CN109459566A - Immuno-chromatographic test paper strip and immuno-chromatography detection device including it - Google Patents
Immuno-chromatographic test paper strip and immuno-chromatography detection device including it Download PDFInfo
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention relates to a kind of immuno-chromatographic test paper strips, comprising: sample pad；Film is detected, the sample pad is connected to, retention line, detection line and nature controlling line are successively arranged on the detection film；Water absorption pad is connected to the detection film.The present invention also provides the immuno-chromatography detection devices including above-mentioned immuno-chromatographic test paper strip.The present invention is by adding a retention line (nature controlling line) in its front end, so that front and back has two nature controlling lines, this retention line (nature controlling line) can retain the marker of most of small aggregation, so that in subsequent chromatography, baseline is greatly reduced, and then improves specificity, sensitivity and the accuracy of product testing.
Description
Technical field
The present invention relates to immuno-chromatographic test paper strip and including its immuno-chromatography detection device, belong to immunoassay technology neck
Domain.
Background technique
Immunochromatography is divided into longitudinal chromatography and two kinds of lateral chromatography.Immune lateral chromatography diagnostic techniques as a kind of stabilization and
Practical technology is suitble to use in the real-time test (POCT) of multiplicity or scene, and being divided by mark part mainly has colloidal gold to exempt from
Epidemic disease lateral chromatography method, common fluorescent be immunized lateral chromatography method, time-resolved fluoroimmunoassay lateral chromatography method, on turn electrochemiluminescent immunoassay side
Lateral chromatography method etc. is immunized to chromatography, quantum dot fluorescence, mainly has nitrocellulose filter, composite material by coating part
(fusion5), micro-fluidic etc..
Immune chromatography method (immunochromatography assay, ICA) is built in nineteen ninety by Oskiowicz etc.
It is vertical.The marker of Oskiowicz application is selenium, thereafter generally using easy colloidal gold, referred to as colloidal gold immunochromatographimethod then
Method.After the development of immunochromatography (immunochromatography) and colloidal gold technique, the especially nineties, colloid
Gold immune lateral chromatography method (Gold immunochromatography assay, GICA) diagnoses the illness in vitro in detection
Extensive use is arrived.
But carry out quantitative detection now, project precision and repeatability are required it is higher and higher, by the hair in many years
Exhibition, nearest all kinds of fluorescence immunoassay lateral chromatography methods emerge one after another.Mainstream advanced technology is had become to be praised highly by many companies.But it is existing
Have there are a certain proportion of " small aggregation marker " in fluorescence (colloidal gold) marker of technology, it is bottom-up to be chromatographed
When, especially All-in-One project, marker is more, and baseline can be relatively high, and T line integral, which is quadratured, will receive interference, and then to spy
The opposite sex, sensitivity, accuracy interfere.
Summary of the invention
In view of this, lateral layer is immunized to reduce the main purpose of the present invention is to provide a kind of immuno-chromatographic test paper strip
Analysis method product, the especially baseline of All-in-One project, decrease T line integral, which is quadratured, will receive interference, and then improve the spy detected
Anisotropic, sensitivity and accuracy.
In order to reach above-mentioned purpose, the present invention provides a kind of immuno-chromatographic test paper strips, comprising:
Sample pad；
Film is detected, the sample pad is connected to, retention line, detection line and nature controlling line are successively arranged on the detection film；
Water absorption pad is connected to the detection film.
As the preferred embodiment of above-mentioned immuno-chromatographic test paper strip, wherein the detection film is nitrocellulose filter, the inspection
It surveys and is successively coated with the retention antibody respectively as retention line, detection line and nature controlling line on film, detect antibody and Quality Control antibody, three
Film concentration ratio of drawing between person is (0.01-0.5): (0.5-3): (0.5-3).
As the preferred embodiment of above-mentioned immuno-chromatographic test paper strip, wherein the retention antibody is goat-anti chicken IgY or biotin-
Avidin.
As the preferred embodiment of above-mentioned immuno-chromatographic test paper strip, wherein being coated with goat-anti chicken IgY conduct on the detection film
Retain line.
As the preferred embodiment of above-mentioned immuno-chromatographic test paper strip, wherein being coated with biotin-avidin on the detection film
As retention line.
As the preferred embodiment of above-mentioned immuno-chromatographic test paper strip, wherein the retention line detection line is closer to described
Sample pad；The nature controlling line detection line is closer to the water absorption pad.
As the preferred embodiment of above-mentioned immuno-chromatographic test paper strip, wherein retention the distance between the line and detection line are 3-
10mm, the distance between the nature controlling line and detection line are 3-6mm.
As the preferred embodiment of above-mentioned immuno-chromatographic test paper strip, wherein the sample pad is glass fibre element film or polyester
Film；The water absorption pad is blotting paper.
As the preferred embodiment of above-mentioned immuno-chromatographic test paper strip, wherein the immuno-chromatographic test paper strip further includes bottom plate, institute
Detection film, sample pad and water absorption pad is stated to be both secured on the bottom plate.
The present invention also provides the preparation methods of above-mentioned immuno-chromatographic test paper strip, comprising the following steps:
1) coating retention line, detection line and nature controlling line on detection film, will test the two sides of film respectively with sample pad and suction
After water cushion bonding and it is fixed on bottom plate, obtains test strips blank；
2) the test strips blank that step 1) obtains is cut into required size up to the immuno-chromatographic test paper strip.
Invention further provides a kind of immuno-chromatography detection devices, including above-mentioned test strips and the package examination
The card plug of paper slip, the test strips are assembled into the card plug, and the card plug is equipped with well, the inspection in the top of sample pad
Survey line is away from well 15-25mm.
As the preferred embodiment of above-mentioned immuno-chromatography detection device, wherein the card plug is made of upper cover and bottom cover；
The bottom cover includes: the multiple first positioning holes and multiple first fastening columns positioned at its surrounding；Multiple described first
Multiple the first limiting sections for limiting test strips transverse shifting and multiple vertical for limiting test strips are equipped between location hole
To the second mobile limiting section, it is equipped between multiple first fastening columns multiple for limiting the first of test strips transverse shifting
Limiting section and multiple the second limiting sections vertically moved for limiting test strips；Multiple first limiting sections and multiple described
Second limiting section, which encloses, is set as test strips placing groove；
The upper cover include with first multiple second location holes for matching of fastening column and with the first positioning hole
The multiple second fastening columns matched；The upper cover further include be set to it is multiple it is described second fastening columns between and observation window and
Multiple fixed parts between well.
As the preferred embodiment of above-mentioned immuno-chromatography detection device, wherein the top of the detection film is equipped with and adopts for data
The observation window of collection, and the observation window is opened in the corresponding position in middle part of the upper Gai Shangyu test strips placing groove.
As the preferred embodiment of above-mentioned immuno-chromatography detection device, wherein the upper cover is corresponding with the sample pad
Well is offered at position.
Invention further provides a kind of manufacturing methods of immuno-chromatography detection device, comprising the following steps:
1) coated antibody: retention antibody, detection antibody and Quality Control antibody are coated with onto detection film, drying for standby；
2) labelled antibody: retention antibody and detection antibody are obtained into labelled antibody with label substance markers, are stored in storing liquid
It is interior, it is spare；
3) marker carrier prepares: above-mentioned marker is dried standby on marker carrier by required concentration specking
With；
4) prepared by sample pad: sample pad is impregnated without pretreatment or with sample treatment liquid or specking sample pad, carries out pre-
Processing, drying for standby；
5) test strips are assembled: will coating it is good needed for antibody detection film and processed sample pad and water absorption pad according to
Bottom plate is pasted together and be fixed on to mold, and is cut into the test strips of required width, is later assembled into the reagent strip accordingly
In card plug.
The preferred embodiment of manufacturing method as above-mentioned immuno-chromatography detection device, the marker be latex fluorescence, when
Between resolved fluorometric, upper transfer shine, quantum dot fluorescence, fluorescent dye, colloidal gold or color micro-sphere；The marker carrier is to survey
Test tube, bottle, suction nozzle, syringe, card plug sample mixing slot, sample pad or detection film；The mode of the drying is evaporation drying, vacuum is dry
Dry or freeze-drying；The width of the test strips is 3-6mm.
Immune its principle of lateral chromatography method is that the antibody (antigen) of specificity is first fixed on to a certain area of nitrocellulose membrane
Band, after sample (urine, serum, blood plasma, whole blood or other samples) are immersed in dry nitrocellulose membrane one end, due to capillary
Effect, sample will be moved forward along the film, when being moved to the region for being fixed with antibody (antigen), corresponding antigen in sample
(antibody) is specifically bound with the antibody (antigen), if the region can be made to show certain color with immune colloid gold,
Visually observe or realized with corresponding readout instrument the immunodiagnosis of specificity.If needing mating readout instrument with fluorescent marker
Data acquisition, processing, checking computations obtain corresponding quantitative concentrations value.
The invention has the following advantages:
The present invention is by adding a retention line (nature controlling line) in its front end, so that front and back has two nature controlling lines, this
Item retention line (nature controlling line) can retain the marker of most of small aggregation, so that baseline is greatly reduced in subsequent chromatography, into
And improve product specificity, sensitivity and accuracy.Since the retention line of front end only plays crown_interception, this retention line
(nature controlling line) must be independent system, cannot influence subsequent detection line.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of one of embodiment of the present invention immuno-chromatographic test paper strip；
Fig. 2 is a kind of schematic diagram of internal structure of bottom cover in immuno-chromatography detection device embodiment of the present invention；
Fig. 3 is a kind of schematic diagram of internal structure of upper cover in immuno-chromatography detection device embodiment of the present invention；
Fig. 4 is immuno-chromatography detection device of the present invention fluorescence curve figure at tester's Procalcitonin (PCT).
Wherein, bottom plate -1；Glass fibre element film -2；Nitrocellulose filter -3；Blotting paper -4；31- detection line；32- Quality Control
Line；33- retains line；
10- bottom cover；11- observation window；12- well；13- first positioning hole；14- first fastens column；15- first is limited
Portion；The second limiting section of 16-；
20- upper cover；23- second location hole；24- second fastens column；25- fixed part；26- fixed part.
Specific embodiment
For a further understanding of the present invention, the preferred embodiment of the invention is described below with reference to embodiment, still
It should be appreciated that these descriptions are only further explanation the features and advantages of the present invention, rather than to the claims in the present invention
Limitation.
As shown in Figure 1, the present invention provides a kind of immuno-chromatographic test paper strips, comprising:
Glass fibre element film 2, size are provided that length is 15-30mm, width 4-6mm, with a thickness of 500-1000
μm；
Nitrocellulose filter 3 is connected to the glass fibre element film 2, and size is provided that length is 20-35mm,
Width is 4-6mm, with a thickness of 0.2-0.3mm；Retention line 33, detection line 31 and matter are successively arranged on the nitrocellulose filter 3
Line 32 is controlled, the distance between the retention line 33 and detection line 31 are 3-10mm, and the nature controlling line 32 is close to blotting paper 4, away from inspection
Survey line 3-6mm；
Blotting paper 4 is connected to the nitrocellulose filter 3, and size is provided that length is 10-30mm, and width is
4-6mm, with a thickness of 900-1800 μm；The material of the blotting paper 4 is glass and velveteen.
Cutting respectively as retention line 33, detection line 31 and nature controlling line 32 is coated on the nitrocellulose filter 3 respectively
Stay antibody, detection antibody and Quality Control antibody.The retention antibody is goat-anti chicken IgY or biotin-avidin.Due to use and mouse
Label of the remote chicken IgY of antibody kind as product retention wire body system, i.e. goat-anti chicken IgY coating, chicken IgY label, to avoid
Cross reaction the case where side occurs.Using biotin-avidin as retention line, i.e. Avidin or streptavidin coating arrives
On nitrocellulose filter, biotinylated protein, albumen reconnects marker or biotin and is directly connected to marker (latex is glimmering
Light, time-resolved fluorescence, it is upper transfer shine, quantum dot, fluorescent dye or other display markers).
The retention line 33 detection line 31 is closer to the glass fibre element film 2；The nature controlling line 32 is relatively described
Detection line 31 is closer to the blotting paper 4.
The immuno-chromatographic test paper strip further includes bottom plate 1, and size is provided that length is 20-30cm, and width is
6-8cm, with a thickness of 0.25-0.5mm；The nitrocellulose filter 3, glass fibre element film 2 and blotting paper 4 are both secured to the bottom
On plate 1.
The preparation method of above-mentioned immuno-chromatographic test paper strip, comprising the following steps:
1) coating retention line 33, detection line 31 and nature controlling line 32 on nitrocellulose filter 3, by nitrocellulose filter 3
After two sides are Nian Jie with glass fibre element film 2 and blotting paper 4 respectively and it is fixed on bottom plate 1, obtains test strips blank；
2) the test strips blank that step 1) obtains is cut into required size up to the immuno-chromatographic test paper strip.
Invention further provides a kind of immuno-chromatography detection devices, including above-mentioned test strips and the package examination
The card plug of paper slip, the test strips are assembled into the card plug, and the card plug is equipped with well in the top of glass fibre element film 2
12.The card plug is made of upper cover 20 and 10 two parts of bottom cover, and the two is to snap connection；The card plug is plastic material.
It fills and confirms that lower base without dirty, completely places the first of lower cover two sides without incompleteness, especially test strips before test strips
Limiting section 15, the second limiting section 16 are complete, can be used normally.
Confirm test strips it is complete, glass fibre element film 2, blotting paper N/D, test strips invariably specification shape, film without draw
Trace, without flash, film surface without signature pen trace etc..
By the site at the top of the card plug of blotting paper one end alignment test strips bottom suction side, then test strips are compacted, it is ensured that
Test strips it is all smooth be placed in test strips placing groove and the contact surface of slot bottom is seamless, no shaking.
Detain test strips lid 20 beyond the Great Wall.
As shown in Fig. 2, the bottom cover 10 includes: multiple first positioning holes 13 and the multiple first fastening columns positioned at its surrounding
14；It is equipped between multiple first positioning holes 13 multiple for limiting the first limiting sections 15 of test strips transverse shifting and more
A the second limiting section 16 vertically moved for limiting test strips, multiple described first fasten between columns 14 equipped with multiple for limiting
Determine the first limiting section 15 and multiple the second limiting sections 16 vertically moved for limiting test strips of test strips transverse shifting；It is right
Multiple first limiting sections 15 and multiple second limiting sections 16 for claiming setting enclose and are set as test strips placing groove, for placing
Test strips.
As shown in figure 3, the upper cover 20 include with first multiple second location holes 23 for matching of fastening column 14 and
The multiple second fastening columns 24 matched with the first positioning hole 13, in this way with the use of with upper cover 20 and bottom cover 10 is fixed
Together；The upper cover 20 further include be set between multiple second fastenings columns 24 and observation window 11 and well 12 it
Between multiple fixed parts 25,26.
The top of the nitrocellulose filter 3 is equipped with the observation window 11 for data acquisition, to expose all detections
Line 31 and nature controlling line 32, for collecting its testing result；And the observation window 11 is opened in the upper cover 20 and puts with test strips
Set the corresponding position in middle part of slot.The upper cover 20 offers at position corresponding with the glass fibre element film 2 to be added
Sample hole 12, for sample to be added dropwise on glass fibre element film 2.The detection line is apart from well 15-25mm.
The present invention also provides the manufacturing methods of above-mentioned immuno-chromatography detection device, comprising the following steps:
1) coated antibody: retention antibody, detection antibody and Quality Control antibody are coated with onto detection film, drying for standby.
In a preferred scheme, it will test antibody 1 with coating buffer and be diluted to fixed concentration (0.5-3mg/mL),
Quality Control antibody 1 is also diluted to fixed concentration (0.5-3mg/mL), and another retain antibody 1 (goat-anti chicken IgY) is also diluted to fixed concentration
Above-mentioned two liquid is coated with to Sai Duolisi company (Sartorius) by (0.01-0.5mg/mL) using special coating equipment
Nitrocellulose filter (NC) on, wherein for nature controlling line between detection line and blotting paper, 37 DEG C of drying boxes are 4 hours dry.Packet
By the sucrose of phosphate buffer (PBS) plus 3wt% that buffer is 0.01M as protective agent.
In another scheme, it will test antibody 1 with coating buffer and be diluted to fixed concentration (0.5-3mg/mL), Quality Control
Antibody 1 is also diluted to fixed concentration (0.5-3mg/mL), and another retain antibody 1 (Avidin or streptavidin) is also diluted to fixation
Above-mentioned two liquid is coated with to Sai Duolisi company by concentration (0.01-0.5mg/mL) using special coating equipment
(Sartorius) on nitrocellulose filter (NC), wherein for nature controlling line between detection line and blotting paper, 37 DEG C of drying boxes are dry
Dry 4 hours.The sucrose of phosphate buffer (PBS) plus 3wt% that buffer is 0.01M is coated with as protective agent.
2) labelled antibody: will retention antibody and detection antibody marker (can for latex fluorescence, time-resolved fluorescence,
It is upper to shift luminous, quantum dot fluorescence, fluorescent dye, colloidal gold or color micro-sphere) label, it is stored in storing liquid, it is spare.
In a preferred scheme, the detection antibody latex fluorescent marker (mass ratio of latex fluorescence and detection antibody
Example is 1:1 to 40:1), and retention antibody (chicken IgY) also uses latex fluorescent marker, is stored in storing liquid (50mMTris (three hydroxyl first
Base aminomethane hydrochloride buffer), 0.5wt%BSA (bovine serum albumin(BSA)), pH 7.8) it is spare.
In another scheme, with latex fluorescent marker, (latex fluorescence and the mass ratio of detection antibody are detection antibody
1:1 to 40:1), retention reagent (biotin) also with identical latex fluorescent marker, be stored in storing liquid (50mM Tris,
0.5wt%BSA (bovine serum albumin(BSA)), pH 7.8) it is spare.
In another scheme, with latex fluorescent marker, (latex fluorescence and the mass ratio of detection antibody are detection antibody
1:1 to 40:1), contrast agents (biotin) also use the latex fluorescence mark of labeled good protein (can use bovine serum albumin(BSA))
Note forms biotin-protein-marker compound or protein-marker-biotin；It is stored in storing liquid
(50mM Tris, 0.5wt%BSA (bovine serum albumin(BSA)), pH 7.8) is spare.
3) marker carrier prepare: by above-mentioned marker by required concentration specking in testing tube, bottle, suction nozzle, syringe,
Card plug sample mixing slot, sample pad or nitrocellulose filter, drying for standby.
In a preferred scheme, by (2-100 μ g fluorescent marker in the sample mixing slot above fluorescent marker object point to card plug
Object/person-portion), drying for standby.
In another scheme, (2-100 μ g fluorescent marker/person-portion) on fluorescent marker object point to suction nozzle inner wall is done
It is dry spare.
It is dry by (2-100 μ g fluorescent marker/person-portion) in fluorescent marker object point to testing tube in another scheme
It is spare.
In another scheme, by fluorescent marker object point or it is dipped into sample pad (2-100 μ g fluorescent marker/person-portion),
Drying for standby.
In another scheme, by fluorescent marker object point or be dipped into nitrocellulose filter (2-100 μ g fluorescent marker/
Person-portion), drying for standby.
4) prepared by sample pad: sample pad is without pretreatment or with sample treatment liquid immersion or specking (manually or by sample
Product processor) in sample pad, it is pre-processed, drying for standby.
The formula of one preferred sample treatment liquid are as follows: 50mM, Tris-HCl (trishydroxymethylaminomethane hydrochloride buffer
Liquid), 0.5wt%Casein (casein), 0.5wt%BSA (bovine serum albumin(BSA)), 0.1wt%Tween-20 (polysorbas20),
0.05wt%PEG (polyethylene glycol), 0.1wt%Tween-80 (Tween 80), 0.05wt%PVP (polyvinylpyrrolidone),
Bis- citric acid monohydrate acid sodium of 0.3wt%, 2wt% sucrose.
The formula of another sample treatment liquid are as follows: required marker, 50mM Tris-HCl (trishydroxymethylaminomethane hydrochloric acid
Buffer), 0.5wt%Casein (casein), 0.5wt%BSA (bovine serum albumin(BSA)), 0.1wt%Tween-20,0.05%
PEG (polyethylene glycol), 0.1%Tween-80 (Tween 80), 0.05wt%PVP (polyvinylpyrrolidone), 0.3wt% bis- hydration
Citric acid sodium, 2wt% sucrose.
5) test strips are assembled: will coating it is good needed for antibody detection film and processed sample pad and water absorption pad according to
Bottom plate is pasted together and be fixed on to " work " pattern tool, and is cut into the test strips of required width, generally the width of 3-6mm.It cuts
The reagent strip cut is assembled into corresponding card plug (Cassette), and card plug is equipped with well in the top of sample pad, detects film
Top be equipped with observation window, for data acquire.
In a preferred scheme, sample diluting liquid, generally physiological saline or 50mM Tris-HCl are needed,
0.9wt%NaCl, 0.1wt%Tween-20.Dilution ratio is had nothing in common with each other by disparity items, and the sample diluted is on card plug
Sample mixing slot in fluorescent marker mix (2-100 μ g fluorescent marker/person-portion), blow and beat repeatedly for several times, dissolve fluorescent marker
Object, and being immunoreacted between measured object in sample, is then added in card plug well, due to capillary action lateral layer forward
Analysis read data in 20 minutes.
In another arrangement, sample is drawn to by the suction nozzle (2-100 μ g fluorescent marker/person-portion) with fluorescent marker
In sample diluting liquid, blows and beats repeatedly for several times, dissolve fluorescent marker, and be immunoreacted between measured object in sample.It is then added to
In card plug well, due to capillary action lateral chromatography forward, data are read in 20 minutes.
In another arrangement, with common suction nozzle pipette samples dilution, (sample diluting liquid is the mixing of more person-portions, i.e. a box produces
One big bottle of sample diluting liquid of product) to (2-100 μ g fluorescent marker/person-portion) in the testing tube for having put fluorescent marker, then
Sample-adding product are blown and beaten for several times repeatedly, complete dissolution fluorescent marker, and are immunoreacted between measured object in sample.It is then added to card plug
In well, due to capillary action lateral chromatography forward, data are read in 20 minutes.
In another arrangement, sample diluting liquid, generally physiological saline or 50mM Tris-HCl, 0.9wt% are needed
NaCl, 0.1wt%Tween-20；Dilution ratio is had nothing in common with each other by disparity items, and the sample diluted is added in well, glimmering
Signal object is contained on sample pad or nitrocellulose filter or interior (2-100 μ g fluorescent marker/person-portion).Due to adding for sample
Enter, measured object reaction in fluorescent marker dissolution, release and sample.Due to capillary action lateral chromatography forward, 20 minutes
Interior reading data.
In another arrangement, do not need sample diluting liquid, sample be applied directly in well (2-100 μ g fluorescent marker/
Person-portion), fluorescent marker is contained on sample pad or nitrocellulose filter or interior.Due to the addition of sample, fluorescent marker dissolution,
Measured object reaction in release and sample.Due to capillary action lateral chromatography forward, data are read in 20 minutes.
The mode of the drying is evaporation drying, vacuum drying or freeze-drying.
1 human calcitonin of embodiment former (PCT) detection
Present embodiments provide a kind of manufacturing method of immuno-chromatography detection device, comprising the following steps:
Coated antibody: human calcitonin former (PCT) detection antibody 1 is diluted to fixed concentration (2.0mg/ with coating buffer
Ml), contrast agents 1 (sheep anti-mouse igg) are diluted to fixed concentration (2.0mg/ml), and retention reagent 1 (goat-anti chicken IgY) is diluted to solid
Determine concentration (0.2mg/ml) and use special coating equipment (XYZ3060 of BIODOT company), by above-mentioned 3 liquid coating (packet
It is measured as on 1 μ l/cm) to Sai Duolisi nitrocellulose filter 140 (nitrocellulose filter), 37 DEG C of drying boxes are 4 hours dry, standby
With.The sucrose of phosphate buffer (PBS) plus 3wt% that buffer is 0.01M is coated with as protective agent.
Labelled antibody: human calcitonin former (PCT) detection antibody 2 and retention antibody 2 (chicken IgY) are marked respectively with latex fluorescence
Remember (mass ratio of latex beads and two kinds of antibody is 1:10), is stored in storing liquid (50mM, Tris, 0.5wt%BSA, pH
7.8) spare in.
Marker prepares: above-mentioned latex fluorescent marker is dried by 5 microlitres of volumes of required concentration specking in testing tube
Spare (vacuum drying).
Sample pad preparation: sample treatment liquid is impregnated by required concentration (can pass through sample treatment machine) or specking is in sample pad
In or on, drying for standby.The specific formula of the sample treatment liquid are as follows: 50mmol/L/l Tris-HCl, 0.5wt%Casein,
Bis- water of 0.5wt%BSA, 0.1wt%Tween-20,0.05wt%PEG, 0.1wt%Tween-80,0.05%PVP, 0.3wt%
Close citric acid sodium, 2wt% sucrose.
Abbreviation: BSA: bovine serum albumin(BSA), Casein: casein, Tris-HCl: trishydroxymethylaminomethane hydrochloride buffer
Liquid, PEG: polyethylene glycol, Tween-20: polysorbas20, Tween-80: Tween 80, PVP: polyvinylpyrrolidone.
Assemble test strips: contrived experiment group and control group are as follows, and wherein the difference of experimental group and control group is only in that, real
Testing group includes retention line, and control group does not include retention line；Experimental group will be coated with the detection film (including retention line) of good required reagent
With processed sample pad and water absorption pad according to " work " character mould paste together with and be fixed on bottom plate, needed for being cut into later
Width test strips, generally 4mm wide, the test strips sheared are assembled into corresponding card plug (Cassette)；Control group first will
Nitrocellulose filter (not including retention line) and processed sample pad and water absorption pad are pasted according to " work " character mould one
Bottom plate is played and be fixed on, is cut into required width test strips, generally 4mm wide later, the test strips sheared are assembled into accordingly
In card plug (Cassette).Card plug is equipped with well in the top of sample pad, and the top of nitrocellulose filter is equipped with observation window, uses
It is acquired in data.Drawing 200 μ L sample dilutions with common suction nozzle, (sample diluting liquid is the mixing of more person-portions, i.e. a box product one
Big bottle sample diluting liquid) to (the 5 every person-portion of μ g/) in the testing tube for having put fluorescent material, then product are loaded, number is blown and beaten repeatedly
It is secondary, fluorescent material is dissolved, and be immunoreacted between measured object in sample.It is then added in the well of card plug, reads within 15 minutes
Data.Or it is first loaded in product to the testing tube for having put fluorescent material (the 5 every person-portion of μ g/), then plus 200 μ L samples dilution
Liquid is blown and beaten for several times repeatedly, dissolves fluorescent material, and be immunoreacted between measured object in sample.It is then added to the well of card plug
Interior, due to capillary action lateral chromatography forward, 15 minutes whens, read data.The Sample dilution specific formula are as follows:
50mmol/L Tris-HCl, 1wt%Tween-80,0.72wt% two citric acid monohydrate trisodiums.
Abbreviation: Tris-HCl: trishydroxymethylaminomethane hydrochloride buffer, Tween-80: Tween 80.
In Procalcitonin of conducting oneself (PCT) project, since front end has added a retention line, behind spectrogram baseline obviously drop
It is low, specifically as shown in figure 4, the baseline that the present invention detects is substantially reduced.If baseline reduces, fluorescent value is when integral is quadratured, energy
It calculates more acurrate, so that sensitivity be made to improve, specifically looks at shown in the following table 1:
It can be seen that detection line numerical value from the data of table 1, between concentration 0.05ng/mL and 0ng/mL, control group ratio
Be 1.68 times, the present invention be 4.6 times (the detection line average value of its detection line average value for being 0.05ng/mL and 0ng/mL it
Than), i.e., sensitivity of the present invention can be turned up at least 1.7 times.
2 human interleukin 6 of embodiment (IL-6) detection
Present embodiments provide a kind of manufacturing method of immuno-chromatography detection device, comprising the following steps:
Coated antibody: human interleukin 6 (IL-6) detection antibody 1 is diluted to fixed concentration (2.0mg/ with coating buffer
Ml), contrast agents 1 (goat-anti chicken IgY) are diluted to fixed concentration (2.0mg/ml), and retention reagent 1 (goat-anti chicken IgY) is diluted to solid
Determine concentration (0.1mg/ml) and above-mentioned 3 liquid is coated with by (coating using special coating equipment (XYZ3060 of BIODOT company)
Amount is 1 μ l/cm) it arrives on Sai Duolisi nitrocellulose filter 140 (nitrocellulose filter), 37 DEG C of drying boxes are 4 hours dry, standby
With.The sucrose of phosphate buffer (PBS) plus 3wt% that buffer is 0.01M is coated with as protective agent.
Labelled antibody: interleukin 6 (IL-6) detection antibody 2 and retention antibody 2 (chicken IgY) are marked respectively with latex fluorescence
(mass ratio of latex beads and two kinds of antibody is 1:10), is stored in storing liquid, spare, (50mM, Tris, 0.5wt%
BSA, pH 7.8).
Marker prepares: above-mentioned marker is dried spare by 5 microlitres of volumes of required concentration specking in testing tube
(vacuum drying).
Sample pad preparation: above-mentioned 2 marker of interleukin 6 antibody is pressed into the every person-portion of 1.5ug, chicken IgY marker presses 0.75ug
Every person-portion is blended in sample pad dilution, and is blended with marker sample treatment liquid by required concentration (at can be by sample
Reason machine) impregnate or specking in or on sample pad, drying for standby.The specific formula of the sample treatment liquid are as follows: 50mMol/l
Tris-HCl, 0.5wt%Casein, 0.5wt%BSA, 0.1wt%Tween-20,0.05wt%PEG, 0.1wt%Tween-
Bis- citric acid monohydrate acid sodium of 80,0.05%PVP, 0.3wt%, 2wt% sucrose.
Abbreviation: BSA: bovine serum albumin(BSA), Casein: casein, Tris-HCl: trishydroxymethylaminomethane hydrochloride buffer
Liquid, PEG: polyethylene glycol, Tween-20: polysorbas20, Tween-80: Tween 80, PVP: polyvinylpyrrolidone.
Assemble test strips: contrived experiment group and control group are as follows, and wherein the difference of experimental group and control group is only in that, real
Testing group includes retention line, and control group does not include retention line；Experimental group will coating it is good needed for the nitrocellulose filter of reagent (including cut
Stay line) and processed sample pad and water absorption pad according to " work " character mould paste together and be fixed on bottom plate, cut later
At required width test strips, generally 4mm wide, the test strips sheared are assembled into corresponding card plug (Cassette)；Control
Group is first glutinous according to " work " character mould by nitrocellulose filter (not including retention line) and processed sample pad and water absorption pad
Bottom plate is sticked together and be fixed on, is cut into required width test strips, generally 4mm wide later, the test strips sheared are assembled into
In corresponding card plug (Cassette).Card plug is equipped with well in the top of sample pad, and the top of nitrocellulose filter, which is equipped with, to be seen
Window is examined, is acquired for data.It draws 75ul sample with common suction nozzle to be added in the well of card plug, 15 minutes reading data.
When doing human interleukin 6 (IL-6) project, since front end has added a retention line, it is substantially reduced baseline, thus
Make fluorescent value when integral is quadratured, can calculate more acurrate, so that sensitivity be made to improve, specifically looks at shown in the following table 2:
It can be seen that detection line numerical value from the data of table 1, between concentration 3pg/mL and 0pg/mL, control group ratio is
1.22 times, the present invention is 3.31 times (the ratio between its detection line average value for being 3pg/mL and the detection line average value of 0pg/mL), i.e.,
Sensitivity of the present invention can be turned up at least 1.7 times.
The above described is only a preferred embodiment of the present invention, be not intended to limit the present invention in any form, according to
According to technical spirit any simple modification, equivalent change and modification to the above embodiments of the invention, this hair is still fallen within
In the range of bright technical solution.
Claims (15)
1. a kind of immuno-chromatographic test paper strip characterized by comprising
Sample pad；
Film is detected, the sample pad is connected to, retention line, detection line and nature controlling line are successively arranged on the detection film；
Water absorption pad is connected to the detection film.
2. immuno-chromatographic test paper strip according to claim 1, which is characterized in that the detection film is nitrocellulose filter,
The retention antibody, detection antibody and Quality Control respectively as retention line, detection line and nature controlling line are successively coated on the detection film
Antibody, the film concentration ratio of drawing between three is (0.01-0.5): (0.5-3): (0.5-3).
3. immuno-chromatographic test paper strip according to claim 2, which is characterized in that the retention antibody be goat-anti chicken IgY or
Biotin-avidin.
4. immuno-chromatographic test paper strip according to claim 1, which is characterized in that be coated with goat-anti chicken on the detection film
IgY is as retention line.
5. immuno-chromatographic test paper strip according to claim 1, which is characterized in that be coated with biotin-on the detection film
Avidin is as retention line.
6. immuno-chromatographic test paper strip according to claim 1, which is characterized in that the retention line detection line is more leaned on
The nearly sample pad；The nature controlling line detection line is closer to the water absorption pad.
7. immuno-chromatographic test paper strip according to claim 6, which is characterized in that it is described retention line and detection line between away from
From for 3-10mm, the distance between the nature controlling line and detection line are 3-6mm.
8. immuno-chromatographic test paper strip according to claim 1, which is characterized in that the sample pad be glass fibre element film or
Polyester film；The water absorption pad is blotting paper；It further include bottom plate, the detection film, sample pad and water absorption pad are both secured to the bottom
On plate.
9. a kind of preparation method of immuno-chromatographic test paper strip according to claim 1-8, which is characterized in that including
Following steps:
1) coating retention line, detection line and nature controlling line on detection film, will test the two sides of film respectively with sample pad and water absorption pad
After bonding and it is fixed on bottom plate, obtains test strips blank；
2) the test strips blank that step 1) obtains is cut into required size up to the immuno-chromatographic test paper strip.
10. a kind of immuno-chromatography detection device, including the described in any item test strips of claim 1-8 and the package test paper
The card plug of item, the test strips are assembled into the card plug, and the card plug is equipped with well, the detection in the top of sample pad
Line-spacing well 15-25mm.
11. immuno-chromatography detection device according to claim 10, which is characterized in that the card plug is by upper cover and bottom cover group
At；
The bottom cover includes: the multiple first positioning holes and multiple first fastening columns positioned at its surrounding；Multiple first positioning
Multiple the first limiting sections for limiting test strips transverse shifting are equipped between hole and multiple for limiting test strips longitudinally shifting
The second dynamic limiting section, multiple described first fasten the first limits between columns equipped with multiple for limiting test strips transverse shifting
Portion and multiple the second limiting sections vertically moved for limiting test strips；Multiple first limiting sections and multiple described second
Limiting section, which encloses, is set as test strips placing groove；
The upper cover includes the multiple second location holes matched with the first fastening column and matches with the first positioning hole
The multiple second fastening columns closed；The upper cover further includes being set between multiple second fastenings columns and observation window and sample-adding
Multiple fixed parts between hole.
12. immuno-chromatography detection device according to claim 11, which is characterized in that the top of the detection film, which is equipped with, to be used
In the observation window of data acquisition, and the observation window is opened in the corresponding position in middle part of the upper Gai Shangyu test strips placing groove
It sets.
13. immuno-chromatography detection device according to claim 12, which is characterized in that the upper cover with the sample pad
Well is offered at corresponding position.
14. a kind of manufacturing method of the described in any item immuno-chromatography detection devices of claim 10-13, which is characterized in that packet
Include following steps:
1) coated antibody: retention antibody, detection antibody and Quality Control antibody are coated with onto detection film, drying for standby；
2) labelled antibody: obtaining labelled antibody with label substance markers for retention antibody and detection antibody, be stored in storing liquid, standby
With；
3) marker carrier prepares: above-mentioned marker is dried spare on marker carrier by required concentration specking；
4) prepared by sample pad: sample pad is impregnated without pretreatment or with sample treatment liquid or specking sample pad, is pre-processed,
Drying for standby；
5) test strips are assembled: by the detection film of coating antibody needed for good and processed sample pad and water absorption pad according to mold
Bottom plate is pasted together and be fixed on, and is cut into the test strips of required width, the reagent strip is assembled into corresponding card plug later
It is interior.
15. manufacturing method described in claim 14, which is characterized in that the marker be latex fluorescence, time-resolved fluorescence,
It is upper to shift luminous, quantum dot fluorescence, fluorescent dye, colloidal gold or color micro-sphere；The marker carrier be testing tube, bottle,
Suction nozzle, syringe, card plug sample mixing slot, sample pad or detection film；The mode of the drying is that evaporation drying, vacuum drying or freezing are dry
It is dry；The width of the test strips is 3-6mm.
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