CN101825633B - Competitive ELISA kit for detecting african swine fever virus antibody and purposes thereof - Google Patents
Competitive ELISA kit for detecting african swine fever virus antibody and purposes thereof Download PDFInfo
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Abstract
The invention discloses a competitive ELISA kit for detecting african swine fever virus antibody and the purposes thereof, which belong to the biological technical field. The kit adopts prokaryotic expression recombinant P30 protein as coating antigen, and detects the african swine fever virus antibody in pig serum according to a competitive ELISA principle. The coating antigen in a 96 pore plate in the kit is the prokaryotic expression recombinant P30 protein, and has good antigenicity. The ELISA kit comprises the 96 pore plate coated by the P30 protein, and monoclonal antibody, concentrated washing liquid, serum dilution, TMB substrate and stopping solution of positive control, negative control and horseradish peroxidase labeling. The kit can be used for screening a large batch of samples, and the main reagent in the kit is provided in the form of working solution, so that 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, and be popular in dozens of countries such as Africa, Europe and America so far, 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 the Poxviridae, places 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 terminal covalence closed unimolecule wire double-stranded DNA, the viral genome total length is 170kb～190kb, there is the conserved region about 125kb in central authorities, two ends are the variable region, contain terminal 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.Experiment showed, P30 albumen for having one of better antigenic albumen, molecular weight of albumen is about 36KD.
Summary of the invention
The objective of the invention is take the recombinant protein of prokaryotic expression as the basis, by a kind of competitive ELISA kit that detects African swine fever virus antibody of Enzyme-multiplied immune technique development.
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.3771 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 P30 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 P30 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 the method that a kind of African swine fever virus antibody accurate, quick, that can carry out a large amount of examinations detects, 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 behind the Recombinant P30 protein purification.
Fig. 2 is western blotting figure behind the Recombinant P30 protein purification.
Embodiment
The hybridoma cell line of anti-African swine fever virus monoclonal antibody is preserved in the common micro-organisms center C GMCC NO.3771 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, sets up clone
1. antigen preparation
A) African swine fever P30 protein expression
According to African swine fever among the GenBank (ASFV) P30 gene order, 1 pair of primer has been synthesized in design, adopt PCR method from ASFV DNA, to amplify the P30 genetic fragment, it is cloned among the expression vector pET28b, made up recombinant plasmid pET-P30, be transformed into expressive host bacterium BL21 (DE3) after the sequence verification, through the IPTG abduction delivering.
The primer of design is:
P30-1-5’-GC
GGATCCTAATTTTAAAATTGAATGGAT-3’
P30-2-5’-GT
CTCGAGCCCAATCAAAATTAGATAACT-3’
Underscore is the restriction enzyme site for introducing partly, and P30-1 is ASFV P30 upstream region of gene amplimer, and the restriction enzyme site of introducing is BamHI; P30-2 is ASFV P30 gene downstream amplimer, and the restriction enzyme site of introducing is XhoI.
B) purifying of African swine fever P30 albumen
After inducing end, rear thalline is induced in centrifugal collection, washs resuspended thalline with PBS, ultrasonic degradation (ultrasonic 1s, interval 1s, altogether 10min), and 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, behind the purifying, identify through SDS-PAGE, western blotting, get access to single band, and possess preferably antigenicity (seeing accompanying drawing 1,2).Protein content behind the use BCA method mensuration purifying, standard items concentration and corresponding OD value thereof see Table 1.The OD value of P30 albumen is 2.25 behind the purifying, with standard items relatively after, its concentration is 1700 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 P30 albumen of purifying as antigen, conventional method immunity BALB/c mouse in 6 age in week, first fundamental immunity hypodermic injection antigen 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 the last fundamental immunity, 25 μ g/, immunity is finished.
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.To the abdominal cavity, washing fluid is reclaimed in repeatedly flushing with injector to inject 10ml RPMI RPMI-1640, and 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 finished, by the neck dislocation mouse that causes death, be soaked in 75% alcohol 5 minutes, take out spleen under the 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, in the 50ml centrifuge tube, wash 1 time with the RPMI1640 nutrient solution, 1200r/min, centrifugal 10min abandons supernatant, with the careful sucking-off residual liquid of dropper.At the bottom of palm touches fusion pipe, make sedimentation cell loose evenly, put preheating in 40 ℃ of water-baths.Adding is preheated to 40 ℃ 50%PEG (PH 8.0) 1ml in the time of 45 seconds, acts on 90 seconds.Add 20ml and be preheated to 37 ℃ RPMI RPMI-1640, room temperature left standstill 10 minutes.1000r/min, centrifugal 5min, supernatant discarded.Add 5ml HAT nutrient culture media, the pressure-vaccum sedimentation cell suspends and mixing it gently, 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 the incubator.
With HAT nutrient culture media 1/2 nutrient culture media that swaps out, change liquid after 48 hours fully after 24 hours.After two weeks with the HT nutrient culture media HAT nutrient culture media that swaps out, kept again for two weeks after, use the RPMI RPMI-1640 instead.
4. the screening of hybridoma
After hybridoma merged for two weeks, carry out the screening of hybridoma 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 10
6-10
7/ ml is transferred in the cryopreservation tube, every bottle of 1ml.Place-70 ℃ of refrigerators, change in the liquid nitrogen next day.
The hybridoma cell line of anti-African swine fever virus monoclonal antibody of preparation is preserved in the common micro-organisms center C GMCC NO.3771 of China Committee for Culture Collection of Microorganisms, preservation day: 2010-04-22, Classification And Nomenclature: hybridoma cell strain.
Two, monoclonal antibody preparation
Among 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 the mouse peritoneal, the hybridoma of in mouse peritoneal, growing, 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 the interval 5 days, observe the mouse ascites production every day, when treating that the many as far as possible and mouse of ascites is on the verge of death, put to death mouse, under the aseptic condition with the ascites sucking-off.The static 30min of room temperature, 1000r/min, centrifugal 10min collects supernatant, packing ,-70 ℃ are frozen for subsequent use.
Three, monoclonal antibody purifying and horseradish peroxidase mark
1. the purifying of monoclonal antibody
The purification scheme 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 the stirring at room, adds in 30 minutes, and 4 ℃ left standstill 2 hours, took out the centrifugal 30min of 12000r/min, abandoned 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; 4 ℃ of lower saturated ammonium sulfate to 45% saturation degrees that add, mixing 30min left standstill 1 hour gently; Centrifugal 30 minutes of 12000r/min abandons supernatant; Precipitation is dissolved among an amount of 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 for subsequent use.
2. the horseradish peroxidase mark of monoclonal antibody
The preparation scheme of the enzyme labeling thing of the monoclonal antibody behind the purifying is the sodium periodate method, and concrete steps are as follows:
Taking by weighing 5mg HRP is dissolved in the 1ml distilled water; To the 0.1M NaIO that wherein adds 0.2ml and newly join
4Solution, the room temperature lucifuge stirred 20 minutes; Dialyse in the sodium-acetate buffer to 1mM pH4.4,4 ℃ are spent the night; To the carbonate buffer solution that wherein adds again 20 μ l 0.2M pH9.5, 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; Add the 4mg/ml NaBH that 0.1ml newly joins
4, mixing was placed 2 hours for 4 ℃; Products therefrom is to 4 ℃ of dialysed overnight among the 0.15M pH7.4PBS.
The dialysis finish after, in stirring, dropwise add the equal-volume saturated ammonium sulfate, 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 among the PBS of a small amount of 0.15M pH 7.4.Mentioned solution is packed in the 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 the 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.Antigen (Recombinant P30 behind the purifying) (hereinafter referred to as FCA-P30) the liquid 1ml of not formula Freund's complete adjuvant (FCA) emulsification is drawn in immunity with syringe for the first time, the subcutaneous 0.5ml that injects of every batter palm.After interval 10-14 days, carry out second time immunity, and inject not formula Freunds incomplete adjuvant (FIA-P30) in the lymph node of Liang Ce popliteal nest and groin enlargement, each lymph node 0.1ml, all the other inject near the subcutaneous altogether 1ml 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, put again 4 ℃ of refrigerators 3 hours.After the blood clotting clot contraction, draw serum with dropper, the centrifugal 15min of 3000r/min gets supernatant, adds final concentration and be 0.01% thimerosal anticorrosion, after the 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 the 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 P30 that recombinates is coated in the 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 the standard items can with coated P30 antigen-reactive in the ELISA Plate, add for the competition that participates in behind the monoclonal antibody linked with peroxidase of P30 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, the content of African swine fever virus antibody is inversely proportional in the size of OD value (depth of color after the color development stopping reaction) and the sample to be tested.
B) composition of kit of the present invention
A) the best preparation method of ELISA Plate
With the coated damping fluid of carbonate buffer solution conduct of pH9.60.05M, after Recombinant P30 albumen dilution behind the purifying of above-mentioned preparation, press the 100ul/ hole and add in the microwell plate, guarantee that the P30 content in every hole is 0.2ug.4 ℃ of coated spending the night discard coating buffer next day, press the confining liquid that the 200ul/ hole adds 1%BSA, and 37 ℃ left standstill 2 hours, and washing dries.The packaging bag of packing into after the 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) establishment of the competitive ELISA kit of detection 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 the sample
A) kit with above-mentioned preparation detects
1) test serum, positive control and negative control are diluted in proportion with antibody diluent, every hole 100 μ l add ELISA Plate, hatch 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.
B) Analysis of test results
When the OD value of negative control sera (NC) is 4 times of positive control serum (PC) at least in i. surveying,
It is effective that detection is considered to.
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
Use multiple hole when ii. detecting, finally using the OD value is two mean value.
When the OD of serum sample value was lower than positive Cut Off value, this sample was the ASFV antibody positive.
That this sample is the ASFV negative antibody when the OD of serum sample value is 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 the 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. the competitive ELISA kit for detection of African swine fever virus is characterized in that, described kit comprises by preserving number being the enzyme labeling thing of the anti-African swine fever virus monoclonal antibody of CGMCC NO.3771 hybridoma cell line secretion.
2. the competitive ELISA kit for detection of African swine fever virus according to claim 1 is characterized in that, also comprises elisa plate bar, serum dilution and concentrated cleaning solution, substrate nitrite ion, stop buffer, positive control, negative control.
3. the competitive ELISA kit for detection of African swine fever virus 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 P30 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;
The enzyme labeling thing of described anti-African swine fever virus monoclonal antibody is the mouse-anti P30 monoclonal antibody of horseradish peroxidase-labeled, uses front 100 times of dilutions;
Positive control;
Negative control.
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| CN102353783B (en) * | 2011-06-29 | 2014-01-15 | 武汉科前动物生物制品有限责任公司 | Kit for detecting pig pseudorabies virus antibodies and block enzyme-linked immuno sorbent assay (ELISA) method |
| CN102539749A (en) * | 2012-02-16 | 2012-07-04 | 江阴康奈尔生物科技有限公司 | Field high-sensitivity immunoassay method and kit |
| CN110904127A (en) * | 2018-09-18 | 2020-03-24 | 瓦赫宁恩研究基金会 | African swine fever virus vaccine |
| CN111072774B (en) * | 2019-10-14 | 2021-03-30 | 中国农业科学院兰州兽医研究所 | anti-African swine fever P30 protein single-domain antibody and ELISA kit for detecting African swine fever virus |
| CN111273028B (en) * | 2020-02-25 | 2023-03-28 | 芜湖天明生物技术有限公司 | rhTSG-6 direct competition ELISA quantitative detection kit and use method and application thereof |
| CN111474367A (en) * | 2020-04-15 | 2020-07-31 | 杭州恒奥科技有限公司 | Kit for screening and detecting African swine fever virus P30 protein monoclonal antibody and preparation method thereof |
| CN111781363A (en) * | 2020-08-12 | 2020-10-16 | 江苏省农业科学院 | Quantum dot microsphere immunochromatography test strip for detecting mucosa sIgA antibody of African swine fever virus and application thereof |
| CN113607952B (en) * | 2021-08-18 | 2022-03-11 | 杭州恒奥科技有限公司 | African swine fever virus blocking ELISA antibody detection kit and preparation method and application thereof |
| CN114167055B (en) * | 2021-10-21 | 2023-07-18 | 山东绿都生物科技有限公司 | Competitive enzyme-linked immunosorbent assay kit for detecting anti-African swine fever antibodies in serum |
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