CN108896765A - A kind of colloidal gold strip and preparation method thereof detecting A subgroup avian leucosis virus - Google Patents
A kind of colloidal gold strip and preparation method thereof detecting A subgroup avian leucosis virus Download PDFInfo
- Publication number
- CN108896765A CN108896765A CN201810721798.7A CN201810721798A CN108896765A CN 108896765 A CN108896765 A CN 108896765A CN 201810721798 A CN201810721798 A CN 201810721798A CN 108896765 A CN108896765 A CN 108896765A
- Authority
- CN
- China
- Prior art keywords
- colloidal gold
- alv
- gold
- antibody
- strip
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57426—Specifically defined cancers leukemia
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Hematology (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Hospice & Palliative Care (AREA)
- Oncology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of colloidal gold strips for detecting A subgroup avian leucosis virus, and the colloidal gold strip includes bottom plate, successively overlap sample pad, gold-labelled pad, nitrocellulose filter and the blotting paper being fixed on bottom plate；The monoclonal antibody of the anti-ALV-A gp85 envelope protein of mouse of colloid gold label is coated in the gold-labelled pad；It is successively arranged detection line and nature controlling line along sample flow direction on the nitrocellulose filter, the detection line is coated with rabbit-anti ALV-A polyclonal antibody, and the nature controlling line is coated with sheep anti-mouse igg antibody.Colloidal gold colloidal gold detection test paper strip of the invention can quickly detect A subgroup avian leucosis virus whether is infected or carried in chicken body, have many advantages, such as reaction news speed, high sensitivity, easy to operate, high specificity, clinic ALV-A be suitble to identify detection.
Description
Technical field
The present invention relates to Preventive Veterinary Medicines to examine field, and in particular to a kind of colloid for detecting A subgroup avian leucosis virus
Gold test paper strip and preparation method thereof.
Background technique
Avian leukosis virus (Avian Leukosis Virus, ALV) belongs to Retroviridae, Oncovirinae,
Fowl Alpharetrovirus can cause the various positive or malignant tumours of birds.According to virus envelope Characteristics of glycoproteins, Viral interference
ALV is classified as 11 subgroups by other molecular biological characteristics such as experiment, host range, and wherein A, B, C, D, E, J, K subgroup are
It is isolated from chicken.A and B subgroup is the most common exogenous virus in commodity egg.
A subgroup avian leucosis (Avian leukosis, AL) is passed caused by A subgroup avian leucosis virus (ALV-A)
Metachromia neoplastic disease, main infection laying hen cause the reduction of egg chicken body immunity, production performance decline, especially Egg Quality and lay eggs
Rate reduces.It is reported that the disease polluted domestic many laying breed fields, and there is certain ascendant trend, China's aviculture is made
At seriously threatening, huge economic losses are caused to China's aviculture.
Currently, can be by clinical dissect and histopathological examination tentative diagnosis for ALV-A infection, further making a definite diagnosis needs
To pass through PCR, immunohistochemistry, the methods of indirect immunofluorescence and ELISA.Above method respectively has advantage and disadvantage, but detects
It is all comparatively laborious, it has not been convenient to.
Therefore the A subgroup avian leucosis immune colloid gold antibody test test paper of fast accurate and easily operated carrying is developed
Item has important practical significance for the purification prevention and treatment of China chicken group's A hypotype avian leukosis.
Summary of the invention
For the above-mentioned prior art, the object of the present invention is to provide a kind of colloidal golds for detecting A subgroup avian leucosis virus
Test strips.It can simply, quickly detect whether chicken group infects, carries ALV-A using the colloidal gold strip, be that chicken group purifies
ALV-A provides LnkTools, reduces purification cost, saves human and material resources.
To achieve the above object, the present invention adopts the following technical scheme that：
The first aspect of the present invention provides a kind of colloidal gold strip for detecting A subgroup avian leucosis virus, the colloid
Gold test paper strip includes bottom plate, successively overlaps sample pad, gold-labelled pad, nitrocellulose filter and the blotting paper being fixed on bottom plate；
The monoclonal antibody of the anti-ALV-Agp85 envelope protein of mouse of colloid gold label is coated in the gold-labelled pad；
Detection line and nature controlling line, the detection line coating are successively arranged along sample flow direction on the nitrocellulose filter
There is rabbit-anti ALV-A polyclonal antibody, the nature controlling line is coated with sheep anti-mouse igg antibody.
Preferably, the monoclonal antibody of the anti-ALV-Agp85 envelope protein of mouse of 3 μ g/cm is coated in the gold-labelled pad.
Preferably, the distance between the detection line and nature controlling line are 7-9mm；Preferably 8mm.
Preferably, the nitrocellulose filter is whatman nitrocellulose filterDiagnose film.
Further, the present invention also provides a kind of test cards for detecting A subgroup avian leucosis virus, including：Above-mentioned glue
Body gold test paper strip and card slot, the card slot have the card chamber of accommodating colloidal gold strip, are provided with observation area and sample-adding on card slot
Area, the card that card slot is arranged in colloidal gold strip is intracavitary, and the observation area of card slot corresponds to the nitrocellulose filter of colloidal gold strip,
Sample application zone corresponds to the sample pad of colloidal gold strip.
The second aspect of the present invention provides the preparation method of above-mentioned colloidal gold strip, includes the following steps：
(1) pH for adjusting colloidal gold solution is 8.8, and the anti-ALV-Agp85 envelope protein of mouse is added into colloidal gold solution
Monoclonal antibody makes the final concentration of 10 μ g/mL of monoclonal antibody；Shaking table mixes, and precipitating is abandoned in BSA closing, centrifugation；Supernatant
Supernatant is abandoned in centrifugation, and precipitating is resuspended with re-suspension liquid, glass fibre is immersed in re-suspension liquid, taking-up makes it dry, and is prepared
Gold-labelled pad；
(2) rabbit-anti ALV-A polyclonal antibody and sheep anti-mouse igg antibody are transferred on nitrocellulose filter respectively, are formed
Nitrocellulose filter containing detection line and nature controlling line；
(3) fixed sample pad, gold-labelled pad, nitrocellulose filter and blotting paper are successively overlapped on bottom plate, that is, are prepared
Colloidal gold strip.
Preferably, in step (1), the partial size of the colloidal gold in the colloidal gold solution is 15-25nm.
Preferably, in step (1), end of the monoclonal antibody of the anti-ALV-Agp85 envelope protein of mouse in colloidal gold solution
Concentration is 3.5mg/ml.
Preferably, in step (1), 1%BSA, 0.5% Sodium azide and 0.16%Tris solution are contained in the re-suspension liquid；
In the Tris solution containing 0.02% Trition X-100, the pH of the Tris solution is 8.5.It is mass percent.
Preferably, in step (2), rabbit-anti ALV-A polyclonal antibody is diluted to 2.0mg/ml with antibody coating buffer, pre-
Fixed detection line position scribing line is detection line after dry；Sheep anti-mouse igg antibody is diluted to 0.6mg/ml, in scheduled matter
Line position scribing line is controlled, is nature controlling line after dry.
The detection method of colloidal gold strip of the invention is：Test strips are laid flat, it is to be checked after taking grinding freeze thawing centrifugation
Tissue, blood or 100 μ L drop of anus wipe paper sample are in well (sample pad), according to showing for the detection line in colour developing area and nature controlling line
Color state, judges testing result：If nature controlling line, detection line occur, determine positive；Nature controlling line occurs, and detection line does not occur,
It is determined as feminine gender；Nature controlling line, detection line do not occur or nature controlling line does not occur and detection line occur, judgement testing result without
Effect.
Beneficial effects of the present invention：
ALV-A colloidal gold colloidal gold detection test paper strip in the present invention, can quickly detect that A subgroup fowl whether is infected or carried in chicken body is white
Blood disease virus has many advantages, such as reaction news speed, high sensitivity, easy to operate, high specificity, clinic ALV-A is suitble to identify detection.
Detailed description of the invention
Fig. 1：The structural schematic diagram of the colloidal gold strip of detection A subgroup avian leucosis virus of the invention；Wherein, 1- sample
Product pad, 2- gold-labelled pad, 3- detection line, 4- nature controlling line, 5- blotting paper, 6- nitrocellulose filter, 7- bottom plate.
Fig. 2：The result judgement figure of colloidal gold strip of the invention；In figure, T- detection line, C- nature controlling line；A:C, T go out
Existing red is determined as the positive；B:Only C occurs red and T red does not occur and is determined as feminine gender；C,D:As long as C line does not occur red knot
Fruit is determined as invalid detection.
Fig. 3：Colloidal gold strip specific detection result of the invention.
Specific embodiment
It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the application.Unless another
It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
As background technology part is introduced, for the detection of A subgroup avian leucosis virus, molecular biology inspection
Measuring tool has the advantages that detection specificity is good, detection sensitivity is high, but time-consuming, needs special instrument and equipment, is not suitable for base
Layer detection；Serology test is shorter compared with molecular Biological Detection time-consuming, and operation is relatively simple, but there are biggish non-specific
Property and be easy that there are false positive issues；So needing to establish quick, sensitive, special and the inspection of A subgroup avian leucosis virus can be used for
The method tested.Based on this, the object of the present invention is to provide a kind of colloidal gold strips for detecting A subgroup avian leucosis virus.
In one embodiment of the present invention, the colloidal gold strip of the detection A subgroup avian leucosis virus provided,
Structure includes：
Bottom plate successively overlaps sample pad, gold-labelled pad, nitrocellulose filter and the blotting paper being fixed on bottom plate；
The monoclonal antibody of the anti-ALV-Agp85 envelope protein of mouse of colloid gold label is coated in the gold-labelled pad；
Detection line and nature controlling line, the detection line coating are successively arranged along sample flow direction on the nitrocellulose filter
There is rabbit-anti ALV-A polyclonal antibody, the nature controlling line is coated with sheep anti-mouse igg antibody.
In above-mentioned colloidal gold strip, the bottom plate can be PVC board, plank, metal plate etc. it is various do not absorb water have
The thin slice of supporting role.
In above-mentioned colloidal gold strip, nitrocellulose filter is also known as NC film, and detection line is used as in colloidal gold strip
With the supporting body of nature controlling line, while be also immune response point, so the type of nitrocellulose filter is for testing result
Specificity, sensitivity, colour developing and background color etc. have significant impact, for specificity, the sensitivity for promoting detection
And convenient for observation, investigation is optimized to a variety of different types of nitrocellulose filters in the present invention, as a result, it has been found that, whatman
Nitrocellulose filterFilm is diagnosed compared with other kinds of nitrocellulose filter, there is Tomography Velocity
Fastly, without background colour developing, detection line colour developing depth, the nothing but advantages such as specific chromogenic, so the present invention selects whatman cellulose nitrate
Plain filmFilm is diagnosed as nitrocellulose filter.
Gold-labelled pad is the selection for combining gold labeling antibody, for gold-labelled pad material therefor, on the one hand to consider material
To the binding capacity of gold labeling antibody, on the other hand it is also contemplated that marking the influence of protein active to gold.Through preferred, the present invention is with glass fibers
Dimension film is Ahlstrom8964 to combine gold labeling antibody, best performance.
In above-mentioned colloidal gold strip, it is the anti-ALV-Agp85's of 3.5mg/mL mouse that concentration is attached in the gold-labelled pad
Monoclonal antibody, i.e. 3 μ g/cm.The antibody can be in conjunction with ALV-A virus-specific.Meet the sensitivity requirement of test strips detection,
The requirement with the more anti-bindings of sheep anti mouse can also be met simultaneously.
In another embodiment of the invention, the preparation method of above-mentioned colloidal gold strip is given, it is specific as follows：
(1) pH for adjusting colloidal gold solution is 8.8, and the anti-ALV-Agp85 envelope protein of mouse is added into colloidal gold solution
Monoclonal antibody makes the final concentration of 3-4mg/mL of monoclonal antibody；Shaking table mixes, and precipitating is abandoned in BSA closing, centrifugation；Supernatant
Supernatant is abandoned in centrifugation, and precipitating is resuspended with re-suspension liquid, glass fibre is immersed in re-suspension liquid, taking-up makes it dry, and is prepared
Gold-labelled pad；
(2) rabbit-anti ALV-A polyclonal antibody and sheep anti-mouse igg antibody are transferred on nitrocellulose filter respectively, are formed
Nitrocellulose filter containing detection line and nature controlling line；
(3) fixed sample pad, gold-labelled pad, nitrocellulose filter and blotting paper are successively overlapped on bottom plate, that is, are prepared
Colloidal gold strip.
The monoclonal antibody and rabbit-anti ALV-A polyclonal antibody of ALV-Agp85 envelope protein anti-for mouse, using such as lower section
It is prepared by method.
In colloidal gold strip of the invention, identify ALV-A monoclonal antibody be it is the most key, the antibody is not just
It can not identify and distinguish ALV-A and other subgroup ALV.Gp85 albumen is protein structure of ALV virus itself, but different subgroups
Gp85 Argine Monohydrochloride the Nomenclature Composition and Structure of Complexes is different, classifies according to albumen difference team ALV, including ALV-A, ALV-B and
ALV-J etc..Distinguishing these different subgroups also will be by identifying gp85 albumen specific amino acids composition and knot in different subgroups
The specific antibodies of structure are.When selecting gold labeling antibody and coated antibody, it is necessary to which at least one kind being capable of the specific recognition Asia
The monoclonal antibody of group just can be with.Polyclonal antibody may identify more on gp85 albumen since ingredient is compared with monoclonal antibody complex
A amino acid sites not can guarantee identification and distinguish the subgroup and other subgroups.Furthermore, it is contemplated that the cost of test strips is developed, this
A kind of labeling of monoclonal antibody colloidal gold is invented, lower-cost polyclonal antibody is coated with detection line, accomplishes that area can be identified
ALV-A is divided to reduce test strips cost again.
Due to test strips reaction principle be related to antigen-antibody combine and chromogenic reaction, these by reaction condition such as pH value,
The influence of antibody concentration content, temperature, colloid gold particle and ingredient etc. is very big, thus prepares reaction condition used by test strips
Also it will have a direct impact on the detection effect of colloidal gold strip.
Trisodium citrate reduction method preparation can be used in preparation for colloidal gold.The colloid gold particle of good quality is to obtain
The premise of the colloidal gold strip of excellent performance is obtained, colloid gold particle size is inhomogenous, in irregular shape, is unfavorable for the mark of albumen
Note, naked gold particle easy to form, prepared gold mark albumen is unstable, causes the generation of nonspecific reaction；And modest size
Colloid gold particle selection and colloidal gold strip preparation key, colloid gold particle is excessive, and Tomography Velocity is slow, hold
Easily assemble, the high sensitivity of test strips, but poor specificity, is easy to appear false positive, and should not save；And partial size is too small,
Its Tomography Velocity is fast, and specificity is good, but the sensitivity of test strips is low, is easy to appear false negative, therefore, it is necessary to comprehensively consider detection
Sample selects the colloid gold particle of appropriate particle size.The present invention verifies discovery, when the partial size of colloid gold particle is 15-25nm, especially
It is suitable for the label of the monoclonal antibody of the anti-ALV-Agp85 envelope protein of mouse.
Closing can prevent and reduce sample or marker to the non-specific adsorption of entire film, and closing can be used for controlling
Tomography Velocity and steady testing processed, control linear protein.In the case where other conditions are constant, different confining liquid sealing effects is poor
Different bigger, non-specificity colour developing is significantly different.The confining liquid that the present invention has investigated different compositions reacts colloidal gold strip
The influence of speed, the background color of film, the gold mark aquation uniformity, specificity and sensitivity etc., it is best therefrom to select
Confining liquid composition.The present invention is found through experiments that, a certain amount of Tween-20 is added in confining liquid, reaction can be improved
Specificity, but the reduction of Tween-20 can then cause reaction speed to slow down, the background color of film is deepened and reaction sensitivity reduces
Deng.By generalized analysis, the present invention be finally found that, using 1%BSA+0.1%Tween-20 as confining liquid of the invention
With optimal detection effect.
The testing principle of colloidal gold strip of the invention is：Test strips are according to " antibody-antigen-antibody " sandwich immunoassay layer
Principle design is analysed, after sample is added dropwise, under capillary action, sample solution migrates upwards, and when reaching gold-labelled pad, gold labeling antibody will be by
Dissolution.When in sample being the A subgroup avian leucosis virus positive, A subgroup avian leucosis virus will be combined with gold labeling antibody, and one
Migration upwards is played, " polyclonal antibody-virus-gold mark when reaching the detection line for being fixed with rabbit-anti ALV-A polyclonal antibody, is formed
Antibody " compound, ultimately forms a red precipitate line, and loose gold labeling antibody continue to go upward to sheep anti-mouse igg it is anti-
At body, " gold labeling antibody-IgG " compound is formed, forms red precipitate line, this line is nature controlling line.When sample is the white blood of A subgroup fowl
When virus is negative, gold labeling antibody dissolves in the sample, and migration upwards together, reaches and is fixed with rabbit-anti ALV-A Anti-TNF-α
It when the detection line of body, cannot be fixed, not occur red stripes at detection line, loose gold labeling antibody continues to go upward to sheep
At dynamics, " gold labeling antibody-IgG " compound is formed, forms red precipitate line, this line is nature controlling line.
Avian leukosis of the present invention divides 11 subgroups such as A, B, C, D, E, F, G, H, J, K, wherein A, B, C, D, E, J, K
Subgroup can infected chicken, A subgroup avian leucosis is a kind of tumour sexually transmitted disease as caused by ALV-A, envelope protein gp85 tool
There is subgroup specific, the chicken infected can produce subgroup specific antibody.There are multiple antigenic determinants on gp85 albumen, it can be with phase
The monoclonal antibody specificity answered combines.In addition, the present invention using rabbit-anti ALV-A polyclonal antibody as detection antibody, with Dan Ke
Grand antibody is compared, and polyclonal antibody is prepared easier, and has stronger reactionogenicity, and sensibility is not found compared with monoclonal
Antibody decline.
In order to enable those skilled in the art can clearly understand the technical solution of the application, below with reference to tool
The technical solution of the application is described in detail in the embodiment of body.
Test material used in the embodiment of the present invention and comparative example is the test material of this field routine, can be passed through
Commercial channel is commercially available.
Embodiment 1：The preparation of ALV-Agp85 antigen protein
According to the ALV-A-SDAU09C1 strain virus gp85 sequence (GenBank delivered：HM452339) design primer,
Upstream primer is：5'-CGCGGATCCCACTTACTCGAGCAGCC-3'；(SEQ ID NO.1)
Downstream primer is：5'-CCCAAGCTTTCACCCTACCGGACGACTG-3'；(SEQ ID NO.2)
Primer upstream and downstream underscore part is respectively BamHI and HindIII restriction enzyme site, and restriction enzyme site and protection is added
Property base.
PCR reaction condition：95 DEG C of 3min are allowed to enter circulating system after being sufficiently denaturalized：95 DEG C of 30s, 61 DEG C of 30s, 72 DEG C
70s is recycled 30 times, last 72 DEG C of 10min.Then PCR product is recycled with DNA gel QIAquick Gel Extraction Kit.By the PCR product of recycling
It connect with pMD18-T cloning vector, overnight in 16 DEG C of connections, is transferred in DH5 α competent E.coli, extract conversion bacteria plasmid,
Plasmid enzyme restriction is accredited as positive bacterium solution sequence verification.By the strain containing recombinant plasmid p MD18-T-gp85 and contain pET-32a
The actication of culture of (+) plasmid, which expands, cultivates and extracts plasmid, and pM D18-T-gp85 and pET-32a (+) plasmid of extraction is carried out
Digestion.37 DEG C of reaction 2h, by the gp85 gene recycled after expression vector pET-32a (+) and double digestion by DNA ligase, 16
DEG C connection 8h, obtained product is transformed into BL21 competent E.coli, and picking positive bacterium colony is inoculated in fluid nutrient medium
It is cultivated in test tube, illustrates to extract plasmid according to OMEGA plasmid extraction kit, double digestion identification is carried out to the recombinant plasmid, and
Sequence verification.
Strain BL21 containing target gene recombinant plasmid is inoculated in containing Amp+The LB solid culture of 100 μ g/mL resistances
On base, 37 DEG C of constant temperature incubation 8-10h, next day picking single colonie is inoculated in containing Amp+The LB liquid medium test tube of 100 μ g/mL
In, 37 DEG C of shaken overnight cultures, by 1:100 ratio transferred speciess are in fluid nutrient medium, 37 DEG C of 220rpm, culture to O D600About
0.6, it takes out the bacterium solution that 1mL is not induced and compares, IPTG, which is added, makes its final concentration of 1mmol/L, and 37 DEG C are continued shaking culture 4h.
By the bacterium solution of upper inducing expression, 5000rpm is centrifuged 10min, abandons supernatant, and bacterial sediment is resuspended with PBS solution；Ultrasonication bacterium
Body, 4 DEG C of centrifugations 12000rpm, 10min abandon supernatant, collect the inclusion body precipitating of bottom；With Buffer B (50mM Na2HPO4,
0.3M NaCl, pH8.0) precipitating collected is dissolved, it is incubated at room temperature 60min, 12000rpm is centrifuged 30min, and it abandons and precipitates, supernatant warp
0.45 μm of filter filtering, obtained liquid is as sample A.The balance of protein purification column：Column is washed with Buffer B；It will be on sample A
Column, trickle be sample B, for improve albumen binding capacity, sample B can be repeated column；Elute foreign protein：With 2 times of cylinders
Long-pending Buffer C (50mM Na2HPO4, 0.3M NaCl, 10mM imidazole pH=8.0) and wash column；Elute destination protein：
With Buffer E (the 50mM Na of minimum2HPO4, 0.3M NaCl, 250mM imidazole pH=8.0) and wash column, efflux
Body is sample D, i.e., required destination protein.By the purity of SDS-PAGE electrophoresis identification sample D, and measure the dense of albumen
Degree.
Embodiment 2：ALV-Agp85 protein monoclonal antibody preparation and purification
ALV-Agp85 antigen protein (preparation of embodiment 1) is diluted to 1.0mg/mL with sterilizing PBS, with Freund's adjuvant etc.
Volume mixture.Inject 6 week old female Blab/C mouse, 0.2mL/ only one exempt from after 3w and 5w booster immunization, after three exempt from 4d
Booster immunization is primary, and the splenocyte of immune mouse and SP2/0 (myeloma cell) is taken to carry out cell fusion after 3d.It is thin according to spleen
Born of the same parents：Cell=10 SP2/0：1 ratio takes suitable SP2/0 cell and splenocyte to mix well the 50mL centrifuge tube in sterilizing
In, 1000rpm is centrifuged 8min；Supernatant is abandoned, centrifuge tube is placed in the beaker for filling 37 DEG C of warm water, is drawn and is preheated with 1mL suction pipe
To 37 DEG C of 50%PEG 1mL, dropwise addition stands 1min after dripping off in cell precipitation；The DMEM base that 5mL is preheated to 37 DEG C is added
Then the culture medium of 10mL is added in basal culture medium, the culture medium of 15mL, 37 DEG C of standing 10min are finally added dropwise；1000rpm centrifugation
5min abandons supernatant；It is slowly added into and is preheated to 37 DEG C of HAT culture medium, gently dispelling precipitating makes it suspend and mix, HAT culture
The dosage of base presses 2 × 106A/mL splenocyte addition；96 porocyte culture plates that cell suspension after mixing is added to are (in every hole
Complete the feeder cells of 100 μ L) in, 100 holes μ L/, 37 DEG C, 5%CO2It is cultivated in incubator；Change liquid：Replacement half is measured after 4d
HAT culture medium more uses HT culture medium after 10d~13d；20%DMEM culture medium is used instead according to cell proliferative conditions later；Training
After supporting 14d, the upgrowth situation of hybridoma is often observed, records and detects in time, to Growth of Hybridoma Cell to every bottom hole
When 1/3 or more area, cell conditioned medium is sucked out and is used for antibody test.By ELISA method preliminary screening hybridoma, then use IFA
Hybridoma is screened in method screening again.The hybridoma of screening expands culture.
The non-immune 10 week old female Balb/c mouse for choosing health, is injected intraperitoneally liquid before being inoculated with oncocyte 1-2w
Paraffin, 0.5mL/ is only；Oncocyte culture is diluted to 5 × 106A/mL, to treated mouse slowly multi-direction intraperitoneal injection
0.2mL cell suspension；After injecting hybridoma 10d, mouse ascites production is observed.If abdomen obviously expands, finger touching
When touching, skin has tension, i.e., available No. 16 needle applicators slowly extract ascites.It generally can continuously adopt 2-3 times, every minor tick
2-3 days, it can once extract 5mL or so；The ascites 10000rpm of collection is centrifuged 10min, removes cell component and other precipitatings
Object collects supernatant, later with 0.45 μm of membrane filtration.
Ascites equilibration buffer (20mM Na2HPO4, 0.15M NaCl, pH=7.0) and carry out 1:1 dilution, sufficiently shakes up
Resin is resuspended, 1mL equilibration buffer is previously added in new chromatographic column, then draws 1ml slurries again into the chromatographic column.It allows
Resin natural subsidence flows out equilibration buffer.5mL equilibration buffer is added and balances resin into chromatographic column, by about 1mL/min's
Flow velocity flows out equilibration buffer.By sample according to the flow velocity loading of about 1mL/min into chromatographic column, with the equilibration buffer of 30mL
Resin is washed, flow velocity maintains about 2mL/min.With 10-15mL elution buffer (0.1M glycine, pH=2.5) antibody elution,
Flow velocity maintains about 1mL/min, collects containing there is the eluent of purpose immunoglobulin, and carve be added 1/10 eluent (1M Tris,
PH=8.5) the neutralization buffer of volume adjusts pH to 7.4.
Embodiment 3：The preparation of the polyclonal antibody of ALV-Agp85 albumen
ALV-Agp85 antigen protein (preparation of embodiment 1) is diluted to 1mg/mL, presses 1 with Freund's complete adjuvant for the first time:
1 mixing it is fully emulsified, after several times with incomplete Freund's adjuvant press 1:1 mixing is fully emulsified, subcutaneously multi-point injection immune rabbit,
Interval 2 weeks, and injected by 1mg/kg rabbit.10 days ear source vein haemospasias after third time, and its antibody effect is measured with ELISA method
Valence, potency reach 10 4 powers, and Culling heart blood after the 4th time collects hyper-immune serum.It is slightly mentioned, then used with sad amine sulfate method
Protein G is purified.Prepared A subgroup avian leucosis polyclonal antibody is used for colloidal gold strip.
The high polyclonal antibody for exempting from rabbit-anti A subgroup avian leucosis gp85 albumen is taken, serum is separated, with octanoic acid-ammonium sulfate
Method slightly mentions, then is purified with protein G.
1. 1 portion of hyper-immune serum first is diluted with the sodium acetate of 3 parts of 60mmol/L, PH to 4.5 is adjusted；Liquid after serum dilution
Every milliliter of body adds 33 microlitres of octanoic acid stirring while adding, and 4 degree of 12000rpm are centrifuged 30min after stirring 30min, takes supernatant, adds 1/10
10 × PBS of volume adjusts pH to 7.4.Ammonium sulfate liquid 0.277g, 4 DEG C of stirring 30min, 5000g centrifugations are added by every milliliter of mixed liquor
15min.Precipitating is taken to be dissolved in a small amount of 0.01M PBS solution.
2. the how anti-protein G purification media after preliminary purification purifies, according to monoclonal antibody-purified side in embodiment 2
Method does not repeat.
Embodiment 4：The preparation of gold-labelled pad
The solution of potassium carbonate that 1% is added in 100mL colloidal gold solution adjusts pH to 8.8, is added while stirring most
Good monoclonal antibody (3.5mg/mL), rocks 30min, is stored at room temperature 10min, and confining liquid (1%BSA+0.1% is added
Tween-20), 40min is closed.Then 1500r/min is centrifuged 15min, takes supernatant 12000r/min to be centrifuged 30min, with 10ml's
Re-suspension liquid (the 0.02%Tris solution (PH8.5) containing 1%BSA, 0.5% Sodium azide and 0.16%Trition X-100) weight
Outstanding precipitating, glass fibre is immersed in re-suspension liquid, and taking-up makes it dry spare, that is, gold-labelled pad is prepared.
Embodiment 5：The coating of nitrocellulose filter
ALV-A polyclonal antibody is diluted to antibody coating buffer (the PB solution (PH8.0) of the 0.01M containing 4% sucrose)
2.0mg/mL.Diluted polyclonal antibody and 0.6mg/mL goat anti-mouse IgG are transferred on NC film and are respectively formed detection line
(T) and nature controlling line (C).The distance between T and C line is 8mm.It is dried 1 hour at 37 DEG C.
Embodiment 6：The assembling of test strips
Nitrocellulose filter is affixed among bottom plate, is then pasted blotting paper and gold-labelled pad in a manner of being overlapped slightly respectively
In nitrocellulose filter two sides.Sample pad is pushed down to gold-labelled pad slightly again and is affixed on bottom plate, the test paper of 4mm is cut into cutting machine
Item.4 DEG C of refrigerators save.Assembled test strips are as shown in Figure 1.
Tissue to be checked, blood or 100 μ L of anus wipe paper sample after taking grinding freeze thawing centrifugation are added in well, 3-5min
Can show as a result, by the color of observation detection line and nature controlling line can determine whether in test sample whether to have ALV-A or by
ALV-A infection.
Result judgement is as shown in Figure 2：
It is positive：Respectively there are a red stripes at detection line (T) and nature controlling line (C), is determined as the positive.Detection line (T)
The depth degree of band color can determine whether the height of the antigenic content of ALV-A, and antigenic content is higher, and band color is deeper, otherwise more
Shallowly.
It is negative：There are a red stripes at nature controlling line (C), detection line (T) does not occur band, illustrates do not have in test sample
There is the presence of ALV-A.
In vain：Only there are band or two lines to occur without band in detection line (T), can determine that test strips detect nothing
Effect.
Embodiment 6：Sensitivity, specificity and the stability test of colloidal gold strip
The gp85 recombinant protein of colloidal gold colloidal gold detection test paper strip prepared by the present invention prokaryotic expression is detected into its sensitivity,
It detects offline for 80ng/mL.Cell virus supernatant is detected, it is similar with colloidal gold colloidal gold detection test paper strip comparing result by ELISA.
ALV-A colloidal gold colloidal gold detection test paper strip can distinguish the strain of ALV-A Yu ALV-B/J/K subgroup, as shown in figure 3, saying
Bright colloidal gold colloidal gold detection test paper strip of the invention has extraordinary specificity.
Valve bag will be packed into a batch of colloidal gold colloidal gold detection test paper strip and be put into desiccant, be put in 4 DEG C, 25 DEG C and 37 respectively
DEG C, the situation of change of the indexs such as periodic detection its sensitivity, specificity.Test strips specificity is good under the conditions of 37 DEG C at the 2nd week,
But sensitivity declines；Test strips specificity is good under the conditions of 25 DEG C at the 4th week, but sensitivity declines；Test strips are steady under the conditions of 4 DEG C
It is fixed, all meet examination criteria in 6 months internal specifics and sensitivity, illustrates that colloidal gold colloidal gold detection test paper strip of the invention has very
Good stability.
Embodiment 7：Clinical sample detection
To 202 parts of cloacal swabs samples of the infection ALV-A chicken of laboratory preservation, 125 parts of blood samples and 64 parts
Egg white sample is utilized respectively colloidal gold strip of the invention, and import ALV P27 antigen ELISA kit is detected.
Testing result shows：This test strips recall rate consistent with ELISA paper box is：Cloacal swabs 94.06%
(190/202)；Blood sample 95.2% (119/125)；Egg white sample 95.31% (61/64).
The foregoing is merely preferred embodiment of the present application, are not intended to limit this application, for the skill of this field
For art personnel, various changes and changes are possible in this application.Within the spirit and principles of this application, made any to repair
Change, equivalent replacement, improvement etc., should be included within the scope of protection of this application.
SEQUENCE LISTING
<110>Shandong Agricultural University
<120>A kind of colloidal gold strip and preparation method thereof detecting A subgroup avian leucosis virus
<130> 2018
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 26
<212> DNA
<213>Artificial sequence
<400> 1
cgcggatccc acttactcga gcagcc 26
<210> 2
<211> 28
<212> DNA
<213>Artificial sequence
<400> 2
cccaagcttt caccctaccg gacgactg 28
Claims (10)
1. a kind of colloidal gold strip for detecting A subgroup avian leucosis virus, which is characterized in that the colloidal gold strip includes
Bottom plate successively overlaps sample pad, gold-labelled pad, nitrocellulose filter and the blotting paper being fixed on bottom plate；
The monoclonal antibody of the anti-ALV-Agp85 envelope protein of mouse of colloid gold label is coated in the gold-labelled pad；
It is successively arranged detection line and nature controlling line along sample flow direction on the nitrocellulose filter, the detection line is coated with rabbit
Anti- ALV-A polyclonal antibody, the nature controlling line are coated with sheep anti-mouse igg antibody.
2. colloidal gold strip according to claim 1, which is characterized in that be coated with the mouse of 3 μ g/cm in the gold-labelled pad
The monoclonal antibody of anti-ALV-A gp85 envelope protein.
3. colloidal gold strip according to claim 1, which is characterized in that the distance between the detection line and nature controlling line
It is 7-9mm；Preferably 8mm.
4. colloidal gold strip according to claim 1, which is characterized in that the nitrocellulose filter is whatman nitre
Acid cellulose filmDiagnose film.
5. a kind of test card for detecting A subgroup avian leucosis virus, which is characterized in that including：Described in claim any one of 1-4
Colloidal gold strip and card slot, the card slot has the card chamber of accommodating colloidal gold strip, be provided on card slot observation area and
Sample application zone, the card that card slot is arranged in colloidal gold strip is intracavitary, and the observation area of card slot corresponds to the cellulose nitrate of colloidal gold strip
Plain film, sample application zone correspond to the sample pad of colloidal gold strip.
6. the preparation method of the described in any item colloidal gold strips of claim 1-4, which is characterized in that include the following steps：
(1) pH for adjusting colloidal gold solution is 8.8, and the Dan Ke of the anti-ALV-A gp85 envelope protein of mouse is added into colloidal gold solution
Grand antibody makes the final concentration of 10 μ g/mL of monoclonal antibody；Shaking table mixes, and precipitating is abandoned in BSA closing, centrifugation；Supernatant centrifugation,
Supernatant is abandoned, precipitating is resuspended with re-suspension liquid, glass fibre is immersed in re-suspension liquid, taking-up makes it dry, and golden mark is prepared
Pad；
(2) rabbit-anti ALV-A polyclonal antibody and sheep anti-mouse igg antibody are transferred on nitrocellulose filter respectively, formation contains
The nitrocellulose filter of detection line and nature controlling line；
(3) fixed sample pad, gold-labelled pad, nitrocellulose filter and blotting paper are successively overlapped on bottom plate, that is, colloid is prepared
Gold test paper strip.
7. preparation method according to claim 6, which is characterized in that the colloid in step (1), in the colloidal gold solution
The partial size of gold is 15-25nm.
8. preparation method according to claim 6, which is characterized in that in step (1), the anti-ALV-Agp85 envelope protein of mouse
Final concentration of 10 μ g/mL of the monoclonal antibody in colloidal gold solution.
9. preparation method according to claim 6, which is characterized in that in step (1), contain 1% in the re-suspension liquid
BSA, 0.5% Sodium azide and 0.16%Tris solution；In the Tris solution containing 0.02% Trition X-100, it is described
The pH of Tris solution is 8.5.
10. preparation method according to claim 6, which is characterized in that in step (2), by rabbit-anti ALV-A polyclonal antibody
It is diluted to 2.0mg/mL with antibody coating buffer, is crossed in scheduled detection line position, is detection line after dry；By sheep anti mouse
IgG antibody is diluted to 0.6mg/mL, crosses in scheduled Quality Control line position, is nature controlling line after dry.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810721798.7A CN108896765A (en) | 2018-07-04 | 2018-07-04 | A kind of colloidal gold strip and preparation method thereof detecting A subgroup avian leucosis virus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810721798.7A CN108896765A (en) | 2018-07-04 | 2018-07-04 | A kind of colloidal gold strip and preparation method thereof detecting A subgroup avian leucosis virus |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108896765A true CN108896765A (en) | 2018-11-27 |
Family
ID=64347579
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810721798.7A Pending CN108896765A (en) | 2018-07-04 | 2018-07-04 | A kind of colloidal gold strip and preparation method thereof detecting A subgroup avian leucosis virus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108896765A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109521199A (en) * | 2018-11-29 | 2019-03-26 | 中国动物疫病预防控制中心(农业农村部屠宰技术中心) | A kind of gold-immunochromatographyreagent reagent for assay box and its application for detecting avian leukosis virus |
| CN111718956A (en) * | 2020-06-30 | 2020-09-29 | 山东农业大学 | Preparation method and application of chicken-derived TRIM25 gene recombinant fluorescent expression plasmid |
| CN111948388A (en) * | 2020-08-18 | 2020-11-17 | 山东农业大学 | Colloidal gold test strip for detecting clostridium putrefactive, preparation method and application thereof |
| WO2021028802A1 (en) * | 2019-08-12 | 2021-02-18 | Instytut Immunologii I Terapii Doświadczalnej Im. Ludwika Hirszfelda Polskiej Akademii Nauk | A strip test for detecting enzootic bovine leukosis and a method for detecting enzootic bovine leukosis with the use of the strip test |
| CN112710832A (en) * | 2020-12-22 | 2021-04-27 | 扬州大学 | Test strip for detecting avian adenovirus serotype 4 based on Fiber2 protein |
| CN113533721A (en) * | 2021-07-15 | 2021-10-22 | 上海伯杰医疗科技有限公司北京分公司 | Colloidal gold method detection test strip for influenza A/B virus antigen and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102636646A (en) * | 2012-05-15 | 2012-08-15 | 扬州大学 | Test paper strip for rapidly detecting avian leukosis antigen and preparation method thereof |
| CN104096241A (en) * | 2014-07-19 | 2014-10-15 | 青岛易邦生物工程有限公司 | ALV (avian leukosis virus)-A genetic engineering subunit vaccine |
| CN104560865A (en) * | 2014-12-25 | 2015-04-29 | 华南农业大学 | Cell line resistant to A subgroup avian leukosis virus as well as construction method and application thereof |
-
2018
- 2018-07-04 CN CN201810721798.7A patent/CN108896765A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102636646A (en) * | 2012-05-15 | 2012-08-15 | 扬州大学 | Test paper strip for rapidly detecting avian leukosis antigen and preparation method thereof |
| CN104096241A (en) * | 2014-07-19 | 2014-10-15 | 青岛易邦生物工程有限公司 | ALV (avian leukosis virus)-A genetic engineering subunit vaccine |
| CN104560865A (en) * | 2014-12-25 | 2015-04-29 | 华南农业大学 | Cell line resistant to A subgroup avian leukosis virus as well as construction method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 张丹丹: "A 亚群禽白血病病毒 gp85 囊膜蛋白原核表达及初步应用", 《中国优秀硕士学位论文全文数据库（农业科技辑）》 * |
| 张恒 等: "禽白血病A亚群病毒gp85的单因子血清制备及其特异性鉴定", 《微生物学报》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109521199A (en) * | 2018-11-29 | 2019-03-26 | 中国动物疫病预防控制中心(农业农村部屠宰技术中心) | A kind of gold-immunochromatographyreagent reagent for assay box and its application for detecting avian leukosis virus |
| WO2021028802A1 (en) * | 2019-08-12 | 2021-02-18 | Instytut Immunologii I Terapii Doświadczalnej Im. Ludwika Hirszfelda Polskiej Akademii Nauk | A strip test for detecting enzootic bovine leukosis and a method for detecting enzootic bovine leukosis with the use of the strip test |
| CN111718956A (en) * | 2020-06-30 | 2020-09-29 | 山东农业大学 | Preparation method and application of chicken-derived TRIM25 gene recombinant fluorescent expression plasmid |
| CN111718956B (en) * | 2020-06-30 | 2022-11-04 | 山东农业大学 | Preparation method and application of chicken-derived TRIM25 gene recombinant fluorescent expression plasmid |
| CN111948388A (en) * | 2020-08-18 | 2020-11-17 | 山东农业大学 | Colloidal gold test strip for detecting clostridium putrefactive, preparation method and application thereof |
| CN112710832A (en) * | 2020-12-22 | 2021-04-27 | 扬州大学 | Test strip for detecting avian adenovirus serotype 4 based on Fiber2 protein |
| CN113533721A (en) * | 2021-07-15 | 2021-10-22 | 上海伯杰医疗科技有限公司北京分公司 | Colloidal gold method detection test strip for influenza A/B virus antigen and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108896765A (en) | A kind of colloidal gold strip and preparation method thereof detecting A subgroup avian leucosis virus | |
| US6649743B1 (en) | Monoclonal antibody against estrogen stimulated leucine aminopeptidase | |
| CN105198986B (en) | Anti- avian leukosis virus p27 protein monoclonal antibodies, including the gold label test strip of the monoclonal antibody and application | |
| KR101782862B1 (en) | Monoclonal antibody for diagnosing MERS virus and immunochromatographic diagnostic kit | |
| CN109187967B (en) | Duplex rapid detection card for detecting and distinguishing O-type and A-type foot-and-mouth disease viruses and preparation method thereof | |
| CN108084255A (en) | A kind of preparation of canine recombinant c reactive protein and its monoclonal antibody | |
| CN111521816A (en) | Time-resolved fluorescence immunochromatographic assay test strip for echinococcosis granulosus of cattle and preparation method thereof | |
| CN103045541A (en) | Hybridoma cell strain and monoclonal antibody of anti-heart-type fatty acid-binding protein generated by same | |
| CN109970851A (en) | The preparation method of monoclonal antibody of CCV virus M protein and preparation method thereof, immunity colloidal gold test paper strip | |
| CN108780095A (en) | Utilize the cancer embryo fibronectin detection method of simple immunoassays | |
| CN113817063B (en) | Anti-human abnormal prothrombin antibody and application thereof | |
| CN107449912A (en) | Anti- H7N9 subtype avian influenza virus monoclonal antibody antigen epitope and its screening technique and application | |
| CN109852588A (en) | The monoclonal antibody and its cell strain of a kind of anti-Tilapia mossambica Immunoglobulin IgM and application | |
| US20030215894A1 (en) | Protein-induced monoclonal receptors to protein ligands | |
| CN109082413A (en) | Anti-human igg monoclonal antibody, its hybridoma cell strain and application | |
| CN108872580A (en) | A kind of colloidal gold strip and preparation method thereof detecting novel goose parvovirus | |
| CN103235127A (en) | Marek's disease virus rapid combined-detection test strip | |
| US6551789B1 (en) | Process for characterizing a biological sample using patterns of oncogene expression | |
| CN108254556A (en) | A kind of pertussis toxin detection kit and its application | |
| CN108152494A (en) | A kind of helicobacter pylori double antibody sandwich method detection kit and preparation method thereof | |
| CN106841606B (en) | Detect colloidal gold immuno-chromatography test paper strip, the kit and preparation method thereof of PCT | |
| KR20120028603A (en) | Antibody for detecting hemagglutinin of swine influenza and use thereof | |
| CN107607718B (en) | A kind of lefteye flounder Edwardsiella tarda disease quick diagnosis test paper and preparation method thereof | |
| CN101196525B (en) | Mycoplasma Gallisepticum immune body immune colloidal gold fast detecting reagent kit and its application | |
| CN105319361B (en) | Scylla paramamosain reovirus colloidal gold immunochromatographimethod reagent strip and preparation method thereof, application method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20181127 |
|
| RJ01 | Rejection of invention patent application after publication |