CN102353783B - Kit for detecting pig pseudorabies virus antibodies and block enzyme-linked immuno sorbent assay (ELISA) method - Google Patents
Kit for detecting pig pseudorabies virus antibodies and block enzyme-linked immuno sorbent assay (ELISA) method Download PDFInfo
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Abstract
The invention discloses a kit for detecting pig pseudorabies virus antibodies and a block enzyme-linked immuno sorbent assay (ELISA) method. The kit for detecting pig pseudorabies virus antibodies comprises pig pseudorabies virus monoclonal antibodies which are labelled by horseradish peroxidase, wherein the pig pseudorabies virus monoclonal antibodies are monoclonal antibodies obtained by pig pseudorabies viruses as immunogens and the pig pseudorabies viruses are pseudorabies virus strain Ea. The kit for detecting pig pseudorabies virus antibodies also comprises an enzyme label plate, a sample diluent, negative and positive contrasts, a coloured solution, a washing solution, and a stopping solution. The block ELISA method comprises the following steps of 1, taking out a detection plate pre-coated with virus antigens from the kit for detecting pig pseudorabies virus antibodies, adding diluted blood serum needing to be detected into the detection plate pre-coated with the virus antigens, and simultaneously, setting negative and positive contrast apertures, 2, shaking up the diluted blood serum in the negative and the positive contrast apertures, shaking off a solution in the negative and the positive contrast apertures, and washing the detection plate by the washing solution, and 3, adding the pig pseudorabies virus monoclonal antibodies labelled by horseradish peroxidase into the negative and the positive contrast apertures, washing, adding the colored solution into the negative and the positive contrast apertures to carry out room-temperature coloration in the dark, adding the stopping solution into the negative and the positive contrast apertures, and determining OD630nm values of the negative and the positive contrast apertures by an ELISA apparatus. The block ELISA method has the advantages of good singularity, high sensitivity, short detection time, and high accuracy because of utilization of an S/N ratio method in result determination.
Description
Technical field
The present invention relates to animal epidemic and detect, be specifically related to a kind of enzyme linked immunological kit that detects PRV antibody, also relate to a kind of blocking-up ELISA detection method of PRV antibody simultaneously.Be applicable to detect the Pseudorabies virus antibody horizontal in pig serum.
Background technology
Pseudoabies (Pseudorabies, PR) be by pseudorabies virus (Pseudorabies Virus, PrV), caused comprise domestic animal and multiple wild animal a kind of take heating, very itch and Important Infectious Diseases that encephalomyelitis is symptom.This disease has caused huge economic loss to pig industry, and pig is this sick natural host and storage person.Prevent, control and finally eradicate the current challenge facing of Gai Bingshi China.In the work anti-processed of pseudoabies, in monitoring and detection pig serum, the level of pseudorabies virus antibody is a very important link.For nonvaccinated pig still, the porcine pseudorabies virus antibody in serum both may reflect the impact of maternal antibody, also may reflect the sign of subclinical infection.Both of these case all can have influence on the effect of inoculation of attenuated live vaccine, even may make vaccine invalid, cannot produce viral immunoprotection, causes immuning failure.For the pig of having inoculated, the porcine pseudorabies virus antibody horizontal in serum has reflected immune effect.
Current domestic detection PRV antibody generally adopts latex agglutination, indirect ELISA method etc., and these method susceptibility are not high, higher to antigen purity requirement, and pure antigen is truly difficult to obtain.Therefore unavoidably can produce nonspecific reaction, result is caused to erroneous judgement.
The present invention's application, for monoclonal antibody and the blocking-up ELISA detection technique of Pseudorabies virus, makes kit have good susceptibility and specificity.
Summary of the invention
The object of the invention is to be to provide a kind of enzyme linked immunological kit that detects PRV antibody, the advantage of this kit be used horseradish peroxidase (HRP) mark monoclonal antibody, improved the sensitivity and the specificity that detect.
Another object of the present invention is the detection method that has been to provide a kind of PRV antibody.The method using PRV as antigen coated on elisa plate, then adding serum to be checked hatches, the PRV monoclonal antibody that adds again enzyme labeling, during colour developing, should there are two kinds of situations, if serum to be checked is positive, the enzyme mark monoclonal antibody so now adding will be by the antibody blocking in positive serum, and microplate reader detects numerical value will be lower.If seronegativity to be checked, so enzyme mark monoclonal antibody will continue with elisa plate on PRV antigen-reactive, the numerical value obtaining will be higher.Its detection method specificity is good, susceptibility is high.With the detection coincidence rate of the pseudorabies antibody ELISA of American I DEXX company kit be 97%.There is good application prospect.
In order to realize above-mentioned object, the present invention adopts following technical measures:
A kind of enzyme linked immunological kit that detects PRV antibody, the monoclonal antibody that comprises the PRV of horseradish peroxidase-labeled, described PRV monoclonal antibody is to take the monoclonal antibody that PRV obtains as immunogene, described PRV is Pseudorabies virus Hubei Province A strain (this Pseudorabies virus Hubei Province A strain source document that sees reference: Chen Huanchun etc., the separation of pseudorabies virus Ea strain is identified, journal of animal science and veterinary medicine, 02 phase in 1998).
Described kit also comprises ELISA Plate, sample diluting liquid, positive control, negative control, nitrite ion A, nitrite ion B, cleansing solution, stop buffer.
Described ELISA Plate is that the sodium bicarbonate solution of pH9.6 is as coated damping fluid with PRV 0.1mol/L when the coated elisa plate.Described positive control is the pig positive control serum of pseudorabies vaccine immunity, and described negative control was not for infecting the health pig negative serum of PRV.
Described cleansing solution is the PBS damping fluid that contains 0.05%Tween-20; Described stop buffer is for containing 0.25% volume ratio hydrofluoric acid solution.
Described nitrite ion A is the citrate buffer that contains 50mg/mL carbamide peroxide, and nitrite ion B liquid is the citric acid/sodium citrate damping fluid that contains 0.2mg/mL TMB pH5.0.
Described sample diluting liquid liquid is the DMEM nutrient culture media of the gelatin containing 2.5mg/mL.
Described monoclonal antibody, its preparation process is to adopt hybridoma cell technology, the immune BALB/c mouse of PRV (Hubei Province A strain) with purifying, get its splenocyte and murine myeloma cell (SP2/0) and carry out Fusion of Cells, after cultivating with HAT selective medium, with the coated plate of PRV of purifying, carry out indirect ELISA screening again, the positive cell strain of screening is cloned through limiting dilution assay, with the pig positive control serum of pseudorabies vaccine immunity and the health pig negative serum that do not infect PRV, block ELISA screening again, finishing screen is chosen the monoclonal antibody that a strain has barrier effect, secrete the cell line called after PRV-GB of this antibody.
A blocking-up ELISA detection method for antibody, the steps include:
1) from kit, take out and pre-coatedly have the check-out console of viral antigen (how many per sample, removable gradation is used), the serum to be checked having diluted (dilution in 1: 1) 100 μ L are added in antigen coated microplate, establish positive and negative control wells simultaneously, respectively establish 2 Kong，Mei hole 100 μ L.
2) sample (not overflowing) in Zhen Yun hole gently, puts 37 ℃ of incubations 30 minutes.Get rid of the solution in plate hole, with cleansing solution, wash plate 5 times, outwell at every turn for standing 3 minutes in 200 μL/ holes, pats dry for the last time on thieving paper.
3) the enzyme-added mark monoclonal antibody 100 μ L in every hole, put 37 ℃ of incubations 30 minutes.Wash 5 times, method is with step 2.Every hole adds nitrite ion A, nitrite ion B each one (50 μ L), mixes room temperature (18～25 ℃, below identical) lucifuge colour developing 10 minutes.Every hole adds one of stop buffer (50 μ L), in 15 minutes, by microplate reader, measures every hole OD
630nmthe value of reading.
Kit criterion of the present invention is: test establishment condition is the average OD of negative control hole
630nmvalue and the average OD in positive control hole
630nmthe difference of value is more than or equal to 0.4.S=sample well OD
630nmvalue, the average OD of N=negative control hole
630nmvalue.If S/N ratio is less than or equal to 0.6, sample is judged to PRV antibody positive.If S/N ratio is less than or equal to 0.7 but be greater than 0.6, this sample must be resurveyed, if come to the same thing, again from animal sampling, detects after a period of time.If S/N ratio is greater than 0.7, sample is judged to PRV negative antibody.
The present invention compared with prior art, has the following advantages and effect:
1. due to the use of monoclonal antibody linked with peroxidase, specificity is good, highly sensitive.
Detection time short, can go out result in general 1.5 hours;
3. result is judged employing S/N ratioing technigue, and scientific and reasonable, accuracy is high.
Embodiment
Embodiment 1: the preparation of antigen coated microplate:
Virus culture and purifying: bhk cell uses the DMEM that contains 10% calf serum in 37 ℃ of cultivations, cell grows up to after individual layer, outwell supernatant, the virus of inoculation 1/50th original volumes, add maintenance medium (containing the DMEM of 3% volume ratio calf serum), liquid has just covered cellular layer, 37 ℃ of absorption are after 1 hour, volume while adding maintenance medium to original cultured cell, 37 ℃ of incubators continue to cultivate, and when pathology appears in 80% above cell, cell bottle are put in-20 ℃ of refrigerators, multigelation 3 times, collects virus liquid.The 4 ℃ of centrifugal 30min of virus liquid 5000r/min that collect go precipitation.Centrifugal 1 hour of 4 ℃ of 27000r/min for supernatant, precipitation is resuspended with 2ml pH7.2 0.01mol/L PBS.With PBS, prepare 20% (W/V, as follows), 35%, 45% and 60% discontinuous gradient sucrose, add sample to gradient interface, 4 ℃ of centrifugal 90min of 27000r/min.Collect the protein band at 45% concentration place to centrifuge tube, resuspended with 30ml PBS dilution, 27000r/min desugar in centrifugal 2 hours.Finally with 2ml PBS, suspend and precipitate.-20 ℃ save backup.
By viral antigen, with the carbonate buffer solution dilution of pH9.6, be 500ng/mL, by 100 μL/ holes, be added in polystyrene micropore plate, 4 ℃ of placements are spent the night. dry; By 4 ℃ of sealings of the phosphate buffer containing 5mg/mL BSA pH7.4 1 hour for 150 μL/ holes, drying; By the phosphate buffer room temperature protection containing 20% sucrose pH7.4 3 hours for 100 μL/ holes; Put hothouse dry after, pack into containing sealing in the packing of drying agent and preserve.
Embodiment 2: the preparation of anti-Pseudorabies virus monoclonal antibody and mark:
The preparation process of anti-Pseudorabies virus monoclonal antibody:
1) preparation of feeder cells: merging the previous day, get one 7 week age female BALB/c mouse, cervical vertebra dislocation is put to death, 75% volume ratio alcohol disinfecting 5min, after drying, move in superclean bench, be fixed on the cystosepiment of ultraviolet disinfection, with sterilizing eye scissors, elbow tweezers, cut off skin (can not damage peritonaeum), through blunt separation, peritonaeum is fully exposed.With 10mL disposable syringe, be filled containing HAT selective medium and inject mouse peritoneal, the right hand fixedly syringe is motionless, and left hand is gently rubbed mouse web portion with tweezers gripping alcohol swab, then extracts Intraabdominal nutrient culture media out with syringe, inject sterilizing plate, add above-mentioned nutrient culture media and adjust cell density to 10
6individual/mL, divides and plants to 4 96 porocyte culture plate ，Mei hole 100uL with multichannel pipettor, puts 37 ℃, cultivates and treat the 4th step use in CO2gas incubator.
2) prepare immune spleen cell:
PRV viral antigen 150 μ L and the equal-volume adjuvant of getting concentration and be 1mg/mL fully mix, the BALB/c mouse in 5 week age of immunity, at interval of two weeks subcutaneous inoculations once, after continuous immunity three times, merge first 3 days abdominal cavity booster immunizations.During immunity for the first time, use not formula Freund's complete adjuvant, not formula Freund's incomplete adjuvant is used in second and third time, does not use adjuvant during booster immunization.The learn from else's experience BALB/c mouse of last booster immunization.Mouse is carried out disinfection as stated above and peel off skin, aseptic taking-up spleen, puts into the plate that fills basic culture solution and cleans, and peels off connective tissue.Spleen is moved in another plate that fills 10mL basic culture solution, with syringe needle, on top, bore a hole, with disposable sterilized injector, draw 5mL basic culture solution, from spleen one end, slowly inject, liquid is flowed out from spleen other end pin hole, repeat 3 times.Splenocyte suspension is gone in the aseptic centrifuge tube of 10mL, the centrifugal 10min of 1000rpm, cell precipitation is with basal medium centrifuge washing once, then that cell is resuspended, treats the 4th step use after counting.
3) prepare myeloma cell:
At fusion the last fortnight, SP2/0 myeloma cell frozen in liquid nitrogen is taken out to recovery, with 1640 complete mediums that are added with 8-anaguanine, select to cultivate three generations, to keep HGPRT deficiency.Select growth vigorous, the good cell of form is made seed cell and is expanded cultivation with 1640 complete mediums.Merge the same day, just get in exponential phase cell (perfectly round, bright, the big or small homogeneous of cell, marshalling that form is good, be half fine and close distribution), the centrifugal 10min of 1000rpm, discards nutrient solution, after washing 2 times with 1640 nutrient culture media, cell is resuspended, after numeration, treat the 4th step use.
4) Fusion of Cells:
The immune spleen cell of above-mentioned preparation and SP2/0 myeloma cell are mixed in 50mL centrifuge tube in 10: 1 ratios, and 1000r/min is centrifugal, and 5min removes supernatant.With the filter paper of sterilizing, blot tube wall, knock gently the pipe end, make cell precipitation loosening slightly.The centrifuge tube that cell mixture is housed is put in 37 ℃ of water-baths.Then in 1min, slowly splash into the PEG0.8mL of pre-temperature to 37 ℃, limit edged stirs with pipette tip gently, adds and continues to stir 1min.Then the 1640 nutrient culture media 40mL that slowly add 37 ℃ of pre-temperature.Concrete grammar is within first minute, dropwise to splash into 1mL.Within second minute, add 1mL, within 3-4 minute, add 3mL, within the 5th minute, add 5mL, finally add 30mL 1640 nutrient culture media, each added-time need slowly add, and constantly stirs lightly.Then the centrifugal 5min of 800r/min, removes supernatant.With 40mLHAT nutrient culture media suspension sedimentation cell, minute kind of containing in 96 well culture plates of feeder cells in the above-mentioned first step.In 37 ℃ of CO2gas incubator, cultivate.After merging, second day starts to observe, and has pollution-freely, adds 1 HAT nutrient culture media in the 5th day, within the 8th day, sucks 100 μ L nutrient culture media and changes HT nutrient culture media 100 μ L.Treat that fused cell colony grows to culture hole 1/4, when nutrient culture media omits flavescence, now emiocytosis, compared with multispecific antibody, can be carried out screening and the clone of positive cell.
5) screening of positive cell and clone:
In the ELISA Plate that is coated with PRV antigen, add the cell conditioned medium on 96 porocyte culture plates, in 37 ℃ of incubations 1 hour, cleansing solution is washed after three times, sheep anti-mouse igg-the HRP that adds dilution in 1: 5000,37 ℃ of reaction 30min, wash after three times, add nitrite ion colour developing, after cessation reaction, by microplate reader Ce Mei hole OD630nm value of reading.Establish the culture supernatant of SP2/0 cell as negative control, 2.1 times of OD630 values, to negative control, are judged to the positive simultaneously.Choose growth vigorous, clone with limiting dilution assay in the good positive cell hole of form.
6) Screening and Identification of blocking effect monoclonal antibody:
The supernatant in the positive cell hole of preliminary screening is collected, in the ELISA Plate of coated good antigen, add the positive and negative pig serum that 100 μ L have diluted, in 37 ℃ of incubation 30min, wash after three times, add the supernatant 100 μ L that state positive cell hole, 37 ℃ of reaction 30min, wash three times, add the sheep anti-mouse igg-HRP having diluted, then wash plate three times, add nitrite ion, after 10 minutes by microplate reader Ce Mei hole OD630nm value of reading.When the OD value that adds positive serum hole is when adding half left and right of OD value, negative serum hole, this monoclonal antibody is both for having the monoclonal antibody of blocking effect.Secrete the cell called after PRV-GB of this antibody.
7) monoclonal antibody is produced:
A large amount of manufacture order clonal antibodies, can adopt and in Mice Body, induce method: first with the above-mentioned hybridoma PRV-GB of a large amount of cultivation of 1640 nutrient culture media.By every mouse peritoneal, inject 10
6individual cell concentration.(mouse is injected cell and need process through freurd incomplete adjuvant for first 5 days), collected ascites after 10 days, the centrifugal solid constituent of removing, and its supernatant contains anti-PRV viral monoclonal antibodies.
The markers step of anti-Pseudorabies virus monoclonal antibody:
1) purifying of antibody: to the acetate buffer that adds two parts of 0.06mol/L (pH=5.0) in a ascites, with the hydrochloric acid adjust pH to 4.7 of 0.1mol/L.Under stirring at room, in 30min, be added dropwise to gradually sad (ascites before every milliliter of dilution adds 33 μ L), 4 ℃ standing 2 hours, the centrifugal 30min of 12000rpm, abandons precipitation.Supernatant is adjusted pH to 7.4 after Filter paper filtering.Under 4 ℃ of ice baths, slowly adding pH is 7.4 saturated ammonium sulfate, makes the final saturation degree of ammonium sulfate be no more than 45%, stirs 30min, within standing 2～4 hours or 4 ℃, spends the night.The centrifugal 20min supernatant discarded of 12000r/min at 4 ℃.Precipitation is resuspended with the PBS (pH=7.4) of 0.01mol/L, the 10mmol/L Tris-Hcl dialysed overnight that is 8.0 with pH.After collecting antibody, with spectrophotometer, detect its concentration.
2) horseradish peroxidase-labeled of Pseudorabies virus monoclonal antibody:
Taking 5mg horseradish peroxidase (HRP) is dissolved in 1mL distilled water, the NaIO4 that adds the 0.06mol/L of the new preparation of 500 μ L, places 30min for 4 ℃, does not surpass this time, now solution is grass green, adds 0.5mL ethylene glycol (0.16mol/L) room temperature lucifuge reaction 30min.The purifying Dan Kelong antibody 1mL that adds again 5mg/mL, 4 ℃ of dialysed overnight in the carbonate buffer solution of 0.05mol/L pH9.5.After sucking-off, add the NaBH40.2mL that 5mg/mL newly joins, place 2 hours, then add isopyknic saturated ammonium sulfate solution for 4 ℃, place 30min for 4 ℃, the centrifugal 10min of 7000r/min, abandons supernatant, resuspended with physiological saline, then in the PBS of 0.15mol/L pH7.4 dialysed overnight.The antibody of collecting mark in bag filter carries out working concentration demarcation, is then sub-packed in 20mL bottle, and 4 ℃ save backup.(enzyme labelled antibody of each batch will carry out working concentration demarcation).
Embodiment 3: kit assembling:
2 of kit Han96 hole ELISA check-out consoles, 1 bottle of the anti-Pseudorabies virus monoclonal antibody of enzyme mark (20mL/ bottle), each 2 pipe (1.5mL/ pipe) of positive and negative contrast, 1 bottle of sample diluting liquid (50mL/ bottle), 20 times of 1 bottle of concentrated cleaning solutions (30mL/ bottle), nitrite ion A, nitrite ion B, stop buffer each 1 bottle (10mL/ bottle), 1 part, attached instructions.
20 times of concentrated cleaning solutions: 160 grams of sodium chloride, 10ml Tween-20 dissolve in 1000ml distilled water.
Confining liquid: containing the phosphate buffer of 5mg/mL BSA.
Sample diluting liquid: the DMEM nutrient culture media that contains the gelatin of 25mg/mL.
Stop buffer: 0.25% volume ratio hydrofluorite.
The preparation of the positive, negative control:
The PRV standard positive serum that screening is obtained adds penicillin and streptomysin by 1000U/ml, and aseptic filtration, as positive control; The pig standard female serum that screening is obtained, adds penicillin and streptomysin by 1000U/ml, and aseptic filtration, as negative control.
The DMEM of 10% described cow's serum and 75% alcohol etc., owing to being all liquid, % represents volume ratio.
Embodiment 4: detect effect experiment
A detection method for PRV antibody, the steps include:
1) from kit, take out and pre-coatedly have the check-out console of viral antigen (how many per sample, removable gradation is used), the serum to be checked having diluted (dilution in 1: 1) 100 μ L are added in antigen coated microplate, establish positive and negative control wells simultaneously, respectively establish 2 Kong，Mei hole 100 μ L.
2) sample (not overflowing) in Zhen Yun hole gently, puts 37 ℃ of incubations 30 minutes.Get rid of the solution in plate hole, with cleansing solution, wash plate 5 times, outwell at every turn for standing 3 minutes in 200 μL/ holes, pats dry for the last time on thieving paper.
3) the enzyme-added mark monoclonal antibody 100 μ L in every hole, put 37 ℃ of incubations 30 minutes.Wash 5 times, method is with step 2.Every hole adds nitrite ion A, nitrite ion B each one (50 μ L), mixes room temperature (18～25 ℃) lucifuge colour developing 10 minutes.Every hole adds one of stop buffer (50 μ L), in 15 minutes, by microplate reader, measures every hole OD
630nmthe value of reading.
Kit criterion of the present invention is: test establishment condition is the average OD of negative control hole
630nmvalue and the average OD in positive control hole
630nmthe difference of value is more than or equal to 0.4.S=sample well OD
630nmvalue, the average OD of N=negative control hole
630nmvalue.If S/N ratio is less than or equal to 0.6, sample is judged to PRV antibody positive.If S/N ratio is less than or equal to 0.7 but be greater than 0.6, this sample must be resurveyed, if come to the same thing, again from animal sampling, detects after a period of time.If S/N ratio is greater than 0.7, sample is judged to PRV negative antibody.
1, specific test detects standard positive serum and the porcine pseudorabies negative serums such as porcine pseudorabies, swine fever, Schweineseuche (O type), pig parvoviral, swine flu and porcine reproductive and respiratory syndrome with porcine pseudorabies virus ELISA antibody assay kit, except PRV standard positive serum S/N value be significantly less than 0.4, all the other serum S/N values are between 0.84～1.08, meet the criterion of negative serum, show that the specificity of this method is good.
The comparison of table 1 specific serum
2, coincidence rate is tested with two kinds of detection kit Parallel testings 800 parts of serum from different pig farms, the results are shown in Table 2.As can be seen from the table, the positive coincidence rate of two kinds of kit detection samples is 96%, and the coincidence rate of negative sample is 99%, and total coincidence rate is 97%.
Table 2 porcine pseudorabies virus ELISA antibody assay kit with
The PRV-gB ELISA of IDEXX company kit testing result
3, kit detects the artificial immunity pseudorabies vaccine serum of different times for the detection of susceptibility, as can be seen from Table 3 after immunity 14 days, and antibody major part test positive, 21 days are all positive afterwards, illustrate that the sensitivity of kit is very high.
The detection of table 3 different times artificial immunity serum antibody
Note: when OD value is greater than 0.902, S/N ratio is greater than 0.7 and is judged to feminine gender, and when OD value is less than 0.773, S/N ratio is less than 0.6 and is judged to the positive.
Claims (2)
1. an enzyme linked immunological kit that detects PRV antibody, is characterized in that: monoclonal antibody, the PRV monoclonal antibody that comprises the PRV of horseradish peroxidase-labeled is that to take monoclonal antibody, the PRV that PRV obtains as immunogene be the A strain of Pseudorabies virus Hubei Province;
Described kit also comprises ELISA Plate, sample diluting liquid, positive control, negative control, nitrite ion A, nitrite ion B, cleansing solution, stop buffer;
Described ELISA Plate is that the sodium bicarbonate solution of pH9.6 is as coated damping fluid with PRV 0.1mol/L when the coated elisa plate;
Described positive control is the hyper-immune serum of PRV immunity;
Described negative control sera was not for infecting the health pig serum of PRV;
Described cleansing solution is the PBS damping fluid that contains 0.05%Tween-20;
Described stop buffer is for containing 0.25% volume ratio hydrofluoric acid solution;
Described nitrite ion A is the citrate buffer that contains 50mg/mL carbamide peroxide, and nitrite ion B liquid is the citric acid/sodium citrate damping fluid that contains 0.2mg/mL TMB pH5.0;
Described sample diluting liquid liquid is the DMEM nutrient culture media of the gelatin containing 2.5mg/mL;
In described ELISA Plate, the preparation method of envelope antigen is:
Virus culture and purifying: bhk cell uses the DMEM that contains 10% calf serum in 37 ℃ of cultivations, cell grows up to after individual layer, outwell supernatant, the virus of inoculation 1/50th original volumes, add the DMEM maintenance medium containing 3% volume ratio calf serum, liquid has just covered cellular layer, 37 ℃ of absorption are after 1 hour, volume while adding maintenance medium to original cultured cell, 37 ℃ of incubators continue to cultivate, when there is pathology in 80% above cell, cell bottle is put in-20 ℃ of refrigerators, multigelation 3 times, collect virus liquid, the virus liquid 5000r/min4 ℃ of centrifugal 30min collecting goes precipitation, supernatant is centrifugal 1 hour with 27000r/min4 ℃, precipitation is resuspended with 2ml pH7.20.01mol/L PBS, with PBS, prepare 20%, 35%, 45% and 60% discontinuous gradient sucrose, add sample to gradient interface, 4 ℃ of centrifugal 90min of 27000r/min, collect the protein band at 45% concentration place to centrifuge tube, resuspended with 30ml PBS dilution, 27000r/min desugar in centrifugal 2 hours, finally with 2ml PBS, suspend and precipitate,-20 ℃ save backup.
2. a kind of non-diagnostic assays method of enzyme linked immunological kit that detects PRV antibody claimed in claim 1, the steps include:
1) from kit, take out the pre-coated check-out console that has viral antigen, the serum 1:1 to be checked having diluted is diluted to 100 μ L and add in antigen coated microplate, establish positive and negative control wells simultaneously, respectively establish 2 Kong，Mei hole 100 μ L;
2) sample in Zhen Yun hole gently, puts 37 ℃ of incubations 30 minutes, gets rid of the solution in plate hole, with cleansing solution, washes plate 5 times, and outwell at every turn for standing 3 minutes in 200 μL/ holes, pats dry for the last time on thieving paper;
3) the enzyme-added mark monoclonal antibody 100 μ L in every hole, put 37 ℃ of incubations 30 minutes, wash 5 times, method is with step 2) ，Mei hole adds each 50 μ L of nitrite ion A, nitrite ion B, mixes, 10 minutes ，Mei holes of room temperature lucifuge colour developing add 50 μ L of stop buffer, in 15 minutes, by microplate reader, measure every hole OD
630nmthe value of reading.
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