CN105779403A - Polypeptide for detecting MGMT (MethylGuanine Methyl Transferase) antibody in serum, detection device and detection kit - Google Patents
Polypeptide for detecting MGMT (MethylGuanine Methyl Transferase) antibody in serum, detection device and detection kit Download PDFInfo
- Publication number
- CN105779403A CN105779403A CN201610191738.XA CN201610191738A CN105779403A CN 105779403 A CN105779403 A CN 105779403A CN 201610191738 A CN201610191738 A CN 201610191738A CN 105779403 A CN105779403 A CN 105779403A
- Authority
- CN
- China
- Prior art keywords
- polypeptide
- serum
- antibody
- mgmt
- detection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
- C12N9/1007—Methyltransferases (general) (2.1.1.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y201/00—Transferases transferring one-carbon groups (2.1)
- C12Y201/01—Methyltransferases (2.1.1)
- C12Y201/01063—Methylated-DNA-[protein]-cysteine S-methyltransferase (2.1.1.63), i.e. O6-methylguanine-DNA methyltransferase
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Hematology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Urology & Nephrology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a polypeptide for detecting an MGMT (MethylGuanine Methyl Transferase) antibody in serum, a detection device and a detection kit. The polypeptide according to the invention is composed of an amino acid sequence as shown in SEQ ID NO: 1. When applied to detecting the MGMT antibody in the serum, the polypeptide according to the invention has high specificity and sensibility, and can provide a laboratory determination evidence for the drug resistance of TMZ (Tribuzone) so as to guide clinical medication for chemotherapy. The polypeptide according to the invention can be applicable to the detection device or the detection kit for detecting the MGMT antibody in the serum.
Description
Technical field
The invention belongs to field of biological detection, relate generally to for detecting the polypeptide of MGMT antibody in serum, detect device and detection kit.
Background technology
Glioblastoma multiforme (GlioblastomamultiformeGBM) is most invasive human brain high malignancy tumor, 12～15 months patient's mean survival time (MST)s after clinical diagnosis, is therefore always up the difficult point of clinical treatment.In clinical treatment, antitumor drug, because in acquired drug-resistance, high toxicity, blood brain barrier (bloodbrainbarrierBBB) and tumor and there is again many reasons such as glioma stem cells (gliomastemcellsGSCs) of some drug resistances around tumor, ultimately results in chemotherapy unsuccessfully.Although up to the present, secondary alkylating agent-temozolomide (TemozolomideTMZ) can effectively pass through BBB, and toxic and side effects is little, the Stupp chemotherapy regimen of International standardization is considered as the chemotherapy regimen of clinical treatment glioblastoma multiforme best at present, and from then on a part of GBM survival of patients phase benefits.But regrettably, even if applying the Comprehensive Treatment of above-mentioned operation, radiation and chemotherapy, the mean survival time (MST) of glioblastoma multiforme, still only has 14.6 months, and within 5 years, survival rate only has 9.8%.Acknowledged, the basic reason causing TMZ standard chemotherapeutic failed is to there is glioma cell and the glioma cell of drug resistance, the O of these mdr cells in tumor6-methyl guanine dnmt rna (O6-methylguanineDNAmethyltransferase, MGMT) on cellular drug resistance, play crucial effect.
MGMT is that a kind of efficient DNA repairs enzyme, it is possible to repair the damage that in chemotherapy, alkylating agent drug on tumor cell DNA causes.Result of study shows, when MGMT high expressed in glioblast tumor tissue, alkylating agent is occurred as soon as drug resistance by tumor cell；Or its MGMT methylates, tumor cell shows as the sensitivity to TMZ.Clinic is using expressing of MGMT in tumor tissues and methylating as judging a new GBM index to TMZ therapeutic sensitivity of MGMT at present.Therefore, it is necessary to collect the tissue of GBM, carry out MGMT methylation analysis, and mgmt protein in tissue is expressed by application immunohistochemistry and Western-blot.But there are two kinds of situations in clinical practice, one is to obtain tumor tissues or only a small amount of tissue, it is impossible to obtain complete drug resistance information；Two is that part GBM secondary resistance can occur in TMZ treatment.Finding that in basic research GBM cell strain can successfully cultivate the cell strain of resistance to TMZ high efficient expression MGMT under TMZ cultivates, this is also simulate the secondary resistance that in clinical practice treatment, some patients occurs.In infecting as clinical antibiotic therapy antibacterial, the bacterial strain of drug resistance occurs, causes the failure of antibiotic therapy.
Therefore, in the clinical practice of glioblastoma multiforme treatment, in the urgent need to developing companion's MGMT detection kit product of a kind of GBM, the clear and definite tumor drug resistance to TMZ is carried out by monitoring the method for patients serum, both solved tumor tissues without or the blindness of Clinical practice TMZ that causes of deficiency, solve again in tumor therapeutic procedure the problem of acquired drug-resistance judging to be likely to occur.
Summary of the invention
It is an object of the invention to provide a kind of for detecting the polypeptide of MGMT antibody in serum, the antibody of this polypeptide anti-, the detection device comprising this polypeptide, the detection kit comprising this polypeptide maybe this detection device and this polypeptide purposes in MGMT antibody, this polypeptide purposes in the detection device prepared for detecting MGMT antibody in serum or detection kit in detection serum.
That is, the present invention comprises following technical proposals:
1., for detecting a polypeptide for serum MGMT antibody, its aminoacid sequence is such as shown in SEQIDNO:1, it may be assumed that TLDSPLGKLELSGCEQGLHE.
2. the nucleic acid of coding polypeptide described in claim 1.
3. comprise the expression vector of nucleic acid described in claim 2.
4. the host cell of importing expression vector described in claim 3.
5. the antibody of the polypeptide described in anti-claim 1.
6. a detection device, comprising:
Solid carrier, and
It is connected to the polypeptide described in the claim 1 on this solid carrier.
7. detection device according to claim 2, wherein, described solid carrier is IPDMS thin film.
8. a detection kit, it includes the polypeptide described in claim 1 or the detection device described in Claims 2 or 3.
9. the polypeptide described in claim 1 is used for detecting the purposes in the test kit of MGMT antibody in serum or detection device in preparation.
The polypeptide of the present invention is applied and MGMT antibody in detection serum, there is high degree of specificity and susceptiveness, TMZ can be produced drug resistance and laboratory judgment basis is provided, thus being used for instructing clinical chemotherapy medication.The polypeptide of the present invention can be applicable to detection device or the detection kit of MGMT antibody in detection serum.
Accompanying drawing explanation
The schematic diagram that the manufacturing process of IPDMS thin film is illustrated by Fig. 1.
Fig. 2 illustrates the schematic diagram of polypeptide microarrays spot sample mode.
Fig. 3 shows the photo to the result that healthy normal person or non-glioblastoma multiforme patients serum detect.
Fig. 4 shows the photo to the result that glioblastoma multiforme patients serum detects.
Detailed description of the invention
The polypeptide of the present invention
The polypeptide of the present invention is 20 peptides.20 peptides of present invention aminoacid sequence shown in SEQIDNO:1 is constituted, it may be assumed that TLDSPLGKLELSGCEQGLHE.As shown in the Examples, it is used for detecting MGMT antibody in serum, and the serum of glioblastoma multiforme patient is positive, and reaction that healthy normal person or non-glioblastoma multiforme patients serum are negative.Therefore, this 20 peptide as detection glioblastoma multiforme patients serum in MGMT antibody detection device and test kit be useful.
The polypeptide of the present invention is when commercially available, it is possible to use commercially available product, adopts the known method such as (1) chemical synthesis process or (2) enzyme reaction synthetic method to obtain further, it is also possible to suitable, and wherein chemosynthesis is more easy.In the polypeptide situation of the chemosynthesis present invention, by using peptide synthesizer synthesis or this polypeptide semi-synthetic to carry out.As chemical synthesis process, it is possible to list such as peptide solid-phase synthesis etc..So the peptide of synthesis can adopt conventional means such as ion exchange chromatography, reverse phase high performance liquid chromatograph, affinity chromatography etc. to be purified.Such peptide solid phase synthesis process with and subsequent peptide purification be all well-known in the art.
Additionally, when being produced the polypeptide of the present invention by enzyme reaction, it is possible to adopt such as No. WO2004/011653 described method of International Publication pamphlet.Namely; can so produce: the aminoacid esterified for the carboxyl terminal of the aminoacid of a side or dipeptides or amidatioon obtained or dipeptides and aminoacid are in the aminoacid (aminoacid of such as carboxy protective) of free state and react under the existence of peptide synthetase, the dipeptides of generation or tripeptides.As peptide synthetase, it is possible to list: have the bacterial disposing thing or this microbe-derived peptide synthetase that generate the culture of microorganism of ability of peptide, this culture microbial cells separated or this microorganism.
And, except above-mentioned enzyme method, chemical synthesis process, in some cases, the polypeptide of the present invention is it is also possible that naturally occur (but not being separated).In naturally occurring situation, it is also possible to be separated.
The nucleic acid of the present invention, expression vector host cell, and the antibody of the polypeptide of the anti-present invention
The invention still further relates to the coding nucleic acid (nucleic acid of the present invention) of this polypeptide, the expression vector (expression vector of the present invention) comprising this nucleic acid, the host cell (host cell of the present invention) that imported this expression vector, they may be preferably used for producing the polypeptide of the present invention.The nucleic acid of the present invention, expression vector, host cell can adopt the method for well known to a person skilled in the art to prepare.The invention still further relates to the antibody of the polypeptide of the anti-present invention, its antibody that can be used for detecting the present invention.The antibody of the present invention can adopt the method for well known to a person skilled in the art to prepare.
The detection device of the present invention
The invention still further relates to a kind of detection device (the detection device of the present invention), it includes the polypeptide of solid carrier and the present invention that is connected on this solid carrier.
In the present invention, solid carrier is not particularly limited, as long as the carrier of solid or insoluble material (such as can be by the material that filtration, precipitation, Magnetic Isolation etc. separate from reactant mixture).
The material constituting solid carrier includes but not limited to: silica gel (polydimethylsiloxane, PDMS), cellulose, polytetrafluoroethyleneTM, NC Nitroncellulose, agarose, glucosan, chitosan, polystyrene, polyacrylamide, polyester, Merlon, polyamide, polypropylene, nylon, polyvinylidene fluoride, latex, silicon dioxide, glass, glass fibre, gold, platinum, silver, copper, ferrum, rustless steel, ferrite, silicon wafer, polyethylene, polymine, polylactic acid, resin, polysaccharide, albumen (albumin etc.), carbon or their combination etc..
The shape of solid carrier includes but is not limited to: pearl, magnetic bead, thin film, micro cautery, filter membrane, plate, micro plate, CNT, sensor chip etc..Just as known in the art, the solid carrier that thin film or plate etc. are smooth can be arranged bottom pit, groove, filter membrane etc..
In the present invention, magnetic bead can have the sphere diameter of about 25nm～about 1mm scope.In a preferred embodiment, magnetic bead has the diameter of about 50nm～about 10 μ m.The size of magnetic bead can select according to specific purposes.
In the present invention, the pearl being made up of the contour crosslinked spherical agarose of Sepharose has the diameter of about 24 μm～about 165 μ m.Preferably, high crosslinked spherical sepharose 4B has the diameter of about 24 μm～about 44 μ m.The size of high crosslinked spherical sepharose 4B can select according to specific purposes.
The polystyrene latex beads such as having that the example of the solid carrier of hydrophobic surface includes can from Polysciences, Warrington, PA or Spherotech, Liberville, the goods that IL buys.
Silicon dioxide (SiO2)-process or silicon dioxide (SiO2) example of solid carrier of base includes the extraordinary magnetic silica pearl etc. that can buy from Polysciences, Warrington, PA, it may be used for catching nucleic acid (such as DNA).Or, it is also possible to using can from the DynalBiotech M-280 etc. bought.
The magnetic bead with hydrophilic surface can be used for catching the bacterial cell of proliferation period, nucleic acid and other composition.Example as this magnetic bead, it is possible to list Polysciences, Warrington, the pearl (title: Biomag (registered trade mark) carboxyl) of PA sale or BangsLaboratory, Inc., Fishers, the name of IN is called the pearl of MC02N/2928.Or, it is possible to use the M-270 etc. that DynalBiotech sells.
In a preferred embodiment of the present invention, described solid carrier is IPDMS thin film.A kind of microarray solid support material (IPDMS thin film, referring to Chinese patent CN101265329A) of the silicone rubber material of Suzhou consor thing Materials Co., Ltd exploitation.This material is based on the PDMS that biological study is commonly used, add specific initiator composition (make this material can pass through surface initiated polymerization (SIP) and realize surface-functionalized modification) wherein, obtain then through polyethylene glycol methacrylate-styrene polymer (poly (oligo (ethyleneglycol) methacrylate), pOEGMA) finishing.IPDMS thin film has outstanding anti-protein non-specific adsorption (Nonspecificproteinadsorption, NPA) ability, non-specific protein absorption in complicated protein immunization detection can be controlled to close to " absolute 0 " level (being near or below the detectable limit of instrument), it is possible not only to exempt the trouble closed and repeatedly clean, it is also possible to by using higher amplification of signal means to improve the susceptiveness of protein microarray.And the essence of its silicone rubber imparts the stronger mechanical performance of this material and good operability.IPDMS thin film has successfully been applied to the combination assay microarray ELISA kit of 11 tumor markers compositions by the poly-company in Suzhou, achieve high flux and high-sensitive detection, it was demonstrated that this material is a kind of outstanding protein microarray solid support material.Meanwhile, this material also has the adjustable characteristic of surface nature, it is possible to adjust its surface topography within the specific limits by the controlled modification response time.
The polypeptide of the present invention and the connection of solid carrier can adopt the method for attachment that well known to a person skilled in the art polypeptide and solid carrier to carry out.Such as, for the connection on protein/polypeptide and modified silica-gel surface, 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides [1-ethyl-3-(3-dimethylami-nopropyl) carbodiimide can be passed through, EDC] and N-hydroxy-succinamide (N-hydroxysuccinimide, NHS) reaction changes the carboxyl (-COOH) group on the macromolecular chain of modified silica-gel surface into activated group, this activated group can react thus realizing protein/polypeptide is fixed on solid carrier surface with the amino (-NH2) be with on protein/polypeptide.
In the sampling liquid used during for point sample, the concentration of the polypeptide of the present invention is not particularly limited, and those skilled in the art can conventionally select, it is preferred to 1 μ g～1000 μ g/mL, more preferably 10 μ g～500 μ g/mL.Additionally, the density being distributed on a solid support for the polypeptide of the present invention is not particularly limited, those skilled in the art can conventionally select, it is preferred to 1～100 points/10mm2, more preferably 5～50 points/10mm2。
The present invention detection device may be used for detection serum in MGMT antibody or preparation for detecting the test kit of MGMT antibody in serum.
The detection kit of the present invention
The invention still further relates to a kind of detection kit (detection kit of the present invention), it includes the polypeptide of the present invention or detects device.This detection kit is preferred for MGMT antibody in detection serum.
The detection device of the present invention or the polypeptide of the present invention are the important documents of the detection kit of the present invention.The detection kit of the present invention can also include:
1. the serum dilution prepared or serum dilution component solution: serum dilution, for instance have the application of sample variable color Sample dilution (production code member bwj010103) etc. of the Sample dilution (production code member 070021-S2) of Sai Chi bio tech ltd, Beijing, Bo Weijia bio tech ltd, Zhengzhou.This serum dilution is used for dilute serum, and the serum of test kit detection to dilute suitable multiple, for instance 2～200 times, it is preferable that 10～100 times.
The detection kit of the present invention can also include:
2. concentration washing liquid: after solid carrier surface hatches serum and ELIAS secondary antibody, the unconjugated antibody of solid carrier surface and ELIAS secondary antibody need to be washed by washing liquid.Concentration washing liquid is such as the polysorbas20 aqueous solution of 1%, need to dilute 2～40 times, preferably 5～20 times during use.
The detection kit of the present invention can also include:
3. ELIAS secondary antibody solution: the MGMT antibody in glioblastoma multiforme patients serum can the polypeptide of the present invention on solid carrier (such as IPDMS thin film) be combined, ELIAS secondary antibody can with antibodies, and two labels resisted can react with luminous substrate, thus sending detectable light.ELIAS secondary antibody can be the goat anti-human igg of such as horseradish peroxidase-labeled.As ELIAS secondary antibody solution, it is possible to list Goat anti human IgG (H+L), the production code member ZB-2304 of the horseradish peroxidase-labeled that Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge produces.ELIAS secondary antibody concentration in ELIAS secondary antibody solution is not particularly limited, it is possible to be such as 1ng～1000ng/mL.
The detection kit of the present invention can also include:
4. luminescent solution component solution: luminescent solution can react with the horseradish peroxidase of labelling in ELIAS secondary antibody so that reaction sends the chemical light that instrument can detect that.Luminescent solution is mixed by two kinds of solution, is A liquid hydrogenperoxide steam generator respectively, and B liquid luminol solution.Luminol (luminol) is only crossed by oxidizer treatment just can luminescence.Generally use the mixed aqueous solution of hydrogen peroxide and a kind of hydroxide bases as exciting agent.Under horseradish peroxidase enzyme catalytic, decomposing hydrogen dioxide solution is oxygen and water:
2H2O2→O2+2H2O
Generating a pairs of anion with hydroxide when luminol reacts, the dioxygen oxidation that it can be gone out by peroxide decomposition, product is an organic peroxide.This peroxide is very unstable, decomposites nitrogen immediately, generates the 3-aminophthalic acid of excited state.During excited state converts to ground state, the energy of release exists with the form of photon, and wavelength is positioned at the blue light components of visible ray.The example of luminescent solution component solution such as ThermoSeientific companyELISAFemtoMaximumSensitivitySubstrate, article No. 37074.
The detection kit of the present invention can also include:
5. one or more reaction cavity (such as Chinese patent Granted publication CN202054829U).
The detection kit of the present invention can also include:
6. other are for detecting the detection molecules (such as polypeptide, protein, nucleic acid etc.) of glioblastoma multiforme relevant disease.
The detection kit of the present invention can also include:
7. operation instructions.
Embodiment
Hereinafter, by embodiment, the present invention carried out more specific description, but be not the restriction to the technology of the present invention scope.By the record of this specification, those skilled in the art can be easy to the present invention is modified/changes, and these are included in the technical scope of the present invention.
The preparation of 1.20 peptides and confirmation
20 peptides used in embodiment have the aminoacid sequence shown in SEQIDNO:1, Shanghai gill biochemistry company limited synthesize, and the sign of this polypeptide can pass through hydrogen spectrum and mass spectrum confirms to have synthesized described polypeptide.Liquid chromatography detection purity: 96.27% (area normalization method): mass spectrography detection (ESI-MS): calculate molecular weight: 2126.37, test value 2126.8.
2. detect the preparation of device
Detection chip is with IPDMS thin film for solid support material, is prepared from by point sample immobilized polypeptide solution thereon.Modified silica-gel is to add with olefin-terminal, surface initiated polymerization initiator in traditional polydimethyl siloxane material, and it is fixed in the three dimensional structure of polydimethylsiloxane by heat cross-linking (si-h bond bonding), obtain a kind of new material and IPDMS thin film.Its manufacturing process is as shown in Figure 1.
A and B therein is two components of polydimethylsiloxane, polydimethylsiloxane (Poly (dimethylsiloxane), Sylgard184) buy from Dow corning (DowCorning) company, comprise liquid composition A (composition is metallic platinum catalyst and the diformazan siloxanes macromolecule precursor mixture with vinyl) and two kinds of compositions of crosslinking agent B (composition is the dimethyl siloxane precursor with vinyl and Si--H).C is the initiator of end strips vinyl, is purchased from Hangzhou Dong Wei company.Macromolecule on finally modifying is that oligomeric ethylene glycol methacrylate monomer (Oligo (ethyleneglycol) methacrylate, hereinafter referred to as OEGMA, molecular weight Mw=526) is bought in Aldrich.Polydimethylsiloxane precursor A and crosslinking agent B and the initiator C with vinyl end are sufficiently mixed with A:B:C=10:1:0.5 ratio.Make transparent elastic silicone rubber by curing reaction, then pass through sip technique and carry out finishing and can obtain IPDMS thin film.Experiments show that, the surface of IPDMS thin film have enough highdensity, by the initiator of covalently immobolization, it can pass through surface initiated polymerization (SIP), and to realize surface macromolecule modified.Use poly (OEGMA) (polyethylene glycol methacrylate-styrene polymer) to react and obtain the surface that Polyethylene Glycol (PolyethyleneGlycol, PEG) is modified, it is achieved the ability of stronger anti-albumen non-specific adsorption.
The IPDMS foamed film made need to be saved in 4 DEG C of refrigerators.
Adopting brilliant PersonalArrayerTM16 people's point sample instrument of core to prepare polypeptide microarrays on modified silica-gel, process is:
1) pretreatment
By IPDMS film sheet (15 × 15mm2) be immersed in activating solution, take out with deionized water drip washing 3 times after 30min, dry up with nitrogen, be immediately used to point sample.
2) point sample
Sampling liquid diluted good and transfer in the 384 corresponding micropores of orifice plate, 384 orifice plates with sample being placed on point sample instrument base station, the modified silica-gel thin slice of pretreatment is placed on the base station of point sample instrument simultaneously, carrying out point sample at once.Point sample environmental condition is room temperature (25 DEG C), and humidity set is 50%.On the polypeptide microarrays made, the point sample amount of each point is about 0.6nL, and sampling point radius is 200 μm.
3) chemistry is fixing
The polypeptide microarrays just made to be placed in climatic chamber (26 DEG C, 60% humidity) and fix at least 6h.Chemistry fixation procedure is as follows.
First pass through point sample instrument and will include the buffer point catching peptide molecule on modified silica-gel thin film, then buffer starts evaporation, catch peptide molecule and IPDMS film surface intimate contact and interact, by chemical bond, the high molecular end-COOH of poly (OEGMA) on modified silica-gel surface and the NH of peptide molecule2Formed and stablize covalent bond, and then chemically active peptide molecule will be had to be fixed on IPDMS film surface.
5) assembling
The polypeptide microarrays of fixing 6h must assemble in two days.First pass through gum IPDMS film sheet to be attached on special reaction column, cover reaction cavity.One reactor is made up of two reaction columns and a reaction cavity.
6) preserve
The polypeptide microarrays assembled, it is necessary to evacuation seals, and is saved in the refrigerator of 4 DEG C, standby.
3. detect with detection device
Testing sequence
1, before starting detection, concentrated cleaning solutions is added purified water in the ratio of 1:10 or distilled water is diluted, directly use after having diluted.Use liquid-transfering gun that 2mL cleanout fluid is added to chip surface, soak chip 3 minutes, it is ensured that chip surface is fully wet out.
2, by test serum sample Sample dilution according to 1:40 dilution mixing.
3, discarding the cleanout fluid soaking chip, when chip surface moistens completely, each serum sample is drawn the serum after 200 μ L dilute and is joined in chip reactor.
4, chip reactor is put into the fixing seat of chip, is put on shaking table, opens shaking table, frequency 150 revs/min, incubated at room 30 minutes.
5, discard the serum sample in chip reactor, clean reaction cavity and chip surface 3 times by 15mL washing liquid.
6, after having cleaned, each chip reactor is separately added into 200 μ L enzyme labelled antibody solution, chip reactor is put into the fixing seat of chip, is put on shaking table, opens shaking table, frequency 150 revs/min, incubated at room 30 minutes.
7, discard the enzyme labelled antibody solution in chip reactor, clean reaction cavity and chip surface 3 times by 15mL washing liquid.
8, after having cleaned, taking off reaction cavity, each chip surface is separately added into 15 μ L luminous substrate liquid, makes luminescent solution can be laid on chip surface uniformly.
9, the chip adding luminescent solution is placed in chemiluminescence imaging in gel imaging instrument sentence read result.
Serum and other disease patient's serum samples of glioblastoma multiforme patient patients serum are provided by chain hospital.Serum is transported by related personnel with parcels such as ice cube/dry ice or is handed to laboratory soon.
Negative control has PBS (namely to hatch without test serum in the 3rd step, and hatch by PBS solution, all the other steps are identical) comparison, the comparison of serum dilution, and the comparison of patients with negative (referring to Healthy People and non-glioblastoma multiforme patient) serum.
The spot sample mode of polypeptide microarrays is as shown in Figure 2: wherein, and the sample behaviour IgM of 16 points that black is circular, as anchor point；The sample of foursquare 4 points is PBS sampling liquid, as the blank of experiment；White circular form point sample be other glioblastoma multiforme patient's mgmt protein antigen protein polypeptide, as the Testing index (these polypeptide have response that the autoantibody having MGMT in detection serum is described) of experiment；The sample of two star point is the polypeptide SEQIDNO:1 of the present invention, and it is mgmt protein antigen polypeptide in glioblastoma multiforme patients serum, MGMT autoantibody in glioblastoma multiforme patient patients serum can be produced response.
This polypeptide microarrays is used according to above-mentioned detecting step, glioblastoma multiforme patients serum and negative control to be detected, response modes is as shown in figs. 34: wherein, Fig. 3 shows the testing result of negative control, and only the sample of the point shown in black circle has response.Fig. 4 shows the testing result of glioblastoma multiforme patients serum, and black is circular, white is circular and star point sample has response.It should be noted that instrument records signal value from low to high, corresponding signaling point color is by dark red white gradual change.
Claims (9)
1., for detecting a polypeptide for MGMT antibody in serum, its aminoacid sequence is such as shown in SEQIDNO:1, it may be assumed that TLDSPLGKLELSGCEQGLHE.
2. the nucleic acid of coding polypeptide described in claim 1.
3. comprise the expression vector of nucleic acid described in claim 2.
4. the host cell of importing expression vector described in claim 3.
5. the antibody of the polypeptide described in anti-claim 1.
6. a detection device, comprising:
Solid carrier, and
It is connected to the polypeptide described in the claim 1 on this solid carrier.
7. detection device according to claim 2, wherein, described solid carrier is IPDMS thin film.
8. a detection kit, it includes the polypeptide described in claim 1 or the detection device described in claim 6 or 7.
9. the polypeptide described in claim 1 is used for detecting the purposes in the test kit of MGMT antibody in serum or detection device in preparation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610191738.XA CN105779403A (en) | 2016-03-30 | 2016-03-30 | Polypeptide for detecting MGMT (MethylGuanine Methyl Transferase) antibody in serum, detection device and detection kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610191738.XA CN105779403A (en) | 2016-03-30 | 2016-03-30 | Polypeptide for detecting MGMT (MethylGuanine Methyl Transferase) antibody in serum, detection device and detection kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105779403A true CN105779403A (en) | 2016-07-20 |
Family
ID=56391143
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610191738.XA Withdrawn CN105779403A (en) | 2016-03-30 | 2016-03-30 | Polypeptide for detecting MGMT (MethylGuanine Methyl Transferase) antibody in serum, detection device and detection kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105779403A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1502630A (en) * | 2002-11-21 | 2004-06-09 | 中国人民解放军军事医学科学院野战输 | Monocloned antibody of O6-methylguanine-DNA methyltransferase preparation process and test kit |
| CN101265329A (en) * | 2007-03-16 | 2008-09-17 | 马雄明 | Polydimethylsiloxane with initiator on surface and its preparation method and use |
-
2016
- 2016-03-30 CN CN201610191738.XA patent/CN105779403A/en not_active Withdrawn
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1502630A (en) * | 2002-11-21 | 2004-06-09 | 中国人民解放军军事医学科学院野战输 | Monocloned antibody of O6-methylguanine-DNA methyltransferase preparation process and test kit |
| CN101265329A (en) * | 2007-03-16 | 2008-09-17 | 马雄明 | Polydimethylsiloxane with initiator on surface and its preparation method and use |
Non-Patent Citations (3)
| Title |
|---|
| KALNINE,N.等: "AAP36645.1", 《GENBANK》 * |
| 屈伸等: "《分子生物学实验技术》", 31 January 2008, 化学工业出版社 * |
| 邢鹏辉等: "HIF-1α、MGMT和CD133在人胶质母细胞瘤中的表达及相关性研究", 《中华临床医师杂志》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103965321B (en) | Polypeptide, the detection device comprising this polypeptide and detection kit | |
| CN103665138B (en) | Polypeptide, the detection means comprising this polypeptide and detection kit | |
| CN103965314B (en) | Polypeptide, the detection device comprising this polypeptide and detection kit | |
| CN103788195B (en) | Polypeptide, the detection means comprising this polypeptide and detection kit | |
| CN103788199B (en) | Polypeptide, the detection means comprising this polypeptide and detection kit | |
| CN104098685B (en) | Polypeptide, the detection means comprising this polypeptide and detection kit | |
| CN104098674B (en) | Polypeptide, the detection means comprising this polypeptide and detection kit | |
| CN103880923A (en) | Polypeptide, detector containing the polypeptide, and detection kit containing the polypeptide | |
| CN105779403A (en) | Polypeptide for detecting MGMT (MethylGuanine Methyl Transferase) antibody in serum, detection device and detection kit | |
| CN105779402A (en) | Polypeptide for detecting MGMT (MethylGuanine Methyl Transferase) antibody in serum, detection device and detection kit | |
| CN105802928A (en) | Polypeptide for detecting MGMT antibody in serum, detection device and detection kit | |
| CN103965322B (en) | Polypeptide, the detection device comprising this polypeptide and detection kit | |
| CN103788183B (en) | Polypeptide, the detection means comprising this polypeptide and detection kit | |
| CN103788203B (en) | Polypeptide, the detection means comprising this polypeptide and detection kit | |
| CN103788201B (en) | Polypeptide, the detection means comprising this polypeptide and detection kit | |
| CN103788196B (en) | Polypeptide, the detection means comprising this polypeptide and detection kit | |
| CN103788202B (en) | Polypeptide, the detection means comprising this polypeptide and detection kit | |
| CN103788198B (en) | Polypeptide, the detection means comprising this polypeptide and detection kit | |
| CN103788194B (en) | Polypeptide, the detection means comprising this polypeptide and detection kit | |
| CN103788200B (en) | Polypeptide, the detection means comprising this polypeptide and detection kit | |
| CN104098680B (en) | Polypeptide, the detection means comprising this polypeptide and detection kit | |
| CN104098684B (en) | Polypeptide, the detection means comprising this polypeptide and detection kit | |
| CN104098686B (en) | Polypeptide, the detection means comprising this polypeptide and detection kit | |
| CN103788204B (en) | Polypeptide, the detection device comprising this polypeptide and detection kit | |
| CN103788205B (en) | Polypeptide, the detection device comprising this polypeptide and detection kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WW01 | Invention patent application withdrawn after publication | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20160720 |