A high-sensitivity detection reagent strip, preparation method and application thereof

Abstract

The present invention relates to the technical field of allergen detection, and discloses a high-sensitivity detection reagent strip, a preparation method, and its application, comprising a shell, the shell comprising at least a sample addition hole, a liquid addition hole, and a display area; a lateral flow test strip is provided inside the shell; the lateral flow test strip comprises a bottom structure, the bottom structure is provided with a marker flow area and a sample flow area; wherein the marker flow area is located above or below the sample flow area; the sample flow area and the marker flow area each comprise at least one sample pad, and a plurality of the sample flow areas and the marker flow areas are physically isolated from each other. The beneficial effects of the present invention are: it can realize combined immunoassay and quantitative detection, solves the problem that the traditional mode cannot be quantified or is too time-consuming, and further increases the sensitivity of the product due to the increase in the sample incubation process.

Description

Technical Field

[0001] The present invention relates to the technical field of detection reagent strips, and in particular to a high-sensitivity detection reagent strip, a preparation method and applications thereof. Background Art

[0002] Allergic diseases are a major global health concern, with morbidity and mortality rates increasing annually, reaching epidemic proportions. Allergen detection methods generally fall into two categories: prick test and serum-specific IgE testing. The latter has a history of over 40 years. The domestic allergen testing market is dominated by three companies: Mediwiss (Germany), Euroimmun (Germany), and Thermo Fisher (USA).

[0003] At present, the methods used in in vitro allergen detection kits at home and abroad, such as enzyme-linked immunosorbent assay, immunoblotting, fluorescent immunoassay, and allergen microarray chip method, have many limitations.

[0004] 1. The sample type is single and can only test serum and plasma; whole blood and fingertip blood cannot be used, which is not suitable for children and emergency mode;

[0005] 2. The detection speed is slow, and it takes 2-4 hours;

[0006] 3. Liquid reagents, the product needs to be refrigerated for storage and transportation;

[0007] 4. High cost of reagents and instruments;

[0008] 5. Unable to remove CCD IgE interference in the sample. Summary of the Invention

[0009] The present invention provides a high-sensitivity detection reagent strip, a preparation method, and its application. Multiple test results can be obtained simultaneously with a single sample loading, thus realizing rapid quantitative detection. The sample volume is small, and whole blood and fingertip blood can be used. The problem of non-quantification in the serial detection process is solved, thus realizing quantitative detection. Compared with the traditional detection mode, the incubation process is added, thereby improving the detection sensitivity.

[0010] To this end, the technical solution adopted by the present invention is as follows: a high-sensitivity detection reagent strip, comprising a shell, the shell including at least a sample addition hole, a liquid addition hole and a display area; a lateral flow test strip is arranged inside the shell; the lateral flow test strip includes a bottom structure; a marker flow area and a sample flow area are provided on the bottom structure, wherein the marker flow area is located above or below the sample flow area; the sample flow area and the marker flow area each include at least one sample pad, and there is physical isolation between the multiple sample flow areas and marker flow areas.

[0011] By adopting the above technical solution: the present invention includes at least one sample addition hole for adding samples; and a liquid addition hole for promoting the release of markers; the positions of the two holes can be adjusted forward and backward according to the structure of the test strip, which can achieve quantitative detection of allergens, solving the problem of the traditional model that cannot be quantified, and because the sample incubation process is added, the sensitivity of the product is further increased.

[0012] Preferably, the housing comprises a bottom shell and an upper shell that are engaged with each other; the sample addition hole, liquid addition hole, and display area are provided on the upper shell, the lateral flow chromatography test strip is fixed between the bottom shell and the upper shell, and the lateral flow chromatography test strip is arranged as a whole at an angle from the liquid addition hole to the display area;

[0013] The lateral flow test strip is fixed between the bottom shell and the upper shell, and the lateral flow test strip is arranged as a whole from the liquid filling hole to the display area.

[0014] A linkage mechanism is provided between the bottom shell and the upper shell of the housing, wherein the linkage mechanism includes a pushing component provided on the upper shell, a driven component provided on the bottom shell, and a linkage component provided between the bottom shell and the upper shell;

[0015] The bottom shell and the upper shell are rotatably connected via a snap-on ear and a snap-on shaft;

[0016] The pushing assembly includes a sliding groove provided on the upper shell and a pushing block sliding in the sliding groove;

[0017] The driven component includes a driven block provided on the bottom shell and having a driven inclined surface;

[0018] The linkage assembly includes a pushing rod connected to the pushing block, a sliding cavity block at the front end of the pushing rod, a sliding block with an inclined surface connected to the interior of the sliding cavity block through an elastic member, a clamping block with an inclined surface is fixedly provided on the side wall of the sliding groove, the inclined directions of the sliding block and the clamping block cooperate with each other, and a pushing rod is provided at the lower part of the pushing block for contacting and cooperating with the passive block.

[0019] By adopting the above technical solution: when conducting the test, by pushing the pushing block, the push rod at the bottom of the pushing block can push the bottom shell to flip a certain angle, wherein the pushing block is arranged on the side opposite to the sample addition hole and the liquid addition hole, so as to facilitate the pouring of the sample. By setting the clamping block and the sliding block, the inclination angle of the bottom shell can be adjusted according to the detection requirements, which solves the problem of slow pouring caused by the flat placement of the reagent strip in the prior art, and the problem of inaccurate manual holding.

[0020] Preferably, the bottom structure includes a bottom plate, and the bottom plate is made of PVC;

[0021] The sample flow area includes absorbent paper arranged at one end of the bottom plate, an NC membrane arranged on the bottom plate, the absorbent paper overlapped on the NC membrane, a sample pad assembly arranged at the other end of the bottom plate, the sample pad assembly overlapped on the NC membrane, and one or more detection lines and quality control lines are sequentially arranged on the NC membrane;

[0022] The marker flow area includes a physical isolation layer arranged on the upper part of the sample pad component 1, a sample pad component 2 is arranged on the upper part of the physical isolation layer, the sample pad component 2 is overlapped with a binding pad, and the binding pad is overlapped on the NC membrane;

[0023] or:

[0024] The marker flow area includes a physical isolation layer arranged on the upper part of the sample pad component 1, a sample pad component 2 is arranged on the upper part of the physical isolation layer, the sample pad component 2 contains an immune marker, and the sample pad component 2 is overlapped on the NC membrane;

[0025] The sample pad component 1 at least includes a sample pad 1, and the sample pad component 2 at least includes a sample pad 2;

[0026] The NC membrane is a nitrocellulose membrane.

[0027] Preferably, the bottom structure includes a bottom plate;

[0028] The marker flow area includes absorbent paper arranged at one end of the bottom plate, an NC membrane arranged on the bottom plate, the absorbent paper overlapped on the NC membrane, and a sample pad second component arranged at the other end of the bottom plate, the sample pad second component overlapped on the conjugate pad, and the conjugate pad overlapped on the NC membrane;

[0029] or:

[0030] The marker flow area includes absorbent paper arranged at one end of the bottom plate, an NC membrane arranged on the bottom plate, the absorbent paper overlapped on the NC membrane, and a sample pad second component arranged at the other end of the bottom plate, the sample pad second component contains an immune marker, and the sample pad second component overlapped on the NC membrane;

[0031] The sample flow area includes a physical isolation layer arranged on the upper portion of the sample pad second component, a sample pad first component is arranged on the upper portion of the physical isolation layer, and the sample pad first component is overlapped on the NC membrane;

[0032] The sample pad component 1 at least includes a sample pad 1, and the sample pad component 2 at least includes a sample pad 2;

[0033] The NC membrane is a nitrocellulose membrane.

[0034] In a second aspect, the present invention provides a method for preparing a high-sensitivity detection reagent strip, comprising the following steps:

[0035] S1, prepare sample flow area;

[0036] S2, preparing a marker flow area;

[0037] S3, preparing NC membrane;

[0038] S4, assembling a lateral flow test strip;

[0039] S5. Attach the lateral flow test strip from step S4 into the housing.

[0040] Preferably, the preparation method in step S1 is as follows:

[0041] The sample pad 1 in the sample flow area is composed of one or more glass fibers or polyester membrane materials with blood filtering function, and the sample pad 1 is treated with a sample pad treatment liquid to resist interference; the sample pad treatment liquid contains a CCD blocker, free biotin and a heterotrophic antibody blocker.

[0042] Preferably, the preparation method in step S2 is as follows:

[0043] The sample pad 2 in the marker flow area is made of one or more glass fibers or polyester film materials, and the material contains immune markers.

[0044] Preferably, the preparation method of the NC membrane is as follows:

[0045] Dilute goat anti-chicken Ig Y antibody to 0.5 mg / mL using coating buffer as the quality control coating solution;

[0046] Use coating buffer to dilute the protein to be coated to 1.0 mg / mL as the test line coating solution;

[0047] Attach the NC film to the base plate, ensuring that the bottom edge is close to the separation line of the bonding pad;

[0048] Use an XYZ three-dimensional film spraying instrument to evenly coat the test line and quality control line coating liquid on the NC membrane.

[0049] Preferably, the lateral flow test strip is assembled as follows:

[0050] Glue the NC membrane to the base plate and attach absorbent paper to one end of the NC membrane. Attach the sample pad assembly 1 to the other end of the NC membrane, attach a physical isolation layer above the sample pad 1, and attach the sample pad assembly 2 above the physical isolation layer.

[0051] Preferably, the lateral flow test strip is assembled as follows:

[0052] The NC membrane was bonded to the bottom plate, and absorbent paper was attached to one end of the NC membrane; the sample pad 2 component was attached to the other end of the NC membrane in sequence, a physical isolation layer was attached above the sample pad 2, and the sample pad 1 component was attached to the physical isolation layer in sequence.

[0053] Preferably, the clamping method in step S5 is as follows:

[0054] Use a strip cutting machine to cut the assembled lateral flow test strips into 0.2-0.8 mm strips, open the housing, fix the strips on the bottom housing, and snap the upper housing onto the bottom housing to complete the snap connection.

[0055] Preferably, the upper shell contains two loading holes, namely the sample flow area loading hole S1 and the marker flow area loading hole S2; during detection, the sample is first added to the S1 hole to complete the binding of the sample and the coated protein on the NC membrane. After the reaction is completed, the diluent is added to the S2 hole to complete the release of the immune marker.

[0056] In a third aspect, the present invention provides the use of the above-mentioned high-sensitivity detection reagent strip for the detection of allergen items and multiple combined immune tests.

[0057] The working principle and beneficial effects of the present invention are:

[0058] 1. Quantum dots are currently a type of nanoscale semiconductor crystal. The emission spectrum of quantum dots can be controlled by changing the size and chemical composition of the quantum dots, and the wavelength can cover the entire optical drive. They have good photostability and strong interference resistance. They have a large Stokes shift, a wide excitation spectrum and a narrow emission spectrum, and can achieve single-element excitation and multi-element emission, without spectral overlap between multi-color quantum dots.

[0059] 2. Compared with the traditional chemiluminescence detection mode, this study greatly reduced the amount of clinical samples used, making it more suitable for promotion in pediatrics and emergency departments (traditional testing requires 200 μL of sample for 5 items, while this study only requires 50 μL of sample for 5 items, an average of 10 μL of sample per item).

[0060] 3. Based on this study, common clinical allergens (house dust mites, house dust, cat dander, dog dander, cockroaches, Penicillium notatum, Aspergillus fumigatus, Alternaria alternata, birch, juniper, elm, sycamore, willow, poplar, fraxinus chinensis, common ragweed, mugwort, quinoa, humulus, Amaranthus retroflexus, Timothy grass, egg white, milk, wheat, sesame, peanuts, soybeans, fish, crab, shrimp, scallops, beef, and lamb) can be randomly combined and coated on the NC membrane to achieve multiple quantitative joint tests.

[0061] 4. Compared with the traditional detection mode (conventional assembly mode and 1 well), the present invention can achieve quantitative detection of allergens, solving the problem of the traditional mode that cannot be quantified. In addition, due to the addition of the sample incubation process, the sensitivity of the product is further increased. It is not only suitable for the quantitative detection of allergen IgE, but also for the combined detection of various types of immune types.

[0062] 5. Adding CCD blocker to the sample pad can eliminate false positives caused by CCD and identify the true allergen. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0064] Figure 1 This is a schematic diagram of the surface structure of the upper shell of the first embodiment of the present invention;

[0065] Figure 2 This is a schematic diagram of the internal structure of the upper shell and the bottom shell after they are matched together in the first embodiment of the present invention;

[0066] Figure 3 For the first embodiment of the present invention Figure 1 A in the figure shows the enlarged structural diagram;

[0067] Figure 4 For the first embodiment of the present invention Figure 2 A schematic diagram of the structure at point B in FIG.

[0068] Figure 5 This is a schematic diagram of the cross-sectional structure of a lateral flow test strip according to Example 1 of the present invention;

[0069] Figure 6 Schematic diagram of the three-dimensional structure of the lateral flow test paper according to Example 1 of the present invention;

[0070] Figure 7 Schematic diagram of the cross-sectional structure of a lateral flow test paper according to Example 2 of the present invention;

[0071] Figure 8 This is a flowchart of the preparation method of Example 1 of the present invention.

[0072] The technical features in the figure are marked as follows:

[0073] 100, shell; 110, bottom shell; 120, upper shell; 200, sample addition hole; 300, liquid addition hole; 400, display area; 500, lateral flow test strip; 510, marker flow area; 520, sample flow area; 521, bottom plate; 522, absorbent paper; 523, NC membrane; 531, sample pad component 1; 532, sample pad component 2; 533, binding pad; 540, physical isolation layer; 550, detection line; 560, quality control line; 600, linkage mechanism; 610, snap-fit ​​ear; 620, snap-fit ​​shaft; 630, sliding groove; 640, pushing block; 641, pushing rod; 642, sliding cavity block; 643, sliding block; 650, driven block; 660, snap-fit ​​block; 670, pushing rod. DETAILED DESCRIPTION

[0074] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0075] Example 1:

[0076] like Figure 1 As shown, the present invention provides a high-sensitivity detection reagent strip, including a shell 100, which is supported by injection molding and includes at least a sample addition hole 200, a liquid addition hole 300 and a display area 400; a lateral flow test strip 500 is provided inside the shell 100; the lateral flow test strip 500 includes a bottom structure, and a marker flow area 510 and a sample flow area 520 are provided on the bottom structure; the marker flow area 510 is located on the upper side or the lower side of the sample flow area 520; the sample flow area 520 and the marker flow area 510 each include at least one sample pad, and multiple sample flow areas 520 and marker flow areas 510 are physically isolated from each other.

[0077] The present invention includes at least one sample addition hole 200 for adding samples; and a liquid addition hole 300 for promoting the release of markers. The positions of the two holes can be adjusted forward and backward according to the structure of the test strip, which can achieve quantitative detection of allergens, solving the problem of the traditional model that cannot be quantified. Moreover, due to the increase in the sample incubation process, the sensitivity of the product is further increased.

[0078] The housing 100 includes a bottom housing 110 and an upper housing 120 that are snap-fitted to each other. Both the bottom housing 110 and the upper housing 120 are injection molded. The sample addition port 200, the liquid addition port 300, and the display area 400 are disposed on the upper housing 120. The lateral flow test strip 500 is secured between the bottom housing 110 and the upper housing 120. The bottom housing 110 and the upper housing 120 are snap-fitted to facilitate assembly. The display area 400 on the upper housing 120 is used to display test results for easy observation.

[0079] The lateral flow test strip 500 is fixed between the bottom shell 110 and the upper shell 120, and the lateral flow test strip is tilted from the liquid filling hole 300 to the display area 400;

[0080] A linkage mechanism 600 is provided between the bottom shell 110 and the upper shell 120 of the housing 100. The linkage mechanism 600 includes a pushing component provided on the upper shell 120, a driven component provided on the bottom shell 110, and a linkage component provided between the bottom shell 110 and the upper shell 120.

[0081] The bottom shell 110 and the upper shell 120 are rotatably connected via a snap-in ear 610 and a snap-in shaft 620;

[0082] The pushing assembly includes a sliding groove 630 formed on the upper shell 120 and a pushing block 640 sliding in the sliding groove 630;

[0083] The actuated assembly includes an actuated block 650 provided on the bottom housing 110 and having an actuated inclined surface;

[0084] The linkage assembly includes a pushing rod 641 connected to the pushing block 640, a sliding cavity block 642 at the front end of the pushing rod 641, a sliding block 643 with an inclined surface connected to the interior of the sliding cavity block 642 through an elastic member, a clamping block 660 with an inclined surface fixedly provided on the side wall of the sliding groove 630, the sliding block 643 and the inclined direction of the clamping block 660 cooperate with each other, and a pushing rod 670 is provided at the lower part of the pushing block 640 for contact and cooperation with the passive block 650.

[0085] During the test, by pushing the push block 640, the push rod 670 at the bottom of the push block 640 can push the bottom shell 110 to flip to a certain angle, wherein the push block 640 is arranged on the side opposite to the sample addition hole 200 and the liquid addition hole 300, so as to facilitate the pouring of the sample. By setting the snap-on block 660 and the sliding block 643, the tilt angle of the bottom shell 110 can be adjusted according to the test requirements, which solves the problem of slow pouring caused by the reagent strip being placed flat in the prior art, and the problem of inaccurate manual holding.

[0086] Reference Figure 2 and Figure 3The bottom structure in this embodiment includes a bottom plate 521; the bottom plate 521 is made of PVC material, wherein the sample flow area 520 includes absorbent paper 522 arranged at one end of the bottom plate 521, an allergen protein-coated nitrocellulose membrane arranged on the bottom plate 521, the absorbent paper 522 overlaps the allergen protein-coated nitrocellulose membrane, and a sample pad 1 arranged at the other end of the bottom plate 521, the sample pad 1 overlaps the allergen protein-coated nitrocellulose membrane, and the allergen protein-coated nitrocellulose membrane is sequentially provided with a plurality of detection lines 550 and quality control lines 560; the marker flow area 510 includes an invisible tape and a rubber plate arranged on the upper part of the sample pad 1, a sample pad 2 is provided on the upper part of the rubber plate, the sample pad 2 is overlapped with a binding pad 533, and the binding pad 533 overlaps the allergen protein-coated nitrocellulose membrane.

[0087] Example 2:

[0088] This embodiment differs from the first embodiment in that the sample flow area 520 and the marker flow area 510 are arranged and combined differently. In this embodiment, the bottom structure includes a base plate 521 made of PVC. The marker flow area 510 includes absorbent paper 522 disposed at one end of the base plate 521 and an allergen protein-coated nitrocellulose membrane (NC membrane 523) disposed on the base plate 521. The absorbent paper 522 overlaps the allergen protein-coated nitrocellulose membrane. A second sample pad assembly 532 is disposed at the other end of the base plate 521 and overlaps a conjugation pad 533. The conjugation pad 533 overlaps the allergen protein-coated nitrocellulose membrane. The sample flow area 520 includes invisible tape and a rubber sheet disposed above the second sample pad assembly 532 and the conjugation pad 533. A first sample pad assembly 531 is disposed on the upper portion of the rubber sheet and overlaps the allergen protein-coated nitrocellulose membrane.

[0089] In a second aspect, the present invention provides a method for preparing the above-mentioned high-sensitivity detection reagent strip, comprising the following steps:

[0090] S1, preparing a sample flow area 520;

[0091] S2, preparing the marker flow area 510;

[0092] S3, preparing allergen protein-coated nitrocellulose membrane;

[0093] S4, assembling a lateral flow test strip 500;

[0094] S5 . Snap the lateral flow test strip 500 from step S4 into the housing 100 .

[0095] The preparation method in step S1 is as follows:

[0096] Sample pad 1: Soak the glass fiber in sample pad treatment solution 1 for 15-30 minutes, then dry it in an oven at 40-45°C for 20-30 hours.

[0097] Sample pad 2: Cut the glass fiber into strips with a width of 8-9 mm. Use a water-soluble pen to draw a blue line on the cut sample pad with uniform depth. Place it in an oven at 40-45°C to dry for 20-30 hours.

[0098] Alternatively, soak the glass fiber in sample pad treatment solution 2 (containing water-soluble pigment), place it in an oven at 40-45°C and dry it for 20-30 hours, then take it out and cut it into strips with a width of 8-9 mm.

[0099] The sample pad treatment solution 1 is a phosphate, borate, or Tris salt ion buffer containing 5%-20% anti-erythrocyte antibody, 0.1%-20% CCD blocking agent, and 0.1%-20% anti-heterophage antibody blocking agent.

[0100] The preparation method of the conjugate pad 533 is as follows:

[0101] Preparation of quantum dot-labeled anti-human IgE antibodies:

[0102] Dissolve EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) in purified water and dilute to a final concentration of 8-12 mg / mL. Dilute 1-3 mg of the anti-human IgE antibody to be labeled to 1-4 mg / mL with 0.1-0.2 mmol / L borate buffer. Add 45-55 mg of quantum dots and pipette several times. Add 150 μL of EDC solution while vortexing (600 rpm). Mix well at room temperature in the dark and react at 280 rpm for 1-3 hours. Centrifuge the quantum dot-labeled anti-human IgE antibody product, transfer to a centrifuge tube, add Solution 1 to a final volume of 1-5 mL, and mix well.

[0103] Preparation of quantum dot labeled chicken IgY antibody:

[0104] Prepare quantum dot-labeled chicken IgY according to the above steps;

[0105] The quantum dot-labeled anti-human IgE antibody and the quantum dot-labeled chicken IgY antibody were mixed in equal proportions, sprayed evenly on the glass fiber using an XYZ three-dimensional film spraying apparatus, and dried at 35-40°C.

[0106] Preferably, one component of the solution is as follows: 100 mmol / L Tris, 2% PVP-K30, 10% sucrose, 1% casein, 1% PEG 200, 1% Triton X-100, 0.1% ProClin 300, pH 8.0.

[0107] The preparation method of the allergen protein-coated nitrocellulose membrane is as follows:

[0108] Dilute goat anti-chicken Ig Y antibody to 0.5 mg / mL using coating buffer as the quality control line 560 coating solution;

[0109] Allergens were diluted to 1.0 mg / mL using coating buffer as the test line 550 coating solution;

[0110] Attach the NC film 523 to the base plate 521, ensuring that the bottom edge is close to the separation line where the bonding pad 533 is located;

[0111] The coating solutions of the test line 550 and the quality control line 560 were evenly coated on the NC membrane 523 using an XYZ three-dimensional film spraying apparatus, and dried at 35-40° C. for 6-8 hours to prepare an allergen protein-coated nitrocellulose membrane.

[0112] The composition of the coating buffer is as follows: 20mmol / L PB+1% trehalose.

[0113] The allergens of the coating buffer at least include ragweed, dog epithelium, humulus, mugwort, and cat epithelium (which are made into different detection lines 550 in turn).

[0114] The assembly method of the lateral flow test strip 500 (for the above-mentioned embodiment 1) is as follows:

[0115] The allergen protein-coated nitrocellulose membrane is bonded to the base plate 521, and absorbent paper 522 is attached to one end of the allergen protein-coated nitrocellulose membrane; sample pad 1 is attached to the other end of the allergen protein-coated nitrocellulose membrane, invisible tape is attached on top of sample pad 1, a plastic sheet is attached on top of the invisible tape, and sample pad 2 is attached to the plastic sheet in sequence.

[0116] The absorbent paper 522 covers the allergen protein-coated nitrocellulose membrane by 1.5 mm ± 0.5 mm; the ends of the allergen protein-coated nitrocellulose membrane and the sample pad 1 overlap and cover each other by 1.5 mm ± 0.5 mm and are pasted together; the binding pad 533 and the sample pad 2 pasted on the adhesive plate in sequence overlap and cover each other by 1.5 mm ± 0.5 mm.

[0117] The assembly method of the lateral flow test strip 500 (for the above-mentioned embodiment 2) is as follows:

[0118] The allergen protein-coated nitrocellulose membrane is bonded to the base plate 521, and absorbent paper 522 is attached to one end of the allergen protein-coated nitrocellulose membrane. A binding pad 533 and a second sample pad are attached to the other end of the allergen protein-coated nitrocellulose membrane in sequence, an invisible tape is attached above the second sample pad, a plastic sheet is attached above the invisible tape, and the first sample pad is attached to the plastic sheet.

[0119] The absorbent paper 522 covers the allergen protein-coated nitrocellulose membrane by 1.5 mm ± 0.5 mm; the ends of the allergen protein-coated nitrocellulose membrane, the conjugate pad 533 and the sample pad 2 overlap and cover each other by 1.5 mm ± 0.5 mm and are pasted together; the sample pad 1 pasted on the adhesive plate overlaps and covers each other by 1.5 mm ± 0.5 mm.

[0120] The clamping method in step S5 is as follows:

[0121] The assembled lateral flow test strip 500 is cut into 0.35-0.45 mm strips using a strip cutting machine. The housing 100 is opened, the strip test strip is fixed to the bottom housing 110 and the upper housing 120 is snap-fitted to the bottom housing 110 to complete the snap-fitting.

[0122] This embodiment further includes the following steps: S6, the detection system quantitatively verifies the reagent strip.

[0123] The quantitative verification method is as follows:

[0124] IgE calibrators (S0: 0 IU / mL, S1: 0.35 IU / mL, S2: 0.7 IU / mL, S3: 3.5 IU / mL, S4: 17.5 IU / mL, S5: 50 IU / mL, S6: 100 IU / mL) were prepared using the ImmuoCAP detection system, and the four-parameter curve fitted by the IgE calibrators was used in the detection system of component-specific IgE antibodies.

[0125] Optimize reaction parameters based on the sensitivity of the detection system. Dilute the sample to be tested with the sample diluent in a 1:1 volume ratio. Mix thoroughly. Pipette 90-100 μL of the diluted sample into well 200. After 5-10 minutes, add 40-50 μL of the allergen sample diluent to well 200. Incubate the plate for 15-20 minutes. Labeling agents include, but are not limited to, quantum dots, quantum dot microspheres, colloidal gold, fluorescein, and fluorescent microspheres.

[0126] The third aspect of the present invention provides an application of the above-mentioned high-sensitivity detection reagent strip for the detection of allergens, which is suitable for various types of combined immune tests. During the test, the sample is first added to the sample addition hole 200S1 of the sample flow area 520 to complete the binding of the sample and the coated protein on the NC membrane 523. After the reaction is completed, the diluent is added to the liquid addition hole 300S2 of the marker flow area 510 to complete the release of the immune marker.

[0127] In the above embodiment, the sample flow zone 520 includes, but is not limited to, a sample pad or a blood filter membrane; the sample pad and conjugate pad 533 are made of, but are not limited to, glass fiber, polyester, or other materials; the marker flow zone 510 includes, but is not limited to, a conjugate pad 533 and a sample pad; the sample flow zone 520 and the marker flow zone 510 are physically separated by a plastic sheet or similar material; the sample flow zone 520 contains a CCD blocker; the sample flow zone 520 contains a material with blood filtering properties or a sample pad treated with anti-red blood cell (RBC) antibodies; the marker flow zone 510 contains a water-soluble pigment, including, but not limited to, various colors such as blue and red; the marker, represented in an embodiment by quantum dot microspheres, includes, but is not limited to, quantum dots, quantum dot microspheres, colloidal gold, fluorescein, fluorescent microspheres, and other substances with tracer functions; the number of detection lines 550 coated on the coating membrane is adjustable, including, but not limited to, five detection lines 550; the coated substances include, but are not limited to, natural allergens, recombinant allergens, and other types of proteins.

[0128] Taking Example 1 as an example, a more specific implementation is as follows:

[0129] Sample pad pretreatment:

[0130] Sample Pad 1: Soak the glass fiber in sample pad treatment solution (containing 5%-20% anti-erythrocyte antibody and 1%-20% CCD blocking agent) for 15-30 minutes. Then dry in an oven at 45°C for 24 hours.

[0131] Sample Pad 2: Cut the glass fiber into 8.5 mm wide strips. Use a water-soluble pen to draw a blue line in the center of the cut sample pad, ensuring a uniform depth. Place the strip in a drying oven at 45°C and dry for 24 hours. Alternatively, soak the glass fiber in a sample pad treatment solution (containing a water-soluble pigment) and dry it in a drying oven at 45°C for 24 hours.

[0132] Preparation of quantum dot-labeled anti-human IgE antibodies:

[0133] Dissolve 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) in purified water and dilute to a final concentration of 10 mg / mL. Dilute 1 mg of the anti-human IgE antibody to be labeled to 2 mg / mL in 0.2 mmol / L borate buffer (pH 7.4). Add 50 mg of quantum dots and gently pipette several times. Add 150 μL of EDC solution while vortexing (600 rpm). Mix thoroughly at room temperature in the dark and incubate at 280 rpm for 2 h. The quantum dot-labeled anti-human IgE antibody product is centrifuged at high speed and transferred to a centrifuge tube. A solution (100 mmol / L Tris, 2% PVP-K30, 10% sucrose, 1% casein, 1% PEG 200, 1% Triton X-100, 0.1% ProClin 300, pH 8.0) is added to a final volume of 2 mL and mixed thoroughly.

[0134] Quantum dot-labeled chicken IgY was prepared according to the above steps. After mixing the two quantum dot markers in equal proportions, the markers were evenly sprayed on glass fiber (8 mm * 30 cm) using an XYZ three-dimensional film spraying device and dried at 37°C for use as conjugate pad 533.

[0135] Preparation of allergen protein-coated nitrocellulose membrane:

[0136] Dilute goat anti-chicken Ig Y antibody to 0.5 mg / mL in coating buffer (20 mmol / L PB + 1% trehalose) to serve as the control line (560) coating solution (C line). Dilute allergens (ragweed, dog epithelium, humulus, mugwort, and cat epithelium) to 1.0 mg / mL in coating buffer to serve as the test line (550) coating solution (T lines: T1-T5). NC membrane 523 was attached to a PVC plate, ensuring the bottom edge was in contact with the dividing line at the conjugate pad 533. Using an XYZ three-dimensional coating sprayer, the T / C line coating solution was evenly applied to the NC membrane 523. Dry at 37°C for 6-8 hours before use as the coating membrane.

[0137] Lateral Flow Test Strips 500 Assembly:

[0138] Attach the coating membrane to the PVC base plate 521, attach absorbent paper 522 to one end of the NC membrane 523, covering the NC membrane 523 by 1.5 mm; attach sample pad 1 to the other end of the NC membrane 523, overlapping by 1.5 mm, attach invisible tape on top of sample pad 1, attach a transparent plastic sheet on top of the invisible tape, attach the binding pad 533 and sample pad 2 on the transparent plastic sheet in sequence, with the coverage limit not exceeding 1.5 mm ± 0.5 mm. All components are firmly attached.

[0139] The above-mentioned set of test paper sheets is cut into test strips of about 4.0 mm using a strip cutting machine and inserted into the interior of the housing 100 to complete the preparation.

[0140] Quantitative verification of the detection system:

[0141] IgE calibrators (S0: 0 IU / mL, S1: 0.35 IU / mL, S2: 0.7 IU / mL, S3: 3.5 IU / mL, S4: 17.5 IU / mL, S5: 50 IU / mL, S6: 100 IU / mL) were prepared using the ImmuoCAP detection system, and the four-parameter curve fitted by the IgE calibrators was used in the detection system of component-specific IgE antibodies.

[0142] Explore and optimize reaction parameters based on the sensitivity of the test system. Dilute the sample to be tested with the sample diluent in a 1:1 volume ratio and mix thoroughly. Use a pipette to transfer 90-100 μL of the diluted sample to well 200 of the test card. After 5-10 minutes, add 40-50 μL of the allergen sample diluent to well 300 of the test card. Incubate the plate for 15-20 minutes.

[0143] The technical effect of this embodiment can be demonstrated by setting the following comparison results:

[0144] Two clinical samples were taken and tested using the traditional detection system and the detection system of the present invention respectively.

[0145] Traditional detection system 1: A quantum dot-labeled mouse anti-human IgE antibody was used to prepare the conjugate pad. Five T lines and one C line were coated on an NC membrane. The sample flow area and the marker flow area were combined into one. The specific results are shown in Tables 1 and 2:

[0146] Table 1:

[0147]

[0148] Table 2:

[0149]

[0150] The results showed that when the sample was multi-positive, the traditional detection system and the detection system of the present invention were used for detection respectively. The deviation between the detection result of the detection system of the present invention and the labeled value was within the range of ±15%; the deviation between the first capture detection result and the labeled value of the traditional detection system was within the range of ±15%, but the deviation between the second capture detection result and the labeled value was greater than ±15%.

[0151] Sensitivity comparison: Two clinical samples were tested using the traditional detection system and the detection system of this application. The specific results are shown in Table 3 and Table 4:

[0152] Table 3:

[0153]

[0154] Table 4:

[0155]

[0156] From the results in the above table, it can be seen that the minimum detection limit (LOD) of the traditional detection system is about 0.4 IU / mL, while the LOD of the detection system of the present invention is about 0.05 IU / mL. It can be seen that the present invention can significantly improve the sensitivity of detection.

[0157] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A high-sensitivity detection reagent strip, comprising a housing (100), characterized in that: The housing (100) comprises at least a sample addition hole (200), a liquid addition hole (300), and a display area (400); A lateral flow test strip (500) is provided inside the housing (100); The lateral flow test strip (500) comprises a bottom structure, on which a marker flow area (510) and a sample flow area (520) are provided; The marker flow area (510) is located above or below the sample flow area (520); The sample flow area (520) and the marker flow area (510) each include at least one sample pad, and one or more physical isolation layers (540) between the sample flow area (520) and the marker flow area (510); The housing (100) comprises a bottom housing (110) and an upper housing (120) that are engaged with each other, and the sample adding hole (200), the liquid adding hole (300) and the display area (400) are arranged on the upper housing (120); The lateral flow test strip (500) is fixed between the bottom shell (110) and the upper shell (120), and the lateral flow test strip as a whole is arranged obliquely from the liquid addition hole (300) toward the display area (400); A linkage mechanism (600) is provided between the bottom shell (110) and the upper shell (120) of the housing (100), the linkage mechanism (600) comprising a pushing component provided on the upper shell (120), a driven component provided on the bottom shell (110), and a linkage component provided between the bottom shell (110) and the upper shell (120); The bottom shell (110) and the upper shell (120) are rotatably connected via a snap-fit ​​ear (610) and a snap-fit ​​shaft (620); The pushing assembly comprises a sliding groove (630) provided on the upper shell (120) and a pushing block (640) sliding in the sliding groove (630); The driven component comprises a driven block (650) provided on the bottom shell (110) and having a driven inclined surface; The linkage assembly includes a push rod (641) connected to a push block (640), a sliding cavity block (642) at the front end of the push rod (641), a sliding block (643) with an inclined surface connected to the interior of the sliding cavity block (642) via an elastic member, a clamping block (660) with an inclined surface fixedly provided on the side wall of the sliding groove (630), the inclined directions of the sliding block (643) and the clamping block (660) cooperate with each other, and a push rod (670) is provided at the lower portion of the push block (640) for contacting and cooperating with the passive block (650).

2. A high-sensitivity detection reagent strip according to claim 1, characterized in that: The bottom structure includes a bottom plate (521); The sample flow area (520) includes an absorbent paper (522) arranged at one end of the bottom plate (521), an NC membrane (523) arranged on the bottom plate (521), the absorbent paper (522) overlapped on the NC membrane (523), and a sample pad assembly (531) arranged at the other end of the bottom plate (521), the sample pad assembly (531) overlapped on the NC membrane (523), and one or more detection lines (550) and quality control lines (560) are sequentially provided on the NC membrane (523); The marker flow zone (510) includes a physical isolation layer (540) disposed on the upper portion of a sample pad component (531), a sample pad component (532) disposed on the upper portion of the physical isolation layer (540), a binding pad (533) overlapped on the sample pad component (532), and the binding pad (533) overlapped on the NC membrane (523); or: The marker flow zone (510) includes a physical isolation layer (540) disposed on the upper portion of a sample pad component (531), a sample pad component (532) disposed on the upper portion of the physical isolation layer (540), the sample pad component (532) containing an immune marker, and the sample pad component (532) overlaps the NC membrane (523); The sample pad 1 component (531) includes at least a sample pad 1, and the sample pad 2 component (532) includes at least a sample pad 2; The NC membrane (523) is a nitrocellulose membrane.

3. A high-sensitivity detection reagent strip according to claim 1, characterized in that: The bottom structure includes a bottom plate (521); The marker flow area (510) includes an absorbent paper (522) arranged at one end of the bottom plate (521), an NC membrane (523) arranged on the bottom plate (521), the absorbent paper (522) overlapped on the NC membrane (523), and a sample pad second component (532) arranged at the other end of the bottom plate (521), the sample pad second component (532) overlapped on the conjugation pad (533), and the conjugation pad (533) overlapped on the NC membrane (523); or: The marker flow area (510) includes an absorbent paper (522) arranged at one end of the bottom plate (521), an NC membrane (523) arranged on the bottom plate (521), the absorbent paper (522) overlapped on the NC membrane (523), and a sample pad second component (532) arranged at the other end of the bottom plate (521), the sample pad second component (532) containing an immune marker, and the sample pad second component (532) overlapped on the NC membrane (523); The sample flow area (520) includes a physical isolation layer (540) disposed on the upper portion of the sample pad second component (532), a sample pad first component (531) is disposed on the upper portion of the physical isolation layer (540), and the sample pad first component (531) is overlapped on the NC membrane (523); The sample pad 1 component (531) includes at least a sample pad 1, and the sample pad 2 component (532) includes at least a sample pad 2; The NC membrane (523) is a nitrocellulose membrane.

4. The method for preparing a high-sensitivity detection reagent strip according to claim 2 or 3, wherein: The following steps are involved: S1, preparing a sample flow area (520); S2, preparing a marker flow area (510); S3, preparation of NC membrane (523); S4, assembling a lateral flow test strip (500); S5. Snap the lateral flow test strip (500) of step S4 into the housing (100).

5. The method for preparing a high-sensitivity detection reagent strip according to claim 4, characterized in that: The preparation method in step S1 is as follows: The sample pad 1 of the sample flow area (520) is composed of one or more glass fibers or polyester membrane materials with blood filtering function, and the sample pad 1 is treated with a sample pad treatment liquid to resist interference; the sample pad treatment liquid contains a CCD blocker, free biotin and a heterotrophic antibody blocker.

6. The method for preparing a high-sensitivity detection reagent strip according to claim 4, characterized in that: The preparation method in step S2 is as follows: The sample pad 2 of the marker flow area (510) is composed of one or more glass fiber or polyester film materials, which contain immune markers. The immune markers are substances with tracing functions such as but not limited to quantum dots, quantum dot microspheres, colloidal gold, fluorescein, and fluorescent microspheres.

7. The method for preparing a high-sensitivity detection reagent strip according to claim 4, characterized in that: The preparation method of the NC membrane (523) is as follows: The goat anti-chicken Ig Y antibody was diluted to 0.5 mg / mL using coating buffer as the quality control line (560) coating solution; The protein to be coated was diluted to 1.0 mg / mL using coating buffer as the test line (550) coating solution; Attach the NC membrane (523) to the base plate (521), ensuring that the bottom edge is close to the separation line at the location of the bonding pad (533); The coating liquid of the detection line (550) and the quality control line (560) is evenly coated on the NC membrane (523) using an XYZ three-dimensional film spraying instrument.

8. The method for preparing a high-sensitivity detection reagent strip according to claim 4, characterized in that: The assembly method of the lateral flow test strip (500) is as follows: The NC membrane (523) is bonded to the base plate (521), and absorbent paper (522) is attached to one end of the NC membrane (523); the sample pad component 1 (531) is attached to the other end of the NC membrane (523), a physical isolation layer (540) is attached above the sample pad 1, and a sample pad component 2 (532) is attached above the physical isolation layer (540).

9. The method for preparing a high-sensitivity detection reagent strip according to claim 4, wherein: The assembly method of the lateral flow test strip (500) is as follows: The NC membrane (523) is bonded to the base plate (521), and absorbent paper (522) is attached to one end of the NC membrane (523); the sample pad component 2 (532) is attached to the other end of the NC membrane (523), a physical isolation layer (540) is attached above the sample pad 2, and the sample pad component 1 (531) is attached to the physical isolation layer (540).

10. The method for preparing a high-sensitivity detection reagent strip according to claim 4, characterized in that: The clamping method in step S5 is as follows: The assembled lateral flow test strip (500) is cut into 0.2-0.8 mm strips using a strip cutting machine. The housing (100) is opened, the strip test strip is fixed on the bottom housing (110) and the upper housing (120) is snap-fitted to the bottom housing (110) to complete the snap-fitting.

11. The method for preparing a high-sensitivity detection reagent strip according to claim 10, characterized in that: The upper shell (120) contains two sample loading holes, namely the sample loading hole (200) S1 of the sample flow area (520) and the liquid loading hole (300) S2 of the marker flow area (510); during detection, the sample is first added to the S1 hole to complete the binding of the sample and the coated protein on the NC membrane (523). After the reaction is completed, the diluent is added to the S2 hole to complete the release of the immune marker.

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