CN107621539A - A kind of method of analyte in detection means and detection liquid sample - Google Patents
A kind of method of analyte in detection means and detection liquid sample Download PDFInfo
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Abstract
The present invention relates to a kind of detection means and detection method, and for detecting analyte in liquid sample, the detection means includes the carrier of support fluid flowing, includes marked region and detection zone on the carrier, the marked region is located at the upstream of detection zone；The upstream of the marked region also includes the calmodulin binding domain CaM for being combined with heterophile antibody blocking agent.The detection means of the present invention and it the method for analyte in sample is detected by the detection means can effectively eliminate the phenomenon of false positive, improve the accuracy of detection.
Description
Technical field
, can be with quick detection sample more particularly to one kind the invention belongs to medical diagnosis class article technical field
Whether a kind of detection means and detection method containing target analytes.
Background technology
Following background technology is used to help reader and understands the present invention, and is not construed as prior art.
Using immune association reaction principle come detect in sample with the presence or absence of analyte this technology it is wide
It is general to apply in every field.Can be detected with it various biological specimens (saliva, blood, urine, serum,
Sweat etc.) analyte detect health status (early pregnancy, tumour, infectious disease, the poison of disease and the mankind
Product etc.).The cardinal principle of this detection technique is the performance with specific binding between immune molecule, such as
Antibody and antigen, haptens and antibody, biotin and antibiotic etc..In addition, many such detections can
Completed on solid dielectric, such as in conventional lateral flow reagent strip, glass or plastic microtiter plates, layer is immunized
Analysis apparatus etc..Generally, some solid particles or chemical substance can be being combined on immune specific binding molecule,
So testing result come qualitative, sxemiquantitative or quantitative can be drawn by naked eyes or Other Instruments equipment.It is this
Solid particle can be colored colloidal solid (latex or gold grain), and this chemical substance can be carried
The material of chromophoric group, these materials it is other it is suitable under the conditions of can send specific wavelength to show detection knot
Fruit.
Typical lateral flow assay test strips include porous sample pad, and liquid is in sample pad
The mark substance pad of circulation and the test agent bar with mark substance pad liquid communication.Generally, mark substance pad bag
Mark substance is included, these mark substances can be colored colloidal solid or the material with chromophoric group.Make
When with detection means, fluid sample is applied on sample pad, then moves to mark substance pad,
It is moved to together with after mark substance combination in the detection zone of reagent strip, the analyte in such sample
It is detected with the presence or absence of can.In such detection process, sample automatic edge in the case where chromatographing active force
Horizontal reagent strip downstream to move, Cucumber can move down with mark substance in sample, in detection zone
With the substance reaction being fixed in detection zone on domain, testing result is shown in detection zone.
Generally, in these test strips or detection means detection liquid sample during analyte, generally use competition
Method or sandwich method.When being detected using competition law, detection zone or result in test strips or detection means are shown
When not having color change or the no colored line to occur on region, testing result is judged to the positive, represents detection
Analyte is there may be in sample；There is color change in detection zone or results display area domain or have color
When lines occur, testing result is judged to feminine gender, represents that analyte may be not present in detection sample.
And when utilizing sandwich method detection, its result presentation method is opposite with competition law, i.e. detection zone or result are shown
When occurring color change on region or have the colored line to occur, testing result is judged to the positive, represents detection sample
Analyte is there may be in this；There is no color change or no color in detection zone or results display area domain
When lines occur, testing result is judged to feminine gender, represents that analyte may be not present in detection sample.
But in some immunoassay procedures, when be commonly present the phenomenon of false positive reaction.That is liquid sample
In and do not contain and need the analysis material that is detected, testing result show should be feminine gender, but reality
Testing result is the positive.Such as in the detection to the communicable disease etiology such as rotavirus and adenovirus,
Often there is the testing result of false positive in the detection of the sample of feminine gender.
The content of the invention
The invention provides a kind of new detection means and detection method, pass through the detection means and detection
Method can effectively improve the phenomenon of the false positive in liquid sample detection.
First, a kind of detection means provided in the present invention, for detecting analyte in liquid sample, wherein,
The detection means includes the carrier of support fluid flowing, includes marked region and detection zone, institute on the carrier
State the upstream that marked region is located at detection zone；The upstream of the marked region also includes being combined with heterophile antibody
The calmodulin binding domain CaM of blocking agent.Liquid sample on carrier by upstream toward downstream direction along calmodulin binding domain CaM, marked region,
Detection zone sequentially flows.
Find during using the detection sample of sandwich method, exist some unknown in the detection of reality
Material, it can be reacted simultaneously with the antibody or antigen of detection zone and the antibody or antigen of marked region,
Cause detection zone colored line occur, so as to be judged as the positive, cause detection to produce the situation of false positive.
According to this problem, consider to introduce heterophile antibody blocking agent, make in liquid sample unknown causes false positive
The material of reaction with before the label and antigen of marked region or antibody binding first with heterophile antibody blocking agent
With reference to so as to the antigen or antibody binding no longer with marked region and detection zone, elimination false positive.
In some preferred embodiments, the heterophile antibody blocking agent be selected from rabbit igg, sheep IgG, mouse IgG,
HBR reagents or MAK33 reagents.
In another preferred embodiment, the concentration of the heterophile antibody blocking agent is 0.5~15mg/mL.
It is more highly preferred to, the concentration of the heterophile antibody blocking agent is 1~10mg/mL.
In other embodiments, the calmodulin binding domain CaM is further fixed on colored indicator.By showing that indicator shows
Border between calmodulin binding domain CaM or calmodulin binding domain CaM and other regions is shown, facilitates operator to observe and operate.
Preferably, colored indicator is selected from light blue, eosin, famille rose, Evans blue.
In some embodiments, the marked region have colored mark substance and with analyte in sample
The antibody of specific bond；The detection zone secure bond has the coating with analyte specific bond in sample to resist
Body.
Preferably, the colored mark substance is selected from colloid gold label particle, latex marking particle or water
Soluble marker's matter.Colored mark substance can be colloid gold label particle, latex marking particle or water-soluble
One or more in property mark substance.
In some preferred embodiments, described device also includes sample application zone, the sample application zone
Positioned at the upstream of calmodulin binding domain CaM.
In some embodiments, the carrier of the support fluid flowing includes label pad, detects film, pad；
The marked region is located in label pad, and on detection film, calmodulin binding domain CaM is located on pad detection zone；
The downstream end of the pad is pressed together on the upstream end thereof of label pad, and the downstream end of label pad is with detecting on film
Swim end connection.
In one preferred embodiment, also there is sample application zone on the pad；The sample region of acceptance
Domain is located at calmodulin binding domain CaM upstream.That is, pad can extend, it is added to sample uncombined different thermophilic
The region of property antibody blocking agent, makes it be entered back into after lateral flow enters row buffering with heterophile antibody blocking agent
Calmodulin binding domain CaM, can so make unknown in sample to cause the material of false positive reaction to block with heterophile antibody
Agent is sufficiently reacted.
In another preferred embodiment, the carrier of the support fluid flowing also includes sample pad；Sample receives
Region is located in sample pad；The downstream end of the sample pad is overlapped on pad upstream end thereof.
In some embodiments, the detection means is test strips.
On the other hand, the present invention also provides a kind of method for detecting analyte in liquid sample, including：
A kind of detection means is provided, the device includes the carrier of support fluid flowing, includes marked region on the carrier
And detection zone, the marked region are located at the upstream of detection zone；The upstream of the marked region also includes knot
Closing has the calmodulin binding domain CaM of heterophile antibody blocking agent；Wherein,
To the calmodulin binding domain CaM adding liquid sample on carrier, liquid sample is flowed through calmodulin binding domain CaM, make unknown in sample
Cause the material of false positive reaction to be combined with the heterophile antibody blocking agent on calmodulin binding domain CaM；
Then, sample is flowed into marker downstream region and detection zone, complete the detection of liquid sample.
In some specific embodiments, the heterophile antibody blocking agent is selected from rabbit igg, sheep IgG, mouse
IgG, HBR reagent MAK33 reagents.
In some embodiments, the concentration of the heterophile antibody blocking agent is 1~10mg/mL.
In some preferred embodiments, sample application zone is also included on the carrier, the sample receives
Region is located at the upstream of calmodulin binding domain CaM；Wherein, sample is made to be added to sample application zone, through sample application zone
Calmodulin binding domain CaM is flowed into after buffering.
Beneficial effect
The detection means of the present invention and the method for detecting analyte in sample by the detection means can have
The phenomenon of the elimination false positive of effect, improve the accuracy of detection.
Brief description of the drawings
Fig. 1 is a kind of detection means schematic diagram of the present invention；
Fig. 2 is another schematic diagram of the detection means of the present invention；
Fig. 3 is another schematic diagram of the detection means of the present invention；
Fig. 4 is another schematic diagram of the detection means of the present invention；
Fig. 5 is material concentration rank to be detected and colored intensity compares figure；
Fig. 6 A- Fig. 6 G are the result display schematic diagram of the detection zone of one embodiment of the invention；
Fig. 7 A- Fig. 7 G are the result display schematic diagram of the detection zone of another embodiment of the present invention.
Reference：
Detection means (carrier) 100, sample application zone 110, calmodulin binding domain CaM 120, marked region 130, inspection
Survey region 140, detection line 141, control line 142, suction zone 150, test strips 200, sample pad 210,
Sample pad downstream end 211, pad 220, pad upstream end thereof 221, pad downstream end 222,
Label pad 230, label pad upstream end thereof 231, pad downstream end 232, film 240 is detected, detected on film
Swim end 243, adsorptive pads 250, sheet material 260, detection line 241,245, control line 242
Embodiment
Structure of the present invention or these used technical terms are described further below.
Carrier
Carrier can be porous or non-infiltration surface.Porous surface can be film, such as detection examination
The water-absorbing material of agent flowing.Paper or pulp product, glass fibre, polymer, such as cellulose nitrate, nylon
It can be used to the absorbent material as the present apparatus.In a mode, the material that is absorbed water can also by with
Do carrier.Unwetted property material, which can be used for producing capillarity between two surfaces, allows fluid to be filled along detection
Put flowing.
Carrier can provide the fluid communication between multiple regions in detection means.These regions can be mark zone
Domain or detection zone.These regions can be arranged on some panels of detection means, can also be arranged on
On one side of detection means or/and following position.For example, marked region can be located on detection zone.
Any three-dimensional structure arranges these regions to be applied to the device that the present invention describes.On carrier also
Liquid sample receiving area can be set, some materials can be handled on liquid sample receiving area, such as buffer
Reagent, filter agent and its reagent reduce the reagent of the interfering material in liquid sample.
Upstream, downstream
" upstream ", " downstream " refer to along liquid flow direction that come what is divided upstream is to be located at liquid flowing side
On, downstream is located under liquid flow direction, and upstream and downstream is a relative concept, and liquid can be with
Downstream position is flowed to from upstream position.
Detection
" detection " represents to chemically examine or test a kind of material or material whether there is, such as, but this is not limited to, change
Learn material, organic compound, inorganic compound, metabolism product, medicine or drug metabolite, have
Machine tissue or the metabolin of organic organization, nucleic acid, protein or polymer.In addition, detection represents test substances
Or the quantity of material.Furtherly, chemical examination is also represented by immune detection, chemical detection, enzyme detection etc..
Marked region 130
The specific binding molecules of analyte carry the mark of reagent area.When specific binding molecule is caught
The analyte for obtaining analyte and being marked with is when detection zone is combined, because label is at this
In gather this area's can and observe." specific binding molecule " refer to be combined with analyte and
Can not be with the binding molecule of other any molecule strong bondeds in sample.The specific combination of analyte point
Son can also be with the presence or the molecule phase associated with presence of analyte that show analyte in sample
With reference to.Strong bonded, which refers to combine to reach, to be changed result of the test or makes the unconspicuous degree of result of the test.One
In a little concrete schemes, specific binding molecule is probably a kind of antibody or a kind of antibody fragment (for example, one
The Fab areas of kind of antibody), a kind of antigen, the fragment or biology of a kind of acceptor of binding partner either acceptor
One composition of element-streptavidin combination pair or other types of combination pair.Such reagent area
Can provides mark, and when sample flows through reagent area, analyte, which combines, can produce detectable letter
Number mark." label pad " refers to the material containing analyte that may be present in mark sample in matrix
Position.Therefore a reagent area is exactly a label pad." mark " can produce detectable signal
Any suitable mark.For example, mark can be sol particle, and fluorescent grain, chemiluminescent molecule, gold
Belong to either alloy (for example, collaurum) or capsule, the particularly liposome comprising visible dyes.It is hydrophobic molten
Glue is also useful, hydrophobic organic dyestuff either insoluble in the water or only very limited small portion of pigment
Divide solvable.Mark can also be polymer particles, such as coloured granules of polystyrene (for example, spherical).
Other useful granular marks include ferritin, phycoerythrin, phycobilin-albumen, precipitation or can
The pigment or derivative of the metal or alloy of dissolubility, fungi, marine alga, or bacterium, such as the leaf of bacterium are green
Plain or other plant materials.In some concrete schemes, mark is coloured particle, such as dextran
Grain.In other concrete schemes, mark and dye selection identical color as positive control, to strengthen
Two kinds of signals are in the interaction in matrix or when Medium Culture produces single obvious symbol.Have in others
In body scheme, mark is probably a kind of specific binding molecules being marked with of analyte (for example, a kind of
Antibody).For example, in a concrete scheme, target analyte is human chorionic gonadotrophin (hCG),
Mark with reference to hCG is the Anti-hCG antisera of aurosol mark.When sample is to label pad, in sample
The Anti-hCG antisera that hCG is marked by aurosol combines.Labelled antibody not the capture molecule in Interference Detection region and
The hCG of mark is combined.For example, mark can combine a part of analyte, and capture molecule can be with
With reference to another part of analyte or binding marker.HCG- Anti-hCG antiseras-Au composite is moved to base
The downstream of matter.When compound reaches detection zone and capture molecule is combined to form gold-anti-anti- hCG- of hCG
Anti-hCG antisera.Capture molecule is probably hCG another specific binding molecules, or with reference to hCG
The specific binding molecules of the halfbody of analyte.As the anti-hCG of gold-anti-hCG specific binding molecules-hCG-
When specific binding molecules compound is attached to detection zone, detection zone by the golden marker coloring on compound simultaneously
And become visually visible in detection zone gold mark.In a concrete scheme, specific binding molecule is anti-
Body or antibody fragment.Mark and capture binding molecule can combine antigen determination section different on analyte
Position, in a concrete scheme, the specific binding molecules of mark combine β-hCG, and capture to combine and divide
Son combines α-hCG.
Detection zone 140
Mark substance on marked region can combine (directly or indirectly) target analyte, so as to
When sample flows through matrix, analyte, which is just labeled to have gone up, can detect ground mark substance.Detection zone is also
Include the reagent that can combine analyte related locus.This moiety be probably analyte itself or
Person combines a kind of reagent of analyte (for example, a kind of reagent can be when analyte passes through reagent area and quilt
Analyte combination) a kind of immunology on epitope.In various concrete schemes, and analyte knot
The reagent of conjunction can be a kind of antibody, a kind of segment of antibody or part, a kind of and be attached to detection zone
Another of the derivative (either segment therein) of the different types of antibody of antibody or specific binding pair
Composition, for example, avidin, streptavidin, or can be with combination analyte
The biotin that halfbody is combined.
In another concrete scheme, detection zone be one along the latitude directional spreding of the test bar longitudinal axis bar
Shape thing, wherein being answered comprising a kind of specific binding molecules of analyte, or the molecule of combination analyte
Compound.
Antibody
" antibody " refers to immunoglobulin, either natural or some or all of synthesis.This
Term also include wherein keep binding ability antibody derivative, also including it is any containing with immunoglobulin
The protein of binding domain homologue or largely homologous binding domain.These protein are derived from day
Right material, it is also possible to some or all of synthesis.A kind of antibody is probably the either polyclonal of monoclonal
's.A kind of antibody is probably a member in any immunoglobulin class, includes the immunoglobulin of any mankind
Type：IgG, IgM, IgA, IgD, IgG and IgE." antibody fragment " is the derivative or antibody of antibody
A part less than total length.Antibody fragment can retain the notable of the binding ability of at least one full length antibody
Site.The example of antibody fragment includes Fab, Fab', F (ab')2, scFv, Fv, dsFv dimers, and
Fd fragments, but not only include the above.
Test strips200
Apply to the horizontal effluent reagent strip that the test strips 100 of the present invention can be known as, these reagents
The concrete structure and Cleaning Principle of bar are technology known to persons skilled in the art in the prior art.Commonly
Reagent strip, as shown in Figure 2 including sample application zone, marked region, detection zone and suction zone, sample
This region of acceptance includes sample pad 210, and marked region includes label pad 230, and suction zone can include water suction
Pad 250, detection zone is on detection film 240.Include to detect whether containing analyte in detection zone
The necessary chemical substance of matter, after detection terminates, can occur the detection line 241 for representing result in detection zone
And control line 242.Typically conventional detection reagent bar is nitrocellulose assay strip, i.e. detection zone bag
Nitrocellulose filter (NC films) is included, specific binding molecule is fixed on nitrocellulose filter to show the knot of detection
Beam, it can also be cellulose acetate film or nylon membrane etc..The width of usual test strips between 3mm-6mm,
In one specific embodiment, the width of test strips is 4mm.
Detection means
Detection means includes a carrier 100 with calmodulin binding domain CaM, marked region and detection zone, such as Fig. 1
It is shown.Fixation is loaded with heterophile antibody blocking agent, the concentration of heterophile antibody blocking agent on calmodulin binding domain CaM 120
For 0.5~15mg/mL, it is preferred that the concentration of heterophile antibody blocking agent is 1~10mg/mL, or is 1-5
mg/mL；Heterophile antibody blocking agent is selected from rabbit igg, sheep IgG, mouse IgG, HBR reagent or MAK33
One or more of combinations in reagent.Mark substance is loaded with marked region 130 and with being analyzed in sample
The antibody or antigen of thing specific binding.Detection zone 140 fixes a kind of detection molecules, and (detection antibody is anti-
It is former).Certainly, can also have positioned at the sample application zone 110, Yi Jiwei of calmodulin binding domain CaM upstream on carrier
Suction zone 150 in most downstream.
In a preferable scheme, detection means includes host material composition reagent strip 200.On reagent strip
Including calmodulin binding domain CaM, marked region and detection zone.Fixation is loaded with heterophile antibody blocking agent on calmodulin binding domain CaM,
Marked region is loaded with mark substance and the antibody or antigen that are specifically bound with analyte in sample, detection zone
A kind of detection molecules (detection antibody or antigen) are fixed in domain.In a concrete scheme, reagent strip includes one kind
Absorbent material, there is provided support the host material of liquid flowing." host material " refers to support liquid flowing and fortune
A kind of defeated material.In a concrete scheme, host material is a kind of absorbent material.Liquid flows through this dress
Put is to be acted on realizing by means of capillary motion.In different concrete schemes, host material can be single
The bar (Fig. 1) or closed by a variety of absorbent materials to interact in a liquid that one material is formed
Into (Fig. 2), " water suction " material refers to that those can stably absorb moisture and moisture is passed through hair wherein
The material of thin vasomotion effect transport.The example of absorbent material includes nitrocellulose, filter paper, glass fibre,
Polyester and other suitable materials.
In a concrete mode, detection means also includes a passage for receiving sample and observes the saturating of testing result
Bright window, the result of detection can be read by window channel.This device and reagent strip containing reagent strip
Position relationship in a device be prior art patent or application disclosed in device can be applied to this hair
On bright reagent strip.
The type of analyte
Can be used in the example of the analyte of stable detection of the present invention includes (but not only including) people's suede
Chorionic Gonadotropin (hCG), lutropin (LH), ovarioestrogen (FSH), hepatitis C virus (HCV),
The medicine of hepatitis B (HBV), hepatitis B surface antigen, AIDS virus and any abuse.Analyte can
Detected in any liquid or liquefied sample, such as urine, saliva, blood, blood plasma or serum.It is other
The example of analyte also have that flesh ammonia acid anhydride is sour, bilirubin, nitrite, protein (nonspecific),
Blood, leucocyte, blood glucose, heavy metal and toxin, bacterial component is (for example, certain types of bacterium is special
Protein and sugar, such as colon bacillus 0157:H7, staphylococcus aureus, salmonella, aerogenesis pod
Film clostridium, campylobacter, listeria monocytogenes, V. parahaemolyticus, or wax bacillus).It is any
The analyte of the suitable lateral flow test format of others can be detected with the present apparatus.
The type of sample
Any kind of sample can be tested with the device of the present invention, including body fluid is (for example, urine
With other body fluid, and clinical sample).Fluid sample may originate from solid or semisolid sample, bag
Include excrement, biological tissue and foodstuff samples.These solids and semisolid sample can pass through any suitable side
Method is transformed into fluid sample, such as is mixed in a kind of appropriate liquid, stamp it is broken, macerate, be incubated, dissolving or
Person digests solid sample (for example, water, phosphate buffer or other buffer solutions)." biological sample " includes
Sample from animal living, plant and food, also including urine, saliva, blood and blood constituent, brain ridge
Liquid, vaginal swab, seminal fluid, excrement, sweat, secretion, tissue, organ, tumour, tissue and organ
It is the conditioned media of culture, cell culture and there, either people or animal.Foodstuff samples include
Finished food ingredients and last product, meat, cheese, wine, milk and drinking water.Plant sample includes
The sample of conditioned media from any plant, plant tissue, plant cell cultures and there." environment sample
Product " be those be derived from environment sample (for example, the sample of lake water sample or other water bodys, sewage sample,
Pedotheque, groundwater sample, seawater sample, the sample of runoff water).Sewage and related waste also may be used
With included in environmental sample.
In the following detailed description, the subsidiary reference word of legend is a part here, and it is with citing
Illustrate the present invention can the mode of actable specific concrete scheme illustrate.We are not precluded from the present invention can be with
Carry out other concrete schemes and change the structure of the present invention in the case of the use range without prejudice to the present invention.
As shown in figure 1, sample testing apparatus includes the carrier 100 for allowing liquid sample to flow, there is upstream on carrier
There is sample application zone 110, calmodulin binding domain CaM 120, marked region 130, detection zone 140 respectively to downstream
With suction zone 150.After liquid sample is added on sample application zone 110, liquid sample is successively from upper
Trip flows through sample application zone 110, calmodulin binding domain CaM 120, marked region 130, detection zone 140, finally arrives
Up to suction zone 150, and it is combined or reacts with the reagent or material being fixed on regional respectively, most
The detection of sample is completed afterwards.Testing result shows in detection zone, wherein, close to the coloured of suction zone
Line is control line (C lines) 142, is used to indicate sample analyte close to the detection line (T lines) of marked region
It whether there is and show negative or positive (being determined according to Cleaning Principle, be specifically shown in background section).Wherein,
In order to prevent the generation of false positive, in of the invention, heterophile antibody blocking agent is fixed with calmodulin binding domain CaM.This
A little heterophile antibody blocking agents can be selected from rabbit igg, sheep IgG, mouse IgG, Scantibodies HBR examinations
Agent or the MAK33 reagents of Roche, these blocking agents can only select a kind of, also choosing multiple combined use.
Specifically, certain density heterophile antibody blocking agent is carried in calmodulin binding domain CaM, these concentration ranges are
0.5~15mg/mL, or 1~10mg/mL, are more highly preferred to, and concentration range is 1~5mg/mL.Combining
Region, colored indicator can also be fixed with, for indicating the position and border of calmodulin binding domain CaM, convenient identification knot
Region is closed, when liquid flows through identification region, colored indicator shows color, so as to very easily area
Separate calmodulin binding domain CaM and other regions.In some embodiments, colored indicator be selected from light blue, eosin, famille rose,
One kind in Evans blue.
As shown in figure 3, the carrier of detection means has multiple absorbent material overlap joints to form, such as：Sample pad 210,
Pad 220, label pad 230 and detection film 240 and absorption pad 250, these absorbing materials are connected to one
Rise, and be sequentially fixed in a bottom sheet 260, form the test strips 200 for being available for fluid to circulate, wherein,
Sample application zone 110 is located in sample pad 210, and marked region 130 is located in label pad 230, detection zone
On detection film 240, calmodulin binding domain CaM 110 is located on pad 240 in domain 140.Sample positioned at most upstream
Pad 210, the downstream pressing of end 212 are overlapped on the upstream end thereof 221 of pad, also, sample pad 210
Pad 220 can be partly or entirely covered, and the pressing of the downstream end 222 of pad is overlapped on label pad
Upstream end thereof 231, the upstream end thereof 243 for detecting film are overlapped on the lower section of label pad downstream end 232 by pressing,
The upstream end thereof of blotting paper 250 is overlapped on the downstream end of detection film 240.
In some preferred embodiments, as shown in Fig. 2 pad 220 extends for the upstream of label pad 230
Section, label pad 230 is the downstream extension of pad 220 in other words, that is to say, that calmodulin binding domain CaM 120 and mark
Remember that region 130 on same absorbent material 220 (230), so, facilitates detection means 100 or test paper
The making of bar 200, meanwhile, liquid sample circulation is also more smooth.
In some preferred embodiments, as shown in figure 4, sample pad 210 extends for the upstream of pad 220
Section, pad 220 is the downstream extension of sample pad 210 in other words, that is to say, that sample application zone 110
With calmodulin binding domain CaM 120 on same absorbent material 220 (210), so, facilitate detection means 100 or
The making of test strips 200, meanwhile, liquid sample circulation is also more smooth.
The method for detecting analyte in liquid sample is described with regard to Fig. 1 and 3 below.First, by liquid-like
Originally it is added in sample pad 210, i.e., on sample application zone, sample arrives along the flow further downstream of test strips 200
Up to pad 220 (calmodulin binding domain CaM), liquid sample flows through calmodulin binding domain CaM 220, and now unknown in sample draws
The material for playing false positive reaction is combined with the heterophile antibody blocking agent on pad 220, the conjugate with reference to after
Pad 220 can be rested on；Flow further downstream can also be continued with sample, reach (the mark of label pad 230
Region), and the label and the antibody response of specific bond in label pad 230, if there is analyzed
Thing, then liquid sample carry combine label and antibody analyte continue to run to detection film 240
(NC films) (detection zone), with being fixed on the antibody response detected on film 240 and being fixed at this, from
And show detection line 241；Remaining liquid sample, which is flowed on the adsorptive pads 250 in downstream, to be absorbed；Complete
The detection of liquid sample.
Embodiment
Experiment 1：Detected with detection means in excrement with the presence or absence of rotavirus, adenovirus
Part I：The reagent strip of the present invention and the assembling of detection means and part make
Reagent strip 200 as shown in Figure 3 is used for part and the making for illustrating reagent strip of the invention used in this experiment
Method.These reagent strips generally comprise label pad 230 and detection film 240；Wherein the upstream of label pad can wrap
The sample pad 210 for receiving sample is included, reagent necessary to completing reaction is included in sample pad；Sample pad 210
Between label pad 230, in addition to pad 220, include heterophile antibody blocking agent on pad 220；
Detecting film 240 includes one or more regions 241 of display test result and the detection positioned at detection zone downstream
As a result control zone 242, general testing result region are linear, and display color becomes on testing result region
Change to represent that analyte whether there is in sample.Include the region of display testing result on detection film 240
With the testing result control zone positioned at detection zone downstream.In addition, also include adsorptive pads 250.
The processing of sample pad 210, from being glass fibre element film, the solution handled on the film is sample pad：
Tris(0.1M).The sample pad handled well is placed in 37 DEG C of baking ovens and dried.
The processing of pad 220, pad are polyester cellulose film, immobilized 1~10mg/mL mouse on pad
IgG solution；Solid support method is to be sprayed on 2.0ul/cm metering on cushion pad, hereafter 37 DEG C of drying.
The processing of labeling pad 230, labeling pad are polyester cellulose film, the C marked by latex particle
After line, rotavirus antibody and adenovirus antibody are using the dilution of Tris buffer solutions, sprayed with 0.2 μ L/cm amount
Point is placed in 37 DEG C of baking ovens and dried on polyester film.Control line (C lines) material can be mouse IgG,
Rabbit igg, bovine serum albumin(BSA) etc..
The processing of film 240 is detected, detection film is nitrocellulose filter (NC), and NC films are along liquid flow direction
On have three lines, wherein, two detection lines 241,245 and the testing result Quality Control positioned at detection line downstream
Line 242, rotavirus antibody is fixed with detection line 241, is fixed with detection line 245 downstream
Adenovirus antibody, antibody is fixed with nature controlling line 242.Processing method be first respectively by rotavirus antibody R,
Adenovirus antibody A and antibody are dissolved separately in PBS, then with 0.1 μ L/cm amount specking in NC
Corresponding position on film.37 DEG C of drying.
Adsorptive pads 250 are filter paper, are dried.
The assembling of detection means-test strips 200, such as Fig. 3, sample pad 210 are located at the upstream of pad 220,
(sample pad portion is completely covered on pad upper surface), the downstream of pad 220 is overlapped on label pad 230
Upstream (or pad connects together with label pad, does not cut off or cuts off), the downstream of label pad 230 are overlapped on inspection
The upstream of film 240 is surveyed, the upstream of adsorptive pads 250 is overlapped on detection film 240 downstream.These parts are placed on one
On the support chip 260 of unwetted property.
Experiment 2：The reagent strip of detection improvement eliminates situation to false positive test results.
2.1 reagent strip：("+" represents there is the material, and "-" represents do not have the material)
| Reagent strip | Sample pad | Pad | Label pad | Detecting pad | Adsorptive pads |
| 1 (common test strip) | + | - | + | + | + |
| 2 (improvement test strips) | + | + | + | + | + |
2.2 sample for testing：
| Sample is numbered | Testing result | Processing | Conclusion |
| 1 | R+/A- | - | Rotavirus positive criteria product |
| 2 | R+/A+ | - | Rotavirus and adenovirus positive criteria product |
| 3 | R-/A+ | - | Adenovirus positive criteria product |
| 4 | R-/A- | - | Negative standards' product |
| 5 | R-/A- | Add false positive sample | False positive sample |
| 6 | R+/A- | Add false positive sample | The false positive sample of rotavirus true positives |
| 7 | R-/A+ | Add false positive sample | The false positive sample of adenovirus true positives |
2.3 colour atla standards (shown in Fig. 5) and testing result
Material concentration rank to be detected and colored intensity compares figure, as shown in figure 5, when there are lines, show
Be shown as land, in Fig. 5 it can be seen from since L4, there is chromogenic line to occur, be shown as positive, profit
Following table is expressed as with form：("+" represents positive, and "-" represents negative)
| As a result | - | - | - | + | + | + | + | + | + | + |
| Color | L1 | L2 | L3 | L4 | L5 | L6 | L7 | L8 | L9 | L10 |
2.4 experimental results such as following table：
Wherein：
Fig. 6 A- Fig. 6 G in the testing result of test strips in form 1 with corresponding, i.e.,：Fig. 6 A are sample number
The testing result schematic diagram of No. 1；Fig. 6 B are the testing result schematic diagram of sample number 2；Fig. 6 C are sample number
The testing result schematic diagram of No. 3；Fig. 6 D are the testing result schematic diagram of sample number 4；Fig. 6 E are sample number
The testing result schematic diagram of No. 5；Fig. 6 F are the testing result schematic diagram of sample number 6；Fig. 6 G are sample number
The testing result schematic diagram of No. 7；
Fig. 7 A- Fig. 7 G and the testing result of test strips 2 in form correspond, i.e.,：Fig. 7 A are sample number 1
Number testing result schematic diagram；Fig. 7 B are the testing result schematic diagram of sample number 2；Fig. 7 C are sample number 3
Number testing result schematic diagram；Fig. 7 D are the testing result schematic diagram of sample number 4；Fig. 7 E are sample number 5
Number testing result schematic diagram；Fig. 7 F are the testing result schematic diagram of sample number 6；Fig. 7 G are sample number 7
Number testing result schematic diagram；
As shown in form and Fig. 7 A- Fig. 7 G, increase is fixed with the examination after the pad of heterophile antibody blocking agent
Paper slip 2 has effectively eliminated false positive.
Experiment 3：Compare elimination situation of the different heterophile antibody blocking agents to false positive sample
3.1 reagent strips ("+" represents there is the material, and "-" represents do not have the material)
Wherein, the equivalent concentration of the heterophile antibody blocking agent added in pad is identical, is
0.15~20mg/ml (different material concentration scope is variant, and this scope is the total size of all blocking agents).
3.2 experimental result
From in form, above-mentioned different heterophile antibody blocking agent, it is to eliminating the effect of false positive all very
It is good, false positive can be effectively eliminated.
Experiment 4：Compare elimination situation of the heterophile antibody blocking agent of various concentrations to false positive sample
4.1 reagent strips ("+" represents there is the material, and "-" represents do not have the material)
4.2 experimental result
By being drawn in form, concentration range 0.5-15mg/mL mouse IgG as heterophile antibody blocking agent pair
The false positive eliminated in detection produces effective effect.
Claims (17)
1. a kind of detection means, for detecting analyte in liquid sample, it is characterised in that the detection means includes supporting
The carrier of flow of fluid, include marked region and detection zone on the carrier, the marked region is located at detection zone
The upstream in domain；The upstream of the marked region also includes the calmodulin binding domain CaM for being combined with heterophile antibody blocking agent.
2. detection means according to claim 1, it is characterised in that the heterophile antibody blocking agent be selected from rabbit igg,
Sheep IgG, mouse IgG, HBR reagent or MAK33 reagents.
3. detection means according to claim 1 or 2, it is characterised in that the concentration of the heterophile antibody blocking agent
For 0.5~15mg/mL.
4. detection means according to claim 1 or 2, it is characterised in that the concentration of the heterophile antibody blocking agent
For 1~10mg/mL.
5. detection means according to claim 1, it is characterised in that the calmodulin binding domain CaM is further fixed on colored indicator.
6. detection means according to claim 5, it is characterised in that colored indicator is selected from light blue, eosin, kermes
One kind in red, Evans blue.
7. detection means according to claim 1, it is characterised in that the marked region has colored label
Matter and the antibody with analyte specific bond in sample；The detection zone secure bond has with being analyzed in sample
The coated antibody of thing specific bond.
8. detection means according to claim 7, it is characterised in that the colored mark substance is selected from colloid
Golden marking particle, latex marking particle or water-soluble marker's material.
9. detection means according to claim 1, it is characterised in that described device also includes sample application zone, institute
State the upstream that sample application zone is located at calmodulin binding domain CaM.
10. according to the detection means described in one of claim 1-9, it is characterised in that the carrier bag of the support fluid flowing
Label pad is included, detects film, pad；The marked region is located in label pad, and detection zone is located on detection film,
Calmodulin binding domain CaM is located on pad；The downstream end of the pad is pressed together on the upstream end thereof of label pad, label pad
Downstream end with detection film upstream end thereof be connected.
11. detection means according to claim 10, it is characterised in that also there is sample region of acceptance on the pad
Domain；The sample application zone is located at calmodulin binding domain CaM upstream.
12. detection means according to claim 10, it is characterised in that the carrier of the support fluid flowing also includes
Sample pad；Sample application zone is located in sample pad；The downstream end of the sample pad is overlapped on pad upstream end
In portion.
13. detection means according to claim 10, it is characterised in that the detection means is test strips.
14. a kind of method for detecting analyte in liquid sample, including：
A kind of detection means is provided, the device includes the carrier of support fluid flowing, include on the carrier marked region and
Detection zone, the marked region are located at the upstream of detection zone；The upstream of the marked region also includes being combined with
The calmodulin binding domain CaM of heterophile antibody blocking agent；Wherein,
To the calmodulin binding domain CaM adding liquid sample on carrier, liquid sample is flowed through calmodulin binding domain CaM, make unknown in sample
The material of false positive reaction is caused to be combined with the heterophile antibody blocking agent on calmodulin binding domain CaM；
Then, sample is flowed into marker downstream region and detection zone, complete the detection of liquid sample.
15. according to the method for claim 14, it is characterised in that the heterophile antibody blocking agent is selected from rabbit igg, sheep
IgG, mouse IgG, HBR reagent or MAK33 reagents.
16. according to the method for claim 15, it is characterised in that the concentration of the heterophile antibody blocking agent is
1~10mg/mL.
17. according to the method for claim 14, it is characterised in that also include sample application zone, institute on the carrier
State the upstream that sample application zone is located at calmodulin binding domain CaM；Wherein, sample is made to be added to sample application zone, through sample
Calmodulin binding domain CaM is flowed into after the buffering of region of acceptance.
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