CN104459144A - Porcine pseudorabies virus virulent strain and vaccine strain identification and detection test paper - Google Patents
Porcine pseudorabies virus virulent strain and vaccine strain identification and detection test paper Download PDFInfo
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Classifications
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- 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
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- 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
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- 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
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
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- Biotechnology (AREA)
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- General Health & Medical Sciences (AREA)
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- Pathology (AREA)
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- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention relates to an appliance for identifying and detecting epidemic disease infection and immunization of livestock and in particular relates to porcine pseudorabies virus virulent strain and vaccine strain identification and detection test paper. The test paper consists of a supporting plate, a sample pad, a gold-labeled pad, a detection film and an absorbent pad, wherein imprints of a virulent virus detection line T1 shaped like a vertical line, a vaccine virus detection line T2 shaped like a vertical line and a quality control line C shaped like a vertical line are arranged on the detection film. During identification and detection, when three red stripes shaped like three vertical lines present on the detection film, the sample is infected with porcine pseudorabies virulent virus; when two red stripes shaped like two vertical lines present on the detection film, the sample is immunized by a porcine pseudorabies virus vaccine; and when only one red stripe shaped like a vertical line presents on the detection film, the sample is negative to porcine pseudorabies virus. The identification and detection test paper is high in specificity, high in sensitivity, wide in response spectrum and easy and convenient and rapid to operate, can be used for detecting the conventional vaccine virus strains and most of popular virulent strains, can be widely applied to identifying and detecting the porcine pseudorabies virus infection and immunization and is easy to popularize and apply in production practice.
Description
Technical field
The present invention relates to a kind of disease of domestic animals and infect the identification detector tool with immunity, poison and vaccine virus differentiate Test paper by force particularly to relate to a kind of PRV.
Background technology
Pseudoabies ((Pseudorabies, PR) is that the many animals caused by Pseudorabies virus (Pseudorabies Virus, PRV) suffers from infectious disease altogether, can infected cattle, pig, dog, sheep, fox and cat etc.Pig is the infection sources and the natural storage host of pseudoabies, and pig shows as heating, piglet nervous symptoms, high mortality, big-and-middle porcine respiratory symptom, the clinical characters such as Sow abortion after infecting PRV.In 2010, pseudoabies was that China's large-scale pig farm carries out one of ideal disease of Blight control, and many pig farms have reached immunity without Infection Status.But in 2011, there is the porcine pseudorabies virus made a variation in the pig farm of some Bartha-K61 vaccine immunities, shows that traditional gE gene-deleted vaccine can not provide enough immunoprotections.The infection rate of Henan Province's swinery pseudoabies since two thousand five presents entire lowering trend, clinically to distribute or atypical case.But, from the end of the year 2011, the pseudo-mad dog epidemic situation of our province selected swine farms burst, very fast in various places outburst inside the province, be widely current; And the animals such as the sheep near selected swine farms, dog, cat, fox also occur infecting death state.Therefore, present stage China's porcine pseudorabies prevention and control become more thorny and complicated.
PRV belongs to herpetoviridae (Herpesviridae), the member of a-herpesviral subfamilies (Alphaherpesvirinae), and genome is linear dsdna, size about 150 kb.Whole PRV genome is at least containing 70 genes, and coding 70-100 is remaining plants albumen.Current research finds PRV codified 11 glycoprotein: gB, gC, gD, gE, gG, gH, gI, gK, gL, gM and gN.Conservative in gB, gH, gL and gM herpetoviridae, and gB, gH and gL to copy in cell culture all herpesvirals be all required.GB albumen can induce the generation of neutralizing antibody, relevant to immunoprotection.PRV gE albumen is called as gI albumen the earliest.All there is gE disappearance in PRV vaccine strain such as Bartha strain and the BUK strain etc. of the screening preparation of generation nineteen sixty.At twentieth century the mid-80, constructed the PRV mutant strain of gE disappearance by DNA recombinant technique; On this basis, TK being carried out disappearance, that PRV virus is caused further is weak.Many gE deletion of vaccine all can prevent to attack poison and infect or alleviate the clinical symptoms of attacking poison infection and causing.Also there is the PRV vaccine of gC and gG disappearance although current, only allow swinery to use gE deletion of vaccine in many countries.From generation nineteen ninety to the end of the year 2011, China more than 80% swinery all employs gE gene-deleted vaccine Bartha and carries out immunity inoculation.The prerequisite that gE vaccine and gE antibody diagnosis support the use for PRV purification is that all epidemic isolates all express gE albumen, and the animal of epidemic strain infection all produces the antibody of gE albumen.Almost gE albumen is all expressed by PRV street strain used at present.By the conbined usage of gE gene-deleted vaccine and gE antibody diagnosis method, some European countries raise pigs in group as Germany, Austria, Sweden, Denmark and Britain and the U.S. are successfully in and have purified porcine pseudorabies.The Chinese government has worked out " national medium-term and long-term animal epidemic control program (2012-2020) " in 2011, propose 2015, original seed Pig Farm pseudoabies reaches purification standard; To the year two thousand twenty, all kinds of whole nation pig farm porcine pseudorabies reaches the target of purification standard.But the purification of pathogen is a long-term and difficult task.If realize purifying completely or may needing for 100 years even longer close to purification state; The porcine pseudorabies of the such as U.S. appears at 1909 first, and its purification plan starts from 1989, is accomplished to most Purification Project in 2005.Nonetheless, in wild boar group, the existence of PRV virus is again infected PRV to family pig and is caused very large threat.Therefore, set up easy, quick, special, responsive differential diagnostic method distinguish vaccine immunity animal and virus-infected animal very necessary.
The differential diagnostic method of pseudoabies comprises PCR (PCR), enzyme linked immunosorbent assay (ELISA) and immunopcroxidase monolayer assay (IPMA) etc., and wherein ELISA kit is antidiastole commodity the most conventional on market.Commercial ELISA kit uses indirectly or stop band restrain respectively, and because clinical serum is comparatively complicated, the specificity of indirect ELISA is slightly poor relative to stop band restrain.Stop band restrain uses clinical Swine serum to block the combination of gE protein-specific monoclonal antibody, specificity and susceptibility higher; But also there is inborn shortcoming in stop band restrain.The monoclonal antibody that stop band restrain uses for be the epi-position in pig body with immunodominance, morphing once PRV virus at gE gene just likely makes monoclonal antibody identification invalid.Although stop band restrain still can go out the PRV antiviral antibody of the emerging variation of China by specific detection, the possibility that monoclonal antibody identification is invalid still exists.So it is imperative that the PRV variation strain according to present stage develops new differential diagnostic method.Immune chromatography test paper is at monoclonal antibody technique, a kind of novel in vitro detection technique that colloidal gold immunochromatographimethod technology and new material technology basis grow up, desirable instant detection (point-of-care test, and onthe technology of site test POCT), it has sensitiveer, special, easy, the advantage such as fast, especially " foolproof " operation can be realized, namely Site Detection can be carried out without any need for auxiliary instrumentation equipment, and in 1-5 minute result of determination, the quantitative and semi-quantitative being widely used in various analysis thing detects fast, comprise antigen, haptens, antibody and nucleic acid etc., become one of immunology detection technology of sensitivity the most fast now.Henan Province Animal Immunology Key Laboratory system from nineteen ninety-five carries out the research of immune test paper Fast Detection Technique, take the lead in succeeding in developing animal epidemic cause of disease and antibody test colloid test paper product in the world--Test Paper for Rapid Diagnosis of IBD and Trichinella sui antibody test paper, establish animal epidemic quick detection test paper research and development technology platform, successfully develop the serial quick detection test paper products such as animal epidemic antigen, antibody and medicament residue detection so far, greatly promote the application and development of this technology.The present invention is directed to the key technical problem of PRV wild virus infection and vaccine immunity antidiastole, it is strong that Screening and Identification can distinguish PRV, the high-affinity pairing monoclonal antibody of vaccine strain, set up the protein chip technology platform based on immune chromatography test paper, development PRV velogen strain and vaccine strain differentiate Test paper, set up and be applicable to cultivate the quick of basic unit and clinical detection, easy pseudorabies wild virus infection and vaccine immunity joint inspection technology, realize the Real-Time Monitoring to PRV wild virus infection, for China's pseudorabies prevention and control and purification provide technical support, to pseudorabies epidemic monitoring and prevention and control significant.
Summary of the invention
The object of the invention is: development is applicable to pseudorabies and infects the strong malicious and vaccine virus of the PRV detected with Immune dctection and differentiate Test paper, and this test paper is special, responsive, quick, easy, easily applies in production practices.
Technical scheme of the present invention is: the strong poison of a kind of PRV and vaccine virus differentiate Test paper, by back up pad, sample pad, gold mark pad, detect film and adsorptive pads formation, gold mark pad is the glass fibre of absorption colloid gold label resisting pstudorabies poison gB protein monoclonal antibody mAb1, detecting film is the strong malicious detection line T1 of trace, the nitrocellulose filter of vaccine virus detection line T2 and nature controlling line C, strong malicious detection line T1/ vaccine virus detection line T2/ nature controlling line C is arranged as to handle end: " │ │ │ " by sample end, strong malicious detection line T1 is resisting pstudorabies poison monoclonal antibody gE albumen mAb2 trace " │ ", vaccine virus detection line T2 is resisting pstudorabies poison polyclonal antibody pAb1 or resisting pstudorabies poison gB protein monoclonal antibody mAb3 trace " │ ", nature controlling line C is anti-mouse IgG antibody pAb2 or staphylococcus aureus SPA trace " │ ".When differentiating to detect, the strong poison of pseudorabies infects and manifests strong malicious detection line T1 at detection film, vaccine virus detection line T2 and nature controlling line C tri-red stripes " │ │ │ ", pseudorabies vaccine virus vaccine immunity manifests vaccine virus detection line T2 and nature controlling line C two red stripes " │ │ " at detection film, then only manifests nature controlling line C red stripes " │ " without PRV.
With PRV gB and gE recombinant protein for immunizing antigen, qualification resisting pstudorabies poison gB and gE protein monoclonal antibody is produced by hybridoma cell technology, immunopcroxidase monolayer assay (IPMA) is utilized to screen the monoclonal antibody of the strong poison of differentiation PRV and vaccine virus, to superpose the monoclonal antibody that enzyme linked immunosorbent assay (ELISA) screening identifies different epitope.For the monoclonal antibody mAb1 of colloid gold label and the monoclonal antibody mAb3 equal specific recognition PRV velogen strain of vaccine virus detection line T2 and vaccine virus vaccine strain, identify the different epitopes of PRV gB albumen respectively, for the monoclonal antibody mAb2 specific recognition PRV velogen strain gE albumen of strong malicious detection line T2, but do not react with vaccine strain.Simultaneously, with the monoclonal antibody that IPMA screening is wide with PRV response spectrum, colloid gold label monoclonal antibody mAb1 and vaccine virus detection line T2 monoclonal antibody mAb3 identifiable design PRV gE gene-deleted vaccine poison vaccine and most popular velogen strain, the popular velogen strain of the most PRVs of strong malicious detection line T1 monoclonal antibody mAb2 identifiable design.With PRV vaccine virus Bartha strain for immunizing antigen prepares resisting pstudorabies poison polyclonal antibody pAb1, can be used for vaccine virus detection line T2 trace.Be that immunizing antigen prepares goat anti-mouse igg polyclonal antibody pAb2 with mouse IgG, pAb2 and staphylococcus aureus SPA all can be used for nature controlling line C trace.
The good effect that the present invention is useful: the strong poison of PRV and vaccine virus differentiate that Test paper achieves the synchronous joint inspection of the strong poison of PRV and vaccine virus, effectively can differentiate that the strong poison of swinery pseudorabies infects and vaccine immunity, and it is simple to operate, everybody can operate, better can meet the needs of different levels personnel, if Disease monitor, customs quarantine control, health and epidemic prevention, intensive culture are to individual cultivation etc., be easy to apply on a large scale, there are wide market outlook and larger economical, societal benefits.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 is detecting on film containing identifying the vaccine virus detection line of the strong poison of PRV and vaccine virus and identifying strong poison but the strong malicious detection line of nonrecognition vaccine virus, synchronously can carry out velogen strain and the vaccine strain joint inspection of PRV, the strong poison and the vaccine virus that realize pseudorabies are differentiated to detect.
(2) high specificity, susceptibility is high.The strong poison of PRV and vaccine virus differentiate that Test paper is prepared from based on the high-affinity pairing monoclonal antibody can distinguishing the strong poison of PRV and vaccine virus, high specificity and the susceptibility of monoclonal antibody are high, formed without covalent bond between gold grain and antibody molecule in gold labeling antibody, the two is combined by the Van der Waals force between the charges of different polarity, the reactivity impact of collaurum on labelled antibody is very little, and has higher mark rate.Therefore, differentiate that Test paper has higher specificity and susceptibility.
(2) fast easy and simple to handle.Without the need to other reagent any during use discriminating Test paper, as long as be inserted into measuring samples about 10 seconds, testing result can be judged in 2 minutes.
(3) testing result is shown vivid, intuitively accurate.Differentiate that Test paper is to show rufous " │ ", " │ │ " and " │ │ │ " trace is as the feminine gender detected, vaccine virus and strong malicious positive mark, namely on detection film, display brownish red band " │ " is PRV feminine gender, represent that detected sample infects without vaccine virus vaccine or strong poison, article two, brownish red band " │ │ " is the PRV vaccine virus positive, represent that test sample is vaccine virus vaccine immunity, article three, brownish red band " │ │ │ " is the strong poison positive of PRV, represent that test sample is that strong poison infects, result judges image, intuitively, accurately, simple and clear, not easily there is false negative and false positive erroneous judgement.
(4) cost is low, small investment.Use and differentiate Test paper, do not need separately to join instrument and equipment and other reagent, Site Detection is settled at one go, with low cost, small investment, instant effect.
Accompanying drawing illustrates that the present invention is further described below in conjunction with drawings and Examples.
Fig. 1 is that the strong poison of PRV and vaccine virus differentiate Test paper side-looking structural representation.
Fig. 2 is that the strong poison of PRV and vaccine virus differentiate Test paper plan structure schematic diagram.
In figure, 1. back up pad, 2. sample pad, 3. gold mark pad; 4. detect film, 5. adsorptive pads, 6. strong malicious detection line T1 trace, 7. vaccine virus detection line T2 trace; 8. nature controlling line C trace, 9-1. sample end diaphragm, 9-2. handle end diaphragm, 10. mark line.
Embodiment PRV by force malicious and vaccine virus differentiates that Test paper can be widely used in the discriminating detection of the strong poison infection of swinery PRV and vaccine immunity.Prepare the strong poison of PRV and vaccine virus discriminating Test paper, first need to prepare PRV immunizing antigen, and then prepare resisting pstudorabies poison polyclonal antibody and monoclonal antibody, and the monoclonal antibody of the strong poison of pseudorabies and vaccine virus is distinguished in screening, identify that the monoclonal antibody mAb1 of the strong poison of pseudorabies and vaccine virus is for the preparation of colloid gold label thing, identify that the strong poison of pseudorabies and the monoclonal antibody mAb2 of nonrecognition vaccine virus are for printing strong malicious detection line trace " │ ", the polyclonal antibody pAb1 of the strong poison of identification pseudorabies and vaccine virus or monoclonal antibody mAb3 is for printing vaccine virus detection line trace " │ ", secondly goat anti-mouse igg antibody or staphylococcus aureus SPA need be prepared, for printing nature controlling line trace " | ".
(1) preparation of PRV immunizing antigen.
(1.1) preparation of PRV gB recombinant protein
With the neutralizing epitope enrichment region of pcr amplification PRVgB albumen, genes of interest is cloned into prokaryotic expression carrier pET-28a, build gB DNA recombinant expression plasmid pET-gB, induce through 1 mol/L IPTG, at E. coli gB recombinant protein, Western blot detects and shows that gB expressing protein all by PRV positive serum specific recognition, can have good reactivity.Ni post affinitive layer purification and dilution refolding technology is adopted to obtain the gB recombinant protein 15mg/L of purity about 90%.
(1.2) preparation of PRV gE recombinant protein
With the epi-position enrichment region of pcr amplification PRVgE albumen, genes of interest is cloned into prokaryotic expression carrier pET-28a, build gB DNA recombinant expression plasmid pET-gE, induce through 0.4 mol/L IPTG, at E. coli gE recombinant protein, Western blot detects and shows that gE expressing protein all by the strong malicious positive serum specific recognition of PRV, can have good reactivity.Ni post affinitive layer purification technology is adopted to obtain the gE recombinant protein 12mg/L of purity about 90%.
(2) preparation of resisting pstudorabies poison monoclonal antibody.
(2.1) foundation of hybridoma cell strain.
Respectively by PRV gB and gE recombinant protein and Fu Shi immunologic adjuvant mixed in equal amounts, fully emulsified, with 50 mg-100 mg/ only immune BALB/c system mouse 3 times, every minor tick 15-30 days; 3-4 days after 3rd booster immunization, by the bloodletting of immune mouse eyeball, draws neck lethal, in 75% alcohol-pickled 5-10 min, asepticly gets its spleen; To shred and through 100 order nylon net filters, centrifugal 10 min of 1000 r/min, collect splenocyte; By 1 × 10
8splenocyte and 2-5 × 10
7sP2/0 myeloma cell mixing, centrifugal 10 min of 1000 r/min, abandon supernatant, by the 40%-50% PEG 4000(pH8.5-9.0 of 0.7-1 ml in the water-bath of 37 DEG C) slowly add cell, after incubation 1 min, slowly add serum-free 1640 nutrient culture media 15 ml, to stop the effect of PEG, 37 DEG C of water-bath 5-10 min, centrifugal 10 min of 1000 r/min, abandon supernatant, are resuspended in by cell in HAT Selective agar medium, and add 96 well culture plates (100 ml-200 ml/ hole), put 37 DEG C of 5% CO
2cultivate in incubator.Cultivate after 7-10 days, get Hybridoma Cell Culture supernatant and screen positive hybridoma cell with enzyme linked immunosorbent assay (ELISA) and immunopcroxidase monolayer assay (IPMA).Infect hamster kidney cell (BHK-21) with the strong poison of PRV standard, after methyl alcohol is fixing, 1 h closed by 5% skimmed milk 37 DEG C; Add cells and supernatant 50mL/ hole to be checked, if HAT nutrient culture media and mouse immune serum are negative and positive control; The goat anti-mouse igg antibody (50 mL/ hole) that the horseradish peroxidase (HRP) adding 1:500 dilution marks, 37 DEG C of effect 30 min; Often all fully wash with the PBS containing 0.05% Tween-20 after step reaction; With substrate A EC color development at room temperature 10-20 min, after rinsing termination colour developing with water, examine under a microscope colour developing result.Continuous 3 limited dilution clonings are carried out in the clone hole choosing strong positive, Growth of Cells vigorous, expand freeze-stored cell after cultivating, and set up resisting pstudorabies poison gB albumen and hybridoma cell strain 8 strain of gE protein monoclonal antibody and 12 strains respectively.
(2.2) preparation of monoclonal antibody: prepare monoclonal antibody to induce ascites in body.The multiparity Balb/c mouse of pristane or the whiteruss sensitization of learning from else's experience, the hybridoma 10 of lumbar injection exponential phase
7individual/only, and extract ascites after 7 d ~ 10 d, get supernatant after centrifugal, packing, frozen.
(2.3) qualification of monoclonal antibody
(2.3.1) antibody titer of monoclonal antibody
The monoclonal antibody measuring cleer and peaceful ascites in Hybridoma Cell Culture with enzyme linked immunosorbent assay (ELISA) and immunopcroxidase monolayer assay (IPMA) is tired, and the ELISA of Mab supernatant and ascites tires respectively in 1:640 and 1:5 × 10
5above, IPMA tires respectively at 1:16 and more than 1:8000.
(2.3.2) specificity of monoclonal antibody
Hamster kidney cell (BHK-21) is infected with PRV standard strain, epidemic isolates and vaccine strain, the reactivity of monoclonal antibody and PRV epidemic isolates is measured with immunopcroxidase monolayer assay (IPMA), screening obtains gB protein monoclonal antibody 6 strain of simultaneously specific recognition PRV standard strain, epidemic isolates and vaccine strain, and specific recognition PRV standard strain and epidemic isolates but monoclonal antibody 8 strain of nonrecognition vaccine strain.The response spectrum of above-mentioned 14 strain monoclonal antibodies and vaccine strain and epidemic isolates is detected with immunopcroxidase monolayer assay (IPMA), prove to be used for the gB protein monoclonal antibody identifiable design PRV vaccine strain of colloid gold label and vaccine virus detection line T2 and most popular velogen strain, for the popular velogen strain of the most PRVs of gE protein monoclonal antibody identifiable design of strong malicious detection line, all there is wider PRV response spectrum.
(2.3.3) monoclonal antibody identification Characterization of antigenic epitopes
With stop band restrain or superposition ELISA, the epitope to the identification of PRV monoclonal antibody is analyzed, screening obtains the monoclonal antibody mAb1 and mAb3 that identify the different epitope of PRV gB albumen respectively, and it is respectively used to colloid gold label and vaccine virus detection line T2 trace; Obtain specific recognition PRV standard strain and epidemic isolates, and the gE protein monoclonal antibody mAb2 do not reacted with vaccine virus vaccine strain, for strong malicious detection line T1 trace.
(2.3.3.1) stop band restrain: with HRP labeled monoclonal antibody respectively, the specificity of ELISA blocking test qualification monoclonal antibody identification epitope, first in the ELISA Plate of gB or gE recombinant protein bag quilt, unlabelled monoclonal antibody is added line by line, if PRV positive serum, negative serum and PBS contrast, 37 DEG C of effect 1 h; Then the monoclonal antibody of HRP mark is added by column, 37 DEG C of effect 1 h; Develop the color with substrate TMB, read the OD of each detect aperture
450value.The epitope that the unmarked monoclonal antibody identification that remarkable inhibitory enzyme mark monoclonal antibody combines is identical or close, combines to enzyme mark monoclonal antibody the unmarked monoclonal antibody had no significant effect and then identifies different epitopes.
(2.3.3.2) superpose ELISA: first utilize the ELISA Plate of envelope antigen to measure the working concentration of each monoclonal antibody with indirect ELISA, draw antigen saturation curve.In superposition ELISA test, suitably dilute monoclonal antibody according to antigen saturation curve, and pairing adds each hole of ELISA Plate, hatches 1 h ~ 2 h for 37 DEG C; Add ELIAS secondary antibody respectively and TMB develops the color, read each hole OD
450value, by following formulae discovery superposition coefficient (AI): AI=[2 ' OD
1+2/ (OD
1+ OD
2)-1] ' 100%, wherein OD
1+2for the OD in pairing monoclonal antibody hole
450value, OD
1and OD
2be the OD in two independent monoclonal antibody holes
450value.The AI value of two monoclonal antibodies is less than 40% and is judged to the identical or close epitope of identification; AI value is greater than 40% and is judged to the different epitope of identification.
(2.4) purifying of monoclonal antibody: with caprylic acid-ammonium purified monoclonal antibody IgG from mouse ascites.Get 1 mL mouse ascites, add 2 mL 0.06 mol/L sodium acetate buffers (pH 5.0), be adjusted to pH 4.5 with 0.1 mol/L HCl; Under stirring at room temperature, it is sad dropwise to add 33 mL, and 4 DEG C leave standstill centrifugal 30 min of 2 h, 15000 r/min, abandon precipitation; In centrifugal supernatant, add 1/10 volume 0.01 mol/L PBS (pH7.4), be adjusted to pH 7.4 with 0.1 mol/L NaOH; Under condition of ice bath, add saturated ammonium sulfate to final concentration 45%, 4 DEG C leave standstill centrifugal 30 min of 2 h, 10000 r/min, abandon supernatant; With the resuspended precipitation of appropriate PBS, to PBS dialysed overnight, change liquid 3 times.Measure the protein content of monoclonal antibody purification IgG at more than 1mg/ml with spectrophotometer method or dying method with coomassie brilliant blue (Bradford method), measure the antibody titer of mouse ascites and IgG purification in 1:5 × 10 with enzyme linked immunosorbent assay (ELISA) and immunopcroxidase monolayer assay (IPMA)
5with more than 1:4000, packing, frozen.
(3) preparation of resisting pstudorabies poison polyclonal antibody.
With the pseudorabies vaccine of 50 mg ~ 100 mg/kg body weight through intramuscular injection immune health piglet 3 ~ 4 times, final immunization is after 10 days, vena cava anterior is taken a blood sample, its serum antibody titer is measured when more than 1:1000 with IPMA, Culling heart blood or arteria carotis bloodletting, collect hyper-immune serum, with anhydrous sodium sulfate (Na
2sO
4) extract immune swine serum IgG, get 1 part of immune serum and add 2 part of 0.01 mol/L PBS(pH7.2) mixing, add anhydrous Na
2sO
4to final concentration 18%, the centrifugal 20min of 37 DEG C of water-bath 30min, 4000 r/min, abandons supernatant, with appropriate PBS (pH7.2) resuspended precipitation, adds the Na of final concentration 16%
2sO
4, 37 DEG C of centrifugal 20min of precipitation 30min, 4000r/min, abandon supernatant, then with appropriate PBS (pH7.2) resuspended precipitation, with the Na of final concentration 14%
2sO
4precipitation, the centrifugal 20min of 4000r/min, abandon supernatant, with appropriate PBS (pH7.2) resuspended precipitation, to PBS (pH7.2) dialyzed overnight, change liquid 2 ~ 3 times, measure 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 trace.
(4) preparation of rabbit anti-mouse IgG polyclonal antibody
With the healthy new zealand rabbit of the mouse IgG of purifying immunity about 2.0 kg, first immunisation is with Freund's complete adjuvant emulsification antigen, subcutaneous multi-point injection 50 mg/ only, each booster immunization interval 3 w, with the intramuscular injection of incomplete Freund's adjuvant emulsification antigen, after last booster immunization 2 w, when measuring immune serum antibody titer higher than 1:40 with agar gel diffusion test (AGP), gather and highly exempt from rabbit whole blood, separation of serum, with caprylic acid-ammonium purified rabbit anti-mouse IgG, method is with (2.4) monoclonal antibody purifying, sad consumption is 45 mL/mL serum, measure antibody titer and protein concentration (10-20 mg/ml), prepared rabbit anti-mouse IgG polyclonal antibody pAb2 can be used for the printing of Test paper nature controlling line C trace.
(5) colloid gold label of monoclonal antibody
(5.1) preparation of collaurum: get the conical flask that 100 mL ultrapure waters are placed in 500 mL cleanings, add 1 mL 1%(w/v) gold chloride boils; Freshly prepared 1 mL 1%(w/v is added rapidly under stirring) sodium citrate solution, boils about 3 min and becomes aubergine to solution colour from yellow, continue to boil 2 min; Treat that solution is cool to room temperature, mend ultrapure water to 100 mL, with 0.2 mol/L K
2cO
3adjust pH to 9.0,4 DEG C of lucifuges can preserve the several months.
(5.2) the suitableeest labelled protein concentration determination: get anti-PRV monoclonal antibody IgG to be marked to 20 mmol/L dobell's solutions (pH 8.0), 4 DEG C of dialyzed overnights.In microwell plate with 25 mL ultrapure waters with 1:2. 1:4. 1:8 ... doubling dilution PRV monoclonal antibody to be marked; Each hole adds 125 mL colloidal gold solutions, and RT leaves standstill 5 min; Add 125 mL 1 mol/L NaCl solution; Each hole color becomes blueness with the reduction of protein concentration from redness.Do not become the protein concentration of the most high dilution of blue monoclonal antibody into the suitableeest label concentration of collaurum with color, during colloid gold label, protein concentration increases by 20%.
(5.3) colloid gold label of monoclonal antibody: the monoclonal antibody IgG to be marked getting the suitableeest protein concentration of 2 mL, adds 10 mL colloidal gold solutions (pH 9.0), mixes rapidly, room temperature effect 10 min ~ 15 min; Add 1/10 volume containing 10%(w/v) 20 mmol/L dobell's solutions of bovine serum albumin(BSA) (BSA), mix rapidly, RT acts on 10 min ~ 15 min; 4 DEG C of 15000 centrifugal 30min of g, carefully removes supernatant; With containing 1%(w/v) the resuspended collaurum of 20 mmol/L dobell's solution of BSA, the same centrifugal, abandon supernatant; Repeated washing 1 time, with 1 mL containing 1%(w/v) the resuspended collaurum of 20mmol/L dobell's solution of BSA, 4 DEG C save backup.
(6) preparation of film is detected
Cellulose nitrate is detected film and be cut into 2.5 × 30 cm
2rectangular, be placed on XYZ 3000 specking instrument platform, and fix with press strip; With PBS(pH 7.2) by specific recognition PRV velogen strain but the gB albumen monoclonal antibody of the gE albumen monoclonal antibody, specific recognition PRV velogen strain and the vaccine virus vaccine strain that do not react with vaccine strain or many anti-igg and rabbit anti-mouse igg are diluted to 1.0 mg/mL, and be put in storage pool respectively; Utilize Biojet Quanti 3000 with 1.0 mL/cm respectively by anti-PRV gE albumen monoclonal antibody, gB albumen monoclonal antibody or PRV many anti-igg solution and rabbit anti-mouse igg solution specking in detection film central authorities, form strong malicious detection line T1, vaccine virus detection line T2 and nature controlling line C trace, detection line and nature controlling line are at a distance of 0.5 cm; Put 42 DEG C of drying box 30 min or natural drying at room temperature; Detection film is put polybag, adds that 4 DEG C, drying agent is airtight to be saved backup.
(7) preparation of pad
Glass wool is cut into 1.5 × 30 cm
2rectangular, be placed on XYZ 3000 specking instrument platform, and fix with press strip; Get 1 mL colloid gold label thing and add the 20 mmol/L dobell's solutions (pH 8.0) of 2 mL containing 2% (w/v) BSA, 3% (w/v) sucrose, 0.6 mol/L NaCl, 0.2% Tween 20 (v/v) and 0.1% (w/v) Sodium azide; Utilize Airjet Quanti 3000 with 15 mL/cm by anti-collaurum label solution specking in glass wool; Put 50 DEG C of drying box 30 min dry; Glue gold pad is put polybag, adds that 4 DEG C, drying agent is airtight to be saved backup.
(8) preparation of sample pad
Glass wool is cut into 1.5 × 30 cm
2rectangular, with will containing the PBS(pH 7.2 of 0.1 mol/L NaCl, 0.2% Tween 20 (v/v) and 0.1% (w/v) Sodium azide) solution soaks glass sliver; Put 50 DEG C of drying box 30 min dry; Sample pad is put polybag, adds that drying agent RT is airtight to be saved backup.
(9) preparation of adsorptive pads
Glass wool is cut into 2.5 × 30 cm
2rectangular, adsorptive pads is put polybag, adds that drying agent RT is airtight to be saved backup.
(10) preparation of back up pad
By double faced adhesive tape in PVC back up pad, be cut into 7.5 × 30 cm
2long slab, preparation back up pad.
(11) assembling of test paper
LM5000 test paper is utilized to assemble instrument or by hand above-mentioned material be assembled into test paper plate.First detection film is pasted on back up pad central authorities, then glue gold pad and sample pad are pasted on the sample end detecting film successively, overlapping 1 mm ~ 2 mm of each interlayer, again adsorptive pads is pasted on the other end detecting film, with overlapping 1 mm ~ 2 mm of detection film, in sample end with white plastic film parcel sample pad and glue gold pad, plastic film is printed on detection side to and detect solution upper limit mark, at handle end with blue plastic film parcel adsorptive pads.
(12) cutting and packaging
Utilize CM4000 cutting device that the test paper plate assembled is cut into the test strips that width is 0.3 cm, test strips is distributed into polybag, add drying agent 4 DEG C of airtight preservations.
(13) PRV velogen strain and vaccine strain differentiate the enforcement structure of Test paper
See in Fig. 1, Fig. 2, figure, 1 is the back up pad strip of foil that do not absorb water, plastic slice bar can be adopted in enforcement or adopt the hard paper sheet material that do not absorb water, reaction reagent carrier absorption layer by 2,3,4,5, combine, from sample end 2, be pasted onto above supporting layer 1 successively to handle end 5, wherein 2 is sample end sample pad, glass fibre cotton can be used in enforcement to be called for short glass wool, 3 for adsorbing the gold mark glass fibre cotton of the strong poison of colloid gold label identification PRV and vaccine strain gB protein monoclonal antibody mAb1, processed glass cellucotton can be adopted, referred to as gold mark pad, 4 for detecting film, nitrocellulose filter can be adopted in enforcement, 5 is handle end adsorptive pads, adopt thieving paper, as filter paper or other thieving paper, test strips overall length 8 cm, width 0.4 cm, 6 for specific recognition PRV velogen strain but the strong malicious detection line trace " | " printed on nitrocellulose filter of the gE protein monoclonal antibody mAb2 do not reacted with vaccine strain, vaccine virus detection line trace " | " that the 7 gB protein monoclonal antibody mAb3 or resisting pstudorabies poison polyclonal antibody pAb1 that are specific recognition PRV velogen strain and vaccine virus vaccine strain print on nitrocellulose filter, the 8 nature controlling line traces " | " printed on nitrocellulose filter for rabbit anti-mouse IgG polyclonal antibody pAb2 or staphylococcus aureus SPA, detecting the strong malicious detection line trace on film, vaccine virus detection line trace and nature controlling line trace assembled arrangement are " || | ".9 is diaphragm, and the diaphragm covering glass wool and gold mark glass wool is 9-1, and the diaphragm covered on water accepting layer filter paper is 9-2.Mark at glass wool and gold on the diaphragm of the white diaphragm of glass wool intersection correspondence position being partial to glass wool one side 0.5 cm place and be printed on a mark line 10; arrow and Max printed words are printed on the right of mark line 10; handle end diaphragm can use yellow or other color; 2; 3; 4,5 each layers each other intersection fiber cross one another infiltration.
(14) PRV velogen strain and vaccine strain differentiate Test paper examinations reaction principle
After PRV velogen strain and vaccine strain differentiate that Test paper sample end inserts detected sample solution, solution to be checked drives PRV antigen to be checked and golden labeling antibody mAb1 together to cellulose nitrate membrane diffusion by syphonic effect, and finally penetrate in filter paper layer, in diffusion process, golden labeling antibody mAb1 combines with PRV velogen strain to be checked or vaccine strain antigen, form golden labeling antibody-antigenic compound, the gold mark compound of PRV velogen strain can simultaneously with detect the strong poison on film and detect trace mAb2 and vaccine virus and detect trace mAb3 and be combined, generate rufous " || " mark, the golden labeling antibody that part is not combined with antigen can not be combined with detection trace and continue to spread, pAb2 or SPA on detection film in Quality Control trace is combined, generate rufous mark " | ", two kinds of marker combination superpositions, form three rufous positive marks " || | ", represent malicious by force containing pseudorabies in sample, and the gold mark compound of PRV vaccine strain can not detect trace mAb2 be combined with detecting the strong poison on film, trace mAb3 can only be detected with vaccine virus to be combined, generate rufous " | " mark, the golden labeling antibody that part is not combined with antigen can not be combined with detection trace and continue to spread, pAb2 or SPA on detection film in Quality Control trace is combined, generate rufous mark " | ", two kinds of marker combination superpositions, form two rufous positive marks " || ", represent in sample only containing pseudorabies vaccine virus, when in sample containing PRV antigen time, do not have golden labeling antibody-antigenic compound to be formed, trace and vaccine virus can not be detected with strong poison and detect trace and be combined, then generate negative marker " | ".If detection film does not have rufous mark display, then show that test strips lost efficacy.
(15) PRV velogen strain and vaccine strain differentiate Test paper detection example method of operating
(15.1) preparation of detection sample solution: gather the different detection such as pig swab to be checked (pharynx is wiped and wiped with anus), sick (extremely) porcine tissue, ight soil and vaccine sample, adds appropriate PBS or water carries out simple suspendible or grinding
(15.2) detection paper: PRV velogen strain and vaccine strain are differentiated that Test paper sample end immerses solution 10 s ~ 20 s to be checked; Take out test paper horizontal positioned 5 min ~ 10 min observations.
(15.3) testing result judges: it is positive as the strong poison of PRV that test paper manifests three rufous bands (strong malicious detection line, vaccine virus detection line and nature controlling line) " || | ", represents in measuring samples containing the strong malicious antigen of PRV; Article two, rufous band (vaccine virus detection line and nature controlling line) " || " is the PRV vaccine virus positive, represents in measuring samples containing PRV vaccine virus antigen; Only manifest a rufous band (nature controlling line) " | " negative for PRV, represent that measuring samples does not detect PRV antigen; Test paper does not manifest any band and shows to detect misoperation or test paper inefficacy, again need detect separately to get Test paper.
Embodiment one, the strong poison of pseudorabies infects quick diagnosis, gather the pathological tissues of sick (extremely) pig, comprise brain, lung, liver,spleen,kidney, tracheae, small intestine, large intestine and lymph node, aborted fetus etc., appropriate PBS is added or water simply grinds with 1:5 or 1:10, prepare solution to be checked, differentiate that Test paper carries out detection and result judgement by (15) method of operating with PRV velogen strain and vaccine strain, the strong poison of antidiastole disease (extremely) pig infects or vaccine immunity.It is that the strong poison of PRV is positive that test paper manifests three rufous bands (strong malicious detection line, vaccine virus detection line and nature controlling line) " || | ", represents that pig to be checked is that the strong poison of PRV infects; Article two, rufous band (vaccine virus detection line and nature controlling line) " || " is the PRV vaccine virus positive, represents that pig to be checked is PRV vaccine immunity; Only manifest a rufous band (nature controlling line) " | " negative for PRV, represent that pig to be checked infects or vaccine immunity without strong poison; Test paper does not manifest any band and shows to detect misoperation or test paper inefficacy, again need detect separately to get Test paper.
Embodiment two, the PRV inspection and quarantine of live pig, gather live pig swab (pharynx is wiped and wiped with anus) or ight soil, appropriate PBS is added or water carries out simple suspendible with 1:5 or 1:10, prepare solution to be checked, differentiate that Test paper carries out detection and result judgement by (15) method of operating with PRV velogen strain and vaccine strain, differentiate that the strong poison detecting live pig infects or vaccine immunity situation.It is that the strong poison of PRV is positive that test paper manifests three rufous bands (strong malicious detection line, vaccine virus detection line and nature controlling line) " || | ", represents that live pig to be checked or environment are that the strong poison of PRV pollutes; Article two, rufous band (vaccine virus detection line and nature controlling line) " || " is the PRV vaccine virus positive, and representing that live pig to be checked or environment pollute without the strong poison of PRV, is PRV vaccine immunity; Only manifest a rufous band (nature controlling line) " | " negative for PRV, represent that live pig to be checked infects or vaccine immunity without strong poison; Test paper does not manifest any band and shows to detect misoperation or test paper inefficacy, again need detect separately to get Test paper.
Embodiment three, the quality testing of pseudorabies vaccine virus vaccine, appropriate PBS or water-soluble solution pseudorabies vaccine virus live vaccine etc. is added with 1:5 or 1:10, prepare vaccine solution to be checked, differentiate that Test paper carries out detection and result judgement by (15) method of operating with PRV velogen strain and vaccine strain, detect the viral level of pseudorabies vaccine virus vaccine and differentiate to detect the strong malicious pollution condition of vaccine.It is that the strong poison of PRV is positive that test paper manifests three rufous bands (strong malicious detection line, vaccine virus detection line and nature controlling line) " || | ", represents that vaccine to be checked is that the strong poison of PRV pollutes; Article two, rufous band (vaccine virus detection line and nature controlling line) " || " is the PRV vaccine virus positive, and represent that vaccine to be checked pollutes without the strong poison of PRV, the colored intensity of vaccine virus detection line is directly proportional to PRV vaccine contg; Only manifest a rufous band (nature controlling line) " | " negative for PRV, represent that vaccine to be checked pollutes without the strong poison of PRV, simultaneously also not containing vaccine virus; Test paper does not manifest any band and shows to detect misoperation or test paper inefficacy, again need detect separately to get Test paper.
Claims (4)
1. the strong poison of PRV and vaccine virus differentiate Test paper, by back up pad, sample pad, gold mark pad, detect film and adsorptive pads formation, it is characterized in that: gold mark pad absorption colloid gold label resisting pstudorabies poison gB protein monoclonal antibody mAb1, the strong malicious detection line T1 detecting film is resisting pstudorabies poison gE protein monoclonal antibody mAb2 trace " │ ", vaccine virus detection line T2 is resisting pstudorabies poison polyclonal antibody pAb1 or gB protein monoclonal antibody mAb3 trace " │ ", nature controlling line C is anti-mouse IgG antibody pAb2 or staphylococcus aureus SPA trace " │ ".
2. discriminating Test paper according to claim 1, it is characterized in that: when the strong poison of pseudorabies infects, manifest strong malicious detection line T1 at detection film, vaccine virus detection line T2 and nature controlling line C tri-red stripes " │ │ │ ", manifest vaccine virus detection line T2 and nature controlling line C two red stripes " │ │ " at detection film during the immunity of pseudorabies vaccine virus, then only manifest nature controlling line C red stripes " │ " without PRV.
3. discriminating Test paper according to claim 1, it is characterized in that: monoclonal antibody mAb1 and mAb3 equal specific recognition PRV velogen strain and vaccine virus vaccine strain, identify the different epitopes of PRV gB albumen respectively, monoclonal antibody mAb2 specific recognition PRV velogen strain gE albumen, but do not react with vaccine strain.
4. the discriminating Test paper as requested described in 1 or 3, it is characterized in that: differentiate that the PRV response spectrum of Test paper is wide, vaccine virus detection line T2 can detect pseudorabies vaccine strain and most popular velogen strain, strong malicious detection line T1 can detect the popular velogen strain of most PRV, can realize detecting the discriminating of PRV Major Epidemic strain and vaccine strain.
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