CN109725150A - A kind of immune joint inspection test strips with sample distribution pad - Google Patents
A kind of immune joint inspection test strips with sample distribution pad Download PDFInfo
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- CN109725150A CN109725150A CN201811591135.4A CN201811591135A CN109725150A CN 109725150 A CN109725150 A CN 109725150A CN 201811591135 A CN201811591135 A CN 201811591135A CN 109725150 A CN109725150 A CN 109725150A
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Abstract
The invention discloses a kind of immune joint inspection test strips with sample distribution pad, belong to technical field of biological.Setting sample distributes pad and test strips on the immune joint inspection test strips bottom plate, sample distribution pad is connected at least 2 test strips, and test strips are sequentially distributed along chromatography direction: labelled antibody bonding pad, the capture antibody being coated in chromatographic film test section, be coated with Quality Control portion, chromatographic film and the water-absorbent material that can capture labelled antibody substance.When detection, sample to be measured is added in sample pad, forms interlayer structure through single layered substrate swimming to detection line, the substance in sample to be tested is detected.By being improved to chromatograph test strip sample application zone (sample pad), so that test strips, after sample-adding, liquid (sample or buffer) is automatically in distribution to the test strips of joint inspection, and easy to operate, speed is faster.
Description
Technical field
The present invention relates to a kind of immune joint inspection test strips with sample distribution pad, belong to technical field of biological.
Background technique
In immunochromatography detection field, it is often necessary to carry out joint inspection to multiple indexs of same sample.For this market
Demand, current existing panimmunity test strips joint inspection technology, including parallel immunity test strip, tandem immunity test strip
Deng.Parallel immunity test strip is that the detection raw material of different markers is prepared into independent test strips, these are independently tried
Paper slip is set side by side；Tandem immunity test strip, which refers to, is sequentially prepared the detection raw material of unlike signal object in same test paper
On item, is formed and form far and near different detection line apart from bonding pad.Since clinical practice application value is larger, two kinds of above-mentioned connection
Paper slip is inspected to be widely applied.
But there is also respective disadvantages for above two joint inspection kit.Parallel joint inspection test strips development difficulty is low, but
Need every test strips that clinical samples are all added when being detection operation；The joint inspection test strips detection of tandem is easy to operate, but not
Testing conditions with marker differ greatly, and cause test strips development difficulty very big.Therefore it provides a kind of development difficulty is low, behaviour
Make easy, easy to detect, high sensitivity immunity test strip, there is important application value for clinical detection field.
Summary of the invention
The first purpose of the invention is to provide a kind of immune joint inspection test strips, setting sample distributes pad and test paper on bottom plate
Item, sample distribution pad are connected at least 2 test strips, and test strips have been sequentially distributed along chromatography direction: labelled antibody bonding pad, packet
By the test section of the capture antibody in chromatographic film, it is coated with and can captures Quality Control portion, chromatographic film and the suction of labelled antibody substance
Water material.
In one embodiment of the invention, test strips and/or bonding pad and/or chromatographic film be set as different in width,
Thickness or length, corresponding different sample sendouts.
In one embodiment of the invention, the ingredient of the described sample distribution pad includes glass fibre or long stapled
Filter paper, sample distribution pad are equipped with well.
In one embodiment of the invention, parallel to each other between test strips, and distribute and pad perpendicular to sample.
In one embodiment of the invention, anti-human erythrocyte antibody is coated on sample distribution pad.
In one embodiment of the invention, the chromatographic film is by nitrocellulose, cellulose acetate film, nylon
Film, pvdf membrane or 5 filter paper of Fusion are made.
In one embodiment of the invention, be coated with can be with the substance in conjunction with marker, institute in the Quality Control portion
The mark substance stated, including colloidal gold, fluorescent microsphere or latex beads.
A second object of the present invention is to provide the preparation methods of above-mentioned immune joint inspection test strips, comprising: first to bonding pad
Specking is carried out, detection line and nature controlling line are prepared；Assembled again, by the good NC film of specking, by nature controlling line above in a manner of paste
Onto bottom plate, absorbent filter is sticked above NC film, bonding pad is sticked below NC film, retains the protection of sample pad area
Film；The different test strips of well cutting are sticked on bottom plate side by side, form test strip.
Third object of the present invention is to provide application of the above-mentioned immune joint inspection test strips in test sample
In one embodiment of the invention, sample to be tested is added on sample distribution pad, extremely through single layered substrate swimming
Detection line forms interlayer structure, detects to sample to be tested.
The present invention is by improving chromatograph test strip sample application zone (sample pad), so that test strips are after sample-adding, liquid
Body (sample or buffer) is automatic to distribute to the test strips of joint inspection because of the capillarity of different test strips；And it is possible to according to
The different structure (e.g., bonding pad, chromatographic film etc.) of test strips is set different width, thickness, length by specific requirements, with
Sample is adjusted to distribute to the amount of different test strips；It can be detected for many index of same sample, operator is not necessarily to
Especially training, it is easy to operate；It needs 15 minutes, examines quickly, and significantly reduce joint inspection test strips from result is loaded onto out only
Development difficulty.
Detailed description of the invention
The top view of Fig. 1 test strip structure, part appended drawing reference: 1,8- bonding pad, 2- direction-indicating arrow, 3- are protected
Cuticula, 4,9- detection lines, 5- nature controlling line, 6- chromatographic film, 7- water-absorbent material, 10- sample distribution pad.
Fig. 2: HCG project performance verification；X-axis is commercially available HCG Test paper box detected value (mIU/mL), and Y-axis is the present invention
Immune joint inspection test strips detected value (mIU/mL)；
Fig. 3: LH project performance verification；X-axis is commercially available LH Test paper barrel detected value (mIU/mL), and Y-axis is this hair
Bright immune joint inspection test strips detected value (mIU/mL).
Specific embodiment
In order to be more clearly understood that technology contents of the invention, spy lifts following embodiment and is described in detail.
In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition, such as Sambrook etc.
People, molecule clone technology Laboratory Manual (New York:Cold Spring Harbor Laboratory Press,
2005) condition described in, or according to the normal condition proposed by manufacturer.
1 test strip structure and working principle of embodiment
It is a kind of to distribute the immune joint inspection test strips padded, including bottom plate with sample, it has been sequentially distributed on bottom plate: sample distribution
Pad 10, bonding pad 1,8, direction-indicating arrow 2, protective film 3, detection line 4,9,5- nature controlling line, chromatographic film 6, water-absorbent material 7；Its
In, sample distributes pad 10, the test strips of connection two or two or more.As shown in Figure 1.
Working principle: by taking two joint inspection test paper with sample distribution pad as an example, when detection, by liquid (sample or buffering
Liquid) it is added dropwise on sample distribution pad；It, will be on liquid chromatography(LC) to two test strips because of the capillarity of test strips itself；And root
It can be not year-on-year by realization of the liquid in two test strips by adjusting the structure of two test strips according to different application demands
The distribution of example.
2 fluorescent marker specific antibody of embodiment
Step 1: the preparation of people's HCG labelled antibody
Cleaning: fluorescent microsphere is taken (to be purchased from Bangs Lab within 2014, article No.: 11233) into centrifuge tube, add 0.1M MES
(pH 5.0) buffer mixes, and with 13000rpm, 15min, 4 DEG C of pelleted by centrifugation, abandons supernatant, slow with 0.1M MES (pH5.0)
Fliud flushing is resuspended stand-by；It activates and cleans: activator EDC, NHS and fluorescent microsphere are activated according to the amount of mass ratio 2:1:2,
Concrete operations are as follows:
It weighs EDC, NHS and is added in 0.1M MES (pH 5.0) buffer and dissolve, appropriate extremely above-mentioned cleaning is taken to finish rapidly
Fluorescent microsphere in, seal with sealing film and be placed on room temperature on 200rpm shaking table and shake up 30min, 13000rpm after taking-up,
30min, 4 DEG C are centrifuged off supernatant, and ultrasound mixing is resuspended with equivalent 0.1M MES (pH 6.5) buffer and cleans, is repeated once
The above operation i.e. cleaning is twice.It is stand-by that supernatant is discarded after the completion of centrifugation.
Label: the latex after activation is resuspended into 0.1M MES (pH 6.5) buffer, is separately added into specificity rapidly
In conjunction with the antibody (mouse monoclonal antibody, 2016 be purchased from Hangzhou Long Ji Bioisystech Co., Ltd) of human chorionic gonadotrophin (HCG),
It mixes, places room temperature, shaken up 4 hours on 200rpm shaking table.
Closing: the microballoon for taking above-mentioned label good is centrifuged off supernatant in 10000rpm, 30min, 4 DEG C, immediately into microballoon
Equivalent confining liquid is added, ultrasound is placed room temperature after being resuspended again, shaken up on 200rpm shaking table 1 hour.10000rpm after the reaction was completed,
20min, 4 DEG C of centrifugations, takes supernatant to be checked.Detection content, quality standard and detection method are as shown in table 1.
1 detection content of table, quality standard and detection method
| Detection content | Quality standard | Detection method |
| Package amount | Package amount >=100 μ g antibody/mg microballoon | BCA determination of protein concentration method |
Cleaning is resuspended: re-suspension liquid is added, ultrasound piping and druming is resuspended, with high speed freezing centrifuge 10000rpm, 20min, 4 DEG C
Centrifugation, discards supernatant；Re-suspension liquid is added, ultrasound piping and druming is resuspended.
Step 2: the preparation of people's LH labelled antibody
Take anti-human interstitialcellstimulating hormone (ICSH) (LH) antibody (mouse monoclonal antibody, 2016 be purchased from the grand limited public affairs of base biotechnology in Hangzhou
Department), by above-mentioned steps, prepare people LH labelled antibody.The microspheres solution being resuspended marks, spare.
The specking of 3 bonding pad of embodiment
The specking method for specifically binding bonding pad filter paper is as follows: the dilution of fluorescent marker solution: using fluorescent marker re-suspension liquid
By fluorescent marker conjugate (coming from embodiment 1) 2 times of dilution of above-mentioned preparation；Point film instrument is set, the power supply of point film instrument is opened, if
Determine specking program, specking amount is 8 μ L/cm；No. 1 pipeline is specking channel；Point film instrument initialization: No. 1 pipeline is placed in fluorescence mark
Note is resuspended in solution, selects initialization program, initializes 6 circulations；Specking: 5 filter paper of Whatman Fusion is pressed into fixed bit
Horizontalization is placed in point film instrument, is pressed on control panel " GO " key and is started specking, removes after having put, and checks the good bonding pad of specking, spray
The fluorescent marker specific antibody band of point is uniform, the continuous and entire straight line of perforation is qualified specking product, and breakpoint occur in straight line is
Unqualified specking product；A piece of bonding pad filter paper is often put, pressing " GO " key on a control panel is that specking is primary (a piece of)；Specking
Terminate, the bonding pad of specking is placed in room temperature and is spontaneously dried 1 hour, specking trace should be can't see on film.
The preparation (specific antibody) of 4 detection line of embodiment and nature controlling line
Step 1: the preparation of HCG detection line
(mouse monoclonal antibody, 2016 limited purchased from the grand base biotechnology in Hangzhou purchased from 2016 for the antibody of specific binding people HCG
Company) it is taken out from refrigerator, room temperature is placed, it is spare.
The preparation method is as follows: the HCG antibody 500ug for taking mouse anti-human, is added in 5ml graduated centrifuge tube, antibody diluent is extremely
1ml, Container Tag T flag.Sheep anti-mouse igg antibody (opening Thailand purchased from Hangzhou in 2016, article No.: B103-GMI01) 10 μ L are taken, are added
Into 5ml graduated centrifuge tube, antibody diluent to 1ml, Container Tag C mark.Point film instrument is set, the power supply of point film instrument is opened,
Specking program is set, specking amount is 1 μ L/cm；No. 1 pipeline is detection line specking channel, and No. 2 pipelines are nature controlling line specking channel；
Point film instrument initialization: No. 1 pipeline being placed in detection line solution, No. 2 pipelines are placed in nature controlling line solution, selection initialization journey
Sequence initializes 6 circulations；Specking: nitrocellulose filter (NC film) is placed in point film instrument by fixed bit horizontalization, by control panel
Upper " GO " key starts specking, removes after having put, and checks the good detection line of specking, and detection line and nature controlling line are two uniform, continuous
Straight line with perforation is qualified specking product, and occurring breakpoint in two straight lines is unqualified specking product；Often put it is a piece of, by a secondary control
" GO " key on panel is that specking is primary (a piece of)；Specking terminates, and the NC film of specking is placed in room temperature and is spontaneously dried 1 hour,
Specking trace should be can't see on film.
Step 2: the preparation of LH detection line
The antibody (mouse monoclonal antibody is purchased from Hangzhou Long Ji Bioisystech Co., Ltd in 2016) for specifically binding people LH presses step
Operation preparation LH detection line in one.
Embodiment 5 assembles
The protection sheet of wider portion on bottom plate is removed, along the lower edge of protection sheet above, the good NC film of specking (is come from
Embodiment 3), by nature controlling line above in a manner of be attached on bottom plate, stick absorbent filter above NC film；It pastes the lower section of NC film
Upper bonding pad retains the protective film of sample pad area.
Embodiment 6 is cut
Cutting electromechanical source is connected, setting cuts film program, sets cutting width as 4mm；Kilocalorie (coming from embodiment 5) is laid flat
Enter in cutting machine platform track, face-up, presses on operation panel " GO " key, start to cut；A piece of kilocalorie qualified product is often put, is pressed
" GO " key is primary on operation panel, until having cut all kilocalorie qualified products；After the completion of cutting, test strips are sticked in into bottom side by side
On plate, test strip is formed.
Embodiment 7 is cut
Cutting electromechanical source is connected, setting cuts film program, sets cutting width as 4mm；By HCG kilocalorie (coming from embodiment 5)
It lays flat in cutting machine platform track, face-up, presses on operation panel " GO " key, start to cut；It is qualified often to put a piece of kilocalorie
It is primary to press on operation panel " GO " key for product, until having cut all kilocalorie qualified products.
Cutting electromechanical source is connected, setting cuts film program, sets cutting width as 2mm；By HCG kilocalorie (coming from embodiment 5)
It lays flat in cutting machine platform track, face-up, presses on operation panel " GO " key, start to cut；It is qualified often to put a piece of kilocalorie
It is primary to press on operation panel " GO " key for product, until having cut all kilocalorie qualified products.
After the completion of cutting, test strips are sticked on bottom plate side by side, forms test strip.
The detection card assembling of embodiment 8
Above-mentioned test strips (coming from embodiment 6) are fitted into cartridge, the guarantor of the sample pad area of two test strips is torn
Cuticula sticks sample pad in the bottom of bottom plate, forms sample distribution pad；Detection card is assembled into according to above procedure.
Take aluminium foil bag and desiccant；Open heat sealing machine, preheating；Detection card to be packed, 1 bag of desiccant are packed into aluminium foil bag
In；According to aluminium foil bag of the length cutting equipped with detection card and desiccant of regulation；Aluminium foil bag is sealed with heat sealing machine；It is labelled.
The detection card assembling of embodiment 9
Above-mentioned test strips (coming from embodiment 7) are fitted into cartridge, the guarantor of the sample pad area of two test strips is torn
Cuticula sticks sample pad in the bottom of bottom plate, forms sample distribution pad；Detection card is assembled into according to above procedure.
Take aluminium foil bag and desiccant；Open heat sealing machine, preheating；Detection card to be packed, 1 bag of desiccant are packed into aluminium foil bag
In；According to aluminium foil bag of the length cutting equipped with detection card and desiccant of regulation；Aluminium foil bag is sealed with heat sealing machine；It is labelled.
10 detection performance of embodiment
By above-mentioned test strips (coming from embodiment 8), carry out verifying its detection performance with following clinical serum sample；Often
After a 100 μ L of blood serum sample loading, loading 15min, divided with the AFS1000 fluorescence that Guangzhou Lan Bo Biotechnology Co., Ltd produces
Analyzer is detected；It purchases the HCG listed and LH commercial kit (from Xiamen Baotai Bio-Technology Co., Ltd.) is made
For control group (sample pad connects single test strips), testing result is as shown in table 2 and table 3:
2 HCG testing result of table
3 LH testing result of table
The result shows that the detected value of joint inspection test strips HCG and LH with sample distribution pad are measured close to commercial reagent box
Value, as shown in Figures 2 and 3, it was demonstrated that test strips performance meets detection demand.
The comparison of 11 detection efficiency of embodiment
By above-mentioned test strips (coming from embodiment 8), carry out verifying its detection performance with 60 clinical serum samples；Each
After blood serum sample loading 100 μ L, loading 15min, with the AFS1000 fluorescence analysis of Guangzhou Lan Bo Biotechnology Co., Ltd production
Instrument is detected；Count sample usage amount and detection efficiency.
The HCG that buying has listed is used as with LH commercial kit (from Xiamen Baotai Bio-Technology Co., Ltd.) and compares
Group (sample pad connects single test strips), carries out verifying its detection performance with above-mentioned identical 60 clinical serum samples；Each
After blood serum sample loading 80 μ L, loading 15min, detected；Sample usage amount and detection efficiency are counted, as a result such as 4 institute of table
Show.
The sample usage amount and detection efficiency of 4 joint inspection test strips of table
| Joint inspection test strips of the present invention | Control group | |
| Amount of samples (μ L) | 100 | 160 |
| Detection efficiency (sample volume/hour, μ L/h) | 120 | 80 |
The result shows that the joint inspection kit with sample distribution pad, can substantially reduce amount of samples, detection effect is improved
Rate.
The sample allocation proportion of 12 joint inspection test strips of embodiment
Above-mentioned test strips (from embodiment 9) 10 are taken, carry out verification sample distribution pad with 1 clinical serum sample
Allocation proportion；After each 100 μ L of blood serum sample loading, loading 15min, produced with Guangzhou Lan Bo Biotechnology Co., Ltd
AFS1000 fluorescence analyser is detected, and the distribution ratio that width in joint inspection kit is respectively the test strips of 4mm and 2mm is calculated
Example, the results are shown in Table 5.
The sample allocation proportion of 5 joint inspection test strips of table
The result shows that the joint inspection kit with sample distribution pad, more controllably, accurately sample can be distributed
In joint inspection test strips.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (10)
1. a kind of immune joint inspection test strips, which is characterized in that sample is arranged on bottom plate and distributes pad and test strips, sample distribution pad connects
At least 2 test strips are connected to, test strips are sequentially distributed along chromatography direction: labelled antibody bonding pad is coated in chromatographic film
It captures the test section of antibody, be coated with Quality Control portion, chromatographic film and the water-absorbent material that can capture labelled antibody substance.
2. immune joint inspection test strips according to claim 1, which is characterized in that test strips, bonding pad, in chromatographic film extremely
Few one kind is set as different in width, thickness or length, corresponding different sample sendouts.
3. immune joint inspection test strips according to claim 1, which is characterized in that the sample distributes the ingredient padded and includes
Glass fibre or long stapled filter paper, sample distribution pad are equipped with well.
4. immune joint inspection test strips according to claim 1 to 3, which is characterized in that it is parallel to each other between test strips,
And it distributes and pads perpendicular to sample.
5. immune joint inspection test strips according to claim 1 to 4, which is characterized in that sprayed on the sample distribution pad
It is coated with anti-human erythrocyte antibody.
6. immune joint inspection test strips according to claim 1, which is characterized in that the chromatographic film by nitrocellulose,
Cellulose acetate film, nylon membrane, pvdf membrane or 5 filter paper of Fusion are made.
7. immune joint inspection test strips according to claim 1, which is characterized in that the Quality Control portion is coated with can be with mark
Remember the substance that object combines, the mark substance, including colloidal gold, fluorescent microsphere or latex beads.
8. the preparation method of any immune joint inspection test strips of claim 1-7.
9. application of the immune joint inspection test strips as claimed in claim 1 to 7 in test sample.
10. application according to claim 9, which is characterized in that sample to be tested is added on sample distribution pad, through single layer base
Matter swimming to detection line forms interlayer structure, detects to sample to be tested.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112285358A (en) * | 2020-09-17 | 2021-01-29 | 上海基灵生物科技有限公司 | Reagent card for dog C-reactive protein and pancreas specific lipase duplex detection |
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