CN104459144B - A kind of PRV velogen strain and vaccine strain differentiate Test paper - Google Patents
A kind of PRV velogen strain and vaccine strain differentiate Test paper Download PDFInfo
- Publication number
- CN104459144B CN104459144B CN201410763138.7A CN201410763138A CN104459144B CN 104459144 B CN104459144 B CN 104459144B CN 201410763138 A CN201410763138 A CN 201410763138A CN 104459144 B CN104459144 B CN 104459144B
- Authority
- CN
- China
- Prior art keywords
- prv
- vaccine
- strain
- test paper
- monoclonal antibody
- 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.)
- Active
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
- G01N33/56994—Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- 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
Abstract
The present invention relates to a kind of infection of disease of domestic animals and immune identification detector tool, differentiate Test paper more particularly to a kind of strong poison of PRV and vaccine virus, by supporting plate, sample pad, gold standard pad, detect film and adsorptive pads are formed, detect on film containing strong malicious detection line T1 " │ ", vaccine virus detection line T2 " │ " and nature controlling line C " │ " trace.When differentiating detection, showing three red stripes in detection film, " │ │ │ " are the strong poison infection of pseudorabies；" │ │ " are pseudorabies vaccine virus vaccine immunity to two red stripes；It is negative for PRV only to show a red stripes " │ ".This discriminating Test paper high specificity, sensitiveness is high, and response spectrum is wide, can detect existing vaccine strain and and most popular velogen strains, and it is easy to operate, quick, it can be widely used for PRV infection and detected with immune discriminating, be easy to the popularization and application in production practices.
Description
Technical field
The present invention relates to a kind of infection of disease of domestic animals and immune identification detector tool, more particularly to a kind of pseudorabies
Virus and vaccine virus differentiate Test paper.
Background technology
Pseudoabies（(Pseudorabies, PR）It is by Pseudorabies virus（Pseudorabies Virus, PRV）Cause
Many animals suffer from infectious disease altogether, can infected cattle, pig, dog, sheep, fox and cat etc..Pig is the infection sources and nature of pseudoabies
Reservoir host, pig show as generating heat after infecting PRV, piglet nervous symptoms, high mortality, big-and-middle porcine respiratory symptom, sow stream
The clinical characters such as production.In 2010, pseudoabies was that China's large-scale pig farm carries out one of ideal disease of Blight control,
Many pig farms have reached immune without Infection Status.But in 2011, the pig farm of some Bartha-K61 vaccine immunities occurs
The porcine pseudorabies virus of variation, shows that traditional gE gene-deleted vaccines can not provide enough immunoprotections.Henan Province
Entire lowering trend is presented in the infection rate of swinery pseudoabies since two thousand five, clinically using distribute or atypical case as
It is main.But since the end of the year 2011, the pseudo- mad dog epidemic situation of our province selected swine farms burst, broken out quickly in various regions inside the province, extensively stream
OK；Moreover, also there is infection death state in the animal such as sheep, dog, cat, fox near selected swine farms.Therefore, China pig at this stage
The prevention and control of pseudoabies become more intractable and complicated.
PRV belongs to herpetoviridae（Herpesviridae）, a- herpesviral subfamilies（Alphaherpesvirinae）'s
Member, genome are linear dsdna, the kb of size about 150.Whole PRV genomes at least contain 70 genes, encode 70-
More than 100 plant albumen.It has now been found that 11 glycoprotein of PRV codifieds：GB, gC, gD, gE, gG, gH, gI, gK, gL, gM and
gN.Guarded in gB, gH, gL and gM herpetoviridae, and gB, gH and gL are carried out to all herpesvirals in cell culture
Duplication is all required.GB albumen can induce the generation of neutralizing antibody, related to immunoprotection.PRV gE albumen is claimed earliest
For gI albumen.GE missings be present in PRV vaccine strains such as Bartha strains and BUK strains prepared by the screening of generation nineteen sixty etc..In 20 generation
Discipline the mid-80, the PRV mutant strains of gE missings are constructed by DNA recombinant techniques；On this basis, TK is carried out into missing makes
PRV viruses are further caused weak.Many gE deletion of vaccine can prevent to attack poison infection or mitigate to attack caused by poison infects to face
Bed symptom.Although currently only swinery is allowed to lack epidemic disease using gE in many countries there is also the PRV vaccines that gC and gG is lacked
Seedling.From generation to the end of the year 2011 nineteen ninety, the swinery of China more than 80% has used gE gene-deleted vaccines Bartha to carry out immune connect
Kind.It is that all popular strains express gE albumen that gE vaccines and gE antibody diagnosis, which are supported the use for the premise of PRV purifications, and
The animal of epidemic strain infection produces the antibody of gE albumen.GE albumen is expressed by current PRV street strains almost used.Pass through gE bases
Because of being used in combination for deletion of vaccine and gE antibody diagnosis methods, some European countries such as Germany, Austria, Sweden, Denmark and English
State and the U.S. have succeeded has purified porcine pseudorabies in domestic pig group.The Chinese government worked out in 2011《It is long-term in country
Animal epidemic control program（2012-2020）》, propose that original seed pig farm porcine pseudorabies reached purification standard by 2015；Arrive
The year two thousand twenty, national all kinds of pig farm porcine pseudorabies reach the target of purification standard.But the purification of pathogen is one long-term
And difficult task.It is fully cleaned up if realized or may need for 100 years even longer close to purification state；Such as
The porcine pseudorabies in the U.S. appear in 1909 first, and its purification plan starts from 1989, most purification items by 2005
Mesh is accomplished.Nonetheless, the presence of PRV viruses infects hog PRV and causes very big threat again in wild boar group.Cause
This, establishes simplicity, quick, special, sensitive differential diagnostic method distinguishes vaccine immunity animal and virus-infected animal very must
Will.
The differential diagnostic method of pseudoabies includes PCR（PCR）, EUSA（ELISA）
And immunopcroxidase monolayer assay（IPMA）Deng wherein ELISA kit is that the most commonly used discriminating of in the market is examined
Disconnected commodity.The ELISA kit of commercialization uses indirectly or blocks ELISA respectively, because clinical serum is complex, indirectly
ELISA specificity is slightly worse relative to blocking ELISA.ELISA is blocked to block gE protein-specific monoclonal antibodies using clinical Swine serum
Combination, specificity it is higher with sensitiveness；However, block ELISA there is also it is congenital the shortcomings that.The monoclonal antibody for blocking ELISA to use
The epitope in pig body with immunodominance is directed to, is possible to once PRV viruses morph in gE genes so that single
Anti- identification is invalid.It is single although blocking ELISA to remain able to the PRV antiviral antibodies that specific detection goes out the emerging variation in China
The invalid possibility of anti-identification is still present.So new differential diagnostic method gesture is developed according to PRV variation strains at this stage
It must go.Immune chromatography test paper is sent out on the basis of monoclonal antibody technique, colloidal gold immunochromatographimethod technology and new material technology
A kind of novel in vitro detection technique that exhibition is got up, it is preferably detection immediately（point-of-care test, POCT）And scene
Detection technique, its have the advantages that it is sensitiveer, special, easy, quick, can especially realize it is " foolproof " operation, that is, be not required to
Want any auxiliary instrumentation equipment to carry out Site Detection, and the result of determination within 1-5 minutes, be widely used in various analytes
Quantitative and semi-quantitative quick detection, including antigen, haptens, antibody and nucleic acid etc., it has also become most quick sensitive now exempts from
One of epidemiology detection technique.Henan Province Animal Immunology Key Laboratory system since nineteen ninety-five is carried out immune test paper and quickly examined
Survey technology is studied, take the lead in succeeding in developing in the world animal epidemic cause of disease and antibody test colloid test paper product-- it is avian infectious
Bursal disease quick diagnosis test paper and Trichinella sui antibody test paper, establish animal epidemic quick detection test paper research and development technology and put down
Platform, the serial quick detection test paper production such as animal epidemic antigen, antibody and medicament residue detection is successfully developed so far
Product, the application and development of the technology have been promoted significantly.The present invention is for PRV wild virus infections and the key of vaccine immunity antidiastole
Technical problem, Screening and Identification can distinguish that PRV is strong, high-affinity pairing monoclonal antibody of vaccine strain, establish based on immune layer
The protein chip technology platform of test paper is analysed, PRV velogen strain is developed and vaccine strain differentiates Test paper, establish suitable
For cultivating quick, the easy pseudorabies wild virus infection and vaccine immunity joint inspection technology of basic unit and clinical detection, realization pair
The real-time monitoring of PRV wild virus infection, technical support is provided for China's pseudorabies prevention and control and purification, it is mad to pig puppet
Dog epidemic monitoring and prevention and control are significant.
The content of the invention
The purpose of the present invention is：Development is applied to pseudorabies infection and the PRV of Immune dctection detection is malicious by force
Differentiate Test paper with vaccine virus, this test paper is special, sensitive, quick, easy, the popularization and application easily in production practices.
The technical scheme is that：A kind of strong poison of PRV and vaccine virus differentiate Test paper, by supporting plate,
Sample pad, gold standard pad, detects film and adsorptive pads are formed, and gold standard pad is absorption colloid gold label resisting pstudorabies poison gB albumen lists
Clonal antibody mAb1 glass fibre, detection film are the nitric acid of trace malicious detection line T1, vaccine virus detection line T2 and nature controlling line C by force
Cellulose membrane, strong malicious detection line T1/vaccine virus detection line T2/ nature controlling lines C is arranged as by sample end to handle end：" │ │ │ ", by force
Malicious detection line T1 is resisting pstudorabies poison monoclonal antibody gE albumen mAb2 traces " │ ", and vaccine virus detection line T2 is that anti-pig is pseudo-
Rabies viruses polyclonal antibody pAb1 or resisting pstudorabies poison gB protein monoclonal antibody mAb3 traces " │ ", nature controlling line C are anti-
Mouse IgG antibody pAb2 or staphylococcus aureus SPA traces " │ ".When differentiating detection, the strong poison infection of pseudorabies is in detection film
Show strong malicious detection line T1, vaccine virus detection line T2 and nature controlling line C tri- red stripes " │ │ │ ", pseudorabies vaccine virus vaccine
It is immunized and shows two red stripes of vaccine virus detection line T2 and nature controlling line C in detection film " │ │ ", no PRV then only show
Existing nature controlling line mono- red stripes " │ " of C.
Using PRV gB and gE recombinant protein as immunizing antigen, anti-pig is identified by hybridoma cell technology production
Pseudorabies virus gB and gE protein monoclonal antibody, utilizes immunopcroxidase monolayer assay（IPMA）Pig is distinguished in screening
The monoclonal antibody of the strong poison of Pseudorabies virus and vaccine virus, to be superimposed EUSA（ELISA）Screening identification is different
The monoclonal antibody of epitope.Monoclonal for the monoclonal antibody mAb1 and vaccine virus detection line T2 of colloid gold label resists
The equal specific recognition PRV velogen strains of body mAb3 and vaccine toxic vaccine strain, PRV gB albumen is identified respectively
Different epitopes, the monoclonal antibody mAb2 specific recognition PRV velogen strain gE eggs for strong malicious detection line T2
In vain, but not reacted with vaccine strain.Meanwhile with IPMA screenings and the wide monoclonal antibody of PRV response spectrum, colloid
Golden labeled monoclonal antibody mAb1 and vaccine virus detection line T2 monoclonal antibodies mAb3 can recognize that PRV gE genes lack
Vaccine virus vaccine and most popular velogen strains are lost, strong malicious detection line T1 monoclonal antibody mAb2 can recognize that most porcine pseudorabies
The popular velogen strain of poison.Resisting pstudorabies poison Anti-TNF-α is prepared by immunizing antigen of PRV vaccine virus Bartha strains
Body pAb1, available for vaccine virus detection line T2 traces.Goat anti-mouse igg polyclonal antibody is prepared by immunizing antigen of mouse IgG
PAb2, pAb2 and staphylococcus aureus SPA are used equally for nature controlling line C traces.
The beneficial good effect of the present invention：It is mad that the strong poison of PRV and vaccine virus differentiate that Test paper realizes pig puppet
It the synchronous joint inspection of dog Virus and vaccine virus, can effectively differentiate the strong poison infection of swinery pseudorabies and vaccine immunity, and grasp
Make simply, everybody is operable, can preferably meet the needs of different levels personnel, as Disease monitor, customs quarantine control, health are anti-
Epidemic disease, intensive culture to individual cultivation etc., are easy to a wide range of popularization and application, have wide market prospects and larger economy,
Social benefit.Test strip has following advantage：
（1）Strong poison and vaccine virus joint inspection.The strong poison of PRV and vaccine virus differentiate that Test paper contains on detection film
There is the strong poison inspection of the strong poison of identification PRV and the vaccine virus detection line of vaccine virus and the strong poison of identification but nonrecognition vaccine virus
Survey line, velogen strain and the vaccine strain joint inspection of PRV can be synchronously carried out, realize the strong poison and vaccine virus of pseudorabies
Differentiate detection.
（2）High specificity, sensitiveness are high.The strong poison of PRV and vaccine virus differentiate Test paper can distinguish pig puppet
It is prepared based on the high-affinity pairing monoclonal antibody of the strong poison of rabies viruses and vaccine virus, the specificity of monoclonal antibody
Strong and sensitiveness is high, is formed in gold labeling antibody between gold grain and antibody molecule without covalent bond, the two passes through between the charges of different polarity
Van der Waals force is combined, and collaurum influences very little to the reactivity of labelled antibody, and has higher mark rate.Therefore, differentiate
Test paper has higher specificity and sensitiveness.
（2）It is easy to operate quick.Using without any other reagent, sample is treated as long as being inserted into when differentiating Test paper
Product 10 seconds or so, testing result was can determine that in 2 minutes.
（3）Show that testing result is vivid, directly perceived accurate.Differentiate Test paper to show rufous " │ ", " │ │ " and " │ │ │ "
Trace shows that a brownish red band " │ " is pig as the feminine gender of detection, vaccine virus and strong malicious positive mark on detection film
Pseudorabies virus is negative, represents to be detected sample without vaccine virus vaccine or strong poison infection, " │ │ " are that pig is pseudo- to two brownish red bands
Hydrophobia poison is positive, and expression test sample is vaccine virus vaccine immunity, and " │ │ │ " are pseudorabies to three brownish red bands
Virus is positive, and expression test sample infects for strong poison, and result judgement is vivid, directly perceived, accurate, simple and clear, is less prone to
False negative and false positive erroneous judgement.
（4）Cost is low, small investment.Using Test paper is differentiated, it is not required to separately match somebody with somebody instrument and equipment and other reagents, makes live inspection
Survey settles at one go, and cost is cheap, and small investment is quick.
The present invention is further described with reference to the accompanying drawings and examples for brief description of the drawings.
Fig. 1 is that the strong poison of PRV and vaccine virus differentiate Test paper side structure schematic view.
Fig. 2 is that the strong poison of PRV and vaccine virus differentiate Test paper overlooking the structure diagram.
In figure, 1. supporting plates, 2. sample pads, 3. gold standard pads, 4. detection films, 5. adsorptive pads, the malicious detection line of the last 6.
T1 traces, 7. vaccine virus detection line T2 traces, 8. nature controlling line C traces, 9-1. sample end diaphragms, the protection of 9-2. handle ends
Film, 10. mark lines.
The strong poison of embodiment PRV and vaccine virus differentiate that Test paper can be widely applied to swinery pig puppet
The strong poison infection of rabies viruses and the discriminating of vaccine immunity detect.Prepare the strong poison of PRV and vaccine virus differentiates detection examination
Paper, need to prepare PRV immunizing antigen first, and then prepare resisting pstudorabies poison polyclonal antibody and monoclonal resists
Body, and the monoclonal antibody for distinguishing the strong poison of pseudorabies and vaccine virus is screened, the Dan Ke of the strong poison of identification pseudorabies and vaccine virus
Grand antibody mAb1 is used to prepare colloid gold label thing, the strong poison of identification pseudorabies and the monoclonal antibody mAb2 of nonrecognition vaccine virus
For printing strong malicious detection line trace " │ ", the polyclonal antibody pAb1 or monoclonal of the strong poison of identification pseudorabies and vaccine virus resist
Body mAb3 is used to print vaccine virus detection line trace " │ ", secondly need to prepare goat anti-mouse igg antibody or staphylococcus aureus
SPA, for printing nature controlling line trace " | ".
（1）The preparation of PRV immunizing antigen.
（1.1）The preparation of PRV gB recombinant proteins
With the neutralizing epitope enrichment region of PCR amplification PRVgB albumen, target gene is cloned into prokaryotic expression carrier pET-
28a, gB DNA recombinant expression plasmid pET-gB are built, induced through 1 mol/L IPTG, in E. coli gB weights
Histone, Western blot detections show that gB expressing proteins can have good reaction by PRV positive serum specific recognitions
Property.The gB recombinant proteins 15mg/L of purity about 90% is obtained using Ni posts affinitive layer purification and dilution refolding technology.
（1.2）The preparation of PRV gE recombinant proteins
With the epitope enrichment region of PCR amplification PRVgE albumen, target gene is cloned into prokaryotic expression carrier pET-28a, structure
GB DNA recombinant expression plasmid pET-gE are built, are induced through 0.4 mol/L IPTG, egg is recombinated in E. coli gE
In vain, Western blot detections show that gE expressing proteins can have good reaction by PRV malicious positive serum specific recognitions by force
Property.The gE recombinant proteins 12mg/L of purity about 90% is obtained using Ni post affinitive layer purifications technology.
（2）The preparation of resisting pstudorabies poison monoclonal antibody.
（2.1）The foundation of hybridoma cell strain.
It is fully emulsified respectively by PRV gB and gE recombinant protein and Freund immunologic adjuvant mixed in equal amounts, with 50
BALB/c systems mouse is only immunized 3 times in mg-100 mg/, every minor tick 15-30 days；, will be immune small 3-4 days after 3rd booster immunization
Rathole ball bloodletting, draw neck lethal, it is sterile to take its spleen in 75% alcohol-pickled 5-10 min；Shred and through 100 mesh nylon wire mistakes
Filter, 1000 r/min centrifuge 10 min, collect splenocyte；By 1 × 108Splenocyte and 2-5 × 107SP2/0 myeloma cell
Mixing, 1000 r/min centrifuge 10 min, supernatant are abandoned, by 0.7-1 ml 40%-50% PEG 4000 in 37 DEG C of water-bath
（pH8.5-9.0）Cell is slowly added into, after incubating 1 min, the ml of 1640 culture medium of serum-free 15 is slowly added to, to terminate PEG's
Effect, 37 DEG C of water-bath 5-10 min, 1000 r/min 10 min of centrifugation, abandons supernatant, cell is resuspended in into HAT Selective agar mediums
In, and add 96 well culture plates（100ml-200ml/ holes）, put 37 DEG C of 5% CO2Cultivated in incubator.After culture 7-10 days, take
Hybridoma Cell Culture supernatant is with EUSA（ELISA）And immunopcroxidase monolayer assay（IPMA）
Screen positive hybridoma cell.With the strong poison infection hamster kidney cell of PRV standard（BHK-21）, after methanol is fixed,
5% 37 DEG C of defatted milk closes 1 h；Add cells and supernatant 50mL/ holes to be checked, if HAT culture mediums and mouse immune serum are feminine gender
And positive control；Add 1:The horseradish peroxidase of 500 dilutions（HRP）The goat anti-mouse igg antibody of mark（50 mL/ holes）, 37
DEG C effect 30 min；Fully washed with the PBS containing 0.05% Tween-20 after often step reaction；With substrate A EC color development at room temperature 10-
20 min, rinsed with water after stopping colour developing, under the microscope observation colour developing result.Choose vigorous gram of strong positive, cell growth
Grand hole carries out continuous 3 limited dilution clonings, expands freeze-stored cell after culture, establishes resisting pstudorabies poison gB albumen respectively
With 8 plants and 12 plants of gE protein monoclonal antibodies hybridoma cell strain.
（2.2）The preparation of monoclonal antibody：Monoclonal antibody is prepared to induce ascites in vivo.Learn from else's experience pristane or atoleine sensitization
Through producing Balb/c mouse, the hybridoma 10 of exponential phase is injected intraperitoneally7Individual/only, ascites is extracted after the d of 7 d~10,
Supernatant is taken after centrifugation, dispenses, freezes.
（2.3）The identification of monoclonal antibody
（2.3.1）The antibody titer of monoclonal antibody
With EUSA（ELISA）And immunopcroxidase monolayer assay（IPMA）Determine hybridoma
The ELISA potency of the monoclonal antibody potency of cells and supernatant and ascites, Mab supernatant and ascites is respectively 1:640 and 1: 5×105
More than, IPMA potency is respectively 1:16 and 1:More than 8000.
（2.3.2）The specificity of monoclonal antibody
Hamster kidney cell is infected with PRV standard strains, popular strain and vaccine strain（BHK-21）, with immunoperoxidase
Enzyme cell monolayer is tested（IPMA）The reactivity of monoclonal antibody and PRV prevalence strains is determined, screening obtains specific recognition PRV standards simultaneously
6 plants of the gB protein monoclonal antibodies of strain, popular strain and vaccine strain, and specific recognition PRV standard strains and popular strain
But 8 plants of the monoclonal antibody of nonrecognition vaccine strain.With immunopcroxidase monolayer assay（IPMA）Detect above-mentioned 14
Strain monoclonal antibody and vaccine strain and the response spectrum of popular strain, it was demonstrated that for colloid gold label and vaccine virus detection line T2 gB
Protein monoclonal antibody can recognize that PRV vaccine strain and most popular velogen strains, the gE for strong malicious detection line
Protein monoclonal antibody can recognize that most PRV prevalence velogen strains, be respectively provided with wider PRV response spectrums.
（2.3.3）Monoclonal antibody identifies Characterization of antigenic epitopes
Analyzed with the epitope for blocking ELISA or superposition ELISA to identify PRV monoclonal antibodies, screening obtains
The monoclonal antibody mAb1 and mAb3 of PRV gB albumen difference epitopes are identified respectively, and it is respectively used to collaurum
Mark and vaccine virus detection line T2 traces；Obtain specific recognition PRV standard strain and popular strain, without with epidemic disease
The gE protein monoclonal antibody mAb2 of seedling toxic vaccine strain reaction, for strong malicious detection line T1 trace.
（2.3.3.1）Block ELISA：Labeled monoclonal antibody is distinguished with HRP, ELISA blocking tests identification monoclonal resists
Body identifies the specificity of epitope, adds unlabelled list line by line in the coated ELISA Plate of gB or gE recombinant proteins first
It is anti-, if PRV positive serums, negative serum and PBS control, 37 DEG C of 1 h of effect；Then the monoclonal antibody that addition HRP is marked by column, 37 DEG C
Act on 1 h；Developed the color with substrate TMB, read the OD of each detection hole450Value.Significantly inhibit the unmarked list of enzyme mark monoclonal antibody combination
The same or like epitope of anti-identification, and enzyme mark monoclonal antibody is combined the unmarked monoclonal antibody having no significant effect then identify it is different
Epitope.
（2.3.3.2）It is superimposed ELISA：The work of each monoclonal antibody is determined with indirect ELISA first with the ELISA Plate of envelope antigen
Make concentration, draw antigen saturation curve.In superposition ELISA experiments, monoclonal antibody is suitably diluted according to antigen saturation curve, and match
Add ELISA Plate each hole, 37 DEG C are incubated the h of 1 h~2；It is separately added into ELIAS secondary antibody and TMB is developed the color, reads each hole OD450Value,
Superposition coefficient is calculated by following equation（AI）：AI=[2´OD1+2/（OD1+OD2）- 1] ' 100%, wherein OD1+2For pairing monoclonal antibody hole
OD450Value, OD1And OD2For the OD in two independent monoclonal antibody holes450Value.The AI values of two monoclonal antibodies be judged to identify less than 40% it is identical or
Close epitope；AI values are judged to identify different epitopes more than 40%.
（2.4）The purifying of monoclonal antibody：With caprylic acid-ammonium from mouse ascites purified monoclonal antibody IgG.Take 1 mL small
Mouse ascites fluid, add the mol/L sodium acetate buffers of 2 mL 0.06（pH 5.0）, pH 4.5 is adjusted to 0.1 mol/L HCl；In room
Under temperature stirring, 33mL octanoic acids are added dropwise, 4 DEG C of standing 2 h, 15000 r/min 30 min of centrifugation, abandon precipitation；In centrifugation supernatant
Middle addition 1/10 volume 0.01 mol/L PBS（pH7.4）, pH 7.4 is adjusted to 0.1 mol/L NaOH；Under condition of ice bath
Saturated ammonium sulfate is added to final concentration 45%, 4 DEG C of standing 2 h, 10000 r/min 30 min of centrifugation, abandons supernatant；With appropriate PBS weights
Outstanding precipitation, stays overnight to PBS, changes liquid 3 times.With spectrophotometer method or dying method with coomassie brilliant blue（Bradford methods）Measure
Monoclonal antibody purification IgG protein content is in more than 1mg/ml, with EUSA（ELISA）With immune peroxidating
Thing enzyme cell monolayer is tested（IPMA）The antibody titer of mouse ascites and IgG purification is determined 1: 5×105With 1:More than 4000,
Packing, freezes.
（3）The preparation of resisting pstudorabies poison polyclonal antibody.
With the pseudorabies vaccine of the mg/kg body weight of 50 mg ~ 100 through intramuscular injection immune health piglet 3 ~ 4 times, last is exempted from
After epidemic disease 10 days, vena cava anterior blood sampling, its serum antibody titer is determined 1 with IPMA:When more than 1000, Culling heart blood or arteria carotis
Bloodletting, hyper-immune serum is collected, with anhydrous sodium sulfate（Na2SO4）Immune swine serum IgG is extracted, takes 1 part of immune serum to add 2 part 0.01
mol/L PBS（pH7.2）Mixing, add anhydrous Na2SO4To final concentration 18%, 37 DEG C of water-bath 30min, 4000 r/min centrifugations
20min, supernatant is abandoned, be resuspended and precipitated with appropriate PBS (pH7.2), add the Na of final concentration 16%2SO4, 37 DEG C of precipitations 30min, 4000r/
Min centrifuges 20min, abandons supernatant, then is resuspended and precipitated with appropriate PBS (pH7.2), with the Na of final concentration 14%2SO4Precipitation, 4000r/
Min centrifuges 20min, abandons supernatant, is resuspended and precipitated with appropriate PBS (pH7.2), PBS (pH7.2) is dialyzed overnight, changes liquid 2 ~ 3 times,
Determine antibody titer and protein concentration（10-20 mg/ml）, prepared resisting pstudorabies poison polyclonal antibody pAb1 can be used for
The printing of Test paper vaccine virus detection line T2 traces.
（4）The preparation of rabbit anti-mouse IgG polyclonal antibodies
2.0 kg or so healthy new zealand rabbit, first immunisation is immunized with the mouse IgG of purifying to emulsify with Freund's complete adjuvant
Antigen, the subcutaneous mg/ of multi-point injection 50 only, each w of booster immunization interval 3, are noted with incomplete Freund's adjuvant emulsification antigen muscle
Penetrate, after the w of last time booster immunization 2, with agar gel diffusion test（AGP）Determine immune serum antibody titer and be higher than 1:When 40, adopt
Collection height exempts from rabbit whole blood, separates serum, purifies rabbit anti-mouse IgG with caprylic acid-ammonium, method is same（2.4）Monoclonal antibody purifies, octanoic acid
Dosage is 45mL/mL serum, determines antibody titer and protein concentration（10-20 mg/ml）, prepared rabbit anti-mouse IgG is polyclonal
Antibody pAb2 can be used for the printing of Test paper nature controlling line C traces.
（5）The colloid gold label of monoclonal antibody
（5.1）The preparation of collaurum：Take 100 mL ultra-pure waters to be placed in the clean conical flasks of 500 mL, add 1 mL 1%（w/
v）Gold chloride boils；1 mL 1% of Fresh is rapidly added under stirring（w/v）Sodium citrate solution, boil about 3
Min is changed into aubergine to solution colour from yellow, continues to boil 2 min；Treat that solution is cool to room temperature, mend ultra-pure water to 100 mL,
With 0.2 mol/L K2CO3PH to 9.0 is adjusted, 4 DEG C of lucifuges can preserve the several months.
（5.2）Most suitable labelled protein concentration mensuration：Anti- PRV monoclonal antibody IgGs to be marked are taken to 20 mmol/L dobell's solutions
（pH 8.0）4 DEG C are dialyzed overnight.With 25 mL ultra-pure waters with 1 in microwell plate:2. 1:4. 1:8 ... doubling dilutions are to be marked
PRV monoclonal antibodies；Each hole adds 125 mL colloidal gold solutions, and RT stands 5 min；Add the mol/L NaCl solutions of 125 mL 1；Each hole
Color is changed into blueness with the reduction of protein concentration from red.Using the protein concentration of the unchanged blue monoclonal antibody highest dilution of color as
The most suitable label concentration of collaurum, during colloid gold label, protein concentration increase by 20%.
（5.3）The colloid gold label of monoclonal antibody：The monoclonal antibody IgG to be marked of the most suitable protein concentrations of 2 mL is taken, adds 10
ML colloidal gold solutions（pH 9.0）, rapid to mix, room temperature acts on the min of 10 min~15；1/10 volume is added containing 10%（w/v）Ox
Seralbumin（BSA）20 mmol/L dobell's solutions, rapid to mix, RT acts on the min of 10 min~15；4℃ 15000
G centrifuges 30min, carefully removes supernatant；With containing 1%（w/v）Collaurum is resuspended in BSA 20 mmol/L dobell's solutions, ibid from
The heart, abandon supernatant；Repeated washing 1 time, contain 1% with 1 mL（w/v）Collaurum, 4 DEG C of guarantors are resuspended in BSA 20mmol/L dobell's solutions
Deposit standby.
（6）Detect the preparation of film
Nitrocellulose detection film is cut into 2.5 × 30 cm2Strip, be placed on the specking instrument platforms of XYZ 3000, and with
Press strip is fixed；Use PBS（pH 7.2）The gE albumen not reacted specific recognition PRV velogen strain but with vaccine strain
The gB albumen monoclonal antibody or more anti-igg and rabbit anti-mouse igg of monoclonal antibody, specific recognition PRV velogen strain and vaccine toxic vaccine strain
1.0 mg/mL are diluted to, and are put in storage pool respectively；Using Biojet Quanti 3000 with 1.0 mL/cm respectively by anti-PRV
GE albumen monoclonal antibody, gB albumen monoclonal antibody or the more anti-igg solution of PRV and rabbit anti-mouse igg solution specking form strong poison in detection film center
Detection line T1, vaccine virus detection line T2 and nature controlling line C traces, detection line is with nature controlling line at a distance of 0.5 cm；Put 42 DEG C of drying boxes 30
Min or natural drying at room temperature；Detection film is put into polybag, adds 4 DEG C of drier is closed to save backup.
（7）The preparation of pad
Mineral wool is cut into 1.5 × 30 cm2Strip, be placed on the specking instrument platforms of XYZ 3000, and fixed with press strip；
Take 1 mL colloid gold labels thing to add 2 mL and contain 2%（w/v） BSA、3% （w/v）Sucrose, 0.6 mol/L NaCl, 0.2%
Tween 20 （v/v）With 0.1%（w/v）20 mmol/L dobell's solutions of Sodium azide（pH 8.0）；Utilize Airjet
Quanti 3000 is with 15 mL/cm by anti-collaurum label solution specking in mineral wool；50 DEG C of min of drying box 30 are put to dry；
Glue gold pad is put into polybag, adds 4 DEG C of drier is closed to save backup.
（8）The preparation of sample pad
Mineral wool is cut into 1.5 × 30 cm2Strip, 0.1 mol/L NaCl, 0.2% Tween 20 will be contained（v/
v）With 0.1%（w/v）The PBS of Sodium azide（pH 7.2）Solution soaks glass sliver；50 DEG C of min of drying box 30 are put to dry；Will
Sample pad puts polybag, adds drier RT is closed to save backup.
（9）The preparation of adsorptive pads
Mineral wool is cut into 2.5 × 30 cm2Strip, adsorptive pads are put into polybag, add drier RT it is closed preserve it is standby
With.
（10）The preparation of supporting plate
By double faced adhesive tape in PVC supporting plates, 7.5 × 30 cm are cut into2Long slab, prepare supporting plate.
（11）The assembling of test paper
Instrument is assembled using LM5000 test paper or above-mentioned material is assembled into test paper plate by hand.Detection film is first pasted on support
Glue gold pad and sample pad, are then pasted on the sample end of detection film by plate center successively, the mm of overlapping 1 mm of each interlayer~2, then will
Adsorptive pads are pasted on the other end of detection film, and with the mm of overlapping 1 mm of detection film~2, sample is wrapped up with white plastic film in sample end
Product pad and glue gold pad, detection direction and detection solution upper limit mark are printed in plastic film, is wrapped up in handle end with blue plastic film
Adsorptive pads.
（12）Cutting and packaging
The test paper plate assembled is cut into the test strips that width is 0.3 cm using CM4000 cutting devices, test strips are dispensed
Enter polybag, add 4 DEG C of closed preservations of drier.
（13）PRV velogen strain and vaccine strain differentiate the implementation structure of Test paper
Referring to Fig. 1, Fig. 2, in figure, 1 is the supporting plate strip of foil that do not absorb water, and using plastic slice bar or can be adopted in implementation
With the hard paper sheets not absorbed water, reaction reagent carrier absorption layer is combined by 2,3,4,5, in one's hands since sample end 2
Pommel 5 is pasted onto above supporting layer 1 successively；Wherein 2 be sample end sample pad, and glass fibre cotton abbreviation glass can be used in implementation
Cotton, 3 identify the strong poison of PRV and vaccine strain gB protein monoclonal antibodies mAb1 gold mark for absorption colloid gold label
Glass fibre cotton, processed glass cellucotton, referred to as gold standard pad can be used, 4 be detection film, and nitrocellulose can be used in implementation
Film, 5 be handle end adsorptive pads, using blotting paper, such as filter paper or other blotting papers, the cm of test strips overall length 8, width 0.4
Cm, the 6 gE protein monoclonal antibodies mAb2 not react with specific recognition PRV velogen strain but with vaccine strain exist
The strong malicious detection line trace " | " printed on nitrocellulose filter, 7 be specific recognition PRV velogen strain and vaccine virus epidemic disease
The gB protein monoclonal antibodies mAb3 or resisting pstudorabies poison polyclonal antibody pAb1 of seedling strain are printed on nitrocellulose filter
Vaccine virus detection line trace " | ", 8 be rabbit anti-mouse IgG polyclonal antibodies pAb2 or staphylococcus aureus SPA fine in nitric acid
The nature controlling line trace " | " printed on plain film is tieed up, strong malicious detection line trace, vaccine virus detection line trace and Quality Control on detection film
Line trace assembled arrangement is " || | ".9 be diaphragm, and the diaphragm for being covered in mineral wool and gold mark mineral wool is 9-1, is covered in
Diaphragm on water accepting layer filter paper is 9-2.On the white diaphragm of mineral wool and gold mark mineral wool intersection correspondence position partially
A mark line 10 is printed on to the diaphragm at the cm of one side of mineral wool 0.5, is printed on arrow and Max words on the right of mark line 10
Sample, handle end diaphragm can use yellow or other colors, and intersection fiber crosses one another infiltration between 2,3,4,5 each layers.
（14）PRV velogen strain and vaccine strain differentiate Test paper examinations reaction principle
After PRV velogen strain and vaccine strain differentiate Test paper sample end insertion detected sample solution,
Solution to be checked drives PRV antigens and gold labeling antibody mAb1 to be checked together to nitrocellulose membrane diffusion, and most by siphonage
Penetrating into eventually in filter paper layer, gold labeling antibody mAb1 is combined with PRV velogen strains to be checked or vaccine strain antigen in diffusion process,
Gold labeling antibody-antigenic compound is formed, the gold mark compound of PRV velogen strains can be simultaneously with detecting the strong poison detection trace on film
MAb2 and vaccine virus detection trace mAb3 are combined, generation rufous " || " mark, part not with the gold labeling antibody of antigen binding not
It can be combined with detection trace and continue to spread, be combined on film is detected with the pAb2 in Quality Control trace or SPA, generate reddish brown colour code
Remember " | ", two kinds of mark combination superpositions, three rufous positive marks of formation " || | ", represent strong containing pseudorabies in sample
Poison；, can only be with vaccine virus and the gold mark compound of PRV vaccine strains can not be combined with the strong poison detection trace mAb2 on detection film
Detect trace mAb3 to combine, generation rufous " | " mark, part can not be with detecting trace knot with the gold labeling antibody of antigen binding
Close and continue to spread, combined on detection film with the pAb2 in Quality Control trace or SPA, generate reddish brown color marker " | ", two kinds of marks
Combination superposition, forms two rufous positive marks " || ", represents to comprise only pseudorabies vaccine virus in sample；When in sample not
During antigen containing PRV, no gold labeling antibody-antigenic compound is formed, it is impossible to is examined with strong poison detection trace and vaccine virus
Survey trace to combine, then generate negative marker " | ".If there is no reddish brown color marker to show on detection film, show that test strips have been lost
Effect.
（15）PRV velogen strain and vaccine strain differentiate Test paper detection example operating method
（15.1）Detect the preparation of sample solution：Gather pig swab to be checked（Pharynx is wiped wipes with anus）, disease（Extremely）Porcine tissue, excrement
The different detection samples with vaccine etc., add appropriate PBS or water be simply suspended or grind
（15.2）Test paper detects：PRV velogen strain and vaccine strain are differentiated into Test paper sample end immerses to treat
Examine the s of solution 10 s~20；Take out the min observation results of test paper horizontal positioned 5 min~10.
（15.3）Testing result judges：Test paper shows three rufous bands（Strong malicious detection line, vaccine virus detection line and matter
Control line）" || | " it is that the strong poison of PRV is positive, represent to contain PRV malicious antigens by force in measuring samples；Two rufous bands（Vaccine virus is examined
Survey line and nature controlling line）" || " is positive for PRV vaccine virus, represents to contain PRV vaccine virus antigens in measuring samples；Only show one it is red
Brown band（Nature controlling line）" | " is negative for PRV, represents that measuring samples do not detect PRV antigens；Test paper does not show any band and shown
Misoperation or test paper failure are detected, need to be separately to take Test paper to detect again.
Embodiment one, the strong poison infection quick diagnosis of pseudorabies, collection disease（Extremely）The pathological tissues of pig, including brain, lung,
Liver,spleen,kidney, tracheae, small intestine, large intestine and lymph node, aborted fetus etc., with 1:5 or 1:10 add appropriate PBS or water simply to be ground
Mill, prepares solution to be checked, differentiates that Test paper is pressed with PRV velogen strain and vaccine strain（15）Operating method is carried out
Detection and result judgement, antidiastole disease（Extremely）The strong poison infection of pig or vaccine immunity.Test paper shows three rufous bands（Strong poison
Detection line, vaccine virus detection line and nature controlling line）" || | " positive for the strong poison of PRV, represent pig to be checked for the strong poison infection of PRV；Two red
Brown band（Vaccine virus detection line and nature controlling line）" || " is positive for PRV vaccine virus, and it is PRV vaccine immunities to represent pig to be checked；Only
Show a rufous band（Nature controlling line）" | " is negative for PRV, represents pig to be checked without strong poison infection or vaccine immunity；Test paper is not
Show any band to show to detect misoperation or test paper failure, need to be separately to take Test paper to detect again.
Embodiment two, the PRV inspection and quarantine of live pig, gather live pig swab（Pharynx is wiped wipes with anus）Or excrement, with
1:5 or 1:10 add appropriate PBS or water to be simply suspended, and solution to be checked are prepared, with PRV velogen strain and vaccine virus
Strain differentiates that Test paper is pressed（15）Operating method is detected and result judgement, and the strong poison infection or vaccine for differentiating detection live pig are exempted from
Epidemic situation condition.Test paper shows three rufous bands（Strong malicious detection line, vaccine virus detection line and nature controlling line）" || | " it is that PRV is malicious by force
The positive, represent that live pig to be checked or environment are the strong poison pollutions of PRV；Two rufous bands（Vaccine virus detection line and nature controlling line）" || "
It is positive for PRV vaccine virus, represent that live pig to be checked or environment are PRV vaccine immunities without the strong poison pollutions of PRV；Only show one it is reddish brown
Vitta band（Nature controlling line）" | " is negative for PRV, represents live pig to be checked without strong poison infection or vaccine immunity；Test paper does not show any
Band shows to detect misoperation or test paper failure, need to be separately to take Test paper to detect again.
Embodiment three, the quality testing of pseudorabies vaccine virus vaccine, with 1:5 or 1:10 add appropriate PBS or water dissolving pig
Pseudo- rabies vaccine virus live vaccine etc., prepares vaccine solution to be checked, differentiates detection with PRV velogen strain and vaccine strain
Test paper is pressed（15）Operating method is detected and result judgement, detects the viral level of pseudorabies vaccine virus vaccine and differentiates and examines
Survey the strong malicious pollution condition of vaccine.Test paper shows three rufous bands（Strong malicious detection line, vaccine virus detection line and nature controlling line）
" || | " positive for the strong poison of PRV, represent vaccine to be checked for the strong poison pollutions of PRV；Two rufous bands（Vaccine virus detection line and matter
Control line）" || " is positive for PRV vaccine virus, represents vaccine to be checked without the strong poison pollutions of PRV, the colored intensity of vaccine virus detection line with
PRV vaccine contgs are directly proportional；Only show a rufous band（Nature controlling line）" | " is negative for PRV, represents vaccine to be checked without PRV
Strong poison pollution, while be also free of vaccine virus；Test paper does not show any band and shows to detect misoperation or test paper failure, need to be with another
Test paper is taken to detect again.
Claims (3)
1. a kind of strong poison of PRV and vaccine virus differentiate Test paper, by supporting plate, sample pad, gold standard pad, film is detected
Formed with adsorptive pads, it is characterized in that：Gold standard pad adsorbs colloid gold label resisting pstudorabies poison gB protein monoclonal antibody mAb1,
The strong malicious detection line T1 of detection film is resisting pstudorabies poison gE protein monoclonal antibody mAb2 traces " │ ", vaccine virus detection line
T2 is resisting pstudorabies poison polyclonal antibody pAb1 or gB protein monoclonal antibody mAb3 traces " │ ", and nature controlling line C is anti-mouse
IgG antibody pAb2 or staphylococcus aureus SPA traces " │ "；The equal specific recognition pigs of monoclonal antibody mAb1 and mAb3 are pseudo-
Rabies viruses velogen strain and vaccine toxic vaccine strain, the different epitopes of PRV gB albumen are identified respectively；The list
Clonal antibody mAb2 specific recognition PRV velogen strain gE albumen, but do not reacted with vaccine strain；The monoclonal resists
Body mAb1, mAb2 and mAb3 are using PRV gB and gE recombinant protein as immunizing antigen, by establishing hybridoma
Method prepare identify PRV gB and gE protein monoclonal antibody, utilize immunoperoxidase cell monolayer examination
The monoclonal antibody that the strong poison of PRV and vaccine virus are distinguished in screening is tested, is known with being superimposed EUSA screening
Do not go out the monoclonal antibody of different epitopes.
2. discriminating Test paper according to claim 1, it is characterized in that：In detection film when the strong poison of PRV infects
Show strong malicious detection line T1, tri- red stripes of vaccine virus detection line T2 and nature controlling line C " │ │ │ ", detect pseudorabies disease vaccine
When malicious detection film show two red stripes of vaccine virus detection line T2 and nature controlling line C " │ │ ", no porcine pseudorabies virus then only
Show mono- red stripes " │ " of nature controlling line C.
3. the discriminating Test paper described in as requested 1 or 2, it is characterized in that：Differentiate the PRV reaction of Test paper
Spectrum is wide, and vaccine virus detection line T2 can detect pseudorabies vaccine strain and most popular velogen strains, and strong malicious detection line T1 can detect
Most PRV prevalence velogen strains, the discriminating inspection to PRV Major Epidemic strain and vaccine strain can be achieved
Survey.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410763138.7A CN104459144B (en) | 2014-12-12 | 2014-12-12 | A kind of PRV velogen strain and vaccine strain differentiate Test paper |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410763138.7A CN104459144B (en) | 2014-12-12 | 2014-12-12 | A kind of PRV velogen strain and vaccine strain differentiate Test paper |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104459144A CN104459144A (en) | 2015-03-25 |
| CN104459144B true CN104459144B (en) | 2017-11-10 |
Family
ID=52905530
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410763138.7A Active CN104459144B (en) | 2014-12-12 | 2014-12-12 | A kind of PRV velogen strain and vaccine strain differentiate Test paper |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104459144B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104459142B (en) * | 2014-12-12 | 2017-11-10 | 河南省农业科学院 | A kind of High Virulent Newcastle Disease Virus and low virulent strain differentiate Test paper |
| CN105137076B (en) * | 2015-09-15 | 2017-03-22 | 北京农学院 | Small-fragment gG antigen and application of small-fragment gG antigen in detecting pseudorabies virus gG antibody |
| CN106405082A (en) * | 2016-08-31 | 2017-02-15 | 洛阳普莱柯万泰生物技术有限公司 | Porcine pseudorabies virus detection test paper |
| CN107831309A (en) * | 2017-10-18 | 2018-03-23 | 武汉科前生物股份有限公司 | A kind of porcine pseudorabies virus gE protein antibodies competitive ELISA detection kits and detection method |
| CN109187968B (en) * | 2018-09-19 | 2022-01-28 | 郑州大学 | Bivalent gold-labeled test paper for detecting classical swine fever virus and porcine pseudorabies virus and preparation method thereof |
| CN110068686A (en) * | 2019-05-15 | 2019-07-30 | 河南百奥生物工程有限公司 | A kind of pseudoabies antibody blocking Test paper |
| CN111781378B (en) * | 2020-07-13 | 2023-04-14 | 祁寒松 | Colloidal gold test strip for pathogen and antibody detection and differential diagnosis thereof |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1477108A (en) * | 2003-06-02 | 2004-02-25 | 中国农业科学院哈尔滨兽医研究所 | Recombinant gE protein and its application for diagnosis of pseudo-rabies virus infection |
| US20060270060A1 (en) * | 2005-05-31 | 2006-11-30 | Smith Henry J | Rapid test for glycated albumin in saliva |
| CN101762705A (en) * | 2010-01-21 | 2010-06-30 | 中国农业科学院哈尔滨兽医研究所 | Colloidal gold immunochromatographic test strip for detecting wild-type classical swine fever virus |
| CN103792373A (en) * | 2014-03-12 | 2014-05-14 | 武汉中博生物股份有限公司 | Colloidal gold immunochromatography test paper for antibodies of pseudorabies viruses of gE, gB and gD and preparation method |
| CN103983781A (en) * | 2014-05-27 | 2014-08-13 | 武汉中博生物股份有限公司 | Porcine pseudorabies virus gE IgM antibody colloidal gold immunochromatograohic assay test strip as well as preparation method and application thereof |
| CN104459142B (en) * | 2014-12-12 | 2017-11-10 | 河南省农业科学院 | A kind of High Virulent Newcastle Disease Virus and low virulent strain differentiate Test paper |
| CN104407137B (en) * | 2014-12-12 | 2017-10-03 | 河南省农业科学院 | A kind of CSFV velogen strain and low virulent strain differentiate Test paper |
-
2014
- 2014-12-12 CN CN201410763138.7A patent/CN104459144B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN104459144A (en) | 2015-03-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104459144B (en) | A kind of PRV velogen strain and vaccine strain differentiate Test paper | |
| CN104407137B (en) | A kind of CSFV velogen strain and low virulent strain differentiate Test paper | |
| CN104198703B (en) | People's mycoplasma pneumoniae gold label silver stain immunochromatographytest test kit and its preparation method and application | |
| CN105461805B (en) | Monoclonal Antibodies against Porcine Epidemic Diarrhea Virus and its application | |
| CN105527437B (en) | A kind of detection kit and its application | |
| CN101339192A (en) | Test paper for one-step detection for pig virus diarrhoea disease pathogen | |
| CN103266088B (en) | A kind of H7 subtype avian influenza virus monoclonal antibody and test kit | |
| CN104316703B (en) | A kind of Mycoplasma bovis test strip and its preparation method | |
| CN110058019A (en) | A kind of hog cholera antibody blocking Test paper | |
| CN110068686A (en) | A kind of pseudoabies antibody blocking Test paper | |
| CN104459142B (en) | A kind of High Virulent Newcastle Disease Virus and low virulent strain differentiate Test paper | |
| CN109970851A (en) | The preparation method of monoclonal antibody of CCV virus M protein and preparation method thereof, immunity colloidal gold test paper strip | |
| CN106405082A (en) | Porcine pseudorabies virus detection test paper | |
| CN109187967A (en) | A kind of detection simultaneously distinguishes O-shaped, the duplex rapid detection card of A type foot and mouth disease virus and preparation method thereof | |
| CN105319359A (en) | Streptococcus pneumoniae quantum dot immunochromatographic assay detection card and preparing method and application thereof | |
| CN105348386B (en) | The monoclonal antibody cocktail and detection kit of the O-shaped virus of resistant to foot and mouth disease | |
| CN101339191A (en) | Test paper for detecting pig breeding disorder virus epidemic pathogen | |
| CN103995136B (en) | Rapid colloidal gold detection test strip for pig porcine reproductive and respiratory syndrome virus | |
| 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 | |
| CN103235127A (en) | Marek's disease virus rapid combined-detection test strip | |
| CN101666800A (en) | Fast test strip of vesicular stomatitis virus (VSV) and preparation method thereof | |
| CN101424689B (en) | Bluetongue virus detection test paper | |
| CN110018304A (en) | A kind of newcastle epidemic disease antibody blocking Test paper | |
| CN204028084U (en) | People's Chlamydia pneumoniae quantum dot immune chromatography test card | |
| CN110058018A (en) | A kind of Animal against infulenza blocking Test paper |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |