CN107090024A - Detection device, detection kit and diagnostic method available for diagnostic system lupus erythematosus - Google Patents
Detection device, detection kit and diagnostic method available for diagnostic system lupus erythematosus Download PDFInfo
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- 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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Abstract
The present invention relates to detection device, detection kit and the detection method available for diagnostic system lupus erythematosus.The kit of the present invention includes detection device, and described detection device includes one or more solid carriers, and the polypeptide being independently connected on one or more of solid carriers.The detection reagent device and detection kit of the present invention can be used for the detection of systemic loupus erythematosus.
Description
Technical field
The invention mainly relates to immunoassay technology.Specifically, the present invention relates to the detection of the polypeptide available for diagnostic system lupus erythematosus device, detection kit and diagnostic method.
Background technology
Systemic loupus erythematosus (systemic lupus erythematosus, SLE it is) that a kind of produced with autoantibody forms the autoimmune disease being characterized with immune complex, clinical manifestation is the symptom that constitutional symptom and multisystem multiple organ are involved, and has a strong impact on life-span and the quality of life of patient.China SLE incidence of disease is 70.1/10 ten thousand, and has the trend risen year by year.SLE early diagnosis, early treatment are to improve the key of prognosis.But China's autoimmune disease diagnostic level is relatively low, and diagnostic reagent relies primarily on import, causes to diagnose somewhat expensive, adds country and the medical treatment cost of patient, be also unfavorable for popularizing to basic hospital.Therefore, detection method that is stable, quick, being adapted to popularization is set up, is the problem of needing to solve at present.
The SLE related auto-antibodies conventional detection projects carried out clinical at present mainly have antinuclear antibodies (ANA), Anti-hCG action, anti-ENA antibody (including anti-sm, anti-U1RNP, anti-SSA/Ro, anti-SSB/La, anti-rRNP, anti-Scl-70 and anti-Jo-1 etc.), antinuclear antibodies and anti-phospholipid antibody etc..Autoantibody detection should be carried out for clinical doubtful SLE patient.In the SLE criteria for classifications that American society of rheumatism is revised, crucial immunological abnormality and autoantibodies include anti-Sm antibody, Anti-hCG action, anti-phospholipid antibody and the ANA positives.Detection method of the prior art is not high due to specificity and sensitivity, there is not yet comparatively ideal Serologic detection index.Antinuclear antibodies positive rate only has 60% or so (Anti-Ro fine specificity defined by multiple antigenic peptides identifies componentsof tertiary epitopes. to such as in the prior art Scofield RH et al. reports SSA in other words
Clin Exp Immunol 109:480-7.).If special " autoantibody repertoire " detection methods of SLE can be set up, detect many strain specific antibodies simultaneously, recall rate and accuracy are improved, reliable foundation will be provided for SLE diagnosis, classification, observation of curative effect and Index for diagnosis, this is by with important clinical meaning.Therefore, find that a kind of sensitiveness is strong, specific high and easy-to-use Serology test, early diagnosis to SLE and treat significant.
Meanwhile, the appearance of a large amount of autoantibodies is one of key character of autoimmune disease, but because its pathogenesis is still not clear, it is difficult to directly detect antibody using autoantigen.Epitope (epitope) is identification, the basis of binding antibody in antigen molecule, therefore is the actually active component for detecting antibody.There is following advantage using epitope peptide or its simulating peptide (mimotope) as diagnostic tool：1) sensitivity and specificity of diagnosis can be improved, it is to avoid non-specific binding (Deroo S et al (2001) Comb Chem High Throughput Screen 4 that large biological molecule is also easy to produce:75–115)；2) scope of medical diagnosis on disease is expanded, for studying autoantigen such as antigen nucleic acid, polysaccharide antigen, lipid antigen less or that be difficult to external application, substitute using simulating peptide as native antigen is diagnosed, it is also possible to obtain result (Bruce G (1997) Proc Natl Acad Sci USA 94 consistent with native antigenic epitopes：1955–1960；Jinaping Q et al(1999)Hyridoma 18(1)：103–109)；3) polypeptide prepare, application cost it is low, be easy to Quality Control.Therefore, auto-antigen epitope peptide or its simulating peptide (Meloen RH et al (2000) J Mol Recognit 13:Acquisition 352-359) is produced from the basis of the polypeptide detection reagent of body antibody.
The content of the invention
It is an object of the invention to provide the method for the new detection device, the diagnostic kit that can be used for diagnosis SLE, and diagnosis SLE.In addition, the present invention also provides purposes of the combination of particular polypeptide in the kit or detection device for SLE diseases is prepared.
There was only 60% or so on SSA or antinuclear antibodies positive rate in the prior art, current inventor provides 11 polypeptide epitopes.The biological specimen originated using subject whether in 11 polypeptides at least two or more have response as sensitivity (and accuracy reaches 92.4%) diagnosis and examination SLE that 89.4% in the case of judging subject whether as the index of SLE patient, can be up to.
Therefore, one aspect of the present invention provides a kind of detection device, and it includes solid carrier, and the following polypeptides being independently connected on the solid carrier：
SEQ ID NO：Polypeptide shown in 1；
SEQ ID NO：Polypeptide shown in 2；
SEQ ID NO：Polypeptide shown in 3；
SEQ ID NO：Polypeptide shown in 4；
SEQ ID NO：Polypeptide shown in 5；
SEQ ID NO：Polypeptide shown in 6；
SEQ ID NO：Polypeptide shown in 7；
SEQ ID NO：Polypeptide shown in 8；
SEQ ID NO：Polypeptide shown in 9；
SEQ ID NO：Polypeptide shown in 10；With
SEQ ID NO：Polypeptide shown in 11.
In a preferred embodiment of the invention, described solid carrier is iPDMS films.
In a preferred embodiment of the invention, it is used for SLE diagnosis.
In addition, another aspect of the present invention is to provide a kind of SLE diagnostic kits, it includes one or more solid carriers, and the following polypeptides being connected on one or more of solid carriers：、
SEQ ID NO：Polypeptide shown in 1；
SEQ ID NO：Polypeptide shown in 2；
SEQ ID NO：Polypeptide shown in 3；
SEQ ID NO：Polypeptide shown in 4；
SEQ ID NO：Polypeptide shown in 5；
SEQ ID NO：Polypeptide shown in 6；
SEQ ID NO：Polypeptide shown in 7；
SEQ ID NO：Polypeptide shown in 8；
SEQ ID NO：Polypeptide shown in 9；
SEQ ID NO：Polypeptide shown in 10；With
SEQ ID NO：Polypeptide shown in 11.
In a preferred embodiment of the invention, the solid carrier is iPDMS films.
In a preferred embodiment of the invention, wherein, SEQ ID NO：Polypeptide shown in 1~11 is independently connected on same solid carrier.
In addition, another aspect of the present invention provides purposes of following polypeptides in SLE diagnostic kits or detection device is prepared：
SEQ ID NO：Polypeptide shown in 1；
SEQ ID NO：Polypeptide shown in 2；
SEQ ID NO：Polypeptide shown in 3；
SEQ ID NO：Polypeptide shown in 4；
SEQ ID NO：Polypeptide shown in 5；
SEQ ID NO：Polypeptide shown in 6；
SEQ ID NO：Polypeptide shown in 7；
SEQ ID NO：Polypeptide shown in 8；
SEQ ID NO：Polypeptide shown in 9；
SEQ ID NO：Polypeptide shown in 10；With
SEQ ID NO：Polypeptide shown in 11.
In addition, another aspect of the present invention provide it is a kind of diagnose subject whether be SLE patient method, this method includes：
SLE detection SEQ ID NO any one of detection device or claim 4~6 any one of usage right requirement 1~3：Whether the polypeptide shown in 1~11 has response to the blood sample that subject originates；Wherein,
When the blood sample that at least two or more than two polypeptides detected in following polypeptides 1~11 are originated to the subject has response, judge that subject is SLE patient；Otherwise, it is determined that subject is not SLE patient.
SEQ ID NO：Polypeptide shown in 1；
SEQ ID NO：Polypeptide shown in 2；
SEQ ID NO：Polypeptide shown in 3；
SEQ ID NO：Polypeptide shown in 4；
SEQ ID NO：Polypeptide shown in 5；
SEQ ID NO：Polypeptide shown in 6；
SEQ ID NO：Polypeptide shown in 7；
SEQ ID NO：Polypeptide shown in 8；
SEQ ID NO：Polypeptide shown in 9；
SEQ ID NO：Polypeptide shown in 10；With
SEQ ID NO：Polypeptide shown in 11.
In a preferred embodiment of the invention, the blood sample is whole blood, blood plasma or serum.
Embodiment
In this specification, following polypeptides 1~11：
SEQ ID NO：1~20 of polypeptide equivalent to 60kDa Ro albumen (60kDa Ro/SSA autoantigen) shown in 1；
SEQ ID NO：221~240 of polypeptide equivalent to 52kDa Ro albumen (52kDa Ro/SSA autoantigen) shown in 2；
SEQ ID NO：421~440 of polypeptide equivalent to 60kDa Ro albumen (60kDa Ro/SSA autoantigen) shown in 3；
SEQ ID NO：451~470 of polypeptide equivalent to 60kDa Ro albumen (60kDa Ro/SSA autoantigen) shown in 4；
SEQ ID NO：186~200 of polypeptide equivalent to cathepsin G (Cathepsin G) shown in 5；
SEQ ID NO：Polypeptide shown in 6 is equivalent to cathepsin G (191 of Cathepsin G~210；
SEQ ID NO：Polypeptide shown in 7 is equivalent to Sm autoantigens (106 of Smith autoantigen~126；
SEQ ID NO：Polypeptide shown in 8 is equivalent to Sm autoantigens (1 of Smith autoantigen~20.
SEQ ID NO：Polypeptide shown in 9 is equivalent to Sm autoantigens (70 of Smith autoantigen~90；
SEQ ID NO：Polypeptide shown in 10 is equivalent to Sm autoantigens (83 of Smith autoantigen~105.
SEQ ID NO：Polypeptide shown in 11 is equivalent to Sm autoantigens (90 of Smith autoantigen~119.
In this manual, recall rate refers to：The positive sample made a definite diagnosis with methods for clinical diagnosis, the ratio of the positive sample determined through this law.False positive rate refers to：The negative sample made a definite diagnosis with methods for clinical diagnosis, the ratio of the positive sample determined through this law.Diagnosis refers to：Make a definite diagnosis or to make a definite diagnosis offer reference information.
As shown in the Examples, the polypeptide set is positive to the serum of SLE disease people, and it is negative to healthy normal person or non-SLE patients serums, therefore, the polypeptide set cooperation is useful for the diagnostic tool of disease caused by SLE (such as SLE).
In this specification, positive reaction refers to：The blood sample that at least any two or two or more polypeptide in the polypeptide 1~11 are originated to the subject has response；Otherwise it is negative reaction.Response refers to：Signal to noise ratio (SNR) is more than or equal to 2, wherein, signal to noise ratio=(polypeptide point signal value-negative control point signal value)/negative control point signal value.
The polypeptide can be obtained suitably using the known method such as (1) chemical synthesis process or (2) enzyme reaction synthetic method, and wherein chemical synthesis is more easy.In the case of the polypeptide of the chemical synthesis present invention, carried out by using peptide synthesizer synthesis or the semi-synthetic polypeptide.As chemical synthesis process, it can include such as peptide solid-phase synthesis.The peptide so synthesized can be purified using conventional meanses such as ion-exchange chromatography, reverse phase high performance liquid chromatogram, affinity chromatography.Such peptide solid phase synthesis process with and subsequent peptide purification be all well-known in the art.
In addition, in the case where producing the polypeptide of the present invention by enzyme reaction, can be using the method for example described in International Publication pamphlet WO2004/011653.I.e., it is possible to so produce：By the carboxyl terminal of the amino acid of a side or dipeptides be esterified or amidatioon obtained from amino acid or dipeptides, the amino acid (amino acid of such as carboxy protective) that with amino acid is in free state reacted in the presence of peptide synthetase, the dipeptides or tripeptides of generation.As peptide synthetase, it can include：The culture of the microorganism of ability with generation peptide, the microbial cells or the bacterial disposing thing of the microorganism or the microbe-derived peptide synthetase separated by the culture.
The chemical synthesis of particularly polypeptide has been realized in commercialization, can be by entrusting the Peptide systhesis company of specialty to synthesize the polypeptide.
Detect device
The present invention provides a kind of detection device, including one or more solid carriers, and the peptide composition being independently connected on one or more of solid carriers.
In the present invention, solid carrier is not particularly limited, as long as it is used as the carrier of solid or insoluble material (such as can be by filtering, precipitate, separated from reactant mixture Magnetic Isolation material).
The material for constituting solid carrier includes but is not limited to：Silica gel (dimethyl silicone polymer, PDMS), cellulose, teflon TM, NC Nitroncellulose, agarose, glucan, chitosan, polystyrene, polyacrylamide, polyester, makrolon, polyamide, polypropylene, nylon, polyvinylidene fluoride, latex, silica, glass, glass fibre, gold, platinum, silver, copper, iron, stainless steel, ferrite, silicon wafer, 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, pit, groove, filter membrane bottom etc. can be set on the flat solid carrier such as film or plate.
In the present invention, magnetic bead can have the sphere diameter of about 25nm~about 1mm scopes.In a preferred embodiment, magnetic bead has the diameter of the μ m of about 50nm~about 10.The size of magnetic bead can be selected according to specific purposes.
In the present invention, the pearl being made up of the high crosslinked spherical agarose such as Sepharose has the diameter of about 24 μm~about 165 μ ms.Preferably, high crosslinked spherical sepharose 4B has the diameter of about 24 μm~about 44 μ ms.The size of high crosslinked spherical sepharose 4B can be selected according to specific purposes.
The example of solid carrier with hydrophobic surface includes can be from Polysciences, Warrington, PA or Spherotech, Liberville, the polystyrene latex beads such as product of IL purchases.
The example of the solid carrier of silica (SiO2)-processing or silica (SiO2) base includes can be from Polysciences, Warrington, extraordinary magnetic silica pearl of PA purchases etc., it can be used for catching nucleic acid (such as DNA).Or, can also use can be from Dynal Biotech M-280 bought etc..
Magnetic bead with hydrophilic surface can be used for bacterial cell, nucleic acid and the other compositions for catching proliferation period.As the example of the magnetic bead, Polysciences, Warrington, the pearl (title of PA sale can be included：Biomag (registration mark) carboxyl) or Bangs Laboratory, Inc., Fishers, IN entitled MC02N/2928 pearl.Or, M-270 that Dynal Biotech sell etc. can be used.
In a preferred embodiment of the present invention, the solid carrier is iPDMS films, it is a kind of microarray solid support material (iPDMS films, referring to Chinese patent CN101265329A) of silicon rubber material of Suzhou Siju Biomaterials Co., Ltd.'s exploitation.Based on this material is the PDMS commonly used by biological study, specific initiator composition (making the material surface-functionalized modification can be realized by surface initiated polymerization (SIP)) is added wherein, obtained again by polyethylene glycol methacrylate-styrene polymer (poly (oligo (ethylene glycol) methacrylate), pOEGMA) surface modification.IPDMS films have outstanding anti-protein non-specific adsorption (Nonspecific protein adsorption, NPA) ability, non-specific protein absorption control during can complicated protein immunization be detected is to close to " absolute 0 " level (detectable limit for being near or below instrument), closing and the trouble being cleaned multiple times can not only be exempted, the sensitivity of protein microarray can also be improved 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.IPDMS films have successfully been applied to the combination assay microarray ELISA kit of 11 tumor markers compositions by the poly- companies of Suzhou Yvonne, realize high flux and highly 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, can adjust its surface topography within the specific limits by the controlled modification reaction time.
In the present invention, the connection of polypeptide and solid carrier can be carried out using well known to a person skilled in the art the connection method of polypeptide and solid carrier.For example, for the connection on protein/polypeptide and modified silica-gel surface, 1- ethyls -3- (3- dimethyl aminopropyls)-carbodiimides [1-ethyl-3- (3-dimethyl ami-nopropyl) carbodiimide can be passed through, EDC] and n-hydroxysuccinimide (N-hydroxysuccinimide, carboxyl (- COOH) group on the macromolecular chain of modified silica-gel surface is changed to activated group by reaction NHS), the amino (- NH that the activated group can be with institute's band on protein/polypeptide2) react and protein/polypeptide is fixed on solid carrier surface so as to realize.
The concentration of polypeptide is not particularly limited in the sampling liquid used during for point sample, and those skilled in the art 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, being not particularly limited for the density that polypeptide is distributed on a solid support, those skilled in the art 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 detection SLE diseases or prepare kit for diagnosing SLE diseases.
SLE
Diagnostic kit
The invention further relates to a kind of SLE diagnostic kits, it includes
One or more solid carriers, and the following polypeptides being connected on one or more of solid carriers：
SEQ ID NO：Polypeptide shown in 1；
SEQ ID NO：Polypeptide shown in 2；
SEQ ID NO：Polypeptide shown in 3；
SEQ ID NO：Polypeptide shown in 4；
SEQ ID NO：Polypeptide shown in 5；
SEQ ID NO：Polypeptide shown in 6；
SEQ ID NO：Polypeptide shown in 7；
SEQ ID NO：Polypeptide shown in 8；
SEQ ID NO：Polypeptide shown in 9；
SEQ ID NO：Polypeptide shown in 10；With
SEQ ID NO：Polypeptide shown in 11.
It is surprising that in the case where the kit of the present invention includes above-mentioned whole polypeptides, kit of the invention can diagnose SLE diseases with the sensitivity (and accuracy reaches 92.4%) for being up to 89.4%.In this specification, sensitivity refers to：In positive sample with the confirmation of " goldstandard " method, the ratio of positive sample is determined as by other method.False positive rate refers to：In negative sample with the confirmation of " goldstandard " method, the ratio of positive sample is determined as by other method.The art detection SLE " goldstandard " method is that clinical symptoms are made a definite diagnosis, the standard that clinical symptoms are made a definite diagnosis is according to American society of rheumatism (American college of rheumatology, ACR new revision) has been done within 1997 on the basis of SLE criteria for classifications, specially 1, cheek erythema；2nd, plate-like erythema；3rd, photosensitization；4th, canker sore；5th, arthritis；6th, scrositis；7th, renal damage：8th, nervous system abnormality：9th, hematological abnormality：10th, crucial immunological abnormality：11st, antinuclear antibodies is positive.Simultaneously or successively at least 4 positives are diagnosable in the above 11.In this specification, " diagnosis " can be made a definite diagnosis or to make a definite diagnosis offer reference information.
The SLE diagnostic kits are preferred for the diagnosis of SLE diseases.
Solid carrier in the SLE diagnostic kits of the present invention can be one or multiple, but preferably one, i.e. SLE diagnostic kits of the invention preferably comprise the detection device of the present invention.
On the solid carrier, the description as described in solid carrier in " detection device " part is referred to.
The SLE diagnostic kits of the present invention can also include：
1. the serum dilution or serum dilution component solution that prepare：Serum dilution, such as the sample-adding discoloration Sample dilution (production code member bwj010103) for the Sample dilution (production code member 070021-S2), Zhengzhou Bo Weijia bio tech ltd for having Beijing Sai Chi bio tech ltd.The serum dilution is used for dilute serum, and the serum of kit detection will dilute suitable multiple, preferably such as 2~200 times, 10~100 times.
The SLE diagnostic kits of the present invention can also include：
2. concentrate washing lotion：Solid carrier surface is incubated after serum and ELIAS secondary antibody, and solid carrier surface uncombined antibody and ELIAS secondary antibody need to be washed with washing lotion.Concentration washing lotion is, for example, the 1% polysorbas20 aqueous solution, and 2~40 times, preferably 5~20 times need to be diluted when using.
The SLE diagnostic kits of the present invention can also include：
3. ELIAS secondary antibody solution：SLE autoantibodies in SLE patients serums can be combined with the polypeptide of the invention on solid carrier (such as iPDMS films), and secondary antibody can be 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 the goat anti-human igg of such as horseradish peroxidase-labeled.As ELIAS secondary antibody solution, the Goat anti human IgG (H+L), production code member ZB-2304 of the horseradish peroxidase-labeled of Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge production can be included.Concentration of the ELIAS secondary antibody in ELIAS secondary antibody solution is not particularly limited, can be such as 1ng~1000ng/mL.
The SLE lupus erythematosus diagnosis kit of the present invention can also include：
4. luminescent solution component solution：Luminescent solution can react with the horseradish peroxidase marked on secondary antibody so that reaction sends the detectable chemical light of instrument.Luminescent solution is mixed by two kinds of solution, is A liquid-hydrogenperoxide steam generator respectively, and B liquid-luminous ammonia solution.Luminol (luminol) is only crossed with oxidizer treatment and can just lighted.Exciting agent is used as usually using hydrogen peroxide and a kind of mixed aqueous solution of hydroxide bases.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, the dioxygen oxidation that it can be decomposited by hydrogen peroxide, and product is an organic peroxide.The peroxide is very unstable, and nitrogen is decomposited immediately, generates the 3- aminophthalic acids of excitation state.During excitation state is converted to ground state, the energy of release exists in the form of photon, and wavelength is located at the blue light components of visible ray.The example of luminescent solution component solution such as Thermo Seientific companiesELISA Femto Maximum Sensitivity Substrate, article No. 37074.
The SLE diagnostic kits of the present invention can also include：
5. one or more reaction cavity (such as Chinese patent Granted publication CN202054829U).
The SLE diagnostic kits of the present invention can also include：
6. other are used for detecting system erythema wolf disease (detection molecules (such as polypeptide, protein, nucleic acid).
The SLE diagnostic kits of the present invention can also include：
7. operation instructions.
The SLE diagnostic kits of the present invention can also include：
8. the positive quality control point (such as H-IgG) being connected on one or more of solid carriers or other independent solid carriers and/or negative Quality Control point (such as PB).
Diagnostic method
The present invention also provide it is a kind of diagnose subject whether be SLE patient method, this method includes：
Use the polypeptide SEQ ID NO in the kit detection peptide composition of the invention described above：Whether the polypeptide shown in 1~11 has response to the blood sample that subject originates；Wherein,
When detecting the blood sample originated respectively from least two or more than two polypeptides in following polypeptides 1~11 to the subject and having response, judge that subject is SLE patient；Otherwise, it is determined that subject is not SLE patient.
In this manual, " response " refers to：Signal to noise ratio (SNR) is more than or equal to 2, wherein, signal to noise ratio=(polypeptide point signal value-negative control point signal value)/negative control point signal value.
In this specification, the positive reaction to the kit of the present invention refers to：The blood sample in subject source meets above-mentioned criterion；Otherwise it is negative reaction.
The blood sample can be whole blood, blood plasma or serum.
Embodiment
1.
The preparation and confirmation of polypeptide
The polypeptide used in embodiment is synthesized by the biochemical (Shanghai) Co., Ltd. of gill, and it is confirmed by mass spectrum.Wherein,
SEQ ID NO：Peptide sequence shown in 1 is MEESVNQMQPLNEKQIANSQ, and molecular weight is 2318；
SEQ ID NO：Peptide sequence shown in 2 is QSQALQELISELDRRCHSSA, and molecular weight is 2271；
SEQ ID NO：Peptide sequence shown in 3 is PCPVTTDMTLQQVLMAMSQI, and molecular weight is 2208；
SEQ ID NO：Peptide sequence shown in 4 is ETFAGGVHPAIALREYRKKM, and molecular weight is 2274；
SEQ ID NO：Peptide sequence shown in 5 is PRRQICVGDRRERKAAFKGD, and molecular weight is 2359；
SEQ ID NO：Peptide sequence shown in 6 is RERKAAFKGDSGGPLLCNNV, and molecular weight is 2132；
SEQ ID NO：Peptide sequence shown in 7 is QVAARGRGRGMGRGNIFQKRR, and molecular weight is 2372；
SEQ ID NO：Peptide sequence shown in 8 is MSLLNKPKSEMTPEELQKRE, and molecular weight is 2389；
SEQ ID NO：Peptide sequence shown in 9 is VKEMWTEVPKSGKGKKKSKPV, and molecular weight is 2372；
SEQ ID NO：Peptide sequence shown in 10 is GKKKSKPVNKDRYISKMFLRGDS, and molecular weight is 2683,
SEQ ID NO：Peptide sequence KKREAVAGRGRGRGRGRGRGRGRGRGGPRR shown in 11, molecular weight is 3244
2.
The preparation of kit
Detection chip is, using iPDMS films as solid support material, to be prepared from thereon by point sample immobilized polypeptide solution.Modified silica-gel is the initiator that surface initiated polymerization with olefin-terminal is added in traditional polydimethyl siloxane material, and be fixed to by heat cross-linking (si-h bond bonding) in the three-dimensional structure of dimethyl silicone polymer, obtain a kind of new material i.e. iPDMS films.Its manufacturing process is referring to International Patent Publication WO2014/044184.
The iPDMS foamed films made can be stored in 4 DEG C of refrigerators.
Using16 people's point sample instruments of PersonalArrayerTM prepare polypeptide microarrays (i.e. detection chip) on modified silica-gel, and process is：
1) pre-process
By iPDMS film sheets (15 × 15mm2) be immersed in activating solution, taking-up is eluted 3 times with deionized water after 30min, 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, 384 orifice plates with sample are 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), and humidity set is 50%.Following polypeptides are put into microarray in iPDMS film sheets, each one point of polypeptide point sample, in addition one H-IgG point of point sample be used as negative Quality Control point as positive quality control point and a PB point.The point sample amount about 0.6nL each put on the polypeptide microarrays being made, sampling point radius is 200 μm.
Here, the polypeptide of point sample is specific as follows on the modified silica-gel thin slice of kit：
SEQ ID NO：Polypeptide shown in 1~11.
3) chemistry is fixed
The polypeptide microarrays just made will be placed in climatic chamber (26 DEG C, 60% humidity) fixed at least 6h.Chemical fixation procedure is referring to International Patent Publication WO2014/044184.
The buffer solution point of capture peptide molecule will be included on modified silica-gel film by point sample instrument first, then buffer solution starts evaporation, capture peptide molecule and the intimate contact of iPDMS film surfaces simultaneously interacts, pass through chemical bond, ploy (OEGMA) high molecular end-COOH and the-NH of peptide molecule on modified silica-gel surface2Stable covalent bond is formed, and then there will be chemically active peptide molecule to be fixed on iPDMS film surfaces.
4) assemble
Fixed 6h polypeptide microarrays must be assembled in two days.IPDMS film sheets are attached on special reaction column by gum first, cover reaction cavity.One reactor is made up of two reaction columns and a reaction cavity.
5) preserve
The polypeptide microarrays that assemble are standby in the refrigerator for being stored in 4 DEG C, it is necessary to vacuumize sealing.
3.
Detected with kit
Checking procedure
(1) start before detection, concentrated cleaning solutions are pressed 1:10 ratio adds purified water or distilled water is diluted, and is directly used after the completion of dilution.2mL cleaning fluids are added to chip surface, immersion chip 3 minutes, it is ensured that chip surface is fully wet out using liquid-transfering gun.
(2) test serum sample is pressed 1 with Sample dilution:200 dilutions are mixed.
(3) cleaning fluid of immersion chip is discarded, in the state of chip surface is moistened completely, the serum that each serum sample draws after 200 μ L dilutions is added in chip reactor.
(4) chip reactor is put into chip fixed seat, be put on shaking table, unlatching shaking table, 150 revs/min of frequency, 4 DEG C are incubated 12-16 hours.
(5) serum sample in chip reactor is discarded, reaction cavity and chip surface 3 times are cleaned with 15mL washing lotions.
(6) after the completion of cleaning, each chip reactor is separately added into 200 μ L enzyme labelled antibody solution, and chip reactor is put into chip fixed seat, is put on shaking table, unlatching shaking table, 150 revs/min of frequency, and 4 DEG C are incubated 12-16 hours.
(7) the enzyme labelled antibody solution in chip reactor is discarded, reaction cavity and chip surface 3 times are cleaned with 15mL washing lotions.
(8) after the completion of cleaning, reaction cavity is removed, each chip surface is separately added into 15 μ L luminous substrate liquid, luminous liquid energy is uniformly laid on chip surface.
(9) chip for adding luminescent solution is placed in chemiluminescence imaging and result of determination in gel imager, as gel imager, using such as day 5500 type Microarray image instrument of energy, the prompt board TN5500 types of Yvonne or the prompt board CLEAR4000 types of Yvonne.
(10) result judgement：Whether for every a serum, each polypeptide in kit is counted respectively a response (that is, signal to noise ratio (SNR) is more than or equal to 2), and the criterion according to described in above-mentioned " diagnostic method " part is judged.That is,
When detecting the blood sample originated respectively from least two or more than two polypeptides in following polypeptides 1~11 to the subject and having response, judge that subject is SLE patient；Otherwise, it is determined that subject is not SLE patient.
Wherein, signal to noise ratio=(polypeptide point signal value-negative control point signal value)/negative control point signal value.Polypeptide point signal value refers to the chemiluminescence intensity value for the polypeptide point that imager software kit is read, and negative control point signal value refers to the chemiluminescence intensity value for the negative control point that imager software kit is read.
Embodiment 1
For 576 parts of serum samples, the method progress contrasting detection that the kit (the step of by above-mentioned 3) prepared in above-mentioned steps 2 and traditional Comprehensive Clinical are confirmed is respectively adopted, testing result is as shown in table 1.
Accuracy rate=(144+388)/576=92.4%；
Sensitivity=144/161=89.4%；
Specificity=388/415=93.5%；
False positive rate=27/415=6.5%
In this 576 parts of serum, made a definite diagnosis using SLE clinical symptoms, patient's SLE positive serum is 161, patient's SLE negative serum is 415；And kit diagnosis of SLE patients positive serum of the invention is used for 144, patient's SLE negative serum is 388, and accuracy rate is=92.4%, and sensitivity is=89.4%, specificity=93.5%, false positive rate=6.5%.It follows that mentioned reagent box and clinical diagnosis data are basically identical, it complies fully with the requirement as diagnostic kit, with good accuracy rate, sensitivity and specificity, it is adaptable to which the clinical examination center that SLE diagnosis is adapted to situation of all-level hospitals is used.
More than, more specific description is carried out to the present invention by embodiment, but be not the restriction to the technology of the present invention scope.By the record of this specification, those skilled in the art readily can be modified/change to present invention progress, and these are included in the technical scope of the present invention.
Claims (9)
1. one kind detection device, it includes solid carrier, and the following polypeptides being independently connected on the solid carrier：
SEQ ID NO：Polypeptide shown in 1；
SEQ ID NO：Polypeptide shown in 2；
SEQ ID NO：Polypeptide shown in 3；
SEQ ID NO：Polypeptide shown in 4；
SEQ ID NO：Polypeptide shown in 5；
SEQ ID NO：Polypeptide shown in 6；
SEQ ID NO：Polypeptide shown in 7；
SEQ ID NO：Polypeptide shown in 8；
SEQ ID NO：Polypeptide shown in 9；
SEQ ID NO：Polypeptide shown in 10；With
SEQ ID NO：Polypeptide shown in 11.
2. detection device according to claim 2, wherein, described solid carrier is iPDMS films.
3. detection device according to claim 1 or 2, it is used for the diagnosis of systemic loupus erythematosus.
4. a kind of systemic lupus erythematosus diagnosis kit, it includes one or more solid carriers, and the following polypeptides being connected on one or more of solid carriers：
SEQ ID NO：Polypeptide shown in 1；
SEQ ID NO：Polypeptide shown in 2；
SEQ ID NO：Polypeptide shown in 3；
SEQ ID NO：Polypeptide shown in 4；
SEQ ID NO：Polypeptide shown in 5；
SEQ ID NO：Polypeptide shown in 6；
SEQ ID NO：Polypeptide shown in 7；
SEQ ID NO：Polypeptide shown in 8；
SEQ ID NO：Polypeptide shown in 9；
SEQ ID NO：Polypeptide shown in 10；With
SEQ ID NO：Polypeptide shown in 11.
5. systemic lupus erythematosus diagnosis kit according to claim 4, wherein, the solid carrier is iPDMS films.
6. the systemic lupus erythematosus diagnosis kit according to claim 4 or 5, wherein, SEQ IDNO：Polypeptide shown in 1~11 is independently connected on same solid carrier.
7. purposes of following polypeptides in preparation system lupus erythematosus diagnosis kit or detection device：
SEQ ID NO：Polypeptide shown in 1；
SEQ ID NO：Polypeptide shown in 2；
SEQ ID NO：Polypeptide shown in 3；
SEQ ID NO：Polypeptide shown in 4；
SEQ ID NO：Polypeptide shown in 5；
SEQ ID NO：Polypeptide shown in 6；
SEQ ID NO：Polypeptide shown in 7；
SEQ ID NO：Polypeptide shown in 8；
SEQ ID NO：Polypeptide shown in 9；
SEQ ID NO：Polypeptide shown in 10；With
SEQ ID NO：Polypeptide shown in 11.
8. it is a kind of diagnose subject whether be Patients with SLE method, this method includes：
Systemic loupus erythematosus detection SEQ ID NO any one of detection device or claim 4~6 any one of usage right requirement 1~3：Whether the polypeptide shown in 1~11 has response to the blood sample that subject originates；Wherein,
When the blood sample that at least two or more than two polypeptides detected in following polypeptides 1~11 are originated to the subject has response, judge that subject is Patients with SLE；Otherwise, it is determined that subject is not Patients with SLE.
SEQ ID NO：Polypeptide shown in 1；
SEQ ID NO：Polypeptide shown in 2；
SEQ ID NO：Polypeptide shown in 3；
SEQ ID NO：Polypeptide shown in 4；
SEQ ID NO：Polypeptide shown in 5；
SEQ ID NO：Polypeptide shown in 6；
SEQ ID NO：Polypeptide shown in 7；
SEQ ID NO：Polypeptide shown in 8；
SEQ ID NO：Polypeptide shown in 9；
SEQ ID NO：Polypeptide shown in 10；With
SEQ ID NO：Polypeptide shown in 11.
9. method according to claim 8, wherein, the blood sample is whole blood, blood plasma or serum.
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