Application of reagent for detecting ITGB5 protein expression in preparation of serum diagnostic kit for ovarian cancer

Abstract

The invention provides the application of the reagent for detecting ITGB5 protein expression in preparation of the kit for ovarian cancer serum diagnosis, the reagent has high specificity and sensitivity, the ITGB5 antigen level in human serum can be quantitatively determined, ovarian cancer diagnosis evaluation can be assisted by testing the expression level of ITGB5 in an ovarian cancer patient, and the kit can be used for diagnosis and evaluation of ovarian cancer. The device has the advantages of being sensitive, reliable and operable.

Description

Application of reagents for detecting ITGB5 protein expression in the preparation of serum diagnostic kits for ovarian cancer Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to the application of a reagent for detecting ITGB5 protein expression in the preparation of a serum diagnostic kit for ovarian cancer. Background Technology

[0002] Ovarian cancer (OC) is a leading cause of death from gynecological malignancies. Globally, there are approximately 239,000 new cases and 152,000 deaths annually. The lifetime risk of a woman developing ovarian cancer is approximately 1 in 78, and the lifetime chance of dying from it is approximately 1 in 108. About four-fifths of ovarian cancer patients are diagnosed at an advanced stage, and only about 30% of these patients survive for more than five years. The lack of effective and practical screening strategies and the asymptomatic nature of ovarian cancer contribute to its late detection and poor prognosis. Therefore, finding tumor markers with both high specificity and sensitivity is crucial for improving the survival of ovarian cancer patients.

[0003] Ideal tumor markers should meet the following conditions: (1) high sensitivity; (2) good specificity; (3) the concentration of tumor markers is related to tumor size, metastasis and malignancy, and can monitor the effect of tumor treatment and tumor recurrence and predict prognosis; (4) short half-life, the concentration drops quickly after effective treatment, and can reflect the actual situation of tumors in the body quickly; (5) exist in body fluids, especially blood, and are easy to detect.

[0004] Integrin β5 (ITGB5) is a member of the integrin family. However, there is currently no clear large-sample data to verify the correlation between its serum expression level and tumor development. This invention found that the sensitivity of serum ITGB5 level in diagnosing ovarian cancer is 82.5% and the specificity is 77.5%. Summary of the Invention

[0005] To address the shortcomings of existing methods, the present invention aims to design and provide a reagent for detecting ITGB5 protein expression in the preparation of a serum diagnostic kit for ovarian cancer. This kit qualitatively or quantitatively detects the level of ITGB5 protein in human serum, serving as an auxiliary means for the early diagnosis of ovarian cancer and significantly improving the sensitivity and specificity of early diagnosis.

[0006] Preferably, the reagent for detecting ITGB5 protein expression is an antibody against human integrin protein ITGB5.

[0007] Preferably, the antibody against human integrin protein ITGB5 is an anti-human ITGB5 monoclonal antibody or an anti-human ITGB5 polyclonal antibody.

[0008] Preferably, the ovarian cancer serum diagnostic kit includes an antibody against human integrin protein ITGB5, an enzyme-labeled antibody (biotin-labeled anti-human ITGB5 detection antibody), and a chromogenic solution (TMB chromogenic solution).

[0009] The beneficial effects of this invention are as follows: the reagent for detecting ITGB5 protein expression prepared in this invention has high specificity and sensitivity, and can quantitatively measure the level of ITGB5 antigen in human serum. By testing the expression level of ITGB5 in ovarian cancer patients, it can assist in the diagnosis and assessment of ovarian cancer. Attached Figure Description

[0010] Figure 1 shows the standard curve of the ELISA experiment in Example 1. As can be seen from Figure 1, the standard curve exhibits good linearity, R0 2 A value close to 1 indicates a certain linear relationship between absorbance and concentration, suggesting high reliability of subsequent sample concentration calculations. Figure 2 shows the ROC curve in Example 1 (based on ELISA detection values, ROC analysis was performed to assess the sensitivity and specificity of ITGB5 as a serum diagnostic marker). As can be seen from Figure 2, the ROC analysis of ITGB5 as a serum marker exhibits good sensitivity and specificity (AUC = 0.8813, P < 0.0001). Figure 3 shows the difference in ITGB5 protein expression in the serum of ovarian cancer patients and normal individuals detected by ELISA in Example 1. As can be seen from Figure 3, human integrin protein ITGB5 is significantly highly expressed in ovarian cancer serum. Detailed Implementation

[0011] To make the present invention easier to understand, specific embodiments are described below to further illustrate the invention. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Specific experimental methods not mentioned in the following embodiments are generally performed using conventional experimental methods.

[0012] Example 11, Collection and Preparation of Serum Samples: Whole blood samples were collected from ovarian cancer patients and normal controls using standard venipuncture. After the blood coagulated at room temperature, it was centrifuged at 3000g / min for 15 minutes. The collected serum could be tested immediately or aliquoted and stored at -80℃ for subsequent analysis.

[0013] 2. Reagents: ELISA kit containing a monoclonal antibody linked to human integrin protein ITGB5.

[0014] 3. Specific steps: (1) Standard dilution: The standard solution is resuspended in 1.0 mL of sample diluent and left to stand for 10 minutes with gentle stirring at room temperature. The standard is diluted to 8 concentration gradients: 10 ng / mL, 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL, 0.625 ng / mL, 0.313 ng / mL, 0.157 ng / mL, and 0 ng / mL.

[0015] (2) Sample addition: Add 100 μL of diluted serum sample and standard to each well and incubate at 37°C for 1 hour; then wash.

[0016] (3) Enzyme-labeled secondary antibody: Add 100 μL of detection reagent A (ITGB5 biotinylated detection antibody) working solution to each well, mix thoroughly, and incubate at 37°C for 1 hour. Discard the liquid and wash three times with 350 μL of washing buffer. Add 100 μL of detection reagent B (streptavidin-horseradish peroxidase complex) working solution to each well and incubate at 37°C for 30 minutes.

[0017] (4) Color development: After cleaning, add 90 μL of methyl ammonium bromide substrate solution, incubate at 37°C for 20 minutes, avoid light and monitor regularly until the optimal color development is achieved.

[0018] (5) Termination: After color development, add 50 μL of stop solution and use an ELISA reader to measure the absorbance of each well at a wavelength of 450 nm.

[0019] (6) Standard curve (as shown in Figure 1): Plot the standard curve with absorbance as the abscissa and the corresponding protein concentration as the ordinate. Calculate the concentration of ITGB5 protein in each sample based on the standard curve.

[0020] (7) Result determination: The protein concentrations of ITGB5 in the serum of normal individuals and patients with ovarian cancer were analyzed by difference and ROC analysis. The ROC curve is shown in Figure 2, and the distribution of ITGB5 points is shown in Figure 3.

[0021] The measurement data are shown in the table below:

[0022] The diagnostic method for ovarian cancer provided by this invention has a sensitivity of 82.5% and a specificity of 77.5%.

[0022] Figure 1 shows the standard curve in the embodiment. As can be seen from Figure 1, the standard curve exhibits good linearity, R... 2 A value close to 1 indicates a certain linear relationship between absorbance and concentration, suggesting high reliability of subsequent sample concentration calculations.

[0023] Figure 2 shows the ROC curve in the example. As can be seen from Figure 2, the ROC analysis of ITGB5 as a serum marker has good sensitivity and specificity (AUC=0.8813, P<0.0001). The sensitivity is 82.5% and the specificity is 77.5%.

[0024] Figure 3 shows the difference in the expression of ITGB5 protein in the serum of ovarian cancer patients and normal individuals detected by ELISA. As can be seen from Figure 3, human integrin protein ITGB5 is significantly highly expressed in the serum of ovarian cancer patients (P=0.0005).

[0025] As can be seen from the above analysis, the reagent for detecting ITGB5 protein expression of the present invention can assist in the diagnosis and assessment of ovarian cancer by testing the expression level of ITGB5 in ovarian cancer patients in the preparation of a serum diagnostic kit for ovarian cancer.

Claims

1. Application of reagents for detecting ITGB5 protein expression in the preparation of serum diagnostic kits for ovarian cancer.

2. The application as described in claim 1, characterized in that: The reagent used to detect ITGB5 protein expression is an antibody against human integrin protein ITGB5.

3. The application as described in claim 1, characterized in that: The antibody against human integrin protein ITGB5 is either an anti-human ITGB5 monoclonal antibody or an anti-human ITGB5 polyclonal antibody.

4. The application as described in claim 2, characterized in that... The ovarian cancer serum diagnostic kit includes an antibody against human integrin protein ITGB5 and an enzyme-labeled antibody.

5. The application as described in claim 4, characterized in that: The enzyme-labeled antibody is a biotin-labeled anti-human ITGB5 detection antibody.

Images