WO2012031374A1 - 用于诊断上皮源性癌症的血液标志物及其单克隆抗体 - Google Patents

用于诊断上皮源性癌症的血液标志物及其单克隆抗体 Download PDF

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WO2012031374A1
WO2012031374A1 PCT/CN2010/001387 CN2010001387W WO2012031374A1 WO 2012031374 A1 WO2012031374 A1 WO 2012031374A1 CN 2010001387 W CN2010001387 W CN 2010001387W WO 2012031374 A1 WO2012031374 A1 WO 2012031374A1
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cancer
antibody
cytokeratin
fragment
epithelial
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PCT/CN2010/001387
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English (en)
French (fr)
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沈恩允
宋扬
任世奇
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北京同为时代生物技术有限公司
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Priority to CN201080068841.6A priority Critical patent/CN103476428B/zh
Priority to US13/821,844 priority patent/US9255142B2/en
Priority to CN201610922143.7A priority patent/CN106706914B/zh
Priority to PCT/CN2010/001387 priority patent/WO2012031374A1/zh
Publication of WO2012031374A1 publication Critical patent/WO2012031374A1/zh
Priority to US14/992,750 priority patent/US9840551B2/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407Specifically defined cancers
    • G01N33/57423Specifically defined cancers of lung
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57496Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving intracellular compounds
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701Details
    • G01N2333/4742Keratin; Cytokeratin

Definitions

  • the present invention relates to the diagnosis of cancer, and more particularly to the use of cytokeratin fragments as markers for the diagnosis of epithelial cancers. Background technique
  • Epithelial cancer refers to cancers that originate in epithelial cells, including but not limited to breast cancer, gastric cancer, oral cancer, esophageal cancer, colon cancer, liver cancer, bladder cancer, pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer, and Skin cancer, prostate cancer, kidney cancer, etc.
  • gastric cancer is the second leading cause of cancer death in the world and one of the most common malignant tumors that threaten human health.
  • the key to the prognosis of patients with gastric cancer is secondary prevention, early detection and early treatment.
  • the timely and accurate detection and treatment of gastric cancer is of great significance for reducing the mortality of gastric cancer.
  • Lung cancer is a common malignant tumor of the lung.
  • the incidence and mortality of lung cancer have risen rapidly in countries all over the world, especially in industrialized countries.
  • the development of highly specific and sensitive serum tumor markers is of great significance for the early detection and treatment of lung cancer.
  • Tumor Markers refers to a substance that is produced or secreted by tumor cells and released into blood, cells, and body fluids during tumorigenesis and proliferation, reflecting the presence and growth of tumors. Tumor markers are usually proteins and have been widely used in the detection and diagnosis of many types of cancer. An elevated level of tumor marker concentration may indicate some form of cancer in the human body. Tumor markers are mainly used for the discovery of primary tumors, screening of high-risk populations, differential diagnosis of benign and malignant tumors, judgment of tumor development, observation and evaluation of tumor treatment effects, and prediction of tumor recurrence and prognosis. Wait.
  • Tumor markers are commonly determined by immunological methods, such as indirect methods, double antibody sandwich methods, and competition methods.
  • the detection means include colloidal gold method, enzyme immunoassay, chemiluminescence method, and electrochemiluminescence method.
  • the purpose of serological testing is to determine The amount of cancer-associated tumor markers in the patient's serum. This method is simple and easy to apply, and is suitable for the census of a large number of people.
  • tumor markers associated with gastric cancer which are currently widely used internationally, include CEA, TPS, and CA72-4.
  • Cytokeratins which comprise more than 20 different proteins, are important components of the cytoskeleton. Although all epithelial cells express a certain level of cytokeratin, certain keratin components such as keratin 8, 18 and 19 are closely related to the development and progression of malignant tumors (M. Nap, Th. Van Wei, C. Andres, et al. Immunohistochemical Profiles of 30 Monoclonal Antibodies against Cytokeratins 8, 18 and 19 [J]. Tumor Biology 2001; 22: 4-10).
  • Cytokeratin 18 is an acidic protein with a molecular weight of 55 kD. It consists of 430 amino acids and has a highly conserved central region of the ⁇ -helical structure with a filamentous structure. It is widely distributed on normal tissue surfaces such as stratified epithelium and squamous epithelium, as well as monolayer epithelial cells such as acinar, tracheal, mammary duct, sweat gland, endometrium, colon and hepatocytes.
  • Cytokeratin 18 expression is relatively stable in normal epithelial cells, and cytokeratin 18 and its fragments are not expressed or expressed in peripheral blood, bone marrow, and lymph nodes, and almost no fragments are released into the blood. Conversely, when epithelial cells are malignantly transformed, cytokeratin 18 expression is dramatically increased. At the same time, the growth process of cytokeratin 18 is abnormal.
  • Cytokeratin 19 is an acidic protein with a molecular weight of 40 kD. It is the smallest member of the keratin family and consists of 400 amino acids. It has a highly conserved central region of the alpha helix and has a filamentous structure. It is widely distributed on normal tissue surfaces such as stratified epithelium and squamous epithelium, as well as monolayer epithelial cells such as acinar, tracheal, mammary duct, sweat gland, endometrium, colon and hepatocytes.
  • Cytokeratin 19 expression is relatively stable in normal epithelial cells, and cytokeratin 19 and its fragments are not expressed or expressed in peripheral blood, bone marrow, and lymph nodes, and almost no fragments are released into the blood circulatory system. Conversely, when epithelial cells are malignantly transformed, cytokeratin 19 expression is dramatically increased. At the same time, the growth process of cytokeratin 19 is abnormal. ; Protease activated during tumor cell apoptosis and necrosis accelerates cell degradation, resulting in the release of a large number of soluble cytokeratin 19 fragments, resulting in increased concentrations of soluble cytokeratin 19 fragments in tissue fluids and body fluids, especially in lung cancer.
  • One aspect of the invention provides a cytokeratin fragment associated with epithelial-derived cancer, wherein the fragment comprises an epitope selected from the group consisting of SEQ ID NOs: 2, 3, 5 and 6.
  • the fragment comprises amino acid residues 200-400 of SEQ ID NO: 1.
  • the fragment comprises amino acid residues 325-400 of SEQ ID NO: 2.
  • Another aspect of the invention provides a monoclonal antibody that specifically binds to the epitope of the antigen.
  • the monoclonal antibody is produced by a hybridoma having the accession number CGMCC No. 1957, CGMCC No. 1956, CGMCC No. 1955 or CGMCC No. 1952.
  • the invention also provides an antigen binding portion of the monoclonal antibody, wherein the antigen binding portion competes with the monoclonal antibody for binding to the antigenic epitope.
  • the antigen binding portion is a humanized antibody.
  • the antigen binding portion is a chimeric antibody.
  • the invention provides a method for early screening, diagnosis or prognosis assessment of an epithelial cancer in a subject, the method comprising: obtaining a biological sample from the subject, detecting the invention in the biological sample The amount of cytokeratin fragment, and the content is compared to a threshold level. If the level exceeds the threshold level, the patient may have cancer.
  • the method further relates to the use in combination with other epithelial-derived tumor markers.
  • the method comprises: obtaining a biological sample from the subject, detecting the content of the cytokeratin fragment of the invention in the biological sample, detecting the content of other epithelial-derived tumor markers in the biological sample, and The levels of the cytokeratin fragments and the other epithelial-derived tumor markers are compared to threshold levels.
  • the other epithelial-derived tumor markers are selected from the group consisting of AFP, CEA, CA242, CA19-9, CA72-4, A group consisting of CA125, CA15-3, NSE, SCCA, Cyfra21-1, PSA, and free PSA.
  • Another aspect of the present invention provides a method of evaluating a therapeutic effect of a drug or therapy for treating an epithelial cancer, comprising: administering the drug or therapy to a subject having an epithelial cancer, administering the drug Collecting a biological sample of the subject before and after the therapy, and detecting the content of the cytokeratin fragment of the invention in the biological sample, wherein the cytokeratin fragment is significantly present before and after administration of the drug or therapy Reduced, indicating that the drug or therapy has a significant effect.
  • the epithelial cancer is selected from the group consisting of gastric cancer, liver cancer, lung cancer, biliary cancer, breast cancer, cervical cancer, ovarian cancer, colon cancer, prostate cancer, kidney cancer, esophageal cancer, intestinal cancer, and bladder cancer.
  • the biological sample is selected from the group consisting of blood, serum, tissue fluid, urine, stool, sputum, cerebrospinal fluid, saliva, tears, and nipple aspirate.
  • kits comprising: a coated antibody immobilized on a solid phase carrier capable of specifically binding to the cytokeratin fragment of the present invention, and a detectably labeled one capable of specifically binding the cytokeratin Fragment detection antibody.
  • the kit can be used to practice the methods of the invention, as well as to detect cytokeratin fragments of the invention.
  • the present invention also provides the use of a specific binding agent for the cytokeratin fragment, such as a monoclonal antibody or antigen-binding fragment of the present invention, in the preparation of a reagent for diagnosing epithelial cancer.
  • a specific binding agent for the cytokeratin fragment such as a monoclonal antibody or antigen-binding fragment of the present invention
  • Figure 1 is a flow chart briefly depicting the steps of preparation and activity analysis of cytokeratin 18 fragments.
  • Figure 2 is an electropherogram showing the results of electrophoresis of the cytokeratin 18 fragment cDNA.
  • Figure 3 shows the structure of the TOPO plasmid. Among them, Ampicillin refers to ampicillin,
  • Neomycin refers to the new sage.
  • Figure 4 is an electropherogram showing the results of restriction enzyme digestion of recombinant expression plasmids TOPO-GY10 and TOPO-GY1 K TOPO-GY12. Among them, marker refers to a marker.
  • Figure 5 is an electropherogram showing the results of SDS-PAGE of recombinant cytokeratin 18 fragments GY10, GY11, and G Y 12 .
  • FIG. 6 shows the results of the optimal antibody pairing experiment.
  • Figure 7 is a schematic diagram showing the positions of the binding epitopes of monoclonal antibodies 3A9 and 2A6. Set.
  • Figure 8 shows the results of immunodetection of monoclonal antibodies 3A9 and 2A6 with other human serum tumor markers.
  • Figure 9 shows the ROC curve for the diagnosis of gastric cancer and non-gastric cancer.
  • specificity refers to specificity
  • sensitivity refers to sensitivity.
  • Figure 10 is a flow chart briefly depicting the steps of preparation and activity analysis of cytokeratin 19 fragments.
  • Figure 11 is an electropherogram showing the results of electrophoresis of the cytokeratin 19 fragment cDNA.
  • Figure 12 shows the structure of the TOPO plasmid. Among them, Ampicillin refers to ampicillin and Neomycin refers to neomycin.
  • Figure 13 is an electrophoresis diagram showing the results of restriction enzyme digestion of recombinant expression plasmids TOPO-GY20, TOPO-GY21 and TOPO-GY22.
  • marker refers to a marker.
  • Figure 14 is an electropherogram showing the results of SDS-PAGE of recombinant cytokeratin 19 fragments GY20, GY2K GY22.
  • FIG. 15 shows the results of the optimal antibody pairing experiment.
  • Figure 16 is a schematic diagram showing the positions of the binding epitopes of the monoclonal antibodies 2G2, 5H2.
  • Figure 17 shows the results of immunodetection of monoclonal antibodies 2G2, 5H2 and other human serum tumor markers.
  • Figure 18 shows the ROC curve for differential diagnosis of lung cancer and non-lung cancer. Among them, specificity refers to specificity, and sensitivity refers to sensitivity. Description of related sequences
  • SEQ ID NO: 1 shows the full-length amino acid sequence of cytokeratin 18 (K18 for short), which is 430 amino acid residues in length.
  • SEQ ID NO: 2 shows the amino acid sequence of the K18 epitope recognized by monoclonal antibody 3A9, 51 amino acids in length, corresponding to the amino acid position of SEQ ID NO: 1 200-250.
  • SEQ ID NO: 3 shows the amino acid sequence of the K18 epitope recognized by monoclonal antibody 2A6, 51 amino acids in length, corresponding to amino acid position 350 to 400 of SEQ ID NO: 1.
  • SEQ ID NO: 4 shows the full length amino acid sequence of cytokeratin 19 (K19 for short), It is 400 amino acid residues in length.
  • SEQ ID NO: 5 shows the amino acid sequence of the K19 epitope recognized by the monoclonal antibody 2G2, which is 26 amino acid residues in length, corresponding to the amino acid position 375 - of SEQ ID NO: 4.
  • SEQ ID NO: 6 shows the amino acid sequence of the K19 epitope recognized by monoclonal antibody 5H2, which is 26 amino acid residues in length, corresponding to amino acid positions 325-350 of SEQ ID NO:4.
  • the present invention provides two epithelial-derived cancer-associated epitopes of cytokeratin 18 (K18). Both cancer-associated epitopes are located at the C-terminus of the cytokeratin 18 amino acid sequence, and the sequences are shown in SEQ ID NOs: 2, 3.
  • the epitope becomes exposed during malignant transformation, particularly during malignant transformation of tissues such as the stomach, colon, breast, ovary, kidney, and the like. Exposure of the cancer-associated epitope is exposed by release of cytokeratin 18 fragments into the blood by continuous proliferation, necrosis, and apoptosis of the tumor cells. Normal tissue and healthy people do not have the expression of these two cancer-associated epitopes in their blood.
  • the present invention provides two smear monoclonal antibodies 3A9 and 2A6 which recognize these two cancer-associated epitopes, respectively, and invents a method for diagnosing cancer using these two monoclonal antibodies.
  • the hybridomas used to produce K18 mAb 3A9 and 2A6 were deposited on March 8, 2007 at the General Microbiology Center of China Microbial Culture Collection Management Committee (CGMCC, No. 13 North Section, Zhongguancun, Haidian District, Beijing).
  • CGMCC General Microbiology Center of China Microbial Culture Collection Management Committee
  • the preservation numbers are CGMCC. NO. 1957 and 1956.
  • the information in the deposit certificate is incorporated herein by reference as part of the original disclosure of the present invention.
  • the sequences are shown in SEQ ID NOs: 5, 6.
  • the present invention provides two monoclonal antibodies 2G2 and 5H2 which recognize these two cancer-associated epitopes, respectively, and uses these two monoclonal antibodies to invent a method for diagnosing cancer.
  • Hybridomas for the production of K19 mAb 2G2 and 5H2 were deposited with the General Microbiology Center of China Microbial Culture Collection Management Committee (CGMCC, No. 13 North Section, Zhongguancun, Haidian District, Beijing) on March 8, 2007.
  • CGMCC General Microbiology Center of China Microbial Culture Collection Management Committee
  • the preservation numbers are CGMCC. NO. 1955 and 1952.
  • the information in the proof of possession is incorporated herein by reference as part of the original disclosure of the present invention.
  • One aspect of the invention provides a cytokeratin fragment associated with epithelial-derived cancer, wherein the fragment comprises an epitope selected from the group consisting of SEQ ID NOs: 2, 3, 5 and 6.
  • epithelial cancer refers to cancer that arises from epithelial cells, including But not limited to breast cancer, basal cell carcinoma, adenocarcinoma, gastrointestinal cancer, lip cancer, oral cancer, esophageal cancer, small intestine and stomach cancer, colon cancer, liver cancer, bladder cancer, pancreatic cancer, ovarian cancer, cervical cancer, lung cancer , breast cancer and skin cancers such as squamous cell and basal cell carcinoma, prostate cancer, renal cell carcinoma and other cancers known to affect epithelial cells through the body.
  • fragment refers to an amino acid sequence that lacks one or more amino acid residues compared to a native full-length protein.
  • a protein fragment can be obtained by deleting a part of a residue from a full-length protein, for example, an N-stage truncated fragment can be obtained by deleting a part of the amino acid residue located in the N segment of the protein.
  • the protein fragment may also contain a foreign sequence to form a chimeric protein fragment. For example, after deletion of the N-terminal domain of protein A, it is linked to the N-terminal domain of protein B to form a chimeric fragment of protein A and protein B. Those skilled in the art will appreciate that the length of the chimeric fragment is likely to exceed the length of protein A.
  • purification tags such as GST (glutathione thiotransferase), His-tag (hexahistidine), and the like can be added to the protein fragment.
  • the cytokeratin fragment of the invention may contain a contiguous amino acid sequence near the epitope of the antigen.
  • a K18 fragment of the invention may comprise at least 60, 70, 80, 90, 100, 120, 150, 180, 200, 210 of SEQ ID NO: 1 encompassing an epitope represented by SEQ ID NO: 2 or 3. 220, 250, 280, 300, 320, 350, 380, 400 or 420 consecutive amino acid residues.
  • the K18 fragment comprises amino acid residues 1-250 of SEQ ID NO: 1 and encompasses an epitope recognized by mAb 3A9; in another embodiment, the K18 fragment comprises SEQ ID NO Amino acid residues 250-430 of 1 encompassing an epitope recognized by mAb 2A6; in yet another embodiment, the K18 fragment comprises amino acid residues 150-430 of SEQ ID NO: 1 while encompassing two epitopes .
  • a fragment comprising one or two epitopes of K18 can be fused to an epitope of K19 to form a chimeric fragment.
  • the K19 fragment of the present invention may comprise at least 30, 50, 60, 70, 80, 90, 100, 120, 150, 180, 200 of the epitope represented by SEQ ID NO: 5 or 6 in SEQ ID NO: 4. 210, 220, 250, 280, 300, 320, 350 or 380 consecutive amino acid residues.
  • the K19 fragment comprises amino acid residues 1-375 of SEQ ID NO: 4, encompassing an epitope recognized by monoclonal antibody 5H2; in another embodiment, the K19 fragment comprises SEQ ID NO:
  • the amino acid residue of 1 is 150-400, and covers the epitopes recognized by the monoclonal antibodies 2G2 and 5H2. Protein fragments comprising specific amino acid residues can be readily prepared by molecular cloning experimental techniques well known in the art.
  • biological sample refers to a sample of biological material obtained from a patient (preferably a patient), including tissue samples, cell samples (eg, tissue biopsy samples, such as aspiration biopsy samples, brush biopsy samples, surface biopsy samples, needle biopsy). Samples, drill biopsy samples, excised biopsy samples, open thoracic biopsy samples, incisional biopsy samples or endoscopic biopsy samples) and tumor samples.
  • tissue samples eg, tissue biopsy samples, such as aspiration biopsy samples, brush biopsy samples, surface biopsy samples, needle biopsy. Samples, drill biopsy samples, excised biopsy samples, open thoracic biopsy samples, incisional biopsy samples or endoscopic biopsy samples
  • the biological sample can also be a biological fluid sample.
  • the biological sample is serum.
  • blood, urine, saliva, cerebrospinal fluid, nipple aspirate, cell lysate supernatant, and the like can also be used.
  • the level of a cytokeratin fragment of the invention can be measured by any means known to those skilled in the art.
  • other methods can be used, for example, by analyzing mRNA transcripts to measure expression of cytokeratin fragments.
  • RNA transcripts can be detected by Northern blotting, where the RNA preparation is performed on a denaturing agarose gel and transferred to a suitable support, such as an active cellulose membrane, a nitrocellulose membrane or a glass membrane. Or nylon membrane. The labeled (e. g., radiolabeled) cDNA or RNA is then hybridized to the preparation, washed, and analyzed by methods such as autoradiography.
  • mRNA expression can be detected on a DNA array, chip or microarray.
  • mRNA is extracted from the biological sample to be tested, and reverse transcription is performed to produce a fluorescent-labeled cDNA probe.
  • the microarray capable of hybridizing with the cytokeratin fragment cDNA was then probed with the labeled cDNA probe, and the slide was scanned to measure the fluorescence intensity. This intensity and intensity of hybridization correlate with expression levels.
  • cytokeratin fragments can also be measured, especially when the biological sample is a fluid sample such as blood or urine.
  • cytokeratin fragment levels are measured by contacting a biological sample with a monoclonal antibody that specifically binds to a cytokeratin fragment.
  • antigen-binding portion of an antibody includes immunoglobulin molecules that specifically bind to an antigenic epitope of a cytokeratin (immunoreactive reaction) and immunologically active determinants of an immunoglobulin molecule, such as a molecule containing an antigen binding site. .
  • the term "antigen-binding portion of an antibody” is intended to include whole antibodies, such as any isotype (IgG, IgA, IgM IgE, etc.), as well as antibody fragments.
  • Antibodies can be fragmented using conventional techniques. Therefore, the term includes A proteolytic fragment or a recombinantly produced portion of an antibody molecule that is capable of selectively reacting with a certain protein.
  • Non-limiting examples of such proteolytic and/or recombinant fragments include Fab, F(ab')2, Fab', Fv, dAbs and single chain antibodies (scFv) comprising a VL region and a VH region joined by a polypeptide linker.
  • the scFv can be covalently or non-covalently linked to form an antibody comprising two or more binding sites.
  • "antigen binding portion of an antibody” includes polyclonal antibodies, monoclonal antibodies or other purified preparations of antibodies and recombinant antibodies.
  • antibody refers to an antibody derived from a non-human antibody (eg, a murine) that retains or substantially retains the antigen binding properties of the parent antibody, but which is less immunogenic in humans.
  • a humanized antibody refers to an antibody derived from a non-human antibody (eg, a murine) that retains or substantially retains the antigen binding properties of the parent antibody, but which is less immunogenic in humans.
  • the advantage of a humanized antibody is that it reduces or eliminates the immunogenicity of the antibody in the host, thereby increasing bioavailability and reducing possible adverse immune responses.
  • Humanized antibodies can be prepared using methods of CDR grafting.
  • labeled antibody includes antibodies that are detectably labeled, including, but not limited to, enzyme-labeled antibodies, radiolabeled antibodies, fluorescently labeled antibodies, and chemiluminescent-labeled antibodies. Antibodies can also be labeled with a detectable label such as HA, HSV, FLAG or HIS. Levels of cytokeratin fragments The presence of cytokeratin in the biological sample is related to the signal intensity emitted by the detectable labeled antibody.
  • the antibody or antigen binding portion of the antibody can be detectably labeled by linking the antibody to an enzyme.
  • the enzyme When the enzyme is contacted with its substrate, it will react with the substrate in a manner that produces a detectable chemical moiety, for example, by spectrophotometry, fluorometry or by visual means.
  • Enzymes useful for detectably labeling antibodies of the invention include, but are not limited to, malate dehydrogenase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, urease, catalase, Glucose amylase and acetylcholinesterase. Chemiluminescence is another method that can be used to detect antibodies or antigen binding portions thereof.
  • Detection can also be accomplished using any of a variety of other immunoassays.
  • the antibody can be detected by radioimmunoassay by radiolabeling the antibody.
  • the radioisotope can be detected by using, for example, a gamma counter or a scintillation counter or by autoradiography.
  • fluorescently labeled antibodies When a fluorescently labeled antibody is exposed to light of a suitable wavelength, its presence can be detected due to fluorescence.
  • fluorescent labeling There are fluorescein isothiocyanate, rhodamine, phycoerythrin and fluorescamine.
  • the invention provides a method for early screening, diagnosis or prognosis assessment of an epithelial cancer in a subject, comprising: obtaining a biological sample from the subject, detecting a cytokeratin fragment in the biological sample The content, and the content is compared to the threshold level. If the level exceeds the threshold level, the patient may have cancer or a poor prognosis.
  • the detection is carried out using the monoclonal antibodies provided herein.
  • K18 or K19 fragments can be detected using only one monoclonal antibody; K18 fragments can be detected using 3A9 and 2A6 in combination; 2K2 and 5H2 can be used in combination to detect K19 fragments; 3A9 and 2G2 can also be used in combination to simultaneously detect K18 fragments. And the K19 fragment; or the combination of the above four monoclonal antibodies for detection. It will be appreciated that the combined use of several antibodies may increase the accuracy of the diagnosis.
  • 3A9 refers to a monoclonal antibody produced by a hybridoma having the accession number CGMCC NO. 1957, which specifically recognizes an epitope on K18.
  • 2A6 refers to a monoclonal antibody produced by a hybridoma having the accession number CGMCC NO. 1956, which specifically recognizes an epitope on K18.
  • 2G2 refers to a monoclonal antibody produced by a hybridoma having the accession number CGMCC NO. 1955, which specifically recognizes an epitope on K19.
  • 5H2 refers to a monoclonal antibody produced by a hybridoma having the accession number CGMCC NO. 1952, which specifically recognizes an epitope on K19.
  • the method further relates to use with other epithelial-derived tumor markers, such as AFP, CEA, CA242, CA19-9, CA72-4, CA125, CA15-3, NSE, SCCA, Cyfra21-PSA , free PSA and other common tumor markers. Combined detection of multiple tumor markers is helpful in improving the accuracy of the diagnosis.
  • epithelial-derived tumor markers such as AFP, CEA, CA242, CA19-9, CA72-4, CA125, CA15-3, NSE, SCCA, Cyfra21-PSA , free PSA and other common tumor markers.
  • Another aspect of the present invention provides a method of evaluating a therapeutic effect of a drug or therapy for treating an epithelial cancer, comprising: administering the drug or therapy to a subject having an epithelial cancer, administering the drug Collecting a biological sample of the subject before and after the therapy, and detecting the content of the cytokeratin fragment of the invention in the biological sample, wherein the cytokeratin fragment is significantly present before and after administration of the drug or therapy Reduced, indicating that the drug or therapy has a significant effect. box.
  • the kit can be a configuration known to the skilled person. One or more of the methods described herein are applied. In addition, it is preferred to carry out the test simultaneously with one or more standards included in the kit so that the test results can be quantified or confirmed.
  • the standard can be a purified K18 or K19 protein.
  • the kit includes means for detecting the level of a cytokeratin fragment, such as an antibody or antibody fragment that selectively binds to a cytokeratin fragment.
  • the diagnostic assay kit is preferably formulated in a standard diabody binding format wherein one specific antibody captures a cytokeratin fragment in a patient sample and another specific antibody is used to detect the captured cytokeratin fragment.
  • the capture antibody (coating antibody) is immobilized on a solid phase such as a test plate or a nitrocellulose membrane.
  • the second antibody, the detection antibody is typically labeled with a detectable label such as horseradish peroxidase or a radioisotope.
  • 3A9 and 2A6 are used in combination in the kit to detect the K18 fragment.
  • 2G2 and 5H2 are used in combination in the kit to detect the K19 fragment. It is also possible to use 3A9 and 2G2 in combination to detect K18 fragments and K19 fragments simultaneously; or to use the above four monoclonal antibodies in combination for detection. It will be appreciated that the combined use of several antibodies may increase the accuracy of the diagnosis.
  • the detection kit can employ, but is not limited to, the following techniques: competitive and non-competitive assays, radioimmunoassay (RIA), bioluminescence and chemiluminescence assays, fluorescence assays, sandwich assays, Immunoradiometric assays, dot blots, enzyme-linked assays (including ELIS A), microtiter plates, and immunocytochemistry.
  • RIA radioimmunoassay
  • bioluminescence and chemiluminescence assays fluorescence assays
  • sandwich assays sandwich assays
  • Immunoradiometric assays dot blots
  • enzyme-linked assays including ELIS A
  • microtiter plates and immunocytochemistry.
  • test kit will also provide instructions for use.
  • Example 1 The invention is further illustrated by the following examples. These examples are provided to aid the understanding of the invention and are not to be construed as limiting the invention.
  • Example 1
  • Recombinant cytokeratin 18 fragment GY10 is expressed by E. coli BL21 (DE3) Escherichia coli. ⁇
  • cDNA encoding various cytokeratin 18 fragments was prepared by RT-PCR: a) template and primers
  • RNA was isolated from a HELA human cancer cell line.
  • the cDNA was then synthesized using a reverse transcription kit (Promega) according to the instructions.
  • the obtained cDNA is a template for PCR. Primers of the three fragments were designed, and each cDNA was subjected to PCR amplification. The fragment number and amino acid sequence are shown in Table 1-1. Number and sequence of recombinant cytokeratin 18 fragments
  • dNTP 2.5 mM each, 4 ⁇ 1 total
  • Taq polymerase (Promega): 5U;
  • the solution was heated to 94 ° C for 2 min and then cycled 40 times with each cycle set to: Heating 94. C 30s , 52 °C lmin, 72 °C 3min. After the procedure was completed, the reaction solution was heated at 72 ° C for 10 min.
  • the amplified DNA fragment was then taken and separated using a 1% agarose gel containing 0.25 g/ml ethidium bromide, as shown in Fig. 2. The results showed that the desired cytokeratin 18 fragment cDNA fragment was contained in the band, and then the DNA fragment was recovered using a Gene Clean kit (BIOIOI, Irvine, CA).
  • the cDNA fragment was cloned with a TOPO100 expression Cloning Kit (Invitrogen, Carlsbad, CA).
  • a TOPO100 expression Cloning Kit Invitrogen, Carlsbad, CA.
  • the surface of the medium can be screened for ampicillin-resistant clones, and a single colony is picked up in a platinum wire coating circle and placed in an L-broth medium (containing ampicillin 10 ( ⁇ g/ml) at 37 ° C for overnight. , shake (200 rpm).
  • the cDNA encoding various cytokeratin 18 has been inserted into the TOPO plasmid.
  • the obtained plasmid was transformed into E. coli BL21 (DE3), and then cultured in an LB medium to an exponential growth phase, and induction was induced with isopropanol ⁇ -thiogalactoside (IPTG) for 3 hours.
  • IPTG isopropanol ⁇ -thiogalactoside
  • the cells were pelleted, resuspended in lysate (8M urea, 20 mM Tris-HCl), and disrupted by ultrasonication.
  • Sephadex G-50 was activated and the recombinant cytokeratin 18 fragment was purified by molecular sieve chromatography.
  • Sephadex G-50 was dissolved in 100 mM Tris-HCl buffer (pH 7.4), boiled at 100 °C for 10 minutes, then blocked with 100 mM Tris-HCl buffer (pH 7.4) and stored at 4 °C. .
  • the bacterial disrupted concentrate was passed through 2 ml of gel beads at a flow rate of 2 ml / min. After the sample was passed, rinse with 50 ml of PBS, elution buffer 0.1 M glycine (pH 2.4), 0.15 M NaCI.
  • the eluent was measured in the ultraviolet OD280nm to determine whether the elution was completed.
  • the effective eluate (OD>0.01) was collected and placed in a dialysis bag and dialyzed against 1 L of phosphate buffer (pH 7.5) at 4 ° C, during which time the dialysis buffer was replaced twice.
  • the purified protein was concentrated to about 1 mg/ml and added to 1%. NaN 3 , stored at 4 ° C. Protein purity was determined by scanning with a 10% SDS-PAGE and scanning with a GDS8000 gel imaging system.
  • the immunogen used to prepare the monoclonal antibody is the recombinant full-length human cytokeratin 18, which is a gift from Professor Zhou Bing of UAB University.
  • Emulsified granules were formed by mixing 1 ml of the antigenic protein solution with the same volume of Freund's complete adjuvant, and each foot was injected with 100 ⁇ l.
  • Emulsified particles were formed by mixing 1 ml of the antigenic protein solution with the same volume of Freund's incomplete adjuvant, and 100 ⁇ l per foot was injected.
  • Lymphocytes were isolated from the axilla and groin of the immunized mice, mixed with NS1 myeloma cells in a ratio of 2:1, washed twice with serum-free RPMI 1640, and added with 1 ml of 37. C pre-warmed PEG1500, the cells were gently mixed at 37 ° C for 1 minute, and then 20 ml of serum-free RPMI 1640 medium preheated to 37 ° C was slowly added dropwise over 3 minutes. After centrifugation, the fused cells were suspended in 12% FBS RPMI 1640 HAT selection medium. The cells were added to 5 96-well cell culture plates, ⁇ /well.
  • the ELISA plate was coated with the above keratin 18 antigen (lg/ml) at 4 ° C overnight; washed 3 times with PBS, and blocked with 3% (w/v) BSA in PBS for 1 h at room temperature.
  • the supernatant of ⁇ cell culture solution was added to each well; the bath was incubated at 37 ° C for 30 minutes, washed with PBST washing solution for 5 times, and patted dry to add peroxidase-conjugated goat anti-mouse immunoglobulin (HRP-GAM Ig, DAKO). Company), warm bath at 37 °C for 30 minutes, remove it and wash it with PBST washing solution for 5 times.
  • substrate solution A and B respectively 50 ⁇ 1 (substrate liquid ⁇ composition: 13.42g Na 2 HP0 4 *12H 2 0, 4.2g citric acid ⁇ ⁇ 2 0 and 0.3g hydrogen peroxide, adjusted volume in deionized water is 700ml; coloring liquid B component is: 0.2g tetramethylbenzidine, 20ml dimethylformamide deionized Adjust the volume in water to 700ml), develop color at 37 °C for 10 minutes, add 50 ⁇ 1 stop solution (2 ⁇ H 2 S0 4 ) to terminate the reaction, and check the OD 45Q value of each well on the microplate reader, with OD 450 value higher than 2.0. The above are considered positive.
  • Protein 18 (aal ⁇ 250) (aa200 - 430) antigen
  • mice 16-week-old healthy Balb/c mice were intraperitoneally injected with 0.5 ml pristane for 5-7 days.
  • the cloned hybridoma cells were collected, centrifuged to remove supernatant, and serum-free medium was added to adjust the cell density to 2x10. 5 ⁇ 2 ⁇ 10 6 /ml, 0.5ml per mouse. After 7 ⁇ 10 days, the abdomen of the mice increased, and the ascites began to be collected. Centrifuge at 3000rpm for 15 minutes, absorb the middle clear part of the liquid, 0.45 ⁇ microporous membrane Filter and sterilize, store at -20 °C after dispensing.
  • the treated ascites was diluted 5 times with 0.02 mol/L PBS (81 ml 0.2 mol/L Na 2 HP0 4 , 19 ml 0.2 mol/L NaH 2 P0 4 plus physiological saline to 100 ml), and 50 ml was taken.
  • the antibody bound to the column was then eluted using 0.1 M Glycine-HCL pH 2.4 buffer, and the eluted fraction 2 ml/tube was collected.
  • HRP horseradish peroxidase
  • the principle is that the glycosyl group of HRP is oxidized to an aldehyde group by sodium periodate, and after adding an antibody IgG, the aldehyde group is combined with an IgG amino group to form a Schiff base.
  • the amino group is blocked with dinitrofluorobenzene before being oxidized by sodium periodate.
  • the Schiff base was stabilized with sodium borohydride.
  • Enzyme amount (mg/ml) OD 4 o3 x 0.4
  • IgG amount (mg/ml) (OD 280 -OD 403 x 0.3 ) ⁇ .62
  • the molar ratio (E/P) the amount of enzyme x4 / IgG, usually between 1-2.
  • Enzyme binding rate enzyme amount X volume / antibody, the labeling rate is generally 0.3-0.6, that is, 1-2 HRP molecules are bound to one antibody molecule, the labeling rate can be greater than 0.6, 0.8, 0.9; OD 403 / OD 280 is equal to 0.4 When E/P is about 1.
  • Enzyme activity and antibody activity can be determined by ELISA, immunodiffusion, DAB-H 2 2 color reaction to determine the enzyme activity of the enzyme conjugate, antibody activity and potency, specificity.
  • Table 1-3 summarizes the results of cross-pairing experiments with 5 pairs of monoclonal antibodies. Among them, antibody pairing with 3A9/2A6-HRP, 2A6/3A9-HRP, 3H7/3A9-HRP showed the stomach The cancer serum has a strong positive reaction and is negative for normal human serum. Therefore, we determined that serum keratin 18 detected by these antibody pairs is highly correlated with gastric cancer. Screening of cross-over experiments of gastric cancer-associated keratin 18 antibody
  • Three pairs of antibody pairs highly correlated with gastric cancer keratin 18 were selected from the above cross-antibody pairing experiments.
  • 3A9/2A6-HRP, 2A6/3A9-HRP, 3H7/3A9-HRP were clinically validated by ELISA.
  • the 96-well plates were coated with purified antibody at l g/ml, overnight at 4 ° C, and then blocked with 3% BSA in PBS for 1 h at room temperature. 10 ⁇ g of gastric cancer serum and 10 normal human serum were separately taken as test samples for detection, and then 50 ⁇ l of 1:1000 enzyme-labeled antibody was added.
  • test data is used as a scatter plot to compare the sensitivity and specificity of the diagnosis. See Figure 6.
  • ELISA plates were coated with purified keratin 18 antigen. Cell supernatants of each monoclonal antibody were added to each well, and incubated at 37 ° C for 30 min; washed with PBST 5 times on a TEC AN automatic plate washer at intervals of 20 seconds, decanted, and added to the appropriate dilution of HRP.
  • the monoclonal antibodies 3A9 and 2A6 are monoclonal antibodies specific for keratin 18.
  • the K18 kit was prepared by using K18 mAb 3A9 as a coating antibody and K18 mAb 2A6 as a labeled antibody.
  • the main components of the kit are: calibrator, coating plate, enzyme conjugate, luminescent solution, concentrated washing solution.
  • the standard is purified keratin 18 .
  • the coated antibody was K18 mAb 3A9.
  • the labeled antibody is K18 mAb 2A6 and is labeled with HRP horseradish peroxidase to form an antibody-HRP complex (enzyme conjugate).
  • the coated board is coated with a monoclonal antibody 3A9, washed, sealed, dried, and vacuum-packed.
  • K18 mAb 3A9 was coated with citric acid buffer pH 4.8 as a coating buffer at a coating concentration of 5 g/mL.
  • the blocking solution was 0.02 M PBS + 0.4% casein +1% BSA + 0.5 M NaCl + 0.2% gelatin + 10% bovine serum + 0.05% Tween-20 + 1%. Preservative + 2.5% sucrose.
  • the labeled antibody is 2A6-HRP.
  • the dilution was 0.02 M PBS + 1% casein +1% BSA +1 %.
  • the luminescent liquid is formed by dispensing luminescent liquid A and luminescent liquid B produced by KPL.
  • the concentrated wash was a 20 X PBS wash.
  • the ROC curve analysis results show that the area under the ROC curve of the keratin 18-3A9 kit (0.743) is larger than the ROC curve of the Roche CA72-4 kit.
  • the surrounding area (0.612), the keratin 18-3 A9 kit for the diagnosis of gastric cancer is better than the CA72-4 kit.
  • Recombinant cytokeratin 19 fragment GY20 is expressed as E. coli BL21 (DE3) E. coli.
  • cDNA encoding various cytokeratin 19 fragments was prepared by RT-PCR: a) template and primers
  • RNA was isolated from a HELA human cancer cell line.
  • the cDNA was then synthesized using a reverse transcription kit (Promega) according to the instructions.
  • the obtained cDNA is a template for PCR. Primers of the three fragments were designed, and each cDNA was subjected to PCR amplification. The fragment number and amino acid sequence are shown in Table 2-1. Number and sequence of recombinant cytokeratin 19 fragments
  • cDNA template 5 ⁇ ⁇ ;
  • dNTP 2.5 mM each, 4 ⁇ 1 total
  • Taq polymerase (Promega): 5U;
  • the solution was heated to 94 ° C for 2 min, then cycled 40 times, each cycle was set to: heating 94 ° C 30s, 52 ° C lmin, 72 ° C 3min. After the procedure is completed, the reaction solution is heated to 72 °C. 10min.
  • the amplified DNA fragment was then taken and separated using a 1% agarose gel containing 0.25 g/ml ethidium bromide, see Figure 11. The results showed that the desired cytokeratin 19 fragment cDNA fragment was contained in the band, and then the DNA fragment was recovered using Gene Clean kit (BIO101, Irvine, CA).
  • the cDNA fragment was cloned using TOPO100 expression Cloning Kit (Invitrogen, Carlsbad, CA).
  • the ampicillin-resistant clones can be screened on the surface of the medium, and a single colony is picked up in a platinum wire coating circle and placed in an L-broth medium (containing ampicillin 100 g/ml), cultured at 37 ° C, overnight, vibrating Shake (200 rpm).
  • L-broth medium containing ampicillin 100 g/ml
  • the recombinant expression plasmids TOPO-GY20, TOPO-GY21, TOPO-GY22 have been obtained by restriction enzyme digestion, as shown in Fig. 13.
  • the cDNA encoding various cytokeratin 19 has been inserted into the TOPO plasmid.
  • the obtained plasmid was transformed into E. coli BL21 (DE3), and then cultured in an LB medium to an exponential growth phase, and induction was induced with isopropanol ⁇ -thiogalactoside (IPTG) for 3 h.
  • IPTG isopropanol ⁇ -thiogalactoside
  • the cells were pelleted, resuspended in lysate (8M urea, 20 mM Tris-HCl), and disrupted by ultrasonication.
  • Protein concentration was measured using BCA reagent (Pierece, Woburn, MA).
  • Sephadex G-50 was activated and the recombinant cytokeratin 19 fragment was purified by molecular sieve chromatography.
  • Sephadex G-50 was dissolved in 100 mM Tris-HCl buffer (pH 7.4), boiled at 100 °C for 10 minutes, then blocked with 100 mM Tris-HCl buffer (pH 7.4), and stored at 4 °C.
  • the bacterial disrupted concentrate was passed through 2 ml of gel beads at a flow rate of 2 ml / min. After the sample was passed, rinse with 50 ml of PBS, elution buffer 0.1 M glycine (pH 2.4), 0.15 M NaCl.
  • the eluent was measured in the ultraviolet OD280nm to determine whether the elution was completed.
  • the effective eluate (OD>0.01) was collected and placed in a dialysis bag and dialyzed against 1 L of phosphate buffer (pH 7.5) at 4 ° C, during which time the dialysis buffer was replaced twice.
  • the purified protein shield was concentrated to about 1 mg/ml and added to 1%. NaN 3 , stored at 4 ° C. Protein purity was determined by scanning with a 10% SDS-PAGE and scanning with a GDS8000 gel imaging system.
  • the immunogen used to prepare the monoclonal antibody is the recombinant full-length human cytokeratin 19, which is a gift from Professor Zhou Bing of UAB University.
  • Emulsified granules were formed by mixing 1 ml of the antigenic protein solution with the same volume of Freund's complete adjuvant, and each foot was injected with 100 ⁇ l.
  • Emulsified particles were formed by mixing 1 ml of the antigenic protein solution with the same volume of Freund's incomplete adjuvant, and 100 ⁇ l per foot was injected.
  • Hybridoma Lymphocytes were isolated from the axilla and groin of immunized mice, mixed with NS1 myeloma cells in a ratio of 2:1, washed twice with serum-free RPMI 1640, and added with 1 ml of pre-warmed PEG1500 at 37 °C, slightly at 37 °C. The cells were mixed for 1 minute, and then 20 ml of serum-free RPMI 1640 medium preheated to 37 ⁇ was slowly added dropwise over 3 minutes. After centrifugation, the fused cells were suspended in 12% FBS RPMI 1640 HAT selection medium. The cells were added to 5 96-well cell culture plates, ⁇ /well.
  • the ELISA plate was coated with the above keratin 19 antigen (1 ⁇ g/ml) at 4 ° C overnight; washed 3 times with PBS, and blocked with 3% (w/v) BSA in PBS for 1 h at room temperature.
  • the supernatant of ⁇ cell culture solution was added to each well during the test; the bath was incubated at 37 ° C for 30 minutes, washed with PBST washing solution for 5 times, and patted dry to add peroxidase-conjugated goat anti-mouse immunoglobulin (HRP-GAM Ig, DAKO company), warm bath at 37 °C for 30 minutes, remove it and wash it with PBST washing solution for 5 times.
  • HRP-GAM Ig peroxidase-conjugated goat anti-mouse immunoglobulin
  • substrate solution A and B each 50 ⁇ 1 (substrate liquid ⁇ composition: 13.42g Na 2 HP0 4 .12H 2 0, 4.2g citric acid ⁇ ⁇ 2 0 and 0.3g hydrogen peroxide, adjusted volume of 700ml in deionized water; color component B component: 0.2g tetradecyl benzidine, 20ml dimercapto amide Adjust the volume in deionized water to 700ml), develop color at 37 °C for 10 minutes, add 50 ⁇ 1 stop solution (2 ⁇ H 2 S0 4 ) to terminate the reaction, and check the OD 45 o value of each well on the microplate reader to OD 45 o Values above 2.0 are considered positive.
  • substrate liquid ⁇ composition 13.42g Na 2 HP0 4 .12H 2 0, 4.2g citric acid ⁇ ⁇ 2 0 and 0.3g hydrogen peroxide, adjusted volume of 700ml in deionized water
  • color component B component 0.2g tetradecyl benzidine, 20ml dimercapto
  • mice 16 weeks old healthy Balb/c mice were injected intraperitoneally with 0.5 ml pristane for 5-7 days.
  • the cloned hybridoma cells were collected, centrifuged to remove the supernatant, and serum-free medium was added to adjust the cell density to 2 ⁇ 10 5 ⁇ 2 ⁇ 10 6 /ml, 0.5ml per mouse. After 7 ⁇ 10 days, the abdomen of the mice increased, and the ascites began to be collected. Centrifuge at 3000rpm for 15 minutes, absorb the middle clear part of the liquid, 0.45 ⁇ microporous membrane Filter and sterilize, store at -20 °C after dispensing.
  • the treated ascites was diluted 5 times with 0.02 mol/L PBS (81 ml 0.2 mol/L Na 2 HP0 4 , 19 ml 0.2 mol/L NaH 2 PO 4 plus physiological saline to 100 ml), and 50 ml was taken.
  • the antibody bound to the column was eluted using 0.1 M Glycine-HCL pH 2.4 buffer, and the eluted fraction 2 ml/tube was collected, and finally all OD 28 was collected. Elution components greater than 0.1 were mixed and then neutralized with 1/10 volume of 1 M Tris-HCL pH 8.5 solution. Dialysis was then carried out overnight in PBS solution during which the dialysate was changed twice.
  • HRP horseradish peroxidase
  • the principle is that the glycosyl group of HRP is oxidized to an aldehyde group by sodium periodate, and after adding an antibody IgG, the aldehyde group is combined with an IgG amino group to form a Schiff base.
  • the amino group is blocked with dinitrofluorobenzene before being oxidized by sodium periodate.
  • the Schiff base was stabilized with sodium borohydride.
  • Enzyme amount (mg/ml) OD 40 3 X 0.4
  • IgG amount (mg/ml) (OD 280 -OD 403 x 0.3 ) ⁇ .62
  • the molar ratio (E/P) the amount of enzyme x4 / IgG, usually between 1-2.
  • Enzyme binding rate enzyme amount X volume / antibody
  • the labeling rate is generally 0.3-0.6, that is, 1-2 HRP molecules are bound to one antibody molecule, and the labeling rate can be greater than 0.6, 0.8, 0.9; OD 4 . 3 / OD 28 .
  • E/P is about 1.
  • Enzyme activity and antibody activity can be determined by ELISA, immunodiffusion, DAB-H 2 2 color reaction to determine the enzyme activity of the enzyme conjugate, antibody activity and potency, specificity.
  • Table 2-3 summarizes the results of cross-pairing experiments with 5 pairs of monoclonal antibodies.
  • antibody pairing with 2G2/5H2-HRP, 5H2/2G2-HRP, 5H2/1D11-HRP showed a strong positive reaction to lung cancer serum, and a negative reaction to normal human serum. Therefore, we determined that serum keratin 19 detected by these antibody pairs is highly correlated with lung cancer.
  • Three pairs of antibodies highly correlated with lung cancer keratin 19 were selected from the above cross-antibody pairing experiments to pair 2G2/5H2-HRP, 5H2/2G2-HRP, 5H2/1D11-HRP, and clinically demonstrated by ELISA.
  • the 96-well plates were coated with lg/ml of purified antibody, overnight at 4 ° C, and then blocked with 3% BSA in PBS for 1 h at room temperature. 50 ⁇ l of each lung cancer serum and 10 normal human serum were separately taken as test samples, and then 50 ⁇ l of 1:1000 enzyme-labeled antibody was added.
  • test data is used as a scatter plot, preliminary observation and comparison diagnosis Sensitivity and specificity, see Figure 15.
  • the serum detection value of 1 lung cancer was within the range of normal human serum detection values, and the serum detection values of other lung cancers were higher, with a specificity of 100%, the sensitivity was 90%; 5H2/ In the 2G2-HRP test, the serum detection values of 2 cases of lung cancer were within the range of normal human serum detection, and the serum detection values of 4 cases of lung cancer were low, with a specificity of 100%, the sensitivity was 80%; 5H2/1D1 1 -HRP was detected The serum detection values of 4 cases of lung cancer were within the range of normal human serum detection values, and the serum detection values of all lung cancers were lower than the previous two tests, with a specificity of 100%, the sensitivity was 60%; therefore, 2G2/5H2- HRP pairing has the best diagnostic performance for serum.
  • the monoclonal antibody 2G2 binds to antigen 1 ⁇ 4 and is positively reactive with all C-terminal deletions. Therefore, the antigen binding epitope of mAb 2G2 is located within a fragment of cytokeratin 19 aa 375-400.
  • the monoclonal antibody 5H2 was positively reacted with all N-terminal deletion fragments and positively reacted with one N-terminal fragment (aal ⁇ 375) and negatively with the other three N-terminal fragments.
  • the monoclonal antibody 5H2 binding epitope is within the fragment of cytokeratin 19 aa 325-350.
  • the binding epitopes of the monoclonal antibody 2G2 and the monoclonal antibody 5H2 are shown in Figure 16:
  • ELISA plates were coated with purified keratin 19 antigen. The supernatant of each monoclonal antibody was added to each well and incubated at 37 ° C for 30 min. Washed with PBST 5 times on a TEC AN automatic plate washer at intervals of 20 seconds, decanted and added to the appropriate dilution of HRP- Goat anti-mouse IgM, IgG1, IgG2a, IgG2b, IgG3 antibody (Serotec) enzyme-labeled secondary antibody, incubated at 37 ° C for 30 min; washed with PBST 5 times on TECAN automatic plate washer, each time interval 20 sec, buckle Dry, add 1 drop of each of the color developing solutions A (H 2 0 2 ) and B (TMB), develop color at 37 ° C for 10 min, add 1 drop of stop solution ( 2M H 2 S0 4 ); measure on TECAN microplate reader OD 45 () nm (reference wavelength is 620 nm), the cutoff
  • Substrate liquid ⁇ composition 13.42g Na 2 HP0 4 12H 2 0 4.2g citric acid ⁇ ⁇ 2 0 and 0.3g hydrogen peroxide, adjusted volume in deionized water is 700ml; color developing liquid B component is: 0.2g tetradecyl benzidine, 20ml dimercapto amide
  • the volume was adjusted to 700 ml in deionized water, and the color was developed at 37 ° C for 10 minutes.
  • the reaction was stopped by adding 50 ⁇ l of stop solution (2M H 2 S0 4 ), and the OD 45 of each well was detected on a microplate reader. value. The results are shown in Figure 17.
  • the monoclonal antibodies 2G2 and 5H2 are monoclonal antibodies specific for keratin 19.
  • Keratin 19-2G2 Assay Kit (Chemical Luminescence Method) A solid-phase 96-well luminescent plate is used as a reaction vector to form a double antibody sandwich method with a pair of monoclonal antibodies against keratin 19 associated with lung cancer, using highly sensitive Chemiluminescence detection technology quantitatively detects keratin 19 fragments in serum of patients with lung cancer.
  • the K19 kit was prepared by using K19 mAb 2G2 as a coating antibody and K19 mAb 5H2 as a labeled antibody.
  • the main components of the kit are: calibrator, coating plate, enzyme conjugate, luminescent solution, concentrated washing solution.
  • the standard is purified keratin 19 .
  • the coated antibody was K19 mAb 2G2.
  • the labeled antibody was K19 mAb 5H2 and was labeled with HRP horseradish peroxidase to form an antibody-HRP complex (enzyme conjugate).
  • the coated board is coated with monoclonal antibody 2G2, which is washed, sealed, dried and vacuum-packed.
  • K19 mAb 2G2 was coated with citrate buffer pH 4.8 as a coating buffer at a coating concentration of 5 g/mL.
  • the blocking solution was 0.02 M PBS + 0.4% casein +1% BSA + 0.5 M NaCl + 0.2% gelatin + 10% bovine serum + 0.05% Tween-20 + 1%. Preservative + 2.5% sucrose.
  • the labeled antibody is 5H2-HRP.
  • the dilution is 0.02M PBS + 0.4% casein
  • the luminescent liquid is formed by dispensing luminescent liquid A and luminescent liquid B produced by KPL.
  • the concentrated wash was a 20 x PBS wash.
  • the false positive rate of the kit for diagnosis of patients such as inflammation is an important factor affecting the diagnosis.
  • 150 patients with pneumonia were tested for serum samples to compare the K19 kit and the CYFRA21-1 kit for differential diagnosis.
  • the results showed that the false positive rate of K19 kit for serum samples of patients with inflammation was 5.3%, and the false positive rate of CYFRA21-1 kit for serum samples of patients with inflammation was 12.0%.
  • the false positive rate of the K19 kit is lower than the CYFRA21-1 kit, see Table 2-6.
  • the test results show that the kit can distinguish the serum of patients with inflammation from the serum of patients with lung cancer, and has good specificity.
  • Table 2-6 Statistics of serum test results in patients with inflammation
  • CYFRA21-1 kit 18 132 From the results of ROC curve analysis in Figure 18, the area under the ROC curve corresponding to the keratin 19-2G2 kit (0.817) is larger than the area under the ROC curve of the CYFRA21-1 kit of Roche (0.766), keratin 19- The 2G2 kit is superior to the CYFRA21-1 kit in diagnosing lung cancer.

Description

用于诊断上皮源性癌症的血液标志物及其单克隆抗体 技术领域
本发明涉及癌症的诊断, 更具体地说, 本发明涉及细胞角蛋白片 段作为诊断上皮源性癌症的标志物的用途。 背景技术
对于癌症患者的成功治疗而言最重要的因素之一是早期检测。 随 着基因分析及蛋白质组学的发展, 在鉴定可用于诊断及预测特定癌症 的分子标记方面已取得了显著的进步。
上皮源性癌症是指起于上皮细胞的癌症, 包括但不限于乳腺癌、 胃癌、 口腔癌、 食管癌、 结肠癌、 肝癌、 膀胱癌、 胰腺癌、 卵巢癌、 宫颈癌、 肺癌、 乳腺癌和皮肤癌、 前列腺癌、 肾癌等。
例如, 胃癌是世界上第二大癌症死因, 是威胁人类健康最常见的 恶性肿瘤之一。 胃癌患者预后的关键是作好二级预防, 即早期发现和 早期治疗。 胃癌的早期及时准确检出和治疗, 对于降低胃癌死亡率具 有十分重要的意义。
肺癌是一种常见的肺部恶性肿瘤, 近年来, 随着吸烟和各种环境 因素的影响,世界各国特别是工业发达国家, 肺癌的发病率和病死率均 迅速上升。 研发具有高度特异性和敏感性的血清肿瘤标记物对肺癌早 期的发现及治疗具有很重要的意义。
分子诊断是诊断癌症最流行的方法。 不同类型的分子如 DNA、 蛋 白质和脂肪被用作诊断工具。 肿瘤标记物 (Tumor Markers,TM ) , 是 指在肿瘤发生和增殖过程中, 由肿瘤细胞所产生或分泌并释放到血液、 细胞、 体液中, 反映肿瘤存在和生长的一类物质。 肿瘤标记物通常是 蛋白质, 已经广泛应用于多种类型癌症的检测和诊断。 肿瘤标记物浓 度水平上升, 可能表明了一种癌症在人体内的某种形式的存在。 肿瘤 标记物在临床上主要用于对原发肿瘤的发现、 肿瘤高危人群的筛选、 良性和恶性肿瘤的鉴别诊断、 肿瘤发展程度的判断、 肿瘤治疗效果的 观察和评价以及肿瘤复发和预后的预测等。 肿瘤标记物常用免疫法测 定, 如间接法、 双抗体夹心法、 竟争法。 检测手段有胶体金法、 酶 免疫法、 化学发光法、 电化学发光法等。 血清学检测的目的在于测定 病人血清中与癌症相关的肿瘤标记物的含量。 此方法简单易行, 适用 于大量人群的普查。 例如, 目前国际上普遍应用的与胃癌相关的肿瘤 标记物包括 CEA, TPS以及 CA72-4等。
细胞角蛋白(Cytokeratins)包括 20多种不同蛋白, 是细胞骨架的重 要成分。 尽管所有上皮源的细胞均表达一定水平的细胞角蛋白, 但是 某些角蛋白的组分例如角蛋白 8、 18以及 19与恶性肿瘤的发生与进展 密切相关 ( M. Nap, Th. Van Wei, C. Andres, et al. Immunohistochemical Profiles of 30 Monoclonal Antibodies against Cytokeratins 8, 18 and 19[J]. Tumor Biology 2001;22:4-10 ) 。
细胞角蛋白 18 (Cytokeratin 18)是一分子量为 55kD的酸性蛋白质, 由 430个氨基酸组成, 具有高度保守的 α螺旋结构中心区域, 呈丝状结 构。 广泛分布于正常组织表面如复层上皮和鳞状上皮, 以及单层上皮 细胞如腺泡、 气管、 乳腺导管、 汗腺、 子宫内膜、 结肠和肝细胞等。 在正常上皮细胞中细胞角蛋白 18表达相对稳定, 细胞角蛋白 18及其片 段在外周血、 骨髓、 淋巴结中无表达或低表达, 几乎没有片段产生释 放入血。 相反的是, 当上皮细胞恶性转化时, 细胞角蛋白 18表达急剧 增高。 同时, 细胞角蛋白 18生长进程发生异常。 在肿瘤细胞凋亡和坏 死过程中激活的蛋白酶加速了细胞的降解, 使得大量可溶性的细胞角 蛋白 18片段被释放, 造成组织液、 体液中可溶性的细胞角蛋白 18片^ 的浓度升高, 尤其在胃癌患者血循环中含量丰富(Stig L, Aleksandra M H ,Takayuki U, et al. Determining tumor apoptosis and necrosis in patient serum using cytokeratin 18 as a biomarker[J]. Cancer Letters 214(2004): 1-9; T. Stigbrand. The Versatility of Cytokeratins as Tumor Markers[J]. Tumor Biology 2001;22: 1-3 ) 。
细胞角蛋白 19是一分子量为 40kD的酸性蛋白质, 是角蛋白家族中 最小的成员, 由 400个氨基酸组成, 具有高度保守的 α螺旋结构中心区 域, 呈丝状结构。 广泛分布于正常组织表面如复层上皮和鳞状上皮, 以及单层上皮细胞如腺泡、 气管、 乳腺导管、 汗腺、 子宫内膜、 结肠 和肝细胞等。 在正常上皮细胞中细胞角蛋白 19表达相对稳定, 细胞角 蛋白 19及其片段在外周血、 骨髓、 淋巴结中无表达或低表达, 几乎没 有片段产生释放入血循环系统。 相反的是, 当上皮细胞恶性转化时, 细胞角蛋白 19表达急剧增高。 同时, 细胞角蛋白 19生长进程发生异常。; 在肿瘤细胞凋亡和坏死过程中激活的蛋白酶加速了细胞的降解, 使得 大量可溶性的细胞角蛋白 19片段被释放, 造成组织液、 体液中可溶性 的细胞角蛋白 19片段的浓度升高, 尤其在肺癌患者血循环中含量丰富 ( J. Niklinski, T. Burzykowski, W. Niklinska,et al. Preoperative CYFRA 21-1 level as a prognostic indicator inresected nonsmall cell lung cancer [J]. Eur Respir J 1998; 12: 1424-1428 ) 。 发明内容
本发明一方面提供了与上皮源性癌症有关的细胞角蛋白片段, 其 中该片段包含选自由 SEQ ID NOs: 2、 3、 5和 6构成的组的抗原表位。 在 一个具体实施方式中,该片段包含 SEQ ID NO: 1的氨基酸残基 200-400。 在另一个具体实施方式中, 该片段包含 SEQ ID NO: 2的氨基酸残基 325-400。
本发明另一方面提供了与所述抗原表位特异性结合的单克隆抗 体。 在一个具体实施方式中, 该单克隆抗体由保藏号为 CGMCC No. 1957、 CGMCC No. 1956、 CGMCC No. 1955或 CGMCC No. 1952的杂交 瘤产生。
本发明还提供了所述单克隆抗体的抗原结合部分, 其中该抗原结 合部分与所述单克隆抗体竟争结合所述抗原表位。 在一个具体实施方 式中, 该抗原结合部分为人源化抗体。 在另一个具体实施方式中, 该 抗原结合部分为嵌合抗体。
本发明一方面提供了用于对受试者中的上皮源性癌症进行早期筛 查、 诊断或预后评估的方法, 该方法包括: 获得来自受试者的生物样 品, 检测该生物样品中本发明的细胞角蛋白片段的含量, 和将该含量 与阈值水平进行比较。 如果该含量超过阈值水平则说明该患者可能患 有癌症。
在一个优选实施方式中, 该方法还涉及与其他上皮源性肿瘤标记 物联用。 具体而言, 该方法包括: 获得来自受试者的生物样品, 检测 该生物样品中本发明的细胞角蛋白片段的含量, 检测该生物样品中其 他上皮源性肿瘤标记物的含量, 和将所述细胞角蛋白片段以及所述其 他上皮源性肿瘤标记物的含量与阈值水平进行比较。 优选地, 所述其 他上皮源性肿瘤标记物选自由 AFP、 CEA、 CA242、 CA19-9、 CA72-4、 CA125、 CA15-3、 NSE、 SCCA、 Cyfra21-1、 PSA, free PSA构成的组。 本发明另一方面提供了评估用于治疗上皮源性癌症的药物或疗法 的治疗效果的方法, 包括: 向患有上皮源性癌症的受试者施用所述药 物或疗法, 在施用所述药物或疗法之前和之后采集所述受试者的生物 样品, 和检测所述生物样品中本发明的细胞角蛋白片段的含量, 其中 在施用所述药物或疗法之前和之后该细胞角蛋白片段含量明显降低 的, 表明所述药物或疗法有明显疗效。
优选地, 所述上皮源性癌症选自由胃癌、 肝癌、 肺癌、 胆嚢癌、 乳腺癌、 宫颈癌、 卵巢癌、 结肠癌、 前列腺癌、 肾癌、 食道癌、 肠癌、 膀胱癌构成的组。
优选地, 所述生物样品选自由血液、 血清、 组织液、 尿液、 大便、 痰液、 脑脊液、 唾液、 眼泪和乳头吸出液构成的组。
本发明另一方面提供了一种试剂盒, 包含: 固定于固相载体上的 能够特异结合本发明的细胞角蛋白片段的包被抗体, 以及被可检测地 标记的能够特异结合该细胞角蛋白片段的检测抗体。 该试剂盒可以用 于实施本发明的方法, 也可用于检测本发明的细胞角蛋白片段。
本发明还提供了所述细胞角蛋白片段的特异结合剂, 例如本发明 的单克隆抗体或者抗原结合片段, 在制备用于诊断上皮源性癌症的试 剂中的用途。 附图说明
图 1是一幅流程图, 简要描述了细胞角蛋白 18片段制备和活性分析 的步骤。
图 2是一幅电泳图, 显示了细胞角蛋白 18片段 cDNA的电泳结果。 图 3显示了 TOPO质粒的结构。 其中, Ampicillin是指氨苄青霉素,
Neomycin是指新尊素。
图 4是一幅电泳图 , 显示了重组表达质粒 TOPO-GY10、 TOPO-GY1 K TOPO-GY12的酶切鉴定结果。 其中, marker是指标志物。
图 5是一幅电泳图, 显示了重组细胞角蛋白 18片段 GY10、 GY11、 G Y 12的 SDS-PAGE结果。
图 6显示了最佳抗体配对实验的结果。
图 7是一幅示意图, 显示了单克隆抗体 3A9、 2A6的结合表位的位 置。
图 8显示了单克隆抗体 3A9及 2A6与其他人血清肿瘤标记物的免疫 检测结果。
图 9显示了胃癌与非胃癌诊断的 ROC曲线。 其中, specificity是指特 异性, sensitivity是指灵敏度。
图 10是一幅流程图, 简要描述了细胞角蛋白 19片段制备和活性分 析的步骤。
图 11是一幅电泳图, 显示了细胞角蛋白 19片段 cDNA的电泳结果。 图 12显示了 TOPO质粒的结构。 其中, Ampicillin是指氨苄青霉素, Neomycin是指新霉素。
图 13是一幅电泳图, 显示了重组表达质粒 TOPO-GY20、 TOPO-GY21 , TOPO-GY22的酶切鉴定结果。其中, marker是指标志物。
图 14是一幅电泳图, 显示了重组细胞角蛋白 19片段 GY20、 GY2K GY22的 SDS-PAGE结果。
图 15显示了最佳抗体配对实验的结果。
图 16是一幅示意图, 显示了单克隆抗体 2G2、 5H2的结合表位的位 置。
图 17显示了单克隆抗体 2G2、 5H2与其他人血清肿瘤标记物的免疫 检测结果。
图 18显示了肺癌与非肺癌鉴别诊断 ROC曲线。 其中, specificity是 指特异性, sensitivity是指灵敏度。 相关序列的描述
SEQ ID ΝΟ:1显示了细胞角蛋白 18 (简称 K18 )的全长氨基酸序列, 长度为 430个氨基酸残基。
SEQ ID NO:2显示了单克隆抗体 3A9识别的 K18抗原表位的氨基酸 序列,长度为 51个氨基酸残基,对应于 SEQ ID NO:l的氨基酸位置 200 ~ 250。
SEQ ID NO:3显示了单克隆抗体 2A6识别的 K18抗原表位的氨基酸 序列,长度为 51个氨基酸残基,对应于 SEQ ID NO:l的氨基酸位置 350 ~ 400。
SEQ ID NO:4显示了细胞角蛋白 19 (简称 K19 )的全长氨基酸序列, 长度为 400个氨基酸残基。
SEQ ID NO:5显示了单克隆抗体 2G2识别的 K19抗原表位的氨基酸 序列,长度为 26个氨基酸残基,对应于 SEQ ID NO:4的氨基酸位置 375 - 稱。
SEQ ID NO:6显示了单克隆抗体 5H2识别的 K19抗原表位的氨基酸 序列,长度为 26个氨基酸残基,对应于 SEQ ID NO:4的氨基酸位置 325 - 350。 具体实施方式
本发明提供了细胞角蛋白 18( K18 )的两个上皮源性癌症相关表位。 两个癌相关表位均位于细胞角蛋白 18氨基酸序列的 C端, 其序列如 SEQ ID NOs: 2、 3所示。 所述表位在恶性转化期间, 特别是在胃, 结肠, 乳 腺, 卵巢, 肾脏等组织的恶性转化期间变得暴露。 所述癌相关表位的 暴露, 是通过肿瘤细胞的不断增殖、 坏死、 凋亡而将细胞角蛋白 18片 段释放于血液中而暴露的。 而正常组织和健康人员血液中没有这两个 癌相关表位的表达。 本发明提供了分别识别这两个癌相关表位的两抹 单克隆抗体 3A9和 2A6, 并应用这两林单抗发明了诊断癌症的方法。 用 于生产 K18单抗 3A9和 2A6的杂交瘤于 2007年 3月 8日保藏于中国微生物 菌种保藏管理委员会普通微生物中心(CGMCC, 北京市海淀区中关村 北一条 13号), 保藏编号分别为 CGMCC NO. 1957和 1956。 保藏证明中 的信息通过援引并入本文, 作为本发明原始公开内容的一部分。 序列如 SEQ ID NOs: 5、 6所示。本发明提供了分别识别这两个癌相关表 位的两株单克隆抗体 2G2和 5H2, 并应用这两株单抗发明了诊断癌症的 方法。 用于生产 K19单抗 2G2和 5H2的杂交瘤于 2007年 3月 8日保藏于中 国微生物菌种保藏管理委员会普通微生物中心(CGMCC, 北京市海淀 区中关村北一条 13号), 保藏编号分别为 CGMCC NO. 1955和 1952。 保 藏证明中的信息通过援引并入本文, 作为本发明原始公开内容的一部 分。
本发明一方面提供了与上皮源性癌症有关的细胞角蛋白片段, 其 中该片段包含选自由 SEQ ID NOs: 2、 3、 5和 6构成的组的抗原表位。
本文所用的术语 "上皮源性癌症" 是指起于上皮细胞的癌症, 包 括但不限于乳腺癌、 基底细胞癌、 腺癌、 胃肠癌、 唇癌、 口腔癌、 食 管癌、 小肠癌和胃癌、 结肠癌、 肝癌、 膀胱癌、 胰腺癌、 卵巢癌、 宫 颈癌、 肺癌、 乳腺癌和皮肤癌例如鳞状细胞和基底细胞癌、 前列腺癌、 腎细胞癌及其它已知通过身体影响上皮细胞的癌症。
本文所用的术语 "片段" 是指与天然的全长蛋白相比, 缺少一个 或多个氨基酸残基的氨基酸序列。 例如, 蛋白片段可以通过从全长蛋 白中删除部分残基而获得, 例如 N段截短片段可以通过将位于蛋白 N段 的部分氨基酸残基删除而获得。
蛋白片段中也可以含有外源序列, 从而形成嵌合蛋白片段。 例如, 将蛋白 A的 N端结构域删除后, 与蛋白 B的 N端结构域连接, 可以形成蛋 白 A和蛋白 B的嵌合片段。 本领域技术人员可以理解, 该嵌合片段的长 度有可能超过蛋白 A的长度。
为了便于纯化,可以在蛋白片段中添加常用的纯化标签, 例如 GST (谷胱甘肽巯基转移酶)、 His-tag (六聚组氨酸) 等。
本发明的细胞角蛋白片段可以含有抗原表位附近的一段连续的氨 基酸序列。
例如, 本发明的 K18片段可以包含 SEQ ID ΝΟ:1中涵盖 SEQ ID NO:2或 3所示抗原表位的至少 60、 70、 80、 90、 100、 120、 150、 180、 200、 210、 220、 250、 280、 300、 320、 350、 380、 400或 420个连续的 氨基酸残基。例如,在本发明一个实施方式中, K 18片段包含 SEQ ID NO: 1的氨基酸残基 1-250, 涵盖单抗 3A9识别的抗原表位; 在另一个实施方 式中, K18片段包含 SEQ ID NO: 1的氨基酸残基 250-430, 涵盖单抗 2A6 识别的抗原表位; 在又一个实施方式中, K18片段包含 SEQ ID NO: 1的 氨基酸残基 150-430, 同时涵盖两个抗原表位。 可以将包含 K18的一个 或两个抗原表位的片段与 K19的抗原表位融合在一起, 形成嵌合片段。
本发明的 K19片段可以包含 SEQ ID NO:4中涵盖 SEQ ID NO:5或 6 所示抗原表位的至少 30、 50、 60、 70、 80、 90、 100、 120、 150、 180、 200、 210、 220、 250、 280、 300、 320、 350或 380个连续的氨基酸残基。 例如, 在本发明一个实施方式中, K19片段包含 SEQ ID NO: 4的氨基酸 残基 1-375, 涵盖单抗 5H2识别的抗原表位; 在另一个实施方式中, K19 片段包含 SEQ ID NO: 1的氨基酸残基 150-400, 同时涵盖单抗 2G2和 5H2 识别的抗原表位。 通过本领域公知的分子克隆实验技术可以容易地制备包含特定氨 基酸残基的蛋白片段。
本文所用的 "生物样品"是指从患者 (优选病人)获得的生物材料样 品, 包括組织样品、 细胞样品 (例如组织活检样品, 例如吸出活检样品、 刷拭活检样品、 表面活检样品、 针吸活检样品、 钻取活检样品、 切除 活检样品、 开胸腹活检样品、 切开式活检样品或内窥镜活检样品)和肿 瘤样品。 生物样品也可为生物流体样品。 在一个优选实施方案中, 生 物样品为血清。 然而, 也可使用血液、 尿液、 唾液、 脑脊液、 乳头吸 出液和细胞裂解物上清液等。
如本文所述, 可通过本领域技术人员所知的任何手段来测量本发 明细胞角蛋白片段的水平。 在本发明中, 一般优选使用抗体或抗体的 抗原结合部分来检测生物样品中的细胞角蛋白片段的水平。 然而, 其 它方法也可使用, 例如通过分析 mRNA转录物来测量细胞角蛋白片段 的表达。
mRNA 水平的评价方法为本领域技术人员所熟知。 举例而言, 通 过 RNA印迹法可完成 RNA转录物的检测,其中 RNA制备物在变性琼 脂糖凝胶上进行, 并转移至合适支持物上, 例如活性纤维素膜、 硝酸 纤维素膜或玻璃膜或尼龙膜。然后使标记 (例如放射性标记)的 cDNA或 RNA与该制备物杂交, 洗涤, 并通过例如放射自显影等方法来分析。 或者, 可在 DNA阵列、 芯片或微阵列上检测 mRNA表达。 举例而言, 为监测 mRNA水平, 从待测生物样品中提取 mRNA, 逆转录, 产生荧 光-标记的 cDNA探针。 然后用标记的 cDNA探针探测能够与细胞角蛋 白片段 cDNA杂交的微阵列, 扫描载玻片, 测量荧光强度。 该强度和 杂交强度与表达水平相关。
也可测量细胞角蛋白片段水平, 尤其是当该生物样品为流体样品 例如血液或尿液时。 在一个实施方案中, 通过让生物样品与特异性结 合细胞角蛋白片段的单克隆抗体接触, 来测量细胞角蛋白片段水平。
术语 "抗体的抗原结合部分" 包括与细胞角蛋白的抗原表位特异 性结合 (起免疫反应)的免疫球蛋白分子和免疫球蛋白分子的免疫学活 性决定簇, 例如含有抗原结合位点的分子。 术语 "抗体的抗原结合部 分" 意指包括全抗体, 例如任何同种型(IgG、 IgA、 IgM IgE 等等), 也包括抗体片段。 可使用常规技术使抗体片段化。 因此, 该术语包括 能够选择性地与某一蛋白反应的抗体分子的蛋白水解片段或重组制备 的部分。此等蛋白水解和 /或重组片段的非限制性实例包括 Fab、F(ab')2、 Fab'、 Fv、 dAbs及含有由多肽接头连接的 VL区和 VH区的单链抗体 (scFv)。 所述 scFv可共价或非共价连接形成含两个以上结合位点的抗 体。 因此, "抗体的抗原结合部分" 包括多克隆抗体、 单克隆抗体或 抗体的其它纯化制备物和重组抗体。 术语 "抗体的抗原结合部分" 还 指包括人源化抗体、 双特异性抗体及含至少一个源自抗体分子的抗原 结合决定簇的嵌合分子。 术语 "人源化抗体" 指源自非人抗体 (例如 鼠) 的保持或基本上保持亲本抗体抗原结合特性, 但在人中具有较低 免疫原性的抗体。 人源化抗体的优点是减少或消除了抗体在宿主中的 免疫原性, 从而增加生物利用度和减少可能的不利免疫反应。 可使用 CDR移植的方法制备人源化抗体。
本文所用的 "标记抗体" 包括以可检测的方式标记的抗体, 包括 但不限于酶标记抗体、 放射性标记抗体、 荧光标记抗体和化学发光标 记抗体。 也可用可检测标签例如 HA、 HSV、 FLAG或 HIS标记抗体。 细胞角蛋白片段的水平 Γ生物样品中存在 细胞角蛋白、片段^平与可 检测的标记抗体发射的信号强度相关。
在一个优选实施方案中, 可通过将抗体与酶连接, 来可检测地标 记抗体或抗体的抗原结合部分。 当该酶与其底物接触时, 将与该底物 反应, 其反应方式能产生可检测化学部分, 举例而言, 通过分光光度 法、 荧光测定法或通过视觉手段来检测。 可用于可检测地标记本发明 抗体的酶包括但不限于苹果酸脱氢酶、 辣根过氧化物酶、 碱性磷酸酶、 天冬酰胺酶、 葡萄糖氧化酶、 尿素酶、 过氧化氢酶、 葡萄糖淀粉酶和 乙酰胆碱酯酶。 化学发光是另一种可用于检测基于抗体或其抗原结合 部分的方法。
使用多种其它免疫测定中的任一种也可完成检测。 举例而言, 通 过放射性标记抗体, 通过使用放射免疫测定可以检测该抗体。 通过使 用例如 γ计数器或闪烁计数器或通过放射自显影等手段可检测放射性 同位素。
用荧光化合物标记抗体也是可行的。 当荧光标记的抗体暴露于合 适波长的光时, 由于荧光于是可检测其存在。 在最常用荧光标记化合 物中有异硫氰酸荧光素、 罗丹明、 藻红蛋白和荧光胺。
本发明一方面提供了用于对受试者中的上皮源性癌症进行早期筛 查、 诊断或预后评估的方法, 包括: 获得来自受试者的生物样品, 检 测该生物样品中细胞角蛋白片段的含量, 和将该含量与阔值水平进行 比较。 如果该含量超过阈值水平则说明该患者可能患有癌症或者预后 不佳。
在一个优选实施方式中, 采用本发明提供的单克隆抗体进行检测。 例如,可以仅使用一种单抗来检测 K18或 K19片段;可以组合使用 3A9 和 2A6来检测 K18片段; 可以组合使用 2G2和 5H2来检测 K19片段; 也可以组合使用 3A9和 2G2来同时检测 K18片段和 K19片段;或者同 时组合使用上述四种单克隆抗体进行检测。 可以理解, 组合使用几种 抗体可能提高诊断的准确性。
本文所用的术语 "3A9" 是指由保藏编号为 CGMCC NO. 1957的杂 交瘤生产的单克隆抗体 , 可以特异识别 K18上的抗原表位。
本文所用的术语 "2A6" 是指由保藏编号为 CGMCC NO. 1956的杂 交瘤生产的单克隆抗体, 可以特异识别 K18上的抗原表位。
本文所用的术语 "2G2" 是指由保藏编号为 CGMCC NO. 1955的杂 交瘤生产的单克隆抗体, 可以特异识别 K19上的抗原表位。
本文所用的术语 "5H2" 是指由保藏编号为 CGMCC NO. 1952的杂 交瘤生产的单克隆抗体, 可以特异识别 K19上的抗原表位。
在一个优选实施方式中, 该方法还涉及与其他上皮源性肿瘤标记 物联用, 例如 AFP、 CEA、 CA242、 CA19-9、 CA72-4、 CA125、 CA15-3、 NSE、 SCCA、 Cyfra21- PSA、 free PSA等常用肿瘤标记物。 对多个 肿瘤标记物进行组合检测对于提高诊断的准确性有帮助。
本发明另一方面提供了评估用于治疗上皮源性癌症的药物或疗法 的治疗效果的方法, 包括: 向患有上皮源性癌症的受试者施用所述药 物或疗法, 在施用所述药物或疗法之前和之后采集所述受试者的生物 样品, 和检测所述生物样品中本发明的细胞角蛋白片段的含量, 其中 在施用所述药物或疗法之前和之后该细胞角蛋白片段含量明显降低 的, 表明所述药物或疗法有明显疗效。 盒。 该试剂盒可以是本领 普道°技术 员所熟知的 何配置 " 用于实 施一种或多种本文所述的方法。 另外, 优选用包括于试剂盒中的一种 或多种标准品同时实施测试, 以便该测试结果可定量或确证。 例如, 标准品可以是纯化的 K18或 K19蛋白。
试剂盒包括检测细胞角蛋白片段水平的手段, 例如选择性结合细 胞角蛋白片段的抗体或抗体片段。 诊断检测试剂盒优选以标准双抗体 结合形式配制, 其中一种特异性抗体捕获患者样品中的细胞角蛋白片 段, 另一种特异性抗体用于检测捕获的细胞角蛋白片段。 举例而言, 将该捕获抗体 (包被抗体) 固定于例如测试板或硝酸纤维素膜等固相 上。 通常用可检测标记例如辣根过氧化物酶或放射性同位素来给第二 抗体即检测抗体作标签。
在一个优选实施方式中,试剂盒中组合使用 3A9和 2A6来检测 K18 片段。 在另一个优选实施方式中, 试剂盒中组合使用 2G2和 5H2来检 测 K19片段。也可以组合使用 3A9和 2G2来同时检测 K18片段和 K19 片段; 或者同时组合使用上述四种单克隆抗体进行检测。 可以理解, 组合使用几种抗体可能提高诊断的准确性。
在其它的实施方案中, 检测试剂盒可采用(但不限于)下列技术: 竟 争性和非竟争性测定、放射免疫测定 (RIA)、生物发光和化学发光测定、 荧光测定、夹层试验、免疫放射测定、斑点印迹、酶联测定(包括 ELIS A)、 微量滴定板和免疫细胞化学法。 对每一试剂盒, 测试的范围、 灵敏度、 准确度、 可靠性、 特异性和再现性均通过本领域技术人员所熟知的方 法来确立。
上述检测试剂盒将还提供使用说明书。
本发明由下列实施例作进一步说明。 提供这些实施例是为了帮助 理解本发明, 不得解释为对本发明的限制。 实施例 1
1. 抗角蛋白 18及片段的单克隆抗体的制备
1.1 免疫原——重组细胞角蛋白 18片段的制备
重组细胞角蛋白 18片段 GY10为 E.coli系 BL21(DE3)大肠扞菌表 达。 ·
1.1.1 研制方案
见图 1。 1.1.2 细胞角蛋白 18片段的 cDNA合成
通过 RT-PCR方法制备编码各种细胞角蛋白 18片段的 cDNA: a) 模板及引物
先从一种 HELA人癌细胞系中分离得到总 RNA。 然后根据说明书 用反转录试剂盒( Promega )合成 cDNA。 所获得的 cDNA即是 PCR的 模板。 设计三种片段的引物, 对各 cDNA进行 PCR扩增。 片段编号及 氨基酸序列见表 1-1。 重组细胞角蛋白 18片段的编号及序列
Figure imgf000013_0001
b) PCR反应
PCR反应溶液组分:
cDNA模板: 5 μ 1;
引物: 5'和 3'引物各 lOpmol
10 X PCR緩冲液: ΙΟ μ Ι;
dNTP: 各 2.5mM, 共 4 μ 1;
Taq聚合酶( Promega ) : 5U;
加入无菌双蒸水至 100 μ 1。
程序如下:
溶液加热至 94°C恒温 2min, 然后循环 40次, 每个循环设置为: 加 热 94。C 30s , 52 °C lmin, 72 °C 3min。 程序完成后, 反应溶液加热 72 °C 10min。 然后获取扩增的 DNA片段, 用含 0.25 g/ml溴化乙锭的 1%琼脂 糖凝胶分离, 见图 2。 结果显示条带中包含所需的细胞角蛋白 18片段 cDNA片段, 然后用 Gene Clean kit ( BIOIOI , Irvine,CA ) 回收 DNA片 段。
1.1.3 构建质粒并筛选
用 TOPO100 expression Cloning Kit(Invitrogen, Carlsbad, CA)克隆 cDNA片段。 (1)将从 PCR反应溶液回收的细胞角蛋白 18的 cDNA与克隆试剂 盒提供的 TOPO质粒 (图 3 ) 50ng混合;
(2)溶液中加入 10 X连接酶反应緩沖液(6mM Tris-HCl(pH7.5), 6mM MgCl, 5mM NaCl, 7mM β -巯基乙醇, 0.1 mM ATP, 2mM DTT, ImM 亚精胺, O.lmg/ml BSA ) ;
(3)再加入 4U T4 DNA连接酶(1 μ 1);
(4)用灭菌去离子水调整溶液体积至 10 μ ΐ;
(5)将其在 14°C温育 15h;
(6)取 2 μ 1加入到 50 μ 1感受态 E.coli 细菌株 TOP10F (由 TA克 隆试剂盒提供, 并按照说明书制成感受态, 混合物冰浴 30min, 然后孵 育 42°C 30s, 再冰浴 5min )
(7)配制 500 μ ΐ培养基, 含 2%(ν/ν)蛋白胨, 0.5%(w/v)酵母膏, 0.05%(w/v)NaCl, 2.5mM KC1, ImM MgCl和 20mM葡萄糖, 将 (6)加 入其中, 37°C温育 lh, 并振摇。
(8)在 L-肉汤琼脂平板 ( 1%(ν/ν)蛋白胨, 0.5%(w/v)酵母膏,
0.5%(w/v)NaCl, 0.1 %葡萄糖, 0.6%(w/v)bacto-agar(Difo, Detroit, MI) ) 涂铺 (7), 10(^g/ml。
(9)培养基表面可筛选出抗氨苄的克隆子, 用铂丝涂布圈挑出单菌 落放入 L-肉汤培养基(含氨苄 10(^g/ml ) 中 37°C培养, 过夜, 振摇 (200rpm)。
(10) 温育过后, 离心收集细菌, 用碱法提取 DNA质粒。
经酶切鉴定,已经得到重组的表达质粒 TOPO-GY10、TOPO-GY11、
TOPO-GY12, 如图 4。
1.1.4 重组蛋白的诱导表达和鉴定
编码各种细胞角蛋白 18的 cDNA已经插入 TOPO质粒中,
(1)所获质粒转化至 E.coli系 BL21(DE3), 然后在 LB培养基中培 养至指数增长期, 用异丙醇 β-硫代半乳糖苷 (IPTG)诱导表达 3h。
(2)沉淀细胞, 用裂解液 (8M urea, 20mM Tris-HCl)重悬, 并用超 声破碎。
(3)后 14,000 x g离心 15min,在 Ni柱纯化上清。用 PBS溶液透析 纯化蛋白, 4°C过夜。
(4)用 BCA试剂(Pierece, Woburn, MA)检测蛋白浓度。 1.1.5 重组细胞角蛋白 18的纯化
活化 Sephadex G-50, 利用分子筛层析纯化重组细胞角蛋白 18片 段。 将 Sephadex G-50溶于 100 mM Tris-HCl緩冲液 (pH 7.4), 在 100 °C煮沸 10分钟, 然后用 100 mM Tris-HCl緩冲液 (pH 7.4)封闭, 并在 4°C保存。 将细菌破碎浓缩液流经 2ml凝胶珠, 流速为 2 ml /min。 样品 通过后, 50毫升 PBS冲洗,洗脱緩冲液 0.1 M甘氨酸 (pH 2.4), 0.15 M NaCI 。 洗脱液于紫外 OD280nm测值判断洗脱是否完毕。 收集有效洗 脱液(OD>0.01 ) 置于透析袋用 1L的磷酸盐緩冲液( pH7.5 ) 4°C透 析, 期间更换 2次透析緩冲液。 将纯化的蛋白质浓缩到约 lmg/ml, 加 入 l %。NaN3, 4°C保存。 用 10 %的 SDS— PAGE检测, 并用 GDS8000 凝胶成像系统扫描分析蛋白纯度。
结果如图 5 , 结果显示获得了高纯度的重组细胞角蛋白 18 片段 GY10、 GY11、 GY12, 电泳测定纯度大于 95 %。
用于制备单克隆抗体的免疫原为基因重组全长人细胞角蛋白 18, 此蛋白由美国 UAB大学周铜教授馈赠。
1.2 免疫流程
动物: 6 ~ 8周龄雌性 Balb/c小鼠。
初次免疫(第 1天) : 采用 1ml抗原蛋白溶液与相同体积的弗氏 完全佐剂混勾形成乳化颗粒, 每只足底注射 100 μ 1。
二次免疫(第 7天) : 采用 1ml抗原蛋白溶液与相同体积的弗氏 不完全佐剂混勾形成乳化颗粒, 每只足底注射 100 μ 1。
强化免疫(第 14、 21、 28天): 采用不含佐剂的抗原(lmg/ml ) , 每只足底注射 100 μ 1。
融合(第 31天) 。
1.3 杂交瘤的建立
取免疫小鼠的胭窝及腹股沟分离淋巴细胞, 将其与 NS1骨髓瘤细 胞按 2:1比例混合, 用无血清 RPMI 1640洗涤 2次, 加入 lml37。C预热 的 PEG1500, 在 37°C轻微混合细胞 1分钟, 然后在 3分钟内緩慢滴加 20ml预热至 37°C的无血清 RPMI 1640培养基。 离心, 将融合细胞悬浮 于 12% FBS RPMI 1640 HAT 选择培养基。 将细胞加入 5块 96孔细胞 培养板中, ΙΟΟμΙ/孔。
1.4 ELISA检测筛选阳性克隆 在细胞融合后 7天, 用间接 ELISA法进行初次筛选, 所有杂交瘤 均用三种重组人细胞角蛋白 18抗原筛选: (1 )全长细胞角蛋白 18; ( 2 ) N端细胞角蛋白 18片段; (3 ) C端细胞角蛋白 18片段。
用上述角蛋白 18抗原 ( l g/ml ) 包被 ELISA板 4°C过夜; 用 PBS 洗涤 3次, 用含 3% (w/v) BSA的 PBS室温封闭 lh。 检测时每孔加入 ΙΟΟμΙ细胞培养液上清; 37°C温浴 30分钟, 用 PBST洗涤液洗 5遍, 拍干后加入过氧化物酶结合的羊抗鼠免疫球蛋白 (HRP-GAM Ig , DAKO公司) , 37°C温浴 30分钟, 取出后用 PBST洗涤液洗 5遍, 拍 干后先后加入底物液 A、 B 各 50μ1 (底物液 Α 成分为: 13.42g Na2HP04*12H20、 4.2g柠檬酸 ·Η20和 0.3g过氧化氢, 用去离子水中调 节体积为 700ml; 显色液 B成分为: 0.2g四甲基联苯胺、 20ml二甲基 甲酰胺用去离子水中调节体积为 700ml ) , 37°C显色 10分钟,加入 50μ1 终止液(2Μ H2S04 )终止反应, 并于酶标仪上检测各孔的 OD45Q值, 以 OD450值高于 2.0以上者视为阳性。
单克隆抗体的初筛结果概括为表 1-2。所有 480孔杂交瘤培养上清 经四种抗原检测后, 我们鉴定出 48个克隆与全长细胞角蛋白 18抗原 反应很强。 其中 5个克隆与角蛋白 18的 N端、 C端片段以及无关对照 抗原呈阳性反应, 这些克隆被视为非特异性克隆; 其中 18个克隆与 N 端细胞角蛋白 18片段呈阳性反应, 定义为 N端特异性克隆; 其中 24 个与 C端细胞角蛋白 18片段呈阳性反应, 定义为 C端特异性克隆。 另 外, 还有 1株克隆与 N端及 C端抗原反应, 其反应特异性未知。 这些 N端和 C端特异性克隆作为候选克隆进行进一步研究。
这些克隆通过有限稀释法进行三次亚克隆。 表 1-2 初次筛选鼠抗人角蛋白 18单抗结果
反应结果 总共 480个克隆 备注 全长角 N-端片段 C-端片段 阴性对照 阳性克隆数
蛋白 18 (aal ~ 250) (aa200 - 430) 抗原
+ + + + 5 非特异性
+ + - - 18 N-端特异
+ ■一 + - 24 C-端特异
+ + + - 1 未知 1.5 单克隆抗体的产生及纯化
取 16周龄的健康 Balb/c小鼠,腹腔注射 0.5ml pristane„ 5 ~ 7天后, 收集克隆化的杂交瘤细胞, 离心去上清, 加入不含血清的培养基, 调 节细胞密度至 2x l05 ~ 2χ 106个 /ml, 每只小鼠注射 0.5ml。 7 ~ 10天后 小鼠腹部增大, 开始收集腹水。 3000rpm离心 15分钟, 吸取中间澄清 部分的液体, 0.45μπι的微孔滤膜过滤除菌, 分装后 -20°C保存。
将处理好的腹水用 0.02mol/L、 pH7.4 的 PBS ( 81ml 0.2mol/L Na2HP04, 19ml 0.2mol/L NaH2P04, 加生理盐水至 100ml ) 5倍稀释, 取 50 ml上样至 2 ml protein-A/G 层析柱, 流速为 1 ml/min. 然后用 PBS 洗涤亲和层析柱至流出液 OD28Q测值小于 0.01。 然后使用 0.1M Glycine-HCL pH2.4 緩冲液洗脱结合在层析柱上的抗体,收集洗脱组分 2ml/管, 最后将所有 OD28Q大于 0.1的洗脱组分混合, 再用 1/10体积的 1M Tris-HCL pH 8.5溶液中和。 最后在 PBS溶液中透析过夜, 期间更 换 2次透析液。
1.6 酶标抗体的制备
辣根过氧化物酶(HRP ) 标记单抗和多克隆抗体的常用方法是过 碘酸钠法。其原理是 HRP的糖基用过碘酸钠氧化成醛基,加入抗体 IgG 后该醛基与 IgG氨基结合, 形成 Schiff氏碱。 为了防止 HRP中糖的醛 基与其自身蛋白氨基发生偶合, 在用过碘酸钠氧化前先用二硝基氟苯 阻断氨基。 氧化反应末了, 用硼氢化钠稳定 Schiff氏碱。
(1) 将 5mg HRP溶于 0.5ml 0.1mol/L NaHC03溶液中; 加 0.5ml 10mmol/L NaIO4溶液, 混匀, 盖紧瓶塞, 室温避光作用 2小时。
(2) 加 0.75ml 0.1mol/L Na2C03混匀。
(3) 加入 0.75ml纯化单抗( 15mg/ml ) , 混匀。
(4) 称取 Sephadex G25干粉 0.3g, 加入一支下口垫玻璃棉的 5ml 注射器外筒内; 随后将上述交联物移入注射器外套; 盖紧, 室温作用
(避光) 3小时或 4°C过夜。
(5) 用少许 PBS将交联物全部洗出, 收集洗出液, 加 1/20V体积 新鲜配制的 5mg/ml NaBH4溶液,混匀,室温作用 30分钟;再加入 3/20V
NaBH4溶液, 混匀, 室温作用 1小时 (或 4°C过夜) 。
(6)将交联物过 Sephadex G200或 Sepharose 6B ( 2.6x50cm )层析纯 化, 分管收集第一峰。
(7) 酶结合物质量鉴定:
克分子比值测定
酶量 (mg/ml ) =OD4o3x0.4
IgG量 ( mg/ml ) = ( OD280-OD403 x 0.3 ) χθ.62
克分子比值(E/P ) =酶量 x4/IgG量, 一般在 1-2之间。 酶结合率= 酶量 X体积 /抗体, 标记率一般为 0.3-0.6, 即 1-2个 HRP分子结合在一 个抗体分子上, 标记率可大于 0.6, 0.8, 0.9; OD403/OD280等于 0.4时, E/P约为 1。
标"己率 =OD403/OD280
酶活性和抗体活性的测定 可应用 ELISA法、免疫扩散、 DAB-H202 显色反应测定酶结合物的酶活性, 抗体活性及效价、 特异性。
(8) HRP抗体结合物的保存: 加入等量甘油后, 小量分装 -20°C存 放, 防止反复冻融; 或加入等量 60%甘油 4°C保存; 不宜加 NaN3或酚 防腐, 否则会影响酶活性。 必要时冻干保存, 以 BSA或脱脂牛奶作保 护剂。
1.7 肿瘤相关角蛋白 18 片段单克隆抗体配对筛选和临床初步验 证
纯化及酶标抗体经 ELISA法确认其抗原反应性后,选取 10株具有 高度亲和力的进行配对实验, 以筛选出与胃癌高度相关的角蛋白 18抗 体配对。
1.7.1 交叉配对试验
为了获取与胃癌血清角蛋白 18具有选择性的单克隆抗体配对, 我 们选取 10份胃癌血清样本及 10份正常人血清样本作为测试样本。 将 10份胃癌血清等量体积混合作为阳性样本,将 10份正常人血清等量体 积混合作为阴性样本。 用 l g/ml的纯化抗体包被 96孔板, 4°C过夜, 然后以含 3%BSA的 PBS封闭, 室温下 lh。 然后在一套 ELISA板中各 孔加入 50μ1的胃癌阳性血清,在另外一套 ELISA板中加入正常人阴性 对照血清, 再各加入 50μ1的 1:1000酶标抗体。 37°C温育后洗涤, 加入 显色底物, 避光 10分钟, 加入终止液, 在 450nm测量 OD值。
表 1-3 概括了 5 对单克隆抗体的交叉配对实验结果。 其中以 3A9/2A6-HRP, 2A6/3A9-HRP, 3H7/3A9-HRP 的抗体配对显示出对胃 癌血清的强阳性反应, 而对正常人血清呈阴性反应。 因此, 我们确定 这些抗体配对所检测的血清角蛋白 18与胃癌高度相关。 筛选胃癌相关角蛋白 18抗体的交叉实验
Figure imgf000019_0001
1.7.2 最佳抗体配对临床验证实验
从上述交叉抗体配对实验中初选出 3对与胃癌角蛋白 18高度相关 的抗体配对 3A9/2A6-HRP, 2A6/3A9-HRP, 3H7/3A9-HRP,使用 ELISA 法进行临床验证。 用 l g/ml的纯化抗体包被 96孔板, 4°C过夜, 然后 以含 3%BSA的 PBS室温下封闭 lh。 分别入 10份胃癌血清和 10份正 常人血清各 50μ1作为检测样本进行检测, 然后加入 50μ1的 1:1000酶 标抗体。 37°C温育后洗涤, 加入显色底物, 避光 10分钟, 加入终止液, 在 450nm测量 OD值。 将检测数据作散点分布图, 初步观察比较诊断 灵敏度及特异性, 见图 6。
结果显示, 3A9/2A6-HRP检测中, 胃癌血清检测值均高于正常人 血清检测值, 灵敏度及特异性均为 100%; 2A6/3A9-HRP检测中, 1例 胃癌血清检测值在正常人血清检测值范围内, 灵敏度及特异性较前者 低; 3H7/3A9-HRP检测中, 4例胃癌血清检测值在正常人血清检测值 范围内, 灵敏度及特异性较前两者低; 所以, 3A9/2A6-HRP配对对血 清的诊断性能最佳。 1.8 肿瘤相关角蛋白 18单克隆抗体的特征性研究
1.8.1 抗体识别肿瘤相关角蛋白 18片段抗原决定簇的确定 为了进一步确定 3A9及 2A6的抗原结合表位, 我们制备了一组重 组角蛋白 18片段, 这些片段带有每 50个氨基酸片段的缺失, 如表 1-4 所示。 采用间接 ELISA法测定抗体与这些抗原的结合活性。
(1) ELISA微孔板包被 l g/ml各种细胞角蛋白 18片段, 4°C过夜;
(2) 用 PBS洗涤 3次, 用含 3% (w/v) BSA的 PBS封闭, 室温, lh;
(3) 微孔中加入检测抗体(终浓度 l g/ml ) , 温育 lh, 37°C ;
(4) 用 PBS洗涤 3次, 未结合抗体即被洗去。 然后加入连接 HRP 的抗 mouse IgG抗体( g/ml), 温育 30min, 37 °C;
(5) 用 PBS洗涤 3次, 加入 TMB底物, 静置 10min, 然后加入 终止液 2N H2S04; 在 ELISA酶标仪上检测样本孔 450 nm/650 nm光密
表 1 -4 抗原识别表位的确定
序号 抗原 3A9 2A6
C1 aal ~ 430 + +
C2 aa50 - - 430 + +
C3 aalOO - 430 + +
C4 aal 50 ~ 430 + +
C5 aa200 - 430 + +
C6 aa250 ~ 430 - +
N7 aal 〜 400 + +
N8 aal ~ 350 +
N9 aal ~ 300 +
N10 aal ~ 250 +
aa 200 ~ 250 aa350 ~ 400 结果显示,单抗 3A9结合抗原 1 ~ 5,但是不能结合抗原 6( aa250 ~ 430 ) , 同时与所有 C端缺失片段呈阳性反应。 因此, 单抗 3A9的抗原 结合表位位于细胞角蛋白 18 aa 200 ~ 250的片段内。 单抗 2A6与所有 的 N端缺失片段均呈阳性反应, 并且与 1个 N端片段(aal - 400 ) 呈 阳性反应, 其它 3个 N端片段均呈阴性反应。 因此, 单抗 2A6结合表 位为细胞角蛋白 18 aa 350 ~ 400的片段内。 单抗 3A9和单抗 2A6的结 合表位如图 7所示。
1.8.2 杂交瘤分泌抗体的类型及亚类鉴定
以纯化角蛋白 18抗原包被 ELISA板。每孔加入各单克隆抗体的细 胞上清 lOO L, 37°C温育 30min; 在 TEC AN全自动洗板机上用 PBST 洗涤 5次, 每次间隔 20秒, 扣干, 加入合适稀释度的 HRP-山羊抗小 鼠 IgM、 IgG IgG2a、 IgG2b、 IgG3抗体(Serotec公司) 酶标二抗, 于 37°C温育 30min; 在 TECAN全自动洗板机上用 PBST洗涤 5次, 每 次间隔 20sec, 扣干 , 加入显色液 A ( H202 )和 B ( TMB )各 1滴, 于 37°C显色 10min, 加入 1滴终止液( 2M H2S04 ); 在 TECAN酶标仪上 测量 OD45Qnm (参比波长为 620nm ) , cutoff值为 2倍的阴性均值, OD 值大于 cutoff值为阳性, OD值小于 cutoff值为阴性。 结果表明, 3A9 为 IgG2a, 2A6为 IgGl。
1.8.3 特异性
为了检测单抗 3A9及 2A6的反应特异性, 我们检测其与其它人血 清肿瘤标记物的免疫结合强度。 首先将 K18、 K8、 K19、 CEA、 CA724 其他人血清肿瘤标记物按 1μβ/πι1 包被 96 孔板, 4°C过夜, 然后以含 3%BSA的 PBS室温下封闭 lh。 检测时每孔加入 50μ1 1 :1000稀释的单 抗 3Α9或 2Α6; 37°C温浴 30分钟, 用 PBST洗涤液洗 5遍, 拍干后 加入过氧化物酶结合的羊抗鼠免疫球蛋白 (HRP-GAM Ig, DAKO公 司) , 37°C温浴 30分钟, 取出后用 PBST洗涤液洗 5遍, 拍干后先后 加入底物液 A、 B各 50μ1 (底物液 A成分为: 13.42g Na2HP04' 12H20、 4.2g柠檬酸 ·Η20和 0.3g过氧化氢, 用去离子水中调节体积为 700ml; 显色液 B成分为: 0.2g四曱基联苯胺、 20ml二曱基甲酰胺用去离子水 中调节体积为 700ml ),37°C显色 10分钟,加入 50μ1终止液( 2M H2S04 ) 终止反应, 并于酶标仪上检测各孔的 OD45Q值。 结果见图 8。
结果显示, 单克隆抗体 3A9、 2A6与 K18呈强阳性反应, 与其它 肿瘤标记物均呈阴性反应。 表明单克隆抗体 3A9、 2A6为角蛋白 18高 度特异的单克隆抗体。
2 角蛋白 18-3A9试剂盒的制备 细胞角蛋白 18-3A9测定试剂盒(化学法发光法) 采用固相 96孔发 光板作为反应载体, 通过与胃癌相关的抗角蛋白 18的一对单克隆抗体 构成双抗体夹心法, 使用高敏感的化学发光检测技术对胃癌病人血清 中.角蛋白 18片段进行定量检测。
以 K18单抗 3A9作为包被抗体, 以 K18单抗 2A6作为标记抗体, 制备 K18试剂盒。
试剂盒主要组分为: 校准品、 包被板、 酶结合物、 发光液、 浓缩 洗涤液。
标准品为纯化的角蛋白 18。 包被抗体为 K18单抗 3A9。 标记抗体 为 K18单抗 2A6, 以 HRP辣根过氧化物酶进行标记, 形成抗体 -HRP 复合物 (酶结合物) 。
包被板是包被单抗 3A9经洗涤、 封闭、 干燥, 真空封装而成。 K18 单抗 3A9 以拧檬酸緩沖液 PH4.8为包被緩冲液, 以 5 g/mL的包被浓 度进行包被。 封闭液为 0.02M PBS+0.4%酪蛋白 +1%BSA+0.5M NaCl+0.2%明胶 +10%牛血清 +0.05%Tween-20+l %。防腐剂 +2.5%蔗糖。
标记抗体为 2A6-HRP。稀释液为 0.02M PBS+1%酪蛋白 +1%BSA +1 %。氨基比啉 +0.05%Tween-20。
发光液由将 KPL生产的发光液 A和发光液 B分装而成。 浓缩洗涤液 为 20 X PBS洗涤液。
3 临床验证和应用
在临床实验的统计分析过程中, 考虑到对治疗(化疗和手术)后处 于恢复期的患者血清中 K18的含量较治疗前会明显减少, 为了排除治 疗对试验结果的干扰, 在临床 1000例样本中选取 24例胃癌初诊患者 (未经治疗)血清标本检测结果进行分析, 结果表明 K18试剂盒的灵 敏度为 62.5 %, CA72-4试剂盒的灵敏度为 37.50%, 详见表 1-5。 表 1-5 K18试剂盒与 CA72-4试剂盒对初诊胃癌患者诊断评价指 标比较
阳性预测值 阴性预测值 诊断试剂 灵敏度% 特异度% 符合率%
% %
K18 62.50 96.00 92.41 65.22 95.52
CA72-4 37.50 95.00 88.84 47.37 84.82 在临床实验的统计分析过程中, 考虑到试剂盒对炎症等患者诊断 的假阳性率是影响诊断结果的重要因素, 本试验对 150例胃炎患者血清 样本进行对比试验, 以比较 K18试剂盒和 CA72-4试剂盒鉴别诊断能力。 结果显示, K18试剂盒对炎症患者血清样本诊断假阳性率为 5.3%, CA72-4试剂盒对炎症患者血清样本诊断假阳性率为 16.7%, K18试剂盒 假阳性率低于 CA72-4。 见表 1-6。 检测结果表明本试剂盒能将炎症患者 血清与胃癌患者血清很好的区分, 具有很好的特异性。 表 1-6 炎症患者血清检测结果统计
、1、,^ 检测结果
试剂盒
阳性 阴性
K18试剂盒 8 142
CA72-4试剂盒 25 125 如图 9所示, 由 ROC曲线分析结果可知, 角蛋白 18-3A9试剂盒对应 的 ROC曲线下面积( 0.743 ) 大于罗氏公司的 CA72-4试剂盒对应 ROC曲 线下所围面积( 0.612 ),角蛋白 18-3 A9试剂盒诊断胃癌的效果较 CA72-4 试剂盒好。
在中国医学科学院肿瘤医院进行的预实验中, 选择癌症患者血清 241例, 其中肺癌 84例, 胃癌 76例, 肠癌 81例, 阴性对照 61例, 正常人 体检血清 61例进行临床验证预实验。 结果如表 1-7, 表明角蛋白 18-3A9 测定试剂盒(化学发光法)对其它上皮源肿瘤也具有较高的检出率。
K18对各种癌症鉴别诊断结果
灵敏度(%) 特异度(%)
肺癌 43 90
胃癌 45 90
肠癌 53 90 实施例 2 1 抗角蛋白 19及片段的单克隆抗体的制备
1.1 免疫原
重组细胞角蛋白 19片段 GY20为 E.coli系 BL21(DE3)大肠杆菌表 达。
通过以下方法研制得到:
1.1.1 研发方案
见图 10
1.1.2 细胞角蛋白 19片段的 cDNA合成
通过 RT-PCR方法制备编码各种细胞角蛋白 19片段的 cDNA: a) 模板及引物
先从一种 HELA人癌细胞系中分离得到总 RNA。 然后根据说明书 用反转录试剂盒( Promega )合成 cDNA。 所获得的 cDNA即是 PCR的 模板。 设计三种片段的引物, 对各 cDNA进行 PCR扩增。 片段编号及 氨基酸序列见表 2-1。 重组细胞角蛋白 19片段的编号及序列
Figure imgf000024_0001
b) PCR反应
PCR反应溶液组分:
cDNA模板: 5 μ ί;
引物: 5,和 3,引物各 lOpmol
10 X PCR緩冲液: 10 μ ΐ;
dNTP: 各 2.5mM, 共 4 μ 1;
Taq聚合酶(Promega ) : 5U;
加入无菌双蒸水至 100 μ 1。
程序如下:
溶液加热至 94°C恒温 2min, 然后循环 40次, 每个循环设置为: 加 热 94 °C 30s , 52 °C lmin, 72 °C 3min。 程序完成后, 反应溶液加热 72 °C 10min。 然后获取扩增的 DNA片段, 用含 0.25 g/ml溴化乙锭的 1%琼脂 糖凝胶分离, 见图 11。 结果显示条带中包含所需的细胞角蛋白 19片段 cDNA片段, 然后用 Gene Clean kit ( BIO101 , Irvine,CA ) 回收 DNA片 段。
1.1.3 构建质粒并筛选
用 TOPO100 expression Cloning Kit(Invitrogen, Carlsbad, CA)克隆 cDNA片段。
(11) 将从 PCR反应溶液回收的细胞角蛋白 19的 cDNA与克隆试 剂盒提供的 TOPO质粒 (图 12 ) 50ng混合;
(12) 溶液中加入 10 X连接酶反应緩沖液( 6mM Tris-HCl(pH7.5),
6mM MgCl, 5mM NaCl, 7mM β -巯基乙醇, 0.1 mM ATP, 2mM DTT, ImM 亚精胺, O.lmg/ml BSA ) ;
(13) 再加入 4U T4 DNA连接酶(1 μ 1);
(14) 用灭菌去离子水调整溶液体积至 10 μ ΐ;
(15) 将其在 14°C温育 15h;
(16) 取 2 μ 1加入到 50 μ 1感受态 E.coli 细菌株 TOP10F (由 TA 克隆试剂盒提供, 并按照说明书制成感受态, 混合物冰浴 30min, 然后 孵育 42°C 30s, 再冰浴 5min )
(17) 配制 500 μ ΐ培养基, 含 2%(ν/ν)蛋白胨, 0.5%(w/v)酵母膏, 0.05%(w/v)NaCl, 2.5mM KC1, ImM MgCl和 20mM葡萄糖, 将 (6)加 入其中, 37°C温育 lh, 并振摇。
(18) 在 L-肉汤琼脂平板 ( 1%(ν/ν)蛋白胨, 0.5%(w/v)酵母膏, 0.5%(w/v)NaCl, 0.1%葡萄糖, 0.6%(w/v)bacto-agar(Difo, Detroit, MI) ) 涂铺 (7), lOO g/mh
(19) 培养基表面可筛选出抗氨苄的克隆子, 用铂丝涂布圈挑出单 菌落放入 L-肉汤培养基(含氨苄 lOO g/ml ) 中 37°C培养, 过夜, 振 摇 (200rpm)。
(20) 温育过后, 离心收集细菌, 用碱法提取 DNA质粒。
经酶切鉴定,已经得到重组的表达质粒 TOPO-GY20、TOPO-GY21、 TOPO-GY22, 如图 13。
1.1.4 重组蛋白的诱导表达和鉴定
编码各种细胞角蛋白 19的 cDNA已经插入 TOPO质粒中, (5)所获质粒转化至 E.coli系 BL21(DE3), 然后在 LB培养基中培 养至指数增长期, 用异丙醇 β-硫代半乳糖苷 (IPTG)诱导表达 3h。
(6)沉淀细胞, 用裂解液 (8M urea, 20mM Tris-HCl)重悬, 并用超 声破碎。
(7)后 14,000 X g离心 15min,在 Ni柱纯化上清。用 PBS溶液透析 纯化蛋白, 4°C过夜。
(8)用 BCA试剂(Pierece, Woburn, MA)检测蛋白浓度。
1.1.5 重组细月包角蛋白 19的纯化
活化 Sephadex G-50, 利用分子筛层析纯化重组细胞角蛋白 19片 段。 将 Sephadex G-50溶于 100 mM Tris-HCl緩冲液 (pH 7.4), 在 100 °C煮沸 10分钟, 然后用 100 mM Tris-HCl緩沖液 (pH 7.4)封闭, 并在 4°C保存。 将细菌破碎浓缩液流经 2ml凝胶珠, 流速为 2 ml /min。 样品 通过后, 50毫升 PBS冲洗,洗脱緩冲液 0.1 M甘氨酸 (pH 2.4), 0.15 M NaCl 。 洗脱液于紫外 OD280nm测值判断洗脱是否完毕。 收集有效洗 脱液(OD>0.01 ) 置于透析袋用 1L的磷酸盐緩沖液( pH7.5 ) 4°C透 析, 期间更换 2次透析緩沖液。 将纯化的蛋白盾浓缩到约 lmg/ml, 加 入 l%。NaN3, 4°C保存。 用 10 %的 SDS— PAGE检测, 并用 GDS8000 凝胶成像系统扫描分析蛋白纯度。
结果如图 14, 结果显示获得了高纯度的重组细胞角蛋白 19 片段 GY20、 GY21、 GY22, 电泳测定纯度大于 95 %。
用于制备单克隆抗体的免疫原为基因重组全长人细胞角蛋白 19, 此蛋白由美国 UAB大学周铜教授馈赠。
1.2 免疫程序
动物: 6 ~ 8周龄雌性 Balb/c小鼠。
初次免疫(第 1天) : 采用 1ml抗原蛋白溶液与相同体积的弗氏 完全佐剂混勾形成乳化颗粒, 每只足底注射 100 μ 1。
二次免疫(第 7天) : 采用 1ml抗原蛋白溶液与相同体积的弗氏 不完全佐剂混勾形成乳化颗粒, 每只足底注射 100 μ 1。
强化免疫(第 14、 21、 28天): 采用不含佐剂的抗原(lmg/ml ) , 每只足底注射 100 μ 1。
融合(第 31天) 。
杂交瘤的建立 取免疫小鼠的胭窝及腹股沟分离淋巴细胞, 将其与 NS1骨髓瘤细 胞按 2: 1比例混合, 用无血清 RPMI 1640洗涤 2次, 加入 lml37°C预热 的 PEG1500, 在 37°C轻微混合细胞 1分钟, 然后在 3分钟内緩慢滴加 20ml预热至 37Ό的无血清 RPMI 1640培养基。 离心, 将融合细胞悬浮 于 12% FBS RPMI 1640 HAT 选择培养基。 将细胞加入 5块 96孔细胞 培养板中, ΙΟΟμΙ/孔。
1.4 ELISA检测筛选阳性克隆
在细胞融合后 7天, 用间接 ELISA法进行初次筛选, 所有杂交瘤 均用三种重组人细胞角蛋白 19抗原筛选: ( 1 )全长细胞角蛋白 19; ( 2 ) Ν端细胞角蛋白 19片段; (3 ) C端细胞角蛋白 19片段。
用上述角蛋白 19抗原( 1 y g/ml )包被 ELISA板 4 °C过夜; 用 PBS 洗涤 3次, 用含 3% (w/v) BSA的 PBS室温封闭 lh。 检测时每孔中加 入 ΙΟΟμΙ细胞培养液上清; 37°C温浴 30分钟,用 PBST洗涤液洗 5遍, 拍干后加入过氧化物酶结合的羊抗鼠免疫球蛋白 (HRP-GAM Ig, DAKO公司) , 37°C温浴 30分钟, 取出后用 PBST洗涤液洗 5遍, 拍 干后先后加入底物液 A、 B 各 50μ1 (底物液 Α 成分为: 13.42g Na2HP04.12H20、 4.2g拧檬酸 ·Η20和 0.3g过氧化氢, 用去离子水中调 节体积为 700ml; 显色液 B成分为: 0.2g四曱基联苯胺、 20ml二曱基 曱酰胺用去离子水中调节体积为 700ml ), 37°C显色 10分钟,加入 50μ1 终止液(2Μ H2S04 )终止反应, 并于酶标仪上检测各孔的 OD45o值, 以 OD45o值高于 2.0以上者视为阳性。
单克隆抗体的初 结果概括与表 2-2。所有 480孔杂交瘤培养上清 经四种抗原检测后, 我们鉴定出 39个克隆与全长细胞角蛋白 19抗原 反应很强。 其中, 3个克隆与角蛋白 19的 N端、 C端片段以及无关对 照抗原呈阳性反应, 这些克隆被视为非特异性克隆; 其中 15个克隆与 N端细胞角蛋白 19 片段呈阳性反应, 定义为 N端特异性克隆; 其中 18个与 C端细胞角蛋白 19片段呈阳性反应, 定义为 C端特异性克隆。 另外, 还有 3株克隆与 N端及 C端抗原反应, 其反应特异性未知。 这 些 N端和 C端特异性克隆作为候选克隆进行进一步研究。
这些克隆通过有限稀释法进行三次亚克隆。 初次筛选角蛋白 19鼠抗人单抗结果
Figure imgf000028_0001
1.5 单克隆抗体的产生及純化
取 16周龄的健康 Balb/c小鼠 ,腹腔注射 0.5ml pristane„ 5 ~ 7天后, 收集克隆化的杂交瘤细胞, 离心去上清, 加入不含血清的培养基, 调 节细胞密度至 2χ 105 ~ 2χ 106个 /ml, 每只小鼠注射 0.5ml。 7 ~ 10天后 小鼠腹部增大, 开始收集腹水。 3000rpm离心 15分钟, 吸取中间澄清 部分的液体, 0.45μπι的微孔滤膜过滤除菌, 分装后 -20°C保存。
将处理好的腹水用 0.02mol/L、 pH7.4 的 PBS ( 81ml 0.2mol/L Na2HP04, 19ml 0.2mol/L NaH2PO4, 加生理盐水至 100ml ) 5倍稀释, 取 50 ml上样至 2 ml protein-A/G 层析柱, 流速为 1 ml/min. 然后用 PBS 洗涤亲和层析柱至流出液 OD28()测值小于 0.01。 使用 0.1M Glycine-HCL pH2.4緩沖液洗脱结合在层析柱上的抗体,收集洗脱组分 2ml/管, 最后将所有 OD28。大于 0.1的洗脱组分混合, 然后用 1/10体积 的 lM Tris-HCL pH 8.5溶液中和。 然后在 PBS溶液中透析过夜, 期间 更换 2次透析液。
1.6 酶标抗体的制备
辣根过氧化物酶(HRP ) 标记单抗和多克隆抗体的常用方法是过 碘酸钠法。其原理是 HRP的糖基用过碘酸钠氧化成醛基,加入抗体 IgG 后该醛基与 IgG氨基结合, 形成 Schiff氏碱。 为了防止 HRP中糖的醛 基与其自身蛋白氨基发生偶合, 在用过碘酸钠氧化前先用二硝基氟苯 阻断氨基。 氧化反应末了, 用硼氢化钠稳定 Schiff氏碱。
(1) 将 5mg HRP溶于 0.5ml 0.1mol/L NaHC03溶液中; 加 0.5ml
10mmol/L NaIO4溶液, 混匀, 盖紧瓶塞, 室温避光作用 2小时。
(2) 加 0,75ml 0.1mol/L Na2C03混匀。 (3) 加入 0.75ml纯化单抗( 15mg/ml ) , 混匀。
(4) 称取 Sephadex G25干粉 0.3g, 加入一支下口垫玻璃棉的 5ml 注射器外筒内; 随后将上述交联物移入注射器外套; 盖紧, 室温作用
(避光) 3小时或 4°C过夜。
(5) 用少许 PBS将交联物全部洗出, 收集洗出液, 加 1/20V体积 新鲜配制的 5mg/ml NaBH4溶液,混匀,室温作用 30分钟;再加入 3/20V NaBH4溶液, 混匀, 室温作用 1小时 (或 4°C过夜) 。
(6)将交联物过 Sephadex G200或 Sepharose 6B ( 2.6x50cm )层析纯 化, 分管收集第一峰。
(7) 酶结合物质量鉴定:
克分子比值测定
酶量( mg/ml ) =OD403 X 0.4
IgG量 ( mg/ml ) = ( OD280-OD403 x 0.3 ) χθ.62
克分子比值(E/P ) =酶量 x4/IgG量, 一般在 1-2之间。 酶结合率= 酶量 X体积 /抗体, 标记率一般为 0.3-0.6, 即 1-2个 HRP分子结合在一 个抗体分子上, 标记率可大于 0.6, 0.8, 0.9; OD43/OD28。等于 0.4时, E/P约为 1。
标记率 =OD403/OD280
酶活性和抗体活性的测定 可应用 ELISA法、免疫扩散、 DAB-H202 显色反应测定酶结合物的酶活性, 抗体活性及效价、 特异性。
(8) HRP抗体结合物的保存: 加入等量甘油后, 小量分装 -20°C存 放, 防止反复冻融; 或加入等量 60%甘油 4°C保存; 不宜加 NaN3或酚 防腐, 否则会影响酶活性。 必要时冻干保存, 以 BSA或脱脂牛奶作保 护剂。
1.7 肿瘤相关角蛋白 19片段单克隆抗体配对的筛选
纯化及酶标抗体经 ELISA法确认其抗原反应性后,选取 10株具有 高度亲和力的进行配对实验, 以筛选出与肺癌高度相关的角蛋白 19抗 体配对。
1.7.1 交叉配对试验选择
为了获取与肺癌血清角蛋白 19具有选择性的单克隆抗体配对, 我 们选取 10份肺癌血清样本及 10份正常人血清样本作为测试样本。 将 10份肺癌血清等量体积混合作为阳性样本,将 10份正常人血清等量体 积混合作为阴性样本。 用 l g/ml的纯化抗体包被 96孔板, 4°C过夜, 然后以含 3%BSA的 PBS室温下封闭 lh。 然后一套 ELISA板中各孔加 入 50μ1的肺癌阳性血清,在另外一套 ELISA板中加入正常人阴性对照 血清, 再各加入 50μ1的 1 :1000酶标抗体。 37°C温育后洗涤, 加入显色 底物, 避光 10分钟, 加入终止液, 在 450nm测量 OD值。
表 2-3 概括了 5 对单克隆抗体的交叉配对实验结果。 其中以 2G2/5H2-HRP, 5H2/2G2-HRP, 5H2/1D11-HRP的抗体配对显示出对肺 癌血清的强阳性反应, 而对正常人血清呈阴性反应。 因此, 我们确定 这些抗体配对所检测的血清角蛋白 19与肺癌高度相关。
Figure imgf000030_0001
1.7.2 初步确认最佳抗体配对实验
从上述交叉抗体配对实验中初选出 3对与肺癌角蛋白 19高度相关 的抗体配对 2G2/5H2-HRP, 5H2/2G2-HRP, 5H2/1D11-HRP,使用 ELISA 法进行临床 r证。 用 l g/ml的纯化抗体包被 96孔板, 4°C过夜, 然后 以含 3%BSA的 PBS室温下封闭 lh。 分别入 10份肺癌血清和 10份正 常人血清各 50μ1作为检测样本进行检测, 然后加入 50μ1的 1 :1000酶 标抗体。 37°C温育后洗涤, 加入显色底物, 避光 10分钟, 加入终止液, 在 450nm测量 OD值。 将检测数据作散点分布图 , 初步观察比较诊断 灵敏度及特异性, 见图 15。
结果显示, 2G2/5H2_HRP检测中, 1 例肺癌血清检测值在正常人 血清检测值范围内,且其它肺癌血清检测值均较高,以特异性 100%计, 灵敏度为 90%; 5H2/2G2-HRP检测中, 2例肺癌血清检测值在正常人 血清检测值范围内, 4例肺癌血清检测值偏低, 以特异性 100%计, 灵 敏度为 80%; 5H2/1D1 1 -HRP检测中, 4例肺癌血清检测值在正常人血 清检测值范围内, 且所有肺癌血清检测值较前两种检测均偏低, 以特 异性 100%计, 灵敏度为 60%; ; 所以, 2G2/5H2-HRP配对对血清的诊 断性能最佳。
1 .8 肿瘤相关角蛋白 19单克隆抗体的特征性研究
1.8.1 肿瘤相关角蛋白 19片段抗原决定簇的确定
为了进一步确定 2G2、 5H2 的抗原结合表位, 我们制备了一组重 组角蛋白 19片段, 这些片段带有每 50个氨基酸片段的缺失, 如表 2.4 所示。 采用间接 ELISA法测定抗体与这些抗原的结合活性。
(1) ELISA微孔板包被 l g/ml各种细胞角蛋白 19片段, 4°C过 夜;
(2) 用 PBS洗涤 3次, 用含 3% (w/v) BSA的 PBS封闭, 室温, lh;
(3) 微孔中加入检测抗体(终浓度 l g/ml ) , 温育 lh, 37 °C ;
(4) 用 PBS洗涤 3次, 未结合抗体即被洗去。 然后加入连接 HRP 的抗 mouse IgG抗体( g/ml) , 温育 30min, 37 °C ;
(5) 用 PBS洗涤 3次, 加入 TMB底物, 静置 10min, 然后加入 终止液 2N H2S04; 在 ELISA酶标仪上检测样本孔 450 nm/650 nm光密 度。
表 2-4 抗原识别表位的确定
序号 抗原 2G2 5H2
CI aal ~ 400 + +
C2 aa50 ~ 400 + +
C3 aal00 ~ 400 + +
C4 aal50 ~ 400 + +
N5 aal ~ 375 - +
N6 aal - 350 - -
N7 aal ~ 300 - -
N8 aal ~ 250 _ _
aa 375 - 400 aa325 - 350 结果显示, 单抗 2G2结合抗原 1 ~ 4, 同时与所有 C端缺失片段呈 阳性反应。因此,单抗 2G2的抗原结合表位位于细胞角蛋白 19 aa 375 ~ 400的片段内。 单抗 5H2与所有的 N端缺失片段均呈阳性反应, 并且 与 1个 N端片段(aal ~ 375 )呈阳性反应, 与其它 3个 N端片段均呈 阴性反应。单抗 5H2结合表位为细胞角蛋白 19 aa 325 ~ 350的片段内。 单抗 2G2和单抗 5H2的结合表位如图 16所示:
1.8.2 杂交瘤分泌抗体的类型及亚类鉴定
以纯化角蛋白 19抗原包被 ELISA板。每孔加入各单克隆抗体的细 胞上清 ΙΟΟμ , 37°C温育 30min; 在 TEC AN全自动洗板机上用 PBST 洗涤 5次, 每次间隔 20秒, 扣干, 加入合适稀释度的 HRP-山羊抗小 鼠 IgM、 IgGl、 IgG2a、 IgG2b、 IgG3抗体(Serotec公司)酶标二抗, 于 37°C温育 30min; 在 TECAN全自动洗板机上用 PBST洗涤 5次, 每 次间隔 20sec, 扣干, 加入显色液 A ( H202 )和 B ( TMB )各 1滴, 于 37°C显色 10min, 加入 1滴终止液( 2M H2S04 ); 在 TECAN酶标仪上 测量 OD45()nm (参比波长为 620nm ) , cutoff值为 2倍的阴性均值, OD 值大于 cutoff值为阳性, OD值小于 cutoff值为阴性。 结果表明 2G2为 IgG2a, 5H2为 IgGl。
1.8.3 特异性
为了检测单抗 2G2及 5H2的反应特异性, 我们检测其与其它人血 清肿瘤标记物的免疫结合强度。 首先将 K18、 K8、 K19、 CEA、 CA724 其他人血清肿瘤标记物按 l g/ml 包被 96 孔板, 4°C过夜, 然后以含 3%BSA的 PBS室温下封闭 lh。 检测时每孔加入 50μ1 1 :1000稀释的单 抗 2G2或 5Η2; 37°C温浴 30分钟, 用 PBST洗涤液洗 5遍, 拍干后加 入过氧化物酶结合的羊抗鼠免疫球蛋白( HRP-GAM Ig, DAKO公司), 37°C温浴 30分钟, 取出后用 PBST洗涤液洗 5遍, 拍干后先后加入底 物液 A、 B各 50μ1 (底物液 Α成分为: 13.42g Na2HP04 12H20 4.2g 柠檬酸 ·Η20和 0.3g过氧化氢, 用去离子水中调节体积为 700ml; 显色 液 B成分为: 0.2g四曱基联苯胺、 20ml二曱基曱酰胺用去离子水中调 节体积为 700ml ) , 37°C显色 10分钟, 加入 50μ1终止液( 2M H2S04 ) 终止反应, 并于酶标仪上检测各孔的 OD45。值。 结果见图 17。
结果显示, 单克隆抗体 2G2、 5H2与 K19呈强阳性反应, 与其它 肿瘤标记物均呈阴性反应。 表明单克隆抗体 2G2、 5H2为角蛋白 19高 度特异的单克隆抗体。
2 角蛋白 19-2G2试剂盒的制备
角蛋白 19-2G2测定试剂盒(化学法发光法) 采用固相 96孔发光板 作为反应载体, 通过与肺癌相关的抗角蛋白 19的一对单克隆抗体构成 双抗体夹心法, 使用高敏感的化学发光检测技术对肺癌病人血清中角 蛋白 19片段进行定量检测。
以 K19单抗 2G2作为包被抗体, 以 K19单抗 5H2作为标记抗体, 制备 K19试剂盒。
试剂盒主要组分为: 校准品、 包被板、 酶结合物、 发光液、 浓缩 洗涤液。
标准品为纯化的角蛋白 19。 包被抗体为 K19单抗 2G2。 标记抗体为 K19单抗 5H2, 以 HRP辣根过氧化物酶进行标记, 形成抗体 -HRP复合物 (酶结合物) 。
包被板是包被单抗 2G2, 经洗涤、 封闭、 干燥, 真空封装而成。 K19单抗 2G2以柠檬酸緩冲液 PH4.8为包被緩沖液, 以 5 g/mL的包 被浓度进行包被。 封闭液为 0.02M PBS+0.4%酪蛋白 +1%BSA+0.5M NaCl+0.2%明胶 +10%牛血清 +0.05%Tween-20+l %。防腐剂 +2.5%蔗糖。
标记抗体为 5H2-HRP。 稀释液为 0.02M PBS+0.4%酪蛋白
+ 1%BSA+0.5M NaCl+1 %。氨基比啉 +0.2%明 胶 +10%牛血清 +0.05%Tween-20。 发光液由将 KPL生产的发光液 A和发光液 B分装而成。 浓缩洗涤液 为 20 x PBS洗涤液。
3 临床验证和应用
在临床实验的统计分析过程中, 考虑到对治疗(化疗和手术)后处 于恢复期的患者, 血清中 K19的含量较治疗前会明显减少。 为了排除 治疗对试验结果的干扰, 在临床 1000例样本中选取 69例肺癌初诊患 者 (未经治疗)血清标本检测结果进行分析, 结果表明 K19试剂盒的 灵敏度为 88.4%, CYFRA21-1试剂盒的灵敏度为 52.17%, 见表 2-5。 表 2-5 K19试剂盒与 CYFRA21-1试剂盒对初诊肺癌患者诊断评 价指标比较
阳性预测值
诊断试剂 灵敏度% 特异度% 符合率% 阴性预测值%
%
K19 88.41 97.50 95.17 92.42 96.06
CYFRA21 -1 52.17 95.50 84.39 80.00 85.27 在临床实验的统计分析过程中, 考虑到试剂盒对炎症等患者诊断 的假阳性率是影响诊断结果的重要因素。 本试验对 150例肺炎患者血 清样本进行对比试验,以比较 K19试剂盒和 CYFRA21-1试剂盒鉴别诊 断能力。 结果显示 K19 试剂盒对炎症患者血清样本诊断假阳性率为 5.3%, CYFRA21-1试剂盒对炎症患者血清样本诊断假阳性率为 12.0%。
K19试剂盒假阳性率低于 CYFRA21-1试剂盒, 见表 2-6。 检测结果表 明本试剂盒能将炎症患者血清与肺癌患者血清很好的区分, 具有很好 的特异性。 表 2-6 炎症患者血清检测结果统计
检测结果
试剂盒 阳性 阴性 K19试剂盒 8 142
CYFRA21-1试剂盒 18 132 由图 18的 ROC曲线分析结果可知, 角蛋白 19-2G2试剂盒对应的 ROC曲线下面积( 0.817 )大于罗氏公司的 CYFRA21-1试剂盒对应 ROC 曲线下所围面积 ( 0.766 ) , 角蛋白 19-2G2 试剂盒诊断肺癌效果优于 CYFRA21-1试剂盒。
在中国医学科学院肿瘤医院进行的预实验中, 选择癌症患者血清 241例, 其中肺癌 84例, 胃癌 76例, 肠癌 81例, 阴性对照 61例, 正 常人体检血清 61 例进行临床验证预实验。 结果如表 2-7,表明角蛋白 19-2G2测定试剂盒(化学发光法)对其它上皮源肿瘤也具有较高的检 出率。
K19对各种癌症鉴别诊断结果
癌种 灵敏度 (%) 特异度(%) 肺癌 75 90
胃癌 63 90
肠癌 62 90

Claims

权 利 要 求
1. 与上皮源性癌症有关的细胞角蛋白片段, 其中该片段包含选自 由 SEQ ID NOs: 2、 3、 5和 6构成的组的抗原表位。
2. 权利要求 1的细胞角蛋白片段, 其中该片段包含 SEQ ID NO: 1 的氨基酸残基 200-400。
3. 权利要求 1的细胞角蛋白片段, 其中该片段包含 SEQ ID NO: 2 的氨基酸残基 325-400。
4. 与权利要求 1中定义的抗原表位特异性结合的单克隆抗体。
5. 权利要求 4的单克隆抗体, 其中该单克隆抗体由保藏号为 CGMCC No. 1957、 CGMCC No. 1956、 CGMCC No. 1955或 CGMCC No. 1952的杂交瘤产生。
6. 权利要求 4或 5的单克隆抗体的抗原结合部分, 其中该抗原结合 部分与权利要求 4或 5的单克隆抗体竟争结合权利要求 1中定义的抗原 表位。
7. 权利要求 6的抗原结合部分, 其中该抗原结合部分为人源化抗 体。
8. 权利要求 6的抗原结合部分, 其中该抗原结合部分为嵌合抗体。
9. 杂交瘤细胞系, 其保藏编号选自由 CGMCC No. 1957、 CGMCC No. 1956、 CGMCC No. 1955和 CGMCC No. 1952构成的组。
10. 用于对受试者中的上皮源性癌症进行早期筛查、诊断或预后评 估的方法, 该方法包括:
获得来自受试者的生物样品,
检测该生物样品中权利要求 1 - 3之任一项的细胞角蛋白片段的含 量, 和
将该含量与阈值水平进行比较。
11. 用于对受试者中的上皮源性癌症进行早期筛查、诊断或预后评 估的方法, 该方法包括:
获得来自受试者的生物样品,
检测该生物样品中权利要求 1 - 3之任一项的细胞角蛋白片段的含 量,
检测该生物样品中其他上皮源性肿瘤标记物的含量, 和 将所述细胞角蛋白片段以及所述其他上皮源性肿瘤标记物的含量 与阈值水平进行比较。
12. 评估用于治疗上皮源性癌症的药物或疗法的治疗效果的方法, 包括:
向患有上皮源性癌症的受试者施用所述药物或疗法,
在施用所述药物或疗法之前和之后采集所述受试者的生物样品, 和
检测所述生物样品中权利要求 1 -3之任一项的细胞角蛋白片段的含 量,
其中在施用所述药物或疗法之前和之后该细胞角蛋白片段含量明 显降低的, 表明所述药物或疗法有明显疗效。
13. 权利要求 10-12之任一项的方法, 其中所述上皮源性癌症选自 由胃癌、 肝癌、 肺癌、 胆嚢癌、 乳腺癌、 宫颈癌、 卵巢癌、 结肠癌、 前列腺癌、 肾癌、 食道癌、 肠癌、 膀胱癌构成的组。
14. 权利要求 10-13之任一项的方法, 其中所述检测采用了权利要 求 4-5之任一项的单克隆抗体或权利要求 6-8之任一项的抗原结合部分。
15. 权利要求 10-14之任一项的方法, 其中所述生物样品选自由血 液、 血清、 组织液、 尿液、 大便、 痰液、 脑脊液、 唾液、 眼泪和乳头 吸出液构成的组。
16. 权利要求 11-15之任一项的方法, 其中所述其他上皮源性肿瘤 标记物选自由 AFP、 CEA、 CA242、 CA19-9、 CA72-4、 CA125、 CA15-3、 NSE、 SCCA、 Cyfra21-1、 PSA、 free PSA构成的组。
17. 试剂盒, 其包含:
固定于固相载体上的能够特异结合权利要求 1-3之任一项的细胞角 蛋白片段的包被抗体, 以及
被可检测地标记的能够特异结合该细胞角蛋白片段的检测抗体。
18. 权利要求 17的试剂盒,其中所述包被抗体和检测抗体各自独立 地为权利要求 4-5之任一项的单克隆抗体或权利要求 6-8之任一项的抗 原结合部分。
19. 权利要求 1-3之任一项的细胞角蛋白片段的特异结合剂在制备 用于诊断上皮源性癌症的试剂中的用途。
20. 权利要求 19的用途, 其中所述特异结合剂为权利要求 4-5之任 一项的单克隆抗体或权利要求 6-8之任一项的抗原结合部分。
21. 权利要求 19-20之任一项的用途, 其中所述上皮源性癌症选自 由胃癌、 肝癌、 肺癌、 胆嚢癌、 乳腺癌、 宫颈癌、 卵巢癌、 结肠癌、 前列腺癌、 腎癌、 食道癌、 肠癌、 膀胱癌构成的组。
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