CN208672650U - Immunofluorescence for detecting canine coronavirus antigen chromatographs detection card - Google Patents
Immunofluorescence for detecting canine coronavirus antigen chromatographs detection card Download PDFInfo
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- CN208672650U CN208672650U CN201820667833.7U CN201820667833U CN208672650U CN 208672650 U CN208672650 U CN 208672650U CN 201820667833 U CN201820667833 U CN 201820667833U CN 208672650 U CN208672650 U CN 208672650U
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Abstract
The immunofluorescence that the utility model discloses a kind of for detecting canine coronavirus antigen chromatographs detection card；It is intended to provide a kind of high sensitivity, testing result is reliably used to detect the immunofluorescence chromatography detection card of canine coronavirus antigen；Its skill main points includes bottom plate, successively linking has sample pad, bonding pad, nitrocellulose membrane and water absorption pad on bottom plate, the bonding pad is equipped with the CCV monoclonal antibody of fluorescent microsphere label and the goat-anti chicken IgY of fluorescent microsphere label, the nitrocellulose membrane is equipped with the Quality Control C line of a coating chicken IgY and the detection T line of a coating CCV monoclonal antibody；Belong to animal epidemic detection field.
Description
Technical field
The utility model discloses a kind of immunofluorescences to chromatograph detection card, specifically, being a kind of coronal for detecting dog
The immunofluorescence chromatography detection card of viral antigen.
Background technique
The pathogen of canine coronavirus disease is canine coronavirus (Canine Corona Virus, CCV).To differ in weight
Gastroenteritis be Clinical symptoms, clinically be in lethal watery diarrhea.Sudden onset, psychotic depression, appetite abolish, vomiting,
The dilute soft and excrement with mucus of stench is discharged.Vomiting often can last for days, until just being alleviated before there is diarrhea.After, excrement
It just is in blood orange or green, that interior mucous sum number amount does not wait by smart shape, half paste to water sample.Rapid dehydration, weight subtract
Gently.The source of infection is mainly sick dog and with malicious dog, and sick dog toxin expelling time is 14 days, and the ability that being kept in contact must infect schedules to last longer disease
Dog is blurted out saliva, nose liquid and the outside toxin expelling of excrement through respiratory tract, alimentary canal, pollution feed, drinking-water, cage tool and ambient enviroment, directly
Or it is transmitted to susceptible animal indirectly.CCV can survive 6-9 days in excrement, can also keep the infectiousness of a few days in water, because
Once this fall ill, then is difficult to prevent spread and epidemic.Canine coronavirus is distributed widely in the whole world, is to China's canine farming, fur
Animal farming industry endangers one of maximum epidemic disease.
Chinese patent ZL201620488080.4 discloses a kind of colloid gold test paper of quickly detection canine coronavirus antibody
Card pastes sample pad in plastic bottom board one end, and one end of sample pad is closely crimped containing the gold for marking anti-canine coronavirus antigen
Mark pad, one end of gold-labelled pad closely crimp the gold mark mouse IgG antibody pad containing label mouse IgG antibody, gold mark mouse IgG antibody pad one
End is close to crimp nitrocellulose NC film, and the other end of the detection line T being separated from each other and nature controlling line C, NC film are coated on NC film
It connects water absorption pad and forms test paper, test paper is packed into plastics and gets stuck interior formation test card.The patent uses traditional colloidal gold chromatographic skill
Art, immune marker rely on Electrostatic Absorption, and that there are sensitivity is lower, is difficult to the defects of quantitative, repeatability and stability are poor, nothing
Method fully meets clinical detection demand.In addition, being clinical special with the gastroenteritis differed in weight after dog infection canine coronavirus
Sign, coronavirus is generally present in the excrement of disease dog, and canine coronavirus antibody is generally there are in the blood of dog, which adopts
With the canine coronavirus antibody in dual-antigen sandwich method detection excrement, it is understood that there may be situations such as recall rate is lower, and accuracy is poor.
Chinese patent ZL201410753855.1 discloses a kind of fluorescence immune chromatography technology detection canine coronavirus
Test strips, including labeling pad and capture film, the labeling pad contain the canine coronavirus detection antibody of fluorescent microsphere label,
The capture film contains capture probe.There are following problems for this method: using the canine coronavirus of reagent bottle packing fluorescent marker
Antibody, rather than be directly sprayed onto labeling pad, so entire detection card non-dry type, need to refrigerate, and when detection, need first containing
The sample solution of canine coronavirus is added in reagent bottle, is operated and is owed convenient, improper on-site test.
Utility model content
In view of the above problems, the object of the present invention is to provide a kind of high sensitivity, the use of testing result favorable reproducibility
In the immunofluorescence chromatography detection card of detection canine coronavirus antigen.
For this purpose, technical solution provided by the present application is such that
A kind of immunofluorescence chromatography detection card for detecting canine coronavirus antigen, including bottom plate, on bottom plate successively
Linking has sample pad, bonding pad, nitrocellulose membrane and water absorption pad, which is characterized in that the bonding pad is equipped with fluorescent microsphere mark
The goat-anti chicken IgY of CCV monoclonal antibody and the fluorescent microsphere label of note, the nitrocellulose membrane are equipped with a coating IgY's
The detection T line of Quality Control C line and a coating CCV monoclonal antibody.
Further, a kind of above-mentioned immunofluorescence for detecting canine coronavirus antigen chromatographs detection card, further includes
Box body, the interior immunofluorescence chromatography detection card equipped with sample dilution bottle and for detecting dog CDV of the box body.
Further, a kind of above-mentioned immunofluorescence for detecting canine coronavirus antigen chromatographs detection card, the box
It is additionally provided with suction pipe in vivo.
Further, a kind of above-mentioned immunofluorescence for detecting canine coronavirus antigen chromatographs detection card, described
Shell is equipped with well compatible with sample pad, watch window compatible with bonding pad.
Further, a kind of above-mentioned immunofluorescence for detecting canine coronavirus antigen chromatographs detection card, described
Housing upper surface is additionally provided with tread plate.
Compared with prior art, technical solution provided by the utility model has the advantages that
1, the utility model operating procedure is simple, optimizes to sample pretreating method, it is only necessary to cat serum is acquired, it is dilute
Be added to after releasing detection card well in can be detected, detection speed it is fast, 10 minutes go out as a result, result can qualitative observation again
It can quantitative determine, substantially reduce testing cost, reduce workload.
2, technical solution provided by the present application, by the optimization to sample pad treatment fluid and mark fluorescent microballoon dilution,
Compared to traditional handicraft, sensitivity is obviously high, and minimum detection limit (LOD) can reach 2.69ng/mL (common process 6.25ng/mL),
Linear more preferable, the coefficient of determination of dose-response curve is promoted by 0.9892 to 0.9953.
3, technical solution provided by the present application is strictly investigated to bonding pad, nitrocellulose membrane preparation process, is optimized, further
Improve the precision of detection card.
Detailed description of the invention
Fig. 1 is Test paper card structure schematic diagram provided by the utility model；
Fig. 2 is test strip structural schematic diagram provided by the utility model；
Fig. 3 is another Test paper card structure schematic diagram provided by the utility model；
Fig. 4 is schematic diagram when detection provided by the utility model card determines result for the positive；
Fig. 5 is that detection card provided by the utility model determines that result is negative schematic diagram；
Fig. 6 is that the utility model detection card provided by the utility model determines a kind of schematic diagram when result is invalid；
Fig. 7 is that the utility model detection card provided by the utility model determines another kind schematic diagram when result is invalid；
Fig. 8 is the canonical plotting of viral antigen Concentration Testing provided by the present application.
Specific embodiment
With reference to embodiment, the claim of the utility model is described in further detail.
Embodiment 1
A kind of immunofluorescence for detecting canine coronavirus antigen provided by the utility model chromatographs detection card, refering to figure
1 to Fig. 2, including shell 7, the interior immunofluorescence being equipped with for detecting canine coronavirus antigen of the shell 7, which chromatographs, to be detected
Card, the immunofluorescence chromatography detection card for detecting canine coronavirus antigen includes bottom plate 1, is successively connected on bottom plate 1
There are sample pad 2, bonding pad 3, nitrocellulose membrane 4 and water absorption pad 5, the bonding pad 3 is equipped with fluorescent microsphere and marks CCV Dan Ke
The goat-anti chicken IgY32 of grand antibody 31 and fluorescent microsphere label, the nitrocellulose membrane 4 are equipped with the Quality Control C of a coating chicken IgY
The detection T line of line and a coating CCV monoclonal antibody.The shell 7 is equipped with sample-adding compatible with sample pad 2
Hole 72, watch window 71 compatible with bonding pad 3,7 upper surface of shell are additionally provided with tread plate 73, facilitate reagent card
It takes out and is put into.
Embodiment 2
Another immunofluorescence chromatography detection card for detecting canine coronavirus antigen provided by the present application, refering to fig. 1
It is anti-equipped with sample diluting liquid bottle 6, suction pipe 8 and for detecting canine coronavirus in the box body 9 to Fig. 3, including box body 9
Former immunofluorescence chromatography detection card, the immunofluorescence chromatography detection for detecting canine coronavirus antigen are positioned in shell
In 7, the immunofluorescence chromatography detection card for detecting canine coronavirus antigen includes bottom plate 1, is successively connected on bottom plate 1
There are sample pad 2, bonding pad 3, nitrocellulose membrane 4 and water absorption pad 5, the bonding pad 3 is equipped with fluorescent microsphere and marks CCV monoclonal
The goat-anti chicken IgY32 of antibody 31 and fluorescent microsphere label, the nitrocellulose membrane 4 are equipped with the Quality Control C line of a coating IgY,
And the detection T line of a coating CCV monoclonal antibody.The shell 7 is equipped with well compatible with sample pad 2
72, watch window 71 compatible with bonding pad 3,7 upper surface of shell is additionally provided with tread plate 73, facilitates taking for reagent card
Out be put into.
Embodiment 3
The system for the immunofluorescence chromatography detection card for detecting canine coronavirus antigen that embodiment 1 and embodiment 2 provide
Preparation Method is assembled in the environment of temperature (18~26) DEG C, humidity≤30%, and successively linking has nitric acid fine on bottom plate 1
Tie up film 4, water absorption pad 5, bonding pad 3 and sample pad 2；The big plate pasted is cut into the test strips of 4.0MM wide, plastics is packed into and gets stuck
It is interior, i.e. CCV detection card；Each detection is placed in aluminum foil bag, desiccant 1 is added and wraps, heat sealing is spare.
It is specific the preparation method is as follows:
The preparation of 1.1 bonding pads
1.1.1 the label of CCV monoclonal antibody
1) select microballoon: microballoon for the fluorescence tramp containing rare-earth europium element polystyrene microsphere；Density: 1.05g/
cm3；Refractive index: 1.59@589nm, 25 DEG C；Diameter 300nm；Functional group: carboxyl；Appearance: white.
2) it dilutes fluorescent microsphere: taking a centrifuge tube, MES label buffer (2- (N- morpholine) second of 1mL0.1M is added
Fluorescent microsphere 1mg is added in sulfonic acid, is vortexed and mixes.
3) activation of microballoon: being added (20-50) μ L (preferably 30 μ L) 50mg/mL in above-mentioned centrifuge tube and mark activator A,
The label activator A is MES buffer (2- (the N- morphine of the 0.05M of HOSu NHS containing 50mg/mLN- (NHS)
Quinoline) ethanesulfonic acid), it is vortexed after mixing and (20-50) μ L (preferably 30 μ L) 50mg/mL label activator B is added, the label is living
Agent B is the 0.05M MES buffer of (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride containing 50mg/mL1- (EDC)
(2- (N- morpholine) ethanesulfonic acid) reacts 30min on rotary incubator；
4) activation is quenched: the fluorescent microsphere 20000g after activation being centrifuged 30min, abandons supernatant, leaves and takes precipitating, 2mL is added
Quencher (the MES buffer of the 0.1M containing 0.25% methanol), ultrasonic 2s；
5) label of antibody: being added 20 μ g CCV monoclonal antibodies in the fluorescent microsphere after ultrasound is resuspended, be vortexed and mix,
It is placed on rotary incubator and reacts 30min；
6) it closes: 200.0 μ L is added and mark confining liquid, ultrasonic 2s, interval 5s are repeated 3 times, and ultrasound is completed to be placed on rotation
30min is reacted on incubator；The label confining liquid is 8.0 ethanol amine of 0.04M pH containing 3% bovine serum albumin(BSA) (BSA)
Solution；
7) purify: after completing above-mentioned reaction, 19000g is centrifuged 30min, sucks supernatant, leaves and takes precipitating, and 600.0 μ L are added
Mark fluorescent microballoon dilution, ultrasound, work 2S, and interval 5S is repeated 3 times.
The fluorescent microsphere dilution is containing 1% bovine serum albumin(BSA) (BSA), 1% glucan T10 and 0.05% junket egg
The 0.05M pH 7.8Tris buffer of the 0.03%Proclin300 of white sodium.
1.1.2 the label of goat-anti chicken IgY
1) select microballoon: microballoon for the fluorescence tramp containing rare-earth europium element polystyrene microsphere；Density: 1.05g/
cm3；Refractive index: 1.59@589nm, 25 DEG C；Diameter 300nm；Functional group: carboxyl；Appearance: white.
2) it dilutes fluorescent microsphere: taking a centrifuge tube, MES label buffer (2- (N- morpholine) second of 1mL0.1M is added
Fluorescent microsphere 1mg is added in sulfonic acid, is vortexed and mixes.
3) activation of microballoon: being added (20-50) μ L (preferably 30 μ L) 50mg/mL in above-mentioned centrifuge tube and mark activator A,
The label activator A is MES buffer (2- (the N- morphine of the 0.05M of n-hydroxysuccinimide containing 50mg/mL (NHS)
Quinoline) ethanesulfonic acid), it is vortexed after mixing and (20-50) μ L (preferably 30 μ L) 50mg/mL label activator B is added, the label is living
Agent B is the 0.05M MES buffer of (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride containing 50mg/mL1- (EDC)
(2- (N- morpholine) ethanesulfonic acid) reacts 30min on rotary incubator；
4) activation is quenched: the fluorescent microsphere 20000g after activation being centrifuged 30min, abandons supernatant, leaves and takes precipitating, 2mL is added
Quencher (the MES buffer of the 0.1M containing 0.25% methanol), ultrasonic 2s；
4) label of antibody: 0.15mg goat-anti chicken IgY is added in the fluorescent microsphere after ultrasound is resuspended, is vortexed and mixes, set
In reacting 30min on rotary incubator；
5) it closes: label confining liquid is added, closes 30min；The label confining liquid is containing 3% bovine serum albumin(BSA)
(BSA) 8.0 ethanolamine solutions of 0.04M pH；
6) purify: after completing above-mentioned reaction, 19000g is centrifuged 30min, sucks supernatant, leaves and takes precipitating, and 1mL label is added
Fluorescent microsphere dilution.
The fluorescent microsphere dilution is containing 1% bovine serum albumin(BSA) (BSA), 1% glucan T10 and 0.05% junket egg
The 0.05M pH 7.8Tris buffer of the 0.03%Proclin300 of white sodium.
1.1.3 film is sprayed
1) glass fiber is cut into (10 ± 1) mm × (300 ± 10) mm size is bonding pad；
2) T, C line antibody after label are uniformly mixed by 20:1 (V:V), 90W ultrasound, work 2s, interval 5s, repeats 3
It is secondary；
3) it is sprayed on the bonding pad after cutting out by 3.5 μ L/cm, 0.02MPa；
4) after having sprayed, (16~18) h is dried overnight in 37 DEG C of drying boxes.
The preparation of 1.2 reaction films
1) IgY is diluted to 1mg/mL, as C line work with the phosphate buffer (PBS) of the 0.01M containing 1% trehalose
Make liquid.
2) another plant of CCV monoclonal antibody is diluted to the phosphate buffer (PBS) of the 0.01M containing 1% trehalose
1mg/mL, as T line working solution.
3) PVC bottom plate is taken, nitrocellulose membrane is pasted.
4) T line and C line are drawn on nitrocellulose membrane, scribing line concentration is 1 μ L/cm.
5) sheet material is placed in 37 DEG C of drying boxes after the completion of and is dried overnight (16~18) h.
The preparation of 1.3 sample pads
1) sample pad treatment fluid is prepared: the pH's 7.4 containing 1%BSA, 0.2%PVP 58000,0.5%Tween-20
The phosphate buffer (PBS) of 0.01M.
2) buffer is sprayed on glass fibre with the concentration of 10 μ L/cm.
Detection method
For the ease of using detection provided by the present application to block, detection card provided by the present application is given below, two kinds of detections are provided
Method.
Method one: using ultraviolet light irradiation, refering to Fig. 4 to Fig. 7: using hand-held ultraviolet light irradiation watch window, works as matter
When control line and the detection line amount of having fluorescence occur (Fig. 4), illustrate that CCV antigen is the positive in sample；When only nature controlling line has fluorescence
And detection line unstressed configuration illustrates that CCV antigen is feminine gender in sample when occurring (Fig. 5)；Nature controlling line does not occur fluorescence (Fig. 6, Fig. 7),
It is invalid then to represent the wrong or testing result of operation, need to repeat to test.
Method two: Immunofluorescence test instrument testing result:
1) sick dog excrement is acquired using cotton swab；
2) take 10 μ L be added containing 1.0mL sample diluting liquid (0.01M's containing 0.8%S21,0.03%Proclin300
PH7.4 phosphate buffer) sample cell in, mix well；
3) detection card is taken out, dry type Immunofluorescence test instrument is opened；
4) the 75 μ L of sample after dilution is drawn, is added in the well of detection card, will test the card that card is put into instrument
In slot, start timing；
5) after 10min, " test " key on instrument is clicked, instrument will start to test；
6) fluorescence intensity is directly proportional to the CCV concentration in sample, is carried out curve fitting by built-in standard curve and concentration
Calculating, dose-effect curve Log (Y)=0.7932Log (X) -1.6475, R=0.9976 (R2=0.9953), refering to Fig. 3.
Immunofluorescence test instrument testing result:
1, the range of linearity
1.1 CCV calibration object is diluted to respectively with negative serum 0ng/mL, 10ng/mL, 30ng/mL, 90ng/mL,
270ng/mL、800ng/mL、2400ng/mL、3000ng/mL、4000ng/mL、5000ng/mL、6000ng/mL、 7000ng/
ML and 8000ng/mL；
Above each concentration samples are taken 75 μ L by 1.2 respectively, and every concentration weight into the cat CCV detection reagent prepared is added
Repetition measurement tries 2 times；
After 1.3 reaction 10min, test paper is put into dry type immunofluorescence analysis instrument, T/C value is read, passes through built-in standard
The calculating that curve carries out curve fitting with concentration.
1.4 test results are shown: reagent is preferable in the CCV linear fit relationship for detecting 10~4000ng/mL, r >
0.9900, when CCV concentration increases to 4000ng/mL, linear fit r value slows down less than 0.9900, T/C growth, and CCV exists
T/C value is gentle when 4000~5000ng/mL, and when CCV reaches 6000ng/mL, T/C value reduces instead.Therefore the maximum of this detection
Detection range can achieve 4000ng/mL.
2, minimum detectability
By negative serum, retest 20 times, T/C mean value is calculated, is substituted into dose-response curve, obtains we
The minimum detectability of method is 2.69ng/mL, is shown in Table 1 and Fig. 8.
Table 1
3, precision
CCV calibration object is diluted to 20ng/mL, 200ng/mL, 2000ng/mL with negative serum, is detected and is blocked with this method
Every concentration retest 10 times, calculates the coefficient of variation of test concentrations.As a result shown in table 2, it can thus be seen that this kit is examined
The coefficient of variation of survey is respectively less than 10% (three concentration are respectively 8.62%, 6.59% and 4.61%), is shown in Table 2, therefore this detection
Method precision with higher.
Table 2
| Concentration | CV | |
| Quality Control I | 20ng/mL | 8.62% |
| Quality Control II | 200ng/mL | 6.59% |
| Quality Control III | 1000ng/mL | 4.61% |
In order to better illustrate the beneficial effects of the utility model, it is given below and is tried using detection provided by the utility model
The method of agent and detection canine coronavirus (CCV) is blood clotting (HA) and hemagglutination-inhibition test (HI) and ELISA method, PCR detection
Method, Comparative result experiment of the colloidal gold method when detecting canine coronavirus antigen, the results are shown in Table 3.
Table 3
Above-described embodiment is the preferable embodiment of the utility model, but the embodiments of the present invention is not by above-mentioned
The limitation of embodiment, it is made under other any spiritual essence and principles without departing from the utility model to change, modify, replacing
In generation, simplifies combination, should be equivalent substitute mode, is included within the protection scope of the utility model.
Claims (3)
1. a kind of immunofluorescence for detecting canine coronavirus antigen chromatographs detection card, including bottom plate (1), on bottom plate (1)
Successively linking has sample pad (2), bonding pad (3), nitrocellulose membrane (4) and water absorption pad (5), which is characterized in that the bonding pad
(3) it is equipped with the CCV monoclonal antibody (31) of fluorescent microsphere label and the goat-anti chicken IgY (32) of fluorescent microsphere label, it is described
Nitrocellulose membrane (4) is equipped with the Quality Control C line of a coating chicken IgY and the detection T line of a coating CCV monoclonal antibody.
2. a kind of immunofluorescence for detecting canine coronavirus antigen according to claim 1 chromatographs detection card, special
Sign is, further includes box body (9), and sample dilution bottle (6) and the immunofluorescence for detecting dog CDV are equipped in the box body (9)
Chromatography detection card.
3. a kind of immunofluorescence for detecting canine coronavirus antigen according to claim 2 chromatographs detection card, special
Sign is, is additionally provided with suction pipe (8) in the box body (9).
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108318685A (en) * | 2018-05-04 | 2018-07-24 | 广州敏捷生物技术有限公司 | Immunofluorescence for detecting canine coronavirus antigen chromatographs detection card and preparation method |
| CN111948381A (en) * | 2020-08-21 | 2020-11-17 | 苏州阿科索生物科技有限公司 | Canine coronavirus fluorescence aptamer test strip |
| CN112798778A (en) * | 2021-03-17 | 2021-05-14 | 广州敏捷生物技术有限公司 | Immunofluorescence chromatography detection card and method for synchronously detecting florfenicol and trimethoprim contents in poultry eggs |
| CN113588949A (en) * | 2021-08-17 | 2021-11-02 | 广州艺得诺生物科技有限公司 | Preparation and use method of canine coronavirus chemiluminescence detection reagent |
-
2018
- 2018-05-04 CN CN201820667833.7U patent/CN208672650U/en active Active
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108318685A (en) * | 2018-05-04 | 2018-07-24 | 广州敏捷生物技术有限公司 | Immunofluorescence for detecting canine coronavirus antigen chromatographs detection card and preparation method |
| CN111948381A (en) * | 2020-08-21 | 2020-11-17 | 苏州阿科索生物科技有限公司 | Canine coronavirus fluorescence aptamer test strip |
| CN112798778A (en) * | 2021-03-17 | 2021-05-14 | 广州敏捷生物技术有限公司 | Immunofluorescence chromatography detection card and method for synchronously detecting florfenicol and trimethoprim contents in poultry eggs |
| CN113588949A (en) * | 2021-08-17 | 2021-11-02 | 广州艺得诺生物科技有限公司 | Preparation and use method of canine coronavirus chemiluminescence detection reagent |
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