A kit for preliminary screening of ovarian cancer and its application

Abstract

This invention belongs to the field of in vitro diagnostic technology and discloses a kit for preliminary screening of ovarian cancer and its application. The kit includes a solid-phase carrier and tumor-associated antigens coated on the solid-phase carrier. The tumor-associated antigens consist of ADHFE1, SAPCD2, ADAM17, REG4, and FLOT1. This kit is the first to combine these five antigens—ADHFE1, SAPCD2, ADAM17, REG4, and FLOT1—for ovarian cancer screening. It can specifically detect the levels of autoantibodies against ADHFE1, SAPCD2, ADAM17, REG4, and FLOT1 in serum, exhibiting high sensitivity and specificity, significantly improving the detection success rate of ovarian cancer, and has broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a reagent kit for preliminary screening of ovarian cancer and its application. Background Technology

[0002] Ovarian cancer is one of the most common malignant tumors of the female reproductive organs, posing a serious threat to women's lives. Because the ovaries are located deep in the pelvis, are small in size, and often lack typical symptoms, early diagnosis of ovarian cancer is difficult. By the time it is clinically discovered, it has often progressed to the middle or late stages. Studies show that only 30% of ovarian cancer patients are found to have tumors confined to the ovary during surgery; most have already spread to the uterus, omentum, and other pelvic organs, with a five-year survival rate hovering between 25% and 30%. If ovarian cancer is detected and treated early, the five-year survival rate exceeds 90%. Therefore, early screening is the most effective means of preventing ovarian cancer.

[0003] Currently, the most common methods for early ovarian cancer screening are ovarian cancer tumor markers and vaginal ultrasound imaging. Ovarian cancer biomarkers refer to abnormal substances or gene mutations produced by tumor cells or the body during the occurrence and development of ovarian cancer. These substances or mutations can be detected in biological samples such as blood, tissue, and urine, and are used for the diagnosis, prognostic assessment, and treatment monitoring of ovarian cancer. CA125 and HE4 are among the most common ovarian cancer tumor markers and are widely used in the diagnosis of epithelial ovarian cancer. However, because approximately 50% of early-stage ovarian cancer patients do not show significant changes in CA125 levels in early screenings, their specificity and sensitivity are not ideal. Furthermore, elevated CA125 levels can also occur in other common gynecological diseases, leading to false-positive results when relying solely on CA125 testing. HE4 testing has better specificity than CA125, but its sensitivity is insufficient when used alone. Vaginal ultrasound imaging is highly dependent on the operator's experience, has limited sensitivity for early-stage ovarian cancer, and is difficult to widely implement for screening. Therefore, CA125 and HE4 testing, along with vaginal ultrasound, cannot improve early screening rates, prognosis, or recurrence rates for ovarian cancer. The development of early ovarian cancer screening kits still faces challenges related to sensitivity, specificity, and cost. Consequently, accurate and reliable tumor markers are currently lacking for the early diagnosis of ovarian cancer, serving as a supplementary means of early detection to improve survival rates and reduce mortality. Recent studies have found that during cancer cell formation, a unique group of autocrine antigens, namely tumor-associated antigens (TAAs), are released, exhibiting measurable changes related to pathophysiological processes. Addressing the limitations of existing testing kits, current research combines TAs with the detection of autoantibody expression in patients to further improve diagnostic sensitivity and specificity, thereby increasing the detection rate of ovarian cancer.

[0004] To achieve the above objectives, the present invention provides a kit for preliminary screening of ovarian cancer. Summary of the Invention

[0005] The first objective of this invention is to provide the application of a combination of tumor-associated antigens ADHFE1, SAPCD2, ADAM17, REG4, and FLOT1 in the preparation of a kit for preliminary screening of ovarian cancer.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] Application of the combination of tumor-associated antigens ADHFE1, SAPCD2, ADAM17, REG4, and FLOT1 in the preparation of a kit for preliminary screening of ovarian cancer.

[0008] A second objective of this invention is to provide a kit for preliminary screening of ovarian cancer.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] A kit for preliminary screening of ovarian cancer, the kit comprising a solid-phase carrier and a tumor-associated antigen coated on the solid-phase carrier; the tumor-associated antigen is composed of a combination of the tumor-associated antigens ADHFE1, SAPCD2, ADAM17, REG4 and FLOT1.

[0011] Furthermore, the solid-phase carrier is an enzyme-labeled plate.

[0012] Furthermore, the kit also includes enzyme-labeled secondary antibody, washing solution, colorimetric solution, stop solution, positive control serum, and negative control serum.

[0013] Furthermore, the enzyme-labeled secondary antibody is horseradish peroxidase-labeled RecA protein.

[0014] Furthermore, the washing solution is PBST; the colorimetric solution is TMB; and the stop solution is sulfuric acid.

[0015] Furthermore, the positive control serum is FLOT1 positive control serum, and the negative control serum is FLOT1 negative control serum;

[0016] Furthermore, the FLOT1 positive control serum is the serum of ovarian cancer patients who tested positive for FLOT1 antibody using indirect ELISA and Western blot methods; the FLOT1 negative control serum is the serum of healthy individuals whose FLOT1 antibody expression level was found to be the average antibody level in the serum of healthy individuals using indirect ELISA and Western blot methods.

[0017] Compared with the prior art, the main advantages of the present invention are as follows:

[0018] The ELISA kit prepared in this invention is the first to combine five antigens—ADHFE1, SAPCD2, ADAM17, REG4, and FLOT1—for ovarian cancer screening, enabling the detection of autoantibody levels of ADHFE1, SAPCD2, ADAM17, REG4, and FLOT1 in serum samples. Compared to using single, two, three, or four tumor-associated antigens for early ovarian cancer detection, the combined use of these five antigens—ADHFE1, SAPCD2, ADAM17, REG4, and FLOT1—shows the best diagnostic efficacy for early ovarian cancer, exhibiting high sensitivity and specificity. Its sensitivity reaches 92.8%, specificity reaches 90.1%, and Youden's index reaches 0.829, significantly improving the detection success rate of ovarian cancer and demonstrating broad application prospects. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the specific testing process for a kit used for preliminary screening of ovarian cancer.

[0020] Figure 2 The relative expression levels of ADHFE1 autoantibodies against tumor-associated antigens in healthy individuals and ovarian cancer;

[0021] Figure 3 The relative expression levels of SAPCD2 autoantibodies against tumor-associated antigens in healthy individuals and ovarian cancer;

[0022] Figure 4 The relative expression levels of ADAM17 autoantibodies against tumor-associated antigens in healthy individuals and ovarian cancer;

[0023] Figure 5 The relative expression levels of REG4 autoantibodies against tumor-associated antigens in healthy individuals and ovarian cancer;

[0024] Figure 6 The relative expression levels of FLOT1 autoantibodies, a tumor-associated antigen, in healthy individuals and ovarian cancer. Detailed Implementation

[0025] The technical solution of the present invention will be further described below with reference to specific embodiments. However, those skilled in the art should understand that the following embodiments are only for illustrating the present invention and should not be regarded as limiting the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used are all conventional products obtained through commercial channels.

[0026] Example 1

[0027] Preparation of ELISA kit:

[0028] (1) Composition of the ELISA kit: ELISA plate, horseradish peroxidase-labeled RecA protein, antigen standard ADHFE1, antigen standard SAPCD2, antigen standard ADAM17, antigen standard REG4, antigen standard FLOT1, washing buffer PBST (0.01M PBST containing 0.05% Tween 20 at pH 7.4), chromogenic solution 2mg / L TMB, stop solution 2M sulfuric acid, positive control serum, and negative control serum constitute the ELISA kit of the present invention. The positive control serum is FLOT1 positive control serum, that is, serum of ovarian cancer patients who are positive for FLOT1 antibody using indirect ELISA and Western blot methods; the negative control serum is FLOT1 negative control serum, that is, serum of healthy individuals whose FLOT1 antibody expression level is the average antibody content of healthy individuals as detected by indirect ELISA and Western blot methods.

[0029] (2) Preparation of six ovarian cancer-related antigen solutions: Antigen standards ADHFE1, SAPCD2, ADAM17, REG4, and FLOT1 were dissolved in coating buffer (50 mM carbonate buffer, pH=9.6), mixed thoroughly, and prepared into recombinant protein solutions of ADHFE1 (1.0 μg / mL), SAPCD2 (1.0 μg / mL), ADAM17 (1.0 μg / mL), REG4 (1.0 μg / mL), and FLOT1 (1.0 μg / mL).

[0030] (3) Preparation of antigen-coated ELISA plates: The ADHFE1 recombinant protein solution, SAPCD2 recombinant protein solution, ADAM17 recombinant protein solution, REG4 recombinant protein solution, and FLOT1 recombinant protein solution prepared in step (2) were added to the sample wells of a 48-well ELISA plate at a volume of 100 μL / well. The plate was incubated at 37°C for 1 hour and then at 4°C overnight. The coating solution was then removed, and the plate was washed three times with PBST washing buffer. Blocking buffer (PBST buffer containing 2% BSA) was added to the wells of the coated 48-well ELISA plate and incubated at room temperature for 1 hour. The blocking solution was then removed. The 48-well ELISA plate was dried in a 37°C oven to obtain antigen-coated 48-well ELISA plates, which were then stored at 4°C for later use.

[0031] (4) Dilution of horseradish peroxidase-labeled RecA protein: Dilute horseradish peroxidase-labeled RecA protein with PBST buffer containing 1% BSA at a ratio of 1:60000 and store at 4°C for later use.

[0032] Experimental Example 1

[0033] Blood sample testing:

[0034] (1) Collect 60 samples, including 30 blood samples from healthy individuals (numbered 1-30) and 30 blood samples from ovarian cancer patients (numbered 31-60). After blood collection, allow it to coagulate at room temperature, then centrifuge at 6000 rpm for 10 min and collect the supernatant. 20 min before the experiment, add the purified serum sample to the antigen-coated ELISA plate prepared in Example 1, incubate at 37°C for 1 h, wash the plate 3 times with PBST washing buffer and spin dry. Add 100 μL of diluted horseradish peroxidase-labeled RecA protein prepared in Example 1 to the reaction wells of a 48-well plate, incubate at 37°C for 1 h, wash 3 times with PBST washing buffer and spin dry. Add 100 μL of 2 mg / L TMB chromogenic solution to the reaction wells, incubate at room temperature in the dark for 10 min, and add 100 μL of 2 M sulfuric acid to stop the reaction. Figure 1 The diagram illustrates the specific detection process of the kit used for preliminary ovarian cancer screening. After the reaction, the OD450 value is detected using an ELISA reader. Serum samples from healthy individuals and ovarian cancer patients are collected to collect the levels of ADHFE1, SAPCD2, ADAM17, REG4, and FLOT1 autoantibodies. For ease of comparison, the autoantibody content in healthy serum samples is set to 1, and the relative expression level of autoantibodies in ovarian cancer patient serum is calculated. Relative expression level = autoantibody content in ovarian cancer patient serum / average autoantibody content in healthy serum samples. The relative expression levels of autoantibodies in the serum of healthy individuals and ovarian cancer patients are represented by a bar chart using Origin software. The results are shown below. Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 As shown.

[0035] Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6The figures show the relative expression levels of autoantibodies against tumor-associated antigens (ADHFE1, SAPCD2, ADAM17, REG4, and FLOT1). As the figures indicate, these autoantibodies are expressed at low levels in healthy individuals but at high levels in ovarian cancer patients. Therefore, the levels of these autoantibodies can significantly differentiate between ovarian cancer patients and healthy individuals. Consequently, ADHFE1, SAPCD2, ADAM17, REG4, and FLOT1 autoantibodies can serve as biomarkers for ovarian cancer and provide new insights and methods for the prediction and diagnosis of early-stage ovarian cancer.

[0036] (2) To further explore the detection effect of different combinations of the above five tumor-associated antigens on ovarian cancer, the sensitivity, specificity, and Youden index of the five tumor-associated antigen autoantibodies in healthy individuals and ovarian cancer patients were calculated based on the results of step (1). The results are shown in Table 1. Sensitivity reflects the ability of the kit to correctly identify patients; specificity reflects the ability of the kit to correctly exclude non-patients; Youden index = sensitivity + specificity - 1. The Youden index is used to evaluate the authenticity of the screening test, also known as the accuracy index. Usually, the Youden index is between 0 and 1. The closer it is to 1, the greater its diagnostic value and the higher its application value.

[0037] Table 1. Results of combined detection of ovarian cancer using different combinations of tumor-associated antigens.

[0038]

[0039] The results are shown in Table 1, which presents the results of combined detection of ovarian cancer using different combinations of tumor-associated antigens. As can be seen from Table 1, the sensitivity of early ovarian cancer diagnosis further increases with the increase in the number of tumor-associated antigen combinations. When five tumor-associated antigens (FLOT1, ADHFE1, SAPCD2, ADAM17, and REG4) are used in combination for early ovarian cancer detection, the sensitivity reaches 92.8%, indicating that the probability of correctly diagnosing early ovarian cancer using the kits coated with the five tumor-associated antigens in Table 1 is 92.8%. When the tumor-associated antigens FLOT1, ADHFE1, SAPCD2, ADAM17, and REG4 are used in combination for early ovarian cancer detection, the specificity reaches 90.1%, indicating that the rate of correctly diagnosing non-early ovarian cancer patients using the kits coated with the five tumor-associated antigens in Table 1 is 90.1%. Furthermore, the sensitivity and specificity of the combined use of five tumor-associated antigens (ADHFE1, SAPCD2, ADAM17, REG4, and REG4) for early ovarian cancer detection, as shown in Table 1, were higher than those of using single, two, three, or four tumor-associated antigens. This result indicates that the combined use of these five tumor-associated antigens for ovarian cancer screening is a relatively ideal method.

[0040] As shown in Table 1, the Youden Index gradually increases with the increase in the number of tumor-associated antigen combinations. When the five tumor-associated antigens FLOT1, ADHFE1, SAPCD2, ADAM17, and REG4 are used in combination for early ovarian cancer detection, the Youden Index reaches 0.829, which is higher than other combinations. This indicates that using the five tumor-associated antigens FLOT1, ADHFE1, SAPCD2, ADAM17, and REG4 as a combination has relatively high application value for early ovarian cancer screening.

[0041] In summary, the ELISA kit prepared in this invention is the first to combine five tumor-associated antigens—ADHFE1, SAPCD2, ADAM17, REG4, and FLOT1—for screening for early ovarian cancer. It exhibits high sensitivity and specificity, significantly improving the detection success rate of ovarian cancer. It has high diagnostic value for early ovarian cancer and has broad application prospects.

[0042] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. Application of the combination of tumor-associated antigens ADHFE1, SAPCD2, ADAM17, REG4 and FLOT1 in the preparation of a kit for preliminary screening of ovarian cancer.

2. A kit for preliminary screening of ovarian cancer, characterized in that, The kit includes a solid-phase carrier and a tumor-associated antigen coated on the solid-phase carrier; the tumor-associated antigen is composed of a combination of tumor-associated antigens ADHFE1, SAPCD2, ADAM17, REG4 and FLOT1 as described in claim 1.

3. The kit for preliminary screening of ovarian cancer according to claim 2, characterized in that, The solid support is an enzyme-labeled plate.

4. A kit for preliminary screening of ovarian cancer according to claim 2, characterized in that, The kit also includes enzyme-labeled secondary antibody, washing solution, colorimetric solution, stop solution, positive control serum, and negative control serum.

5. A kit for preliminary screening of ovarian cancer according to claim 4, characterized in that, The enzyme-labeled secondary antibody is horseradish peroxidase-labeled RecA protein.

6. A kit for preliminary screening of ovarian cancer according to claim 4, characterized in that, The washing solution is PBST; the colorimetric solution is TMB; and the stop solution is sulfuric acid.

7. A kit for preliminary screening of ovarian cancer according to claim 4, characterized in that, The positive control serum was FLOT1 positive control serum, and the negative control serum was FLOT1 negative control serum.

8. A kit for preliminary screening of ovarian cancer according to claim 7, characterized in that, The FLOT1 positive control serum is the serum of ovarian cancer patients who tested positive for FLOT1 antibody using indirect ELISA and Western blot methods; the FLOT1 negative control serum is the serum of healthy individuals whose FLOT1 antibody expression level was found to be the average level of antibodies in the serum of healthy individuals using indirect ELISA and Western blot methods.

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