Kit for ovarian cancer detection and detection method

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a kit for detecting ovarian cancer and a detection method. The kit comprises an elisa plate and a tumor-associated antigen coated on the elisa plate, the tumor associated antigen is composed of a combination of Sui1, NDE1 and PDLIM1. According to the kit, the three antigens Sui1, NDE1 and PDLIM1 serve as a combination for screening the ovarian cancer for the first time, the content of the autoantibodies Sui1, NDE1 and PDLIM1 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] This invention belongs to the field of biomedical technology, specifically relating to a reagent kit and detection method for ovarian cancer detection. Background Technology

[0002] Currently, common methods for early ovarian cancer screening involve ovarian cancer tumor markers and pelvic examinations. However, the specificity and sensitivity of CA125, a tumor marker, are not ideal because it is not detected in approximately 50% of early-stage ovarian cancer patients, only increasing in 90% of patients with advanced disease. Furthermore, pelvic examinations are not sensitive for detecting ovarian masses. Therefore, identifying suitable biomarkers to improve the sensitivity and specificity of early ovarian cancer screening is crucial.

[0003] With the advancement of tumor immunotherapy, the correlation between tumors and immunity has received increasing attention. Studies have shown that early-stage tumor cells can express certain abnormal proteins that can be recognized by the body's immune system, leading to the production of specific antibodies. These proteins are called tumor-associated antigens (TAAs), and their antibodies are called autoantibodies. Detecting autoantibodies can indirectly reflect the presence of TAs. Many studies have shown that autoantibodies produced from tumor antigens are important indicators for early tumor diagnosis and can be used for monitoring tumor recurrence. Therefore, kits for specifically detecting autoantibodies can serve as a supplementary means of early cancer detection in medicine, enabling simple, rapid, and sensitive detection of cancer. Generally, the diagnostic sensitivity of currently available single autoantibodies is relatively low; combining multiple autoantibodies can effectively improve sensitivity. Therefore, the discovery of new ovarian cancer autoantibodies, and the subsequent development of better detection protocols, is of significant value in improving diagnostic accuracy.

[0004] To achieve the above objectives, the present invention provides a reagent kit and detection method for ovarian cancer detection. Summary of the Invention

[0005] The primary objective of this invention is to provide a reagent kit for ovarian cancer detection.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: An ovarian cancer detection kit, the kit comprising an ELISA plate and a tumor-associated antigen coated on the ELISA plate; the tumor-associated antigen is composed of a combination of Sui1, NDE1 and PDLIM1.

[0007] Furthermore, the coating concentration of tumor-associated antigen Sui1 on the ELISA plate is 1-2 μg / mL, the coating concentration of tumor-associated antigen NDE1 is 1-3 μg / mL, and the coating concentration of tumor-associated antigen PDLIM1 is 3-5 μg / mL.

[0008] Furthermore, the kit also includes enzyme-labeled secondary antibody, positive control serum, negative control serum, washing solution, colorimetric solution, and stop solution.

[0009] Furthermore, the enzyme-labeled secondary antibody is a horseradish peroxidase-labeled goat anti-human IgG antibody.

[0010] Furthermore, the positive control serum is PDLIM1 positive control serum; the negative control serum is PDLIM1 negative control serum; the washing solution is PBST washing solution; the colorimetric solution is TMB colorimetric solution; and the stop solution is sulfuric acid.

[0011] Furthermore, the PDLIM1 positive control serum is the serum of ovarian cancer patients who tested positive for PDLIM1 antibody using indirect ELISA and Western blot methods; the PDLIM1 negative control serum is the serum of healthy individuals whose PDLIM1 antibody expression level was found to be the average antibody level in the serum of healthy individuals using indirect ELISA and Western blot methods.

[0012] A second objective of this invention is to provide a detection method for a kit used in ovarian cancer detection.

[0013] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The detection method of the ovarian cancer detection kit described in any one of the above claims is as follows: (1) Prepare enzyme-labeled plates coated with tumor-associated antigens Sui1, NDE1 and PDLIM1, wash the plates with washing solution, add the samples to be tested, incubate at room temperature for 1-3 hours, and then wash with washing solution. (2) Add horseradish peroxidase-labeled goat anti-human IgG antibody to the system of step (1), incubate at room temperature for 1-3 hours, and then wash with washing solution; (3) Add colorimetric solution to the system after the reaction in step (2), develop color at room temperature in the dark for 5-10 minutes, and then add stop solution to terminate the reaction; (4) Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the OD value at an absorption wavelength of 450 nm.

[0014] Furthermore, the sample to be tested is serum.

[0015] Compared with the prior art, the main advantages of the present invention are as follows: This invention is the first to experimentally discover differences in the expression of autoantibodies against tumor-associated antigens Sui1, NDE1, and PDLIM1 in the serum of healthy individuals and ovarian cancer patients, suggesting their potential as biomarkers for ovarian cancer. The kit provided by this invention is the first to combine Sui1, NDE1, and PDLIM1 antigens for ovarian cancer screening, capable of detecting the levels of Sui1, NDE1, and PDLIM1 autoantibodies in serum samples. Compared to the use of single or two tumor-associated antigens for early ovarian cancer detection, the combined use of Sui1, NDE1, and PDLIM1 demonstrates optimal efficacy in the early diagnosis of ovarian cancer, exhibiting high sensitivity and specificity, significantly improving the efficiency of ovarian cancer detection and diagnosis, and showing broad application prospects. Attached Figure Description

[0016] Figure 1 The relative expression levels of tumor-associated antigen Sui1 autoantibodies in healthy individuals and ovarian cancer; Figure 2 The relative expression levels of tumor-associated antigen NDE1 autoantibodies in healthy individuals and ovarian cancer; Figure 3 The relative expression levels of tumor-associated antigen PDLIM1 autoantibodies in healthy individuals and ovarian cancer; Figure 4 ROC curves for the combined diagnosis of ovarian cancer using different tumor-associated antigens. Detailed Implementation

[0017] 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.

[0018] Example 1 Screening for autoantibodies associated with ovarian cancer: (1) Forty samples were collected, including blood samples from 20 ovarian cancer patients (numbered 1-20) and serum samples from 20 healthy individuals (numbered 21-40). The blood samples from the 20 ovarian cancer patients were mixed in pairs to obtain 10 mixed ovarian cancer serum samples; the blood samples from the 20 healthy controls were mixed in pairs to obtain 10 mixed healthy control blood samples. All blood samples were centrifuged at 4000 rpm for 10 min, and the supernatant was separated into clean EP tubes and stored at -80℃ for later use.

[0019] (2) Take the human proteome chip out of the -80℃ freezer, first place it in the 4℃ freezer for 30 min to warm up, and then continue to warm up at room temperature for 15 min; place the warmed chip face up in the incubation box, add 10 mL of blocking solution (3 mL of 10% BSA, add 7 mL of 1xPBST solution), place it in a shaker, and block at room temperature for 2 h at 80 rpm.

[0020] (3) After blocking, discard the blocking solution. Dilute the mixed serum sample obtained in step (1) with diluent (1 mL 10% BSA added to 9 mL 1xPBST) at a ratio of 1:200 to obtain the diluted serum incubation solution. Add the serum incubation solution to the chip incubation box and incubate overnight at 4°C and 20 rpm. After incubation, place the chip in the washing solution and wash it 3 times.

[0021] (4) After cleaning, place the chip in a dryer for centrifugal drying, perform a standard fluorescence scan on the dried chip according to the scanner's instructions, and record the fluorescence signal.

[0022] (5) According to the SNR calculation formula, the SNR values ​​(signal-to-noise ratio) of serum samples from 10 mixed ovarian cancer patients and 10 mixed healthy control serum samples were calculated to evaluate the quality of the detected autoantibody signals. The SNR values ​​of the 10 mixed ovarian cancer serum samples and 10 mixed healthy control serum samples were linearly normalized to the median. For each autoantibody, the fold difference between the ovarian cancer group and the healthy control group was calculated (fold difference = mean SNR after linear normalization of the median value of the ovarian cancer group / mean SNR after linear normalization of the median value of the healthy control group). The screening conditions were set as fold difference > 1.5 and positive rate difference > 60%, and the anti-tumor related antigen autoantibodies that met the conditions were screened out. The specific results are shown in Table 1.

[0023] Table 1. Screening for autoantibodies associated with ovarian cancer The results are shown in Table 1. By using human proteome chip technology to screen autoantibodies associated with ovarian cancer, and through a series of experimental steps and data analysis, three tumor-associated antigen autoantibodies were successfully screened: tumor-associated antigen Sui1 autoantibody, tumor-associated antigen NDE1 autoantibody, and tumor-associated antigen PDLIM1 autoantibody.

[0024] Example 2 Preparation of ELISA kit: (1) Preparation of three ovarian cancer-related antigen solutions: Antigen standard Sui1, antigen standard NDE1 and antigen standard PDLIM1 were dissolved in coating solution (50mM carbonate buffer, pH=9.6) and mixed thoroughly to prepare Sui1 recombinant protein solution with a concentration of 1μg / mL, NDE1 recombinant protein solution with a concentration of 2μg / mL and PDLIM1 recombinant protein solution with a concentration of 4μg / mL.

[0025] (2) Preparation of antigen-coated ELISA plates: The prepared Sui1 recombinant protein solution, NDE1 recombinant protein solution, and PDLIM1 recombinant protein solution were added to each well of a 48-well ELISA plate. PDLIM1 recombinant protein solution was added to the positive control well and negative control well, and coating buffer (50 mM carbonate buffer, pH=9.6) was added to the blank control well. The sample volume was 50 μL / well. The plates were coated overnight at 4℃. The remaining coating buffer was removed, and the plates were washed three times with PBST and then patted dry. Blocking buffer was added to the reaction wells of the coated 48-well ELISA plate at a sample volume of 100 μL / well. The plates were blocked in a 37℃ water bath for 2 h. The blocking buffer was then removed, and the plates were washed three times with PBST and then patted dry. ELISA plates coated with tumor-associated antigen Sui1, tumor-associated antigen NDE1, and tumor-associated antigen PDLIM1 were obtained, respectively.

[0026] (3) Composition of the ELISA kit: The ELISA kit of the present invention consists of an antigen-coated ELISA plate, an enzyme-labeled secondary antibody (horseradish peroxidase-labeled goat anti-human IgG antibody), a washing buffer PBST (PBST buffer containing 0.2% Tween 20), a chromogenic solution of 2 mg / L TMB, a stop solution of 10% sulfuric acid, a positive control serum, and a negative control serum. The positive control serum is a PDLIM1 positive control serum, that is, the serum of ovarian cancer patients who are positive for PDLIM1 antibody by indirect ELISA and Western blot methods; the negative control serum is a PDLIM1 negative control serum, that is, the serum of healthy individuals whose PDLIM1 antibody expression level is the average level of antibody in the serum of healthy individuals by indirect ELISA and Western blot methods.

[0027] Experimental Example 1 Blood sample testing: Fifty samples were collected, including 25 blood samples from healthy individuals (numbered 1-25) and 25 blood samples from ovarian cancer patients (numbered 26-50). After collection, blood samples were allowed to coagulate at room temperature, then centrifuged at 6000 rpm for 10 min, and the supernatant was collected. Twenty min before the experiment, the purified blood samples were added to the antigen-coated ELISA plate prepared in Example 1 and incubated at room temperature for 1 h. The plate was washed three times with PBST and then dried. 100 μL of horseradish peroxidase-labeled goat anti-human IgG antibody was added to the wells of a 48-well plate and incubated at room temperature for 1 h. The plate was washed three times with PBST and then dried. 100 μL of 2 mg / L TMB chromogenic solution was added to the wells and incubated at room temperature in the dark for 10 min. The reaction was terminated by adding 100 μL of 10% sulfuric acid. After the reaction, the OD value at 450 nm was measured using an ELISA reader. The levels of Sui1, NDE1, and PDLIM1 autoantibodies were collected from serum samples of healthy individuals and ovarian cancer patients. For ease of comparison, the autoantibody content in healthy serum samples was set to 1, and the relative expression levels of autoantibodies in the serum of ovarian cancer patients were 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 were plotted using Origin software, and the results are shown below. Figure 1 , Figure 2 , Figure 3 As shown.

[0028] Figure 1 , Figure 2 , Figure 3 The figures show the relative expression levels of Sui1, NDE1, and PDLIM1 autoantibodies, representing tumor-associated antigens. As the figures indicate, Sui1, NDE1, and PDLIM1 autoantibodies are expressed at low levels in healthy individuals but at high levels in ovarian cancer patients. Therefore, the levels of these three autoantibodies can significantly differentiate between ovarian cancer patients and healthy individuals. Consequently, Sui1, NDE1, and PDLIM1 autoantibodies can serve as biomarkers for ovarian cancer, providing new insights and methods for the prediction and diagnosis of early-stage ovarian cancer.

[0029] Experimental Example 2 ROC curve assessment of the diagnostic value of the kit in ovarian cancer: One hundred samples were collected, including 50 serum samples from healthy individuals as a control group and 50 serum samples from individuals with early-stage ovarian cancer as an observation group. The levels of Sui1, NDE1, and PDLIM1 autoantibodies were detected using the procedures outlined in Experiment 1. Statistical analysis was performed using ROC curves to evaluate the predictive ability of the selected Sui1, NDE1, and PDLIM1 autoantibody serum markers for ovarian cancer. ROC curves for the combined detection of different tumor-associated antigens in early-stage ovarian cancer were plotted, and the results are shown below. Figure 4 As shown.

[0030] Figure 4 The ROC curves for the combined diagnosis of early ovarian cancer using different tumor-associated antigens (TAAs) are shown. A larger ROC curve area indicates greater diagnostic value. As the number of TAA combinations increases, the ROC curve area for ovarian cancer diagnosis significantly increases, indicating that using Sui1, NDE1, and PDLIM1 as a combination has a higher diagnostic value than using one or two TAAs for early ovarian cancer. Therefore, the combined use of all three is more effective in diagnosing ovarian cancer. This result demonstrates that the combined use of Sui1, NDE1, and PDLIM1 for ovarian cancer screening has high sensitivity and specificity, greatly improving the detection success rate of ovarian cancer. It provides strong experimental evidence for the development of in vitro diagnostic kits for early ovarian cancer screening and has broad application prospects.

[0031] In summary, this invention is the first to experimentally discover differences in the expression of tumor-associated antigens Sui1, NDE1, and PDLIM1 autoantibodies in the serum of healthy individuals and ovarian cancer patients, suggesting their potential as biomarkers for ovarian cancer. Furthermore, this invention is the first to use a combination of Sui1, NDE1, and PDLIM1 antigens for ovarian cancer screening, demonstrating high sensitivity and specificity, significantly improving the detection rate of ovarian cancer, and possessing high diagnostic value for early-stage ovarian cancer, with broad application prospects.

[0032] 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. A reagent kit for ovarian cancer detection, characterized in that, The kit includes an ELISA plate and a tumor-associated antigen coated on the ELISA plate; the tumor-associated antigen is composed of a combination of Sui1, NDE1 and PDLIM1.

2. The ovarian cancer detection kit according to claim 1, characterized in that, The coating concentrations of tumor-associated antigen Sui1, tumor-associated antigen NDE1, and tumor-associated antigen PDLIM1 on the ELISA plate are 1-2 μg / mL, 1-3 μg / mL, and 3-5 μg / mL, respectively.

3. The ovarian cancer detection kit according to claim 1, characterized in that, The kit also includes enzyme-labeled secondary antibody, positive control serum, negative control serum, washing solution, colorimetric solution, and stop solution.

4. The ovarian cancer detection kit according to claim 3, characterized in that, The enzyme-labeled secondary antibody is a horseradish peroxidase-labeled goat anti-human IgG antibody.

5. The ovarian cancer detection kit according to claim 3, characterized in that, The positive control serum was PDLIM1 positive control serum; the negative control serum was PDLIM1 negative control serum; the washing solution was PBST washing solution; the chromogenic solution was TMB chromogenic solution; and the stop solution was sulfuric acid.

6. The ovarian cancer detection kit according to claim 5, characterized in that, The PDLIM1 positive control serum is the serum of ovarian cancer patients who tested positive for PDLIM1 antibody using indirect ELISA and Western blot methods; the PDLIM1 negative control serum is the serum of healthy individuals whose PDLIM1 antibody expression level was found to be the average antibody level in the serum of healthy individuals using indirect ELISA and Western blot methods.

7. The detection method of the ovarian cancer detection kit according to any one of claims 1-6, characterized in that, The detection method is as follows: (1) Prepare enzyme-labeled plates coated with tumor-associated antigens Sui1, NDE1 and PDLIM1, wash the plates with washing solution, add the samples to be tested, incubate at room temperature for 1-3 hours, and then wash with washing solution. (2) Add horseradish peroxidase-labeled goat anti-human IgG antibody to the system of step (1), incubate at room temperature for 1-3 hours, and then wash with washing solution; (3) Add colorimetric solution to the system after the reaction in step (2), develop color at room temperature in the dark for 5-10 minutes, and then add stop solution to terminate the reaction; (4) Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the OD value at an absorption wavelength of 450 nm.

8. The detection method of the ovarian cancer detection kit according to claim 7, characterized in that, The sample to be tested is serum.

Images