CN110501499A - Effluent reagent strip with indicating area and preparation method thereof - Google Patents

Effluent reagent strip with indicating area and preparation method thereof Download PDF

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Publication number
CN110501499A
CN110501499A CN201910726877.1A CN201910726877A CN110501499A CN 110501499 A CN110501499 A CN 110501499A CN 201910726877 A CN201910726877 A CN 201910726877A CN 110501499 A CN110501499 A CN 110501499A
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CN
China
Prior art keywords
line
reagent strip
antibody
carrier film
sample
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Pending
Application number
CN201910726877.1A
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Chinese (zh)
Inventor
成晶
李明睿
陈超
尚京京
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SHANGHAI AIRUIDE BIOTECHNOLOGY Co Ltd
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SHANGHAI AIRUIDE BIOTECHNOLOGY Co Ltd
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Priority to CN201910726877.1A priority Critical patent/CN110501499A/en
Publication of CN110501499A publication Critical patent/CN110501499A/en
Pending legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

Abstract

The present invention provides a kind of effluent reagent strip and preparation method thereof with indicating area.Specifically, reagent strip provided by the invention includes: sample pad (2), bonding pad (3), antibody carrier film (4) and blotting paper (5)；And the antibody carrier film is equipped with and is used to indicate the whether used indicating area (43) of the reagent strip, detection zone (41) and Quality Control region (42)；Wherein, the sample pad, bonding pad, antibody carrier film and blotting paper are arranged successively on sample to be tested chromatography direction.Reagent strip of the invention can discern whether to have used without using special installation.

Description

Effluent reagent strip with indicating area and preparation method thereof
Technical field
The invention belongs to detection fields, and in particular to a kind of effluent reagent strip and preparation method thereof.
Background technique
Fluorescence immune chromatography method is that immunofluorescence technique is combined to combine a kind of detection skill to be formed with traditional chromatographic technique Art, the increased characteristic of fluorescent tracing of existing high sensitivity also has the characteristics that convenient and efficient, can be realized the standard to testing result It determines amount, is one of current main clinical detection means.
Although traditional fluorescence immune chromatography product (for example, detection reagent item) belongs to although indicating these products in explanation It is disposable, it is not possible to it reuses, but due to being this non-based on fluorescence on these fluorescence immune chromatography principle of products Visible light wave range is detected, these products used after have occurred be immunoreacted etc. position (such as detection line and On nature controlling line) phenomena such as being not in visual visible response line or spot, this results in these products in actual use, Under conditions of not by other special examined tools or instrument, it is difficult to distinguish whether the reagent strip taken out has used. The reagent strip used reuses its result inaccuracy, does not have Clinical significance of detecting, this will lead to once used reagent strip Obscure with not used reagent strip, not only needs to take a substantial amount of time to distinguish, also increase reuse Probability, to increase the risk of clinical misdiagnosis.
In conclusion can quickly and conveniently distinguish whether the reagent strip has used there is an urgent need in the art to develop one kind Reagent strip with and preparation method thereof.
Summary of the invention
The object of the invention is to solve conventional fluorescent immunochromatography platform cannot be distinguished analoids using whether, easily cause The defect that analoids are reused, and one kind is provided can quickly and conveniently distinguish the reagent strip whether used effluent examination Agent item, and its preparation method and application.
In the first aspect of the present invention, a kind of reagent strip is provided, the reagent strip includes: sample pad (2), bonding pad (3), antibody carrier film (4) and blotting paper (5)；And the antibody carrier film includes: detection zone (41), Quality Control region (42), And it is used to indicate the whether used indicating area (43) of the reagent strip；
Wherein, the indicating area is equipped with the index line (I line) for being coated with pigmented mixture, and the detection zone is equipped with inspection Survey line (T line) and the Quality Control region are equipped with nature controlling line (C line).
In another preferred example, the pigmented mixture is the mixture comprising the first component, wherein first component For colored dyes.
In another preferred example, the colored dyes are Ponceaux.
In another preferred example, the pigmented mixture also includes the second component, wherein described second group is selected from and includes: Polyol, water-soluble polymer, or combinations thereof.
In another preferred example, the polyol is selected from the group: alcohol compound (preferably, being polyalcohol), Saccharide compound (preferably, being oligosaccharide), or combinations thereof.
In another preferred example, the alcohol compound is glycerol.
In another preferred example, the saccharide compound is selected from the group: sucrose, trehalose, or combinations thereof.
In another preferred example, the polyol is selected from the group: sucrose, trehalose, glycerol, or combinations thereof.
In another preferred example, the water soluble polymer is selected from the group: glucan, albumin are (preferably, be cow's serum Albumin (BSA)), polyvinylpyrrolidone (PVP), polyethylene glycol (PEG) (preferably, PEG8000), or combinations thereof.
In another preferred example, second component is selected from the group: sucrose, trehalose, glucan, glycerol, PEG are (preferably Ground, PEG8000), PVP, BSA, or combinations thereof；Preferably, described second group is selected from and is selected from the group: sucrose, trehalose, Portugal are poly- Sugar, PVP, or combinations thereof.
In another preferred example, the indicating area, which is equipped with, has been coated with containing the first component, and including polyhydroxy chemical combination The index line of the pigmented mixture of second component of object and/or water-soluble polymer；Wherein, it is divided into coloured dye for described first group Material.
In another preferred example, the indicating area, which is equipped with, has been coated with comprising Ponceaux and selected from by sucrose, trehalose, Portugal The index line of the pigmented mixture of the second component for the group that glycan, glycerol, PEG8000, PVP, BSA and a combination thereof form.
In another preferred example, in the pigmented mixture, the mass ratio of the colored dyes and second component For 1:(0.1~100)；Preferably, being 1:(0.1~50)；It more preferably, is 1:(0.2~50)；Most preferably, for 1:(0.2~ 40)。
In another preferred example, described in the pigmented mixture when being divided into polyol for described second group The mass ratio of colored dyes and polyol is 1:(0.1~50)；Preferably, being 1:(5~40)；It more preferably, is 1:(5 ~20)；It most preferably, is 1:(10 ± 2).
In another preferred example, described in the pigmented mixture when being divided into water-soluble polymer for described second group The mass ratio of colored dyes and the water-soluble polymer is 1:(0.1~20)；Preferably, 1:(0.2~10).
In another preferred example, when being divided into the combination of polyol and water-soluble polymer for described second group, In In the pigmented mixture, the mass ratio of the colored dyes and polyol is 1:(5~50)；And the colored dyes Mass ratio with the water-soluble polymer is 1:(0.1~20)；Preferably, being 1:(0.2~10).
In another preferred example, the index line is coated with every meter of 4~5mg (preferably, every meter of 4.2~4.6mg；More preferably Ground, every meter of 4.4 ± 0.1mg) pigmented mixture.
In another preferred example, the index line is coated with every meter of 0.3~0.5mg (preferably, every meter of 0.4 ± 0.01mg) Dyestuff.
In another preferred example, the index line is coated with to antibody carrier film for I line coating buffer and is formed by index line, In, the I line coating buffer is the coating buffer containing pigmented mixture.
In another preferred example, the I line coating buffer be pigmented mixture in dilution (preferably, PBS dilution；More Goodly, 0.01M PBS dilution) in coating buffer.
In another preferred example, in the I line coating buffer, the concentration of the first component is 3~20mg/ml；Preferably, 5~ 10mg/ml。
In another preferred example, the I line coating buffer is that the first component is diluted to packet with the dilution containing the second component It is the obtained coating buffer of c1, and c1=3~20mg/ml (preferably, 5~10mg/ml) by the concentration of the first component in liquid.
In another preferred example, in the dilution for containing the second component, the concentration of the second component is 0.5~250mg/ Ml dilution (i.e. 0.05~25% (w/v))；Preferably, 1~200mg/ml dilution (i.e. 0.1~20% (w/v)).
In another preferred example, when being divided into polyol for described second group, in the dilution for containing the second component In liquid, the concentration of the second component is 1~250mg/ml dilution；Preferably, being 1~200mg/ml dilution；It more preferably, is 50 ~200mg/ml dilution.
In another preferred example, when being divided into water-soluble polymer for described second group, in the dilution for containing the second component In liquid, the concentration of the second component is 0.5~100mg/ml dilution；Preferably, being 1~50mg/ml dilution.
In another preferred example, when being divided into the combination of polyol and water-soluble polymer for described second group, In In the dilution for containing the second component, the concentration of the polyol is 50~250mg/ml dilution；And the water The concentration of soluble polymer is 5~100mg/ml dilution.
In another preferred example, the sample pad connect or be directly connected to the bonding pad interval, the bonding pad with Antibody carrier film interval connects or is directly connected to, the antibody carrier film connect with the blotting paper interval or directly connects It connects.
In another preferred example, the sample pad is directly connected to the bonding pad, the bonding pad is held with the antibody Film carrier is directly connected to, the antibody carrier film is directly connected to the blotting paper.
In another preferred example, the sample pad, bonding pad, antibody carrier film and blotting paper are arranged successively.
In another preferred example, the indicating area is located at antibody carrier film close to one end of blotting paper.
In another preferred example, the detection zone (41), Quality Control region (42) and indicating area (43) are arranged successively.
It in another preferred example, is 4 ± 0.5mm of spacing between the index line and the nature controlling line；Preferably, 4 ± 0.3mm；It more preferably, is 4 ± 0.1mm；It most preferably, is 4mm.
In another preferred example, the spacing between the detection line and the nature controlling line is 3 ± 0.5mm；Preferably, being 3 ±0.2mm；It more preferably, is 3 ± 0.1mm；It most preferably, is 3mm.
In another preferred example, the reagent strip further includes bottom plate (1).
In another preferred example, the bottom plate is polyester bottom plate or plastic bottom board；It preferably, is PVC bottom plate.
In another preferred example, the sample pad, bonding pad, antibody carrier film and/or blotting paper be set to bottom plate (1) it On.
In another preferred example, the sample pad, bonding pad, antibody carrier film and blotting paper chromatograph direction in sample to be tested On be sequentially arranged on the bottom plate (1).
In another preferred example, the length of the reagent strip is 58 ± 1mm；Preferably, being 58 ± 0.2mm；More preferably, it is 58±0.1mm；Most preferably, 58mm.
In another preferred example, the width of the reagent strip is 3.2 ± 0.5mm；Preferably, being 3.2 ± 0.3mm；More preferably Ground is 3.2 ± 0.1mm；It most preferably, is 3.2mm.
In another preferred example, the antibody carrier film is NC film.
In another preferred example, the bonding pad is fluorescent microsphere-first antibody compound bonding pad that tiled.
In another preferred example, the detection line is equipped with the detection line for being coated with secondary antibody.
In another preferred example, the nature controlling line is equipped with the nature controlling line for being coated with third antibody.
In another preferred example, the first antibody can be specifically bound with target substance.
In another preferred example, the first antibody is selected from the group: D-Dimer monoclonal antibody, CRP monoclonal antibody, SAA monoclonal antibody.
In another preferred example, the secondary antibody can be specifically bound with target substance.
In another preferred example, the secondary antibody is selected from the group: D-Dimer monoclonal antibody, CRP monoclonal antibody, SAA monoclonal antibody.
In another preferred example, the first antibody and secondary antibody are same or different；Preferably, being different 's.
In another preferred example, the third antibody can capture marker-first antibody compound.
In another preferred example, the third antibody is selected from the group: sheep anti-mouse igg antibody.
In the second aspect of the present invention, a kind of method for preparing reagent strip as described in relation to the first aspect, the side are provided Method comprising steps of
(1) provide includes indicating area, the detection zone equipped with detection line and the matter equipped with nature controlling line equipped with index line Control the antibody carrier film in region；
Wherein, the index line is prepared via a method which:
(i) I line coating buffer is provided, wherein stating I line coating buffer is the coating buffer containing pigmented mixture；With
(ii) pigmented mixture in I line coating buffer is coated with to antibody carrier film and forms index line；
(2) sample pad, bonding pad, antibody carrier film and blotting paper are assembled, the reagent strip is obtained.
In another preferred example, in the I line coating buffer, the concentration of the first component is 3~10mg/ml；Preferably, 5 ±1mg/ml。
In another preferred example, the I line coating buffer in first aspect the same as defining.
In another preferred example, I line coating buffer is coated with to antibody carrier film formation index line by drawing film instrument.
In another preferred example, in step (2), by the way that sample pad, bonding pad, antibody carrier film and blotting paper are affixed to Bottom plate is assembled；Preferably, bottom plate is successively affixed to be assembled.
In another preferred example, the detection zone is prepared via a method which:
(i) T line coating buffer is provided, wherein the T line coating buffer is the coating buffer containing secondary antibody；With
(ii) secondary antibody in the T line coating buffer is coated with to antibody carrier film and forms detection line.
In another preferred example, T line coating buffer is coated with to antibody carrier film formation detection line by drawing film instrument.
In another preferred example, the Quality Control region is prepared via a method which:
(i) C line coating buffer is provided, wherein the C line coating buffer is the coating buffer of the antibody containing third；With
(ii) the third antibody in the C line coating buffer is coated with to antibody carrier film and forms nature controlling line.
In another preferred example, C line coating buffer is coated with to antibody carrier film formation nature controlling line by drawing film instrument.
In another preferred example, while by the coating buffer containing colored dyes, the coating buffer containing secondary antibody and third antibody Coating buffer be coated with to antibody carrier film.
In another preferred example, described stroke of film instrument is to draw film gold spraying instrument.
In another preferred example, the bonding pad is prepared via a method which:
(i) it provides containing marker-first antibody compound liquid；With
(ii) liquid is tiled to blank bonding pad and drying, obtains the bonding pad.
In the third aspect of the present invention, a kind of reagent card is provided, the reagent card includes: examination as described in relation to the first aspect Agent item.
In the fourth aspect of the present invention, a kind of reagent strip as described in relation to the first aspect is provided and/or such as third aspect institute The purposes for the kit stated, for detecting target substance, and the target substance is antigen.
In another preferred example, the target substance is selected from the group: D-Dimer, CRP, SAA, or combinations thereof.
In the fifth aspect of the invention, a kind of detection method is provided, comprising steps of
(1) sample to be tested is contacted with the sample pad (2) of reagent strip as described in relation to the first aspect；
(2) make the liquid chromatography(LC) by antibody carrier film, and indicating area is made used mark occur；With
(3) used reagent strip is analyzed by detection system；
Wherein, the sample to be tested is that possible contain the liquid of target substance.
In another preferred example, the dosage of the sample is 60 ± 10 μ L；Preferably, being 60 ± 5 μ L；It more preferably, is 60 ±1μL；It most preferably, is 60 μ L.
In another preferred example, the detection system is fluorescence detecting system.
In another preferred example, the sample to be tested is the liquid that sample to be tested obtains after processing and/or dilution.
In another preferred example, the sample to be tested is selected from the group: blood, blood plasma, serum.
In another preferred example, it before step (1), further comprises the steps of: and judges that the reagent strip is by the indicating area It is no to have used.
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, In This no longer tires out one by one states.
Detailed description of the invention
The side schematic view of reagent strip of the invention is shown in Fig. 1.
The schematic top plan view of reagent strip of the invention is shown in Fig. 2.
It is respectively identified in figure as follows:
1 it is bottom plate, 2 be sample pad, 3 be bonding pad, 4 be antibody carrier film, 5 is blotting paper；
41 it is detection zone, 42 be Quality Control region, 43 is indicating area.
It is that traditional fluorescence immune chromatography product reagent strip (no indicating area) uses forward and backward situation that Fig. 3, which is shown,.
It is forward and backward that Fig. 4 shows that the reagent strip with instruction structure of the invention uses.
Fig. 5 shows the situation about reacting after 15min when the reagent strip detection prepared with embodiment 10.
Fig. 6 shows the unreacted when reagent strip detection with embodiment 6 (PEG8000 concentration is 5% (w/v)) preparation After (right side) and reaction 10min the case where (left side).
When Fig. 7 shows the reagent strip detection with embodiment 6 (PEG8000 concentration is 0.1%, 1% (w/v)) preparation, add 5~10min of sample, index line still have the case where minor residual.
Specific embodiment
The present inventor's in-depth study by long-term develops that a kind of whether have been subjected to indicator item makes for the first time With the reagent strip of function, enable experimenter simply and not by other by unique indicating area on the reagent strip Instrument can differentiate whether reagent strip has used by naked eyes, so as to avoid the misuse for having used reagent strip.Particularly, originally Invention is by the special formulation of pigmented mixture, so that the finger for being coated with pigmented mixture that the indicating area of reagent strip is equipped with Timberline have stablize it is not vulnerable, be not easy to spread and the advantages such as sensitivity height, and reagent strip of the invention be used in it is micro The detection of sample is also enough to clearly indicate whether reagent strip has been subjected to use.Based on this, inventor completes the present invention.
Term
As used herein, " interval connection " refers to, in corresponding two components (for example, sample pad, bonding pad, antibody are held Film carrier and blotting paper) between be additionally provided with third component；For example, being additionally provided with reacting pad between bonding pad and antibody carrier film.
As used herein, term " Ponceaux " refers to Ponceau S, and No. CAS is 6226-79-5, and structural formula is C2H12N4Na4O3S4Dyestuff.
Herein, unless stated otherwise, used abridge represents the conventional sense of this field, for example, BSA refers to cow's serum Albumin；DMF refers to dimethylformamide；DMSO refers to dimethyl sulfoxide；PEG refers to PEG 8000；PVP refers to polyvinylpyrrolidine Ketone.
Herein, " % (w/v) " refers to every 100mL solvent solute containing 1g unless stated otherwise, is equivalent to, for example, containing The pH7.3 of 5% (w/v) sucrose, 0.01M PBS dilution refers to be contained in the PBS dilution (pH7.3,0.01M) of every 100ml 5g sucrose.
Reagent strip
Fluorescence immune chromatography method reagent strip is generally by bottom plate, sample pad, bonding pad, antibody carrier film and water absorption pad totally 5 Part forms.Its test philosophy is mainly filtration of the sample Jing Guo sample pad, antigen therein when flowing through bonding pad, The identification and combination of the antibody specificity marked on fluorescent microsphere, the fluorescent microsphere being subsequently formed-Antibody-antigen complex layer When analysis to detection line on film (T line) position, since another epitope of antigen is by the specific effect power of the antibody on T line, in T It will form the double antibodies sandwich immune response structure of microballoon-antibody-antigen-antibody (on T line), and extra fluorescent microsphere-on line Antibody complex and a small amount of fluorescent microsphere-Antibody-antigen complex continuation chromatograph on film, can be caught by nature controlling line (C line) It obtains, the fluorescence intensity of the enrichment of T line and C line is then scanned using fluorescence detecting system, obtains sample by certain analytical calculation The concentration of antigen to be detected in this.
The present invention provides a kind of reagent strip with indicating area, which is equipped with visual visible index line. The index line region can mainly be formed by some colored dyes, such as Ponceaux etc., these dyestuffs by certain solvent dissolution after, Cooperation detection line and nature controlling line are coated on the solid phase carriers such as nitrocellulose filter by drawing film gold spraying instrument.It is certain density to have Color dyestuff, such as Ponceaux dyestuff have the visual identification of height, can play good indicative function；What these dyestuffs were formed Index line can act in reagent strip use process with solution chromatography, within a short period of time (in general 1~3min time), Coloration can gradually weaken until disappearance, thus plays the role of differentiation.And (the primary inspection of sample size needed for the detection of this law reagent strip Survey about 60 μ L samples) it is enough to wash away the dyestuff on index line, solvent or the dilution etc. without additional addition for washing away dyestuff.
Typically, the present invention provides a kind of effluent reagent strips, and as depicted in figs. 1 and 2, the reagent strip includes: to be set to Antibody carrier film 4 is simultaneously used to indicate the whether used indicating area of the reagent strip 43 (indicating area is equipped with and has been coated with color contamination Close the index line I line of object), the antibody carrier film is additionally provided with detection zone 41 (detection zone is equipped with detection line T line) and Quality Control Region 42 (Quality Control region is equipped with nature controlling line C line)；
The reagent strip further include sample pad 2, bonding pad 3 (bonding pad tiling have fluorescent microsphere-antibody complex), For absorbing the blotting paper 5 of extra sample.
Preferably, reagent strip of the invention further includes bottom plate 1；Sample pad 2, bonding pad 3, antibody carrier film 4 and blotting paper 5 It is sequentially arranged on the bottom plate 1.
In the present invention, the bottom plate can be made of any stable, non-porous known materials, and intensity should be enough to support Sample pad, bonding pad, antibody carrier film and/or the blotting paper adhered thereto.Preferably, the bottom plate is substantially waterproof 's.In a preferred embodiment, the bottom plate is made of polychloroethylene film, and preferably the bottom plate is PVC bottom plate.
In the present invention, sample pad can be made of any of absorbent material.Workable example of material includes: fiber Element, nitrocellulose, cellulose acetate, glass fibre, nylon, polyelectrolyte ion exchange membrane, propylene copolymer/nylon and Polyether sulfone.
In the present invention, bonding pad or antibody carrier film can be made of any of material, as long as the material has enough Porosity is to allow on surface and the internal capillarity that fluid occurs.Bonding pad or antibody carrier film should have enough holes Porosity, so that the particle for allowing to be coated with antibody or antigen is mobile.Bonding pad or antibody carrier film can also be contained analyte to be detected Sample used in liquid wetting (for example, for waterborne liquid have hydrophily, for organic solvent have hydrophobicity).It can Example of material for manufacturing bonding pad or antibody carrier film include: polymer PET, cellulose, nitrocellulose, cellulose acetate, Glass fibre, nylon, polyelectrolyte ion exchange membrane, propylene copolymer/nylon and polyether sulfone (polyethersulfone). In a preferred embodiment, bonding pad is made of polymer PET, and antibody carrier film is made of nitrocellulose (NC).
In the present invention, absorption pad can use any known materials that can be absorbed as sample and the liquid of buffer to be made. The absorbability of absorption pad is sufficiently large, to absorb the liquid for being added to testing piece.The example of material suitable for absorption pad Including cellulose, glass fibre and thick blotting paper.
Detection method
In a specific embodiment, it provides a kind of method detected using reagent strip of the invention: taking out inspection Card (including reagent strip of the invention) is surveyed, sample is loaded the (liquid after the dilution of 60 μ L is about added from adding mouth after dilution Sample, that is, sample), liquid sample (i.e. sample) chromatography after dilution will resist to antibody microballoon bonding pad in antibody microballoon bonding pad Body microballoon is multiple blend-out next from bonding pad, and the Ag-Ab microsphere compound then formed is chromatographed under capillary action to antibody On film, the fluorescence intensity of fluorogenic quantitative detection system detection T line and the enrichment of C line region is utilized.Single detection usually requires 5~10 Minute (and it is expected that the time spent needed for single detection is shorter as far as possible such as 5 minutes such as within 10 minutes).
Main advantages of the present invention include:
1) present invention prepared by instruction structure have it is very high can identification, not by additional instrument or equipment Preferable indicative function can be played；
2) the index line stability in the present invention is good, even if will not expand at least 3 days exposing in air Phenomena such as scattered, color is desalinated, the gray value of index line is not substantially reduced.
3) the index line high sensitivity in the present invention, is enough micro sample to take off index line completely or almost completely Color, so that whether indicator item has been subjected to use.
4) colored dyes used in the present invention be easy to get, cost is relatively low and avirulent chemical substance.
6) preparation method of reagent strip of the present invention is simple, it is only necessary to common to draw film gold spraying instrument it is achieved that and be with The scribing line of detection line and nature controlling line carries out simultaneously.
7) indicating area that pigmented mixture used in is formed on film after spraying or coating is uniform and diffusion path It spends small.
8) index line of the invention will not have an impact to the test result of reagent strip.
9) reagent strip applicability of the present invention is wide, (only normal to detect required place without specially treated is carried out again to sample Reason), it can be widely applied to lateral flow chromatography platform.
10) section the time required to instruction, the time required to conventional detection in range (such as 5-10min) can indicator item whether It has used.
In addition, the reagent strip in the present invention with preferred index line (or instruction structure) is (as prepared by embodiment 1-5 Reagent strip) it can be under conditions of shorter reaction time (or detection time) (reaction time needed for such as only 5min) Not used reagent strip obviously distinguishes (instruction heading line off or only minor residual), so that reagent strip of the invention can be used In the shorter detection architecture of required detection time.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number are weight/volume percents With weight/volume number.
It unless otherwise instructed, is to be loaded about 60 μ L samples from adding mouth, and observe in each embodiment and comparative example Using the variation of the index line of the reagent strip after preceding and use, to investigate the effect of different index lines.
Sample used is blood, blood plasma, serum or any buffer.
The preparation of 1 effluent reagent strip of embodiment
1) preparation of Sidestream chromatography reagent strip
1. the preparation of antibody membrane 5
A) preparation of T line coating buffer: a kind of anti-monoclonal antibody of mouse is diluted to pH7.3,0.01M PBS dilution 2mg/mL, 2-8 DEG C spare；
B) preparation of C line coating buffer: with pH7.3,0.01M PBS dilution that a kind of polyclonal antibody of mouse antiantibody is dilute It releases to 1mg/mL, 2-8 DEG C spare.
C) preparation of I line indicating area coating buffer: with the pH7.3 for containing 5% (w/v) sucrose, 0.01M PBS dilution will be beautiful Spring is red to be diluted to 5mg/mL, and 2-8 DEG C spare.
D) it is coated with line: being respectively coated with T line coating buffer, C line coating buffer, I line coating buffer extremely simultaneously by drawing film gold spraying instrument On NC film, later by the NC film after coating in dry by class vacuum 2h.
2. prepared by antibody microballoon bonding pad 3: select the carboxyl of fluorescent microsphere after EDC/NHS is activated, in mass ratio 10: 1 is added the anti-D-Dimer monoclonal antibody of another mouse, and the amino on antibody can be coupled with the carboxyl after activating on microballoon, shape At fluorescent microsphere in the coupling labeled complex of antibody；With the pH7.3 for containing 0.6% (w/v) BSA, 50 times of 0.01M PBS solution dilute Release antibody microsphere compound, and evenly laid out on bonding pad, it is dry by class vacuum 2h.
3. assembling reagent strip: according to shown in Fig. 2 and Fig. 3 by sample pad, antibody microballoon bonding pad, antibody membrane and blotting paper It successively pastes and is assembled on PVC bottom plate, be cut into the reagent strip that width is 3.2mm with cutting machine after being completed, put into reagent strip, Pressure shell is prepared into detection card.
As shown in figure 4, having apparent visually visible index line, test using preceding reagent strip of the present invention as shown in Fig. 4 a) Reagent strip of the invention such as Fig. 4 b after the completion) instruction heading line off, joined the sample of about 60 μ L in Fig. 4, the reaction time (i.e. plus The time of plate is run after sample) about 10min.
Embodiment 2
Substantially with embodiment 1, difference is preparation method: the step of c) preparation of I line indicating area coating buffer, is changed to:
Ponceaux is diluted to 10mg/mL with containing 5% (w/v) sucrose pH7.3,0.01M PBS dilution, 2-8 DEG C spare.
Embodiment 3
Substantially with embodiment 1, difference is preparation method: the step of c) preparation of I line indicating area coating buffer, is changed to:
Respectively with containing (a) 10% (w/v) or (b) pH7.3 of 20% (w/v) sucrose, the PBS dilution of 0.01M is by the beautiful spring Red to be diluted to 5mg/mL, 2-8 DEG C spare.
Embodiment 4
Substantially with embodiment 1, difference is preparation method: the step of c) preparation of I line indicating area coating buffer, is changed to:
(a) pH7.3 for containing 5% (w/v) trehalose is used, Ponceaux is diluted to 5mg/mL, 2-8 by 0.01M PBS dilution It is DEG C spare；Or
Be changed to: Ponceaux is diluted to by (b) with the PBS dilution (pH7.3,0.01M) containing 2.5% (w/v) glucan 5mg/ml, 2-8 DEG C spare.
Embodiment 5
Substantially with embodiment 1, difference is preparation method: the step of c) preparation of I line indicating area coating buffer, is changed to:
Respectively with containing (a) 0.1% (w/v), (b) 1% (w/v) or (c) 5% (w/v) polyvinylpyrrolidone (PVP) Ponceaux is diluted to 5mg/mL by PBS dilution (pH7.3,0.01M), and 2-8 DEG C spare.
Embodiment 6
Substantially with embodiment 1, difference is preparation method, and the step of c) preparation of I line indicating area coating buffer is changed to:
Respectively with containing (a) 0.1% (w/v), (b) 1% (w/v) or (c) 5% (w/v) PEG 8000 (PEG8000) PBS dilution (pH7.3,0.01M) Ponceaux is diluted to 5mg/mL, 2-8 DEG C is spare.
It is respectively the reagent strip test of the dilution preparation of 0.1% (w/v), 1% (w/v) with PEG8000 concentration, has been loaded Finish, sample is chromatographed to blotting paper direction, 5~10min after sample-adding, and discovery index line still has minor residual, referring to Fig. 7.Sample-adding After 10min, index line noresidue.
The reagent strip made from the dilution that PEG8000 concentration is 5% (w/v) is detected, and sample-adding finishes, and sample is to suction Water paper direction chromatographs, and 10min or more after sample-adding, index line still has more visible residual.Referring to Fig. 6.
Embodiment 7
Substantially with embodiment 1, difference is preparation method, and the step of c) preparation of I line indicating area coating buffer is changed to:
Respectively with containing (a) 0.1% (w/v), (b) 1% (w/v) or (c) PBS of 5% (w/v) bovine serum albumin(BSA) (BSA) Ponceaux is diluted to 5mg/mL by dilution (pH7.3,0.01M), and 2-8 DEG C spare.
It being detected with reagent strip prepared by embodiment 7, sample-adding finishes, and sample is chromatographed to blotting paper direction, 5 after sample-adding~ 10min, index line still have minor residual.After being loaded 10min, index line noresidue.
Embodiment 8
Substantially with embodiment 1, difference is preparation method, and the step of c) preparation of I line indicating area coating buffer is changed to:
Respectively with containing (a) 0.1% (w/v), (b) 1% (w/v) or (c) 5% (w/v) glycerol PBS dilution (pH7.3, Ponceaux 0.01M) is diluted to 5mg/mL, 2-8 DEG C spare.
The reagent strip made from embodiment 8 is detected.Sample-adding finishes, and sample is chromatographed to blotting paper direction, 5 after sample-adding~ 10min, index line still have minor residual.After being loaded 10min, index line noresidue.
Embodiment 9
Substantially with embodiment 1, difference is preparation method, and the step of c) preparation of I line indicating area coating buffer is changed to:
With containing 20% (w/v) sucrose, 3% (w/v) BSA, 0.5% (w/v) glycerol, 0.5% (w/v) PEG and 0.5% (w/ V) Ponceaux is diluted to 5mg/ml by the PBS dilution (pH7.3,0.01M) of PVP.
It is detected with the reagent strip of comparative example 5.Sample-adding finishes, and sample is chromatographed to blotting paper direction, 10min after sample-adding, Index line still has minor residual.
Embodiment 10
Substantially with embodiment 1, difference is preparation method: the step of c) preparation of I line indicating area coating buffer, is changed to:
Ponceaux is diluted to 2.5mg/ml with the 0.01M PBS dilution containing 2.5% (w/v) DMF.
It is detected with reagent strip prepared by comparative example 4, after sample-adding when 10min or more, single detects required for discovery Sample size is not enough to wash away index line, and index line still has clear residual after 15min.Referring to Fig. 5.
The traditional fluorescence immune chromatography product reagent strip (no indicating area) of comparative example 1
Substantially with embodiment 1, difference is preparation method: the coating without I line.
As shown in figure 3, (as shown in Figure 3a) and nothing (as shown in Figure 3b) after use are obvious before conventional fluorescent reagent strip use Observable difference.
Comparative example 2
Substantially with embodiment 1, difference is preparation method: the step of c) preparation of I line indicating area coating buffer, is changed to:
Ponceaux is diluted to 5mg/mL with PBS dilution (pH7.3,0.01M), 2-8 DEG C spare.
After by I line coating buffer coating to NC film, discovery index line diffusion.
Comparative example 3
Substantially with embodiment 1, difference is preparation method: the step of c) preparation of I line indicating area coating buffer, is changed to: Ponceaux is diluted to 2.5mg/ml, 1mg/ml respectively with PBS dilution (pH7.3,0.01M), 2-8 DEG C spare.
After the I line coating buffer of the Ponceaux containing various concentration is coated with onto NC film respectively, it is found that the color of index line is equal It is shallower, it is difficult to play indicative function.
Table A lists index line parameter and service condition in each embodiment and comparative example:
Table A
Unless stated otherwise, the index line of the reagent strip prepared in each embodiment in the present invention is in the aerial item of exposure Under part, will not occur at least 3 days diffusion, color desalination phenomena such as, the gray value of index line is not substantially reduced.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

Claims (10)

1. a kind of reagent strip, which is characterized in that the reagent strip includes: sample pad (2), bonding pad (3), antibody carrier film (4) With blotting paper (5)；And the antibody carrier film includes: detection zone (41), Quality Control region (42), and is used to indicate the reagent The whether used indicating area (43) of item；
Wherein, the indicating area is equipped with the index line (I line) for being coated with pigmented mixture, and the detection zone is equipped with detection line (T line) and the Quality Control region are equipped with nature controlling line (C line).
2. reagent strip as described in claim 1, which is characterized in that the pigmented mixture is the mixing comprising the first component Object, wherein described first group is divided into colored dyes (preferably, the colored dyes are Ponceaux).
3. reagent strip as claimed in claim 2, which is characterized in that the pigmented mixture also includes the second component, wherein institute Stating second group and being selected from includes: polyol, water-soluble polymer, or combinations thereof.
4. reagent strip as claimed in claim 3, which is characterized in that second component is selected from the group: sucrose, trehalose, Portugal Glycan, glycerol, PEG (preferably, PEG8000), PVP, BSA, or combinations thereof；Preferably, described second group is selected from and is selected from down Group: sucrose, trehalose, glucan, PVP, or combinations thereof.
5. reagent strip as described in claim 1, which is characterized in that the sample pad, bonding pad, antibody carrier film and blotting paper It is arranged successively；And the indicating area is located at antibody carrier film close to one end of blotting paper.
6. reagent strip as described in claim 1, which is characterized in that
(i) bonding pad is fluorescent microsphere-first antibody compound bonding pad that tiled；
(ii) detection line is equipped with the detection line for being coated with secondary antibody；With
(iii) nature controlling line is equipped with the nature controlling line for being coated with third antibody.
7. a kind of method for preparing reagent strip as described in claim 1, which is characterized in that the method includes the steps:
(1) provide includes indicating area, the detection zone equipped with detection line and the quality control region equipped with nature controlling line equipped with index line The antibody carrier film in domain；
Wherein, the index line is prepared via a method which:
(i) I line coating buffer is provided, wherein stating I line coating buffer is the coating buffer containing pigmented mixture；With
(ii) pigmented mixture in I line coating buffer is coated with to antibody carrier film and forms index line；
(2) sample pad, bonding pad, antibody carrier film and blotting paper are assembled, the reagent strip is obtained.
8. a kind of reagent card, which is characterized in that the reagent card includes: reagent strip as described in claim 1.
9. the purposes of a kind of reagent strip as described in claim 1 or kit as claimed in claim 8, which is characterized in that For detecting target substance, and the target substance is antigen.
10. a kind of detection method, which is characterized in that comprising steps of
(1) sample to be tested is contacted with the sample pad (2) of reagent strip as described in claim 1；
(2) make the liquid chromatography(LC) by antibody carrier film, and indicating area is made used mark occur；With
(3) used reagent strip is analyzed by detection system；
Wherein, the sample to be tested is that possible contain the liquid of target substance.
CN201910726877.1A 2019-08-07 2019-08-07 Effluent reagent strip with indicating area and preparation method thereof Pending CN110501499A (en)

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CN212031502U (en) * 2019-08-07 2020-11-27 上海艾瑞德生物科技有限公司 Lateral flow reagent strip with indicating area and detection card comprising same

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Publication number Priority date Publication date Assignee Title
US20040161859A1 (en) * 2003-02-13 2004-08-19 Huiyan Guo Lateral flow immunoassay controls
CN1690711A (en) * 2004-04-23 2005-11-02 中国人民解放军军事医学科学院微生物流行病研究所 Immune chromatographic test paper bar based on up conversion luminescence technology
CN107709479A (en) * 2015-06-15 2018-02-16 株式会社丸保 Water soluble dyestuffs system is printed black liquid and printing process and the method for printing and dyeing of black liquid is printed using the water soluble dyestuffs system
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