CN100455662C - Avianinflu virus H5 subtype emulsion agglutination kit and its use - Google Patents
Avianinflu virus H5 subtype emulsion agglutination kit and its use Download PDFInfo
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Abstract
This invention discloses a latex agglutination kit to quickly detect H5 subtype of avian influenza virus. The anti monoclonal antibodies of H5 subtype of avian influenza virus hemagglutinin protein were coupled to the surface of carboxyl polystyrene latex particles using water-soluble carbodiimide, the methods about detection of H5 subtype of avian influenza virus were established, the latex agglutination detection kits of H5 subtype of avian influenza virus were prepared supplemented by other matching reagents. This kit was made up of box body and the body latex diagnostic reagents in the box, sample handling liquid A, sample handling liquid B, the positive control samples and negative control samples. This invention kit can directly detect the H5 subtype avian flu virus, with high specificity, high sensitivity, simple and rapid diagnosis, and other significant advantages.
Description
Technical field
The invention belongs to the bird immunity field that learns a skill, particularly, the present invention relates to be applicable to a kind of development and the application in the H5 subtype avian influenza virus detects thereof of avian influenza virus H5 subtype emulsion agglutination assay kit.
Background technology
As everyone knows, the H5N1 subtype highly pathogenic avian influenza is a kind of serious harm aviculture and the deadly infectious disease that jeopardizes human health." Hong Kong bird flu " incident in 1997 is that bird flu is for the first time directly striden kind of propagation and given the people, and South East Asia bird flu in 2004 causes that more the whole world strides the very big concern of kind of propagation to bird flu.China also had in 2004 50 epidemic places of 16 provinces and cities also big area broken out bird flu epidemic situation, caused the heavy losses of China's aviculture.Since in July, 2005, the Vietnam in South East Asia, Thailand, Cambodia, Indonesia, Philippines and China, the Greece in Europe, Romania, Croatia, Britain, Sweden and Russia have reported the generation of H5N1 subtype highly pathogenic avian influenza in succession.The H5N1 subtype highly pathogenic avian influenza is along with the migration of migratory bird has become the world to distribute.Human infection H5N1 bird flu case according to The World Health Organization (WHO) announced on October 24th, 2005 since the end of the year 2003, has had 121 cases, 62 people's death.
At present, detect the main virulent isolation identification of etiology method, neutralization test (SN), agar diffusion test (AGP), RT-PCR, immunofluorescent test (IF) and the enzyme linked immunosorbent assay (ELISA) etc. of bird flu.The isolation identification of virus is to detect the most basic, the classic methods of AIV, but this method exists inherent some shortcomings part, and the times loaded down with trivial details relatively such as operation, that need are long, is polluted easily and problem such as biological safety.Neutralization test is more loaded down with trivial details, and the personnel that need know a thing or two operate.Agar diffusion and immunofluorescent test are all unsatisfactory aspect susceptibility and specificity, and the field sample is complicated and changeable, disturb greatly, and the sample process difficulty makes the application of these methods in clinical detection be subjected to certain limitation.Though enzyme linked immunosorbent assay more relatively easily, but still need certain plant and instrument and operating skill.Latex agglutination test (Latex Aagglutination Test is called for short LAT) is owing to its technical ability and any special instruments and equipment rapid, simple and need not be special have huge advantage.
Since being used as the material of agglutination test since the middle of last century polystyrene latex particle, because the distinct advantages that it had, as: easy and simple to handle, do not need special instrument, naked eyes are judged, are not also needed operating process is giveed training; Time is short, generally can go out the result within 2 minutes; Cheap, detect single duplicate samples than other serology, etiology method considerably cheaper; Be suitable for on-the-spot detection etc., in Clinical Laboratory, obtained using widely.Its meaning has: the 1. diagnosis of communicable disease: the infection of bacterium, fungi, parasite, rickettsia, virus detects; 2. autoimmune disease diagnosis: as detections such as antinuclear antibody, Rheumatoid factors, polyclonal; Traditional latex agglutination test (LAT) be with inertia polystyrene latex particle as carrier, adsorb specific antigen or antibody, when running into corresponding antibody and antigen composition, can form macroscopic agglutinating particle.When application latex agglutination technology is set up the etiology detection method, generally be to the latex particle surface with specific antibody sensitization, the physics electrostatic adhesion that is combined into non-selectivity of ordinary polystyrene latex and antibody, because the molecular weight of antibody is little, be difficult to be attached to the latex surface, even the antibody on the sensitization also comes off or the sex change inactivation from latex particle easily, cause the good latex reagent preservation period of sensitization short, be difficult to be fit to the needs of product development.For this reason, the applicant (Carboxylated Polystyrene latex COOH) is carrier, is medium with bifunctional reagent water-soluble carbodiimide (EDAC), through chemical reaction antibody (IgG) molecule is coupled to the latex surface to have carboxylic group.When method of the present invention has overcome the generic latex sensitizing antibody, sensitizing dose is few, unstable, shortcomings such as antibody comes off easily, improved the joint efficiency of antibody, and the antibody on the sensitization is not easy to come off from latex particle, and then improves the susceptibility and the quality guaranteed period of latex, has been fit to the requirement of product development and scale operation.
Summary of the invention
First purpose of the present invention is the monoclonal antibody of anti-H5 subtype avian influenza virus hemagglutinin of core reagent that can be used for assembling detection kit that obtains a kind of high specificity.
Second purpose of the present invention is a kind of latex agglutination test kit that is used to be applicable to detection H5 subtype avian influenza virus of assembling.
The 3rd purpose of the present invention is the application of test kit in the H5 subtype avian influenza virus detects.
General technical route map of the present invention is seen shown in Figure 1.
The present invention is achieved in that
The applicant has prepared a kind of monoclonal antibody of high specificity, it is the monoclonal antibody of anti-H5 subtype avian influenza virus hemagglutinin, the hybridoma cell strain ZJLXB-1 that secretes this monoclonal antibody is deposited in Chinese typical culture collection center (CCTCC) on November 28th, 2005, and deposit number is CCTCC-C200518.The applicant utilizes the quick Carboxylated Polystyrene latex of described monoclonal anti system to obtain the latex diagnostic reagent, and is that core reagent has been assembled a kind of latex agglutination test kit that is used for rapid detection H5 subtype avian influenza virus with this latex diagnostic reagent.Test kit of the present invention is by box body and is included in the intravital latex diagnostic reagent of box, sample preparation liquid A, sample preparation liquid B, and positive control sample and negative control sample are formed.
The component of described sample preparation liquid A and B and content by weight/volume is:
Sample preparation liquid A
Na
2B
4O
7·10H
2O 13.3g/L
H
3BO
3 16.08g/L
NaCl 8.5g/L
Replenish distilled water to 1L.
Sample preparation liquid B
Na
2B
4O
7·10H
2O 13.3g/L
H
3BO
3 16.08g/L
NaCl 8.5g/L
N-acetyl-L-cysteine 5g/L
Nonidet P-40(NP-40) 5ml/L
Replenish distilled water to 1L.
Described positive control sample is a bird flu H5 hypotype hemagglutinin 0.5ml/ pipe;
Described negative control sample is a chick embryo allantoic liquid 0.5ml/ pipe.
The present invention has prepared 1 strain and has secreted the hybridoma cell strain ZJLXB-1 of the monoclonal antibody of anti-H5 subtype avian influenza virus hemagglutinin, and this hybridoma cell strain is deposited in CCTCC, and deposit number is CCTCC-C200518.
Obviously, the test kit of the present invention's preparation is applicable in the application that detects on the avian influenza virus, particularly in the application that detects on the H5 subtype highly pathogenic avian influenza virus, application wherein comprises a kind of application that is applicable to avian influenza virus H5 subtype emulsion agglutination assay kit of preparation.
Detection kit of the present invention can directly detect the H5 subtype avian influenza virus in the sample.If contain the H5 subtype avian influenza virus in the sample, then the aggegation of obvious uniformity appearred in latex reagent in 30 seconds, can be judged to positive findings; If do not contain the H5 subtype avian influenza virus in the sample, then latex reagent drop in 30 seconds still is original even emulsus, can be judged to negative findings.
Compared with prior art the present invention has following advantage:
1, test kit of the present invention can directly detect the H5 subtype avian influenza virus, has high specificity, and is highly sensitive, detection time short (going out the result within 30 seconds), the characteristics of test sample wide ranges (chick embryo allantoic liquid, larynx swab and various viscera tissue);
2, test kit of the present invention is applicable to the H5 subtype avian influenza virus is detected, and (H1-H4 H6-H15) has specificity to the avian influenza virus of other hypotype.
3, detection method of the present invention has the low characteristics of the cost of detection without any need for specific apparatus, equipment.Test kit is easy and simple to handle, does not need to be operated by the professional.Test kit long preservative period of the present invention, placing under 4 ℃ of conditions can not influence its susceptibility half a year.
Description of drawings
Fig. 1: be general technical route map of the present invention;
Fig. 2: the Western-blotting of expressing protein detects
Fig. 3: the SF9 cellular form that is untransfected
Fig. 4: the cell of pathology takes place in the reorganization Bacmid that has been transfection
Embodiment
Embodiment 1 anti-H5 subtype avian influenza virus hemagglutinin MONOCLONAL ANTIBODIES SPECIFIC FOR and evaluation
1, MONOCLONAL ANTIBODIES SPECIFIC FOR
With reference to Xue Qingshan, " philosophy and technique of vitro culture ", Science Press, method in the calendar year 2001 version: with bird flu H5 hypotype oil-emulsion inactivated vaccine (producing available from Chinese Harbin biotechnology development company of dimension section) is antigen immune Balb/C mouse (available from Hubei Prov. Academy of Medical Sciences's Experimental Animal Center), immune programme for children is to select 4 BALB/c mouse to carry out immunity, dosage is 200ul/, every subcutaneous multi-point injection in mouse carotid back.Two week backs with the same dose booster immunization once, after two weeks again booster immunization once, dosage be 400ul/ only, at interval around the back in mouse peritoneal injection 400ul antigen.Getting its splenocyte after three days merges by the laboratory ordinary method.During fusion, one of the Balb/C mouse of the last reinforced immunological of learning from else's experience, eye socket sacrificed by exsanguination (collect serum, be positive serum) is soaked the 5min sterilization in 75% alcohol.Aseptic taking-up mouse spleen is isolated splenocyte, with SP2/0 myeloma cell's (SP2/0 myeloma cell is available from Ministry of Health's Wuhan institute of Biological Products) of prepared fresh by 1～2 * 10
7Individual SP2/0 and 10
8Ratio mixing in the 50mL centrifuge tube of individual immunocyte (1: 10～1: 15), 1500r/m, centrifugal 10min.Evacuation supernatant (filter paper of available sterilization blots) knocks the pipe end gently, makes cell precipitation loosening slightly.The centrifuge tube that cell mixture is housed is put in 37 ℃ of water-baths.Slowly splash into 50% polyoxyethylene glycol (PEG) 0.8mL (available from sigma company product) of pre-temperature to 37 ℃ then in 1min, the limit edged stirs with pipette tip gently, continues to stir 1min.1640 (available from the commercial substratum of sigma company) the basal liquid 10mL that slowly adds 37 ℃ of pre-temperature then.Concrete steps are: dropwise splashed into 1mL in first minute.Added 1mL in second minute, added 3mL on the 3rd～4 minute, added remaining 5mL on the 5th minute, each added-time needs slowly to add, and constantly stirs lightly.Add 30mL 1640 liquid at last, also need slowly to add.The centrifugal 5min of 800r/m removes supernatant, places 5～8min in 37 ℃.Suspend with HAT (available from Sigma company commercial product culture medium) substratum, simultaneously also with the HAT substratum suspend the raising splenocyte for preparing and with fusion after cytomixis, add an amount of HAT substratum as required, divide and plant in 96 well culture plates, about 250 μ L/ holes.Once merge and to inoculate 4～8 96 orifice plates.Also can plant less as required, generally press the cell count of SP2/0 and calculate, every hole inoculum size contains 10 approximately
4About SP2/0 cell.In 37 ℃, cultivate in the 5%CO2 incubator.Merging to begin in back second day to observe had pollution-freely, added 1 HAT substratum in the 4th day, and suction in the 8th～10 day goes 100 μ L substratum to change HT (available from sigma company) substratum 100 μ L.Treat that the fused cell colony grows to culture hole 1/4, when substratum omits flavescence, carry out antibody titer and detect.Employing as screening antigen, utilizes HI (blood clotting inhibitions) method to filter out the positive hole of secreting anti-H5 subtype avian influenza virus hemagglutinin antibody available from the bird flu H5 HI antigen of biotechnology development company of Harbin dimension section.To the positive hole that screens use at once limiting dilution assay (with reference to Xue Qingshan, " philosophy and technique of vitro culture ", Science Press, calendar year 2001 version) clone, screen.Through 3～4 time clonings, finishing screen is selected the monoclonal hybridoma strain of the anti-H5 subtype avian influenza virus hemagglutinin of secretion.
2, the evaluation of monoclonal antibody
(1) the HI evaluation of tiring
Employing is measured the tiring of Hybridoma Cell Culture supernatant and mouse ascites as antigen with the method that blood clotting suppresses available from the bird flu H5 HI antigen of biotechnology development company of Harbin dimension section, and wherein to tire be 2 to hybridoma cell line 96 porocyte culture plate cells and supernatant HI
7, it is 2 that Balb/C mouse ascites HI tires
18
(2) evaluation of Ig subclass (type)
Use mouse source monoclonal antibody hypotype identification kit (Mouse Mab Isotyping Test Kit) the resulting monoclonal antibody of the present invention to be identified the monoclonal antibody antibody of gained is through being accredited as IgG 2a subclass available from ROCKLAND company.
(3) specificity is identified
Measure monoclonal antibody and bird flu H9 HI antigen, bird flu H7 HI antigen (available from biotechnology development company of Harbin dimension section) respectively with hemagglutination-inhibition test, NDV, IBV, and the cross reaction of negative urine, the result is all negative, illustrates that the monoclonal antibody that obtains has excellent specificity.
(4) chromosome counting
Carry out the hybridoma chromosome counting according to a conventional method, (SP2/0 myeloma cell's modal number is 70 to the hybridoma modal number of all acquisitions between 85-97, Balb/c mice spleen cell chromosome number is 40), the chromosome number that all is higher than two parent's cells, the hybridoma that proves all acquisitions really is the heterozygote of SP2/0 cell and immune spleen cell.
Through evaluation to monoclonal antibody, determine that this monoclonal hybridoma that filters out is that secreted antibody is the monoclonal antibody of anti-H5 subtype avian influenza virus hemagglutinin, the applicant is with its called after ZJLXB-1, and sending Chinese typical culture collection center (CCTCC) preservation on November 17th, 2005, deposit number is CCTCC-C200501.
3, Purification of Monoclonal Antibodies
With reference to Zhu Li equality, " immunology common experimental method ", method in the People's Medical Officer Press 2000 editions: the mouse ascites 5ml that gets gained mixes with isopyknic silicon-dioxide, add isopyknic barbitol buffer solution, behind the room temperature vibration 1h, room temperature leaves standstill 30min, gets supernatant in clean centrifuge tube, 4 ℃, the centrifugal 10min of 3000r/m; Get supernatant liquor 8ml, add 16ml 0.06mol sodium-acetate buffer, with HCL adjust pH to 4.5, after fully stirring slowly adds sad 132 μ l down, stirring at room 30min, change 4 ℃ of refrigerators then over to and fully precipitate 2h, 4 ℃, the centrifugal 30min of 15000r/m gets supernatant liquor 22ml, the phosphoric acid buffer that adds 2.2ml 0.1M, with NaOH adjust pH to 7.6, slowly adding ammonium sulfate to final concentration under stirring is 0.277g/ml, after 4 ℃ of refrigerators fully precipitate 2h, 4 ℃, the centrifugal 30min of 12000r/m abandons supernatant, and precipitation is resuspended with the phosphoric acid buffer of 5ml 0.1M, the dialysis tubing of packing into, after the abundant dialysis of 5000ml 0.01M pH7.2 phosphate buffered saline buffer (PBS),, at last 3000ml three is boiled off the ionized water dialysis again to the 2000ml distill water dialysis, the protein solution that fully dialysis is good is concentrated into 3ml with polyoxyethylene glycol-20000 (PEG-20000), 4 ℃ then, the centrifugal 30min of 12000r/m abandons precipitation, collect supernatant liquor, recording protein concentration is 1.6mg/ml.Be accredited as the monoclonal antibody of purifying through the SDS-PAGE electrophoresis, purity is 98%.
The foundation of embodiment 2 avian influenza virus H5 subtype emulsion aggegation detection methods
Get 125 μ L, 10% carboxylated latex and put into the EP pipe, pH9.6,0.1M carbonic acid buffer is washed 3 times, use pH4.5 again, 0.01M phosphoric acid buffer selects 3 times, adds water-soluble carbodiimide room temperature (25 ℃) effect 4h in phosphoric acid buffer of 0.5% then, back pH8.5,0.01M borate buffer is washed 3 times, latex is suspended with the 1ml borate buffer adds the terminator termination reaction after the back adds an amount of IgG sensitization certain hour.Its concrete steps are as follows:
1, best coupling IgG amount determines
In the carboxylated latex that the 1mL borate buffer suspends, add the IgG of different amounts respectively, be incremented to 1800 μ g from 200 μ g, 400 μ g, 600 μ g, collect supernatant, remaining protein content in UV-120 spectrophotometric determination supernatant, the coupling efficiency of calculating IgG, formula is as follows:
Initial add-on * 100% of the coupling efficiency of IgG (%)=(the initial add-on of IgG-remaining IgG amount)/IgG
Through verification experimental verification, in 1mL concentration was 1% carboxylated latex, when the IgG amount that adds during for 800-1000 μ g, it is maximum that coupling efficiency reaches, and the sensitivity that this moment, latex detected is the highest, and stability is best.Concrete outcome sees Table 1:
Coupling efficiency during table 1 different I gG add-on
2, the selection of best coupling time
In the carboxylated latex that the 1mL borate buffer suspends, add 1000 μ g IgG, the different time of room temperature effect (0.5,1,2,4,, 6,8,10,12,14h), use up the back and collect supernatant, remaining protein content in UV-120 spectrophotometric determination supernatant, the coupling efficiency of calculating IgG, formula is as follows:
Initial add-on * 100% of the coupling efficiency of IgG (%)=(the initial add-on of IgG-remaining IgG amount)/IgG
Through verification experimental verification, when coupling time reached 8h, the IgG amount that is coupled on the latex beads no longer changed basically, and this moment, coupling efficiency reached maximum, so select for use 8h as best coupling time.Concrete outcome sees Table 2:
The coupling efficiency of the different sensitization of table 2 during the time
3, the selection of linked reaction terminator
Antibody and the excessive coupling of latex beads meeting cause the antibody inactivation, thereby reduce antibody-microballoon matrix and antigen bonded ability.So need in reaction system, introduce other amino group to stop linked reaction.Described in the present invention terminator is thanomin or glycine.The effect of making terminator with thanomin through a large amount of verification experimental verifications is better than glycine, therefore the present invention preferably uses thanomin as terminator, used concentration is 0.1M, and the dosage that uses is to add 50 μ L 0.1M thanomins in 1% the carboxylated latex as 1mL concentration, and the reaction times is 15min.
The preparation of embodiment 3 avian influenza virus H5 subtype emulsion agglutination assay kit positive controls
Select the positive control sample of the H5 subtype avian influenza virus hemagglutinin of the expression in insect cell SF9 for use as test kit.The hemagglutinin gene of H5 subtype avian influenza virus is cloned in the Pfastbac plasmid in the baculovirus BAC TO BAC expression system, has made up Pfastbac-HA intermediate transfer plasmid.In intestinal bacteria DH10BAC, make Pfastbac-HA and transformed clover californica nuclear polyhedrosis virus (ACNPY) genome (BACMID) that homologous recombination in the bacterium take place, thereby obtain to include the recombinant shuttle vector of complete HA gene, and transfection SF9 cell obtains recombinant baculovirus ACNPY-HA., analyze band big or small about visible 70kd through SDS-PAGE electrophoresis and Western-blotting, show that hemagglutinin gene has obtained expression in insect cell.Hemagglutination test (HA test) and indirect immunofluorescence assay prove that further expression product has good hemagglutination activity and immunogenicity, can be used as the positive control sample of test kit.Its concrete preparation process is as follows:
1, the structure of Pfastbac-HA intermediate transfer plasmid
(click in the Genebank website: A/chicken/Hubei/489/2004) strain isolated hemagglutinin gene sequences Design is also synthesized a pair of primer to the H5N1 subtype avian influenza virus of including according to Genebank, and the sequence of this primer is as follows: P1:TT ACT AGT ATG GAG AAA ATA GTGC; P2:GC AAG CTT TTA AAT GCA AAT TCT G, carry out PCR reaction (concrete operations step reference: Sambrook J, Molecular Cloning:A Laboratry Manual. such as Fritsch EF, New York, Cold Spring HarborLaboratory PreSS, 2001).With speI+HindIII difference double digestion PCR product and pfast bac I carrier (available from Invitrogen company), obtain the segment of size respectively for 1.7kb and 0.4kb size, after ligation, transformed into escherichia coli DH5a promptly obtains called after Pfastbac-HA plasmid.
2, the swivel base of HA gene and blue hickie screening
(concrete steps are with reference to Sambrook J according to a conventional method, Molecular Cloning:A LaboratryManual. such as Fritsch EF, New York, Cold Spring Harbor Laboratory Press, 2001) preparation DH10BAC competent cell (available from Invitrogen company), the Pfastbac-HA plasmid is transformed the DH10BAC competent cell, and with converted product with 10
-1, 10
-2, 10
-3Different extent of dilution are coated with three anti-plates, this plate shifts to an earlier date 30min coating 25mg/ml x-gal, and (English name is: IPTG (sec.-propyl-b-d-thiogalactoside) 5ul of 40ul and 200ug/ml B-casomorphin fragment1-5*hydrochloride bovineB-casomorphin fragment 1-5*hydrochloride bovine) makes its inactivation present white colony owing to carrying HA expression of gene box insertion LacZ gene.After selecting white colony and the blue hickie purifying of process three-wheel, (provide according to the bac-to-bac handbook by Invitrogen company, a kind of pamphlet that extensively distributes in public Biotechnology Experiment chamber) extract bacmid (a kind of baculovirus genome), the performing PCR of going forward side by side is identified.Converted product is with 10
-2The visible uniform blue hickie of extent of dilution coating plate, the white colony that the picking colony form is bigger, line purifying three times carries out PCR and identifies.
3, the acquisition of recombinant virus and proteic expression
SF9 cell (available from China typical culture collection center) uses go down to posterity the day before yesterday, when treating that cell confluency reaches 60%-70%, prepares reorganization Bacmid-HA DNA in a small amount, through liposome-mediated direct transfection SF9 cell, and with normal cell in contrast.The observation of cell form if after covering with individual layer after cell 4-5 days obvious cytopathy does not take place still, was collected culture supernatant and was continued and go down to posterity, to cell generation pathology every day.Collect cell culture supernatant and the sick cell of pathology after three days.Because the AcNPY polyhedrosis gene is destroyed, so the reorganization Bacmid that obtains behind the swivel base can not express polyhedrin.Cell cultures to the five days microscopically occur tangible pathology (Cytopathic effect, CPE), referring to Fig. 3, shown in Figure 4, it is big that cell becomes, particularly nucleus expands, the strong irregular particle of visible many refractivities in cell.Collect culture supernatant and the centrifugal cell debris of removing, be saved to-80 ℃ and be recombinant baculovirus ACNPY-HA (this virus is that those skilled in the art can realize according to preparation method of the present invention).
4, the evaluation of expressing protein
With the ACNPY-HA virus infection SF9 cell of collecting, and set up wild poison contrast simultaneously.Collect the sick cell of metainfective 72h, behind SDS-PAGE, the band about a treaty 70KD appears in coomassie brilliant blue staining, and is consistent with the expressed molecular weight of albumen of hemagglutinin gene.According to a conventional method (referring to: Sambrook J, Fritsch EF, Maniatis T.Molecular Cloning:A Laboratry Manual, NewYork:Cold Spring Harbor Laboratory Press, 2001) carrying out Western-blotting detects, anti-with anti-H5 hypotype hemagglutinin gene monoclonal antibody as one, the sheep anti-mouse igg enzyme labelled antibody is as two anti-detections, the Idiotype band occurs at the 70KD place, do not have this band (as shown in Figure 2) behind the contrast wild virus infection cell.The result shows: hemagglutinin gene has obtained expression, and can react with anti-avian influenza hemagglutinin gene monoclonal antibody.
5, the preparation of test kit positive control sample
Recombinant virus AcNPY-HA is infected SF9 cell 2000ml, infect back 72h and collect sick cell, pH7.4; With suspending with 40ml PBS after the 0.01M PBS washed twice, in-20 ℃ of multigelations 2 times, 4 ℃, 12, collect supernatant as positive control sample behind the centrifugal 30min of 000r/min, to divide to install in the 1.5mlEP pipe, branchs loading amount is the every pipe of 0.5ml/, in-80 ℃ of preservations down.Note avoiding multigelation and pollution.
6, the titration of positive control
Get positive control sample and carry out hemagglutination test with avian influenza virus H5 HI antigen, in the present invention, it is qualified that the blood clotting valency reaches 1: 128 and is decided to be.
The sensitivity test of embodiment 4 avian influenza virus H5 subtype emulsion agglutination assay kits and specificity test
1, sensitivity test
With each hypotype of bird flu (H1～15) allantoic fluid virus doubling dilution, detect with the good latex diagnostic reagent of sensitization respectively, discovery can with the H5 subtype avian influenza virus allantoic fluid reaction of dilution in 1: 64 (+++), reached susceptibility (this test-results is by the checking of China national bird flu reference laboratory) preferably.The results are shown in Table 3
Table 3 sensitivity test (n=5)
Annotate:: " ++ ++ " whole latex agglutinations, particle gathers in drop edge, and liquid is transparent fully; " +++" most of latex agglutination, particle is obvious, and liquid is muddy slightly; " ++ " about 50% latex agglutination, but particle is thinner, and liquid is muddy; "+" has a little aggegation, and it is muddy that liquid is; "-" drop is original even emulsus.
2, specificity test
Latex reagent that sensitization is good and the reaction of H5 subtype avian influenza virus are strong positive, with the main Respirovirus of chicken as: newcastle disease virus (NDV), infectious bursal disease virus (IBDV), infectious bronchitis virus (52,120 strain), chicken egg-decreasing syndrome virus (EDSV-76), avian paramyxoviruses 2 types (PMV-2), SPF chick embryo allantoic liquid all do not react, and have reached specificity preferably.The results are shown in Table 4.
Table 4 specificity test (n=5)
The replica test of embodiment 5 avian influenza virus H5 subtype emulsion agglutination assay kits
1, criticizes interior replica test
Prepare 3 batches of avian influenza virus H5 subtype emulsion agglutination assay kits (the test kit lot number is respectively 0411,0412,0413), 20 every batch, in every batch of test kit, randomly draw 3 test kits the H9N2 subtype avian influenza virus and the newcastle disease virus (NDV) of positive control sample, negative control sample, deactivation detected, observe its susceptibility, specificity and stability.The result shows that its susceptibility, specificity and stability do not change, and illustrates that batch interior repeatability of this LAT method is good.Its detected result sees Table shown in 5,6,7:
Revision test (test kit lot number 0411) in the table 5 batch
| The test kit numbering | Positive control sample | The negative control sample | H9N2AIV | NDV |
| 0411-02 | ++++ | - | - | - |
| 0411-11 | ++++ | - | - | - |
| 0411-16 | ++++ | - | - | - |
Revision test (test kit lot number 0412) in the table 6 batch
| The test kit numbering | Positive control sample | The negative control sample | H9N2AIV | NDV |
| 0412-04 | ++++ | - | - | - |
| 0412-12 | ++++ | - | - | - |
| 0412-19 | ++++ | - | - | - |
Revision test (test kit lot number 0413) in the table 7 batch
| The test kit numbering | Positive control sample | The negative control sample | H9N2AIV | NDV |
| 0413-06 | ++++ | - | - | - |
| 0413-07 | ++++ | - | - | - |
| 0413-09 | ++++ | - | - | - |
2, criticize between replica test
At different time preparation 3 batches of avian influenza virus H5 subtype emulsion agglutination assay kits (the test kit lot number is respectively 0411,0412,0413), to detecting, observe its susceptibility, specificity and stability with a H9N2 subtype avian influenza virus and newcastle disease virus (NDV) to positive control sample, negative control sample, deactivation.The result shows, its susceptibility, specificity and stability do not change, illustrate this LAT method batch between repeatability good.Detected result is as shown in table 8.
Revision test between table 8 batch
| The test kit numbering | Positive control sample | The negative control sample | H9N2AIV | NDV |
| 0411 | ++++ | - | - | - |
| 0412 | ++++ | - | - | - |
| 0413 | ++++ | - | - | - |
The preservation period test of embodiment 6 avian influenza virus H5 subtype emulsion agglutination assay kits
1, high temperature is to the destruction (37 ℃ act on 3 days down) of test kit
Descend effect after 3 days in 37 ℃ the avian influenza virus H5 subtype emulsion agglutination assay kit of 3 different batches, with the same batch of test kit while examination criteria feminine gender, the positive control that are kept at 4 ℃, result's (as shown in table 9) shows that high temperature (37 ℃ act on 3 days) is not enough to destroy its stability to the destruction of test kit.
Table 9 high temperature (37 ℃ act on 3 days) is to the destruction of test kit
2, the preservation period of test kit experiment (4 ℃)
To be distributed into aliquot with the latex reagent of a collection of sensitization, under 4 ℃ and room temperature, preserved 1 to 9 months respectively, regularly carried out the LAT test in every month, react with the H9N2 subtype avian influenza virus and the Avian pneumo-encephalitis virus (NDV) of positive control sample, negative control sample, deactivation respectively, observe its susceptibility, specificity and stability, to determine the preservation period of latex reagent.In this test, the applicant has prepared three batches latex diagnostic reagent (lot number 0411,0412,0413), carries out parallel test simultaneously.By this test, latex reagent is preserved its specificity more than 6 months through 4 ℃, and stability, susceptibility all do not change.The results are shown in Table 10,11,12:
Table 10 latex diagnostic reagent preservation period test (lot number 0411)
Annotate: the aggegation degree of " # " expression " ++ ++ "
Table 11 latex diagnostic reagent preservation period test (lot number 0412)
Annotate: the aggegation degree of " # " expression " ++ ++ "
Table 12 latex diagnostic reagent preservation period test (lot number 0413)
Embodiment 7 avian influenza virus H5 subtype emulsion agglutination assay kits are to the detection of the different samples of experimental infection H5 subtype avian influenza virus animal
1, zoogenetic infection testing program
Animal experiment all carries out in the efficient shield retaining of chicken in the animalcule room in three grades of laboratories of Biosafety (PIII), and all superseded animals are all through the cinerator burning disposal.
1) test materials avian influenza virus H5 hypotype strain provides (GeneBank accession number: AY684708.1) by Hua Zhong Agriculture University, because former reason China's agriculture university's animal medicine institute animal virus chamber (contriver of the application unit) special-purpose holding room preservation under the measure of strictness of Biosafety, this material only only limits to biological test effect of the present invention is carried out parallel evaluation, do not relate to the own core technology of the present invention and the structure of material (product), do not relate to environment yet and discharge and externally granting.
2) testing program: measure all chicken serum antibody before the experiment blood clotting inhibition (HI) of H5 subtype avian influenza virus is tired, be divided into 3 classes according to the Ab level: Ab level＞2
5Be the I class, Ab level≤2
5Be the II class, what the Ab level was negative fully is the III class.When attacking poison, the parallel 100EID that establishes of while
50, 10EID
50, 1EID
50And 0.1EID
50Attack the toxic agent amount for 4,12 of each dosage groups, wherein I class, II class, III class are respectively 4.With 100 EID
50Attacking malicious chicken mixing raising but not attacking malicious chicken has 8, and wherein I class, II class are each 4.Attacking malicious mode is 0.3mL/ of chest muscle injection.
2, attack the death condition of poison back chicken
As shown in table 13:
Table 13 infected chicken death condition
As can be seen from Table 13, attack poison after, chicken the earliest is dead within 24 hours, the slowest chicken still survived 30 days after.When the Ab level of attacking malicious chicken greater than 2
5The time, can resist 1EID
50And 0.1EID
50The toxic agent amount of attacking; Less than 2
5The time, can only resist 0.1EID
50The toxic agent amount of attacking, the Ab level is entirely the attack that negative chicken can not be resisted the H5N1 Highly Pathogenic Avian Influenza Virus (HPAIV).Do not attack poison but and 100EID
50Attack the foster mortality ratio of chicken within 96 hours of the mixed group feeding of malicious chicken and reached 87.5%.
The detected result of 3 larynx swabs the results are shown in Table 14:
The detected result of table 14 larynx swab
As can be seen from Table 14, the latex reagent of applicant's preparation can detect the H5N1 avian influenza virus from the larynx swab of attacking malicious chicken.Promptly can detect avian influenza virus after attacking poison back 12h, and also can not detect avian influenza virus the larynx swab of dead chicken yet after attacking malicious 30d, be 1EID when attacking the toxic agent amount
50The time, recall rate is higher relatively, has reached 80.5% and 78.4% respectively.Attacking the toxic agent amount is 100EID
50The time, recall rate is minimum, only has 45.5%.In addition, also can detect the H5N1 avian influenza virus from the larynx swab of natural infection chicken, average recall rate is 65.4%.
4, the detected result of each tissue, internal organs the results are shown in Table 15:
Table 15 is respectively organized the detected result of internal organs
As can be seen from Table 15, the latex reagent of applicant's preparation can detect the H5N1 avian influenza virus from attacking organizing of malicious chicken the internal organs, wherein the recall rate of lungs is the highest, can reach 96.4%, next is a kidney, the recall rate of spleen, liver, pancreas is suitable, then detects not come out from chest muscle.
Embodiment 8 avian influenza virus H5 subtype emulsion agglutination assay kits are to the detection of the different samples of other hypotype of experimental infection avian influenza virus (for example H9, H1, H3, H4 type) animal
1, zoogenetic infection testing program
Animal experiment all carries out in by the efficient shield retaining of chicken in the animalcule room with negative pressure device of checking and accepting permission, and all superseded animals are all through the cinerator burning disposal.
1) fowl influenza virus strain type: (HA tires 2 to the former poison of H9N2 allantoic fluid
10); (HA tires 2 to the former poison of H1N1 allantoic fluid
10); (HA tires 2 to the former poison of H3N2 allantoic fluid
11); (HA tires 2 to the former poison of H4N6 allantoic fluid
11); (HA tires 2 to the former poison of NDV allantoic fluid
8);
2) testing program: the blood clotting of measuring all chickens before the experiment suppresses (HI) tires, and chooses 120 of the negative chickens of antibody horizontal and carries out challenge test.Test is divided into H9N2, H1N1, four artificial infected group of H3N2, H4N6, and 30 every group, the parallel 1000EID that establishes of while
50, 100EID
50, 10EID
50, 1EID
50And 0.1EID
50Attack the toxic agent amount for 5,6 of each dosage groups.Attacking malicious mode is 0.3mL/ of chest muscle injection.
2, the test sample detected result the results are shown in Table 16:
Table 16 test sample detected result
The influenza virus of four different subtypes is suitable to the virulence of chicken, is 1000EID when attacking the toxic agent amount
50The time, 4 chicken deaths were arranged on the 3rd day, and mortality ratio is 3.3% (4/120), but other chicken can both anti-mistake and shows the good mental status, death still appears in chicken in the time of 20 days, and the applicant carries out the LAT detection with the chicken execution and the collection viscera tissue of all survivals after 20 days.As can be seen from Table 16, the latex reagent of applicant's preparation has excellent specificity, and is all negative to the avian flu virus detection result of four hypotypes.
3, negative match-rate
80 parts of negative larynx swabs and 15 parts of viscera tissues of being used to test all pick up from the SPF chicken, and the result of viral isolation identification is all negative, the results are shown in Table 17:
Table 17 negative match-rate detected result
As can be seen from Table 17, for not with the sample of healthy chicken of poison, its negative match-rate of detected result reaches 100% respectively, has eliminated basically by the caused nonspecific agglutination of the impurity in the sample.
4, Avian pneumo-encephalitis virus (NDV) infected chicken LAT detected result
(HA 2 with newcastle disease allantoic fluid virus
11) attack 20 chickens of poison, chest muscle is injected the former malicious 0.5mL of allantoic fluid.By the result of table 18 as can be seen, its specificity coincidence rate has reached 100%.
Table 18 Avian pneumo-encephalitis virus (NDV) infected chicken LAT detected result
Embodiment 9 avian influenza virus H5 subtype emulsion agglutination assay kits and the isolating comparison of viral chicken embryo
1, detects sample: preserve with clinical collection during the H5 subtype avian influenza of 2003-2004 outburst and by animal medicine institute of Hua Zhong Agriculture University Preventive Veterinary Medicine laboratory.
2, the comparative result of bird flu H5 subtype emulsion agglutination reagent box and viral isolation identification
Clinical collection organized pathological material of disease (heart, liver, spleen, lung, kidney, tracheae, cloaca) to do latex agglutination to detect and to separate with the chicken embryo, the results are shown in Table 19.The recall rate of each tissue sample latex agglutination is 50-100%.Wherein cloacal recall rate is minimum to be 50%, and the recall rate of liver, spleen, lung, kidney is up to 100%.And viral chicken embryo separation recall rate is 100%.The results are shown in Table 19,20
The latex agglutination of organizing pathological material of disease of the clinical collection of table 19 detects and chicken embryo separating resulting
The comparative result of table 20 bird flu H5 subtype emulsion agglutination reagent box and viral isolation identification (coincidence rate is 97.8%)
3, interpretation of result
Avian influenza virus H5 subtype emulsion agglutination assay kit separated with the chicken embryo of virus make comparisons, detect totally 500 parts of the various pathological material of diseases collected during the 2003-2004 outburst H5 subtype avian influenza, by chicken embryo isolation identification, 145 parts positive, and 355 parts negative.Avian influenza virus H5 subtype emulsion agglutination assay kit detects 134 parts of positives, 366 parts of feminine genders.Positive rate is 93.1%, and coincidence rate is 93.1%.Negative recall rate is 100%, and coincidence rate is 97%.Total coincidence rate is 97.8%.
The comparison of embodiment 10 avian influenza virus H5 subtype emulsion agglutination assay kits and bird flu H5 hypotype fast diagnose test paper bar
1, susceptibility relatively
Avian influenza virus H5 subtype emulsion agglutination assay kit positive control sample done behind the doubling dilution detect with avian influenza virus H5 subtype emulsion agglutination assay kit and bird flu fast diagnose test paper bar simultaneously, result's (table 21) shows that the susceptibility of avian influenza virus H5 subtype emulsion agglutination assay kit and bird flu fast diagnose test paper bar is suitable.
Table 21 susceptibility comparative result
| H9N2AIV | 1∶2 | 1∶4 | 1∶8 | 1∶16 | 1∶32 | 1∶64 | 1∶128 |
| Latex agglutination | + | + | - | - | - | - | - |
| Test strip | + | + | - | - | - | - | - |
2, specificity relatively
Use avian influenza virus H5 subtype emulsion agglutination assay kit and bird flu fast diagnose test paper bar to detect avian influenza virus H9N2 simultaneously and mainly cause the cause of disease (Avian pneumo-encephalitis virus, infectious bursa of Fabricius virus, infectious bronchitis virus etc.) of respiratory system disease with some, detected result is all negative.Illustrate that two kinds of methods all have specificity preferably.See Table 22.
Table 22 specific detection result
3, coincidence rate relatively
Use avian influenza virus H5 subtype emulsion agglutination assay kit and bird flu H5 hypotype fast diagnose test paper bar to detect 107 parts of clinical tissue samples simultaneously, the bird flu fast diagnose test paper bar detects positive 23 parts altogether, negative 84 parts.Avian influenza virus H5 subtype emulsion agglutination assay kit detects positive 20 parts altogether, negative 87 parts.Both coincidence rates are 97.2%.The results are shown in Table 23.
The coincidence rate comparative result (coincidence rate is 97.2%) of table 23LAT test kit and bird flu H5 hypotype fast diagnose test paper bar test kit
The assembling of embodiment 11 avian influenza virus H5 subtype emulsion agglutination assay kits
1, avian influenza virus H5 subtype emulsion agglutination assay kit comprises:
1) latex antigen 1 pipe (1ml/ pipe)
2) positive control 1 pipe (0.5ml/ pipe)
3) negative control 1 pipe (0.5ml/ pipe)
4) 1 bottle of sample preparation liquid A (100ml/ bottle)
5) 1 bottle of sample preparation liquid B (100ml/ bottle)
2, the preparation of related solution
1) preparation of sample preparation liquid A: take by weighing Na2B4O710H2O 13.3g, H3BO3 16.08g, NaCl 8.5g, ddH2O 800mL, adjust pH are that 8.4 backs are settled to 1000mL with distilled water.4 ℃ of storages are put in autoclaving (121 ℃, 30 minutes high pressure steam) back packing, are used to handle viscera tissue.
2) preparation of sample preparation liquid B: take by weighing Na2B4O710H2O 13.3g, H3BO3 16.08g, NaCl 8.5g, ddH2O 800mL, adjust pH are that 8.4 backs are settled to 1000mL with distilled water.Autoclaving (121 ℃, 30 minutes high pressure steam) back adds the 5g N-acetyl-L-cysteine, 5mL NP-40, and 4 ℃ of storages are put in packing behind the mixing, are used to handle the larynx swab.
3) preparation of positive control: recombinant virus AcNPY-HA is infected SF9 cell 2000ml with 5pfu, infect back 72h and collect sick cell, pH7.4 suspends with 40ml PBS after the 0.01M PBS washed twice, in-20 ℃ of multigelations 2 times, 4 ℃, 12, collect behind the centrifugal 30min of 000r/min behind the supernatant as positive control sample, divide to install in the 1.5mlEP pipe, the branch loading amount is 0.5ml, preserves down for-80 ℃.Note avoiding multigelation and pollution.
4) preparation of negative control: the aseptic SPF chick embryo allantoic liquid of collecting, 4 ℃, 12, adding 0.02%NaN3 is anticorrosion behind the centrifugal 30min of 000r/min.Be distributed into the 0.5mL/ pipe, put-20 ℃ and store down.
The using method of embodiment 12 test kits of the present invention
1, specimen preparation
1) treatment process of clinical chicken embryo isolated viral allantoic fluid sample: it is to be checked that the allantoic fluid sample is got supernatant in the centrifugal 5min of 12000rpm.
2) viscera tissue detects the treatment process of sample: take by weighing viscera tissue piece 0.5 gram, place homogenizer, add sample treatment solution A 1ml, take out after the grinding fully and organize leach liquor, and freeze thawing 3 times, then in 4 ℃, the centrifugal 15min of 12000rpm, it is to be checked to get supernatant.
3) the larynx swab detects the treatment process of sample: pick transudate with aseptic cotton swab in chicken larynx portion, be soaked in then among the 0.8ml sample treatment solution B, on snail whirlpool instrument, extrude the liquid in the cotton swab behind the concuss 1min, abandon cotton swab, in 37 ℃ of incubators, act on 15min after the liquid portion freeze thawing 3 times, back in 4 ℃, the centrifugal 15min of 12000rpm, it is to be checked to get supernatant.
2, detect
Get test sample, positive control, each 8-10 μ l of negative control, on the sheet glass that is placed in, respectively add the latex of equivalent, stirring and evenly mixing shakes observations after 30 seconds.
3, the result judges
1) controlled trial: occur following result in 30 seconds, test can be set up: the aggegation of obvious uniformity appears in positive control and the effect of latex diagnostic reagent, negative control and the not aggegation of latex diagnostic reagent.
2) the aggegation person of the obvious uniformity of appearance is judged to the positive in 30 seconds, and it is negative that drop is original even emulsus in 30 seconds.
Although content of the present invention is to describe in conjunction with present embodiment, can not think limitation of the scope of the invention, scope of the present invention is limited by appended claims.In addition, those skilled in the art carries out various changes or modification to the present invention in the appended claims restricted portion, and these changes or modified forms drop in protection scope of the present invention equally.
Claims (6)
1, a kind of hybridoma cell strain ZJLXB-1 is deposited in Chinese typical culture collection center, and its deposit number is CCTCC-C200518.
2, a kind of is the monoclonal antibody that the hybridoma cell strain excretory of CCTCC-C200518 can be discerned the H5 subtype avian influenza virus specially by deposit number.
3, the latex agglutination assay kit that comprises the monoclonal antibody of claim 2.
4, a kind of latex agglutination assay kit that is applicable to the H5 subtype avian influenza virus, it comprises box body and is located at the intravital latex detection reagent of box, sample preparation liquid A, sample preparation liquid B, positive control sample and negative control sample, it is characterized in that, described latex detection reagent be can with the latex reagent of H5 subtype avian influenza virus specific reaction, be to be that the hybridoma cell strain excretory monoclonal antibody sensitization carboxylated latex of CCTCC-C200518 obtains by deposit number, described sample preparation liquid A, sample preparation liquid B by weight/component of volumeter is as follows:
Sample preparation liquid A:
Na
2B
4O
7·10H
2O 13.3g/L，
H
3BO
3 16.08g/L，
NaCl 8.5g/L，
Replenish distilled water to 1L;
Sample preparation liquid B:
Na
2B
4O
7·10H
2O 13.3g/L，
H
3BO
3 16.08g/L，
NaCl 8.5g/L，
N-acetyl-L-cysteine 5g/L,
Nonidet P-40(NP-40) 5ml/L，
Replenish distilled water to 1L;
Described positive control sample is a bird flu H5 hypotype hemagglutinin 0.5ml/ pipe;
Described negative control sample is a chick embryo allantoic liquid 0.5ml/ pipe.
5, the application of the described monoclonal antibody of claim 2 in preparation H5 subtype avian influenza virus latex agglutination assay kit.
6, the application of the described latex agglutination assay kit of claim 4 in vitro detection H5 subtype avian influenza virus.
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| CN101550188B (en) * | 2008-04-01 | 2012-05-02 | 南方医科大学 | H5 subtype bird-flue virus H5N1 hemagglutinin monoclonal antibody and nucleic acid sequence and preparation method thereof |
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| 免疫金探针快速检测禽流感病毒研究. 刘永德等.上海交通大学学报，第23卷第3期. 2005 * |
| 禽流感的诊断技术研究. 梁锐萍等.畜禽业，第11期. 2004 * |
| 高致病性H5亚型禽流行性感冒病毒血凝素单克隆抗体的制备与初步应用. 陈毅歆等.病毒学报，第21卷第6期. 2005 * |
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