Triple marker for diagnosing recurrent breast cancer and application thereof
By constructing a triple biomarker detection system of Her2, Hsp90α and TK1, the problem of insufficient sensitivity of existing breast cancer biomarkers in early diagnosis and recurrence detection is solved, and efficient monitoring and diagnosis of breast cancer recurrence is achieved.
Patent Information
- Application Number
- CN202511105383.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-07
- Publication Date
- 2025-12-12
AI Technical Summary
Existing breast cancer markers lack sufficient sensitivity in early diagnosis and recurrence detection, resulting in a high rate of missed diagnoses and an inability to effectively monitor abnormal cell proliferation and assess cell proliferation trends.
A triple biomarker for diagnosing recurrent breast cancer, comprising Her2, Hsp90α, and TK1, was constructed and detected using an ELISA kit. The biomarker levels were accurately detected by combining the preparation of coating solution, PBST washing buffer, washing buffer, calibrator diluent, enzyme diluent, and chromogenic solution.
It improves the sensitivity and specificity of breast cancer recurrence detection. The combined detection of triple biomarkers can achieve a sensitivity of 88.24% and a specificity of 90.70%, which significantly improves the diagnostic efficiency of recurrent breast cancer.
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Figure CN121114435A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of breast cancer, and particularly relates to a triple marker for diagnosing recurrent breast cancer and application thereof. BACKGROUND
[0002] Thymidine kinase 1 (TK1) is an enzyme that plays an important role in the cell cycle, mainly expressed in the cytoplasm during the cell division phase. It is a catalytic enzyme that converts deoxythymidine into thymidylic acid, and is a key enzyme in the DNA repair synthesis pathway. Its concentration is positively correlated with DNA synthesis and changes with the cell cycle, and is an internationally recognized specific marker of cell proliferation.
[0003] Healthy cells are mostly in a resting state, and the TK1 enzyme content in the serum is extremely small. Tumor cells lose normal growth regulation and will undergo malignant proliferation, at which time the TK1 enzyme content in the serum will significantly increase, which can be more than 2 times or even more than 100 times the average level of healthy people. Therefore, by detecting the change in the concentration level of TK1 in the serum, cell proliferation abnormalities can be sensitively detected, and the development trend of cell proliferation can be dynamically evaluated, thereby providing important information for early diagnosis, efficacy monitoring, prognosis judgment, etc. of breast cancer.
[0004] Her2 / neu is a protein encoded by an oncogene (erbB-2) with a molecular weight of 185,000 daltons (p185 for short). The cell surface receptors encoded by the oncogene family contain intercellular tyrosine kinase activity and are structurally similar to epidermal growth factor receptors (erbB-1), and are one of the members of the human epidermal growth factor receptor family. Since the Her2 / neu oncogene and the protein it encodes were reported in the mid-1980s, they have played an important role in the development and metastasis of some breast cancers. Detecting Her2 / neu can be used to monitor breast cancer metastasis patients with Her2 levels higher than 15 ng / mL in the blood, and the detection results should be combined with other diagnostic procedures and information of the patient for breast cancer management.
[0005] The early symptoms of breast cancer are occult, and the sensitivity of existing clinical markers (such as CA15-3, CEA) to early patients is less than 50%, which easily leads to missed diagnosis. HSP90α is abnormally elevated due to cell stress at an early stage of tumor occurrence, and can be detected in the serum of patients with stage I / II breast cancer, with a significantly higher sensitivity than traditional markers.
[0006] Breast cancer detection, as the key defense line of disease prevention and control, plays an irreplaceable role in reducing mortality, guiding precise treatment, saving medical resources, and improving the quality of life of patients. Building an early detection system with molecular marker detection as the core and multi-modal technology coordination has become an inevitable trend in the field of breast cancer prevention and treatment, and has far-reaching significance for improving the health level of the whole people. SUMMARY
[0007] The present application aims to provide a triplex marker for diagnosing recurrent breast cancer and its application.
[0008] A triplex marker for diagnosing recurrent breast cancer, the marker being Her2, Hsp90a and TK1.
[0009] Application of the triplex marker in preparing an ELISA detection kit.
[0010] The ELISA detection kit comprises TK1 antibody, Her2 antibody and Hsp90a antibody.
[0011] The ELISA detection kit comprises coating solution, PBST washing solution, washing buffer, calibration diluent, enzyme diluent, blocking solution and color developing solution.
[0012] Coating solution preparation: sodium dihydrogen phosphate (NaH2PO4·2H2O) 14.1 mM; sodium hydrogen phosphate (Na2HPO4·12H2O) 36.0 mM; sodium chloride (NaCl) 0.9%; 800 mL of deionized water is added to deionized water to 1 L, pH 7.2-7.6;
[0013] 20xPBST washing solution preparation method: sodium dihydrogen phosphate 4.0 g; sodium phosphate dibasic 58.0 g; sodium chloride 175.0 g; add 800 mL of tertiary water to the beaker, stir well to dissolve; add 10 mL of Tween-20 (Tween-20); add 1.0 mL of preservative (Proclin-300), stir well to dissolve; adjust the pH to 7.2-7.6, then dilute to 1 L with tertiary water, mix well; transfer to a stock tank and store at 4℃ for standby.
[0014] 1x washing buffer preparation: add 800 mL of tertiary water to the beaker, then add 20 mL of 20x washing buffer, stir well to dissolve, adjust the pH to 7.2-7.6, then dilute to 1 L with tertiary water, mix well. Transfer to a stock tank and store at 4℃ for standby.
[0015] Blocking solution preparation: sodium dihydrogen phosphate (NaH2PO4·2H2O) 0.6 g; sodium phosphate dibasic (Na2HPO4·12H2O) 5.8 g; sodium chloride (NaCl) 9.0 g; bovine serum albumin (BSA) 10 g; sucrose (C 12 H 22 O 11 ) 25 g; add 800 mL of purified water to the beaker, stir well to dissolve; transfer to a stock tank and store at 4℃ for standby.
[0016] Calibrator diluent preparation: weigh 2.2 g of NaH2PO4·2H2O, 12.9 g of Na2HPO4·12H2O, 9.0 g of sodium chloride, 10 g of BSA into a 1 L beaker; add 500 mL of tertiary purified water to the beaker, stir to dissolve, then add 1.0 mL of biological preservative, stir evenly, and then make up to 1 mL; transfer to a stock solution tank for storage at 4°C for standby.
[0017] Enzyme diluent preparation: weigh 0.6 g of NaH2PO4·2H2O, 5.8 g of Na2HPO4·12H2O, 0.76 g of KCl, 10 g of bovine serum albumin, 25 g of sucrose, 5.0 g of hydrolyzed gelatin, and 1.0 g of AES complex enzyme stabilizer into a 1 L beaker; add 500 mL of tertiary purified water to the beaker, stir to dissolve, then add 1.0 mL of biological agent 4, 80 mL of biological agent 5, 1.0 mL of biological preservative, 1.0 mL of food red, stir evenly, and then make up to 1 L; transfer to a stock solution tank for storage at 4°C for standby.
[0018] The chromogenic solution is purchased from Huzhou Yingchuang Biotechnology Co., Ltd. Qualitative TMB substrate TMB-S-004.
[0019] The method for detecting the expression level of the triad marker comprises the following steps:
[0020] (1) Collect human serum specimens, coat TK1 antibody, Her2 antibody and Hsp90α antibody;
[0021] (2) Blocking, washing plate, and adding sample;
[0022] Blocking: Use a multichannel pipettor to install a high-pressure steam sterilized pipette tip, add 300 μL / well of blocking solution to the coated and washed enzyme-labeled plate, cover the enzyme-labeled plate with a sealing film, place it flat, and incubate at 2-8°C for 20-28 h; then pour out the liquid in the enzyme-labeled plate and dry it on a clean absorbent paper.
[0023] Washing plate: discard the blocking solution in the wells, place the enzyme-labeled plate in an automatic plate washer, wash with 300 μL / well of washing buffer for 10 seconds each time, repeat for 3 times, and dry after washing;
[0024] Adding sample: calibrator wells: take 50 μL from the calibrator tube, add the calibrators to the enzyme-labeled plate in order of low to high concentration, and change the pipette tip for each concentration; sample wells: add 50 μL of diluted sample.
[0025] (3) Add enzyme: add 40-60 μL of HRP to each well, and shake to mix;
[0026] (4) Incubation: Cover the ELISA plate with sealing film, shake for 20-30 seconds to mix the liquid, and incubate at 20-26℃ for 1-2 hours;
[0027] (5) Washing: Remove the liquid in the wells, wash the plate with washing solution 2-4 times, 3-5 min each time, 200-400 μL / well, and finally pat dry on absorbent paper;
[0028] (6) Color development: Add 80-120 μL of color development solution to each well, shake to mix, and develop color at 20-26℃ in the dark for 15-25 min;
[0029] (7) Termination: Add stop solution, 80-120 μL / well;
[0030] (8) Measurement and calculation.
[0031] The serum sample collection method is as follows: 5 mL of blood is collected from the vein, allowed to stand, and then centrifuged at 3000 rpm / min for 5 min. After centrifugation, the supernatant is collected into a 1.5 mL Eppendorf tube for retention. The samples are then numbered and included in the specimen bank. After aliquoting, the samples are frozen at -80℃ for later use.
[0032] The coating solution was diluted to a final concentration of 4 μg / mL and coated onto 96-well plates at a rate of 100 μL / well. The plates were then sealed with plastic wrap to prevent evaporation at a rate of 100 μL / well and incubated horizontally at 0-8℃ for 20-28 hours.
[0033] The assay was performed using an enzyme-linked immunosorbent assay (ELISA) instrument at a wavelength of 450 nm to measure the OD value of each well.
[0034] The calculation process is as follows: Using the absorbance (OD) value of the calibrator as the abscissa (X) and the corresponding calibrator concentration as the ordinate (Y), the corresponding standard curve y = ax is obtained. 3 +bx 2 +cx+d; The concentration of the sample can be obtained by converting the OD value X of the sample into the corresponding concentration Y from the standard curve.
[0035] The beneficial effects of this invention are:
[0036] (1) The ELISA kit constructed can be used to detect the levels of TK1, Her2 and Hsp90α antigens, which can be used as an effective indicator to assist in the diagnosis and monitoring of relapse.
[0037] (2) By comparing the Her2 / Hsp90α / TK1 levels in the serum of 34 patients with recurrent metastatic breast cancer and 43 patients without recurrence or metastasis, it was found that the levels of Her2, Hsp90α, and TK1 alone in patients with recurrent metastatic breast cancer were slightly higher than those in patients without recurrence or metastasis. The area under the curve for recurrent metastatic breast cancer and patients without recurrence or metastasis was Her2 AUC 0.764.
[0038] (0.655-0.873); Hsp90α AUC 0.659 (0.531-0.788); TK1: AUC 0.683 (0.556-0.811). The sensitivity, specificity, and concordance rate of individual biomarkers were as follows: Her2: sensitivity: 47.06%, specificity: 93.02%, concordance rate: 72.73%; Hsp90α: sensitivity: 41.18%, specificity: 93.02%, concordance rate: 70.13%; TK1: sensitivity: 35.29%, specificity: 95.35%, concordance rate: 68.83%. This demonstrates that individual detection of TK1, Her2, and Hsp90α levels has a certain ability to differentiate patients with recurrent breast cancer.
[0039] (3) ROC curve analysis of recurrent and metastatic breast cancer samples and samples without recurrence revealed that the combined detection of two biomarkers Her2+Hsp90α: AUC 0.820 (0.717-0.924); Her2+TK1: AUC 0.865 (0.781-0.948); Hsp90α+TK1: AUC 0.846 (0.752-0.940); and the combined detection of three biomarkers Her2+Hsp90α+TK1: AUC 0.925 (0.862-0.989). The combined detection of two biomarkers, Her2+Hsp90α, yielded the following results: sensitivity: 67.65%, specificity: 90.70%, and concordance rate: 80.52%; Her2+TK1, sensitivity: 70.59%, specificity: 90.70%, and concordance rate: 81.82%; Hsp90α+TK1, sensitivity: 73.53%, specificity: 93.02%, and concordance rate: 84.42%. The combined detection of three biomarkers, Her2+Hsp90α+TK1, yielded the following results: sensitivity: 88.24%, specificity: 90.70%, and concordance rate: 89.61%. These results indicate that combining the detection of two of the three biomarkers (TK1, Her2, and Hsp90α) can improve the detection rate of recurrent breast cancer to a certain extent, and combining the detection of all three biomarkers can significantly improve the diagnostic efficiency of recurrent breast cancer. Attached Figure Description
[0040] Figure 1 This is a schematic diagram of Her2 ROC curve analysis.
[0041] Figure 2 This is a schematic diagram of the Hsp90αROC curve analysis.
[0042] Figure 3 This is a schematic diagram of the TK1 ROC curve analysis.
[0043] Figure 4 This is a schematic diagram of the Her2+Hsp90α ROC curve analysis.
[0044] Figure 5 This is a schematic diagram of the Her2+TK1 ROC curve analysis.
[0045] Figure 6 This is a schematic diagram of the ROC curve analysis for Hsp90α+TK1.
[0046] Figure 7 This is a schematic diagram of the ROC curve analysis for Her2+Hsp90α+TK1. Detailed Implementation
[0047] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.
[0048] Example 1: Preparation of ELISA Detection Reagents
[0049] The ELISA test kit includes coating solution, PBST washing solution, washing buffer, calibrator diluent, enzyme diluent, blocking solution, and colorimetric solution.
[0050] Preparation of buffer solution:
[0051] Coating solution preparation: Sodium dihydrogen phosphate (NaH2PO4·2H2O) 14.1mM; Sodium hydrogen phosphate (Na2HPO4·12H2O) 36.0mM; Sodium chloride (NaCl) 0.9%; 800mL deionized water, diluted to 1L.
[0052] Preparation of 20xPBST washing solution: Sodium dihydrogen phosphate 4.0 g; disodium hydrogen phosphate 58.0 g; sodium chloride 175.0 g; add 800 mL of grade III water to a beaker and stir thoroughly to dissolve. Add 10 mL of Tween-20. Add 1.0 mL of preservative (Proclin-300) and stir thoroughly to dissolve. Adjust the pH to 7.4, then bring the volume to 1 L with grade III water and mix well. Transfer to a storage tank and store at 4°C for later use.
[0053] Preparation of 1× Wash Buffer: Add 800 mL of grade III water to a beaker, then add 20 mL of 20× Wash Buffer, stir thoroughly to dissolve, adjust the pH to 7.4, and then bring the volume to 1 L with grade III water. Mix well. Transfer to a storage tank and store at 4°C for later use.
[0054] Blocking solution preparation: Sodium dihydrogen phosphate (NaH2PO4·2H2O) 0.6g; disodium hydrogen phosphate (Na2HPO4·12H2O) 5.8g; sodium chloride (NaCl) 9.0g; bovine serum albumin (BSA) 10g; sucrose (C 12 H 22 O 11 25g. Add 800mL of purified water to a beaker and stir thoroughly to dissolve. Transfer to a storage container and store at 4℃ for later use.
[0055] Preparation of calibrator diluent: Accurately weigh 2.2g NaH2PO4·2H2O, 12.9g Na2HPO4·12H2O, 9.0g sodium chloride, and 10g BSA into a 1L beaker; add 500mL of purified water to the beaker, stir thoroughly to dissolve, then add 1.0mL of biological preservative, stir evenly, and bring the volume to 1mL; transfer to a storage tank and store at 4℃ for later use.
[0056] Enzyme dilution preparation: Accurately weigh 0.6g NaH2PO4·2H2O, 5.8g Na2HPO4·12H2O, 0.76g KCl, 10g bovine serum albumin; 25g sucrose, 5.0g hydrolyzed gelatin; and 1.0g of compound enzyme stabilizer AES into a 1L beaker; add 500mL of tertiary purified water to the beaker, stir thoroughly to dissolve, then add 1.0mL of biological agent 4, 80mL of biological agent 5, 1.0mL of biological preservative, and 1.0mL of food red, stir evenly, and bring the volume to 1L; transfer to a storage tank and store at 4℃ for later use.
[0057] The colorimetric reagent was purchased from Huzhou Yingchuang Qualitative TMB Substrate TMB-S-004.
[0058] Example 2 Detection
[0059] Using the reagents prepared in Example 1, the expression levels of TK1, Her2, and Hsp90α proteins in the serum of 34 patients with recurrent metastatic breast cancer and 43 patients with non-recurrent metastatic breast cancer were detected.
[0060] The TK1 antibody used in the experiment was purchased from Chongqing Tansheng Technology Co., Ltd. (FAB-T030-4G4); the Her2 antibody was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd. (10004-R511); and the Hsp90α antibody was purchased from Beijing Mycroft Biotechnology Co., Ltd. (MPT021).
[0061] The specific operating steps are as follows:
[0062] (1) The test sample is human serum. The serum sample collection method is as follows: 5 mL of blood is collected from the vein, and after standing, it is centrifuged at 3000 rpm / min for 5 min. The supernatant is then collected into a 1.5 mL Eppendorf tube for retention. The tubes are numbered and included in the specimen library. After aliquoting, the tubes are frozen at -80℃ for later use, avoiding repeated freeze-thaw cycles.
[0063] (2) Coating antibody: Dilute the coating solution to a final concentration of 4ug / mL, coat 96-well plates, 100μL / well, seal with plastic wrap to prevent evaporation, 100μL / well, and incubate horizontally at 4℃ for 20-28h.
[0064] (3) Blocking: Using a multichannel pipette equipped with a pipette tip sterilized by autoclaving, add 300 μL of blocking solution per well to the coated and washed ELISA plate. Cover the ELISA plate with a sealing film, lay it flat, and let it stand at 4°C for 24 hours. Then, pour out the liquid from the ELISA plate, pat it dry on clean absorbent paper, and prepare it for drying.
[0065] (4) Washing: Discard the blocking solution in the wells, place the microplate in an automatic plate washer, wash with 300 μL of washing buffer per well, 10 seconds per wash, repeat 3 times, and pat dry after washing.
[0066] (5) Sample addition: Calibration wells: Take 50 μL from each calibration tube and add the calibrator to the microplate in order of increasing concentration. Replace the pipette tip for each concentration. Sample wells: Add 50 μL of diluted sample.
[0067] (6) Add enzyme: Add 50 μL of HRP enzyme-labeled antibody to each well above and gently shake to mix.
[0068] (7) Incubation: Cover the ELISA plate with the sealing film, shake for 25 seconds to mix the liquid, and incubate at 22°C for 1.5 hours.
[0069] (8) Washing: Remove the liquid in the wells, wash the plate 3 times with 1× washing solution for 4 min each time, 300 μL / well, and finally pat dry on clean absorbent paper.
[0070] (9) Color development: Add 100 μL of color development solution to each well, gently shake to mix, and develop color at 22℃ in the dark for 2 min.
[0071] (10) Termination: Add stop solution, 100 μL / well (the timing of adding stop solution can be referenced to the highest concentration calibrator; termination is indicated when it turns dark blue).
[0072] (11) Reading: Immediately measure the OD value of each well using an enzyme-linked immunosorbent assay (ELISA) at a wavelength of 450 nm.
[0073] (12) Calculation: Using the absorbance OD value of the calibrator as the abscissa (X) and the corresponding calibrator concentration as the ordinate (Y), the corresponding standard curve y = ax² is obtained. 3 +bx 2 +cx+d (does not pass through zero). The concentration (Y) of the measured sample can be obtained by converting the sample's OD value (X) to the corresponding concentration (Y) using the standard curve.
[0074] This invention uses SPSS 17.0 software for ROC analysis, and the area under the curve (AUC) is used to determine the antibody's ability to differentiate between different types of recurrent and metastatic breast cancer. The reference range for TK1 is 0-2.0 pmol / L, the reference range for Her2 is 0-15.0 ng / mL, and the reference range for Hsp90α is 0-82.0 ng / mL. Values exceeding the upper limit of normal are considered to indicate a risk of breast cancer.
[0075] 3. Application of combined detection of TK1, Her-2 and Hsp90α protein levels in the diagnosis of recurrent breast cancer
[0076] (1) By comparing the Her2 / Hsp90α / TK1 levels in the serum of 34 patients with recurrent metastatic breast cancer and 43 patients without recurrence or metastasis, it was found that the levels of Her2, Hsp90α, and TK1 alone in patients with recurrent metastatic breast cancer were slightly higher than those in patients without recurrence or metastasis. The area under the curve for recurrent metastatic breast cancer and patients without recurrence or metastasis was Her2 AUC 0.764.
[0077] (0.655-0.873); Hsp90α AUC 0.659 (0.531-0.788); TK1: AUC 0.683 (0.556-0.811). The sensitivity, specificity, and concordance rate of individual biomarkers were as follows: Her2: sensitivity: 47.06%, specificity: 93.02%, concordance rate: 72.73%; Hsp90α: sensitivity: 41.18%, specificity: 93.02%, concordance rate: 70.13%; TK1: sensitivity: 35.29%, specificity: 95.35%, concordance rate: 68.83%. This demonstrates that individual detection of TK1, Her2, and Hsp90α levels has a certain ability to differentiate patients with recurrent breast cancer.
[0078] (2) ROC curve analysis of recurrent and metastatic breast cancer samples and samples without recurrence revealed that the combined detection of two biomarkers Her2+Hsp90α: AUC 0.820 (0.717-0.924); Her2+TK1: AUC 0.865 (0.781-0.948); Hsp90α+TK1: AUC 0.846 (0.752-0.940); and the combined detection of three biomarkers Her2+Hsp90α+TK1: AUC 0.925 (0.862-0.989). The combined detection of two biomarkers, Her2+Hsp90α, yielded the following results: sensitivity: 67.65%, specificity: 90.70%, and concordance rate: 80.52%; Her2+TK1, sensitivity: 70.59%, specificity: 90.70%, and concordance rate: 81.82%; Hsp90α+TK1, sensitivity: 73.53%, specificity: 93.02%, and concordance rate: 84.42%. The combined detection of three biomarkers, Her2+Hsp90α+TK1, yielded the following results: sensitivity: 88.24%, specificity: 90.70%, and concordance rate: 89.61%. These results indicate that combining the detection of two of the three biomarkers, TK1, Her2, and Hsp90α, can improve the detection rate of recurrent breast cancer to a certain extent, and combining the detection of all three biomarkers can significantly improve the diagnostic efficiency of recurrent breast cancer (Table 1).
[0079] Table 1. Diagnostic value of Her2, HSP90α, and TK1 for breast cancer recurrence and metastasis (%)
[0080]
[0081] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.
Claims
1. A triple biomarker for diagnosing recurrent breast cancer, characterized in that, The markers are TK1, Her2, and Hsp90α.
2. The application of the triple biomarker as described in claim 1 in the preparation of an ELISA detection kit.
3. The application of the triple biomarker according to claim 2 in the preparation of an ELISA detection kit, characterized in that, The ELISA test kit includes TK1 antibody, Her2 antibody and Hsp90α antibody.
4. The application of the triple biomarker according to claim 2 in the preparation of an ELISA detection kit, characterized in that, The ELISA detection kit includes coating solution, PBST washing solution, washing buffer, calibrator diluent, enzyme diluent, blocking solution, and colorimetric solution.
5. A method for detecting the expression level of the triple marker as described in claim 1, characterized in that, Includes the following steps: (1) Collect human serum samples and coat them with TK1 antibody, Her2 antibody and Hsp90α antibody; (2) Seal, wash plate, and add sample; (3) Add enzyme: Add 40-60 μL of HRP to each well and shake to mix. (4) Incubation: Cover the ELISA plate with sealing film, shake for 20-30 seconds to mix the liquid, and incubate at 20-26℃ for 1-2 hours; (5) Washing: Remove the liquid in the wells, wash the plate with washing solution 2-4 times, 3-5 min each time, 200-400 μL / well, and finally pat dry on absorbent paper; (6) Color development: Add 80-120 μL of color development solution to each well, shake to mix, and develop color at 20-26℃ in the dark for 15-25 min; (7) Termination: Add stop solution, 80-120 μL / well; (8) Measurement and calculation.
6. The method for expressing levels using a triple marker according to claim 5, characterized in that, The serum sample collection method is as follows: 5 mL of blood is collected from the vein, allowed to stand, and then centrifuged at 3000 rpm / min for 5 min. After centrifugation, the supernatant is collected into a 1.5 mL Eppendorf tube for retention. The samples are then numbered and included in the specimen bank. After aliquoting, the samples are frozen at -80℃ for later use.
7. The method for expressing levels using three markers according to claim 5, characterized in that, The coating solution was diluted to a final concentration of 4 μg / mL and coated onto 96-well plates at a rate of 100 μL / well. The plates were then sealed with plastic wrap to prevent evaporation at a rate of 100 μL / well and incubated horizontally at 0-8℃ for 20-28 hours.
8. The method for expressing levels using the triple markers according to claim 5, characterized in that, The assay was performed using an enzyme-linked immunosorbent assay (ELISA) instrument at a wavelength of 450 nm to measure the OD value of each well.
9. The method for expressing levels using a triple marker according to claim 5, characterized in that, The calculation process is as follows: Using the absorbance (OD) value of the calibrator as the abscissa (X) and the corresponding calibrator concentration as the ordinate (Y), the corresponding standard curve y = ax is obtained. 3 +bx 2 +cx+d; The concentration of the sample can be obtained by converting the OD value X of the sample into the corresponding concentration Y from the standard curve.