Kit for preliminary screening of ovarian cancer and application thereof

Abstract

The invention belongs to the technical field of in-vitro diagnosis, and discloses a kit for preliminary screening of ovarian cancer and application of the kit. The kit comprises a solid phase carrier and a tumor associated antigen coated on the solid phase carrier, wherein the tumor associated antigen is composed of ADHFE1, SAPCD2, ADAM17, REG4 and FLOT1. According to the kit, the five antigens ADHFE1, SAPCD2, ADAM17, REG4 and FLOT1 serve as a combination for screening the ovarian cancer for the first time, the content of ADHFE1, SAPCD2, ADAM17, REG4 and FLOT1 autoantibodies in serum can be specifically detected, high sensitivity and specificity are achieved, the detection success rate of ovarian cancer diseases is greatly increased, and wide application prospects are achieved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a kit for preliminary screening of ovarian cancer and application thereof. BACKGROUND

[0002] Ovarian cancer is one of the common malignant tumors of female reproductive organs, which seriously threatens women's lives. Since the ovary is deep in the pelvic cavity and small in size, there are no typical symptoms when it occurs, so it is difficult to diagnose early ovarian cancer. When found clinically, it is often in the middle and late stages. Studies have shown that only 30% of ovarian cancer patients found during surgery have tumors confined to the ovaries, and most have spread to the uterus, greater omentum and organs in the pelvic cavity, with a five-year survival rate hovering between 25% and 30%. If ovarian cancer is found early and treated, the five-year survival rate is about 90%. Therefore, early screening is the most effective means of preventing ovarian cancer.

[0003] At present, the most commonly used method for early screening of ovarian cancer is through ovarian cancer tumor markers and vaginal ultrasound imaging examination. Ovarian cancer biomarkers refer to abnormal substances or genetic variations produced by tumor cells or the body during the occurrence and development of ovarian cancer. These substances or variations can be detected in biological samples such as blood, tissue, and urine, and are used for the diagnosis, prognosis evaluation, and treatment monitoring of ovarian cancer. CA125 and HE4 are currently one of the most common ovarian cancer tumor markers, and are widely used in the diagnosis of ovarian epithelial cancer. However, about 50% of early ovarian cancer patients do not show significant changes in CA125 during early detection, and the specificity and sensitivity are not ideal. Moreover, CA125 can also be elevated in other common gynecological diseases, and the detection of CA125 alone can result in false positive test results. The specificity of HE4 detection is better than that of CA125, but the sensitivity is insufficient when used alone. Vaginal ultrasound imaging examination is highly dependent on the experience of the operator, and the sensitivity of early ovarian cancer is limited, making it difficult to popularize screening. Therefore, CA125, HE4 detection, and vaginal ultrasound examination cannot improve the early screening rate and prognosis of ovarian cancer recurrence, and the development of an ovarian cancer early screening kit still faces challenges in sensitivity, specificity, and cost. Therefore, there is still a lack of accurate and reliable tumor markers for the early diagnosis of ovarian cancer, and as a supplementary means for early detection of ovarian cancer in medicine, to achieve the purpose of improving survival rate and reducing mortality. In recent years, research has found that during the formation of cancer cells, a unique group of autocrine antigens, tumor-associated antigens, are released, which have measurable changes related to the pathophysiological process. In view of the problems existing in the detection kit, tumor-associated antigens have been used in combination to detect the expression of autoantibodies in patients, further improving the sensitivity and specificity of diagnosis, and improving the detection rate of ovarian cancer.

[0004] Based on the above purpose, the application provides a kit for preliminary screening of ovarian cancer. SUMMARY

[0005] The first object of the application is to provide a combination of tumor-related antigens ADHFE1, SAPCD2, ADAM17, REG4 and FLOT1 for preparing a kit for preliminary screening of ovarian cancer.

[0006] To achieve the above object, the technical scheme adopted by the application is: The application of a combination of tumor-related antigens ADHFE1, SAPCD2, ADAM17, REG4 and FLOT1 in preparing a kit for preliminary screening of ovarian cancer.

[0007] The second object of the application is to provide a kit for preliminary screening of ovarian cancer.

[0008] To achieve the above object, the technical scheme adopted by the application is: A kit for preliminary screening of ovarian cancer, comprising a solid phase carrier and tumor-related antigens coated on the solid phase carrier; the tumor-related antigens are composed of a combination of tumor-related antigens ADHFE1, SAPCD2, ADAM17, REG4 and FLOT1.

[0009] Further, the solid phase carrier is an enzyme-labeled plate.

[0010] Further, the kit further comprises enzyme-labeled secondary antibodies, a washing solution, a color developing solution, a termination solution, positive control serum and negative control serum.

[0011] Further, the enzyme-labeled secondary antibodies are horseradish peroxidase-labeled RecA proteins.

[0012] Further, the washing solution is PBST; the color developing solution is TMB; and the termination solution is sulfuric acid.

[0013] Further, the positive control serum is FLOT1 positive control serum, and the negative control serum is FLOT1 negative control serum. Further, the FLOT1 positive control serum is serum of an ovarian cancer patient with positive FLOT1 antibody detected by indirect ELISA and Western blot methods; and the FLOT1 negative control serum is serum of a healthy person with an FLOT1 antibody expression level equal to the average content of serum antibodies of healthy people.

[0014] Compared with the prior art, the application has the following beneficial effects: The prepared ELISA kit first uses the five antigens ADHFE1, SAPCD2, ADAM17, REG4 and FLOT1 as a combination for screening of ovarian cancer, and can detect the levels of ADHFE1, SAPCD2, ADAM17, REG4 and FLOT1 autoantibodies in serum samples. Compared with single, two, three and four tumor-related antigens for early ovarian cancer detection, the five tumor-related antigens ADHFE1, SAPCD2, ADAM17, REG4 and FLOT1 combined for early ovarian cancer detection have the best effect on diagnosing ovarian cancer, with high sensitivity and specificity, the sensitivity is as high as 92.8%, the specificity is as high as 90.1%, the Youden index is 0.829, which greatly improves the detection success rate of ovarian cancer disease, and has a broad application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The specific detection process diagram of the kit for preliminary screening of ovarian cancer is shown in the figure. Figure 2 The relative expression amount of the tumor-related antigen ADHFE1 autoantibody in healthy people and ovarian cancer is shown in the figure. Figure 3 The relative expression amount of the tumor-related antigen SAPCD2 autoantibody in healthy people and ovarian cancer is shown in the figure. Figure 4 The relative expression amount of the tumor-related antigen ADAM17 autoantibody in healthy people and ovarian cancer is shown in the figure. Figure 5 The relative expression amount of the tumor-related antigen REG4 autoantibody in healthy people and ovarian cancer is shown in the figure. Figure 6 The relative expression amount of the tumor-related antigen FLOT1 autoantibody in healthy people and ovarian cancer is shown in the figure. DETAILED DESCRIPTION

[0016] The technical solutions of the present application are further described below in combination with the specific embodiments. However, those skilled in the art should understand that the following examples are only used to illustrate the present application, and should not be regarded as limiting the present application. The specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, such as those not specifically mentioned, are conventional products obtained through market channels.

[0017] Example 1 Preparation of ELISA kit: (1) The composition of the ELISA kit: enzyme-labeled plate, horseradish peroxidase-labeled RecA protein, antigen standard ADHFE1, antigen standard SAPCD2, antigen standard ADAM17, antigen standard REG4, antigen standard FLOT1, washing solution PBST (0.01M PBST containing 0.05% Tween 20, pH 7.4), color developing solution 2mg / L TMB, stop solution 2M sulfuric acid, positive control serum, negative control serum constitute the ELISA kit of the present application. The positive control serum is FLOT1 positive control serum, i.e. the serum of an ovarian cancer patient whose FLOT1 antibody is positive as detected by indirect ELISA and Western blot method; the negative control serum is FLOT1 negative control serum, i.e. the serum of a healthy person whose FLOT1 antibody expression level is the average content of serum antibodies of healthy people as detected by indirect ELISA and Western blot method.

[0018] (2) Preparation of six kinds of ovarian cancer related antigen solutions: dissolve the antigen standard ADHFE1, antigen standard SAPCD2, antigen standard ADAM17, antigen standard REG4, antigen standard FLOT1 in coating solution (50mM carbonate buffer, pH=9.6) respectively, mix thoroughly, and configure ADHFE1 recombinant protein solution with a concentration of 1.0μg / mL, SAPCD2 recombinant protein solution with a concentration of 1.0μg / mL, ADAM17 recombinant protein solution with a concentration of 1.0μg / mL, REG4 recombinant protein solution with a concentration of 1.0μg / mL, and FLOT1 recombinant protein solution with a concentration of 1.0μg / mL respectively.

[0019] (3) Preparation of antigen-coated enzyme-labeled plate: add 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) to the sample wells of a 48-well enzyme-labeled plate, with a sample amount of 100μL / well; incubate in a 37℃ constant temperature incubator for 1 hour, remove the coating solution after overnight incubation at 4℃, and then wash with PBST washing solution for 3 times. Add blocking solution (PBST buffer containing 2% BSA) to the wells of the coated 48-well enzyme-labeled plate, incubate at room temperature for 1 hour, and remove the blocking solution. Place the 48-well enzyme-labeled plate after blocking in a 37℃ drying oven to dry, obtain the antigen-coated 48-well enzyme-labeled plate, and store at 4℃ for standby use.

[0020] (4) Dilution of horseradish peroxidase-labeled RecA protein: dilute the horseradish peroxidase-labeled RecA protein with PBST buffer containing 1% BSA at a ratio of 1:60000, and store at 4℃ for standby use.

[0021] Test Example 1 Blood sample detection: (1) 60 samples were collected, including 30 samples of healthy people (No. 1-30) and 30 samples of ovarian cancer patients (No. 31-60). After blood collection, the blood was placed at room temperature to coagulate, and then centrifuged at 6000 rpm for 10 min to obtain the supernatant. 20 min before the experiment, the serum sample after separation and purification was added to the antigen-coated enzyme-labeled plate prepared in Example 1, incubated at 37°C for 1 h, washed the plate 3 times with PBST and spun dry. 100 μL of diluted horseradish peroxidase-labeled RecA protein prepared in Example 1 was added to the reaction wells of the 48-well plate, incubated at 37°C for 1 h, washed 3 times with PBST and spun dry. 100 μL of 2 mg / L TMB color developing solution was added to the reaction wells, and color development was carried out at room temperature for 10 min in the dark. 100 μL of 2M sulfuric acid was added to stop the reaction, and the OD450 value was detected by an enzyme-labeled instrument. The ADHFE1 autoantibody, SAPCD2 autoantibody, ADAM17 autoantibody, REG4 autoantibody and FLOT1 autoantibody contents in the serum samples of healthy people and ovarian cancer patients were collected. In order to facilitate comparison, the autoantibody content of the serum sample of healthy people was set to 1, and the relative expression amount of autoantibody in the serum of ovarian cancer patients was calculated. Relative expression amount = autoantibody content in serum of ovarian cancer patients / average content of autoantibody in serum of healthy people. The relative expression amount of autoantibody in the serum of healthy people and ovarian cancer patients was realized by drawing a column chart, and the Origin software was used for drawing, and the results are shown in Figure 1 Figure 2 、 Figure 3 、 Figure 4 、 Figure 5 、 Figure 6

[0022] Figure 2 、 Figure 3 、 Figure 4 、 Figure 5 、 Figure 6 ​​The relative expression amounts of ADHFE1 autoantibody, SAPCD2 autoantibody, ADAM17 autoantibody, REG4 autoantibody and FLOT1 autoantibody, which are tumor-associated antigens, respectively. It can be seen from the figure that ADHFE1 autoantibody, SAPCD2 autoantibody, ADAM17 autoantibody, REG4 autoantibody and FLOT1 autoantibody are low expression in healthy people and high expression in ovarian cancer patients, and ovarian cancer and healthy people can be distinguished by the contents of tumor-associated antigen ADHFE1 autoantibody, tumor-associated antigen SAPCD2 autoantibody, tumor-associated antigen ADAM17 autoantibody, tumor-associated antigen REG4 autoantibody and tumor-associated antigen FLOT1 autoantibody. Therefore, ADHFE1, SAPCD2, ADAM17, REG4 and FLOT1 autoantibodies can be used as ovarian cancer biomarkers, and can provide new ideas and means for the prediction and diagnosis of early ovarian cancer.

[0023] (2) In order 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 people and ovarian cancer patients were calculated respectively, taking the results of step (1) as the standard, and 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, Youden index is used to evaluate the authenticity of screening experiments, also known as correct index, usually Youden index is between 0 and 1, the closer to 1, the greater the diagnostic value, the higher the application value.

[0024] Table 1 Results of combined detection of different tumor-associated antigen combinations for ovarian cancer The results are shown in Table 1, which are the results of the combined detection of different tumor associated antigens for ovarian cancer. As can be seen from the results in Table 1, with the increase of the number of tumor associated antigen combinations, the sensitivity of early ovarian cancer diagnosis is further increased; when five tumor associated antigens FLOT1, ADHFE1, SAPCD2, ADAM17 and REG4 are combined for early ovarian cancer detection, the sensitivity is 92.8%; it is shown that when the five tumor associated antigens in Table 1 are used for early ovarian cancer diagnosis, the probability of correctly diagnosing early ovarian cancer is 92.8%; when the tumor associated antigens FLOT1, ADHFE1, SAPCD2, ADAM17 and REG4 are combined for early ovarian cancer detection, the specificity is 90.1%; it is shown that when the five tumor associated antigens in Table 1 are used for early ovarian cancer diagnosis, the ratio of correctly diagnosing non-ovarian cancer patients is 90.1%. Furthermore, the sensitivity and specificity of the five tumor associated antigens ADHFE1, SAPCD2, ADAM17, REG4 and FLOT1 combined for early ovarian cancer detection are higher than those of single, two, three or four tumor associated antigens for early ovarian cancer detection. This result shows that the five tumor associated antigens FLOT1, ADHFE1, SAPCD2, ADAM17 and REG4 combined for ovarian cancer screening are an ideal screening method.

[0025] As can be seen from the Youden index in Table 1, with the increase of the number of tumor associated antigen combinations, the Youden index gradually increases; when five tumor associated antigens FLOT1, ADHFE1, SAPCD2, ADAM17 and REG4 are combined for early ovarian cancer detection, the Youden index is 0.829, which is higher than that of other combinations, indicating that the five tumor associated antigens FLOT1, ADHFE1, SAPCD2, ADAM17 and REG4 as a combination have relatively high application value for early ovarian cancer screening.

[0026] In summary, the ELISA kit prepared by the present application first combines five tumor associated antigens ADHFE1, SAPCD2, ADAM17, REG4 and FLOT1 for early ovarian cancer screening, has high sensitivity and specificity, greatly improves the detection success rate of ovarian cancer disease, has high diagnostic value for early ovarian cancer, and has broad application prospects.

[0027] Finally, it should be pointed out that the above embodiments are only used to illustrate the technical solutions of the present application, but not to limit them. The basic principles and main features of the present application have been described above with specific embodiments, and some modifications or replacements can be made on the basis of the present application, but these modifications or replacements do not make the corresponding technical solutions deviate from the scope of the present application.

Claims

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

2. A kit for the primary screening of ovarian cancer, characterized in that, The kit comprises a solid support and tumor-associated antigens coated on the solid support; the tumor-associated antigens consist of a combination of tumor-associated antigens ADHFE1, SAPCD2, ADAM17, REG4 and FLOT1 as claimed in claim 1.

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

4. The kit for the preliminary screening of ovarian cancer according to claim 2, characterized in that, The kit further comprises enzyme-labeled secondary antibodies, washing solution, color developing solution, termination solution, positive control serum, and negative control serum.

5. The kit for the primary screening of ovarian cancer according to claim 4, characterized in that, The enzyme-labeled secondary antibodies are horseradish peroxidase-labeled RecA protein.

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

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

8. The kit for the primary screening of ovarian cancer according to claim 7, characterized in that, The FLOT1 positive control serum is serum from an ovarian cancer patient whose FLOT1 antibody is positive as detected by indirect ELISA and Western blot methods; and the FLOT1 negative control serum is serum from a healthy person whose FLOT1 antibody expression level is the average content of serum antibodies in a healthy population as detected by indirect ELISA and Western blot methods.

Images