CN101833005B - Competitive ELISA (Enzyme-Linked Immuno Sorbent Assay) kit for detecting antibody of African swine fever virus and application thereof - Google Patents
Competitive ELISA (Enzyme-Linked Immuno Sorbent Assay) kit for detecting antibody of African swine fever virus and application thereof Download PDFInfo
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Abstract
The invention discloses a competitive ELISA (Enzyme-Linked Immuno Sorbent Assay)kit for detecting an antibody of an African swine fever virus and application thereof, belonging to the technical field of organisms. The kit is used for detecting an antibody of the African swine fever virus in pig serum by adopting prokaryotically expressed recombinant P54 protein as an envelope antigen according to a competitive ELISA principle. The envelope antigen in a 96 pore plate in the kit is prokaryotically expressed recombinant P54 protein and has favorable antigenicity. The enzyme-linked immuno kit provided by the invention comprises the P54 protein enveloped 96 pore plate, positive control, negative control, a horseradish peroxidase marked monoclonal antibody, a concentrated cleaning solution, serum diluent, a TMB substrate and a stopping solution. The kit can be used for screening samples in bulk, main reagents in the kit are provided in a working solution way, and the use is convenient.
Description
Technical field
The present invention belongs to biological technical field for detecting African swine fever virus (ASFV) antibody assay kit.Particularly, the present invention relates in a kind of animal quarantine ELISA kit of detecting antibody and uses thereof.
Background technology
African swine fever (African swine fever, ASF) is a kind of acute, hot, the strong communicable disease of contact highly of the pig that caused by African swine fever virus (ASFV).It is characterized by that the course of disease is short, the case fatality rate high rate, can be up to 100%, clinical symptoms and pathological change all are similar to acute swine fever, very easily mistaken diagnosis when diagnosis, the high heat of performance, dermohemia cyanosis, miscarriage, oedema and internal organs are hemorrhage.(William, Hess Adv.African swine fever:a reassessment[J] .Vet Sci Comp Med, 1981,25:39～69) world animal tissue (OIE) classifies the category-A epidemic disease as, China is defined as animal one class disease, be subject to countries in the world great attention (Sun Huaichang. Chinese Preventive Veterinary Medicine newspaper, 1999,21 (2): 117～119).
This disease from nineteen twenty-one since Kenya finds, be present in the African country on the south the Sahara always, nineteen fifty-seven successively spreads to West Europe and Latin American countries, majority is in time put out, singly in Portugal, the Spain west and south and gondola Sardinia still have popular, so far in Africa, Europe and America etc. dozens of country popular, and continuous spreading trend is arranged.2007, Armenia recurred six African swine fevers, and China there is no this disease.
African swine fever is attributed to Iridoviridae in the 4th report of ICTV, in the 5th report of this council, it is listed under Poxviridae, is placed in outside the Chordopoxvirinae and Entomopoxvirinae of this section.But DNA sequence analysis shows, ASF virus has the feature between poxvirus and irido virus, this characteristic of ASFV shows that it does not belong to any section that ICTV appraises and decides, individual new, the 6th report of nineteen ninety-five the 9th international virus taxis committee member, African swine fever virus is listed in " class African swine fever virus genus ", African swine fever is unique known representative species.
African swine fever virus is a kind of large, double-stranded DNA virus that cyst membrane is arranged, is unique entomophila DNA virus.Its genome is the covalence closed unimolecule wire double-stranded DNA of end, the viral genome total length is 170kb～190kb, there is the conserved region of 125kb left and right in central authorities, two ends are the variable region, contain the end Inverted repeat, the increase of these repetitive sequences or disappearance are to cause the main cause (Rafacl of different isolates genome difference in length, Yancz, Javier M, et al.Analysis of the completeNucleotide Sequence of Afican Swine Fever Virus[J] .Virology, 1995,208:249～279).The ASF viral genome has 5 encoding genes, comprise putative membrane protein, secreted protein, participation nucleic acid and nucleic acid metabolism (DNA reparation) and protein modified enzyme, whole genome contains 151 ORF, 150～200 kinds of protein of can encoding have separated identifying 86 kinds of virus protein polypeptide (Qu Liandong, Yu Kangzhen from the cell that ASFV infects, the African swine fever progress, China animal doctor science and technology, 1998,28 (11): 42～43).
The virulence of the most of strains of African swine fever virus is all very strong, but immunogenicity is very low, only has a few albumen to have immunogenicity, and wherein P54 albumen is for having one of better antigenic albumen.
ASFV P54 albumen is by the E183L gene code, and the about polypeptide of 25kD contains one section cross-film zone, mainly concentrates on derivative endoplasmic reticulum place.P54 albumen can be in vitro culture, and can infection cell.And after infection cell at endoplasmic reticulum place transient expression.The cross-film structure of P54 albumen plays a very important role (RodriguezJM when endoplasmic reticulum changes into the peplos precursor at virus protein, Garcia Escudero African Swine Fever Virus Structural Protein p54Is Essential for the Recruitment of Envelope Precursors to AssemblySites.Journal of Virology, 2004,78 (8): 4299～4313).There is special cross reaction in the light chain tenuigenin dynein DLC8 of ASFVP54 albumen and 8kD in addition, and plays an important role in endocytosis and viral process.Copy in early days after infecting virus, but virus active cell apoptotic proteins enzyme and bring out Apoptosis effect (Alonso C, J Miskin AfricanSwine Fever Virus Protein p54Interacts with the Microtubular MotorComplex through Direct Binding to Light-Chain Dynein.Journal ofVirology 2001,75:9819～9827).For analytical structure albumen P54 role in Apoptosis, Hernaez B in 2004 and Diaz-Gil G are with its transient expression in African green monkey kidney cell, but experiment shows active cell apoptotic proteins enzyme, bring out Apoptosis effect (HernaezB, Diaz Gil G.The African swine fever virus dynein-binding proteinp54induces infected cell apoptosis.FEBS Letters 2004,569 (123): 224～228).But lacking 13aa, the mutant of P54 loses function (the Alejo A of active cell apoptotic proteins enzyme, GAndr é s, ML Salas.African Swine Fever Virus Proteinase Is Essential for Core Maturation and Infectivity Journalof Virology, 2003,77:5571～557).
Summary of the invention
The objective of the invention is to develop a kind of competitive ELISA kit that detects African swine fever virus antibody take the recombinant protein of prokaryotic expression as the basis by Enzyme-multiplied immune technique.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is: a kind of competitive ELISA kit for detection of African swine fever virus antibody, described kit comprise by preserving number being the enzyme labeling thing of the anti-African swine fever virus monoclonal antibody of CGMCC NO.3750 hybridoma cell line secretion.
Also comprise elisa plate bar, serum dilution and concentrated cleaning solution, substrate nitrite ion, stop buffer, positive control, negative control.
Above-mentioned competitive ELISA kit for detection of African swine fever virus antibody comprises:
Described elisa plate bar: each kit is equipped with 2 or 5 ELISA microwell plates, and every elisa plate bar is the ELISA microwell plate of envelope antigen that can realizing self disassembling, and envelope antigen is the P54 albumen of prokaryotic expression, and specification is 8 holes * 12;
Described serum dilution and concentrated cleaning solution: serum dilution is instant, and cleansing solution is 25 times and concentrates, dilution before using;
Described substrate nitrite ion: instant TMB nitrite ion, 50mL;
The H of described stop buffer: 2mol/L
2SO
4Solution, 50mL;
Described monoclonal antibody linked with peroxidase: the mouse-anti P54 monoclonal antibody of described horseradish peroxidase-labeled, use front 100 times of dilutions;
Positive control;
Negative control.
The application of described competitive ELISA kit in detecting African swine fever virus.
The invention has the beneficial effects as follows: set up a kind of accurately, fast, security is good, can carry out the method that a large amount of examination African swine fever virus antibodies detect, and this kit envelope antigen used is recombinant expression protein, can be in a large number, steady production, for the animal test quarantine departments provides a cover diagnostic kit product practical, reliable for effect.
Description of drawings
Fig. 1 is SDS-PAGE figure after restructuring P54 protein purification.
Fig. 2 is western blotting figure after restructuring P54 protein purification.
Fig. 3 is for adopting ELISA kit of the present invention to detect sample African swine fever antibody lab diagram.
Embodiment
The hybridoma cell line of anti-African swine fever virus monoclonal antibody is preserved in the common micro-organisms center C GMCC NO.3750 of China Committee for Culture Collection of Microorganisms, preservation day: 2010-04-22, Classification And Nomenclature: hybridoma cell strain.
Below in conjunction with embodiment, the present invention is described in further detail:
Technical scheme of the present invention is as follows:
One, set up clone
1. antigen preparation
A) African swine fever P54 protein expression
According to African swine fever in GenBank (ASFV) P54 gene order, 1 pair of primer has been synthesized in design, adopt PCR method to amplify the P54 genetic fragment from ASFV DNA, it is cloned in expression vector pET28b, built recombinant plasmid pET-P54, be transformed into expressive host bacterium BL21 (DE3) after sequence verification, through the IPTG abduction delivering.
The primer of design is:
P54-1-5’-GC
GGATCCCATTATTATCATCGTT-3’
P54-2-5’-AG
CTCGAGCAAGGAGTTTTCTA-3’
Underscore is the restriction enzyme site for introducing partly, and P54-1 is ASFV P54 upstream region of gene amplimer, and the restriction enzyme site of introducing is BamHI; P54-2 is ASFV P54 gene downstream amplimer, and the restriction enzyme site of introducing is XhoI.
B) purifying of African swine fever P54 albumen.
After inducing end, rear thalline is induced in centrifugal collection, with the resuspended thalline of PBS washing, and ultrasonic degradation (ultrasonic 1s, interval 1s, 10min altogether), 12000rpm is centrifugal, collects respectively upper cleer and peaceful precipitation, carries out SDS-PAGE and analyzes.Use the nickel ion affinity chromatograph post during protein purification, after purifying, identify through SDS-PAGE, western blotting, get single band, and possess antigenicity (seeing accompanying drawing 1,2) preferably.Protein content after use BCA method mensuration purifying, standard items concentration and corresponding OD value thereof see Table 1.After purifying, the OD value of P54 albumen is 1.583, with standard items relatively after, its concentration is 900 μ g/ml.
Table 1BCA method bioassay standard product protein content
| The sample title | A (μg/ml) | B (μg/ml) | C (μg/ml) | D (μg/ml) | E (μg/ml) | F (μg/ml) | G (μg/ml) | H (μg/ml) | 0 (μg/ml) |
| Concentration | 2000 | 1500 | 1000 | 750 | 500 | 250 | 125 | 25 | 0 |
| The OD value | 2.6894 | 2.2378 | 1.6493 | 1.3278 | 1.0414 | 0.6454 | 0.4062 | 0.1893 | 0.1251 |
2. antigen immune
Take the African swine fever virus P54 albumen of purifying as antigen, conventional method immunity BALB/c mouse in 6 age in week, the antigen of fundamental immunity hypodermic injection first 200 μ g use complete Freund's adjuvant; Carry out primary immune response every 3 weeks, totally three times, 50 μ g//times, intrasplenic injection booster immunizations after 3 weeks after last fundamental immunity, 25 μ g/, immunity is completed.
3. the preparation of hybridoma
A) preparation of feeder cells
With the BALB/c mouse peritoneal macrophage as feeder cells.Merged front 1 day, and drew neck to put to death mouse, the body surface sterilization and fixing after, cut tweezer with sterilization and start skin of abdomen from venter posterior, the exposure peritonaeum.Sterilize with cotton ball soaked in alcohol wiping peritonaeum., repeatedly rinse to the abdominal cavity with injector to inject 10ml RPMI RPMI-1640, reclaim washing fluid, centrifugal 10 minutes of 1000r/min abandons supernatant.With the resuspended precipitation of RPMI RPMI-1640 that contains 20% hyclone, adjusting cell concentration is 2 * 10
5/ ml.Above-mentioned cell suspension is added 96 orifice plates, and every hole 0.1ml puts 37 ℃, 6%CO
2Incubator in overnight incubation.
B) preparation of immune spleen cell
Get the BALB/c mouse after immunity is completed, by the neck lethal mouse of dislocating, be soaked in 75% alcohol 5 minutes, take out spleen under aseptic condition, in plate, the RPMI RPMI-1640 cleans 1 time.Spleen is moved in another plate that fills 10ml RPMI RPMI-1640, make splenocyte enter nutrient solution.With suction pipe piping and druming for several times.Filter, the results splenocyte suspension, centrifugal 10 minutes of 1000r/min uses RPMI RPMI-1640 centrifuge washing 2 times, and is then that cell is resuspended, and counting is used for Fusion of Cells.
C) myeloma cell
Before merging 48-36 hour, the myeloma cell is enlarged cultivation, with connector bend dropping tube, cell is blown down gently from the bottle wall on fusion same day, be collected in the 50ml centrifuge tube.Centrifugal 10 minutes of 1000r/min, supernatant discarded.Add the incomplete nutrient culture media of 30ml, with the method centrifuge washing once.Then cell is resuspended to the incomplete nutrient culture media of 10ml, mixing.Get myeloma cell's suspension, counting is used for Fusion of Cells.
D) Fusion of Cells and HAT select hybridoma
The myeloma cell is mixed in 1: 10 ratio with immune spleen cell, wash 1 time with the RPMI1640 nutrient solution in the 50ml centrifuge tube, 1200r/min, centrifugal 10min abandons supernatant, with the careful sucking-off residual liquid of dropper.At the bottom of touching fusion pipe on palm, make sedimentation cell loose evenly, put preheating in 40 ℃ of water-baths.Add 50%PEG (PH 8.0) 1ml that is preheated to 40 ℃ in the time of 45 seconds, act on 90 seconds.Add 20ml to be preheated to the RPMI RPMI-1640 of 37 ℃, standing 10 minutes of room temperature.1000r/min, centrifugal 5min, supernatant discarded.Add 5ml HAT nutrient culture media, pressure-vaccum sedimentation cell gently suspends and mixing it, then add contain feeder cells the HAT nutrient culture media to 80ml.Packing 96 porocyte culture plates, then every hole 0.1ml puts culture plate 37 ℃, 6%CO
2Cultivate in incubator.
With HAT nutrient culture media 1/2 nutrient culture media that swaps out, change liquid after 48 hours fully after 24 hours.With the HT nutrient culture media HAT nutrient culture media that swaps out, then after keeping for two weeks, use the RPMI RPMI-1640 instead after two weeks.
4. the screening of hybridoma
Hybridoma carries out the screening of hybridoma after merging for two weeks according to the routine immunization zymotechnic.Filter out the hybridoma hole that to secrete anti-African swine fever virus antibody.
5. the cloning of hybridoma and frozen
A) clone of hybridoma
The cloning scheme is limiting dilution assay, carries out according to conventional hybridization oncocyte cloning process, and the clone carries out three times with method.
B) hybridoma is frozen
Cells frozen storing liquid: 50% calf serum+40%RPMI RPMI-1640+10% dimethyl sulfoxide (DMSO)
Hybridoma is centrifugal, and Eddy diffusion is in the cryopreserving liquid of precooling, and concentration is 106-107/ml, is transferred in cryopreservation tube every bottle of 1ml.Be placed in-70 ℃ of refrigerators, change in liquid nitrogen next day.
The hybridoma cell line of preparation anti-African swine fever virus monoclonal antibody is preserved in the common micro-organisms center C GMCC NO.3750 of China Committee for Culture Collection of Microorganisms, preservation day: 2010-04-22, Classification And Nomenclature: hybridoma cell strain.
Two, monoclonal antibody preparation
In the present invention, the scheme of a large amount of manufacture order clonal antibodies is the mouse ascites method, and step is as follows:
This programme is inoculated in the hybridoma of above-mentioned acquisition in mouse peritoneal, the hybridoma of growing in mouse peritoneal, and produce ascites, obtain a large amount of ascites monoclonal antibodies.
Concrete grammar is: BALB/c mouse intraperitoneal inoculation whiteruss, every mouse 0.5ml.After two weeks, the hybridoma that intraperitoneal inoculation dilutes with serum free medium, every mouse 5 * 10
5/ 0.2ml.Behind interval 5 days, observe the mouse ascites production every day, when the many as far as possible and mouse of ascites is on the verge of death, put to death mouse, under aseptic condition with the ascites sucking-off.The static 30min of room temperature, 1000r/min, centrifugal 10min collects supernatant, packing ,-70 ℃ are frozen standby.
Three, monoclonal antibody purifying and horseradish peroxidase mark
1. the purifying of monoclonal antibody
The purification schemes of the ascites monoclonal antibody of above-mentioned acquisition is sad-ammonium sulfate precipitation method.
Get 1 part of pretreated ascites and add 2 parts of 0.06mol/L PH5.0 acetate buffer solutions, transfer PH to 4.8 with 1mol/LHCl; Add the sad ratio of 11ul in every milliliter of dilution ascites, it is sad dropwise to add under stirring at room, added in 30 minutes, 4 ℃ standing 2 hours, take out the centrifugal 30min of 12000r/min, abandon precipitation; Supernatant filters (125um) through nylon mesh, adds the 0.01mol/L PBS of 1/10 volume, transfers PH to 7.2 with 1mol/L NaOH; Add saturated ammonium sulfate to 45% saturation degree under 4 ℃, mixing 30min gently, standing 1 hour; Centrifugal 30 minutes of 12000r/min abandons supernatant; Precipitation is dissolved in appropriate PBS, and to the PBS dialysis of 50-100 times of volume, 4 ℃ are spent the night; Take out the centrifugal 30min of 12000r/min, remove infusible precipitate, packing, frozen standby.
2. the horseradish peroxidase mark of monoclonal antibody
The preparation scheme of the enzyme labeling thing of the monoclonal antibody after purifying is the sodium periodate method, and concrete steps are as follows:
Taking 5mg HRP is dissolved in 1ml distilled water; The 0.1MNaIO that adds wherein 0.2ml newly to join
4Solution, the room temperature lucifuge stirred 20 minutes; Dialyse in sodium-acetate buffer to 1mM pH4.4,4 ℃ are spent the night; Add wherein the carbonate buffer solution of 20 μ l 0.2M pH9.5 again, make the pH of above hydroformylation thing be elevated to 9.0, then add immediately the monoclonal antibody sterling 5mg (10mg/ml) that is dissolved in the 0.01M carbonate buffer solution, the room temperature lucifuge is soft to be stirred 2 hours; The 4mg/ml NaBH that adds 0.1ml newly to join
4, mixing was placed 2 hours for 4 ℃; Products therefrom is to 4 ℃ of dialysed overnight in 0.15M pH7.4PBS.
The dialysis complete after, dropwise add the equal-volume saturated ammonium sulfate in stirring, put 4 ℃ 1 hour.The centrifugal 30min of 3000r/min abandons supernatant.Sediment is washed secondary with semi-saturation ammonium sulfate, and last sediment is dissolved in the PBS of a small amount of 0.15M pH 7.4.Mentioned solution is packed in bag filter, to the PBS damping fluid dialysis of 0.15M pH7.4, take out ammonium ion, the centrifugal 30min of 10000r/min removes precipitation, and supernatant is enzyme conjugates, and is after packing, frozen.
Four, the preparation of standard A SFV positive serum and standard A SFV negative serum
In the ELISA testing process, can there be certain error between different operating personnel and different detections batch, operate miss can cause the error of test sample OD value.The present invention has developed standard A SFV antibody positive control serum and standard A SFV negative antibody control serum, for detection of the optimization of condition and the judgement of testing result.
A) preparation of standard positive serum
Get the healthy adult rabbit, body weight 2-3kg cuts off the part rabbit hair of two hind paws, uses iodine disinfection skin.Immunity, draw antigen (P54 recombinates after purifying) (hereinafter referred to as FCA-P54) liquid 1ml of not formula Freund's complete adjuvant (FCA) emulsification with syringe for the first time, the subcutaneous 0.5ml that injects of every batter palm.After interval 10-14 days, carry out for the second time immunity, and inject not formula Freunds incomplete adjuvant (FIA-P54) in the lymph node of Liang Ce popliteal nest and groin enlargement, each lymph node 0.1ml, all the other inject near the subcutaneous 1ml altogether lymph nodes.After interval 7-10 days, from ear vein blood sampling 0.5-1.0ml, separation of serum adopts the ELISA method to detect serum titer, and antibody titer can reach more than 1: 100.
Adopt the blood sampling of heart blood collection method, the blood that extracts is injected aseptic Erlenmeyer flask immediately.The blood of Erlenmeyer flask is put 37 ℃ of incubators 1 hour, then put 4 ℃ of refrigerators 3 hours.After the blood clotting clot contraction, draw serum with dropper, the centrifugal 15min of 3000r/min gets supernatant, and adding final concentration is that 0.01% thimerosal is anticorrosion, after packing ,-20 ℃ of preservations.
B) preparation of standard female serum
The SPF rabbit of learning from else's experience and being up to the standards, heart blood sampling, separation of serum, add ten thousand/ thimerosal anticorrosion.Be sub-packed in sterile tube-20 ℃ of preservations with 0.5ml.
Five, the foundation of kit
A) the detection principle of kit of the present invention
Adopt competition law, the African swine fever virus structural proteins P54 that recombinates is coated in microwell plate, then with 1%BSA, ELISA Plate is sealed, add sample to be tested and standard positive control, negative control.African swine fever virus antibody in sample or standard items can with coated P54 antigen-reactive in ELISA Plate, add for the competition that participates in after the monoclonal antibody linked with peroxidase of P54 in conjunction with epi-position.Subsequently, add horseradish peroxidase substrate TMB colour developing, the stop buffer cessation reaction, by microplate reader under the 450nm wavelength, measure each hole absorbance, in the size of OD value (depth of color after the color development stopping reaction) and sample to be tested, the content of African swine fever virus antibody is inversely proportional to.
B) composition of kit of the present invention
A) the best preparation method of ELISA Plate
,, add in microwell plate by the 100ul/ hole after restructuring P54 albumen dilution after the purifying of above-mentioned preparation as coated damping fluid with the carbonate buffer solution of pH9.60.05M, guarantee that the P54 content in every hole is 0.2ug.4 ℃ of coated spending the night discard coating buffer next day, add the confining liquid of 1%BSA by the 200ul/ hole, 37 ℃ standing 2 hours, washing dries.The packaging bag of packing into after drying at room temperature adds drying agent, and vacuum is preserved.
B) configuration of work reagent
Cleansing solution (pH 7.4,0.15M PBS): KH
2PO
40.2g, Na
2HPO
4-12H
2O 2.9g, NaCl 8.0g, KCl 0.2g, Tween-20 (0.05%) 0.5ml, adding distil water is to 1000ml.Be condensed into 25 times as storage liquid.
Serum dilution: bovine serum albumin(BSA) 0.1g adds lavation buffer solution to 100ml
Substrate buffer solution (pH 5.0 phosphoric acid citric acids): 0.2M Na
2HPO
425.7ml, 0.1M citric acid 24.3ml, adding distil water 50ml
TMB (tetramethyl benzidine) uses liquid: TMB (10mg/5ml absolute ethyl alcohol) 0.5ml, substrate buffer solution 10ml, 0.75%H
2O
232ul
Stop buffer (2M H
2SO
4): distilled water 178.3ml dropwise adds the concentrated sulphuric acid (98%) 21.7ml
C) detect the establishment of the competitive ELISA kit of African swine fever
Set up the enzyme linked immunological kit that detects African swine fever, comprise following component:
96 hole ELISA Plate
The monoclonal antibody of horseradish peroxidase-labeled
Standard positive control
Standard negative control
Concentrated cleaning solution
Serum dilution
Tmb substrate
Stop buffer
Product description
Six, the detection of African swine fever virus antibody in sample
A) kit with above-mentioned preparation detects
1) test serum, positive control and negative control are diluted in proportion every hole 100 μ l with antibody diluent
Add ELISA Plate, hatched 1 hour for 37 ℃.Cleansing solution cleans 3-5 time.
2) every hole adds the rear monoclonal antibody linked with peroxidase 100 μ l of dilution, hatches 30min for 37 ℃, and cleansing solution cleans 3-5 time.
3) every hole adds tmb substrate liquid 100 μ l, room temperature lucifuge reaction 10-15 minute.
4) every hole adds 100 μ l 2M H
2SO
4Cessation reaction, microplate reader detects the 450nm absorbance.(lab diagram is seen accompanying drawing 3)
B) Analysis of test results
When in i. surveying, the OD value of negative control sera (NC) was 4 times of positive control serum (PC) at least, detection was considered to effective.(experimental data sees Table 2)
NC/PC≥4
Positive Cut Off=NC-[(NC-PC) * 0.5]
Negative Cut Off=NC-[(NC-PC) * 0.4]
NC=negative control sera OD value wherein
PC=positive control serum OD value
In his-and-hers watches 2, data are calculated, and drawing positive Cut Off is 0.800, and negative Cut Off is 0.930.
Table 2 adopts ELISA kit of the present invention to detect sample African swine fever antibody data
| 1 | 2 | 3 | 4 | 5 | 6 | |
| A | Negative control 1.483 | Negative control 1.417 | 0.195 | 0.196 | Negative serum 1.215 | Negative serum 1.223 |
| B | Positive control 0.145 | Positive control 0.154 | 0.229 | 0.219 | Negative serum 1.158 | Negative serum 1.201 |
| C | 0.215 | 0.227 | 0.34 | 0.328 | Negative serum 1.121 | Negative serum 1.159 |
| D | 0.203 | 0.204 | 0.531 | 0.518 | Negative serum 1.106 | Negative serum 1.152 |
| E | 0.183 | 0.185 | Suspicious sample 0.797 | Suspicious sample 0.813 | ||
| F | 0.184 | 0.177 | Negative serum 1.035 | Negative serum 1.067 | ||
| G | 0.184 | 0.18 | 0.21 | 0.202 | ||
| H | 0.191 | 0.192 | Negative serum 1.201 | Negative serum 1.195 |
Use multiple hole when ii. detecting, finally using the OD value is the mean value of two.
During lower than positive Cut Off value, this sample is the ASFV antibody positive when the OD of serum sample value.
When the OD of serum sample value is that this sample is the ASFV negative antibody higher than negative Cut Off value.
When the OD of serum sample value is between two Cut Off values, be considered to suspicious, should retest for this sample, perhaps adopt other detection methods to confirm.
In sum, content of the present invention is not limited in the above-described embodiment, and the knowledgeable people in same area can propose other embodiment easily within technological guidance's thought of the present invention, but this embodiment comprises within the scope of the present invention.
Claims (3)
1. competitive ELISA kit for detection of African swine fever virus antibody, it is characterized in that, described kit comprises the enzyme labeling thing of anti-African swine fever virus monoclonal antibody, and wherein said anti-African swine fever virus monoclonal antibody is the secretion of CGMCC NO.3750 hybridoma cell line by preserving number.
2. the competitive ELISA kit for detection of African swine fever virus antibody according to claim 1, is characterized in that, also comprises elisa plate bar, serum dilution and concentrated cleaning solution, stop buffer, substrate nitrite ion, positive control, negative control.
3. the competitive ELISA kit for detection of African swine fever virus antibody according to claim 2, is characterized in that,
Described elisa plate bar: each kit is equipped with 2 or 5 ELISA microwell plates, and every elisa plate bar is the ELISA microwell plate of envelope antigen that can realizing self disassembling, and envelope antigen is the African swine fever virus P54 albumen of prokaryotic expression, and specification is 8 holes * 12;
Described serum dilution: bovine serum albumin(BSA) 0.1g adds lavation buffer solution to 100ml;
The 0.15M PBS of described concentrated cleaning solution: pH 7.4 is condensed into 25 times as storage liquid;
Described substrate nitrite ion: instant TMB nitrite ion, 50mL, it is formulated as follows:
10mg TMB/5ml absolute ethyl alcohol 0.5ml
Substrate buffer solution 10ml
0.75％H
2O
2 32ul；
Described substrate buffer solution is the phosphoric acid citric acid of pH 5.0;
The H of described stop buffer: 2mol/L
2SO
4Solution, 50mL;
The enzyme labeling thing of described anti-African swine fever virus monoclonal antibody: horseradish peroxidase-labeled be the monoclonal antibody of the mouse-anti African swine fever virus P54 albumen of CGMCC NO.3750 hybridoma cell line secretion by preserving number, use front 100 times of dilutions.
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| WO2008129103A1 (en) * | 2007-04-17 | 2008-10-30 | Centre De Recerca En Sanitat Animal (Cresa) | Use of african swine pest haemoglutinin as an adjuvant |
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|---|---|---|---|---|
| WO2008129103A1 (en) * | 2007-04-17 | 2008-10-30 | Centre De Recerca En Sanitat Animal (Cresa) | Use of african swine pest haemoglutinin as an adjuvant |
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