CN105223349B - A kind of device for detecting sample - Google Patents
A kind of device for detecting sample Download PDFInfo
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- CN105223349B CN105223349B CN201410309729.7A CN201410309729A CN105223349B CN 105223349 B CN105223349 B CN 105223349B CN 201410309729 A CN201410309729 A CN 201410309729A CN 105223349 B CN105223349 B CN 105223349B
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- 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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Abstract
The present invention relates to a kind of device for detecting sample, including, test strips, test strips include sample pad, label pad and detecting pad, and label pad is located at the downstream of sample pad, and detecting pad is located at the downstream of label pad；Wherein, the device of the detection sample also includes that capture is padded, and the capture pad is located at the upstream of test strips and is circulated with test strips liquid phase；The capture pad is connected or is not connected to test strips.The device of detection sample of the invention is divided into two parts and separates because of the antibody on capture pad and capture pad, increased time and the space of the combination of analyte in antibody and sample, so that with reference to more abundant, especially for special sample, the possibility of the false positive of exclusion, improves the accuracy of detection.
Description
Technical field
Device the present invention relates to detect sample, particularly for detecting the test strips of sample.
Background technology
Detect that this technology in sample with the presence or absence of analyte is used extensively using immune association reaction principle
In every field.The analyte of various biological specimens (saliva, blood, urine, serum, sweat etc.) can be detected with it
Matter carrys out the health status (early pregnancy, tumour, infectious disease, drugs etc.) of monitoring of diseases and the mankind.The basic original of this detection technique
Reason is built upon between immune molecule the performance with specific bond, such as antibody and antigen, haptens/antibody, biotin with
Antibiotin etc..In addition, many such detections can be completed on solid dielectric, such as conventional lateral flow reagent
In bar, glass or plastic microtiter plates, device for immunochromatography etc..Generally, can be in conjunction with one on immune specific binding molecule
A little solid particles or chemical substance, so can come qualitative by naked eyes or other instruments equipment, quantitatively or semi-quantitatively must
Go out testing result.This solid particle can be colored colloidal solid (latex or gold grain), and this chemical substance can be with
It is the material with chromophoric group, these materials can send specific wavelength to show detection knot under the conditions of other are suitable
Really.
Can be found in the prior art using the detection reagent bar or device of these principles, such as the following patent
The reagent strip of description or the device containing reagent strip：US4857453；US5073484；US5119831；US5185127；
US5275785；US5416000；US5504013；US5602040；US5622871；US5654162；US5656503；
US5686315；US5766961；US5770460；US5916815；US5976895；US6248598；US6140136；
US6187269；US6187598；US6228660；US6235241；US6306642；US6352862；US6372515；
US6379620；And US6403383.
Immune detection generally includes two kinds of principles, sandwich and competition law, wherein in detecting sample with competing method
Haptens small-molecule substance it is most commonly seen.The method and reagent and detection means detected using competition law are in United States Patent (USP)
US4235601；It is described later in detail in US4442204, US5208535, US5229073.These devices are all descriptions in detection
When not having color change or no colored line to occur on region or in result reading area, testing result is judged to the positive,
Represent that detection sample there may be analyte, conversely, when occur in detection zone or result reading area color change or
When person has colored line to occur, testing result is judged to feminine gender, represents in detection sample may not exist analyte.
In some detection means, sample process region separates with test strips, such as Chinese patent
A kind of detection means described in CN200610052628.1, including second reagent areas.Second reagent areas and examination
Paper slip is separated, and in advance and sample contact, the analyte in sample is sufficiently combined with its antibody；Then, it is processed
The sample crossed flows to test strips and is detected again.
In actual operation, there is problems with this detection means：For it is sticky or more than the low foam of water content
Saliva sample cannot completely be eluted the antibody in most early stage capture pad (the second reagent areas), i.e. sample cannot be with
Antibody on capture pad is sufficiently combined, and causes the antigen binding in antibody and label pad without q.s, therefore nothing
Method develops the color, so as to cause false positive.
The content of the invention
In order to solve these problems, the present invention provides an improved detection means and the side using the improved device
Method, the analyte and the molecule for capturing the specific bond on pad in sample can be made by improved detection means and method
(antibody) is more fully combined, so as to effectively overcome the problem of false positive, improves the accuracy rate of Product checking.
On the one hand, it is provided by the present invention it is a kind of detect sample device, including, test strips, test strips include sample pad,
Label pad and detecting pad, label pad are located at the downstream of label pad, and detecting pad is located at the downstream of label pad；Characterized in that, the inspection
The device of test sample sheet also includes that capture is padded, and the capture pad is located at the upstream of test strips and is circulated with test strips liquid phase；The capture
Pad can be connected or be not connected to test strips.
One preferred embodiment in, the capture pad include first capture pad and second capture pad.
In the mode being more highly preferred to, the first capture pad is located at the second capture pad upstream, and the two is not connected with, but liquid is connected
It is logical.
In one specific embodiment, the second capture pad is positioned at the upstream of test strips, the two and liquid communication.This
When, the second capture pad can be connected with test strips, it is also possible to be not connected with.
Capture pad includes two, and is not connected with, and the time for flowing through sample lengthens, i.e. total stop on capture pad
Time lengthens.When the reagent reacted with analyte in sample is included on capture pad, analyte can be made with examination
Agent has more fully time and space to be combined.
In some implementation methods, the second capture pad is connected positioned at the upstream of the sample pad of test strips with sample pad.
In one specific embodiment, the second capture pad is the prolongation of sample pad.
In other implementation methods, capture mat material matter is polyester fiber film or glass fibre membrane.
In preferred embodiment, the first capture mat material matter is polyester fiber film；Second capture mat material matter is glass fibre
Film.
In some implementation methods, dividing comprising a kind of specific bond analyte that can be flowed with liquid on capture pad
Son.
In other implementation methods, capture pad also include with the incoherent specific binding molecule of analyte to one kind
Molecule.
The first capture pad and the second capture pad for reagent (the specific bond analyte that is combined with analyte
Molecule and with the incoherent specific binding molecule of analyte to a kind of molecule) have certain proportioning after, can
More fully reacted with the analyte in sample.Some preferred embodiment in, second capture pad on special knot
Close analyte molecule and with the incoherent specific binding molecule of analyte to a kind of molecule content be first
With the 15%-50% of the second capture pad total amount.
One more specifically in implementation method, the molecule of the specific bond analyte on the second capture pad and with quilt
Analysis the incoherent specific binding molecule of material to a kind of molecule content be the first and second capture pad total amounts 20%-
35%.
Include in some implementation methods, on detecting pad a kind of fixed uncorrelated to the upper analyte of capture pad
Specific binding molecule to another molecule.
In other implementation methods, the molecule bag of the first upper specific bond analyte captured with second on pad of capture pad
Include the first antibody of analyte；Label pad includes the SA of analyte.
Preferably, it is described with the incoherent specific binding molecule of analyte to selected from following molecule to one of：
Biotin/avidin (biotin/avidin)；Biotin/Streptavidin (biotin/streptavidin)；If rhodamine/
Red bright antibody (rhodamine/anti-rhodamine)；Antibody (the Mouse IgG/anti-mouse of mouse IgG/ mouse IgG
IgG)。
Be included in capture pad on reagent (molecule of specific bond analyte and with the incoherent spy of analyte
Different binding molecule to a kind of molecule) be processed and be fixed on capture pad, when liquid sample is added on capture pad
And when flowing through capture pad, can be flowed with liquid after being combined with analyte by reagent of the fixing process on capture pad.
Wherein, reagent be processed at capture pad upper type it is various.In some specific embodiments, the molecule of specific bond analyte
And with the incoherent specific binding molecule of analyte to a kind of molecule be processed at whole first capture pad and the
On two capture pads.In this mode, analyte can be made sufficiently to be reacted with reagent.
Other preferred embodiment in, the molecule of specific bond analyte and incoherent with analyte
Specific binding molecule to a kind of molecule be processed on whole first capture pad；The molecule of specific bond analyte and
With the incoherent specific binding molecule of analyte to a kind of molecule the second capture is processed in linearly aligned mode
On one line of pad.Linearly aligned reagent arrangement on second capture pad is more concentrated, and all of sample is flowed through line
Property at carry out sufficient liquid contact, make to combine it is more abundant so that the also more efficient way false positive of product.
On the other hand, the present invention also provide it is a kind of detect liquid sample in the presence or absence of analyte method, including：
A kind of device for detecting sample is provided, the detection means includes that reagent strip and capture are padded, and described reagent strip test strips include sample
This pad, label pad and detecting pad, label pad are located at the downstream of sample pad, and detecting pad is located at the downstream of label pad；Capture pad includes
First capture pad and the second capture pad；First capture pad is located at the second capture pad upstream, and the two is not connected with but liquid phase circulates；
Positioned at the upstream of the sample pad of test strips, the two cocurrent body phase that is connected is connected second capture pad；It is characterized in that：Caught to first
The upper adding liquid sample of pad is obtained, is allowed the liquid sample first and the first reagent reacting captured on pad is after -60 minutes 30 seconds；Then allow
Liquid sample flow to successively sample pad in the second capture pad and test strips, label pad, detecting pad and with each pad on reagent
Reaction completes detection；Read testing result.
In some implementation methods, the reagent on capture pad includes the molecule and and analyte of specific bond analyte
The incoherent specific binding molecule of matter to a kind of molecule；The molecule is connected with the molecule of described specific bond analyte
Or combine and can be flowed with liquid.
Some preferred embodiment in, the molecule of the specific bond analyte on the second capture pad and with it is analyzed
The incoherent specific binding molecule of material to a kind of molecule content be the first and second capture pad total amounts 15%-50%.
Other preferred embodiment in, to first capture pad adding liquid sample after, allow liquid sample and first to catch
Obtain the reagent reacting -30 minutes 30 seconds on pad.
The present invention also provides a kind of kit for detecting sample, including：The device for detecting sample and the device for collecting sample,
Wherein, the device of detection sample includes reagent strip, and described test strips include sample pad, label pad and detecting pad, label pad position
In the downstream of sample pad, detecting pad is located at the downstream of label pad；Wherein, collecting the device of sample includes that liquid sample can be absorbed
Collection head；Characterized in that, also including that capture is padded in the device of described detection sample, capture pad is positioned at the upstream of test strips
And circulated with test strips liquid phase；The capture pad can be connected or be not connected to test strips.
Preferably, capture pad includes the first capture pad and the second capture pad；First capture pad is located at the second capture pad upstream,
The two is not connected with but liquid phase circulates；Positioned at the upstream of the sample pad of test strips, the two is connected and fluid second capture pad
It is connected.
Other preferred embodiment in, include specific bond analyte on the first capture pad and the second capture pad
Molecule and with the incoherent specific binding molecule of analyte to a kind of molecule.
In some specific embodiments, the molecule of the specific bond analyte on the second capture pad and with it is analyzed
The incoherent specific binding molecule of material to a kind of molecule content be the first and second capture pad total amounts 15%-50%.
Beneficial effect
The device of detection sample of the invention is divided into two parts and separates because of the antibody on capture pad and capture pad, increased
The time of the combination of analyte and space in antibody and sample, so that with reference to more abundant, especially for special sample
This, the possibility of the false positive of exclusion improves the accuracy of detection.
Brief description of the drawings
Fig. 1 is the decomposition texture schematic diagram of common test strip；
Fig. 2 is the top view of common test strip；
Fig. 3 is the structural representation of the device of the detection sample before improvement of the invention；
Fig. 4 is the structural representation of the device of the detection sample after improvement of the invention；
Fig. 5 is that the detection means of one concrete scheme of the present invention detects result schematic diagram after preceding and detection；
Fig. 6 is that the detection means of another concrete scheme of the invention detects result schematic diagram after preceding and detection；
Fig. 7 is that the detection means of another concrete scheme of the invention detects result schematic diagram after preceding and detection；
Fig. 8 is the decomposing schematic representation of detection box of the present invention comprising the device that sample is detected after improving；
Fig. 9 is the generalized section for part that Fig. 8 detects box.
Description of reference numerals：
100,300 reagent strips；101 sample pads；102 label pads；103 detecting pads；132 testing result lines；133 testing results
Control line；104 adsorptive pads；105 support chips；210 capture pads；310 first capture pads；320 second capture pads；400 detection means；
500 collection devices；501 collect handle；502 collect head；4011 first collecting chambers；4012 second collecting chambers；401 collecting chambers；
The through hole of the collecting chamber of 4011-1,4011-2 first；403,403-1,403-2 sealing ring；406 sample ingress areas；406-1 samples
Import through hole；3101 paste-sides；3102 reagent ends；404 bodies；4041 read window；Confirm chamber 407；4071 bases；4073 lead
Go out through hole；4072 stoppers；4043 upper plates；4045 lower plates.
Specific embodiment
Structure of the present invention or these technical terms for being used are described further below.
Test strips 100
General reagent strip, as depicted in figs. 1 and 2, reagent strip 100 includes sample application zone and marked region and inspection
Region is surveyed, detection zone includes a kind of specific binding molecule.The reagent strip for more optimizing also includes the suction positioned at detection zone downstream
Aqua region, sample can flow along the signified direction of arrow on reagent strip.Include completing to react on sample application zone
Necessary material, such as some buffer reagents, pretreatment sample reagent etc.；Include fluorescence labeling material in marked region,
Colloid gold label particle, or to colour gel marking particle, can also be water-soluble marker's material, these mark substances can be with
Connection antibody, antigen, haptens or analyte similar substance etc., include fixed special knot in detection zone
Molecule is closed, it is analyzed whether there is in representing sample to occur color change in detection zone by specific binding molecule
Material.Can also include testing result control area 133 in the downstream of detection zone.Reagent strip can be made up of following part,
Sample pad 101 and label pad 102, detecting pad 103, adsorptive pads 104, these materials are assembled on support chip 105 and constitute whole
Reagent strip 100 (such as Fig. 1)；Include detection zone 132 wherein on detecting pad 103, preferential scheme is under detection zone
Trip also includes testing result control area 133.Liquid sample can along the signified direction of arrow from sample pad 101 sequentially through
Label pad 102, detecting pad 103 finally reach adsorptive pads 104.The all parts for constituting reagent strip can be by absorbent material structure
Into generally, detecting pad 103 can be constituted for one of nitrocellulose filter, CAM or nylon membrane.These constitute examination
The part and material of agent bar and to process some conventional chemical reagent and processing method on these parts be all prior art
It is disclosed.
Capture pad 210
Include the molecule of the specific bond analyte that can be flowed with liquid, 210, the capture pad on capture pad 210
Separated in the upstream of reagent strip 100 and with reagent strip 100.A scheme as shown in Figure 3, capture pad 210 is located at reagent strip 100
Sample pad 101 upstream, liquid sample firstly flow through capture pad 210 then reach reagent strip 100 on sample pad on 101 complete
Whole detection.
Detection means 400
Such as Fig. 8, shown in 9, detection means 400 includes body 404 and collection well 401, wherein reagent strip 100 or 300
In body 404, capture pad is located in collection well 401；Capture pad and reagent strip are in liquid communication state.Here institute
" the liquid communication state " said refers to that liquid can be flowed on reagent strip from collecting chamber 401, and this flowing can be due to liquid sheet
The Action of Gravity Field of body and flow, it is also possible to flowed by being provided with through hole between collecting chamber 401 and reagent strip 300, at the same receive
Some flow guiding structures can also be installed between collection chamber and reagent strip, can so allow liquid more smoothly to be flowed to from collecting chamber
On reagent strip.More specifically, include reading window 4041 and sample ingress area 406, sample ingress area on body 404
406 import through hole 406-1 etc., the correspondence of reading window 4041 in the detection zone and body 404 on reagent strip, examination including sample
It is corresponding that the sample application zone (sample pad) of agent bar imports through hole with sample.The sample that through hole 406-1 is flowed into is imported by sample
The sample application zone on reagent strip can be flowed to and then detection zone is reached and complete whole detection reaction.Collecting chamber 401 includes
First collecting chamber 4011 and the second collecting chamber 4012, the one end open of the first collecting chamber 4011 be used for receive need detection sample or
Person receives the collection device with sample, and several liquid through-hole 4011-1 that liquid can be allowed to pass through are provided with an opposite end,
4011-2 etc. (Fig. 9)；There is a vertical bar beam on the correspondence position of liquid through-hole 4011-1, the beam one end can be fixed on the first receipts
Collect on the side wall in chamber 4011, the other end extends downward and through liquid through-hole 4011-1, capture pad is located on vertical bar beam.Capture pad
One end can be fixed on the surface of vertical beam, and the other end can pass through liquid through-hole to reach the bottom of the second collecting chamber 4012,
Capture the specific bond analyte for including being flowed with liquid on one end of pad molecule (Y1) and with analyte not phase
The specific binding molecule of pass plants molecule (M1) to one of (M1/M2)；The material of the capture pad can be some water-absorbing materials
Composition, some protein substances, such as glass fibre, filter paper, acetate fiber etc. can be processed above, and the one end for capturing pad can
To be bonded at the surface of vertical beam by organic nothing or machine glue.The capture pad can also be adhesive in liquid through-hole 4011-2.Capture pad
Can also other forms be present in collecting chamber 401, such as capture pad can be placed directly in the bottom of the first collecting chamber 4011,
Can be placed in the insulating space formed between the first collecting chamber bottom and the second collecting chamber bottom, as long as capture pad is placed on receipts
In collection chamber 401, can allow sample fully and its contact just can be so that in collecting chamber 401, capture pad 210 can be completely soaked
In sample, it is also possible to be partly immersed in sample.Some other auxiliary reagents can also be included on capture pad, such as buffering is made
Buffer reagent, some protein moleculars etc..Those of ordinary skill in the art with reference to the present invention be all readily apparent that other
Form or mode can serve as one embodiment of the present of invention.First collecting chamber 4011 and the second collecting chamber 4012 are assembled into
Collecting chamber 401 (Fig. 8).Second collecting chamber 4012 includes one end open, and the other end is provided with several liquid through-holes, logical in these liquid
Hole is separately installed with corresponding sealing ring 403,403-1,403-2 etc..Similar other specific structure descriptions with the present apparatus
There is specific description in U.S. Patent application 2005/0180882A1.
In specific embodiment, can also include and the incoherent specific bond of analyte on capture pad
Molecule to a kind of molecule M1, the molecule is connected or combines with the molecule Y1 of specific bond analyte.Capture pad can be with position
In the upstream of sample pad on reagent strip.More specifically, it is exactly that capture is padded positioned at the upstream of sample pad 201, sample pad is located at mark
Remember the upstream of pad 202, label pad is located at the upstream of detecting pad；Specific bond is fixed with detection zone on detecting pad to be divided
Another molecule Y2 of material is analysed, mark substance L is included in label pad and is divided with the incoherent specific bond of analyte
Son to another molecule M2 (Fig. 5 A).When being reacted, if containing analyte A in sample, it is analyzed
Specific binding molecule Y1 in substance A and capture pad is combined to form compound substance A-Y1-M1；When the compound substance passes through sample
This pad is reached in label pad, just forms new compound substance A-Y1-M1-M2-L；When new compound substance is flowed through on detecting pad
When, the new compound substance of the specific binding molecule Y2 specific bonds that are fixed in detection zone, so as to go out in detection zone
Existing color represents positive findings (Fig. 5 B).
In another specific embodiment, special knot incoherent with analyte can also be included on capture pad
Close molecule to a kind of molecule M1, the molecule is connected or combines with the molecule Y1 of specific bond analyte, in detecting pad
Be fixed with detection zone with the incoherent specific binding molecule of analyte to another molecule M2, in label pad wrap
Include the mark substance L and another molecule Y1 (Fig. 6 A) with specific bond analyte.When being reacted, if sample
Contain analyte A in this, then the specific binding molecule Y1 on analyte A and capture pad carries out specific bond and forms multiple
Compound matter A-Y1-M1；When the compound substance is reached in label pad by sample pad, new compound substance M1-Y1-A- is just formed
Y2-L；When new compound substance is flowed through on detecting pad, it is fixed on incoherent with analyte in detection zone
Specific binding molecule to the new compound substance of another molecule M2 specific bonds, M2-M1-Y1-A-Y2-L is formed, so as in inspection
There is color on survey region and represent positive findings (Fig. 6 B).
In another specific embodiment, based on competition law, include on capture pad incoherent with analyte
Specific binding molecule to a kind of molecule M1, the molecule is connected or combines with the molecule Y1 of specific bond analyte, in inspection
Survey pad detection zone on be fixed with the incoherent specific binding molecule of analyte to another molecule M2, mark
Include the similar substance A* (Fig. 7 A) of mark substance L and analyte on pad.Reagent concentration on capture pad and test strips can
Arbitrarily adjusted with different with the requirement of detection；, it is necessary to make the concentration of certain drugs in sample big for example in illicit drugs inspection
When the concentration C for pre-setting, allow colored line is occurred without in detection zone represent positive findings, less than concentration C
When, in detection zone colored line occur represents negative findings.
When being reacted, if contain analyte A in sample, and more than the concentration C for pre-setting
When, then the specific binding molecule Y1 on capture pad almost carries out specific bond and forms compound substance with analyte A completely
A-Y1-M1, is now likely present the analyte A of remaining excess；When the compound substance reaches label pad by sample pad
On, because the reagent Y1-M1 on capture pad is reacted completely, the reagent A *-L in label pad cannot be attached on Y1-M1；When
Compound substance A-Y1-M1 is flowed through when on detecting pad, be fixed in detection zone with the incoherent spy of analyte
Different binding molecule to another molecule M2 specific bonds compound substances, M2-M1-Y1-A is formed, so as in detection zone
Occur without color and represent positive findings (Fig. 7 B).
If conversely, containing analyte A in sample, and when less than the concentration C for pre-setting, then capturing pad
On specific binding molecule Y1 be part and analyte A carry out specific bond formed compound substance A-Y1-M1, now
It is likely present the Y1-M1 of remaining excess；When the compound substance is reached in label pad by sample pad, the reagent in label pad
A*-L is just and Y1-M1 combines to form M1-Y1-A*-L；When these compound substances A-Y1-M1, M1-Y1-A*-L are flowed through on detecting pad
When, be fixed in detection zone with the incoherent specific binding molecule of analyte to another molecule M2 it is special
With reference to the compound substance, M2-M1-Y1-A and M2-Y1-A*-L is formed, negative knot is represented so as to occur color in detection zone
Really (Fig. 7 B).
The incoherent specific binding molecule of analyte include but not limited to this to (M1/M2), biotin/affinity
Plain (biotin/avidin), biotin/Streptavidin (biotin/streptavidin), antibody/antigen (antibody/
Antigen) (antibody and analyte not including anti-analyte are in itself), the antibody of rhodamine/rhodamine
(rhodamine/anti-rhodamine), antibody (Mouse IgG/anti-mouse IgG) of mouse IgG/ mouse IgG etc..
Analyte A can be antibody or antigen, Y1 and Y2 can be on analyte the corresponding specific antigen of different loci or
Antibody molecule or antibody fragment.Mark substance L includes but are not limited to this, colloid gold particle, latex particle, water-soluble
Mark substance etc., the mark substance antigen special with some, antibody or other specific binding molecules are connected or combine
Technology is existing known technology.Certainly, other of some regulation reaction system conditions can also be included in sample application zone
Chemical reagent, such as phosphoric acid buffer reagent, borate buffer reagent, carbonic acid buffer reagent or other albumen, macromolecular etc. come
Optimizing reaction system.These Optimized Measures are all that persons skilled in the art combine the present invention and prior art is readily apparent that
With implementation.
The device in example is applied using upper mask body can detect low concentration or small-molecule substance in sample, because some
The molecular weight very little at important analyte concentration in the sample very bottom or analyte, using traditional detection
Device often can not be detected because the analyte concentration in sample is very low or reach the requirement on clinical meaning.Utilize
Device of the invention in advance need and sample reaction agent treatment on capture pad, one can be to allow sample and reagent reacting
Fully, the degree that reaction will not be influenceed to complete because liquid to flow in time, another is exactly that operator need not be still further
The other reagents of addition.
But in actual detection, the above-mentioned detection means including capture pad is directed to some especially sticky or water content
Sample more than low foam, such as sticky saliva sample still occurs following problem：Because the viscosity of sample is higher, contain
Water is few, or show bubble state, can not sufficiently be reacted with the reagent on capture pad when causing sample on capture pad,
Cause the reagent (molecule of antibody or specific bond) captured on pad can not be completely combined and with the examination in sample inflow downstream
On paper slip, then in the detection process of test strips, the antigen binding in antibody and label pad without q.s, nothing can be caused
Method develops the color, so as to cause false positive (in particular for the product of competition law detection).
This problem is solved, then the reagent for needing to enable the analyte in sample fully to be padded with capture is (special
With reference to analyte molecule Y1 and with the incoherent specific binding molecule of analyte to a kind of molecule M1) combine,
One preferred embodiment in, increase by one capture pad, make capture pad include 2, and 2 capture pad be separately not attached to
Connect, treatment has a reagent (Y1, M1) combined with analyte in sample respectively on separate two captures pad, the reagent it is total
Measure identical with the total amount of an original capture pad.So, sample is firstly flowed through the first capture pad, enter with the reagent on capture pad
Row sufficiently after reaction, passes through the second capture pad, is reacted again with reagent thereon.So, in reagent total amount identical
In the case of, the time and space that analyte and reagent are combined all increase, so that the combination of the two is more abundant, because
This, in the detection process of follow-up test strips, it is to avoid because the deficiency of the antibody that analyte is carried cannot be in detection zone
The false positive that colour developing is caused.
In some specific embodiments, the first capture pad 310 is identical with original capture pulvilliform shape, and difference is to locate thereon
Reagent (Y1, M1 etc.) amount of reason is reduced；And the first capture pad 310 is still located on the first collecting chamber 401 of detection means.The
Two capture pads 320 are located in test strips 100, more specifically, in the sample pad 101 of test strips, with the part of sample pad 101
Overlap joint, as shown in Figure 4；Treatment has the reagent (Y1, M1) of another part on second capture pad 320, and sample first flows through the second capture
Pad and be simultaneously combined with reagent thereon, then flow through sample pad in test strips, label pad, detecting pad and water suction successively again
Pad, completes detection.
In some specific embodiments, the reagent content on the first capture capture pad 320 of pad 310 and second has one
Fixed proportioning, in some specific embodiments, second capture pad on reagent (the molecule Y1 of specific bond analyte and
With the incoherent specific binding molecule of analyte to a kind of molecule M1) between the 15-50% of reagent total amount.At this
Under proportioning, the combination of analyte and reagent in sample is more abundant.In another particularly preferred embodiment, the second capture
On pad reagent (the molecule Y1 of specific bond analyte and with the incoherent specific binding molecule of analyte to one of
Plant molecule M1) between the 20-35% of reagent total amount.
In a specific embodiment, the production of test strips for convenience, the second capture pad can directly and sample pad
It is identical, it is the prolongation of sample pad.After the part treatment of extension has Y1 and M1, i.e., equivalent to the function of the second capture pad,
Play a part of the second capture pad.
The material for capturing pad can be polyester fiber film, glass fibre membrane and can stream material selected from capture mat material matter
Filter paper etc..First capture pad can be with identical with the material of the second capture pad, it is also possible to different.One preferred embodiment
In, the first capture mat material matter is polyester fiber film；Second capture mat material matter is glass fibre membrane.
Detected with saliva is collected in saliva below and be described in detail how to use the present invention come as a example by with the presence or absence of drugs
Detection means and detection box.As shown in figure 8, being the collection phase before detection, the first collecting chamber 4011 is located at the second collecting chamber
Inside 4012, and liquid through-hole on the second collecting chamber is not and the sample on sample ingress area 406 imports through hole
406-1 is communicated, but is sealed by the baseplane of sample ingress area 406, in order to increase collecting chamber 401 and sample ingress area
The sealing of 306 baseplane, can install sealing ring in the liquid through-hole correspondence position of the bottom of the second collecting chamber 4012.It is first
The saliva to be detected first is collected, the saliva sample for needing detection is first collected with sample collection device 500, be put into collecting first 502
To in the mouth of detected person, 502 are made up of absorbent material, for example sponge material, so collection head can absorb and need detection
Saliva sample.Then in the first collecting chambers 4011 collection first 502 that suction has saliva sample put into collecting chamber 401,
Collection first 502 is extruded by rotating the handle 501 of collection device, the saliva sample at this time collected on head is just extruded to the
In one collecting chamber 4011, and it is flowed into two collecting chambers 4012 by liquid through-hole.In this process, due to the first collecting chamber
The first capture pad 310 in 4011 extends to the bottom of the second collecting chamber 4012 by liquid through-hole, while sample is extruded,
Reagent of the saliva sample just and on the first capture pad 310 fully reacts, after reacting suitable time, such as 10,20 or 30
Second, after 1,2,3,4,5,6,7,8,9,10,15,20,30,45,60 minutes, collecting chamber 401 is rotated, allow the liquid of the second collecting chamber
Liquid on through hole and liquid ingress area imports through hole and communicates, and liquid through-hole and liquid import through hole and communicates.This when, saliva
Liquid sample just imports through hole and flows on reagent strip by liquid, and liquid is arrived first at the second capture pad of test strips upstream end,
Liquid sample continues sufficiently to be combined with the reagent on the second capture pad, and then, liquid is flowed into sample pad again, successively
Flow further downstream, reaches label pad and detecting pad, finally reaches adsorptive pads, carries out detection reaction, if existed in saliva certain
The drugs of amount, colored line are occurred without in the detection zone of reagent strip and represent positive findings, conversely occur as soon as colored line table
Show negative findings, this testing result in detection zone can observe result, read by the reading window 4041 on body
Taking on window 4041 can be that transparent material covering detection zone can also directly allow detection zone exposed outside.Saliva sample
Originally through hole can also be imported by liquid and enters confirmation chamber 407, saliva sample is detected to further confirm.Confirm chamber
407 are surrounded by the side wall of base 4071 and surrounding, can be taken out by deriving through hole 4073 into the liquid for confirming chamber, are derived logical
Hole 4073 is sealed before sampling by a stopper 4072.When the testing result further by other means of needs are thought, for example, use liquid
Phase color is general, gas chromatography or other more accurate instruments are confirmed when testing result, removes stopper 4073, from confirming chamber
407 take out part saliva samples to detect (such as Fig. 8 and 9).Here be only with saliva sample as an example come specifically
It is bright how to use detection means of the invention, in addition to saliva, can also be other liquid samples, such as blood, urine, sweat
Liquid, excrement etc..
Embodiment
Illustrate how the present invention realizes that illustrated with limited experiment now, these explanations only exist in order to clearer
Limited citing is done under the marrow of claim of the invention, limiting to the claimed invention is not constituted.
Experiment：With the present invention improve before and after detection means detect saliva in the presence or absence of certain density anxiolytic with
Hypnotic sedative agent (BZO).
Part I：The assembling of reagent strip of the invention and detection means and part make (has the first and second captures
Pad)
Reagent strip 100 as shown in Figure 3 is used for illustrating the part and preparation method of reagent strip of the invention used by this experiment.
The treatment of nitrocellulose filter.Detecting pad 103 is nitrocellulose filter (NC), there is two on nitrocellulose filter
Lines, one is detection line 132, positioned at the testing result control line 133 in detection line downstream.It is fixedly connected with detection line
The Streptavidin (streptavidin-IgG) of IgG；The fixed foster anti-rabbit IgG on testing result control line.Fixed method
Can be automatically processed with automatic spray film process machine, wherein the concentration in processing detection lines is 1.0 mg/mls, dilution
Buffering liquid is phosphate buffer (PBS)；Goat anti-rabbit igg antibody is fixed on testing result control line；Concentration be 1.0 milligrams/
Milliliter, dilution buffer liquid is phosphate buffer (PBS).The nitrocellulose filter handled well is placed in 37 DEG C of baking ovens and is dried i.e.
Can.
The treatment of label pad 102.Marker slip is polymer PET, and BSA is divided with coupled protein by what colloid gold particle was marked
BZO haptens be processed on polymer PET.The OD values of processing solution are 57, and dilute solution is 1 times of PBS cushioning liquid, wherein
Also contain 1% BSA.Also treatment has the rabbit igg antibody of colloid gold label on marker slip.The marker slip handled well is placed on 37
Dried in DEG C baking oven.
The treatment of sample pad 101.Sample blank film is glass fibre element film, and the solution processed on the film is：Borax
(0.07M/L)；Tween20 (1%)；Cholic Acid (1%)；Tris(0.1M).The sample blank film handled well is placed on 37
Dried in DEG C baking oven.
The treatment of the first capture pad 310.Capture pad includes the reagent end of the paste-side 3101 of unwetted and water imbibition
3102.The material at reagent end is polyester film, and the chemical solution of reagent end treatment includes：The anti-of the upper biotin (Biotin) of connection
BZO antibody materials are dissolved in 1 times of PBS cushioning liquid, make ultimate density be 1.0 mg/mls；Wherein also contain 1%
BSA, it is 0.0025 milligram to make handle well first to capture the anti-BZO antibody materials content on pad.The capture pad handled well is put
Dried in 37 DEG C of baking ovens.Then the polymer PET small pieces (3102) containing reagent are pasted onto on the small pieces of unwetted
(3101) on.
The treatment of the second capture pad 320.The capture of a part second pad processes chemical solution on whole capture pad：Connection
The anti-BZO antibody materials of upper biotin (Biotin) are dissolved in 1 times of PBS cushioning liquid, make ultimate density for 0.2 milligram/
Milliliter；Wherein also contain 1% BSA；It is 0.0005 milli to make handle well second to capture the anti-BZO antibody materials content on pad
Gram.The capture pad of another part second, the second capture for being sprayed on the anti-BZO antibody materials of 0.2 mg/ml in the way of a spray
Pad on the position of the linear series at a port 2mm.All of second capture pad is placed in 37 DEG C of baking ovens and is dried.
The content ratio about 5 that the final anti-BZO antibody materials content made on first sample pad is padded with the second capture：1.
The assembling of reagent strip 300.The all parts handled well are assembled according to the appearance shown in Fig. 4, the second capture pad is allowed
(two kinds) upstreams for being located at sample pad, allow sample pad to be located at the upstream of label pad, and label pad is located between detecting pad and sample pad,
The filter paper 104 of water suction is placed in the downstream of detecting pad.These parts are all placed on a support chip for unwetted 105.
The assembling of detection means.Detection means such as Fig. 8, shown in 9.The paste-side 3101 of the first capture pad is pasted onto first
On the surface of the vertical bar beam on collecting chamber 4011, the other end 3102 passes through liquid through-hole and reaches the bottom of the second collecting chamber 4012
Portion.Then whole collecting chamber 401 is arranged in the snap ring of the sample Lead-In Area 406 of body 404, and allows the second collecting chamber
4012 bottom is in the position of basal surface sealing (Fig. 8) for being imported into region.Reagent strip 300 with capture pad is placed on
Between upper plate 4043 and lower plate 4045, make the detection zone of reagent strip 100 corresponding with reading window 4041, sample application zone and
Liquid imports through hole 306-1 correspondences.Upper plate and lower plate are fastened as one is overall.Thus it has been assembled into this experiment detection dress used
Put.
Part II：The assembling and making (there is capture to pad) of control stripes bar 100 and detection means 400.
Compared with reagent strip recited above and detection means, contrast agents bar is substantially the same with detection means.With
Unlike, install on detection means be capture pad, while in test strips without second capture pad.Wherein, catch
Obtain the treatment of pad 210.The chemical solution for capturing the reagent end treatment of pad includes：The anti-BZO of the upper biotin (Biotin) of connection
Antibody materials are dissolved in 1 times of PBS cushioning liquid, make ultimate density be 1.0 mg/mls；Wherein also containing 1% BSA,
The anti-BZO antibody materials content on the capture pad handled well is set to be about 0.003 milligram.
Testing result record all reads after saliva sample is added 10 minutes.Generally, set the concentration of BZO in saliva sample as
10ng/ml (nanograms/milliliter) is detection threshold value.If detection threshold value it is meant that analyte BZO in sample
Concentration is more than detection threshold value, and in theory, testing result should be positive, whereas if the concentration of analyte BZO is less than
Detection threshold value, testing result is feminine gender.
Operating method：
The bottom for first allowing each detection means to be in the second collecting chamber 4012 of collecting chamber 401 is imported into region 406 and closes
State.
The sample to be detected is subsequently adding, allows in sample and collecting chamber 401 reagent captured on pad (the first capture pad) anti-
Answer 1 minute；
Rotating collection chamber 401, allows saliva fluid that the sample application zone of reagent strip is flowed to from collecting chamber and completes reaction；
The actual result of detection is recorded after 10 minutes.
Explanation：
Control is the detection means before improving；Lot1 is device after improving, wherein the second capture pad disposed of in its entirety examination
Agent；Lot2 is device after improving, wherein the second capture pad linear process reagent
1. the detection sensitivity of detection means before and after improving is compared using standard solution.Set respectively in master sample
The concentration for putting BZO is 0ng/ml (negative sample)；30ng/ml, is detected using each device.
Analysis：
Detection means after improvement in the detection sensitivity than improving before it is higher.
2. the detection of pair negative sample：The saliva sample for gathering our company employee is detected
Analysis：
False positive sample before improvement, after being detected using the detection means after improvement, is completely eliminated.Therefore, should
Detection means after improvement can avoid the presence of the false positive of detection, improve the accuracy rate of detection.
3. the detection of pair positive sample：
The multiple feminine gender saliva samples of collection, are separately added into the BZO (seeing attached list) of various concentration in these salivas, enter respectively
Row detection
Analysis：
From in terms of upper table, the detection means after improvement, when positive sample is detected, does not have any changing compared with before improvement
Become.Therefore, the detection means after improvement does not have any influence when positive sample is detected.
In summary interpretation：
1. the detection means after improving increases compared with before-improvement in detection sensitivity；
2. the detection means after improving can effectively avoid the false positive issue of negative sample, improve the accurate of detection
Rate；
3. the detection means after improving is identical with before improvement when positive sample is detected, does not influence what positive sample was detected
Accuracy.
Claims (18)
1. it is a kind of detect sample device, including, test strips, test strips include sample pad, label pad and detecting pad, label pad position
In the downstream of sample pad, detecting pad is located at the downstream of label pad；Characterized in that, the device of the detection sample also includes capture
Pad, the capture pad is located at the upstream of test strips and is circulated with test strips liquid phase；The capture pad is connected or is not connected to test strips；
The capture pad includes the first capture pad and the second capture pad；First capture pad is located at the second capture pad upstream, and the two is not connected with,
But liquid communication；Second capture pad is positioned at the upstream of test strips, the two liquid communication.
2. it is according to claim 1 detection sample device, it is characterised in that second capture pad positioned at test strips sample
The upstream of pad, and be connected with sample pad.
3. it is according to claim 2 detection sample device, it is characterised in that second capture pad for sample pad extension
Point.
4. the device of detection sample according to claim 1, it is characterised in that capture mat material matter is polyester fiber film or glass
Glass tunica fibrosa.
5. the device of detection sample according to claim 4, it is characterised in that the first capture mat material matter is polyester fiber
Film；Second capture mat material matter is glass fibre membrane.
6. according to the device of one of claim 1-5 described detection sample, it is characterised in that can be with comprising one kind on capture pad
The molecule of the specific bond analyte flowed with liquid.
7. the device of detection sample according to claim 6, it is characterised in that described capture pad also includes and analyzed
The incoherent specific binding molecule of material to a kind of molecule.
8. it is according to claim 7 detection sample device, it is characterised in that second capture pad on specific bond is divided
Analyse thing molecule and with the incoherent specific binding molecule of analyte to a kind of molecule content be first and second
The 15%-50% of capture pad total amount.
9. it is according to claim 8 detection sample device, it is characterised in that second capture pad on specific bond is divided
Analyse thing molecule and with the incoherent specific binding molecule of analyte to a kind of molecule content be first and second
The 20%-35% of capture pad total amount.
10. it is according to claim 9 detection sample device, it is characterised in that the molecule of specific bond analyte with
And with the incoherent specific binding molecule of analyte to a kind of molecule be processed at whole first capture pad and second
On capture pad.
11. it is according to claim 9 detection samples devices, it is characterised in that the molecule of specific bond analyte with
And with the incoherent specific binding molecule of analyte to a kind of molecule be processed on whole first capture pad；Specifically
With reference to analyte molecule and with the incoherent specific binding molecule of analyte to a kind of molecule with linear array
Mode be processed on a line of the second capture pad.
With the presence or absence of the method for analyte in a kind of 12. detection liquid samples, including：
A kind of device for detecting sample is provided, the detection means includes that test strips and capture are padded, and described test strips include sample
Pad, label pad and detecting pad, label pad are located at the downstream of sample pad, and detecting pad is located at the downstream of label pad；Capture pad includes the
One capture pad and the second capture pad；First capture pad is located at the second capture pad upstream, and the two is not connected with but liquid phase circulates；The
Positioned at the upstream of the sample pad of test strips, the two cocurrent body phase that is connected circulates two capture pads；It is characterized in that：To the first capture
Adding liquid sample on pad, allows the liquid sample first and the first reagent reacting captured on pad is after -60 minutes 30 seconds；Then liquid is allowed
Body sample flows to sample pad in the second capture pad and test strips, label pad, detecting pad and anti-with reagent on each pad successively
Detection should be completed；Read testing result.
13. methods according to claim 12, it is characterised in that the reagent on capture pad includes specific bond analyte
Molecule and with the incoherent specific binding molecule of analyte to a kind of molecule；The molecule and described specific bond
The molecule of analyte is connected or combines and can be flowed with liquid.
14. methods according to claim 13, it is characterised in that second captures dividing for the specific bond analyte on pad
Son and with the incoherent specific binding molecule of analyte to a kind of molecule content for the first and second capture pads it is total
The 15%-50% of amount.
15. methods according to claim 14, it is characterised in that after padding adding liquid sample to the first capture, allow liquid
Reagent reacting on sample and the first capture pad -30 minutes 30 seconds.
A kind of 16. kits for detecting sample, including：The device for detecting sample and the device for collecting sample, wherein, detect sample
Device include test strips, described test strips include sample pad, label pad and detecting pad, and label pad is located under sample pad
Trip, detecting pad is located at the downstream of label pad；Wherein, collecting the device of sample includes absorbing the collection head of liquid sample；Its
It is characterised by, also include that capture is padded in the described device for detecting sample, capture is padded positioned at the upstream of test strips and and test strips
Liquid phase circulates；The capture pad can be connected or be not connected to test strips；Capture pad includes the first capture pad and the second capture pad；
First capture pad is located at the second capture pad upstream, and the two is not connected with but liquid phase circulates；Second capture pad is positioned at test strips
The upstream of sample pad, the two cocurrent body phase connection that is connected.
17. kits as claimed in claim 16, it is characterised in that include special knot on the first capture pad and the second capture pad
Close analyte molecule and with the incoherent specific binding molecule of analyte to a kind of molecule.
18. kits as claimed in claim 17, it is characterised in that second captures dividing for the specific bond analyte on pad
Son and with the incoherent specific binding molecule of analyte to a kind of molecule content for the first and second capture pads it is total
The 15%-50% of amount.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410309729.7A CN105223349B (en) | 2014-07-01 | 2014-07-01 | A kind of device for detecting sample |
| CN201710274311.0A CN106996973A (en) | 2014-07-01 | 2014-07-01 | A kind of sample testing apparatus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201410309729.7A CN105223349B (en) | 2014-07-01 | 2014-07-01 | A kind of device for detecting sample |
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| CN201410309729.7A Active CN105223349B (en) | 2014-07-01 | 2014-07-01 | A kind of device for detecting sample |
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| JP6926319B2 (en) * | 2018-03-16 | 2021-08-25 | 株式会社日立ハイテク | Automatic analyzer and analysis method |
| GB201902810D0 (en) * | 2019-03-01 | 2019-04-17 | Vidya Holdings Ltd | Disposition of reagents in assay device |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU713409B2 (en) * | 1995-07-12 | 1999-12-02 | Charm Sciences, Inc. | Test apparatus, system and method for the detection of test samples |
| GB0016813D0 (en) * | 2000-07-07 | 2000-08-30 | Lee Helen | Improved dipstick assays (4) |
| US20050119589A1 (en) * | 2003-11-14 | 2005-06-02 | Tung Hsiaoho E. | Rapid sample collection and analysis device and methods of use |
| DE102004009012A1 (en) * | 2004-02-25 | 2005-09-15 | Roche Diagnostics Gmbh | Test element with a capillary for transporting a liquid sample |
| ATE479096T1 (en) * | 2004-07-29 | 2010-09-15 | Relia Diagnostic Systems Llc | CROSS-FLOW SYSTEM AND ASSAY |
| CN101078726A (en) * | 2005-09-05 | 2007-11-28 | 因韦尔尼斯医药瑞士股份有限公司 | Device for detecting immune globulin |
| CN101017169B (en) * | 2006-07-26 | 2012-07-18 | 艾博生物医药(杭州)有限公司 | Analysis equipment of biological sample |
| CN200968955Y (en) * | 2006-07-26 | 2007-10-31 | 艾博生物医药(杭州)有限公司 | Biological liquid sample analytical equipment |
| CN101358969A (en) * | 2007-08-02 | 2009-02-04 | 孙东旭 | Novel method for quantitatively determining analyte by scavenger with single specificity |
| CN103154735B (en) * | 2009-05-11 | 2018-10-02 | 连接Dx股份有限公司 | Method and composition for testing and analyzing object |
| US9128039B2 (en) * | 2011-10-05 | 2015-09-08 | Joinsoon Medical Technology Co., Ltd. | Biosensor test strip |
| CN102998440A (en) * | 2012-12-28 | 2013-03-27 | 闫志文 | Dry chemical multinomial detecting device |
| CN203083995U (en) * | 2013-01-29 | 2013-07-24 | 艾博生物医药(杭州)有限公司 | Sample detection device |
| CN204214874U (en) * | 2014-07-01 | 2015-03-18 | 艾博生物医药(杭州)有限公司 | A kind of device detecting sample |
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