CN1687406A - Monoclonal antibody, testing kit including the monoclonal antibody and application - Google Patents
Monoclonal antibody, testing kit including the monoclonal antibody and application Download PDFInfo
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- CN1687406A CN1687406A CN 200510011521 CN200510011521A CN1687406A CN 1687406 A CN1687406 A CN 1687406A CN 200510011521 CN200510011521 CN 200510011521 CN 200510011521 A CN200510011521 A CN 200510011521A CN 1687406 A CN1687406 A CN 1687406A
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Abstract
The present invention discloses a reagent box for quickly detect chicken and birds flu virus antibody. This reagent box includes body, 96 aperture enzyme mark bar in the box, test paper and sample diluted liquid. The test paper consist of sample cushion, absorb cushion, besmear IgG Fc golden cushion, bundled with NP decect string and IgG quality string pyroxylin film, in turn absorb cushion, pyroxylin film, golden cushion, sample cushion plaster unsopped water supported slice. This invention relates to a kind of special monoclonal antibody cross bred tumour cell BDRPDP and special identify IgGFc monoclonal antibody preparation. The reagent box which detect chicken and birds flu virus has strong diathesis, higher sensitivity, simply manipulation and diagnose quickly distinct characteristic.
Description
Technical field
The invention belongs to immune Application Areas, relate to association areas such as animal molecular biology, immunology.The present invention relates to a kind of preparation of monoclonal antibody specific specifically, comprise the test kit and the application in avian influenza antiviral antibody diagnostic detection thereof of this monoclonal antibody.
Background technology
As everyone knows, eqpidemic diseases such as bird flu, newcastle disease, the fabricius bursa, infectious bronchitis, infectious laryngotracheitis, horse Garrick, egg-decreasing syndrome are the important eqpidemic diseases of the global aviculture of harm, wherein especially with bird flu for very.At the beginning of 2004, the H5N1 subtype highly pathogenic avian influenza has taken place in Asia part countries and regions in succession, China also fails to escape by luck, epidemic situation has in various degree taken place in more than ten province in the whole nation (district) in succession in a short time, caused enormous economic loss for the aviculture of part countries and regions, Asia, therefore government of countries in Asia all pays much attention to the anti-system of bird flu, and has proposed a series of anti-system measure in succession.In existing research, lay particular emphasis on the novel method of exploring diagnosis and identifying avian influenza virus mostly.For example in the patent documentations such as 200410014289.9,200310107732.2 that retrieve, introduced this class detection method respectively.And the monitoring and the detection of avian influenza antiviral antibody still rested on original classical way, as agar diffusion test (Agarose gelimmunodiffusion AGID), hemagglutination-inhibition test (Hemagglutination inhibition HI) and enzyme linked immunosorbent assay (Enzyme-linked Immunosorbent Assay; ELISA), neuraminidase inhibition test (Neuraminidaseinhibitor test NIT), virus neutralization tests (Virus Neutralization Test; VNT) etc., the length that expends time in that these methods have (needs 24～48 hours as agar diffusion test; Hemagglutination-inhibition test needs 2～3 hours; The ELISA test needs 3～5 hours with first-class), the plant and instrument of the needs costliness that has and loaded down with trivial details operative technique can only be finished in the laboratory, are not easy in good time and quick diagnosis.
Immunochromatography (immunochromatography) is immunoassay mode (the Glad C that comes across a kind of uniqueness at the eighties initial stage, Grubb AO.Immunocapillarymigration with enzyme-labeled antibodies:rapidquantification of C-reactive protein in human plasma.Anal Biochem, 1981,116:335-340), the core technology of this method is to be solid phase with the fibre strip chromatographic material, make sample solution swimming on chromatography strip by capillary action, and make simultaneously that the acceptor (as antigen or antibody) at determinand takes place high special on determinand in the sample and the chromatographic material, the immune response of high-affinity.Colloidal gold immunochromatographimethod is analyzed (gold-immunochromatography assay GICA) and is used colloidal gold-labeled method, with Radioactive colloidal gold as tracer, be applied to a kind of novel immunolabelling technique (the Osikowicz G et al One-step chromatographic immunoassay for qualitative determination ofchoricogonadotropin in urine.Clin Chem.1990 of antigen antibody reaction, 36,1586).In the chromatography process, gold marker and prior immobilization are in chromatographic material (nitrocellulose filter for example, be the NC film) on antigen or antibody (detection line) specific immune response takes place and be trapped, further enrichment, form the visible red-purple band of naked eyes, thereby obtain experimental result intuitively, reach the purpose of detection.This method is had relatively high expectations to employed antigen-antibody, requires to have good specificity and high purity.
According to this principle, developed and a lot of colloidal gold immune chromatography rapid detecting test paper strips.Cure application facet the people, used the gold-marking immunity chromatograph test strip at the antigen and the aspects such as antibody, venereal disease cause of disease, bacterium, parasite, tumor marker, cardiovascular diseases mark, medicine and some other protein of hormone, transmissible disease cause of disease both at home and abroad.In animal doctor's application facet, for example also used the gold-marking immunity chromatograph test strip at aspects such as swine fever, canine parvovirus diseases.In diagnosis of chicken eqpidemic disease and context of detection, Chinese invention patent prospectus 99101537.1, disclose a kind of preparation and application of fast diagnostic test paper strip for livestock and poultry pestilence, but the main points of this invention are the detections at the livestock and poultry pestilence cause of disease, do not relate to the detection to the avian influenza antiviral antibody.
Summary of the invention
First purpose of the present invention is the monoclonal antibody that obtains a kind of anti-chicken IgG Fc of high specificity.
Second purpose of the present invention is a kind of test kit that is used to detect the avian influenza antiviral antibody of assembling.
The 3rd order of the present invention is the application of test kit of the present invention in avian influenza virus antibody 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-chicken IgG Fc, the hybridoma daughter cell of secreting this monoclonal antibody is that BDRPDP is deposited in Chinese typical culture collection center (CCTCC) on March 17th, 2005, and deposit number is CCTCC-C200501.Monoclonal antibody that applicant's utilization obtains and avian influenza virus reorganization nucleocapsid protein (NP albumen) be a kind of test kit that is used for rapid detection avian influenza antiviral antibody for core reagent has made up.Test kit of the present invention is by box body and be included in the intravital 96 hole ELIAS strips of box, test strip, sample diluting liquid and form.Test strip (structure iron is shown in Fig. 2,3) is the core of test kit of the present invention, and it is sticked to successively by illustrated order by absorption pad (1), nitrocellulose filter (2), gold mark pad (3), sample pad (4) and assembles on the support slice (7) that does not absorb water.Be coated with the monoclonal antibody of the present invention's preparation of colloid gold label on the gold mark pad; Be coated with detection line (5) and nature controlling line (6) on the nitrocellulose filter, wherein detection line is the avian influenza virus reorganization nucleocapsid protein (NP albumen) of the present invention's preparation, and nature controlling line is the anti-mouse IgG antibody of the present invention's preparation.Sample diluting liquid is 8% salt solution.Described nitrocellulose filter, absorption pad, gold mark pad, sample pad, the support slice that do not absorb water are all available from Millipore company.Described affinity column is available from AmershamBiosciences company.The described chicken IgG Fc segment that is used to screen anti-chicken IgG Fc monoclonal antibody, mouse source monoclonal antibody hypotype identification kit are all available from ROCKLAND company.
Principle of the present invention be select affinity purification for use the avian influenza antiviral antibody is had specific reorganization nucleocapsid protein (NP albumen) as detection line, anti-chicken IgG Fc segment monoclonal antibody is as the antibody of colloid gold label, utilize indirect method [Shyu RH etc., Colloidal gold-based immunochromatographic assay for detection of ricin J Toxicon2002,40 (3): 255-258] detect whether contain the avian influenza antiviral antibody in the tested material.During detection, the Fc of all chicken IgG molecules elder generation and the combination of the anti-chicken IgG of golden mark Fc monoclonal antibody in the sample, because capillary action, the reaction mixture is along coated film swimming forward, if the specific antibody of anti-avian influenza virus is arranged in the sample, when arriving detection line, run into the reorganization nucleocapsid protein (NP albumen) that is coated on the nitrocellulose filter, will form NP albumen-antibody-gold labeled monoclonal antibody mixture, thereby be enriched on the detection line, form the red precipitate line; The antibody-gold labeled monoclonal antibody mixture that is not the non-specific antibody formation of anti-avian influenza virus then passes through detection line, is enriched on the nature controlling line, forms the red precipitate line, is judged to positive findings.If do not contain the avian influenza antiviral antibody in the sample, when the reaction mixture arrives detection line, run into capture antigen and just can not form NP albumen-antibody-gold labeled monoclonal antibody mixture, the reaction mixture passes through detection line, be enriched on the nature controlling line, form the red precipitate line, be judged to negative findings.
Compared with prior art the present invention has following advantage:
1, to the detection of avian influenza antiviral antibody, have high specificity, highly sensitive, detection time short (15～20 minutes), the characteristics of test sample wide ranges (serum, yolk, muscle tissue leach liquor all can);
2, detection method of the present invention has the low characteristics of the cost of detection without any need for specific apparatus, equipment;
3, detection kit of the present invention is easy and simple to handle, does not need to be operated by the professional;
4, test kit of the present invention stores conveniently, and is not high to temperature requirement, and effective preservation period can reach 1 year under 4～8 ℃; At room temperature can preserve six months.
Description of drawings
Fig. 1: general technical route map of the present invention
Fig. 2: the side-view of test strip of the present invention, among the figure:
1 is absorption pad, and 2 is nitrocellulose filter, and 3 is gold mark pad, and 4 is sample pad, and 5 is detection line, and 6 is nature controlling line, and 7 is the support slice bar that does not absorb water
Fig. 3: the vertical view of test strip of the present invention, among the figure:
1 is absorption pad, and 2 is nitrocellulose filter, and 3 is gold mark pad, and 4 is sample pad, and 5 is detection line, and 6 is nature controlling line, and 7 is the support slice bar that does not absorb water
Fig. 4: the physical build-up figure of the NP expression of recombinant proteins plasmid that the present invention relates to
Fig. 5: test strip result of the present invention judges synoptic diagram, among the figure:
1 positive result, 2 negative results, 3,4 are the test strip inefficacy.
Embodiment
1, anti-chicken IgG Fc MONOCLONAL ANTIBODIES SPECIFIC FOR:
With reference to Xue Qingshan " philosophy and technique of vitro culture " Science Press calendar year 2001 version, in method: the chicken IgG immunity Balb/C mouse (available from Hubei Province's medical courses in general institute Experimental Animal Center) that utilizes the microorganism at contriver place and immunization experiment chamber to extract, immune programme for children is to get protein solution and isopyknic Fo Shi Freund's complete adjuvant (available from the sigma company) emulsification that contains chicken IgG 50～100 μ g, the injection mouse peritoneal, later every interval was strengthened once in ten days, used Freund emulsification (sigma) instead.At last in merging first three day, (preferably and immunity last time be separated by more than 4 weeks), the abdominal cavity reinforced immunological, the antigen amount doubles, and does not add adjuvant.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
7U 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 grammar is: 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 commodity) substratum, simultaneously also with the HAT substratum suspend the raising splenocyte for preparing and with merge 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 ℃, 5%CO
2Cultivate in the 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 test.Employing as screening antigen, utilizes the ELISA method to filter out the positive hole of the anti-chicken IgGFc segment antibody of secretion available from the chicken IgG Fc segment of ROCKLAND company.Use limiting dilution assay (with reference to Xue Qingshan " philosophy and technique of vitro culture " Science Press calendar year 2001 version) to clone, screen at once to the positive hole that screens.Through 3～4 time clonings, finishing screen is selected the pulsating monoclonal hybridoma of the anti-chicken IgG Fc of secretion system.To this monoclonal hybridoma system that filters out, the applicant is its called after BDRPDP, and send Chinese typical culture collection center (CCTCC) preservation on March 17th, 2005, and deposit number is CCTCC-C200501.This clone has been carried out chromosome counting, and the result shows that the chromosomal mean number of SP2/0 is 70, and splenocyte karyomit(e) is 40, and the chromosome number of hybridoma is between 80～94.The chromosome number that all is higher than two parent's cells illustrates the cell of SP2/0 really of fused cell and the hybridization product of splenocyte, and the chromosome number of hybridoma is obviously more than the karyomit(e) of myeloma cell SP2/0.With this clone injection Balb/C mouse peritoneal, manufacture order clonal antibody.Employing is carried out the hypotype evaluation available from the mouse source monoclonal antibody hypotype identification kit (MouseMab Isotyping Test Kit) of ROCKLAND company to the resulting monoclonal antibody of the present invention, and the result is a mouse IgG1 subclass.
2, Purification of Monoclonal Antibodies
With reference to the method in Zhu Li equality " the immunology common experimental method " People's Medical Officer Press 2000 editions: the mouse ascites 5ml that gets gained mixes with an amount of silicon-dioxide, add isopyknic barbitol buffer solution, behind the room temperature vibration 1h, room temperature leaves standstill 30min, get supernatant in clean centrifuge tube, 4 ℃, the centrifugal 10min of 3000r/m; Get supernatant liquor 8ml, add the 16ml0.06mol 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 2.2ml0.1M, 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%.This monoclonal antibody can be used for preparing the reagent that detects the avian influenza antiviral antibody.
Embodiment 2
The proteic preparation of avian influenza virus N P:
Employed molecular biology method is referring to document: Sa nurse Brooker J, and Ritchie EF not, Manny A Disi T edits (Jin Dongyan, Li Mengfeng etc. translate).The molecular cloning experiment guide.Second edition, Beijing Science Press, the method that provides in 1992 is carried out.
1, the clone of NP gene and order-checking
NP gene involved in the present invention is that applicant oneself clone obtains, and the sequence of this gene is logined in the GeneBank database, and accession number is AY788915.This sequence and known array have 97% homology.The clone of NP gene with the order-checking concrete grammar is: [the GeneBank accession number is AY788915 to separate a strain bird flu strain A/chicken/China/HSS2004 (H9N2) who obtains with the immunization experiment chamber with the microorganism at contriver place, the separation of virus is seen document with evaluation: the separating and preliminary evaluation of the diagnosis of Hubei Province's bird flus such as Li Zili and virus strain, China's Preventive Veterinary Medicine newspaper, 2001,23 (3): 146-148] allantoic cavity inoculation AIV is in 9-11 age in days healthy chicken embryo, abandon dead germ before 24 hours, collect 24-96 hour chick embryo allantoic liquid; Under 4 ℃ of conditions 4, the centrifugal 30min of 000rpm gets supernatant; Under 4 ℃ of conditions 30, the centrifugal 60min concentrating virus of 000rpm.With PBS (preparation of the DEPC treating water) dissolution precipitation of pH7.2 ,-70 ℃ of preservations are standby after the packing.According to reference (Deng Guohua etc., avian influenza virus isolate A/Chicken/Xinjiang/1/96 (H14N5) NP gene sequencing China Preventive Veterinary Medicine 1998 06 phases of newspaper) design primer, amplification NP gene.Upstream primer P1:AG
GGTACCTAA TCACTCACTGA; Downstream primer P2:TG
GAATTCTTTAATTGTCGTACTCCTC.P1 is positioned at the promotor upstream, is added with the KpnI site, and P2 includes terminator codon, has added the EcoRI site, and two restriction enzyme sites mark with underscore.The reading frame that includes the NP gene complete between two primers.Get an amount of viral suspension and extract RNA, by the method that provides in TaKaRa RNA PCR Kit (AMV) the Ver.2.1 specification sheets, amplify DNA, adopt UNIQ-10 pillar DNA glue to reclaim test kit (E.Z.N.A Gel Extraction Kit) and reclaim purifying RT-PCR amplified production, cloning vector pMD18-T direct and available from TaKaRa company carries out ligation with the RT-PCR product that reclaims, to connect product then and be transformed in the middle competent cell bacillus coli DH 5 alpha of test kit (available from the commercial reagents box of TaKaRa company), with the complementary changing effect of checking of α.The picking positive colony, (Manny A Disi T edits (Jin Dongyan, Li Mengfeng etc. translate) for Sa nurse Brooker J, Ritchie EF not to adopt alkaline lysis.The molecular cloning experiment guide.Second edition, Beijing Science Press, 1992 editions) extract recombinant plasmid dna, and recombinant plasmid dna is identified.To identify correct recombinant plasmid called after pMD-NP, and send Chinese Shanghai Bo Ya biotech company to check order, and then the relevant data among sequencing result and the GeneBank be carried out homology and compare and divide folding.
2, the structure of expression vector pKG-NP
After pMD-NP cuts with NcoI and SalI enzyme, reclaim the fragment about 1.5kb, then this fragment is connected construction of expression vector with the expression vector pGEX-KG of the Amersham Biosciences company of cutting with same enzyme enzyme.Recombinant expression vector transforms the DH5 α competent cell of prepared fresh, and the transformed bacteria coating is contained 37 ℃ of overnight incubation of LB agar plate of penbritin.Prepare plasmid in a small amount with alkaline lysis after choosing several single bacterium colony overnight incubation, carry out restriction analysis and pcr amplification and identify.With the positive colony called after pKG-NP that obtains.The intestinal bacteria (Escherichia coliBL21/pKG-NP) that comprise this recombinant plasmid are deposited in Chinese typical culture collection center (CCTCC), preservation day: on March 29th, 2005; Deposit number: CCTCC M205026.Positive colony is prepared plasmid DNA in a large number with alkaline lysis, and packing is frozen in-20 ℃.Recombinant expression plasmid pKG-NP makes up flow process as shown in Figure 4
3, the proteic expression of NP
Change the expression plasmid pKG-NP that builds over to expression with among the colibacillary competent cell BL21 codon plus (BL21codon plus is available from Stratagene company), be applied to and contain corresponding microbiotic (paraxin 25 μ g/mL, tsiklomitsin 10 μ g/mL, penbritin 60 μ g/mL) on the LB plate, cultivate 16-18h, grow single bacterium colony for 37 ℃.Several single bacterium colonies of picking are overnight incubation in the 3mL LB that is added with corresponding microbiotic (paraxin 25 μ g/mL, tsiklomitsin 10 μ g/mL, penbritin 60 μ g/mL), carries out the bacterium activation.Dilute go in the fresh LB substratum at 1: 50 overnight culture in proportion, when shaking culture in 37 ℃ (250-300rpm) arrives OD600=0.5-0.8, adding final concentration is 1mmol/L isopropylthio-(IPTG), continues to cultivate abduction delivering 3h.Under 4 ℃ of conditions 8, the centrifugal 15min of 000rpm collects bacterium.Once the back is centrifugal with 0.01M pH7.2 phosphate buffered saline buffer (PBS) washing for bacterium, and fast freeze-thaw is 3-4 time repeatedly, and is resuspended with appropriate amount of buffer solution STE (prescription: 100.0mmol/LNaCl, 10.0mmol/L Tris-Cl, 1.0mmol/L EDTA pH8.0).Use big ultrasonic head, set the pitch time of 10 circulations and 50%, ultrasonication 1min in ice bath.Under 4 ℃ of conditions 12, the centrifugal 15min of 000rpm, abandon precipitation, being concentrated into volume after supernatant fully dialysed to 0.01M pH7.2 PBS is 5mL, press protein purification post (HiTrap affiniy columns, Amersham Biosciences company product) program that provides of specification sheets is carried out affinitive layer purification, obtains the NP albumen of purifying.This albumen can be used for preparing the reagent that detects avian influenza virus antibody.
Embodiment 3
The preparation of rabbit anti-mouse igg antibody:
Referring to chief editors such as He Zhaoyang, " animal immunology experimental technique " Changchun: Jilin science tech publishing house, method in 2002: utilize the microorganism at contriver place and the Balb/C mouse IgG immune health rabbit that extract the immunization experiment chamber, the rabbit anti-mouse igg hyper-immune serum that preparation high specific, height are tired, adopt the saturated ammonium sulphate method slightly to carry to hyper-immune serum, after G-200 crosses post, obtain highly purified rabbit anti-mouse igg antibody.Utilize this antibody to can be used as nature controlling line.
Embodiment 4
The preparation of nitrocellulose filter
1, bag is cushioned the preparation of liquid: the 0.01M pH7.2PBS damping fluid that contains 6% methyl alcohol is cushioned liquid for bag, 0.22 μ membrane filtration mistake, put 4 ℃ standby, one week of validity period.The 0.01M pH7.2PBS buffer formulation of 1000ml 6% methyl alcohol: NaCl8g, KCl 0.2g, Na
2HPO
412H
2O 2.9g, KH
2PO
40.2g, methyl alcohol 60ml, two ionized waters that boil off are settled to 1000ml.
2, the preparation of nitrocellulose filter: be cushioned liquid with bag NP albumen is diluted to 5～10 μ g/ml, adjust machine, be scribed ss the T line, be detection line, the T line is near gold mark pad end, apart from the about 5mm of gold mark pad end; Be cushioned liquid with rabbit anti-mouse igg antibody dilution to 5～10 μ g/ml with bag, adjust machine, be scribed ss the C line, be control line, the C line is near absorption pad, apart from the about 3mm of absorption pad.Two linear distances, 5～8mm, line should be careful, even.37 ℃ of oven dry encapsulate standby.
Embodiment 5
The preparation of Radioactive colloidal gold, golden labeled monoclonal antibody
1, the preparation of solution
(1) preparation of hydrochloro-auric acid: boil off ionized water dissolved chlorine auric acid with two, be made into 1% solution, put 4 ℃ standby, validity period four months.1000ml1% chlorauric acid solution prescription: 10g hydrochloro-auric acid; Two ionized waters that boil off are settled to 1000ml.
(2) preparation of trisodium citrate: with two ionized waters dissolving trisodium citrates that boil off, be made into 1% solution, 0.22 μ membrane filtration mistake, put 4 ℃ standby, validity period is held to 1000ml.
(3) preparation of 0.1M salt of wormwood: boil off the ionized waters preparation with two, 0.22 μ membrane filtration mistake, put 4 ℃ standby, validity period four months.1000ml 0.1M solution of potassium carbonate prescription: 13.8g salt of wormwood; Two ionized waters that boil off are settled to 1000ml.
(4) preparation of 2%PEG-20000: boil off the ionized waters preparation with two, 0.22 μ membrane filtration mistake, put 4 ℃ standby, validity period four months.1000ml2%PEG-20000 solution formula: 20g PEG-20000; Two ionized waters that boil off are settled to 1000ml.
(5) preparation of mark washing preservation liquid: 2% bovine serum albumin (BSA), 0.05% sodium azide (NaN
3), 0.01M pH7.2 PBS solution, 0.22 μ membrane filtration mistake, put 4 ℃ standby, validity period four months.Liquid formula: 20gBSA, 0.5g NaN are preserved in the washing of 1000ml mark
3, 0.01M pH7.2 PBS solution is settled to 1000ml.
2, the preparation of Radioactive colloidal gold:
With two ionized waters that boil off 1% hydrochloro-auric acid is diluted to 0.01%, puts electric furnace and boil, add 2ml 1% trisodium citrate, continue to boil, be shiny red up to liquid and promptly stop heating, supply dehydration after being cooled to room temperature by every 100ml 0.01% hydrochloro-auric acid.The Radioactive colloidal gold outward appearance for preparing should be pure, bright, do not have precipitation and floating matter, one week of validity period.
3, colloid gold label MONOCLONAL ANTIBODIES SPECIFIC FOR:
PH value to 8.2 with 0.1M salt of wormwood is transferred Radioactive colloidal gold adds anti-chicken IgG Fc monoclonal antibody by 8～10 μ g antibody/ml Radioactive colloidal golds, magnetic stirring apparatus mixing 30min, stir adding BSA to final concentration be 1%, left standstill 1 hour.13000rpm, 4 ℃ of centrifugal 30min abandon supernatant, and precipitation is preserved the liquid washed twice with mark washing, with the mark of 1/10th initial Radioactive colloidal gold volumes wash preservation liquid will precipitate resuspended, put 4 ℃ standby, one week of validity period.
Embodiment 6
The preparation of gold mark pad
1, the preparation of confining liquid:
2% BSA, 0.1% Triton X-100,0.05% NaN
3, 0.01M pH7.2 PBS solution, 0.22 μ m millipore filtration filter, put 4 ℃ standby, validity period four months.1000ml confining liquid prescription: 20g BSA, 0.5g NaN
3, 1ml Triton X-100,0.01M pH7.2PBS solution be settled to 1000ml.
2, the preparation of gold mark pad;
Gold mark pad is soaked in the confining liquid behind the 30min, in 37 ℃ of oven dry.The golden traget antibody that will prepare then is layered on the gold mark pad uniformly, 20 square centimeters of every ml soln shops, lyophilize, encapsulation, put 4 ℃ standby.
Embodiment 7
The preparation of test strip sample pad
1, the preparation of confining liquid:
2% BSA, 0.1%Triton X-100,0.05% NaN
3, 0.01M pH7.2 PBS solution, 0.22 μ m millipore filtration filter, put 4 ℃ standby, validity period four months.1000ml confining liquid prescription: 20g BSA, 0.5g NaN
3, 1ml Triton X-100,0.01M pH7.2PBS solution be settled to 1000ml.
2, the preparation of sample pad:
Sample pad is soaked in the confining liquid behind the 30min, in 37 ℃ of oven dry, encapsulation, put 4 ℃ standby.
Embodiment 8
The assembling of test strip
Nitrocellulose filter, glass fibre, absorption pad, sample pad are stacked gradually by figure, be cut into wide little of 3mm.Per ten little one bags add siccative, Vacuum Package.4～8 ℃ of preservations are valid for one year; Normal temperature is preserved, validity period 6 months.
Embodiment 9
The test kit of rapid detection avian influenza antiviral antibody is formed
1, the test kit of rapid detection avian influenza antiviral antibody comprises:
1., test strip one bag (10/bag)
2., one bottle of sample diluting liquid (10ml/ bottle)
3., one of the 96 hole ELIAS strip (12 holes/bar) of band base
2, the preparation of related solution
Sample diluting liquid: sample diluting liquid is 8% NaCl solution.Compound method: 80g NaCl, adding distil water is settled to 1000ml.
Embodiment 10
The using method of test kit of the present invention:
1, specimen preparation: the preparation of chicken serum sample, get 5 μ l serum to be picked up and place in 96 orifice bores, be diluted to 100 μ l with 8% salt solution; After perhaps serum sample being diluted 20 times with 8% salt solution, get 100 μ l and place in 96 orifice bores.The preparation of Ovum Gallus domesticus Flavus sample is got 5 μ l yolk to be picked up and is placed in 96 orifice bores, is diluted to 100 μ l with 8% salt solution; Or with the yolk sample with 20 times of 8% salt solution dilutions after, get 100 μ l and place in 96 orifice bores.Chicken muscle is organized the preparation of leach liquor sample, gets about 1g muscle tissue and places in the glass homogenizer, adds 2ml 8% salt solution, after the abundant homogenate, and 3, the centrifugal 15min of 000rpm gets supernatant liquor 100 μ l and places in 96 orifice bores.
2, detect: take out test kit, equilibrium at room temperature 20 minutes; Open the package, take out test strip, the sample pad end of test strip is immersed in the sample for preparing result of determination in 15～20 minutes.
3, the result judges: as shown in Figure 5, when macroscopic red-purple nature controlling line appears in test strip, macroscopic red-purple detection line do not occur, the result is judged to feminine gender, is designated as "-"; When macroscopic red-purple nature controlling line appears in test strip, simultaneously macroscopic red-purple detection line also appears, and the result is judged to the positive, is designated as "+"; The antibody horizontal of the detected sample of the dark more explanation of detection line color is high more; When macroscopic red-purple nature controlling line does not appear in test strip, no matter macroscopic red-purple detection line whether occurs, the result is judged to test strip and lost efficacy, and should discard.
Embodiment 11
The preparation and the application of the test kit of rapid detection avian influenza antiviral antibody
1, the preparation of the test kit of rapid detection avian influenza antiviral antibody
The test kit for preparing rapid detection avian influenza antiviral antibody by the method for embodiment 1-10.
2, the application of the test kit of rapid detection avian influenza antiviral antibody
2.1, the detection of chicken standard serum
1. specificity test: respectively avian influenza virus H5, H7, H9 hypotype positive serum, newcastle disease (ND) positive serum, chicken infectious bronchitis (IB) positive serum, infections chicken cloacal bursa (IBD) positive serum (above each standard serum is all available from Harbin Veterinary Medicine Inst., China Academy of Agriculture) of standard are tested by embodiment 8 described the inventive method (GICA).The results are shown in Table 1.Table 1 shows that after avian influenza virus H5, the H7 of test strip of the present invention and standard, the test of H9 hypotype positive serum, tangible red-purple band all appears in nature controlling line and detection line; And with physiological saline (blank), SPF chicken negative serum, standard newcastle disease virus (ND), infections chicken cloacal bursa virus (IBD), the test of avian infectious bronchitis virus positive serums such as (IB) after, the red-purple band does not appear in detection line.This shows test strip specificity height of the present invention, does not have any cross reaction with other respiratory tract eqpidemic disease antiviral antibodies of chicken.
Table 1 is used test strip of the present invention to chicken standard serum detected result
| Test sample | Physiological saline | The SPF chicken serum | The ND positive serum | The IB positive serum | The IB positive serum | The H5 positive serum | The H7 positive serum | The H9 positive serum |
| Detected result | - | - | - | - | - | + | + | + |
2. sensitivity test is carried out doubling dilution respectively with avian influenza virus H5, H7, H9 hypotype standard positive serum (available from Harbin Veterinary Medicine Inst., China Academy of Agriculture), final extent of dilution is 1: 2048, respectively gets 100 μ l and dilutes good sample and test by the inventive method (GICA).Simultaneously, suppress (HI) and agar diffusion method (AGID) [Shen Guanxin, Zhou Rulin with conventional blood clotting.Modern immunological experiment technology (second edition) [M], Hubei science tech publishing house, 2002] measure tiring of H5, H7, H9 standard positive serum.Test-results sees Table 2.The result shows, test strip method of the present invention highly sensitive in agar diffusion test, and the effect that suppresses method with blood clotting is suitable.
The sensitivity test result that table 2 the present invention and conventional sense method detect avian influenza virus antibody
| GICA detects | HI detects | AGID detects | |
| H5 hypotype standard positive serum | ????1∶512 | ????1∶1024 | ????1∶4 |
| H7 hypotype standard positive serum | ????1∶1024 | ????1∶1024 | ????1∶8 |
| H9 hypotype standard positive serum | ????1∶1024 | ????1∶1024 | ????1∶8 |
2.2, the detection of censorship chicken serum sample
Gather 166 parts of chicken serums altogether from 3 chicken houses on ground such as Hubei, Henan, (GICA) tests by the inventive method.Simultaneously, tire with conventional blood clotting inhibition (HI) and agar diffusion method (AGID) mensuration.The results are shown in 3.Table 3 shows that GICA method recall rate is 56% (93/166), and the HI method is 58% (97/166), and the two coincidence rate is 84% (78/93, the HI positive/GICA positive); AGID method recall rate is 30% (50/166), lower than GICA method recall rate, be that the AGID method is negative because the susceptibility of GICA method than AGID high 128 times (8/1024), makes when the GICA method is positive, the coincidence rate of the two is 90% (45/50, the AGID positive/GICA positive).
The relatively detection effect of table 3 test strip method of the present invention and several ordinary methods
| The HI detection method | The AGID detection method | The GICA detection method | |
| The negative number of number positive (part) (part) positive rate (%) | ????97 ????69 ????58 | ????50 ????116 ????30 | ????93 ????73 ????56 |
2.3 the detection of Ovum Gallus domesticus Flavus sample
In general, when detecting avian influenza antibody from the ovum gallinaceum yellow liquor, tiring after yolk liquid dilutes 1 times is suitable with serum titer.Therefore, also can be directly come substitute blood serum to carry out the detection that avian influenza antibody is tired with the yolk liquid of chicken.Randomly draw 100 pieces in adult laying hen egg from the chicken house on ground such as Henan, Hubei, (GICA) tests by the inventive method.Simultaneously, tire with inhibition of ordinary method blood clotting and agar diffusion mensuration.The results are shown in Table 4, show that GICA method recall rate is 68% (68/100), the HI method is 72% (72/100), and the two coincidence rate is 96% (65/68, the HI positive/GICA positive); AGID method recall rate is 44% (44/100), lower than GICA method recall rate, be that the AGID method is negative because the susceptibility of GICA method than AGID high 128 times (8/1024), makes when the GICA method is positive, the coincidence rate of the two is 98% (43/44, the GICA positive/AGID positive).
The comparison of table 4 test strip method of the present invention and several conventional sense methods
| The HI detection method | The AGID detection method | The GICA detection method | |
| Number positive (part) | ????72 | ????44 | ????68 |
| Negative number (part) positive rate (%) | ????28 ????72 | ????56 ????44 | ????32 ????68 |
2.4 chicken muscle is organized the detection of leach liquor sample;
Randomly draw laying hen 100 plumages of growing up from the Hubei chicken house, the peace ordinary method is carried out HI test and AGID test behind the collection serum, gets muscle tissue after chicken being put to death again, and (GICA) tests by the inventive method, the results are shown in Table 5.The result shows that GICA method recall rate is 52% (52/100), and the HI method is 55% (55/100), and the two coincidence rate is 100% (52/52, the HI positive/GICA positive); AGID method recall rate is 27% (27/100), lower than GICA method recall rate, be that the AGID method is negative because the susceptibility of GICA method than AGID high 128 times (8/1024), makes when the GICA method is positive, the coincidence rate of the two is 93% (25/27, the GICA positive/AGID positive).Illustrate that test strip of the present invention can be used as the rapid detection that ordinary method is used for chicken meat product bird flu on the market fully.
Table 5 test strip method of the present invention and several ordinary method are organized the comparison of leach liquor sample detection effect to chicken muscle
| The HI detection method | The AGID detection method | The GICA detection method | |
| The negative number of number positive (part) (part) positive rate (%) | ????55 ????45 ????55 | ????27 ????73 ????27 | ????52 ????48 ????52 |
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 (8)
1, a kind of hybridoma cell line BDRPDP is deposited in CCTCC, and deposit number is CCTCC-C200501.
2, a kind of monoclonal antibody that can discern chicken IgG Fc specially.
3, the detection kit that comprises claim 2.
4, the described test kit of claim 3 is characterized in that said test kit is the test kit that detects the avian influenza antiviral antibody.
5, a kind of test kit that detects the avian influenza antiviral antibody that is applicable to, it comprises box body, is located at the intravital 96 hole ELIAS strips of box, test strip and sample diluting liquid.
6, the described a kind of test kit that detects the avian influenza antiviral antibody that is applicable to of claim 5, test strip wherein are to stick on upward formation of the support slice (7) that do not absorb water by the order of absorption pad (1), nitrocellulose filter (2), gold mark pad (3), sample pad (4) successively by sample pad, absorption pad, the gold mark pad that is coated with the anti-chicken IgG of golden mark Fc monoclonal antibody, the nitrocellulose filter that is coated with the detection line (4) of NP recombinant protein and is coated with the nature controlling line (5) of anti-mouse IgG.
7, claim 5 and 6 described a kind of test kits that detect the avian influenza antiviral antibody that are applicable to, sample diluting liquid wherein is 8% salt solution.
8, the application of each described test kit of claim 3-7 in detecting the avian influenza antiviral antibody.
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