CN102353783A - 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 the Pseudorabies virus antibody horizontal that detects in the porcine blood serum.
Background technology
Pseudoabies (Pseudorabies, PR) be by pseudorabies virus (Pseudorabies Virus, PrV) cause comprise domestic animal and multiple wild animal a kind of with heating, very itch and encephalomyelitis is the important infectious disease of symptom.This disease has caused enormous economic loss to pig industry, and pig is this sick natural host and storage person.Preventing, controlling and finally eradicate this disease is the current challenge that faces of China.In the anti-system work of pseudoabies, the level of pseudorabies virus antibody is a very important link in monitoring and the detection porcine blood serum.For nonvaccinated pig still only, the porcine pseudorabies virus antibody in the serum both possibly reflect the influence of maternal antibody, also possibly reflect the sign of subclinical infection.Both of these case all can have influence on the effect of inoculation of attenuated live vaccine, even possibly make vaccine invalid, can't produce the immunoprotection to virus, causes immuning failure.For the pig that has inoculated only, the porcine pseudorabies virus antibody horizontal in the serum has then reflected immune effect.
Present domestic detection PRV antibody generally adopts latex agglutination, indirect ELISA method etc., and these method susceptibility are not high, and the antigen purity requirement but than higher, is obtained and pure antigen truly is very difficult.Therefore unavoidably can produce nonspecific reaction, the result is caused erroneous judgement.
The present invention uses to monoclonal antibody of Pseudorabies virus and blocking-up ELISA detection technique, makes kit have good susceptibility and specificity.
Summary of the invention
The objective 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 to use horseradish peroxidase (HRP) mark monoclonal antibody, improved the sensitivity and the specificity that detect.
Another object of the present invention is to be to provide a kind of PRV detection of antibodies method.This method with PRV as antigen coated on elisa plate; Adding serum to be checked then hatches; Two kinds of situation should appear in the PRV monoclonal antibody that adds enzyme labeling again during colour developing, if serum to be checked is positive; The enzyme mark monoclonal antibody that adds this moment so will be by the antibody blocking in the positive serum, and ELIASA detects numerical value will be low more.If seronegativity to be checked, so enzyme mark monoclonal antibody will continue with elisa plate on the PRV antigen-reactive, the numerical value that obtains will be high more.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%.Good prospects for application is arranged.
In order to realize above-mentioned purpose, 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 be the monoclonal antibody that immunogene obtains with the PRV, and 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 isolation identification of porcine pseudorabies virus Hubei Province A strain; Journal of animal science and veterinary medicine, 1998 02 phases).
Described kit also comprises ELISA Plate, sample diluting liquid, positive control, negative control, colour developing liquid A, colour developing liquid B, cleansing solution, stop buffer.
Described ELISA Plate is to use 0.1mol/L when the coated elisa plate with PRV, and the sodium bicarbonate solution of pH9.6 is as encapsulating damping fluid.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; Said stop buffer is for containing 0.25% volume ratio hydrofluoric acid solution.
Described colour developing liquid A is the citrate buffer that contains the 50mg/mL carbamide peroxide, and colour developing liquid 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 that contains the gelatin of 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 the HAT selective medium, encapsulate plate with the PRV of purifying again and carry out the indirect ELISA screening; The positive cell strain of screening is cloned through limiting dilution assay; Block the ELISA screening with the pig positive control serum of pseudorabies vaccine immunity and the health pig negative serum that do not infect PRV again, finishing screen is chosen the monoclonal antibody that a strain has barrier effect, secretes the cell line called after PRV-GB of this antibody.
A kind of blocking-up ELISA detection method of PRV antibody the steps include:
1) from kit, take out the check-out console be coated with viral antigen in advance (per sample what; Removable gradation is used), the serum to be checked that dilution is good (dilution in 1: 1) 100 μ L add in the antigen coated microplate, establish the positive and negative control wells simultaneously; Respectively establish 2 holes, every hole 100 μ L.
2) sample (not overflowing) in the even hole that shakes was gently put 37 ℃ of incubations 30 minutes.Get rid of the solution in the plate hole, wash plate 5 times with cleansing solution, 200 μ L/ holes are left standstill at every turn and were outwelled in 3 minutes, do at the thieving paper arsis for the last time.
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 colour developing liquid A, colour developing liquid B each (50 μ L), mixing, room temperature (18～25 ℃, below identical) lucifuge colour developing 10 minutes.Every hole adds one of stop buffer (50 μ L), measures every hole OD with ELIASA in 15 minutes
630nmThe value of reading.
Kit criterion of the present invention is: the 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 the PRV antibody positive.If S/N ratio is less than or equal to 0.7 but greater than 0.6, this sample must be resurveyed,, then detect from the animal sampling again after a period of time if come to the same thing.If S/N ratio is greater than 0.7, sample is judged to the PRV negative antibody.
The present invention compared with prior art has the following advantages and effect:
1. owing to the use of monoclonal antibody linked with peroxidase, specificity is good, and is highly sensitive.
2. lack detection time, can go out the result in general 1.5 hours;
3. the result judges 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 in 37 ℃ of cultivations, after cell grows up to individual layer, is outwelled supernatant with the DMEM that contains 10% calf serum, the virus of inoculation 1/50th original volumes; Add and keep liquid (DMEM that contains 3% volume ratio calf serum), liquid has just covered cellular layer and has got final product, and 37 ℃ of absorption are after 1 hour; Add the volume when keeping liquid to original cultured cell, 37 ℃ of incubators continue to cultivate, when treating that pathology appears in 80% above cell; The cell bottle is put in-20 ℃ of refrigerators, and multigelation 3 times is collected viral liquid.The 4 ℃ of centrifugal 30min of viral liquid 5000r/min that collect go deposition.Supernatant precipitates resuspended with 2ml pH7.2 0.01mol/L PBS with centrifugal 1 hour of 4 ℃ of 27000r/min.With PBS preparation 20% (W/V, as follows), 35%, 45% and 60% discontinuous gradient sucrose, add sample to gradient interface, 4 ℃ of centrifugal 90min of 27000r/min.The protein band of collecting 45% concentration place is to centrifuge tube, and is resuspended with 30ml PBS dilution, 27000r/min desugar in centrifugal 2 hours.Suspend with 2ml PBS at last and precipitate.-20 ℃ of preservations are subsequent use.
Use the carbonate buffer solution dilution of pH9.6 to be to be added on 500ng/mL in the polystyrene micropore plate by 100 μ L/ holes viral antigen, 4 ℃ of placements are spent the night. dry; Seal 1 hour, drying by 150 μ L/ holes with 4 ℃ of the phosphate buffers that contains 5mg/mL BSA pH7.4; Protect 3 hours by 100 μ L/ holes with the phosphate buffer room temperature that contains 20% sucrose pH7.4; After putting the hothouse drying, sealing is preserved in the packing that contains drying agent of packing into.
Embodiment 2: anti-Pseudorabies virus MONOCLONAL ANTIBODIES SPECIFIC FOR and mark:
Anti-Pseudorabies virus MONOCLONAL ANTIBODIES SPECIFIC FOR step:
1) preparation of feeder cells: merging the previous day; Get one 7 age in week female BALB/c mouse, cervical vertebra dislocation is put to death, 75% volume ratio alcohol disinfecting 5min; Drying the back moves in the superclean bench; Be fixed on the cystosepiment of ultraviolet disinfection, cut off skin (can not damage peritonaeum), peritonaeum is fully exposed through blunt separation with sterilization eye scissors, elbow tweezers.Be filled with the 10mL disposable syringe and contain the 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, extracts Intraabdominal nutrient culture media out with syringe again; Inject the sterilization plate, add above-mentioned nutrient culture media adjustment cell density to 10
6Individual/mL, plant to 4 96 porocyte culture plates with the multichannel pipettor branch, every hole 100uL puts 37 ℃, cultivates in the CO2gas incubator and treats the use of the 4th step.
2) preparation immune spleen cell:
Getting concentration is PRV viral antigen 150 μ L and the abundant mixing of equal-volume adjuvant of 1mg/mL, the BALB/c mouse in 5 ages in week of immunity, and the two all subcutaneous immunity of every interval are once, behind the continuous immunity three times, merge preceding 3 days abdominal cavity booster immunizations.Use not formula Freund's complete adjuvant during immunity for the first time, not formula Freund 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 peels off skin, and aseptic taking-up spleen is put into the plate that fills basic culture solution and is cleaned, and peels off connective tissue.Spleen is moved in another plate that fills the 10mL basic culture solution, bore a hole on the top, draw the 5mL basic culture solution, slowly inject, liquid is flowed out from spleen other end pin hole, repeat 3 times from spleen one end with disposable sterilized injector with syringe needle.Splenocyte suspension is gone in the aseptic centrifuge tube of 10mL, and the centrifugal 10min of 1000rpm, cell precipitation are with the basal medium centrifuge washing once, and be then that cell is resuspended, treats the use of the 4th step behind the counting.
3) preparation myeloma cell:
To in liquid nitrogen, frozen SP2/0 myeloma cell take out recovery at fusion the last fortnight, select to cultivate the three generations, to keep the HGPRT deficiency with 1640 complete mediums that are added with the 8-azaguanine.Select growth vigorous, the form good cell is made seed cell and is carried out enlarged culture with 1640 complete mediums.Merge the same day, get and just be in exponential phase, form good cell (perfectly round, bright, the big or small homogeneous of cell, marshalling; Be half fine and close the distribution), the centrifugal 10min of 1000rpm discards nutrient solution; After washing 2 times with 1640 nutrient culture media that cell is resuspended, treat after the numeration that the 4th step uses.
4) Fusion of Cells:
With the immune spleen cell of above-mentioned preparation and SP2/0 myeloma cell in 10: 1 ratio mixings in the 50mL centrifuge tube, 1000r/min is centrifugal, and 5min removes supernatant.Filter paper with sterilization blots tube wall, knocks the pipe end gently, makes cell precipitation loosening slightly.The centrifuge tube that cell mixture is housed is put in 37 ℃ of water-baths.In 1min, slowly splash into the PEG0.8mL of preparatory temperature to 37 ℃ then, the limit edged stirs with pipette tip gently, adds to continue to stir 1min.Slowly add 37 ℃ of 1640 nutrient culture media 40mL of temperature in advance then.Concrete grammar is dropwise to splash into 1mL in first minute.Added 1mL in second minute, added 3mL in 3-4 minute, added 5mL on the 5th minute, add 30mL 1640 nutrient culture media at last, each added-time needs slowly to add, and constantly stirs lightly.The centrifugal 5min of 800r/min removes supernatant then.With 40mLHAT nutrient culture media suspension sedimentation cell, divide and plant in 96 well culture plates that contain feeder cells of the above-mentioned first step.In 37 ℃ of CO2gas incubators, cultivate.Merge and began in back second day to observe, have pollution-freely, added 1 HAT nutrient culture media in the 5th day, suction in the 8th day goes 100 μ L nutrient culture media to change HT nutrient culture media 100 μ L.Treat that the fused cell colony grows to culture hole 1/4, when nutrient culture media omited flavescence, emiocytosis this moment can be carried out the screening and the clone of positive cell than multispecific antibody.
5) screening of positive cell and clone:
At the cell conditioned medium that adds on the ELISA Plate that is coated with PRV antigen on the 96 porocyte culture plates, in 37 ℃ of incubations 1 hour, after cleansing solution is given a baby a bath on the third day after its birth time; Sheep anti-mouse igg-the HRP that adds dilution in 1: 5000; 37 ℃ of reaction 30min, wash three times after, add colour developing liquid and develop the color; After the cessation reaction, survey every hole OD630nm value of reading with ELIASA.The culture supernatant of establishing the SP2/0 cell simultaneously is as negative control, and 2.1 times of OD630 values are judged to the positive to negative control.It is vigorous to choose growth, and 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, add the good positive and negative porcine blood serum of 100 μ L dilution on the ELISA Plate of antigen having encapsulated, in 37 ℃ of incubation 30min; After washing three times, add the supernatant 100 μ L that state the positive cell hole, 37 ℃ of reaction 30min; Wash three times, add the good sheep anti-mouse igg-HRP of dilution, wash plate again three times; Add colour developing liquid, survey every hole OD630nm value of reading with ELIASA after 10 minutes.When the OD value that adds the positive serum hole was lower than adding half left and right sides of OD value, negative serum hole, this monoclonal antibody was both for having the monoclonal antibody of blocking effect.Secrete the cell called after PRV-GB of this antibody.
7) monoclonal antibody production:
A large amount of manufacture order clonal antibodies can adopt the method that induces in the mouse body: at first with a large amount of above-mentioned hybridoma PRV-GB of cultivation of 1640 nutrient culture media.Inject 10 by every mouse peritoneal
6Individual cell concentration.(mouse is injected cell to be needed through not exclusively not formula adjuvant processing in preceding 5 days) collected ascites after 10 days, and the centrifugal solid constituent of removing, its supernatant promptly contain anti-PRV viral monoclonal antibodies.
The markers step of anti-Pseudorabies virus monoclonal antibody:
1) purifying antibody: in a ascites, add the acetate buffer of two parts of 0.06mol/L (pH=5.0), with the hydrochloric acid adjust pH to 4.7 of 0.1mol/L.In 30min, be added dropwise to sad (every milliliter of preceding ascites of dilution adds 33 μ L) under the stirring at room gradually, 4 ℃ left standstill 2 hours, and the centrifugal 30min of 12000rpm abandons deposition.Supernatant is transferred pH to 7.4 behind filter paper filtering.Under 4 ℃ of ice baths, slowly add pH and be 7.4 saturated ammonium sulfate, make the final saturation degree of ammonium sulfate be no more than 45%, stir 30min, leave standstill and spent the night in 2～4 hours or 4 ℃.4 ℃ of centrifugal 20min supernatant discarded of following 12000r/min.Precipitate resuspendedly with the PBS (pH=7.4) of 0.01mol/L, using pH is 8.0 10mmol/L Tris-Hcl dialysed overnight.Detect its concentration with spectrophotometer after collecting antibody.
2) horseradish peroxidase-labeled of Pseudorabies virus monoclonal antibody:
Taking by weighing 5mg horseradish peroxidase (HRP) is dissolved in the 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; This moment, solution was grass green, added 0.5mL monoethylene glycol (0.16mol/L) room temperature lucifuge reaction 30min.The purifying Dan Kelong antibody 1mL that adds 5mg/mL again, 4 ℃ of dialysed overnight in the carbonate buffer solution of 0.05mol/L pH9.5.Add the NaBH40.2mL that 5mg/mL newly joins after the sucking-off, placed 2 hours, and added isopyknic saturated ammonium sulfate solution again for 4 ℃; Place 30min for 4 ℃, the centrifugal 10min of 7000r/min abandons supernatant; Resuspended with physiological saline, dialysed overnight in the PBS of 0.15mol/L pH7.4 again.The antibody of collecting mark in the bag filter carries out working concentration to be demarcated, and is sub-packed in then in the 20mL bottle, and 4 ℃ of preservations are subsequent use.(enzyme labelled antibody of each batch all will carry out working concentration and demarcate).
Embodiment 3: the kit assembling:
Kit contains 2 of 96 hole ELISA check-out consoles; Enzyme is marked 1 bottle of anti-Pseudorabies virus monoclonal antibody (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); Colour developing liquid A, colour developing liquid B, stop buffer each 1 bottle (10mL/ bottle) attach 1 part in instructions.
20 times of concentrated cleaning solutions: 160 gram sodium chloride, 10ml Tween-20 dissolve in the 1000ml distilled water.
Confining liquid: the phosphate buffer that contains 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 obtains is pressed 1000U/ml adding penicillin and streptomysin, and aseptic filtration is as positive control; With the pig standard female serum that screening obtains, to press 1000U/ml and add penicillin and streptomysin, aseptic filtration is as negative control.
The DMEM of described 10% cow's serum and 75% alcohol etc., owing to be liquid all, % representes volume ratio.
Embodiment 4: detect the effect experiment
A kind of PRV detection of antibodies method the steps include:
1) from kit, take out the check-out console be coated with viral antigen in advance (per sample what; Removable gradation is used), the serum to be checked that dilution is good (dilution in 1: 1) 100 μ L add in the antigen coated microplate, establish the positive and negative control wells simultaneously; Respectively establish 2 holes, every hole 100 μ L.
2) sample (not overflowing) in the even hole that shakes was gently put 37 ℃ of incubations 30 minutes.Get rid of the solution in the plate hole, wash plate 5 times with cleansing solution, 200 μ L/ holes are left standstill at every turn and were outwelled in 3 minutes, do at the thieving paper arsis for the last time.
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 colour developing liquid A, colour developing liquid B each (50 μ L), mixing, room temperature (18～25 ℃) lucifuge colour developing 10 minutes.Every hole adds one of stop buffer (50 μ L), measures every hole OD with ELIASA in 15 minutes
630nmThe value of reading.
Kit criterion of the present invention is: the 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 the PRV antibody positive.If S/N ratio is less than or equal to 0.7 but greater than 0.6, this sample must be resurveyed,, then detect from the animal sampling again after a period of time if come to the same thing.If S/N ratio is greater than 0.7, sample is judged to the PRV negative antibody.
1, the specificity test detects standard positive serum and 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 that the PRV standard positive serum the S/N value 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, the coincidence rate test is with the 800 part serum of two kinds of parallel detections of detection kit from different pig farms, and the result sees table 2.From table, can find out that the positive coincidence rate of two kinds of kit test sample is 96%, 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, the detection of susceptibility can be found out from table 3 after immunity 14 days with the artificial immunity pseudorabies vaccine serum of kit detection different times, and the most of test positive of antibody is all positive after 21 days, explains that the sensitivity of kit is very high.
The detection of table 3 different times artificial immunity serum antibody
Annotate: when OD value S/N ratio greater than 0.902 time is judged to feminine gender greater than 0.7, when OD value S/N ratio less than 0.773 time is judged to the positive less than 0.6.
Claims (3)
1. enzyme linked immunological kit that detects PRV antibody, it is characterized in that: monoclonal antibody, the PRV monoclonal antibody that comprises the PRV of horseradish peroxidase-labeled is to be that monoclonal antibody, the PRV that immunogene obtains is the A strain of Pseudorabies virus Hubei Province with the PRV;
Described kit also comprises ELISA Plate, sample diluting liquid, positive control, negative control, colour developing liquid A, colour developing liquid B, cleansing solution, stop buffer;
Described ELISA Plate is to use 0.1mol/L when the coated elisa plate with PRV, and the sodium bicarbonate solution of pH9.6 is as encapsulating damping fluid;
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;
Said stop buffer is for containing 0.25% volume ratio hydrofluoric acid solution;
Described colour developing liquid A is the citrate buffer that contains the 50mg/mL carbamide peroxide, and colour developing liquid 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 that contains the gelatin of 2.5mg/mL.
2. a kind of enzyme linked immunological kit that detects PRV antibody according to claim 1; It is characterized in that: described monoclonal antibody, its preparation process are to adopt hybridoma cell technology, with PRV Hubei Province A strain of purifying; The immunity BALB/c mouse; Get its splenocyte and murine myeloma cell and carry out Fusion of Cells, after cultivating with the HAT selective medium, encapsulate plate with the PRV of purifying again and carry out the indirect ELISA screening; The positive cell strain of screening is cloned through limiting dilution assay; Block the ELISA screening with the pig positive control serum of pseudorabies vaccine immunity and the health pig negative serum that do not infect PRV again, finishing screen is chosen a strain monoclonal antibody, secretes the cell line called after PRV-GB of this antibody.
3. the blocking-up ELISA detection method of the described a kind of PRV antibody of claim 1 the steps include:
1) from kit, take out the check-out console that is coated with viral antigen in advance, the serum 1:1 to be checked that dilution is good dilutes 100 μ L and adds in the antigen coated microplate, establishes the positive and negative control wells simultaneously, respectively establishes 2 holes, every hole 100 μ L;
2) sample in the even hole that shakes was gently put 37 ℃ of incubations 30 minutes, got rid of the solution in the plate hole, washed plate 5 times with cleansing solution, and 200 μ L/holes are left standstill at every turn and outwelled in 3 minutes, do at the thieving paper arsis for the last time;
3) the enzyme-added mark monoclonal antibody 100 μ L in every hole put 37 ℃ of incubations 30 minutes, washed 5 times; The same step of method (2), every hole add colour developing liquid A, each one 50 μ L of colour developing liquid B, mixing; Room temperature lucifuge colour developing 10 minutes, every hole adds one 50 μ L of stop buffer, measures every hole OD with ELIASA in 15 minutes
630nmThe value of reading.
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