CN105784997B - Kit and detection method for detecting enterovirns type 71 IgM antibody - Google Patents
Kit and detection method for detecting enterovirns type 71 IgM antibody Download PDFInfo
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Abstract
The invention discloses the kit and detection method for detecting enterovirns type 71 IgM antibody.It includes one or more solid carriers, and the following polypeptide set 1 being independently connected on one or more of solid carriers.Detection method is, when the enterovirns type 71 antibody IgM in the blood sample that any one or more than one polypeptide detected in the polypeptide set 1 is originated to subject has response, to judge the subject as enterovirns type 71 infected patient.The kit accuracy of the present invention is good, and specificity is high, for detecting that enterovirns type 71 IgM antibody infects, and then for the auxiliary diagnosis of hand-foot-and-mouth disease.
Description
Technical field
The invention mainly relates to Measurement for Biotechnique and medical science.It specifically, the present invention relates to detection
The kit and detection method of enterovirns type 71 IgM antibody.
Background technology
The infection of enterovirus EV 71 type is the general name of the disease as caused by human enterovirus 71 (EV71), clinically mainly
Hand-foot-and-mouth disease is shown as, many eruption and prevalences in infant of hand-foot-and-mouth disease, part EV71 infection infants show as herpetic isthmus faucium
There is viral encephalitis, viral cerebrospinal meningitis, pulmonary edema, empsyxis etc. in inflammation, patient with severe symptoms.Cause the disease of hand-foot-and-mouth disease
Original, most commonly coxsackie virus A 16 and enterovirns type 71;Severe occurs for the disease caused by enterovirns type 71 infection
The large percentage of infection, case fatality rate is also higher, and severe cases case fatality rate is up to 10%-25%, therefore etiological diagnosis meaning weight
Greatly.
Enterovirns type 71 (EV71) genome is the single-stranded positive RNA containing about 7.5Kb, including 5' ends non-coding
Area, 3' ends noncoding region and by Pl, P2, the polyprotein that P3 is constituted encodes 11 kinds of albumen altogether, wherein 4 structural proteins VPl,
VP2, VP3, VP4,7 NS2 Proteins A, 2B, 2C, 3A, 3B, 3C, 3D.Wherein VP 1, VP 2, VP 3 are outer exposed to virus
The surface of shell, is the main antigenic variation positions of EV71;The inner side that VP4 is embedded in virus coat is combined with internal RNA, its work(
The anchor of the similar virus coat of energy, the structure to stablize virus protein.NS2 Protein A is division pioneer at initial stage protein,
It is relevant with terminating albumen needed for host cell synthesizes itself;3C then participates in most protein cleavage reaction;3D polymerize for RNA
Enzyme, transcription and replication reaction are dominated when viral RNA is replicated;Four kinds of albumen such as 2B, 2C, 3A, 3B are then replicated with viral RNA
Close.
Three aspects of clinical diagnosis Main Basiss of enterovirus EV 71:One is clinical manifestation, and two be serological evidence, three
It is Etiological Evidence.Due to enterovirus EV 71 type infection clinical manifestation it is many, therefore the Serologic detection of enterovirus EV 71 and
Pathogeny detection is current topmost foundation.
The detection of enterovirns type 71 IgM antibody has had pertinent literature report, substantially to use colloidal gold method or enzyme more
Linked immunosorbent assay, still has many weak points in terms of cost, specificity.Protein-chip due to technology it is advanced into
The trend developed for technology, but in the market is to have no the enterovirns type 71 IgM antibody inspection based on protein-chip method
The report of test agent box.
The content of the invention
Good it is an object of the invention to provide a kind of accuracy, specificity is high to be used to detect that enterovirns type 71 IgM resists
The kit and detection method of body.
Current inventor provides 4 polypeptides.It is surprising that the inventors discovered that, although 4 polypeptides are individually examined
Sensitivity when surveying the IgM antibody in hand-foot-and-mouth disease infection is not up to used as between 19.7%~78.5%, much to be detected
The requirement of instrument, still, the biological specimen that the present invention is originated with subject whether at least one in 4 polypeptides or
One or more have response as judgement subject whether by the index of Infected With Plasmodium in the case of, can be to be up to 93.0%
Sensitivity (false positive rate as little as 1.9%) detection enterovirns type 71 IgM antibody infection, and then for the auxiliary of hand-foot-and-mouth disease
Diagnosis.
Therefore, the present invention includes:
1. a kind of kit for being used to detect enterovirns type 71 IgM antibody, it includes one or more solid carriers, with
And it is independently connected to following polypeptide set 1 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;With
SEQ ID NO:Polypeptide shown in 4.
2. kit according to claim 1, it is used to detect enterovirns type 71 antibody IgM.
3. kit according to claim 1, wherein, whole polypeptides are independently connected on same solid carrier.
4. following polypeptide set 1 are preparing the purposes in being used to detect the kit of enterovirns type 71 antibody IgM:
SEQ ID NO:Polypeptide shown in 1;
SEQ ID NO:Polypeptide shown in 2;
SEQ ID NO:Polypeptide shown in 3;With
SEQ ID NO:Polypeptide shown in 4.
5. it is a kind of detect subject whether be enterovirns type 71 infected patient method, this method includes:
The polypeptide in the kit detection polypeptide set 1 any one of usage right requirement 1~3 is to subject
Whether the enterovirns type 71 antibody IgM in the blood sample in source has response;
When in the blood sample that any one or more than one polypeptide detected in the polypeptide set 1 is originated to subject
Enterovirns type 71 antibody IgM when having response, judge the subject as enterovirns type 71 infected patient.
6. method according to claim 5, wherein, the blood sample is whole blood, blood plasma or serum.
7 methods according to claim 5, wherein, the subject is with the tested of hand-foot-and-mouth disease clinical symptoms
Person.
The embodiment of invention
Polypeptide set
In this specification, polypeptide set 1 refers to whole polypeptides positioned at the first detection zone, and the polypeptide set 1 is comprising only
Vertical following polypeptides:
SEQ ID NO:Peptide sequence shown in 1 is APTGQNTQVSSHRLDTGKVP;
SEQ ID NO:Peptide sequence shown in 2 is HPYVLDAGIPISQLTVCPHQ;
SEQ ID NO:Sequence shown in 3 is polypeptide DKFANPVKDIFTEMAAPLKS;With
SEQ ID NO:Sequence shown in 4 is polypeptide DPDHFDGYKQQVVTVMDDLC.
The polypeptide can be obtained suitably using the known method such as (1) chemical synthesis process or (2) enzyme reaction synthetic method
, wherein chemical synthesis is more easy.Chemical synthesis the present invention polypeptide in the case of, by using peptide synthesizer synthesis or
The semi-synthetic polypeptide is carried out.As chemical synthesis process, it can include such as peptide solid-phase synthesis.The peptide so synthesized
It 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, such as International Publication pamphlet can be used
Method described in No. WO2004/011653.I.e., it is possible to so produce:By the amino acid of a side or the carboxyl terminal quilt of dipeptides
The amino acid of amino acid or dipeptides and amino acid in free state obtained from esterification or amidatioon be (such as carboxy protective
Amino acid) reacted in the presence of peptide synthetase, the dipeptides or tripeptides of generation.As peptide synthetase, it can include:Tool
There are the culture, the microbial cells that are separated by the culture or the thalline of the microorganism of the microorganism of the ability of generation peptide
Manage thing or the microbe-derived peptide synthetase.
The chemical synthesis of particularly polypeptide have been realized in commercialization, can by entrust specialty Peptide systhesis company come
Synthesize the polypeptide.
Kit
The present invention provides a kind of detection kit (kit of the invention), and it includes one or more solid carriers, with
And it is independently connected to following polypeptide set 1 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;With
SEQ ID NO:Polypeptide shown in 4.
In this specification, sensitivity refers to:In positive sample with the confirmation of " goldstandard " method, it is determined as by other method
The ratio of positive sample.False positive rate refers to:In negative sample with the confirmation of " goldstandard " method, sun is determined as by other method
The ratio of property sample." goldstandard " method of the art detection human enterovirus 71 infection is nucleic acid detection method, i.e. PCR
Method.In this specification, " detection " can be made a definite diagnosis or to make a definite diagnosis offer reference information.
In the present invention, solid carrier is not particularly limited, as long as solid or insoluble material, (being, for example, can
Material to be separated by filtering, precipitation, Magnetic Isolation etc. from reactant mixture) carrier.
The material for constituting solid carrier includes but is not limited to:Silica gel (dimethyl silicone polymer, PDMS), cellulose, Teflon
Grand TM, NC Nitroncellulose, agarose, glucan, chitosan, polystyrene, polyacrylamide, polyester, makrolon, polyamides
It is amine, 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 they
Combination etc..
The shape of solid carrier includes but should not be limited to:Pearl, magnetic bead, film, microcapillary, filter membrane, plate, micro plate, carbon
Nanotube, sensor chip etc..Just as known in the art, it can be set on the flat solid carrier such as film or plate
Put pit, groove, filter membrane bottom etc..
Magnetic bead can have the sphere diameter of about 25nm~about 1mm scopes.In a preferred embodiment, magnetic bead has about
The diameter of the μ m of 50nm~about 10.The size of magnetic bead can be selected according to specific purposes.It is contour by Sepharose
The pearl that crosslinked spherical agarose is made has the diameter of about 24 μm~about 165 μ ms.Preferably, high crosslinked spherical agarose
Pearl has the diameter of about 24 μm~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 includes can be from Polysciences, Warrington, PA or
The polystyrene latex beads such as 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 nucleic acid (for example
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 magnetic bead, can include Polysciences, Warrington, the pearl (title of PA sale:Biomag (registrars
Mark) carboxyl) or Bangs Laboratory, Inc., Fishers, IN entitled MC02N/2928 pearl, Huo Zheke
To use Dynal Biotech M-270 sold etc..
In a preferred embodiment, the solid carrier is SJ modified silica-gels, and it is that Yvonne poly- biomaterials in Suzhou have
A kind of microarray solid support material (the iPDMS films, referring to Chinese patent of silicon rubber material of limit company exploitation
CN101265329A).This material be by biological study commonly use PDMS based on, add wherein specific initiator into
Divide (material surface-functionalized modification can be realized by surface initiated polymerization (SIP)), then by polyethylene glycol methyl
What acrylate (poly (oligo (ethylene glycol) methacrylate), pOEGMA) surface modification was obtained.SJ changes
Property silica gel has outstanding anti-protein non-specific adsorption (Nonspecific protein adsorption, NPA) ability,
Non-specific protein absorption during can complicated protein immunization be detected is controlled to close to " absolute 0 " level (is near or below
The detectable limit of instrument), it can not only exempt closing and the trouble being cleaned multiple times, can also be by using stronger amplification of signal
Means improve the sensitivity of protein microarray.And the essence of its silicon rubber impart the stronger mechanical performance of the material and
Good operability.SJ modified silica-gels are successfully applied to many of 11 tumor markerses composition by the poly- companies of Suzhou Yvonne
Index joint inspection microarray ELISA kit, 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 pass through control
The modification reaction time adjusts its surface topography within the specific limits.
The connection of polypeptide and solid carrier can be using well known to a person skilled in the art the connection of polypeptide and solid carrier
Method is carried out.For example, for the connection on protein/polypeptide and modified silica-gel surface, 1- ethyl -3- (3- can be passed through
Dimethyl aminopropyl)-carbodiimides [1-ethyl-3- (3-dimethyl ami-nopropyl) carbodiimide, EDC]
Reaction with n-hydroxysuccinimide (N-hydroxysuccinimide, NHS) is by the macromolecular chain of modified silica-gel surface
Carboxyl (- COOH) group is changed to activated group, the activated group can with protein/polypeptide institute's band amino (- NH2) reaction so as to
Protein/polypeptide is fixed on solid carrier surface by realization.
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 normal
Rule selection, the μ g/mL of preferably 1 μ g~1000 μ g/mL, more preferably 10 μ g~500.In addition, dividing on a solid support for polypeptide
The density of cloth is not particularly limited, and those skilled in the art can be according to conventional selection, preferably 1~100 point/10mm2, more preferably
5~50 points/10mm2。
In this manual, " independent " refers to:The polypeptide forms a point on the solid carrier, is not wrapped in the point
Containing other polypeptides on the influential amount of detection, the polypeptide is preferably only included.
Solid carrier in the kit of the present invention can be one or multiple, preferably two or more.
The kit can also include:
1. the serum dilution or serum dilution component solution that prepare:Serum dilution, for example, have Beijing to match biology of speeding
Sample dilution (production code member 070021-S2), the sample-adding of Zhengzhou Bo Weijia bio tech ltd of Science and Technology Ltd.
Change colour Sample dilution (production code member bwj010103) etc..The serum dilution is used for dilute serum, the serum of kit detection
Dilute suitable multiple, preferably such as 2~200 times, 10~100 times.
The kit can also include:
2. concentrate washing lotion:Solid carrier surface is incubated after serum and ELIAS secondary antibody, and solid carrier table need to be washed with washing lotion
Face uncombined antibody and ELIAS secondary antibody.Concentration washing lotion be, for example, 1% the polysorbas20 aqueous solution, need to be diluted when using 2~40 times,
It is preferred that 5~20 times.
The kit can also include:
3. ELIAS secondary antibody solution:IgM and solid carrier (such as SJ modified silicons in human enterovirus 71 infected person anteserum
Glue) on polypeptide set I in polypeptide combine, ELIAS secondary antibody can be combined with IgM, and the label on ELIAS secondary antibody can be with lighting
Substrate reactions, so as to send detectable light.ELIAS secondary antibody can be the goat-anti people IgM of such as horseradish peroxidase-labeled.
Concentration of the ELIAS secondary antibody in ELIAS secondary antibody solution is not particularly limited, can be a 1ng~1000ng/mL.
The kit can also include:
4. luminescent solution component solution:Luminescent solution can react with the horseradish peroxidase marked on ELIAS secondary antibody so that anti-
The detectable chemical light of instrument should be sent.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.Usually using hydrogen peroxide and one
The mixed aqueous solution for planting hydroxide bases is used as exciting agent.Under horseradish peroxidase enzyme catalytic, decomposing hydrogen dioxide solution be oxygen and
Water:
2H2O2→O2+2H2O
Luminol generates a pairs of anion when being reacted with hydroxide, the oxygen oxygen that it can be decomposited by hydrogen peroxide
Change, product is an organic peroxide.The peroxide is very unstable, and nitrogen is decomposited immediately, generates the 3- ammonia of excitation state
Base phthalic acid.During excitation state is converted to ground state, the energy of release exists in the form of photon, and wavelength is located at the indigo plant of visible ray
Light part.The example of luminescent solution component solution such as Thermo Seientific companiesELISA
Femto Maximum Sensitivity Substrate, article No. 37074.
The kit can also include:
5. the biological incubation reactor more than one or two, according to demand can be using for example two-sided biological incubation
Reactor, Chinese patent ZL 201120177686.3 or ZL201110142518.5;Or the biological incubation reactor of one side
ZL201220430886.x。
The kit can also include:
6. other are used for the detection molecules for detecting human enterovirus 71 infection (such as polypeptide, protein, nucleic acid).
The kit can also include:
7. operation instructions.
The kit can also include:
8. the positive quality control point being connected on one or more of solid carriers or other independent solid carriers (can
Positive quality control point is used as using e.g. people IgM) and/or negative Quality Control point (such as phosphate buffer, abbreviation PB).
Detection method
In another aspect, the present invention provide it is a kind of detect subject whether be human enterovirus 71 infection method
(detection method of the invention), this method includes:
The blood sample that the polypeptide in the polypeptide set I is originated to subject is detected using the kit of the invention described above
In enterovirns type 71 antibody IgM whether have response;
When in the blood sample that any one or more than one polypeptide detected in the polypeptide set 1 is originated to subject
Enterovirns type 71 antibody IgM when having response, the subject is judged as enterovirns type 71 infected patient, so that for brothers
The auxiliary diagnosis of stomatosis.
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 is believed
Number 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 is met
State criterion;Otherwise it is negative reaction.
The blood sample can be whole blood, blood plasma or serum.
In one preferred embodiment, the subject can be the subject with hand-foot-and-mouth disease clinical symptoms.
In the present invention, hand-foot-and-mouth disease clinical symptoms, which are embodied in the positions such as heating and hand, foot, oral cavity, fash or bleb.
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 has been carried out really by mass spectrum
Recognize.Wherein,
SEQ ID NO:1 (APTGQNTQVSSHRLDTGKVP), molecular weight is 2093;
SEQ ID NO:2 (HPYVLDAGIPISQLTVCPHQ), molecular weight is 2189;
SEQ ID NO:3 (DKFANPVKDIFTEMAAPLKS), molecular weight is 2223;
SEQ ID NO:4 (DPDHFDGYKQQVVTVMDDLC), molecular weight is 2325;
2. the preparation of kit
Detection chip is, for solid support material, to fix many by point sample thereon with SJ modified silica-gels (iPDMS films)
Peptide solution is prepared from.SJ modified silica-gels are that with olefin-terminal, surface is added in traditional polydimethyl siloxane material
The initiator of initiated polymerization, and pass through three-dimensional structure of the heat cross-linking (si-h bond bonding) fixed to dimethyl silicone polymer
In, obtain a kind of new material i.e. SJ modified silica-gels.Its manufacturing process is referring to International Patent Publication WO2014/044184.
The SJ modified silica-gels film made can be stored in 4 DEG C of refrigerators.
Using brilliant core16 people's point sample instruments of PersonalArrayerTM prepare polypeptide microarrays (i.e. on modified silica-gel
Detection chip), process is:
1) pre-process
By SJ modified silica-gel thin slices (15 × 15mm2) be immersed in activating solution, taking-up elutes 3 with deionized water after 30min
It is secondary, dried up with nitrogen, be 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 in point sample instrument
On 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
For room temperature (25 DEG C), humidity set is 50%.Following polypeptides are put into microarray, polypeptide set I on SJ modified silica-gel thin slices
In each polypeptide one point of point sample, in addition one people IgM point of point sample be used as negative matter as positive quality control point and a PB point
Control 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 set 1 of point sample is specific as follows on the modified silica-gel thin slice of kit:
SEQ ID NO:Polypeptide shown in 1;
SEQ ID NO:Polypeptide shown in 2;
SEQ ID NO:Polypeptide shown in 3;With
SEQ ID NO:Polypeptide shown in 4;
3) chemistry is fixed
The polypeptide microarrays just made will be placed in climatic chamber (26 DEG C, 60% humidity) fixed at least 6h.Chemistry is solid
Process is determined referring to International Patent Publication WO2014/044184.
The buffer solution point for capturing peptide molecule will be included on modified silica-gel film by point sample instrument first, then buffered
Liquid starts evaporation, and capture peptide molecule and the surface intimate contact of SJ modified silica-gels simultaneously interacts, and passes through chemical bond, modified silicon
Ploy (OEGMA) high molecular end-COOH on glue surface and peptide molecule-- NH2Stable covalent bond is formed, and then will be had
Chemically active peptide molecule is fixed on SJ modified silica-gels surface.
4) assemble
Fixed 6h polypeptide microarrays must be assembled in two days.SJ modified silica-gel thin slices are attached to by gum first
On special reaction column, reaction cavity is covered.Biological incubation reactor can use two-sided biological incubation reactor according to demand,
That is, one reactor is made up of two reaction columns and a reaction cavity, or one side biological incubation reactor, i.e. one anti-
Device is answered to be made up of a reaction column 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
Checking procedure
1) temperature of reagent is balanced:All reagents are balanced to room temperature.
2) preparation of washing lotion:Washing lotion purified water will be concentrated or distilled water presses 1:9 dilutions, for example:450mL purified waters or steaming
50mL concentration washing lotions are added in distilled water, are fully mixed.The washing lotion being not used is placed on 2~8 DEG C of preservations, can preserve 3 months.
3) dilution of sample:Testing sample is pressed 1 with serum dilution:100 dilutions are (such as:2 μ L serum are added to 198 μ L
Serum dilution, is fully mixed).Sample detection needs the μ L of sample 200 after diluting.
4) wetted chip:Take out the biochip incubation reaction device needed for detection.1mL washing lotions are added, chip surface 3 is infiltrated
Minute.Discard the washing lotion of immersion chip.
5) it is loaded:In the present embodiment, it is loaded with one side biological incubation reactor, it is to be measured by what is diluted using liquid-transfering gun
Sample is added in one side biological incubation reactor by liquid feeding passage (any one in two), and each one side biological incubation is anti-
Device plus 200 μ L are answered, liquid feeding passage (two) is closed with sealing paste.
6) sample incubation:The one side biological incubation reactor for adding sample is placed in 150rpm on horizontal shaker, is incubated at room temperature
30 minutes.
7) clean:Reaction column is taken out from the reaction cavity of one side biological incubation reactor, liquid in reactor is discarded,
Rinse repeatedly inside reaction column top chip surface and reaction cavity 3 times (about using 12mL washing lotions every time, rinse 1-2 minutes).
8) enzyme labelled antibody is incubated:Reaction column is reconfigured with reaction cavity, clamping.Throw off sealing paste, each one side life
Thing incubation reaction device adds the μ L of goat-anti people IgM solution 200 of horseradish peroxidase-labeled.Again led to sealing paste closing liquid feeding
Stomata.150rpm on shaking table is placed in, is incubated at room temperature 30 minutes.
9) clean again:Reaction cavity is discarded, chip surface is carefully cleaned with washing lotion 3 times.
10) luminescent solution is added:Each chip surface is separately added into 30 μ L luminescent solutions, luminous liquid energy is uniformly laid on chip
Surface
11) chip is imaged:The chip chemiluminescence image analysis system for adding luminous substrate liquid liquid is entered to chip
Row exposure is imaged for 1 minute.During as chemiluminescence image analysis system, it is not particularly limited, it is micro- using such as day 5500 types of energy
The prompt board TN5500 type Microarray image instrument of array image-forming instrument, Yvonne or the prompt board CLEAR4000 type Microarray image instrument of Yvonne etc..
Result judgement:For every a serum, whether each polypeptide counted respectively in kit has response (that is, to believe
Make an uproar and be more than or equal to 2 than (SNR)), and criterion according to described in above-mentioned " detection method " part judged.That is,
When detect in the polypeptide in the polypeptide set 1 any one or more than one to subject originate blood sample
Enterovirns type 71 antibody IgM in this has response, judges the subject as enterovirns type 71 infected patient, i.e. hand-foot-and-mouth disease
Infected 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 imaging
The chemiluminescence intensity value for the negative control point that instrument software kit is read.
Embodiment
To being collected into 332 parts of clinical serum, using the present invention kit testing result with using traditional goldstandard method-
The testing result of nucleic acid detection method (using Takara fluorescent quantificationally PCR detecting kit) is compared, as a result such as the institute of table 1
Show.
The ratio of the kit and the fluorescent quantificationally PCR detecting kit using Takara of the present invention of table 1
Accuracy rate=(159+158)/332 × 100%=95.5%
Sensitivity=159/171 × 100%=93.0%
Specificity=158/161 × 100%=98.1%
False positive=3/161 × 100%=1.9%
Result above shows that the present invention is used to detect that the infection of hand enterovirns type 71 has good accuracy rate, sensitivity
And specificity, it is adaptable to the aetology laboratory auxiliary detection of enterovirns type 71 infection, and it is simple to operate, it is adapted to medical treatment at different levels
Used with disease control department.
As a comparison, for any bar in above-mentioned 4 polypeptides, implement according to International Patent Publication WO2014/044184
The description of example division header 4 is prepared for detecting device, using above-mentioned 332 parts of serum samples, according to International Patent Publication WO2014/
The description of the title 3 of 044184 embodiment part is detected, in the case of SNR=2, as a result shows above-mentioned 4 polypeptides
Individually detection hand-foot-and-mouth disease infection in IgM antibody when sensitivity between 19.7%~78.5%, (being shown in Table 2) can not expire
The sufficient requirement as detection kit.
Sensitivity results unit of 24 polypeptides of table in 332 parts of serum:%
Sequence number | 1 | 2 | 3 | 4 |
IgM sensitivity | 35.2 | 78.5 | 33.6 | 19.7 |
More than, more specific description is carried out to the present invention by embodiment, but be not the limit to the technology of the present invention scope
It is fixed.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 (4)
1. a kind of kit for being used to detect enterovirns type 71 IgM antibody, it includes one or more solid carriers, and solely
On the spot it is connected to following polypeptide set 1 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;With
SEQ ID NO:Polypeptide shown in 4.
2. kit according to claim 1, it is used to detect enterovirns type 71 antibody IgM.
3. kit according to claim 1, wherein, whole polypeptides are independently connected on same solid carrier.
4. following polypeptide set 1 are preparing the purposes in being used to detect the kit of enterovirns type 71 antibody IgM:
SEQ ID NO:Polypeptide shown in 1;
SEQ ID NO:Polypeptide shown in 2;
SEQ ID NO:Polypeptide shown in 3;With
SEQ ID NO:Polypeptide shown in 4.
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CN108663517A (en) * | 2017-03-31 | 2018-10-16 | 深圳市儿童医院 | It can be used for diagnosing the kit of human enterovirus infection |
CN107255713B (en) * | 2017-03-31 | 2019-01-08 | 深圳市儿童医院 | It can be used for diagnosing the kit of human enterovirus infection |
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CN101891805A (en) * | 2010-05-18 | 2010-11-24 | 北京凯悦宁科技有限公司 | Human enterovirus 71 type specific polypeptide and application thereof |
CN102351947A (en) * | 2011-10-17 | 2012-02-15 | 湖北新纵科病毒疾病工程技术有限公司 | Ev71 early diagnosis method and reagent kit |
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CN103539839A (en) * | 2012-07-13 | 2014-01-29 | 厦门大学 | Neutralizing epitope peptide of enterovirus 71-type VP2 antigen and application thereof |
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CN102702348A (en) * | 2010-06-09 | 2012-10-03 | 财团法人卫生研究院 | Monoclonal antibodies against Enterovirus 71 and uses thereof |
CN102351947A (en) * | 2011-10-17 | 2012-02-15 | 湖北新纵科病毒疾病工程技术有限公司 | Ev71 early diagnosis method and reagent kit |
CN102558313A (en) * | 2012-01-12 | 2012-07-11 | 东南大学 | Enterovirus 71 type specific recombinant protein antigen and application thereof |
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