CN106290839A - Plasmodium falciparum emulsion technique detection kit - Google Patents
Plasmodium falciparum emulsion technique detection kit Download PDFInfo
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- CN106290839A CN106290839A CN201510242736.4A CN201510242736A CN106290839A CN 106290839 A CN106290839 A CN 106290839A CN 201510242736 A CN201510242736 A CN 201510242736A CN 106290839 A CN106290839 A CN 106290839A
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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
- G01N33/56905—Protozoa
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- G01—MEASURING; TESTING
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- 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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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
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Abstract
The present invention relates to emulsion technique immunochromatography field, specifically disclose a kind of Plasmodium falciparum 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-plasmodium falciparum Histidine rich protein Ⅱ 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-plasmodium falciparum Histidine rich protein Ⅱ monoclonal antibody II and the nature controlling line being coated goat anti-rabbit igg.The Plasmodium falciparum 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 Plasmodium falciparum emulsion technique detection kit and preparation thereof
With application.
Background technology
Malaria is through infecting the arthropod borne infection caused by plasmodium as mosquito bite or the blood of input tape plasmodium person.Parasitic
Plasmodium in human body has four kinds, i.e. Plasmodium vivax, malariae, Plasmodium falciparum and Plasmodium ovale.At me
State is mainly Plasmodium vivax and Plasmodium falciparum；Other two kinds rare, some cases of the most accidental external input.Different
Plasmodium cause tertian malaria, quartan malaria, subtertian malaria and oval malaria respectively.
Malaria is one of big parasitic disease in the world five, has the highest M & M, is prevalent in the torrid zone and subtropical zone
Area, the roomy belt-like zone being positioned at around equator.Major Epidemic area is central africa, South Asia, Southeast Asia and North of South America
The torrid areas in portion, the most again with African epidemic situation the most very.On the Chinese side, the popular area that malaria is main is Central China China
The jungle mountainous region in south, but epidemic situation is light far beyond Africa.Malaria is to be main feature with periodically cold and hot outbreak, splenomegaly
Greatly, the impaired various syndromes caused such as anemia and brain, liver, kidney, the heart, intestinal, stomach.Patient every year 300,000,000～500,000,000 it
Between, because suffering from malaria and the number of death is between 1,000,000～3,000,000, wherein most is child.
Early, fast and effeciently detection plasmodium is the key effectively preventing and treating malaria.Traditional detection method is to pass through blood smear
Microscopy judges, this method simplicity, economy, accuracy rate are high, and shortcoming is that judged result is by reviewer's microscopy water
The impact of the factors such as gentle asthenopia degree, is easily caused mistaken diagnosis and missing inspection and checks inefficient, being not suitable for extensive scene
Investigation and short time mass disposal sample.
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 Plasmodium falciparum emulsion technique detection kit.
A first aspect of the present invention discloses a kind of Plasmodium falciparum emulsion technique detection kit, including reagent strip, described reagent
Bar 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 are also fixed on described substrate, described sensitization breast
It is coated with the anti-plasmodium falciparum Histidine rich protein Ⅱ monoclonal antibody I of colored latex particles labelling on glue polyester film, exempts from
Epidemic disease nitrocellulose filter is provided with detection line and the bag being coated anti-plasmodium falciparum Histidine rich protein Ⅱ monoclonal antibody II
By the nature controlling line of 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-plasmodium falciparum Histidine rich protein Ⅱ Dan Ke of labelling chromatic colour latex particle on sensitization latex polyester film of the present invention
Grand antibody I and immunity nitrocellulose filter detection line (T line) coated anti-plasmodium falciparum Histidine rich protein Ⅱ monoclonal
Antibody II is monoclonal antibody.
Another aspect of the present invention, it is provided that the preparation method of a kind of Plasmodium falciparum emulsion technique detection kit, including following
Step:
1) preparation of sensitization latex particle: with carboxylated polystyrene labelling anti-plasmodium falciparum histidine rich protein
II 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-plasmodium falciparum Histidine rich protein Ⅱ monoclonal antibody II and goat anti-rabbit antibody are sprayed on nitric acid respectively
On the position of cellulose membrane detection line (T line) and nature controlling line (C line), dry for standby, prepare immunity nitre
Acid cellulose film；
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 Plasmodium falciparum 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-plasmodium falciparum Histidine rich protein Ⅱ 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-plasmodium falciparum histidine egg with enriched carboxylated in described step c latex suspension
The amount of white II monoclonal antibody Ι is: 0.5mg anti-plasmodium falciparum Histidine rich protein Ⅱ monoclonal antibody Ι/10mg polyphenyl
Ethylene granule.
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, is sprayed on the anti-plasmodium falciparum Histidine rich protein Ⅱ monoclonal antibody on nitrocellulose filter detection line (T line)
II concentration is 1.0～2.0mg/ml.Preferably, the long nitrocellulose filter of every 50m be coated with respectively 6ml goat anti-rabbit igg and
Anti-plasmodium falciparum Histidine rich protein Ⅱ monoclonal antibody II solution of 6ml, detection line and the spacing of nature controlling line be 4.0～
6.0mm。
Plasmodium falciparum emulsion technique detection kit prepared by the present invention is highly sensitive, high specificity；In addition the reagent of the present invention
Box also has simple to operate, and price is low, stores and the advantage such as convenient transportation.
The using method of Plasmodium falciparum emulsion technique detection kit of the present invention:
1. the process of sample: take whole blood, standby；The sample that suggestion is handled well detects in time, handles well as do not detected immediately
Sample can store 48 hours 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-plasmodium falciparum group with on nitrocellulose filter
There is immunoreation in propylhomoserin rich protein II monoclonal antibody-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, expression sample has pernicious
The existence of plasmodium antigens.
Negative: red lines only to occur in nature controlling line position, represents the existence without Plasmodium Falciparum Antigens 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 Plasmodium falciparum 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: Plasmodium falciparum emulsion technique detection kit reagent strip schematic diagram (1. exempt from by filter sample paper 2. sensitization latex polyester film 3.
Epidemic disease 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 Plasmodium falciparum 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-plasmodium falciparum Histidine rich protein Ⅱ 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:
By 2.0mg/ml goat anti-rabbit antibody and 1.5mg/ml anti-plasmodium falciparum Histidine rich protein Ⅱ monoclonal antibody II difference
It is sprayed on the position of nitrocellulose filter nature controlling line and detection line, dries 2 hours at 37 DEG C, keep in Dark Place at sealing 4-30 DEG C,
Prepare immunity nitrocellulose filter.Every long nitrocellulose filter of 50m is coated with the goat anti-rabbit igg of 6ml respectively and anti-plasmodium is former
Worm Histidine rich protein Ⅱ monoclonal antibody 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 Plasmodium falciparum 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 6.020mM 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 7.00.2M in the thing to be marked that step b obtains makes latex suspension, and to
This latex suspension adds 2.5mg anti-plasmodium falciparum Histidine rich protein Ⅱ 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:
By 1.5mg/ml goat anti-rabbit antibody and 2.0mg/ml anti-plasmodium falciparum Histidine rich protein Ⅱ monoclonal antibody antibody II
Being sprayed on respectively on the position of nitrocellulose filter nature controlling line and detection line, dry 2 hours at 37 DEG C, at sealing 4-30 DEG C, lucifuge is protected
Deposit, prepare immunity nitrocellulose filter.Every long nitrocellulose filter of 50m is coated with the goat anti-rabbit igg of 6ml respectively and resists pernicious
Plasmodium Histidine rich protein Ⅱ monoclonal antibody antibody 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 6.020mM 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 7.00.2M in the thing to be marked that step b obtains makes latex suspension, and
3.75mg anti-plasmodium falciparum Histidine rich protein Ⅱ 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:
By 1.0mg/ml goat anti-rabbit antibody and 1.0mg/ml anti-plasmodium falciparum Histidine rich protein Ⅱ monoclonal antibody II difference
It is sprayed on the position of nitrocellulose filter nature controlling line and detection line, dries 2 hours at 37 DEG C, keep in Dark Place at sealing 4-30 DEG C,
Prepare immunity nitrocellulose filter.Every long nitrocellulose filter of 50m is coated with the goat anti-rabbit igg of 6ml respectively and anti-plasmodium is former
Worm Histidine rich protein Ⅱ monoclonal antibody 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 Plasmodium falciparum emulsion technique detection kit
Detection method:
1. Example 1 preparation Plasmodium falciparum emulsion technique detection kit and the contrast agent box of embodiment 2, by test kit place
On level table.
2. the preparation of testing sample: by four class experimental subjecies of table 1, take whole blood as testing sample.
3. every class experimental subject is 100 people, testing result such as table 1:
Table 1 Plasmodium falciparum emulsion technique detection kit specific test result
As seen from the above table, the Plasmodium falciparum emulsion technique detection kit no cross reaction equal to hepatitis B and syphilis of the present invention,
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 Plasmodium falciparum 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 nitric acid are fine
Tie up element film and absorbent paper tandem array successively and be fixed on described substrate, it is characterised in that described sensitization latex polyester film
On be coated with the anti-plasmodium falciparum Histidine rich protein Ⅱ monoclonal antibody I of colored latex particles labelling, immunity nitric acid
Cellulose membrane is provided with and has been coated the detection line of anti-plasmodium falciparum Histidine rich protein Ⅱ monoclonal antibody II and has been coated
The nature controlling line of 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 Plasmodium falciparum emulsion technique detection kit described in claim 1-2 any claim, including following
Step:
1) preparation of sensitization latex particle: with carboxylated polystyrene labelling anti-plasmodium falciparum histidine rich protein
II 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-plasmodium falciparum Histidine rich protein Ⅱ monoclonal antibody II and goat anti-rabbit antibody are sprayed on nitric acid respectively
On the position of cellulose membrane detection line and 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 Plasmodium falciparum 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-plasmodium falciparum Histidine rich protein Ⅱ list
Clonal 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-plasmodium falciparum Histidine rich protein Ⅱ monoclonal antibody Ι is: 0.5mg anti-plasmodium falciparum group
Propylhomoserin rich protein II 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 preparation Plasmodium falciparum detectable of the test kit described in claim 1-2 any claim.
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| CN201510242736.4A CN106290839A (en) | 2015-05-13 | 2015-05-13 | Plasmodium falciparum emulsion technique detection kit |
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