CN106908602A - A kind of preparation method of plasmodium pf/pan Test papers - Google Patents
A kind of preparation method of plasmodium pf/pan Test papers Download PDFInfo
- Publication number
- CN106908602A CN106908602A CN201710009907.8A CN201710009907A CN106908602A CN 106908602 A CN106908602 A CN 106908602A CN 201710009907 A CN201710009907 A CN 201710009907A CN 106908602 A CN106908602 A CN 106908602A
- Authority
- CN
- China
- Prior art keywords
- pan
- plasmodium
- serum albumin
- bovine serum
- monoclonal antibody
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- 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
- 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
- G01N2333/445—Plasmodium
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
It is the invention discloses a kind of preparation method of plasmodium pf/pan Test papers, including step：With the Immuno gold of plasmodium falciparum lactic dehydrogenase monoclonal antibody and pLDH labeling of monoclonal antibody respectively to glass fibre element film metal spraying, obtain pf gold pads and pan gold pads, the polyvinyl chloride bottom lining out of nitrocellulose filter is being stained with respectively with plasmodium falciparum lactic dehydrogenase monoclonal antibody detection line coating buffer, pLDH monoclonal antibody detection line coating buffer and control line coating buffer, the polyvinyl chloride base plate of a film is being obtained；Sample pad, gold pad, the polyvinyl chloride base plate of point film and blotting paper are fitted together, Test paper is cut into.Whether the Test paper that the present invention is obtained can be read by manual operations, naked eyes and be judged contain plasmodium pf/pan in blood sample based on immune lateral flow chromatographic technique, and diagnosis is quick and result is accurate, and person under inspection's body will not be damaged.
Description
Technical field
The invention belongs to biologic applications technical field, more particularly to a kind of preparation side of plasmodium pf/pan Test papers
Method.
Background technology
Malaria be infect plasmodium through the blood as mosquito bite or input tape plasmodium person caused by insect-borne infectious disease,
First of the lethal parasitic disease in its residence whole world.The plasmodium for parasitizing human body has four kinds, i.e. Plasmodium vivax, malarlae malaria
Protozoon, plasmodium falciparum and Plasmodium ovale.It is mainly Plasmodium vivax and plasmodium falciparum in China, other two kinds are rare,
In recent years some cases of accidental external input.Different plasmodiums causes tertian fever, malarlae malaria, malignant malaria and oval malaria respectively.
Malaria is mainly shown as that periodic regularity breaks out, and goes cold all over, generates heat, hidrosis, after long-term repeatedly breaking-out, can cause anaemia and spleen
Enlargement.
According to French State Scientific Research Centre introduction, the annual whole world there are about 1,000,000 people and die from malaria, but medical field is so far not yet
Develop the effective vaccine for this infectious disease.Used as one kind of plasmodium, plasmodium falciparum is pathogenic extremely strong, and it is mainly deposited
It is the torrid areas in Africa, South America and Asia, and human malaria to 80% is related.For over ten years, medical field uses green grass or young crops always
Artemisin and its derived product are treated to patient, it not only cure rate it is high, and take effect quickly.But current scientists hair
Existing minority plasmodium falciparum shows the resistance to the action of a drug to artemisinin-based drug, and this has beaten alarm bell for the preventing and controlling of malaria.
Have for the method that predominantly detects of malaria both at home and abroad at present：Microscopical Method For Detection, polymerase chain reaction（PCR）, Immunological Method.
Laboratory diagnosis includes malaria aetology diagnostic techniques and Malaria Antibodies diagnostic techniques, and some methods are present in these diagnostic techniques
There is the defect in detection.Etiological diagnosis in malaria aetology diagnostic techniques（Microscope Blood piece is checked）Microscopical Method For Detection is time-consuming to be taken
Power, and need professional to operate, easily there is mistaken diagnosis and fail to pinpoint a disease in diagnosis when parasitemia is relatively low.Malaria aetology is examined
Pathogenic genes detection in disconnected technology（PCR）Detection needs laboratory apparatus and the personnel of specialty.Malaria Antibodies diagnostic techniques
（IFA）Fluorescence microscope, fluorescence results is needed to judge there is certain subjective factor.Cause of disease in malaria aetology diagnostic techniques is exempted from
Epidemiology is diagnosed（Rapid immunodiagnosis strip）Rapid immunodiagnosis strip is similar to microexamination plasmodium method, is mainly used in
Clinical labororatory's diagnosis of malaria patient and resident investigate with worm, with easily and fast, simply, do not need and instrument and equipment and have
The advantages of reviewer of experience.
The content of the invention
The present invention solves the technical problem of a kind of preparation method of plasmodium pf/pan Test papers of offer, the system
The test paper that Preparation Method is obtained is using a trace sample（5 microlitres）Can not only detect whether to have infected malignant malaria, can also examine
Whether survey has other three kinds of malaria in addition to malignant malaria（Malarlae malaria, ovale malaria, tertian fever）Individual event or mixed infection, examine
Breaking, quick and result is accurate.
In order to solve the above technical problems, one aspect of the present invention is：A kind of plasmodium pf/pan inspections are provided
The preparation method of test paper, including step is：
（1）Prepare pf re-suspension liquids：The pf re-suspension liquids include trishydroxymethylaminomethane, sucrose, trehalose, bovine serum albumin
White and polysorbas20, water and constant volume are dissolved in by trishydroxymethylaminomethane, sucrose, trehalose, bovine serum albumin(BSA) and polysorbas20, are adjusted
Section pH value is obtained to 8-9；Prepare pan re-suspension liquids：The pan re-suspension liquids include trishydroxymethylaminomethane, sucrose, marine alga
Sugar, bovine serum albumin(BSA) and casein, trishydroxymethylaminomethane, sucrose, trehalose, bovine serum albumin(BSA) and casein is molten
In water and constant volume, regulation pH value to 8-9 is obtained；Prepare coating base fluid：Trehalose be dissolved in pH value be 7-8 phosphate buffer solution simultaneously
Constant volume is obtained；
（2）Collaurum is mixed with potassium carbonate and pH value is adjusted, plasmodium falciparum lactic dehydrogenase is added（pfLDH）Monoclonal
Antibody, bovine serum albumin solution mixing, centrifugation obtain the first precipitation, and first precipitation is resuspended with the pf re-suspension liquids, obtains
To pf Immuno golds；Collaurum is mixed with potassium carbonate and pH value is adjusted, pLDH is added（panLDH）Dan Ke
Grand antibody, bovine serum albumin solution mixing, centrifugation obtain the second precipitation, and second precipitation is resuspended with the pan re-suspension liquids,
Obtain pan Immuno golds；
（3）The plasmodium falciparum lactic dehydrogenase is diluted with the coating base fluid（pfLDH）Monoclonal antibody obtains malignant malaria
Protozoon lactic dehydrogenase（pfLDH）Monoclonal antibody detection line coating buffer；The plasmodium lactic acid is diluted with the coating base fluid
Dehydrogenase（panLDH）Monoclonal antibody obtains pLDH（panLDH）Monoclonal antibody detection line coating buffer；With
Resist the coating base fluid dilution sheep anti-mouse igg more and obtain control line coating buffer；
（4）The immune gold pads of pf are obtained to gold pad metal spraying with the pf Immuno golds；Gold pad metal spraying is obtained with the pan Immuno golds
Pan is immunized gold pad；With the plasmodium falciparum lactic dehydrogenase（pfLDH）Monoclonal antibody detection line coating buffer, the malaria are former
Worm lactic dehydrogenase（panLDH）Monoclonal antibody detection line coating buffer and the control line coating buffer are being affixed on polychlorostyrene second respectively
Rule on the nitrocellulose filter of alkene base plate, obtain the polyvinyl chloride base plate of a film；
（5）By sample pad, the immune gold pads of the pf, the immune gold pads of the pan, described film polyvinyl chloride base plate and blotting paper
Fit together, cut into plasmodium pf/pan Test papers.
In a preferred embodiment of the present invention, step（1）Described in trishydroxymethylaminomethane described in pf re-suspension liquids,
The ratio of the sucrose, the trehalose, the bovine serum albumin(BSA), the polysorbas20 and constant volume cumulative volume is 0.242g：
10g：5g：1g：0.2 mL：100mL；Trishydroxymethylaminomethane, the sucrose, the marine alga described in the pan re-suspension liquids
The ratio of sugared, described bovine serum albumin(BSA), the casein and constant volume cumulative volume is 0.242g：10g：5g：1g：0.2g：
100mL。
In a preferred embodiment of the present invention, step（1）Described in pf re-suspension liquids pH value with 1M hydrochloric acid adjust to
8.5；The pH value of the pan re-suspension liquids is adjusted to 9.0 with 1M hydrochloric acid；The phosphate buffer solution is 0.01M, pH value is 7.4
Phosphate buffer solution.
In a preferred embodiment of the present invention, step（2）It is middle to prepare concretely comprising the following steps for pf Immuno golds：By collaurum with
Potassium carbonate mixes and stirs 5min after adjusting pH value, adds plasmodium falciparum lactic dehydrogenase（pfLDH）Monoclonal antibody is simultaneously stirred
5min is mixed, bovine serum albumin solution is added and is mixed and stirred for 5min, be centrifuged under conditions of 4 DEG C, rotating speed are for 6000r/min
30min obtains the first precipitation, and first precipitation is resuspended to being centrifuged the 1/10 of front volume with the pf re-suspension liquids, obtains pf and is immunized
Gold；Prepare concretely comprising the following steps for pan Immuno golds：5min is stirred after collaurum to be mixed with potassium carbonate and adjusted pH value, malaria is added
Protozoon lactic dehydrogenase（panLDH）Monoclonal antibody simultaneously stirs 5min, adds bovine serum albumin solution and is mixed and stirred for
5min, 30min is centrifuged under conditions of 4 DEG C, rotating speed are for 6000r/min and obtains the second precipitation, and second precipitation uses described
Pan re-suspension liquids are resuspended to being centrifuged the 1/20 of front volume, obtain pan Immuno golds.
In a preferred embodiment of the present invention, step（2）Described in potassium carbonate be 0.2M potassium carbonate, the 0.2M carbonic acid
Potassium and the plasmodium falciparum lactic dehydrogenase（pfLDH）The ratio of monoclonal antibody is 3uL/mL:10 μ g/mL, the 0.2M
Potassium carbonate and the pLDH（panLDH）The ratio of monoclonal antibody is 4uL/mL:5µg/mL；The ox blood
Pure protein solution is the bovine serum albumin solution that mass fraction is 10%, and the mass fraction is 10% bovine serum albumin(BSA)
The addition volume of solution is the 1/10 of the collaurum volume；Stirring is placed in what is realized on magnetic stirring apparatus.
In a preferred embodiment of the present invention, step（3）Described in plasmodium falciparum lactic dehydrogenase（pfLDH）Dan Ke
Plasmodium falciparum lactic dehydrogenase described in grand antibody detection line coating buffer（pfLDH）The concentration of monoclonal antibody is 2mg/mL；
The pLDH（panLDH）PLDH described in monoclonal antibody detection line coating buffer
（panLDH）The concentration of monoclonal antibody is 2mg/mL；Concentration anti-more than sheep anti-mouse igg is described in the control line coating buffer
2mg/mL。
In a preferred embodiment of the present invention, step（4）Described in gold pad use glass fibre membrane；The metal spraying is used
Point film metal-spraying equipment is completed.
In a preferred embodiment of the present invention, step（4）The polyvinyl chloride base plate of the middle point film that will be obtained is put into condition
To dry 2h, sealing preserve in 60 DEG C ± 3 DEG C, the environment of relative humidity≤30%.
In a preferred embodiment of the present invention, step（6）Described in sample pad be glass fibre membrane；The sample pad exists
Dried after soaking 10min using preceding use sample pad treatment fluid, wherein the sample pad treatment fluid includes trihydroxy methyl amino first
Alkane, TritonX-100, surfactant S7, surfactant S9 and BSA.
In a preferred embodiment of the present invention, the sample pad treatment fluid be by ratio be 1.21g：0.3mL：0.3g：
0.4g：S9 and BSA are soluble in water for the trishydroxymethylaminomethane of 1g, TritonX-100, surfactant S7, surfactant
100 mL are settled to, pH value is adjusted to 9.0.
The beneficial effects of the invention are as follows：The preparation method of plasmodium pf/pan Test papers of the invention, the detection for obtaining
Test paper is based on immune lateral flow chromatographic technique, is not required to any instrument and equipment, can be read by manual operations, naked eyes and judge whole blood
Whether plasmodium pf/pan is contained in sample, and diagnosis is quick and result is accurate, and person under inspection's body will not be damaged.The examination
Paper is using a trace sample（5μL）Plasmodium pf/pan can simultaneously be detected point out whether to have infected malignant malaria or removed
Other three kinds of malaria beyond malignant malaria（Malarlae malaria, ovale malaria, tertian fever）Individual event or mixed infection, it is adaptable to malaria doubt
Like the examination of the auxiliary diagnosis or malaria area of patient.
Specific embodiment
The technical scheme in the embodiment of the present invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of embodiment of the invention, rather than whole embodiments.Based on the embodiment in the present invention, this area is common
All other embodiment that technical staff is obtained under the premise of creative work is not made, belongs to the model of present invention protection
Enclose.
（One）Container and material
Tankage：Beaker, graduated cylinder, volumetric flask, glass bar, magnetic stirrer, electronic balance, liquid-transfering gun, point film metal-spraying equipment,
Guillotine, cutting machine, sealing machine, case pressing machine, pH meter, air dry oven.
Reagent auxiliary material：Gold chloride, trisodium citrate, K2CO3, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, Tris（Three
Hydroxymethyl aminomethane）、HCl、BSA（Bovine serum albumin(BSA)）, surfactant S7, surfactant S9, TritonX-100
（Qula is led to）, casein, Tween-20（Polysorbas20）, sucrose, trehalose.
Material：It is glass fibre membrane, nitrocellulose filter, polyvinyl chloride base plate, blotting paper, plastic clip, drier, disposable
Dropper, aluminium foil bag.
Environment：Cleaning, room temperature, dry area's relative humidity≤30%, wet area's relative humidity 45-65%, water quality：High purity water.
（Two）Preparation process
1st, the preparation of collaurum：Using trisodium citrate reduction method
50 mL mass fractions are 2% chlorauric acid solution preparation：The gold chloride for taking 1g/ bottles is dissolved in high purity water, volumetric flask constant volume
To 50 mL, 2-8 DEG C saves backup.
100 mL mass fractions are 5% citric acid three sodium solution preparation：Weigh 5.00g trisodium citrates and be dissolved in high purity water
In, volumetric flask constant volume to 100 mL is filtered with 0.22 μm of filter, now with the current.
Collaurum preparation method：Two clean beakers are taken, 500mL and 500mL high purity waters are separately added into, the former adds 2% chlorine
Auric acid solution, the latter adds 5% citric acid three sodium solution, in being heated to boiling on magnetic stirrer, treats that two kinds of solution all seethe with excitement
When, citric acid three sodium solution is quickly poured into the chlorauric acid solution of boiling, continue to boil 30min, pure water is increased after cooling dilute
Release, 2-8 DEG C saves backup.
Each component adds volume：
2nd, the pretreatment of sample pad：
1L sample pads treatment fluid is prepared：12.1g Tris, 3g surfactant S7,4g surfactant S9,1g BSA is weighed, it is molten
In high purity water, 3mL TritonX-100 are injected, volumetric flask constant volume to 1L adjusts pH for 9.0,2-8 DEG C of closed preservation with 1M hydrochloric acid
It is standby.
The glass fibre membrane of model SB08 is infiltrated in sample pad treatment fluid, 10min is soaked, taken out, relative humidity
Air-dry 4h for≤30% time, sanction goes to the edge to prevent edge effect, and room temperature preservation is standby in being fitted into aluminium foil bag.
3rd, re-suspension liquid, coating base fluid are prepared
100mL 1M hydrochloric acid is prepared：Measure 8.59 mL concentrated hydrochloric acids to be dissolved in high purity water, volumetric flask constant volume to 100 mL, room temperature is protected
Deposit standby.
100mL pf re-suspension liquids are prepared：Weigh 0.24g Tris, 10g sucrose, 5g trehaloses and 1g BSA and be dissolved in high purity water,
0.2mL Tween-20, the constant volume in 100mL volumetric flasks is added to adjust pH to be 8.5,2-8 DEG C with 1M hydrochloric acid and closed save backup.
100mL pan re-suspension liquids are prepared：Weigh 0.24g Tris, 10g sucrose, 5g trehaloses, 1g BSA and 0.2g junket eggs
High purity water is dissolved in vain, the constant volume in 100mL volumetric flasks is adjusted pH to be 9.0,2-8 DEG C and closed saved backup with 1M hydrochloric acid.
Phosphate buffer（PB）Prepare：Weigh 2.84g disodium hydrogen phosphates and be dissolved in high purity water, volumetric flask constant volume to 100 mL,
As 0.2M disodium phosphate solns；Weigh 2.40g sodium dihydrogen phosphates and be dissolved in high purity water, volumetric flask constant volume to 100 mL, as
0.2M sodium dihydrogen phosphates；Filtered with 0.22 μm of filter, 2-8 DEG C saves backup.Measure 19 mL0.2M sodium dihydrogen phosphates molten
Liquid, 81 mL0.2M disodium phosphate solns add 1900 mL high purity waters, mix, as 0.01M, the phosphoric acid buffer of pH=7.4
Liquid（PB）, 2-8 DEG C closed to save backup.
Coating base fluid is prepared：Weigh 1.00g trehaloses and be dissolved in phosphate buffer（PB）, volumetric flask constant volume to 100 mL, 2-8
It is DEG C closed to save backup.
4th, immuno-gold labeling
The M solution of potassium carbonate of 100mL 0.2 is prepared：Weigh 2.76 g potassium carbonate and be dissolved in high purity water, volumetric flask constant volume to 100mL, 2-
8 DEG C closed to save backup.
100mL mass fractions are 10% BSA solution preparation：Weigh 10 g BSA to be dissolved in high purity water, volumetric flask constant volume
To 100mL, 2-8 DEG C closed to save backup.
Mark plasmodium falciparum lactic dehydrogenase（pfLDH）Monoclonal antibody：Beaker is taken, collaurum is added, magnetic force is put and is stirred
Mix and stirred on machine, add the 0.2M potassium carbonate of 3uL/mL, adjust pH and stir 5min, the malignant malaria for being slowly added to 10 μ g/mL is former
Worm lactic dehydrogenase（pfLDH）Monoclonal antibody, stirs 5min, is slowly added to 10% BSA protective agents, to final concentration 1%, stirring
5min, rotating speed is that 30min is centrifuged under conditions of 6000r/min at 4 DEG C, and abandoning supernatant, precipitation is resuspended to former with re-suspension liquid
The 1/10 of volume, obtains pf Immuno golds, and 2-8 DEG C saves backup.
Mark pLDH（panLDH）Monoclonal antibody：Beaker is taken, collaurum is added, is put and stirred on magnetic stirrer
Mix, add the 0.2M potassium carbonate of 4uL/mL, adjust pH and stir 5min, be slowly added to the pLDH of 5 μ g/mL
（panLDH）Monoclonal antibody, stirs 5min, is slowly added to 10% BSA protective agents, to final concentration 1%, 5min is stirred, at 4 DEG C
Rotating speed under conditions of 6000 r/min to be centrifuged 30min, and abandoning supernatant, precipitation is resuspended to the 1/20 of original volume with re-suspension liquid,
Pan Immuno golds are obtained, 2-8 DEG C saves backup.
5th, point film metal spraying operation
Metal spraying：Opening point film metal-spraying equipment, metal spraying module is subdued, and line module is carried on to be come, and prevents scribe head from touching plate
Face, opens vavuum pump to 0.5 atmospheric pressure.
Enter operation picture by [operation], selection [program] is transformed into metal spraying mode of operation.Into after routine data picture
Program number is chosen, the parameters such as " length ", " concentration ", " speed ", " origin X, Y, Z " are editted, after having changed parameter, by [guarantor
Deposit] data are preserved, by [persistence] by persistence data.
Enter manual picture by [manual], by No. 2 pumps（Metal spraying pattern）In sample pipe insertion high purity water test tube, pipeline end
End prepares the vessel of water receiving.Selection [cleaning] is cleaned 5 times using high purity water.After high purity water test tube is taken away, selection [discharge opeing] is straight
No moisture in pipeline.Now by No. 2 pump sample pipes insertion test tubes, pf Immuno golds are full of up to pipeline is interior by [liquid feeding].
Gold pad is placed on the correct position in plate face, operation picture is entered by [operation], metal spraying is carried out by [beginning].
Metal spraying is finished, and by No. 2 pump sample pipes insertion high purity water test tubes, pipe end prepares the vessel of water receiving.Selection
[cleaning] is cleaned 5 times using high purity water.After high purity water test tube is taken away, selection [discharge opeing] is up to no moisture in pipeline.Now by 2
In number pump sample pipe insertion test tube, by [liquid feeding] until being full of pan Immuno golds in pipeline.It is suitable in plate face that gold pad is placed on
Position, operation picture is entered by [operation], and metal spraying is carried out by [beginning].
Metal spraying is finished, and by No. 2 pump sample pipes insertion high purity water test tubes, pipe end prepares the vessel of water receiving.Selection
[cleaning] is cleaned 5 times using high purity water.It is zero to open gas to air pressure by [the gas operation] of [main menu].
Pf gold pads after metal spraying treatment are with pan gold pads in the lower air-dried 1h of relative humidity≤30%.Immuno gold after dried process
Pad is placed in aluminium foil bag, plus drier room temperature sealing preserve is standby.
Prepare detection line coating buffer：Plasmodium falciparum lactic dehydrogenase is diluted with coating base fluid（pfLDH）Monoclonal antibody
To final concentration of 2mg/mL, detection line coating buffer i.e. T1 lines working solution is obtained；PLDH is diluted with coating base fluid
（panLDH）Monoclonal antibody obtains detection line coating buffer i.e. T2 lines working solution to final concentration of 2mg/mL, respectively by two kinds of T lines
2-8 DEG C of working solution is saved backup.
Prepare control line coating buffer：To be resisted sheep anti-mouse igg with coating base fluid more and be diluted to final concentration of 2mg/mL, obtain right
It is C line working solutions according to line coating buffer, 2-8 DEG C saves backup.
Point film：Line module is subdued, metal spraying module is carried on to come, form certain drop, prevent metal spraying head from touching
Plate face.
Enter operation picture by [operation], selection [program] is transformed into line mode of operation.Into after routine data picture
Program number is chosen, the parameters such as " length ", " concentration ", " speed ", " origin X, Y, Z " are editted, wherein " speed " can not be too fast,
It is 80mm/s, after having changed parameter, data is preserved by [preservation], by [persistence] by persistence data.
Enter manual picture by [manual], by 1, No. 3 pumps（Line pattern）In sample pipe insertion high purity water test tube, pipeline
End prepares the vessel of water receiving.Selection [cleaning] is cleaned 5 times using high purity water.After high purity water test tube is taken away, select [discharge opeing]
Until no moisture in pipeline.Now by No. 1 pump（Line pattern）In sample pipe insertion C line working solutions, by No. 3 pumps（Line mould
Formula）In sample pipe insertion T1 line working solutions, by [liquid feeding] until being full of coating buffer in pipeline.Nitric acid is pasted with by ready
The polyvinyl chloride base plate of cellulose membrane is placed on the correct position in plate face, and operation picture is entered by [operation], is carried out by [beginning]
Line.
Scribing operation is finished, and by 1, No. 3 pump sample pipes insertion high purity water test tubes, pipe end prepares the device of water receiving
Ware.Selection [cleaning] is cleaned 5 times using high purity water.After high purity water test tube is taken away, selection [discharge opeing] is up to no moisture in pipeline.
Close No. 1 pump.By No. 3 pumps（Line pattern）In sample pipe insertion T2 line working solutions, by [liquid feeding] until full of bag in pipeline
By liquid.The ready polyvinyl chloride base plate for being pasted with nitrocellulose filter is placed on the correct position in plate face, by [operation]
Into operation picture, rule by [beginning].Scribing operation is finished, and by No. 3 pump sample pipes insertion high purity water test tubes, is managed
Road end prepares the vessel of water receiving.Selection [cleaning] is cleaned 5 times using high purity water.Close point film gold spraying instrument.After the completion of line
Nitrocellulose filter is reaction film, and the polyvinyl chloride base plate that will be stained with reaction film is placed in drying box, 60 DEG C ± 3 DEG C, relatively wet
Degree≤30%, dries 2h.The polyvinyl chloride base plate is placed in aluminium foil bag after the completion of drying, plus drier room temperature sealing preserve
It is standby.
6th, assembling cutting, assembling, packaging
Sample pad, the immune gold pads of pf, the immune gold pads of pan, the polyvinyl chloride base plate and blotting paper that are stained with reaction film are assembled in one
Rise, 4mm test strips are cut into using automatic cutting machine.Every test strips are assembled in plastic clip, are compressed using case pressing machine.Add
It is fitted into preservation, as finished product in aluminium foil bag after one drier, a disposable dropper.
7th, sample lysate is prepared
100mL lysates are prepared：Weigh 0.24g Tris, 0.1g casein and 1.5g sodium chloride is dissolved in high purity water, inject
1.5mL TritonX-100, the constant volume in 100mL volumetric flasks is adjusted pH to be 9.0,2-8 DEG C and closed is saved backup with 1M hydrochloric acid.
The plasmodium pf/pan Test papers secure plasmodium falciparum lactic acid and take off in the detection zone of nitrocellulose filter
Hydrogen enzyme（pfLDH）Monoclonal antibody, pLDH（panLDH）Monoclonal antibody, sheep anti mouse is secured in check plot
IgG resists more, the pre-coated plasmodium falciparum lactic dehydrogenase on glass fibre element film（pfLDH）Monoclonal antibody-collaurum, malaria
Protozoon lactic dehydrogenase（panLDH）Monoclonal antibody-collaurum.During test, 5uL whole bloods are added dropwise, then a drop is added dropwise（About 50 μ L）
Buffer solution, horizontal positioned.Plasmodium falciparum lactic dehydrogenase in immune gold pad（pfLDH）Monoclonal antibody-collaurum and malaria
Protozoon lactic dehydrogenase（panLDH）Monoclonal antibody-collaurum can be dissolved, and plasmodium falciparum lactic dehydrogenase is contained such as in sample
Enzyme（pfLDH）Or pLDH（panLDH）, plasmodium falciparum lactic dehydrogenase（pfLDH）Monoclonal antibody-glue
Plasmodium falciparum lactic dehydrogenase in body gold and sample（pfLDH）It is combined together, forms " Ag-Ab-gold " compound,
PLDH（panLDH）PLDH in monoclonal antibody-collaurum and sample（panLDH）Knot
It is combined, forms " Ag-Ab-gold " compound, compound rearward moves along test strips, arrives first at and be coated with evil
Property pLDH（pfLDH）The detection zone of monoclonal antibody（T1）, " Ag-Ab-gold " compound will be with pernicious
PLDH（pfLDH）Monoclonal antibody is combined, and is detained in detection line, forms a line for red,
This represents positive findings.The depth of line color does not have proportionate relationship with the antigen levels in sample.If not no band in the area
The lines of color are free of plasmodium falciparum lactic dehydrogenase in representing sample（pfLDH）Or plasmodium falciparum lactic dehydrogenase
（pfLDH）Less than this test paper minimum detectability, free " Ag-Ab-gold " compound continues to be moved to test paper rear, reaches
It has been coated with pLDH（panLDH）The detection zone of monoclonal antibody（T2）, Ag-Ab-gold " and compound will be same
PLDH（panLDH）Monoclonal antibody is combined, and is detained in detection line, forms a line for red,
This represents positive findings.The depth of line color does not have proportionate relationship with the antigen levels in sample.If not no band in the area
The lines of color are free of pLDH in representing sample（panLDH）Or pLDH（panLDH）It is dense
Degree is less than this test paper minimum detectability.Compound is continued to move to, and arrival has been coated with the check plot resisted sheep anti-mouse igg more, free
" antibody-gold " compound will be combined together with sheep anti-mouse igg and be enriched in check plot（C）, form a line for red.Control
The appearance of line proves that ELISA test strip function is normal.Whether no matter plasmodium falciparum lactic dehydrogenase is contained in sample（pfLDH）
Or pLDH（panLDH）, in efficiency test, the check plot of test strips should all occur light red to aubergine
Control line.Sample is continued to move to, and uptake zone is entered by check plot.The effect of uptake zone is the remaining compound of absorption, is made
It is moved in test strips, and eliminates the color of background.Timing, 15 ~ 20 from the sample mixed liquor after test strips add dilution
Result can be read in minute.
Embodiments of the invention are the foregoing is only, the scope of the claims of the invention is not thereby limited, it is every to utilize this hair
Equivalent structure or equivalent flow conversion that bright description is made, or directly or indirectly it is used in other related technology necks
Domain, is included within the scope of the present invention.
Claims (10)
1. a kind of preparation method of plasmodium pf/pan Test papers, it is characterised in that be including step：
（1）Prepare pf re-suspension liquids：The pf re-suspension liquids include trishydroxymethylaminomethane, sucrose, trehalose, bovine serum albumin
White and polysorbas20, water and constant volume are dissolved in by trishydroxymethylaminomethane, sucrose, trehalose, bovine serum albumin(BSA) and polysorbas20, are adjusted
Section pH value is obtained to 8-9；Prepare pan re-suspension liquids：The pan re-suspension liquids include trishydroxymethylaminomethane, sucrose, marine alga
Sugar, bovine serum albumin(BSA) and casein, trishydroxymethylaminomethane, sucrose, trehalose, bovine serum albumin(BSA) and casein is molten
In water and constant volume, regulation pH value to 8-9 is obtained；Prepare coating base fluid：Trehalose be dissolved in pH value be 7-8 phosphate buffer solution simultaneously
Constant volume is obtained；
（2）Collaurum is mixed with potassium carbonate and pH value is adjusted, plasmodium falciparum lactic dehydrogenase pfLDH monoclonals is added and is resisted
Body, bovine serum albumin solution mixing, centrifugation obtain the first precipitation, and first precipitation is resuspended with the pf re-suspension liquids, obtains
Pf Immuno golds；Collaurum is mixed with potassium carbonate and pH value is adjusted, pLDH panLDH monoclonals is added and is resisted
Body, bovine serum albumin solution mixing, centrifugation obtain the second precipitation, and second precipitation is resuspended with the pan re-suspension liquids, obtains
Pan Immuno golds；
（3）The plasmodium falciparum lactic dehydrogenase pfLDH monoclonal antibodies are diluted with the coating base fluid obtain malignant malaria original
Worm lactic dehydrogenase pfLDH monoclonal antibody detection line coating buffers；The plasmodium lactic dehydrogenase is diluted with the coating base fluid
Enzyme panLDH monoclonal antibodies obtain pLDH panLDH monoclonal antibody detection line coating buffers；Use the coating
Resist base fluid dilution sheep anti-mouse igg more and obtain control line coating buffer；
（4）The immune gold pads of pf are obtained to gold pad metal spraying with the pf Immuno golds；Gold pad metal spraying is obtained with the pan Immuno golds
Pan is immunized gold pad；With the plasmodium falciparum lactic dehydrogenase pfLDH monoclonal antibody detection lines coating buffer, the plasmodium
Lactic dehydrogenase panLDH monoclonal antibody detection line coating buffers and the control line coating buffer are being affixed on polyvinyl chloride bottom respectively
Rule on the nitrocellulose filter of plate, obtain the polyvinyl chloride base plate of a film；
（5）By sample pad, the immune gold pads of the pf, the immune gold pads of the pan, described film polyvinyl chloride base plate and blotting paper
Fit together, cut into plasmodium pf/pan Test papers.
2. the preparation method of plasmodium pf/pan Test papers according to claim 1, it is characterised in that step（1）In
It is trishydroxymethylaminomethane described in the pf re-suspension liquids, the sucrose, the trehalose, the bovine serum albumin(BSA), described
The ratio of polysorbas20 and constant volume cumulative volume is 0.242g：10g：5g：1g：0.2 mL：100mL；Three described in the pan re-suspension liquids
Hydroxymethyl aminomethane, the sucrose, the trehalose, the bovine serum albumin(BSA), the casein and constant volume cumulative volume
Ratio is 0.242g：10g：5g：1g：0.2g：100mL.
3. the preparation method of plasmodium pf/pan Test papers according to claim 1, it is characterised in that step（1）In
The pH value of the pf re-suspension liquids is adjusted to 8.5 with 1M hydrochloric acid；The pH value of the pan re-suspension liquids is adjusted to 9.0 with 1M hydrochloric acid；It is described
Phosphate buffer solution is 0.01M, the phosphate buffer solution that pH value is 7.4.
4. the preparation method of plasmodium pf/pan Test papers according to claim 1, it is characterised in that step（2）In
Prepare concretely comprising the following steps for pf Immuno golds：5min is stirred after collaurum to be mixed with potassium carbonate and adjusted pH value, malignant malaria is added
Protozoon lactic dehydrogenase pfLDH monoclonal antibodies simultaneously stir 5min, add bovine serum albumin solution and are mixed and stirred for 5min,
30min is centrifuged under conditions of 4 DEG C, rotating speed are for 6000r/min and obtains the first precipitation, first precipitation is resuspended with the pf
Liquid is resuspended to being centrifuged the 1/10 of front volume, obtains pf Immuno golds；Prepare concretely comprising the following steps for pan Immuno golds：By collaurum and carbon
Sour potassium mixes and stirs 5min after adjusting pH value, adds pLDH panLDH monoclonal antibodies and stirs 5min,
Add bovine serum albumin solution and be mixed and stirred for 5min, 30min is centrifuged under conditions of 4 DEG C, rotating speed are for 6000r/min
The second precipitation is obtained, second precipitation is resuspended to being centrifuged the 1/20 of front volume with the pan re-suspension liquids, obtains pan Immuno golds.
5. the preparation method of the plasmodium pf/pan Test papers according to claim 1 or 4, it is characterised in that step（2）
Described in potassium carbonate be 0.2M potassium carbonate, the 0.2M potassium carbonate and the plasmodium falciparum lactic dehydrogenase（pfLDH）Dan Ke
The ratio of grand antibody is 3uL/mL:10 μ g/mL, the 0.2M potassium carbonate and the pLDH panLDH monoclonals
The ratio of antibody is 4uL/mL:5µg/mL；The bovine serum albumin solution is molten for the bovine serum albumin(BSA) that mass fraction is 10%
Liquid, the mass fraction is that the addition volume of 10% bovine serum albumin solution is the 1/10 of the collaurum volume；Stirring is
It is placed in what is realized on magnetic stirring apparatus.
6. the preparation method of plasmodium pf/pan Test papers according to claim 1, it is characterised in that step（3）In
Plasmodium falciparum lactic dehydrogenase described in the plasmodium falciparum lactic dehydrogenase pfLDH monoclonal antibody detection line coating buffers
The concentration of pfLDH monoclonal antibodies is 2mg/mL；The pLDH panLDH monoclonal antibodies detection line coating
The concentration of the monoclonal antibody of pLDH panLDH described in liquid is 2mg/mL；Described in the control line coating buffer
The concentration resisted sheep anti-mouse igg is 2mg/mL more.
7. the preparation method of plasmodium pf/pan Test papers according to claim 1, it is characterised in that step（4）In
The gold pad uses glass fibre membrane；The metal spraying is completed using point film metal-spraying equipment.
8. the preparation method of plasmodium pf/pan Test papers according to claim 1, it is characterised in that step（4）In
The polyvinyl chloride base plate of the point film that will be obtained is put into condition to dry 2h in 60 DEG C ± 3 DEG C, the environment of relative humidity≤30%, close
Envelope is preserved.
9. the preparation method of plasmodium pf/pan Test papers according to claim 1, it is characterised in that step（6）In
The sample pad is glass fibre membrane；The sample pad is dried after 10min is soaked using preceding use sample pad treatment fluid, wherein institute
State sample pad treatment fluid include trishydroxymethylaminomethane, TritonX-100, surfactant S7, surfactant S9 and
BSA。
10. the preparation method of plasmodium pf/pan Test papers according to claim 9, it is characterised in that the sample
Pad treatment fluid be by ratio be 1.21g：0.3mL：0.3g：0.4g：The trishydroxymethylaminomethane of 1g, TritonX-100, surface
Activating agent S7, surfactant S9 and BSA are soluble in water to be settled to 100 mL, and pH value is adjusted to 9.0.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710009907.8A CN106908602A (en) | 2017-01-06 | 2017-01-06 | A kind of preparation method of plasmodium pf/pan Test papers |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710009907.8A CN106908602A (en) | 2017-01-06 | 2017-01-06 | A kind of preparation method of plasmodium pf/pan Test papers |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106908602A true CN106908602A (en) | 2017-06-30 |
Family
ID=59207402
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710009907.8A Pending CN106908602A (en) | 2017-01-06 | 2017-01-06 | A kind of preparation method of plasmodium pf/pan Test papers |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106908602A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109738637A (en) * | 2019-01-17 | 2019-05-10 | 南方医科大学 | A kind of quick detection kit and detection method of skin care item mesocuticle Porcine HGF |
| CN109932508A (en) * | 2019-03-14 | 2019-06-25 | 河南省人民医院 | A kind of syphilis helicoid antibody detection card, kit and detection method |
| CN110146697A (en) * | 2019-06-03 | 2019-08-20 | 中国疾病预防控制中心寄生虫病预防控制所 | A kind of immunity test strip of quick detection falciparum malaria, plasmodium vivax, plasmodium vivax and ovale malaria |
| WO2020049444A1 (en) * | 2018-09-03 | 2020-03-12 | Module Innovations Private Limited | A device and a lateral flow assay method for differential identification of plasmodium species |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997024141A1 (en) * | 1995-12-29 | 1997-07-10 | Piper Robert C | Monoclonal antibodies and immuno-capture method for quantitation and speciation of malaria parasites |
| CN101806799A (en) * | 2009-06-09 | 2010-08-18 | 北京金沃夫生物工程科技有限公司 | Reagent kit for fast detecting and identifying ignant malaria and tertian malaria and preparation method thereof |
| CN104450625A (en) * | 2014-11-17 | 2015-03-25 | 深圳市菲鹏生物股份有限公司 | Hybridoma cells capable of secreting anti-plasmodial lactate dehydrogenase monoclonal antibody, monoclonal antibody and application of monoclonal antibody |
| CN104678098A (en) * | 2015-03-13 | 2015-06-03 | 苏州万木春生物技术有限公司 | Preparation method of joint-inspection test paper for cardiac troponin I and heart-type fatty acid binding protein |
-
2017
- 2017-01-06 CN CN201710009907.8A patent/CN106908602A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997024141A1 (en) * | 1995-12-29 | 1997-07-10 | Piper Robert C | Monoclonal antibodies and immuno-capture method for quantitation and speciation of malaria parasites |
| CN101806799A (en) * | 2009-06-09 | 2010-08-18 | 北京金沃夫生物工程科技有限公司 | Reagent kit for fast detecting and identifying ignant malaria and tertian malaria and preparation method thereof |
| CN104450625A (en) * | 2014-11-17 | 2015-03-25 | 深圳市菲鹏生物股份有限公司 | Hybridoma cells capable of secreting anti-plasmodial lactate dehydrogenase monoclonal antibody, monoclonal antibody and application of monoclonal antibody |
| CN104678098A (en) * | 2015-03-13 | 2015-06-03 | 苏州万木春生物技术有限公司 | Preparation method of joint-inspection test paper for cardiac troponin I and heart-type fatty acid binding protein |
Non-Patent Citations (1)
| Title |
|---|
| 唐凤 等: "万孚疟原虫检测试剂盒检测卵形疟原虫效果评价及影响因素分析", 《中国血吸虫病防治杂志》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020049444A1 (en) * | 2018-09-03 | 2020-03-12 | Module Innovations Private Limited | A device and a lateral flow assay method for differential identification of plasmodium species |
| CN109738637A (en) * | 2019-01-17 | 2019-05-10 | 南方医科大学 | A kind of quick detection kit and detection method of skin care item mesocuticle Porcine HGF |
| CN109932508A (en) * | 2019-03-14 | 2019-06-25 | 河南省人民医院 | A kind of syphilis helicoid antibody detection card, kit and detection method |
| CN110146697A (en) * | 2019-06-03 | 2019-08-20 | 中国疾病预防控制中心寄生虫病预防控制所 | A kind of immunity test strip of quick detection falciparum malaria, plasmodium vivax, plasmodium vivax and ovale malaria |
| CN110146697B (en) * | 2019-06-03 | 2022-02-11 | 中国疾病预防控制中心寄生虫病预防控制所 | Immune test strip for rapidly detecting malignant malaria, vivax malaria, triumphaniae and oval malaria |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104198703B (en) | People's mycoplasma pneumoniae gold label silver stain immunochromatographytest test kit and its preparation method and application | |
| CN106908602A (en) | A kind of preparation method of plasmodium pf/pan Test papers | |
| CN106706908A (en) | Preparation method of hepatitis B virus surface antigen test paper | |
| CN107942061A (en) | A kind of test card, preparation and its detection method for detecting transmissible gastro-enteritis virus antibody | |
| CN211148669U (en) | S-antigen-based novel coronavirus 2019-nCoV antibody rapid detection kit | |
| CN101762705A (en) | Colloidal gold immunochromatographic test strip for detecting wild-type classical swine fever virus | |
| CN103048459B (en) | Immune detection reagent for detecting respiratory syncytial virus | |
| CN106771123A (en) | A kind of preparation method of dengue virus IgM/IgG antibody Test paper | |
| CN113533721B (en) | Colloidal gold method detection test strip for influenza A/B virus antigen and preparation method thereof | |
| CN101377493A (en) | Kidney syndrome blooding diagnosis test paper strip, preparing method and detection reagent kit thereof | |
| CN101074956B (en) | Immune chromatography test paper for inspecting legionella pneumophilia antibody and its production | |
| CN105044367A (en) | Colloidal gold immunochromatographic assay test strip for detecting dexamethasone residues in milk | |
| CN101738475B (en) | Hepatitis E virus antibody detection kit and preparation method thereof | |
| CN110007095A (en) | A kind of dengue virus NS 1 antigen test strip, kit and preparation method thereof | |
| CN107957497A (en) | A kind of bird flu H5 subtype virus antibody rapid quantitative detection reagent box and its application | |
| CN110133279A (en) | A kind of joint inspection colloidal gold strip detecting transgenosis BT albumen and CP4-EPSPS albumen | |
| CN106501513B (en) | A kind of Potyvirus Rapid detection test strip and its preparation method and application | |
| CN109187968A (en) | A kind of swine fever virus and the bigeminy gold mark detection test paper of porcine pseudorabies virus and preparation method thereof | |
| CN105753981B (en) | The immune chromatography reagent kit of anti-human Respiratory Syncytial Virus(RSV) N protein antibody and the application antibody | |
| CN102226811A (en) | Preparation method of diagnostic test paper for detecting H-FABP (heart-type fatty acid binding protein) | |
| CN101706499A (en) | FLAG fusion tag colloidal gold test strip and preparation method thereof | |
| CN101587122A (en) | Swine Hemagglutinating encephalomyelitis virus colloidal gold antigen detecting test paper and preparing method thereof | |
| CN104360068A (en) | Semi-quantitative rapid detection test paper for white spot syndrome virus (WSSV), and preparation method of test paper | |
| CN102539768A (en) | EB (Epstein-Barr) virus Zta IgA (Immunoglobulin A) antibody colloidal gold detection kit and preparation method thereof | |
| CN108226514A (en) | A kind of newcastle disease virus antibody rapid quantitative detection reagent box and its application |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170630 |