Lateral flow test strip with indicator area and preparation method thereof

Abstract

The present invention provides a lateral flow reagent strip with an indication area and a preparation method thereof. Specifically, the reagent strip provided by the present invention comprises: a sample pad (2), a conjugation pad (3), an antibody-carrying membrane (4) and blotting paper (5); and the antibody-carrying membrane is provided with an indication area (43) for indicating whether the reagent strip has been used, a detection area (41) and a quality control area (42); wherein the sample pad, the conjugation pad, the antibody-carrying membrane and the blotting paper are arranged in sequence in the chromatographic direction of the sample to be tested. The reagent strip of the present invention can identify whether it has been used without using special equipment.

Description

Technical Field

[0001] The invention belongs to the field of detection, and particularly relates to a lateral flow reagent strip and a preparation method thereof. Background Art

[0002] Fluorescent immunochromatography is a detection technology that combines immunofluorescence technology with traditional chromatography technology. It has the characteristics of high-sensitivity fluorescent tracer increase and is convenient and fast. It can achieve accurate quantification of test results and is currently one of the main clinical detection methods.

[0003] Although traditional fluorescent immunochromatographic products (e.g., test strips) are marked in the instructions as single-use and cannot be reused, these fluorescent immunochromatographic products are based on the principle of fluorescence, a non-visible light band, for detection. After these products are used, no visible reaction lines or spots will appear at the locations where immune reactions occur (such as test lines and quality control lines). This makes it difficult to distinguish whether the removed test strips have been used without the help of other special inspection tools or instruments during the actual use of these products. The results of using the used test strips again are inaccurate and have no detection significance. This will result in the confusion of used test strips with unused test strips, which will not only take a lot of time to distinguish, but also increase the chance of reuse, thereby increasing the risk of clinical misdiagnosis.

[0004] In summary, there is an urgent need in the art to develop a reagent strip that can quickly and conveniently identify whether the reagent strip has been used and a preparation method thereof. Summary of the invention

[0005] The purpose of the present invention is to solve the defect that the traditional fluorescent immunochromatography platform cannot distinguish whether the reagent strip has been used or not, which easily causes the repeated use of the reagent strip, and to provide a lateral flow reagent strip that can quickly and conveniently distinguish whether the reagent strip has been used, and its preparation method and application.

[0006] In a first aspect of the present invention, a reagent strip is provided, the reagent strip comprising: a sample pad (2), a conjugate pad (3), an antibody-carrying membrane (4) and absorbent paper (5); and the antibody-carrying membrane comprises: a detection area (41), a quality control area (42), and an indication area (43) for indicating whether the reagent strip has been used;

[0007] 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).

[0008] In another preferred example, the colored mixture is a mixture comprising a first component, wherein the first component is a colored dye.

[0009] In another preferred embodiment, the colored dye is Ponceau red.

[0010] In another preferred example, the colored mixture further comprises a second component, wherein the second component is selected from: a polyhydroxy compound, a water-soluble polymer, or a combination thereof.

[0011] In another preferred embodiment, the polyhydroxy compound is selected from the following group: alcohol compounds (preferably, polyols), sugar compounds (preferably, oligosaccharides), or a combination thereof.

[0012] In another preferred embodiment, the alcohol compound is glycerol.

[0013] In another preferred embodiment, the sugar compound is selected from the group consisting of sucrose, trehalose, or a combination thereof.

[0014] In another preferred embodiment, the polyhydroxy compound is selected from the following group: sucrose, trehalose, glycerol, or a combination thereof.

[0015] In another preferred embodiment, the water-soluble polymer is selected from the following group: dextran, albumin (preferably bovine serum albumin (BSA)), polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG) (preferably PEG8000), or a combination thereof.

[0016] In another preferred embodiment, the second component is selected from the group consisting of sucrose, trehalose, dextran, glycerol, PEG (preferably PEG8000), PVP, BSA, or a combination thereof; preferably, the second component is selected from the group consisting of sucrose, trehalose, dextran, PVP, or a combination thereof.

[0017] In another preferred embodiment, the indicator area is provided with an indicator line coated with a colored mixture containing a first component and a second component including a polyhydroxy compound and / or a water-soluble polymer; wherein the first component is a colored dye.

[0018] In another preferred example, the indicator area is provided with an indicator line coated with a colored mixture comprising Ponceau red and a second component selected from the group consisting of sucrose, trehalose, dextran, glycerol, PEG8000, PVP, BSA and a combination thereof.

[0019] 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, 1:(0.1-50); more preferably, 1:(0.2-50); most preferably, 1:(0.2-40).

[0020] In another preferred embodiment, when the second component is a polyhydroxy compound, in the colored mixture, the mass ratio of the colored dye to the polyhydroxy compound is 1:(0.1-50); preferably, 1:(5-40); more preferably, 1:(5-20); optimally, 1:(10±2).

[0021] In another preferred embodiment, when the second component is a water-soluble polymer, in the colored mixture, the mass ratio of the colored dye to the water-soluble polymer is 1:(0.1-20); preferably, 1:(0.2-10).

[0022] In another preferred embodiment, when the second component is a combination of a polyhydroxy compound and a water-soluble polymer, in the colored mixture, the mass ratio of the colored dye to the polyhydroxy compound is 1:(5-50); and the mass ratio of the colored dye to the water-soluble polymer is 1:(0.1-20); preferably, it is 1:(0.2-10).

[0023] 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±0.1 mg per meter) of the colored mixture.

[0024] In another preferred embodiment, the indicator line is coated with 0.3-0.5 mg per meter (preferably, 0.4±0.01 mg per meter) of dye.

[0025] In another preferred embodiment, the indicator line is an indicator line formed by coating an I-line coating solution onto an antibody-carrying membrane, wherein the I-line coating solution is a coating solution containing a color mixture.

[0026] In another preferred embodiment, the I-line coating solution is a coating solution of a colored mixture in a diluent (preferably, a PBS diluent; more preferably, a 0.01 M PBS diluent).

[0027] In another preferred embodiment, the concentration of the first component in the I-line coating solution is 3 to 20 mg / ml; preferably, 5 to 10 mg / ml.

[0028] In another preferred embodiment, the I-line coating solution is a coating solution obtained by diluting the first component with a diluent containing the second component until the concentration of the first component in the coating solution is c1, and c1 = 3-20 mg / ml (preferably, 5-10 mg / ml).

[0029] In another preferred embodiment, in the diluent containing the second component, the concentration of the second component is 0.5-250 mg / ml diluent (i.e. 0.05-25% (w / v)); preferably, 1-200 mg / ml diluent (i.e. 0.1-20% (w / v)).

[0030] In another preferred embodiment, when the second component is a polyhydroxy compound, the concentration of the second component in the diluent containing the second component is 1-250 mg / ml of diluent; preferably, 1-200 mg / ml of diluent; more preferably, 50-200 mg / ml of diluent.

[0031] 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 to 100 mg / ml of the diluent; preferably, 1 to 50 mg / ml of the diluent.

[0032] In another preferred embodiment, when the second component is a combination of a polyhydroxy compound and a water-soluble polymer, in the diluent containing the second component, the concentration of the polyhydroxy compound is 50-250 mg / ml of the diluent; and the concentration of the water-soluble polymer is 5-100 mg / ml of the diluent.

[0033] In another preferred embodiment, the sample pad is connected to the conjugate pad at intervals or directly connected, the conjugate pad is connected to the antibody-carrying membrane at intervals or directly connected, and the antibody-carrying membrane is connected to the absorbent paper at intervals or directly connected.

[0034] In another preferred embodiment, the sample pad is directly connected to the conjugate pad, the conjugate pad is directly connected to the antibody-carrying membrane, and the antibody-carrying membrane is directly connected to the absorbent paper.

[0035] In another preferred embodiment, the sample pad, conjugate pad, antibody-carrying membrane and absorbent paper are arranged in sequence.

[0036] In another preferred embodiment, the indicator area is located at one end of the antibody-carrying membrane close to the absorbent paper.

[0037] In another preferred embodiment, the detection area (41), the quality control area (42) and the indication area (43) are arranged in sequence.

[0038] In another preferred embodiment, the distance between the indicator line and the quality control line is 4±0.5 mm; preferably, 4±0.3 mm; more preferably, 4±0.1 mm; and most preferably, 4 mm.

[0039] In another preferred embodiment, the spacing 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; and most preferably, 3 mm.

[0040] In another preferred embodiment, the reagent strip further comprises a bottom plate (1).

[0041] In another preferred embodiment, the bottom plate is a polyester bottom plate or a plastic bottom plate; preferably, it is a PVC bottom plate.

[0042] In another preferred embodiment, the sample pad, the conjugate pad, the antibody-carrying membrane and / or the absorbent paper are arranged on the bottom plate (1).

[0043] In another preferred embodiment, the sample pad, the conjugate pad, the antibody-carrying membrane and the absorbent paper are sequentially arranged on the bottom plate (1) in the chromatography direction of the sample to be tested.

[0044] In another preferred embodiment, 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.

[0045] In another preferred embodiment, the width of the reagent strip is 3.2±0.5 mm; preferably, 3.2±0.3 mm; more preferably, 3.2±0.1 mm; and most preferably, 3.2 mm.

[0046] In another preferred embodiment, the antibody-carrying membrane is a NC membrane.

[0047] In another preferred embodiment, the binding pad is a binding pad paved with a fluorescent microsphere-first antibody complex.

[0048] In another preferred embodiment, the detection line is a detection line coated with a second antibody.

[0049] In another preferred embodiment, the quality control line is provided with a quality control line coated with a third antibody.

[0050] In another preferred example, the first antibody can specifically bind to the target substance.

[0051] In another preferred embodiment, the first antibody is selected from the following group: D-Dimer monoclonal antibody, CRP monoclonal antibody, SAA monoclonal antibody.

[0052] In another preferred example, the second antibody can specifically bind to the target substance.

[0053] In another preferred embodiment, the second antibody is selected from the following group: D-Dimer monoclonal antibody, CRP monoclonal antibody, SAA monoclonal antibody.

[0054] In another preferred embodiment, the first antibody and the second antibody are the same or different; preferably, they are different.

[0055] In another preferred embodiment, the third antibody is capable of capturing the marker-first antibody complex.

[0056] In another preferred embodiment, the third antibody is selected from the following group: goat anti-mouse IgG antibody.

[0057] In a second aspect of the present invention, a method for preparing the reagent strip according to the first aspect is provided, the method comprising the steps of:

[0058] (1) providing an antibody-carrying membrane including an indication region with an indication line, a detection region with a detection line, and a quality control region with a quality control line;

[0059] Wherein, the indicator line is prepared by the following method:

[0060] (i) providing an I-line coating solution, wherein the I-line coating solution is a coating solution containing a color mixture; and

[0061] (ii) coating the colored mixture in the line coating solution onto the antibody-carrying membrane to form an indicator line;

[0062] (2) Assembling a sample pad, a conjugate pad, an antibody-carrying membrane and absorbent paper to obtain the reagent strip.

[0063] In another preferred embodiment, in the I-line coating solution, the concentration of the first component is 3-10 mg / ml; preferably, 5±1 mg / ml.

[0064] In another preferred embodiment, the I-line coating solution is the same as defined in the first aspect.

[0065] In another preferred embodiment, the I-line coating solution is coated onto the antibody-carrying membrane by a membrane scribing instrument to form an indicator line.

[0066] In another preferred embodiment, in step (2), the sample pad, the conjugate pad, the antibody-carrying membrane and the absorbent paper are assembled by pasting them to the bottom plate; preferably, they are assembled by pasting them to the bottom plate in sequence.

[0067] In another preferred embodiment, the detection area is prepared by the following method:

[0068] (i) providing a T-line coating solution, wherein the T-line coating solution is a coating solution containing a second antibody; and

[0069] (ii) coating the second antibody in the T-line coating solution onto the antibody-carrying membrane to form a detection line.

[0070] In another preferred embodiment, the T-line coating solution is coated onto the antibody-carrying membrane by a membrane scribing instrument to form a detection line.

[0071] In another preferred embodiment, the quality control area is prepared by the following method:

[0072] (i) providing a C-line coating solution, wherein the C-line coating solution is a coating solution containing a third antibody; and

[0073] (ii) coating the third antibody in the C-line coating solution onto the antibody-carrying membrane to form a quality control line.

[0074] In another preferred embodiment, the C-line coating solution is coated onto the antibody-carrying membrane by a membrane scribing instrument to form a quality control line.

[0075] In another preferred embodiment, a coating solution containing a color dye, a coating solution containing a second antibody, and a coating solution containing a third antibody are coated onto the antibody-carrying membrane at the same time.

[0076] In another preferred embodiment, the film scratching instrument is a film spraying instrument.

[0077] In another preferred embodiment, the conjugate pad is prepared by the following method:

[0078] (i) providing a liquid containing a marker-first antibody complex; and

[0079] (ii) spreading the liquid onto a blank conjugate pad and drying the pad to obtain the conjugate pad.

[0080] In a third aspect of the present invention, a reagent card is provided, the reagent card comprising: the reagent strip as described in the first aspect.

[0081] In a fourth aspect of the present invention, there is provided a use of the reagent strip as described in the first aspect and / or the kit as described in the third aspect for detecting a target substance, wherein the target substance is an antigen.

[0082] In another preferred embodiment, the target substance is selected from the following group: D-Dimer, CRP, SAA, or a combination thereof.

[0083] In a fifth aspect of the present invention, a detection method is provided, comprising the steps of:

[0084] (1) contacting a sample to be tested with a sample pad (2) of the reagent strip as described in the first aspect;

[0085] (2) allowing the liquid to chromatograph through the antibody-carrying membrane and causing a mark indicating that the liquid has been used to appear in the indicator area; and

[0086] (3) analyzing the used reagent strips through the detection system;

[0087] Wherein, the sample to be tested is a liquid that may contain the target substance.

[0088] In another preferred embodiment, the amount of the sample is 60±10 μL; preferably, 60±5 μL; more preferably, 60±1 μL; and most preferably, 60 μL.

[0089] In another preferred embodiment, the detection system is a fluorescence detection system.

[0090] In another preferred embodiment, the sample to be tested is a liquid obtained after the sample to be tested is processed and / or diluted.

[0091] In another preferred embodiment, the sample to be tested is selected from the following group: blood, plasma, and serum.

[0092] In another preferred embodiment, before step (1), the method further includes the step of: determining whether the reagent strip has been used by the indicator area.

[0093] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1 Shown is a schematic side view of a reagent strip of the present invention.

[0095] Figure 2 Shown is a schematic top view of the reagent strip of the present invention.

[0096] The symbols in the figure are as follows:

[0097] 1 is the base plate, 2 is the sample pad, 3 is the conjugate pad, 4 is the antibody carrier membrane, and 5 is the absorbent paper;

[0098] 41 is the detection area, 42 is the quality control area, and 43 is the indication area.

[0099] Figure 3 It shows the traditional fluorescent immunochromatography product reagent strip (without indicator area) before and after use.

[0100] Figure 4 The test strip with an indicator structure of the present invention is shown before and after use.

[0101] Figure 5 The results of the test using the reagent strip prepared in Example 10 after 15 minutes of reaction are shown.

[0102] Figure 6 The results are shown in the figure before the reaction (right) and after the reaction for 10 min (left) using the reagent strip prepared in Example 6 (PEG8000 concentration is 5% (w / v)).

[0103] Figure 7 It shows that when the reagent strip prepared in Example 6 (PEG8000 concentration of 0.1% and 1% (w / v)) is used for testing, there is still a weak residue on the indicator line 5 to 10 minutes after adding the sample. DETAILED DESCRIPTION

[0104] The inventor has developed a reagent strip with a function of indicating whether a reagent strip has been used for the first time through long-term and in-depth research, and a unique indication area on the reagent strip allows the experimenter to simply and without the aid of other instruments to distinguish by naked eyes whether the reagent strip has been used, thereby avoiding the misuse of the used reagent strip. In particular, the present invention, through the special formula of colored mixture, makes the indicator line coated with the colored mixture provided in the indication area of ​​the reagent strip have the advantages of being stable and not easy to fail, not easy to diffuse and high sensitivity, and the reagent strip of the present invention is also enough to clearly indicate whether the reagent strip has been used even if it is used for the detection of trace samples. Based on this, the inventor has completed the present invention.

[0105] the term

[0106] As used herein, "interval connection" means that a third component is provided between two corresponding components (eg, sample pad, conjugate pad, antibody-carrying membrane and absorbent paper); for example, a reaction pad is provided between the conjugate pad and the antibody-carrying membrane.

[0107] As used herein, the term "Ponceau S" refers to Ponceau S, CAS No. 6226-79-5, with a structural formula of C2H 12 Dye of N4Na4O3S4.

[0108] Unless otherwise specified, all abbreviations used herein represent common meanings in the art, for example, BSA refers to bovine serum albumin; DMF refers to dimethylformamide; DMSO refers to dimethyl sulfoxide; PEG refers to polyethylene glycol 8000; and PVP refers to polyvinyl pyrrolidone.

[0109] In this document, unless otherwise specified, "% (w / v)" means 1 g of solute per 100 mL of solvent, which is equivalent to, for example, 5% (w / v) sucrose in pH 7.3, 0.01 M PBS dilution means 5 g of sucrose per 100 ml of PBS dilution (pH 7.3, 0.01 M).

[0110] Reagent Strips

[0111] Fluorescent immunochromatography reagent strips are generally composed of five parts: a base plate, a sample pad, a conjugate pad, an antibody-carrying membrane, and an absorbent pad. The testing principle is that the sample is filtered through the sample pad, and the antigen in it is specifically recognized and bound by the antibody labeled on the fluorescent microsphere when it flows through the conjugate pad. Then, when the fluorescent microsphere-antibody-antigen complex formed is chromatographed to the detection line (T line) on the membrane, due to the specific force of the antibody on the T line on the other epitope of the antigen, a double antibody sandwich immune reaction structure of microsphere-antibody-antigen-antibody (on the T line) is formed on the T line, and the excess fluorescent microsphere-antibody complex and a small amount of fluorescent microsphere-antibody-antigen complex continue to chromatograph on the membrane, which will be captured by the quality control line (C line). Then, the fluorescence detection system is used to scan the enriched fluorescence intensity of the T line and the C line, and the concentration of the antigen to be detected in the sample is obtained after certain analysis and calculation.

[0112] The present invention provides a reagent strip with an indication area, and the indication area is provided with a visually visible indication line. The indication line area can be mainly formed by some colored dyes, such as Ponceau red, etc. After these dyes are dissolved in a certain solvent, the coordinated detection line and the quality control line are coated on a solid phase carrier such as a nitrocellulose membrane through a film spray gold instrument. Colored dyes of a certain concentration, such as Ponceau red and other dyes, have a high degree of visual recognition and can play a good indicating role; during the use of the reagent strip, the indicator lines formed by these dyes will gradually weaken the chromaticity until they disappear in a short time (generally within 1 to 3 minutes) as the solution chromatographically acts, thus playing a distinguishing role. And the sample amount required for the detection of the reagent strip of this method (about 60 μL of sample for one detection) is sufficient to wash away the dye on the indicator line, and there is no need to add additional solvents or diluents for washing away the dye.

[0113] Typically, the present invention provides a lateral flow test strip, such as Figure 1 and Figure 2 As shown, the reagent strip comprises: an indication area 43 (the indication area is provided with an indication line I line coated with a colored mixture) provided on the antibody carrying film 4 and used to indicate whether the reagent strip has been used, and the antibody carrying film is further provided with a detection area 41 (the detection area is provided with a detection line T line) and a quality control area 42 (the quality control area is provided with a quality control line C line);

[0114] The reagent strip further comprises a sample pad 2, a conjugate pad 3 (the conjugate pad is paved with a fluorescent microsphere-antibody complex), and absorbent paper 5 for absorbing excess sample.

[0115] Preferably, the reagent strip of the present invention further comprises a base plate 1 ; a sample pad 2 , a conjugate pad 3 , an antibody-carrying membrane 4 and a blotting paper 5 are sequentially arranged on the base plate 1 .

[0116] In the present invention, the base plate can be made of any stable, non-porous known material, and its strength should be sufficient to support the sample pad, conjugate pad, antibody-carrying membrane and / or absorbent paper adhered thereto. Preferably, the base plate is substantially impermeable. In a preferred embodiment, the base plate is made of a polyvinyl chloride film, and preferably the base plate is a PVC base plate.

[0117] In the present invention, the sample pad can be made of any known absorbent material. Examples of usable materials include cellulose, cellulose nitrate, cellulose acetate, glass fiber, nylon, polyelectrolyte ion exchange membrane, propylene copolymer / nylon, and polyethersulfone.

[0118] In the present invention, the conjugate pad or antibody-carrying membrane can be made of any known material, as long as the material has enough porosity to allow the capillary action of fluid to occur on the surface and inside. The conjugate pad or antibody-carrying membrane should have enough porosity to allow the particles coated with antibodies or antigens to move. The conjugate pad or antibody-carrying membrane can also be wetted by the liquid used in the sample containing the analyte to be detected (for example, having hydrophilicity for aqueous liquids and having hydrophobicity for organic solvents). The material examples that can be used to make the conjugate pad or antibody-carrying membrane include: polyester film, cellulose, nitrocellulose, cellulose acetate, glass fiber, nylon, polyelectrolyte ion exchange membrane, propylene copolymer / nylon and polyethersulfone. In a preferred embodiment, the conjugate pad is made of polyester film, and the antibody-carrying membrane is made of nitrocellulose (NC).

[0119] In the present invention, the absorbent pad can be made of any known material that can absorb liquids as samples and buffers. The absorbent pad should have a large enough absorption capacity to absorb the liquid added to the test piece. Examples of materials suitable for the absorbent pad include cellulose, glass fiber, and thick absorbent paper.

[0120] Detection Methods

[0121] In a specific embodiment, a method for testing using the reagent strip of the present invention is provided: take out the test card (including the reagent strip of the present invention), add the sample from the sample loading port after dilution (add about 60 μL of the diluted liquid sample, i.e., the sample), chromatograph the diluted liquid sample (i.e., the sample) to the antibody microsphere binding pad, remelt the antibody microspheres in the antibody microsphere binding pad from the binding pad, and then the formed antigen-antibody microsphere complex is chromatographed to the antibody membrane under capillary action, and the fluorescence intensity enriched in the T line and C line regions is detected by a fluorescence quantitative detection system. A single test usually takes 5 to 10 minutes (and it is expected that the time required for a single test is as short as possible, such as within 10 minutes, such as 5 minutes).

[0122] The main advantages of the present invention include:

[0123] 1) The indicator structure prepared by the present invention has a high degree of recognizability and can play a good indicating role without the aid of additional instruments or equipment;

[0124] 2) The indicator line of the present invention has good stability. Even if it is exposed to air, it will not diffuse or fade in color for at least 3 days, and the gray value of the indicator line will not be significantly reduced.

[0125] 3) The indicator line of the present invention has high sensitivity, and a trace amount of sample is sufficient to completely or substantially completely fade the indicator line, thereby indicating whether the reagent strip has been used.

[0126] 4) The colored dye used in the present invention is a chemical substance that is easily available, low in cost and non-toxic.

[0127] 6) The preparation method of the reagent strip of the present invention is simple and can be realized by only using a common film-scribing and gold-spraying instrument, and can be carried out simultaneously with the scribing of the test line and the quality control line.

[0128] 7) The indication area formed on the membrane by the colored mixture after spraying or coating is uniform and has a small diffusion degree.

[0129] 8) The indicator line of the present invention will not affect the test results of the reagent strip.

[0130] 9) The reagent strip of the present invention has wide applicability, does not require special processing of the sample (only the processing required for normal detection), and can be widely used in lateral flow chromatography platforms.

[0131] 10) Indicates the required time period. It can indicate whether the reagent strip has been used within the time range required for routine testing (such as 5-10 minutes).

[0132] In addition, the reagent strips of the present invention having the preferred indicator line (or indicator structure) (such as the reagent strips prepared in Examples 1-5) can be clearly distinguished from unused reagent strips (the indicator line disappears or only faintly remains) even under conditions of a shorter reaction time (or detection time) (for example, the required reaction time is only 5 minutes), thereby enabling the reagent strips of the present invention to be used in detection systems requiring shorter detection times.

[0133] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for the unrecorded specific conditions in the following examples are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight / volume percentages and weight / volume parts.

[0134] Unless otherwise specified, in each embodiment and comparative example, about 60 μL of sample was added from the sample injection port, and the changes in the indicator line of the reagent strip before and after use were observed to examine the effects of different indicator lines.

[0135] The samples used were blood, plasma, serum or any buffer.

[0136] Example 1 Preparation of lateral flow test strips

[0137] 1) Preparation of lateral flow chromatography reagent strips

[0138] ① Preparation of antibody-carrying membrane 4

[0139] a) Preparation of T-line coating solution: dilute a mouse anti-monoclonal antibody to 2 mg / mL in 0.01M PBS, pH 7.3, and store at 2-8°C for later use;

[0140] b) Preparation of C-line coating solution: Dilute a mouse anti-antibody polyclonal antibody to 1 mg / mL with pH 7.3, 0.01M PBS diluent and store at 2-8℃ for use.

[0141] c) Preparation of coating solution for I-line indicator area: dilute Ponceau Red to 5 mg / mL with 0.01 M PBS, pH 7.3, containing 5% (w / v) sucrose and store at 2-8°C for later use.

[0142] d) Coating lines: The T-line coating solution, C-line coating solution and I-line coating solution are coated onto the NC membrane simultaneously by a film-scratch gold spraying apparatus, and then the coated NC membrane is dried in a quasi-vacuum environment for 2 hours.

[0143] ② Preparation of antibody microsphere binding pad 3: After the carboxyl groups of the fluorescent microspheres are activated by EDC / NHS, another mouse anti-D-Dimer monoclonal antibody is added at a mass ratio of 10:1. The amino groups on the antibody can be coupled with the activated carboxyl groups on the microspheres to form a coupling labeling complex of the fluorescent microspheres and the antibody; the antibody microsphere complex is diluted 50 times with a pH 7.3, 0.01M PBS solution containing 0.6% (w / v) BSA, and evenly spread on the binding pad, and dried in a vacuum-like manner for 2 hours.

[0144] ③Assemble the reagent strip: Figure 2 and Figure 3 As shown, the sample pad, antibody microsphere binding pad, antibody membrane and absorbent paper are sequentially pasted and assembled on the PVC base plate. After the assembly is completed, a strip cutter is used to cut it into a reagent strip with a width of 3.2 mm, which is then loaded into the reagent strip and pressed into a shell to prepare a test card.

[0145] like Figure 4 As shown, before use, the reagent strip of the present invention is as follows Figure 4As shown in a), there is a clear visual indicator line. After the test is completed, the reagent strip of the present invention is as follows Figure 4 b) The indicator line disappears, Figure 4 About 60 μL of sample was added, and the reaction time (i.e., the time for running the plate after adding the sample) was about 10 min.

[0146] Example 2

[0147] The preparation method is basically the same as in Example 1, except that the step of preparing the coating solution of the line indicator region c) is changed to:

[0148] Dilute Ponceau to 10 mg / mL with 0.01 M PBS containing 5% (w / v) sucrose, pH 7.3, and store at 2-8°C for later use.

[0149] Example 3

[0150] The preparation method is basically the same as in Example 1, except that the step of preparing the coating solution of the line indicator region c) is changed to:

[0151] Ponceau red was diluted to 5 mg / mL using PBS diluent at pH 7.3, 0.01 M containing (a) 10% (w / v) or (b) 20% (w / v) sucrose and stored at 2-8°C for later use.

[0152] Example 4

[0153] The preparation method is basically the same as in Example 1, except that the step of preparing the coating solution of the line indicator region c) is changed to:

[0154] (a) diluting Ponceau S to 5 mg / mL with 0.01 M PBS, pH 7.3, containing 5% (w / v) trehalose and incubating at 2-8°C for later use; or

[0155] Change to: (b) dilute Ponceau S to 5 mg / ml with PBS diluent (pH 7.3, 0.01 M) containing 2.5% (w / v) dextran and store at 2-8°C for later use.

[0156] Example 5

[0157] The preparation method is basically the same as in Example 1, except that the step of preparing the coating solution of the line indicator region c) is changed to:

[0158] Ponceau red was diluted to 5 mg / mL with PBS diluent (pH 7.3, 0.01 M) containing (a) 0.1% (w / v), (b) 1% (w / v), or (c) 5% (w / v) polyvinylpyrrolidone (PVP) and stored at 2-8°C for later use.

[0159] Example 6

[0160] The preparation method is basically the same as in Example 1, except that the step of preparing the coating solution of the line indicator region c) is changed to:

[0161] Ponceau red was diluted to 5 mg / mL with PBS diluent (pH 7.3, 0.01 M) containing (a) 0.1% (w / v), (b) 1% (w / v), or (c) 5% (w / v) polyethylene glycol 8000 (PEG8000) and stored at 2-8°C for later use.

[0162] The test strips were prepared with PEG8000 diluents of 0.1% (w / v) and 1% (w / v) concentrations. After the sample was added, the sample was chromatographed toward the absorbent paper. 5 to 10 minutes after the sample was added, it was found that there was still a faint residue on the indicator line. Figure 7 . 10 minutes after adding the sample, there is no residue on the indicator line.

[0163] The test strips were prepared with a 5% (w / v) diluent of PEG8000. After the sample was added, the sample was chromatographed toward the absorbent paper. More than 10 minutes after the sample was added, the indicator line still had a clear residue. Figure 6 .

[0164] Example 7

[0165] The preparation method is basically the same as in Example 1, except that the step of preparing the coating solution of the line indicator region c) is changed to:

[0166] Ponceau red was diluted to 5 mg / mL with PBS diluent (pH 7.3, 0.01 M) containing (a) 0.1% (w / v), (b) 1% (w / v), or (c) 5% (w / v) bovine serum albumin (BSA) and stored at 2-8°C for later use.

[0167] The test strip prepared in Example 7 was used for testing. After the sample was added, the sample was chromatographed toward the absorbent paper. 5 to 10 minutes after the sample was added, there was still a slight residue on the indicator line. 10 minutes after the sample was added, there was no residue on the indicator line.

[0168] Example 8

[0169] The preparation method is basically the same as in Example 1, except that the step of preparing the coating solution of the line indicator region c) is changed to:

[0170] Ponceau red was diluted to 5 mg / mL with PBS diluent (pH 7.3, 0.01 M) containing (a) 0.1% (w / v), (b) 1% (w / v), or (c) 5% (w / v) glycerol and stored at 2-8°C for later use.

[0171] The test was performed using the reagent strip prepared in Example 8. After the sample was added, the sample was chromatographed in the direction of the absorbent paper. 5 to 10 minutes after the sample was added, there was still a slight residue on the indicator line. 10 minutes after the sample was added, there was no residue on the indicator line.

[0172] Example 9

[0173] The preparation method is basically the same as in Example 1, except that the step of preparing the coating solution of the line indicator region c) is changed to:

[0174] Ponceau red was diluted to 5 mg / ml using PBS diluent (pH 7.3, 0.01 M) 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.

[0175] The test was performed using the reagent strip of Comparative Example 5. After the sample was added, the sample was chromatographed toward the absorbent paper. Ten minutes after the sample was added, there was still a faint residue on the indicator line.

[0176] Example 10

[0177] The preparation method is basically the same as in Example 1, except that the step of preparing the coating solution of the line indicator region c) is changed to:

[0178] Ponceau S was diluted to 2.5 mg / ml using 0.01 M PBS containing 2.5% (w / v) DMF.

[0179] The test strip prepared in Comparative Example 4 was used for testing. It was found that the sample volume required for a single test was insufficient to wash away the indicator line more than 10 minutes after the sample was added, and the indicator line still remained clear after 15 minutes. Figure 5 .

[0180] Comparative Example 1: Traditional fluorescent immunochromatography product reagent strip (without indicator area)

[0181] The preparation method is basically the same as in Example 1, except that the coating of line I is not performed.

[0182] like Figure 3 As shown, before using the traditional fluorescent reagent strip (such as Figure 3 a) and after use (as shown in Figure 3 b) No obvious difference is visible.

[0183] Comparative Example 2

[0184] The preparation method is basically the same as in Example 1, except that the step of preparing the coating solution of the line indicator region c) is changed to:

[0185] Dilute Ponceau S to 5 mg / mL with PBS diluent (pH 7.3, 0.01 M) and store at 2-8°C for later use.

[0186] After coating the I-line coating solution onto the NC membrane, the indicator line was found to spread.

[0187] Comparative Example 3

[0188] The preparation method is basically the same as Example 1, except that the step of preparing the coating solution of the line indicator region c) is changed to: dilute Ponceau red to 2.5 mg / ml and 1 mg / ml respectively with PBS diluent (pH 7.3, 0.01 M) and store at 2-8°C for later use.

[0189] After coating the NC membrane with I-line coating solutions containing different concentrations of Ponceau red, it was found that the colors of the indicator lines were all light and could hardly play an indicating role.

[0190] Table A lists the parameters and usage of the indicator lines in various embodiments and comparative examples:

[0191] Table A

[0192]

[0193]

[0194] Unless otherwise specified, the indicator line of the reagent strip prepared in each embodiment of the present invention will not diffuse or fade in color within at least 3 days when exposed to air, and the gray value of the indicator line will not decrease significantly.

[0195] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A fluorescent immunochromatography reagent strip, characterized in that: The reagent strip comprises: a sample pad (2), a conjugation pad (3), an antibody-carrying membrane (4) and absorbent paper (5); the sample pad, the conjugation pad, the antibody-carrying membrane and the absorbent paper are arranged in sequence; and the antibody-carrying membrane comprises: a detection area (41), a quality control area (42), and an indication area (43) for indicating whether the reagent strip has been used; and the detection area (41), the quality control area (42) and the indication area (43) are arranged in sequence; and the indication area is located at one end of the antibody-carrying membrane close to the absorbent paper; The indicator area is provided with an indicator line coated with a colored mixture, namely, an I line, the detection area is provided with a detection line, namely, a T line, and the quality control area is provided with a quality control line, namely, a C line; The colored mixture is a mixture comprising a first component and a second component, wherein the first component is a colored dye, and the second component is selected from: polyhydroxy compounds, water-soluble polymers, or a combination thereof; wherein the colored dye is Ponceau red, and the second component is selected from the following group: sucrose, trehalose, dextran, glycerol, PEG8000, PVP, and BSA, or is a combination of sucrose, BSA, glycerol, PEG8000 and PVP.

2. The fluorescent immunochromatography reagent strip according to claim 1, characterized in that: The colored mixture is a mixture consisting of a first component and a second component.

3. The fluorescent immunochromatography reagent strip according to claim 1, characterized in that: In the colored mixture, the mass ratio of the colored dye to the second component is 1:(0.1-100).

4. The fluorescent immunochromatography reagent strip according to claim 1, characterized in that: In the colored mixture, the mass ratio of the colored dye to the second component is 1:(0.1-50).

5. The fluorescent immunochromatography reagent strip according to claim 1, characterized in that: In the colored mixture, the mass ratio of the colored dye to the second component is 1:(0.2-50).

6. The fluorescent immunochromatography reagent strip according to claim 1, characterized in that: In the colored mixture, the mass ratio of the colored dye to the second component is 1:(0.2-40).

7. The fluorescent immunochromatography reagent strip according to claim 1, characterized in that: When the second component is a polyhydroxy compound, in the colored mixture, the mass ratio of the colored dye to the polyhydroxy compound is 1:(0.1-50); wherein the polyhydroxy compound is selected from the following group: sucrose, trehalose, or glycerol.

8. The fluorescent immunochromatography reagent strip according to claim 1, characterized in that: When the second component is a polyhydroxy compound, in the colored mixture, the mass ratio of the colored dye to the polyhydroxy compound is 1:(5-40); wherein the polyhydroxy compound is selected from the following group: sucrose, trehalose, or glycerol.

9. The fluorescent immunochromatography reagent strip according to claim 1, characterized in that: When the second component is a polyhydroxy compound, in the colored mixture, the mass ratio of the colored dye to the polyhydroxy compound is 1:(5-20); wherein the polyhydroxy compound is selected from the following group: sucrose, trehalose, or glycerol.

10. The fluorescent immunochromatography reagent strip according to claim 1, characterized in that: When the second component is a polyhydroxy compound, in the colored mixture, the mass ratio of the colored dye to the polyhydroxy compound is 1:(10±2); wherein the polyhydroxy compound is selected from the following group: sucrose, trehalose, or glycerol.

11. The fluorescent immunochromatography reagent strip according to claim 1, characterized in that: When the second component is a water-soluble polymer, in the colored mixture, the mass ratio of the colored dye to the water-soluble polymer is 1:(0.1-20); wherein the water-soluble polymer is selected from the following group: dextran, BSA, PVP or PEG8000.

12. The fluorescent immunochromatography reagent strip according to claim 1, characterized in that: When the second component is a water-soluble polymer, in the colored mixture, the mass ratio of the colored dye to the water-soluble polymer is 1:(0.2-10); wherein the water-soluble polymer is selected from the following group: dextran, BSA, PVP or PEG8000.

13. The fluorescent immunochromatography reagent strip according to claim 1, characterized in that: When the second component is a combination of sucrose, BSA, glycerol, PEG8000 and PVP, in the colored mixture, the mass ratio of the colored dye to the polyhydroxy compound is 1:(5-50); and the mass ratio of the colored dye to the water-soluble polymer is 1:(0.1-20); wherein the polyhydroxy compound is a combination of sucrose and glycerol; and the water-soluble polymer is selected as a combination of BSA, PVP and PEG8000.

14. The fluorescent immunochromatography reagent strip according to claim 1, characterized in that: When the second component is a combination of sucrose, BSA, glycerol, PEG8000 and PVP, in the colored mixture, the mass ratio of the colored dye to the polyhydroxy compound is 1:(5-50); and the mass ratio of the colored dye to the water-soluble polymer is 1:(0.2-10); wherein the polyhydroxy compound is a combination of sucrose and glycerol; and the water-soluble polymer is selected as a combination of BSA, PVP and PEG8000.

15. The fluorescent immunochromatography reagent strip according to claim 1, characterized in that: The second component is selected from the group consisting of sucrose, trehalose, dextran, PVP, or a combination thereof; and the mass ratio of the colored dye to the second component is 1:(0.2-40).

16. The fluorescent immunochromatography reagent strip according to claim 1, characterized in that: (i) the binding pad is a binding pad on which a fluorescent microsphere-first antibody complex is laid; (ii) the test line is a test line coated with a second antibody; and (iii) The quality control line is provided with a quality control line coated with a third antibody.

17. A method for preparing the fluorescent immunochromatography reagent strip according to claim 1, characterized in that: The method comprises the steps of: (1) providing an antibody-carrying membrane including an indication region with an indication line, a detection region with a detection line, and a quality control region with a quality control line; Wherein, the indicator line is prepared by the following method: (i) providing an I-line coating solution, wherein the I-line coating solution is a coating solution containing a color mixture; and (ii) coating the colored mixture in the line coating solution onto the antibody-carrying membrane to form an indicator line; (2) Assembling a sample pad, a conjugate pad, an antibody-carrying membrane and absorbent paper to obtain the reagent strip.

18. The method according to claim 17, characterized in that The I-line coating solution is obtained by diluting the first component with a diluent containing the second component until the concentration of the first component in the coating solution is c1, and c1=3-20 mg / ml; and in the diluent containing the second component, the concentration of the second component is 0.5-250 mg / ml of the diluent.

19. A reagent card, characterized in that: The reagent card comprises: the fluorescent immunochromatography reagent strip as described in claim 1.

20. A use of the fluorescent immunochromatography reagent strip as claimed in claim 1 or the reagent card as claimed in claim 19, characterized in that: Used to detect a target substance, and the target substance is an antigen.

21. A detection method, characterized in that: Includes steps: (1) contacting a sample to be tested with a sample pad (2) of the fluorescent immunochromatography reagent strip as claimed in claim 1; (2) allowing the sample to be tested to pass through the antibody-supporting membrane through chromatography, and causing a mark indicating that the sample has been used to appear in the indicator area; and (3) analyzing the used reagent strips through the detection system; Wherein, the sample to be tested is a liquid that may contain the target substance.

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