CN108148813A - The anti-infectious bovine rhinotrachetis virus monoclonal antibody and application of one strain of hybridoma strain and its secretion - Google Patents
The anti-infectious bovine rhinotrachetis virus monoclonal antibody and application of one strain of hybridoma strain and its secretion Download PDFInfo
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Abstract
The invention belongs to cell engineering fields, especially belong to veterinary biologics field, more particularly relate to one plant can the anti-infectious bovine rhinotrachetis virus monoclonal antibody of stably excreting hybridoma cell strain and its application.Hybridoma cell strain 5F9, in August in 2016 31 days in China General Microbiological culture presevation administrative center preservation, deposit number is：CGMCC No.12700.The present invention passes through the anti-infectious bovine rhinotrachetis virus monoclonal antibody and fluorescein isothiocynate of secreting this hybridoma（FITC）With reference to, and then establish quick, sensitive IBRV detection methods.Ox is imported and exported for China and its meat products IBRV detections are of great significance.
Description
Technical field
The invention belongs to cell engineering fields, especially belong to veterinary biologics field, more particularly relate to
The anti-infectious bovine rhinotrachetis virus monoclonal antibody and application of one strain of hybridoma strain and its secretion.
Background technology
Infectious bovine rhinotrachetis (Infectious bovine rhinotracheitis, IBR) is by Niu Chuanran
One kind of ox caused by property rhinotracheitis virus (Infectious bovine rhinotracheitis virus, IBRV)
Acute, hot, contagious disease.IBRV be also known as bovine herpes virus-1, bovid herpesvirus 1 and contagious ecthyma vulva-
Vaginitis virus.The disease can also cause reproductive system characterized by high fever, expiratory dyspnea, rhinitis, sinusitis and inflammation of upper respiratory tract
There is miscarriage and stillborn foetus and the symptoms such as enteritis and calf encephalitis occurs, eye conjunctivitis and keratitis also occur sometimes in damage.
Secondary bacterium infection can lead to more serious breathing problem.
China provides that IBRV must be detected by importing and exporting ox and its meat products, this causes the detection method of the disease to send out rapidly
Exhibition is got up.The detection of the disease is first had to combine epidemiology and clinical examination in practice and makes tentative diagnosis, then root again
Room method makes exact judgement according to the experiment.
Detection for infectious bovine rhinotrachetis virus, conventional method are mostly serological method, such as neutralization test, indirectly
Hemagglutination test etc..The shortcomings that these method various degrees or sensitivity is not high or need the time longer.PCR is current
Detect infectious bovine rhinotrachetis virus than faster, one of sensitive, simple method, particularly to infectious bovine rhinotrachetis
Virus lays dormant infection is made a definite diagnosis, and high specificity can also do biopsy, but false positive rate is higher.In addition to the PCR side tentatively established
Method, it is domestic at present there is not yet the report of other diagnostic antigen methods, is examined for this purpose, carrying out infectious bovine rhinotrachetis virus antigen
The research of disconnected method, is China's infectious bovine rhinotrachetis virus epidemiological survey, provides a kind of good antigen detection hand
Section, prevention and control and elimination to epidemic disease from now on are all of great significance.
As laboratory diagnosis reagent, monoclonal antibody with its high specificity, purity is high, homogeneity is good the advantages that, extensively
Applied to technologies such as enzyme-linked immunosorbent assay, radiommunoassay, immunohistochemistry and flow cytometers.
It prepares for the monoclonal antibody of IBRV both at home and abroad at present and carries out IBRV detections using related monoclonal antibody
Technique study has not been reported.Therefore the monoclonal antibody that the energy anti-infectious bovine rhinotrachetis virus of stably excreting is prepared is miscellaneous
Oncocyte is handed over, is of great significance for quick, the sensitive IBRV detection methods of commercialized development production, is quick, sensitive
The solid foundation of IBRV detection method researchs.
Invention content
The object of the present invention is to provide one plant can the anti-infectious bovine rhinotrachetis virus monoclonal antibody of stably excreting it is miscellaneous
Hand over tumor cell strain.
The hybridoma cell strain of the energy anti-infectious bovine rhinotrachetis virus monoclonal antibody of stably excreting is miscellaneous in the present invention
Tumor cell strain 5F9 is handed over, in August in 2016 31 days in China General Microbiological culture presevation administrative center preservation, preservation is compiled
Number it is：CGMCC No.12700.
The preparation method of the hybridoma cell strain 5F9 described in the present invention is：By the infectious bovine rhinotrachetis purified
BALB/c mouse is immunized for antigen in viral (IBRV), after cell fusion, with indirect IBRV-ELISA and cellmediated immunity fluorescence
Carry out the cell strain that screening obtains the energy anti-infectious bovine rhinotrachetis virus monoclonal antibody of stably excreting.The cell strain passes in vitro
It is commissioned to train foster stabilization, culture supernatant ELISA potency is 10000, and chromosome number is 99.
Also, the monoclonal antibody that present invention protection is generated by the hybridoma cell strain 5F9.
Further, it protects and is prepared by the hybridoma cell strain 5F9 monoclonal antibodies generated in the present invention
Anti- infectious bovine rhinotrachetis virus monoclonal fluorescence antibody.
The preparation method of this fluorescence antibody is the anti-infectious bovine rhinotrachetis virus monoclonal for obtaining preceding method
Antibody and fluorescein isothiocynate（FITC）It is combined into.
Its specific preparation method is：Monoclonal antibody is prepared using method is induced in Mice Body, uses Freund within 7 ~ 10 days in advance
SPF grades of female Balb/c mouse of 8 ~ 10 week old of Freund's incomplete adjuvant intraperitoneal inoculation are pre-processed, will be in the miscellaneous of exponential phase
Oncocyte intraperitoneal inoculation mouse is handed over, hybridoma is grown in mouse peritoneal, and generate ascites, is acquired when mouse web portion significantly swells
Ascites is monoclonal antibody from intermediate faint yellow supernatant is taken.
Protect the monoclonal antibody of the hybridoma cell strain 5F9 generations simultaneously in the present invention and by the monoclonal antibody
Application of the obtained monoclonal fluorescence antibody prepared in infectious bovine rhinotrachetis virus is detected.
Application mode mainly includes two kinds, and one kind is to establish indirect immunofluorescence method using the monoclonal antibody, this side
Formula is primarily adapted for use in the monoclonal antibody of hybridoma cell strain 5F9 generations；Another kind is the list prepared using the monoclonal antibody
Clone's fluorescence antibody establishes direct immuno-fluorescent antibody assay method, can further assemble immune reagent kit, immunity test strip, immune examination
Agent etc., this mode are primarily adapted for use in the fluorescence antibody being prepared by the hybridoma cell strain 5F9 monoclonal antibodies generated；
Wherein, in the second way, detection method is using the 5F9 monoclonal antibodies that FITC is marked as detection antibody.It is preferred that
Testing conditions be following any one or multinomial condition：（1）Antigen fixative be+20% methanol of 80% acetone and 10% formaldehyde,
（2）Fixed temperature is -20 DEG C,（3）Set time is 30 minutes,（4）Labelled antibody working concentration is 1：100；（5）Optimum antibody
Incubation time is 45 min.
The present invention obtains the hybridoma of the energy anti-infectious bovine rhinotrachetis virus monoclonal antibody of stably excreting by screening
Cell strain, and prepare to form irrelevant monoclonal fluorescence antibody with the monoclonal antibody that the cell strain is secreted, so establish it is quick,
Sensitive IBRV detection methods.Ox is imported and exported for China and its meat products IBRV detections are of great significance.
Description of the drawings
Fig. 1 is 5F9 hybridoma forms under microscope；
Fig. 2 is that the antigentic specificity of 5F9 culture supernatants reacts fluorescence picture.
Specific embodiment
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and
It is apparent.But these embodiments are only exemplary, do not form any restrictions to the scope of the present invention.People in the art
Member it should be understood that without departing from the spirit and scope of the invention can to the details of technical solution of the present invention and form into
Row modifications or substitutions, but these modifications and replacement are each fallen in protection scope of the present invention.
The hybridoma cell strain 5F9 arrived involved in the present invention, in August in 2016 31 days in China General Microbiological strain
Preservation administrative center preservation, deposit number are：CGMCC No.12700, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Number.
Foundation, identification and the passage of 1 hybridoma of embodiment
1st, the preparation of viral antigen liquid
（1）IBRV NM strain virus is inoculated with individual layer MDBK cells by the proliferation of virus, and virus liquid is harvested when CPE is up to 80%.It presses
It is 108.5 that Spearman Karber formula, which calculate the IBRV NM strain virus TCID50/mL obtained,.
（2）The concentrating and purifying of virus removes cell fragment to 0.45 μm of membrane filtration of virus liquid of harvest, takes filtered solution
35000 r/min ultracentrifugations, 2 h, precipitation are suspended in suitable PBS buffer solution（pH 7.2）In, 4 DEG C make it fully molten overnight
Viral solution, is then carefully added into sucrose gradient centrifugation pipe upper strata liquid level, sucrose density gradient is respectively from top to bottom by solution
25%, 45%, 65%, require interface apparent between different saccharose gradients, 25000 r/min centrifuge 2 h, are carefully sucked out with elbow minute hand
The viral level of white between 45%~65% sucrose, is dissolved in 35mL PBS buffer solution, then 35000 r/min exceed the speed limit from
2 h of the heart, takes precipitation to be dissolved in 1mL PBS, this solution is the virus liquid after concentrating and purifying, and the purifying antigen liquid of collection is led to
0.22 μm of membrane filtration degerming is crossed, is frozen in -20 DEG C.
（3）The inactivation of virus and the BEI for examining addition 3nM are uniformly mixed, and are put 37 DEG C and are inactivated 36 hours, during which do not stop to stir
It mixes, puts 2-8 DEG C of preservation, should be no more than 30.Inactivation of viruses liquid stoste is taken to be inoculated with 75cm2 square vase MDBK cell monolayers, 37 DEG C of senses
Make 1h, add cell growth medium, while set one bottle of MDBK cell controls, 72h is cultivated in 37 DEG C, 5%CO2 incubators, observe
CPE, it should occur without CPE.In 1 generation of blind passage, 37 DEG C are continued to cultivate 72h, observe CPE, should occur without CPE, then sentence inactivation and examine
It is qualified.
（4）Virus protein assay spectrophotometric determination virus protein content, the calculation formula of protein concentration
For：Protein concentration (mg/mL)=(1.45 × D280nm-0.74 × D260nm) × extension rate.Measure IBRV NM plants after purification
Protein content be 4.84mg/mL.
（5）Animal immune head exempts from NM plants of the IBRV with purifying with after Freund's complete adjuvant emulsification, being injected intraperitoneally immune 6 ~ 8
Week old female BAl BIc/c mouse, two exempt to exempt from the 14th d after head exempts from and the 28th d progress with three, incomplete with Freund
Adjuvant emulsion, three exempt from rear 14th d dockings blood sampling separation serum, detect the antibody titer of serum.3 d are noted through tail vein before fusion
Penetrate reinforced immunological 1 time.
2nd, the screening and purifying of cell fusion and hybridoma
（1）24 h take the peritoneal macrophage of healthy BALB/c mouse to prepare feeder cells before fusion, and 37 DEG C of cultures are routinely square
Method is sterile to prepare splenocyte, carries out cell fusion under PEG effects with SP2/0 cells, selective culture is carried out with HAT culture mediums
7~10d.When there is fused cell colony to occur, replace HT culture mediums and continue to cultivate.When fused cell colony covers with the 1/3 of bottom hole
When ~ 1/2, its supernatant is detected with indirect ELISA.Method is as follows：
1. NM plants of concentration for being diluted to 10ug/ml of IBRV of purifying, abundant mixing are coated with by coating antigenic dilution per hole
100 μ l are measured, 2 ~ 8 DEG C of coatings of microwell plate of coating buffer will be filled overnight.
Make cleaning solution by 7.4 PBST of 0.01mol/L pH value 2. washing, per 300 μ l of hole, room temperature acts on 2 ~ 3 minutes,
It is repeated 5 times, pats dry until no-watermark.
3. PBST of the closing containing 5% skimmed milk power, injects in microwell plate, 37 DEG C are incubated 1 hour, according to 1.2.3.2 side
Method is washed.
4. plus detection sample, per 100 μ l of hole, 37 DEG C are incubated 0.5 hour, make positive control with immune serum, are immunized
Preceding serum makees negative control.It is washed according to 1.2.3.2 methods.
5. make 1 with PBST：5000 times of dilution HRP label goat anti-mouse igg ELIAS secondary antibodies, per 100 μ l of hole, 37 DEG C of incubations
It 0.5 hour, is washed according to 1.2.3.2 methods.
6. colour developing adds 50 μ l of color developing agent per hole, 37 DEG C are protected from light effect 15 minutes.
Add 50 μ l of terminate liquid, the readings under microplate reader 450nm wavelength per hole 7. terminating.
8. result judgement is with positive A value >=1.0, positive A values/feminine gender A values>It is Positive judgement standards when 2.1.
（2）ELISA detection positive cell hole supernatants is taken to use cellmediated immunity fluorescent test again（IFA）Further detection,
Method is as follows：
1. the culture and preparation of cell：MDBK cells, after trypsin digestion, to contain the DMEM culture mediums point of 6% newborn bovine serum
It dissipates, adjusts 1.5 × 105/ml of cell density, be added in each hole of 96 porocyte culture plates according to 100 μ l/ holes, 37 DEG C, 5%CO2
Under the conditions of, culture 12 ~ for 24 hours, it is made to grow up to individual layer.
2. virus inoculation：Virus is diluted to certain dilution with DMEM culture mediums, after abundant mixing, with multichannel pipettor plus
To each hole of cell monolayer, if negative cells compare.
3. 37 DEG C are incubated, culture 48 ~ 60 hours in 5%CO2 incubators.
4. fixation abandons each hole culture solution of 96 porocyte culture plates in waste liquid cylinder（Containing sodium hydroxide）, liquid in plate is emptied, is used
PBS, 200L/ hole are cleaned 1 time.Precooling fixative 100L/ holes are added in each hole, plank is placed in -20C effect 30min rapidly,
Room temperature naturally dry.
PBS is added in 5. cleaning in each hole, 200 L/ holes are cleaned 1 time.
6. by working concentration, antiviral positive serum or monoclonal antibody are diluted with PBS, added with multichannel pipettor per hole
Enter 50 μ l, incubated 45 minutes ~ 1 hour, while set negative control in 37 DEG C of wet box（Malicious hole is not connect）And blank control（Malicious hole is connect,
Primary antibody adds PBS holes）.
PBS is added in 7. cleaning in each hole, 200 μ L/ holes are cleaned 2 times.
8. by working concentration, with the secondary antibody of PBS dilution fluorescent markers, 50 μ l are added in per hole with multichannel pipettor, 37 DEG C wet
It is incubated 45 minutes ~ 1 hour in box.
It is cleaned 3 times with PBS, 200 μ l/ holes in compared with dark situation 9. cleaning.
For 10 result judgements under fluorescence microscope, positive cell is in specificity fluorescent, and staining cell should be endochylema coloring,
Cell outline is clear and legible, negative cells control wells and blank control should without specificity fluorescent, as long as being colored there are one cell,
It can determine that detection sample for the positive, is otherwise denoted as " feminine gender ".
ELISA and IFA detections are all that positive monoclonal hole can be confirmed as positive cell clone strain.The sun that will be screened
Property cell clone cloning, until all monoclonal cell holes Positive rate be 100% when, can determine obtain stablize point
Secrete the hybridoma cell strain of monoclonal antibody.The hybridoma cell clone strain of acquisition is enlarged culture respectively, and freeze in
In liquid nitrogen.
5th, the identification of hybridoma cell strain
（1）Cellular morphology is observed：With the DMEM cell culture fluids containing 10% newborn bovine serum, 37 DEG C, 5%CO2 incubator cultures 2h
Can gently wall, individual layer can be covered with for 24 hours.Micro- Microscopic observation cellular morphology.Hybridoma form is as shown in Figure 1.
（2）Pure property detection：By existing《Chinese veterinary pharmacopoeia》Annex is detected, no bacterium, mould, mycoplasma and external source
Virus pollution.
（3）Chromosome analysis：The hybridoma in cell log growth period is chosen, adds in 0.08 μ g/ml colchicums
Element is in 37 DEG C of incubators.37 DEG C are continued 8 ~ 10h of culture.Collect dividing cell, 1000r/min centrifugations 10min.Supernatant is absorbed, is added
Enter pre-temperature to the KCl solution 10ml of 38 DEG C of 0.075mol/L, static 20 ~ 30min in incubator.Then it is added in into suspension new
The acetic acid of fresh preparation-methanol fixer（1：3）1.0ml is blown and beaten uniformly with suction pipe, cell surface is made slightly to fix, so as to prevent
Cytoadherence is blocking when fixed.Then 10min is centrifuged through 1000r/min, sucks supernatant.In addition 5 ~ 10ml of fixer is stated, gently
Piping and druming is uniform, puts room temperature and fixes 15 ~ 20min.It is then centrifuged for, absorbs supernatant, repeat and fix 1 time.Above-mentioned cell is centrifuged, it is young
Supernatant carefully is sucked, stays fixer about 0.5ml, by cell mixing.Cold glass slide 1 is taken to open, is dripped carefully to glass slide in about 30cm height
Born of the same parents' suspension 1 ~ 2 drips, and then puts in room temperature and enables its natural drying.It is dyed with Ji's nurse Sa.1 part of mother liquor Ji's nurse Sa is taken, is used（PBS
pH6.8）9 parts of dilutions, dye 10min.Then it washes, dries.Chromosome cytometry analysis is carried out under oil mirror, 5F9 hybridomas are thin
Born of the same parents are averaged chromosome number as 99, meet the sum of chromosome number of SP2/0 cells and splenocyte, it was demonstrated that 5F9 hybridomas
It is merged for SP2/0 with splenocyte.
（4）The activity of secrete monoclonal antibody：Hybridoma Cell Culture supernatant is taken, does 1：10,1：100,1：1000,1：10
000,1：100 000,1：1000 000 are serially diluted, and are detected with indirect IBRV-ELISA.The culture supernatant of 5F9 hybridomas
ELISA potency >=1: 10000.
（5）The specificity of secrete monoclonal antibody
Antigenic cross-reaction is tested：Hybridoma Cell Culture supernatant is taken, the MDBK cells of BVDV, IBRV infection are exempted from indirectly
Epidemic disease fluoroscopic examination, the hybridoma generation culture supernatant of screening should only with IBRV idiosyncrasies.As a result show 5F9 culture supernatants only with
IBRV specific reactions have stronger specificity fluorescent, and significantly positive, MDBK cells and SP2/0 cells and supernatants work is presented
Negative control is set up, and as a result sees Fig. 2.
From the cross reaction of different IBRV separation strains：The culture supernatant of hybridoma generation is taken, to different IBRV isolated strains：
The MDBK cell monolayers of LN plants of 67 plants of IBRV Bartha Nu, IBRV infection carry out indirect immunofluorescene assay, screening it is miscellaneous
Hand over knurl secondary culture supernatant that should be able to be reacted with these IBRV isolated strains.As a result show that 5F9 can be with IBRV-M, IBRV-LN, IBRV
67 specific reactions of Bartha Nu have stronger specificity fluorescent, and significantly positive, MDBK cells and the training of SP2/0 cells is presented
Foster supernatant is made negative control and is set up, and the results are shown in Table 1.
The cross reaction result of 1 5F9 monoclonal antibodies of table and different IBRV separation strains
The stability of secrete monoclonal antibody：Subculture in vitro separately culture takes culture supernatant indirect immunofluorescene assay every 5 generations,
Culture supernatant is taken to detect 1 time every 10 generation cryopreservation resuscitations, continue to reach 30 generations, indirect ELISA detection culture supernatant.As a result it shows
Hybridoma can be with stably excreting monoclonal antibody.
6th, hybridoma secondary culture method：Choosing growth the cell for having formed individual layer on the 3rd, with suction pipe softly cell from
It is blown and beaten in bottle wall, is allowed to be dispersed into individual cells, by 1:5~1:6 dispersion rates pass to cell in new cell bottle, add
Growth-promoting media in 37 DEG C of cultures, forms good individual layer in 24 ~ 48 hours.
The preparation and inspection of 2 monoclonal antibody of embodiment
1st, the preparation of monoclonal antibody ascites
（1）Pretreatment selection SPF grades of BALB/c mouses of 8 ~ 10 week old of BALB/c mouse, are inoculated with 7 ~ 10 before IBR hybridomas
It is to BALB/c mouse intraperitoneal inoculation incomplete Freund's adjuvant, and 0.5ml/ is only.
（2）The IBR hybridomas that the culture and processing of hybridoma will cover with individual layer, discard nutrient solution, dispel
It is even, by 1:3~1:5 ratios pass on, in 37 DEG C, containing 5%CO224 ~ 48h is cultivated in incubator and is in exponential phase, it is miscellaneous to collect IBR
Oncocyte is handed over, 1000r/min centrifugation 10min use PBS（PH value 7.4,0.01mol/l）Adjust IBR hybridomas density for 5 ~
10×106A/ml.
（3）The inoculation of hybridoma and ascites are collected thin to the BALB/c mouse intraperitoneal inoculation IBR hybridomas of pretreatment
Born of the same parents, 0.5ml/ is only.Ascites is acquired when mouse peritoneal significantly swells, 2 ~ 8 DEG C stand overnight, and 8000r/min centrifugation 10min are abandoned
The impurity such as upper strata grease and membrane substance, lower floor's cell fragment, IBR monoclonal antibodies are primarily present in intermediate faint yellow or pale red
In liquid, preservative Proclin300 to final concentration of 0.02% is added in, is dispensed after less than -70 DEG C preservations.
2nd, the inspection of monoclonal antibody ascites
（1）Character weak yellow liquid.
（2）It is pure by existing《Chinese veterinary pharmacopoeia》Annex is tested, no bacterium, mould contamination.
（3）Antibody titer detection takes IBR monoclonal antibody ascites to do 1:4000,1:5000,1:6000,1:7000,1:
8000...... it is serially diluted, carries out indirect immunofluorescene assay, the antibody titer of IBR monoclonal antibody ascites is 1：
100000。
3rd, the purifying of monoclonal antibody
（1）Enough glass fibres are padded in the removal of grease, membrane substance and cell fragment in funnel（Band gloves when taking）, add
Enter ascites, collect the ascites after filtering, finally squeeze glass fibre with glove hand to obtain all samples；In room temperature (15
~25 DEG C) under the conditions of, the ascites after filtering is centrifuged into 30 min in 20000g, supernatant is taken, abandons membrane substance and cell in precipitation
Fragment.
（2）The removal of foreign protein takes 1 part of ascites to add in 2 parts of acetate buffer solutions（4.8,0.06 mol/l of pH value）, adjust pH value
To 4.8；Caprylic acid, room temperature is added dropwise under (15~25 DEG C) stirrings of room temperature in the ratio for adding 33 μ l caprylic acids in every milliliter of ascites
Mixing 30min under the conditions of (15~25 DEG C)；2 ~ 8 DEG C of standing at least 2h, 20000g centrifugation 35min, take supernatant；If in supernatant
There are still precipitation, repeated centrifugation is primary.
（3）Ammonium sulfate precipitation adds in PBS (10X, 7.2,0.1 mol/l of pH value) according to the 1/10 of supernatant volume, adjusts pH
It is worth to 7.2；Ammonium sulfate is slowly added into ascites by 0.277~0.310g/ml in ice-water bath, it is stirring while adding, until 45%~
50% saturation degree, mixing 30min in ice-water bath, 2 ~ 8 DEG C of standing at least 2h, 20000g centrifugation 1h abandon supernatant.
（4）Desalination of dialysing is dissolved with appropriate PBS to be precipitated, and is fitted into bag filter after mixing, with 1000 under the conditions of 2 ~ 8 DEG C
The PBS of times volume（7.4,0.01 mol/l of pH value）Dialyse 24 ~ 48h, during which replaces buffer solution at least 3 times, 20000g centrifugations
10min removes insoluble sediment, adds in 5% volume glycerine, is sub-packed in less than -70 DEG C and freezes.
The label of 3 monoclonal antibody of embodiment
1st, the preparation of 1mg/mL FITC solution adds 1mL carbonate buffer solutions (pH value 9.0,0.1mol/ by 1mg FITC pulvis
L) ratio mixing, spiral to being completely dissolved, this solution must matching while using, be finished in 5min.
2nd, paddling process label adds the ratio of 250uL FITC solution (1mg/mL) according to 1mL IgG solution (5mg/mL), to
FITC solution (1mg/mL) is added dropwise in the monoclonal antibody of purifying, it is stirring while adding（90rpm/min）, room temperature (15~25
DEG C) it is protected from light gentle agitation 2h.
3rd, the removal of free fluorescein is in charge of collection eluent using the free fluorescein of glucan G-25M pillars removal.
With the absorption value of the every pipe eluent of ultraviolet specrophotometer 280nm wavelength detectings, it is seen that have 2 eluting peaks, conjugate appears in
First eluting peak；Collect main conjugate, collect the eluent of A280 >=0.4.
The foundation of 4 infectious bovine rhinotrachetis virus direct immuno-fluorescent antibody assay method of embodiment and detection kit group
Dress
1st, the foundation of infectious bovine rhinotrachetis virus direct immuno-fluorescent antibody assay method
（1）The culture and preparation of cell：MDBK cells, after trypsin digestion, to contain the DMEM culture mediums of 6% newborn bovine serum
Dispersion adjusts 1.5 × 105/ml of cell density, is added in each hole of 96 porocyte culture plates according to 100 μ l/ holes, is placed in 37 DEG C,
The interior culture 12 of 5%CO2 incubators ~ for 24 hours, it is made to grow up to 80 ~ 90% individual layers.
（2）The inoculation of virus：Virus to be checked is taken, with the DMEM culture mediums dilution virus containing 2% newborn bovine serum extremely
100TCID50/50 μ l add to corresponding cell hole by 50 μ l/ holes.37 DEG C are placed in, is cultivated 48 ~ 60 hours in 5%CO2 incubators, and
If negative cells compare.
（3）The PBS for washing 0.01mol/L PH7.4 is cleaned 1 time, and 200L/ holes, static 2 ~ 3 minutes of room temperature gets rid of washing lotion.
（4）Fixation abandons each hole culture solution of 96 porocyte culture plates in waste liquid cylinder（Containing sodium hydroxide）, liquid in plate is emptied,
It is cleaned 1 time with PBS.Pre- cold antigen fixative 100ul/ holes are added in each hole, plank is placed in -20 DEG C rapidly acts on 30 points
Clock.Discard fixer, natural drying at room temperature.The wherein described antigen fixative is+20% methanol of 80% acetone and 10% formaldehyde.
（5）The same step of washing methods（3）.
（6）The 5F9 monoclonal antibodies of FITC labels are diluted with PBS by working concentration, corresponding cell is added to by 50 μ l/ holes
Hole.This fluorescence antibody is prepared in 5 minutes before use, is avoided strong light direct projection, is preferably carried out in dark environment.Wherein institute
It is 1 to state labelled antibody working concentration:100.
（7）It is incubated 37 DEG C and is protected from light water-bath incubation 45 minutes.
（8）PBS is washed 3 times, and method is the same as step 3.
（9）There is specificity fluorescent in result judgement fluorescence microscopy Microscopic observation, positive hole, and staining cell is clear-cut, endochylema
Coloring, negative control hole should be judged to " positive ", otherwise be judged to without specificity fluorescent, all holes for having no less than 1 fluorecyte
" feminine gender ".
2nd, the assembling of the detection kit based on direct immunofluorescence method
（1）The ratio that addition protective agent is dissolved in 100ml fluorescence antibody semi-finished product in 1g balf serum albumins is uniformly mixed,
Add preservative Proclin300 to final concentration of 0.02%, 0.45 μm of filtering.
（2）Dispense aseptic subpackaged in the brown pipe that sterilizes, 2 ~ 8 DEG C of preservations.
（3）The inspection of finished product
1. character this product is yellow green transparency liquid, odorless, tasteless, deposit-free.
2. steriling test is by existing《Chinese veterinary pharmacopoeia》Annex is tested, asepsis growth.
3. antibody titer detects this product and does 1：100,1：200,1：300,1：400,1：500,1：600...... it is serial dilute
It releases, direct immuno-fluorescent antibody assay is carried out to IBRV infection cells, measures finished product antibody titer.With visible special under fluorescence microscope
Property fluorescence maximum dilution multiple be finished product antibody titer.Finished product antibody titer is 1：2000.
4. sensitivity assays are respectively with 103.0、102.0、101.0, tetra- dilutions of 1TCID50/ml IBRV infection cells
The direct immuno fluorescence test of finished product is carried out, measures the sensibility of fluorescence antibody.All dilutions connect poison cell, and CPE is positive
Hole all detects specificity fluorescent.
5. specific assay is to 1 type of bovine viral diarrhea virus（BVDV1）, 2 type of bovine viral diarrhoea virus（BVDV2）、
Infectious bovine rhinotrachetis virus（IBRV）, bovine parainfluenza type-3 virus（PIV3）It is glimmering that the MDBK cells of infection carry out direct immunization
Phototesting measures the specificity of finished product.Only with IBRV infection cells specific reaction occurs for finished product, with BVDV1, BVDV2, PIV3
Do not react.
The foundation of 5 infectious bovine rhinotrachetis virus indirect immunofluorescene assay method of embodiment
1st, the preparation MDBK cells of cell are disperseed after trypsin digestion with the DMEM culture mediums containing 6% horse serum, and adjustment is thin
Born of the same parents' density is 2.0 × 105A/ml is added in 96 porocyte culture plates, 0.1ml/ holes, puts 37 DEG C, containing 5%CO2It is cultivated in incubator
16h ~ 20h makes it grow up to 70%~90%MDBK cell monolayers.
2nd, virus inoculation uses the DMEM culture mediums containing 3.5% horse serum that BVDV is diluted to 10 NM01 plants3.0TCID50/ ml,
Inoculation has grown up to 96 porocyte culture plates of good MDBK cell monolayers, 0.1ml/ holes, while sets negative cells control.Put 37
DEG C, 48h ~ 72h is cultivated in cell incubator containing 5%CO2.
3rd, liquid is abandoned in washing, uses PBST（PBS containing 0.5 ‰ Tween-20,7.4,0.01 mol/l of pH value）Washing 1 time,
200ml/ holes.
4th, fixed to add in 4% formaldehyde, 0.1ml/ holes fix 10min at 25 ± 1 DEG C, abandon liquid.
5th, washing methods is the same as 3.
6th, it is penetrating that 0.5% TritonX-100 0.1ml are added in per hole, 10min is acted at 25 ± 1 DEG C, abandons liquid.
7th, washing methods is the same as 3.
8th, sample-adding is by above-mentioned 96 porocyte culture plates of sample inoculation, if PBS is compares, 0.1ml/ holes are put in 37 DEG C of wet box
45~60min is incubated, abandons liquid.
9th, washing PBST（PBS containing 0.5 ‰ Tween-20,7.4,0.01 mol/l of pH value）Washing 2 times, 200ml/
Hole.
10th, secondary antibody is added to add in the PBS with 1% BSA per hole（PH value 7.4,0.01mol/l）It is diluted to working concentration
（0.0025mg/ml）Sheep anti mouse fluorescence antibody（Secondary antibody）0.1 ml puts 45~60min of incubation in 37 DEG C of wet box, abandons liquid.
11st, washing methods is the same as 9.
12nd, observation is under fluorescence microscope, and PBS control hole is without specificity fluorescent, IBR Hybridoma Cell Cultures supernatant dye
The visible specificity fluorescent of cytochrome, staining cell are coloured for endochylema, and cell outline is clear and legible.
Claims (8)
1. one plant can the anti-infectious bovine rhinotrachetis virus monoclonal antibody of stably excreting hybridoma cell strain 5F9, in
In China General Microbiological culture presevation administrative center preservation, deposit number was August in 2016 on 31st：CGMCC
No.12700。
2. the preparation method of hybridoma cell strain 5F9 described in claim 1, it is characterized in that, by the ox infectious rhinotracheitis purified
BALB/c mouse is immunized for antigen in scorching virus (IBRV), after cell fusion, with indirect ELISA and cellmediated immunity fluorescence method into
Row screening obtains the cell strain of the energy anti-infectious bovine rhinotrachetis virus monoclonal antibody of stably excreting.
3. the monoclonal antibody that the hybridoma cell strain 5F9 as described in claim 1 is generated.
4. the anti-ox infectiousness nose gas that the monoclonal antibody that the hybridoma cell strain 5F9 as described in claim 1 is generated is prepared
Pipe inflammation viral monoclonal fluorescence antibody.
5. the monoclonal antibody that the hybridoma cell strain 5F9 described in claim 2 is generated is in detection infectious bovine rhinotrachetis disease
Application in poison.
6. application according to claim 5, it is characterized in that, establish indirect immunofluorescence method using the monoclonal antibody.
7. the anti-infectious bovine rhinotrachetis virus monoclonal fluorescence antibody described in claim 4 is in detection ox infectious rhinotracheitis
Application in scorching virus.
8. application according to claim 7, it is characterized in that：The monoclonal fluorescence antibody prepared using the monoclonal antibody is built
Vertical direct immuno-fluorescent antibody assay method.
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| CN110763838A (en) * | 2019-09-23 | 2020-02-07 | 华威特（江苏）生物制药有限公司 | Immunofluorescence reagent for detecting bovine parainfluenza virus type 3 and detection kit thereof |
| CN114878819A (en) * | 2022-07-12 | 2022-08-09 | 北京市农林科学院 | Rapid quantitative detection method for infectious bovine rhinotracheitis virus |
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| CN110763838A (en) * | 2019-09-23 | 2020-02-07 | 华威特（江苏）生物制药有限公司 | Immunofluorescence reagent for detecting bovine parainfluenza virus type 3 and detection kit thereof |
| CN114878819A (en) * | 2022-07-12 | 2022-08-09 | 北京市农林科学院 | Rapid quantitative detection method for infectious bovine rhinotracheitis virus |
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