CN105445457A - Porcine circovirus II competition ELISA antibody detection kit - Google Patents
Porcine circovirus II competition ELISA antibody detection kit Download PDFInfo
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Abstract
The invention discloses a porcine circovirus II competition ELISA antibody detection kit. The kit contains a peroxidase labeled monoclonal antibody secreted by a hybridoma cell strain with the preservation No. being CGMCC No.10205. The invention also discloses a method for utilizing the monoclonal antibody to establish a competition ELISA for detecting porcine serum PCV2 antibody. The method comprises the following steps: coating ELISA plate with PCV2 positive serum, thereby capturing PCV2 antigen, and then reacting with to-be-detected porcine serum antibody and HRP-marked monoclonal antibody 3A5 and displaying a competition ELISA detection result by measuring the peroxidase labeled monoclonal antibody. The method and the IPMA method are utilized to perform parallel test on 237 porcine serum samples; the detection coincidence rate is 94.1% according to the two methods; the sensibility is 92.6% and the specificity is 98.4% according to the method; the kit has no cross reaction with other porcine circovirus reference positive serums; the kit provided by the invention has the characteristics of simple and easy operation, high specificity, high sensibility, and the like; an effective technical method is supplied for PCV2 epidemiological investigation and serum antibody detection.
Description
Technical field
The present invention relates to a kind of kit, particularly a kind of porcine circovirus 2 type competitive ELISA antibody assay kit, belongs to field of immunology.
Background technology
Porcine circovirus 2 type (Porcinecircovirustype2, PCV2) belongs to PCV-II section Circovirus member, and be one of minimum in animal virus up to now virus, diameter is 17nm, and icosahedral symmetry, without cyst membrane.Viral genome is made up of sub-thread closed hoop DNA, containing 1766 ~ 1769 nucleotide (OlveraA, CorteyM, Segal é sJ.Molecularevolutionofporcinecircovirustype2genomes:Phy logenyandclonality [J] .Virology, 2007,357 (2): 175 ~ 185; HuangL.P., LuY.H., WeiY.W., etal.Identificationofonecriticalaminoacidthatdeterminesa conformationalneutralizingepitopeinthecapsidproteinofpor cinecircovirustype2.BMCMicrobiol.2011a, 11:188.).PCV2 genome mainly comprises 2 large open reading frames (ORF1 and ORF2) (CheungAK.Transcriptionalanalysisofporcinecircovirustype2 .Virology, 2003,305:168-180.).ORF1 coding replicase associated protein, participate in copying of virus, ORF2 encodes Cap protein, the exclusive architecture albumen of virus, there is good immunogenicity, the desirable target antigen (LiuC of this viral diagnosis method development, IharaT, NunoyaT, etal.DevelopmentofanELISAbasedonthebaculovirus-expressed capsidproteinofporcinecircovirustype2asantigen [J] .JVetMedSci., 2004,66 (3): 237 ~ 242.).Although PCV2 only has a serotype, but is divided into Multi-genotype, comprise PCV2a, PCV2b, PCV2c and PCV2d etc.These genotypic differences make its Cap protein antigenicity there are differences.Resist of PCV2 can not identify these differences more, monoclonal antibody is only had to distinguish (SahaD., HuangL.P., BussalleuE., etal.Antigenicsubtypingandepitopes ' competitionanalysisofporcinecircovirustype2usingmonoclon alantibodies.Vet.Microbiol.2012,157:13-22; HuangL.P., LuY.H., WeiY.W., etal.Identificationofonecriticalaminoacidthatdeterminesa conformationalneutralizingepitopeinthecapsidproteinofpor cinecircovirustype2.BMCMicrobiol.2011a, 11:188.).
PCV2 causes the main pathogens that PMWS disease occurs clinically, but its mechanism of causing a disease is illustrated not yet completely.(PeterMeerts, GeraldMisinzo, DavidLefebvre, JensNielsen, the Anette such as Meerts
charlotteSKristensen, HansJNauwynck.Correlationbetweenthepresenceofneutralizin gantibodiesagainstporcinecircovirus2 (PCV2) andprotectionagainstreplicationofthevirusanddevelopmento fPCV2-associateddisease [J] .BMCVetRes2006, 2:6 – 16.) research show, lacking PCV2 neutralizing antibody in pig body strengthens relevant with Virus reproductivity, because in body, NAT is lower, can not completely in and body inner virus propagation, finally develop into clinical PMWS disease.In order to confirm this viewpoint further, (the FortM such as Fort, OlveraA, SibilaM, Segal é sJ, MateuE:Detectionofneutralizingantibodiesinpostweaningmul tisystemicwastingsyndrome (PMWS)-affectedandnon-PMWS-affectedpigs [J] .VetMicrobiol2007, 125:244 – 255.) carry out system test, application serum neutralization test, IPMA and quantitative PCR detection PMWS pig, PMWS is not had to show and the positive pig of PCV2 and the negative pig of PCV2, found that: in neutralizing antibody and serum, virus load is negative correlation, while neutralizing antibody increases, viral load obviously declines, PMWS Swine serum neutralizing antibody level is starkly lower than does not have PMWS to show and the positive pig of PCV2.Visible neutralizing antibody plays a significant role in PCV2 and rehabilitation process in removing blood circulation.Therefore, humoral immunity may occupy critical role in PCV2 relevant disease.
The main IIF of method for PCV2 Serum Antibody Detection, IPMA, indirect ELISA, stop band restrain, competitive ELISA and immune colloidal gold technique etc., wherein classical and conventional with IIF and IPMA, although these two kinds of methods have plurality of advantages, but there is certain subjectivity in result judgement, being difficult to realize high flux and Aulomatizeted Detect needs to make it promote and is restricted.The indirect ELISA set up based on recombinant protein has good specificity, but its susceptibility is lower than IPMA (Zhang Chaoxia, Liu Changming, to endanger gorgeous force, Deng, the development of porcine circovirus 2 type ELISA antibody assay kit and application [J]. Chinese Preventive Veterinary Medicine report, 2008,30 (7): 548-551; Zhang Zhi is intelligent, gorgeous force of endangering, Wu Hongli, Deng, comparison and application [J] of several ELISA of porcine circovirus 2 type antibody assay kit. Chinese Preventive Veterinary Medicine report, 2014,36 (2): 129-133), above-mentioned several method testing result can not directly react PCV2 neutralizing antibody.The Inhibition ELISA that the present invention utilizes PCV2 neutralizing monoclonal antibodies to set up can direct-detection serum neutralizing antibody; possesses the advantage of scale batch production; Aulomatizeted Detect requirement can be realized; high specificity; susceptibility is suitable with IPMA, for the detection of this virus vaccine and clinical onset animal blood serum antibody provides a kind of effective means.
Summary of the invention
First object of the present invention is to provide a kind of porcine circovirus 2 type competitive ELISA antibody assay kit, and this kit is easy and simple to handle, and specificity is good, and susceptibility is high, for PCV2 epidemiology survey and Serum Antibody Detection provide a kind of effective technology means;
Second object of the present invention is that providing a kind of uses above-described kit to detect the application in Swine serum porcine circovirus 2 type antibody reagent in preparation;
3rd object of the present invention is be provided for setting up the monoclonal antibody of the above kit and secrete the hybridoma cell strain of this monoclonal antibody.
In order to realize object of the present invention, the invention provides following technical scheme:
The present invention, using the PCV2 of purifying as immunogene, carries out 3 immunity to female BAl BIc/c mouse in 6 week age respectively.Head exempts from, the purified virus of every mouse injection abdominal cavity 50 μ g (0.5ml); Interval is carried out two for 3 weeks and is exempted from, and immunogene, dosage and method are the same; The 2 weeks same procedure booster immunizations in interval again.
Asepticly according to a conventional method take immune mouse spleen cell, merge with PEG1450.IPMA method is adopted to screen anti-PCV2 monoclonal antibody, concrete operation method reference literature (Liu Changming, Zhang Chaofan, to endanger gorgeous force, Deng. the research and apply [J] of porcine circovirus 2 type immunopcroxidase monolayer assay antibody assay kit. Chinese Preventive Veterinary Medicine report, 2007, 29 (8): 621 ~ 624.), doma supernatant is as primary antibodie, the rabbit anti-mouse igg (H+L) of HRP mark resists as detecting two, after testing, the hybridoma of IPMA test positive is expanded and cultivates, carry out the clone of positive hybridoma cell with limiting dilution assay simultaneously, clone 3 times, by frozen in time for the positive hybridoma cell obtained.
Detect screening through IPMA, obtain the positive hybridoma cell strain of 1 strain secretion resisting porcine circovirus 2 type monoclonal antibody, called after PCV2-Cap-3A5, Classification And Nomenclature is the hybridoma cell strain secreting anti-PCV2-Cap-3A5 monoclonal antibody.Be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, and its microbial preservation number is: CGMCCNO.10205, and the preservation time is on Dec 22nd, 2014.
Further, present invention also offers the monoclonal antibody of being secreted generation by described hybridoma cell strain.
The present invention utilizes the monoclonal antibody obtained to establish a kind of porcine circovirus 2 type competitive ELISA antibody assay kit, it is characterized in that, comprise employing and indirectly catch bag by method bag by the resisting porcine circovirus 2 type Cap protein monoclonal antibody of the ELISA Plate of porcine circovirus 2 type antigen and enzyme labeling.
Described monoclonal antibody is secreted by hybridoma cell strain and is produced, and described hybridoma cell strain called after PCV2-Cap-3A5, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCCNO.10205.
In the present invention, preferably, described employing indirectly catch bag by method envelope antigen for first with porcine circovirus 2 type positive serum bag by elisa plate to catch porcine circovirus 2 type antigen; Described porcine circovirus 2 type antigen is PCV2/LG strain culture, and malicious valency is 1 × 10
5tCID
50/ ml; Described enzyme is horseradish peroxidase.
In the present invention, preferably, the bag of described porcine circovirus 2 type positive serum is 1:1000 by extension rate; Described action time of catching porcine circovirus 2 type antigen is 1h.
In the present invention, preferably, described kit also comprises negative control, positive control, dilution, nitrite ion, cleansing solution and stop buffer.
In the present invention, preferably, described negative control is porcine circovirus 2 type negative serum; Described positive control is porcine circovirus 2 type positive serum; Described dilution is the PBST damping fluid containing 1% bovine serum albumin(BSA); Described nitrite ion is ABTS working fluid; Described cleansing solution is PBST damping fluid; Described stop buffer is the NaF solution of 1%.
In one particular embodiment of the present invention, preferably, described kit comprises:
ELISA Plate: adopt and indirectly catch bag by method bag by porcine circovirus 2 type antigen;
The resisting porcine circovirus 2 type Cap protein monoclonal antibody of enzyme labeling: the resisting porcine circovirus 2 type Cap protein monoclonal antibody of horseradish peroxidase mark, monoclonal antibody is that the hybridoma cell strain being CGMCCNO.10205 by deposit number secretes generation;
Dilution: the PBST damping fluid containing 1% bovine serum albumin(BSA);
Cleansing solution: PBST damping fluid;
Positive control: porcine circovirus 2 type positive serum;
Negative control: porcine circovirus 2 type negative serum;
Nitrite ion: ABTS working fluid;
Stop buffer: the NaF solution of 1%.
The kit that present invention also offers described in utilization detects the application in Swine serum porcine circovirus 2 type antibody reagent in preparation.
In the present invention, preferably, detect with the Swine serum antibody of Inhibition ELISA to porcine circovirus type 2 infection or vaccine immunity, concrete comprises the following steps:
(1) take out adopt indirectly catch bag by method bag by the ELISA Plate of porcine circovirus 2 type antigen, wash plate with cleansing solution;
(2) add Swine serum to be measured and the contrast of diluted, hatch for 37 DEG C;
(3) then add the resisting porcine circovirus 2 type Cap protein monoclonal antibody of enzyme labeling, hatch, wash plate with cleansing solution for 37 DEG C;
(4) add nitrite ion, 37 DEG C of colour developings, finally add stop buffer cessation reaction, measure OD
405nmvalue.
In order to determine optimum reaction condition, the present invention is under differential responses condition, square formation titration is done to after porcine circovirus 2 type positive serum, viral cultures and enzyme mark monoclonal antibody respectively serial dilution, high for evaluation index with S/N value, determine their best working concentration; Then the working concentration fixing antiserum, viral cultures and enzyme mark monoclonal antibody detects Swine serum to be measured incubation time (30min, 45min and 60min) different from contrast, viral cultures and enzyme labelled antibody respectively on the impact of Detection results, chooses the incubation time of S/N value the higher person as the best; Finally measure different developing time (10min, 20min and 30min) detects porcine circovirus 2 type antibody effect impact on competitive ELISA, choose the developing time of S/N value the higher person as the best equally; Select and demarcate PCV2 weak positive serum (IPMA tires between 100 ~ 400) 5 parts through IPMA, after serial dilution (1:50,1:100 and 1:200), carry out competitive ELISA detection, determine the optimum diluting multiple of test serum.Determine that the bag of porcine circovirus 2 type positive serum is 1:1000 by extension rate through test; The working concentration of the resisting porcine circovirus 2 type Cap protein monoclonal antibody of enzyme labeling is 1:4000; The extension rate of described Swine serum to be measured is 1:100; Described porcine circovirus 2 type antigen, Swine serum to be measured and contrast, the resisting porcine circovirus 2 type Cap protein monoclonal antibody of enzyme labeling and be respectively 1h, 45min, 30min and 20min the action time of nitrite ion;
In the present invention, preferably, described contrast comprises positive control and negative control, and positive control is PCV2 positive serum, and negative control is PCV2 negative serum; Described porcine circovirus 2 type antigen is PCV2/LG strain culture, and malicious valency is 1 × 10
5tCID
50/ ml; Described enzyme is horseradish peroxidase; Described cleansing solution is PBST damping fluid; Described dilution is the PBST damping fluid containing 1% bovine serum albumin(BSA); Described nitrite ion is ABTS working fluid; Described stop buffer is the NaF solution of 1%.
3A5 monoclonal antibody subclass of the present invention is IgG2a, and light chain is κ type.Westernblot result shows, and 3A5 monoclonal antibody does not react with the PCV2 of purifying.But IPMA and virus neutralization tests result show, this monoclonal antibody can react with PCV2, and with PCV1 no cross reaction; This monoclonal antibody has Neutralization effect, and Neutralization effect is 94.8%, is the monoclonal antibody for PCV2-Cap albumen neutralizing epitope; Catch the display of ELISA result, this monoclonal antibody can react from different PCV2 isolated strains.This strain monoclonal antibody is utilized to establish a kind of competitive ELISA method, for detecting the Swine serum antibody of this virus infections or vaccine immunity.The method first with porcine circovirus 2 type positive serum bag by elisa plate to catch PCV2 antigen, then react with the monoclonal antibody 3A5 of Swine serum antibody to be measured and horseradish peroxidase-labeled, by measuring enzyme mark monoclonal antibody display competitive ELISA testing result.
Compared with prior art, beneficial effect of the present invention is embodied in:
(1) competitive ELISA that the present invention utilizes PCV2 neutralizing monoclonal antibodies to set up can direct-detection serum neutralizing antibody; possesses the advantage of scale batch production; Aulomatizeted Detect requirement can be realized; method high specificity; susceptibility is suitable with IPMA, for the detection of this virus vaccine and clinical onset animal blood serum antibody provides a kind of effective means.This competitive ELISA is assembled into complete kit, can preserve 1 year at 4 DEG C.
(2) the present invention adopts virus-positive serum bag by elisa plate, and catch known viruse antigen with it, this mode is easy, quick, and can obtain good specificity.Inventor also once the PCV2-Cap recombinant protein of trial recombinant baculovirus expression as envelope antigen, but Detection results undesirable (data do not show), illustrates using natural virion as detectable antigens still have the unrivaled advantage of recombinant protein.In view of the enzyme mark monoclonal antibody that competitive ELISA is used is tired height, active good, improve ELISA specificity and susceptibility, and operation is more simple and quick.
Accompanying drawing explanation
Fig. 1 is the Electronic Speculum result figure of purifying PCV2 virion;
Fig. 2 is the response characteristic figure that IPMA method detects monoclonal antibody and PCV2;
Fig. 3 is that Western-blot method carries out analysis of immunogenicity result figure to PCV2 monoclonal antibody, wherein M: standard protein sample; 3A5:PCV2 monoclonal antibody; 2C10:PCV1 monoclonal antibody; 2E8:PCV2 monoclonal antibody;
Fig. 4 is that virus neutralization tests detects 3A5 monoclonal antibody to the Neutralization effect figure of PCV2 strain;
Fig. 5 catches the response characteristic figure that ELISA detects 3A5 monoclonal antibody isolated strain different from PCV2;
Fig. 6 is that IPMA method detects PCV2 positive and negative serum result figure, wherein A: test group, B: control group;
Fig. 7 is the response characteristic figure that Western-blot method detects PCV2 positive and negative, wherein M: standard protein sample; 1 swimming lane: PCV2 positive serum; 2 swimming lanes: PCV2 negative serum;
Fig. 8 is the SDS-PAGE analysis chart of 3A5 monoclonal antibody purifying, wherein M: standard protein sample; 1: the 3A5 monoclonal antibody sample of purifying; 2:3A5 odd contradictive hydroperitoneum crude product;
Fig. 9 is competitive ELISA specific detection result figure;
Figure 10 is the testing result figure of competitive ELISA to the blood serum sample of different antibodies level.
Figure 11 is competitive ELISA kit storage life test findings figure.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But embodiment is only exemplary, does not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
The preparation of embodiment 1 resisting porcine circovirus 2 type (PCV2) Cap protein monoclonal antibody
1, materials and methods
1.1 cells, seed culture of viruses, experimental animal, antibody and reagent
SP2/0 and PK15 clone (PCV1 and PCV2 detects feminine gender), PCV1/G strain, PCV2 strain (LG, JF, JF2, CL, YJ, SH, BDH, AH etc.) is preserved by pig circular ring virus seminar of Harbin Veterinary Medicine Inst., China Academy of Agriculture; 6 week age, female BAl BIc/c mouse was provided by same institute Experimental Animal Center.ABTS is purchased from BBI company, HAT and HT salt-mixture, PEG1450,3-amino-9-ethyl imidazol(e) (AEC), horseradish peroxidase (HRP, TypeVI-A) and HRP mark rabbit anti-mouse igg (H+L) all available from Sigma; DMEM nutrient culture media and calf serum (FBS) are all purchased from Gibco company; Pre-dyed albumen Marker is purchased from Fermentas company; MouseMonoAb-IDKit (HRP), HRP-GoatAnti-MouseIgG (H+L) are purchased from Invitrogen company; Horseradish peroxidase (HRP)-ProteinA label (HRP-SPA) is Zymed product; ProteinG chromatographic column is GEHealthcare product; Other reagent is that domestic analysis is pure.
1.2 porcine circovirus 2 types (PCV2) purifying
Adopt CsCl gradient density centrifugal method purifying PCV2a/LG strain.The malicious also freeze thawing of results 300mlLG strain tissue cultures 3 times, virus multiplication method is see document (Liu Changming, Zhang Chaofan, Liu Huanzhang, Deng. the cultivation of the cell adapted strain of porcine circovirus 2 type and qualification [J]. Chinese Preventive Veterinary Medicine report, 2006,28 (3): 248 ~ 252.).4 DEG C of centrifugal 30min of 12000rpm, results supernatant; Supernatant is loaded in centrifuge tube, adds 30% sucrose at the bottom of pipe, 4 DEG C of centrifugal 3h of 45000rpm (50tirotor, beckman), and supernatant discarded, by resuspended for precipitation 50mMHepes liquid; Finally re-suspension liquid is placed in 35%CsCl solution upper strata, 4 DEG C of centrifugal 18h of 36000rpm (SW41rotor, beckman), collects milky band in the middle part of centrifuge tube, in 50mMHepes liquid, dialysis removes CsCl ,-80 DEG C of preservations.Electronic microscope photos after the viral negative staining of purifying, ultraviolet method measures the concentration of purified virus.
The preparation of 1.3 monoclonal antibodies
Using the PCV2 of purifying as immunogene, respectively 3 immunity are carried out to female BAl BIc/c mouse in 6 week age.Head exempts from, the purified virus of every mouse injection abdominal cavity 50 μ g (0.5ml); Interval is carried out two for 3 weeks and is exempted from, and immunogene, dosage and method are the same; The 2 weeks same procedure booster immunizations in interval again.
Asepticly according to a conventional method take immune mouse spleen cell, merge with PEG1450.IPMA method is adopted to screen anti-PCV2 monoclonal antibody, concrete operation method reference literature (Liu Changming, Zhang Chaofan, to endanger gorgeous force, Deng. the research and apply [J] of porcine circovirus 2 type immunopcroxidase monolayer assay antibody assay kit. Chinese Preventive Veterinary Medicine report, 2007, 29 (8): 621 ~ 624.), doma supernatant is as primary antibodie, the rabbit anti-mouse igg (H+L) of HRP mark resists as detecting two, after testing, the hybridoma of IPMA test positive is expanded and cultivates, carry out the clone of positive hybridoma cell with limiting dilution assay simultaneously, clone 3 times, by frozen in time for the positive hybridoma cell obtained.
The qualification of 1.4 monoclonal antibodies
1.4.1 monoclonal antibody is tired and subgroup identification
Prepare 3A5 monoclonal antibody by two kinds of methods: cultivate PCV2-Cap-3A5 hybridoma, when 50% cell occurs dead after growing to individual layer, collect supernatant; Prepared by ascites, adopt BALB/c Female Rats in 8 week age, first through lumbar injection sterilizing paraffin oil (0.5mL/ is only), and 7d pneumoretroperitoneum injection hybridoma 1 × 10
5individual/only, after mouse web portion expands, extract its ascites, 3000r/min is centrifugal, and 10min gets supernatant.Adopt IPMA method to detect upper cleer and peaceful ascites antibody to tire.Adopt MouseMonoAb-IDKit (HRP) to identify monoclonal antibody subclass, operate and undertaken by kit instructions.
1.4.2 monoclonal antibody Western-blot activity analysis
The PCV2a/LG strain virus getting purifying carries out SDS-PAGE analysis, method detailed is see document (Huang Liping, Liu Changming, to endanger gorgeous force, Deng. the preparation of resisting porcine circovirus 2 type Cap protein neutralizing monoclonal antibody and qualification [J]. Chinese Preventive Veterinary Medicine report, 2009,31 (2): 132 ~ 136.).Then be transferred on nitrocellulose filter (NC); Close for 2% skimmed milk PBST4 DEG C and spend the night, then according to loading hole, NC is cut into strip; Using the 3A5 monoclonal antibody screened, anti-PCV1 monoclonal antibody 2C10 (negative control) and anti-PCV2 monoclonal antibody 2E8 (positive control) as primary antibodie, 1:500 dilutes, and 37 DEG C of reactions 1h, PBST wash 3 times, each 5min; Mark rabbit anti-mouse IgG (H+L) using HRP to resist as two, 1:5000 dilutes, and 37 DEG C of reactions 1h, PBST wash 5 times, each 5min; DAB substrate solution develops the color, takes pictures.
1.4.3 monoclonal antibody Neutralization effect detects
Virus neutralization tests is carried out for detecting the Neutralization effect of monoclonal antibody by the method for fixed virus dilution antibody.PCV2-Cap-3A5 Hybridoma Cell Culture supernatant 1:10 is diluted, respectively with containing 10
4.3tCID
50the PCV2/LG strain of (200 μ L), PCV2/CL strain, PCV2/YJ strain or the mixing of PCV2/JF strain equal-volume, with establishing PCV2 monoclonal antibody 1D2 and 2E8 cells and supernatant as positive and negative control, putting 37 DEG C of senses and making 2h.Then the PK15 cell of 60% individual layer is grown in inoculation, potpourri Simultaneous vaccination 4 holes of often kind of virus and antibody, put 37 DEG C and cultivate 60 ~ 72h, fixed cell, carry out IPMA with PCV2 positive serum and detect the viral antigen reaction of each hole, the antibody of 50% virus multiplication can be suppressed to be defined as having Neutralization effect, repressed for PCV2 replication capacity percentage to be defined as the Neutralization effect of monoclonal antibody.
1.4.4 the detection of monoclonal antibody and PCV2 strain response spectrum
Employing is caught ELISA method and is detected different PCV2 strain, comprising: PCV2a genotype strain LG, CL, JF2,1010 and 1121; PCV2b genotype strain: JF, YJ, SH and 48285; PCV2d genotype strain: BDH and AH; PCV2a/b hybridizes poison: PCV2b (JF)/2a (CL).Being diluted by above-mentioned strain is 10
4.5tCID50/ml, carry out catching ELISA and detect, using PCV1/G strain as negative control, each sample does three holes and repeats.Catch the trace routine of ELISA see document (Huang Liping, Liu Changming, to endanger gorgeous force, Deng. the preparation of resisting porcine circovirus 2 type Cap protein neutralizing monoclonal antibody and qualification [J]. Chinese Preventive Veterinary Medicine report, 2009,31 (2): 132 ~ 136.), but there is a bit little change, detect monoclonal antibody and change 3A5 into.
2, result
2.1PCV2a/LG strain purifying
Adopt CsCl gradient density centrifugal method purifying PCV2/LG strain, the viral negative staining of purifying the results are shown in Figure 1, and virion is spherical, diameter is about 17nm, and foreign protein is fewer, and it is 265ng/ul that ultraviolet method measures purified product concentration, therefore, purified virus may be used for the immunity of BALB/c mouse.
The qualification result of 2.2 monoclonal antibodies
2.2.1 monoclonal antibody subgroup identification and titration
Detect screening through IPMA, obtain the positive hybridoma cell that anti-PCV2 monoclonal antibody is secreted in 1 strain, called after PCV2-Cap-3A5, Classification And Nomenclature is the hybridoma cell strain secreting anti-PCV2-Cap-3A5 monoclonal antibody.Be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, and its microbial preservation number: CGMCCNO:10205, the preservation time is on Dec 22nd, 2014.This strain monoclonal antibody and PCV2 virus strain infection PK15 cell are positive (Fig. 2 left), and are negative with PCV1 virus strain infection cell and reacts on (Fig. 2 right side).By cell clone, subculture in vitro separately and frozen with recovery, prove that this strain cell can stably excreting specific monoclonal antibody.Carried out subgroup identification with MouseMonoAb-IDKit (HRP) to the monoclonal antibody secreted by this strain of hybridoma, this monoclonal antibody subclass is IgG2a, and light chain is κ type.By IPMA method, antibody titer mensuration is carried out to PCV2-Cap-3A5 Hybridoma Cell Culture supernatant and prepared ascites thereof, this strain monoclonal antibody cells and supernatant and ascites antibody are tired and are respectively 1:1000 and 1:1000000, show that the monoclonal antibody prepared has higher tiring.
2.2.2 monoclonal antibody response characteristic is identified
Adopt the response characteristic of Western-blot method qualification 3A5 monoclonal antibody, test findings as shown in Figure 3, positive control monoclonal antibody 2E8 can produce specific band with PCV2-Cap albumen at about 29kD place, and 3A5 monoclonal antibody does not produce reaction, with the PCV1 monoclonal antibody 2C10 that sets as negative reaction.As can be seen here, 3A5 monoclonal antibody may be the monoclonal antibody for Porcine circovirus type 2 Cap comformational epitope.
2.2.3 monoclonal antibody Neutralization effect measures
The method of neutralization test is adopted to identify the Neutralization effect of 3A5 monoclonal antibody to PCV2a/LG, PCV2a/CL, PCV2b/YJ and PCV2b/JF strain respectively, the results are shown in Figure 4, Fig. 4 shows, 3A5 monoclonal antibody all shows good Neutralization effect to above-mentioned 4 strain separated strains, and the Neutralization effect of its cells and supernatant (1:10 dilution) is respectively 91.1%, 94.2%, 85.4% and 88.8%; And PCV2 monoclonal antibody 2E8 is without the ability of the corresponding virus of neutralization.
2.2.4 the detection of monoclonal antibody and PCV2 strain response spectrum
The response spectrum of ELISA is caught in order to detect PCV2, choose this laboratory to represent strain and carry out catching ELISA and detect, result as shown in Figure 5, this catches ELISA can detect that PCV2a genotype 4 strain is separated poison, PCV2b genotype 4 strain is separated poison, can also hybridize strain be positive with laboratory one strain PCV2a/2b.Can prove thus, this is caught ELISA and can detect each genotype in this laboratory and represent strain, detects spectrum relatively wider, so can infer that the method can detect most of PCV2 isolated strain.
The present invention, with the PCV2 of purifying immunity BALB/c mouse, obtains the hybridoma cell strain of the anti-PCV2 of 1 strain stably excreting, called after PCV2-Cap-3A5.In this Hybridoma Cell Culture, cleer and peaceful ascites antibody is tired and is respectively 1:1000 and 1:1, and 000,000.3A5 monoclonal antibody subclass is IgG2a, and light chain is κ type.Westernblot result shows, and 3A5 monoclonal antibody does not react with the PCV2 of purifying.But IPMA and virus neutralization tests result show, this monoclonal antibody can react with PCV2, and with PCV1 no cross reaction; This monoclonal antibody has Neutralization effect, and Neutralization effect is 94.8%, is the monoclonal antibody for PCV2-Cap albumen neutralizing epitope.
The preparation of embodiment 2 porcine circovirus 2 type (PCV2) positive serum and negative serum
1 materials and methods
1.1 test material
Porcine circovirus 2 type (LG strain) inactivated vaccine is provided by Harbin Wei Ke biotechnology development company, PCV2/YJ strain (10
5.0tCID
50/ ml) to be preserved by pig circular ring virus seminar of Harbin Veterinary Medicine Inst., China Academy of Agriculture, 6 30-40 age in days piglets (PCV2-IPMA serum antibody titer is less than or equal to 1:50 doubly) are provided by Harbin Veterinary Medicine Inst., China Academy of Agriculture's Experimental Animal Center.
1.2 test method
1.2.1 animal immune experiment
Piglet 6 is divided into test group and control group at random, often organizes 3, separately raise.1st immunity, piglet musculi colli injection porcine circovirus 2 type (LG strain) inactivated vaccine 1mL only
-1; Interval 21d carries out the 2nd immunity, immunization method and dosage the same; Carry out PCV2/YJ strain Experimental infection after the 14d of interval again, route of infection and dosage as follows: nasal cavity and musculi colli inject each 1mL only
-1.Two exempt from after after 14d and artificial challenge 21d take a blood sample through vena cava anterior, separation of serum, adopts PCV2-IPMA method to detect antibody titer.Control group adopts PBS immunity, immunization method and the same immune group of dosage.
1.2.2 antiserum prepare method
When the IPMA of test group serum antibody tire >=1:25600 time, cut open and kill test pig and get blood, 37 DEG C of 1h, 4 DEG C are spent the night, the centrifugal 5min of 1500rpm, draw upper serum, are designated as PCV2 positive serum, and-80 DEG C of packing are preserved.In control group PCV2 serum antibody IPMA tire < 1:50 times time, same method cuts open and kills pig separation of serum, is designated as PCV2 negative serum, and-80 DEG C of packing are preserved.
1.2.3IPMA method measures antibody titer
Adopt IPMA method to measure antibody titer, synchronously inoculate PK-15 cell suspension by 5%PCV2/LG kind toxic agent amount, with 100 μ l/ hole dispensings in 96 orifice plates, put 37 DEG C of 5%CO
2cultivate under condition and form individual layer, fix with 33% acetone-PBS, dry rearmounted-20 DEG C save backup.With PCV2 standard positive and negative serum in contrast, after immune serum and control group serum are made 2 times of gradient dilutions simultaneously, detect with IPMA plate, concrete steps are as follows.By serum to be checked according to after 1:50,1:100,1:200,1:400,1:800,1:1600,1:3200,1:6400,1:12800 and 1:25600 dilution, every hole adds the serum to be checked that 100 μ L have diluted, and after 37 DEG C of placement 1h, PBS washes 3 times; Every hole adds proteinA (1:3000 dilution) the 100 μ L that horseradish peroxidase (HRP) marks, and places 1h, PBS for 37 DEG C and washes 3 times; Every hole adds the AEC chromophoric solution 100 μ L of new preparation, and after color development at room temperature 20min, discard nitrite ion, every hole adds 100 μ L deionized waters, observations under inverted microscope.
1.2.4 result judges
PCV2 positive serum and PCV2 infection cell are brownish red, PCV2 negative serum and virus infected cell non-coloring react, above-mentioned two as positive and negative serum criterion, be that the inverse of the most highly diluted multiple of the positive is tired as the IPMA of detection serum with IPMA.
1.2.5PCV2 the qualification of positive and negative serum
By the PCV2 of purifying after SDS-PAGE electrophoresis, turn 2h and be transferred on nitrocellulose filter through the mini transfer core of BG-transBLOT is wet, the phosphate buffer 4 DEG C of the 50mmol/LpH7.2 containing 2% skimmed milk is closed and is spent the night.PBST washes 5 times, doubly dilutes the positive and negative serum of preparation, 37 DEG C of 1h with the Tween-20 confining liquid 1:100 of interpolation 0.1%.PBST washes 5 times, and immerse the proteinA of the HRP mark of the Tween-20 confining liquid 1:3000 dilution containing 0.1%, place 1h, PBST for 37 DEG C and wash 5 times, DAB develops the color, and takes pictures.
2 test findings
2.1 antibody test results
The sun adopting IPMA method to detect tested serum turns situation and antibody titer.Test findings shows: inactivated vaccine two exempts from the serum antibody sun turn (Fig. 6 A) gathered for latter 14 days, and Mean antibody titer can reach 800 times, and control group serum 1:50 times of testing result is negative (Fig. 6 B); After experimental group attacks poison, 21 balance antibody titers are all greater than 25600 times, and control group serum 1:50 times of testing result is negative.
2.2Westernblot testing result
Westernblot analyzes display, PCV2 positive serum can the restructuring PCV2-Cap albumen of specific recognition purifying, present a positive reaction band (Fig. 7,1 swimming lane), control group PCV2 negative serum control is then without this reaction band (Fig. 7,2 swimming lanes), show that the PCV2 positive serum prepared has good reactivity.
The foundation of embodiment 3 porcine circovirus 2 type competitive ELISA method
1, materials and methods
1.1 material
PCV2 positive and negative serum is made by oneself by this laboratory and is preserved, and other several swine disease hemlock is examined positive serum and provided by relevant seminar of this institute.3-amino-9-ethyl imidazol(e) (AEC) and horseradish peroxidase (HRP, TypeVI-A) are Sigma product; Two-3-ethyl-phenylpropyl alcohol thiazoline the sulfanilamide (SN) (ABTS) of 2,2-azine group is BBI product; Horseradish peroxidase (HRP)-ProteinA label (HRP-SPA) is Zymed product; ProteinG chromatographic column is GEHealthcare product; DMEM nutrient culture media and hyclone (FBS) are all purchased from Gibco company; Other reagent is that domestic analysis is pure.
The preparation of ABTS working fluid: (1) colour developing A liquid (0.1mol/L citrate buffer) preparation: claim 29.41g sodium citrate (Na
3c
6h
5o
72H
2o) be dissolved in 1800mL deionized water, with citric acid, pH be adjusted to 4.3, then add 200 μ L30%H
2o
2, be finally settled to 2000mL with deionized water; (2) develop the color B liquid (2.19%ABTS solution) preparation: claim 2.19gABTS powder, be settled to 100mL after fully dissolving with 0.1mol/L citrate buffer; (3) prepare ABTS working fluid: add 0.5ml to develop the color B liquid at the 9.5mL A liquid that develops the color, use after mixing, instantly to join.
1.2 monoclonal antibody purifying and HRP mark
The purifying of monoclonal antibody: adopt ProteinG affinitive layer purification 3A5 odd contradictive hydroperitoneum, monoclonal antibody after purifying, see instructions, is carried out SDS-PAGE analysis by concrete operations, measures its antibody protein purity and content through thin layer scanning and UV Absorption method.
The preparation of horseradish peroxidase-labeled monoclonal antibody: by document (Jin Baiquan. cell and molecular immunology experimental technique [M]. Xi'an: publishing house of The Fourth Military Medical University, 2002 (156 ~ 158) .) the simple and easy Over-voltage protection introduced carries out HRP-monoclonal antibody mark test, measures the OD of labelled antibody
280nmand OD
403nm, calculate grammol ratio.
1.3 competitive ELISA art formulas
Dilute PCV2 positive serum with 50mmol/L carbonate buffer solution (pH9.6), wrap by 96 hole ELISA Plate, put 4 DEG C and spend the night.3 times are washed, each 3min with the phosphate buffer (PBST) of the 50mmol/LpH7.2 containing 0.05%Tween-20.Close with containing 5% sucrose and 1% bovine serum albumin(BSA) (BSA)-PBST fluid-tight, put 37 DEG C of 1h, wash the same.Add the PCV2/LG strain culture with confining liquid dilution, 100 μ L/ holes, 1h is made in 37 DEG C of senses, washs the same.Add the test serum, negative control and the positive control that dilute with the PBST damping fluid containing 1% bovine serum albumin(BSA), 100 μ L/ holes, 1h is made in 37 DEG C of senses, serum to be checked in retaining holes, then enzyme-added mark monoclonal antibody, and 50 μ L/ holes, 1h is made in 37 DEG C of senses, washs the same.Add ABTS working fluid in every hole, 100 μ L/ holes, 37 DEG C of colour developings 15min, 1%NaF stop, 50 μ L/ holes.Each hole OD is read by microplate reader
405nmvalue.With positive and negative reference serum OD
405nmbe worth as a reference, according to following formulae discovery, S/N (%)=100 × (tested Virus monitory value/negative serum detected value).Judge as a result with S/N.Positive control is PCV2 positive serum, and negative control is PCV2 negative serum.
The determination of 1.4 competitive ELISA reaction conditionss
Porcine circovirus 2 type positive serum, viral cultures and enzyme mark monoclonal antibody do square formation titration after serial dilution respectively, high for evaluation index with S/N value, determine their best working concentration; Then the working concentration fixing antiserum, viral cultures and enzyme mark monoclonal antibody detects Swine serum to be measured incubation time (30min, 45min and 60min) different from contrast, viral cultures and enzyme labelled antibody respectively on the impact of Detection results, chooses the incubation time of S/N value the higher person as the best; Finally measure different developing time (10min, 20min and 30min) detects porcine circovirus 2 type antibody effect impact on competitive ELISA, choose the developing time of S/N value the higher person as the best equally.Select and demarcate PCV2 weak positive serum (IPMA tires between 100 ~ 400) 5 parts through IPMA, after serial dilution (1:50,1:100 and 1:200), carry out competitive ELISA detection, determine the optimum diluting multiple of test serum.
1.5 competitive ELISA critical values are determined
The IPMA that learns from else's experience is demarcated as the negative pig anteserum sample 80 parts of PCV2, detects its S/N, calculating mean value with competitive ELISA
with standard deviation (SD), determine to judge critical value.
1.6 competitive ELISAs and IPMA detection kit contrast test
Select 237 parts of test pig serum, application competitive ELISA and IPMA carry out Parallel testing simultaneously, take IPMA as standard, calculate coincidence rate, the Sensitivity and Specificity of competitive ELISA and its testing result.
The correlation test of 1.7 competitive ELISAs and neutralization test
Choose 24 parts of test pig serum and carry out 2 times of serial dilutions (1:50 ~ 1:12800), application competitive ELISA and neutralization test carry out Parallel testing respectively simultaneously.The inverse of the most highly diluted multiple of the antibody of 50% virus multiplication can be suppressed to be decided to be antibody Neutralizing titer, the inverse of the most highly diluted multiple of competitive ELISA test positive is decided to be competitive ELISA antibody titer, finally calculates the related coefficient of competitive ELISA and neutralization test testing result.
1.8 competitive ELISA detection specificity tests
Cross reaction test is carried out with reference to positive serum by known pig circular ring virus 1 type (PCV1), pig parvoviral 1 type (PPV1), porcine pseudorabies virus (PRV), transmissible gastro-enteritis virus (TGEV), Porcine epidemic diarrhea virus (PEDV), porcine rotavirus (PRoV), CSFV (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV) and pig O type foot and mouth disease virus (FMDV-O).
1.9 competitive ELISA detection sensitivity tests
Choose 12 parts have been demarcated different PCV2 antibody titer serum through IPMA, wherein strong positive 3 parts (IPMA tires and is greater than 1:6400), positive 3 parts (IPMA tires between 1:800 to 1:3200), weak positive 3 parts (IPMA tires between 1:100 to 1:400) and negative 3 parts (IPMA tires and is less than 1:100).With sample diluting liquid by above-mentioned 12 parts of serum by 1: 50,1: 100,1: 200,1: 400,1: 800,1: 1600,1: 3200 and 1: 6400 gradient dilute, then detect according to competitive ELISA running program, according to testing result determination porcine circovirus 2 type ELISA antibody assay kit to the maximum detection extension rate of strong positive blood serum sample, positive serum samples, weak positive serum sample and negative serum sample, to determine the susceptibility of this kit.
1.10 detect blood serum sample source
The immunity of PCV2 inactivated vaccine is with challenge test pig from the nonimmune sodium selenite of 30 age in days 8, and detect PCV2 through PCR negative, No.3,19,21,26 and No. 32 pigs are set to immune group, and No.1,9 and No. 10 pigs are set to blank group.Immune group is through intramuscular injection PCV2 (LG strain) inactivated vaccine, 1mL/ head, interval 3w booster immunization 1 time, carries out challenge test in 6w time vaccines immune group together with blank group pig, and Infection route is in nose and intramuscular routes inoculation PCV2 velogen strain culture (1 × 10
5tCID
50/ mL) each 1mL/ head.Vaccine immunity and challenge test pig taking periodic blood weekly, blood serum sample is used for competitive ELISA antibody test.In addition, to 862 parts of clinical serum samples of censorship from various parts of the country be also at war with ELISA detect.
1.11 competitive ELISA kit storage life tests
Preserve under overall for competitive ELISA kit is positioned over 4 DEG C of conditions, adopt this kit to detect 8 parts of pig anteserum samples (each 2 parts of PCV2 strong positive, the positive, the weak positive and negative serum) at quarterly intervals, detect 12 months.
2, result
The purifying of 2.1 monoclonal antibody 3A5 and HRP mark
With the monoclonal antibody of ProteinG affinity column purifying from ascites sample, the results are shown in Figure 8 through SDS-PAGE electrophoretic analysis, detect monoclonal antibody purity through thin layer scanning and reach 89.2%, it is 10.2mg/mL that ultraviolet method surveys protein concentration.Carry out HRP mark test to the monoclonal antibody of purifying, the enzyme labelled antibody of mark is at OD
280nmand OD
403nmdetect optical density value and be respectively 5.5 and 3.9, grammol ratio is 2.1, shows that the enzyme mark monoclonal antibody mark effect prepared is better, can use.
The determination of 2.2 competitive ELISA reaction conditionss
Warp is to the working concentration of various reagent and the test of reaction conditions, and finally determine that anti-pig PCV2 positive serum bag is 1:1000 by extension rate, 4 DEG C are spent the night; Viral cultures poison valency is 1 × 10
5tCID
50/ mL, 37 DEG C of effect 1h; Tested Swine serum extension rate is 1:100,37 DEG C of effect 45min; Enzyme mark monoclonal antibody working concentration is 1:4000,37 DEG C of effect 30min; ABTS working fluid colour developing 20min, the NaF of 1% stops; Microplate reader reads each hole OD
405 nmvalue.
The determination of 2.3 competitive ELISA critical values
Statistical analysis is carried out to the known negative test pig serum 80 parts ELISA testing result that is at war with, calculates the average S/N of blood serum sample
be 87.3%, standard deviation (SD) is 11.0%,
be judged to serum antibody positive reaction when S/N≤54.3%, during 54.3% < S/N < 65.3%, serum is suspicious, needs duplicate detection once, if be still more than or equal to 65.3%, is serum antibody negative reaction.
2.4 competitive ELISAs and IPMA accordance are tested
Carry out Parallel testing with competitive ELISA and IPMA to 237 parts of serum, the results are shown in Table 1, IPMA detection sample has 175 parts of positives, 62 parts of feminine genders, and competitive ELISA detection has 163 parts of positives, 74 parts of feminine genders.In IPMA positive, competitive ELISA is detected as negative 13 parts, 1 part of competitive ELISA test positive in IPMA negative sample.Two kinds of methods detect and are positive 162 parts, and be negative 61 parts, total coincidence rate is 94.1% (223/237).Using IPMA as reference standard, competitive ELISA susceptibility is 92.6% (162/175), and specificity is 98.4% (61/62), shows that competitive ELISA has higher Sensitivity and Specificity.
Table 1 competitive ELISA and IPMA accordance test findings
2.5 competitive ELISAs and neutralization test relevance detection results
Adopt competitive ELISA and neutralization test Parallel testing 24 parts of test pig serum, and calculate the PCV2 antibody titer of these two kinds of detection methods respectively, testing result is as shown in table 2, competitive ELISA antibody titer is between 100 ~ 12800, NAT is between 100 ~ 6400, can infer that the susceptibility that these two kinds of methods detect is suitable thus, related coefficient is 0.9438, therefore can estimate neutralization test result by competitive ELISA result.
Table 2 adopts neutralization test and competitive ELISA method to detect Swine serum antibody titer
The specific test of 2.6 competitive ELISAs
Examine positive serum (PPV1, CSFV, PRRSV, PRV, PEDV, TGEV, PRoV, FMDV-O, SIV and PCV1) cross reaction result with competitive ELISA to several swine disease hemlock to show, except PCV2 positive serum is positive reaction, other several swine disease poison reference serums are feminine gender, prove the method specific detection PCV2 antibody, with other several viral serum-free cross reactions (Fig. 9).
2.7 competitive ELISA sensitivity Detection tests
12 parts of Swine serum of tiring to different antibodies ELISA that is at war with measures, the results are shown in Figure 10, increase progressively with serum diluting multiple, strong positive, positive and weak positive serum S/N% curve is smoothly increasing progressively in various degree, negative serum S/N% changes little, wherein the extension rate of strong positive serum competitive ELISA test positive is greater than 1:3200 doubly, namely competitive ELISA is tired and is greater than 3200, the competitive ELISA of positive serum is tired between 1600 ~ 3200, it is 100 ~ 400 that the competitive ELISA of weak positive serum is tired, show that competitive ELISA all can obtain the blood serum sample of different antibodies level and good detect susceptibility.
The detection of 2.8 pairs of test pig serum antibodies
The testing result that application competitive ELISA carries out test pig serum, in table 3, be positive according to S/N≤54.3%, has 100% (5/5) at immune 3w antibody male rotary, establish the antibody test of control group Swine serum to be feminine gender together in vaccine immunity group.Attack malicious rear 2w, namely during immune rear 7w, attack malicious control group Swine serum 33.3% (1/3) antibody male rotary, attack the rear 3w of poison, attack malicious control group Swine serum 100% (3/3) antibody male rotary; Vaccine immunity group antibody level of serum is in higher level from 4w to attacking the rear antibody of poison always.Result shows, and the neutralizing antibody that inactivated vaccine immune swine produces is morning comparatively, and the duration is longer
Table 3 competitive ELISA is to the mensuration of inactivated vaccine immune swine serum antibody Fluctuation
The detection of 2.9 pairs of clinical censorship Swine serum antibody
With competitive ELISA, the 862 part serum submitted samples regional from 9, the whole nation are detected, the results are shown in Table 4.All be positive from the antibody test of all regions blood serum sample, recall rate is 44.7% ~ 87.7%, and total recall rate is 70.8%, and test findings shows, in China pig farm, PCV2 infects ubiquity, and positive rate is higher.
Table 4 competitive ELISA is to PCV2 antibody test result in pig farm censorship serum
2.10 competitive ELISA kit storage life tests
Adopt the PCV2 competitive ELISA kit preserved under 4 DEG C of conditions to detect the Swine serum of 8 parts of different PCV2 antibody titers at quarterly intervals, testing result is shown in Figure 11.This kit along with the prolongation of holding time, although the S/N% of 8 parts of serum changes to some extent, do not have influence on the judgement of result, so at least can preserve 1 year at 4 DEG C.
The assembling of embodiment 4 kit
Kit described in the present invention comprises:
ELISA Plate: adopt and indirectly catch bag by method bag by porcine circovirus 2 type antigen;
The resisting porcine circovirus 2 type Cap protein monoclonal antibody of enzyme labeling: the resisting porcine circovirus 2 type Cap protein monoclonal antibody of horseradish peroxidase mark, monoclonal antibody is that the hybridoma cell strain being CGMCCNO.10205 by deposit number secretes generation;
Dilution: the PBST damping fluid containing 1% bovine serum albumin(BSA);
Cleansing solution: PBST damping fluid;
Positive control: porcine circovirus 2 type positive serum;
Negative control: porcine circovirus 2 type negative serum;
Nitrite ion: ABTS working fluid;
Stop buffer: the NaF solution of 1%.
Claims (10)
1. a porcine circovirus 2 type competitive ELISA antibody assay kit, is characterized in that, comprises employing and indirectly catches bag by method bag by the resisting porcine circovirus 2 type Cap protein monoclonal antibody of the ELISA Plate of porcine circovirus 2 type antigen and enzyme labeling;
Described monoclonal antibody is secreted by hybridoma cell strain and is produced, and described hybridoma cell strain called after PCV2-Cap-3A5, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCCNO.10205.
2. kit according to claim 1, is characterized in that, described employing indirectly catch bag by method bag by porcine circovirus 2 type antigen for first with porcine circovirus 2 type positive serum bag by elisa plate to catch porcine circovirus 2 type antigen; Described porcine circovirus 2 type antigen is PCV2/LG strain culture, and malicious valency is 1 × 10
5tCID
50/ ml; Described enzyme is horseradish peroxidase.
3. kit according to claim 2, is characterized in that, the bag of described porcine circovirus 2 type positive serum is 1:1000 by extension rate; Described action time of catching porcine circovirus 2 type antigen is 1h.
4. the kit according to any one of claim 1-3, is characterized in that, described kit also comprises negative control, positive control, dilution, nitrite ion, cleansing solution and stop buffer.
5. kit according to claim 4, is characterized in that, described negative control is porcine circovirus 2 type negative serum; Described positive control is porcine circovirus 2 type positive serum; Described dilution is the PBST damping fluid containing 1% bovine serum albumin(BSA); Described nitrite ion is ABTS working fluid; Described cleansing solution is PBST damping fluid; Described stop buffer is the NaF solution of 1%.
6. the kit described in any one of claim 1-5 detects the application in Swine serum porcine circovirus 2 type antibody reagent in preparation.
7. application according to claim 6, is characterized in that, detect with the Swine serum antibody of Inhibition ELISA to porcine circovirus type 2 infection or vaccine immunity, concrete comprises the following steps:
(1) take out adopt indirectly catch bag by method bag by the ELISA Plate of porcine circovirus 2 type antigen, wash plate with cleansing solution;
(2) add Swine serum to be measured and the contrast of diluted, hatch for 37 DEG C;
(3) then add the resisting porcine circovirus 2 type Cap protein monoclonal antibody of enzyme labeling, hatch, wash plate with cleansing solution for 37 DEG C;
(4) add nitrite ion, 37 DEG C of colour developings, finally add stop buffer cessation reaction, measure OD
405nmvalue.
8. application according to claim 7, is characterized in that, the working concentration of the resisting porcine circovirus 2 type Cap protein monoclonal antibody of described enzyme labeling is 1:4000; The extension rate of described Swine serum to be measured is 1:100; Described contrast comprises negative control and positive control, and negative control is porcine circovirus 2 type negative serum; Positive control is porcine circovirus 2 type positive serum; Described porcine circovirus 2 type antigen is PCV2/LG strain culture, and malicious valency is 1 × 10
5tCID
50/ ml; Described Swine serum to be measured and contrast, the resisting porcine circovirus 2 type Cap protein monoclonal antibody of enzyme labeling and be respectively 45min, 30min and 20min the action time of nitrite ion; Described enzyme is horseradish peroxidase; Described dilution is the PBST damping fluid containing 1% bovine serum albumin(BSA); Described cleansing solution is PBST damping fluid; Described nitrite ion is ABTS working fluid; Described stop buffer is the NaF solution of 1%.
9. the hybridoma cell strain of a secretion resisting porcine circovirus 2 type Cap protein monoclonal antibody according to claim 1, called after PCV2-Cap-3A5, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCCNO.10205.
10. the monoclonal antibody produced is secreted by hybridoma cell strain according to claim 9.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112595846A (en) * | 2020-12-07 | 2021-04-02 | 新乡学院 | IPMA antibody detection method of PCV2 |
| CN112611865A (en) * | 2020-12-07 | 2021-04-06 | 新乡学院 | Detection method of IPMA (ionic polymer A) neutralizing antibody of PCV2 |
| CN114958776A (en) * | 2022-06-30 | 2022-08-30 | 天康制药(苏州)有限公司 | PCV2 monoclonal antibody and hybridoma cell strain 1A6 secreting same |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009000459A1 (en) * | 2007-06-22 | 2008-12-31 | Ghent University | Methods and compositions in the treatment of porcine circoviral infection |
| WO2009058285A2 (en) * | 2007-10-29 | 2009-05-07 | Virginia Tech Intellectual Properties, Inc. | PORCINE DC-SIGN, ICAM-3 AND LSECtin AND USES THEREOF |
| CN101768218A (en) * | 2010-03-02 | 2010-07-07 | 福建省农业科学院畜牧兽医研究所 | Preparation method for PCV-II Cap protein monoclonal antibody, antibody and application |
| WO2012083837A1 (en) * | 2010-12-22 | 2012-06-28 | 福又达生物科技股份有限公司 | Porcine circovirus type 2, immune composition containing the same, assay kit, and use thereof |
| CN103033622A (en) * | 2011-12-26 | 2013-04-10 | 武汉中博生物股份有限公司 | PCV2 (porcine circovirus 2) ELISA (enzyme-linked immuno sorbent assay) antigen detection kit as well as preparation method and applications thereof |
| CN103695376A (en) * | 2013-11-13 | 2014-04-02 | 南京农业大学 | Hybridoma cell strain secreting porcine circovirus type 2 Cap protein monoclonal antibody |
| CN104749361A (en) * | 2015-01-21 | 2015-07-01 | 中国农业科学院哈尔滨兽医研究所 | Porcine circovirus type 2 antigen capture ELISA kit |
| CN105223359A (en) * | 2015-04-13 | 2016-01-06 | 中国农业科学院哈尔滨兽医研究所 | Porcine circovirus 2 type competitive ELISA antibody assay kit |
-
2015
- 2015-11-10 CN CN201510762194.3A patent/CN105445457B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009000459A1 (en) * | 2007-06-22 | 2008-12-31 | Ghent University | Methods and compositions in the treatment of porcine circoviral infection |
| WO2009058285A2 (en) * | 2007-10-29 | 2009-05-07 | Virginia Tech Intellectual Properties, Inc. | PORCINE DC-SIGN, ICAM-3 AND LSECtin AND USES THEREOF |
| CN101768218A (en) * | 2010-03-02 | 2010-07-07 | 福建省农业科学院畜牧兽医研究所 | Preparation method for PCV-II Cap protein monoclonal antibody, antibody and application |
| WO2012083837A1 (en) * | 2010-12-22 | 2012-06-28 | 福又达生物科技股份有限公司 | Porcine circovirus type 2, immune composition containing the same, assay kit, and use thereof |
| CN103033622A (en) * | 2011-12-26 | 2013-04-10 | 武汉中博生物股份有限公司 | PCV2 (porcine circovirus 2) ELISA (enzyme-linked immuno sorbent assay) antigen detection kit as well as preparation method and applications thereof |
| CN103695376A (en) * | 2013-11-13 | 2014-04-02 | 南京农业大学 | Hybridoma cell strain secreting porcine circovirus type 2 Cap protein monoclonal antibody |
| CN104749361A (en) * | 2015-01-21 | 2015-07-01 | 中国农业科学院哈尔滨兽医研究所 | Porcine circovirus type 2 antigen capture ELISA kit |
| CN105223359A (en) * | 2015-04-13 | 2016-01-06 | 中国农业科学院哈尔滨兽医研究所 | Porcine circovirus 2 type competitive ELISA antibody assay kit |
Non-Patent Citations (4)
| Title |
|---|
| DIPONGKOR SAHA ET AL: "Antigenic subtyping and epitopes’ competition analysis of porcine circovirus type 2 using monoclonal antibodies", 《VETERINARY MICROBIOLOGY》 * |
| IAN W. WALKER ET AL: "Development and Application of a Competitive Enzyme-Linked Immunosorbent Assay for the Detection of Serum Antibodies to Porcine Circovirus Type 2", 《JOURNAL OF VETERINARY DIAGNOSTIC INVESTIGATION》 * |
| 刘长明等: "猪圆环病毒2型细胞培养适应毒株的培育和鉴定", 《中国预防兽医学报》 * |
| 黄立平等: "抗猪圆环病毒2型Cap蛋白中和性单克隆抗体的制备及鉴定", 《中国预防兽医学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112595846A (en) * | 2020-12-07 | 2021-04-02 | 新乡学院 | IPMA antibody detection method of PCV2 |
| CN112611865A (en) * | 2020-12-07 | 2021-04-06 | 新乡学院 | Detection method of IPMA (ionic polymer A) neutralizing antibody of PCV2 |
| CN114958776A (en) * | 2022-06-30 | 2022-08-30 | 天康制药(苏州)有限公司 | PCV2 monoclonal antibody and hybridoma cell strain 1A6 secreting same |
| CN114958776B (en) * | 2022-06-30 | 2024-02-06 | 天康制药股份有限公司 | PCV2 monoclonal antibody and hybridoma cell strain 1A6 secreting same |
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