CN113267626A

CN113267626A - Test strip for early screening of cardia adenocarcinoma of high risk group

Info

Publication number: CN113267626A
Application number: CN202110535035.5A
Authority: CN
Inventors: 王立东; 徐瑞华; 李志强; 韩文莉; 胡传松; 罗宏; 郭贵周; 李承宽; 李爱丽; 魏梦霞; 钟侃; 胡景峰; 杨苗苗; 王盼盼; 宋昕; 赵学科; 雷玲玲
Original assignee: Guangshan People's Hospital; Huixian Third People's Hospital; Linzhou Cancer Hospital; First Affiliated Hospital of Zhengzhou University
Current assignee: Guangshan People's Hospital; Huixian Third People's Hospital; Linzhou Cancer Hospital; First Affiliated Hospital of Zhengzhou University
Priority date: 2021-05-17
Filing date: 2021-05-17
Publication date: 2021-08-17
Anticipated expiration: 2041-05-17
Also published as: CN116087519A; CN113267626B

Abstract

The invention belongs to the technical field of medical biology, and discloses a test strip for early screening of cardia adenocarcinoma of high risk groups, which comprises a bonding pad and a chromatographic pad, wherein the bonding pad is coated with a capture antibody, the capture antibody is a mixture of a GAGE12B antibody A, AGPAT4 antibody A, P2RX4 antibody A, and both the GAGE12B antibody A, AGPAT4 antibody A, P2RX4 antibody A are provided with detectable markers; the chromatography pad is provided with three detection lines and a quality control line, and detection antibodies fixed by the three detection lines are GAGE12B antibody B, AGPAT4 antibody B, P2RX4 antibody B respectively; rabbit anti-mouse IgG is fixed on the quality control line. The test strip can rapidly detect GAGE12B, AGPAT4 and P2RX4 in human serum, realizes the joint detection of three tumor markers, can effectively detect the cardiac adenocarcinoma according to the detection result, and greatly improves the detection rate of the early cardiac adenocarcinoma.

Description

Test strip for early screening of cardia adenocarcinoma of high risk group

Technical Field

The invention belongs to the technical field of medical biology, and particularly relates to a test strip for early screening of cardia adenocarcinoma of high risk groups.

Background

Cardia adenocarcinoma is one of the most common digestive tract malignant tumors in northern areas of China. A significant epidemiological feature of cardiac adenocarcinoma is the co-occurrence of esophageal cancer in the same high incidence area. The cardia adenocarcinoma is very similar to the esophageal squamous cell carcinoma in geographical distribution, and is highly developed in Taihang mountainous areas in the north of China, in particular to Linzhou province (original Lin county) in Henan province of high-developed esophageal carcinoma areas. In recent 20 years, the incidence rate of distal gastric cancer is in a significant decline trend worldwide, particularly in the European and American areas, the incidence rate of cardia adenocarcinoma is sharply increased, the incidence rate of esophageal-gastric boundary cancer is increased by 6 times at a rate of 4% per year, the esophageal-gastric boundary cancer is one of the most rapidly-growing malignant tumors, and the pathogenesis of the esophageal-gastric boundary cancer is not clear. The cardia adenocarcinoma is obviously different from the distal gastric cancer in epidemiology, etiology, histogenesis, clinical characteristics and the like, and should be classified as a unique clinical disease. The early stage cardiac adenocarcinoma lacks sensitive biomarkers and diagnosis technology, lacks effective and specific therapeutic and preventive reagents, is mostly found in the middle and late stages, and results in poor prognosis and high fatality rate. The incidence of diseases of Taihang mountain areas in northern China, particularly in junctions of Henan, Hebei and Shanxi, is 190/10 ten thousand, and is still the main cause of tumor-related death in the areas.

The patients with cardiac adenocarcinoma are mostly found in the middle and late stages, the prognosis is very poor, the 5-year survival rate is only about 15%, and the 5-year survival rate of early cardiac adenocarcinoma can be improved to more than 95%. The main reasons for this phenomenon are that patients with early-stage cardia adenocarcinoma have no obvious specific symptoms, the molecular basis for predicting the onset risk of cardia adenocarcinoma is unclear, and the molecular targets screened at early stage by asymptomatic high-risk groups are lacked. Traditionally, "asymptomatic population with high incidence, over 40 years old, male, smoking, drinking, and positive family history" is defined as population at high risk for cardiac adenocarcinoma, which is the main target for early screening of cardiac adenocarcinoma. The traditional screening of cardia adenocarcinoma relies on the traditional examination of esophageal hauling net cast-off cytology, and at present, the examination of esophageal hauling net cast-off cytology is replaced by the examination of pigment endoscopy and mucosa pathological biopsy due to the rapid popularization and application of pigment endoscopy, biopsy pathology and endoscopic treatment technology. However, it is particularly pointed out that endoscopy requires not only experienced physicians, but also complicated equipment and procedures, and is uncomfortable to patients, costly and inefficient, and not readily acceptable to the general public. These limitations limit the popularization and application of endoscopes in early warning screening and early detection of cardia adenocarcinoma in asymptomatic high risk groups.

In order to reduce the burden of a patient, improve the examination compliance and the screening efficiency of the patient and find an effective specific molecular diagnosis marker for early detection of the cardiac adenocarcinoma of high risk groups, a liquid biopsy technology is adopted for early detection and early treatment of the cardiac adenocarcinoma, so that an important technical support is provided for finally reducing the incidence and mortality of the cardiac adenocarcinoma.

The development of cardiac adenocarcinoma is a result of the interaction of genetic, environmental, etc. factors and is a process of multiple evolutions. Tumor cells synthesize and release a group of antigens during their development and progression, i.e., tumor-associated antigens, which are not tumor-specific, but, in the presence of tumors, their secretion is greatly increased, and tumors can be identified or diagnosed based on their biochemical or immunological properties. Like alpha-fetoprotein, it is known that the content of alpha-fetoprotein in blood of liver cancer patients is greatly increased, and it has been used for diagnosis of liver cancer. Tumor cells have heterogeneity, even if the same tumor may have different antigen protein expression, and at present, no single molecular marker for a certain tumor has been found. Therefore, compared with a single tumor-associated antigen detection method, the method for detecting the tumor-associated antigens in the body of the patient by using a plurality of autoantibodies in a combined way is beneficial to improving the detection rate of a certain tumor. At present, a combined detection method for multiple tumor markers for early diagnosis of cardiac adenocarcinoma and application of corresponding test strips are rare.

The research team detects and analyzes the early cardia adenocarcinoma patients, asymptomatic high risk groups and normal human serum in a high incidence area by adopting a protein chip technology on the basis of a plurality of groups of chemical databases established in the past by utilizing proteomics, genomics, transcriptomics, epigenetics and the like, and screens out three tumor serum molecular markers which are differentially expressed in the cardia adenocarcinoma patients and the normal human, namely GAGE12B protein, AGPAT4 protein and P2RX4 protein.

GAGE12B is often expressed in many different types of tumors, while expression in normal tissues is limited to the germ cells, testis, and ovary of immune privileged organs. When the GAGE12B protein is expressed in tumor cells, cellular and humoral immune responses can be caused, and the GAGE12B antibody can be used as a potential immune target for tumor treatment and an index for early screening.

AGPAT4 is an abbreviation for 1-acylglycerol-3-phosphate acyltransferase 4, and is involved in lipid metabolism and is closely related to tumor progression. It has been reported that it is abnormally expressed in tissues of bladder cancer and colorectal cancer, and it is predicted that the survival rate of colorectal cancer patients is low.

The P2RX4 purinergic receptor is a member of the purinergic channel family, is an ionogenic adenosine triphosphate receptor subtype, and plays an important role in the development of tumors. Evidence suggests that P2X4R, expressed in the rat C6 glioma model, plays an important role in cell growth and apoptosis in human brain glioblastoma multiforme.

The GAGE12B protein, AGPAT4 protein and P2RX4 protein are low expressed in the serum of normal people and high expressed in the serum of patients with gastric cardia adenocarcinoma, and further statistical tests show that the positive rates of the GAGE12B protein, the AGPAT4 protein and the P2RX4 protein in the population of patients with gastric cardia adenocarcinoma are obviously higher than those of the normal population, and the positive rates are obviously different. Therefore, the GAGE12B protein, AGPAT4 protein and P2RX4 protein can be used for diagnosing the cardia adenocarcinoma. Aiming at the discovery, the research team uses GAGE12B protein, AGPAT4 protein and P2RX4 protein as detection indexes to prepare a product for early screening of the cardiac adenocarcinoma of high risk groups, and the product is used for early screening of asymptomatic people in the cardiac adenocarcinoma high incidence area so as to improve the early detection rate of the cardiac adenocarcinoma, improve the life quality of patients and reduce the burden of families and society.

Disclosure of Invention

Aiming at the problems and the defects in the prior art, one purpose of the invention is to provide a marker for screening cardiac adenocarcinoma of high risk groups, the other purpose of the invention is to provide an application of a detection reagent of the marker for screening cardiac adenocarcinoma of high risk groups at an early stage, the third purpose of the invention is to provide an application of the marker for screening cardiac adenocarcinoma of high risk groups in preparing a product for screening cardiac adenocarcinoma of high risk groups, and the fourth purpose of the invention is to provide a test strip for screening cardiac adenocarcinoma of high risk groups at an early stage.

In order to realize the purpose of the invention, the technical scheme adopted by the invention is as follows:

the invention provides a marker for screening cardiac adenocarcinoma of high risk people, which is any one or combination of more of GAGE12B protein, AGPAT4 protein and P2RX4 protein.

In a second aspect, the invention provides a use of the detection reagent for the marker of the first aspect in preparing a product for screening cardiac adenocarcinoma of high risk group.

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

According to the above use, preferably, the detection reagent is an antibody that specifically binds to the marker.

According to the above application, preferably, the product detects the marker in the sample by immunochromatography or enzyme-linked immunoassay.

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

In a third aspect, the invention provides a use of the marker of the first aspect in the preparation of a product for screening cardiac adenocarcinoma in high risk groups, wherein a detection sample of the product is serum. Since the expression abundance of the tumor associated antigen in serum is directly proportional to the expression abundance of the corresponding autoantibody, the tumor associated antigen is highly expressed, and the corresponding autoantibody is also highly expressed. Therefore, the GAGE12B protein, AGPAT4 protein and P2RX4 protein can also be used as detection reagents for detecting autoantibodies corresponding to GAGE12B protein, AGPAT4 protein and P2RX4 protein in serum, and the autoantibodies of the three tumor-associated antigens can be used as diagnosis and screening indexes of cardia adenocarcinoma.

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 comprising a solid support coated with a marker according to the first aspect.

The invention provides a test strip for early screening of cardia adenocarcinoma in high risk groups, which comprises a binding pad and a chromatographic pad, wherein the binding pad is coated with a capture antibody, the capture antibody is a mixture of GAGE12B antibody A, AGPAT4 antibody A, P2RX4 antibody A, and both GAGE12B antibody A, AGPAT4 antibody A, P2RX4 antibody A are provided with detectable markers; the chromatography pad is provided with three detection lines and a quality control line, detection antibodies are fixed on the detection lines, and the detection antibodies fixed on the three detection lines are GAGE12B antibody B, AGPAT4 antibody B, P2RX4 antibody B; the antibody A and the antibody B of the same antigen recognize different epitopes of the antigen; and rabbit anti-mouse IgG is fixed on the quality control line.

According to the above test strip for early screening of cardia adenocarcinoma of high risk group, preferably, the marker is colloidal gold particles, and the GAGE12B antibody A, AGPAT4 antibody A, P2RX4 antibody A is respectively marked with colloidal gold particles with different particle sizes.

According to the above test strip for early screening of cardia adenocarcinoma of high risk group, preferably, the GAGE12B antibody A, AGPAT4 antibody A, P2RX4 antibody a is GAGE12B monoclonal antibody, AGPAT4 monoclonal antibody, P2RX4 monoclonal antibody, respectively; wherein, the GAGE12B monoclonal antibody is marked by colloidal gold particles with the particle size of 24.5nm, the AGPAT4 monoclonal antibody is marked by colloidal gold particles with the particle size of 41nm, the P2RX4 monoclonal antibody is marked by colloidal gold particles with the particle size of 71nm, and the corresponding colors are respectively orange red, red and purple red.

According to the above test strip for early screening of cardiac adenocarcinoma of high risk group, preferably, the GAGE12B antibody B, AGPAT4 antibody B, P2RX4 antibody B is a GAGE12B polyclonal antibody, an AGPAT4 polyclonal antibody, and a P2RX4 polyclonal antibody, respectively.

According to the above test strip for the early screening of cardiac adenocarcinoma of high risk group, preferably, the test strip further comprises a sample pad, a sample sucking pad and a bottom plate, wherein the sample pad, the combination pad, the chromatography pad and the sample sucking pad are sequentially fixed on the bottom plate; wherein, one end of the combination pad is pressed below the sample pad, and the other end of the combination pad is pressed above the chromatography pad; one end of the chromatographic pad is pressed below the conjugate pad, and the other end of the chromatographic pad is pressed below the sample suction pad.

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

According to the test strip for the early screening of cardia adenocarcinoma of high risk group, 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 sucking pad to one end close to the combination pad, a quality control line C coated with rabbit anti-mouse IgG, a detection line T3 fixed with P2RX4 polyclonal antibody, a detection line T2 fixed with AGPAT4 polyclonal antibody and a detection line T1 fixed with GAGE12B polyclonal antibody are sequentially arranged.

According to the test strip for the early screening of cardia adenocarcinoma of high risk group, 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.

According to the test strip for the early screening of cardia adenocarcinoma of high risk group, preferably, the sample pad is made of water-absorbent glass fiber, the bonding pad is made of glass fiber membrane, and the chromatographic pad is made of nitrocellulose membrane; the sample absorbing pad is made of absorbent paper or an absorbent glass fiber membrane; the bottom plate is a PVC bottom plate, a hard board or a hard fiberboard.

The fifth aspect of the invention provides another test strip for early screening of cardiac adenocarcinoma of high risk groups, which comprises a binding pad and a chromatographic pad, wherein the binding pad is coated with a capture antibody, the capture antibody is a mixture of GAGE12B antibody A, AGPAT4 antibody A, and both GAGE12B antibody A, AGPAT4 antibody A carry a detectable marker; the chromatography pad is provided with two detection lines and a quality control line, detection antibodies are fixed on the detection lines, and the detection antibodies fixed on the two detection lines are GAGE12B antibody B, AGPAT4 antibody B respectively; the antibody A and the antibody B of the same antigen recognize different epitopes of the antigen; and rabbit anti-mouse IgG is fixed on the quality control line.

According to the above test strip for early screening of cardia adenocarcinoma of high risk group, preferably, the marker is colloidal gold particles, and the GAGE12B antibody A, AGPAT4 antibody a is respectively marked with colloidal gold particles with different particle sizes.

According to the test strip for the early screening of cardia adenocarcinoma of high risk group, preferably, the GAGE12B antibody A, AGPAT4 antibody A is GAGE12B monoclonal antibody and AGPAT4 monoclonal antibody respectively; wherein, the GAGE12B monoclonal antibody is marked by colloidal gold particles with the particle size of 24.5nm, the AGPAT4 monoclonal antibody is marked by colloidal gold particles with the particle size of 41nm, and the corresponding colors are respectively orange red and red.

According to the above test strip for early screening of cardiac adenocarcinoma of high risk group, preferably, the antibody B, AGPAT 4B of the GAGE12B is a GAGE12B polyclonal antibody, an AGPAT4 polyclonal antibody, and a P2RX4 polyclonal antibody, respectively.

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

According to the test strip for the early screening of cardia adenocarcinoma of high risk group, 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 combination pad, a quality control line C coated with rabbit anti-mouse IgG, a detection line T2 fixed with AGPAT4 polyclonal antibody and a detection line T1 fixed with GAGE12B polyclonal antibody are arranged in sequence.

According to the test strip for the early screening of cardia adenocarcinoma of high risk group, 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.

In a fifth aspect, the invention provides a kit comprising the test strip for early screening of cardiac adenocarcinoma in high risk groups in the third or fourth aspect.

According to the kit, the kit preferably further comprises a pipette, a serum sample cup, a disposable syringe and the like.

The use method of the test strip for early screening of cardia adenocarcinoma of high risk groups comprises the following steps:

and dropwise adding the serum sample to be detected on the sample pad of the test strip or inserting the test strip sample pad into the serum sample to be detected, taking out the test strip, observing the color change of the detection line and the quality control line within 5-15 min, and recording the result. Wherein, the serum sample can be diluted by a sample diluent before detection.

The result judgment method of the test strip for the early screening of the cardia adenocarcinoma of the high risk group comprises the following steps:

positive results: at least one detection line in the three or two detection lines of the chromatography pad of the test strip is colored, and the quality control line C is colored, so that the detection result is judged to be positive.

Negative results: and (4) the detection lines on the chromatographic pad of the test strip are not colored, only the quality control line C is colored, and the detection result is judged to be negative.

Invalid result: the quality control line C is not developed, and the detection result is judged to be invalid and needs to be detected again.

The detection principle of the test strip for early screening of cardia adenocarcinoma of high risk groups is as follows:

the test strip takes the cardiac adenocarcinoma markers GAGE12B protein, AGPAT4 protein and P2RX4 protein as detected substances, and utilizes the antigen-antibody reaction principle and the 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) existing in human blood. When the serum sample to be detected is dripped on the sample pad of the test strip, the serum sample to be detected flows to the direction of the sample absorption pad under the capillary action of the water absorption material of the sample absorption pad, when the flow-on binding pad was reached, the gold-labeled GAGE12B monoclonal antibody, the gold-labeled AGPAT4 monoclonal antibody, and the gold-labeled P2RX4 monoclonal antibody coated on the binding pad were dissolved, wherein, the colloidal gold-labeled GAGE12B monoclonal antibody is combined with GAGE12B antigen possibly contained in a serum sample to form a colloidal gold-labeled GAGE12B monoclonal antibody-GAGE 12B antigen complex, the colloidal gold-labeled AGPAT4 monoclonal antibody is combined with AGPAT4 antigen possibly contained in the serum sample to form a colloidal gold-labeled AGPAT4 monoclonal antibody-AGPAT 4 antigen complex, and the colloidal gold-labeled P2RX4 monoclonal antibody is combined with P2RX4 antigen possibly contained in the serum sample to form a colloidal gold-labeled P2RX4 monoclonal antibody-P2 RX4 antigen complex; due to capillary effect, the three colloidal gold-labeled capture antibody-antigen complexes continue to move towards the chromatographic pad, and when moving to the detection line T1, the colloidal gold-labeled GAGE12B monoclonal antibody-GAGE 12B antigen complex specifically binds with the GAGE12B polyclonal antibody to form a solidified immune complex, which is trapped on the detection line T1; when moving to the detection line T2, the colloidal gold labeled AGPAT4 monoclonal antibody-AGPAT 4 antigen complex is specifically combined with AGPAT4 polyclonal antibody to form a solidified immune complex, and the immobilized immune complex is trapped on the detection line T2; when moving to the detection line T3, the colloidal gold-labeled P2RX4 monoclonal antibody-P2 RX4 antigen complex is specifically combined with the P2RX4 polyclonal antibody to form an immobilized immune complex, and the immobilized immune complex is trapped on the detection line T3; the free colloidal gold labeled capture antibody continuously moves forwards due to the capillary effect, and is combined with the rabbit anti-mouse IgG coated on the quality control line C and trapped on the quality control line; excess unbound material continues to move to the draw pad by capillary action. Therefore, by detecting the color development of the lines T1, T2 and T3 and the color development of the quality control line, the tumor associated antigens GAGE12B, AGPAT4 and P2RX4 in the sample to be tested can be qualitatively judged.

Compared with the prior art, the invention has the following positive beneficial effects:

(1) according to the invention, three proteins, namely GAGE12B protein, AGPAT4 protein and P2RX4 protein, are used for early screening detection of the cardia adenocarcinoma for the first time, and the expression levels of GAGE12B protein, AGPAT4 protein and P2RX4 protein in human serum are detected, so that the cardia adenocarcinoma can be effectively detected, and particularly the detection rate of the early cardia adenocarcinoma can be improved; moreover, when the GAGE12B protein, AGPAT4 protein and P2RX4 protein are used as a combination for the early screening detection of cardiac adenocarcinoma of high risk population, the detection sensitivity is up to 87% (namely the ratio of correctly diagnosing early cardiac adenocarcinoma when 3 proteins are applied to early cardiac adenocarcinoma patients for diagnosis is 87%), the specificity is up to 77% (namely the ratio of correctly diagnosing the patients without cardiac adenocarcinoma when 3 proteins are applied to non-cardiac adenocarcinoma patients for diagnosis is 77%), therefore, the marker of the invention has higher sensitivity and specificity, greatly improves the detection rate of early cardiac adenocarcinoma, and the detection rate of cardiac adenocarcinoma is far higher than that of the existing clinical endoscope for screening cardiac adenocarcinoma (2% -3%), can be used for the large-scale screening of asymptomatic high risk population of cardiac adenocarcinoma high risk population, and is beneficial to the early discovery of asymptomatic cardiac adenocarcinoma, thereby greatly reducing the mortality of the patient with the cardia adenocarcinoma, improving the survival rate of the patient with the cardia adenocarcinoma and lightening the burden of families and society.

(2) The test strip can rapidly detect GAGE12B protein, AGPAT4 protein and P2RX4 protein in human serum, realizes the joint detection of three tumor markers, has higher sensitivity and specificity, has high detection accuracy for early-stage cardia adenocarcinoma, greatly improves the detection rate of the early-stage cardia adenocarcinoma, is beneficial to the early-stage discovery of asymptomatic cardia adenocarcinoma high-risk groups, provides an important detection means for realizing the long-term tracking of asymptomatic groups in a cardia adenocarcinoma high-incidence area, and has wide market prospect and social benefit.

(3) The test strip for early screening of the cardia adenocarcinoma of high risk groups is simple and convenient to operate, convenient to use and short in detection result time, the test end of the test strip is only required to be inserted into a sample liquid to be detected for about 10s, then the detection result can be judged within 15min, and the diagnosis efficiency of the early cardia adenocarcinoma is greatly improved.

(4) The three capture antibodies coated on the combination pad are respectively marked by the colloidal gold particles with different particle sizes, so that each tumor marker on the chromatography pad displays different colors, and the result judgment is more visual.

(5) The test strip for early screening of cardia adenocarcinoma of high risk group does not need other instruments and reagents, and non-professional personnel can also detect at any time, so that the detection cost and the detection expense can be greatly reduced, and the test strip has small manual operation error and good stability.

(6) The test sample of the test strip for early screening of cardia adenocarcinoma of high risk group is serum, the blood demand is less, the pain of the tested person is less, and the compliance is high.

Drawings

Fig. 1 is a schematic structural diagram of a test strip for early screening of cardiac adenocarcinoma of high risk group prepared in embodiment 1 of the present invention.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the following detailed description and accompanying drawings. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention.

Tumor markers are abnormally expressed bioactive substances produced by tumor tissues and cells, and can identify or diagnose tumors according to the immune characteristics after biochemistry. On the basis of a genomic database established by the research team in the early stage by using technologies such as whole genome correlation analysis, whole genome sequencing, whole genome exon sequencing and the like, the protein chip technology is adopted to detect and analyze the serum of early-stage cardia adenocarcinoma patients, asymptomatic high risk groups in high incidence areas and normal people, and three tumor serum markers which are differentially expressed in the cardia adenocarcinoma patients and the normal people are screened out: GAGE12B protein, AGPAT4 protein, P2RX4 protein. The GAGE12B protein, AGPAT4 protein and P2RX4 protein are low expressed in the serum of normal people and high expressed in the serum of patients with gastric cardia adenocarcinoma, and further statistical tests show that the positive rates of the GAGE12B protein, the AGPAT4 protein and the P2RX4 protein in the population of patients with gastric cardia adenocarcinoma are all obviously higher than those of the normal population, and the positive rates are obviously different. Therefore, the GAGE12B protein, AGPAT4 protein and P2RX4 protein can be used for diagnosing the cardia adenocarcinoma. Aiming at the discovery, the research team uses the GAGE12B protein, AGPAT4 protein and P2RX4 protein as detection indexes to prepare a test strip for early detection of cardiac adenocarcinoma of high risk groups, and the test strip is used for detecting the expression levels of the GAGE12B protein, AGPAT4 protein and P2RX4 protein in a sample to realize early screening of cardiac adenocarcinoma of high risk groups.

The experimental procedures described in the following examples, unless otherwise specified, are conventional in the art or according to the conditions recommended by the manufacturers; the reagents, materials and instruments used are not indicated by manufacturers, and are all conventional products commercially available.

Example 1: preparation of test strip for early screening of cardia adenocarcinoma of high risk group

1. Experimental Material

The bioactive raw materials used in the invention are all commercial products. Wherein, the GAGE12B monoclonal antibody is purchased from Shanghai Aibixin Biotech limited company with the product number of abs 101001; GAGE12B polyclonal antibody was purchased from Shanghai Sum Biotech, Inc., Cat. AP11254 a; AGPAT4 monoclonal antibody was purchased from Shanghai Biotech limited, cat # bs-5033R; AGPAT4 polyclonal antibody is purchased from Shanghai Hai Ji Biotech limited, having a product number of H00056895-B01; the P2RX4 monoclonal antibody is purchased from Shanghai Kemin Biotech, Inc., with the product number of bs-7690R; the P2RX4 polyclonal antibody was purchased from wuhan yi pu biotechnology limited, cat No. ATA 34839.

PVC base plate, nitrocellulose membrane, glass fiber membrane, absorbent paper, etc. are all the products sold in the market.

2. Preparation of the bonding pad

(1) Preparing colloidal gold:

preparing the colloidal gold solution by a trisodium citrate reduction method. The preparation of colloidal gold by the trisodium citrate reduction method is a conventional method in the art and will not be described in detail herein.

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

dialyzing the GAGE12B monoclonal antibody solution, AGPAT4 monoclonal antibody solution and P2RX4 monoclonal antibody solution respectively for desalting (overnight at4 ℃), and adjusting the protein concentration to 1 mg/ml; under electromagnetic stirring, the P2RX4 monoclonal antibody was labeled with colloidal gold having a particle size of 24.5nm, the AGPAT4 monoclonal antibody was labeled with colloidal gold having a particle size of 41nm, and the GAGE12B monoclonal antibody was labeled with colloidal gold having a particle size of 71nm, to obtain three kinds of colloidal gold-labeled monoclonal antibodies. And then mixing the three colloidal gold labeled monoclonal antibodies according to the mass ratio of 1:1:1 to obtain a capture antibody solution.

(3) Preparation of the bonding pad:

selecting a glass fiber membrane as a bonding pad material, soaking the glass fiber membrane in a pretreatment solution (a borate buffer solution containing 2% BSA, 3% sucrose, 0.6M NaCl and 0.2% Tween-20 in the pretreatment solution) for 10-15 min, and then drying at 36-38 ℃ or vacuum freeze drying; and (3) immersing the pretreated glass fiber membrane into the capture antibody solution, taking out after fully immersing, and freeze-drying to obtain the bonding pad.

3. Preparation of a chromatography pad

A nitrocellulose membrane is selected as a chromatography pad material, and the positions of 3 detection lines T1, T2, T3 and 1 quality control line C are marked on the nitrocellulose membrane, and are spaced by 6 mm. Diluting the GAGE12B polyclonal antibody, the AGPAT4 polyclonal antibody and the P2RX4 polyclonal antibody to 1mg/mL and the rabbit anti-mouse IgG to 1.5mg/mL, respectively carrying out scribing on the positions of 3 detection lines T1, T2, T3 and a quality control line C on a nitrocellulose membrane by using a scribing instrument according to the dosage of 0.1-0.5 mu L/mm, and drying at 37 ℃ for overnight; then, the nitrocellulose membrane was immersed in 0.01mol/L PBS buffer (pH8.0) containing 1% BSA, and then taken out, washed with the PBS buffer, and dried to obtain a chromatography pad.

4. Sample pad preparation

The sample pad is made of a glass fiber membrane, the glass fiber membrane is placed into a sample pad sealing solution to be soaked for 30min, and the sample pad is dried at 37 ℃ to obtain the sample pad; wherein the sample pad blocking solution is 0.01mol/LPBS buffer solution (pH 7.4) containing 1% BSA, 1.0% -2.0% sucrose, 0.1% -0.5% Tween-20, and 0.1-1.0% PVP polyvinylpyrrolidone.

5. Test strip assembly

The test strip consists of a sample pad, a combination pad, a chromatography pad, a sample absorption pad and a bottom plate, wherein the sample absorption pad is made of water absorption filter paper.

Attaching the chromatographic pad to the middle of the bottom plate to form a detection area and a quality control area, namely arranging detection lines T1, T2, T3 and a quality control line C for interpreting results on the chromatographic pad; the right end (upper section) of the chromatography pad is a handheld part, and a sample absorbing pad (water absorbing filter paper) is fixed on the right end (upper section) of the chromatography pad and absorbs redundant liquid in a detection sample; a binding pad is fixed at the left end of the chromatographic pad, and the binding pad is coated with a gold colloidal labeled GAGE12B monoclonal antibody, AGPAT4 monoclonal antibody and P2RX4 monoclonal antibody; the combination pad and the sample sucking pad are respectively overlapped with the two ends of the chromatography pad; and a sample pad is fixed at one end (lower section) of the combination pad, which is far away from the chromatography pad, and is used for contacting a sample to be detected, the sample is cut into test strips with the width of 4mm after the assembly is finished, and the test strips are dried to obtain the test strips for screening the early-stage cardia adenocarcinoma of the high risk group (see figure 1).

6. Using method of test strip

And dropwise adding the serum sample to be detected on a sample pad of the test strip, flatly placing the test strip, observing the color changes of the detection line and the quality control line within 5-15 min, and recording the result.

7. Determination of test result of test strip

Positive results: at least one detection line among the detection lines T1, T2 and T3 of the chromatographic pad of the test strip is colored, and the quality control line C is colored, so that the detection result is judged to be positive.

Negative results: and the detection lines T1, T2 and T3 on the chromatographic pad of the test strip are not colored, only the quality control line C is colored, and the detection result is judged to be negative.

8. Attention points in test paper strip detection

(1) The sample to be tested is tested at room temperature (about 20 ℃).

(2) The sample to be tested is required to be a fresh sample. And the sample is collected for detection within 1h, so that the reliability of the result is ensured.

(3) The test strip is only used for in vitro detection and coarse screening, cannot be used as a confirmation reagent, and the positive result needs to be subjected to further auxiliary diagnosis of gastrointestinal endoscopy.

(4) After 30min the observation was invalid.

(5) The test strip should be stored in the dark.

Example 2: diagnostic value analysis of test strips

The test paper prepared in the embodiment 1 of the invention is used for detecting serum samples of early stage cardia adenocarcinoma patients and normal persons which are pathologically diagnosed, and evaluating and analyzing the value of the test paper used for screening and diagnosing early stage cardia adenocarcinoma.

Meanwhile, in order to compare the detection performance of the test strip prepared in the embodiment 1 of the invention, 6 kinds of comparison test strips are prepared, and the 6 kinds of comparison test strips are used for respectively testing serum samples of early-stage cardia adenocarcinoma patients and normal persons diagnosed by pathology.

The preparation method of 6 kinds of comparison test strips is basically the same as that of the test strip disclosed in embodiment 1 of the invention, and the difference is that: the 6 types of control test strips have different capture antibodies coated on the binding pads, and different detection lines and detection antibodies fixed on the detection lines.

Wherein, the 1 st control test strip: the detection index of the 1 st comparison test strip is P2RX4 protein, and the capture antibody coated on the binding pad of the test strip is P2RX4 monoclonal antibody marked by colloidal gold with the particle size of 24.5 nm; the test strip chromatography pad is provided with 1 detection line and 1 quality control line, wherein the detection line is fixed with a P2RX4 polyclonal antibody, and the quality control line is fixed with rabbit anti-mouse IgG.

Comparative test strip No. 2: the detection index of the 2 nd comparative test strip is AGPAT4 protein, and the capture antibody coated on the binding pad of the test strip is AGPAT4 monoclonal antibody labeled by colloidal gold with the particle size of 41 nm; the test strip chromatography pad is provided with 1 detection line and 1 quality control line, wherein the detection line is fixed with AGPAT4 polyclonal antibody, and the quality control line is fixed with rabbit anti-mouse IgG.

Comparative test strip No. 3: the detection index of the 3 rd comparative test strip is GAGE12B protein, and the capture antibody coated on the binding pad of the test strip is a GAGE12B monoclonal antibody marked by colloidal gold with the particle size of 71 nm; the test strip chromatographic pad is provided with 1 detection line and 1 quality control line, wherein the detection line is fixed with a GAGE12B polyclonal antibody, and the quality control line is fixed with rabbit anti-mouse IgG.

Comparative test strip No. 4: the detection indexes of the 4 th contrast test strip are P2RX4 protein and AGPAT4 protein, the capture antibody coated on the binding pad of the test strip is a mixture of a P2RX4 monoclonal antibody and an AGPAT4 monoclonal antibody, the P2RX4 monoclonal antibody is labeled by colloidal gold with the particle size of 24.5nm, the AGPAT4 monoclonal antibody is labeled by colloidal gold with the particle size of 41nm, and the mass ratio of the P2RX4 monoclonal antibody to the AGPAT4 monoclonal antibody in the capture antibody is 1: 1; the test strip chromatography pad is provided with 2 detection lines and 1 quality control line, wherein the 1 st detection line is fixed with P2RX4 polyclonal antibody, the 2 nd detection line is fixed with AGPAT4 polyclonal antibody, and the quality control line is fixed with rabbit anti-mouse IgG.

Comparative test strip No. 5: the detection indexes of the 5 th contrast 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 by colloidal gold with the particle size of 41nm, the GAGE12B monoclonal antibody is labeled by colloidal gold with the particle size of 71nm, and the mass ratio of the AGPAT4 monoclonal antibody to the GAGE12B monoclonal antibody in the capture antibody is 1: 1; the test strip chromatographic pad is provided with 2 detection lines and 1 quality control line, wherein the 1 st detection line is fixed with AGPAT4 polyclonal antibody, the 2 nd detection line is fixed with GAGE12B polyclonal antibody, and the quality control line is fixed with rabbit anti-mouse IgG.

Comparative test strip No. 6: the detection indexes of the 6 th contrast test strip are P2RX4 protein and GAGE12B protein, the capture antibody coated on the binding pad of the test strip is a mixture of a P2RX4 monoclonal antibody and a GAGE12B monoclonal antibody, the P2RX4 monoclonal antibody is marked by colloidal gold with the particle size of 24.5nm, the GAGE12B monoclonal antibody is marked by colloidal gold with the particle size of 71nm, and the mass ratio of the P2RX4 monoclonal antibody to the GAGE12B monoclonal antibody in the capture antibody is 1: 1; the test strip chromatographic pad is provided with 2 detection lines and 1 quality control line, wherein the 1 st detection line is fixed with a P2RX4 polyclonal antibody, the 2 nd detection line is fixed with a GAGE12B polyclonal antibody, and the quality control line is fixed with rabbit anti-mouse IgG.

1. Sample source

200 serum samples from the national key laboratory for prevention and treatment of esophageal cancer in provincial co-construction of the first subsidiary hospital of Zhengzhou university were collected, wherein 100 serum samples were obtained from normal persons (control group) and 100 serum samples were obtained from patients with early stage cardia adenocarcinoma (cardia adenocarcinoma group). 100 normal human sera were obtained from healthy physical population in the laboratory cooperative hospital health center and were free of any tumor and immune-related diseases. Of the 100 normal persons, 64 men and 36 women had an average age of 57.36 ± 7.59 years, with the age range of 40-75 years. 100 sera of early stage cardia adenocarcinoma patients were obtained from histopathologically confirmed early stage (stage 0 + stage I) cardia adenocarcinoma patients who received no radiation or chemotherapy treatment. Of 100 patients with cardiac adenocarcinoma, 63 men and 37 women had an average age of 57.48 ± 8.29 years, with the age range of 40-75 years.

2. Experimental methods

The test paper strip prepared by the invention is adopted to respectively detect the blood serum samples of the cardia adenocarcinoma group and the control group, and the specific detection method comprises the following steps: and dropwise adding the serum sample to be detected on a sample pad of the test strip, flatly placing the test strip, observing the color changes of the detection line and the quality control line within 5-15 min, and recording the result.

According to the result judgment standard, respectively calculating the positive rates of the 3 antigens in the cardia adenocarcinoma group and the control group (dividing the number of the positive objects detected in each group by the total number of the detected objects in the group to obtain the positive rate); the statistical test is carried out by applying the sps 26.0 software, the antigen positive rates in the cardia adenocarcinoma group and the control group are compared by adopting a two-independent sample chi-square test method, the test level alpha is 0.05, when the p is less than 0.05, the result has statistical significance, and then the diagnostic value of detecting the cardia adenocarcinoma by the autoantibody is evaluated by adopting a screening test method (Table 1).

3. Analysis of results

The test paper strip is evaluated for the screening and diagnosis value of the test paper strip on the cardia adenocarcinoma according to the detection result, and the result is shown in table 1.

TABLE 1 Authenticity evaluation of the diagnostic value of different antigen combinations for cardia adenocarcinoma

As shown in Table 1, the positive rates of the P2RX4 protein, AGPAT4 protein and GAGE12B protein in the cardia adenocarcinoma group are 51%, 44% and 48%, respectively, the positive rates of the P2RX4 protein, AGPAT4 protein and GAGE12B protein in the control group are 12%, 9% and 8%, respectively, the positive rates of the P2RX4 protein, AGPAT4 protein and GAGE12B protein in the cardia adenocarcinoma group are higher than those in the control group, and the difference between the cardia adenocarcinoma group and the control group has statistical significance (P < 0.05). Therefore, the P2RX4 protein, the AGPAT4 protein and the GAGE12B protein can be used as an index for diagnosing and detecting early-stage cardia adenocarcinoma, are used for detecting the early-stage cardia adenocarcinoma and have important diagnostic value.

Moreover, as can be seen from table 1, with the increase of the test strip detection index (antigen), the positive rate (i.e. sensitivity) of the antigen in the serum of the patient with early stage cardia adenocarcinoma gradually increases, and the specificity gradually decreases; when the test strip jointly detects three antigens, namely P2RX4 protein, AGPAT4 protein and GAGE12B protein, the detection sensitivity of early-stage cardia adenocarcinoma reaches 87%, namely the percentage of cardia adenocarcinoma patients who can be correctly diagnosed by the detection method is 87%. With the increase of the detection indexes of the test strip, the detection specificity is reduced, but when three antigens of P2RX4 protein, AGPAT4 protein and GAGE12B protein are jointly detected, the detection specificity can still reach 77 percent, namely when the method is adopted for detecting patients with non-cardia adenocarcinoma, the percentage of correctly diagnosed healthy people is 77 percent.

Compared with the test strip for detecting one tumor-associated antigen alone, the test strip for jointly detecting 3 antigens of the P2RX4 protein, the AGPAT4 protein and the GAGE12B protein has the sensitivity of 1.71 times, 1.98 times and 1.81 times of that of the test strip for detecting the single indexes of the P2RX4 protein, the AGPAT4 protein and the GAGE12B protein respectively when the early diagnosis of the cardiac adenocarcinoma is carried out.

Therefore, the P2RX4 protein, the AGPAT4 protein and the GAGE12B protein in the serum sample are jointly detected to screen and diagnose the early-stage cardia adenocarcinoma, so that the sensitivity of diagnosis can be greatly improved on the premise of ensuring the specificity of diagnosis; the risk assessment of the cardia adenocarcinoma of the object to be detected has good diagnosis and application values.

In addition, the john index statistically means that 1 is subtracted from the sum of sensitivity and specificity, and the range of the john index is 0-1, and the closer the john index is to 1, the higher the diagnostic value is. According to the invention, with the increase of the antigen index detected by the test strip, the john's index is continuously increased and gradually approaches to 1, which indicates that the 3 antigens have better diagnostic value when being jointly used for diagnosing and screening early-stage cardia adenocarcinoma.

In conclusion, the early-stage cardia adenocarcinoma screening and diagnosing method can ensure higher specificity and sensitivity by jointly detecting the P2RX4 protein, the AGPAT4 protein and the GAGE12B protein in the serum sample, and has better diagnosis and application values for the risk assessment of cardia adenocarcinoma of a to-be-detected object.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the present invention, but rather as the following description is intended to cover all modifications, equivalents and improvements falling within the spirit and scope of the present invention.

Claims

1. A marker for screening cardiac adenocarcinoma of high risk group, wherein the marker is any one or more of GAGE12B protein, AGPAT4 protein and P2RX4 protein.

2. The use of the detection reagent for the marker of claim 1 in the preparation of a product for screening cardiac adenocarcinoma in high risk group, wherein the detection sample of the product is serum.

3. The use of claim 2, wherein the detection reagent is an antibody that specifically binds to the marker.

4. Use according to claim 2, wherein the product is used for detection of the marker in a sample by immunochromatography or enzyme-linked immunoassay.

5. The use of any one of claims 2 to 4, wherein the product is a test strip, a kit or a detection chip.

6. The test strip for the early screening of the cardiac adenocarcinoma of the high risk group comprises a binding pad and a chromatographic pad, wherein the binding pad is coated with a capture antibody, the capture antibody is a mixture of a GAGE12B antibody A, AGPAT4 antibody A, P2RX4 antibody A, and both the GAGE12B antibody A, AGPAT4 antibody A, P2RX4 antibody A carry detectable markers; the chromatography pad is provided with three detection lines and a quality control line, detection antibodies are fixed on the detection lines, and the detection antibodies fixed on the three detection lines are GAGE12B antibody B, AGPAT4 antibody B, P2RX4 antibody B; the antibody A and the antibody B of the same antigen recognize different epitopes of the antigen; and rabbit anti-mouse IgG is fixed on the quality control line.

7. The test strip for the early screening of cardiac adenocarcinoma of high risk group according to claim 6, wherein the marker is colloidal gold particles, and the GAGE12B antibody A, AGPAT4 antibody A, P2RX4 antibody A is labeled with colloidal gold particles of different particle sizes respectively.

8. The test strip for the early screening of the cardia adenocarcinoma of the high risk group comprises a binding pad and a chromatographic pad, wherein a capture antibody is coated on the binding pad, the capture antibody is a mixture of a GAGE12B antibody A, AGPAT4 antibody A, and both the GAGE12B antibody A, AGPAT4 antibody A carry a detectable marker; the chromatography pad is provided with two detection lines and a quality control line, detection antibodies are fixed on the detection lines, and the detection antibodies fixed on the two detection lines are GAGE12B antibody B, AGPAT4 antibody B respectively; the antibody A and the antibody B of the same antigen recognize different epitopes of the antigen; and rabbit anti-mouse IgG is fixed on the quality control line.

9. A kit comprising the test strip for early screening of cardiac adenocarcinoma of high risk group according to any one of claims 6 to 8.

10. The use of the marker of claim 1 in the preparation of a product for screening cardiac adenocarcinoma in high risk groups, wherein the test sample of the product is serum.