CN209400545U - Rabies viruses gold-immunochromatographyreagent reagent for assay and kit - Google Patents
Rabies viruses gold-immunochromatographyreagent reagent for assay and kit Download PDFInfo
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- CN209400545U CN209400545U CN201820862260.3U CN201820862260U CN209400545U CN 209400545 U CN209400545 U CN 209400545U CN 201820862260 U CN201820862260 U CN 201820862260U CN 209400545 U CN209400545 U CN 209400545U
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Abstract
The utility model discloses a kind of rabies viruses gold-immunochromatographyreagent reagent for assay, the detection reagent includes a detection card (effluent piece) and a cartridge 8, the detection card includes backing 7 and the sample pad 1 on backing, gold-labelled pad 2, detection zone 3, quality control region 4, nitrocellulose filter 5, absorption pad 6, gold-labelled pad 2 are coated with the rabies virus antibodies -1 of colloid gold label；And detection zone 3 is coated with rabies virus antibodies -2.This detection reagent can detect rabies viruses, the diagnosis and prevention and control suitable for animal rabies.
Description
Technical field
The utility model relates to detect inspection technology field, more particularly to a kind of rabies viruses gold-immunochromatographyreagent reagent for assay.
Background technique
Rabies (Rabies Virus, RV) virus belongs to Rhabdoviridae, lyssavirus, and form is in bullet shaped,
Virion is made of shell and core two parts.Viral genome is made of 11930 or so nucleotide, for it is single-stranded, regardless of
The strand RNA of segment, virion are made of 5 structural proteins, nucleoprotein (N), phosphoprotein (P), membrane matrix albumen (M), sugar
Albumen (G) and transcription large protein (L).Rabies viruses is a kind of Zoonosis infectious agent, which is the mankind so far
Unique case fatality rate is up to 100% acute infectious disease, worldwide causes about 50,000 or more deaths every year, Asia is
Rabies continue hotspot, and China is Yang Quan big country and the higher country of rabies disease incidence.Prevention rabies uniquely have
The method of effect is timely inoculation rabies vacciness, while reinforcing detecting immune effect of vaccine, to grasp Immunity.
Hydrophobin the Methods of Detection of Pathogens common at present is that rapid fluorescence stove inhibits test (RFFIT) method, fluorescence anti-
Precursor virus neutralization test (FAVN) method, indirect immunofluorescence assay (IF) or molecular biology method etc., these methods all have
The advantages that high specificity, high sensibility.But there is detection time length, operation skill high to laboratory condition requirement in these methods
The disadvantages of high, cumbersome and single part of sample size of needs can be required more.In consideration of it, developing immune-gold labeled detection method, establish to dog
The viral diagnosis of sample is secreted, to prevent the propagation of control virus to be very important.
Therefore, this field is badly in need of exploitation stability is good, high sensitivity, quick specific detection rabies viruses easy to use
Gold-immunochromatographyreagent reagent for assay.The above-mentioned characteristic requirements of the utility model rabies viruses gold-immunochromatographyreagent reagent for assay reality.
Utility model content
The purpose of this utility model is to provide a kind of rabies viruses gold-immunochromatographyreagent reagent for assay.
Specifically, the purpose of this utility model is to provide a kind of detection card of immune quickly detection rabies viruses, this realities
With novel detection card sensitivity with higher and accuracy.
In the utility model in a first aspect, providing a kind of rabies viruses gold-immunochromatographyreagent reagent for assay, the detection reagent
Including a detection card (effluent piece), the detection card includes backing (7), and proximally arrives distal end on the backing
With flowering structure: sample pad (1), gold-labelled pad (2), detection zone (3), quality control region (4), nitrocellulose filter (5) and absorption pad
(6)；
And the gold-labelled pad (2) is coated with the rabies virus antibodies -1 of colloid gold label；
And the detection zone (3) is coated with rabies virus antibodies -2.
In another preferred example, the antibody -1 and the antibody -2 are incorporated into the different epitopes of rabies viruses.
In another preferred example, the antibody -1 and the antibody -2 are rabies viruses specific antibody.
In another preferred example, the quality control region (4) is coated with the secondary antibody that can be incorporated into the rabies virus antibodies -1.
In another preferred example, the secondary antibody is sheep anti-mouse igg.
In another preferred example, the 40nm colloid gold particle.
In another preferred example, the backing (7) is PVC board.
In another preferred example, the sample pad (1) is glass fibre.
In another preferred example, the absorption pad (6) is blotting paper.
In another preferred example, the sample pad (1), gold-labelled pad (2), nitrocellulose filter (5) and absorption pad (6)
It is pasted on the backing.
In another preferred example, the sample pad (1) and gold-labelled pad (2) partly overlap.
In another preferred example, the detection zone (3) is positioned close to the side of gold-labelled pad (2), and the Quality Control
Area (4) is positioned close to the side of absorption pad (6).
In another preferred example, the detection reagent further includes cartridge (8), and the well in the cartridge
(9) and observation window (10).
In another preferred example, the detection card is completely set up in cartridge (8), and the well (9) corresponds to institute
The sample pad (1) on detection card is stated, and the observation window (10) corresponds to detection zone (3) and quality control region on the detection card
(4)。
In another preferred example, the length of the cartridge is 80-90mm, width 10-20mm, and height is 4-5mm.
In another preferred example, the length of the detection card is 60-70mm, and width is 3-4mm.
In another preferred example, the detection zone (3) and quality control region (4) are located on nitrocellulose filter (5).
In another preferred example, the sample of the detection reagent detection includes whole blood, blood plasma, serum, juice (such as saliva
Liquid) or tissue samples.
In the second aspect of the utility model, provide a kind of for rabies viruses gold-immunochromatographyreagent reagent for assay box, the examination
Agent box includes:
(i) container；And
(ii) the rabies viruses gold-immunochromatographyreagent reagent for assay as described in the utility model first aspect.
In another preferred example, the kit further includes specification.
It should be understood that in the scope of the utility model, above-mentioned each technical characteristic of the utility model and (strictly according to the facts below
Apply example) in specifically describe each technical characteristic between can be combined with each other, to form a new or preferred technical solution.Limit
In length, not repeated them here.
Detailed description of the invention
Fig. 1 is a kind of structure sectional view of the detection card of preference of the utility model.Wherein, 1 is sample pad, and 2 be gold mark
Pad, 3 be detection zone, and 4 be quality control region, and 5 be nitrocellulose filter, and 6 be absorption pad, and 7 be backing.
Fig. 2 is a kind of vertical view of the rabies viruses gold-immunochromatographyreagent reagent for assay (detection card and cartridge) of preference of the utility model
Figure.Wherein, 8 be cartridge, and 9 be well, and 10 be observation window.
Specific embodiment
The utility model people develops a kind of rabies viruses colloidal gold detection examination by depth studying extensively for the first time
Agent, the detection reagent include a detection lug and a cartridge, and detection lug includes backing 7 and the sample pad 1 on backing, gold
Mark pad 2, detection zone 3, quality control region 4, nitrocellulose filter 5, absorption pad 6, the gold-labelled pad (2) are coated with colloid gold label
Rabies virus antibodies -1；And the detection zone (3) is coated with rabies virus antibodies -2.The detection reagent of the utility model can be special
Opposite sex detection rabies viruses, stability is good, high sensitivity, easy to use.On this basis, the utility model is completed.
Detection method and working principle
The testing principle of the utility model detection reagent is provided for the ease of understanding the utility model,.It should be understood that this is practical
Novel protection scope is not influenced or limitation by the principle.
The immunologic function test reagent of a kind of quickly detection rabies viruses provided by the invention comprising a detection card and a card
Box detects rabies viruses using double antibody immuno-sandwich method principle.Specifically by the specific rabies of colloid gold label
The antibody -1 of poison is simultaneously adsorbed on bonding pad, and the antibody -2 of specific rabies poison is fixed on nitrocellulose membrane, then will
Bonding pad and nitrocellulose membrane are assembled into PVC board, and sample pad is pasted at both ends respectively and blotting paper just forms detector bar.When detection,
After sample to be examined is added to the sample pad of test strips (detection card) one end, liquid-like moves forward through capillary action, dissolution knot
The colloid gold label object on pad is closed, if contain detected virus in sample, virus is by the antibody capture of colloid gold label
Compound is formed, at the detection line that compound is moved to film, and it is compound to be fixed on the formation double fastener heart of the antibody capture on film
Object, that is, the viral conjugate with colloidal label antibody (antibody -1) are detected the specific antibody (antibody -2) at survey line again
In conjunction with and retain and accumulate in detection and take to form Yan style colour band, color degree of strength reflects in sample virus is how much；If sample
In without virus, dissolved gum body gold is not detected among line retention, color belt does not occur in detection zone, and sample is negative sample
Product.Carried out for the ease of judgement reaction and the quality of reagent, in quality control region (C) fixed nature controlling line, so regardless of result how, matter
There is colour band always in control line C, to prove reagent validity.Thus the detection reagent is easy to carry and save, be easy to operate,
It is completed within detection process 5-10min.
Sensitivity
The sensitivity of the rabies viruses gold-immunochromatographyreagent reagent for assay of the utility model is 103.0FCID50/ ml or more.
Specificity
The hydrophobin detection reagent of the utility model is to canine parvovirus, canine distemper virus, hepatitis infectiosa canis virus, dog pair stream
The Pathogen tests result such as Influenza Virus, canine coronavirus is feminine gender.
The manufacture of detection reagent
In the present invention, the existing material system in this field can be selected in each component (or component) of the detection reagent
At.
Backing 7 can be made of any stable, non-porous material, and intensity should be enough supporting material and adhere thereto
Each component.Because many measurements use water as dispersive medium, backing 7 is preferably substantially water-impervious.It is excellent at one
It selects in example, it is more preferably made of polychloroethylene film (such as PVC offset plate) that backing 7, which is made of polymer film,.
Sample pad 1 can be made of any absorbent material.Workable example of material includes: cellulose, glass fibre etc..
Absorption pad 6 can be made of any material that can absorb sample detection liquid.The absorbability of absorption pad 6 should be enough
Greatly, the whole areas after testing of liquid that detection blocks are added to absorb.The example of material suitable for absorption pad 6 includes fiber
Element and absorbent filter.
Colloid gold label
Colloidal gold is by gold chloride (HAuCl4) the reducing agent such as white phosphorus, ascorbic acid, sodium citrate, tannic acid the effects of under,
It can be grouped to a certain size gold particle, and since electrostatic interaction becomes a kind of stable colloidal state, form electronegative dredge
Aqueous colloidal solution becomes stable colloidal state due to electrostatic interaction, therefore claims colloidal gold.
Colloidal gold is negatively charged under mild alkaline conditions, can form firm combination with the positive charge group of protein molecule,
Since this combination is electrostatical binding, so not influencing the biological nature of protein.Colloidal gold other than in conjunction with protein,
Can also be in conjunction with many other large biological molecules, such as SPA, PHA, ConA.According to some physical behaviors of colloidal gold, such as
High electron density, granular size, shape and color reaction, in addition the immune and biological characteristics of conjugate, thus make colloidal gold
It is widely used in the fields such as immunology, histology, pathology and cell biology.
The preparation of detection reagent
The each component (or component) of the utility model detection reagent is using technical method manufacture generally in the art and assembling.
Preferably, rabies virus antibodies -2 and Quality Control antibody are diluted with coating buffer, by two kinds of capture liquid after dilution
It is equably coated on the nitrocellulose membrane as detection part respectively, two kinds of capture liquid penetrate into after nitrocellulose filter respectively
Form detection zone and quality control region.
Sample pad, gold-labelled pad, nitrocellulose filter, absorption pad are pasted on backing by adhesive respectively.With slitting
Machine-cut is placed in cartridge at the detection card of 4mm, well alignment detection card sample pad location, is assembled into detection reagent after compression
Box is added desiccant with foil sealing and packs preservation.
The structure of detection reagent
The rabies viruses gold-immunochromatographyreagent reagent for assay of the utility model includes cartridge and the detection card (effluent in cartridge
Piece, test strips, detector bar or test-strips).
A kind of structure of preferred detection card is as shown in Figure 1 comprising backing 7, and on backing: sample pad 1,
Gold-labelled pad 2, detection zone 3, quality control region 4 (or check plot, control line, nature controlling line), nitrocellulose filter 5 and absorption pad 6.
A kind of structure of preferred cartridge 8 as shown in Fig. 2, include well 9 and observation window 10 thereon.Also, it is described
Detection card is completely set up in cartridge 8, and the well 9 corresponds to the sample pad 1 on the detection card, and the observation window 10
Corresponding to the detection zone 3 and quality control region 4 on the detection card.
Specifically, the detection reagent of the utility model has the following characteristics that
The length of backing 7 is identical as detection card.
Sample pad 1 is located at one end of detection card, and absorption pad 6 is located at the other end of detection card.
Load has the rabies virus antibodies -1 of colloid gold label in gold-labelled pad 2.
Between sample pad 1 and absorption pad 6, detection zone 3 is located at close to sample pad 1 detection zone 3 (also referred to as detection line)
One end, usual detection zone are arranged on nitrocellulose filter 5, pass through 2 He of gold-labelled pad of the nitrocellulose filter linkage flag
Absorption pad 6.Preferably, it is additionally provided with quality control region 4 (also referred to as nature controlling line) on the nitrocellulose filter 5, quality control region 4 is located at detection
Between area 3 and absorption pad 6.
Wherein, the detection zone 3 is coated with rabies virus antibodies -2, and quality control region 4 is coated with secondary antibody, and the secondary antibody can be tied
Together in the rabies virus antibodies -1.
The advantages of the utility model:
The utility model detection reagent can specific detection rabies viruses, stability is good, high sensitivity, easy to use, normal
Warm storage and transport.
The present invention will be further illustrated below in conjunction with specific embodiments.It should be understood that these embodiments are merely to illustrate this
Utility model rather than limitation the scope of the utility model.In the following examples, the experimental methods for specific conditions are not specified, leads to
Often according to normal conditions, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number be by weight
It calculates.
Embodiment 1
Rabies virus antibodies preparation
1. experimental animal
2-3 monthly age health beasle dog, hydrophobin, canine distemper virus, canine parvovirus, canine parainfluenza virus, dog gland
The antigens such as virus, canine coronavirus and antibody are feminine gender.
2. animal immune
Cultivate obtained mad dog antigen with Flury-LEP plants, CVS-11 plants, PV plants and SAD plants of rabies disease, this antigen with
Isometric Freund's complete adjuvant (Freund's complete adjuvant and incomplete Freund's adjuvant are purchased from Sigma company) is uniformly mixed, and cream
Change completely, 3 beasle dogs is immunized respectively, branch dorsal sc injection, every dog is immune, 1ml/ point at 5 points.Second is carried out after 14 days
Secondary immune, two when exempting from, will every kind of antigen thaw after be uniformly mixed respectively with isometric incomplete Freund's adjuvant, and emulsified
Entirely, immunization method is same exempts from.Hereafter exempted from respectively at interval of 14 days according to Freund's complete adjuvant and incomplete Freund's adjuvant
Epidemic disease, three, which exempt from rear preceding blood sampling immune every time, measures serum neutralizing antibody, until when neutralize antibody titers reach 1:512 or more, one week
Culling heart blood afterwards, blood collection are set 37 DEG C and are incubated for 2 hours, 2~8 DEG C overnight in sterilization container.Sterile extraction serum.It will be every
With 0.45 μm of filter filtration sterilization after the dog serum mixing of kind antigen.Packing, sets -20 DEG C or less preservations by 2ml/ bottles, or freeze-drying is protected
It deposits.
3. antibody purification, saturated ammonium sulfate and immune serum are precipitated by 1:2, supernatant, precipitating 10mM pH7.2 are abandoned in centrifugation
PBS dialysis, then be further purified to obtain anti-rabies virus antibody with affinity column.
Embodiment 2
Detect the preparation of the detection card of rabies viruses
1, prepared by gold-labelled pad
40nm colloidal gold is selected, with 0.1M K2CO3It adjusts near pH to isoelectric point, is carried out with every milliliter of 5 μ g antibody -1 of colloidal gold
Label must precipitate after marking fluid centrifugation, and precipitating 10mM pH7.2PBS dissolves, and light absorption value OD is measured at 650nm, with base
Plinth liquid is diluted to 1-3OD, applies on paving polyester material, at 37 DEG C, dries 10 hours in relative humidity less than 20% hothouse, i.e., mad
Dog antibody-Au gold-labelled pad.
2, the preparation of nitrocellulose filter detection zone and check plot
0.01M pH7.2 phosphate of the peridium concentration 0.4mg/ml containing 2% sucrose is slow on the detection zone of nitrocellulose membrane
Antibody -2 in fliud flushing, while being the 0.01M pH7.3 phosphate-buffered that 0.8mg/ml contains 2% sucrose in check plot peridium concentration
Sheep anti-mouse igg, package amount be 10 μ l/cm2, at 37 DEG C, relative humidity is dried 15 hours less than 20% hothouse.
3, the preparation of sample pad
20mM pH7.4 phosphate buffer is pressed containing sodium taurocholate 0.5%, S17 1% is prepared, and is uniformly layered on sample pad polyester
On fiber and maintain 37 DEG C, relative humidity is less than 20% drying.
4, test card assembles
By the nitrocellulose filter of coated antibody and sheep anti-mouse igg, blotting paper, one fixed width colloid gold label pad, sample
Product pad is successively attached to composition test kilocalorie in PVC board, and kilocalorie is cut 3-4mm item and is fitted into card, is fitted into aluminium foil bag and seals, be exactly
The complete corresponding viral test card of composition.
Embodiment 3
The detection of sample
1. acquisition testing sample, sample can be whole blood, blood plasma, serum, juice (such as saliva, hydrocrania), excreta
Or tissue samples, the present embodiment is using the dilution of fresh saliva or hydrocrania as detection sample；
2. will test card to lay flat, sample to be examined liquid is drawn with dropper, the vertical 2-3 drop that is added dropwise is opened after sample-adding in well
Beginning timing.
3.5-10 minute reads testing result.
4. result judges:
A: detection zone and quality control region change colour (red) simultaneously, show to detect in sample and contain rabies viruses, testing result is
It is positive.
B: quality control region changes colour (red), and detection zone is non-discolouring, shows to detect in sample without rabies viruses or rabies
The concentration of poison is lower than minimum detected value, and testing result is feminine gender.
C: quality control region is non-discolouring, and no matter whether detection zone changes colour, and shows that this detection is invalid.
It is incorporated herein by reference in all documents that the utility model refers to, just as each document quilt
It is individually recited as with reference to such.In addition, it should also be understood that, after having read the above-mentioned teaching content of the utility model, this field skill
Art personnel can make various changes or modifications the utility model, and such equivalent forms equally fall within the application appended claims
Book limited range.
Claims (8)
1. a kind of rabies viruses gold-immunochromatographyreagent reagent for assay, which is characterized in that the detection reagent includes a detection card, the inspection
Survey card include backing (7), and on the backing proximally arrive distal end with flowering structure: sample pad (1), gold-labelled pad
(2), detection zone (3), quality control region (4), nitrocellulose filter (5) and absorption pad (6)；
And the sample pad (1) and gold-labelled pad (2) partly overlap；
And the backing (7) is PVC board；
And the gold-labelled pad (2) is coated with the rabies virus antibodies -1 of colloid gold label, and the gold-labelled pad (2) is to apply
It is covered with the polyester material of the rabies virus antibodies -1 of the colloid gold label；
And the sample pad (1) is glass fibre；
And the detection zone (3) is coated with rabies virus antibodies -2；
Wherein, the detection zone (3) and quality control region (4) are located on nitrocellulose filter (5)；And the detection zone (3) is set
It sets in the side close to gold-labelled pad (2), and the quality control region (4) is positioned close to the side of absorption pad (6)；
And the absorption pad (6) is cellulosic absorbent pad or absorbent filter.
2. detection reagent as described in claim 1, which is characterized in that the quality control region (4) be coated with can be incorporated into it is described
The secondary antibody of rabies virus antibodies -1.
3. detection reagent as described in claim 1, which is characterized in that the detection reagent further includes cartridge (8), and is located at
Well (9) and observation window (10) in the cartridge.
4. detection reagent as claimed in claim 3, which is characterized in that the detection card is completely set up in cartridge (8), institute
The sample pad (1) that well (9) correspond on the detection card is stated, and the observation window (10) corresponds on detection card
Detection zone (3) and quality control region (4).
5. detection reagent as claimed in claim 4, which is characterized in that the length of the cartridge is 80-90mm, width 10-
20mm, and height is 4-5mm.
6. detection reagent as described in claim 1, which is characterized in that the length of the detection card is 60-70mm, and width is
3-4mm。
7. a kind of rabies viruses gold-immunochromatographyreagent reagent for assay box, which is characterized in that the kit includes:
(i) container；And
(ii) detection reagent as described in claim 1-6 is any.
8. kit as claimed in claim 7, which is characterized in that the kit further includes specification.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201820862260.3U CN209400545U (en) | 2018-06-05 | 2018-06-05 | Rabies viruses gold-immunochromatographyreagent reagent for assay and kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201820862260.3U CN209400545U (en) | 2018-06-05 | 2018-06-05 | Rabies viruses gold-immunochromatographyreagent reagent for assay and kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN209400545U true CN209400545U (en) | 2019-09-17 |
Family
ID=67876652
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201820862260.3U Active CN209400545U (en) | 2018-06-05 | 2018-06-05 | Rabies viruses gold-immunochromatographyreagent reagent for assay and kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN209400545U (en) |
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2018
- 2018-06-05 CN CN201820862260.3U patent/CN209400545U/en active Active
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