A marker for early screening of cardiac adenocarcinoma in high-risk population and its application

Abstract

The present invention belongs to the field of biomedicine technology, and discloses a marker for early screening of cardiac adenocarcinoma in high-risk populations, the marker being GAGE12B protein or a combination of GAGE12B protein and AGPAT4 protein or a combination of GAGE12B protein and P2RX4 protein, and by detecting the expression level of the above marker in serum, cardiac adenocarcinoma can be effectively detected. The present invention also discloses a test strip for early screening of cardiac adenocarcinoma in high-risk populations, the test strip binding pad is coated with a capture antibody, the capture antibody is a mixture of GAGE12B antibody A and AGPAT4 antibody A; two detection lines and a quality control line are provided on the chromatography pad, and detection antibodies are fixed on the detection lines, and the detection antibodies fixed on the two detection lines are GAGE12B antibody B and AGPAT4 antibody B respectively; rabbit anti-mouse IgG is fixed on the quality control line. The test strip of the present invention can quickly detect GAGE12B and AGPAT4 in human serum, and can effectively detect cardiac adenocarcinoma according to the detection results, greatly improving the detection rate of early cardiac adenocarcinoma.

Description

[0001] This application is a divisional application of the invention patent with application number CN 2021105350355, application date 2021-05-17, and name "A test strip for early screening of cardiac adenocarcinoma in high-risk populations". Technical Field

[0002] The present invention belongs to the field of biomedicine technology, and specifically relates to a marker for early screening of cardiac adenocarcinoma in a high-risk population and an application thereof. Background Art

[0003] Cardiac adenocarcinoma is one of the most common digestive tract malignancies in northern my country. A notable epidemiological feature of cardiac adenocarcinoma is that it occurs in the same high-incidence area as esophageal cancer. The geographical distribution of cardiac adenocarcinoma is very similar to that of esophageal squamous cell carcinoma. It is highly prevalent in the Taihang Mountains in northern my country, especially in Linzhou (formerly Lin County), Henan Province, a high-incidence area for esophageal cancer. In the past 20 years, the incidence of distal gastric cancer has shown a significant downward trend worldwide, especially in Europe and the United States, while the incidence of cardiac adenocarcinoma has risen sharply. The incidence of esophageal-gastric junction cancer has increased sixfold at a rate of 4% per year, making it one of the fastest growing malignant tumors, and its pathogenesis is still unclear. Cardiac adenocarcinoma is significantly different from distal gastric cancer in terms of epidemiology, etiology, tissue occurrence, and clinical characteristics. Cardiac adenocarcinoma should be listed as a unique clinical disease. Cardiac adenocarcinoma lacks sensitive biomarkers and diagnostic techniques in the early stage, and lacks effective and specific treatment and prevention agents. It is often discovered in the middle and late stages, resulting in poor prognosis and high mortality. It is highly prevalent in northern my country, especially in the Taihang Mountains at the junction of Henan, Hebei and Shanxi, with an incidence rate of up to 190 / 100,000, and is still the leading cause of cancer-related deaths in the region.

[0004] Patients with cardia adenocarcinoma are usually found in the middle or late stage, with a very poor prognosis, and a 5-year survival rate of only about 15%, while the 5-year survival rate of early cardia adenocarcinoma can be increased to more than 95%. The main reason for this phenomenon is that patients with early cardia adenocarcinoma have no obvious specific symptoms, the molecular basis for predicting the risk of cardia adenocarcinoma is unclear, and there is a lack of molecular targets for early screening of asymptomatic high-risk groups. Traditionally, "asymptomatic people in high-incidence areas, over 40 years old, male, smoking, drinking, and with a positive family history" are defined as high-risk groups for cardia adenocarcinoma, and these people are the main targets for early screening of cardia adenocarcinoma. In the past, screening for cardia adenocarcinoma relied on traditional esophageal cytology. At present, due to the rapid promotion and application of chromoendoscopy, biopsy pathology, and endoscopic treatment technology, esophageal cytology has been replaced by chromoendoscopy and mucosal pathology biopsy technology. However, it should be pointed out that endoscopic examination not only requires experienced doctors, but also requires complex equipment and procedures, and will cause discomfort to patients, high cost and low efficiency, which is not easily accepted by the public. These limitations limit the promotion and application of endoscopy in early detection and early detection of cardiac adenocarcinoma in asymptomatic high-risk populations.

[0005] In order to reduce the burden on patients, improve their examination compliance and screening efficiency, it is crucial to find effective specific molecular diagnostic markers for early detection of cardiac adenocarcinoma in high-risk populations. The use of liquid biopsy technology for early detection and early treatment of cardiac adenocarcinoma will provide important technical support for ultimately reducing the incidence and mortality of cardiac adenocarcinoma.

[0006] The occurrence of cardia adenocarcinoma is the result of the interaction of genes, heredity, environment and other factors, and is a multi-stage evolutionary process. During its occurrence and development, tumor cells synthesize and release a group of antigens, namely tumor-associated antigens. This antigen is not unique to the tumor, but in the presence of a tumor, its secretion will greatly increase, and the tumor can be identified or diagnosed based on its biochemical or immune characteristics. Like alpha-fetoprotein, we all know that in patients with liver cancer, the content of alpha-fetoprotein in the blood will increase significantly, and it has been used for the diagnosis of liver cancer. Tumor cells are heterogeneous, and even the same tumor may have different antigen protein expressions. At present, people have not found a single molecular marker for a certain tumor. Therefore, compared with the single tumor-associated antigen detection method, the use of multiple autoantibodies to jointly examine the expression of tumor-associated antigens in patients is conducive to improving the detection rate of a certain tumor. At present, the combined detection method of multiple tumor markers for the early diagnosis of cardia adenocarcinoma and the application of corresponding test strips are relatively rare.

[0007] Based on the multi-omics database established in the early stage using proteomics, genomics, transcriptomics, epigenetics, etc., this research team used protein chip technology to detect and analyze the sera of patients with early gastric cardia adenocarcinoma in high-incidence areas, asymptomatic high-risk people, and normal people, and screened out three tumor serum molecular markers that are differentially expressed in patients with gastric cardia adenocarcinoma and normal people, namely GAGE12B protein, AGPAT4 protein and P2RX4 protein.

[0008] GAGE12B is often expressed in many different types of tumors, while its expression in normal tissues is limited to the germ cells, testes, and ovaries of immune privileged organs. When GAGE12B protein is expressed in tumor cells, it can induce cellular and humoral immune responses and produce GAGE12B antibodies, which can be used as a potential immune target for tumor treatment and an indicator for early screening.

[0009] AGPAT4 is short for 1-acylglycerol-3-phosphate acyltransferase 4, which is involved in lipid metabolism and closely related to tumor progression. It has been reported to be abnormally expressed in bladder cancer and colorectal cancer tissues, and predicts a low survival rate in colorectal cancer patients.

[0010] The P2RX4 purinergic receptor is a member of the purinergic channel family and is an ionotropic ATP receptor subtype that plays an important role in the occurrence and development of tumors. There is evidence that P2X4R is expressed in the rat C6 glioma model and plays an important role in the cell growth and apoptosis of human brain glioblastoma multiforme.

[0011] GAGE12B protein, AGPAT4 protein and P2RX4 protein are lowly expressed in normal human serum and highly expressed in the serum of patients with cardiac adenocarcinoma. Moreover, further statistical tests found that the positive rates of GAGE12B protein, AGPAT4 protein and P2RX4 protein in patients with cardiac adenocarcinoma were significantly higher than those in the normal population, with significant differences. This shows that GAGE12B protein, AGPAT4 protein and P2RX4 protein can be used for the diagnosis of cardiac adenocarcinoma. In response to this finding, this research team used GAGE12B protein, AGPAT4 protein and P2RX4 protein as detection indicators to prepare products for early screening of cardiac adenocarcinoma in high-risk populations, and for early screening of asymptomatic people in areas with high incidence of cardiac adenocarcinoma, in order to improve the early detection rate of cardiac adenocarcinoma, improve the quality of life of patients, and reduce the burden on families and society. Summary of the invention

[0012] In view of the problems and shortcomings in the prior art, one of the objects of the present invention is to provide a marker for screening of cardiac adenocarcinoma in a high-risk population. The second object of the present invention is to provide an application of a detection reagent for a marker for early screening of cardiac adenocarcinoma in a high-risk population. The third object of the present invention is to provide an application of a marker for screening of cardiac adenocarcinoma in a high-risk population in the preparation of a screening product for cardiac adenocarcinoma in a high-risk population. The fourth object of the present invention is to provide a test strip for early screening of cardiac adenocarcinoma in a high-risk population.

[0013] To achieve the purpose of the invention, the technical solution adopted by the present invention is as follows:

[0014] In a first aspect, the present invention provides a marker for screening cardiac adenocarcinoma in a high-risk population, wherein the marker is any one or more combinations of GAGE12B protein, AGPAT4 protein and P2RX4 protein.

[0015] The second aspect of the present invention provides a use of a detection reagent for the marker described in the first aspect in the preparation of a screening product for cardiac adenocarcinoma in a high-risk population.

[0016] According to the above application, preferably, the test sample of the product is serum.

[0017] According to the above application, preferably, the detection reagent is an antibody that specifically binds to the marker.

[0018] According to the above application, preferably, the product detects the marker in the sample by immunochromatography or enzyme-linked immunosorbent assay.

[0019] According to the above application, preferably, the product is a test strip, a test kit or a detection chip.

[0020] The third aspect of the present invention provides an application of the marker described in the first aspect in the preparation of a screening product for cardiac adenocarcinoma in a high-risk population, wherein the test sample of the product is serum. Since the expression abundance of tumor-associated antigens in serum is proportional to the expression abundance of their corresponding autoantibodies, if the tumor-associated antigens are highly expressed, their corresponding autoantibodies will also be highly expressed. Therefore, GAGE12B protein, AGPAT4 protein and P2RX4 protein can also be used as detection reagents to detect the autoantibodies corresponding to GAGE12B protein, AGPAT4 protein and P2RX4 protein in serum, and the autoantibodies to the three tumor-associated antigens are used as diagnostic and screening indicators for cardiac adenocarcinoma.

[0021] According to the above application, preferably, the product detects the expression level of the antibody of the marker in the sample by enzyme-linked immunosorbent assay. More preferably, the product is a kit, which comprises a solid phase carrier coated with the marker described in the first aspect above.

[0022] The fourth aspect of the present invention provides a test strip for early screening of cardiac adenocarcinoma in a high-risk population, the test strip comprising a conjugation pad and a chromatography pad, the conjugation pad being coated with a capture antibody, the capture antibody being a mixture of GAGE12B antibody A, AGPAT4 antibody A, and P2RX4 antibody A, and the GAGE12B antibody A, AGPAT4 antibody A, and P2RX4 antibody A all carry a detectable marker; three detection lines and one quality control line are provided on the chromatography pad, the detection lines are all fixed with detection antibodies, and the detection antibodies fixed on the three detection lines are GAGE12B antibody B, AGPAT4 antibody B, and P2RX4 antibody B, respectively; antibodies A and B of the same antigen recognize different epitopes of the antigen; and rabbit anti-mouse IgG is fixed on the quality control line.

[0023] According to the above-mentioned test strip for early screening of cardiac adenocarcinoma in high-risk populations, preferably, the marker is colloidal gold particles, and GAGE12B antibody A, AGPAT4 antibody A, and P2RX4 antibody A are respectively labeled with colloidal gold particles of different particle sizes.

[0024] According to the above-mentioned test strip for early screening of cardiac adenocarcinoma in high-risk populations, preferably, GAGE12B antibody A, AGPAT4 antibody A, and P2RX4 antibody A are GAGE12B monoclonal antibody, AGPAT4 monoclonal antibody, and P2RX4 monoclonal antibody, respectively; wherein, GAGE12B monoclonal antibody is labeled with colloidal gold particles with a particle size of 24.5 nm, AGPAT4 monoclonal antibody is labeled with colloidal gold particles with a particle size of 41 nm, and P2RX4 monoclonal antibody is labeled with colloidal gold particles with a particle size of 71 nm, and the corresponding colors are orange-red, red, and purple-red, respectively.

[0025] According to the above-mentioned test strip for early screening of cardiac adenocarcinoma in high-risk populations, preferably, GAGE12B antibody B, AGPAT4 antibody B, and P2RX4 antibody B are GAGE12B polyclonal antibody, AGPAT4 polyclonal antibody, and P2RX4 polyclonal antibody, respectively.

[0026] According to the above-mentioned test strip for early screening of cardiac adenocarcinoma in high-risk populations, preferably, the test strip also includes a sample pad, a sample suction pad and a base plate, and the sample pad, conjugation pad, chromatography pad and sample suction pad are fixed on the base plate in sequence; wherein one end of the conjugation pad is pressed under the sample pad, and the other end of the conjugation pad is pressed above the chromatography pad; one end of the chromatography pad is pressed under the conjugation pad, and the other end of the chromatography pad is pressed under the sample suction pad.

[0027] According to the above-mentioned test strip for early screening of cardiac adenocarcinoma in high-risk populations, preferably, the mass ratio of GAGE12B monoclonal antibody, AGPAT4 monoclonal antibody and P2RX4 monoclonal antibody in the capture antibody is 1:1:1.

[0028] According to the above-mentioned test strip for early screening of cardiac adenocarcinoma in high-risk populations, preferably, the three detection lines and one quality control line on the chromatographic pad are arranged in the following order: from one end close to the sample suction pad to one end close to the binding pad, they are the quality control line C coated with rabbit anti-mouse IgG, the detection line T3 fixed with P2RX4 polyclonal antibody, the detection line T2 fixed with AGPAT4 polyclonal antibody, and the detection line T1 fixed with GAGE12B polyclonal antibody.

[0029] According to the above-mentioned test strip for early screening of cardiac adenocarcinoma in high-risk populations, preferably, the intervals between the detection lines T1, T2, T3 and the quality control line C on the chromatographic pad are not less than 5 mm.

[0030] According to the above-mentioned test strip for early screening of cardiac adenocarcinoma in high-risk populations, preferably, the material of the sample pad is absorbent glass fiber, the material of the binding pad is glass fiber membrane, and the material of the chromatography pad is nitrocellulose membrane; the material of the sample absorption pad is absorbent paper or absorbent glass fiber membrane; the bottom plate is a PVC bottom plate, cardboard or hard fiberboard.

[0031] The fifth aspect of the present invention provides another test strip for early screening of cardiac adenocarcinoma in high-risk populations, the test strip comprising a conjugation pad and a chromatography pad, the conjugation pad being coated with a capture antibody, the capture antibody being a mixture of GAGE12B antibody A and AGPAT4 antibody A, both of which carry a detectable marker; two detection lines and a quality control line being arranged on the chromatography pad, the detection lines being fixed with detection antibodies, the detection antibodies fixed on the two detection lines being GAGE12B antibody B and AGPAT4 antibody B respectively; antibodies A and B of the same antigen recognize different epitopes of the antigen; and rabbit anti-mouse IgG being fixed on the quality control line.

[0032] According to the above-mentioned test strip for early screening of cardiac adenocarcinoma in high-risk populations, preferably, the marker is colloidal gold particles, and GAGE12B antibody A and AGPAT4 antibody A are respectively labeled with colloidal gold particles of different particle sizes.

[0033] According to the above-mentioned test strip for early screening of cardiac adenocarcinoma in high-risk populations, preferably, GAGE12B antibody A and AGPAT4 antibody A are GAGE12B monoclonal antibody and AGPAT4 monoclonal antibody, respectively; wherein, GAGE12B monoclonal antibody is labeled with colloidal gold particles with a particle size of 24.5 nm, and AGPAT4 monoclonal antibody is labeled with colloidal gold particles with a particle size of 41 nm, and the corresponding colors are orange-red and red, respectively.

[0034] According to the above-mentioned test strip for early screening of cardiac adenocarcinoma in high-risk populations, preferably, GAGE12B antibody B and AGPAT4 antibody B are GAGE12B polyclonal antibody, AGPAT4 polyclonal antibody and P2RX4 polyclonal antibody, respectively.

[0035] According to the above-mentioned test strip for early screening of cardiac adenocarcinoma in high-risk populations, preferably, the test strip also includes a sample pad, a sample suction pad and a base plate, and the sample pad, conjugation pad, chromatography pad and sample suction pad are fixed on the base plate in sequence; wherein one end of the conjugation pad is pressed under the sample pad, and the other end of the conjugation pad is pressed above the chromatography pad; one end of the chromatography pad is pressed under the conjugation pad, and the other end of the chromatography pad is pressed under the sample suction pad.

[0036] According to the above-mentioned test strip for early screening of cardiac adenocarcinoma in high-risk populations, preferably, the mass ratio of the GAGE12B monoclonal antibody to the AGPAT4 monoclonal antibody in the capture antibody is 1:1.

[0037] According to the above-mentioned test strip for early screening of cardiac adenocarcinoma in high-risk populations, preferably, the two detection lines and one quality control line on the chromatographic pad are arranged in the following order: from one end close to the sample suction pad to one end close to the binding pad, they are the quality control line C coated with rabbit anti-mouse IgG, the detection line T2 fixed with AGPAT4 polyclonal antibody, and the detection line T1 fixed with GAGE12B polyclonal antibody.

[0038] According to the above-mentioned test strip for early screening of cardiac adenocarcinoma in high-risk populations, preferably, the intervals between the detection lines T1, T2 and the quality control line C on the chromatographic pad are not less than 5 mm.

[0039] According to the above-mentioned test strip for early screening of cardiac adenocarcinoma in high-risk populations, preferably, the material of the sample pad is absorbent glass fiber, the material of the binding pad is glass fiber membrane, and the material of the chromatography pad is nitrocellulose membrane; the material of the sample absorption pad is absorbent paper or absorbent glass fiber membrane; the bottom plate is a PVC bottom plate, cardboard or hard fiberboard.

[0040] The fifth aspect of the present invention provides a kit comprising the test strip for early screening of cardiac adenocarcinoma in high-risk populations as described in the third aspect or the fourth aspect.

[0041] According to the above-mentioned kit, preferably, the kit further comprises a pipette, a serum sample adding cup, a disposable syringe and the like.

[0042] The method of using the test strip for early screening of cardiac adenocarcinoma in high-risk populations of the present invention is as follows:

[0043] Drop the serum sample to be tested on the sample pad of the test strip or insert the sample pad of the test strip into the serum sample to be tested, and observe the color changes of the test line and the quality control line within 5 to 15 minutes after taking out the test strip and record the results. The serum sample can be diluted with a sample diluent before testing.

[0044] The method for determining the result of the test strip used for early screening of cardiac adenocarcinoma in high-risk populations of the present invention is as follows:

[0045] Positive result: At least one of the three or two detection lines on the chromatographic pad of the test strip is colored, and the quality control line C is colored, and the test result is determined to be positive.

[0046] Negative result: None of the test lines on the chromatographic pad of the test strip show color, only the quality control line C shows color, and the test result is determined to be negative.

[0047] Invalid result: The quality control line C did not show color, and the test result was judged to be invalid and needs to be retested.

[0048] The detection principle of the test strip for early screening of cardiac adenocarcinoma in high-risk populations of the present invention is:

[0049] The test strip of the present invention uses the cardiac adenocarcinoma markers GAGE12B protein, AGPAT4 protein and P2RX4 protein as detected objects, and utilizes the antigen-antibody reaction principle and colloidal gold immunochromatography technology to detect the GAGE12B protein (also called GAGE12B antigen), AGPAT4 protein (also called AGPAT4 antigen) and P2RX4 protein (also called P2RX4 antigen) present in human blood. When the serum sample to be tested is dropped onto the sample pad of the test strip, the serum sample to be tested will flow toward the sample pad under the capillary action of the water-absorbent material of the sample pad, and when it flows onto the conjugation pad, the colloidal gold-labeled GAGE12B monoclonal antibody, the colloidal gold-labeled AGPAT4 monoclonal antibody, and the colloidal gold-labeled P2RX4 monoclonal antibody coated on the conjugation pad will dissolve, wherein the colloidal gold-labeled GAGE12B monoclonal antibody will bind to the GAGE12B antigen that may be contained in the serum sample to form a colloidal gold-labeled GAGE12B monoclonal antibody-GAGE12B antigen complex, the colloidal gold-labeled AGPAT4 monoclonal antibody will bind to the AGPAT4 antigen that may be contained in the serum sample to form a colloidal gold-labeled AGPAT4 monoclonal antibody-AGPAT4 antigen complex, and the colloidal gold-labeled P2RX4 monoclonal antibody will bind to the P2RX4 antigen that may be contained in the serum sample to form a colloidal gold-labeled P2RX4 monoclonal antibody-P2RX4 antigen complex; due to the capillary effect, the three The capture antibody-antigen complex labeled with colloidal gold continues to move toward the chromatography pad. When it moves to the detection line T1, the colloidal gold-labeled GAGE12B monoclonal antibody-GAGE12B antigen complex specifically binds to the GAGE12B polyclonal antibody to form a solidified immune complex, which is retained on the detection line T1; when it moves to the detection line T2, the colloidal gold-labeled AGPAT4 monoclonal antibody-AGPAT4 antigen complex specifically binds to the AGPAT4 polyclonal antibody to form a solidified immune complex, which is retained on the detection line T2; when it moves to the detection line T3, the colloidal gold-labeled P2RX4 monoclonal antibody-P2RX4 antigen complex specifically binds to the P2RX4 polyclonal antibody to form a solidified immune complex, which is retained on the detection line T3; the free colloidal gold-labeled capture antibody continues to move forward due to the capillary effect, binds to the rabbit anti-mouse IgG coated on the quality control line C, and is retained on the quality control line; the excess unbound substances continue to move to the sample suction pad under the capillary action. Therefore, the color development of the detection lines T1, T2, and T3 as well as the color development of the quality control line can be used to qualitatively determine the GAGE12B, AGPAT4, and P2RX4 tumor-associated antigens in the sample to be tested.

[0050] Compared with the prior art, the present invention has the following positive and beneficial effects:

[0051] (1) The present invention uses the three proteins, GAGE12B protein, AGPAT4 protein and P2RX4 protein, for the early screening and detection of cardiac adenocarcinoma for the first time. By detecting the expression levels of GAGE12B protein, AGPAT4 protein and P2RX4 protein in human serum, cardiac adenocarcinoma can be effectively detected, and in particular, the detection rate of early cardiac adenocarcinoma can be improved. Moreover, when GAGE12B protein, AGPAT4 protein and P2RX4 protein are used as a combination for the early screening and detection of cardiac adenocarcinoma in high-risk populations, the detection sensitivity is as high as 87% (i.e., 10.3% of patients with early cardiac adenocarcinoma are correctly diagnosed as having early cardiac adenocarcinoma when these three proteins are used for diagnosis). The detection rate of the markers is 87%, and the specificity is 77% (i.e., the rate of patients without cardiac adenocarcinoma who are diagnosed with the three proteins is 77%). Therefore, the markers of the present invention have high sensitivity and specificity, greatly improving the detection rate of early cardiac adenocarcinoma. Moreover, the detection rate of cardiac adenocarcinoma is much higher than the detection rate of cardiac adenocarcinoma by existing clinical endoscopic screening (2%-3%), and can be used for large-scale screening of asymptomatic high-risk populations in areas with high incidence of cardiac adenocarcinoma, which is conducive to the early detection of cardiac adenocarcinoma in asymptomatic high-risk populations, thereby greatly reducing the mortality rate of patients with cardiac adenocarcinoma, improving the survival rate of patients with cardiac adenocarcinoma and reducing the burden on families and society.

[0052] (2) The test strip of the present invention can quickly detect GAGE12B protein, AGPAT4 protein, and P2RX4 protein in human serum, realizing the combined detection of three tumor markers. Moreover, the test strip has high sensitivity and specificity, and has high detection accuracy for early cardiac adenocarcinoma, greatly improving the detection rate of early cardiac adenocarcinoma, which is beneficial for the early detection of asymptomatic people at high risk of cardiac adenocarcinoma. At the same time, it also provides an important detection means for the long-term tracking of asymptomatic people in areas with a high incidence of cardiac adenocarcinoma, and has broad market prospects and social benefits.

[0053] (3) The test strip for early screening of cardiac adenocarcinoma in high-risk populations of the present invention is simple to operate, easy to use, and has a short detection result time. It only needs to insert its test end into the sample liquid to be tested for about 10 seconds, and then the test result can be determined within 15 minutes, which greatly improves the diagnostic efficiency of early cardiac adenocarcinoma.

[0054] (4) The three capture antibodies coated on the conjugate pad of the present invention are respectively labeled with colloidal gold particles of different particle sizes, so that each tumor marker on the chromatographic pad displays a different color, and the result judgment is more intuitive.

[0055] (5) The test strip for early screening of cardiac adenocarcinoma in high-risk groups of the present invention does not require other instruments and reagents, and non-professionals can also perform the test at any time, which can greatly reduce the test cost and test fee, and the human operation error is small and the stability is good.

[0056] (6) The test sample of the test strip for early screening of cardiac adenocarcinoma in high-risk populations of the present invention is serum, which requires a small amount of blood, causes little pain to the person being tested, and has high compliance. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 This is a schematic diagram of the structure of a test strip for early screening of cardiac adenocarcinoma in high-risk populations prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0058] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings. It will be understood by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.

[0059] Tumor markers are abnormally expressed bioactive substances produced by tumor tissues and cells. Tumors can be identified or diagnosed based on their biochemical and post-immunological characteristics. Based on the genomic database established in the early stage using whole genome association analysis, whole genome sequencing, and whole genome exome sequencing, this research team used protein chip technology to detect and analyze the serum of patients with early gastric cardia adenocarcinoma, asymptomatic high-risk people in high-incidence areas, and normal people, and screened out three tumor serum markers that are differentially expressed in patients with gastric cardia adenocarcinoma and normal people: GAGE12B protein, AGPAT4 protein, and P2RX4 protein. GAGE12B protein, AGPAT4 protein, and P2RX4 protein are lowly expressed in normal human serum and highly expressed in the serum of patients with gastric cardia adenocarcinoma. Moreover, further statistical tests found that the positive rates of GAGE12B protein, AGPAT4 protein, and P2RX4 protein in patients with gastric cardia adenocarcinoma were significantly higher than those in the normal population, with significant differences. This shows that GAGE12B protein, AGPAT4 protein, and P2RX4 protein can be used for the diagnosis of cardiac adenocarcinoma. In response to this discovery, the research team used GAGE12B protein, AGPAT4 protein, and P2RX4 protein as detection indicators to prepare test strips for early detection of cardiac adenocarcinoma in high-risk populations, which are used to detect the expression levels of GAGE12B protein, AGPAT4 protein, and P2RX4 protein in samples, and achieve early screening of cardiac adenocarcinoma in high-risk populations.

[0060] The experimental methods described in the following examples, unless otherwise specified, are all conventional techniques in the art, or are based on the conditions recommended by the manufacturers; the reagents, materials and instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0061] Example 1: Preparation of a test strip for early screening of cardiac adenocarcinoma in high-risk populations

[0062] 1. Experimental Materials

[0063] The biologically active raw materials used in the present invention are all commercially available products. Among them, GAGE12B monoclonal antibody was purchased from Shanghai Aibixin Biotechnology Co., Ltd. with the item number abs101001; GAGE12B polyclonal antibody was purchased from Shanghai Qunji Biotechnology Co., Ltd. with the item number AP11254a; AGPAT4 monoclonal antibody was purchased from Shanghai Kemin Biotechnology Co., Ltd. with the item number bs-5033R; AGPAT4 polyclonal antibody was purchased from Shanghai Qunji Biotechnology Co., Ltd. with the item number H00056895-B01; P2RX4 monoclonal antibody was purchased from Shanghai Kemin Biotechnology Co., Ltd. with the item number bs-7690R; P2RX4 polyclonal antibody was purchased from Wuhan Yipu Biotechnology Co., Ltd. with the item number ATA34839.

[0064] The PVC base plate, nitrocellulose membrane, glass fiber membrane, absorbent paper, etc. are all existing commercially available products.

[0065] 2. Prepare the conjugate pad

[0066] (1) Preparation of colloidal gold:

[0067] The colloidal gold solution was prepared by the trisodium citrate reduction method, which is a conventional method in the art and will not be described in detail here.

[0068] (2) Preparation of colloidal gold labeled GAGE12B monoclonal antibody, colloidal gold labeled AGPAT4 monoclonal antibody, and colloidal gold labeled P2RX4 monoclonal antibody:

[0069] GAGE12B monoclonal antibody solution, AGPAT4 monoclonal antibody solution, and P2RX4 monoclonal antibody solution were dialyzed for desalting (4°C overnight) to adjust the protein concentration to 1 mg / ml; under electromagnetic stirring, P2RX4 monoclonal antibody was labeled with colloidal gold with a particle size of 24.5 nm, AGPAT4 monoclonal antibody was labeled with colloidal gold with a particle size of 41 nm, and GAGE12B monoclonal antibody was labeled with colloidal gold with a particle size of 71 nm to obtain three colloidal gold-labeled monoclonal antibodies. Then the three colloidal gold-labeled monoclonal antibodies were mixed at a mass ratio of 1:1:1 to obtain a capture antibody solution.

[0070] (3) Preparation of conjugate pad:

[0071] A glass fiber membrane is selected as the binding pad material, and the glass fiber membrane is immersed in a pretreatment solution (pretreated with a borate buffer solution containing 2% BSA, 3% sucrose, 0.6M NaCl, and 0.2% Tween-20) for 10 to 15 minutes, and then dried at 36 to 38°C or vacuum freeze-dried; the pretreated glass fiber membrane is immersed in the above-mentioned capture antibody solution, taken out after sufficient soaking, and freeze-dried to obtain a binding pad.

[0072] 3. Preparation of chromatography pad

[0073] A nitrocellulose membrane is selected as a chromatographic pad material, and the positions of three detection lines T1, T2, T3 and a quality control line C are marked on the nitrocellulose membrane, and the intervals between each other are 6 mm. GAGE12B polyclonal antibody, AGPAT4 polyclonal antibody, and P2RX4 polyclonal antibody are all diluted to 1 mg / mL, and rabbit anti-mouse IgG is diluted to 1.5 mg / mL. The three detection lines T1, T2, T3 and the quality control line C on the nitrocellulose membrane are respectively marked with a film marker at a dosage of 0.1-0.5 μL / mm, and dried at 37°C overnight; then the nitrocellulose membrane is immersed in a 0.01 mol / L PBS buffer (PH8.0) containing 1% BSA, and then the nitrocellulose membrane is taken out and washed with PBS buffer, and the chromatographic pad is obtained after drying.

[0074] 4. Sample pad preparation

[0075] The material of the sample pad is glass fiber membrane. The glass fiber membrane is immersed in the sample pad blocking solution for 30 minutes and dried at 37°C to obtain the sample pad. The sample pad blocking solution is 0.01 mol / L PBS buffer (pH = 7.4) containing 1% BSA, 1.0% to 2.0% sucrose, 0.1%-0.5% Tween-20, and 0.1-1.0% PVP polyvinyl pyrrolidone.

[0076] 5. Test strip assembly

[0077] The test strip consists of a sample pad, a binding pad, a chromatography pad, a sample suction pad and a bottom plate, and the material of the sample suction pad is absorbent filter paper.

[0078] The chromatography pad is attached to the middle of the bottom plate, which is the detection area and the quality control area, that is, the detection lines T1, T2, T3 and the quality control line C for judging the results are set on the chromatography pad; the right end (upper section) of the chromatography pad is the hand-held part, and a sample absorption pad (water-absorbing filter paper) is fixed to absorb the excess liquid in the test sample; the left end of the chromatography pad is fixed with a binding pad, and the binding pad is coated with colloidal gold-labeled GAGE12B monoclonal antibody, AGPAT4 monoclonal antibody and P2RX4 monoclonal antibody; the binding pad and the sample absorption pad overlap with the two ends of the chromatography pad respectively; the end of the binding pad away from the chromatography pad (lower section) is fixed with a sample pad for contacting the sample to be tested. After the assembly is completed, it is cut into a test strip with a width of 4 mm. The test strip is dried to obtain a test strip for early cardiac adenocarcinoma screening in high-risk populations (see Figure 1 ).

[0079] 6. How to use the test strips

[0080] Add the serum sample to be tested onto the sample pad of the test strip, lay the test strip flat, observe the color changes of the test line and quality control line within 5 to 15 minutes and record the results.

[0081] 7. Determination of test results of test strips

[0082] Positive result: At least one of the detection lines T1, T2, and T3 on the test strip is colored, and the quality control line C is colored, and the test result is determined to be positive.

[0083] Negative result: The T1, T2, and T3 test lines on the test strip chromatography pad are not colored, only the quality control line C is colored, and the test result is determined to be negative.

[0084] Invalid result: The quality control line C did not show color, and the test result was judged to be invalid and needs to be retested.

[0085] 8. Precautions for testing with test strips

[0086] (1) The samples to be tested are tested at room temperature (around 20°C).

[0087] (2) The samples to be tested must be fresh and tested within 1 hour of sample collection to ensure the reliability of the results.

[0088] (3) This test strip is only for in vitro screening and cannot be used as a confirmation reagent. Positive results must be further confirmed by upper gastrointestinal endoscopy.

[0089] (4) Observation results after 30 minutes are invalid.

[0090] (5) The test strips should be stored away from light.

[0091] Example 2: Analysis of the diagnostic value of test strips

[0092] The test strip prepared in Example 1 of the present invention was used to detect serum samples of patients with early cardiac adenocarcinoma confirmed by pathology and normal subjects, and the value of the test strip of the present invention for screening and diagnosing early cardiac adenocarcinoma was evaluated and analyzed.

[0093] At the same time, in order to compare the detection performance of the test strip prepared in Example 1 of the present invention, the present invention prepared 6 comparison test strips, and used the 6 comparison test strips to test serum samples of patients with early cardiac adenocarcinoma diagnosed by pathology and normal subjects respectively.

[0094] The preparation methods of the six comparison test strips are basically the same as the preparation method of the test strips disclosed in Example 1 of the present invention, except that the capture antibodies coated on the binding pads of the six comparison test strips are different, and the number of detection lines on the chromatographic pads and the detection antibodies fixed on the detection lines are different.

[0095] Among them, the first comparison test strip: the detection index of the first comparison test strip is P2RX4 protein, and the capture antibody coated on the binding pad of the test strip is a P2RX4 monoclonal antibody labeled with colloidal gold with a particle size of 24.5 nm; the chromatography pad of the test strip is provided with a detection line and a quality control line, the detection line is fixed with P2RX4 polyclonal antibody, and the quality control line is fixed with rabbit anti-mouse IgG.

[0096] The second comparative test strip: The detection index of the second comparative test strip is AGPAT4 protein, and the capture antibody coated on the binding pad of the test strip is AGPAT4 monoclonal antibody labeled with colloidal gold with a particle size of 41 nm; a detection line and a quality control line are arranged on the chromatography pad of the test strip, the detection line is fixed with AGPAT4 polyclonal antibody, and the quality control line is fixed with rabbit anti-mouse IgG.

[0097] The third comparison test strip: The detection index of the third comparison test strip is GAGE12B protein, and the capture antibody coated on the binding pad of the test strip is GAGE12B monoclonal antibody labeled with colloidal gold with a particle size of 71nm; the chromatography pad of the test strip is provided with 1 detection line and 1 quality control line, the detection line is fixed with GAGE12B polyclonal antibody, and the quality control line is fixed with rabbit anti-mouse IgG.

[0098] The fourth comparison test strip: The detection indicators of the fourth comparison test strip are P2RX4 protein and AGPAT4 protein. The capture antibody coated on the binding pad of the test strip is a mixture of P2RX4 monoclonal antibody and AGPAT4 monoclonal antibody. The P2RX4 monoclonal antibody is labeled with colloidal gold with a particle size of 24.5 nm, and the AGPAT4 monoclonal antibody is labeled with colloidal gold with a particle size of 41 nm. The mass ratio of the P2RX4 monoclonal antibody to the AGPAT4 monoclonal antibody in the capture antibody is 1:1. Two detection lines and one quality control line are provided on the chromatography pad of the test strip, the first detection line is fixed with P2RX4 polyclonal antibody, the second detection line is fixed with AGPAT4 polyclonal antibody, and the quality control line is fixed with rabbit anti-mouse IgG.

[0099] The fifth comparison test strip: The detection indicators of the fifth comparison test strip are AGPAT4 protein and GAGE12B protein. The capture antibody coated on the binding pad of the test strip is a mixture of AGPAT4 monoclonal antibody and GAGE12B monoclonal antibody. The AGPAT4 monoclonal antibody is labeled with colloidal gold with a particle size of 41 nm, and the GAGE12B monoclonal antibody is labeled with colloidal gold with a particle size of 71 nm. The mass ratio of the AGPAT4 monoclonal antibody to the GAGE12B monoclonal antibody in the capture antibody is 1:1. Two detection lines and one quality control line are provided on the chromatography pad of the test strip, the first detection line is fixed with AGPAT4 polyclonal antibody, the second detection line is fixed with GAGE12B polyclonal antibody, and the quality control line is fixed with rabbit anti-mouse IgG.

[0100] The 6th comparison test strip: The detection indicators of the 6th comparison test strip are P2RX4 protein and GAGE12B protein. The capture antibody coated on the binding pad of the test strip is a mixture of P2RX4 monoclonal antibody and GAGE12B monoclonal antibody. The P2RX4 monoclonal antibody is labeled with colloidal gold with a particle size of 24.5nm, and the GAGE12B monoclonal antibody is labeled with colloidal gold with a particle size of 71nm. The mass ratio of P2RX4 monoclonal antibody to GAGE12B monoclonal antibody in the capture antibody is 1:1; 2 detection lines and 1 quality control line are provided on the chromatographic pad of the test strip, the first detection line is fixed with P2RX4 polyclonal antibody, the second detection line is fixed with GAGE12B polyclonal antibody, and the quality control line is fixed with rabbit anti-mouse IgG.

[0101] 1. Sample source

[0102] A total of 200 serum samples were collected from the National Key Laboratory for the Prevention and Treatment of Esophageal Cancer, the First Affiliated Hospital of Zhengzhou University, including 100 normal human sera (control group) and 100 sera from patients with early cardiac adenocarcinoma (cardiac adenocarcinoma group). The 100 normal human sera were from the healthy physical examination population of the physical examination center of the laboratory's cooperative hospital, without any tumor or immune-related diseases. Among the 100 normal people, there were 64 males and 36 females, with an average age of 57.36±7.59 years old, ranging from 40 to 75 years old. The 100 sera from patients with early cardiac adenocarcinoma were from patients with early (stage 0+stage I) cardiac adenocarcinoma confirmed by histopathology, who did not receive radiotherapy or chemotherapy. Among the 100 patients with cardiac adenocarcinoma, there were 63 males and 37 females, with an average age of 57.48±8.29 years old, ranging from 40 to 75 years old.

[0103] 2. Experimental methods

[0104] The test strip prepared by the present invention is used to detect serum samples of a cardiac adenocarcinoma group and a control group, respectively. The specific detection method is: drop the serum sample to be tested on the sample pad of the test strip, lay the test strip flat, observe the color changes of the test line and the quality control line within 5 to 15 minutes, and record the results.

[0105] According to the result judgment criteria, the positive rates of the three antigens in the cardiac adenocarcinoma group and the control group were calculated respectively (the number of positive objects detected in each group divided by the total number of objects detected in the group was the positive rate); SPSS26.0 software was used for statistical testing, and the two independent sample chi-square test method was used to compare the antigen positive rates in the cardiac adenocarcinoma group and the control group. The test level α = 0.05, when p < 0.05, the result was statistically significant, and then the screening test method was used to evaluate the diagnostic value of autoantibody detection of cardiac adenocarcinoma (Table 1).

[0106] 3. Results Analysis

[0107] The screening and diagnostic value of the test strip for cardiac adenocarcinoma was evaluated based on the test results. The results are shown in Table 1.

[0108] Table 1 Evaluation of the authenticity of different antigen combinations in the diagnosis of cardiac adenocarcinoma

[0109]

[0110] As shown in Table 1, the positive rates of P2RX4 protein, AGPAT4 protein, and GAGE12B protein in the cardiac adenocarcinoma group were 51%, 44%, and 48%, respectively, and the positive rates of P2RX4 protein, AGPAT4 protein, and GAGE12B protein in the control group were 12%, 9%, and 8%, respectively. The positive rates of P2RX4 protein, AGPAT4 protein, and GAGE12B protein in the cardiac adenocarcinoma group were higher than those in the control group, and the difference between the cardiac adenocarcinoma group and the control group was statistically significant (P < 0.05). It can be seen that P2RX4 protein, AGPAT4 protein, and GAGE12B protein can be used as diagnostic indicators for early cardiac adenocarcinoma and have important diagnostic value for the detection of early cardiac adenocarcinoma.

[0111] Moreover, it can be seen from Table 1 that, as the test strip detects the increase of the index (antigen), in the early stage cardiac adenocarcinoma patient serum, the positive rate (i.e. sensitivity) of the antigen gradually increases, while the specificity gradually decreases; when the test strip jointly detects the three antigens of P2RX4 protein, AGPAT4 protein, and GAGE12B protein, the detection sensitivity of early cardiac adenocarcinoma has reached 87%, that is to say, the percentage of cardiac adenocarcinoma that can be correctly diagnosed by the detection method in the cardiac adenocarcinoma patient is 87%. As the test strip detects the increase of the index, the specificity of the detection is reduced, but when jointly detecting the three antigens of P2RX4 protein, AGPAT4 protein, and GAGE12B protein, the detection specificity can still reach 77%, that is to say, when the non-cardia adenocarcinoma patient adopts this method to detect, the percentage of correctly diagnosing as a healthy person is 77%.

[0112] Moreover, compared with the test strip that detects only one tumor-associated antigen, the sensitivity of the test strip that jointly detects three antigens, P2RX4 protein, AGPAT4 protein, and GAGE12B protein, for the diagnosis of early cardiac adenocarcinoma is 1.71 times, 1.98 times, and 1.81 times that of detecting a single indicator, P2RX4 protein, AGPAT4 protein, and GAGE12B protein, respectively.

[0113] Therefore, the combined detection of P2RX4 protein, AGPAT4 protein, and GAGE12B protein in serum samples for screening and diagnosis of early cardiac adenocarcinoma can greatly improve the diagnostic sensitivity while ensuring the diagnostic specificity; it has good diagnostic and application value for the risk assessment of cardiac adenocarcinoma in the subjects to be tested.

[0114] In addition, the Youden Index refers to the sum of sensitivity and specificity minus 1 in statistics, and its range is 0 to 1. The closer the Youden Index is to 1, the higher its diagnostic value. In the present invention, as the antigen index detected by the test strip increases, the Youden Index continues to increase and gradually tends to 1, indicating that the combination of these three antigens has a good diagnostic value for diagnosing and screening early cardiac adenocarcinoma.

[0115] In summary, the combined detection of P2RX4 protein, AGPAT4 protein, and GAGE12B protein in serum samples can ensure high specificity and sensitivity for the screening and diagnosis of early cardiac adenocarcinoma, and has good diagnostic and application value for the risk assessment of cardiac adenocarcinoma in the subjects to be tested.

[0116] The above description is only a preferred embodiment of the present invention, but is not limited to the above examples. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of a marker detection reagent in the preparation of a product for assisting in screening for cardiac adenocarcinoma in high-risk populations, wherein the detection sample of the product is serum, and the marker is GAGE12B protein or a combination of GAGE12B protein and AGPAT4 protein or a combination of GAGE12B protein and P2RX4 protein.

2. The use according to claim 1, characterized in that: The detection reagent is an antibody that specifically binds to the marker.

3. The use according to claim 1, characterized in that: The product detects the marker in the sample by immunochromatography or enzyme-linked immunosorbent assay.

4. The use according to any one of claims 1 to 3, characterized in that: The product is a test strip, a test kit or a detection chip.

5. A test strip for assisting in the early screening of cardiac adenocarcinoma in high-risk populations, characterized in that: The test strip comprises a conjugation pad and a chromatography pad, wherein the conjugation pad is coated with a capture antibody, wherein the capture antibody is a mixture of GAGE12B antibody A and AGPAT4 antibody A, and both GAGE12B antibody A and AGPAT4 antibody A carry detectable markers; two detection lines and a quality control line are arranged on the chromatography pad, wherein the detection lines are fixed with detection antibodies, and the detection antibodies fixed on the two detection lines are GAGE12B antibody B and AGPAT4 antibody B respectively; antibodies A and B of the same antigen recognize different epitopes of the antigen; and rabbit anti-mouse IgG is fixed on the quality control line.

6. A kit comprising the test strip for assisting early screening of cardiac adenocarcinoma in high-risk populations as described in claim 5.

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