CN101196523A - Chicken infectious Fabricius bursa immune body immune colloidal gold fast detecting and its application - Google Patents
Chicken infectious Fabricius bursa immune body immune colloidal gold fast detecting and its application Download PDFInfo
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- CN101196523A CN101196523A CNA2007101691045A CN200710169104A CN101196523A CN 101196523 A CN101196523 A CN 101196523A CN A2007101691045 A CNA2007101691045 A CN A2007101691045A CN 200710169104 A CN200710169104 A CN 200710169104A CN 101196523 A CN101196523 A CN 101196523A
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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 respectively used for fast testing the IBDV antibody, 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 VP2 recombinant proteins coated with the IBDV as the detection line and the VP2 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.Specifically, the present invention is used for detecting chicken serum infectious bursa of Fabricius antibody to determine whether tested chicken infected in infections chicken cloacal bursa virus or the immune chicken body of crossing whether produced corresponding antibody.
Background technology
(Infectious bursal disease is by chicken infectivity bursa of Fabricius virus (Infectiousbursal disease virus, a kind of height contagious disease of chicken that IBDV) causes and turkey IBD) to gumboro disease.IBDV mainly influences the bursa of farbricius, and (bursa of fabricius, the BF) differentiation of bone-marrow-derived lymphocyte causes immunosupress in various degree, thereby increases the neurological susceptibility to concurrency or Secondary cases virus and bacterial infection.And then, cause very big loss to aviculture.And the monitoring and the detection of infections chicken cloacal bursa serum antibody still rested on original classical way, as agar gel diffusion test (Agarose gel immuno-diffusion; AGID), enzyme linked immunosorbent assay (Enzyme-linked Immunosorbent Assay; ELISA) and serum virus microneutralization test etc., these methods have expends time in longly (needs 24～48 hours as agar gel diffusion test; ELISA test needed 3～5 hours etc.), the instrument and equipment and the complex operating steps of the needs costliness that has can only be finished 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 by capillarity, and make the immune response that high specific, high-affinity take place at the acceptor (as antigen or antibody) of determinand on determinand in the sample and the chromatographic material 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 chromatographic immunoassay for qualitative determination ofchoricogonadotropin in urine.Clin Chem.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 specific immune response takes place and be trapped, further enrichment, form 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.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 (number of patent application: 99101537.1), a kind of preparation and application of fast diagnostic test paper strip for livestock and poultry pestilence are disclosed, but the main points of this invention are the detections at the livestock and poultry pestilence cause of disease, do not relate to the diagnostic detection to infections chicken cloacal bursa antibody.Bursal Disease antibody assay kit utility application (number of patent application: 02203069.7) be the ELISA method, and the unexposed biological material specimens that it adopts, also do not have to submit to the preservation of the biological material specimens that is used for proprietary program, those skilled in the art can't implement this invention and reach the effect shown in this invention.
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 infections chicken cloacal bursa antibody of assembling; Second purpose is the application of kit of the present invention in the infections chicken cloacal bursa 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 shown in Fig. 2,3) is the core of kit of the present invention, 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 (NC film) (3), gold mark pad (2), sample pad (1) and goes up formation.Gold mark pad (2) on be coated with the anti-chicken IgG Fc monoclonal antibody of colloid gold label (the hybridoma cell line BDRPDP that secretes this antibody be published in the patent documentation that the applicant formerly authorizes, the patent No.: ZL200510011521.8, this hybridoma cell line be deposited in Chinese typical culture collection center (CCTCC) in the Wuhan University of Chinese Wuhan City, Hubei Province on March 17th, 2005, deposit number is CCTCC NO:C200501); Be coated with nature controlling line (C line) (6) that detection line (T line) (5) that recombinant VP 2 albumen constitutes and rabbit anti-mouse igg constitute on the nitrocellulose filter (3) with the test strips formation test card in the test card (8) of packing into.For the infections chicken cloacal bursa virus antibody assay kit, recombinant protein is an infections chicken cloacal bursa virus reorganization serotype specificity antigen (VP2 albumen), and sample diluting liquid is respectively 0.8% sodium chloride solution.Described 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.Described affinity column is available from Amersham Biosciences company.Recombinant VP 2 albumen of the present invention is expressed by Escherichia coli (Escherichia coli) BL21 (DE3) the plys S/pKG-VP2 of the applicant's preparation, the Escherichia coli that comprise this recombinant plasmid are deposited in the Chinese typical culture collection center (CCTCC) that is positioned at Chinese Wuhan City, Hubei Province Wuhan University on Dec 20th, 2007, and deposit number is CCTCC NO:M207204.
Principle of the present invention is to select for use the infections chicken cloacal bursa virus reorganization serotype specificity antigen VP2 albumen of affinitive layer purification 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 infections chicken cloacal bursa antibody in the tested serum.During detection, the Fc fragment of all chicken serum IgG molecules earlier and the combination of the anti-chicken IgG of golden mark Fc section monoclonal antibody forms the compound of golden labeled monoclonal antibody and chicken serum IgG in the sample, owing to capillarity, reacts compound along the swimming forward of NC film.If the specific antibody of anti-chicken IBD is arranged in the sample, when arriving detection line, run into the recombinant VP 2 albumen that is coated on the detection line, will form the compound of specific antibody-Jin labeled monoclonal antibody-chicken serum IgG of recombinant VP 2 albumen-anti-chicken IBD, thereby be enriched on the detection line, form specific red precipitate line; The antibody-gold labeled monoclonal antibody compound that is not the non-specific antibody formation of anti-infections chicken cloacal bursa then can pass through detection line, close with rabbit anti-mouse igg two resistive connections that are coated on the nature controlling line when arriving nature controlling line, thereby be enriched on the nature controlling line, form the red precipitate line, promptly be judged to positive findings.If do not contain infections chicken cloacal bursa virus antibody in the sample, when the reaction compound arrives detection line, the reaction compound just can not combine with the recombinant protein on being coated on detection line, thereby cross detection line, close with rabbit anti-mouse igg two resistive connections that are coated on the nature controlling line when arriving nature controlling line, thereby 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 the biological safety height.Involved in the present invention to expression vector pGEX-KG be expression vector commonly used in the molecular biology, do not have bio-hazard, the expression vector pKG-VP2 of Gou Jianing is also without any bio-hazard on this basis.Recombination bacillus coli BL21 (DE3) plys S/pKG-VP2 is converted in the molecular biology e. coli bl21 competent cell commonly used with expression vector pKG-VP2 after ampicillin and chlorampenicol resistant screening acquisition, does not also have a bio-hazard.Test strips detection line bag quilt among the present invention be the infections chicken cloacal bursa virus reorganization serotype specificity antigen VP2 albumen that the present invention prepares, do not relate to the infections chicken cloacal bursa live virus in the preparation process, therefore the potential danger that does not have the infections chicken cloacal bursa live virus to escape, spread.
2, two of advantage of the present invention is that production cost is low.The required core reagent of kit provided by the present invention is anti-chicken IgGFc monoclonal antibody and infections chicken cloacal bursa virus reorganization serotype specificity antigen (VP2 albumen).Anti-chicken IgG Fc monoclonal antibody can be by 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 by the sad-ammonium sulfate method purifying of economy.VP2 albumen can obtain a large amount of expression external by recombination bacillus coli BL21 (DE3) plys S/pKG-VP2, is fit to large-scale production.
3, compare with the disclosed ELISA method that is used for the infections chicken cloacal bursa virus antibody test, kit of the present invention have many ELISA methods the advantage that can not compare, as detect fast (10～15min); Without any need for special instruments and 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% sodium chloride solution) in the testing process, human body is not had harm, environmentally safe; It is convenient to store, not high to temperature requirement, and effective storage life can reach 1 year under 4 ℃; At room temperature can preserve six months (table 1).
The comparison of table 1 kit of the present invention and disclosed ELISA method
| ELISA method (prior art) | The inventive method (colloidal gold immunochromatographimethod method) |
| High specificity, highly sensitive detection time is long, and (2～4h) need equipment such as microplate reader, and it is loaded down with trivial details to be not suitable for the execute-in-place operation, needs multistep reaction, needs be operated by the professional; Detect and need plurality of reagents, especially benzidine (TMB) in the higher testing process of cost, harmful human body, contaminated environment.Be stored in 4 ℃, the room temperature preservation phase is very short. | High specificity, highly sensitive detection quick (10min) is without any need for specific apparatus, equipment, and it is easy and simple to handle to can be used for execute-in-place, only needs single step reaction, does not need to be operated by the professional; Detect in the low testing process of cost and only need a kind of reagent (8%NaCl), human body is not had harm, environmentally safe.It is convenient to store, not high to temperature requirement, and 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, be gold mark pad; 3, be nitrocellulose filter (NC film); 4, be absorption pad (handle end); 5, be detection line, i.e. the T line; 6, be nature controlling line, i.e. the C line; 7, the support slice bar for not absorbing water; 8, be test card; 9, be well.
Fig. 3: the vertical view of test card of the present invention, among the figure:
1, sample pad (sample end); 2, be gold mark pad; 3, be nitrocellulose filter (NC film); 4, be absorption pad (handle end); 5, be detection line, i.e. the T line; 6, be nature controlling line, i.e. the C line; 8, be test card; 9 is well.
Fig. 4: invention test card result judges synoptic diagram, among the figure:
The positive result of a ++ ++; B 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 VP2 expression of recombinant proteins plasmid that the present invention relates to
Embodiment
The preparation of infections chicken cloacal bursa virus VP2 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 version in 1992 provides is carried out.
1, the clone of VP2 gene and order-checking
VP2 gene involved in the present invention is that the applicant includes a pair of primer of IBDV-STC strain A segment cDNA sequences Design of (AJ632141) according to reference GenBank; amplification length is the VP2 gene of 1.37kb; the synthetic of primer finished by Shanghai China promise bio tech ltd, and primer sequence is as follows:
Upstream primer P1:5 '-C
GGATCCATGACAAACCTGCAAGAT-3 '
Downstream primer P2:5 '-CC
AAGCTTCACAGCTATCCTCCTTAGG-3 '
Get an amount of infectious bursa of Fabricius virus (Zheng Lingyan, the foundation [D] of the clone of infectious bursal disease virus VP 2 gene, expression and ELISA antibody detection method, 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﹠amp; CurRec=1) suspension extracts RNA, by the method that provides in TaKaRa RNA PCR Kit (AMV) the Ver.2.1 kit instructions, amplify DNA, adopt UNIQ-10 pillar DNA glue to reclaim kit (purchase) and reclaim purifying RT-PCR amplified production from E.Z.N.A Gel Extraction Kit company, cloning vector pMD18-T direct and available from TaKaRa company carries out coupled reaction 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 kit (available from the commercial reagents box of TaKaRa company), with the complementary changing effect of checking of α.The picking positive colony adopts alkaline lysis (Sa nurse Brooker J, not Ritchie EF, Manny A Disi T edits (Jin Dongyan, Li Mengfeng etc. translate), 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-VP2, 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-VP2
After pMD-VP2 cuts with HindIII and BamHI enzyme, reclaim the fragment about 1368bp, 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.Transform the DH5 α competent cell of prepared fresh with recombinant expression carrier, and transformed bacteria is coated with 37 ℃ of overnight incubation of LB agar plate of chloromycetin and ampicillin (chloromycetin 25 μ g/mL, 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 plasmid called after pKG-VP2 that obtains.Escherichia coli (Escherichia coli) BL21 (DE3) the plys S/pKG-VP2 that comprises this recombinant plasmid) is deposited in the Chinese typical culture collection center (CCTCC) that is positioned at Chinese Wuhan City, Hubei Province Wuhan University, preservation day: on Dec 20th, 2007; Deposit number: CCTCC NO:M207204.Positive colony is prepared plasmid DNA in a large number with alkaline lysis, and packing is frozen in-20 ℃.Recombinant expression plasmid pKG-VP2 makes up flow process as shown in Figure 5.
3, the expression of infections chicken cloacal bursa virus VP2 albumen
Change the expression plasmid pKG-VP2 that builds over to expression with among colibacillary competent cell Escherichia coli BL21 (DE3) the plys S (Escherichia coli BL21 (DE3) plys S is available from Stratagene company), be applied to and contain corresponding microbiotic (chloromycetin 25 μ g/mL, ampicillin 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 (chloromycetin 25 μ g/mL, ampicillin 60 μ g/mL), carries out the bacterium activation.Overnight culture is gone in the fresh LB nutrient culture media by 1: 50 dilution proportion, (250～300rpm) to OD600=0.5～0.8 o'clock for shaken cultivation in 37 ℃, adding final concentration is 1mmol/L isopropylthio-(IPTG), and regulating the shaking table temperature is that abduction delivering 6h is cultivated in 30 ℃ of continuation.Under 4 ℃ of conditions 8, the centrifugal 15min of 000rpm collects bacterium.(be PBS, it is as follows to fill a prescription: 140mM NaCl, 2.7mM KCl, 10mM Na with 0.01M pH7.2 phosphate buffer for bacterium
2HPO
4, 1.8mM KH
2PO
4PH7.2) the centrifugal supernatant of abandoning after the washing once, to be added with the 0.01M pH7.2 phosphate buffer (PBS) of 1/100 dithiothreitol (DTT) (DTT) resuspended with accounting for culture 1/20 volume for precipitation, use big ultrasonic head, 400 watts, 90 circulations of setting power and fragmentation 4 seconds, interval 10 seconds, ultrasonication is to limpid in ice bath.Under 4 ℃ of conditions 12, the centrifugal 15min of 000rpm abandons precipitation, and it is 5mL that supernatant is concentrated into volume, presses protein purification post (Glutathione Sepharose
TM4B HiTrap affinity columns, Amersham Biosciences company product) program that provides of instructions is carried out affinitive layer purification, obtains the VP2 albumen of purifying.This albumen can be used for as the reagent that detects infections chicken cloacal bursa 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. described 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 precipitation; 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 precipitation repeats aforesaid operations, precipitation 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 ℃ down 12, 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 determined the kind of selected sephadex and the volume of post bed.
The volume of post bed is calculated as follows:
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 uses sephadex (Sephadex G-200) as chromatographic material.
The detailed process of purifying is as follows:
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 have soaked rearmounted 4 ℃ of refrigerators and have preserved standby.
The chromatographic column vertical fixing of cleaning on experiment frame, is added a small amount of eluent (0.01mol/L, pH7.2 PBS), checks the unobstructed property of drain pipe, qualified after, close liquid outlet.Take out soaked Sephadex G-200 from 4 ℃ of refrigerators, balance can chromatographic column to the room temperature.Put the filter paper suitable with column internal diameter gently on the chromatographic column gel that installs, the PBS equilibrate overnight of 0.01mol/L pH7.2 is reserved the high liquid layer of 1cm～2cm above gel, prepares 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 enters 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, the first immunisation Balb/C mouse IgG of Freund's complete adjuvant emulsification, immunizing dose is a 2mgIgG/ rabbit, the Balb/C mouse IgG immunity of incomplete Freund emulsification of each time later on.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, as serum AGID (agar gel immunodiffusion test (agar gel immuno-diffusion, AGID) be called for short agar diffusion test) tiring reaches at 1: 32 o'clock, 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.
Carry out the extraction and purification of the anti-Balb/C mouse of rabbit IgG according to the method for present embodiment step 1 and step 2.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 bag is cushioned the preparation of liquid: the 0.01M pH7.2 PBS damping fluid that contains 3% methyl alcohol (volume ratio) is cushioned liquid for bag, 0.22 μ membrane filtration mistake, put 4 ℃ standby, one week of the term of validity.The 0.01M pH7.2 PBS buffer formulation of 1000ml 3% methyl alcohol: NaCl 8g, KCl 0.2g, Na
2HPO
412H
2O 2.9g, KH
2PO
40.2g, methyl alcohol 30ml, boil off ionized water and be settled to 1000ml with two.
The preparation of 2 nitrocellulose filters: be cushioned liquid with the bag in the present embodiment step 1 VP2 albumen is diluted to 600 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 50～100 μ g/ml with bag, adjust machine, be scribed ss the C line, be nature controlling 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.
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 (mass volume ratio), put 4 ℃ standby, the term of validity four months.1000ml 1% chlorauric acid solution prescription: the 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, now with the current.The prescription of 100ml 1% citric acid three sodium solution: the 1g trisodium citrate, two ionized waters that boil off are settled to 100ml.
(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 ℃ standby, the term of validity four months.1000ml 0.1M 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 ℃ standby, 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), 0.01M pH7.2 PBS solution, 0.22 μ m membrane filtration mistake, put 4 ℃ standby, 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:
With two ionized waters that boil off 1% gold chloride 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 10,0m1 0.01% gold chloride.The collaurum outward appearance for preparing should be pure, bright, do not have precipitation and floating thing, one week of the term of validity.
3 colloid gold label MONOCLONAL ANTIBODIES SPECIFIC FOR:
PH value to 8.2 with 0.1Mol/L sal tartari is transferred collaurum adds anti-chicken IgG Fc monoclonal antibody by 8～10 μ g antibody/ml collaurums, magnetic stirring apparatus mixing 30min, stir adding BSA to final concentration be 1% (m/V), 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 collaurum volumes wash preservation liquid will precipitate resuspended, put 4 ℃ standby, 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), 0.5%Tween-20,0.05%NaN
3, 0.01M pH7.2 PBS solution, 0.22 μ m miillpore filter filters, put 4 ℃ standby, the term of validity four months.1000ml confining liquid prescription: 20g BSA, 0.5g NaN
3, 5mlTween-20,0.01M 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 in the present embodiment step 1 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 ℃ standby
The preparation of test strips sample pad
The preparation of 1 confining liquid:
2%BSA, 0.5%Tween-20,0.05%NaN
3, 0.01M pH7.2 PBS solution, 0.22 μ m miillpore filter filters, put 4 ℃ standby, the term of validity four months.1000ml confining liquid prescription: 20g BSA, 0.5g NaN
3, 5ml Tween-20,0.01M pH7.2 PBS solution be settled to 1000ml.
The preparation of 2 sample pad:
Sample pad is soaked in the confining liquid in the present embodiment step 1 behind the 30min, in 37 ℃ of oven dry, encapsulation, put 4 ℃ standby.
The assembling of test card
Nitrocellulose filter, absorption pad, glass fibre, 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 fast detecting infections chicken cloacal bursa virus antibody is formed
The kit of 1 fast detecting infections chicken cloacal bursa 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 infections chicken cloacal bursa virus antibody: sample diluting liquid is 0.8% sodium chloride solution.Compound method: 8g NaCl, adding distil water is settled to 1000ml.
Embodiment 9
The using method 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 results judge: as shown in Figure 4, when macroscopic aubergine nature controlling line appears in test card, macroscopic aubergine detection line do not occur, 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 tested serum is proportionate, and the antibody titer of the detected sample of the dark more explanation of color 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 tested 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 | February | April | June | August | October | Dec | |
| 0℃ 20℃ | ?1∶4096 ?1∶4096 | ?1∶4096 ?1∶4096 | ?1∶4096 ?1∶4096 | ?1∶4096 ?1∶4096 | ?1∶4096 ?1∶4096 | ?1∶4096 ?1∶1024 | ?1∶4096 ?1∶512 | Lost efficacy at 1: 1024 | 1∶256 | ?1∶64 |
Embodiment 11
The preparation and the application of the kit of fast detecting infections chicken cloacal bursa antibody
The preparation of the kit of 1 fast detecting infections chicken cloacal bursa antibody
The kit for preparing fast detecting infections chicken cloacal bursa antibody by the method for embodiment 1-9.
The application of the kit of 2 fast detecting infections chicken cloacal bursa antibody
2.1 the detection of chicken standard serum
1. specificity test: respectively infections chicken cloacal bursa (IBD) positive serum, chicken poison Mycoplasma (MG) positive serum, newcastle disease (ND) positive serum, infective bronchitis (IB) positive serum (available from China Veterinery Drug Inspection Office), avian influenza virus H5, H9 hypotype positive serum, SPF chicken serum (available from Harbin Veterinary Medicine Inst., China Academy of Agriculture) are tested by the embodiment of the invention 9 described methods (GICA).The results are shown in Table 3.Table 3 shows that after detection test card of the present invention and the test of infections chicken cloacal bursa positive serum (IBD) standard positive serum, tangible aubergine band all appears in nature controlling line and detection line; And with 0.8% sodium chloride solution (blank), chicken poison Mycoplasma (MG) positive serum, newcastle disease (ND) positive serum, chicken synovia mycoplasma (MD) positive serum chicken, infective bronchitis (IB) positive serum, avian influenza virus H5, H9 hypotype positive serum, SPF chicken serum.This shows test card specificity height of the present invention, does not have any cross reaction with other important diseases antibody of chicken.
Table 3 kit of the present invention is to chicken standard serum testing result
| Test sample | 0.8% sodium chloride solution | The SPF chicken serum | The ND positive serum | The IB positive serum | The H5 positive serum | The H9 positive serum | The MG positive serum | The IBD positive serum |
| Test sample | - | - | - | - | - | - | - | ++++ |
2. sensitivity tests is carried out doubling dilution respectively with infections chicken cloacal bursa (IBD) positive serum (available from China Veterinery Drug Inspection Office), and final dilutability is 1: 4096, respectively gets 100 μ l and dilutes good sample and test by the inventive method (GICA).Simultaneously, agar diffusion method (AGID) (Shen Guanxin, Zhou Rulin, modern immunological experiment technology (second edition) [M], Hubei science tech publishing house, 2002) is measured tire (test findings sees Table 4) of infections chicken cloacal bursa positive serum.The result shows that it is high 128 times that the present invention detects the remolding sensitivity agar gel diffusion test of test paper block-regulations.
Table 4 kit of the present invention and conventional sense method are to the sensitivity tests result of infections chicken cloacal bursa antibody test
| Kit of the present invention detects | AGID detects | |
| Chicken IBD standard |
1∶4096 | 1∶32 |
2.2 the detection of censorship chicken serum sample
Gather 216 parts of chicken serums altogether from 4 chicken houses on the ground such as Xinzhou, Jiangxia and Qianjiang in Hubei, (GICA) tests by the inventive method.Agar method of diffusion (AGID) is measured simultaneously.The results are shown in 5.Table 5 shows, method recall rate 56.48% (122/216) of the present invention, AGID method recall rate is 28.70% (62/154), lower than GICA method recall rate, be because the susceptibility of GICA method than AGID high 128 times (4096/32), when making the GICA method positive and AGID method feminine gender, the coincidence rate 64.81% of the two ((54+86)/216, ((AGID and GICA with positive+AGID 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) agar method of diffusion (AGID) | 122 62 | 94 154 | 56.48％ 28.70％ |
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 (2)
1. one kind is applicable to the kit that detects infections chicken cloacal bursa 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, is 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 the special VP2 antigen protein formation of infections chicken cloacal bursa virus reorganization serotype and the nature controlling line (6) that rabbit anti-mouse igg constitutes on the described nitrocellulose filter (3) respectively; Described sample diluting liquid is a distilled water.
2. the application of the described kit of claim 1 in detecting infections chicken cloacal bursa antibody.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007101691045A CN101196523A (en) | 2007-12-29 | 2007-12-29 | Chicken infectious Fabricius bursa immune body immune colloidal gold fast detecting and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007101691045A CN101196523A (en) | 2007-12-29 | 2007-12-29 | Chicken infectious Fabricius bursa immune body immune colloidal gold fast detecting and its application |
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| Publication Number | Publication Date |
|---|---|
| CN101196523A true CN101196523A (en) | 2008-06-11 |
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|---|---|---|---|
| CNA2007101691045A Pending CN101196523A (en) | 2007-12-29 | 2007-12-29 | Chicken infectious Fabricius bursa immune body immune colloidal gold fast detecting and its application |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110320364A (en) * | 2018-03-30 | 2019-10-11 | 洛阳普莱柯万泰生物技术有限公司 | A kind of double antibodies sandwich gold-immunochromatographyreagent reagent for assay box, and the preparation method and application thereof |
| CN112194709A (en) * | 2020-12-07 | 2021-01-08 | 北京纳百生物科技有限公司 | Immunochromatographic test strip for detecting feline panleukopenia syndrome virus antibody, and preparation method and application thereof |
| CN113156112A (en) * | 2021-04-25 | 2021-07-23 | 渤海大学 | Fluorescence immunochromatography kit for simultaneously detecting four nitrofuran metabolites and preparation method and application thereof |
| CN115248243A (en) * | 2021-12-15 | 2022-10-28 | 重庆工程职业技术学院 | Method for detecting AFP (alpha fetoprotein) based on sandwich electrochemical immunoassay mode of hyperbranched polyethyleneimine |
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2007
- 2007-12-29 CN CNA2007101691045A patent/CN101196523A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110320364A (en) * | 2018-03-30 | 2019-10-11 | 洛阳普莱柯万泰生物技术有限公司 | A kind of double antibodies sandwich gold-immunochromatographyreagent reagent for assay box, and the preparation method and application thereof |
| CN112194709A (en) * | 2020-12-07 | 2021-01-08 | 北京纳百生物科技有限公司 | Immunochromatographic test strip for detecting feline panleukopenia syndrome virus antibody, and preparation method and application thereof |
| CN113156112A (en) * | 2021-04-25 | 2021-07-23 | 渤海大学 | Fluorescence immunochromatography kit for simultaneously detecting four nitrofuran metabolites and preparation method and application thereof |
| CN115248243A (en) * | 2021-12-15 | 2022-10-28 | 重庆工程职业技术学院 | Method for detecting AFP (alpha fetoprotein) based on sandwich electrochemical immunoassay mode of hyperbranched polyethyleneimine |
| CN115248243B (en) * | 2021-12-15 | 2024-03-15 | 重庆工程职业技术学院 | Method for detecting AFP (alpha-fetoprotein) based on sandwich electrochemical immunoassay mode of hyperbranched polyethyleneimine |
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Application publication date: 20080611 |