CN113109563B - Marker for screening early esophageal squamous carcinoma of high risk group and application thereof - Google Patents
Marker for screening early esophageal squamous carcinoma of high risk group and application thereof Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
Abstract
The invention belongs to the technical field of medical biology, and discloses a group of markers for screening early esophageal squamous cell carcinoma of high risk population, wherein the markers are C1QTNF6 protein or a combination of C1QTNF6 protein and RFC2 protein. The invention also discloses an application of the detection reagent of the marker in preparing a product for screening early esophageal squamous cell carcinoma, wherein the product is a test strip or a kit. According to the invention, the combination of the C1QTNF6 protein or the C1QTNF6 protein and the RFC2 protein is used as the marker of the early esophageal squamous carcinoma screening product for the first time, the marker has high sensitivity and specificity, the detection rate of early esophageal squamous carcinoma is greatly improved, the detection rate of esophageal squamous carcinoma is far higher than that of the existing clinical endoscope screening of esophageal carcinoma, and the marker can be used for large-scale screening of asymptomatic high-risk population in an esophageal squamous carcinoma high-incidence area and is beneficial to early discovery of the asymptomatic esophageal squamous carcinoma high-risk population.
Description
The application is a divisional application of an invention patent with the application number of CN 2020104612093, the application date of 2020-05-27 and the name of 'a test strip for screening early esophageal squamous carcinoma of high risk group'.
Technical Field
The invention belongs to the technical field of medical biology, and particularly relates to a test strip for screening early esophageal squamous cell carcinoma of high risk group.
Background
Esophageal squamous carcinoma is one of the six most common malignant tumors in the world, the global morbidity and mortality rate account for 6 th, China is the country with the highest esophageal squamous carcinoma morbidity and mortality rate, the national morbidity accounts for 6 th, the mortality rate accounts for 4 th, and the morbidity rate is 100 times higher than that of the western countries. More than half of 50 new esophageal squamous carcinoma patients occur in China every year around the world. The prevalence characteristics of the esophageal squamous cell carcinoma in China have obvious regional distribution difference (the incidence rates of high and low incidence areas are 500 times, particularly, the areas with the highest incidence rate and death rate of esophageal squamous cell carcinoma in Henan province, Linzhou and adjacent areas such as Hui county and Anyang in Henan province are the areas with the highest incidence rate and death rate of esophageal squamous cell carcinoma in the world) and obvious family aggregation phenomenon (40 percent of patients with positive family history), and the method prompts that environmental and genetic factors play an important role in the occurrence of the esophageal squamous cell carcinoma in China.
The prognosis of the middle and late stage esophageal squamous cell carcinoma patients is very poor, the 5-year survival rate is only about 15%, and the 5-year survival rate of the early stage esophageal squamous cell carcinoma patients can be improved to more than 90%. However, more than 95% of patients with esophageal squamous cell carcinoma who are clinically diagnosed for the first time at present are in middle and advanced stages. The main reasons for this phenomenon are that patients with early-stage esophageal squamous cell carcinoma have no obvious specific symptoms, the molecular basis for predicting the onset risk of esophageal squamous cell carcinoma is unclear, and a molecular target for early screening of asymptomatic high-risk groups is lacked. Traditionally, a "high-risk area, an asymptomatic population over 40 years old, male, smoking, drinking and having a positive family history" is generally defined as a "high risk population of esophageal squamous cell carcinoma", and is also a main object for early screening of esophageal squamous cell carcinoma. Because of the rapid popularization and application of pigment endoscopes, biopsy pathologies and endoscopic treatment technologies, the traditional early-stage detection screening means of esophageal pull-net cast-off cytology is completely replaced by the pigment endoscopes. It is important to note, however, that endoscopy requires experience, complicated equipment and procedures, and is not readily accepted by the general public due to minimal invasiveness and discomfort, particularly high cost and inefficiency. These limitations limit the popularization and application of endoscopes in early warning screening and early detection of esophageal squamous cell carcinoma in asymptomatic high risk groups.
Therefore, effective molecular diagnosis markers specific to esophageal squamous cell carcinoma are searched for early diagnosis of esophageal squamous cell carcinoma and early detection and treatment of esophageal squamous cell carcinoma, so that important technical support is provided for finally reducing morbidity and mortality of esophageal squamous cell carcinoma.
The occurrence of esophageal squamous carcinoma is a multi-stage evolutionary process which is jointly participated by a plurality of cancer suppressor genes and oncogenes and interacts with environmental factors. Cancer cells synthesize and release a group of unique secreted antigens, i.e., Tumor-associated antigens (TAAs), during their development and progression, which can identify or diagnose tumors based on their biochemical or immunological properties, such as alpha-fetoprotein (AFP), which has been used well-established in liver cancer diagnosis. Meanwhile, because tumors have heterogeneity and even the same tumor may have different antigen expression, 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 patient body by using a plurality of autoantibodies is beneficial to improving the detection rate of a certain tumor. However, the combined detection method of multiple tumor markers for early diagnosis of esophageal squamous cell carcinoma and the application of corresponding kit are still rare.
On the basis of a genomic database established by the technologies of whole genome correlation analysis, whole genome sequencing, whole genome exon sequencing and the like in the previous stage, the research team detects and analyzes early esophageal squamous carcinoma patients, asymptomatic high-risk groups in high-incidence areas and normal human serum by adopting a protein chip technology, and screens out three tumor serum markers, namely RFC2 protein, C1QTNF6 protein and CFHR3 protein, which are differentially expressed in the esophageal squamous carcinoma patients and normal people.
RFC2 is responsible for loading PCNA onto chromatin during DNA replication, is involved in DNA replication repair and cell cycle checkpoint signaling, and is involved in PCNA-associated mismatch and damage repair mechanisms following DNA damage. RFC2 can also interact with RFC3 with the oncogene c-MYC to induce cell division and proliferation; the mechanisms of C1QTNF 6-mediated tumorigenesis include cell cycle, G2M checkpoint, epithelial-mesenchymal transition, and angiogenic signaling pathways.
C1QTNF6 (recombined Human Complement C1q Tumor Necrosis Factor-Related Protein 6, Recombinant Human Complement C1q Tumor Necrosis Factor-Related Protein 6), high-expression C1QTNF6 can activate Akt signal pathway and increase Tumor angiogenesis, inhibition of C1QTNF6 can block migration and survival of liver cancer by inactivating AKT signal pathway, and over-expression of C1QTNF6 is helpful for proliferation and migration of gastric cancer cells.
CFHR3(Recombinant complementary Factor H Related Protein 3) is a Protein coding gene that plays a role in a variety of diseases, is a key component of innate immunity, and can promote or inhibit tumorigenesis. The low expression phenotype of CFHR3 is significantly enriched in important biological functions and pathways, and is associated with tumorigenesis, such as regulation of the cell activation cycle, WNT and NOTCH signaling pathways, and the like.
The RFC2 protein, the C1QTNF6 protein and the CFHR3 protein are low expressed in the serum of normal people and high expressed in the serum of esophageal squamous cell carcinoma patients, and further statistical tests show that the positive rates of the RFC2 protein, the C1QTNF6 protein and the CFHR3 protein in the population of the esophageal squamous cell carcinoma patients are obviously higher than those of the normal population, and the significant differences exist. Therefore, the RFC2 protein, the C1QTNF6 protein and the CFHR3 protein can be used for diagnosing esophageal squamous cell carcinoma. Aiming at the discovery, the research team uses RFC2 protein, C1QTNF6 protein and CFHR3 protein as detection indexes to prepare products for detecting early esophageal squamous cell carcinoma of high-risk groups, and is used for carrying out early esophageal squamous cell carcinoma screening (non-invasive, simple and economical) on asymptomatic groups in regions with high esophageal squamous cell carcinoma incidence so as to improve the early detection rate of esophageal squamous cell carcinoma, improve the survival 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 early esophageal squamous carcinoma, the other purpose of the invention is to provide an application of a detection reagent of the marker for screening early esophageal squamous carcinoma, and the third purpose of the invention is to provide a test strip for screening early esophageal squamous carcinoma of high risk population.
In order to realize the purpose of the invention, the technical scheme adopted by the invention is as follows:
the invention firstly provides a marker for screening early esophageal squamous carcinoma, wherein the marker is any one or combination of more of CFHR3 protein, C1QTNF6 protein and RFC2 protein.
The invention also provides application of the detection reagent of the marker in preparing a product for screening early esophageal squamous carcinoma.
According to the above-mentioned use, preferably, the detection reagent is an antibody specifically binding to the marker. More preferably, the antibody is a monoclonal antibody or a polyclonal antibody.
According to the above-mentioned application, preferably, the product is used for detecting the marker in a sample by immunochromatography or enzyme-linked immunoassay.
Preferably, the sample is serum, according to the above-mentioned use.
According to the above application, preferably, the product is a test strip or a kit.
The invention also provides a test strip for screening early esophageal squamous cell carcinoma of high risk population, which comprises a binding pad and a chromatography pad, wherein the binding pad is coated with a capture antibody, the capture antibody is a mixture of CFHR3 antibody A, C1QTNF6 antibody A, RFC2 antibody A, and CFHR3 antibody A, C1QTNF6 antibody A, RFC2 antibody A is provided with a detectable marker; the chromatography pad is provided with three detection lines and a quality control line, detection antibodies are fixed on the detection lines, the detection antibodies fixed on the three detection lines are respectively CFHR3 antibody B, C1QTNF6 antibody B, RFC2 antibody B, and antibody A and antibody B of the same antigen recognize different epitopes of the antigen; rabbit anti-mouse IgG is fixed on the quality control line.
According to the test strip for screening early esophageal squamous carcinoma of high risk group, preferably, the marker is colloidal gold particles; CFHR3 antibody A, C1QTNF6 antibody A, RFC2 antibody a was labeled with colloidal gold particles of different particle sizes, respectively.
According to the test strip for screening early esophageal squamous cell carcinoma of high risk population, preferably, the antibody A of the CFHR 3A, C1QTNF6 antibody A, RFC2 is respectively a CFHR3 monoclonal antibody, a C1QTNF6 monoclonal antibody and a RFC2 monoclonal antibody; wherein, the RFC2 monoclonal antibody is marked by colloidal gold particles with the particle size of 24.5nm, the C1QTNF6 monoclonal antibody is marked by colloidal gold particles with the particle size of 41nm, the CFHR3 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 test strip for screening early esophageal squamous cell carcinoma of high risk population, preferably, antibody B of CFHR 3B, C1QTNF6 and antibody B of B, RFC2 are CFHR3 polyclonal antibody, C1QTNF6 polyclonal antibody and RFC2 polyclonal antibody respectively.
According to the test strip for screening early esophageal squamous carcinoma 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 test strip for screening early esophageal squamous cell carcinoma of high risk population, preferably, the mass ratio of the CFHR3 monoclonal antibody, the C1QTNF6 monoclonal antibody and the RFC2 monoclonal antibody in the capture antibody is 1:1: 1.
According to the test strip for screening early esophageal squamous carcinoma of high risk group, preferably, 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 RFC2 polyclonal antibody, a detection line T2 fixed with C1QTNF6 polyclonal antibody and a detection line T1 fixed with CFHR3 polyclonal antibody are sequentially arranged.
According to the test strip for screening early esophageal squamous carcinoma of high risk population, 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 screening early esophageal squamous carcinoma of high risk group, preferably, the sample pad is made of water-absorbent glass fiber, the combination 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 invention also provides a kit containing the test strip for screening early esophageal squamous cell carcinoma of high risk group.
According to the kit, preferably, the kit further comprises a pipette, a serum sample adding cup and a disposable syringe.
The use method of the test strip for screening the early esophageal squamous carcinoma of the high risk group 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 screening early esophageal squamous carcinoma of high risk group comprises the following steps:
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 (3) detecting 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.
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 screening early esophageal squamous carcinoma of high risk group is as follows:
the test strip takes esophageal squamous carcinoma markers RFC2, C1QTNF6 and CFHR3 as detected objects, and utilizes the principle of antigen-antibody reaction and the colloidal gold immunochromatography technology to detect RFC2, C1QTNF6 and CFHR3 antigens 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 flowing to the conjugate pad, the colloidal gold-labeled CFHR3 monoclonal antibody, the colloidal gold-labeled C1QTNF6 monoclonal antibody and the colloidal gold-labeled RFC2 monoclonal antibody coated on the conjugate pad are dissolved, wherein, the colloidal gold-labeled CFHR3 monoclonal antibody is combined with CFHR3 antigen (CFHR3 protein) possibly contained in a serum sample to form a colloidal gold-labeled CFHR3 monoclonal antibody-CFHR 3 antigen complex, the colloidal gold-labeled C1QTNF6 monoclonal antibody is combined with C1QTNF6 antigen (C1QTNF6 protein) possibly contained in the serum sample to form a colloidal gold-labeled C1QTNF6 monoclonal antibody-C1 QTNF6 antigen complex, and the colloidal gold-labeled RFC2 monoclonal antibody is combined with RFC2 antigen (RFC2 protein) possibly contained in the serum sample to form a colloidal gold-labeled RFC2 monoclonal antibody-RFC 2 antigen complex; due to capillary effect, the three colloidal gold-labeled capture antibody-antigen complexes continue to move towards the chromatographic pad, and when the three colloidal gold-labeled capture antibody-antigen complexes move to the detection line T1, the colloidal gold-labeled CFHR3 monoclonal antibody-CFHR 3 antigen complexes are specifically combined with CFHR3 polyclonal antibody to form a solidified immune complex, and the immobilized immune complex is trapped on the detection line T1; when the test line is moved to a test line T2, the colloidal gold labeled C1QTNF6 monoclonal antibody-C1 QTNF6 antigen compound is specifically combined with the C1QTNF6 polyclonal antibody to form a solidified immune compound, and the solidified immune compound is intercepted on the test line T2; when moving to the detection line T3, the colloidal gold labeled RFC2 monoclonal antibody-RFC 2 antigen complex is specifically combined with the RFC2 polyclonal antibody to form a solidified 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, the color development conditions of the lines T1, T2 and T3 and the color development conditions of the quality control lines are detected, so that the tumor-associated antigens of CFHR3, C1QTNF6 and RFC2 in the sample to be detected can be qualitatively judged.
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 CFHR3 protein, the C1QTNF6 protein and the RFC2 protein can also be applied as detection reagents for detecting autoantibodies corresponding to the CFHR3 protein, the C1QTNF6 protein and the RFC2 protein in serum, and the autoantibodies of the three tumor-associated antigens are used as diagnosis and screening indexes of esophageal squamous cell carcinoma.
Compared with the prior art, the invention has the following positive beneficial effects:
(1) the invention uses the CFHR3 protein, the C1QTNF6 protein and the RFC2 protein for early screening and detection of the esophageal squamous carcinoma for the first time, and can effectively detect the esophageal squamous carcinoma, especially the early esophageal squamous carcinoma by detecting the expression levels of the CFHR3 protein and the C1QTNF6 protein in human serum; moreover, when the CFHR3 protein and the C1QTNF6 protein are used as a combination for screening and detecting early esophageal squamous carcinoma, the detection sensitivity is up to 88.8 percent (namely, the ratio of early esophageal squamous carcinoma to be correctly diagnosed when 3 proteins are applied to early esophageal squamous carcinoma patients are diagnosed is 88.8 percent), the specificity is up to 77.5 percent (namely, the ratio of patients without esophageal squamous carcinoma to be diagnosed when 3 proteins are applied to non-esophageal squamous carcinoma patients are diagnosed is 77.5 percent), therefore, the marker of the invention has higher sensitivity and specificity, greatly improves the detection rate of early esophageal squamous carcinoma, and the detection rate of esophageal squamous carcinoma is far higher than that of the existing clinical endoscope for screening esophageal squamous carcinoma (2 to 3 percent), can be used for large-scale screening of people at high risk of asymptomatic regions of esophageal squamous carcinoma, and is beneficial to discovering of the high risk of asymptomatic early esophageal squamous carcinoma, thereby greatly reducing the death rate of the esophageal squamous carcinoma patients and bringing great welfare for the esophageal squamous carcinoma patients and families.
(2) The test strip can rapidly detect the CFHR3 protein, the C1QTNF6 protein and the RFC2 protein in human serum, realizes the joint detection of three tumor markers, has higher sensitivity and specificity, has high detection accuracy for early cancer, greatly improves the detection rate of early esophageal squamous cell carcinoma, is favorable for the early discovery of asymptomatic high risk groups of the esophageal squamous cell carcinoma, provides an important detection means for realizing the long-term tracking of the asymptomatic high risk groups in an esophageal squamous cell carcinoma high incidence area, and has wide market prospect and social benefit.
(3) The test strip for screening early esophageal squamous carcinoma 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, and then the detection result can be judged within 15min, so that the diagnosis efficiency of early esophageal squamous carcinoma 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 screening early esophageal squamous carcinoma does not need other instruments and reagents, can be used for detection at any time by non-professional personnel, can greatly reduce the detection cost and the detection expense, and has small manual operation error and good stability.
(6) The test sample of the test strip for screening early esophageal squamous carcinoma is serum, so that the test strip has the advantages of less blood consumption, less pain for the masses and high acceptance.
Drawings
Fig. 1 is a schematic structural diagram of a test strip for screening early esophageal squamous carcinoma 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 the 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 technologies of whole genome correlation analysis, whole genome sequencing, whole genome exon sequencing and the like in the previous stage, the research team detects and analyzes early esophageal squamous carcinoma patients, asymptomatic high-risk groups in high-incidence areas and normal human serum by adopting a protein chip technology, and screens out three tumor serum markers, namely RFC2 protein, C1QTNF6 protein and CFHR3 protein, which are differentially expressed in the esophageal squamous carcinoma patients and normal people. The RFC2 protein, the C1QTNF6 protein and the CFHR3 protein are low expressed in the serum of normal people and high expressed in the serum of esophageal squamous cell carcinoma patients, and further statistical tests show that the positive rates of the RFC2 protein, the C1QTNF6 protein and the CFHR3 protein in the population of the esophageal squamous cell carcinoma patients are obviously higher than those of the normal population, and the significant differences exist. Therefore, the RFC2 protein, the C1QTNF6 protein and the CFHR3 protein can be used for diagnosing esophageal squamous cell carcinoma. Aiming at the discovery, the research team uses CFHR3 protein, C1QTNF6 protein and RFC2 protein as detection indexes to prepare a test strip for detecting early esophageal squamous cell carcinoma, and the test strip is used for detecting the expression levels of CFHR3 protein, C1QTNF6 protein and RFC2 protein in a sample to realize early screening of esophageal squamous cell carcinoma.
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 paper strip
1. Experimental Material
The bioactive raw materials used in the invention are all commercial products. Wherein, the CFHR3 monoclonal antibody is purchased from Shanghai Biotechnology, Inc. with the product number of MAL329Hu 21; the CFHR3 polyclonal antibody was purchased from Shanghai Kogymin Biotech, Inc., cat # K005454P; the C1QTNF6 monoclonal antibody is purchased from Shanghai group Hei Biotech limited, Cat MAB 16078; the C1QTNF6 polyclonal antibody is purchased from Eimeria technologies, Inc., and has a product number of H00114904-B01P; the RFC2 monoclonal antibody is purchased from Xiamen, research and science and technology Co., Ltd, and has a product number of YKA-AT3620 a; the polyclonal antibody RFC2 was purchased from Eimei technologies, Inc., under the designation H00005982-B01P.
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) Preparing a colloidal gold-labeled CFHR3 monoclonal antibody, a colloidal gold-labeled C1QTNF6 monoclonal antibody and a colloidal gold-labeled RFC2 monoclonal antibody:
dialyzing and desalting the CFHR3 monoclonal antibody solution, the C1QTNF6 monoclonal antibody solution and the RFC2 monoclonal antibody solution respectively (overnight at 4 ℃), and adjusting the protein concentration to 1 mg/ml; under electromagnetic stirring, the RFC2 monoclonal antibody is marked by colloidal gold with the particle size of 24.5nm, the C1QTNF6 monoclonal antibody is marked by colloidal gold with the particle size of 41nm, and the CFHR3 monoclonal antibody is marked by colloidal gold with the particle size of 71nm, so that three kinds of colloidal gold-marked monoclonal antibodies are obtained. 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 CFHR3 polyclonal antibody, C1QTNF6 polyclonal antibody and RFC2 polyclonal antibody to 1mg/mL, diluting rabbit anti-mouse IgG to 1.5mg/mL, respectively scribing at the positions of 3 detection lines T1, T2, T3 and quality control line C on a nitrocellulose membrane by using a scribing instrument according to the dosage of 0.1-0.5 muL/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 in a sample pad sealing solution to be soaked for 30min, and the sample pad is obtained after drying at 37 ℃; wherein the sample pad blocking solution is 0.01mol/L PBS 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 chromatography 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 chromatography 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 chromatography pad, and the binding pad is coated with a CFHR3 monoclonal antibody, a C1QTNF6 monoclonal antibody and an RFC2 monoclonal antibody which are marked by colloidal gold; 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 chromatographic 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 early esophageal squamous carcinoma of 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 strip test result
And (4) positive result: 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 (3) detecting 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.
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.
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 away from light.
Example 2: diagnostic value analysis of test strips
The test strip prepared in the embodiment 1 of the invention is used for detecting serum samples of early esophageal squamous cell carcinoma patients and normal persons which are pathologically diagnosed, and evaluating and analyzing the value of the test strip for screening and diagnosing early esophageal squamous cell carcinoma.
1. Sample source
Serum samples from an important open laboratory of esophageal squamous carcinoma in Henan, province, the first subsidiary hospital of Zhengzhou university were collected, wherein 80 parts of serum of normal persons (control group) and 80 parts of serum of patients with early esophageal squamous carcinoma (esophageal squamous carcinoma group) were collected. 80 normal human sera were from healthy physical population in the laboratory cooperative hospital physical center without any evidence of tumor association. Among 80 normal persons, 42 men and 38 women had an average age of 55.9 ± 6.2 years, with the age range of 40-75 years. 80 parts of early esophageal squamous carcinoma patient serum is from histopathologically confirmed early (stage 0 + stage I) esophageal squamous carcinoma patients, and is not treated by radiotherapy or chemotherapy. Of the 80 patients with esophageal squamous carcinoma, 57 men and 23 women had an average age of 58.5 ± 7.0 years, with the age range of 45-78 years.
2. Experimental methods
The test strip prepared in the embodiment 1 of the invention is used for respectively detecting the serum samples of the esophageal squamous cell carcinoma 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.
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 detection indexes of the 6 kinds of comparison test strips are respectively RFC2 protein, C1QTNF6 protein, CFHR3 protein, RFC2 protein + C1QTNF6 protein, C1QTNF6 protein + CFHR3 protein and RFC2 protein + CFHR3 protein. And 2 kinds of contrast test paper strips are adopted to respectively detect the serum samples of the esophageal squamous carcinoma group and the contrast group.
Respectively calculating the positive rates of the 3 antigens in the esophageal cancer group and the control group according to a result judgment standard (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); statistical tests are carried out by using the sps 26.0 software, the antigen positive rates of the esophageal squamous cell carcinoma group and the control group are compared by using a two-independent sample chi-square test method, the test level alpha is 0.05, when p is less than 0.05, the result has statistical significance, and then the diagnostic value of detecting the esophageal squamous cell carcinoma by using the autoantibody is evaluated by using a screening test method (Table 1).
3. Analysis of results
The value of the test strip in screening and diagnosing esophageal squamous cell carcinoma is evaluated according to the detection result of the test strip, and the result is shown in table 1.
TABLE 1 Authenticity evaluation of diagnostic value of different antigen combinations for esophageal squamous cell carcinoma
As can be seen from Table 1, the positive rates of the RFC2 protein, the C1QTNF6 protein and the CFHR3 protein in the esophageal squamous cell carcinoma group are respectively 43.8%, 50% and 47.5%, the positive rates of the RFC2 protein, the C1QTNF6 protein and the CFHR3 protein in the control group are respectively 13.8%, 11.3% and 7.5%, the positive rates of the RFC2 protein, the C1QTNF6 protein and the CFHR3 protein in the esophageal squamous cell carcinoma group are all higher than the positive rates in the control group, and the difference between the esophageal squamous cell carcinoma group and the control group has statistical significance (P < 0.05). Therefore, RFC2 protein, C1QTNF6 protein and CFHR3 protein can be used as an early esophageal squamous cell carcinoma diagnosis and detection index, are used for detecting early esophageal squamous cell carcinoma and have diagnosis 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 esophageal squamous cell carcinoma gradually increases, and the specificity gradually decreases; when the test strip jointly detects three antigens, namely RFC2 protein, C1QTNF6 protein and CFHR3 protein, the detection sensitivity of early esophageal squamous cell carcinoma reaches 88.8 percent, namely the percentage of esophageal squamous cell carcinoma which can be correctly diagnosed by applying the detection method to esophageal squamous cell carcinoma patients is 88.8 percent. With the increase of the test indexes of the test strip, the detection specificity is reduced, but when three antigens, namely RFC2 protein, C1QTNF6 protein and CFHR3 protein are jointly detected, the detection specificity can still reach 77.5 percent, namely when the method is adopted to detect the patients with the non-esophageal squamous cell carcinoma, the percentage of the patients who are correctly diagnosed as healthy people is 77.5 percent.
Moreover, compared with the test strip for detecting one tumor-associated antigen, when the test strip for jointly detecting 3 antigens, namely the RFC2 protein, the C1QTNF6 protein and the CFHR3 protein, is used for diagnosing early esophageal squamous cell carcinoma, the sensitivity is respectively 2.03 times, 1.78 times and 1.87 times of that for detecting single indexes, namely the RFC2 protein, the C1QTNF6 protein and the CFHR3 protein.
Therefore, the RFC2 protein, the C1QTNF6 protein and the CFHR3 protein in the serum sample are jointly detected to screen and diagnose the early esophageal squamous carcinoma, so that the sensitivity of diagnosis can be greatly improved on the premise of ensuring the diagnosis specificity; the method has good diagnosis and application values for evaluating the risk of esophageal squamous cell carcinoma of a subject to be detected.
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 indexes detected by the test strip, the yotans index is continuously increased and gradually approaches to 1, which indicates that the combination of the 3 antigens has better diagnostic value in diagnosing and screening early esophageal squamous cell carcinoma.
In conclusion, the screening and diagnosis of early esophageal squamous carcinoma are carried out by jointly detecting RFC2 protein, C1QTNF6 protein and CFHR3 protein in a serum sample, so that higher specificity and sensitivity can be ensured, and the method has better diagnosis and application values for esophageal squamous carcinoma risk assessment of a to-be-detected object.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.
Claims (4)
1. The application of a detection reagent for detecting the expression of a marker for screening early esophageal squamous carcinoma in preparing a product for screening early esophageal squamous carcinoma is disclosed, wherein the marker is the combination of C1QTNF6 protein and RFC2 protein; the detection sample of the product is serum.
2. The use of claim 1, wherein the detection reagent is an antibody that specifically binds to the marker.
3. The use of claim 1, wherein said product is used for detection of said marker in a sample by immunochromatography or enzyme-linked immunoassay.
4. The use of any one of claims 1 to 3, wherein the product is a test strip or a kit.
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