CN108181476A - Test strips based on fluorescent micro-ball immune chromatography method detection avian leukosis virus and preparation method thereof - Google Patents
Test strips based on fluorescent micro-ball immune chromatography method detection avian leukosis virus and preparation method thereof Download PDFInfo
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Abstract
The present invention " test strips based on fluorescent micro-ball immune chromatography method detection avian leukosis virus and preparation method thereof ", 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.Available for field quick detection, step is simple and fast, and sensitivity and specificity are all suitable with Elisa coating plate detections, prevention and control and the purification of chicken group available for chicken group's epidemic disease.
Description
Technical field
The invention belongs to animal virus detection technique field, more particularly to white based on fluorescent micro-ball immune chromatography method detection fowl
Test strips of blood disease virus and preparation method thereof.
Background technology
Avian leukosis (Avian Leukosis, AL) is by Retroviridae A type retrovirus fowl reverse transcription disease
A kind of general designation of birds kinds of tumors disease caused by malicious (Avian Leukosis Virus, ALV).Avian leukosis virus oneself
10 subgroups are classified to, are respectively designated as A subgroups to J subgroups.Wherein A~D subgroups are exogenous ALV, and E subgroups are endogenous
ALV；A, B subgroups be once considered as business laying hen group in (especially Leghorn) most common exogenous virus subgroup, C subgroups
It is extremely rare with the report of D subgroups infection, and E subgroup virus then includes extremely universal low pathogenicity endogenous avian leukosis viruses；F
Subgroup is isolated from Chinese ring-necked pheasant, and G subgroups are located away from golden yellow pheasant, and the I of H subgroups and Gambia quail in addition with Hungary chicken is sub-
Group；J subgroups are to be located away from a kind of new virus of broiler chicken early 1990s；E subgroups are ubiquitous, but mainly with endogenic
Provirus type exists.
In past more than 30 years due to apply some eradicate ALV methods, by leukemia virus or sarcoma virus with
And tumor incidence caused by their correlated virus is generally all very low, virus shows relative stability genetically.This disease is in
Worldwide distribution, infection rate is high, can cause the death of chicken, become thin, and reduces the production capacity of chicken group, is to seriously endanger aviculture hair
One of principal disease of exhibition.The main method for preventing this disease is to cultivate the new varieties with heredity resistance, eliminates the positive
Chicken purifies population, and no leukaemia chicken group is purified by several generations selection and breeding, and the short time consumption period is long, and new chicken group still may
It infects at any time.Therefore extremely it is necessary to establish it is a kind of efficiently and sensitive Methods Used In The Detection of Avian Leukosis to the prevention of avian leukosis with
Monitoring.
At present, the method for detecting ALV includes：Virus neutralization tests, agar gel diffusion test, complement fixation test (CFT), ELISA,
Radio-immunity, immunofluorescence technique etc., however these methods are time-consuming and laborious, need special instrument and equipment and technical staff, only
It can be detected in laboratory, and not be suitable for Site Detection, quick detection and base's Veterinary office clinical detection.
Fluorescence immune chromatography technology is one kind that immunofluorescence technique and Traditional immunochromatographic technology are combined development innovation
Quantitative novel detection technique.Immunochromatography technique is to be fixed with the fibre strip chromatographic material of detection line and control line as fixation
Phase, test fluid are mobile phase, determinand are made to be moved on chromatography strip through capillary action, and determinand occurs specifically at detection line
Property immune response.The technology remains the advantages of colloidal gold immunochromatographimethod technical operation is easy, detection is quick, portability is strong, also
Enhancing technology by fluorescent tracing realizes the accurate quantification of testing result.
About the test paper and its technology of preparing using fluorescence immune chromatography technology, there are many reports in the prior art.
It can be seen that arriving many links and parameter involved in fluorescence immune chromatography test paper and its preparation process, for different types of inspection
Target or different testing goals are surveyed, is respectively had between the fluorescence immune chromatography test paper reported in the prior art and preparation process
Difference；The report or product being detected using fluorescence immune chromatography technology for avian leukosis virus are had not seen at present.
Invention content
Technical need and blank based on above-mentioned field, the present invention establish the fluorescence immunoassay layer for avian leukosis virus
Analyse test strip and preparation method thereof.Test strips can be used for field quick detection, in addition its accessible high sensitivity and
Specificity can be used for the investigation of virus infected flow row disease, so as to the prevention for chicken group's epidemic disease and control and chicken group
Purification offer science foundation and method, be conducive to the sound development of animal farming industry, solve those skilled in the art's letter
Technical barrier to be solved.Technical solution provided by the invention is as follows：
A kind of test strips based on fluorescent micro-ball immune chromatography method detection avian leukosis virus, it is characterised in that：
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 anti-avian leukosis virus P27 protein monoclonal antibodies
Conjugate；Avian leukosis P27 genes are one section of highly conserved genes between avian leukosis virus difference subgroup.The white blood of fowl
Sick P27 albumen is the main component of ALV core capsid albumen, is a kind of highly conserved non-glycosylated protein, and there are many viruses
Antigen point.
The detection line is coated with by goat-anti avian leukosis virus P27 protein polyclone antibodies, and the control line is by Portugal
Grape pneumococcal proteins (Staphylococal Protein A, SPA) are coated with；
The fluorescent microsphere grain size is 180-240nm, preferably 210nm.
In some embodiments, on the bonding pad, resisted in the fluorescent microsphere labelled antibody compound with monoclonal
Weight calculates, and coating density is every square centimeter for 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 anti-avian leukosis virus P27 protein monoclonal antibodies 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 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, adds in a concentration of 10mg/ml's
EDC solution adds anti-avian leukosis virus P27 protein monoclonal antibodies, is slowly stirred 2h at room temperature, adds in final concentration of
After 2% BSA solution closing 30min, with 10000r/min, 4 DEG C of centrifugation 10min, supernatant is removed, is contained in gained precipitation
The fluorescent microsphere labelled antibody compound.
In some embodiments, the grain size 180-240nm of the fluorescent microsphere, preferably 210nm.
In some embodiments, the fluorescent microsphere uses rare-earth europium fluorescent microsphere.
In some embodiments, the test strips width is 3mm, and the width of the detection line and control line is also 3mm；Institute
It states in detection line, the goat-anti avian leukosis virus P27 protein polyclone antibodies package amount is 0.6ug/ items；On control line, institute
The protein staphylococcus package amount stated is 0.4ug/ items；Preferably, detection line and control line are at a distance of 5mm.
In some embodiments, the upstream of the bonding pad is additionally provided with sample pad；For receiving fluid sample, and by sample
Substance in product is transported on bonding pad；
In some embodiments it may be preferred that further including shell, there is opening in the position that the shell corresponds to the sample pad,
The position of corresponding detection line and nature controlling line is set as transparent or hatch frame.
Another aspect of the present invention provides the above-mentioned test paper based on fluorescent micro-ball immune chromatography method detection avian leukosis virus
The preparation method of item, which is characterized in that including
Prepare the fluorescent microsphere labelled antibody compound：By fluorescent microsphere and anti-avian leukosis virus P27 albumen Dan Ke
Grand antibody is with 1~6mg：The ratio of 1mg, preferably 2~5mg:1mg, 2~4mg:1mg or 1mg:The ratio of 1mg be coupled anti-
It answers and obtains；
Fluorescent microsphere labelled antibody complex solution is sprayed on 5ul/cm on bonding pad, vacuum is drained；
Bonding pad, chromatographic film, water absorption pad are assembled to successively on test strips bottom liner.
In preparation method, further include the goat-anti avian leukosis virus P27 protein polyclone antibodies of a concentration of 2mg/ml
Be coated on nitrocellulose membrane respectively with the protein staphylococcus of a concentration of 2mg/ml, formed at a distance of 5mm the detection 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 sequentially add the fluorescent microsphere, the EDC solution for adding in a concentration of 10mg/ml, add anti-avian leukosis disease
Malicious P27 protein monoclonal antibodies are slowly stirred 2h at room temperature, after adding in final concentration of 2% BSA solution closing 30min, with
10000r/min, 4 DEG C of centrifugation 10min, remove supernatant, and it is compound to contain the fluorescent microsphere labelled antibody in gained precipitation
Object；
Preferably, before the fluorescent microsphere labelled antibody compound is encountered bonding pad, first using pH8.2, containing 2%
The borate buffer solution immersion treatment bonding pad of a concentration of 0.05M of tween.
The present invention passes through the optimization of the grain size of fluorescent microsphere, type, fluorescent microsphere and antibody coupling condition and embedding amount
Etc. factors optimization and selection, be successfully established can high special, in high sensitivity detection avian leukosis virus fluorescence exempt from
Epidemic disease chromatography detecting test paper strip.Compared with ELISA detection method, two methods testing result coincidence rate is 98.9%.The test strips
Have the advantages that detect it is quick, easy to operate, do not need to special instrument and equipment and professional technician.In addition, the test strips
It is easy to storage and transport, small using detection sample size, testing cost is relatively low, and safe operation does not cause environmental pollution.Therefore the party
Method is especially suitable for quickly detecting, base's Disease Diagnosis of Veterinary department clinical detection and breeding enterprise voluntarily detect.The test strips
It develops, detection, quarantine and epidemiological survey for ALV provide good tool.
Testing principle, result judgement standard and the detecting step of test strips of the present invention：
When preparing test strips, fluorescent microsphere labeled monoclonal antibody compound is fixed on compound pad, detection line is
Goat-anti ALV polyclonal antibodies, control line SPA.During detection, ALV and fluorescent microsphere labeled monoclonal antibody in positive
Specifically combine, when being moved on NC films, be detected survey line on ALV polyclonal antibodies capture, fluorescent microsphere particle aggregation in
This, under the excitation for reading instrument excitation light source in fluorescence, forms visible green fluorescence band, i.e. detection line.On the contrary, negative sample
In without ALV, do not form green fluorescence band at detection line.Detection line reagent SPA and fluorescent microsphere labeled monoclonal antibody
Fc segments combine, and capture gold mark compound, in contrast line.Whether no matter ALV, fluorescent microsphere label monoclonal is contained in sample
Antibody can be combined with control line reagent, 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
Colour developing, then be feminine gender；It is invalid knot 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
Fruit needs to change test strips and detects again.
Description of the drawings
Fig. 1 show a kind of avian leukosis virus P27 protein fluorescences immunochromatographydetecting detecting test strip structural representation of the present invention
Figure.
1- backings, 2- sample pads, 3- bonding pads, 4- chromatographic films, 41- detection lines, 42- control lines, 5- water absorption pads.
Specific embodiment
The present invention is carefully described, but not as limiting the scope of the invention below in conjunction with Fig. 1 and specific embodiment.
The reagent and instrument used in the implementation example of the present 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)
Avian leukosis virus P27 protein monoclonal antibodies：Corresponding hybridoma cell strain ALV-P27, in April, 2013
China typical culture collection center is deposited within 23rd, deposit number is CGMCC No.7456, is recorded in CN103243077A
In.The monoclonal antibody of anti-avian leukosis virus P27 albumen commercially available from this can also be used
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, buy from SIGMA
The 15 species specificity standard serum antigen samples that Dutch GD companies provide：Poultry infectious bursal disease (IBD), fowl pass
Metachromia bronchitis (IBV, Beaudette plants), newcastle disease (NDV), avian encephalomyelitis (AE), the fowl anaemia factor (CAV) infect
Property laryngotracheitis (ILT), synovia mycoplasma (MS), bird flu (AI, 0126 plant), reovirus (REO), avian infectious branch gas
Guan Yan (IBV, Mass plants), aviadenovirus (Phelp plants), aquatic bird/bird pox virus (FPV), fowl subtract egg virus (EDS-76), sepsis
Mycoplasma (MG), paramyxovirus (APMV)；
Fluorescent microsphere：The microballoon of time-resolved fluorescence containing europium, grain size 210nm, excitation wavelength 340nm, launch wavelength are
610nm, purchased from Merk companies.
Albumin A/G antibody purification columns：It buys from SIGMA
Protein staphylococcus：It buys from SIGMA
1. avian leukosis virus P27 protein expression and purifications of embodiment
Avian leukosis virus P27 albumen：Using being avian leukosis virus P27 disclosed in embodiment 1 in CN103243077A
Gene recombination plasmid expression and purification and obtain.
The LB containing ampicillin (Amp) resistance in recombinant plasmid transformed to e. coli bl21 bacterium, will be inoculated in consolidate
In body culture medium, after 37 DEG C are incubated overnight, 3~4 single bacterium colonies is taken to be inoculated in the LB fluid nutrient mediums containing Amp, 37 is Celsius
Degree oscillation (250rpm) is cultivated to OD600=0.6, it is extremely final concentration of to add in IPTG (isopropyl-beta D-thio galactopyranoside)
1mmol/l, 37 degrees Celsius are continued culture 3 hours, obtain expression albumen, and avian leukosis P27 expressing quantities are 565mg/L.
The strain liquid screened, adds in GuNTA-0Buffer and PMSF sonicated cells at low temperature, 4 DEG C,
12000r/min is centrifuged 15 minutes, takes supernatant, -20 DEG C of preservations.Affinity chromatography is carried out to cracking thalline with Ni-NTA Purification Resins,
Sample is added in chromatographic column, and flow velocity 15ml/h collects penetrating component.The GuNTA-60Buffer of 5 times of NTA volumes of chromatography is eluted,
Flow velocity 15ml/h collects eluent.With the dialysis eluent of 0.5mol/L NaCl and 20mmol/L Tris-HCl solution 24 hours,
Avian leukosis P27 albumen after purification carries out SDS-PAGE and Western-blot testing goal purity of protein and is up to 97%, point
Son amount size is 36KD.
Embodiment 2 prepares the fluorescence immune chromatography test strip of detection avian leukosis
The preparation of step 1 avian leukosis virus P27 protein monoclonal antibodies
Prepare ascites：20 week old or so BALB/C mice is chosen, norphytane, 7d pneumoretroperitoneums injection 5*10 is injected intraperitoneally5It is a
Hybridoma ALV-P27, the visible mouse web portions of 7-10d significantly expand after injection, acquire ascites, and 6000r/min centrifuges 5min,
Collect supernatant, as monoclonal antibody ascites.It is detected with avian leukosis virus P27 albumen-ELISA, titer of ascites 1:
512000, after packing -80 DEG C freeze it is spare.
The purifying of monoclonal antibody：The ascites of preparation using albumin A/G antibody purification columns is purified, uses SDS-PAGE
Monoclonal antibody after purification is analyzed.It is detected with nucleic acid-protein analyzer, after purification a concentration of 1.8mg/ml of monoclonal antibody,
The monoclonal antibody of purifying is diluted to 1mg/ml with the 0.02mol/L PBS of pH7.2, -70 DEG C save backup.
The preparation of step 2 fluorescent labeled antibody compound and bonding pad
3mg fluorescent microspheres are sequentially added into the 0.02mol/L PBS solutions of 5ml pH5.0,15ul now matches a concentration of
The EDC solution of 10mg/ml adds 1mg monoclonal antibodies, is slowly stirred 2h at room temperature, with the closing of final concentration of 2%BSA
After 30min, 10000r/min, 4 DEG C of centrifugation 10min remove supernatant, add in 500ul0.05M borate buffer solutions pH8.2 and (contain
2% tween), sediment is resuspended, abundant mixing saves backup fluorescent microsphere labelled antibody compound in 4 DEG C.
The preparation of fluorescent labeled antibody compound pad:The monoclonal that fluorescent microsphere marks is resisted with XYZ3050 sample application platforms
Nanocrystal composition solution is sprayed on 300mm*5mm glass fibre cottons, discharge rate 5ul/cm, and is drained in 4 DEG C of vacuum.
The preparation of step 3 goat-anti avian leukosis virus P27 protein polyclone antibodies
With avian leukosis virus P27 3 monthly age of the protein immunization health sheep of purifying, immunizing dose only, is spaced 2 weeks for 3mg/
It is 1 time immune, it is immunized 4 times altogether, the preceding emulsion mixed in equal volume for Freund's complete adjuvant and P27 albumen twice, for the third time for not
The emulsion that complete Freund's adjuvant and P27 albumen mix in equal volume, does not have to adjuvant for the 4th time, last time immune 15 day after tomorrow, warp
Ear vein takes a blood sample and detaches serum.After detecting antibody titer, by arteria carotis bloodletting, serum is detached, 30min is inactivated in 56 DEG C,
Purified with antibody A/G purification columns to polyclonal antibody, measuring IgG albumen with DU-800 foranalysis of nucleic acids/protein analyzer contains
It measures as 4.1mg/ml, 2mg/ml is diluted to the 0.02mol/LPBS of ph7.2.
The preparation of step 4 chromatographic film
Chromatographic film：Nitrocellulose filter
Nitrocellulose filter (35mm*300mm) is pasted on backing (70mm*300mm) center surface, such as Fig. 1 first.
The goat-anti polyclonal antibody (2mg/ml) purified and protein staphylococcus (2mg/ml) are selected respectively as detection line
With control line reagent.Two kinds of reagents are put on nitrocellulose membrane respectively, albumen package amount is respectively 0.6ug/ items and 0.4ug/
Item (according to point sample speed and the concentration of detection line reagent and control line reagent, obtains detection line and control line reagent package amount point
It Wei 0.6ug/ items and 0.4ug/ items).
Detection line 41 is located among nitrocellulose filter, perpendicular with chromatography direction, between control line 42 and detection line 41
Distance is 5mm.37 DEG C of dry 2h, 4 DEG C be sealed it is standby.
The assembling and cutting of step 5 test strips
As shown in Figure 1, by backing 1 and, the chromatographic film 4 of 42 reagent of point sample detection line 41 and control line, fluorescent microsphere mark
Avian leukosis virus P27 protein monoclonal antibody compounds pad 3, sample pad 2, water absorption pad 5 stick together, after compacting, in
In CM4000 cutting machines, it is cut into the test strips of 3mm wide.
The testing principle of test strips, result judgement standard：
When preparing test strips, fluorescent microsphere labeled monoclonal antibody compound is fixed on compound pad, detection line is
Goat-anti ALV polyclonal antibodies, control line SPA.During detection, ALV and fluorescent microsphere labeled monoclonal antibody in positive
Specifically combine, when being moved on NC films, be detected survey line on ALV polyclonal antibodies capture, fluorescent microsphere particle aggregation in
This, under the excitation for reading instrument excitation light source in fluorescence, forms visible green fluorescence band, i.e. detection line.On the contrary, negative sample
In without ALV, do not form green fluorescence band at detection line.Detection line reagent SPA and fluorescent microsphere labeled monoclonal antibody
Fc segments combine, and capture gold mark compound, in contrast line.No matter whether contain in sample, fluorescent microsphere label monoclonal resists
Body can be combined with control line reagent, in contrast line.When judging result, if detection line and control line develop the color simultaneously, then for
The positive, if detection line does not develop the color, control line colour developing, then be feminine gender.If detection line and control line do not develop the color or detection line colour developing
And control line does not develop the color, and is null result, needs to change test strips and detects again.
During detection, test strips are lain against on experimental bench, take 100ul with etc. samples product be added in sample pad, be stored at room temperature
Reaction reads instrument observation detection line and control line colour developing situation using fluorescence in 15min, and carries out result judgement.
1 fluorescence immune chromatography test strip of experimental example specificity, sensibility and coincidence rate experiment
(1) specificity of test strips
Detect a part avian leukosis virus P27 albumen i.e. standard positive sample, other correlated virus serum respectively with test strips
Sample include poultry infectious bursal disease (IBD), avian infectious bronchitis (IBV, Beaudette plants), newcastle disease (NDV),
Avian encephalomyelitis (AE), the fowl anaemia factor (CAV), infectious laryngotracheitis (ILT), synovia mycoplasma (MS), bird flu (AI,
0126 plant), reovirus (REO), avian infectious bronchitis (IBV, Mass plants), aviadenovirus (Phelp plants), aquatic bird/
Bird pox virus (FPV), fowl subtract egg virus (EDS-76), Frustrate blood and mycoplasma (MG), paramyxovirus (APMV) positive and feminine gender
Control sample SP.
During detection, test strips are lain against on experimental bench, 100ul measuring samples is taken to be added dropwise in sample pad, are stored at room temperature
Reaction reads instrument observation detection line and control line colour developing situation using fluorescence in 15min, and carries out result judgement.As a result see
The following table 1：
Table 1 detects approximate virus results
It is positive reaction when as a result showing ELISA test strip ALV positives, it is similar viral and negative right detecting remaining
Product is feminine gender in the same old way, shows that test strips specificity is good.
(2) sensitivity tests of test strips
Avian leukosis virus P27 albumen stoste 5mg/ml (standard items), are serially diluted 101-107Times, exempted from fluorescence of the present invention
Epidemic disease chromatograph test strip detects, while is compared with ELISA method, as a result shows that test strips can detect 105Diluted positive sample again
Product, consistent with ELISA testing results, testing result see the table below 2：
2 examination criteria product of table verify test strips sensitivity
Contrast method：Using the P27ELISA detection kits and its operating method described in CN103243077A.It is above-mentioned
The result shows that test strips of the invention are in examination criteria product,Sensitivity is suitable with ELISA method,But detection it is only necessary to
A large amount of laboratory operation step is omitted in sample-adding-waiting 10 to 15 minutes-reading result, fast and convenient.
(3) ELISA test strip actual sample
825 parts of chicken house serum samples are detected simultaneously with test strips and ELISA method.As a result such as following table, according to testing result
It is calculated,
Compared with ELISA, test strips sensibility is 98.9%, and specificity is 98.7%, test strips and ELISA testing results
Between coincidence rate be 98.9%：Statistical data see the table below 3
Table 3 detects chicken house sample test test strips sensitivity
It is detected again after all diluting 103 with batch 825 parts of serum samples, as a result such as the following table 4：
Table 4 detects chicken house sample test test strips sensitivity
Testing result shows that test strips of the invention can detect the virus of low concentration content in serum, but existing
ELISA is for the serum virus of low concentration content, and sensibility decreases, and ELISA test strip result performance of the present invention is stablized.
Embodiment 3 selects influence of the different fluorescent microspheres to testing result
In one group of parallel laboratory test of embodiment 2, three kinds of grain sizes of comparison are the fluorescent microsphere label monoclonal antibody of 210nm：
The microballoon of time-resolved fluorescence containing europium, fluorescent microsphere containing rhodamine isothiocyanate, containing 1,8- benzene-naphthalene diimide class fluorescence
Microballoon, remaining making step with embodiment 2, make 3 kinds of test strips completely.
Negative serum and the serum containing the sick virus P27 albumen of 0.5ug blood are detected, as a result such as the following table 5：
Table 5, using the testing result difference of different fluorescent microspheres
It can be seen that the sensitivity of 210nm time-resolved fluorescences containing europium microballoon is significantly higher than other two kinds of microballoons.
Carry out in experimental example 2 specificity, the duplicate experiment of sensitivity experiment, the results show that 210 times containing europium
The specificity of resolved fluorometric microballoon test strips, sensitivity are all significantly better than remaining two groups.
In one group of parallel embodiment, 180nm, 200nm, 220nm are tested, the time-resolved fluorescence containing europium of 240nm is micro-
Test strips made of ball.Specificity, the duplicate experiment of sensitivity experiment in progress experimental example 2, the results show that grain size
The specificity of the test strips of the microballoon of time-resolved fluorescence containing europium between 180-240nm, sensitivity is all suitable with ELISA method,
210nm behaves oneself best.
Influence of the rate of charge of 3 fluorescent microsphere of embodiment and monoclonal antibody to testing result
By in the step of embodiment 2 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 6
The ELISA test strip result difference of the different rate of charges of table 6, fluorescent microsphere and monoclonal antibody
As can be seen that the rate of charge of fluorescent microsphere and monoclonal antibody is 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.
Carry out in experimental example 1 specificity, the duplicate experimental example of sensitivity experiment, the results show that fluorescent microsphere
3mg is designed as with the rate of charge of monoclonal antibody：Specificity, the sensitivity statistical result of the test strips of 1mg are all significantly better than remaining two groups.
ALV P27 protein specific monoclonal antibodies prepared by the present invention have the activity specifically bound with ALV,
And with other correlated virus no cross reactions, show that the monoclonal antibody can be used for immunological detection method.Base is conducive to fluorescence
The monoclonal antibody and SPA of microballoon label are as main material, and the immunochromatographydetecting detecting test strip of foundation is with good special
Property and sensibility, compared with ELISA detection method, two methods testing result coincidence rate be 98.9%.The test strips have inspection
Survey it is quick, easy to operate, do not need to professional technician.In addition, which is easy to storage and transport, uses detection sample size
Small, testing cost is relatively low, and safe operation does not cause environmental pollution.Therefore quickly detection, base of this method especially suitable for ALV
The detection of layer veterinary clinic and breeding enterprise voluntarily detect.The development of the test strips is detection, quarantine and epidemiological survey carry
For 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 and replacement in details.Certainly, these spirit is done according to the present invention various modifications, addition and replacements
Deng variation, should all include within scope of the present invention.
Claims (10)
1. a kind of test strips based on fluorescent micro-ball immune chromatography method detection avian leukosis virus, it is characterised in that：
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 is the idol of fluorescent microsphere and anti-avian leukosis virus P27 protein monoclonal antibodies
Join object；
The detection line is coated with by goat-anti avian leukosis virus P27 protein polyclone antibodies, and the control line is by grape ball
Mycoprotein (Staphylococal Protein A, SPA) is coated with；
The fluorescent microsphere grain size is 180-240nm, preferably 210nm.
2. test strips according to claim 1, which is characterized in that on the bonding pad, the fluorescent microsphere labelled antibody
With monoclonal antibody Mass Calculation in compound, it is every square centimeter for 7.5ug/ to be coated with density.
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：By fluorescent microsphere and anti-avian leukosis virus P27 protein monoclonal antibodies with 2~6mg：The ratio of 1-3mg
Example, preferably 2~5mg:1mg, 2~4mg:1mg or 3mg:The ratio of 1mg carries out coupling reaction and is 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, adds in concentration
For the EDC solution of 10mg/ml, anti-avian leukosis virus P27 protein monoclonal antibodies are added, 2h is slowly stirred at room temperature, adds
After entering final concentration of 2% BSA solution closing 30min, with 10000r/min, 4 DEG C of centrifugation 10min, supernatant is removed, gained sinks
Contain the fluorescent microsphere labelled antibody compound in shallow lake.
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 micro- for rare-earth europium fluorescence
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 avian leukosis virus P27 protein polyclone antibody packets
It is measured as 0.6ug/ items；On control line, the protein staphylococcus package amount is 0.4ug/ items；Preferably, detection line and right
According to line 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；For receiving sample, and the substance in sample is transported to bonding pad
On；
Preferably, shell is further included, 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. the system of any test strips based on fluorescent micro-ball immune chromatography method detection avian leukosis virus of claim 1-8
Preparation Method, which is characterized in that including
Prepare the fluorescent microsphere labelled antibody compound：Fluorescent microsphere and anti-avian leukosis virus P27 protein monoclonals are resisted
Body 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；
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 the goat-anti fowl of a concentration of 2mg/ml is white
The protein staphylococcus of blood disease virus P27 protein polyclone antibodies and a concentration of 2mg/ml are coated in chromatographic film respectively, are formed
The detection line and the control line at a distance of 5mm；
Preferably, the preparation process of the fluorescent microsphere labelled antibody compound is as follows：To pH5.0, concentration 0.02mol/L's
The fluorescent microsphere, the EDC solution for adding in a concentration of 10mg/ml are sequentially added in PBS solution, adds anti-avian leukosis virus
P27 protein monoclonal antibodies are slowly stirred 2h at room temperature, after adding in final concentration of 2% BSA solution closing 30min, with
10000r/min, 4 DEG C of centrifugation 10min, remove supernatant, and it is compound to contain the fluorescent microsphere labelled antibody in gained precipitation
Object；Preferably, before the fluorescent microsphere labelled antibody compound is encountered bonding pad, first using pH8.2, containing 2% tween
A concentration of 0.05M borate buffer solution immersion treatment bonding pad.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810015962.2A CN108181476A (en) | 2018-01-08 | 2018-01-08 | Test strips based on fluorescent micro-ball immune chromatography method detection avian leukosis virus and preparation method thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810015962.2A CN108181476A (en) | 2018-01-08 | 2018-01-08 | Test strips based on fluorescent micro-ball immune chromatography method detection avian leukosis virus and preparation method thereof |
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| CN201810015962.2A Pending CN108181476A (en) | 2018-01-08 | 2018-01-08 | Test strips based on fluorescent micro-ball immune chromatography method detection avian leukosis virus and preparation method thereof |
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| CN110470836A (en) * | 2019-05-14 | 2019-11-19 | 广州维佰生物科技有限公司 | A kind of detection avian leukosis virus fluorescent micro-ball immune chromatography test paper and preparation method thereof |
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