CN212031502U - Lateral flow reagent strip with indicating area and detection card comprising same - Google Patents
Lateral flow reagent strip with indicating area and detection card comprising same Download PDFInfo
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- CN212031502U CN212031502U CN201921272900.6U CN201921272900U CN212031502U CN 212031502 U CN212031502 U CN 212031502U CN 201921272900 U CN201921272900 U CN 201921272900U CN 212031502 U CN212031502 U CN 212031502U
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Abstract
The utility model provides a side flow reagent strip with indicate regional and including the test card of this side flow reagent strip. Specifically, the utility model provides a reagent strip includes: the kit comprises a sample pad (2), a combination pad (3), an antibody bearing film (4) and absorbent paper (5); the antibody bearing film is provided with an indicating area (43) for indicating whether the reagent strip is used or not, a detection area (41) and a quality control area (42); the sample pad, the combination pad, the antibody bearing membrane and the absorbent paper are sequentially arranged in the chromatographic direction of the sample to be detected. The reagent strip of the utility model can distinguish whether the reagent strip is used without using special equipment.
Description
Technical Field
The utility model belongs to the field of detection, concretely relates to side flow reagent strip reaches test card including this side flow reagent strip with indicate regional.
Background
The fluorescence immunochromatography is a detection technology formed by combining an immunofluorescence technology and a traditional chromatography technology, has the characteristics of high sensitivity, fluorescence tracing increase and convenience and rapidness, can realize accurate quantification of a detection result, and is one of the main clinical detection means at present.
Although the conventional fluorescence immunochromatographic products (e.g., detection reagent strips) are indicated to be disposable and not reusable in the description, since the fluorescence immunochromatographic products are detected based on the invisible light band, no visible reaction line or spot occurs at the position (e.g., detection line or quality control line) where the immunoreaction occurs after the products are used, which results in that it is difficult to distinguish whether the reagent strips taken out are used without other special inspection tools or instruments during the actual use of the products. The used reagent strip has inaccurate result and no detection significance when being reused, so that once the used reagent strip is mixed with the unused reagent strip, a great deal of time is consumed for distinguishing, the probability of repeated use is increased, and the risk of clinical misdiagnosis is increased.
In view of the above, there is a need in the art to develop a reagent strip and a method for preparing the same, which can quickly and conveniently identify whether the reagent strip is used.
SUMMERY OF THE UTILITY MODEL
The utility model aims at solving the defect that the traditional fluorescence immunochromatography platform can not distinguish whether the reagent sheet is used or not and can easily cause the reagent sheet to be reused, and providing a lateral flow reagent strip which can quickly and conveniently distinguish whether the reagent strip is used or not, and a preparation method and application thereof.
In a first aspect of the present invention, there is provided a reagent strip, comprising: the kit comprises a sample pad (2), a combination pad (3), an antibody bearing film (4) and absorbent paper (5); and the antibody-bearing membrane comprises: a detection region (41), a quality control region (42), and an indication region (43) for indicating whether the reagent strip has been used;
wherein, the indication area is provided with an indication line (I line) coated with a colored mixture, the detection area is provided with a detection line (T line), and the quality control area is provided with a quality control line (C line).
In another preferred embodiment, the colored mixture is a mixture comprising a first component, wherein the first component is a colored dye.
In another preferred embodiment, the colored dye is ponceau.
In another preferred embodiment, the colored mixture further comprises a second component, wherein the second component is selected from the group consisting of: a polyol, a water soluble polymer, or a combination thereof.
In another preferred embodiment, the polyol is selected from the group consisting of: an alcohol compound (preferably, a polyol), a saccharide compound (preferably, an oligosaccharide), or a combination thereof.
In another preferred embodiment, the alcohol compound is glycerol.
In another preferred embodiment, the saccharide compound is selected from the group consisting of: sucrose, trehalose, or a combination thereof.
In another preferred embodiment, the polyol is selected from the group consisting of: sucrose, trehalose, glycerol, or a combination thereof.
In another preferred embodiment, the water-soluble polymer is selected from the group consisting of: dextran, albumin (preferably, Bovine Serum Albumin (BSA)), polyvinylpyrrolidone (PVP), polyethylene glycol (PEG) (preferably, PEG8000), or combinations thereof.
In another preferred embodiment, the second component is selected from the group consisting of: sucrose, trehalose, dextran, glycerol, PEG (preferably PEG8000), PVP, BSA, or combinations thereof; preferably, the second component is selected from the group consisting of: sucrose, trehalose, dextran, PVP, or a combination thereof.
In another preferred embodiment, the indicator region is provided with an indicator line coated with a colored mixture comprising a first component and a second component comprising a polyol and/or a water-soluble polymer; wherein the first component is a colored dye.
In another preferred embodiment, the indicator region is provided with an indicator line coated with a colored mixture comprising ponceau and a second component selected from the group consisting of sucrose, trehalose, dextran, glycerol, PEG8000, PVP, BSA and combinations thereof.
In another preferred embodiment, in the colored mixture, the mass ratio of the colored dye to the second component is 1 (0.1-100); preferably, the ratio is 1 (0.1-50); more preferably, it is 1 (0.2 to 50); most preferably 1 (0.2-40).
In another preferred embodiment, when the second component is a polyhydroxy compound, the mass ratio of the colored dye to the polyhydroxy compound in the colored mixture is 1 (0.1-50); preferably, the ratio is 1 (5-40); more preferably, 1 (5-20); most preferably 1: 10. + -. 2.
In another preferred embodiment, when the second component is a water-soluble polymer, the mass ratio of the colored dye to the water-soluble polymer in the colored mixture is 1 (0.1-20); preferably 1 (0.2-10).
In another preferred embodiment, when the second component is the combination of the polyhydroxy compound and the water-soluble polymer, the mass ratio of the colored dye to the polyhydroxy compound in the colored mixture is 1 (5-50); the mass ratio of the colored dye to the water-soluble polymer is 1 (0.1-20); preferably 1 (0.2-10).
In another preferred embodiment, the indicator line is coated with 4-5 mg per meter (preferably, 4.2-4.6 mg per meter; more preferably, 4.4 ± 0.1mg per meter) of the colored mixture.
In another preferred embodiment, the indicator line is coated with 0.3-0.5 mg per meter (preferably, 0.4 ± 0.01mg per meter) of dye.
In another preferred example, the indicator line is formed by coating an I-line coating solution onto an antibody-bearing film, wherein the I-line coating solution is a coating solution containing a colored mixture.
In another preferred embodiment, the I-line coating solution is a coating solution of a colored mixture in a diluent (preferably, PBS diluent; more preferably, 0.01M PBS diluent).
In another preferred example, the concentration of the first component in the I-line coating solution is 3-20 mg/ml; preferably, the concentration is 5 to 10 mg/ml.
In another preferred example, the coating solution for the I-line is obtained by diluting the first component with a diluent containing the second component to a concentration of the first component in the coating solution of c1, and c1 is 3-20 mg/ml (preferably, 5-10 mg/ml).
In another preferred embodiment, the concentration of the second component in the diluent containing the second component is 0.5-250 mg/ml of the diluent (i.e. 0.05-25% (w/v)); preferably, the concentration of the diluent is 1 to 200mg/ml (i.e., 0.1 to 20% (w/v)).
In another preferred embodiment, when the second component is a polyol, the concentration of the second component in the diluent containing the second component is 1-250 mg/ml of the diluent; preferably, the concentration is 1-200 mg/ml of diluent; more preferably, the concentration is 50 to 200 mg/ml.
In another preferred embodiment, when the second component is a water-soluble polymer, the concentration of the second component in the diluent containing the second component is 0.5-100 mg/ml of the diluent; preferably, the concentration is 1-50 mg/ml.
In another preferred embodiment, when the second component is a combination of a polyhydroxy compound and a water-soluble polymer, the concentration of the polyhydroxy compound in the diluent containing the second component is 50-250 mg/ml of the diluent; and the concentration of the water-soluble polymer is 5-100 mg/ml of diluent.
In another preferred embodiment, the sample pad is connected with the binding pad at intervals or directly, the binding pad is connected with the antibody bearing membrane at intervals or directly, and the antibody bearing membrane is connected with the absorbent paper at intervals or directly.
In another preferred embodiment, the sample pad is directly connected to the binding pad, the binding pad is directly connected to the antibody-bearing membrane, and the antibody-bearing membrane is directly connected to the absorbent paper.
In another preferred embodiment, the sample pad, the conjugate pad, the antibody-bearing membrane and the absorbent paper are arranged in this order.
In another preferred embodiment, the indication area is located at one end of the antibody bearing membrane close to the absorbent paper.
In another preferred example, the detection region (41), the quality control region (42) and the indication region (43) are arranged in sequence.
In another preferred example, the distance between the indication line and the quality control line is 4 +/-0.5 mm; preferably, 4 ± 0.3 mm; more preferably, 4 + -0.1 mm; most preferably 4 mm.
In another preferred example, the distance between the detection line and the quality control line is 3 +/-0.5 mm; preferably, 3 + -0.2 mm; more preferably, 3 + -0.1 mm; most preferably 3 mm.
In another preferred example, the reagent strip further comprises a bottom plate (1).
In another preferred embodiment, the bottom plate is a polyester bottom plate or a plastic bottom plate; preferably a PVC base plate.
In another preferred embodiment, the sample pad, the conjugate pad, the antibody-bearing membrane and/or the absorbent paper are provided on the base plate (1).
In another preferred example, the sample pad, the combination pad, the antibody bearing membrane and the absorbent paper are sequentially arranged on the bottom plate (1) in the chromatographic direction of the sample to be detected.
In another preferred example, the length of the reagent strip is 58 +/-1 mm; preferably, 58 + -0.2 mm; more preferably, 58 + -0.1 mm; most preferably 58 mm.
In another preferred example, the width of the reagent strip is 3.2 +/-0.5 mm; preferably, 3.2mm plus or minus 0.3 mm; more preferably, 3.2 + -0.1 mm; most preferably 3.2 mm.
In another preferred embodiment, the antibody-bearing membrane is an NC membrane.
In another preferred embodiment, the conjugate pad is a conjugate pad tiled with fluorescent microsphere-first antibody complexes.
In another preferred embodiment, the detection line is a detection line provided with a second antibody coated thereon.
In another preferred example, the control line is provided with a control line coated with a third antibody.
In another preferred embodiment, the first antibody is capable of specifically binding to a substance of interest.
In another preferred embodiment, the first antibody is selected from the group consisting of: D-Dimer monoclonal antibody, CRP monoclonal antibody, SAA monoclonal antibody.
In another preferred embodiment, the second antibody is capable of specifically binding to a target substance.
In another preferred embodiment, the second antibody is selected from the group consisting of: D-Dimer monoclonal antibody, CRP monoclonal antibody, SAA monoclonal antibody.
In another preferred embodiment, the first and second antibodies are the same or different; preferably, it is different.
In another preferred embodiment, the third antibody is capable of capturing a label-first antibody complex.
In another preferred embodiment, the third antibody is selected from the group consisting of: goat anti-mouse IgG antibodies.
In a second aspect of the present invention, there is provided a method of preparing a reagent strip according to the first aspect, the method comprising the steps of:
(1) providing an antibody bearing film comprising an indicating area provided with an indicating line, a detecting area provided with a detecting line and a quality control area provided with a quality control line;
wherein the indicator line is prepared by the following method:
(i) providing I thread coating liquid, wherein the I thread coating liquid is coating liquid containing a colored mixture; and
(ii) coating the colored mixture in the I-line coating solution on an antibody bearing film to form an indicating line;
(2) and assembling the sample pad, the combination pad, the antibody bearing film and the absorbent paper to obtain the reagent strip.
In another preferred example, the concentration of the first component in the I-line coating liquid is 3-10 mg/ml; preferably, 5. + -. 1 mg/ml.
In another preferred example, the I-line coating solution is coated on the antibody bearing film by a scribing instrument to form an indicating line.
In another preferred embodiment, the I-line package is defined as in the first aspect.
In another preferred example, in the step (2), the sample pad, the conjugate pad, the antibody-bearing film and the absorbent paper are adhered to the base plate for assembly; preferably, the assembly is performed by sequentially adhering to the base plate.
In another preferred embodiment, the detection area is prepared by the following method:
(i) providing a T-line coating solution, wherein the T-line coating solution is a coating solution containing a second antibody; and
(ii) and coating the second antibody in the T-line coating solution on an antibody bearing membrane to form a detection line.
In another preferred example, the detection line is formed by coating the T-line coating solution on the antibody bearing membrane by a membrane scribing instrument.
In another preferred embodiment, the quality control region is prepared by the following method:
(i) providing a C line coating solution, wherein the C line coating solution is a coating solution containing a third antibody; and
(ii) and coating the third antibody in the C line coating solution on an antibody bearing film to form a quality control line.
In another preferred example, the coating solution of the C line is coated on the antibody bearing film by a scribing instrument to form a quality control line.
In another preferred example, the coating solution containing the color dye, the coating solution containing the second antibody and the coating solution containing the third antibody are coated onto the antibody-bearing membrane at the same time.
In another preferred example, the scribing instrument is a scribing and gold spraying instrument.
In another preferred embodiment, the conjugate pad is prepared by the following method:
(i) providing a liquid comprising a label-first antibody complex; and
(ii) spreading the liquid onto a blank bonding pad and drying to obtain the bonding pad.
In a third aspect of the present invention, there is provided a detection card, comprising: the reagent strip of the first aspect.
In a fourth aspect of the present invention there is provided a use of a reagent strip according to the first aspect and/or a kit according to the third aspect for detecting a target substance, and the target substance is an antigen.
In another preferred embodiment, the target substance is selected from the group consisting of: D-Dimer, CRP, SAA, or a combination thereof.
In a fifth aspect of the present invention, there is provided a detection method, comprising the steps of:
(1) contacting a sample to be tested with a sample pad (2) of a reagent strip according to the first aspect;
(2) passing the liquid chromatography through an antibody-bearing membrane and allowing the indicator region to appear as a used label; and
(3) analyzing the used reagent strip by the detection system;
wherein, the sample to be tested is liquid possibly containing target substances.
In another preferred embodiment, the amount of the sample is 60 +/-10 muL; preferably 60 +/-5 mu L; more preferably, 60. + -. 1. mu.L; most preferably 60 μ L.
In another preferred embodiment, the detection system is a fluorescence detection system.
In another preferred example, the sample to be tested is a liquid obtained by processing and/or diluting a sample to be tested.
In another preferred embodiment, the sample to be tested is selected from the group consisting of: blood, plasma, serum.
In another preferred example, before the step (1), the method further comprises the steps of: and judging whether the reagent strip is used or not through the indicating area.
It is understood that within the scope of the present invention, the above-mentioned technical features of the present invention and those specifically described below (e.g. in the examples) can be combined with each other to constitute new or preferred technical solutions. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 is a schematic side view of a reagent strip of the present invention.
Fig. 2 is a schematic top view of the reagent strip of the present invention.
The various designations in the figure are as follows:
1 is a bottom plate, 2 is a sample pad, 3 is a combination pad, 4 is an antibody bearing film, and 5 is absorbent paper;
FIG. 3 shows the conventional fluorescence immunochromatographic product reagent strip (no indication area) before and after use; wherein FIG. 3a) shows the conventional fluorescent reagent strip before use, and FIG. 3b) shows the conventional fluorescent reagent strip after use.
FIG. 4 shows the reagent strip with indicating structure of the present invention before and after use; wherein, fig. 4a) shows the reagent strip of the present invention before use, and fig. 4b) shows the reagent strip of the present invention after completion of the test.
FIG. 5 shows the results after 15min of reaction when the test strip prepared in example 10 was used.
FIG. 6 shows the unreacted (right) and after 10min (left) of reaction in the test using the reagent strip prepared in example 6(PEG8000 concentration of 5% (w/v)).
FIG. 7 shows that when the reagent strip prepared in example 6(PEG8000 concentration of 0.1%, 1% (w/v)) is used for detection, the indicator line still remains weakly after sample application for 5-10 min.
Detailed Description
The inventor of the present invention has conducted extensive and intensive studies for a long time to develop a reagent strip having a function of indicating whether the reagent strip has been used, which enables an experimenter to distinguish whether the reagent strip has been used by naked eyes simply and without the aid of other instruments through a unique indicating region on the reagent strip, thereby preventing misuse of the used reagent strip. In particular, the utility model discloses a reagent strip of the regional indicator of the colored mixture of cladding has special formulation for the regional indicator line of indicator of reagent strip has advantages such as stable difficult inefficacy, difficult diffusion, and sensitivity height, moreover the utility model discloses a reagent strip is used for the detection of trace sample also enough clearly to indicate whether the reagent strip has used. Based on this, the inventors have completed the present invention.
Term(s) for
As used herein, "spaced apart" means that a third component is also disposed between the respective two components (e.g., the sample pad, the conjugate pad, the antibody-bearing membrane, and the absorbent paper); for example, a reaction pad is further provided between the conjugate pad and the antibody-bearing membrane.
As used herein, the term "ponceau" refers to ponceau S having a CAS number of 6226-79-5 and a structural formula of C2H12N4Na4O3S4The dye of (4).
In this context, the abbreviations used represent the conventional meaning in the art unless otherwise specified, e.g., BSA means bovine serum albumin; DMF means dimethylformamide; DMSO refers to dimethyl sulfoxide; PEG means polyethylene glycol 8000; PVP refers to polyvinylpyrrolidone.
In this context, "% (w/v)" means 1g of solute per 100mL of solvent, corresponding, for example, to 5% (w/v) sucrose in pH7.3, and 0.01M PBS dilution means 5g of sucrose per 100mL of PBS dilution (pH7.3, 0.01M).
Reagent strip
The fluorescence immunochromatography reagent strip generally consists of 5 parts, namely a bottom plate, a sample pad, a combination pad, an antibody bearing membrane and a water absorption pad. The testing principle is that the sample is filtered by the sample pad, the antigen is specifically identified and combined by the antibody marked on the fluorescent microsphere when flowing through the combination pad, then when the formed fluorescent microsphere-antibody-antigen complex is chromatographed to the position of a detection line (T line) on the membrane, because the other epitope of the antigen is acted by the specificity of the antibody on the T line, a double-antibody sandwich immunoreaction structure of microsphere-antibody-antigen-antibody (on the T line) is formed on the T line, while the redundant fluorescent microsphere-antibody complex and a small amount of fluorescent microsphere-antibody-antigen complex are continuously chromatographed on the membrane and can be captured by a quality control line (C line), and then scanning the enriched fluorescence intensity of the T line and the C line by using a fluorescence detection system, and obtaining the concentration of the antigen to be detected in the sample through certain analysis and calculation.
The utility model provides a reagent strip with indicate regional, this indicate regional be equipped with the visible instruction line. The indicator line area can be mainly formed by some colored dyes, such as ponceau and the like, after the dyes are dissolved by a certain solvent, the cooperative detection line and the quality control line are coated on a solid phase carrier, such as a nitrocellulose membrane and the like, through a membrane-scribing metal spraying instrument. Colored dyes with certain concentration, such as ponceau and other dyes, have high visible identification degree and can play a good indicating role; the indicating lines formed by the dyes can gradually weaken and disappear in a short time (generally within 1-3 min) along with the solution chromatography effect in the using process of the reagent strip, so that the distinguishing effect is achieved. And the sample amount required by the test of the reagent strip (about 60 mu L of sample in one test) is enough to wash off the dye on the indicating line, and no extra solvent or diluent for washing off the dye is needed.
Typically, the present invention provides a lateral flow reagent strip, as shown in fig. 1 and 2, comprising: an indicating area 43 (an indicating line I coated with a colored mixture) which is arranged on the antibody bearing film 4 and is used for indicating whether the reagent strip is used or not, a detecting area 41 (a detecting line T line is arranged on the detecting area) and a quality control area 42 (a quality control line C line is arranged on the quality control area) are also arranged on the antibody bearing film;
the reagent strip also comprises a sample pad 2, a combination pad 3 (the combination pad is flatly paved with fluorescent microsphere-antibody compound), and absorbent paper 5 for absorbing redundant samples.
Preferably, the reagent strip of the present invention further comprises a bottom plate 1; the sample pad 2, the combination pad 3, the antibody bearing film 4 and the absorbent paper 5 are sequentially arranged on the bottom plate 1.
In the present invention, the base plate may be made of any stable, non-porous known material, and should have sufficient strength to support the sample pad, conjugate pad, antibody-bearing membrane and/or absorbent paper adhered thereto. Preferably, the floor is substantially impermeable to water. In a preferred embodiment, the base plate is made of a polyvinyl chloride film, preferably the base plate is a PVC base plate.
In the present invention, the sample pad can be made of any known absorbent material. Examples of materials that can be used include: cellulose, nitrocellulose, cellulose acetate, glass fiber, nylon, polyelectrolyte ion-exchange membranes, propylene copolymer/nylon, and polyethersulfone.
In the present invention, the conjugate pad or antibody-bearing membrane can be made of any known material, as long as the material has sufficient porosity to allow capillary action of the fluid to occur on the surface and inside. The conjugate pad or antibody-bearing membrane should be sufficiently porous to allow movement of the antibody or antigen coated particles. The conjugate pad or antibody-bearing membrane can also be wetted by the liquid used in the sample containing the analyte to be detected (e.g., hydrophilic for aqueous liquids and hydrophobic for organic solvents). Examples of materials that can be used to make the conjugate pad or antibody-bearing membrane include: polyester membranes, cellulose, nitrocellulose, cellulose acetate, glass fiber, nylon, polyelectrolyte ion exchange membranes, propylene copolymer/nylon, and polyethersulfone (polyethersulfone). In a preferred embodiment, the conjugate pad is made of a mylar, and the antibody-bearing membrane is made of Nitrocellulose (NC).
In the present invention, the absorbent pad can be made of any known material that can absorb the liquid as the sample and the buffer solution. The absorbent capacity of the absorbent pad should be large enough to absorb the liquid added to the test strip. Examples of suitable materials for the absorbent pad include cellulose, glass fibers, and thick absorbent paper.
Detection method
In one embodiment, a method for performing a test using the reagent strip of the present invention is provided: take out the detection card (including the utility model discloses a reagent strip), the sample is from the application of sample mouth application of sample (about adding 60 μ L's the liquid sample after dilution promptly the sample) after diluting, liquid sample (promptly the sample) chromatography after diluting to antibody microsphere combination pad, melts out antibody microsphere from the combination pad in the antibody microsphere combination pad again, then the antigen-antibody microsphere complex that forms on chromatography to antibody membrane under capillary, utilizes fluorescence quantitative determination system to detect the fluorescence intensity of T line and the regional enrichment of C line. A single test typically takes 5-10 minutes (and it is desirable that the time taken for a single test is as short as possible, e.g., within 10 minutes, e.g., 5 minutes).
The utility model discloses a main advantage includes:
1) the prepared indicating structure of the utility model has high identifiability, and can play a good indicating role without the help of additional instruments or equipment;
2) the utility model provides an indicating line stability is good, even expose in the air, can not take place phenomenons such as diffusion, colour desalination in at least 3 days, and the grey level value of indicating line does not obviously reduce.
3) The utility model provides an indicator sensitivity is high, is enough to make the indicator completely or basically fade completely to the sample of trace to indicate whether the reagent strip has used.
4) The colored dye used in the utility model is a chemical substance which is easy to obtain, low in cost and nontoxic.
6) The utility model discloses the preparation method of reagent strip is simple, only needs ordinary drawing the membrane and spouts the gold appearance just can realize, and can go on simultaneously with the marking off of detection line and quality control line.
7) The colored mixture used forms a uniform and diffuse indicator area on the film after spraying or coating.
8) The utility model discloses an indicator line can not be influential to the test result of reagent strip.
9) The utility model discloses reagent strip extensive applicability need not carry out special treatment again to the sample (only normally detect required processing), but wide application in lateral flow chromatography platform.
10) Indicating the required time period, and indicating whether the reagent strip is used within the time range required by the conventional detection (such as 5-10 min).
Furthermore, the reagent strip of the present invention having the preferred indicator line (or indicator structure) (e.g., the reagent strip prepared in examples 1 to 5) can be clearly distinguished from the unused reagent strip (the indicator line disappears or only slightly remains) even in the case of a shorter reaction time (or detection time) (e.g., the required reaction time is only 5min), so that the reagent strip of the present invention can be used in a detection system requiring a shorter detection time.
The present invention will be further described with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Percentages and parts are weight/volume percentages and weight/volume parts, unless otherwise indicated.
In each of examples and comparative examples, about 60. mu.L of a sample was applied from an application port, and changes in the indicator lines of the reagent strip before and after use were observed to examine the effects of the different indicator lines, unless otherwise specified.
The sample used is blood, plasma, serum or any buffer.
EXAMPLE 1 preparation of lateral flow reagent strips
1) Preparation of lateral flow chromatography reagent strips
Preparation of antibody film 5
a) Preparing a T-line coating liquid: diluting a mouse anti-monoclonal antibody to 2mg/mL by using PBS (phosphate buffer solution) with pH7.3 and 0.01M, and keeping the temperature at 2-8 ℃ for later use;
b) c, preparing a coating solution for the yarn C: a polyclonal antibody against mouse antibody was diluted to 1mg/mL with 0.01M PBS (pH7.3), and kept at 2-8 ℃ until use.
c) Preparation of coating solution in the indicated area of line I: ponceau was diluted to 5mg/mL with 5% (w/v) sucrose in 0.01M PBS diluent ph7.3, 2-8 ℃ for use.
d) Coating the wires: coating the T-line coating liquid, the C-line coating liquid and the I-line coating liquid onto the NC film by a film-scribing metal spraying instrument, and drying the coated NC film in vacuum for 2 h.
Preparing an antibody microsphere bonding pad 3: selecting carboxyl of a fluorescent microsphere, activating by EDC/NHS, adding another mouse anti-D-Dimer monoclonal antibody according to the mass ratio of 10:1, wherein amino on the antibody can be coupled with the activated carboxyl on the microsphere to form a coupling labeling compound of the fluorescent microsphere and the antibody; the antibody microsphere complex was diluted 50-fold with 0.6% (w/v) BSA in PBS, pH7.3,0.01M, and spread evenly onto the conjugate pad, and dried under vacuum-like conditions for 2 h.
Assembling reagent strips: according to the figure 2 and figure 3, the sample pad, the antibody microsphere combination pad, the antibody membrane and the absorbent paper are sequentially adhered and assembled on the PVC base plate, and are cut into reagent strips with the width of 3.2mm by a slitter after the assembly is finished, and the reagent strips are loaded and pressed into shells to prepare the detection card.
As shown in figure 4, before using the utility model reagent strip as shown in figure 4a), there is obvious visual indicator line, after the test is completed the utility model reagent strip as shown in figure 4b) indicator line disappears, about 60 uL of sample is added in figure 4, the reaction time (i.e. the time of running the plate after the sample is added) is about 10 min.
Example 2
The preparation method is basically the same as example 1, except that: the step of c) preparing the coating liquid of the I line indication area is changed as follows:
ponceau was diluted to 10mg/mL with a 0.01M PBS diluent containing 5% (w/v) sucrose, ph7.3, and was kept at 2-8 ℃ until use.
Example 3
The preparation method is basically the same as example 1, except that: the step of c) preparing the coating liquid of the I line indication area is changed as follows:
ponceau was diluted to 5mg/mL with PBS (pH7.3, 0.01M) containing (a) 10% (w/v) or (b) 20% (w/v) sucrose, respectively, and kept at 2-8 ℃ until use.
Example 4
The preparation method is basically the same as example 1, except that: the step of c) preparing the coating liquid of the I line indication area is changed as follows:
(a) diluting ponceau with 5% (w/v) trehalose in PBS (pH7.3, 0.01M) to 5mg/mL at 2-8 deg.C; or
Instead, the method comprises the following steps: (b) ponceau was diluted to 5mg/ml with 2.5% (w/v) dextran in PBS (pH7.3, 0.01M) and kept at 2-8 ℃ until needed.
Example 5
The preparation method is basically the same as example 1, except that: the step of c) preparing the coating liquid of the I line indication area is changed as follows:
ponceau was diluted to 5mg/mL with PBS diluent (pH7.3, 0.01M) containing (a) 0.1% (w/v), (b) 1% (w/v), or (c) 5% (w/v) polyvinylpyrrolidone (PVP), respectively, and kept at 2-8 ℃ until use.
Example 6
The preparation method is basically the same as that of the example 1, except that the step of c) preparing the coating liquid of the I line indicating area is changed to:
ponceau was diluted to 5mg/mL with PBS diluent (pH7.3, 0.01M) containing (a) 0.1% (w/v), (b) 1% (w/v), or (c) 5% (w/v) polyethylene glycol 8000 (PEG8000), respectively, and kept at 2-8 ℃ until use.
And (3) testing the test strip prepared by using the diluent with the PEG8000 concentration of 0.1% (w/v) and 1% (w/v) respectively, after the sample is added, carrying out chromatography on the sample in the direction of absorbent paper, and after the sample is added, 5-10min, finding that an indication line still has weak residue, and referring to fig. 7. After sample application for 10min, no indicator line remained.
And (3) detecting by using a reagent strip prepared from a diluent with PEG8000 concentration of 5% (w/v), after the sample is added, carrying out chromatography on the sample in the direction of absorbent paper, wherein the indicator line still has clear residue after the sample is added for more than 10 min. See fig. 6.
Example 7
The preparation method is basically the same as that of the example 1, except that the step of c) preparing the coating liquid of the I line indicating area is changed to:
ponceau was diluted to 5mg/mL with PBS diluent (pH7.3, 0.01M) containing (a) 0.1% (w/v), (b) 1% (w/v), or (c) 5% (w/v) Bovine Serum Albumin (BSA) at 2-8 ℃ for use.
And (3) detecting by using the reagent strip prepared in the example 7, after the sample is added, carrying out chromatography on the sample in the direction of the absorbent paper, and after the sample is added, keeping the indicator line still slightly remained for 5-10 min. After sample application for 10min, no indicator line remained.
Example 8
The preparation method is basically the same as that of the example 1, except that the step of c) preparing the coating liquid of the I line indicating area is changed to:
ponceau was diluted to 5mg/mL with PBS diluent (pH7.3, 0.01M) containing (a) 0.1% (w/v), (b) 1% (w/v), or (c) 5% (w/v) glycerol, respectively, and kept at 2-8 ℃.
The test was carried out using the reagent strip prepared in example 8. And (3) after the sample is added, carrying out chromatography on the sample in the direction of the absorbent paper, and keeping the indicator line still weakly remained after 5-10min after the sample is added. After sample application for 10min, no indicator line remained.
Example 9
The preparation method is basically the same as that of the example 1, except that the step of c) preparing the coating liquid of the I line indicating area is changed to:
ponceau was diluted to 5mg/ml with PBS diluent (pH7.3, 0.01M) containing 20% (w/v) sucrose, 3% (w/v) BSA, 0.5% (w/v) glycerol, 0.5% (w/v) PEG and 0.5% (w/v) PVP.
The test was carried out using the reagent strip of comparative example 5. And (3) after the sample is added, carrying out chromatography on the sample in the direction of the absorbent paper, and keeping the indicator line still weakly remained after 10min after the sample is added.
Example 10
The preparation method is basically the same as example 1, except that: the step of c) preparing the coating liquid of the I line indication area is changed as follows:
ponceau was diluted to 2.5mg/ml with a 0.01M PBS dilution containing 2.5% (w/v) DMF.
When the test was carried out using the reagent strip prepared in comparative example 4, it was found that the amount of sample required for a single test was not sufficient to wash off the indicator line 10min after the sample application, and the indicator line remained clear after 15 min. See fig. 5.
COMPARATIVE EXAMPLE 1 conventional fluorescence immunochromatographic product reagent strip (non-indication area)
The preparation method is basically the same as example 1, except that: the coating of the I-line is not performed.
As shown in FIG. 3, there is no obvious difference between the conventional fluorescent reagent strip before use (as shown in FIG. 3a) and after use (as shown in FIG. 3 b).
Comparative example 2
The preparation method is basically the same as example 1, except that: the step of c) preparing the coating liquid of the I line indication area is changed as follows:
ponceau was diluted to 5mg/mL with PBS diluent (pH7.3, 0.01M) and kept at 2-8 ℃ until use.
After coating the I-line with the coating solution onto the NC film, the indicator line was found to spread.
Comparative example 3
The preparation method is basically the same as example 1, except that: the step of c) preparing the coating liquid of the I line indication area is changed as follows: ponceau was diluted to 2.5mg/ml and 1mg/ml with PBS diluent (pH7.3, 0.01M), respectively, at 2-8 ℃ for further use.
After I-line coating liquids containing ponceau red with different concentrations are coated on an NC film, the colors of the indicating lines are all lighter, and the indicating function is difficult to play.
Table A lists the indicated line parameters and usage in each of the examples and comparative examples:
TABLE A
Unless otherwise stated, the indicator lines of the reagent strips prepared in the examples of the present invention were not diffused, faded in color, etc. for at least 3 days under the condition of exposure to air, and the gray values of the indicator lines were not significantly decreased.
All documents mentioned in this application are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention may be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the appended claims.
Claims (8)
1. A lateral flow reagent strip having an indicator region, said lateral flow reagent strip comprising: the kit comprises a sample pad (2), a combination pad (3), an antibody bearing film (4) and absorbent paper (5); and the antibody-bearing membrane comprises: a detection zone (41), a quality control zone (42), and an indicator zone (43) for indicating whether the lateral flow reagent strip has been used;
the indicating area is provided with an indicating line coated with a colored mixture, the detection area is provided with a detection line, and the quality control area is provided with a quality control line.
2. A lateral flow reagent strip having an indicator region according to claim 1, wherein the sample pad, conjugate pad, antibody-bearing membrane and absorbent paper are arranged in sequence.
3. A lateral flow reagent strip having an indicator region according to claim 1 wherein the indicator region is located at an end of the antibody-bearing membrane adjacent to the bibulous paper.
4. A lateral flow reagent strip having an indicator region according to claim 1 wherein the indicator line is spaced from the control line by a distance of 4 ± 0.5 mm; and/or the distance between the detection line and the quality control line is 3 +/-0.5 mm.
5. A lateral flow reagent strip having an indicator region according to claim 1, wherein the lateral flow reagent strip has a length of 58 ± 1 mm; and/or a width of 3.2 + -0.5 mm.
6. A lateral flow reagent strip having an indicator region according to claim 1, wherein said lateral flow reagent strip further comprises a bottom panel (1); and the sample pad, the combination pad, the antibody bearing film and the absorbent paper are arranged on the bottom plate (1).
7. A lateral flow reagent strip having an indicator region according to claim 1,
(i) the combination pad is a combination pad tiled with fluorescent microsphere-first antibody compound;
(ii) the detection line is provided with a second antibody coated detection line; and
(iii) the quality control line is provided with a quality control line coated with a third antibody.
8. A test card, comprising: a lateral flow reagent strip having an indicator region according to claim 1.
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| CN201921272900.6U CN212031502U (en) | 2019-08-07 | 2019-08-07 | Lateral flow reagent strip with indicating area and detection card comprising same |
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| CN201921272900.6U CN212031502U (en) | 2019-08-07 | 2019-08-07 | Lateral flow reagent strip with indicating area and detection card comprising same |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110501499A (en) * | 2019-08-07 | 2019-11-26 | 上海艾瑞德生物科技有限公司 | Effluent reagent strip with indicating area and preparation method thereof |
| CN113866426A (en) * | 2021-09-26 | 2021-12-31 | 上海艾瑞德生物科技有限公司 | Kit for detecting D-dimer and preparation method thereof |
-
2019
- 2019-08-07 CN CN201921272900.6U patent/CN212031502U/en active Active
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110501499A (en) * | 2019-08-07 | 2019-11-26 | 上海艾瑞德生物科技有限公司 | Effluent reagent strip with indicating area and preparation method thereof |
| CN113866426A (en) * | 2021-09-26 | 2021-12-31 | 上海艾瑞德生物科技有限公司 | Kit for detecting D-dimer and preparation method thereof |
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