WO2024208185A1 - Biomarker combination, diagnostic kit comprising same, and use thereof - Google Patents

Abstract

Provided are a biomarker combination, a diagnostic kit comprising same, and use thereof. The biomarker combination comprises CCL18, C1D, FXR1, ZNF573, ESO1, and p53 antigens or autoantibodies binding thereto; and the biomarker further comprises one or more antigens selected from CA125, TM4SF1, IGFBP1, FUBP1, ARP3, KRAS, SPINT1, and YKL-40, or autoantibodies binding thereto. The diagnostic kit comprises the biomarker combination, shows excellent diagnostic performance for ovarian cancer, and can be used for diagnosing or assisting in the diagnosis of early-stage ovarian cancer, or for recurrence monitoring and prognosis monitoring after ovarian cancer treatment.

Description

A biomarker combination, a diagnostic kit containing the same and its application

This application claims the priority of Chinese patent application No. 2023103622710 filed on April 4, 2023. This application cites the entire text of the above Chinese patent application.

Technical Field

The present invention relates to the field of early cancer diagnosis kits, and in particular to a biomarker combination, a diagnostic kit containing the biomarker, and applications thereof.

Background Art

Ovarian cancer is known as the "first gynecological cancer" and is a major malignant tumor that seriously affects women's health. Its mortality rate ranks first among female malignant tumors. Worldwide, the five-year survival rate of ovarian cancer is not optimistic, and ovarian cancer has become an increasingly prominent public health topic. Compared with other female tumors, ovarian cancer is difficult to detect early, has a high recurrence rate, and has the highest overall mortality rate among all types of female tumors, reaching 40% to 50%.

At present, the main clinical examination methods for monitoring ovarian cancer are as follows: 1. Pelvic examination: Bimanual examination is the most important examination in pelvic examination, but its diagnostic specificity is poor, and the diagnostic results of different doctors vary greatly. 2. B-ultrasound examination: B-ultrasound has become a routine examination for pelvic masses. For patients with ovarian cancer, vaginal B-ultrasound examination has the highest sensitivity, but it is difficult to judge the benign or malignant nature of the tumor. 3. CT examination: This examination method can detect the cystic solidity of ovarian masses. Generally speaking, the larger the tumor and the more solid components, the greater the possibility of malignancy. However, if the tumor tissue is too small or the lesion is relatively hidden, its detection sensitivity is less than 30%. 4. Immunological examination: The immunological examination used in clinical diagnosis of ovarian cancer refers to tumor marker detection. Common tumor markers include CA125, HE4, etc. However, the ovarian cancer diagnostic kit for such common markers has low sensitivity and specificity, which is difficult to meet the requirements of early rapid diagnosis and is not the best ideal screening method for early diagnosis of ovarian cancer. Therefore, the above-mentioned examination methods have more or less limitations of insufficient characteristics.

Tumor markers (TM) refer to a class of substances that are synthesized or released by tumor cells themselves, or produced by the body in response to tumor cells, during the occurrence and proliferation of tumors, and that indicate the presence and growth of tumors. These substances do not exist in normal adults or appear at significantly higher levels in cancer patients than in normal people. Currently, tumor marker detection technology is considered to be the only way to detect asymptomatic micro-tumors at an early stage. This detection technology can detect tumors before physical examinations such as X-rays, ultrasound, CT, MRI or PET-CT. It can be used for screening of malignant tumors in high-risk populations, tumor diagnosis and differential diagnosis, evaluation of treatment effectiveness, and prediction or monitoring of tumor recurrence or metastasis. Research reports indicate that the 5-year survival rate of patients with advanced ovarian cancer is less than 40%, but if it is detected in the early stages of the patient (stages I to II), the survival rate will increase to more than 71%. Early diagnosis of ovarian cancer has a great impact on the patient's survival rate, and early diagnosis of ovarian cancer is crucial to the survival of patients. The key to improving early diagnosis capabilities lies in the sensitivity and specificity of tumor markers for ovarian cancer.

The detection of autoantibody spectrum for ovarian cancer has more advantages. Tumor-specific antigens are specific protein products released, shed or exocrine after cell necrosis during the occurrence and progression of tumors. In the early stages of cancer, the body's immune system can recognize tumor-specific antigens expressed by tumor cells and produce autoantibodies against these antigens. In the blood of patients with early ovarian cancer, the level of autoantibodies is much higher than the level of antigens. Autoantibodies have the characteristics of immune surveillance, immune amplification, and circulation and diffusion. In addition, compared with other tumor antigen markers and DNA markers, autoantibodies have the following characteristics: early appearance in serum, long blood detection time window, high blood signal intensity, and high sensitivity of early detection. Therefore, the combined detection of multiple autoantibody markers is a means to obtain highly sensitive and specific detection.

In addition, epithelial cancer accounts for 50-70% of ovarian malignancies in clinic, and is more common in middle-aged and elderly women. The incidence of ovarian germ cell tumors is second only to epithelial tumors, and is more common in adolescents or young women. Epithelial tumors plus germ cell tumors account for more than 80% of ovarian malignancies. Therefore, if there is a better combination of biomarkers for early detection of ovarian cancer autoantibody spectrum, which can be used for early ovarian cancer diagnosis or its prognosis, it will greatly improve the survival rate of ovarian cancer patients and greatly reduce the clinical misdiagnosis rate and missed diagnosis rate.

Summary of the invention

In order to solve the technical problem of low sensitivity and specificity of biomarker combinations or kits used for early ovarian cancer diagnosis or prognosis in the prior art, the present invention provides a biomarker combination, a diagnostic kit containing the same and applications thereof.

To solve the above technical problems, one of the technical solutions of the present invention is: a biomarker combination, which comprises CCL18, C1D, FXR1, ZNF573, ESO1 and p53 antigens or autoantibodies binding thereto; and the biomarker further comprises one or more antigens selected from: CA125, TM4SF1, IGFBP1, FUBP1, ARP3, KRAS, SPINT1 and YKL-40 or autoantibodies binding thereto.

By detecting the biomarker combination, for example, when the biomarker is an antigen (usually a protein), the corresponding antibody molecule is used to detect the protein; and when the biomarker is an antibody to a certain antigen, the antigen can be used to detect the antibody. Thus, based on the test results, it can be comprehensively determined whether the sample, such as a serum sample, is from an early ovarian cancer patient, or the prognosis of the patient.

The biomarker combination as described in one of the technical solutions comprises CCL18, C1D, FXR1, ZNF573, ESO1, p53 and TM4SF1 antigens or autoantibodies binding thereto.

In a preferred embodiment of the present invention, the biomarker combination further comprises IGFBP1 and/or FUBP1 antigens or autoantibodies binding thereto.

In a more preferred embodiment of the present invention, the biomarker combination further comprises ARP3 and/or KRAS antigens or autoantibodies binding thereto.

In a further preferred embodiment of the present invention, the biomarker combination further comprises a member selected from: An antigen of one or more of SPINT1, YKL-40 and CA125 or an autoantibody binding thereto.

The biomarker combination as described in one of the technical solutions, wherein the biomarker combination comprises CCL18, C1D, FXR1, p53, SPINT1, ZNF573, YKL-40 and ESO1 antigens or autoantibodies binding thereto.

In a preferred embodiment of the present invention, the biomarker combination further comprises one or more antigens selected from the group consisting of CA125, KRAS, ARP3, IGFBP1, FUBP1 and TM4SF1, or autoantibodies binding thereto.

The biomarker combination as described in one of the technical solutions, wherein the biomarker combination is a biomarker combination in serum, which is selected from any one of the following groups:

1) ESO1 autoantibodies, p53 autoantibodies, FXR1 autoantibodies, ZNF573 autoantibodies, TM4SF1 autoantibodies, C1D autoantibodies, and CCL18;

2) ESO1 autoantibodies, p53 autoantibodies, FXR1 autoantibodies, ZNF573 autoantibodies, TM4SF1 autoantibodies, C1D autoantibodies, CCL18, and IGFBP1 autoantibodies;

3) ESO1 autoantibodies, p53 autoantibodies, FXR1 autoantibodies, ZNF573 autoantibodies, TM4SF1 autoantibodies, C1D autoantibodies, CCL18, and FUBP1 autoantibodies;

4) ESO1 autoantibody, p53 autoantibody, FXR1 autoantibody, ZNF573 autoantibody, TM4SF1 autoantibody, C1D autoantibody, CCL18, IGFBP1 autoantibody, and FUBP1 autoantibody;

5) ESO1 autoantibody, p53 autoantibody, FXR1 autoantibody, ZNF573 autoantibody, TM4SF1 autoantibody, C1D autoantibody, CCL18, ARP3 autoantibody, IGFBP1 autoantibody and FUBP1 autoantibody;

6) ESO1 autoantibody, p53 autoantibody, FXR1 autoantibody, ZNF573 autoantibody, TM4SF1 autoantibody, C1D autoantibody, CCL18, KRAS autoantibody, IGFBP1 autoantibody and FUBP1 autoantibody;

7) ESO1 autoantibody, p53 autoantibody, FXR1 autoantibody, ZNF573 autoantibody, TM4SF1 autoantibody, C1D autoantibody, CCL18, ARP3 autoantibody, KRAS autoantibody, IGFBP1 autoantibody and FUBP1 autoantibody;

8) ESO1 autoantibodies, p53 autoantibodies, FXR1 autoantibodies, ZNF573 autoantibodies, TM4SF1 autoantibodies, C1D autoantibodies, CCL18 and ARP3 autoantibodies;

9) ESO1 autoantibodies, p53 autoantibodies, FXR1 autoantibodies, ZNF573 autoantibodies, TM4SF1 autoantibodies, C1D autoantibodies, CCL18 and KRAS autoantibodies;

10) ESO1 autoantibodies, p53 autoantibodies, FXR1 autoantibodies, ZNF573 autoantibodies, TM4SF1 autoantibodies, C1D autoantibodies, CCL18, ARP3 autoantibodies, and KRAS autoantibodies;

11) ESO1 autoantibody, p53 autoantibody, FXR1 autoantibody, ZNF573 autoantibody, TM4SF1 autoantibody, C1D autoantibody, CCL18, ARP3 autoantibody and IGFBP1 autoantibody;

12) ESO1 autoantibody, p53 autoantibody, FXR1 autoantibody, ZNF573 autoantibody, TM4SF1 autoantibody, C1D autoantibody, CCL18, ARP3 autoantibody and FUBP1 autoantibody;

13) ESO1 autoantibody, p53 autoantibody, FXR1 autoantibody, ZNF573 autoantibody, TM4SF1 autoantibody, C1D autoantibody, CCL18, KRAS autoantibody and IGFBP1 autoantibody;

14) ESO1 autoantibody, p53 autoantibody, FXR1 autoantibody, ZNF573 autoantibody, TM4SF1 autoantibody, C1D autoantibody, CCL18, KRAS autoantibody and FUBP1 autoantibody;

15) ESO1 autoantibody, p53 autoantibody, FXR1 autoantibody, ZNF573 autoantibody, TM4SF1 autoantibody, C1D autoantibody, CCL18, ARP3 autoantibody, KRAS autoantibody, and IGFBP1 autoantibody;

16) ESO1 autoantibody, p53 autoantibody, FXR1 autoantibody, ZNF573 autoantibody, TM4SF1 autoantibody, C1D autoantibody, CCL18, ARP3 autoantibody, KRAS autoantibody, and FUBP1 autoantibody;

17) ESO1 autoantibody, p53 autoantibody, FXR1 autoantibody, ZNF573 autoantibody, TM4SF1 autoantibody, C1D autoantibody, CCL18 and SPINT1;

18) CCL18, SPINT1, C1D autoantibodies, FXR1 autoantibodies, ZNF573 autoantibodies, ESO1 autoantibodies, and p53 autoantibodies;

19) CCL18, SPINT1, YKL-40, C1D autoantibody, FXR1 autoantibody, ZNF573 autoantibody, ESO1 autoantibody and p53 autoantibody;

20) CCL18, SPINT1, YKL-40, C1D autoantibody, FXR1 autoantibody, ZNF573 autoantibody, ESO1 autoantibody, p53 autoantibody, and KRAS autoantibody;

21) CCL18, SPINT1, YKL-40, C1D autoantibody, FXR1 autoantibody, ZNF573 autoantibody, ESO1 autoantibody, p53 autoantibody, KRAS autoantibody, and TM4SF1 autoantibody;

22) CCL18, SPINT1, YKL-40, C1D autoantibody, FXR1 autoantibody, ZNF573 autoantibody, ESO1 autoantibody, p53 autoantibody, KRAS autoantibody, TM4SF1 autoantibody, and ARP3 autoantibody; and,

23) ESO1 autoantibody, p53 autoantibody, FXR1 autoantibody, ZNF573 autoantibody, TM4SF1 autoantibody, C1D autoantibody, CCL18, ARP3 autoantibody, KRAS autoantibody, IGFBP1 autoantibody, FUBP1 autoantibody, SPINT1, and YKL-40;

In a preferred embodiment of the present invention, any one of the biomarker combinations 1) to 23) further comprises CA125.

In order to solve the above technical problems, the second technical solution of the present invention is: to provide a diagnostic kit for early ovarian cancer diagnosis or prognosis thereof, wherein the diagnostic kit comprises the biomarker combination as described in one of the technical solutions.

In a preferred embodiment of the present invention, the biomarkers in the biomarker combination are antigens or antibodies; And/or, the diagnostic kit further comprises a reagent for detecting the biomarker, such as a detection antibody for antigen detection, or a detection antigen for antibody detection.

In a more preferred embodiment of the present invention, the antigen or antibody is from a serum sample.

As in the diagnostic kit of the second technical solution, the reagent for detecting the biomarker combination is selected from any one of the following groups:

1) Antibodies to ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, and CCL18;

2) Antibodies against ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, CCL18, and IGFBP1;

3) Antibodies against ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, CCL18, and FUBP1;

4) Antibodies against ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, CCL18, IGFBP1, and FUBP1;

5) Antibodies against ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, CCL18, ARP3, IGFBP1, and FUBP1;

6) ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, CCL18 antibodies, KRAS, IGFBP1, and FUBP1;

7) ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, CCL18 antibodies, ARP3, KRAS, IGFBP1, and FUBP1;

8) Antibodies against ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, CCL18, and ARP3;

9) Antibodies against ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, CCL18, and KRAS;

10) Antibodies against ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, CCL18, ARP3, and KRAS;

11) Antibodies against ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, CCL18, ARP3, and IGFBP1;

12) Antibodies against ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, CCL18, ARP3, and FUBP1;

13) Antibodies against ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, CCL18, KRAS, and IGFBP1;

14) ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, CCL18 antibodies, KRAS, and FUBP1;

15) ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, CCL18 antibodies, ARP3, KRAS, and IGFBP1;

16) ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, CCL18 antibodies, ARP3, KRAS, and FUBP1;

17) Antibodies against ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, CCL18, and SPINT1;

18) Antibodies against CCL18, SPINT1, C1D, FXR1, ZNF573, ESO1, and p53;

19) Antibodies against CCL18, SPINT1, YKL-40, C1D, FXR1, ZNF573, ESO1, and p53;

20) Antibodies against CCL18, SPINT1, YKL-40, C1D, FXR1, ZNF573, ESO1, p53, and KRAS;

21) Antibodies against CCL18, SPINT1, YKL-40, C1D, FXR1, ZNF573, ESO1, p53, KRAS, and TM4SF1;

22) CCL18 antibody, SPINT1 antibody, YKL-40 antibody, C1D, FXR1, ZNF573, ESO1, p53, KRAS, TM4SF1 and ARP3; and,

23) ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, CCL18 antibodies, ARP3, KRAS, IGFBP1, FUBP1, SPINT1 antibodies and YKL-40 antibodies;

In a preferred embodiment of the present invention, any one of the groups 1) to 23) of reagents for detecting the biomarker combination further comprises a CA125 antibody.

The CCL18 antibody of the present invention is used to detect CCL18 in serum;

The SPINT1 antibody of the present invention is used to detect SPINT1 in serum;

The C1D of the present invention is used to detect C1D autoantibodies in serum;

The FXR1 of the present invention is used to detect FXR1 autoantibodies in serum;

The ZNF573 of the present invention is used to detect ZNF573 autoantibodies in serum;

The ESO1 of the present invention is used to detect ESO1 autoantibodies in serum;

The p53 of the present invention is used to detect p53 autoantibodies in serum;

The IGFBP1 of the present invention is used to detect IGFBP1 autoantibodies in serum;

The FUBP1 of the present invention is used to detect FUBP1 autoantibodies in serum;

The YKL-40 antibody of the present invention is used to detect YKL-40 in serum;

The KRAS of the present invention is used to detect KRAS autoantibodies in serum;

The TM4SF1 of the present invention is used to detect TM4SF1 autoantibodies in serum;

The ARP3 of the present invention is used to detect ARP3 autoantibodies in serum;

The CA125 antibody of the present invention is used for detecting CA125 in serum.

The antibody is preferably hIgG.

The above biomarker combination can be used as a calibrator in the kit. The calibrator of the antigen in the biomarker combination is preferably a full-length peptide or a fragment thereof, which can be a natural product purified into serum, or expressed and purified in vitro using a prokaryotic or eukaryotic expression system; the calibrator of the autoantibody in the biomarker combination is preferably a recombinant human anti-tag peptide immunoglobulin G, and more preferably a 9E10 chimeric antibody hIgG.

In some preferred embodiments of the above technical solution, the biomarker, detection antigen or detection antibody further contains a label peptide; preferably, the label peptide includes: one or more of a His tag, a streptavidin tag, an avidin tag, a biotin tag, a GST tag, a C-myc tag, a Flag tag and an HA tag; more preferably, the biomarker, detection antigen or detection antibody can be expressed by Escherichia coli, yeast, insect cells or animal cells; and/or, the biomarker, detection antigen or detection antibody is purified by Ni affinity chromatography, ion exchange chromatography, molecular sieve, Purify by dialysis, ultrafiltration or hydrophobic chromatography.

Preferably, the kit further comprises phycoerythrin and its fragments, phycoerythrin-labeled anti-human immunoglobulin G and its fragments, biotinylated antibodies or biotinylated antigens against protein molecules in human serum, and one or more of a dilution buffer, a washing solution and an analysis buffer.

More preferably, the biomarker is fixed on a solid phase carrier; preferably, the solid phase carrier includes: microspheres, micropores of an ELISA plate, an affinity membrane or a liquid phase chip.

Further preferably, the biomarker, detection antigen or detection antibody is directly or indirectly coupled to the microsphere, and the label peptide is 6×His tag and/or C-Myc tag;

Preferably, the direct coupling is: microsphere-NH2-(C-Myc tag)-Linker-(6×His tag)-Linker-biomarker, detection antigen or detection antibody amino acid sequence; or microsphere-NH2-(C-Myc tag)-Linker-biomarker, detection antigen or detection antibody amino acid sequence-(6×His tag). The Linker is, for example, (G ₄ S) _n , where n is an integer of 1 to 5.

The indirect coupling step comprises:

(1) The microspheres are coupled to biotinylated bovine serum albumin (BSA-biotin), and the biomarker, detection antigen, or detection antibody is bound to streptavidin;

(2) Biotin binds to streptavidin, thereby allowing the biomarker, detection antigen or detection antibody to specifically bind to the microspheres.

More preferably, the dilution buffer is NaCl 300mM, KCl 2.7mM, Na ₂ HPO ₄ 8.1mM, KH ₂ PO ₄ 1.5mM, 5% donkey serum or 5w/v% BSA, 0.05% prcolin300 or 0.05w/v% sodium azide, pH 7.0-8.0; the washing solution is NaCl 137mM, KCl 2.7mM, Na ₂ HPO ₄ 8.1mM, KH ₂ PO ₄ 1.5mM, 0.05% Tween-20, 0.03% prcolin300, pH 7.6; the analysis buffer is NaCl 137mM, KCl 2.7mM, Na ₂ HPO ₄ 8.1mM, KH ₂ PO ₄ 1.5 mM, 1% donkey serum or 1 w/v% BSA, 0.05% prcolin300 or 0.05 w/v% sodium azide; wherein the % is volume percentage, and the w/v% is mass volume ratio.

To solve the above technical problems, the third technical solution of the present invention is: use of a biomarker combination as described in one of the technical solutions or a diagnostic kit as described in the second technical solution in the preparation of reagents and/or kits for detecting specific proteins or antibody molecules in human serum.

In a preferred embodiment of the present invention, the reagent and/or kit is used for diagnosing or assisting in the diagnosis of early ovarian cancer, screening of high-risk populations for ovarian cancer, assisting in the early diagnosis of benign or malignant ovarian tumors, or for recurrence monitoring and/or prognosis monitoring after treatment of ovarian cancer.

The present invention provides a biomarker combination, a kit containing the same and its application, wherein the detection kit comprises a set of optimized recombinant antibodies for detecting any 7 or more, preferably 8 or more, of multiple proteins or antibodies in human serum, wherein the antibodies for detection may be IgG antibodies, and the amino acids of the antigens for detection may be The acid sequence is a full-length or ectopically sheared sequence; the antigenic proteins are all connected with a tag peptide, and the tag peptide can be selected from: His tag, streptavidin tag, avidin tag, c-Myc tag, Flag tag, HA tag and biotin tag, and the positive quality control product is a recombinant human anti-tag peptide immunoglobulin G or a fragment thereof.

The in vitro diagnostic kit product prepared using the biomarker combination of the present invention has a precision coefficient of variation (CV) of less than 15%, and stability test results show that the shelf life of the product is not less than 12 months.

To solve the above technical problems, the fourth technical solution of the present invention is: an ovarian cancer diagnosis system, the ovarian cancer diagnosis system comprises the following modules:

(1) an input module, which is used to input the detection value of the biomarker combination as described in one of the technical solutions contained in the sample to be tested;

(2) Analysis module, which is used to calculate p, where

X＝0.008576*CCL18+0.002106*CA125+0.001736*C1D+0.000499*FXR1-0.001239*ZNF573+0.000357*ESO1+0.000126*p53-0.000229*KRAS+0.001062*IGFBP1+0.001421*FUBP1+0.000556*TM4SF1-0.000401*ARP3- 3.363822;

in:

1) CCL18 is the CCL18 antigen content measured in serum;

2) CA125 is the CA125 antigen content measured in serum;

3) C1D is the C1D autoantibody content measured in serum;

4) FXR1 is the level of FXR1 autoantibodies measured in serum;

5) ZNF573 is the level of ZNF573 autoantibodies measured in serum;

6) ESO1 is the ESO1 autoantibody content measured in serum;

7) p53 is the p53 autoantibody content measured in serum;

8) KRAS is the level of KRAS autoantibodies measured in serum;

9) IGFBP1 is the IGFBP1 autoantibody content measured in serum;

10) FUBP1 is the FUBP1 autoantibody content measured in serum;

11) TM4SF1 is the TM4SF1 autoantibody content measured in serum;

12) ARP3 is the ARP3 autoantibody content measured in serum;

p is the predicted probability of cancer;

(3) Judgment module, cutoff is 0.6011; when p≥cutoff, it is judged as positive, and when p＜cutoff, it is judged as negative.

In a preferred embodiment of the present invention, the ovarian cancer diagnosis system further comprises a login module, and/or the ovarian The cancer diagnosis system also includes a printing module; the login module requires input of a user name and a user password, and the printing module can print the results generated by the input module, the analysis module and the judgment module.

In a more preferred embodiment of the present invention, the sample to be tested is serum.

According to the embodiment of the present invention, in the present invention, when the regression value (p value) is less than the cutoff value, it is diagnosed as "negative"; when the regression value is greater than or equal to the cutoff value, it is diagnosed as "positive". The present invention analyzes and processes the test results of the ovarian cancer early diagnosis test kit and outputs them in the form of a report, which has the characteristics of convenient data analysis and intuitive result reporting.

In order to solve the above technical problems, the fifth technical solution of the present invention is: a computer-readable medium, wherein the computer-readable medium stores a computer program, and when the computer program is executed by a processor, it can realize the functions of the ovarian cancer diagnosis system as described in the fourth technical solution.

In order to solve the above technical problems, the sixth technical solution of the present invention is: an ovarian cancer diagnosis device, the ovarian cancer diagnosis device comprising:

(1) The computer-readable medium as described in technical solution 5;

(2) A processor, configured to execute a computer program to implement the functions of the ovarian cancer diagnosis system.

To solve the above technical problems, the seventh technical solution of the present invention is: the biomarker combination as described in one of the technical solutions, the diagnostic kit as described in the second technical solution, the ovarian cancer diagnostic system as described in the fourth technical solution, the computer-readable medium as described in the fifth technical solution, or the ovarian cancer diagnostic device as described in the sixth technical solution is used for diagnosing or assisting in the diagnosis of early ovarian cancer, screening of high-risk groups for ovarian cancer, and early assisting in the diagnosis of benign and malignant ovarian tumors.

To solve the above technical problems, the eighth technical solution of the present invention is: a method for recurrence monitoring and/or prognosis monitoring after ovarian cancer treatment, the method comprising using the diagnostic kit as described in the second technical solution to detect the biomarker combination as described in one of the technical solutions in the sample to be tested, and using the detection value as described in the fourth technical solution The system for ovarian cancer diagnosis, the computer-readable medium as described in the fifth technical solution, or the ovarian cancer diagnostic device as described in the sixth technical solution to determine the result.

On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

The reagents and raw materials used in the present invention are commercially available.

The positive and progressive effects of the present invention are as follows:

The present invention utilizes the advantages of autoantibody spectrum in early diagnosis and screening of ovarian cancer and prognosis monitoring of ovarian cancer treatment, utilizes the high specificity and high sensitivity of antigen-antibody reaction, adopts liquid suspension chip technology, and performs high-throughput detection of ovarian cancer autoantibody spectrum by flow cytometry, which is superior to traditional detection technology, and the biomarker combination of the present invention has the following advantages: high sensitivity and/or high specificity.

More specifically, the biomarker combination described in the present invention shows excellent diagnostic performance for ovarian cancer. The specificity in the healthy group, benign group, and interference malignant group was greater than 90%, and there was no significant difference in the detection rate between early (stage I) and mid-to-late (stage II-IV) ovarian cancer (p>0.05). In the malignant interference group, it had a specificity of 90.2%. It had very high specificity for non-ovarian malignant tumors such as cervical cancer, gastric cancer, and colorectal cancer. That is, the marker combination described in the present invention can only specifically differentiate and diagnose ovarian cancer, thereby greatly reducing the misdiagnosis rate of non-ovarian malignant tumors in clinical practice.

In addition, the combined system disclosed in the prior art, such as CN106248940B, can only detect epithelial cancer (serous and mucinous), and its effect on the diagnosis of ovarian germ cell tumors is unknown. The biomarker combination described in the present invention has a sensitivity of 82.6% to ovarian germ cell tumors, which is not significantly different from the sensitivity of epithelial cancer (86.5%) (p>0.05). In summary, the above fully shows that the biomarker combination described in the present invention has excellent differential diagnosis performance for ovarian cancer, and the early diagnosis of ovarian malignant tumors such as germ cell tumors is included in the clinical diagnosis, which helps to cover the entire population of ovarian cancer and greatly reduce the misdiagnosis rate and missed diagnosis rate of ovarian cancer patients in the clinic.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows SDS-PAGE analysis (left) and Western blot analysis (right, anti-His antibody) of ESO1 protein expressed in E. coli.

FIG2 is the SDS-PAGE detection of the purified ESO1 protein.

Figure 3 shows SDS-PAGE analysis (left) and Western blot analysis (right, anti-His antibody) of ZNF573 protein expressed in E. coli.

FIG4 is SDS-PAGE detection of purified ZNF573 protein.

Figure 5 shows the expression levels of CCL18, SPINT1, YKL-40, and C1D autoantibodies in different populations, where interference represents the interference malignant group, Benign represents the benign group, healthy represents the healthy group, and oc represents the ovarian cancer group.

Figure 6 shows the expression levels of FXR1 autoantibodies, ZNF573 autoantibodies, p53 autoantibodies, and ESO1 autoantibodies in different populations, where interference represents the interference malignant group, Benign represents the benign group, healthy represents the healthy group, and oc represents the ovarian cancer group.

Figure 7 shows the ROC curve of ovarian cancer vs. healthy subjects.

Figure 8 shows the ROC curve for ovarian cancer vs benign disease.

FIG9 shows the ROC curve of ovarian cancer vs. interfering malignancy.

Figure 10 shows the ROC curve of eight combined tests.

FIG. 11 is the ROC curve of CA125 detection alone.

FIG. 12 is the ROC curve of the 12 markers alone for the diagnosis of ovarian cancer.

FIG13 is the ROC curve of the combined diagnosis of 11 markers.

FIG14 is the ROC curve of the combined diagnosis of 12 markers.

DETAILED DESCRIPTION

In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and/or" used herein includes any and all combinations of one or more related listed items.

1. PBS: NaCl 137mM; KCl 2.7mM; Na ₂ HPO ₄ 8.1mM; KH ₂ PO ₄ 1.5mM; pH 7.6, filtered through 0.22μm membrane;

2. Serum/antibody dilution buffer: NaCl 300 mM; KCl 2.7 mM; Na ₂ HPO ₄ 8.1 mM; KH ₂ PO ₄ 1.5 mM; 5% donkey serum (volume ratio); 0.05% proclin 300 (volume ratio); pH 7.6; filtered through a 0.22 μm filter membrane.

3. 5× washing solution: NaCl 685mM; KCl 13.5mM; Na ₂ HPO ₄ 40.5mM; KH ₂ PO ₄ 7.5mM; 0.25% Tween-20 (volume ratio), 0.25% proclin 300 (volume ratio), pH 7.6; filtered through a 0.22 μm filter membrane;

4. 5× assay buffer: NaCl 685 mM; KCl 13.5 mM; Na ₂ HPO ₄ 40.5 mM; KH ₂ PO ₄ 7.5 mM; 5% BSA (mass volume ratio); 0.25% proclin 300 (volume ratio); pH 7.6; filtered through a 0.22 μm filter membrane.

The ESO1 antigen, C1D antigen, FXR1 antigen, p53 antigen, KRAS antigen, TM4SF1 antigen, ARP3 antigen, Anti-CCL18 antibody, etc. used in the embodiments of the present invention are expressed by conventional means in the art or can be purchased commercially.

Example 1 Expression and purification of recombinant ESO1 containing Streptavidin tag

(I) Construction of ESO1 recombinant genetically engineered bacteria

1. Artificially synthesize the target gene ESO1 and recombinant it into the PUC-57 vector according to the sequence 1 (Myc tag+Linker+His tag+Linker+ESO1+Linker+Streptavidin), where Linker is (G ₄ S) _n , and n is an integer of 1-5.

2. Take 5 μL of correctly sequenced PUC-57-ESO1 recombinant bacteria and inoculate it into liquid culture medium containing 30 mg/mL Amp+, and culture it at 37℃ and 200 r/min overnight.

3. Extract the plasmid from the PUC-57-ESO1 recombinant bacteria. Take 1.5 ml of the recombinant bacterial solution and use the QIAGEN plasmid extraction kit (LOT: 154014885) to extract the plasmid.

4. Take 1.5 μg of PUC-57-ESO1 plasmid and pET-30a vector respectively and incubate with HindⅢ and Nde I endonucleases at 37℃ for 1h Perform enzyme digestion. Use the OMEGA gel recovery kit to recover the target fragment and vector: First, cut the gel under ultraviolet light according to the size of the PCR product. When cutting the gel, be careful not to cut it too large or too small. Too large or too small will lead to a decrease in the recovery rate. Put the cut gel in a 1.5 or 2.0mL centrifuge tube and act with the yellow Bingding Buffer in a water bath at about 70℃ until the gel block is completely melted (shake from time to time during the period). Transfer the melted liquid to the adsorption column to allow the gene fragments in the gel block to bind to the adsorption column. Rinse the impurities with the eluent to ensure that all impurities are removed, then elute the target gene with deionized water.

The restriction enzyme digestion reaction system of PUC-57-ESO1 plasmid is as follows:

5. Recover the target fragment and vector at a molar ratio of 5:1, connect them at 16℃ for 4h under the action of T4 ligase, construct a prokaryotic expression vector, and transform it into BL21 (DE3) competent cells. Take out a 100μl competent cell from the -70℃ refrigerator, put it in an ice box immediately after the palm of your hand melts, add the entire amount of the ligation product to the 100μl competent cell, swirl gently to mix, place on ice for 30min, then heat shock at 42℃ for 90s, be careful not to shake, immediately ice bath for 5min, add 1ml of LB liquid culture medium that has been pre-bathed at 37℃ to the above tube in an ultra-clean workbench, and then fix it to the spring frame of an air shaker and shake at 37℃ for 1h. After the incubation, the cells were collected by centrifugation at 3000rpm for 5min, and about 200μl of the supernatant was retained and the rest was discarded. The cells were mixed by vortexing and spread on LB/agar plate medium with a final concentration of Amp+0.1mg/ml. The cells were spread evenly with a glass spreader rod burned by an alcohol lamp, and cultured in a 37℃ incubator overnight. Single colonies were selected for culture, plasmids were extracted, and positive clones were screened. The positive recombinant plasmid pET-30a-ESO1(DE3) was sent to GenScript Sequencing Company for sequencing.

The pET-30a-ESO1 ligase catalyzed reaction system is as follows:

The pET-30a-ESO1 double enzyme digestion identification reaction system is as follows:

(II) Cultivation and expression of recombinant ESO1 genetically engineered bacteria

1. Take 20 μL of recombinant ESO1 genetically engineered bacteria and inoculate them into 100 mL of sterile LB liquid culture medium containing 0.1% ampicillin.

2. Culture at 37°C, 220 rpm for 16 h, then inoculate into 1 L LB liquid culture medium containing 0.1% ampicillin and expand the culture at 37°C, 220 rpm for 3 h.

3. Add 1 mL IPTG and continue to induce expression at 37°C, 220 rpm for 4 hours.

4. After the induction of expression is completed, the bacterial solution is centrifuged at 5000 rpm and the bacteria are collected at 15 min.

5. Take the uninduced and induced bacterial cultures and analyze the expression of BRAF protein by SDS-PAGE and Western blot (as shown in Figure 1). There is a big difference in the electrophoresis of bacterial proteins before and after induction (the arrow in the figure indicates the target protein). The recombinant protein expression accounts for about 40% of the total bacterial protein.

Figure 1 illustrates SDS-PAGE analysis (left) and Western blot analysis (right, anti-His antibody) of ESO1 protein expressed in E. coli, where lane M1: protein marker and lane M2: Western blot marker

Lane PC1: BSA (1 μg); Lane PC2: BSA (2 μg); Lane NC: uninduced whole cell lysate; Lane NC1: supernatant of uninduced cell lysate; Lane NC2: precipitate of uninduced cell lysate; Lane 1: whole cell lysate induced at 37°C for 4 hours; Lane 2: supernatant of cell lysate induced at 37°C for 4 hours; Lane 3: precipitate of cell lysate induced at 37°C for 4 hours.

3. Purification of recombinant ESO1

1. Ultrasonic disruption of bacteria

Add 50 ml of lysis buffer (3 g Tris-base, 14.61 g sodium chloride, 0.74 g disodium EDTA, dissolved in 450 mL ddH ₂ O, adjust pH to 8.0, add water to 500 mL, filter with 0.45 μm filter), mix the cells, and then ultrasonically disrupt the cells in an ice bath (300 W ultrasonic 4S, 4S interval, ultrasonic 25min). After ultrasonic disruption of the cells, centrifuge at 4°C 10000 rpm for 15 min, discard the supernatant, and collect the inclusion body precipitate.

2. Inclusion body cleaning

Add inclusion body washing solution (20mM PBS, pH 8.0) at a ratio of inclusion body: inclusion body washing buffer = 1:10 (g:ml). After thorough oscillation and washing at 4℃, centrifuge at 10000rpm for 15min, discard the supernatant, and collect the precipitate. Repeat once.

3. Inclusion body dissolution

Add 100 ml of inclusion body dissolution solution (7.8 g sodium dihydrogen phosphate dihydrate, 29.22 g sodium chloride, 573 g urea, 1.4 g imidazole, dissolved in 800 mL ddH2O, adjust pH to 8.0, dilute to 1000 mL, filter with 0.45 um filter) to the inclusion body precipitate collected by centrifugation, fully resuspend the precipitate, and shake on a shaker in an ice bath for 3 h. Centrifuge the fully dissolved inclusion bodies at 4°C 10000 rpm for 30 min, and collect the inclusion body supernatant.

4. Protein Purification

a) Load 10 ml of Ni-NTA filler into a chromatographic column (chromatographic column specification: 16 mm × 20 cm).

b) Equilibrate the column with 2 column volumes of inclusion body solubilization buffer.

c) Load the collected inclusion body solution supernatant onto the equilibrated Ni-NTA column at a flow rate of 1 ml/min.

d) After the sample is loaded, the filler is washed with 4 column volumes of washing buffer (50 mM Na ₂ HPO 4 , 10 mM Tris·Cl , 10 mM imidazole , 8 M Urea , pH 8.0) until the A280 value detected by UV is stable.

e) The target protein on the chromatographic column was eluted with elution buffer (50 mM Na ₂ HPO ₄ , 10 mM Tris·Cl, 250 mM imidazole, 8 M Urea, pH 8.0), and the chromatographic peaks were collected according to the A280 value detected by ultraviolet light.

5. Protein Renaturation

The purified target protein sample was dialyzed against PBS solution at 4°C for 10 hours. The dialyzed sample was concentrated by ultrafiltration or PEG20000 to obtain the renatured target protein. BRAF was quantified using the Folin-phenol method, and the protein concentration was measured to be 0.5 mg/ml.

(IV) SDS-PAGE detection of recombinant ESO1

The purified target protein was renatured and then detected by SDS-PAGE. The results are shown in Figure 2, and the purity was greater than 90%. Lane M: protein marker, lanes 1, 2, and 3 are after ESO1 renatured.

The expression and purification methods of other marker antigens or autoantibodies containing a Streptavidin tag in the present invention are the same as the expression and purification methods of the recombinant ESO1 containing a Streptavidin tag in this example.

Example 2 Expression and purification of recombinant ZNF573 without Streptavidin tag

(I) Construction of ZNF573 recombinant engineered bacteria

1. Artificially synthesize the target gene ZNF573 and recombinant it into the PUC-57 vector according to sequence 2 (NdeI-Myc tag-Linker-His tag-Linker-ZNF573-Stop codon-HindIII), wherein Linker is (G ₄ S) _n , and n is an integer of 1-5.

2. Take 5 μL of correctly sequenced PUC-57-ZNF573 recombinant bacteria and inoculate it into liquid culture medium containing 30 mg/mL Amp+, and culture it at 37°200 r/min overnight.

3. Extract the plasmid from the PUC-57-ZNF573 recombinant bacteria. Take 1.5 ml of the recombinant bacterial solution and use the QIAGEN plasmid extraction kit (LOT: 154014885) to extract the plasmid.

4. Take 1.5 μg of PUC-57-ZNF573 plasmid and pET-30a vector respectively and digest them with HindⅢ and NdeI endonucleases at 37° for 1 hour. Use OMEGA gel recovery kit to recover the target fragment and vector: First, under ultraviolet light, Cut the gel according to the size of the PCR product. Be careful not to cut it too big or too small when cutting the gel. Too big or too small will lead to a decrease in recovery rate. Put the cut gel in a 1.5 or 2.0mL centrifuge tube and act with the yellow Bingding Buffer in a water bath at about 70℃ until the gel block is completely melted (shake from time to time during the period). Transfer the melted liquid to the adsorption column to allow the gene fragments in the gel block to bind to the adsorption column. Rinse the impurities with the eluent to ensure that all impurities are removed and then elute the target gene with deionized water.

The PUC-57-ZNF573 plasmid restriction enzyme digestion reaction system is as follows:

5. Recover the target fragment and vector at a molar ratio of 5:1, connect them at 16℃ for 4h under the action of T4 ligase, construct a prokaryotic expression vector, and transform it into BL21 (DE3) competent cells. Take out a 100μl competent cell from the -70℃ refrigerator, put it in an ice box immediately after the palm of your hand melts, add the entire amount of the ligation product to the 100μl competent cell, swirl gently to mix, place on ice for 30min, then heat shock at 42℃ for 90s, be careful not to shake, immediately ice bath for 5min, add 1ml of LB liquid culture medium that has been pre-bathed at 37℃ to the above tube in an ultra-clean workbench, and then fix it to the spring frame of an air shaker and shake at 37℃ for 1h. Place the cells on a centrifuge and centrifuge at 3000rpm for 5 minutes. Keep about 200μl of the supernatant and discard the rest. Vortex the cells and spread them on LB/agar plates with a final concentration of Amp+0.1mg/ml. Use a glass coating rod burned by an alcohol lamp to spread them evenly. Culture them in a 37℃ incubator overnight. Pick a single colony for culture, extract the plasmid, and screen the positive clones. The positive recombinant plasmid pET-30a-ZNF573(DE3) was sequenced to confirm that it was correct.

The pET-30a-ZNF573 ligase catalyzed reaction system is as follows:

The pET-30a-ZNF573 double enzyme digestion identification reaction system is as follows:

(II) Cultivation and expression of recombinant ZNF573 genetically engineered bacteria

1. Take 20 μl of recombinant ZNF573 genetically engineered bacteria and inoculate them into 100 mL of sterile LB liquid culture medium containing 0.1% ampicillin.

2. Culture at 37℃, 220rpm for 16h, then inoculate into 1L LB liquid culture medium containing 0.1% ampicillin, and expand the culture at 37℃, 220rpm for 3h.

3. Add 1 mL IPTG and continue to induce expression at 37°C, 220 rpm for 4 hours.

4. After the induction of expression is completed, the bacterial solution is centrifuged at 5000 rpm and the bacteria are collected at 15 min.

5. The expression of ZNF573 protein was analyzed by SDS-PAGE and Western blot in the bacterial suspension before and after induction (as shown in Figure 3). In Figure 3, electrophoresis lane M1: protein marker; lane PC1: BSA (1μg); lane PC2: BSA (2μg); lane M1: protein marker; lane NC: uninduced whole cell lysate; lane NC1: supernatant of uninduced cell lysate; lane NC2: precipitate of uninduced cell lysate; lane 1: cell lysate induced at 37℃ for 4 hours; lane 2: supernatant of cell lysate induced at 16℃ for 4 hours; lane 3: precipitate of cell lysate induced at 16℃ for 4 hours;

(III) Purification and SDS-PAGE detection of recombinant ZNF573

The purification method of recombinant ZNF573 is the same as that of Example 1 "Purification of recombinant ESO1". The purified target protein is renatured and then detected by SDS-PAGE. The purity is greater than 90%, as shown in Figure 4. Lane M is a protein marker, and lanes 1, 2, and 3 are ZNF573 after renaturation.

The expression and purification methods of other marker antigens or autoantibodies without a Streptavidin tag in the present invention are the same as the expression and purification methods of the recombinant ZNF573 without a Streptavidin tag in this example.

Example 3 Preparation method of indirectly coupled antibody or antigen microspheres

In the present invention, biotin-labeled BSA (BSA-biotin) is first directly coupled to the surface of the microspheres, and the antigen or antibody protein containing the streptavidin tag prepared in Example 1 is then coated on the surface of the microspheres coupled with BSA-biotin by utilizing the high affinity and high specificity of biotin-streptavidin. The antigen or antibody protein can specifically bind to and capture the corresponding antibody or antigen in ovarian cancer serum, and the phycoerythrin-labeled anti-c-Myc human immunoglobulin G or anti-c-Myc human immunoglobulin M can specifically bind to the captured antibody or antigen, and finally form "BSA-biotin+biotin-antigen or antibody protein+serum antibody or antigen+fluorescent secondary antibody (phycoerythrin-labeled anti-c-Myc human immunoglobulin G or anti-c-Myc human immunoglobulin M, and their fragments)" against ovarian cancer serum autoantibodies or antigens. The complex, the phycoerythrin-labeled fluorescent secondary antibody, can be excited by the reporter laser of the liquid phase chip instrument to emit fluorescence and be received by the reporter molecule detector. The fluorescence of the serum sample is converted into a standard curve generated by the fluorescence of the standard, and the content of ovarian cancer autoantibodies or antigens in the serum bound to the PE-labeled fluorescent secondary antibody can be detected.

1. Activation of Microspheres

a) Take out Microspheres (Luminex) were vortexed and sonicated for about 20 seconds to fully mix and resuspend the microspheres. According to the number of microspheres required for coupling, an appropriate volume of microsphere suspension (about 5×10 ⁶ microspheres) was pipetted into the coupling reaction tube.

b) Place the coupling reaction tube on a magnetic separator for 30-60 seconds to separate the microspheres from the liquid. Carefully remove the supernatant with a pipette to avoid sucking away the microbead precipitate.

c) Remove the magnetic separator, add 500 μL activation buffer (0.1M NaH ₂ PO ₄ , pH 6.2) to the reaction tube, vortex and sonicate to resuspend the microspheres. Place the reaction tube on the magnetic separator to magnetically separate the microspheres, and carefully remove the supernatant with a pipette. Repeat this step to wash the microspheres 1-2 times.

d) Add 480 μL of activation buffer to the washed microspheres, vortex and ultrasonically mix, add 10 μL of NHS and 10 μL of EDC to the microspheres in sequence, vortex and mix, and incubate the reaction tube at room temperature at 300 rpm on a rotating mixer for 20-30 minutes away from light.

2. Carrier protein (biotinylated BSA) coupled to microspheres

a) Place the activated microsphere reaction tube on a magnetic separator for magnetic separation for 30-60 seconds. Carefully remove the supernatant with a pipette, remove the magnetic separator, add 400 μL of coupling buffer (50 mM MES, pH 6.0) to the reaction tube, vortex and sonicate for 20 seconds to resuspend the microspheres.

b) Add biotion-BSA to the suspended microsphere solution in the ratio of carrier protein biotion-BSA: number of microspheres = 5 μg: 1×10 ^6. Vortex and sonicate. Incubate the reaction tube at room temperature at 300 rpm in a rotating mixer away from light for 1-2 hours. Then place the reaction tube on a magnetic separator for 30-60 seconds to separate the microspheres. Carefully remove the supernatant with a pipette.

3. Cleaning of Microspheres

Add 500 μL of washing solution (10 mM PBS, 0.05% Tween-20) to the reaction tube, vortex and sonicate to resuspend the microspheres after coupling with the carrier protein. Then place the reaction tube on a magnetic separator for 30-60 seconds to separate the microspheres, and carefully remove the supernatant with a pipette.

4. Closure of microspheres

Add 500 μL of blocking buffer (10 mM PBS, 5% donkey serum, 0.03% Proclin 300) to the washed microspheres, vortex and sonicate to resuspend the microspheres. Incubate the reaction tube at room temperature at 300 rpm on a rotating mixer in the dark for 30 min.

5. Coating of antigen or antibody protein

a) Place the reaction tube with enclosed microspheres on a magnetic separator for 30-60 seconds, carefully remove the supernatant with a pipette, add 500 μL of washing solution to the reaction tube, oscillate and ultrasonically wash for about 20 seconds to resuspend the microspheres, and then place the reaction tube on a magnetic separator. Carefully remove the supernatant using a pipette.

b) Add 200 μL of coating buffer (10 mM PBS, 1% donkey serum, 0.03% Proclin300) to the reaction tube, vortex for 20 seconds, and add the antigen or antibody to be coated according to the ratio of antigen or antibody to be coated: number of microspheres = 40 μg: 1×10 ^6. After vortexing and sonicating for about 20 seconds to resuspend the microspheres, incubate the reaction tube at room temperature at 300 rpm in a rotating mixer in the dark for 60 minutes.

c) Place the reaction tube on a magnetic separator, carefully remove the supernatant with a pipette, add 500 μL of washing solution to the reaction tube, vortex and sonicate for about 20 seconds, place the reaction tube on a magnetic separator, carefully remove the supernatant with a pipette, and repeat this washing step to wash the microspheres 2-3 times. Finally, resuspend the microspheres with 1 mL of microsphere storage solution (10 mM PBS, 3% donkey serum, 5% trehalose, 0.03% Proclin300) and store at 2-8°C in the dark.

6. Counting magnetic beads after coupling

After the magnetic beads are coupled, they are counted with a hemacytometer under a microscope to determine their concentration and calculate the yield and loss of the coupling process; the hemacytometer calculation uses the following formula: total number of microspheres = number of microspheres in 4×4 grids×(1×10 ⁴ )×dilution factor×suspension volume.

Example 4 Preparation method of directly coupled antibody or antigen microspheres

In this embodiment, the antigen or antibody protein is directly coated onto the surface of the microsphere.

1. Activation of Microspheres

Same as in Example 3, "1 Activation of Microspheres"

2. Antigen or antibody coupled to microspheres

a) Place the activated microsphere reaction tube on a magnetic separator for magnetic separation for 30-60 seconds. Carefully remove the supernatant with a pipette, remove the magnetic separator, add 400 μL of coupling buffer (50 mM MES, pH 6.0) to the reaction tube, vortex and sonicate for 20 seconds to resuspend the microspheres.

b) Add the antibody or antigen to be coupled to the suspended microsphere solution at a ratio of antibody or antigen: microsphere number = 5 μg: 1×10 ^6. After vortexing and sonication, incubate the reaction tube at room temperature at 300 rpm on a rotating mixer in the dark for 1-2 hours.

3. Cleaning of Microspheres

After the microspheres are coupled, place the reaction tube on a magnetic separator for 30-60 seconds to separate the microspheres. Carefully remove the supernatant with a pipette. Remove the magnetic separator and resuspend the microspheres in 1 mL of PBST solution by vortexing and sonicating for about 20 seconds. Repeat this step. Wash twice with a total of 1 mL of PBST solution.

4. Closure of microspheres

Add 500 μL of blocking buffer (10 mM PBS, 5% donkey serum, 0.03% Proclin 300) to the washed microspheres, vortex and sonicate to resuspend the microspheres. Incubate the reaction tube at room temperature at 300 rpm on a rotating mixer in the dark. 30min.

5. Storage of Microspheres

Follow the steps in 3 above to wash the magnetic beads 2 to 3 times, resuspend the microspheres in 1 mL of microsphere storage solution (10 mM PBS, 3% donkey serum, 5% trehalose, 0.03% Proclin 300), and store at 2-8°C in the dark.

6. Counting of Microspheres

After the magnetic beads are coupled, they are counted with a hemacytometer under a microscope to determine their concentration and calculate the yield and loss of the coupling process; the hemacytometer calculation uses the following formula: total number of microspheres = number of microspheres in 4×4 grids×(1×10 ⁴ )×dilution factor×suspension volume.

Example 5 Preparation of flow immunofluorescence kit for detecting tumor antigens or autoantibodies and flow fluorescence detection

1. Composition of flow immunofluorescence kit for detecting autoantibodies or antigens

The flow immunofluorescence kit for detecting autoantibodies or antigens (fixed antigen) includes the following components:

1) Using a group of detection microspheres coupled or coated with different antigens or antibodies;

2) Phycoerythrin-labeled donkey anti-human immunoglobulin G and phycoerythrin-labeled donkey anti-human immunoglobulin M, concentration 500μg/ml; phycoerythrin, concentration 500μg/ml; biotinylated antibody for corresponding antigen protein detection or biotinylated antigen for corresponding antibody protein detection, 1mg/mL. The working solution is 5μg/mL;

3) Positive quality control (standard): high concentration of human anti-c-Myc tag immunoglobulin G and human anti-c-Myc tag immunoglobulin M, or antigen protein (such as CCL18, SPINT1, YKL-40);

4) Negative quality control: low concentration of human anti-c-Myc tag immunoglobulin G and human anti-c-Myc tag immunoglobulin M, or antigen protein (such as CCL18, SPINT1, YKL-40);

5) serum dilution buffer (NaCl 300 mM, KCl 2.7 mM, Na ₂ HPO ₄ 8.1 mM, KH ₂ PO ₄ 1.5 mM, 5% v/v donkey serum or 5% w/v BSA, 0.05% prcolin 300 or 0.05% w/v sodium azide, pH 7.0-8.0);

6) 20× washing solution (1× washing solution: NaCl 137 mM, KCl 2.7 mM, Na ₂ HPO ₄ 8.1 mM, KH ₂ PO ₄ 1.5 mM, 0.05% Tween-20, 0.03% v/v prcolin 300, pH 7.6);

7) Assay buffer (NaCl 137 mM, KCl 2.7 mM, Na ₂ HPO ₄ 8.1 mM, KH ₂ PO ₄ 1.5 mM, 0.2% v/v donkey serum or 1% w/v BSA, 0.05% prcolin 300 or 0.05% w/v sodium azide);

8) 96-well round bottom plate (Corning);

9) Sealing film.

2. Preparation of detection microspheres and flow immunofluorescence detection

2.1 Preparation of detection microspheres:

The preparation of microspheres is the same as in Example 3 and/or Example 4.

Each antibody or antigen is coupled to a differently encoded microsphere.

2.2 Flow immunofluorescence detection method of microspheres

2.2.1 Detection methods of specific autoantibodies in serum

(I) Packaging of microspheres into 96-well plates and washing of microspheres

Add a group of microspheres coupled with coated antigens to a 96-well plate (2500 microspheres/type/well), and add 120 μl of washing solution to each well. Place the 96-well plate on a rotary mixer and shake at 1000 rpm for 1 min at room temperature. Place the 96-well plate on a magnetic plate and let it stand for 1 min. Keep the 96-well plate fixed on the magnetic plate and facing upward, quickly and forcefully flip the magnetic plate downward to shake off the liquid in the wells, keep the magnetic plate facing downward, and quickly shake it vertically downward for 3 to 4 times until no liquid drips in the reaction plate.

(II) Incubation of microspheres with serum to be tested

1. Add the standard, quality control and diluted serum samples to the corresponding positions of the 96-well plate, adding 100 μl to each well; place the 96-well plate on a rotary mixer, shake and mix at room temperature, 1000 rpm for 1 min, apply the sealing film, place in a 37°C constant temperature incubator, and incubate for 90 min; at the end of the incubation, place the 96-well plate on a magnetic plate, let it stand for 1 min, and then shake off the liquid in the wells.

2. Remove the 96-well plate from the magnetic plate, add 120 μl of washing solution to each well of the reaction plate, place the 96-well plate on a rotary mixer, shake and mix at room temperature and 1000 rpm for 1 minute; remove the reaction plate from the rotary mixer, place it on the magnetic plate, let it stand for 1 minute, and then discard the liquid in the wells. Repeat this step to wash the microspheres twice.

(III) Secondary Antibody Incubation

1. Add the phycoerythrin-labeled fluorescent secondary antibody working solution to each well, place the 96-well plate on a rotating mixer, shake and mix at 1000 rpm for 1 min at room temperature, apply a sealing film, place in a 37°C constant temperature incubator, and incubate for 60 min; at the end of incubation, place the 96-well plate on a magnetic plate, let it stand for 1 min, and then shake off the liquid in the wells.

2. Remove the 96-well plate from the magnetic plate, add 120 μl of washing solution to each well of the reaction plate, place the 96-well plate on a rotary mixer, shake and mix at room temperature and 1000 rpm for 1 minute; remove the reaction plate from the rotary mixer, place it on the magnetic plate, let it stand for 1 minute, and then discard the liquid in the wells. Repeat this step to wash the microspheres twice.

(IV) Detection of serum autoantibody expression levels

The serum autoantibody content was detected using the Luminex 200 liquid phase chip system (Luminex Corporation), and the concentration of each indicator in the serum was reflected by the median fluorescent intensity (MFI). After incubation with the secondary antibody, 100 μl of analysis buffer was added to each well, placed in a rotating mixer at 1000 rpm for 3 minutes, and immediately put on the machine to read the plate.

2.2.2 Detection methods of specific antigenic proteins in serum

(I) Packaging of microspheres into 96-well plates and washing of microspheres

A group of microspheres to be tested coupled with specific antibodies were added to a 96-well plate (2500 microspheres/type/well), and 120 μl of washing solution was added to each well. The 96-well plate was placed on a rotating mixer and shaken at 1000 rpm for 1 min at room temperature. Place the 96-well plate on the magnetic plate and let it stand for 1 min. Keep the 96-well plate fixed on the magnetic plate and facing upward. Quickly and forcefully flip the magnetic plate downward to shake off the liquid in the wells. Keep the magnetic plate facing downward and quickly shake it vertically downward 3 to 4 times until there is no liquid dripping in the reaction plate.

(II) Incubation of microspheres with serum to be tested

1. Add the standard, quality control and diluted serum samples to the corresponding positions of the 96-well plate, adding 100 μl to each well; place the 96-well plate on a rotary mixer, shake and mix at room temperature, 1000 rpm for 1 min, apply the sealing film, place in a 37°C constant temperature incubator, and incubate for 90 min; place the 96-well plate on a magnetic plate, let it stand for 1 min, and then shake off the liquid in the wells.

2. Remove the 96-well plate from the magnetic plate, add 120 μl of washing solution to each well of the reaction plate, place the 96-well plate on a rotary mixer, shake and mix at room temperature and 1000 rpm for 1 minute; remove the reaction plate from the rotary mixer, place it on the magnetic plate, let it stand for 1 minute, and then discard the liquid in the wells. Repeat this step to wash the microspheres twice.

(III) Incubation of biotinylated detection antibody

1. Add the biotinylated detection antibody working solution to each well, place the 96-well plate on a rotating mixer, shake and mix at 1000 rpm for 1 min at room temperature, apply the sealing film, place in a 37°C constant temperature incubator, and incubate for 60 min; place the 96-well plate on a magnetic plate, let it stand for 1 min, and then shake off the liquid in the well.

2. Remove the 96-well plate from the magnetic plate, add 120 μl of washing solution to each well of the reaction plate, place the 96-well plate on a rotary mixer, shake and mix at room temperature and 1000 rpm for 1 minute; remove the reaction plate from the rotary mixer, place it on the magnetic plate, let it stand for 1 minute, and then discard the liquid in the wells. Repeat this step to wash the microspheres twice.

(IV) Incubation of Phycoerythrin

1. Add phycoerythrin working solution to each well, place the 96-well plate on a rotary mixer, shake and mix at 1000 rpm for 1 min at room temperature, apply a sealing film, place in a 37°C constant temperature incubator, and incubate for 15 min;

2. Remove the 96-well plate from the magnetic plate, add 120 μl of washing solution to each well of the reaction plate, place the 96-well plate on a rotary mixer, shake and mix at room temperature and 1000 rpm for 1 minute; remove the reaction plate from the rotary mixer, place it on the magnetic plate, let it stand for 1 minute, and then discard the liquid in the wells. Repeat this step to wash the microspheres twice.

(V) Detection of serum specific antigen protein expression level

The serum specific antigen protein content was detected using the American Luminex 200 liquid phase chip system (Luminex Company), and the concentration of each indicator in the serum was reflected by the median fluorescent intensity (MFI). After incubation with the secondary antibody, 100 μl of analysis buffer was added to each well, placed in a rotating mixer at 1000 rpm for 3 minutes, and immediately put on the machine to read the plate.

3 Statistical analysis

Graph Pad 8 and SPSS 25 software were used for statistical analysis. Receiver operating characteristic (ROC) curve was used to analyze the best cut-off value for clinical diagnosis, as well as the corresponding sensitivity and specificity.

Example 6 Analysis of the expression levels of the eight biomarkers of the present invention in samples from different groups

According to the flow immunofluorescence detection method of the microspheres in Example 5, CCL18 antigen, SPINT1 antigen, YKL-40 antigen, C1D autoantibody, FXR1 autoantibody, ZNF573 autoantibody, ESO1 autoantibody, and p53 autoantibody in serum samples were detected. The serum samples consisted of: (1) 64 ovarian cancer positive sera, including 28 cases of stage I, 36 cases of stage II to IV, including 30 cases of serous carcinoma, 16 cases of mucinous carcinoma, and 18 cases of ovarian germ cell tumor; (2) 76 healthy women; 61 cases of gynecological benign tumors (hereinafter referred to as benign group); 53 cases of interfering malignant tumors (hereinafter referred to as interfering malignant), including 23 cases of cervical cancer, 16 cases of colorectal cancer, and 14 cases of gastric cancer. The clinical information of the samples is shown in Table 1 below:

Table 1 Clinical information of serum samples

The expression levels of CCL18 antigen, SPINT1 antigen, YKL-40 antigen, C1D IgG autoantibody, FXR1 IgG autoantibody, ZNF573 IgG autoantibody, ESO1 IgG autoantibody, and p53 IgG autoantibody in different populations are shown in Figures 5 and 6; non-parametric tests were performed between each group of samples, and the results showed that the expression levels of each marker described in the present application in ovarian cancer were significantly higher than those in other control groups, and it has very good differential diagnostic performance for ovarian cancer. The specific data are as follows:

1) CCL18 antigen: compared with ovarian cancer and other groups, p was < 0.0001, with extremely significant differences; compared with the interference malignant group and the healthy group, p was = 0.0017, with extremely significant differences; there were no significant differences among the other groups (p > 0.05).

2) SPINT1 antigen: The difference between ovarian cancer and other groups was extremely significant (p<0.0001); there was no significant difference between the other groups (p>0.05).

3) YKL-40 antigen: The difference between ovarian cancer and other groups was extremely significant (p＜0.0001); there was no significant difference between the other groups (p＞0.05).

4) C1D IgG autoantibodies: ovarian cancer compared with other groups p < 0.0001, the difference is extremely significant; the other groups There was no significant difference between the two groups (p＞0.05).

5) FXR1 IgG autoantibody: compared with the other groups, p of ovarian cancer was less than 0.0001, which was a very significant difference; compared with the interference group and the healthy group, p was ＝0.0292, which was a significant difference; there was no significant difference among the other groups (p＞0.05).

6) ZNF573 IgG autoantibody: compared with other groups, the p values of ovarian cancer were all < 0.0001, which was a very significant difference; there was no significant difference between the other groups (p > 0.05).

7) ESO1 IgG autoantibody: compared with other groups, the p value of ovarian cancer was less than 0.0001, which was a very significant difference; there was no significant difference between the other groups (p>0.05).

8) p53 IgG autoantibody: compared with other groups, the p value of ovarian cancer was less than 0.0001, which was a very significant difference; there was no significant difference between the other groups (p>0.05).

Example 7 Diagnostic performance of a diagnostic kit containing eight items including CCL18 antibody, SPINT1 antibody, YKL-40 antibody, C1D, FXR1, ZNF573, ESO1, and p53 for ovarian cancer

The contents of the eight indicators in the serum samples of Example 6 above were used to establish a combined Logistics regression model of the eight indicators. The ROC curves of ovarian cancer versus healthy group, ovarian cancer versus benign group, and ovarian cancer versus interfering malignant group are shown in Figures 7 to 9. The area under the ROC curve of ovarian cancer for the healthy group is AUC = 0.953, the maximum Youden index is 0.824, the accuracy is 91.2%, the specificity is 90.2%, and the sensitivity is 92.2%, of which the sensitivity of stage I is 92.9%, and the sensitivity of stage II to IV is 91.7%. The area under the ROC curve of ovarian cancer for the benign group is AUC = 0.927, the maximum Youden index is 0.746, the accuracy is 87.2%, the specificity is 90.2%, and the sensitivity is 84.4%, of which the sensitivity of stage I is 82.1%, and the sensitivity of stage II to IV is 86.1%. The area under the ROC curve of ovarian cancer for the malignant group is AUC = 0.893, the maximum Youden index is 0.652, the accuracy is 82.4%, the specificity is 90.2%, and the sensitivity is 75%, of which the sensitivity of stage I is 75%, and the sensitivity of stage II to IV is 75%. The detailed diagnostic performance of ovarian cancer for each group of samples is shown in Table 2 below:

Table 2 Detailed diagnostic performance of ovarian cancer for each group of samples

Note: The accuracy rate is: (number of samples with correct positive judgment + number of samples with correct negative judgment) / (total number of negative samples + total number of positive samples) × 100%

The above results show that the combination of the eight markers of the present invention has excellent diagnostic performance for ovarian cancer. The specificity in the healthy group, benign group, and interference malignant group is greater than 90%, and the specificity for early (stage I) and mid- and late-stage There was no significant difference in the detection rate of ovarian cancer in the first stage (II to IV) (p>0.05). In the malignant interference group, it had a specificity of 90.2%. It had very high specificity for non-ovarian malignant tumors such as cervical cancer, gastric cancer, and colorectal cancer. That is, the marker combination described in the present invention can only specifically differentiate and diagnose ovarian cancer, thus greatly reducing the clinical misdiagnosis rate of non-ovarian malignant tumors. The combined system disclosed in CN106248940B has a high diagnostic efficiency for ovarian cancer, breast cancer, liver cancer, and lung cancer, and it may have a high misdiagnosis rate for non-ovarian malignant tumors in clinical applications. Therefore, the biomarker combination described in the present invention is much better than the existing marker combination in terms of technical effect.

Example 8 Comparison of the diagnostic performance of the eight biomarkers combined with CA125 alone for ovarian cancer

The eight biomarkers described in Example 6 of the present invention were used to perform a combined detection on 75 cases of ovarian cancer (38 cases of serous, 14 cases of mucinous, 23 cases of germ cell tumor; 34 cases of stage I, 41 cases of stage II to IV), and 74 controls (including 38 healthy women; 36 benign cases: 10 cases of uterine fibroids, 6 cases of fallopian tube cysts, 11 cases of ovarian cysts, and 9 cases of teratomas) according to the flow immunofluorescence detection method of the microspheres in Example 5, and the CA125 values of these samples were detected at the same time. Results: The area under the ROC curve of the combined 8 markers of the present invention was AUC = 0.932, the maximum Youden index = 0.772, the sensitivity was 85.3%, the specificity was 91.9%, and in the classification of various tissues of ovarian cancer, the sensitivity of serous was 86.8%, the sensitivity of mucinous was 85.7%, the sensitivity of germ cell tumor was 82.6%, the sensitivity of stage I was 82.4%, and the sensitivity of stage II to IV was 87.8%; the specificity of healthy was 92.1%, and the specificity of benign was 91.7%. CA125 has an area under the ROC curve of AUC = 0.75, a maximum Youden index of 0.465, a sensitivity of 62.7%, a specificity of 83.8%, and in the classification of various tissues of ovarian cancer, the sensitivity of serous is 63.2%, the sensitivity of mucinous is 64.3%, and the sensitivity of germ cell tumor is 60.9%; the sensitivity of stage I is 61.8%, and the sensitivity of stage II to IV is 63.4%; the specificity of healthy is 92.1%, and the specificity of benign is 75%. The diagnostic performance of the combined detection of the eight markers of the present invention and the detection of CA125 alone is shown in Table 3 or Figures 10 and 11 below:

Table 3 Diagnostic performance of the combined detection of 8 markers and CA125 alone

From the above results, it can be seen that the sensitivity of CA125 alone is 62.7%, the specificity is 83.8%, and the diagnostic performance is low. Among them, the specificity of CA125 in the healthy group is 92.1%, but in the benign group, the specificity drops to 75%, indicating that CA125 has a high misdiagnosis rate for benign gynecological lesions and is only suitable for screening healthy women. The combined detection of the eight markers of the present invention is The sensitivity of ovarian cancer was 85.3% and the specificity was 91.9%, of which the specificity was 92.1% in the healthy group and 91.7% in the benign group. There was no significant difference in specificity between the healthy group and the benign group. The combined system disclosed in CN106248940B can only detect epithelial cancer (serous and mucinous), and the effect of diagnosing ovarian germ cell tumors is unknown. The biomarker combination of the present invention has a sensitivity of 82.6% to ovarian germ cell tumors, which is not significantly different from the sensitivity of epithelial cancer (86.5%) (p>0.05). In summary, the biomarker combination of the present invention shows excellent differential diagnosis performance for ovarian cancer. It is included in the early diagnosis of ovarian malignant tumors such as germ cell tumors in clinical practice, which helps to cover the entire population of ovarian cancer and greatly reduce the misdiagnosis rate and missed diagnosis rate of ovarian cancer patients in clinical practice.

Example 9 The diagnostic effect of the combination of CCL18, CA125, C1D autoantibody, FXR1 autoantibody, ZNF573 autoantibody, ESO1 autoantibody, p53 autoantibody, KRAS autoantibody, IGFBP1 autoantibody, FUBP1 autoantibody, TM4SF1 autoantibody, and ARP3 autoantibody on ovarian cancer

The CCL18 antibody, CA125 antibody, C1D, FXR1, ZNF573, ESO1, p53, KRAS, IGFBP1, FUBP1, FXR1, and TM4SF1 of the present invention were used to treat 316 cases of ovarian cancer (202 cases of high-grade serous carcinoma, 34 cases of endometrial carcinoma, 38 cases of clear cell carcinoma, 19 cases of mucinous carcinoma, 12 cases of low-grade serous carcinoma, and 11 cases of mixed carcinoma; 97 cases in stage I, 219 cases in stages II to IV), 281 cases of benign ovarian cancer (including 42 cases of uterine fibroids; 3 6 cases of mature teratoma; 157 cases of ovarian cysts; 46 cases of fallopian tube cysts) were jointly detected for CCL18, CA125, C1D autoantibody, FXR1 autoantibody, ZNF573 autoantibody, ESO1 autoantibody, p53 autoantibody, KRAS autoantibody, IGFBP1 autoantibody, FUBP1 autoantibody, FXR1 autoantibody, and TM4SF1 autoantibody according to the flow immunofluorescence detection method of the microspheres in Example 5, and the clinical information of the detected samples is shown in Table 4 below.

Table 4 Clinical information of samples tested

The ROC curves of the 12 markers of the present invention for ovarian cancer diagnosis are shown in FIG12 .

The diagnostic performance of each marker at 90% specificity is shown in Table 5 below.

Table 5 Diagnostic performance of each marker for ovarian cancer

As shown in Table 5 above, at a specificity of 90%, the sensitivity of each marker to ovarian cancer is between 32.3% and 51.6%, among which CCL18, CA125, p53 autoantibody, and ESO1 autoantibody have the highest diagnostic performance among the 12 markers.

IBM SPSS25.0 software was used to establish a logistics regression model for the joint determination of 11 markers, including CCL18 antigen, C1D autoantibody, FXR1 autoantibody, ZNF573 autoantibody, ESO1 autoantibody, p53 autoantibody, KRAS autoantibody, IGFBP1 autoantibody, FUBP1 autoantibody, FXR1 autoantibody, and TM4SF1 autoantibody (excluding CA125 antigen). The ROC curve of the 11-marker model is shown in Figure 13, AUC = 0.946, maximum Youden index = 0.817, accuracy 91.1%, specificity 88.3%, sensitivity 93.7%, and sensitivity in various tissue classifications of 316 cases of ovarian cancer: high-grade serous carcinoma 95.5%, endometrial carcinoma 94.1%, clear cell carcinoma 94.7%, mucinous carcinoma 89.5%, low-grade serous carcinoma 91.7%, mixed carcinoma 90.9%, stage I sensitivity 91.8%, and stage II to IV sensitivity 94.5%.

Furthermore, CA125 antigen was included on the basis of the above 11 markers, and a Logistics regression model for the joint determination of 12 markers including CA125 antigen was established using IBM SPSS25.0 software. The ROC curve of the 12-marker model is shown in Figure 14, AUC = 0.982, maximum Youden index = 0.896, accuracy = 94.8%, sensitivity = 94.6%, specificity = 95%, and the sensitivity in various tissue classifications of 316 cases of ovarian cancer: high-grade serous carcinoma 94.1%, endometrial carcinoma 94.1%, clear cell carcinoma 92.1%, mucinous carcinoma 94.7%, low-grade serous carcinoma 91.7%, mixed carcinoma 90.9%, stage I sensitivity 93.8%, stage II to IV sensitivity 95%.

The diagnostic efficacy of the 12-marker combination formed by incorporating CA125 antigen is better than that of the 11-marker combination. The difference in diagnostic performance between the two is shown in Table 6 below:

Table 6 Diagnostic performance of 11 or 12 markers combined

The Omnibus Tests of Model Coefficients of the 12 markers established was P < 0.001, and the Hosmer-Lemeshow Test was significant = 0.256 > 0.05, indicating that the model can fit the observed data well. The model formula of the 12 markers is as follows:

P = EXP[Logit(p)]/[1+EXP(Logit(p)]; Cut off = 0.6011. When the predicted probability of cancer P ≥ Cut off, it is judged as positive; when P < Cut off, it is judged as negative.

Wherein, Logit(p)＝0.008576*CCL18+0.002106*CA125+0.001736*C1D+0.000499*FXR1-0.001239*ZNF573+0.000357*ESO1+0.000126*p53-0.000229*KRAS+0.001062*IGFBP1+0.001421*FUBP1+0.000556*TM4SF1-0.000401*ARP3-3.363822,

in:

(1) CCL18 is the CCL18 antigen content measured in serum;

(2) CA125 is the CA125 antigen content measured in serum;

(3) C1D is the C1D autoantibody content measured in serum;

(4) FXR1 is the level of FXR1 autoantibodies measured in serum;

(5) ZNF573 is the level of ZNF573 autoantibodies measured in serum;

(6) ESO1 is the ESO1 autoantibody content measured in serum;

(7) p53 is the p53 autoantibody content measured in serum;

(8) KRAS is the KRAS autoantibody content measured in serum;

(9) IGFBP1 is the IGFBP1 autoantibody content measured in serum;

(10) FUBP1 is the FUBP1 autoantibody content measured in serum;

(11) TM4SF1 is the TM4SF1 autoantibody content measured in serum;

(12) ARP3 is the ARP3 autoantibody content measured in serum.

Example 10 Precision evaluation of the kit prepared by the present invention for detecting CCL18, CA125, C1D autoantibodies, FXR1 autoantibodies, ZNF573 autoantibodies, ESO1 autoantibodies, p53 autoantibodies, KRAS autoantibodies, IGFBP1 autoantibodies, FUBP1 autoantibodies, TM4SF1 autoantibodies, and ARP3 autoantibodies in serum samples

According to the method in Example 5, a flow immunofluorescence kit for detecting CCL18, CA125, C1D autoantibodies, FXR1 autoantibodies, ZNF573 autoantibodies, ESO1 autoantibodies, p53 autoantibodies, KRAS autoantibodies, IGFBP1 autoantibodies, FUBP1 autoantibodies, TM4SF1 autoantibodies, and ARP3 autoantibodies was prepared, and one serum sample with high, medium, and low values was tested (according to the flow immunofluorescence detection method of the microspheres in Example 5), and each test sample was tested 10 times, and the coefficient of variation of the results of 10 repeated tests of each sample was calculated to evaluate the precision of the kit detection. The serum sample data detected are shown in Table 7 below, and the test results are shown in Table 8 below.

Table 7 Precision evaluation sample information

Table 8 Precision evaluation sample test results (unit: U/mL)

Continued from the table above

The average value (AV), standard deviation (SD) and coefficient of variation (CV) of 10 replicates of each sample were calculated, and the results are shown in Table 9 below:

Table 9 Precision evaluation of the mean value (AV), standard deviation (SD) and coefficient of variation (CV) of each sample

From the above results, it can be seen that for the detection of high-value samples, the coefficient of variation CV of the detection results of the 12 markers is between 4.07% and 6.29%; for the detection of median samples, the coefficient of variation CV of the detection results of the 12 markers is between 4.37% and 6.45%; for the detection of low-value samples, the coefficient of variation CV of the detection results of the 12 markers is between 2.94% and 5.97%; the coefficient of variation CV of the detection results of the three samples is all <10%, indicating that the flow immunofluorescence kit prepared by the present invention has good precision for sample detection.

Example 11 Evaluation of the stability of the kit prepared by the present invention for detecting CCL18, CA125, C1D autoantibodies, FXR1 autoantibodies, ZNF573 autoantibodies, ESO1 autoantibodies, p53 autoantibodies, KRAS autoantibodies, IGFBP1 autoantibodies, FUBP1 autoantibodies, TM4SF1 autoantibodies, and ARP3 autoantibodies in serum samples

According to the method in Example 5, a flow immunofluorescence kit for detecting CCL18, CA125, C1D autoantibodies, FXR1 autoantibodies, ZNF573 autoantibodies, ESO1 autoantibodies, p53 autoantibodies, KRAS autoantibodies, IGFBP1 autoantibodies, FUBP1 autoantibodies, TM4SF1 autoantibodies, and ARP3 autoantibodies was prepared, and the kit was placed in a 37°C incubator for accelerated aging test, and taken out on the 0th day, 3rd day, 5th day, and 7th day of placement, and one serum sample of high, medium, and low values was tested respectively (according to the flow immunofluorescence detection method of the microspheres in Example 5), and each sample was repeated, and the average value (AV) and standard deviation (SD) of each marker detected by each sample on the 0th day, the 3rd day, the 5th day, and the 7th day were calculated, and the coefficient of variation (CV) was calculated to evaluate the stability of the kit. The serum samples tested were consistent with those in Example 10, and the test results are shown in Table 10 below.

Table 10 37℃ accelerated aging test sample test results (unit: U/mL)

Continued from the table above

The average values of two replicate wells of each marker of each sample at each accelerated day were calculated, and the results are shown in Table 11 below:

Table 11 Average values of samples tested in 37℃ accelerated aging test (unit: U/mL)

Continued from the table above

Taking the sample test results on day 0 as the control, the relative deviations of the accelerated test results on days 3, 5, and 7 were compared with the control test results (relative deviation = (experimental group - control data) / (experimental group + control group) / 2 × 100%). The results are shown in Table 12 below:

Table 12 Relative deviations of the results of the 37℃ accelerated aging test on each day and the control test on day 0

From the results in the above table, we can see that for high-value samples: the relative deviation of each marker between the 3rd day of acceleration and the 0th day is between -0.92% and -7.83%; the relative deviation of each marker between the 5th day of acceleration and the 0th day is between -3.86% and -9.73%; the relative deviation of each marker between the 7th day of acceleration and the 0th day is between -8.02% and -13.23%. For the median sample: the relative deviation of each marker between the 3rd day of acceleration and the 0th day is between -0.13% and -9.16%; the relative deviation of each marker between the 5th day of acceleration and the 0th day is between -4.34% and -10.87%; the relative deviation of each marker between the 7th day of acceleration and the 0th day is between -8.62% and -13.20%. For low-value samples: the relative deviation of each marker between the 3rd day and the 0th day is between -1.04% and -7.11%; the relative deviation of each marker between the 5th day and the 0th day is between -3.22% and -8.95%; the relative deviation of each marker between the 7th day and the 0th day is between -6.18% and -11.34%. In summary, the flow immunofluorescence kit prepared by the present invention can still maintain stable test results after being placed at 37°C for 7 days, and the relative deviation is <±20%, indicating that the flow immunofluorescence reagent prepared by the present invention has high stability. According to the general understanding in the industry, 7 days at 37°C is equivalent to 1 year at 2-8°C, so it is shown that the flow immunofluorescence kit prepared by the present invention can be stably stored at 2-8°C for 1 year.

Claims (10)

A biomarker combination, characterized in that the biomarker combination comprises CCL18, C1D, FXR1, ZNF573, ESO1 and p53 antigens or autoantibodies binding thereto; and the biomarker further comprises one or more antigens or autoantibodies binding thereto selected from: CA125, TM4SF1, IGFBP1, FUBP1, ARP3, KRAS, SPINT1 and YKL-40. The biomarker combination according to claim 1, characterized in that the biomarker combination satisfies the following condition (1) or condition (2): (1) The biomarker combination comprises CCL18, C1D, FXR1, ZNF573, ESO1, p53 and TM4SF1 antigens or autoantibodies binding thereto; preferably, the biomarker combination further comprises IGFBP1 and/or FUBP1 antigens or autoantibodies binding thereto; more preferably, the biomarker combination further comprises ARP3 and/or KRAS antigens or autoantibodies binding thereto; further more preferably, the biomarker combination further comprises one or more antigens selected from: SPINT1, YKL-40 and CA125 or autoantibodies binding thereto; (2) The biomarker combination comprises CCL18, C1D, FXR1, p53, SPINT1, ZNF573, YKL-40 and ESO1 antigens or autoantibodies binding thereto; preferably, the biomarker combination further comprises one or more antigens selected from: CA125, KRAS, ARP3, IGFBP1, FUBP1 and TM4SF1 or autoantibodies binding thereto. The biomarker combination according to claim 1 or 2, characterized in that the biomarker combination is a biomarker combination in serum, which is selected from any one of the following groups: 1) ESO1 autoantibodies, p53 autoantibodies, FXR1 autoantibodies, ZNF573 autoantibodies, TM4SF1 autoantibodies, C1D autoantibodies, and CCL18; 2) ESO1 autoantibodies, p53 autoantibodies, FXR1 autoantibodies, ZNF573 autoantibodies, TM4SF1 autoantibodies, C1D autoantibodies, CCL18, and IGFBP1 autoantibodies; 3) ESO1 autoantibodies, p53 autoantibodies, FXR1 autoantibodies, ZNF573 autoantibodies, TM4SF1 autoantibodies, C1D autoantibodies, CCL18, and FUBP1 autoantibodies; 4) ESO1 autoantibody, p53 autoantibody, FXR1 autoantibody, ZNF573 autoantibody, TM4SF1 autoantibody, C1D autoantibody, CCL18, IGFBP1 autoantibody, and FUBP1 autoantibody; 5) ESO1 autoantibody, p53 autoantibody, FXR1 autoantibody, ZNF573 autoantibody, TM4SF1 autoantibody, C1D autoantibody, CCL18, ARP3 autoantibody, IGFBP1 autoantibody and FUBP1 autoantibody; 6) ESO1 autoantibodies, p53 autoantibodies, FXR1 autoantibodies, ZNF573 autoantibodies, TM4SF1 autoantibodies autoantibodies to C1D, CCL18, KRAS, IGFBP1, and FUBP1; 7) ESO1 autoantibody, p53 autoantibody, FXR1 autoantibody, ZNF573 autoantibody, TM4SF1 autoantibody, C1D autoantibody, CCL18, ARP3 autoantibody, KRAS autoantibody, IGFBP1 autoantibody and FUBP1 autoantibody; 8) ESO1 autoantibodies, p53 autoantibodies, FXR1 autoantibodies, ZNF573 autoantibodies, TM4SF1 autoantibodies, C1D autoantibodies, CCL18 and ARP3 autoantibodies; 9) ESO1 autoantibodies, p53 autoantibodies, FXR1 autoantibodies, ZNF573 autoantibodies, TM4SF1 autoantibodies, C1D autoantibodies, CCL18 and KRAS autoantibodies; 10) ESO1 autoantibodies, p53 autoantibodies, FXR1 autoantibodies, ZNF573 autoantibodies, TM4SF1 autoantibodies, C1D autoantibodies, CCL18, ARP3 autoantibodies, and KRAS autoantibodies; 11) ESO1 autoantibody, p53 autoantibody, FXR1 autoantibody, ZNF573 autoantibody, TM4SF1 autoantibody, C1D autoantibody, CCL18, ARP3 autoantibody and IGFBP1 autoantibody; 12) ESO1 autoantibody, p53 autoantibody, FXR1 autoantibody, ZNF573 autoantibody, TM4SF1 autoantibody, C1D autoantibody, CCL18, ARP3 autoantibody and FUBP1 autoantibody; 13) ESO1 autoantibody, p53 autoantibody, FXR1 autoantibody, ZNF573 autoantibody, TM4SF1 autoantibody, C1D autoantibody, CCL18, KRAS autoantibody and IGFBP1 autoantibody; 14) ESO1 autoantibody, p53 autoantibody, FXR1 autoantibody, ZNF573 autoantibody, TM4SF1 autoantibody, C1D autoantibody, CCL18, KRAS autoantibody and FUBP1 autoantibody; 15) ESO1 autoantibody, p53 autoantibody, FXR1 autoantibody, ZNF573 autoantibody, TM4SF1 autoantibody, C1D autoantibody, CCL18, ARP3 autoantibody, KRAS autoantibody, and IGFBP1 autoantibody; 16) ESO1 autoantibody, p53 autoantibody, FXR1 autoantibody, ZNF573 autoantibody, TM4SF1 autoantibody, C1D autoantibody, CCL18, ARP3 autoantibody, KRAS autoantibody, and FUBP1 autoantibody; 17) ESO1 autoantibody, p53 autoantibody, FXR1 autoantibody, ZNF573 autoantibody, TM4SF1 autoantibody, C1D autoantibody, CCL18 and SPINT1; 18) CCL18, SPINT1, C1D autoantibodies, FXR1 autoantibodies, ZNF573 autoantibodies, ESO1 autoantibodies, and p53 autoantibodies; 19) CCL18, SPINT1, YKL-40, C1D autoantibody, FXR1 autoantibody, ZNF573 autoantibody, ESO1 autoantibody and p53 autoantibody; 20) CCL18, SPINT1, YKL-40, C1D autoantibodies, FXR1 autoantibodies, ZNF573 autoantibodies autoantibodies, ESO1 autoantibodies, p53 autoantibodies, and KRAS autoantibodies; 21) CCL18, SPINT1, YKL-40, C1D autoantibody, FXR1 autoantibody, ZNF573 autoantibody, ESO1 autoantibody, p53 autoantibody, KRAS autoantibody, and TM4SF1 autoantibody; 22) CCL18, SPINT1, YKL-40, C1D autoantibody, FXR1 autoantibody, ZNF573 autoantibody, ESO1 autoantibody, p53 autoantibody, KRAS autoantibody, TM4SF1 autoantibody, and ARP3 autoantibody; and, 23) ESO1 autoantibody, p53 autoantibody, FXR1 autoantibody, ZNF573 autoantibody, TM4SF1 autoantibody, C1D autoantibody, CCL18, ARP3 autoantibody, KRAS autoantibody, IGFBP1 autoantibody, FUBP1 autoantibody, SPINT1, and YKL-40; Preferably, any one of the biomarker combinations 1) to 23) further comprises CA125. A diagnostic kit for early ovarian cancer diagnosis or prognosis thereof, characterized in that the diagnostic kit comprises the biomarker combination according to any one of claims 1 to 3; Preferably, the biomarker in the biomarker combination is an antigen or an antibody; and/or, the diagnostic kit further comprises a reagent for detecting the biomarker, such as a detection antibody for antigen detection, or a detection antigen for antibody detection; More preferably, the antigen or antibody is from a serum sample; Further preferably, the reagent for detecting the biomarker combination is selected from any one of the following groups: 1) Antibodies to ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, and CCL18; 2) Antibodies against ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, CCL18, and IGFBP1; 3) Antibodies against ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, CCL18, and FUBP1; 4) Antibodies against ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, CCL18, IGFBP1, and FUBP1; 5) Antibodies against ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, CCL18, ARP3, IGFBP1, and FUBP1; 6) ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, CCL18 antibodies, KRAS, IGFBP1, and FUBP1; 7) ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, CCL18 antibodies, ARP3, KRAS, IGFBP1, and FUBP1; 8) Antibodies against ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, CCL18, and ARP3; 9) Antibodies against ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, CCL18, and KRAS; 10) Antibodies against ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, CCL18, ARP3, and KRAS; 11) Antibodies against ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, CCL18, ARP3, and IGFBP1; 12) Antibodies against ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, CCL18, ARP3, and FUBP1; 13) Antibodies against ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, CCL18, KRAS, and IGFBP1; 14) ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, CCL18 antibodies, KRAS, and FUBP1; 15) ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, CCL18 antibodies, ARP3, KRAS, and IGFBP1; 16) ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, CCL18 antibodies, ARP3, KRAS, and FUBP1; 17) Antibodies against ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, CCL18, and SPINT1; 18) Antibodies against CCL18, SPINT1, C1D, FXR1, ZNF573, ESO1, and p53; 19) Antibodies against CCL18, SPINT1, YKL-40, C1D, FXR1, ZNF573, ESO1, and p53; 20) Antibodies against CCL18, SPINT1, YKL-40, C1D, FXR1, ZNF573, ESO1, p53, and KRAS; 21) Antibodies against CCL18, SPINT1, YKL-40, C1D, FXR1, ZNF573, ESO1, p53, KRAS, and TM4SF1; 22) CCL18 antibody, SPINT1 antibody, YKL-40 antibody, C1D, FXR1, ZNF573, ESO1, p53, KRAS, TM4SF1 and ARP3; and, 23) ESO1, p53, FXR1, ZNF573, TM4SF1, C1D, CCL18 antibodies, ARP3, KRAS, IGFBP1, FUBP1, SPINT1 antibodies and YKL-40 antibodies; Preferably, any one of the groups 1) to 23) of reagents for detecting the biomarker combination further comprises a CA125 antibody. The diagnostic kit according to claim 4, characterized in that in the biomarker combination, the biomarker, detection antigen or detection antibody further contains a tag peptide; Preferably, the tag peptide includes one or more of: His tag, streptavidin tag, avidin tag, biotin tag, GST tag, C-myc tag, Flag tag and HA tag; More preferably, the biomarker, detection antigen or detection antibody can be expressed by Escherichia coli, yeast, insect cells or animal cells; and/or, the biomarker, detection antigen or detection antibody is purified by Ni affinity chromatography, ion exchange chromatography, molecular sieve, dialysis, ultrafiltration or hydrophobic chromatography; Further preferably, the diagnostic kit also includes phycoerythrin and its fragments, phycoerythrin-labeled anti-human immunoglobulin G and its fragments, biotinylated antibodies or biotinylated antigens against protein molecules in human serum, and one or more of a dilution buffer, a washing solution and an analysis buffer; preferably, the biomarker is fixed on a solid phase carrier; more preferably, the solid phase carrier includes: microspheres, ELISA plate microwells, affinity membranes or liquid phase chips. The diagnostic kit according to claim 5, characterized in that the biomarker, detection antigen or detection The detection antibody is directly or indirectly coupled to the microsphere, and the label peptide is 6×His tag and/or C-Myc tag; Preferably, the direct coupling is: microsphere-NH2-(C-Myctag)-Linker-(His6tag)-Linker-biomarker, detection antigen or detection antibody amino acid sequence; or microsphere-NH2-(C-Myctag)-Linker-biomarker, detection antigen or detection antibody amino acid sequence-(His6tag); the Linker is, for example, (G ₄ S) _n , where n is an integer of 1 to 5; The indirect coupling step comprises: (1) The microspheres are coupled to biotinylated bovine serum albumin (BSA-biotin), and the biomarker, detection antigen, or detection antibody is bound to streptavidin; (2) Biotin binds to streptavidin, thereby allowing the biomarker, detection antigen or detection antibody to specifically bind to the microspheres; More preferably, the dilution buffer is NaCl 300mM, KCl 2.7mM, Na ₂ HPO ₄ 8.1mM, KH ₂ PO ₄ 1.5mM, 5% donkey serum or 5w/v% BSA, 0.05% prcolin300 or 0.05w/v% sodium azide, pH 7.0-8.0; the washing solution is NaCl 137mM, KCl 2.7mM, Na ₂ HPO ₄ 8.1mM, KH ₂ PO ₄ 1.5mM, 0.05% Tween-20, 0.03% prcolin300, pH 7.6; the analysis buffer is NaCl 137mM, KCl 2.7mM, Na ₂ HPO ₄ 8.1mM, KH ₂ PO ₄ 1.5 mM, 1% donkey serum or 1 w/v% BSA, 0.05% prcolin300 or 0.05 w/v% sodium azide; wherein the % is volume percentage, and the w/v% is mass volume ratio. A use of the biomarker combination according to any one of claims 1 to 3 or the diagnostic kit according to any one of claims 4 to 6 in the preparation of a reagent and/or a kit for detecting a specific protein or antibody molecule in human serum; Preferably, the reagent and/or kit is used for diagnosing or assisting in the diagnosis of early ovarian cancer, screening high-risk populations for ovarian cancer, assisting in the early diagnosis of benign or malignant ovarian tumors, or for recurrence monitoring and/or prognosis monitoring after ovarian cancer treatment. An ovarian cancer diagnosis system, characterized in that the ovarian cancer diagnosis system comprises the following modules: (1) an input module, which is used to input the detection value of the biomarker combination according to any one of claims 1 to 3 contained in the sample to be tested; (2) Analysis module, which is used to calculate p, where
X＝0.008576*CCL18+0.002106*CA125+0.001736*C1D+0.000499*FXR1-0.001239*ZN F573+0.000357*ESO1+0.000126*p53-0.000229*KRAS+0.001062*IGFBP1+0.001421*FUB P1+0.000556*TM4SF1-0.000401* ARP3-3.363822; (3) Judgment module, cutoff is 0.6011; when p≥cutoff, it is judged as positive, and when p＜cutoff, it is judged as negative; Preferably, the ovarian cancer diagnosis system further comprises a login module, and/or the ovarian cancer diagnosis system further comprises a print module. Print module; the login module requires input of a user name and a user password, and the printing module can print the results generated by the input module, the analysis module and the judgment module; More preferably, the sample to be tested is serum. A computer-readable medium, characterized in that the computer-readable medium stores a computer program, and when the computer program is executed by a processor, it can realize the functions of the ovarian cancer diagnosis system as claimed in claim 8. An ovarian cancer diagnostic device, characterized in that the ovarian cancer diagnostic device comprises: (1) The computer-readable medium of claim 9; (2) A processor, configured to execute a computer program to implement the functions of the ovarian cancer diagnosis system.