CN103808927B - A kind of enzyme linked immunological kit detecting porcine reproductive and respiratory syndrome virus - Google Patents
A kind of enzyme linked immunological kit detecting porcine reproductive and respiratory syndrome virus Download PDFInfo
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- CN103808927B CN103808927B CN201410039309.1A CN201410039309A CN103808927B CN 103808927 B CN103808927 B CN 103808927B CN 201410039309 A CN201410039309 A CN 201410039309A CN 103808927 B CN103808927 B CN 103808927B
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
Abstract
The invention discloses a kind of enzyme linked immunological kit detecting porcine reproductive and respiratory syndrome virus, belong to animal viral disease surveillance and diagnostic reagent field.This test kit includes being coated the ELISA Plate of the polyclonal antibody of anti-porcine reproductive and respiratory syndrome virus, the monoclonal antibody of biotin labeled anti-porcine reproductive and respiratory syndrome virus, enzyme mark Streptavidin, positive control, negative control, TMB nitrite ion, stop buffer, cleaning mixture and diluent.Test kit of the present invention is not merely with the more virus antigen epitope of polyclonal antibody identification, and utilizes biotin-labeled monoclonal antibody, is further increased the sensitivity of this test kit by the signal amplification of enzyme mark enzyme mark Streptavidin.This test kit and pig parvoviral, PRV (Pseudorabies virus), pig circular ring virus and swine fever virus no cross reaction, reproducible simultaneously, it is adaptable to the quick diagnosis of porcine reproductive and respiratory syndrome virus infection and research work.
Description
Technical field
The invention belongs to animal viral disease surveillance and diagnostic reagent field, be more specifically a kind of enzyme linked immunological kit detecting porcine reproductive and respiratory syndrome virus.
Background technology
Porcine reproductive and respiratory syndrome (Porcinereproductiveandrespiratorysyndrome, PRRS) it is commonly called as reproductive and respiratory syndrome, it is a kind of with sow breeding difficulty, piglet, the infectious disease that respiratory tract disease is feature is occurred by what porcine reproductive and respiratory syndrome virus (Porcinereproductiveandrespiratorysyndromevirus, PRRSV) caused.It it is one of animal epidemic of 93 kinds of statutory reports of listing in of OIE (OfficeInternationalDesEpizooties, OIE).According to statistics, this disease causes the economic loss (ChoandDee, 2006) of nearly 5.6 hundred million dollars to every year the pig industry of the U.S..China was separated to PRRSV in 1996 from aborted fetus, it was demonstrated that China there is also Porcine reproductive and respiratory syndrome.Along with viral heredity and variation, 2006, it was the high-pathogenicity blue ear disease (TianKetal., 2007) of cause of disease that China has been broken out again with the american type virus that makes a variation, and makes China's pig industry suffer huge economic loss.
The method detecting PPRSV at present mainly has the diagnostic methods such as RT-PCR method, Virus Isolation, Immunohistochemical Method, indirect immunofluorescence assay and serum neutralization test, but these methods otherwise need operation live virus, complicated operation, length consuming time, specificity and sensitivity are not enough, are unfavorable for application and the popularization of basic unit.And existing ELISA method is mainly using artificial recombination albumen as antigen, and seldom uses enzyme mark Streptavidin when detecting antigen with double-antibody method and amplify the effect of signal, so that specificity and sensitivity are not enough all to some extent.
Summary of the invention
The primary and foremost purpose of the present invention is in that the shortcoming overcoming prior art is with not enough, it is provided that a kind of enzyme linked immunological kit detecting porcine reproductive and respiratory syndrome virus.This test kit is under the premise ensureing specificity, sensitivity and repeatability, it is possible to simple and quick accurate detection porcine reproductive and respiratory syndrome virus.
Another object of the present invention is to provide the using method of the enzyme linked immunological kit of above-mentioned detection porcine reproductive and respiratory syndrome virus.
To achieve these goals, technical scheme provided by the invention is:
A kind of enzyme linked immunological kit detecting porcine reproductive and respiratory syndrome virus, its composition includes being coated the ELISA Plate of the polyclonal antibody of anti-porcine reproductive and respiratory syndrome virus, the monoclonal antibody of biotin labeled anti-porcine reproductive and respiratory syndrome virus, enzyme mark Streptavidin, positive control, negative control, TMB nitrite ion, stop buffer, cleaning mixture and diluent.
The polyclonal antibody of described anti-porcine reproductive and respiratory syndrome virus is prepared preferably by the method comprised the steps of: utilize Marc-145 cell to cultivate PPRSVJXA1-R strain, with the PPRSV of ultracentrifugation purification for antigen, after fully emulsified with Freund's complete adjuvant, immunity 60 age in days rabbit, 15 days, 30 days respectively with the fully emulsified rear each booster immunization of same antigen and incomplete Freund's adjuvant once, take a blood sample after 45 days, separate serum；The polyclonal antibody of anti-porcine reproductive and respiratory syndrome virus is obtained with ProteinA affinitive layer purification.
The ELISA Plate of the described polyclonal antibody being coated anti-porcine reproductive and respiratory syndrome virus is prepared preferably by the method comprised the steps of: by the polyclonal antibody of anti-porcine reproductive and respiratory syndrome virus with being coated liquid with every hole 100ng/100 μ L coated elisa plate, 4 DEG C are coated after overnight, close 2 hours with the PBST solution containing 2%BSA 37 DEG C, wash 3 times with the PBST of pH7.5 again, pat dry standby；Described is coated the carbonate buffer solution that liquid is 0.1M, pH9.6.
The concentration of the monoclonal antibody of described biotin labeled anti-porcine reproductive and respiratory syndrome virus is preferably 1mg/mL.The monoclonal antibody of biotin labeled anti-porcine reproductive and respiratory syndrome virus is prepared preferably by the method comprised the steps of:
(1) 10mg/mL biotin N-hydroxy-succinamide ester solution is prepared with anhydrous DMSO；
(2) antibody-solutions of 1～3mg/mL it is at least with the borate buffer solution compound concentration of 0.1mol/L, pH8.8；
(3) being added in antibody by biotin N-hydroxy-succinamide ester by the ratio of 25～100 μ g/mg, mix homogeneously also at room temperature hatches 4h；
(4) ammonium chloride of 20 μ L1mol/L, incubated at room 10min are added in every 250 μ g biotin N-hydroxy-succinamide esters；
(5) by antibody-solutions PBS or other required buffer dialysis, to remove unconjugated biotin.
The concentration of described enzyme mark Streptavidin is preferably 1mg/mL.
Described enzyme mark Streptavidin is prepared preferably by the method comprised the steps of: weighs 5mgHRP and is dissolved in 0.5mL distilled water, adds freshly prepared 0.06mol/LNaIO4Aqueous solution 0.5mL, mixing is put 4 degree of refrigerator and is reacted 30 minutes, take out and add 0.16mol/L glycol water 0.5mL, the aqueous solution 1mL containing 5mg Streptavidin is added after room temperature lucifuge is reacted 30 minutes, mix and fill the carbonate buffer solution of bag filter and 0.05mol/L, pH9.5 and be slowly stirred dialysis 6h or overnight, so as to combine；Then solution in sucking-off bag filter, adds the NaBH of 5mg/mL4Solution 0.2mL, puts refrigerator 2h, takes out and adds equal-volume saturated ammonium sulfate；It is centrifuged after putting refrigerator 30min, gained precipitate is dissolved in the PBS of a small amount of 0.02mol/L, pH7.4, and dialysed overnight；Next day is centrifuged off insoluble matter again, obtains enzyme mark Streptavidin.Adding to after 5mL is measured with the PBS of 0.02mol/L, pH7.4, lyophilization preserves.
Described positive control is preferably the PPRSV of 10ng/mL purification；Negative control is the PBS of 0.01mol/L, pH7.2.
Described TMB nitrite ion is preferably from commercial product；Stop buffer is preferably 2MH2SO4Solution；Cleaning mixture is preferably the 0.1MPBS solution containing 0.05%Tween-20, and pH is 7.2,10 times of dilutions during use；Diluent is preferably the PBS of 0.01mol/L, pH7.2 containing 1%BSA.
The using method of the enzyme linked immunological kit of above-mentioned detection porcine reproductive and respiratory syndrome virus, comprises the steps: that the every hole 100 μ L of ELISA Plate adds testing sample, hatches 30 minutes for 37 DEG C, PBST washs；Every hole adds the monoclonal antibody of the 100 μ L1:1000 times biotin labeled anti-porcine reproductive and respiratory syndrome virus diluted, and hatches 30 minutes for 37 DEG C, and PBST washs；Every hole adds the 100 μ L1:2000 times enzyme mark Streptavidin diluted, and hatches 30 minutes for 37 DEG C, and PBST washs；Every hole adds 100 μ LTMB nitrite ions, and 37 DEG C are reacted 10 minutes；Every hole adds 50 μ L stop buffers；Survey light absorption value by microplate reader at 450nm place, calculate S/P value result of determination (when S/P value >=0.18 is positive, be negative as S/P value < 0.18).S is the OD450nm readings of testing sample, and P is the OD450nm readings of positive control.
Compared with prior art, the test kit of the present invention has the following advantages and effect:
(1) present invention is not merely with the more virus antigen epitope of polyclonal antibody identification, and utilize biotin-labeled monoclonal antibody, and employ BAS(biotin-avidin detection system)-Elisas detects system, is substantially increased sensitivity and the stability of test kit by the signal amplification of enzyme mark enzyme mark Streptavidin.
(2) present invention is by adopting the single solution TMB nitrite ion of instant, and it is more convenient that bi-component nitrite ion used by more conventional test kit uses, and result is more stable.
(3) this test kit is simple to operation, and pig parvoviral, PRV (Pseudorabies virus), pig circular ring virus and swine fever virus no cross reaction, reproducible, it is adaptable to quick diagnosis that porcine reproductive and respiratory syndrome virus infects and research work.
Accompanying drawing explanation
Fig. 1 is present invention process flow chart.
Detailed description of the invention
Below in conjunction with embodiment and accompanying drawing, the present invention done further detailed description, but embodiments of the present invention are not limited to this.
Present invention process flow chart is as shown in Figure 1.
The preparation of embodiment 1 ELISA Plate
1, the preparation of porcine reproductive and respiratory syndrome virus antigen
Recovery Marc-145 cell, grows up to after monolayer until cell, outwells supernatant, inoculates a small amount of JXA1-R strain, adds maintenance medium (2% hyclone, 98%DMEM high glucose medium), after 37 DEG C of absorption 1 hour, adds maintenance medium to the original volume cultivating cell, 37 DEG C, 6%CO2Incubator is cultivated, and when cytopathy more than 80%, is placed in-20 DEG C of refrigerators by cell bottle, multigelation 3 times, collects virus liquid.The virus liquid 5000r/min, the 4 DEG C of centrifugal 30min that collect go precipitation.Supernatant 27000r/min, 4 DEG C centrifugal 1 hour, precipitate resuspended with a small amount of pH7.2,0.01MPBS.Prepare the discontinuous gradient sucrose of 15%, 25%, 35%, 45% and 55% mass volume ratio with PBS, add sample to gradient interface, 4 DEG C, the centrifugal 15h of 35900r/min.The albumen collecting 25% concentration place brings in centrifuge tube, and resuspended with PBS dilution, 36000r/min is centrifuged desaccharide in 3 hours.Finally suspending with PBS and precipitate ,-20 DEG C save backup.
2, the preparation of anti-porcine reproductive and respiratory syndrome virus polyclonal antibody
Take the antigen (about about 3mg) of appropriate purification fully emulsified with Freund's complete adjuvant after, adopt back multiple spot immunity 60 age in days rabbit；15 days, 30 days respectively with the fully emulsified rear each booster immunization of same antigen and incomplete Freund's adjuvant once；Collecting systemic blood after 45 days, 4 DEG C stand the centrifugal 15min of 3000g after overnight, collect serum.The polyclonal antibody of anti-porcine reproductive and respiratory syndrome virus is obtained with ProteinA affinitive layer purification.
3, it is coated polyclonal antibody
With every hole 100ng/100 μ L coated elisa plate, being coated liquid is 0.1M, pH9.6 carbonate buffer solution, and 4 DEG C are coated after overnight, closes 2 hours with the PBST solution containing 2%BSA 37 DEG C, then washs 3 times with the PBST of pH7.5, pats dry standby.
The preparation of the monoclonal antibody of the anti-porcine reproductive and respiratory syndrome virus of embodiment 2
1, the preparation of feeder cells
BALB/c mouse being extractd eyeball blood-letting, and separates serum for negative control, draw neck dislocation to put to death mice, be soaked in 75% alcoholic solution 5min, put into superclean bench immediately, abdominal part is fixed on dissection plate upward.Mention mouse part skin with tweezers, cut an osculum with shears, then with mosquito forceps to upper and lower both sides direction tearing skin, fully expose peritoneum.Sterilize with cotton ball soaked in alcohol wiping peritoneum.To draw incomplete DMEM in high glucose culture fluid 5mL with syringe, inject mouse peritoneal. the right hand fixes syringe, makes syringe needle be retained in intraperitoneal, and left hand holds the cotton ball soaked in alcohol l of abdomen massage gently～2min, it is also possible to this syringe repeatedly aspirates at intraperitoneal, injects for several times.Draw back intraperitoneal liquid by original annotation emitter, inject centrifuge tube.1000rpm is centrifuged 5min, abandons supernatant.First being suspended by sedimentation cell with 10mLHAT culture fluid and mix, making cell counting, then according to cell counts, add HAT culture fluid, making cell concentration is 2 × 105/ mL.Being added by cell suspension in 96 well culture plates, every hole 0.1mL, culture plate puts 37 DEG C containing 5%CO2Incubator in cultivate.
2, the preparation of splenocyte
The Purification of Pig Reproductive and respiratory syndrome virus antigen taking suitable concn mixes completely with equal-volume adjuvant, immune 6 week old Balb/c mices, and injection dosage is 60～100 μ g.At interval of three weeks subcutaneous inoculations once, after continuous immunity three times, merge first 3 days abdominal cavity booster immunizations.Using Freund's complete adjuvant during first time immunity, second and third time uses incomplete Freund's adjuvant, and booster immunization does not use adjuvant.Take the Balb/c mice of last booster immunization, extract eyeball blood-letting, separate positive serum for detection antibody, and by sacrifice, be soaked in 5min in 75% ethanol, put into super-clean bench immediately.Opening abdomen with aseptic operation, take out spleen, put into oneself and fill in the plate of the incomplete culture fluid of 5～10mL (serum-free DMEM high glucose medium, purchased from Hyclone company) and wash gently once, surrounding connective tissue is peelled off in carefulness.Spleen is moved into 200 eye mesh screens, puts in another plate filling the incomplete culture fluid of 10mL.Shred with aseptic eye scissors and ophthalmic tweezers, after complete, rock plate 2min, make the tissue caking precipitation do not pulverized.Sucking-off supernatant 1000r/min is centrifuged 10min, is the immune spleen cell prepared after removing supernatant.
3, the preparation of myeloma cell's suspension
Merge recovery the last week SP2/0 myeloma cell, cultivate in the hyclone culture fluid (serum-free DMEM high glucose medium, purchased from Hyclone company) containing 10%.Select in the myeloma cell of logarithmic growth, before merging in T75 Tissue Culture Flask amplification culture, every bottle adds 10mL culture fluid.Put 37 DEG C, containing 5%CO2Cultivate in incubator.Merge the same day, with connector bend dropping tube, the myeloma cell in logarithmic growth is blown down gently from bottle wall, be collected in 50mL centrifuge tube.And write down volume number, mixing.Take myeloma cell's suspension, add platform and expect that blue dye liquor is standby after making viable count.
4, cell fusion
Ready myeloma cell is washed twice with serum-free DMEM in high glucose culture fluid, spleen cell is also carried out same washing simultaneously, counts after washing.It is preheated to 37 DEG C for the PEG solution of cell fusion, serum-free DMEM in high glucose culture fluid.Draw respectively containing 1 × 108Individual splenocyte and 2～3 × 107The suspension of individual myeloma cell, adds in 50mL centrifuge tube, adds incomplete culture fluid to 30mL, fully mix.1000rpm is centrifuged 10min, is discarded by supernatant as far as possible.Gently at the bottom of attack pipe, make the loose uniform one-tenth pasty state of sedimentation cell, and this centrifuge tube is placed in 37 DEG C of water-baths.Uniform rotation centrifuge tube on the other hand, the tube wall (as far as possible close to cell place) that another hands 1mL suction pipe draws the 50% of 1.0mL preheating, the PEG solution edge of pH7.2 rotates adds.Control at about 60s from joining the time added, after static 1 minute, add incomplete culture fluid (stop buffer) and start to terminate merging.Concrete addition is that 1min adds 1mL, 2min add 2mL(all should limit edged rotate centrifuge tube gently), 3min subsequently adds 3mL, subsequently often increase by 1 minute stop buffer add 1mL until adding 20mL stop buffer.1000rpm is centrifuged 5min, supernatant discarded.Add HAT culture fluid (10mL/ plate), gently the cell of pressure-vaccum precipitation so that it is suspend and mix.Cell suspension is added in 96 well culture plates being covered with feeder cells, every hole 0.1mL.Culture plate puts 37 DEG C, containing 5%CO2Incubator in cultivate.
5, the screening of positive cell and clone
Marc-145 cell is incubated on 96 porocyte culture plates, when growing up to monolayer soon, one piece of Pigs Inoculated Reproductive and respiratory syndrome virus JXA1-R strain, another block not virus inoculation.After 48 hours, fix with acetone when pathological changes occurs.Then each culture supernatant adding hybridoma to be screened, 37 DEG C of incubations 1 hour, wash 3 times with pH7.2,0.01MPBS, add goat anti-mouse igg-FITC, 37 DEG C of lucifuges reaction 60min, wash 3 rearmounted fluorescence microscopy Microscopic observations.When there is fluorescence in virus inoculation strain hole, and when the non-virus inoculation of correspondence does not have fluorescence, this cell hole inner cell is judged to positive cell.Choosing growth vigorous, the good positive cell limiting dilution assay of form is cloned, and finally gives monoclonal hybridoma strain.
6, the preparation of odd contradictive hydroperitoneum and purification
Adopt the method that ascites produces monoclonal antibody that induces in animal body.Take healthy Balb/c female mice 5. every lumbar injection 500 μ L liquid norphytane, standby after 1～2 week.Blowing down by the positive colony hybridoma of cultivation, 1000rpm is centrifuged 10min, abandons supernatant, collects cell.Cannot be used up full culture fluid by cell suspension, mixing, and cell number is adjusted to 106Individual/mL, the mouse peritoneal of every pretreatment injects 500 μ L positive colony hybridomies；Collecting ascites after 7～10d, 3000rpm is centrifuged 10min, abandons fat deposit and cellular layer, and clear layer in the middle of collecting, tubule subpackage after purification ,-20 DEG C frozen.Take ascites 1 part pretreated and add 2 parts of 0.06mol/L, pH5.0 acetate buffer solutions, adjust pH to 4.8 with 1mol/LHCl；Diluting ascites in every milliliter and add 11 ratios sad for μ L, be stirred at room temperature down and be added dropwise over sad, added in 30 minutes, 4 DEG C stand 2 hours, take out 15000g centrifugal 30 minutes, abandon precipitation；Supernatant filters (125 μm) through nylon mesh, adds the 0.01mol/LPBS of 1/10 volume, adjusts pH to 7.2 with 1mol/LNaOH；At 4 DEG C, add saturated ammonium sulfate to 45% saturation, act on 30 minutes, stand 1 hour；Centrifugal 30 minutes of 10000g, abandons supernatant；Precipitation is dissolved in appropriate PBS(containing 137mmol/LNaCl, 2.6mol/LKCl, 0.2mmol/LEDTA) in, the PBS to 50-100 times of volume, 4 DEG C are overnight, change water therebetween more than 3 times；Take out 10000g centrifugal 30 minutes, remove insoluble sediment, after measuring protein content, subpackage, frozen standby.
The monoclonal antibody of embodiment 3 biotin labeling porcine reproductive and respiratory syndrome virus
(1) 10mg/mL biotin N-hydroxy-succinamide ester (biotin ester) solution is prepared with anhydrous DMSO；
(2) antibody-solutions of 1～3mg/mL it is at least with borate buffer solution (0.1mol/L, pH8.8) compound concentration；
(3) being added in antibody by biotin ester by the ratio of 25～100 μ g/mg, mix homogeneously also at room temperature hatches 4h；
(4) ammonium chloride of 20 μ L1mol/L, incubated at room 10min are added in every 250 μ g biotin esters；
(5) by antibody-solutions PBS or other required buffer dialysis, to remove unconjugated biotin.
Embodiment 4 one kinds detects the application of the enzyme linked immunological kit of porcine reproductive and respiratory syndrome virus
A kind of enzyme linked immunological kit detecting porcine reproductive and respiratory syndrome virus, including the ELISA Plate (embodiment 1) of the polyclonal antibody being coated anti-porcine reproductive and respiratory syndrome virus, the monoclonal antibody (embodiment 3) of biotin labeled anti-porcine reproductive and respiratory syndrome virus, enzyme mark Streptavidin, positive control, negative control, TMB nitrite ion, stop buffer, cleaning mixture and diluent.Positive control is the PPRSV of 10ng/mL purification；Negative control is the PBS of 0.01mol/L, pH7.2；TMB nitrite ion is from commercial product；Stop buffer is 2MH2SO4Solution；Cleaning mixture is the 0.1MPBS solution containing 0.05%Tween-20, and pH is 7.2,10 times of dilutions during use；Diluent is the PBS of 0.01mol/L, pH7.2 containing 1%BSA.
The preparation of enzyme mark Streptavidin: weigh 5mgHRP and be dissolved in 0.5mL distilled water, add freshly prepared 0.06mol/LNaIO4Aqueous solution 0.5mL, mixing is put 4 degree of refrigerator and is reacted 30 minutes, take out and add 0.16mol/L glycol water 0.5mL, the aqueous solution 1mL containing 5mg Streptavidin (Strepavidin) is added after room temperature lucifuge is reacted 30 minutes, mix and fill bag filter and 0.05mol/L, pH9.5 carbonate buffer solution and be slowly stirred dialysis 6h or overnight, so as to combine；Then solution in sucking-off bag filter, adds NaBH4Solution (5mg/mL) 0.2mL, puts refrigerator 2h, takes out and adds equal-volume saturated ammonium sulfate；It is centrifuged after putting refrigerator 30min, gained precipitate is dissolved in the PBS of a small amount of 0.02mol/L, pH7.4, and dialysed overnight；Next day is centrifuged off insoluble matter again, obtains enzyme mark Streptavidin.Adding to after 5mL is measured with the PBS of 0.02mol/L, pH7.4, lyophilization preserves.
The concrete steps of the enzyme linked immunological kit of application porcine reproductive and respiratory syndrome virus:
(1) taking out ELISA Plate from test kit, every hole 100 μ L adds testing sample, and positive control and negative control respectively add 2 holes, every hole 100 μ L simultaneously.Put 37 DEG C and hatch 30 minutes.
(2) wash 3 times with PBST, add 350 μ L/ holes every time, stand 3min.
(3) every hole adds the biotinylation monoclonal antibody that original content is 1mg/mL of 100 μ L1:1000 times diluted, hatches 30 minutes for 37 DEG C.
(4) wash 3 times with PBST, add 200 μ L/ holes every time, stand 3min.
(5) original content that every hole adds 100 μ L1:2000 times diluted is 1mg/mL enzyme mark Streptavidin, hatches 30 minutes for 37 DEG C.
(6) wash 5 times with PBST, add 200 μ L/ holes every time, stand 3min.
(7) every hole adds 100 μ LTMB nitrite ions, and after 37 DEG C of reactions 10 minutes, every hole adds 50 μ L stop buffers.By microplate reader at 450nm place survey light absorption value.Calculate S/P value result of determination (when S/P value >=0.18 is positive, be negative as S/P value < 0.18).S is the OD450nm readings of test serum sample, and P is the OD450nm readings of positive control.
The judgement of ELISA marginal value: 50 parts of positive serum samples and 50 parts of negative serum samples are carried out ELISA detection, (S is the OD450nm readings of test serum sample to calculate S/P value, P is the OD450nm readings of positive control), SPSS software is utilized to do the correlation analysis of the two S/P value, determine that the maximum marginal value met is S/P=0.18, therefore, it is determined that when S/P value >=0.18 is positive, be negative as S/P value < 0.18.
Embodiment 5 specifically applies effect
(1) cross reaction test:
The enzyme linked immunological kit of porcine reproductive and respiratory syndrome virus is used to carry out cross reaction experiment with pig parvoviral, PRV (Pseudorabies virus), pig circular ring virus, swine fever virus, porcine reproductive and respiratory syndrome virus positive control, porcine reproductive and respiratory syndrome virus negative control respectively.Result is as shown in table 1, it was shown that be absent from cross reaction between this test kit and other viral diseases of pig.
Table 1. cross reaction is tested
| Antigen | Parvovirus | Pseudorabies virus | Porcine circovirus | Swine fever virus | Positive control | Negative control |
| OD450nmValue | 0.032 | 0.036 | 0.037 | 0.033 | 0.825 | 0.026 |
(2) sensitivity tests
Utilizing the concentration that this test kit detects minimum porcine reproductive and respiratory syndrome virus is 20ng/mL.
Table 2. sensitivity tests
| PRRSV concentration (ng/mL) | 320 | 160 | 80 | 40 | 20 | 10 | Blank |
| OD450nmValue | 3.423 | 1.885 | 1.053 | 0.528 | 0.314 | 0.132 | 0.036 |
(3) specific test
Take 200 parts of clinical samples (including serum and each tissue samples), detect with RT-PCR with this test kit after cracking simultaneously simultaneously.Result shows that the coincidence rate of the method and RT-PCR is 96%.
Wherein, RT-PCR correlated condition is as follows:
Forward primer: 5'-GAATGGCCAGCCAGTCAATC-3'；
Downstream primer: 5'-GTCGCCCTAATTGAATAGGTGAC-3'.
Positive control: the JXA1-R strain cultivated with Marc145 cell；
Negative control: the DEPC water of sterilizing ,-20 DEG C of preservations.
Response procedures: 42 DEG C of 45min, 95 DEG C of 3min, amplification condition is 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s；After 35 circulations, 72 DEG C extend 10min.
Table 3. specific test
Above-described embodiment is the present invention preferably embodiment; but embodiments of the present invention are also not restricted to the described embodiments; the change made under other any spirit without departing from the present invention and principle, modification, replacement, combination, simplification; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
SEQUENCELISTING
<110>Wuhan Chopper Biology Co., Ltd.
<120>a kind of enzyme linked immunological kit detecting porcine reproductive and respiratory syndrome virus
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gtcgccctaattgaataggtgac23
Claims (2)
1. the enzyme linked immunological kit detecting porcine reproductive and respiratory syndrome virus, it is characterised in that including: be coated the ELISA Plate of the polyclonal antibody of anti-porcine reproductive and respiratory syndrome virus, the monoclonal antibody of biotin labeled anti-porcine reproductive and respiratory syndrome virus, enzyme mark Streptavidin, positive control, negative control, TMB nitrite ion, stop buffer, cleaning mixture and diluent；
The polyclonal antibody of described anti-porcine reproductive and respiratory syndrome virus is prepared by the method comprised the steps of: utilize Marc-145 cell to cultivate PRRSVJXA1-R strain, with the PRRSV of ultracentrifugation purification for antigen, after fully emulsified with Freund's complete adjuvant, immunity 60 age in days rabbit, 15 days, 30 days respectively with the fully emulsified rear each booster immunization of same antigen and incomplete Freund's adjuvant once, take a blood sample after 45 days, separate serum；The polyclonal antibody of anti-porcine reproductive and respiratory syndrome virus is obtained with ProteinA affinitive layer purification；
The ELISA Plate of the described polyclonal antibody being coated anti-porcine reproductive and respiratory syndrome virus is prepared by the method comprised the steps of: by the polyclonal antibody of anti-porcine reproductive and respiratory syndrome virus with being coated liquid with every hole 100ng/100 μ L coated elisa plate, 4 DEG C are coated after overnight, close 2 hours with the PBST solution containing 2%BSA 37 DEG C, wash 3 times with the PBST of pH7.5 again, pat dry standby；Described is coated the carbonate buffer solution that liquid is 0.1M, pH9.6；
The concentration of the monoclonal antibody of described biotin labeled anti-porcine reproductive and respiratory syndrome virus is 1mg/mL；The monoclonal antibody of biotin labeled anti-porcine reproductive and respiratory syndrome virus is prepared by the method comprised the steps of:
(1) 10mg/mL biotin N-hydroxy-succinamide ester solution is prepared with anhydrous DMSO；
(2) antibody-solutions of 1～3mg/mL it is at least with the borate buffer solution compound concentration of 0.1mol/L, pH8.8；
(3) being added in antibody by biotin N-hydroxy-succinamide ester by the ratio of 25～100 μ g/mg, mix homogeneously also at room temperature hatches 4h；
(4) ammonium chloride of 20 μ L1mol/L, incubated at room 10min are added in every 250 μ g biotin N-hydroxy-succinamide esters；
(5) by antibody-solutions PBS or other required buffer dialysis, to remove unconjugated biotin；
The concentration of described enzyme mark Streptavidin is 1mg/mL；Enzyme mark Streptavidin is prepared by the method comprised the steps of: weighs 5mgHRP and is dissolved in 0.5mL distilled water, adds freshly prepared 0.06mol/LNaIO4Aqueous solution 0.5mL, mixing is put 4 degree of refrigerator and is reacted 30 minutes, take out and add 0.16mol/L glycol water 0.5mL, the aqueous solution 1mL containing 5mg Streptavidin is added after room temperature lucifuge is reacted 30 minutes, mix and fill the carbonate buffer solution of bag filter and 0.05mol/L, pH9.5 and be slowly stirred dialysis 6h or overnight, so as to combine；Then solution in sucking-off bag filter, adds the NaBH of 5mg/mL4Solution 0.2mL, puts refrigerator 2h, takes out and adds equal-volume saturated ammonium sulfate；It is centrifuged after putting refrigerator 30min, gained precipitate is dissolved in the PBS of a small amount of 0.02mol/L, pH7.4, and dialysed overnight；Next day is centrifuged off insoluble matter again, obtains enzyme mark Streptavidin；
Described positive control is the PPRSV of 10ng/mL purification；Negative control is the PBS of 0.01mol/L, pH7.2；
Described stop buffer is 2MH2SO4Solution；Cleaning mixture is the 0.1MPBS solution containing 0.05%Tween-20, and pH is 7.2,10 times of dilutions during use；Diluent is the PBS of 0.01mol/L, pH7.2 containing 1%BSA.
2. the using method of the enzyme linked immunological kit of the detection porcine reproductive and respiratory syndrome virus described in claim 1, it is characterised in that comprising the steps: that the every hole 100 μ L of ELISA Plate adds testing sample, hatch 30 minutes for 37 DEG C, PBST washs；Every hole adds the monoclonal antibody of the 100 μ L1:1000 times biotin labeled anti-porcine reproductive and respiratory syndrome virus diluted, and hatches 30 minutes for 37 DEG C, and PBST washs；Every hole adds the 100 μ L1:2000 times enzyme mark Streptavidin diluted, and hatches 30 minutes for 37 DEG C, and PBST washs；Every hole adds 100 μ LTMB nitrite ions, and 37 DEG C are reacted 10 minutes；Every hole adds 50 μ L stop buffers；Surveying light absorption value by microplate reader at 450nm place, calculate S/P value result of determination, wherein S is the OD450nm readings of testing sample, and P is the OD450nm readings of positive control, when S/P value >=0.18 is positive, is negative as S/P value < 0.18.
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| CN104593522A (en) * | 2014-09-12 | 2015-05-06 | 黄光东 | Method for fast detection of porcine viral infectious diseases |
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| CN112946285B (en) * | 2021-02-03 | 2022-07-01 | 广东永顺生物制药股份有限公司 | ELISA kit for detecting highly pathogenic PRRSV infection |
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