CN104407137B - A kind of CSFV velogen strain and low virulent strain differentiate Test paper - Google Patents
A kind of CSFV velogen strain and low virulent strain differentiate Test paper Download PDFInfo
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- CN104407137B CN104407137B CN201410763168.8A CN201410763168A CN104407137B CN 104407137 B CN104407137 B CN 104407137B CN 201410763168 A CN201410763168 A CN 201410763168A CN 104407137 B CN104407137 B CN 104407137B
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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
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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
- G01N33/558—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
- G01N2333/08—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
Abstract
The present invention relates to a kind of infection of disease of domestic animals and immune identification detector tool, the strong malicious and weak poison of more particularly to a kind of CSFV differentiates Test paper, by supporting plate, sample pad, gold standard pad, detect that film and adsorptive pads are constituted, strong malicious detection line T1 " │ ", weak malicious detection line T2 " │ " and nature controlling line C " │ " trace are contained on detection film.When differentiating detection, showing three red stripes in detection film, " │ │ │ " are the strong poison infection of swine fever, and " │ │ " are swine fever attenuated vaccine immunity to two red stripes, only show a red stripes " │ " negative for CSFV.This discriminating Test paper high specificity, sensitiveness is high, and response spectrum is wide, can detect existing attenuated vaccine strain and and most popular velogen strains, and it is easy to operate, quick, it can be widely used for swine fever virus infection and detected with immune discriminating, it is easy to the popularization and application in production practices.
Description
Technical field
The present invention relates to a kind of infection of disease of domestic animals and immune identification detector tool, more particularly to a kind of CSFV
Strong malicious and weak poison differentiates Test paper.
Background technology
Swine fever（classical swine fever, CSF）It is by CSFV（classical swine fever
virus, CSFV）A kind of caused highly contagious disease being characterized with hyperpyrexia, bleeding and high mortality, world animal
Health organization（OIE）OIE epidemic disease registers are included in, are notifiable zoonosis, China is classified as a class animal epidemic.I
State is using immune hog cholera lapinised virus poison（Hog cholera lapinized vaccine, HCLV）Vaccine prevention and control CSF
Extensive prevalence, but new change occurs again for CSF prevalence and characteristics of incidence in recent years, shows as atypia, chronic and hidden more
Sexuality dye, occurs in that so-called " non-typical swine fever ", " mild swine fever " and " the malicious sow syndrome of band ", is still harm pig industry
One of Infectious Diseases.CSFV belongs to flaviviridae（Flaviviridae）Pestivirus（Pestivirus）, with the ox belonged to together
The type of viral diarrhea virus 1（bovine diarrhea virus 1, BVDV-1）, the type of bovine viral diarrhea virus 2（BVDV-
2）, border disease virus（border disease virus, BDV）With giraffe pestivirus（pestivirus of
giraffe）With very high homology, and there is serological cross reaction.CSFV genomes are single-stranded positive RNA, and size is about
1.23 ´104Nt, by 5 ' end noncoding regions（5’-untranslated region, 5’-UTR）, an ORFs
（open reading frame, ORF）With 3 '-end noncoding region（3’-UTR）Constitute, and 5 ' ends are without " cap " knot that methylates
Structure, 3 ' ends are without polyadenylic acid (poly A) structure.The big ORF is encoded before the polyprotein of about 3899 amino acid residues
Body, is processed by virus and host cell proteins enzyme and forms 12 kinds of ripe virus proteins, i.e. C, Erns, 4 kinds of structure eggs such as E1 and E2
White and Npro, 8 kinds of non-structural proteins such as p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B.According to 5 '-UTR, E2 and NS5B bases
Because of sequence difference, CSFV points are 3 gene groups, 10 gene subgroups, i.e., 1.1,1.2,1.3,2.1,2.2,2.3,3.1,3.2,
3.3 and 3.4.Epidemiology survey shows, the popular strains of CSFV of the China after 2000 are divided into 3 gene subgroups, i.e., 1.1,
2.1 and 2.2, wherein 2.1 gene subgroups are occupied an leading position in the groove in China's swine fever.
At present, the strategy based on vaccine immunity is still taken in the prevention and control of China's swine fever, and HCLV vaccines are China's last century
The universally acknowledged good vaccine that the fifties are developed, security is good, immunogenicity is good, stabilization characteristics of genetics, can induce simultaneously
Humoral immunity and cellular immunity, can be to different genes subgroup（1.1-3.4 subgroup）Immunoprotection is produced, but is due to shortage serum
Mark and supporting differential diagnostic method are learned, its large-scale application in China so that by antibody test it is difficult to distinguish immune
Weak poison and wild virus infection, are unfavorable for CSF purification.With separate sources, different regions CSFV street strains and the full base of vaccine strain
Because of the completion and the development of molecular Biological Detection technology of sequence, domestic and foreign scholars differentiate that context of detection has been carried out largely in CSFV
Research, by analyzing strong, the low virulent strain characteristic site of CSFV genomes, using 5 '-UTR, NS5B or 3 '-UTR conserved sequences as
Target spot is detected, strong, low virulent strain specific primer or probe is separately designed, establishes and distinguishes the wild malicious and rabbitization attenuated vaccines of CSFV
Multiple or Nest RT-PCR, dual or composite fluorescence quantitative RT-PCR equimolecular differentiate detection method, with higher sensitiveness
And specificity, but these detection methods need to rely on PCR instrument or quantitative real time PCR Instrument, exist operation it is relatively cumbersome, it is time-consuming compared with
The deficiencies such as length, testing cost height, limit its application in epidemiology survey and veterinary clinic.Immune chromatography test paper be
A kind of novel in vitro detection grown up on the basis of monoclonal antibody technique, colloidal gold immunochromatographimethod technology and new material technology
Technology, is preferably detection immediately（point-of-care test, POCT）And onthe technology of site test, its have it is sensitiveer,
Specifically, easy, quick the advantages of, " foolproof " operation can be especially realized, that is, does not need any auxiliary instrumentation equipment to enter
Row Site Detection, and the result of determination in 1-5 minutes, are widely used in the quantitative and semi-quantitative quick detection of various analytes,
Including antigen, haptens, antibody and nucleic acid, it has also become one of current most quick sensitive immunology detection technology.Move in Henan Province
Thing immunology key lab system since nineteen ninety-five carries out immune test paper Fast Detection Technique research, takes the lead in grinding in the world
Work(animal epidemic cause of disease and antibody test colloid test paper product-- Test Paper for Rapid Diagnosis of IBD and pig is made
Trichina antibody test paper, establishes animal epidemic quick detection test paper research and development technology platform, successfully develops so far
The serial quick detection test paper products such as animal epidemic antigen, antibody and medicament residue detection, have promoted answering for the technology significantly
With and development.The present invention is for CSFV wild virus infections and the key technical problem of vaccine immunity antidiastole, and Screening and Identification can area
Point CSFV is strong, the high-affinity of low virulent strain pairing monoclonal antibody, sets up the protein chip technology based on immune chromatography test paper and puts down
Platform, develops CSFV velogen strain and low virulent strain and differentiates Test paper, set up be applied to cultivation basic unit and clinical detection it is quick,
Easy hog cholera field virus infection and vaccine immunity joint inspection technology, realize the real-time monitoring infected wild-type classical swine fever virus, are China
Swine fever prevention and control provide technical support with purification, significant to swine fever epidemic monitoring and prevention and control.
The content of the invention
The purpose of the present invention is：Develop the strong malicious and weak poison mirror of CSFV for being applied to swine fever infection with Immune dctection is detected
Other Test paper, this test paper is special, sensitive, quick, easy, the popularization and application easily in production practices.
The technical scheme is that：A kind of strong malicious and weak poison of CSFV differentiates Test paper, by supporting plate, sample
Pad, gold standard pad, detection film and adsorptive pads are constituted, and gold standard pad is absorption colloid gold label swine fever virus resistant monoclonal antibody mAb1's
Glass fibre, detection film is the strong malicious detection line T1 of trace, weak malicious detection line T2 and nature controlling line C nitrocellulose filter, by sample
Hold to handle end and be arranged as strong poison detection line T1/weak malicious detection line T2/ nature controlling lines C：" │ │ │ ", strong poison detection line T1 is anti-pig
Pestivirus monoclonal antibody mAb2 traces " │ ", weak malicious detection line T2 is swine fever virus resistant polyclonal antibody pAb1 or anti-hog choleras
Malicious monoclonal antibody mAb3 traces " │ ", nature controlling line C is anti-mouse IgG antibody pAb2 or staphylococcus aureus SPA traces " │ ".
When differentiating detection, the strong poison infection of swine fever shows strong malicious detection line T1, weak malicious tri- red of detection line T2 and nature controlling line C in detection film
Band " │ │ │ ", swine fever attenuated vaccine immunity detection film show two red stripes of weak malicious detection line T2 and nature controlling line C " │ │ ",
Then only show mono- red stripes " │ " of nature controlling line C without CSFV.
Using the strong malicious Strain Shimen of CSFV standard as immunizing antigen, anti-hog cholera is identified by hybridoma cell technology production
Malicious monoclonal antibody, utilizes immunopcroxidase monolayer assay（IPMA）It is strong malicious and weak malicious that CSFV is distinguished in screening
Monoclonal antibody, to be superimposed EUSA（ELISA）The monoclonal antibody of the different epitopes of screening identification.For
The monoclonal antibody mAb1 of the colloid gold label and weak malicious detection line T2 equal specific recognition CSFVs of monoclonal antibody mAb3 are strong
Strain and attenuated vaccine strain, recognize the different epitopes of CSFV respectively, the monoclonal antibody for strong malicious detection line T2
MAb2 specific recognition CSFV velogen strains, but do not reacted with low virulent strain.Meanwhile, screen wide with CSFV response spectrum with IPMA
Monoclonal antibody, colloid gold label monoclonal antibody mAb1 and weak malicious detection line T2 monoclonal antibodies mAb3 can recognize that hog cholera
Malicious attenuated vaccine and most popular velogen strains, the strong recognizable most CSFVs of poison detection line T1 monoclonal antibody mAb2 are popular
Velogen strain.With CSFV attenuated vaccine, HCLV plants are that immunizing antigen prepares swine fever virus resistant polyclonal antibody pAb1, be can be used for
Weak malicious detection line T2 traces.Goat anti-mouse igg polyclonal antibody pAb2, pAb2 are prepared by immunizing antigen of mouse IgG and golden yellow
Color staphylococcus SPA is used equally for nature controlling line C traces.
The beneficial good effect of the present invention, it is malicious by force that the strong malicious and weak poison of CSFV differentiates that Test paper realizes CSFV
With weak malicious synchronous joint inspection, it can effectively differentiate the strong poison infection of swinery swine fever and vaccine immunity, and it is simple to operate, and everybody can grasp
Make, the need for preferably meeting different levels personnel, such as Disease monitor, customs quarantine control, health and epidemic prevention, intensive culture to individual
Cultivation etc., it is easy to a wide range of popularization and application, with wide market prospects and larger economical, societal benefits.Test strip
With following advantage：
（1）Strong malicious and weak malicious joint inspection.The strong malicious and weak poison of CSFV differentiates Test paper on detection film containing identification pig
The strong malicious and weak malicious weak malicious detection line of pestivirus and the strong poison of identification but the weak malicious strong malicious detection line of nonrecognition, can synchronously carry out swine fever
The velogen strain of virus and low virulent strain joint inspection, realize that the strong malicious and weak poison of swine fever differentiates detection.
（2）High specificity, sensitiveness is high.The strong malicious and weak poison of CSFV differentiates that Test paper is strong can distinguish CSFV
It is prepared from based on malicious and weak malicious high-affinity pairing monoclonal antibody, the high specificity and sensitiveness of monoclonal antibody
Formed in height, gold labeling antibody between gold grain and antibody molecule without covalent bond, the two passes through the Van der Waals force phase between the charges of different polarity
With reference to, collaurum influences very little to the reactivity of labelled antibody, and with higher mark rate.Therefore, Test paper tool is differentiated
There is higher specificity and sensitiveness.
（2）It is easy to operate quick.Using without any other reagent, sample is treated as long as being inserted into when differentiating Test paper
Product 10 seconds or so, testing result was can determine that in 2 minutes.
（3）Show that testing result is vivid, directly perceived accurate.Differentiate Test paper to show rufous " │ ", " │ │ " and " │ │ │ "
Trace is as negative, the weak malicious and strong malicious positive mark of detection, i.e., one brownish red band " │ " of display is swine fever on detection film
It is viral negative, represent to be detected sample without attenuated vaccine or strong poison infection, " │ │ " are the weak poison of CSFV to two brownish red bands
The positive, expression test sample is attenuated vaccine immunity, and " │ │ │ " are that the strong poison of CSFV is positive to three brownish red bands, represent quilt
Sample product are strong poison infection, and result judgement is vivid, directly perceived, accurate, simple and clear, is less prone to false negative and false positive erroneous judgement.
（4）Cost is low, small investment.Using Test paper is differentiated, it is not required to separately match somebody with somebody instrument and equipment and other reagents, makes live inspection
Survey is settled at one go, with low cost, small investment, instant effect.
The present invention is further described with reference to the accompanying drawings and examples for brief description of the drawings.
Fig. 1 is that the strong malicious and weak poison of CSFV differentiates Test paper side structure schematic view.
Fig. 2 is that the strong malicious and weak poison of CSFV differentiates Test paper overlooking the structure diagram.
In figure, 1. supporting plates, 2. sample pads, 3. gold standard pads, 4. detection films, 5. adsorptive pads, the last 6. poison detection
Line T1 traces, 7. weak malicious detection line T2 traces, 8. nature controlling line C traces, 9-1. sample end diaphragms, the protection of 9-2. handle ends
Film, 10. mark lines.
It is strong that the strong malicious and weak poison of embodiment CSFV differentiates that Test paper can be widely applied to swinery CSFV
Poison infection and the discriminating of vaccine immunity are detected.Prepare the strong malicious and weak poison of CSFV and differentiate Test paper, need to prepare swine fever first
Virus immunity antigen, and then swine fever virus resistant polyclonal antibody and monoclonal antibody are prepared, and it is strong malicious and weak to screen differentiation swine fever
The monoclonal antibody of poison, the strong malicious and weak malicious monoclonal antibody mAb1 of identification swine fever is used to prepare colloid gold label thing, recognizes pig
Pest are strong malicious and the weak malicious monoclonal antibody mAb2 of nonrecognition is used to print strong malicious detection line trace " │ ", and identification swine fever is malicious and weak by force
The polyclonal antibody pAb1 or monoclonal antibody mAb3 of poison are used to print weak malicious detection line trace " │ ", and it is small secondly need to prepare goat-anti
Mouse IgG antibody or staphylococcus aureus SPA, for printing nature controlling line trace " | ".
（1）The preparation of CSFV immunizing antigen.
Porcine kidney cell is inoculated with CSFV standard velogen strain Strain Shimen（PK-15）, collect Virus culture within 48-60 hours
Liquid, 4 DEG C of 3000 min of r/min low-speed centrifugals 30 goes the removal of impurity, and ultrafiltration is carried out to virus-culturing fluid using 50 kDa milipore filters
Concentration, gel filtration chromatography is carried out with the Fast Flow agarose Gel columns of Sepherose 4 to CSFV, is determined
The half cell culture infective amount of CSFV（TCID50）Up to 10-7More than, quantitative fluorescent PCR determines purified virus copy number
Up to 1010More than.
（2）The preparation of swine fever virus resistant monoclonal antibody.
（2.1）The foundation of hybridoma cell strain.
It is fully emulsified by CSFV immunizing antigen and Freund immunologic adjuvant mixed in equal amounts, only exempted from 50 mg-100 mg/
Epidemic disease BALB/c systems mouse 3 times, every minor tick 15-30 days；3-4 days after 3rd booster immunization, immune eyeball of mouse bloodletting is drawn
Neck is lethal, sterile to take its splenocyte in 75% alcohol-pickled 5-10min；Shred and through 100 mesh nylon net filters, 1000 r/min
10 min are centrifuged, splenocyte is collected；By 1 × 108Splenocyte and 2-5 × 107SP2/0 myeloma cell mixing, 1000 r/
Min centrifuges 10 min, supernatant is abandoned, by 0.7-1 ml 40%-50% PEG 4000 in 37 DEG C of water-bath（pH8.5-9.0）It is slow
It is slow to add cell, incubate after 1 min, the ml of 1640 culture medium of serum-free 15 is slowly added to, to terminate PEG effect, 37 DEG C of water-baths
5-10 min, 1000 r/min centrifuge 10 min, abandon supernatant, cell is resuspended in HAT Selective agar mediums, and add the training of 96 holes
Support plate（100 ml-200 ml/ holes）, put 37 DEG C of 5% CO2Cultivated in incubator.After culture 7-10 days, hybridoma is taken to train
Supernatant is supported with EUSA（ELISA）And immunopcroxidase monolayer assay（IPMA）The positive hybridization of screening
Oncocyte.With the strong malicious Strain Shimen infection porcine kidney cell of CSFV standard（PK-15）, after being fixed through methanol, 5% 37 DEG C of defatted milk
Close 1 h；Plus cells and supernatant 50mL/ holes to be checked, if HAT culture mediums and mouse immune serum are negative and positive control；
Plus 1:500 horseradish peroxidases（HRP）Mark goat anti-mouse igg antibody（50 mL/ holes）, 37 DEG C of 30 min of effect；Often walk
Fully washed with the PBS containing 0.05% Tween-20 after reaction；With substrate A EC color development at room temperature 10-20 min, in being rinsed with water
After only developing the color, colour developing result is observed under the microscope.The vigorous clone hole of strong positive, cell growth is chosen have for continuous 3 times
Limit dilution cloning, expands freeze-stored cell after culture, sets up 8 plants of swine fever virus resistant monoclonal antibody hybridoma cell strain.
（2.2）The preparation of monoclonal antibody：Monoclonal antibody is prepared to induce ascites in vivo.Learn from else's experience pristane or atoleine sensitization
Through produce Balb/c mouse, be injected intraperitoneally exponential phase hybridoma 107Individual/only, ascites is extracted after the d of 7 d~10,
Supernatant is taken after centrifugation, dispenses, freezes.
（2.3）The identification of monoclonal antibody
（2.3.1）The antibody titer of monoclonal antibody
With EUSA（ELISA）And immunopcroxidase monolayer assay（IPMA）Determine hybridoma
The ELISA potency of the monoclonal antibody potency of cells and supernatant and ascites, Mab supernatant and ascites is respectively 1:640 and 1:105More than,
IPMA potency is respectively 1:16 and 1:More than 1600.
（2.3.2）The specificity of monoclonal antibody
With CSFV standard strains Strain Shimen, HCLV plants of infection porcine kidney cells of popular strain and attenuated vaccine strain（PK-15）, with
Immunopcroxidase monolayer assay（IPMA）The reactivity of monoclonal antibody and the popular strains of CSFV is determined, screening obtains simultaneously special
6 plants of the monoclonal antibody of different identification CSFV standard strains, popular strain and attenuated vaccine strain, and specific recognition CSFV standard strains
With 2 plants of the monoclonal antibody of popular strain but nonrecognition attenuated vaccine strain.With immunopcroxidase monolayer assay
（IPMA）Detect the response spectrum of above-mentioned 8 plants of monoclonal antibodies and vaccine strain and prevalence strain, it was demonstrated that for colloid gold label and weak
Malicious detection line T2 monoclonal antibody can recognize that CSFV attenuated vaccine and most popular velogen strains, for strong malicious detection line
The popular velogen strains of the recognizable most CSFVs of monoclonal antibody, be respectively provided with wider CSFV response spectrums.
（2.3.3）Monoclonal antibody recognizes Characterization of antigenic epitopes
Analyzed with the epitope for blocking ELISA or superposition ELISA to recognize CSFV monoclonal antibodies, screening is obtained
The monoclonal antibody mAb1 and mAb3 of the different epitopes of CSFV are recognized respectively, and it is respectively used to colloid gold label and weak poison
Detection line T2 traces；Specific recognition CSFV standard strain and popular strain are obtained, without the list reacted with attenuated vaccine strain
Clonal antibody mAb2, for strong malicious detection line T1 trace.
（2.3.3.1）Block ELISA：Labeled monoclonal antibody is distinguished with HRP, ELISA blocking tests identification monoclonal resists
Body recognizes the specificity of epitope, adds unlabelled monoclonal antibody line by line in the coated ELISA Plate of E0-E2 recombinant proteins first,
If CSFV positive serums and PBS control, 37 DEG C of 1 h of effect；Then the monoclonal antibody of HRP marks, 37 DEG C of 1 h of effect are added by column；With
Substrate TMB is developed the color, and reads the OD of each detection hole450Value.The unmarked monoclonal antibody identification for significantly inhibiting the combination of enzyme mark monoclonal antibody is identical
Or close epitope, and the unmarked monoclonal antibody having no significant effect is combined to enzyme mark monoclonal antibody and then recognizes different epitopes.
（2.3.3.2）It is superimposed ELISA：The work of each monoclonal antibody is determined with indirect ELISA first with the ELISA Plate of envelope antigen
Make concentration, draw antigen saturation curve.In superposition ELISA experiments, monoclonal antibody is suitably diluted according to antigen saturation curve, and match
Add ELISA Plate each hole, 37 DEG C are incubated the h of 1 h~2；It is separately added into ELIAS secondary antibody and TMB is developed the color, reads each hole OD450Value,
Superposition coefficient is calculated by following equation（AI）：AI=[2´OD1+2/（OD1+OD2）- 1] ' 100%, wherein OD1+2For pairing monoclonal antibody hole
OD450Value, OD1And OD2For the OD in two independent monoclonal antibody holes450Value.The AI values of two monoclonal antibodies be judged to less than 40% identification it is identical or
Close epitope；AI values are judged to recognize different epitopes more than 40%.
（2.4）The purifying of monoclonal antibody：With caprylic acid-ammonium from mouse ascites purified monoclonal antibody IgG.Take 1 mL small
Mouse ascites fluid, adds the mol/L sodium acetate buffers of 2 mL 0.06（pH 5.0）, pH 4.5 is adjusted to 0.1 mol/L HCl；In room
Under temperature stirring, 33 mL octanoic acids are added dropwise, 4 DEG C of standing 2 h, 15000 r/min 30 min of centrifugation abandon precipitation；In centrifugation supernatant
Middle addition 1/10 volume 0.01 mol/L PBS（pH7.4）, pH 7.4 is adjusted to 0.1 mol/L NaOH；In under condition of ice bath
Saturated ammonium sulfate is added to final concentration 45%, 4 DEG C of standing 2 h, 10000 r/min 30 min of centrifugation abandon supernatant；With appropriate PBS weights
Outstanding precipitation, stays overnight to PBS, changes liquid 3 times.With spectrophotometer method or dying method with coomassie brilliant blue（Bradford methods）Determine
Monoclonal antibody purification IgG protein content is in more than 1mg/ml, with EUSA（ELISA）With immune peroxidating
Thing enzyme cell monolayer is tested（IPMA）The antibody titer of mouse ascites and IgG purification is determined 1:105With 1:More than 1000, point
Dress, freezes.
（3）The preparation of swine fever virus resistant polyclonal antibody.
With the swine fever attenuated vaccine of the mg/kg body weight of 50 mg ~ 100 through intramuscular injection immune health piglet 3 ~ 4 times, last is exempted from
After epidemic disease 10 days, venous blood collection determines its serum antibody titer 1 with IPMA:When more than 1000, Culling heart blood or arteria carotis are put
Blood, collects hyper-immune serum, with anhydrous sodium sulfate（Na2SO4）Immune swine serum IgG is extracted, 1 part of immune serum plus 2 part 0.01 is taken
mol/L PBS（pH7.2）Mixing, adds anhydrous Na2SO4To final concentration 18%, 37 DEG C of water-bath 30min, 4000 r/min centrifugations
20min, abandons supernatant, with the Na of appropriate PBS (pH7.2) resuspension precipitations, plus final concentration 16%2SO4, 37 DEG C of precipitations 30min, 4000r/
Min centrifuges 20min, abandons supernatant, then precipitation is resuspended with appropriate PBS (pH7.2), with the Na of final concentration 14%2SO4Precipitation, 4000r/
Min centrifuges 20min, abandons supernatant, is resuspended and precipitated with appropriate PBS (pH7.2), to PBS (pH7.2) dialyzed overnight, changes liquid 2 ~ 3 times,
Determine antibody titer and protein concentration（10-20 mg/ml）, prepared swine fever virus resistant polyclonal antibody pAb1 can be used for detect
The printing of the weak malicious detection line T2 traces of test paper.
（4）The preparation of rabbit anti-mouse IgG polyclonal antibodies
The healthy new zealand rabbits of 2.0 kg or so are immunized with purified mouse IgG, first immunisation is anti-with Freund's complete adjuvant emulsification
Original, only, each w of booster immunization interval 3 emulsifies antigen intramuscular injection to the subcutaneous mg/ of multi-point injection 50 with incomplete Freund's adjuvant,
After the w of last time booster immunization 2, with agar gel diffusion test（AGP）Determine immune serum antibody titer and be higher than 1:When 40, collection
Height exempts from rabbit whole blood, separates serum, and rabbit anti-mouse IgG is purified with caprylic acid-ammonium, and method is same（2.4）Monoclonal antibody is purified, and octanoic acid is used
Measure as 45 mL/mL serum, measure antibody titer and protein concentration（10-20 mg/ml）, prepared rabbit anti-mouse IgG is polyclonal
Antibody pAb2 can be used for the printing of Test paper nature controlling line C traces.
（5）The colloid gold label of monoclonal antibody
（5.1）The preparation of collaurum：Take 100 mL ultra-pure waters to be placed in the clean conical flasks of 500 mL, add the % of 1 mL 1
（w/v）Gold chloride boils；1 mL 1% of Fresh is rapidly added under stirring（w/v）Sodium citrate solution, boils about
3 min are changed into aubergine to solution colour from yellow, continue to boil 2 min；Treat that solution is cool to room temperature, mend ultra-pure water to 100
ML, with 0.2 mol/L K2CO3PH to 9.0 is adjusted, 4 DEG C of lucifuges can preserve the several months.
（5.2）Most suitable labelled protein concentration mensuration：Anti- CSFV monoclonal antibody IgGs to be marked are taken to 20 mmol/L dobell's solutions
（pH 8.0）4 DEG C of dialyzed overnights.With 25 mL ultra-pure waters 1 in microwell plate:2、1:4、1:8 ... doubling dilutions CSFV to be marked
Monoclonal antibody；Each hole adds 125 mL colloidal gold solutions, and RT stands 5 min；Add the mol/L NaCl solutions of 125 mL 1；Each Kong Yan
Color is changed into blueness with the reduction of protein concentration from red.Protein concentration using the unchanged blue monoclonal antibody highest dilution of color is glue
The most suitable label concentration of body gold, during colloid gold label, protein concentration increase by 20%.
（5.3）The colloid gold label of monoclonal antibody：The monoclonal antibody IgG to be marked of the most suitable protein concentrations of 2 mL is taken, 10 are added
ML colloidal gold solutions（pH 9.0）, rapid to mix, room temperature acts on the min of 10 min~15；1/10 volume is added containing 10%（w/v）
Bovine serum albumin(BSA)（BSA）20 mmol/L dobell's solutions, rapid to mix, RT acts on the min of 10 min~15；4℃
15000 g centrifuge 30min, carefully remove supernatant；With containing 1%（w/v）Colloid is resuspended in BSA 20 mmol/L dobell's solutions
Gold, is ibid centrifuged, and abandons supernatant；Repeated washing 1 time, contains 1% with 1 mL（w/v）Glue is resuspended in BSA 20mmol/L dobell's solutions
Body gold, 4 DEG C save backup.
（6）Detect the preparation of film
Nitrocellulose detection film is cut into 2.5 × 30 cm2Strip, be placed on the specking instrument platforms of XYZ 3000, and with
Press strip is fixed；Use PBS（pH 7.2）Monoclonal antibody by specific recognition CSFV velogen strain but not with low virulent strain reaction, specific recognition
The monoclonal antibody or many anti-igg and rabbit anti-mouse igg of CSFV velogen strain and attenuated vaccine strain are diluted to 1 mg/mL, and are put in respectively
Storage pool；Using Biojet Quanti 3000 with 1 mL/cm respectively by anti-CSFV monoclonal antibodies or many anti-igg solution and rabbit-anti mouse
IgG solution specking forms strong malicious detection line T1, weak malicious detection line T2 and nature controlling line C traces, detection line and matter in detection film center
Line is controlled at a distance of 0.5 cm；Put 42 DEG C of min of drying box 30 or natural drying at room temperature；Detection film is put into polybag, plus 4 DEG C of drier
It is closed to save backup.
（7）The preparation of pad
Mineral wool is cut into 1.5 × 30 cm2Strip, be placed on the specking instrument platforms of XYZ 3000, and fixed with press strip；
Take 1 mL colloid gold labels thing to add 2 mL and contain 2%（w/v） BSA、3% （w/v）Sucrose, 0.6 mol/L NaCl, 0.2%
Tween 20 （v/v）With 0.1%（w/v）20 mmol/L dobell's solutions of Sodium azide（pH 8.0）；Utilize Airjet
Quanti 3000 is with 15 mL/cm by anti-collaurum label solution specking in mineral wool；Put the 50 DEG C of min of drying box 30 dryings；
Glue gold pad is put into polybag, plus 4 DEG C of drier is closed saves backup.
（8）The preparation of sample pad
Mineral wool is cut into 1.5 × 30 cm2Strip, 0.1 mol/L NaCl, 0.2% Tween 20 will be contained（v/
v）With 0.1%（w/v）The PBS of Sodium azide（pH 7.2）Solution soaks glass sliver；Put the 50 DEG C of min of drying box 30 dryings；Will
Sample pad puts polybag, plus drier RT is closed saves backup.
（9）The preparation of adsorptive pads
Mineral wool is cut into 2.5 × 30 cm2Strip, adsorptive pads are put into polybag, plus drier RT is closed preserves standby
With.
（10）The preparation of supporting plate
By double faced adhesive tape in PVC supporting plates, 7.5 × 30 cm are cut into2Long slab, prepare supporting plate.
（11）The assembling of test paper
Test paper plate is assembled into using LM5000 test paper assembling instrument or by hand by above-mentioned material.Detection film is first pasted on support
Glue gold pad and sample pad, are then pasted on the sample end of detection film by plate center successively, the mm of overlapping 1 mm of each interlayer~2, then will
Adsorptive pads are pasted on the other end of detection film, and with the mm of overlapping 1 mm of detection film~2, sample is wrapped up with white plastic film in sample end
Detection direction and detection solution upper limit mark are printed in product pad and glue gold pad, plastic film, is wrapped up in handle end with blue plastic film
Adsorptive pads.
（12）Cutting and packaging
The test paper plate assembled is cut into 0.3 cm test strips using CM4000 cutting devices, test strips are distributed into plastics
Bag, plus the closed preservation of 4 DEG C of drier.
（13）CSFV velogen strain and low virulent strain differentiate the implementation structure of Test paper
Referring to Fig. 1, Fig. 2, in figure, 1 is the supporting plate strip of foil that do not absorb water, and plastic slice bar can be used in implementation or be adopted
With the hard paper sheets not absorbed water, reaction reagent carrier absorption layer is combined by 2,3,4,5, in one's hands since sample end 2
Pommel 5 is pasted onto above supporting layer 1 successively；Wherein 2 be sample end sample pad, and glass fibre cotton abbreviation glass can be used in implementation
Glass cotton, 3 recognize that the strong poison of CSFV and low virulent strain monoclonal antibody mAb1 gold mark glass fibre cotton for absorption colloid gold label,
Processed glass cellucotton, referred to as gold standard pad can be used, 4 be detection film, nitrocellulose filter can be used in implementation, 5 be handle
Adsorptive pads are held, using blotting paper, such as filter paper or other blotting papers, the cm of test strips overall length 8, the cm of width 0. 4,6 be with special
Different identification CSFV velogen strain but not with low virulent strain react monoclonal antibody mAb2 printed on nitrocellulose filter it is strong
Malicious detection line trace " | ", 7 be the monoclonal antibody mAb3 or anti-swine fevers of specific recognition CSFV velogen strain and attenuated vaccine strain
The weak malicious detection line trace " | " that viral polyclonal antibody pAb1 is printed on nitrocellulose filter, 8 be many grams of rabbit anti-mouse IgG
The nature controlling line trace " | " that grand antibody pAb2 or staphylococcus aureus SPA are printed on nitrocellulose filter, on detection film
Strong poison detection line trace, weak malicious detection line trace and nature controlling line trace assembled arrangement are " || | ".9 be diaphragm, is covered in glass
The diaphragm of cotton and gold mark mineral wool is 9-1, and the diaphragm being covered on water accepting layer filter paper is 9-2.In mineral wool and gold mark glass
It is inclined to be printed on a mark on the diaphragm at the cm of one side of mineral wool 0.5 on the white diaphragm of glass cotton intersection correspondence position
Line 10, arrow and Max printed words are printed on the right of mark line 10, and handle end diaphragm can use yellow or other colors, and 2,3,4,5 is each
Intersection fiber crosses one another infiltration between layer.
（14）CSFV velogen strain and low virulent strain differentiate Test paper examinations reaction principle
It is to be checked molten after CSFV velogen strain and low virulent strain differentiate Test paper sample end insertion detected sample solution
Liquid drives CSFV antigens to be checked and gold labeling antibody mAb1 together to nitrocellulose membrane diffusion by siphon, and finally penetrates into filter
In ply of paper, gold labeling antibody mAb1 is combined with CSFV velogen strains to be checked or low virulent strain antigen in diffusion process, forms gold mark anti-
Body-antigenic compound, the gold mark compound of CSFV velogen strains can be simultaneously with detecting strong poison detection trace mAb2 and weak poison on film
Detect that trace mAb3 is combined, generation rufous " || " mark, part can not be with detection trace with the gold labeling antibody of antigen binding
With reference to and continue to spread, with the pAb2 or SPA in Quality Control trace on detection film, generate reddish brown color marker " | ", two kinds of mark groups
Close and be superimposed, three rufous positive marks of formation " || | ", represent malicious by force containing swine fever in sample；And the gold of CSFV vaccine strains
Mark compound can not be combined with the strong poison detection trace mAb2 on detection film, can only be combined, generated with weak poison detection trace mAb3
Rufous " | " mark, part can not be combined with detection trace with the gold labeling antibody of antigen binding and continue diffusion, in detection film
Upper and pAb2 or PSA in Quality Control trace, generate reddish brown color marker " | ", and two kinds of mark combination superpositions form two rufous sun
Property mark " || ", represent sample in comprise only hog cholera vaccine poison；It is anti-without gold mark when being free of CSFV antigen in sample
Body-antigenic compound is formed, it is impossible to is combined with strong poison detection trace and weak poison detection trace, then generates negative marker " | ".If
There is no reddish brown color marker to show on detection film, then show that test strips have failed.
（15）CSFV velogen strain and low virulent strain differentiate Test paper detection example operating method
（15.1）Detect the preparation of sample solution：Gather pig swab to be checked（Pharynx is wiped wipes with anus）, disease（Extremely）Porcine tissue, excrement
The different detection samples with vaccine etc., plus appropriate PBS or water be simply suspended or grind
（15.2）Test paper is detected：CSFV velogen strain and low virulent strain are differentiated that Test paper sample end immerses solution to be checked
The s of 10 s~20；Take out the min observation results of the min of test paper horizontal positioned 5~10.
（15.3）Testing result judges：Test paper shows three rufous bands（Strong poison detection line, weak malicious detection line and Quality Control
Line）" || | " positive for the strong poison of CSFV, represent in measuring samples containing the strong malicious antigens of CSFV；Two rufous bands（Weak poison detection
Line and nature controlling line）" || " is positive for the weak poison of CSFV, represents to contain CSFV vaccine virus antigens in measuring samples；Only show one it is reddish brown
Vitta band（Nature controlling line）" | " is negative for CSFV, represents that measuring samples do not detect CSFV antigens；Test paper does not show any band and shown
Misoperation or test paper failure are detected, need to be separately to take Test paper to detect again.
Embodiment one, the strong poison infection quick diagnosis of swine fever, collection disease（Extremely）The pathological tissues of pig, including lung, liver,spleen,kidney,
Tracheae, small intestine, large intestine and lymph node etc., with 1:5 or 1:10 add appropriate PBS or water simply to be ground, and prepare solution to be checked, with
CSFV velogen strain and low virulent strain differentiate that Test paper is pressed（15）Operating method is detected and result judgement that antidiastole is sick
（Extremely）The strong poison infection of pig or vaccine immunity.Test paper shows three rufous bands（Strong poison detection line, weak malicious detection line and Quality Control
Line）" || | " positive for the strong poison of CSFV, it is the strong poison infection of CSFV to represent pig to be checked；Two rufous bands（Weak malicious detection line and matter
Control line）" || " is positive for the weak poison of CSFV, and it is CSFV vaccine immunities to represent pig to be checked；Only show a rufous band（Nature controlling line）
" | " is negative for CSFV, represents pig to be checked without strong poison infection or vaccine immunity；Test paper does not show any band and shows detection operation not
When or test paper failure, need to be separately to take Test paper to detect again.
Embodiment two, the CSFV inspection and quarantine of live pig gathers live pig swab（Pharynx is wiped wipes with anus）Or excrement, with 1:5
Or 1:10 add appropriate PBS or water to be simply suspended, and prepare solution to be checked, are differentiated with CSFV velogen strain and low virulent strain and detected
Test paper is pressed（15）Operating method is detected and result judgement, differentiates strong poison pollution or the vaccine immunity situation of detection live pig.Examination
Paper shows three rufous bands（Strong poison detection line, weak malicious detection line and nature controlling line）" || | " positive for the strong poison of CSFV, represent to treat
It is the strong poison pollutions of CSFV to examine live pig or environment；Two rufous bands（Weak malicious detection line and nature controlling line）" || " is the weak poison sun of CSFV
Property, represent that live pig to be checked or environment, without the strong poison pollutions of CSFV, are CSFV vaccine immunities；Only show a rufous band（Quality Control
Line）" | " is negative for CSFV, represents live pig to be checked without strong poison infection or vaccine immunity；Test paper does not show any band and shows detection
Misoperation or test paper failure, need to be separately to take Test paper to detect again.
Embodiment three, the quality testing of swine fever attenuated vaccine, with 1:5 or 1:10 add appropriate PBS or water the dissolving weak poison of swine fever
Live vaccine, including newborn rabbit seedling, cell vaccine and pouring spleen seedling etc., vaccine solution to be checked is prepared, with CSFV velogen strain and low virulent strain
Differentiate that Test paper is pressed（15）Operating method is detected and result judgement, detects viral level and the discriminating of swine fever attenuated vaccine
Detect the strong malicious pollution condition of vaccine.Test paper shows three rufous bands（Strong poison detection line, weak malicious detection line and nature controlling line）
" || | " positive for the strong poison of CSFV, it is the strong poison pollutions of CSFV to represent vaccine to be checked；Two rufous bands（Weak malicious detection line and matter
Control line）" || " is positive for the weak poison of CSFV, represents vaccine to be checked without the strong poison pollutions of CSFV, the colored intensity and CSFV of weak malicious detection line
Vaccine contg is directly proportional；Only show a rufous band（Nature controlling line）" | " is negative for CSFV, represents that vaccine to be checked is strong without CSFV
Poison pollution, while being also free of vaccine virus；Test paper does not show any band and shows detection misoperation or test paper failure, need to be separately to take
Test paper is detected again.
Claims (3)
1. a kind of strong malicious and weak poison of CSFV differentiates Test paper, by supporting plate, sample pad, gold standard pad, detection film and water suction
Pad is constituted, it is characterized in that：Gold standard pad adsorbs colloid gold label swine fever virus resistant monoclonal antibody mAb1, the strong poison detection of detection film
Line T1 is swine fever virus resistant monoclonal antibody mAb2 traces " │ ", and weak malicious detection line T2 is swine fever virus resistant polyclonal antibody pAb1
Or monoclonal antibody mAb3 traces " │ ", nature controlling line C is anti-mouse IgG antibody pAb2 or staphylococcus aureus SPA traces " │ "；
The equal specific recognition CSFV velogen strains of monoclonal antibody mAb1 and mAb3 and attenuated vaccine strain, recognize CSFV respectively
Different epitopes；The monoclonal antibody mAb2 specific recognitions CSFV velogen strain, and do not reacted with low virulent strain；It is
Using the strong malicious Strain Shimen of CSFV standard as immunizing antigen, anti-hog cholera is identified by the method preparation for setting up hybridoma
Malicious monoclonal antibody, the strong malicious and weak malicious Dan Ke of CSFV is distinguished using immunopcroxidase monolayer assay screening
Grand antibody, to be superimposed the monoclonal antibody that EUSA screening identifies different epitopes.
2. discriminating Test paper according to claim 1, it is characterized in that：Swine fever strong poison shows strong poison when infecting in detection film
Detection line T1, " │ │ │ " show weak malicious tri- red stripes of detection line T2 and nature controlling line C during swine fever attenuated vaccine immunity in detection film
" │ │ ", no CSFV then only shows mono- red stripes of nature controlling line C to existing weak malicious two red stripes of detection line T2 and nature controlling line C
“│”。
3. the discriminating Test paper described in as requested 1 or 2, it is characterized in that：Differentiate that the CSFV response spectrum of Test paper is wide,
Weak malicious detection line T2 can detect hog cholera lapinised virus（HCLV）And most popular velogen strains, the strong detectable majority of poison detection line T1
CSFV prevalence velogen strain, can be achieved the discriminating to CSFV Major Epidemic strain and vaccine strain and detects.
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