CN106483291A - A kind of Asia1 type antibodies against foot-and-mouth disease virus solid phase competitive ELISA kit based on type specificity monoclonal antibody - Google Patents
A kind of Asia1 type antibodies against foot-and-mouth disease virus solid phase competitive ELISA kit based on type specificity 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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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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Abstract
The invention discloses a kind of Asia1 type antibodies against foot-and-mouth disease virus solid phase competitive ELISA kit based on type specificity monoclonal antibody.Asia1 type foot and mouth disease specific monoclonal antibody containing HRP labelling.The test kit of the present invention can be used for Asia1 type antibodies against foot-and-mouth disease virus level monitoring.Substitute in existing ELISA foot and mouth disease rabbit resist, guinea pig antiserum and anti-Cavia porcelluss enzyme labelled antibody, simplify ELISA method step, improve stability, detection time is greatly shortened, foreshorten in 1.5 hours from 24h, breaching original ELISA needs one to resist the course of reaction such as anti-with two.
Description
Technical field
The present invention relates to biotinylation kit field, more particularly, to a kind of Asia1 type foot and mouth disease based on type specificity monoclonal antibody
Antiviral antibody solid phase competitive ELISA kit.
Background technology
Foot and mouth disease is acute, the hot, contagious disease suffered from altogether by the artiodactyls that foot and mouth disease viruses cause, and can give
Animal husbandry production and foreign trade cause huge economic loss, and this disease not only has a strong impact on the development of animal husbandry, also to people
Class health constitutes very big harm.China cultivates big country as cattle and sheep, and foot and mouth disease is that harm cattle and sheep cultivate particularly important disease,
Strengthen to the diagnosis of foot and mouth disease and the research and development of new detecting technique, break through foot and mouth disease diagnostic reagent industrialization key technology with
Technique, is great country and the social need solving the fast-developing bottleneck problem of restriction China animal husbandry, will be to improving me
The overall development level of state's animal husbandry and overall control and prevention of disease ability, promote the sound development of animal husbandry to produce positive, far-reaching
Impact, and safe to food and human health has special meaning.And to fundamentally control the generation of foot and mouth disease with
Popular it is necessary to strengthen the research of the diagnosticss to primary disease first, for controlling China foot and mouth disease to provide technical support.
At present, the serotype of foot and mouth disease viruses is many, and persistent infection asymptomatic band poison is serious, Accurate Diagnosis serotype and precisely
Monitoring carriers, are this sick primary links of prevention and control.Develop synchronous, foot and mouth disease diagnosis with the diagnostic monitoring technology in life field
The development course of monitoring technology can be divided into 3 stages, i.e. the complement fixation test of early stage and virus neutralization tests, is subsequently joined with enzyme
Imnnoadsorption (ELISA) is the cause of disease of core and antibody detection method, now based on polymerase chain reaction (PCR)
Different kinds of molecules diagnostic techniquess.However, with the development of biotechnology, existing method shows such or such deficiency,
Or loaded down with trivial details time-consuming, or it is not reaching to very accurately degree.Be applied at present foot and mouth disease diagnosis have virus purification, ELISA, PCR,
Agglutination test, immune colloidal gold chromatography technology etc., most widely used is LPB-ELISA, but LPB-ELISA program is relatively
Many, operating procedure is complicated, and detection at least needs the time of one day could obtain result, therefore cannot realize big when epidemic situation happens suddenly
The instant detection of the sample of amount.Present situation to be overcome is necessary for development of new ELISA diagnostic techniquess.
Solid phase competitive ELISA method is the standard method that OIE recommends.By screening and preparing diagnosis key mark thing (type
Specific monoclonal antibody), solve the problems, such as that between type, intersection, stability are poor, breach using multi-resistance intersection, unstable problem；Pass through
HRP labelling monoclonal antibody, substitute the foot and mouth disease rabbit in existing LPB-ELISA resist, guinea pig antiserum and anti-Cavia porcelluss enzyme labelled antibody, letter
Change ELISA method step, thus reduce can laboratory operating procedures, substrate directly and an anti-reflective should, save the time, improve
Sensitivity and Specificity.Improve stability, breaching original ELISA needs one to resist the course of reaction such as anti-with two, is greatly shortened
Detection time.
Content of the invention
It is an object of the invention to provide a kind of Asia1 type antibodies against foot-and-mouth disease virus solid phase based on type specificity monoclonal antibody is competing
Strive ELISA kit.
For achieving the above object, the present invention provides a kind of Asia1 type antibodies against foot-and-mouth disease virus based on type specificity monoclonal antibody
Solid phase competitive ELISA kit is it is characterised in that the Asia1 type foot and mouth disease specific monoclonal antibody containing HRP labelling.
Further, the Asia1 type foot and mouth disease specific monoclonal antibody preparation method of described HRP labelling is,
The preparation of the cell suspension of splenocyte：Immunity is carried out to Balb/c mice using routine immunization method, totally 4 times, first
First use bacillus calmette-guerin vaccine sensitized mice, 200 μ l/ are only；Will be complete with Fu Shi for the Asia1 type foot and mouth disease viruses inactivation antigen of purification after one week
The quality such as adjuvant mix, fully emulsified after, by 0.3ml/ immune mouse, axillary fossa, groin is injected, altogether immunity 5；Exempt from for the first time
After epidemic disease at interval of 3 weeks again immunity once, the quality such as the Asia1 type foot and mouth disease viruses inactivation antigen of purification and freund 's incomplete adjuvant
Mix, in mouse carotid dorsal sc gradation multi-point injection, first 3 days abdominal cavity booster immunizations of cell fusion are once；This mice is plucked after 3 days
Eyeball takes blood, obtains the cell suspension of splenocyte；
Cell fusion：The cell suspension of the myeloma cell and described splenocyte that are in exponential phase is mixed, centrifugation
Abandon supernatant；Fusion pipe is put in palm and rubs back and forth, so that cell block is loosened, be slowly added to 0.7ml 37 toward in glass tubing with dropper
The 50%PEG1000 of DEG C preheating, adds in 90s, stands 60s, first slow in 5min after be added dropwise over the depletion of blood of 37 DEG C of preheatings soon
Clear DMEM, altogether plus 10ml, after jog mixes, supernatant is abandoned in centrifugation；With HAT culture fluid re-suspended cell, move into 60mlHAT culture immediately
In liquid, gently mix, then proceed in 96 porocyte culture plates by 100 μ l/ holes, be placed in 5%CO2, in 37 DEG C of constant incubators
Culture；
The screening of hybridoma：Cultivate the 6th day and carry out half amount with HAT culture fluid and change liquid, treat that cell grows to the 1/ of bottom hole
Suction out supernatant when 4～1/3 and carry out indirect ELISA detection；The cell of P/N >=2.1 is hybridoma；
The colonized culture of hybridoma；
The preparation of monoclonal antibody：Every mouse peritoneal injects 0.5mL1x106～6x106The hybridoma of/mL, treats little
Mus abdominal part slowly swells, and mice spirit is depressed, when not walking about, being at death's door, and cervical dislocation puts to death mice, collects ascites, room temperature,
10000r/min is centrifuged 10min, collects middle bright liquid and is monoclonal antibody；
The monoclonal antibody of HRP labelling purification：Concentration after saturated ammonium sulfate purification is the monoclonal of 5.284mg/ml
Antibody 0.3mL and 0.1mL peroxidase add in pipe, and gently piping and druming mixes, and 4 DEG C overnight；Add the triethanolamine of 40 μ L,
Mix, add the sodium borohydride of 50 μ L, mix, after 4 DEG C are placed 30min, add 10 μ L glycine to mix；Afterwards mixed liquor is put
Dialyse in bag filter, dialysis solution is the PBS of PH 7.2, gained is the ASIA1 type foot and mouth disease specific monoclonal antibody of HRP labelling.
Further, in described cell fusion step, the ratio of spleens cell number and myeloma cell's number is 5:1 to 10:1.
The present invention also protects and carries out ASIA1 type antibodies against foot-and-mouth disease virus level using described solid phase competitive ELISA kit
The purposes of monitoring.
Detection method involved in the present invention is simplified or the accurate new qualitative, quantitative technology as target with light.
There is the technological deficiencies such as time-consuming, serious, the stability difference of intersection for the existing diagnostic techniquess of foot and mouth disease in the present invention, pass through
Screening and preparation diagnosis key mark thing (type specificity monoclonal antibody), intersection, poor, the time-consuming, complex steps of stability etc. between solution type
Problem, breaches using multi-resistance intersection, unstable, time-consuming, complex steps problem；By this monoclonal antibody of HRP labelling, substitute existing
In ELISA foot and mouth disease rabbit resist, guinea pig antiserum and anti-Cavia porcelluss enzyme labelled antibody, simplify ELISA method step, improve stability, big
Amplitude shortens detection time, foreshortens in 1.5 hours from 24h, and breaching original ELISA needs that one is anti-and two anti-grades were reacted
Journey.The method can provide a kind of more preferable diagnosis method for the immunity and infection state that detect ASIA1 type foot and mouth disease.
Detectable prepared by the present invention can be adjusted for ASIA1 type foot and mouth disease stream and antibody surveillance provides and supports.
Brief description
Fig. 1 is that antibody titer measures 10 parts of sample layout figures of (quantitative) detection.
Fig. 2 is TPPA 20 parts of sample layout figures of (qualitative) detection.
Fig. 3 is the specific outcome figure of ascites monoclonal antibody in detection embodiment 1.
Fig. 4 is the SDS-PAGE of the ascites monoclonal antibody of non-purification and purification.
Fig. 5 is the specific ELISA testing result figure of 5E5-HRP.
Fig. 6 is the testing result figure of the potency of 5E5-HRP.
Fig. 7 is the antigen-reactive spectrum testing result figure of type specificity monoclonal antibody 5E5.
Specific embodiment
Embodiments of the invention are described below in detail, the example of described embodiment is shown in the drawings, wherein from start to finish
The element that same or similar label represents same or similar element or has same or like function.Below with reference to attached
The embodiment of figure description is exemplary it is intended to be used for explaining the present invention, and is not considered as limiting the invention.Embodiment
In unreceipted particular technique or condition person, according to the technology described by document in the art or condition or according to the description of product
Book is carried out.Agents useful for same or the unreceipted production firm person of instrument, be can by city available from conventional products.
Embodiment 1：
1.1 material
1.1.1 laboratory animal, antigen and cell
8 week old SPF level BALB/c raettins, buy in Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences；SP/20 and BHK-21
Cell is purchased from the national classical collection thing of Wuhan virus institute of the Chinese Academy of Sciences and preserves center.O-shaped foot and mouth disease viruses inactivation antigen, A type hoof-and-mouth disease
Malicious inactivation antigen, Asia1 type foot and mouth disease viruses inactivation antigen is purchased from middle peasant Witter biotech inc.
1.1.2 main agents
Liquid paraffin, goat anti-mouse IgG-HRP is purchased from Sigma company, and 96 hole elisa Plates are purchased from CORNING company,
The H of 2mol/L2SO4There is provided by Lanzhou veterinary institute detection group, hyclone is purchased from GIBICO company, TMB is purchased from
SurModics company, modified form RPM1-1640 is purchased from Hyclone company, and carbonate buffer solution capsule is purchased from Sigma company, not
Pre-dyed albumen Marker is purchased from Fermentas.
1.2 method
2.1 animal immune
Immunity is carried out to Balb/c mice using routine immunization method, totally 4 times, uses bacillus calmette-guerin vaccine sensitized mice, 200 μ first
L/ is only.After one week, the Asia1 type foot and mouth disease viruses inactivation antigen of purification and the quality such as Freund's complete adjuvant are mixed, fully emulsified
Afterwards, by 0.3ml/ immune mouse, axillary fossa, groin is injected, altogether immunity 5；At interval of immunity one again in 3 weeks after initial immunity
Secondary, the Asia1 type foot and mouth disease viruses inactivation antigen of immunity is mixed with quality such as freund 's incomplete adjuvants, in mouse carotid dorsal sc
Gradation multi-point injection, first 3 days abdominal cavity booster immunizations of cell fusion are once.
1.2.2 the foundation of screening technique
Adopt indirect ELISA detection method during screening, select optimal antigen coat concentration with square formation burette test.Radix Cochleariae officinalises peroxide
The goat anti-mouse igg changing enzyme (HRP) labelling is anti-as two, and optimal two anti-concentration adopt this laboratory general operations concentration 1:1
000.Comprise the following steps that：
(1) antigen is made 1:250,1:500 and 1:1 000 dilution, 100 μ l/ holes are coated, 37 DEG C of effect 2h, 4 DEG C of mistakes
Night.
(2) next day, pat dry being coated liquid in ELISA Plate, press 120 μ l/ hole closings with confining liquid, after 37 DEG C of effect 2h, pat dry
To be measured.
(3) positive serum is from 1:40 start doubling dilution to the 9th row (1:10240), set 1 group of negative control (as simultaneously
The mouse serum of not immune Asia1 type foot-and-mouth disease antigen), one group of blank (being as not added with the control wells of any serum)；Plus
After sample, 37 DEG C of effect 0.5h, wash 3 times.
(4) enzyme mark horseradish peroxidase-labeled sheep anti-mouse igg two is added to resist, 50 μ l/ holes, 37 DEG C of effect 30min, washing
3 times.
(5) substrate A, each 50 μ l/ holes of B are added, develop the color at 37 DEG C 10min, uses terminate liquid terminating reaction, 50 μ l/ holes.With
Enzyme mark detector measures OD450 value.
When being screened with indirect ELISA detection method, select the ratio maximum of positive serum and negative serum, and OD450 value connects
It is bordering on 1 antigen diluent degree as the best effort concentration of indirect ELISA reaction system.This system is used during screening hybridoma
Cell supernatant is detected, when P/N >=2.1, is judged to the positive.
1.2.3 the mensure of Mouse titers
After indirect ELISA method is set up, from the antibody titer being most preferably coated Concentration Testing immune mouse.Choose immunity effect
Valency detected value highest mouse peritoneal supplementary immunization once, takes the spleen of this Mus to carry out cell fusion after three days.
1.2.4. the foundation of hybridoma cell strain
1.2.4.1 the preparation of myeloma cell
(preserve purchased from from the national classical collection thing of Wuhan virus institute of the Chinese Academy of Sciences in merging the last fortnight recovery myeloma cell SP/20
Center).
Cryopreservation tube is taken out from liquid nitrogen, is put into immediately in 37 DEG C of warm water, allow it to dissolve, until completely dissolved, 1000rpm
Centrifugation 10min, abandons supernatant, and precipitation is carefully suspended with the DMEM complete culture solution containing 20% hyclone, adds cell after mixing
In culture bottle, it is placed in 5%CO2, Secondary Culture in 37 DEG C of constant incubators.Incubation adds 8- nitrogen bird fast in culture fluid
Purine, is added by 100 μ l/ bottle cells, processes once daily, continuous processing three times.Make SP/20 cell quicker to HAT culture fluid
Sense.Reach 8 bottles, and make the myeloma cell on the fusion same day be in exponential phase, standby.
1.2.4.2 the preparation of feeder cells
The cell fusion same day prepares feeder cells, and method is as follows：
(1) take 8～10 week old health Balb/c mices, pluck eyeball and take blood, prepare negative serum.Disconnected neck is put to death, and uses
75% alcohol disinfecting is processed.
(2) mice is placed in superclean bench, the outside of belly upwards, lifts mouse part skin with tweezers, cuts in hypogastric region
One osculum, longitudinally peels off, and fully exposes peritoneum, and disinfects in alcohol.
(3) primary sterilization syringe is used to extract in 5ml HAT culture fluid injection mouse peritoneal, right hand syringe keeps not
Dynamic, jog mice, subsequently extracts cell suspension.
(4) cell suspension is added in 60ml HAT culture medium, count after mixing, adjustment cell concentration to 1～5 × 105
Individual/ml.As cell quantity is very few, feeder cells can be prepared by above-mentioned steps again.
(5) be transferred in 96 porocyte culture plates by 100 μ l/ holes, be placed in 5%CO2, cultivate in 37 DEG C of constant incubators standby
With.
1.2.4.3 the preparation of splenocyte
(1) before taking three days, the mice of booster immunization, plucks eyeball and takes blood, prepare positive serum.Disconnected neck is put to death, with 75% wine
Essence is disinfected.
(2) in superclean bench, aseptic win spleen, remove fat and connective tissue, be placed in 200 mesh sterilizing wire nettings
On, it is lightly ground spleen with sterilizing glass syringe core, be slowly added dropwise serum-free DMEM simultaneously.
(3) collect splenocyte suspension, and transfer in 10ml centrifuge tube, 1 000rpm centrifugation 5min, abandon supernatant, resuspended
In 5ml serum-free DMEM culture fluid, splenocyte is standby after counting.
1.2.4.4 the fusion of cell
(1) select in good condition and in logarithmic growth 6～8 bottles of SP/20 cell, under cell is gently blown and beaten from bottle wall
Come and transfer in 50ml centrifuge tube, 1 000rpm centrifugation 10min.Abandon supernatant, suspended with 5ml serum-free DMEM, count.
(2) SP/20 cell and immune spleen cell suspension are mixed, the ratio of spleens cell number and myeloma cell's number should maintain
5:1 to 10:Between 1.
(3) 1 000rpm centrifugation 5min, abandon supernatant.
(4) fusion pipe is put in palm and rub back and forth, so that cell block is loosened, this walks as merging committed step.
(5) it is slowly added to the 50%PEG (1000) of 37 DEG C of preheatings of 0.7ml with dropper toward in glass tubing, add in 90s, quiet
Put 60s.
(6) it is added dropwise over the serum-free DMEM of 37 DEG C of preheatings after first slow in 5min soon, altogether plus 10ml, after jog mixes, 1
000rpm is centrifuged 5min, abandons supernatant.
(7) use HAT culture fluid re-suspended cell, move in 60mlHAT culture fluid immediately, gently mix, then press 100 μ l/
Hole proceeds in 96 porocyte culture plates, is placed in 5%CO2, cultivates in 37 DEG C of constant incubators.
1.2.4.5 the screening of hybridoma
Note after fusion observing Growth of Hybridoma Cell situation, general culture has little clone to occur for three days about.6th
It carries out the 1st half amount and changes liquid with HAT culture fluid, the 8th～10 day about, suctions out when cell grows to the 1/4～1/3 of bottom hole
Carry out clearly indirect ELISA detection.Using P/N >=2.1 as positive criterion, with SP/20 cells and supernatant and normal mouse
Negative control made by serum.The cell of test positive is enlarged cultivating, carries out colonized culture simultaneously.First time sub-clone
Shi Huanyong HT culture fluid, is changed to common complete culture solution after two weeks.
1.2.4.6 the colonized culture of hybridoma
The hybridoma positive to ELISA detection carries out colonized culture, in time using limiting dilution assay [59].Step
As follows：
(1) feeder cells are prepared, method is as previously mentioned.In HT culture fluid, prepare feeder layer, every hole 100 μ l.
(2) with pipettor, ELISA is detected that in positive hole, hybridoma gently blows and beats mixing, sampling, in hemocytometer
Make cell counting on number plate, then use HT culture fluid to adjust cell number.First during sub-clone general adjustment cell number to 15～20
Individual/ml, during second, third sub-clone, general adjustment cell number is to 10/ml.
(3) by hybridoma suspension inoculation in 96 porocyte culture plates containing feeder layer, every hole 100 μ l.
At 37 DEG C, cultivate under the conditions of 5%CO2 and saturated humidity.
(4) observe daily and record every hole number of cell clones, half amount changes liquid when cell grows to the 1/4～1/3 of bottom hole, and
Indirect ELISA detection is carried out to cell conditioned medium.
(5) select clone number is few, OD450 value is high positive hole, by its time cloning again.Operate through 3～4 time cloningizations, directly
When extremely all cloning cell hole Positive rates reach 100%, you can determine the hybridoma obtaining secretion specific monoclonal antibody
Strain is the hybridoma (2D6) of anti-Asia1 type FMDV monoclonal antibody, should timely amplification culture frozen.
1.2.5 the recovery of hybridoma
From liquid nitrogen container, the hybridoma (2D6) of the anti-Asia1 type FMDV monoclonal antibody of one specificity of taking-up is frozen
Pipe (in preparation method reference implementation example 1 1.2), puts into rapidly after being clamped with tweezers in 37 DEG C of water-baths, ceaselessly rocking makes
Cell melts rapidly, aseptic open cryopreservation tube, by Cell sap move into fill in 10mL modified form RPM1-1640 culture medium, be placed in
Containing 5%CO237 DEG C of incubator moderate hybridization oncocytes completely adherent after cell is carried out changing liquid.
1.2.6 a large amount of preparations of monoclonal antibody
Choose the Balb/c raettin of 8 week old, aseptically, the liquid paraffin of every lumbar injection 0.5mL sterilizing.Two
Zhou Hou, ready Hybridoma Cell Culture liquid (modified form RPMI-1640) is injected the intraperitoneal of mice.
The preparation of hybridoma：Select the good hybridoma of upgrowth situation and adopt culture bottle Secondary Culture, aseptic
Under the conditions of, outwell culture medium in cell bottle, add appropriate trypsin digestion cell so that it is come off, add 5mL serum-free modified form
RPM1-1640 culture medium, gently blows afloat cell, all moves in centrifuge tube, room temperature, 1000r/min centrifugation 10min, abandons supernatant,
Appropriate serum-free modified form RPM1-1640 culture medium is added to hang cell, platform expects blue cell counting, and adjustment cell quantity is extremely
1x106～6x106/ mL, every mouse peritoneal injects 0.5mL gained cell, observes mice daily, and mouse web portion slowly swells, about
7～10d mice spirit is depressed, when not walking about, being at death's door, cervical dislocation puts to death mice, collects ascites, room temperature, 10000r/
Min is centrifuged 10min, collects middle bright liquid as 2D6 and is also monoclonal antibody.
1.2.7 the specific detection of ascites
Antigen O, A and Asia1 of inactivation are coated in ELISA Plate, 100 μ L/ holes, 4 DEG C are overnight.Using indirect ELISA method
The specificity of ascites is measured：Every hole adds the 2D6 (1 of 100 μ L PBS dilutions:2000), 37 DEG C of incubation 1h.Wash plate, often
Hole adds the goat anti-mouse IgG-HRP (1 that 100 μ L PBS have diluted:10000), 37 DEG C of incubation 1h.Wash plate, every hole adds 50 μ
L TMB, 37 DEG C of incubation 15min, add 2M H2SO4, read OD with microplate reader450.
1.2.8 the spectrum method of type specificity monoclonal antibody 2D6
By Asia1 type foot and mouth disease inactivation antigen (purchased from middle peasant Witter bio tech ltd) popular for China in recent years
It is coated elisa plate, 100 μ L/ holes, 4 DEG C are overnight.Surveyed using response spectrum in the type to Asia1 type monoclonal antibody for the indirect ELISA method
Fixed：Every hole adds the 2D6 (1 of 100 μ L PBS dilutions:2000), 37 DEG C of incubation 1h.Wash plate, every hole adds 100 μ L PBS to dilute
Goat anti-mouse IgG-HRP (1:10000), 37 DEG C of incubation 1h.Wash plate, every hole adds 50 μ L TMB, 37 DEG C of incubation 15min,
Add 2MH2SO4, read OD with microplate reader450Value.
1.2.9 the monoclonal antibody of HRP labelling purification
The antibody 0.3mL that concentration after saturated ammonium sulfate purification is 5.284mg/mL is added with 0.1mL peroxidase
In the EP pipe of 1.5mL, gently piping and druming mixes, and 4 DEG C overnight.Add the triethanolamine of 40 μ L in mixture, mix, add 50 μ L
Sodium borohydride, mix, 4 DEG C placement 30min.Add 10 μ L glycine solutions again, mix.Afterwards mixed liquor is placed in dialysis
(PBS of PH 7.2) is dialysed in bag.
1.2.10 the preparation of Asia1 type foot and mouth disease viruses rabbit anti-serum
1.2.10.1 the viral purification no sucrose of RNase (productions of Sigma company) prepare 150,250,350,450g/L
Sucrose density gradient, 4 DEG C standing balance overnight.Sample is added on top by next day, 10 DEG C of 35000r/min (Hitachi CP70VB from
Scheming) centrifugation 150min, with 0.5mL for 1 fraction, it is collected from low concentration to high concentration.According to grinding of Bachrach etc.
Study carefully, take sucrose density to be in 350～450g/L, herein for foot and mouth disease viruses effective inactivation antigen rich region, -80 DEG C of preservations are standby
With
1.2.10.2 immune programme for children takes above-mentioned adequately emulsified Freund's complete adjuvant antigen, in every rabbit two back leg foot
Subcutaneous, at lymph node and the inoculation of dorsal sc multiple spot, inoculation total amount is 2ml, after 14 days still with above-mentioned antigen and dosage by carrying on the back
Portion's subcutaneous multiple spot inoculation, carries out second immunity；After 11 days, with above-mentioned incomplete Freund's adjuvant antigen, by every rabbit shoulder flesh
2 points of meat, dorsal sc multiple spot is inoculated, and inoculation total amount is 2ml, for its third time immunity.
1.2.10.3 take a blood sample after the immunity of examination blood third time, rabbit is measured by Asia1 type foot and mouth disease LPB-ELISA
Antiserum titre, if potency reaches 1:1024, then adopting carotid artery separation rabbit anti-serum, if being not reaching to, carrying out the 4th time
Immunity, until serum titer reaches 1:1024, it is standby that blood sampling separates serum.
1.3 foundation based on the solid phase competitive ELISA method of enzyme labelled antibody
1.3.1 the determination of each reagent optimal use concentration
The present invention utilizes Asia1 type foot and mouth disease viruses rabbit anti-serum (the O-shaped rabbit anti-serum of 1.2.10 preparation) and HRP labelling
Asia1 type foot and mouth disease specific monoclonal antibody (monoclonal antibody of the HRP labelling purification that namely 1.2.9 prepares) set up solid
Phase competitive ELISA method.Establish the optimal use of coated multi-resistance in the method, antigen and competition antibody using square formation titrimetry
Concentration.
1.3.2 the foundation of O-shaped foot and mouth disease solid phase competitive ELISA method
◆ prepare
According to different needs, one of figure below layout is selected to set corresponding testing goal.Fig. 1. antibody titer
Measure 10 parts of sample layout figures of (quantitative) detection；Fig. 2. TPPA 20 parts of sample layout figures of (qualitative) detection.
◆ dilute serum
According to testing demand dilute serum on serum-dilution plate, overall moving to is coated in plate Sptting plate afterwards for suggestion.
In serum-dilution plate, with the amount in 60 μ L/ holes 2 times of serial dilution serum of sample diluting liquid, tested serum is the 1st
～10 arrange from 1:4 (i.e. A1～A10 holes, 75 μ L sample diluents add the tested serum of 25 μ L) are diluted to 1:512；Dilute in the 11st row
Release positive control serum, method is with dilution serum to be checked；In the 12nd row dilution negative control sera, from A12～D12, method is same
Dilute serum to be checked；E12～H12 is blank (only sample diluting liquid).
Note：Because this method is competitive ELISA method, after dilute serum moves to Sptting plate, add the enzyme mark of equivalent
Antibody working solution, now the dilution factor of serum double, tested serum is from 1:8～1:1024, because positive control serum is pre-
First do 1:8 times of dilutions, then be changed into 1:32～1:4096.
◆ sample-adding
By dilution plate integral translation to being coated in Sptting plate, every hole 50 μ L；Subsequently add 50 μ L, labeling antibody to all reacting holes
Working solution, gently shakes, and 37 DEG C are reacted 30 minutes.
◆ ELISA Plate is taken out in washing, is dried, is washed with cleaning mixture 3～5 times, dries in absorbent paper.
◆ the every hole of colour developing adds substrate solution 50 μ L, stands lucifuge in 37 DEG C of incubators and reacts 5～10 minutes.
◆ terminate every hole and add 50 μ L terminate liquids.
◆ result calculates and judges to measure every hole absorbance value of ELISA Plate at microplate reader 450nm wavelength, obtains every
The tested serum of part and the average light absorption value with the comparison of plate monoclonal antibody, calculate each sample suppression ratio (PI) value, PI=according to formula
[1- sample well OD/ competes antibody control hole OD] x100%.To compete the average OD in antibody control hole450Value between 0.9～2.2,
Negative control sera PI value ＜ 40%, during positive control serum PI value ＞ 50%, blank PI value ＜ 5%, test is set up.
Criterion：PI ＞ 60%, antibody positive；PI ＜ 60%, negative antibody；PI=60%, judges positive suspicious, weight
Rechecking and survey once, if remaining as 60, being judged to the positive, if be not equal to 60 need to detect again, or being detected with other methods.
1.3.3 solid phase competes the determination of marginal value
22 parts of positive serum samples and 20 parts of negative serum samples are carried out and times dilution respectively, with the competition of foundation
ELISA method detects, counts the different dilution PI value (suppression ratio) of each sample, determines the most preferably dilute of serum i.e. antigen
Release multiple.According to each dilution testing result of each sample in result, it is ultimately determined to optimal marginal value.
1.3.4 the specificity of solid phase competitive ELISA method
Using the ELISA method detection foot-and-mouth disease a type set up, O-shaped, swine fever, the pig indigo plant infectious disease such as ear and sore mouth virus sun
Property blood serum sample.Meanwhile, with the positive serum (10 parts) of Asia1 type foot and mouth disease known to the detection of this ELISA method, statistic mixed-state is tied
Really, assay kit specificity.
1.3.5 the coincidence rate of solid phase competitive ELISA method
Application commercialization detection Asia1 type foot and mouth disease viruses serum antibody test kit detection Asia1 type foot and mouth disease positive and negative
Serum, 60 parts altogether, the test kit of commercialization is the PrioCHECK foot-and-mouth disease antibody detection kit of Holland.
1.3.6 the repeatability of solid phase competitive ELISA method
Repeat in batch：With the multi-resistance of same batch preparation, antigen, the competition antibody of HRP labelling and other same reagent, in phase
With under the conditions of with the method set up, 7 parts of blood serum samples are carried out detecting with independent detection 5 times, statistic mixed-state result, calculating standard
Difference and the coefficient of variation.
Repeat between batch：With the multi-resistance of three batch preparations, antigen, the competition antibody of HRP labelling and other same reagent, in phase
With under the conditions of with the method set up, 7 parts of blood serum samples are carried out detecting with independent detection 1 time, statistic mixed-state result, calculating standard
Difference and the coefficient of variation
2. result
2.1 detection ascites specific outcome
Detection through indirect ELISA, the Mab and Asia1 type antigen reactivity of preparation very well, with A and Asia1 type antigen and
BHK-21 cell does not all react, and has good specificity, and result is shown in Fig. 3.
2.2 the SDS-PAGE analysis of 2D6 monoclonal antibody after purification
2D6 monoclonal antibody through saturated ammonium sulphate purification is carried out SDS-PAGE electrophoretic analysiss, result is shown in Fig. 2, wherein swims
Road M is protein molecule Marker；Swimming lane 1 is unpurified 2D6 ascites electrophoresis result, and swimming lane 2 is the 2D6 ascites electrophoresis of purification
As a result, it can be seen that 2D6 monoclonal antibody after purification has two bands, it is heavy chain and the light chain of IgG respectively, size is respectively about
For 45kDa and 25kDa, be consistent (Fig. 4) with expected resultss, the use of the concentration that ultraviolet spectrophotometer records antibody is 5.927mg/
mL.
The testing result of Mab specificity and potency after 2.3 labellings
Result is shown in Fig. 5 and Fig. 6.From figure 5 it can be seen that after Asia1 type specificity monoclonal antibody labelling HRP, only with Asia1 type
Foot and mouth disease viruses react, and all do not react with A type, O-shaped foot and mouth disease and bhk cell culture supernatant, illustrate that this labelling resists
Body specificity is very high.From fig. 6 it can be seen that the Asia1 type monoclonal antibody after labelling is being diluted to 1:When 12800, its OD450 value is still
Close to 1.5.
The spectrum method of the type specificity monoclonal antibody 2D6 of 2.4 anti-Asia1 type foot and mouth disease
As can be seen from Figure 7 2D6 all can be able to react with Asia1 type foot and mouth disease viruses popular in recent years, by
This can be seen that 2D6 not only has specificity between good type, and has good reaction broad spectrum activity in same type, is
Optimum antibody as detection.X-axis represents O-shaped not synantigen, and Y-axis represents the OD value at 492nm.
2.5 each reagent optimal use concentration and the determination of marginal value
The solid phase competitive ELISA side that rabbit anti-Asia1 type foot and mouth disease polyvalent antibody and HRP enzyme mark monoclonal antibody are set up is applied in this research
Method.The rabbit anti-multi-resistance (potency 1 being determined with square formation titrimetry:1024) optimal use concentration (1:1000), antigen (TCID50
Optimum dilution degree 0.1mL=7.2) is 1:The optimal use concentration dilution (1 of 15 detection monoclonal antibodies:7000),
With the method for foundation, 22 parts of positive serum samples and 20 parts of negative serum samples are carried out detecting and carry out respectively, root
According to sensitivity and the specific outcome at each point of contact in result, so be ultimately determined to optimal marginal value being：Work as antibody dilution
≥1:64, and PI is more than 60%, judges antibody positive；Less than 60%, judge negative antibody.
The specificity of 2.6 solid phase competitive ELISA methods
Foot and mouth disease is O-shaped using the ELISA method detection set up, A type, Asia1 type, swine fever, pig indigo plant ear and sore mouth virus etc.
Infectious disease positive serum samples, are negative (table 1) according to decision method.These as shown by datas, the ELISA method tool set up
There is good specificity, between serum antibody, there is not cross reaction.Meanwhile, with Asia1 type mouth known to the detection of this ELISA method
The positive serum (10 parts) of fever aphthous is the positive.The Asia1 type aftosa serum antibody detection method tool set up as can be seen here
There is good specificity.
Table 1. specific detection result
The coincidence rate of 2.7 solid phase competitive ELISA methods
Application commercialization detection Asia1 type foot and mouth disease viruses serum antibody test kit detection Asia1 type foot and mouth disease positive and negative
Serum, 60 parts altogether, the test kit of commercialization is the coincidence rate of the PrioCHECK of Holland is 96.7%, coincidence rate=two kind phase
Mutually consistent serum number/serum number × 100% to be checked of the test kit testing result of comparison, shows detection examination involved in the present invention
Agent box testing result is reliable.Specifically it is shown in Table 2.
Table 2. coincidence rate tests table
The repeatability of 2.8 solid phase competitive ELISA methods
Repeat in batch：With the multi-resistance of same batch preparation, antigen, the competition antibody of HRP labelling and other same reagent, in phase
With set up method, independent detection is carried out to 7 parts of blood serum samples (wherein 5 parts of positive serum, 2 parts of negative serum) under the conditions of same,
Statistic mixed-state result, calculates standard deviation and the coefficient of variation.The test kit variation within batch coefficient of the present invention is respectively less than 10%, and it is described
Reproducible.
Repeat experimental result table in 3. batches, table
Repeat between batch：With the multi-resistance of three batch preparations, antigen, the competition antibody of HRP labelling and other same reagent, in phase
With set up method, independent detection is carried out to 7 parts of blood serum samples (wherein 5 parts of positive serum, 2 parts of negative serum) under the conditions of same,
Statistic mixed-state result, calculates standard deviation and the coefficient of variation.The results are shown in Table 4, as can be seen from Table 4, interassay coefficient of variation is all little
In 10%, illustrate that the test kit of the present invention is reproducible between criticizing.
Repeat experimental result table between 4. batches, table
Although embodiments of the invention have been shown and described above it is to be understood that above-described embodiment is example
Property it is impossible to be interpreted as limitation of the present invention, those of ordinary skill in the art is in the principle without departing from the present invention and objective
In the case of above-described embodiment can be changed within the scope of the invention, change, replace and modification.
Claims (4)
1. a kind of Asia1 type antibodies against foot-and-mouth disease virus solid phase competitive ELISA kit based on type specificity monoclonal antibody, its feature exists
In the Asia1 type foot and mouth disease specific monoclonal antibody containing HRP labelling.
2. solid phase competitive ELISA kit described in claim 1 is it is characterised in that the Asia1 type foot and mouth disease of described HRP labelling
Specific monoclonal antibody preparation method is,
The preparation of the cell suspension of splenocyte：Immunity is carried out to Balb/c mice using routine immunization method, totally 4 times, uses first
Bacillus calmette-guerin vaccine sensitized mice, 200 μ l/ are only；By the Asia1 type foot and mouth disease viruses inactivation antigen of purification and Freund's complete adjuvant after one week
Mix etc. quality, fully emulsified after, by 0.3ml/ immune mouse, axillary fossa, groin is injected, altogether immunity 5；After initial immunity
At interval of 3 weeks again immunity once, the quality such as the Asia1 type foot and mouth disease viruses inactivation antigen of purification and freund 's incomplete adjuvant is mixed
Even, in mouse carotid dorsal sc gradation multi-point injection, first 3 days abdominal cavity booster immunizations of cell fusion are once；This little rathole is plucked after 3 days
Ball takes blood, obtains the cell suspension of splenocyte；
Cell fusion：The cell suspension of the myeloma cell and described splenocyte that are in exponential phase is mixed, centrifugation is abandoned
Clearly；Fusion pipe is put in palm and rubs back and forth, so that cell block is loosened, be slowly added to 0.7ml37 DEG C toward in glass tubing in advance with dropper
The 50%PEG1000 of heat, adds in 90s, stands 60s, first slow in 5min after be added dropwise over the serum-frees of 37 DEG C of preheatings soon
DMEM, altogether plus 10ml, after jog mixes, supernatant is abandoned in centrifugation；With HAT culture fluid re-suspended cell, move into 60mlHAT culture fluid immediately
In, gently mix, then proceed in 96 porocyte culture plates by 100 μ l/ holes, be placed in 5%CO2, train in 37 DEG C of constant incubators
Support；
The screening of hybridoma：Cultivate the 6th day and carry out half amount with HAT culture fluid and change liquid, treat cell grow to bottom hole 1/4～
Suction out supernatant when 1/3 and carry out indirect ELISA detection；The cell of P/N >=2.1 is hybridoma；
The colonized culture of hybridoma；
The preparation of monoclonal antibody：Every mouse peritoneal injects 0.5mL1x106～6x106The hybridoma of/mL, treats mice abdomen
Portion slowly swells, and mice spirit is depressed, when not walking about, being at death's door, and cervical dislocation puts to death mice, collects ascites, room temperature,
10000r/min is centrifuged 10min, collects middle bright liquid and is monoclonal antibody；
The monoclonal antibody of HRP labelling purification：Concentration after saturated ammonium sulfate purification is the monoclonal antibody of 5.284mg/ml
0.3mL and 0.1mL peroxidase add in pipe, and gently piping and druming mixes, and 4 DEG C overnight；Add the triethanolamine of 40 μ L, mix
Even, add the sodium borohydride of 50 μ L, mix, after 4 DEG C are placed 30min, add 10 μ L glycine to mix；Afterwards mixed liquor is placed in
Dialyse in bag filter, dialysis solution is the PBS of PH7.2, gained is the ASIA1 type foot and mouth disease specific monoclonal antibody of HRP labelling.
3. solid phase competitive ELISA kit described in claim 2 is it is characterised in that in described cell fusion step, spleens cell number
Ratio with myeloma cell's number is 5:1 to 10:1.
4. usage right requires solid phase competitive ELISA kit described in any one of 1-3 to carry out Asia1 type antibodies against foot-and-mouth disease virus water
The purposes of flat monitoring.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109765365A (en) * | 2019-01-31 | 2019-05-17 | 中国农业科学院兰州兽医研究所 | A kind of detection kit of Asia1 type antibodies against foot-and-mouth disease virus |
| CN109799344A (en) * | 2018-12-07 | 2019-05-24 | 中国农业科学院兰州兽医研究所 | A kind of competitive ELISA antibody assay kit based on Asia I type foot and mouth disease virus sample particle |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101096657A (en) * | 2007-06-14 | 2008-01-02 | 中国农业科学院兰州兽医研究所 | Preparation method of monoclonal antibody against Asial type foot-and-mouth disease virus and usage |
| CN101241138A (en) * | 2007-11-29 | 2008-08-13 | 中国农业科学院兰州兽医研究所 | Asia 1 type foot-and-mouth disease antibody level detection test paper and preparation method |
-
2016
- 2016-08-31 CN CN201610778727.1A patent/CN106483291A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101096657A (en) * | 2007-06-14 | 2008-01-02 | 中国农业科学院兰州兽医研究所 | Preparation method of monoclonal antibody against Asial type foot-and-mouth disease virus and usage |
| CN101241138A (en) * | 2007-11-29 | 2008-08-13 | 中国农业科学院兰州兽医研究所 | Asia 1 type foot-and-mouth disease antibody level detection test paper and preparation method |
Non-Patent Citations (6)
| Title |
|---|
| EMILIANA BROCCHI ET AL.: "Validation of a Solid Phase Competitive ELISA (SPBE) based on the use a single neutralising monoclonal antibody for the measurement of antibodies to FMDV type Asia 1", 《REPORT OF THE SESSION OF THE RESEARCH GROUP OF THE STANDING TECHNICAL COMMITTEE OF THE EUROPEAN COMMISSION FOR THE CONTROL OF FOOT-AND-MOUTH DISEASE (EUFMD)》 * |
| 党双锁: "《医学常用实验技术精编》", 30 June 2004 * |
| 李万等: "抗Asial型口蹄疫病毒单克隆抗体的制备及生物学特性鉴定", 《中国预防兽医学报》 * |
| 李影林: "《临床医学检验手册》", 31 August 1987 * |
| 石正旺: "O型FMD固相竞争ELISA及双抗夹心ELISA方法的建立与应用", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 魏孔福: "Asia-I 型口蹄疫抗体固相竞争ELISA检测方法研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109799344A (en) * | 2018-12-07 | 2019-05-24 | 中国农业科学院兰州兽医研究所 | A kind of competitive ELISA antibody assay kit based on Asia I type foot and mouth disease virus sample particle |
| CN109765365A (en) * | 2019-01-31 | 2019-05-17 | 中国农业科学院兰州兽医研究所 | A kind of detection kit of Asia1 type antibodies against foot-and-mouth disease virus |
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