CN111551545B - Liquid biopsy ELISA kit for early screening of high risk group of esophageal cancer - Google Patents
Liquid biopsy ELISA kit for early screening of high risk group of esophageal cancer Download PDFInfo
- Publication number
- CN111551545B CN111551545B CN202010450305.8A CN202010450305A CN111551545B CN 111551545 B CN111551545 B CN 111551545B CN 202010450305 A CN202010450305 A CN 202010450305A CN 111551545 B CN111551545 B CN 111551545B
- Authority
- CN
- China
- Prior art keywords
- esophageal cancer
- tvp23c
- liquid biopsy
- serum
- elisa kit
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
Abstract
The invention belongs to the technical field of biological engineering and biological medicine, particularly belongs to the field of molecular biology and oncology, and more particularly relates to a group of liquid biopsy ELISA kits for early screening of high risk groups of esophageal cancer. The kit comprises a solid phase carrier and tumor-associated antigens TVP23C, PAQR5 and NUBPL coated on the solid phase carrier, and further comprises a sample diluent, a second antibody diluent, a negative control serum, a positive control serum, a washing solution, a developing solution and a stopping solution. The kit disclosed by the invention has detection sensitivity of 91.46% and detection specificity of 83.75% on esophageal cancer, and is suitable for large-scale screening of asymptomatic people in high-incidence regions of esophageal cancer.
Description
Technical Field
The invention belongs to the technical field of biological engineering and biological medicine, relates to the field of molecular biology and oncology, and more particularly relates to a group of liquid biopsy ELISA kits for early screening of high risk groups of esophageal cancer.
Background
Esophageal cancer is a common tumor of the digestive tract, has high incidence rate, and is second only to gastric cancer. China is one of the high-incidence areas of esophageal cancer in the world, and the average death rate of people is about 15 ten thousand every year. In rural areas of China, esophageal cancer is the tumor with the highest incidence. Esophageal cancer is a malignant tumor which occurs in esophageal epithelial tissues, and the occurrence of esophageal cancer is related to chronic stimulation of nitrosamine compounds, inflammation and trauma, genetic factors and the content of trace elements in drinking water, food and vegetables. Generally, the pathogenesis of esophageal cancer can be divided into two categories, namely environment and gene. With the improvement of living standard of people, living environment is improved, the influence of environmental factors on the pathogenesis of esophageal cancer is smaller and smaller, and the pathogenic factor of gene is more and more important. The occurrence and development of the esophageal cancer are a multi-factor, multi-stage and slow development process, the genes are related to early esophageal cancer and precancerous lesion, and the combined detection change of a plurality of tumor-related genes is beneficial to playing a certain early warning role for asymptomatic high-risk groups of the esophageal cancer. Traditionally, "asymptomatic population with high incidence, over 40 years old, male, smoking, drinking, and positive family history" is generally defined as "high risk or high risk population of esophageal cancer", and is also the main object for early screening of esophageal cancer. The resection rate of esophageal cancer in early detection and surgical treatment can reach 100 percent, the survival rate in 5 years can reach more than 90 percent, but most of patients in current clinic have reached middle and late stages, and the long-term curative effect is not satisfactory. And various combined detection methods aiming at early diagnosis of esophageal cancer and application of corresponding kits are still rare. Early detection of asymptomatic high risk groups of esophageal cancer is particularly important, but when the esophageal cancer is screened by gastroscope and the like, people of examinees need to bear certain pain, are not easy to comply, and the detection machine is expensive, so that the gastroscope examination cannot be popularized in remote areas. Therefore, there is an urgent need for new low-cost detection techniques and devices to meet the general investigation of high incidence of esophageal cancer in the whole country.
Esophageal cancer is a complex multi-stage evolution process involving proteins encoded by various genes, is the result of environmental and genetic interactions, and is stimulated by various physical or biochemical factors, such as DNA alkylation damage caused by nitrosamine, trace element deficiency and the like, which can cause excessive hyperplasia of esophageal squamous epithelial basal cells, further become atypical hyperplasia and finally develop into early invasive cancer. The generation and development process of the esophageal cancer involves the mutation of a plurality of oncogenes and cancer suppressor genes, and various enzymological changes are generated and expressed as the synergistic action of multiple genes, multiple factors and multiple steps.
The inventor's research institution collects the related information of 30 more than ten thousand cases of esophageal squamous cell carcinoma patients over the years, utilizes molecular mechanism to screen esophageal carcinoma specific markers, establishes a molecular marker screening system for high risk groups and early esophageal carcinoma discovery, screens the molecular markers on the basis, simultaneously combines a study recently carried out by the inventor's research institution, adopts protein microarray chip technology to carry out pairing study on peripheral blood of ten thousand cases of esophageal carcinoma patients and healthy volunteers, discovers a plurality of antigen proteins related to esophageal carcinoma occurrence, screens three antigens related to early esophageal squamous cell carcinoma lesion, namely TVP23C, PAQR5 and BPNUL, as early diagnosis indexes, and develops a kit for early esophageal carcinoma screening with high sensitivity and high specificity based on the three antigen markers, can effectively carry out early warning of esophageal cancer, and has important significance for improving the discovery rate of early esophageal cancer and reducing the death rate of esophageal cancer.
Disclosure of Invention
Aiming at the problems and the defects in the prior art, the invention aims to provide a liquid biopsy ELISA kit for early screening of high risk groups of esophageal cancer.
Based on the purpose, the invention adopts the following technical scheme:
the invention provides application of a combination of tumor-associated antigens TVP23C, PAQR5 and NUBPL in preparation of a liquid biopsy kit for early screening of high risk groups of esophageal cancer.
The invention also provides a liquid biopsy ELISA kit for early screening of high risk groups of esophageal cancer, which comprises a solid phase carrier and a tumor-associated antigen coated on the solid phase carrier, wherein the tumor-associated antigen consists of TVP23C, PAQR5 and NUBPL.
According to the above liquid biopsy ELISA 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 a PBST buffer containing 1% (W/V) BSA; the second antibody dilution was PBST buffer containing 1% (W/V) BSA; the color developing solution consists of a color developing solution A and a color developing solution B, wherein the color developing solution A is 0.02% (W/V) TMB (3,3 ', 5, 5' -tetramethyl benzidine), and the color developing solution B is 0.006% (W/V) urea peroxide; the stop solution is concentrated sulfuric acid with the concentration of 10 wt%; the wash was PBST buffer pH =7.4 and containing 0.05 v% tween-20.
Preferably, the second antibody carries a detectable label according to the liquid biopsy ELISA kit described above.
Preferably, the marker is horseradish peroxidase according to the above liquid biopsy ELISA kit.
Preferably, the second antibody is RecA protein according to the liquid biopsy ELISA kit described above.
Preferably, the positive control serum is TVP23C positive control serum and the negative control serum is TVP23C negative control serum according to the above liquid biopsy ELISA kit.
According to the liquid biopsy ELISA kit, preferably, the TVP23C positive control serum is esophageal cancer patient serum which is positive for TVP23C antibody detected by using indirect ELISA and Western blot method; the TVP23C negative control serum is the serum of a normal person, the expression level of TVP23C antibody of which is detected by indirect ELISA and Western blot method is the average content of the serum antibody of the normal person. Recent research in the laboratory finds that the TVP23C antigen plays an important role in regulation in the occurrence and development processes of esophageal cancer, and an anti-TVP 23C antibody has a high positive rate in the serum of esophageal cancer patients. The invention selects TVP23C antibody positive serum as positive control, and the strength of other antigen antibody reaction of the same ELISA kit can be used as reference, thereby achieving the purpose of quality control.
Preferably, the solid support is an ELISA plate according to the above liquid biopsy ELISA kit. More preferably, the solid phase carrier is preferably a 48-well enzyme label plate (8 rows and 6 columns in total); the 48-well microplate was coated with three tumor-associated antigens, TVP23C, PAQR5, NUBPL, in the layout shown in FIG. 1, with one antigen coated in every two rows, each in a total of 14 wells. Serum samples of the same detection object are diluted and then added into the sample application holes of the ELISA kit, so that the purpose of simultaneously detecting the expression levels of the three tumor-associated antigens in the serum samples is achieved. The 48-hole enzyme label plate is provided with a blank control hole, a positive control hole and a negative control hole, wherein 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 TVP23C antigen.
In the antigen coating layout (figure 1) of the 48-hole enzyme label plate in the liquid biopsy ELISA kit, the antigen name indicates that the hole is coated with the antigen, and the serum to be detected is added into the hole to be detected so as to detect the expression level of the corresponding antibody in the serum to be detected; "positive" means positive control wells to which positive control serum was added; "negative" means negative control wells to which negative control serum was added; blank indicates a blank control well, no serum to be tested is added to the blank control well, all other operations are the same in all the wells, and the blank control is used for reflecting the background value in the test process.
According to the above liquid biopsy ELISA kit, preferably, the detection object of the liquid biopsy ELISA kit is human serum. The same serum sample is diluted and added into an enzyme label plate, so that the expression level of antibodies corresponding to three antigens of TVP23C, PAQR5 and NUBPL in the serum sample can be detected simultaneously.
Compared with the prior art, the invention has the following positive beneficial effects:
(1) the invention combines three tumor-related antigens TVP23C, PAQR5 and NUBPL as a combination for the first time, and is jointly used for detecting the expression levels of antibodies corresponding to the three antigens in human serum, so that the esophageal cancer, especially early esophageal cancer can be effectively detected, the detection sensitivity of the kit for early esophageal cancer reaches as high as 91.46 percent (namely, the ratio of early esophageal cancer to accurately diagnosed by using the three tumor-related antigens in early esophageal cancer patients is 91.46 percent), the detection specificity reaches 83.75 percent (namely, the ratio of non-esophageal cancer to determined as non-esophageal cancer to be 83.75 percent when using the three tumor-related genes for joint detection) and is higher than the detection rate of the existing clinical endoscopic esophageal cancer screening, therefore, the kit has higher sensitivity and specificity, and can be used for large-scale screening of asymptomatic people in high-incidence regions of esophageal cancer, can greatly improve the detection rate of early esophageal cancer, is beneficial to screening and early discovery of asymptomatic high-risk groups, and greatly reduces the death rate of esophageal cancer patients.
(2) The ELISA kit prepared by the invention can synchronously detect the expression levels of three esophageal cancer TAA antibodies in a serum sample, and compared with the method for singly detecting one antibody, the kit provided by the invention adopts the combined detection of the antibodies of three antigens, namely TVP23C, PAQR5 and NUBPL, has the advantages of high detection success rate, good technical reproducibility, less material consumption, low cost, simple operation, convenience and rapidness in use, greatly improves the detection efficiency and the diagnosis efficiency of clinical esophageal cancer, and can be popularized and used in common laboratories.
Drawings
FIG. 1 is an antigen coating distribution diagram of a 48-well ELISA plate in the autoantibody joint detection ELISA kit of the present invention;
FIG. 2 is a schematic diagram of an indirect enzyme-linked immunosorbent assay;
FIG. 3 shows the OD values of three tumor-associated antigens in the esophageal cancer group and the control group;
FIG. 4 is a ROC curve for the detection of early esophageal cancer with different combinations of three tumor-associated antigens.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the reagents used are commercially available.
The experimental procedures, for which specific conditions are not indicated in the examples, are generally conventional in the art, according to conventional conditions such as those described in Sambrook et al, molecular cloning, A laboratory Manual (third edition) (scientific Press, 2002), or according to conditions recommended by the reagent manufacturers. The reagents, materials and instruments used are not indicated by manufacturers, and are all conventional products commercially available.
Example 1 liquid biopsy ELISA kit for early screening of high risk group of esophageal cancer
1. Composition of the kit
The kit comprises the following components:
(1) the three antigens are coated on the ELISA plate by three antigens of TVP23C, PAQR5 and NUBPL, and the layout of the three antigens on the ELISA plate is shown in figure 1;
(2) sample diluent: PBST (phosphate Tween) buffer containing 1% (W/V) BSA;
(3) secondary antibody dilution: PBST (phosphate Tween) buffer containing 1% (W/V) BSA;
(4) enzyme-labeled secondary antibody: horse radish peroxidase-labeled RecA protein (purchased from Invitrogen);
(5) color development liquid: the color developing solution consists of a color developing solution A and a color developing solution B, wherein the color developing solution A is 0.02% (W/V) TMB (3,3 ', 5, 5' -tetramethylbenzidine), and the color developing solution B is 0.006% (W/V) urea peroxide; when the color developing solution is used, the color developing solution A and the color developing solution B are uniformly mixed according to the volume ratio of 1:1 for use;
(6) stopping liquid: 10 wt% of concentrated sulfuric acid;
(7) washing liquid: PBST buffer pH =7.4 and containing 0.05 v% tween-20
(8) Positive control serum: TVP23C positive control serum, namely esophageal cancer patient serum with positive TVP23C antibody detected by indirect ELISA and Western blot method;
(9) negative control serum: TVP23C negative control serum, namely serum of healthy people with TVP23C antibody expression level as the average content of serum antibodies of healthy people detected by indirect ELISA and Western blot method.
And packaging the reagents (2) - (9) respectively, and then forming a kit with the antigen-coated ELISA plate.
Preparation method of antigen-coated enzyme label plate in kit
2.1 sources of antigen and ELISA plate
Three tumor-associated antigens, TVP23C, PAQR5 and NUBPL, were purchased from Wuhan Eimei technologies, Inc.; enzyme-labeled plates were purchased from Corning.
2.2 preparation of antigen-coated ELISA plates
The preparation process of the antigen-coated ELISA plate is as follows:
(1) three tumor-associated gene solutions were prepared:
three tumor-associated antigen proteins, namely TVP23C, PAQR5 and NUBPL, are respectively dissolved in the coating solution, and are fully and uniformly mixed to prepare three antigen solutions. Wherein the coating solution is carbonate buffer solution with pH =9.6 and 50 mM; the concentration of the TVP23C solution is 0.125 mug/mL; the concentration of the PAQR5 solution is 0.25 mu g/mL; the concentration of NUBPL solution was 1.0. mu.g/mL.
(2) Coating an enzyme label plate:
adding the prepared three tumor-associated antigen solutions into the sample wells of the ELISA plate according to the layout shown in FIG. 1, wherein the sample addition amount is 100 μ L/well; adding serum antigen solution into the positive control hole and the negative control hole, adding coating solution into the blank control hole, incubating in a constant temperature incubator at 37 ℃ for 1h, removing the coating solution after overnight at 4 ℃, and washing with washing solution for three times, wherein each time is 3 min.
(3) And (3) sealing:
adding a blocking solution into the sample application holes of the coated enzyme label plate, wherein the sample application amount is 300 mu L/hole, incubating for 2 hours at room temperature, and then removing the blocking solution, wherein the coating solution is pH =9.6 and 50mM carbonate buffer solution, and the blocking solution is PBST buffer solution containing 2% (W/V) BSA.
(4) And (3) drying and packaging:
and (3) placing the enzyme label plate subjected to sealing treatment in a drying box at 37 ℃, drying, packaging to obtain an antigen-coated enzyme label plate, and storing at 4 ℃ for later use.
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: diluting at the ratio of 500, adding the diluted serum sample into a sample application hole of an enzyme label plate coated with the antigen, wherein the sample application amount is 100 mu L/hole, placing the sample in a constant-temperature incubator at 37 ℃ for incubation for 1h, then discarding the liquid in the sample application hole, and washing the sample with a washing solution five times for 3min each time.
2. Incubation with enzyme-labeled secondary antibody:
horseradish peroxidase-labeled RecA protein was diluted with secondary antibody at 1: 8000, adding the diluted RecA protein marked by horseradish peroxidase into a spot hole of an enzyme label plate, wherein the sample adding amount is 100 mu L/hole, placing the plate in a constant temperature incubator at 37 ℃ for incubation for 50min, then discarding liquid in the spot hole, and washing the plate for three times with washing liquid, wherein each time is 3 min.
3. Color development and termination reaction:
uniformly mixing the color development solution A and the color development solution B in an equal volume according to a ratio of 1:1, quickly adding the mixed color development solution into a sample application hole of an enzyme label plate, wherein the sample application amount is 100 mu L/hole, placing the sample plate at 37 ℃ in a dark place for color development reaction for 15min, then adding 50 mu L of stop solution into each sample application hole, stopping the color development reaction, reading OD450 optical density values at 450nm (detection wavelength) and 595nm (reference wavelength) by using an automatic enzyme label reader, and zeroing by using a blank control hole.
4. And (4) judging a result:
the Mean of the OD values in the positive control well and the negative control well plus two standard deviations (Mean +2 SD) were used as a cutoff value (Cut off value), and above this value, positive was judged, and below this value, negative was judged.
Detection principle of kit
The liquid biopsy ELISA kit for early screening of high risk groups of esophageal cancer carries out detection of antibodies to be detected based on the principle of indirect enzyme-linked immunosorbent assay. The principle of indirect enzyme-linked immunosorbent assay is shown in figure 2, wherein an antigen is linked to a solid phase carrier, an antibody to be detected in a sample is combined with the solid phase antigen-detected antibody complex, an enzyme-labeled secondary antibody is combined with an antibody in the solid phase antigen-detected antibody complex to form a solid phase antigen-detected antibody-enzyme-labeled secondary antibody complex, and then the color development degree after adding a substrate (color development liquid) is measured to determine the content of the antibody to be detected.
Example 2 analysis of the effectiveness of the kit of the invention in detecting esophageal cancer
The kit provided by the embodiment 1 of the invention is used for detecting serum samples of early esophageal cancer patients and normal people so as to evaluate and analyze the value of the kit provided by the invention for screening and diagnosing early esophageal cancer.
Sample source
Serum samples from esophageal cancer focus open laboratory of Henan province, the first subsidiary hospital of Zhengzhou university were collected, wherein 480 parts of serum of normal persons (control group) and 480 parts of serum of patients with early esophageal cancer (early esophageal cancer group) were collected. 480 normal human sera were from healthy physical population in the laboratory cooperative hospital physical center without any evidence of tumor association. Of the 480 normal individuals, 228 males and 252 females had a mean age of 58.7 ± 9.2 years, with the age range being 35-82 years. 480 early esophageal cancer patients were serum from histopathologically confirmed early (stage 0 + stage I) esophageal cancer patients who received no radiation or chemotherapy treatment. Of the 480 patients with esophageal cancer, 228 men and 252 women had a mean age of 58.5 ± 6.4 years, with the age range of 48-81 years.
Serum preparation
5mL of fasting venous blood is extracted into a centrifuge tube, the centrifuge tube is kept still for 30 minutes at room temperature, centrifugation (2000 r/min) is carried out, upper serum is sucked and subpackaged, 100 mu L of each tube is stored in a refrigerator at minus 80 ℃ to be tested.
Test method and result analysis
The kit prepared in the embodiment 1 and the using method of the kit are adopted to detect the contents of three antigens, namely TVP23C, PAQR5 and NUBPL, in 480 normal population serums (a control group) and 480 esophageal cancer patient serums (an esophageal cancer group). A prism software is used for drawing a scatter diagram of the content of the three tumor-associated antigen autoantibodies in the early esophageal cancer group and the control group, and as shown in figure 3, the result has statistical significance.
Fig. 3 is a scatter diagram showing the content of the three tumor-associated antigen autoantibodies TVP23C, PAQR5, and NUBPL in the early esophageal cancer group and the control group, and it can be seen from fig. 3 that the average expression level of the three tumor-associated antigen autoantibodies in the early esophageal cancer group is higher than that in the healthy control group, which suggests that the three tumor-associated antigens TVP23C, PAQR5, and NUBPL can be used for screening early esophageal cancer.
In order to further explore the effectiveness of the three tumor-associated antigens in the invention in screening early esophageal cancer, the diagnostic value of esophageal cancer detection by different antigen combinations is evaluated by adopting an evaluation method of a screening test, if any antigen in the combination is detected to be positive, the detection result is judged to be positive, and the result is shown in table 1 and figure 4, wherein the table 1 is the detection sensitivity and specificity analysis of different antigen combinations on an esophageal cancer group and a control group; FIG. 4 shows ROC curves for esophageal cancer detection with different antigen combinations.
TABLE 1 analysis of detection sensitivity and specificity of different antigen combinations for esophageal cancer group and control group
Table 1 shows the detection results of any combination of three different tumor-associated antigens on the esophageal cancer group and the control group, and it can be seen from table 1 that when the three antigens TVP23C, PAQR5, and NUBPL are used alone for detecting esophageal cancer, the detection sensitivity is between 27.71% and 35.83%, and the detection specificity is between 97.08% and 95.00%; the number range of the johnsen index is 0-1, the closer the johnsen index is to 1, the higher the diagnostic value is, the higher the application value of the method is, and when the three antigens are independently used for detecting the esophageal cancer, the higher the specificity is, but the detection sensitivity and the overall diagnostic effect are not obvious, so that the diagnostic value of the three antigens of TVP23C, PAQR5 and NUBPL to the esophageal cancer is limited.
With the increase of the number of the antigens, the detection sensitivity of the test strip for the esophageal cancer is increased from 27.71% to 91.46%, and when the TVP23C, PAQR5 and NUBPL are jointly used for detecting the esophageal cancer, the sensitivity is up to 91.46%, namely when the test strip is used for detecting early esophageal cancer, the percentage of esophageal cancer can be correctly diagnosed as 91.46%. Although the detection specificity for early esophageal cancer is gradually reduced along with the increase of the number of specific detection reagents of the four antigens to be detected, the detection specificity for esophageal cancer still can reach 83.75% when the three antigens are used in combination, and the result shows that the percentage of esophageal cancer which is correctly diagnosed as not suffering from esophageal cancer is 83.75% when the test strip is used for detecting esophageal cancer. Therefore, when the combination of the three antigens TVP23C, PAQR5 and NUBPL 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 combination of the three antigens TVP23C, PAQR5 and NUBPL for esophageal cancer detection can maintain higher specificity and improve diagnosis sensitivity, and has good diagnosis and application values for evaluating the risk of esophageal cancer of a to-be-detected object. In addition, the jotan index is continuously increased along with the increase of the number of the antigens in the kit, and the fact that the three antigens have high diagnostic value for early esophageal squamous cell carcinoma when used in combination is shown.
FIG. 4 shows ROC curves of seven different antigen combinations for detecting esophageal cancer, wherein seven points marked with numbers in FIG. 4 represent 7 different gene combinations, and the numbers indicate the sensitivity of the corresponding combinations. 0.28, 0.32 and 0.36 respectively represent the sensitivity of detecting the three antigens of TVP23C, PAQR5 and NUBPL for early esophageal cancer independently; 0.58, 0.61 and 0.66 respectively represent the detection sensitivity of the TVP23C + PAQR5 combination, TVP23C + NUBPL combination and PAQR5+ NUBPL combination on early esophageal cancer; 0.91 represents the detection sensitivity of the combination of three tumor-related genes TVP23C + PAQR5+ NUBPL on early esophageal cancer, and as can be seen from FIG. 4, the area under the ROC curve is obviously increased along with the increase of the number of the genome combinations, which indicates that the three antigens are jointly used for detecting esophageal cancer, so that the specificity can be kept higher and the diagnosis sensitivity can be improved, and indicates that the kit provided by the invention adopts the three antigens TVP23C, PAQR5 and NUBPL to be jointly used for detecting esophageal cancer, so that the kit has higher judgment accuracy and diagnosis value, and is suitable for large-scale screening of asymptomatic people in a high-incidence area of esophageal cancer.
Claims (10)
1. The application of the combination of the tumor associated antigens TVP23C, PAQR5 and NUBPL in preparing a liquid biopsy kit for early screening of high risk groups of esophageal cancer.
2. A liquid biopsy ELISA kit for early screening of esophageal cancer high risk population 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 TVP23C, PAQR5 and NUBPL.
3. The liquid biopsy ELISA kit of claim 2, wherein the kit further comprises a sample diluent, a second antibody diluent, a negative control serum, a positive control serum, a wash solution, a color developing solution, and a stop solution.
4. The liquid biopsy ELISA kit of claim 3 wherein the second antibody is detectably labeled.
5. The liquid biopsy ELISA kit of claim 4, wherein the marker is horseradish peroxidase.
6. The liquid biopsy ELISA kit of any one of claims 3 to 5, wherein the second antibody is RecA protein.
7. The liquid biopsy ELISA kit of claim 6, wherein the positive control serum is TVP23C positive control serum; the negative control serum is TVP23C negative control serum.
8. The liquid biopsy ELISA kit of claim 7, wherein the TVP23C positive control serum is esophageal cancer patient serum positive for both TVP23C antibody detected using indirect ELISA and Western blot; the TVP23C negative control serum is the serum of a healthy person, wherein the TVP23C antibody expression level is the average content of the serum antibodies of the healthy person detected by using an indirect ELISA and Western blot method.
9. The liquid biopsy ELISA kit of claim 8 wherein the solid support is an ELISA plate.
10. The liquid biopsy ELISA kit of claim 9, wherein the detection object of the liquid biopsy ELISA kit is human serum.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010450305.8A CN111551545B (en) | 2020-05-25 | 2020-05-25 | Liquid biopsy ELISA kit for early screening of high risk group of esophageal cancer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010450305.8A CN111551545B (en) | 2020-05-25 | 2020-05-25 | Liquid biopsy ELISA kit for early screening of high risk group of esophageal cancer |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111551545A CN111551545A (en) | 2020-08-18 |
| CN111551545B true CN111551545B (en) | 2020-11-06 |
Family
ID=72003607
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010450305.8A Active CN111551545B (en) | 2020-05-25 | 2020-05-25 | Liquid biopsy ELISA kit for early screening of high risk group of esophageal cancer |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111551545B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112213487B (en) * | 2020-10-23 | 2021-10-29 | 郑州大学第一附属医院 | Application of ASTE1 in detection of esophageal squamous carcinoma |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002223765A (en) * | 2001-01-31 | 2002-08-13 | Keio Gijuku | Human malignant melanoma antigen |
| CN101273144A (en) * | 2005-07-27 | 2008-09-24 | 肿瘤疗法科学股份有限公司 | Method of diagnosing esophageal cancer |
| CN105219715A (en) * | 2015-10-20 | 2016-01-06 | 上海隆耀生物科技有限公司 | A kind of test kit for activating esophageal carcinoma specific immune response |
| CN105277709A (en) * | 2014-06-27 | 2016-01-27 | 汕头大学医学院附属肿瘤医院 | ALDOA autoantibody andapplications thereof in diagnosis of esophageal squamous cell carcinoma |
| CN109837340A (en) * | 2017-11-24 | 2019-06-04 | 顾万君 | Peripheral blood gene marker for lung cancer non-invasive diagnosis |
| CN110187108A (en) * | 2019-05-31 | 2019-08-30 | 郑州大学第一附属医院 | A kind of autoantibody joint-detection ELISA kit for early stage cancer of the esophagus screening |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20180353445A1 (en) * | 2017-01-12 | 2018-12-13 | Whitehead Institute For Biomedical Research | Methods and compositions relating to proteasome inhibitor resistance |
| CN109880894A (en) * | 2019-03-05 | 2019-06-14 | 杭州西合森医学检验实验室有限公司 | The construction method of tumour immunity microenvironment prediction model based on RNAseq |
-
2020
- 2020-05-25 CN CN202010450305.8A patent/CN111551545B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002223765A (en) * | 2001-01-31 | 2002-08-13 | Keio Gijuku | Human malignant melanoma antigen |
| CN101273144A (en) * | 2005-07-27 | 2008-09-24 | 肿瘤疗法科学股份有限公司 | Method of diagnosing esophageal cancer |
| CN105277709A (en) * | 2014-06-27 | 2016-01-27 | 汕头大学医学院附属肿瘤医院 | ALDOA autoantibody andapplications thereof in diagnosis of esophageal squamous cell carcinoma |
| CN105219715A (en) * | 2015-10-20 | 2016-01-06 | 上海隆耀生物科技有限公司 | A kind of test kit for activating esophageal carcinoma specific immune response |
| CN109837340A (en) * | 2017-11-24 | 2019-06-04 | 顾万君 | Peripheral blood gene marker for lung cancer non-invasive diagnosis |
| CN110187108A (en) * | 2019-05-31 | 2019-08-30 | 郑州大学第一附属医院 | A kind of autoantibody joint-detection ELISA kit for early stage cancer of the esophagus screening |
Non-Patent Citations (1)
| Title |
|---|
| Identification of Four Oxidative Stress-Responsive MicroRNAs,miR-34a-5p,miR-1915-3p,miR-638,and miR-150-3p,in Hepatocellular Carcinoma;Yong Wan 等;《Oxidative medicine and cellular longevity》;20171231;全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111551545A (en) | 2020-08-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110187113B (en) | Autoantibody joint detection ELISA kit for early screening of esophageal squamous cell carcinoma | |
| CN110187109B (en) | Autoantibody joint detection ELISA kit for early screening of cardia adenocarcinoma | |
| CN110187108B (en) | Autoantibody joint detection ELISA kit for early esophageal cancer screening | |
| CN104650234B (en) | Anti- AKR1B10 protein monoclonal antibodies and its application | |
| CN111218509B (en) | New diagnosis marker PPEF1 for breast cancer and application thereof | |
| CN110283909B (en) | Application of ZBTB20 protein or specific antibody thereof in cardiac cancer detection kit | |
| CN104136630A (en) | Breast cancer diagnosis and indication marker | |
| CN110187111B (en) | ELISA kit for screening early cardiac cancer | |
| CN109266740A (en) | For pulmonary cancer diagnosis or the marker and diagnostic reagent of prognosis | |
| US20230408520A1 (en) | Arteriosclerosis and cancer detection method using deoxyhypusine synthase gene as indicator | |
| CN111551545B (en) | Liquid biopsy ELISA kit for early screening of high risk group of esophageal cancer | |
| CN111077312B (en) | Application of group of tumor-associated antigens in preparation of cardiac cancer early screening kit | |
| CN101493463B (en) | Stomach cancer diagnosis reagent and uses thereof | |
| Mahmoodi et al. | Determination of serum survivin for prognostic role in esophageal cancer | |
| CN113075413B (en) | Early esophageal squamous carcinoma screening kit based on group of tumor-associated antigens | |
| CN111308090B (en) | Esophageal cancer multi-joint rapid detection ELISA kit | |
| CN111323588B (en) | Application of esophageal cancer related antigen-protein combination or specific antibody thereof in esophageal cancer detection kit | |
| CN111323587B (en) | Autoantibody joint detection ELISA kit for early-stage cardia adenocarcinoma screening | |
| US20230400466A1 (en) | Methods and systems for risk stratification and management of bladder cancer | |
| US20170029898A1 (en) | Novel method for screening for prostate cancer | |
| WO2023004626A1 (en) | Myeloma biomarker serpinf2 and use thereof | |
| WO2023004624A1 (en) | Myeloma biomarker lgals3bp and use thereof | |
| WO2009154510A1 (en) | Test-system for diagnosing prostate cancer and a prostate cancer diagnosis method | |
| CN109799347B (en) | Leukemia biomarker IGFBP3 and application thereof | |
| WO2015026893A1 (en) | Predictive biomarkers for metabolic syndrome |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |