CN104120109A - ELISA blocking kit for detecting infectious bovine rihinotracheitis virus gB antibody and application of kit - Google Patents
ELISA blocking kit for detecting infectious bovine rihinotracheitis virus gB antibody and application of kit Download PDFInfo
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Abstract
The invention discloses an ELISA blocking kit for detecting an infectious bovine rihinotracheitis virus gB antibody and application of the kit. The kit adopts inactivated purified BHV-1 virion as envelope antigen for detecting the antibodies of the infectious bovine rihinotracheitis viruses in bovine serum according to the blocking ELISA principle. The envelope antigen in a 96-pore plate in the kit is inactivated purified BHV-1 virion which has good antigenicity. The kit comprises a hybridoma cell strain, an ELISA plate strip, blood serum diluent and washing concentrate, stop buffer, substrate developing liquor A, substrate developing liquor B, a positive control group and a negative control group. The kit disclosed by the invention is strong in sensitivity, high in specificity, convenient to apply, easy for standardization, especially suitable for detecting large-scale samples, and can be taken as gold standard for investigating a prevalence state of the infectious bovine rihinotracheitis viruses. Compared with American IDEXXgE blocking kit, the ELISA blocking kit disclosed by the invention has a very high positive coincidence rate and a very high negative coincidence rate, and can be taken as a standard method for diagnosing infectious bovine rihinotracheitis.
Description
Technical field
The invention belongs to biological technical field.Be specifically related to a kind of blocking-up ELISA test kit for infectious bovine rhinotrachetis virus gB antibody test, also relate to a kind of purposes with detecting infectious bovine rhinotrachetis virus gB antibody test blocking-up ELISA test kit.
Background technology
Infectious bovine rhinotrachetis (Infectious Bovine Rhinotracheitis, IBR) be by infectious bovine rhinotrachetis virus (Infectious Bovine Rhinotracheitis viruse, IBRV) be ox 1 type simplexvirus (Bovine herpe svirus-1, BHV-1) a kind of bovine respiratory contagious disease causing, claims again " gangrenosum acne rhinitis ", " red rhinopathy ".
Ox 1 type simplexvirus (BHV-1) is the member of herpetoviridae Alphaherpesvirinae, and it is a kind of distrand DNA virus that has cyst membrane.The virus particle of BHV-1 is ball-shaped, and ripe particle dia approximately has 150～220nm, mainly comprises core, capsid tunicle and cyst membrane three parts.Wherein core is to be mutually entwined by viral DNA and protein, and nucleocapsid is an icosahedron, and its outward appearance is sexangle, three-dimensional symmetrical on space.Its outside surface has 162 capsomeres, is around the cyst membrane that an astragal has lipid.
The clinical symptom of infectious bovine rhinotrachetis is divided into five kinds, respiratory tract type, reproductive tract type, miscarriage type, encephalitis type and ophthalmia type.Respiratory tract type shows as rhinotracheitis, is modal one type of this disease; Reproductive tract type shows as dam vulvovaginitis, claims again contagious ecthyma vulvovaginitis, and therefore male animal balanoposthitis claim contagious ecthyma balanoposthitis; First tire heifer any stage of period of pregnancy is generally shown in by miscarriage type, also can betide multiparity cow; Encephalitis type: easily betide 4～6 the monthly age calf; Ophthalmia type shows as conjunctiva keratitis, and normal and respiratory tract type merges generation.Although IBRV does not demonstrate very high mortality ratio after infecting, after infecting, can cause latent infection and infect throughout one's life, and sick ox can be all with poison for a long time even throughout one's life; when the resistibility of body reduces; activate again, outwards toxin expelling, makes the extremely difficult radical cure of this disease.
This disease found first in nineteen fifty-five, and nearly all country all detects IBR antibody so far.Only in Australia, Denmark, Finland, Norway, Sweden, Switzerland, eradicated this disease at present.China finds this disease in import cows in 20 century 70s.From Zelanian Imported Holstein isolate this disease virus by Zhou Taichong Deng Shenzhen customs the eighties in 20th century, in 1986, by Deng Peilin, 1987, by Wang Zexing etc., from cow, buffalo, ox, be separated to again this virus afterwards, confirm really to exist in IBR China cows.In this laboratory, within 2012, by Fan Qiang etc., utilize the method for latex agglutination antibody test to detect 521 parts of clinical samples that Liao Cong China Inner Mongol, Xinjiang and Jiangsu San Sheng gather, positive rate reaches 34.57%.
Virus neutralization tests (VN) and enzyme linked immunosorbent assay (ELISA) are to detect the ordinary method of antibody, and some blocking-up ELISA methods have been set up in the laboratory in Europe, are mainly used in the epidemiology survey of infectious bovine rhinotrachetis.The present invention has prepared the monoclonal antibody of infectious bovine rhinotrachetis virus, with this monoclonal antibody of horseradish peroxidase (HRP) mark, has set up blocking-up ELISA antibody detection method, for infectious bovine rhinotrachetis provides reliable Data support.
Current many developed countries are all carrying out the control of infectious bovine rhinotrachetis and are eliminating plan, its Basic practice method is to promote the use of gene-deleted vaccine and supporting differential diagnosis kit, by vaccine immunity, reduce sickness rate and infection rate on the one hand, by differential diagnosis, filter out the band poison ox after immunity on the other hand, by being with malicious ox carry out isolated rearing or directly eliminate, progressively make cows be purified.China is as cowboying big country, and the harm of infectious bovine rhinotrachetis is very serious, and way and experience that cattle farm with good conditionsi should Developed Countries, progressively implement, to the control of infectious bovine rhinotrachetis and purification, to increase economic efficiency.
Prior art has latex agglutination and neutralization test etc., although latex agglutination susceptibility easy and simple to handle is very low, false negative rate is very high, and neutralization test complex operation is high to technician's experimental implementation Capability Requirement, is not easy to the detection of extensive sample.And susceptibility of the present invention is strong, specificity is high, easy to use, be easy to stdn, set up a kind of accurately, fast, security is good, the method that can carry out the antibody test of a large amount of examination infectious bovine rhinotrachetis virus, and this test kit and American I DEXX test kit have very high coincidence rate, positive coincidence rate is 96.12%, negative match-rate is 100%, total coincidence rate is 97.57%, almost non-false positive and false negative occur, for animal test sanitary authority provides a set of practical, reliable for effect diagnostic kit product.Following table for the comparison result of the test kit of setting up and American I DEXX test kit be sensitivity test result:
And cross reactivity test-results shows that the method specificity is very high, draw the method except having cross reaction with BHV-5, with the equal no cross reaction of other virus, because the two homology of BHV-1 and BHV-5 is up to more than 82%, so this result is also within expecting, following table is specific test result:
Summary of the invention
The object of the invention is to be to provide a kind of and detect blocking-up ELISA test kit for infectious bovine rhinotrachetis gB antiviral antibody, this test kit susceptibility is strong, specificity is high, easy to use, be easy to stdn, compare with American I DEXX gE blocking-up test kit, have very high positive coincidence rate and negative match-rate, can be used as the standard method of Diagnosis of Cattle infectious bovine rhinotracheitis.
Another object of the present invention is to be to provide the application of a kind of infectious bovine rhinotrachetis virus gB antibody test blocking-up ELISA test kit in detecting infectious bovine rhinotrachetis virus antibody, this application is for detection of having or not the antibody of infectious bovine rhinotrachetis virus to occur in bovine serum, detect tested ox and whether infect infectious bovine rhinotrachetis virus, susceptibility is extremely strong, almost non-false positive and false negative occur, be particularly useful for the detection of extensive sample, can be used as the gold standard of investigation infectious bovine rhinotrachetis popularity.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is:
For an infectious bovine rhinotrachetis virus gB antibody test blocking-up ELISA test kit, test kit adopts the purifying BHV-1 virus particle of deactivation as envelope antigen, detects the antibody of infectious bovine rhinotrachetis virus in bovine serum according to blocking-up ELISA principle.Envelope antigen in test kit in 96 orifice plates is the BHV-1 virus particle of purifying, and it has good antigenicity.The enzyme labelling thing of the anti-infectious bovine rhinotrachetis virus gB protein monoclonal antibody that the hybridoma cell line that it is CCTCC NO:C201345 that described test kit comprises by preserving number is secreted.Applicant delivers to the center preservation of Chinese Typical Representative culture collection by this hybridoma cell strain, address on April 3rd, 2013: Wuhan, China Wuhan University, deposit number CCTCC NO:C201345, Classification And Nomenclature: hybridoma cell strain 1H3.
For an infectious bovine rhinotrachetis virus gB antibody test blocking-up ELISA test kit, it is characterized in that: hybridoma cell strain, elisa plate bar, serum dilution and concentrated cleaning solution, stop buffer, substrate nitrite ion A, substrate nitrite ion B, positive control, negative control, monoclonal antibody linked with peroxidase: the mouse-anti infectious bovine rhinotrachetis virus gB protein monoclonal antibody of described horseradish peroxidase-labeled.
Described elisa plate bar: each test kit is equipped with 2 or 5 ELISA microwell plates, every elisa plate bar is the ELISA microwell plate of the envelope antigen of energy realizing self disassembling, the BHV-1 virus particle that envelope antigen is purifying, specification is 8 hole * 12;
Described serum dilution: serum dilution is instant;
Described concentrated cleaning solution: washings is 10 times and concentrates, dilution before using;
Described substrate nitrite ion A:0.006%H
2o
2damping fluid;
Described substrate nitrite ion B:TMB nitrite ion;
Described stop buffer: 0.025% hydrofluoric acid (HF);
Positive control; Negative control.
A mouse-anti infectious bovine rhinotrachetis virus gB protein monoclonal antibody for horseradish peroxidase-labeled, its preparation process is:
One, set up clone:
1. antigen preparation:
The wild poison of MDBK cell inoculation BHV-1, treats that crack releasing appears in 80% cell, and-78--82 ℃ of freeze thawing three times, at 4 ℃, the centrifugal 40min of 6000rpm, gets supernatant, at 4 ℃, the centrifugal 2h of 25000rpm, the resuspended precipitation of TEN, utilizes sucrose gradient centrifugation to obtain the virus particle of purifying.
2. antigen immune:
After deactivation purified virus particle, immunity BALB/C mice in 6 week age, the antigen 1 of fundamental immunity subcutaneous injection first 00 μ g/ only, is used complete Freund's adjuvant; Every 2 weeks, carry out primary immune response, totally three times, subcutaneous injection antigen 1 00 μ g//time, use incomplete Freund's adjuvant; Merge first 3 days abdominal injection booster immunizations, 200 μ g/ only, do not add adjuvant.
3. the preparation of hybridoma:
A) preparation of feeder cell:
1 of blank mouse, eye socket bloodletting, collects negative serum, 75%(massfraction) alcohol-pickled 5min, mouse is fixed, the aseptic spleen of getting, add 3ml basic 1640 and grind, add basic 1640 to 10ml, standing 2min, suct layer standby with 50ml centrifuge tube, add again basic 1640 to 10ml, repeat the centrifugal 10min of twice, 1000rpm and remove supernatant, 100mlHAT substratum is resuspended, and 37 ℃ standby.
B) preparation of immune spleen cell:
Reinforced immunological mouse, positive serum, 75%(massfraction are collected in eye socket bloodletting) alcohol-pickled 5min, mouse is fixed, and the aseptic spleen of getting adds 3ml basic 1640 and grinds, and adds basic 1640 to 10ml, and standing 2min, sucts layer standby with 50ml centrifuge tube, then adds base
C) myeloma cell's preparation:
Draw neck to put to death, 75%(massfraction) alcohol-pickled 5min, mouse is fixed, the aseptic tumour of getting, put homogenizer, adding 5ml basic 1640 grinds, add basic 1640 to 10ml, standing 2min, suct layer standby with 50ml centrifuge tube, add again basic 1640 to 10ml, repeat twice, 1000rpm is centrifugal, and 10min removes supernatant, basic 1640 resuspended myeloma cells, cumulative volume reaches 20ml, another 50ml centrifuge tube adds 20ml lymphocyte separation medium, myeloma cell's suspension is added on parting liquid along tube wall lightly, the centrifugal 10min of 1000rpm, attract the position the white cellular layer (can suitably draw upper strata substratum) in interface densification, add appropriate basic 1640, the centrifugal 10min of 1000rpm, abandon supernatant, add 10ml basic 1640 resuspended, count rear 4 ℃ standby.
D) cytogamy and HAT select hybridoma:
Myeloma cell (1-2*10
7) and immunocyte (1*10
8) in 50ml centrifuge tube, mix, the centrifugal 10min of 1000rpm, turned letter supernatant (sterilizing filter paper blots to place of settling), rap the pipe end, make cell precipitation loosening slightly, in 37 ℃ of water-baths, in 1mi n, slowly splash into the 50%PEG0.8ml of pre-temperature to 37 degree, limit edged stirs with suction pipe, standing 1m in after continuation stirring 30s, slowly add the basic 164040ml(of 37 ℃ of pre-temperature to pack in advance centrifuge tube into), concrete grammar following (completing in 37 ℃ of water-baths): 1min dropwise splashes into 1ml, 2min adds 1ml, 3-4min adds 3ml, 5min adds 5ml, each added-time need slowly add, and constantly stir gently, finally slowly add basis 1640, supply 40ml, total time is 10min, 1000rp m is centrifugal, and 10min removes supernatant, resuspended have the HAT substratum of feeder cell resuspended, divide and plant in 4 96 orifice plates, 37 ℃, 5%CO2 incubator is cultivated.
Hybridoma merged after two weeks, carried out the screening of hybridoma according to routine immunization zymotechnic, filtered out the fused cell that can secrete for ox infectivity nose tracheae antiviral antibody.
4. the cloning of hybridoma and frozen:
A) clone of hybridoma:
(1) prepare feeder layer: according to 2.2.4.2, prepare feeder cell, the resuspended splenocyte of complete 1640 substratum of 18-20ml, inoculation 96 porocyte culture plates.
(2) suction pipe piping and druming hybridoma is suspended in substratum it, and sampling, counting, adjust concentration to 50,20,10/ml, adopts limiting dilution assay, and concrete steps are:
1. after complete 1640 substratum dilution countings cell to make its concentration be 10
3cells/ml, this cell suspension is considered as A liquid;
2. get A liquid 0.2ml in complete 640 substratum of 3.8ml, its concentration is 50cells/ml, and this cell suspension is considered as B liquid;
3. get B liquid 1.6ml in complete 640 substratum of 2.4ml, its concentration is 20cells/ml, and this cell suspension is considered as C liquid;
4. get C liquid 2ml in complete 640 substratum of 2ml, its concentration is 10cells/ml, and this cell suspension is considered as D liquid;
(3) respectively by above-mentioned B, C, D hybridoma suspension inoculation in containing in the culture plate of feeder cell, every hole 100 μ l, make every hole be respectively 5,2,1 cells, each extent of dilution is done two row.
(4) be placed in 37 ℃, the cultivation of 5%CO2 incubator, within 3 days, later half amount is changed liquid or fluid infusion, and after 7 days, cell conditioned medium is tired.
(5) choose OD
630nmhigher time cloning again, generally carries out 3 time clonings, be mono-clonal and grow, and cell conditioned medium is tired higher to hybridoma.
B) hybridoma is frozen:
Cells frozen storing liquid: containing 10%(volume ratio) foetal calf serum of dimethyl sulfoxide (DMSO).
By the cell strain of enlarged culturing in 24 orifice plates, at cell log during vegetative period, collecting cell suspension, the centrifugal 10min of 1000rpm, discards cell conditioned medium, resuspended with appropriate cells frozen storing liquid, after mixing, be sub-packed in cell cryopreservation tube, 1ml/ pipe, successively 4 ℃ of 30min ,-20 ℃ of 2h ,-80 ℃ spend the night, move into next day in liquid nitrogen container, carry out frozen.
Two, the preparation of monoclonal antibody:
In the present invention, the scheme of a large amount of manufacture order clonal antibodies is mouse ascites method.
Method is: by five days in advance abdominal injection Freund's incomplete adjuvant 500 μ l/ of mouse only, then the supernatant of positive cell is collected in centrifuge tube, cell lays from hole wall blowing up with 1640 basic mediums, suck in 15ml centrifuge tube, the centrifugal 10min of 1000rpm, and then resuspended with 1640 basal liquids, wash twice, finally use 1640 appropriate basal liquids resuspended, mouse peritoneal is only injected 500 μ l/.After 9 to 10 days, it is large that mouse web portion obviously becomes, and alcohol swab is sterilized at mouse web portion, with 10ml syringe needle, inserts mouse web portion, collects the ascites automatically flowing out from syringe needle with 15ml centrifuge tube simultaneously.The centrifugal 10min of 1500rpm, removes cell, and supernatant is collected in another 15ml centrifuge tube, frozen in-20 ℃ of refrigerators.
Three, monoclonal antibody purifying and horseradish peroxidase mark:
1. the purifying of monoclonal antibody:
The purification schemes of the ascites monoclonal antibody of above-mentioned acquisition is a sad ammonium sulfate precipitation method.
Step is: by centrifugal 5 minutes of the ascites 12000rpm collecting, get supernatant x ml; The acetate buffer solution that adds 4x ml; The above-mentioned ascites of every x ml adds sad 33ul, and limit edged stirs (slowly); Add rear continuation and stir 30min, the then centrifugal 30min of 12000rpm at 4 ℃; Supernatant filters with filter paper, and the supernatant of collection is adjusted PH7.4; ≤ 45%SAS precipitation once, after stirring 30min, spend the night by 4 ℃ of precipitations; At 4 ℃, the centrifugal 30min of 12000rpm, removes supernatant, uses 10mM Tris(pH9.0) resuspended precipitation, then use 10mM Tris(pH9.0) dialyse 3 days, change dialyzate every day one time; Sucking-off packing, the every pipe of 1ml ,-20 ℃ of preservations.
The preparation scheme of the monoclonal antibody horseradish peroxidase mark after purifying is improvement sodium periodate method.
Step is: get 5mg HRP and be dissolved in 0.5mL distilled water; Add 0.5mL0.06mol/L NaIO4,4 ℃ are gently stirred 30min, and solution is yellow-green colour; Add 0.5mL0.16mol/L ethylene glycol, room temperature lucifuge is gently stirred effect 30min, stops oxidizing reaction; Add 5mg antibody (IgG), pack dialysis tubing into, set to 0 .05mol/L, in pH9.6 carbonate buffer solution 1000mL, 4 ℃ of dialysed overnight, change 3 times; Take out liquid in dialysis tubing, add 5mg/mL NaHB40.2mL4 ℃ 2h or spend the night; Add equal-volume saturated ammonium sulphate solution precipitation binding substances, put after 4 ℃ of 30min, the centrifugal 15min of 3000rpm, abandons supernatant; Throw out is dissolved in to PBS(0.02M pH7.4) in, pack dialysis tubing into, and with this damping fluid dialysis equilibrium 6-12h, change liquid 3 times; After dialysis, sucking liquid is sub-packed in-20 ℃ of preservations.
The hybridoma cell line of the anti-infectious bovine rhinotrachetis virus monoclonal antibody of preparation is carried out to preservation, applicant delivers to the center preservation of Chinese Typical Representative culture collection on April 1st, 2013 by this hybridoma cell strain, address: Wuhan, China Wuhan University, deposit number CCTCC NO:C201345, Classification And Nomenclature: hybridoma cell strain 1H3.
Four, the evaluation of monoclonal antibody antigen epi-position
1. immunoprecipitation
(1) get 200 μ l purifying BHV-1 virus particle, add 133 μ l Extraction buffer(to contain PMSF and Aptot inin), fully mix dissolving.
By above-mentioned solution more than-80 ℃ of frozen 4h.
(3) take out and thaw rapidly, adding 25%Triton-1003.3 μ l, resuspended after frozen water placement 30min.
(4) add the blank mice serum of approximately 1 μ g and 20 μ l Protein A+G Agarose, jolt 4h at 4 ℃.
(5) the centrifugal 5min of 2500rpm, gets supernatant totally 300 μ l, packing 150 μ l/ pipes.
(6) two pipes add respectively approximately 2 μ g monoclonal antibody 1H3, jolt at 4 ℃ and spend the night.
(7) add 40 μ l Protein A+G Agarose, jolt 3h at 4 ℃.
(8) the centrifugal 5min of 2500rpm, abandons supernatant.
(9) PBS washes precipitation 5 times.
(10) the resuspended precipitation of 40 μ l1 * SDS-PAGE sample buffer, instantaneous high speed centrifugation.
(11) process 5min at 95 ℃.
(12) carry out SDS-PAGE and Western blot.
2. Mass Spectrometric Identification
By in SDS-PAGE glue figure (accompanying drawing 2A) with Western blot(accompanying drawing 2B) band on corresponding albumin glue cuts, send words and deeds biology in Wuhan to carry out Mass Spectrometric Identification, qualification result and Western blot result all show that monoclonal antibody 1H3 is for infectious bovine rhinotrachetis virus gB albumen.
A kind of application at detection infectious bovine rhinotrachetis virus antibody for infectious bovine rhinotrachetis virus gB antibody test blocking-up ELISA test kit.Its application process is:
1. the detection principle of test kit of the present invention:
Adopt blocked method, the purifying BHV-1 virus particle of deactivation is coated in microwell plate, then with 1%BSA, enzyme plate is sealed, add testing sample and standard positive control, negative control, 37 ℃ add monoclonal antibody linked with peroxidase after hatching, after continuation is hatched at 37 ℃, add the colour developing of horseradish peroxidase substrate, stop buffer termination reaction, by microplate reader under 630nm wavelength, measure each hole absorbance, in the size of OD value (depth of color after color development stopping reaction) and sample to be tested, the content of infectious bovine rhinotrachetis virus antibody is inversely proportional to.
2. the composition of test kit of the present invention:
A) the best preparation method of enzyme plate:
With the carbonate buffer solution of pH9.60.05M as coated damping fluid, the purifying BHV-1 virus particle of above-mentioned preparation is diluted to 4 μ g/ml, by 100 μ l/ holes, add in microwell plate coated spending the night, next day, discard coating buffer, the confining liquid that adds 1%BSA by 200ul/ hole, 37 ℃ standing 2 hours, after washing dries, pack packing bag into, add siccative, vacuum is preserved.
B) configuration of work reagent:
Washings (pH7.4,0.15M PBS): KH
2pO
40.2g, Na
2hPO
4-12H
2o2.9g, NaCl8.0g, KCl0.2g, Tween-20 (0.1%) 1ml, adding distil water, to l000ml, is condensed into 10 times as storage liquid.
Serum dilution: bovine serum albumin 0.1g, adds lavation buffer solution to 100ml.
Substrate buffer solution: pH5.0 phosphoric acid salt citrate buffer solution, 0.2M Na
2hPO
425.7ml, 0.1M citric acid 24.3ml, adding distil water 50ml.
Substrate nitrite ion A:0.006%H
2o
2damping fluid;
Substrate nitrite ion B: get Na
2hPO
412H
2o14.2g, citric acid 10.5g, uses ddH
2o is settled to 500m and is made into 0.1mL phosphoric acid salt citrate buffer solution, and pH5.0 is that 20mg/L adds benzidine by final concentration;
Stop buffer: 0.025% hydrofluoric acid (HF); HF (40%) 625 μ L, uses ddH
2o is settled to 100mL.
C) preparation of standard BHV-1 positive serum and standard BHV-1 negative serum:
In ELISA testing process, between different operator and different detection batch, can have certain error, operate miss can cause detecting the error of sample OD value.The present invention has developed standard BHV-1 antibody positive control serum and standard BHV-1 negative antibody control serum, for detection of the optimization of condition and the judgement of detected result.
The preparation of standard positive serum:
Select healthy adult ox, after being infectious bovine rhinotrachetis negative antibody after testing before immunity, quarantine 7.By after the deactivation of infectious bovine rhinotrachetis virus suspension, add after the adjuvant emulsion of equivalent as immunogen.By musculi colli injecting pathway, divide 3 immunoprophylaxis former, every minor tick 21 days, after last immunity, blood sampling on the 7th～10 detects.Blood sampling separation of serum, carry out ELISA test with serum to be checked and detect antibody.Get standby serum and with sample diluting liquid, suitably dilute after 30 minutes through 56 ℃ of deactivations, by 0.01% of serum amount, add Thiomersalate, 0.45 μ m membrane filtration degerming.Aseptic subpackaged, every pipe 1ml, 4 ℃ of storages.
The preparation of standard female serum:
Selecting healthy adult ox, is the negative negative antibody of infectious bovine rhinotrachetis after testing, quarantines after 7 days, takes neck arteries blood sampling mode, and aseptic collection blood is centrifugal and separation of serum is standby (2～8 ℃ of preservations, validity period is 12 months).Get standby serum and with sample diluting liquid, suitably dilute after 30 minutes through 56 ℃ of deactivations, by 0.01% of serum amount, add Thiomersalate, 0.45 μ m membrane filtration degerming, aseptic subpackaged, every pipe 1ml, 4 ℃ of storages.
D) detect the establishment of the blocking-up ELISA test kit of infectious bovine rhinotrachetis:
The enzyme linked immunological kit of setting up infectious bovine rhinotrachetis, comprises following component:
96 hole enzyme plates; The monoclonal antibody of horseradish peroxidase-labeled; Standard positive control; Standard negative control; Concentrated cleaning solution;
Serum dilution; Stop buffer.
3. the detection method of test kit of the present invention
A) with the test kit of above-mentioned preparation, detect:
1) test serum, positive control and negative control are diluted in proportion with antibody diluent, every hole 100 μ l, add enzyme plate, hatch 1 hour for 37 ℃;
2) washings cleans 3-5 time;
3) every hole adds the monoclonal antibody linked with peroxidase 100 μ l after dilution, hatches 1h for 37 ℃;
4) washings cleans 3-5 time;
5) every hole adds substrate A, each 50 μ l of B, hatches 15min for 37 ℃;
6) every hole adds stop buffer 50 μ l;
7) microplate reader detects 630nm absorbance.
B) Analysis of test results:
1. in surveying, to be at least 0.4 be NC-PC≤0.4 for the OD value of negative control sera (NC) and the OD value difference value of positive control serum (PC), and detection is considered to effective.
S=sample well OD630 value, N=negative control hole OD630 value.
If S/N ratio≤0.6, sample is judged to be BHV-1 antibody positive.
If S/N ratio≤0.7 but >0.6, sample is resurveyed.
If S/N ratio >0.7, sample is judged to be BHV-1 negative antibody.
2. while detecting, the positive and negative control are used multiple hole, and finally using OD value is the mean value of two.
The present invention compared with prior art, has the following advantages and effect:
Accompanying drawing explanation
Fig. 1 is a kind of whole preparation flow of infectious bovine rhinotrachetis virus gB antibody test blocking-up ELISA test kit.
Fig. 2 A:SDS-PAGE and B:Western blot
Embodiment
Embodiment 1:
A kind of for infectious bovine rhinotrachetis virus gB antibody test blocking-up ELISA test kit, comprise hybridoma cell strain, hybridoma cell strain applicant delivers to the center preservation of Chinese Typical Representative culture collection on April 3rd, 2013, address: Wuhan, China Wuhan University, deposit number CCTCC NO:C201345, Classification And Nomenclature: hybridoma cell strain 1H3.
For an infectious bovine rhinotrachetis virus gB antibody test blocking-up ELISA test kit, it is characterized in that: hybridoma cell strain, elisa plate bar, serum dilution and concentrated cleaning solution, stop buffer, substrate nitrite ion A, substrate nitrite ion B, positive control, negative control, monoclonal antibody linked with peroxidase: the mouse-anti infectious bovine rhinotrachetis virus gB protein monoclonal antibody of described horseradish peroxidase-labeled.
Described elisa plate bar: each test kit is equipped with 2 or 5 ELISA microwell plates, every elisa plate bar is the ELISA microwell plate of the envelope antigen of energy realizing self disassembling, the BHV-1 virus particle that envelope antigen is purifying, specification is 8 hole * 12;
Described serum dilution: serum dilution is instant;
Described concentrated cleaning solution: washings is 10 times and concentrates, dilution before using;
Described substrate nitrite ion A:0.006%H
2o
2damping fluid;
Described substrate nitrite ion B:TMB nitrite ion;
Described stop buffer: 0.025% hydrofluoric acid (HF);
Described monoclonal antibody linked with peroxidase: the mouse-anti infectious bovine rhinotrachetis virus gB protein monoclonal antibody of described horseradish peroxidase-labeled; Positive control; Negative control.
A mouse-anti infectious bovine rhinotrachetis virus gB protein monoclonal antibody for horseradish peroxidase-labeled, its preparation process is:
One, set up clone:
1. antigen preparation:
The wild poison of MDBK cell inoculation BHV-1, treats that crack releasing appears in 80% cell, and-80 ℃ of freeze thawing three times, at 4 ℃, the centrifugal 40min of 6000rpm, gets supernatant, at 4 ℃, the centrifugal 2h of 25000rpm, the resuspended precipitation of TEN, utilizes sucrose gradient centrifugation to obtain the virus particle of purifying.
2. antigen immune:
After deactivation purified virus particle, ordinary method immunity BALB/C mice in 6 week age, the antigen 1 of fundamental immunity subcutaneous injection first 00 μ g/ only, is used complete Freund's adjuvant; Every 2 weeks, carry out primary immune response, totally three times, subcutaneous injection antigen 1 00 μ g//time, use incomplete Freund's adjuvant; Merge first 3 days abdominal injection booster immunizations, 200 μ g/ only, do not add adjuvant.
3. the preparation of hybridoma:
A) preparation of feeder cell:
1 of blank mouse, eye socket bloodletting, collects negative serum, 75%(massfraction) alcohol-pickled 5min, mouse is fixed, the aseptic spleen of getting, add 3ml basic 1640 and grind, add basic 1640 to 10ml, standing 2min, suct layer standby with 50ml centrifuge tube, add again basic 1640 to 10ml, repeat the centrifugal 10min of twice, 1000rpm and remove supernatant, 100mlHAT substratum is resuspended, and 37 ℃ standby.
B) preparation of immune spleen cell:
Reinforced immunological mouse, positive serum, 75%(massfraction are collected in eye socket bloodletting) alcohol-pickled 5min, mouse is fixed, and the aseptic spleen of getting adds 3ml basic 1640 and grinds, and adds basic 1640 to 10ml, and standing 2min, sucts layer standby with 50ml centrifuge tube, then adds base
C) myeloma cell's preparation:
Draw neck to put to death, 75%(massfraction) alcohol-pickled 5min, mouse is fixed, the aseptic tumour of getting, put homogenizer, adding 5ml basic 1640 grinds, add basic 1640 to 10ml, standing 2min, suct layer standby with 50ml centrifuge tube, add again basic 1640 to 10ml, repeat twice, 1000rpm is centrifugal, and 10min removes supernatant, basic 1640 resuspended myeloma cells, cumulative volume reaches 20ml, another 50ml centrifuge tube adds 20ml lymphocyte separation medium, myeloma cell's suspension is added on parting liquid along tube wall lightly, the centrifugal 10min of 1000rpm, attract the position the white cellular layer (can suitably draw upper strata substratum) in interface densification, add appropriate basic 1640, the centrifugal 10min of 1000rpm, abandon supernatant, add 10ml basic 1640 resuspended, count rear 4 ℃ standby.
D) cytogamy and HAT select hybridoma:
Myeloma cell (1-2*10
7) and immunocyte (1*10
8) in 50ml centrifuge tube, mix, the centrifugal 10min of 1000rpm, turned letter supernatant (sterilizing filter paper blots to place of settling), rap the pipe end, make cell precipitation loosening slightly, in 37 ℃ of water-baths, in 1mi n, slowly splash into the 50%PEG0.8ml of pre-temperature to 37 degree, limit edged stirs with suction pipe, standing 1m in after continuation stirring 30s, slowly add the basic 164040ml(of 37 ℃ of pre-temperature to pack in advance centrifuge tube into), concrete grammar following (completing in 37 ℃ of water-baths): 1min dropwise splashes into 1ml, 2min adds 1ml, 3-4min adds 3ml, 5min adds 5ml, each added-time need slowly add, and constantly stir gently, finally slowly add basis 1640, supply 40ml, total time is 10min, 1000rp m is centrifugal, and 10min removes supernatant, resuspended have the HAT substratum of feeder cell resuspended, divide and plant in 4 96 orifice plates, 37 ℃, 5%CO2 incubator is cultivated.
Hybridoma merged after two weeks, carried out the screening of hybridoma according to routine immunization zymotechnic, filtered out secretion for the fused cell of infectious bovine rhinotrachetis virus antibody.
5. the cloning of hybridoma and frozen:
A) clone of hybridoma: cloning scheme is limiting dilution assay, carries out according to conventional hybridization oncocyte cloning process, and with method, clone carries out three times.
B) hybridoma is frozen: cells frozen storing liquid: containing 10%(volume ratio) foetal calf serum of dimethyl sulfoxide (DMSO).
By the cell strain of enlarged culturing in 24 orifice plates, at cell log during vegetative period, collecting cell suspension, the centrifugal 10min of 1000rpm, discards cell conditioned medium, resuspended with appropriate cells frozen storing liquid, after mixing, be sub-packed in cell cryopreservation tube, 1ml/ pipe, successively 4 ℃ of 30min ,-20 ℃ of 2h ,-80 ℃ spend the night, move into next day in liquid nitrogen container, carry out frozen.
The hybridoma cell line of the anti-infectious bovine rhinotrachetis virus monoclonal antibody of preparation is preserved in to Chinese Typical Representative culture collection center, preservation day: on April 3rd, 2013, address: Wuhan, China Wuhan University, deposit number CCTCC NO:C201345, Classification And Nomenclature: hybridoma cell strain 1H3.
Two, monoclonal antibody preparation:
In the present invention, the scheme of a large amount of manufacture order clonal antibodies is mouse ascites method.
Concrete grammar is: by five days in advance abdominal injection Freund's incomplete adjuvant 500 μ l/ of mouse only, then the supernatant of positive cell is collected in centrifuge tube, cell lays from hole wall blowing up with 1640 basic mediums, suck in 15ml centrifuge tube, the centrifugal 10min of 1000rpm, and then resuspended with 1640 basal liquids, wash twice, finally use 1640 appropriate basal liquids resuspended, mouse peritoneal is only injected 500 μ l/.After 9 to 10 days, it is large that mouse web portion obviously becomes, and alcohol swab is sterilized at mouse web portion, with 10ml syringe needle, inserts mouse web portion, collects the ascites automatically flowing out from syringe needle with 15ml centrifuge tube simultaneously.The centrifugal 10min of 1500rpm, removes cell, and supernatant is collected in another 15ml centrifuge tube, frozen in-20 ℃ of refrigerators.
Three, monoclonal antibody purifying and horseradish peroxidase mark:
1. the purifying of monoclonal antibody:
The purification schemes of the ascites monoclonal antibody of above-mentioned acquisition is sad-ammonium sulfate precipitation method.
Concrete steps are: by centrifugal 5 minutes of the ascites 12000rpm collecting, get supernatant x ml; The acetate buffer solution that adds 4x ml; The above-mentioned ascites of every x ml adds sad 33ul, and limit edged stirs (slowly); Add rear continuation and stir 30min, the then centrifugal 30min of 12000rpm at 4 ℃; Supernatant filters with filter paper, and the supernatant of collection is adjusted PH7.4; ≤ 45%SAS precipitation once, after stirring 30min, spend the night by 4 ℃ of precipitations; At 4 ℃, the centrifugal 30min of 12000rpm, removes supernatant, uses 10mM Tris(pH9.0) resuspended precipitation, then use 10mM Tris(pH9.0) dialyse 3 days, change dialyzate every day one time; Sucking-off packing, the every pipe of 1ml ,-20 ℃ of preservations.
The preparation scheme of the monoclonal antibody horseradish peroxidase mark after purifying is improvement sodium periodate method.
Concrete steps are: get 5mg HRP and be dissolved in 0.5mL distilled water; Add 0.5mL0.06mol/L NaIO4,4 ℃ are gently stirred 30min, and solution is yellow-green colour; Add 0.5mL0.16mol/L ethylene glycol, room temperature lucifuge is gently stirred effect 30min, stops oxidizing reaction; Add 5mg antibody (IgG), pack dialysis tubing into, set to 0 .05mol/L, in pH9.6 carbonate buffer solution 1000mL, 4 ℃ of dialysed overnight, change 3 times; Take out liquid in dialysis tubing, add 5mg/mL NaHB40.2mL4 ℃ 2h or spend the night; Add equal-volume saturated ammonium sulphate solution precipitation binding substances, put after 4 ℃ of 30min, the centrifugal 15min of 3000rpm, abandons supernatant; Throw out is dissolved in to PBS(0.02M pH7.4) in, pack dialysis tubing into, and with this damping fluid dialysis equilibrium 6-12h, change liquid 3 times; After dialysis, sucking liquid is sub-packed in-20 ℃ of preservations.
Four, the evaluation of monoclonal antibody antigen epi-position
1. immunoprecipitation
(1) get 200 μ l purifying BHV-1 virus particle, add 133 μ l Extraction buffer(to contain PMSF and Aptot inin), fully mix dissolving.
By above-mentioned solution more than-80 ℃ of frozen 4h.
(3) take out and thaw rapidly, adding 25%Triton-1003.3 μ l, resuspended after frozen water placement 30min.
(4) add the blank mice serum of approximately 1 μ g and 20 μ l Protein A+G Agarose, jolt 4h at 4 ℃.
(5) the centrifugal 5min of 2500rpm, gets supernatant totally 300 μ l, packing 150 μ l/ pipes.
(6) two pipes add respectively approximately 2 μ g monoclonal antibody 1H3, jolt at 4 ℃ and spend the night.
(7) add 40 μ l Protein A+G Agarose, jolt 3h at 4 ℃.
(8) the centrifugal 5min of 2500rpm, abandons supernatant.
(9) PBS washes precipitation 5 times.
(10) the resuspended precipitation of 40 μ l1 * SDS-PAGE sample buffer, instantaneous high speed centrifugation.
(11) process 5min at 95 ℃.
(12) carry out SDS-PAGE and Western blot.
2. Mass Spectrometric Identification
By in SDS-PAGE glue figure (accompanying drawing 2A) with Western blot(accompanying drawing 2B) band on corresponding albumin glue cuts, send words and deeds biology in Wuhan to carry out Mass Spectrometric Identification, qualification result and Western blot result all show that monoclonal antibody 1H3 is for infectious bovine rhinotrachetis virus gB albumen.
Embodiment 2:
A kind of application at detection infectious bovine rhinotrachetis virus antibody for infectious bovine rhinotrachetis virus gB antibody test blocking-up ELISA test kit.Its application process is:
1. the detection principle of test kit of the present invention:
Adopt blocked method, the BHV-1 virus particle of the purifying of deactivation is coated in microwell plate, then with 1%BSA, enzyme plate is sealed, add testing sample and standard positive control, negative control, 37 ℃ add monoclonal antibody linked with peroxidase after hatching, after continuation is hatched at 37 ℃, add the colour developing of horseradish peroxidase substrate, stop buffer termination reaction, by microplate reader under 630nm wavelength, measure each hole absorbance, in the size of OD value (depth of color after color development stopping reaction) and sample to be tested, the content of infectious bovine rhinotrachetis virus antibody is inversely proportional to.
2. the composition of test kit of the present invention:
A) the best preparation method of enzyme plate:
With the carbonate buffer solution of pH9.60.05M as coated damping fluid, the purifying BHV-1 virus particle of above-mentioned preparation is diluted to 4 μ g/ml, by 100 μ l/ holes, add in microwell plate coated spending the night, next day, discard coating buffer, the confining liquid that adds 1%BSA by 200ul/ hole, 37 ℃ standing 2 hours, after washing dries, pack packing bag into, add siccative, vacuum is preserved.
B) configuration of work reagent:
Washings (pH7.4,0.15M PBS): KH
2pO
40.2g, Na
2hPO
4-12H
2o2.9g, NaCl8.0g, KCl0.2g, Tween-20 (0.1%) 1ml, adding distil water, to l000ml, is condensed into 10 times as storage liquid.
Serum dilution: bovine serum albumin 0.1g, adds lavation buffer solution to 100ml.
Substrate buffer solution: pH5.0 phosphoric acid salt citrate buffer solution, 0.2M Na
2hPO
425.7ml, 0.1M citric acid 24.3ml, adding distil water 50ml.
Substrate nitrite ion A:0.006%H
2o
2damping fluid;
Substrate nitrite ion B: get Na
2hPO
412H
2o14.2g, citric acid 10.5g, uses ddH
2o is settled to 500m and is made into 0.1mL phosphoric acid salt citrate buffer solution, and pH5.0 is that 20mg/L adds benzidine by final concentration;
Stop buffer: 0.025% hydrofluoric acid (HF); HF (40%) 625 μ L, uses ddH
2o is settled to 100mL.
C) preparation of standard BHV-1 positive serum and standard BHV-1 negative serum:
In ELISA testing process, between different operator and different detection batch, can have certain error, operate miss can cause detecting the error of sample OD value.The present invention has developed standard BHV-1 antibody positive control serum and standard BHV-1 negative antibody control serum, for detection of the optimization of condition and the judgement of detected result.
The preparation of standard positive serum:
Select healthy adult ox, after being infectious bovine rhinotrachetis negative antibody after testing before immunity, quarantine 7.By after the deactivation of infectious bovine rhinotrachetis virus suspension, add after the adjuvant emulsion of equivalent as immunogen.By musculi colli injecting pathway, divide 3 immunoprophylaxis former, every minor tick 21 days, after last immunity, blood sampling on the 7th～10 detects.Blood sampling separation of serum, carry out ELISA test with serum to be checked and detect antibody.Get standby serum and with sample diluting liquid, suitably dilute after 30 minutes through 56 ℃ of deactivations, by 0.01% of serum amount, add Thiomersalate, 0.45 μ m membrane filtration degerming.Aseptic subpackaged, every pipe 1ml, 4 ℃ of storages.
The preparation of standard female serum:
Selecting healthy adult ox, is the negative negative antibody of infectious bovine rhinotrachetis after testing, quarantines after 7 days, takes neck arteries blood sampling mode, and aseptic collection blood is centrifugal and separation of serum is standby (2～8 ℃ of preservations, validity period is 12 months).Get standby serum and with sample diluting liquid, suitably dilute after 30 minutes through 56 ℃ of deactivations, by 0.01% of serum amount, add Thiomersalate, 0.45 μ m membrane filtration degerming, aseptic subpackaged, every pipe 1ml, 4 ℃ of storages.
D) detect the establishment of the blocking-up ELISA test kit of infectious bovine rhinotrachetis
The enzyme linked immunological kit of setting up infectious bovine rhinotrachetis, comprises following component:
96 hole enzyme plates; The monoclonal antibody of horseradish peroxidase-labeled; Standard positive control; Standard negative control; Concentrated cleaning solution;
Serum dilution; Stop buffer.
3. the detection method of test kit of the present invention
A) with the test kit of above-mentioned preparation, detect:
1) test serum, positive control and negative control are diluted in proportion with antibody diluent, every hole 100 μ l, add enzyme plate, hatch 1 hour for 37 ℃;
2) washings cleans 3-5 time;
3) every hole adds the monoclonal antibody linked with peroxidase 100 μ l after dilution, hatches 1h for 37 ℃;
4) washings cleans 3-5 time;
5) every hole adds substrate A, each 50 μ l of B, hatches 15min for 37 ℃;
6) every hole adds stop buffer 50 μ l;
7) microplate reader detects 630nm absorbance.
B) Analysis of test results:
1. in surveying, to be at least 0.4 be NC-PC≤0.4 for the OD value of negative control sera (NC) and the OD value difference value of positive control serum (PC), and detection is considered to effective.
S=sample well OD630 value, N=negative control hole OD630 value.
If S/N ratio≤0.6, sample is judged to be BHV-1 antibody positive.
If S/N ratio≤0.7 but >0.6, sample is resurveyed.
If S/N ratio >0.7, sample is judged to be BHV-1 negative antibody.
2. while detecting, the positive and negative control are used multiple hole, and finally using OD value is the mean value of two.
In sum, content of the present invention is not limited in the above-described embodiment, and the knowledgeable people in same area can propose easily other embodiment within technical director's thought of the present invention, but this embodiment comprises within the scope of the present invention.
Claims (4)
1. a hybridoma for infectious bovine rhinotrachetis virus monoclonal antibody, is characterized in that: hybridoma can be secreted the antibody for BHV-1 gB albumen, and hybridoma cell strain is 1H3, CCTCC NO:C201345.
2. a blocking-up ELISA test kit that detects infectious bovine rhinotrachetis virus antibody, is characterized in that: hybridoma cell strain, elisa plate bar, serum dilution and concentrated cleaning solution, stop buffer, substrate nitrite ion A, substrate nitrite ion B, positive control, negative control, monoclonal antibody linked with peroxidase: the monoclonal antibody of the mouse-anti infectious bovine rhinotrachetis virus gB albumen of described horseradish peroxidase-labeled.
3. ELISA test kit is blocked in a kind of infectious bovine rhinotrachetis virus gB antibody test according to claim 2, it is characterized in that: described elisa plate bar: each test kit is equipped with 2 or 5 ELISA microwell plates, every elisa plate bar is the ELISA microwell plate of the envelope antigen of energy realizing self disassembling, the purifying BHV-1 virus particle that envelope antigen is deactivation;
Described serum dilution: serum dilution is instant;
Described concentrated cleaning solution: washings is 10 times and concentrates, dilution before using;
Described substrate nitrite ion A:0.006%H
2o
2damping fluid;
Described substrate nitrite ion B:TMB nitrite ion;
Described stop buffer: 0.025% hydrofluoric acid.
4. the application of a kind of infectious bovine rhinotrachetis virus gB antibody test blocking-up ELISA test kit claimed in claim 2 in detecting infectious bovine rhinotrachetis virus antibody.
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Cited By (5)
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| CN109374886A (en) * | 2018-10-12 | 2019-02-22 | 北京纳百生物科技有限公司 | Infectious bovine rhinotrachetis virus antibody assay kit and its application |
| CN113461808A (en) * | 2021-09-01 | 2021-10-01 | 北京市农林科学院 | Competitive ELISA antibody detection kit for infectious bovine rhinotracheitis virus and application thereof |
| CN114878819A (en) * | 2022-07-12 | 2022-08-09 | 北京市农林科学院 | Rapid quantitative detection method for infectious bovine rhinotracheitis virus |
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| CN108918869A (en) * | 2018-06-15 | 2018-11-30 | 新兴县国研科技有限公司 | The application of fiber2 albumen and its recombinant protein in terms of detecting 4 type aviadenovirus antibody of serum |
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| CN113461808A (en) * | 2021-09-01 | 2021-10-01 | 北京市农林科学院 | Competitive ELISA antibody detection kit for infectious bovine rhinotracheitis virus and application thereof |
| CN114878819A (en) * | 2022-07-12 | 2022-08-09 | 北京市农林科学院 | Rapid quantitative detection method for infectious bovine rhinotracheitis virus |
| CN114878819B (en) * | 2022-07-12 | 2022-09-20 | 北京市农林科学院 | Rapid quantitative detection method for infectious bovine rhinotracheitis virus |
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Application publication date: 20141029 |