CN107475203A - A kind of H7 avian influenza virus monoclonal antibody and application - Google Patents
A kind of H7 avian influenza virus monoclonal antibody and application Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1018—Orthomyxoviridae, e.g. influenza virus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/35—Valency
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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- Engineering & Computer Science (AREA)
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- Communicable Diseases (AREA)
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- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Tropical Medicine & Parasitology (AREA)
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Abstract
The invention belongs to clinical immunology technology neck, a kind of H7 avian influenza virus monoclonal antibody and application are specifically disclosed, described monoclonal antibody is secreted to obtain by hybridoma 1H11, and the hybridoma has delivered to CCTCC preservations, and deposit number is：CCTCC NO：C2017112.Described antibody titer is high, specificity is good, and there is barrier effect, ELISA antibody assay kits are blocked available for H7 subtype avian influenzas are prepared, measuring samples are tested using the kit, has more than 98% coincidence rate with HI experiments, is tested with respect to HI, test period is shortened, operating procedure is simpler.
Description
Technical field
The present invention is to belong to clinical immunology technical field, is related to detection and the identification technology field of H7 avian influenza virus,
More particularly to a kind of H7 avian influenza virus monoclonal antibody, a kind of hybridization of secretion H7 avian influenza virus monoclonal antibodies is further related to
Oncocyte, a kind of preparation method of H7 avian influenza virus monoclonal antibody is also related to, further relates to H7 avian influenza virus Dan Ke
Grand antibody is preparing the application in detecting H7 avian influenza virus kits.
Background technology
Bird flu is a kind of Acute respiratory infectious disease as caused by avian influenza virus, has that infectiousness is strong, spread speed
It hurry up, the features such as antigen easily makes a variation.Avian influenza virus belongs to orthomyxovirus section Influenza Virus, and its genome has the list of 8 sections
Stock strand RNA, encodes 11 known albumen.Wherein, memebrane protein hemagglutinin is to discriminate between the foundation of a variety of virus subtypes.According to fowl
The pathogenic difference of influenza virus, highly pathogenic bird flu (HPAI) and low pathogenicity bird flu (LPAI) can be classified as.Closely
The H7 subtype avian influenzas to be taken place frequently over year all over the world just belong to highly pathogenic bird flu.
H7 subtype avian influenzas epidemic situation has never been controlled effectively since 2013 occur.Although H7N9 bird flus
Virus does not evolve the ability of human-to-human transmission also, but the example of birds-to-human transmission happens occasionally.2016-2017 Winter-Springs join when I
There is the 5th H7 subtype avian influenza outburst peak in state, and current epidemic situation is even more serious than in the past, show as disease time it is early and
The incidence of disease is high, and resistance mutation and the increased phenomenon of virulence occurs in virus.For H7 subtype avian influenza epidemic situation present situations,
6 general offices of Yue Chu Ministry of Agriculture issue in 2017 carries out H7N9 immunizations in advance in Guangdong and Guangxi Provinces area, is that Guangdong and Guangxi Provinces area and the whole nation are high
Effect carries out safety control of bird flu work and provides strong technical guarantee.
After the popularization of H7 subtype avian influenzas is immune, the monitoring of immune rear antibody level is just faced.Conventional detection bird flu
The method of antibody is that blood clotting suppresses (HI) experiment.HI tests requires higher to the specialized capability of operating personnel, and process is cumbersome, to knot
The judgement of fruit also has stronger subjectivity.Therefore, a kind of H7 subtype avian influenzas antibody assay kit is developed to evaluate us
Immune effect of vaccine and timely monitoring chicken group's antibody level seem particularly important.
At present, the application on the monoclonal antibody of H7 subtype avian influenza virus has been reported that in succession, mainly in collaurum
The application of test strips, indirect immunofluorescence, SABC etc., and the monoclonal antibody with barrier effect yet there are no report
Road.The present invention provides a kind of monoclonal antibody for having barrier effect to H7 subtype avian influenza virus antibody, have high sensitivity and
Specificity.The kit assembled by the monoclonal antibody, up to 98.9%, is faced on a large scale with HI agreement with experimental rate for us
Field detection provides facility.
The content of the invention
It is an object of the invention to provide a kind of monoclonal of the good anti-H7 subtype avian influenza virus of high specificity, sensitivity
Antibody, the antibody have barrier effect to H7 subtype avian influenza virus antibody, and described monoclonal antibody is by hybridoma 1H11
Secretion is obtained, and the hybridoma cell strain delivers to China typical culture collection center preservation on July 18th, 2017, and preservation is compiled
Number it is：CCTCC NO：C2017112, Classification And Nomenclature：Hybridoma cell strain 1H11, address：Wuhan, China Wuhan University.
It is another object of the present invention to provide a kind of application of the monoclonal antibody of anti-H7 subtype avian influenza virus,
H7 subtype avian influenzas can be prepared using the antibody of the present invention and block ELISA antibody assay kits, for H7 subtype avian influenza epidemic diseases
The evaluation of seedling immune effect and the monitoring of chicken group's antibody level.
In order to achieve the above object, the present invention takes following technical measures：
A kind of hybridoma cell strain for secreting anti-H7 subtype avian influenza virus monoclonal antibody, hybridoma cell strain 1H11
Acquisition be using inactivation H7-AIV antigen immune BALB/c mouses, anti-H7-AIV monoclonals are established by hybridoma technology and resisted
What body hybridoma storehouse obtained.Described hybridoma cell strain has delivered to Chinese Typical Representative culture guarantor on July 18th, 2017
The preservation of Tibetan center, deposit number are：CCTCC NO：C2017112, Classification And Nomenclature：Hybridoma cell strain 1H11, address：It is Chinese military
Chinese Wuhan University.
Hybridoma 1H11 is in RPMI-1640 complete mediums, containing 5%CO2, culture 48 is small under the conditions of 37 DEG C
When, micro- Microscopic observation, cell growth is good, perfectly round bright in thyrsiform, colony, and the culture for meeting hybridoma is special
Property.
Mitotic stability detection is carried out to the cell of hybridoma 1H11 maximum limits generation (F25 generations), as a result every generation
Cell the energy specific monoclonal antibody of stably excreting, antibody positive rate 100%, and the HI antibody of cells and supernatant
Potency is 27More than, illustrate the ability that the hybridoma has stably excreting antibody.
A kind of application of the monoclonal antibody of anti-H7 subtype avian influenza virus, described monoclonal antibody can be used for preparing H7 hypotype fowl
Influenza blocks ELISA antibody assay kits, and whether identification tested animal sample infects H7 subtype avian influenza virus or to be checked
Whether contain H7 subtype avian influenza virus antibody in serum.
The features and advantages of the invention are as follows：
1. the H7-AIV immunogenes that the present invention uses are available from Ha Weike standard antigen, carrying out popularization with country exempts from
The H7 subtype avian influenza vaccines of epidemic disease are homologous, and the kit evaluation vaccine immunity that the monoclonal antibody prepared with the antigen assembles is imitated
Fruit and monitoring chicken group's antibody level result are more accurate.
2. the present invention filters out one plant of life from the hybridoma of 16 plants of obtained anti-H7-AIV monoclonal antibodies of secretion
The best hybridoma of thing characteristic, it is 18log2 with blood clotting suppression HI potency of the ascites of its preparation after purification to H7-AIV,
ELISA potency is up to 4 × 105。
3. the 1H11 monoclonal antibodies that the present invention obtains, it is that the H7 subtype avian influenza virus obtained first has the monoclonal antibody of barrier effect,
ELISA antibody assay kits are blocked available for H7 subtype avian influenzas are prepared, kit application blocks ELISA method detection blood
H7 subtype avian influenza antibody in final proof sheet, improve the specificity and sensitiveness of testing result.It is that have detected greatly with this method
Chicken serum is measured, and serum blocking rate is calculated with formula (1- samples OD values/negative control OD value), it is final to determine：Chicken serum
Blocking rate is judged to the positive more than or equal to 0.6, and feminine gender is judged to less than 0.6.This method has more than 98% coincidence rate with HI experiments,
Tested with respect to HI, shorten test period, operating procedure is simpler.
Embodiment
Technical scheme described in the embodiment of the present invention, it is this area routine techniques if not otherwise specified；The reagent or material
Material, if not otherwise specified, derives from commercial channel.
Embodiment 1：
Hybridoma cell strain 1H11 acquisition：
The foundation of monoclonal antibody hybridoma cell strain
Animal immune
Take and be resuspended purchased from the Ha Weike μ L physiological saline of one bottle of use of H7 standard antigens 500, helped completely with isometric Freund
Agent is fully emulsified, and subcutaneous branch injects the μ L/ of 8 week old BALB/c mouse 200 only；After 2 weeks, 4 weeks in the same way using Freund not
The antigen of Freund's complete adjuvant emulsification is each again to be immunized once；Take same dose antigen to be injected intraperitoneally after 6 weeks, be not added with adjuvant；Melt after 4 days
Close.
Cell fusion
Myeloma cell's prepares growth selection SP2/0 cells in good condition, abandons during density length to blake bottle bottom 75%
Supernatant, after washed once with incomplete DMEM culture mediums, cell is gently blown down with l0mL incomplete DMEM culture mediums.
The preparation of splenic lymphocytes takes the mouse of 4 days after booster immunization, extracts eyeball blood sampling and is used as positive serum；Cervical vertebra
Dislocate lethal mouse, puts 10min in 75% alcohol, and belly is fixed on the dissection plate in super-clean bench upward；Sterile opening abdominal cavity takes
Go out spleen, cannot be used up full DMEM culture mediums washing, remove unnecessary connective tissue and fat；Spleen is then transferred to another Sheng
In the plate for having incomplete DMEM culture mediums.Spleen is ground in cell screen clothes with grinding rod, collects splenocyte suspension.
The feeder cells that prepare of feeder cells can be as follows in fusion evening before that day preparation, method：Take a ripe health
ICR mouse, pluck eyeball blood sampling and be used as negative serum, cervical dislocation execution；10min in 75% alcohol is put, fixing limbs, is cut
Opening skin exposes peritonaeum, and peritonaeum is wiped with cotton ball soaked in alcohol；With l0mL syringes, 12# syringe needles, 10mL HAT culture mediums are drawn
Abdominal cavity carefully is expelled to, cotton ball soaked in alcohol gently abdomen massage is held, draws back intraperitoneal liquid, is injected in oneself ready container, is counted
Count and added after diluting in 96 porocyte culture plates.
The splenocyte of above-mentioned preparation and myeloma cell are mixed in 50mL fusion pipe by fusion, 1000g centrifugations
10min, abandon supernatant.Fusion pipe is placed in palm, gently friction bottom, two kinds of cells is fully mixed；In 37 DEG C of water-baths
The PEG1500 of 1mL preheatings is slowly added in 45s into fusion pipe, side edged gently shakes up；Fast drop after first slow in 90s immediately
Add the incomplete DMEM culture mediums terminating reaction of 37 DEG C of preheatings of 30mL, 10min, 1000g centrifugations are stood in 37 DEG C of water-baths
10min；Supernatant is abandoned, is gently suspended sedimentation cell with 60mL HAT culture mediums, is sub-packed in the 96 hole cells for having completed feeder cells
Culture plate, culture plate is then put into 37 DEG C of 6%CO2Cultivated in incubator；After 5d liquid is partly changed with fresh HAT culture mediums；
Swapped out HAT culture mediums with the HT culture mediums of preheating after 10d；Growth of Hybridoma Cell situation is observed, treats that its cells and supernatant becomes
When yellow or clone is distributed to more than the 1/10 of bottom hole area, draws 100 μ L cell conditioned mediums and carry out antibody test.
The screening of hybridoma
Using the H7 standard antigens purchased from Ha Weike as detection antigen, two methods are tested to sieve using indirect ELISA and HI
Select positive colony.
Determine that (the H7 standard antigens that antigen used in the present embodiment is available from Ha Weike are inspection to detection antigen with square formation test method(s)
Survey antigen) coating concentration, standard antigen is done into gradient dilution with coating buffer solution, often row makees a dilution factor, 100 μ L/ holes coating
ELISA Plate, 37 DEG C of coating 2h；Washed 3 times, each 5min, patted dry with PBST；The μ L/ holes of calf serum 200 for adding 10%PBS to dilute
Closing, 4 DEG C of closings overnight, are ibid washed 3 times；The μ L/ holes of positive serum 100 of gradient dilution are added, 37 DEG C of incubation 1h, ibid wash 3
Time；Add working concentration (1：10000 dilution) the μ L/ holes of sheep anti mouse HRP-IgG 50,37 DEG C incubation 1h, ibid wash 3 times；Add
The μ L/ holes of TMB nitrite ions 50,37 DEG C of 10~15min of effect；Add 2M H2SO450 μ L/ holes terminating reactions.Determine OD450Readings.
The antigen concentration 1 determined by square formation:100 coated elisa plates, -20 DEG C save backup.Detect the ELISA of hybridoma supernatant
Method is same as above.Positive control when after the concentration dilution that immune serum is determined by square formation as screening, does feeder cells
ICR mice serums set up blank zeroing hole as negative control.HI experiment sieving methods：This method is directed to avian flu
Malicious H7 hypotypes hemagglutinin, the hybridoma for screening the positive are cloned.The positive hole of detection is subcloned (using limited
Dilution method carries out cloning to the hybridoma of ELISA test positive), until the antibody of cell line secretion can be stably
Untill specific reaction occurs with H7 subtype avian influenza virus liquid.1 plant of positive hybridoma is obtained through 3 subclones
System, is named as 1H11.
The hybridoma cell strain delivers to China typical culture collection center preservation on July 18th, 2017, and preservation is compiled
Number it is：CCTCC NO：C2017112, Classification And Nomenclature：Hybridoma cell strain 1H11, address：Wuhan, China Wuhan University.
Embodiment 2：
The preparation and titration of ascites：
The BALB/c mouse of 10 week old is taken, sterilized liquid paraffin is injected intraperitoneally, 0.5mL/ is only；1 week pneumoretroperitoneum injection PBS
The hybridoma 1H11 in exponential phase of dilution, 5 × 105Individual/only；When mouse web portion substantially swells, 16# is used
Syringe needle gathers ascites from abdominal cavity, 2500g centrifugation 10min, removes adipose tissue, draws supernatant, -70 DEG C save backup.
The identification of monoclonal antibody characteristic：
Titer of ascites measure is as a result as follows using indirect ELISA and HI experiment two methods detection titer of ascites：
Subgroup identification is carried out by the method for monoclonal antibody subclass kit specification introduction, as a result shows monoclonal antibody 1H11
For IgG1Subclass.
Embodiment 3：
Hybridoma 1H11 repeated prunings
In the generation of hybridoma cell strain 1H11 continuous passages 15 that will be obtained, the potency for determining its cell conditioned medium is tested with HI,
As a result the cell line energy stably excreting antibody is shown, and the HI antibody titers of cells and supernatant are 27More than.
Embodiment 4：
1H11 monoclonal antibody specificity identifications
1H11 hybridoma supernatants are taken, using two methods of HI experiments and indirect ELISA to avian influenza virus H7 hypotypes
Hemagglutination-inhibition test antigen (H7-AIAg), avian influenza virus H1 hypotype hemagglutination-inhibition test antigens (H1-AIAg), avian flu
Malicious H5 hypotypes hemagglutination-inhibition test antigen (H5-AIAg), avian influenza virus H9 hypotype hemagglutination-inhibition test antigens (H9-AIAg),
NDV (NDV), infectious bronchitis virus (IBV), egg drop syndrome viral (EDS-76), chicken are infected
Property bursal disease virus (IBDV) carry out cross matching, identify the specificity of monoclonal antibody.
As a result show：Either tested using ELISA method or HI, monoclonal antibody 1H11 is only (i.e. above-mentioned to H7 hypotypes strain
H7 hypotype hemagglutination-inhibition tests antigen) there is good reactivity, to beyond H7 subtype avian influenza virus other are any
Virus is reactionless, illustrates the specific monoclonal antibody that this plant of monoclonal antibody is anti-H7 subtype avian influenzas.
Embodiment 5：
The purifying of the monoclonal antibody of anti-H7 subtype avian influenza virus
Odd contradictive hydroperitoneum prepared by embodiment 2 is purified using Protein G affinity chromatography methods, Protein G purifying
Method is as follows：Container of the gentle inversion equipped with Protein G affine resins makes it suspend completely with hybrid resin for several times.Will be appropriate
Protein G resins are fitted into chromatographic column, add 5mL combination buffer balance pillar.Use identical or more volumes
Combination buffer dilutes odd contradictive hydroperitoneum, loading.After loading pillar is washed with 10ml combination buffers.After combination buffer is flow to end, use
10mL elution buffer antibody elution, collect eluted product.The pH value of eluent is detected in elution process in real time, is used in time
1M Tris-Cl (pH 8.5) neutralize eluent, and the pH for making the eluent containing eluted product is 7.4.Eluent loads bag filter,
Using 0.01M 4 DEG C of dialysed overnights of Tris-Cl buffer solutions (pH 7.4), liquid is changed halfway 2~3 times.Monoclonal antibody concentration is after dialysis
11.2mg/ml.Ascites before purification has 8ml, and 150mg monoclonal antibodies are obtained after purification, and equivalent to every milliliter ascites contains 18 milligrams
The monoclonal antibody of left and right, this shows the very strong monoclonal antibody secrete ability of the hybridoma cell strain that the present invention filters out.
With two methods of the titer of ascites of ELISA and HI experiment detection after purification, HI potency can reach 218, ELISA potency
Show that antibody is diluted to 1：It is still the positive at 400000 times, shows that the antibody has the characteristics of high sensitivity.
Embodiment 6：
A kind of monoclonal antibody of anti-H7 subtype avian influenza virus blocks the inspection of ELISA antibody in the H7 subtype avian influenzas of preparation
Applied in test agent box：
1. the preparation of ELISA Plate
Determine that H7 hypotype hemagglutination-inhibition tests antigen (resists purchased from Ha Weike H7 hypotype hemagglutination-inhibition tests by square formation method
It is former) coating concentration be 1:160, it is coated with 14 hours at 2-8 DEG C.Coating buffer is discarded, adds 0.5% bovine serum albumin(BSA) (BSA)
Closed 2 hours for 37 DEG C as confining liquid.Confining liquid is discarded, is vacuumized after natural air drying and is packaged into finished product kit ELISA Plate.
2. the preparation of kit other components
Sample diluting liquid：NaCl 8g, KCl 0.2g, Na2HPO412H2O 2.9g, KH2PO4 0.2g, Tween-20
0.5ml, 1000ml (pH=7.4) is settled to deionized water；
The preparation of enzyme labelled antibody：10mgHRP is dissolved in 2ml injection waters, in brownish red, adds 1ml concentration 0.06M/L
NaIO4, in grass green, 4 DEG C of 30min add 0.16M/L ethylene glycol 1ml terminating reactions, room temperature lucifuge 30min, and solution becomes palm fibre
Yellow, add 4 DEG C of dialysis in 1H11 antibody 1ml, the 0.05M PH=9.5CB that concentration prepared by embodiment 5 is 11.2mg/ml
Overnight.Suction out and add 5mg/ml NaBH4 0.4ml, 4 DEG C of 2h, add isometric saturated ammonium sulfate, 4 DEG C of 30min, 10000r/min
5min, then it is resuspended and is dialysed with 2ml 20mM PH=7.4PB, harvested enzyme labelled antibody 4.5ml, add isometric glycerine, -20 DEG C
Save backup.The working concentration of enzyme labelled antibody is determined by square formation method, concentration is 0.02mg/ml.
Substrate solution A：Na2HPO4·12H2O 14.60g, citric acid 9.33g, 30% hydrogen peroxide 2ml, add deionization water-soluble
Solve and be settled to 1000ml, adjust pH to 5.0~5.4, packing, 10ml/ bottles.
Substrate solution B：Tetramethyl benzidine (TMB) 20.00mg, absolute ethyl alcohol 10.00ml, add deionized water dissolving to be settled to
1000ml, it is distributed into 10ml/ bottles.
Terminate liquid：2.5ml hydrofluoric acid (HF) is added in 900ml deionized waters, is settled to 1000ml, packing, 10ml/ bottles.
20 times of concentrated cleaning solutions：NaCl 160g, KCl 4g, Na2HPO4·12H2O 58g, KH2PO44g, Tween-20
10ml, 1000ml (pH=7.4) is settled to deionized water.
Positive control：As positive right after H7 subtype avian influenza standard positive serums purchased from Ha Weike are diluted in proportion
According to.
Negative control：SPF chicken serums are used as negative control after diluting in proportion.
3. specifically used step is as follows：
Sample treatment takes chicken whole blood, and 4000 revs/min centrifuge 10 minutes after blood clotting, collect supernatant.Also can gather
Blood, serum is separated out naturally after to be solidified, it is desirable to which serum is limpid, no haemolysis.
Cleaning solution is prepared before use, the cleaning solution of concentration is taken out from kit, is balanced to room temperature (20~25 DEG C),
And shake, make precipitation dissolve (heat 5~10 minutes preferably in 37 DEG C of water-baths), then with distilled water make 20 times dilute (such as：
Per two boards 570ml distilled water is added with 30ml 20 times of concentrated cleaning solutions), mix, the cleaning solution diluted can at 2~8 DEG C
With storage 7 days.
Blood serum sample to be checked is pressed 1 by sample preliminarily diluted in serum-dilution plate:50 dilution proportion (such as：In serum
196 μ l sample diluting liquids are first added in dilution plate, then add 4 μ l serum to be checked), pay attention to that control can not be diluted.Different samples will
Pay attention to changing suction nozzle.Sample will be mixed fully in dilution.
【Operating procedure】
1 take antigen coated microplate (according to sample how much, removable gradation use), with the cleaning solution board-washing diluted 1 time
Patted dry after (200 μ l/ holes).The blood serum sample that 100 μ l have diluted is added in antigen coated microplate, control serum directly takes 100 μ l
It is added in antigen coat plate hole.Control serum respectively sets 2 holes, and measuring samples set 1 hole, sample (not overflowing) in the even hole that gently shakes,
Put and incubated 30 minutes at 37 DEG C.
2 discard the liquid in hole, and the μ l of cleaning solution 200 diluted are added per hole, after standing 3 minutes, discard cleaning solution, and
Pat dry on blotting paper, wash 3 times altogether.
3, per the enzyme-added μ l of labeling antibody 100 in hole, put and are incubated 30 minutes at 37 DEG C.
4 discard the liquid in hole, wash 5 times, and method is the same as 2.
5 first add the μ l of substrate solution A 50 per hole, add the μ l of substrate solution B 50, mix, put and kept away under room temperature (20~25 DEG C)
Light develops the color 10 minutes.
6 add the μ l of terminate liquid 50, measurement result after mixing per hole.
【Result judgement】
The OD in each hole is surveyed on ELIASA630nmValue.Testing the condition set up is：The average OD of negative control hole630nmValue answers ＞
0.5, the average OD of Positive control wells630nmValue answers ＜ 0.5.
S is sample well OD630nmValue, N are the average OD of negative control hole630nmValue, calculate samples blocking rate (1-S/N)
Value.
If it is judged to the positive during blocking rate ＞ 0.6；If it is judged to feminine gender during blocking rate≤0.6.
(3) H7 subtype avian influenzas antibody blocking ELISA detection kit and the contrast of HI experiments
Applicant is detected simultaneously with two methods of H7 subtype avian influenza antibody blocking ELISA detection kits and HI experiments
356 parts of chicken serums for picking up from clinic, comparative test result, calculate coincidence rate.Positive coincidence rate is 100%, and negative match-rate is
97.8%, total coincidence rate is 98.9%, illustrates that the kit and HI agreement with experimental rates are good, is adapted in basic unit is detected on a large scale
Popularization and application.
Claims (4)
1. the hybridoma of one plant of separation, described hybridoma is hybridoma 1H11, and deposit number is：CCTCC
NO：C2017112.
2. the monoclonal antibody of the hybridoma secretion described in claim 1.
3. the monoclonal antibody described in hybridoma or claim 2 described in claim 1 is preparing detection H7 bird flus
Application in virus agent box.
4. the monoclonal antibody described in hybridoma or claim 2 described in claim 1 is preparing H7 subtype avian influenzas
Block the application in ELISA antibody assay kits.
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Cited By (5)
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| CN108303529A (en) * | 2018-01-23 | 2018-07-20 | 武汉科前生物股份有限公司 | A kind of immunity colloidal gold test paper strip and preparation method thereof for detecting avian influenza virus H7 hypotypes |
| CN108872593A (en) * | 2018-07-04 | 2018-11-23 | 北京索莱宝科技有限公司 | It is a kind of for assessing the blocking ELISA kit of HPV16 immune effect of vaccine |
| 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 |
| CN110879293A (en) * | 2019-11-05 | 2020-03-13 | 暨南大学 | Method for screening hybridoma cell strain secreting pairing monoclonal antibody and application |
| CN111172117A (en) * | 2020-01-20 | 2020-05-19 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Monoclonal antibody capable of identifying N9 subtype avian influenza virus neuraminidase protein in broad spectrum manner and application thereof |
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| CN108303529A (en) * | 2018-01-23 | 2018-07-20 | 武汉科前生物股份有限公司 | A kind of immunity colloidal gold test paper strip and preparation method thereof for detecting avian influenza virus H7 hypotypes |
| CN108303529B (en) * | 2018-01-23 | 2020-08-25 | 武汉科前生物股份有限公司 | Immune colloidal gold test strip for detecting avian influenza virus H7 subtype and preparation method thereof |
| 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 |
| CN108872593A (en) * | 2018-07-04 | 2018-11-23 | 北京索莱宝科技有限公司 | It is a kind of for assessing the blocking ELISA kit of HPV16 immune effect of vaccine |
| CN110879293A (en) * | 2019-11-05 | 2020-03-13 | 暨南大学 | Method for screening hybridoma cell strain secreting pairing monoclonal antibody and application |
| CN111172117A (en) * | 2020-01-20 | 2020-05-19 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Monoclonal antibody capable of identifying N9 subtype avian influenza virus neuraminidase protein in broad spectrum manner and application thereof |
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