CN102532309B - Colloid gold test strip for detecting porcine reproductive and respiratory syndrome antibody - Google Patents
Colloid gold test strip for detecting porcine reproductive and respiratory syndrome antibody Download PDFInfo
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Abstract
The invention belongs to the technical field of animal immunology, and in particular relates to a colloid gold immunochromatographic test strip for quickly detecting Porcine Reproductive And Respiratory Syndrome Virus (PRRSV) antibody. The test strip consists of an absorbent pad, a nitrocellulose membrane, a glass fiber membrane, a sample pad and a polyvinyl chloride (PVC) back board, wherein the nitrocellulose membrane is marked with lines, namely the PRRSV ecombinant N protein -enveloped lines are taken as a detection line and rabbit anti-rat IgG antibody is taken as a quality control line; and the glass fiber membrane is combined with a colloid gold labeled rat anti-pig IgG Fc monoclonal antibody (with the collection number of CCTCC C2010105). The PRRSV antibody in porcine serum is detected by an indirect method. When the test strip is used for detecting the PRRSV antibody, the test strip is easy to operate, a result is quickly and accurately obtained, intuitive, and easily distinguished, the repeatability is high, the production cost is low, special instruments are not required, and the test strip is suitable for large-scale on-site detection and epidemiology survey in a basic level, and has an auxiliary diagnosis effect on PRRSV infection.
Description
Technical field
The invention belongs to animal immunology detection technique field, be specifically related to a kind of porcine reproductive and respiratory syndrome virus colloidal gold immunochromatographimethod antibody test test strip, it comprises a kind of preparation and application of specific monoclonal antibody.
Background technology
Porcine reproductive and respiratory syndrome (Porcine Reproductive and Respiratory Syndrome, be called for short PRRS), claim again blue otopathy, it is a kind of transmissible disease that is caused by porcine reproductive and respiratory syndrome virus (PRRSV), with Adult Pig premature labor, miscarriage, stillborn foetus, the piglet dyspnoea, take high hot, highly pathogenic and high mortality as feature.Because virus constantly makes a variation, the development of world's pig industry in serious harm in recent years.
This sick diagnostic method is a lot, according to clinical symptom and pathological change, can make tentative diagnosis, makes a definite diagnosis and must rely on laboratory diagnosis.Antibody detection method has indirect enzyme-linked immunosorbent assay (ELISA), serum neutralization test etc.As patent publication No. CN1525170, " detect ELISA test kit and the usage of porcine reproductive and respiratory syndrome antibody ", a kind of special, responsive indirect ELISA antibody detection method is provided.But these methods exist various shortcoming, and instrument as special as needs, complicated operation, detection time are grown, operator need pass through professional training, the technical requirements condition is high, somewhat expensive etc.
The colloidal gold immunochromatographimethod technology is a kind of method for quick that grows up on the basis of immunity percolation, its principle is that colloid gold label albumen or antibody are coated on to gold mark pad, coated antibody or albumen on immobilon-p, and be grouped together with the sample pad absorbent pad, prepare colloidal gold immuno-chromatography test paper strip.The method is reproducible, highly sensitive, and is simple to operate, quick and precisely, intuitively easily distinguish, and can prepare in a large number by result, and technique is simple, low production cost, and the mass field that is suitable for basic unit and accident detects and epidemiology survey.Porcine reproductive and respiratory syndrome virus colloidal gold antigen detection method existing relevant patent report, as publication number CN101363865 " a kind of pig blue-ear disease poison colloidal gold colloidal gold detection test paper strip, its preparation method and application thereof ", publication number CN201285399, " detect the test strip of pig breeding disorder virus epidemic pathogen ".and relevant colloidal gold antibody detection method such as publication number CN101363858 " a kind of test paper strip for detecting PRRSV antibody colloidal gold, its preparation method and application thereof ", it is the N protein polyclone antibody at the upper coated pig blue-ear disease poison N albumen of nitrocellulose membrane (NC film) and anti-pig blue-ear disease poison, but this patent documentation the detailed openly genome sequence of PRRS virus, the nucleotide sequence of the specific fragment of this virus N albumen of amplification is not provided yet, structure physical map and the restriction enzyme site of its plasmid vector are not provided simultaneously, cause those skilled in the art can't implement this invention and reach the effect shown in this invention.In addition, the unsuitable stdn of polyclonal antibody, cause the coated condition of golden labeling antibody repeatedly to grope, and is also the shortcoming that this patent exists.
Summary of the invention
One object of the present invention is to overcome the defect of prior art, utilize the monoclonal antibody that builds to prepare porcine reproductive and respiratory syndrome colloidal gold immunochromatographimethod antibody test test strip, can be used for fast, conveniently detecting porcine reproductive and respiratory syndrome virus antibody in serum.Especially suitable veterinary station of basic unit and the epidemic prevention department that does not possess the under-developed country of laboratory condition of test strip of the present invention uses.
the present invention is with indirect ratio juris, utilize colloidal gold immunochromatographimethod technology and porcine reproductive and respiratory syndrome recombinant N protein and a kind of mouse-anti pig IgG Fc monoclonal antibody, prepared a kind of colloidal gold immune chromatography rapid detecting test paper strip that detects pig PRRSV antibody, its characteristics are to have used a kind of mouse-anti pig IgG Fc monoclonal antibody, because the existing method of this monoclonal antibody utilization cannot repeat to obtain, therefore before the applying date, the preservation of patented procedure has been carried out at the Chinese Typical Representative culture collection center (CCTCC) that the applicant has delivered the hybridoma cell strain 4F9 of this monoclonal antibody of secretion of acquisition in the Wuhan University of Wuhan City, Hubei Province, its preservation day is on November 4th, 2010, preserving number is CCTCC NO:C2010105.From obviously different from the technological line of the polyclonal antibody of above-mentioned patent literature in the screening of antibody.
The present invention implements by the following technical programs:
A kind of porcine reproductive and respiratory syndrome virus colloidal gold antibody test strip, by absorbent pad, nitrocellulose filter, glass fibre membrane, sample pad, PVC backing plate, carefully dressed up, on described glass fibre membrane, be combined with the mouse-anti pig IgG Fc monoclonal antibody of colloid gold label.Detection line position line on described nitrocellulose filter is coated with the porcine reproductive and respiratory syndrome virus recombinant N protein.Line is coated with rabbit anti-mouse igg antibody at described nature controlling line position.Described porcine reproductive and respiratory syndrome virus recombinant N protein prepares (the detailed technology scheme is shown in the embodiment part) by prokaryotic expression.
A kind of preparation method of porcine reproductive and respiratory syndrome virus colloidal gold antibody test strip, its step comprises:
1) prepare the porcine reproductive and respiratory syndrome virus recombinant N protein, its nucleotide sequence is as described in sequence table SEQ ID NO:1.Utilize existing method not reproducible acquisition rabbit anti-mouse igg antibody and mouse-anti pig IgG Fc monoclonal antibody, its preserving number is CCTCC NO:C2010105;
2) preparation of sample pad and gold mark pad: glass fibre membrane is soaked to the oven dry of spending the night with the confining liquid that has prepared under 37 ℃;
3) preparation of gold mark pad: use 0.1mol/L K
2CO
3Amount by 13 μ L/mL is regulated Radioactive colloidal gold pH, by every milliliter of colloid gold label 10 μ g mouse-anti pig IgG Fc monoclonal antibodies, by the amount of the 10 μ L/cm Radioactive colloidal gold that mark is good, sprays on the glass fibre membrane that has sealed, and is standby after vacuum lyophilization;
4) preparation of nitrocellulose filter: nitrocellulose filter is adhered to appropriate location on backing plate, the porcine reproductive and respiratory syndrome virus recombinant N protein is diluted to 800 μ g/ml; Rabbit anti-mouse igg antibody 1.0mg/ml, interval 5mm is sprayed on nitrocellulose filter, and is respectively as detection line and nature controlling line, standby after oven dry 2h under 37 ℃;
5) will pad with sample pad and gold mark that glass fibre is made, nitrocellulose filter, and absorbent pad mutually overlaps and adheres on backing plate successively;
While 6) using, only need insert the test strip that makes in test serum, in several minutes, get final product observations.
More detailed technical scheme is shown in that " embodiment " is described.
The accompanying drawing explanation
Sequence table SEQ ID NO:1 is the partial nucleotide sequence of the present invention's porcine reproductive and respiratory syndrome virus ORF7 gene of cloning, and sequence length is 372bp.
Fig. 1: the front and the side schematic view that are porcine reproductive and respiratory syndrome virus antibody test test strip of the present invention.In figure: 1 is absorbent pad; 2 are nature controlling line (being coated with rabbit anti-mouse igg antibody); 3 are detection line (being coated with the porcine reproductive and respiratory syndrome virus recombinant N protein); 4 are gold mark pad (containing colloid gold label mouse-anti pig IgG Fc monoclonal antibody); 5 is sample pad; For the PVC backing plate; 7 is nitrocellulose filter.
Fig. 2: be ELISA test strip result schematic diagram prepared by the present invention.Be followed successively by from top to bottom: 1. T, two lines of C are arranged, be judged to be the positive; 2. only have a C line, be judged to be feminine gender; 3. all it's too late only has the T line for T, C line, judges that test strip is invalid.
Fig. 3: the physical build-up figure that is the recombinant N protein expression plasmid that the present invention relates to.
Embodiment
Embodiment 1: the preparation and purification of anti-pig IgG Fc monoclonal antibody
1. the preparation of anti-pig IgG Fc monoclonal antibody
Method (Xue Qingshan with reference to the report of Xue Qingshan, the philosophy and technique of vitro culture, Science Press, 2001), utilize the microorganism at contriver place and the pig IgG immunity Balb/C mouse (purchased from Hubei Province Preventive Medicine Academy's Experimental Animal Center) of immunization experiment chamber extraction, immune programme for children is to get protein solution and isopyknic Freund's complete adjuvant (purchased from the Sigma company) emulsification that contains pig IgG 100 μ g, intraperitoneal injection of mice, at interval of ten days, strengthen once later, use Freund's incomplete adjuvant (purchased from Sigma company) emulsification instead.Finally in merging first three day, the abdominal cavity reinforced immunological, the antigen amount doubles, and does not add adjuvant.During fusion, 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), soak the 5min sterilization in 75% alcohol.Aseptic taking-up mouse spleen, separating Morr. cell, press 1 * 10 with SP2/0 myeloma cell's (purchased from Ministry of Health's Wuhan institute of Biological Products) of fresh preparation
7Individual SP2/0 and 10
8The ratio of individual immunocyte (1: 10～1: 15) mixes in the 50mL centrifuge tube, the centrifugal 10min of 1500r/min.The evacuation supernatant, knock the pipe end gently, makes cell precipitation loosening slightly.The centrifuge tube that cell mixture is housed is put in 37 ℃ of water-baths.Then in 1min, slowly add 50% Polyethylene glycol-2000 (PEG-2000, purchased from the Sigma company) 0.8mL that is incubated to 37 ℃, the limit edged stirs gently, continues 1min.Then add RMPI-1640 basic culture solution (purchased from the GIBCO company) 10mL that is incubated to 37 ℃, cell mass is disperseed.Concrete grammar is: first add the 1mL basic culture solution, then add the 2mL basic culture solution, then add the 3mL basic culture solution, finally add remaining 5mL basic culture solution, each added-time need slowly add, and constantly stirs lightly.Finally slowly add 40mL RMPI-1640 basic culture solution, tighten lid, repeatedly put upside down and mix.The centrifugal 5min of 1500r/min, abandon supernatant, and is with the RMPI-1640 complete culture solution (purchased from GIBCO company) that the resuspended 65mL that feeder cell are arranged contains 1%HAT (purchased from GIBCO company), that fused cell is resuspended.Resuspended cell is added drop-wise in 96 well culture plates to about 250 μ L/ holes, 37 ℃ of 5%CO
2In incubator, cultivate.After merging, namely can be observed microcolony on the 3rd day, added the RMPI-1640 complete culture solution that contains 1%HAT on the 4th day, 1/hole, sucked 100 μ L substratum and change the RMPI-1640 complete culture solution 100 μ L that contain 1%HT (purchased from GIBCO company) in 7th～10 days.Treating that the fused cell colony grows to accounts for 1/4～1/3 size at the bottom of culture hole, and nutrient solution slightly during flavescence, carries out antibody test.
Adopt pig IgG Fc segment (purchased from Sigma company) as envelope antigen, carry out the positive hole that the ELISA testing sieve is selected the anti-pig IgG Fc segment antibody of secretion.To the positive hole that screens, use at once limiting dilution assay (Xue Qingshan, the philosophy and technique of vitro culture, Science Press, 2001) to clone, screen.Through 3～4 time clonings, finishing screen is selected the monoclonal hybridoma system of the anti-pig IgG Fc segment of secretion.To this monoclonal hybridoma strain that filters out, the applicant is by its called after 4F9, and sending Chinese Typical Representative culture collection center (CCTCC) preservation in the Wuhan University of Wuhan City, Hubei Province on November 4th, 2010, its preserving number is CCTCCNO:C2010105.This hybridoma cell strain has been carried out to chromosome counting, and its chromosome number is approximately 90.By this cell strain abdominal injection Balb/C mouse, manufacture order clonal antibody.Adopt mouse source monoclonal antibody hypotype identification kit (purchased from Hycult company) to carry out the hypotype evaluation to this monoclonal antibody, result is mouse IgG 1 subclass.
2. the purifying of monoclonal antibody
Adopt caprylic acid-ammonium (Zhu Li equality, immunology common experimental method, People's Medical Officer Press, 2000): get mouse ascites 5mL and mix with appropriate silicon-dioxide, add isopyknic barbitol buffer solution (Zhu Li equality, immunology common experimental method, People's Medical Officer Press, 2000), after room temperature vibration 1h, the standing 30min of room temperature, get supernatant in clean centrifuge tube, 4 ℃, the centrifugal 10min of 3000r/min.get supernatant liquor 8mL, add 16mL 0.06mol/L sodium-acetate buffer (Zhu Li equality, immunology common experimental method, People's Medical Officer Press, 2000), with HCl adjust pH to 4.5, after under fully stirring, slowly adding sad 132 μ L, stirring at room 30min, 4 ℃ of standing 2h, 4 ℃ of centrifugal 30min of 15000r/min, supernatant liquor adds phosphoric acid buffer (the Zhu Li equality of the 0.1mol/L pH7.2 of 1/10 volume, immunology common experimental method, People's Medical Officer Press, 2000), with 1ml/LNaOH adjust pH to 7.6, under stirring, slowly adding ammonium sulfate to final concentration is 0.277g/mL, after 4 ℃ of standing 2h, 4 ℃ of centrifugal 30min of 12000r/min, abandon supernatant, precipitation phosphoric acid buffer (the Zhu Li equality of 5mL 0.1mol/L, immunology common experimental method, People's Medical Officer Press, 2000) resuspended, the dialysis tubing of packing into.With 4 ℃ of dialysis 3d of 5L 0.01mol/L pH7.2 phosphate buffered saline buffer (Zhu Li equality, immunology common experimental method, People's Medical Officer Press, 2000).The protein solution of fully having dialysed is concentrated into to 3mL with PEG-400 (PEG-20000), and 4 ℃ of centrifugal 30min of 12000r/min, abandon precipitation, collects supernatant liquor, measures protein concentration.
Embodiment 2: the Cloning and Expression of porcine reproductive and respiratory syndrome virus recombinant N protein
The molecular biology method that uses is referring to document: Pehanorm Brooker J, Ritchie EF not, Manny A Disi T chief editor, Jin Dongyan, Li Mengfeng etc. translate), molecular cloning experiment guide, second edition, Beijing Science Press, the method that version in 1992 is introduced.Concrete steps are:
1.ORF7 the clone of gene and order-checking
Porcine reproductive and respiratory syndrome virus HA strain (Jiang Zenghai with the clinical separation in Preventive Veterinary Medicine laboratory at contriver place; the separating of porcine reproductive and respiratory syndrome virus and porcine pseudorabies virus, evaluation; Hua Zhong Agriculture University's master thesis; 2004, see the middle http://epub.cnki.net/grid2008/detail.aspx of National IP Network? QueryID=3& CurRec=1) with Growth of Cells maintenance medium (the DMEM solution that contains 2% new-born calf serum), be to be inoculated in MARC-45 cell (namely at 1: 10 by volume, African green monkey kidney cell M104 clone strain, Bavaria, Germany state animal diseases diagnosis center C doctor zerny present), 37 ℃ when being cultured to 80% cell and the characteristic pathology occurring, draw supernatant liquor as the material that extracts the PRRSV geneome RNA, extracting method extracts by RNA the method that provides in test kit (purchased from Qiagen company) specification sheets to carry out.Adopt RT-PCR test kit (purchased from Qiagen company) amplification ORF7 gene.(the Genbank accession number: U87392) design primer, the upstream primer sequence is 5 '-CGAT according to porcine reproductive and respiratory syndrome virus ATCC VR-2332 strain complete genome sequence
GAATTCATATGCCAAATAACAACGGC-3 ' (underscore is EcoR I restriction enzyme site); The downstream primer sequence is 5 '-CTTA
CTCGAGAATGCCAGCCCATCATG-3 ' (underscore is Xho I restriction enzyme site).adopt DNA glue to reclaim test kit (giving birth to work biotechnology company limited purchased from Shanghai) and reclaim purifying RT-PCR amplified production, will with the RT-PCR product of receiving directly and pMD18-T carrier (purchased from precious biotechnology (Dalian) company limited) carry out ligation, then will connect product is transformed in competent cell bacillus coli DH 5 alpha (a kind of business intestinal bacteria commonly used), the picking positive colony, adopt alkaline lysis (Pehanorm Brooker J, Ritchie EF not, Manny A Disi T chief editor, Jin Dongyan, Li Mengfeng etc. translate), the molecular cloning experiment guide, second edition, Beijing Science Press, version in 1992) extract recombinant plasmid dna, and recombinant plasmid dna is identified.To identify that correct recombinant plasmid pMD-N send precious biotechnology (Dalian) company limited to check order, then relevant data in sequencing result and GeneBank is carried out to homology relatively.The nucleotide sequence of the specific fragment of the present invention clone's ORF7 gene is shown in that sequence table SEQ ID NO:1 is described, and sequence length is 372bp.
2. the structure of recombinant expression plasmid pKG-N
After pMD-N cuts with EcoR I and Xho I enzyme, reclaim the fragment of 400bp left and right, the pGEX-KG expression vector (purchased from Amersham Biosciences company) that this fragment and the enzyme with same are cut is connected, and builds recombinant expression vector.With recombinant expression vector, transform the bacillus coli DH 5 alpha competent cell of fresh preparation, and the transformed bacteria coating is contained on the LB agar plate of 100 μ g/mL penbritins to 37 ℃ of overnight incubation.After choosing several single bacterium colony overnight incubation, use alkaline lysis (Pehanorm Brooker J, Ritchie EF not, Manny A Disi T chief editor, Jin Dongyan, Li Mengfeng etc. translate), the molecular cloning experiment guide, second edition, Beijing Science Press, version in 1992) prepare plasmid in a small amount, carry out restriction analysis and pcr amplification and identify.By the positive colony called after pKG-N that obtains.Comprise positive colony is prepared to plasmid DNA in a large number with alkaline lysis, and packing is frozen in-20 ℃.Recombinant expression plasmid pKG-N of the present invention builds flow process as shown in Figure 3.
3. the abduction delivering of fusion rotein
Recombinant expression plasmid pKG-N is transformed and expresses in e. coli bl21 codon plus competent cell (purchased 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 LB plate, cultivate 8-10h, screen positive bacterium colony for 37 ℃.In the LB substratum after the jolting overnight incubation, the bacterium that will spend the night is to be seeded in 1000mL LB liquid nutrient medium at 1: 100 according to volume ratio, and 3h is cultivated in 37 ℃ of joltings (250-300r/min).Adding isopropylthio-β-D-galactoside (IPTG) to final concentration is that 1.0mmol/L carries out abduction delivering 3h.4 ℃ 8, the centrifugal 15min of 000r/min, collect bacterium.After ultrasonic disruption, extract inclusion body, then (sex change of inclusion body and the refolding method of recombinant protein are with reference to Pehanorm Brooker J to carry out the sex change of inclusion body and the renaturation of recombinant protein, Ritchie EF not, Manny A Disi T chief editor, Jin Dongyan, Li Mengfeng etc. translate, the molecular cloning experiment guide, second edition, Beijing Science Press, version in 1992), the purifying of dialysing obtains fusion rotein.
Embodiment 3: the preparation of rabbit anti-mouse igg antibody
The Balb/C mouse IgG immune health rabbit that utilizes laboratory, contriver place to extract, the rabbit anti-mouse igg hyper-immune serum for preparing high specific, high-titer, to hyper-immune serum, adopt the saturated ammonium sulphate method slightly to carry, after dextrane gel (Sephadex-G200) (purchased from Pharmacia company) is crossed column purification, obtain highly purified rabbit anti-mouse igg antibody.Utilize this antibody to can be used as the nature controlling line (working method reference literature: He Zhaoyang etc., animal immunology experimental technique, Changchun: Jilin science tech publishing house, 2002) of test strip of the present invention.
Embodiment 4: the preparation of porcine reproductive and respiratory syndrome colloidal gold antibody test strip
(1) preparation of Radioactive colloidal gold: adopt trisodium citrate reduction method to prepare Radioactive colloidal gold, get three of 200mL and boil off ionized water, be heated with stirring to boiling.Add again 2.0mL 1% hydrochloro-auric acid (HAuCl
4) solution, continue heating 2min, then disposable 3.6ml 1% citric acid three sodium solution that adds rapidly, continue stirring heating, lurid aqueous solution of chloraurate after trisodium citrate adds gradually by the xanthochromia ash again blackening finally redden or be orange red.Until solution become shiny red or orange red after, be chilled to room temperature and supplement tri-distilled water to 200ml.
(2) preparation of colloid gold label mouse-anti pig Fc monoclonal antibody: determine to use 0.1mol/l K through testing
2CO
3Amount by 13 μ l/ml is regulated Radioactive colloidal gold pH, and is with the monoclonal antibody mark Radioactive colloidal gold of 10 μ g/ml, stable through bovine serum albumin, and liquid (0.1%PEG-20000,0.05%NaN are preserved in the mark washing
3, being dissolved in 0.002mol/L pH9.0 borate buffer, the compound method of this borate buffer is referring to Pehanorm Brooker J, Ritchie EF not, Manny A Disi T chief editor, Jin Dongyan, Li Mengfeng etc. translate, the molecular cloning experiment guide, second edition, Beijing Science Press, version in 1992) after washed twice, 1/10,4 ℃ of the simmer down to original volume saves backup.
(3) preparation of sample pad and gold mark pad: glass fibre membrane is used to confining liquid (2% bovine serum albumin that has prepared, 0.1%Triton X-100,0.05% sodium azide (NaN3), 0.01mol/LpH 7.2 phosphoric acid buffers) soak, in 37 ℃ of oven dry of spending the night.The preparation of gold mark pad: the amount by 10 μ l/cm sprays colloid gold label mouse-anti pig IgG Fc monoclonal antibody on the glass fibre membrane that has sealed, and vacuum lyophilization is standby.
(4) preparation of nitrocellulose filter: at first nitrocellulose filter is adhered to appropriate location on the PVC backing plate, by porcine reproductive and respiratory syndrome recombinant N protein coated damping fluid (NaCl 8.0g, 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) be diluted to 800 μ g/mL, rabbit anti-mouse igg antibody 1.0mg/mL, interval 5mm is sprayed on nitrocellulose filter, respectively as detection line and the nature controlling line of test strip, is placed in 37 ℃ of oven dry 2h standby.
(5) will mutually overlap and adhere on backing plate successively with sample pad and gold mark pad, nitrocellulose filter, absorbent pad that glass fibre is made,
Embodiment 5: the using method of test strip of the present invention (seeing Fig. 2)
Detect: test strip sample pad place is inserted in sample 100 μ L to be checked to the 5-10min result of determination
Result is judged: if in serum, contain porcine reproductive and respiratory syndrome virus antibody, with gold mark monoclonal antibody, form corresponding gold mark mixture, upwards the porcine reproductive and respiratory syndrome recombinant N protein of chromatography on being coated in nitrocellulose filter is combined and formed red stripes, namely at T line place, form red stripes, be judged to be positive findings.If do not contain this antiviral antibody, do not form red stripes, be judged to be feminine gender.
No matter whether contain porcine reproductive and respiratory syndrome virus antibody in serum, gold mark monoclonal antibody all continues upwards chromatography and is coated on the rabbit anti-mouse igg antibodies on nitrocellulose filter, forms red stripes, namely at C line place, forms the red precipitate line, is nature controlling line.If the redfree band, show that this test strip lost efficacy, result is invalid.
Embodiment 6: the Preliminary Applications of test strip of the present invention
1. specific test: the porcine reproductive and respiratory syndrome virus positive serum of standard, PRV (Pseudorabies virus) positive serum, Latex agglutination test positive serum, pig parvoviral positive serum, porcine circovirus 2 type positive serum (by biological products Science and Technology Ltd. present before the section of Wuhan) are tested by the described method of embodiment 5.After result demonstration, test strip of the present invention were only tested with standard P RRSV positive serum, obvious red-purple band all appearred in nature controlling line and detection line.And after with physiological saline, PRRSV negative serum, other cause of disease positive serums, testing, the red-purple band does not appear in detection line.Show that test strip specificity prepared by the present invention is high.
2. sensitivity test: the PRRS standard positive serum is carried out respectively to doubling dilution, and final extent of dilution is 1: 2048, respectively gets the sample that 100 μ l have diluted and tests by the inventive method.Simultaneously, with PRRS indirect ELISA reagent kit (purchased from biological products Science and Technology Ltd. before the section of Wuhan), detect.Result shows, the sensitivity of test strip method of the present invention, near indirect ELISA, all reaches 2
8(dilution in 1: 256 by volume).
Above embodiment, for the present invention is described, is not limited to scope of the present invention.If do not specialize, the conventional means that in embodiment, technique means used is well known to the skilled person.
Claims (2)
1. the hybridoma cell strain 4F9 of the anti-pig IgG Fc fragment monoclonal antibody of secretion, be deposited in Chinese Typical Representative culture collection center (CCTCC), and its preserving number is CCTCC NO:C2010105.
2. porcine reproductive and respiratory syndrome virus colloidal gold antibody test strip, comprise sample pad, pad, nitrocellulose filter, absorbent pad and PVC backing plate, it is characterized in that, on the PVC backing plate, be stained with successively in order sample pad, pad, nitrocellulose filter and absorbent pad; On described pad, be coated with preserving number and be the monoclonal antibody of the secreted mouse-anti pig IgG Fc of the hybridoma cell strain 4F9 of CCTCC NO:C2010105 and the marker of Radioactive colloidal gold; On described nitrocellulose filter, be coated with respectively the detection line of porcine reproductive and respiratory syndrome virus recombinant N protein and the nature controlling line that rabbit anti-mouse igg antibody forms.
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| CN103995136B (en) * | 2014-06-06 | 2017-01-25 | 武汉中博生物股份有限公司 | Rapid colloidal gold detection test strip for pig porcine reproductive and respiratory syndrome virus |
| CN111308072A (en) * | 2020-03-25 | 2020-06-19 | 中山生物工程有限公司 | Colloidal gold immunochromatography kit for rapidly detecting novel coronavirus IgG antibody and preparation method thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108872601A (en) * | 2018-07-10 | 2018-11-23 | 安徽九川生物科技有限公司 | A kind of the immunofluorescence immue quantitative detection reagent box and its application method of porcine reproductive and respiratory syndrome antibody |
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