CN110261618B

CN110261618B - Application of SPRR4 protein as gastric cancer serum biomarker and kit thereof

Info

Publication number: CN110261618B
Application number: CN201910516525.3A
Authority: CN
Inventors: 王靖方; 庞世超; 徐泓淋; 杨俊晨
Original assignee: Shanghai Sinuo Biological Technology Co ltd
Current assignee: Shanghai Sinuo Biological Technology Co ltd
Priority date: 2019-06-14
Filing date: 2019-06-14
Publication date: 2021-08-31
Anticipated expiration: 2039-06-14
Also published as: CN110261618A

Abstract

The invention discloses an application of SPRR4 protein as a gastric cancer serum biomarker and a kit thereof, wherein the kit comprises: SPRR4 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 immunoglobulin G antibody for resisting SPRR4 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, can be used for quantitatively determining the level of the immunoglobulin G antibody of the protein SPRR4 in human serum and reflecting the level of SPRR4 protein, and is used for gastric cancer diagnosis.

Description

Application of SPRR4 protein as gastric cancer serum biomarker and kit thereof

Technical Field

The invention relates to the field of bioscience, and particularly relates to application of SPRR4 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 SPRR4(small proline-rich protein 4) is encoded by SPRR4 gene on chromosome 1, 9p21.3, and contains 79 amino acids in total, and the molecular weight is 8.79 kDa. At present, no report that the SPRR4 protein is used as a gastric cancer biomarker is found.

Disclosure of Invention

The invention aims to provide application of SPRR4 protein as a gastric cancer serum biomarker and a kit thereof. The specificity and the sensitivity of the serum marker for detecting gastric cancer are 82% and 83%, 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 protein SPRR4 in human serum, thereby reflecting the level of SPRR4 protein.

The kit adopts Enzyme linked immunosorbent assay (ELISA) technology to qualitatively detect the immunoglobulin G antibody level of protein SPRR4 in human serum by indirect method. The method comprises the steps of coating a microporous plate with purified human protein SPRR4 to prepare a solid phase antigen, sequentially adding serum to be detected and a related reagent into the micropore coated with the antigen, combining with a secondary antibody marked by HRP to form an SPRR 4-antibody-enzyme-labeled secondary antibody compound, and finally adding a substrate TMB for color development after thorough washing. TMB is converted to blue by the HRP enzyme and to a final yellow color by the action of an acid, the shade of which is positively correlated with the level of igg antibodies to the protein SPRR4 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 SPRR4 protein as a gastric cancer serum biomarker.

Preferably, the SPRR4 protein is obtained by inducing overexpression of genetically engineered Saccharomyces cerevisiae through galactose, and then separating and purifying through agarose affinity medium glutathione.

Preferably, the SPRR4 protein is a human SPRR4 protein.

In a second aspect, the present invention provides a serum biomarker for gastric cancer, comprising SPRR4 protein.

In a third aspect, the present invention provides a kit for detecting serum markers of gastric cancer, comprising: SPRR4 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 TMB200, 5-30mL of DMSO and 3.2-7.9g of citric acid.

Preferably, in the preparation of the SPRR4 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 immunoglobulin G antibody of protein SPRR4 in serum based on the aforementioned kit, comprising the steps of:

(1) coating: diluting the purified human protein SPRR4 to 1 μ g/mL with a coating buffer solution, adding the diluted human protein SPRR4 into a well ELISA plate, coating the well ELISA plate for 2 hours at 37 ℃, washing for 3 times, and spin-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) The optical density of each well is measured sequentially by an enzyme-linked instrument at the wavelength of 450nm, and then calculation is carried out, thus obtaining the immunoglobulin G antibody concentration of the protein SPRR4 in the serum.

Compared with the prior art, the invention has the following beneficial effects:

1. the SPRR4 protein provided by the invention has high specificity as a gastric cancer serum biomarker;

2. the invention provides a sensitive, safe and simple-to-operate commercialized kit, which can be used for qualitatively and quantitatively determining the level of an immunoglobulin G antibody of a protein SPRR4 in human serum and reflecting the level of SPRR4 protein, so that the kit can be used for gastric cancer diagnosis.

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 the SPRR4 protein:

the human protein SPRR4 is obtained by using galactose to induce over-expression from genetically engineered Saccharomyces cerevisiae by a conventional method, 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)

4, ELISA method for determining the concentration of human immunoglobulin G antibody of protein SPRR4 in serum and diagnosis of gastric cancer:

the specific operation steps are as follows:

(1) coating: the purified human protein SPRR4 was diluted to 1. mu.g/mL with coating buffer, added to a 96-well microplate, coated for 2 hours at 37 ℃, washed 3 times with washing solution, and spun-dried.

(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 direct purchase) 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 solution into the 96-hole enzyme label plate coated with the human protein SPRR4 prepared in the step (1), shaking uniformly, and covering or laminating a membrane. 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.

(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 SPRR4 in the serum (i.e., the unit value in the following equation) was obtained.

A450 is an abbreviation for absorbance at 450 nm.

Currently, there is no internationally agreed reference standard for the SPRR4 antibody, so the assay results were calibrated using relative units.

Determination of anti-SPRR 4 values in serum:

anti-SPRR 4 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 carcinoma are 82% and 83%.

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

1. An application of SPRR4 protein in preparing a gastric cancer serum biomarker detection kit, wherein the gastric cancer serum biomarker is an immunoglobulin G antibody of SPRR4 protein.

2. The use of claim 1, wherein the SPRR4 protein is obtained from genetically engineered Saccharomyces cerevisiae by galactose-induced overexpression followed by purification by agarose affinity media glutathione separation.

3. The use according to claim 1, wherein the kit comprises: SPRR4 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 of the enzyme labeling reagent is a human immunoglobulin G antibody labeled by horseradish peroxidase.

4. The use according to claim 3, wherein the enzyme substrate solution is a TMB use solution comprising colour reagent A and B;

the developer B comprises the following components in every 500 mL: 200-650mg of TMB, 5-30mL of DMSO, and 3.2-7.9g of citric acid.

5. The use as claimed in claim 3, wherein the SPRR4 protein coated on the microplate is prepared by using 0.05M carbonate buffer as coating buffer; 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 use of claim 3, wherein the blocking solution is 0.01mol/L PBS buffer 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 and sodium hydrogen phosphate dodecahydrate 2.9g/L, and the pH value is 7.4.

7. The use of claim 3, wherein the sample diluent is 0.01mol/L PBS buffer, 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 use according to claim 3, wherein the washing solution is 0.01mol/L PBST phosphate buffer solution, comprising: potassium chloride 0.2g/L, sodium chloride 8g/L, dipotassium hydrogen phosphate 0.2g/L, sodium hydrogen phosphate dodecahydrate 2.9g/L and Tween-200.5 mL/L, and the pH value is 7.4;

the stop solution is a 2mol/L sulfuric acid solution.

9. A method for determining the concentration of the igg antibody of the protein SPRR4 in serum, for non-diagnostic purposes, based on the kit of claim 3, comprising the steps of:

(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 (3) sequentially measuring the optical density of each hole at the wavelength of 450nm by using an enzyme-linked analyzer, and then calculating to obtain the concentration of the immunoglobulin G antibody of the anti-protein SPRR4 in the serum.