CN108362877A - Feline calicivirus fluorescence immune chromatography test strip and preparation method thereof - Google Patents
Feline calicivirus fluorescence immune chromatography test strip and preparation method thereof Download PDFInfo
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Abstract
" feline calicivirus fluorescence immune chromatography test strip and preparation method thereof " of the invention, is related to animal virus detection technique, it is characterised in that：Bonding pad, chromatographic film and the water absorption pad set gradually including backing and on backing, composition can make fluid sample from bonding pad chromatography to the chromatography path of water absorption pad；Fluorescent microsphere labelled antibody compound is combined on the bonding pad；The detection line and control line with the chromatography path orthogonal are provided in the chromatographic film.It can be used for field quick detection, step is simple and fast, and sensitivity and specificity are all suitable with PCR method, can be used for the detection and prevention of feline calicivirus.
Description
Technical field
The invention belongs to animal virus detection technique field, more particularly to feline calicivirus (Feline calicivirus,
FCV) fluorescence immune chromatography test strip and preparation method thereof.
Background technology
Feline calicivirus (Feline calicivirus, FCV) is Caliciviridae, and water sore Tobamovirus is a kind of list
Strand rna virus.FCV is that cat disease toxicity respiratory infectious disease is mainly shown as canker sore, eye nasal secretions, salivation, knot
Film inflammation, photophobia, stomatitis, tracheitis, bronchitis, with diphasic fever.It is the frequently-occurring disease of cat, incidence is high, velogen strain infection
Pneumonia, expiratory dyspnea, even death can occur.For major source of infection for sick cat and with malicious cat, sick cat can be with secretion in acute stage
A large amount of viruses, the susceptible cat of direct contagion is discharged with excreta.Through treatment, symptom can disappear the malicious cat of band, but still long after clinical rehabilitation
Phase toxin expelling is the infection sources of danger.
At present, the method for detecting FCV includes：Electronic Speculum observational technique, immunofluorescent test, PCR detections of viral nucleic acid etc.
Experiment, however these methods are time-consuming and laborious, although so be difficult to promote specificity and sensibility is all very high, need special instrument
Device equipment and technical staff, are only used for test in laboratory.These methods are not suitable for Site Detection, quickly detection and base
Veterinary office clinical detection.Existing to be directed to the method that animal virus detection method includes fluorescence immune chromatography detection, fluorescence is exempted from
Epidemic disease chromatographic technique is a kind of quantitative novel detection that immunofluorescence technique work and Traditional immunochromatographic technology are combined development innovation
Technology.For immunochromatography technique to be fixed with the fibre strip chromatographic material of detection line and control line as stationary phase, test fluid is stream
Dynamic phase, makes determinand be moved on chromatography strip through capillary action, and determinand locates that specific immune response occurs online.Educt
Immune response occurs for online place.The technology retain colloidal gold immunochromatographimethod technical operation is easy, detection quickly, portability it is strong
Outside advantage, also enhancing technology by fluorescent tracing realizes the accurate quantification of testing result.
Fluorescence immune chromatography Test paper and detection are established for feline calicivirus (Feline calicivirus, FCV)
Technology is used for field quick detection, is conducive to the investigation to infection epidemiology, to provide science for prevention i.e. and control
Foundation, be conducive to the sound development of animal, become those skilled in the art's letter technical barrier to be solved.It yet there are no
Report.
Invention content
Technical need based on above-mentioned field and blank, the present invention are established for feline calicivirus (Feline
Calicivirus, FCV) fluorescence immune chromatography test strip and preparation method thereof, can be used for FCV quick detection and
Site Detection：
In order to reach above-mentioned technique effect, technical scheme of the present invention includes
A kind of feline calicivirus (Feline calicivirus, FCV) fluorescence immune chromatography test strip, feature exist
In：
Bonding pad, chromatographic film and the water absorption pad set gradually including backing and on backing, composition can make sample from
Bonding pad is chromatographed to the chromatography path of water absorption pad；
Fluorescent microsphere labelled antibody compound is combined on the bonding pad；It is provided in the chromatographic film and the chromatography
The detection line and control line of path orthogonal；
The fluorescent microsphere labelled antibody compound is fluorescent microsphere and rabbit-anti feline calicivirus (Feline
Calicivirus, FCV) F protein polyclonal antibody conjugate；
The detection line is coated with by goat-anti feline calicivirus (Feline calicivirus, FCV) polyclonal antibody,
The control line is coated with by protein staphylococcus (Staphylococal Protein A, SPA)；
The fluorescent microsphere grain size is 180-240nm.
In some embodiments, on the bonding pad, which is characterized in that on the bonding pad, the fluorescent microsphere label
The coating density of antibody complex：With rabbit-anti feline calicivirus (Feline calicivirus, FCV) F protein polyclonal antibody
It is every square centimeter that quality is calculated as 7.5ug/.
In some embodiments, the fluorescent microsphere labelled antibody compound is to be made by the steps and obtain：It will be glimmering
Light microballoon is with rabbit-anti feline calicivirus (Feline calicivirus, FCV) F protein polyclonal antibody with 2~6mg：1~3mg
Ratio, preferably 2~5mg:1mg, 2~4mg:1mg or 3mg:The ratio of 1mg carries out coupling reaction and is made.
Preferably, in some embodiments, the fluorescent microsphere labelled antibody compound be made by the steps and
：To pH5.0, the fluorescent microsphere is sequentially added in the PBS solution of concentration 0.02mol/L, is added a concentration of 10mg/ml's
EDC solution adds rabbit-anti feline calicivirus (Feline calicivirus, FCV) F protein polyclonal antibody, delays at room temperature
Slow stirring 2h, after final concentration of 2% BSA solution closing 30min is added, with 10000r/min, 4 DEG C of centrifugation 10min, in removal
Clear liquid, gained contain the fluorescent microsphere labelled antibody compound in precipitating.
In some embodiments, the grain size 210nm of the preferably described fluorescent microsphere.
In some embodiments, the fluorescent microsphere is rare-earth europium fluorescent microsphere.
Any of the above-described test strips, which is characterized in that the test strips width is 3mm, the detection line and control
The width of line is also 3mm；In the detection line, more grams of the goat-anti feline calicivirus (Feline calicivirus, FCV)
Grand antibody package amount is 0.5ug/ items, and the protein staphylococcus package amount is 0.3ug/ items；Preferably, detection line and control
Line is at a distance of 5mm.
Any of the above-described test strips, the upstream of the bonding pad is additionally provided with sample pad；For receiving sample, and will
Substance in sample is transported on bonding pad；
Preferably, further include shell, there are opening, corresponding detection line and Quality Control in the position that the shell corresponds to the sample pad
The position of line is set as transparent or hatch frame.
Another aspect of the present invention provides above-mentioned feline calicivirus (Feline calicivirus, FCV) fluorescence immunoassay
The preparation method of chromatography detecting test paper strip, which is characterized in that including
Prepare the fluorescent microsphere labelled antibody compound：By fluorescent microsphere and rabbit-anti feline calicivirus (Feline
Calicivirus, FCV) F protein polyclonal antibody is with 2~6mg：The ratio of 1~3mg, preferably 2~5mg:1mg, 2~4mg:
1mg or 3mg:The ratio of 1mg carries out coupling reaction；
Fluorescent microsphere labelled antibody complex solution is sprayed on 5ul/cm on bonding pad, vacuum is drained；
By bonding pad, chromatographic film, water absorption pad is assembled to successively on test strips bottom liner.
Preferably, further include that the goat-anti feline calicivirus (Feline calicivirus, FCV) of a concentration of 2mg/ml is more
The protein staphylococcus of clonal antibody and a concentration of 1mg/ml are coated in chromatographic film respectively, form the detection at a distance of 5mm
Line and the control line；
Preferably, the preparation process of the fluorescent microsphere labelled antibody compound is as follows：To pH5.0, concentration 0.02mol/L
PBS solution in the EDC solution that sequentially adds the fluorescent microsphere, a concentration of 10mg/ml is added, add rabbit-anti cat cup-shaped disease
Malicious (Feline calicivirus, FCV) F protein polyclonal antibody, is slowly stirred 2h at room temperature, is added final concentration of 2%
After BSA solution closes 30min, with 10000r/min, 4 DEG C of centrifugation 10min, supernatant is removed, is containing described in gained precipitation
Fluorescent microsphere labelled antibody compound；
Preferably, before the fluorescent microsphere labelled antibody compound is encountered bonding pad, pH8.2 is first used, contains 2%
The borate buffer solution immersion treatment bonding pad of a concentration of 0.05M of tween；
Preferably, further include preparing rabbit-anti feline calicivirus (Feline calicivirus, FCV) F protein Anti-TNF-α
Body：With feline calicivirus (Feline calicivirus, FCV) F protein immunizing rabbit of purifying, takes a blood sample and detaches serum,
FCV F-ELISA detect antibody titer, if potency reaches 1:5000 or more, by arteria carotis bloodletting, serum is detached, is used after inactivation
Antibody purification column purifies polyclonal antibody, and 1mg/ml is diluted to PBS；
Preferably, further include the preparation of goat-anti feline calicivirus (Feline calicivirus, FCV) polyclonal antibody:
It with feline calicivirus (Feline calicivirus, FCV) immune sheep of purifying, takes a blood sample through ear vein and detaches serum, use
FCV-ELISA detects antibody titer, and potency reaches 1:When 5000 or more, by arteria carotis bloodletting, serum is detached, with anti-after inactivation
Body purification column purifies polyclonal antibody, and 2mg/ml is diluted to PBS.
Optimization and embedding amount of the present invention by the grain size of fluorescent microsphere, type, fluorescent microsphere and antibody coupling condition
Etc. factors optimization and selection, be successfully established can high special, in high sensitivity detect feline calicivirus fluorescence immunoassay
Chromatography detecting test paper strip.Compared with ELISA detection method, two methods testing result coincidence rate is 98.9%.The test strips have
Have detection it is quick, easy to operate, do not need the advantages of special instrument and equipment and professional technician.In addition, the test strips are easy
In storage and transport, small using detection sample size, testing cost is relatively low, and safe operation does not cause environmental pollution.Therefore this method
Especially suitable for quickly detecting, base's Disease Diagnosis of Veterinary department clinical detection and breeding enterprise voluntarily detect.The test strips are ground
System, good tool is provided for the detection, quarantine and epidemiological survey of FCV.
Description of the drawings
Fig. 1 show a kind of feline calicivirus (Feline calicivirus, FCV) fluorescence immune chromatography detection of the present invention
Test strips structure schematic diagram：
1- backings, 2- sample pads, 3- bonding pads, 4- chromatographic films, 41 1 detection lines, 42 1 control lines, 5 one water absorption pads.
Specific implementation mode
The present invention is carefully described below in conjunction with Fig. 1 and specific embodiment, but not as limiting the scope of the invention.
XYZ3050 sample application platforms (BioDot companies of the U.S.)
CM4000 cutting machines (BioDot companies of the U.S.)
Centrifuge (Hunan instrument)
Microplate reader (Thermo Scientific)
3 monthly age health sheep, 20 week old BALB/c mouse inbred lines are purchased from Experimental Animal Center.
EDC:1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides are bought from SIGMA
With feline panleucopenia virus, cat hcrpesviruses, cat infectiousness rhino-bronchitis virus, feline infectious peritonitis virus and
Standard positive sample is bought from GD companies of Holland.
Fluorescent microsphere：The microballoon of time-resolved fluorescence containing europium, grain size 210nm, excitation wavelength 340nm, launch wavelength are
610nm is purchased from Merk companies.
Albumin A/G antibody purification columns：It buys from SIGMA
Protein staphylococcus：It buys from SIGMA
A kind of feline calicivirus (Feline calicivirus, FCV) fluorescence immune chromatography test strip as shown in Figure 1
Structural schematic diagram, including 1. sample pad of backing；2. bonding pad；3. detection line 41 and control line 42；4. water absorption pad；The detection line
41 are coated with goat-anti feline calicivirus (Feline calicivirus, FCV) polyclonal antibody, and the control line 42 is coated with Portugal
Grape pneumococcal proteins, the rabbit-anti feline calicivirus (Feline calicivirus, FCV) F protein polyclonal antibody are a concentration of
1mg/ml。
Embodiment 1. prepares the system of rabbit-anti feline calicivirus (Feline calicivirus, FCV) F protein polyclonal antibody
It is standby
(1) with the feline calicivirus F protein of the gene engineering expression of purifying：
Reference Master's thesis in 2016, Jiang Yanmei's《Feline calicivirus VP1 antigenicity analysis and indirect ELISA diagnosis side
The foundation of method》Described in method.
(2) 3 monthly age healthy rabbits are immunized in purifying feline calicivirus F protein, and immunizing dose is 0.5mg/, and interval is exempted from for 2 weeks
Epidemic disease 1 time is immunized 4 times altogether, and last time was immunized for 15 day after tomorrow, takes a blood sample through ear vein and detaches serum, after detecting antibody titer；.
(3) rabbit-anti feline calicivirus (Feline calicivirus, FCV) F protein polyclonal antibody specificity and
ELISA potency:It is total with feline panleucopenia virus, cat hcrpesviruses, cat infectiousness rhino-bronchitis virus, feline infectious peritonitis virus
4 parts of virus-positive serum establish indirect ELISA method detection respectively.As a result it shows, the ELISA not established with these antigens
There is specific binding, show that the antibody has good specificity, is 1 with FCV detection antibody titers:5000.
(4) by arteria carotis bloodletting, serum is detached, 30min is inactivated in 56 DEG C, with antibody A/G purification columns to Anti-TNF-α
Body is purified, and it is 2mg/ml to measure IgG protein contents with mono- 800 foranalysis of nucleic acids of DU/protein analyzer, with ph7.2's
0.02mol/lPBS is diluted to 1mg/ml.
Prepare goat-anti feline calicivirus (Feline calicivirus, FCV) polyclonal antibody：
3 monthly age health sheep, immunizing dose is immunized with the feline calicivirus (Feline calicivirus, FCV) of purifying
Be 3mg/ only, be spaced 2 weeks immune 1 time, be immunized 4 times altogether, last time immune 15 day after tomorrow take a blood sample through ear vein and detach serum,
After detecting antibody titer, by arteria carotis bloodletting, serum is detached, 30min is inactivated in 56 DEG C, with antibody A/G purification columns to more grams
Grand antibody is purified, and it is 3.1mg/ml to measure IgG protein contents with mono- 800 foranalysis of nucleic acids of DU/protein analyzer, uses ph7.2
0.02mol/lPBS be diluted to 2mg/ml.
Embodiment 3. prepares test strips
(1) fluorescent microsphere marks rabbit-anti feline calicivirus (Feline calicivirus, FCV) F protein polyclonal antibody
Using EDC mediated methods, (grain size 210nm, excitation wavelength are by the monoclonal antibody of purifying and fluorescent microsphere
470nm, launch wavelength 525nm, Merk Products) it is coupled.Into the 0.02mol/lPBS solution of 5mlph5.0 according to
Secondary addition 3mg fluorescent microspheres, the EDC solution for a concentration of 10mg/ml that 15ul now matches add a concentration of 1mg/ml rabbit-antis cat cup
Shape virus F protein polyclonal antibody 1ml, is slowly stirred 2h at room temperature, after the closing 30min of final concentration of 2%BSA,
10000r/min, 4 DEG C of centrifugation 10min, remove supernatant, 500ul are added, sediment is resuspended, mix well, by fluorescent microsphere mark
Note antibody complex saves backup in 4 DEG C
(2) preparation of bonding pad:The polyclonal antibody complex solution for being marked fluorescent microsphere with XYZ3050 sample application platforms
It is sprayed on 300mm*5mm glass fibre cottons, discharge rate 5ul/cm, and is drained in 4 DEG C of vacuum.
(3) preparation of chromatographic film
Nitrocellulose filter (35mm*300mm) is pasted on backing (70mm* by the preparation of detection line and control line first
Surface 300mm) is hit exactly, with the goat-anti polyclonal antibody (2mg/ml) and protein staphylococcus (1mg/ml) of purifying respectively as inspection
Survey line and control line reagent.Two kinds of reagents are put on nitrocellulose membrane respectively, albumen package amount be respectively 0.5ug/ items and
0.3ug/ items.When point sample, detection line 41 is located at nitrocellulose filter center line, and distance is 5mm between control line 42 and detection line 41.
37 DEG C of dry 2h, 4 DEG C be sealed it is standby.
(4) assembling and cutting of paper slip
As shown in Figure 1, by backing 1 and nitrocellulose membrane 4, the fluorescent microsphere of 42 reagent of point sample detection line 41 and control line
It marks rabbit-anti feline calicivirus (Feline calicivirus, FCV) F protein Anti-TNF-α nanocrystal composition pad 3, sample pad 2, inhale
Water cushion 5 sticks together, and after compacting, in CM4000 cutting machines, is cut into the test strips of 3mm wide.
According to the concentration of point sample speed and detection line reagent and control line reagent, detection line and control line reagent coating are obtained
Amount is respectively 0.5ug/ items and 0.3ug/ items.
When testing principle, result judgement standard and the detecting step of test strips prepare test strips, fluorescent microsphere is marked into rabbit
Anti- feline calicivirus (Feline calicivirus, FCV) F protein Anti-TNF-α nanocrystal composition is fixed on compound pad, inspection
Survey line is goat-anti FCV polyclonal antibodies, control line SPA.When detection, the FCV in positive marks rabbit-anti with fluorescent microsphere
Feline calicivirus (Feline calicivirus, FCV) F protein polyclonal antibody specifically combines, when being moved on NC films,
FCV polyclonal antibodies capture on tested survey line, fluorescent microsphere particle aggregation read the excitation of instrument excitation light source in this, in fluorescence
Under, form visible green fluorescence band, i.e. detection line.On the contrary, FCV is free of in negative sample, is not formed at detection line green
Color fluorescent bands.Detection line reagent SPA marks rabbit-anti feline calicivirus (Feline calicivirus, FCV) F with fluorescent microsphere
Protein polyclone antibody Fc segments combine, and capture gold mark compound, in contrast line.No matter whether contain in sample, fluorescence is micro-
Ball marks rabbit-anti feline calicivirus (Feline calicivirus, FCV) F protein polyclonal antibody can be with control line reagent knot
It closes, in contrast line.Then it is the positive if detection line and control line develop the color simultaneously when judging result, if detection line does not develop the color,
Control line develops the color, then is feminine gender.It is nothing if detection line and control line do not develop the color or detection line develops the color and control line does not develop the color
Result is imitated, test strips is needed to change and detects again.When detection, test strips are lain against on experimental bench, take 100ul with etc. samples product add
In in sample pad, being stored at room temperature reaction, instrument observation detection line and control line colour developing situation are read using fluorescence in 15min, and
Carry out result judgement.
1 fluorescence immune chromatography test strip specificity of experimental example, sensibility and coincidence rate experiment
(1) specificity of test strips
Detected respectively with test strips part feline calicivirus (Feline calicivirus, FCV) i.e. standard positive sample, its
Its correlated virus includes feline panleucopenia virus, cat hcrpesviruses, cat infectiousness rhino-bronchitis virus, feline infectious peritonitis virus
Positive and negative control sample.
Table 1 detects approximate virus results
It is positive reaction when as a result showing ELISA test strip FCV positives, with other virus-positives and negative control sample
It is feminine gender when product.Show that test strips specificity is good.
(2) sensibility of test strips
It is serially diluted 10 with ELISA test strip1-107FCV positives, while as control,
The primer upstream：cgGGATCCatgtgctcaacctgcgctaacg
Downstream：cgGAATTCtcataacttagtcatgggactcct
2 examination criteria product of table verify test strips sensitivity
As a result it shows that test strips can detect 105 diluted positives, PCR detection sensitivities can be reached.
(3) test strips and PCR method testing result
550 parts of clinical sample is detected simultaneously with test strips and PCR, as a result such as following table.
It is calculated according to testing result, compared with PCR, test strips sensibility is 98.9%, and specificity is 98.7%, examination
Coincidence rate between paper slip and PCR testing results is 98.9%
Detailed data is shown in Table 1:
3 clinical sample testing result of table
Embodiment 4 selects influence of the different fluorescent microspheres to testing result
In one group of parallel laboratory test of embodiment 3, fluorescent microsphere is using the time-resolved fluorescence containing europium that grain size is 210nm
Microballoon, fluorescent microsphere containing rhodamine isothiocyanate, three kinds of microballoons containing 1,8- benzene-naphthalene diimide classes fluorescent microsphere mark monoclonal antibody, remaining
Making step with embodiment 1, makes 3 kinds of test strips completely.
Negative serum and the positive serum containing 0.5ug FCV albumen are detected, as a result such as the following table 4：
Table 4, using the testing result difference of different fluorescent microspheres
It can be seen that the sensitivity of the 210nm microballoons of time-resolved fluorescence containing europium is obviously higher than other two kinds of microballoons.
It carries out with the specificity in experimental example 1, the duplicate experimental example of sensitivity experiment, the results show that 210 whens containing europium
Between resolved fluorometric microballoon test strips specificity, sensitivity is all significantly better than remaining two groups.
The influence of 5 fluorescent microsphere of embodiment and the rate of charge of how anti-coupling to testing result
In another group of parallel laboratory test, compare,
By in the step of embodiment 1 two, the rate of charge of fluorescent microsphere (microballoon of time-resolved fluorescence containing europium) and monoclonal antibody designs
For 6mg：1mg；3mg：1mg；3mg：Tri- kinds of schemes of 3mg, other operations and reagent make three kinds of test strips completely with embodiment 1
The fluorescent value of sample (negative serum, the serum containing the sick virus P27 albumen of 0.5ug blood) is detected, it is each to repeat detection 8 times, gained
The results are shown in Table 5.
The ELISA test strip result difference of the different rate of charges of 5 fluorescent microsphere of table and monoclonal antibody
As can be seen that fluorescent microsphere and mostly anti-rate of charge are 3：Sensitivity higher when 1；It has also been found that 2~5mg:1mg or 2
~4mg:The rate of charge of 1mg can obtain and 3:Effect similar in 1,
It carries out with the specificity in experimental example 1, the duplicate experimental example of sensitivity experiment, the results show that fluorescent microsphere
It is designed as 3mg with the rate of charge of monoclonal antibody：The specificity of the test strips of 1mg, sensitivity are all significantly better than remaining two groups.
Rabbit-anti feline calicivirus (Feline calicivirus, FCV) F protein polyclonal antibody prepared by the present invention,
With the activity specifically bound with FCV, and with other correlated virus no cross reactions, show the rabbit-anti feline calicivirus
(Feline calicivirus, FCV) F protein polyclonal antibody can be used for immunological detection method.Base is conducive to fluorescent microsphere
Rabbit-anti feline calicivirus (Feline calicivirus, FCV) the F protein polyclonal antibody and SPA of label as main material,
Establishing immunochromatographydetecting detecting test strip has good specificity and sensibility, compared with PCR detection method, two methods detection
As a result coincidence rate is 98.9%.The test strips are quick, easy to operate with detection, do not need professional technician.In addition, the examination
Paper slip is easy to storage and transport, and small using detection sample size, testing cost is relatively low, and safe operation does not cause environmental pollution.Therefore
This method is especially suitable for the quick detection of FCV, the detection of base veterinary clinic etc..The development of the test strips be detection, quarantine and
Epidemiological survey provides good tool.
Above-mentioned detailed description is illustrating for the possible embodiments invented, which is not to limit this hair
Bright the scope of the claims, all equivalence enforcements or change without departing from the present invention should all be contained in the scope of the claims of the present invention.
In addition, those skilled in the art can also the claims in the present invention scope of disclosure and spirit in do other forms and
Various modifications, addition in details and replacement.Certainly, these spirit is done according to the present invention various modifications, addition and replacements
All should include within scope of the present invention Deng variation.
Claims (10)
1. a kind of feline calicivirus (Feline calicivirus, FCV) fluorescence immune chromatography test strip, feature exist
In：
Bonding pad, chromatographic film and the water absorption pad set gradually including backing and on backing, composition can make sample from combination
Bed course is analysed to the chromatography path of water absorption pad；
Fluorescent microsphere labelled antibody compound is combined on the bonding pad；It is provided in the chromatographic film and the chromatography path
Vertical detection line and control line；
The fluorescent microsphere labelled antibody compound be fluorescent microsphere with rabbit-anti feline calicivirus (Feline calicivirus,
FCV) the conjugate of F protein polyclonal antibody；
The detection line is coated with by goat-anti feline calicivirus (Feline calicivirus, FCV) polyclonal antibody, described
Control line is coated with by protein staphylococcus (Staphylococal Protein A, SPA)；
The fluorescent microsphere grain size is 180-240nm.
2. test strips according to claim 1, which is characterized in that on the bonding pad, the fluorescent microsphere labelled antibody
The coating density of compound：With rabbit-anti feline calicivirus (Feline calicivirus, FCV) F protein Anti-TNF-α weight
It is every square centimeter to be calculated as 7.5ug/.
3. test strips according to claim 2, which is characterized in that the fluorescent microsphere labelled antibody compound is by such as
Lower step is prepared：Fluorescent microsphere and rabbit-anti feline calicivirus (Feline calicivirus, FCV) F protein is polyclonal
Antibody is with 2~6mg：The ratio of 1~3mg, preferably 2~5mg:1mg, 2~4mg:1mg or 3mg:The ratio of 1mg be coupled anti-
It should be made.
4. test strips according to claim 3, which is characterized in that the fluorescent microsphere labelled antibody compound is by such as
Lower step is prepared：To pH5.0, the fluorescent microsphere is sequentially added in the PBS solution of concentration 0.02mol/L, concentration is added
For the EDC solution of 10mg/ml, rabbit-anti feline calicivirus (Feline calicivirus, FCV) F protein Anti-TNF-α is added
Body is slowly stirred 2h at room temperature, after final concentration of 2% BSA solution closing 30min is added, with 10000r/min, 4 DEG C of centrifugations
10min removes supernatant, contains the fluorescent microsphere labelled antibody compound in gained precipitation.
5. test strips according to claim 3, which is characterized in that the grain size 210nm of the fluorescent microsphere.
6. according to any test strips of claim 1-5, which is characterized in that the fluorescent microsphere is that rare-earth europium fluorescence is micro-
Ball.
7. according to any test strips of claim 1-5, which is characterized in that the test strips width is 3mm, the detection
The width of line and control line is also 3mm；In the detection line, the goat-anti feline calicivirus (Feline calicivirus,
FCV) polyclonal antibody package amount is 0.5ug/ items, and the protein staphylococcus package amount is 0.3ug/ items；Preferably, it detects
Line and control line are at a distance of 5mm.
8. test strips according to claim 1, which is characterized in that
The upstream of the bonding pad is additionally provided with sample pad；It is transported to bonding pad for receiving sample, and by the substance in sample
On；
Preferably, further include shell, there is an opening in the position that the shell corresponds to the sample pad, corresponding detection line and nature controlling line
Position is set as transparent or hatch frame.
9. any feline calicivirus (Feline calicivirus, FCV) fluorescence immune chromatography detections of claim 1-8
The preparation method of test strips, which is characterized in that including
Prepare the fluorescent microsphere labelled antibody compound：By fluorescent microsphere and rabbit-anti feline calicivirus (Feline
Calicivirus, FCV) F protein polyclonal antibody is with 2~6mg：The ratio of 1~3mg, preferably 2~5mg:1mg, 2~4mg:
1mg or 3mg:The ratio of 1mg carries out coupling reaction；
Fluorescent microsphere labelled antibody complex solution is sprayed on 5ul/cm on bonding pad, vacuum is drained；
By bonding pad, chromatographic film, water absorption pad is assembled to successively on test strips bottom liner.
10. preparation method according to claim 9, which is characterized in that further include by the goat-anti cat cup of a concentration of 2mg/ml
The protein staphylococcus of shape virus (Feline calicivirus, FCV) polyclonal antibody and a concentration of 1mg/ml are coated with respectively
In chromatographic film, the detection line at a distance of 5mm and the control line are formed；
Preferably, the preparation process of the fluorescent microsphere labelled antibody compound is as follows：To pH5.0, concentration 0.02mol/L's
The EDC solution for sequentially adding the fluorescent microsphere in PBS solution, a concentration of 10mg/ml being added, adds rabbit-anti feline calicivirus
(Feline calicivirus, FCV) F protein polyclonal antibody is slowly stirred 2h at room temperature, and final concentration of 2% BSA is added
After solution closes 30min, with 10000r/min, 4 DEG C of centrifugation 10min, supernatant is removed, contains the fluorescence in gained precipitation
Microballoon labelled antibody compound；
Preferably, before the fluorescent microsphere labelled antibody compound is encountered bonding pad, pH8.2 is first used, contains 2% tween
A concentration of 0.05M borate buffer solution immersion treatment bonding pad；
Preferably, further include preparing rabbit-anti feline calicivirus (Feline calicivirus, FCV) F protein polyclonal antibody：With
Feline calicivirus (Feline calicivirus, FCV) F protein immunizing rabbit of purifying, takes a blood sample and detaches serum, FCV F-
ELISA detects antibody titer, if potency reaches 1:5000 or more, by arteria carotis bloodletting, serum is detached, it is pure with antibody after inactivation
Change column to purify polyclonal antibody, 1mg/ml is diluted to PBS；
Preferably, further include the preparation of goat-anti feline calicivirus (Feline calicivirus, FCV) polyclonal antibody:With pure
Feline calicivirus (Feline calicivirus, FCV) immune sheep of change, takes a blood sample through ear vein and detaches serum, use FCV-
ELISA detects antibody titer, if potency reaches 1:5000 or more, by arteria carotis bloodletting, serum is detached, it is pure with antibody after inactivation
Change column to purify polyclonal antibody, 2mg/ml is diluted to PBS.
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