CN108398565A - Immunofluorescence for detecting dog C reactive protein chromatographs detection card and preparation method - Google Patents
Immunofluorescence for detecting dog C reactive protein chromatographs detection card and preparation method Download PDFInfo
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- CN108398565A CN108398565A CN201810417752.6A CN201810417752A CN108398565A CN 108398565 A CN108398565 A CN 108398565A CN 201810417752 A CN201810417752 A CN 201810417752A CN 108398565 A CN108398565 A CN 108398565A
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4737—C-reactive protein
Abstract
The immunofluorescence chromatography detection card and preparation method that the invention discloses a kind of for detecting dog C reactive proteins；It is intended to provide a kind of high sensitivity, testing result reliably is used to detect the immunofluorescence chromatography detection card of dog C reactive proteins；Technical points include 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 CRP monoclonal antibody 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 CRP monoclonal antibody 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 that one kind being used to detect dog C- reaction eggs
White immunofluorescence chromatography detection card, the invention also discloses the preparation methods that the immunofluorescence chromatographs detection card.
Background technology
C reactive protein (CRP) is the constituent of body non-immunological systems, is had in the acute stage of a variety of mammalian diseases
Important diagnostic alerts effect.CRP is mainly adjusted by inflammatory factors such as IL-1, IL-6 and TNF-α in the synthetic reaction of liver.
CRP concentration is influenced little by age, gender and weight.In human clinical medicine diagnoses, CRP is as inflammatory or non-inflammatory disease
The diagnosis marker of disease was detected more than 30 years.On veterinary clinic, the detection of dog CRP is also relatively wider in clinical application
It is general, including the monitoring of diagnosis and differential diagnosis, post-operative infection of acute infectious diseases, the observation of antibiotic curative effect, the course of disease
Monitoring and Index for diagnosis etc..Dog CRP is high to disease susceptibility, can quickly be increased in disease early stage, reflect the state of an illness in time；And
The concentration of CRP is not influenced by antibiotic and immunosuppressor, can accurately be supervised in the treatment to the development of disease
It surveys, there is very high value in clinic diagnosis.Therefore it is used as Inflammation Marker, effects of the CRP on veterinary clinic increasingly aobvious
It writes, detection dog CRP has important value to clinical diagnosis, curative effect and Observation On The Prognosis etc..
CRP is to form the acute-phase response of compound by a kind of can be reacted with streptococcus pneumonia C polysaccharide that liver synthesizes
Albumen.It is coupled by 5 identical subunit units with non-covalent fashion, different from Human C-reactiveprotein, in 5 subunits of dog CRP there are two
It is glycosylated.Studies have shown that the various antiserums for resisting other species CRP albumen, it all cannot be with acute stage dog serum or separation
Dog CRP cross reaction occurs, illustrate that the uncommon antigenicities of other species CRP and dog CRP, this phenomenon also illustrate dog
The rapid detection method of CRP cannot rely upon the commercialization CRP Rapid detection test strips of people.Currently, quickly detecting dog in clinic
The method of CRP is to rely on clinical detection instrument more, this does not have in average pet hospital.Moreover, being examined for animal epidemic
Line-break industry needs to carry out in the wild, this is not have for existing dog CRP detection methods for the diagnosis of disease in many cases
What method was completed.Time-resolved fluoroimmunoassay chromatographic technique uses rare earth element (such as Eu3+Deng) overcome fluorescent microsphere sensitivity
Poor disadvantage.Compared with radioimmunoassay, time-resolved fluoroimmunoassay chromatographic technique "dead" pollution has sensitive
The advantages that degree is high, anti-matrix interference is good, and stability is strong.
Chinese patent ZL201710468614.6 discloses a kind of dog c reactive protein colloidal gold immuno-chromatography test paper strip, by
Sample absorption pad, conjugate release pad, reaction film, water absorption pad, bottom plate form successively, and mouse is coated in the conjugate release pad
Anti- dog c reactive protein monoclonal antibody-colloid gold label object includes detection zone and quality control region, the detection on the reaction film
Area is coated with the anti-dog c reactive protein polyclonal antibody of chicken, and the quality control region is coated with sheep anti mouse antiantibody.This method has selected glue
For body gold as tracer, immune marker relies on Electrostatic Absorption, and that there are sensitivity is relatively low, is difficult to quantitative, repeatability and stability
The defects of poor, can not fully meet clinical detection demand.
Chinese patent ZL201710468621.6 discloses a kind of dog c reactive protein fluorescence detection test strip, is inhaled by sample
Receipts pad, conjugate release pad, reaction film, water absorption pad, bottom plate form successively, and the anti-dog C of mouse is coated in the conjugate release pad
Reactive protein monoclonal antibody-Fluorescent microsphere marker includes detection zone and quality control region, the detection zone packet on the reaction film
There are the anti-dog c reactive protein polyclonal antibody of chicken, the quality control region to be coated with sheep anti mouse antiantibody；But there are following for this method
Disadvantage:Detection line coated antibody is polyclonal antibody, may influence whether the specificity of detection；In addition reaction film detection zone and matter
When controlling area's generation immune response, a kind of marker is shared：The anti-dog c reactive protein monoclonal antibody-Fluorescent microsphere marker of mouse, when
C reactive protein variation is that C lines fluorescent value is by respective change in sample, so as to influence the stability of C values and T/C values, to
Seriously affect quantitative accuracy.
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 reproducibility is used to detect
The immunofluorescence chromatography detection card of dog C reactive protein.
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 dog C reactive protein, including bottom plate, hold in the mouth successively on bottom plate
It is connected to sample pad, bonding pad, nitrocellulose membrane and water absorption pad, the bonding pad is equipped with the CRP monoclonals of fluorescent microsphere label
The goat-anti chicken IgY of antibody and fluorescent microsphere label, the nitrocellulose membrane are equipped with the Quality Control C lines of a coating chicken IgY, and
The detection T lines of one coating CRP monoclonal antibody.
Further, the above-mentioned immunofluorescence chromatography detection card for detecting dog C reactive protein, further includes that sample is dilute
Release liquid bottle.
Further, the above-mentioned immunofluorescence chromatography detection card for detecting dog C reactive protein, the sample are dilute
The pH7.8 Tris- hydrochloric acid for releasing the 0.1M that the sample diluting liquid contained in liquid bottle is the X-100 containing 1%S9 and 0.1%Triton is slow
Fliud flushing.
Second technical solution of the present invention is to provide the preparation method of above-mentioned detection reagent card.
A kind of preparation method of immunofluorescence chromatography detection card for detecting dog C reactive protein, holds in the mouth successively on bottom plate
It is connected to sample 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：Containing 1% bovine serum albumin(BSA), 2% Macrogol 4000 and 0.5%Triton X-
The glycine buffer of 100 pH9.0 0.1M；
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) CRP monoclonal antibody and the goat-anti chicken IgY marked with fluorescent microsphere will be marked according to volume ratio with fluorescent microsphere
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 matter with the phosphate buffer of the 0.01M of the pH 7.4 containing 3% sucrose
Control line working solution；
2) CRP monoclonal antibody is diluted to 1mg/ with the phosphate buffer of the 0.01M of the pH 7.4 containing 3% sucrose
ML, as T detection lines working solution；
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, the preparation side of above-mentioned a kind of immunofluorescence chromatography detection card for detecting dog C reactive protein
Method, fluorescent microsphere label CRP monoclonal antibody preparation method be：
1) fluorescent microsphere is diluted：A centrifuge tube is taken, 1mL is added and marks buffer solution 0.1M MES buffer solutions, it is micro- that fluorescence is added
Ball 1mg, vortex 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 CRP 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 dog C reactive protein
The preparation method of goat-anti chicken IgY of method, fluorescent microsphere label is：
1) microballoon is diluted：A centrifuge tube is taken, the label buffer solution label buffer solution 0.1M 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 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：1000 μ g 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：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 dog C reactive protein
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 dog C reactive protein
Method, the label confining liquid are 8.0 ethanolamine solutions of 0.04M pH containing 3% bovine serum albumin(BSA)；The mark fluorescent
Microballoon dilution be containing 1% bovine serum albumin(BSA), 0.5% bovine gamma globulin(BGG), 3% sucrose pH9.0 0.1M glycine it is slow
Fliud flushing；The quencher is the MES buffer solutions of the 0.1M containing 0.25% methanol.
Further, the preparation side of above-mentioned a kind of immunofluorescence chromatography detection card for detecting dog C reactive protein
Method, the label activator A are the 0.05MES buffer solutions containing 50mg/mLNHS；The label activator B is containing 50mg/mL
The 0.05M MES buffer solutions of EDC.
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, is optimized to sample pretreating method, it is only necessary to adopt
Collect dog serum, be added to after dilution detection card well in i.e. can detect, detection speed is fast, go out within 10 minutes as a result, result i.e.
Can qualitative observation can quantitative determine again, substantially reduce testing cost, reduce workload.
2, technical solution provided by the invention, 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 0.86mg/L (former technique 1.56mg/L), linearly
More preferably, 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 present invention is more abundant using treated the fluorescent microsphere release of sample pad treatment fluid, reproducible, low middle high by three
A horizontal Quality Control coefficient of variation is below 10% (7.21%, 6.24% and 4.26%)；In addition biological antiseptic is added in buffer solution
Agent Proclin 300 can effectively keep the stability of detection reagent, and prevent from reducing conventional catalyst to testing result
Influence, it is ensured that the accuracy of testing result, 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 dog C reactive protein provided by the invention, extremely schemes refering to fig. 1
2, including shell 7, the interior immunofluorescence being equipped with for detecting dog C reactive protein of the shell 7, which chromatographs, detects card, the use
In detection dog C reactive protein immunofluorescence chromatography detection card include card bottom plate 1, on bottom plate 1 successively be connected have sample pad 2,
Bonding pad 3, nitrocellulose membrane 4 and water absorption pad 5, the bonding pad 3 are equipped with the CRP monoclonal antibody and glimmering of fluorescent microsphere label
The goat-anti chicken IgY of light microballoon label；The nitrocellulose membrane 4 is equipped with the Quality Control C lines and a packet of a coating chicken IgY
By the detection T lines of CRP monoclonal antibody.The shell 7 is equipped with well 72 compatible with sample pad 2, with bonding pad 3
Compatible watch window 71,7 upper surface of shell are additionally provided with tread plate, facilitate the taking-up of reagent card and are put into.
Embodiment 2
Another immunofluorescence chromatography detection card for detecting dog C reactive protein provided by the present application, refering to fig. 1 extremely
Fig. 3, including box body 9, the body 9 is interior to be equipped with sample diluting liquid bottle 6, and suction pipe 8 and detection card, the detection are positioned in shell
In 7, the immunofluorescence chromatography detection card for detecting dog C reactive protein includes bottom plate 1, and linking has successively on bottom plate 1
Sample pad 2, bonding pad 3, nitrocellulose membrane 4 and water absorption pad 5, the bonding pad 3 are equipped with the CRP monoclonals of fluorescent microsphere label
The goat-anti chicken IgY of antibody and fluorescent microsphere label.The nitrocellulose membrane 4 is equipped with the Quality Control C lines of a coating chicken IgY, with
And the detection T lines of a coating CRP 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, facilitate the taking-up of reagent card with
It is put into.
More specifically, sample diluting liquid is containing 1%S9,1%SDS, 0.1% in the sample diluting liquid bottle
The Tris- hydrochloride buffers of the 0.1M pH7.8 of Triton X-100.
Embodiment 3
A kind of system of the immunofluorescence chromatography detection card for detecting dog C reactive protein provided in embodiment 1 or 2
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. CDV 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
1.1.1 the label of CRP 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, the MES that 1mL 0.1M are added marks buffer solution (2- (N- morpholines) second
Fluorescent microsphere 1mg, vortex mixing is added in sulfonic acid.
3) activation of microballoon：(20-50) μ l (preferably 30 μ l) 10mg/mL label activators A is added in above-mentioned centrifuge tube
[the MES buffer solutions (2- (N- morpholines) ethanesulfonic acid) of the 0.05M of 10mg/mLN- HOSu NHSs (NHS)], vortex mixing
(20-50) μ l (preferably 30 μ l) 50mg/mL is added afterwards and marks activator B [10mg/mL1- (3- dimethylamino-propyls) -3- ethyls
The 0.05M MES buffer solutions (2- (N- morpholines) ethanesulfonic acid) of carbodiimide hydrochloride (EDC)], it reacts on rotary incubator
30min；
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 g CRP 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 and [contain 8.0 ethyl alcohol of 0.04M pH of 3% bovine serum albumin(BSA) (BSA)
Amine aqueous solution], ultrasonic 2s, interval 5s are repeated 3 times, and ultrasound is completed to be placed on rotary incubator to react 30min；
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 [the pH9.0 0.1M containing 1% bovine serum albumin(BSA) (BSA), 0.5% bovine gamma globulin(BGG), 3% sucrose
Glycine buffer], ultrasound, work 2S, and interval 5S is repeated 3 times.
1.1.2 the label of Quality Control C lines antibody
1) microballoon is diluted：A centrifuge tube is taken, the label buffer solution of 1mL is added, takes fluorescent microsphere 0.5mg, vortex mixing；
2) activation of microballoon：20 μ l-50 μ l (preferably 30 μ l) label activators A is added in above-mentioned centrifuge tube and [contains 10mg/
The MES buffer solutions (2- (N- morpholines) ethanesulfonic acid) of the 0.05M of mLN- HOSu NHSs (NHS)] and 20 μ l-50 μ l it is (excellent
Select 30 μ l) label activator B [(3- the dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides Han 10mg/mL1- (EDC)
0.05M MES buffer solutions (2- (N- morpholines) ethanesulfonic acid)], 30min is reacted on rotary incubator；
3) activation is quenched：Fluorescent microsphere 19000g after activation is centrifuged into 30min, supernatant is abandoned, leaves and takes precipitation, 1mL is added
Quencher (the MES buffer solutions of the 0.1M containing 0.25% methanol), 90W ultrasounds, work 2s, and interval 5s is repeated 1 times；
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, and [8.0 ethanol amines of 0.04M pH containing 3% bovine serum albumin(BSA) (BSA) are molten
Liquid], close 30min；
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 [containing 1% bovine serum albumin(BSA) (BSA), 0.5% bovine gamma globulin(BGG), 3% sucrose pH9.0 0.1M it is sweet
Propylhomoserin buffer solution], 90W ultrasounds, work 2s, and interval 5s is repeated 3 times.
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 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 pH 7.4 containing 3% sucrose, i.e.,
For C line working solutions.
2) with the phosphate buffer (PBS) of the 0.01M of the pH 7.4 containing 3% sucrose by another plant of CRP monoclonal antibody
It is diluted to 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：Containing 1% bovine serum albumin(BSA) (BSA), 2% Macrogol 4000 and 0.5%
The glycine buffer of the pH9.0 0.1M of Triton X-100；
2) buffer solution is sprayed on glass fibre with the concentration of 5.0 μ l/cm.
Contrast test
The detection reagent card structure that the detection reagent card structure embodiment 1 that this comparative example provides provides is almost the same, no
It is with place, fluorescent microsphere dilution and sample pad treatment fluid will be marked to be changed in embodiment 3 in preparation process conventional slow
Fliud flushing, such as sample pad treatment fluid are containing 0.5%Tween-20,0.1% bovine serum albumin(BSA), 0.5%Trtion-100
8.0 10mmol/L Tris-HCl buffer solutions of 50mM pH；For example mark fluorescent microballoon dilution is containing 2%BSA, 0.1%
The 8.0 10mmol/L Tris-HCl buffer solutions of pH of PEG4000,5% trehalose, 5% sucrose, 0.05% Sodium azide, Qi Tacan
Number and condition are consistent with embodiment 3.
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 CRP antigens are the positive in sample；When only nature controlling line has fluorescence
And detection line unstressed configuration illustrates that CRP 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) dog serum is collected；
2) take 10 μ l be added containing 1.0mL sample diluting liquids (0.1M's of the X-100 containing 1%S9 and 0.1%Triton
In the sample cell of pH7.8 Tris- hydrochloride buffers, 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 CRP concentration in sample, is carried out curve fitting by built-in standard curve and concentration
Calculating, dose-effect curve Log (Y)=0.59476Log (X) -0.5267, R=0.9994 (R2=0.9989), refering to Fig. 3.
Immunofluorescence test instrument testing result：
1, the range of linearity
1.1.1 CRP calibration objects are diluted to respectively with negative serum 0mg/L, 2.5mg/L, 7.5mg/L, 20mg/L,
50mg/L, 100mg/L, 200mg/L, 300mg/L, 400mg/L, 500mg/L, 600mg/L, 700mg/L and 800mg/L；
1.1.2 above each concentration samples are taken into 75 μ L respectively, be added into the dog CRP detection reagents prepared, per concentration
Retest 2 times；
1.1.3 after reacting 10min, test paper is put into dry type immunofluorescence analysis instrument, T/C values is read, passes through built-in mark
The calculating that directrix curve carries out curve fitting with concentration.
1.1.4 test result is shown：Reagent is preferable in the CRP linear fit relationships for detecting 2.5~300mg/L, r ＞
0.9900, when CRP concentration increases to 400mg/L, linear fit r values are less than 0.9900, T/C growths and slow down, CRP 400~
T/C values are gentle when 500mg/L, and when CRP reaches 600mg/L, T/C values reduce instead.Therefore the maximum detection range of this detection can
To reach 300mg/L.
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 0.86mg/L.Specifically it is shown in Table 1 and Fig. 8；
Table 1
Detection card prepared by comparison contrast test is detected with same method, detection is specifically shown in Table 2 and Fig. 9
Table 2
3, precision
CRP calibration objects are diluted to 10mg/L, 30mg/L, 100mg/L with negative serum, with this method detection card per concentration
Retest 10 times, calculates the coefficient of variation of test concentrations.As a result shown in table 3, it can thus be seen that the change of this kit detection
Different coefficient is respectively less than 10% (three concentration are respectively 7.21%, 6.24% and 4.26%), therefore this detection method is with higher
Precision.
Table 3
| Concentration | CV | |
| Quality Control I | 10mg/L | 7.21% |
| Quality Control II | 30mg/L | 6.24% |
| Quality Control III | 100mg/L | 4.26% |
In order to better illustrate beneficial effects of the present invention, it is given below using detection reagent provided by the invention and other
The detection performance when detecting dog C reactive protein such as method (colloidal gold method, fluorescent chromatographic method) compares, and 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, 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 (9)
1. a kind of immunofluorescence chromatography detection card for detecting dog C reactive protein, including bottom plate (1), on bottom plate (1) according to
Secondary 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 goat-anti chicken IgY (32) of the CRP monoclonal antibody (31) and fluorescent microsphere label of fluorescent microsphere label, the nitric acid
Tunica fibrosa (4) is equipped with the Quality Control C lines and a detection T line for being coated with CRP monoclonal antibody of a coating chicken IgY.
2. the immunofluorescence chromatography detection card according to claim 1 for detecting dog C reactive protein, which is characterized in that
Further include sample diluting liquid bottle (6).
3. the immunofluorescence chromatography detection card according to claim 2 for detecting dog C reactive protein, which is characterized in that
0.1M of the sample diluting liquid containing 1%S9 and 0.1% Triton X-100 contained in the sample diluting liquid bottle (6)
PH7.8 Tris- hydrochloride buffers.
4. the method for preparing a kind of immunofluorescence chromatography detection card for detecting dog C reactive protein described in claim 1,
Linking has sample pad (2), bonding pad (3), nitrocellulose membrane (4) and water absorption pad (5) successively on bottom plate (1), which is characterized in that
A the preparation method of the sample pad described in) is：
1) sample pad treatment fluid is prepared：Containing 1% bovine serum albumin(BSA), 2% Macrogol 4000 and 0.5%Triton X-100
The glycine buffer of pH9.00.1M；
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) CRP monoclonal antibody and the goat-anti chicken IgY marked with fluorescent microsphere will be marked according to volume ratio 20 with fluorescent microsphere:1
It is uniformly mixed, 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 with the phosphate buffer of the 0.01M of the pH 7.4 containing 3% sucrose
Working solution；
2) CRP monoclonal antibody is diluted to 1mg/mL with the phosphate buffer of the 0.01M of the pH 7.4 containing 3% sucrose, i.e.,
For T detection line working solutions；
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 preparation side of immunofluorescence chromatography detection card for detecting dog C reactive protein according to claim 4
Method, which is characterized in that fluorescent microsphere label CRP monoclonal antibody preparation method be：
1) fluorescent microsphere is diluted：A centrifuge tube is taken, 1mL is added and marks buffer solution 0.1M MES buffer solutions, fluorescent microsphere is added
1mg, 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 20000g after activation is centrifuged into 30min, supernatant is abandoned, leaves and takes precipitation, 500 μ l are added and quench
It goes out agent, ultrasonic 2s；
4) label of antibody：The addition 20-200 μ g CRP 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.
6. a kind of preparation side of immunofluorescence chromatography detection card for detecting dog C reactive protein according to claim 4
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 solution label buffer solution 0.1M MES buffer solutions of 1mL are added, take fluorescence
Microballoon 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.
What 7. a kind of immunofluorescence chromatography detection for detecting dog C reactive protein according to claim 5 or 6 blocked
Preparation method, which is characterized in that the microballoon is the polystyrene microsphere of the fluorescence tramp containing rare-earth europium element；It is close
Degree：1.05g/cm3；Refractive index：1.59@589nm, 25 DEG C；Diameter 300nm；Functional group：Carboxyl；Appearance：White.
What 8. a kind of immunofluorescence chromatography detection for detecting dog C reactive protein according to claim 5 or 6 blocked
Preparation method, which is characterized in that the label confining liquid is that 8.0 ethanol amines of 0.04M pH containing 3% bovine serum albumin(BSA) are molten
Liquid；The mark fluorescent microballoon dilution is containing 1% bovine serum albumin(BSA), 0.5% bovine gamma globulin(BGG), 3% sucrose
The glycine buffer of pH9.00.1M；The quencher is the MES buffer solutions of the 0.1M containing 0.25% methanol.
9. a kind of system of immunofluorescence chromatography detection card for detecting dog C reactive protein according to claim 5 or 6
Preparation Method, which is characterized in that the label activator A is the 0.05MES buffer solutions containing 50mg/mLNHS；The label activation
Agent B is the 0.05M MES buffer solutions containing 50mg/mLEDC.
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