WO2016037460A1 - Utilisation de n-acétylglucosamine dans la préparation d'un kit pour détecter les tumeurs - Google Patents

Utilisation de n-acétylglucosamine dans la préparation d'un kit pour détecter les tumeurs Download PDF

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WO2016037460A1
WO2016037460A1 PCT/CN2015/071941 CN2015071941W WO2016037460A1 WO 2016037460 A1 WO2016037460 A1 WO 2016037460A1 CN 2015071941 W CN2015071941 W CN 2015071941W WO 2016037460 A1 WO2016037460 A1 WO 2016037460A1
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cancer
acetylglucosamine
tumor
detecting
biological sample
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范振符
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张昊岩
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    • 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
    • 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
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • 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
    • G01N33/577—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 the use of N-acetylglucosamine in the preparation of a kit for detecting tumors.
  • 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
  • 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.
  • N-acetylglucosamine in the preparation of a kit for detecting tumors.
  • the new serum marker N-acetylglucosamine established in the present disclosure is the first tumor antigen that can be written into a molecular formula and a structural formula, and the clear structure makes the preparation problem of the combined body easy to solve, and the significance is extraordinary.
  • a first aspect of the present disclosure provides the use of N-acetylglucosamine in the preparation of a kit for detecting a tumor; wherein the kit for detecting a tumor is a kit using serum as a test sample; the tumor is a malignant tumor.
  • the tumor is 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.
  • pancreatic cancer liver cancer
  • lung cancer esophageal cancer
  • oral cancer colon cancer
  • cholangiocarcinoma ovarian cancer
  • cervical cancer uterine cancer
  • bladder cancer ovarian cancer
  • stomach cancer stomach cancer
  • kidney cancer and cardiac cancer.
  • cardiac cancer One or several.
  • 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 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 application 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).
  • a second aspect of 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.
  • the substance for detecting N-acetyl-D-glucosamine 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
  • 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%.
  • a method of detecting a tumor comprises the steps of: acquiring a biological sample; detecting N-acetylaminoglucosamine in the biological sample The concentration of glucose; and comparing the concentration of the N-acetylglucosamine to a predetermined upper limit to determine if the biological sample has a tumor.
  • the biological sample is serum or plasma.
  • the predetermined upper limit is 1 U/mL.
  • the step of detecting the concentration of N-acetylglucosamine in the biological sample further comprises: detecting an N-acetylglucosamine specific binding partner in the biological sample.
  • the N-acetylglucosamine specific binder is formed from N-acetylglucosamine with a monoclonal antibody, polyclonal antibody, aptamer or lectin.
  • the aptamer that specifically binds to N-acetylglucosamine is a nucleic acid having the sequence set forth in SEQ ID NO: 1.
  • the tumor is 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
  • a method of screening for a medicament for treating or preventing a tumor comprises the steps of: detecting a concentration of N-acetylglucosamine in a biological sample; administering a candidate agent to the biological sample, and detecting N-acetylglucosamine in the biological sample after administration Concentration; and determining the change in concentration of N-acetylglucosamine in the biological sample before and after administration.
  • the step of detecting the concentration of N-acetylglucosamine in the biological sample : detecting the N-acetylglucosamine specific binding partner in the biological sample.
  • the N-acetylglucosamine specific binder is formed from N-acetylglucosamine with a monoclonal antibody, polyclonal antibody, aptamer or 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 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 mimetic 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 are provided.
  • the kit for detecting a tumor is a kit using serum as a test sample; the tumor is a malignant tumor.
  • the tumor is 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.
  • pancreatic cancer liver cancer
  • lung cancer esophageal cancer
  • oral cancer colon cancer
  • cholangiocarcinoma ovarian cancer
  • cervical cancer uterine cancer
  • bladder cancer ovarian cancer
  • stomach cancer stomach cancer
  • kidney cancer and cardiac cancer.
  • cardiac cancer One or several.
  • 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 application 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 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 with 0.05M pH 9.2 phosphate buffer.
  • the antigen can react with a specific binding body of N-acetylglucosamine to form a serum tumor marker assay
  • 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
  • a 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 ACGCT AGC- 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 1010 L/M, antigenic determinant GlcNAc, murine resistance, hybridoma 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.
  • 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 glycoprotein linked products can be used as antigens in substances for detecting tumors, such as highly purified MA153 (Mu).
  • MA153 Mo
  • -GlcNAc -GlcNAc antigen as a starting antigen, target, ligand, to prepare, find, screen, and breed specific binding partners such as: aptamers, monoclonal antibodies, polyclonal antibodies, lectins, lectins, receptors, so that Establish a tumor detection method; use MA153 (Mu-GlcNAc) as an analyte for tumor 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 found GlcNAc (N-acetylglucosamine, N-acetylglucosamine) special glycosyl, and then the target sugar is separated and purified by polyacrylamide electrophoresis, high pressure liquid chromatography and lectin affinity chromatography. protein.
  • 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 preparing aptamers is our first choice. It can be used for tumor diagnosis as well. It can be used for tumor treatment, its molecular size is smaller, and it has more advantages than antibody 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
  • NHS-Biotin 1 mg/mL 50 ⁇ L was added quickly.
  • Into the aptamer solution shake at room temperature (25 degrees Celsius) for 2 hours, dialyze in 0.05 M pH 7.5 PBS for two days, and take out for use.
  • MA153-B Mab E111, IgG1, Ka 10 10 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.
  • reaction solution and washing solution Add 0.05% CaCl 2 , 0.05% MgCl 2 and 6% BSA to 0.05 mol/L PBS buffer at pH 7.5 to form a reaction solution; add 0.01% Tween 20 to the reaction solution. It is the washing liquid.
  • 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.
  • 2.5Mu-GlcNAc Assay Procedure Add 0.1 ml of the standard solution and the test serum sample to the polystyrene small test tube coated with the monoclonal antibody MA695, and then add 0.1 mL of biotin-Aptamer (biotin-linked Aptamer). About 0.01 ⁇ g), react at 4 ° C overnight (not less than 12 hours), discard the reaction solution, wash 3 times with washing solution, add 125 I-streptavidin 0.1mL (about 150,000 cpm), react at room temperature for 2h, discard the reaction solution, Wash 3 times with washing solution, measure the radioactivity count, plot the standard curve, and calculate the Mu-GlcNAc content in the sample, expressed in U/mL.
  • biotin-Aptamer biotin-linked Aptamer
  • 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 right In terms of the 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), therefore, 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.
  • 2.1 125 I Streptavidin preparation Take a tube of Iodogen coated tube, add 0.05 ⁇ L of phosphate buffer containing 100 ⁇ g of streptavidin in pH 7.5, and add 10 ⁇ L of 125 I (3.7 ⁇ 10 7 Bq) while stirring. The reaction was carried out for 5 minutes at room temperature, and the reaction solution was transferred to a Sephdex G25 column, and the first radioactive peak was collected and set at 4 ° C until use.
  • reaction solution and washing solution Add 0.05% CaCl 2 , 0.05% MgCl 2 and 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
  • NHS-Biotin 1 mg/mL 50 ⁇ L was added quickly.
  • Into the aptamer solution shake at room temperature (25 degrees Celsius) for 2 hours, dialyze in 0.05 M pH 7.5 PBS for two days, and take out for use.
  • the serum to be tested is diluted to 1/10 by pH 9.2 0.05M phosphate buffer. 200 ⁇ L is directly added to the polystyrene 96-well plate, reacted at room temperature for 4 hours, and the reaction 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. Discard the reaction solution Wash twice with PBS and dry 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

L'invention concerne l'utilisation de N-acétylglucosamine dans la préparation d'un kit pour détecter les tumeurs.
PCT/CN2015/071941 2014-09-12 2015-01-30 Utilisation de n-acétylglucosamine dans la préparation d'un kit pour détecter les tumeurs WO2016037460A1 (fr)

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CN104198707B (zh) * 2014-09-12 2016-09-14 范飞舟 N-乙酰氨基葡萄糖在制备检测肿瘤的试剂盒中的应用
CN104267185B (zh) * 2014-09-12 2016-05-18 范飞舟 检测肿瘤的试剂盒及其专用识别n-乙酰氨基葡萄糖的物质
WO2017139975A1 (fr) * 2016-02-19 2017-08-24 Huiru Wang Anticorps dirigés contre la n-acétylglucosamine et la n-acétylgalactosamine
CN107361360A (zh) * 2017-07-24 2017-11-21 冯纪敏 一种组合物及其应用
CN109879967A (zh) * 2017-12-15 2019-06-14 苏州和锐生物科技有限公司 一种乙酰氨基葡萄糖偶联物的制备方法及应用
CN111549035B (zh) * 2020-05-27 2021-08-03 武汉大学 能特异性识别O-GlcNAc修饰的单链DNA适配体及其筛选方法和应用

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