CN101995465A - Peste des petits ruminants virus IgG antibody colloidal gold detection card and production and using methods thereof - Google Patents
Peste des petits ruminants virus IgG antibody colloidal gold detection card and production and using methods thereof Download PDFInfo
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- CN101995465A CN101995465A CN2009101094842A CN200910109484A CN101995465A CN 101995465 A CN101995465 A CN 101995465A CN 2009101094842 A CN2009101094842 A CN 2009101094842A CN 200910109484 A CN200910109484 A CN 200910109484A CN 101995465 A CN101995465 A CN 101995465A
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Abstract
The invention relates to a peste des petits ruminants virus IgG antibody colloidal gold detection card and production and using methods thereof, and belongs to the technical field of immunology. The rapid diagnostic card comprises a shell and test paper, and is characterized in that: the shell is provided with a sample feeding hole and an observation window; the test paper is arranged in the shell; nitrocellulose films are stacked in the middle of a PVC lining plate, and water absorption pads and sample pads are respectively stacked at two ends; the nitrocellulose films are lapped with the water absorption pads and the sample pads; a section of membrane containing a staphylococcal protein A immune colloidal gold compound is additionally arranged in a sample area; and a detection belt containing inactivated peste des petits ruminants virus antigen and a quality control belt containing goat anti-rabbit IgG are formed on the nitrocellulose films. The invention provides a rapid, accurate and sensitive diagnostic card without special instruments, which is easy to operate, and can meet the detection requirements of food safety, slaughter and detection mechanisms.
Description
Technical field
The present invention relates to a kind of virus IgG antibody collaurum test card of ruminating for a short time, belong to the immunological technique field.
Background technology
PPR is caused by paramyxovirus section Morbillivirus PPR virus (PPRV), is the acute contagious disease of feature with heating, stomatitis, diarrhoea, pneumonia, goat and sheep susceptible, and the goat incidence of disease and case fatality rate are all higher.OIE (OIE) classifies it as statutory report animal epidemic, and China classifies it as class animal epidemic.This disease nineteen forty-two at first takes place in the Cote d'lvoire in Africa, 25 national report generation PPRs is arranged in 2003, mainly is distributed in Asia, Africa, also has the Turkey in Europe in addition.China from July 25th, 2007 after the PPR epidemic situation takes place in Tibet Autonomous Region Ritu County, a lot of pestilences have taken place again in succession.This epidemic situation is propagated rapidly, serious threat China herding safety, and the research of therefore carrying out this epidemic situation fast detecting is significant.
PPR virus belongs to paramyxovirus section Morbillivirus.To rinderpest virus similar physical chemistry and immunological characteristic are arranged.Virus is pleomorphism, is generally coarse sphere.Virion is big than rinderpest virus, and nucleocapsid is the spiral hollow rod shape and distinctive subunit is arranged, cyst membrane is arranged.Virus can be bred on the emerald green ball cell of tire sheep kidney, tire sheep and newborn sheep, Vero cell, and produces cytopathy (CPE), forms plasomidum.PPRV and other Morbillivirus pathogenic similar has the lymph of becoming and the epithelium that becomes.Therefore, it is the easiest duplicating in the lymphoid tissue of animal body and epithelial tissue, and is also serious to the injury of these tissues.Respiratory tract may be the door that virus enters body.Virus is at first duplicated at throat, lymphoodi mandibulares and tonsillotome after entering body by respiratory tract, and 2～3d forms viremia virusemia, and clinical symptoms appears in 2～3d subsequently first.Viremia virusemia causes virus to arrive lymphoid organ spleen, marrow and the intestines and stomach of whole body and the mucous membrane of respiratory system continues increment.
Usually the detection method for PPR mainly contains viral separating experiment, counter immunoelectrophoresis, enzyme-linked immuno assay, quantitative fluorescent PCR etc.All need certain condition and technology, or because need specific apparatus, or because expense costliness etc., its on-the-spot detection is promoted and is repeatedly repeated to follow the trail of check and is restricted.The colloidal gold immunochromatographimethod that rises the eighties in 20th century detects, and detects principle based on serology, and complicated experimental skill and specific installation are judged, be need not to clear being easy to of simple and quick, result, is particularly suitable for on-the-spot the detection.And the domestic and international report that does not still have this technology monitoring PPR.
Summary of the invention
At the appeal problem, the invention provides a kind of virus IgG antibody collaurum test card of ruminating for a short time, it is quick, accurate, stable that this quick diagnosis card detects.
Another object of the present invention provides the application of this quick diagnosis card.
Above-mentioned preparation of ruminating virus IgG antibody collaurum test card for a short time may further comprise the steps:
(1) preparation of colloid gold particle: get 0.5% chlorauric acid solution 2ml, add the chlorauric acid solution that the 98ml ultrapure water becomes final concentration 0.01%, behind the ebuillition of heated, get in the chlorauric acid solution that the disposable rapid adding of 1% trisodium citrate 2.1ml boils, continue to be heated to solution and transfer the black-and-blue shiny red that finally becomes to by faint yellow, continue heating 5min behind the colour stable, the room temperature cooling replenishes dehydration to original volume.
(2) preparation of colloid gold label staphylococcal protein A compound: (pH is modulated to 5.9～6.2) in a certain amount of protein solution adding colloidal gold solution carried out cross-linking reaction.
(3) purifying of colloid gold label staphylococcal protein A compound: supercentrifugation purifying immune colloid gold compound.
(4) preparation of sample pad: preserve liquid dilution colloid gold label staphylococcal protein A compound stoste to working concentration, evenly be dipped in the plain film of glass fibre in proportion ,-20 ℃ frozen, and after the vacuum freezedrying, sealing is preserved.
(5) ruminate the preparation of eqpidemic disease poison inactivation antigen for a short time: will ruminate in eqpidemic disease poison and the liquid nitrogen multigelation for a short time to inactivation.
(6) ruminate the preparation of eqpidemic disease poison part solid phase nitrocellulose filter for a short time: ruminate eqpidemic disease poison inactivation antigen bag for a short time by in the test reaction district of the observations of nitrocellulose filter, be defined as and detect band (T), bag, is defined as and accuses band (C) in the Quality Control district by normal rabbit igg (2mg/ml).
(7) processing of sample pad: select suitable closed reagent, surfactant and (or) non-ionic detergent separately or evenly to be dipped in the plain film of glass fibre after the proper proportion combination, the vacuum drying of room temperature cleaning condition is standby.
(8) test strips assembling: as backing, at the stacked nitrocellulose filter in the middle part of PVC liner plate, two ends are stacked adsorptive pads and sample pad respectively with PVB, and nitrocellulose filter connects mutually with adsorptive pads, sample pad.The test strips that assembles is placed in housing to have in the shell of well and view window.
Invention is ruminated virus IgG antibody for a short time by following operation steps and is detected:
(1) adds 6ul serum earlier at the well place, add the 74ul distilled water again, be flat on room temperature (the available dropper of on-the-spot detection is got a droplet serum and added in the sample well earlier, adds 2～3 distilled waters again).
(2) 10-15 minute observations.
Compared with prior art, the present invention has following beneficial effect: low price, testing cost is cheap, and detection speed is fast, and whole testing process only needs 15-20 minute, simple to operate, need not specialized equipment, the naked-eye observation result, the result is stable, can satisfy food security, butcher, the detection requirement of testing agency, be more convenient for on-the-spot the detection.
Description of drawings
Fig. 1 ruminates virus IgG antibody collaurum test card for a short time and detects synoptic diagram
Wherein:
1 is observation window
2 is well
3 is quality control band
4 for detecting band
Fig. 2 ruminates virus IgG antibody collaurum test card structural drawing for a short time
Wherein
5 is sample pad
6 is colloid gold label staphylococcal protein A glass fibre membrane
7 is absorbing membrane
8 is cellulose membrane
Embodiment
The preparation of embodiment 1 colloid gold particle
Get 1% chlorauric acid solution 1ml, add the chlorauric acid solution that the 99ml ultrapure water becomes final concentration 0.01%, behind the ebuillition of heated, get in the chlorauric acid solution that the disposable rapid adding of 1% trisodium citrate 1.6ml boils, continue to be heated to solution and transfer the black-and-blue shiny red that finally becomes to by faint yellow, continue heating 5min behind the colour stable, the room temperature cooling replenishes dehydration to original volume.
The preparation of embodiment 2 colloid gold label staphylococcal protein A compounds:
After being determined, the optimal pH of the suitableeest stable quantity of protein and mark just can carry out mark.Concrete steps are as follows: by the 120% quantitative total amount that calculates required protein to be marked of minimum steady.Under magnetic agitation, protein solution is added (pH is modulated to 5.9～6.2) in the colloidal gold solution, should dropwise add when adding protein, the about 5min of the protein of 1mg adds.Get 1ml collaurum-staphylococcal protein A bond liquid (experimental group) and 1ml collaurum stoste (control group) respectively and in test tube, add 10% sodium chloride solution 0.1ml, room temperature leaves standstill 1h, observations: if control group test tube solution transfers blueness to by redness, even can see polymer precipitation, and experimental group solution still keeps red, do not have precipitation, can continue next step experiment, otherwise need add mark albumen staphylococcal protein A.Add final concentration at last and be 0.2% polyglycol (PEG MW20000), continue to stir 30min.
The purifying of embodiment 3 colloid gold label staphylococcal protein A compounds:
Supercentrifugation purifying immune colloid gold compound.Earlier with the centrifugal 20min of 3500rpm (4 ℃), abandon precipitation with supernatant with the centrifugal 35min of 13500rpm (4 ℃), abandon supernatant, hang than the red deposit of loosening with preserving liquid; Again with the centrifugal 35min of 11000rpm (4 ℃), carefully move abandon supernatant after, hang than the red deposit of loosening to original volume 1/10 with preserving liquid, be the immune colloid gold compound of preliminary purification.
The preparation of embodiment 4 sample pad:
Preserve liquid dilution colloid gold label staphylococcal protein A compound stoste to working concentration, evenly be dipped in the plain film of glass fibre in proportion ,-20 ℃ frozen, and after the vacuum freezedrying, sealing is preserved.
According to the cell cryopreservation principle, adopt and freeze the slow method of melting slowly.Sample 1ml in every cell cryopreservation pipe.Multigelation is 10 times in-70 ℃ of refrigerators and liquid nitrogen.Respectively take a sample 1 during freeze thawing the 2nd, 4,6,8,10 times, detect and ruminate eqpidemic disease poison antigen titre and infection titer for a short time.And clasmatosis situation and chip size after the apparent emblem of optics mirror is observed each freeze thawing pulverizing down.
For a short time after ruminating the thawing of eqpidemic disease poison antigen room temperature, the centrifugal 10min of 12000r/min, getting supernatant 2mg/ml uses the PB damping fluid (pH 7.2) of 0.01mol/L with 250 μ g/ml interval, through the linear bag of BIO-DOT type XYZ3000 point sample instrument dispenser by in the test reaction district of the observations of nitrocellulose filter, be defined as and detect band, distance detecting band 5mm quality control band far away uses the linear bag of dispenser by normal rabbit igg (2mg/ml).37 ℃ of dry 2h, (see figure 2) is preserved in 4 ℃ of sealings.
The assembling of embodiment 7 test strips:
As backing, at the stacked nitrocellulose filter in the middle part of PVC liner plate, two ends are stacked adsorptive pads and sample pad respectively with PVB, and nitrocellulose filter connects (see figure 2) mutually with adsorptive pads, sample pad.The test strips that assembles is placed in housing has (see figure 1) in the shell of well and view window.
The use of embodiment 8 test card
Add the 6ul test serum earlier at the well place, add the 74ul distilled water again, be flat on room temperature (the available dropper of on-the-spot detection is got a droplet serum and added in the sample well earlier, adds 2～3 distilled waters again).10-15 minute observations.Referring to Fig. 1, red stripes appears as nature controlling line (numbering 3) position, and not developing the color in the position, then sample is positive for detection line (numbering 4); Red stripes all occurs as nature controlling line and detection line, then sample is negative; Not developing the color as nature controlling line, then testing result is invalid.
Claims (5)
1. for a short time virus IgG antibody collaurum test card of ruminating, it is characterized in that: it comprises test strips and shell two parts, and test strips places in the shell.
2. the virus IgG antibody collaurum test card of ruminating for a short time according to claim 1, it is characterized in that: described test strips uses PVB as backing, at the stacked nitrocellulose filter in the middle part of PVC liner plate, two ends are stacked adsorptive pads and sample pad respectively, nitrocellulose filter connects mutually with adsorptive pads, sample pad, be installed with one section film that contains staphylococcal protein A immune colloid gold compound at sample area, on nitrocellulose filter, have one to contain and ruminate inactivation of viruses detection of antigens band and a quality control band that contains goat anti-rabbit igg for a short time.
3. the virus IgG antibody collaurum test card of ruminating for a short time according to claim 1, it is characterized in that: described case body has well and view window.Well is over against sample pad, and view window is over against nitrocellulose filter.
4. for a short time production method of ruminating virus IgG antibody collaurum test card is characterized in that being made up of following process and step:
(1) preparation of colloid gold particle;
(2) preparation of colloid gold label staphylococcal protein A compound;
(3) supercentrifugation purifying colloid gold label staphylococcal protein A compound;
(4) preparation of sample pad, this pad is installed with the film of one section colloid gold label staphylococcal protein A;
(5) will ruminate in eqpidemic disease poison and the liquid nitrogen multigelation for a short time to inactivation, eqpidemic disease poison inactivation antigen is ruminated in preparation for a short time;
(6) ruminate the preparation of eqpidemic disease poison part solid phase nitrocellulose filter for a short time, this film bag is contained by one ruminates inactivation of viruses detection of antigens band and a bag for a short time by the quality control band of goat anti-rabbit igg;
(7) processing of sample pad: select suitable closed reagent, surfactant and (or) non-ionic detergent separately or evenly to be dipped in the plain film of glass fibre after the proper proportion combination, drying at room temperature is standby;
(8) detection of virus IgG antibody collaurum is ruminated in assembling for a short time, and as backing, at the stacked nitrocellulose filter in the middle part of PVC liner plate, two ends are stacked adsorptive pads and sample pad respectively with PVB for this test card, and nitrocellulose filter connects mutually with adsorptive pads, sample pad.
5. for a short time using method of ruminating virus IgG antibody collaurum test card is characterized in that at the well place adding 6ul serum earlier, adds the 74ul distilled water again, is flat on room temperature, 10-15 minute observations.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2009101094842A CN101995465A (en) | 2009-08-21 | 2009-08-21 | Peste des petits ruminants virus IgG antibody colloidal gold detection card and production and using methods thereof |
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| CN2009101094842A CN101995465A (en) | 2009-08-21 | 2009-08-21 | Peste des petits ruminants virus IgG antibody colloidal gold detection card and production and using methods thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107937352B (en) * | 2017-08-02 | 2020-09-29 | 北京世纪元亨动物防疫技术有限公司 | Colloidal gold immunochromatographic test strip for detecting peste des petits ruminants virus H protein antibody |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2604687Y (en) * | 2003-03-24 | 2004-02-25 | 中山大学 | Clonorchis sinensis immunocolloidal gold detecting box |
| CN101055273A (en) * | 2007-06-08 | 2007-10-17 | 中国人民解放军军事医学科学院军事兽医研究所 | Animal brucella antibody colloidal gold test paper film detection reagent kit |
-
2009
- 2009-08-21 CN CN2009101094842A patent/CN101995465A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2604687Y (en) * | 2003-03-24 | 2004-02-25 | 中山大学 | Clonorchis sinensis immunocolloidal gold detecting box |
| CN101055273A (en) * | 2007-06-08 | 2007-10-17 | 中国人民解放军军事医学科学院军事兽医研究所 | Animal brucella antibody colloidal gold test paper film detection reagent kit |
Non-Patent Citations (2)
| Title |
|---|
| RAJ等: "Detection of peste des petits ruminants virus antigen using immunofiltration and antigen-competition ELISA methods", 《VETERINARY MICROBIOLOGY》 * |
| 李刚等: "小反刍兽疫快速诊断技术及其疫苗的研究进展", 《中国畜牧兽医》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107937352B (en) * | 2017-08-02 | 2020-09-29 | 北京世纪元亨动物防疫技术有限公司 | Colloidal gold immunochromatographic test strip for detecting peste des petits ruminants virus H protein antibody |
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Application publication date: 20110330 |