CN113267626B - Test strip for early screening of cardia adenocarcinoma of high risk group - Google Patents

Abstract

The invention belongs to the technical field of medical biology, and discloses a test strip for early screening cardiac adenocarcinoma of high risk groups, which comprises a combination pad and a chromatography pad, wherein the combination pad is coated with a capture antibody, the capture antibody is a mixture of a GAGE12B antibody A, an AGPAT4 antibody A and a P2RX4 antibody A, and the GAGE12B antibody A, the AGPAT4 antibody A and the P2RX4 antibody A are all 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 a GAGE12B antibody B, an AGPAT4 antibody B and a 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 the three tumor markers, can effectively detect the cardia adenocarcinoma according to the detection result, and greatly improves the detection rate of the early-stage cardia adenocarcinoma.

Description

A test strip for early screening of cardiac adenocarcinoma in high-risk groups

technical field

The invention belongs to the technical field of medicine and biology, and in particular relates to a test strip for early screening of cardiac adenocarcinoma in high-risk groups.

Background technique

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 and esophageal squamous cell carcinoma is very similar, and its incidence is high in the Taihang Mountains in northern my country, especially in Linzhou (formerly Lin County), Henan Province, which is 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, the incidence of cardia adenocarcinoma has risen sharply, and the incidence of esophagogastric junction cancer has increased by 4% per year6 Times, is one of the fastest growing malignant tumors, its pathogenesis is still unclear. Cardia adenocarcinoma is significantly different from distal gastric cancer in terms of epidemiology, etiology, histogenesis, and clinical features. Cardia 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 reagents. Most of them are found in the middle and late stages, resulting in poor prognosis and high mortality. In northern my country, especially in the Taihang Mountains at the junction of Henan, Hebei, and Shanxi, the incidence is high, and the incidence rate can reach 1.90/100,000. It is still the main cause of tumor-related deaths in this area.

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

In order to reduce the burden of patients, improve patient inspection compliance and screening efficiency, it is very important to find effective specific molecular diagnostic markers for early detection of cardiac adenocarcinoma in high-risk groups. Treatment, thus providing important technical support for ultimately reducing the morbidity and mortality of cardiac adenocarcinoma.

The occurrence of cardia adenocarcinoma is the result of the interaction of genes, heredity, environment and other factors, and it is a multi-stage evolution process. Tumor cells will synthesize and release a group of antigens during their occurrence and development, that is, tumor-associated antigens. This antigen is not unique to tumors, but its secretion will greatly increase in the presence of tumors. According to its biochemical Or immune properties can identify or diagnose tumors. 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 in the diagnosis of liver cancer. Tumor cells are heterogeneous, and even the same tumor may have different antigenic protein expressions. So far, no single molecular marker for a certain tumor has been found. Therefore, compared with a single tumor-associated antigen detection method, using multiple autoantibodies to jointly examine the expression of tumor-associated antigens in patients is beneficial to improve the detection rate of a certain tumor. At present, the combined detection method of multiple tumor markers and the application of corresponding test strips for the early diagnosis of cardia adenocarcinoma are relatively rare.

Based on the multi-omics database established earlier by using proteomics, genomics, transcriptomics, and epigenetics, the research team used protein chip technology to study patients with early cardiac adenocarcinoma in high-incidence areas, asymptomatic high-risk groups, and Normal human serum was detected and analyzed, and three tumor serum molecular markers that were differentially expressed in cardia adenocarcinoma patients and normal controls were screened out, namely GAGE12B protein, AGPAT4 protein and P2RX4 protein.

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

AGPAT4, short for 1-acylglycerol-3-phosphate acyltransferase 4, is involved in lipid metabolism and is closely related to tumor progression. It has been reported to be aberrantly expressed in bladder cancer and colorectal cancer tissues and predicts poor survival in colorectal cancer patients.

P2RX4 purinergic receptor, a member of the purinergic channel family, is a subtype of ionotropic adenosine triphosphate receptor, which 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 cell growth and apoptosis in human brain glioblastoma multiforme.

GAGE12B protein, AGPAT4 protein and P2RX4 protein are lowly expressed in normal human serum, but highly expressed in serum of patients with cardia adenocarcinoma The positive rates were significantly higher than the normal population, and there were significant differences. This shows that GAGE12B protein, AGPAT4 protein and P2RX4 protein can be used in the diagnosis of cardia adenocarcinoma. In response to this discovery, the 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 groups, and for early screening of asymptomatic people in high-incidence areas 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.

Contents of the invention

In view of the problems and deficiencies in the prior art, one of the purposes of the present invention is to provide a marker for the screening of cardiac adenocarcinoma in high-risk groups, and the second purpose of the present invention is to provide a marker for cardiac adenocarcinoma screening in high-risk groups. The application of detection reagents for markers for early cancer screening, the third purpose of the present invention is to provide a marker for high-risk population cardia adenocarcinoma screening in the preparation of products for high-risk population cardiac adenocarcinoma screening The fourth purpose of the application of the present invention is to provide a test strip for early screening of cardia adenocarcinoma in high-risk groups.

For realizing the purpose of the invention, the technical scheme adopted in the present invention is as follows:

The first aspect of the present invention provides a marker for screening high-risk populations for cardia adenocarcinoma, wherein the marker is any one or a combination of GAGE12B protein, AGPAT4 protein and P2RX4 protein.

The second aspect of the present invention provides an application of a detection reagent for the marker described in the first aspect in the preparation of products for screening cardiac adenocarcinoma in high-risk groups.

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

According to the above application, 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 test kit or a detection chip.

The third aspect of the present invention provides an application of the marker described in the first aspect above in preparing a product for screening cardiac adenocarcinoma in high-risk groups, and the detection sample of the product is serum. Since the expression abundance of tumor-associated antigens in serum is directly proportional to the expression abundance of their corresponding autoantibodies, if 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 autoantibodies corresponding to GAGE12B protein, AGPAT4 protein, and P2RX4 protein in serum. diagnostic and screening indicators.

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, and the kit includes a solid-phase carrier coated with the marker described in the first aspect above.

The fourth aspect of the present invention provides a test strip for early screening of cardiac adenocarcinoma in high-risk groups, the test strip includes a binding pad and a chromatography pad, the binding pad is coated with a capture antibody, and the capture The antibody is a mixture of GAGE12B antibody A, AGPAT4 antibody A, and P2RX4 antibody A, and GAGE12B antibody A, AGPAT4 antibody A, and P2RX4 antibody A all have detectable markers; the chromatography pad is provided with three detection lines and a quality control Lines, detection antibodies are immobilized on the detection lines, and the detection antibodies fixed on the three detection lines are GAGE12B antibody B, AGPAT4 antibody B, and P2RX4 antibody B; antibody A and antibody B of the same antigen recognize different epitopes of the antigen; Rabbit anti-mouse IgG is immobilized on the quality control line.

According to the above-mentioned test strips for early screening of cardia adenocarcinoma in high-risk groups, preferably, the markers are colloidal gold particles, and GAGE12B antibody A, AGPAT4 antibody A, and P2RX4 antibody A use colloidal gold particles of different particle sizes respectively. mark.

According to the above test strips for early screening of cardiac adenocarcinoma in high-risk groups, preferably, GAGE12B antibody A, AGPAT4 antibody A, and P2RX4 antibody A are GAGE12B monoclonal antibody, AGPAT4 monoclonal antibody, and P2RX4 monoclonal antibody, respectively; , the GAGE12B monoclonal antibody was labeled with colloidal gold particles with a particle size of 24.5nm, the AGPAT4 monoclonal antibody was labeled with colloidal gold particles with a particle size of 41nm, and the P2RX4 monoclonal antibody was labeled with colloidal gold particles with a particle size of 71nm. The colors are orange-red, red and purple-red.

According to the aforementioned test strips for early screening of cardiac adenocarcinoma in high-risk groups, preferably, GAGE12B antibody B, AGPAT4 antibody B, and P2RX4 antibody B are GAGE12B polyclonal antibody, AGPAT4 polyclonal antibody, and P2RX4 polyclonal antibody, respectively.

According to the above-mentioned test strip for early screening of cardiac adenocarcinoma in high-risk groups, preferably, the test strip also includes a sample pad, a suction pad and a bottom plate, and the sample pad, binding pad, chromatography pad and suction pad The sample pads are fixed on the bottom plate in turn; wherein, one end of the binding pad is pressed under the sample pad, and the other end of the binding pad is pressed above the chromatography pad; one end of the chromatography pad is pressed under the binding pad, and the The other end is pressed under the suction pad.

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

According to the above-mentioned test strips for early screening of cardiac adenocarcinoma in high-risk groups, preferably, the three detection lines and one quality control line on the chromatography pad are arranged in the following order: from the end near the suction pad to the end near the One end of the binding pad is the quality control line C coated with rabbit anti-mouse IgG, the detection line T3 immobilized with P2RX4 polyclonal antibody, the detection line T2 immobilized with AGPAT4 polyclonal antibody, and the detection line T1 immobilized with GAGE12B polyclonal antibody .

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

According to the above-mentioned test strips for early screening of cardiac adenocarcinoma in high-risk groups, preferably, the material of the sample pad is water-absorbent glass fiber, the material of the binding pad is glass fiber membrane, and the material of the chromatography pad is The material is nitrocellulose membrane; the material of the sample suction pad is water-absorbing paper or water-absorbing glass fiber membrane; the bottom plate is PVC bottom plate, cardboard or hard fiberboard.

The fifth aspect of the present invention provides another test strip for early screening of cardiac adenocarcinoma in high-risk groups, the test strip includes a binding pad and a chromatography pad, the binding pad is coated with a capture antibody, the The capture antibody is a mixture of GAGE12B antibody A and AGPAT4 antibody A, and both GAGE12B antibody A and AGPAT4 antibody A have detectable markers; the chromatography pad is provided with two detection lines and a quality control line, and the detection line There are detection antibodies immobilized on both detection lines, and the detection antibodies fixed on the two detection lines are GAGE12B antibody B and AGPAT4 antibody B respectively; antibody A and antibody B of the same antigen recognize different epitopes of the antigen; rabbits are fixed on the quality control line Anti-mouse IgG.

According to the above-mentioned test strips for early screening of cardiac adenocarcinoma in high-risk groups, preferably, the markers are colloidal gold particles, and GAGE12B antibody A and AGPAT4 antibody A are respectively labeled with colloidal gold particles of different particle sizes.

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

According to the aforementioned test strips for early screening of cardiac adenocarcinoma in high-risk groups, preferably, the GAGE12B antibody B and the AGPAT4 antibody B are polyclonal antibodies to GAGE12B, AGPAT4, and P2RX4, respectively.

According to the above-mentioned test strip for early screening of cardiac adenocarcinoma in high-risk groups, preferably, the test strip also includes a sample pad, a suction pad and a bottom plate, and the sample pad, binding pad, chromatography pad and suction pad The sample pads are fixed on the bottom plate in turn; wherein, one end of the binding pad is pressed under the sample pad, and the other end of the binding pad is pressed above the chromatography pad; one end of the chromatography pad is pressed under the binding pad, and the The other end is pressed under the suction pad.

According to the aforementioned test strips for early screening of cardiac adenocarcinoma in high-risk groups, preferably, the mass ratio of GAGE12B monoclonal antibody to AGPAT4 monoclonal antibody in the capture antibody is 1:1.

According to the above-mentioned test strips for early screening of cardiac adenocarcinoma in high-risk groups, preferably, the two detection lines and one quality control line on the chromatography pad are arranged in the following order: from one end close to the suction pad to Near the end of the binding pad are the quality control line C coated with rabbit anti-mouse IgG, the detection line T2 immobilized with the AGPAT4 polyclonal antibody, and the detection line T1 immobilized with the GAGE12B polyclonal antibody.

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

According to the above-mentioned test strips for early screening of cardiac adenocarcinoma in high-risk groups, preferably, the material of the sample pad is water-absorbent glass fiber, the material of the binding pad is glass fiber membrane, and the material of the chromatography pad is The material is nitrocellulose membrane; the material of the sample suction pad is water-absorbing paper or water-absorbing glass fiber membrane; the bottom plate is PVC bottom plate, cardboard or hard fiberboard.

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 or fourth aspect.

According to the above kit, preferably, the kit further includes a suction dropper, a serum sampling cup, a disposable syringe and the like.

The method of using the test strip for the early screening of cardia adenocarcinoma of high-risk groups in the present invention is as follows:

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, observe the color change of the test line and quality control line within 5 to 15 minutes after taking out the test strip, and record the results. Wherein, the serum sample can be diluted with a sample diluent before detection.

The method for judging the results of the test strips used in the early screening of cardia adenocarcinoma of high-risk groups in the present invention is as follows:

Positive result: if at least one of the three or two detection lines on the chromatography pad of the test strip develops color, and the quality control line C develops color, the test result is determined to be positive.

Negative result: none of the test lines on the chromatography pad of the test strip develops color, only the quality control line C develops color, and the test result is judged to be negative.

Invalid result: The quality control line C does not develop color, and the test result is judged to be invalid and needs to be tested again.

The detection principle of the test strip used for the early screening of cardia adenocarcinoma in high-risk groups in the present invention is as follows:

The test strip of the present invention uses cardia adenocarcinoma markers GAGE12B protein, AGPAT4 protein, and P2RX4 protein as the detected objects, and uses the principle of antigen-antibody reaction and colloidal gold immunochromatography technology to detect the GAGE12B protein (also known as GAGE12B antigen) present in human blood. , AGPAT4 protein (also known as AGPAT4 antigen), P2RX4 protein (also known as P2RX4 antigen). When the serum sample to be tested is dropped on the sample pad of the test strip, the serum sample to be tested will flow to the direction of the suction pad under the action of the capillary of the absorbent material of the pad, and when it flows to the binding pad, the binding pad contains The colloidal gold-labeled GAGE12B monoclonal antibody, colloidal gold-labeled AGPAT4 monoclonal antibody, and colloidal gold-labeled P2RX4 monoclonal antibody are dissolved, wherein the colloidal gold-labeled GAGE12B monoclonal antibody binds to the GAGE12B antigen that may be contained in the serum sample to form colloidal gold Labeled GAGE12B monoclonal antibody-GAGE12B antigen complex, colloidal gold-labeled AGPAT4 monoclonal antibody binds to AGPAT4 antigen that may be contained in serum samples to form a colloidal gold-labeled AGPAT4 monoclonal antibody-AGPAT4 antigen complex, colloidal gold-labeled P2RX4 monoclonal antibody Combine with 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 colloidal gold-labeled capture antibody-antigen complexes continue to move to the chromatography pad, when moving When it reaches 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 trapped 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 trapped on the detection line T2; when it moves to the detection line T3, the colloidal gold-labeled P2RX4 monoclonal The antibody-P2RX4 antigen complex specifically binds to the P2RX4 polyclonal antibody to form a solidified immune complex, which is trapped on the detection line T3; the free colloidal gold-labeled capture antibody continues to move forward due to the capillary effect, and the quality control line C The coated rabbit anti-mouse IgG is bound and trapped on the quality control line; the excess unbound material continues to move to the suction pad under capillary action. Therefore, the tumor-associated antigens of GAGE12B, AGPAT4 and P2RX4 in the sample to be tested can be qualitatively judged by the color development of the detection lines T1, T2, and T3 and the color development of the quality control line.

Compared with the prior art, the positive beneficial effect that the present invention obtains is:

(1) In the present invention, GAGE12B protein, AGPAT4 protein and P2RX4 protein are used for the early screening and detection of cardia adenocarcinoma for the first time, and the expression levels of GAGE12B protein, AGPAT4 protein and P2RX4 protein in human serum can be effectively detected Adenocarcinoma, especially can improve the detection rate of early cardia adenocarcinoma; moreover, when GAGE12B protein, AGPAT4 protein and P2RX4 protein are used as a combination for early screening detection of cardia adenocarcinoma in high-risk groups, the detection sensitivity is as high as 87% (ie In patients with early cardia adenocarcinoma, the rate of correct diagnosis of early cardia adenocarcinoma was 87% when these three proteins were used for diagnosis), and the specificity reached 77% (that is, in non-cardia adenocarcinoma patients, these three proteins were used for diagnosis). The ratio of not suffering from cardia adenocarcinoma during diagnosis is 77%), therefore, the marker of the present invention has higher sensitivity and specificity, greatly improves the detection rate of early cardia adenocarcinoma, and, to cardia adenocarcinoma The detection rate of adenocarcinoma is much higher than that of the existing clinical endoscopic screening for cardiac adenocarcinoma (2%-3%), and it can be used for large-scale screening of asymptomatic high-risk populations in high-incidence areas of cardiac adenocarcinoma. It is beneficial to the early detection of cardiac adenocarcinoma in asymptomatic high-risk groups, thereby greatly reducing the mortality of cardiac adenocarcinoma patients, improving the survival rate of cardiac adenocarcinoma patients and reducing the burden on families and society.

(2) The test strip of the present invention can quickly detect GAGE12B protein, AGPAT4 protein, and P2RX4 protein in human serum, and realize the combined detection of three tumor markers, and the test strip has higher sensitivity and specificity , the detection accuracy of early cardia adenocarcinoma is high, which greatly improves the detection rate of early cardia adenocarcinoma, which is conducive to the early detection of asymptomatic high-risk groups of cardia adenocarcinoma, and also provides a long-term goal for asymptomatic people in high-incidence areas of cardia adenocarcinoma. Tracking provides an important means of detection and has broad market prospects and social benefits.

(3) The test strip used for the early screening of cardiac adenocarcinoma in high-risk groups is easy to operate and easy to use, and the time for detecting the result is short. It only needs to insert the test end into the sample liquid to be tested for about 10 seconds, and then The detection result can be judged within 15 minutes, which greatly improves the diagnostic efficiency of early cardiac adenocarcinoma.

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

(5) The test strip used in the early screening of cardiac adenocarcinoma in high-risk groups does not require other instruments and reagents, and non-professionals can also detect it at any time, which can greatly reduce the detection cost and detection cost, and the human error is small , Good stability.

(6) The detection sample of the test strip used in the early screening of cardiac adenocarcinoma of high-risk groups in the present invention is serum, which requires less blood volume, less pain for the tested personnel, and high compliance.

Description of drawings

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

detailed description

In order 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. Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be considered as limiting the scope of the present invention.

Tumor markers are abnormally expressed biologically active substances produced by tumor tissues and cells, and tumors can be identified or diagnosed according to their biochemical and postimmune properties. Based on the genomics database established by genome-wide association analysis, whole-genome sequencing, and whole-genome exome sequencing, the research team used protein chip technology to analyze patients with early cardiac adenocarcinoma, asymptomatic high-risk groups in high-incidence areas, and Normal human serum was detected and analyzed, and three tumor serum markers that were differentially expressed in cardiac adenocarcinoma patients and normal controls were screened out: GAGE12B protein, AGPAT4 protein, and P2RX4 protein. GAGE12B protein, AGPAT4 protein and P2RX4 protein are lowly expressed in normal human serum, but highly expressed in serum of patients with cardiac adenocarcinoma The positive rates were significantly higher than the normal population, and there were significant differences. This shows that GAGE12B protein, AGPAT4 protein, and P2RX4 protein can be used for the diagnosis of cardia 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, and to detect the expression levels of GAGE12B protein, AGPAT4 protein, and P2RX4 protein in samples , to achieve early screening of cardiac adenocarcinoma in high-risk groups.

The experimental methods described in the following examples, unless otherwise specified, are conventional techniques in this technical field, or according to the conditions suggested by the manufacturer; the reagents, materials and instruments used are not indicated by the manufacturer, all of which can be purchased from the market. Obtained conventional products.

Example 1: Preparation of test strips for early screening of cardiac adenocarcinoma in high-risk groups

1. Experimental materials

The bioactive raw materials used in the present invention are all commercially available products. Among them, the GAGE12B monoclonal antibody was purchased from Shanghai Aibison Biotechnology Co., Ltd., the article number is abs101001; the GAGE12B polyclonal antibody was purchased from Shanghai Qunji Biotechnology Co., Ltd., the article number was AP11254a; the AGPAT4 monoclonal antibody was purchased from Shanghai Kemin Biotechnology Co., Ltd. Company, Cat. No. bs-5033R; AGPAT4 polyclonal antibody was purchased from Shanghai Qunji Biotechnology Co., Ltd., Cat. Purchased from Wuhan Yipu Biotechnology Co., Ltd., article number ATA34839.

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

2. Preparation of binding pads

(1) Preparation of colloidal gold:

Colloidal gold solution was prepared by trisodium citrate reduction method. The reduction of trisodium citrate to prepare colloidal gold is a conventional method in the art, and will not be described in detail here.

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

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

(3) Preparation of binding pad:

The glass fiber membrane was selected as the bonding pad material, and the glass fiber membrane was soaked in the pretreatment solution (pretreatment was borate buffer solution containing 2% BSA, 3% sucrose, 0.6M NaCl, 0.2% Tween-20) for 10 ~15min, then dry at 36-38°C or freeze-dry in vacuum; immerse the pretreated glass fiber membrane in the above-mentioned capture antibody solution, take it out after fully soaking, and freeze-dry to obtain a binding pad.

3. Preparation of chromatography pad

Choose nitrocellulose membrane as the chromatography pad material, mark the position of 3 detection lines T1, T2, T3 and 1 quality control line C on the nitrocellulose membrane, with an interval of 6mm between each other. Dilute GAGE12B polyclonal antibody, AGPAT4 polyclonal antibody, P2RX4 polyclonal antibody to 1mg/mL, rabbit anti-mouse IgG to 1.5mg/mL, and draw three detection lines T1 , T2, T3 and the position of the quality control line C were streaked at 0.1-0.5 μL/mm, and dried at 37°C overnight; then the nitrocellulose membrane was immersed in 0.01mol/L PBS buffer containing 1% BSA ( pH 8.0), then the nitrocellulose membrane was taken out, washed with PBS buffer solution, and dried to obtain a chromatographic pad.

4. Sample pad preparation

The material of the sample pad is glass fiber membrane, soak the glass fiber membrane in the sample pad sealing solution for 30 minutes, and dry at 37°C to obtain the sample pad; the sample pad sealing solution contains 1% BSA, 1.0% ~ 2.0% sucrose , 0.1%-0.5% Tween-20, 0.1-1.0% PVP polyvinylpyrrolidone in 0.01mol/LPBS buffer solution (pH=7.4).

5. Assembly of test strips

The test strip is composed 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 water-absorbing filter paper.

Attach the chromatography pad in 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 quality control line C for interpreting the results are set on the chromatography pad; the right end of the chromatography pad (upper section) It is a hand-held part, fixed with a suction pad (absorbent filter paper) to absorb 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. Cloned antibody; the binding pad and the suction pad overlap the two ends of the chromatography pad respectively; the end (lower section) of the binding pad away from the chromatography pad is fixed with a sample pad, which is used to contact the sample to be tested. After assembly, it is cut into a 4mm wide Test strips, after drying, the test strips can be used for early cardia adenocarcinoma screening in high-risk groups (see Figure 1).

6. How to use the test strip

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.

7. Judgment of the test result of the test strip

Positive result: if at least one of the detection lines T1, T2, and T3 of the chromatography pad of the test strip develops color, and the quality control line C develops color, the test result is determined to be positive.

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

Invalid result: The quality control line C does not develop color, and the test result is judged to be invalid and needs to be tested again.

8. Precautions when testing strips

(1) The tested samples were tested at room temperature (around 20°C).

(2) The sample to be inspected is required to be a fresh sample. The samples are collected and tested within 1 hour to ensure the reliability of the results.

(3) This test strip is only for rough screening in vitro and cannot be used as a confirmatory reagent. If the result is positive, further upper gastrointestinal endoscopy must be carried out for consultation.

(4) After 30 minutes, the observation result is invalid.

(5) The test strips should be kept away from light.

Embodiment 2: the diagnostic value analysis of test strip

Use the test strip prepared in Example 1 of the present invention to detect the serum samples of pathologically confirmed early cardiac adenocarcinoma patients and normal people, and evaluate and analyze the value of the test strip of the present invention for screening and diagnosis of early cardiac adenocarcinoma.

At the same time, in order to compare the detection performance of the test strips prepared in Example 1 of the present invention, the present invention prepared 6 kinds of contrast test strips, and used 6 kinds of contrast test strips to test the pathologically diagnosed early cardia adenocarcinoma patients and normal people respectively. of serum samples.

The preparation methods of the 6 kinds of comparative test strips are basically the same as the preparation methods of the test strips disclosed in Example 1 of the present invention, except that the capture antibodies coated on the binding pads of the 6 kinds of comparative test strips are different, and the The number of detection lines on the assay pad and the detection antibodies immobilized on the detection lines are different.

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 24.5nm ; The test strip chromatography pad is provided with a detection line and a quality control line, the detection line is immobilized with P2RX4 polyclonal antibody, and the quality control line is immobilized with rabbit anti-mouse IgG.

The second comparison test strip: the detection index of the second comparison 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 diameter of 41nm; One detection line and one quality control line are arranged on the paper strip chromatography pad, the detection line is immobilized with AGPAT4 polyclonal antibody, and the quality control line is immobilized with rabbit anti-mouse IgG.

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 a GAGE12B monoclonal antibody labeled with colloidal gold with a particle size of 71nm; One detection line and one quality control line are arranged on the paper strip chromatography pad, the detection line is immobilized with GAGE12B polyclonal antibody, and the quality control line is immobilized with rabbit anti-mouse IgG.

The fourth comparison test strip: the detection indicators of the fourth comparison test strip are P2RX4 protein and AGPAT4 protein, and the capture antibody coated on the binding pad of the test strip is a mixture of P2RX4 monoclonal antibody and AGPAT4 monoclonal antibody, P2RX4 monoclonal antibody The cloned antibody is labeled with colloidal gold with a particle size of 24.5nm, and the AGPAT4 monoclonal antibody is labeled with colloidal gold with a particle size of 41nm. The mass ratio of P2RX4 monoclonal antibody to AGPAT4 monoclonal antibody in the capture antibody is 1:1; the test paper There are 2 detection lines and 1 quality control line on each chromatography pad, the first detection line is immobilized with P2RX4 polyclonal antibody, the second detection line is immobilized with AGPAT4 polyclonal antibody, and the quality control line is immobilized with There is rabbit anti-mouse IgG.

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, AGPAT4 monoclonal antibody The cloned antibody is labeled with colloidal gold with a particle size of 41nm, and the GAGE12B monoclonal antibody is labeled with colloidal gold with a particle size of 71nm. The mass ratio of AGPAT4 monoclonal antibody to GAGE12B monoclonal antibody in the capture antibody is 1:1; the test strip There are 2 detection lines and 1 quality control line on the chromatography pad. AGPAT4 polyclonal antibody is immobilized on the first detection line, GAGE12B polyclonal antibody is immobilized on the second detection line, and GAGE12B polyclonal antibody is immobilized on the quality control line. Rabbit anti-mouse IgG.

The sixth comparison test strip: the detection indicators of the sixth comparison test strip are P2RX4 protein and GAGE12B protein, and the capture antibody coated on the binding pad of the test strip is a mixture of P2RX4 monoclonal antibody and GAGE12B monoclonal antibody. The cloned 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; the test paper There are 2 detection lines and 1 quality control line on each chromatography pad, the first detection line is immobilized with P2RX4 polyclonal antibody, the second detection line is immobilized with GAGE12B polyclonal antibody, and the quality control line is immobilized with There is rabbit anti-mouse IgG.

1. Sample source

200 serum samples were collected from the State Key Laboratory of Esophageal Cancer Prevention and Control jointly established by the First Affiliated Hospital of Zhengzhou University, including 100 samples of normal human serum (control group) and 100 serum samples of patients with early cardiac adenocarcinoma (cardia adenocarcinoma group). Serum from 100 cases of normal people came from the healthy physical examination center of the laboratory's cooperative hospital without any tumor and immune-related diseases. Among the 100 normal subjects, there were 64 males and 36 females, with an average age of 57.36±7.59 years old and a range of 40-75 years old. 100 serum samples from patients with early cardiac adenocarcinoma were obtained from patients with early (stage 0+stage I) cardiac adenocarcinoma confirmed by histopathology, and none of them received 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 and a range of 40-75 years old.

2. Experimental method

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

According to the result judgment standard, calculate the positive rate of these three antigens in the cardia adenocarcinoma group and the control group respectively (the number of positive subjects detected in each group divided by the total number of tested subjects in this group is the positive rate); The spss26.0 software was used for the statistical test, and the two independent sample chi-square test method was used to compare the positive rate of the antigen 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 used Methods of screening tests evaluated the diagnostic value of autoantibodies in the detection of cardiac adenocarcinoma (Table 1).

3. Analysis of results

According to the detection results of the test strips, its value in screening and diagnosis of cardia adenocarcinoma was evaluated, and the results are shown in Table 1.

Table 1 Authenticity evaluation of different antigen combinations in the diagnosis of cardiac adenocarcinoma

It can be seen from Table 1 that 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%, 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 early diagnosis and detection indicators of cardia adenocarcinoma, and have important diagnostic value for the detection of early cardia adenocarcinoma.

Moreover, it can also be seen from Table 1 that with the increase of the detection index (antigen) of the test strip, in the serum of patients with early cardia adenocarcinoma, the positive rate (ie sensitivity) of the antigen gradually increases, while the specificity gradually decreases. Small; when the test strip combined detection of P2RX4 protein, AGPAT4 protein, GAGE12B protein three antigens, the detection sensitivity of early cardia adenocarcinoma reached 87%, that is to say, the application of this detection method in patients with cardia adenocarcinoma can correctly diagnose cardia The percentage of adenocarcinoma was 87%. With the increase of test strip detection indicators, the specificity of detection has decreased, but when combined detection of P2RX4 protein, AGPAT4 protein, and GAGE12B protein three antigens, the detection specificity can still reach 77%, that is to say, non-cardia adenocarcinoma When patients were tested this way, the percentage of correctly diagnosed healthy people was 77%.

Moreover, compared with the test strips for the detection of one tumor-associated antigen alone, the sensitivity of the test strips for the combined detection of P2RX4 protein, AGPAT4 protein, and GAGE12B protein in the diagnosis of early cardiac adenocarcinoma was Index P2RX4 protein, AGPAT4 protein, GAGE12B protein 1.71 times, 1.98 times, 1.81 times.

Therefore, the joint detection of P2RX4 protein, AGPAT4 protein, and GAGE12B protein in serum samples can screen and diagnose early cardiac adenocarcinoma, which can greatly improve the sensitivity of diagnosis under the premise of ensuring diagnostic specificity; Good diagnostic and application value.

In addition, the Youden index statistically refers to the sum of sensitivity and specificity minus 1, and its range is 0-1. The closer the Youden index is to 1, the higher its diagnostic value. In the present invention, with the increase of the antigen index detected by the test strip, the Youden index increases continuously and gradually tends to 1, indicating that the combined use of these three antigens for diagnosis and screening of early cardiac adenocarcinoma has good diagnostic value.

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 a good effect in risk assessment of cardiac adenocarcinoma. diagnostic and application value.

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

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

Claims (8)

1. The application of the detection reagent of P2RX4 protein or the combination of P2RX4 protein and AGPAT4 protein or the combination of P2RX4 protein, AGPAT4 protein and GAGE12B protein in the preparation of products for high-risk population cardia adenocarcinoma screening, and the detection sample of the product is serum . 2. The application according to claim 1, wherein the detection reagent is an antibody specifically binding to a marker. 3. The application according to claim 1, characterized in that the product detects the markers in the sample by immunochromatography or enzyme-linked immunoassay. 4. The application according to any one of claims 1-3, wherein the product is a test strip, a kit or a detection chip. 5. A test strip for early screening of cardiac adenocarcinoma in high-risk groups, characterized in that, the test strip includes a binding pad and a chromatography pad, and the binding pad is coated with a capture antibody, and the capture antibody It is a mixture of GAGE12B antibody A, AGPAT4 antibody A, and P2RX4 antibody A, all of which have detectable markers; the chromatography pad is provided with three detection lines and one quality control line , detection antibodies are immobilized on the detection lines, and the detection antibodies fixed on the three detection lines are GAGE12B antibody B, AGPAT4 antibody B, and P2RX4 antibody B respectively; antibody A and antibody B of the same antigen recognize different epitopes of the antigen; Rabbit anti-mouse IgG was immobilized on the quality control line. 6. The test strip for early screening of cardiac adenocarcinoma in high-risk groups according to claim 5, wherein the marker is colloidal gold particles, and GAGE12B antibody A, AGPAT4 antibody A, and P2RX4 antibody A are used respectively Colloidal gold particles of different sizes are labeled. 7. A test kit comprising the test strip for early screening of cardiac adenocarcinoma in high-risk groups according to claim 5 or 6. 8. The application of P2RX4 protein or the combination of P2RX4 protein and AGPAT4 protein or the combination of P2RX4 protein, AGPAT4 protein and GAGE12B protein in the preparation of products for high-risk population cardiac adenocarcinoma screening, and the detection sample of the products is serum.

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