CN102608315A - Enzyme linked immunosorbent assay kit for porcine pseudorabies virus gE protein antibody and application thereof - Google Patents
Enzyme linked immunosorbent assay kit for porcine pseudorabies virus gE protein antibody and application thereof Download PDFInfo
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Abstract
The invention discloses an enzyme linked immunosorbent assay (ELISA) kit for porcine pseudorabies virus gE protein antibody and application of the ELISA kit. The ELISA kit comprises porcine pseudorabies virus gE protein monoclonal antibody labeled with horseradish peroxidase, an ELISA plate, sample diluent, negative (positive) controls, developer, washing solution and stop buffer, wherein the porcine pseudorabies virus is porcine pseudorabies virus Ea-strain. With respect to the application of the kit in detecting the porcine pseudorabies virus gE protein antibody, the method comprises: a. taking the ELISA plate out from the kit, adding diluted serum to be detected into an antigen coated plate, and setting negative and positive control wells; b. shaking the sample in the wells uniformly, throwing solution in the plate wells away, and washing with the washing solution; c. adding the monoclonal body labeled by the horseradish peroxidase into each well, washing, adding the developer, developing at room temperature away from light, adding the stop buffer, and determining OD630 reading of each wall with a microplate reader. The kit disclosed by the invention has the advantages of good specificity, high sensitivity and short detection time. In addition, an S/N ratio method is adopted for result determination, and the accuracy is high.
Description
Technical field
The present invention relates to animal epidemic detects; Be specifically related to a kind of enzyme linked immunological kit of PRV gE protein antibodies; The kit purposes that also relates to a kind of enzyme linked immunological of PRV gE protein antibodies simultaneously is applicable to the Pseudorabies virus gE protein antibodies level that detects in the porcine blood serum.
Technical background
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 elimination of pseudoabies in the works, monitoring and detect that pseudorabies virus gE protein antibodies is a very important link in the porcine blood serum.Because pseudo-rabies vaccine at present on the market all is the vaccine of gE gene delection basically, vaccinated pig does not only contain PRV gE protein antibodies in the serum.If detected the gE protein antibodies, explain that pig is by the wild virus infection of pseudorabies virus.Can the pig that infect be eliminated, thoroughly eradicate pseudoabies.
Present domestic detection PRV gE protein antibodies generally adopts indirect ELISA method etc., and these method susceptibility are not high, and dimension unavoidably can cause erroneous judgement to actual result.
The present invention uses the monoclonal antibody and the blocking-up ELISA detection technique of PRV gE albumen, 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 of PRV gE protein antibodies, 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 the application that has been to provide a kind of enzyme linked immunological kit of PRV gE protein antibodies.With PRV as antigen coated on elisa plate; Adding serum to be checked then hatches; Add the PRV gE monoclonal antibody of enzyme labeling again, should occur two kinds of situation 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.This kit specificity is good, susceptibility is high.With the detection coincidence rate of the PRV gI of American I DEXX company protein antibodies ELISA 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 kit of enzyme linked immunological of PRV gE protein antibodies; The monoclonal antibody that comprises the PRV of horseradish peroxidase-labeled; Described PRV monoclonal antibody is to be the monoclonal antibody of the hybridoma cell strain 2E6 secretion that obtains of immunogene with the PRV; 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, cleansing solution, colour developing liquid A, colour developing liquid B, 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 that PRV infects, 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% volume ratio Tween-20; Described 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 are that (referring to document: Shen Guanxin, Zhou Rulin, modern immunological experiment is technological for the employing hybridoma cell technology; Wuhan: Hubei science tech publishing house;, 1998), with the immune BALB/c mouse of PRV (Hubei Province A strain) of purifying; Get its splenocyte and murine myeloma cell (SP2/0) and carry out Fusion of Cells; After cultivating with HAT (available from SIGMA company) selective medium, use A strain of PRV Hubei Province and pseudoabies gE/gI gene-deleted strain (referring to the Chinese invention patent ublic specification of application, number of patent application 200510019513.8) to carry out the indirect immunofluorescence screening again; The positive cell strain of screening is cloned through limiting dilution assay; The pig positive control serum and the immune negative control sera of PRV gene-deleted vaccine that infect with PRV are blocked the ELISA screening again, and finishing screen is chosen the monoclonal antibody that a strain has barrier effect, secretes the cell line called after 2E6 of this antibody.
The application of a kind of kit of enzyme linked immunological of PRV gE protein antibodies in the enzyme linked immunological that detects PRV gE protein antibodies the steps include:
1) from kit, takes out ELISA Plate (being coated with the check-out console of viral antigen); With sample diluting liquid serum to be checked is done 2 times of dilutions (60 μ L sample diluting liquids and 60 μ L serum to be checked mixes); Getting 100 μ L mixed liquors adds in the ELISA Plate; Positive control and negative control respectively add 2 holes, every hole 100 μ L simultaneously.
2) sample (not overflowing) in the even hole that shakes was gently put 37 ℃ of incubations 45 minutes.
3) discard solution in the hole, add cleansing solution, 200 μ L/ holes are left standstill and were outwelled in 3 minutes, repeat to wash plate 5 times, do at the thieving paper arsis for the last time.
4) the enzyme-added mark monoclonal antibody 100 μ L in every hole put 37 ℃ of incubations (same step 3) of method of washing plate after 20 minutes 5 times.
5) every hole adds colour developing liquid A, colour developing liquid B each (about 50 μ L), mixing, room temperature (18～25 ℃, below identical) lucifuge colour developing 10 minutes.Every hole adds one of stop buffer (about 50 μ L), measures every hole OD with ELIASA in 10 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 0.3.S=sample well OD
630nmValue, the average OD of N=negative control hole
630nmValue.If S/N ratio is less than or equal to 0.35, sample is judged to the PRV-gE antibody positive.If S/N ratio is less than or equal to 0.4 but greater than 0.35, 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 0.4, sample is judged to the PRV-gE 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 1:
A kind of enzyme linked immunological kit that detects PRV gE protein antibodies; The monoclonal antibody that comprises the PRV of horseradish peroxidase-labeled; Described PRV monoclonal antibody is to be the monoclonal antibody of the hybridoma cell strain 2E6 secretion that obtains of immunogene with the PRV; 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, cleansing solution, colour developing liquid A, colour developing liquid B, 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 that PRV infects, 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% volume ratio Tween-20; Described 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 are that (referring to document: Shen Guanxin, Zhou Rulin, modern immunological experiment is technological for the employing hybridoma cell technology; Wuhan: Hubei science tech publishing house;, 1998), with the immune BALB/c mouse of PRV (Hubei Province A strain) of purifying; Get its splenocyte and murine myeloma cell (SP2/0) and carry out Fusion of Cells; After cultivating with HAT (available from SIGMA company) selective medium, use A strain of PRV Hubei Province and pseudoabies gE/gI gene-deleted strain (referring to the Chinese invention patent ublic specification of application, number of patent application 200510019513.8) to carry out the indirect immunofluorescence screening again; The positive cell strain of screening is cloned through limiting dilution assay; The pig positive control serum and the immune negative control sera of PRV gene-deleted vaccine that infect with PRV are blocked the ELISA screening again, and finishing screen is chosen the monoclonal antibody that a strain has barrier effect, secretes the cell line called after 2E6 of this antibody.
The kit assembling:
Kit contains 2 of 96 hole ELISA check-out consoles; Enzyme is marked 1 bottle of anti-Pseudorabies virus gE protein 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); Substrate solution A, substrate solution B, stop buffer each 1 bottle (10mL/ bottle) attach 1 part in instructions.
20 times of concentrated cleaning solution 160 gram sodium chloride, 10ml Tween-20 dissolve in the 1000ml distilled water.
Confining liquid contains the phosphate buffer of 0.5%BSA.
Sample diluting liquid is to contain the DMEM nutrient culture media that the quality percentage composition is 0.25% gelatin.
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.
Embodiment 2: 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% volume ratio calf serum, a small amount of virus of inoculation; 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 a small amount of pH7.20.01mol/L PBS with centrifugal 1 hour of 4 ℃ of 27000r/min.Discontinuous gradient sucrose with PBS preparation quality mark 20%, 35%, 45% and 60% mass volume ratio adds 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 the PBS dilution, 27000r/min desugar in centrifugal 2 hours.Suspend with 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 0.5%BSA pH7.4; Protect 3 hours by 100 μ L/ holes with the phosphate buffer room temperature that contains 20% mass volume ratio sucrose pH7.4; After putting the hothouse drying, sealing is preserved in the packing that contains drying agent of packing into.
Embodiment 3: the preparation and the mark of anti-Pseudorabies virus gE protein monoclonal antibody
The preparation of feeder cells
Merging the previous day; Get a normal female BALB/c mouse, 75% volume ratio alcohol disinfecting 5min is put to death in the cervical vertebra dislocation; 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.Being filled the HAT that contains 20% volume ratio hyclone with the 10mL disposable syringe selects nutrient solution to inject mouse peritoneal; The right hand fixedly syringe is motionless; Left hand is gently rubbed mouse web portion with tweezers gripping alcohol swab; Extract Intraabdominal nutrient solution out with syringe again, inject the sterilization plate, add above-mentioned nutrient solution 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 5%CO2 incubator.
The preparation immune spleen cell:
Get the purifying PRV viral antigen and the abundant mixing of equal-volume adjuvant of suitable concn, the Balb/C mouse in immune 5 ages in week, ID is 60～100 micrograms.The two all subcutaneous immunity of every interval 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, the eyeball excise bloodletting, separation of serum is made positive control and is used.Mouse is carried out disinfection by last method 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, and the counting back is subsequent use.
The 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 basic culture solution that cell is resuspended, the numeration back is subsequent use.
Fusion of Cells:
Earlier the centrifugal 5min of immune spleen cell 1000r/min is removed supernatant, subsequent use in 37 ℃ of placements.With 1 * 10
7Individual SP2/0 and 10
8Individual immune spleen cell is mixing in the 50mL centrifuge tube, centrifugal 10min.The turned letter supernatant blots tube wall with the filter paper of sterilizing, and 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 50% volume ratio PEG 0.8mL (Sigma) of preparatory temperature to 37 ℃ then, the limit edged stirs with pipette tip gently.Continue to stir 1min.Slowly add 37 ℃ of basal liquid 10mL 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 remaining 5mL on the 5th minute, each added-time needs slowly to add, and constantly stirs lightly.Add 30mL 1640 liquid at last, also need slowly to add.The centrifugal 5min of 800r/min goes supernatant to place 5min in 37 ℃.Suspend with the HAT nutrient culture media, the mixing with cells after also raising splenocyte and merge with the suspension of HAT nutrient culture media is simultaneously added an amount of HAT nutrient culture media as required, divides and plants in 96 porocyte culture plates, about 250 μ L/ holes.Once merge and to inoculate 4 96 orifice plates.In 37 ℃ of 5%CO
2Cultivate in the incubator.Merge and began in back second day to observe, have pollution-freely, added 1 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 omits flavescence, carry out antibody test.
The screening of positive cell and clone:
Positive cell is with IIF method preliminary screening: bhk cell is incubated in 2 96 porocyte culture plates, and when growing up to individual layer soon, Hubei Province A strain of an inoculation Pseudorabies virus, another piece inoculation gE/gI gene-deleted strain Pseudorabies virus.After 36 hours, when pathology occurring, use acetone fixed.Each adds the culture supernatant of hybridoma to be screened then, and 37 ℃ of incubations 1 hour are given a baby a bath on the third day after its birth time with the PBS of pH7.20.01mol/L, add the sheep anti-mouse igg-FITC of dilution in 1: 200 again, 37 ℃ of lucifuges reaction 30min, wash three times after, put observation under the fluorescent microscope.When the cell hole of inoculation Hubei Province A strain Pseudorabies virus has green fluorescence, and the cell hole of corresponding inoculation gE/gI gene-deleted strain Pseudorabies virus is not when having fluorescence, and this cell hole inner cell is judged to positive cell.It is vigorous to choose growth, and the good positive cell of form is cloned with limiting dilution assay.
Monoclonal antibody is identified:
The supernatant in the positive cell hole of preliminary screening is collected, add the good contrast 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 the substrate solution colour developing, after HF acid cessation reaction, measured value under the wavelength of 630nm.When the OD value that adds the positive serum hole was starkly lower than adding negative serum hole OD value, this monoclonal antibody was both for having the monoclonal antibody of blocking effect.
Monoclonal antibody is produced:
Cultivate the strain of above-mentioned evaluation positive hybridoma cell with conventional nutrient solution.A large amount of manufacture order clonal antibodies can adopt in the mouse body and induce method.Inject female mice (6～8 age in week) intraperitoneal through hybridoma through not exclusively not formula adjuvant processing with the anti-Pseudorabies virus antibody of the above-mentioned generation that obtains; Collect ascites after 10 days; The centrifugal solid constituent of removing, its supernatant promptly contain the monoclonal antibody of anti-PRV virus gE albumen.
Purifying antibody adopts sad-ammonium sulfate method, and is specific as follows: in the pretreated ascites of portion, 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.
The horseradish peroxidase-labeled of Pseudorabies virus monoclonal antibody:
Taking by weighing 5mg horseradish peroxidase (HRP) is dissolved in the lmL 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 antibody purification 1mL that adds 5mg/mL again, 4 ℃ of dialysed overnight in the carbonate buffer solution of 0.05M pH9.5.Add the NaBH40.2mL that 5mg/mL newly joins after the sucking-off and placed 2 hours for 4 ℃, add isopyknic saturated ammonium sulfate solution again, place 30min for 4 ℃, the centrifugal 10min of 7000r/min abandons supernatant, and is resuspended with physiological saline, again dialysed overnight in the PBS of 0.15M pH7.4.The antibody of collecting mark in the bag filter carries out working concentration to be demarcated, packing then (enzyme labelled antibody of each batch all will carry out working concentration and demarcate).
Embodiment 4:
A kind of application that detects the enzyme linked immunological kit of PRV gE protein antibodies the steps include:
1) from kit, takes out ELISA Plate (being coated with the check-out console of viral antigen); With sample diluting liquid serum to be checked is done 2 times of dilutions (60 μ L sample diluting liquids and 60 μ L serum to be checked mixes); Getting 100 μ L mixed liquors adds in the ELISA Plate; Positive control and negative control respectively add 2 holes, every hole 100 μ L simultaneously.
2) sample (not overflowing) in the even hole that shakes was gently put 37 ℃ of incubations 45 minutes.
3) discard solution in the hole, add cleansing solution, 200 μ L/ holes are left standstill and were outwelled in 3 minutes, repeat to wash plate 5 times, do at the thieving paper arsis for the last time.
4) the enzyme-added mark monoclonal antibody 100 μ L in every hole put 37 ℃ of incubations and wash plate after 20 minutes 5 times, (the same step 3) of method.
5) every hole adds colour developing liquid A, colour developing liquid B each (about 50 μ L), mixing, room temperature (18～25 ℃, below identical) lucifuge colour developing 10 minutes.Every hole adds one of stop buffer (about 50 μ L), measures every hole OD with ELIASA in 10 minutes
630nmThe value of reading.The situation of institute's result of use experiment is following in the application:
The test of 1 specificity:
Detect standard positive serum and porcine pseudorabies negative serums such as porcine pseudorabies, swine fever, Schweineseuche (0 type), pig japanese b encephalitis 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 0.35; All the other serum S/N values are greater than 0.4; Meet the criterion of negative serum, show that the specificity of this method is good.
The detection of table 1 specific serum
| PRV | CSFV | FMD | JEV | PRRSV | Negative control | |
| S/N | 0.133 | 0.972 | 0.904 | 0.923 | 0.845 | 1 |
| OD630 | 0.146 | 1.065 | 0.991 | 1.012 | 0.926 | 1.096 |
2, the detection of susceptibility:
Detect the serum of the infected pigs Pseudorabies virus of different extension rates simultaneously with self-control kit and import IDEXX kit; From table 3, can find out when No. 948 serum dilute 20 times; Two kinds of method detection antibody are all positive; When 40 times of No. 950 and the dilutions of No. 955 serum, it is all positive that two kinds of methods detect antibody, explains that homemade kit is suitable with import IDEXX kit susceptibility.
The infection serum testing result of the different extension rates of table 3
Annotate: the criterion of IDEXX kit: S/N is judged to feminine gender greater than 0.7, and S/N is less than or equal to 0.6 and is judged to the positive.0.6 it is suspicious that＜S/N≤0.7 is judged to. the criterion of self-control kit is: S/N is judged to feminine gender greater than 0.4, and S/N is less than or equal to 0.35 and is judged to the positive.0.35 it is suspicious that＜S/N≤0.4 is judged to.
3, coincidence rate is relatively:
With the 979 part serum of two kinds of parallel detections of detection kit from different pig farms, the result sees table 2.From table, can find out that the positive coincidence rate of two kinds of kit test sample is 91%, the coincidence rate of negative sample is 98%, and total coincidence rate is 97%.
Table 2 PRV gE protein antibodies detection kit and the PRV-gI ELISA of IDEXX company antibody assay kit coincidence rate are relatively
Claims (3)
1. the kit of the enzyme linked immunological of a PRV gE protein antibodies, it is characterized in that: monoclonal antibody, the PRV gE protein monoclonal antibody that comprises the PRV gE albumen 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, cleansing solution, colour developing liquid A, colour developing liquid B, 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 that PRV infects;
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% volume ratio Tween-20;
Described 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. the kit of the enzyme linked immunological of a kind of PRV gE protein antibodies according to claim 1; It is characterized in that: described monoclonal antibody; Its preparation process is to adopt hybridoma cell technology, with PRV Hubei Province A strain immunity BALB/c mouse of purifying, gets its splenocyte and murine myeloma cell and carries out Fusion of Cells; After cultivating with the HAT selective medium; Carry out the indirect immunofluorescence screening with A strain of PRV Hubei Province and Pseudorabies virus gE/ gI gene-deleted strain again, the positive cell strain of screening is cloned through limiting dilution assay, and the pig positive control serum and the immune negative control sera of PRV gene-deleted vaccine that infect with PRV are blocked the ELISA screening again; Finishing screen is selected monoclonal antibody, secretes the cell line called after 2E6 of this antibody.
3. the application of the kit of the enzyme linked immunological of the described a kind of PRV gE protein antibodies of claim 1 in the enzyme linked immunological that detects PRV gE protein antibodies the steps include:
1) from kit, takes out ELISA Plate; With sample diluting liquid serum to be checked is done 2 times of dilutions: 60 μ L sample diluting liquids and 60 μ L serum to be checked mixes; Get 100 μ L mixed liquors and add in the ELISA Plate, positive control and negative control respectively add 2 holes, every hole 100 μ L simultaneously;
2) sample in the even hole that shakes was gently put 37 ℃ of incubations 45 minutes;
3) discard solution in the hole, add cleansing solution, 200 μ L/holes are left standstill and were outwelled in 3 minutes, repeat to wash plate 5 times, do at the thieving paper arsis for the last time;
4) the enzyme-added mark monoclonal antibody 100 μ L in every hole put 37 ℃ of incubations and wash plate after 20 minutes 5 times;
5) every hole adds each one of colour developing liquid A, colour developing liquid B, mixing, and room temperature lucifuge colour developing 10 minutes, every hole adds one of stop buffer, measures every hole OD with ELIASA in 10 minutes
630nmThe value of reading.
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Application publication date: 20120725 |