CN108148814A - A kind of double-antibody sandwich elisa diagnostic kit and its application for being used to detect bovine rota - Google Patents
A kind of double-antibody sandwich elisa diagnostic kit and its application for being used to detect bovine rota Download PDFInfo
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- CN108148814A CN108148814A CN201711353547.XA CN201711353547A CN108148814A CN 108148814 A CN108148814 A CN 108148814A CN 201711353547 A CN201711353547 A CN 201711353547A CN 108148814 A CN108148814 A CN 108148814A
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Classifications
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- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- 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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- C—CHEMISTRY; METALLURGY
- 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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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
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- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
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Abstract
The invention discloses a kind of for detecting double-antibody sandwich elisa diagnostic kit and its application of bovine rota.Contain the monoclonal antibody 4D10 that the hybridoma cell strain secretion for being CGMCC NO.14726 by culture presevation number generates in the kit.In addition, the present invention is resisted with the rabbit-anti BRV purified to capture antibody more, using 4D10 monoclonal antibodies as detection antibody, BRV double-antibodies sandwich ELISAs are established.Sensitivity experiments show that this method is 9.884 × 10 to the limit of identification of BRV4TCID50/mL；Specificity experiments the result shows that, this method have good specificity, with the equal no cross reaction of PoRV, PEDV, BPV and TGEV；Between batch and batch interior repeated experiment is the results show that this method has good repeatability.95 parts of clinical samples are detected using the double-antibodies sandwich ELISA of foundation, compared with RT PCR methods, coincidence rate 100%.It can be seen that the double-antibodies sandwich ELISA that the present invention establishes has good specificity, sensibility and repeatability, the quick detection available for BRV.
Description
Technical field
The present invention relates to a kind of virus detection kits, more particularly to a kind of to be based on double-antibody sandwich elisa testing principle
The double-antibody sandwich elisa diagnostic kit for bovine rota detection prepared.The invention belongs to pharmaceutical technology fields.
Background technology
Rotavirus (Rotavirus, RV) is the member of Reoviridae (Reoviridae), rotavirus, it
Seven kinds of Different groups of shared A, B, C, D, E, F, G.Wherein A groups is to cause one of important pathogen of scouring, and colyliform disease
The severity of poison infection is age-dependent, is clinically most commonly in young animal.Calf infection rotavirus is being undergone
After the incubation period of 2-4 days, symptom is subsequent to continue the watery diarrhea of 3-8 days usually since unexpected fever and vomiting, and causes
Dehydration, electrolyte imbalance infect serious calf and even have a convulsion symptom.The infection of BRV causes the production performance of ox to decline
With the different degrees of death rate, while treatment be also required to certain cost, huge economic loss can be brought to China's cattle-raising.
Nineteen sixty-eight, Mebus et al., electricity consumption sem observation to RV, and was isolated for the first time, it was demonstrated that is that RV causes newborn calf
Ox diarrhea is named as NCDV plants (Nebraska calfdiarrha Virus, NCDV), finally determines that it is standard strain.
Hereafter, there is the case of diarrhea due to rotavirus infection, and in addition to calf in Europe, America and Australia various countries, other
There is the young animal such as lamb, cat of diarrhea phenomenon and young coltfoal etc. and also detect that the virus.External pertinent literature represents that BRV feels
Calf diarrhea caused by dye, morbidity risk rate are 60%~80%, and it is 0%~50% dead probability occur.
China found a kind of acute infectious disease in 1974 in South Fujian Province for the first time, and diarrhea is in spurting watery diarrhea, passes through electricity
The reason of sem observation shows to cause diarrhea is that calf has infected BRV.Then also there is cattle infected BRV in China In Fujian Province
Case, locality has herded the collected diarrheic stools of buffalo of doctor and has detected rotavirus；1990, Heilungkiang was each
Also there is symptom of diarrhea in the ox of local farm, and 33 parts of pathological material of diseases, and therefrom separation disease are randomly selected from these farms
Poison, by these virus infected cells, and in electric Microscopic observation cytopathy situation.East China cow's serum is detected, finds ox
Rotavirus antibody positive rate can reach 80% or so.More than investigation result illustrates that BRV infection is relatively common in China.
At present, the method for being usually used in detect and diagnose BRV has：Electron microscopy (DEM), enzyme linked immunosorbent assay (ELISA),
Latex agglutination test, RT-PCR methods etc..DEM is earliest for the detection method of BRV in excrement, is laboratory diagnosis virus
Conventional method.RV particles can be observed directly, as a result reliably.But the cost of electron microscope is higher, and needs profession
Maintenance and relevant technical staff, this method can not extensive use.On the other hand, this method sensitivity is poor, to virus
Population has certain requirement, every milliliter of virus at least 106A virion can be just detected.It is viral also in excrement
Particle shape can also influence Electronic Speculum as a result, therefore, this method is not particularly suited for large-scale sample detection.At present, it is suitable for taking turns
The cell line of shape viral growth breeding is more rare, and the cell line of common culture bovine rota is MA104.Only A at present
Group BRV can adapt to cell culture, other BRV breedings can only be realized by susceptible animal for model.And BRV is in cell
In duplication and passage before, pancreatin is needed to handle, virus can be realized after pancreatin activates duplication in cell culture and
Passage.The method is cumbersome, and condition of culture is more demanding, spends the time longer, so the method being separately cultured also is not suitable for
It is widely used in clinical detection.RT-PCR is one of most common Protocols in Molecular Biology, and this method is in vitro in analogue body
Gene duplication process.A large amount of amplifications of nucleic acid molecules are completed by the cycle for being denaturalized, annealing and sprawl.Although PCR method is special
Different in nature strong, sensitivity is high, but easily pollution, leads to the result of false positive.
ELISA method is easy to operate, sensitivity is strong, specificity is high, can batched operation, therefore, establish a kind of quick inspection
It surveys and the ELISA method of diagnosis BRV is very important for clinical detection.
Invention content
An object of the present invention is to provide a kind of double-antibody sandwich elisa diagnosis examination for being used to detect bovine rota
Agent box.
The second object of the present invention is to provide a kind of double-antibody sandwich using the kit detect and diagnose BRV
ELISA method.
In order to achieve the above object, present invention employs following technological means：
The present invention selects bovine rota VP6 albumen, by cell fusion, screening and clone, finally to be obtained as immunogene
The monoclonal antibody of 5 plants of anti-BRV VP6 albumen is obtained, is respectively designated as 5C9,3G10,6E11,4D10 and 5A3.Subclass of antibody qualification result
Show：3G10,6E11 and 5C9 are IgM subclass, and 4D10 and 5A3 are IgG2a subclass.5 strain of hybridoma 3G10,6E11,
5C9,4D10 and 5A3 cell conditioned medium potency are respectively 1:500、 1:800、1：800、1：1000,1：800.Western blot and
Indirect immunofluorescence experiment result shows that 5 plants of monoclonal antibodies have good compatibility with BRV；Specificity experiments are the results show that 5
Strain monoclonal antibody and the equal no cross reaction of PoRV, PEDV, BPV and TGEV illustrate that monoclonal antibody specificity is good.In addition, the present invention is to purify
Rabbit-anti BRV more resist for capture antibody, using 4D10 monoclonal antibodies for detect antibody, tentatively establish BRV double-antibody sandwich elisa methods.
Sensitivity experiments show the double-antibodies sandwich ELISA established of the present invention to the limit of identification of BRV for 9.884 ×
104TCID50/mL；Specificity experiments the result shows that, the double-antibody sandwich elisa method established of the present invention has good special
Property, with the equal no cross reaction of PoRV, PEDV, BPV and TGEV；Between batch and batch interior repeated experiment is the results show that this method has
Good repeatability.The double-antibody sandwich elisa method of foundation is detected 95 parts of clinical samples, with RT-PCR method
It compares, coincidence rate 100%.
Therefore, on the basis of the studies above, first, the present invention proposes the anti-bovine rota VP6 of one plant of stably excreting
The hybridoma cell strain of protein monoclonal antibody, is named as 4D10, and Classification And Nomenclature is miscellaneous for secretion bovine rota monoclonal antibody
Oncocyte is handed over, the hybridoma cell strain is deposited in China Committee for Culture Collection of Microorganisms microorganism center, address
In Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, culture presevation number is CGMCC
NO.14726, preservation time are on October 18th, 2017.
Anti- bovine rota VP6 protein monoclonal antibodies are generated also in the present invention by the hybridoma cell strain secretion
Protection domain within.
Further, the invention also provides the monoclonal antibody is preparing detection or diagnosis bovine rota sense
Application in the reagent of dye.
Further, the invention also provides a kind of for detecting the diagnosis of the double-antibody sandwich elisa of bovine rota
Kit contains monoclonal antibody of the present invention.
In kit of the present invention, it is preferred that it is polyclonal that the kit further includes rabbit-anti bovine rota
Antibody, HRP- sheep anti-mouse iggs, cleaning solution, confining liquid, dilution, positive control, negative control, developing solution and terminate liquid.
When detecting bovine rota using kit of the present invention, it is preferred that follow the steps below：
(1) by after rabbit-anti bovine rota polyclonal antibody diluted, ELISA Plate is added according to 100 μ L/ holes
In, 37 DEG C of coating 1-2h；
(2) cleaning solution washing ELISA Plate 3 times, each 5min, adds in confining liquid, per hole 200 μ L, 37 DEG C of closing 2h；
(3) cleaning solution washing ELISA Plate 3 times, each 5min, sample serum to be checked is added in ELISA Plate, and setting is parallel
Sample, 37 DEG C of incubations, while positive control and negative control are set；
(4) cleaning solution washing ELISA Plate 3 times, each 5min adds in the monoclonal antibody after dilution, 37 DEG C of incubation 1h；
(5) cleaning solution washing ELISA Plate 3 times, each 5min, adds in 1:5000 diluted HRP- sheep anti-mouse iggs, 37 DEG C incubate
Educate 1h；
(6) cleaning solution washing ELISA Plate 3 times, each 5min adds in the tmb substrate developing solution newly prepared, 37 DEG C of colour developings
10min；
(7) 2M H are added in2SO4Terminate liquid, reading numerical values, yin and yang attribute critical value is 0.148355, if sample OD450nmValue>
When 0.148335, it is judged to the positive；OD450nmValue<Feminine gender is judged to when 0.148335.
Wherein, it is preferred that the cleaning solution contains KH2PO40.2g/L, Na2HPO4·12H2O 2.9g/L, sodium chloride
8.0g/L, KCl 0.2g/L, Tween-20 0.5mL L, remaining is deionized water, and pH is adjusted to 7.4；The dilution contains
NaHCO32.93g/L Na2CO31.5g/L, remaining is deionized water, and pH is adjusted to 9.6；The confining liquid is contains 5w/
The PBS of v% skimmed milks；The positive control be inoculated with BRV viruses MA104 cells and supernatants, the negative control
For MA104 cells.
Wherein, it is preferred that in step (1), a concentration of 8.0 μ g/ of the rabbit-anti bovine rota polyclonal antibody after dilution
ML, 37 DEG C of coating 2h.
Wherein, it is preferred that in step (3), the incubation time of sample serum to be checked is 2h.
Wherein, it is preferred that in step (4), a concentration of 10.0 μ g/mL of the monoclonal antibody after dilution.
The double-antibodies sandwich ELISA that the present invention establishes has good specificity, sensibility and repeatability, can use
In the quick detection of BRV.
Description of the drawings
Fig. 1 is BRV VP6 protein expression SDS-PAGE qualification results；
M. low molecular weight protein Marker；After 1.pProHTa-BRV-VP6/Rosetta (DE3) inductions；2. pProHTa-
Before BRV-VP6/Rosetta (DE3) inductions；
Fig. 2 is the identification of VP6 albumen after purification；
M. low molecular weight protein Marker；1. BRV VP6 albumen after purification；
Fig. 3 is counted for 4D10,5A3,6E11,3G10,5C9 and SP2/0 cell chromosome；
Fig. 4 detects monoclonal antibody for Western-blot；
M：Albumen Marker；1：BRV；2：MA104；
Fig. 5 is 5 strain of hybridoma indirect immunofluorescence results；
Fig. 6 is monoclonal antibody specificity identification result；
Fig. 7 is the SDS-PAGE electrophoresis of serum after purification；
M. low molecular weight protein Marker；1. the more antivenom purifications of rabbit-anti BRV VP6；
Fig. 8 is the selection of the mostly anti-best coating conditions of rabbit-anti BRV VP6；
Fig. 9 is the selection of confining liquid；
Figure 10 is determining for Best Times；
Figure 11 is determining for measuring samples action time；
Figure 12 is specific test result；
Figure 13 is positive clinical sample RT-PCR results M.600DNA molecular mass standard；
1~4. detection sample；5. positive control；6. negative control.
Specific embodiment
The present invention is further described with reference to specific embodiments and the drawings, and the advantages and features of the present invention will be with
Description and it is apparent.But embodiment is only exemplary, does not form any restrictions to the scope of the present invention.This field
Technical staff should be understood that without departing from the spirit and scope of the invention can be to the details of technical solution of the present invention
It modifies or replaces with form, but these modifications and replacement are each fallen in protection scope of the present invention.
Embodiment 1 secretes the screening and foundation of anti-bovine rota VP6 protein monoclonal antibody hybridomas
1 test material
1.1 bacterial strains, cell and virus
The recombinant bacterium pProHTa-NCDV-VP6/Rosetta of expression bovine rota VP6 albumen is built by this laboratory
And preserve that (recombinant bacterium has been recorded in the following documents：" bovine rota VP6 albumen indirect ELISA detection methods are built
It is vertical ", Zhang Yiting etc., Chinese veterinary science, 2010,40 (10), 1039-1043)；BRV NCDV cell adapted strains are purchased from China
Veterinary medicament supervises institute, and MA104 cells are preserved by this laboratory.
1.2 experimental animal
SPF grades of BALB/c mouses, 2~3 kilograms of weight Healthy female rabbit to be purchased from the long-living biotechnology in Liaoning limited
Company.
1.3 reagent
(1) solution I：12g urea, 1.56g NaH2PO4·2H2O and 0.12g Tris-Base are dissolved in 100mL tri-distilled waters
In, pH is adjusted in 8.0, -4 DEG C of preservations.
(2) solution II：24g urea, 1.56g NaH2PO4·2H2O and 0.12g Tris-Base are dissolved in 100mL tri-distilled waters
In, pH is adjusted in 8.0, -4 DEG C of preservations.
(3) solution III：36g urea, 1.56g NaH2PO4·2H2O and 0.12g Tris-Base are dissolved in 100mL tri- and steam
PH is adjusted in water in 8.0, -4 DEG C of preservations.
2 test methods
The preparation of 2.1 immunogenes
(1) recombinant bacterium pProHTa-NCDV-VP6/Rosetta 1mL. is taken to be activated, passed on.And it is inoculated in ammonia
In the LB culture mediums of benzyl resistance, expand culture.An OD is detected per half an hour600Value；
(2) as bacterium solution OD600When value is up to 0.4~0.6, the IPTG derivants of 1.0mmol/L, 37 DEG C of inductions, per half are added in
Hour detection is primary, treats bacterium solution OD600When value is up to 0.8, induction can terminate；
(3) after inducing, 100mL bacterium solutions are taken, 4 DEG C of centrifugation 10min of 5000r/min abandon supernatant, and 10mL PBS are resuspended；
(4) bacterium is resuspended in 4 DEG C, 4 DEG C of centrifugation 10min of 5000r/min abandon supernatant, 8mL PBS are resuspended；
(5) step 2 is repeated；
(6) 1.6mL lysozymes, 37 DEG C of effect 30min are added in per 100mL bacterium solutions in aforesaid liquid；
(7) ultrasonication 40min；
(8) 4 DEG C after ultrasound, 5000r/min centrifugation 10min abandon supernatant, add 8ml solution Is per 100mL bacterium solutions, will precipitate
It hangs；
(9) 4 DEG C, 5000r/min centrifugation 10min abandon supernatant, add 8mL solution IIs per 100mL bacterium solutions, precipitation is hanged；
(10) 4 DEG C, 5000r/min centrifugation 10min abandon supernatant, add 1.6ml solution IIIs per 100mL bacterium solutions, and precipitation is outstanding
It rises, 1：1 adds in 2 × sample-loading buffers of SDS-PAGE (containing DTT), boils 15min, -20 DEG C save backup.
(11) albumen of extraction is purified using the method for cutting glue purification.The KCl colour developings of precooling, cut albumen one
With rear electroelution.- 40 DEG C of preservations, record protein name and concentration.
2.2 animal immune
Using the BRV VP6 albumen of purifying as immunogene, specific immune programme uses for reference bibliography：Anti- porcine rotavirus
The preparation of monoclonal antibody and preliminary foundation [D] Meng Liu of double-antibody sandwich elisa detection method, Northeast Agricultural University,
2014。
The screening and foundation of 2.3 hybridoma cell strains
2.3.1 the recovery of myeloma SP2/0 cells
(1) after determining the cell fusion time, 2-3 weeks in advance recovery SP2/0 cell.SP2/0 cells are taken out from -140 DEG C,
It is put at once in 37 DEG C of water-baths, gently shaking makes it quickly melt as far as possible.
(2) cell suspension is gently sucked out after melting, 8mL bases 1640 culture medium is then added dropwise by slow-to-fast, pays attention to light
Soft operation.
(3) 1000r/min centrifuges 8min after mixing, discards supernatant liquid, is slowly blown afloat with the complete 1640 culture mediums of 5mL
Cell makes cell be evenly distributed in culture solution, moves into cell bottle, is placed in constant temperature cell incubator culture.
2.3.2 the preparation of feeder cells
(1) BALB/c mouse of 4-6 week old is selected, the neck that breaks is put to death, and sterilizes 5min.
(2) after sterilizing, mouse is fixed, mouse is dissected with the scissors and tweezers of sterilizing, its peritonaeum is completely exposed
Afterwards, the HAT culture solutions of intraperitoneal injection 5mL precoolings
(3) mass rearing confluent monolayer cells are generated with alcohol swab stimulation abdominal cavity, then culture solution is sucked out again.Pay attention to sterile behaviour
Make.
(4) it is added in HAT culture solutions, mixing.
(5) it is added in 96 orifice plates with every hole 0.1mL, is closed with adhesive tape, in 37 DEG C, 5%CO2Under the conditions of cultivate.
2.3.3 the preparation of splenocyte
(1) immune rear BALB/c mouse is taken to carry out cell fusion.
(2) eyeball blood sampling obtains positive serum, and after having collected positive serum, the neck that breaks is put to death, after alcohol disinfecting 5min.
(3) mouse peritoneal is fixed upward, pushes skin, peritonaeum under aseptic condition aside successively, taken out spleen, be put into and fill
In the plate on 10mL bases 1640, the tissue of adhesion is removed as possible.
(4) spleen is turned to be placed in new plate, it is repeated multiple times with basic 1640 culture medium from spleen one end by splenocyte
It is blown out from the other end, until spleen becomes transparent.
(5) splenocyte is centrifuged, rotating speed 1000r/min abandons supernatant, and splenocyte is resuspended with 5mL bases 1640 culture medium,
Carefully blow it is even, 1:200 times of dilutions count.
2.3.4 the preparation of myeloma SP2/0 cells
In Microscopic observation cell state, the smooth of the edge, complete and translucency and the good SP2/0 cells of form are selected, with
1000r/min centrifuges 10min, discards supernatant liquid, takes about 5mL bases 1640 culture medium that cell is resuspended, and is uniformly mixed, and counts.
2.3.5 cell fusion
(1) splenocyte and SP2/0 cells are according to 8：1 ratio carries out mixing, centrifuges 10min, is trained with 10mL bases 1640
The precipitation of two kinds of mixing with cells is resuspended again for nutrient solution, and mixing action is soft, then primary, the removal training by two kinds of cell cleanings
Serum in nutrient solution, to prevent stopping blooding, the clear fusion agent that influences plays a role.
(2) 10min is centrifuged again, abandons supernatant.Flick tube bottom, it is ensured that two kinds of cell mixings are uniform.
Under (3) 37 DEG C of water bath conditions, the fusion agent PEG in 1min by 600 μ L preheatings is at the uniform velocity added in cell, and
Side is added dropwise and just gently shakes, and 90s is stood after adding.
(4) the basic 1640 culture medium of 10ml preheatings is added in 5min, it is slow again from slow to fast to pay attention to the speed being added dropwise
Mode.Later, slow mixing cell centrifuges 10min.It repeats to be washed once with basic 1640 culture medium, abandons supernatant.
(5) cell after fusion is resuspended in HAT culture solutions, is finally added drop-wise in 96 orifice plates for being covered with feeder cells, uses glue
After band closing, it is placed in quiescent culture in cell incubator.
2.3.6 the screening of positive hybridoma cell and clone
(1) it needs to observe cell, and first three day had better not move cell after fusion before screening and clone.It is seen after 4 days
It examines cell state, whether pollute.
(2) merge 7 days after, if cell state well if can partly be changed liquid.Change liquid sequence be followed successively by HAT culture solutions,
HT culture solutions, complete 1640 culture medium.After about merging 12~15 days in Microscopic observation fused cell state, mark and be fused into
The hole of work(.
(3) when cell covers with 1/3 bottom hole, 100ul cell culture supernatants progress indirect ELISA method is taken out from hole
Detect cell conditioned medium potency.Wherein using BRV VP6 albumen as detection antigen, while control group is set.Testing result works as P/N>2
It is determined as the positive.
(4) hybridoma is cloned using limiting dilution assay.Subclone generally requires 3-4 times, until screening
As a result all positives, cell strain at this time is monoclonal cell strain.
2.3.7 hybridoma freezes and recovers
(1) after being determined as positive hybridoma cell, it is enlarged culture.Cell state is observed, if cellular morphology is mellow and full,
Size is uniform, and form is complete, and growth conditions are good, and in exponential phase when, frozen.Cell is gently dispelled, with
The rotating speed centrifugation about 8min of 1000r/min, abandons supernatant, frozen stock solution is added in into cell precipitation.Relevant information has been marked, has been convenient for
It preserves.Cell is put into freezing storing box, -80 DEG C overnight after preserve to -140 DEG C.
(2) cell recovery：Process is the same as recovery myeloma SP2/0 cells.
The measure of 2.4 antibody titers
Observe positive hybridoma cell growth, when culture solution in apparent yellow and also it is in good condition when collect culture on
Clear liquid.Antigen selection is BRV cell cultures, for detecting the potency of monoclonal antibody.
2.5 monoclonal antibody Identification of Biological Characteristics
2.5.1 the identification of hybridoma chromosome number
(1) hybridoma of passage and SP2/0 cells are taken out from cell incubator.
(2) the colchicine 0.05mL of 20 μ g/mL is added in into cell bottle, makes its final concentration of 0.1 μ g/mL, is put into 37
DEG C, 5%CO2Continue to cultivate 2.5h in incubator, its chromosome is made to stop at metaphase.
(3) cell from cell bottle is sucked out and be put into centrifuge tube, 1000r/min centrifugation 7min abandon cell conditioned medium reservation
Cell precipitation.With 2mL, 0.075mol/mL KCl solution is resuspended, the Hypotonic treatment 30min in 37 DEG C of water-baths.
(4) 1mL cell fixer (methanol is added in：Glacial acetic acid=3:1) after pre-fixing 2~3min, 1000r/min, centrifugation
7min abandons supernatant and stays precipitation.1mL cell fixers are added, room temperature fixes 2~3min, 1000r/min, centrifuges 7min, then
It repeats fixed primary.
(5) fixer is added according to the amount of cell precipitation and suspension is made, cell concentration is excessive, influences to observe.Concentration is too small
It can not find target cell.Ordinary circumstance adds in μ L of 200 μ L~500 or so.
(6) cell suspension of 1-2 drops is added dropwise on glass slide, glass slide dries rear pre- cold standby.By cell Giemsa
Dyeing liquor dyes, and the time is 15~20min.It finally with distilled water flushing and dries, observes under the microscope.
(7) count hybridoma and 10, SP/20 cells respectively form is complete, Chromosome spread is good, is non-overlapping,
It is observed without lost individual cells.Chromosome counting is analyzed, average chromosome number is obtained.
2.5.2 monoclonal antibody subgroup identification
By 5 strain of hybridoma supernatants of preservation, identified using monoclonal antibody immunity immunoglobulin subclass identification kit
Protein subclass, specific steps are operated according to kit specification.
2.5.3 hybridoma secretory antibody repeated pruning
The hybridoma cell strain continuous passage 3 months of screening is carried out on indirect ELISA method detection hybridoma
Clear liquid potency.It recovers after 3 months the hybridoma frozen, adjusts cell growth state, when its is in good condition, collect thin
Born of the same parents' supernatant detects hybridoma supernatant potency by indirect ELISA method.Finally, the two antibody titer is carried out pair
Than analyzing the stability of its secretory antibody.
2.5.4 monoclonal antibody Western-blot is identified
(1) preparation of protein sample
The viral sample of purifying gained is taken to be put into EP pipes, adds in 5 × SDS buffer of 1/4 volume.Boiling water boiling sample
10min, ice bath 5min.Albumen sample is directly used in Western blot.
(2) SDS-PAGE
(1) offset plate is assembled：Determine that electrophoresis tank and glass plate are rinsed with water totally, and by protein adhesive blackboard eraser it is clean or
Drying finally assembles albumen offset plate, and hunts leak.1.5mm model offset plates are selected, corresponding adhesive tape is respectively grey.Adhesive tape is put
In mother glass groove, adhesive tape is put straight, covers otic placode.Assembled offset plate is carefully moved on on foot stool, otic placode to
Outside, offset plate is fixed with fixed plate.Add 3mL or so distilled water in leak detection.
(2) separation gel：This experiment is detached using 12%SDS-PAGE, finally adds TEMED (coagulant).It is shaken before adding TEMED
Dynamic fine taper bottle about 10s, makes the abundant mixing of coagulant liquid.Observe assembled offset plate whether leak, if water-tight, water is discarded,
Carefully the water in offset plate is blotted and (is careful not to distort adhesive tape) with filter paper.After getting out offset plate, added in coagulant liquid
TEMED shakes about 10s mixings immediately after addition.
(3) encapsulating：Mixed coagulant liquid 6mL is rapidly added in offset plate, be rapidly added distilled water moulding (with pipettor come
Return at the uniform velocity mobile addition distilled water), gel 40min.
(4) concentration glue (spacer gel)：With 5%SDS-PAGE spacer gels, before adding TEMED, the moulding water in offset plate is abandoned
Fall, the water (not encounter spacer gel carefully) in offset plate is blotted with filter paper, gets out clean comb.In lamination sol solution
TEMED is added in, abundant mixing fills it up with offset plate.Careful comb of plugging (inserts that comb process is slow, in order to avoid overflow excessive lamination
Glue), gel 30min.
(5) electrophoresis：Comb is slowly extracted, fixed plate is extracted, carefully takes out two offset plates, take adhesive tape, otic placode is inside
It installs back on glue frame, mounting plate.Appropriate electrophoresis liquid is added in glue groove, foot stool is put into electrophoresis tank at a slant, offset plate
Try not have bubble in lower section.Inside groove is filled it up with into electrophoresis liquid.
Offset plate comb is pulled up, unloads offset plate, carefully removes adhesive tape, otic placode puts back to foot stool in the inner part.On 1.5mm offset plates
Sample amount is less than 40 μ L.Albumen Marker is mixed in advance with 1 × SDS Buffer, and applied sample amount is consistent with sample size, without sample
1 × SDS Buffer occupy-places of hole equivalent.Upper strata glue 80V, lower floor glue 120V, until bromine Finland swims out.
(3) transferring film
The sequence of transferring film from bottom to top is successively：Cathode (-), cotton net, five layers of filter paper, glue, NC film or PVDF films, six layers
Filter paper, cotton net, anode (+) pay attention to：Transfer film and glue are in the same size, excessive too small all to influence transfer efficiency.Pvdf membrane
It is activated before use with formaldehyde.
(4) it closes
4 DEG C of closings of confining liquid (PBS of the skimmed milk containing 5w/v%) are overnight or 37 DEG C are incubated 1h or incubation at room temperature 2h.
(5) film is washed
Pvdf membrane is put into PBST solution, is placed on shaking table and carries out washing film, each 10min is washed 3 times.
(6) primary antibody, secondary antibody are incubated
Pvdf membrane is placed in sealed plastic bag, hybridoma supernatant, is incubated at room temperature 2h or 4 DEG C overnight.Wash film,
Method is same as above.Skimmed milk equally 5% degreasing milk solution of selection, the mountain sheep anti-mouse igg extension rate of diluted FITC labels is 1:
5000, it slowly shakes on shaking table, is incubated at room temperature 1h.Incubation time can not be long, and otherwise background is deepened.PBST rinsings 3 times, often
Secondary 10min.
(7) ECL develops the color
Film is placed on colour plate, the PBST liquid on film is sucked with filter paper, adds 200 μ L of developing solution on film.It is put into instrument
It develops the color in device.
2.5.5 the specificity identification of monoclonal antibody
Selection BRV and PoRV, TGEV, PEDV and BPV are reacted with 5 plants of monoclonal antibodies, and SP2/0 supernatants make negative control.It is logical
It crosses indirect ELISA method and detects 5 plants of Mab supernatants and the reactivity of above-mentioned virus.Criterion is：P/N>2 be negative, table
With above-mentioned virus cross reaction does not occur for bright 5 plants of monoclonal antibodies.
2.5.6 the Characterization of antigenic epitopes that monoclonal antibody is directed to
Preliminary analysis is carried out to the epitope of monoclonal antibody using superposition ELISA method, method is as follows：
BRV virocytes culture is per 200 μ L of hole, and 4 DEG C of coatings are overnight or 37 DEG C are incubated 2h；PBST washes 3 times, every time
5min；Being superimposed the primary antibody of ELISA needs plus twice, first adds in one kind and add another Mab supernatant, 37 DEG C of incubations every time
1h；Washing, method are same as above；The mountain sheep anti-mouse igg of 5% skimmed milk dilution horseradish peroxidase-labeled is selected, extension rate is
1：1000,100 μ L/ holes, 37 DEG C of incubation 1h；PBST is washed 3 times, each 5min；TMB develops the color, microplate reader OD450Readings.Finally tie
Fruit calculates AI=(A1.2--A1)/A2 × 100% (the OD values of A1, A2 for independent monoclonal antibody, the OD values of A1.2 superposition primary antibodies).
Criterion：AI>50% shows to identify that antigen site is different, AI<50% site is similar or identical.
3 results
3.1 induced expression VP6 protein SDS-PAGEs are analyzed
Recombinant bacterium pProHTa-NCDV-VP6/Rosetta, which expands, to be cultivated, and IPTG induced expressions is added in, with inducing before induction
SDS-PAGE results such as Fig. 1 afterwards.
Through cutting glue purification, the VP6 protein concentrations for obtaining purifying are 1.125mg/mL, and the results are shown in Figure 2 by SDS-PAGE,
VP6 albumen after purification is compared with before purification, no foreign protein band, it is seen that has clear band at 52kDa, with expected size phase
Symbol.
The screening and foundation of 3.2 hybridoma cell strains
The BRV VP6 protein immunizations BALB/c mouse of purifying three times, after being immunized three times, measures the sero-fast potency of VP6,
When potency is higher than 1:When more than 25600, to mouse booster immunization, cell fusion experiment, the 3- after fusion are carried out after three days
5 days, in Microscopic observation cell fusion situation, and determine that cell confluency is 85%.The hybridoma in positive hole is carried out
Clone clones about 3-4 times.Finally obtain 5 plants of positive hybridoma cell strains, be respectively designated as 5A3,6E11,5C9,4D10 and
3G10。
The measure of 3.3 antibody titers
5 strain of hybridoma strains of acquisition are expanded into culture, adjust cell state, collect 90% cell bottle of cell monolayer
Mab supernatant liquid.Using BRV cell toxicants as antigen, SP2/0 cells are negative control, are detected through indirect ELISA method.Detection
As a result it is：The potency of 3G10,6E11,5C9,4D10 and 5A3 hybridoma supernatant is respectively 1:500、1:800、1：800、
1：1000,1：800.So selection 4D10 is used for the foundation of double-antibodies sandwich ELISA, and by the hybridoma preservation
In China Committee for Culture Collection of Microorganisms microorganism center, culture presevation number is CGMCC NO.14726.
3.4 hybridoma Identification of Biological Characteristics
3.4.1 monoclonal antibody subgroup identification
Using commercialization monoclonal antibody immunoglobulin subclass identification kit, the hypotype of hybridoma secretory antibody is identified.
It is IgG2a that 4D10 and two strain of hybridoma of 5A3, which generate Antibody types, and tri- strain of hybridoma of 3G10,6E11 and 5C9 is
IgM。
3.4.2 hybridoma chromosome counting
Myeloma cell and mouse boosting cell chromosome number are respectively 55~70 and 40.As shown in figure 3, hybridoma
The chromosome number number ratio of cell is about 86~100 by chromosome counting with SP2/0 showed increaseds.Close to myeloma
The sum of cell and mouse boosting cell chromosome number.
3.4.3 hybridoma repeated pruning
By 3G10,6E11,5C9,4D10 and 5A3 hybridoma continuous passage 3 months, and by freezing repeatedly and
It recovers, before detection monoclonal antibody cell cryopreservation and freezes rear cell conditioned medium, using BRV VP6 as detection antigen.Testing result is shown, miscellaneous
The numerical value that oncocyte freezes front and rear secretion supernatant is handed over to be all remarkably higher than SP2/0.It can be seen that hybridoma has preferably surely
It is qualitative.
The repeated pruning result of 1 hybridoma secretory antibody of table
3.4.4 monoclonal antibody Western-blot is identified
As a result Fig. 4 is seen, it can be found that 5 plants are handed over oncocyte culture supernatant to be reacted with BRV cell cultures, in transfer film
Occur apparent specific band at 52ku, illustrate that with BRV specific reaction occurs for 5 plants of monoclonal antibodies, and reactionogenicity is good
It is good.
3.4.5 indirect immunofluorescence assay
MA104 cell effect of 5 plants of Mab supernatants respectively with infection BRV is collected, adds in fluorescence secondary antibody.It is immunized indirectly glimmering
Light result figure 5 is shown：5 plants of Mab supernatants are reacted with BRV, generate fluorescence, and MA104 unstressed configurations occur, it is known that generation it is glimmering
Light is specificity fluorescent.
3.4.6 the specificity analysis of monoclonal antibody
5 plants of Mab supernatants and SP2/0 supernatants are reacted respectively with BRV and above-mentioned 4 kinds of virus.Fig. 6 is the results show that 5 plants of lists
The P/N ratios that anti-supernatant is reacted with BRV all reach 9 or so, P/N values and are much larger than 2.5 plants of Mab supernatants and BPV, TGEV, PEDV
1 is respectively less than with the P/N of PoRV, illustrates that with above-mentioned cross reaction does not occur for 5 plants of Mab supernatants, specificity is very high.
3.4.7 it is superimposed ELISA result of the tests
It is superimposed ELISA method and preliminary analysis is carried out to monoclonal antibody epitope, as a result calculation formula：AI=(A1.2--
A1)/A2 × 100%.A1, A2 are the OD values of independent monoclonal antibody, and A1.2 is the OD values of two plants of superposition primary antibodies.Criterion：AI>
50% shows to identify that antigen site is different, AI<50% site is similar or identical.Such as the following table 2, illustrate that being directed to for 5 strain antibodies is anti-
Former epitope is different from.
2 monoclonal antibody epitope Preliminary Analysis Results of table
The foundation of 2 double-antibodies sandwich ELISA of embodiment
1 test material
1.1 bacterial strains, cell and virus
BRV NCDV cell adapted strains are purchased from China Veterinery Drug Inspection Office, and MA104 cells are preserved by this laboratory.
1.2 main agents
(1) PBST cleaning solutions：KH2PO40.2g, Na2HPO4·12H2O 2.9g, sodium chloride 8.0g, KCl 0.2g, 1L are gone
Ionized water, Tween-20 0.5mL, pH are adjusted to 7.4.
(2)Na2CO3It is coated with dilution：NaHCO32.93g Na2CO31.5g, 1000mL deionized water dissolving, dense HCl
With NaOH tune pH to 9.6.
(3) confining liquid：1g skimmed milks are dissolved in 20mL PBS buffer solution (KH2PO40.2g, Na2HPO4·12H2O 2.9g, chlorine
Change sodium 8.0g, KCl 0.2g, 1L deionized water) dissolving, 4 DEG C of preservations.
(4) PBS buffer solution：KH2PO40.2g, Na2HPO4·12H2O 2.9g, sodium chloride 8.0g, KCl 0.2g, 1L are gone
Ionized water.
(4) tmb substrate developing solution：A liquid, B liquid 1:1 mixing, matching while using pay attention to being protected from light.10mL systems add in before reaction
4 μ L C liquid, mixing are protected from light spare.
(5) terminate liquid：2M H2SO4, the configuration concentrated sulfuric acid into distilled water it is noted that slowly add in the dense H of 10mL2SO4,
Constantly rotation, until total volume reaches 100mL.
(6) 4% paraformaldehydes：4g paraformaldehydes are dissolved in 1 × PBS solutions of 100mL, add a small amount of NaOH hydrotropies, -4 DEG C
It preserves.
(7) 0.2%Triton-100：0.2mL Triton-100 are dissolved in 1 × PBS solutions of 100mL, matching while using.
(8) 0.3%BSA：0.3g BSA are dissolved in 1 × PBS solutions of 100mL, matching while using.
(9) 3%BSA：3g BSA are dissolved in 1 × PBS solutions of 100mL, matching while using.
2 methods
The preparation of 2.1 rabbit-anti bovine rota polyclonal antibodies
2.1.1 the preparation of rabbit-anti bovine rota polyclonal antibody
Experimental animal is Adult female White Rabbit, and antigen is NCDV plants of the BRV of purifying, and injection dosage is 1mg//times.
The BRV antigen 1 mL that 1mg/mL is taken to purify, add in isometric Freund's complete adjuvant (FCA) 1mL, inject house after emulsification completely
Rabbit.Subcutaneous multi-point injection virus, is spaced 14 days, with doses of virus liquid, adds in isometric incomplete Freund's adjuvant (FIA) breast
Change, subcutaneous multi-point injection, every injection 0.1mL；Interval 14 days, injection site is with dosage with second；After third time is immune
5-7 days, ear edge vein exploitating blood 0.5mL, serum antibody titer is surveyed using indirect ELISA method, potency will reach 1:10000 with
Upper ability Culling heart blood, collects viral polyvalent antibody.
2.1.2 the purifying of rabbit-anti bovine rota polyclonal antibody
Rabbit Heart blood 90mL is acquired, 1h in 37 DEG C of incubators is put into, places into 4 DEG C of refrigerator overnights, is divided after clot contraction
From serum.By 4 DEG C of 5000r/min centrifugation 30min of serum, supernatant is taken, supernatant is carried out using the proteinG purification columns of GE companies
Purifying, surveys how anti-protein concentration after purification.It is as follows：
(1) taking-up is stored in -40 DEG C of polyclonal antibody, is filtered after thawing with 0.45 μm of filter.It removes big
Molecular impurity；
(2) polyclonal antibody after filtering, by 1:9 volume ratios are diluted with combination buffer, 4 DEG C of balance 8h；
(3) purification column is taken out, is connected with the connector and purification column provided in kit, the combination for drawing precooling is delayed
Fliud flushing removes the bubble in syringe in syringe, in order to avoid damage purification column；
(4) purification column is rinsed with the combination buffer of 10 times of purifying column volumes.It is slow during flushing, flow velocity 1mL/
min；
(5) it after syringe draws filtered polyvalent antibody, is connected on purification column, is slowly injected into purification column.For
The amount of purification column binding antibody IgG is improved, serum can be incubated 10min with purification column；
(6) purification column is rinsed with the combination buffer of 10mL or 20mL.The elution for purifying column volume with 2-5 times again
Buffer is eluted, and recycles the IgG antibody of combination；
(7) EP pipes recycling eluent, the EP pipes for recycling eluent add in the Tris-Cl of 60-200 μ L 1mol/L in advance
(pH 9.0) buffer solution；
(8) purification column after eluting 10mL combination buffers continue to rinse purification column, then with 5mL deionized waters and
20% ethyl alcohol rinses purification column, in case reusing；
(9) IgG antibody of collection is packed into bag filter, dialyse 48h in PBS solution.Antibody concentration is measured, is preserved anti-
Bulk concentration will be less than 3mg/mL, in order to avoid antibody inactivates, albumen precipitation occur.Antibody is dispensed -40 DEG C to save backup.
2..1.3 the identification of polyclonal antibody after purification
(1) preparation of protein sample：Appropriate how anti-protein sample after purification is taken to add in the 5 of 1/4 volume in EP pipes
× SDS buffer, boiling water boiling sample 30min.
(2) offset plate is assembled：Electrophoresis tank and glass plate are eluted with water, and dry in an oven or dry glass plate with filter paper, and
It is assembled.Select 1.5mm models offset plate and corresponding adhesive tape.Adhesive tape is put in mother glass groove, adhesive tape is put flat
Directly, otic placode is covered.Assembled offset plate is carefully moved on on foot stool, otic placode is outside, fixes offset plate with fixed plate, adds 3ml or so
Distilled water checks whether leak.
(3) separation gel：It is detached using 12%SDS-PAGE, finally adds TEMED (coagulant).Conelet is shaken before adding TEMED
Shape bottle about 10s makes the abundant mixing of coagulant liquid.Water in offset plate is discarded, is dried with filter paper.5 μ L are added in coagulant liquid
TEMED mixings.
(4) encapsulating：The coagulant liquid 6ml of mixing is rapidly added in offset plate, distilled water moulding is rapidly added and (uses pipettor
It is at the uniform velocity mobile back and forth to add in distilled water).Foot stool is placed on to horizontal local gel 40min.
(5) concentration glue (spacer gel)：Moulding water in offset plate is discarded, is inverted one minute, is dried (carefully not with filter paper
Encounter spacer gel), get out clean comb.Offset plate is uniformly added into 5%SDS-PAGE spacer gels, carefully plugs comb,
Gel 30min.
(6) electrophoresis：Comb is slowly extracted, fixed plate is extracted, carefully takes out two offset plates, take adhesive tape, otic placode is inside
It installs back on glue frame, mounting plate.Appropriate electrophoresis liquid is added in first glue groove, foot stool is put into electrophoresis tank, it is electric in supplemental tank
Swimming liquid, but do not exceed electrode tip.Offset plate comb is pulled up, unloads offset plate, carefully removes adhesive tape, otic placode puts back to foot in the inner part
Frame.1.5mm offset plates applied sample amount is less than 40 μ L.Albumen Marker is mixed in advance with 1 × SDS Buffer, the same sample size of applied sample amount
Unanimously, without the equivalent 1 × SDS Buffer occupy-places of the hole of sample.Upper strata glue 80V, lower floor glue 120V, until bromophenol blue is swum out.
(7) protein adhesive is put into Coomassie brilliant blue, after dyeing 40min, is put into destainer and is destained overnight.Work as background
After color takes off totally, scanning preserves result.
The preparation and purifying of 2.2 monoclonal antibody ascites
2.2.1 the preparation of monoclonal antibody ascites
SPF grades of 10-12 week old BALB/c female mices are taken, 500 μ L sterilized liquid paraffin are injected intraperitoneally, pneumoretroperitoneum injection in 7 days is miscellaneous
Hand over oncocyte 1 × 106-1×107It is a.Observation mouse abdominal circumference daily, mouse abdominal circumference significantly increases within about 7-14 days, according to mouse shape
State is every other day or two days extraction ascites is primary.4 DEG C of centrifugation 10min of ascites 3000r/min of collection draw the abdomen of middle layer
Water.The ascites of acquisition is dispensed, -80 DEG C save backup.
2.2.2 the purifying of monoclonal antibody ascites
The purifying of monoclonal antibody ascites equally uses the proteinG purification columns of GE companies, the same polyclonal antibody of method
Purification step.
2.2.3 the identification of ascites after purification
Monoclonal antibody ascites identification method is the same as polyclonal antibody identification method after purification.
2.3 rabbit-anti BRV are mostly anti-determining with McAb best effort concentration
(1) rabbit-anti BRV polyclonal antibodies after purification are made 1:100、1:200、1:400、1:800、1:1600、 1:
3200 dilutions, add according to 100 μ L/ holes in ELISA Plate, 37 DEG C of coating 2h；
(2) cleaning solution washing ELISA Plate 3 times, each 5min.Confining liquid is added in, per hole 200 μ L, 37 DEG C of closing 2h；
(3) cleaning solution washing ELISA Plate 3 times, each 5min.The MA104 supernatants (i.e. positive control) of BRV viruses will be inoculated with
With normal MA104 cells (i.e. negative control) while add in elisa plate, Duplicate Samples, 37 DEG C of effect 2h are set；
(4) cleaning solution washing ELISA Plate 3 times, each 5min, monoclonal antibody after purification (contain 5w/v% with confining liquid
The PBS of skimmed milk), made 1:10、1:20、1:40、1:80、1:160、1:320 dilutions, add in elisa plate, 100 μ L/
Hole sets parallel hole, 37 DEG C of effect 2h；
(5) cleaning solution washing ELISA Plate 3 times, each 5min, addition are pressed with confining liquid (PBS of the skimmed milk containing 5w/v%)
According to 1:5000 diluted HRP- sheep anti-mouse iggs, 37 DEG C of effect 1h；
(6) cleaning solution washing ELISA Plate 3 times, each 5min, add in the tmb substrate developing solution newly prepared, 37 DEG C of colour developings
10min；
(7) 2M H are added in2SO4Terminate liquid, reading numerical values.Wherein OD values are close to 1, P/N>When 2, i.e., by corresponding rabbit-anti
Mostly anti-and monoclonal antibody the dilutions of BRV are determined as best effort concentration.
2.4 double-antibodies sandwich ELISAs optimize
2.4.1 the selection of how anti-coating condition
It is coated with according to fixed coating concentration mostly anti-.There are four types of coating conditions：After 37 DEG C of 1h 4 DEG C overnight, 37 DEG C
4 DEG C of overnight, 37 DEG C of 1h, 37 DEG C of 2h, remaining step are same as above after 2h.According to OD450nmValue and P/N values determine best coated item
Part.
2.4.2 confining liquid determines
The coating concentration and condition of polyvalent antibody are coated with according to the result groped.Confining liquid selects to contain respectively
PBS solution (the KH of 2w/v%BSA, 5w/v%BSA, 2w/v% skimmed milk and 5w/v% skimmed milks2PO40.2g, Na2HPO4·
12H2O 2.9g, sodium chloride 8.0g, KCl 0.2g, 1L deionized water), 37 DEG C of closing 2h, remaining step same 2.3, according to
OD450nmValue and P/N values determine the selection of confining liquid.
2.4.3 off-period determines
It is coated with and is closed according to above-mentioned determining condition, off-period is respectively set as 37 DEG C of 120min, 37 DEG C
90min, 37 DEG C of 60min.
2.4.4 the measuring samples reaction time is determining
The reaction time of measuring samples is identified as 37 DEG C of 120min, 37 DEG C of 90min, 37 DEG C of 60min.Remaining step
It is operated according to 2.3 method for building up.
2.4.5 double-antibody sandwich elisa Positive judgement standards is determining
The detection method established using 2.3, while to 91 parts, negative cow dung is detected, and measures OD450nm.Calculate sample
OD450nmAverage value and standard variance (SD), according to principle of statistics, average value+3SD is set as to the positive and negative blood of sample
Clear critical value.As the OD of sample450nm>During average value+3SD, the positive can be judged in 99.9% level.Conversely, work as sample
This OD450nm<During average value+2SD, feminine gender can be judged to.
The sensitivity experiment of 2.5 double-antibodies sandwich ELISAs
The MA104 cells and supernatants PBS for being inoculated with BRV viruses is diluted 1 respectively:2、1:4、1:8、1:16、 1:32、
1:64、1:128、1:It 256 times, is carried out according to the double-antibodies sandwich ELISA after optimization.Analyze the dual anti-of this experiment foundation
The sensitivity of body sandwich ELISA method.
The specific test of 2.6 double-antibodies sandwich ELISAs
This research has selected the specificity of four kinds of viral diagnosis double-antibodies sandwich ELISAs, respectively PoRV, TGEV,
PEDV and BPV.It is thin with the double-antibodies sandwich ELISA detection BRV and PoRV, TGEV, PEDV and BPV, MA140 that have optimized
Born of the same parents' supernatant is as negative control.
The repetitive test of 2.7 double-antibodies sandwich ELISAs
2.7.1 repetitive test in batch
Using the rabbit anteserum and MAb of same Batch purification, the double-antibody sandwich elisa detection method established with 2.3, point
It is other to carrying out repeating to detect three times with a batch of BRV, compare their result difference, evaluation repeatability.
2.7.2 repetitive test between batch
The rabbit anteserum and MAb purified using different batches, the double-antibody sandwich elisa detection method established with 2.3, point
It is other to carrying out repeating to detect three times with a batch of BRV, compare their result difference, evaluation repeatability.
Detection of 2.8 double-antibodies sandwich ELISAs to clinical sample
The cow dung of 95 parts of suspected infection BRV is had collected from Heilungkiang, Liaoning and three, Inner Mongol province cattle farm just, wherein black
21 parts of Longjiang province, 21 parts of Liaoning Province, 53 parts of Jilin Province.It, will with PH7.0 0.2M PBS buffer solution after the sample of collection is ground
Suspension is made in it, through multigelation 3 times, after 1500rpm centrifugations 10min, supernatant is taken, respectively with the double-antibody sandwich established
ELISA method and RT-PCR method detection excrement sample compare the testing result of the two.
3 results
The polyclonal antibody qualification result of 3.1 purifying
The rabbit-anti BRV prepared is how anti-through GE purifying column purifications, measure its a concentration of 5.12mg/mL.VP6 after purification
Mostly anti-SDS-PAGE results such as Fig. 7 observes band, it is seen that it is a concentration of to measure its for the apparent heavy chains of IgG and light chain bands
5.12mg/mL。
3.6.2 it is how anti-determining with monoclonal antibody best effort concentration
It is final to determine by optimizing the working concentration of two kinds of antibody：Optimum dilution degree mostly anti-rabbit-anti BRV is 1: 800
(8.0 μ g/mL), the optimum dilution degree of 4D10 monoclonal antibodies is 1:20 (10.0 μ g/mL), the P/N values under the conditions of this are 2.102 (tables
3)。
Table more than 3 resists and monoclonal antibody best effort concentration
3.6.3 the optimization of double-antibodies sandwich ELISA
3.6.3.1 coating condition is determining
Resisted to capture antibody with rabbit-anti BRV more, to be coated with time and temperature as variable, carry out double-antibody sandwich elisa inspection
It surveys.Testing result is as shown in figure 8, best coating condition is 37 DEG C of 2h.
3.6.3.2 confining liquid determines
With reference to lot of documents, several different confining liquids have been selected：Contain 2w/v%BSA, 5 w/v%BSA, 2w/ respectively
The PBS sealase mark versions of v% skimmed milks, 5w/v% skimmed milks carry out double-antibody sandwich elisa detection.Testing result shows,
When containing the PBS of 5w/v% skimmed milks as confining liquid, P/N values are maximum.Therefore the PBS closing effects containing 5w/v% skimmed milks
Fruit is best, selects confining liquids (Fig. 9) of the PBS for containing 5w/v% skimmed milks as double-antibody sandwich elisa.
3.6.3.3 off-period determines
60min, 90min, 120min, which are closed off, using the PBS containing 5w/v% skimmed milks carries out double-antibody sandwich
ELISA is tested, and when off-period is 90min, P/N is maximum, and feminine gender value is low compared to other off-periods, accordingly, it is determined that most preferably
Off-period is 90min (Figure 10).
3.6.3.4 antigen incubation time is determining
After testing, when the reaction time of measuring samples is 120min, P/N values are maximum, thus may determine that measuring samples are anti-
It is 120min between seasonable.The result is shown in Figure 11.
3.6.3.5 Positive judgement standards determine
90 parts of negative excrement are collected, after treatment, are detected using double-antibody sandwich elisa.Calculate OD450Average value is
0.101867, standard deviation (S) is 0.015496, and it is 0.148355 to determine yin and yang attribute critical value (average value+3S), if sample
OD450nmValue>When 0.148335, it is judged to the positive；OD450nmValue<Feminine gender is judged to when 0.148335.
3.6.4 the sensitivity detection of double-antibodies sandwich ELISA
By BRV cell culture doubling dilutions, detected, while set up feminine gender with the ELISA method of foundation, positive control,
Experimental result is shown in Table 4.
4 sensitivity result of the test of table
Sensitivity result of the test shows when virus 1：During 32 dilution, result is the positive (OD450Value 0.332), and dilution
It is 1:When 64, result is feminine gender (OD450Be worth for 0.172), represent this method viral limit of identification 9.884 ×
104TCID50/mL。
3.6.5 the specific detection of double-antibodies sandwich ELISA
BRV, TGEV, PoRV, PEDV and BPV are detected using the double-antibodies sandwich ELISA that this research is established, as a result
As shown in figure 12, in addition to BRV test positive, TGEV, PoRV, PEDV and BPV virus are feminine gender.Thus this research is proved
The double-antibodies sandwich ELISA specificity of foundation is good, cross reaction does not occur with other viruses.
3.6.6 the repeatability detection of double-antibodies sandwich ELISA
The BRV cell toxicants of 5 batches are randomly selected, carry out repetitive test.Five groups of repetitions are carried out in same ELISA Plate
Property experiment, calculate five groups of result of the tests the coefficient of variation between 1.7%~3.6% (table 5)；Different ELISA Plates carries out five
Group repetitive test, calculates its coefficient of variation in 2.8%~8.5% (table 6).The coefficient of variation of two groups of repetitive tests exists
10% hereinafter, illustrate that the double antibody sandwich ELISA that this experiment is established has good repeatability.
Repetitive test in 5 plate of table
Note：S.D. standard deviation is represented；Mean represents arithmetic mean of instantaneous value；CV represents the coefficient of variation, CV%=S.D./Mean
Repetitive test between 6 plate of table
Note：S.D. standard deviation is represented；Mean represents arithmetic mean of instantaneous value；CV represents the coefficient of variation, V%=S.D./Mean
3.6.7 the result of double-antibodies sandwich ELISA detection clinical sample
95 parts of cow dungs of collection are just ground, suspension is made, with this research establish double-antibodies sandwich ELISA with
And RT-PCR method is detected 95 parts of fecal specimens.Testing result is shown in Table 7,8, there is the OD of 4 parts of samples450>0.148335,
It can determine and detect that 4 parts of samples are positive pathological material of disease from this 95 parts of samples.RT-PCR results are as shown in figure 13, also examine
4 parts of positive pathological material of diseases are measured, purpose band size is consistent with expection, and negative control does not locate any band occur.Therefore it can obtain
Go out conclusion, the double-antibodies sandwich ELISA of foundation is 100% with the coincidence rate of RT-PCR method testing result.
The testing result of 7 clinical sample of table
The testing result of 8 clinical sample of table
Claims (10)
1. the hybridoma cell strain of the anti-bovine rota VP6 protein monoclonal antibodies of one plant of stably excreting, is named as 4D10, described
Hybridoma cell strain be deposited in China Committee for Culture Collection of Microorganisms microorganism center, culture presevation number is
CGMCC NO.14726。
2. anti-bovine rota VP6 protein monoclonal antibodies, which is characterized in that the monoclonal antibody is by claim 1 institute
The hybridoma cell strain secretion stated generates.
3. application of the monoclonal antibody in the reagent of detection or Diagnosis of Cattle rotavirus infection is prepared described in claim 2.
4. a kind of double-antibody sandwich elisa diagnostic kit for being used to detect bovine rota, which is characterized in that wanted containing having the right
Seek the monoclonal antibody described in 2.
5. kit as claimed in claim 4, which is characterized in that the kit further includes more grams of rabbit-anti bovine rota
Grand antibody, HRP- sheep anti-mouse iggs, cleaning solution, confining liquid, dilution, positive control, negative control, developing solution and terminate liquid.
6. kit as described in claim 4 or 5, it is characterised in that when being detected for bovine rota, according to following steps
It carries out：
(1) it by after rabbit-anti bovine rota polyclonal antibody diluted, is added in ELISA Plate according to 100 μ L/ holes, 37 DEG C
It is coated with 1-2h；
(2) cleaning solution washing ELISA Plate 3 times, each 5min, adds in confining liquid, per hole 200 μ L, 37 DEG C of closing 2h；
(3) cleaning solution washing ELISA Plate 3 times, each 5min, sample serum to be checked is added in ELISA Plate, setting Duplicate Samples, and 37
It DEG C is incubated, while positive control and negative control is set；
(4) cleaning solution washing ELISA Plate 3 times, each 5min adds in the monoclonal antibody after dilution, 37 DEG C of incubation 1h；
(5) cleaning solution washing ELISA Plate 3 times, each 5min, adds in 1:5000 diluted HRP- sheep anti-mouse iggs, 37 DEG C of incubation 1h；
(6) cleaning solution washing ELISA Plate 3 times, each 5min adds in the tmb substrate developing solution newly prepared, 37 DEG C of colour developing 10min；
(7) 2M H are added in2SO4Terminate liquid, reading numerical values, yin and yang attribute critical value is 0.148355, if sample OD450nmValue>
When 0.148335, it is judged to the positive；OD450nmValue<Feminine gender is judged to when 0.148335.
7. kit as claimed in claim 6, which is characterized in that the cleaning solution contains KH2PO40.2g/L,
Na2HPO4·12H2O 2.9g/L, sodium chloride 8.0g/L, KCl 0.2g/L, Tween-20 0.5mL L, remaining is deionized water,
PH is adjusted to 7.4；The dilution contains NaHCO32.93g/L Na2CO31.5g/L, remaining is deionized water, and pH is adjusted to
9.6；The confining liquid is the PBS solution of the skimmed milk containing 5w/v%；The positive control is the MA104 for being inoculated with BRV viruses
Cells and supernatant, the negative control are MA104 cells.
8. kit as claimed in claim 6, which is characterized in that in step (1), more grams of the rabbit-anti bovine rota after dilution
A concentration of 8.0 μ g/mL of grand antibody, 37 DEG C of coating 2h.
9. kit as claimed in claim 6, which is characterized in that in step (3), the incubation time of sample serum to be checked is
2h。
10. kit as claimed in claim 6, which is characterized in that in step (4), the concentration of the monoclonal antibody after dilution
For 10.0 μ g/mL.
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| CN110763843A (en) * | 2019-11-06 | 2020-02-07 | 华中农业大学 | Mycoplasma bovis double-antibody sandwich ELISA (enzyme-Linked immuno sorbent assay) detection kit and application thereof |
| CN110903403A (en) * | 2019-12-16 | 2020-03-24 | 中国农业大学 | Bovine rotavirus chimeric antigen and colloidal gold immunochromatographic test paper card for detecting bovine rotavirus antibody |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110763843A (en) * | 2019-11-06 | 2020-02-07 | 华中农业大学 | Mycoplasma bovis double-antibody sandwich ELISA (enzyme-Linked immuno sorbent assay) detection kit and application thereof |
| CN110763843B (en) * | 2019-11-06 | 2020-09-15 | 华中农业大学 | Mycoplasma bovis double-antibody sandwich ELISA (enzyme-Linked immuno sorbent assay) detection kit and application thereof |
| CN111073859A (en) * | 2019-12-09 | 2020-04-28 | 东北农业大学 | Double-antibody sandwich ELISA kit for detecting bovine parvovirus and application thereof |
| CN111073859B (en) * | 2019-12-09 | 2021-09-28 | 东北农业大学 | Double-antibody sandwich ELISA kit for detecting bovine parvovirus and application thereof |
| CN110903403A (en) * | 2019-12-16 | 2020-03-24 | 中国农业大学 | Bovine rotavirus chimeric antigen and colloidal gold immunochromatographic test paper card for detecting bovine rotavirus antibody |
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