CN108318685A - Immunofluorescence for detecting canine coronavirus antigen chromatographs detection card and preparation method - Google Patents
Immunofluorescence for detecting canine coronavirus antigen chromatographs detection card and preparation method Download PDFInfo
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- CN108318685A CN108318685A CN201810417581.7A CN201810417581A CN108318685A CN 108318685 A CN108318685 A CN 108318685A CN 201810417581 A CN201810417581 A CN 201810417581A CN 108318685 A CN108318685 A CN 108318685A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Abstract
The immunofluorescence chromatography detection card and preparation method that the invention discloses a kind of for detecting canine coronavirus antigen；It is intended to provide a kind of high sensitivity, testing result reliably is used to detect the immunofluorescence chromatography detection card of canine coronavirus antigen；Its skill main points includes bottom plate, linking has sample pad, bonding pad, nitrocellulose membrane and water absorption pad successively on bottom plate, the bonding pad is equipped with the goat-anti chicken IgY of the CCV monoclonal antibodies and fluorescent microsphere label of fluorescent microsphere label, the nitrocellulose membrane is equipped with the Quality Control C lines and a detection T line for being coated with CCV monoclonal antibodies of a coating chicken IgY；Belong to animal epidemic detection field.
Description
Technical field
The invention discloses a kind of immunofluorescences to chromatograph detection card, specifically, being a kind of for detecting canine coronavirus
The immunofluorescence chromatography detection card of antigen, the invention also discloses the preparation methods that the immunofluorescence chromatographs detection card.
Background technology
The pathogen of canine coronavirus disease is canine coronavirus (Canine Corona Virus, CCV).Differed with weight
Gastroenteritis be Clinical symptoms, clinically be in lethal watery diarrhea.The useless exhausted, vomiting of sudden onset, psychotic depression, appetite,
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
Just it 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 quick detection canine coronavirus antibody
Card pastes sample pad in plastic bottom board one end, and closely crimping contains the gold for marking anti-canine coronavirus antigen for one end of sample pad
One end of mark pad, gold-labelled pad closely crimps the gold mark mouse IgG antibody pad containing label mouse IgG antibody, golden mark mouse IgG antibody pad one
End is close to crimp nitrocellulose NC films, 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 films
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 relatively low, is difficult to the defects of quantitative, repeatability and stability are poor, nothing
Method fully meets clinical detection demand.It is clinical special with the gastroenteritis that weight differs in addition, 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
The canine coronavirus antibody in excrement is detected with dual-antigen sandwich method, it is understood that there may be situations such as recall rate is relatively low, and accuracy is poor.
Chinese patent ZL201410753855.1 is disclosed a kind of detecting canine coronavirus with fluorescence immune chromatography technology
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：The canine coronavirus of fluorescent marker is dispensed using reagent bottle
Antibody, rather than be directly sprayed onto in 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 Site Detection.
Invention content
In view of the above-mentioned problems, the object of the present invention is to provide a kind of high sensitivity, testing result favorable reproducibility is used to examine
Survey the immunofluorescence chromatography detection card of canine coronavirus antigen.
Second object of the present invention is to provide the preparation method of above-mentioned detection reagent card.
For this purpose, first technical solution provided by the present application is such:
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 antibodies and the fluorescent microsphere label of note, the nitrocellulose membrane are equipped with one and are coated with IgY's
The detection T lines of Quality Control C lines and a coating CCV monoclonal antibody.
Further, a kind of above-mentioned immunofluorescence chromatography detection for detecting canine coronavirus antigen blocks, and further includes
Sample dilution bottle.
Further, above-mentioned a kind of immunofluorescence chromatography detection card for detecting canine coronavirus antigen, feature
It is, sample diluting liquid is housed in the sample dilution bottle, and the sample diluting liquid is containing 0.8%S21,0.03%
The pH7.4 phosphate buffers of the 0.01M of Proclin300.
Second technical solution provided by the present application is such：
A kind of immunofluorescence chromatography detection card for detecting canine coronavirus antigen, linking has sample successively on bottom plate
Pad, bonding pad, nitrocellulose membrane and water absorption pad,
A the preparation method of the sample pad described in) is：
1) sample pad treatment fluid is prepared：The 0.01M of pH7.4 containing 1%BSA, 0.2%PVP58000,0.5%Tween-20
Phosphate buffer；
2) concentration of the 5.0 μ L/cm of buffer solution prepared in step 1) is sprayed on glass fibre；
B the preparation method of the bonding pad described in) is：
1) by the CCV monoclonal antibodies marked with fluorescent microsphere and the goat-anti chicken IgY marked with fluorescent microsphere according to volume
Than 20:1 is uniformly mixed, and ultrasonic 2s, interval 5s are repeated 3 times；
2) it is sprayed onto on the bonding pad after cutting out by 3.5 μ L/cm, 0.02MPa；
C the preparation method of the reaction film described in) is：
1) chicken IgY is diluted to 1mg/mL, as C nature controlling lines working solution with the coating dilution containing antibody；
2) CCV monoclonal antibodies are diluted to 1mg/mL, as T detection lines working solution with antibody coating dilution；
3) base plate sticking nitrocellulose membrane is taken；
4) T lines and C lines 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.
Further, above-mentioned a kind of immunofluorescence chromatography detection card for detecting canine coronavirus antigen, it is described
Antibody is coated with the phosphate buffer that dilution is the 0.01M containing 1% trehalose.
Further, the preparation side of above-mentioned a kind of immunofluorescence chromatography detection card for detecting canine coronavirus antigen
Method, fluorescent microsphere label CCV monoclonal antibodies preparation method be：
1) fluorescent microsphere is diluted：A centrifuge tube is taken, 1mL0.05M MES buffer solutions are added, fluorescent microsphere 1mg is added, is vortexed
Mixing；
2) activation of microballoon：20 μ L-50 μ L label activator A are added in above-mentioned centrifuge tube and 20 μ L-50 μ L labels are lived
Agent B reacts 30min on rotary incubator；
3) activation is quenched：Fluorescent microsphere 20000g after activation is centrifuged into 30min, supernatant is abandoned, leaves and takes precipitation, be added 5000
μ L quenchers, ultrasonic 2s；
4) label of antibody：20-200 μ g CCV monoclonal antibodies are added in the fluorescent microsphere after ultrasound is resuspended, are vortexed
Mixing is placed on rotary incubator and reacts 30min；
5) it closes：2mL, which is added, marks confining liquid, ultrasonic 2s, interval 5s to be repeated 3 times, and ultrasound is completed to be placed on rotating and culturing
30min is reacted on device；
6) it purifies：After completing above-mentioned reaction, 19000g centrifuges 30min, sucks supernatant, leaves and takes precipitation, and it is glimmering that 2mL labels are added
Light microballoon dilution, ultrasound, work 2S, and interval 5S is repeated 3 times.
Further, the preparation side of above-mentioned a kind of immunofluorescence chromatography detection card for detecting canine coronavirus antigen
The preparation method of method, fluorescent microsphere label goat-anti chicken IgY is：
1) microballoon is diluted：A centrifuge tube is taken, the 0.05M MES buffer solutions of 1mL are added, takes fluorescent microsphere 1.0mg, is vortexed mixed
It is even；
2) activation of microballoon：20 μ L-50 μ L label activator A are added in above-mentioned centrifuge tube and 20 μ L-50 μ L labels are lived
Agent B reacts 30min on rotary incubator；
3) activation is quenched：Fluorescent microsphere 19000g after activation is centrifuged into 30min, supernatant is abandoned, leaves and takes precipitation, 2mL is added
Quencher, 90W ultrasounds, work 2s, and interval 5s is repeated 1 times；
4) label of antibody：100 μ g goat-anti chicken IgY are added in the fluorescent microsphere after ultrasound is resuspended, vortex mixing is placed in
30min is reacted on rotary incubator；
5) it closes：500 μ L label confining liquids are added, close 30min；
6) it purifies：After completing above-mentioned reaction, 19000g centrifuges 30min, sucks supernatant, leaves and takes precipitation, and it is glimmering that 2mL labels are added
Light microballoon dilution, 90W ultrasounds, work 2s, and interval 5s is repeated 3 times.
Further, the preparation side of above-mentioned a kind of immunofluorescence chromatography detection card for detecting canine coronavirus antigen
Method, the microballoon are the polystyrene microsphere of the fluorescence tramp containing rare-earth europium element；Density：1.05g/cm3；Refraction
Rate：1.59@589nm, 25 DEG C；Diameter 300nm；Functional group：Carboxyl；Appearance：White.
Further, the preparation side of above-mentioned a kind of immunofluorescence chromatography detection card for detecting canine coronavirus antigen
Method, the label activator A are the MES buffer solutions of the 0.05M of the n-hydroxysuccinimide containing 50mg/mL；The label activation
Agent B is the 0.05M MES buffer solutions of 1- containing 50mg/mL (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides.
Further, the preparation side of above-mentioned a kind of immunofluorescence chromatography detection card for detecting canine coronavirus antigen
Method, the MES buffer solutions that the activation quencher is the 0.1M containing 0.25% methanol；The label confining liquid is containing 3% cow's serum
8.0 ethanolamine solutions of 0.04M pH of albumin；The mark fluorescent microballoon dilution is containing 1% bovine serum albumin(BSA), 1%
The 0.05M pH7.8Tris buffer solutions of the 0.03%Proclin300 of glucan T10 and 0.05% casein-sodium.
Compared with prior art, technical solution provided by the invention has the following advantages that：
1, operating procedure of the present invention is simple, is optimized to sample pretreating method, it is only necessary to cat serum is acquired, after dilution
Be added to detection card well in i.e. can detect, detection speed is fast, go out within 10 minutes as a result, result can qualitative observation can determine again
It is fixed to measure, and substantially reduces testing cost, reduces 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.
4, the application is different according to detection target, explores and pads treatment fluid using Special sample, immunochromatography is more abundant, exempts from
Epidemic disease reaction efficiency is more preferable；Reproducible, low middle high three horizontal Quality Control coefficient of variation are below 10% (8.62%, 6.59% and
4.61%)；In addition biological preservative Proclin300 is added in buffer solution, can effectively keep the stability of detection reagent, and
Protect harm of the tester not by conventional preservatives such as Sodium azide.
Description of the drawings
Fig. 1 is Test paper card structure schematic diagram provided by the invention；
Fig. 2 is test strip structural schematic diagram provided by the invention；
Fig. 3 is another Test paper card structure schematic diagram provided by the invention；
Fig. 4 be detection provided by the invention card judgement result for the positive when schematic diagram；
Fig. 5 is that detection card judgement result provided by the invention is negative schematic diagram；
Fig. 6 is that the detection card that invention provides judges a kind of schematic diagram when result is invalid；
Fig. 7 is that the detection card that invention provides judges another kind schematic diagram when result is invalid；
Fig. 8 is the canonical plotting of viral antigen Concentration Testing provided by the present application.
Fig. 9 is the canonical plotting for the viral antigen Concentration Testing that comparative example provides.
Specific implementation mode
With reference to embodiment, the claim of the present invention is described in further detail.
Embodiment 1
A kind of immunofluorescence chromatography detection card for detecting canine coronavirus antigen provided by the invention, refering to fig. 1 extremely
Fig. 2, including shell 7, the interior immunofluorescence being equipped with for detecting canine coronavirus antigen of the shell 7 chromatograph detection card, institute
It includes bottom plate 1 to state the immunofluorescence for detecting canine coronavirus antigen and chromatograph detection card, and linking has sample successively on bottom plate 1
Pad 2, bonding pad 3, nitrocellulose membrane 4 and water absorption pad 5, the bonding pad 3 are equipped with fluorescent microsphere label CCV monoclonal antibodies 31
With the goat-anti chicken IgY32 of fluorescent microsphere label, the nitrocellulose membrane 4 is equipped with the Quality Control C lines of a coating chicken IgY, and
The detection T lines of one coating CCV monoclonal antibody.The shell 7 is equipped with well 72 compatible with sample pad 2, with
3 compatible watch window 71 of bonding pad, 7 upper surface of shell are additionally provided with tread plate 73, facilitate the taking-up of reagent card with
It 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 connected successively on bottom plate 1
There are sample pad 2, bonding pad 3, nitrocellulose membrane 4 and water absorption pad 5, the bonding pad 3 to be equipped with fluorescent microsphere and mark CCV monoclonals
The goat-anti chicken IgY32 of antibody 31 and fluorescent microsphere label, the nitrocellulose membrane 4 are equipped with the Quality Control C lines of a coating IgY,
And the detection T lines 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
Go out and is 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 linking has nitric acid fine successively 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 cards；Each detection is placed in aluminum foil bag, drier 1 is added and wraps, heat sealing is spare.
Specific preparation method is as follows：
The preparation of 1.1 bonding pads
1.1.1CCV the label of monoclonal antibody
1) microballoon is selected：Microballoon is the polystyrene microsphere of the fluorescence tramp containing rare-earth europium element；Density：1.05g/
cm3；Refractive index：1.59@589nm, 25 DEG C；Diameter 300nm；Functional group：Carboxyl；Appearance：White.
2) fluorescent microsphere is diluted：A centrifuge tube is taken, MES label buffer solution (2- (N- morpholines) second sulphurs of 1mL0.1M are added
Fluorescent microsphere 1mg, vortex mixing is added in acid.
3) activation of microballoon：(20-50) μ L (preferably 30 μ L) 50mg/mL is added in above-mentioned centrifuge tube and marks activator A,
The label activator A is MES buffer solutions (2- (the N- morphines of the 0.05M of HOSu NHS containing 50mg/mLN- (NHS)
Quinoline) ethanesulfonic acid), (200-500) (preferably 300 μ L) μ L50mg/mL are added after vortex mixing and mark activator B, the label
Activator B is that the 0.05MMES of (3- the dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides containing 50mg/mL1- (EDC) is buffered
Liquid (2- (N- morpholines) ethanesulfonic acid) reacts 30min on rotary incubator；
4) activation is quenched：Fluorescent microsphere 20000g after activation is centrifuged into 30min, supernatant is abandoned, leaves and takes precipitation, 2mL is added
Quencher (the MES buffer solutions of the 0.1M containing 0.25% methanol), ultrasonic 2s；
5) label of antibody：It is added 20 μ gCCV monoclonal antibodies in the fluorescent microsphere after ultrasound is resuspended, vortex mixing,
It is placed on rotary incubator and reacts 30min；
6) it closes：200.0 μ L label confining liquids are added, 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 that the 0.04MpH8.0 ethanol amines containing 3% bovine serum albumin(BSA) (BSA) are molten
Liquid；
7) it purifies：After completing above-mentioned reaction, 19000g centrifuges 30min, sucks supernatant, leaves and takes precipitation, and 600.0 μ L marks are added
Remember fluorescent microsphere 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 7.8 Tris buffer solutions of 0.05M pH of the 0.03%Proclin300 of white sodium.
1.1.2 the label of goat-anti chicken IgY
1) microballoon is selected：Microballoon is the polystyrene microsphere of the fluorescence tramp containing rare-earth europium element；Density：1.05g/
cm3；Refractive index：1.59@589nm, 25 DEG C；Diameter 300nm；Functional group：Carboxyl；Appearance：White.
2) fluorescent microsphere is diluted：A centrifuge tube is taken, MES label buffer solution (2- (N- morpholines) second sulphurs of 1mL0.1M are added
Fluorescent microsphere 1mg, vortex mixing is added in acid.
3) activation of microballoon：(20-50) μ L (preferably 30 μ L) 50mg/mL is added in above-mentioned centrifuge tube and marks activator A,
The label activator A is MES buffer solutions (2- (the N- morphines of the 0.05M of HOSu NHS containing 50mg/mLN- (NHS)
Quinoline) ethanesulfonic acid), (20-50) μ L (preferably 30 μ L) 50mg/mL is added after vortex mixing and marks activator B, label activation
Agent B is the 0.05M MES buffer solutions of (3- the dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides containing 50mg/mL1- (EDC)
(2- (N- morpholines) ethanesulfonic acid) reacts 30min on rotary incubator；
4) activation is quenched：Fluorescent microsphere 20000g after activation is centrifuged into 30min, supernatant is abandoned, leaves and takes precipitation, 2mL is added
Quencher (the MES buffer solutions of the 0.1M containing 0.25% methanol), ultrasonic 2s；
4) label of antibody：0.25mg goat-anti chicken IgY are added in the fluorescent microsphere after ultrasound is resuspended, vortex mixing is 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) it purifies：After completing above-mentioned reaction, 19000g centrifuges 30min, sucks supernatant, leaves and takes precipitation, and it is glimmering that 1mL labels are added
Light microballoon dilution.
The fluorescent microsphere dilution is containing 1% bovine serum albumin(BSA) (BSA), 1% glucan T10 and 0.05% junket egg
The 7.8 Tris buffer solutions of 0.05M pH of the 0.03%Proclin300 of white sodium.
1.1.3 film is sprayed
1) it is bonding pad glass fiber to be cut into (10 ± 1) mm × (300 ± 10) mm sizes；
2) T, C line antibody after label are pressed 20:1(V：V it) is uniformly mixed, 90W ultrasounds, work 2s, interval 5s, repeats 3
It is secondary；
3) it is sprayed onto 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 lines 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 lines working solution.
3) PVC bottom plates are taken, nitrocellulose membrane is pasted.
4) T lines and C lines 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.
It is prepared by 1.3 sample pads
1) sample pad treatment fluid is prepared：The 0.01M of pH7.4 containing 1%BSA, 0.2%PVP58000,0.5%Tween-20
Phosphate buffer (PBS).
2) buffer solution is sprayed on glass fibre with the concentration of 5.0 μ L/cm.、
Contrast test
By the pH7.4's of sample pad treatment fluid 1%BSA, 0.2%PVP58000,0.5%Tween-20 in embodiment 3
The phosphate buffer (PBS) of 0.01M changes 0.5%Tween-20,0.1% bovine serum albumin(BSA), 0.5%Trtion-100 into
8.0 10mmol/L Tris-HCl buffer solutions of 50mM pH；Mark fluorescent microballoon dilution is contained into 1% bovine serum albumin(BSA)
(BSA), the 7.8 Tris buffer solutions of 0.05M pH of the 0.03%Proclin300 of 1% glucan T10 and 0.05% casein-sodium
It is changed to 8.0 10mmol/L of pH containing 2%BSA, 0.1%PEG4000,5% trehalose, 5% sucrose, 0.05% Sodium azide
Tris-HCl buffer solutions, other conditions and technological parameter are constant.
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 ultra violet lamp, refering to Fig. 4 to Fig. 7：With hand-held ultraviolet light irradiation watch window, work as matter
When control line and the detection line amount of having fluorescence occur (Fig. 4), illustrate that CCV antigens are the positive in sample；When only nature controlling line has fluorescence
And detection line unstressed configuration illustrates that CCV antigens are 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) cotton swab is used to acquire sick dog excrement；
2) take 10 μ L be added containing 1.0mL sample diluting liquids (0.01M's containing 0.8%S21,0.03%Proclin300
PH7.4 phosphate buffers) 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 are drawn, are added in the well of detection card, detection card is put into the card of instrument
In slot, start timing；
5) after 10min, " test " button 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 objects are 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, are added into the cat CCV detection reagents prepared, per concentration weight
Repetition measurement tries 2 times；
After 1.3 reaction 10min, test paper is put into dry type immunofluorescence analysis instrument, T/C values 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 relationships for detecting 10~4000ng/mL, r ＞
0.9900, when CCV concentration increases to 4000ng/mL, linear fit r values are less than 0.9900, T/C growths and slow down, and CCV is 4000
T/C values are gentle when~5000ng/mL, and when CCV reaches 6000ng/mL, T/C values reduce instead.Therefore the maximum detection of this detection
Range can reach 4000ng/mL.
2, minimum detectability
By negative serum, retest 20 times calculates T/C mean values, is substituted into dose-response curve, obtain we
The minimum detectability of method is 2.69ng/mL, is shown in Table 1 and Fig. 8.
Table 1
With same method detection comparative example experiment, minimum detectability 6.25ng/mL is shown in Table 2 and Fig. 9.
Table 2
3, precision
CCV calibration objects are diluted to 20ng/mL, 200ng/mL, 2000ng/mL with negative serum, is detected and is blocked with this method
Per concentration retest 10 times, the coefficient of variation of test concentrations is calculated.As a result shown in table 3, 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 3, therefore this detection
Method has higher precision.
Table 3
| 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 beneficial effects of the present invention, it is given below using detection reagent provided by the invention and detection
The method of canine coronavirus (CCV) is blood clotting (HA) and hemagglutination-inhibition test (HI) and ELISA method, PCR detection methods, colloidal gold
Comparative result experiment of the method when detecting canine coronavirus antigen, the results are shown in Table 4.
Table 4
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from the spirit and principles of the present invention made by changes, modifications, substitutions, combinations, simplifications,
Equivalent substitute mode is should be, is included within the scope of the present invention.
Claims (10)
1. a kind of immunofluorescence chromatography detection card for detecting canine coronavirus antigen, including bottom plate (1), on bottom plate (1)
Linking has sample pad (2), bonding pad (3), nitrocellulose membrane (4) and water absorption pad (5) successively, which is characterized in that the bonding pad
(3) it is equipped with the goat-anti chicken IgY (32) of the CCV monoclonal antibodies (31) and fluorescent microsphere label of fluorescent microsphere label, it is described
Nitrocellulose membrane (4) is equipped with the Quality Control C lines and a detection T line for being coated with CCV monoclonal antibodies of a coating chicken IgY.
2. a kind of immunofluorescence chromatography detection card for detecting canine coronavirus antigen according to claim 1, special
Sign is, further includes sample dilution bottle (6).
3. a kind of immunofluorescence chromatography detection card for detecting canine coronavirus antigen according to claim 2, special
Sign is, sample diluting liquid is housed in the sample dilution bottle (6), and the sample diluting liquid is containing 0.8%S21,0.03%
The pH7.4 phosphate buffers of the 0.01M of Proclin300.
4. preparing a kind of side of immunofluorescence chromatography detection card for detecting canine coronavirus antigen described in claim 1
Method, linking has sample pad (2), bonding pad (3), nitrocellulose membrane (4) and water absorption pad (5) successively on bottom plate (1), and feature exists
In：
A the preparation method of the sample pad described in) is：
1) sample pad treatment fluid is prepared：The 0.01M's of pH 7.4 containing 1%BSA, 0.2%PVP 58000,0.5%Tween-20
Phosphate buffer；
2) concentration of the 5.0 μ L/cm of buffer solution prepared in step 1) is sprayed on glass fibre；
B the preparation method of the bonding pad (3) described in) is：
1) the goat-anti chicken IgY marked by the CCV monoclonal antibodies marked with fluorescent microsphere and with fluorescent microsphere is according to volume ratio 20:
1 is uniformly mixed, and ultrasonic 2s, interval 5s are repeated 3 times；
2) it is sprayed onto on the bonding pad after cutting out by 3.5 μ L/cm, 0.02MPa；
C the preparation method of the reaction film described in) is：
1) chicken IgY is diluted to 1mg/mL, as C nature controlling lines working solution with the coating dilution containing antibody；
2) CCV monoclonal antibodies are diluted to 1mg/mL, as T detection lines working solution with antibody coating dilution；
3) bottom plate (1) is taken to paste nitrocellulose membrane；
4) T lines and C lines 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.
5. a kind of immunofluorescence chromatography detection card for detecting canine coronavirus antigen according to claim 4, special
Sign is that the antibody is coated with the phosphate buffer that dilution is the 0.05M containing 1% trehalose.
6. a kind of preparation of immunofluorescence chromatography detection card for detecting canine coronavirus antigen according to claim 5
Method, which is characterized in that the preparation method of CCV monoclonal antibodies of fluorescent microsphere label is：
1) fluorescent microsphere is diluted：A centrifuge tube is taken, 1mL0.1M MES buffer solutions are added, fluorescent microsphere 1mg, vortex mixing is added；
2) activation of microballoon：20 μ L-50 μ L label activator A and 20 μ L-50 μ L20 μ L-50 μ L marks are added in above-mentioned centrifuge tube
Remember activator B, reacts 30min on rotary incubator；
3) activation is quenched：Fluorescent microsphere 20000g after activation is centrifuged into 30min, supernatant is abandoned, leaves and takes precipitation, 5000 μ L are added and quench
It goes out agent, ultrasonic 2s；
4) label of antibody：The addition 20-200 μ g CCV monoclonal antibodies in the fluorescent microsphere after ultrasound is resuspended, vortex mixing,
It is placed on rotary incubator and reacts 30min；
5) it closes：2mL, which is added, marks confining liquid, ultrasonic 2s, interval 5s to be repeated 3 times, and ultrasound is completed to be placed on rotary incubator
React 30min；
6) it purifies：After completing above-mentioned reaction, 19000g centrifuges 30min, sucks supernatant, leaves and takes precipitation, and it is micro- that 2mL mark fluorescents are added
Ball dilution, ultrasound, work 2S, and interval 5S is repeated 3 times.
7. a kind of preparation of immunofluorescence chromatography detection card for detecting canine coronavirus antigen according to claim 5
Method, which is characterized in that the preparation method of goat-anti chicken IgY of fluorescent microsphere label is：
1) microballoon is diluted：A centrifuge tube is taken, the 0.05M MES buffer solutions of 1mL are added, take fluorescent microsphere 1.0mg, vortex mixing；
2) activation of microballoon：20 μ L-50 μ L label activator A and 20 μ L-50 μ L label activators are added in above-mentioned centrifuge tube
B reacts 30min on rotary incubator；
3) activation is quenched：Fluorescent microsphere 19000g after activation is centrifuged into 30min, supernatant is abandoned, leaves and takes precipitation, 2mL is added and is quenched
Agent, 90W ultrasounds, work 2s, and interval 5s is repeated 1 times；
4) label of antibody：100 μ g goat-anti chicken IgY are added in the fluorescent microsphere after ultrasound is resuspended, vortex mixing is placed in rotation
30min is reacted on incubator；
5) it closes：500 μ L label confining liquids are added, close 30min；
6) it purifies：After completing above-mentioned reaction, 19000g centrifuges 30min, sucks supernatant, leaves and takes precipitation, and it is micro- that 2mL mark fluorescents are added
Ball dilution, 90W ultrasounds, work 2s, and interval 5s is repeated 3 times.
8. a kind of immunofluorescence chromatography detection card for detecting canine coronavirus antigen according to claim 6 or 7
Preparation method, which is characterized in that the microballoon be the fluorescence tramp containing rare-earth europium element polystyrene microsphere；It is close
Degree：1.05g/cm3；Refractive index：1.59@589nm, 25 DEG C；Diameter 300nm；Functional group：Carboxyl；Appearance：White.
9. a kind of immunofluorescence chromatography detection card for detecting canine coronavirus antigen according to claim 6 or 7
Preparation method, which is characterized in that the label activator A is the MES of the 0.05M of the HOSu NHS containing 50mg/mLN-
Buffer solution；The label activator B is containing 50mg/mL1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides
0.05M MES buffer solutions.
10. a kind of immunofluorescence chromatography detection card for detecting canine coronavirus antigen according to claim 6 or 7
Preparation method, which is characterized in that it is described activation quencher be the 0.1M containing 0.25% methanol MES buffer solutions；The label
Confining liquid is 8.0 ethanolamine solutions of 0.04M pH containing 3% bovine serum albumin(BSA)；The mark fluorescent microballoon dilution be containing
The 0.05M pH 7.8 of the 0.03%Proclin300 of 1% bovine serum albumin(BSA), 1% glucan T10 and 0.05% casein-sodium
Tris buffer solutions.
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