CN116539881A - Serum tumor marker and application thereof in preparation of cancer diagnosis reagent - Google Patents

Abstract

The invention 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 invention discloses a new tumor marker Endophilin A2 antibody for the first time, and experiments prove that the Endophilin A2 antibody is obviously increased in serum of patients with breast cancer, liver cancer and gastric cancer. The discovery of the Endophilin A2 antibody supplements the defects of the existing tumor markers and is more beneficial to early diagnosis of breast cancer. The Endophilin A2 antibody is used as a new tumor marker, has highest diagnostic value for breast cancer, is more beneficial to stage diagnosis and treatment guidance of breast cancer patients, solves the problem that the existing tumor marker of breast cancer is not ideal, and achieves the aim of improving the sensitivity and accuracy of early diagnosis of breast.

Description

Serum tumor marker and application thereof in preparation of cancer diagnosis reagent

Technical Field

The invention 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

Cancer is a serious disease that threatens human life and social development, with an increasing number of Cancer attacks since the last century (brain, f., cancer,2021, 127:3029-3030). Malignant tumors have an increasing incidence and a tendency to be young year by year, and early diagnosis of malignant tumors is relatively difficult, and the malignant tumors have a high mortality rate, which poses a serious threat to human health (Chen w., CA: A Cancer Journal for Clinicians,2015,66: 115-132). Recent data indicate that the number of new cases of breast Cancer has exceeded lung Cancer, becoming the most frequently occurring malignancy worldwide (Sung, h., CA Cancer J Clin,2021, 71:209-249). On the one hand, malignant tumors such as breast cancer are highly developed due to environment, living habit and the like; on the other hand, with the increasing awareness of self-health care and the increasing proportion of people actively checking up, the clinical breast cancer detection rate is higher and higher. Early discovery, early diagnosis and early treatment can obviously reduce the death 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 early stages of breast cancer, and finding a simple, feasible and effective examination means is of great importance for early diagnosis of breast (Jafari, s.h., J Cell Physiol,2018, 233:5200-5213).

The diagnosis of breast cancer needs to be comprehensively judged according to the results of medical history, physical signs, imaging examination, biomarker monitoring, histopathology and the like of patients. Tumor marker detection, imaging examination and histopathology are three important methods for diagnosing malignant tumors. Among them, tumor marker detection has 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. (seal, k.n., clin Breast Cancer,2022,22:e319-e 331). With the deep research of tumor markers, the application of the tumor marker is not limited to the diagnosis and follow-up of malignant tumors, and the tumor marker plays an irreplaceable role in various aspects such as diagnosis, differential diagnosis, curative effect evaluation, prognosis, recurrence monitoring and the like.

In recent years, a large number of novel tumor markers have been discovered, which have also become a hotspot for research. CA15-3, CA125 and carcinoembryonic antigen are the most common serum markers for breast cancer, but they lack sensitivity to early disease and are more suitable for providing prognostic information (Duffy, M.J., clin Chem,2006,52:345-351; barzaman, K., int Immuno pharmacol,2020, 84:106535). Due to the advent and development of next generation sequencing, mutated 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 lower concentration of these genes, amplification is required first, which makes the detection scheme more time consuming and complex (Bettegogda, C., sci Transl Med,2014, 6:224). In clinical application, the tumor can not be diagnosed according to the detection result of a single tumor marker, and simultaneously, the combined detection and dynamic observation of a plurality of tumor markers aiming at different diseases are advocated to improve the accuracy, the sensitivity and the specificity of diagnosis.

The research progress of the existing tumor markers is slow, and the clinical application value is limited. Clinical laboratories are urgently required to develop specific and sensitive tumor markers as targets, strengthen the foundation and transformation research of the tumor markers, and play an important role in particular to tumors with atypical histology, difficult differential diagnosis and difficult tissue source determination.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a novel serum tumor marker and application thereof in preparing breast cancer diagnostic reagents so as to assist in early diagnosis and differential diagnosis of various cancers.

In order to achieve the above purpose, the invention is realized by adopting the following technical scheme:

the invention discloses a serum tumor marker which is an Endophilin A2 antibody, and the amino acid sequence of an identified antigen Endophilin A2 is shown as SEQ ID NO. 1.

Preferably, the tumor comprises breast cancer, liver cancer, gastric cancer, intestinal cancer, lung cancer and leukemia.

Further preferably, the tumor is breast cancer.

The invention also discloses application of the serum tumor marker in preparation of a reagent/kit for diagnosing cancer.

Preferably, the cancer is breast cancer for early diagnosis, staged diagnosis, monitoring of the effect of treatment or prognostic assessment of breast cancer.

The invention also discloses a kit for diagnosing cancer or evaluating cancer risk, which comprises:

ELISA plates coated with Endophilin A2 antigen;

1% BSA blocking solution;

a positive standard and a negative standard for detecting serum to be detected;

HRP conjugated goat anti-human IgG;

ABTS color developing solution;

the amino acid sequence of the Endophilin A2 antigen is shown as SEQ ID NO. 1;

the cancer comprises breast cancer, liver cancer, gastric cancer, intestinal cancer, lung cancer and leukemia.

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

Preferably, the working concentration of the coating antigen is 20 mug/mL, the dilution of the serum to be tested is 1:100, and the dilution of the goat anti-human IgG conjugated with the ELISA antibody HRP is 1:5000.

Preferably, the kit using method comprises the following steps:

1) ELISA plate preparation: coating the enzyme-linked plate with Endophilin A2 antigen solution at 4 ℃ overnight, and washing;

2) Closing: blocking with 1% BSA blocking solution at 37deg.C for 2 hr, washing;

3) Sample collection and processing: drawing peripheral blood to be detected, and separating serum to be detected;

4) Sample adding: adding diluted serum to be detected, setting up negative and positive references at the same time, incubating for 2 hours at 37 ℃, and washing;

5) Adding a detection antibody: adding HRP-labeled goat anti-human IgG secondary antibody, incubating for 1h at 37 ℃, and washing;

6) Color development: adding ABTS substrate color development liquid, and carrying out light-proof reaction at room temperature for 10-20 min;

7) Reading a plate: reading an OD value at a wavelength of 405nm under an enzyme label instrument;

8) According to the OD value judgment result: and drawing a standard curve by using a positive reference, and obtaining the relative concentration of serum of different patients according to the standard curve corresponding to the OD value of serum to be detected of the ELISA plate after using a blank control Kong Diaoling.

Compared with the prior art, the invention has the following beneficial effects:

the invention discloses a new tumor marker Endophilin A2 antibody for the first time, and experiments prove that the Endophilin A2 antibody is obviously increased in serum of patients with breast cancer, liver cancer and gastric cancer. Experimental results show that the marker has the best diagnosis effect on breast cancer, and the area under the working curve (AUC) of a subject can reach 0.8014. The detection result of a large sample breast cancer patient shows that the index has better diagnosis effect on early and remote metastatic breast cancer patients, and AUC is 0.8014 and 0.7885 respectively. At present, most tumor markers are tumor antigens, and detection of autoantibodies in serum can precede tumor antigens, so that early diagnosis is facilitated. At the same time, a small amount of antigen can cause the body to release high-titer antibodies, and has an amplifying effect. Therefore, the discovery of the Endophilin A2 antibody supplements the defects of the existing tumor markers and is more beneficial to early diagnosis of breast cancer.

In addition, the application also establishes an indirect ELISA detection method of the Endophilin A2 antibody in serum. A mixed serum sample of 5 patients with higher OD value in early antibody detection is selected as a positive standard substance to draw a standard curve, so that the antibody detection result can be relatively quantified, and the method standardization is realized. Compared with antigen, the antibody has stronger stability in serum and is more beneficial to clinical detection. Therefore, the Endophilin A2 antibody is used as a new tumor marker, has highest diagnostic value for breast cancer, is more beneficial to stage diagnosis and treatment guidance of breast cancer patients, solves the problem that the existing tumor marker of breast cancer is not ideal, and achieves the aim of improving the sensitivity and accuracy of early diagnosis of breast.

Drawings

FIG. 1 shows the results of specific expression of Endophilin A2 protein;

FIG. 2 shows the expression of Endophilin A2 in different cancer tissues in TIMER database;

FIG. 3 is a standard curve of ELISA detection;

FIG. 4 shows straight line segments in standard curve of ELISA assay.

FIG. 5 is a dilution linearity test chart;

FIG. 6 is a graph showing the analysis of the differences in serum of six cancer patients versus serum of healthy females; wherein A is breast cancer, B is liver cancer, C is gastric cancer, D is intestinal cancer, E is acute myelogenous leukemia, and F is lung cancer;

FIG. 7 is a ROC curve for six different cancers diagnosed with this index; wherein A is breast cancer, B is liver cancer, C is gastric cancer, D is intestinal cancer, E is acute myelogenous leukemia, and F is lung cancer;

FIG. 8 is a graph showing the analysis of the difference in serum between breast cancer patients and healthy females; wherein A is all breast cancer patients, and B is different stage breast cancer patients;

FIG. 9 shows the differential analysis of the index for breast cancer patients at different tumor sizes (T), lymph node metastasis (N), distant metastasis (M); wherein A is breast cancer patients with different tumor sizes (T), B is breast cancer patients with different degrees of lymph node metastasis (N), and C is breast cancer patients with distant metastasis (M);

FIG. 10 is a graph showing the difference analysis of the index for breast cancer patients with different treatment conditions;

FIG. 11 is a graph showing the differential analysis of the index for patients with breast cancer with different Her2 expression;

FIG. 12 is a ROC curve of the index for diagnosing breast cancer and various stages; wherein A is all breast cancer patients, and B is different stage breast cancer patients.

Detailed Description

In order that those skilled in the art will better understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.

It should be noted that the terms "first," "second," and the like in the description and the claims of the present invention and the above figures are used for distinguishing between similar objects and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used may be interchanged where appropriate such that the embodiments of the invention described herein may be implemented in sequences other than those illustrated or otherwise described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

The invention is described in further detail below with reference to the attached drawing figures:

in one aspect, the invention provides a novel tumor marker, antibodies to Endophilin A2 protein in serum of a patient; in another aspect, methods of detecting the markers are also provided.

The detection kit comprises: (1) An ELISA plate coated with Endophilin A2, wherein the ELISA plate is an ELISA plate coated with Endophilin A2 antigen which is specifically expressed or commercially purchased; (2) 1% BSA blocking solution; (3) Positive standard and negative standard for detecting serum to be detected by using an Endophilin A2ELISA plate; (4) HRP conjugated goat anti-human IgG; (5) ABTS color development solution.

The detection method comprises the following steps:

(1) Antigen preparation: preparing antigen liquid by using the specific expression Endophilin A2 antigen, or directly purchasing commercial Endophilin A2 antigen liquid;

(2) ELISA plate preparation: endophilin A2 antigen was diluted to 20. Mu.g/mL with coating solution, and coated overnight at 4℃on each well of the ELISA plate at 100. Mu.L. Drying the protein coating liquid every other day, adding 200 mu L of PBST into each hole, washing for three times, and finally beating on absorbent paper;

(3) Closing: adding 300 mu L of 1% BSA blocking solution into each hole, blocking for 2 hours at 37 ℃, drying the blocking solution in the hole, and washing the hole by the same method;

(4) Sample collection and processing: drawing peripheral blood to be detected, and separating serum to be detected;

(5) Sample adding: the serum to be tested is diluted according to the proportion of 1:100, and is added into ELISA coating plates after dilution, and meanwhile, negative and positive references are established, 100 mu L/hole is acted for 2 hours at 37 ℃. The washing method is the same as above;

(6) Adding a detection antibody: HRP-labeled goat anti-human IgG secondary antibody diluted at 1:5000 fold was added at a volume of 100. Mu.L/well and allowed to act at 37℃for 1h. The washing method is the same as above.

(7) Color development: adding ABTS substrate color development liquid according to the volume of 100 mu L/hole, and reacting for 10-20 min at room temperature in a dark place.

(8) Reading a plate: reading an OD value at a wavelength of 405nm under an enzyme label instrument;

(9) According to the OD value judgment result: uncoated, coated non-blood sample wells were used as blank control wells, wells with different dilutions of cancer patient mixed blood samples were used as positive control wells and standard curves were drawn, wells with healthy people mixed blood samples were used as negative control wells. After using a blank control Kong Diaoling, the relative concentration of serum of different patients is obtained according to the standard curve corresponding to the OD value of serum to be detected of the ELISA plate.

The theoretical basis of the method is as follows: in some types of cancer cells, the Endophilin A2 protein is highly expressed, and in early stages of cancer, symptoms are not yet present, and even when no change can be found in pathological detection, along with the rupture of a large number of cancer cells, antibodies against the Endophilin A2 protein are present in the blood of cancer patients. Since antibodies occur earlier in serum and antibody production has an amplifying effect, a small amount of tumor antigen can induce a large amount of antibody production. By adopting the technical scheme, the effect of early diagnosis can be achieved.

ELISA kit, wherein the working concentration of the coating antigen is 20 mug/mL, the dilution of the serum to be detected is 1:100, and the dilution of the ELISA antibody is 1:5000.

Serum samples of six cancer patients of different stages and healthy persons of the same age group and sex were collected, and the relative concentrations of the Endophilin A2 antibodies in all samples were detected. In the application, the detected tumor is breast cancer, liver cancer, gastric cancer, intestinal cancer, leukemia or lung cancer. The tumor is preferably breast cancer. The application of the index in clinical characteristics such as breast cancer stage, tumor size, lymph node metastasis, distal metastasis and the like is analyzed.

The starting reagent materials in the examples of the present invention are commercially available, and the experimental methods without specifying the specific conditions are conventional methods and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

1. Expression of full Length Endophilin A2 protein

To obtain recombinant plasmid pET28a-FL-Endophilin A2, a sequence encoding the full length of Endophilin A2 (shown as SEQ ID NO: 1) was synthesized and cloned into the BamHI-XhoI cleavage site of pET28a (+). The recombinant plasmid was transformed into competent E.coli BL21 (DE 3) cells. Endophilin A2 protein was expressed in E.coli BL21 (DE 3) cells and positive colonies were selected. Protein expression was induced with 0.5mM IPTG, overnight at 18℃and samples were taken 8, 12, 16 and 20h after IPTG induction and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein purification is carried out by using a 5mL HisTrap HP IMAC chromatographic column, and finally the target protein after the purification is identified by Western blot.

2. Analysis of expression level variation of Endophilin A2mRNA levels in different kinds of cancers using TIMER data

Entering into TIMER database official network (https:// cistome. Shinyapps. Io/TIMER /), inputting gene name Sh3gl1 and tumor item name, and searching the expression data of the gene in the tumor tissue relative to the normal tissue. Clicking on the left navigation bar selects tumor species, differential expression gene cutoff, minimum unit, up-or down-regulation of expression. Non-parametric tests were used for each set of data to compare mRNA levels expression in tumor patients and normal tissues with significant differences of P < 0.05.

3. Subject clinical data collection

Through the agreement of the inventor and family members, the study includes 854 qualified participants, and blood samples of cancer patients meeting the standard, including breast cancer, liver cancer, gastric cancer, intestinal cancer, leukemia and lung cancer, are taken from 2021, 9 months and 2023, 2 months of clinical laboratory of a first affiliated hospital of the western traffic university; healthy control subjects matched to the age and sex of the cancer participants were also selected. Peripheral blood was collected with EDTA anticoagulant tubes and centrifuged at 3000g/min for 15 min at room temperature over 30 min. After centrifugation, the serum was transferred to a 1.5mL EP tube and stored immediately at-80 ℃. The specific clinical data are shown in Table 1.

4. ELISA detection of Endophilin A2 antibodies in serum

(1) ELISA plate preparation: endophilin A2 antigen was diluted to 20. Mu.g/mL with coating solution, and coated overnight at 4℃on each well of the ELISA plate at 100. Mu.L. Drying the protein coating liquid every other day, adding 200 mu L of PBST into each hole, washing for three times, and finally beating on absorbent paper;

(2) Closing: adding 300 mu L of 1% BSA blocking solution into each hole, blocking for 2 hours at 37 ℃, drying the blocking solution in the hole, and washing the hole by the same method;

(3) Sample collection and processing: drawing peripheral blood to be detected, and separating serum to be detected;

(4) Sample adding: the serum to be tested is diluted according to the proportion of 1:100, and is added into ELISA coating plates after dilution, and meanwhile, negative and positive references are established, 100 mu L/hole is acted for 2 hours at 37 ℃. The washing method is the same as above;

(5) Adding a detection antibody: HRP-labeled goat anti-human IgG secondary antibody diluted at 1:5000 fold was added at a volume of 100. Mu.L/well and allowed to act at 37℃for 1h. The washing method is the same as above.

(6) Color development: adding ABTS substrate color development liquid according to the volume of 100 mu L/hole, and reacting for 10-20 min at room temperature in a dark place.

(7) Reading a plate: reading an OD value at a wavelength of 405nm under an enzyme label instrument;

(8) According to the OD value judgment result: uncoated, coated non-blood sample wells were used as blank control wells, wells with different dilutions of cancer patient mixed blood samples were used as positive control wells and standard curves were drawn, wells with healthy people mixed blood samples were used as negative control wells. After using a blank control Kong Diaoling, the relative concentration of serum of different patients is obtained according to the standard curve corresponding to the OD value of serum to be detected of the ELISA plate.

5. ELISA Performance measurement

(1) Standard curve drawing

The mixed serum sample of 5 patients with higher OD value in early antibody detection is selected as a positive standard, and the titer of the Endophilin A2 antibody is set as 1 unit. For each ELISA test, PBS was used to serially dilute the positive standards (1:25, 1:50,1:100,1:200,1:400,1:800,1:1600, 1:3200) to measure the OD values, and a calibration curve was drawn to obtain the relative concentrations according to the corresponding standard curves of the OD values measured for each serum sample to be tested.

(2) Serum dilution linearity test

Serum samples were serially diluted with PBS and ELISA was used to determine if the relative concentration of Endophilin A2 antibody was linear with dilution when the titer was in the range of 0.03U/ml to 1U/ml.

(3) Repeatability test

The in-batch and inter-batch reproducibility of the Endophilin A2 antibody ELISA assay with 2 Quality Control (QC) samples corresponded to the high and low regions of the calibration curve, respectively. Intra-batch reproducibility was calculated by 3 repeated measurements of 2 QC samples in 1 ELISA assay. Batch-to-batch reproducibility was calculated by measuring 2 QC samples in 3 different ELISA assays.

6. Statistical method

Using GraphPad Prism statistical software, continuous variables are represented by median (IQR). The comparison between the two groups was performed by t-test, the normal distribution of the multiple groups of continuous variables was performed by One-way analysis of variance (One-way ANOVA) and Bonferroni test, and the quantitative data of the abnormal distribution was performed by Kruskal-Wallis and Dunn's test. The ratio difference of the classification variables is analyzed by using chi-square test or fisher accurate test. Subject work characteristics (ROC) curves were plotted using GraphPad Prism software. There was a statistical difference with P < 0.05.

7. Experimental results

(1) The full-length Endophilin A2 protein was expressed successfully, and the results are shown in FIG. 1.

(2) The TIMER database analyzed elevated expression of Endophilin A2 at mRNA levels in different cancers, as shown in FIG. 2. Among them, BLCA (bladder urothelial cancer), BRCA (breast invasive cancer), CHOL (cholangiocarcinoma), COAD (colon adenocarcinoma), ESCA (esophageal cancer), KICH (renal chromophobe cancer), KIRC (renal clear cell carcinoma), KIRP (renal papillary carcinoma), LIHC (hepatocellular carcinoma), LUAD (lung adenocarcinoma), luc (lung squamous cell carcinoma), READ (rectal adenocarcinoma), THCA (thyroid cancer) and UCEC (endometrial cancer).

(3) Clinical data of study object

The experimental group included six cancer patients meeting the requirements, and the healthy subjects selected in the control group were matched with the age and sex of each cancer patient, respectively (as shown in table 1).

Table 1 clinical data for groups of cancer patients and healthy persons

(4) ELISA Performance measurement

Standard curves can be drawn according to the OD values of positive standards of different dilutions, and the dilution of the serum sample is determined to be 1:100 by selecting a linear relative concentration range (see figures 3 and 4).

The results of the dilution linear assay showed that the relative concentration of endosphilin A2 antibody in the same serum sample was linear with serum dilution when the serum dilution was 1:50 to 1:400 (see fig. 5).

The coefficient of variation in the in-batch reproducibility test for the high QC samples was 6.055% and 3.195% for the low QC samples. The variation coefficient of the high QC samples was 2.852% for the batch-to-batch reproducibility test, and 3.159% for the low QC samples.

(5) The levels of Endophilin A2 antibodies in serum of different cancer patients and the diagnostic efficacy thereof were significantly increased (P < 0.05) in breast cancer, liver cancer and gastric cancer patients as shown in fig. 6. The ROC curve results of each group showed that the level of Endophilin A2 antibody in serum was the highest for diagnosis of breast cancer (auc= 0.8046), followed by liver cancer (auc= 0.6635), stomach cancer (auc= 0.6298), intestinal cancer (auc= 0.5850), and had substantially no diagnostic effect on leukemia (auc= 0.5061), lung cancer (auc= 0.5170), as shown in fig. 7.

The Endophilin A2 antibody titres were significantly increased in the breast cancer group 2 compared to the healthy control group 6, which is consistent with the case of the breast cancer group 1 (fig. 8).

(6) Endophilin A2 antibody levels in serum of patients with different staged breast cancer

Endophilin A2 antibody levels in serum were significantly elevated in stage I, II, IV breast cancer patients, as shown in FIG. 8. According to the tumor size (T), the level of Endophilin A2 antibodies in serum was significantly elevated in stage i, ii breast cancer patients; according to lymph node metastasis (N), endophilin A2 antibody levels in serum were significantly elevated in stage 0, stage i breast cancer patients; according to distant metastasis (M), the levels of Endophilin A2 antibodies in serum were significantly elevated in both stage 0, stage i breast cancer patients; as in fig. 9. The above results indicate that early patients had significantly higher levels of Endophilin A2 antibodies, including stage I and stage II. Accordingly, patients with smaller tumor sizes (T1 and T2) and less regional lymph node metastasis (N0 and N1) will have higher levels of Endophilin A2 antibodies. Furthermore, the level of Endophilin A2 antibodies in serum of stage IV breast cancer patients was significantly elevated, which may be associated with distant metastasis.

Endophilin A2 antibody levels in serum were significantly elevated in both post-chemotherapy and post-operative breast cancer patients, as shown in FIG. 10. According to the results of cancer tissue organization of breast cancer patients, the levels of Endophilin A2 antibodies in serum were significantly increased in patients with different expression levels of Her2, as shown in fig. 11.

ROC curve results show that the level of Endophilin A2 antibodies in serum was higher for diagnosis of breast cancer (auc= 0.7665, 95% ci= 0.7254-0.8076), as shown in panel a of fig. 12; for patients with breast cancer of different stages, the diagnosis effect on patients with breast cancer of stage i was optimal (auc= 0.8014), followed by stage iv (auc= 0.7885), stage ii (auc= 0.7605), stage iii (auc= 0.7066), as shown in panel B of fig. 12, which is consistent with previous antibody level results.

The above is only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited by this, and any modification made on the basis of the technical scheme according to the technical idea of the present invention falls within the protection scope of the claims of the present invention.

Claims (9)

1. The serum tumor marker is characterized in that the serum tumor marker is an Endophilin A2 antibody, and the amino acid sequence of the identified antigen Endophilin A2 is shown as SEQ ID NO. 1.

2. The serum tumor marker according to claim 1, wherein said tumor comprises breast cancer, liver cancer, stomach cancer, intestinal cancer, lung cancer and leukemia.

3. The serum tumor marker according to claim 2, wherein said tumor is breast cancer.

4. Use of the serum tumor marker of claim 1 in the preparation of a reagent/kit for diagnosing cancer.

5. The use according to claim 4, wherein the cancer is breast cancer for early diagnosis, staged diagnosis, monitoring of the effect of treatment or prognostic assessment of breast cancer.

6. A kit for diagnosing cancer or evaluating the risk of cancer, comprising:

ELISA plates coated with Endophilin A2 antigen;

1% BSA blocking solution;

a positive standard and a negative standard for detecting serum to be detected;

HRP conjugated goat anti-human IgG;

ABTS color developing solution;

the amino acid sequence of the Endophilin A2 antigen is shown as SEQ ID NO. 1;

the cancer comprises breast cancer, liver cancer, gastric cancer, intestinal cancer, lung cancer and leukemia.

7. The kit for diagnosing cancer or evaluating the risk of cancer according to claim 6, wherein the Endophilin A2 antigen-coated ELISA plate is an ELISA plate coated with specifically expressed or commercially available Endophilin A2 antigen.

8. The kit for diagnosing cancer or evaluating the risk of cancer according to claim 6, wherein the working concentration of the coating antigen is 20 μg/mL, the dilution of serum to be measured is 1:100, and the dilution of goat anti-human IgG conjugated with enzyme-linked antibody HRP is 1:5000.

9. The kit for diagnosing cancer or evaluating the risk of cancer according to claim 6, wherein the method of using the kit comprises:

1) ELISA plate preparation: coating the enzyme-linked plate with Endophilin A2 antigen solution at 4 ℃ overnight, and washing;

2) Closing: blocking with 1% BSA blocking solution at 37deg.C for 2 hr, washing;

3) Sample collection and processing: drawing peripheral blood to be detected, and separating serum to be detected;

4) Sample adding: adding diluted serum to be detected, setting up negative and positive references at the same time, incubating for 2 hours at 37 ℃, and washing;

5) Adding a detection antibody: adding HRP-labeled goat anti-human IgG secondary antibody, incubating for 1h at 37 ℃, and washing;

6) Color development: adding ABTS substrate color development liquid, and carrying out light-proof reaction at room temperature for 10-20 min;

7) Reading a plate: reading an OD value at a wavelength of 405nm under an enzyme label instrument;

8) According to the OD value judgment result: and drawing a standard curve by using a positive reference, and obtaining the relative concentration of serum of different patients according to the standard curve corresponding to the OD value of serum to be detected of the ELISA plate after using a blank control Kong Diaoling.