CN105461785A - Polypeptide composition capable of being used for assisting in diagnosing systemic lupus erythematosus, detection device and detection kit - Google Patents
Polypeptide composition capable of being used for assisting in diagnosing systemic lupus erythematosus, detection device and detection kit Download PDFInfo
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- CN105461785A CN105461785A CN201510954808.8A CN201510954808A CN105461785A CN 105461785 A CN105461785 A CN 105461785A CN 201510954808 A CN201510954808 A CN 201510954808A CN 105461785 A CN105461785 A CN 105461785A
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- polypeptide
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- systemic lupus
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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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
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
-
- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
- G01N2800/104—Lupus erythematosus [SLE]
Abstract
The invention relates to polypeptide capable of being used for assisting in diagnosing systemic lupus erythematosus, a detection device and a detection kit. The detection kit comprises a detection device. The detection device comprises a solid carrier and polypeptide connected to the solid carrier. The detection reagent device and the detection kit can be used for assisting in diagnosing and screening systemic lupus erythematosus.
Description
Technical field
The present invention relates generally to immunoassay technology.Specifically, the present invention relates to the peptide composition that can be used for assistant diagnosis system lupus erythematosus, detection means and detection kit.
Background technology
Systemic lupus erythematous (systemiclupuserythematosus, SLE) be a kind of autoimmune disorder being formed as feature with autoantibody generation and immunocomplex, clinical manifestation is the symptom that constitutional symptom and multisystem multiple organ are got involved, and has a strong impact on life-span and the quality of life of patient.The sickness rate of China SLE is 70.1/10 ten thousand, and has the trend risen year by year.The early diagnosis of SLE, early treatment are the keys improving prognosis.But China's autoimmune disorder diagnostic level is lower, and the main dependence on import of diagnostic reagent, cause diagnosis somewhat expensive, add the medical treatment cost of country and patient, be also unfavorable for popularizing to basic hospital.Therefore, setting up detection method that is stable, quick, that be applicable to popularization, is the problem needing at present to solve.
SLE clinical criteria comprises autoantibody such as antinuclear antibody, and ((ANA), anti-dsDNA antibody (ds-DNA antibody) and anti-Sm antibody detect, but not high due to specificity and sensitivity is not also desirable Serologic detection index.If " not holding any government official post's antibody repertoire " detection method that SLE is special can be set up, detect multiple specific antibody simultaneously, improve recall rate and accuracy, provide reliable foundation by for the diagnosis of SLE, classification, observation of curative effect and Index for diagnosis, there is important clinical meaning.Therefore, find that a kind of susceptibility is strong, the high and adopted simple and easy to do Serology test of specificity, to the early diagnosis of systemic lupus erythematous and treat significant.
The appearance of a large amount of autoantibody is one of key character of autoimmune disorder, but is still not clear due to its pathogenesis, is difficult to directly utilize autoantigen to detect antibody.Epitope (epitope) is the basis of identification, binding antibody in antigen molecule, is therefore the actual active principle detecting antibody.There is following advantage using epitope peptide or its simulating peptide (mimotope) as diagnostic tool: sensitivity and the specificity that diagnosis 1) can be improved, avoid the non-specific binding (DerooSetal (2001) CombChemHighThroughputScreen4:75 – 115) that biomacromolecule easily produces; 2) scope of medical diagnosis on disease is expanded, less for research or be difficult to external application autoantigen as antigen nucleic acid, polysaccharide antigen, lipid antigen, diagnose using simulating peptide as the surrogate of natural antigen, also can obtain result (BruceG (1997) the ProcNatlAcadSciUSA94:1955 – 1960 consistent with native antigenic epitopes; JinapingQetal (1999) Hyridoma18 (1): 103-109); 3) cost of polypeptide preparation, application low, be easy to Quality Control.Therefore, the acquisition of auto-antigen epitope peptide or its simulating peptide (MeloenRHetal (2000) JMolRecognit13:352-359) is the basis of the polypeptide detection reagent preparing autoantibody.
Summary of the invention
The nucleic acid that the object of the present invention is to provide a kind of polypeptide useful to the diagnosis of systemic lupus erythematous, this polypeptide of encoding, the expression vector comprising this nucleic acid, the host cell having imported this expression vector, the antibody of this polypeptide anti-, the detection means comprising this polypeptide, the detection kit comprising this polypeptide or this detection means and this polypeptide are detecting the purposes in sjogren syndrome, this polypeptide for the preparation of the purposes in the test kit of detection system lupus erythematosus or detection means.That is, the present invention comprises following technical proposals:
1. a peptide species, its aminoacid sequence as shown in SEQIDNO:1, that is: PRRQICVGDRRERKAAFKGD.
2. the nucleic acid of coding polypeptide according to claim 1.
3. comprise the expression vector of nucleic acid according to claim 2.
4. imported the host cell of expression vector according to claim 3.
5. a detection means, it comprises:
Solid carrier, and
Be connected to the polypeptide according to claim 1 on this solid carrier.
6. detection means according to claim 5, wherein, described solid carrier is iPDMS film.
7. a detection kit, it comprises the detection means described in polypeptide according to claim 1 or claim 5 or 6.
8. the antibody of anti-polypeptide according to claim 1.
9. polypeptide according to claim 1 is for the preparation of the purposes in the test kit of detection system lupus erythematosus or detection means.
Accompanying drawing explanation
The schematic diagram that the making processes of Fig. 1 iPDMS film is described.
Fig. 2 illustrates the schematic diagram of polypeptide microarrays spot sample mode.
Fig. 3 is the photo of display to the result that healthy normal people or non-systemic lupus erythematosus patients serum detect.
Fig. 4 is the photo of display to the result that lupus erythematosus patients serum detects.
Embodiment
Polypeptide of the present invention
In one aspect of the method, the invention provides the early screening of systemic lupus erythematous and the useful polypeptide of clinical assistant diagnosis, be made up of the aminoacid sequence shown in SEQIDNO:1, that is: PRRQICVGDRRERKAAFKGD.As shown in the Examples, the serum of polypeptide of the present invention to lupus erythematosus patients is positive, and to be negative reaction to healthy normal people or non-systemic lupus erythematosus patients serum.Therefore, polypeptide of the present invention is useful as the early screening of systemic lupus erythematous and clinical assistant diagnosis instrument.Polypeptide of the present invention may be used for manufacturing detection means (example detection means of the present invention described as follows) or the detection kit (example detection kit of the present invention described as follows) of early screening and the clinical assistant diagnosis that can be used for systemic lupus erythematous.
Polypeptide of the present invention can be suitable for adopting the known method such as (1) chemical synthesis process or (2) enzyme reaction synthetic method to obtain, but is not limited thereto, and wherein chemosynthesis is more easy.In chemosynthesis polypeptide situation of the present invention, undertaken by using peptide synthesizer synthesis or this polypeptide semi-synthetic.As chemical synthesis process, such as peptide solid-phase synthesis etc. can be listed.The peptide of such synthesis can adopt conventional means such as ion-exchange chromatography, reverse phase high performance liquid chromatogram, affinity chromatography etc. to carry out purifying.Such peptide solid phase synthesis process with and subsequent peptide purification be all well-known in the art.
In addition, when producing polypeptide of the present invention by enzyme reaction, the method such as described in No. WO2004/011653, International Publication brochure can be adopted.Namely; can produce like this: by the amino acid of a side or the C-terminal of dipeptides is esterified or amidation and the amino acid that obtains or dipeptides, the amino acid (amino acid of such as carboxy protective) that is in unbound state with amino acid react under the existence of peptide synthetase, the dipeptides of generation or tripeptides.As peptide synthetase, can list: there is the culture of microorganism of the ability generating peptide, the bacterial disposing thing of the microbial cells be separated by this culture or this microorganism or this microbe-derived peptide synthetase.
And, except above-mentioned enzyme method, chemical synthesis process, such as gene engineering method can also be adopted to produce polypeptide of the present invention.
The aminoacid sequence of polypeptide of the present invention can adopt conventional method to carry out analyzing and confirming, such as, can utilize the method such as mass spectrum, chromatogram to carry out and analyze and confirm.
Detection means of the present invention
In another aspect, the present invention also provides a kind of detection means (detection means of the present invention), and it comprises solid carrier and the protein of the present invention that is connected on this solid carrier or polypeptide of the present invention.Described detection means is useful for the early screening of systemic lupus erythematous and clinical assistant diagnosis.
In the present invention, solid carrier is not particularly limited, as long as the carrier of solid or insoluble material (be such as can pass through filtration, material that precipitation, magnetic resolution etc. are separated from reaction mixture).
The material forming solid carrier includes but not limited to: silica gel (polydimethylsiloxane, PDMS), Mierocrystalline cellulose, teflon TM, Nitrocellulose, agarose, dextran, chitosan, polystyrene, polyacrylamide, polyester, polycarbonate, polymeric amide, polypropylene, nylon, poly(vinylidene fluoride), latex, silicon-dioxide, glass, glass fibre, gold, platinum, silver, copper, iron, stainless steel, ferrite, silicon wafer, polyethylene, polymine, poly(lactic acid), resin, polyose, albumen (albumin etc.), carbon or their combination etc.
The shape of solid carrier comprises but is not limited to: pearl, magnetic bead, film, microcapillary, filter membrane, plate, micro plate, carbon nanotube, sensor chip etc.Just as known in the art, the solid carrier that 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 that about 25nm-is about 1mm scope.In a preferred embodiment, magnetic bead has the diameter of about 50nm-about 10 μm of scopes.The size of magnetic bead can be selected according to specific purposes.
In the present invention, the pearl be made up of the contour crosslinked spherical agarose of Sepharose has the diameter of about 24 μm of-Yue 165 μm of scopes.Preferably, high crosslinked spherical sepharose 4B has the diameter of about 24 μm of-Yue 44 μm of scopes.The size of high crosslinked spherical sepharose 4B can be selected according to specific purposes.
The example with the solid carrier of water repellent surface comprises can from Polysciences, Warrington, PA or Spherotech, the polystyrene latex beads such as the goods that Liberville, IL buy.
Silicon-dioxide (SiO
2)-process or silicon-dioxide (SiO
2) example of solid carrier of base comprises the extraordinary magnetic silica pearl etc. can bought from Polysciences, Warrington, PA, it may be used for catching nucleic acid (such as DNA).Or, the M-280 etc. that can buy from DynalBiotech can also be used.
The magnetic bead with hydrophilic surface can be used for the bacterial cell of seizure proliferation period, nucleic acid and other composition.As the example of this magnetic bead, can list Polysciences, the pearl (title: Biomag (registered trademark) carboxyl) that Warrington, PA sell or the name of BangsLaboratory, Inc., Fishers, IN are called MC0
2the pearl of N/2928.Or, the M-270 etc. that DynalBiotech sells can be used.
In a preferred embodiment of the present invention, described solid carrier is SJ modified silica-gel, it is the microarray solid support material (iPDMS film, see the open CN101265329A of Chinese patent) of a kind of silicon rubber material of Suzhou Siju Biomaterials Co., Ltd.'s exploitation.This material is based on the conventional PDMS of biological study, add specific initiator composition (making this material realize surface-functionalized modification by surface initiated polymerization (SIP)) wherein, obtain through polyethylene glycol methacrylate-styrene polymer (poly (oligo (ethyleneglycol) methacrylate), pOEGMA) finishing again.SJ modified silica-gel has outstanding anti-protein non-specific adsorption (Nonspecificproteinadsorption, NPA) ability, non-specific protein absorption in complicated protein immunization can being detected controls to close to " absolute 0 " level (being near or below the limit of detection of instrument), not only can exempt the trouble closed and repeatedly clean, can also by the susceptibility using stronger amplification of signal means to improve protein microarray.And the essence of its silicon rubber imparts the stronger mechanical property of this material and good operability.Suzhou Yvonne gathers the combination assay microarray ELISA kit successfully SJ modified silica-gel being applied to 11 tumor markers compositions, achieve high-throughput and high-sensitive detection, demonstrating this material is a kind of outstanding protein microarray solid support material.Meanwhile, this material also has the adjustable characteristic of surface properties, can adjust its surface topography within the specific limits by the controlled modification reaction times.
The connection of polypeptide of the present invention and 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)-carbodiimide [1-ethyl-3-(3-dimethylami-nopropyl) carbodiimide can be passed through, EDC] and N-hydroxy-succinamide (N-hydroxysuccinimide, NHS) reaction by modified silica-gel surface macromolecular chain on carboxyl (-COOH) group change activating group into, this activating group can with protein/polypeptide on amino (-NH
2) react thus realize protein/polypeptide to be fixed on solid carrier surface (see such as HongweiMa, YuanziWu, XiaoliYang, XingLiu, Jian ' anHe, LongFu, JieWang, HongkeXu, YiShiandRenqianZhong, Integratedpoly (dimethysiloxane) withanintrinsicnonfoulingpropertyapproaching " Absolute " zerobackgroundinimmunoassays, Anal.Chem., 82,6338 – 6342,2010).
Concentration for polypeptide of the present invention in the sampling liquid used during point sample is not particularly limited, and those skilled in the art can conventionally select, and is preferably 1 μ g-1000 μ g/mL, more preferably 10 μ g-500 μ g/mL.In addition, the density distributed on a solid support for polypeptide of the present invention is not particularly limited, and those skilled in the art can conventionally select, and is preferably 1-100 point/10mm
2, more preferably 5-50 point/10mm
2.
Detection means of the present invention is useful for the early screening of systemic lupus erythematous and clinical assistant diagnosis, or it may be used for manufacturing and can be used for the early screening of systemic lupus erythematous and the detection kit of clinical assistant diagnosis.
Detection kit of the present invention
In one aspect of the method, the present invention also provides a kind of detection kit (detection kit of the present invention), and it comprises detection means of the present invention.This detection kit can be used for early screening and the clinical assistant diagnosis of systemic lupus erythematous.
Detection kit of the present invention must comprise detection means of the present invention.Detection kit of the present invention can also comprise:
1. the serum sample diluent prepared or serum sample diluent component solution: Sample dilution, such as, have the Sample dilution (production code member 070021-S2) of Sai Chi bio tech ltd, Beijing, the application of sample variable color Sample dilution (production code member bwj010103) etc. of Bo Weijia bio tech ltd, Zhengzhou.Such Sample dilution is the PBS solution containing the composition such as BSA, sucrose, and containing sanitas, clarified liq, can directly use.This Sample dilution is used for dilute serum, and the serum that test kit detects will dilute suitable multiple, and such as 2-200 doubly, and preferred 10-100 doubly.
Detection kit of the present invention can also comprise:
2. concentrated washing lotion and washing lotion: after solid carrier surface hatches serum and ELIAS secondary antibody, the unconjugated antibody of solid carrier surface and ELIAS secondary antibody need be washed by washing lotion.Concentrated washing lotion is such as the polysorbas20 aqueous solution of 1%, need dilute 2-40 times, preferably 5-20 times during use.Can dilute by 1:9 by concentrating washing lotion (10 ×) purified water or distilled water, configure washing lotion.Such as, add 50mL in 450mL purified water or distilled water and concentrate washing lotion (10 ×), fully mix.The washing lotion do not used is placed on 2-8 DEG C of preservation, can preserve 3 months.
Detection kit of the present invention can also comprise:
3. ELIAS secondary antibody solution: the IgM in robot system lupus erythematosus person serum can be combined by the polypeptide of the present invention on solid carrier (such as SJ modified silica-gel), ELIAS secondary antibody can with antibodies, and the marker in ELIAS secondary antibody can react with luminous substrate mixing solutions, thus send detectable light.ELIAS secondary antibody can be the goat-anti people IgM of such as horseradish peroxidase-labeled.Being not particularly limited the concentration of ELIAS secondary antibody in ELIAS secondary antibody solution, can be such as 1ng-1000ng/mL.
Detection kit of the present invention can also comprise:
4. luminous substrate mixing solutions: luminous substrate mixing solutions can react with the horseradish peroxidase that marks in ELIAS secondary antibody, make to react the chemical light sending instrument and can detect, luminous substrate mixing solutions comprises luminous substrate liquid A-superoxol, and luminous substrate liquid B-luminol (luminol,3-aminophthalic acid cyclic hydrazide) solution.Adopt two kinds of luminous substrate liquid to mix with equal-volume during use in advance, obtain luminous substrate mixing solutions (using in 45 minutes).Luminol only has crosses just meeting luminescence by oxidizer treatment.The mixed aqueous solution of usual use hydrogen peroxide and a kind of hydroxide bases is as exciting agent.Under horseradish peroxidase enzyme catalytic, decomposing hydrogen dioxide solution is oxygen G&W:
2H
2O
2→O
2+2H
2O
Luminol,3-aminophthalic acid cyclic hydrazide and oxyhydroxide generate a pairs of anion when reacting, the dioxygen oxidation that it can be gone out by peroxide decomposition, and product is an organo-peroxide.This superoxide is very unstable, decomposites nitrogen immediately, generates the 3-aminophthalic acid of excited state.During excited state to ground state transforms, the energy of release exists with the form of photon, and wavelength is positioned at the blue light components of visible ray.The example such as ThermoSeientific company of luminous substrate mixing solutions
eLISAFemtoMaximumSensitivi-tySubstrate, article No. 37074.
Detection kit of the present invention can also comprise:
5. one or more reaction cavity, can adopt such as two-sided biological incubation reactor, Chinese patent ZL201120177686.3 or ZL201110142518.5 according to demand; Or the biological incubation reactor ZL201220430886.x of one side.
Detection kit of the present invention can also comprise:
6. other detection molecules for detection system lupus erythematosus (such as polypeptide, protein, nucleic acid etc.).
Detection kit of the present invention can also comprise:
7. working instructions, wherein describe early screening and clinical assistant diagnosis that this detection kit may be used for systemic lupus erythematous.
Reaction member and reaction kit can such as adopt gel imaging instrument to carry out chemiluminescence imaging.As gel imaging instrument, such as GELAS4000mini type or sky energy 5500 type Microarray image instrument can be adopted.
Embodiment
Below, by embodiment, more specific description is carried out to the present invention, but be not the restriction to the technology of the present invention scope.By the record of this specification sheets, those skilled in the art can be easy to modify the present invention/change, and these are included in technical scope of the present invention.Scope of the present invention is determined by claims.
1. polypeptide preparation and confirmation
The polypeptide used in embodiment has the aminoacid sequence shown in SEQIDNO:1, is synthesized by Chengdu Kaijie Biological Medicine Science and Technology Development Co., Ltd, and the sign of this polypeptide confirms to have synthesized described polypeptide by hydrogen spectrum and mass spectrum.Liquid phase chromatography detects purity: 96.2% (area normalization method): mass spectroscopy detects (ESI-MS): calculate molecular weight: 2358.71, test value 2359.12.
2. the preparation of detection means
Detection chip be with SJ modified silica-gel (iPDMS film) for solid support material, be prepared from by point sample immobilized polypeptide solution thereon.Modified silica-gel be add in traditional polydimethyl siloxane material band olefin-terminal, the initiator of surface initiated polymerization, and be fixed in the three-dimensional structure of polydimethylsiloxane by heat cross-linking (si-h bond bonding), obtain SJ modified silica-gel, its making processes as shown in Figure 1.
A and B is wherein 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 polymer precursor mixture being with vinyl) and crosslinking agent B (composition is the dimethyl siloxane precursor with vinyl and Si-H group) two kinds of compositions.C is the initiator of end strips vinyl, is purchased from Hangzhou Dong Wei company.Polymer 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 are fully mixed with A:B:C=10:1:0.5 ratio with the initiator C of band vinyl end.Make transparent elastic silicone rubber by curing reaction, then carry out finishing by sip technique and can obtain SJ modified silica-gel.Experiment shows, the surface of SJ modified silica-gel have enough highdensity, by the initiator of covalently immobolization, it can pass through surface initiated polymerization (SIP), and to realize surface macromolecule modified.The surface that reaction acquisition polyoxyethylene glycol (PolyethyleneGlycol, PEG) is modified is carried out in use poly (OEGMA) (polyethylene glycol methacrylate-styrene polymer), realizes the ability of stronger anti-albumen non-specific adsorption.
The SJ modified silica-gel film made need be kept in 4 DEG C of refrigerators.
Adopt
personalArrayerTM16 people's point sample instrument prepares polypeptide microarrays on modified silica-gel, and process is:
1) pre-treatment
SJ modified silica-gel thin slice (15 × 15mm2) is immersed in activation solution, takes out with deionized water drip washing 3 times after 30min, dry up with nitrogen, be used for point sample at once.
2) point sample
Sampling liquid is diluted and gets well and transfer in the corresponding micropore of 384 orifice plate, 384 orifice plates of band sample are placed on point sample instrument base station, pretreated modified silica-gel thin slice are placed on the base station of point sample instrument simultaneously, carry out point sample at once.Point sample envrionment conditions 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 will be placed on fixing at least 6h in climatic chamber (26 DEG C, 60% humidity).Chemistry fixation procedure is as follows:
First by point sample instrument by include catch peptide molecule damping fluid point on modified silica-gel film, then damping fluid starts evaporation, catch peptide molecule and the surface intimate contact interacting of SJ modified silica-gel, pass through Chemical bond, ploy (OEGMA) high molecular end-COOH on modified silica-gel surface and peptide molecule--NH2 forms stable covalent linkage, and then chemically active peptide molecule will be had to be fixed on SJ modified silica-gel surface.
4) assemble
The polypeptide microarrays of fixing 6h must assemble in two days.First by gum, SJ modified silica-gel thin slice is attached on special reaction column, covers reaction cavity.A reactor is made up of two reaction columns and a reaction cavity.
5) preserve
The polypeptide microarrays assembled, needs to vacuumize sealing, is kept in the refrigerator of 4 DEG C, for subsequent use.
3. detect by detection means
Checking procedure
1, before starting to detect, concentrated washing lotion is added purified water in the ratio of 1:10 or distilled water dilutes, must washing lotion after dilute, direct use, use liquid-transfering gun that 2mL washing lotion is added to chip surface, soak chip 3 minutes, ensure that chip surface is fully wet out.
2, test serum sample Sample dilution is mixed according to 1:40 dilution.
3, discard the washing lotion of soaking chip, under the state that chip surface is completely moistening, the serum after each serum sample draws 200 μ L dilutions joins in chip reactor.
4, chip reactor is put into chip permanent seat, be put on shaking table, open shaking table, frequency 150 revs/min, incubated at room 30 minutes.
5, the serum sample in chip reactor is discarded, with 15mL washing lotion cleaning reaction cavity and chip surface 3 times.
6, after having cleaned, each chip reactor adds 200 μ L ELIAS secondary antibody solution respectively, chip reactor is put into chip permanent seat, is put on shaking table, opens shaking table, frequency 150 revs/min, incubated at room 30 minutes.
7, the ELIAS secondary antibody solution in chip reactor is discarded, with 15mL washing lotion cleaning reaction cavity and chip surface 3 times.
8, in advance luminous substrate liquid A and luminous substrate liquid B can be mixed with equal-volume, obtain luminous substrate mixing solutions (using in 45 minutes).To be cleaned complete after, take off reaction cavity, each chip surface adds 15 μ L luminous substrate mixing solutionss respectively, makes luminous substrate mixing solutions can be laid on chip surface uniformly.
9, the chip adding luminescent solution is placed in gel imaging instrument chemiluminescence imaging, and sentence read result.
Healthy normal human serum, lupus erythematosus patients serum and other disease patient's serum samples are provided by chain hospital, and disease criterion all through Clinical Laboratory confirmation, all obtains the Informed Consent Form of supplier.
Negative control has PBS damping fluid (namely to hatch without test serum in the 3rd step, and hatch by PBS solution, all the other steps are identical) contrast, the contrast of serum dilution, and the contrast of negative serum (referring to the serum of Healthy People and non-systemic lupus erythematosus patient).
The spot sample mode of polypeptide microarrays is as shown in Figure 2: wherein, the sample of leg-of-mutton 4 points is human IgG, as locating point; The sample of foursquare 1 point is PB sampling liquid, as sky from contrast; The sample of circular point is other systemic lupus erythematous autoantigen protein polypeptide, as Testing index (these polypeptide have response to illustrate in detected serum have the Autoantibodies of Systemic Lupus Erythematosus); The sample polypeptides of star point is polypeptide of the present invention, and it is systemic lupus erythematous autoantigen protein polypeptide, can produce response to Serum in Patients with SLE.
This polypeptide microarrays is used to detect lupus erythematosus patients serum and negative control according to above-mentioned detecting step, response modes is as shown in Figure 3-4: wherein, Fig. 3 shows the detected result of negative control, only has the sample of the point shown in trilateral to have response.Fig. 4 shows the detected result of lupus erythematosus patients serum, and the sample of trilateral, circle and star point has response.It should be noted that, instrument records signal value from low to high, and corresponding signaling point color is by black-red-white gradual change.
Above, by embodiment, more specific description is carried out to the present invention, but be not the restriction to the technology of the present invention scope.By the record of this specification sheets, those skilled in the art can be easy to modify the present invention/change, and these are included in technical scope of the present invention.
Claims (9)
1. a peptide species, its aminoacid sequence as shown in SEQIDNO:1, that is:
PRRQICVGDRRERKAAFKGD。
2. the nucleic acid of coding polypeptide according to claim 1.
3. comprise the expression vector of nucleic acid according to claim 2.
4. imported the host cell of expression vector according to claim 3.
5. a detection means, it comprises:
Solid carrier, and
Be connected to the polypeptide according to claim 1 on this solid carrier.
6. detection means according to claim 5, wherein, described solid carrier is iPDMS film.
7. a detection kit, it comprises the detection means described in polypeptide according to claim 1 or claim 5 or 6.
8. the antibody of anti-polypeptide according to claim 1.
9. polypeptide according to claim 1 is for the preparation of the purposes in the test kit of detection system lupus erythematosus or detection means.
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| CN104098674A (en) * | 2013-04-03 | 2014-10-15 | 苏州偲聚生物材料有限公司 | Polypeptide, and detection member and detection kit both containing same |
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| WO2008156807A2 (en) * | 2007-06-19 | 2008-12-24 | The Johns Hopkins University | Antithrombotic agents and methods of use thereof |
| CN104098674A (en) * | 2013-04-03 | 2014-10-15 | 苏州偲聚生物材料有限公司 | Polypeptide, and detection member and detection kit both containing same |
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