CN101067627A - Asial type foot-and-mouth disease virus fast diagnose test paper bar and producing method thereof - Google Patents
Asial type foot-and-mouth disease virus fast diagnose test paper bar and producing method thereof Download PDFInfo
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- CN101067627A CN101067627A CN 200710126176 CN200710126176A CN101067627A CN 101067627 A CN101067627 A CN 101067627A CN 200710126176 CN200710126176 CN 200710126176 CN 200710126176 A CN200710126176 A CN 200710126176A CN 101067627 A CN101067627 A CN 101067627A
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Abstract
The invention discloses a fast diagnosis test paper which is used to detect the Asia1 foot-and-mouth disease virus antigen and its preparation method. The invention joins the single antibody of the foot-and-mouth disease virus to the colloid gold solution, the mix solution of the colloid gold single antibody soaks by the porous fibrous material, set the examination line and the comparison line position on the nitric acid fibrous membrane which spurts another foot-and-mouth disease virus single antibody and the rabbit anti-mouse antibody forms the examination line and the comparison line, then pastes the dry nitric acid fibrous membrane on the impervious material, the porous fibrous material which infiltrates with the golden sign mix solution and the another porous fibrous material separately pastes on the impervious material and which are located on the cellulose nitrate membrane beginnings and ends, sets porous absorbing water material on the other end of the porous fibrous material to make the diagnosis test paper.
Description
Technical field
The present invention relates to a kind of fast diagnose test paper bar that is used to detect Asial type foot-and-mouth disease virus antigen, with and preparation method thereof.
Background technology
Aftosa is as great animal epidemic, and countries in the world are all attached great importance to this sick inspection and quarantine, prevention and control.The main method of detection, diagnostics port fever aphthous is to detect morbidity or with foot and mouth disease virus and antibody horizontal thereof in the malicious animal body, measure methods such as antibody as isolated viral, ELISA at present.Isolated viral is the reliable diagnostic to aftosa, but the long cycle can be incured loss through delay the Optimal Control time to this deadly infectious disease; Though it is simple but to need to distinguish be natural infection or because the antibody that vaccine immunity produces can not in time be made diagnosis that ELISA measures antibody; In addition, above experimental technique also needs many conditions such as expensive instrument and equipment, professional and technical personnel.Therefore, the detection kit quick, convenient, that be fit to use at the scene that development directly detects foot-and-mouth disease virus antigen not only has very strong practical value and wide application prospect, and significant to the prevention and control of China's aftosa.
China's large-area Asial type aftosa that once happened suddenly in 2005, epidemic situation involves tens provinces and cities, causes heavy economic losses for the animal husbandry production of China.This breaks out not only longer duration, and the epidemic situation complexity.This breaks out convenient, quick, responsive, special fast diagnosis reagent of our development research early of caution and development quick diagnosis reagent kit.Therefore, the test substances of developing a kind of fast detecting Asial type foot-and-mouth disease virus antigen is very necessary.
Summary of the invention
Fundamental purpose of the present invention is for the high detection Asial type foot-and-mouth disease virus antigen gold mark fast diagnose test paper bar of a species specificity and the preparation method of this test strips are provided.
Detection of the present invention Asial type foot-and-mouth disease virus fast diagnose test paper bar is by being fixed in the application of sample section that constitutes with multiporous fiber on the impermeable material, being positioned at the detection segment that is made of nitrocellulose membrane of application of sample section end and being positioned at the absorber portion that constitutes with multiporous fiber of detection segment end, wherein: the bond that is loaded with Asial type foot and mouth disease virus monoclonal antibody and collaurum on the application of sample section, its detection line is made of another strain Asial type foot and mouth disease virus monoclonal antibody, and its control line is made of the rabbit anti-mouse antibody.
In the detection Asial type foot-and-mouth disease virus fast diagnose test paper bar of the present invention, impermeable material is PVC, and used multiporous fiber is a nonwoven fabrics.
The preparation method of detection Asial type foot-and-mouth disease virus fast diagnose test paper bar of the present invention is:
The monoclonal antibody that at first prepares Asial type foot and mouth disease virus.
The monoclonal antibody preparation process: the total mRNA with Asial type foot and mouth disease virus (this Strain is preserved in country of Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences aftosa reference laboratory) is a template, the design primer carries out reverse transcription and PCR, obtains the gene VP1 of Asial type hoof-and-mouth disease virus capsid protein.VP1 inserted obtain recombinant expression plasmid in the prokaryotic expression carrier, obtain the coli strain of recombinant expression carrier again with the recombinant expression plasmid transformed into escherichia coli.Foreign protein will be removed after the aforementioned thalline ultrasonication, obtain the recombinant protein that purifying is expressed with chromatography, with recombinant protein and the abundant mixing of equal-volume adjuvant, carry out immunity for the healthy mice hypodermic injection, wherein the adjuvant of immune usefulness is a complete Freund's adjuvant for the first time, immune used adjuvant is an incomplete Freund's adjuvant for the second time, immunity is without adjuvant for the third time, the spleen of getting mouse in 3～4 days after the immunity obtains splenocyte and carries out dispersion treatment, added RPMI-1640 then in 1: 30 by volume, piping and druming for several times gently, again the centrifugal supernatant that goes of splenocyte is obtained the splenocyte precipitation, get the X-63 that from mouse, has obtained, NS-1 or SP2/0 clone, after in conventional nutrient solution, cultivating 3～4 days, the centrifugal supernatant that goes obtains myeloma cell's precipitation, precipitate with suspend above-mentioned splenocyte precipitation and myeloma cell of RPMI-1640, and with splenocyte and myeloma cell by quantity than 5～10: two kinds of cells of 1 mixing, the centrifugal then supernatant that goes, fully disperse sediment, in sediment, added concentration in 1: 2 by volume and be 50% polyglycol PEG4000, leave standstill the centrifugal supernatant that goes after 1～2 minute again, 25～35 times the HAT nutrient solution that in sediment, slowly adds the sediment volume, again fused cell is added in 96 well culture plates by 100 μ l/ holes, every Kong Zhongzai adds 100 μ l and washes the Turnover of Mouse Peritoneal Macrophages of getting as feeder cells with the HAT nutrient solution, obtain anti-Asial type foot and mouth disease virus antibody positive, the hybridoma that titre is high, again hybridoma is transferred in another piece 96 orifice plates and cloned, obtain hybridoma cell strain, cultivate above-mentioned resulting hybridoma cell strain with conventional nutrient solution again, a large amount of manufacture order clonal antibodies.
Perhaps adopt another kind of method preparation, this preparation method's preceding step and preceding method are basic identical, but is to adopt in the mouse body to induce method in the later stage during a large amount of manufacture order clonal antibody, that is: elder generation is a template with total mRNA of Asial type foot and mouth disease virus, the design primer carries out reverse transcription and PCR, obtain the VP1 gene of Asial type hoof-and-mouth disease virus capsid protein, again the VP1 gene is inserted in the prokaryotic expression carrier and obtain recombinant expression plasmid, obtain the coli strain of recombinant expression carrier again with the recombinant expression plasmid transformed into escherichia coli, foreign protein will be removed after the thalline ultrasonication, obtain the recombinant protein that purifying is expressed with chromatography, with recombinant protein and the abundant mixing of equal-volume adjuvant, carry out immunity for the healthy mice hypodermic injection, wherein the adjuvant of immune usefulness is a complete Freund's adjuvant for the first time, immune used adjuvant is an incomplete Freund's adjuvant for the second time, immunity is without adjuvant for the third time, the spleen of getting mouse in 3～4 days after the immunity obtains splenocyte and carries out dispersion treatment, added RPMI-1640 then in 1: 30 by volume, piping and druming for several times gently, again the centrifugal supernatant that goes of splenocyte is obtained the splenocyte precipitation, get the X-63 that from mouse, has obtained, NS-1 or SP2/0 clone, after in conventional nutrient solution, cultivating 3～4 days, the centrifugal supernatant that goes obtains myeloma cell's precipitation, resulting hybridoma is injected the female mice intraperitoneal of handling through norphytane, 2 to 3 weeks back collection ascites, the centrifugal solid constituent of removing, get its supernatant ammonium sulfate precipitation and gel chromatography purifying, obtain the monoclonal antibody of anti-Asial type foot and mouth disease virus.
Monoclonal antibody by the anti-Asial type of preceding method gained foot and mouth disease virus can adopt ammonium sulfate precipitation and gel chromatography to carry out purifying.
Simultaneously can go out colloidal gold solution according to prior art for preparing.
Can carry out the preparation of test strips after preparing monoclonal antibody and collaurum, its process is as follows:
Getting Asial type foot and mouth disease virus monoclonal antibody 0.24mL (it is 1mg/mL that monoclonal antibody stores concentration) is dissolved in the 100mL colloidal gold solution, rapid mixing, stir, after adding 1mg bovine serum albumin(BSA) fully dissolves in the mixed liquor of monoclonal antibody and collaurum again, with the colloidal gold solution centrifugal treating, supernatant discarded, to precipitate with containing 0.02M sodium chloride, 0.02M Tris, 0.4mL glacial acetic acid pH is 8.6 diluted is 8% of original volume, the colloid gold label monoclonal antibody mixed solution that dilution is good soaks into porous fibrous material, then with porous fibrous material in the ambient temperature drying, on nitrocellulose membrane, set out detection line and control line position, another strain Asial type foot and mouth disease virus monoclonal antibody and the rabbit anti-mouse antibody of spraying 0.2mg/L with Membrane jetter respectively in its position form detection line and control line, again with dry under the nitrocellulose membrane room temperature, nitrocellulose membrane is affixed on the impermeable material, the porous fibrous material that again infiltration is had gold to mark mixed solution is affixed on the impermeable material respectively with another porous fibrous material and makes both be positioned at the two ends of nitrocellulose membrane, have on the porous fibrous material of gold mark mixed solution in infiltration and an end that does not link to each other with nitrocellulose membrane is provided with another piece porous water-absorbing material, with as handle and adsorptive pads.
The present invention can be directly, fast Asial type foot and mouth disease virus is made diagnosis, and the evaluation that do not need to finalize the design again, Asial type foot-and-mouth disease virus antigen colloid gold label fast diagnose test paper bar of the present invention have sensitivity, special, detect convenient, with be suitable for the advantage that the scene is detected and diagnosed, and do not need special equipment, also need not special technician.
Description of drawings
Fig. 1 is a test strips vertical section structure synoptic diagram of the present invention, Fig. 2 is the perspective view of test strips of the present invention, among the figure: 1 is the PVC plate, 2 is the reaction application of sample section of colloid gold label antibody, 3 detection segment for the nitrocellulose membrane formation, 4 is absorber portion, and 5 for being positioned at the handling section of application of sample section end; A is a detection line, and B is a control line.
Embodiment
One, the preparation of the monoclonal antibody of Asial type foot and mouth disease virus
(1) the total mRNA that extracts from Asial type foot and mouth disease virus (annotate: this strain is preserved in country of Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences aftosa reference laboratory) with RNeasy Mini Kit (Qiagen) kit is a template, use Auele Specific Primer to carry out reverse transcription and pcr amplification, obtain Asial type foot and mouth disease virus VP1 gene at Asial type VP1.Used in the present invention primer is:
Upstream primer 5 '-CCG
GAATTCGCTTACCAGTGGATGCTCG-3 '
Downstream primer 5-' CCC
AAGCTTCGCTAGCTTGAGCAGGTC-3 '
Be connected with pGEM-T easy carrier according to the genes of interest of common method of attachment, adopt usual method will connect product transformed into escherichia coli DH5a competent cell, make up the coli strain that contains recombinant cloning vector, with agarose plate (amp pcr amplification
+Resistance) and blue hickie screening positive clone, picking hickie clone increases.Cut and sequential analysis evaluation positive colony with PCR, enzyme, adopt double digestion to downcut genes of interest from being accredited as positive clone, cut target gene fragment with the method recovery enzyme that molecular biology adopts usually, with the directed expression vector pP that inserts of the genes of interest that obtains
ROEX HTb makes up recombinant expression plasmid, and transformed into escherichia coli competent cell (DH5 α) makes up the coli strain of recombinant expression carrier, adopts bacterium coated plate (Kan
+Resistance) screening positive clone.Cut through PCR, enzyme and to be accredited as positive recombinant expression plasmid with method such as sequential analysis and to carry out prokaryotic expression.When recombinant expressed bacteria growing to mid-log phase A
550During=0.5-1.0, common A
550=0.6 o'clock is 1mmol/L IPTG abduction delivering with final concentration.Induce results expression bacterium after 6 hours, the centrifugal 10min collecting precipitation of 8000rpm is abandoned supernatant.After adding 5mL 20mM/L Tris damping fluid (pH8.0) suspension thalline by every grammes per square metre, the ultrasonication thalline, 12000rpm stays precipitation to abandon supernatant after centrifugal 15 minutes, with inclusion body lavation buffer solution (1M Tris pH8.0,0.5M EDTA pH8.0,3M NaCl, 0.5% Triton X-100) fully after the washing, the centrifugal 30min of 12000rpm repeats 4-5 time to remove foreign protein.With the 8M urea buffer solution (0.5M NaCl, 0.02M Tris, 8M urea, the pH8.0) precipitation that suspends, with the recombinant protein that affinity chromatography (Ni-NTA His) purifying is expressed, the recombinant protein of acquisition with ultraviolet spectrophotometer measure behind the content-20 ℃ frozen.
(2) preparation immune spleen cell: get aforesaid purifying Asial type foot and mouth disease virus VP1 gene prokaryotic antigen and the abundant mixing of equal-volume adjuvant, inject healthy mice nape portion both sides respectively, pars inguinalis is subcutaneous; The viral dosage of every per injection is 20～80 μ g antigens, after 2～4 weeks, uses the same method and repeats once more to inject at least 1 time at interval; The adjuvant of immune usefulness is a complete Freund's adjuvant for the first time, immune used adjuvant changes incomplete Freund's adjuvant into for the second time, immunity for the third time is 1: 1 (volume ratio) last immunity 2 weeks of back without the ratio of adjuvant, adjuvant and antigen, survey the antibody titer of the foot-and-mouth disease virus resistant gene VP1 prokaryotic expression antigen in the mice serum, will show the source of the mouse of enough antibody titers as immune spleen cell.
Measure the method for antibody titer, available radiommunoassay (RIA) or enzyme linked immunological absorption (ELISA) determination method etc. adopt common ELISA method among the present invention.
(3) preparation myeloma cell: the myeloma cell uses X-63, NS-1 and the SP2/0 clone that has obtained usually from mouse.Before carrying out Fusion of Cells, it was cultivated 3～4 days in the conventional nutrient solution of the DMEM that is added with 10% hyclone (100u/mL penicillin, 100ug/mL streptomysin), so that when merging, can obtain 2 * 10
7Or more fused cell.The present invention selects SP2/0 myeloma cell usually for use.
(4) Fusion of Cells: to the mouse peritoneal immunoprophylaxis of the mode immunity inoculation of (2) set by step, and the spleen of getting mouse in 3～4 days after immunity obtains splenocyte with the purifying Asial type foot and mouth disease virus VP1 gene prokaryotic antigen of the aforementioned gained of 20～80 μ g.Disperse splenocyte, added RPMI-1640 then in 1: 30 by volume, piping and druming obtains the splenocyte precipitation with the centrifugal supernatant that goes of splenocyte for several times more gently.The centrifugal supernatant that goes of bone marrow cells in mice knurl SP2/0 that above-mentioned (3) are cultivated obtains myeloma cell's precipitation.Precipitate with suspend above-mentioned splenocyte precipitation and myeloma cell of RPMI-1640, and with splenocyte and myeloma cell by quantity than (5～10): 1 mixes two kinds of cells, the centrifugal then supernatant that goes, fully disperse sediment, in sediment, added concentration in 1: 2 by volume and be 50% polyglycol PEG4000, leave standstill the centrifugal supernatant that goes after 1～2 minute again, slowly add 25～35 times HAT nutrient solution of sediment volume in sediment, used HAT solution is: hypoxanthine 10
-6～10
-3M, aminopterin-induced syndrome 10
-8～10
-7M, thymidine 10
-6～10
-4M, the suspension fused cell is added HAT nutrient solution 140～160 times to the sediment volume at last.Fused cell is added in 96 well culture plates by 100 μ l/ holes, every Kong Zhongzai adds 100 μ l and washes the Turnover of Mouse Peritoneal Macrophages of getting with the HAT nutrient solution as feeder cells (about 1～2 * 10 again
5Individual/hole).It is 3～7% CO that above-mentioned culture hole is placed concentration
2In the incubator, be to cultivate 10～14 hours under 35～38 ℃ of conditions,, changed liquid once with every Kong Banliang HAT nutrient solution every 2～3 days since the 3rd day in temperature.
When in observing culture hole, the hybrid cell colony occurring, get culture supernatant earlier, and then add half amount HAT nutrient solution in every hole.Detect the antibody and the titre of anti-Asial type foot and mouth disease virus VP1 gene prokaryotic antigen again with ELISA.To show that it is considered as when tangible rising is arranged compared with the control qualified when detection.
Above-mentioned anti-Asial type foot and mouth disease virus antibody positive, hybridoma that titre is high transferred in another piece 96 orifice plates clone, obtain hybridoma cell strain.Can make the method that only contains a single hybridoma in each hole through this hybridoma of limiting dilution by a kind of, or a kind of soft agar method of from soft agar medium, separating colony, or a kind of employing micromanipulator, the method of the single hybridoma of picking, or a kind of " sorter clone " method that adopts cell sorter to separate single hybridoma.
(5) preparation monoclonal antibody: cultivate the hybridoma cell strain that obtains in above-mentioned (4) with conventional nutrient solution.A large amount of manufacture order clonal antibodies can adopt big rolling bottle rotary culturing.Or induce method in the homology mouse body.Wherein big rolling bottle rotary culturing can obtain the foot-and-mouth disease virus resistant monoclonal antibody by cultivating above-mentioned (3) hybridoma purifying.The method that induces in homology mouse (the present invention the uses the Balb/c mouse) body can be by the hybridoma (5 * 10 of generation foot-and-mouth disease virus resistant antibody that above-mentioned (3) are obtained
5～10
6) inject female mice (4～8 age in the week) intraperitoneal of handling through norphytane, collect ascites after 2 to 3 weeks, the centrifugal solid constituent of removing, its supernatant promptly contain anti-Asial type foot and mouth disease virus monoclonal antibody.Be further purified if desired, can adopt ammonium sulfate precipitation and gel chromatography purifying.
The monoclonal antibody of anti-Asial type foot and mouth disease virus VP1 gene prokaryotic antigen is carried out the indirect ELISA test with bag by Asial, O, A and C type foot-and-mouth disease virus antigen respectively, the result shows the serotype specificity with height, not with other serotype generation cross reaction, can be used to prepare the gold-immunochromatographyreagent reagent for assay box that detects Asial type foot and mouth disease virus.
Two, the preparation of collaurum:
(1) the 1g chlorauride is dissolved in makes 1% aqueous solution in the 100mL distilled water, get 1% chlorauride aqueous solution 1mL, add distilled water 99mL and be configured to 0.01% chlorauride aqueous solution.Be heated to boiling, adding concentration rapidly is 1% trisodium citrate aqueous solution 1mL, continues to boil, and observes the liquid color situation of change simultaneously, cessation reaction when boiling about 7～10min liquid and transparent claret occurs.Measure the size and the shape thereof of the colloid gold particle of preparation with Electronic Speculum.Use 0.1mol/L K
2CO
2Or the pH value to 8.6 of 0.1N HCl adjustment collaurum, 4 ℃ keep in Dark Place.
(2) preparation of monoclonal antibody to be marked: get the good monoclonal antibody of the purifying of-20 ℃ of preservations and place room temperature, treat that it melts the back in the centrifugal 20min of 15000rpm, get supernatant, adjust antibody concentration and be 1mg/mL put 4 ℃ standby.
(3) the colloid gold label antibody mensuration of suitable stable quantity: (10-80 μ g/mL behind the monoclonal antibody stepwise dilution with mark, other establishes contrast), respectively get in the Eppendorf pipe that equal-volume adds a series of 1mL of being equipped with collaurums in proper order, behind the 5mim, the NaCl solution that in each pipe, adds 0.1mL 10% (W/V) respectively, blank does not add the monoclonal antibody dilution, adds the 0.1mL damping fluid, fully leaves standstill 2 hours observationss behind the mixing.Get that to add monoclonal antibody concentration minimum and pipe that dyeing does not change is the suitableeest labelled amount, adding 10% (V/V) concentration on this basis again is the monoclonal antibody solution of 1mg/mL (1mg/mL stores concentration for monoclonal antibody among the present invention), be the required antibody actual amount of stable colloid gold, be defined as every 100mL colloidal gold solution according to correlation test and add the monoclonal antibody solution 0.24mL that concentration is 1mg/mL, its monoclonal antibody final concentration is 0.24mg/100mL (colloidal gold solution).
(4) colloid gold label of antibody: calculate the consumption of required monoclonal antibody according to the amount of collaurum to be marked, under magnetic stirrer stirs, slowly monoclonal antibody solution is added in the colloidal gold solution, the antibody of 1mg approximately adds with 5min, adds the back and continues to stir 20min.It is 1% that the bovine serum albumin(BSA) (BSA) that continue to add filters under stirring makes its final concentration, slowly stir 20min again after, use 0.45 membrane filtration, directly use or-20 ℃ are frozen.
(5) colloid gold label purifying antibody: colloid gold label antibody in the centrifugal 15min of 1500rpm, is abandoned precipitation and stayed supernatant.15000rpm abandoned supernatant and stays precipitation in 4 ℃ of centrifugal 40min after supernatant moved into new centrifuge tube.With with centrifugal before equal volume 0.02M TBS (0.02M sodium chloride, 0.02M Tris, the 0.4mL glacial acetic acid, pH8.6) dissolution precipitation, after repeated centrifugation 2-3 time, the 0.02M TBS dissolution precipitation of usefulness original volume 8%, 4 ℃ of preservations are standby.
(6) colloid gold label antibody absorption glass fibre or nonwoven fabrics: with 0.02M pH8.6TBS dilution gold mark monoclonal antibody, fully behind the mixing, 1cm * 5cm size glass fiber filter paper or nonwoven fabrics are immersed in the gold mark monoclonal antibody solution, dry in the shade after the taking-up, be cut into the rectangular standby of 5mm * 5cm.
(7) trace of detection line and control line: another strain monoclonal antibody (0.2mg/L) and rabbit anti-mouse antibody (0.2mg/L) are sprayed on the NC film 37 ℃ of dried overnight with the test strips Membrane jetter.
(8) assembling of colloidal gold mark test paper: after placing the PVC base plate on the operator's console and opening adhesive sticker, stick (7) described NC film, subsequently that drying is the good nonwoven fabrics that is adsorbed with collaurum is connected with T line one end of NC film and sticks on the PVC base plate, on the collaurum pad, cover absorbing membrane then, make handle with thick adsorptive pads at last and be pressed in the end of NC film, with cutting machine test strips is cut into 2.5mm * 5cm after all these are finished and gets final product by the C line.
This Asial type foot-and-mouth disease virus fast diagnose test paper bar detects the animal bubble skin suspension (grinding with 0.04M PBS damping fluid) of 70 parts of grindings that national aftosa reference laboratory provides, the result shows, yin and yang attribute is set up, positive two bands, a negative band, result and other classical way testing result basically identical.
Claims (3)
1, Asia1 type foot-and-mouth disease virus fast diagnose test paper bar, comprise the detection segment that constitutes by nitrocellulose membrane that is linearly aligned and is fixed in the application of sample section that constitutes with multiporous fiber on the impermeable material, is positioned at application of sample section end and be positioned at the absorber portion that constitutes with multiporous fiber of detection segment end, it is characterized in that being loaded with on the application of sample section bond of Asia1 type foot and mouth disease virus monoclonal antibody and collaurum, its detection line is made of another strain Asia1 type foot and mouth disease virus monoclonal antibody, and its control line is made of the rabbit anti-mouse antibody.
2, Asia1 type foot-and-mouth disease virus fast diagnose test paper bar according to claim 1 is characterized in that used impermeable material is PVC, and used multiporous fiber is a nonwoven fabrics.
3, the preparation method of Asia1 type foot-and-mouth disease virus fast diagnose test paper bar according to claim 1 and 2, the monoclonal antibody that at first prepares Asia1 type foot and mouth disease virus, prepare colloidal gold solution, it is characterized in that: 0.24mg Asia1 type foot and mouth disease virus monoclonal antibody is dissolved in the 100mL colloidal gold solution, adding 5% bovine serum albumin(BSA) more therein, to make its final concentration be 1%, again with the colloidal gold solution centrifugal treating, supernatant discarded, to precipitate with containing 0.02M sodium chloride, 0.02M Tris, 0.4mL glacial acetic acid pH is 8.6 diluted is 8% of original volume, the gold mark mixed solution that dilution is good soaks into porous fibrous material, then with porous fibrous material in the ambient temperature drying, on nitrocellulose membrane, set out detection line and control line position, another strain Asia1 type foot and mouth disease virus monoclonal antibody and the rabbit anti-mouse antibody of spraying 0.2mg/L with Membrane jetter respectively in its position form detection line and control line, again with dry under the nitrocellulose membrane room temperature, nitrocellulose membrane is affixed on the impermeable material, the porous fibrous material that again infiltration is had gold to mark mixed solution is affixed on the impermeable material respectively with another porous fibrous material and makes both be positioned at the two ends of nitrocellulose membrane, have on the porous fibrous material of gold mark mixed solution in infiltration and an end that does not link to each other with nitrocellulose membrane is provided with another piece porous water-absorbing material, with as handle and adsorptive pads.
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| CN 200710126176 CN101067627A (en) | 2007-06-14 | 2007-06-14 | Asial type foot-and-mouth disease virus fast diagnose test paper bar and producing method thereof |
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|---|---|---|---|
| CN 200710126176 CN101067627A (en) | 2007-06-14 | 2007-06-14 | Asial type foot-and-mouth disease virus fast diagnose test paper bar and producing method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102043057A (en) * | 2010-11-11 | 2011-05-04 | 哈尔滨工业大学 | Test strip for testing oxytetracycline residue in meat and aquatic products and preparation method thereof |
| CN101236204B (en) * | 2007-11-29 | 2012-09-05 | 中国农业科学院兰州兽医研究所 | O -type foot-and-mouth disease antibody horizontal detection test paper and preparation method |
| CN101609093B (en) * | 2009-07-17 | 2013-05-15 | 田伏洲 | Test card for rapid diagnosis of pancreatictrauma and acute pancreatitis and test method thereof |
| CN110297088A (en) * | 2019-07-17 | 2019-10-01 | 中国农业科学院兰州兽医研究所 | Foot and mouth disease virus quantitative testing test paper card and preparation method thereof |
-
2007
- 2007-06-14 CN CN 200710126176 patent/CN101067627A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101236204B (en) * | 2007-11-29 | 2012-09-05 | 中国农业科学院兰州兽医研究所 | O -type foot-and-mouth disease antibody horizontal detection test paper and preparation method |
| CN101609093B (en) * | 2009-07-17 | 2013-05-15 | 田伏洲 | Test card for rapid diagnosis of pancreatictrauma and acute pancreatitis and test method thereof |
| CN102043057A (en) * | 2010-11-11 | 2011-05-04 | 哈尔滨工业大学 | Test strip for testing oxytetracycline residue in meat and aquatic products and preparation method thereof |
| CN110297088A (en) * | 2019-07-17 | 2019-10-01 | 中国农业科学院兰州兽医研究所 | Foot and mouth disease virus quantitative testing test paper card and preparation method thereof |
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