CN104459142A - Test paper for distinguishing virulent strain from attenuated strain of newcastle disease virus - Google Patents
Test paper for distinguishing virulent strain from attenuated strain of newcastle disease virus Download PDFInfo
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- CN104459142A CN104459142A CN201410763127.9A CN201410763127A CN104459142A CN 104459142 A CN104459142 A CN 104459142A CN 201410763127 A CN201410763127 A CN 201410763127A CN 104459142 A CN104459142 A CN 104459142A
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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
-
- 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
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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
- G01N2333/08—RNA viruses
- G01N2333/18—Togaviridae; Flaviviridae
- G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
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- Hematology (AREA)
- Biomedical Technology (AREA)
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- General Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
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Abstract
The invention relates to a detecting appliance for distinguishing infection and immunization of diseases in poultry, and particularly relates to a test paper for distinguishing a virulent strain from an attenuated strain of a newcastle disease virus. The test paper is composed of a support plate, a sample pad, a gold pad, a detection membrane and an absorbent pad, wherein the detection membrane contains prints of a virulent detection line T1 '|', an attenuated detection line T2 '|' and a quality control line C '|'; when distinguishing detection is carried out, infection of the virulent newcastle disease virus is determined when three red strips '|||' appear on the detection membrane; immunization of a newcastle attenuated vaccine is determined when two red strips '||' appear; and a negative newcastle disease virus is determined when only one red strip '|' appears. The distinguishing detection test paper has the advantages of high specificity, high sensitivity, and wide response spectrum, is simple, convenient and fast to operate, can be widely applied to distinguishing detection of infection and immunization of the newcastle disease virus, and is easy to popularize and apply in production practice; and existing attenuated vaccine strains and most of popular virulent strains can be detected.
Description
Technical field
The present invention relates to a kind of poultry disease and infect the identification detector tool with immunity, particularly relate to the strong malicious and weak poison of a kind of Avian pneumo-encephalitis virus and differentiate Test paper.
Background technology
Newcastle disease (Newcastle disease, ND) also known as philippine fowl disease, be by Avian pneumo-encephalitis virus (Newcastle disease virus, NDV) cause a kind of with respiratory tract, the hemorrhage high degree in contact for typical cytopathic of gastrointestinal mucosal, acute septic bird infectious disease.Except poultry, having at least more than 200 kind of bird can nature or laboratory infection, be one of the most serious infectious disease of harm world aviculture.This disease is since reported first in 1926, still popular all over the world so far, and time has and breaks out, and cause serious economic loss to aviculture, the World Health Organization (WHO) (OIE) is classified as should report epidemic disease, and China is classified as a class zoonosis.China adopts that attenuated vaccine prevention and control ND's is popular on a large scale, but there is new change again in the popular and characteristics of incidence of ND, show as atypia and chronic infection more, occur so-called " non typical newcastle disease " and " mild ewcastle disease ", other respiratory pathogens mixed infection such as NDV and avian influenza virus is also very general simultaneously, ND is risen one after another, and provisions fowl industry brings no small loss, also makes the anti-system of ewcastle disease more difficult.NDV for having cyst membrane, non-segmented negative, sub-thread minus-stranded rna virus, belong to paramyxovirus section (
paramyxoviridae) avian paramyxovirus genus (
paramyxovirus), its serotype is avian paramyxovirus I type (APMV-1), can be divided into again 16 genotype.NDV genome contains 15186, 15192 or 15198 nucleotide, Organization of viral genome pattern is 3 '-NP-P-M-F-HN-L-5 ', encode nucleocapsid protein (Nucleocapsid protein successively, NP), phosphoprotein (Phosphoprotein, P), stromatin (Matrix protein, M), fusion (Fusion protein, F), hemagglutinin-neuraminidase albumen (Heamagglutinin-Neuraminidase protein, and large rna dependent rna polymerase (Large RNA dependent RNA-polymerase HN), L), wherein F and HN albumen is important two membrane glycoproteins in NDV surface, they play important effect in virus infection.NDV virus virulence depends primarily on F precursor protein partial amino-acid series, and highly pathogenic NDV is usually containing C-
112r/K-RQ/K/R-R/K-R-F
117-N, low pathogenicity NDV are then C-
112g/EK/R-Q-G/E-R-L
117-N, NDV is divided into addicted to visceratonia and nervous system type (high mortality), middle hair style (low actual, moderate Respiratory symptoms), weak malicious type (gentle infection in respiratory system, not dead, vaccine strain) and asymptomatic (asymptomatic or enteron aisle subclinical infection) by OIE.Genotype I strain is low virulent strain, in gene II type virus, also have the velogen strain of medium virulence strain and highly pathogenicity, and other 7 genotype (III-IX type) NDV occurred after gene I and II type strain is velogen strain except low virulent strain.Epidemic data shows, the eighties in last century, the NDV strain of Major Epidemic in China chicken group was VAnd if VIg hypotype, 90 mid-nineties 90 China there is NDV genotype VII, and become the preponderant genotype that China NDV is popular, current VIId hypotype has overwhelming superiority in the popular velogen strain of China, and the attenuated vaccine strain LaSota generally used in China fowl group belongs to gene II type, the genotype therefore between newcastle disease vaccine strain and current popular strain and antigenic specificity are the theoretical foundations differentiating Newcastle Disease Virus Vaccine strain and velogen strain.
At present, the strategy based on vaccine immunity is still taked in the prevention and control of China's ewcastle disease, since the nineties in last century, ND attenuated vaccine strain obtains on a large scale in China, the frequent use of high dose, though the conventional strong malicious epidemic strain of the ND of vaccine strain LaSota to different genotype can produce clinical protection completely, just the infection duplication of epidemic strain in immune chicken and discharge can not be prevented, and the strong poison infection of ND still happens occasionally in vaccinated flock, simultaneously owing to lacking serologic marker and supporting differential diagnostic method, it makes to be difficult to distinguish immune weak poison and wild virus infection by antibody test technology such as hemagglutination-inhibition test (HI) and enzyme linked immunosorbent assay (ELISA) in the large-scale application of China, be unfavorable for control and the purification of ND.Along with separate sources, different regions NDV street strain and vaccine strain complete genome sequence complete the development with molecular Biological Detection technology, at NDV, Chinese scholars differentiates that context of detection has carried out large quantity research, by force genomic by analyzing NDV, low virulent strain characteristic site, design is strong respectively, low virulent strain Auele Specific Primer or probe, establish the RT-PCR and quantitative fluorescent PCR equimolecular discriminating detection method that distinguish the strong poison of NDV and weak poison, there is higher Sensitivity and Specificity, but these detection methods need to depend on PCR instrument or quantitative real time PCR Instrument, there is operation relatively loaded down with trivial details, consuming time longer, the high deficiency of testing cost, limit its application in epidemiology survey and veterinary clinic.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, 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 NDV wild virus infection and vaccine immunity antidiastole, it is strong that Screening and Identification can distinguish NDV, the high-affinity pairing monoclonal antibody of low virulent strain, set up the protein chip technology platform based on immune chromatography test paper, development High Virulent Newcastle Disease Virus and low virulent strain differentiate Test paper, set up and be applicable to cultivate the quick of basic unit and clinical detection, easy newcastle disease virus infects and vaccine immunity joint inspection technology, realize the Real-Time Monitoring to bird Avian pneumo-encephalitis virus wild virus infection, for China's ewcastle disease prevention and control and purification provide technical support, to ewcastle disease epidemic monitoring and prevention and control significant.
Summary of the invention
The object of the invention is: development is applicable to poultry ewcastle disease and infects the strong malicious and weak poison of the Avian pneumo-encephalitis virus 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: Test paper differentiated by the strong poison of a kind of Avian pneumo-encephalitis virus and weak poison, 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 anti-new castle disease virus monoclonal antibody mAb1, detecting film is the strong malicious detection line T1 of trace, the nitrocellulose filter of weak malicious detection line T2 and nature controlling line C, the weak malicious detection line T2/ nature controlling line C of strong malicious detection line T1/ is arranged as to handle end: " │ │ │ " by sample end, strong malicious detection line T1 is anti-new castle disease virus monoclonal antibody mAb2 trace " │ ", weak malicious detection line T2 is anti-new castle disease virus polyclonal antibody pAb1 or anti-new castle disease virus monoclonal antibody mAb3 trace " │ ", nature controlling line C is anti-mouse IgG antibody pAb2 or staphylococcus aureus SPA trace " │ ".When differentiating to detect, Virulent Newcastle Disease Virus infects and manifests strong malicious detection line T1 at detection film, weak malicious detection line T2 and nature controlling line C tri-red stripes " │ │ │ ", Newcastle disease attenuated vaccine immunity manifests weak malicious detection line T2 and nature controlling line C two red stripes " │ │ " at detection film, then only manifests nature controlling line C red stripes " │ " without Avian pneumo-encephalitis virus.
With the strong malicious F48E8 strain of Avian pneumo-encephalitis virus standard for immunizing antigen, qualification anti-new castle disease virus monoclonal antibody is produced by hybridoma cell technology, immunopcroxidase monolayer assay (IPMA) is utilized to screen the monoclonal antibody distinguishing the strong poison of Avian pneumo-encephalitis virus and weak poison, 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 equal specific recognition High Virulent Newcastle Disease Virus of monoclonal antibody mAb3 of weak malicious detection line T2 and attenuated vaccine strain, identify the different epitopes of Avian pneumo-encephalitis virus respectively, for the monoclonal antibody mAb2 specific recognition High Virulent Newcastle Disease Virus of strong malicious detection line T2, but do not react with low virulent strain.Simultaneously, with the monoclonal antibody that IPMA screening is wide with Avian pneumo-encephalitis virus response spectrum, colloid gold label monoclonal antibody mAb1 and the weak malicious detection line T2 monoclonal antibody mAb3 medium virulence of identifiable design Avian pneumo-encephalitis virus and attenuated vaccine and most popular velogen strain, the popular velogen strain of the most Avian pneumo-encephalitis virus of strong malicious detection line T1 monoclonal antibody mAb2 identifiable design.With Avian pneumo-encephalitis virus attenuated vaccine LaSota strain for immunizing antigen prepares chicken anti-new castle disease virus polyclonal antibody pAb1, can be used for weak malicious 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 Avian pneumo-encephalitis virus and weak poison differentiate that Test paper achieves the synchronous joint inspection of the strong poison of Avian pneumo-encephalitis virus and weak poison, effectively can differentiate that poultry Virulent Newcastle Disease Virus 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 weak malicious joint inspection.The strong poison of Avian pneumo-encephalitis virus and weak poison differentiate that Test paper is detecting on film containing identifying the weak malicious detection line of the strong poison of Avian pneumo-encephalitis virus and weak poison and identifying strong poison but the strong malicious detection line of the weak poison of nonrecognition, synchronously can carry out velogen strain and the low virulent strain joint inspection of Avian pneumo-encephalitis virus, the strong poison and the weak poison that realize ewcastle disease are differentiated to detect.
(2) high specificity, susceptibility is high.The strong poison of Avian pneumo-encephalitis virus and weak poison differentiate that Test paper is prepared from based on the high-affinity pairing monoclonal antibody can distinguishing the strong poison of Avian pneumo-encephalitis virus and weak poison, 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, weak poison and strong malicious positive mark, namely on detection film, display brownish red band " │ " is Avian pneumo-encephalitis virus feminine gender, represent that detected sample infects without attenuated vaccine or strong poison, article two, brownish red band " │ │ " is the weak poison positive of Avian pneumo-encephalitis virus, expression test sample is attenuated vaccine immunity, article three, brownish red band " │ │ │ " is the strong poison positive of Avian pneumo-encephalitis virus, 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 Test paper side-looking structural representation differentiated by the strong poison of Avian pneumo-encephalitis virus and weak poison.
Fig. 2 is that Test paper plan structure schematic diagram differentiated by the strong poison of Avian pneumo-encephalitis virus and weak poison.
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. weak malicious detection line T2 trace, 8. nature controlling line C trace, 9-1. sample end diaphragm, 9-2. handle end diaphragm, 10. mark line.
The strong poison of embodiment Avian pneumo-encephalitis virus and weak poison differentiate that Test paper can be widely used in the discriminating detection of multiple poultry (as chicken, duck, goose, dove etc.) virulent newcastle disease virus infection and vaccine immunity.Test paper differentiated by the strong poison of preparation Avian pneumo-encephalitis virus and weak poison, first preparation Avian pneumo-encephalitis virus immunizing antigen is needed, and then prepare anti-new castle disease virus polyclonal antibody and monoclonal antibody, and the monoclonal antibody of Virulent Newcastle Disease Virus and weak poison is distinguished in screening, identify that the monoclonal antibody mAb1 of Virulent Newcastle Disease Virus and weak poison is for the preparation of colloid gold label thing, identify that the monoclonal antibody mAb2 of Virulent Newcastle Disease Virus and the weak poison of nonrecognition is for printing strong malicious detection line trace " │ ", the polyclonal antibody pAb1 of identification Virulent Newcastle Disease Virus and weak poison or monoclonal antibody mAb3 is for printing weak malicious 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 Avian pneumo-encephalitis virus immunizing antigen.
With Avian pneumo-encephalitis virus standard velogen strain F48E8 through allantoic cavity inoculation 9-11 age in days SPF chicken embryo, within 36-48 hour, collect allantoic fluid, differential centrifugation purified virus antigen, 4 DEG C of 3000 r/min low-speed centrifugal 30 min removes impurity, 4 DEG C of 60000 r/min ultracentrifugation 1 h, by 7 ml 0.01 mol/L PBS (pH value 7.2) resuspended precipitations.The HA measuring Avian pneumo-encephalitis virus with hemagglutination test (HA) tires and reaches 2
-12above.
(2) preparation of anti-new castle disease virus monoclonal antibody.
(2.1) foundation of hybridoma cell strain.
By Avian pneumo-encephalitis virus immunizing antigen and Fu Shi immunologic adjuvant mixed in equal amounts, fully emulsified, with 50mg-100mg/ 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 5-10min, asepticly gets its splenocyte; 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 immunopcroxidase monolayer assay (IPMA).With strong malicious F48E8 strain infected chicken embryo fibroblast (CEF) of Avian pneumo-encephalitis virus standard or hamster kidney cell (BHK-21) cell, 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; Add 1:500 horseradish peroxidase (HRP) and mark goat anti-mouse igg antibody (50 mL/ hole), 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, set up anti-new castle disease virus monoclonal antibody hybridoma cell strain 28 strain.
(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 immunopcroxidase monolayer assay (IPMA) is tired, and the IPMA of Mab supernatant and ascites tires respectively at 1:16 and more than 1:1600.The HI measuring monoclonal antibody ascites with hemagglutination-inhibition test (HI) tires, and screening has the strain of blood clotting inhibit activities monoclonal antibody 5, and its HI tires at more than 1:128.
(2.3.2) specificity of monoclonal antibody
Use NDV standard strain, epidemic isolates and attenuated vaccine strain I system (Mukteswar strain), II system (B1 strain), III system (F strain), IV system (Lasota strain), strain that V4(is high temperature resistant) and clone strain (Clone-30, N-29, Clone-83 and N-88) infected chicken embryo fibroblast (CEF) or hamster kidney cell (BHK-21), the reactivity of monoclonal antibody and NDV epidemic isolates is measured with immunopcroxidase monolayer assay (IPMA), screening obtains specific recognition NDV standard strain simultaneously, monoclonal antibody 22 strain of epidemic isolates and attenuated vaccine strain, and specific recognition NDV standard strain and epidemic isolates but monoclonal antibody 6 strain of nonrecognition attenuated vaccine strain.The response spectrum of above-mentioned 28 strain monoclonal antibodies and vaccine strain and epidemic isolates is detected with immunopcroxidase monolayer assay (IPMA), prove to be used for the medium virulence of monoclonal antibody identifiable design Avian pneumo-encephalitis virus of colloid gold label and weak malicious detection line T2 and attenuated vaccine and most popular velogen strain, for the popular velogen strain of the most Avian pneumo-encephalitis virus of monoclonal antibody identifiable design of strong malicious detection line, all there is wider NDV 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 NDV monoclonal antibody is analyzed, screening obtains the monoclonal antibody mAb1 and mAb3 that identify the different epitope of Avian pneumo-encephalitis virus respectively, and it is respectively used to colloid gold label and weak malicious detection line T2 trace; Obtain specific recognition Avian pneumo-encephalitis virus standard strain and epidemic isolates, and the monoclonal antibody mAb2 do not reacted with attenuated 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 that NDV standard positive is antigen coated, unlabelled monoclonal antibody is added line by line, if NDV positive 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.The protein content of monoclonal antibody purification IgG is measured at more than 1mg/ml with spectrophotometer method or dying method with coomassie brilliant blue (Bradford method), the antibody titer of mouse ascites and IgG purification is measured at more than 1:1000 with immunopcroxidase monolayer assay (IPMA), packing, frozen.
(3) preparation of anti-new castle disease virus polyclonal antibody.
With the Avian pneumo-encephalitis virus immunizing antigen of 50mg ~ 100mg/kg body weight with immunologic adjuvant through subcutaneous and intramuscular injection immunity SPF chicken 3 ~ 4 times, final immunization is after 10 days, venous blood collection, 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 poultry blood serum IgY, 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 anti-new castle disease virus polyclonal antibody pAb1 can be used for the printing of the weak malicious detection line T2 trace of Test paper.
(4) preparation of rabbit anti-mouse IgG polyclonal antibody
With the healthy new zealand rabbit of purified mouse IgG 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, adds 1 mL 1 % (w/v) gold chloride and 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-NDV monoclonal antibody IgG to be marked to 20 mmol/L dobell's solutions (pH 8.0), 4 DEG C of dialyzed overnights.With 25 mL ultrapure water 1:2,1:4,1:8 in microwell plate ... doubling dilution NDV 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 the 20 mmol/L dobell's solutions of 1/10 volume containing 10% (w/v) 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 the resuspended collaurum of 20 mmol/L dobell's solution containing 1% (w/v) BSA, the same centrifugal, abandon supernatant; Repeated washing 1 time, with the 20mmol/L dobell's solution resuspended collaurum of 1 mL containing 1% (w/v) 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 High Virulent Newcastle Disease Virus but the monoclonal antibody of the monoclonal antibody, specific recognition High Virulent Newcastle Disease Virus and the attenuated vaccine strain that do not react with low virulent strain or many anti-igg and rabbit anti-mouse igg are diluted to 1 mg/mL, and be put in storage pool respectively; Utilize Biojet Quanti 3000 with 1 mL/cm respectively by anti-NDV monoclonal antibody or many anti-igg solution and rabbit anti-mouse igg solution specking in detection film central authorities, form strong malicious detection line T1, weak malicious 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, so that glass sliver will be soaked containing PBS (pH 7.2) solution of 0.1 mol/L NaCl, 0.2% Tween 20 (v/v) and 0.1% (w/v) Sodium azide; 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 of 0.3 cm, test strips is distributed into polybag, add drying agent 4 DEG C of airtight preservations.
(13) High Virulent Newcastle Disease Virus and low virulent 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 Avian pneumo-encephalitis virus and low virulent strain 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 High Virulent Newcastle Disease Virus but the strong malicious detection line trace " | " printed on nitrocellulose filter of the monoclonal antibody mAb2 do not reacted with low virulent strain, the weak malicious detection line trace " | " that the 7 monoclonal antibody mAb3 or anti-new castle disease virus polyclonal antibody pAb1 that are specific recognition High Virulent Newcastle Disease Virus and attenuated 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, weak malicious 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) High Virulent Newcastle Disease Virus and low virulent strain differentiate Test paper examinations reaction principle
After High Virulent Newcastle Disease Virus and low virulent strain differentiate that Test paper sample end inserts detected sample solution, solution to be checked drives ndv antigen to be checked and golden labeling antibody mAb1 together to cellulose nitrate membrane diffusion by siphon, and finally penetrate in filter paper layer, in diffusion process, golden labeling antibody mAb1 combines with NDV velogen strain to be checked or low virulent strain antigen, form golden labeling antibody-antigenic compound, the gold mark compound of NDV velogen strain can simultaneously with detect the strong poison on film and detect trace mAb2 and weak poison 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, with pAb2 or SPA in Quality Control trace on detection film, generate rufous mark " | ", two kinds of marker combination superpositions, form three rufous positive marks " || | ", represent in sample containing Virulent Newcastle Disease Virus, and the gold mark compound of NDV vaccine strain can not detect trace mAb2 be combined with detecting the strong poison on film, trace mAb3 can only be detected with weak poison 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, with pAb2 or PSA in Quality Control trace on detection film, generate rufous mark " | ", two kinds of marker combination superpositions, form two rufous positive marks " || ", represent in sample only containing newcastle disease vaccine poison, when in sample containing Avian pneumo-encephalitis virus antigen time, do not have golden labeling antibody-antigenic compound to be formed, trace and weak poison 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) High Virulent Newcastle Disease Virus and low virulent strain differentiate Test paper detection example method of operating
(15.1) preparation of detection sample solution: gather the different samples that detect such as live-bird swab to be checked (pharynx is wiped and wiped with anus), sick (extremely) fowl tissue, ight soil and vaccine, adds appropriate PBS or water carries out simple suspendible or grinding
(15.2) detection paper: High Virulent Newcastle Disease Virus and low virulent 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 NDV that test paper manifests three rufous bands (strong malicious detection line, weak malicious detection line and nature controlling line) " || | ", represents in measuring samples containing the strong malicious antigen of NDV; Article two, rufous band (weak malicious detection line and nature controlling line) " || " is the weak poison positive of NDV, represents in measuring samples containing NDV vaccine virus antigen; Only manifest a rufous band (nature controlling line) " | " negative for NDV, represent that measuring samples does not detect ndv 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, poultry Virulent Newcastle Disease Virus infects quick diagnosis, gather the pathological tissues of sick (extremely) fowl, comprise glandular stomach, lung, liver,spleen,kidney, tracheae, small intestine, large intestine and lymph node 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 High Virulent Newcastle Disease Virus and low virulent strain, the strong poison of antidiastole disease (extremely) fowl infects or vaccine immunity.It is that the strong poison of NDV is positive that test paper manifests three rufous bands (strong malicious detection line, weak malicious detection line and nature controlling line) " || | ", represents that poultry to be checked is that the strong poison of NDV infects; Article two, rufous band (weak malicious detection line and nature controlling line) " || " is the weak poison positive of NDV, represents that poultry to be checked is NDV vaccine immunity; Only manifest a rufous band (nature controlling line) " | " negative for NDV, represent that poultry 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 Avian pneumo-encephalitis virus inspection and quarantine of live-bird, gather live-bird 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 High Virulent Newcastle Disease Virus and low virulent strain, differentiate that the strong poison detecting live-bird pollutes or vaccine immunity situation.It is that the strong poison of NDV is positive that test paper manifests three rufous bands (strong malicious detection line, weak malicious detection line and nature controlling line) " || | ", represents that poultry to be checked or environment are that the strong poison of NDV pollutes; Article two, rufous band (weak malicious detection line and nature controlling line) " || " is the weak poison positive of NDV, and representing that poultry to be checked or environment pollute without the strong poison of NDV, is NDV vaccine immunity; Only manifest a rufous band (nature controlling line) " | " negative for NDV, represent that poultry 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 Newcastle disease attenuated vaccine, appropriate PBS or water-soluble solution Newcastle disease attenuated vaccine is added with 1:5 or 1:10, comprise the medium virulence I system of Avian pneumo-encephalitis virus (Mukteswar strain) and attenuated vaccine strain II system (B1 strain), III system (F strain), IV system (Lasota strain), strain that V4(is high temperature resistant) and clone strain (Clone-30, N-29, Clone-83 and N-88) etc., prepare vaccine solution to be checked, differentiate that Test paper carries out detection and result judgement by (15) method of operating with High Virulent Newcastle Disease Virus and low virulent strain, detect the viral level of Newcastle disease attenuated vaccine and differentiate to detect the strong malicious pollution condition of vaccine.It is that the strong poison of NDV is positive that test paper manifests three rufous bands (strong malicious detection line, weak malicious detection line and nature controlling line) " || | ", represents that vaccine to be checked is that the strong poison of NDV pollutes; Article two, rufous band (weak malicious detection line and nature controlling line) " || " is the weak poison positive of NDV, and represent that vaccine to be checked pollutes without the strong poison of NDV, the colored intensity of weak malicious detection line is directly proportional to NDV vaccine contg; Only manifest a rufous band (nature controlling line) " | " negative for NDV, represent that vaccine to be checked pollutes without the strong poison of NDV, 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. Test paper differentiated by the strong poison of Avian pneumo-encephalitis virus and weak poison, 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 anti-new castle disease virus monoclonal antibody mAb1, the strong malicious detection line T1 detecting film is anti-new castle disease virus monoclonal antibody mAb2 trace " │ ", weak malicious detection line T2 is anti-new castle disease virus polyclonal antibody pAb1 or monoclonal antibody mAb3 trace " │ ", and 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 Virulent Newcastle Disease Virus infects, manifest strong malicious detection line T1 at detection film, weak malicious detection line T2 and nature controlling line C tri-red stripes " │ │ │ ", manifest weak malicious detection line T2 and nature controlling line C two red stripes " │ │ " at detection film during Newcastle disease attenuated vaccine immunity, then only manifest nature controlling line C red stripes " │ " without Avian pneumo-encephalitis virus.
3. discriminating Test paper according to claim 1, it is characterized in that: the equal specific recognition High Virulent Newcastle Disease Virus of monoclonal antibody mAb1 and mAb3 and attenuated vaccine strain, identify the different epitopes of Avian pneumo-encephalitis virus respectively, monoclonal antibody mAb2 specific recognition High Virulent Newcastle Disease Virus, but do not react with low virulent strain.
4. the discriminating Test paper as requested described in 1 or 3, it is characterized in that: differentiate that the Avian pneumo-encephalitis virus response spectrum of Test paper is wide, weak malicious detection line T2 can detect the medium virulence I system of Avian pneumo-encephalitis virus (Mukteswar strain) and attenuated vaccine strain II system (B1 strain), III system (F strain), IV system (Lasota strain), strain that V4(is high temperature resistant) and clone strain (Clone-30, N-29, Clone-83 and N-88) and most popular velogen strain, strong malicious detection line T1 can detect the popular velogen strain of most Avian pneumo-encephalitis virus, can realize detecting the discriminating of Avian pneumo-encephalitis virus Major Epidemic strain and vaccine strain.
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