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WO2024208185A1 - Combinaison de biomarqueurs, kit de diagnostic la comprenant et son utilisation - Google Patents

Combinaison de biomarqueurs, kit de diagnostic la comprenant et son utilisation Download PDF

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Publication number
WO2024208185A1
WO2024208185A1 PCT/CN2024/085479 CN2024085479W WO2024208185A1 WO 2024208185 A1 WO2024208185 A1 WO 2024208185A1 CN 2024085479 W CN2024085479 W CN 2024085479W WO 2024208185 A1 WO2024208185 A1 WO 2024208185A1
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WIPO (PCT)
Prior art keywords
autoantibody
autoantibodies
eso1
znf573
fxr1
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PCT/CN2024/085479
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English (en)
Chinese (zh)
Inventor
胡海
赵峰
黄朝远
黄璐
林当
王增松
陈嘉雯
莫楚婵
周腊梅
李洪锋
秦伟闻
刘紫莹
王兴勇
廖丽影
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Guangzhou Bioblue Technology Co Ltd
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Guangzhou Bioblue Technology Co Ltd
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Publication of WO2024208185A1 publication Critical patent/WO2024208185A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57449—Specifically defined cancers of ovaries
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5302—Apparatus specially adapted for immunological test procedures

Definitions

  • 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.
  • 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%.
  • 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 used in clinical diagnosis of ovarian cancer refers to tumor marker detection.
  • Common tumor markers include CA125, HE4, etc.
  • 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 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.
  • 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.
  • 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.
  • the body's immune system can recognize tumor-specific antigens expressed by tumor cells and produce autoantibodies against these antigens.
  • 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.
  • autoantibodies 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.
  • 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.
  • the present invention provides a biomarker combination, a diagnostic kit containing the same and applications thereof.
  • 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.
  • 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.
  • the sample such as a serum sample
  • the prognosis of the patient 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.
  • the biomarker combination further comprises IGFBP1 and/or FUBP1 antigens or autoantibodies binding thereto.
  • the biomarker combination further comprises ARP3 and/or KRAS antigens or autoantibodies binding thereto.
  • 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.
  • 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.
  • 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.
  • 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:
  • ESO1 autoantibodies ESO1 autoantibodies, p53 autoantibodies, FXR1 autoantibodies, ZNF573 autoantibodies, TM4SF1 autoantibodies, C1D autoantibodies, and CCL18;
  • ESO1 autoantibodies p53 autoantibodies, FXR1 autoantibodies, ZNF573 autoantibodies, TM4SF1 autoantibodies, C1D autoantibodies, CCL18, and IGFBP1 autoantibodies;
  • ESO1 autoantibodies p53 autoantibodies, FXR1 autoantibodies, ZNF573 autoantibodies, TM4SF1 autoantibodies, C1D autoantibodies, CCL18, and FUBP1 autoantibodies;
  • ESO1 autoantibody ESO1 autoantibody, p53 autoantibody, FXR1 autoantibody, ZNF573 autoantibody, TM4SF1 autoantibody, C1D autoantibody, CCL18, IGFBP1 autoantibody, and FUBP1 autoantibody;
  • ESO1 autoantibody ESO1 autoantibody, p53 autoantibody, FXR1 autoantibody, ZNF573 autoantibody, TM4SF1 autoantibody, C1D autoantibody, CCL18, ARP3 autoantibody, IGFBP1 autoantibody and FUBP1 autoantibody;
  • ESO1 autoantibody ESO1 autoantibody, p53 autoantibody, FXR1 autoantibody, ZNF573 autoantibody, TM4SF1 autoantibody, C1D autoantibody, CCL18, KRAS autoantibody, IGFBP1 autoantibody and FUBP1 autoantibody;
  • ESO1 autoantibody p53 autoantibody, FXR1 autoantibody, ZNF573 autoantibody, TM4SF1 autoantibody, C1D autoantibody, CCL18, ARP3 autoantibody, KRAS autoantibody, IGFBP1 autoantibody and FUBP1 autoantibody;
  • ESO1 autoantibodies ESO1 autoantibodies, p53 autoantibodies, FXR1 autoantibodies, ZNF573 autoantibodies, TM4SF1 autoantibodies, C1D autoantibodies, CCL18 and ARP3 autoantibodies;
  • ESO1 autoantibodies p53 autoantibodies, FXR1 autoantibodies, ZNF573 autoantibodies, TM4SF1 autoantibodies, C1D autoantibodies, CCL18 and KRAS autoantibodies;
  • ESO1 autoantibodies p53 autoantibodies, FXR1 autoantibodies, ZNF573 autoantibodies, TM4SF1 autoantibodies, C1D autoantibodies, CCL18, ARP3 autoantibodies, and KRAS autoantibodies;
  • ESO1 autoantibody p53 autoantibody, FXR1 autoantibody, ZNF573 autoantibody, TM4SF1 autoantibody, C1D autoantibody, CCL18, ARP3 autoantibody and IGFBP1 autoantibody;
  • ESO1 autoantibody ESO1 autoantibody, p53 autoantibody, FXR1 autoantibody, ZNF573 autoantibody, TM4SF1 autoantibody, C1D autoantibody, CCL18, ARP3 autoantibody and FUBP1 autoantibody;
  • ESO1 autoantibody p53 autoantibody, FXR1 autoantibody, ZNF573 autoantibody, TM4SF1 autoantibody, C1D autoantibody, CCL18, KRAS autoantibody and IGFBP1 autoantibody;
  • ESO1 autoantibody ESO1 autoantibody, p53 autoantibody, FXR1 autoantibody, ZNF573 autoantibody, TM4SF1 autoantibody, C1D autoantibody, CCL18, KRAS autoantibody and FUBP1 autoantibody;
  • ESO1 autoantibody ESO1 autoantibody, p53 autoantibody, FXR1 autoantibody, ZNF573 autoantibody, TM4SF1 autoantibody, C1D autoantibody, CCL18, ARP3 autoantibody, KRAS autoantibody, and IGFBP1 autoantibody;
  • ESO1 autoantibody 16) ESO1 autoantibody, p53 autoantibody, FXR1 autoantibody, ZNF573 autoantibody, TM4SF1 autoantibody, C1D autoantibody, CCL18, ARP3 autoantibody, KRAS autoantibody, and FUBP1 autoantibody;
  • any one of the biomarker combinations 1) to 23) further comprises CA125.
  • 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.
  • 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.
  • a reagent for detecting the biomarker such as a detection antibody for antigen detection, or a detection antigen for antibody detection.
  • the antigen or antibody is from a serum sample.
  • the reagent for detecting the biomarker combination is selected from any one of the following groups:
  • CCL18 antibody CCL18 antibody, SPINT1 antibody, YKL-40 antibody, C1D, FXR1, ZNF573, ESO1, p53, KRAS, TM4SF1 and ARP3; and,
  • 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.
  • 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.
  • 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.
  • 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.
  • 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;
  • 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.
  • microspheres are coupled to biotinylated bovine serum albumin (BSA-biotin), and the biomarker, detection antigen, or detection antibody is bound to streptavidin;
  • BSA-biotin biotinylated bovine serum albumin
  • Biotin binds to streptavidin, thereby allowing the biomarker, detection antigen or detection antibody to specifically bind to the microspheres.
  • 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.
  • 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.
  • 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.
  • CV precision coefficient of variation
  • the fourth technical solution of the present invention is: an ovarian cancer diagnosis system, the ovarian cancer diagnosis system comprises the following modules:
  • 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;
  • CCL18 is the CCL18 antigen content measured in serum
  • CA125 is the CA125 antigen content measured in serum
  • C1D is the C1D autoantibody content measured in serum
  • FXR1 is the level of FXR1 autoantibodies measured in serum
  • ZNF573 is the level of ZNF573 autoantibodies measured in serum
  • ESO1 is the ESO1 autoantibody content measured in serum
  • p53 is the p53 autoantibody content measured in serum
  • KRAS is the level of KRAS autoantibodies measured in serum
  • IGFBP1 is the IGFBP1 autoantibody content measured in serum
  • FUBP1 is the FUBP1 autoantibody content measured in serum
  • TM4SF1 is the TM4SF1 autoantibody content measured in serum
  • ARP3 is the ARP3 autoantibody content measured in serum
  • p is the predicted probability of cancer
  • 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.
  • the sample to be tested is serum.
  • 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.
  • 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.
  • the sixth technical solution of the present invention is: an ovarian cancer diagnosis device, the ovarian cancer diagnosis device comprising:
  • a processor configured to execute a computer program to implement the functions of the ovarian cancer diagnosis system.
  • 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.
  • 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.
  • the reagents and raw materials used in the present invention are commercially available.
  • 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.
  • 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).
  • 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.
  • the combined system disclosed in the prior art 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).
  • 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.
  • 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.
  • PBS NaCl 137mM; KCl 2.7mM; Na 2 HPO 4 8.1mM; KH 2 PO 4 1.5mM; pH 7.6, filtered through 0.22 ⁇ m membrane;
  • 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 ⁇ m filter membrane.
  • 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 ⁇ m filter membrane;
  • 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.
  • the restriction enzyme digestion reaction system of PUC-57-ESO1 plasmid is as follows:
  • the cells were collected by centrifugation at 3000rpm for 5min, and about 200 ⁇ l 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°C 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:
  • the bacterial solution is centrifuged at 5000 rpm and the bacteria are collected at 15 min.
  • 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 ⁇ g); Lane PC2: BSA (2 ⁇ g); 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.
  • 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 ⁇ m 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.
  • 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.
  • washing buffer 50 mM Na 2 HPO 4 , 10 mM Tris ⁇ Cl , 10 mM imidazole , 8 M Urea , pH 8.0
  • 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.
  • elution buffer 50 mM Na 2 HPO 4 , 10 mM Tris ⁇ Cl, 250 mM imidazole, 8 M Urea, pH 8.0
  • 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.
  • 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 PUC-57-ZNF573 plasmid restriction enzyme digestion reaction system is as follows:
  • the pET-30a-ZNF573 ligase catalyzed reaction system is as follows:
  • the pET-30a-ZNF573 double enzyme digestion identification reaction system is as follows:
  • the bacterial solution is centrifuged at 5000 rpm and the bacteria are collected at 15 min.
  • electrophoresis lane M1 protein marker
  • lane PC1 BSA (1 ⁇ g);
  • lane PC2 BSA (2 ⁇ g);
  • 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°C for 4 hours;
  • lane 2 supernatant of cell lysate induced at 16°C for 4 hours;
  • lane 3 precipitate of cell lysate induced at 16°C for 4 hours;
  • 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.
  • 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
  • the complex 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.
  • Microspheres Luminex
  • 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.
  • Carrier protein biotinylated BSA
  • 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.
  • total number of microspheres number of microspheres in 4 ⁇ 4 grids ⁇ (1 ⁇ 10 4 ) ⁇ dilution factor ⁇ suspension volume.
  • the antigen or antibody protein is directly coated onto the surface of the microsphere.
  • 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.
  • microsphere storage solution (10 mM PBS, 3% donkey serum, 5% trehalose, 0.03% Proclin 300), and store at 2-8°C in the dark.
  • total number of microspheres number of microspheres in 4 ⁇ 4 grids ⁇ (1 ⁇ 10 4 ) ⁇ dilution factor ⁇ suspension volume.
  • the flow immunofluorescence kit for detecting autoantibodies or antigens includes the following components:
  • Positive quality control 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);
  • 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);
  • 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;
  • 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
  • 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.
  • a group of microspheres coupled with coated antigens to a 96-well plate (2500 microspheres/type/well), and add 120 ⁇ l of washing solution to each well.
  • 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 ⁇ l 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.
  • MFI median fluorescent intensity
  • a group of microspheres to be tested coupled with specific antibodies were added to a 96-well plate (2500 microspheres/type/well), and 120 ⁇ l 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.
  • 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 ⁇ l 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.
  • MFI median fluorescent intensity
  • ROC Receiver operating characteristic
  • Example 6 Analysis of the expression levels of the eight biomarkers of the present invention in samples from different groups
  • 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:
  • 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).
  • 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).
  • 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).
  • 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).
  • 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).
  • 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.
  • Table 2 The detailed diagnostic performance of ovarian cancer for each group of samples is shown in Table 2 below:
  • 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).
  • 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.
  • 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.
  • 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:
  • the sensitivity of CA125 alone is 62.7%, the specificity is 83.8%, and the diagnostic performance is low.
  • 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).
  • 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 auto
  • the ROC curves of the 12 markers of the present invention for ovarian cancer diagnosis are shown in FIG12 .
  • 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).
  • 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 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:
  • the model formula of the 12 markers is as follows:
  • CCL18 is the CCL18 antigen content measured in serum
  • CA125 is the CA125 antigen content measured in serum
  • C1D is the C1D autoantibody content measured in serum
  • FXR1 is the level of FXR1 autoantibodies measured in serum
  • ZNF573 is the level of ZNF573 autoantibodies measured in serum
  • ESO1 is the ESO1 autoantibody content measured in serum
  • p53 is the p53 autoantibody content measured in serum
  • KRAS is the KRAS autoantibody content measured in serum
  • IGFBP1 is the IGFBP1 autoantibody content measured in serum
  • FUBP1 is the FUBP1 autoantibody content measured in serum
  • TM4SF1 is the TM4SF1 autoantibody content measured in serum
  • 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
  • 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.
  • 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
  • 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)
  • AV average value
  • 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%.
  • 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%.
  • 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%.
  • 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.

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Abstract

L'invention concerne une combinaison de biomarqueurs, un kit de diagnostic la comprenant, et son utilisation. La combinaison de biomarqueurs comprend les antigènes CCL18, C1D, FXR1, ZNF573, ESO1 et p53 ou les auto-anticorps se liant à ceux-ci ; et le biomarqueur comprend en outre un ou plusieurs antigènes choisis parmi CA125, TM4SF1, IGFBP1, FUBP1, ARP3, KRAS, SPINT1 et YKL-40, ou les auto-anticorps se liant à ceux-ci. Le kit de diagnostic comprend la combinaison de biomarqueurs, présente d'excellentes performances de diagnostic pour le cancer de l'ovaire, et peut être utilisé pour diagnostiquer ou aider au diagnostic du cancer de l'ovaire à un stade précoce, ou pour une surveillance de la récidive et une surveillance du pronostic après un traitement du cancer de l'ovaire.
PCT/CN2024/085479 2023-04-04 2024-04-02 Combinaison de biomarqueurs, kit de diagnostic la comprenant et son utilisation Pending WO2024208185A1 (fr)

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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102590531A (zh) * 2012-03-24 2012-07-18 广西壮族自治区肿瘤防治研究所 卵巢癌相关抗原自身抗体谱液相芯片检测试剂盒及其制备方法
CN106248940A (zh) * 2016-07-07 2016-12-21 广西医科大学 多指标联合诊断卵巢癌和/或非卵巢来源的恶性肿瘤的系统
US20190302122A1 (en) * 2016-07-11 2019-10-03 Benjamin Katchman Autoantibody biomarkers for the early detection of ovarian cancer
CN111735949A (zh) * 2020-07-17 2020-10-02 北京信诺卫康科技有限公司 Wnt7a和CA125联合用作早期卵巢癌生物标志物以及试剂盒
CN112834748A (zh) * 2019-11-22 2021-05-25 广州市丹蓝生物科技有限公司 一种生物标志物组合、含其的试剂盒及其应用
WO2022063156A1 (fr) * 2020-09-23 2022-03-31 杭州凯保罗生物科技有限公司 Biomarqueur du cancer du sein et son application
CN115128272A (zh) * 2022-07-01 2022-09-30 广州市丹蓝生物科技有限公司 一种与肺癌相关的生物标志物组合、含其的试剂盒及其用途
CN115877006A (zh) * 2022-12-20 2023-03-31 杭州凯保罗生物科技有限公司 卵巢癌相关的生物标志物及其应用

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102590531A (zh) * 2012-03-24 2012-07-18 广西壮族自治区肿瘤防治研究所 卵巢癌相关抗原自身抗体谱液相芯片检测试剂盒及其制备方法
CN106248940A (zh) * 2016-07-07 2016-12-21 广西医科大学 多指标联合诊断卵巢癌和/或非卵巢来源的恶性肿瘤的系统
US20190302122A1 (en) * 2016-07-11 2019-10-03 Benjamin Katchman Autoantibody biomarkers for the early detection of ovarian cancer
CN112834748A (zh) * 2019-11-22 2021-05-25 广州市丹蓝生物科技有限公司 一种生物标志物组合、含其的试剂盒及其应用
CN111735949A (zh) * 2020-07-17 2020-10-02 北京信诺卫康科技有限公司 Wnt7a和CA125联合用作早期卵巢癌生物标志物以及试剂盒
WO2022063156A1 (fr) * 2020-09-23 2022-03-31 杭州凯保罗生物科技有限公司 Biomarqueur du cancer du sein et son application
CN115128272A (zh) * 2022-07-01 2022-09-30 广州市丹蓝生物科技有限公司 一种与肺癌相关的生物标志物组合、含其的试剂盒及其用途
CN115877006A (zh) * 2022-12-20 2023-03-31 杭州凯保罗生物科技有限公司 卵巢癌相关的生物标志物及其应用

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