CN107271667A - Blue tongue virus type specificity competitive ELISA detection kit and its application - Google Patents
Blue tongue virus type specificity competitive ELISA detection kit and its application Download PDFInfo
- Publication number
- CN107271667A CN107271667A CN201710415171.4A CN201710415171A CN107271667A CN 107271667 A CN107271667 A CN 107271667A CN 201710415171 A CN201710415171 A CN 201710415171A CN 107271667 A CN107271667 A CN 107271667A
- Authority
- CN
- China
- Prior art keywords
- btv
- blue tongue
- tongue virus
- serum
- virus type
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Tropical Medicine & Parasitology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of blue tongue virus type specificity competitive ELISA detection kit and its application.The kit includes following two groups of components：Component I includes the ELISA Plate for being coated with bluetongue viral antigen I and the monoclonal antibody I detected for blue tongue virus type specificity, and described monoclonal antibody I is secreted by hybridoma cell strain 8B3G11 to be produced；Component II includes the ELISA Plate for being coated with bluetongue viral antigen II and the monoclonal antibody II detected for blue tongue virus type specificity, and described monoclonal antibody II is secreted by hybridoma cell strain 8B5D4 to be produced.The present invention establishes the joint competition ELISA detection method of blue tongue virus type specificity detection using two plant of 8 type BTV VP2 monoclonal antibodies 8B5D4 and 8B3G11 preparing, it can be used for the preliminary animal blood serum for distinguishing the infection of different shaped blue tongue disease in the detection method short time, experiment condition requires low, is suitable for large-scale BTV serum antibodies inspection.
Description
Technical field
It is more particularly to a kind of to be used for blue tongue virus type the present invention relates to a kind of kit detected for blue tongue rims
The competitive ELISA detection kit of specific detection, further relates to its application in blue tongue rims detect, the invention belongs to disease
Malicious detection technique field.
Background technology
Blue tongue disease (bluetongue, BT) is by caused by Reoviridae Orbivirus blue tongue virus (BTV)
The zoonosis propagated through insect vector (Kuku midge, yellow-fever mosquito etc.).Susceptible animal is mainly ox, sheep, goat, deer, camel
Deng.The World Health Organization (OIE) provides it for that must report epidemic disease, and China provides that it is a class infectious disease.BTV has found earliest
In 1876, in worldwide distribution.At present from Africa, Europe, Asia, North America, South America and Oceanian multiple torrid zones, Asia
The torrid zone and Temperate Region in China country are separated to BTV, and its distribution constantly expands.
Blue tongue virus (bluetongue virus, BTV) is diplornavirus, and its genome is by 10 linear double-strands
RNA fragments are constituted, and encode 10 kinds of albumen altogether, including 7 kinds of structural proteins (VP1-VP7) and 4 non-structural proteins (NS1, NS2,
NS3/NS3a、NS4).VP2 albumen be BTV be BTV serotype main determining factor.VP2 amino acid sequence between different blood groups
Row difference is from 22.4% to 73%.
As the continuous variation of blue tongue virus has now been found that 27 kinds of blue tongue virus serotypes, different serotypes it
Between cross-protection it is poor.At present both at home and abroad without the vaccine that can be effectively prevented and treated BT, therefore prevention and control BT important measures
It is to set up the differential diagnostic method for different serotypes.Blue tongue disease has a variety of detection methods, but every kind of method respectively has its excellent scarce
Point, for example：Immunofluorescent test and virus neutralization tests need to be separately cultured to virus, and operating process is cumbersome time-consuming, and
There are problems that easily dissipating the bio-safeties such as poison, fluorescence quantifying PCR method cost is higher, have to detection device and personnel ability
High requirement, Ago-Gel diffusion test specificity is relatively low.And the competitive ELISA inspection based on monoclonal antibody at present
Survey method (C-ELISA) is the optimum detection method that OIE recommends.
The present invention is resisted using two plants of monoclonal antibodies for 8 type BTV VP2 albumen as competitive antibody with to be detected
Body is competed, so that the C-ELISA detection methods detected for blue tongue virus type specificity are established, and it is big by inhibiting rate
The small quantity to judge detection antibody, while verifying the sensitiveness of this method, specificity and repeatability.The present invention is established
A set of sensitive, special, high-throughout detection method, the quick detection for China's blue tongue disease provides technical support, to ensure China
The sound development of animal husbandry.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of blue tongue virus type specificity competitive ELISA detection examination
Agent box and its application in the detection of BTV type specificities.
In order to achieve the above object, present invention employs following technological means：
A kind of blue tongue virus type specificity competitive ELISA detection kit of the present invention, including following two groups of components：
Component I includes the ELISA Plate for being coated with bluetongue viral antigen I and for the detection of blue tongue virus type specificity
Monoclonal antibody I, described monoclonal antibody I is secreted by hybridoma cell strain 8B3G11 to be produced, described hybridoma cell strain
8B3G11 is deposited in China typical culture collection center, and address is in Wuhan University, its microbial preservation number：CCTCC
No.C201770；Described bluetongue viral antigen I is the type VP2 albumen of blue tongue virus 8 or containing SEQ ID NO.1 institutes
Show the fragment of the VP2 albumen of epitope；
Component II includes the ELISA Plate for being coated with bluetongue viral antigen II and for the detection of blue tongue virus type specificity
Monoclonal antibody II, described monoclonal antibody II is secreted by hybridoma cell strain 8B5D4 to be produced, and described hybridoma is thin
Born of the same parents' strain 8B5D4 is deposited in China typical culture collection center, and address is in Wuhan University, its microbial preservation number：CCTCC
No.C201769；Described bluetongue viral antigen II is the type VP2 albumen of blue tongue virus 8 or containing SEQ ID NO.2 institutes
Show the fragment of the VP2 albumen of epitope.
In competitive ELISA detection kit of the present invention, it is preferred that the kit also includes the enzyme of anti-mouse
Mark secondary antibody, negative control, positive control, dilution, TMB nitrite ions, cleaning solution and terminate liquid.
In competitive ELISA detection kit of the present invention, it is preferred that the ELIAS secondary antibody of described anti-mouse is HRP
The mountain sheep anti-mouse igg of mark.
In competitive ELISA detection kit of the present invention, it is preferred that the negative control is blue tongue virus
Negative serum；The positive control is the type positive serum of blue tongue virus 8；The dilution is to contain 5w/v% skimmed milks
PBS；The cleaning solution is PBS.
In competitive ELISA detection kit of the present invention, it is preferred that described PBS includes NaCl
8g, KCl 0.2g, Na2HPO41.44g, KH2PO4PH value is adjusted to 7.4 after 0.24g, dissolving, is settled to deionized water
1000mL。
In competitive ELISA detection kit of the present invention, it is preferred that described contains shown in SEQ ID NO.1
The fragment of the VP2 albumen of epitope is by 1021- of the Genbank accession number for 195,972,621 8 type BTV L2 genes
2091bp's is nucleotide sequence coded；The described fragment for having the VP2 albumen of epitope shown in SEQ ID NO.2 by
Genbank accession number is nucleotide sequence coded for the 1021-2091bp's of 195,972,621 8 type BTV L2 genes.
When being used for the detection of blue tongue virus type specificity using competitive ELISA detection kit of the present invention, respectively
Using component I and component II be at war with ELISA detection, judged according to testing result：If two methods testing result
Be feminine gender, then measuring samples be BTV-2, BTV-3, BTV-5, BTV-9, BTV-11, BTV-12, BTV-13, BTV-15,
The serum or negative serum of BTV-16, BTV-17, BTV-20, BTV-24 infection；If two methods testing result is sun
Property, then measuring samples are the serum that BTV-7 or BTV-8 infects；If use component I be at war with ELISA detection result for
It is positive and use component II be at war with ELISA detections result for feminine gender, then measuring samples for BTV-10 infection serum；
If use component I be at war with ELISA detection result for feminine gender use component II be at war with ELISA detect knot
Fruit is the positive, and measuring samples may be BTV-1, BTV-4, BTV-6, BTV-14, BTV-18, BTV-19, BTV-21, BTV-23
The serum of infection.
Wherein, it is preferred that using component I or component II be at war with ELISA detection when, carry out in accordance with the following methods：
(1) coating bluetongue viral antigen I or coating bluetongue viral antigen II ELISA Plate are taken out, is washed with cleaning solution
Plate；
(2) the monoclonal antibody I for adding the test serum of diluted and being detected for blue tongue virus type specificity
Or the monoclonal antibody II detected for blue tongue virus type specificity, 37 DEG C of incubations；While to be separately added into diluted
Feminine gender or the hole of positive serum be used as control；
(3) with cleaning solution board-washing, the ELIAS secondary antibody of anti-mouse is added；
(4) with cleaning solution board-washing, TMB nitrite ions are added, 37 DEG C of colour developings finally add terminate liquid terminating reaction, determine OD
Value.
Wherein, it is preferred that coated bluetongue viral antigen I's or bluetongue viral antigen II is dense on the ELISA Plate
Spend for 5 μ g/ holes, 37 DEG C of incubation 1h；The extension rate of described test serum is 20 times, and blue tongue virus type is used in component I
The extension rate of the monoclonal antibody I of specific detection and the ELIAS secondary antibody of anti-mouse is 1000 times, and blue tongue is used in component II
The extension rate of the monoclonal antibody II of sick virus type specific detection and the ELIAS secondary antibody of anti-mouse is 2000 times, is incubated temperature
Spend for 37 DEG C, the time is 2h；The TMB chromogenic reaction times are 10min.
Further, the invention also provides described competitive ELISA detection kit is preparing blue tongue virus type spy
Application in specific detecting agents.
Compared to prior art, the beneficial effects of the present invention are：
1st, a kind of competitive ELISA method and reagent detected present invention firstly provides type specificity available for BTV
Box.At present, the domestic main competitive ELISA method that have studied the detection of BTV group specificities, there are no the type specificity inspection to BTV
The competitive ELISA method of survey is set up.In embodiments of the present invention, 8 type BTV VP2-B albumen are respectively adopted for envelope antigen, point
It is competition antibody not with anti-BTV-8VP2 monoclonal antibodies 8B5D4,8B3G11, establishes joint competition ELISA method；
2nd, high specificity and sensitiveness height are the great advantages of ELISA detection method, and micro sample is with regard to that can cause efficiently
Antigen-antibody reaction, therefore, the coating concentration of accurate selection antigen and the dilution factor of blood serum sample are the weights that ELISA is tested
Want one of link.The present invention titrates the dilution factor of further adjustment primary antibody and secondary antibody by square formation, fully optimizes reaction condition,
Improve detection sensitivity.The C-ELISA detections that analysis shows are carried out using monoclonal antibody 8B5D4,8B3G11 have good
Specificity and sensitiveness.The sensitivity to two kinds of C-ELISA methods is detected respectively, determines two kinds of C-ELISA sides
The sensitivity of method detection is respectively 1:160,1:80.The sensitiveness of the competitive ELISA method set up in this experiment, specificity and
Repeatability meets《Mammal, birds and honeybee A and B class disease diagnostic tests and vaccine standards handbook》In related rule
It is fixed.
3rd, 8B3G11 C-ELISA can detect tri- kinds of positive serums of BTV-7, BTV-8, BTV-10 in specificity experiments.
8B5D4 C-ELISA can detect BTV-1, BTV-4, BTV-6, BTV-7, BTV-8, BTV-14, BTV-18, BTV-19, BTV-
21st, ten kinds of positive serums of BTV-23.8B5D4 C-ELISA and 8B3G11 C-ELISA methods use in conjunction can distinguish BTV not
Homologous serotype.
4th, the present invention sets up joint C- using for 8 type BTV VP2 specific monoclonal antibody of excreting 8B5D4 and 8B3G11
ELISA detection method, can be used for the preliminary animal blood serum for distinguishing the infection of different shaped blue tongue disease in the detection method short time,
And experiment condition requires low, with type specificity, it is easy for large-scale serum antibody generaI investigation, and this research is next
Step is set up BTV type specificity detection methods and laid the foundation.
Brief description of the drawings
Fig. 1 is His-8A, His-8B SDS-PAGE analysis results；
Before 1.pET-28a-8A/BL21 inductions；After 2.pET-28a-8A/BL21 inductions；3. protein molecular weight standard；
Before 4.pET-28a-8B/BL21 inductions；After 5.pET-28a-8B/BL21 inductions；
Fig. 2 is His-4C SDS-PAGE analysis results；
After 1.pET-28a-8C/BL21 inductions；2. protein molecular weight standard；Before 3.pET-28a-8C/BL21 inductions；
Fig. 3 is the SDS-PAGE analysis results of restructuring MBP-8A, MBP-8B albumen；
Before 1.pMAL-c5x-8A/BL21 inductions；After 2.pMAL-c5x-8A/BL21 inductions；3. protein molecular weight mark
It is accurate；Before 4.pMAL-c5x-8B/BL21 inductions；After 5.pMAL-c5x-8B/BL21 inductions；
Fig. 4 is the SDS-PAGE analysis results of restructuring MBP-8C albumen；
1. protein molecular weight standard；Before 2.pMAL-c5x-8C/BL21 inductions；3.pM AL-c5x-8C/BL21 are induced
Afterwards；
Fig. 5 is 8B3G11 Mab supernatant Western blot qualification results；
1. albumen Marker；2.His-8A albumen；3.His-8B albumen；4.His-8Cization albumen；
Fig. 6 is 8B5D4 Mab supernatant Western blot qualification results；
1. albumen Marker；2.His-8A albumen；3.His-8B albumen；4.His-8Cization albumen；
Fig. 7 is 8B3G11 indirect immunofluorescence qualification results；
A.8B3G11 indirect immunofluorescence qualification result；B. positive control；The BHK- of C.SP2/0 supernatants and inoculation BTV8
21 cellmediated immunity Fluorescence Identification results；
Fig. 8 is 8B5D4 indirect immunofluorescence qualification results；
A.8B5D4 indirect immunofluorescence qualification result；B. positive control；The BHK-21 of C.SP2/0 supernatants and inoculation BTV8
Cellmediated immunity Fluorescence Identification result；
Fig. 9 is 8B3G11 C-ELISA detection methods to negative serum inhibiting rate and correspondence serum number distribution map；
Figure 10 is 8B5D4 C-ELISA detection methods to negative serum inhibiting rate and correspondence serum number distribution map.
The preservation information of biomaterial：
The hybridoma cell strain of the anti-BTV8-VP2 protein monoclonal antibodies of one plant of stably excreting, is named as hybridoma
Strain 8B3G11, Classification And Nomenclature is hybridoma cell strain 8B3G11；China typical culture collection center is deposited in, address is in
State Wuhan Wuhan Universitys, its microbial preservation number is：CCTCC No.C201770, the preservation time is on May 8th, 2017.
The hybridoma cell strain of the anti-BTV8-VP2 protein monoclonal antibodies of one plant of stably excreting, is named as hybridoma
Strain 8B5D4, Classification And Nomenclature is hybridoma cell strain 8B5D4；China typical culture collection center is deposited in, address is in Chinese
Wuhan Wuhan Universitys, its microbial preservation number is：CCTCC No.C201769, the preservation time is on May 8th, 2017.
Embodiment
The invention will now be further described with reference to specific embodiments, and advantages of the present invention and feature will be with description
And it is apparent.But these embodiments are only exemplary, do not constitute any limitation to the scope of the present invention.This area skill
Art personnel should be understood that without departing from the spirit and scope of the invention can to the details of technical solution of the present invention and
Form is modified or replaced, but these modifications and replacement are each fallen within protection scope of the present invention.
Embodiment 18 type BTV VP2 monoclonal antibodies 8B5D4 and 8B3G11 preparation
1st, main agents and medicine
Various restriction enzymes, T4 ligases, reverse transcriptase, Taq enzyme, DNA m arker are precious bioengineering
(Dalian) Co., Ltd product；Small amount plasmid extraction agent He Gou AXYGE companies；Glue reclaim small volume of reagent box is auspicious purchased from middle section
Safe Co., Ltd；Horseradish peroxidase (HRP) mark sheep anti-mouse igg is purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge；
O-phenylenediamine (OPD) is purchased from SIGMA companies；Hyclone is purchased from GIBCO companies；Superfine hyclone is purchased from Tianjin Hao sun
Company；1640 basal mediums, dimethyl sulfoxide (DMSO) (DMSO), trypsase, PEG3350, Freund's complete adjuvant and Freund are endless
Full adjuvant, HAT (50 ×) culture medium, HT (l00 ×) culture medium, diaminobenzidine (DAB), o-phenylenediamine (OPD), antibody
Subgroup identification kit is purchased from SIGMA companies.
2nd, experimental animal
4-6 week old BALB/C mice is provided by the long-living Bioisystech Co., Ltd in Liaoning.
3rd, the prokaryotic expression of BTV8-VP2 genes and purifying
3.1 design of primers
According to the L2 gene orders of listed 8 type BTV in Genbank (accession number is 195972621).Utilize albumen
Analysis software etc. is analyzed and predicted to L2 genes, and the L2 genes of 8 types are divided into A (1-1230bp), B (1021-
2091bp), 3 sections of C (1831-2886bp)；Pcr amplification primer thing is designed, the primer point designed for pET-28a-c carriers
It is not：8A (P1-P2), 8B (P3-P4), 8C (P5-P6) pcr amplification primer thing sequence it is as shown in table 1 below, table 2 be for
The primer sequence of pMAL-c5x carriers, 8A (P7-P8), 8B (P9-P10), 8C (P11-P12).
Table 1 pET-28a-c (+) vector primer sequence
The pM AL-c5x vector primer sequences of table 2
3.2 gene magnification
BTV8 L2 genes using synthesis is templates, and using specific upstream and downstream primer pair, it enters performing PCR amplification.Reaction
System is as follows：
Reaction condition is：Enter PCR cycle, 94 DEG C of denaturation 40s, 54 DEG C of anneal 40s, 72 DEG C after 95 DEG C of pre-degeneration 5min
Extend 1min, 30 circulations, 72 DEG C of extension 10min.1 μ L products are taken to carry out 0.8% agarose gel electrophoresis, analysis result.
The structure of 3.3 BTV8-VP2 albumen pronucleus expression recombinant vectors
PCR primer and l0 × Loading Buffer are mixed, after 1.0% agarose gel electrophoresis, shone in uviol lamp
Penetrate down and cut the gel containing purpose fragment, be placed in the EP pipes weighed, the weight of gel calculated, according to a small amount of
DNA glue reclaims kit method is operated：300 μ L Buffer S1 are added in per l00mg nucleic acid glue, are placed in 56 DEG C of water-baths
Dissolving, during mix once every 2-3min concussions are reverse, it is solidifying according to 150 μ L isopropanols/l00mg when it melts completely
Colloid product adds isopropanol and mixed, and gained liquid is added in adsorption column, 12000rpm centrifugation 1min, discards filtrate.Add
700 μ L rinsing liquids are in adsorption column, and 12000rpm centrifugation 1min discard filtrate.Add 600 μ L rinsing liquids repeated washings one
Secondary, 12000rpm centrifugations 1min discards filtrate.Repeat above-mentioned code, sky centrifugation 2min.40 μ L preheatings are added in adsorbed film center
Deionized water, staticaccelerator adsorption 2min, 12000rpm centrifugation 2min collect eluent in new centrifuge tube.By after purification
PCR primer is connected with pMD18-T simple carriers, and is transferred in TG1.
Target gene 8A, 8B, 8C after purification is attached with carrier pET-28a-c (+), pMAL-c5x respectively.
Coupled reaction total system is 10 μ L：The μ L of I 5 μ L, pMD18-T simple carriers of Solution 0.3, what purifying was reclaimed
The μ L of PCR primer 4.7.It is put into after mixing in connection instrument, 16 DEG C of connection 4h.
3.4 conversions are with choosing bacterium
Product after connection is transformed into competent escherichia coli cell TG1：Competence is taken out from -80 DEG C of refrigerators thin
Born of the same parents, are placed on ice to melt 3min, and the μ L of product after connection 10 are added in the μ L of TG1 100, mix, 20min is stood on ice.42
DEG C thermal shock 100s, ice bath 5min.LB liquid recovery media 700 μ L, 37 DEG C of shaking table shaken cultivation 40min are added thereto.Take
Go out bacterium solution after 200 μ L cultures to be spread evenly across on the LB flat boards containing Amp+, be all cultured after base absorbs after bacterium solution and be put into 37
In DEG C incubator, culture 12h is inverted.
Digestion identification and the sequencing of 3.5 recombinant expression plasmids
Recombinant plasmid is extracted with the small extraction reagent kit of plasmid.The bacterium solution after 1mL cultures is taken in EP pipes, 12000rpm centrifugations
1min, supernatant is discarded, and adds 250 μ L Buffer P1 and fully shaking mixing makes bacterial sediment suspend fully；Add
250 μ L Buffer P2, gentle mixing 6-8 times of turning upside down；350 μ L Buffer P3 are added, gentle turning upside down is mixed
It is even 5-6 times, 12000rpm centrifugations 10min；Gained supernatant is added in adsorption column, 12000rpm centrifugation 1min discard filter
Liquid.700 μ L rinsing liquids are added in adsorption column, 12000rpm centrifuges 1 min, discards filtrate.Add the repetition of 500 μ L rinsing liquids
It washed once, 12000rpm centrifugation 1min, discard filtrate, sky centrifugation 2min.Adsorption column is put into a new EP pipe,
Adsorbed film center adds the deionized water of 60 μ L preheatings, and staticaccelerator adsorption 2min, 12000rpm centrifugation 1min collects DNA and washed
De- liquid is in new centrifuge tube.
Single endonuclease digestion system (10 μ L)：
Double digestion system (10 μ L)：
The plasmid of extraction carries out single double digestion identification, and 37 DEG C of water-baths act on 30min.Product is through 0.8% fine jade after single double digestion
Sepharose electroresis appraisal.Digestion is identified that correct positive colony plasmid is sent to Suzhou Jin Weizhi bio tech ltd
It is sequenced, compares objective gene sequence completely correct.
The prokaryotic expression of 3.6 BTV8-VP2 genes and purifying
Positive recombinant plasmid pET-28a-c (+), pMAL-c5x are transformed into prokaryotic expression according to method for transformation described in 3.4
BL21 competent cells are used, 100 μ L bacterium solutions is then taken out and is coated on the LB flat boards containing ammonia section penicillin (100 μ g/mL),
White on 37 DEG C of overnight incubations, picking LB plating mediums isolates bacterium colony, is inoculated in containing ammonia section penicillin (100 μ g/mL)
LB fluid nutrient mediums in 37 DEG C of overnight incubations.Following expand with 1 100 of induced expression concrete operations is cultivated, 37 DEG C of vibration trainings
Support to when determining OD600=0.4~0.6, adding derivant IPTG is induced its final concentration of 1.0mmol/L, inducing
Afterwards after 4h, albumen sample is handled.Bacterium solution after 1mL inductions is taken to be placed in EP pipes, 12 000r/min centrifugation 2min abandon supernatant, by every
Milliliter OD600=1 adds the 2 × sample-loading buffers of μ L and SDS-PAGE of PBS 50 (containing DTT) 50 μ L, and 100 DEG C are boiled 10min,
SDS-PAGE identifications are carried out with 12% separation gel.
Expression vector pET-28a identifies with target gene 8A, 8B and 8C recombinant bacterium built through SDS-PAGE, as a result
As illustrated in fig. 1 and 2, there is specific band at 47kD in recombinant bacterium pET-28a (+)-VP2-8B/BL21 after induction, and pre-
Phase size is consistent, and its expression-form is expressed for inclusion body；Expression vector pMAL-c5x and target gene 8A, 8B and 8C structure
The recombinant bacterium built identifies through SDS-PAGE, as a result as shown in Figures 3 and 4, the recombinant bacterium pMAL-c5x-VP2-8B/BL21 after induction
Occur specific band at 82kD, be consistent with expected size, and predominantly solubility expression.
Recombinant bacterium pET-28a (+)-VP2-8B/BL21 is largely induced, inclusion body is extracted.Washed by cutting glue, electricity
De- mode is purified to albumen.Albumen after purification, determines packing after the concentration of albumen and is positioned over -80 DEG C of cryopreservations.
Recombinant bacterium pGEX-6P-1-8B/BL21 largely after induction, is carried out pure using the affinity column of GST label proteins
Change.Albumen after purification determines protein concentration, -80 DEG C of cryopreservations after packing using micro-spectrophotometer.
4th, the preparation of monoclonal antibody
4.1 mouse immune
The restructuring BTV8-VP2-B albumen of prokaryotic expression of the mouse immune to cut glue purification is as immunizing antigen, and dosage is
100 μ g/ are only.By 1：1 ratio is mixed with Freund's complete adjuvant, and fully emulsified pneumoretroperitoneum injects 4-6 week old BALB/c mouses；
One exempt from after the 14th day by immunogene and the isometric mixing and emulsifying of incomplete Freund's adjuvant, it is the same to be immunized position and dosage, progress the
Secondary immunity；Two exempt from latter 7th day, mouse tail blood sampling detection serum antibody titer；Repetition two in the 14th day was exempted from after exempting from two
Journey, is immunized position and dosage is the same, is that third time is immune；Afterbody blood sampling determines potency, last booster immunization, by abdominal cavity after 7 days
The multiple dose immunogene of direct injection 2, without using adjuvant, dosage is the same, and cell fusion is carried out after 3 days.
4.2 cell fusion
Fusion prepares feeder cells in first 1 day, and BALB/C mice peritoneal macrophage is laid in 96 porocyte culture plates
It is stand-by.Eyeball is taken a blood sample, and is taken off neck and is put to death mouse, spleen and separating Morr. cell is taken, by splenocyte and SP2/0 myeloma cell 8：1
Ratio carries out cell fusion with PEG, and the cell after fusion is laid on ready feeder cells.
The screening of 4.3 positive hybridoma cell strains and clone
Indirect ELISA detection method is set up using the BTV8-VP2 albumen with MBP labels of prokaryotic expression after purification
Positive hybridoma cell strain is screened, hybridoma is subcloned using limiting dilution assay, after being subcloned 3 times, is detected miscellaneous
Hand over the stability of oncocyte secretory antibody.The positive hybridoma cell being subcloned is frozen in time.It is final obtain two plants can be with
The hybridoma cell strain of the anti-BTV8-VP2 protein monoclonal antibodies of stably excreting, is respectively designated as hybridoma cell strain 8B3G11
And hybridoma cell strain 8B5D4, described hybridoma cell strain is deposited in China typical culture collection center, and address exists
Wuhan University, its microbial preservation number is respectively：CCTCC No.C201770 and CCTCC No.C201769.
5th, the identification of monoclonal antibody
Subclass and the epitope identification of 5.1 monoclonal antibodies
Identified through Subclass of antibody identification kit, in the monoclonal antibody of anti-BTV8VP2 albumen, 8B3G11 and 8B5D4
Subclass be respectively IgG2b and IgG2a.Anti- 8 type BTV VP2 monoclonal antibody 8B3G11 epitope is283LL284
(shown in SEQ ID NO.1), 8B5D4 epitope is192LKP194(shown in SEQ ID NO.2),
5.2 Western blot are tested
Tested by Western blot and the atopic of monoclonal antibody is identified.By the weight of prokaryotic expression
Group BTV8-VP2-A, BTV8-VP2-B and BTV8-VP2-C albumen be transferred on NC films, 8B3G11 culture supernatants or
8B5D4 culture supernatants carry out primary antibody incubation as primary antibody, and cell supernatant is not diluted, and 4 DEG C of closings are stayed overnight.NC films are taken out to use
PBST wash three times, 10min/ times, be then placed in through 5% skimmed milk by 1:The HRP mark mountain sheep anti-mouse iggs of 2000 dilutions
In, secondary antibody incubation is carried out, room temperature effect 2.5h takes out NC films and washed with PBST three times, 10min/ times, ECL colour developing colour developings are seen
Examine result.
Result of the test confirms that the monoclonal antibody 8B3G11 and 8B5D4 prepared by the present invention can be with BTV8-VP2-B
Specific reaction occurs for albumen, and (such as Fig. 5, Fig. 6) is not reacted with BTV8-VP2-A, BTV8-VP2-C.
5.3 indirect immunofluorescence experiments detect the reaction of monoclonal antibody 8B3G11 and 8B5D4 and BTV-8 viruses
BHK21 cells are reached in 12 orifice plates for placing creep plate, 8 type BTV are inoculated with after cell covers with individual layer, are set simultaneously
Put negative control hole.It is placed in saturated humidity, 37 DEG C, the interior culture quiescent culture 24h of 5%CO2 incubators；Take out creep plate light with PBS
Fine laundering washs 3 times, 5min/ times.3% paraformaldehyde fixer room temperature 30min is fixed；PBS is washed three times, 5min/ times；Often
The BSA37 DEG C of closing 1h that hole adds 3% is closed；Discard confining liquid and three times, 5min/ times are washed with PBS；With preservation
8B3G11 or 8B5D4 cell supernatants are primary antibody, while cell controls group is set, 37 DEG C of incubation 1h；Washed with PBS three times,
5min/ times；Add 1:37 DEG C of effect 1h under the conditions of the FITC mark mountain sheep anti-mouse iggs of 2000 dilutions, 200 μ L/ holes, lucifuge；Keep away
Three times, 5min/ times are washed under optical condition with PBS；Observation experiment result such as Fig. 7 and Fig. 8 institutes under inverted fluorescence microscope
Show.
Result of the test confirms that monoclonal antibody 8B3G11 and 8B5D4 has positive reaction with BTV-8 type viruses.
The foundation of the blue tongue virus type specificity competitive ELISA detection method of embodiment 2
1 materials and methods
1.1 cell
Anti- 8 type BTV VP2 albumen MAb 8B3G11,8B5D4 are prepared by embodiment 1.
1.2 main agents
8 type BTV VP2-B albumen are prepared according to the method for embodiment 1；1-24 type BTV standard positive serums and negative serum
Purchased from blue tongue disease reference laboratory Pirbright In stitute；70 portions of sheep, goat and cow's serum sample are respectively from
Certain plant in the Inner Mongol and Heilungkiang；The mountain sheep anti-mouse igg of HRP marks is purchased from Co., Ltd of Zhong Shan Golden Bridge；TMB nitrite ions
Alto, which is won, purchased from Beijing reaches Science and Technology Ltd.；Bluetongue antibody ELISA kit is purchased from the limited public affairs of Shanghai Ya Ji biotechnologies
Department.PBS：NaCl 8g, KCl 0.2g, Na2HPO41.44g, KH2PO4PH value is adjusted to 7.4, use after 0.24g, dissolving
Deionized water is settled to 1000mL；Dilution：Skimmed milk 5g, PBS 100mL.
1.3 optimization C-ELISA reaction conditions
Using 8 type BTV VP2-B albumen as antigen, respectively with 1 μ g/ holes, 2 μ g/ holes, 5 μ g/ holes, 10 μ g/ holes coating enzyme mark
Plate, the coating time is respectively 1h, 2h, 3h；The mountain sheep anti-mouse igg for again marking ascites after purification and HRP is dilute with dilution
Release 1:500、1:1000、1:2000、1:4000 times；8 type BTV standard positive serums carry out 1 with PBS dilutions:10、1:20、1:
50、1:100、1:200 times, optimum diluting multiple is determined, ascites dilutions and blood serum sample are mixed and added in ELISA Plate；Instead
1h, 2h, 3h are respectively set between seasonable, each step optimum reacting time is determined.The TMB chromogenic reaction times be respectively 7min,
10min, 15min, it is determined that optimal developing time.According to optimization amounts of reactants and the result of time, the detection method is determined most
Good working concentration and reaction time.
The identification of 1.4 blood serum samples
According to bluetongue antibody ELISA kit specification, 70 portions of sheep, goat and cow's serum sample are detected,
Determine whether the source animal of Sample serum infects BTV.
The determination of 1.4 criterions
Using 1.3 detection methods set up, while being detected to 50 parts of negative sheep, goat and ox negative serum, survey
Serum OD450 is obtained, blocking rate is calculated, calculation formula is PI (blocking rate)={ 1- (ODx-ODmin)/(ODmax-ODmin) } *
100% (ODmax:To be not added with light absorption value during sample, ODx is the light absorption value of sample, and ODmin is the extinction of blank control wells
Value.).The average value () and standard variance (SD) of 50 parts of serum sample PI values are calculated, according to principle of statistics ,+2SD is set
For the critical value of the positive and negative serum of sample.The PI values of sample>During+2SD, the positive can be judged in 99.9% level.
1.5 sensitivity assessment
8 type BTV standard positives are diluted 1: 5,1: 10,1: 20,1: 40,1: 80,1: 160 and 1:320 times anti-as detection
Body, is detected with the competitive ELISA detection method set up in 1.3.
1.6 Evaluation on specificity
1-24 type BTV standard positive serums and each 10 parts of negative sample are detected respectively using two kinds of C-ELISA, according to 1:
20 extension rates, per the μ L of hole 100.The specificity to two kinds of detection methods of 8B3G11,8B5D4 C-ELISA is identified respectively.
1.7 replica test
Using with reagent used in a batch of recombinant VP 2 albumen and 1.3, with the competitive ELISA detection side of foundation
10 parts of serum are carried out three repetitions respectively and detected, compared their result difference and calculate its coefficient of variation, evaluate and repeat by method
Property.
1.8 clinical samples are detected
With 1.3 kinds of detection methods set up, 20 parts of positive serums that BTV standard reagent boxes are detected are detected.
2 results
2.1 optimization reaction conditions
The optimal diluted concentration of reaction and reaction time are determined according to square formation titration, as a result as shown in table 3, table 4.With
The coating condition of the detection method set up based on MAb 8B3G11,8B5D4 most preferably is 5 μ g/ holes, 37 DEG C of incubation 1h；Sample
Dilution ratio is 1：20, the dilution ratio of ascites (primary antibody) is 1：1000 and 1：2000,37 DEG C of incubation 2h, mountain sheep anti-mouse igg
The dilution ratio of (secondary antibody) is 1：1000 and 1：2000,37 DEG C of incubation 2h；100 μ L TMB nitrite ions, 37 DEG C of lucifuges are added per hole
Develop the color 10min.
The reaction condition optimization result of the 8B3G11 C-ELISA detection methods of table 3
The reaction condition optimization result of the 8B5D4 C-ELISA detection methods of table 4
The identification of 2.2 blood serum samples
70 parts of serum samples are detected using blue tongue disease group specificity ELISA kit, wherein 50 parts of Sample serum performances
For feminine gender, 20 parts of serum show as the positive.
The determination of 2.3 criterions
50 portions of sheep and ox BTV negative serums are examined using the MAb 8B3G11 C-ELISA detection methods after optimization
Survey, Fig. 9 is shown in the serum number distribution of different inhibiting rates.Determine OD450Nm values, the blocking rate average value of detection is 20%, standard
Difference is 12%, according to formula：Average value+2S is calculated, and the critical value that positive and negative findings judges is 44%.That is blocking rate
44% and above be then judged to BTV positive serums.
50 portions of sheep and ox BTV negative serums are examined using the MAb 8B5D4 C-ELISA detection methods after optimization
Survey, Figure 10 is shown in the serum number distribution of different inhibiting rates.Determine OD450Nm values, the blocking rate average value of detection is 27%, standard
Difference is 11%, according to formula：Average value+2S is calculated, and the critical value that positive and negative findings judges is 49%.That is blocking rate
49% and above be then judged to BTV positive serums.
2.4 sensitivity analysis
It is different using the MAb 8B3G11 C-ELISA after optimization, two kinds of detection method detections of MAb 8B5D4 C-ELISA
The standard BTV-8 positive serums of diluted concentration, MAb 8B3G11 C-ELISA testing results show dilution factor 1：When 80, PI>
48%；MAb 8B5D4 C-ELISA testing results show dilution factor 1：When 160, PI>54%.
2.5 specificity analyses
Using MAb 8B3G11 C-ELISA, two kinds of detection method detection 1-24 types BTV marks of MAb 8B5D4 C-ELISA
Quasi- positive serum and standard female serum, as a result such as table 5.Two kinds of detection methods can detect 8 type BTV standard positive blood
Clearly, but two methods can detect other serum for the positive, may be relevant with non-specific binding.Two methods are total to
Nonspecific reaction can be largely excluded when using together.Two methods can detect BTV-7, BTV-8 when being used in combination.
The BTV competitive ELISA specific test testing results of table 5
2.6 repeated experiment
The detection reagent of the two methods prepared using 3 batches is detected respectively to 10 parts of serum, is as a result shown, profit
With the coefficient of variation of different batches reagent detection same sample between 4%-10%, no significant difference.
2.7 clinical samples are detected
It will be examined through blue tongue disease group specificity ELISA kit testing result for 20 parts of positive BTV blood serum samples
Survey.The positive detected with the C-ELISA detection methods based on 8B3G11 has 1, therefore the detection sample may be
The animal blood serum of BTV-7, BTV-8 and BTV-10 infection；The positive sample detected with the C-ELISA detection methods based on 8B5D4
Product have 4, therefore the detection sample may be BTV-1, BTV-4, BTV-6, BTV-7, BTV-8, BTV-14, BTV-18, BTV-
19th, the animal blood serum of BTV-21 and BTV-23 infection.Two methods detection be it is positive have 1 sample, the positive can
It can be the blue tongue disease serum of infection BTV-7 or BTV-8 types, detect and tie with import group specificity bluetongue antibody ELISA kit
Fruit is consistent.
Sequence table
<110>Northeast Agricultural University
<120>Blue tongue virus type specificity competitive ELISA detection kit and its application
<130> KLPI170260
<160> 2
<170> PatentIn 3.5
<210> 1
<211> 2
<212> PRT
<213> Bluetongue disease virus
<400> 1
Leu Leu
<210> 2
<211> 3
<212> PRT
<213> Bluetongue disease virus
<400> 2
Leu Lys Pro
Claims (10)
1. blue tongue virus type specificity competitive ELISA detection kit, it is characterised in that including following two groups of components：
Component I includes the ELISA Plate for being coated with bluetongue viral antigen I and the Dan Ke detected for blue tongue virus type specificity
Grand antibody I, described monoclonal antibody I is secreted by hybridoma cell strain 8B3G11 to be produced, described hybridoma cell strain
8B3G11 is deposited in China typical culture collection center, and address is in Wuhan University, its microbial preservation number：CCTCC
No.C201770；Described bluetongue viral antigen I is the type VP2 albumen of blue tongue virus 8 or containing shown in SEQ ID NO.1
The fragment of the VP2 albumen of epitope；
Component II includes the ELISA Plate for being coated with bluetongue viral antigen II and the list detected for blue tongue virus type specificity
Clonal antibody II, described monoclonal antibody II is secreted by hybridoma cell strain 8B5D4 to be produced, described hybridoma cell strain
8B5D4 is deposited in China typical culture collection center, and address is in Wuhan University, its microbial preservation number：CCTCC
No.C201769；Described bluetongue viral antigen II is the type VP2 albumen of blue tongue virus 8 or containing SEQ ID NO.2 institutes
Show the fragment of the VP2 albumen of epitope.
2. competitive ELISA detection kit as claimed in claim 1, it is characterised in that the kit also includes anti-mouse
ELIAS secondary antibody, negative control, positive control, dilution, TMB nitrite ions, cleaning solution and terminate liquid.
3. competitive ELISA detection kit as claimed in claim 1 or 2, it is characterised in that the ELIAS secondary antibody of described anti-mouse
The mountain sheep anti-mouse igg marked for HRP.
4. competitive ELISA detection kit as claimed in claim 3, it is characterised in that the negative control is blue tongue disease disease
Malicious negative serum；The positive control is the type positive serum of blue tongue virus 8；The dilution is to contain 5w/v% skimmed milks
PBS；The cleaning solution is PBS.
5. competitive ELISA detection kit as claimed in claim 4, it is characterised in that described PBS includes
NaCl 8g, KCl 0.2g, Na2HPO41.44g, KH2PO4PH value is adjusted to 7.4 after 0.24g, dissolving, is settled to deionized water
1000mL。
6. competitive ELISA detection kit as claimed in claim 1, it is characterised in that described contains SEQ ID NO.1 institutes
Show the fragment of VP2 albumen of epitope by 1021- of the Genbank accession number for 195,972,621 8 type BTV L2 genes
2091bp's is nucleotide sequence coded；The described fragment containing the VP2 albumen of epitope shown in SEQ ID NO.2 by
Genbank accession number is nucleotide sequence coded for the 1021-2091bp's of 195,972,621 8 type BTV L2 genes.
7. competitive ELISA detection kit as claimed in claim 1, it is characterised in that for blue tongue virus type specificity
During detection, be respectively adopted component I and component II be at war with ELISA detection, judged according to testing result：If two kinds of sides
Method testing result is feminine gender, then measuring samples be BTV-2, BTV-3, BTV-5, BTV-9, BTV-11, BTV-12, BTV-13,
The serum or negative serum of BTV-15, BTV-16, BTV-17, BTV-20, BTV-24 infection；If two methods testing result is equal
For the positive, then measuring samples are the serum that BTV-7 or BTV-8 infects；If the knot for the ELISA detections that are at war with using component I
Fruit for it is positive use component II be at war with ELISA detections result for feminine gender, then measuring samples are the blood of BTV-10 infection
Clearly；If use component I be at war with ELISA detection result for feminine gender use component II be at war with ELISA detect
As a result it is the positive, measuring samples may be BTV-1, BTV-4, BTV-6, BTV-14, BTV-18, BTV-19, BTV-21, BTV-23
The serum of infection.
8. the competitive ELISA detection kit as described in claim any one of 1-7, it is characterised in that use component I or component
II be at war with ELISA detection when, carry out in accordance with the following methods：
(1) coating bluetongue viral antigen I or coating bluetongue viral antigen II ELISA Plate are taken out, with cleaning solution board-washing；
(2) the monoclonal antibody I or use for adding the test serum of diluted and being detected for blue tongue virus type specificity
The monoclonal antibody II detected in blue tongue virus type specificity, 37 DEG C of incubations；While the moon to be separately added into diluted
The hole of property or positive serum is used as control；
(3) with cleaning solution board-washing, the ELIAS secondary antibody of anti-mouse, 37 DEG C of incubations are added；
(4) with cleaning solution board-washing, TMB nitrite ions are added, 37 DEG C of colour developings finally add terminate liquid terminating reaction, determine OD values.
9. competitive ELISA detection kit as claimed in claim 8, it is characterised in that coated blue tongue on the ELISA Plate
Sick viral antigen I or bluetongue viral antigen II concentration are 5 μ g/ holes, 37 DEG C of incubation 1h；The dilution of described test serum times
Number is 20 times, and the dilute of monoclonal antibody I that blue tongue virus type specificity detects and the ELIAS secondary antibody of anti-mouse is used in component I
Multiple is released for 1000 times, monoclonal antibody II that blue tongue virus type specificity detects and the enzyme mark of anti-mouse are used in component II
The extension rate of secondary antibody is 2000 times, and incubation temperature is 37 DEG C, and the time is 2h；The TMB chromogenic reaction times are 10min.
10. the competitive ELISA detection kit described in claim any one of 1-9 is preparing the detection of blue tongue virus type specificity
Application in reagent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710415171.4A CN107271667B (en) | 2017-06-05 | 2017-06-05 | Blue tongue virus type specificity competitive ELISA detection kit and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710415171.4A CN107271667B (en) | 2017-06-05 | 2017-06-05 | Blue tongue virus type specificity competitive ELISA detection kit and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107271667A true CN107271667A (en) | 2017-10-20 |
| CN107271667B CN107271667B (en) | 2019-01-22 |
Family
ID=60065637
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710415171.4A Active CN107271667B (en) | 2017-06-05 | 2017-06-05 | Blue tongue virus type specificity competitive ELISA detection kit and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107271667B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108588275A (en) * | 2018-03-23 | 2018-09-28 | 重庆出入境检验检疫局检验检疫技术中心 | The multiple RT-PCR kit and its detection method of BTV-10 types, 20 types, 23 type genotypings |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2966458A1 (en) * | 2010-10-20 | 2012-04-27 | Id Vet | USE OF BTV / 8 ANTI-VP2 ANTIBODIES FOR SEROTYPING AND DIAGNOSIS OF BTV INFECTIONS |
| CN105842450A (en) * | 2016-05-13 | 2016-08-10 | 云南省畜牧兽医科学院 | Bluetongue antibody competition liquid and corresponding quick ELISA detection method |
| CN106190986A (en) * | 2016-07-07 | 2016-12-07 | 东北农业大学 | The monoclonal antibody of resistant to bluetongue virus serum 4 type VP2 albumen, secretes hybridoma cell strain and the purposes of this monoclonal antibody |
-
2017
- 2017-06-05 CN CN201710415171.4A patent/CN107271667B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2966458A1 (en) * | 2010-10-20 | 2012-04-27 | Id Vet | USE OF BTV / 8 ANTI-VP2 ANTIBODIES FOR SEROTYPING AND DIAGNOSIS OF BTV INFECTIONS |
| CN105842450A (en) * | 2016-05-13 | 2016-08-10 | 云南省畜牧兽医科学院 | Bluetongue antibody competition liquid and corresponding quick ELISA detection method |
| CN106190986A (en) * | 2016-07-07 | 2016-12-07 | 东北农业大学 | The monoclonal antibody of resistant to bluetongue virus serum 4 type VP2 albumen, secretes hybridoma cell strain and the purposes of this monoclonal antibody |
Non-Patent Citations (2)
| Title |
|---|
| J. FEHRSEN ET AL: "Serogroup-reactive and type-specific detection of bluetongue virus antibodies using chicken scFvs in inhibition ELISAs", 《JOURNAL OF VIROLOGICAL METHODS》 * |
| 刘琦: "蓝舌病病毒VP7和VP2蛋白的原核表达及其单克隆抗体的制备", 《万方数据库》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108588275A (en) * | 2018-03-23 | 2018-09-28 | 重庆出入境检验检疫局检验检疫技术中心 | The multiple RT-PCR kit and its detection method of BTV-10 types, 20 types, 23 type genotypings |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107271667B (en) | 2019-01-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105461805B (en) | Monoclonal Antibodies against Porcine Epidemic Diarrhea Virus and its application | |
| CN105527437B (en) | A kind of detection kit and its application | |
| CN110964102B (en) | Monoclonal antibody capable of simultaneously combining with canine, feline and mink parvoviruses, variable region sequence thereof, hybridoma cell strain and application | |
| CN108486064A (en) | The monoclonal antibody and application of hybridoma cell strain Anti-CLasMcAb1 and its secretion | |
| CN103048459B (en) | Immune detection reagent for detecting respiratory syncytial virus | |
| CN108486063A (en) | The monoclonal antibody and application of hybridoma cell strain Anti-CLas McAb2 and its secretion | |
| CN105695420A (en) | Mouse bone marrow hybridoma cell strains, monoclonal antibody generated by same and application | |
| CN104459144B (en) | A kind of PRV velogen strain and vaccine strain differentiate Test paper | |
| CN102731615A (en) | Detection reagent and detection method for PRRSV | |
| CN107937352A (en) | For detecting the colloidal gold immuno-chromatography test paper strip of PPR virus H protein antibodies | |
| CN107312088A (en) | Porcine epidemic diarrhea virus specificity SIgA ELISA detection kits and its application | |
| CN111999497A (en) | Enzyme linked immunosorbent assay kit for detecting rabies virus glycoprotein antigen and application thereof | |
| CN106190986B (en) | The monoclonal antibody of 4 type VP2 albumen of resistant to bluetongue virus serum, secretes the hybridoma cell strain and purposes of the monoclonal antibody | |
| CN101597334A (en) | Monoclonal antibody of bluetongue virus (BTV) and preparation method and application | |
| CN107449912A (en) | Anti- H7N9 subtype avian influenza virus monoclonal antibody antigen epitope and its screening technique and application | |
| CN108918869B (en) | Application of fiber2 protein and recombinant protein thereof in detecting serum type 4 avian adenovirus antibody | |
| CN111073859B (en) | Double-antibody sandwich ELISA kit for detecting bovine parvovirus and application thereof | |
| CN105974119B (en) | G kinds enterovirus direct immunofluorescence reagent and its kit | |
| CN107271667B (en) | Blue tongue virus type specificity competitive ELISA detection kit and its application | |
| CN106701687A (en) | Hybridoma cell strain and rabies virus phosphoprotein monoclonal antibody generated by same | |
| CN105296435B (en) | The monoclonal antibody specific and application of hybridoma cell strain and its O-shaped (O/GX/09-7) virus of the resistant to foot and mouth disease of secretion | |
| CN105137076B (en) | Small-fragment gG antigen and application of small-fragment gG antigen in detecting pseudorabies virus gG antibody | |
| CN110484510A (en) | Hybridoma cell strain, its secretion identification cover his viral monoclonal antibodies and application | |
| CN107664694B (en) | A kind of ELISA kit based on E2 Protein Detection pig atypia pestivirus antibody | |
| CN105543177A (en) | Hybridoma cell strain secreting citrus yellow vein clearing virus-resistant monoclonal antibodies and monoclonal antibody application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |