CN107475213A - For detecting the polypeptide of MGMT antibody in serum, device and detection kit are detected - Google Patents

For detecting the polypeptide of MGMT antibody in serum, device and detection kit are detected Download PDF

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CN107475213A
CN107475213A CN201610403519.3A CN201610403519A CN107475213A CN 107475213 A CN107475213 A CN 107475213A CN 201610403519 A CN201610403519 A CN 201610403519A CN 107475213 A CN107475213 A CN 107475213A
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polypeptide
serum
antibody
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mgmt
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马宏伟
忻寅强
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Suzhou Institute of Nano Tech and Nano Bionics of CAS
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Suzhou Institute of Nano Tech and Nano Bionics of CAS
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    • C12N9/1007Methyltransferases (general) (2.1.1.)
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    • C07ORGANIC CHEMISTRY
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    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
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    • C12Y201/00Transferases transferring one-carbon groups (2.1)
    • C12Y201/01Methyltransferases (2.1.1)
    • C12Y201/01063Methylated-DNA-[protein]-cysteine S-methyltransferase (2.1.1.63), i.e. O6-methylguanine-DNA methyltransferase
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    • G01MEASURING; TESTING
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    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854Immunoglobulins

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Abstract

The present invention relates to the polypeptide for detecting MGMT antibody in serum, detects device and detection kit.The polypeptide of the present invention is by SEQ ID NO:Amino acid sequence shown in 1 is formed.The polypeptide of the present invention is applied and detects MGMT antibody in serum, there is high degree of specificity and sensitivity, drug resistance can be produced to TMZ and laboratory judgment basis is provided, so as to for instructing clinical chemotherapy medication.The polypeptide of the present invention can be applied to detect the detection device or detection kit of MGMT antibody in serum.

Description

For detecting the polypeptide of MGMT antibody in serum, device and detection kit are detected
Technical field
The invention belongs to field of biological detection, relate generally to for detecting the more of MGMT antibody in serum Peptide, detect device and detection kit.
Background technology
Glioblastoma (Glioblastoma multiforme GBM) is that most aggressive human brain is high Spend malignant tumour, 12~15 months patient's mean survival time (MST)s after clinical diagnosis, therefore always clinical treatment Difficult point.In clinical treatment, antineoplastic is because acquired resistance, high toxicity, blood-brain barrier (blood Brain barrier BBB) and intra-tumor and tumour around exist again certain amount resistance glioma it is dry thin Many reasons such as born of the same parents (glioma stem cells GSCs), ultimately result in chemotherapy failure.Although at present Untill, two generation alkylating agents-Temozolomide (Temozolomide TMZ) can effectively pass through BBB, and poison Small side effects, the Stupp chemotherapy regimens of International standardization are considered as clinical treatment colloid best at present The chemotherapy regimen of blastoma, a part of GBM patient survivals benefit from this.But regrettably, i.e., Make to apply above-mentioned operation, the complex treatment of radiation and chemotherapy, the mean survival time (MST) of glioblastoma is still So only 14.6 months, survival rate only had 9.8% within 5 years.It is acknowledged, cause TMZ standard chemotherapeutics to fail Basic reason be the glioma cell and glioma cell for existing in tumour resistance, these mdr cells O6- methyl guanine dnmt rna (O6- methylguanine DNAmethyltransferase, MGMT the effect of key) is played on cellular drug resistance.
MGMT is a kind of efficient DNA repair enzymes, and it is thin to tumour can to repair alkylating agent medicine in chemotherapy Damage caused by born of the same parents DNA.Result of study shows, as MGMT in glioblast tumor tissue high table Reach, tumour cell occurs as soon as resistance to alkylating agent;Or its MGMT methylates, tumour cell performance For the sensitiveness to TMZ.The expression and MGMT methyl MGMT in tumor tissues clinical at present It is turned to judge indexs of the new hair GBM to TMZ therapeutic sensitivities.Therefore, it is necessary to collect GBM's Tissue, MGMT methylation analysis, and application immunohistochemistry and Western-blot are carried out to tissue Middle mgmt protein expression.But two kinds of situations in clinical practice be present, when can not obtain tumor tissues or Only a small amount of tissue, can not obtain complete drug resistance information;Second, in part GBM meeting TMZ treatments There is secondary resistance.Find that GBM cell lines can successfully be trained under TMZ cultures in basic research Resistance to TMZ cell line, and high efficient expression MGMT are brought out, this is also to simulate clinical practice treatment middle part The secondary resistance for dividing patient to occur.As in clinical antibiotic therapy bacterium infection, there is the thin of resistance Bacterial strain, cause the failure of antibiotic therapy.
Therefore, in the clinical practice of glioblastoma treatment, there is an urgent need to develop a kind of GBM companion MGMT detection kit products, pass through the resistance for monitoring the method for patients serum come clear and definite tumour to TMZ Property, both solved tumor tissues without or deficiency caused by Clinical practice TMZ blindness, solve again The problem of acquired resistance being likely to occur is judged in tumor therapeutic procedure.
The content of the invention
It is an object of the invention to provide a kind of for detecting the polypeptide of MGMT antibody in serum, resisting and be somebody's turn to do The antibody of polypeptide, the detection device comprising the polypeptide, the detection reagent comprising the polypeptide or the detection device Purposes in MGMT antibody in detecting serum of box and the polypeptide, the polypeptide are used to examine preparing Survey the purposes detected in device or detection kit of MGMT antibody in serum.
That is, the present invention includes following technical proposals:
1. a kind of polypeptide for being used to detect serum MGMT antibody, its amino acid sequence such as SEQ ID NO: Shown in 1, i.e.,:VPALHHPVFQQESFTRQVLW..
2. encode the nucleic acid of the polypeptide described in claim 1.
3. include the expression vector of the nucleic acid described in claim 2.
4. import the host cell of the expression vector described in claim 3.
5. the antibody of the polypeptide described in anti-claim 1.
6. one kind detection device, it includes:
Solid carrier, and
It is connected to the polypeptide described in the claim 1 on the solid carrier.
7. detection device according to claim 2, wherein, the solid carrier is IPDMS thin Film.
8. a kind of detection kit, it includes polypeptide or the claim 2 described in claim 1 Or the detection device described in 3.
9. the polypeptide described in claim 1 is preparing the kit for detecting MGMT antibody in serum Or the purposes in detection device.
The polypeptide of the present invention is applied with detecting MGMT antibody in serum, there is high degree of specificity and spirit Quick property, drug resistance can be produced to TMZ and laboratory judgment basis is provided, so as to for instructing clinical chemotherapy Medication.The polypeptide of the present invention can be applied to detect the detection device of MGMT antibody or detection examination in serum Agent box.
Brief description of the drawings
The schematic diagram that Fig. 1 illustrates to the manufacturing process of IPDMS films.
Fig. 2 illustrates the schematic diagram of polypeptide microarrays spot sample mode.
Fig. 3 is to show the result detected to healthy normal person or non-glioblastoma patients serum Photo.
Fig. 4 is the photo for showing the result detected to glioblastoma patients serum.
Embodiment
The polypeptide of the present invention
The polypeptide of the present invention is 20 peptides.20 peptides of the present invention are by SEQ ID NO:Amino acid shown in 1 Sequence composition, i.e.,:VPALHHPVFQQESFTRQVLW.As shown in the Examples, it is used to examine MGMT antibody in serum is surveyed, the serum of glioblastoma patient is positive, and to health Normal person or non-glioblastoma patients serum are negative.Therefore, 20 peptide is as detection glue The detection device of MGMT antibody and kit are useful in matter blastoma patients serum.
The polypeptide of the present invention can use commercially available product, further, it is also possible to suitably in the case of commercially available Obtained using the known method such as (1) chemical synthesis process or (2) enzyme reaction synthetic method, wherein chemistry Synthesis is more easy.Chemical synthesis the present invention polypeptide in the case of, by using peptide synthesizer synthesis or Person semi-synthetic polypeptide is carried out.As chemical synthesis process, such as peptide solid-phase synthesis can be included Deng.The peptide so synthesized can use conventional meanses for example ion-exchange chromatography, reverse phase high performance liquid chromatogram, Affinity chromatography etc. is purified.Such peptide solid phase synthesis process with and subsequent peptide purification be all this technology Known to field.
In addition, in the case where producing the polypeptide of the present invention by enzyme reaction, can use for example international public Open the method described in pamphlet WO2004/011653.I.e., it is possible to so produce:By a side's The carboxyl terminal of amino acid or dipeptides be esterified or amidatioon obtained from amino acid or dipeptides and amino acid Amino acid (such as amino acid of carboxy protective) in free state is carried out instead in the presence of peptide synthetase Should, the dipeptides or tripeptides of generation.As peptide synthetase, can include:Ability with generation peptide The culture of microorganism, the microbial cells separated by the culture or the microorganism bacterial disposing thing, Or the microbe-derived peptide synthetase.
Moreover, in addition to above-mentioned enzyme method, chemical synthesis process, it is in some cases, of the invention Polypeptide is also possible to be naturally occurring (but not being separated)., can be with the case of naturally occurring It is separated.
Nucleic acid, the expression vector host cell of the present invention, and the antibody of anti-polypeptide of the invention
The invention further relates to encode the nucleic acid (nucleic acid of the invention) of the polypeptide, the expression load comprising the nucleic acid Body (expression vector of the invention), the host cell (host cell of the invention) for having imported the expression vector, They may be preferably used for the polypeptide of the production present invention.Nucleic acid, expression vector, the host cell of the present invention can To be prepared using well known to a person skilled in the art method.The invention further relates to anti-polypeptide of the invention Antibody, it can be used for the antibody of the detection present invention.The antibody of the present invention can use those skilled in the art It is prepared by known method.
The detection device of the present invention
The invention further relates to one kind detection device (detection device of the invention), it include solid carrier, with And it is connected to the polypeptide of the invention on the solid carrier.
In the present invention, solid carrier is not particularly limited, as long as solid or insoluble material The load of (be, for example, can by filtering, precipitating, the material that Magnetic Isolation etc. separates from reactant mixture) Body.
The material for forming solid carrier includes but is not limited to:Silica gel (dimethyl silicone polymer, PDMS), Cellulose, teflonTM, it is NC Nitroncellulose, agarose, glucan, chitosan, polystyrene, poly- Acrylamide, polyester, makrolon, polyamide, polypropylene, nylon, polyvinylidene fluoride, latex, Silica, glass, glass fibre, gold, platinum, silver, copper, iron, stainless steel, ferrite, silicon wafer Piece, polyethylene, polyethyleneimine, PLA, resin, polysaccharide, albumen (albumin etc.), carbon or Combinations thereof etc..
The shape of solid carrier includes but is not limited to:Pearl, magnetic bead, film, microcapillary, filter membrane, Plate, micro plate, CNT, sensor chip etc..Just as known in the art, film Or pit, groove, filter membrane bottom etc. can be set on the flat solid carrier such as plate.
In the present invention, magnetic bead can have the sphere diameter of about 25nm~about 1mm scopes.Preferred Embodiment in, magnetic bead has the diameter of the μ m of about 50nm~about 10.The size of magnetic bead can root Selected according to specific purposes.
In the present invention, the pearl made of the high crosslinked spherical agarose such as Sepharose has about The diameter of 24 μm~about 165 μ ms.Preferably, high crosslinked spherical sepharose 4B have about 24 μm~ The diameter of about 44 μ ms.The size of high crosslinked spherical sepharose 4B can be entered according to specific purposes Row selection.
The example of solid carrier with hydrophobic surface include can from Polysciences, Warrington, The polystyrene latex beads such as PA or the product of Spherotech, Liberville, IL purchase.
Silica (SiO2)-processing or silica (SiO2) include can be from for the example of solid carrier of base Extraordinary magnetic silica pearl of Polysciences, Warrington, PA purchase etc., it can be used for catching Catch nucleic acid (such as DNA).Or the M-280 etc. that can be bought from Dynal Biotech can also be used.
Magnetic bead with hydrophilic surface can be used for catching the bacterial cell of proliferation period, nucleic acid and it is other into Point.As the example of the magnetic bead, Polysciences can be included, Warrington, PA sale Pearl (title:Biomag (registration mark) carboxyl) or Bangs Laboratory, Inc., Fishers, IN entitled MC02N/2928 pearl.Or it can use what Dynal Biotech were sold M-270 etc..
In a preferred embodiment of the present invention, the solid carrier is IPDMS films.Suzhou A kind of microarray solid support material of silicone rubber material of Yvonne consor things Materials Co., Ltd exploitation (IPDMS films, referring to Chinese patent CN101265329A).This material is normal with biological study Based on PDMS, adding specific initiator composition wherein (makes the material to draw by surface Hair polymerisation (SIP) realizes surface-functionalized modification), then by polyethylene glycol methacrylate-styrene polymer What (poly (oligo (ethylene glycol) methacrylate), pOEGMA) surface modification obtained.IPDMS Film has outstanding anti-protein non-specific adsorption (Nonspecific protein adsorption, NPA) Ability, the non-specific protein absorption control during can complicated protein immunization be detected are arrived close to " definitely 0 " horizontal (detectable limit for being near or below instrument), can not only exempt what is closed and be cleaned multiple times Trouble, can also improve the sensitivity of protein microarray by using stronger amplification of signal means. And the essence of its silicon rubber imparts the stronger mechanical performance of the material and good operability.Suzhou The multi objective that IPDMS films have successfully been applied to 11 tumor markers compositions by the poly- companies of Yvonne joins Microarray ELISA kit is examined, realizes high flux and highly sensitive detection, it was demonstrated that this material It is a kind of outstanding protein microarray solid support material.Meanwhile this material also has surface nature Adjustable characteristic, its surface topography can be adjusted within the specific limits by the controlled modification reaction time.
The connection of the polypeptide and solid carrier of the present invention can be using well known to a person skilled in the art polypeptide Connection method with solid carrier is carried out.For example, for protein/polypeptide and the company on modified silica-gel surface For connecing, 1- ethyls -3- (3- dimethyl aminopropyls)-carbodiimides can be passed through [1-ethyl-3- (3-dimethyl ami-nopropyl) carbodiimide, EDC] and n-hydroxysuccinimide The reaction of (N-hydroxysuccinimide, NHS) is by the carboxyl on the macromolecular chain of modified silica-gel surface (- COOH) group is changed to activated group, and the activated group can be with the amino of institute's band on protein/polypeptide Protein/polypeptide is fixed on solid carrier surface by (- NH2) reaction so as to realize.
It is not particularly limited for the concentration of polypeptide of the invention in the sampling liquid that is used during point sample, this area Technical staff can be according to conventional selection, the μ g/mL of preferably 1 μ g~1000 μ g/mL, more preferably 10 μ g~500. In addition, the density being distributed on a solid support for the polypeptide of the present invention is not particularly limited, this area skill Art personnel can be according to conventional selection, preferably 1~100 point/10mm2, more preferably 5~50 points/10mm2
The detection device of the present invention can be used for detecting MGMT antibody in serum or prepare for examining Survey the kit of MGMT antibody in serum.
The detection kit of the present invention
The invention further relates to a kind of detection kit (detection kit of the invention), and it includes the present invention's Polypeptide or detection device.The detection kit is preferred for detecting MGMT antibody in 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.This hair Bright detection kit can also include:
1. the serum dilution or serum dilution component solution that prepare:Serum dilution, such as have Beijing Sample dilution (production code member 070021-S2), the Zhengzhou Bo Weijia of Sai Chi bio tech ltd Sample-adding discoloration Sample dilution (production code member bwj010103) of bio tech ltd etc..The blood Clear dilution is used for dilute serum, and the serum of kit detection will dilute suitable multiple, such as 2~200 times, It is preferred that 10~100 times.
The detection kit of the present invention can also include:
2. concentrate washing lotion:After solid carrier surface is incubated serum and ELIAS secondary antibody, it need to be washed with washing lotion solid Body carrier surface uncombined antibody and ELIAS secondary antibody.Concentration washing lotion is, for example, the 1% polysorbas20 aqueous solution, 2~40 times, preferably 5~20 times need to be diluted during use.
The detection kit of the present invention can also include:
3. ELIAS secondary antibody solution:MGMT antibody in glioblastoma patients serum can carry with solid Polypeptide of the invention on body (such as IPDMS films) combines, ELIAS secondary antibody can with antibody binding, And the label on secondary antibody can react with luminous substrate, so as to send detectable light.ELIAS secondary antibody can be with It is the goat anti-human igg of such as horseradish peroxidase-labeled.As ELIAS secondary antibody solution, can include The Goat anti human of the horseradish peroxidase-labeled of Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge production IgG (H+L), production code member ZB-2304.To concentration of the ELIAS secondary antibody in ELIAS secondary antibody solution without spy Different limitation, can 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 marked on ELIAS secondary antibody, So that reaction sends the detectable chemical light of instrument.Luminescent solution is mixed by two kinds of solution, is respectively A liquid-hydrogenperoxide steam generator, and B liquid-luminous ammonia solution.Luminol (luminol) is only with oxidation Agent is treated just to light.Usually using hydrogen peroxide and a kind of mixed aqueous solution of hydroxide bases as sharp Send out agent.Under horseradish peroxidase enzyme catalytic, decomposing hydrogen dioxide solution is oxygen and water:
2H2O2→O2+2H2O
Luminol generates a pairs of anion when being reacted with hydroxide, and it can be decomposited by hydrogen peroxide Dioxygen oxidation, product is an organic peroxide.The peroxide is very unstable, decomposites immediately Nitrogen, generate the 3- aminophthalic acids of excitation state.During excitation state converts to ground state, the energy of release Exist in the form of photon, wavelength is located at the blue light components of visible ray.The example example of luminescent solution component solution Such as Thermo Seientific companiesELISA Femto Maximum Sensitivity Substrate, 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 be used for detect glioblastoma relevant disease detection molecules (such as polypeptide, protein, Nucleic acid etc.).
The detection kit of the present invention can also include:
7. operation instructions.
Embodiment
Hereinafter, more specific description is carried out to the present invention by embodiment, but is not to the technology of the present invention The restriction of scope.By the record of this specification, those skilled in the art readily can enter to the present invention Row modification/change, these are included in the technical scope of the present invention.
The preparation and confirmation of 1.20 peptides
20 peptides used in embodiment have SEQ ID NO:Amino acid sequence shown in 1, by Shang Haiji Your biochemical Co., Ltd synthesis, the sign of the polypeptide can be composed by hydrogen and mass spectrum confirm to have synthesized it is described more Peptide.Liquid chromatography detects purity:97.32% (area normalization method):Mass spectrography detects (ESI-MS):Meter Calculate molecular weight:2419.77, test value 2420.12.
2. detect the preparation of device
Detection chip is using IPDMS films as solid support material, is passing through point sample immobilized polypeptide thereon Solution is prepared.Modified silica-gel is that band olefin-terminal is added in traditional polydimethyl siloxane material , the initiator of surface initiated polymerization, and be fixed to poly- two by heat cross-linking (si-h bond bonding) In the three-dimensional structure of methylsiloxane, a kind of new material i.e. IPDMS films are obtained.Its manufacturing process As shown in Figure 1.
A and B therein be dimethyl silicone polymer two components, dimethyl silicone polymer (Poly (dimethylsiloxane), Sylgard 184) is bought from Dow corning (DowCorning) Company, comprising liquid composition A, (composition is metallic platinum catalyst and the diformazan siloxanes high score with vinyl Sub- precursor mixture) and crosslinking agent B (composition is the dimethyl silica with vinyl and Si -- H Alkane precursor) two kinds of compositions.C is initiator of the end with vinyl, is purchased from Hangzhou Dong Wei companies.Finally Macromolecule in modification is oligomeric ethylene glycol methacrylate monomers (Oligo (ethylene glycol) Methacrylate, hereinafter referred to as OEGMA, molecular weight Mw=526) buy in Aldrich.Will be poly- Dimethyl siloxane precursor A and crosslinking agent B and the initiator C with vinyl end with A:B:C=10:1:0.5 ratio is sufficiently mixed.Transparent elastic silicone rubber is made up of curing reaction, then Surface modification is carried out by sip technique and can obtain IPDMS films.Experiment shows, IPDMS films Surface have initiator that is highdensity enough, being fixed by covalent bond, it can be triggered poly- by surface Close reaction (SIP) and realize that surface is macromolecule modified.Use poly (OEGMA) (polyethylene glycol methyl-prop Olefin(e) acid ester) surface that reaction obtains polyethylene glycol (Polyethylene Glycol, PEG) modification is carried out, Realize the ability of stronger anti-albumen non-specific adsorption.
The IPDMS foamed films made need to be stored in 4 DEG C of refrigerators.
UsingIt is micro- that 16 people's point sample instruments of PersonalArrayerTM prepare polypeptide on modified silica-gel Array, process are:
1) pre-process
By IPDMS film sheets (15 × 15mm2) be immersed in activating solution, take out and spend after 30min Ion water wash 3 times, is dried up with nitrogen, is immediately used to point sample.
2) point sample
Sampling liquid is diluted good and is transferred in the corresponding micropore of 384 orifice plates, by 384 holes with sample Plate is placed on point sample instrument base station, while the modified silica-gel thin slice of pretreatment is placed on the base station of point sample instrument, Point sample is carried out at once.Point sample environmental condition is room temperature (25 DEG C), humidity set 50%.It is manufactured more The point sample amount each put on peptide microarray is about 0.6nL, and sampling point radius is 200 μm.
3) chemistry is fixed
The polypeptide microarrays just made will be placed in climatic chamber (26 DEG C, 60% humidity) and fix at least 6h.Chemical fixation procedure is as follows.
The buffer solution point for capturing peptide molecule will be included on modified silica-gel film by point sample instrument first, Then buffer solution starts to evaporate, capture peptide molecule and the intimate contact of IPDMS film surfaces and phase interaction With, by chemical bond, poly (OEGMA) high molecular end-COOH on modified silica-gel surface with Peptide molecule-NH2Stable covalent bond is formed, and then there will be chemically active peptide molecule to be fixed on IPDMS film surfaces.
5) assemble
Fixed 6h polypeptide microarrays must assemble in two days.It is by gum that IPDMS is thin first Film thin slice is attached on special reaction column, covers reaction cavity.One reactor is by two reaction columns and one Individual reaction cavity composition.
6) preserve
The polypeptide microarrays that assemble are stored in 4 DEG C of refrigerator, it is necessary to vacuumize sealing, standby.
3. detected with detection device
Checking procedure
1st, before starting detection, concentrated cleaning solutions are pressed 1:10 ratio adds purified water or distilled water enters Row dilution, is directly used after the completion of dilution.2mL cleaning fluids are added to chip surface using liquid-transfering gun, Soak chip 3 minutes, ensure that chip surface is fully wet out.
2nd, by test serum sample Sample dilution according to 1:40 dilutions mix.
3rd, the cleaning fluid of immersion chip, in the state of chip surface moistens completely, each blood are discarded Serum after the μ L dilutions of final proof this absorption 200 is added in chip reactor.
4th, chip reactor is put into chip fixed seat, be put on shaking table, open shaking table, frequency 150 Rev/min, it is incubated at room temperature 30 minutes.
5th, the serum sample in chip reactor is discarded, reaction cavity and core are cleaned with 15mL washing lotions Piece surface 3 times.
6th, after the completion of cleaning, each chip reactor is separately added into 200 μ L enzyme labelled antibody solution, will Chip reactor is put into chip fixed seat, is put on shaking table, opens shaking table, 150 revs/min of frequency, room Temperature is incubated 30 minutes.
7th, the enzyme labelled antibody solution in chip reactor is discarded, reaction cavity is cleaned with 15mL washing lotions With chip surface 3 times.
8th, after the completion of cleaning, reaction cavity is removed, each chip surface is separately added into 15 μ L and lighted Substrate solution, luminous liquid energy is set uniformly to be laid on chip surface.
9th, the chip for adding luminescent solution is placed in chemiluminescence imaging in gel imager, and interpretation As a result.
The serum of glioblastoma patient patients serum and other disease patient's serum samples are cured by cooperation Institute provides.Serum is handed to the transport of the parcels such as ice cube/dry ice or soon laboratory by related personnel.
Negative control has PBS (not have to test serum i.e. in the 3rd step to be incubated, and use PBS Solution is incubated, and remaining step is identical) control, the control of serum dilution, and patients with negative (refers to strong Health people and non-glioblastoma patient) serum control.
The spot sample mode of polypeptide microarrays is as shown in Figure 2:Wherein, the sample of 16 circular points of black For people IgM, as anchor point;The sample of 4 points of square is PBS sampling liquids, as experiment Blank control;The sample of white circular dot is that other glioblastoma patient mgmt proteins resist Former polypeptide, as experiment Testing index (these polypeptides have response explanation detection serum in have MGMT autoantibody);The sample of two star points is polypeptide SEQ ID NO of the invention:1, It is mgmt protein antigen polypeptide in glioblastoma patients serum, can be to glioblastoma disease MGMT autoantibodies produce response in people patient serum.
Using the polypeptide microarrays according to above-mentioned detecting step to glioblastoma patients serum and the moon Property control detected, response modes are as shown in figs. 34:Wherein, Fig. 3 shows negative control Testing result, the sample of the point only shown in black circle have response.Fig. 4 shows glioblastoma The testing result of patients serum, black is circular, white is circular and the sample of star point has response.Need Bright, instrument measures signal value from low to high, and corresponding signaling point color is by black-red-white gradual change.

Claims (9)

1. a kind of polypeptide for being used to detect MGMT antibody in serum, its amino acid sequence such as SEQ ID NO:Shown in 1, i.e.,:VPALHHPVFQQESFTRQVLW.
2. encode the nucleic acid of the polypeptide described in claim 1.
3. include the expression vector of the nucleic acid described in claim 2.
4. import the host cell of the expression vector described in claim 3.
5. the antibody of the polypeptide described in anti-claim 1.
6. one kind detection device, it includes:
Solid carrier, and
It is connected to the polypeptide described in the claim 1 on the solid carrier.
7. detection device according to claim 2, wherein, the solid carrier is IPDMS thin Film.
8. a kind of detection kit, it includes polypeptide or the claim 6 described in claim 1 Or the detection device described in 7.
9. the polypeptide described in claim 1 is preparing the kit for detecting MGMT antibody in serum Or the purposes in detection device.
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