WO2016037459A1 - 检测肿瘤的试剂盒及其专用识别特定糖基结构的物质 - Google Patents

检测肿瘤的试剂盒及其专用识别特定糖基结构的物质 Download PDF

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WO2016037459A1
WO2016037459A1 PCT/CN2015/071940 CN2015071940W WO2016037459A1 WO 2016037459 A1 WO2016037459 A1 WO 2016037459A1 CN 2015071940 W CN2015071940 W CN 2015071940W WO 2016037459 A1 WO2016037459 A1 WO 2016037459A1
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cancer
acetylglucosamine
tumor
glcnac
kit
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范振符
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张昊岩
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

Definitions

  • the present disclosure relates to kits for detecting tumors and materials thereof that specifically identify a particular glycosyl structure.
  • tumor-specific macromolecular glycoprotein antigen CA
  • CA tumor-specific macromolecular glycoprotein antigen
  • sandwich analysis From the perspective of laboratory testing, tumor markers are classified into serum/plasma tumor markers and tissue cell tumor markers. Serum/plasma tumor markers are broadly classified into three categories: 1) embryonic antigens. Embryonic expression, antigens not expressed in normal adults, re-expressed with tumorigenesis, such as AFP, CEA. 2) Tumor-associated glycolipids and glycoproteins.
  • ELISA Immunosorbent assay
  • CLIA chemiluminescence immunoassay
  • ECLI electrochemiluminescence immunoassay
  • TRFIA time-resolved fluoroimmunoassay
  • CEA cancer anti-proliferative adenosarcoma
  • the above markers are not broad-spectrum, most of them can only diagnose one or several tumors, and CEA, for example, has the best diagnostic effect on primary colon cancer. 80%, although effective for pancreatic cancer, stomach cancer, lung cancer, breast cancer, but the lowest specificity is only 25%, in other words, 75% will miss the diagnosis.
  • the early detection and diagnosis of tumors is crucial for improving the cure rate of patients and prolonging the life of patients.
  • Scholars engaged in cancer research have never relaxed in exploring and searching for effective tumor markers.
  • the current commercial kits are divided into two categories, the sandwich mode and the competition mode, and there is only a sandwich mode of the double combination in the field of tumor markers.
  • kits for detecting a tumor comprising a substance that can specifically bind to N-acetylglucosamine.
  • the present invention utilizes a substance which specifically binds N-acetylglucosamine to prepare a test kit which is simple in operation, high in detection specificity and sensitivity, and suitable for an early stage of tumor onset.
  • the kit for detecting a tumor is a kit using serum as a test sample; the tumor is a malignant tumor.
  • the tumor is one or more of breast cancer, pancreatic cancer, liver cancer, lung cancer, esophageal cancer, oral cancer, colon cancer, cholangiocarcinoma, ovarian cancer, cervical cancer, uterine cancer, bladder cancer, stomach cancer, kidney cancer and cardiac cancer.
  • breast cancer pancreatic cancer
  • liver cancer lung cancer
  • esophageal cancer oral cancer
  • colon cancer cholangiocarcinoma
  • ovarian cancer cervical cancer
  • uterine cancer bladder cancer
  • stomach cancer stomach cancer
  • kidney cancer kidney cancer
  • cardiac cancer cardiac cancer
  • the above-mentioned substance capable of specifically binding to N-acetylglucosamine is a monoclonal antibody having a N-acetylglucosamine as an antigenic determinant, a polyclonal antibody, an aptamer specifically binding to N-acetylglucosamine or N- Acetylglucosamine specifically binds to a lectin.
  • the aptamer that specifically binds to N-acetylglucosamine is a nucleic acid having the sequence set forth in SEQ ID NO: 1.
  • the present disclosure also protects an aptamer that specifically binds to N-acetylglucosamine, which is a nucleic acid having the sequence set forth in SEQ ID NO: 1.
  • N-acetylglucosamine molecular formula: C 8 H 15 NO 6 , molecular weight: 2221.21, chemical code - GlcNAc, its structure can be coupled with carrier protein to obtain antigen as shown in FIG. 1A, and a specific binding body capable of detecting tumor can be prepared.
  • specific binding partners can specifically bind to N-acetylglucosamine, such as antibodies, nucleic acid aptamers, lectins and the like.
  • the above carrier protein may be a commonly used carrier protein such as ovalbumin (OVA) or bovine serum white.
  • OVA ovalbumin
  • BSA bovine serum white
  • KLH hemocyanin
  • glycoproteins such as mucin and other proteins.
  • the inventors isolated the tumor antigen Mu-GlcNAc (code MA153) purified by the present disclosure formed by N-acetylglucosamine linked mucin 1 (Mucin1, code Mu) in serum. The position of its linkage is at the beginning of the sugar chain in mucin 1, see Figure 1B.
  • the amino acid sequence of the mucin 1 (Mu) structure is (-PDTRPAPGSTAPPAHGVTSA-) ⁇ N, ie the amino acid sequence of mucin 1 (Mu-) is : - Multiple repeat alignment of -PDTRPAPGSTAPPAHGVTSA- (SEQ ID NO: 2).
  • the present disclosure utilizes a specific binding complex of N-acetylglucosamine including a purified new highly sensitive serum tumor marker antigen, 2-deoxy-N-acetyl-D-glucosamine-mucin1 (referred to as Mu-GlcNAc, also referred to as MA153).
  • N-acetylglucosamine including a purified new highly sensitive serum tumor marker antigen, 2-deoxy-N-acetyl-D-glucosamine-mucin1 (referred to as Mu-GlcNAc, also referred to as MA153).
  • Specific binding partners for N-acetylglucosamine include aptamers (nucleic acids such as MA153-A) and other binding partners such as monoclonal antibodies, polyclonal antibodies, plant lectins (PHA) and/or receptors, related ligands, And methods for detecting early tumors.
  • the method for directly detecting N-acetylglucosamine includes two modes: 1) Ma695-Mu-GlcNAc-MA153-A sandwich, and 2) single binding, which directly recognizes GlcNAc by a specific binding of N-acetylglucosamine. Using this method, 97% of lung cancers can be detected with a tumor specificity of 96%.
  • the present disclosure provides the above method for detecting a tumor by detecting the concentration of N-acetylglucosamine in the serum of the patient to be tested, and using 1 U/mL as the upper limit value of the normal boundary value, wherein U is an N-acetylglucosamine activity unit.
  • Clinical serological tests show that the detection rate of many tumors in the technical solution of the present disclosure is above 90%, and the tumor specificity is above 95%. This will greatly improve the early diagnosis rate of tumors.
  • the present disclosure is suitable for early oncology screening in a normal population.
  • the present disclosure opens up a new mode, named Single Binder, which directly recognizes N-acetylglucosamine from a specific binding partner of N-acetylglucosamine. It saves antibodies and reduces costs by 80%, increasing sensitivity without reducing specificity.
  • the single-combination mode improves the detection rate of lung cancer by 22% compared to the sandwich mode: the former has a sensitivity of 97% and a specificity of 96%; the latter has a sensitivity of 75% and a specificity of 95%.
  • Figure 1A shows a structural diagram of N-acetylglucosamine
  • Figure 1B shows the relationship between N-acetylglucosamine and mucin-1; the relationship between N-acetylglucosamine and aptamer MA153-A; the relationship between mucin 1 and monoclonal antibody Ma695; Mu-GlcNAc simulation structure;
  • Figure 2 shows the principle of the Mu-GlcNAc sandwich assay
  • Figure 3 shows a standard curve of the Mu-GlcNAc sandwich assay
  • Figure 4A shows the principle of the determination of N-acetylglucosamine monoconjugate
  • Figure 4B is similar to Figure 4A except that the portion of the protein to be tested that is linked to N-acetylglucosamine has no -PDTR-peptide segment. It is indicated that there may be other glycoproteins carrying -GlcNAc other than Mucin1 in MA153;
  • Figure 5 is a standard curve for the determination of N-acetylglucosamine monoconjugate.
  • kits for detecting a tumor comprising a substance that can specifically bind to N-acetylglucosamine.
  • the disclosure discloses that the substance specifically bound by N-acetylglucosamine is simple to prepare, has high detection specificity and sensitivity, and is suitable for detection in the early stage of tumor onset.
  • the kit for detecting a tumor is a kit using serum as a test sample; the tumor is a malignant tumor.
  • the tumor is one or more of breast cancer, pancreatic cancer, liver cancer, lung cancer, esophageal cancer, oral cancer, colon cancer, cholangiocarcinoma, ovarian cancer, cervical cancer, uterine cancer, bladder cancer, stomach cancer, kidney cancer and cardiac cancer.
  • breast cancer pancreatic cancer
  • liver cancer lung cancer
  • esophageal cancer oral cancer
  • colon cancer cholangiocarcinoma
  • ovarian cancer cervical cancer
  • uterine cancer bladder cancer
  • stomach cancer stomach cancer
  • kidney cancer kidney cancer
  • cardiac cancer cardiac cancer
  • the above-mentioned substance capable of specifically binding to N-acetylglucosamine is a monoclonal antibody having a N-acetylglucosamine as an antigenic determinant, a polyclonal antibody, an aptamer specifically binding to N-acetylglucosamine or N- Acetylglucosamine specifically binds to a lectin.
  • the aptamer that specifically binds to N-acetylglucosamine is a nucleic acid having the sequence set forth in SEQ ID NO: 1.
  • the present disclosure also protects an aptamer that specifically binds to N-acetylglucosamine, a nucleic acid of the sequence set forth in SEQ ID NO: 1.
  • N-acetylglucosamine molecular formula: C 8 H 15 NO 6 , molecular weight: 2221.21, chemical code - GlcNAc, its structure can be coupled with carrier protein to obtain antigen as shown in FIG. 1A, and a specific binding body capable of detecting tumor can be prepared.
  • specific binding partners can specifically bind to N-acetylglucosamine, such as antibodies, nucleic acid aptamers, lectins and the like.
  • GlcNAc N-acetylglucosamine
  • N-acetylglucosamine can be coupled as a hapten to a carrier protein to obtain an antigen, and is used to prepare a monoclonal antibody or a polyclonal antibody for detecting the same.
  • the carrier protein may be a commonly used carrier protein such as ovalbumin (OVA), bovine serum albumin (BSA) and hemocyanin (KLH), or may be one of glycoproteins, such as mucin and other proteins.
  • OVA ovalbumin
  • BSA bovine serum albumin
  • KLH hemocyanin
  • the inventors isolated the tumor antigen Mu-GlcNAc (code MA153) purified by the present disclosure formed by N-acetylglucosamine linked mucin 1 (Mucin1, code Mu) in serum. It is linked at the exposed end of the sugar chain in mucin 1, as shown in Figure 1B.
  • the amino acid sequence of the mucin 1 (Mu) structure is (-PDTRPAPGSTAPPAHGVTSA-) ⁇ N, ie the amino acid sequence of mucin 1 (Mu-) is : - Multiple repeat alignment of -PDTRPAPGSTAPPAHGVTSA- (SEQ ID NO: 2).
  • the tumor antigen obtained by the N-acetylglucosamine-linked mucin 1 of the present disclosure can be isolated and purified from the serum of tumor cells or tumor patients, and its main features are as follows:
  • Mu-GlcNAc was ligated to the monoclonal antibody Ma695 affinity chromatography column by determinant-PDTR-, thus obtaining the purified tumor antigen Mu-GlcNAc (see Figure 1B). );
  • GlcNAc antigen can be recognized by N-acetylglucosamine specific binding
  • F in the serum of breast cancer, pancreatic cancer, liver cancer, lung cancer, esophageal cancer, oral cancer, colon cancer, cholangiocarcinoma, ovarian cancer, cervical cancer, uterine cancer, bladder cancer, stomach cancer, kidney cancer, cardiac cancer;
  • the antigen can be coated on the surface of the polystyrene material as a solid phase antigen by using 0.05M pH 9.2 phosphate buffer solution;
  • this antigen can react with N-acetylglucosamine specific binding partner to form serum tumor Marker determination
  • K, Mu-GlcNAc antigen can form a sandwich mode assay with Ma695 monoclonal antibody, and specific binding complex of N-acetylglucosamine;
  • this antigen can react with a specific binding body of N-acetylglucosamine to form a single binder assay
  • N-acetylglucosamine can also link other glycoproteins to form tumor antigens, that is, -GlcNAc can also be linked with other glycoproteins to form Glycoprotein-N-acetyl-glucosamine (glycoprotein-N-acetyl-glucosamine) structure, also known as Gp. -GlcNAc; its characteristics are:
  • glycoprotein described in B and A carries -GlcNAc without the -PDTR-peptide segment
  • this glycoprotein can react with any of the binding bodies A, B, C, D, but not with Ma695 mAb;
  • N-acetylglucosamine (-GlcNAc) has a higher tumor recognition rate than the specific binding partner of PDTR-; therefore, N-acetylglucosamine (- GlcNAc) can be referred to as a new serum tumor marker for screening tumor-detecting substances (aptamers, antibodies, lectins).
  • the present disclosure provides the use of a substance for detecting N-acetylglucosamine in the preparation of a kit for detecting a tumor.
  • the substance for detecting N-acetylglucosamine is a substance capable of specifically binding to N-acetylglucosamine (N-acetylglucosamine specific binding body).
  • the substance for detecting N-acetylglucosamine is a monoclonal antibody, polyclonal antibody, aptamer or lectin capable of specifically binding to N-acetylglucosamine.
  • the monoclonal antibody or polyclonal antibody capable of specifically binding to N-acetylglucosamine is a monoclonal antibody or a polyclonal antibody having N-acetylglucosamine as an antigen.
  • the kit for detecting a tumor is a kit using serum as a test sample; the tumor is a malignant tumor.
  • the tumor is one or more of breast cancer, pancreatic cancer, liver cancer, lung cancer, esophageal cancer, oral cancer, colon cancer, cholangiocarcinoma, ovarian cancer, cervical cancer, uterine cancer, bladder cancer, stomach cancer, kidney cancer and cardiac cancer.
  • breast cancer pancreatic cancer
  • liver cancer lung cancer
  • esophageal cancer oral cancer
  • colon cancer cholangiocarcinoma
  • ovarian cancer cervical cancer
  • uterine cancer bladder cancer
  • stomach cancer stomach cancer
  • kidney cancer kidney cancer
  • cardiac cancer cardiac cancer
  • aptamer, MA153-A the nucleotide sequence is P1(18)-N60-P2(18), and the MA153-A gene clone A1 used in this experiment has the sequence: 5'-biotin-CGTAC GGTCG ACGCTAGC- 3'-gggtt cagtg gtcga cgact ttgtt tgtgt cctca catgc attac taacg gtcat ccctg-5'-GGATC CGAGC TCCAC GTG-3' (SEQ ID NO: 1), Kd 20nM, starting target ("antigen”): N-acetyl Glucosamine (GlcNAc), technique used: SELEX.
  • MA153-B Mab E111, IgG1, Ka 10 10 L/M, antigenic determinant GlcNAc, murine resistance, hybridoma technique.
  • C polyclonal antibody, MA153-C; Pab anti-GlcNAc, Ka 10 10 L/M, corresponding hapten GlcNAc, rabbit anti-subcutaneous injection technique.
  • N-acetylglucosamine assay has two modes, sandwich mode and single combination mode;
  • Ma695 is a capture body
  • MA153 is a central antigen
  • N-acetylglucosamine specifically binds one of ABCD in the above-mentioned substance for detecting N-acetylglucosamine, the same below
  • any " GlcNAc-binding" linkage analysis for the detection of tumors such as Ma695-Mu-GlcNAc-aptamer sandwich analysis for tumor detection.
  • Single-combined assay is also called “single-combination non-competitive analysis” or “direct-recognition analysis” to distinguish it from current competitive analysis; single-combination assay is to use only N-acetylglucosamine-specific combination.
  • the recognition body is a single-combination assay that directly recognizes N-acetylglucosamine in the test subject with an N-acetylglucosamine-specific binder; the essence of the single-combination assay is the specific binding of the binder to the formation of -GlcNAc.
  • -GlcNAc-MA153-A is ligated into a single binding assay for tumor detection
  • -GlcNAc-MA153-B (Mab E111) is ligated into a single binding assay for tumor detection
  • -GlcNAc-MA153-C(Pab) The junction is made into a single binding body for tumor detection
  • -GlcNAc-MA153-D biotin-ATV new plant lectin Triticum Vulgaris
  • the present disclosure uses -GlcNAc as an initial antigen, target, and ligand to prepare, find, culture, and screen for specific binding partners, such as: aptamers, monoclonal antibodies, polyclonal antibodies, lectins, lectins, receptors.
  • specific binding partners such as: aptamers, monoclonal antibodies, polyclonal antibodies, lectins, lectins, receptors.
  • N-acetylglucosamine and a glycoprotein linked product can be used as an antigen, such as a highly purified MA153 (Mu-GlcNAc) antigen as a starting antigen, a target, a ligand, to prepare, find, Screening and breeding specific binding partners such as aptamers, monoclonal antibodies, polyclonal antibodies, lectins, lectins, and receptors to establish tumor detection methods; analytes for MA153 (Mu-GlcNAc), tumors Marker determination
  • the antigenic structure (Mu-GlcNAc) is used as a guide, or these routes are used as clues to obtain a combination, and a tumor marker is detected;
  • glycoprotein-N-acetyl-glucosamine Gp-GlcNAc is used as an initial antigen, target, and ligand to prepare, find, culture, and screen for specific binding partners, such as aptamers, monoclonal antibodies, and polyclonal antibodies.
  • specific binding partners such as aptamers, monoclonal antibodies, and polyclonal antibodies.
  • the present disclosure utilizes N-acetylglucosamine as a marker for tumor detection to screen tumor detection substances in the following ways:
  • the inventors of the present disclosure found that there is a significant difference in the amount of N-acetylglucosamine in the blood of a tumor patient compared to the amount in the blood of a healthy person.
  • the present disclosure directly uses SELDI and MELDI mass spectrometry in the blood of a tumor patient to obtain a special glycan structure map of the tumor patient, and finds a specific line of the tumor, thereby finding a tumor-specific sugar structure from the detection results of the two types of mass spectrometers, Glycoprotein, we have discovered GlcNAc (N-acetylglucosamine) special glycosyl, and then purified and purified target glycoprotein by polyacrylamide electrophoresis, high pressure liquid chromatography and lectin affinity chromatography.
  • GlcNAc N-acetylglucosamine
  • Mucin1 (mucin 1, a glycoprotein) was obtained from MCF-7 tumor cell line.
  • Mucin1 mucin 1, a glycoprotein
  • the glycoprotein extracted above was named MA153, and its chemical structure was N-acetylglucosamine linked mucin 1 (Mucin1, code Mu), which was linked at the exposed end of the sugar chain in mucin 1, see Figure 1B.
  • the amino acid sequence of the mucin 1 (Mu) structure is (-PDTRPAPGSTAPPAHGVTSA-) ⁇ N, that is, the amino acid sequence of mucin 1 (Mu-) is: - multiple repeat arrangement of -PDTRPAPGSTAPPAHGVTSA- (SEQ ID NO: 2). It was also found to form a sandwich reaction with the monoclonal antibody Ma695 and the related Aptamer (aptamer) to form Ma695-Mu-GlcNAc-Aptamer.
  • tumors can be identified by detection of N-acetylglucosamine.
  • the new tumor marker-GlcNAc N-acetylglucosamine structure (see Figure 1A) and its relationship to the glycoprotein in which it is located, and its relationship to the relevant binder (see Figure 1B).
  • the amino acid sequence of the linked mucin 1 (Mu-) is: - multiple repeat arrangement of -PDTRPAPGSTAPPAHGVTSA- (SEQ ID NO: 2).
  • N-acetylglucosamine-specific bindings after the discovery of new markers is the second critical step.
  • the purified antigen provides extremely convenient conditions for the preparation of specific binding partners.
  • SELEX's new technology for the preparation of aptamers is our first choice. It can be used for both tumor diagnosis and tumor therapy. It has a small molecule and has more advantages than antibodies in tumor-targeted therapy. This is followed by monoclonal antibodies, polyclonal antibodies, plant lectins (PHAs) and/or receptors and other ligands.
  • aptamer MA153-A monoclonal antibody MA153-B (E111), polyclonal antibody MA153-C, new plant lectin MA153-D (biotin-ATV, biotin-phyto-Agglutinin from Triticum Vulgaris): Specifically as described in AD:
  • a aptamer a nucleic acid library consisting of a single-stranded DNA fragment of more than one hundred million different structures was synthesized on a Biosearch 8600 DNA synthesizer. PCR amplification, becoming a DNA duplex. Dissociate the double strands and label them for use as an isotope experiment. Separate the complementary strand into the screen.
  • Starting target (“antigen"): N-acetylglucosamine (GlcNAc), technique used: SELEX.
  • the candidate Aptamer was captured by solid phase Mu-GlcNAc to form a Mu-GlcNAc-DNA single-stranded complex. Dissociation of the DNA single strand from the complex. Re-enter PCR amplification (step 2).
  • aptamer SEQ ID NO: 1
  • 0.1 M pH 8.8 borate buffer 1 mg was dialyzed against 0.1 M pH 8.8 borate buffer for more than 12 hours, taken out and placed in a clean glass bottle to prepare NH-Biotin 1 mg/mL, and 50 ⁇ L was added quickly.
  • Into the aptamer solution shake at room temperature (25 degrees Celsius) for 2 hours, dialyze in 0.5% BSA 0.05 M pH 7.5 PBS for two days, and take it out.
  • MA153-B Mab E111, IgG1, Ka 1010 L/M, antigenic determinant GlcNAc, murine resistance, hybridoma technique.
  • Example 2 Method for screening tumors by screening specific binding bodies using N-acetylglucosamine (sandwich method)
  • the sandwich mode was used to determine the Mu-GlcNAc antigen, and the principle is shown in FIG.
  • the Mu-GlcNAc stock solution was diluted with the reaction solution to a standard of different concentrations of 0, 1, 3, 10, 30, 100, 300 U/mL.
  • U is an N-acetylglucosamine active unit.
  • Mu-GlcNAc antigen is positive in the serum of more than 10 tumor patients, and the tumor specificity is as high as 95%, which is a good new tumor marker. From its broad spectrum of tumors, it may be closely related to the malignant nature shared by the tumor, not just individual tumors. Further research on it is of great significance.
  • the monoclonal antibody Ma695 recognizes the -PDTR-peptide on the Mucin1 (mucin-1) protein, and the epitope recognized by the aptamer used above, via a lectin chip (Tateno H, Uchiyama N, Kuno A, Et al.A novel strategy for mammalian cell surface glycome profiling using lectin microarray. Glycobiology 2007, 17(10): 1138-1146.) approved as GlcNAc (N-acetyl-Glucosamine, N-acetylglucosamine), thus in the reaction
  • the basic structure of the sandwich antigen is Mu-GlcNAc provided by the present disclosure.
  • Protein glycosylation is a key factor in protein function, and the process of carcinogenesis is accompanied by abnormal glycosylation (Fan Zhenfu, Chen Zhizhou, Fan Feizhou. Basic research on glycosylation changes and tumors. Labeled immunoassays and clinical, 2010, 17 (2 ): 122-126.), the abnormal glycosyl group formed constitutes an immunological determinant of tumor mAb. So far, carbohydrate antigen (CA) has occupied a prominent position in clinical tumor detection. The study of glycobiology is an important source of new tumor marker development. This study is expected to be produced in the near future. New anti-cancer vaccine.
  • CA carbohydrate antigen
  • Example 3 single binding body assay method for detecting tumor
  • Single-combined assays use only N-acetylglucosamine-specific binders as identifiers; single-binding assays use N-acetylglucosamine-specific binders to directly recognize N-acetylglucosamine in test subjects; single-binding
  • the essence of the bulk assay is the specific binding of the binder to the formation of -GlcNAc.
  • reaction solution and washing solution Add 0.05% CaCl 2 , 0.05% MgCl 2 , 6% BSA to the reaction solution in pH 7.5, 0.05 M PBS buffer; add 0.01% Tween 20 to the reaction solution. It is the washing liquid.
  • Standard and serum sample dilution Standard and serum samples were diluted 10-fold with 0.05 M pH 9.2 phosphate buffer prior to assay.
  • 2.4 GlcNAc antigen determination step 0.1 mL of the diluted standard and the test serum sample were sequentially added to the polystyrene small test tube, and the solid phase antigen was formed at 4 ° C overnight to discard the reaction solution. Add 0.1 mL of 6% BSA, react at room temperature for 2 h, coat with saturation, and discard the reaction solution.
  • the current commercial kits are divided into two categories, the sandwich mode and the competition mode, and there is only a sandwich mode of the double combination in the field of tumor markers.
  • the present disclosure opens up a new mode, named the Single binder assay. It saves antibodies and reduces costs by 80%, increasing sensitivity without reducing specificity.
  • the single-combination mode improves the detection rate of lung cancer by 22% compared to the sandwich mode: the former has a sensitivity of 97% and a specificity of 96%; the latter has a sensitivity of 75% and a specificity of 95%.
  • Kits can be prepared using a single conjugate mode, which kits can include reagents as shown in the following 1-4, as indicated by 5.
  • aptamer SEQ ID NO: 1
  • 0.1 M pH 8.8 borate buffer 1 mg was dialyzed against 0.1 M pH 8.8 borate buffer for more than 12 hours, taken out and placed in a clean glass bottle to prepare NHS-Biotin 1 mg/mL, and 50 ⁇ L was added quickly.
  • Into the aptamer solution shake at room temperature (25 degrees Celsius) for 2 hours, dialyze in PBS containing 0.05 M pH 7.5 for two days, and take it out for use.
  • the serum to be tested is diluted to 1/10 by pH 9.20.05M phosphate buffer solution, 200 ⁇ L is directly added to the polystyrene 96-well plate, reacted at room temperature for 4 hours, and the reaction mixture is discarded and washed with 0.05 M pH 7.5 PBS for 3 times. 250 ⁇ L of PBS containing 1.5% bovine serum albumin, one hour at 37 °C. The reaction solution was discarded and washed twice with PBS for use.
  • the experiment proves that the GlcNAc tumor-specific adaptive analysis kit of the present disclosure is simple and suitable for screening, and one test can cover more than ten kinds of tumors, and its detection efficiency, that is, the total positive rate of cancer exceeds any existing tumor. Detection kit

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Abstract

公开了检测肿瘤的试剂盒和与N-乙酰氨基葡萄糖特异性结合的适配体。所述试剂盒包含检测肿瘤的物质，该检测肿瘤的物质与N-乙酰氨基葡萄糖特异性结合的物质。

Description

检测肿瘤的试剂盒及其专用识别特定糖基结构的物质
相关申请的交叉引用
本公开主张在2014年9月12日在中国提交的中国专利申请号No.201410464565.5的优先权，其全部内容通过引用包含于此。
技术领域
本公开涉及检测肿瘤的试剂盒及其专用识别特定糖基结构的物质。
背景技术
自20世纪80年代，专家利用杂交瘤技术获得了能识别肿瘤特异性大分子糖蛋白抗原(carbohydrate antigen,CA)，并研制了单克隆抗体识别系统。发展至今在目前肿瘤体外早期诊断试剂领域，存在竞争性分析和非竞争性夹心分析两种分析方法。从实验室检测的角度，肿瘤标志物分为血清/血浆肿瘤标志物与组织细胞肿瘤标志。血清/血浆肿瘤标志物大致分为三类：1)胚胎抗原。胚胎期表达、正常成人不表达、伴随肿瘤发生又重新表达的抗原，如AFP、CEA。2)肿瘤相关糖脂及糖蛋白。CA199、CA50、CA242、CA724、CA125、CA153等。3)激素肽、酶、蛋白。这些在正常组织中有表达，但是在肿瘤组织中只是过量表达的抗原。PSA、NSE、TPA等。对这些肿瘤标志物的检测也发展出很多微量免疫检测技术，比如：1959年Berson与Yalow创立了放射免疫测定(radioimmunoassay,RIA)，1971年瑞典的Engvall建立了酶联免疫吸附实验(enzyme-linked immunosorbent assay,ELISA)以及近期的化学发光免疫测定(chemluminescence immunoassay,CLIA)、电化学发光免疫测定(electrochemiluminesence immunoassay,ECLI)、时间分辨荧光免疫测定(time-resolved fluoroimmunoassay,TRFIA)。目前对于任何一个肿瘤标志物不管采用那种测定技术，鉴定肿瘤和非肿瘤患者不是100％准确的。比如AFP对原发性肝癌的特异性为70％；CEA对原发性结肠癌特异度为45-80％，对胰腺癌、胃癌、肺癌、乳腺癌为25-70％；CA125对卵巢癌为82％；CA153对乳腺癌早期为75％；CA724对胃癌为42％，NSE对小细胞肺癌为70％。以上的标志物并不是广谱的，大都只能诊断一种或几种肿瘤，并且以CEA为例，其对原发性结肠癌诊断效果最好， 为80％，虽然对胰腺癌、胃癌、肺癌、乳腺癌也有效，但最低的特异性仅有25％，换句话说，75％是会漏诊的。肿瘤的早期发现与诊断，对于提高患者治愈率、延长患者生命是至关重要的，致力于肿瘤研究的学者在探索和寻找有效的肿瘤标志从未松懈。
肿瘤早期诊断至今仍然是世界性难题，现用的肿瘤标志物只能检出约70％的肿瘤，且绝大多数已属晚期。现行肿瘤标志物大都确切结构尚不明确，难于复制。
现行商品试剂盒的模式分为两类，夹心模式和竞争模式，且在肿瘤标志物领域只有双结合体的夹心模式。
发明内容
基于上述缺陷，本公开的目的是提供检测肿瘤的试剂盒，该试剂盒包含可以与N-乙酰氨基葡萄糖特异性结合的物质。本公开利用N-乙酰氨基葡萄糖特异性结合的物质制备操作简单，检测特异性和灵敏性高，适于肿瘤发病初期的检测试剂盒。
所述检测肿瘤的试剂盒为以血清为检测样本的试剂盒；所述肿瘤为恶性肿瘤。
所述肿瘤为乳腺癌、胰腺癌、肝癌、肺癌、食管癌、口腔癌、结肠癌、胆管癌、卵巢癌、宫颈癌、子宫癌、膀胱癌、胃癌、肾癌和贲门癌的一种或几种。
上述能与N-乙酰氨基葡萄糖特异性结合的物质为以N-乙酰氨基葡萄糖为抗原决定簇的单克隆抗体、多克隆抗体、与N-乙酰氨基葡萄糖特异性结合的适配体或与N-乙酰氨基葡萄糖特异性结合的凝集素。
所述与N-乙酰氨基葡萄糖特异性结合的适配体为具有如SEQ ID NO：1所示序列的核酸。
本公开还保护与N-乙酰氨基葡萄糖特异性结合的适配体，为具有如SEQID NO：1所示序列的核酸。
N-乙酰氨基葡萄糖分子式：C8H15NO6，分子量：221.21，化学代号-GlcNAc，其结构如图1A所示它可以与载体蛋白偶联得到抗原，制备可以检测肿瘤的特异性结合体，如抗体、核酸适配体、凝集素等，这些特异性结合体可以与N-乙酰氨基葡萄糖特异性结合。
上述载体蛋白可以为常用的载体蛋白，如卵白蛋白(OVA)、牛血清白 蛋白(BSA)与血蓝蛋白(KLH)，也可以为糖蛋白，如粘蛋白及其他的蛋白中的一种或几种。
例如，在本公开的实施例中，发明人在血清中分离出了N-乙酰氨基葡萄糖链接粘蛋白1(Mucin1，代号Mu)形成的本公开所提纯的肿瘤抗原Mu-GlcNAc(代号MA153)，它链接的位置在粘蛋白1内糖链的起始端，见图1B，粘蛋白1(Mu)结构的氨基酸序列为(-PDTRPAPGSTAPPAHGVTSA-)×N，即粘蛋白1(Mu-)的氨基酸序列为：-PDTRPAPGSTAPPAHGVTSA-(SEQ ID NO：2)的多次重复排列。
本公开利用N-乙酰氨基葡萄糖的特异结合体包括提纯的新的高灵敏血清肿瘤标志物抗原即2-deoxy-N-acetyl-D-glucosamine-mucin1(简称Mu-GlcNAc，也称为MA153)和N-乙酰氨基葡萄糖的特异结合体：适配体(核酸，如MA153-A)和其它结合体如单克隆抗体，多克隆抗体，植物凝集素(PHA)和/或受体，相关配体，以及检测早期肿瘤的方法。直接检测N-乙酰氨基葡萄糖的方法包括两个模式：1)Ma695-Mu-GlcNAc-MA153-A夹心式，2)单结合体式，由N-乙酰氨基葡萄糖的特异结合体直接识别GlcNAc。用此方法可以检测出97％的肺癌，肿瘤特异度96％。
本公开提供上述检测肿瘤的方法，是检测待测患者血清中的N-乙酰氨基葡萄糖的浓度，以1U/mL为正常界值的上限值，其中U为N-乙酰氨基葡萄糖活性单位。
临床血清学检测表明，本公开的技术方案对很多肿瘤的检出率都在90％以上，肿瘤特异性在95％以上。这必将大大提高肿瘤的早期诊断率。本公开适于在正常人群中做肿瘤早期普查。
本公开开辟了一种新模式，命名为单结合体模式(Single binder assay)，由N-乙酰氨基葡萄糖的特异结合体直接识别N-乙酰氨基葡萄糖。它节省抗体，可降低成本80％，提高灵敏度而不降低特异度。在本公开中，单结合体模式比夹心模式将肺癌的检出率提高了22％：前者灵敏度97％，特异度96％；后者灵敏度75％，特异度95％。
附图说明
图1A表示N-乙酰氨基葡萄糖的结构图；
图1B表示N-乙酰氨基葡萄糖与粘蛋白1的关系；N-乙酰氨基葡萄糖与适配体MA153-A的关系；粘蛋白1与单抗Ma695的关系；Mu-GlcNAc模拟 结构；
图2表示Mu-GlcNAc夹心测定分析的原理；
图3表示Mu-GlcNAc夹心式测定标准曲线；
图4A表示N-乙酰氨基葡萄糖单结合体测定原理；
图4B与图4A相似，不同的是待测血清中与N-乙酰氨基葡萄糖链接的蛋白部分中没有-PDTR-肽段。表明在MA153中可能存在Mucin1以外其它携带-GlcNAc的糖蛋白；
图5为N-乙酰氨基葡萄糖单结合体测定标准曲线。
具体实施方式
下述实施例中的百分含量，如无特别说明，均为质量百分含量。
基于本公开，提供本公开的目的是提供检测肿瘤的试剂盒，该试剂盒包含可以与N-乙酰氨基葡萄糖特异性结合的物质。本公开利用N-乙酰氨基葡萄糖特异性结合的物质制备简单，检测特异性和灵敏性高，适于肿瘤发病初期的检测。
所述检测肿瘤的试剂盒为以血清为检测样本的试剂盒；所述肿瘤为恶性肿瘤。
所述肿瘤为乳腺癌、胰腺癌、肝癌、肺癌、食管癌、口腔癌、结肠癌、胆管癌、卵巢癌、宫颈癌、子宫癌、膀胱癌、胃癌、肾癌和贲门癌的一种或几种。
上述能与N-乙酰氨基葡萄糖特异性结合的物质为以N-乙酰氨基葡萄糖为抗原决定簇的单克隆抗体、多克隆抗体、与N-乙酰氨基葡萄糖特异性结合的适配体或与N-乙酰氨基葡萄糖特异性结合的凝集素。
所述与N-乙酰氨基葡萄糖特异性结合的适配体为具有如SEQ ID NO：1所示序列的核酸。
本公开还保护与N-乙酰氨基葡萄糖特异性结合的适配体，为SEQ ID NO：1所示序列的核酸。
N-乙酰氨基葡萄糖分子式：C8H15NO6，分子量：221.21，化学代号-GlcNAc，其结构如图1A所示它可以与载体蛋白偶联得到抗原，制备可以检测肿瘤的特异性结合体，如抗体、核酸适配体、凝集素等，这些特异性结合体可以与N-乙酰氨基葡萄糖特异性结合。
N-乙酰氨基葡萄糖(GlcNAc)肿瘤抗原的特性：
N-乙酰氨基葡萄糖分子式：C8H15NO6，分子量：221.21，化学代号-GlcNAc，其结构如图1A所示，它可以用于筛选、制备可以检测肿瘤的特异性结合体，如抗体、核酸适配体、凝集素等，这些特异性结合体可以与N-乙酰氨基葡萄糖特异性结合。N-乙酰氨基葡萄糖可以作为半抗原与载体蛋白偶联得到抗原，用于制备检测其的单克隆抗体或多克隆抗体。
上述载体蛋白可以为常用的载体蛋白，如卵白蛋白(OVA)、牛血清白蛋白(BSA)与血蓝蛋白(KLH)，也可以为糖蛋白，如粘蛋白及其他的蛋白中的一种或几种。
例如，在本公开的实施例中，发明人在血清中分离出了N-乙酰氨基葡萄糖链接粘蛋白1(Mucin1，代号Mu)形成的本公开所提纯的肿瘤抗原Mu-GlcNAc(代号MA153)，它链接的位置在粘蛋白1内糖链的暴露端，见图1B，粘蛋白1(Mu)结构的氨基酸序列为(-PDTRPAPGSTAPPAHGVTSA-)×N，即粘蛋白1(Mu-)的氨基酸序列为：-PDTRPAPGSTAPPAHGVTSA-(SEQ ID NO：2)的多次重复排列。本公开N-乙酰氨基葡萄糖链接粘蛋白1得到的肿瘤抗原，可以从肿瘤细胞或者肿瘤患者血清中分离纯化得到，其主要特点如下：
A、Mu-GlcNAc的模拟结构及与相关结合体的关系：Mu-GlcNAc通过决定簇-PDTR-挂到单抗Ma695亲和层析柱上，从而得到纯化的肿瘤抗原Mu-GlcNAc(见图1B)；
B、Mu-GlcNAc分子量约140kD；
C、由肿瘤细胞所产生；
D、在粘蛋白1上有-PDTR-肽段抗原决定簇；此决定簇可被Ma695单抗识别(见图1B)；
E、作为GlcNAc抗原可被N-乙酰氨基葡萄糖特异性结合体所识别；
F、存在于乳腺癌、胰腺癌、肝癌、肺癌、食管癌、口腔癌、结肠癌、胆管癌、卵巢癌、宫颈癌、子宫癌、膀胱癌、胃癌、肾癌、贲门癌患者的血清中；
G、不存在于正常人的血清中；
H、不存在于良性疾病患者血清中；
I、用0.05M pH 9.2磷酸缓冲液可以将此抗原包被于聚苯乙烯材料表面成为固相化抗原；
J、此抗原可与N-乙酰氨基葡萄糖特异性结合体反应，而形成血清肿瘤 标志物测定；
K、Mu-GlcNAc抗原可与Ma695单抗，以及N-乙酰氨基葡萄糖的特异性结合体形成夹心模式测定；
N、此抗原可与N-乙酰氨基葡萄糖的特异性结合体反应，形成单结合体测定(Single binder assay)；
N-乙酰氨基葡萄糖还可以链接其他糖蛋白形成肿瘤抗原，即-GlcNAc也可以和其它糖蛋白链接形成Glycoprotein-N-acetyl-glucosamine(糖蛋白-N-乙酰-氨基葡萄糖)结构，可又名Gp-GlcNAc；其特征是：
A、通过单结合体测定的灵敏度高于夹心模式测定的事实说明在肿瘤抗原中与N-乙酰氨基葡萄糖链接的可能还含有Mucin1以外的少部分糖蛋白；
B、A所述的此糖蛋白带有-GlcNAc，而不带有-PDTR-肽段；
C、此糖蛋白可与结合体A、B、C、D中的任何一个反应，而不与Ma695单抗反应；
通过本公开的实验表明，N-乙酰氨基葡萄糖(-GlcNAc)的特异性结合体对比-PDTR-的特异性结合体而言，前者的肿瘤识别率更高；因此，N-乙酰氨基葡萄糖(-GlcNAc)可以被称为一个新的血清肿瘤标志物，用于筛选检测肿瘤的物质(适配体、抗体、凝集素)。
本公开提供检测N-乙酰氨基葡萄糖的物质在制备检测肿瘤的试剂盒中的应用。
其中，所述检测N-乙酰氨基葡萄糖的物质是能与N-乙酰氨基葡萄糖特异性结合的物质(N-乙酰氨基葡萄糖特异性结合体)。
所述检测N-乙酰氨基葡萄糖的物质是能与N-乙酰氨基葡萄糖特异性结合的单克隆抗体、多克隆抗体、适配体或凝集素。
所述能与N-乙酰氨基葡萄糖特异性结合的单克隆抗体、多克隆抗体是以N-乙酰氨基葡萄糖为抗原的单克隆抗体或多克隆抗体。
所述检测肿瘤的试剂盒为以血清为检测样本的试剂盒；所述肿瘤为恶性肿瘤。
所述肿瘤为乳腺癌、胰腺癌、肝癌、肺癌、食管癌、口腔癌、结肠癌、胆管癌、卵巢癌、宫颈癌、子宫癌、膀胱癌、胃癌、肾癌和贲门癌的一种或几种。
上述检测N-乙酰氨基葡萄糖的物质分列如下：
A：适配体，MA153-A，核苷酸序列为P1(18)-N60-P2(18)通式，本实验所用MA153-A属克隆A1其序列：5‘-biotin-CGTAC GGTCG ACGCTAGC-3’-gggtt cagtg gtcga cgact ttgtt tgtgt cctca catgc attac taacg gtcat ccctg-5’-GGATC CGAGC TCCAC GTG-3’(SEQ ID NO：1)，Kd 20nM，起始靶的(“抗原”)：N-乙酰氨基葡萄糖(GlcNAc)，所用技术：SELEX。
B：单克隆抗体，MA153-B：Mab E111，IgG1，Ka 1010L/M，抗原决定簇GlcNAc，鼠抗，杂交瘤技术。
C：多克隆抗体，MA153-C；Pab anti-GlcNAc，Ka 1010L/M，所对应的半抗原GlcNAc，兔抗，皮下多点注射免疫技术。
D：新植物凝集素/或受体，MA153-D：生物素-植物凝集素Triticum Vulgaris，ATV，Sigma USA，Catalogue No 9604。
N-乙酰氨基葡萄糖检测方法有下列特点：
N-乙酰氨基葡萄糖测定有两种模式，夹心模式和单结合体模式；
A、在夹心模式中，Ma695是扑捉体，MA153是中心抗原，N-乙酰氨基葡萄糖特异性结合(上述检测N-乙酰氨基葡萄糖的物质中A.B.C.D之一，下同)是报告体；任何“GlcNAc-结合体”联接式分析，用以检测肿瘤；如Ma695-Mu-GlcNAc-适配体夹心式分析，做肿瘤检测。
B、单结合体式分析又名为“单结合体非竞争性分析”或“直接识别式分析”以区别于现行的竞争性分析；单结合体式分析是只用N-乙酰氨基葡萄糖特异性结合体做识别体；单结合体分析是用N-乙酰氨基葡萄糖特异性结合体直接识别检测对象中的的N-乙酰氨基葡萄糖；单结合体分析的本质是结合体与-GlcNAc形成的特异性结合。如-GlcNAc-MA153-A联接做成单结合体测定，做肿瘤检测；-GlcNAc-MA153-B(Mab E111)联接做成单结合体测定，做肿瘤检测；-GlcNAc-MA153-C(Pab)联接做成单结合体测定，做肿瘤检测；-GlcNAc-MA153-D(biotin-ATV新植物凝集素Triticum Vulgaris)联接做成单结合体测定，做肿瘤检测。
本公开以-GlcNAc用作初始抗原、靶标、配体，来制备、寻找、培育、筛选特异结合体，如：适配体、单克隆抗体、多克隆抗体、植物凝集素、凝集素、受体，以建立肿瘤标志物检测分析。以-GlcNAc为目的分析物，做肿瘤标志物测定；
由于在肿瘤患者的血清中，N-乙酰氨基葡萄糖经常与糖蛋白链接，因 此，在检测肿瘤的物质中，可以使用N-乙酰氨基葡萄糖与糖蛋白链接产物作为抗原，如高度纯化的MA153(Mu-GlcNAc)抗原作为起始抗原、靶标、配体、来制备、寻找、筛选、培育特异结合体如：适配体、单克隆抗体、多克隆抗体、植物凝集素、凝集素、受体，以便建立肿瘤检测方法；以MA153(Mu-GlcNAc)为目的分析物，做肿瘤标志物测定；
抗原结构(Mu-GlcNAc)为指导，或以这些路线为线索，来获得结合体，做成肿瘤标志物检测；
又如，糖蛋白-N-乙酰-氨基葡萄糖Gp-GlcNAc用作初始抗原、靶标、配体，来制备、寻找、培育、筛选特异结合体，如：适配体、单克隆抗体、多克隆抗体、植物凝集素、凝集素、受体，以建立肿瘤标志物检测分析；以-Gp-GlcNAc为目的分析物，做肿瘤标志物测定；
基于上述方法，本公开利用N-乙酰氨基葡萄糖作为肿瘤检测的标志物进行肿瘤检测物质的筛选应用有下述几种方式：
a、1)Mu-GlcNAc、或者Gp-GlcNAc作为整体纯靶标制备适配体；2)小分子的GlcNAc可以单独做靶标直接筛选、制备适配体，用于肿瘤检测；
b、1)Mu-GlcNAc、或者Gp-GlcNAc作为整体纯抗原制备单克隆抗体；2)小分子GlcNAc作为半抗原(hapten)联接载体蛋白成为有效抗原，制备单克隆抗体，用于肿瘤检测；
c、1)Mu-GlcNAc、或者Gp-GlcNAc作为整体纯抗原制备多克隆抗体；2)小分子GlcNAc作为半抗原(hapten)联接载体蛋白成为有效抗原，制备多抗，用于肿瘤检测。
实施例
实施例1、N-乙酰氨基葡萄糖用于肿瘤检测的发现和鉴定
本公开的发明人发现在肿瘤患者的血液中有一种N-乙酰氨基葡萄糖的含量与健康人的血液中的含量相比具有显著的差异。
本公开用肿瘤患者的血液在体外直接做SELDI、MELDI质谱分析，得到肿瘤患者特别的糖结构图谱，找到肿瘤的特异谱线，从而从两种类型质谱仪的检测结果中发现肿瘤特异糖结构、糖蛋白，目前我们发现的就是GlcNAc(N-乙酰氨基葡萄糖)特殊糖基，进而以聚丙酰胺电泳、高压液相层析、凝集素亲和层析分离纯化得到的目标糖蛋白。
我们试用亲和层析的方法(Mu-GlcNAc通过决定簇-PDTR-挂到单抗 Ma695亲和层析柱上，从而得到纯化的肿瘤抗原Mu-GlcNAc)从MCF-7肿瘤细胞株得到Mucin1(粘蛋白1，一个糖蛋白)。我们用凝集素芯片技术证实，上述新提取的Mucin1恰巧携带有密集的-GlcNAc，我们判断这正是我们寻找的新标志物，并在本公开的实验中得到了证实。
将上述提取的这种糖蛋白命名为MA153，其化学结构中N-乙酰氨基葡萄糖链接粘蛋白1(Mucin1，代号Mu)它链接的位置在粘蛋白1内糖链的暴露端，见图1B，粘蛋白1(Mu)结构的氨基酸序列为(-PDTRPAPGSTAPPAHGVTSA-)×N，即粘蛋白1(Mu-)的氨基酸序列为：-PDTRPAPGSTAPPAHGVTSA-(SEQ ID NO：2)的多次重复排列。并且发现它可以与单克隆抗体Ma695和相关Aptamer(适配体)形成Ma695-Mu-GlcNAc-Aptamer的夹心反应。因此，可以通过对N-乙酰氨基葡萄糖的检测，来鉴定肿瘤。新肿瘤标志物-GlcNAc(N-乙酰氨基葡萄糖)结构(见图1A)及其与所在糖蛋白的关系，和与相关结合体的关系(见图1B)。其中，其连接的粘蛋白1(Mu-)的氨基酸序列为：-PDTRPAPGSTAPPAHGVTSA-(SEQ ID NO：2)的多次重复排列。
新标志物发现后建立N-乙酰氨基葡萄糖特异性结合体是第二个关键步骤。明确的结构，纯化的抗原为制备特异结合体，提供了极为便利的条件。SELEX新技术制备适配体(aptamer)是我们的首选，它既可用于肿瘤诊断也可用于肿瘤治疗，它的分子较小，在肿瘤靶向治疗方面具有比抗体更多的优越性。其后是单克隆抗体、多克隆抗体、植物凝集素(PHA)和/或受体及其他配体。最后我们得到了4种针对-GlcNAc抗原/决定簇的最适结合体：适配体MA153-A、单克隆抗体MA153-B(E111)、多克隆抗体MA153-C、新植物凝集素MA153-D(biotin-ATV，biotin-phyto-Agglutinin from Triticum Vulgaris)：具体如下A-D所述：
A适配体，在Biosearch 8600DNA合成仪上合成含有百万亿以上个不同结构的单链DNA片段构成的核酸文库。PCR扩增，成为DNA双链。解离双链，标记其一，用做同位素实验。分离互补链进入筛选。起始靶的(“抗原”)：N-乙酰氨基葡萄糖(GlcNAc)，所用技术：SELEX。用固相Mu-GlcNAc扑捉候补Aptamer，形成Mu-GlcNAc-DNA单链复合物。从复合物中解离DNA单链。再次进入PCR扩增(步骤2)。如此往复循环11周，得到想要的Aptamer。共获得A1-A3030个克隆，核苷酸序列为P1(18)-N60-P2(18)通式；本实验所用MA153-A属克隆A1为筛选获得的特异 性效果最好的克隆，其序列：5‘-CGTAC GGTCG ACGCT AGC-3’-gggtt cagtggtcga cgact ttgtt tgtgt cctca catgc attac taacg gtcat ccctg-5’-GGATC CGAGCTCCAC GTG-3’(SEQ ID NO：1)，Kd 20nM。
生物素标记适配体
将适配体(SEQ ID NO：1)1mg在0.1M pH 8.8硼酸缓冲液中透析12小时以上，取出后放置在干净的玻璃瓶中，配制NH-Biotin 1mg/mL，取50微升快速加入到适配体溶液中，室温(25摄氏度)震荡2小时，在含0.5％BSA 0.05M pH 7.5PBS中透析两天，取出备用。
B单克隆抗体，MA153-B：Mab E111，IgG1，Ka 1010L/M，抗原决定簇GlcNAc，鼠抗，杂交瘤技术。
C多克隆抗体，MA153-C；Pab anti-GlcNAc，Ka 1010L/M，所对应的半抗原GlcNAc，兔抗，皮下多点注射免疫技术。
D新植物凝集素/或受体，MA153-D：生物素-植物凝集素Triticum Vulgaris，ATV，Sigma USA，Catalogue No 9604。
实施例2、利用N-乙酰氨基葡萄糖筛选特异性结合体检测肿瘤的方法(夹心法)
夹心模式测定Mu-GlcNAc抗原，其原理如图2所示。
(一)材料和方法
1试剂和肿瘤患者血清标本
1.1试剂Ma695单克隆抗体(Mab)，CanAg Diagnostics AB，瑞典，生物素(biotin)联结的Aptamer(如实施例1制备的SEQ ID NO：1所示的适配体，生物素标记)，Mu-GlcNAc抗原，自行亲和层析提取(见实施例1)；也可购自北京永瀚星港生物科技股份有限公司。
1.2癌症患者血清标本肿瘤患者血请来自中国医学科学院肿瘤医院和解放军301医院，患者诊断均经病理证实。
2方法
2.1固相抗体制备将2mg Ma695单抗溶解在200mL pH9.2的磷酸缓冲液中，分装于聚苯乙烯小试管内，每管0.2mL，4℃放置过夜，弃去磷酸缓冲液，加入0.2mL 6％的牛血清白蛋白溶液进行饱和包被，4℃放置5小时，弃牛血清白蛋白溶液，放置4℃备用。
2.2125I标记链酶亲和素(Streptavidin)的制备取Iodogen涂管一支(陈 智周，范振符.肿瘤相关抗原CA-50的免疫放射分析.中国医学科学院学报，1988,4(1):60-64.)，加入0.05mol/L pH 7.5含有100μg streptavidin的磷酸缓冲液100μL，一边搅拌一边加入10μL 125I(3.7×107Bq)，室温下反应5min，将反应液转移至Sephdex G25柱，收集第一个放射性峰，置4℃备用。
2.3反应溶液和洗涤液的配制于pH 7.5，0.05mol/L PBS缓冲液中加入0.05％CaCl2、0.05％MgCl2、6％BSA，成为反应溶液；于反应溶液中加入0.01％的吐温20即为洗涤液。
2.4标准液配制将Mu-GlcNAc原液用反应溶液稀释成0、1、3、10、30、100、300U/mL不同浓度的标准。U为N-乙酰氨基葡萄糖活性单位。
2.5Mu-GlcNAc测定步骤向包被好单抗Ma695的聚苯乙烯小试管内加入标准液、被测血清样品各0.1mL，然后加入biotin-Aptamer(生物素(biotin)联结的Aptamer)0.1mL(约0.01μg)，4℃反应过夜(不少于12小时)，弃反应液，用洗涤液洗3次，加入125I-streptavidin 0.1mL(约15万cpm)，室温反应2h，弃反应液，用洗涤液洗3次，测定放射性计数，绘出标准曲线，并计算样品中Mu-GlcNAc含量，以U/mL表示。
2.6临床测定结果判定取100例正常人血清，Mu-GlcNAc抗原测定结果
Figure PCTCN2015071940-appb-000001
(该公式中s是指标准差)，设定为正常值上限。
(二)结果
1标准曲线
Mu-GlcNAc测定标准曲线数据见表1和图3。
表1 不同浓度Mu-GlcNAc抗原的放射性计数
Figure PCTCN2015071940-appb-000002
2临床测定
正常人和不同肿瘤患者血清Mu-GlcNAc临床测定结果见表2
Figure PCTCN2015071940-appb-000003
Figure PCTCN2015071940-appb-000004
(三)讨论
从临床检测结果可以看出，Mu-GlcNAc抗原在10种以上受试肿瘤患者血清中呈现阳性，肿瘤特异性高达95％，是个良好的新肿瘤标志物。从其对肿瘤的广谱性来看，它可能与肿瘤共有的恶性本质密切相关，而不是仅对个别肿瘤。对它进一步研究意义重大。
已知单克隆抗体Ma695识别的是Mucin1(粘蛋白1)蛋白上的-PDTR-肽段，而上述所用的适配体识别的表位，经凝集素芯片(Tateno H,Uchiyama N,Kuno A,et al.A novel strategy for mammalian cell surface glycome profiling using lectin microarray.Glycobiology 2007,17(10):1138-1146.)核定为GlcNAc(N-acetyl-Glucosamine,N-乙酰氨基葡萄糖)，因此反应中的夹心抗原基本结构是本公开提供的Mu-GlcNAc。
蛋白质糖基化是蛋白质功能的关键因素，癌变过程伴有糖基化异常(范振符，陈智周，范飞舟.糖基化改变与肿瘤的基础研究.标记免疫分析与临床，2010,17(2):122-126.)，所形成的异常糖基构成了肿瘤单抗的免疫决定簇。迄今，糖类抗原(carbohydrate antigen，简称为CA)已在临床肿瘤检测中占有突出重要地位，糖生物学的研究是新肿瘤标志物开发的重要源泉，本研究预期在不远的将来还可能产生新的抗癌疫苗。
实施例3、单结合体测定方法检测肿瘤
单结合体测定方法原理如图4A和图4B，利用N-乙酰氨基葡萄糖的特异性结合体适配体(SEQ ID NO：1)。单结合体式分析是只用N-乙酰氨基葡萄糖特异性结合体做识别体；单结合体分析是用N-乙酰氨基葡萄糖特异性结合体直接识别检测对象中的的N-乙酰氨基葡萄糖；单结合体分析的本质是结合体与-GlcNAc形成的特异性结合。
具体方法如下所示：
(一)材料和方法：
1试剂和肿瘤患者血清标本
1.1试剂：适配体biotin-MA153-A，北京永瀚星港生物科技股份有限公司；MA153抗原，北京永瀚星港生物科技股份有限公司；Streptavidin，Sigma USA。
1.2癌症患者血清标本肿瘤患者血清来自中国医学科学院肿瘤医院和解放军301医院，患者诊断均经病理证实。
2方法
2.1125I标记链酶亲和素(Streptavidin)的制备：取Iodogen涂管一支，加入0.05M pH7.5含有100ug streptavidin的磷酸缓冲液100μL，一边搅拌一边加入10μL 125I(3.7×107Bq)，室温下反应5分钟，将反应液转移至Sephdex G25柱，收集第一个放射性峰，置4℃备用。
2.2反应溶液和洗涤液的配制：于pH 7.5，0.05M PBS缓冲液中，加入0.05％CaCl2，0.05％MgCl2，6％BSA，成为反应溶液；于反应溶液中加入0.01％的吐温20即为洗涤液。
2.3标准配制：将Mu-GlcNAc原液用反应溶液稀释成0、1、3、10、30、100、300U/mL，U为N-乙酰氨基葡萄糖活性单位。
标准和血清样品的稀释：测定前用0.05M pH 9.2的磷酸缓冲液将标准和血清样品做10倍稀释。
2.4GlcNAc抗原测定步骤：向聚苯乙烯小试管内依次加入稀释好的标准、被测血清样品各0.1mL，4℃过夜，形成固相抗原，弃去反应液。加入0.1mL 6％BSA，室温反应2h，以饱和包被，弃反应液。加入biotin-Aptamer(生物素(biotin)联结的Aptamer)0.1mL，约0.01μg，室温反应3h，弃反应液，用洗涤液洗3次，加入125-I-streptavidin 0.1mL，约15万cpm，室温反应2h，弃反应液，用洗涤液洗3次，测定放射性，绘出标准曲线，并计算样品中GlcNAc含量，表示为U/mL。
2.5取100例正常人血清测定其MA153(GlcNAc)标准抗原含量结果X+2SD＝1U/mL设定为正常值上限。
(二)结果
1.正常人血清CA153(GlcNAc)标准抗原测定结果
平均值：0.8U/mL
SD：0.1U/mL
正常值上限：X+2SD＝0.8U/mL+2x 0.1U/mL＝1U/mL
2.单结合体测定临床结果
正常人和不同肿瘤患者血清GlcNAc临床测定结果见表3
Figure PCTCN2015071940-appb-000005
现行商品试剂盒的模式分为两类，夹心模式和竞争模式，且在肿瘤标志物领域只有双结合体的夹心模式。本公开开辟了一种新模式，命名为单结合体模式(Single binder assay)。它节省抗体，可降低成本80％，提高灵敏度而不降低特异度。在本公开中，单结合体模式比夹心模式将肺癌的检出率提高了22％：前者灵敏度97％，特异度96％；后者灵敏度75％，特异度95％。
利用单结合体模式可制备试剂盒，该试剂盒可包括如下1-4所示的试剂，操作方法如5所示。
1、0.05M磷酸缓冲液，pH 9.2
2、96孔板(聚苯乙烯材质，透明或白色均可)
3、辣根过氧化物酶标记链酶亲和素
(按常规标记方法，《标记免疫分析和临床》2004年12:162)
4、生物素标记适配体
将适配体(SEQ ID NO：1)1mg在0.1M pH 8.8硼酸缓冲液中透析12小时以上，取出后放置在干净的玻璃瓶中，配制NHS-Biotin 1mg/mL，取50微升快速加入到适配体溶液中，室温(25摄氏度)震荡2小时，在含0.05MpH 7.5PBS中透析两天，取出备用。
5操作说明
5.1待测血清经pH 9.20.05M磷酸缓冲液稀释成1/10，取200μL直接加入聚苯乙烯96孔板中，室温反应4小时，弃去反应物用0.05M pH 7.5PBS洗3次，加入含1.5％牛血清蛋白的PBS 250μL，37℃一小时。弃反应液用PBS洗2次甩干待用。
5.2包被血清的聚苯乙烯板条每孔加入200μL稀释好的Biotin-Aptamer，室温反应2小时后弃反应液，用PBST洗板3次后每孔加入HRP-SA继续室温反应2小时，弃反应液，用PBST洗板6次，控干板条，每孔加入底物200μL，室温反应20-30分钟置于相应仪器中测量。
实验证明，本公开GlcNAc肿瘤特异性适配分析试剂盒操作简便、适合筛查，一次检测可以覆盖十种以上的肿瘤，它的检出效率，即癌症总阳性率超过目前已有的任何一个肿瘤检测试剂盒
以上所述仅是本公开的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本公开原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视本公开的保护范围。

Claims (8)

  1. 一种检测肿瘤的试剂盒，包含：与N-乙酰氨基葡萄糖特异性结合的物质。
  2. 根据权利要求1所述的试剂盒，其中所述检测肿瘤的试剂盒为以血清为检测样本的试剂盒；所述肿瘤为恶性肿瘤。
  3. 根据权利要求2所述的试剂盒，其中所述肿瘤为乳腺癌、胰腺癌、肝癌、肺癌、食管癌、口腔癌、结肠癌、胆管癌、卵巢癌、宫颈癌、子宫癌、膀胱癌、胃癌、肾癌和贲门癌的一种或几种。
  4. 根据权利要求1-3任一项所述的试剂盒，其中与N-乙酰氨基葡萄糖特异性结合的物质为以N-乙酰氨基葡萄糖为抗原决定簇的单克隆抗体、多克隆抗体、与N-乙酰氨基葡萄糖特异性结合的适配体或与N-乙酰氨基葡萄糖特异性结合的凝集素。
  5. 根据权利要求4所述的试剂盒，其中与N-乙酰氨基葡萄糖特异性结合的适配体为具有如SEQ ID NO：1所示序列的核酸。
  6. 根据权利要求5所述的试剂盒，其中所述试剂盒包括生物素标记适配体、酶标记链酶亲和素、聚苯乙烯酶标板和pH 9.20.1M的碳酸缓冲液。
  7. 根据权利要求5所述的试剂盒，其中所述试剂盒包括Ma695单抗、生物素标记适配体、酶标记链酶亲和素。
  8. 一种与N-乙酰氨基葡萄糖特异性结合的适配体，其为具有如SEQ ID NO：1所示序列的核酸。
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