CN101029894A - Double-antigen sandwiched colloidal golden inspecting test paper of rabies virus antibody and its production - Google Patents
Double-antigen sandwiched colloidal golden inspecting test paper of rabies virus antibody and its production Download PDFInfo
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- CN101029894A CN101029894A CN 200710055487 CN200710055487A CN101029894A CN 101029894 A CN101029894 A CN 101029894A CN 200710055487 CN200710055487 CN 200710055487 CN 200710055487 A CN200710055487 A CN 200710055487A CN 101029894 A CN101029894 A CN 101029894A
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Abstract
An antibody double-antigen sandwiched collaurum test paper used fro detecting animal hydrophobin is prepared as arranging a filtering pad under sample adding hole, encapsulating hydrophobin GP/GN antigen collaurum coupling labeled matter in reaction region, encapsulating purified hydrophobin antigen on detection line, encapsulating rabbit resisting hydrophobin specific immune globulin on quality control line and sticking water absorption pad on side of quality control line.
Description
Technical field:
The present invention relates to a kind of rabies virus antibody double-antigen sandwiched colloidal golden inspecting test paper and preparation method, be used for the rabies virus antibodies fast detecting of animal rabies immunologic surveillance and epidemiology survey, belong to animal epidemic antibody test technical field.
Background technology:
Rabies are promptly on the books as far back as B.C. fourth century, are direct contact, the viral infectious that is caused by rabies viruses, and the equal susceptible of all warm-blooded animals is a kind of important people beast property the suffered from eqpidemic disease altogether of present harm humans health.According to statistics, the existing whole world is died from rabic patient and reach the 35000-50000 example every year, about 102280 examples of 1950-2005 China rabies total toll, and total morbidity and death toll are only second to India, occupy the 2nd in the world.Have rabic dog, cat and wild animal and be the beastly rabic propagation host of people and store up malicious host.Therefore to fundamentally control and eliminate rabies, must carry out comprehensive prevention and control measure dog, cat and wild animal.For the domestic and rabic prevention and control of wild animal, most important means are immunity inoculations of rabies vaccine at present, but whether have produced the antibody that reaches the immunoprotection level in the dog body after the vaccine inoculation, need certain detection method.At present commonly used in the world have method such as the interior neutralization test of immunofluorescence kitchen range inhibition test, enzyme linked immunosorbent assay and mouse brain, but these methods all need certain instrument and equipment and higher operative technique, have influenced it and have applied.
Collaurum is a kind of detection technique that development in recent years is got up, at first be used for immuno-electron microscope by Faulk and Taylor (1971), has become additional greatly beyond fluorescein, radioactive isotope and the enzyme labeling now.Colloid gold test paper is to utilize antigen-antibody reaction and immunochromatography principle, with antigen or antibody with colloid gold label, when containing in the sample to be checked with the antibody of antigen or antibody specific bond or antigen, gathering will take place and produce macroscopic colour band, have easy, quick, visual result, advantage such as accurate.
Summary of the invention:
The invention provides the rabies virus antibody double-antigen sandwiched colloidal golden inspecting test paper, can detect hydrophobin antibody easy, quickly and accurately.
The invention also discloses the preparation technology of above-mentioned detection test paper, be suitable for suitability for industrialized production.
Solution of the present invention is the immunology ultimate principle according to antigen-antibody energy specific bond, the rabies virus glycoprotein (GP) or the nucleoprotein (NP) of purifying are used colloid gold label, specking is on the glass fibre membrane of reaction zone, the rabies virus antigen of purifying and rabbit rabies poison specific immune globulin are coated on detection line place and nature controlling line place on the nitrocellulose filter (NC film) respectively, when containing the rabies viruses specific antibody in the test sample, then combine with the GP/NP antigen of colloid gold label in reaction zone, and under the effect of adsorptive pads, permeate swimming forward, combine once more with the rabies virus antigen on the detection line, macroscopic colour band on the NC film, occurs.
Concrete preparation technology is as follows: be by position shown in the accompanying drawing respectively with corresponding reagent and material specking or bag by in plain film of glass fibre and nitrocellulose filter, form rabies virus antibody immune colloid gold dual-antigen sandwich method detection test paper.Wherein well (S) has and filters pad, be coated with the rabies viruses GP/NP antigen colloidal gold coupling label of purifying on the plain film of the glass fibre of reaction zone (R), detection line (T) is coated with the rabies virus antigen of purifying, nature controlling line (C) is coated with rabbit rabies poison specific immune globulin, and nature controlling line (A) side is posted adsorptive pads.During detection, extract 1 of the anticoagulation of detected animal or serum, drop in the well (S) of this test strips, observe detection line (T) and nature controlling line (C) and red ribbon whether occurs, judge whether produce the antibody that reaches the protection level in this animal body.
In addition, the present invention also provides a kind of preparation method of rabies virus antibody immune colloid gold double antigens sandwich detection test paper, relates generally to the content of the following aspects:
1. purifying rabies virus antigen preparation: the rabies viruses of using Vero cell and adaptation thereof adopts rolling bottle or microcarrier culture technique, preparation rabies viruses cell culture fluid, adopt chemical reagent to add the concentrated and purified rabies viruses of cultivating of column chromatography then successively, make the purifying rabies virus antigen.
2. the preparation of purifying rabies viruses nucleoprotein (NP) and glycoprotein (GP) antigen: adopt technique for gene engineering, clone, expression or direct its nucleoprotein and the glycoprotein of from the rabies virus antigen of above-mentioned purifying, extracting by nucleoprotein and glycoprotein gene, and concentrate and purifying, make the rabies viruses GP/NP antigen of purifying.
3. the preparation of rabbit rabies poison immunoglobulin (Ig): use above-mentioned prepared purifying rabies viruses or its GP/NP antigen immune rabbit respectively, make rabbit rabies poison immunoglobulin (Ig).
4. the preparation of rabies viruses GP/NP antigen colloidal gold coupling label: use above-mentioned purifying rabies virus antigen and commercially available chloric acid gold, adopt the sodium citrate labelling method to make rabies virus antigen collaurum coupling label.
5. rabies virus antigen collaurum double antigens sandwich detects the assembling of test paper: the plain film of glass fibre of using prepared mentioned reagent and choosing, nitrocellulose filter, filtration pad, adsorptive pads, plastic clip isocolloid gold detection test paper material, carry out specking, bag quilt and lamination assembling.Be about to filter pad and place sample application zone; The plain film of the glass fibre that is adsorbed with purifying rabies viruses GP/NP antigen colloidal gold coupling label is placed reaction zone; With purifying rabies virus antigen bag by in the detection line place of nitrocellulose filter; With rabbit rabies poison immunoglobulin (Ig) bag by in the nature controlling line place of nitrocellulose filter; Adsorptive pads is placed the nature controlling line outside, in plastic clip, make rabies virus antigen collaurum double antigens sandwich and detect test paper with laminating machine laminating.
The present invention demarcates with the control serum standard; when the contained neutralizing antibody of serum reaches 0.5IU/ml or when above; in 10-20 minute can wine-colored positive reaction colour band appear in detection line; there is not this red ribbon as detection line; showing does not have rabies antibody or antibody amount to be lower than the protection level in the serum, nature controlling line shows that test strips is effective no matter whether there is rabies antibody red positive reaction colour band all should occur in the serum; if the reactionless colour band of nature controlling line illustrates that test strips lost efficacy.According to the WHO required standard, when neutralizing antibody reached 0.5IU, animal just can reach effective immunoprotection, otherwise timely booster immunization.
The present invention's existing technology of comparing has following advantage: the present invention detects easy, suitable, quick, economic, pollution-free, the easy to operate and interpretation of rabies virus antibodies test card; need be by Other Instruments equipment; can detect the rabies virus antibodies more than the 0.5IU (the minimum immunoprotection antibody titer of rabies) in 10-20 minute, the scene that is specially adapted to rabies virus antibodies is detected and epidemiology survey etc.Preparation method's science of the present invention, cost is low, stability is high, good reproducibility, is easy to make, storage and transport.
Description of drawings
Fig. 1, be the structural representation that the present invention detects test paper:
Fig. 2 is that the present invention detects the test paper sectional view.
1-plastic clip, 2-well, 3-reaction zone (made by the plain film of glass fibre, on be sprayed with the purifying GP/NP antigen of colloid gold label), 4-detection line (be sprayed on nitrocellulose filter on make the rabies virus antigen of purifying), 5-viewport, 6-nature controlling line (be sprayed on nitrocellulose filter on make rabbit rabies poison immunoglobulin (Ig)), 7-adsorptive pads, 8-liner plate, 9-filter pad.
Embodiment
By following examples the present invention is described for example further, and do not limit the present invention in any way, under the prerequisite that does not deviate from technical solution of the present invention, any change or change that those of ordinary skills that the present invention did are realized easily all will fall within the claim scope of the present invention.
Embodiment 1
According to shown in Figure 1, it is as follows that the present invention detects the test paper structure:
1, rabies virus antigen preparation: the rabies viruses of using Vero cell and adaptation thereof adopts rolling bottle or microcarrier culture technique, preparation rabies viruses cell culture fluid, adopt chemical reagent to add the concentrated and purified rabies viruses of cultivating of column chromatography then successively, make the purifying rabies virus antigen.
2, the preparation of purifying rabies viruses nucleoprotein (NP) and glycoprotein (GP): adopt technique for gene engineering, clone, expression or direct its nucleoprotein and the glycoprotein of from the rabies virus antigen of above-mentioned purifying, extracting by nucleoprotein and glycoprotein gene, and concentrate and purifying, make the rabies viruses GP/NP antigen of purifying.
3, the preparation of rabbit rabies poison immunoglobulin (Ig): use above-mentioned prepared purifying rabies virus antigen or its GP/NP antigen immune rabbit respectively, make rabbit rabies poison immunoglobulin (Ig).
4, the preparation of rabies viruses GP/NP antigen colloidal gold coupling label: use above-mentioned purifying rabies virus antigen and commercially available chloric acid gold, adopt the sodium citrate labelling method to make rabies virus antigen collaurum coupling label.
The technological process of production of test paper:
1, the preparation of preparation (1) collaurum of rabies viruses GP/NP collaurum coupling label: getting concentration is 0.01% gold chloride (HAuCl
4) accurately add a certain amount of 1% trisodium citrate aqueous solution behind the solution 100mL ebuillition of heated rapidly, keep boiling 10-15min again, the cooling back returns to original volume with distilled water, again through glycerine or sucrose density gradient centrifugation, can make size and be the uniform colloidal gold solution of 20-40 nano particle; (2) colloid gold label rabies viruses GP/NP: in colloidal gold solution, use 0.1M sal tartari (K
2CO
3) solution transfers pH to transfer to about the isoelectric point of GP/NP albumen, adds a certain amount of GP/NP, press 1g/100ml adding bovine serum albumin(BSA) (BSA) through mixing again after stirring 2-3 minute in solution, 4 ℃ left standstill 2-4 hour.With above-mentioned colloidal gold solution through 2000 rev/mins centrifugal 20 minutes, remove sediment and get supernatant.Again with supernatant through 10000 rev/mins centrifugal 40 minutes sediment.Sediment is got golden mark antigenic solution in 1/10 ratio with 10mM PBS pH 7.0+1%BSA+1% sucrose damping fluid resuspension, contain the Sodium azide of 0.01-0.06% in this damping fluid.(3) mark is good gold mark antigenic solution is placed on reaction zone 4 so that the binding capacity spraying of the best is dry in the plain film of glass fibre.Standby in 2-4 ℃ of preservation.
2, the preparation of test paper tears the non cohesive gel on the liner plate 2 off, stick the NC film respectively?, be coated with gold mark antigen the plain film of glass fibre, filter pad 1, adsorptive pads 7.
3, the spraying bag is provided with two lines by nitrocellulose filter (NC film): detection line 5 and nature controlling line 6.Detection line 5 spraying bags are purified rabies virus antigen, press 0.8-1.2mg/ml and spray the tested survey line 5 of bag, live width 0.8-1mm in NC film stage casing with Membrane jetter.Nature controlling line 6 bag is by rabbit rabies poison immunoglobulin (Ig), press 1.0-1.5mg/ml and sprays apart from the 0.5cm place of detection line 5 in NC film stage casing with Membrane jetter and wrap by nature controlling line 6, and live width also is 0.8-1mm, 37 ℃ of dryings 2 hours.The PBS that contains 5%BSA again with 0.01mM pH7.0 sealed 30 minutes the PBS rinsing of 0.01mM pH7.0,37 ℃ of dryings down at 37 ℃.
4, the gold mark detection test paper bar of long 6-8cm, wide about 1cm will the above-mentioned gold mark detection test paper film that is assembled into be cut into earlier in assembling with cutting cutter, will go up test paper with laminating machine again and be laminated in the plastic clip, makes rabies virus antibody colloidal gold double antigens sandwich detection test paper.
Claims (2)
1, a kind of rabies virus antibody double-antigen sandwiched colloidal golden inspecting test paper, it is characterized in that: liner plate (8) big envelope is in plastic clip (1), the surface of plastic clip (1) offers well (2) and viewport (5), be compounded with treated filtration pad (9) on the surface of liner plate (8), reaction zone (3), detection line (4), nature controlling line (6) and adsorptive pads (7), wherein, reaction zone (3) is made by the plain film of glass fibre, on be sprayed with the purifying GP/NP antigen colloidal gold coupling label of colloid gold label, detection line (4) is sprayed on the nitrocellulose filter by the rabies virus antigen of purifying and makes, nature controlling line (6) is sprayed on the nitrocellulose filter by rabbit rabies poison immunoglobulin (Ig) and makes, nature controlling line (6) side is posted adsorptive pads (7), well (2) is corresponding to filtering on pad (9) position, but the red blood cell in the filtered sample, viewport (5) is positioned on the reaction zone (4).
2, according to the preparation method of the described detection test card of claim 1, comprise the steps:
1) mark is good gold mark antigenic solution is placed on reaction zone so that the binding capacity spraying of the best is dry in the plain film of glass fibre, and is standby in 2-4 ℃ of preservation;
2) on liner plate, stick respectively nitrocellulose filter, be coated with gold mark antigen the plain film of glass fibre, filter pad, adsorptive pads;
3) nitrocellulose filter is provided with two lines: detection line and nature controlling line, and detection line spraying bag is purified rabies virus antigen, presses 0.8-1.2mg/ml and sprays the tested survey line of bag, live width 0.8-1mm in the nitrocellulose filter stage casing with Membrane jetter; The nature controlling line bag is by rabbit rabies poison immunoglobulin (Ig), press 1.0-1.5mg/ml and sprays apart from the 0.5cm place of detection line in NC film stage casing with Membrane jetter and wrap by nature controlling line, and live width also is 0.8-1mm, 37 ℃ of dryings 2 hours; The PBS that contains 5%BSA again with 0.01mMpH7.0 sealed 30 minutes the PBS rinsing of 0.01mM pH7.0,37 ℃ of dryings down at 37 ℃;
4) will the above-mentioned gold mark detection test paper film that is assembled into be cut into earlier the gold mark detection test paper bar of long 6-8cm, wide about 1cm, will go up test paper with laminating machine again and be laminated in the plastic clip, make rabies virus antibody colloidal gold double antigens sandwich detection test paper with cutting cutter.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103149361A (en) * | 2013-02-06 | 2013-06-12 | 河南科技大学 | Canine early pregnancy factor colloidal gold detection test paper card, its preparation method and application |
| CN103226143A (en) * | 2013-04-07 | 2013-07-31 | 南京基蛋生物科技有限公司 | Dry-type immunoassay test strip and preparation method and application thereof |
| CN103954777A (en) * | 2014-05-20 | 2014-07-30 | 北京凯思百奥科技发展有限公司 | Rabies virus monoclonal antibody and application thereof |
| CN108872572A (en) * | 2018-05-30 | 2018-11-23 | 广州优迪生物科技股份有限公司 | It is a kind of for detecting the kit of rabies virus antibodies |
| CN109844531A (en) * | 2016-10-10 | 2019-06-04 | 房东河 | For detecting the device and method of dog cancer |
| CN110736830A (en) * | 2019-11-18 | 2020-01-31 | 威尚生物技术（合肥）有限公司 | detection cards for autoimmune colloidal gold |
| CN112166323A (en) * | 2018-07-02 | 2021-01-01 | 美国西门子医学诊断股份有限公司 | Direct immunoassay measurement of autoantibodies |
| CN113252893A (en) * | 2021-06-23 | 2021-08-13 | 北京市动物疫病预防控制中心 | Method for rapidly and quantitatively detecting rabies virus antibody by applying rabies virus G protein, encoding gene of G protein and test paper |
-
2007
- 2007-04-04 CN CN 200710055487 patent/CN101029894A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103149361A (en) * | 2013-02-06 | 2013-06-12 | 河南科技大学 | Canine early pregnancy factor colloidal gold detection test paper card, its preparation method and application |
| CN103226143A (en) * | 2013-04-07 | 2013-07-31 | 南京基蛋生物科技有限公司 | Dry-type immunoassay test strip and preparation method and application thereof |
| CN103954777A (en) * | 2014-05-20 | 2014-07-30 | 北京凯思百奥科技发展有限公司 | Rabies virus monoclonal antibody and application thereof |
| CN109844531A (en) * | 2016-10-10 | 2019-06-04 | 房东河 | For detecting the device and method of dog cancer |
| CN108872572A (en) * | 2018-05-30 | 2018-11-23 | 广州优迪生物科技股份有限公司 | It is a kind of for detecting the kit of rabies virus antibodies |
| CN112166323A (en) * | 2018-07-02 | 2021-01-01 | 美国西门子医学诊断股份有限公司 | Direct immunoassay measurement of autoantibodies |
| CN110736830A (en) * | 2019-11-18 | 2020-01-31 | 威尚生物技术（合肥）有限公司 | detection cards for autoimmune colloidal gold |
| CN113252893A (en) * | 2021-06-23 | 2021-08-13 | 北京市动物疫病预防控制中心 | Method for rapidly and quantitatively detecting rabies virus antibody by applying rabies virus G protein, encoding gene of G protein and test paper |
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Application publication date: 20070905 |