGEA10 protein, and MAGEA12 protein were prepared separately.

[0083] Taking the preparation of an ELISA plate coated with the tumor-associated antigen MAGEA1 protein as an example, the preparation method includes the following steps:

[0084] 1) Preparation of tumor-associated antigen MAGEA1 protein solution: MAGEA1 protein was prepared into a MAGEA1 protein solution with a concentration of 0.25 μg / mL using a coating solution.

[0085] 2) Coating the microplate: Add the MAGEA1 protein solution from 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.

[0086] 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 MAGEA1 protein.

[0087] The preparation methods for ELISA plates coated with tumor-associated antigen MAGEA3 protein, MAGEA4 protein, MAGEA6 protein, MAGEA10 protein, and MAGEA12 protein are the same as those for ELISA plates coated with tumor-associated antigen MAGEA1 protein, except that: step 1) uses different tumor-associated antigens, and the concentrations of the prepared tumor-associated antigen solutions are different. Specifically, the concentrations of the tumor-associated antigen MAGEA3 protein solution are 0.125 μg / mL, MAGEA4 protein solution is 0.25 μg / mL, MAGEA6 protein solution is 0.25 μg / mL, MAGEA10 protein solution is 0.125 μg / mL, and MAGEA12 protein solution is either 0.25 μg / mL or 0.125 μg / mL.

[0088] (2) Detection of autoantibody expression levels of six antitumor-associated antigens MAGEA in serum samples:

[0089] For the same serum sample, the expression levels of IgG / IgM autoantibodies against the six tumor-associated antigens MAGEA1, MAGEA3, MAGEA4, MAGEA6, MAGEA10, and MAGEA12 in the serum sample were detected by ELISA using enzyme-linked immunosorbent assay (ELISA) plates coated with the six tumor-associated antigens prepared above.

[0090] Taking the detection of the expression level of anti-tumor-associated antigen MAGEA1-IgG autoantibody as an example, the specific operation steps are as follows:

[0091] 1) Serum sample incubation

[0092] The serum sample to be tested was diluted with serum sample diluent at a volume ratio of 1:100. The diluted serum sample was added to the reaction wells of the 96-well microplate coated with MAGEA1 protein prepared in step (1) above, in columns 1 to 11, with a sample volume of 50 μl / well; the quality control serum diluted at 1:100 was added to the reaction wells of the 96-well microplate coated with MAGEA1 protein in column 12, in columns 1 to 6, with a sample volume of 50 μl / well. The quality control serum was used as a quality control for standardization between different microplates; the antibody diluent without serum (sample volume of 50 μl / well) was added to the reaction wells of the 96-well microplate coated with MAGEA1 protein in column 12, in columns 7 to 8 as a blank control; then the 96-well microplate was incubated in a water bath at 37°C for 1 h, and then the liquid in the reaction wells was discarded. The wells were washed 5 times with washing buffer (sample volume of 300 μl / well) and patted dry.

[0093] 2) Secondary antibody incubation

[0094] The HRP-labeled mouse anti-human IgG antibody was diluted with antibody dilution buffer at a ratio of 1:5000 (v / v). The diluted HRP-labeled mouse anti-human IgG antibody was then added to the corresponding reaction wells of a 96-well microplate at a volume of 50 μl / well. The plate was incubated at 37°C for 1 h. The liquid in the reaction wells was then discarded, and the plate was washed 5 times with washing buffer (300 μl / well) and then patted dry.

[0095] 3) Color development and termination reaction

[0096] Mix chromogenic solution A and chromogenic solution B in a 1:1 ratio until homogeneous. Then, quickly add 50 μl of the mixture to each well of a 96-well microplate. Incubate at room temperature in the dark for 5-15 minutes. Finally, add 25 μl of stop solution to each well to terminate the reaction. Use a microplate reader to read the absorbance (OD) at 450 nm and 620 nm wavelengths. 450 OD 620 Among them, the absorbance OD at a wavelength of 620 nm 620 As background value, in OD 450 With OD 620 The difference is used as the final result of the detected absorbance value.

[0097] The expression levels of antitumor-associated antigen MAGEA3-IgG autoantibodies, antitumor-associated antigen MAGEA3-IgM autoantibodies, antitumor-associated antigen MAGEA4-IgG autoantibodies, antitumor-associated antigen MAGEA6-IgG autoantibodies, antitumor-associated antigen MAGEA10-IgG autoantibodies, antitumor-associated antigen MAGEA12-IgG autoantibodies, and antitumor-associated antigen MAGEA12-IgM autoantibodies in serum samples were detected. The procedure was the same as described above for detecting antitumor-associated antigen MAGEA1-IgG autoantibodies, except that: in step 1), the enzyme-linked immunosorbent assay (ELISA) plates used for detection were respectively ELISA plates coated with tumor-associated antigen MAGEA3 protein, tumor-associated antigen MAGEA4 protein, tumor-associated antigen MAGEA6 protein, tumor-associated antigen MAGEA10 protein, and tumor-associated antigen MAGEA12 protein; in step 2), for the tumor-associated antigen-coated plates... For the reaction wells coated with the MAGEA3 antigen, HRP-labeled mouse anti-human IgG antibody was diluted 1:5000 (v / v) or HRP-labeled mouse anti-human IgM antibody was diluted 1:2500 (v / v). For the reaction wells coated with the tumor-associated antigen MAGEA4 protein, HRP-labeled mouse anti-human IgG antibody was diluted 1:5000 (v / v). For the reaction wells coated with the tumor-associated antigen MAGEA6 protein, HRP-labeled mouse anti-human IgG antibody was diluted 1:5000 (v / v). For the reaction wells coated with the tumor-associated antigen MAGEA10 protein, HRP-labeled mouse anti-human IgG antibody was diluted 1:5000 (v / v). For the reaction wells coated with the tumor-associated antigen MAGEA12 protein, HRP-labeled mouse anti-human IgG antibody was diluted 1:5000 (v / v) or HRP-labeled mouse anti-human IgM antibody was diluted 1:2500 (v / v).

[0098] 4. Data Processing

[0099] Kolmogorov-Smirnova test was performed on the absorbance values ​​of serum samples from the esophageal squamous cell carcinoma group and the normal control group. The results showed that the expression levels of eight anti-tumor-associated antigen MAGEA autoantibodies in the serum samples of the subjects did not conform to a normal distribution. P <0.05), therefore, the 25th percentile (P25), median (P50), and 75th percentile (P75) were used to describe the expression level distribution of autoantibodies against eight antitumor-associated antigens; then, the nonparametric test (Mann-Whitney U) was used to compare whether there was a difference in the expression level of autoantibodies between the esophageal squamous cell carcinoma group and the normal control group.

[0100] 5. Experimental Results

[0101] The distribution of expression levels of eight anti-tumor-associated antigen MAGEA autoantibodies in serum samples from the training set esophageal squamous cell carcinoma group and the normal control group is shown in the figure below. Figure 3-1 As shown.

[0102] Depend on Figure 3-1 It was found that the expression levels of antitumor-associated antigens MAGEA1-IgG, MAGEA3-IgG, MAGEA3-IgM, MAGEA4-IgG, MAGEA6-IgG, MAGEA10-IgG, and MAGEA12-IgG autoantibodies in the serum samples of the esophageal squamous cell carcinoma group were significantly higher than those in the normal control group, and the differences were statistically significant. P <0.05).

[0103] The validation set further validated the expression levels of seven MAGEA autoantibodies with potential diagnostic value from the training set. The distribution of MAGEA autoantibodies expression levels in serum samples from the esophageal squamous cell carcinoma group and the normal control group in the validation set is shown below. Figure 3-2 As shown.

[0104] Depend on Figure 3-2 It can be seen that the expression levels of the seven anti-tumor-associated antigens MAGEA autoantibodies in the validation set were significantly higher in the serum samples of the esophageal squamous cell carcinoma group than in the normal control group, and the differences were statistically significant. P <0.05). This indicates that the seven anti-tumor-associated antigen MAGEA autoantibodies can be used as an adjunct to the diagnosis of esophageal squamous cell carcinoma.

[0105] Example 3: Assessment of the diagnostic capabilities of anti-tumor-associated antigen MAGEA family autoantibodies for esophageal squamous cell carcinoma

[0106] 1. Experimental Samples

[0107] In Example 2, serum samples from 230 patients with esophageal squamous cell carcinoma (ESCC) in the training set (referred to as the ESCC group) and 230 normal controls (referred to as the normal control group) were used. In the validation set, serum samples from 193 patients with ESCC and 193 normal controls were used. Based on the expression levels of antitumor-associated antigen (MAA) 1-IgG, MAGEA3-IgG, MAGEA3-IgM, MAGEA4-IgG, MAGEA6-IgG, MAGEA10-IgG, and MAGEA12-IgG antibodies detected by ELISA in the training and validation sets in Example 2, ROC curves were plotted using GraphPad Prism 8.0 to analyze the diagnostic value of these seven MAGEA autoantibodies for esophageal squamous cell carcinoma.

[0108] 2. The ability of a single anti-tumor-associated antigen (MAGEA) autoantibody to diagnose and differentiate esophageal squamous cell carcinoma patients from normal individuals.

[0109] (1) The training set was used to evaluate the ability of autoantibodies against a single tumor-associated antigen to diagnose and differentiate esophageal squamous cell carcinoma from normal controls.

[0110] Based on the expression levels of anti-tumor-associated antigen (MAA) MAGEA1-IgG antibodies (denoted as anti-MAGEA1-IgG antibodies), MAGEA3-IgG antibodies (denoted as anti-MAGEA3-IgG antibodies), MAGEA3-IgM antibodies (denoted as anti-MAGEA3-IgM antibodies), MAGEA4-IgG antibodies (denoted as anti-MAGEA4-IgG antibodies), MAGEA6-IgG antibodies (denoted as anti-MAGEA6-IgG antibodies), MAGEA10-IgG antibodies (denoted as anti-MAGEA10-IgG antibodies), and MAGEA12-IgG antibodies (denoted as anti-MAGEA12-IgG antibodies) in serum samples from 230 MAA patients and 230 normal controls in the training set, ROC curves were plotted for each MAGEA autoantibody. The ROC curves were used to evaluate the ability of each MAGEA autoantibody to independently diagnose and distinguish between MAA patients and normal individuals.

[0111] ROC curves for anti-MAGEA1-IgG antibodies, anti-MAGEA3-IgG antibodies, anti-MAGEA3-IgM antibodies, anti-MAGEA4-IgG antibodies, anti-MAGEA6-IgG antibodies, anti-MAGEA10-IgG antibodies, and anti-MAGEA12-IgG antibodies in differentiating esophageal squamous cell carcinoma patients from normal individuals are shown in the following figures. Figure 4 As shown. Based on the ROC curve, the cutoff value is the absorbance with a specificity greater than 80% and the highest Youden index (Youden index = sensitivity + specificity - 1). At the same time, the corresponding AUC, 95% confidence interval, sensitivity, and specificity are calculated.

[0112] Depend on Figure 4 It was found that when the seven anti-tumor-associated antigen MAGEA autoantibodies were used alone to differentiate esophageal squamous cell carcinoma patients from normal individuals, their ROC curve AUC all reached above 0.55, with a specificity of over 80%. Among them, the anti-MAGEA1-IgG antibody had the highest diagnostic value, with an AUC of 0.684, a sensitivity of 43.5%, and a specificity of 85.7%. This indicates that all seven anti-tumor-associated antigen MAGEA autoantibodies can be used for the auxiliary diagnosis of esophageal squamous cell carcinoma.

[0113] (2) The validation set was used to validate the diagnostic value of a single anti-tumor-associated antigen MAGEA antibody in esophageal squamous cell carcinoma.

[0114] Based on the expression levels of anti-MAGEA1-IgG, anti-MAGEA3-IgG, anti-MAGEA3-IgM, anti-MAGEA4-IgG, anti-MAGEA6-IgG, anti-MAGEA10-IgG, and anti-MAGEA12-IgG antibodies in serum samples from 193 esophageal squamous cell carcinoma patients and 193 normal controls in the validation set, ROC curves for each anti-tumor-associated antigen autoantibody were plotted, as follows: Figure 5 As shown in the figure. The corresponding AUC, 95% confidence interval, sensitivity, and specificity were calculated based on the ROC curve to verify the diagnostic value of each anti-tumor-associated antigen MAGEA autoantibody alone in diagnosing esophageal squamous cell carcinoma.

[0115] Depend on Figure 5 It can be seen that in the validation set, the AUC of the seven anti-tumor-associated antigen MAGEA autoantibodies in distinguishing between esophageal squamous cell carcinoma patients and normal individuals can all reach above 0.55, which is basically consistent with the conclusions of the training set.

[0116] 3. The ability of the anti-tumor-associated antigen MAGEA autoantibody combination to diagnose and differentiate esophageal squamous cell carcinoma patients from normal individuals.

[0117] (1) The training set was used to evaluate the ability of the combination of anti-tumor-associated antigen MAGEA autoantibodies to diagnose esophageal squamous cell carcinoma.

[0118] Using the expression levels of seven anti-tumor-associated antigen MAGEA autoantibodies in serum samples from 230 esophageal squamous cell carcinoma patients and 230 normal controls in the training set as independent variables and whether an event was esophageal squamous cell carcinoma as the dependent variable, logistic regression analysis was performed on the expression levels of the seven anti-tumor-associated antigen MAGEA autoantibodies in serum samples from the esophageal squamous cell carcinoma group and the normal control group to construct a diagnostic model to distinguish between esophageal squamous cell carcinoma patients and normal controls. The diagnostic model is: PRE (P=ESCC)=1 / (1+EXP(-(-2.438+8.255×OD)) MAGEA1-IgG +3.718×OD MAGEA4-IgG +2.119 ×OD MAGEA6-IgG -4.411 ×OD MAGEA12-IgG In this diagnostic model: OD MAGEA1-IgG The expression level of anti-MAGEA1-IgG antibody in the subject's serum (expression level is measured by the absorbance value detected by the ELISA method described in Example 2), OD MAGEA4-IgG This indicates the expression level of anti-MAGEA4-IgG antibody in the subject's serum (expression level is measured by the absorbance value detected by the ELISA method described in Example 2), OD. MAGEA6-IgG This indicates the expression level of anti-MAGEA6-IgG antibody in the subject's serum (expression level is measured by the absorbance value detected by the ELISA method described in Example 2), OD. MAGEA12-IgG This indicates the expression level of anti-MAGEA12-IgG antibody in the subject's serum (expression level is measured by the absorbance value detected by the ELISA method described in Example 2). Substituting the expression levels of anti-MAGEA1-IgG, anti-MAGEA4-IgG, anti-MAGEA6-IgG, and anti-MAGEA12-IgG antibodies in each serum sample into the diagnostic model yields the predicted probability (PRE value) for each serum sample. ROC curves are then plotted based on the predicted probabilities. The ROC curves are shown below. Figure 6 As shown. According to the ROC curve, the optimal cutoff value for diagnosing and distinguishing esophageal squamous cell carcinoma patients from normal individuals is set as the predictive probability value with a specificity greater than 80% and the highest Youden index (Youden index = sensitivity + specificity - 1). (If the PRE value is greater than or equal to the cutoff value, the subject is initially determined to be an esophageal squamous cell carcinoma patient; if the PRE value is less than the cutoff value, the subject is initially determined to be a normal individual.)

[0119] Depend on Figure 6It can be seen that the combined anti-MAGEA1-IgG antibody, anti-MAGEA4-IgG antibody, anti-MAGEA6-IgG antibody and anti-MAGEA12-IgG antibody had an area under the ROC curve (AUC) of 0.725, corresponding to a sensitivity of 55.2% and a specificity of 80.4% in distinguishing between patients with esophageal squamous cell carcinoma and normal individuals.

[0120] (2) The value of the combination of anti-tumor-associated antigen MAGEA autoantibodies in the diagnosis of esophageal squamous cell carcinoma was validated using a validation set.

[0121] The expression levels of anti-MAGEA1-IgG, anti-MAGEA4-IgG, anti-MAGEA6-IgG, and anti-MAGEA12-IgG antibodies in serum samples from 193 patients with esophageal squamous cell carcinoma and 193 normal controls in the validation set were substituted into the diagnostic model constructed in step (1) above to obtain the predicted probability for each serum sample. ROC curves were then plotted based on the predicted probabilities. The ROC curves are shown below. Figure 7 As shown. According to the ROC curve, the optimal cutoff value for diagnosing and distinguishing esophageal squamous cell carcinoma patients from normal individuals is set as the predictive probability value with a specificity greater than 80% and the highest Youden index (Youden index = sensitivity + specificity - 1). (If the PRE value is greater than or equal to the cutoff value, the subject is initially determined to be an esophageal squamous cell carcinoma patient; if the PRE value is less than the cutoff value, the subject is initially determined to be a normal individual.)

[0122] Depend on Figure 7 As can be seen from the validation set, the area under the ROC curve (AUC) of the combination of anti-MAGEA1-IgG antibody, anti-MAGEA4-IgG antibody, anti-MAGEA6-IgG antibody, and anti-MAGEA12-IgG antibody in diagnosing and distinguishing esophageal squamous cell carcinoma patients from normal individuals is 0.698, which is consistent with the conclusions of the training set.

Claims

1. Use of reagents for detecting serum protein markers in the manufacture of a product for the early screening and diagnosis of esophageal squamous cell carcinoma, characterized in that, The serum protein marker is any one of anti-tumor associated antigen MAGEA1 autoantibody, anti-tumor associated antigen MAGEA3 autoantibody, anti-tumor associated antigen MAGEA4 autoantibody, anti-tumor associated antigen MAGEA6 autoantibody, anti-tumor associated antigen MAGEA10 autoantibody and anti-tumor associated antigen MAGEA12 autoantibody or a combination of anti-tumor associated antigen MAGEA1-IgG antibody, anti-tumor associated antigen MAGEA4-IgG antibody, anti-tumor associated antigen MAGEA6-IgG antibody and anti-tumor associated antigen MAGEA12-IgG antibody; The anti-tumor associated antigen MAGEA1 autoantibody is anti-tumor associated antigen MAGEA1-IgG antibody; the anti-tumor associated antigen MAGEA3 autoantibody is one of anti-tumor associated antigen MAGEA3-IgG antibody and anti-tumor associated antigen MAGEA3-IgM antibody; the anti-tumor associated antigen MAGEA4 autoantibody is anti-tumor associated antigen MAGEA4-IgG antibody; The anti-tumor associated antigen MAGEA6 autoantibody is anti-tumor associated antigen MAGEA6-IgG antibody; the anti-tumor associated antigen MAGEA10 autoantibody is anti-tumor associated antigen MAGEA10-IgG antibody; and the anti-tumor associated antigen MAGEA12 autoantibody is anti-tumor associated antigen MAGEA12-IgG antibody.

2. Use according to claim 1, wherein The product for early screening and diagnosis of esophageal squamous cell carcinoma is a kit.

3. Use according to claim 2, wherein the compound is ###0002### The kit comprises reagents for detecting the serum protein marker; the reagents are antigens for detecting the serum protein marker.

4. Use according to claim 3, wherein the compound is ###0002### The antigens are any one of tumor associated antigen MAGEA1 protein, tumor associated antigen MAGEA3 protein, tumor associated antigen MAGEA4 protein, tumor associated antigen MAGEA6 protein, tumor associated antigen MAGEA10 protein and tumor associated antigen MAGEA12 protein; and the antigens are coated on a solid carrier.

5. The use according to claim 4, wherein the compound is ###0002### The solid carrier is made of polyvinyl chloride, polystyrene, polyacrylamide or cellulose; and the kit further comprises any one of positive control serum, negative control serum, blocking solution, sample diluent, secondary antibody, secondary antibody diluent, washing solution, color developing solution or termination solution or a combination of two or more thereof.

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