Fluorescence percolation kit for detecting allergen specific IgE (Immunoglobulin E) antibody

Abstract

The invention relates to the technical field of allergen detection. The invention provides a fluorescent percolation kit for detecting an allergen-specific IgE antibody. The kit comprises a detection film, a microporous film, a plastic film, absorbent paper, a cleaning solution and an IgE secondary antibody working solution. According to the invention, latex microsphere-allergen protein is coated on a nitrocellulose membrane, when serum containing an allergen sIgE antibody is added into the detection card, the sIgE antibody is combined with an antigen fixed on the membrane, so that a latex microsphere-antigen-sIgE antibody immune complex is formed and fixed on the membrane, and the detection card is used for detecting the allergen sIgE antibody. And the other substances which are not combined are removed through the washing step. An IgE secondary antibody working solution is added into the detection card to form a latex microsphere-antigen-slgE antibody-anti-human IgE antibody-fluorescence labeling compound, and after uncombined substances are removed through washing, the fluorescence intensity of a detectable point represents the concentration of the sIgE antibody contained in serum.

Description

Technical Field

[0001] The present invention relates to the technical field of allergen detection, and in particular to a fluorescent percolation kit for detecting allergen-specific IgE antibodies. Background Art

[0002] Allergic diseases are caused by inhalation, ingestion or contact with allergens. Allergens are usually proteins or peptides, and there are many types. There are more than 3,000 major allergens. Most patients are not aware of the types of allergens that cause the disease. They hope to screen as many allergen items as possible at one time if their financial conditions permit. However, the cost of single quantitative testing of allergens is high, and large-scale single testing of allergens is very expensive and time-consuming.

[0003] The main detection methods of allergen-specific IgE antibody kits are enzyme-linked immunosorbent assay (ELISA), immunoblotting (IBT), fluorescent enzyme-linked immunosorbent assay (FEIA), chemiluminescent immunoassay (CLIA), microarray chip method, etc. These methods are complicated to operate, take a long time to detect, and are expensive. The time and cost of detecting allergens are high; the existing detection methods are complicated to operate, take a long time to detect, and the amount of clinical samples increases with the increase of test items. The use of peripheral blood is not supported due to the difficulty in blood collection, and it is difficult to use in pediatrics; the sensitivity of current detection reagents is generally around 0.35 IU / ml, which is low.

[0004] At present, there is a lack of detection reagents suitable for routine screening of allergens in emergency and outpatient clinics; at the same time, the problems of reagent sensitivity, specificity, and repeatability must be addressed. Summary of the invention

[0005] The purpose of the present invention is to provide a fluorescent filtration kit for detecting allergen-specific IgE antibodies, which is easy to operate, shortens the detection time, can quickly produce results, and can be used in conjunction with a fluorescent reader to accurately identify protein sites, effectively analyze the concentration value of sIgE, and improve accuracy.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The invention provides a fluorescent percolation kit for detecting allergen-specific IgE antibodies. The fluorescent percolation kit comprises a detection membrane, a microporous membrane, a plastic membrane, absorbent paper, a washing solution and an IgE secondary antibody working solution.

[0007] Preferably, the detection membrane is a nitrocellulose membrane coated with latex microspheres-allergen protein and latex microspheres-anti-mouse IgG.

[0008] Preferably, the method for preparing the detection membrane comprises the following steps: (1) Activate the latex microspheres with EDC, add allergen protein after activation, incubate with rotation for 1 h~2 h, add blocking agent, block for 1 h~2 h, centrifuge, and resuspend in PBS buffer to obtain a latex microsphere-allergen protein suspension; (2) Activate the latex microspheres with EDC, add the quality control antibody after activation, incubate with rotation for 1 h~2 h, add the blocking agent, block for 1 h~2 h, centrifuge, and resuspend in PBS buffer to obtain a latex microsphere-anti-mouse IgG suspension; (3) Spot the latex microsphere-allergen protein suspension and the latex microsphere-anti-mouse IgG suspension onto a nitrocellulose membrane and obtain a detection membrane after drying.

[0009] Preferably, the allergen proteins 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, mugwort w6, amaranth w14, humulus 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, mango f91, the type of the blocking agent is PBS buffer containing 5% to 50% BSA, and the centrifugation conditions are: 16000 to 18000 rpm, and centrifugation for 15 to 25 min.

[0010] 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: 16000 to 18000 rpm, and centrifugation for 15 to 25 min.

[0011] 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; The plastic film is a hard plastic film with a thickness of 0.05-0.2 mm.

[0012] Preferably, the preparation method of the washing solution comprises the following steps: adding 0.01% to 0.5% Tween-20 by total volume to a Tris-HCl buffer having a pH value of 7.3 to 7.5 to obtain a concentrated solution, and diluting the concentrated solution 23 to 27 times with water to obtain the washing solution.

[0013] Preferably, the preparation method of the IgE secondary antibody working solution comprises the following steps: activating the fluorescent substance using EDC, adding anti-human IgE antibody after activation, incubating with rotation for 1h~2h, adding blocking agent, blocking for 1h~2h, centrifuging to obtain anti-human IgE antibody labeled with fluorescent substance, and resuspending the anti-human IgE antibody labeled with fluorescent substance to obtain IgE secondary antibody working solution.

[0014] Preferably, the fluorescent substance includes fluorescein, fluorescent microspheres, time-resolved fluorescent microspheres, quantum dots or quantum dot microspheres, the anti-human IgE antibody is mouse anti-human IgE antibody, the type of the blocking agent is 5%~50% BSA PBS buffer, the centrifugation conditions are: 16000~18000rpm, centrifugation for 15~25min, the microsphere preservation solution is used for resuspension, the microsphere preservation solution is a PBS buffer containing 0.1%~2% BSA, 0.01%~0.5% Tween-20 and 0.01%~0.1% Proclin-300, and the concentration of the anti-human IgE antibody labeled with the fluorescent substance in the IgE secondary antibody working solution is 0.1~10μg / mL.

[0015] Preferably, the detection membrane, microporous membrane, plastic film and absorbent paper are combined with the upper shell and the lower shell to form a detection card when in use, and then used. The detection card is composed of an upper shell, a detection membrane, a microporous membrane, a plastic film, absorbent paper and a lower shell from top to bottom.

[0016] The present invention provides a fluorescent percolation kit for detecting allergen-specific IgE antibodies, the kit comprising: a detection membrane, a microporous membrane, a plastic film, absorbent paper, a washing solution and an IgE secondary antibody working solution. The kit of the present invention can be used for rapid and convenient detection, has high sensitivity, high efficiency, good accuracy, and low cost, and can detect 5 to 30 allergen sIgE antibodies at one time.

[0017] The present invention is used for quantitatively detecting the level of allergen-specific IgE (sIgE) antibodies in human serum and is clinically used for I In vitro auxiliary diagnosis of type 2 hypersensitivity diseases, using protein-microsphere coupling technology, the allergen proteins are coated on the nitrocellulose membrane: 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, humulus 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, mango f91 (protein source: allergens obtained by genetic engineering expression and purification or natural purification), which can detect 5 to 30 items at a time with good stability.

[0018] The present invention adds a layer of plastic film between the NC membrane, the microporous membrane and the absorbent paper, which can prevent the liquid from filtering downward, thereby controlling the sample incubation time, allowing the antigen and the antibody to fully react, and improving the detection sensitivity.

[0019] The present invention provides a method for detecting allergen sIgE antibodies in human serum using a fluorescent filtration kit. The method is simple to operate, shortens the detection time, and can quickly produce results. When used in conjunction with a fluorescent reader, the method can accurately identify protein sites, effectively analyze the concentration value of sIgE, and improve accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a side view of the test card; Figure 2 This is a schematic diagram of the test card lying flat. DETAILED DESCRIPTION

[0021] The invention provides a fluorescent percolation kit for detecting allergen-specific IgE antibodies. The fluorescent percolation kit comprises a detection membrane, a microporous membrane, a plastic membrane, absorbent paper, a washing solution and an IgE secondary antibody working solution.

[0022] In the present invention, the detection membrane is preferably a nitrocellulose membrane coated with latex microspheres-allergen protein and latex microspheres-anti-mouse IgG.

[0023] In the present invention, the method for preparing the detection membrane preferably comprises the following steps: (1) Activate the latex microspheres with EDC, add allergen protein after activation, incubate with rotation for 1 h~2 h, add blocking agent, block for 1 h~2 h, centrifuge, and resuspend in PBS buffer to obtain a latex microsphere-allergen protein suspension; (2) Activate the latex microspheres with EDC, add the quality control antibody after activation, incubate with rotation for 1 h~2 h, add the blocking agent, block for 1 h~2 h, centrifuge, and resuspend in PBS buffer to obtain a latex microsphere-anti-mouse IgG suspension; (3) Spot the latex microsphere-allergen protein suspension and the latex microsphere-anti-mouse IgG suspension onto a nitrocellulose membrane and obtain a detection membrane after drying.

[0024] In the present invention, the allergen proteins 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, mugwort w6, amaranth w14, humulus 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, mango f91, and the type of the blocking agent is 5% to 50% BSA in PBS buffer, more preferably 10% The BSA PBS buffer solution is preferably centrifuged at 16000-18000 rpm for 15-25 min, and more preferably centrifuged at 17000 rpm for 20 min.

[0025] In the present invention, the quality control antibody in step (2) is preferably goat anti-mouse IgG, the type of the blocking agent is preferably 5%~50% BSA PBS buffer, and more preferably, the centrifugation conditions are preferably: 16000~18000rpm, centrifugation 15~25min.

[0026] In the present invention, the microporous membrane is a plastic microporous plate with a pore size of preferably 10 to 100 μm, more preferably 50 μm, and a thickness of preferably 0.5 to 1 mm, more preferably 0.7 to 0.8 mm; The plastic film is a hard plastic film with a thickness of preferably 0.05 to 0.2 mm, more preferably 0.1 to 0.15 mm.

[0027] In the present invention, the preparation method of the washing solution preferably comprises the following steps: adding 0.01% to 0.5% of the total volume of Tween-20 to a Tris-HCl buffer having a pH value of 7.3 to 7.5 to obtain a concentrated solution, diluting the concentrated solution 23 to 27 times with water to obtain the washing solution, and further preferably: adding 0.2 to 0.3% of the total volume of Tween-20 to a Tris-HCl buffer having a pH value of 7.4 to obtain a concentrated solution, and diluting the concentrated solution 25 times with water to obtain the washing solution.

[0028] In the present invention, the preparation method of the IgE secondary antibody working solution preferably comprises the following steps: activating the fluorescent substance using EDC, adding anti-human IgE antibody after activation, incubating with rotation for 1h~2h, adding blocking agent, blocking for 1h~2h, centrifuging to obtain anti-human IgE antibody labeled with fluorescent substance, resuspending the anti-human IgE antibody labeled with fluorescent substance to obtain IgE secondary antibody working solution, and further preferably: activating the fluorescent substance using EDC, adding anti-human IgE antibody after activation, incubating with rotation for 1.5h, adding blocking agent, blocking for 1.5h, centrifuging to obtain anti-human IgE antibody labeled with fluorescent substance, and resuspending the anti-human IgE antibody labeled with fluorescent substance to obtain IgE secondary antibody working solution.

[0029] In the present 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 a host mouse that can specifically bind to human IgE.

[0030] In the present invention, the type of the blocking agent is preferably 5% to 50% BSA PBS buffer, more preferably 10% BSA PBS buffer, the centrifugation conditions are preferably: 16000 to 18000 rpm, centrifugation for 15 to 25 min, more preferably: 17000 rpm, centrifugation for 20 min, the resuspension is preferably carried out using a microsphere preservation solution, the microsphere preservation solution is preferably a PBS buffer containing 0.1% to 2% BSA, 0.01% to 0.5% Tween-20 and 0.01% to 0.1% Proclin-300, more preferably a PBS buffer containing 0.1 to 1.5% BSA, 0.25 to 0.3% Tween-20 and 0.04 to 0.05% Proclin-300, the concentration of the fluorescent substance-labeled anti-human IgE antibody in the IgE secondary antibody working solution is preferably 0.1 to 10 μg / mL, more preferably 5 μg / mL.

[0031] In the present invention, the detection membrane, microporous membrane, plastic film and absorbent paper are preferably combined with the upper shell and the lower shell to form a detection card before use. The detection card is composed of an upper shell, a detection membrane, a microporous membrane, a plastic film, absorbent paper and a lower shell from top to bottom.

[0032] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0033] Example 1 Latex microspheres labeling allergen proteins

[0034] (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, solid content 10%). (2) Take 10 μL of microspheres (solid content 10%) and dissolve them in 1 mL of ultrapure water. Centrifuge at 17,000 rpm for 20 min (first pass). Remove the supernatant and resuspend the microspheres in 1000 μL of ultrapure water (use a water bath sonicator with a power of 600 W and an ultrasonic duration of 3 min, the same below). Centrifuge at 17,000 rpm for 20 min (second pass). Remove the supernatant and resuspend the microspheres in 900 μL of 0.05 M MES buffer (pH 6.0) for later use.

[0035] (3) Mix 900 μL of resuspended microspheres, 100 μL of 10 mg / mL LEDC solution, and 100 μg of labeled protein thoroughly, and incubate at room temperature for 2 h. After incubation, centrifuge at 17,000 rpm for 20 min (first time); remove the supernatant and resuspend the microsphere conjugate with 1000 μL of 0.05 M MES buffer (pH 6.0); centrifuge at 17,000 rpm for 20 min (second time); remove the supernatant and resuspend the microsphere conjugate with 900 μL of 0.05 M MES buffer (pH 6.0).

[0036] (4) Add 100 μL of blocking solution (10% BSA) to the resuspended microsphere conjugate, mix and incubate at room temperature for 1 hour. After incubation, centrifuge at 17,000 rpm for 20 minutes, remove the supernatant, and resuspend the protein-microsphere complex in 1000 μL of 0.01M pH 7.4 PBS buffer. It can be used for spotting (use a commercially available spotter to spot the protein on the nitrocellulose membrane).

[0037] Example 2 Preparation of test card

[0038] (1) The detection card is composed of an upper shell 1, a detection membrane 2, a microporous membrane 3, a plastic film 4, an 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 through which a portion of the plastic film 4 can be extended to form a pull-out strip 7. The upper shell 1 and the lower shell 6 are fixedly connected by a buckle 9.

[0039] (2) The detection membrane 2 is a nitrocellulose membrane with a protein-microsphere complex prepared in Example 1, which is coated on a 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 coated on the plastic film 4, the function of which is mainly to slow down the liquid infiltration rate and to isolate the plastic film 4 from the detection membrane 2, so that the detection membrane 2 will not be damaged when the plastic film 4 is pulled; the main function of the plastic film 4 is to prevent the liquid from infiltrating downward, thereby controlling the sample incubation time. After the incubation is completed, the plastic film 4 can be pulled out of the gap 8 by pinching the pull-out strip 7, so that the liquid can infiltrate downward to the absorbent paper 5, the function of the absorbent paper 5 is to absorb excess unreacted liquid and washing liquid; the above structures are placed in the lower shell 6 in sequence, and finally the upper shell 1 is tightly covered with the corresponding buckle 9, and the detection card is ready.

[0040] Upper shell and lower shell: a plastic shell with a sample adding slot and an opening in the middle for installing a pull-out plastic film.

[0041] Detection membrane: Nitrocellulose membrane coated with latex microspheres-allergen protein and latex microspheres-anti-mouse IgG.

[0042] Microporous membrane: a plastic microporous plate with a pore size of 100 μm and a thickness of 1 mm.

[0043] Plastic film: hard plastic film, thickness between 0.1mm.

[0044] Absorbent paper: filter paper with high water absorbency.

[0045] Example 3

[0046] A fluorescent percolation kit for detecting allergen-specific IgE antibodies comprises a detection card, a washing solution, and an IgE secondary antibody working solution.

[0047] (1) Preparation of test card See Example 2 (2) Washing solution: Use Tris-HCl buffer with a pH value 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 obtain a washing solution.

[0048] (3) Preparation of IgE secondary antibody working solution: Activate fluorescent substances (including but not limited to fluorescein, fluorescent microspheres, time-resolved fluorescent microspheres, quantum dots, and quantum dot microspheres) with EDC. After activation, add the anti-human IgE antibody to be labeled (Abcam, USA, catalog number ab106494). After rotating and incubating for 2 hours, add the blocking agent (10% BSA in PBS buffer). After blocking for 2 hours, centrifuge and resuspend the microspheres in the preservation solution to obtain the anti-human IgE antibody labeled with the fluorescent substance. The concentration of the IgE secondary antibody working solution is 5 μg / mL.

[0049] ① The fluorescent microspheres used for labeling are time-resolved fluorescent microspheres with a particle size of 300 nm and surface modified with carboxyl groups (purchased from Suzhou Weidu, with a solid content of 1%).

[0050] ② Take 100μL microspheres (solid content 1%) in 1mL ultrapure water, centrifuge at 17000rpm for 20min, the first time; remove the supernatant, resuspend the microspheres with 1000μL ultrapure water (use a water bath sonicator, recommended power 700W, ultrasonic time 5min, the same below); centrifuge at 17000rpm for 20min, the second time; remove the supernatant, resuspend the microspheres with 900μL 0.05M pH 6.0 MES buffer for later use.

[0051] ③ Mix 900μL resuspended microspheres, 100μL 10mg / mL LEDC solution, and 100ug anti-human IgE thoroughly, incubate at room temperature for 2h, centrifuge at 17000rpm for 20min after incubation, remove the supernatant, and resuspend the microsphere conjugate with 1000μL 0.05M pH6.0 MES buffer; centrifuge at 17000rpm for 20min, second time; remove the supernatant, and resuspend the microsphere conjugate with 900μL 0.05M pH 6.0 MES buffer.

[0052] ④ Add 100 μL of blocking solution (10% BSA) to the resuspended microsphere conjugate, mix and incubate at room temperature for 1 hour. After incubation, centrifuge at 17,000 rpm for 20 minutes, remove the supernatant, and resuspend the anti-human IgE antibody-microsphere complex with 1,000 μL of microsphere storage solution (0.01M pH 7.4 PBS, 0.1% BSA, 0.01% Tween-20, 0.01% Proclin-300).

[0053] ⑤ Prepare 5 μg / mL IgE secondary antibody working solution by combining the anti-human IgE antibody-microsphere complex and microsphere preservation solution.

[0054] Example 4

[0055] Detection of allergen sIgE antibodies in human serum using a fluorescent filtration test kit with controlled incubation time (with plastic film 4).

[0056] See Example 2 for the preparation of the test card.

[0057] See Example 3 for the preparation of the kit.

[0058] The detection method is as follows: (1) Take out the test card and place it on a stable table.

[0059] (2) Add 500 μL of serum to the test card and let it stand for 5 min.

[0060] (3) After incubation, remove the plastic film inside the test card to allow the serum to penetrate into the absorbent paper underneath.

[0061] (4) After infiltration, add 500 μL of washing solution to the test card.

[0062] (5) After infiltration, add 500 μL of IgE secondary antibody working solution into the test card and let it stand for 10 minutes.

[0063] (6) Add 500 μL of washing solution into the test card and wait for the test result after penetration.

[0064] (7) Scan the membrane strip with a reader, read the fluorescence value at the corresponding position, and then calculate the concentration value based on the standard curve. The results are shown in Table 1.

[0065] Example 5

[0066] Detection of allergen sIgE antibodies in human serum using a fluorescent filtration kit with uncontrolled incubation test cards (without plastic film 4).

[0067] Preparation of the test card: The test card consists of an upper shell 1, a test membrane 2, a microporous membrane 3, an 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 a buckle 9; the test membrane 2 is a nitrocellulose membrane with a protein-microsphere complex prepared as described in Example 1, which is coated on the microporous membrane 3 with tiny pores, and 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 coated on the absorbent paper 5, and the function of the absorbent paper 5 is to absorb excess unreacted liquid and washing liquid; the above structures are placed in the lower shell 6 in sequence, and finally the upper shell 1 is tightly covered with the corresponding buckles 9, and the test card is ready.

[0068] See Example 3 for the preparation of the kit.

[0069] The detection method is as follows: (1) Take out the test card and place it on a stable table.

[0070] (2) Add 500 μL of serum to the test card and let it stand for 5 minutes.

[0071] (3) After infiltration, add 500 μL of washing solution to the test card.

[0072] (4) After infiltration, add 500 μL of IgE secondary antibody working solution into the test card and let it stand for 10 minutes.

[0073] (5) Add 500 μL of washing solution into the test card and test the result after penetration.

[0074] (6) Scan the membrane strip with a reader, read the fluorescence value at the corresponding position, and then calculate the concentration value based on the standard curve. The results are shown in Table 1.

[0075] Table 1

[0076] Comparing the test results in Table 1, it can be seen that since Example 5 did not use a controllable incubation test card, the sample incubation was incomplete, resulting in undetectable 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, with level 0 being negative and levels 1 to 6 being positive, and the higher the concentration, the higher the level).

[0077] The relationship between international specific IgE antibody concentration and grading standards is shown in Table 2.

[0078] Table 2 International specific IgE antibody concentration and grading standards

[0079] As can be seen from the above embodiments, the present invention provides a fluorescent percolation kit for detecting allergen-specific IgE antibodies, the kit comprising: a detection membrane, a microporous membrane, a plastic film, a wicking paper, a washing solution and an IgE secondary antibody working solution. The present invention coats latex microspheres-allergen proteins on a nitrocellulose membrane, and when serum containing allergen sIgE antibodies is added to the detection card, the sIgE antibodies are combined with the antigens fixed on the membrane, thereby forming an immune complex of latex microspheres-antigens-slgE antibodies fixed on the membrane, and the remaining substances that are not combined are removed by the washing step. Then, the IgE secondary antibody working solution is added to the detection card to form a latex microsphere-antigen-slgE antibody-anti-human IgE antibody-fluorescent labeling complex, and after removing the unbound substances by washing, the fluorescence intensity of the detectable point represents the sIgE antibody concentration contained in the serum.

[0080] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A fluorescent filtration kit for detecting allergen-specific IgE antibodies, characterized in that: The fluorescent percolation 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 infiltration 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 infiltration kit according to claim 2, characterized in that: The preparation method of the detection membrane comprises the following steps: (1) Activate the latex microspheres with EDC, add allergen protein after activation, incubate with rotation for 1 h~2 h, add blocking agent, block for 1 h~2 h, centrifuge, and resuspend in PBS buffer to obtain a latex microsphere-allergen protein suspension; (2) Activate the latex microspheres with EDC, add the quality control antibody after activation, incubate with rotation for 1 h~2 h, add the blocking agent, block for 1 h~2 h, centrifuge, and resuspend in PBS buffer to obtain a latex microsphere-anti-mouse IgG suspension; (3) Spot the latex microsphere-allergen protein suspension and the latex microsphere-anti-mouse IgG suspension onto a nitrocellulose membrane and obtain a detection membrane after drying.

4. The fluorescent infiltration kit according to claim 3, characterized in that: The allergen proteins 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, mugwort w6, amaranth w14, humulus 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, mango f91, the type of the blocking agent is 5%~50% BSA PBS buffer, and the centrifugation conditions are: 16000~18000rpm, centrifugation 15~25min.

5. The fluorescent infiltration kit according to claim 4, characterized in that: In step (2), the quality control antibody is goat anti-mouse IgG, the blocking agent is 5% to 50% BSA in PBS buffer, and the centrifugation conditions are: 16000 to 18000 rpm, and centrifugation for 15 to 25 min.

6. The fluorescent infiltration 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 hard plastic film with a thickness of 0.05-0.2 mm.

7. The fluorescent infiltration kit according to claim 1, characterized in that: The preparation method of the washing liquid comprises the following steps: adding 0.01% to 0.5% of Tween-20 in total volume to a Tris-HCl buffer having a pH value of 7.3 to 7.5 to obtain a concentrated solution, and diluting the concentrated solution 23 to 27 times with water to obtain the washing liquid.

8. The fluorescent infiltration kit according to claim 1, characterized in that: The preparation method of the IgE secondary antibody working solution comprises the following steps: activating the fluorescent substance with EDC, adding anti-human IgE antibody after activation, incubating with rotation for 1h~2h, adding blocking agent, blocking for 1h~2h, centrifuging to obtain anti-human IgE antibody labeled with fluorescent substance, and resuspending the anti-human IgE antibody labeled with fluorescent substance to obtain IgE secondary antibody working solution.

9. The fluorescent infiltration 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 type of the blocking agent is a PBS buffer solution containing 5% to 50% BSA. The centrifugation conditions are: 16000 to 18000 rpm, centrifugation for 15 to 25 minutes. Microsphere preservation solution is used for resuspension. The microsphere preservation solution is a PBS buffer solution containing 0.1% to 2% BSA, 0.01% to 0.5% Tween-20 and 0.01% to 0.1% Proclin-300. The concentration of the anti-human IgE antibody labeled with the fluorescent substance in the IgE secondary antibody working solution is 0.1 to 10 μg / mL.

10. The kit fluorescent infiltration 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 shell and the lower shell to form a detection card when in use, and then used. The detection card is composed of an upper shell, a detection membrane, a microporous membrane, a plastic film, absorbent paper and a lower shell from top to bottom.

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