Allergen specific IgE antibody detection card, kit and application

Abstract

The invention belongs to the technical field of biological detection, and discloses an allergen-specific IgE antibody detection card, a kit and application. The allergen specificity IgE antibody detection card comprises a combination pad and a nitrocellulose membrane, wherein the combination pad is coated with a mouse anti-human IgE antibody marked by AIE polystyrene fluorescent microspheres and a rabbit IgG antibody marked by AIE polystyrene fluorescent microspheres; a detection line of the nitrocellulose membrane is coated with a specific antigen, and a quality control line of the nitrocellulose membrane is coated with a goat anti-rabbit IgG antibody. The kit prepared from the reagent card is used for detecting the allergen-specific IgE antibody, has the characteristics of high detection sensitivity, high specificity, simplicity in operation, cost saving and the like, and has a very large market and a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, in particular to an allergen-specific IgE antibody detection card, kit and application. BACKGROUND

[0002] IgE-mediated allergic reaction is a type of immune response induced by B cells in the body after contacting allergens. The allergic reaction is based on the release of histamine, often occurs rapidly, and can cause a series of allergic reactions, which are manifested as skin system inflammation such as skin redness and itching in clinical practice. 90% of allergic symptoms in allergic people are caused by food allergens.

[0003] The current common immunological methods are: (1) enzyme-linked immunoassay (ELISA) is a commonly used technology, which is to combine solid-phase antigen or antibody with enzyme-labeled antigen and antibody, and the common combination methods are double-antibody sandwich method and indirect method. The main principle of enzyme-linked immunoassay is to detect by double-antibody sandwich method, and the coating antibody is adsorbed on the surface of the solid-phase carrier to combine with the specific antigen. When the specific antigen is contained in the test substance, an antigen-antibody complex is formed. Then, another enzyme-labeled antibody is added as a detection antibody, which can retain the activity of the enzyme and the immunological activity of the antibody to improve the sensitivity of the ELISA method. The enzyme-labeled antibody will combine with different epitopes of the antigen in the test substance to form a double-antibody sandwich complex, and the complex is also combined on the solid-phase carrier. After adding the enzyme reaction substrate, the substrate is catalyzed by the enzyme to form a colored product, and the color depth of the colored product can be used to judge the concentration of the antigen in the test substance, so as to carry out qualitative or quantitative experiments. However, this method has a long detection time and a narrow detection range. (2) Immunocolloidal gold method (ICA) is to label high-electron-density colloidal gold on the antibody or antigen as a marker. When it aggregates at the corresponding ligand, it will produce a color visible to the naked eye, so it can be used for qualitative or semi-quantitative detection in research. With the development of colloidal gold research, the surface of the gold core can be modified with ligands containing sulfur, phosphorus oxide, phosphorus oxide, amino and carboxyl. However, this method has low sensitivity and cannot be used for quantitative detection, which cannot meet the requirements of clinical diagnosis. (3) Radioimmunoassay (RIA) is a method of using isotope-labeled and unlabeled antigens to compete with antibodies for inhibition. However, this method has cross-reactions, long operation time, high instrument price, and certain radiation damage to operators. (4) Chemiluminescence immunoassay (CLIA) is a combination of high-sensitivity chemiluminescence measurement technology and high-specificity immunoassay for the detection and analysis of various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins and drugs. However, the cost of the instrument equipment is high.

[0004] Therefore, there is an urgent need to develop new, highly sensitive, specific, simple, and rapid methods for the immunodetection of food allergens. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide an allergen-specific IgE antibody detection card, kit, and application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides an allergen-specific IgE antibody detection card, comprising a card shell and a test strip, wherein the test strip comprises a sample pad, a conjugate pad, a nitrocellulose membrane, absorbent paper, and a PVC base plate; wherein the conjugate pad is coated with mouse anti-human IgE antibody and rabbit IgG antibody labeled with AIE polystyrene fluorescent microspheres. The detection line of the nitrocellulose membrane is coated with a specific antigen, and the control line is coated with goat anti-rabbit IgG antibody.

[0007] The allergen-specific IgE antibody detection card of this invention is based on immunological and chromatographic techniques to qualitatively detect and analyze specific IgE antibodies in human serum or whole blood. It features high sensitivity, strong specificity, simple and rapid operation, and low cost, and has broad application prospects.

[0008] In a preferred embodiment of the allergen-specific IgE antibody detection card of the present invention, the concentration of AIE polystyrene fluorescent microspheres in the antibody solution labeled with the detection line during the preparation of the conjugate pad is 0.5 mg / mL-2.0 mg / mL, preferably 1.0 mg / mL; the concentration of AIE polystyrene fluorescent microspheres in the antibody solution labeled with the control line is 0.5 mg / mL-2.0 mg / mL, preferably 1.0 mg / mL; and the final concentration of the goat anti-rabbit IgG antibody coating is 0.5 mg / mL-1.5 mg / mL, preferably 1.0 mg / mL.

[0009] In a preferred embodiment of the allergen-specific IgE antibody detection card of the present invention, the mass ratio of the microspheres to the mouse anti-human IgE antibody or rabbit IgG antibody is 10-30:1, preferably 20:1; the detection lines of the nitrocellulose membrane include four parallel detection lines, which are coated with specific antigens, and can simultaneously detect four different allergens.

[0010] As a preferred embodiment of the allergen-specific IgE antibody detection card of the present invention, the specific antigens include inhaled allergens and food allergens; inhaled allergens include at least one of mites, animal dander, and pollen; food allergens include at least one of eggs, milk, peanuts, soybeans, tomatoes, wheat, crabs, shrimp and fish, mangoes, cashews, sesame seeds, and almonds.

[0011] In a preferred embodiment of the allergen-specific IgE antibody detection card of the present invention, the final concentration of the specific antigen-coated antigen on the detection card is 0.25 mg / mL - 0.75 mg / mL; and the final concentration of the goat anti-rabbit IgG antibody is 0.5 mg / mL - 1.5 mg / mL.

[0012] Secondly, the present invention provides a method for preparing the aforementioned allergy-specific IgE antibody detection card, comprising the following steps: (1) Treat the sample pad with sample pad treatment solution, dry it overnight, and then cut it into a certain size for later use; (2) Microspheres were activated by adding EDC solution and NHS solution; after centrifugation to remove supernatant, they were washed; the mouse anti-human IgE antibody was coupled; and the microspheres were blocked to obtain T-line labeled antibody solution. (3) Microspheres were activated by adding EDC solution and NHS solution; after centrifugation to remove supernatant, they were washed; the rabbit IgG antibody was coupled; and the microspheres were blocked to obtain C-line labeled antibody solution; (4) Mix the T-line labeled antibody solution, C-line labeled antibody solution, and blank labeled reconstitution solution; spray onto the cut conjugate pad; The coupling buffer used to prepare the conjugate pad was a 0.05 mol / L, pH 8.0 boric acid-borax buffer solution. The volume percentages of the T-line labeled antibody solution, C-line labeled antibody solution, and labeled reconstituted solution are (10%-20%):(1%-2%):(78%-89%). Preferably, the volume ratio of the detection line labeled antibody solution to the quality control line labeled antibody solution is 10:1. (5) Cut and paste the NC membrane, coat the C and T lines with working solution, and prepare the reaction plate; (6) Cut absorbent paper to prepare absorbent pads; (7) Assemble the sample pad, binding pad, absorption pad and reaction plate.

[0013] In a preferred embodiment of the preparation method of the allergy-specific IgE antibody detection card of the present invention, the sample pad treatment solution includes 0.05 mg / mL-0.15 mg / mL of mouse IgG, 0.1 mg / mL-0.2 mg / mL of HBR-5, 0.1 mg / mL-0.2 mg / mL of HBR-7, 0.2 mg / mL-0.3 mg / mL of RBC antibody and sample pad buffer matrix solution; Preferably, the sample pad treatment solution comprises 0.10 mg / mL mouse IgG, 0.15 mg / mL HBR-5 (heterophile antibody blocker-5), 0.15 mg / mL HBR-7 (heterophile antibody blocker-7), 0.25 mg / mL RBC antibody, and sample pad buffer matrix solution.

[0014] The sample pad buffer matrix comprises 2.5 mg / mL-7.5 mg / mL casein sodium salt, 7.5 mg / mL-12.5 mg / mL disodium ethylenediaminetetraacetate, 25 mg / mL-75 mg / mL trehalose dihydrate, 1.25 mg / mL-2.5 mg / mL PVP-K30, 2.5 mg / mL-7.5 mg / mL ethyl phenyl polyethylene glycol (NP-40), 0.0025 mL / mL-0.0075 mL / mL Tween-20, 0.1 μL / mL-0.3 μL / mL ProClin300, and 0.05 mol / L Tris-HCl buffer at pH 8.0. Preferably, the sample pad buffer matrix comprises 5 mg / mL casein sodium salt, 10 mg / mL disodium ethylenediaminetetraacetate, 50 mg / mL trehalose dihydrate, 2 mg / mL PVP-K30, 5 mg / mL ethyl phenyl polyethylene glycol (NP-40), 0.005 mL / mL Tween-20, 0.2 μL / mL ProClin300, and 0.05 mol / L Tris-HCl buffer at pH 8.0.

[0015] The EDC solution contains 10 mg / mL EDC, 0.02 mol / L MES buffer at pH 6.0; the NHS solution contains 10 mg / mL NHS, 0.02 mol / L MES buffer at pH 6.0.

[0016] The labeled reconstitution solution comprises 5 mg / mL-15 mg / mL casein sodium salt, 10 mg / mL-30 mg / mL anhydrous trehalose, 5 mg / mL-15 mg / mL S9 (propylene oxide-ethylene oxide-vinyl diamine copolymer), 0.1 μL / mL-0.3 μL / mL ProClin 300, and 0.05 mol / L borate-borax (BB) buffer at pH 8.0. Preferably, the labeling reconstitution solution comprises 10 mg / mL casein sodium salt, 20 mg / mL anhydrous trehalose, 10 mg / mL S9 (propylene oxide-ethylene oxide-vinyl diamine copolymer), 0.2 μL / mL ProClin300, and 0.05 mol / L borate-borax (BB) buffer at pH 8.0.

[0017] The labeling buffer is a 0.02 mol / L, pH 6.0 MES buffer; the blocking solution contains 10% BSA, 0.02% ProClin300, and purified water.

[0018] Thirdly, the present invention provides an allergen-specific IgE antibody detection kit, comprising the allergen-specific IgE antibody detection card and sample diluent; The sample diluent was a 0.01 mol / L phosphate buffer.

[0019] Fourthly, the present invention provides a method for detecting allergen-specific IgE antibodies, comprising the following steps: During testing, the sample is mixed with diluent and added to the reagent well. Chromatography is performed under capillary effect. Specific IgE antibodies in the sample bind to AIE fluorescently labeled mouse anti-human IgE antibodies, diffuse to the test area, are captured by the specific antigen coated on the detection line, and form an "antigen-antibody-AIE fluorescent secondary antibody" complex. The concentration of specific IgE antibodies in the sample is directly proportional to the fluorescence intensity of the complex. According to a set standard curve, the immunofluorescence detector converts the fluorescence signal value into the concentration of specific IgE antibodies in the sample. The concentration of specific IgE antibodies in the sample is detected, and the result is expressed in IU / mL. The sample can be serum, venous blood, or finger-prick blood. The sample diluent was a 0.01 mol / L phosphate buffer.

[0020] Fifthly, the present invention applies the allergen-specific IgE antibody detection card or the allergen-specific IgE antibody detection kit to the detection of specific IgE antibodies.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention relates to an allergen-specific IgE antibody detection card that uses immunochromatography to qualitatively detect and analyze specific IgE antibodies in human blood. Allergens include eggs, milk, crab, house dust mites, etc. During testing, a diluted serum or whole blood sample is added to the sample well of the detection card, and chromatography proceeds forward under capillary action. The specific IgE antibodies in the sample first bind to mouse anti-human IgE monoclonal antibodies labeled with red fluorescent microspheres. When chromatography reaches the detection line (T), they bind to the specific antigen coated on the NC membrane, forming a "microsphere-labeled polyclonal antibody-antibody-antigen" complex. The presence of specific IgE antibodies in the patient's serum can be determined by observing the appearance of a red band on the detection line (T). This detection technology features high sensitivity, strong specificity, simple operation, and cost-effectiveness, and has a large market potential and promising application prospects. Detailed Implementation

[0022] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0023] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0024] Example 1: An allergen-specific IgE antibody detection card This allergen-specific IgE antibody detection card includes a card case and a test strip; The test strip includes a sample pad, a conjugate pad, a reaction membrane, an absorbent pad, and a base plate; The base plate has a long strip structure, the reaction membrane is disposed on the surface of the base plate, one end of the binding pad is overlapped on one end of the reaction membrane, one end of the absorbent pad is overlapped on the other end of the reaction membrane, one end of the sample pad is overlapped on the other end of the binding pad, and the sample pad and the absorbent pad are respectively disposed at both ends of the base plate; The reaction membrane is provided with a first detection line, a second detection line, a third detection line, a fourth detection line, and a quality control line that are parallel to each other. The binding pad is coated with mouse anti-human IgE antibody and rabbit IgG antibody labeled with AIE polystyrene fluorescent microspheres; the multiple detection lines of the reaction membrane are coated with different allergen-specific antigens, and the control line is coated with goat anti-rabbit IgG antibody.

[0025] The casing includes a receiving cavity, a sample application port, and an observation window; the sample application port and the observation window are respectively connected to the receiving cavity; The test strip is placed in the storage cavity, the sample application hole corresponds to the sample pad area, and the observation window corresponds to the reaction membrane area.

[0026] Allergen-specific antigens include inhaled allergens and food allergens; inhaled allergens include at least one of mites, animal dander, and pollen; food allergens include at least one of eggs, milk, peanuts, soybeans, tomatoes, wheat, crab, shrimp, fish, mango, cashews, sesame seeds, and almonds.

[0027] The final coating concentration of the allergen-specific antigen on the test card is 0.5 mg / mL to 2.5 mg / mL.

[0028] In the embodiments, the first detection line is coated with egg allergy-specific antigen, the second detection line is coated with milk allergy-specific antigen, the third detection line is coated with crab allergy-specific antigen, and the fourth detection line is coated with house dust mite / dry dust mite allergy-specific antigen, with a final coating concentration of 0.5 mg / mL.

[0029] Example 2: A method for preparing an allergen-specific IgE antibody detection card The preparation method of this allergen-specific IgE antibody detection card is as follows: A sample pad, a conjugation pad, a reaction membrane, and an absorbent pad are prepared. The sample pad is made of glass cellulose membrane treated with surfactant buffer; the conjugation pad is made of glass cellulose membrane; the reaction membrane is made of nitrocellulose membrane; and the absorbent pad is made of H-1 absorbent paper.

[0030] The reaction membrane is set on the base plate, one end of the conjugate pad is overlapped on the end of the reaction membrane near the detection line, one end of the absorbent pad is overlapped on the other end of the reaction membrane near the quality control line, and one end of the sample pad is attached to the other end of the conjugate pad to form a test strip.

[0031] The test strip is assembled with the card to obtain the test card.

[0032] The specific preparation method is as follows: (1) Preparation of sample pad The glass cellulose membrane was soaked in a sample pad treatment solution and then dried at a temperature of 18–26°C and a relative humidity of ≤30% to obtain the sample pad. (2) Preparation of the binding pad 1) Preparation of antibody solution for detection line labeling: a. Activation: Add AIE polystyrene fluorescent microspheres to labeling buffer, mix well, centrifuge, remove supernatant, add labeling buffer to reconstitute, and sonicate to resuspend; add EDC solution and NHS solution successively, mix quickly and thoroughly, and mix at room temperature in the dark for 20 min.

[0033] b. Washing: Centrifuge, remove supernatant, add coupling buffer, and sonicate to resuspend.

[0034] c. Coupling: Centrifuge, remove supernatant, add coupling buffer, and sonicate to resuspend; add mouse anti-human IgE, mix at room temperature in the dark for 2 hours.

[0035] d. Blocking: Add blocking solution and mix at room temperature in the dark for 1 hour.

[0036] e. Reconstitution: Centrifuge, discard the supernatant, add reconstitution solution to reconstitute, sonicate to resuspend, and obtain the antibody solution labeled with the detection line.

[0037] 2) Preparation of control line labeled antibody solution: a. Activation: Add AIE polystyrene fluorescent microspheres to labeling buffer, mix well, centrifuge, remove supernatant, add labeling buffer to reconstitute, and sonicate to resuspend; add EDC solution and NHS solution successively, mix quickly and thoroughly, and mix at room temperature in the dark for 20 min.

[0038] b. Washing: Centrifuge, remove supernatant, add coupling buffer, and sonicate to resuspend.

[0039] c. Coupling: Centrifuge, remove supernatant, add coupling buffer, and resuspend by sonication; add rabbit IgG, mix at room temperature in the dark for 2 hours.

[0040] d. Blocking: Add blocking solution and mix at room temperature in the dark for 1 hour.

[0041] e. Reconstitution: Centrifuge, remove supernatant, add reconstitution solution to reconstitute, sonicate to resuspend, and obtain control line labeled antibody solution.

[0042] 3) Mixing the working solution of the spray pad: Mix the antibody solution labeled with the detection line, the antibody solution labeled with the control line, and the blank labeled reconstitution solution; 4) The working solution of the spray pad is uniformly sprayed onto the pretreated glass cellulose membrane using a gold spraying and film-coating machine, and the bonding pad is obtained after drying. (3) Preparation of reaction membrane The first detection line allergen-specific antigen, the second detection line allergen-specific antigen, the third detection line allergen-specific antigen, and the fourth detection line allergen-specific antigen were diluted to a preset concentration using coating solution A to obtain the corresponding detection line coating working solution. The goat anti-rabbit IgG antibody was diluted to the preset concentration using coating solution B to obtain the quality control line coating working solution. Using a gold spraying and coating machine, the working solution for coating the detection lines and the working solution for coating the quality control lines are evenly sprayed onto the nitrocellulose membrane, forming parallel first detection lines, second detection lines, third detection lines, and fourth detection and quality control lines. After drying, a reaction membrane is obtained.

[0043] In the above preparation steps: The centrifugation conditions were 13,000 rpm and 4°C for 20 min.

[0044] The sample pad processing solution includes 0.05 mg / mL-0.15 mg / mL of mouse IgG, 0.1 mg / mL-0.2 mg / mL of HBR-5, 0.1 mg / mL-0.2 mg / mL of HBR-7, 0.2 mg / mL-0.3 mg / mL of RBC antibody, and sample pad buffer matrix solution. Preferably, the sample pad treatment solution comprises 0.10 mg / mL mouse IgG, 0.15 mg / mL HBR-5 (heterophile antibody blocker-5), 0.15 mg / mL HBR-7 (heterophile antibody blocker-7), 0.25 mg / mL RBC antibody, and sample pad buffer matrix solution.

[0045] The sample pad buffer matrix comprises 2.5 mg / mL-7.5 mg / mL casein sodium salt, 7.5 mg / mL-12.5 mg / mL disodium ethylenediaminetetraacetate, 25 mg / mL-75 mg / mL trehalose dihydrate, 1.25 mg / mL-2.5 mg / mL PVP-K30, 2.5 mg / mL-7.5 mg / mL ethyl phenyl polyethylene glycol (NP-40), 0.0025 mL / mL-0.0075 mL / mL Tween-20, 0.1 μL / mL-0.3 μL / mL ProClin300, and 0.05 mol / L Tris-HCl buffer at pH 8.0. Preferably, the sample pad buffer matrix comprises 5 mg / mL casein sodium salt, 10 mg / mL disodium ethylenediaminetetraacetate, 50 mg / mL trehalose dihydrate, 2 mg / mL PVP-K30, 5 mg / mL ethyl phenyl polyethylene glycol (NP-40), 0.005 mL / mL Tween-20, 0.2 μL / mL ProClin300, and 0.05 mol / L Tris-HCl buffer at pH 8.0.

[0046] The concentration of AIE polystyrene fluorescent microspheres in the detection line-labeled antibody solution is 0.5 mg / mL to 2.0 mg / mL; preferably 1.0 mg / mL.

[0047] The concentration of AIE polystyrene fluorescent microspheres in the control line labeled antibody solution is 0.5 mg / mL-2.0 mg / mL; preferably 1.0 mg / mL.

[0048] The mass ratio of AIE polystyrene fluorescent microspheres to mouse anti-human IgE antibody is 30-10:1, preferably 20:1.

[0049] The mass ratio of AIE polystyrene fluorescent microspheres to rabbit IgG antibody is 30-10:1, preferably 20:1.

[0050] The labeling buffer was a 0.02 mol / L, pH 6.0 MES buffer.

[0051] The coupling buffer was a 0.05 mol / L, pH 8.0 borate-borax (BB) buffer.

[0052] The blocking solution contains 10% BSA, 0.02% ProClin300, and purified water.

[0053] The EDC solution contains 10 mg / mL EDC and 0.02 mol / L MES buffer at pH 6.0.

[0054] The NHS solution contains 10 mg / mL NHS, 0.02 mol / L MES buffer, and pH 6.0.

[0055] The volume ratio of the detection line labeled antibody solution, the quality control line labeled antibody solution, and the blank labeled reconstituted solution is (10%-20%):(1%-2%):(78%-89%). Preferably, the volume ratio of the detection line labeled antibody solution to the control line labeled antibody solution is 10:1.

[0056] The reconstitution solution consists of 5 mg / mL–15 mg / mL casein sodium salt, 10 mg / mL–30 mg / mL anhydrous trehalose, 5 mg / mL–15 mg / mL S9 (propylene oxide-ethylene oxide-vinyl diamine copolymer), 0.1 μL / mL–0.3 μL / mL ProClin 300, and 0.05 mol / L borate-borax (BB) buffer at pH 8.0. Preferably, the reconstitution solution comprises 10 mg / mL casein sodium salt, 20 mg / mL anhydrous trehalose, 10 mg / mL S9 (propylene oxide-ethylene oxide-vinyl diamine copolymer), 0.2 μL / mL ProClin300, and 0.05 mol / L borate-borax (BB) buffer at pH 8.0.

[0057] Coating solution A consists of 0.05 g / mL anhydrous trehalose, 0.2 μL / mL ProClin300, and 0.01 mol / L pH 7.4 PBS buffer. Coating solution B consists of 0.5 mg / mL BSA, 0.05 g / mL anhydrous trehalose, 0.2 μL / mL ProClin300, and 0.01 mol / L pH 7.4 PBS buffer.

[0058] The final concentration of goat anti-rabbit IgG antibody coating is 0.5 mg / mL-1.5 mg / mL, preferably 1.0 mg / mL.

[0059] Example 3: An allergen-specific IgE antibody detection card This reagent card, based on fluorescent microsphere immunochromatography, can qualitatively determine allergen-specific IgE antibodies in human serum and whole blood samples, achieving highly sensitive, specific, and rapid detection. The specific implementation steps are as follows: 1. Preparation of sample pad (1) Prepare sample pad treatment solution at a rate of 50 mL / sheet, and use the sample pad treatment solution to soak the glass cellulose membrane. Sample pad preparation solution: 0.10 mg / mL mouse IgG, 0.15 mg / mL HBR-5 (heterophile antibody blocker-5), 0.15 mg / mL HBR-7 (heterophile antibody blocker-7), 0.25 mg / mL RBC antibody and sample pad buffer matrix solution. The sample pad buffer matrix consists of 5 mg / mL casein sodium salt, 10 mg / mL disodium ethylenediaminetetraacetate, 50 mg / mL trehalose dihydrate, 2 mg / mL PVP-K30, 5 mg / mL ethyl phenyl polyethylene glycol (NP-40), 0.005 mL / mL Tween-20, 0.2 μL / mL ProClin300, and 0.05 mol / L Tris-HCl buffer at pH 8.0.

[0060] Remove the prepared sample pad and place it on a clean, dry, and flat drying rack.

[0061] (2) Place the above drying rack at a temperature of (18-26)℃ and relative humidity of ≤30% overnight for (16-24)h.

[0062] (3) Under the conditions of temperature (18-26)℃ and relative humidity ≤30%, cut off 5mm from each of the two long sides of the dried sample pad, and then cut it into a size of (17±1)mm×(300±10)mm for later use.

[0063] 2. Preparation of the binding pad (1) Preparation of T-line labeled antibody solution: Mouse anti-human IgE antibody labeled with AIE polystyrene fluorescent microspheres (taking 1.0 mg fluorescent microspheres as an example), the specific labeling steps are as follows: a. Activation: Take 100 μL (i.e., 1.0 mg) of 10 mg / mL (1% solid content) AIE polystyrene fluorescent microspheres, add 400 μL of labeling buffer and mix well. Centrifuge at 13000 rpm and 4℃ for 20 min. Remove the supernatant and add 500 μL of labeling buffer to resuspend. Sonicate to resuspend. Weigh EDC and NHS, dissolve them in labeling buffer to prepare 10 mg / mL EDC and 10 mg / mL NHS. Note that they should be prepared and used immediately. Quickly add 50 μL of EDC and 50 μL of NHS one after the other, mix quickly and thoroughly, and mix at room temperature in the dark for 20 min.

[0064] The labeling buffer is a 0.02 mol / L, pH 6.0 MES buffer.

[0065] b. Washing: After activation, centrifuge at 13000 rpm and 4℃ for 20 min, remove the supernatant, add 500 μL of coupling buffer and sonicate to resuspend.

[0066] The coupling buffer is a 0.05 mol / L, pH 8.0 borate-borax (BB) buffer.

[0067] c. Conjugation: After washing, centrifuge at 13000 rpm and 4℃ for 20 min, remove the supernatant, add 500 μL of conjugation buffer and sonicate to resuspend; add 50 μg of mouse anti-human IgE antibody to be labeled at a mass ratio of AIE polystyrene fluorescent microspheres: antibody = 20:1, and mix at room temperature in the dark for 2 h.

[0068] The coupling buffer is a 0.05 mol / L, pH 8.0 borate-borax (BB) buffer.

[0069] d. Blocking: After coupling is complete, add 100 μL of blocking solution and mix at room temperature in the dark for 1 h.

[0070] The blocking solution contains 10% BSA, 0.02% ProClin300, and purified water; e. Reconstitution: After blocking, centrifuge at 12000 rpm and 4℃ for 20 min, remove the supernatant, add 1000 μL of reconstitution solution to reconstitute, and sonicate to resuspend. The final microsphere concentration is 1.0 mg / mL.

[0071] The reconstitution solution comprises 10 mg / mL casein sodium salt, 20 mg / mL anhydrous trehalose, 10 mg / mL S9 (propylene oxide-ethylene oxide-vinyl diamine copolymer), 0.2 μL / mL ProClin300, and 0.05 mol / L borate-borax (BB) buffer at pH 8.0.

[0072] After mixing, store at 2–8°C.

[0073] (2) Preparation of C-line labeled antibody solution: Rabbit IgG antibody labeled with AIE polystyrene fluorescent microspheres (taking 1.0 mg fluorescent microspheres as an example), the specific labeling steps are as follows: a. Activation: Take 100 μL (i.e., 1.0 mg) of 10 mg / mL (1% solid content) AIE polystyrene fluorescent microspheres, add 400 μL of labeling buffer and mix well. Centrifuge at 13000 rpm and 4℃ for 20 min. Remove the supernatant and add 500 μL of labeling buffer to reconstitute and resuspend by sonication. Weigh EDC and NHS, dissolve them in labeling buffer to prepare 10 mg / mL EDC and 10 mg / mL NHS. Note that they should be prepared and used immediately. Quickly add 50 μL of EDC and 50 μL of NHS one after the other, mix quickly and thoroughly, and mix at room temperature in the dark for 20 min.

[0074] The labeling buffer is a 0.02 mol / L, pH 6.0 MES buffer.

[0075] b. Washing: After activation, centrifuge at 13000 rpm and 4℃ for 20 min, remove the supernatant, add 500 μL of coupling buffer and sonicate to resuspend.

[0076] The coupling buffer is a 0.05 mol / L, pH 8.0 borate-borax (BB) buffer.

[0077] c. Conjugation: After washing, centrifuge at 13000 rpm and 4℃ for 20 min, remove the supernatant, add 500 μL of conjugation buffer and sonicate to resuspend; add 50 μg of rabbit IgG antibody to be labeled at a mass ratio of AIE polystyrene fluorescent microspheres: antibody = 20:1, and mix at room temperature in the dark for 2 h.

[0078] The coupling buffer is a 0.05 mol / L, pH 8.0 borate-borax (BB) buffer.

[0079] d. Blocking: After coupling is complete, add 100 μL of blocking solution and mix at room temperature in the dark for 1 h.

[0080] The blocking solution contains 10% BSA, 0.02% ProClin300, and purified water; e. Reconstitution: After blocking, centrifuge at 12000 rpm and 4℃ for 20 min, remove the supernatant, add 1000 μL of reconstitution solution to reconstitute, and sonicate to resuspend. The final microsphere concentration is 1.0 mg / mL.

[0081] The reconstitution solution comprises 10 mg / mL casein sodium salt, 20 mg / mL anhydrous trehalose, 10 mg / mL S9 (propylene oxide-ethylene oxide-vinyl diamine copolymer), 0.2 μL / mL ProClin300, and 0.05 mol / L borate-borax (BB) buffer at pH 8.0.

[0082] After mixing, store at 2–8°C.

[0083] (3) T-line and C-line mixed spray pad working solution: Mix the labeled T and C-line antibody markers with the blank reconstitution solution at a volume ratio of 20%:2%.

[0084] The reconstitution solution comprises 10 mg / mL casein sodium salt, 20 mg / mL anhydrous trehalose, 10 mg / mL S9 (propylene oxide-ethylene oxide-vinyl diamine copolymer), 0.2 μL / mL ProClin300, and 0.05 mol / L borate-borax (BB) buffer at pH 8.0.

[0085] (4) Blank bonding pad cutting: Cut the glass fiber into (12±0.5)mm×(300±10)mm.

[0086] (5) Spraying pads: The fluorescent microsphere-labeled antibody solution is loaded into the gold spraying machine and sprayed onto the cut conjugate pads at a spraying volume of 3.0 μL / cm. The sprayed conjugate pads are placed in an electric heating drying oven at a temperature of (50±2)℃ and a humidity of ≤30% for (16-24)h. The dried conjugate pads are then removed, a suitable amount of desiccant is placed in them, and they are sealed in a self-sealing bag.

[0087] 3. Coating of the reaction plate (1) Cutting and pasting of NC film Take the PVC base plate and NC film. Cut the NC film into 25mm × (300±5)mm pieces and set aside. Gently peel off the protective film at the NC film adhesion point on the PVC base plate, and push the NC film evenly from left to right to make it secure.

[0088] (3) Prepare the coating working solutions for the C-line and T-line according to the following table: The required amount is calculated using one NC membrane coating as an example, as shown in Table 1: Table 1. Preparation of C-line and T-line coating working fluids Coating solution A consists of 0.05 g / mL anhydrous trehalose, 0.2 μL / mL ProClin300, and 0.01 mol / L pH 7.4 PBS buffer. Coating solution B includes 0.5 mg / mL BSA, 0.05 g / mL anhydrous trehalose, 0.2 μL / mL ProClin300, and 0.01 mol / L pH 7.4 PBS buffer; Turn on the gold spraying and marking machine, set the marking volume to 1μL / cm, change the marking length to 300mm, adjust the marking needle position, and operate with reference to the four marking position standard plate for the distance between the C line and the T line. After the NC film is etched, it is placed in an electric heating drying oven at a temperature of (50±2)℃ and a humidity of ≤30% to dry for (16-24) hours. Remove the dried reaction plate, seal it in a self-sealing bag, and store it at 4-30℃ under conditions of relative humidity ≤30%.

[0089] 4. Preparation of absorbent pads Prepare H-1 absorbent paper, cut it into pieces of (22±0.5) mm × (300±10) mm, put in an appropriate amount of desiccant, and seal it in a resealable bag.

[0090] 5. Assemble the large panels Sample pads, binding pads, absorption pads, and reaction plates should be prepared at a temperature of (18-26)℃ and a humidity of ≤30%.

[0091] (1) Adhesion of the absorbent pad: Lay the reaction plate flat on the workbench, gently peel off the protective film at the place where the absorbent pad is to be attached on the reaction plate, and adhere the absorbent pad on it. When attaching, operate gently and roll evenly to avoid scratches or bubbles. Ensure that the top of the absorbent pad is aligned with the top of the base plate, and the other side partially covers the NC film by 1-2mm.

[0092] (2) Adhesion of the binding pad and the sample pad: Lay the reaction plate flat on the workbench and gently peel off the protective film at the bonding area between the binding pad and the sample pad on the reaction plate; Adhere the binding pad to its upper end. When adhere, operate gently and roll evenly to avoid scratches or air bubbles. Ensure that the front of the binding pad is facing up and one end of the binding pad covers the NC film by 1-2 mm; Adhere the sample pad to its upper end. When adhere, operate gently and roll evenly to avoid scratches or air bubbles. Ensure that the lower end of the sample pad is aligned with the bonding area of ​​the PVC base plate sample pad and that the upper end of the sample pad covers the binding pad.

[0093] Place the pasted board into an aluminum foil bag, add desiccant, and seal it with a sealing machine.

[0094] Test card: Place the prepared test strip into the card holder to obtain the test card.

[0095] During testing, the sample is mixed with the diluent and added to the reagent well. Chromatography is performed under capillary action. Specific IgE antibodies in the sample bind to AIE fluorescently labeled mouse anti-human IgE antibodies, diffuse to the test area, are captured by the specific antigen coated on the detection line, and form an "antigen-antibody-AIE fluorescent secondary antibody" complex. The concentration of specific IgE antibodies in the sample is directly proportional to the fluorescence intensity of the complex. Based on a set standard curve, the immunofluorescence analyzer converts the fluorescence signal value into the concentration of specific IgE antibodies in the sample. The concentration of specific IgE antibodies in the sample can be detected using an instrument purchased from Guangzhou Lanbo Biotechnology Co., Ltd. The instrument's default measurement unit is IU / mL.

[0096] Example 4: An Allergen-Specific IgE Antibody Detection Kit 1. Kit Components The allergen-specific IgE antibody detection kit includes an allergen-specific IgE antibody detection card and sample diluent.

[0097] The allergen-specific IgE antibody test card consists of a card shell and a test strip. The test strip consists of a sample pad, a conjugate pad (containing mouse anti-human IgE antibody and rabbit IgG antibody labeled with AIE polystyrene fluorescent microspheres), a nitrocellulose membrane (the test line is coated with specific antigen, and the control line is coated with goat anti-rabbit IgG antibody), absorbent paper, and a PVC backing.

[0098] The sample diluent was 0.01 mol / L pH 7.4 phosphate buffer.

[0099] 2. Rapid detection method using this kit During testing, the sample is mixed with the diluent and added to the reagent well. Chromatography is performed under capillary action. Specific IgE antibodies in the sample bind to AIE fluorescently labeled mouse anti-human IgE antibodies, diffuse to the test area, are captured by the specific antigen coated on the detection line, and form an "antigen-antibody-AIE fluorescent secondary antibody" complex. The concentration of specific IgE antibodies in the sample is directly proportional to the fluorescence intensity of the complex. Based on a set standard curve, the immunofluorescence detector converts the fluorescence signal value into the concentration of specific IgE antibodies in the sample. Using suitable instruments manufactured by Guangzhou Lanbo Biotechnology Co., Ltd., the concentration of specific IgE antibodies in the sample can be detected. The instrument's default measurement result is expressed in IU / mL.

[0100] The sample can be serum, venous blood, or finger prick blood.

[0101] Sample collection: Serum collection: Collect venous blood from the subject using serum tubes or rapid serum tubes containing a coagulant. The serum should be separated as soon as possible after blood collection to avoid hemolysis. Whole blood collection: Collect venous blood from the subject using an anticoagulant tube containing lithium heparin, EDTA-K2 or sodium citrate anticoagulant, and shake the collected blood sample well before use. Finger-tip blood collection: First, use a disposable lancet to routinely collect blood from the subject's ring finger, then use a disposable blood collection tube to collect capillary blood from the subject for immediate testing.

[0102] 3. Preparation of detection kits A kit for preparing allergen-specific IgE antibody detection cards comprising the examples described herein.

[0103] 4. Performance evaluation experiment of the reagent kit All test samples were simultaneously tested using the Inhalation-Food Allergen Specific Antibody IgE Detection Kit 2 (Immunoblotting) produced by Hangzhou Zhejiang University Disun Biotechnology Co., Ltd. as a control reagent. The detection process and interpretation of the test results of the control reagent were carried out in accordance with the instructions of the kit.

[0104] Clinical sample validation results: (1) Eggs Table 2 Description of Egg Test Results Table 3 Analysis of Egg Test Results Eighty-eight cases were tested using both the control reagent and the test reagent. Among them, 44 cases were negative and 44 cases were positive using the control reagent, while 43 cases were negative and 45 cases were positive using the test reagent.

[0105] The positive concordance rate was 100.00% (95% CI: 91.97%-100.00%), the negative concordance rate was 97.73% (95% CI: 88.19%-99.60%), and the overall concordance rate was 98.86% (95% CI: 93.84%-99.80%). The Kappa value was 0.9773, and a Kappa value ≥ 0.75 indicates a good degree of agreement.

[0106] (2) Milk Table 4. Description of Milk Test Results Table 45 Analysis of Milk Test Results Eighty-eight cases were tested using both the control reagent and the test reagent. Among them, 44 cases were negative and 44 cases were positive using the control reagent, while 42 cases were negative and 46 cases were positive using the test reagent.

[0107] The positive concordance rate was 100.00% (95% CI: 91.97%-100.00%), the negative concordance rate was 95.45% (95% CI: 84.86%-98.74%), and the overall concordance rate was 97.73% (95% CI: 92.09%-99.37%). The Kappa value was 0.9545, and a Kappa value ≥ 0.75 indicates a good degree of agreement.

[0108] (3) Crab Table 6. Description of Crab Detection Results Table 7 Analysis of Crab Detection Results Ninety cases were tested using both the control reagent and the test reagent. Among them, 45 cases were negative and 45 cases were positive using the control reagent, while 46 cases were negative and 44 cases were positive using the test reagent.

[0109] The positive concordance rate was 97.78% (95% CI: 88.43%-99.61%), the negative concordance rate was 100.00% (95% CI: 92.13%-100.00%), and the overall concordance rate was 98.89% (95% CI: 93.97%-99.80%). The Kappa value was 0.9778, and a Kappa value ≥ 0.75 indicates a good degree of agreement.

[0110] (4) House dust mites / dry dust mites Table 8. Description of House Dust Mite / Dry Mite Detection Results Table 9 Analysis of House Dust Mite / Dry Mite Detection Results Eighty-eight cases were tested using both the control reagent and the test reagent. Among them, 44 cases were negative and 44 cases were positive using the control reagent, while 43 cases were negative and 45 cases were positive using the test reagent.

[0111] The positive concordance rate was 100.00% (95% CI: 91.97% - 100.00%), the negative concordance rate was 97.73% (95% CI: 88.19% - 99.60%), and the overall concordance rate was 98.86% (95% CI: 93.84% - 99.80%). The Kappa value was 0.9773, and a Kappa value ≥ 0.75 indicates a good degree of agreement.

[0112] Comparative Example 1: An Allergen-Specific IgE Antibody Detection Card The only difference between this allergen-specific IgE antibody test card and the allergen-specific IgE antibody test card in Example 3 is that: The coupling buffer is 0.01 mol / L phosphate-buffered saline (PBS) at pH 7.4.

[0113] Experimental Example 3: Thirty samples were tested using the allergen-specific IgE antibody detection cards prepared in Example 3 and Comparative Example 1. All samples were simultaneously tested using a milk allergen-specific IgE antibody detection kit (enzyme-linked immunosorbent assay) produced by Jiangsu Haooubo Biomedical Co., Ltd. as a control reagent. The detection process and interpretation of the results for the control reagent were performed in accordance with the kit's instructions.

[0114] Clinical sample validation results (taking milk as an example): Example 3, positive concordance rate 100.00%, negative concordance rate 100.00%, total concordance rate 100.00%; Comparative Example 1, positive concordance rate 96.67%, negative concordance rate 93.33%, total concordance rate 95.00%. Therefore, Example 3 showed the best effect and consistency.

[0115] This invention combines AIE polystyrene fluorescent microsphere immunochromatography with traditional immunochromatography. While retaining the advantages of traditional immunochromatography—simple operation, rapid detection, and portability—this technology also achieves precise detection results through microsphere labeling enhancement. Compared with traditional rapid detection technologies, this technology offers higher sensitivity, a wider detection range, and lower cost. This invention uses the capture method in AIE polystyrene fluorescent microsphere immunochromatography to qualitatively detect specific IgE antibodies in serum or whole blood samples. Through optimized process conditions, performance evaluation, and clinical application, an effective latex immunochromatographic assay kit for detecting specific IgE antibodies has been established, offering higher sensitivity, a wider detection range, and lower cost.

[0116] This invention, based on the principles of immunology and chromatography, utilizes AIE polystyrene fluorescent microspheres as a label to develop a highly sensitive, specific, simple, and rapid in vitro quantitative immunoassay method for detecting specific IgE antibodies in human serum or whole blood samples. It has significant market potential and promising application prospects.

[0117] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An allergen-specific IgE antibody detection card, comprising a card casing and a test strip, characterized in that, The test strip includes a sample pad, a conjugate pad, a nitrocellulose membrane, absorbent paper, and a PVC base plate; the conjugate pad is coated with mouse anti-human IgE antibody and rabbit IgG antibody labeled with AIE polystyrene fluorescent microspheres. The detection line of the nitrocellulose membrane is coated with a specific antigen, and the control line is coated with goat anti-rabbit IgG antibody.

2. The allergen-specific IgE antibody detection card according to claim 1, characterized in that, The concentration of AIE polystyrene fluorescent microspheres in the detection line-labeled antibody solution during the preparation of the binding pad is 0.5 mg / mL-2.0 mg / mL; the concentration of AIE polystyrene fluorescent microspheres in the quality control line-labeled antibody solution is 0.5 mg / mL-2.0 mg / mL; and the final concentration of the goat anti-rabbit IgG antibody coating is 0.5 mg / mL-1.5 mg / mL.

3. The allergen-specific IgE antibody detection card according to claim 1, characterized in that, The mass ratio of the microspheres to the mouse anti-human IgE antibody or rabbit IgG antibody is 10-30:

1.

4. The allergen-specific IgE antibody detection card according to claim 1, characterized in that, The nitrocellulose membrane has four parallel detection lines, each coated with a specific antigen. The specific antigen includes inhaled allergens and food allergens. Inhaled allergens include at least one of mites, animal dander, and pollen. Food allergens include at least one of eggs, milk, peanuts, soybeans, tomatoes, wheat, crabs, shrimp, fish, mangoes, cashews, sesame seeds, and almonds.

5. The allergen-specific IgE antibody detection card according to claim 4, characterized in that, The final concentration of the specific antigen-coated antigen on the test card is 0.25 mg / mL - 0.75 mg / mL; the final concentration of the goat anti-rabbit IgG antibody is 0.5 mg / mL - 1.5 mg / mL.

6. The method for preparing the allergy-specific IgE antibody detection card according to any one of claims 1-5, characterized in that, Includes the following steps: (1) Treat the sample pad with sample pad treatment solution, dry it overnight, and then cut it into a certain size for later use; (2) Take microspheres and add them to EDC solution and NHS solution for activation; After centrifugation to remove the supernatant, wash the product. Conjugate with the mouse anti-human IgE antibody; block treatment; Obtain the T-line labeled antibody solution; (3) Take microspheres and add them to EDC solution and NHS solution for activation; After centrifugation to remove the supernatant, wash the product. Conjugate with the rabbit IgG antibody; block treatment; Obtain the C-line labeled antibody solution; (4) Mix the T-line labeled antibody solution, the C-line labeled antibody solution, and the blank labeled reconstituted solution; Spray onto the cut bonding pad; The coupling buffer used to prepare the conjugate pad was a 0.05 mol / L, pH 8.0 boric acid-borax buffer solution. The volume percentages of the T-line labeled antibody solution, C-line labeled antibody solution, and labeled reconstituted solution are (10%-20%):(1%-2%):(78%-89%). (5) Cut and paste the NC membrane, coat the C and T lines with working solution, and prepare the reaction plate; (6) Cut absorbent paper to prepare absorbent pads; (7) Assemble the sample pad, binding pad, absorption pad and reaction plate.

7. The method for preparing the allergy-specific IgE antibody detection card according to claim 6, characterized in that, The sample pad treatment solution comprises 0.05 mg / mL-0.15 mg / mL mouse IgG, 0.1 mg / mL-0.2 mg / mL HBR-5, 0.1 mg / mL-0.2 mg / mL HBR-7, and 0.2 mg / mL-0.3 mg / mL HBR-7. The sample pad buffer contains 2.5 mg / mL RBC antibody and sample pad buffer matrix; the sample pad buffer matrix includes 2.5 mg / mL-7.5 mg / mL casein sodium salt, 7.5 mg / mL-12.5 mg / mL EDTA disodium salt, 25 mg / mL-75 mg / mL trehalose dihydrate, 1.25 mg / mL-2.5 mg / mL PVP-K30, 2.5 mg / mL-7.5 mg / mL ethylphenyl polyethylene glycol (NP-40), 0.0025 mL / mL-0.0075 mL / mL Tween-20, 0.1 μL / mL-0.3 μL / mL ProClin300, and 0.05 mol / L, pH 8.0 Tris-HCl buffer; the EDC solution contains 10 mg / mL EDC and 0.02 mol / L, pH 6.0 MES buffer; the NHS solution contains 10 mg / mL... The labeling and reconstitution solution comprises 5 mg / mL-15 mg / mL casein sodium salt, 10 mg / mL-30 mg / mL anhydrous trehalose, 5 mg / mL-15 mg / mL S9 (propylene oxide-ethylene oxide-vinyl diamine copolymer), 0.1 μL / mL-0.3 μL / mL ProClin300, and 0.05 mol / L borate-borax (BB) buffer at pH 8.0; the labeling buffer is 0.02 mol / L MES buffer at pH 6.0; the blocking solution comprises 10% BSA, 0.02% ProClin300, and purified water.

8. An allergen-specific IgE antibody detection kit, characterized in that, Includes the allergen-specific IgE antibody detection card and sample diluent as described in any one of claims 1-5; The sample diluent was a 0.01 mol / L phosphate buffer.

9. A method for detecting allergen-specific IgE antibodies, characterized in that, Includes the following steps: During testing, the sample is mixed with the diluent and added to the reagent well. Chromatography is performed under capillary action. The specific IgE antibody in the sample binds to the AIE fluorescently labeled mouse anti-human IgE antibody, diffuses to the test area, is captured by the specific antigen coated on the detection line, and forms an "antigen-antibody-AIE fluorescent secondary antibody" complex. The concentration of the specific IgE antibody in the sample is directly proportional to the fluorescence intensity of the complex. According to the set standard curve, the immunofluorescence detector converts the fluorescence signal value into the concentration of the specific IgE antibody in the sample. The concentration of the specific IgE antibody in the sample is detected, and the result is expressed in IU / mL. The sample can be serum, venous blood, or finger-prick blood. The sample diluent was a 0.01 mol / L phosphate buffer.

10. The use of the allergen-specific IgE antibody detection card according to any one of claims 1-5 or the allergen-specific IgE antibody detection kit according to claim 8 in the detection of specific IgE antibodies.

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