CN104267185B - Detect the kit of tumour and the material of special identification 2-Acetamido-2-deoxy-D-glucose thereof - Google Patents
Detect the kit of tumour and the material of special identification 2-Acetamido-2-deoxy-D-glucose thereof Download PDFInfo
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- CN104267185B CN104267185B CN201410464565.5A CN201410464565A CN104267185B CN 104267185 B CN104267185 B CN 104267185B CN 201410464565 A CN201410464565 A CN 201410464565A CN 104267185 B CN104267185 B CN 104267185B
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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
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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
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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
- G01N33/577—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
Abstract
The invention discloses a kind of kit that detects tumour. The material of its inclusion test tumour, the material of this detection tumour be can with the material of 2-Acetamido-2-deoxy-D-glucose specific binding. The material of detection tumour of the present invention is to the recall rate of a lot of tumours all more than 90%, and tumour-specific is more than 95%. This will improve the early diagnostic rate of tumour greatly. The present invention is suitable for doing tumour in normal population and generally investigates in early days.
Description
Technical field
The present invention relates to detect the kit of tumour and the material of special identification 2-Acetamido-2-deoxy-D-glucose thereof.
Background technology
From the eighties in 20th century, expert utilizes hybridoma technology to obtain to identify the large molecular saccharides albumen of tumour-specific anti-Former (carbohydrateantigen, CA), and developed monoclonal antibody recognition system. Be developed so far at current Tumor in Vitro, there are two kinds of analytical methods of competitive analysis and noncompetitive sandwich assay in early diagnosis reagent field. Detect from laboratoryAngle, tumor markers is divided into blood serum/blood plasma tumor markers and histocyte tumor-marker. Blood serum/blood plasma tumor markersRoughly be divided three classes: 1) EA. Expression embryonic period, embryonic phase, normal adult do not express, follow tumour that the anti-of expression again occursFormer, as AFP, CEA. 2) Tumor-assaciated glycolipid and glycoprotein. CA199, CA50, CA242, CA724, CA125, CA153 etc. 3) swashElement peptide, enzyme, albumen. These have expression in normal structure, but are the antigen of overexpression in tumor tissues. PSA,NSE, TPA etc. The detection of these tumor markerses is also developed to a lot of skeptophylaxis detection techniques, such as: nineteen fifty-nineBerson and Yalow have founded radiommunoassay (radioimmunoassay, RIA), and the Engvall of Sweden in 1971 sets upEnzyme-linked immunosorbent assay (enzyme-linkedimmunosorbentassay, ELISA) and recent chemiluminescenceImmunoassays (chemluminescenceimmunoassay, CLIA), electrochemiluminescent immunoassay(electrochemiluminesenceimmunoassay, ECLI), time-resolved fluorescent immunoassay (time-resolvedFluoroimmunoassay, TRFIA). At present for any one tumor markers no matter adopt that determination techniques, qualificationTumour and non-tumor patient are not 100% accurately. Such as AFP is 70% to the specificity of primary carcinoma of liver; CEA is to colon cancerSpecificity is 60%, cancer of pancreas is 80%, cancer of the stomach is 60%, lung cancer be 75% and breast cancer be 60%; CA125 to oophoroma is82%; CA153 is 75% to breast cancer in early days; CA724 is that 42%, NSE is 70% to ED-SCLC to cancer of the stomach. Above markWill thing is not wide spectrum, mostly can only diagnose one or more tumours, and taking CEA as example, it is to diagnosis of pancreatic cancer effectBest, be 80%, although also effective to colon cancer, cancer of the stomach, lung cancer, breast cancer, minimum specificity only has 60%, in other wordsSay, the 40%th, can fail to pinpoint a disease in diagnosis. The early detection of tumour and diagnosis are to pass for improving patient's cure rate, extending patient's lifeImportant, the scholar who is devoted to tumor research is exploring and is finding effective tumor-marker and never relax.
Early diagnosis of tumor remains global problem so far, used tumor markers can only detect approximately 70% swollenKnurl, and the overwhelming majority has belonged to late period. Existing tumor markers mostly precise structure is still not clear, and is difficult to copy.
The pattern of existing commercially available reagent box is divided into two classes, sandwich mode and competitive mode, and in tumor markers field onlyThere is the sandwich mode of binode zoarium.
Summary of the invention
Based on above-mentioned defect, the object of this invention is to provide the kit that detects tumour, this kit comprise can with N-The material of acetylglucosamine specific binding. The present invention utilizes the material preparation of 2-Acetamido-2-deoxy-D-glucose specific bindingSimply, detection specificity and sensitivity are high, are suitable for the detection at tumor invasion initial stage.
The kit of described detection tumour is taking serum as detecting the kit of sample; Described tumour is malignant tumour.
Described tumour be breast cancer, cancer of pancreas, liver cancer, lung cancer, the cancer of the esophagus, carcinoma of mouth, colon cancer, cholangiocarcinoma, oophoroma,One or more of cervical carcinoma, the cancer of the uterus, carcinoma of urinary bladder, cancer of the stomach, kidney and cardia cancer.
Above-mentioned can be taking 2-Acetamido-2-deoxy-D-glucose as antigen certainly with the material of 2-Acetamido-2-deoxy-D-glucose specific bindingThe monoclonal antibody of fixed bunch, polyclonal antibody, with the aptamers of 2-Acetamido-2-deoxy-D-glucose specific binding or with N-acetyl ammoniaThe agglutinin of base glucose specific binding.
Described and aptamers 2-Acetamido-2-deoxy-D-glucose specific binding is the nucleic acid shown in sequence 1 in sequence table.
The present invention also protects the aptamers with 2-Acetamido-2-deoxy-D-glucose specific binding, shown in sequence in sequence table 1Nucleic acid.
2-Acetamido-2-deoxy-D-glucose molecular formula: C8H15NO6, molecular weight: 221.21, chemical code name-GlcNAc, its structure asShown in Figure 1A, it can obtain antigen with carrier protein couplet, and preparation can detect the specific binding body of tumour, as antibody, coreAcid aptamers, agglutinin etc., these specific binding bodies can with 2-Acetamido-2-deoxy-D-glucose specific binding.
Above-mentioned carrier protein can be conventional carrier protein, as ovalbumin (OVA), bovine serum albumin(BSA) (BSA) withMultitube carrier hemocyanin (KLH), can be also glycoprotein, as one or more in mucoprotein and other albumen.
For example, in an embodiment of the present invention, it is sticky that applicant has isolated 2-Acetamido-2-deoxy-D-glucose link in serumThe tumour antigen Mu-GlcNAc (code name MA153) that this patent that albumen 1 (Mucin1, code name Mu) forms is purified, its linkThe exposed ends of position sugar chain in MUC-1, is shown in Figure 1B, the amino acid sequence of MUC-1 (Mu) structure be (PDTRPAPGSTAPPAHGVTSA-) xN, the amino acid sequence of MUC-1 (Mu-) is :-PDTRPAPGSTAPPAHGVTSA-Repeatedly repeated arrangement.
The present invention utilizes the specific bond body of 2-Acetamido-2-deoxy-D-glucose to comprise the new highly sensitive blood serum tumor mark of purificationWill thing antigen be 2-deoxy-N-acetyl-D-glucosamine-mucin1 (be called for short Mu-GlcNAc, also referred to as MA153) andThe specific bond body of 2-Acetamido-2-deoxy-D-glucose: aptamers (nucleic acid, as MA153-A) and other combination be as monoclonal antibody,Polyclonal antibody, phytolectin (PHA) and/or acceptor, associated ligands, and the method for detection infantile tumour. Direct-detectionThe method of 2-Acetamido-2-deoxy-D-glucose comprises two patterns: 1) Ma695 – Mu-GlcNAc – MA153-A sandwich, 2) unijunction zoariumFormula, by the specific bond body Direct Recognition GlcNAc of 2-Acetamido-2-deoxy-D-glucose. Can detect 97% lung cancer by the method,Tomour specific degree 96%.
The invention provides the method for above-mentioned detection tumour, is to detect 2-Acetamido-2-deoxy-D-glucose in patients serum to be measuredConcentration, the higher limit taking 1U/mL as normal dividing value, wherein, U is 2-Acetamido-2-deoxy-D-glucose active unit.
Clinical serology detects and shows, technical scheme of the present invention to the recall rate of a lot of tumours all more than 90%, swollenKnurl specificity is more than 95%. This will improve the early diagnostic rate of tumour greatly. The present invention is suitable for doing in normal population swollenKnurl is generally investigated in early days.
The present invention has opened up a kind of new model, and the fit pattern of called after unijunction (Singlebinderassay), by N-secondThe specific bond body Direct Recognition 2-Acetamido-2-deoxy-D-glucose of acylamino-glucose. It saves antibody, can reduce costs 80%, carriesHigh sensitivity and do not reduce specificity. In the present invention, the fit pattern of unijunction has improved the recall rate of lung cancer than sandwich mode22%: the former sensitivity 97%, specificity 96%; The latter's sensitivity 75%, specificity 95%.
Brief description of the drawings
Figure 1A represents the structure chart of 2-Acetamido-2-deoxy-D-glucose;
Figure 1B represents the relation of 2-Acetamido-2-deoxy-D-glucose and MUC-1; 2-Acetamido-2-deoxy-D-glucose and aptamersThe relation of MA153-A; The relation of MUC-1 and monoclonal antibody Ma695; Mu-GlcNAc model configuration;
Fig. 2 represents the principle that Mu-GlcNAc sandwich assay is analyzed;
Fig. 3 represents Mu-GlcNAc sandwich bioassay standard curve.
Fig. 4 A represents the fit measuring principle of 2-Acetamido-2-deoxy-D-glucose unijunction
Fig. 4 B is similar to Fig. 4 A, and different is not have in the protein part linking with 2-Acetamido-2-deoxy-D-glucose in test serumHave-PDTR-peptide section. Show may exist the mucin1 glycoprotein of other carry-GlcNAc in addition in MA153.
Fig. 5 is the fit bioassay standard curve of 2-Acetamido-2-deoxy-D-glucose unijunction.
Detailed description of the invention
Percentage composition in following embodiment, if no special instructions, is quality percentage composition.
Based on the present invention, provide and the object of this invention is to provide the kit that detects tumour, this kit comprises can be withThe material of 2-Acetamido-2-deoxy-D-glucose specific binding. The present invention utilizes the material system of 2-Acetamido-2-deoxy-D-glucose specific bindingStandby simple, detection specificity and sensitivity are high, are suitable for the detection at tumor invasion initial stage.
The kit of described detection tumour is taking serum as detecting the kit of sample; Described tumour is malignant tumour.
Described tumour be breast cancer, cancer of pancreas, liver cancer, lung cancer, the cancer of the esophagus, carcinoma of mouth, colon cancer, cholangiocarcinoma, oophoroma,One or more of cervical carcinoma, the cancer of the uterus, carcinoma of urinary bladder, cancer of the stomach, kidney and cardia cancer.
Above-mentioned can be taking 2-Acetamido-2-deoxy-D-glucose as antigen certainly with the material of 2-Acetamido-2-deoxy-D-glucose specific bindingThe monoclonal antibody of fixed bunch, polyclonal antibody, with the aptamers of 2-Acetamido-2-deoxy-D-glucose specific binding or with N-acetyl ammoniaThe agglutinin of base glucose specific binding.
Described and aptamers 2-Acetamido-2-deoxy-D-glucose specific binding is the nucleic acid shown in sequence 1 in sequence table.
The present invention also protects the aptamers with 2-Acetamido-2-deoxy-D-glucose specific binding, shown in sequence in sequence table 1Nucleic acid.
2-Acetamido-2-deoxy-D-glucose molecular formula: C8H15NO6, molecular weight: 221.21, chemical code name-GlcNAc, its structure asShown in Figure 1A, it can obtain antigen with carrier protein couplet, and preparation can detect the specific binding body of tumour, as antibody, coreAcid aptamers, agglutinin etc., these specific binding bodies can with 2-Acetamido-2-deoxy-D-glucose specific binding.
The characteristic of 2-Acetamido-2-deoxy-D-glucose (GlcNAc) tumour antigen:
2-Acetamido-2-deoxy-D-glucose molecular formula: C8H15NO6, molecular weight: 221.21, chemical code name-GlcNAc, its structure asShown in Figure 1A, it can be for the specific binding body screening, preparation can detect tumour, as antibody, aptamer, aggegationElement etc., these specific binding bodies can with 2-Acetamido-2-deoxy-D-glucose specific binding. 2-Acetamido-2-deoxy-D-glucose can be doneFor haptens and carrier protein couplet obtain antigen, for the preparation of detecting its monoclonal antibody or polyclonal antibody.
Above-mentioned carrier protein can be conventional carrier protein, as ovalbumin (OVA), bovine serum albumin(BSA) (BSA) withMultitube carrier hemocyanin (KLH), can be also glycoprotein, as one or more in mucoprotein and other albumen.
For example, in an embodiment of the present invention, it is sticky that applicant has isolated 2-Acetamido-2-deoxy-D-glucose link in serumThe tumour antigen Mu-GlcNAc (code name MA153) that this patent that albumen 1 (Mucin1, code name Mu) forms is purified, its linkThe exposed ends of position sugar chain in MUC-1, is shown in Figure 1B, the amino acid sequence of MUC-1 (Mu) structure be (PDTRPAPGSTAPPAHGVTSA-) xN, the amino acid sequence of MUC-1 (Mu-) is :-PDTRPAPGSTAPPAHGVTSA-The repeatedly repeated arrangement of (sequence 2). The tumour antigen that 2-Acetamido-2-deoxy-D-glucose link MUC-1 of the present invention obtains, Ke YicongIn tumour cell or Serum of Cancer Patients, separation and purification obtains, and its main feature is as follows:
The model configuration of A, Mu-GlcNAc and hanging by determinant-PDTR-to the relation of relevant combination: Mu-GlcNAcTo monoclonal antibody Ma695 affinity column, thereby obtain the tumour antigen Mu-GlcNAc (seeing Figure 1B) of purifying;
B, the about 140kd of Mu-GlcNAc molecular weight;
C, produced by tumour cell;
D, have on MUC-1-PDTR-peptide section antigenic determinant; This determinant can (be shown in figure by the identification of Ma695 monoclonal antibody1B)；
E, can be identified by 2-Acetamido-2-deoxy-D-glucose specific binding body as GlcNAc antigen;
F, be present in breast cancer, cancer of pancreas, liver cancer, lung cancer, the cancer of the esophagus, carcinoma of mouth, colon cancer, cholangiocarcinoma, oophoroma, palaceIn the serum of neck cancer, the cancer of the uterus, carcinoma of urinary bladder, cancer of the stomach, kidney, patients with cardiac cancer;
G, be not present in normal person's serum;
H, be not present in benign disease patients serum;
I, this antigen coatedly can be become to immobilization in polystyrene material surface with 0.05MpH9.2 phosphate bufferAntigen;
J, this antigen can with 2-Acetamido-2-deoxy-D-glucose specific binding precursor reactant, survey and form blood serum tumor markersFixed;
K, Mu-GlcNAc antigen can with Ma695 monoclonal antibody, and the specific binding body of 2-Acetamido-2-deoxy-D-glucose forms folderHeart pattern is measured;
N, this antigen can with the specific binding precursor reactant of 2-Acetamido-2-deoxy-D-glucose, form that unijunction is fit to be measured(Singlebinderassay)；
2-Acetamido-2-deoxy-D-glucose can also link other glycoprotein and form tumour antigen ,-GlcNAc also can and itsThe link of its glycoprotein forms Glycoprotein-N-acetyl-glucosamine (glycoprotein-N-acetyl-Glucosamine) knotStructure, can have another name called Gp-GlcNAc; It is characterized in that:
A, by the fit highly sensitive statement of facts of measuring in sandwich mode of measuring of unijunction in tumour antigen with N-What acetylglucosamine linked may also contain Mucin1 small part glycoprotein in addition;
This glycoprotein described in B, A is with-GlcNAc, and without-PDTR-peptide section;
C, this glycoprotein can react with any one in combination A, B, C, D, and do not react with Ma695 monoclonal antibody;
Show by experiment of the present invention, the contrast of the specific binding body of 2-Acetamido-2-deoxy-D-glucose (GlcNAc)-The specific binding body of PDTR-, the former tumour discrimination is higher; Therefore, 2-Acetamido-2-deoxy-D-glucose (GlcNAc) canTo be called as a new blood serum tumor markers, for screening the material (aptamers, antibody, agglutinin) that detects tumour.
The invention provides the material that detects 2-Acetamido-2-deoxy-D-glucose in the application of preparing in the kit that detects tumour.
Wherein, the material of described detection 2-Acetamido-2-deoxy-D-glucose can with 2-Acetamido-2-deoxy-D-glucose specific bindingMaterial (2-Acetamido-2-deoxy-D-glucose specific binding body).
The material of described detection 2-Acetamido-2-deoxy-D-glucose be can with the Dan Ke of 2-Acetamido-2-deoxy-D-glucose specific bindingGrand antibody, polyclonal antibody, aptamers or agglutinin.
Described can be with N-acetyl ammonia with the monoclonal antibody of 2-Acetamido-2-deoxy-D-glucose specific binding, polyclonal antibodyBase glucose is monoclonal antibody or the polyclonal antibody of antigen.
The kit of described detection tumour is taking serum as detecting the kit of sample; Described tumour is malignant tumour.
Described tumour be breast cancer, cancer of pancreas, liver cancer, lung cancer, the cancer of the esophagus, carcinoma of mouth, colon cancer, cholangiocarcinoma, oophoroma,One or more of cervical carcinoma, the cancer of the uterus, carcinoma of urinary bladder, cancer of the stomach, kidney and cardia cancer.
The material apportion of above-mentioned detection 2-Acetamido-2-deoxy-D-glucose is as follows:
A aptamers, MA153-A, nucleotides sequence is classified P1 (18)-N60-P2 (18) general formula as, and this tests MA153-A usedBelong to its sequence of clone A1: 5 ' biotin-CGTACGGTCGACGCTAGC-3 '-gggttcagtggtcgacgactttgtttgtgtcctcacatgcattactaacggtcatccctg-5’-GGATCCGAGCTCCACGTG-3’,Kd20nM, (" antigen ") of initial target: 2-Acetamido-2-deoxy-D-glucose (GlcNAc), technology used: SELEX.
B monoclonal antibody, MA153-B:MabE111, IgG1, Ka1010L/M, antigenic determinant GlcNAc, mouse-anti,Hybridoma technology.
C polyclonal antibody, MA153-C; Pabanti-GlcNAc, Ka1010L/M, corresponding haptens GlcNAc,Rabbit is anti-, subcutaneous multi-point injection immunological technique.
D new plant agglutinin/or acceptor, MA153-D: biotin-phytolectin TriticumVulgaris, ATV,SigmaUSA，CatalogueNo9604。
2-Acetamido-2-deoxy-D-glucose detection method has following feature:
2-Acetamido-2-deoxy-D-glucose is measured two kinds of patterns, the fit pattern of sandwich mode and unijunction;
A, in sandwich mode, Ma695 flutters to catch body, MA153 is center antigen, 2-Acetamido-2-deoxy-D-glucose specificity knotClosing (one of A.B.C.D in the material of above-mentioned detection 2-Acetamido-2-deoxy-D-glucose, lower same) is Report Body; Any " GlcNAc-combinationBody " articulated analysis, in order to detect tumour; Analyze as Ma695-Mu-GlcNAc – aptamers sandwich, do lesion detection.
B, the analysis of unijunction combination type have another name called for " the fit noncompetitive analysis of unijunction " or " analysis of Direct Recognition formula " is to distinguishIn existing competitive analysis; The analysis of unijunction combination type is only to do identification body with 2-Acetamido-2-deoxy-D-glucose specific binding body;Unijunction is fit analyze be with in 2-Acetamido-2-deoxy-D-glucose specific binding body Direct Recognition detected object N-acetylaminoGlucose; . the fit essence of analyzing of unijunction is combination and-specific binding that GlcNAc forms. As-GlcNAc – MA153-AConnect and make the fit mensuration of unijunction, do lesion detection; The fit survey of unijunction is made in-GlcNAc – MA153-B (MabE111) connectionFixed, do lesion detection; The fit mensuration of unijunction is made in-GlcNAc – MA153-C (Pab) connection, does lesion detection;-GlcNAc –The fit mensuration of unijunction is made in MA153-D (biotin-ATV new plant agglutinin TriticumVulgaris) connection, does tumour inspectionSurvey.
This invention Yi – GlcNAc, as initial antigen, target, part, prepares, finds, cultivates, screens specific bondBody, as: aptamers, monoclonal antibody, polyclonal antibody, phytolectin, agglutinin, acceptor, to set up tumor-marker quality testingCls analysis. Taking-GlcNAc as goal analysis thing, do tumor markers and measure;
Due in the serum of tumor patient, 2-Acetamido-2-deoxy-D-glucose often links with glycoprotein, therefore, swollen in detectionIn the material of knurl, can use 2-Acetamido-2-deoxy-D-glucose and glycoprotein chains to practice midwifery thing as antigen, as highly purified MA153(Mu-GlcNAc) antigen as initial antigen, target, part, prepare, find, screen, cultivate specific bond body as adaptationBody, monoclonal antibody, polyclonal antibody, phytolectin, agglutinin, acceptor, to set up lesion detection approach; With MA153(Mu-GlcNAc) be goal analysis thing, do tumor markers and measure;
Antigenic structure (Mu-GlcNAc) is guidance, or taking these routes as clue, obtains combination, makes tumour markWill quality testing is surveyed;
And for example, glycoprotein-N-acetyl-Glucosamine Gp-GlcNAc is used as initial antigen, target, part, prepare,Find, cultivate, screening specific bond body, as: aptamers, monoclonal antibody, polyclonal antibody, phytolectin, agglutinin, be subject toBody, detects analysis to set up tumor markers; Taking – Gp-GlcNAc as goal analysis thing, do tumor markers and measure;
Based on said method, the present invention utilizes 2-Acetamido-2-deoxy-D-glucose to carry out tumour inspection as the mark of lesion detectionThe screening application of surveying material has following several mode:
A, 1) Mu-GlcNAc or the pure as a whole target of Gp-GlcNAc prepare aptamers; 2) micromolecular GlcNAcCan make separately target and directly screen, prepare aptamers, for lesion detection;
B, 1) Mu-GlcNAc or the pure as a whole antigen of Gp-GlcNAc prepares monoclonal antibody; 2) little moleculeGlcNAc connects carrier protein as haptens (hapten) becomes Effective Antigens, and preparation monoclonal antibody, for lesion detection;
C, 1) Mu-GlcNAc or the pure as a whole antigen of Gp-GlcNAc prepares polyclonal antibody; 2) little moleculeGlcNAc connects carrier protein as haptens (hapten) becomes Effective Antigens, and how anti-preparation is, for lesion detection.
Embodiment
Embodiment 1,2-Acetamido-2-deoxy-D-glucose are used for discovery and the qualification of lesion detection
The present inventor finds to have a kind of content of 2-Acetamido-2-deoxy-D-glucose with strong in the blood of tumor patientContent in health people's blood is compared has significant difference.
The present invention directly does SELDI, MELDI mass spectral analysis in vitro with the blood of tumor patient, obtains tumor patient spyOther sugared structure collection of illustrative plates, finds the special spectral line of tumour, thereby find tumour spy from two types of mass spectrometric testing resultHeterosugar structure, glycoprotein, that we find at present is exactly GlcNAc (2-Acetamido-2-deoxy-D-glucose, 2-Acetamido-2-deoxy-D-glucose) spyDifferent glycosyl, and then the target sugar egg obtaining to gather propionamide electrophoresis, high pressure liquid chromatography (HPLC), lectin affinity chromatography separation and purificationIn vain.
(Mu-GlcNAc is suspended to monoclonal antibody Ma695 affinity chromatography by determinant-PDTR-to our method of affinity chromatography on probationOn post, thereby obtain the tumour antigen Mu-GlcNAc of purifying) from MCF-7 tumor cell line Mucin1 (MUC-1, a sugared eggIn vain). We use lectin chip technology to confirm, the Mucin1 of above-mentioned new extraction by chance carries intensive-GlcNAc, and we sentenceDisconnected this new mark that we find just, and obtained confirmation in experiment of the present invention.
By this glycoprotein called after MA153 of said extracted, in its chemical constitution, 2-Acetamido-2-deoxy-D-glucose link is stickyThe exposed ends of position sugar chain in MUC-1 of its link of albumen 1 (Mucin1, code name Mu), is shown in Figure 1B, MUC-1 (Mu) knotThe amino acid sequence of structure is (PDTRPAPGSTAPPAHGVTSA-) xN, and the amino acid sequence of MUC-1 (Mu-) is :-The repeatedly repeated arrangement of PDTRPAPGSTAPPAHGVTSA-. And find it can to monoclonal antibody Ma695 and relevantAptamer (aptamers) forms the sandwich reaction of Ma695 – Mu-GlcNAc – Aptamer. Therefore, can pass through N-acetyl ammoniaThe detection of base glucose, identifies tumour. New tumor markers-GlcNAc (2-Acetamido-2-deoxy-D-glucose) structure (seeing Figure 1A) andThe relation of itself and place glycoprotein, and to the relation (seeing Figure 1B) of relevant combination. Wherein, the MUC-1 of its connection (Mu-)Amino acid sequence is: the repeatedly repeated arrangement of-PDTRPAPGSTAPPAHGVTSA-.
It is second committed step that new mark is set up 2-Acetamido-2-deoxy-D-glucose specific binding body after finding. Clear and definiteStructure, the antigen of purifying, for preparation specific bond body, provides condition very easily. SELEX new technology is prepared aptamers(aptamer) be our first-selection, it both can be used for diagnosing tumor and also can be used for oncotherapy, and its molecule is less, in tumourTargeted therapy aspect has than the more superiority of antibody. Monoclonal antibody, polyclonal antibody, phytolectin thereafterAnd/or acceptor and other parts (PHA). Finally we have obtained 4 kinds of the suitableeest combinations for-GlcNAc antigen/determinant:Aptamers MA153-A, monoclonal antibody MA153-B (E111), polyclonal antibody MA153-C, new plant agglutinin MA153-D(biotin-ATV, biotin-phyto-AgglutininfromTriticumVulgaris): described in A-D specific as follows:
A aptamers, synthetic 100,000,000,000,000 lists with last different structure that contain on Biosearch8600DNA synthesizerThe nucleic acid library that chain DNA fragment forms. Pcr amplification, becomes DNA double chain. The two strands of dissociating, mark one, is used as isotope experiment.Separate complementary strand and enter screening. (" antigen ") of initial target: 2-Acetamido-2-deoxy-D-glucose (GlcNAc), technology used: SELEX.Flutter and catch candidate Aptamer with solid phase Mu-GlcNAc, form Mu-GlcNAc-DNA strand compound. DNA dissociates from compoundStrand. Again enter pcr amplification (step 2). Circulate and so forth 11 weeks, the Aptamer that obtains wanting. Obtain altogether A1 – A3030Individual clone, nucleotides sequence is classified P1 (18)-N60-P2 (18) general formula as; This is tested MA153-A used and belongs to clone A1 for screening acquisitionThe best clone of specificity effect, its sequence: 5 ' CGTACGGTCGACGCTAGC-3 '-gggttcagtggtcgacgactttgtttgtgtcctcacatgcattactaacggtcatccctg-5’-GGATCCGAGCTCCACGTG-3 ' (shown in sequence 1), Kd20nM.
Biotin labeling aptamers
Aptamers (shown in sequence 1) 1mg is dialysed more than 12 hours in 0.1MPH8.8 borate buffer, after taking-up, putPut in clean vial, preparation NH-Biotin1mg/mL, gets 50 microlitres and joins fast in aptamers solution, room temperature(25 degrees Celsius) concussion 2 hours, dialysing two days containing in 0.5BSA0.05MPH7.5PBS, takes out for subsequent use.
B monoclonal antibody, MA153-B:MabE111, IgG1, Ka1010L/M, antigenic determinant GlcNAc, mouse-anti,Hybridoma technology.
C polyclonal antibody, MA153-C; Pabanti-GlcNAc, Ka1010L/M, corresponding haptens GlcNAc,Rabbit is anti-, subcutaneous multi-point injection immunological technique.
D new plant agglutinin/or acceptor, MA153-D: biotin-phytolectin TriticumVulgaris, ATV,SigmaUSA，CatalogueNo9604。
Embodiment 2, utilize the health check-up of 2-Acetamido-2-deoxy-D-glucose screening specific binding to survey the method (sandwich method) of tumour
Sandwich mode is measured Mu-GlcNAc antigen, and its principle as shown in Figure 2.
(1) materials and methods
1 reagent and Serum of Cancer Patients sample
1.1 reagent Ma695 monoclonal antibodies (Mab), CanAgDiagnosticsAB, Sweden, biotin(biotin) Aptamer (aptamers as shown in sequence 1 prepared by embodiment 1, biotin labeling) connecting, Mu-GlcNAcAntigen, affinity chromatography is extracted (seeing embodiment 1) voluntarily; Also can be purchased from Yong Hanxing port, Beijing bio tech ltd.
1.2 cancer patient's serum sample Blood of Tumor Patients are please from Cancer Hospital of Chinese Academy of Medical Sciences and PLA301 Hospital, patient diagnosis is all through proved by pathology.
2 methods
2.1 insolubilized antibody preparations are dissolved in 2mgMa695 monoclonal antibody in the phosphate buffer of 200mLpH9.2, packingIn polystyrene small test tube, every pipe 0.2mL, 4 DEG C of placements are spent the night, and discard phosphate buffer, add the cow's serum of 0.2mL6%Albumin solution carries out saturated coated, places 5 hours for 4 DEG C, abandons bovine serum albumin solution, place 4 DEG C for subsequent use.
2.2125The preparation of I mark streptavidin (Streptavidin) is got Iodogen and is coated with Guan Yizhi (Chen Zhizhou, modelSymbol shakes. the immunoradiometric assay of tumor associated antigen CA-50. and Chinese Academy of Medical Sciences's journal, 1988,4 (1): 60-64.), addEnter the phosphate buffer 100 μ L that 0.05mol/LpH7.5 contains 100 μ gstreptavidin,, stir while add 10 μL125I(3.7x107Bq), under room temperature, react 5min, reactant liquor is transferred to SephdexG25 post, collect first radioactivityPeak, put 4 DEG C for subsequent use.
2.3 reaction solutions and cleaning solution be formulated in pH7.5, in 0.05mol/LPBS buffer solution, add 0.05%CaCl2、0.05％MgCl2, 6%BSA, become reaction solution; In reaction solution, add 0.01% polysorbas20 to be washingLiquid.
2.4 titers preparations by Mu-GlcNAc stoste with reaction solution be diluted to 0,1,3,10,30,100,300U/mLThe standard of variable concentrations. U is 2-Acetamido-2-deoxy-D-glucose active unit.
2.5Mu-GlcNAc determination step to add in the polystyrene small test tube of coated good monoclonal antibody Ma695 titer,The each 0.1mL of tested blood serum sample, then adds biotin-Aptamer (Aptamer that biotin (biotin) connects) 0.1mL(approximately 0.01 μ g), spend the night (being no less than 12 hours), abandons reactant liquor by 4 DEG C of reactions, washes 3 times with cleaning solution, adds 125I-Streptavidin0.1mL (approximately 150,000 cpm), room temperature reaction 2h, abandons reactant liquor, washes 3 times with cleaning solution, measures radioactivity meterNumber, draws calibration curve, and Mu-GlcNAc content in calculation sample, represents with U/mL.
2.6 judging, clinical assays result gets 100 routine normal human serums, Mu-GlcNAc antigen measuring result(in this formula, s refers to standard deviation), is set as Upper Limit of Normal Value.
(2) result
1 calibration curve
Mu-GlcNAc bioassay standard curve data is in table 1 and Fig. 3.
The radiocounting of table 1 variable concentrations Mu-GlcNAc antigen
2 clinical assays
Normal person and different Serum of Cancer Patients Mu-GlcNAc clinical assays the results are shown in Table 2.
Table 2.Mu-GlcNAc measures detection sensitivity and the specificity to different tumor patients, normal person
(3) discuss
Can find out from Clinical detection result, Mu-GlcNAc antigen presents in tested Serum of Cancer Patients more than 10 kindsThe positive, tumour-specific, up to 95%, is a good new tumor markers. Broad spectrum activity from it to tumour, it mayClosely related with the pernicious essence that tumour is total, instead of only to indivedual tumours. Great to its further Research Significance.
Known monoclonal antibody Ma695 identification be on Mucin1 (MUC-1) albumen-PDTR-peptide section, and above-mentioned instituteWith the epi-position of aptamers identification, through lectin chip (TatenoH, UchiyamaN, KunoA, etal.AnovelstrategyformammaliancellsurfaceglycomeprofilingusinglectinMicroarray.Glycobiology2007,17 (10): 1138-1146.) appraise and decide (N-acetyl-into GlcNAcGlucosamine, 2-Acetamido-2-deoxy-D-glucose), therefore, the sandwich antigen basic structure in reaction is Mu-provided by the inventionGlcNAc。
Protein glycosylation is the key factor of protein function, and Carcinogenesis is with glycosylation abnormal (Fan Zhenfu, Chen ZhiWeek, model swift boat. the basic research of glycosylation change and tumour. labelled immune analysis and clinical, 2010,17 (2): 122-126.),The abnormal glycosyl forming has formed the immunologic determinants of tumour monoclonal antibody. So far, sugar antigen (carbohydrateAntigen, referred to as CA) in detecting, clinical tumor occupies outstanding critical role, and the research of glycobiology is new tumour markThe important source of will thing exploitation, this research expection also may produce new anti-cancer vaccine in the near future.
Embodiment 3, the fit assay method of unijunction detect tumour
The fit assay method principle of unijunction, as Fig. 4 A and Fig. 4 B, utilizes the specific binding body of 2-Acetamido-2-deoxy-D-glucose suitablePart (shown in sequence 1). The analysis of unijunction combination type is only to do identification body with 2-Acetamido-2-deoxy-D-glucose specific binding body; UnijunctionFit analyze be with in 2-Acetamido-2-deoxy-D-glucose specific binding body Direct Recognition detected object N-acetylamino grapeSugar; . the fit essence of analyzing of unijunction is combination and-specific binding that GlcNAc forms.
Concrete grammar is as follows:
(1) materials and methods:
1 reagent and Serum of Cancer Patients sample
1.1 reagent: aptamers biotin-MA153-A, Beijing Nymphavn Biotechnology Co., Ltd.; MA153Antigen, Beijing Nymphavn Biotechnology Co., Ltd.; Streptavidin, SigmaUSA.
1.2 cancer patient's serum sample Serum of Cancer Patients are from Cancer Hospital of Chinese Academy of Medical Sciences and PLA 301Hospital, patient diagnosis is all through proved by pathology.
2 methods
2.1125The preparation of I mark streptavidin (Streptavidin): get Iodogen and be coated with Guan Yizhi, add 0.05MThe phosphate buffer 100uL that pH7.5 contains 100ugstreptavidin,, stir while add 10uL125I(3.7x107Bq), under room temperature, react 5 minutes, reactant liquor is transferred to SephdexG25 post, collect first radioactivity peak, put4 DEG C for subsequent use.
The preparation of 2.2 reaction solutions and cleaning solution: in pH7.5, in 0.05MPBS buffer solution, add 0.05%CaCl2,0.05%MgCl2,6%BSA, becomes reaction solution; In reaction solution, add 0.01% polysorbas20 to be cleaning solution.
2.3 standard preparation: Mu-GlcNAc stoste is diluted to 0,1,3,10,30,100,300u/mL with reaction solution, UFor 2-Acetamido-2-deoxy-D-glucose active unit.
The dilution of standard and blood serum sample: use the phosphate buffer of 0.05MpH9.2 by standard and blood serum sample before measuringDo 10 times of dilutions.
2.4-GlcNAc antigen measuring step: add successively the standard of having diluted, tested blood in polystyrene small test tubeThe each 0.1mL of final proof product, 4 DEG C are spent the night, and form solid phase antigen, discard reactant liquor. Add 0.1mL6%BSA, room temperature reaction 2h, withSaturated coated, abandon reactant liquor. Add biotin-Aptamer (Aptamer that biotin (biotin) connects) 0.1mL, approximately0.01ug, room temperature reaction 3h, abandons reactant liquor, washes 3 times with cleaning solution, adds 125-I-streptavidin0.1mL, approximately 150,000Cpm, room temperature reaction 2h, abandons reactant liquor, washes 3 times with cleaning solution, measures radioactivity, draws calibration curve, and in calculation sample-GlcNAc content, is expressed as u/mL.
2.5 clinical assays results are judged: get 100 routine normal human serum GlcNAc antigen measuring result X+2SD=1u/mL,Be set as Upper Limit of Normal Value.
(2) result
1 calibration curve
Mu-GlcNAc bioassay standard curve data is in table 3 and Fig. 5.
The radiocounting of table 3 variable concentrations Mu-GlcNAc antigen
The fit clinical effectiveness of measuring of 2 unijunctions
Normal person and different Serum of Cancer Patients GlcNAc clinical assays the results are shown in Table 4.
Table 4MA153 measures sensitivity and the specificity to different tumor patients and normal person
The pattern of existing commercially available reagent box is divided into two classes, sandwich mode and competitive mode, and in tumor markers field onlyThere is the sandwich mode of binode zoarium. The present invention has opened up a kind of new model, the fit pattern (Singlebinder of called after unijunctionAssay). It saves antibody, can reduce costs 80%, improves sensitivity and do not reduce specificity. In the present invention, unijunction zoariumPattern has improved 22% than sandwich mode by the recall rate of lung cancer: the former sensitivity 97%, specificity 96%; The latter's sensitivity75%, specificity 95%.
Utilize the fit pattern of unijunction can prepare kit, this kit can comprise the reagent shown in following 1-4, method of operatingAs shown in 5.
1,0.1M carbonic acid buffer, PH9.2
2,96 orifice plates (polystyrene material, transparent or white all can)
3, horseradish peroxidase-labeled streptavidin
(labeling method routinely, " labelled immune analysis and clinical " 12:162 in 2004)
4, biotin labeling aptamers
Aptamers (shown in sequence 1) 1mg is dialysed more than 12 hours in 0.1MPH8.8 borate buffer, after taking-up, putPut in clean vial, preparation NH-Biotin1mg/mL, gets 50 microlitres and joins fast in aptamers solution, room temperature(25 degrees Celsius) concussion 2 hours, dialysing two days containing in 0.05MPH7.5PBS, takes out for subsequent use.
5, operating instruction
5.1 test serums are diluted to 1/10 through PH9.20.1M carbonic acid buffer, get 200uL and directly add polystyrene 96In orifice plate, room temperature reaction 4 hours, discards reactant 0.05MPH7.5PBS and washes 3 times, adds containing 1.5% bovine serum albuminPBS250ul, 37 DEG C one hour. Abandoning reactant liquor PBS, to wash 2 dryings stand-by.
The Biotin-Aptamer that the every hole of polystyrene batten of 5.2 coated serum adds 200uL to dilute, room temperature is anti-Answer after 2 hours and abandon reactant liquor, wash plate 3 times with PBST after every hole add HRP-SA to continue room temperature reaction 2 hours, abandon reactant liquor, usePBST washes plate 6 times, control dry plate bar, and every hole adds substrate 200uL, and room temperature reaction 20-30 minute is placed in corresponding instrument and measures.
Experiment showed, that GlcNAc tumour-specific fitting analysis kit of the present invention is easy and simple to handle, be applicable to examination, once inspectionSurvey can cover ten kinds of above tumours, its detector efficiency, and cancer total positives rate exceedes that at present existing any one is swollenKnurl detection kit.
Claims (6)
1. detect the kit of tumour, comprise can with the material of 2-Acetamido-2-deoxy-D-glucose specific binding, can with N-acetylThe material of Glucosamine specific binding is aptamers, and described aptamers is the nucleic acid shown in sequence 1 in sequence table.
2. kit according to claim 1, is characterized in that: the kit of described detection tumour is taking serum as detectionThe kit of sample; Described tumour is malignant tumour.
3. kit according to claim 2, is characterized in that: described tumour be breast cancer, cancer of pancreas, liver cancer, lung cancer,One of the cancer of the esophagus, carcinoma of mouth, colon cancer, cholangiocarcinoma, oophoroma, cervical carcinoma, the cancer of the uterus, carcinoma of urinary bladder, cancer of the stomach, kidney and cardia cancerKind or several.
4. kit according to claim 3, is characterized in that: described kit comprises described in biotin labeling adaptiveBody, enzyme labeling streptavidin, polystyrene ELISA Plate and pH9.2,0.1M carbonic acid buffer.
5. kit according to claim 1, is characterized in that: described kit comprises Ma695 monoclonal antibody, biotin labelingAptamers, enzyme labeling streptavidin.
6. with the aptamers of 2-Acetamido-2-deoxy-D-glucose specific binding, be the nucleic acid shown in sequence in sequence table 1.
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| PCT/CN2015/071940 WO2016037459A1 (en) | 2014-09-12 | 2015-01-30 | Kit for detecting tumours and dedicated substance thereof for identifying specific carbohydrate structures |
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| CN104267185B (en) * | 2014-09-12 | 2016-05-18 | 范飞舟 | Detect the kit of tumour and the material of special identification 2-Acetamido-2-deoxy-D-glucose thereof |
| CN104198707B (en) * | 2014-09-12 | 2016-09-14 | 范飞舟 | 2-Acetamido-2-deoxy-D-glucose application in the test kit of preparation detection tumor |
| CN105891517A (en) * | 2015-11-15 | 2016-08-24 | 陈博 | Kit for specific detection of oral cancer |
| WO2017139975A1 (en) | 2016-02-19 | 2017-08-24 | Huiru Wang | Antibodies against n-acetylglucosamine and n-acetyl-galactosamine |
| CN111549035B (en) * | 2020-05-27 | 2021-08-03 | 武汉大学 | Single-stranded DNA aptamer capable of specifically recognizing O-GlcNAc modification, and screening method and application thereof |
| CN113462744B (en) * | 2021-05-24 | 2023-06-23 | 中国药科大学 | Application of N-acetylglucosamine as marker in preparation of reagent for diagnosing ischemic cerebral apoplexy |
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