Fluorescence filtration kit for detecting allergen-specific ige antibody
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2025-02-06
- Publication Date
- 2026-08-13
Smart Images
Figure CN2025075905_13082026_PF_FP_ABST
Abstract
Description
A fluorescent percolation kit for detecting allergen-specific IgE antibodies Technical Field
[0001] This invention relates to the field of allergen detection technology, and more particularly to a fluorescent percolation kit for detecting allergen-specific IgE antibodies. Background Technology
[0002] Allergic diseases are caused by the inhalation, ingestion, or contact with allergens. Allergens are usually proteins or peptides, and there are many types, with more than 3,000 major allergens. Most patients are unaware of the types of allergens that cause their illness and hope to undergo screening for as many allergens as possible at once if their financial situation allows. However, the cost of quantitative testing for a single allergen is high, and large-scale testing for a single allergen is very expensive and time-consuming.
[0003] The main detection methods for allergen-specific IgE antibody kits include enzyme-linked immunosorbent assay (ELISA), Western blotting (IBT), fluorescence enzyme-linked immunosorbent assay (FEIA), chemiluminescent immunoassay (CLIA), and microarray chip methods. These methods are complex to operate, time-consuming, and expensive, resulting in high time and cost for allergen detection. Existing detection methods are complex to operate, time-consuming, and require larger clinical sample volumes as the number of tests increases. They also do not support the use of capillary blood, and are difficult to use in pediatric settings due to the difficulty of blood collection. The sensitivity of current test reagents is generally around 0.35 IU / ml, which is low.
[0004] Currently, there is a lack of testing reagents suitable for routine allergen screening of emergency and outpatient patients; at the same time, the issues of reagent sensitivity, specificity, and repeatability need to be addressed. Summary of the Invention
[0005] The purpose of this invention is to provide a fluorescent percolation kit for detecting allergen-specific IgE antibodies. It is easy to operate, shortens the detection time, and can produce results quickly. When used with a fluorescence reader, it can accurately identify protein sites, effectively analyze the concentration of sIgE, and improve accuracy.
[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:
[0007] This invention provides a fluorescent percolation kit for detecting allergen-specific IgE antibodies, the fluorescent percolation kit comprising: a detection membrane, a microporous membrane, a plastic membrane, absorbent paper, a washing solution, and an IgE secondary antibody working solution.
[0008] Preferably, the detection membrane is a nitrocellulose membrane coated with latex microspheres-allergen protein and latex microspheres-anti-mouse IgG.
[0009] Preferably, the method for preparing the detection membrane includes the following steps:
[0010] (1) Activate the latex microspheres using EDC, add allergen protein after activation, incubate by rotation for 1-2 hours, add blocking agent, block for 1-2 hours, centrifuge, resuspend in PBS buffer to obtain latex microsphere-allergen protein suspension;
[0011] (2) The latex microspheres were activated using EDC. After activation, quality control antibody was added, and the mixture was rotated and incubated for 1-2 hours. Then, blocking agent was added, and the mixture was blocked for 1-2 hours. After centrifugation, the mixture was resuspended in PBS buffer to obtain latex microsphere-anti-mouse IgG suspension.
[0012] (3) The latex microsphere-allergen protein suspension and the latex microsphere-anti-mouse IgG suspension were spotted onto the nitrocellulose membrane and dried to obtain the detection membrane.
[0013] Preferably, the allergens mentioned in step (1) include: house dust h1, cockroach i6, house dust mite d1, dust mite d2, cat epithelium e1, dog epithelium e5, common ragweed w1, artemisia w6, amaranth w14, hop w22, cotton poplar t14, willow t12, mulberry t70, juniper t6, common birch t3, sycamore t11, Aspergillus fumigatus m3, Alternaria alternata m6, wheat flour f4, milk f2, egg white f1, cod f3, scallop f338, sesame f10, soybean f14, peanut f13, shrimp f24, crab f23, cashew f202, and mango f91. The blocking agent is 5%–50% BSA in PBS buffer, and the centrifugation conditions are: 16000–18000 rpm for 15–25 min.
[0014] Preferably, the quality control antibody in step (2) is goat anti-mouse IgG, the blocking agent is 5% to 50% BSA in PBS buffer, and the centrifugation conditions are 16,000 to 18,000 rpm for 15 to 25 minutes.
[0015] Preferably, the microporous membrane is a plastic microporous plate with a pore size of 10-100 μm and a thickness of 0.5-1 mm;
[0016] The plastic film is a rigid plastic film with a thickness of 0.05 to 0.2 mm.
[0017] Preferably, the washing solution is prepared by the following steps: adding 0.01% to 0.5% of Tween-20 by total volume to a Tris-HCl buffer solution with a pH of 7.3 to 7.5 to obtain a concentrated solution, and diluting the concentrated solution with water 23 to 27 times to obtain the washing solution.
[0018] Preferably, the preparation method of the IgE secondary antibody working solution includes the following steps: activating the fluorescent substance using EDC, adding the anti-human IgE antibody after activation, incubating by rotation for 1-2 hours, adding the blocking agent, blocking for 1-2 hours, centrifuging to obtain the fluorescently labeled anti-human IgE antibody, and resuspending the fluorescently labeled anti-human IgE antibody to obtain the IgE secondary antibody working solution.
[0019] Preferably, the fluorescent substance includes fluorescein, fluorescent microspheres, time-resolved fluorescent microspheres, quantum dots, or quantum dot microspheres; the anti-human IgE antibody is a mouse anti-human IgE antibody; the blocking agent is a PBS buffer containing 5%–50% BSA; the centrifugation conditions are 16000–18000 rpm for 15–25 min; the resuspension uses a microsphere preservation solution containing 0.1%–2% BSA, 0.01%–0.5% Tween-20, and 0.01%–0.1% Proclin-300; and the concentration of the fluorescently labeled anti-human IgE antibody in the IgE secondary antibody working solution is 0.1–10 μg / mL.
[0020] Preferably, the detection membrane, microporous membrane, plastic film, and absorbent paper are combined with the upper and lower shells to form a detection card before use. The detection card consists of the upper shell, detection membrane, microporous membrane, plastic film, absorbent paper, and lower shell from top to bottom.
[0021] This invention provides a fluorescent filtration kit for detecting allergen-specific IgE antibodies. The kit comprises a detection membrane, a microporous membrane, a plastic membrane, absorbent paper, washing solution, and IgE secondary antibody working solution. This kit allows for rapid and convenient detection, exhibiting high sensitivity, high efficiency, good accuracy, and low cost. It can simultaneously detect 5-30 allergen sIgE antibodies.
[0022] This invention is used for the quantitative detection of allergen-specific IgE (sIgE) antibody levels in human serum. Clinically, it is used for the in vitro auxiliary diagnosis of type I hypersensitivity diseases. It employs protein-microsphere conjugation technology to coat nitrocellulose membranes with allergen proteins: house dust h1, cockroach i6, house dust mite d1, flour mite d2, cat epithelial e1, dog epithelial e5, common ragweed w1, artemisia w6, amaranth w14, hop w22, cotton poplar t14, willow t12, and mulberry. T70, Juniper T6, Common Birch T3, Sycamore T11, Aspergillus fumigatus M3, Alternaria M6, Wheat Flour F4, Milk F2, Egg White F1, Cod F3, Scallop F338, Sesame F10, Soybean F14, Peanut F13, Shrimp F24, Crab F23, Cashew F202, Mango F91 (Protein source: allergens obtained through genetic engineering expression and purification or natural purification). It can detect 5-30 items at once and has good stability.
[0023] This invention adds a plastic film between the NC membrane, the microporous membrane, and the absorbent paper to prevent liquid from seeping downwards, thereby controlling the sample incubation time, allowing the antigen and antibody to react fully, and improving detection sensitivity.
[0024] This invention provides a method for detecting allergen sIgE antibodies in human serum using a fluorescent percolation kit. The method is simple to operate, shortens the detection time, and can quickly produce results. When used with a fluorescence reader, it can accurately identify protein sites, effectively analyze the concentration of sIgE, and improve accuracy. Attached Figure Description
[0025] Figure 1 is a side view of the test card;
[0026] Figure 2 shows a schematic diagram of the test card being laid flat. Detailed Implementation
[0027] This invention provides a fluorescent percolation kit for detecting allergen-specific IgE antibodies, the fluorescent percolation kit comprising: a detection membrane, a microporous membrane, a plastic membrane, absorbent paper, a washing solution, and an IgE secondary antibody working solution.
[0028] In this invention, the detection membrane is preferably a nitrocellulose membrane coated with latex microspheres-allergen protein and latex microspheres-anti-mouse IgG.
[0029] In this invention, the method for preparing the detection membrane preferably includes the following steps:
[0030] (1) Activate the latex microspheres using EDC, add allergen protein after activation, incubate by rotation for 1-2 hours, add blocking agent, block for 1-2 hours, centrifuge, resuspend in PBS buffer to obtain latex microsphere-allergen protein suspension;
[0031] (2) The latex microspheres were activated using EDC. After activation, quality control antibody was added, and the mixture was rotated and incubated for 1-2 hours. Then, blocking agent was added, and the mixture was blocked for 1-2 hours. After centrifugation, the mixture was resuspended in PBS buffer to obtain latex microsphere-anti-mouse IgG suspension.
[0032] (3) The latex microsphere-allergen protein suspension and the latex microsphere-anti-mouse IgG suspension were spotted onto the nitrocellulose membrane and dried to obtain the detection membrane.
[0033] In this invention, the allergen proteins mentioned in step (1) preferably include: house dust h1, cockroach i6, house dust mite d1, dust mite d2, cat epithelium e1, dog epithelium e5, common ragweed w1, artemisia w6, amaranth w14, hop w22, cotton poplar t14, willow t12, mulberry t70, juniper t6, common birch t3, sycamore t11, Aspergillus fumigatus m3, Alternaria alternata m6, wheat flour f4, milk f2, egg white f1, cod f3 The ingredients are: scallop f338, sesame f10, soybean f14, peanut f13, shrimp f24, crab f23, cashew f202, and mango f91. The blocking agent is a PBS buffer containing 5% to 50% BSA, more preferably a PBS buffer containing 10% BSA. The centrifugation conditions are preferably 16000 to 18000 rpm for 15 to 25 min, more preferably 17000 rpm for 20 min.
[0034] In this invention, the quality control antibody in step (2) is preferably goat anti-mouse IgG, the blocking agent is preferably 5% to 50% BSA PBS buffer, and more preferably, the centrifugation conditions are preferably 16000 to 18000 rpm for 15 to 25 min.
[0035] In this invention, the microporous membrane is preferably a plastic microporous plate with a pore size of 10-100 μm, more preferably 50 μm, and a thickness of 0.5-1 mm, more preferably 0.7-0.8 mm;
[0036] The plastic film is preferably a rigid plastic film with a thickness of 0.05 to 0.2 mm, and more preferably 0.1 to 0.15 mm.
[0037] In this invention, the preferred method for preparing the washing solution includes the following steps: adding 0.01% to 0.5% of Tween-20 by total volume to a Tris-HCl buffer solution with a pH of 7.3 to 7.5 to obtain a concentrated solution; diluting the concentrated solution with water 23 to 27 times to obtain the washing solution; more preferably: adding 0.2% to 0.3% of Tween-20 by total volume to a Tris-HCl buffer solution with a pH of 7.4 to obtain a concentrated solution; diluting the concentrated solution with water 25 times to obtain the washing solution.
[0038] In this invention, the preferred method for preparing the IgE secondary antibody working solution includes the following steps: activating the fluorescent substance using EDC, adding anti-human IgE antibody after activation, incubating by rotation for 1-2 hours, adding blocking agent, blocking for 1-2 hours, centrifuging to obtain fluorescently labeled anti-human IgE antibody, and resuspending the fluorescently labeled anti-human IgE antibody to obtain the IgE secondary antibody working solution. More preferably, the method involves: activating the fluorescent substance using EDC, adding anti-human IgE antibody after activation, incubating by rotation for 1.5 hours, adding blocking agent, blocking for 1.5 hours, centrifuging to obtain fluorescently labeled anti-human IgE antibody, and resuspending the fluorescently labeled anti-human IgE antibody to obtain the IgE secondary antibody working solution.
[0039] In this invention, the fluorescent substance preferably includes fluorescein, fluorescent microspheres, time-resolved fluorescent microspheres, quantum dots or quantum dot microspheres, and the anti-human IgE antibody is preferably a mouse anti-human IgE antibody, that is, an antibody produced in the host mouse that can specifically bind to human IgE.
[0040] In this invention, the blocking agent is preferably a PBS buffer containing 5%–50% BSA, more preferably a PBS buffer containing 10% BSA. The centrifugation conditions are preferably 16000–18000 rpm for 15–25 min, more preferably 17000 rpm for 20 min. The resuspension is preferably performed using a microsphere preservation solution, which is preferably a PBS buffer containing 0.1%–2% BSA, 0.01%–0.5% Tween-20, and 0.01%–0.1% Proclin-300, more preferably a PBS buffer containing 0.1%–1.5% BSA, 0.25%–0.3% Tween-20, and 0.04%–0.05% Proclin-300. The concentration of the fluorescently labeled anti-human IgE antibody in the IgE secondary antibody working solution is preferably 0.1–10 μg / mL, more preferably 5 μg / mL.
[0041] In this invention, the detection membrane, microporous membrane, plastic membrane, and absorbent paper are preferably combined with the upper and lower shells to form a detection card before use. The detection card consists of the upper shell, detection membrane, microporous membrane, plastic membrane, absorbent paper, and lower shell from top to bottom.
[0042] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0043] Example 1: Latex microspheres labeled with allergen proteins
[0044] (1) The white latex microspheres used for labeling are polystyrene microspheres with a particle size of 100-300 nm and surface modified with carboxyl groups (purchased from Suzhou Weidu, with a solid content of 10%).
[0045] (2) Take 10 μL of microspheres (10% solids content) into 1 mL of ultrapure water, centrifuge at 17000 rpm for 20 min for the first time; remove the supernatant and resuspend the microspheres in 1000 μL of ultrapure water (using a water bath sonicator, power 600W, sonication time 3 min, the same below); centrifuge at 17000 rpm for 20 min for the second time; remove the supernatant and resuspend the microspheres in 900 μL of 0.05M pH 6.0 MES buffer for later use.
[0046] (3) Mix 900 μL of resuspended microspheres, 100 μL of 10 mg / mLEDC solution and 100 μg of labeled protein thoroughly, and incubate at room temperature for 2 h. After incubation, centrifuge at 17000 rpm for 20 min for the first time; remove the supernatant and resuspend the microsphere conjugate with 1000 μL of 0.05 M pH 6.0 MES buffer; centrifuge at 17000 rpm for 20 min for the second time; remove the supernatant and resuspend the microsphere conjugate with 900 μL of 0.05 M pH 6.0 MES buffer.
[0047] (4) Add 100 μL of blocking solution (10% BSA) to the resuspended microsphere conjugate, mix and incubate at room temperature for 1 h. After incubation, centrifuge at 17000 rpm for 20 min, remove the supernatant, and resuspend the protein-microsphere complex in 1000 μL of 0.01 M pH 7.4 PBS buffer. It can then be used for spotting (use a commercially available spotting instrument to spot the protein onto the nitrocellulose membrane).
[0048] Example 2: Preparation of the test card
[0049] (1) The test card is composed of an upper shell 1, a test membrane 2, a microporous membrane 3, a plastic film 4, absorbent paper 5, and a lower shell 6 from top to bottom. There is a gap 8 between the upper shell 1 and the lower shell 6, which allows the plastic film 4 to extend out to form a pull strip 7. The upper shell 1 and the lower shell 6 are fixedly connected by a buckle 9.
[0050] (2) The detection membrane 2 is a nitrocellulose membrane with protein-microsphere complexes prepared in Example 1. It is covered on the microporous membrane 3 with tiny pores. The detection membrane 2 is slightly smaller than the microporous membrane 3, but not smaller than the window 10 of the upper shell 1. The microporous membrane 3 is covered on the plastic membrane 4. The main function of the microporous membrane 3 is to slow down the liquid seepage rate and isolate the plastic membrane 4 from the detection membrane 2, so that the detection membrane 2 will not be damaged when the plastic membrane 4 is pulled. The main function of the plastic membrane 4 is to prevent the liquid from seeping downward, thereby controlling the sample incubation time. After the incubation is completed, the plastic membrane 4 can be pulled out from the gap 8 by pinching the pull strip 7, so that the liquid seeps downward to the absorbent paper 5. The function of the absorbent paper 5 is to absorb the excess unreacted liquid and washing liquid. All the above structures are placed in the lower shell 6 in sequence. Finally, the upper shell 1 is closed with the corresponding buckle 9, and the detection card is completed.
[0051] Upper and lower shells: Plastic outer shells with sample loading slots and openings in the middle for installing pull-out plastic films.
[0052] Detection membrane: Nitrocellulose membrane coated with latex microspheres containing allergen proteins and latex microspheres containing anti-mouse IgG.
[0053] Microporous membrane: a plastic microporous plate with a pore size of 100μm and a thickness of 1mm.
[0054] Plastic film: Rigid plastic film with a thickness of 0.1mm.
[0055] Absorbent paper: Filter paper with high absorbency.
[0056] Example 3
[0057] A fluorescent percolation kit for detecting allergen-specific IgE antibodies includes: a test card, a washing solution, and an IgE secondary antibody working solution.
[0058] (1) For the preparation of the test card, please refer to Example 2.
[0059] (2) Washing solution: Using Tris-HCl buffer solution with a pH of 7.4, add 0.5% surfactant (Tween-20) to obtain a concentrated washing solution. The concentrated washing solution is diluted with distilled water or deionized water at a ratio of 1:25 to prepare a washing solution.
[0060] (3) Preparation of IgE secondary antibody working solution: Fluorescent substances (including but not limited to fluorescein, fluorescent microspheres, time-resolved fluorescent microspheres, quantum dots, and quantum dot microspheres) are activated using EDC. After activation, anti-human IgE antibody (Abcam, USA, catalog number ab106494) to be labeled is added. After rotating and incubating for 2 hours, blocking agent (PBS buffer of 10% BSA) is added. After blocking for 2 hours, the mixture is centrifuged and resuspended in microsphere preservation solution to obtain fluorescently labeled anti-human IgE antibody. The concentration of the IgE secondary antibody working solution is 5 μg / mL.
[0061] ① The labeling microspheres are time-resolved fluorescent microspheres with a particle size of 300 nm and a surface modified with carboxyl groups (purchased from Suzhou Weidu, with a solid content of 1%).
[0062] ② Take 100 μL of microspheres (1% solid content) and place them in 1 mL of ultrapure water. Centrifuge at 17000 rpm for 20 min for the first time. Remove the supernatant and resuspend the microspheres in 1000 μL of ultrapure water (using a water bath sonicator, recommended power 700W, sonication time 5 min, the same below). Centrifuge at 17000 rpm for 20 min for the second time. Remove the supernatant and resuspend the microspheres in 900 μL of 0.05 M pH 6.0 MES buffer for later use.
[0063] ③ Thoroughly mix 900 μL of resuspended microspheres, 100 μL of 10 mg / mL LEDC solution, and 100 μg of anti-human IgE. Incubate at room temperature for 2 h. After incubation, centrifuge at 17000 rpm for 20 min for the first time. Remove the supernatant and resuspend the microsphere conjugate in 1000 μL of 0.05 M pH 6.0 MES buffer. Centrifuge at 17000 rpm for 20 min for the second time. Remove the supernatant and resuspend the microsphere conjugate in 900 μL of 0.05 M pH 6.0 MES buffer.
[0064] ④ Add 100 μL of blocking buffer (10% BSA) to the resuspended microsphere conjugate, mix and incubate at room temperature for 1 h. After incubation, centrifuge at 17000 rpm for 20 min, remove the supernatant, and resuspend the anti-human IgE antibody-microsphere complex in 1000 μL of microsphere preservation buffer (0.01 M pH 7.4 PBS, 0.1% BSA, 0.01% Tween-20, 0.01% Proclin-300).
[0065] ⑤ Prepare a 5 μg / mL IgE secondary antibody working solution by combining the anti-human IgE antibody-microsphere complex with microsphere preservation solution.
[0066] Example 4
[0067] The fluorescent percolation kit used to detect allergen sIgE antibodies in human serum was developed using a controlled-time incubation test card (with plastic film 4).
[0068] For the preparation of the test card, please refer to Example 2.
[0069] See Example 3 for reagent kit preparation.
[0070] The detection method is as follows:
[0071] (1) Take out the test card and place it on a stable table.
[0072] (2) Add 500 μL of serum into the test card and let it stand for 5 minutes.
[0073] (3) After incubation, remove the plastic film from the test card to allow the serum to seep into the lower absorbent paper.
[0074] (4) After penetration, add 500 μL of washing solution into the test card.
[0075] (5) After infiltration, add 500 μL of IgE secondary antibody working solution into the test card and let it stand for 10 min.
[0076] (6) Add 500 μL of washing solution to the test card and test the results after it has penetrated.
[0077] (7) Scan the membrane strip with a reader, read the fluorescence value at the corresponding position, and then calculate the concentration value according to the standard curve. The results are shown in Table 1.
[0078] Example 5
[0079] The fluorescent percolation kit for detecting allergen sIgE antibodies in human serum was used with an uncontrolled incubation test card (without plastic film 4).
[0080] Preparation of the test card: The test card consists of an upper shell 1, a test membrane 2, a microporous membrane 3, absorbent paper 5, and a lower shell 6 from top to bottom; there is no gap between the upper shell 1 and the lower shell 6; the upper shell 1 and the lower shell 6 are fixedly connected by snap fasteners 9; the test membrane 2 is a nitrocellulose membrane with protein-microsphere complex dots prepared in Example 1, which is covered on the microporous membrane 3 with tiny pores. The test membrane 2 is slightly smaller than the microporous membrane 3, but not smaller than the window 10 of the upper shell 1; the microporous membrane 3 is directly covered on the absorbent paper 5, and the function of the absorbent paper 5 is to absorb excess unreacted liquid and washing liquid; all the above structures are placed in the lower shell 6 in sequence, and finally the upper shell 1 is closed tightly with the corresponding snap fasteners 9, and the test card is completed.
[0081] See Example 3 for reagent kit preparation.
[0082] The detection method is as follows:
[0083] (1) Take out the test card and place it on a stable table.
[0084] (2) Add 500 μL of serum into the test card and let it stand for 5 minutes.
[0085] (3) After penetration, add 500 μL of washing solution into the test card.
[0086] (4) After infiltration, add 500 μL of IgE secondary antibody working solution into the test card and let it stand for 10 min.
[0087] (5) Add 500 μL of washing solution to the test card and wait for it to penetrate before testing the results.
[0088] (6) Scan the membrane strip with a reader to read the fluorescence value at the corresponding position, and then calculate the concentration value according to the standard curve. The results are shown in Table 1.
[0089] Table 1
[0090] Comparing the test results in Table 1, it can be seen that because the controlled incubation test card was not used in Example 5, the sample incubation was incomplete, resulting in the failure to detect sIgE antibody concentrations below level 2 (including some level 2). (The semi-quantitative results of allergen sIgE detection are expressed in a graded form, which is divided into 0 to 6 levels according to the international grading standard. Level 0 is negative, and levels 1 to 6 are positive. The higher the concentration, the higher the level.)
[0091] The relationship between the concentration of specific IgE antibodies and the grading standards internationally is shown in Table 2.
[0092] Table 2. International Standards for the Concentration and Grading of Specific IgE Antibodies
[0093] As shown in the above embodiments, the present invention provides a fluorescent filtration kit for detecting allergen-specific IgE antibodies. The kit comprises a detection membrane, a microporous membrane, a plastic membrane, absorbent paper, washing solution, and IgE secondary antibody working solution. In this invention, latex microspheres containing allergen proteins are coated onto a nitrocellulose membrane. When serum containing allergen sIgE antibodies is added to the detection card, the sIgE antibodies bind to the antigen immobilized on the membrane, forming an immune complex of latex microspheres, antigen, and sIgE antibody immobilized on the membrane. Any unbound material is removed through a washing step. Then, IgE secondary antibody working solution is added to the detection card, forming a latex microsphere-antigen-sIgE antibody-anti-human IgE antibody-fluorescently labeled complex. After washing to remove unbound material, the fluorescence intensity at the detectable points represents the concentration of sIgE antibodies in the serum.
[0094] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A fluorescent percolation kit for detecting allergen-specific IgE antibodies, characterized in that, The fluorescence permeation kit comprises: a detection membrane, a microporous membrane, a plastic membrane, absorbent paper, a washing solution, and an IgE secondary antibody working solution.
2. The fluorescent percolation kit according to claim 1, characterized in that, The detection membrane is a nitrocellulose membrane coated with latex microspheres-allergen protein and latex microspheres-anti-mouse IgG.
3. The fluorescent percolation kit according to claim 2, characterized in that, The method for preparing the detection membrane includes the following steps: (1) Activate the latex microspheres using EDC, add allergen protein after activation, incubate by rotation for 1-2 hours, add blocking agent, block for 1-2 hours, centrifuge, resuspend in PBS buffer to obtain latex microsphere-allergen protein suspension; (2) The latex microspheres were activated using EDC. After activation, quality control antibody was added, and the mixture was rotated and incubated for 1-2 hours. Then, blocking agent was added, and the mixture was blocked for 1-2 hours. After centrifugation, the mixture was resuspended in PBS buffer to obtain latex microsphere-anti-mouse IgG suspension. (3) The latex microsphere-allergen protein suspension and the latex microsphere-anti-mouse IgG suspension were spotted onto the nitrocellulose membrane and dried to obtain the detection membrane.
4. The fluorescent percolation kit according to claim 3, characterized in that, The allergens mentioned in step (1) include: house dust h1, cockroach i6, house dust mite d1, dust mite d2, cat epithelium e1, dog epithelium e5, common ragweed w1, artemisia w6, amaranth w14, hop w22, cotton poplar t14, willow t12, mulberry t70, juniper t6, common birch t3, sycamore t11, Aspergillus fumigatus m3, Alternaria alternata m6, wheat flour f4, milk f2, egg white f1, cod f3, scallop f338, sesame f10, soybean f14, peanut f13, shrimp f24, crab f23, cashew f202, and mango f91. The blocking agent is 5%–50% BSA in PBS buffer. The centrifugation conditions are: 16000–18000 rpm for 15–25 min.
5. The fluorescent percolation kit according to claim 4, characterized in that, The quality control antibody in step (2) is goat anti-mouse IgG, the blocking agent is 5% to 50% BSA in PBS buffer, and the centrifugation conditions are: 16,000 to 18,000 rpm for 15 to 25 minutes.
6. The fluorescent percolation kit according to claim 1, characterized in that, The microporous membrane is a plastic microporous plate with a pore size of 10-100 μm and a thickness of 0.5-1 mm; The plastic film is a rigid plastic film with a thickness of 0.05 to 0.2 mm.
7. The fluorescent percolation kit according to claim 1, characterized in that, The preparation method of the washing solution includes the following steps: adding 0.01% to 0.5% of Tween-20 by total volume to a Tris-HCl buffer solution with a pH of 7.3 to 7.5 to obtain a concentrated solution, and diluting the concentrated solution with water 23 to 27 times to obtain the washing solution.
8. The fluorescent percolation kit according to claim 1, characterized in that, The preparation method of the IgE secondary antibody working solution includes the following steps: activating the fluorescent substance using EDC, adding the anti-human IgE antibody after activation, incubating by rotation for 1-2 hours, adding the blocking agent, blocking for 1-2 hours, centrifuging to obtain the fluorescently labeled anti-human IgE antibody, and resuspending the fluorescently labeled anti-human IgE antibody to obtain the IgE secondary antibody working solution.
9. The fluorescent percolation kit according to claim 8, characterized in that, The fluorescent substance includes fluorescein, fluorescent microspheres, time-resolved fluorescent microspheres, quantum dots, or quantum dot microspheres. The anti-human IgE antibody is a mouse anti-human IgE antibody. The blocking agent is a PBS buffer containing 5%–50% BSA. The centrifugation conditions are 16000–18000 rpm for 15–25 min. The resuspension uses a microsphere preservation solution, which is a PBS buffer containing 0.1%–2% BSA, 0.01%–0.5% Tween-20, and 0.01%–0.1% Proclin-300. The concentration of the fluorescently labeled anti-human IgE antibody in the IgE secondary antibody working solution is 0.1–10 μg / mL.
10. The fluorescent percolation kit according to any one of claims 1 to 9, characterized in that, The detection membrane, microporous membrane, plastic film, and absorbent paper are combined with the upper and lower shells to form a detection card before use. The detection card consists of the upper shell, detection membrane, microporous membrane, plastic film, absorbent paper, and lower shell from top to bottom.