CN105859880B - Anti-A type foot and mouth disease virus neutralizing monoclonal antibody and its application - Google Patents
Anti-A type foot and mouth disease virus neutralizing monoclonal antibody and its application Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1009—Picornaviridae, e.g. hepatitis A virus
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
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- Biophysics (AREA)
- Immunology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
The invention discloses the preparation of the monoclonal antibody of anti-A type foot and mouth disease virus and applications, belong to the prevention and control field of aftosa.The present invention constructs the hybridoma cell line of the neutralizing monoclonal antibody of secretion anti-A type FMDV, and microbial preservation number is: CGMCC 12049, and the monoclonal antibody of secretion is A type FMDV serotype specificity monoclonal antibody, there is high-intensitive neutralising capacity；With the detection method for the A type FMDV antibody that the monoclonal antibody is established, good competitiveness is presented with the strong positive of A type FMDV, weakly positive and doubtful serum in A type FMDV antibody detection method in monoclonal antibody of the present invention, and with O-shaped and Asia1 type FMDV strong positive serum and FMDV negative serum without competitiveness, be capable of specificity distinguish A type FMDV yin and yang attribute serum, the diagnosis of A type aftosa, prevention or in terms of it is with important application prospects.
Description
Technical field
The present invention relates to the neutralizing monoclonal antibody of monoclonal antibody more particularly to anti-A type foot and mouth disease virus and point
The hybridoma cell line for secreting the neutralizing monoclonal antibody, the invention further relates to the monoclonal antibodies in diagnosis, in advance
Application in anti-or treatment A type aftosa, belongs to the preparation and application field of A type foot and mouth disease virus monoclonal antibody.
Background technique
Foot and mouth disease virus (FootandMouthDiseaseVirus, FMDV) belongs to Picornaviridae, foot and mouth disease virus
Belong to, FMDV geneome RNA overall length about 8.5kb.Aftosa (Foot and mouth disease, FMD) is by FMD virus
(FMDV) caused by, domestic animals and a variety of wild highly contagious diseases artiodactylous such as ox, pig, sheep are mainly encroached on.The disease
Outburst and it is popular usually bring huge loss to Animal husbandry production and economic development, seriously affected economic trade and herding
The development of industry, by the concern of countries in the world.World Organization for Animal Health (OIE) and FAO (Food and Agriculture Organization of the United Nation) (FAO) are arranged
For A class zoonosis.
FMDV has 7 serotypes, respectively O, A, C, SAT1, SAT2, SAT3 and Asial, without intersection between each serotype
Immunoprotection.The antigenicity of different subtype in same serotype has the difference without degree, the journey of serological cross reaction
Degree is also had nothing in common with each other, so that the diagnosis and control of aftosa have very big difficulty.Monoclonal antibody, which has, identifies single antigen
The ability in site, strong with antigen-binding specificity, homogenieity is high, and bioactivity is single, is easy to standardize, the spy that can be mass
Point, is widely used in field of biomedicine.Currently, Chinese Major Epidemic is O-shaped, Asial type and A type FMD.In recent years, Chinese
Some areas have occurred A type FMD in succession, therefore the preparation of A type foot and mouth disease virus monoclonal antibody is for the diagnosis of the disease and pre-
It is anti-to be of great significance.
Summary of the invention
An object of the present invention is to provide a Zhu Neng stably excreting senior middle school and active anti-A type foot and mouth disease virus monoclonal
The hybridoma cell line of antibody；
The second object of the present invention is to provide a kind of to have senior middle school and active as secreted by above-mentioned hybridoma cell line
Anti-A type foot and mouth disease virus monoclonal antibody；
The third object of the present invention is that above-mentioned anti-A type foot and mouth disease virus monoclonal antibody is applied to diagnosis A type aftosa；
The fourth object of the present invention is to be applied to above-mentioned anti-A type foot and mouth disease virus monoclonal antibody to prevent or treat A type
Aftosa.
The present invention reaches above-mentioned purpose and is achieved through the following technical solutions:
The present invention discloses a Zhu Neng stably excreting senior middle school and active anti-A type foot and mouth disease virus neutrality monoclonal first
The hybridoma cell line of antibody and the monoclonal antibody as secreted by the hybridoma cell line.
BALB/c mouse is immunized using the A type foot and mouth disease virus for inactivating and purifying in the present invention, prepares anti-A type FMDV A/JLYS/
CHA/2014 plants of monoclonal antibody, antibody positive hybridoma are subcloned through 3 limiting dilution assays, and obtaining three plants can stablize
The hybridoma of secretory antibody, is respectively designated as 9A9,8G9 and 9H4: through IFA specificity identification, monoclonal antibody 9A9 heavy chain type is
IgG2a, light chain are κ type；Monoclonal antibody 8G9 heavy chain type is IgG2a, and light chain is κ type；Monoclonal antibody 9H4 heavy chain type is IgG2b,
Light chain is κ type.
Few cells neutralization test shows that monoclonal antibody secreted by hybridoma 9A9 has very high neutralization activity, abdomen
Water neutralization titer is up to 1:4096；The neutralization titer of monoclonal antibody secreted by hybridoma 8G9 is only 1:89, hybridoma
Monoclonal antibody secreted by 9H4 is then without neutralization activity.Neutralization test the result shows that, the neutralization of monoclonal antibody secreted by hybridoma 9A9
Activity is significantly higher than the neutralization activity of monoclonal antibody secreted by hybridoma cell line 8G9.
The present invention further carries out Western blot analysis to totivirus with monoclonal antibody 9A9, shows that apparent specificity is anti-
Band is answered, it is thus regarded that a linear epitope of monoclonal antibody 9A9 identification A type FMDV.By monoclonal antibody 9A9 culture supernatant respectively with infection A,
The BHK-21 cell of O and Asia1 type FMDV carries out indirect immunofluorescene assay, the results show that 9A9 is only positive with A type strain
Reaction, and with the multiple O-shaped and equal no cross reaction of Asia1 type FMDV separation strains, can assert 9A9 for A type FMDV serotype it is special
Property monoclonal antibody.
It is A type FMDV serotype specificity list in view of anti-A type foot and mouth disease virus monoclonal antibody secreted by hybridoma cell line 9A9
It is anti-, and neutralization activity with higher, the mechanism that hybridoma cell line 9A9 is submitted patent to approve by the present invention carry out preservation,
Its microbial preservation number are as follows: CGMCC 12049；It is recommended that classification naming are as follows: the neutrality monoclonal of secretion anti-A type FMDV is anti-
Body hybridoma cell strain；Depositary institution: China Microbiological preservation administration committee common micro-organisms center；The preservation time is:
On March 21st, 2016；Preservation address: the institute 3 of BeiJing, China, North Star West Road, Chaoyang District 1, Institute of Microorganism, Academia Sinica.
It is that A type FMDV serotype is special in view of anti-A type foot and mouth disease virus monoclonal antibody secreted by hybridoma cell line 9A9
The monoclonal antibody can be applied to diagnosis, prevention or treatment A type mouth hoof by anisotropic monoclonal antibody, and neutralization activity with higher
Reagent or drug of epidemic disease etc.；It for example, can be with being prepared into based on anti-A type foot and mouth disease virus monoclonal antibody of the invention
To the ELISA kit of diagnosis A type foot and mouth disease virus
The present invention further discloses a kind of as a result, for diagnosing the ELISA detection kit of A type foot and mouth disease virus, wraps
It includes: A type foot-and-mouth disease virus antigen, sheep anti-mouse igg, the sun that microwell plate, dilution, cleaning solution, developing solution, terminate liquid, enzyme mark
Property control serum, negative control sera and enzyme label monoclonal antibody；Wherein, the monoclonal antibody is offer of the present invention
The high neutrality of anti-A type foot and mouth disease virus monoclonal antibody.
Wherein, the microwell plate is preferably 96 hole microwell plates；The enzyme is horseradish peroxidase；The dilution
Liquid is the Na of pH9.62CO3/NaHCO3Buffer；The cleaning solution is the PBST of pH7.4；The developing solution is tmb substrate
Solution；The terminate liquid is 2M H2SO4；The positive control serum is A type foot and mouth disease virus positive serum；The yin
Property control serum be A type foot and mouth disease virus negative serum.
The present invention purifies the monoclonal antibody 9A9 ascites of preparation through octanoic acid-saturated ammonium sulfate method, and ultraviolet specrophotometer is surveyed
Determining 9A9 concentration is 7.64mg/ml.Antibody 9A9 is further purified through DEAE-sephadex a-50 ion-exchange chromatography again, with changing
Good Over-voltage protection marks HRP.A type aftosa in A type antibodies against foot-and-mouth disease virus detection system is primarily determined with square matrix titration
The peridium concentration of viral purification antigen is 6.4 μ g/ml, extension rate 1:1000；The most suitable extension rate of HRP-9A9 is 1:
1500。
In order to verify diagnostic value of the monoclonal antibody 9A9 in the detection of A type antibodies against foot-and-mouth disease virus, with the A type mouth tentatively established
Aphtovirus antibody detection method detect respectively the A type FMDV strong positives of different extension rates, weakly positive, doubtful (critical value) and
Negative serum, while O-shaped and Asia1 type FMDV strong positive serum is set up as control, to verify monoclonal antibody 9A9 in A type aftosa
The energy of A type foot and mouth disease virus yin and yang attribute serum is distinguished in antiviral antibody detection method with the binding ability of antigen and monoclonal antibody 9A9
Power.The antibody titer curve of strong positive, weakly positive, doubtful (critical value), negative serum is drawn with above-mentioned testing result；Detection knot
Fruit shows: monoclonal antibody 9A9 presents in A type FMDV antibody detection method with the strong positive of A type FMDV, weakly positive and doubtful serum good
Good competitiveness, and with O-shaped and Asia1 type FMDV strong positive serum and FMDV negative serum without competitiveness, Neng Goute
The opposite sex distinguishes the yin and yang attribute serum of A type FMDV, it is determined that monoclonal antibody 9A9 is in establishing A type antibodies against foot-and-mouth disease virus detection method
Value and application potential.
Technical solution of the present invention has the advantages that compared with prior art
Anti-A type foot and mouth disease virus monoclonal antibody secreted by hybridoma cell line 9A9 of the present invention is A type FMDV serotype
Specific monoclonal antibody has high-intensitive neutralising capacity, can apply to the diagnosis, prevention or treatment of A type aftosa, for A type mouth hoof
The diagnosis or prevention of epidemic disease all have important meaning.
Detailed description of the invention
Fig. 1 IFA detects monoclonal antibody 9A9 and A type, O-shaped, Asia1 type FMDV reacts；
The monoclonal antibody 9A9 that Fig. 2 is purified through octanoic acid-saturated ammonium sulfate method and ion exchange chromatography；
Fig. 3 with A type antibodies against foot-and-mouth disease virus detection method detect A type FMDV strong positive, weakly positive, doubtful, FMDV is negative,
The titre change curve of O-shaped FMDV strong positive and Asia1 type FMDV strong positive serum.
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.It should be understood that described, examples are merely exemplary, does not constitute 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 details to technical solution of the present invention and
Form is modified or is replaced, but these modifications or substitutions each fall within protection scope of the present invention.
1. material
1.1 viruses, cell, bacterial strain and experimental animal
The strain that the analysis of monoclonal antibody specificity is carried out for immunofluorescence includes: A FMDV A/KT/58 (GenBank
accession number:AJ131665),Asia1FMDV Asia1/YS/CHA/05(GU931682),O/YS/CHA/05
(HM008917),O FMDV O/Tibet/CHA/99(AJ539138),O/GD/86(AJ131468),O/Akesu/58
(AF511039)(Wang H,Zhao L,Li W,Zhou G,Yu L(2011)Identification of a
conformational epitope on the VP1G-H Loop of type Asia1foot-and-mouth disease
virus defined by a protective monoclonal antibody.Veterinary microbiology
148:189-199.) in addition, hoof-and-mouth disease strain A/JLYS/CHA/2014 belongs to 97 pedigree (Sea-97) of the Southeast Asia A type FMDV,
The homology of VP1 sequence and A/GDMM/CHA/2013 (GenBank accession number:KF450794) (Zheng H,
Lian K,Yang F,Jin Y,Zhu Z,Guo J,Cao W,Liu H,He J,Zhang K,Li D,Liu X(2015)
Cross-protective efficacy of engineering serotype A foot-and-mouth disease
virus vaccine against the two pandemic strains in swine.Vaccine 33:5772-5778)
Up to 99%, belong on source with primary popular strain.Above-mentioned strain is saved by this laboratory.
Newborn hamster kidney cell BHK-21, SP2/0 myeloma cell is the preservation of this laboratory；
Plasmid pGEX-6p-1 and recipient bacterium BL21 are saved by this laboratory；
Cleaning grade female BAl BIc/c mouse is purchased from Harbin Veterinary Medicine Inst., China Academy of Agriculture's Experimental Animal Center.
1.2 main agents
The sheep anti mouse that fusion agent PEG/DMSO (Mw, 1450), HAT salt (50x), HT salt (50x), HRP or FITC are marked
IgG, Freund's complete adjuvant and incomplete Freund's adjuvant are purchased from Sigma company；Monoclonal antibody subgroup identification kit is purchased from
Southern Biotech company；
L-Glutamine (glutamine), glycine are purchased from Amresco company；
Dimethyl sulfoxide (DMSO), o-phenylenediamine (OPD), PEG6000 are purchased from Solarbio company；
Restriction enzyme EcoRI, XhoI are purchased from TaKaRa company；
T4DNA ligase is purchased from New England Biolabs company；
DMEM in high glucose dry powder is purchased from GIBCO company；
Import top grade fetal calf serum (PAA) is purchased from Spain Nalgene company；
96 porocyte culture plates are purchased from JET Biochemical company, Canada.
The preparation of experimental example 1A type foot and mouth disease virus neutralizing monoclonal antibody
1, test method
The immune preparation with monoclonal antibody of 1.1 mouse
(1) A type FMDV is inoculated in the BHK-21 cell monolayer covered with, poison is received after lesion occurs in 75% or more cell.
Three times by the cell culture fluid multigelation of harvest, preliminary lytic cell, releasing virus.Virus liquid after freeze thawing is to be added 1:
1000TritonX-100 and the cracking of 4: 10000 formaldehyde and inactivation 48h or more, take BHK-21 cell on the virus liquid 1mL inactivated
Whether blind passage three generations, detection inactivation are thorough.By after freezing-thawing and cracking culture solution 9000rpm (Beckman supercentrifuge,
JA-10 rotor), 4 DEG C of centrifugation 90min, recycle virus liquid supernatant, discard cell precipitation.It is added by the volume of virus liquid supernatant
The NaCl of the PEG6000 and 40g/L of 80g/L, are stirred at room temperature 2h to being completely dissolved, 4 DEG C of overnight precipitations.Next day is by supernatant
9000rpm, 4 DEG C of centrifugation 90min abandon supernatant, recycling precipitating.Precipitating is gently resuspended under low temperature with NET buffer,
29000rpm, 4 DEG C of centrifugation 2h, are gently resuspended precipitating with suitable NET buffer in ice chest.Prepare 15%~45% uniform line
Property saccharose gradient, sucrose density gradient ultracentrifugation is carried out to viral concentration liquid, using with UV spectrophotometer measuring OD259
Value calculates the antigenic content of 146S according to formula.
(2) with the A type FMDV antigen of purifying, 100 μ g/200uL add isometric Freund's complete adjuvant, it is fully emulsified after through carrying on the back
6 week old female BAl BIcs/c mouse is subcutaneously injected in portion；It carries out the 2nd time after 2 weeks to be immunized, adjuvant is incomplete Freund's adjuvant；After 2 weeks,
Be not added the equivalent amount of antigen of adjuvant carry out the 3rd time it is immune.Strong immunization reaction is quickly generated for stimulation mouse, 3d is adopted before merging
Booster immunization is carried out with the immunization ways that tail vein and spleen injection combine, injects the antigen of doubling dose.
(3) cell fusion
A) preparation of feeder cells, eye scissors, ophthalmic tweezers the preparation of feeder cells: are carried out on the day before cell fusion
The test toolss such as son, plate do steriling test with preceding high temperature hot air sterilization, HAT complete culture solution.2 BALB/c mouses are taken to extract
Eyeball bloodletting, separates negative serum according to a conventional method.It draws neck dislocation to put to death mouse, is soaked in 75% alcohol, after 10min
Move into superclean bench.Mouse web portion is fixed on upwards on mouse frame, lifts the positive middle part skin of abdomen with tweezers, eye scissors are lateral
An osculum is cut, is sure not to break peritonaeum, skin is torn up and down with scissors and tweezers, sufficiently exposes peritonaeum.Gently lift abdomen with tweezers
The 8-10mL HAT complete culture solution drawn in 10mL syringe is injected abdominal cavity, is sure not to puncture internal organs and enteron aisle, syringe by film
It not extract, aspirate 5-10 times, then massaged two sides abdomen 30 seconds or so with tweezers, then aspirated, weight back and forth intraperitoneal
Multiple the above operation 2-3 times.With the intraperitoneal liquid of syringe resorption.Pay attention to avoiding mesenterium and adipose tissue, in order to avoid blocking syringe needle.
It will be added in 55mL HAT culture solution from the abdominal cavity cell taken out in 2 mouse, dispel mixing cell, be sub-packed in 6 piece of 96 hole culture
Plate, 100 holes μ L/.It is placed in 37 DEG C, 5%CO2It is cultivated in incubator.
B) preparation of myeloma cell: myeloma cell is expanded and is cultivated by 36-48h before merging, and so that cell is in logarithm raw
For a long time.On the fusion same day, cell is blown down from bottle wall with 15mL DMEM basic culture solution, is collected in 50mL centrifuge tube.
1000rpm is centrifuged 10min.Cell precipitation resuspension is set in 20mL DMEM basic culture solution, is mixed.Take a small amount of myeloma cell
Suspension, platform expects blue dyeing counting, spare.
C) preparation of immune spleen cell: eyeball of mouse blood sampling is extractd before fusion, prepares positive serum.Mouse is drawn into neck dislocation
It is lethal, it is soaked in 75% alcohol, super-clean bench is put in after 10min.Sterile opening abdominal cavity separates connective tissue and takes out spleen,
Spleen is put into equipped with sterilizing nylon wire and is filled in the plate of 15mLDMEM basic culture solution, with sterilizing glass syringe inner core
Spleen is ground, so that splenocyte is all passed through mesh and enters in plate.Spleen cell solutions are transferred in 50mL centrifuge tube, DMEM base is added
Plinth culture solution about to 30mL, mixes.1000rpm is centrifuged 8min, abandons supernatant.Cell precipitation is suspended from the training of the basis 10mL DMEM
In nutrient solution, mix.Cell suspension is taken, platform expects blue dyeing counting, spare.
D) splenocyte is merged with myeloma cell: taking 65mLHAT culture solution, the basis 15mL DMEM culture solution and 1mL50%
PEG is preheated in 37 DEG C of water-baths, the another standby 200mL beaker for filling 37 DEG C of water.Add 1 part of myeloma cell's number by 5 parts of spleens cell numbers
Corresponding cell suspension amount is taken, is added in 50mL glass centrifuge tube, DMEM basic culture solution is added to 30mL, mixes.1 000rpm
It is centrifuged 10min, abandons supernatant, is fallen as far as possible dry.Centrifugation bottom of the tube is tapped with palm, keeps sedimentation cell loose uniformly in the pasty state.It will
Centrifuge tube is put into the 200mL beaker for filling 37 DEG C of water, proficiency uniform rotation centrifuge tube, and the other hand is drawn with 1mL pasteur pipet
It is added in 37 DEG C of 50%PEG solution 1mL, 1min, stands 2min.Be added fastly after then first slow DMEM basic culture solution terminate it is anti-
It answers, 37 DEG C of water-baths stand 10min.1000rpm is centrifuged 10min, abandons supernatant, and 65mLHAT culture medium is added, and lightly dispels thin
Born of the same parents, every hole 0.1mL, which is inoculated in, has cultivated have feeder cells 6 piece of 96 well culture plate, sets 37 DEG C, trains in 5% CO2 incubator
It supports.The later half amount of 5d changes liquid, changes liquid entirely after 8d, wait clone it is long to hole floor space 1/4-1/3 when take supernatant to be detected and change HT training
Nutrient solution.
1.2 antibody test ELISA
It is coated in 96 orifice plates with the viral antigen of purification, 100uL Hybridoma Cell Culture supernatant, 37 DEG C of incubation 1h is added
After wash, the sheep anti mouse secondary antibody of 1:5000 diluted HRP label is added, substrate OPD is added after washing and is protected from light colour developing 10min, in
Wavelength 492nm measures light absorption value.
1.3 indirect immunofluorescences (IFA)
Microwell plate culture BHK-21 cell is inoculated with foot and mouth disease virus 4 × 10 to single layer3TCID50/ well, before there is CPE
It is fixed with cold dehydrated alcohol, 50uL Hybridoma Cell Culture supernatant is added, PBS washing three times, adds after 37 DEG C of incubation 40min
Enter the diluted FITC label sheep anti-mouse igg antibody of 1:200,37 DEG C of incubation 40min are observed under inverted fluorescence microscope after washing
Fluorescence intensity.
1.4 few cells neutralization tests
(1) measurement of viral titer: maintaining liquid is added after cell monolayer, 37 DEG C of absorption 1h in virus inoculation, sets incubator
Culture；Day by day it observes, to cytopathy (CPE) up to 75% or more, harvests viral suspension freeze thawing 3 times, with 3 000r/min centrifugation
10min takes supernatant, and quantitative separating is set -70 DEG C at 1ml bottle and saved backup, and the virus of selection must be had to cell relatively surely
Fixed pathogenicity.One bottle of virus for setting the preservation of -70 DEG C of refrigerators is taken, virus is made to 10 times of progressive dilutions on 96 well culture plates is
10-1, 10-2, 10-11..., every hole viral suspension amount is 50 μ l, and each dilution makees 8 holes, and 100 cell suspensions are added in every hole, often
The last line of block plate sets 8 hole cell controls, prepares the concentration of cell suspension so that cell covers with single layer in for 24 hours as degree.?
Culture plate sets 5%CO237 DEG C of incubator culture, from 48-72h day by day from cytopathy, record result.By Reed and Muench two
Family name's method calculates TCID50。
(2) neutralization test: taking odd contradictive hydroperitoneum on 96 hole Microtitration plates, is made with the DMEM without serum a series of
Doubling dilution, making its dilution is respectively 1:4,1:8,1:16,1:32,1:64 etc. of former ascites, and every hole content is 50 μ l, often
A dilution makees 4 holes.The virus liquid for taking -70 DEG C of refrigerators to save, makees 200TCID by malicious valence after measured50Dilution is (with equivalent abdomen
Water mixing, malicious valence are 100TCID50).50 μ l virus liquids are added in every hole, seal lid, are placed in 37 DEG C of incubators and 1h.It is preparing
When cell suspension, concentration is to be degree in the interior single layer that covers with for 24 hours: taking out in serum-virus and after 1h, 100 μ l cells are added in every hole
Suspension.Set 5%CO237 DEG C of incubator cultures, since culture 48h observe and record day by day, 120h sentences eventually.Fixed virus dilutes abdomen
The result of water neutralization test calculates, and is to calculate the ascites dilution that 50% cell hole can be protected not generate cytopathy, this is dilute
Degree of releasing is the neutralize antibody titers of this part of serum.With Reed and Muench Liang Shi method (or Karber method) calculated result.
2, test result
2.1 are directed to the screening and identification of the linear neutralizing epitope monoclonal antibody of A type FMDV
The hybridoma of immune mouse spleen cell and SP2/0 cell fusion, culture supernatant FMDV specific antibody are used
Indirect ELISA detection and IFA verifying, Positive judgement standards are as follows: with Hybridoma Cell Culture supernatant to the OD of FMDV antigen492Light
Absorption value and normal BALB/c mouse serum, SP2/0 cells and supernatant are to FMDV antigen OD492The ratio between value is all larger than 2.1, and
Hybridoma supernatant and normal BHK-21 cell do not generate fluorescence reaction, are judged to the positive.
Antibody positive hybridoma is subcloned through 3 limiting dilution assays, obtains the hybridization of three plants of energy stably excreting antibody
Oncocyte is respectively designated as 9A9,8G9 and 9H4.Immunoglobulin subclass identification display, monoclonal antibody 9A9 heavy chain type are IgG2a,
Light chain is κ type.Monoclonal antibody 8G9 heavy chain type is IgG2a, light chain is κ type.Monoclonal antibody 9H4 heavy chain type is IgG2b, light chain κ
Type.Few cells neutralization test shows that 9A9 has very high neutralization activity, and ascites neutralization titer is up to 1:4096.8G9 abdomen
Water neutralization titer is only 1:89, and 9H4 is then without neutralization activity.
It is A type FMDV senior middle school and active monoclonal antibody in view of above-mentioned 9A9, chooses the research that monoclonal antibody 9A9 carries out next step.
Monoclonal antibody 9A9 culture supernatant is immunized with the BHK-21 cell of infection A, O and Asia1 type FMDV indirectly respectively glimmering
Light detection (Fig. 1), the results show that 9A9 only be positive with A type strain, and with multiple O-shaped and Asia1 type FMDV separation strains
Equal no cross reaction can assert that 9A9 is A type FMDV serotype specificity monoclonal antibody.
The above results show monoclonal antibody secreted by hybridoma cell line 9A9 be A type foot and mouth disease virus specificity, senior middle school and
The monoclonal antibody of property.
The mechanism that hybridoma cell line 9A9 is submitted patent to approve by the present invention carries out preservation, microbial preservation number
Are as follows: CGMCC 12049；Depositary institution: China Microbiological preservation administration committee common micro-organisms center；The preservation time is:
On March 21st, 2016；Preservation address: the institute 3 of BeiJing, China, North Star West Road, Chaoyang District 1, Institute of Microorganism, Academia Sinica.
The foundation of experimental example 2A type antibodies against foot-and-mouth disease virus detection method
1, test method
The purifying of 1.1 monoclonal antibodies
The ascites of preparation is purified using octanoic acid-saturated ammonium sulfate method, operating procedure is summarized as follows:
(1) ascites for taking 3ml pretreated adds 6ml 0.06mol/L pH4.8 acetate buffer solution；In ascites plus 99ul is pungent
Acid is stirred at room temperature 30 minutes, and 4 DEG C stand 2 hours or more；It takes out 11000rpm to be centrifuged 30 minutes, abandons precipitating；Supernatant 2mol/L
NaOH tune pH to 7.4.
(2) saturated ammonium sulfate is added under the conditions of 4 DEG C to 50% saturation degree, ice bath stirring acts on 30 minutes, and 4 DEG C stand 2
Hour or more；11000 turns are centrifuged 30 minutes, abandon supernatant；In the PBS for precipitating the pH7.4 for being dissolved in 5ml0.1M；
(3) add appropriate saturated ammonium sulfate solution while stirring into precipitating suspended matter, make concentration in ammonium sulfate
30%--40%, ice bath stirring act on 30 minutes, and 4 DEG C 2 hours or more static；11000 turns are centrifuged 30 minutes, take precipitating, will sink
It forms sediment and incorporates in the PBS of the pH7.4 of 2ml 0.1M；
(4) precipitation suspension is fitted into dialysis band, dialyses in the PBS of 4 DEG C of pH7.4 with 0.1M, changes liquid one within 2 hours
It is secondary, it dialyses 2 days.After the completion of dialysis, the purifying ascites in bag filter is recycled, ultraviolet specrophotometer measures protein concentration, uses
SDS-PAGE carries out purity detecting.
It is chromatographed again with DEAE-Sephadex A-50 (GE company) column and monoclonal antibody is further purified, after recovery purifying
Monoclonal antibody, ultraviolet specrophotometer measure protein concentration, carry out purity detecting with SDS-PAGE.
1.2 horseradish peroxidase-labeled monoclonal antibodies
Monoclonal antibody after purified qualification is marked with Over-voltage protection.Operating procedure is as follows:
1. take 5mg HRP to be dissolved in the 0.06M HAc-NaAc buffer of 0.5ml pH5.6, what addition was newly prepared
0.06Mol/L NaIO4Solution 0.5ml mixes, sets 4 DEG C of 20-30min；
2. 0.16Mol/L glycol water (10mlH is added after taking out2O+0.09ml ethylene glycol) 0.5ml, it is placed at room temperature for
30min；
3. the solution 1ml of the antibody purification containing 10mg is added, mix, adjusts pH value 9 or so, and be packed into bag filter, use 0.05M/L
The carbonate buffer solution of pH9.5 slowly stirs dialysis 16-24h, is allowed to combine；
4. NaBH is added4Solution (5mg/ml) 0.2ml mixes, sets 4 DEG C of 2h；
5. being slowly added to isometric saturated ammonium sulfate solution in the above solution, mix, 4 DEG C of effect 30min,
3000rpm is centrifuged 30 minutes, removes supernatant, and precipitating is packed into bag filter with a little 0.02Mol/L pH7.4PBS dissolution, with
0.02Mol/L pH7.4PBS is stayed overnight in 4 DEG C of dialysis desalinations；
6. next day takes out centrifugation, 10000rpm is centrifuged 30 minutes to remove insoluble matter to get enzyme-antibody (HRP-IgG) knot
Object is closed, it is molten to 1-2ml with 0.02Mol/L pH 7.4PBS；
7. measuring OD respectively with ultraviolet specrophotometer403And OD280, it is qualified after by the IgG- horseradish peroxidating of label
Object enzyme adds equivalent glycerol, -20 DEG C of preservations.
The titration of the optimal use concentration of each reagent in 1.3A type antibodies against foot-and-mouth disease virus detection method
Determine the use concentration of the peridium concentration of A type foot and mouth disease virus purifying antigen and the detection monoclonal antibody of HRP label.Test
ELISA Plate used is 96 hole flat bottom microtiter plates (Costar ELISA Plate, high-affinity), and test uses 50ul reaction system.Respectively
With the Na of pH9.62CO3/NaHCO3The A type FMDV antigen coat elisa plate of buffer dilution purifying, 4 DEG C overnight, with pH7.4's
PBST board-washing 5 times, drying.Then with 2 times of PBST HRP being serially diluted label detection monoclonal antibodies (1:100,1:150~1:
12800,1:19200) be added elisa plate hole, 37 DEG C incubate 1 hour, after board-washing be added tmb substrate solution be protected from light colour developing 15~
After 20min, with 2M H2SO4Reaction is terminated, 450nm reads OD value (OD value), reach 1.6 with the OD value of enzyme conjugates~
The highest extension rate of reagent uses concentration for it when 2.0.
The use concentration of each reagent is primarily determined by square matrix titration, is detected for subsequent A type antibodies against foot-and-mouth disease virus
Method.
1.4A type antibodies against foot-and-mouth disease virus detection method test procedure
A type foot and mouth disease virus purifying antigen pH 9.6Na2CO3Buffer, which is diluted to, is coated with elisa plate, 50 μ using concentration
The hole l/, sealing plate or sets in wet box, and 4 DEG C overnight.Elisa plate is taken out, wherein liquid is discarded, with pH 7.4PBST board-washing 5 times, gets rid of
It is dry.50 holes μ l/ are added in elisa plate after being initiated with the multiple proportions serial dilution of 1:2 to serum to be checked with PBST.In elisa plate
In be added use the diluted HRP-9A9 of PBST immediately, 50 holes μ l/, sealing plate, abundant shake mixes, 37 DEG C of incubation 1h.Ibid board-washing 5
It is secondary, substrate solution is added, 50 holes μ l/, sealing plate, room temperature, which is protected from light, is incubated for 15min.Terminate liquid is added, 50 holes μ l/ terminate reaction,
450nmOD value is read in microplate reader.
2, test result
The purifying of 2.1 monoclonal antibody ascites and label
It by monoclonal antibody 9A9 ascites prepared by the present invention, is purified through octanoic acid-saturated ammonium sulfate method, ultraviolet specrophotometer is surveyed
Determining 9A9 concentration is 7.64mg/ml.(figure is further purified through DEAE-sephadex a-50 ion-exchange chromatography again in antibody 9A9
2), HRP is marked with improvement Over-voltage protection.
The preliminary foundation of 2.2A type antibodies against foot-and-mouth disease virus detection method
Primarily determine that A type foot and mouth disease virus is purified in A type antibodies against foot-and-mouth disease virus detection system with square matrix titration first
The use concentration of peridium concentration and HRP-9A9 the detection monoclonal antibody of antigen.The result shows that: the packet of A type foot and mouth disease virus purifying antigen
It is 6.4 μ g/ml, extension rate 1:1000 by concentration；The most suitable extension rate of HRP-9A9 is 1:1500.
In order to verify diagnostic value of the monoclonal antibody 9A9 in the detection of A type antibodies against foot-and-mouth disease virus, with the A type mouth tentatively established
Aphtovirus antibody detection method detect respectively the A type FMDV strong positives of different extension rates, weakly positive, doubtful (critical value) and
Negative serum, while O-shaped and Asia1 type FMDV strong positive serum is set up as control, to verify monoclonal antibody 9A9 in A type aftosa
A type foot and mouth disease virus yin and yang attribute serum is distinguished with the binding ability of antigen and monoclonal antibody 9A9 in antiviral antibody detection method
Ability.
The antibody titer curve of strong positive, weakly positive, doubtful (critical value), negative serum is drawn with above-mentioned testing result
(Fig. 3), testing result shows: monoclonal antibody 9A9 in A type FMDV antibody detection method with the strong positive of A type FMDV, weakly positive and doubt
Good competitiveness is presented like serum, and it is not competing with O-shaped and Asia1 type FMDV strong positive serum and FMDV negative serum
Striving property is capable of the yin and yang attribute serum of specificity differentiation A type FMDV, it is determined that monoclonal antibody 9A9 is establishing the inspection of A type antibodies against foot-and-mouth disease virus
Value and application potential in survey method.
Claims (8)
1. the hybridization of one plant of secretion anti-A type foot and mouth disease virus (FootandMouthDiseaseVirus) neutralizing monoclonal antibody
Oncocyte system, microbial preservation number are: CGMCC 12049.
2. the monoclonal antibody of the secretion of the hybridoma cell line as described in claim 1.
3. purposes of the monoclonal antibody as claimed in claim 2 in the reagent or drug of preparation diagnosis aftosa.
4. purposes of the monoclonal antibody as claimed in claim 2 in the reagent or drug of preparation prevention or treatment aftosa.
5. diagnosing the ELISA detection kit of A type foot and mouth disease virus, comprising: A type foot-and-mouth disease virus antigen, microwell plate, dilution
Sheep anti-mouse igg, positive control serum and the yin that liquid, cleaning solution, developing solution, terminate liquid, the monoclonal antibody of enzyme label, enzyme mark
Property control serum, it is characterised in that: the monoclonal antibody be monoclonal antibody as claimed in claim 2.
6. kit according to claim 5, it is characterised in that: the enzyme is horseradish peroxidase.
7. according to kit described in claim 5 or 6, it is characterised in that: the microwell plate is 96 hole microwell plates.
8. kit according to claim 7, it is characterised in that: the dilution is the Na of pH9.62CO3/NaHCO3
Buffer；The cleaning solution is the PBST of pH7.4；The developing solution is tmb substrate solution；The terminate liquid is 2M
H2SO4；The positive control serum is A type foot and mouth disease virus positive serum；The negative control sera is A type aftosa
Viral negative serum.
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| CN107541500A (en) * | 2017-07-27 | 2018-01-05 | 武汉科前生物股份有限公司 | A kind of A types foot and mouth disease virus monoclonal antibody and application |
| CN109295005B (en) * | 2018-09-26 | 2022-03-04 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Hybridoma cell line for secreting foot-and-mouth disease virus non-structural protein monoclonal antibody 2H1 and application thereof |
| CN109799343A (en) * | 2018-12-07 | 2019-05-24 | 中国农业科学院兰州兽医研究所 | A type antibodies against foot-and-mouth disease virus detection kit based on virus-like particle |
| CN109655623A (en) * | 2019-01-31 | 2019-04-19 | 中国农业科学院兰州兽医研究所 | A kind of the visualization quick detection kit and its application of A type antibodies against foot-and-mouth disease virus |
| CN110144329B (en) * | 2019-05-29 | 2020-12-29 | 北京标驰泽惠生物科技有限公司 | Hybridoma cell strain 6B1, monoclonal antibody secreted by same and used for resisting foot-and-mouth disease A type virus and application of monoclonal antibody |
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| Develope Monoclonal Antibody against Foot-and-mouth Disease Virus A Type;Tong Lin等;《VIROLOGICA SINICA》;20110831;第26卷(第4期);273-278 * |
| 抗A 型口蹄疫病毒单克隆抗体的制备及抗原捕获ELISA 检测方法的建立;孙静等;《中国预防兽医学报》;20130228;第35卷(第2期);146-150 * |
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