CN101196522B - Newcastle disease immune body immune colloidal gold fast detecting reagent kit and its application - Google Patents
Newcastle disease immune body immune colloidal gold fast detecting reagent kit and its application Download PDFInfo
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- CN101196522B CN101196522B CN2007101691030A CN200710169103A CN101196522B CN 101196522 B CN101196522 B CN 101196522B CN 2007101691030 A CN2007101691030 A CN 2007101691030A CN 200710169103 A CN200710169103 A CN 200710169103A CN 101196522 B CN101196522 B CN 101196522B
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Abstract
The invention belongs to the immunity application field, which relates to the related fields of animal molecular biochemistry and the immunology, etc. The invention discloses a reagent kit used for fast testing the antibody of Newcastle Disease, which comprises a case body, and the test paper card and sample diluent equipped inside the case body, wherein, the test paper card is formed by the absorption pad, nitrocellulose membrane, gold-label pad and sample pad affixed in turn on the non-absorbent supporting flake by taking the sample pad, absorption pad and the HN recombinant proteins coated with the Newcastle Disease as the detection line and the HN recombinant proteins coated with anti-mouse IgG as nitrocellulose membrane of the quality control line. The reagent kit used for testing the corresponding antibody has the obvious advantages of strong specificity, high sensitivity, easy operation and fast diagnosis.
Description
Technical field
The invention belongs to immune application, relate to association areas such as animal molecular biology, immunology.Concretely, the invention belongs to a kind of chicken infectious disease antibody fast detecting kit, be used for chicken serum newcastle disease detection of antibodies, whether infected in newcastle disease virus or the immune chicken body of crossing whether produced corresponding antibody to confirm seized chicken.
Background technology
(Newcastle disease ND) has another name called for checken pest, philippine fowl disease etc. ewcastle disease, is one of main bird infectious disease that endangers at present China's aviculture; Main infringement chicken, turkey, other bird and wild fowl class cause respiratory tract infection and immunosupress; In all kinds of fowl crowds, extensively exist for many years, though respectively there is vaccine to carry out immunity inoculation, vaccinated flock is morbidity again and again still always; It is continuous without cease to cause the state of an illness to be prolonged, and produces for China's aviculture and has caused enormous economic loss.
Domestic most chicken house carries out antibody detection seldom after the immunity of having carried out newcastle disease vaccine, mainly be because there is not simple and efficient method.Monitoring and detection to newcastle disease still rest on original classical way, like agar gel diffusion test (Agarose gel immunodiffusion; AGID), hemagglutination-inhibition test (Hemagglutinationinhibition; HI) and EUSA (Enzyme-linked Immunosorbent Assay; ELISA), neuraminidase inhibition test (Neuraminidase inhibitor test; NIT), virus neutralization tests (Virus Neu-tralization Test; VNT) etc., the length that expends time in that these methods have (needs 24～48 hours like agar gel diffusion test; Hemagglutination-inhibition test needs 2～3 hours; ELISA test needed 3～5 hours etc.), instrument and equipment and complex operating steps that the needs that have are expensive, higher technical operation personnel can only accomplish in the laboratory, are not easy in good time and quick diagnosis.
Immunochromatography (immunochromatography) is the immune analysis method that comes across a kind of uniqueness at the eighties initial stage; The core technology of this method is to be solid phase with the fibre strip chromatographic material; Make sample solution swimming on chromatography strip through capillarity, and make on determinand and the chromatographic material in the sample immune response that high specific, high-affinity take place to the acceptor (like antigen or antibody) of determinand simultaneously.Colloidal gold immunochromatographimethod is analyzed (gold-immunochromatography assay; GICA) be to use colloidal gold-labeled method; With collaurum as tracer; Be applied to a kind of novel immunolabelling technique (the Osikowicz G et al.One-step chroma-tographic immunoassay for qualitative determination of choricogonadotropin in urine.ClinChem.1990 of antigen-antibody reaction; 36,1586).In the chromatography process; Gold label 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 forms macroscopic aubergine band; Thereby obtain experimental result intuitively, reach the purpose of fast detecting.This method is had relatively high expectations to employed antigen-antibody, requires to have good specificity and high-purity.
According to this principle, a lot of colloidal gold immune chromatography rapid detecting test paper strips have been developed.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 disease mark, medicine and some other protein of hormone, infectious disease cause of disease both at home and abroad.Aspect veterinary applications, 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 (patent No. ZL99101537.1); 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 to the livestock and poultry pestilence cause of disease, do not relate to the newcastle disease detection of antibodies.Newcastle epidemic disease antibody semiquantitative fast detection gold mark test paper bar process patent application (application number 200610042408.0); Be to adopt the colloid gold label NDV as gold mark pad; NDV is as detection line, and some drawbacks of this method: the purifying of virus is difficult for standardization, and; It is malicious to loose easily, and biological safety is relatively poor; Its specificity, sensitivity are also wayward; Batch between, batch in difference also bigger; Be difficult to standardized production; And the unexposed biological material specimens that it adopts does not have to submit to the preservation of the biological material specimens that is used for proprietary program yet, and those skilled in the art can't implement this invention and reach the effect shown in this invention.And the principle that the present invention adopts is different with patented claim 200610042408.0; We adopt the Fc section monoclonal antibody of the anti-chicken IgG of colloid gold label as gold mark pad, and the gene engineering method expressed proteins is as detection line, and have some tangible advantages like this: specificity is better, sensitivity is higher; Differences between batches are also less; Do not have the problem of the poison that looses, biological safety is good, produces and also is easy to standardization (table 1).
The comparison of table 1 kit of the present invention and patented claim 200610042408.0
| Detection method of the present invention | Patented claim 200610042408.0 | |
| Colloid gold label quality testing survey line encrusting substance specificity production performance biological safety | The Fc section monoclonal antibody reorganization HN protein-specific of gold mark anti-chicken IgG is better produced easy standardization, between batch, batch in difference less high not have the poison of loosing dangerous | The relatively poor production of NDV new castle disease virus specific is not easy standardization, between batch, batch in differ greatly and low have the poison of loosing dangerous |
Summary of the invention
The objective of the invention is to overcome the prior art defective.Its first purpose is a kind of kit that is used to detect newcastle disease antibody of assembling; Second purpose is the application of kit of the present invention in the newcastle disease antibody test.
General technical route map of the present invention is seen shown in Figure 1.
The present invention is achieved in that
Kit of the present invention is made up of box body and the test card, the sample diluting liquid that are included in the box body.Test card (structural drawing is like Fig. 2, shown in 3) is the core of kit of the present invention, is made up of test strips and test card.Test strips goes up formation by the support slice (7) that does not absorb water that sticks on successively of absorption pad (4), nitrocellulose filter (3), gold mark pad (2), sample pad (1).Be coated with the anti-chicken IgG Fc monoclonal antibody (secrete the hybridoma cell line BDRPDP patent No. of this antibody: ZL200510011521.8, deposit number are CCTCCNO:C200501) of colloid gold label on the gold mark pad (2); Be coated with reorganization newcastle disease virus reorganization hemagglutinin neuraminidase HN albumen on the nitrocellulose filter (NC film) (3) respectively, the nature controlling line (C line) (6) that detection line of formation (T line) (5) and rabbit anti-mouse igg constitute; Test strips packed into constitute test card in the test card (8).Sample diluting liquid is 0.8% sodium chloride solution.Said nitrocellulose filter (NC film), absorption pad, gold mark pad, sample pad, the support slice that do not absorb water are all available from Millipore company.Said affinity column is available from Amersham Biosciences company.Escherichia coli (Escherichia coli) BL21/pKG-6p-1-HN that comprises recombinant plasmid pKG-6p-1-HN has been deposited on Dec 20th, 2007 and has been positioned at Chinese Wuhan City, Hubei Province Wuhan University Chinese typical culture collection center (CCTCC), and deposit number is CCTCCNO:M207205.
Principle of the present invention is to select newcastle disease virus reorganization hemagglutinin (HN) albumen of affinitive layer purification respectively for use; 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 newcastle disease antibody in the seized material.During detection, the Fc fragment of all chicken IgG molecules elder generation and the anti-chicken IgG of golden mark Fc section monoclonal antibody combine in the sample, because capillarity; The reaction compound is along coated film swimming forward, if the specific antibody of anti-newcastle disease is arranged in the sample, when arriving detection line; Run into the recombinant protein that is coated on the nitrocellulose filter; Will form recombinant protein-antibody-gold labeled monoclonal antibody compound, thereby be enriched on the detection line, form specific red precipitate line; If not the antibody-gold labeled monoclonal antibody compound that the specific antibody of anti-newcastle disease forms then can directly pass through detection line, be enriched on the nature controlling line, form the red precipitate line, promptly be judged to positive findings.If do not contain newcastle disease virus antibody in the sample, when the reaction compound arrives detection line, run into capture antigen and just can not form recombinant protein-antibody-gold labeled monoclonal antibody compound; The reaction compound passes through detection line; Be enriched on the nature controlling line, form the red precipitate line, promptly be judged to negative findings.
Advantage of the present invention:
1, one of advantage of the present invention is that biological safety is high.Involved in the present invention to expression vector pGEX-KG be expression vector commonly used in the molecular biology, do not have bio-hazard property, the expression vector pKG-6p-1-HN that makes up on this basis also has no bio-hazard property.Recombination bacillus coli BL21/pKG-6p-1-HN is converted in the molecular biology e. coli bl21 competent cell commonly used after the amicillin resistance screening obtains with expression vector pKG-6p-1-HN, does not also have a bio-hazard property.What the test strips detection line among the present invention encapsulated is the newcastle disease virus reorganization non-structural protein that the present invention prepares, and does not relate to the newcastle disease live virus in the preparation process, the potential danger that does not therefore have the newcastle disease live virus to escape, spread.
2, two of advantage of the present invention is that production cost is low.The required core reagent of newcastle disease antibody assay kit provided by the present invention is anti-chicken IgG Fc monoclonal antibody and newcastle disease virus reorganization hemagglutinin neuraminic acid zymoprotein (HN albumen).Anti-chicken IgG Fc monoclonal antibody can be through secreting the monoclonal hybridoma system (patent No.: ZL200510011521.8 of anti-chicken IgG Fc segment; Deposit number is CCTCC NO:C200501) injection Balb/C mouse peritoneal; Induce ascites, a large amount of acquisition through the sad-ammonium sulfate method purifying of economy.HN albumen can obtain a large amount of expression external through recombination bacillus coli BL21/pKG-6p-1-HN, is fit to large-scale production.
3, with the disclosed data by MoM and MEI that is used for the newcastle disease antibody test, kit of the present invention has the advantage that many additive methods institutes can not compare, as detects quick (10～15min); Without any need for specific apparatus, equipment, can be used for execute-in-place; Easy and simple to handle, only need single step reaction, do not need to operate by the professional; The detection cost is low; Only need a kind of reagent (0.8%NaCl solution) in the testing process, human body is not had harm, environmentally safe; Convenient for storing, not high to temperature requirement, effective storage life can reach 1 year under 4 ℃; 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 card of the present invention, among the figure:
1 sample pad (sample end); 2 is gold mark pad; 3 is nitrocellulose filter (NC film); 4 is absorption pad (handle end); 5 is detection line, i.e. the T line; 6 is nature controlling line, i.e. the C line; 7 is the support slice bar that does not absorb water; 8 is test card; 9 is well.
Fig. 3: the vertical view of test card of the present invention, among the figure:
1 sample pad (sample end); 2 is gold mark pad; 3 is nitrocellulose filter (NC film); 4 is absorption pad (handle end); 5 is detection line, i.e. the T line; 6 is nature controlling line, i.e. the C line; 8 is test card; 9 is well.
Fig. 4: invention test card result judges synoptic diagram, among the figure:
The positive result of a ++ ++; B property result +++; The positive result of c ++; The positive result of d+; The negative result of e; F, g are that test card lost efficacy
Fig. 5: the physical build-up figure of the HN expression of recombinant proteins plasmid that the present invention relates to
Embodiment
The preparation of newcastle disease virus HN albumen
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 HN gene and order-checking
HN gene involved in the present invention is that the NDV La Sota that the applicant includes according to GenBank is that HN gene order oneself clone obtains, and this sequence and known array have 98% homology.The clone of HN gene with the order-checking concrete grammar is: Losota is a strain with newcastle disease (NDV); Buy from Chinese Academy of Agricultural Sciences's Lanzhou veterinary institute; Perhaps with reference to list of references: Sun Shuna, the preliminary foundation [D] of the clonal expression of F gene of NDV strain and HN gene and ELISA antibody detection method thereof, Hua Zhong Agriculture University; 2006; Middle National IP Network, Chinese outstanding master thesis full-text database, http://dlib.edu.cnki.net/kns50/detail.aspx? QueryID=23&CurRec=1.Allantoic cavity is inoculated in 9-11 age in days healthy chicken embryo, abandons dead germ before 24 hours, collects 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 (DEPC the handles the water preparation) dissolution precipitation of pH7.2 ,-70 ℃ of preservations are subsequent use after the packing.The NDV LaSota that includes according to GenBank is a HN gene order design primer, amplification HN gene.Primer sequence is following:
Upstream primer P1:5 '-ATA
GGATCCGGGGCTAGCACACTTA-3 ';
Downstream primer P2:5 '-AGC
CTCGAGCTAGCCAGACTTGGCT-3 '.
P1 is positioned at the promoter upper reaches, is added with the BamHI site, and P2 includes terminator codon, has added Xho I site, and two restriction enzyme sites mark with underscore.Include disappearance cytoplasmic region and the HN gene reading frame of striding the film region sequence 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 instructions; Amplify DNA; Adopt UNIQ-10 pillar DNA glue to reclaim kit (E.Z.N.A Gel Extraction Kit) and reclaim purifying RT-PCR amplified production; The cloning vector pMD18-T RT-PCR product that reclaims is direct and available from TaKaRa company carries out coupled reaction, will connect product then and be transformed in the middle competent cell bacillus coli DH 5 alpha of kit (available from the commercial reagents box of TaKaRa company), with the complementary inspection of α changing effect.The picking positive colony adopts alkaline lysis (Sa nurse Brooker J, not Ritchie EF; Manny A Disi T chief editor (Jin Dongyan etc. translate, the molecular cloning experiment guide. second edition, Beijing Science Press; 1992 editions) extract recombinant plasmid dna, and recombinant plasmid dna is identified.The recombinant plasmid called after pMD-HN that evaluation is correct, and send Chinese Shanghai Bo Ya biotech company to check order, then the relevant data among sequencing result and the GeneBank is carried out homology and compare and divide folding.
2, the structure of expression vector pKG-6P-1-HN
After pMD-HN cuts with BamH I and Xho I enzyme, reclaim the fragment about 1.6kb, then this fragment is connected construction of expression vector with the expression vector pGEX-6P-1-HN of the Amersham Biosciences company of cutting with same enzyme enzyme.Recombinant expression carrier transforms the DH5 α competent cell of prepared fresh, and transformed bacteria is coated with 37 ℃ of overnight incubation of LB agar plate of ampicillin (ampicillin 60 μ g/mL).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-6P-1-HN that obtains (Escherichia coli that comprise this plasmid are deposited in the Chinese typical culture collection center (CCTCC) in the Wuhan University of Chinese Wuhan City, Hubei Province on Dec 20th, 2007, and deposit number is CCTCC NO:M207205).Positive colony is prepared DNA in a large number with alkaline lysis (Manny A Disi T edits (Jin Dongyan etc. translate), molecular cloning experiment guide, second edition, Beijing Science Press, 1992 for Sa nurse Brooker J, Ritchie EF not), and packing is frozen in-20 ℃.It is as shown in Figure 5 that recombinant expression plasmid pKG-6P-1-HN makes up flow process
3, the expression of newcastle disease virus HN albumen
Change the expression plasmid pKG-6P-1-HN that builds over to expression with among the colibacillary competent cell Escherichiacoli BL21 (DE3) (Escherichia coli BL21 (DE3) is available from Stratagene company); Be applied on the LB plate that contains microbiotic (ampicillin 60 μ g/mL); Cultivate 10～12h for 37 ℃; Grow single bacterium colony, several single bacterium colonies of picking are overnight incubation in the 3mL LB that is added with microbiotic (ampicillin 60 μ g/mL), carries out the bacterium activation.Overnight culture (V/V) dilution in 1: 50 is in proportion gone in the fresh LB nutrient culture media; (250～300rpm) to OD600=0.6～0.8 o'clock for shaken cultivation in 37 ℃; Adding final concentration is 1mmol/L isopropylthio-(IPTG), continues to cultivate abduction delivering 4h.Under 4 ℃ of conditions 8, the centrifugal 15min of 000rpm collects bacterium.Bacterium is with 0.01M pH7.2 phosphate buffer (PBS prescription: 140mM NaCl, 2.7mM KCl, 10mMNa
2HPO
4, 1.8mM KH
2PO
4, pH7.2) once the back is centrifugal in washing, and fast freeze-thaw is 3～4 times repeatedly, and is resuspended with appropriate amount of buffer solution STE (prescription: 100.0mmol/L NaCl, 10.0mmol/L Tris-Cl, 1.0mmol/L EDTA pH8.0).Use big ultrasonic head, 400 watts, 20 circulations of setting power and fragmentation 5 seconds, interval 10 seconds, ultrasonication in ice bath.Under 4 ℃ of conditions 12, the centrifugal 15min of 000rpm abandons deposition, and after supernatant was fully dialysed with 0.01M pH7.2 PBS, being concentrated into volume was 5mL, presses protein purification post (Glutathione Sepharose
TM4B HiTrap affinitycolumns, Amersham Biosciences Company products) program that provides of instructions is carried out affinitive layer purification, obtains the HN albumen of purifying.This albumen can be used for preparing the reagent that detects newcastle disease virus antibody.
The preparation of rabbit anti-mouse igg antibody:
1, the extraction of Balb/C mouse IgG
Extraction and the purifying of IgG are pressed (Shen Guanxin etc. such as Shen Guanxin; Modern immunological experiment technology (second edition) [M]; Hubei science tech publishing house, 2002) etc. said method is carried out: get the healthy Balb/C mice serum of 10.0mL and after equivalent 0.8% sodium chloride solution mixes, dropwise add saturated (NH
4)
2SO
4Solution 20.0mL makes into (the NH of 50% saturation degree
4)
2SO
4Solution.4 ℃ are taken out after leaving standstill 30min; 4 ℃ are descended 3, and the centrifugal 30min of 000r/min abandons supernatant, adds the 20.0mL0.8% sodium chloride solution in the deposition; After treating resolution of precipitate, slowly add the saturated (NH of 10.0mL
4)
2SO
4Solution makes into (the NH of 33% saturation degree
4)
2SO
4Solution.Take out behind 4 ℃ of placement 30min; 4 ℃ are descended 3, the centrifugal 30min of 000r/min.Abandon supernatant, after deposition repeats aforesaid operations, deposition be dissolved in 1.0mL0.8% sodium chloride solution (former serum amount volume 1/10), in the bag filter of packing into the 0.8% sodium chloride solution desalination of dialysing.Use 2%BaCl
2Solution detects (NH
4)
2SO
4Whether fully by dialysis.After the dialysis fully, protein solution is concentrated into proper volume with polyglycol (PEG)-20,000, and 4 ℃ are descended 12, and the centrifugal 10min of 000r/min gets supernatant, measures protein content, the Balb/C mouse IgG that is promptly slightly carried.
2, the purifying of Balb/C mouse IgG
Kind per sample and volume and selected chromatographic column are confirmed the kind of selected sephadex and the volume of post bed.The volume of post bed calculates by following formula:
Vt＝πr
2h
(the volume of Vt-post bed; π-circular constant; The r-radius; The h-post height of bed)
Kind per sample, the present invention with sephadex (Sephadex G-200) as chromatographic material.
The detailed process of purifying is following:
Taking by weighing 3.0g Sephadex G-200 pours in the distilled water of several times volume and mixes; Put soaking at room temperature 3d, change distilled water every day 2 times, after the part that slowly upper strata is contained floating particle is outwelled; The distilled water that adds original volume again, gel soak good rearmounted 4 ℃ of refrigerators and preserve subsequent use.
The chromatographic column vertical fixing of cleaning on experiment frame, is added a small amount of eluent (0.01mol/L, pH7.2 PBS), the unobstructed property of inspection drain pipe, qualified after, close liquid outlet.Take out soaked Sephadex G-200 from 4 ℃ of refrigerators, can chromatographic column after balance to the room temperature.On the chromatographic column gel that installs, put the filter paper suitable with column internal diameter gently, the phosphate buffer of 0.01mol/L (is PBS, fills a prescription as follows: 140mM NaCl, 2.7mM KCl, 10mMNa
2HPO
4, 1.8mM KH
2PO
4, pH7.2) equilibrate overnight is reserved the high liquid layer of 1cm～2cm above gel, prepare application of sample.
During application of sample, the liquid layer of gel top is emitted, when treating just to expose filter paper, close liquid outlet immediately.Draw sample with dropper, flowing down along tube wall, get a little 0.8% sodium chloride solution flushing sample bottle, washing lotion is added, clean tube wall with eluent at last near the filter paper place.Open liquid outlet, when sample all gets into the post bed and just filter paper occurred, add eluent immediately, make post bed top form the thick liquid layer of 5cm～10cm.Behind the sample upper prop; Detect eluate with 20% sulfosalicylic acid liquid frequently, when slight milkiness reaction appears in eluate, collect eluate with the 1.5mL EP pipe of having numbered immediately; Every pipe 1mL finishes to collect when eluate can not detect muddiness with 20% sulfosalicylic acid.
After finishing to collect; Measure protein content in every pipe effluent with ultraviolet spectrophotometer, be numbered horizontal ordinate with the EP pipe, the effluent protein content is ordinate curve plotting figure in the pipe; Required solution is collected in distribution according to protein concentration; Pack in the bag filter, PEG-20,000 is concentrated into proper volume.4 ℃ 12, the centrifugal 10min of 000r/min collects supernatant, measures through the Balb/C mouse IgG protein content after concentrating with ultraviolet spectrophotometer, put-20 ℃ frozen.
3, the extraction and purification of the anti-Balb/C mouse of immunity of rabbit and rabbit IgG
Choose the bull healthy rabbits of body weight about 2.5kg; Antigen with preparation in 1 and 2; First immunisation is with the Balb/C mouse IgG of Freund's complete adjuvant emulsification, and immunizing dose is a 2mgIgG/ rabbit, and each time later on is with the Balb/C mouse IgG immunity of incomplete Freund emulsification.Except that head exempts from and two exempts from interval 3 weeks, all the other each immunity 10 days at interval, and the more preceding once high 0.8mg of content of Balb/C mouse IgG in each immunizing dose, each immunization route all adopts subcutaneous multi-point injection.After the immunity 3 times; Blood sampling detects serum titer, and (tire reaches at 1: 32 o'clock to agar gel immunodiffusion test (agar gel immunodiffusion AGID) is called for short agar diffusion test) as serum AGID; Booster immunization is 1 time again; Rear neck artery bloodletting in 10 days, separation of serum is used for the extraction of the anti-Balb/C mouse of rabbit IgG.Method according to 1 and 2 is carried out the extraction and purification of the anti-Balb/C mouse of rabbit IgG.Can obtain the rabbit anti-mouse igg antibody that high specific required for the present invention, height are tired.Utilize this antibody to can be used as the nature controlling line (C line) of colloidal gold test paper card of the present invention.
The preparation of nitrocellulose filter
1 encapsulates the preparation of damping fluid: the 0.01M pH7.2 PB damping fluid that contains 3% methyl alcohol is for encapsulating damping fluid, 0.22 μ membrane filtration mistake, put 4 ℃ subsequent use, one week of the term of validity.The 0.01M pH7.2 PB buffer formulation of 1000ml 3% methyl alcohol: KCl 0.2g, Na
2HPO
412H
2O 2.9g, KH
2PO
40.2g, methyl alcohol 30ml, two ionized waters that boil off are settled to 1000ml.
The preparation of 2 nitrocellulose filters: with encapsulating damping fluid HN albumen is diluted to 50～100 μ g/ml, the adjustment machine is scribed ss the T line, is detection line (T line) near gold mark pad end, apart from the about 5mm of gold mark pad end; With encapsulating damping fluid with rabbit anti-mouse igg antibody dilution to 50～100 μ g/ml, the adjustment machine is scribed ss the C line, is nature controlling line, and 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 subsequent use.
The preparation of collaurum, golden labeled monoclonal antibody
1, the preparation of solution
(1) preparation of gold chloride: boil off ionized water dissolved chlorine auric acid with two, be made into 1% solution, put 4 ℃ subsequent use, the term of validity four months.1000ml 1% chlorauric acid solution prescription: 10g gold chloride; Two ionized waters that boil off are settled to 1000ml.
The preparation of (2) 1% trisodium citrates: with two ionized water dissolving trisodium citrates that boil off, be made into 1% solution, 0.22 μ m membrane filtration mistake is joined existing usefulness at present.
(3) preparation of 0.1Mol/L sal tartari: boil off the ionized waters preparation with two, 0.22 μ m membrane filtration mistake, put 4 ℃ subsequent use, the term of validity four months.1000ml 0.1Mol/L solution of potassium carbonate prescription: 13.8g sal tartari; 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 m membrane filtration mistake, put 4 ℃ subsequent use, the term of validity four months.1000ml 2%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) (BSA), 0.05% Sodium azide (NaN
3), 0.01MpH7.2 PBS solution, 0.22 μ m membrane filtration mistake, put 4 ℃ subsequent use, the term of validity four months.Formula of liquid is preserved in the washing of 1000ml mark: 20g BSA, 0.5g NaN
3, 0.01M pH7.2 PBS solution is settled to 1000ml.
2, the preparation of collaurum:
Boil off ionized water 1% gold chloride is diluted to 0.01% with two, put 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% gold chloride.The collaurum outward appearance for preparing should be pure, bright, do not have deposition and floating thing, one week of the term of validity.
3, colloid gold label MONOCLONAL ANTIBODIES SPECIFIC FOR:
PH value to 8.2 with 0.1M sal tartari is transferred collaurum adds anti-chicken IgG Fc section monoclonal antibody by 8～10 μ g antibody/ml collaurums, 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 deposition is preserved the liquid washed twice with mark washing, with the mark of 1/10th initial collaurum volumes wash preservation liquid will precipitate resuspended, put 4 ℃ subsequent use, one week of the term of validity.
The preparation of gold mark pad
The preparation of 1 confining liquid:
2% bovine serum albumin(BSA) (BSA), 1%Tween-20,2.5% sucrose, 0.05%NaN
3, 0.01M pH7.2PB solution, 0.22 μ m miillpore filter filters, put 4 ℃ subsequent use, the term of validity four months.1000ml confining liquid prescription: 20g BSA, 0.5g NaN
3, 10ml Tween-20,25g sucrose, 0.01M pH7.2 PBS solution be settled to 1000ml.
The preparation of 2 gold medals mark pad:
Gold mark pad is soaked in the confining liquid behind the 30min, in 37 ℃ of oven dry.The golden labelled antibody that will prepare then is layered on the gold mark pad uniformly, 20 square centimeters of every ml soln shops, freeze drying, encapsulation, put 4 ℃ subsequent use.
The preparation of test strips sample pad
The preparation of 1 confining liquid:
2%BSA, 0.5%Tween-20,2.5% sucrose, 0.05%NaN
3, 0.01M pH7.2 PBS solution, 0.22 μ m miillpore filter filters, put 4 ℃ subsequent use, the term of validity four months.1000ml confining liquid prescription: 20g BSA, 0.5g NaN
3, 5ml Tween-20,2.5g sucrose, 0.01M pH7.2 PBS solution be settled to 1000ml.
The preparation of 2 sample pad:
Sample pad is soaked in the confining liquid behind the 30min, in 37 ℃ of oven dry, encapsulation, put 4 ℃ subsequent use.
The assembling of test card
Nitrocellulose filter, absorption pad, spun glass, sample pad are stacked gradually by figure, be cut into wide little of 3.6mm, the test card of packing into then.Per ten one bags add drying agent, Vacuum Package.4～8 ℃ of preservations are valid for one year; Normal temperature is preserved, the term of validity 6 months.
The kit of 1 fast detecting newcastle disease virus antibody comprises:
1. test card one wraps (10/bag)
2. one bottle of sample diluting liquid (10ml/ bottle)
The preparation of 2 related solution
The sample diluting liquid of the kit of fast detecting newcastle disease virus antibody: sample diluting liquid is a 0.8%NaCl solution.Compound method: 8gNaCl, adding distil water is settled to 1000ml.
Embodiment 9
The method of application of kit of the present invention:
1, specimen preparation: the preparation of chicken serum sample, with blood serum sample with 20 times of 0.8% sodium chloride solution dilutions.
2, detect: take out kit, equilibrium at room temperature 20 minutes; Open the package, take out test card, get the sample that 100 μ l prepare and splash in the well of test card result of determination in 10～15 minutes.
3, the result judges: as shown in Figure 4, and when macroscopic aubergine nature controlling line appears in test card, macroscopic aubergine detection line does not appear, and the result is judged to feminine gender, is designated as "-"; When macroscopic aubergine nature controlling line appears in test card, simultaneously macroscopic aubergine detection line also appears, and the result is judged to the positive, is designated as "+"; Antibody titer in detection line color depth and the seized serum is proportionate, and color explains that more deeply the antibody titer of sample to be detected is high more; The detection line color is dense, with the nature controlling line solid colour or near the time be judged to ++ ++, color depth between+and ++ ++ between the time take the circumstances into consideration to declare ++ or +++.When reaching+reaching above color depth, the antibody that is judged to the seized serum of this part is positive.Nature controlling line does not have band and the inefficacy of detection test card occurs then being judged to.
Embodiment 10
The kit stability assessment of fast detecting infections chicken cloacal bursa antibody
Design temperature: 20 ℃, 4 ℃
Design time: 0 day, 10 days, 20 days, 30 days, February, April, June, August, October, Dec
Store method: test card Vacuum Package (be stored in 4 ℃ the refrigerator or 20 ℃ room temperature in)
Performance assessment criteria: susceptibility
Table 2 kit test card of the present invention stability test result
| 0 day | 10 days | 20 days | 30 days | February | April | June | August | October | Dec | |
| 0℃ 20 |
1∶512 1∶512 | ?1∶512?1∶512 | 1∶5121∶512 | ?1∶512?1∶512 | ?1∶512?1∶512 | ?1∶512?1∶256 | ?1∶512?1∶128 | Lost efficacy at 1: 256 | 1∶128 | 1∶64 |
Embodiment 11
The preparation and the application of the kit of fast detecting newcastle disease antibody
1, the preparation of the kit of fast detecting newcastle disease antibody
The kit for preparing fast detecting newcastle disease antibody by the method for embodiment 1-9.
2, the application of the kit of fast detecting newcastle disease antibody
2.1 the detection of chicken standard serum
1. specificity test: respectively newcastle disease (ND) positive serum, infectious bronchitis of chicken (IB) positive serum, infections chicken cloacal bursa (IBD) positive serum (available from China Veterinery Drug Inspection Office), avian influenza virus H5, H7, H9 hypotype positive serum (available from Harbin Veterinary Medicine Inst., China Academy of Agriculture) are made an experiment by the embodiment of the invention 9 said methods (GICA).The result sees table 3.Table 3 shows that the present invention detects test card (GICA) and tangible aubergine band all occurs with newcastle disease (ND) standard positive serum test back nature controlling line and detection line; And with distilled water (blank), SPF chicken negative serum, avian influenza virus H5, H7, H9 hypotype; After infections chicken cloacal bursa (IBD), the test of avian infectious bronchitis virus positive serums such as (IB); The aubergine band does not appear in detection line, and obvious aubergine band appears in nature controlling line.This shows that test card specificity of the present invention is high, does not have any cross reaction with other respiratory tract eqpidemic disease antiviral antibodies of chicken.
Table 3 kit of the present invention is to chicken standard serum testing result
| Test sample | Distilled water | The SPF chicken serum | The ND positive serum | The IB positive serum | The IBD positive serum | The H5 positive serum | The H7 positive serum | The H9 positive serum |
| Testing result | - | - | ++++ | - | - | - | - | - |
2. sensitivity tests suppresses (HI) [Shen Guanxin with newcastle disease standard positive serum (available from Harbin Veterinary Medicine Inst., China Academy of Agriculture) with conventional blood clotting; Zhou Rulin. modern immunological experiment technology (second edition) [M]; Hubei science tech publishing house; 2002] detecting its blood clotting inhibition valency is 1: 512, carries out doubling dilution.Good sample makes an experiment by the inventive method (GICA) to get 100 μ l dilution, and test card still is judged to be the positive when the newcastle disease standard positive serum is diluted to 1: 512.Test findings is seen table 4.The result shows, it is suitable with effect that blood clotting suppresses method that the present invention detects the sensitivity of test paper block-regulations.
Table 4 kit of the present invention and conventional sense method are to the sensitivity tests result of newcastle disease antibody test
| GICA detects | HI detects | |
| Chicken ND standard |
1∶512 | 1∶512 |
2.2 the detection of censorship chicken serum sample
Gather 150 parts of chicken serums altogether from 3 chicken houses on ground such as Hubei, Henan, (GICA) makes an experiment by the inventive method.Simultaneously, suppress (HI) with conventional blood clotting and measure tire (result sees 5).Table 5 shows that the inventive method (GICA) recall rate is 84.7% (127/150), and the HI method is 87.3% (131/150), and the two coincidence rate is 97.33% ((127+19)/150, (HI and GICA with positive+HI and GICA with negative)/gross sample number).
Table 5 kit of the present invention and conventional method detect the comparison of effect
| Positive | Negative | Positive rate | |
| Method of the present invention (GICA) blood clotting suppresses (HI) | 127 131 | 23 19 | 84.7％ 87.3％ |
Although content of the present invention is to combine present embodiment to describe, can not think limitation of the scope of the invention, scope of the present invention is limited 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 (1)
1. one kind is applicable to the kit that detects newcastle disease antibody, and it comprises box body, is located at test card and sample diluting liquid in the box body, it is characterized in that test card wherein is made up of test strips and test card; Test strips sticks on the support slice (7) that does not absorb water successively by absorption pad (4), nitrocellulose filter (3), gold mark pad (2), sample pad (1) and goes up formation; Be coated with the anti-chicken IgG Fc fragment monoclonal antibody of colloid gold label on the described gold mark pad (2); Be coated with the detection line (5) of newcastle disease virus reorganization hemagglutinin HN albumen formation and the nature controlling line (6) that rabbit anti-mouse igg constitutes on the described nitrocellulose filter (3) respectively; Described sample diluting liquid is 0.8% sodium chloride solution; Wherein, the deposit number of secreting the hybridoma cell line of anti-chicken IgG Fc fragment monoclonal antibody is CCTCC NO:C200501; The Escherichia coli that comprise the recombinant expression plasmid pKG-6P-1-HN of newcastle disease virus reorganization hemagglutinin HN albumen are deposited in Chinese typical culture collection center (CCTCC) on Dec 20th, 2007, and deposit number is CCTCC NO:M207205.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007101691030A CN101196522B (en) | 2007-12-29 | 2007-12-29 | Newcastle disease immune body immune colloidal gold fast detecting reagent kit and its application |
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| CN2007101691030A CN101196522B (en) | 2007-12-29 | 2007-12-29 | Newcastle disease immune body immune colloidal gold fast detecting reagent kit and its application |
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| CN101196522A CN101196522A (en) | 2008-06-11 |
| CN101196522B true CN101196522B (en) | 2012-01-11 |
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Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101701959A (en) * | 2009-03-05 | 2010-05-05 | 中国检验检疫科学研究院 | Fluorescent test strip for rapidly testing and immunizing Newcastle disease virus and application |
| CN102426231B (en) * | 2011-09-15 | 2014-09-03 | 长沙三诺生物传感技术股份有限公司 | Immunochromatography reagent strip and sealing agent composition used for same |
| CN102507946B (en) * | 2011-11-09 | 2014-09-03 | 湖北省农业科学院畜牧兽医研究所 | Subgroup J avian leukosis antibody quick test paper card, and application thereof |
| CN102608331A (en) * | 2012-03-16 | 2012-07-25 | 吉林大学 | Colloidal gold labeled quick-diagnosis test strip for virulent strains and low-virulent strains of Newcastle disease |
| CN104730261A (en) * | 2015-03-17 | 2015-06-24 | 孙素玲 | Colloidal gold test strip for detecting chicken infectious anemia virus |
| CN105759046A (en) * | 2016-04-08 | 2016-07-13 | 重庆理工大学 | Newcastle disease virus double-antibody sandwich AlphaLISA detection kit and detection method thereof |
| CN108226514A (en) * | 2017-08-07 | 2018-06-29 | 华中农业大学 | A kind of newcastle disease virus antibody rapid quantitative detection reagent box and its application |
| CN110320364B (en) * | 2018-03-30 | 2023-03-31 | 洛阳普泰生物技术有限公司 | Double-antibody sandwich colloidal gold detection kit, and preparation method and application thereof |
| CN110488010B (en) * | 2018-05-14 | 2022-11-01 | 洛阳中科生物芯片技术有限公司 | Newcastle disease virus antibody detection kit |
| CN110488011B (en) * | 2018-05-14 | 2022-11-01 | 洛阳中科生物芯片技术有限公司 | Newcastle disease virus antibody detection kit |
| CN110018304A (en) * | 2019-05-15 | 2019-07-16 | 河南省农业科学院 | A kind of newcastle epidemic disease antibody blocking Test paper |
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Application publication date: 20080611 Assignee: HUBEI HUADA REAL TECHNOLOGY Co.,Ltd. Assignor: Wuhan Huazhong Agricultural University Assets Management Co.,Ltd. Contract record no.: 2014420000113 Denomination of invention: Newcastle disease immune body immune colloidal gold fast detecting reagent kit and its application Granted publication date: 20120111 License type: Exclusive License Record date: 20140618 |
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