Serum tumor marker and its use in preparing cancer diagnostic reagent

Abstract

The application discloses a serum tumor marker and application thereof in preparation of a cancer diagnosis reagent, and belongs to the technical field of tumor diagnosis reagents. The application discloses a new tumor marker Endophilin A2 antibody for the first time, and experiments prove that the Endophilin A2 antibody is significantly increased in serum of patients with breast cancer, liver cancer and gastric cancer. The discovery of the Endophilin A2 antibody supplements the deficiency of existing tumor markers, and is more beneficial to early diagnosis of breast cancer. As a new tumor marker, the Endophilin A2 antibody has the highest diagnosis value for breast cancer, is more beneficial to stage diagnosis and treatment guidance of breast cancer patients, solves the problem that existing tumor markers for breast cancer are not ideal, and achieves the goal of improving the sensitivity and accuracy of early diagnosis of breast cancer.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of tumor diagnosis reagents, and particularly relates to a serum tumor marker and application thereof in preparation of a cancer diagnosis reagent. BACKGROUND

[0002] Cancer is a major disease that seriously threatens human life and social development. Since the last century, the number of cancer patients has been increasing (Bray, F., Cancer, 2021, 127: 3029-3030). The incidence of malignant tumors is increasing year by year and showing a trend of youth, and early diagnosis of malignant tumors is relatively difficult, with a high mortality rate, which seriously threatens human health (Chen W., CA: A Cancer Journal for Clinicians, 2015, 66: 115-132). The latest data show that the number of new cases of breast cancer has exceeded that of lung cancer, becoming the highest incidence of malignant tumors in the world (Sung, H., CA Cancer J Clin, 2021, 71: 209-249). On the one hand, due to the environment and living habits, etc., the incidence of breast cancer and other malignant tumors is high; on the other hand, as people's self-health awareness is increasing, the proportion of people who actively undergo physical examination is increasing, and the detection rate of clinical breast cancer is increasing. Early detection, early diagnosis and early treatment can significantly reduce the mortality rate of breast cancer patients and improve the cure rate (Harbeck, N., Lancet, 2017, 389: 1134-1150). However, there are no obvious clinical symptoms or signs in the early stage of breast cancer, and it is of great significance to find a simple, feasible and effective examination method for early diagnosis of breast (Jafari, S. H., J Cell Physiol, 2018, 233: 5200-5213).

[0003] The diagnosis of breast cancer needs to be comprehensively judged according to the patient's medical history, signs, imaging examination, biomarker monitoring, histopathology, etc. Tumor marker detection, imaging examination and histopathology are the three important methods for diagnosing malignant tumors. Among them, tumor marker detection has the advantages of high efficiency, convenience and small trauma, and plays an important role in assisting tumor screening, early diagnosis, histological typing, clinical staging, monitoring tumor recurrence and metastasis, etc. (Seale, K. N., Clin Breast Cancer, 2022, 22: e319-e331). With the in-depth study of tumor markers, their application is not limited to the diagnosis and follow-up of malignant tumors. They play an irreplaceable role in diagnosis, differential diagnosis, efficacy evaluation, prognosis and recurrence monitoring.

[0004] In recent years, a large number of new tumor markers have been found, and these markers have also become a research hotspot. CA15-3, CA125 and carcinoembryonic antigen are the most commonly used serum markers for breast cancer, but they lack sensitivity for early disease and are more suitable for providing prognostic information (Duffy, M.J., Clin Chem, 2006, 52: 345-351; Barzaman, K., Int Immunopharmacol, 2020, 84: 106535). Due to the emergence and development of next-generation sequencing, mutant cancer gene sequences can now be detected in blood samples using techniques such as liquid biopsy (Diaz, L.A., J Clin Oncol, 2014, 32: 579-586). However, due to the low concentration of these genes, amplification is required, which makes the detection scheme more time-consuming and complex (Bettegowda, C., Sci Transl Med, 2014, 6: 224ra224). In clinical applications, tumors cannot be diagnosed solely based on the detection results of a single tumor marker, and simultaneous detection of multiple tumor markers for different diseases and dynamic observation are also recommended to improve the accuracy, sensitivity and specificity of diagnosis.

[0005] The existing tumor marker research is slow, and the clinical application value is limited. The clinical laboratory urgently needs to develop specific and sensitive tumor markers as the goal, and to strengthen the basic and translational research of tumor markers, especially for tumors with atypical histological morphology, difficult differential diagnosis and uncertain tissue origin. SUMMARY

[0006] In order to overcome the shortcomings of the prior art, the purpose of the present application is to provide a new serum tumor marker and its application in the preparation of a breast cancer diagnostic reagent, to assist in the early diagnosis and differential diagnosis of various cancers.

[0007] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0008] The present application discloses a serum tumor marker, which is an Endophilin A2 antibody, and the amino acid sequence of the antigen Endophilin A2 recognized by the antibody is shown as SEQ ID NO: 1.

[0009] Preferably, the tumor includes breast cancer, liver cancer, gastric cancer, intestinal cancer, lung cancer and leukemia.

[0010] Further preferably, the tumor is breast cancer.

[0011] The present application also discloses the application of the above-mentioned serum tumor marker in the preparation of a reagent / kit for diagnosing cancer.

[0012] Preferably, the cancer is breast cancer, for early diagnosis, staging diagnosis, monitoring treatment effect or prognosis evaluation of breast cancer.

[0013] The present application also discloses a kit for diagnosing cancer or evaluating cancer risk, comprising:

[0014] Endophilin A2 antigen coated ELISA plate;

[0015] 1% BSA blocking solution;

[0016] positive and negative standard samples for detecting the serum to be tested;

[0017] HRP coupled goat anti-human IgG;

[0018] ABTS color developing solution;

[0019] The amino acid sequence of the Endophilin A2 antigen is shown in SEQ ID NO: 1;

[0020] The cancer includes breast cancer, liver cancer, gastric cancer, intestinal cancer, lung cancer and leukemia.

[0021] Preferably, the Endophilin A2 antigen coated ELISA plate is an enzyme labeled plate coated with Endophilin A2 antigen specifically expressed or commercially available.

[0022] Preferably, the working concentration of the coated antigen is 20 μg / mL, the dilution of the serum to be tested is 1:100, and the dilution of the enzyme-linked antibody HRP coupled goat anti-human IgG is 1:5000.

[0023] Preferably, the kit uses method comprises:

[0024] 1) ELISA plate preparation: coating the enzyme-linked plate with Endophilin A2 antigen solution overnight at 4°C, and washing;

[0025] 2) Blocking: blocking with 1% BSA blocking solution at 37°C for 2 hours, and washing;

[0026] 3) Sample collection and processing: drawing the peripheral blood to be tested, and separating the serum to be tested;

[0027] 4) Sample addition: adding the serum to be tested after dilution, and setting up negative and positive references, incubating at 37°C for 2 hours, and washing;

[0028] 5) Addition of detection antibody: adding HRP labeled goat anti-human IgG secondary antibody, incubating at 37°C for 1 hour, and washing;

[0029] 6) Color development: add ABTS substrate color developing solution, react at room temperature for 10-20 min in dark;

[0030] 7) Read plate: read OD value at 405 nm wavelength under the enzyme label instrument;

[0031] 8) Determine the result according to OD value: draw a standard curve using positive reference, after zero adjustment with blank control hole, obtain relative concentration of serum of different patients according to OD value of serum to be tested of the ELISA plate corresponding to the standard curve.

[0032] Compared with the prior art, the present application has the following beneficial effects:

[0033] The present application discloses a new tumor marker Endophilin A2 antibody for the first time, and experiments prove that the tumor marker is significantly increased in serum of breast cancer, liver cancer and gastric cancer patients. The experimental results show that the tumor marker has the best diagnosis effect on breast cancer, and the area under the receiver operating characteristic curve (AUC) can reach 0.8014. The detection results of a large sample of breast cancer patients show that the index has good diagnosis effect on early breast cancer patients and breast cancer patients with distant metastasis, and the AUCs are 0.8014 and 0.7885 respectively. At present, most tumor markers are tumor antigens, and the detection of autoantibodies in serum can precede tumor antigens, which is beneficial to early diagnosis. At the same time, a small amount of antigen can cause the body to release high-titer antibodies, which has an amplification effect. Therefore, the discovery of Endophilin A2 antibody supplements the deficiency of existing tumor markers, and is more conducive to early diagnosis of breast cancer.

[0034] In addition, the present application also establishes an indirect ELISA detection method for Endophilin A2 antibody in serum. The mixed serum samples of 5 patients with high OD value in early antibody detection are selected as positive standard samples to draw a standard curve, so that the antibody detection result can be relatively quantified, and the method is standardized. Compared with antigens, antibodies have stronger stability in serum, and are more conducive to clinical detection. Therefore, Endophilin A2 antibody as a new tumor marker has the highest diagnosis value for breast cancer, and is more conducive to the staging diagnosis and treatment guidance of breast cancer patients, so as to solve the problem that the existing tumor markers for breast cancer are not ideal, and achieve the goal of improving the sensitivity and accuracy of early diagnosis of breast cancer. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 is the result of specific expression of Endophilin A2 protein;

[0036] Figure 2 is the expression of Endophilin A2 in different cancer tissues in the TIMER database;

[0037] Figure 3 Standard curve for ELISA detection;

[0038] Figure 4 Linear segment in standard curve for ELISA detection.

[0039] Figure 5 Dilution linear test chart;

[0040] Figure 6 Difference analysis of the index in serum of six cancer patients and serum of healthy women; wherein, A is breast cancer, B is liver cancer, C is gastric cancer, D is intestinal cancer, E is acute myeloid leukemia, and F is lung cancer;

[0041] Figure 7 ROC curve of the index in diagnosis of six different cancers; wherein, A is breast cancer, B is liver cancer, C is gastric cancer, D is intestinal cancer, E is acute myeloid leukemia, and F is lung cancer;

[0042] Figure 8 Difference analysis of the index in serum of breast cancer patients and serum of healthy women; wherein, A is all breast cancer patients, and B is breast cancer patients in different stages;

[0043] Figure 9 Difference analysis of the index in breast cancer patients in different tumor size (T), lymph node metastasis (N), and distant metastasis (M) stages; wherein, A is breast cancer patients in different tumor size (T), B is breast cancer patients in different lymph node metastasis (N) degree, and C is breast cancer patients with or without distant metastasis (M);

[0044] Figure 10 Difference analysis of the index in breast cancer patients in different treatment conditions;

[0045] Figure 11 Difference analysis of the index in breast cancer patients with different Her2 expression;

[0046] Figure 12 ROC curve of the index in diagnosis of breast cancer and different stages; wherein, A is all breast cancer patients, and B is breast cancer patients in different stages. DETAILED DESCRIPTION

[0047] In order for those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be described clearly and completely in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor should be within the scope of protection of the present application.

[0048] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, unless the contrary is indicated herein or otherwise apparent from the foregoing description. For a better overview of the present application, reference is made to the accompanying drawings, which form a part of this disclosure, and, in which are shown by way of illustration, and not of limitation, specific embodiments contemplated for carrying out the present application. It is to be understood that other specific arrangements can be utilized and that the generic description set forth in this disclosure is exemplary and not restrictive of the application. Moreover, unlike names of components, e.g., "first," "second," etc., designations such as "top," "bottom," and "side" are merely used for convenience and are not intended to implicate absolute orientation or direction.

[0049] The application will be further described in conjunction with the drawings, in which:

[0050] In one aspect, the present application provides a new tumor marker, i.e., an antibody against Endophilin A2 protein in serum of a patient. In another aspect, the present application also provides a method for detecting the marker.

[0051] The detection kit comprises: (1) an Endophilin A2-coated ELISA plate, which is an enzyme-labeled plate coated with a specific expression or commercially available Endophilin A2 antigen; (2) 1% BSA blocking solution; (3) positive and negative standard samples for detecting serum to be tested by the Endophilin A2 ELISA plate; (4) HRP-conjugated goat anti-human IgG; and (5) ABTS developing solution.

[0052] The detection method is as follows:

[0053] (1) Antigen preparation: specific expression Endophilin A2 antigen is used to prepare an antigen solution, or a commercially available Endophilin A2 antigen solution is directly purchased;

[0054] (2) ELISA plate preparation: dilute the Endophilin A2 antigen with a coating solution to 20 μg / mL, 100 μL per well of the enzyme-linked plate, and coat at 4°C overnight. The next day, dry the protein coating solution, add 200 μL of PBST per well, wash three times, and finally dry on a blotting paper;

[0055] (3) Blocking: add 300 μL of 1% BSA blocking solution per well, and block at 37°C for 2 hours. Then, dry the blocking solution in the well, and wash as above;

[0056] (4) Sample collection and processing: draw peripheral blood to be tested, and separate the serum to be tested;

[0057] (5) Sample adding: dilute the serum to be detected at a ratio of 1:100, and then add into the ELISA coating plate, and set up negative and positive references, 100 μL / well, 37℃ for 2h. The washing method is the same as above.

[0058] (6) Adding detection antibody: add HRP labeled goat anti-human IgG secondary antibody diluted by 1:5000 into the plate at a volume of 100 μL / well, and 37℃ for 1h. The washing method is the same as above.

[0059] (7) Color developing: add ABTS substrate color developing liquid into the plate at a volume of 100 μL / well, and react at room temperature for 10-20min.

[0060] (8) Reading plate: read OD value at 405nm wavelength under an enzyme labeler.

[0061] (9) Judging result according to OD value: the sample adding hole without coating and without adding blood sample is taken as a blank control hole, the sample adding hole of mixed blood sample of cancer patients with different dilution degrees is taken as a positive control hole and a standard curve is drawn, and the sample adding hole of mixed blood sample of healthy people is taken as a negative control hole. After zero adjustment of the blank control hole, the relative concentration of serum of different patients is obtained according to the OD value of the serum to be detected of the ELISA plate corresponding to the standard curve.

[0062] The theoretical basis of the method is that in some types of cancer cells, Endophilin A2 protein is highly expressed, in the early stage of cancer, even when the pathological examination cannot find changes, with the rupture of a large number of cancer cells, there are antibodies against Endophilin A2 protein in the blood of cancer patients. Since the antibody appears earlier in the serum, and the antibody production has an amplification effect, a small amount of tumor antigen can induce a large amount of antibody production. The technical scheme can achieve the effect of earlier diagnosis.

[0063] The working concentration of the coating antigen of the ELISA kit is 20 μg / mL, the dilution degree of the serum to be detected is 1:100, and the dilution degree of the enzyme-linked antibody is 1:5000.

[0064] Collect serum samples of patients with six types of cancer in different stages and healthy people of the same age and gender, and detect the relative concentration of Endophilin A2 antibody in all samples. In the application, the detected tumors are breast cancer, liver cancer, gastric cancer, intestinal cancer, leukemia and lung cancer. The tumor is preferably breast cancer. The application of the index in the clinical characteristics of breast cancer staging, tumor size, lymph node metastasis and distant metastasis is analyzed.

[0065] All the original reagent materials in the embodiments of the application can be commercially available, and the experimental methods without specific conditions are conventional methods and conventional conditions familiar to the field, or according to the conditions recommended by the instrument manufacturer.

[0066] 1. Expression of full-length Endophilin A2 protein

[0067] To obtain the recombinant plasmid pET28a-FL-Endophilin A2, the sequence encoding the full-length Endophilin A2 (as shown in SEQ ID NO: 1) was synthesized and cloned into the BamHI-Xhol enzyme digestion site of pET28a(+). The recombinant plasmid was transformed into competent Escherichia coli BL21 (DE3) cells. Endophilin A2 protein was expressed in Escherichia coli BL21 (DE3) cells, and positive colonies were selected. Protein expression was induced with 0.5 mM IPTG, induced overnight at 18°C, and samples were collected at 8, 12, 16, and 20 h after IPTG induction for analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein purification was performed using a 5 mL HisTrap HP IMAC column, and the final purified target protein was identified by Western blotting.

[0068] 2. Analysis of changes in the expression level of Endophilin A2 mRNA in different cancers using TIMER data

[0069] Enter the TIMER database website (https: / / cistrome.shinyapps.io / timer / ), input the gene name Sh3gl1 and the tumor project name, and you can retrieve the expression data of the gene in the tumor tissue relative to normal tissue. Click the left navigation bar to select the tumor type, differential expression gene threshold, minimum unit, and whether the expression is up-regulated or down-regulated. Non-parametric test is used for each group of data to compare the expression of mRNA levels in tumor patients and normal tissues, with P<0.05 as the significant difference.

[0070] 3. Collection of clinical data of research subjects

[0071] With the consent of the subject and his family, a total of 854 qualified participants were included in this study, all of which were taken from the blood samples of cancer patients who met the criteria in the Clinical Laboratory of the First Affiliated Hospital of Xi'an Jiaotong University from September 2021 to February 2023, including breast cancer, liver cancer, gastric cancer, intestinal cancer, leukemia, and lung cancer. At the same time, healthy control subjects matched in age and gender with cancer participants were selected. Peripheral blood was collected in EDTA anticoagulant tubes and centrifuged at 3000g / min for 15 minutes at room temperature within 30 minutes. After centrifugation, the serum was transferred to a 1.5 mL EP tube and immediately stored at -80°C. See Table 1 for specific clinical data.

[0072] 4. ELISA detection of Endophilin A2 antibodies in serum

[0073] (1) ELISA plate preparation: dilute Endophilin A2 antigen with coating solution to 20 μg / mL, 100 μL per well of enzyme-linked plate, 4°C coating overnight. The next day, control the protein coating solution dry, add 200 μL PBST per well, wash three times, and finally dry on the blotting paper;

[0074] (2) Blocking: add 300 μL of 1% BSA blocking solution per well, 37°C blocking for 2 hours, then control the blocking solution in the well dry, and wash as above;

[0075] (3) Sample collection and processing: draw the peripheral blood to be tested, and separate the serum to be tested;

[0076] (4) Sample addition: dilute the serum to be tested at a ratio of 1:100, add to the ELISA coating plate after dilution, and set up negative and positive references at the same time, 100 μL per well, 37°C for 2 hours. Wash as above;

[0077] (5) Add detection antibody: add 100 μL per well of 1:5000 dilution of HRP labeled goat anti-human IgG secondary antibody, 37°C for 1 hour. Wash as above.

[0078] (6) Color development: add 100 μL per well of ABTS substrate color development solution, and react at room temperature for 10-20 min.

[0079] (7) Read plate: read OD value at 405 nm wavelength under the enzyme labeler;

[0080] (8) Determine the result according to the OD value: the sample addition well without coating and without blood is used as a blank control well, the sample addition well of mixed blood of cancer patients with different dilutions is used as a positive control well and a standard curve is drawn, and the sample addition well of mixed blood of healthy people is used as a negative control well. After zero adjustment with the blank control well, the relative concentration of different patient serum is obtained according to the OD value of the serum to be tested on the standard curve.

[0081] 5. ELISA performance determination

[0082] (1) Standard curve drawing

[0083] Select 5 cases of mixed serum samples with higher OD values in early antibody detection as positive standard, and set the Endophilin A2 antibody titer as 1 unit. Each time ELISA detection is performed, the positive standard is diluted with PBS (1:25, 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200) to measure the OD value and draw a calibration curve. The relative concentration of each serum sample to be tested is obtained by corresponding to the standard curve according to the measured OD value.

[0084] (2) Serum dilution linearity test

[0085] The serum sample was serially diluted with PBS, and the ELISA could detect whether the relative concentration of Endophilin A2 antibody was linear with the dilution when the titer was in the range of 0.03 U / ml to 1 U / ml.

[0086] (3) Reproducibility test

[0087] The intra-assay and inter-assay reproducibility of Endophilin A2 antibody ELISA detection was performed with 2 quality control (QC) samples, corresponding to the high and low regions of the calibration curve, respectively. The intra-assay reproducibility was calculated by 3 repeated measurements of 2 QC samples in 1 ELISA test. The inter-assay reproducibility was calculated by measuring 2 QC samples in 3 different ELISA tests.

[0088] 6. Statistical method

[0089] The continuous variables were represented by median (interquartile, IQR) using GraphPad Prism statistical software. The t test was used for comparison between 2 groups, the one-way ANOVA and Bonferroni test were used for normal distribution of multiple continuous variables, the Kruskal-Wallis and Dunn's test were used for non-normal distribution of quantitative data. The proportion difference of categorical variables was analyzed by chi-square test or fisher's exact test. The receiver operating characteristic (ROC) curve was drawn by GraphPad Prism software. P<0.05 was considered statistically significant.

[0090] 7. Experimental results

[0091] (1) The specific expression of full-length Endophilin A2 protein was successful, and the results were as follows Figure 1 .

[0092] (2) The TIMER database analysis showed that the expression of Endophilin A2 at the mRNA level was increased in different cancers, and the results were as follows Figure 2 . Among them, including BLCA (bladder urothelial carcinoma), BRCA (breast invasive carcinoma), CHOL (cholangiocarcinoma), COAD (colon adenocarcinoma), ESCA (esophageal carcinoma), KICH (kidney chromophobe carcinoma), KIRC (kidney renal clear cell carcinoma), KIRP (kidney renal papillary carcinoma), LIHC (liver hepatocellular carcinoma), LUAD (lung adenocarcinoma), LUSC (lung squamous cell carcinoma), READ (rectum adenocarcinoma), THCA (thyroid carcinoma) and UCEC (uterine corpus endometrial carcinoma).

[0093] (3) Clinical data of research subjects

[0094] The experimental group included six cancer patients who met the requirements, and the control group selected healthy people matched in age and gender with each cancer patient (Table 1).

[0095] Table 1 Clinical data of cancer patients and healthy people in each group

[0096]

[0097]

[0098] (4) ELISA performance test

[0099] According to the OD values of the positive standard samples at different dilutions, a standard curve can be drawn, and the relative concentration range showing a straight line is selected to determine the dilution of the serum sample as 1:100 (such as Figure 3 , 4).

[0100] The dilution linearity test results show that when the serum dilution is 1:50 to 1:400, the relative concentration of Endophilin A2 antibody in the same serum sample is linearly related to the serum dilution (such as Figure 5 ).

[0101] The batch repeatability test coefficient of variation of the high QC sample was 6.055%, and that of the low QC sample was 3.195%. The batch repeatability test coefficient of variation of the high QC sample was 2.852%, and that of the low QC sample was 3.159%.

[0102] (5) Endophilin A2 antibody levels in serum of patients with different cancers and their diagnostic efficiency The Endophilin A2 antibody levels in serum were significantly increased in breast cancer, liver cancer, and gastric cancer patients (P<0.05), such as Figure 6 . The ROC curve results of each group showed that the diagnostic level of Endophilin A2 antibody in serum for breast cancer was the highest (AUC=0.8046), followed by liver cancer (AUC=0.6635), gastric cancer (AUC=0.6298), intestinal cancer (AUC=0.5850), and leukemia (AUC=0.5061) and lung cancer (AUC=0.5170) had basically no diagnostic effect, such as Figure 7 .

[0103] Compared with the six groups of healthy controls, the Endophilin A2 antibody titer in the breast cancer 2 group was significantly increased, which was consistent with the case of the breast cancer 1 group ( Figure 8 ).

[0104] (6) Endophilin A2 antibody levels in serum of breast cancer patients at different stages

[0105] The serum Endophilin A2 antibody level was significantly increased in breast cancer patients in stages I, II, and IV, as shown in Figure 8 According to the tumor size (T), the serum Endophilin A2 antibody level was significantly increased in breast cancer patients in stages I and II; according to the lymph node metastasis (N), the serum Endophilin A2 antibody level was significantly increased in breast cancer patients in stages 0 and I; according to the distant metastasis (M), the serum Endophilin A2 antibody level was significantly increased in breast cancer patients in stages 0 and I; as shown in Figure 9 The above results show that the Endophilin A2 antibody level is significantly higher in early-stage patients, including stages I and II. Accordingly, patients with smaller tumor size (T1 and T2) and less regional lymph node metastasis (N0 and N1) will have a higher Endophilin A2 antibody level. In addition, the serum Endophilin A2 antibody level is significantly increased in breast cancer patients in stage IV, which may be related to distant metastasis.

[0106] The serum Endophilin A2 antibody level was significantly increased in breast cancer patients after chemotherapy and after surgery, as shown in Figure 10 According to the histological results of the cancer tissues of the breast cancer patients, the serum Endophilin A2 antibody level was significantly increased in patients with different expression levels of Her2, as shown in Figure 11

[0107] The ROC curve results show that the serum Endophilin A2 antibody level has a high diagnostic level for breast cancer (AUC = 0.7665, 95% CI = 0.7254-0.8076), as shown in Figure 12 A of FIG. 6; for breast cancer patients in different stages, the diagnostic effect is best for patients in stage I (AUC = 0.8014), followed by stages IV (AUC = 0.7885), II (AUC = 0.7605), and III (AUC = 0.7066), as shown in Figure 12 B of FIG. 6, which is consistent with the previous antibody level results.

[0108] The above merely illustrates the technical idea of the present application and cannot limit the protection scope of the present application. Any modification made according to the technical idea of the present application on the basis of the technical solution falls within the protection scope of the claims of the present application.​

Claims

1. Use of a serum tumor marker in the manufacture of a reagent / kit for early diagnosis, staging diagnosis, monitoring therapeutic effect or prognosis evaluation of breast cancer, characterized in that, The serum tumor marker is an Endophilin A2 antibody, and the amino acid sequence of the antigen Endophilin A2 recognized by the antibody is shown as SEQ ID NO:

1.

2. Use of a kit for the manufacture of a reagent for the early diagnosis, the staging diagnosis, the monitoring of the therapeutic effectiveness or the prognosis evaluation of breast cancer, characterized in that, The kit comprises: an Endophilin A2 antigen-coated ELISA plate; 1% BSA blocking solution; positive and negative standard samples for detecting the serum to be tested; HRP-conjugated goat anti-human IgG; ABTS developing solution; The amino acid sequence of the Endophilin A2 antigen is shown as SEQ ID NO:

1.

3. Use according to claim 2, characterized in that, The working concentration of the coated antigen is 20 μg / mL, the dilution of the serum to be tested is 1:100, and the dilution of the HRP-conjugated goat anti-human IgG is 1:5000.

4. Use according to claim 2, characterized in that, The method for using the kit comprises: 1) ELISA plate preparation: coat the enzyme-linked plate with the Endophilin A2 antigen solution at 4°C overnight, and wash; 2) Blocking: block with 1% BSA blocking solution at 37°C for 2 h, and wash; 3) Sample collection and processing: draw the peripheral blood to be tested, and separate the serum to be tested; 4) Sample addition: add the diluted serum to be tested, and set up negative and positive references, incubate at 37°C for 2 h, and wash; 5) Addition of detection antibody: add HRP-labeled goat anti-human IgG secondary antibody, incubate at 37°C for 1 h, and wash; 6) Color development: add ABTS substrate developing solution, react at room temperature for 10-20 min in the dark; 7) Plate reading: read the OD value at 405 nm wavelength under the enzyme labeler; 8) Determine the result according to the OD value: use the positive reference to draw a standard curve, adjust the zero with the blank control hole, and obtain the relative concentration of the serum of different patients according to the OD value of the serum to be tested on the ELISA plate corresponding to the standard curve.

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