CN111562379A - Multi-index joint detection colloidal gold test strip for early esophageal cancer screening - Google Patents
Multi-index joint detection colloidal gold test strip for early esophageal cancer screening Download PDFInfo
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Abstract
The invention belongs to the technical field of bioengineering and biomedicine, particularly relates to the field of oncology, and more particularly relates to a group of multi-index joint detection colloidal gold test strips for early esophageal cancer screening. The colloidal gold test strip jointly utilizes specific detection reagents of four antigens, namely KDM2A, SLC2A3, HOXC13 and TMEM132C to detect early esophageal cancer, the detection sensitivity is up to 93.7%, and the specificity is up to 71.4%.
Description
Technical Field
The invention belongs to the technical field of bioengineering and biomedicine, particularly relates to the field of oncology, and more particularly relates to a group of multi-index joint detection colloidal gold test strips for early esophageal cancer screening.
Background
Since the 20 th century, the incidence and mortality of esophageal cancer have been on the rise, and their geographical distribution is unbalanced. According to GLOBOCAN2018, 57.2 ten thousand new cases of global esophageal cancer are shown to be located at the 6 th position of malignant tumor; the death case is 50.9 thousands of people, and the 7 th position of malignant tumor is located. China is one of the countries with high incidence of esophageal cancer. The national statistical data show that the incidence of esophageal cancer in 2018 is 18.85/10 ten thousand, the 5 th position of malignant tumor, the mortality rate is 14.11/10 ten thousand, and the 4 th position of the cause of death of malignant tumor. And 90% of domestic esophageal cancer is squamous carcinoma, which seriously threatens the health and life of people. Most patients with esophageal cancer are in the middle and late stages at the time of treatment, are not suitable for surgical treatment, and have a 5-year survival rate of less than 20%, while for patients diagnosed with early esophageal cancer, the 10-year survival rate can reach 95% through active comprehensive treatment. Therefore, early diagnosis and treatment of esophageal cancer is very critical. The early diagnosis method of esophageal cancer mainly comprises imaging examination, cytology examination, endoscopy examination and colloidal gold test paper detection. The first three methods are easy to cause discomfort for general population, so that the compliance of patients is poor, and the colloidal gold test paper has the characteristics of convenience and economy in detection, no wound, timely response of disease changes and the like. However, the existing colloidal gold test paper can only detect one index, so that in the process of detecting multiple indexes, the detection efficiency is relatively low, the detection efficiency is seriously influenced, and a brand-new multi-index joint detection colloidal gold test paper strip is urgently needed.
The research team is dedicated to the research of the esophageal cancer for a long time, a large database and a biological sample library of patients with the esophageal cancer and healthy people are established, four tumor-associated antigens (TAAs) are screened out by using an autoantibody chip technology on the basis of a genomic database established by using technologies such as whole genome association analysis, whole genome sequencing, whole genome exon sequencing and the like, the four TAAs are KDM2A, SLC2A3, HOXC13 and TMEM132C respectively, and the four TAA antigens can be detected before the esophageal mucosa squamous epithelium canceration occurs, and the expression products have higher specific sensitivity on patients with the esophageal cancer and precancerous lesion, so the esophageal cancer early screening colloidal gold test strip prepared by using the antibodies specifically combined with the four TAA antigens can be applied to the screening of the early esophageal cancer of asymptomatic people in high-incidence areas, has important significance for improving the survival rate of patients with esophageal cancer.
Disclosure of Invention
Aiming at the problems and the defects of the prior art, the invention aims to provide a group of multi-index joint detection colloidal gold test strips for early esophageal cancer screening.
Based on the purpose, the invention adopts the following technical scheme:
the invention provides application of a detection reagent capable of specifically detecting KDM2A, SLC2A3, HOXC13 and TMEM132C or gene expression products of the group in a colloidal gold test strip for early esophageal cancer screening.
According to the above application, preferably, the detection reagent comprises a molecule for detecting whether the expression product of KDM2A is expressed, a molecule for detecting whether the expression product of SLC2A3 is expressed, a molecule for detecting whether the expression product of HOXC13 is expressed and a molecule for detecting whether the expression product of TMEM132C is expressed.
According to the above application, preferably, the molecule for detecting whether the KDM2A expression product is expressed is KDM2A antibody capable of specifically binding to KDM2A expression product; the molecule for detecting whether the expression product of the SLC2A3 is expressed is SLC2A3 antibody capable of being specifically combined with the expression product of SLC2A 3; the molecule for detecting whether the expression product of the HOXC13 is expressed is a HOXC13 antibody capable of specifically binding to the HOXC13 expression product; the molecule for detecting whether the expression product of TMEM132C is expressed is TMEM132C antibody capable of specifically binding with the expression product of TMEM 132C.
According to the application, preferably, the detection object of the colloidal gold test strip for early esophageal cancer screening is a serum sample.
The invention also provides a colloidal gold test strip for screening early esophageal cancer, which contains specific detection reagents for detecting KDM2A expression products, SLC2A3 expression products, HOXC13 expression products and TMEM132C expression products.
According to the above colloidal gold test strip for screening early esophageal cancer, preferably, the detection reagent comprises a molecule for detecting whether a KDM2A expression product is expressed, a molecule for detecting whether an SLC2A3 expression product is expressed, a molecule for detecting whether an HOXC13 expression product is expressed, and a molecule for detecting whether a TMEM132C expression product is expressed.
According to the colloidal gold test strip for screening early esophageal cancer, preferably, the molecule for detecting whether the KDM2A expression product is expressed is a KDM2A antibody capable of being specifically bound with the KDM2A expression product; the molecule for detecting whether the expression product of the SLC2A3 is expressed is SLC2A3 antibody capable of being specifically combined with the expression product of SLC2A 3; the molecule for detecting whether the expression product of the HOXC13 is expressed is a HOXC13 antibody capable of specifically binding to the HOXC13 expression product; the molecule for detecting whether the expression product of TMEM132C is expressed is TMEM132C antibody capable of specifically binding with the expression product of TMEM 132C.
According to the above colloidal gold test strip for screening early esophageal cancer, preferably, the test strip comprises a bottom plate, and a sample pad, a gold-labeled pad, a nitrocellulose membrane and a water absorption pad which are sequentially laid on the bottom plate along the length direction; four test lines and a control line are sequentially arranged on the nitrocellulose membrane; the gold label pad is adsorbed with a colloidal gold-labeled monoclonal antibody which can be specifically combined with a gene expression product to be detected; KDM2A polyclonal antibody capable of being specifically combined with KDM2A expression product, SLC2A3 polyclonal antibody capable of being specifically combined with SLC2A3 expression product, HOXC13 polyclonal antibody capable of being specifically combined with HOXC13 expression product and TMEM132C polyclonal antibody capable of being specifically combined with TMEM132C expression product are respectively fixed on the four test line positions; and a goat anti-mouse secondary antibody which can be specifically combined with the monoclonal antibody is fixed on the control line, and the goat anti-mouse secondary antibody is a goat anti-mouse polyclonal antibody.
According to the above colloidal gold test strip for early esophageal cancer screening, preferably, the monoclonal antibodies include KDM2A monoclonal antibody, SLC2A3 monoclonal antibody, HOXC13 monoclonal antibody and TMEM132C monoclonal antibody.
The invention provides an application of a detection reagent capable of specifically detecting KDM2A expression products in a colloidal gold test strip for screening early esophageal cancer.
The invention provides an application of a detection reagent capable of specifically detecting an SLC2A3 expression product in a colloidal gold test strip for screening early esophageal cancer.
The invention provides an application of a detection reagent capable of detecting a HOXC13 expression product in early esophageal cancer screening colloidal gold test strips.
The invention provides an application of a detection reagent capable of specifically detecting an expression product of TMEM132C in a colloidal gold test strip for screening early esophageal cancer.
Compared with the prior art, the invention has the following beneficial effects:
(1) the monoclonal antibodies corresponding to the four antigens KDM2A, SLC2A3, HOXC13 and TMEM132C are combined for the first time and are jointly used for detecting the esophageal cancer, particularly early esophageal cancer, and the detection sensitivity of the test strip for the early esophageal cancer is up to 93.7 percent, and the specificity of the test strip for the early esophageal cancer is up to 71.4 percent.
(2) The test strip prepared by the invention can be used for simultaneously detecting four antigens in a serum sample, has high detection success rate, good technical reproducibility, less material consumption, low cost, simple operation, convenient and quick use, greatly improves the detection efficiency and the diagnosis efficiency of esophageal cancer and early esophageal cancer, and is easy to popularize and apply.
Drawings
FIG. 1 is a schematic structural diagram of the test strip of the present invention;
FIG. 3 is a graph showing the difference in expression of four antigens in early esophageal cancer groups and healthy subjects;
FIG. 2 is a ROC plot of specific detection reagents for KDM2A, SLC2A3, HOXC13, TMEM132C used alone for early esophageal cancer screening;
FIG. 4 is a ROC plot of different combinations of antigen-specific detection reagents for early esophageal cancer screening.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the scope of the present invention is not limited thereto.
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 colloidal gold test strip for early esophageal cancer screening
1. Structural composition of colloidal gold test strip
A colloidal gold test strip for early esophageal cancer screening is shown in figure 1 and comprises a PVC base plate, a sample pad, a gold label pad, a nitrocellulose membrane and a water absorption pad, wherein the sample pad, the gold label pad, the nitrocellulose membrane and the water absorption pad are sequentially paved on the PVC base plate along the length direction; the initial end of the water absorption pad is lapped at the tail end of the nitrocellulose membrane; four test lines (T1, T2, T3 and T4) and a control line (C line) are sequentially arranged on the nitrocellulose membrane, the T1 line is arranged at one end, close to the gold-labeled pad, of the nitrocellulose membrane, and the C line is arranged at one end, close to the absorption pad, of the nitrocellulose membrane.
The length of the PVC plate is 9cm, the lengths of the sample pad, the gold mark pad, the nitrocellulose membrane and the water absorption pad are sequentially 2cm, 1cm, 5cm and 2cm, the overlapping length of the sample pad and the gold mark pad is 0.3cm, the overlapping length of the gold mark pad and the nitrocellulose membrane is 0.3cm, and the overlapping length of the nitrocellulose membrane and the water absorption pad is 0.4 cm. The distances between the T1, the T2, the T3, the T4 and the C line and the starting end of the water absorption pad are 3.9cm, 3.2cm, 2.5cm, 1.8cm and 1.1cm in sequence.
The gold-labeled pad is a glass fiber material layer, the glass fiber is fully soaked in a mixed liquid containing a colloidal gold-labeled KDM2A monoclonal antibody, a colloidal gold-labeled SLC2A3 monoclonal antibody, a colloidal gold-labeled HOXC13 monoclonal antibody and a colloidal gold-labeled TMEM132C monoclonal antibody, the gold-labeled pad is taken out after full soaking, and then the gold-labeled pad is prepared by freeze drying, wherein the concentration of each monoclonal antibody in the mixed liquid is 10 mug/mL. After the treatment, the gold label pad is adsorbed with the colloidal gold labeled monoclonal antibody which can be specifically combined with the gene expression product to be detected; the monoclonal antibodies include KDM2A monoclonal antibody, SLC2A3 monoclonal antibody, HOXC13 monoclonal antibody and TMEM132C monoclonal antibody.
KDM2A polyclonal antibody, SLC2A3 polyclonal antibody, HOXC13 polyclonal antibody, TMEM132C polyclonal antibody and goat anti-mouse secondary antibody are sprayed into five lines in sequence on a nitrocellulose membrane by a film spraying machine, wherein the five lines are a test line T1, a test line T2, a test line T3, a test line T4 and a control line (line C), and then the nitrocellulose membrane is dried in vacuum. Namely, KDM2A polyclonal antibody capable of being specifically combined with KDM2A expression product is fixed on the T1 test line, and the concentration of KDM2A polyclonal antibody sprayed at the position of the T1 test line is 6 mug/mL; the test line of T2 is fixed with SLC2A3 polyclonal antibody which can be specifically combined with the expression product of SLC2A3, and the concentration of the SLC2A3 polyclonal antibody sprayed at the position of the test line of T2 is 6 mug/mL; the HOXC13 polyclonal antibody capable of being specifically combined with the HOXC13 expression product is fixed on the T3 test line, and the concentration of the HOXC13 polyclonal antibody sprayed at the position of the T3 test line is 6 mug/mL; TMEM132C polyclonal antibody capable of specifically binding with the TMEM132C expression product is fixed on the T4 test line, and the concentration of the TMEM132C polyclonal antibody sprayed at the position of the T4 test line is 6 mu g/mL. The control line position is fixed with a goat anti-mouse polyclonal antibody which can be specifically combined with the monoclonal antibody, namely a goat anti-mouse secondary antibody, and the concentration of the goat anti-mouse secondary antibody sprayed at the control line position is 1 mg/mL.
Reagent sources or methods of preparation
2.1 sources of antibodies
2.1.1 preparation of monoclonal antibodies
The monoclonal antibodies include a KDM2A monoclonal antibody, an SLC2A3 monoclonal antibody, an HOXC13 monoclonal antibody and a TMEM132C monoclonal antibody, the preparation processes of the four monoclonal antibodies are the same, and the preparation process of the monoclonal antibodies is described below by taking the KDM2A monoclonal antibody as an example. The preparation method of the monoclonal antibody comprises the following steps:
(1) animal immunization: balb/c mice were immunized with the recombinant bovine KDM2A protein by four immunizations. Immunizing Balb/c mice with 0.2mL Freund's Complete Adjuvant (FCA) and 0.2mg recombinant bovine KDM2A protein for the first time; immunizing Balb/c mice with 0.2mL Freund's Incomplete Adjuvant (FIA) and 0.2mg recombinant bovine KDM2A protein for the second time; immunizing Balb/c mice with 0.2mL Freund's Incomplete Adjuvant (FIA) and 0.2mg of recombinant bovine KDM2A protein for the third time; mice were immunized the fourth time with 0.4mg of recombinant bovine KDM2A protein.
(2) Preparation and cell fusion of myeloma cell SP 2/0: one week before cell fusion, myeloma cells SP2/0 frozen in liquid nitrogen were thawed. The myeloma cell SP2/0 is cultured in DMEM medium containing 20% fetal calf serum and 1% HT, and the myeloma cell SP2/0 cultured for one week is fused with spleen cells of an immunized mouse to form a hybridoma.
(3) Screening of positive hybridoma cells and purification of monoclonal antibodies: screening the grown hybridoma cells by adopting an indirect ELISA method, and coating the hybridoma cells with recombinant bovine KDM2A protein of 6 microgram/mL. And (3) carrying out secondary subcloning on the hybridoma cells which are detected to be positive by adopting a limiting dilution method, and carrying out amplification culture on the monoclonal cells which are good in form and grow vigorously to prepare the ascites monoclonal antibody.
The specific process is as follows: culturing the hybridoma cells in a selective medium (HAT medium), dying unfused cells, surviving the hybridoma cells, screening positive clones to obtain a plurality of different monoclonal antibodies, screening the monoclonal antibodies by enzyme-linked immunosorbent assay (ELISA) according to different antigenic determinants of the monoclonal antibodies, and selecting the hybridoma cells of the KDM2A monoclonal antibody.
Injecting the selected hybridoma cell of KDM2A monoclonal antibody into guinea pig abdominal cavity, culturing for 72h, collecting whole blood by heart blood collection method, naturally coagulating at room temperature, placing in ice at 4 deg.C for blood coagulation, separating serum, and inactivating at 56 deg.C for 30 min. The inactivation treated serum was subjected to crude extraction of monoclonal antibody using DEAE-cellulose (DE 32) in the following manner: weighing 50g of DEAE-cellulose, placing the DEAE-cellulose in a 1000mL beaker, washing with distilled water to remove floating fine particles, adjusting the pH to 7.4 with 0.05mol/L phosphate buffer solution, mixing serum and the DEAE-cellulose after pH adjustment, placing the mixture at 4 ℃ for standing and adsorption for 1h, then performing suction filtration with a Brinell filter funnel, and collecting filtrate to obtain the crude monoclonal antibody.
The crude monoclonal antibody is eluted and separated by a sephadex packing chromatographic column, and the specific process comprises the following steps: adding 0.1mol/L glycine 0.1mL into the crude polyclonal antibody, adding sodium bicarbonate to adjust the pH value to 7.4, adding distilled water until the total volume of the crude monoclonal antibody is 15mL, eluting and separating by adopting a sephadex packing chromatographic column with the inner diameter of 1cm and the outer diameter of 1.5cm, wherein the eluent is 0.1mol/L Tris-HCl buffer salt solution with the pH value of 8.0, the salt solution is 0.14mol/L NaCl aqueous solution, the elution flow rate is 0.47mL/min, and the KDM2A monoclonal antibody is collected in a time period of 5-11 min, and the KDM2A monoclonal antibody is prepared by separation and purification.
(4) Identification of monoclonal antibodies: and (3) carrying out immunoblotting analysis on the prepared ascites monoclonal antibody by using the recombinant bovine KDM2A protein, carrying out DAB color development and chemiluminescence color development, and identifying the monoclonal antibody.
Polyclonal antibody origin or preparation
The polyclonal antibodies include KDM2A polyclonal antibody, SLC2A3 polyclonal antibody, HOXC13 polyclonal antibody and TMEM132C polyclonal antibody. The KDM2A polyclonal antibody is purchased from Thermo Fisher company, and has the product number of PA 5-40421; SLC2A3 polyclonal antibody was purchased from Thermo Fisher, cat # OSG 00013G; HOXC13 polyclonal antibody was purchased from LI-COR, cat # 926-42212; the polyclonal antibody TMEM132C was purchased from Atlas under the designation HPA 015633.
Source of goat anti-mouse secondary antibody
The goat anti-mouse secondary antibody was purchased from Shanghai West Tang Biotech Co., Ltd, cat # C0252.
Preparation method of colloidal gold labeled monoclonal antibody
The preparation method of the colloidal gold labeled monoclonal antibody is described below by taking the KDM2A monoclonal antibody as an example, and comprises the following steps:
(1) preparing a colloidal gold solution: heating 100mL of 0.01 wt% chloroauric acid aqueous solution to boiling, adding 1mL of 1wt% citric acid aqueous solution into the boiling solution, stirring continuously until the gold chloroauric acid aqueous solution turns red within 2min, stirring at medium speed, boiling for 5min until the color is stable, cooling, recovering to original volume with ultrapure water, adding 0.02 wt% NaN3And (4) performing corrosion prevention, filtering and sterilizing through a filter membrane of 0.22 mu m to obtain a colloidal gold solution, and storing in a dark place at 4 ℃. Wherein the particle size of the colloidal gold is 30-80 nm, and the colloidal gold particles in the particle size range are purple red.
(2) Preparation of colloidal gold-labeled monoclonal antibody: and (2) taking 10mL of the colloidal gold solution prepared in the step (1), adjusting the pH value to 5-9, slowly dropwise adding a KDM2A monoclonal antibody into the colloidal gold solution, dropwise adding an lmg KDM2A monoclonal antibody for about 5min, then stirring for 30min by magnetic force, then adding 1mL of 1wt% BSA solution for sealing, and centrifugally resuspending the sealed solution to prepare the colloidal gold-labeled KDM2A monoclonal antibody.
Detection principle of colloidal gold test strip
Dropping a serum solution to be detected on a sample pad, moving the serum solution to be detected towards the water absorption pad under the capillary siphon effect, forming a conjugate by an antigen to be detected possibly contained in the sample and a colloidal gold-labeled monoclonal antibody when the serum solution passes through a gold-labeled pad, continuously migrating the conjugate to the positions of detection lines T1, T2, T3 and T4, and combining polyclonal antibodies fixed at the positions of the detection lines T1, T2, T3 and T4 with other sites on the antigen to be detected to form a monoclonal antibody-antigen-polyclonal antibody conjugate, so that the detection lines are colored (red); and if the liquid to be detected does not contain the antigen to be detected, the detection line does not develop color.
Meanwhile, the colloidal gold labeled monoclonal antibody is electrophoresed to the position of the control line along with the liquid and is captured by the goat anti-mouse secondary antibody which is fixed at the position of the control line and can be specifically combined with the monoclonal antibody, so that the control line is developed (red); if the control line is colored, a valid result is obtained; if the control line does not develop a color, a false result is required and re-inspection is required.
Application method of colloidal gold test strip
Taking 0.5mL of serum of an individual to be detected, and mixing the serum with a sample diluent according to a volume ratio of 1: 50 are mixed and diluted to be used as a test solution, wherein the sample diluent is PBS buffer solution containing 1wt% BSA and 0.05% (v/v) Tween 20.
And then directly dipping the serum sample diluent by using the sample pad or dropwise adding the serum sample diluent to the sample pad, standing for 5min, and observing a color development result.
Determination of detection result
Positive results: the control line is colored, and any one of the four test lines of T1, T2, T3 and T4 is colored, namely positive.
Negative results: the control line is colored, and the four test lines of T1, T2, T3 and T4 are not colored, namely negative.
Invalid result: the control line is not colored, i.e., is invalid and needs to be re-detected.
Example 2 analysis of the effectiveness of colloidal gold test strips for early esophageal cancer screening
The test strip is used for detecting serum samples of early esophageal cancer patients and healthy subjects so as to evaluate and analyze the effectiveness analysis of the test strip on early esophageal cancer screening.
Serum sample collection
255 healthy physical examiners from the laboratory cooperation hospital physical examination center where the inventor is located are collected, and the selected healthy physical examiners do not find any tumor-related lesion through physical examination. Meanwhile, the same number of early-stage (stage 0 + stage I) esophageal cancer patients from the laboratory cooperation hospital are collected, and the selected early-stage esophageal cancer patients are early-stage esophageal cancer patients diagnosed by pathological sections and have not received radiotherapy or chemotherapy treatment.
And (3) extracting 5mL of venous blood of a person to be detected on an empty stomach, placing the blood into a centrifuge tube, standing the tube for 30 minutes at room temperature, centrifuging the tube for 5 minutes at 2000rpm/min, sucking upper serum, subpackaging the tube with 0.5mL per tube, and storing the tube in a refrigerator at the temperature of-80 ℃ for later use.
Effectiveness evaluation of colloidal gold test strip for early esophageal cancer screening
2.1 differential expression analysis of four antigens in esophageal cancer patients and healthy subjects
The expression levels of four tumor-associated antigens including KDM2A, SLC2A3, HOXC13 and TMEM132C in the serum of 255 healthy subjects and the serum of 255 early esophageal cancer patients are detected by adopting chromatography, and the absorbance is recorded, wherein the absorbance can reflect the expression levels of the four antigens. The mean expression level distribution of the four tumor-associated antigens in the early esophageal cancer patients and the healthy subjects is drawn by using the MedCalc software, and the result is shown in fig. 2, as can be seen from fig. 2, the mean mAU value of the four tumor-associated antigens in the early esophageal cancer patients is about 150, and the mean mAU value of the four tumor-associated antigens in the healthy subjects is about 45, which indicates that the mean expression level of the four tumor-associated antigens in the early esophageal cancer patients is higher than that of the healthy subjects, thereby reflecting that the four tumor-associated antigens of KDM2A, SLC2A3, HOXC13 and TMEM132C can be used as detection markers of early esophageal cancer, namely the four tumor-associated antigens can be used as detection indexes for early esophageal cancer screening.
2.2 effectiveness analysis of four antigen-specific detection reagents separately used for early esophageal cancer detection
Referring to the preparation and application methods of the test paper strip in example 1, the sera of the 255 healthy subjects and the 255 early esophageal cancer patients were tested for esophageal cancer by using the specific detection reagents of the four antigens, namely KDM2A, SLC2A3, HOXC13 and TMEM132C, and the test results are shown in Table 1.
As can be seen from Table 1, when the four antigen-specific detection reagents KDM2A, SLC2A3, HOXC13 and TMEM132C are detected separately, the positive rate of the early esophageal squamous cell carcinoma diagnosis is between 30.6% and 47.1%, the detection sensitivity is between 30.6% and 43.1%, and the detection specificity is between 90.2% and 94.1%; wherein, the jotans index is the sum of the sensitivity and specificity minus 1 in statistics, the numerical range is 0-1, the more the jotans index is close to 1, the higher the diagnostic value is, the higher the application value of the method is, and the jotans index of the four specific detection reagents of the invention used alone is between 0.208 and 0.347, and as can be seen from fig. 3, the area under the ROC curve of the detection of early esophageal squamous cell carcinoma of the four specific detection reagents used alone is between 0.639 and 0.698. It can be seen that when the four antigen-specific detection reagents, namely KDM2A, SLC2A3, HOXC13 and TMEM132C, are used for detecting early esophageal cancer independently, although the four antigen-specific detection reagents have better specificity, the sensitivity and the overall diagnostic efficacy are not obvious, so that the four antigen-specific detection reagents, namely KDM2A, SLC2A3, HOXC13 and TMEM132C, have limited diagnostic value on early esophageal cancer when being used for detecting the four antigen-specific detection reagents independently.
2.3 the effectiveness analysis of the combination of four antigen-specific detection reagents for early esophageal cancer detection
To further investigate the diagnostic value of different combinations of specific detection reagents for early esophageal cancer, 255 cases of sera of healthy subjects and 255 cases of sera of early esophageal cancer patients were tested by using different combinations of antigen-specific detection reagents according to the preparation and application methods of the test paper strip in example 1, and the test results are shown in table 2 and fig. 4.
As shown in Table 2, as the number of specific detection reagents for the antigen to be detected increases, the positive rate of diagnosis of early esophageal cancer increases from 30.6% to 93.7%, i.e., the detection sensitivity increases from 30.6% to 93.7%. When the specific detection reagents of four antigens to be detected, namely KDM2A, SLC2A3, HOXC13 and TMEM132C, are used together, the detection sensitivity of the test strip to early esophageal cancer reaches 93.7 percent, namely when the test strip is used for detecting early esophageal cancer, the percentage of esophageal cancer can be correctly diagnosed to be 93.7 percent.
Although the detection specificity for early esophageal cancer is gradually reduced along with the increase of the number of specific detection reagents for the four antigens to be detected, the detection specificity for early esophageal cancer can still reach 71.4% when the specific detection reagents for the four antigens to be detected are used in combination, and the result shows that the test strip can be used for correctly diagnosing early esophageal cancer, and the percentage of esophageal cancer which is not suffered from the esophageal cancer is 71.4%. Therefore, when the monoclonal antibody combination corresponding to the four antigens of KDM2A, SLC2A3, HOXC13 and TMEM132C is used for early esophageal cancer detection, the sensitivity of diagnosis can be greatly improved on the premise of ensuring the specificity of diagnosis. Therefore, the monoclonal antibodies corresponding to the four antigens KDM2A, SLC2A3, HOXC13 and TMEM132C are combined for detecting the four antigens in the serum to be detected, so that the high specificity can be maintained, the diagnosis sensitivity can be improved, and the kit has good diagnosis and application values for evaluating the risk of esophageal cancer of a to-be-detected object.
In addition, with the increase of the number of the specific detection reagents corresponding to the autoantibody markers, the john's index is continuously increased and gradually tends to 1, and the fact that the four specific detection reagents are used together has higher diagnostic value for early esophageal squamous cell carcinoma is shown.
In addition, as can be seen from fig. 4, with the increase of the number of the four antigen detection reagents, the area under the ROC curve is increased from 0.64 to 0.85, which indicates that the specific detection reagents of the four antigens are jointly used for detecting early esophageal cancer, so that the high specificity can be maintained and the diagnostic sensitivity can be improved, and that the test strip provided by the invention adopts the specific detection reagents of the four antigens, namely KDM2A, SLC2A3, HOXC13 and TMEM132C, to jointly detect early esophageal cancer, so that the test strip has high judgment accuracy and diagnostic value and is suitable for large-scale screening of asymptomatic people in a high esophageal cancer incidence area.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the present invention, but rather as the following description is intended to cover all modifications, equivalents and improvements falling within the spirit and scope of the present invention.
Claims (9)
1. The application of the detection reagent capable of specifically detecting KDM2A, SLC2A3, HOXC13 and TMEM132C or gene expression products of the group in the colloidal gold test strip for early esophageal cancer screening.
2. The use according to claim 1 wherein the detection reagents comprise a molecule which detects the expression of KDM2A expression product, a molecule which detects the expression of SLC2A3 expression product, a molecule which detects the expression of HOXC13 expression product and a molecule which detects the expression of TMEM132C expression product.
3. The use according to claim 2, characterized in that the molecule for detecting the expression of the KDM2A expression product is a KDM2A antibody capable of binding specifically to the KDM2A expression product; the molecule for detecting whether the expression product of the SLC2A3 is expressed is an SLC2A3 antibody capable of being specifically combined with the expression product of the SLC2A 3; the molecule for detecting whether the HOXC13 expression product is expressed is HOXC13 antibody capable of specifically binding with the HOXC13 expression product; the molecule for detecting whether the expression product of the TMEM132C is expressed is TMEM132C antibody capable of being specifically bound with the expression product of the TMEM 132C.
4. The use of claim 3, wherein the colloidal gold test strip for early esophageal cancer screening is used for detecting serum samples.
5. The colloidal gold test strip for early esophageal cancer screening is characterized by comprising specific detection reagents for detecting KDM2A expression products, SLC2A3 expression products, HOXC13 expression products and TMEM132C expression products.
6. The colloidal gold test strip for screening early esophageal cancer according to claim 5, wherein the detection reagent comprises a molecule for detecting whether KDM2A expression product is expressed, a molecule for detecting whether SLC2A3 expression product is expressed, a molecule for detecting whether HOXC13 expression product is expressed, and a molecule for detecting whether TMEM132C expression product is expressed.
7. The colloidal gold test strip for screening early esophageal cancer according to claim 6, wherein the molecule for detecting whether the KDM2A expression product is expressed is KDM2A antibody capable of specifically binding with KDM2A expression product; the molecule for detecting whether the expression product of the SLC2A3 is expressed is an SLC2A3 antibody capable of being specifically combined with the expression product of the SLC2A 3; the molecule for detecting whether the HOXC13 expression product is expressed is HOXC13 antibody capable of specifically binding with the HOXC13 expression product; the molecule for detecting whether the expression product of the TMEM132C is expressed is TMEM132C antibody capable of being specifically bound with the expression product of the TMEM 132C.
8. The colloidal gold test strip for screening early esophageal cancer according to claim 7, wherein the test strip comprises a bottom plate, and a sample pad, a gold label pad, a nitrocellulose membrane and a water absorption pad which are sequentially laid on the bottom plate along the length direction; four test lines and a control line are sequentially arranged on the nitrocellulose membrane; the gold label pad is adsorbed with a colloidal gold-labeled monoclonal antibody which can be specifically combined with a gene expression product to be detected; the four test lines are respectively fixed with a KDM2A polyclonal antibody capable of being specifically combined with a KDM2A expression product, an SLC2A3 polyclonal antibody capable of being specifically combined with an SLC2A3 expression product, an HOXC13 polyclonal antibody capable of being specifically combined with an HOXC13 expression product and a TMEM132C polyclonal antibody capable of being specifically combined with a TMEM132C expression product; and a goat anti-mouse secondary antibody which can be specifically combined with the monoclonal antibody is fixed on the control line.
9. The colloidal gold test strip for screening of early esophageal cancer according to claim 8, wherein the monoclonal antibodies comprise KDM2A monoclonal antibody, SLC2A3 monoclonal antibody, HOXC13 monoclonal antibody and TMEM132C monoclonal antibody.
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