CN110261611B - Application of ZNF709 protein as gastric cancer serum biomarker and kit thereof - Google Patents
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Abstract
The invention discloses an application of ZNF709 protein as a gastric cancer serum biomarker and a kit thereof, wherein the kit comprises: ZNF709 protein coated on an enzyme label plate, 0U/mL standard serum 1, 100U/mL standard serum 2, an enzyme labeling reagent, an enzyme substrate solution, a confining liquid, a sample diluent, a washing liquid and a stop solution. The anti-ZNF 709 immunoglobulin G antibody provided by the invention has high specificity as a gastric cancer serum biomarker; the kit is sensitive and safe in detection and simple in operation, and can be used for quantitatively determining the level of the immunoglobulin G antibody of the protein ZNF709 in human serum and reflecting the level of the ZNF709 protein, so that the kit is used for diagnosing gastric cancer.
Description
Technical Field
The invention relates to the field of bioscience, and particularly relates to application of ZNF709 protein as a gastric cancer serum biomarker and a kit thereof.
Background
Gastric cancer is a common malignant tumor of the digestive tract, and the mortality rate of gastric cancer is second and second to that of lung cancer according to the statistics of the World Health Organization (WHO). According to the statistics of the national cancer center and the national tumor registration center, the incidence rate of the gastric cancer in China is about thirty-one-hundred-ten-thousand. Taking 2014 as an example, the number of newly diagnosed gastric cancer cases in China is about 41 ten thousand, and the number of stomach cancer death cases is 29 ten thousand (the death rate is twenty-one of one hundred thousand). The incidence rate of the stomach cancer of the male is far higher than that of the female, and the standardized incidence rate is about 2.4 times of that of the female. At present, the overall 5-year survival rate of gastric cancer in China is 43.4%, but the five-year survival rate of gastric cancer patients in stage III and IV is only 28.01% and 8.42%. The early diagnosis rate of the gastric cancer is improved, and the treatment effect of the gastric cancer patient is greatly improved. The biomarker can be used as an auxiliary means for imaging examination, and the combined use of the biomarker and the biomarker can greatly improve the early diagnosis rate of the gastric cancer.
An ideal biomarker should meet the following characteristics: (1) high specificity, i.e., the expression of the marker specifically in the corresponding tissue; (2) the concentration of the marker is related to the size, metastasis and malignancy of the tumor, and can assist in staging the tumor and judging prognosis; (3) the half-life period is short, the sensitivity is high, the physiological condition in vivo can be quickly reflected, the concentration is quickly increased under the diseased state, but the concentration is quickly reduced after effective treatment; (4) easy to detect.
Currently, some gastric cancer biomarkers are applied in clinic: (1) carcinoembryonic antigen (CEA) is acidic Tang-white with human embryo antigen characteristics, exists in the serum of patients with gastric cancer and other adenocarcinoma, has small diagnostic significance on early gastric cancer, and is mainly used for dynamic observation before and after gastric cancer treatment; (2) glycoproteins such as CA125, CA19-9, CA50, CA724, CA242 and the like have sensitivity of only 20-40% although the glycoprotein antigens are increased in part of patients with gastric cancer; (3) tumor genes such as DDC, c-myc, c-erb-2, p53, nm23 and the like have certain significance for the generation and metastasis of gastric cancer, but the wide application in clinic is still limited. In view of the above, none of the existing biomarkers have ideal performance in gastric cancer diagnosis, and therefore, a new gastric cancer biomarker needs to be found.
The human protein ZNF709(zinc finger 709) is encoded by ZNF709 gene on chromosome 19p13.2, contains 641 amino acids in total, and has a molecular weight of 74.65 kDa. At present, no report that ZNF709 protein is taken as a gastric cancer biomarker is found.
Disclosure of Invention
The invention aims to provide application of ZNF709 protein as a gastric cancer serum biomarker and a kit thereof. The specificity of the serum marker for detecting gastric cancer is 85 percent, the sensitivity is 81 percent, and the provided kit is a sensitive, safe and simple-to-operate gastric cancer diagnostic kit, and can be used for qualitatively detecting the immunoglobulin G antibody level of the protein ZNF709 in human serum, thereby reflecting the level of the ZNF709 protein.
The kit adopts Enzyme linked immunosorbent assay (ELISA) technology and adopts an indirect method to qualitatively detect the immunoglobulin G antibody level of protein ZNF709 in human serum. The method comprises the steps of coating a microporous plate with purified human protein ZNF709 to prepare a solid-phase antigen, sequentially adding to-be-detected serum and a related reagent into the antigen-coated microporous plate, combining with a secondary antibody marked by HRP to form a ZNF 709-antibody-enzyme-labeled secondary antibody compound, and finally washing thoroughly and adding a substrate TMB for color development. TMB is converted to blue by the catalysis of HRP enzyme and to a final yellow color by the action of acid, the shade of which is positively correlated with the level of igg antibodies to the protein ZNF709 in the test sample. Finally, an absorbance value (OD value) is measured by using an enzyme-labeling instrument at the wavelength of 450nm to serve as a quantitative detection result.
The purpose of the invention is realized by the following technical scheme:
in a first aspect, the invention provides an application of ZNF709 protein as a serum biomarker of gastric cancer.
Preferably, the ZNF709 protein is obtained by performing galactose-induced over-expression on genetically engineered saccharomyces cerevisiae and then separating and purifying through agarose affinity medium glutathione.
Preferably, the ZNF709 protein is a human ZNF709 protein.
In a second aspect, the invention provides a gastric cancer serum biomarker comprising ZNF709 protein.
In a third aspect, the present invention provides a kit for detecting serum markers of gastric cancer, comprising: ZNF709 protein coated on an enzyme label plate, 0U/mL standard serum 1, 100U/mL standard serum 2, an enzyme labeling reagent, an enzyme substrate solution, a confining liquid, a sample diluent, a washing liquid and a stop solution.
Preferably, the enzyme substrate solution is TMB application solution which comprises a color-developing agent A and a color-developing agent B;
the developer A is counted by every 500mL and comprises the following components: 10.7-14.2g of sodium acetate, 0.3-1.9g of citric acid and 0.2-0.6mL of 30% hydrogen peroxide;
the developer B comprises the following components in every 500 mL: 650mg of TMB 200, 5-30mL of DMSO and 3.2-7.9g of citric acid.
Preferably, in the preparation of the ZNF709 protein coated on the ELISA plate, the coating buffer solution is 0.05M carbonate buffer solution; the carbonate buffer comprises the following components: 1.59g/L of sodium carbonate and 2.93g/L of sodium bicarbonate; the pH of the carbonate buffer was 7.4.
Preferably, the blocking solution is 0.01mol/L PBS buffer solution containing 0.5% bovine serum albumin, and specifically comprises: bovine serum albumin BSA 5g/L, sodium chloride 8g/L, potassium chloride 0.2g/L, dipotassium hydrogen phosphate 0.2g/L, sodium hydrogen phosphate dodecahydrate 2.9g/L, pH 7.4.
Preferably, the sample diluent is 0.01mol/L PBS buffer solution, and specifically comprises: 0.2g/L of potassium chloride, 8g/L of sodium chloride, 0.2g/L of disodium hydrogen phosphate, 2.9g/L of sodium hydrogen phosphate dodecahydrate and the pH value of 7.4.
Preferably, the washing solution is 0.01mol/L PBST phosphate buffer solution, and specifically comprises: potassium chloride 0.2g/L, sodium chloride 8g/L, dipotassium hydrogen phosphate 0.2g/L, sodium hydrogen phosphate dodecahydrate 2.9g/L, Tween-200.5 mL/L, pH value is 7.4;
the stop solution is a 2mol/L sulfuric acid solution;
the enzyme label is a human immunoglobulin G antibody labeled by horseradish peroxidase.
In a fourth aspect, the present invention provides a method for determining the concentration of an immunoglobulin G antibody of the protein ZNF709 in serum based on the aforementioned kit, comprising the steps of:
(1) coating: diluting purified human protein ZNF709 with coating buffer solution to 1 μ g/mL, adding into a well enzyme label plate, coating for 2 hours at 37 ℃, then washing for 3 times, and drying;
(2) and (3) sealing: adding 200 μ L of confining liquid, standing at room temperature for 2 hr, washing for 3 times, and spin-drying;
(3) sample adding: diluting 0U/ml standard serum 1, 100U/ml standard serum 2 and the serum sample to be detected to 100 mu L according to the ratio of 1:100 respectively, adding the diluted serum sample into each antigen detection pore plate, uniformly shaking, and covering or laminating a film;
(4) and (3) incubation: placing the enzyme label plate at 37 ℃ for reaction for 120 minutes, and then completely throwing off liquid in the hole without washing;
(5) enzyme labeling: and adding 100 mu L of human immunoglobulin G antibody marked by horseradish peroxidase into each hole, placing the mixture at 37 ℃ for 60 minutes, then completely throwing off liquid in the holes, washing the plate for 5 times, and then drying the plate.
(6) Color development: and dripping 50 mu L of color-developing agent A and 50 mu L of color-developing agent B into each hole after the holes are patted dry, shaking and uniformly mixing, placing at 37 ℃ in a dark place for color development for 15 minutes, adding 50 mu L of stop solution, and stopping reaction.
(7) And sequentially measuring the optical density of each hole at the wavelength of 450nm by using an enzyme linked instrument, and then calculating to obtain the concentration of the immunoglobulin G antibody of the protein ZNF709 in the serum.
Compared with the prior art, the invention has the following beneficial effects:
1. the ZNF709 protein provided by the invention has high specificity as a gastric cancer serum biomarker;
2. the invention provides a commercial kit which is sensitive, safe and simple to operate, and can be used for qualitatively and quantitatively determining the level of an immunoglobulin G antibody of a protein ZNF709 in human serum and reflecting the level of the ZNF709 protein, thereby being used for diagnosing gastric cancer.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit of the invention. All falling within the scope of the present invention.
The present invention is further illustrated by reference to examples.
Example 1
1. Expression, purification and identification of ZNF709 protein:
the human protein ZNF709 is obtained by adopting a conventional method, inducing over-expression by galactose from genetically engineered saccharomyces cerevisiae, separating and purifying by agarose affinity medium glutathione, and identifying by Western-Blotting.
2. Preparation of serum samples:
the whole blood specimen is placed at room temperature for 2 hours, then centrifuged for 20 minutes, and the supernatant is taken for split charging and stored at-80 ℃. The thawed serum sample may be centrifuged again before being used for detection.
Preparation methods of various buffers and reagents of ELISA method:
(1) coating buffer solution: 0.05M sodium carbonate-sodium bicarbonate (pH 9.6)
(2) Sample diluent: PBS solution pH 7.4
(3) Washing liquid: PBST solution pH 7.4
(4) Sealing liquid: 0.5% BSA in PBS (pH 7.4)
(5) Enzyme substrate solution: color-developing agent A and color-developing agent B
(for use as ready-to-use)
(for use as ready-to-use)
(6) Stopping liquid: 2mol/L sulfuric acid solution
(concentrated sulfuric acid is slowly dropped into distilled water during the time distribution, and the concentrated sulfuric acid is mixed evenly while being added)
ELISA method for determining human immunoglobulin G antibody concentration of protein ZNF709 in serum and diagnosis of gastric cancer:
the specific operation steps are as follows:
(1) coating: the purified human protein ZNF709 is diluted to 1 mu g/mL by using a coating buffer solution, then is added into a 96-hole enzyme label plate, is coated for 2 hours at 37 ℃, and then is washed for 3 times by using a washing solution and is dried by spin.
(2) And (3) sealing: adding 200 μ L of confining liquid, standing at room temperature for 2 hr, washing with washing solution for 3 times, and drying.
(3) Sample adding: diluting the standard substance (0U/ml standard serum 1, 100U/ml standard serum 2; obtained by the method in the step 2) and the serum sample to be detected (obtained by the method in the step 2) according to the ratio of 1:100 respectively to 100 mu L, adding the diluted sample into the 96-well enzyme label plate coated with the human protein ZNF709 prepared in the step (1), shaking uniformly, and covering or laminating a film. The standard substance and the sample to be detected need to be prepared within 15 minutes before use and are discarded after use.
(4) And (3) incubation: the enzyme label plate is placed at 37 ℃ for reaction for 120 minutes, and then liquid in the hole is completely thrown away without washing.
(5) Enzyme labeling: and adding 100 mu L of human immunoglobulin G antibody marked by horseradish peroxidase into each hole, placing the mixture at 37 ℃ for 60 minutes, then completely throwing off liquid in the holes, washing the plate for 5 times, and then drying the plate.
(6) Color development: and dripping 50 mu L of color-developing agent A and 50 mu L of color-developing agent B into each hole after the holes are patted dry, shaking and uniformly mixing, placing at 37 ℃ in a dark place for color development for 15 minutes, adding 50 mu L of stop solution, and stopping reaction.
(7) And (4) judging a result:
i. the optical density (OD value) of each well was measured sequentially at a wavelength of 450nm by an enzyme-linked analyzer, and then calculated by the following formula
The concentration of the immunoglobulin G antibody against ZNF709 in the serum (i.e., the unit value of the following formula) was obtained.
A450 is an abbreviation for absorbance at 450 nm.
Currently, no international standard for reference exists for the ZNF709 antibody, so that relative units are adopted for calibration of the detection result.
Determination of anti-ZNF 709 values in serum:
| anti ZNF709 value (U/mL) | Determination |
| Greater than 5 | Stomach cancer |
| 2-5 | High risk |
| Less than 2 | Health care |
Quality control
Each test result must meet the following criteria:
a450 of standard serum 1: less than or equal to 0.100
A450 of standard serum 2: not less than 0.700
If the above criteria are not met, the result is deemed invalid and must be retested.
interpretation of test results
The above reference values were established by ROC analysis of 50 healthy human sera, 37 gastric cancer sera, and 14 high-risk sera.
5. Specificity and sensitivity detection: the specificity and sensitivity of the invention were tested by using 101 serum samples (50 healthy human sera, 37 gastric cancer sera, 14 high-risk sera). The specificity and the sensitivity of the invention for diagnosing the gastric cancer are respectively 85% and 81%.
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention. The embodiments and features of the embodiments of the present application may be combined with each other arbitrarily without conflict.
Claims (9)
1. An application of an immunoglobulin G antibody of ZNF709 protein in preparing a kit for detecting a gastric cancer serum marker.
2. The use as claimed in claim 1 wherein the ZNF709 protein is obtained from genetically engineered Saccharomyces cerevisiae by galactose-induced over-expression and glutathione separation and purification in agarose affinity media.
3. A kit for detecting serum markers of gastric cancer, comprising: ZNF709 protein coated on an enzyme label plate, 0U/mL standard serum 1, 100U/mL standard serum 2, an enzyme labeling reagent, an enzyme substrate solution, a confining liquid, a sample diluent, a washing liquid and a stop solution; the enzyme label is a human immunoglobulin G antibody labeled by horseradish peroxidase.
4. The gastric cancer serum marker detection kit according to claim 3, wherein the enzyme substrate solution is TMB application solution comprising color reagent A and color reagent B;
the developer A is counted by every 500mL and comprises the following components: 10.7-14.2g of sodium acetate, 0.3-1.9g of citric acid and 0.2-0.6mL of 30% hydrogen peroxide;
the developer B comprises the following components in every 500 mL: 650mg of TMB 200, 5-30mL of DMSO and 3.2-7.9g of citric acid.
5. The kit for detecting the gastric cancer serum marker as claimed in claim 3, wherein the ZNF709 protein coated on the enzyme label plate is prepared by using a coating buffer solution of 0.05M carbonate buffer solution; the carbonate buffer comprises the following components: 1.59g/L of sodium carbonate and 2.93g/L of sodium bicarbonate; the pH of the carbonate buffer was 7.4.
6. The kit for detecting the serum marker of gastric cancer according to claim 3, wherein the blocking solution is 0.01mol/L PBS buffer solution containing 0.5% bovine serum albumin, and specifically comprises: bovine serum albumin BSA 5g/L, sodium chloride 8g/L, potassium chloride 0.2g/L, dipotassium hydrogen phosphate 0.2g/L, sodium hydrogen phosphate dodecahydrate 2.9g/L, pH 7.4.
7. The kit for detecting the serum marker of gastric cancer according to claim 3, wherein the sample diluent is 0.01mol/L PBS buffer solution, and specifically comprises: 0.2g/L of potassium chloride, 8g/L of sodium chloride, 0.2g/L of disodium hydrogen phosphate, 2.9g/L of sodium hydrogen phosphate dodecahydrate and the pH value of 7.4.
8. The kit for detecting the serum marker of gastric cancer according to claim 3, wherein the washing solution is 0.01mol/L PBST phosphate buffer solution, and specifically comprises: potassium chloride 0.2g/L, sodium chloride 8g/L, dipotassium hydrogen phosphate 0.2g/L, sodium hydrogen phosphate dodecahydrate 2.9g/L, Tween-200.5 mL/L, pH value is 7.4;
the stop solution is a 2mol/L sulfuric acid solution.
9. A method for determining the concentration of igg antibodies to the protein ZNF709 in serum, for non-diagnostic purposes, based on the kit of claim 3, comprising the steps of:
(1) coating: diluting purified human protein ZNF709 with coating buffer solution to 1 μ g/mL, adding into a well enzyme label plate, coating for 2 hours at 37 ℃, then washing for 3 times, and drying;
(2) and (3) sealing: adding 200 μ L of confining liquid, standing at room temperature for 2 hr, washing for 3 times, and spin-drying;
(3) sample adding: diluting 0U/ml standard serum 1, 100U/ml standard serum 2 and the serum sample to be detected to 100 mu L according to the ratio of 1:100 respectively, adding the diluted serum sample into each antigen detection pore plate, uniformly shaking, and covering or laminating a film;
(4) and (3) incubation: placing the enzyme label plate at 37 ℃ for reaction for 120 minutes, and then completely throwing off liquid in the hole without washing;
(5) enzyme labeling: adding 100 mu L of human immunoglobulin G antibody marked by horseradish peroxidase into each hole, placing the mixture at 37 ℃ for 60 minutes, then completely throwing off liquid in the holes, washing the plate for 5 times, and then beating the plate to be dry;
(6) color development: dripping 50 mu L of color-developing agent A and 50 mu L of color-developing agent B into each hole after the holes are patted dry, shaking and uniformly mixing, placing the mixture at 37 ℃ in a dark place for color development for 15 minutes, adding 50 mu L of stop solution, and stopping reaction;
(7) and sequentially measuring the optical density of each hole at the wavelength of 450nm by using an enzyme linked instrument, and then calculating to obtain the concentration of the immunoglobulin G antibody of the anti-protein ZNF709 in the serum.
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| WO2013166264A2 (en) * | 2012-05-02 | 2013-11-07 | University Of Georgia Research Foundation, Inc. | Methods for altering virus replication |
| CN104792894A (en) * | 2015-04-21 | 2015-07-22 | 首都医科大学附属北京儿童医院 | Protein characteristic spectrum of active tuberculosis in children and method for creating protein characteristic spectrum |
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