CN106290841A - Vibrio cholerae O 1 emulsion technique detection kit - Google Patents
Vibrio cholerae O 1 emulsion technique detection kit Download PDFInfo
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- CN106290841A CN106290841A CN201510242765.0A CN201510242765A CN106290841A CN 106290841 A CN106290841 A CN 106290841A CN 201510242765 A CN201510242765 A CN 201510242765A CN 106290841 A CN106290841 A CN 106290841A
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- latex
- vibrio cholerae
- polyester film
- sensitization
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- 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
- G01N33/56911—Bacteria
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- 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
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/18—Togaviridae; Flaviviridae
- G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The present invention relates to emulsion technique immunochromatography field, specifically disclose a kind of vibrio cholerae O 1 emulsion technique detection kit, including reagent strip, described reagent strip includes substrate, filter sample paper, sensitization latex polyester film, immunity nitrocellulose filter and absorbent paper, described filter sample paper, sensitization latex polyester film, immunity nitrocellulose filter and absorbent paper tandem array successively being fixed on described substrate, the anti-vibrio cholerae O 1 type monoclonal antibody I of colored latex particles labelling it is coated with on described sensitization latex polyester film, immunity nitrocellulose filter is provided with the detection line being coated anti-vibrio cholerae O 1 type monoclonal antibody II and the nature controlling line being coated goat anti-rabbit igg.The vibrio cholerae O 1 emulsion technique detection kit of the present invention is quick on the draw, easy to detect, testing result is stable, cost-effective.
Description
Technical field
The present invention relates to immunochromatography technique field, be specifically related to a kind of vibrio cholerae O 1 emulsion technique detection kit and system thereof
Standby with application.
Background technology
Vibrio cholera (Vibrio cholerae) is the pathogen of cholera, can be divided into more than 200 serum according to the difference of its O antigen
Group, wherein Ol group is the dominant serogroup causing mankind's cholera.Cholera be a kind of ancient and popular deadly infectious disease widely it
One.The mankind are unique susceptible persons of vibrio cholera.Vibrio cholera enters digestive tract and arrives small intestinal, adsorbs also at intestinal mucosal surface
Breeding rapidly.The cholera enterotoxin produced acts on mucous membrane of small intestine, causes the secretion that intestinal fluid is excessive.Cause the most in the world
Repeatedly being very popular, mainly show as vomiting and dirarrhea, cause dehydration and metabolic acidosis, circulatory failure, mortality rate is the highest.
Belong to international quarantine infectious disease.
Cholera is the category A infectious disease that China is legal, and national requirements quotes assay in the 24h that sees and treat patients.Therefore, reflect early
Make vibrio cholera and the popular and treatment controlling this disease is had extremely important meaning.The fast diagnosis method of vibrio cholera at present
As undesirable in effects such as fast braking test and immunofluorescence fungus ball tests, it is primarily due to during the vibrio cholera in patient's feces many
Time few, time minimum check result be negative.
Latex chromatography is the immunochromatographic method with color latex as label, its Cleaning Principle and colloidal gold immunity chromatography phase
Similar, it need not instrument, the most with the naked eye sentence read result, has simple to operate, quick, and reagent stability is good, Yi Bao
Deposit, single part independent packaging, the advantages such as reagent loss is few, be suitable for the rapid screening of the specimen such as clinical patients, physical examination of healthy population.
Summary of the invention
It is an object of the invention to overcome prior art defect, from improving detection sensitivity, it is provided that a kind of detection sensitivity height,
High specificity, detect quick vibrio cholerae O 1 emulsion technique detection kit.
A first aspect of the present invention discloses a kind of vibrio cholerae O 1 emulsion technique detection kit, including reagent strip, described examination
Agent bar includes substrate, filter sample paper, sensitization latex polyester film, immunity nitrocellulose filter and absorbent paper, described filter sample paper, cause
Quick latex polyester film, immunity nitrocellulose filter and absorbent paper tandem array successively are also fixed on described substrate, described sensitization
The anti-vibrio cholerae O 1 type monoclonal antibody I of colored latex particles labelling, immunity celluloid it is coated with on latex polyester film
Film is provided with the detection line being coated anti-vibrio cholerae O 1 type monoclonal antibody II and the nature controlling line being coated goat anti-rabbit igg.
Preferably, described colored latex particles is the granules of polystyrene of particle diameter 290～300nm.
Preferably, the color of described colored latex particles is red.
The anti-vibrio cholerae O 1 type monoclonal antibody I of labelling chromatic colour latex particle and exempting from sensitization latex polyester film of the present invention
Epidemic disease nitrocellulose filter detection line (T line) coated anti-vibrio cholerae O 1 type monoclonal antibody II is monoclonal antibody.
Another aspect of the present invention, it is provided that the preparation method of a kind of vibrio cholerae O 1 emulsion technique detection kit, including following
Step:
1) preparation of sensitization latex particle: by carboxylated polystyrene labelling anti-vibrio cholerae O 1 type monoclonal antibody
Ι, it is thus achieved that sensitization latex particle；
2) preparation of sensitization latex polyester film: by step 1) the sensitization latex particle that obtains is coated polyester film, it is thus achieved that cause
Quick latex polyester film；
3) anti-vibrio cholerae O 1 type monoclonal antibody II and goat anti-rabbit antibody are sprayed on respectively nitrocellulose filter detection line
On the position of (T line) and nature controlling line (C line), dry for standby, prepare immunity nitrocellulose filter；
4) filter sample paper, sensitization latex polyester film, immunity nitrocellulose filter, absorbent paper are pasted onto PVC sheet successively
On, cutting prepares reagent strip；
5) reagent strip is loaded plastic casing, it is thus achieved that vibrio cholerae O 1 emulsion technique detection kit.
Preferably, described step 1) being prepared as of sensitization latex particle:
A. taking the carboxylated polystyrene of certain volume, the carbonate buffer solution with 2～3 times of volume 20mM pH 6.0 is many
Secondary flushing, it is centrifuged, abandons supernatant, it is thus achieved that carboxylated granules of polystyrene precipitation；
B. the carboxylated polystyrene obtained by the phosphate buffer solution suspension previous step of 1～1.5 times of volume 20mM pH6.0
Granule precipitates, and adds the water solublity carbonization diimine of 1～1.5 times of volume, stirs 20～30 minutes under room temperature,
Finally repeatedly rinse with the borate buffer solution of 2～3 times of volume 0.01M pH 8.0, be centrifuged, abandon supernatant, it is thus achieved that
Thing to be marked；
C. the borate buffer adding 2～3 times of volume 0.2M pH 7.0 in the thing described to be marked that step b obtains makes breast
Glue suspension, and in described latex suspension, add anti-vibrio cholerae O 1 type monoclonal antibody Ι；
D. fully reaction overnight, adds terminator and terminates reaction, it is thus achieved that sensitization latex particle.
In the preparation process of sensitization latex particle, the water solublity carbonization diimine of addition, each buffer solution volume all with just
On the basis of the volume of the carboxylated polystyrene that the beginning is taken, add by volume multiple of the present invention: such as the carboxyl taken
The volume of granules of polystyrene changed is 1ml, then the phosphate buffer solution volume needing pH 6.0 20mM added can be
1～1.5ml, pH 7.0 the volume of borate buffer of 0.2M be 2～3ml.
More excellent, in described step a, carbonate buffer solution is the 20mM pH 6.0 carbonate buffer solution containing 0.01%SDS.
The carbonate buffer solution being added with SDS can preferably wash away the surface-active substance of latex, and cleaning performance is more preferable.
More excellent, polystyrene concentrations carboxylated in described step a is 6～12w/v%.
More excellent, the centrifugal condition in described step a is 12000～14000rpm, centrifugal 10min.
More excellent, in described step b, the concentration of water solublity carbonization diimine is 9～10mg/ml.
More excellent, the centrifugal condition in described step b is 12000～14000rpm, centrifugal 10min.
More excellent, granules of polystyrene coupling anti-vibrio cholerae O 1 type monoclonal anti carboxylated in described step c latex suspension
The amount of body Ι is: 0.5mg anti-vibrio cholerae O 1 type monoclonal antibody Ι/10mg granules of polystyrene.
More excellent, in described step d, terminator is the Tris-HCl of 50mM pH 8.2.
In the present invention, v% refers to percent by volume；W/v% refers to percent weight in volume, as 1w/v% is 100ml solution
In the material Han 1g.
Preferably, described step 2) in the preparation of sensitization latex polyester film concretely comprise the following steps:
A. sensitization latex particle is diluted with latex buffer, it is thus achieved that the sensitization latex solution of 0.3～0.5w/v%；
B. the sensitization latex solution spraying polyester film prepared by step A, is dried, prepares sensitization latex polyester film.
More excellent, the latex buffer formulation in described step A is as follows: 9～11v%1.0M Tris liquid, and 0.25～0.35w/v
% PEG 20000,0.18～0.20w/v% bovine serum albumin, 0.15～0.25w/v% skim milk, 0.28～0.30
W/v% casein, and 0.04～0.06w/v% Hydrazoic acid,sodium salt, with salt acid for adjusting pH to 8.0 ± 0.02, surplus is water.
Optimum, the latex buffer formulation in described step A is as follows: 10v%1.0M Tris liquid, the poly-second of 0.3w/v% two
Alcohol 20000,0.2w/v% bovine serum albumin, 0.2w/v% skim milk, 0.3w/v% casein, and 0.05w/v% are folded
Sodium nitride, with salt acid for adjusting pH to 8.0 ± 0.02, surplus is water.
The most preferably, so that test kit has more preferably sensitivity and specificity, latex of the present invention buffers
Liquid also includes following component: fructose, potassium chloride and glycine, and the total concentration of fructose, potassium chloride and glycine is
3.25～4.75g/L, the pH value of buffer is 7.3-7.5.
It is furthermore preferred that the concentration that each component is in latex buffer is:
Fructose 1.0-2.0g/L；Potassium chloride 0.25-0.5g/L；Glycine 2.0-2.25g/L.
More excellent, step B sprays polyester film with sensitization latex solution, every 261mm × 220mm polyester film sprays 1.28
Ml sensitization latex solution, is dried, and prepares sensitization latex polyester film.
Preferably, described step 3) in, be sprayed on the concentration of the goat anti-rabbit igg on nitrocellulose filter nature controlling line (C line) be 1.0～
2.0mg/ml, anti-vibrio cholerae O 1 type monoclonal antibody II concentration being sprayed on nitrocellulose filter detection line (T line) be 1.0～
2.0mg/ml.Preferably, the long nitrocellulose filter of every 50m is coated with the goat anti-rabbit igg of 6ml and the anti-cholera of 6ml respectively
Vibrio O1 type monoclonal antibody II solution, the spacing of detection line and nature controlling line is 4.0～6.0mm.
Vibrio cholerae O 1 emulsion technique detection kit prepared by the present invention is highly sensitive, high specificity；In addition the examination of the present invention
Agent box also has simple to operate, and price is low, stores and the advantage such as convenient transportation.
The using method of vibrio cholerae O 1 emulsion technique detection kit of the present invention:
1. the process of sample: sampling from the different parts of Feces of Patients at random with special-purpose taking lining bar, sampling amount is to be stained with special sampling
The small circle ring of nose is as the criterion；Prepare a sample processing tube, be vertically put on process pipe holder, and add in sample cell
0.6ml distilled water；The distilled water that the sample taken on special-purpose taking lining bar inserts in sample cell is stirred, standby；Build
The sample that view is handled well detects in time, can store 48 hours as do not detected the sample handled well immediately at 2～8 DEG C.
2. the detection of sample: being added dropwise in sample cell with suction pipe pipette samples liquid 2-3, measuring samples is under capillary action to suction
Water paper end layer is analysed, and passes sequentially through polyester film, nitrocellulose filter.After arriving polyester film, by the monoclonal antibody of latex labelling
Fully identify and combine, continuing to chromatograph on nitrocellulose filter, the coated anti-vibrio cholerae O 1 type with on nitrocellulose filter
There is immunoreation in monoclonal antibody monoclonal-II, shows corresponding red lines.
3. the interpretation of result: 8-15 minute sentence read result after dropping sample.
Positive: in detection line and the nature controlling line position of detectable bar, one red lines respectively to occur, represent in sample have cholera
The existence of vibrio O1 type antigen.
Negative: red lines only to occur in nature controlling line position, represents the existence without vibrio cholerae O 1 type antigen in sample.
Invalid: nature controlling line position occurs without red line, represent that result is invalid, should retry.
The beneficial effects of the present invention is: the vibrio cholerae O 1 emulsion technique detection kit of the present invention is highly sensitive, high specificity；
In addition the test kit of the present invention also has simple to operate, and price is low, stores and the advantage such as convenient transportation.
Accompanying drawing explanation
Fig. 1: vibrio cholerae O 1 emulsion technique detection kit reagent strip schematic diagram (1. filter sample paper 2. sensitization latex polyester film 3.
Immunity nitrocellulose filter 4. absorbent paper 5. detects line 6. nature controlling line)
Detailed description of the invention
Below by way of specific instantiation, embodiments of the present invention being described, those skilled in the art can be taken off by this specification
The content of dew understands other advantages and effect of the present invention easily.The present invention can also be by the most different specific embodiment parties
Formula is carried out or applies, and the every details in this specification can also be based on different viewpoints and application, without departing from this
Various modification or change is carried out under bright spirit.
Before further describing the specific embodiment of the invention, it should be appreciated that protection scope of the present invention is not limited to following spy
Fixed specific embodiments；It is also understood that the term used in the embodiment of the present invention is specifically to be embodied as to describe
Scheme rather than in order to limit the scope of the invention；In description of the invention and claims, unless another in literary composition
Explicitly pointing out outward, singulative " ", " one " and " this " include plural form.
When embodiment provides numerical range, it should be appreciated that unless the present invention is otherwise noted, two end points of each numerical range
And any one numerical value all can be selected between two end points.Unless otherwise defined, all technology used in the present invention and section
The same meaning that technics and those skilled in the art of the present technique are generally understood that.Except in embodiment use concrete grammar, equipment,
Outside material, according to those skilled in the art's grasp to prior art and the record of the present invention, it is also possible to use and this
Any method, equipment and the material of the prior art that the method described in inventive embodiments, equipment, material are similar or equivalent comes
Realize the present invention.
Unless otherwise indicated, the experimental technique that disclosed in this invention, detection method, preparation method all use the art
Molecular biology, biochemistry, chromatin Structure and the analysis of routine, analytical chemistry, cell cultivation, recombinant DNA technology
And the routine techniques of association area.These technology have improved explanation in existing document, specifically can be found in Sambrook etc.
MOLECULAR CLONING:A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley&Sons, New York, 1987and periodic updates；the
Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN
STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS
IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic
Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin
Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
The preparation of embodiment 1 vibrio cholerae O 1 emulsion technique detection kit
One, reagent source:
Goat anti-rabbit igg polyclonal antibody is (purchased from Mei Di Bioisystech Co., Ltd of U.S. MAXMED LABORATORIES
INC.)
Cellulose nitrate film, polyester film, granules of polystyrene (purchased from Sartorius AG SARTORIUS)
Granules of polystyrene (purchased from WHATMAN company)
Two, preparation process:
Method 1:
1. the preparation of sensitization latex particle:
A. take polystyrene latex carboxylated for 0.4ml 10% and put in centrifuge tube, add 1ml pH 6.0 20mM Han 0.01%SDS
Carbonic acid buffer rinse twice, 14000g/min is centrifuged 10min, abandons supernatant, it is thus achieved that carboxylated granules of polystyrene
Precipitation.
B. the carboxylated granules of polystyrene precipitation obtained by the phosphate buffer solution suspension previous step of 0.5ml pH 6.020mM,
And add 0.5ml 10% water solublity carbonization diimine, stir under room temperature 30 minutes, finally with 0.8ml 0.01M pH 8.0
Borate buffer solution flush three times, be centrifuged, abandon supernatant, it is thus achieved that thing to be marked.
C. the borate buffer adding 1ml 0.2M pH 7.0 in the thing to be marked that step b obtains makes latex suspension, and to this breast
Glue suspension adds 2mg anti-vibrio cholerae O 1 type monoclonal antibody Ι.
After the most fully reacting 12 hours, the Tris-HCl adding 50mM pH8.2 terminates reaction as terminator, it is thus achieved that sensitization breast
Glue granule.
2. the preparation of sensitization latex polyester film:
(1) preparation of latex buffer: take 800ml purified water, add 100ml 1.0M Tris liquid inward, accurately weigh 3.0g
PEG 20000,2.0g bovine serum albumin, 2.0g skim milk, 3.0g casein solution, 0.5g Hydrazoic acid,sodium salt,
1.5g fructose, 0.4g potassium chloride, 2.20g glycine, add in solution, fully dissolve, mix homogeneously, regulate with hydrochloric acid
PH to 8.0, adds purified water to cumulative volume 1000ml.
(2) with the latex buffer dilution sensitization latex particle of previous step, it is thus achieved that the sensitization latex solution of 0.4w/v%.
(3) take on 1.28ml sensitization latex solution point latex machine specking polyester film after the pre-treatment, be placed at a temperature of 37 DEG C
Dry 2 hours, keep in Dark Place at sealing 4-30 DEG C, prepare sensitization latex polyester film.
3. the preparation of immunity nitrocellulose filter:
Anti-to 2.0mg/ml goat anti-rabbit antibody and 1.5mg/ml vibrio cholerae O 1 type monoclonal antibody II is sprayed on celluloid respectively
On the position of film nature controlling line and detection line, dry 2 hours at 37 DEG C, keep in Dark Place at sealing 4-30 DEG C, prepare immunity nitric acid fine
Dimension element film.Every long nitrocellulose filter of 50m is coated with the goat anti-rabbit igg of 6ml and anti-vibrio cholerae O 1 type monoclonal anti respectively
Body II solution, the spacing of detection line and nature controlling line is 5mm.
4. filter sample paper, sensitization latex polyester film, immunity nitrocellulose filter, absorbent paper are pasted onto on PVC sheet successively, cutting system
Obtain reagent strip；Reagent strip is loaded plastic casing and prepares detection kit, it is thus achieved that vibrio cholerae O 1 emulsion technique detection kit.
(as shown in Figure 1).
Method 2:
1. the preparation of sensitization latex particle:
A. take polystyrene latex carboxylated for 1ml 6% and put in centrifuge tube, add 2ml pH 6.0 containing 0.01%SDS
The carbonic acid buffer of 20mM flushes three times, and 12000g/min is centrifuged 10min, abandons supernatant, it is thus achieved that carboxylated polyphenyl second
Alkene granule precipitates.
B. the carboxylated granules of polystyrene obtained by the phosphate buffer solution suspension previous step of 1.5ml pH 6.0 20mM sinks
Form sediment, and add 1.5ml 9% water solublity carbonization diimine, stir under room temperature 20 minutes, finally with 3ml 0.01M pH 8.0
Borate buffer solution flush three times, be centrifuged, abandon supernatant, it is thus achieved that thing to be marked.
C. the borate buffer adding 2ml pH 7.0 0.2M in the thing to be marked that step b obtains makes latex suspension, and to
This latex suspension adds 2.5mg anti-vibrio cholerae O 1 type monoclonal antibody Ι.
D., after fully reacting overnight, add terminator and terminate reaction, it is thus achieved that sensitization latex particle.
2. the preparation of sensitization latex polyester film:
(1) preparation of latex buffer: take 800ml purified water, add 90ml 1.0M Tris liquid inward, accurately weigh 3.5g
PEG 20000,1.8g bovine serum albumin, 1.5g skim milk, 3.0g casein solution, 0.6g Hydrazoic acid,sodium salt
1.0g fructose, 0.25g potassium chloride, 2.00g glycine, add in solution, fully dissolve, mix homogeneously, adjust with hydrochloric acid
Joint pH to 8.0, adds purified water to cumulative volume 1000ml.
(2) with the latex buffer dilution sensitization latex particle of previous step, it is thus achieved that the sensitization latex solution of 0.2w/v%.
(3) take on 1.28ml sensitization latex solution point latex machine specking polyester film after the pre-treatment, be placed in the temperature of 37 DEG C
Lower drying 2 hours, keeps in Dark Place at sealing 4-30 DEG C, prepares sensitization latex polyester film.
3. the preparation of immunity nitrocellulose filter:
Anti-to 1.5mg/ml goat anti-rabbit antibody and 2.0mg/ml vibrio cholerae O 1 type monoclonal antibody antibody II is sprayed on respectively nitric acid fine
On the position of dimension element film nature controlling line and detection line, dry 2 hours at 37 DEG C, keep in Dark Place at sealing 4-30 DEG C, prepare immunity nitre
Acid cellulose film.Every long nitrocellulose filter of 50m is coated with the goat anti-rabbit igg of 6ml and anti-vibrio cholerae O 1 type Dan Ke respectively
Grand antibodies Antibodies II solution, the spacing of detection line and nature controlling line is 5mm.
4. with method 1.
Method 3:
1. the preparation of sensitization latex particle:
A. take polystyrene latex carboxylated for 0.5ml 12% and put in centrifuge tube, add 1.5ml pH 6.0 containing 0.01%SDS
The carbonic acid buffer of 20mM rinses twice, and 13000g/min is centrifuged 10min, abandons supernatant, it is thus achieved that carboxylated polyphenyl second
Alkene granule precipitates.
B. the carboxylated granules of polystyrene obtained by the phosphate buffer solution suspension previous step of 0.5ml pH 6.0 20mM sinks
Form sediment, and add 0.5ml 10% water solublity carbonization diimine, stir under room temperature 25 minutes, finally with 1.5ml 0.01M pH8.0
Borate buffer solution rinse twice, be centrifuged, abandon supernatant, it is thus achieved that thing to be marked.
C. the borate buffer adding 1.5ml pH 7.0 0.2M in the thing to be marked that step b obtains makes latex suspension, and
3.75mg anti-vibrio cholerae O 1 type monoclonal antibody Ι is added in this latex suspension.
D., after fully reacting overnight, add terminator and terminate reaction, it is thus achieved that sensitization latex particle.
2. the preparation of sensitization latex polyester film:
(1) preparation of latex buffer: take 800ml purified water, add 110ml 1.0M Tris liquid inward, accurately weigh 2.5g
PEG 20000,1.9g bovine serum albumin, 2.5g skim milk, 2.8g casein solution and 0.4g Hydrazoic acid,sodium salt
2.0g fructose, 0.5g potassium chloride, 2.25g glycine adds in solution, fully dissolves, mix homogeneously, regulate with hydrochloric acid
PH to 8.0, adds purified water to cumulative volume 1000ml.
(2) with the latex buffer dilution sensitization latex particle of previous step, it is thus achieved that the sensitization latex solution of 0.6w/v%.
(3) take on 1.28ml sensitization latex solution point latex machine specking polyester film after the pre-treatment, be placed in the temperature of 37 DEG C
Lower drying 2 hours, keeps in Dark Place at sealing 4-30 DEG C, prepares sensitization latex polyester film.
3. the preparation of immunity nitrocellulose filter:
Anti-to 1.0mg/ml goat anti-rabbit antibody and 1.0mg/ml vibrio cholerae O 1 type monoclonal antibody II is sprayed on celluloid respectively
On the position of film nature controlling line and detection line, dry 2 hours at 37 DEG C, keep in Dark Place at sealing 4-30 DEG C, prepare immunity nitric acid fine
Dimension element film.Every long nitrocellulose filter of 50m is coated with the goat anti-rabbit igg of 6ml and anti-vibrio cholerae O 1 type monoclonal anti respectively
Body II solution, the spacing of detection line and nature controlling line is 4mm.
4. with method 1.
The preparation of embodiment 2 contrast agent box
The preparation method of contrast agent box: without fructose, potassium chloride and glycine, other reagent and experiment in latex buffer
Method is all with method 1～3 in embodiment 1.
The specificity experiments of embodiment 3 vibrio cholerae O 1 emulsion technique detection kit
Detection method:
1. Example 1 preparation vibrio cholerae O 1 emulsion technique detection kit and the contrast agent box of embodiment 2, test kit is put
Put on level table.
2. the preparation of testing sample: by four class experimental subjecies of table 1, take from the different parts of tester's feces at random with special-purpose taking lining bar
Sample, sampling amount is as the criterion with the small circle ring being stained with special-purpose taking lining bar front end；Prepare a sample processing tube, be vertically put in process pipe
On support, and in sample cell, add 0.6ml distilled water；The sample taken on special-purpose taking lining bar is inserted the distillation in sample cell
Water stirs as testing sample.
3. every class experimental subject is 100 people, testing result such as table 1:
Table 1 vibrio cholerae O 1 emulsion technique detection kit specific test result
As seen from the above table, the vibrio cholerae O 1 emulsion technique detection kit of the present invention to escherichia coli and dysentery bacterium all without intersecting
Reaction, specificity is good.
Above embodiment illustrates that embodiment disclosed by the invention, can not be interpreted as limitation of the present invention.This
Outward, method, the change of compositions in various amendments listed herein and invention, without departing from the scope of the present invention and essence
It is apparent from for those skilled in the art on the premise of god.Although having combined the multiple concrete preferred of the present invention
Embodiment has carried out concrete description to the present invention, it is to be understood that, the present invention should not be limited only to these specific embodiments.Thing
In reality, various as above for those skilled in the art obvious amendment obtain invention and be intended to be included in
In the scope of the present invention.
Claims (10)
1. a vibrio cholerae O 1 emulsion technique detection kit, including reagent strip, described reagent strip includes substrate, filter sample paper, cause
Quick latex polyester film, immunity nitrocellulose filter and absorbent paper, described filter sample paper, sensitization latex polyester film, immunity nitric acid
Cellulose membrane and absorbent paper tandem array successively is also fixed on described substrate, it is characterised in that described sensitization latex polyester
The anti-vibrio cholerae O 1 type monoclonal antibody I of colored latex particles labelling it is coated with, on immunity nitrocellulose filter on film
It is provided with the detection line being coated anti-vibrio cholerae O 1 type monoclonal antibody II and the nature controlling line being coated goat anti-rabbit igg.
2. test kit as claimed in claim 1, it is characterised in that described colored latex particles is the poly-of particle diameter 290～300nm
Styrene pellets.
3. the preparation method of the vibrio cholerae O 1 emulsion technique detection kit described in claim 1-2 any claim, including with
Lower step:
1) preparation of sensitization latex particle: by carboxylated polystyrene labelling anti-vibrio cholerae O 1 type monoclonal antibody
Ι, it is thus achieved that sensitization latex particle；
2) preparation of sensitization latex polyester film: by step 1) the sensitization latex particle that obtains is coated polyester film, it is thus achieved that cause
Quick latex polyester film；
3) anti-vibrio cholerae O 1 type monoclonal antibody II and goat anti-rabbit antibody are sprayed on respectively nitrocellulose filter detection line
With on the position of nature controlling line, dry for standby, prepare immunity nitrocellulose filter；
4) filter sample paper, sensitization latex polyester film, immunity nitrocellulose filter, absorbent paper are pasted onto on PVC sheet successively,
Cutting prepares reagent strip；
5) reagent strip is loaded plastic casing, it is thus achieved that vibrio cholerae O 1 emulsion technique detection kit.
4. preparation method as claimed in claim 3, it is characterised in that described step 1) being prepared as of sensitization latex particle:
A. take the carboxylated polystyrene of certain volume, buffer with the carbonic acid of 2～3 times of volume 20mM pH 6.0
Solution repeatedly rinses, is centrifuged, abandons supernatant, it is thus achieved that carboxylated granules of polystyrene precipitation；
B. carboxylated the gathering obtained by the phosphate buffer solution suspension previous step of 1～1.5 times of volume 20mM pH6.0
Styrene pellets precipitates, and adds the water solublity carbonization diimine of 1～1.5 times of volume, stirs under room temperature
20～30 minutes, finally with the borate buffer solution of 2～3 times of volume 0.01M pH 8.0 repeatedly rinse,
It is centrifuged, abandons supernatant, it is thus achieved that thing to be marked；
C. in the thing described to be marked that step b obtains, add the borate buffer of 2～3 times of volume 0.2M pH 7.0
Make latex suspension, and in described latex suspension, add anti-vibrio cholerae O 1 type monoclonal antibody Ι；
D. fully reaction overnight, adds terminator and terminates reaction, it is thus achieved that sensitization latex particle.
5. preparation method as claimed in claim 4, it is characterised in that in described step a, carbonate buffer solution is 20mM pH 6.0
Carbonate buffer solution containing 0.01%SDS.
6. preparation method as claimed in claim 4, it is characterised in that polystyrene concentrations carboxylated in described step a be 6～
12w/v%, in described step b, the concentration of water solublity carbonization diimine is 9～10mg/ml.
7. preparation method as claimed in claim 4, it is characterised in that polystyrene carboxylated in described step c latex suspension
The amount of granule coupling anti-vibrio cholerae O 1 type monoclonal antibody Ι is: 0.5mg anti-vibrio cholerae O 1 type monoclonal antibody
Ι/10mg granules of polystyrene.
8. preparation method as claimed in claim 4, it is characterised in that described step 2) in the preparation tool of sensitization latex polyester film
Body step is:
A. sensitization latex particle is diluted with latex buffer, it is thus achieved that the sensitization latex solution of 0.3～0.5w/v%；
B. the sensitization latex solution spraying polyester film prepared by step A, is dried, prepares sensitization latex polyester film.
9. preparation method as claimed in claim 8, it is characterised in that the latex buffer formulation in described step A is as follows: 9～
11v%1.0M Tris liquid, 0.25～0.35w/v% PEG 20000,0.18～0.20w/v% bovine serum albumin,
0.15～0.25w/v% skim milk, 0.28～0.30w/v% casein, and 0.04～0.06w/v% Hydrazoic acid,sodium salt, use
Salt acid for adjusting pH is to 8.0 ± 0.02, and surplus is water.
10. the application in preparing vibrio cholerae O 1 detectable of the test kit described in claim 1-2 any claim.
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| CN201510242765.0A CN106290841A (en) | 2015-05-13 | 2015-05-13 | Vibrio cholerae O 1 emulsion technique detection kit |
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| CN201510242765.0A CN106290841A (en) | 2015-05-13 | 2015-05-13 | Vibrio cholerae O 1 emulsion technique detection kit |
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