CN103266088A - H7 subtype avian influenza virus monoclonal antibody of and kit - Google Patents
H7 subtype avian influenza virus monoclonal antibody of and kit Download PDFInfo
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Abstract
The invention provides an H7 subtype avian influenza virus monoclonal antibody of and kit, and obtains hybridoma cell strain that secretes H7 subtype avian influenza virus monoclonal antibody, wherein the accession number of the hybridoma cell strain is CGMCC NO. 7308, and the obtained H7 subtype avian influenza virus monoclonal antibody is provided with advantages like high sensitivity, strong singularity and high bioactivity etc., which can be used to identify if the virus to be tested is H7 subtype avian influenza virus, and if the sample to be tested is infected by H7 subtype avian influenza virus, and if the serum to be tested contains H7 subtype avian influenza virus. The monoclonal antibody above also could be used to prepare immunological diagnostic reagent of H7 subtype avian influenza virus or its antibody, such as Enzyme-linked diagnostic reagent and colloidal gold immunoassay kit or the like, and also could detect if the poultry feces, the oral and nasal secreta or the stool contains H7 subtype avian influenza virus and if the poultry serum contains H7 subtype avian influenza virus antibody.
Description
Technical field
The invention belongs to Measurement for Biotechnique, relate to a kind of H7 subtype avian influenza virus monoclonal antibody and test kit.
Background technology
(Avian Influenza AI) is called for short bird flu to avian influenza, is the bird transmissible disease that is caused by the Influenza Virus A of orthomyxoviridae family type influenza virus.(Avian Influenza virus AIV) can infect nearly all wild fowl and poultry to avian influenza virus, according to the difference of AIV virulence, it can be divided into highly pathogenic, low pathogenicity and no pathogenicity AIV.In recent years, bird flu is popular at world wide, causes enormous economic loss to aviculture.Avian influenza virus very easily makes a variation, and can stride kind of a propagation and infect the mankind, becomes one of important cause of disease that threatens human health.Hong Kong in 1997, Holland in 2003, Yadu, the southeast people occurred and has infected high pathogenic avian influenza and death cases since 2004.(Hihgly Pathogenic Avian Influenza HPAI) is classified as the statutory report eqpidemic disease by OIE (OIE) to high pathogenic avian influenza, and China also classifies it as a class animal epidemic.
Mexico outburst H1N1 epidemic situation caused hundred people to infect to April in 2009 3, and epidemic situation bamboo telegraph thereafter is to the whole world.In morning on April 30th, 2009, the World Health Organization brings up to the 5th grade to global flu outbreak warning level.On June 11st, 2009, the World Health Organization is promoted to the 6th grade of highest ranking with global flu outbreak warning level.This time H1N1 influenza being very popular worldwide caused very big fear to the whole world, influenced normal economy, the trade order in the whole world, also causes very big threat to public health security.Although China's popular high pathogenic avian influenza at present mainly is the H5 hypotype, be separated to the H7 subtype avian influenza virus on China Hebei and other places, it is popular that the H7 subtype avian influenza has also appearred in the Korea adjacent with China.So China is necessary to carry out epidemiology detection, diagnosis and the prevention and control of H7 subtype avian influenza virus.Quick, the hypospecificity detection kit of therefore foundation detection H7 subtype highly pathogenic avian influenza, and H7 subtype influenza virus are conducive to this sick prevention and control, also help to eliminate this disease to the threat of human health.
Generally, the diagnosis of high pathogenic avian influenza can be according to clinical symptom, cut open inspection etc. substantially makes tentative diagnosis, specifically " the high pathogenic avian influenza Prevention Technique standard " that can promulgate with reference to country.Making a definite diagnosis with somatotype then needs the chamber of experimentizing diagnosis, comprising: Pathogen Isolation and evaluation, serodiagnosis.But high pathogenic avian influenza need could be operated in 3 grades of laboratories of Biosafety, and operator must be through the training of specialty.Therefore separation and evaluation H7 subtype avian influenza virus need experiment equipment, the operative technique of specialty, and need the time than length, so the application of this method is subjected to great restriction.Along with development of molecular biology and application, it is more and more to use Protocols in Molecular Biology to carry out the method that high pathogenic avian influenza detects, especially based on reverse transcription (RT-) PCR of polymerase chain reaction (PCR), and real-time fluorescence RT-PCR.But this method needs expensive and professional plant and instrument, especially real-time fluorescence RT-PCR, and used consumptive material is also expensive, needs simultaneously could be competent at operation through the personnel of professional training, and occurs owing to sample contamination causes false positive easily.HI test-results reliability height, be that WHO carries out the method that global influenza monitoring is adopted, use the standard antiserum(antisera) of known hypotype, can carry out hypotype identifies, but the shortcoming of this method is to remove the non-specific agglutination element that is prevalent in the serum, need carry out the antigen stdn simultaneously when each experiment, and need the relatively technical ability of the correct interpretation of specialty, therefore this method also only can be used in relevant professional, is unsuitable for penetration and promotion.And based on the catching methods such as ELISA, colloidal gold strip, IiT, immunohistochemical methods and can detect the H7 subtype avian influenza fast, delicately of antibody, be suitable in large-scale Monitoring systems, using and the terrain detection.And the application-dependent of catching methods such as ELISA, colloidal gold strip, IiT, immunohistochemical methods is in the development at the monoclonal antibody specific of H7 subtype avian influenza antigen.
But so far, Shang Weiyou sees that sensitivity is good, high specificity, tires the monoclonal antibody of H7 subtype avian influenza virus high and the report of corresponding detection kit.
Summary of the invention
Demand and deficiency according to above-mentioned field, the invention provides that a kind of sensitivity is good, high specificity, the monoclonal antibody of tiring the H7 subtype avian influenza virus high, monoclonal antibody of the present invention can be applied to detect H7 subtype avian influenza virus or H7 subtype avian influenza virus antibody.
A kind of hybridoma cell strain, its preserving number is CGMCC No.7308, the acquisition of described hybridoma cell strain is by the H7N2 avian influenza virus being seeded on the 9 ages in days fertilization SPF chicken embryo, H7N2 strain virus after obtaining increasing, get H7N2 avian influenza virus alive deactivation, prepare H7N2 avian influenza virus antigen refined solution with deactivation H7N2 avian influenza venom, behind the immune mouse, get immune mouse spleen cell and the SP2/0 murine myeloma cell merges, cultivate above-mentioned cell the back, and further subclone filters out a strain at the monoclonal antibody of H7 subtype avian influenza virus, and hybridoma, purifying and the preservation of this monoclonal antibody of secretion.
A kind of H7 subtype avian influenza virus monoclonal antibody is got by above-mentioned hybridoma cell strain secretion, and this monoclonal anti physical efficiency is specific in conjunction with H7 subtype avian influenza virus hemagglutinin (HA) albumen.
The application of above-mentioned monoclonal antibody in detecting H7 subtype avian influenza virus or H7 subtype avian influenza virus antibody.
A kind of reaction carriers for detection of the H7 subtype avian influenza virus is characterized in that described reaction carriers directly or indirectly is coated with said monoclonal antibody.
A kind of test kit that detects the H7 subtype avian influenza virus is characterized in that, includes the above-mentioned reaction carriers for detection of the H7 subtype avian influenza virus.
A kind of reaction carriers for detection of H7 subtype avian influenza virus antibody is characterized in that described reaction carriers directly or indirectly is coated with said monoclonal antibody.
A kind of test kit that detects H7 subtype avian influenza virus antibody is characterized in that, includes the above-mentioned reaction carriers for detection of H7 subtype avian influenza virus antibody.
A kind of colloidal gold kit that detects the H7 subtype avian influenza virus is characterized in that, marks the H7 subtype avian influenza virus monoclonal antibody that fiber mat is coated with colloid gold label, i.e. Au-McAb at gold; It is how anti-that detection zone is coated with the anti-H7N2 avian influenza virus of rabbit, i.e. H7N2-PcAb; During detection, in sample, there is the H7 subtype avian influenza virus, then forms immune complex with Au-McAb, and then be combined with the H7N2-PcAb of detection zone, colour developing.
A kind of colloidal gold kit that detects H7 subtype avian influenza virus antibody is characterized in that, is coated with the monoclonal antibody of the H7 subtype avian influenza virus of colloid gold label at gold mark fiber mat; Detection zone is coated with H7N2 avian influenza virus purifying antigen; The check plot bag is resisted by sheep anti-mouse igg two; During detection, in sample, there is H7 subtype avian influenza virus antibody, then the H7 subtype avian influenza virus antibody monoclonal antibody that suppresses the H7 subtype avian influenza virus of colloid gold label is combined with H7N2 avian influenza virus purifying antigen, and the colourless or color of detection zone obviously shoals.
Technique effect
Test kit of the present invention can be for the identification of the H7 subtype avian influenza virus, and whether assistant identification virus to be measured is whether H7 subtype avian influenza virus, assistant identification tested animal sample infect the H7 subtype avian influenza virus or identify in the test serum whether contain H7 subtype avian influenza virus antibody.
Monoclonal antibody provided by the invention is for detection of H7 subtype avian influenza virus or its antibody, has that specificity is good, susceptibility strong, the advantages of higher of tiring.The H7 subtype avian influenza virus monoclonal antibody of the present invention's development can be used for developing the immunological diagnostic reagent of H7 subtype avian influenza virus or its antibody, as enzyme connection diagnostic reagent, colloid gold immune test paper bar etc.
H7 subtype avian influenza virus antibody ELISA immunity detection reagent uses the H7 subtype influenza virus HA antigen specific antibody in the competition law detection serum specimen.On the polyethylene micropore plate of test kit, wrap by H7 subtype avian influenza virus purifying antigen in advance earlier.If the serum specimen that adds can obviously suppress the combination of monoclonal antibody linked with peroxidase and antigen, then show the specific antibody that contains avian influenza virus H7 hypotype HA antigen in the sample.When not containing avian influenza virus antibody in the sample or be not H7 subtype avian influenza virus antibody, just can not suppress the combination of monoclonal antibody linked with peroxidase and antigen, it is more dark to develop the color, on the contrary colour developing is more shallow.
H7 subtype avian influenza virus HA antigen enzyme-linked immunologic detecting kit uses double antibody sandwich method to detect the HA antigen of the H7 subtype avian influenza virus in the sample.On the polyethylene micropore lath of test kit, wrap by the polyclonal antibody of the anti-H7N2 avian influenza virus of rabbit HA albumen in advance earlier, after the H7 subtype avian influenza virus after the cracking is added to micropore, the polyclonal antibody of pre-bag quilt can be caught it, the monoclonal antibody linked with peroxidase of Jia Ruing combination with it subsequently comes judged result by the demonstration degree of substrate for enzymatic activity then.When not containing influenza antigen in the sample or be not H7 subtype influenza virus, substrate can not develop the color.The detectable sample of this test kit comprises the intact virus of animal excrements, mouth and nose solinocrine thing, chicken embryo culture or lytic virus etc.
Immune colloid gold reagent box of the present invention adopts the diagnostic reagent of new generation of colloidal gold immunochromatographimethod technology development, colloidal gold kit of the present invention is easy and simple to handle, reliable, need not plant and instrument, carry the Quality Control contrast, without any need for additive reagent, display result is clear and definite, be swift in response, the entire operation time only needs 30min.
The present invention detects H7 subtype avian influenza virus colloidal gold kit (golden mark method), and the detection zone bag is by the polyclonal antibody of the anti-H7N2 avian influenza virus of rabbit (H7N2-PcAb) on the nitrocellulose filter of colloidal gold strip respectively, and the check plot bag is by sheep anti-mouse igg (two is anti-).During detection in the sample H7 subtype avian influenza virus monoclonal antibody (Au-McAb) of H7 subtype avian influenza virus and colloid gold label form immune complex (Au-McAb-Ag), because this mixture of chromatography effect moves along film, be combined with the H7N2-PcAb of detection zone bag quilt and form mixture (Au-McAb-Ag-PcAb), close with two resistive connections of control line bag quilt and form mixture (Au-McAb-Ag-two is anti-).As positive sample, then can respectively form a red line at detection zone and check plot respectively; As negative sample, then only form a red line in the check plot.This test kit can be used for detecting whether contain the H7 subtype avian influenza virus in bird movement, mouth and nose solinocrine thing and the ight soil.
The colloidal gold kit (golden mark method) that the present invention detects H7 subtype avian influenza virus antibody respectively on the nitrocellulose filter of colloidal gold strip the detection zone bag by H7N2 avian influenza virus purifying antigen, the check plot bag is by sheep anti-mouse igg (two is anti-), during detection, the antibody in the sample is combined with the H7N2 avian influenza virus purifying antigen of detection zone bag quilt with the H7 of colloid gold label subtype avian influenza virus monoclonal antibody competitiveness.If the sample that adds can obviously suppress the H7 subtype avian influenza virus monoclonal antibody of colloid gold label and the combination of purifying antigen, then show the specific antibody that contains avian influenza virus H7 hypotype in the mark product.When not containing avian influenza virus antibody in the sample or be not H7 subtype avian influenza virus antibody, just can not suppress this combination, the color of detection line is more dark, on the contrary color is more shallow.Be that test sample is positive, then only form a red line in the check plot; As negative sample, then can respectively form a red line at detection zone and check plot respectively.This test kit can be used for detecting whether contain H7 subtype avian influenza virus antibody in the bird serum.
Monoclonal antibody of the present invention has following advantage:
1. specificity is good, susceptibility strong, the height of tiring;
2. can be used for reagent and test kit based on immunoreactive detection H7 subtype avian influenza virus or H7 subtype avian influenza virus antibody;
3. can be applied to the detection that the H7 subtype avian influenza virus is carried out in various places;
4. use monoclonal antibody of the present invention and special, quick and convenient based on immunoreactive test kit sensitivity;
5. economical and practical, do not need the professional and technical personnel you, be easy to popularize;
6. can be used for using in the large-scale Monitoring systems and the terrain detection.
Hybridoma preservation information:
Hybridoma cell strain title: the hybridoma cell strain 2D4 strain of H7 subtype avian influenza virus monoclonal antibody
Strain classification: mouse hybridoma cell system
Deposit number is: CGMCC No.7308
Preservation date: on March 1st, 2013
Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC)
The preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Description of drawings
Fig. 1 detects the detected result synoptic diagram of H7 subtype avian influenza virus colloidal gold kit for the present invention
Fig. 2 detects the detected result synoptic diagram of H7 subtype avian influenza virus antibody colloidal gold test kit for the present invention
Embodiment
It is in order further to understand the present invention better that following embodiment is provided; be not limited to described preferred forms; content of the present invention and protection domain are not construed as limiting; anyone makes up with the feature of other prior aries under enlightenment of the present invention or with the present invention and the identical or akin method of any and the present invention and the product that draw, all drops within protection scope of the present invention.
The value of the concentration of reagent of the present invention, temperature and its dependent variable just illustrates application of the present invention, and is not construed as limiting the invention.
The source of experiment material of the present invention:
Biomaterial:
Immunity is provided by Ministry of Agriculture's animal doctor's diagnositc center with avian influenza virus (Alv): A/Chicken/Hebei/1/2002 (H7N2 is called for short CK/H B/1/02), A/Chicken/Hebei/2/2002 (H7N2 is called for short CK/HB/2/02) strain isolated;
Newcastle disease virus (NDV), egg drop syndrome virus (EDSV): provided by Ministry of Agriculture's animal doctor's diagnositc center;
Applicant's statement, above biomaterial all has preservation in the applicant laboratory, can provide to the public to be used for proof test in 20 years applyings date.
Avian influenza virus (Alv) type strain: H1～H15 type strain is provided by Ministry of Agriculture's animal doctor's diagnositc center;
Avian influenza virus (Alv) strain isolated: 2 strains of H5 hypotype, 2 strains of H7 hypotype, 2 strains of H9 hypotype are provided by Ministry of Agriculture's animal doctor's diagnositc center;
The SP2/0 cell is so kind as to give by Military Medical Science Institute;
The Bslb/c mouse is available from Beijing dimension tonneau China company;
SPF kind egg Beijing logical experimental animal technology company limited of Cimmeria dimension.
The source of reagent and specification:
| Mercaptoethanol | The 100ml/ bottle | Invitrogen company |
| PEG | 5ml/ props up | Simga company |
| The DMEM high glucose medium | The 1L/ bag | GIBCO company |
| Foetal calf serum | The 500ml/ bottle | HycLone company |
| HAT selects substratum | 10ml/ props up | Simga company |
| HT selects substratum | 10ml/ props up | Simga company |
| The inferior maple (DMSO) of dimethyl | The 500ml/ bottle | Simga company |
| Freund's complete adjuvant (FCA) | The 10ml/ bottle | Simga company |
| Freund's incomplete adjuvant (FICA) | The 10ml/ bottle | Simga company |
| The yellow soda ash analytical pure | The 500g/ bottle | Beijing chemical reagents corporation |
| Sodium bicarbonate | The 500g/ bottle | Beijing chemical reagents corporation |
| Sodium-chlor | The 500g/ bottle | Beijing chemical reagents corporation |
| SODIUM PHOSPHATE, MONOBASIC | The 500g/ bottle | Beijing chemical reagents corporation |
| Sodium phosphate dibasic | The 500g/ bottle | Beijing chemical reagents corporation |
| Sucrose | The 500g/ bottle | Beijing chemical reagents corporation |
| Enzyme plate | ? | The worker is given birth in Shanghai |
| Horseradish peroxidase HRP | The 10g/ bottle | Simga company |
| Sodium periodate NaIO | The 100g/ bottle | Beijing chemical reagents corporation |
| Ethylene glycol | The 500ml/ bottle | Beijing chemical reagents corporation |
| Sodium borohydride | The 50g/ bottle | Beijing chemical reagents corporation |
The acquisition of embodiment 1, H7 subtype avian influenza virus HA gene monoclonal antibody hybridoma
1, the breeding of H7N2 avian influenza virus and purifying
The H7N2 avian influenza virus is inoculated 9 ages in days fertilizations SPF chicken embryo, 30 ℃ hatch 2 days after, collect chick embryo allantoic liquid, the H7N2 strain virus after obtaining increasing.Collect the H7N2 avian influenza virus, at 4 ℃ with 0.03% formalin deactivation, deactivation H7N2 avian influenza poisons HA detects, (concrete grammar that HA titer determination and HI detect is referring to WHO to determine the titre of deactivation H7N2 avian influenza venom, we select HA=1024, and this H7N2 avian influenza strain is provided by China Animal Disease Control And Prevention Center).
With centrifugal 30 minutes of H7N2 avian influenza virus inactivation of viruses liquid 8000rpm, stay supernatant, will precipitate with ultrasonic treatment (impacting 3sec, intermittently 5sec) 3 minutes at every turn, 8000rpm got supernatant in centrifugal 30 minutes again.Above-mentioned supernatant is mixed through 8000 centrifugal 25 hours, pour out supernatant, precipitate with about 3ml physiological saline piping and druming sucking-off, the cleer and peaceful HA that blows and beats out precipitation tires in the survey.Carry out 40%, 50%, 60% sucrose density gradient according to ordinary method, centrifugal 120 minutes of 25000～30000rpm draws each band survey HA from top to bottom and tires.Get the band that HA tires high and add the dilution of 30ml physiological saline, 5000～30000rpm is after centrifugal 60 minutes, and supernatant discarded will precipitate to dilute with 1.5ml sterilization PBS and evenly be H7N2 avian influenza virus antigen refined solution.The protein concentration of purifying H7N2 avian influenza venom is measured 6.35mg/ml by ultraviolet spectrophotometer, and it is 2 that its HA of HA test survey tires
10
2, Polyclonal Antibody Preparation
H7N2 avian influenza virus antigen refined solution (1000ug/ only) is diluted to 1.5ml and 1.5ml complete Freund's adjuvant equal-volume mixing and emulsifying, adopts back leg muscle multi-point injection method that rabbit is carried out immunity after the emulsification.The 2nd～4 the same dosage H7N2 of usefulness avian influenza virus antigen refined solution 6.35mg/ml) add the emulsification of equal-volume incomplete Freund's adjuvant, with back leg muscle multi-point injection method immunity, each immunity is 2～4 weeks at interval.In 2 weeks after the 4th immunity, be diluted to 2ml with 300ug/ H7N2 avian influenza virus antigen refined solution and carry out booster immunization through abdominal injection.1 week blood sampling behind the booster immunization is used indirect elisa method to detect polyclonal antibody and is tired, and the result is 1:320000.
3, the preparation of hybridoma
1) mouse immune:
With above-mentioned H7N2 avian influenza virus antigen refined solution (6.35mg/ml) and Freund's complete adjuvant equal-volume mixing and emulsifying, through the limb muscle multi-point injection, every per injection 100ul.15d, 29d after the first immunisation add Freund's incomplete adjuvant with the H7N2 avian influenza virus antigen refined solution of same dosage respectively and carry out the second time and immunity for the third time.Immunity back blood sampling for the third time detects HI and tires, reach 1:640 when tiring after, do not add freund's adjuvant and carry out booster immunization through abdominal injection.3～5d gets mouse spleen cell and SP2/0 cytogamy behind the booster immunization.
2) cytogamy:
Getting the highest mouse spleen cell of serum HI titre merges mutually with the SP2/0 murine myeloma cell, earlier spleen is ground and obtain splenocyte suspension, press the 10:1 mixed with the SP2/0 murine myeloma cell of logarithmic phase then, through the PEG1500 effect with two kinds of cytogamy, cell after the fusion is resuspended in the complete screening culture medium of the DMEM that contains HAT and 20%FBS, is positioned in 37 ℃ 5% the CO2gas incubator to cultivate.Merge the 4th day half amount in back and change the complete screening culture medium of DMEM that contains HAT and 20%FBS, amount was changed the complete screening culture medium of DMEM that contains HT and 20%FBS in the 8th day half.
3) cell screening
Above-mentioned cell is drawn each hole culture supernatant and is detected by hemagglutination-inhibition test (HI) after cultivating 10 days on the 96 porocyte plates.This screening method, at avian influenza virus H7 hypotype hemagglutinin, the positive hybridoma of screening is cloned, with the positive hole that detects time cloning and subclone again, all take a sample behind each clone and carry out the HI detection, can stablize inhibition H7 subtype avian influenza virus antigen purification liquid up to the antibody of cell strain secretion, till chicken red blood cell generation aggegation.
4, The selection result:
Obtain a strain at H7 subtype avian influenza virus monoclonal antibody, and the hybridoma of this monoclonal antibody of secretion.
5, the cultivation of hybridoma:
The hybridoma cell strain of stably excreting monoclonal antibody is amplification cultivation in CO2gas incubator earlier, is transferred to 24 holes through 96 holes, transfers to 50ml cell bottle through amplification cultivation.Injection cell in the collecting cell bottle is drawn ascites after 7～10 days from mouse peritoneal in mouse peritoneal then.
6, Purification of Monoclonal Antibodies:
Above-mentioned ascites is handled with 50% ammonium sulfate precipitation earlier, use 20mM phosphate buffered saline buffer (PBS then, pH7.2), use DEAE post purifying under HPLC afterwards, obtain the monoclonal antibody behind the purifying, monoclonal antibody purity after the SDS-PAGE purification Identification, the monoclonal anti bulk concentration of purifying is 0.26ug/ml.
7, the preparation of ascites monoclonal antibody
Induce the ascites legal system in the employing body and be equipped with McAb, concrete steps are as follows: select the above BALB/c mouse of 20g, every intraperitoneal injection sterilising liq paraffin 0.5mL; Behind 14～18d, every abdominal cavity inoculation hybridoma 1～5 * 10
5Individual.Rose in the 7th day behind the implantation cell, observe mouse web portion every day, treat that mouse web portion obviously expands, with 9# syringe needle puncture abdominal cavity, gather ascites, the centrifugal 10mni of 3000prm collects supernatant liquor.
8, H7 subtype avian influenza virus monoclonal antibody culture supernatant and the ascites mensuration of tiring
Prepare 8 unit standard antigens with H7N2 avian influenza virus antigen refined solution, logical blood clotting suppresses experiment (HI) experiment and detects, and measures and tires as following table 1-20 ℃ of preservations.Measurement result shows that H7 subtype avian influenza virus monoclonal antibody ascites is tired very high.
The titration of table 1 monoclonal antibody
9, specific assay
Preparation AlV H1～H15 standard strain, AIV H5, AIV H7 hypotype, AIV H9 hypotype strain isolated, 8 unit antigens of virus such as newcastle disease virus and chicken egg drop syndrome virus, with H7 subtype avian influenza virus monoclonal antibody culture supernatant and ascites it is carried out blood clotting respectively and suppress cross matching, whether detect between this monoclonal antibody specific and other hypotypes exists antigen to intersect, criterion is: have blood clotting to suppress to be judged to cross reaction, no blood clotting suppresses to be judged to no cross reaction, the result shows: H7 subtype avian influenza virus monoclonal antibody does not have the HI activity to other any subtype avian influenza virus standard strains and the isolated strain beyond the H7 hypotype, strain has good reactivity to H7 hypotype different virus, explanation, this strain monoclonal antibody is the specific monoclonal antibody of anti-H7 subtype avian influenza virus, and different H7 subtype virus is all had good specificity.As following table 2:
The test of table 2 antigen-specific
| ? | Cell conditioned medium | Ascites |
| AlV H1 type strain | – | – |
| AlV H2 type strain | – | – |
| AlV H3 type strain | – | – |
| AlV H4 type strain | – | – |
| AlV H5 type strain | – | – |
| AlV H6 type strain | – | – |
| AlV H7 type strain | + | + |
| AlV H8 type strain | – | – |
| AlV H9 type strain | – | – |
| AlV H10 type strain | – | – |
| AlV H11 type strain | – | – |
| AlV H12 type strain | – | – |
| AlV H13 type strain | – | – |
| AlV H14 type strain | – | – |
| AlV H15 type strain | – | – |
| AlV H5 strain isolated 1 | – | – |
| AlV H5 strain isolated 2 | – | – |
| AlV H7 strain isolated 1 | + | + |
| AlV H7 strain isolated 2 | + | + |
| AlV H9 strain isolated 1 | – | – |
| AlV H9 strain isolated 2 | – | – |
| A/Chicken/Hebei/1/2002 | + | + |
| Newcastle disease virus | – | – |
| Chicken egg drop syndrome virus | – | – |
Embodiment 2, H7 subtype avian influenza virus HA antigen enzyme-linked immunologic detecting kit (euzymelinked immunosorbent assay (ELISA), ELISA)
1, the preparation of enzyme plate:
Wrap by the polyclonal antibody of the anti-H7N2 avian influenza virus of rabbit in advance on the polyethylene micropore plate of test kit, this polyclonal antibody bag is used 10mM carbonate salts damping fluid (pH9.6), wraps down at 4 ℃ and is spent the night; Use PBST(10mMPBS+0.05%Tween20 then) washed twice, add again confining liquid (10mM PBS+2% bovine serum albumin, BSA), 37 ℃ the sealing 2 hours, dry final vacuum is drained and is packaged into finished product test kit enzyme plate.
2, the configuration of other compositions of test kit:
A) employing virus cracking liquid is formed:
Lysate A(LB-A): 6%CHAPS+2%Tween-20+1%Tween-80.
Lysate B(LB-B): 100mM PMSF, use the Virahol dissolving, the work final concentration is 2mM.
Lysate C(LB-C): 10mM PBS, PH7.4
B) enzyme marking reagent:
Prepare enzyme mark monoclonal antibody with the sodium periodate oxidation style, concrete steps are as follows.Take by weighing 5mg HRP, be dissolved in the 1ml pure water, it is brown that solution is; Add the freshly prepared 0.06mol/LNaIO4 solution of 1ml (12.8mg/ml), stir 30min with molecular hybridization furnace chamber temperature lucifuge, solution is by the brown deep green that transfers to; Add 1ml and newly join 0.08mol/L ethylene glycol solution (450 μ l ethylene glycol solutions are diluted in the 550 μ l pure water), room temperature is placed 30min, intermittently shakes, and solution recovers light brown; Add the 0.1mol/L carbonate buffer solution 1ml dissolved H7 subtype avian influenza virus monoclonal antibody behind the 5.8mg purifying, the room temperature lucifuge is gently stirred 2～3h; Add 0.2ml sodium borohydride (5mg/ml, fresh preparation), place 2h for 4 ℃, with the stable bond thing; Move into dialysis tubing again, in the pH7.2PBS dialysed overnight, the monoclonal antibody of the anti-H7 subtype avian influenza virus of HRP mark of acquisition adds equal-volume glycerine, and-20 ℃ of preservations are standby after the packing.Be to add the SPF chick embryo allantoic liquid during 1 μ g/ml wraps by plate to do contrast in how anti-concentration then, add the H7N2 virus antigen refined solution through doubling dilution simultaneously, hatch the adding of washing back through the HRP mark monoclonal antibody of doubling dilution, obtain the suitable enzyme marking reagent of extent of dilution.
C) positive control: use the H7 subtype avian influenza inactivation of viruses of suitable titre as positive control.
D) negative control: with the negative contrast of lysate A.
E) developer A liquid:
F) developer B liquid:
G) stop buffer:
H) concentrated cleaning solution: 20 * PBST
I) shrouding film: 2
J) valve bag: 1
K) specification sheets: 1 part
4, testing process:
A) dosing: it is standby that 50ml concentrated solution (20 *) is diluted to 1000ml with distilled water or deionized water.
B) numbering: the corresponding microwell plate of sample is numbered according to the order of sequence, and every plate should be established negative control 3 holes, positive control 2 holes and blank 1 hole (the blank hole does not add sample and enzyme marking reagent, and all the other respectively go on foot identical).
C) sample preparation machine application of sample:
When being liquid, sample (comprises type specimen, chicken embryo culture sample, cell cultures sample): need the prewired an amount of LB-A of 100ul LB-A+4ul LB-B to mix with mixed solution and the vibration of LB-B by every hole.Every hole adds 100ul sample to be measured earlier on micropore then, adds the above-mentioned lysate that 100ul prepares again and reacts.
When sample is dried swab type specimen:
After getting 1ml LB-A+40ul LB-B+1ml PBS and mixing, adds in the specimen tube, vibration dissolving sample, after room temperature leaves standstill 30min, shake again outstanding after, the centrifugal 5min of 6000rpm draws the supernatant detection, every hole adds 100ul and reacts.
When sample is argol just during type specimen:
After getting 1ml LB-A+40ul LB-B+1ml PBS and mixing, add in the specimen tube, argol just be configured to 10%(w/v) excrement suspension sample, vibration dissolving sample, after room temperature left standstill 30min, it was outstanding to shake again, the centrifugal 5min of 6000rpm draws supernatant and detects, and every hole adds 100ul and reacts.
Each detection all needs to arrange the yin and yang attribute control wells, and every hole adds 100ul contrast liquid.
D) hatch: with sealing moderate speed's room temperature (25～28 ℃) vibration 60min on the rearmounted micro oscillator of film shrouding.
E) washing: carefully take the shrouding film off, wash 5 times with washing the plate machine washing, last button is as far as possible done.
F) enzyme-added: as in respective aperture, to add enzyme marking reagent 100ul respectively.
G) hatch: after sealing the film shrouding, put 37 ℃ of incubation 30min.
H) repeating step 6.
I) colour developing: every hole adds developer A, each 50ul of B liquid, the mixing that vibrates gently, 37 ℃ of lucifuge colour developing 30min.
J) measure: every hole adds 1 of stop buffer (50ul), and the mixing that vibrates gently, single wavelength 450nm(need establish the blank hole with microplate reader) or dual wavelength 450nm/630nm measure each hole OD value.
5, the result judges:
A) normal range of negative control: under the normal circumstances, negative control hole≤0.1(negative control hole OD value is if should give up greater than 0.1, if all negative control hole OD values are answered repeated experiments all greater than 0.1.If negative control hole less than 0.03, then calculates by 0.03).
B) normal range of positive control: under the normal circumstances, positive control hole OD value 〉=0.5.
C) threshold value is calculated: negative control hole OD value+0.15
D) positive judgement: sample OD value 〉=threshold value person is the avian influenza virus H7 type HA antigen-reactive positive.
E) negative judgement: sample OD value＜threshold value person is avian influenza virus H7 type HA antigen-reactive feminine gender.
The intact virus that samples such as this test kit can animal excrements sensitive, that detect specifically, mouth and nose solinocrine thing, chicken embryo are cultivated or lytic virus etc.
Embodiment 3, H7 subtype avian influenza virus antibody ELISA immunity detection reagent (euzymelinked immunosorbent assay (ELISA), ELISA)
1, the preparation of enzyme plate:
Wrap by H7N2 avian influenza virus purifying antigen in advance on the polyethylene micropore plate of test kit, coating buffer uses 10mM carbonate buffer solution (pH9.6), wraps down at 4 ℃ and is spent the night; Use PBST(10mM PBS+0.05%Tween20 then) washed twice, and then adding confining liquid (10mM PBS+2% bovine serum albumin BSA), 37 ℃ of sealings 2 hours are dried after removing liquid, vacuumize after to be dried and are packaged into finished product test kit enzyme plate.
3, the configuration of other compositions of test kit:
A) enzyme marking reagent: prepare enzyme mark monoclonal antibody with the sodium periodate oxidation style, concrete steps are as follows.Take by weighing 5mg HRP, be dissolved in the 1ml pure water, it is brown that solution is; Add the freshly prepared 0.06mol/LNaIO4 solution of 1ml (12.8mg/ml), stir 30min with molecular hybridization furnace chamber temperature lucifuge, solution is by the brown deep green that transfers to; Add 1ml and newly join 0.08mol/L ethylene glycol solution (450 μ l ethylene glycol solutions are diluted in the 550 μ l pure water), room temperature is placed 30min, intermittently shakes, and solution recovers light brown; Add the 0.1mol/L carbonate buffer solution 1ml dissolved H7 monoclonal antibody behind the 5.8mg purifying, the room temperature lucifuge is gently stirred 2～3h; Add 0.2ml sodium borohydride (5mg/ml, fresh preparation), place 2h for 4 ℃, with the stable bond thing; Move into dialysis tubing again, in the pH7.2PBS dialysed overnight, the monoclonal antibody of the anti-H7 subtype avian influenza virus of HRP mark of acquisition adds equal-volume glycerine, and-20 ℃ of preservations are standby after the packing.Be to add the SPF chick embryo allantoic liquid during 1 μ g/ml wraps by plate to do contrast in how anti-concentration then, add the H7N2 virus antigen refined solution through doubling dilution simultaneously, hatch the adding of washing back through the HRP mark monoclonal antibody of doubling dilution, obtain the suitable enzyme marking reagent of extent of dilution.
B) positive control: use the polyclonal antibody of anti-H7N2 avian influenza virus of suitable titre as positive control.
C) negative control: use 100% calf serum (NBS) as negative control.
D) developer A liquid:
E) developer B liquid:
F) stop buffer:
G) concentrated cleaning solution: 20 * PBST
H) shrouding film: 2
I) valve bag: 1
J) specification sheets: 1 part
4, testing process:
A) dosing: it is standby that 50ml concentrated solution (20 *) is diluted to 1000ml with distilled water or deionized water.
B) numbering: the corresponding microwell plate of sample is numbered according to the order of sequence, and every plate should be established negative control 3 holes, positive control 2 holes and blank 2 holes (the blank hole does not add sample and enzyme marking reagent, and all the other respectively go on foot identical).
C) application of sample: in respective aperture, add testing sample or feminine gender, positive control 50ul respectively.
D) enzyme-added: as in respective aperture, to add enzyme marking reagent 50ul.
E) hatch: with sealing the film shrouding, put 37 ℃ of incubation 60min behind the mixing.
F) washing: carefully take the shrouding film off, wash 5 times with washing the plate machine washing, last button is as far as possible done.
G) colour developing: every hole adds developer A, each 50ul of B liquid, the mixing that vibrates gently, 37 ℃ of lucifuge colour developing 15min.
H) measure: every hole adds 1 of stop buffer (50ul), and the mixing that vibrates gently, single wavelength 450nm(need establish the blank hole with microplate reader) or dual wavelength 450nm/630nm measure each hole OD value.
5, the result judges:
A) normal range of negative control: under the normal circumstances, negative control hole 〉=0.1
B) normal range of positive control: under the normal circumstances, positive control hole OD value≤0.1.
C) threshold value is calculated: negative control hole OD value average/2
D) positive judgement: sample OD value＜threshold value person is the avian influenza virus H7 hypotype antibody response positive.
E) negative judgement: sample OD value 〉=threshold value person is avian influenza virus H7 hypotype antibody response feminine gender.
This test kit uses the H7 subtype influenza virus HA antigen specific antibody in the competition law detection serum specimen, and it is good to have susceptibility, the characteristics of high specificity.
The colloidal gold kit (golden mark method) of embodiment 4, detection H7 subtype avian influenza virus.
Detection zone (T) bag is by the polyclonal antibody of the anti-H7N2 avian influenza virus of rabbit (H7N2-PcAb) on nitrocellulose filter respectively for this test kit, and check plot (C) bag is by sheep anti-mouse igg (two is anti-).During detection in the sample N7 subtype avian influenza virus monoclonal antibody (Au-McAb) of H7 subtype avian influenza virus and colloid gold label form immune complex (Au-McAb-Ag), because this mixture of chromatography effect moves along film, be combined with the H7N2-PcAb of detection line bag quilt and form mixture (Au-McAb-Ag-PcAb), close with two resistive connections of control line bag quilt and form mixture (Au-McAb-Ag-two is anti-).As positive sample, then can respectively form a red line at detection zone and check plot respectively; As negative sample, then only form a red line in the check plot.
1, the preparation of Radioactive colloidal gold
The 1%HAuCl4 solution adding of getting 1ml fills in the reagent bottle of 100ml distilled water, is heated to boiling, adds 1% trisodium citrate aqueous solution of 5ml rapidly, continues heating, treats that solution presents transparent grape wine redness, cooling, and place 4 ℃ to keep in Dark Place standby.
2, the preparation of Radioactive colloidal gold binding substances pad
With the colloid gold label H7 subtype avian influenza virus monoclonal antibody for preparing, and be sprayed on equably on the glass fibre membrane of strip, treat to be placed on immediately after it soaks fully lyophilize in the Freeze Drying Equipment, add the siccative sealing after the taking-up and preserve standby.
3, the preparation of reaction film
Respectively sheep anti mouse two is resisted and the anti-H7N2 avian influenza virus of rabbit many anti-line, detection lines in contrast according to setting program on nitrocellulose filter with Membrane jetter, and will dry in its placement loft drier, it is standby that taking-up adds siccative sealing preservation.
4, the preparation of sample diluting liquid
Add 1ml Tween20 in 1L0.01M pH value to 7.2PBS, fully mixing is aseptic subpackaged behind 0.45 μ m membrane filtration, every pipe 180 μ l
5, test strip preparation
Sample pad, Radioactive colloidal gold binding substances pad, reaction film, absorbent pad are sticked on (as figure) on the colloidality backboard successively.With the Bio-dot cutting machine colloidality backboard that pastes is cut into the wide test strip of 4mm according to setting program.With the test strip that is up to the standards, in the plastic casing that cleans up of packing into and compress, in the aluminium foil bag of packing into, put into 1 siccative, 1 plastic dropper again, after continuous sealing machine sealing, room temperature preservation gets final product.(notice that will control temperature in assembling, packing and the whole process of sealing is no more than 30 ℃, humidity is no more than 30%)
6, the composition of test kit, effect and purposes
This test kit comprises test strip (30), sample diluting liquid (30), sampling cotton swab (30) and specification sheets (1 part); Mainly for detection of whether containing the H7 subtype avian influenza virus in bird movement, mouth and nose solinocrine thing and the ight soil.
7, using method and result judge
A) specimen preparation:
1) dip in the movement of getting fowl, mouth and nose solinocrine thing with the sampling cotton swab after, insert in the sample diluting liquid pipe, fully stand for standby use behind the mixing.
2) sample should be housed in-20 ℃, sample should be deposited in-70 ℃ if can not detect within the week, avoids multigelation.
3) sample collecting, transportation need to be undertaken by national regulation.
B) operation steps:
1) test strip is taken out from airtight packing bag, be put on the horizontal table top;
2) with dropper from dilute good sample hose, draw sample,
3) dropwise join in the sample well of test strip 4;
4) leave standstill 20 minutes, observations.
8, the result judges (see figure 1):
1) positive: test paper occurs parallel red-purple (or red) line at C with the T zone position, detects the H7 subtype avian influenza virus in the interpret sample;
2) feminine gender: one red-purple (or red) line only appears in test paper in the C zone position, do not detect the H7 subtype avian influenza virus in the interpret sample;
3) invalid: any line does not appear in test paper, illustrates to detect failure, and what please more renew detects.
9, the advantage of colloidal gold kit of the present invention
This test strip is to adopt the diagnostic reagent of new generation of colloidal gold immunochromatographimethod technology development, and detectable sample comprises the intact virus of animal excrements, mouth and nose solinocrine thing, chicken embryo culture or lytic virus etc.This product is easy and simple to handle, reliable, need not plant and instrument, carries the Quality Control contrast, and without any need for additive reagent, display result is clear and definite, is swift in response, and the entire operation time only needs 30min.
The colloidal gold kit (golden mark method) of embodiment 5, detection H7 subtype avian influenza virus antibody.
This test strip respectively on nitrocellulose filter detection zone (T) bag by H7N2 avian influenza virus purifying antigen, check plot (C) bag is by sheep anti-mouse igg (two is anti-), during detection, the antibody in the sample is combined with the H7N2 avian influenza virus purifying antigen of detection zone bag quilt with the H7 of colloid gold label subtype avian influenza virus monoclonal antibody competitiveness.If the sample that adds can obviously suppress the combination of H7 subtype avian influenza virus monoclonal antibody and the H7N2 avian influenza virus purifying antigen of colloid gold label, then show the specific antibody that contains avian influenza virus H7 hypotype in the sample.When not containing avian influenza virus antibody in the sample or be not H7 subtype avian influenza virus antibody, just can not suppress this combination, the color of detection zone is more dark, on the contrary color is more shallow.Be that test sample is positive, then only form a red line in the check plot; As negative sample, then can respectively form a red line at detection zone and check plot respectively.
1, the preparation of Radioactive colloidal gold
The 1%HAuCl4 solution adding of getting 1ml fills in the reagent bottle of 100ml distilled water, is heated to boiling, adds 1% trisodium citrate aqueous solution of 5ml rapidly, continues heating, treats that solution presents transparent grape wine redness, cooling, and place 4 ℃ to keep in Dark Place standby.
2, the preparation of Radioactive colloidal gold binding substances pad
With the colloid gold label H7 subtype avian influenza virus monoclonal antibody for preparing, and be sprayed on equably on the glass fibre membrane of strip, treat to be placed on immediately after it soaks fully lyophilize in the Freeze Drying Equipment, add the siccative sealing after the taking-up and preserve standby.
3, the preparation of reaction film
With Membrane jetter on nitrocellulose filter according to setting program anti-and H7N2 avian influenza virus purifying antigen line, the detection line in contrast with sheep anti mouse two respectively, and it is placed in loft drier and dry, take out and add the siccative sealing and preserve standby.
4, the preparation of sample diluting liquid
Add 1ml Tween20 in 1L0.01M pH value to 7.2PBS, fully mixing is aseptic subpackaged behind 0.45 μ m membrane filtration, every pipe 180 μ l
5, test strip preparation
Sample pad, Radioactive colloidal gold binding substances pad, reaction film, absorbent pad are sticked on (as figure) on the colloidality backboard successively.With the Bio-dot cutting machine colloidality backboard that pastes is cut into the wide test strip of 4mm according to setting program.With the test strip that is up to the standards, in the plastic casing that cleans up of packing into and compress, in the aluminium foil bag of packing into, put into 1 siccative, 1 plastic dropper again, after continuous sealing machine sealing, room temperature preservation gets final product.(notice that will control temperature in assembling, packing and the whole process of sealing is no more than 30 ℃, humidity is no more than 30%)
6, the composition of test kit, effect and purposes
This test kit comprises test strip (30), sample diluting liquid (30), sampling cotton swab (30) and specification sheets (1 part); This test kit can be used for detecting whether contain H7 subtype avian influenza virus antibody in the bird serum.
7, using method and result judge
A) specimen preparation:
1) take blood sample, after leaving standstill, separation of serum is standby.
2) sample should be housed in 4 ℃, sample should be deposited in-20 ℃ if can not detect within the week, avoids multigelation.
3) sample collecting, transportation need to be undertaken by national regulation.
B) operation steps:
1) test strip is taken out from airtight packing bag, be put on the horizontal table top;
2) with dropper from dilute good sample hose, draw sample,
3) dropwise join in the sample well of test strip 4;
4) leave standstill 20 minutes, observations.
8, the result judges (see figure 2):
1) positive: one red-purple (or red) line only appears in test paper in the C zone position, detect H7 subtype avian influenza virus antibody in the interpret sample;
2) feminine gender: test paper occurs parallel red-purple (or red) line at C with the T zone position, does not detect H7 subtype avian influenza virus antibody in the interpret sample;
3) invalid: any line does not appear in test paper, illustrates to detect failure, and what please more renew detects.
9, the advantage of colloidal gold kit of the present invention
This test strip is to adopt the diagnostic reagent of new generation of colloidal gold immunochromatographimethod technology development, can detect whether contain H7 subtype avian influenza virus antibody in the serum.This product manual dexterity, special, easy, reliable, need not plant and instrument, carry Quality Control contrast, without any need for additive reagent, display result is clear and definite, is swift in response, the entire operation time only needs 30min.
Claims (9)
1. hybridoma cell strain, its preserving number is CGMCC No.7308.
2. a H7 subtype avian influenza virus monoclonal antibody is got by the described hybridoma cell strain secretion of claim 1.
3. the application of the described monoclonal antibody of claim 2 in detecting H7 subtype avian influenza virus or H7 subtype avian influenza virus antibody.
4. the reaction carriers for detection of the H7 subtype avian influenza virus is characterized in that, described reaction carriers directly or indirectly is coated with the described monoclonal antibody of claim 2.
5. a test kit that detects the H7 subtype avian influenza virus is characterized in that, includes the described reaction carriers of claim 4.
6. the reaction carriers for detection of H7 subtype avian influenza virus antibody is characterized in that, described reaction carriers directly or indirectly is coated with the described monoclonal antibody of claim 2.
7. a test kit that detects H7 subtype avian influenza virus antibody is characterized in that, includes the described reaction carriers of claim 6.
8. a colloidal gold kit that detects the H7 subtype avian influenza virus is characterized in that, marks the H7 subtype avian influenza virus monoclonal antibody that fiber mat is coated with colloid gold label, i.e. Au-McAb at gold; It is how anti-that detection zone is coated with the anti-H7N2 avian influenza virus of rabbit, i.e. H7N2-PcAb; During detection, in sample, there is the H7 subtype avian influenza virus, then forms immune complex with Au-McAb, and then be combined with the H7N2-PcAb of detection zone, colour developing.
9. a colloidal gold kit that detects H7 subtype avian influenza virus antibody is characterized in that, is coated with the monoclonal antibody of the H7 subtype avian influenza virus of colloid gold label at gold mark fiber mat; Detection zone is coated with H7N2 avian influenza virus purifying antigen; The check plot bag is resisted by sheep anti-mouse igg two; During detection, have H7 subtype avian influenza virus antibody in sample, then the monoclonal antibody of the H7 subtype avian influenza virus of H7 subtype avian influenza virus antibody inhibition colloid gold label is combined with H7N2 avian influenza virus purifying antigen.
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| CN107831306A (en) * | 2017-10-10 | 2018-03-23 | 武汉科前生物股份有限公司 | A kind of H7 subtype avian influenza virus double-antibody sandwich elisa kit and its detection method |
| CN107831306B (en) * | 2017-10-10 | 2020-04-24 | 武汉科前生物股份有限公司 | H7 subtype avian influenza virus double-antibody sandwich ELISA kit and detection method thereof |
| CN107904209A (en) * | 2017-11-03 | 2018-04-13 | 武汉科前生物股份有限公司 | A kind of H7 subtype avian influenza virus monoclonal antibody and application |
| CN107904209B (en) * | 2017-11-03 | 2021-06-15 | 武汉科前生物股份有限公司 | H7 subtype avian influenza virus monoclonal antibody and application thereof |
| 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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| CN115873104B (en) * | 2022-08-09 | 2023-07-14 | 华南农业大学 | Nanometer antibody M124 for H7 subtype avian influenza virus and application thereof |
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