CN101833005A - Competitive ELISA (Enzyme-Linked Immuno Sorbent Assay) kit for detecting antibody of African swine fever virus and application thereof - Google Patents

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

A kind of competitive ELISA kit of detecting African swine fever virus antibody and uses thereof that is used to

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 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) to African swine fever.It is characterized by course of disease weak point, 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 eqpidemic disease as, China is defined as animal one class disease, be subjected 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 does not still have this disease.

African swine fever belongs 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 any section that it does not belong to ICTV and is appraised and decided, be 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 big, 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 counter-rotating repetitive sequence, 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, isolation identification goes out 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, has only 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 polypeptide of about 25kD contains one section and strides diaphragm area, mainly concentrates on the endoplasmic reticulum place of deriving.P54 albumen can be in vitro culture, and can infection cell.And behind infection cell at endoplasmic reticulum place transient expression.The membrane structure of striding of P54 albumen plays crucial effect (RodriguezJM at virus protein when endoplasmic reticulum changes into the peplos precursor, 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.Duplicate early stage behind the infective virus, but virus active cell apoptotic proteins enzyme and bring out cell apoptosis (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 taking off at cell, analytical structure albumen P54 bites middle role, 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 cell apoptosis (HernaezB, Diaz Gil G.The African swine fever virus dynein-binding proteinp54induces infected cell apoptosis.FEBS Letters 2004,569 (123): 224～228)., the mutant of P54 loses function (the Alejo A of active cell apoptotic proteins enzyme but lacking 13aa, GAndr é s, ML Salas.African Swine Fever VirusProteinase Is Essential for Core Maturation and Infectivity Journalof Virology, 2003,77:5571～557).

Summary of the invention

The objective of the invention is based on prokaryotic expression recombinant albumen, 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 that is used to detect 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 colour developing liquid, stop buffer, positive control, negative control.

The above-mentioned competitive ELISA kit that is used to detect 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 the envelope antigen that can dismantle voluntarily, 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 an instant, and cleansing solution is 25 times and concentrates, dilution before using;

Described substrate colour developing liquid: the instant TMB liquid that develops the color, 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 preceding 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, the method that can carry out the antibody test of a large amount of examination African swine fever virus, and the used envelope antigen of this kit is a 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 behind the reorganization P54 protein purification.

Fig. 2 is western blotting figure behind the reorganization P54 protein purification.

Fig. 3 detects sample African swine fever antibody lab diagram for adopting ELISA kit of the present invention.

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 name: 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) expression of African swine fever P54 albumen

According to African swine fever among the GenBank (ASFV) P54 gene order, 1 pair of primer has been synthesized in design, adopt PCR method from ASFV DNA, to amplify the P54 genetic fragment, it is cloned among the expression vector pET28b, made up recombinant plasmid pET-P54, be transformed into expressive host bacterium BL21 (DE3) after the 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 an ASFV P54 upstream region of gene amplimer, and the restriction enzyme site of introducing is BamHI; P54-2 is an 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, the back thalline is induced in centrifugal collection, washs resuspended thalline with PBS, ultrasonic degradation (ultrasonic 1s, 1s, 10min altogether at interval), and 12000rpm is centrifugal, collects respectively and goes up cleer and peaceful precipitation, carries out SDS-PAGE and analyzes.Use the nickel ion affinity chromatograph post during protein purification, behind the purifying, identify, get access to single band, and possess antigenicity (seeing accompanying drawing 1,2) preferably through SDS-PAGE, western blotting.Protein content behind the use BCA method mensuration purifying, standard items concentration and corresponding OD value thereof see Table 1.The OD value of P54 albumen is 1.583 behind the purifying, 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

African swine fever virus P54 albumen with purifying is antigen, conventional method immunity BALB/c mouse in 6 age in week, and the antigen of fundamental immunity hypodermic injection first 200 μ g use complete Freund's adjuvant; Immune every carrying out in 3 weeks once, 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 preceding 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, the exposure peritonaeum from venter posterior.Sterilize with cotton ball soaked in alcohol wiping peritonaeum.To the abdominal cavity, washing fluid is reclaimed in flushing repeatedly 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, aseptic condition takes out spleen down, and 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 then that cell is resuspended, counting is used for Fusion of Cells.

C) myeloma cell

Before merging 48-36 hour, with myeloma cell's enlarged culture, cell is blown down gently from the bottle wall on fusion same day with the elbow dropper, be collected in the 50ml centrifuge tube.Centrifugal 10 minutes of 1000r/min, supernatant discarded.Add the incomplete nutrient culture media of 30ml, once with the method centrifuge washing.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 touching fusion pipe on the palm, 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, add then contain feeder cells the HAT nutrient culture media to 80ml.Packing 96 porocyte culture plates, every hole 0.1ml puts culture plate 37 ℃, 6%CO then 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.Two week backs are with the HT nutrient culture media HAT nutrient culture media that swaps out, kept for two weeks again after, use the RPMI RPMI-1640 instead.

4. hybridoma screening

After hybridoma merged for two weeks, carry out hybridoma screening 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 a 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, be suspended in again in the cryopreserving liquid of precooling, concentration is 106-107/ml, is transferred in the frozen pipe every bottle of 1ml.Place-70 ℃ of refrigerators, change in the 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 name: hybridoma cell strain.

Two, Monoclonal Antibody

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 abdominal cavity inoculation liquid paraffin body, every mouse 0.5ml.After two weeks, the hybridoma that the abdominal cavity inoculation is diluted with serum free medium, every mouse 5 * 10 ⁵/ 0.2ml.At interval after 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 standby.

Three, monoclonal antibody purifying and horseradish peroxidase mark

1. Purification of Monoclonal Antibodies

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; Add saturated ammonium sulfate to 45% saturation degree down at 4 ℃, 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 standby.

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 wherein adding the 0.1MNaIO that 0.2ml newly joins ₄Solution, the room temperature lucifuge stirred 20 minutes; Dialyse in the sodium-acetate buffer to 1mM pH4.4,4 ℃ are spent the night; Carbonate buffer solution to wherein adding 20 μ l 0.2M pH9.5 again makes the pH of above hydroformylation thing be elevated to 9.0, adds the pure product 5mg of monoclonal antibody (10mg/ml) that is dissolved in the 0.01M carbonate buffer solution then immediately, and 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.Above-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, is used for the optimization of testing conditions 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 is drawn antigen (P54 recombinates behind the purifying) (to call FCA-P54 in the following text) the 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.At interval after 10-14 days, carry out immunity second time, and inject not formula Freund (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 of being total to lymph nodes.After 7-10 days, from ear vein blood sampling 0.5-1.0ml, separation of serum adopts the ELISA method to detect serum titer at interval, 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 4 ℃ of refrigerators again 3 hours.After treating the blood clotting clot contraction, draw serum with dropper, the centrifugal 15min of 3000r/min gets supernatant, and it is anticorrosion to add final concentration and be 0.01% thimerosal, after the packing, and-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 P54 that recombinates is wrapped by in microwell plate, with 1%BSA ELISA Plate is sealed then, add sample to be tested and standard positive control, negative control.The P54 antigen-reactive of bag quilt in African swine fever virus antibody capable in sample or the standard items and the ELISA Plate adds at the competition that participates in behind 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, the content of African swine fever virus antibody is inversely proportional in size of OD value (depth of color development stopping reaction back color) and the sample to be tested.

B) composition of kit of the present invention

A) the best preparation method of ELISA Plate

Carbonate buffer solution with pH9.60.05M is cushioned liquid as bag, after reorganization P54 albumen dilution behind the purifying of above-mentioned preparation, presses the 100ul/ hole and adds in the microwell plate, guarantees that the P54 content in every hole is 0.2ug.4 ℃ of bags are spent the night, and 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, African swine fever virus detection of antibodies in the 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 dilution back monoclonal antibody linked with peroxidase 100 μ l, 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) testing result analysis

When the OD value of negative control sera (NC) was 4 times of positive control serum (PC) at least in i. surveying, 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

Data computation in the his-and-hers watches 2, drawing positive Cut Off is 0.800, 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

	??1	??2	??3	??4	??5	??6
							??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 two mean value.

When the OD of serum sample value was lower than positive Cut Off value, this sample was the ASFV antibody positive.

When the OD of serum sample value is higher than negative Cut Off value is that this sample is the ASFV negative antibody.

When the OD of serum sample value is between two Cut Off values, be considered to suspicious, should test again 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 (4)

1. a competitive ELISA kit that is used to detect African swine fever virus antibody 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.3750 hybridoma cell line secretion.

2. the competitive ELISA kit that is used to detect 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 colour developing liquid, positive control, negative control.

3. the competitive ELISA kit that is used to detect 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 the envelope antigen that can dismantle voluntarily, 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 an instant, and cleansing solution is 25 times and concentrates, dilution before using;

Described substrate colour developing liquid: the instant TMB liquid that develops the color, 50mL;

The H of described stop buffer: 2mol/L ₂5O ₄Solution, 50mL;

Described monoclonal antibody linked with peroxidase: the mouse-anti P54 monoclonal antibody of described horseradish peroxidase-labeled, use preceding 100 times of dilutions;

Positive control;

Negative control.

4. the application of the described competitive ELISA kit of claim 1 in detecting African swine fever virus.

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