CN108680750A - The ELISA detection method and kit of TROP2 expressing quantities - Google Patents

Abstract

The present invention relates to biomedicine technical field, the specifically ELISA detection method and kit of TROP2 expressing quantities, the ELISA kit of TROP2 expressing quantities includes that TROP2 antibody is coated with 96 hole reaction plates；TROP2 antibody HRP enzyme-labeled secondary antibodies；TMB developing solutions；H2SO4 terminate liquids.The ELISA detection method and kit of a kind of TROP2 expressing quantities provided by the invention, the detection method can quickly detect TROP2 expressing quantities in serum, the high expression in various Serum of Cancer Patients that researches show that CD151 albumen.It prompts TROP2 albumen as a kind of potential serodiagnosis marker, can judge for clinical quick diagnosis, therapeutic effect and Index for diagnosis provides reference.

Description

ELISA detection method and kit for TROP2 protein expression

technical field

The invention relates to the technical field of biomedicine, in particular to an ELISA detection method and kit for TROP2 protein expression.

Background technique

Malignant tumors have become a major disease that seriously threatens human health. The number of tumors in my country has been increasing, and the mortality rate has risen from 83.65/100,000 in the 1970s to 108.26/100,000 in the 1990s, an increase of 29.42%. The mortality rate of malignant tumors in Guangxi is also on the rise. In 1996 alone (34,700 deaths) it was 12,700 more than in 1991, an increase of nearly 57.73%. Calculated from birth to 74 years old), the risk of death from malignant tumors is about 1/10 (cumulative risk 10.66%); malignant tumors reduce the average life expectancy of the population in Guangxi by 2.13 years. Malignant tumors have caused a heavy social and economic burden. It is estimated that the annual medical expenses for cancer patients are about 80 billion yuan, accounting for about 20% of the total health expenses. The national disability-adjusted life-years lost due to cancer was 1.851 million, and the estimated economic loss was as high as 143.23 billion yuan. Early diagnosis and early treatment of tumors is an effective means of prevention and treatment of tumors.

TROP2 is a glycoprotein receptor on the surface of epithelial cell membrane, one of its functions is as a transmembrane transducer of intracellular calcium signal. TROP2 has no expression or very low expression in normal tissues, but overexpression in various cancer tissues, so it is a very meaningful marker for tumor diagnosis, treatment and prognosis evaluation. Studies in recent years have found that the expression of TROP2 is associated with the invasion and metastasis of endometrial cancer, ovarian cancer, prostate cancer, pancreatic cancer, colorectal cancer, gastric and esophageal cancer, bile duct cancer, oral cancer, glioma and breast cancer. are closely related. In addition to the expression of TROP2 protein on the cell membrane, the extracellular fragments of the molecule can be hydrolyzed under the action of enzymes and released into the blood circulation, which may reflect the development status of tumors and is a potential serological diagnostic marker. The detection of TROP2 protein expression level and serological indicators is conducive to the research and application of rapid clinical diagnosis, treatment effect judgment and prognosis judgment. At present, the TROP2 detection methods reported at home and abroad are not very mature, and they are only used in scientific research experiments, and have not yet been developed into kits suitable for clinical detection, which is not conducive to routine clinical use.

Contents of the invention

The object of the present invention is to provide an ELISA detection method and a kit for detecting the expression level of TROP2 protein aiming at the above-mentioned defects in the prior art.

In order to realize the above-mentioned purpose of the invention, the present invention has adopted following technical scheme:

An ELISA kit for TROP2 protein expression, including TROP2 antibody-coated 96-well reaction plate; TROP2 antibody-HRP enzyme-labeled secondary antibody; TMB chromogenic solution; H ₂ SO ₄ stop solution; washing solution; ELISA kit composition :

In addition, the present invention also provides the following subsidiary technical solutions:

Preferably, the ELISA detection method of the TROP2 protein expression level comprises the steps of:

(1) Preparation of ELISA reaction reagents and optimization of reaction conditions: preparation of monoclonal antibody hybridoma cells-mouse ascites model; monoclonal antibody hybridoma-mouse ascites fluid TROP2 antibody purification; TROP2 antibody-HRP enzyme marker screening; purification of TROP2 antibody-HRP Enzyme-labeled conjugates;

(2) Purification of monoclonal antibody hybridoma-mouse ascites TROP2 antibody; TROP2 antibody-HRP enzyme marker screening; purification of TROP2 antibody-HRP enzyme marker conjugate;

(3) ELISA technology is used to detect the TROP2 protein in the normal control and test sample serum;

(4) The antigen and antibody remain on the solid phase carrier after binding;

(5) react with the enzyme-labeled antibody to form a product;

(6) After adding the chromogenic solution to react, add H ₂ SO ₄ to terminate the reaction, and analyze the result according to the depth of the colored product.

Preferably, the ELISA detection method of the TROP2 protein expression level uses ELISA technology to detect normal serum or serum from a sample to be tested, and the specific method is:

A. Take normal serum or the serum of the specimen to be tested, and incubate with the TROP2 antibody on the surface of the solid phase carrier for the first time, and separate the antigen-antibody complex formed on the solid phase carrier from other substances in the liquid by washing;

B. Add HRP enzyme-labeled antibody for the second incubation.

Preferably, in step A, the TROP2 antibody is diluted to 2 μg/mL, 100 uL is coated, and the coating condition is 2-5° C. for 8-15 hours.

Preferably, in step A, 100 uL of the serum to be tested is added to the well plate, washed with PBST for 3 to 5 times after 40 to 50 minutes, and the plate is tapped after each washing.

Preferably, in step A, the TROP2 antibody is incubated for the first time at 37° C. for 40-50 minutes, washed with PBST for 3-5 times, and tapped after each wash.

Preferably, the temperature of the second incubation of the TROP2 antibody in step B is 37° C. for 40-50 min, and the plate is washed with PBST for 3-5 times, and the plate is tapped after each wash.

Preferably, in the ELISA detection method of TROP2 protein expression, 90-110 uL of LTMB chromogenic solution is added after washing, and 40-60 uL of 2M H2SO4 is added after 4-8 minutes to terminate the reaction and obtain the experimental results.

TROP2 protein is not expressed or expressed very low in normal tissues, but overexpressed in various cancer tissues. The extracellular fragments of its molecules can be hydrolyzed under the action of enzymes and released into the blood circulation, which may reflect the development status of tumors and are a potential marker for serological diagnosis and monitoring.

The beneficial effect of the present invention lies in: the ELISA detection method and kit for the expression of TROP2 protein provided by the present invention, the detection method can rapidly detect the expression of TROP2 protein in serum, and studies have shown that TROP2 protein is highly expressed in serum of various tumor patients. It is suggested that TROP2 protein, as a potential serological diagnostic marker, can provide reference for rapid clinical diagnosis, judgment of treatment effect and prognosis.

Description of drawings

Fig. 1 is the ELISA result of TROP2 standard antigen;

Figure 2 shows the sensitivity detection of the TROP2 double-antibody sandwich method;

Figure 3A is the expression of TROP2 in the serum samples of breast cancer patients;

Figure 3B is the expression of TROP2 in normal healthy control serum samples.

Detailed ways

The technical solution of the present invention will be described in further non-limiting detail below in conjunction with preferred embodiments.

Example 1

ELISA kit for TROP2 protein expression, including TROP2 antibody-coated 96-well reaction plate; TROP2 antibody-HRP enzyme-labeled secondary antibody; TMB chromogenic solution; H2SO4 stop solution; washing solution; ELISA kit composition:

The ELISA detection method of described TROP2 protein expression amount, comprises the steps:

(1) Preparation of ELISA reaction reagents and optimization of reaction conditions: preparation of monoclonal antibody hybridoma cells-mouse ascites model; monoclonal antibody hybridoma-mouse ascites fluid TROP2 antibody purification; TROP2 antibody-HRP enzyme marker screening; purification of TROP2 antibody-HRP Enzyme-labeled conjugates;

(2) Purification of monoclonal antibody hybridoma-mouse ascites TROP2 antibody; TROP2 antibody-HRP enzyme marker screening; purification of TROP2 antibody-HRP enzyme marker conjugate;

(3) ELISA technology is used to detect the TROP2 protein in the normal control and test sample serum;

(4) The antigen and antibody remain on the solid phase carrier after binding;

(5) react with the enzyme-labeled antibody to form a product;

(6) After adding the chromogenic solution for reaction, add H2SO4 to terminate the reaction, and analyze the result according to the depth of the colored product.

The ELISA detection method of the TROP2 protein expression level uses ELISA technology to detect normal serum or serum from a sample to be tested, and the specific method is:

A. Take normal serum or the serum of the specimen to be tested, and incubate with the TROP2 antibody on the surface of the solid phase carrier for the first time, and separate the antigen-antibody complex formed on the solid phase carrier from other substances in the liquid by washing;

B. Add HRP enzyme-labeled antibody for the second incubation.

In step A, the TROP2 antibody was diluted to 2 μg/mL, and 100 uL was coated, and the coating condition was 2°C for 8 hours.

In step A, take 100uL of the serum to be tested and add it to the well plate, wash with PBST 3 times after 40min, and shake the plate after each wash.

In step A, the TROP2 antibody was incubated for the first time at 37°C for 45 minutes, washed 4 times with PBST, and tapped after each wash.

In step B, the second incubation temperature of TROP2 antibody was 37°C for 40 min, and the plate was washed 4 times with PBST, and the plate was tapped after each wash.

In the ELISA detection method for the expression of TROP2 protein, 110 uL TMB chromogenic solution was added after washing, and 40 uL 2M H2SO4 was added after 4 minutes to terminate the reaction, and the experimental results were obtained.

Example 2

ELISA kit for TROP2 protein expression, including TROP2 antibody-coated 96-well reaction plate; TROP2 antibody-HRP enzyme-labeled secondary antibody; TMB chromogenic solution; H2SO4 stop solution; ELISA kit composition:

The ELISA detection method of described TROP2 protein expression amount, comprises the steps:

(1) Preparation of ELISA reaction reagents and optimization of reaction conditions: preparation of monoclonal antibody hybridoma cells-mouse ascites model; monoclonal antibody hybridoma-mouse ascites fluid TROP2 antibody purification; TROP2 antibody-HRP enzyme marker screening; purification of TROP2 antibody-HRP Enzyme-labeled conjugates;

(2) Purification of monoclonal antibody hybridoma-mouse ascites TROP2 antibody; TROP2 antibody-HRP enzyme marker screening; purification of TROP2 antibody-HRP enzyme marker conjugate;

(3) ELISA technology is used to detect the TROP2 protein in the normal control and test sample serum;

(4) The antigen and antibody remain on the solid phase carrier after binding;

(5) react with the enzyme-labeled antibody to form a product;

(6) After adding the chromogenic solution for reaction, add H2SO4 to terminate the reaction, and analyze the result according to the depth of the colored product.

The ELISA detection method of the TROP2 protein expression level uses ELISA technology to detect normal serum or serum from a sample to be tested, and the specific method is:

A. Take normal serum or the serum of the specimen to be tested, and incubate with the TROP2 antibody on the surface of the solid phase carrier for the first time, and separate the antigen-antibody complex formed on the solid phase carrier from other substances in the liquid by washing;

B. Add HRP enzyme-labeled antibody for the second incubation.

In step A, the TROP2 antibody was diluted to 2 μg/mL, and 100 uL was coated, and the coating condition was 3°C for 10 hours.

In step A, take 100uL of the serum to be tested and add it to the well plate, wash with PBST 5 times after 50min, and shake the plate after each wash.

In Step A, the TROP2 antibody was incubated for the first time at 37°C for 50 min, washed 5 times with PBST, and tapped after each wash.

In step B, the second incubation temperature of TROP2 antibody was 37°C for 50 min, and the plate was washed 5 times with PBST, and the plate was tapped after each wash.

In the ELISA detection method for the expression of TROP2 protein, 100 uL TMB chromogenic solution was added after washing, and 60 uL 2M H2SO4 was added after 5 minutes to terminate the reaction, and the experimental results were obtained.

Example 3

ELISA kit for TROP2 protein expression, including TROP2 antibody-coated 96-well reaction plate; TROP2 antibody-HRP enzyme-labeled secondary antibody; TMB chromogenic solution; H2SO4 stop solution; washing solution; ELISA kit composition:

The ELISA detection method of described TROP2 protein expression amount, comprises the steps:

(1) Preparation of ELISA reaction reagents and optimization of reaction conditions: preparation of monoclonal antibody hybridoma cells-mouse ascites model; monoclonal antibody hybridoma-mouse ascites fluid TROP2 antibody purification; TROP2 antibody-HRP enzyme marker screening; purification of TROP2 antibody-HRP Enzyme-labeled conjugates;

(2) Purification of monoclonal antibody hybridoma-mouse ascites TROP2 antibody; TROP2 antibody-HRP enzyme marker screening; purification of TROP2 antibody-HRP enzyme marker conjugate;

(3) ELISA technology is used to detect the TROP2 protein in the normal control and test sample serum;

(4) The antigen and antibody remain on the solid phase carrier after binding;

(5) react with the enzyme-labeled antibody to form a product;

(6) After adding the chromogenic solution for reaction, add H2SO4 to terminate the reaction, and analyze the result according to the depth of the colored product.

The ELISA detection method of the TROP2 protein expression level uses ELISA technology to detect normal serum or serum from a sample to be tested, and the specific method is:

A. Take normal serum or the serum of the specimen to be tested, and incubate with the TROP2 antibody on the surface of the solid phase carrier for the first time, and separate the antigen-antibody complex formed on the solid phase carrier from other substances in the liquid by washing;

B. Add HRP enzyme-labeled antibody for the second incubation.

In step A, the TROP2 antibody was diluted to 2 μg/mL, and 100 uL was coated, and the coating condition was 5°C for 10 hours.

In step A, take 100uL of the serum to be tested and add it to the well plate, wash with PBST 4 times after 45min, and shake the plate after each wash.

In step A, the TROP2 antibody was incubated for the first time at 37°C for 45 minutes, washed 4 times with PBST, and tapped after each wash.

In step B, the second incubation temperature of TROP2 antibody was 37°C for 45 minutes, and the plate was washed 4 times with PBST, and the plate was tapped after each wash.

In the ELISA detection method for the expression of TROP2 protein, 110 uL TMB chromogenic solution was added after washing, and 60 uL 2M H2SO4 was added after 8 minutes to terminate the reaction, and the experimental results were obtained.

Example 4

ELISA kit for TROP2 protein expression, including TROP2 antibody-coated 96-well reaction plate; TROP2 antibody-HRP enzyme-labeled secondary antibody; TMB chromogenic solution; H2SO4 stop solution; ELISA kit composition:

.

The ELISA detection method of described TROP2 protein expression amount, comprises the steps:

(1) Preparation of ELISA reaction reagents and optimization of reaction conditions: preparation of monoclonal antibody hybridoma cells-mouse ascites model; monoclonal antibody hybridoma-mouse ascites fluid TROP2 antibody purification; TROP2 antibody-HRP enzyme marker screening; purification of TROP2 antibody-HRP Enzyme-labeled conjugates;

(2) Purification of monoclonal antibody hybridoma-mouse ascites TROP2 antibody; TROP2 antibody-HRP enzyme marker screening; purification of TROP2 antibody-HRP enzyme marker conjugate;

(3) ELISA technology is used to detect the TROP2 protein in the normal control and test sample serum;

(4) The antigen and antibody remain on the solid phase carrier after binding;

(5) react with the enzyme-labeled antibody to form a product;

(6) After adding the chromogenic solution for reaction, add H2SO4 to terminate the reaction, and analyze the result according to the depth of the colored product.

The ELISA detection method of the TROP2 protein expression level uses ELISA technology to detect normal serum or serum from a sample to be tested, and the specific method is:

A. Take normal serum or the serum of the specimen to be tested, and incubate with the TROP2 antibody on the surface of the solid phase carrier for the first time, and separate the antigen-antibody complex formed on the solid phase carrier from other substances in the liquid by washing;

B. Add HRP enzyme-labeled antibody for the second incubation.

In step A, the TROP2 antibody was diluted to 2 μg/mL, and 100 uL was coated, and the coating condition was 2°C for 8 hours.

In step A, take 100uL of the serum to be tested and add it to the well plate, wash with PBST 4 times after 45min, and shake the plate after each wash.

In step A, the TROP2 antibody was incubated for the first time at 37°C for 45 minutes, washed 4 times with PBST, and tapped after each wash.

In step B, the second incubation temperature of TROP2 antibody was 37°C for 45 minutes, and the plate was washed 4 times with PBST, and the plate was tapped after each wash.

In the ELISA detection method for the expression of TROP2 protein, 90-110 uL TMB chromogenic solution is added after washing, and 40-60 uL 2M H2SO4 is added after 4-8 minutes to terminate the reaction, and the experimental results are obtained.

Test Example 1

Positive in the present invention means that if the color reaction of the serum to be tested is higher than that of the blank control and negative control, then the expression of TROP2 protein is positive.

The high expression in the present invention means that the color reaction of the serum to be tested is higher than that of the blank control and the negative control, and the deeper the color reaction, the higher the expression of TROP2 protein.

The biological materials, reagents or instruments used in the ELISA detection method and kit for TROP2 protein expression provided by the present invention can be purchased from the market.

1. Materials and methods

1. Patient serum samples

For EILSA detection, serum samples from 46 breast cancer patients and 46 normal healthy controls were collected from the People's Hospital of Guangxi Zhuang Autonomous Region from January 2014 to December 2016. The collection criteria were pathological diagnosis of breast cancer, newly diagnosed and untreated, and no history of other tumors. Carcinoma in situ was excluded from the study. Clinical samples were used for research only and were approved by the Ethics Committee of the People's Hospital of Guangxi Zhuang Autonomous Region.

The clinical sample included 46 female breast cancer patients aged 35-55 with an average age of 42.6 years. All patients underwent mastectomy. The histopathological grade and clinical stage of this study were defined according to the World Health Organization (WHO) and the sixth edition pTMN International Union Against Cancer (UICC, 2002). Histological types and grades of all tumors were evaluated by two pathologists from the People's Hospital of Guangxi Zhuang Autonomous Region.

2. ELISA

Reagent 1: 96-well enzyme-labeled antibody-coated plate was taken out from 4°C and placed at room temperature for 5-10 minutes; Reagent 2: TROP2 antigen standard of known concentration was diluted with PBS, and the diluted concentrations were: 0, 3.125, 6.25, 12.5, 25, 50, 100ng/ml. Dilute the known concentration of TROP2 antigen, 10ug/ml, with 1×PBS 1:100. Each ELISA plate was diluted 1:100 with 5ul, the final concentration of the first tube was 100ng/ml, and then serially diluted 1:2, so that the final concentration of the last tube was 3.125ng/ml. Add 100uL each of TROP2 antigen of known concentration and the sample to be tested into the ELISA microwell plate, and incubate at room temperature for 45 minutes; take out reagent 3: 10×PBS and reagent 4: Tween-20 from the refrigerator, and press reagent preparation The preparation method of PBST is prepared, one plate needs about 360ml, PBST; wash the ELISA microplate 4 times with PBST, 300ul per well, and shoot the plate after each wash; after washing, add reagent 5: TROP2-37 cloning enzyme Labeled antibody, 100ul per well. The enzyme-labeled antibody was diluted with 1XPBS, and the accurate dilution factor was 1/40 (final concentration 2ug/ml). Improper dilution factor would cause background, and incubated at room temperature for 45 minutes; washed 4 times with PBST, and clapped the plate after each wash; , add reagent 6: TMB chromogenic solution for color development, 100ul per well, incubate at room temperature for 5min; accurately incubate at room temperature for 3.5min Add reagent 7: 2M H ₂ SO ₄ to stop the reaction, 50ul per well, incubation time exceeds 3.5min will produce Background: Measure the OD _450nm value of each well at OD _450nm with a microplate reader.

3. Effect evaluation

TROP2 shows a good linear relationship within a certain concentration range, and it will show an "S" curve beyond this range. Therefore, if the antigen concentration of TROP2 in some samples is higher than the above range, showing that the OD value is higher than the highest point, it is necessary to perform ELISA reaction after diluting the samples. Typical results are shown in Figure 1 below: different concentrations (0, 3.125, 6.25, 12.5, 25, 50, 100ng/ml, in order from left to right)

4. Statistical analysis

The t test was used to evaluate the expression of TROP2 protein in the serum of breast cancer patients and normal healthy controls. Statistical analysis was performed with SPSS22.0 software. P<0.05, the difference was considered significant.

2. Results

1. High expression of TROP2 in breast cancer patients

In order to analyze the expression difference of TROP2 between breast cancer patients and normal healthy controls, this study used ELISA method to detect the expression level of TROP2 in the serum samples of 46 breast cancer patients and 46 normal healthy controls. Among them, 25 cases (54.3%) of breast cancer patients showed high expression of TROP2 in serum samples (Fig. 3A); 6 cases of normal healthy control serum samples showed low expression of TROP2 (13.0%) and 3 cases showed moderate expression (6.5%) ( FIG. 3B ), (P<0.05).

The OD values shown in Figures 3A and 3B were compared by t test, and there was a significant difference in the expression of TROP2 protein between breast cancer patients and normal people (P<0.05).

It should be pointed out that the above-mentioned preferred embodiments are only to illustrate the technical conception and characteristics of the present invention, the purpose of which is to enable those familiar with this technology to understand the content of the present invention and implement it accordingly, and cannot limit the scope of the present invention. protected range. All equivalent changes or modifications made according to the spirit of the present invention shall fall within the protection scope of the present invention.

Claims (8)

1. The ELISA kit of 1.TROP2 expressing quantities, it is characterised in that：It is coated with 96 hole reaction plates including TROP2 antibody； TROP2 antibody-HRP enzyme-labeled secondary antibodies；TMB developing solutions；H2SO4 terminate liquids；Cleaning solution；ELISA kit forms：

   。
2. 2. the ELISA detection method of TROP2 expressing quantities according to claim 1, it is characterised in that：Including walking as follows Suddenly：

   （1）Prepare ELISA reaction reagents and optimization reaction condition：Prepare monoclonal antibody hybridoma-mouse ascites model；Monoclonal antibody is miscellaneous Hand over the purifying of tumor-mouse ascites TROP2 antibody；TROP2 antibody-HRP enzyme label screenings；Purify TROP2 antibody-HRP enzymes label Conjugate；

   （2）The purifying of monoclonal antibody hybridoma-mouse ascites TROP2 antibody；TROP2 antibody-HRP enzyme label screenings；It is anti-to purify TROP2 Body-HRP enzymes mark conjugate；

   （3）TROP2 albumen in normal control and sample to be tested serum is detected using elisa technique；

   （4）Antigen-antibody stays after combining in solid phase carrier；

   （5）Product is formed after being reacted with enzyme labelled antibody；

   （6）After developing solution reaction is added, H is added₂SO₄Reaction is terminated, interpretation of result is carried out according to the depth of color products.
3. 3. the ELISA detection method of TROP2 expressing quantities according to claim 1, it is characterised in that：The use Elisa technique is detected normal serum or sample serum to be checked, and specific method is：

   A, normal serum or sample serum to be checked are taken, first time incubation is carried out with the TROP2 antibody of surface of solid phase carriers, with washing Method so that the antigen-antibody complex formed on solid phase carrier is separated with other substances in liquid；

   B, HRP enzymic-labelled antibodies are added and carry out second of incubation.
4. 4. the ELISA detection method of TROP2 expressing quantities according to claim 3, it is characterised in that：In step A TROP2 antibody is diluted to 2 μ g/mL, wrapper sheet 100uL, and wrapper sheet condition is 2 ~ 5 DEG C and places 8 ~ 15 hours.
5. 5. the ELISA detection method of TROP2 expressing quantities according to claim 4, it is characterised in that：It is taken in step A 100uL serum to be checked is added in orifice plate, and PBST is cleaned 3 ~ 5 times after 40 ~ 50min, has the final say after each board-washing.
6. 6. the ELISA detection method of TROP2 expressing quantities according to claim 3, it is characterised in that：In step A TROP2 antibody first time incubation temperatures are 37 DEG C, and the time is 40 ~ 50min, is cleaned 3 ~ 5 times using PBST, is clapped after each board-washing Plate.
7. 7. the ELISA detection method of TROP2 expressing quantities according to claim 3, it is characterised in that：In step B Second of incubation temperature of TROP2 antibody is 37 DEG C, and the time is 40 ~ 50min, is cleaned 3 ~ 5 times using PBST, is clapped after each board-washing Plate.
8. 8. according to the ELISA detection method of claim 5 ~ 7 any one of them TROP2 expressing quantities, it is characterised in that： 90 ~ 110uLTMB developing solutions are added after cleaning, 40 ~ 60uL2M H2SO4, which are added, after 4 ~ 8min terminates reaction, is tested As a result.