CN206038685U - Immunity side direction chromatography detecting system - Google Patents
Immunity side direction chromatography detecting system Download PDFInfo
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- CN206038685U CN206038685U CN201620709049.9U CN201620709049U CN206038685U CN 206038685 U CN206038685 U CN 206038685U CN 201620709049 U CN201620709049 U CN 201620709049U CN 206038685 U CN206038685 U CN 206038685U
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Abstract
The utility model discloses an immunity side direction chromatography detecting system belongs to medical field, detecting system include the bottom plate, it includes nearly sample end and sample end far away, the bottom plate on hold sample far away end to set gradually the sample pad by nearly sample, the sample stack up and be provided with the application of sample hole, nitrocellulose membranes with the pad absorbs water, nitrocellulose membranes on be provided with matter accuse area and detect the area, matter accuse area set up and be in detection area and sample pad between, matter accuse area for the biotin the matter accuse system of the different kinds of the avidin system inclusive AND mouse -anti body, nitrocellulose membranes on be provided with and be used for data collection's observation window. The utility model discloses a detecting system has higher sensitivity, precision and the degree of accuracy.
Description
Technical field
The utility model is related to medical domain, and in particular to a kind of immune lateral chromatography detecting system.
Background technology
Immune its principle of lateral chromatography method is that the antibody (antigen) of specificity is first fixed on a certain area of nitrocellulose membrane
Band, after dry nitrocellulose membrane one end immersion sample (urine, serum, blood plasma, whole blood or other samples), due to capillary
Effect, sample will be moved forward along the film, when the region for being fixed with antibody (antigen) is moved to, corresponding antigen in sample
(antibody) occurs to specifically bind 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 using fluorescent marker, supporting readout instrument is needed
Data acquisition, is processed, and checking computations draw corresponding quantitative concentrations value.
However as the development of society, the sensitivity of current detection detecting system, preci-sion and accuracy are all not
Existing needs can be reached.
Utility model content
The utility model embodiment provides a kind of immune lateral chromatography detecting system, substantially increases the sensitive of detection
Degree, preci-sion and accuracy.
The utility model embodiment additionally provides a kind of preparation method of immune lateral chromatography detecting system.
The utility model embodiment additionally provides a kind of application of immune lateral chromatography detecting system.
The utility model embodiment provides a kind of immune lateral chromatography detecting system, and including base plate, which includes nearly sample
End and remote sample end；Sample pad is disposed with by nearly sample end to remote sample end on described base plate, in described sample pad
It is provided with well, nitrocellulose filter and adsorptive pads；Quality control band and detection band, institute is provided with described nitrocellulose filter
The quality control band stated is arranged between described detection band and sample pad；Described quality control band be biotin-avidin system or with
The system of quality control of mouse antibody different genera；The observation window for gathered data is provided with described nitrocellulose filter.
Further, labeling pad is provided between described sample pad and nitrocellulose filter.
Further, it is described to be coated with using goat-anti chicken IgY with mouse antibody different genera system of quality control, chicken IgY marks.
Further, described biotin-avidin system is coated with to celluloid by Avidin or Streptavidin
Obtain on film.
On the other hand, the utility model embodiment additionally provides a kind of preparation method of immune lateral chromatography detecting system,
Comprise the steps：
(1) by first antibody or antigen coat on nitrocellulose filter；Simultaneously by contrast agents coating to cellulose nitrate
On plain film；
(2) prepare label：SA or antigen are marked into rear specking in testing tube, bottle, suction nozzle, syringe
Interior, jam sample mixing groove, sample pad, labeling pad or nitrocellulose filter obtain described label；
(3) prepare sample pad；
(4) assemble described immune lateral chromatography detecting system：Fix sample pad, nitrocellulose filter on base plate successively
And adsorptive pads；Quality control band and detection band is provided with described nitrocellulose filter, and described quality control band is arranged on described inspection
Between measuring tape and sample pad；Described quality control band is biotin-avidin system or the system of quality control with mouse antibody different genera；
The observation window for gathered data is provided with described nitrocellulose filter.
Further, described step (1) specifically includes following steps：
Described antibody or antigen coating buffer solution are diluted to into 0.1-5.0mg/ml, coating to the celluloid
On film, drying for standby；Described coating buffer solution for 0.01-0.1M phosphate buffer add 1-10% (w/v, such as
It is sucrose 3%) as protective agent that 3% i.e. 3 grams solutes are dissolved in 100ml solvents.
Further, labeling pad is provided between described sample pad and nitrocellulose filter, its label is latex
Fluorescence, time-resolved fluorescence, luminous upper transfer, quantum dot fluorescence, fluorescent dye or collaurum.
Further, it is with the sample pad described in sample treatment liquid immersion or specking, drying for standby to prepare sample pad；It is described
Sample treatment liquid in Tris-CL molar concentration be 10-100mMol/l, according to quality percentage in described sample treatment liquid
Number meter includes following component：The Tween-20 of the BSA of the Casein of 0.1-2%, 0.1-5%, 0.05-1%, 0.05%-0.5%
PEG, the Tween-80 of 0.05%-1%, the PVP of 0.05%-0.5%, 0.1%-1% two citric acid monohydrates acid sodium, 1-
10% sucrose.
Another further aspect, the utility model embodiment also provide a kind of application of immune lateral chromatography detecting system, described
Immune lateral chromatography detecting system is detecting system prepared by above-mentioned method.
Further, detected after dilute detected sample；The dilution that described dilution is selected is physiological saline
Or sample diluting liquid, in described sample diluting liquid the molar concentration of Tris-CL be 10-100mMol/L, the mass concentration of NaCl
For 0.8-1%, the mass concentration of Tween-20 is 0.05-1%.
Compared with prior art, the utility model immunity lateral chromatography detecting system at least has the advantages that：
Quality control band and detection band location swap, in the case where detecting system point overall length is constant, extend well
The distance between with detection band, extend the reaction time, improve sensitivity.Such as some are not high to sensitivity requirement, can be in identical spirit
Under sensitivity, the shorter or reagent dosage that detecting system is done is reduced, and saves production cost.Meanwhile, determining in viscous samples
Measure in examination, the data of quality control band are more accurately and controllable, so as to improve the preci-sion and accuracy of product.
Using with the diverse system of traditional quality control band system, it is to avoid cross reaction.Using remote with mouse antibody kind
Chicken IgY is coated with as product quality control band system, goat-anti chicken IgY, chicken IgY marks (or be used as using biotin-avidin system
Quality control band system)；So as to the situation side for avoiding cross-blocks reaction occurs.
Description of the drawings
Fig. 1 is the immune lateral chromatography detecting system structural representation in embodiment 1 and 2 in the utility model；
Fig. 2 is the immune lateral chromatography detecting system structural representation in embodiment 3 in the utility model.
Specific embodiment
The utility model scheme is described further with reference to preferred embodiment, it will be appreciated that preferred embodiment is
Understanding of the those skilled in the art to the utility model scheme for convenience, not as the restriction of the utility model scheme.
Colloid gold immune lateral chromatography method no doubt has the advantages that his such as simplicity, quick etc.；But due to collaurum itself
The characteristics of cause sensitivity to be unable to reach the clinical requirement of some projects.And then immunofluorescence technique is derived, which is mainly in glue
In the case of the above-mentioned advantage of body gold, the shortcoming of collaurum method is compensate for well, good sensitivity is obtained, become now i.e.
When check (POCT) field prefered method.
But even with more sensitive fluorescent method and mix corresponding readout instrument go improve sensitivity, still cannot arrive
The requirement of some projects.It is because immune lateral chromatography technology should ask the reaction time oversize, long to be unable to reach inspection immediately
The purpose tested；Require again very high in some project sensitivity.It is well known that what is played a decisive role in immune response is time, temperature
Degree and ionic strength, in the case where ensureing that reaction buffer fully optimizes, in room temperature or 37 degree or so reactions, have reaction only
Time can optimize, but the reaction time can not long be the requirement of real-time test, while too extend the reaction time can also lead
Cause the unfavorable factors such as back flow of sample, background increasing.In addition to sensitivity if sample is the sticky sample such as whole blood,
The sample of lateral chromatography reagent strip can be caused excessively slow by the speed that capillary action is moved with immobilon-p, caused in the stipulated time
The compound of interior sample and contained label and measured object fully cannot discharge, and reagent reacting cmpletely cannot affect last
As a result, the particularly result of definite value product.Can so cause precision bad, and cause as quality control band data are inaccurate
Detected value is forbidden, so as to affect the degree of accuracy, precision.
According to above-mentioned analysis, it can be found that cross reaction can be just avoided from suitable quality control band system, such that it is able to
By quality control band and detection band location swap, it is achieved thereby that while existing detecting system length is kept, extending well
With the distance of detection band, so as to improve sensitivity, precision and the degree of accuracy.
The principle of the utility model method：This method it is creative by quality control band position and detection band location swap, make matter
Control band is located between detection band and well；Extend to exchange the distance between detection band position and well for, distance extends can
To extend the reaction time, so as to improve product sensitivity in the case where product total length is constant.As some are to sensitivity requirement
It is not high, can be under same sensitivity, the shorter or reagent dosage that reagent strip does is reduced, and saves production cost.
Cannot be originally that this can be formed instead with labelled antibody as quality control band is typically using sheep anti mouse by location swap
Should, affect detection band reading value.Or using the close goat-anti rabbit-rabbit-anti body method of kind, although the method is generally
Will not directly affect reaction, but due to kind it is excessively near, or easily and the more or less cross reaction of some mouse antibody, impact
Properties of product.
Specific embodiment is presented herein below：
Embodiment 1 (marker free pad, label have Sample dilution in testing tube)
Coated antibody：HFABP (HFABP) detection antibody 1 is diluted to into fixation with coating buffer solution
Concentration (2.0mg/ml), contrast agents 1 (goat-anti chicken IgY) are diluted to fixed concentration (2.0mg/ml), using BioDot companies
By above-mentioned two liquid coating on Sai Duolisi nitrocellulose filters 140 (NC films), wherein quality control band is located at detection to XYZ3060
Between band and well, 37 DEG C of drying boxes are dried 4 hours, standby.Phosphate buffer of the coating buffer solution for 0.01Mol/l
(PBs) sucrose for Jia 3% is used as protective agent.
Labelled antibody：By hFABP (HFABP) detection antibody 2 and contrast agents 2 (chicken IgY) breast
Glue fluorescence labeling, is stored in storing liquid, standby, and (50mMol/l Tris, 0.5%BSA, pH is 7.8).
Label prepares：Above-mentioned labelled antibody is pressed into 5 microlitres of volumes of desired concn specking in testing tube, is dried standby
With.
It is prepared by sample pad：By sample treatment liquid press desired concn immersion or specking in sample pad or on, drying for standby.Place
Reason liquid composition is as follows：50mMol/l Tris-CL, 0.5%Casein, 0.5%BSA, 0.1%Tween-20,0.05%PEG,
0.1%Tween-80,0.05%PVP, 0.3% 2 citric acid monohydrate acid sodium, 2% sucrose.
Wherein, BSA：Bovine serum albumin(BSA), Casein：Casein, Tris-CL：Trishydroxymethylaminomethane hydrochloride buffer
Liquid, PEG：Polyethylene glycol, Tween-20：Polysorbas20, Tween-80：Tween 80, PVP：Polyvinylpyrrolidone, 0.5% attaches most importance to
The amount gram solute of volume ratio, i.e., 0.5 is dissolved in 100ml solvents.
Sample diluting liquid is dispensed：By 50mMol/l Tris-CL, 0.9%NaCl, 0.1%Tween-20,3.0%BSA, point
It is attached in big bottle (5ml-50ml), it is standby.
Assembling reagent strip：By the nitrocellulose filter and processed good sample pad and adsorptive pads of coating reagent needed for good
Paste together according to mould, and be cut into required width reagent strip, generally 4mm width.The reagent strip sheared is assembled into phase
In the jam (Cassette) answered, jam has well above sample pad, and jam has observation window above nitrocellulose filter,
For data acquisition.With common suction nozzle pipette samples dilution, (sample diluting liquid mixes for many person-portions, i.e. one big bottle of a box product
Sample diluting liquid) to having put in the testing tube of fluorescent material, then product are loaded, blow and beat repeatedly for several times, dissolve fluorescent material, and
With immune response between measured object in sample.It is then added in jam well, reads data within 15 minutes.Or product are first loaded to
In the testing tube of the good fluorescent material of Jing points, then plus sample diluting liquid, blow and beat repeatedly for several times, dissolve fluorescent material, and with sample in
Immune response between measured object.It is then added in jam well, due to capillary action lateral chromatography forward, reads when 15 minutes
Fetch data.
Table 1 is the testing result Data Comparison of this method and control group in the present embodiment.
Table 1
As can be seen from Table 1, it is when hFABP (HFABP) project is done, due to position adjustment, sensitive
Degree significantly improves that (either detection band numerical value or fluorescent value, ratio all differs from 1.5 between concentration 3.3ng/ml and 0ng/ml
Times, i.e. this method sensitivity can heighten at least 1.5 times).
Embodiment 2 (marker free pad, label have Sample dilution in suction nozzle)
Coated antibody：C reactive protein (CRP) detection antibody 1 is diluted to into fixed concentration (0.5mg/ with coating buffer solution
Ml), contrast agents 1 (goat-anti chicken IgY) are diluted to fixed concentration (2.0mg/ml), will be upper using the XYZ3060 of BioDot companies
Two liquid coatings are stated on Sai Duolisi nitrocellulose filters (NC), wherein quality control band is located between detection band and well,
37 DEG C of drying boxes are dried 4 hours, standby.Coating buffer solution is that the sucrose that the phosphate buffer (PBs) of 0.01Mol/l Jia 3% is made
For protective agent.
Labelled antibody：C reactive protein (CRP) detection antibody 2 and contrast agents 2 (chicken IgY) are used into latex fluorescence labeling, is stored up
Exist in storing liquid, standby, (50mMol/l Tris, 0.5%BSA, pH is 7.8).
Label prepares：Above-mentioned labelled antibody is pressed into 5 microlitres of volumes of desired concn specking in suction nozzle inwall, is dried
It is standby.
It is prepared by sample pad：By sample treatment liquid press desired concn immersion or specking in sample pad or on, drying for standby.Place
Reason liquid composition is as follows：50mMol/l Tris-CL, 0.5%Casein, 0.5%BSA, 0.1%Tween-20,0.05%PEG,
0.1%Tween-80,0.05%PVP, 0.3% 2 citric acid monohydrate acid sodium, 2% sucrose.
Wherein：BSA：Bovine serum albumin(BSA), Casein：Casein, Tris-CL：Trishydroxymethylaminomethane hydrochloride buffer
Liquid, PEG：Polyethylene glycol, Tween-20：Polysorbas20, Tween-80：Tween 80, PVP：Polyvinylpyrrolidone, 0.5% attaches most importance to
The amount gram solute of volume ratio, i.e., 0.5 is dissolved in 100ml solvents.
Sample diluting liquid is dispensed：By 50mMol/l Tris-CL, 0.9%NaCl, 0.1%Tween-20,3%BSA, dispense
It is in reaction buffer bottle (0.2ml-3ml), standby.
Assembling reagent strip：By the nitrocellulose filter and processed good sample pad and adsorptive pads of coating reagent needed for good
Paste together according to mould, and be cut into required width reagent strip, generally 4mm width.The reagent strip sheared is assembled into phase
In the jam (Cassette) answered, jam has well above sample pad, and jam has observation window above nitrocellulose filter,
For data acquisition.Number is blown and beaten in reaction buffer bottle, repeatedly with the suction nozzle pipette samples with label fluorescent material
It is secondary, dissolve fluorescent material, and the immune response between measured object in sample.It is then added in jam well, due to capillary
Lateral chromatography forward is acted on,
Data are read when 3 minutes.Table 2 is the testing result contrast table of this method and prior art in the present embodiment.
Table 2
Can be drawn by table 2, when c reactive protein (CRP) project is done, due to position adjustment, sensitivity is significantly improved
(explain：Either detection band numerical value or fluorescent value, between concentration 0.0315mg/L and 0mg/L ratio all differ from 1.5 times with
On, i.e. this method sensitivity can heighten at least 1.5 times).
Embodiment 1 and 2 prepare immune lateral chromatography detecting system structural representation as shown in figure 1, wherein, detecting system
Including PVC offset plates 1, sample pad 2 is disposed with by left-to-right on PVC offset plates, has well (not shown) thereon；Nitric acid
Cellulose membrane 3, is provided with quality control band 5, detection band 6 and observation window (not shown)；Adsorptive pads 4.
Embodiment 3. (has a labeling pad, coating reagent is antigen, n.s dilution)
Coated antibody：HCV (hepatitis C) detection antigen 1 coating buffer solutions are diluted to into fixed concentration (3.0mg/
Ml), contrast agents 1 (streptavidin SA) are diluted to fixed concentration (2.0mg/ml), will using the XYZ3060 of BioDot companies
To on nitrocellulose filter (NC films), wherein quality control band is located between detection band and well above-mentioned two liquid coating, 37 DEG C
Drying box is dried 4 hours.Coating buffer solution for 0.01Mol/l phosphate buffer (PBs) Jia 3% sucrose as protective agent.
Labelled antibody：HCV (hepatitis C) is detected into that antigen 2 and control antibodies 2 (BSA-Biotin) use colloid gold label,
It is stored in gold mark storing liquid, (50mMol/l Tris, 0.5%BSA, pH 7.8),
It is prepared by labeling pad：By above-mentioned labelled antibody press desired concn immersion or specking in gold standard pad or on, be dried, cut
Cut standby.Available evaporation drying, vacuum drying or freeze-drying.
It is prepared by sample pad：By sample treatment liquid press desired concn immersion or specking in gold standard pad or on, drying for standby.Can
With evaporation drying, vacuum drying or freeze-drying.
Treatment fluid composition is as follows：50mM Tris-CL, 0.5%Casein, 0.5%BSA, 0.1%Tween-20,0.05%
PEG, 0.1%Tween-80,0.05%PVP, 0.3% 2 citric acid monohydrate acid sodium, 2% sucrose.
Abbreviation：BSA：Bovine serum albumin(BSA), Casein：Casein, Tris-CL：Trishydroxymethylaminomethane hydrochloride buffer
Liquid, PEG：Polyethylene glycol, Tween-20：Polysorbas20, Tween-80：Tween 80, PVP：Polyvinylpyrrolidone, Biotin are biological
Element, SA streptavidins.
Assembling reagent strip：By the nitrocellulose filter (NC films) of coating antibody needed for good, labeling pad, adsorptive pads and sample
Product pad is pasted together according to mould, and is cut into required width reagent strip, generally 4mm width.The reagent strip assembling sheared
To in corresponding jam (Cassette), jam has well above sample pad, and jam has sight above nitrocellulose filter
Window is examined, for data acquisition.120 microlitres of volume samples are drawn with common suction nozzle to be added in well, due to capillary action
Lateral chromatography, read data in 15 minutes forward.
Data are read when 15 minutes.Table 3 is the testing result contrast table of this method and prior art in the present embodiment.
Remarks：Collaurum result is estimated,：For feminine gender ,+：For the positive, estimating has simple red line, ++：For the positive, mesh
Survey has obvious red line.
Can be drawn by table 3, when HCV (HCV) antibody diagnosing reagent (colloidal gold method) project is done, due to
Position adjustment, sensitivity is significantly improved (to be explained：Calibration object 1:During 256 diluted concentration, contrast method is feminine gender, so sensitive
Degree improves about 2 times).
Embodiment 3 prepare immune lateral chromatography detecting system structural representation as shown in Fig. 2 wherein, detecting system bag
PVC offset plates 1 are included, and sample pad 2 are disposed with by left-to-right on PVC offset plates, are had well (not shown) thereon；Label
Pad 7；Nitrocellulose filter 3, is provided with quality control band 5, detection band 6 and observation window (not shown)；Adsorptive pads 4.
The addition of above example label fluorescent material can also be in the following ways：Label particle is to above jam
Sample mixing groove in；Label particle is on suction nozzle inwall；Label particle is in testing tube；Label particle is dipped into sample
Pad.
The not most part of the utility model application, those skilled in the art can carry out conventional selection according to existing knowledge,
Such as：Drying can be using evaporation drying, vacuum drying or freeze-drying etc..
The above, specific embodiment only of the present utility model, but protection domain of the present utility model do not limit to
In this, any those familiar with the art can readily occur in change in the technical scope that the utility model is disclosed
Or replace, should all cover within protection domain of the present utility model.Therefore, protection domain of the present utility model should be with the power
The protection domain that profit is required is defined.
Claims (4)
1. a kind of immune lateral chromatography detecting system, it is characterised in that including base plate, which includes nearly sample end and remote sample end；
Sample pad is disposed with by nearly sample end to remote sample end on described base plate, in described sample pad, well is provided with,
Nitrocellulose filter and adsorptive pads；Quality control band and detection band is provided with described nitrocellulose filter, and described quality control band sets
Put between described detection band and sample pad；Described quality control band is biotin-avidin system or not of the same race with mouse antibody
The system of quality control of category；The observation window for gathered data is provided with described nitrocellulose filter.
2. immune lateral chromatography detecting system according to claim 1, it is characterised in that described sample pad is fine with nitric acid
Labeling pad is provided between the plain film of dimension.
3. immune lateral chromatography detecting system according to claim 1, it is characterised in that described is not of the same race with mouse antibody
The system of quality control of category is coated with using goat-anti chicken IgY, chicken IgY marks.
4. immune lateral chromatography detecting system according to claim 1, it is characterised in that described biotin-avidin
System is obtained on nitrocellulose filter by Avidin or Streptavidin coating.
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Cited By (4)
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| CN109521207A (en) * | 2018-12-28 | 2019-03-26 | 广州菲康生物技术有限公司 | A kind of IGF-1 fluorescence immune chromatography detection kit |
| CN110488016A (en) * | 2019-08-14 | 2019-11-22 | 江南大学 | A kind of zearalenone-vomitoxin binary channels immune quantitative test paper item |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107290549A (en) * | 2017-07-13 | 2017-10-24 | 深圳市亚辉龙生物科技股份有限公司 | A kind of kit, its preparation method and detection method for determining anti-Miao Le Shi pipe hormones |
| CN107389929A (en) * | 2017-09-06 | 2017-11-24 | 深圳市检验检疫科学研究院 | A kind of H7 subtype avian influenza virus fluorescent microsphere detection card and its application |
| CN109521207A (en) * | 2018-12-28 | 2019-03-26 | 广州菲康生物技术有限公司 | A kind of IGF-1 fluorescence immune chromatography detection kit |
| CN110488016A (en) * | 2019-08-14 | 2019-11-22 | 江南大学 | A kind of zearalenone-vomitoxin binary channels immune quantitative test paper item |
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