CN105348372A - Method for detecting porcine pseudorabies virus - Google Patents
Method for detecting porcine pseudorabies virus Download PDFInfo
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- CN105348372A CN105348372A CN201510697228.5A CN201510697228A CN105348372A CN 105348372 A CN105348372 A CN 105348372A CN 201510697228 A CN201510697228 A CN 201510697228A CN 105348372 A CN105348372 A CN 105348372A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
- C07K16/085—Herpetoviridae, e.g. pseudorabies virus, Epstein-Barr virus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
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- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/01—DNA viruses
- G01N2333/03—Herpetoviridae, e.g. pseudorabies virus
- G01N2333/032—Pseudorabies virus, i.e. Aujetzky virus
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Abstract
The invention provides a method for detecting porcine pseudorabies virus. The method uses a high-specificity monoclonal antibody of gE protein having an amino acid sequence being SEQ ID NO: 1, a blocking ELISA method established based on the monoclonal antibody has the characteristics of high specificity, strong sensitivity and no false positive detection, so that the method provides a scientific basis for PR accurate diagnosis.
Description
Technical field
The invention belongs to disease of domestic animals diagnostic techniques field, be specifically related to a kind of porcine pseudorabies virus detection method.
Background technology
Porcine pseudorabies (PseudorabiesPR) is a kind of high degree in contact, the acute infectious disease of the multiple domestic animal such as pig, ox, sheep and the wildlife caused by porcine pseudorabies virus (PseudorabiesvirusPRV).Pig is the main natural host of PR and topmost contagium.The pig of each age level all can be infected, and this disease, in the many breaks out and spread of swinery, brings huge financial loss to the pig industry in China and even the whole world.At present, the main Bartha-K61 attenuated vaccine of inoculation gE genetically deficient that relies on prevents this disease.But new virulence Geng Qiang variation strain has appearred in the many provinces of China since 2011, causes very large financial loss, even if the pig of inoculation can not escape death by sheer luck, there is the death of Different age group pig.Show that Bartha-K61 attenuated vaccine can not provide protection completely.
And, because viral genome is easy to morph, the existing antibody for detecting is caused to lose effect, this just increases the difficulty of PRV being carried out to correct diagnosis, therefore carry out the method for differential diagnosis in the urgent need to setting up the high PRV of a species specificity, thus correctly diagnose the means that science is provided for PR.
Summary of the invention
The object of this invention is to provide a kind of detection method of PRV (Pseudorabies virus), with the highly specific monoclonal antibody for PRVgE albumen prepared by the present invention, the stop band restrain method set up based on this monoclonal antibody has the advantages that specificity is high, sensitivity strong, detect without false sun, thus provides the foundation of science for PR Accurate Diagnosis.
The present invention first provide a kind of have new there is good immunogenic gE albumen, the gE albumen of the PRV reported than before, the insertion of 1 aspartic acid is respectively had the 48th and 492nd ~ 496, this region is in the structural domain residing for gE Protein Epitopes just, amino acid whose insertion probably changes the epitope that known strain exists, cause its pathogenic enhancing, its aminoacid sequence is SEQIDNO:1;
Encode the gene of above-mentioned gE albumen, its a kind of nucleotide sequence is SEQIDNO:2;
Above-mentioned gE albumen as antigen for the preparation of antibody;
Another aspect of the present invention provides a kind of stop band restrain method detecting ill swinery antibody, comprises following concrete steps:
By gE proteantigen through diluted, be quantitatively coated in ELISA96 orifice plate, 4 DEG C are spent the night;
With PBST washings, unconjugated antigen is cleaned;
Respectively negative serum, positive serum and serum to be checked are added in corresponding detect aperture, slightly shake, make the liquid blending in Sptting plate;
Micro-reaction plate sealing is placed on 37 DEG C hatch;
Vertically outwell the liquid in Sptting plate, and carry out cleaning each hole with washings;
With confining liquid, Sptting plate is closed, be placed in 37 DEG C and hatch;
Remove the liquid in Sptting plate, and carry out cleaning each hole with washings;
The enzyme mark monoclonal antibody diluted is joined in each reacting hole, is placed in 37 DEG C and hatches;
Outwell the liquid in Sptting plate, and carry out cleaning each hole with washings;
Add substrate, be placed in 37 DEG C of reactions;
Add stop buffer, termination reaction, and read 450nm absorbance in microplate reader, sample value/feminine gender value is greater than 2 and is judged to be the positive, is less than 1 for negative.
Wherein the preparation method of negative serum is: be negative serum without immune serum;
Positive serum is the separation of serum of gE protein immunization mouse.
Described confining liquid is: 5% skim-milk
Washings is: containing the PBS solution of 0.5 ‰ tween 20s;
The enzyme mark monoclonal antibody used, its preparation method is:
To be used for after enzyme target monoclonal antibody carries out purifying, carry out horseradish peroxidase (HRP) to monoclonal antibody and carry out enzyme mark, concrete steps are as follows:
Taking 5mgHRP is dissolved in 0.5ml distilled water;
Add 0.5ml0.06MNaIO4,4 DEG C of jog 30min, solution presents yellow-green colour;
Add 0.5ml0.16M ethylene glycol, lucifuge jog 30min under room temperature, stop oxidizing reaction;
Add 5mg antibody, load in dialysis tubing, be placed in 0.05M, pH is in the carbonate buffer solution (CBS) of 9.6,4 DEG C of dialysed overnight, and period changes at least 3 CBS;
Take out the liquid in dialysis tubing, add 5mg/mlNaHB40.2ml, 4 DEG C are spent the night;
Add equal-volume saturated ammonium sulphate solution precipitation binding substances, after 4 DEG C of 30min, the centrifugal 15min of 3000rpm, abandons supernatant;
Throw out is dissolved in PBS, loads in dialysis tubing, and be that damping fluid is dialysed 6-12h with PBS, change 3 dialyzates;
Finally, liquid-20 DEG C of packing obtained are preserved.
Stop buffer is: 2MH
2sO
4.Preparation method is distilled water 178.3ml, dropwise adds vitriol oil 21.7ml.
Of the present invention have the high monoclonal antibody of specificity by preparation, and set up the method detecting PR strain based on this monoclonal antibody, thus PR strain popular is at present had to the effect of good differential diagnosis.
Embodiment
Come to describe in detail income of the present invention further below in conjunction with specific embodiment, those of ordinary skill in the art is on the basis of technical solution of the present invention, the method steps that this area is conventional can be selected, and be not limited only to the concrete record of specification sheets embodiment of the present invention.
The structure of the clone of embodiment 1:PRV major antigen gE gene plasmid, conversion and expression vector
Adopt bioinformatics method, and combine relevant bibliographical information, the present invention have chosen gE gene and comprises the gene order that major antigenic sites film exterior domain 1bp ~ 1275bp amounts to 1275bp and increase, the masterplate DNA that amplification adopts for this laboratory preferred and be sent to the bacterial classification center of containing and be numbered CGMCCNo.10266 strain through early stage.
The object fragment of 1275bp is obtained after pcr amplification, and it is connected with pMD-19T carrier, after order-checking is correct, after BamHI and XhoI double digestion, be connected to after glue recovery on the pGEX-6p-1 expression vector cut through same enzyme, after order-checking is correct, called after pGEX-gE carries out fungi preservation, and tests for downstream.Be classified as SEQIDNO:1 to the plasmid nucleotides sequence obtaining object fragment that checks order, the aminoacid sequence of coding is SEQIDNO:2;
Comparing to the new gE nucleotide sequence be separated, it is relatively far away with the sibship of the strain be separated in the past.GE gene the 48th and 492nd ~ 496 respectively has the insertion of 1 aspartic acid.And the strain be separated in the past only has individually a position to insert amino acid, the overwhelming majority is insertion not.This is likely that existing vaccine can not provide the reason protected completely place.
Table 1: the primer used in amplification
The prokaryotic protein expression of embodiment 2:gE gene, analysis and purifying
Recombinant plasmid pGEX-gE is transformed and expresses competence BL21 (DE3), picking K
+white list bacterium colony on LB flat board, is inoculated in 5ml containing A
+lB nutrient solution in 37 DEG C of incubated overnight.Next day, bacterium liquid is inoculated in containing K in 1:100 ratio
+lB nutrient solution in, 37 DEG C of shaking table shaking culture are to OD
600nmwhen reaching 0.4 ~ 0.6, and the optimal expression condition determining albumen is groped to the condition such as temperature, IPTG induced concentration, induction time expressed.By the analysis of the albumen of expression through SDS-PAGE expression, and analyze through the antigenicity of agarose diffusion experiment to expressing protein.A large amount of amplification target protein, and utilize GST protein purification test kit to carry out purifying to soluble proteins, and by BCA test kit and nanodrop2000, protein content is measured.
Embodiment 3: the preparation of monoclonal antibody
1. animal immune
With the prokaryotic expression recombinant protein gE after purifying for immunogen, immunity 4 ~ 6 week age female Balb/c mouse.Immunity 3 times altogether, every minor tick two weeks.One exempts from the recombinant protein gE albumen after by purifying and isopyknic Freund's complete adjuvant mixing and emulsifying, and immunization route is subcutaneous inoculation; Second and third immunity is all by purifying gE and isopyknic Freund's incomplete adjuvant mixing and emulsifying, and immunization route is peritoneal immunity.Three times immunizing dose is only 100 μ g/.3d before cytogamy, injects 100 μ g purifying gE albumen (not adding adjuvant) to the Balb/c mouse peritoneal of good immune effect, carries out booster immunization.
2.PRV purifying
PRV is inoculated Vero cell, MOI=1.37 DEG C of incubations, harvested cell after 48h ~ 72h.
After the cell multigelation three times of results, 4 DEG C of centrifugal 30min of 4000rpm, the then centrifugal 60min of 8000rpm again, abandon precipitation.
By the supernatant ultracentrifugation collected, 4 DEG C of centrifugal 1h30min of 25000rpm, abandon supernatant.
With appropriate PBS dissolution precipitation, through 20%, 40% and 60% sucrose density gradient, 4 DEG C of centrifugal 2.5h of 30000rpm, abandon supernatant.
Again use appropriate PBS dissolution precipitation, 4 DEG C of centrifugal 3h of 100000g carry out desugar, abandon supernatant.
Add appropriate PBS dissolution precipitation, packing is stored in-70 DEG C.Its concentration is surveyed by BCA method.
3. the foundation of indirect ELISA method
By 7 ~ 9d mouse docking blood sampling after two, three immunity, separation of serum.With the PRV of sucrose density gradient purifying be antigen, with immune mouse serum be positive serum, non-immune mouse serum for negative serum, set up screening hybridoma indirect ELISA detection method.Concrete steps are as follows:
Started to do 2 times of gradient dilutions with the concentration of 10 μ g/ml by PRV after purifying with CBS damping fluid, 100 μ l/ holes, 4 DEG C of bags are spent the night;
Abandon coating buffer, PBST vibration washes plate 3 times, 3min/ time, pats dry;
The PBS added containing 5% skimming milk closes, 200 μ l/ holes, 37 DEG C hatch 2h after wash plate as aforesaid method;
Mouse yin and yang attribute serum is l:100, l:200, l:400,1:800 doubly dilute, 100 μ l/ holes, establish blank simultaneously.Plate is washed after 37 DEG C of effect 1h;
Sheep anti mouse HRP-IgG is done 20000 times of dilutions, 100 μ l/ holes, after 37 DEG C of effect 45min, wash plate;
Every hole adds 100 μ lTMB nitrite ions, 37 DEG C of lucifuge colour developing 15min.2MH
2sO
4after (50 μ l/ hole) termination reaction, measure OD in microplate reader
450nmvalue.
4. cytogamy
1) preparation of feeder layer cells
1d before cytogamy, by lethal for not immune for health in 8 ~ 12 week age BALB/c mouse eyeball blood sampling, be separated the serum obtained and can be used as negative control.Put in 75% alcohol and soak 5min, be put in Biohazard Safety Equipment and fix;
Mention its skin of abdomen with aseptic nipper, carefully will cut off and the stripping of pausing property herein, and make peritonaeum fully and intactly expose.Simultaneously with cotton ball soaked in alcohol cleaning disinfection gently;
Draw 5mlDMEM basic culture solution with disposable sterilized injector and inject mouse peritoneal, fixing syringe also massages its belly 1-2min gently with cotton ball soaked in alcohol.Nutrient solution subsequently in sucking-off abdominal cavity injects HAT nutrient solution.
Be added in 96 porocyte culture plates after cell is mixed, 100 μ l/ holes.96 porocyte culture plates are placed in 37 DEG C, 5%CO
2cultivate in incubator.
Observation of cell upgrowth situation after 12 ~ 24h.Cell is polymorphism, adherent closely and refractivity is good, then can carry out follow-up test.
2) preparation of SP2/0 cell
1 ~ 2d before cytogamy, SP2/0 cell expansion growth conditions is good, full, bright, refractivity is good is cultivated.Merge the same day with DMEM basic culture solution by SP2/0 cell harvesting in centrifuge tube, the centrifugal 10min of 1000rpm in horizontal centrifuge.Abandon supernatant liquor, then use DMEM basic culture solution that cell is resuspended, for subsequent use after cell counting.
3) preparation of splenocyte
By lethal for the blood sampling of the BALB/c mouse eyeball of 3d after booster immunization, be separated the serum obtained and can be used as positive control.Be soaked in 75% alcohol the 5min that sterilizes, be put in Biohazard Safety Equipment and fix.
Cut off its skin of abdomen and peritonaeum successively with sterile scissors, tweezers, take out spleen, put into the sterilized petri dishes being added with DMEM basic culture solution in advance and clean one time, peel off the reticular tissue on tunicle and fat as far as possible.
Spleen after cleaning is moved into another to be equipped with in the sterilized petri dishes of DMEM basic culture solution, slowly inserts, and inject nutrient solution simultaneously with sterilizing syringe after drawing substratum from spleen one end.Repeatedly for several times until spleen color is transparent.By the centrifugal 10min of splenocyte 1000rpm in horizontal centrifuge under washing.Abandon supernatant liquor.
Use DMEM basic culture solution that cell is resuspended again, for subsequent use after cell counting.
4) cytogamy
SP2/0 cell after counting and mouse boosting cell are moved into 50ml centrifuge tube according to 1:5-1:10 ratio, and the centrifugal 10min of 1000rpm, abandons supernatant, avoids residual liquid as far as possible.
Pat bottom centrifuge tube in the centre of the palm, make that two kinds of cells are loose is mixed into pasty state, be placed in 37 DEG C of warm water.
The PEG4000 getting 1ml37 DEG C of preheating slowly instills within 1min, drips while rotate centrifuge tube, leaves standstill 1min.
Drip DMEM basic culture solution and stop PEG effect.1min instills 1mlDMEM basic culture solution, and 2min instills 2ml, and 3min instills 3ml, progressively dilutes PEG4000 successively, adds 25mlDMEM basic culture solution altogether.Whole process is wanted to drip while rotate centrifuge tube.The centrifugal 10min of 1000rmp, abandons supernatant.
Hanged cell precipitation with HAT nutrient solution, 100 μ l/ holes add the 96 porocyte culture plates that are covered with feeder cell and are placed in 37 DEG C, cultivate in 5%CO2 incubator.
5) screening of positive hybridoma cell, clone
After merging, 4d starts observation of cell state, and namely 4-6d can be observed the cell merged, and 7d adopts the mode of partly changing liquid to change nutrient solution, namely carries out changing liquid with HT nutrient solution.About 10d just can naked-eye observation to hybridoma colonies.The screening of positive hybridoma cell can be carried out when cell colony grows to culture hole 1/5-1/3.
About about 10d after cytogamy, gets 100 μ l cell conditioned medium liquid carry out positive clone strain screening according to the indirect ELISA method of the detection hybridoma above set up.Arrange mouse positive serum, negative serum and SP2/0 supernatant liquor to contrast as yin and yang attribute simultaneously.
The cell strain of limiting dilution assay to positive colony hole is adopted to clone.Blowing afloat mixing by detecting positive hybridoma colonies, taking a morsel and carrying out cell counting.According to cell counts, draw a certain amount of cell and start to carry out 10 times of dilutions in 1.5mlEP pipe (often pipe 1mlDMEM basic culture solution), until be diluted to 1ml containing 100-200 cell.This 1ml nutrient solution is joined 20mlHT nutrient solution, after mixing, adds 96 porocyte culture plates, 200 μ l/ holes.Remaining cell after cloning is carried out enlarged culturing frozen.
6) preparation of monoclonal antibody ascites and purifying thereof
Get female Balb/c mouse in 8 ~ 12 week age, first abdominal injection 500 μ l/ Freund's incomplete adjuvant, one to two all pneumoretroperitoneum injections 1 × 10
6an individual/hybridoma.When mouse web portion obviously expands and be handicapped, extract its ascites.By centrifugal for ascites 3000rpm 10min, get its supernatant packing mark, in-20 DEG C of preservations.
ProteinG is utilized to carry out purifying to ascites, MAb concentration after BCA method mensuration purifying, the purity of monoclonal antibody after SDS-PAGE mensuration purifying.
7) monoclonal antibody immunogenicity and specificity analyses
After being induced by recombinant protein gE and pGEX-6P-1, thalline carries out SDS-PAGE electrophoresis, electric transfer printing afterwards.With the positive hybridoma nutrient solution supernatant of screening for primary antibodie, be two anti-carry out Western detection with sheep anti mouse HRP-IgG.
Utilize the elisa plate of PRV, PEDV, PPV bag quilt to detect the specificity of prepared monoclonal antibody.Criterion is P/N >=2.1 item is the positive, and namely monoclonal antibody has cross reactivity with it; P/N < 2.1 is negative, i.e. monoclonal antibody and its no cross reaction.
The foundation of embodiment 4PRV stop band restrain method
1 material
1.1gE albumen
Be masterplate amplification with the PRV genome that deposit number is CGMCCNo.10266, carry out expressions also purifying by prokaryotic expression system.
1.2 experimental animal
BALB/C small white mouse, New Zealand white rabbit is all purchased from Shandong University's Experimental Animal Center.
1.3 positive serum
By the PRV of deactivation virus after white oil emulsification, with hypodermic mode immunize New Zealand white rabbits, immunity 3 times altogether, every minor tick 14 days, the 3rd immunity be ear vein blood sampling mensuration antibody titer after 10 days, when agarose diffusion test is tired and is reached more than 1:16, from heart bloodletting, serum can be collected, i.e. obtained PRV positive serum, packing after 0.22um membrane filtration ,-20 DEG C of preservations.
1.4 negative serum
Negative serum for not saying recombinant protein immunity, and is detected as PRV negative antibody through indirect ELISA method, the serum obtained after eye socket blood sampling is centrifugal, packing after 0.22um membrane filtration ,-20 DEG C of preservations.
1.5TMB nitrite ion
Purchased from Beijing Tian Gen biochemical technology company limited
1.6 stop buffer
Described stop buffer is 2M sulfuric acid, by the vitriol oil of 22.2ml, dropwise joins configuration in the distilled water of 177.8ml and forms.
1.7 concentrated cleaning solutions, sample diluting liquid
Described washings is 0.1M phosphate buffered saline buffer, NaCl80g, KCl2g, KH
2pO
42.4g, Na
2hPO
4× 12H
2o36.28g, be dissolved in 800ml distilled water, be 7.4 with hydrochloric acid adjust pH, distilled water is settled to 1000ml, autoclaving, room temperature preservation.During use, by distilled water 10 times dilution use, and add the Tween-20 of 0.05%.
Sample diluting liquid, on the basis of above-mentioned washings working concentration, adds the BSA of 0.3%.
The monoclonal antibody of 1.8 peroxidase labellings
The specificity of 1.9PRV stop band restrain method
The stop band restrain method adopting this test to set up, porcine circovirus 2 type, pig parvoviral, the sick virus of pig virus diarrhoea, PRV (Pseudorabies virus), Pestivirus suis, swine foot-and-mouth disease virus positive serum are detected, found that the method detects except having cross reaction with porcine circovirus 2 type, with other viruses all no cross reactions, show that the method that the present invention sets up has good specificity.
The susceptibility of 1.10PRV stop band restrain method
Detect 165 parts of porcine blood serum respectively with porcine pseudorabies virus ELISA antibody kit before the section of Wuhan and American I DEXXgE antibody assay kit, detected result shows, before the section of Wuhan, test kit detects that 103 parts for positive serum, and 62 parts is negative serum.It is 100 parts that U.S. Ai Deshi (IDEXX) test kit detects positive serum, and negative serum is 65 parts.The stop band restrain method adopting this test to set up, is tested with 100 parts of positive serums, be 97.1% is 100% with American I DEXX coincidence rate before positive coincidence rate and Wuhan section; 65 parts of negative serums detect to be all negative with this experimental technique, and negative match-rate is 100%.
Therefore, the stop band restrain method that the present invention sets up avoids domestic detection method and there is false-positive result, improves the susceptibility of detected result, can make diagnosis more exactly to PR.
| Positive number | Negative sample number | Positive control | Negative control | |
| Before the section of Wuhan | 103 | 62 | Positive | Negative |
| American I DEXX | 100 | 65 | Positive | Negative |
| The present invention | 100 | 65 | Positive | Negative |
And, use existing porcine pseudorabies virus ELISA antibody to detect CGMCCNo.10266 strain, found that and occur obvious Problem of False Negative, and use the antibody that gE albumen of the present invention is prepared as antigen, then can not produce false positive; This also demonstrates the epitope that amino acid whose insertion changes the existence of known strain, causes the Detection results of existing detection reagent to CGMCCNo.10266 strain bad.
Claims (10)
1. a gE albumen, is characterized in that, the aminoacid sequence of described gE albumen is SEQIDNO:1.
2. the gene of coding gE albumen according to claim 1, it is characterized in that, the nucleotides sequence of described gene is classified as SEQIDNO:2.
3. gE albumen according to claim 1 is as the application of antigen.
4. an antibody, is characterized in that, described antibody carries gE albumen according to claim 1 to be prepared as antigen.
5. detect a stop band restrain method for ill swinery antibody, it is characterized in that, described method comprises following step:
By gE proteantigen through diluted, be quantitatively coated in ELISA96 orifice plate, 4 DEG C are spent the night;
With PBST washings, unconjugated antigen is cleaned;
Respectively negative serum, positive serum and serum to be checked are added in corresponding detect aperture, slightly shake, make the liquid blending in Sptting plate;
Micro-reaction plate sealing is placed on 37 DEG C hatch;
Vertically outwell the liquid in Sptting plate, and carry out cleaning each hole with washings;
With confining liquid, Sptting plate is closed, be placed in 37 DEG C and hatch;
Remove the liquid in Sptting plate, and carry out cleaning each hole with washings;
The enzyme mark monoclonal antibody diluted is joined in each reacting hole, is placed in 37 DEG C and hatches;
Outwell the liquid in Sptting plate, and carry out cleaning each hole with washings;
Add substrate, be placed in 37 DEG C of reactions;
Add stop buffer, termination reaction, and read 450nm absorbance in microplate reader, sample value/feminine gender value is greater than 2 and is judged to be the positive, is less than 1 for negative.
6. method as claimed in claim 5, it is characterized in that, described negative serum is the serum without immune mouse.
7. method as claimed in claim 5, is characterized in that, described positive serum is by the separation of serum of PRV virus immune mouse after deactivation.
8. method as claimed in claim 5, it is characterized in that, described confining liquid is 5% skim-milk.
9. method as claimed in claim 5, is characterized in that, described washings is the PBS solution containing 0.5 ‰ tween 20s.
10. method as claimed in claim 5, is characterized in that, described enzyme mark monoclonal antibody, carries out enzyme mark by antibody horseradish peroxidase according to claim 4 and prepare.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106771179A (en) * | 2016-11-22 | 2017-05-31 | 无锡艾科瑞思产品设计与研究有限公司 | A kind of detection method of PRV |
| CN107163108A (en) * | 2017-06-05 | 2017-09-15 | 中国农业科学院上海兽医研究所 | A kind of preparation and application of the epitope, antibody of pseudorabies virus gE albumen |
| CN108931642A (en) * | 2017-05-27 | 2018-12-04 | 中国农业大学 | Detect the kit and detection method of the wild malicious antibody of porcine pseudorabies virus |
| CN109900902A (en) * | 2019-03-29 | 2019-06-18 | 中牧实业股份有限公司 | A kind of porcine pseudorabies virus gB blocks ELISA antibody assay kit and its application |
| CN109900903A (en) * | 2019-03-29 | 2019-06-18 | 中牧实业股份有限公司 | A kind of porcine pseudorabies virus gE blocks ELISA antibody assay kit and its application |
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| CN107163108A (en) * | 2017-06-05 | 2017-09-15 | 中国农业科学院上海兽医研究所 | A kind of preparation and application of the epitope, antibody of pseudorabies virus gE albumen |
| CN109900902A (en) * | 2019-03-29 | 2019-06-18 | 中牧实业股份有限公司 | A kind of porcine pseudorabies virus gB blocks ELISA antibody assay kit and its application |
| CN109900903A (en) * | 2019-03-29 | 2019-06-18 | 中牧实业股份有限公司 | A kind of porcine pseudorabies virus gE blocks ELISA antibody assay kit and its application |
| CN109900903B (en) * | 2019-03-29 | 2020-06-09 | 中牧实业股份有限公司 | Porcine pseudorabies virus gE blocking ELISA antibody detection kit and application thereof |
| CN109900902B (en) * | 2019-03-29 | 2020-06-09 | 中牧实业股份有限公司 | Porcine pseudorabies virus gB blocking ELISA antibody detection kit and application thereof |
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