CN107286236A - A kind of anti-PEDV monoclonal antibody and its chemiluminescence method detection method - Google Patents
A kind of anti-PEDV monoclonal antibody and its chemiluminescence method detection method Download PDFInfo
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- 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
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- 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
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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Abstract
The present invention provides a kind of porcine epidemic diarrhea resisting virus（PEDV）Monoclonal antibody and chemical luminescence detection method, from the triple attenuated vaccine of pig epidemic diarrhea transmissible gastroenteritis of swine rotavirus purifying obtain PEDV strains, be used as the antigen for preparing PEDV monoclonal antibodies；By Virus culture, immune Balb/c mouse, using cell-fusion techniques, anti-PEDV IgG2a monoclonal antibodies are obtained；Using the monoclonal antibody, concentration, confining liquid, ELIAS secondary antibody concentration and the optimization in reaction time are coated with by coating buffer, monoclonal antibody, a kind of detection PEDV chemical luminescence detection method is established.This method has high specificity, and sensitiveness is high, disposable detection limit is big and the features such as good stability, can detect 0.26ng virus.
Description
Technical field
The invention belongs to biological monitoring field, and in particular to a kind of anti-PEDV monoclonal antibody and its chemiluminescence method
Detection method.
Background technology
Pig epidemic diarrhea（PED）By Porcine epidemic diarrhea virus（PEDV）Cause pig occur vomiting, diarrhoea, enteritis,
The high mortality of dehydration and suckling pig is characterized.The swinery of all different phases can be infected, and the incidence of disease is fed up to 100%
The death rate of suckling piglet is up to more than 90%.The flourishing country of many pig industrys all oneself have the report that this disease is broken out, especially exist
The area such as Asia, Europe causes great economic loss.
PED is then reflected first first in 1971 in Britain's report in the disease in 1978 in Belgium and Britain
It is fixed, China confirmed in 1984 the disease presence, most of pig-raising countries have the sick report in the world at present, especially
It is serious with the morbidity of the country such as South Korea, Japan, China, Thailand and Vietnam.Since the autumn in 2010, south China part province occurs
Serious piglet epidemic diarrhea, incidence causes young substantial amounts of suckling pig morbidity death, especially than previously more violent
It is that the piglet death rate within 7 ages in days is up to 100%, and huge economic loss is caused to raiser, and turns into and influence me
One of important epidemic disease that state's pig industry develops in a healthy way.Because PED and transmissible gastroenteritis of swine (TGE) are in epidemiology, clinical condition
This upper closely similar quick diagnosis sick to this of the change such as shape and preventing and treating all bring certain difficulty.Further, since PEDV has
The characteristics of difficulty is big is separately cultured in vitro, passes through some egg of prokaryotic expression virus mostly in terms of monoclonal antibody is developed
In vain, the monoclonal antibody for resisting corresponding albumen is prepared, and by PEDV vaccine low virulent strains, develops anti-PEDV IgG2a monoclonals
Antibody, and monoclonal antibody is utilized, chemical method detection PEDV methods are set up, report is had no.Therefore, this research and utilization plaque is pure
Change technology, PEDV low virulent strains are obtained from the triple attenuated vaccine of pig epidemic diarrhea-transmissible gastroenteritis of swine-rotavirus,
As antigen, PEDV monoclonal antibodies are prepared, and set up chemiluminescence detection technology and the diagnosis sick applied to this, to the disease
Efficient diagnosis is significant with prevention and control.
The content of the invention
It is main it is an object of the invention to provide the monoclonal antibody of anti-PEDV a kind of and its chemiluminescence method detection method
Plaque-purified technology is utilized, from the trigeminal live vaccine of Porcine epidemic diarrhea virus-transmissible gastroenteritis of swine-porcine rotavirus
Purifying obtains the weak poison of PEDV, by cell culture, gradient centrifugation, immune small white mouse and preparation monoclonal antibody core technology, obtains
The strain of PEDV monoclonal antibody hybridoma cells and PEDV IgG2a monoclonal antibodies must be resisted, monoclonal antibody has potency high, special
The features such as opposite sex is by force and stability is good.It is excellent by sets of conditions such as coating buffer, monoclonal antibody coating concentration using PEDV monoclonal antibodies
Change, establish a kind of detection PEDV chemical luminescence detection method.This method has high specificity, sensitiveness height, disposable inspection
The features such as measurement is greatly and stability is good, can detect 0.26ng virus.The chemical luminescence detection method of foundation, using show with
The coincidence rate of RT-PCR detection techniques, with fluorescence quantitative RT-PCR detecting method coincidence rate 100%, is adapted to PEDV more than 95%
Quick diagnosis.
To achieve the above object, the present invention is adopted the following technical scheme that：
A kind of chemical luminescence detection method that detection PEDV is set up using the PEDV monoclonal antibodies, specifically includes following step
Suddenly：
（1）Antibody is coated with：Phosphate buffer is used for coating buffer, is coated in 4ug/ml PEDV MAb concentrations solid
On phase carrier microwell plate, 100ul/ holes, 37 DEG C are incubated 2h or 4 DEG C overnight, wash 3 times；
（2）Closing：The closing covering microwell plate using 2.5%BSA as sealing liquid, per hole 300ul/ holes, 4 DEG C overnight, board-washing 3 times；
（3）Sample-adding：Sample is sequentially added, 37 DEG C of 100ul/ holes are incubated 1 hour, board-washing 3 times；
（4）Plus ELIAS secondary antibody：The PEDV monoclonal antibodies of HRP enzymes mark are added, with 1:Liquid after 4000 dilutions adds 100ul/
Hole, 37 DEG C of 45min, board-washing 6 times；
（5）Plus detection substrate：Add Chemoluminescent substrate Thermo Scientific SuperSignal ELISA Femto
Substrate solution（Purchased from ThermoFisher）100ul/ holes, lucifuge is reacted 3 minutes；
（6）Detection：Luminous count is determined with luminometer.
The method of HRP enzymes mark PEDV monoclonal antibodies is marked by conventional method to be obtained.
The advantage of the invention is that：
Obtained monoclonal antibody has high specificity, and stability is good, the features such as antibody titer is high（ELISA and IFA potency exists
10-5）, and be the monoclonal antibody for PEDV M albumen, while the strain of PEDV monoclonal antibodies, which has, neutralizes virus characteristic, it is imitated
Valency is 10-3.Using PEDV monoclonal antibodies, a kind of detection PEDV chemical luminescence detection method is established, this method has spy
It is different in nature strong（It is only able to detect PEDV）, sensitiveness it is high (0.26ng virus can be detected, 10 times more sensitive than conventional RT-PCR with
On), it is simple to operate（Equivalent to common ELISA detection method）, disposable detection limit it is big（80 can once be detected with last sample
Product）And stability it is good the features such as, with absolute predominance more unrivaled than other detection methods.Using show and RT-PCR detect
The coincidence rate of technology is more than 95%, with fluorescence quantitative RT-PCR detecting method coincidence rate 100%.It is adapted to the quick diagnosis to PEDV
And scientific research and testing.
Brief description of the drawings
Fig. 1 is that PEDV inoculation Vero cells cause cell the lesion that atrophy comes off occur.
Fig. 2 is the plaque form that PEDV is inoculated with Vero cells.
Fig. 3 is PEDV-E1 hybridoma supernatant IFA coloration results.
Embodiment
The present invention mainly uses Plaque-purified technology, from Porcine epidemic diarrhea virus-transmissible gastroenteritis of swine-pig colyliform
Purifying obtains the weak poison of PEDV in the trigeminal live vaccine of virus, and virus is by gradient centrifugation, immune small white mouse and prepares monoclonal and resists
Body core technology, obtains anti-PEDV IgG2a monoclonal antibody hybridoma cells strain and corresponding monoclonal antibody.Utilize monoclonal
Antibody, is optimized by sets of conditions, and chemiluminescence diagnostic techniques is set up first, for preventing and treating PED diseases.Illustrate the present invention below
The chemical luminescence detection method for preparing anti-PEDV IgG2a monoclonal antibodies and foundation is applied in disease diagnosis prevention and control, from reason
By and its concrete operation method：
Embodiment 1
（One）Purifying culture PED viruses
Using Plaque-purified technology, by Vero cells from the three of pig epidemic diarrhea-transmissible gastroenteritis of swine-rotavirus
In attenuated vaccine, purifying obtains PEDV strains.The virus can cause Vero cells typical pathological change occur（See accompanying drawing 1）, disease
Toxic effect valency is up to 10-7.5/ 0.1ml, lays the foundation to prepare PEDV monoclonal antibodies.Its Plaque-purified step is as follows：
1st, 6 orifice plate 3 times for covering with Vero cell monolayer is rinsed with DMEM；Add 400 μ L doubling dilutions（10-2、10-3、10-4、
10-5、10-6、10-7）Virus liquid, and set negative control to add 400 μ L DMEM, 37 DEG C of incubation 45min, shaken up per 15min
Fully to contact；Virus liquid is suctioned out after incubation, is rinsed 3 times with DMEM, 4mL low melting point agar mixed liquors are added per hole（2.0% is low
Melt agarose is mixed with 2 × DMEM equal proportions, and adds 1.5% FBS）；After after its solidification, culture is inverted in 37 DEG C of insulating boxs
4-6d, notes observation；Plaque slowly becomes big with the time, it has been found that virus is 10-6During dilution factor, its plaque form is easiest to
Observation and discrimination, then abandon covering agar, after 0.5% violet staining, are clear that typical PEDV plaques are (attached
Fig. 2).
2nd, the suitable plaque of picking is in 400 μ L DMEM（Serum-free）In, freeze thawing 4 times carries out virus liquid the plaque of 5 wheels
Purifying.When treating that plaque length puts suitable size, covering agar is abandoned, 20min, 0.5% crystal violet dye liquor dye are fixed with 10% formaldehyde
Color 10min, PBS flushing 3 times, observes and records plaque form.
3rd, the virus liquid for purifying 5 wheels expands culture in Vero cells, collects virus liquid and enters performing PCR identification, it is determined that
Virus liquid is with the presence or absence of other thermophilic cell line viruses.As a result transmissible gastroenteritis of swine and rotavirus in the virus liquid of purifying are shown
It is feminine gender Deng other thermophilic cell line Viral diagnosis, and PEDV test positive.Illustrate that experiment obtains single PEDV strains.
(2) PEDV IgG2a monoclonal antibodies are prepared
1st, prepared by PED antigens
PEDV is inoculated in Vero cell monolayers, 2~3d is cultivated, when cytopathy is up to more than 85%, harvesting poison.Cell
Poisons ultrasonic degradation, 10 000 rmin-160min is centrifuged, supernatant, 45 000rmin is taken-1180min is centrifuged, is used
0.01mol·L-1PBS（pH 7.2）Suspend precipitation, is slightly to put forward virus；Take it is thick carry antigen paving be padded on successively 30%-60% sucrose-
PBS, 33 000rmin-1150min is centrifuged, the viral purpose bands of 30-40% is collected, is resuspended in PBS, 45 000 rmin-1180min elution sucrose is centrifuged, it is the viral antigen purified that precipitation, which is resuspended in PBS, and PEDV is accredited as through W-B and RT-PCR
Afterwards, it is 4mg/mL to determine protein concentration, for Balb/c mouse to be immunized.
2nd, Balb/c mouse are immunized
Long-range is carried out using routine immunization method to be immunized.Purified virus antigen (1mg/mL) and isometric Freund's complete adjuvant will be taken
After emulsification, the subcutaneous week old Babl/C mouse of multi-point injection 6,0.2ml/ is only.The 14th day, 28 days and the 42nd day after initial immunity, take anti-
Former mixed with incomplete Freund's adjuvant equivalent emulsifies, and two exempt from, three exempt from and four exempt from, when serum ELISA antibody titers are up to 1:
When more than 10000, take within first 3 days purifying antigen through tail vein booster immunization in fusion, 100 μ g/ are only.
3rd, cell fusion
It is i.e. sterile to take immune mouse spleen, disperse splenic lymphocytes, 1000 rpm centrifuge 10 min, by cell it is loose after plus
NH4CL, the min of ice bath 10, is ibid centrifuged, loose, be resuspended in 10 mL DMEM, collects SP2/O cells, carry out splenocyte and
SP2/O cell counts, extracting spleen cell and SP2/O cells press 6:1 ratio mixing, 1200 rmin-1Centrifuge 5 min, it is loose after
The PEG that 37 DEG C of 1 ml preheating is slowly added into 1min carries out cell fusion, and twisted 5 min is slowly added to DMEM, ibid from
After the heart, loose cell, hybridoma is resuspended in the HAT-DMEM culture mediums containing 20% cow's serum, splenocyte quantity is adjusted, plus
To 96 porocyte culture plates, 0.1 mL/ holes are put in 37 DEG C, 5%CO2 incubators and cultivated, and add within the 5th day 0.1mL containing 15% ox blood
Clear HT-DMEM, observes growing state daily, treats cell length to the 1/3 of bottom hole, or during nutrient solution flavescence, supernatant carries out antibody
Detection.
4th, the foundation of PEDV indirect ELISA methods
PEDV antigens are diluted with pH 9.6 carbonate buffer solution, are coated in elisa plate bar, 100 μ L/hole put 37 DEG C of 2 h,
After 0.01 M PBST cleaning solutions are washed 3 times, closed with 1.5%BSA-PBST, 300 μ L/ holes, 37 DEG C of 2 h is ibid washed, plus
Enter Hybridoma Cell Culture supernatant, 100 μ L/ holes, 37 DEG C of reaction 1h Jia 1 after ibid washing:The sheep anti mouse of 40 000 dilutions
IgG horseradish peroxidases, 100 μ L/ holes, 37 DEG C of reaction 1h, after ibid washing, addition OPD, 100 μ L/ holes, 37 DEG C are kept away
Light colour developing 15min, 2 mol/L H2SO4Terminating reaction, determines the OD in each hole in automatic enzyme detector490Value, with P:N≥
When 2.1, it is judged as that hybridoma secretory antibody is positive.Through square formation titration measuring, when antigen coat concentration is 2.5 μ g/
0.1ml/hole, serum dilution is 1:2000 or antigen coat concentration be 3.5 μ g/0.1ml/hole, serum dilution is 1:
When 4000, positive serum OD490Value is up to 1.0, the OD of negative serum490It is worth smaller, OD490Value is below 0.08, and P/N values reach
More than 15, it is determined that optimal antigen coat concentration is 2.5-3.5 μ g/0.1ml/hole.
5th, positive hybridoma cell strain is screened
With indirect ELISA screening Growth of Hybridoma Cell hole specific antibody, from the positive hole of detection, OD values are selected high, thin
Born of the same parents' form quite grows positive hole that is vigorous and not intersecting with normal cell culture, by feeder cells culture, carries out limited dilute
Interpretation of the law is cloned.After multiple limiting dilution, the hybridoma cell strain of 1 plant of energy anti-PEDV antibody of stably excreting is obtained, is named as
PEDV-E1.This plant of cell is continuously trained 3 months in vitro, energy stably excreting antibody, and after liquid nitrogen cryopreservation, recovery, cell growth is good
It is good, and supernatant antibody titer is constant.
6th, prepared by ascites
6.1 Babl/C mouse induce
The atoleine of 8 week old Balb/c mouse peritoneal injections sterilizing is induced, sub-cage rearing 8 days.Take the logarithm growth period
Hybridoma, 5 min sedimentation cells are centrifuged through 1500 r/min, are resuspended with the about 1ml of the DMEM without serum, and stone is injected intraperitoneally
The Babl/C mouse of wax induction, every mouse injection 1 × 106Hybridoma.
6.2 collect ascites
Mouse injection hybridoma observes mouse web portion after about 10 days, has ascitogenous mouse web portion substantially to expand.This
Ascites is collected in Shi Caiyong drainages, and 1000 r/min are centrifuged 15 min, collect the faint yellow ascites in upper strata, given birth to using Thermo companies
The NAbTMSpin Kits kits of production are purified, and determine ELISA potency and indirect immunofluorescence assay（IFA）Potency, does
Good mark, freezes standby in -20 DEG C.
The preparation and immunofluorescent test (IFA) of 6.3 PEDV infection cells
PEDV is inoculated in the porocyte plates of Vero 96 for having grown up to individual layer, 37 DEG C of 5% CO2, which is cultivated to 30% cell, lesion occurs
When, supernatant is abandoned, PBS is washed 1 time, the pre- μ l of cold methanol 100 is added per hole, 4 DEG C are fixed 30 min, abandon methanol, PBS washings 1
It is secondary, after airing, -30 DEG C freeze it is standby.The Vero cells that same processing does not connect poison are used for negative control.When IFA is detected, in above-mentioned
30ul Hybridoma Cell Cultures supernatant is added per hole in cell plates and act on 30 min in 37 DEG C, with 0.85% brine 3
It is secondary, 5 minutes every time, dry, plus sheep anti-mouse igg-FITC is per hole 30ul, 37 DEG C act on 30 minutes, and washing ibid, dries, plus 50%
PBS-glycerine, per hole 30ul, in observing IFA coloration results under inverted fluorescence microscope, with fluorescence reaction（See accompanying drawing 3）.
7th, monoclonal antibody specificity analysis
The measure of 7.1 monoclonal antibody substates part
The method introduced according to monoclonal antibody parting kit is carried out.Identify that monoclonal antibody is in subclass by antibody subtype kit
PEDV-E1 plants belong to IgG2a in identification.
7.2 cell line stability tests
By the hybridoma in-vitro cultivation 2 months with secretion characteristic, every antibody titer for detecting its supernatant for 15 days
It is stable；By positive hybridoma cell recovery every month of liquid nitrogen cryopreservation once, continuous 3 months, supernatant antibody titer is determined basic
It is constant.
7.3 monoclonal antibodies are specific to be determined
Monoclonal antibody can only recognize PEDV；Indirect immunofluorescence assay（IFA）As a result show：Monoclonal antibody not with Vero cells, PRRSV,
PRV、PCV2Reacted with SCFV, illustrate the specific good of monoclonal antibody.
7.4 MAb mediated ELISA potency and monoclonal antibody IFA valencys are determined
The potency of Hybridoma culture supernatants and IFA ascites antibodies is detected using indirect ELISA, hybridoma supernatant is used anti-
Body diluted is into 2-1~2-10 , ascites antibody diluent is diluted to 10 by 10 times-2~10-8.As a result ELISA potency
The culture supernatant of PEDV-E1 plants of monoclonal antibodies is 2-5, ascites antibody potency is 10-5, IFA potency is 10-5。
8 Western blot are tested
SDS-PAGE electrophoresis is carried out with PEDV antigens.It is transferred to NC films to be closed with TBS-T, 4 DEG C overnight, and TBS-T washes film 3
It is secondary, plus 1:The ascites monoclonal antibody of 100 times of dilutions, 37 DEG C of effect 2h, washing adds sheep anti-mouse igg alkaline phosphatase mark
Remember antibody (1:10000) 2 h, are reacted at room temperature, are ibid washed, substrate colour developing is placed in, it is to be directed to M albumen as a result to show PEDV-E1
Monoclonal antibody.
(3) PEDV chemical luminescence detection methods are set up
1st, chemiluminescent workflow
This experiment is marked using the method for enzyme-catalyzed chemical luminescence immune detection using PEDV monoclonal antibodies as coated antibody, with HRP
The PEDV monoclonal antibodies of note are labelled antibody.Specific workflow is as follows：（1）Coating：PEDV monoclonal antibodies are coated in
On solid phase carrier microwell plate, 100ul/ holes, 37 DEG C are incubated 2h or 4 DEG C overnight, wash 3 times；（2）Closing：Microwell plate is covered per hole
Add 2.5%BSA to be closed, 300ul/ holes, 4 DEG C overnight, board-washing 3 times;（3）Sample-adding：Sample 100ul/ holes are sequentially added, it is dense
Spend for 80ug/ml（S5）, 37 DEG C of incubation 1h, board-washing 3 times；（4）Plus secondary antibody：The PEDV monoclonal antibodies of HRP enzymes mark（With 1:
Liquid after 4000 dilutions）100ul/ holes, 37 DEG C of 40min, board-washing 6 times；（5）Plus detection substrate：Add chemiluminescence detection
Thermo Scientific SuperSignal ELISA Femto substrate solutions are purchased from ThermoFisher）100ul/ holes, lucifuge
Reaction 3 minutes；(6) detect：Luminous count is determined with luminometer.
2nd, the optimization of chemiluminescence condition of work
The selection of 2.1 coating buffers
By chemiluminescence workflow, 3 kinds of buffer solutions are selected, the first is 5%BSA phosphate buffers, the 2nd kind is phosphate
Buffer solution, the third is carbonate buffer solution.Dilution coating monoclonal antibody, other conditions are constant, according to monoclonal antibody bag
By concentration 80ug（S5）/ 0ug (S0) value determines optimal coating buffer.When as a result using phosphate buffer as coating buffer solution, its
The luminous value of S0 points is significantly lower than other coating buffers, and S5/S0 value is coated with buffer solution apparently higher than other, therefore, selects phosphorus
Phthalate buffer is used as coating buffer solution.It see the table below 1
S0, S5 of the different coating buffer solutions of table 1 luminous value and S5/S0 value
The selection of 2.2 confining liquids
By chemiluminescent workflow, confining liquid is respectively 2.5%BSA and 5%BSA, 2.5% gelatin and 5% gelatin 300ul/ holes,
Other 2 conditions are constant, and optimal dilution is determined according to the value of experimental result and S5/S0.As a result with 2.5%BSA phosphate buffers
During as dilution, and S5/S0 value apparently higher than other groups, therefore, selection 2.5%BSA phosphate buffers are used as closing
Liquid.It see the table below 2.
S0, S5 of the different confining liquids of table 2 luminous value and S5/S0 value
The selection of 2.3 coated antibody concentration
By chemiluminescent workflow, monoclonal antibody is diluted to 1ug/ml, 2ug/ml, 4ug/ml by coating buffer respectively, 8ug/ml holes,
Other conditions are constant, and optimal coating concentration is determined according to S5/S0 value.As a result concentration is coated with using 4ug/ml as monoclonal antibody
When, the luminous value of its S0 point is significantly lower than other, and S5/S0 value apparently higher than other groups, therefore, select monoclonal antibody bag
It is 4ug/ml by concentration.It see the table below 3.
The luminous value of different monoclonal antibody coating concentration S0, S5 of table 3 and S5/S0 value
The selection of 2.4 enzyme labelled antibody working concentrations
By chemiluminescence workflow, by horseradish peroxidase target monoclonal antibody respectively with 1:2000、1:4000 and 1:6000 times
Dilution, other conditions are constant, determine that optimal enzyme marks the diluted concentration of monoclonal antibody according to according to S5/S0 value.As a result marked with enzyme
The concentration of monoclonal antibody is 1:When 4000, the luminous value of its S0 point is significantly lower than other, and S5/S0 value is also higher, therefore, choosing
The concentration for selecting enzyme labeled monoclonal antibody is 1:4000.It see the table below 4.
The luminous value and S5/S0 value of concentration S0, S5 of the different enzyme labeled monoclonal antibodies of table 4
2.5 enzymes mark the selection of monoclonal antibody best effort time
By chemiluminescent workflow, according to the best operating condition of above-mentioned determination, react respectively 30min, 45min and
60min, other conditions are constant, determine that enzyme marks the best effort time of monoclonal antibody according to S5/S0 value.As a result Dan Ke is marked with enzyme
The working time of grand antibody is 45min, and S5/S0 value apparently higher than other groups, therefore, selection enzyme labeled monoclonal antibody
Working time 45min, see the table below 5.
Working time S0, S5 of the different enzyme labeled monoclonal antibodies of table 5 luminous value and S5/S0 value
The determination of 2.6 chemiluminescence reaction systems and condition
By the optimization of above series of condition, chemiluminescence optimal reaction system and reaction condition are established：Using phosphate
Buffer solution is diluent liquid, and 100ul/ holes, 37 on solid phase carrier microwell plate are coated in 4ug/ml PEDV MAb concentrations
DEG C be incubated 2h or 4 DEG C overnight, wash 3 times；Closing：The closing covering microwell plate using 2.5%BSA as sealing liquid, 300ul/ holes,
4 DEG C overnight, board-washing 3 times;Sample-adding：Sequentially add sample, 37 DEG C of 100ul/ holes incubation 1h, board-washing 3 times；Plus secondary antibody：HRP enzyme marks
The PEDV monoclonal antibodies of note（With 1:Liquid after 4000 dilutions）100ul/ holes, 37 DEG C of 45min, board-washing 6 times；
(5）Plus detection substrate：Add chemiluminescence detection Thermo Scientific SuperSignal ELISA Femto bottoms
Thing liquid
（Purchased from ThermoFisher）100ul/ holes, lucifuge reaction 3min；(6) detect：Luminous count is determined with luminometer.
3rd, the measure of chemical luminescence detection method specificity, sensitiveness and repeatability
3.1 specific measure
According to the chemical luminescence detection method of foundation, Porcine epidemic diarrhea virus is respectively adopted（PEDV）, CSFV (SCFV),
Porcine reproductive and respiratory syndrome virus (PRRSV), PRV (PRV), porcine circovirus 2 type (PCV2), pig transmissible
Stomach and intestine virus (TEGV) and porcine rotavirus (RV) are to be detected sample, the specificity of assay method.As a result there was only PEDV detections
It is positive, other Viral diagnosis are feminine gender to strong values of chemiluminescence 421975, illustrates that the chemiluminescence method set up has very
Good specificity.It see the table below 6.
The specific assay of table 6
Note：+ represent positive, expression-feminine gender
The measure of 3.2 sensitiveness
According to the chemical luminescence detection method of foundation, PEDV is carried out 1:10、1:100、1:1000、1:10000 successively multiple proportions it is dilute
Release, determine the sensitiveness of detection method.As a result this method can detect 2.6ng viral level, and illustration method has preferable
Sensitivity.It see the table below 7.
The sensitivity testing of table 7
+ represent positive, expression-feminine gender
The measure of 3.3 repeatability
According to the chemical luminescence detection method of foundation, PEDV samples are subjected to 5 repetitions, the repeatability of detection method is determined.Knot
Fruit chemiluminescence intensity is basically identical, and illustration method has good repeatability, see the table below 8.
The repeatability of table 8 is determined
Note：+ represent positive, expression-feminine gender
4th, the clinical principium application of chemiluminescence method
By detecting that 56 parts of doubtful pathological material of diseases are detected, and be compared with conventional RT-PCR detection technique, coincidence rate 95% with
On, it is compared with fluorescence quantitative RT-RCR detection technique, coincidence rate is 100%, illustrates the PEDV chemical methodes detection side set up
Method, is adapted to the quick diagnosis to PEDV, good technical support can be provided for PED quick diagnosis.
The foregoing is only presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, should all belong to the covering scope of the present invention.
Claims (4)
1. a kind of monoclonal antibody of porcine epidemic diarrhea resisting virus, it is characterised in that：From pig epidemic diarrhea-pig transmissible stomach
Purifying obtains Porcine epidemic diarrhea virus strain in the trigeminal live vaccine of enteritis-porcine rotavirus, as preparing pig epidemic
The antigen of diarrhea virus monoclonal antibody；By Virus culture, immune Balb/c mouse, monoclonal antibody technology of preparing, through clone, obtains pig
Epidemic diarrhea virus monoclonal antibody.
2. a kind of monoclonal antibody of porcine epidemic diarrhea resisting virus according to claim 1, it is characterised in that：It is
IgG2a hypotype monoclonal antibodies.
3. a kind of chemiluminescence detection side of the detection Porcine epidemic diarrhea virus of monoclonal antibody described in utilization claim 1
Method, it is characterised in that：Using the monoclonal antibody, concentration, confining liquid, ELIAS secondary antibody concentration are coated with by coating buffer, monoclonal antibody
And the optimization in reaction time, establish a kind of chemical luminescence detection method for detecting Porcine epidemic diarrhea virus.
4. chemical luminescence detection method according to claim 3, it is characterised in that：Specifically include following steps：
（1）Antibody is coated with：Phosphate buffer is used for coating buffer, with 4ug/ml Porcine epidemic diarrhea virus monoclonal antibodies
Concentration is coated on solid phase carrier microwell plate, 100ul/ holes, and 37 DEG C are incubated 2h or 4 DEG C overnight, wash 3 times；
（2）Closing：The closing covering microwell plate using 2.5%BSA as sealing liquid, per hole 300ul/ holes, 4 DEG C overnight, board-washing 3 times；
（3）Sample-adding：Sequentially add sample, 37 DEG C of 100ul/ holes incubation 1h, board-washing 3 times；
（4）Enzyme-added mark 2 resists：The Porcine epidemic diarrhea virus monoclonal antibody of HRP enzymes mark is added, with 1:Liquid after 4000 dilutions
Body adds 100ul/ holes, 37 DEG C of 45min, board-washing 6 times；
（5）Plus detection substrate：Add Chemoluminescent substrate 100ul/ holes, lucifuge reaction 3min；
（6）Detection：Luminous count is determined with luminometer.
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| CN107099506A (en) * | 2017-04-24 | 2017-08-29 | 江苏省农业科学院 | Porcine epidemic diarrhea virus double-antibody sandwich elisa immue quantitative detection reagent box and its application |
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