CN113075413B - Early esophageal squamous carcinoma screening kit based on group of tumor-associated antigens - Google Patents
Early esophageal squamous carcinoma screening kit based on group of tumor-associated antigens Download PDFInfo
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Abstract
The invention belongs to the technical field of medical biology, and particularly discloses an early esophageal squamous carcinoma screening kit based on a group of tumor-associated antigens, which comprises a solid-phase carrier and the tumor-associated antigens coated on the solid-phase carrier, wherein the tumor-associated antigens consist of a P53 protein, a ZBTB20 protein, an ERICH3 protein and a C15orf57 protein. Further, the kit further comprises a sample diluent, a second antibody diluent, positive control serum, negative control serum, a color development liquid, a stop solution and a washing solution. The ELISA kit can effectively detect esophageal cancer, particularly early esophageal cancer, has the detection sensitivity as high as 82% and the specificity as high as 84%, can be used for large-scale screening of asymptomatic people in esophageal cancer high-incidence areas, and is beneficial to screening and early discovery of asymptomatic high-risk people.
Description
Technical Field
The invention belongs to the technical field of medical biology, and particularly relates to application of FCRL1 protein or a specific antibody thereof in cardia cancer screening.
Background
Esophageal cancer is one of the six most common malignant tumors in the world, has obvious regional distribution difference and family aggregation phenomenon, is the country with the highest esophageal cancer incidence and mortality in the world, has about 50 ten thousand cases of cardiac esophageal cancer patients every year in the world, and more than half of the cases occur in China. In China, half of the patients occur in the Taihang mountain area where Henan, Hebei and Shanxi three provinces are bordered, wherein Henan Linxian county is the area in the world where the incidence and death rate of esophageal cancer are the highest and is hundreds of times higher than that of western countries. The esophageal cancer is a disease with Chinese characteristics, the pathological type of the esophageal cancer is mainly squamous cell carcinoma in China, the proportion of the esophageal cancer is up to 97 percent, and the esophageal cancer is greatly different from the main pathological type in the west (80 percent of adenocarcinoma). In addition, the method has great difference with the population of western countries in terms of circulation characteristics, disease risk factors and the like.
The prognosis of esophageal cancer is poor, and the 5-year survival rate of patients in middle and late stages after operation is only about 10 percent; the 5-year survival rate of early esophageal cancer (the focus is limited to a mucous membrane layer and a submucosa layer and is not accompanied with lymph node metastasis) can reach 95 percent, so that early diagnosis and early treatment of the esophageal cancer are important influencing factors for the prognosis of the esophageal cancer. However, because early patients have no specific symptoms, most of the patients are in the middle and late stages when the patients visit, and the detection rate of early esophageal cancer is less than 2%, so that the death rate of the patients in the high-incidence region of the esophageal cancer in China is not obviously improved. An important method for finding early esophageal cancer in endoscopic screening is that the national generally defines 'asymptomatic people over 40 years old, male, smoking, drinking and having positive family history' as 'high-risk or high-risk people' in esophageal cancer high-incidence areas, and the people are screened early by pigment endoscopy and mucosa biopsy. However, endoscopy is invasive and expensive, the detection rate of early cancer is only 2%, and more than 90% of asymptomatic people are accompanied by inspection, and the early screening and understanding of esophageal cancer are seriously hindered by the factors. Meanwhile, a detection method with low invasiveness and low cost is searched, and the improvement of the early screening efficiency of the esophageal cancer is an important trend of the prevention and treatment work of the esophageal cancer.
The tumor marker is a substance which is synthesized and secreted by the gene expression of tumor cells or abnormally generated and/or increased by the body to the tumor reaction in the process of malignant tumor generation and proliferation and reflects the existence and growth of the tumor. The compound is present in blood, body fluid, cells or tissues of patients, can be measured by methods such as biochemistry, immunology, molecular biology and the like, and has certain value on auxiliary diagnosis, differential diagnosis, curative effect observation, relapse monitoring and prognosis evaluation of tumors. The ideal tumor marker has high sensitivity and good specificity, and can clearly distinguish tumor patients from non-tumor patients; the tumor-specific gene has tissue and organ specificity, can be used for positioning tumors and predicting the prognosis of the tumors. Among the tumor markers most rapidly studied are tumor-associated antigens (TAAs). The discovery that tumor-associated antigens can stimulate the immune system of the human body to generate immune responses, thereby triggering a large number of autoantibodies corresponding to the tumor-associated antigens to be released into serum, and the autoantibodies of the tumor-associated antigens can be detected 3-5 years before the clinical symptoms of cancer patients appear makes it possible to utilize serological methods for early diagnosis of tumors. On the basis of a genomic database established by the technologies of whole genome association analysis, whole genome sequencing and the like in the previous period, the research team screens 4 TAAs (P53, ZBTB20, ERICH3 and C15orf57) by using an autoantibody tissue chip, and finds that the autoantibodies of the 6 TAAs can be detected before the esophageal mucosa squamous epithelium cancerates, and the autoantibodies have high specificity and sensitivity to esophageal squamous cell carcinoma and precancerous patients. However, there is no report of the use of these TAAs in combination for early esophageal squamous carcinoma screening. Therefore, the research jointly utilizes the 4 screened TAAs to develop an ELISA kit with high sensitivity and specificity for screening early esophageal squamous cell carcinoma.
Disclosure of Invention
In view of the problems and disadvantages of the prior art, it is an object of the present invention to provide the use of a combination of tumor associated antigens P53 protein, ZBTB20 protein, ERICH3 protein and C15orf57 protein for the preparation of a product for screening esophageal squamous cell carcinoma; the invention also aims to provide a kit for screening early esophageal squamous carcinoma; the invention also aims to provide an application of a combined detection reagent of a tumor associated antigen P53 protein, a ZBTB20 protein, an ERICH3 protein and a C15orf57 protein in preparing a product for screening esophageal squamous cell carcinoma.
Based on the above purpose, the technical scheme adopted by the invention is as follows:
the invention provides an application of a combination of a tumor associated antigen P53 protein, a ZBTB20 protein, an ERICH3 protein and a C15orf57 protein in preparing a product for screening esophageal squamous cell carcinoma.
According to the above application, preferably, the product detects the expression level of antibodies against P53 protein, ZBTB20 protein, ERICH3 protein and C15orf57 protein in a sample by enzyme-linked immunosorbent assay.
Preferably, the sample is serum, according to the above-mentioned use.
According to the above-mentioned use, preferably, the product is a chip, a preparation or a kit.
The invention provides a kit for screening early esophageal squamous carcinoma, which comprises a solid phase carrier and a tumor-associated antigen coated on the solid phase carrier, wherein the tumor-associated antigen consists of P53 protein, ZBTB20 protein, ERICH3 protein and C15orf57 protein.
According to the kit, preferably, the detection sample of the kit is serum.
According to the above kit, preferably, the kit is used for detecting autoantibodies to the P53 protein, ZBTB20 protein, ERICH3 protein, C15orf57 protein in a serum sample.
According to the above kit, preferably, the kit further comprises a sample diluent, a second antibody diluent, negative control serum, positive control serum, a washing solution, a color-developing solution and a stop solution. More preferably, the sample diluent is PBST (phosphate tween) buffer containing 1% (W/V) BSA; the second antibody diluent is PBST (phosphate Tween) buffer containing 1% (W/V) BSA; the color development liquid consists of a color development liquid A and a color development liquid B, wherein the color development liquid A is 0.02% (W/V) TMB (3,3 ', 5, 5' -tetramethylbenzidine), and the color development liquid B is 0.006% (W/V) urea peroxide; the stop solution is 2mol/L sulfuric acid; the wash was 0.01M PBST (phosphate Tween) buffer pH7.4 containing 0.2% Tween 20.
Preferably, the second antibody is provided with a detectable label according to the kit described above.
According to the above kit, preferably, the label is horseradish peroxidase.
According to the above kit, preferably, the second antibody is RecA protein.
Preferably, the positive control serum is a P53 positive control serum and the negative control serum is a P53 negative control serum according to the above-mentioned kit. More preferably, the P53 positive control serum is the serum of an esophageal squamous carcinoma patient which is positive for P53 antibody by using an indirect ELISA and a Western blot method, and the P53 negative control serum is the serum of a normal human which is positive for P53 antibody expression level by using an indirect ELISA and a Western blot method. A large number of studies clearly show that the P53 antigen plays a very important regulatory role in the occurrence and development processes of esophageal squamous cell carcinoma, and the P53 antibody has higher expression in the serum of patients with esophageal squamous cell carcinoma. The invention selects P53 antibody positive serum as positive control, P53 antibody negative serum as negative control, the positive control serum and the negative control serum are carefully screened serum, and the strength of other antigen-antibody reactions of the same ELISA kit can be used as reference, thereby achieving the purpose of quality control.
According to the kit, preferably, the solid phase carrier is an enzyme label plate. More preferably, the microplate is a 96-well microplate (8 rows and 12 columns total), which 96-well microplate is coated with 4 tumor-associated antigens, P53 protein, ZBTB20 protein, ERICH3 protein and C15orf57 protein, according to a well-designed layout (see fig. 2), wherein each row is coated with one antigen, and each antigen is coated in 11 spot wells. The serum sample of the same detection object is diluted and then added into 4 continuous holes in the same column of the 96-hole enzyme label plate (such as an A-D hole in the 1 st column, an E-H hole in the 1 st column, an A-D hole in the 2 nd column, an E-H hole in the 2 nd column, and the like), and the ELISA detection kit can be simultaneously used for detecting the expression levels of 4 tumor-associated antigen autoantibodies in 22 serum samples to be detected, and can carry out large-scale sample detection. A blank control hole, a positive control hole and a negative control hole are arranged in the 12 th column of the 96-well enzyme label plate, the blank control hole is coated with coating liquid without antigen, and the positive control hole and the negative control hole are both coated with P53 antigen.
The application is to use the tumor-associated antigens P53 protein, ZBTB20 protein, ERICH3 protein and C15orf57 protein as detection reagents and to screen esophageal squamous cell carcinoma by detecting autoantibodies of five tumor-associated antigens in serum. Because the human immune system can generate humoral immune response aiming at the tumor-associated antigen, an autoantibody (called tumor-associated antigen autoantibody for short) for resisting the tumor-associated antigen is generated, and the tumor-associated antigen autoantibody is released into blood and can circulate in the blood; moreover, the concentration of the tumor-associated antigen autoantibodies in blood is directly proportional to the abundance of the corresponding tumor-associated antigens, and higher concentration of the tumor-associated antigen autoantibodies indicates higher expression of the tumor-associated antigens, so that the detection of the contents of the tumor-associated antigens P53 protein, ZBTB20 protein, ERICH3 protein and C15orf57 protein in human serum by using a detection reagent can be used for realizing the screening and diagnosis of esophageal squamous cell carcinoma.
The third aspect of the invention provides an application of a combined detection reagent of a tumor associated antigen P53 protein, a ZBTB20 protein, an ERICH3 protein and a C15orf57 protein in preparing a product for screening esophageal squamous cell carcinoma.
According to the above application, preferably, the product detects the expression levels of tumor-associated antigens P53 protein, ZBTB20 protein, ERICH3 protein and C15orf57 protein in a sample by immunohistochemistry, Western blotting, enzyme-linked immunosorbent assay or protein chip.
According to the above-mentioned use, preferably, the detection reagent is a nucleic acid or a protein. More preferably, the detection reagent is a protein.
According to the above-mentioned use, preferably, the protein is a specific antibody that binds to the P53 protein, the ZBTB20 protein, the ERICH3 protein and the C15orf57 protein.
The basic information for the four tumor-associated antigens of the present invention is as follows:
the P53 gene is one of the earliest discovered cancer suppressor genes as a transcription factor, the expressed P53 protein plays an important role in regulating the process of apoptosis, and different researches show that the P53 mutation is related to the occurrence and development of various tumors. The expression of ERICH3 protein has been linked to a number of cancers, such as gliomas and cutaneous sarcomas. ERICH3 has been shown to have specific mutations in relapsed leukemia. It has been shown by researchers that pathway analysis shows that ERICH3 mutation can cause change of drug target or affect drug intake through cell membrane, and such drug resistance-related pathway mutation may affect survival of tumor patients. The ZBTB20 protein plays a role in transcriptional inhibition in many aspects such as tumor and lymphoid lineage differentiation, and recent studies show that the expression of ZBTB20 protein is detected in various immune cells and is involved in the immune regulation of the body. The ZBTB20 protein is expressed in various immune cells, such as monocytes, DC cells, T cells and B cells, etc., suggesting that it may be involved in the regulation of innate and adaptive immunity. The C15orf57 protein is also named as CCDC32 protein, is a member of a CCDC family, and researches report that the CCDC family protein can participate in biological processes such as signal conduction, genetic signal transcription, molecular recognition, cell cycle regulation, cell differentiation, apoptosis and the like in cells; in recent years, the expression of CCDC family proteins in malignant tumor tissues and tumor cell lines has gradually gained much attention, and studies have shown that abnormal expression of CCDC proteins can promote the movement and migration of tumor cells by modulating the rearrangement of cytoskeleton to activate intracellular signal phospholipase C and mitogen-activated protein.
Compared with the prior art, the invention has the following positive beneficial effects:
(1) the invention detects the expression level of the 4 TAAs in human serum by combining the 4 TAAs of the P53 protein, ZBTB20 protein, ERICH3 protein and C15orf57 protein as a combination for the first time, can effectively detect esophageal squamous carcinoma, especially early esophageal squamous carcinoma, the detection sensitivity is as high as 82% (namely the ratio of the 4 tumor-associated antigens to be correctly diagnosed as early esophageal squamous carcinoma in patients with early esophageal squamous carcinoma is 82%), the specificity (also called true negative rate, namely the percentage of actually no disease correctly diagnosed as no disease according to the diagnosis standard, and higher specificity means lower misdiagnosis rate) reaches 84% (namely the ratio of the patients without esophageal squamous carcinoma to be determined as patients without esophageal squamous carcinoma is 84% when the 4 tumor-associated antigens are used for combined detection).
Therefore, the kit disclosed by the invention combines the P53 protein, the ZBTB20 protein, the ERICH3 protein and the C15orf57 protein for use, so that on the basis of ensuring the detection sensitivity, the detection specificity is greatly improved, the misdiagnosis rate is greatly reduced while the low missed diagnosis rate is ensured, the early esophageal squamous carcinoma is screened out to the greatest extent, physical and psychological pains of patients and waste of medical resources of the country caused by misdiagnosis can be well avoided, and a more accurate and highly balanced scientific basis is provided for effectively judging the early esophageal squamous carcinoma.
(2) The kit has higher sensitivity and specificity, greatly improves the detection rate of early esophageal squamous cell carcinoma, can be used for large-scale screening of asymptomatic crowds in high incidence areas of esophageal squamous cell carcinoma, is far higher than the detection rate of current clinical endoscope screening of cardia adenocarcinoma, and is beneficial to screening and early discovery of asymptomatic esophageal squamous cell carcinoma high-risk crowds, thereby greatly reducing the death rate of patients with esophageal squamous cell carcinoma and bringing great welfare for the patients with esophageal squamous cell carcinoma and families.
(3) The kit prepared by the invention can simultaneously detect the expression levels of 4 TAA antibodies in a serum sample, and compared with the independent detection of the 4 TAA antibodies, the combined detection of the 4 TAA antibodies has the advantages of 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 clinical esophageal squamous cell carcinoma, can be popularized and used in common laboratories, and has good application prospect.
Drawings
FIG. 1 is a diagram showing the antigen coating layout of a 96-well microplate in the kit of the present invention (wherein the name of the antigen indicates that the well is coated with the antigen, to-be-detected serum is added to detect the expression level of the corresponding antibody in the to-be-detected serum, "+" indicates a positive control well to which a positive control serum is added, "-" indicates a negative control well to which a negative control serum is added, and "blank" indicates a blank control well to which a sample diluent without serum is added, and the other operations are the same, and the blank control is used for the background value in the reaction experiment process);
FIG. 2 is a graph of the profile of 4 TAA autoantibodies in esophageal squamous carcinoma serum;
FIG. 3 is a profile of 4 TAA autoantibodies in control sera;
FIG. 4 is a graph showing the results of the positive rates of 4 TAA autoantibodies in the esophageal cancer group and the control group;
FIG. 5 is a ROC graph showing the detection of esophageal squamous carcinoma by 4 TAA autoantibodies.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are not intended to limit the scope of the present invention.
The experimental methods in the following examples, which do not indicate specific conditions, all employ conventional techniques in the art, or follow the conditions suggested by the manufacturers; the reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1: preparation of the kit
The invention prepares an autoantibody combined detection ELISA kit which can be used for early esophageal squamous cell carcinoma screening and diagnosis according to the principle of indirect enzyme-linked immunosorbent assay. The indirect enzyme-linked immunity method is characterized by that the antigen is connected to solid-phase carrier, the antibody to be tested in the sample is combined with the solid-phase antigen-detected antibody compound, then the enzyme-labeled secondary antibody is combined with the antibody in the solid-phase antigen-detected antibody compound to form the solid-phase antigen-detected antibody-enzyme-labeled secondary antibody compound, then the chromogenic degree after adding substrate is measured, and the content of antibody to be tested is determined.
1. Experimental materials and reagents:
(1)4 tumor-associated antigen proteins (P53, ZBTB20, ERICH3 and C15orf57) purchased from Eimei technologies, Inc., Wuhan;
(2) 96-well enzyme label plate: 3590(costar. us);
(3) coating liquid: 50mM carbonate buffer, pH 9.6;
(4) sealing liquid: PBST buffer containing 2% (W/V) BSA;
(5) sample diluent: PBST buffer containing 1% (W/V) BSA;
(6) secondary antibody dilution: PBST buffer containing 1% (W/V) BSA;
(7) enzyme-labeled secondary antibody: horse radish peroxidase-labeled RecA protein (Invitrogen corporation);
(8) washing liquid: PBST (phosphate tween) buffer containing 0.2% tween 20;
(9) positive control serum: p53 positive control serum, namely esophageal squamous carcinoma patient serum with positive P53 antibody detected by indirect ELISA and Western blot method;
(10) negative control serum: p53 negative control serum, namely the serum of normal people with P53 antibody expression level being the average content of normal people serum antibody by indirect ELISA and Western blot method;
(11) color developing solution A: 0.02% (W/V) TMB, formulation: dissolving 0.005g of methylbenzidine (TMB) in 25ml of deionized water;
(12) color developing solution B: 0.006% (W/V) of urea peroxide, formulation: fully dissolving 4.665g of citric acid and 4.40g of Na2HPO418 in 400ml of deionized water, adding 3.2ml of 0.75% urea hydrogen peroxide, adjusting the pH value to 5.0-5.5, adding deionized water to a constant volume of 500ml, uniformly mixing, and storing at 4 ℃;
(13) stopping liquid: 2mol/L sulfuric acid;
(14) an enzyme-labeling instrument: star Fax 2100 (aware. us).
2. Preparing an antigen-coated ELISA plate:
(1)4 tumor-associated antigen solutions were prepared:
4 tumor-associated antigen proteins are respectively dissolved in the coating liquid, and are fully and uniformly mixed to prepare 4 antigen solutions with the concentration of 0.5 mu g/mu L.
(2) Coating an enzyme label plate:
respectively adding the prepared 4 tumor-associated antigen solutions into sample application holes of a 96-hole enzyme label plate according to the layout shown in figure 1, wherein the sample application amount is 100 mu l/hole; adding p53 antigen solution into positive control well and negative control well, adding coating solution into blank control well, incubating at 37 deg.C for 1h, removing coating solution after overnight at 4 deg.C, and washing with washing solution for 3 times (each for 3 min).
(3) And (3) sealing:
blocking solution (the amount of the blocking solution added is 300. mu.l/well) is added to the spotting wells of the coated 96-well microplate, incubated at room temperature for 2 hours, and then removed.
(4) And (3) drying and packaging:
and (3) placing the 96-hole enzyme label plate subjected to sealing treatment in a drying box at 37 ℃, drying, and packaging to obtain the antigen-coated 96-hole enzyme label plate, and storing at 4 ℃ for later use.
3. The kit comprises the following components:
(1) the 96-hole enzyme label plate coated by the antigen prepared in the step 2;
(2) sample diluent: PBST buffer containing 1% (W/V) BSA;
(3) secondary antibody dilution: PBST buffer containing 1% (W/V) BSA;
(4) enzyme-labeled secondary antibody: horse radish peroxidase-labeled RecA protein (Invitrogen corporation);
(5) color development liquid: the color development liquid consists of a color development liquid A and a color development liquid B, wherein the color development liquid A is 0.02% (W/V) TMB, and the color development liquid B is 0.006% (W/V) urea peroxide; when in use, the color development liquid A and the color development liquid B are uniformly mixed in equal volume according to the ratio of 1: 1;
(6) stopping liquid: 2mol/L sulfuric acid;
(7) washing liquid: PBST (phosphate tween) buffer containing 0.2% tween 20;
(8) positive control serum: p53 positive control serum;
(9) negative control serum: p53 negative control serum.
And the reagents (2) - (9) are packaged respectively and then form a kit with the 96-hole enzyme label plate coated with the antigen.
Example 2: method of using kit
1. Incubation of serum samples:
and (3) diluting the serum sample to be detected with a sample diluent according to the ratio of 1: 500, adding the diluted serum sample into a reaction hole of a 96-hole enzyme label plate coated with the antigen, wherein the sample adding amount is 100 mu l/hole, placing the reaction hole in a constant-temperature incubator at 37 ℃ for incubation for 1h, then discarding the liquid in the reaction hole, and washing the reaction hole for 3 times by using a washing solution, wherein the washing time is 3min each time.
2. Incubation with enzyme-labeled secondary antibody:
horseradish peroxidase-labeled RecA protein was diluted with secondary antibody at 1: 40000, adding the diluted RecA protein labeled by horseradish peroxidase into the reaction well of a 96-well enzyme label plate, adding the sample at 100 μ l/well, placing the plate in a constant-temperature incubator at 37 ℃ for incubation for 50min, then discarding the liquid in the sample well, and washing the plate for 3 times with washing liquid, 3min each time.
3. Color development and termination reaction:
and (3) uniformly mixing the color development liquid A and the color development liquid B in an equal volume according to a ratio of 1:1, then quickly adding the mixed color development liquid into reaction holes of a 96-hole enzyme label plate, wherein the sample addition amount is 100 mu l/hole, placing the plate at 37 ℃ in a dark place for reaction for 15min, then adding 50 mu l of stop solution into each reaction hole, stopping the reaction, reading the OD450 optical density value by using an enzyme label instrument within 20 min, and zeroing by using a blank control hole.
4. And (3) judging the detection result of the kit:
and taking the average number of the measured OD values of the negative control wells plus two standard deviations (Mean +2SD) as a cut-off value (cut-off value), judging that the reading of the OD value in the reaction well is more than or equal to the cut-off value is positive, and judging that the reading of the OD value in the reaction well is less than the cut-off value is negative.
Example 3: diagnostic value analysis of the kit of the invention
In this example, the kit prepared in example 1 is used to detect serum samples of patients with early esophageal squamous cell carcinoma and normal persons according to the method described in example 2, so as to evaluate and analyze the value of the kit of the present invention for screening and diagnosing early esophageal squamous cell carcinoma.
1. Sample source
200 serum samples from the national focus laboratory of esophageal cancer prevention and treatment of the first subsidiary hospital of Zhengzhou university were collected, wherein 100 serum samples were obtained from normal persons (control group) and 100 serum samples were obtained from patients with early esophageal squamous cell carcinoma (esophageal squamous cell carcinoma group). 100 normal human sera were from healthy physical population in the laboratory cooperative hospital physical center without any evidence of tumor association. Among 100 normal persons, 61 men and 39 women had an average age of 58.9 ± 6.3 years, and the age range was 40-70 years. 100 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 100 patients with esophageal squamous carcinoma, 57 men and 43 women had an average age of 58.5 ± 7.0 years, with the age range of 45-75 years. All subjects were randomized by gender, age and clinical stage match.
Serum preparation: 5ml of fasting venous blood is extracted into a centrifuge tube, kept stand for 30 minutes at room temperature, centrifuged (2000 rpm), and the upper serum is sucked and subpackaged, wherein each tube is 100 mu l, and the tubes are stored in a refrigerator at minus 80 ℃.
2. Experimental methods
The kit prepared in the embodiment 1 and the use method of the kit in the embodiment 2 are adopted to detect the contents of 4 tumor-associated antigen autoantibodies in the sera of 100 cases of esophageal squamous cell carcinoma patients (esophageal squamous cell carcinoma group) and 100 cases of normal human sera (control group). The mean expression level distribution diagram of 4 tumor-associated antigen autoantibodies in the esophageal squamous carcinoma group and the control group is drawn by using MedCalc software (the result is shown in figure 2 and figure 3)
Taking the result judgment standard in the step 4 of the embodiment 2 as a standard, respectively calculating the positive rates of the 4 tumor-associated antigen autoantibodies in the esophageal squamous carcinoma group and the control group (the positive rate is obtained by dividing the number of the positive objects detected in each group by the total number of the detected objects in the group), and drawing bar graphs of the positive rates of the 4 tumor-associated antigen autoantibodies in the early esophageal squamous carcinoma group and the control group by using Excel software (the result is shown in fig. 4). And (3) performing statistical test by using SPSS22.0 software, comparing the antibody positive rates of the esophageal squamous cell carcinoma group and the control group by using a two-independent sample chi-square test method, wherein the test level alpha is 0.05, and when P is less than 0.05, the result has statistical significance, and then evaluating the diagnostic value of detecting the esophageal squamous cell carcinoma by using the autoantibody by using an evaluation method of a screening test (the result is shown in Table 1 and figure 5).
Meanwhile, in order to compare with the diagnostic effect of the kit of the present invention, the inventors also prepared a comparison kit of different antigen combinations. The comparative kit was prepared in substantially the same manner as in example 1, except that: the contrast kit has different combinations of solid phase carriers and tumor antigens coated on the ELISA plate; wherein, the tumor-associated antigen coated on the spotting hole of the ELISA plate of the first contrast kit is only ERICH 3; the tumor-associated antigens coated on the wells of the enzyme label plate of the second contrast kit are ERICH3 and C15orf57(ERICH3 and C15orf57 respectively coat different wells); the tumor-associated antigens coated on the wells of the ELISA plate of the third comparative kit are ERICH3, C15orf57 and P53(ERICH3, C15orf57 and P53 respectively coat different wells).
3. Analysis of results
FIG. 2 is a distribution diagram of 4 tumor-associated antigen autoantibodies in the serum of the esophageal squamous cell carcinoma group, and it can be seen from the diagram that the average expression level of the 4 tumor-associated antigen autoantibodies in the esophageal squamous cell carcinoma group is higher than that of the 4 tumor-associated antigen autoantibodies, and the average OD value is floated around 0.5. FIG. 3 is a distribution graph of 4 tumor-associated antigen autoantibodies in the serum of 150 control groups, and it can be seen from the graph that the 4 tumor-associated antigen autoantibodies have a low average expression level in the control group, and the average OD value fluctuates around 0.08. As can be seen from FIG. 2 and FIG. 3, the mean level of 4 TAA autoantibodies in the serum of patients with esophageal squamous cell carcinoma was significantly higher than that of the control group, indicating that 4 TAA autoantibodies can be used for diagnosis of esophageal squamous cell carcinoma.
FIG. 4 is a graph showing the results of the positive rate of 4 tumor-associated antigen autoantibodies in the esophageal squamous cell carcinoma group and the control group, and it can be seen from FIG. 4 that the positive expression rate of 4 TAA autoantibodies, P53, ZBTB20, ERICH3 and C15orf57 in the serum of patients with early esophageal squamous cell carcinoma group is in the range of 35% to 54%, while the positive expression rate of 4 TAA autoantibodies in the control group is only 3% to 6%. Furthermore, the 4 TAA autoantibodies were statistically higher in the esophageal squamous cell carcinoma group than in the control group. Therefore, the 4 TAA autoantibodies can be used as a detection index for early diagnosis of esophageal squamous cell carcinoma and are used for early diagnosis of esophageal squamous cell carcinoma.
TABLE 1 detection results of different tumor-associated antigens autoantibody combination kit
As shown in Table 1, as the number of antigen combinations increases, the sensitivity of diagnosis of early esophageal squamous cell carcinoma increases; when 4 tumor-associated antigens are combined, the sensitivity is up to 82 percent, namely the ratio of the esophageal squamous cell carcinoma to the esophageal squamous cell carcinoma which can be correctly diagnosed in patients with the esophageal squamous cell carcinoma by applying the method is 82 percent; although the detection specificity is gradually reduced along with the increase of the number of the antigens, when the 4 tumor-associated antigens are combined, the specificity can still reach 84 percent, and the result shows that the percentage of correctly diagnosed non-esophageal squamous cell carcinoma patients who adopt the 4 tumor-associated antigens for combined detection is 84 percent; therefore, the combination of 4 tumor-associated antigens P53, ZBTB20, ERICH3 and C15orf57 is used for early esophageal squamous cell carcinoma diagnosis, so that the diagnosis sensitivity can be greatly improved on the premise of ensuring the diagnosis specificity. In addition, the jotan index is obtained by subtracting 1 from the sum of sensitivity and specificity in statistics, the numerical range is 0-1, and the closer the jotan index is to 1, the higher the diagnostic value is, and the higher the application value of the method is. With the increase of the number of antigens, the john index is continuously increased and gradually trends to 1, which indicates that the method for diagnosing and screening early esophageal squamous cell carcinoma by combining 4 tumor-associated antigens has better diagnostic value. Therefore, the method for detecting the expression level of the corresponding autoantibody in the serum to be detected by adopting the combination of the autoantigens of the 4 tumor-associated antigens P53, ZBTB20, ERICH3 and C15orf57 can keep higher specificity and improve the sensitivity of diagnosis, has good diagnosis and application values for evaluating the risk of the object to be detected suffering from esophageal squamous cell carcinoma, and is an ideal early esophageal squamous cell carcinoma screening method and means.
FIG. 5 is a ROC curve diagram of 4 tumor-associated antigen autoantibodies for detecting esophageal squamous cell carcinoma. As can be seen from FIG. 5, when a single TAA is used for detection, the areas under the ROC curves are all low, and the maximum area is only 0.75(ERICH 3); the area under the ROC curve of 2 tumor-associated antigens in combined detection is 0.79, the area under the ROC curve of 3 tumor-associated antigens in combined detection is 0.81, and the area under the ROC curve of 4 tumor-associated antigens in combined detection is 0.83 to the maximum. Therefore, the ELISA kit has higher judgment accuracy and higher diagnosis value for the esophageal cancer, and further proves that the ELISA kit is an ideal early diagnosis and screening method and means for the esophageal cancer.
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 (10)
1. The application of the combination of a tumor associated antigen P53 protein, a ZBTB20 protein, an ERICH3 protein and a C15orf57 protein in preparing a product for screening esophageal squamous cell carcinoma.
2. The use of claim 1, wherein the product is used for detecting the expression level of antibodies against P53 protein, ZBTB20 protein, ERICH3 protein and C15orf57 protein in a sample by ELISA.
3. The use of claim 2, wherein the sample is serum.
4. Use according to claim 2, wherein the product is a chip, a preparation or a kit.
5. A kit for screening early esophageal squamous carcinoma is characterized by comprising a solid phase carrier and a tumor-associated antigen coated on the solid phase carrier, wherein the tumor-associated antigen consists of a P53 protein, a ZBTB20 protein, an ERICH3 protein and a C15orf57 protein.
6. The kit according to claim 5, wherein the detection sample of the kit is serum.
7. The kit according to claim 5, wherein the kit further comprises a sample diluent, a second antibody diluent, a negative control serum, a positive control serum, a washing solution, a color-developing solution, and a stop solution.
8. The application of a combined detection reagent of a tumor associated antigen P53 protein, a ZBTB20 protein, an ERICH3 protein and a C15orf57 protein in preparing a product for screening esophageal squamous cell carcinoma.
9. The use of claim 8, wherein the product detects the expression levels of tumor associated antigens P53 protein, ZBTB20 protein, ERICH3 protein and C15orf57 protein in a sample by immunohistochemistry, Western blotting, enzyme-linked immunosorbent assay or protein chip.
10. The use of claim 8, wherein the detection reagent is a nucleic acid or a protein.
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