CN105974116A - Multi-linked rapid detection test strip for pig diseases and preparation method thereof - Google Patents
Multi-linked rapid detection test strip for pig diseases and preparation method thereof Download PDFInfo
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- CN105974116A CN105974116A CN201610544386.1A CN201610544386A CN105974116A CN 105974116 A CN105974116 A CN 105974116A CN 201610544386 A CN201610544386 A CN 201610544386A CN 105974116 A CN105974116 A CN 105974116A
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- G—PHYSICS
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- 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
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- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
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- G01N2333/085—Picornaviridae, e.g. coxsackie virus, echovirus, enterovirus
- G01N2333/09—Foot-and-mouth disease virus
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Abstract
The invention discloses a multi-linked rapid detection test strip for pig diseases and a preparation method thereof. The test strip adopts a double-antigen sandwiched method, takes staphylococcus A protein (SPA) as a capturing antigen and takes a yeast expressed main antigen region (rED) of swine fever, porcine blue-ear disease and O-shaped foot and mouth disease virus as a detection antigen. The test strip does need to be operated by professionals, any instrument does not need to be used, the detection time is short and the preparation is simple. When one blood serum sample is collected, antibodies of the swine fever, the porcine blue-ear disease and the -shaped foot and mouth disease virus can be detected at the same time. The requirements on diagnosis of important diseases by primary-level organizations and farmers can be met.
Description
Technical field
The present invention relates to a kind of animal epidemic detectable, be specifically related to one and can detect pig simultaneously
Pestilence, reproductive and respiratory syndrome and the immuno-chromatographic test paper strip of O type antibodies against foot-and-mouth disease virus, the present invention also relates to
And the preparation method of this test strips, belong to biological technical field.
Background technology
Swine fever (classical swine fever, CSF) is commonly called as rinderpest, is the one of infected pigs
Acute, hot, high degree in contact sexually transmitted disease.Swine fever can cause ill pig fever, anorexia, abdomen
Rush down, dead etc., the large quantities of death of Sow abortion, piglet can be caused.Porcine reproductive and respiratory syndrome
(porcine reproductive and respiratory syndrome, PRRS) is due to morbidity
There is ear cyanosis symptom in pig, also known as reproductive and respiratory syndrome.Reproductive and respiratory syndrome main infection brood sow and piglet,
Cause immunosuppressant, pig streptococcicosis is had fall out effect.Foot and mouth disease (foot and mouth
Disease, FMD) it is a kind of many hosts infectious disease, the artiodactyls such as pig, cattle, sheep is the easiest
Sense.Pig O type foot and mouth disease sickness rate is high, propagates rapidly, Sow abortion, piglet can be caused big
Criticize death.Swine fever, highly pathogenic PRRS and foot and mouth disease are the most serious to pig industry harm
Three kinds of swine diseasess, are classified as A class infectious disease by OIE (OIE) and legal declare
Animal epidemic, China is also classified as a class infectious disease.At present, China is for these three swine diseases
Taking the purification strategy that compulsory immunization combines with epidemic monitoring, vaccine immunity becomes affects epidemic disease
The key of prevention and control, and it is significant to set up suitable evaluation means for vaccine.
At present, vaccine immunity effect evaluation relies primarily on ELISA method.Have many dynamic both at home and abroad
Thing epidemic disease Serum Antibody Detection reagent, such as U.S. Ai Deshi (IDEXX) and Korea S's gold promise (JBT)
Swine fever block ELISA kit, the hog cholera indirect ELISA of China Veterinery Drug Inspection Office
Test kit.ELISA sensitivity is high, can Parallel testing multiple sample, but the method is time-consuming
Longer, indirect ELISA needs about 2h, and blocking ELISA needs 2～3h.Additionally, ELISA
Detection needs to use the instruments such as microplate reader, may be only available for having the mechanism that Experimental Hardware is supported,
Immunologic surveillance cannot be carried out under the conditions of clinical line etc. is simple and crude.Therefore, research and development antiviral antibody is fast
Speed detection means is significant with immunologic surveillance for swine diseases clinical diagnosis, and based on immunity
The colloidal gold strip detection technique of chromatographic theory is simple to operate, and the detection time is short, becomes important
Serological surveillance means.It is similar to the detection of patent CN103293306B gold colloidal at present
Card, but used escherichia coli expression antigen, and prokaryotic expression system lacks protein folding
Cofactor, conformational antigen epi-position can be affected and formed, and finally affect protein active.Bi Chi
Yeast expression system belongs to eukaryotic expression system, has complete Protein processing modified mechanism, and
Solubility expression can be realized, be the important tool of immunological investigation.
Swine fever virus (CSFV) is a kind of togavirus, and genome contains a big opening
Reading frame (ORF), encodes about 3898 amino acid whose polyproteins of one.This poly egg
During translation process, be processed into 12 kinds of ripe virus proteins by protease in vain, including C,
4 kinds of structural protein such as E0, E1 and E2, wherein envelope glycoprotein E2 is main protectiveness
Antigen protein.E2 albumen is started by ORF 5,690 amino acids of end, to 1060 ammonia
Base acid terminates, and contains 370 aminoacid altogether, and its 4 main antigen sites are respectively positioned on N
Between 690 and 866 amino acids of end.E2 albumen carries the protection that swine fever virus is main
Property antigenic determinant it is considered to be hog cholera genetic engineering bacterin and serodiagnosis goods research and development
Important target.Reproductive and respiratory syndrome virus (PRRSV) is a kind of tiny RNA togavirus, genome
Close 8 overlapping ORF, wherein ORF5-7 coding structure albumen, respectively GP5, M
And N protein.Wherein Nucleocapsid protein effect in terms of immunoprotection is little, but conservative
Property is good, expression is high, antigenicity is strong, is the desired target of reproductive and respiratory syndrome diagnostic detection.Foot and mouth disease
Virus (FMDV) is without cyst membrane structure, and capsid protein is by VP1, VP2, VP3 and VP4
Four kinds of structural protein compositions.Research shows, 5 neutralizations are at least contained on O type FMDV surface
Epitope, 3 epi-positions of most important of which concentrate on VP1 albumen.VP1 is protein induced
Neutralizing antibody produces, and is the important research object of FMD immunoprophylaxis, Genetic evolution.
Summary of the invention
Problem to be solved by this invention is to overcome the deficiencies in the prior art, it is provided that one can be simultaneously
Detection swine fever, reproductive and respiratory syndrome, the colloidal gold strip of O type antibodies against foot-and-mouth disease virus.This test strips
It is not required to professional's operation, is not required to by any instrument, detect the shortest and preparation is simple.This
Invention test strips utilize staphylococcal protein A (SPA) as capture antigen, can be in combination with
Multiple epidemic disease cause of disease IgG antibody.Additionally, test strips of the present invention is respectively with the pig of yeast expression
Pestilence, reproductive and respiratory syndrome, pig O type foot and mouth disease virus major antigen district (rED) are detection antigen,
Possesses specificity high, reproducible feature.
The technical problem to be solved is achieved through the following technical solutions:
A kind of multi-joint Rapid detection test strip of pig disease, test strips includes base plate, coated film, glass
Glass fibrous membrane, absorbent paper and sample pad.Wherein, the bottom surface of coated film is pasted onto on base plate, bag
The two ends in tunicle front sticking glass fibrous membrane and absorbent paper respectively.At glass fibre membrane away from suction
Sample pad is pasted in the front of water paper one end.Coated film is provided with detection line I, II, III and matter
Control line, detection line I, II, III are coated swine fever, reproductive and respiratory syndrome, pig O type hoof-and-mouth disease respectively
Poison rED albumen, nature controlling line is coated rabbit anti-pig IgG antibody.Spray on described glass fibre membrane
SPA protein-colloid gold grain production.
Described rED albumen expresses preparation, pig by yeast expression system GS115-pGAPZ α A
Pestivirus major antigen district (rED1) derives from raq gene, PRRS virus major antigen
District (rED2) derives from N gene, pig O type foot and mouth disease virus major antigen district (rED3)
Derive from VP1 gene.
Test strips of the present invention uses dual-antigen sandwich method, when containing swine fever, blue ear in test sample
During sick and pig O type antibodies against foot-and-mouth disease virus, then at sample pad with SPA colloid gold particle mark
Note conjugate combines, and forms " SPA conjugate-virus antibody complex " respectively, and in water suction
Swimming is permeated forward under the effect of paper.When complex arrive detection line I, antibody against swine fever virus with
RED1 albumen occurs specific binding, forms " SPA conjugate-antibody against swine fever virus
-rED1 complex ", coated film occurs macroscopic colour band.When complex arrives inspection
Survey line II, reproductive and respiratory syndrome virus antibody occurs specific binding with rED2 albumen, forms " SPA
Conjugate-reproductive and respiratory syndrome virus antibody-rED2 complex ", coated film occurs naked eyes are visible
Colour band.When complex arrives detection line III, O type antibodies against foot-and-mouth disease virus and rED3 egg
Poliosis is raw specific binding, and " SPA conjugate-antibodies against foot-and-mouth disease virus-rED3 is multiple in formation
Compound,, coated film occurs macroscopic colour band.When in test sample without swine fever,
When reproductive and respiratory syndrome, O type antibodies against foot-and-mouth disease virus, SPA colloid gold particle production swimming
To nature controlling line, pig IgG antibodies anti-with rabbit, coated film occurs macroscopic color
Band.During detection, draw blood serum sample to be checked 1, add well, observe detection line I,
Whether II, III and nature controlling line there is red ribbon, thus whether contain pig in judging animal body
Pestivirus, PRRS virus and pig O type antibodies against foot-and-mouth disease virus.
The positive effect of the present invention is:
1, test strips association colloid technology for gold of the present invention and film chromatographic technique, simple operation, be not required to
Wanting instrument, result interpretation is simple.Relative to ELISA, test strips supplementary material is the simplest,
Low cost, is of value to popularization and application.Gather a blood serum sample, swine fever, pig can be detected simultaneously
Reproductive and respiratory syndrome and pig O type antibodies against foot-and-mouth disease virus.Disclosure satisfy that grassroots organization, peasant household are for weight
The demand of big disease diagnosis.
2, the present invention uses yeast eukaryotic expression system to prepare the detection antigen egg in test strips
In vain.Yeast is eukaryotic expression host, and protein translation post-treatment modified mechanism is more perfect, expresses egg
White activity is high.
3, the present invention uses dual-antigen sandwich method, is not limited by the Hosts of sample, except detection
Outside swine fever, reproductive and respiratory syndrome and pig O type antibodies against foot-and-mouth disease virus, it may also be used for many animals O type
Foot and mouth disease virus detects.
Accompanying drawing explanation
Fig. 1 is the side structure schematic diagram of test strips of the present invention.In figure 1: base plate；2: be coated
Film；3: glass fibre membrane；4: absorbent paper；5: sample pad.
Fig. 2 is the Facad structure schematic diagram of Fig. 1.In figure 6: detection line I；7: detection line II；
8: detection line III；9: nature controlling line.
Fig. 3 is that swine fever virus major antigen district (rED1) Western blot identifies figure.
Fig. 4 is that reproductive and respiratory syndrome virus major antigen district (rED2) Western blot identifies figure.
Fig. 5 is that O type foot and mouth disease virus major antigen district (rED3) Western blot identifies figure.
Detailed description of the invention
The most specifically introduce the present invention the multi-joint Rapid detection test strip of pig disease and
Preparation method.It should be appreciated by those skilled in the art that the embodiments described below is intended merely to more
Understand the present invention well, be not intended to limit the present invention.
As it is shown in figure 1, include base plate according to the multi-joint Rapid detection test strip of pig disease of the present invention
1, coated film 2, glass fibre membrane 3, absorbent paper 4 and sample pad 5.Wherein, coated film 2
It is pasted onto above base plate 1, the two ends in coated film 2 front sticking glass fibrous membrane 3 respectively and suction
Water paper 4.Glass fibre membrane 3 away from one end of absorbent paper 4 front paste sample pad 5.
Base plate 1 usually PVC material, coated film 2 is nitrocellulose filter (NC film).At glass
Colloid gold label SPA albumen it is coated with on glass fibrous membrane 3.
As in figure 2 it is shown, be provided with detection line I6, detection line II7, detection line III on coated film
8 and nature controlling line 9, detection line I6 is coated swine fever virus major antigen district (rED1), detects line
II7 is coated reproductive and respiratory syndrome virus major antigen district (rED2), and detection line III8 is coated O type mouth
Aphtovirus major antigen district (rED3), nature controlling line 9 is coated rabbit anti-pig IgG antibody.Described
Detect line I6, detection line II7, detection line III8 and nature controlling line 9 on coated film 2 in 4
Bar line is arranged in parallel, according to detection line I6, detection line II7, detection line III8 and nature controlling line
Whether 9 there is red ribbon to carry out result judgement.
Main raw material(s) source in following embodiment is: swine fever virus major antigen district
(rED1), reproductive and respiratory syndrome virus major antigen district (rED2), O type foot and mouth disease virus mainly resist
Former district (rED3), antibody against swine fever virus positive serum, reproductive and respiratory syndrome virus Positive Sera,
Pig O type foot and mouth disease virus positive serum is that Zhengzhou Zhong Dao Bioisystech Co., Ltd prepares and right
Outer offer.Wherein 3 kinds of antigens are that Pichia sp. eukaryotic system is expressed, and 3 kinds of positive serums are
Commercialized vaccine is prepared by immune animal.Other reagent and material are all derived from commercial sources.Institute
State experimental technique, unless otherwise noted, be normal experiment method.
The concrete preparation method of the test strips of the present invention is as follows:
1.1 swine fevers, reproductive and respiratory syndrome, the preparation of O type foot and mouth disease virus major antigen district (rED)
According to the pathogen detected, retrieval ncbi database also selects corresponding with reference to gene:
Swine fever virus (AF531433.1), reproductive and respiratory syndrome virus (EU807840.1), O type foot and mouth disease
Virus (JQ973889).Design 3, to primer, expands swine fever, reproductive and respiratory syndrome and O type mouth respectively
Aphtovirus major antigen district gene:
(1) swine fever virus rED1:
P1:5 '-TTTGAATTCAGACTAGCCTGCAAGGAAGATTAC-3′；
P2:5 '-TATGCGGCCGCGTTGAAGTCGAAGCCACACC-3′；
(2) PRRS virus rED2:
P3:5 '-TTTGAATTCATGCCAAACAACAACGGTAA-3′；
P4:5 '-TTTGCGGCCGCAGCAGATGGGGAAGCAGTGA-3′；
(3) pig O type foot and mouth disease virus rED3:
P5:5 '-TTTGAATTCACCAGCACCGGTGAAAGCGC-3′；
P6:5 '-TTTGCGGCCGCCAGGCTCTGTTTCACCGGGG-3′。
According to Pichia sp. codon preference, genes of interest is carried out codon optimized, optimize
After gene order by the synthesis of Jin Wei intelligence biotech firm.Build recombinant yeast
PGAPZ α A-GS115, and in YPD culture medium, carry out the fermentation expression of 1L volume.Receive
Collection fermented liquid supernatant dialysed overnight, then carries out protein active analysis.Such as Fig. 3, Fig. 4 and Tu
Shown in 5, three kinds of viral major antigen districts have good antigen active, warp after yeast expression
Western blot identifies and can occur specific binding with corresponding positive serum.
The preparation of 1.2 coated films 2
Nitrocellulose filter sprays swine fever virus major antigen district (rED1), blue ear respectively
Virus major antigen district (rED2), O type foot and mouth disease virus major antigen district (rED3)
Pig IgG antibody anti-with rabbit, and arranged in parallel in 4 lines, packet composition detection line I6, inspection
Survey line II7, detection line III8 and nature controlling line 9.37 DEG C of drying and processing 2h, put equipped with being dried
The sealing bag of agent saves backup.
Detection line I6: debugging spray film instrument, spouting liquid is 1 μ L/cm, with being coated buffer dilution
Swine fever virus major antigen district (rED1), concentration is 2.5mg/mL, draws with spray film instrument spraying
Line.
Detection line II7: debugging spray film instrument, spouting liquid is 1 μ L/cm, with being coated buffer dilution
Reproductive and respiratory syndrome virus major antigen district (rED2), concentration is 3.0mg/mL, with spray film instrument spraying
Line.
Detection line II8: debugging spray film instrument, spouting liquid is 1 μ L/cm, with being coated buffer dilution
O type foot and mouth disease virus major antigen district (rED3), concentration is 3.0mg/mL, with spray film instrument
Spraying line.
Nature controlling line 9: debugging spray film instrument, spouting liquid is 1 μ L/cm, with being coated buffer dilution rabbit
Anti-pig IgG antibody, concentration is 1mg/mL, with spray film instrument spraying line.
The described buffer formulation that is coated: weigh bovine serum albumin 0.01g, 12 hypophosphite monohydrate hydrogen
Disodium 30.072g, potassium dihydrogen phosphate 2.176g, dissolve with ultra-pure water, and be settled to 1000mL.
Being drawn 4 lines on coated film 2 and answer fine uniform, adjacent line-to-line is every 0.4-1.0cm.
The preparation of 1.3 glass fibre membranes 3
(1) preparation of colloidal gold solution
Being placed on magnetic stirring apparatus by distilled water and be heated to boiling, being rapidly added concentration is 1%
Chlorauric acid solution is to final concentration of 0.02%.Boiling 5min, adding concentration afterwards is 1%
Citric acid three sodium solution, injection volume is 1.8 times of chlorauric acid solution.After boiling 10min,
Being cooled to room temperature, then be 0.02% by distilled water adjustment gold chloride concentration, room temperature keeps in Dark Place standby
With.
(2) preparation of colloid gold label SPA glass fibre membrane 3
Colloidal gold solution PH to 7.5-8.0 is regulated, by 60ug antibody/mL glue with 5M potassium carbonate
The ratio of body gold solution adds SPA albumen, stands 5min after mixing.Again by 5% volume
Amount adds 10% bovine serum albumin solution, stands 5min after mixing.10000r/min is centrifuged
30min, supernatant discarded, wash 1 time with labelling cleaning mixture.Precipitate with 1/10th initial glue
The gold mark albumen of body gold volume preserves liquid and dissolves, then according to every milliliter of solution paving 10cm2Amount
It is sprayed on glass fibre membrane 3.Vacuum drying 3h, puts and protects equipped with in the sealing bag of desiccant
Deposit standby.
Described 10% bovine serum albumin solution: take bovine serum albumin 100g, fixed with distilled water
Hold to 1000mL.
Described labelling cleaning mixture: take bovine serum albumin 4g, PEG 8000 2g, poly-second
Alkene pyrrolidone 2g, sucrose 2g, Tris 1.211g, be settled to 1000mL with distilled water, and
Regulation PH to 8.1.
Described gold mark albumen preserves liquid: take bovine serum albumin 25g, PEG 8000 5g,
Tris 10.2g, sodium hydroxide 2.5g, polysorbas20 7.5mL, be settled to 1000mL with distilled water,
And regulate PH to 8.5.
1.4 big plate group bars
Each component specification (long × wide): base plate 1 (30cm × 7.6cm), coated film 2
(30cm × 2.0cm), sample pad 3 (30cm × 3.0cm), coating colloid gold label SPA egg
White glass fibre membrane 3 (30cm × 0.7cm) and absorbent paper 4 (30cm × 3.2cm).
Each component is as follows by compound mode: be pasted onto by coated film 2 above base plate 1, coated film
The two ends in 2 fronts sticking glass fibrous membrane 3 and absorbent paper 4 respectively.Remote at glass fibre membrane 3
Sample pad 5 is pasted from the front of one end of absorbent paper 4.I.e. glue the most successively on base plate 1
Patch coated film 2, glass fibre membrane 3, absorbent paper 4, sample pad 5.
1.5 cutting
With cutting cutter, big plate being cut into wall scroll, every width is 3-5mm.
The ELISA test strip principle and the result that describe the present invention the most again in detail judge.
2.1 ELISA test strip principles
Test strips of the present invention uses dual-antigen sandwich method, when containing swine fever, blue ear in test sample
During sick and pig O type antibodies against foot-and-mouth disease virus, then at sample pad 5 with SPA colloid gold particle
Production combines, and forms " SPA conjugate-virus antibody complex " respectively, and is inhaling
Swimming is permeated forward under the effect of water paper 4.When complex arrives detection line I6, swine fever virus
Antibody occurs specific binding with rED1 albumen, and " SPA conjugate-swine fever virus resists in formation
Body-rED1 complex ", coated film 2 occurs macroscopic colour band.When complex arrives
Reaching detection line II7, reproductive and respiratory syndrome virus antibody occurs specific binding with rED2 albumen, is formed
" SPA conjugate-reproductive and respiratory syndrome virus antibody-rED2 complex ", occurs on coated film 2
Macroscopic colour band.When complex arrives detection line III8, O type antibodies against foot-and-mouth disease virus
Occur specific binding with rED3 albumen, form " SPA conjugate-antibodies against foot-and-mouth disease virus
-rED3 complex ", coated film 2 occurs macroscopic colour band.When SPA colloid
Gold grain production swimming, to nature controlling line 9, pig IgG antibodies anti-with rabbit, is being coated
Macroscopic colour band occurs on film.During detection, drawing blood serum sample to be checked 1, addition adds
Sample hole, observes detection line and whether nature controlling line red ribbon occurs, thus in judging animal body is
No containing swine fever virus, PRRS virus and pig O type antibodies against foot-and-mouth disease virus.
2.2 test strips results judge
Positive findings judges: when red stripes occurs in nature controlling line 9, detect line I6, II7, III8
When respectively red stripes occurring, represent swine fever, reproductive and respiratory syndrome and O type foot and mouth disease virus respectively anti-
Body is positive.
Negative findings judges: when red stripes occurs in nature controlling line 9, and detect line I6, II7,
When III8 redfree band occurs, show swine fever, reproductive and respiratory syndrome and O type antibodies against foot-and-mouth disease virus
Negative.
Null result: when nature controlling line 9 occurs without red stripes, testing result is invalid.
Claims (3)
1. the multi-joint Rapid detection test strip of pig disease, this test strips includes base plate, is coated
Film, glass fibre membrane, absorbent paper and sample pad；
Wherein, the bottom surface of coated film is pasted onto on base plate, and the two ends in coated film front are pasted respectively
Glass fibre membrane and absorbent paper；Glass fibre membrane away from absorbent paper one end front paste sample
Pad；Coated film is provided with detection line I, II, III and nature controlling line, detects line I, II, III
Being coated swine fever, reproductive and respiratory syndrome, pig O type foot and mouth disease virus rED albumen respectively, nature controlling line is coated
Rabbit anti-pig IgG antibody, glass fibre membrane sprays SPA protein-colloid gold grain labelling coupling
Thing.
The multi-joint Rapid detection test strip of pig disease the most according to claim 1, Qi Zhongjian
Survey line I, II, III and nature controlling line are in being arranged in parallel, and adjacent line-to-line is every 0.4-1.0cm.
3. a preparation method for the multi-joint Rapid detection test strip of pig disease, comprises the following steps:
(1) by swine fever, reproductive and respiratory syndrome, pig O type foot and mouth disease virus rED albumen, the anti-pig of rabbit
IgG antibody respectively be coated buffer dilution after formed concentration be the spraying of 1.0-3.0mg/mL
Liquid；With spray film instrument on coated film respectively spraying rule corresponding spray coating liquor, formed detection line I,
II, III and nature controlling line；
(2) distilled water is placed on magnetic stirring apparatus be heated to boiling, be rapidly added gold chloride
Solution；Boiling and add citric acid three sodium solution afterwards, injection volume is the 1.5-3 of chlorauric acid solution
Times；After boiling 10-30 minute, it is cooled to room temperature and forms colloidal gold solution；
(3) with potassium carbonate regulation colloidal gold solution pH value to 7.5-8.0, by 60ug antibody/mL
The ratio of colloidal gold solution adds SPA albumen, stands after mixing；Again by the amount of 5% volume
Add 10% bovine serum albumin solution, stand after mixing；Centrifugal supernatant discarded, uses labelling
Cleaning mixture washs；The precipitate gold mark albumen of 1/10th initial colloid gold solution volumes preserves
Liquid dissolves, then according to every milliliter of solution paving 10-15cm2Amount be sprayed on glass fibre membrane,
After vacuum drying standby；
(4) coated film made in step (1) is pasted onto substrate, then by step (3)
In the glass fibre membrane made be pasted onto one end of coated film, absorbent paper is pasted onto coated film
The other end, glass fibre membrane away from one end of absorbent paper front paste sample pad；
(5) with cutting cutter, the big plate made in step (4) being cut into width is 3-5mm's
Wall scroll, forms the multi-joint Rapid detection test strip of pig disease.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610544386.1A CN105974116A (en) | 2016-07-12 | 2016-07-12 | Multi-linked rapid detection test strip for pig diseases and preparation method thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610544386.1A CN105974116A (en) | 2016-07-12 | 2016-07-12 | Multi-linked rapid detection test strip for pig diseases and preparation method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107064488A (en) * | 2017-05-02 | 2017-08-18 | 中国农业科学院兰州兽医研究所 | A kind of total antibody solid phase of CSFV serum blocks the preparation method of antigen used in ELISA kit |
| CN107064502A (en) * | 2017-05-02 | 2017-08-18 | 中国农业科学院兰州兽医研究所 | A kind of multiple ELISA detection kit of pig virus infectious disease serum IgG antibody and its detection method |
| CN109187969A (en) * | 2018-09-19 | 2019-01-11 | 天康生物股份有限公司 | Detect immune chromatography test paper, kit and the purposes of antibody against swine fever virus |
| CN111351927A (en) * | 2020-03-17 | 2020-06-30 | 陈韬 | Antibody matrix detection method (MEGA method) aiming at pathogen antigen and multi-connected detection card |
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| CN109187969A (en) * | 2018-09-19 | 2019-01-11 | 天康生物股份有限公司 | Detect immune chromatography test paper, kit and the purposes of antibody against swine fever virus |
| CN111351927A (en) * | 2020-03-17 | 2020-06-30 | 陈韬 | Antibody matrix detection method (MEGA method) aiming at pathogen antigen and multi-connected detection card |
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