CN108663514A - A kind of toxoplasma nano-colloid carbon diagnosis test paper and preparation method - Google Patents
A kind of toxoplasma nano-colloid carbon diagnosis test paper and preparation method Download PDFInfo
- Publication number
- CN108663514A CN108663514A CN201810476893.5A CN201810476893A CN108663514A CN 108663514 A CN108663514 A CN 108663514A CN 201810476893 A CN201810476893 A CN 201810476893A CN 108663514 A CN108663514 A CN 108663514A
- Authority
- CN
- China
- Prior art keywords
- esa
- nano
- carbon
- toxoplasma
- colloid
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56905—Protozoa
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/44—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from protozoa
- G01N2333/45—Toxoplasma
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Urology & Nephrology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a kind of toxoplasma nano-colloid carbon diagnosis test paper and preparation method, which includes：Water absorption pad, nitrocellulose filter, nano-colloid carbon emissions pad, sample pad, PVC offset plates；The preparation method of the toxoplasma nano-colloid carbon diagnosis test paper includes：The preparation of toxoplasma antigen ESA, the preparation of rabbit-anti ESA serum, purifying rabbit-anti ESA antibody, the best pH value for determining nano-colloid carbon ESA solution and the dosage of ESA, the preparation of nano-colloid carbon mother liquor, the determination of nano-colloid carbon solution optimum diluting multiple, the assembling of test strips, the toxoplasmosis colloidal-carbon diagnosis test paper sensitivity and specificity that this research is established are preferable, with easy to operate, quick, test strip is clear, the advantages of greatly being reduced relative to traditional colloidal gold diagnosis test strips cost of manufacture, is suitable for the quick diagnosis of clinical toxoplasmosis.
Description
Technical field
The invention belongs to toxoplasmosis detection technique field, it is related to a kind of toxoplasma nano-colloid carbon diagnosis test paper and system
Preparation Method.
Background technology
Toxoplasma can infect almost all of warm-blooded animal, and great economic loss is brought to aquaculture, is infected simultaneously
The pregnant woman of toxoplasma can lead to miscarriage, fetal anomaly, birth defects or stillborn foetus.The serological diagnostic method of toxoplasmosis at present
Mainly there are improvement agglutination experiment (MAT), indirect hemagglutination experiment (IHA), indirect immunofluorescence experiment (IFA), Enzyme-linked Immunosorbent Assay
It tests (ELISA) etc., but there is these method some needs the technical staff of professional knowledge could grasp in laboratory conditions
It completes, it is time-consuming and laborious；Some costs of manufacture are higher to be unfavorable for being widely applied.Nano-colloid carbon overcomes drawbacks described above, can
With the mark substance effectively as quick diagnosis test strips, and cost is very low.Toxoplasma excretory-secretory antigen (ESA) is main
Including Microneme protein (MIC), studies on rhoptry proteins (ROP) and dense granule protein, have good immunogenicity and reaction former
Property.The relatively traditional gold mark colloid test strips inspection of the nano-colloid carbon test paper strip that is occurred as marker based on nano carbon particle
Survey background is relatively sharp, and the cost of nano carbon particle is a ten thousandth of gold mark particle, this greatly reduces test strips and grinds
The cost of system, the test strips developed currently based on the marker have been successfully applied to the detection of Schistosoma japonicum and plasmodium.
This research does chromogenic label using nano-sized carbon, and toxoplasma ESA is as detectable substance, it is intended to develop a kind of quick, sensitive arch
Parasitosis diagnosis test paper.
Invention content
To achieve the above object, a kind of toxoplasma nano-colloid carbon diagnosis test paper of present invention offer and preparation method, solution
It has determined problems of the prior art.
The technical solution adopted in the present invention is：A kind of toxoplasma nano-colloid carbon diagnosis test paper includes nature controlling line 1, inspection
Survey line 2, water absorption pad 3, nitrocellulose filter 4, nano-colloid carbon emissions pad 5, sample pad 6, PVC offset plates 7, which is characterized in that institute
It is that ESA IgG will be resisted to be sprayed on nitrocellulose filter 4 to be formed to state nature controlling line 1, and the detection line 2 is will to purify ESA to be sprayed on nitric acid fibre
The plain formation of film 4 of dimension, detection line 2 and nature controlling line 1 are arranged at the both sides of nitrocellulose filter 4, the top both ends of nitrocellulose filter 4
The top of respectively nano-colloid carbon emissions pad 5 and water absorption pad 3, nano-colloid carbon emissions pad 5 is sample pad 6, the sample pad
6, nano-colloid carbon emissions pad 5, nitrocellulose filter 4, water absorption pad 3 array from left to right and are pasted onto on PVC offset plates 7.
Preferably, the sample pad 6 is glass fibre element film.
Preferably, the nano-colloid carbon emissions pad 5 is the polyester film that colloidal carbon solution is handled well.
Preferably, the water absorption pad 3 and 4 joint of nitrocellulose filter are overlapped 1.0-1.5mm, nano-colloid carbon emissions pad
5 1.0-1.5mms Chong Die with 4 joint of nitrocellulose filter, the sample pad 6 are Chong Die with nano-colloid carbon emissions 5 joints of pad
1.0-1.5mm。
A kind of preparation method of toxoplasma nano-colloid carbon diagnosis test paper, which is characterized in that the toxoplasma nano-colloid
The preparation method of carbon diagnosis test paper specifically includes following steps：
Step 1: the preparation of toxoplasma antigen ESA：
The toxoplasma tachyzoite preserved from liquid nitrogen is put into 37 DEG C of water-baths and thaws, and after about 5min melts, uses
5mL sterile salines are added to the 1mL toxoplasma tachyzoites after melting and preserve in liquid, blow and beat 5000rpm/ after mixing repeatedly
Min centrifuges 10min, abandons supernatant after centrifugation, precipitation again plus the sterile saline of 5mL mix well after at a same speed from
The heart is washed 2~3 times, finally with the solution sluicing pipe bottom for being mixed with 1000U antibiotic, is noted by the dosage of every mouse 0.2mL repeatedly
Enter in mouse peritoneal, after 3 recovery worm strain activity of continuous passage and virulence, when mouse invasion, is rushed with 2mL sterile salines
Wash the abdominal cavity of infected mice, collect abdominal cavity washing lotion, 3000rpm/min centrifuges 30min, collect supernatant microscopy determine no tachyzoite and
Freeze after Peritoneal Cells of Mice in -20 DEG C of refrigerator overnights, next day, which takes, freezes the thawing of supernatant room temperature, and 3000rpm/min centrifugations 30min is removed
It goes from condensate, it is ESA antigens to collect supernatant；
Through2.0 fluorescent quantitation instruments measure toxoplasma antigen a concentration of 1.56mg/mL of ESA albumen, take 50 μ LESA
It is added after 10 μ L5X Loading buffer are mixed well and boils 5-10min in boiling water, sample is then added to polyacrylamide
Loading in the comb hole of amine gel carries out the ESA results that SDS-PAGE electrophoresis determines purifying；
Step 2: the preparation of rabbit-anti ESA serum：
Using toxoplasma ESA after purification as antigen, SPF new zealand white rabbits are immunized, the ESA antigens 0.5mL of purifying is taken to use
Normal saline dilution, be added isometric complete Freund's adjuvant it is fully emulsified after, noted in rabbit leg muscle, dorsal sc
Penetrate, be spaced 2 weeks after first time is immune again with the isometric incomplete Freund's adjuvant of 0.5mLESA antigens addition it is fully emulsified after
Family's rabbit back, leg is subcutaneous and muscle is injected, then is spaced 2 weeks and with second of identical method be immunized for the third time,
Its potency is surveyed in blood sampling, is not up to required, and directly takes injection after 0.5mLESA antigens normal saline dilution to carry out booster immunization, most
The 10th day method using Culling heart blood acquires blood after booster immunization afterwards, and the rabbit blood of acquisition is put in 37 DEG C of insulating boxs and is put
Supernatant is collected by centrifugation in 4000rpm/min after setting 1h；
Step 3: concentration mensuration and the SDS-PAGE detection of rabbit-anti ESA antibody purifications；
With2.0 fluorescent quantitation instruments measure purified antibodies albumen concentration, then pass through SDS-PAGE electrophoresis detections
Antibody protein after purification purifies situation；
Step 4: determining the best pH value of nano-colloid carbon ESA solution and the dosage of ESA：
Configuration pH is respectively 4,5,6,7,8,9,10 phosphate buffer solution, and the suspension that nano-sized carbon is made into 0.1% is added,
Add 0.1%Triton-100, Ultrasonic Cell Disruptor works 6S, stops 3S, and after ultrasonic 30min, 250rpm centrifuges 10min, and it is heavy to abandon
It forms sediment, after supernatant is stood for 24 hours, the pH value for not occurring precipitating is optimum pH；
After rabbit-anti ESA protein solutions are serially diluted, 0.2mL/20-100 μ g is taken to be added to 1mL0.1% colloidal-carbons
In solution, separately sets a pipe and be not added with ESA as blank control, 100 μ L sodium chloride solutions are added after 3h, are stood for 24 hours after mixing, it is unstable
Fixed colloidal carbon solution can precipitate, and the minimum ESA dosages that colloidal carbon solution is stablized can be made to add 10% as most suitable rabbit-anti again
ESA dosages；
Step 5: the preparation of nano-colloid carbon mother liquor；
It determines optimum mark pH, after rabbit-anti ESA dosages, rabbit-anti ESA carbon standard liquids is mixed well with turbula shaker, 10 DEG C
Shaking table shakes 3h, and colloidal-carbon is made to be come into full contact with rabbit-anti ESA, be added BSA make its final concentration of 1%, add 10% polyethylene glycol
(PEG 6000) to final concentration of 0.2%, 10 DEG C of shaking tables shake 3h, then will examination liquid in pipe with the speed of 13000rpm/min, 4
DEG C centrifugation 20min, abandons supernatant, precipitation is suspended with the phosphate buffer (PB) containing 0.4%BSA, 0.2%PEG, repeated centrifugation two
It is secondary, precipitation with the carbon mark dilution of original volume 1/10 (containing 1%BSA, 0.2%PEG, 3% sucrose, 0.5%triton-100,
It is mother liquor 0.5PVP) to suspend, and 0.02% Sodium azide anti-corrosion is added, it is spare to be put in 4 DEG C of refrigerators；
Step 6: the preparation of carbon mark ESA：
Polyester film and glass fibre element film are dried for standby after being impregnated with 0.05% Tween-20, take out the polyester handled well
Film immerses in the carbon mark ESA colloidal carbon solutions that marks, after 37 DEG C of drying boxes dryings taking-up be put in 4 DEG C it is spare；
Step 7: the determination of nano-colloid carbon solution optimum diluting multiple：
The colloidal-carbon mother liquor prepared is diluted 2 times, 6 times, 10 times, 14 times respectively, is coated with polyester film, group after 37 DEG C of drying
The extension rate of dress test strips, test strip and clear background is best mother liquor extension rate；
Step 8: the assembling of test strips：
Anti- ESA IgG and purifying ESA are sprayed on nitrocellulose filter (4) and form nature controlling line (1) and detection line (2), nitric acid
The top both ends of cellulose membrane (4) are respectively nano-colloid carbon emissions pad (5) and water absorption pad (3), nano-colloid carbon emissions pad (5)
Top be sample pad (6), all of above part is all pasted onto on PVC offset plates (7), cuts the ribbon into the small item of 0.5cm wide, in
4 DEG C are sealed；
Preferably, which detects animal blood serum sample, is detected and is tied with IHA kits
Fruit is standard, and the two positive and negative match-rate are respectively 80.0% (24/30) and 93.3% (42/45), test strip spirit
Sensitivity is 88.9%, accuracy rate 88%, and test strips specificity is good.
Preferably, the rabbit anteserum potency of acquisition is detected up to carrying out Culling heart blood when 8000.
Preferably, the PH of the nano-colloid carbon ESA solution most just when the optimum dose for 9, ESA be 66 μ g/mL.
Preferably, nano-colloid carbon mother liquor optimum diluting multiple is 10 times of dilutions.
A kind of application method of toxoplasma nano-colloid carbon diagnosis test paper is：Blood serum sample after dilution is added on sample
On pad, allows sample to fully absorb and incorporate in sample pad；Judgement is observed in 5-10min, detection line 2 does not develop the color, and nature controlling line 1 is aobvious
Color is considered as feminine gender；Detection line 2 and nature controlling line 1 develop the color, and are considered as the positive；Nature controlling line 1 does not develop the color, and no matter whether detection line 2 develops the color
All it is considered as that test paper is invalid, test paper is invalid, need to do again.
The beneficial effects of the invention are as follows：The present invention a kind of toxoplasma nano-colloid carbon diagnosis test paper this use nano-sized carbon
Chromogenic label is done, toxoplasma ESA is as detectable substance, susceptibility 1：64, have well specificity not with pork measles and rotation
Caterpillar positive serum reacts, and it is respectively 80.0% and 93.3% to detect positive and negative match-rate with IHA, and relative to tradition
Colloidal gold toxoplasma test strip cost of manufacture it is lower, relative to ELISA, MAT, IHA, the diagnostic methods such as PCR do not need
Professional person operates, interior in 5-10min to obtain testing result, has quick and convenient, the clear advantage of test strip, fits
For clinical detection application, it is thus determined that the toxoplasma colloidal-carbon diagnosis test paper that the present invention establishes applies valence with certain
Value.
Description of the drawings
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technology description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention for those of ordinary skill in the art without creative efforts, can be with
Obtain other attached drawings according to these attached drawings.
Fig. 1 is the structural schematic diagram of toxoplasma nano-colloid carbon diagnosis test paper of the present invention；
In figure：1, nature controlling line, 2, detection line, 3, water absorption pad, 4, nitrocellulose filter, 5, nano-colloid carbon emissions pad, 6,
Sample pad, 7, PVC offset plates；
Fig. 2 is IgG SDS-PAGE electrophoresis schematic diagrames of the present invention；
Fig. 3 is ESA SDS-PAGE electrophoresis schematic diagrames；
Fig. 4 is difference ESA dosage result schematic diagrams of the invention；
Fig. 5 makes various concentration colloidal carbon solution coating result schematic diagram of the present invention；
Fig. 6 is test strips sensibility schematic diagram of the present invention；
Fig. 7 is specific schematic diagram of the invention；
Fig. 8 is colloidal carbon test paper strip schematic diagram of the present invention；
Fig. 9 is colloidal carbon test paper strip sample detection result of the present invention
Specific implementation mode
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation describes, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
Below in conjunction with the accompanying drawings and specific embodiment is further described the application principle of the present invention.
Key instrument used in the present invention：Spray film instrument (BioDot companies),2.0 fluorescent quantitation instruments (the silent winged generation of match
Your company), general refrigerator BCD-2156KALM (Co., Ltd of Haier Group), -80 DEG C of 702 (Thermo of refrigerator
Scientific), high-pressure steam sterilizing pan HVE-50 (Japanese Hirayama companies), high speed tabletop centrifuge SIGMA1-14 are (beautiful
SIGMA companies of state), superclean bench SW-CJ-IFD (safe and sound company of Su Jing groups), gel imaging system (UVP companies of the U.S.),
Instantaneous mini centrifuge Mini-6K (Changsha Xiang Yi Co., Ltds), micropipette rifle (German Eppendorf companies), micro-wave oven
(Glanz Co., Ltd), electronic analytical balance BSA124S types (Sartorius AG), vortex mixer QL -901 (Qi
Unicorn medical apparatus factory), constant incubator and constant-temperature table (Beijing innovation is thought into Science and Technology Ltd.), Ultrasonic Cell Disruptor 250-F
(Ningbo Hai Shu Ke Sheng ultrasonic devices company), imark microplate reader (BIO-RAD companies of the U.S.).
Main material：German import nano carbon particle (SB4) is purchased from Gansu benefit people's livelihood object Science and Technology Development Co., Ltd., nitric acid
Cellulose membrane (NC films -140) is purchased from Sartorius companies, polyester film (VL98), glass fibre element film (SB06), blotting paper
(SX18), PVC offset plates (SM31-40) are purchased from Shanghai Jinbiao Bio-Tech Co., Ltd., and bag filter is purchased from Solarbio companies,
Protein A purification medias are purchased from Jin Sirui companies.
Main agents：Goat anti-rabbit igg-HRP, tmb substrate developing solution, Triton X-100, PVP-K30, PEG6000, sugarcane
Sugar is purchased from Solarbio companies, BSA, skimmed milk power, and Sodium azide is purchased from Shanghai Sheng Gong Co., Ltds, and Freund is completely and not exclusively
Adjuvant is purchased from Sigma companies, other reagents are domestic analysis net product, and toxoplasma ESA antigens are prepared by this laboratory.
Experimental animal：SPF grades of mouse 20 are served only for the preparation of toxoplasma ESA, and new zealand white rabbit 2 is served only for preparing anti-
The serum of ESA, two kinds of animals are purchased from Lanzhou veterinary institute Experimental Animal Center
The configuration of main agents
(1) configuration of albumen related solution
Separation gel buffer solution：Tris 36.3g add 1mol/L HCl 48mL, redistilled water 80mL are added to make it dissolve, and adjust
PH8.9 is settled to 100mL, 4 DEG C of preservations；
Concentrate glue buffer solution：Tris 5.98g add 1mol/L HCl 48mL, redistilled water 80mL are added to make it dissolve, and adjust
PH6.7 is settled to 100mL, 4 DEG C of preservations；
5XSDS-PAGE electrophoretic buffers：Tris 15.1g are taken, Glycine 94g, SDS 5g are in the burning of 1L deionized waters
It is sufficiently mixed in cup spare；
Coomassie brilliant blue staining liquid：It weighs in 1g coomassie brilliant blue R250s to 1L beakers, measures 250mL isopropanols and be added
State in beaker, add 100mL glacial acetic acid, be eventually adding 650mL deionized waters be sufficiently stirred it is spare；
Coomassie brilliant blue destainer:Acetic acid 100mL is added in 1L beakers, acetic acid 50mL, distilled water 850mL are sufficiently mixed
It is spare；
(2) configuration of ELSIA related solutions
Antigen coat liquid, that is, carbonate buffer solution (pH 9.6)：1.59g NaHCO3,2.93g NaHCO3 are weighed, is added and steams
It is spare to be settled to 1000mL after adjusting pH to 9.6 by distilled water 800mL；
Washing buffer PBST (pH 7.4)：Take KH2PO40.2g, Na2HPO412H2O 2.9g, NaCl 8g, KCl
Distilled water 800mL is added after 0.2g, 0.05%Tween-200.5mL, it is spare to be settled to 1000mL after tune pH to 7.4；
Terminate liquid：178mL distilled water is taken to be slowly added in the dense H2SO4 of 22mL.
Embodiment one,
With reference to figure 1-9, a kind of toxoplasma nano-colloid carbon diagnosis test paper include nature controlling line 1, detection line 2, water absorption pad 3,
Nitrocellulose filter 4, nano-colloid carbon emissions pad 5, sample pad 6, PVC offset plates 7, which is characterized in that the nature controlling line 1 is will to resist
ESA IgG are sprayed on the formation of nitrocellulose filter 4, and the detection line 2 is will purify ESA and be sprayed on nitrocellulose filter 4 to be formed, detection
Line 2 and nature controlling line 1 are arranged in the both sides of nitrocellulose filter 4, and the top both ends of nitrocellulose filter 4 are respectively nano-colloid carbon
Release pad 5 and water absorption pad 3, the 1.0-1.5mm Chong Die with 4 joint of nitrocellulose membrane of the water absorption pad 3, the nano-colloid carbon are released
The 1.0-1.5mm Chong Die with 4 joint of nitrocellulose membrane of pad 5 is put, the top of nano-colloid carbon emissions pad 5 is sample pad 6, described to receive
Rice glue body carbon emissions pad 5 is the polyester film that colloidal carbon solution is handled well, and the sample pad 6 is glass fibre element film, nano-colloid
The 1.0-1.5mm Chong Die with 6 joint of sample pad of carbon emissions pad 5, the sample pad 6, nano-colloid carbon emissions pad 5, cellulose nitrate
Plain film 4, water absorption pad 3 array from left to right and are pasted onto on PVC offset plates 7.
The application method of the present invention：Blood serum sample after dilution is added in sample pad 6, sample is allowed to fully absorb involvement sample
In product pad 6；Judgement is observed in 5-10min, detection line 2 does not develop the color, and nature controlling line 1 develops the color, and is considered as feminine gender；Detection line 2 and Quality Control
Line 1 develops the color, and is considered as the positive；Nature controlling line 1 does not develop the color, and no matter whether detection line 2, which develops the color, is all considered as that test paper is invalid, and test paper is invalid
, it need to do again.
Testing result judges：
It is positive：Serum after dilution is added in sample pad, observation judgement in 5-10min, if existed in tested serum anti-
The antibody of ESA, then serum in sample pad 6 first can be in conjunction with the carbon mark ESA on nano-colloid carbon emissions pad 5, when flowing through
The other end can make detection line 2 develop the color in conjunction with coated ESA in detection line 2 when detection line 2, and the serum being then added dropwise continues forward
Flowing, the anti-ESA IgG being coated on nature controlling line 1 can make nature controlling line 1 develop the color, be considered as the positive in conjunction with the carbon mark ESA in sample liquid；
It is negative：Anti- ESA antibody is not present if be detected in serum, will not develop the color when serum flows through detection line 2, and
Since a part of carbon mark ESA can be driven when serum flows through nano-colloid carbon emissions pad 5, so when the serum stream with carbon mark ESA
When through nature controlling line 1, the anti-ESA IgG on nature controlling line 1 still can be combined colour developing with carbon mark ESA, be considered as feminine gender；
In vain：It is treated as in vain as long as nature controlling line 1 does not develop the color.
A kind of preparation method of toxoplasma nano-colloid carbon diagnosis test paper of the present invention specifically includes following steps：
Step 1: the preparation of toxoplasma antigen ESA：
Why the toxoplasma tachyzoite preserved from liquid nitrogen is placed in liquid nitrogen, is the toxoplasma due to being preserved under room temperature
The activity of tachyzoite will receive influence, and the time preserved is not also grown, and is put into 37 DEG C of water-baths and thaws, is placed on 37 herein
It is since toxoplasma is the parasite of cytozoicus to thaw in DEG C water-bath, and the body temperature of people is 37 degree, so being taken from liquid nitrogen
It is to keep its vigor unaffected to go out the defrosting at 37 degree, and extremes of temperature can all make polypide be affected, and about 5min melts
After change, it is added to the 1mL toxoplasma tachyzoites after melting with 5mL sterile salines and preserves in liquid, after blowing and beating mixing repeatedly
5000rpm/min centrifuges 10min, and supernatant is abandoned after centrifugation, precipitates after adding the sterile saline of 5mL to mix well again with same
Speed centrifugation, wash repeatedly 2~3 times, finally with the solution sluicing pipe bottom of 1000U antibiotic is mixed with, by every mouse 0.2mL
Dosage injection mouse peritoneal in, after continuous passage 3 times restores worms strain activity and virulence, when mouse invasion, sterilized and given birth to 2mL
The abdominal cavity of normal saline washing infected mice is managed, abdominal cavity washing lotion is collected, 3000rpm/min centrifuges 30min, and collecting the determination of supernatant microscopy does not have
After tachyzoite and Peritoneal Cells of Mice freeze in -20 DEG C of refrigerator overnights, next day take freezes supernatant room temperature thawing, 3000rpm/min from
Heart 30min is removed from condensate, and it is ESA antigens to collect supernatant；
Through2.0 fluorescent quantitation instruments measure toxoplasma antigen a concentration of 1.56mg/mL of ESA albumen, take 50 μ LESA
It is added after 10 μ L5X Loading buffer are mixed well and boils 5-10min in boiling water, sample is then added to polyacrylamide
Loading in the comb hole of amine gel carries out SDS-PAGE electrophoresis；
Step 2: the preparation of rabbit-anti ESA serum：
Using toxoplasma ESA after purification as antigen, SPF new zealand white rabbits are immunized, the ESA antigens 0.5mL of purifying is taken to use
Normal saline dilution, be added isometric complete Freund's adjuvant it is fully emulsified after noted in rabbit leg muscle, dorsal sc
It penetrates, is spaced 2 weeks after adding isometric incomplete Freund's adjuvant fully emulsified with 0.5mLESA antigens again and is in after first time is immune
Rabbit back, leg is subcutaneous and muscle is injected, then is spaced 2 weeks and with second of identical method be immunized for the third time, adopts
Blood surveys its potency, does not reach requirement, directly takes injection after 0.5mLESA antigens normal saline dilution to carry out booster immunization, most
The 10th day method using Culling heart blood acquires blood after booster immunization afterwards, and the rabbit blood of acquisition is put in 37 DEG C of insulating boxs and is put
Supernatant is collected by centrifugation in 4000rpm/min after setting 1h；
Step 3: concentration mensuration and the SDS-PAGE detection of rabbit-anti ESA antibody purifications；
(1) blood serum sample is according in the flow velocity loading to chromatographic column of about 1mL/min；
(2) the equilibration buffer solution resin of 30mL, flow velocity is used to maintain about 2mL/min；
(3) it uses 10-15mL elution buffer antibody elutions, flow velocity to maintain about 1mL/min, collects eluent, and add at once
Enter the neutralization buffer of 1/10 elution buffer volume, adjusts pH to 7.4；
(4) eluent 4000rpm/min centrifugations 40min after purification collects precipitation, with 10mL PBS dilution precipitations, is packed into
In bag filter, for 24 hours with 10 times of volume PBS dialysis desalinations, liquid is changed for several times in centre；
With2.0 fluorescent quantitation instruments measure purified antibodies albumen concentration.Then pass through SDS-PAGE electrophoresis detections
Antibody protein after purification purifies situation；
Step 4: determining the best pH value of nano-colloid carbon ESA solution and the dosage of ESA：
Configuration pH is respectively 4,5,6,7,8,9,10 phosphate buffer solution, and the suspension that nano-sized carbon is made into 0.1% is added,
Add 0.1%Triton-100, Ultrasonic Cell Disruptor works 6S, stops 3S, and after ultrasonic 30min, 250rpm centrifuges 10min, and it is heavy to abandon
It forms sediment, after supernatant is stood for 24 hours, the pH value for not occurring precipitating is optimum pH；
After rabbit-anti ESA protein solutions are serially diluted, 0.2mL (20-100 μ g) is taken to be added to 1mL0.1% colloidal-carbons
In solution, separately sets a pipe and be not added with ESA as blank control, 100 μ L sodium chloride solutions are added after 3h, are stood for 24 hours after mixing, it is unstable
Fixed colloidal carbon solution can precipitate, and the minimum ESA dosages that colloidal carbon solution is stablized can be made to add 10% as most suitable rabbit-anti again
ESA dosages；
Step 5: the preparation of nano-colloid carbon mother liquor；
It determines optimum mark pH, after rabbit-anti ESA dosages, rabbit-anti ESA carbon standard liquids is mixed well with turbula shaker, 10 DEG C
Shaking table shakes 3h, and colloidal-carbon is made to be come into full contact with rabbit-anti ESA, be added rabbit-anti BSA make its final concentration of 1%, add 10% poly- second
For glycol (PEG 6000) to final concentration of 0.2%, 10 DEG C of shaking tables shake 3h, then by examination liquid in pipe with the speed of 13000rpm/min
Degree, 4 DEG C of centrifugation 20min, abandons supernatant, and precipitation is suspended with the phosphate buffer (PB) containing 0.4%BSA, 0.2%PEG, repeat from
Twice, precipitation is with the carbon mark dilution of original volume 1/10 (containing 1%BSA, 0.2%PEG, 3% sucrose, 0.5%triton- for the heart
100,0.5PVP) suspend be mother liquor, be added 0.02% Sodium azide anti-corrosion, it is spare to be put in 4 DEG C of refrigerators；
Step 6: the processing of carbon mark ESA coating polyester films and glass fibre element film：
Polyester film and glass fibre element film are dried for standby after being impregnated with 0.05% Tween-20, take out the polyester handled well
Film immerses in the carbon mark ESA colloidal carbon solutions that marks, after 37 DEG C of drying boxes dryings taking-up be put in 4 DEG C it is spare；
Step 7: the determination of nano-colloid carbon solution optimum diluting multiple：
The colloidal-carbon mother liquor prepared is diluted into (2 times, 6 times, 10 times, 14 times) coating polyester films of different multiples respectively,
Test strips are assembled after 37 DEG C of drying, and the extension rate of test strip and clear background is best mother liquor extension rate；
Step 8: the assembling of test strips：
Anti- ESA IgG and purifying ESA are sprayed on nitrocellulose filter (4) and form nature controlling line (1) and detection line (2), nitric acid
The upper end of cellulose membrane (4) is respectively nano-colloid carbon emissions pad (5) and water absorption pad (3), nano-colloid carbon emissions pad (5)
Top be sample pad (6), all of above part is all pasted onto on PVC offset plates, the small item of 0.5cm wide is cut the ribbon into, in 4 DEG C
It is sealed；
The toxoplasma nano-colloid carbon diagnosis test paper detects animal blood serum sample, is mark with IHA kit testing results
Standard, the two positive and negative match-rate are respectively 80.0% (24/30) and 93.3% (42/45), calculate the detection for understanding to prepare
Test strips sensitivity is 88.9%, and accuracy rate 88% does not have with the ELISA test strip pork measles and trichina positive serum of preparation
It detects, shows to prepare test strips specificity well.
Preferably, it passes through2.0 fluorescent quantitation instruments measure a concentration of 1.56mg/mL of ESA albumen.
Preferably, the rabbit anteserum potency of acquisition is detected up to carrying out Culling heart blood when 8000.
Preferably, it passes through2.0 fluorescent quantitation instruments measure a concentration of 3.55mg/ of rabbit-anti ESA antibody proteins after purification
mL。
Preferably, the PH of nano-colloid carbon ESA solution is most just when being 9.
Preferably, the optimum dose of ESA is 66 μ g/mL.
Preferably, nano-colloid carbon mother liquor optimum diluting multiple is 10 times of dilutions.
Embodiment two,
The toxoplasma antigen ESA determination of protein concentration and SDS-PAGE of preparation are analyzed：
Through2.0 fluorescent quantitation instruments measure ESA albumen concentration, take 50 μ LESA that 10 μ L5X Loading are added
Buffer boils 5-10min in boiling water after mixing well, then sample is added in the comb hole of polyacrylamide gel
Sample carries out SDS-PAGE electrophoresis, after electrophoresis, polyacrylamide gel is immersed in coomassie brilliant blue staining liquid, level is shaken
After bed dyeing 40min, dyeing liquor is discarded, destainer is changed and carries out elution until clear background.
As the toxoplasma ESA antigens of Fig. 2, preparation pass throughIt is a concentration of that 2.0 fluorescent quantitation instruments measure its albumen
1.56mg/mL。
Embodiment three,
Indirect ELISA detects rabbit-anti ESA serum titers：
Coating：Toxoplasma ESA antigens are diluted with coating buffer, are added in 96 hole elisa Plates, per 100 μ L of hole, 4 DEG C of refrigerators are put
It sets overnight.
Closing：Next day clappers drying ELISA Plate liquid, is then washed 3 times with PBST, stands 5min every time.It is last to add per hole
Enter 100 μ L skimmed milk powers, 2h is incubated in 37 DEG C of insulating boxs.
Sample-adding：After PBST board-washings 3 times, the serum diluted with PBST is added, per 100 μ L of hole, is incubated in 37 DEG C of insulating boxs
Educate 2h.
ELIAS secondary antibody is added：After PBST board-washings 3 times, it is added 1:The goat anti-rabbit igg of the HRP labels of 3000 dilution proportions is anti-
Body, per 100 μ L of hole, 37 DEG C of insulating boxs are incubated 1h.
Substrate is added：100 μ LTMB developing solutions, 37 DEG C of incubation 15-30min are added after PBS board-washings 3 times per hole.
Terminate liquid is added：100 μ L concentrated sulfuric acid terminate liquids are added per hole.
Survey OD values：Microplate reader measures OD450nm values.
Comparative test result is shown：The rabbit anteserum potency of indirect ELISA detection acquisition is up to adopted to progress heart when 8000
Blood.
Example IV,
The concentration mensuration and SDS-PAGE of rabbit-anti ESA antibody purifications detect：
With2.0 fluorescent quantitation instruments measure purified antibodies albumen concentration.Then pass through SDS-PAGE electrophoresis detections
Antibody protein after purification purifies situation.
Such as Fig. 3, rabbit-anti ESA IgG antibodies warp after purificationIt is a concentration of that 2.0 fluorescent quantitation instruments measure its albumen
3.55mg/mL。
Embodiment five,
The condition optimizing of nano-colloid carbon mark ESA solution Optimal pH and ESA dosages：
Configuration pH is respectively 4,5,6,7,8,9,10 phosphate buffer solution, and the suspension that nano-sized carbon is made into 0.1% is added,
Add 0.1%Triton-100, Ultrasonic Cell Disruptor works 6S, stops 3S, and after ultrasonic 30min, 250rpm centrifuges 10min, and it is heavy to abandon
It forms sediment, after supernatant is stood for 24 hours, the pH value for not occurring precipitating is optimum pH.
Comparative test result is shown：PH value is respectively discovery pH after 4,5,6,7,8,9,10 nano-sized carbon solution left standstill 24
9 nano-sized carbon phosphate buffer does not precipitate, thus determine pH 9 be most just when.
After ESA protein solutions are serially diluted, 0.2mL (20-100 μ g) is taken to be added to 1mL0.1% colloidal carbon solutions
In, it separately sets a pipe and is not added with ESA as blank control, 100 μ L sodium chloride solutions are added after 3h, are stood for 24 hours after mixing, it is unstable
Colloidal carbon solution can precipitate, and the minimum ESA dosages that colloidal carbon solution is stablized can be made to add 10% as most suitable ESA dosages again.
If Fig. 4 is when being added the ESA albumen more than 60 μ g in the nanometer carbon solution of 1mL0.1%, 100 μ L chlorinations are added
Sodium solution does not precipitate, therefore determines that ESA albumen optimum doses are 66 μ g/mL.
Embodiment six,
The determination of nano-colloid carbon solution optimum diluting multiple
The colloidal-carbon mother liquor prepared is diluted into (2 times, 6 times, 10 times, 14 times) coating polyester films of different multiples respectively,
Test strips are assembled after 37 DEG C of drying, and the extension rate of test strip and clear background is best mother liquor extension rate.
There is Fig. 5 it is found that test strip and background are more clear when mother liquor dilutes 10 times, therefore select 10 times to be diluted to mother liquor most
Good extension rate.
Embodiment seven,
Coating protein determines optimum detection line and nature controlling line spray film concentration on NC films
The determination of ELISA test strip line (2) most preferably spray film concentration：Purified toxoplasma ESA protein solutions are divided with PBS
It is not diluted to the concentration of 1.4,1.2,1.0,0.8,0.6,0.4mg/mL, is sprayed on NC films and is made with the speed of 1 μ L/cm with spray film instrument
For T lines, film concentration is most preferably sprayed according to T lines definition judgment.
The determination of test strips nature controlling line (1) most preferably spray film concentration：IgG antibody protein solution after purification is diluted with PBS
For the concentration of 3.0,2.5,2.0,1.5,1.0,0.5mg/mL, the speed of spray film instrument using 1 μ L/cm is used to be sprayed on NC films as C lines,
Best spray film concentration is determined according to C line clarity.
Comparative test result is shown：When detection line (2) and nature controlling line (1) concentration are respectively 0.8mg/mL and 2.0mg/mL
Test strip is more clear.
Embodiment eight,
The judgement of sensibility and specificity
By toxoplasma positive control serum (1:8,1:16,1:32,1:64,1:128,1:256,1:512) doubling dilution is surveyed
Its sensibility is tried, test strips specificity is determined as a contrast with 1 part of pork measles positive serum and 1 part of Trichinella sui positive serum.
Such as Fig. 6,7：Serum is diluted to 1：Result shows and still can detect at 64 times, shows to prepare test strips sensibility
Preferably.It is not detected with ELISA test strip pork measles and trichina positive serum, shows to prepare test strips specificity well.
Embodiment nine,
Repeatability and stability test
It randomly selects the test strips produced between 3 batches batches to be detected, determines test strips repeatability.4 DEG C of dryings are sealed
Test strips 2 weeks 1 month, determine detection stability for 2 months.
Comparative test result is shown：The test strips produced between 3 batches randomly selected batch, yin and yang attribute testing result are all normal.
Detection is without significant change after 4 DEG C of dryings of test strips are sealed 2 weeks, 1 month, 2 months, it is thus determined that test strips can be protected with 4 DEG C
It deposits 2 months
Embodiment ten,
The detection of sample
With the pig toxoplasma positive serum and 45 parts of pig toxoplasma feminine gender blood of 30 parts of IHA detection determinations that this laboratory preserves
It is clear to determine ELISA test strip effect.
The comparison of table 3-1 colloidal carbon test paper strips and IHA testing results
With reference to figure 8,9：The positive of the test strips and IHA kits of preparation and negative match-rate are respectively known to table 3-1
80.0% (24/30) and 93.3% (42/45).
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the scope of the present invention.It is all
Any modification, equivalent replacement, improvement and so within the spirit and principles in the present invention, are all contained in protection scope of the present invention
It is interior.
Claims (9)
1. a kind of toxoplasma nano-colloid carbon diagnosis test paper includes nature controlling line (1), detection line (2), water absorption pad (3), nitric acid fibre
The plain film (4) of dimension, nano-colloid carbon emissions pad (5), sample pad (6), PVC offset plates (7), which is characterized in that the nature controlling line (1) is
Anti- ESA IgG are sprayed on nitrocellulose filter (4) formation, the detection line (2) is will to purify ESA to be sprayed on nitrocellulose filter
(4) it is formed, detection line (2) and nature controlling line (1) are arranged in the both sides of nitrocellulose filter (4), the top of nitrocellulose filter (4)
Both ends are respectively nano-colloid carbon emissions pad (5) and water absorption pad (3), and the top of nano-colloid carbon emissions pad (5) is sample pad
(6), the sample pad (6), nano-colloid carbon emissions pad (5), nitrocellulose filter (4), water absorption pad (3) are arranged successively from left to right
It arranges and is pasted onto on PVC offset plates (7).
2. a kind of toxoplasma nano-colloid carbon diagnosis test paper according to claim 1, which is characterized in that the sample pad
(6) it is glass fibre element film.
3. a kind of toxoplasma nano-colloid carbon diagnosis test paper according to claim 1, which is characterized in that the nanometre glue
Body carbon emissions pad (5) is the polyester film that colloidal carbon solution is handled well.
4. a kind of toxoplasma nano-colloid carbon diagnosis test paper according to claim 1, which is characterized in that the water absorption pad
(3) and nitrocellulose filter (4) joint is overlapped 1.0-1.5mm, nano-colloid carbon emissions pad (5) and nitrocellulose filter (4)
Joint is overlapped 1.0-1.5mm, sample pad (6) 1.0-1.5mm Chong Die with nano-colloid carbon emissions pad (5) joint.
5. a kind of preparation method of toxoplasma nano-colloid carbon diagnosis test paper as described in claim 1, which is characterized in that should
The preparation method of toxoplasma nano-colloid carbon diagnosis test paper specifically includes following steps：
Step 1: the preparation of toxoplasma antigen ESA：
The toxoplasma tachyzoite preserved from liquid nitrogen is put into 37 DEG C of water-baths and thaws, and after about 5min melts, is gone out with 5mL
Bacterium physiological saline is added to the 1mL toxoplasma tachyzoites after melting and preserves in liquid, and 5000rpm/min is centrifuged after blowing and beating mixing repeatedly
10min abandons supernatant after centrifugation, precipitation centrifuges at a same speed after adding the sterile saline of 5mL to mix well again, repeatedly
Washing 2~3 times injects mouse finally with the solution sluicing pipe bottom for being mixed with 1000U antibiotic by the dosage of every mouse 0.2mL
In abdominal cavity, after 3 recovery worm strain activity of continuous passage and virulence, when mouse invasion, infection is rinsed with 2mL sterile salines
Abdominal cavity washing lotion is collected in the abdominal cavity of mouse, and 3000rpm/min centrifuges 30min, is collected supernatant microscopy and is determined no tachyzoite and mouse abdomen
Freeze after chamber cell in -20 DEG C of refrigerator overnights, next day, which takes, freezes the thawing of supernatant room temperature, and 3000rpm/min centrifuges 30min and removes self-solidifying
Object, it is ESA antigens to collect supernatant；
Through2.0 fluorescent quantitation instruments measure toxoplasma antigen a concentration of 1.56mg/mL of ESA albumen, and 50 μ LESA is taken to be added
10 μ L5X Loading buffer boil 5-10min after mixing well in boiling water, and sample, which is then added to polyacrylamide, coagulates
Loading in the comb hole of glue carries out the ESA results that SDS-PAGE electrophoresis determines purifying；
Step 2: the preparation of rabbit-anti ESA serum：
Using toxoplasma ESA after purification as antigen, SPF new zealand white rabbits are immunized, take the ESA antigen 0.5mL physiology of purifying
Brine dilute, be added isometric complete Freund's adjuvant it is fully emulsified after, injected in rabbit leg muscle, dorsal sc,
Be spaced 2 weeks after first time is immune again with the isometric incomplete Freund's adjuvant of 0.5mLESA antigens addition it is fully emulsified after in rabbit
Back, leg is subcutaneous and muscle is injected, then is spaced 2 weeks and with second of identical method be immunized for the third time, blood sampling
Its potency is surveyed, is not up to required, directly takes injection after 0.5mLESA antigens normal saline dilution to carry out booster immunization, finally exists
The 10th day method using Culling heart blood acquires blood after booster immunization, and the rabbit blood of acquisition is put in 37 DEG C of insulating boxs and places 1h
Supernatant is collected by centrifugation in 4000rpm/min afterwards；
Step 3: concentration mensuration and the SDS-PAGE detection of rabbit-anti ESA antibody purifications；
With2.0 fluorescent quantitation instruments measure purified antibodies albumen concentration, are then purified by SDS-PAGE electrophoresis detections
Antibody protein afterwards purifies situation；
Step 4: determining the best pH value of nano-colloid carbon ESA solution and the dosage of ESA：
Configuration pH is respectively 4,5,6,7,8,9,10 phosphate buffer solution, the suspension that nano-sized carbon is made into 0.1% is added, then add
Entering 0.1%Triton-100, Ultrasonic Cell Disruptor works 6S, stops 3S, and after ultrasonic 30min, 250rpm centrifuges 10min, abandons precipitation, on
After quietness is set for 24 hours, the pH value for not occurring precipitating is optimum pH；
After rabbit-anti ESA protein solutions are serially diluted, 0.2mL/20-100 μ g is taken to be added to 1mL0.1% colloidal carbon solutions
In, it separately sets a pipe and is not added with ESA as blank control, 100 μ L sodium chloride solutions are added after 3h, are stood for 24 hours after mixing, it is unstable
Colloidal carbon solution can precipitate, can make colloidal carbon solution stablize minimum ESA dosages add 10% as most suitable rabbit-anti ESA again
Dosage；
Step 5: the preparation of nano-colloid carbon mother liquor；
It determines optimum mark pH, after rabbit-anti ESA dosages, rabbit-anti ESA carbon standard liquids, 10 DEG C of shaking tables is mixed well with turbula shaker
Shake 3h, colloidal-carbon made to be come into full contact with rabbit-anti ESA, be added BSA make its final concentration of 1%, add 10% polyethylene glycol (PEG
6000) to final concentration of 0.2%, 10 DEG C of shaking tables shake 3h, then will examination liquid in pipe with the speed of 13000rpm/min, 4 DEG C from
Heart 20min, abandons supernatant, and precipitation is suspended with the phosphate buffer (PB) containing 0.4%BSA, 0.2%PEG, repeated centrifugation twice,
Precipitation with the carbon mark dilution of original volume 1/10 (containing 1%BSA, 0.2%PEG, 3% sucrose, 0.5%triton-100,
It is mother liquor 0.5PVP) to suspend, and 0.02% Sodium azide anti-corrosion is added, it is spare to be put in 4 DEG C of refrigerators；
Step 6: the preparation of carbon mark ESA：
Polyester film and glass fibre element film are dried for standby after being impregnated with 0.05% Tween-20, take out the polyester film leaching handled well
Enter in the carbon mark ESA colloidal carbon solutions marked, taken out after 37 DEG C of drying boxes are dried be put in 4 DEG C it is spare；
Step 7: the determination of nano-colloid carbon solution optimum diluting multiple：
The colloidal-carbon mother liquor prepared is diluted 2 times, 6 times, 10 times, 14 times respectively, polyester film is coated with, examination is assembled after 37 DEG C of drying
The extension rate of paper slip, test strip and clear background is best mother liquor extension rate；
Step 8: the assembling of test strips：
Anti- ESA IgG and purifying ESA are sprayed on nitrocellulose filter (4) and form nature controlling line (1) and detection line (2), cellulose nitrate
The top both ends of plain film (4) are respectively nano-colloid carbon emissions pad (5) and water absorption pad (3), nano-colloid carbon emissions pad (5) it is upper
Side is sample pad (6), and all of above part is all pasted onto on PVC offset plates (7), the small item of 0.5cm wide is cut the ribbon into, in 4 DEG C
It is sealed.
6. a kind of preparation method of toxoplasma nano-colloid carbon diagnosis test paper according to claim 5, which is characterized in that
The toxoplasma nano-colloid carbon diagnosis test paper detects animal blood serum sample, using IHA kits testing result as standard, the two sun
Property and negative match-rate be respectively 80.0% (24/30) and 93.3% (42/45), test strip sensitivity is 88.9%, accurate
True rate is 88%, and test strips specificity is good.
7. a kind of preparation method of toxoplasma nano-colloid carbon diagnosis test paper according to claim 5, which is characterized in that
The rabbit anteserum potency of detection acquisition is up to carrying out Culling heart blood when 8000.
8. a kind of preparation method of toxoplasma nano-colloid carbon diagnosis test paper according to claim 5, which is characterized in that
The PH of the nano-colloid carbon ESA solution most just when the optimum dose for 9, ESA be 66 μ g/mL.
9. a kind of preparation method of toxoplasma nano-colloid carbon diagnosis test paper according to claim 5, which is characterized in that
Nano-colloid carbon mother liquor optimum diluting multiple is 10 times of dilutions.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810476893.5A CN108663514A (en) | 2018-05-18 | 2018-05-18 | A kind of toxoplasma nano-colloid carbon diagnosis test paper and preparation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810476893.5A CN108663514A (en) | 2018-05-18 | 2018-05-18 | A kind of toxoplasma nano-colloid carbon diagnosis test paper and preparation method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108663514A true CN108663514A (en) | 2018-10-16 |
Family
ID=63776585
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810476893.5A Pending CN108663514A (en) | 2018-05-18 | 2018-05-18 | A kind of toxoplasma nano-colloid carbon diagnosis test paper and preparation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108663514A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110907638A (en) * | 2020-02-14 | 2020-03-24 | 北京纳百生物科技有限公司 | Antibody dispersant for immune colloidal gold homogeneous phase labeling and application thereof |
| CN112305216A (en) * | 2020-10-26 | 2021-02-02 | 杭州电子科技大学 | Carbon nano-immune label chromatography test strip and method for detecting antibiotics in animal hair |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002001229A1 (en) * | 2000-06-26 | 2002-01-03 | Beckman Coulter, Inc. | Opposable-element chromatographic assay device for detection of analytes in whole blood samples |
| CN101169413A (en) * | 2006-10-29 | 2008-04-30 | 中国农业科学院兰州兽医研究所 | Toxoplasma immune-gold labeled quick detection reagent and its preparation method |
| CN101393216A (en) * | 2008-10-23 | 2009-03-25 | 车宏莉 | Colloidal carbon test paper strip for rapid diagnosis of schistosomiasis japanica |
| CN102435744A (en) * | 2011-09-19 | 2012-05-02 | 厦门大学附属中山医院 | Colloidal gold immunochromatography assay reagent strip for total antibodies to Toxoplasma gondii and preparation method thereof |
| CN105759030A (en) * | 2016-03-15 | 2016-07-13 | 中国农业大学 | Colloidal gold test strip for testing toxoplasma gondii antibodies |
-
2018
- 2018-05-18 CN CN201810476893.5A patent/CN108663514A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002001229A1 (en) * | 2000-06-26 | 2002-01-03 | Beckman Coulter, Inc. | Opposable-element chromatographic assay device for detection of analytes in whole blood samples |
| CN101169413A (en) * | 2006-10-29 | 2008-04-30 | 中国农业科学院兰州兽医研究所 | Toxoplasma immune-gold labeled quick detection reagent and its preparation method |
| CN101393216A (en) * | 2008-10-23 | 2009-03-25 | 车宏莉 | Colloidal carbon test paper strip for rapid diagnosis of schistosomiasis japanica |
| CN102435744A (en) * | 2011-09-19 | 2012-05-02 | 厦门大学附属中山医院 | Colloidal gold immunochromatography assay reagent strip for total antibodies to Toxoplasma gondii and preparation method thereof |
| CN105759030A (en) * | 2016-03-15 | 2016-07-13 | 中国农业大学 | Colloidal gold test strip for testing toxoplasma gondii antibodies |
Non-Patent Citations (1)
| Title |
|---|
| 张改平: "《免疫层析试纸快速检测技术》", 31 August 2015 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110907638A (en) * | 2020-02-14 | 2020-03-24 | 北京纳百生物科技有限公司 | Antibody dispersant for immune colloidal gold homogeneous phase labeling and application thereof |
| CN112305216A (en) * | 2020-10-26 | 2021-02-02 | 杭州电子科技大学 | Carbon nano-immune label chromatography test strip and method for detecting antibiotics in animal hair |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105527437B (en) | A kind of detection kit and its application | |
| CN102621329B (en) | Bird's-nest enzyme linked immunosorbent assay kit | |
| CN104655847A (en) | Enzyme linked immunosorbent assay kit (ELISA kit) for detecting adprin and detection method thereof | |
| CN105158480A (en) | Kit for detecting peste des petits ruminants virus hemagglutinin protein antibody and application method of kit | |
| CN101581726A (en) | New-generation brucellosis antibody competition enzyme-linked immunosorbent adsorption test detection kit | |
| CN103173420A (en) | Hybridoma cell capable of secreting anti-cardiac troponin I monoclonal antibodies and applications thereof | |
| CN101592661B (en) | Brucellosis antibody competitive enzyme-linked immunosorbent assay reagent kit | |
| CN109387627A (en) | A kind of Reagent Protocol of screening for cancer and early diagnosis based on placenta sample chondroitin sulfate A (CSA) | |
| CN107271682A (en) | A kind of dog c reactive protein fluorescence detection test strip | |
| CN108663514A (en) | A kind of toxoplasma nano-colloid carbon diagnosis test paper and preparation method | |
| CN101592660B (en) | Brucellosis indirect enzyme-linked immunosorbent assay milk liquid antibody reagent kit | |
| CN105181958B (en) | A kind of method that 80nmAuNPs is quantitatively detected based on Indirect cELISA | |
| CN102495215A (en) | Kit for quantitatively detecting tumor necrosis factor alpha | |
| CN1880961A (en) | Immunochromatographic assay test paper for detecting staphylococcal enterotoxin B and preparation method thereof | |
| CN101666800B (en) | Fast test strip of vesicular stomatitis virus (VSV) and preparation method thereof | |
| CN1159580C (en) | Reagent for colloidal gold chromatographic analysis of SARS coronavirus antigen | |
| CN102368070A (en) | Kit for detecting content of MR-1S (Myofibrillogenesis Regulator-1S) in human plasma and preparation method thereof | |
| CN101710117A (en) | Testing kit for enrofloxacin and testing method thereof | |
| CN107656057A (en) | A kind of ELISA kit and its application for being used to detect brucellosis IgM antibody | |
| Njayou et al. | Purification of measles virus by affinity chromatography and by ultracentrifugation: a comparative study | |
| Avraham et al. | A highly sensitive solid-phase radioimmunoassay for the assay of Plasmodium falciparum antigens and antibodies | |
| CN109374886A (en) | Infectious bovine rhinotrachetis virus antibody assay kit and its application | |
| CN105403706A (en) | Heat shock protein 90 (HSP90) chemiluminescence detection kit | |
| CN108872580A (en) | A kind of colloidal gold strip and preparation method thereof detecting novel goose parvovirus | |
| CN110045123A (en) | A kind of carbohydrate antigen 242 magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof |
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: 20181016 |
|
| RJ01 | Rejection of invention patent application after publication |