CN105859880A - Neutralizing monoclonal antibody of A-type foot and mouth disease virus (FMDV) and application of neutralizing monoclonal antibody - Google Patents
Neutralizing monoclonal antibody of A-type foot and mouth disease virus (FMDV) and application of neutralizing monoclonal antibody Download PDFInfo
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- 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
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Abstract
The invention discloses preparation and application of a monoclonal antibody of an A-type foot and mouth disease virus (FMDV) and belongs to the field of prevention and treatment of foot and mouth diseases. According to the preparation and the application, a hybridoma cell line secreting the neutralizing monoclonal antibody of the A-type FMDV is constructed, the microbiological collection number of the hybridoma cell line is CGMCC 12049, and the secreted monoclonal antibody is an A-type FMDV serotype specific monoclonal antibody and has high-strength neutralizing ability; and a detection method for the A-type FMDV antibody is created by using the monoclonal antibody. The monoclonal antibody prepared by the invention shows good competitive ability with strongly-positive, weakly-positive and suspected serum of the A-type FMDV in the detection method for the A-type FMDV antibody and is free of competitiveness with strongly-positive serum of an O-type FMDV, strongly-positive serum of an Asia1-type FMDV and negative serum of an FMDV, so that the monoclonal antibody can be used for specifically discriminating the negative and positive serum of the A-type FMDV and has an important application prospect in aspects such as the diagnosis, prevention and treatment of the A-type foot and mouth diseases.
Description
Technical field
The present invention relates to monoclonal antibody, particularly relate to the neutralizing monoclonal antibody of anti-A type foot and mouth disease virus and divide
Secrete the hybridoma cell line of this neutralizing monoclonal antibody, the invention further relates to described monoclonal antibody and diagnosing, in advance
Prevent or treat the application in A type foot and mouth disease, belonging to preparation and the application of A type foot and mouth disease virus monoclonal antibody.
Background technology
Foot and mouth disease virus (FootandMouthDiseaseVirus, FMDV) belongs to Picornaviridae, foot and mouth disease virus
Belong to, FMDV geneome RNA total length about 8.5kb.Foot and mouth disease (Foot and mouth disease, FMD) is by FMD virus
(FMDV) cause, mainly encroach on domestic animal and the multiple wild high degree in contact sexually transmitted diseases artiodactylous such as cattle, pig, sheep.This disease
Outburst and popular bring huge loss usually to Animal husbandry production and economic development, had a strong impact on economic trade and herding
The development of industry, enjoys the concern of countries in the world.OIE (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 intersecting between each serotype
Immunoprotection.The antigenicity of the different subtype in same serotype all has the difference without degree, the journey of serological cross reaction
Degree is also had nothing in common with each other so that diagnosis and the control of foot and mouth disease exist the biggest difficulty.Monoclonal antibody has the single antigen of identification
The ability in site, strong with antigen-binding specificity, homogeneity is high, and biological activity is single, it is easy to standardization, the spy that can be mass
Point, is widely used in biomedical sector.At present, China's Major Epidemic O type, Asial type and A type FMD.In recent years, China
Some areas there occurs A type FMD in succession, and therefore the preparation of A type foot and mouth disease virus monoclonal antibody is for the diagnosis of this disease and pre-
Anti-significant.
Summary of the invention
An object of the present invention is to provide Zhu Neng stably excreting senior middle school and an anti-A type foot and mouth disease virus monoclonal for activity
The hybridoma cell line of antibody；
The two of the purpose of the present invention be to provide a kind of by secreted by above-mentioned hybridoma cell line have senior middle school and activity
Anti-A type foot and mouth disease virus monoclonal antibody；
The three of the purpose of the present invention are to be applied to above-mentioned anti-A type foot and mouth disease virus monoclonal antibody diagnose A type foot and mouth disease；
The four of the purpose of the present invention are that above-mentioned anti-A type foot and mouth disease virus monoclonal antibody is applied to prevention or treatment A type
Foot and mouth disease.
The present invention reaches above-mentioned purpose and is achieved through the following technical solutions:
First the present invention discloses Zhu Neng stably excreting senior middle school and an anti-A type foot and mouth disease virus neutrality monoclonal for activity
The hybridoma cell line of antibody and by the monoclonal antibody secreted by this hybridoma cell line.
The present invention utilizes the A type foot and mouth disease virus immunity BALB/c Mus of inactivation purification, prepares anti-A type FMDV A/JLYS/
The monoclonal antibody of CHA/2014 strain, antibody positive hybridoma is through 3 limiting dilution assay sub-clones, it is thus achieved that three strains can be stable
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 is 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, the monoclonal antibody secreted by hybridoma 9A9 has the highest neutralization activity, its abdomen
Water is up to 1:4096 with titer；The neutralization titer of the monoclonal antibody secreted by hybridoma 8G9 is only 1:89, hybridoma
Monoclonal antibody secreted by 9H4 is then without neutralizing activity.Neutralization test result shows, the neutralization of the monoclonal antibody secreted by hybridoma 9A9
Activity is significantly higher than the neutralization activity of the monoclonal antibody secreted by hybridoma cell line 8G9.
The present invention carries out Western blot analysis with monoclonal antibody 9A9 to totivirus further, shows that obvious specificity is anti-
Answer band, it is thus regarded that monoclonal antibody 9A9 identifies a linear epitope of A type FMDV.By monoclonal antibody 9A9 culture supernatant respectively with infect A,
The BHK-21 cell of O and Asia1 type FMDV carries out indirect immunofluorescene assay, and result shows, 9A9 is only positive with A type strain
Reaction, and the equal no cross reaction of strain is separated with multiple O types and Asia1 type FMDV, can assert that 9A9 is that A type FMDV serotype is special
Property monoclonal antibody.
In view of the anti-A type foot and mouth disease virus monoclonal antibody secreted by hybridoma cell line 9A9 is A type FMDV serotype specificity list
Anti-, and there is higher neutralization activity, this hybridoma cell line 9A9 is submitted to the mechanism of patent accreditation to carry out preservation by the present invention,
Its microbial preservation is numbered: CGMCC 12049；The Classification And Nomenclature of suggestion is: the neutrality monoclonal anti of secretion anti-A type FMDV
Body hybridoma cell strain；Depositary institution: China Microbiological preservation administration committee's common micro-organisms center；The preservation time is:
On March 21st, 2016；Preservation address: North Star West Road, Chaoyang District, BeiJing, China 1 institute 3, Institute of Microorganism, Academia Sinica.
In view of the anti-A type foot and mouth disease virus monoclonal antibody secreted by hybridoma cell line 9A9 is that A type FMDV serotype is special
Opposite sex monoclonal antibody, and there is higher neutralization activity, this monoclonal antibody can be applied to diagnosis, prevent or treat A type mouth hoof
The aspects such as the reagent of epidemic disease or medicine；Such as, can be prepared into based on the anti-A type foot and mouth disease virus monoclonal antibody of the present invention
ELISA kit to diagnosis A type foot and mouth disease virus
Thus, the present invention further discloses a kind of ELISA detection kit for diagnosing A type foot and mouth disease virus, bag
Include: A type foot-and-mouth disease virus antigen, microwell plate, diluent, cleaning mixture, nitrite ion, stop buffer, the sheep anti-mouse igg of enzyme labelling, sun
Property control serum, negative control sera and the monoclonal antibody of enzyme labelling；Wherein, described monoclonal antibody is that the present invention provides
The monoclonal antibody of anti-A type foot and mouth disease virus high neutrality.
Wherein, described microwell plate is preferably 96 hole microwell plates；Described enzyme is horseradish peroxidase；Described dilution
Liquid is the Na of pH9.62CO3/NaHCO3Buffer；Described cleaning mixture is the PBST of pH7.4；Described nitrite ion is tmb substrate
Solution；Described stop buffer is 2M H2SO4；Described positive control serum is A type foot and mouth disease virus positive serum；Described the moon
Property control serum is A type foot and mouth disease virus negative serum.
The present invention is by the monoclonal antibody 9A9 ascites of preparation, and through octanoic acid-saturated ammonium sulfate method purification, ultraviolet spectrophotometer 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 labelling HRP.A type foot and mouth disease in A type antibodies against foot-and-mouth disease virus detecting system is primarily determined that by square formation titrimetry
The concentration that is coated of viral purification antigen is 6.4 μ g/ml, and extension rate is 1:1000；The suitableeest extension rate of HRP-9A9 is 1:
1500。
In order to verify monoclonal antibody 9A9 diagnostic value in A type antibodies against foot-and-mouth disease virus detects, with the preliminary A type mouth set up
Aphtovirus antibody detection method detect respectively the different A type FMDV strong positive of extension rate, the weak positive, doubtful (marginal value) and
Negative serum, set up simultaneously O type and Asia1 type FMDV strong positive serum as comparison, to verify that monoclonal antibody 9A9 is in A type foot and mouth disease
Antiviral antibody detection method distinguishes the energy of A type foot and mouth disease virus yin and yang attribute serum with the binding ability of antigen and monoclonal antibody 9A9
Power.Strong positive, the weak positive, doubtful (marginal value), the antibody titer curve of negative serum is drawn by above-mentioned testing result；Detection knot
Fruit shows: monoclonal antibody 9A9 presents good with the strong positive of A type FMDV, weak positive and doubtful serum in A type FMDV antibody detection method
Good competitiveness, and there is no competitiveness with O type and Asia1 type FMDV strong positive serum and FMDV negative serum, it is possible to special
The opposite sex distinguishes the yin and yang attribute serum of A type FMDV, it is determined that monoclonal antibody 9A9 is in setting up A type antibodies against foot-and-mouth disease virus detection method
It is worth and application potential.
Technical solution of the present invention compared with prior art has the advantages that
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 the neutralising capacity of high intensity, can apply to the diagnosis of A type foot and mouth disease, prevents or treat, for A type mouth hoof
The diagnosis of epidemic disease or prevention are respectively provided with important meaning.
Accompanying drawing explanation
Fig. 1 IFA detection monoclonal antibody 9A9 and A type, O type, the reaction of Asia1 type FMDV；
Fig. 2 is through octanoic acid-saturated ammonium sulfate method and the monoclonal antibody 9A9 of ion exchange chromatography purification；
Fig. 3 A type antibodies against foot-and-mouth disease virus detection method detection A type FMDV strong positive, the weak positive, doubtful, FMDV is negative,
O type FMDV strong positive and the titre change curve of Asia1 type FMDV strong positive serum.
Detailed description of the invention
Below in conjunction with specific embodiment further describe the present invention, advantages of the present invention and feature will be with describe and
Apparent.It should be understood that described embodiment is only exemplary, the scope of the present invention is not constituted any restriction.This area
Skilled artisans appreciated that, lower without departing from the spirit and scope of the present invention can to the details of technical solution of the present invention and
Form is modified or replaces, but these amendments or replacement each fall within protection scope of the present invention.
1. material
1.1 viruses, cell, bacterial strain and laboratory animal
The strain carrying out monoclonal antibody specificity analyses 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.) another, hoof-and-mouth disease strain A/JLYS/CHA/2014 belongs to A type FMDV Southeast Asia 97 pedigree (Sea-97), its
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%, source belongs to the most popular strain.Above-mentioned strain is preserved by this laboratory.
Breast hamster kidney cell BHK-21, SP2/0 myeloma cell preserves for this laboratory；
Plasmid pGEX-6p-1 and recipient bacterium BL21 is preserved by this laboratory；
Cleaning grade female BAl BIc/c mice is purchased from Harbin Veterinary Medicine Inst., China Academy of Agriculture's Experimental Animal Center.
1.2 main agents
Fusion agent PEG/DMSO (Mw, 1450), HAT salt (50x), HT salt (50x), the sheep anti mouse of HRP or FITC labelling
IgG, Freund's complete adjuvant and incomplete Freund's adjuvant are purchased from Sigma company；Monoclonal antibody subgroup identification test kit is purchased from
Southern Biotech company；
L-glutaminate (glutamine), glycine are purchased from Amresco company；
Dimethyl sulfoxide (DMSO), o-phenylenediamine (OPD), PEG6000 are purchased from Solarbio company；
Restricted 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 hyclone (PAA) is purchased from Nalgene company of Spain；
96 porocyte culture plates are purchased from JET Biochemical company of Canada.
The preparation of experimental example 1A type foot and mouth disease virus neutralizing monoclonal antibody
1, test method
1.1 Mus immunity and the preparations of monoclonal antibody
(1) A type FMDV is inoculated in the BHK-21 cell monolayer covered with, after pathological changes occurs in more than 75% cell, receives poison.
By the cell culture fluid multigelation three times of results, preliminary cell lysis, releasing virus.Virus liquid after freeze thawing is to add 1:
1000TritonX-100 and 4: 10000 formaldehyde crack and inactivation more than 48h, take BHK-21 cell on the virus liquid 1mL inactivated
Blind passage three generations, detection inactivate the most thorough.By after freezing-thawing and cracking culture fluid 9000rpm (Beckman high speed centrifuge,
JA-10 rotor), 4 DEG C of centrifugal 90min, reclaim virus liquid supernatant, discard cell precipitation.Add by the volume of virus liquid supernatant
The NaCl of PEG6000 and 40g/L of 80g/L, is stirred at room temperature 2h to being completely dissolved, 4 DEG C of overnight precipitation.Next day is by supernatant
9000rpm, 4 DEG C of centrifugal 90min, abandon supernatant, reclaims precipitation.To precipitate the most resuspended under low temperature with NET buffer,
29000rpm, 4 DEG C of centrifugal 2h, by the appropriate the most resuspended precipitation of NET buffer in ice chest.Preparation 15%～45% uniform line
Property saccharose gradient, carries out sucrose density gradient ultracentrifugation, uses UV spectrophotometer measuring OD259 viral concentration liquid
Value, calculates the antigenic content of 146S according to formula.
(2) with the A type FMDV antigen of purification, 100 μ g/200uL add equal-volume Freund's complete adjuvant, fully emulsified after through the back of the body
Portion's subcutaneous injection 6 week old female BAl BIc/c mice；Carrying out the 2nd immunity after 2 weeks, adjuvant is incomplete Freund's adjuvant；After 2 weeks,
Carry out the 3rd immunity being not added with the equivalent amount of antigen of adjuvant.For stimulating mice quickly to produce strong immunization reaction, before merging, 3d adopts
Inject, with tail vein and spleen, the immunization ways combined and carry out booster immunization, the antigen of injection doubling dose.
(3) cell merges
A) preparation of feeder layer cells: cell merges and carries out the preparation of feeder layer cells, eye scissors, ophthalmic tweezers the previous day
Test tools front high temperature dry heat sterilization, the HAT complete culture solutions such as son, plate do steriling test.Take 2 BALB/c mouse to extract
Eyeball blood-letting, separates negative serum according to a conventional method.Draw neck dislocation to put to death mice, be soaked in 75% ethanol, after 10min
Move into superclean bench.Upwards being fixed on by mouse web portion on Mus frame, mention abdomen positive middle part skin with tweezers, eye scissors is horizontal
Cut an osculum, be sure not to break peritoneum, tear skin up and down with shears and tweezers, fully expose peritoneum.Abdomen is mentioned gently with tweezers
Film, injects abdominal cavity by the 8-10mL HAT complete culture solution drawn in 10mL syringe, is sure not to puncture internal organs and intestinal, syringe
Do not extract, aspirate 5-10 time back and forth at intraperitoneal, then with tweezers massage both sides abdominal part about 30 seconds, then aspirate, weight
Multiple above operation 2-3 time.With syringe resorption intraperitoneal liquid.Note avoiding mesentery and fatty tissue, in order to avoid blocking syringe needle.
The abdominal cavity cell taken out from 2 Mus is added in 55mL HAT culture fluid, dispels mixing cell, be sub-packed in 6 piece of 96 hole and cultivate
Plate, 100 μ L/ holes.It is placed in 37 DEG C, 5%CO2Incubator is cultivated.
B) preparation of myeloma cell: 36-48h before merging, by myeloma cell's amplification culture, makes cell be in logarithm raw
For a long time.Merge the same day, with 15mL DMEM basic culture solution, cell is blown down from bottle wall, be collected in 50mL centrifuge tube.
1000rpm is centrifuged 10min.Cell is precipitated and resuspended puts in 20mL DMEM basic culture solution, mixing.Take a small amount of myeloma cell
Suspension, platform expects blue dyeing counting, standby.
C) preparation of immune spleen cell: extract eyeball of mouse blood sampling before merging, prepare positive serum.Neck is drawn to dislocate mice
Lethal, it is soaked in 75% ethanol, after 10min, is put in super-clean bench.Aseptic abdominal cavity of opening, separates connective tissue and takes out spleen,
Spleen put into equipped with sterilizing nylon wire and fill the plate of 15mLDMEM basic culture solution, with sterilizing glass syringe inner core
Grind spleen, make splenocyte all be entered in plate by mesh.Proceed to Spleen cell solutions, in 50mL centrifuge tube, add DMEM base
Plinth culture fluid is about to 30mL, mixing.1000rpm is centrifuged 8min, abandons supernatant.Cell precipitation is suspended from the training of 10mL DMEM basis
In nutrient solution, mixing.Obtained cell suspension, platform expects blue dyeing counting, standby.
D) splenocyte and myeloma cell fusion: take 65mLHAT culture fluid, 15mL basis DMEM culture fluid and 1mL50%
PEG preheats in 37 DEG C of water-baths, the another standby 200mL beaker filling 37 DEG C of water.1 part of myeloma cell's number is added by 5 parts of spleens cell numbers
Take corresponding cell suspension amount, add in 50mL glass centrifuge tube, add DMEM basic culture solution to 30mL, mixing.1 000rpm
Centrifugal 10min, abandons supernatant, falls dry as far as possible.Touch bottom centrifuge tube with palm, make sedimentation cell loose the most in the pasty state.Will
Centrifuge tube is put in the 200mL beaker filling 37 DEG C of water, uniform rotation centrifuge tube on the other hand, and another hands 1mL pasteur pipet is drawn
37 DEG C of 50%PEG solution 1mL, add in 1min, stand 2min.Add DMEM basic culture solution after elder generation is slow subsequently soon and terminate anti-
Should, 37 DEG C of water-baths stand 10min.1000rpm is centrifuged 10min, abandons supernatant, adds 65mLHAT culture medium, dispels thin lightly
Born of the same parents, every hole 0.1mL is inoculated in and cultivates 6 piece of 96 well culture plate having feeder cells, puts 37 DEG C, trains in the CO2 incubator of 5%
Support.The later half amount of 5d changes liquid, entirely changes liquid after 8d, takes supernatant and carry out detecting and change HT training when clone's length is to hole floor space 1/4-1/3
Nutrient solution.
1.2 antibody test ELISA
It is coated in 96 orifice plates with the virus antigen purified, adds 100uL Hybridoma Cell Culture supernatant, 37 DEG C of incubation 1h
Rear washing, the sheep anti mouse two of the HRP labelling adding 1:5000 dilution resists, and adds substrate OPD lucifuge colour developing 10min after washing, in
Wavelength 492nm measures light absorption value.
1.3 indirect immunofluorescences (IFA)
Microwell plate cultivates BHK-21 cell to monolayer, inoculation foot and mouth disease virus 4 × 103TCID50, before there is CPE in/well
Being fixed with cold dehydrated alcohol, add 50uL Hybridoma Cell Culture supernatant, after 37 DEG C of incubation 40min, PBS washs three times, adds
Enter the FITC labelling sheep anti-mouse igg antibody of 1:200 dilution, 37 DEG C of incubation 40min, observe under inverted fluorescence microscope after washing
Fluorescence intensity.
1.4 few cells neutralization tests
(1) mensuration of viral titer: by virus inoculation in cell monolayer, adds after 37 DEG C of absorption 1h and maintains liquid, put incubator
Cultivate；Day by day observe, treat that cytopathy (CPE) reaches more than 75%, gather in the crops viral suspension freeze thawing 3 times, be centrifuged with 3 000r/min
10min, takes supernatant, and quantitative separating becomes 1ml bottle to put-70 DEG C to save backup, the virus of selection must be cell is had more steady
Fixed pathogenicity.Take one bottle of the virus putting-70 DEG C of Refrigerator stores, virus is made on 96 well culture plates 10 times and goes forward one by one dilution i.e.
10-1, 10-2, 10-11..., every hole viral suspension amount is 50 μ l, and each dilution factor makees 8 holes, and every hole adds 100 cell suspension, often
Last column of block plate sets 8 porocyte comparisons, prepares the concentration of cell suspension so that cell covers with monolayer for degree in 24h.?
Culture plate puts 5%CO2Incubator 37 DEG C cultivation, from 48-72h observation of cell pathological changes day by day, records result.By Reed and Muench two
Family name's method calculates TCID50。
(2) neutralization test: take odd contradictive hydroperitoneum on 96 hole Microtitration plates, make a series of with the DMEM without serum
Doubling dilution so that it is dilution factor is respectively 1:4,1:8,1:16,1:32,1:64 of former ascites etc., every hole content is 50 μ l, often
Individual dilution factor makees 4 holes.Take the virus liquid of-70 DEG C of Refrigerator stores, make 200TCID by malicious valency after measured50Dilution is (with equivalent abdomen
Water mixes, and its poison valency is 100TCID50).Every hole adds 50 μ l virus liquids, seals lid, is placed in 37 DEG C of incubators and 1h.In preparation
During cell suspension, its concentration is to cover with monolayer for degree in 24h: taking out in serum-virus and after 1h, every hole adds 100 μ l cells
Suspension.Put 5%CO237 DEG C of incubators are cultivated, and start observed and recorded day by day from cultivating 48h, and 120h sentences eventually.Fixed virus dilution abdomen
The result of water neutralization test calculates, and is to calculate to protect 50% cell hole not produce cytopathic ascites dilution factor, and this is dilute
Degree of releasing is the NAT of this part of serum.By Reed and Muench Liang Shi method (or Karber method) result of calculation.
2, result of the test
2.1 for the screening of A type FMDV linear neutralizing epitope monoclonal antibody and qualification
The hybridoma that immune mouse spleen cell merges with SP2/0 cell, its culture supernatant FMDV specific antibody is used
Indirect ELISA detection and IFA verify, Positive judgement standards is: with the Hybridoma Cell Culture supernatant OD to FMDV antigen492Light
Absorption value and normal BALB/c mouse serum, SP2/0 cells and supernatant are to FMDV antigen OD492The ratio of value is all higher than 2.1, and
Hybridoma supernatant and normal BHK-21 cell do not produce fluorescence reaction, are judged to the positive.
Antibody positive hybridoma is through 3 limiting dilution assay sub-clones, it is thus achieved that the hybridization of three strain energy stably excreting antibody
Oncocyte, is respectively designated as 9A9,8G9 and 9H4.Immunoglobulin subclass identifies display, and monoclonal antibody 9A9 heavy chain type is 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 is κ
Type.Few cells neutralization test shows, 9A9 has the highest neutralization activity, is up to 1:4096 with titer in ascites.8G9 abdomen
Water is only 1:89,9H4 with titer then without neutralizing activity.
In view of above-mentioned 9A9 is A type FMDV senior middle school and the monoclonal antibody of activity, chooses monoclonal antibody 9A9 and carry out next step research.
Monoclonal antibody 9A9 culture supernatant is carried out respectively immunity indirectly glimmering with the BHK-21 cell infecting A, O and Asia1 type FMDV
Light detection (Fig. 1), result shows, 9A9 is only positive with A type strain, and separates strain with multiple O types and Asia1 type FMDV
All no cross reaction, can assert that 9A9 is A type FMDV serotype specificity monoclonal antibody.
The above results shows, the 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.
This hybridoma cell line 9A9 is submitted to the mechanism of patent accreditation to carry out preservation by the present invention, and its microbial preservation is numbered
For: CGMCC 12049；Depositary institution: China Microbiological preservation administration committee's common micro-organisms center；The preservation time is:
On March 21st, 2016；Preservation address: North Star West Road, Chaoyang District, BeiJing, China 1 institute 3, 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 purification of 1.1 monoclonal antibodies
Using octanoic acid-saturated ammonium sulfate method to be purified the ascites of preparation, operating procedure is summarized as follows:
(1) take ascites pretreated for 3ml and add 6ml 0.06mol/L pH4.8 acetate buffer solution；Ascites adds 99ul pungent
Acid is stirred at room temperature 30 minutes, and 4 DEG C stand more than 2 hours；Take out 11000rpm to be centrifuged 30 minutes, abandon precipitation；Supernatant 2mol/L
NaOH adjusts pH to 7.4.
(2) under the conditions of 4 DEG C, saturated ammonium sulfate is added to 50% saturation, ice bath stirring action 30 minutes, 4 DEG C of standings 2
More than hour；11000 leave the heart 30 minutes, abandon supernatant；In the PBS of the pH7.4 that precipitation is dissolved in 5ml0.1M；
(3) in precipitation float, add appropriate saturated ammonium sulfate solution while stirring, make the concentration in ammonium sulfate be
30%--40%, ice bath stirring action 30 minutes, 4 DEG C static more than 2 hours；11000 leave the heart 30 minutes, take precipitation, will be heavy
Form sediment in the PBS of the pH7.4 incorporating 2ml 0.1M；
(4) precipitation suspension is loaded in dialysis band, at 4 DEG C of PBS with the pH7.4 of 0.1M, within 2 hours, change liquid one
Secondary, dialyse 2 days.After having dialysed, reclaiming the purification ascites in bag filter, ultraviolet spectrophotometer measures protein concentration, uses
SDS-PAGE carries out purity detecting.
It is further purified monoclonal antibody with DEAE-Sephadex A-50 (GE company) column chromatography again, reclaims after purification
Monoclonal antibody, ultraviolet spectrophotometer measures protein concentration, carries out purity detecting with SDS-PAGE.
1.2 horseradish peroxidase-labeled monoclonal antibodies
Purified qualified after monoclonal antibody Over-voltage protection be marked.Operating procedure is as follows:
1. take 5mg HRP to be dissolved in the 0.06M HAc-NaAc buffer of 0.5ml pH5.6, add new preparation
0.06Mol/L NaIO4Solution 0.5ml, mixing, put 4 DEG C of 20-30min；
2. 0.16Mol/L glycol water (10mlH is added after taking out2O+0.09ml ethylene glycol) 0.5ml, room temperature is placed
30min；
3. add the solution 1ml containing 10mg antibody purification, mixing, adjust pH value about 9, and load 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, mixing, put 4 DEG C of 2h；
5. in above solution, it is slowly added to isopyknic saturated ammonium sulfate solution, mixing, 4 DEG C of effect 30min,
3000rpm is centrifuged 30 minutes, removes supernatant, precipitates and dissolves with a little 0.02Mol/L pH7.4PBS, loads bag filter, with
0.02Mol/L pH7.4PBS is at 4 DEG C of dialysis desalinations overnight；
6. take out centrifugal next day, 10000rpm be centrifuged 30 minutes to remove insoluble matter, obtain enzyme-antibody (HRP-IgG) and tie
Compound, molten to 1-2ml with 0.02Mol/L pH 7.4PBS；
7. OD is measured respectively with ultraviolet spectrophotometer403And OD280, qualified after by the IgG-Radix Cochleariae officinalis peroxidating of labelling
Thing enzyme adds equivalent glycerol ,-20 DEG C of preservations.
The titration of the optimal concentration of each reagent in 1.3A type antibodies against foot-and-mouth disease virus detection method
Determine the concentration of the detection monoclonal antibody being coated concentration and HRP labelling of A type foot and mouth disease virus purifying antigen.Test
ELISA Plate used is 96 hole flat bottom microtiter plate (Costar ELISA Plate, high-affinities), and test uses 50ul reaction system.Respectively
With the Na of pH9.62CO3/NaHCO3The antigen coated elisa plate of A type FMDV of buffer dilution purification, 4 DEG C overnight, with pH7.4's
PBST washes plate 5 times, dries.Then with the HRP marker detection monoclonal antibody of 2 times of serial dilutions of PBST (1:100,1:150～1:
12800,1:19200) add elisa plate hole, 37 DEG C of incubations 1 hour, add after washing plate tmb substrate solution lucifuge colour developing 15～
After 20min, use 2M H2SO4Terminate reaction, 450nm read optical density value (OD value), with the OD value of enzyme conjugates reach 1.6～
When 2.0, the highly diluted multiple of reagent is its concentration.
The concentration of each reagent primarily determines that through square formation titration, for follow-up A type antibodies against foot-and-mouth disease virus detection
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 is diluted to concentration and is coated elisa plate, 50 μ
L/ hole, shrouding or put in wet box, 4 DEG C overnight.Take out elisa plate, discard wherein liquid, wash plate 5 times with pH 7.4PBST, get rid of
Dry.After serum to be checked is initiated with the multiple proportions serial dilution of 1:2 with PBST, 50 μ l/ holes add in elisa plates.At elisa plate
In add immediately with PBST dilution HRP-9A9,50 μ l/ holes, shrouding, abundant shake mix, hatch 1h for 37 DEG C.Ibid wash plate 5
Secondary, add substrate solution, 50 μ l/ holes, shrouding, room temperature lucifuge hatches 15min.Add stop buffer, 50 μ l/ holes, terminate reaction,
450nmOD value is read in microplate reader.
2, result of the test
The purification of 2.1 monoclonal antibody ascites and labelling
The monoclonal antibody 9A9 ascites present invention prepared, through octanoic acid-saturated ammonium sulfate method purification, ultraviolet spectrophotometer is surveyed
Determining 9A9 concentration is 7.64mg/ml.Antibody 9A9 is further purified (figure through DEAE-sephadex a-50 ion-exchange chromatography again
2), with improvement Over-voltage protection labelling HRP.
The preliminary foundation of 2.2A type antibodies against foot-and-mouth disease virus detection method
First primarily determine that in A type antibodies against foot-and-mouth disease virus detecting system, A type foot and mouth disease virus is purified by square formation titrimetry
Antigen be coated concentration and HRP-9A9 detection monoclonal antibody concentration.Result shows: the bag of A type foot and mouth disease virus purifying antigen
Being 6.4 μ g/ml by concentration, extension rate is 1:1000；The suitableeest extension rate of HRP-9A9 is 1:1500.
In order to verify monoclonal antibody 9A9 diagnostic value in A type antibodies against foot-and-mouth disease virus detects, with the preliminary A type mouth set up
Aphtovirus antibody detection method detect respectively the different A type FMDV strong positive of extension rate, the weak positive, doubtful (marginal value) and
Negative serum, set up simultaneously O type and Asia1 type FMDV strong positive serum as comparison, to verify that monoclonal antibody 9A9 is in A type foot and mouth disease
In antiviral antibody detection method, binding ability and monoclonal antibody 9A9 with antigen distinguish A type foot and mouth disease virus yin and yang attribute serum
Ability.
Strong positive, the weak positive, doubtful (marginal value), the antibody titer curve of negative serum is drawn by 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, the weak positive and doubt
Good competitiveness is presented like serum, and the most competing with O type and Asia1 type FMDV strong positive serum and FMDV negative serum
Striving property, it is possible to specificity distinguishes the yin and yang attribute serum of A type FMDV, it is determined that monoclonal antibody 9A9 is setting up the inspection of A type antibodies against foot-and-mouth disease virus
Value in survey method and application potential.
Claims (8)
1. the hybridization of strain secretion anti-A type foot and mouth disease virus (FootandMouthDiseaseVirus) neutralizing monoclonal antibody
Oncocyte system, its microbial preservation number is: CGMCC 12049.
2. by the monoclonal antibody of hybridoma cell line secretion described in claim 1.
3. the purposes in the reagent or medicine of preparation diagnostics port fever aphthous of the monoclonal antibody described in claim 2.
4. the purposes in preparation prevention or the reagent for the treatment of foot and mouth disease or medicine of the monoclonal antibody described in claim 2.
5. diagnose the ELISA detection kit of A type foot and mouth disease virus, including: A type foot-and-mouth disease virus antigen, microwell plate, dilution
Liquid, cleaning mixture, nitrite ion, stop buffer, the monoclonal antibody of enzyme labelling, the sheep anti-mouse igg of enzyme labelling, positive control serum and the moon
Property control serum, it is characterised in that: described monoclonal antibody is the monoclonal antibody described in claim 2.
6. according to the test kit described in claim 5, it is characterised in that: described enzyme is horseradish peroxidase.
7. according to the test kit described in claim 5 or 6, it is characterised in that: described microwell plate is 96 hole microwell plates.
8. according to the test kit described in claim 7, it is characterised in that: described diluent is the Na of pH9.62CO3/NaHCO3
Buffer；Described cleaning mixture is the PBST of pH7.4；Described nitrite ion is tmb substrate solution；Described stop buffer is 2M
H2SO4；Described positive control serum is A type foot and mouth disease virus positive serum；Described negative control sera is A type foot and mouth disease
Virus negative serum.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107253979A (en) * | 2017-05-17 | 2017-10-17 | 中国农业科学院哈尔滨兽医研究所 | The hoof-and-mouth disease serotypes sharing epitope of monoclonal antibody 10B10 identifications and its application |
| 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 |
| CN109295005A (en) * | 2018-09-26 | 2019-02-01 | 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） | Secrete hybridoma cell line and its application of foot and mouth disease virus non-structural protein monoclonal antibody 2H1 |
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| CN110144329A (en) * | 2019-05-29 | 2019-08-20 | 北京标驰泽惠生物科技有限公司 | The monoclonal antibody and application of hybridoma cell strain 6B1 and its resistant to foot and mouth disease A type virus of secretion |
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Cited By (9)
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| CN107253979A (en) * | 2017-05-17 | 2017-10-17 | 中国农业科学院哈尔滨兽医研究所 | The hoof-and-mouth disease serotypes sharing epitope of monoclonal antibody 10B10 identifications and its application |
| CN107253979B (en) * | 2017-05-17 | 2020-09-29 | 中国农业科学院哈尔滨兽医研究所 | Foot-and-mouth disease virus serotype shared epitope recognized by monoclonal antibody 10B10 and application thereof |
| 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 |
| CN109295005A (en) * | 2018-09-26 | 2019-02-01 | 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） | Secrete hybridoma cell line and its application of foot and mouth disease virus non-structural protein monoclonal antibody 2H1 |
| 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 |
| CN110144329A (en) * | 2019-05-29 | 2019-08-20 | 北京标驰泽惠生物科技有限公司 | The monoclonal antibody and application of hybridoma cell strain 6B1 and its resistant to foot and mouth disease A type virus of secretion |
| 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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