CN104804081B - For detecting the monoclonal antibody and kit of hand-foot-and-mouth disease enterovirus albumen - Google Patents
For detecting the monoclonal antibody and kit of hand-foot-and-mouth disease enterovirus albumen Download PDFInfo
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Abstract
The present invention relates to the ELISA detection kits for detecting the monoclonal antibody of hand-foot-and-mouth disease enterovirus albumen and being prepared using the monoclonal antibody specific.The monoclonal antibody of anti-hand-foot-and-mouth-disease enterovirus albumen of the present invention be by preserving number be CCTCC NO:The hybridoma cell strain HEV71 vP1 4G12 of C2013161 are generated, the Serial No. SEQ ID NO in energy specific recognition HEV71 VP1 albumen:1 amino acid sequence.The ELISA detection kit of the present invention for being used to detect hand-foot-and-mouth disease enterovirus albumen, include ELISA ELISA Plates, capture antibody, envelope antigen, ELIAS secondary antibody, streptavidin horseradish peroxidase, developing solution, dilution, terminate liquid, wherein, the capture antibody is monoclonal antibody as described in the present invention, and the coated antibody is anti-HEV71 VP1 polyclonal antibodies.The kit provides quick, special, sensitive detection method for hand-foot-and-mouth disease enterovirus albumen.
Description
Technical field
The invention belongs to biological technical fields, are related to antibody engineering technology, and in particular to for detecting hand-foot-and-mouth disease enteron aisle
The monoclonal antibody of virus protein and the ELISA detection kit prepared using the monoclonal antibody specific.
Background technology
Hand-foot-and-mouth disease（Hand foot and mouth disease, HFMD）It is a kind of common as caused by enterovirus
Children's infectious disease also known as eruptive vesicular stomatitis, mostly occur in 3 years old and 3 years old Infants Below child, can cause hand, foot, mouth
The bleb or fash at the positions such as chamber, a small number of infants can cause the complication such as myocarditis, pulmonary edema, aseptic meningitis.Individually
If children with serious disease progression of the disease is fast, death is easily led to.The disease formed using after hand, foot and oral mucosa bleb or ulceration ulcer as
Main clinic symptoms.Hand-foot-and-mouth disease is the infectious disease as caused by enterovirus, and infectiousness is strong, and route of transmission is complicated, is propagated soon,
It can cause to be very popular in a short time.Triggering the enterovirus of hand-foot-and-mouth disease has more than 20 kinds（Type）, wherein with Coxsackie virus
A16 types（Cox A16）And enterovirns type 71（EV71）It is most commonly seen.
For the Virus Type of HFMD, Common Diagnostic Methods is in serology and experiment, aetology cell inoculation, divide at present
Sub- biology reverse transcription polymerase chain reaction（RT-PCR）Or real-time RT-PCR（rRT-PCR）Deng.These methods are not
Same field plays corresponding important function, but also exposes some problems accordingly：These methods are all comparatively laborious and right
Corresponding reagent and clinical sample demand are larger, and pharynx swab and anus swab etc. specimen components are complicated, and virus load is low, easily occur
False negative, that in addition detects is costly, is also easy to produce cross reaction, and professional person is needed to be operated, otherwise it is difficult to ensure that most
The accuracy of termination fruit.
The content of the invention
In order to overcome problem above and meet the market needs of detection hand-foot-and-mouth disease, it is an object of the invention to provide one
Kind for detecting the monoclonal antibody of hand-foot-and-mouth disease enterovirus albumen.
It is of the present invention be for the monoclonal antibody that detects hand-foot-and-mouth disease enterovirus albumen by preserving number be CCTCC
NO:The hybridoma cell strain HEV71-vP1-4G12 of C2013161 is generated, the sequence in energy specific recognition HEV71-VP1 albumen
Number be SEQ ID NO:1 amino acid sequence.
Hybridoma cell strain HEV71-vP1-4G12 of the present invention, preserving number are CCTCC NO:C2013161 is protected
Tibetan unit is China typical culture collection center（CCTCC）, address is Wuhan University of Wuhan City of Hubei China province；Preservation date
For on October 17th, 2013.
The present invention is that the epitope of the monoclonal antibody is determined by indirect elisa method.Exempted from by recombinant protein
The monoclonal antibody that epidemic disease obtains, detection derive from 7 groups of difference peptide fragments of HEV71-VP1 protein sequences, this 7 groups of difference peptide fragments pass through
The Bioinformatics Predictions epitope such as interaction of molecules power, hydrophobicity principle statistics draws and by DNASTAR,
The prediction of the bioinformatics softwares such as SWISSMODEL is drawn.From chromogenic reaction, the epitope of antibody of the present invention is at it
In second group of peptide section sequence in（Gly-Glu-Ile-Asp-Leu-Pro-Leu-Lys-Gly-Thr-Thr-Asn-Pro-Asn-
Gly-Tyr-Ala-Asn-Tr p-Asp-Ile-Asp）, which is SEQ ID NO:1.
The further feature of monoclonal antibody according to the present invention, the monoclonal antibody energy specific recognition SEQ
ID NO:5 to 22 amino acid in 1 amino acid sequence.It is 5 amino acid since epitope identification sequence is most short, and
SEQ ID NO:1 amino acid sequence overall length be 22 amino acid, it is known that the epitope of the monoclonal antibody the amino acid sequence 5 to
Among 22 amino acid.
The present invention further provides a kind of for detecting the ELISA detection kit of hand-foot-and-mouth disease enterovirus albumen.
The ELISA detection kit of the present invention for being used to detect hand-foot-and-mouth disease enterovirus albumen, includes ELISA enzymes
Target, capture antibody, envelope antigen, ELIAS secondary antibody, streptavidin-horseradish peroxidase, developing solution, dilution, terminate liquid,
Wherein, the capture antibody is monoclonal antibody as described in the present invention, and the coated antibody is anti-HEV71-VP1 Anti-TNF-αs
Body.
The further feature of ELISA detection kit according to the present invention, the optimal dilution of the monoclonal antibody
Concentration is 100 to 200ng/ml, and the optimal coating concentration of the polyclonal antibody is 100ng/ml, the streptavidin-horseradish
The optimal diluted concentration of peroxidase is 1 to 100ng/ml.
The advantages and features of the present invention is：
（1）Since the extraction comparison of hand-foot-and-mouth disease enterovirus albumen in itself is difficult, the present invention constructs Enterovirus 71
The HEV71-VP1 fusion proteins of type, and prepare the monoclonal antibody of the antigen.The monoclonal antibody is by preserving number
The hybridoma cell strain secretion of C2013161 can specifically identify the special epitope of HEV71-VP1 protein sequences, i.e. sequence
Row number is SEQ ID NO:1 amino acid sequence.Our experiments show that monoclonal antibody of the present invention is to enteron aisle type virus 71
Type VP1 albumen has higher affinity, can preferably meet the requirement to hand-foot-and-mouth disease reagent for clinical diagnosis, is also brothers mouthful
The exploitation of sick reagent for clinical diagnosis provides good technical foundation.
（2）It is of the present invention to be used to detect hand-foot-and-mouth disease Enterovirus 71 in above-mentioned recombinant antigen and monoclonal antibody
The ELISA detection kit of type needs to provide quick, special, sensitive detection method, the party for the clinical difference with practice
Method is of less demanding to instrument and equipment, and cause of disease examination is carried out suitable for community hospital.
Description of the drawings
Fig. 1 is the specificity identification figure of monoclonal antibody of the present invention, and colour developing represents there is spy for HEV71-VP1
The opposite sex.
Fig. 2 is knot of the ELISA detection kit of the present invention using the IgM in Dot hybridization detection person under inspection's serum
Fruit.
Specific embodiment
Embodiment 1：The preparation of HEV71-VP1 antigens
（1）The structure of pET28a-HEV71-VP1 recombinant vectors
According to the DNA sequence dna of the people HEV71-VP1 provided in Genbank（AB524277）, the artificial synthesized sequence removes
N-terminal signal peptide designs pair of primers, and by the 5 of two primers, ' end introduces NdeI+XhoI restriction enzyme sites respectively, uses Standard PCR
Method expands to obtain the HEV71-VP1 genes of overall length 891bp.The HEV71-VP1 protein sequences of the gene expression are 297 amino
Acid.
The target gene of HEV71-VP1 is obtained by PCR amplification, by carrier pET-28a and by agarose gel purification
HEV71-VP1 genetic fragments, carry out double digestion processing with NdeI+XhoI, with T4DNA ligases will after purification digestion products connect
It connects, obtains recombinant plasmid pET-28a-HEV71-VP1, and connection product conversion is containing ammonia benzyl green grass or young crops into bacillus coli DH 5 alpha
Selected clone on the LB tablets of mycin, prepares plasmid in a small amount, goes out positive colony, sequencing result by double digestion/PCR evaluation and screenings
Show that HEV71-VP1 segments and the sequence designed of restructuring are completely the same.
（2）The expression of HEV71-VP1 recombinant proteins
For HEV71-VP1 plasmids after sequence verification, conversion enters Escherichia coli（BL21）, in the LB containing ampicillin
It is cultivated in culture medium, positive colony can be selected on LB tablets and carries out plasmid enzyme restriction identification, prepared plasmid in a small amount, use double digestion
PCR evaluation and screenings go out positive colony, final to obtain the recombinant plasmid engineering bacteria containing HEV71-VP1.
In expression, by the recombinant plasmid engineering bacteria of HEV71-VP1 in the LB culture mediums containing 100 μ g/ml ampicillins
Middle culture between A600 reaches 0.5-0.6, then adds in the Isopropyl β-D-1- of final concentration of 0.5mM
thiogalactopyranoside（IPTG）4h, the bacterium solution 4 after the completion of inducing are induced in 37 DEG C, 000rpm centrifugation 10min are received
Collect thalline, and precipitation is washed with PBS；PBS is resuspended precipitation and is placed in ice bath, and 12000rpm centrifuges 20min after carrying out ultrasonic bacteria breaking, on
Cleer and peaceful precipitation carries out SDS-PAGE electrophoresis respectively, the results showed that：The HEV71-VP1 recombinant proteins of expression are endochylema insolubility table
It reaches, which is named as BL21 (DE3), molecular weight is 45kDa or so.
（3）The purifying of HEV71-VP1 recombinant proteins and quantitative
The thalline that great expression is obtained, centrifuges after ultrasonication, then carries out inclusion body washing, and GE is used after the completion of washing
The His Trap FF purification columns of Healthcare companies purify albumen（According to product description carry out preparation of reagents and
Purifying）.The albumen finally obtained is analyzed with SDS-PAGE electrophoresis, and measuring its concentration with BCA protein quantification kits is
0.23mg/ml。
Embodiment 2：The preparation of the anti-HEV71-VP1 monoclonal antibodies of mouse
（1）The foundation of the anti-HEV71-VP1 hybridoma cell strains of mouse and the identification of monoclonal antibody hypotype
A. immune programme uses 4 fundamental immunities and 1 booster immunization.Select 8 week old, weight 18g or so and health
Female BAl BIc/c mouse 2 after adaptability is raised 1 week, gathers negative blood and is used as control；
B. intermediate range immunization protocol is used（0.3ml/, 2 weeks/time）, during first immunisation（50 μ g/ are only）By immunogene and wait bodies
Hereafter long-pending Freund's complete adjuvant stirring and emulsifying, dorsal sc multi-point injection are not exclusively helped by immunogene and isometric Freund
Agent stirring and emulsifying carries out routine immunization；
C.3 when secondary immune, general 50 μ g antigen+TiterMax mixed in equal amounts emulsifies back part multi-point injection, and effect is surveyed after 7 days
Valency.Mouse titers substantially reach booster immunization after certain requirement, and booster immunization is not added with adjuvant, and booster immunization dosage is 50 μ g, is added
It is strong immune 3 days latter, eyeball blood sampling is plucked, separates serum keeping.Simultaneously spleen is taken to be merged.It is immunized with identical immunization method
SPF grades of new zealand rabbits, the polyclonal antibody of acquisition cardiac blood clearly after purification is as capture antibody.
D. during cell fusion, splenocyte and myeloma cell are pressed 4:1 or so is mixed, and in polyethylene glycol（PEG,
Molecular weight is 1450）Rush melt and merged under effect, fused cell is cultivated in HAT selectivity culture solution again, after 10 days
The positive hybridoma cell that can be reacted with HEV71-VP1 albumen, and the sun that primary dcreening operation is obtained are filtered out by indirect ELISA method
Property hybridoma expand culture, carry out label protein two days later（His-tag）The exclusion of hybridoma goes out to be directed to secondary screening
The hybridoma of HEV71-VP1 albumen rather than label；
E. the positive hybridoma cell of acquisition is continuously subcloned more than at least twice with limiting dilution assay, be subcloned every time
It is cultivated with HT selective mediums, subclone carries out ELISA screenings after 8-10 days, until monoclonal cell positive rate is
Until 100%, the monoclonal cell strain HEV71-vP1-4G12 of the antibody of 1 plant of energy anti-HEV71-VP1 albumen of stably excreting is obtained.
F. Application mouse monoclonal antibody hypotype authentication chip kit（Purchased from Raybiotech companies of the U.S.）Identification is single
Monoclonal cell strain supernatant is carried out 80-100 times by anti-hypotype with DMEM serum free mediums respectively by chip agent box specification
Dilution, is added in array, the array for measuring all hypotypes is contained in a hole, and each hypotype has 4 repetitions, the results show：It is miscellaneous
The antibody subtype for handing over oncocyte HEV71-vP1-4G12 supernatants is IgM.
（2）The ascites antibody of the anti-HEV71-VP1 of mouse prepares and purifying
a）The female sex-health BALB/c mouse of 8-12 week old is selected, norphytane is injected intraperitoneally, 0.5ml/ is only；After 7-10 days, give
Every mouse peritoneal injection 1 × 106~5 × 106A monoclonal hybridoma notices that PBS need to be used by blowing down cell or diluting cells
Or serum free medium；
b）By ascites 10,000r/min centrifugation 15min remove cell component and other sediments, fat and oil reservoir
Deng, collect interlayer, measure antibody titer, packing, put -70 DEG C freeze it is spare.
C) saturated ammonium sulphate：It draws the ascites that 5ml is handled well to move into small beaker, under stiring, be added dropwise
The PBS5.0ml of 0.22 μm of filter membrane；After mixing, then it is added dropwise 10ml saturated ammonium sulfate solution（pH7.4）, continue slow
Stir 30min；10,000r/min is centrifuged 15 minutes after standing 2h, supernatant discarding, the PBS weights of the used 0.22 μm of filter membrane of sediment
It is outstanding, the re-suspension liquid is then crossed into 0.22 μm of filter membrane again；
D) according to antibody different subtype, the purification column of different GE Healthcare companies is selected, IgM pillars have accordingly
Specification configures different buffer solutions according to its specification and is purified：It is first slow with combination buffer, elution buffer and regeneration
Pillar is balanced by fliud flushing, 5 column volumes is usually balanced, then by the ascites buffer solution of resuspension in the speed of 1ml/min
Sample is balanced with combination buffer after completion of the sample, then is washed till baseline position with eluent, collects antibody peak；It will with PBS buffer solution
Antibody is dialysed, and with BCA protein quantification kit measurement antibody concentrations, and antibody is dispensed and is preserved, and with indirect ELISA into
Row titration.
The hybridoma cell strain HEV71-vP1-4G12 for generating the antibody is sent to China typical culture collection center
（CCTCC）Preservation is carried out, address is Wuhan University of Wuhan City of Hubei China province, and preservation date is on October 17th, 2013, preservation
Number be CCTCC NO:C2013161.
Embodiment 3：The foundation of the indirect ELISA detection method of HEV71-VP1
In the present embodiment, the peptide fragment of the HEV71-VP1 obtained in Application Example 1（Or other cross-reacting proteins）
As envelope antigen, the monoclonal antibody prepared using in embodiment 2 establishes the indirect of detection HEV71-VP1 as detection antibody
The method of ELISA.
a）The coating of ELISA Plate
Use coating buffer（Na2CO31.5g, NaHCO32.9g, Na2N31.2g adds ddH2O to 1L adjusts pH to 9.6）Antigen is dilute
1 μ g/ml are interpreted into, are added to after mixing in 96 hole elisa Plates, per 100 μ l of hole, plate is sealed in 4 DEG C overnight.
b）The closing of ELISA Plate
By the use of containing the PBS of 5% skim milk as confining liquid.Overnight ELISA Plate will be coated with first to pat dry, and adds in 200 μ l/
The confining liquid in hole, 37 DEG C of closing 2h are patted dry ELISA Plate with after board-washing machine washing plate 6 times, and spare in 4 DEG C or -20 DEG C long-term protect
It deposits.
c）The monoclonal antibody for preparing in the present invention is added in, 100 μ l/ holes incubate 1h in 37 DEG C, with board-washing machine washing plate 6 times
ELISA Plate is patted dry afterwards, adds the sheep anti mouse IgM antibody of certain density biotin labeling（Purchased from U.S. Raybiotech）,
100 μ l/ holes incubate 1h in 37 DEG C；The horseradish peroxidase of streptomysin mark is added in after board-washing（HRP）, 100 μ l/ holes, in 37
DEG C incubate 1h；TMB developing solutions are added in after board-washing, after colour developing completely, the concentrated sulfuric acid color development stopping of 2M is added in, and uses microplate reader
（U.S. Biotek）OD450 is measured, the results are shown in Table 1.
Table 1.HEV71-VP1 purified antibodies titrations
| Antibody concentration (ng/ml) | HEV71-VP1-HEV71-vP1-4G12 |
| 100 | 1.631 |
| 50 | 1.592 |
| 25 | 1.27 |
| 12.5 | 0.912 |
| 6.25 | 0.643 |
| 3.125 | 0.477 |
| 1.562 | 0.291 |
| Negative serum | 0.152 |
Wherein when antibody concentration is 3.125, detection light absorption value is the 2 times or more of negative serum, it is known that detects antibody
Potency is 3.125ng/ml.
Embodiment 4：The epitope of monoclonal antibody of the present invention determines
HEV71-VP1 protein sequences are imported into SWISS MODEL online softwares first, simulate the three-dimensional of similar protein sequence
Structural model will come out positioned at protein conformation extra amino acid sequences.Full HEV71-VP1 protein sequences are imported again
DNASTAR softwares count peptide fragment by Epitope prediction instrument and combine.Two groups of data are compared and draws and is most possibly
7 groups of peptide fragments of epitope.7 groups of peptide fragments are synthesized by Shanghai gill biochemistry biotech firm, indirect elisa method is carried out and confirms this list
Anti- recognition site.
The epitope of this monoclonal antibody is detected using indirect elisa method.The different peptide fragment of 100ng7 groups is coated in elisa plate
On, for coating 100ng HEV71-VP1 recombinant proteins as positive control, PBS sets up two repetitions as blank control.Monoclonal antibody is made
To detect antibody, as a result such as following table：
Table 2：
| 1 | 2 | |
| Peptide fragment 1 | 0.182 | 0.137 |
| Peptide fragment 2 | 0.583 | 0.545 |
| Peptide fragment 3 | 0.105 | 0.15 |
| Peptide fragment 4 | 0.156 | 0.125 |
| Peptide fragment 5 | 0.118 | 0.112 |
| Peptide fragment 6 | 0.191 | 0.18 |
| Peptide fragment 7 | 0.151 | 0.127 |
| Blank control | 0.12 | 0.09 |
| Positive control | 0.696 | 0.716 |
It thereby determines that, the recognition site of the monoclonal antibody therefore of peptide fragment 2（Epitope）, the amino acid sequence of the peptide fragment is：
Gly-Glu-Ile-Asp-Leu-Pro-Leu-Lys-Gly-Thr-Thr-Asn-Pro-Asn-Gly-Tyr-Ala-
Asn-Trp-Asp-Ile-Asp）.
Embodiment 5：The specificity identification of monoclonal antibody of the present invention
Using Dot hybridization point 100ng different virus albumen, if one repeats cross reaction and compares analysis, most terminate
Fruit shows such as Fig. 1, using its detected value of gray count, as a result such as table 3.
Table 3：Antibody specificity is identified（Dot hybridization）
| Ag | HEV71-VP1 | HCV-CORE | P24 | ORFV |
| Detected value 1 | 18.16 | 5.35 | 6.61 | 3.3 |
| Detected value 2 | 17.5 | 7.21 | 6.3 | 2.17 |
| Average value | 17.83 | 6.28 | 6.455 | 2.735 |
Fig. 1 and table 3 the result shows that, monoclonal antibody of the present invention has significant special for HEV71-VP1
Property, with other virus proteins（Such as hepatitis C virus HCV-CORE, running sore virus ORFV, HIV P 24）There is no cross reactions.
Embodiment 5：The composition of the body kit of detection hand-foot-and-mouth disease enterovirus albumen of the present invention and application
1st, the composition of kit
（1）8 × 12 detachable 96 hole microwell plates；
（2）Recombinant protein HEV71-VP1 dry powder one is managed；
（3）Anti- HEV71-VP1 detects antibody dry powder, i.e. monoclonal antibody prepared by embodiment 2；
（4）Blank control, positive control, each pipe of negative control dry powder；
（5）HRP marks secondary antibody（Anti- HEV71-VP1 polyclonal antibodies）, coating buffer solution, confining liquid, antibody diluent,
TMB developing solutions, terminate liquid, each one bottle of cleaning solution.
2nd, the application method of kit
2.1 coating：Packaging detachable 96 hole microwell plate is opened, recombinant protein HEV71-VP1 is micro- by 100 nanograms every 100
It rises and is added in micropore, 4 spend night or 37 degree of 4 hours incubations.It is washed on board-washing machine.
2.2 confining liquids are incubated with every 200 microlitres of additions in hole, 37 degree of 1 hours.Washing.
2.3 add in sample to be tested, blank control, the positive, negative control in microwell plate after diluting by a certain percentage, every hole
100 microlitres of 1 hours of incubation at room temperature, washing.
Detection antibody is pressed 1 by 2.4：1000 dilutions, which add in, is incubated 1 hour in microwell plate, washing.
2.5 sequentially add ELIAS secondary antibody, TMB developing solutions, terminate liquid.In ELISA Plate reading.
Experimentally determined, in the kit, the optimal diluted concentration of monoclonal antibody is 100 to 200ng/ml, more
The optimal coating concentration of clonal antibody is 100ng/ml, the optimal diluted concentration of streptavidin-horseradish peroxidase be 1 to
100ng/ml。
3rd, the application of kit
Infant acute phase serum 136 is made a definite diagnosis in collection, and the recall rate of 100 detection HEV71-VP1 of Healthy Human Serum is to be measured
Sample OD values subtract negative control OD value of the blank control OD values more than 2 times and are judged as HEV71-VP1IgM antibody positives.Detection
The result is shown in Fig. 2.
ELISA method detection HEV71-VP1 positive rate results see the table below：
Table 4：
| Positive rate | Negative recall rate |
| 121/136（89%） | 97/100（97%） |
Claims (3)
1. a kind of monoclonal antibody for being used to detect hand-foot-and-mouth disease enterovirus albumen, it is characterised in that：The monoclonal antibody
Be by preserving number be CCTCC NO:The hybridoma cell strain HEV71-vP1-4G12 of C2013161 is generated, can specific recognition
Serial No. SEQ ID NO in HEV71-VP1 albumen:1 amino acid sequence.
2. it is a kind of for detecting the ELISA detection kit of hand-foot-and-mouth disease enterovirus albumen, include ELISA ELISA Plates, capture
Antibody, envelope antigen, ELIAS secondary antibody, developing solution, dilution, terminate liquid, it is characterised in that：The capture antibody is that right such as will
Seek the monoclonal antibody described in 1, the antibody in the ELIAS secondary antibody is anti-HEV71-VP1 polyclonal antibodies.
3. ELISA detection kit according to claim 2, it is characterised in that：The diluted concentration of the monoclonal antibody
It is 100 to 200 ng/ml, the coating concentration of the polyclonal antibody is 100ng/ml.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102690327A (en) * | 2010-11-12 | 2012-09-26 | 厦门大学 | Enterovirus 71 neutralized epitope polypeptide and application thereof |
| CN102854317A (en) * | 2011-09-27 | 2013-01-02 | 上海博沃生物科技有限公司 | EV71 (human enterovirus 71) antigen enzyme-linked reaction detection kit and its preparation method |
| WO2013032404A1 (en) * | 2011-08-26 | 2013-03-07 | Temasek Life Sciences Laboratory Limited | Human enterovirus specific antibodies and their uses in diagnostics |
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2014
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102690327A (en) * | 2010-11-12 | 2012-09-26 | 厦门大学 | Enterovirus 71 neutralized epitope polypeptide and application thereof |
| WO2013032404A1 (en) * | 2011-08-26 | 2013-03-07 | Temasek Life Sciences Laboratory Limited | Human enterovirus specific antibodies and their uses in diagnostics |
| CN102854317A (en) * | 2011-09-27 | 2013-01-02 | 上海博沃生物科技有限公司 | EV71 (human enterovirus 71) antigen enzyme-linked reaction detection kit and its preparation method |
Non-Patent Citations (1)
| Title |
|---|
| 人类肠道病毒71型研究进展;李静等;《公共卫生与预防医学》;20120229;第23卷(第1期);59-62 * |
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