CN102465116B - Latex agglutination detection method for porcine circovirus type 2 and application thereof - Google Patents
Latex agglutination detection method for porcine circovirus type 2 and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of livestock immunodetection, relating to the technical field of immunochemistry, in particular to a latex agglutination reaction method for porcine circovirus type 2 and application thereof. According to the invention, the latex agglutination detection method for the porcine circovirus type 2 is established by utilizing the electrostatic adsorption function of sulfonic groups on surfaces of terpolymer latex particles to couple screened monoclonal antibodies to the surfaces of the latex particles so as to support sensitizing latex to serve as a diagnostic reagent. The method disclosed by the invention has the outstanding advantages of simplicity in sample processing, high detection speed, strong specificity, high sensitivity, visibility, convenience for popularization and the like. The monoclonal antibody prepared in the invention is preserved in the China Center for Type Culture Collection, with the collection number of CCTCC NO:C201043.
Description
Technical field
The invention belongs to livestock immunological technique field, relate to the association areas such as immunology and immunochemistry.The present invention relates to concretely a kind of porcine circovirus 2 type latex agglutination detection method and application.
Background technology
Porcine circovirus desease be a kind of receive much attention in recent years in pandemic disease all over the world, be the general name of a series of diseases of being caused by porcine circovirus 2 type.Find first pig circular ring virus (Porcine circovirus from Tischer in 1974 etc., PCV) since, along with to the going deep into of PCV research, it can be divided into again pig circular ring virus 1 type (PCV1) of no pathogenicity and pathogenic porcine circovirus 2 type (PCV2) is arranged according to the pathogenic and genome difference of PCV.PCV1 is to the pig no pathogenicity, but can produce serum antibody, ubiquity in swinery.PCV2 can cause the symptoms such as pmws, pig interstitial pneumonia, the scorching nephrotic syndrome of pigskin and sow breeding difficulty.PCV2 can cause sow to return the feelings rate increasing, and stillborn foetus, mummy tire and weakon etc. are produced in miscarriage, the institute's pig Preweaning death rate of farrowing rising.After boar infects PCV2, can by mating infect to join sow, thereby cause its breeding difficulty.In addition, PCV2 can cause the immunizing power of infected pigs greatly to reduce, thereby has created condition for the invasion of other viruses or germ.Such as PCV and reproductive and respiratory syndrome virus synergy, when making pig secondary infection gold-coloured staphylococci, suis, thereby make the disease pig symptoms such as fervescence, congested fash, vomiting, diarrhoea, expiratory dyspnea occur.PCV2 has caused great financial loss to the development of pig industry, and therefore, the detection method of research PCV2 all has great theoretical and practical significance for early diagnosis, control, prevention, treatment and the minimizing financial loss of these diseases.Ubiquity PCV antibody in the swinery, and single antibody positive can not be diagnosed as the positive, makes a definite diagnosis to rely on laboratory method to detect PCV2 antigen or nucleic acid.Because the virus replication difficulty, so detection and prevention and control research are very difficult.Simultaneously, this disease easily and bacteriosis viral with some is mixed or secondary infection, therefore only is difficult to make accurate judgement with symptom, pathology and epidemiology survey.For this reason, veterinary work person and scientist have carried out a large amount of research both at home and abroad, virusology, immunology and the diagnosis of molecular biology method of multiple high degree of specificity have been set up, but up to now, although the method for multiple detection PCV is arranged, but the whole bag of tricks has its limitation, and detected result and judgement thereof often are subjected to the impact of many factors.As: viral isolation technique, because cytopathy does not appear in virus on culturing cell, the result can appear in 3 talentes after the pathological material of disease inoculation usually; By immunohistochemistry staining method (IHC), polymerase chain reaction (PCR) or electron microscope observation, confirm viral separation case, virus is separated wastes time and energy, and is unsuitable for disease detection; Immunohistochemical Method (IHC) operates more loaded down with trivial detailsly, and the time is longer, and must remove endogenous peroxidase to the interference of reaction in experimentation; In situ hybridization (ISH), susceptibility is high, can accurately locate the position of PCV viral DNA in cell, and for the pathogenesis of further furtheing investigate PCV provides help, but shortcoming is to waste time and energy, and is non-specific more intense; Indirect immunofluorescence experiment (IFA), the method is widely used in the antibody test of PCV, external existing stdn test kit comes out by detecting the antiviral antibody in the porcine blood serum, determines the PCV infection conditions, but this method can not effectively to distinguish antibody be for PCV1 or PCV2; Immunoperoxidase monolayer cell experiment (IPMA), the method can not be distinguished two kinds of serotypes of PCV, uses comparatively limitation, the domestic the method for seldom using; Enzyme-linked immunosorbent assay (ELISA) although, antigen capture ELISA etc. attracted recently very much, some advantage in some aspects, but still can not generally use relevant for the reason of the aspects such as instrument, expense.Also can't benefit in many ways in addition the development of the pig industry of the world and China owing to there are some defectives.So be badly in need of now a kind of sensitivity, fast, low-cost, pig itself is free from side effects and the detection method that can also effectively be popularized.Latex agglutination test (LAT) owing to its fast, simple and need not special technical ability and any plant and instrument has huge advantage.
Since doing the material of agglutination test since the middle of last century polystyrene latex particle, because its particular advantages that has, as: easy and simple to handle, do not need specific apparatus, naked eyes are judged, are not also needed operating process is giveed training; Time is short, generally can go out the result in 2 minutes; Cheap, detect the single sera sample than cheap many of other serology, etiology method; Be applicable to Site Detection etc., in Clinical Laboratory, be widely used.Traditional latex agglutination test is to make carrier with inertia polystyrene latex particle, adsorbs specific antigen or antibody, when running into corresponding antibody and antigenic component, can form macroscopic agglutinating particle.When utilizing the latex agglutination technology to set up the etiology detection method, generally be to the latex particle surface with specific antibody sensitization, the combination of ordinary polystyrene latex and antibody is the physics electrostatic adhesion of non-selectivity, because the molecular weight of antibody is little, be difficult to be attached to the latex surface, even the antibody on the sensitization also comes off or deactivation from latex particle easily, cause the good latex reagent preservation period of sensitization short, be difficult to be fit to the needs of product development.For this reason, the applicant take with sulfonic ter-polymers latex particle as carrier, with antibody coupling to latex surface.When method of the present invention has overcome the generic latex sensitizing antibody, sensitizing dose is few, unstable, the shortcomings such as antibody comes off easily, improved the joint efficiency of antibody, and the antibody on the sensitization is not easy to come off from latex, and then improves susceptibility and the quality guaranteed period of latex, has been fit to the requirement of product development and scale operation.
Summary of the invention
The object of the invention is to overcome the defective of prior art, set up a kind of latex agglutination method and application of rapid detection porcine circovirus 2 type.
(the general technical route map is as shown in Figure 1) that the present invention is achieved through the following technical solutions:
The applicant uses porcine circovirus 2 type WH strain, immune mouse obtains immune serum, from this immune serum, extract monoclonal antibody (the hybridoma cell strain PCV2/3E5 of carrying monoclonal antibody, be deposited in Chinese Typical Representative culture collection center (CCTCC) in the Wuhan University of Wuhan City, Hubei Province on April 29th, 2010, its deposit number is CCTCC NO:C201043), obtain the latex diagnostic reagent with the quick latex of this monoclonal anti system, and set up a kind of latex agglutination method for the rapid detection porcine circovirus 2 type take this latex diagnostic reagent as core reagent.
The invention provides a kind of quick latex agglutination detection method of porcine circovirus 2 type, it comprises preparation latex solution, latex diagnostic reagent, positive control sample, negative control sample, it is characterized in that it is prepared by the following step:
A, obtain immune serum with the porcine circovirus 2 type immune mouse, from this immune serum, extract and obtain the monoclonal antibody that preserving number is CCTCC-C201043, obtain described latex diagnostic reagent with the quick latex of this monoclonal anti system; With
The preparation of B, latex solution is obtained by the following step:
1) gets 14.1mL vinylbenzene, 0.78mL methyl methacrylate and 100mL deionized water in the 250mL there-necked flask, mix and blend, logical nitrogen protection;
When 2) treating that fluid temperature rises to 70 ℃ in the bottle, mixed liquor A was added in the bottle fast after 70 ℃ of lower polymerization 2-4 hours, add mixed liquid B, repolymerization 4-6 hour, obtain the homogeneous latex;
3) it is centrifugal under the condition of 10000r/min to get the latex of 1mL, removes supernatant liquor; In precipitation, add pH and be 7.4 phosphoric acid buffer 1mL, obtain uniform latex solution;
Wherein
Step 3) described phosphoric acid buffer fluid component is: sodium-chlor 8g, and dipotassium hydrogen phosphate 0.2g, Sodium phosphate dibasic 1.15g,
Repone K 0.2g replenishes deionized water to 1000mL, pH7.4;
Step 2) described mixed liquor A component is: bicarbonate of ammonia 0.506g, and methacrylic acid propyl sulfonic acid potassium 0.1-0.4g, ammonium thiosulfate 0.73g adds deionized water 10mL;
Step 2) the mixed liquid B component is: vinylbenzene 2.81mL, methyl methacrylate 0.6mL methacrylic acid propyl group sulphur
Acid potassium 0.5g, bicarbonate of ammonia 0.1g adds deionized water 10mL;
Described positive is the porcine circovirus 2 type WH of deactivation, and described negative sample is not for containing the porcine blood serum of porcine circovirus 2 type.
The concrete steps that the present invention uses are:
1) in the latex solution of above-mentioned gained, add 10 μ l preserving numbers be CCTCC-C201043 the secreted monoclonal antibody of hybridoma cell strain PCV2/3E5, with this latex-2 times of volumes of monoclonal antibody mixing solutions dilution, place 25 ℃ of thermostat containers to place 70min, obtain the latex diagnostic reagent, for subsequent use;
2) pig blood that collection is obtained is deposited 1 hour in 37 ℃, and is centrifugal in 5000r/min, obtains porcine blood serum to be checked;
3) on clean slide glass, drip respectively 10 μ l porcine blood serum, positive and negative sample to be checked, in described porcine blood serum, positive, negative sample, drip the described latex diagnostic reagent of 10 μ l again, stir, shake observations after 30 seconds.
4) result judges: occur following result in 30 seconds, test can be set up: the aggegation of obvious uniformity appears in positive control and the effect of latex antibody complex, negative control and the not aggegation of latex diagnostic reagent, uniformity appears in serum to be checked in 30 seconds aggegation is judged to be the positive, presents original even emulsus and is judged to be feminine gender.
The method can directly detect the porcine circovirus 2 type in the sample.If contain porcine circovirus 2 type in the sample, then the aggegation of obvious uniformity appearred in latex reagent in 30 seconds, was judged to positive findings; If do not contain porcine circovirus 2 type in the sample, then latex reagent drop in 30 seconds still is original even emulsus, is judged to negative findings.
Compared with prior art, the present invention has following advantage:
Directly porcine circovirus 2 type is detected, have high specificity, highly sensitive, the characteristics of detection time short (going out the result within the 1min);
2. detection method of the present invention is conducive to popularize without any need for special instrument, equipment, and has the low characteristics of testing cost.
Description of drawings
Fig. 1: be general technical route map of the present invention.
Fig. 2: the electromicroscopic photograph (* 100,000) that is the present invention's porcine circovirus 2 type negative staining virus particle of separating, identifying.
Fig. 3: be negative, positive control sample detected result photo.
Embodiment
The preparation of embodiment 1 monoclonal antibody and latex solution
The preparation of 1 monoclonal antibody
1) antigen preparation
The recombinant salmonella (Chen Donghuan that contains porcine circovirus 2 type open reading frame 2 (PCV2-ORF2) gene with the LB solid plate recovery that contains 50 μ g/ml penbritins (being called for short Amp), express structure and the evaluation [D] of pig 2 type PCV-II Cap Protein reconstitution Salmonellass. the .2009 of Hua Zhong Agriculture University, http://epub.cnki.net/grid2008/detail.aspx? QueryID=4﹠amp; CurRec=1) picking list colony inoculation contains in the antibiotic LB substratum of 50 μ g/ml Amp, in 37 times about 12h of shaking culture in 5mL.Be that dilution in 1: 100 is inoculated in fresh containing in the antibiotic LB substratum of 50 μ g/ml Amp by volume with bacterium liquid, when 30 ℃ of lower low temperature are cultured to OD600 and reach 0.5-2.0, adding final concentration is the 2mmol/L isopropyl-β-D-thiogalactoside(IPTG), 30 ℃ of low temperature induction 6h; 4 ℃ of centrifugal 10min of 8000r/min, the collection thalline also places on ice, (its composition is: sodium-chlor 8g, dipotassium hydrogen phosphate 0.2g, Sodium phosphate dibasic 1.15g, Repone K 0.2g to add 1 * phosphoric acid buffer by the amount of every milliliter of substratum 50 μ L, add deionized water 1000mL, be phosphoric acid buffer, pH is 7.4) resuspended thalline, with ultrasonic disruption (operating frequency: 18KHz); 4 ℃ of centrifugal 10min of 12000r/min abandon precipitation, with the glutathione agarose gel-purified of soluble proteins with 50% concentration.Add this suspension by the amount that adds 2mL50% glutathione agarose gel suspension in the 100mL ultrasonic disruption lysate, shake gently 30min under the room temperature, the sweet peptide S of Guang transferring enzyme tag fusion protein fully is attached on the glutathione agarose gel; Centrifugal 5min abandons supernatant under 500 * g; Amount by 10 * sepharose volume adds 1 * PBS damping fluid detergent gel, and centrifugal 5min abandons supernatant under 500 * g, and repeats this washing process 3-4 time, with the foreign protein of removal with glutathione agarose gel non-specific binding; The amount that adds 1mL by every milliliter of gel volume adds elutriant (prescription: the 0.154g reductive glutathione is dissolved among the 50mL 50mmol/L Tris-HCl (pH 8.0) and is elutriant) wash-out, mixing gently, make gel resuspended, under room temperature, act on 10min, fusion rotein is eluted from the glutathione agarose gel, centrifugal 5min under 500 * g collects supernatant again.Repeat above-mentioned elution process more than 2 times, fusion rotein is fully eluted, and pass through wash-out situation and the purity of SDS-PAGE electrophoresis detection fusion rotein.
2) mouse immune
With step 1) purifying protein that obtains is diluted to 300 μ g/mL with phosphoric acid buffer, with the emulsification of Freund's complete adjuvant equal-volume mixing, select 6 week female BALB/c mouse in age (available from Disease Prevention Control Center, Hubei Prov) 5, the subcutaneous multi-point injection 0.2mL of nape section.Booster immunization 1 time (adjuvant is used Freund's incomplete adjuvant instead, the same first immunisation in immunizing dose and position) during 14d.Tail vein blood when 24d, separation of serum, with the indirect elisa method (Master degree candidate of Hua Zhong Agriculture University paper, Chen Donghuan, express structure and the evaluation [D] of pig 2 type PCV-II Cap Protein reconstitution Salmonellass. the .2009 of Hua Zhong Agriculture University) the detection antibody horizontal, when 42d, to the best BALB/c mouse of immune effect with the antigen 0.2mL tail vein injection booster immunization that does not add adjuvant once, can merge behind the 3d.
3) cytogamy
(1) with SP2/0 myeloma cell and immune spleen cell take volume ratio as 1: 10 ratio mixing in the 50mL centrifuge tube, the centrifugal 10min of 1500r/min.
(2) the turned letter supernatant blots with the filter paper of sterilizing, and knocks gently the pipe end, makes cell precipitation slightly loosening.
The centrifuge tube that (3) cell mixture will be housed is put in 37 ℃ of water-baths.Then slowly splash into the last Macrogol 4000 0.8mL of 37 ℃ of pre-temperature in 1min, the limit edged stirs gently.
(4) continue stirring and leave standstill 1min after 30 seconds.Then 10mL 1640 basic mediums (available from U.S. GBICO company) that slowly add 37 ℃ of pre-temperature.Concrete grammar is: respectively added 1mL in the 1st minute and the 2nd minute, 3-4min adds 3mL, adds remaining 5mL on the 5th minute, needs slowly to add at every turn, and constantly stirs lightly.
(5) in the centrifugal 5min of 1000r/min, supernatant discarded.
(6) add 30mL 1640 basic medium re-suspended cells precipitation, in the centrifugal 5min of 1000r/min, supernatant discarded.
(7) use 10mL HAT substratum (available from U.S. GBICO company.) cell after suspend merging, suspend with 90mL HAT substratum simultaneously and raise splenocyte, after the two is mixed, drip in 96 porocyte culture plates about 250 μ L/ holes.Place 37 ℃, cultivate in the 5%CO2 incubator.
(8) add 1 HAT substratum in 3d after the fusion, suck 96 orifice plates, 100 μ L substratum during 5d and change HT substratum 100 μ L.When treating that the fused cell colony grows to culture hole 1/4, can carry out antibody test.
4) monoclonal antibody production
Get the BALB/c mouse in 8 ages in week, abdominal injection Freund's incomplete adjuvant 0.5mL/, 7d pneumoretroperitoneum injection hybridoma (hybridoma cell strain PCV2/3E5, deposit number are CCTCC NO:C201043) 5 * 10
5-5 * 10
6/ only, gather mouse ascites behind the 7-10d, get supernatant in the centrifugal 10min of 1200r/min, frozen for subsequent use.
5) purifying of Monoclonal Antibodies in Mice Ascites
Adopt ascites caprylic acid-ammonium purifying.Concrete steps are as follows:
(1) with step 4) mouse ascites and the centrifugal 10min of cell 1200r/min of gained, get supernatant in 4 ℃ of centrifugal 30min of 12000r/min, discard impurity.
(2) add the acetic acid solution of 2 times of volume 0.06mol/L (pH 5.0) in every milliliter of ascites of step (1).
(3) then be added dropwise to gradually sad (every milliliter of ascites adds 33 μ L), stirring at room 30min, the centrifugal 30min of 12000r/min abandons precipitation.
(4) supernatant is transferred pH to 7.4 behind filter paper filtering.
(5) slowly adding pH under 4 ℃ of ice baths is 7.4 saturated ammonium sulphate, makes the final saturation ratio 50% of ammonium sulfate, stirs 30min, and 4 ℃ are spent the night.
(6) 4 ℃ of centrifugal 20min of lower 12000r/min, supernatant discarded.
(7) precipitation is resuspended with the PBS damping fluid (pH 7.4) of 0.01mol/L, and the 3d that dialyses in pH is 7.4 PBS damping fluid is frozen for subsequent use.
The preparation of 2 latex solutions
1) gets 14.1ml vinylbenzene, 0.78ml methyl methacrylate, 100ml deionized water in the 250ml there-necked flask, mix and blend, logical nitrogen protection.
When 2) treating that fluid temperature rises to 70 ℃ in the bottle, mixed liquor A (is contained following composition: bicarbonate of ammonia 0.506g, methacrylic acid propyl sulfonic acid potassium 0.1-0.4g, ammonium thiosulfate 0.73g, add deionized water 10ml, mix this composition and be mixed liquor A), add fast in the bottle with 70 ℃ of lower polymerization 2-4 hours after, add again mixed liquid B and (contain following composition: vinylbenzene 2.81ml, methyl methacrylate 0.6ml methacrylic acid propyl sulfonic acid potassium 0.5g, bicarbonate of ammonia 0.1g, add deionized water 10ml, be mixed liquid B), repolymerization 4-6 hour, obtain the homogeneous latex.
3) get 1ml step 2) latex centrifugal under the condition of 10000r/min, remove supernatant liquor; In precipitation, add the phosphoric acid buffer 1ml (its composition is: sodium-chlor 8g, dipotassium hydrogen phosphate 0.2g, Sodium phosphate dibasic 1.15g, Repone K 0.2g add deionized water to 1000mL, are phosphoric acid buffer) of pH=7.4, obtain uniform latex solution.
Adding 10 μ l preserving numbers in above-mentioned latex solution is the secreted monoclonal antibody of hybridoma cell strain PCV2/3E5 of CCTCC-C201043, and with this latex-2 times of volumes of monoclonal antibody mixing solutions dilution, place 25 ℃ of thermostat containers to place 70min, be latex diagnostic reagent of the present invention.
The foundation of embodiment 2 porcine circovirus 2 type latex agglutination detection methods
Get the good latex solution of dilution, add the monoclonal antibody that embodiment 1 prepares, under room temperature, place 70min, form the latex diagnostic reagent.
Determining of 1 best coupling antibody amount
In latex solution, add the antibody (doubling dilution 1: 1,1: 2,1: 4,1: 8) of 10 μ L different concns, place 25 ℃ of thermostat containers to place 60min.The latex antibody complex good with sensitization detects porcine circovirus 2 type.Through verification experimental verification, in latex solution, add the antibody of 2 times of dilutions, it is maximum that its coupling efficiency reaches, and detection sensitivity is the highest.Concrete outcome sees Table 1.
Determining of the best coupling antibody of table 1 amount
Annotate: " ++ ++ " whole latex agglutinations, particle aggregation is in drop edge, and liquid is fully transparent;
" +++" about 75% latex agglutination, particle is obvious, and liquid is slightly muddy;
The latex agglutination of " ++ " about 50%, liquid are muddy;
"+" has a little aggegation, and it is muddy that liquid is;
"-" drop is original even emulsus, not aggegation.
The selection of 2 best coupling times
Add the monoclonal antibody that 10 μ L dilute good embodiment in latex solution, the different time of effect is finished rear detection porcine circovirus 2 type in 25 ℃ of thermostat containers.When verification experimental verification is found latex and antibody effect 70min, reach preferably coupling effect, so select 70min as best coupling time.Concrete outcome sees Table 2.
The selection of the best coupling time of table 2
The foundation of 3 porcine circovirus 2 type latex agglutination detection methods
(1) in latex solution, add the monoclonal antibody (diluting 2 times) of 10 μ L embodiment 1, place 25 ℃ of thermostat containers to place 70min, form the latex antibody complex.
(2) pig is carried out venous blood collection, the blood of collection was deposited 1 hour in 37 ℃, and 5000r/min is centrifugal, collected serum.
(3) on clean slide glass, drip respectively 10 μ L serum to be checked, positive serum sample and (contain the porcine circovirus 2 type WH strain of deactivation (referring to (Chen Donghuan, express structure and the evaluation [D] of pig 2 type PCV-II Cap Protein reconstitution Salmonellass. the .2009 of Hua Zhong Agriculture University
Http:// epub.cnki.net/grid2008/detail.aspx? QueryID=4﹠amp; CurRec=1, the gene order of porcine circovirus 2 type WH strain number is: FJ598044), negative serum sample (not containing porcine circovirus type 2 strain), respectively add 10 μ L latex antibody complexes, stir, shake observations after 30 seconds.
(4) result judges: occur following result in 30 seconds, test can be set up: the aggegation of obvious uniformity appears in positive serum sample and the effect of latex diagnostic reagent, negative serum sample and the not aggegation of latex diagnostic reagent.Uniformity appears in serum to be checked in 30 seconds aggegation is judged to the positive, and it is negative to present original even emulsus.Detected result as shown in Figure 3.
Sensitivity test and the specific test of embodiment 3 latex agglutination detection methods
1 sensitivity test
With the porcine circovirus 2 type doubling dilution, detect with the good latex diagnostic reagent of sensitization respectively, discovery can react with the virus of dilution in 1: 128, reaches preferably susceptibility.Detected result sees Table 3.
Table 3 sensitivity experiments
2 specific tests
The latex antibody diagnosing reagent that sensitization is good and porcine circovirus 2 type reaction are strong positive, with other viruses of pig as: pig breeding is not all reacted with respiratory complication virus (PRRSV), pig parvoviral (PPV), transmissible gastro-enteritis virus (TGEV), negative serum, has reached preferably specificity.The results are shown in Table 4
The experiment of table 4 specificity
The comparison that embodiment 4 latex agglutination detection methods separate with virus
Detect altogether 27 parts of clinical samples, compare with Virus Isolation, both coincidence rates are 96%.Concrete outcome sees Table 5.
The comparative result that table 5 latex agglutination detection method separates with virus
The application method of embodiment 5 porcine circovirus 2 type latex agglutination detection methods in clinical
The preparation of 1 sample
Pig (kind: Large White, a kind of market pig of public offering) is carried out venous blood collection, and the blood of collection was deposited 1 hour in 37 ℃, and 5000r/min is centrifugal, and it is to be checked to collect serum.
2 detect
Get serum to be checked, positive, each 10 μ L of negative sample, place respectively on the sheet glass, respectively add 10 μ L latex antibody complexes, stir, shake observations after 30 seconds.
3 results judge
(1) controlled trial: occur following result in 30 seconds, test can be set up: the aggegation of obvious uniformity appears in positive control and the effect of latex antibody complex, negative control and the not aggegation of latex antibody complex.
The aggegation that occurs uniformity in (2) 30 seconds is judged to the positive, presents original even emulsus negative (the results are shown in Figure 3).
Claims (4)
1. hybridoma cell strain PCV2/3E5, it is deposited in Chinese Typical Representative culture collection center, and deposit number is CCTCCNO:C201043.
2. one kind is the monoclonal antibody that can identify specially porcine circovirus 2 type of the hybridoma cell strain secretion of CCTCC-C201043 by deposit number.
3. the latex diagnostic reagent that comprises monoclonal antibody claimed in claim 2, described monoclonal antibody are to be the hybridoma cell strain secretion of CCTCC-C201043 by preserving number.
4. the application of monoclonal antibody claimed in claim 2 in the latex agglutination assay kit of preparation porcine circovirus 2 type.
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| CN103529204B (en) * | 2013-10-22 | 2016-04-27 | 武汉市畜牧兽医科学研究所 | Detect the latex agglutination kit of milk cow Streptococcusagalactiae antibody |
| CN104407145B (en) * | 2014-10-28 | 2016-06-01 | 绍兴市疾病预防控制中心 | Enterovirns type 71 latex agglutination assay kit preparations and applicatio |
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| CN101025420A (en) * | 2005-09-29 | 2007-08-29 | 华中农业大学 | Avian influenza virus latex agglutination assay kit and its use |
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