CN109187993A - A kind of foot and mouth disease A-type virus sIgA antibody ELISA detection kit and its application - Google Patents
A kind of foot and mouth disease A-type virus sIgA antibody ELISA detection kit and its application Download PDFInfo
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- CN109187993A CN109187993A CN201811069024.7A CN201811069024A CN109187993A CN 109187993 A CN109187993 A CN 109187993A CN 201811069024 A CN201811069024 A CN 201811069024A CN 109187993 A CN109187993 A CN 109187993A
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Abstract
The invention discloses the ELISA detection kits and its application of a kind of foot and mouth disease A-type virus sIgA antibody.The kit includes the coated enzyme reaction plate of foot and mouth disease A-type virus broad spectrum multi-epitope recombination antigen, 100 × concentration enzyme labelled antibody, enzyme labelled antibody dilution, sample diluting liquid, concentrated cleaning solution, developing solution, terminate liquid, positive control sample and negative control sample.The foot and mouth disease A-type virus broad spectrum multi-epitope recombination antigen is made of the dominant antigen epitope (TB/A) that 3 plants of foot-and-mouth disease a types of isolated in China represent strain, therefore the sensitivity and specificity of kit detection are improved, and is suitable for the detection that different foot and mouth disease A-type virus infects.Kit provided by the invention is adapted to detect for pig, ox, the sIgA antibody in three kinds of susceptible animal mucosal secretions of sheep, has great importance for the propagation and infection that prevent and treat foot and mouth disease A-type virus.
Description
Technical field
The present invention relates to a kind of foot and mouth disease A-type virus sIgA antibody indirect ELISA detection kit and its detection methods.This
Invention belongs to technical field of biological.
Background technique
Aftosa (Foot-and-Mouth Disease, FMD) is caused by foot and mouth disease virus with the mouth of infected animal, hoof
There is the communicable disease that blister disease is characterized in portion.Foot and mouth disease virus (Foot-and-Mouth Disease Virus,
FMDV) belong to Picornaviridae, viral center is a single stranded RNA.According to serotype characteristic can be divided into A type, O-shaped, c-type,
SAT1 type, SAT2 type, SAT3 type (i.e. 1,2,3 type of South Africa) and 7 serotypes of Asial (Type Asia 1), it is several between different serotypes
There is no immune protective efficiency, the animal for having infected One serotype FMDV still can infect the FMDV of another serotype and fall ill.
FMDV infects object up to more than 70, and incubation period is 1~7d, and spread speed is fast, and morbidity is anxious, and virus mainly by respiratory tract and disappears
Change road to enter in animal body, initially enters animal throat and tonsillotome, subsequently into the animal blood circulatory system, clinical symptoms are
There is bubble in infection animal lassitude, sialic acid, mouth, hoof, nose and nipple area, in FMD incubation period, almost all of group
It knits, organ, secretion, secretion etc. all contain FMDV.The disease is moved for 15 kinds specified by International Animal Health tissue (OIE)
The first place of logistics row name of disease list, results in significant economic losses aquaculture and international animal trade.
About 8500 bases of MDV full-length genome, contain only a big open reading frame, express the polyprotein of generation by
Grade cracking generate virus structural proteins (VP1, VP2, VP3, VP4) and non-structural protein (Lab, 2A, 2B, 2C, 3A, 3B, 3C,
3D), structural proteins assembling generates the capsid structure of virus, and non-structural protein then participates in the interaction of virus and host, inhibits
The transcription and translation mechanism of host cell, the reproduction process for participating in virus.Viral capsid proteins are the masters that induction generates neutralizing antibody
Immunogene is wanted, there are multiple conforma-tional with linear neutralizing epitope on structural proteins.Four kinds of Structural protein VP1s of FMDV,
The amino acid of the G-H ring of VP1 is easy to make a variation in VP2, VP3, VP4, but arginine-glycine-aspartic acid therein acid
(Arg-Gly-Asp (RGD)) is highly conserved.The span of G-H ring is about 20 amino acid residues of the position 140-160 of VP1.
There is only main antigen site (141-160aa, 200-213aa and 21-40aa) on VP1 albumen, and contain FMD virus
Host's recognition site.Therefore the albumen of the regional gene construction of expression vector expression and purification is selected to can be used as good preparation
The alternative envelope antigen of antibody assay kit.It is not related to additionally by multi-epitope antigen production process prepared by prokaryotic expression system
And live virus, there is no the risks of scattered poison；Both epitope sequences can be adjusted according to the variation of popular strain at any time, it is possible to use different
Antigen sequence, increase the broad spectrum activity of antigen.
The main effects factor of the sIgA antibody as mucosa-immune, is widely present in the juices such as colostrum, tear, saliva
In, it not only can be in first time and viral, but also further activation system can be immunoreacted by way of mucous membrane, into
And prevent the intrusion of virus.Its major function has: suppress and stick, be immunized exclude, dissolution of bacteria, neutralize virus, mediate antibody according to
Bad cell-mediated cytotoxic effect (ADCC, antibody-dependent cell-mediated
Cytotoxicity), anti-inflammatory promotes native factor effect and adjusts mucosa-immune reaction.Foot-and-mouth disease virus resistant sIgA antibody
Be generate the mark of specificity in respiratory tract and alimentary canal first after mouth disease virus infection or vaccine mucosal immune, while
The effect of mucosa-immune vaccine is reacted.
There are no the kits that can effectively detect foot and mouth disease virus sIgA antibody currently on the market, in view of in recent years to glutinous
Film immunologic mechanism and mucosa-immune vaccine research go deep into, and develop foot and mouth disease virus sIgA antibody in a kind of detection mucosal secretions
Horizontal ELISA kit can more accurately react mouth disease virus infection and mucosa-immune state, while utilize the party
Method monitors foot and mouth disease virus sIgA Antibody dynamics changing rule, for aftosa mucosa-immune vaccine and establishes mating detection method and mentions
Theoretical and experimental basis is supplied.
Summary of the invention
The purpose of the present invention is to above-mentioned defects in the prior art, provide a kind of foot and mouth disease A-type virus sIgA
The detection kit and its method of antibody.
To achieve the goals above, technical solution provided by the invention are as follows:
The present invention represents strain (A/GDMM/ with 3 plant foot and mouth disease virus A types of the DNAStar software to isolated in China
2013, A/HuBWH/2009, A/GSLX/62) VP1 gene carry out sequence analysis, it is determined that dominant antigen epitope, by phase
Introducing between adjacent two epitopes can guarantee spacer sequence KKK, (G4S) 2 and GPGPG that each epitopic structures are correctly shown, thus
Building obtains the tandem sequence of multi-epitope gene (TB/A), and series sequence is A/GDMM/2013 [(20-85)-KKK- (133-
172)-(G4S)2-(193-212)]-GPGPG-A/HuBWH/2009[(20-85)-KKK-(133-172)-(G4S)2-(193-
212)]-GPGPG-A/GSLX/62[(20-85)-KKK-(133-173)-(G4S)2-(194-213)].The core of multi-epitope gene
The synthesis of nucleotide sequence trust money Si Rui biotechnology company.
A kind of foot and mouth disease A-type virus broad spectrum multi-epitope recombination antigen that the present invention obtains, the foot and mouth disease A-type virus are wide
Spectrum multi-epitope recombination antigen includes that foot and mouth disease A-type virus represents strain A/GDMM/2013, A/HuBWH/2009, A/GSLX/62's
20-85,133-172 and 193-212 main neutrality epitope sequences in VP1 albumen, it is described
Foot and mouth disease A-type virus broad spectrum multi-epitope recombination antigen amino acid sequence as shown in SEQ ID No.2.
The nucleotide sequence of the coding foot and mouth disease A-type virus broad spectrum multi-epitope recombination antigen is also in protection of the invention
Within the scope of, it is preferred that the nucleotide sequence is as shown in SEQ ID No.1.
Further, the invention also provides the foot and mouth disease A-type virus broad spectrum multi-epitope recombination antigens examines in preparation
Survey the purposes in foot and mouth disease A-type virus sIgA antibody reagent.
A kind of foot and mouth disease A-type virus sIgA antibody ELISA detection kit, the kit include foot and mouth disease A-type virus
It is the coated ELISA Plate of broad spectrum multi-epitope recombination antigen, 100 × concentration enzyme labelled antibody, enzyme labelled antibody dilution, sample diluting liquid, dense
Contracting cleaning solution, developing solution, terminate liquid, positive control sample and negative control sample.
Wherein, it is preferred that the foot and mouth disease A-type virus broad spectrum multi-epitope recombination antigen is obtained through prokaryotic expression
?.
Wherein, it is preferred that the coated ELISA Plate of foot and mouth disease A-type virus broad spectrum multi-epitope recombination antigen is according to following
Method is prepared:
(1) preparation and coating of envelope antigen
The recombinant protein inclusion body that prokaryotic expression obtains purifies to obtain foot-and-mouth disease a type disease through renaturation and Ni-NTA
Malicious broad spectrum multi-epitope recombination antigen, amino acid sequence is as shown in SEQ ID No.2, with the carbonate buffer of pH9.6 when coating
Liquid is diluted to 3 μ g/ml, and according to 100 hole μ l/ coated elisa plates, 4 DEG C of refrigerators are stood overnight；
(2) closing and preservation of ELISA Plate
It is coated with overnight ELISA Plate and washs 4 times with PBST and pat dry, every hole adds 10mM PBS of the 100 μ l containing 0.5w/v%BSA
Confining liquid, 37 DEG C of placement 2h discard confining liquid, and PBST is washed 3 times, obtain the ELISA Plate of pre-coated antigen, every hole adds 100 μ l to contain
The ELISA Plate stabilizer of 6v/v% horse serum, is placed at room temperature for 30min, discards naturally dry after liquid, with aluminium foil bag vacuum seal,
4 DEG C of preservations；
Wherein, the ELISA Plate stabilizer is to be added to 5g BSA, 10g sucrose, 20g trehalose
In 1000ml0.01mol/L PBS (pH7.4) gently concussion to dissolve, add 0.05w/v%Procline300, be stored in 4 DEG C it is standby
With.
Wherein, it is preferred that the positive control sample is that acquisition foot and mouth disease A-type virus attacks 7-21d pig, ox and sheep after poison
Nose swab sample, through mixing, a series of sample that detection OD450 value is 1.5 ± 0.05 after dilutions；The negative sample isFMDV NS kit detect foot and mouth disease virus non-structural protein antibody be feminine gender, foot-and-mouth disease a type, it is O-shaped,
AsiaI Liquid-phase blocking ELISA kit detect serum antibody titer < 1/4, FMDV specific PCR detection antigen be negative pig,
The nose swab of ox and sheep detects the sample that OD450 value is 0.1 ± 0.05 after being mixed, being diluted, aseptic subpackaged spare.
Wherein, it is preferred that described 100 × concentration enzyme labelled antibody is that horseradish peroxidase (HRP) marks the anti-pig of mouse, ox
Or the monoclonal antibody of sheep IgA, in use, with being used after 100 times of enzyme labelled antibody dilution dilutions, the enzyme labelled antibody dilution
Liquid is containing 1v/v% glycerol, 0.5w/v% bovine serum albumin(BSA), 1w/v% casein and 0.05w/v%Procline300
0.01mol/L PBS buffer solution, pH7.4.
Wherein, it is preferred that the sample diluting liquid is NaCl containing 0.5M, 2.68mM KCl, 2.79mM KH2PO4、
8.1mM Na2HPO4, 5g/L casein, 0.05%Tween20 and 10mM PBS mixed solution；The developing solution is aobvious for TMB
Color liquid, the terminate liquid are 1moL/L H2SO4Solution；The concentrated cleaning solution is 10 × PBST solution, that is, contains 0.5v/
The 0.1mol/L PBS solution of v%Tween20, pH 7.6 dilute 10 times when use.
Wherein, it is preferred that when carrying out foot and mouth disease A-type virus sIgA antibody test using the kit, according to following
Step carries out:
(1) Sample Dilution
By sample to be tested, positive control and negative control, with sample diluting liquid, 1:2 is diluted by volume；
(2) board-washing
The coated ELISA Plate of foot and mouth disease A-type virus broad spectrum multi-epitope recombination antigen is taken out from 4 DEG C of refrigerators, opens aluminium foil
Bag takes out ELISA Plate, and with cleaning solution board-washing 3 times diluted, blotting paper is patted dry, each 3min；
(3) it is loaded
Sample to be tested, positive control and negative control after dilution is separately added into ELISA Plate, 100 μ L of every hole, 37
DEG C be incubated for 45min；
(4) it washs
ELISA Plate is taken out, sample is got rid of, is rinsed 4 times with cleaning solution, blotting paper pats dry；
(5) enzyme labelled antibody is added
The anti-pig of mouse, ox or the sheep IgA monoclonal antibody for the horseradish peroxidase-labeled that addition has diluted, 100 holes μ L/,
37 DEG C of work 30min；
(6) add substrate:
Enzyme labelled antibody is got rid of, is rinsed 4 times with cleaning solution, blotting paper pats dry, and 37 DEG C of colour developing 15min of developing solution are added, and takes out
Terminate liquid is added immediately afterwards；
(7) result judgement
OD450nm numerical value is read in microplate reader, the OD450nm value of sample to be tested is average more than or equal to negative control sample
2.5 times of value are judged to the positive, and 2.5 times less than negative control sample average value are judged to feminine gender.
Technical key point of the present invention are as follows:
1) anti-using the foot and mouth disease A-type virus dominant antigen neoepitope Western (TB/A) of prokaryotic expression as coating
Original after measuring samples are reacted with envelope antigen, passes through enzyme using the anti-pig of mouse of enzyme label, ox, sheep IgA monoclonal antibody as secondary antibody
The further reaction of labelled antibody is finally compared with reference positive nose swab and negative nose swab, to evaluate foot and mouth disease virus
SIgA antibody level.
2) envelope antigen is through Escherichia coli pET-30a (+)) the multi-epitope albumen that purifies after prokaryotic expression, which contains
Have foot and mouth disease virus major antigenic sites (141-160aa, 200-213aa and 21-40aa), and the place containing foot and mouth disease virus
Main recognition site (tri- peptide motif of RGD).
3) secondary antibody is that the anti-pig of mouse, ox, sheep IgA monoclonal marked with horseradish peroxidase (HRP) labelling kit resists
Body.
It 4) is that laboratory is attacked after poison and infects within 7-21 days the nose swab of the pig of aftosa, ox, sheep with reference to positive sample；Negative sample
Product be throughFMDV NS kit detect foot and mouth disease virus non-structural protein antibody be feminine gender, A type, it is O-shaped,
It is negative that AsiaI aftosa Liquid-phase blocking ELISA kit, which detects antibody titer < 1/4, FMDV specific PCR detection antigen,
The nose swab of pig, ox, sheep.
Compared to the prior art, the invention has the benefit that
1) the invention discloses a kind of method and its detection kit for detecting foot and mouth disease A-type virus sIgA antibody, pass through
The anti-pig of mouse, the ox, sheep IgA monoclonal antibody of envelope antigen, HRP label, provide a kind of evaluation aftosa mucosa-immune effect
Effective ways, and provide a kind of new method for the early diagnosis of infection of foot-and-mouth disease, can fast and accurately detect pig, ox,
Foot and mouth disease A-type virus sIgA antibody in sheep nose swab；
2) since popular malicious mutation occurrence frequency is higher and higher in recent years for foot and mouth disease virus, the present invention passes through prokaryotic expression
3 plants of foot-and-mouth disease a types of system expression represent the dominant antigen epitope of poison as envelope antigen, and it is anti-to prepare the more full coating of update
Original improves kit to the Detection capability of existing strain；
3) acquisition of sample is easy to operate, and manpower consumption is small, to animal stress be small；
4) this method is easy to operate, and required time is short, can be used for the detection of a large amount of samples and commenting for mucosa-immune effect
Valence.
Detailed description of the invention
Fig. 1 is TB/A protein SDS-PAGE figure provided in an embodiment of the present invention；
In figure: swimming lane M1: Protein Marker；Swimming lane 1:BSA albumen；Swimming lane 2:TB/A albumen
Fig. 2 is purifying protein result provided in an embodiment of the present invention.
In figure: swimming lane M2: Protein Marker；Swimming lane 3: elution albumen.
Specific embodiment
The present invention will be further described combined with specific embodiments below, but following embodiments will not limit in any way
Protection scope of the present invention.
The preparation and assembling of 1 foot and mouth disease A-type virus sIgA antibody ELISA detection kit of embodiment
(1) preparation of kit:
1, the design of the screening of foot and mouth disease A-type virus dominant antigen epitope (TB/A):
Foot-and-mouth disease VP1 is the dominant antigen of virus, and the natural VP1 albumen either isolated and purified is still
Recombination expression product can induce body to generate protectiveness neutralizing antibody, have type specificity.VP 1 Gene of Foot-and-Mouth Disease virus overall length
It is made of 639 nucleotide, one albumen with 213 amino acid of coding, Main Antigenic concentrates on 140-160
The amino acid and 200-213 amino acid sections of position.The present invention is with DNAStar biosoftware to 3 plants of mouths of isolated in China
Aphtovirus A type represents strain A/GDMM/2013 (GenBank accession number: KF450794.1), A/HuBWH/2009 (GenBank
Accession number: JF792355.1), the VP1 gene sequencing of A/GSLX/62 (GenBank accession number: AJ131666.1) determines
The amino acid section that dominant antigen epitope is 20-85,133-172,193-212.Occur in order to prevent when constructing gene
New epitope, introducing between adjacent two epitope can guarantee the correct spacer sequence KKK of each epitopic structures, (G4S) 2 He
GPGPG, the series sequence of multi-epitope gene are as follows:
A/GDMM/2013/[(20-85)-KKK-(133-172)-(G4S)2-(193-212)]-GPGPG-A/HuBWH/
2009/[(20-85)-KKK-(133-172)-(G4S)2-(193-212)]-GPGPG-A/GSLX/62/[(20-85)-KKK-
(133-173)-(G4S)2-(194-213)]
The nucleotide sequence of multi-epitope gene is as shown in SEQ ID No.1, encoded amino acid sequence such as SEQ ID
Shown in No.2, the nucleotide sequence trust money Si Rui biotechnology company of multi-epitope gene is synthesized.
2, the expression of foot and mouth disease A-type virus dominant antigen neoepitope Western:
The encoding gene 1287bp (shown in SEQ ID NO.1) of foot and mouth disease A-type virus dominant antigen neoepitope Western is cloned
To pET-30a (+) prokaryotic expression carrier, after digestion, sequencing are correct, positive plasmid is converted to BL21DE3plysS bacterial strain, card
That chloramphenicol resistance TB plate screening monoclonal, 37 DEG C, TB culture medium shakes bacterium and stays overnight；Bacterium solution (v/v) 1:100 by volume will be stayed overnight
It is forwarded to fresh TB culture medium, 37 DEG C, 200rpm shakes 3h and reaches 4 or so to OD600 value, and the IPTG of final concentration 1mmol/L is added
Continue to shake bacterium 3h, bacterium solution is collected by centrifugation in 6000rpm.Cultivation temperature is reduced to 30 DEG C；IPTG inducer is added to final concentration
0.5mM, 30 DEG C of continuation shake culture 3-4h；8000rpm is centrifuged 3min and collects thallus, is resuspended in 50mL pre-cooling NTA-0 buffer
In, ice bath 30min；Ultrasonication thallus, 4 DEG C of centrifugation 50min of 16000rpm collect supernatant and precipitating；Take a small amount of supernatant and
Precipitating carries out SDS-PAGE detection, and protein expression result is as shown in Figure 1.Remaining supernatant and precipitating be placed in 4 DEG C it is spare.
3, protein purification, renaturation:
DTT to final concentration 1mM is added with the resuspension of 50mL NTA-0 buffer in precipitating, and ultrasound promotes foreign protein dissolution, centrifugation
Precipitating is collected, supreme clear bright in triplicate, precipitating is resuspended with PBS, and supernatant is removed in ultrasound, centrifugation, is forgiven with the resuspension of 6M guanidine hydrochloride
Body adds DTT to final concentration 5mM；37 DEG C of concussion 3h are all dissolved to inclusion body, and supernatant is removed in centrifugation.Use protein renaturation liquid by egg again
White low temperature dialysis renaturation is added dropwise to 200mL renaturation solution with the 3M guanidine hydrochloride diluted protein solution of 2 times of volumes at 4 DEG C
(pH8.0) in, rotational speed regulation to maximum, stirring for 24 hours, takes protein solution in bag filter, slow with PBS after being concentrated with PEG60000
Fliud flushing dialysed overnight.Albumen after renaturation is purified again with anion-exchange column Ni-NTA, collects albumen.Protein purification result is such as
Shown in Fig. 2.
4, the preparation and the selection of best peridium concentration of envelope antigen:
Purifying protein is serially diluted into 6 μ g/mL, 3 μ g/mL, 1.5 μ g/mL, 0.75 μ g/ with carbonate buffer solution (pH9.6)
ML, 0.38 μ g/mL, 0.19 μ g/mL, each two column of concentration coating are added in 96 hole elisa Plates, 4 DEG C of coatings according to 100 holes μ L/
Overnight.The result shows that positive value OD450 value is greater than 1.0 when envelope antigen concentration is 3 μ g/mL, and the value of P/N is maximum, therefore really
The fixed concentration is best peridium concentration (table 1).
1 Checkerboard titration antigen working concentration (OD450nm) of table
5, the preparation of standard positive and negative nose swab:
The positive control sample is the nose swab sample for acquiring foot and mouth disease A-type virus and attacking 7-21d pig, ox and sheep after poison
Product, a series of sample that detection OD450 value is 1.5 ± 0.05 after mixing, dilutions；The negative control sample isFMDV NS kit detect foot and mouth disease virus non-structural protein antibody be feminine gender, foot-and-mouth disease a type, it is O-shaped,
AsiaI Liquid-phase blocking ELISA kit detect serum antibody titer < 1/4, FMDV specific PCR detection antigen be negative pig,
The nose swab of ox and sheep detects the sample that OD450 value is 0.1 ± 0.05 after being mixed, being diluted, aseptic subpackaged spare.
6, the determination of best confining liquid:
ELISA Plate is closed with different confining liquids, 37 DEG C of closing 1h screen best confining liquid, the results are shown in Table 2.
Table 2 is closed (OD450nm) to ELISA Plate with different confining liquids
The result shows that positive value OD450 value is greater than when selecting the 10mM PBS for containing 0.5w/v%BSA as confining liquid
1.0, and the value of P/N is maximum, it is thus determined that the confining liquid is best confining liquid.
7, the closing of ELISA Plate:
Above-mentioned ELISA Plate washes 4 times with PBST (the 0.01mol/L PBS solution containing 0.5v/v%Tween20, pH 7.6), often
Hole adds 10mM PBS confining liquid of the 100 μ l containing 0.5w/v%BSA, 37 DEG C of placement 2h.Confining liquid is discarded, PBST is washed 3 times, obtained
The ELISA Plate of pre-coated antigen, every hole add the ELISA Plate stabilizer of 100 μ l horse serums containing 6v/v%, are placed at room temperature for 30min, discard
Naturally dry after liquid, using aluminium foil bag vacuum seal, 4 DEG C of preservations.
Wherein, the ELISA Plate stabilizer is to be added to 5g BSA, 10g sucrose, 20g trehalose
In 1000ml0.01mol/L PBS (pH7.4) gently concussion to dissolve, add 0.05w/v%Procline300, be stored in 4 DEG C it is standby
With.
8, enzyme labelled antibody (10 ×)
The anti-pig of the mouse of horseradish peroxidase-labeled, ox, sheep IgA secondary antibody, 10 times of uses of dilution when use.
9, sample diluting liquid
Sample diluting liquid is NaCl containing 0.5M, 2.68mM KCl, 2.79mM KH2PO4、8.1mM Na2HPO4, 5g/L junket
The mixed solution of albumen, 0.05%v/vTween20 and 10mM PBS.
10、PBST(10×)
0.1mol/LPBS solution containing 0.5v/v%Tween20, pH 7.6.10 times of uses of dilution when use.
11, developing solution and terminate liquid
Developing solution is TMB developing solution, and terminate liquid is 1moL/L H2SO4Solution.
(2) assembling and preservation of kit
According to kit contents listed by table 3, kit is assembled, is saved after composition loaded on 4 DEG C.
3 ELISA detection kit content of table
(3) application method (detection method) of kit
(1) it dilutes: PBST (10 ×) washing lotion being diluted to 250ml with aqua sterilisa, with sample diluting liquid by enzyme labelled antibody (10
×) it is diluted to 5ml.
(2) board-washing: opening the package, and takes out the ELISA Plate of pre-coated antigen, with PBST board-washing 4 times diluted, water suction
Paper pats dry, each 3min.
(3) it is loaded: sample is diluted with 1:2 times, every hole 100ul is added in reaction plate, and positive control and feminine gender is added
Control, 100 holes μ L/, the positive control, negative control do two repetitions respectively.37 DEG C of incubation 45min.
(4) enzyme labeling antibody: getting rid of sample, is rinsed 4 times with PBST, and blotting paper pats dry, and the horseradish peroxide diluted is added
The anti-pig of mouse, ox or the sheep IgA secondary antibody of compound enzyme label, 100 holes μ L/, 37 DEG C of incubation 30min.
(5) add substrate: getting rid of enzyme labelled antibody, rinsed 4 times with PBST, blotting paper pats dry.37 DEG C of TMB developing solution incubations are added
Then the reaction terminating liquid in 100 holes μ l/ is added in 15min.
(6) OD is read in microplate reader450Numerical value.
OD is read in microplate reader450Numerical value, the OD of sample to be tested450nmValue is more than or equal to negative control sera average value
2.5 times are judged to the positive, and 2.5 times less than negative control sera average value are judged to feminine gender.
Sensibility, the specificity, repeated experiment of 2 foot and mouth disease A-type virus sIgA antibody ELISA detection kit of embodiment
1, sensitivity experiments:
Kit is prepared using embodiment 1 and detects 90 parts of nose swab samples, these samples are received after poison is attacked in experiment
The nose swab sample of collection, does not carry out any immune before carrying out attacking poison, and detection method is with embodiment 1 by calculating positive detection
Rate analyzes the sensibility of this method.ELISA testing result shows 88 parts of the above Sample Positive, 2 parts negative, and positive rate is
97.78%.Illustrate that kit of the invention has preferable sensibility, the results are shown in Table 4.
Table 4ELISA kit sensitivity tests
| Detection method | Sample number (part) | Number positive (part) | Negative number (part) | Positive rate (%) |
| Indirect ELISA | 90 | 88 | 2 | 97.78% |
2, specificity experiments:
Kit is prepared using embodiment 1 and has detected Porcine epidemic diarrhea virus (PEDV) infection piglet intestinal mucosa punching
Wash Samples, porcine reproductive and respiratory syndrome virus (PRRSV) antibody positive sample, pig circular ring virus (PCV) specificity IgA are anti-
Body positive sample, swine fever virus (CSFV) Specific IgA antibody positive sample increase for A type, O-shaped FMDV cross-infection situation
84 parts of O-shaped FMDV infection hog snout swab samples are added to be detected, it is right using A type aftosa sIgA antibody positive sample as the positive
According to analyzing the specificity of this method.ELISA testing result shows that 84 parts of O-shaped FMDV infection hog snout swab samples are 1 part positive, can
7 parts are doubted, 76 parts negative, coincidence rate 90.5%.And it is sick with Porcine epidemic diarrhea virus (PEDV), porcine reproductive and respiratory syndrome
Malicious (PRRSV), pig circular ring virus (PCV), swine fever virus (CSFV) IgA antibody all without cross reaction, ensure that the diagnosis
Kit has good sensibility and specificity simultaneously, fundamentally ensure that the accuracy and reliability of result.
3, repeated experiment:
The positive schneiderian membrane swab sample and 1 part of negative schneiderian membrane swab sample for choosing 6 parts of different antibodies levels, using same
One batch antigen coat elisa plate carries out repeating to test in 4 times batches in different time, and statistic mixed-state is as a result, variation within batch coefficient
Less than 7% (1.53%~6.41%), show the antigen coat prepared with a schneiderian membrane swab sample in same batch
Coefficient of variation very little in elisa plate has good repeatability.Using the antigen coat elisa plate of 4 batches preparation same
Time carries out repeating to test between criticizing, and statistic mixed-state is as a result, interassay coefficient of variation less than 8% (1.27%~7.92%), shows together
A schneiderian membrane swab sample coefficient of variation also very little in the elisa plate of antigen coat prepared by different batches, illustrates this hair
The indirect ELISA method of bright foundation has repeated well (table 5).
5 repeated experiment result of table
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Sequence table
<110>Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences
<120>a kind of foot and mouth disease A-type virus sIgA antibody ELISA detection kit and its application
<160>2
<170>Patent-In 3.5
<210>1
<211>1287
<212>DNA
<213> artificial sequence
<400>1
atgggcgaga cccaggcgca gcgtcgttac cacaccgatg ttggtttcct gatggaccgt 60
ttcgtgcaga tcaagccggt tggcccgacc cacgtgatcg acctgatgca gacccaccaa 120
catggtctgg ttggcgcgat gctgcgtgcg gcgacctact atttcagcga tctggagatt 180
gtggttaacc acaccggtaa caagaaaaag accagcaaat atagcgcgcc gcagaaccgt 240
cgtggtgacc tgggtccgct ggcggcgcgt ctggcggcgc aactgccggc gagcttcaac 300
tttggcgcga tccgtgcgac cgaaatccgt ggtggcggtg gcagcggtgg cggtggcagc 360
gaagtgagca gccaggatcg tcacaaacaa aagatcattg cgccggcgaa gcaactgctg 420
ggtccgggtc cgggtggtga aacccaggtt caacgtcgtc accacaccga cgtgagcttc 480
atcatggatc gttttgtgca aattaagccg gttagcccga cccatgttat tgatctgatg 540
caaacccatc agcatggtct ggttggcgct atgctgcgcg cggcgaccta ttacttcagc 600
gatctggaaa tcgttgttaa ccacaccggt cgtaaaaaga aaaccagcaa atatagcgct 660
cctgcgactc gtcgtggtga cctgggcagc ctggcggcgc gcctggcggc gcagctgccg 720
gcgagcttta actatggtgc gatccgtgcg accgagattc aaggtggcgg tggcagcggt 780
ggcggtggca gcgaagtgac cagccaagac cgccataagc agaaaattat cgctcctgcg 840
aagcaactgc tgggcccggg cccgggtggt gaaacccaag ttcaacgtcg tcaacacacc 900
aacgtgggtt tcatcatgga ccgttttgtt aagattccga gccagagccc gacccacgtg 960
atcgatctga tgcaaaccca ccagcatggt ctggttggcg cattattacg cgcggcgacc 1020
tattatttta gcgacctgga aatcgtggtt cgtcacgacg ataacaagaa aaagaccacc 1080
aaatatagca ccggtaacgc gggtcgtcgt ggtgatctgg gcagcctggc tgctcgtgtt 1140
gctgctcagc tgccggcgag cttcaatttc ggcgcgattc gcgcgaccgt tatccatggt 1200
ggcggtggca gcggtggcgg tggcagcaag gttaccagcc aggaccgtca taaacaacgc 1260
atcatcgcgc cggcgaaaca actgctg 1287
<210>2
<211>429
<212>PRT
<213>artificial sequence
<400>2
Met Gly Glu Thr Gln Ala Gln Arg Arg Tyr His Thr Asp Val Gly Phe Leu Met Asp Arg 20
Phe Val Gln Ile Lys Pro Val Gly Pro Thr His Val Ile Asp Leu Met Gln Thr His Gln 40
His Gly Leu Val Gly Ala Met Leu Arg Ala Ala Thr Tyr Tyr Phe Ser Asp Leu Glu Ile 60
Val Val Asn His Thr Gly Asn Lys Lys Lys Thr Ser Lys Tyr Ser Ala Pro Gln Asn Arg 80
Arg Gly Asp Leu Gly Pro Leu Ala Ala Arg Leu Ala Ala Gln Leu Pro Ala Ser Phe Asn 100
Phe Gly Ala Ile Arg Ala Thr Glu Ile Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 120
Glu Val Ser Ser Gln Asp Arg His Lys Gln Lys Ile Ile Ala Pro Ala Lys Gln Leu Leu 140
Gly Pro Gly Pro Gly Gly Glu Thr Gln Val Gln Arg Arg His His Thr Asp Val Ser Phe 160
Ile Met Asp Arg Phe Val Gln Ile Lys Pro Val Ser Pro Thr His Val Ile Asp Leu Met 180
Gln Thr His Gln His Gly Leu Val Gly Ala Met Leu Arg Ala Ala Thr Tyr Tyr Phe Ser 200
Asp Leu Glu Ile Val Val Asn His Thr Gly Arg Lys Lys Lys Thr Ser Lys Tyr Ser Ala 220
Pro Ala Thr Arg Arg Gly Asp Leu Gly Ser Leu Ala Ala Arg Leu Ala Ala Gln Leu Pro 240
Ala Ser Phe Asn Tyr Gly Ala Ile Arg Ala Thr Glu Ile Gln Gly Gly Gly Gly Ser Gly 260
Gly Gly Gly Ser Glu Val Thr Ser Gln Asp Arg His Lys Gln Lys Ile Ile Ala Pro Ala 280
Lys Gln Leu Leu Gly Pro Gly Pro Gly Gly Glu Thr Gln Val Gln Arg Arg Gln His Thr 300
Asn Val Gly Phe Ile Met Asp Arg Phe Val Lys Ile Pro Ser Gln Ser Pro Thr His Val 320
Ile Asp Leu Met Gln Thr His Gln His Gly Leu Val Gly Ala Leu Leu Arg Ala Ala Thr 340
Tyr Tyr Phe Ser Asp Leu Glu Ile Val Val Arg His Asp Asp Asn Lys Lys Lys Thr Thr 360
Lys Tyr Ser Thr Gly Asn Ala Gly Arg Arg Gly Asp Leu Gly Ser Leu Ala Ala Arg Val 380
Ala Ala Gln Leu Pro Ala Ser Phe Asn Phe Gly Ala Ile Arg Ala Thr Val Ile His Gly 400
Gly Gly Gly Ser Gly Gly Gly Gly Ser Lys Val Thr Ser Gln Asp Arg His Lys Gln Arg 420
Ile Ile Ala Pro Ala Lys Gln Leu Leu 429
Claims (10)
1. a kind of foot and mouth disease A-type virus broad spectrum multi-epitope recombination antigen, which is characterized in that the foot and mouth disease A-type virus wide spectrum
Multi-epitope recombination antigen includes that foot and mouth disease A-type virus represents strain A/GDMM/2013, A/HuBWH/2009, A/GSLX/62's
20-85,133-172 and 193-212 main neutrality epitope sequences in VP1 albumen, it is described
Foot and mouth disease A-type virus broad spectrum multi-epitope recombination antigen amino acid sequence as shown in SEQ ID No.2.
2. encoding the nucleotide sequence of foot and mouth disease A-type virus broad spectrum multi-epitope recombination antigen described in claim 1, it is preferred that
The nucleotide sequence is as shown in SEQ ID No.1.
3. foot and mouth disease A-type virus broad spectrum multi-epitope recombination antigen described in claim 1 detects foot and mouth disease A-type virus in preparation
Purposes in sIgA antibody reagent.
4. a kind of foot and mouth disease A-type virus sIgA antibody ELISA detection kit, which is characterized in that the kit includes right
It is required that the coated ELISA Plate of foot and mouth disease A-type virus broad spectrum multi-epitope recombination antigen described in 1,100 × concentration enzyme labelled antibody, enzyme mark
Antibody diluent, sample diluting liquid, concentrated cleaning solution, developing solution, terminate liquid, positive control sample and negative control sample.
5. foot and mouth disease A-type virus sIgA antibody ELISA detection kit as claimed in claim 4, which is characterized in that the mouth
Fever aphthous A type virus broad spectrum multi-epitope recombination antigen is obtained through prokaryotic expression.
6. foot and mouth disease A-type virus sIgA antibody ELISA detection kit as claimed in claim 4, which is characterized in that described
The coated ELISA Plate of foot and mouth disease A-type virus broad spectrum multi-epitope recombination antigen is prepared in accordance with the following methods:
(1) preparation and coating of envelope antigen
The recombinant protein inclusion body that prokaryotic expression obtains purifies to obtain foot and mouth disease A-type virus wide through renaturation and Ni-NTA
Multi-epitope recombination antigen is composed, for amino acid sequence as shown in SEQ ID No.2, when coating is dilute with the carbonate buffer solution of pH9.6
It releases to 3 μ g/ml, according to 100 hole μ l/ coated elisa plates, 4 DEG C of refrigerators are stood overnight；
(2) closing and preservation of ELISA Plate
It is coated with overnight ELISA Plate and washs 4 times with PBST and pat dry, every hole adds 10mM PBS closing of the 100 μ l containing 0.5w/v%BSA
Liquid, 37 DEG C of placement 2h discard confining liquid, and PBST is washed 3 times, obtain the ELISA Plate of pre-coated antigen, and every hole adds 100 μ l containing 6v/
The ELISA Plate stabilizer of v% horse serum, is placed at room temperature for 30min, discards naturally dry after liquid, with aluminium foil bag vacuum seal, 4 DEG C
It saves；
Wherein, the ELISA Plate stabilizer is that 5g BSA, 10g sucrose, 20g trehalose are added to 1000ml0.01mol/L
In PBS (pH7.4) gently concussion to dissolve, add 0.05w/v%Procline300, be stored in 4 DEG C it is spare.
7. foot and mouth disease A-type virus sIgA antibody ELISA detection kit as claimed in claim 4, which is characterized in that the sun
Property control sample be to acquire foot and mouth disease A-type virus to attack the nose swab sample of 7-21d pig, ox and sheep after poison, through mixing, a series of
The sample that OD450 value is 1.5 ± 0.05 is detected after dilution；The negative sample isThe inspection of FMDV NS kit
Surveying foot and mouth disease virus non-structural protein antibody is feminine gender, and foot-and-mouth disease a type, O-shaped, AsiaI Liquid-phase blocking ELISA kit detect blood
Clear antibody titer < 1/4, FMDV specific PCR detection antigen is the nose swab of negative pig, ox and sheep, after being mixed, being diluted
The sample that OD450 value is 0.1 ± 0.05 is detected, it is aseptic subpackaged spare.
8. foot and mouth disease A-type virus sIgA antibody ELISA detection kit as claimed in claim 4, which is characterized in that described
100 × concentration enzyme labelled antibody is the monoclonal antibody that horseradish peroxidase (HRP) marks the anti-pig of mouse, ox or sheep IgA, is used
When, with using after 100 times of enzyme labelled antibody dilution dilutions, the enzyme labelled antibody dilution is containing 1v/v% glycerol, 0.5w/
The 0.01mol/LPBS buffer of v% bovine serum albumin(BSA), 1w/v% casein and 0.05w/v%Procline300,
pH7.4。
9. foot and mouth disease A-type virus sIgA antibody ELISA detection kit as claimed in claim 4, which is characterized in that described
Sample diluting liquid is NaCl containing 0.5M, 2.68mM KCl, 2.79mM KH2PO4、8.1mM Na2HPO4, 5g/L casein,
The mixed solution of 0.05%Tween20 and 10mM PBS；The developing solution is TMB developing solution, and the terminate liquid is 1moL/
L H2SO4Solution；The concentrated cleaning solution is 10 × PBST solution, i.e. the 0.1mol/L PBS containing 0.5v/v%Tween20
Solution, pH 7.6 dilute 10 times when use.
10. foot and mouth disease A-type virus sIgA antibody ELISA detection kit as described in claim 1, which is characterized in that utilize
When the kit carries out foot and mouth disease A-type virus sIgA antibody test, follow the steps below:
(1) Sample Dilution
By sample to be tested, positive control and negative control, with sample diluting liquid, 1:2 is diluted by volume；
(2) board-washing
The coated ELISA Plate of foot and mouth disease A-type virus broad spectrum multi-epitope recombination antigen is taken out from 4 DEG C of refrigerators, is opened aluminium foil bag, is taken
ELISA Plate out, with cleaning solution board-washing 3 times diluted, blotting paper is patted dry, each 3min；
(3) it is loaded
Sample to be tested, positive control and negative control after dilution is separately added into ELISA Plate, every 100 μ L of hole, 37 DEG C incubate
Educate 45min；
(4) it washs
ELISA Plate is taken out, sample is got rid of, is rinsed 4 times with cleaning solution, blotting paper pats dry；
(5) enzyme labelled antibody is added
The anti-pig of mouse, ox or the sheep IgA monoclonal antibody for the horseradish peroxidase-labeled that addition has diluted, 100 holes μ L/, 37 DEG C
Work 30min；
(6) add substrate:
Enzyme labelled antibody is got rid of, is rinsed 4 times with cleaning solution, blotting paper pats dry, and 37 DEG C of colour developing 15min of developing solution are added, and stands after taking-up
Terminate liquid is added；
(7) result judgement
OD450nm numerical value is read in microplate reader, the OD450nm value of sample to be tested is more than or equal to negative control sample average value
2.5 times are judged to the positive, and 2.5 times less than negative control sample average value are judged to feminine gender.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109655612A (en) * | 2019-01-31 | 2019-04-19 | 中国农业科学院兰州兽医研究所 | A kind of detection kit and detection method of Asia1 type foot and mouth disease virus |
| CN110526959A (en) * | 2019-08-29 | 2019-12-03 | 中国农业科学院兰州兽医研究所 | A kind of foot-and-mouth disease virus antigen, the gene for expressing foot-and-mouth disease virus antigen and its recombinant vector and Recombinant Lactococcus lactis |
| CN112358544A (en) * | 2020-11-17 | 2021-02-12 | 中国农业科学院兰州兽医研究所 | FMDV A type bovine-derived broad-spectrum neutralizing monoclonal antibody W145 and neutralizing antibody competition ELISA detection kit |
| CN113063941A (en) * | 2021-03-25 | 2021-07-02 | 哈尔滨国生生物科技股份有限公司 | ELISA detection method and detection kit for detecting IgA antibody of African swine fever virus |
| CN113267621A (en) * | 2021-05-14 | 2021-08-17 | 北京金诺百泰生物技术有限公司 | Stabilizer for ELISA kit coated plate, preparation method of stabilizer, kit coated plate and kit |
| CN114705857A (en) * | 2022-05-16 | 2022-07-05 | 北京亿森宝生物科技有限公司 | Microporous plate type chemiluminescence detection kit for pig foot-and-mouth disease virus O-type and A-type antibodies and application thereof |
Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102180952A (en) * | 2011-05-10 | 2011-09-14 | 申联生物医药（上海）有限公司 | Foot and mouth disease virus antigen polypeptide, fusion antigen polypeptide and vaccine |
| CN103193869A (en) * | 2013-03-29 | 2013-07-10 | 中国农业科学院兰州兽医研究所 | Cattle food-and-mouth disease virus A type synthetic peptide and preparation and application thereof |
| CN103472222A (en) * | 2013-08-26 | 2013-12-25 | 河北省科学院生物研究所 | Long-acting ELISA plate stabilizing agent |
| CN103554234A (en) * | 2013-09-05 | 2014-02-05 | 广西壮族自治区动物疫病预防控制中心 | Competitive ELISA method based on foot-and-mouth disease A type VP1 protein and its monoclonal antibody |
| CN104693310A (en) * | 2015-02-17 | 2015-06-10 | 江苏省农业科学院 | Chimeric protein, virus-like particle and application thereof |
| CN105056227A (en) * | 2015-07-21 | 2015-11-18 | 复旦大学 | Anti-FMDV (foot and mouth disease virus) VLP (virus-like particles) vaccine and preparation method thereof |
| CN105777909A (en) * | 2016-03-03 | 2016-07-20 | 中国农业科学院兰州兽医研究所 | A-type foot-and-mouth disease targeting composite epitope protein mediated by pig chemotactic factors and vaccine |
| CN106706923A (en) * | 2016-11-25 | 2017-05-24 | 中国农业科学院兰州兽医研究所 | Method for detecting specific IgA antibody of O-type foot-and-mouth disease virus of pig and detection kit thereof |
| CN106706903A (en) * | 2016-11-25 | 2017-05-24 | 中国农业科学院兰州兽医研究所 | Detection method and detection kit for porcine type A foot-and-mouth disease virus specific IgA antibody |
| CN107365367A (en) * | 2017-08-16 | 2017-11-21 | 山东省农业科学院奶牛研究中心 | A types FMDV VP1 Protein Epitopes gene polypeptide and its application |
| US20180169216A1 (en) * | 2015-11-23 | 2018-06-21 | Merial Inc. | FMDV and E2 Fusion Proteins and Uses Thereof |
| KR20180068937A (en) * | 2018-06-14 | 2018-06-22 | 대한민국(농림축산식품부 농림축산검역본부장) | Soluble Multi-Epitope Antigen of Foot-and-Mouth Disease Virus and Uses Thereof |
-
2018
- 2018-09-13 CN CN201811069024.7A patent/CN109187993B/en active Active
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102180952A (en) * | 2011-05-10 | 2011-09-14 | 申联生物医药（上海）有限公司 | Foot and mouth disease virus antigen polypeptide, fusion antigen polypeptide and vaccine |
| CN103193869A (en) * | 2013-03-29 | 2013-07-10 | 中国农业科学院兰州兽医研究所 | Cattle food-and-mouth disease virus A type synthetic peptide and preparation and application thereof |
| CN103472222A (en) * | 2013-08-26 | 2013-12-25 | 河北省科学院生物研究所 | Long-acting ELISA plate stabilizing agent |
| CN103554234A (en) * | 2013-09-05 | 2014-02-05 | 广西壮族自治区动物疫病预防控制中心 | Competitive ELISA method based on foot-and-mouth disease A type VP1 protein and its monoclonal antibody |
| CN104693310A (en) * | 2015-02-17 | 2015-06-10 | 江苏省农业科学院 | Chimeric protein, virus-like particle and application thereof |
| CN105056227A (en) * | 2015-07-21 | 2015-11-18 | 复旦大学 | Anti-FMDV (foot and mouth disease virus) VLP (virus-like particles) vaccine and preparation method thereof |
| US20180169216A1 (en) * | 2015-11-23 | 2018-06-21 | Merial Inc. | FMDV and E2 Fusion Proteins and Uses Thereof |
| CN105777909A (en) * | 2016-03-03 | 2016-07-20 | 中国农业科学院兰州兽医研究所 | A-type foot-and-mouth disease targeting composite epitope protein mediated by pig chemotactic factors and vaccine |
| CN106706923A (en) * | 2016-11-25 | 2017-05-24 | 中国农业科学院兰州兽医研究所 | Method for detecting specific IgA antibody of O-type foot-and-mouth disease virus of pig and detection kit thereof |
| CN106706903A (en) * | 2016-11-25 | 2017-05-24 | 中国农业科学院兰州兽医研究所 | Detection method and detection kit for porcine type A foot-and-mouth disease virus specific IgA antibody |
| CN107365367A (en) * | 2017-08-16 | 2017-11-21 | 山东省农业科学院奶牛研究中心 | A types FMDV VP1 Protein Epitopes gene polypeptide and its application |
| KR20180068937A (en) * | 2018-06-14 | 2018-06-22 | 대한민국(농림축산식품부 농림축산검역본부장) | Soluble Multi-Epitope Antigen of Foot-and-Mouth Disease Virus and Uses Thereof |
Non-Patent Citations (6)
| Title |
|---|
| 娄慧，等。: "口蹄疫病毒A/WH/09株结构蛋白P1基因的克隆及其B细胞抗原表位的预测。", 《中国兽医科学》 * |
| 尚延丽。: "口蹄疫病毒A/GDMM/CHA/2013株VP1 H-2d限制性CTL表位预测与鉴定。", 《中国优秀硕士学位论文全文数据库》 * |
| 张昱 等。: "口蹄疫病毒株AF72 VP1的结构构建与B细胞表位预测。", 《生物技术通报》 * |
| 李世芳 等。: "口蹄疫表位疫苗的研究进展。", 《中国兽牧兽医》 * |
| 李登科，等。: "A型口蹄疫病毒多抗原表位杆状病毒表达载体的构建及表达。", 《中国兽医科学》 * |
| 杜以军 等。: "猪口蹄疫病毒多抗原表位的原核表达与纯化。", 《兽牧与兽医》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109655612A (en) * | 2019-01-31 | 2019-04-19 | 中国农业科学院兰州兽医研究所 | A kind of detection kit and detection method of Asia1 type foot and mouth disease virus |
| CN110526959A (en) * | 2019-08-29 | 2019-12-03 | 中国农业科学院兰州兽医研究所 | A kind of foot-and-mouth disease virus antigen, the gene for expressing foot-and-mouth disease virus antigen and its recombinant vector and Recombinant Lactococcus lactis |
| CN112358544A (en) * | 2020-11-17 | 2021-02-12 | 中国农业科学院兰州兽医研究所 | FMDV A type bovine-derived broad-spectrum neutralizing monoclonal antibody W145 and neutralizing antibody competition ELISA detection kit |
| CN113063941A (en) * | 2021-03-25 | 2021-07-02 | 哈尔滨国生生物科技股份有限公司 | ELISA detection method and detection kit for detecting IgA antibody of African swine fever virus |
| CN113267621A (en) * | 2021-05-14 | 2021-08-17 | 北京金诺百泰生物技术有限公司 | Stabilizer for ELISA kit coated plate, preparation method of stabilizer, kit coated plate and kit |
| CN114705857A (en) * | 2022-05-16 | 2022-07-05 | 北京亿森宝生物科技有限公司 | Microporous plate type chemiluminescence detection kit for pig foot-and-mouth disease virus O-type and A-type antibodies and application thereof |
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