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CN108845128A - Immunofluorescence for detecting canine distemper virus antigen chromatographs detection card and preparation method - Google Patents

Immunofluorescence for detecting canine distemper virus antigen chromatographs detection card and preparation method Download PDF

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CN108845128A
CN108845128A CN201810417584.0A CN201810417584A CN108845128A CN 108845128 A CN108845128 A CN 108845128A CN 201810417584 A CN201810417584 A CN 201810417584A CN 108845128 A CN108845128 A CN 108845128A
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label
added
preparation
fluorescent microsphere
canine distemper
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汤永平
梁展鹏
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Guangzhou Agile Biotechnology Co Ltd
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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
    • 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
    • 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

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Abstract

The immunofluorescence that the invention discloses a kind of for detecting canine distemper virus antigen chromatographs detection card and preparation method thereof；It is intended to provide a kind of high sensitivity, testing result is reliably used to detect the immunofluorescence chromatography detection card of canine distemper virus antigen；Technical points include 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 CDV 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 CDV monoclonal antibody；Belong to animal epidemic detection field.

Description

Immunofluorescence for detecting canine distemper virus antigen chromatographs detection card and preparation method
Technical field
The invention discloses a kind of immunofluorescences to chromatograph detection card, specifically, being a kind of for detecting canine distemper virus The immunofluorescence chromatography detection card of antigen, the invention also discloses the preparation methods that the immunofluorescence chromatographs detection card.
Background technique
Canine distemper (CD) is a kind of viral disease of acute, the high contact of the dog occurred extensively in world wide, Cause of disease is canine distemper virus (CDV), is under the jurisdiction of paramyxovirus section Morbillivirus, natural reservoir (of bird flu viruses) includes most Carnivora Animal.
Early stage two-phase heat type, acute coryza and subsequent bronchitis, catarrhal pneumonia, serious gastroenteritis and nerve Symptom is characterized, and the nose and foot pad of some infected animals also show hyperkeratosis.It is also that a kind of infectiousness is strong, and clinical symptoms are more Sample is easy the mixed infection or secondary infection of secondary bacterial and other viruses, and disease incidence is up to 80%, and infected animal is after rehabilitation Also easily there are the sequelae such as paralysis, twitch, epilepsy seizure.Routine immunization is a kind of highly effective under normal circumstances Prevention & protection measure, but it is reported that canine distemper is fine in the national routine immunization execution such as Germany, France, Japan, the U.S. and Finland Area also have different degrees of prevalence, this shows that the dog of immunization campaign may also fall ill, and pathogenesis there may be New variation.
In recent years, with China army dog, police dog, experimental dog and pet dog breeding amount be significantly increased and strange land hand over Stream increases, and disease incidence and lethality of the canine distemper in China dog have raised trend, and clinical manifestation was also and in the past It is different.Sick dog is characterized by being presented two-phase heat type, rhinitis, serious digestive disorder and respiratory inflammation etc..Puppy The death rate is 90% or more, at the death rate of dog 50% or more.Encephalitis can occur for a small number of cases.In addition to canid, Mustelidae, The many animals such as Procyonidae and giant panda section can also infection morbidity.Therefore, CDV is currently to support to raising pets industry, economic animal It grows industry and zoo watches industry and endangers maximum epidemic disease.
Chinese patent 201711054386.4 discloses a kind of canine distemper virus antibody rapid quantitative detection card and user Method, including detection card shell and the test strips being assemblied in detection card shell, the test strips include the plastic bottom with pressure sensitive adhesive Plate successively pastes sample pad, labeling pad, nitrocellulose membrane and blotting paper on bottom plate, and the labeling pad is by carrier substrate It is formed with marker, marker is that spraying lanthanide fluoro detects microballoon and lanthanide fluoro Quality Control microballoon are formed one in carrier substrate Tunic, coating canine distemper virus recombinant antigen is detection line on the nitrocellulose membrane, and coating rabbit-anti chicken lgY antibody is Quality Control Line, marker are that the fluorescence detection microballoon for being marked with the recombinant antigen of canine distemper virus structural proteins H protein and label chicken lgY resist The fluorescence Quality Control microballoon of body.But the detection reagent opens needs preparation H protein in advance, complex steps, required reagent original Expect higher cost, and needs the synthesis of professional's progress albumen.In addition detection reagent detection is the hundstaupe pyreticosis of sample Malicious antibody cannot directly detect the viral antigen in dog excrement, be unfavorable for disease Rapid&Early diagnosis and treatment.
Chinese patent 201310562376.7 discloses a kind of canine distemper virus antigen chemiluminescence detection kit, side Case includes being arranged in the intracorporal coating plate of box and reagent, and the reagent includes the antibody of horseradish peroxidase label sheep anti mouse, packet By solution, lock solution, cleaning solution and chemical luminescence for liquid, the coating plate is the luminescent screen for being coated with canine distemper virus antibody, The dilution of the canine distemper virus antibody is 1：700-1000, it is 1 that horseradish peroxidase, which marks the antibody dilution of sheep anti mouse,： 2500；The luminescent screen is the opaque 96 hole Chemiluminescent plate of polystyrene of milky；The coating of the canine distemper virus antibody It is that canine distemper virus antibody is placed in the coating solution of 0.1mol/L, in 4 DEG C of at a temperature of reaction coating 12h, then uses Lock solution is sealed in the hole on luminescent screen；The coating solution is sodium carbonate and sodium bicarbonate manufactured carbonic acid soluble in water Salt buffer, and the concentration of carbonate is 0.1mol/L；The lock solution is to be added made of the BSA of 10g in every 100mL water BSA solution, then sequentially add thereto the skimmed milk power of BSA solution weight 5%, 1% gelatin and 0.05% NaN3.But Be the detection kit the range of linearity it is not high, patent fails the dose-effect curve of open reagent；Using horseradish peroxidating Enzyme marks the antibody of sheep anti mouse, and specificity may be affected.In addition whole operation is very cumbersome time-consuming, sample is needed to dilute, Sample-adding, the enzyme antibody for marking sheep anti mouse, washing, adds the series of steps such as luminescent solution, detection at washing, and it is fast to be unfavorable for disease early stage Fast Clinics and Practices.
Summary of the invention
In view of the above-mentioned problems, testing result is reliably used to detect dog the object of the present invention is to provide a kind of high sensitivity The immunofluorescence chromatography detection card of distemper virus antigen.
A second object of the present invention is to provide the preparation methods 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 distemper virus antigen, including bottom plate, on bottom plate successively Linking has sample pad, bonding pad, nitrocellulose membrane and water absorption pad, and the bonding pad is equipped with the CDV Dan Ke of fluorescent microsphere label The goat-anti chicken IgY of grand antibody and fluorescent microsphere label, the nitrocellulose membrane are equipped with the Quality Control C line of a coating chicken IgY, with And the detection T line of a coating CDV monoclonal antibody.
Further, the immunofluorescence chromatography detection card of above-mentioned a kind of detection canine distemper virus antigen, further includes sample Dilution bottle.
Further, a kind of immunofluorescence chromatography detection card of detection canine distemper virus antigen on, which is characterized in that institute It states sample dilution bottle and is provided with sample diluting liquid, the sample diluting liquid is containing 0.8%S21,0.03%Proclin300 The pH7.4 phosphate buffer of 0.01M.
Second technical solution provided by the present application is such：
A kind of immunofluorescence for detecting canine distemper virus antigen chromatographs the preparation method of detection card, on bottom plate successively Linking has sample pad, bonding pad, nitrocellulose membrane and water absorption pad, wherein：
A the preparation method of the sample pad described in) is：
3) sample pad treatment fluid is prepared：Containing 0.5%S9,0.05% casein-sodium, 0.3%PVP and 0.5%TritonX- 305 0.01MpH8.0 borate buffer solution；
4) concentration of the 5 μ l/cm of buffer prepared in step 1) is sprayed on glass fibre；
B the preparation method of the bonding pad described in) is：
1) by the CDV monoclonal antibody 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 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 matter with the phosphate buffer of the 0.01M of the pH7.4 containing 3% sucrose Control line working solution；
2) CDV monoclonal antibody is diluted to 1mg/mL with the phosphate buffer of the 0.01M of the pH7.4 containing 3% sucrose, As T detection line working solution；
3) base plate sticking nitrocellulose membrane is taken；
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.
Further, a kind of above-mentioned immunofluorescence for detecting canine distemper virus antigen chromatographs the preparation side of detection card The preparation method of method, the CDV monoclonal antibody of the described fluorescent microsphere label is：
1) fluorescent microsphere is diluted：A centrifuge tube is taken, 1mL is added and marks buffer, fluorescent microsphere 1mg is added, is vortexed and mixes；
2) activation of microballoon：20 μ l-50 μ l label activator A and 20 μ l-50 μ l label is added in above-mentioned centrifuge tube to live Agent B reacts 30min on rotary incubator；
3) activation is quenched：Fluorescent microsphere 20000g after activation is centrifuged 30min, abandons supernatant, leaves and takes precipitating, is added 5000 μ l quencher, ultrasonic 2s；
4) label of antibody：20-200 μ gCDV monoclonal antibody is added in the fluorescent microsphere after ultrasound is resuspended, is vortexed mixed It is even, it is placed on rotary incubator and reacts 30min；
5) it closes：2mL is added and marks confining liquid, ultrasonic 2s, interval 5s are 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 is centrifuged 30min, sucks supernatant, leaves and takes precipitating, and it is glimmering that 2mL label is added Light microballoon dilution, ultrasound, work 2S, and interval 5S is repeated 3 times.
Further, a kind of above-mentioned immunofluorescence for detecting canine distemper virus antigen chromatographs the preparation side of detection card The preparation method of method, the goat-anti chicken IgY of the described fluorescent microsphere label is：
1) microballoon is diluted：A centrifuge tube is taken, the label buffer of 1mL is added, takes fluorescent microsphere 1.0mg, is vortexed and mixes；
2) activation of microballoon：20 μ l-50 μ l label activator A and 20 μ l-50 μ l label is added in above-mentioned centrifuge tube to live Agent B reacts 30min on rotary incubator；
3) activation is quenched：Fluorescent microsphere 19000g after activation is centrifuged 30min, abandons supernatant, leaves and takes precipitating, 2mL is added Quencher, 90W ultrasound, 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, is vortexed and mixes, be placed in 30min is reacted on rotary incubator；
5) it closes：500 μ l label confining liquid is added, closes 30min；
6) it purifies：After completing above-mentioned reaction, 19000g is centrifuged 30min, sucks supernatant, leaves and takes precipitating, and it is glimmering that 2mL label is added Light microballoon dilution, 90W ultrasound, work 2s, and interval 5s is repeated 3 times.
Further, a kind of above-mentioned immunofluorescence for detecting canine distemper virus antigen chromatographs the preparation side of detection card 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, a kind of above-mentioned immunofluorescence for detecting canine distemper virus antigen chromatographs the preparation side of detection card Method, the label buffer are 0.05MMES buffer；The label confining liquid is containing 3% bovine serum albumin(BSA) 0.04MpH8.0 ethanolamine solutions；The mark fluorescent microballoon dilution is containing 0.05% casein-sodium, 0.5%S9,0.3% The pH8.0 borate buffer of PVP, 0.05%Tween-20 and 0.03%Proclin300；The quencher is containing 0.25% The MES buffer of the 0.1M of methanol.
Further, a kind of above-mentioned immunofluorescence for detecting canine distemper virus antigen chromatographs the preparation side of detection card Method, the label activator A are the MES buffer of the 0.05M of 50mg/mLN- HOSu NHS；The label is living Agent B is the 0.05MMES buffer containing 50mg/mL1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride.
Compared with prior art, technical solution provided by the invention has the following advantages that：
1, technical solution operating procedure provided by the invention is simple, optimizes to sample pretreating method, it is only necessary to adopt Collect dog serum, being added in the well of detection card after dilution can be detected, and detection speed is fast, go out as a result, result is within 10 minutes Can qualitative observation can quantitative determine again, 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 0.86ng/mL (original process 1.56ng/mL), line Property it is more preferable, the coefficient of determination of dose-response curve is promoted by 0.9874 to 0.9989.
3, technical solution provided by the invention is strictly investigated to bonding pad, nitrocellulose membrane preparation process, is optimized, further Improve the precision of detection card.
4, the sample pad treatment fluid that technical solution provided by the invention uses, fluorescent microsphere release is more abundant, reproducible, Low middle high three horizontal Quality Control coefficient of variation (CV) are below 10% (8.45%, 5.64% and 5.85%)；In addition in buffer Biological preservative Proclin300 is added, can effectively keep the stability of detection reagent, and tester is protected not prevented routinely The harm of rotten agent such as Sodium azide etc., and the sensitivity for ensuring to check.
Detailed description of the invention
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 is schematic diagram when detection provided by the invention card determines result for the positive；
Fig. 5 is that detection card provided by the invention determines that result is negative schematic diagram；
Fig. 6 is that the detection card that invention provides determines a kind of schematic diagram when result is invalid；
Fig. 7 is that the detection card that invention provides 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.
Fig. 9 is the canonical plotting for the viral antigen Concentration Testing that comparative example provides.
Specific embodiment
With reference to embodiment, claim of the invention is described in further detail.
Embodiment 1
A kind of immunofluorescence for detecting canine distemper virus antigen provided by the invention chromatographs detection card, refering to fig. 1 extremely Fig. 2, including shell 7, the interior immunofluorescence being equipped with for detecting canine distemper virus antigen of the shell 7 chromatograph detection card, institute The immunofluorescence chromatography detection card stated for detecting canine distemper virus antigen includes bottom plate 1, and successively linking has sample on bottom plate 1 Pad 2, bonding pad 3, nitrocellulose membrane 4 and water absorption pad 5, the bonding pad 3 are equipped with the CDV monoclonal antibody of fluorescent microsphere label 31 and fluorescent microsphere label goat-anti chicken IgY32, the nitrocellulose membrane 4 be equipped with one coating chicken IgY Quality Control C line, with And the detection T line of a coating CDV monoclonal antibody.The shell 7 is equipped with well 72 compatible with sample pad 2, Watch window 71 compatible with bonding pad 3,7 upper surface of shell are additionally provided with tread plate 73, facilitate the taking-up of reagent card Be put into.
Embodiment 2
Another immunofluorescence chromatography detection card for detecting canine distemper virus 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 distemper virus in the box body 9 to Fig. 3, including box body 9 Former immunofluorescence chromatography detection card, the immunofluorescence chromatography detection for detecting canine distemper virus antigen are positioned in shell In 7, the immunofluorescence chromatography detection card for detecting canine distemper virus 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 the CDV Dan Ke of fluorescent microsphere label 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 CDV 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 3
The system for the immunofluorescence chromatography detection card for detecting canine distemper virus 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. CDV detection card；Each detection is placed in aluminum foil bag, desiccant 1 is added and wraps, heat sealing is spare.
Specific preparation method is as follows：
The preparation of 1.1 bonding pads
1.1.1 the label of CDV 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, 1mL is added and marks buffer, fluorescent microsphere 1mg is added, is vortexed and mixes； The label buffer is 0.05MMES buffer (2- (N- morpholine) ethanesulfonic acid).
3) activation of microballoon：20-50 μ l (preferably 30 μ l) mg/mL50mg/mL label activation is added in above-mentioned centrifuge tube Agent A is vortexed after mixing and 20-50 μ l (preferably 30 μ l) 50mg/mL label activator B is added, reacts 30min on rotary incubator；
The label activator A is the MES buffer of the 0.05M of 50mg/mLN- HOSu NHS；The mark Note activator B is that the 0.05MMES of 50mg/mL50mg/mL1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride is slow Fliud flushing.
4) activation is quenched：Fluorescent microsphere 20000g after activation is centrifuged 30min, abandons supernatant, leaves and takes precipitating, 2mL is added Quencher, ultrasonic 2s；The quencher is the MES buffer of the 0.1M containing 0.25% methanol.
5) label of antibody：20 μ gCDV monoclonal antibodies are added in the fluorescent microsphere after ultrasound is resuspended, is vortexed and mixes, It is placed on rotary incubator and reacts 30min；
6) it closes：200.0 μ l label confining liquid is 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 contains the 0.04MpH8.0 ethanolamine solutions of 3% bovine serum albumin(BSA) (BSA)；
7) it purifies：After completing above-mentioned reaction, 19000g is centrifuged 30min, sucks supernatant, leaves and takes precipitating, and 600.0 μ l mark is added Remember fluorescent microsphere dilution, ultrasound, work 2S, and interval 5S is repeated 3 times.
The mark fluorescent microballoon dilution is containing 0.05% casein-sodium, 0.5%S9,0.3%PVP, 0.05% The pH8.0 borate buffer of Tween-20 and 0.03%Proclin300.
1.1.2 the label of Quality Control C line 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) microballoon is diluted：Dilute fluorescent microsphere：A centrifuge tube is taken, 1mL is added and marks buffer, fluorescent microsphere 1mg is added, It is vortexed and mixes；The label buffer is 0.05MMES buffer (2- (N- morpholine) ethanesulfonic acid)；
3) activation of microballoon：20 μ l-50 μ l (preferably 30 μ l) label activator A and 20 μ l- is added in above-mentioned centrifuge tube 50 μ l (preferably 30 μ l) mark activator B, react 30min on rotary incubator；The label activator A is 50mg/mLN- The MES buffer of the 0.05M of HOSu NHS；The label activator B is containing 50mg/mL1- (3- dimethylamino third Base) -3- ethyl-carbodiimide hydrochloride 0.05MMES buffer.
4) activation is quenched：Fluorescent microsphere 19000g after activation is centrifuged 30min, abandons supernatant, leaves and takes precipitating, 1mL is added Quencher, 90W ultrasound, work 2s, and interval 5s is repeated 1 times；The MES that the quencher is the 0.1M containing 0.25% methanol is slow Fliud flushing.
5) label of antibody：0.1mg goat-anti chicken IgY is added in the fluorescent microsphere after ultrasound is resuspended, is vortexed and mixes, be placed in 30min is reacted on rotary incubator；
6) it closes：Label confining liquid is added, closes 30min；
The label confining liquid contains the 0.04MpH8.0 ethanolamine solutions of 3% bovine serum albumin(BSA) (BSA)；
7) it purifies：After completing above-mentioned reaction, 19000g is centrifuged 30min, sucks supernatant, leaves and takes precipitating, and it is glimmering that 1mL label is added Light microballoon dilution, 90W ultrasound, work 2s, and interval 5s is repeated 3 times.
The mark fluorescent microballoon dilution is containing 0.05% casein-sodium, 0.5%S9,0.3%PVP, 0.05% The pH8.0 borate buffer of Tween-20 and 0.03%Proclin300.
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 pressed 20:1(V：V it) is uniformly mixed, 90W ultrasound, work 2s, interval 5s, repeats 3 It is secondary；
3) it is sprayed on the bonding pad after cutting out by 5.0 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) chicken IgY is diluted to 1mg/mL with the phosphate buffer (PBS) of the 0.01M of the pH7.4 containing 3% sucrose, i.e., For C line working solution.
2) phosphate buffer (PBS) with the 0.01M of the pH7.4 containing 3% sucrose is dilute by another plant of CDV monoclonal antibody Release 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：Containing 0.5%S9,0.05% casein-sodium, 0.3%PVP and 0.5%TritonX- 305 0.01MpH8.0 borate buffer solution.
2) buffer is sprayed on glass fibre with the concentration of 5 μ l/cm.
Comparative test
The detection reagent card structure that this comparative experiments provides and the detection reagent card structure that embodiment 1 provides are almost the same, The difference is that being during the preparation process containing 0.5%S9,0.05% casein by the sample pad treatment fluid in embodiment 3 The 0.01MpH8.0 borate buffer solution of sodium, 0.3%PVP and 0.5%TritonX-305 replace with containing 0.5%Tween-20, The 50mMpH8.010mmol/LTris-HCl buffer of 0.1% bovine serum albumin(BSA), 0.5%Trtion-100；By mark fluorescent Microballoon dilution contains 0.05% casein-sodium, 0.5%S9,0.3%PVP, 0.05%Tween-20 and 0.03%Proclin300 PH8.0 borate buffer replace with containing 2%BSA, 0.1%PEG4000,5% trehalose, 5% sucrose, 0.05% Sodium azide PH8.010mmol/LTris-HCl buffer, other conditions and technological parameter are constant.
Detection method
Blocked for the ease of those skilled in the art using detection provided by the present application, detection provided by the present application is given below Card provides two kinds of detection methods.
Method one：Using ultraviolet light irradiation, 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 CDV antigen is the positive in sample；When only nature controlling line has fluorescence And detection line unstressed configuration illustrates that CDV 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 saliva, urine, discharge of eye etc. are 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 CDV concentration in sample, is carried out curve fitting by built-in standard curve and concentration Calculating, dose-effect curve Log (Y)=0.6151Log (X) -1.3923, R=0.9971 (R2=0.9942), refering to Fig. 3.
Immunofluorescence test instrument testing result：
1, the range of linearity
1.1 CDV calibration object is diluted to respectively with negative serum 0ng/mL, 10ng/mL, 40ng/mL, 150ng/mL, 600ng/mL, 2000ng/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 dog CDV 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 CDV linear fit relationship for detecting 5~300ng/mL, r > 0.9900, When CDV concentration increases to 400ng/mL, linear fit r value slows down less than 0.9900, T/C growth, and CDV is in 400~500ng/ T/C value is gentle when mL, and when CDV reaches 800ng/mL, T/C value reduces instead.Therefore the maximum detection range of this detection can reach To 400ng/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 4.26ng/mL, is shown in Table 1 and Fig. 8.
Table 1
The detection reagent card provided with same method detection comparative experiments, minimum detectability 7.56ng/mL are shown in Table 2 and Fig. 9.
Table 2
3, precision
CDV calibration object is diluted to 20ng/mL, 200ng/mL, 100ng/mL with negative serum, it is every with this method detection card Concentration retest 10 times, calculate the coefficient of variation of test concentrations.As a result shown in table 3, it can thus be seen that this kit detects The coefficient of variation be respectively less than 10% (three concentration are respectively 8.45%, 5.64% and 5.85%), be shown in Table 3, therefore this detection side Method precision with higher.
Table 3
Quality-control product Concentration The coefficient of variation
Quality Control I 20ng/mL 8.45%
Quality Control II 200ng/mL 5.64%
Quality Control III 1000ng/mL 5.85%
Beneficial effect in order to better illustrate the present invention, be given below using detection reagent provided by the invention with it is enzyme-linked The detection performance of immunization, chemoluminescence method and other fluorescence immune chromatography methods when detecting canine distemper virus original is compared, It is 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, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention, It should be equivalent substitute mode, be included within the scope of the present invention.

Claims (9)

1. a kind of immunofluorescence for detecting canine distemper virus 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 CDV 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 CDV monoclonal antibody.
2. a kind of immunofluorescence chromatography detection card for detecting canine distemper virus antigen according to claim 1, feature exist In further including sample dilution bottle (6).
3. a kind of immunofluorescence chromatography detection card for detecting canine distemper virus antigen according to claim 2, feature exist In the sample dilution bottle (6) is provided with sample diluting liquid, and the sample diluting liquid is containing 0.8%S21,0.03% The pH7.4 phosphate buffer of the 0.01M of Proclin300.
4. preparing the side that a kind of immunofluorescence for detecting canine distemper virus antigen described in claim 1 chromatographs detection card Method, successively linking has sample pad (2), bonding pad (3), nitrocellulose membrane (4) and water absorption pad (5) 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：Containing 0.5%S9,0.05% casein-sodium, 0.3%PVP and 0.5%TritonX-305 0.01MpH8.0 borate buffer solution；
2) concentration of the 10 μ l/cm of buffer prepared in step 1) is sprayed on glass fibre；
B the preparation method of the bonding pad (3) described in) is：
1) by the CDV monoclonal antibody marked with fluorescent microsphere and the goat-anti chicken IgY marked with fluorescent microsphere according to volume ratio 20: 1 is uniformly mixed, and ultrasonic 2s, interval 5s are repeated 3 times；
2) it is sprayed 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 line with the phosphate buffer of the 0.01M of the pH7.4 containing 3% sucrose Working solution；
2) CDV monoclonal antibody is diluted to 1mg/mL with the phosphate buffer of the 0.01M of the pH7.4 containing 3% sucrose, as T detection line working solution；
3) bottom plate (1) is taken to paste nitrocellulose membrane；
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.
5. the preparation that a kind of immunofluorescence for detecting canine distemper virus antigen according to claim 4 chromatographs detection card Method, which is characterized in that the preparation method of CDV monoclonal antibody of fluorescent microsphere label is：
1) fluorescent microsphere is diluted：A centrifuge tube is taken, 1mL is added and marks buffer, fluorescent microsphere 1mg is added, is vortexed and mixes；
2) activation of microballoon：20 μ l-50 μ l label activator A and 20 μ l-50 μ l label activator is added in above-mentioned centrifuge tube B reacts 30min on rotary incubator；
3) activation is quenched：Fluorescent microsphere 20000g after activation is centrifuged 30min, abandons supernatant, leaves and takes precipitating, 500 μ l are added and quench It goes out agent, ultrasonic 2s；
4) label of antibody：20-200 μ g CDV monoclonal antibody is added in the fluorescent microsphere after ultrasound is resuspended, is vortexed and mixes, It is placed on rotary incubator and reacts 30min；
5) it closes：2mL is added and marks confining liquid, ultrasonic 2s, interval 5s are repeated 3 times, and ultrasound is completed to be placed on rotary incubator React 30min；
6) it purifies：After completing above-mentioned reaction, 19000g is centrifuged 30min, sucks supernatant, leaves and takes precipitating, and it is micro- that 2mL mark fluorescent is added Ball dilution, ultrasound, work 2S, and interval 5S is repeated 3 times.
6. the preparation that a kind of immunofluorescence for detecting canine distemper virus antigen according to claim 4 chromatographs detection card 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 label buffer of 1mL is added, takes fluorescent microsphere 1.0mg, is vortexed and mixes；
2) activation of microballoon：20 μ l-50 μ l label activator A and 20 μ l-50 μ l label activator is added in above-mentioned centrifuge tube B reacts 30min on rotary incubator；
3) activation is quenched：Fluorescent microsphere 19000g after activation is centrifuged 30min, abandons supernatant, leaves and takes precipitating, 2mL is added and is quenched Agent, 90W ultrasound, 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, is vortexed and mixes, be placed in rotation 30min is reacted on incubator；
5) it closes：500 μ l label confining liquid is added, closes 30min；
6) it purifies：After completing above-mentioned reaction, 19000g is centrifuged 30min, sucks supernatant, leaves and takes precipitating, and it is micro- that 2mL mark fluorescent is added Ball dilution, 90W ultrasound, work 2s, and interval 5s is repeated 3 times.
7. a kind of immunofluorescence chromatography detection card for detecting canine distemper virus antigen according to claim 5 or 6 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.
8. a kind of immunofluorescence chromatography detection card for detecting canine distemper virus antigen according to claim 5 or 6 Preparation method, which is characterized in that the label buffer be 0.05M MES buffer；The label confining liquid be containing The 0.04MpH8.0 ethanolamine solutions of 3% bovine serum albumin(BSA)；The mark fluorescent microballoon dilution is containing 0.05% junket egg The pH8.0 borate buffer of white sodium, 0.5%S9,0.3%PVP, 0.05%Tween-20 and 0.03%Proclin300；It is described Quencher be the 0.1M containing 0.25% methanol MES buffer.
9. a kind of immunofluorescence chromatography detection card for detecting canine distemper virus antigen according to claim 5 or 6 Preparation method, which is characterized in that the described label activator A is the MES of the 0.05M of 50mg/mL n-hydroxysuccinimide Buffer；The label activator B is 1- containing 50mg/mL (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride 0.05M MES buffer.
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