An allergen-specific antibody test kit

Abstract

This invention relates to an allergen-specific antibody detection kit, which includes a reaction plate, an enzyme-labeled secondary antibody solution, and a chromogenic substrate solution. The reaction plate has multiple independent microwells with inner surfaces coated with allergens. The enzyme-labeled secondary antibody solution is formulated as follows: 1~50 μg / mL polyHRP-labeled anti-human antibody, the remainder being diluent. The diluent is formulated as follows: 10~50 mM biological buffer, 50~250 mM NaCl, 1~10% trehalose, 0.1~5% bovine serum albumin, 0.01~0.1% nonionic surfactant, and the remainder being water. The chromogenic substrate solution is formulated as follows: 0.01~0.5 M biological buffer, 0.1~2 mM EDTA-2Na, 0.01~1% polyvinylpyrrolidone, 0.005~0.1% benzalkonium chloride, 0.1~0.5% carrageenan, 0.01~0.05 mM sodium thiosulfate, 0.01~0.5% polyacrylamide, 0.01~0.1% preservative, 20~50% H2O2, and 1~5 mM... TMB, 0.1-1% (v / v) DMSO, balance water. This invention enables micro-sampling, visual interpretation, high sensitivity and specificity, and long-term room temperature storage.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostic reagents, specifically relating to an allergen-specific antibody detection kit. Background Technology

[0002] In recent years, with the increasing incidence of allergic diseases, in vitro diagnostic reagents have been increasingly widely used in the field of allergen detection. Common detection platforms currently available include enzyme-linked immunosorbent assay (ELISA), immunoblotting, microfluidics, and chemiluminescence immunoassay. These technologies have achieved qualitative and quantitative detection of various allergens to a certain extent, but the current sample types are still mainly venous whole blood or serum.

[0003] However, existing clinically used allergen detection methods still have the following major shortcomings: 1. Inconvenient sampling: Traditional venous blood collection usually requires ≥5 mL of blood sample, which is uncomfortable for patients, especially children, the elderly, and patients with chronic diseases, leading to poor compliance. In addition, skin prick tests are easily interfered with by antihistamines, glucocorticoids, and other drugs, which may lead to false negative results. 2. Strong equipment dependence: Mainstream chemiluminescence and ELISA methods require large, precise detection instruments and a stable laboratory environment, limiting their application in primary healthcare institutions or in areas without laboratories. 3. Poor timeliness: Although conventional immunochromatographic test strips can achieve rapid detection, their sensitivity is generally low. The test results often require professional technicians to interpret them based on clinical experience, which is not conducive to on-site immediate diagnosis. 4. High storage and transportation costs: Most existing in vitro diagnostic reagents require cold chain transportation and storage at 2–8℃ to ensure their biological activity. This not only increases logistics and storage costs but also makes it more difficult to promote their application in resource-limited areas.

[0004] In summary, given the shortcomings of existing allergen detection technologies in terms of sample collection, equipment dependence, detection timeliness, and storage and transportation conditions, it is necessary to develop a new type of test kit that can reduce sample volume, decrease equipment dependence, improve detection sensitivity and stability, and be suitable for room temperature storage and transportation, so as to meet the diverse application needs of multiple scenarios such as clinical, primary healthcare, and home use. Summary of the Invention

[0005] The purpose of this invention is to provide an allergen-specific antibody detection kit that enables micro-sampling, visual interpretation, high sensitivity and specificity, and long-term room temperature storage.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides an allergen-specific antibody detection kit, which includes a reaction plate, an enzyme-labeled secondary antibody solution, and a chromogenic substrate solution.

[0007] The reaction plate has multiple independent micropores, the inner surface of which is coated with an allergen. Physical isolation is formed between the micropores, and each micropore is independently coated with an allergen to prevent cross-contamination. Preferably, each micropore is coated with only one type of allergen. The micropores serve as reaction containers and solid-phase carriers for allergen coating, blood sample incubation, and colorimetric reactions.

[0008] The enzyme-labeled secondary antibody solution is formulated as follows: 1~50 μg / mL enzyme-labeled secondary antibody, with the remainder being diluent. The diluent is formulated as follows: 10~50 mM biological buffer, 50~250 mM NaCl, 1~10% (w / v) trehalose, 0.1~5% (w / v) bovine serum albumin, 0.01~0.1% (w / v) nonionic surfactant, with the remainder being water. The enzyme-labeled secondary antibody includes polyHRP-labeled anti-human antibody.

[0009] The formulation of the chromogenic substrate solution is as follows: 0.01~0.5M biological buffer, 0.1~2mM EDTA-2Na, 0.01~1% (w / v) polyvinylpyrrolidone, 0.005~0.1% (v / v) benzalkonium chloride, 0.1~0.5% (w / v) carrageenan, 0.01~0.05mM sodium thiosulfate, 0.01~0.5% (w / v) polyacrylamide, 0.01~0.1% (v / v) preservative, 20~50% (v / v) H2O2, 1~5mM TMB (3,3',5,5'-tetramethylbenzidine), 0.1~1% (v / v) DMSO, with the balance being water.

[0010] In some embodiments, the concentration of the enzyme-labeled secondary antibody in the enzyme-labeled secondary antibody solution is 1~10 μg / mL, for example 1 μg / mL, 0.5 μg / mL, 2 μg / mL, 2.5 μg / mL, 3 μg / mL, 3.5 μg / mL, 4 μg / mL, 4.5 μg / mL, 5 μg / mL, 5.5 μg / mL, 6 μg / mL, 6.5 μg / mL, 7 μg / mL, 7.5 μg / mL, 8 μg / mL, 8.5 μg / mL, 9 μg / mL, 9.5 μg / mL, and 10 μg / mL.

[0011] According to some preferred embodiments, the diluent is formulated as follows: 10-30 mM biological buffer, 100-200 mM NaCl, 2-8% (w / v) trehalose, 0.5-2% (w / v) bovine serum albumin, 0.03-0.08% (w / v) nonionic surfactant, and the balance being water.

[0012] In some embodiments, the enzyme-labeled secondary antibody is a polyHRP-labeled anti-human IgG antibody or a polyHRP-labeled anti-human IgE antibody. Preferably, it includes both an enzyme-labeled secondary antibody solution containing polyHRP-labeled anti-human IgG antibody and an enzyme-labeled secondary antibody solution containing polyHRP-labeled anti-human IgE antibody, thereby providing two independent detection modules simultaneously: an IgG detection module using the polyHRP-labeled anti-human IgG antibody solution and an IgE detection module using the polyHRP-labeled anti-human IgE antibody solution. Users can select a single module for detection according to their needs, and only need to select the appropriate enzyme-labeled secondary antibody solution to switch detection types.

[0013] In some embodiments, the biological buffer in the diluent is HEPES.

[0014] In some embodiments, the nonionic surfactant is Pluronic F68.

[0015] According to some preferred embodiments, the formulation of the chromogenic substrate solution is as follows: 0.05~0.2M biological buffer, 0.1~1mM EDTA-2Na, 0.05~0.5% (w / v) polyvinylpyrrolidone, 0.005~0.05% (v / v) benzalkonium chloride, 0.1~0.3% (w / v) carrageenan, 0.01~0.03mM sodium thiosulfate, 0.01~0.2% (w / v) polyacrylamide, 0.01~0.05% (v / v) preservative, 25~35% (v / v) H2O2, 1~4mM TMB, 0.1~1% (v / v) DMSO, and the balance being water.

[0016] In some embodiments, the biological buffer in the chromogenic substrate solution is MES, and the organic solvent is dimethyl sulfoxide.

[0017] The enzyme-labeled secondary antibody solution and the chromogenic substrate solution constitute an ultrasensitive rapid chromogenic system with the following chromogenic characteristics: extremely rapid response: the chromogenic reaction is completed within 1 minute, and a stable state is reached in 2 minutes; gradient visualization: the color changes from light blue to blue to dark blue according to the antibody concentration; comparative advantages: compared with traditional TMB soluble substrates, the sensitivity is increased by 5 times, background interference is reduced by 60%, and the reaction time is shortened by 80%. In addition, the reagents have good stability, especially the chromogenic substrate solution, which has significantly improved stability. The kit can be transported and stored at room temperature.

[0018] In some embodiments, the coating method for the allergen is as follows: the allergen is diluted to the working concentration using an allergen diluent, added to the micropores, dried in an environment of 25~37°C, sealed with a blocking solution, washed, and dried.

[0019] Furthermore, the formula for the allergen diluent is: 1~2g / L anhydrous sodium carbonate, 2~4g / L sodium bicarbonate, and the remainder water.

[0020] Furthermore, the blocking solution is formulated as follows: 0.05~0.5M MES, 0.5~1.5% (w / v) sodium chloride, 0.5~1.5% (w / v) bovine serum albumin, and the balance being water, with a pH value of 7~7.5.

[0021] In some embodiments, the allergen is a food allergen, selected from corn allergens, rice allergens, milk allergens, egg allergens, wheat allergens, soybean allergens, tomato allergens, beef allergens, chicken allergens, pork allergens, crab allergens, shrimp allergens, cod allergens, or mushroom allergens.

[0022] In some embodiments, the reaction plate is provided with multiple microwells coated with the same allergen, allowing for simultaneous detection of the same item on multiple samples.

[0023] In other embodiments, the reaction plate has multiple micropores coated with different allergens, allowing for the simultaneous detection of multiple substances.

[0024] In some embodiments, the reaction plate is made of modified polystyrene.

[0025] Specifically, the reaction plate is white. The micropores are cylindrical with open tops, serving as observation windows. The white material makes it easier to observe the color changes after the micropore reaction with the naked eye, and facilitates comparison of the intensity of the blue-purple color after a positive reaction.

[0026] Specifically, the inner diameter of the microwell is 2-5 mm. Compared with traditional 96-well plates, the volume of the microwell can be reduced by up to 80%, enabling the detection of food-specific antibodies based on finger-prick blood, achieving integrated detection of "one drop of blood → multiple indicators".

[0027] Specifically, the reaction plate is further provided with micropores with positive quality control coating on the inner surface and / or micropores with negative quality control coating on the inner surface.

[0028] In some embodiments, the kit further includes a sample diluent formulated as follows: 5-10 mM disodium hydrogen phosphate dodecahydrate, 2-4 mM sodium dihydrogen phosphate dihydrate, 0.5-1.2% (w / v) sodium chloride, 0.5-1.5% (w / v) bovine serum albumin, 0.05-0.2% (v / v) nonionic surfactant, 0.01-0.1% (v / v) preservative, with the balance being water, and the pH of the sample diluent being 7-7.5.

[0029] Furthermore, the nonionic surfactant in the sample diluent is selected from Triton X-100 and Tween-20.

[0030] Furthermore, the preservative in the sample diluent is Proclin-300.

[0031] In some embodiments, the kit further includes a washing solution formulated with: 0.1-0.2% (w / v) trihydroxyaminomethane, 0.5-1.5% (w / v) sodium chloride, 0.001-0.005% (w / v) nonionic surfactant, and the balance being water.

[0032] Furthermore, the nonionic surfactant in the washing liquid is Triton X-100.

[0033] In some embodiments, the kit further includes an antibody concentration-color gradient colorimetric card, which can be used for qualitative analysis of positive or negative or semi-quantitative assessment of antibody levels in samples. The colorimetric card has multiple color levels, each corresponding to a concentration range, which is determined by the detection of the corresponding antibody sample.

[0034] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art: This invention, through a combination of a specially formulated enzyme-labeled secondary antibody solution and a specially formulated substrate reaction solution, along with a reaction plate design, significantly enhances the signal intensity and reagent stability of the colorimetric reaction. It can achieve highly sensitive and visualized qualitative / semi-quantitative detection of allergens with only 50 μL of blood sample. This not only greatly reduces the difficulty of sampling and equipment dependence, but also solves the pain points of traditional detection in terms of sampling, cost and universality. It can be widely used in home, community and primary healthcare, and has the potential to expand into open platform fields such as autoimmune diseases and tumor markers, with significant social and clinical value. Attached Figure Description

[0035] Figure 1 This is a three-dimensional structural diagram of the reaction plate in the kit of the present invention; Figure 2 This is a top view of the reaction plate in the kit of the present invention; Figure 3 This is a schematic diagram showing the state of the reaction plate in the kit of the present invention during use. In the attached diagrams above, 1 represents the reaction plate, and 11 represents the micropores. Detailed Implementation

[0036] To address the pain points of traditional allergen-specific antibody testing, such as large sample volumes, difficult blood collection, expensive equipment, narrow range of indicators, and difficulties in storage and transportation, and to promote the era of home-based, real-time, and intelligent allergen testing, the inventors of this application optimized the reagent formulation in the kit and conducted extensive experimental screening and verification, designed a corresponding reaction plate, and obtained the allergen-specific antibody detection kit based on micro-samples of this application. The technical solution is as follows: An allergen-specific antibody detection kit includes a reaction plate, an enzyme-labeled secondary antibody solution, and a chromogenic substrate solution. The reaction plate has multiple independent microwells, the inner surface of which is coated with an allergen. The enzyme-labeled secondary antibody solution is formulated as follows: 1-50 μg / mL enzyme-labeled secondary antibody, with the remainder being diluent. The diluent is formulated as follows: 10-50 mM biological buffer, 50-250 mM NaCl, 1-10% (w / v) trehalose, 0.1-5% (w / v) bovine serum albumin, 0.01-0.1% (w / v) nonionic surfactant, with the remainder being water. The enzyme-labeled secondary antibody includes a poly(HRP)-labeled anti-human antibody. The formulation of the chromogenic substrate solution is as follows: 0.01~0.5M biological buffer, 0.1~2mM EDTA-2Na, 0.01~1% (w / v) polyvinylpyrrolidone, 0.005~0.1% (v / v) benzalkonium chloride, 0.1~0.5% (w / v) carrageenan, 0.01~0.05mM sodium thiosulfate, 0.01~0.5% (w / v) polyacrylamide, 0.01~0.1% (v / v) preservative, 20~50% (v / v) H2O2, 1~5mM TMB, 0.1~1% (v / v) DMSO, with the balance being water.

[0037] By combining specially formulated enzyme-labeled secondary antibody solutions and substrate reaction solutions, the signal intensity and reagent stability of the colorimetric reaction are significantly enhanced. Stable reagent storage at room temperature for up to 18 months is achieved. This technology addresses the industry pain point of high cold chain transportation costs, significantly reducing logistics barriers and usage costs. Combined with a self-developed colorimetric interpretation algorithm, qualitative or semi-quantitative analysis can be completed simply by observing color gradient changes, without relying on large instruments.

[0038] Thanks to improvements in the colorimetric system, detection sensitivity is significantly enhanced. The kit of this invention requires only a tiny 50 μL of fingertip blood sample, far lower than the amount required for traditional venous blood collection (≥5 mL). This not only significantly reduces pain and psychological burden on the examinee but also greatly improves compliance among children, the elderly, and those prone to needle phobia. It also allows for self-administration via home blood collection devices, effectively shortening waiting time for diagnosis and treatment.

[0039] By setting different enzyme-labeled secondary antibody solutions, a dual detection platform was established that can simultaneously detect specific IgE (immediate-type allergy) and specific IgG (delayed-type / chronic allergy), breaking through the limitation of traditional detection that can only cover a single type of allergy and realizing a comprehensive analysis of the allergy mechanism.

[0040] This invention systematically solves the bottlenecks of traditional allergen testing in terms of sampling convenience, equipment dependence, indicator coverage, and storage and transportation costs. The technology has extremely high versatility and is suitable for scenarios lacking large-scale equipment, such as pediatrics, emergency rooms, primary care clinics, and health checkup centers. Its open platform architecture also has broad research expansion potential, extending to point-of-care testing (POCT) in multiple fields such as autoimmune disease antibodies and tumor markers. This technology promotes the development of allergen testing towards home-based, real-time, and intelligent approaches, with significant socio-economic benefits and clinical application value.

[0041] The present invention will be further described below with reference to embodiments and comparative examples. However, the present invention is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to different requirements of specific applications, and the implementation conditions not specified are conventional conditions in the industry. The technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0042] In the following examples and comparative examples, the implementation conditions not specified are standard conditions in the industry, and the reagents not specified are all commercially available products.

[0043] Corn allergen: purchased from Greer Laboratories, Inc. (USA), catalog number XPF102D3A25, working concentration 1.50 μg / mL.

[0044] Rice allergen: purchased from Greer Laboratories, Inc. (USA), catalog number XPF200D3A25, working concentration 2.00 μg / mL.

[0045] Milk allergen: Purchased from Greer Laboratories, Inc. (USA), catalog number XPF395D3A25, working concentration 2.00 μg / mL.

[0046] Egg allergen: Purchased from Greer Laboratories, Inc. (USA), catalog number XPF271D3A2.5, working concentration 0.80 μg / mL.

[0047] Wheat allergen: purchased from Greer Laboratories, Inc. (USA), catalog number XPF235D3A2.5, working concentration 1.80 μg / mL.

[0048] Soybean allergen: purchased from Greer Laboratories, Inc. (USA), catalog number XPF209D3A2.5, working concentration 3.00 μg / mL.

[0049] Tomato allergen: purchased from Greer Laboratories, Inc. (USA), catalog number XPF225D3A5, working concentration 0.10 μg / mL.

[0050] Beef allergen: purchased from Greer Laboratories, Inc. (USA), catalog number XPF241D3A2.5, working concentration 8.00 μg / mL.

[0051] Chicken allergen: Purchased from Greer Laboratories, Inc. (USA), catalog number XPF265D3A25, working concentration 6.00 μg / mL.

[0052] Pork allergen: Purchased from Greer Laboratories, Inc. (USA), catalog number XPF258D3A25, working concentration 7.00 μg / mL.

[0053] Crab allergen: purchased from Greer Laboratories, Inc. (USA), catalog number XPF12D3A2.5, working concentration 4.00 μg / mL.

[0054] Shrimp allergen: purchased from Greer Laboratories, Inc. (USA), catalog number XPF34D3A25, working concentration 2.50 μg / mL.

[0055] Cod allergen: purchased from Greer Laboratories, Inc. (USA), catalog number XPF11D3A2.5, working concentration 3.00 μg / mL.

[0056] Mushroom allergen: purchased from Greer Laboratories, Inc. (USA), catalog number XPF150D3A2.5, working concentration 0.25 μg / mL.

[0057] Example 1: This example provides a multiplex allergen-specific antibody (IgE / IgG) detection kit based on a small amount of blood sample.

[0058] The kit in this embodiment includes reaction plate 1, reagent 1, reagent 2, reagent 3, reagent 4, and colorimetric card.

[0059] The reaction plate has multiple independent micropores 11, each of which is a micro-reaction cell with an inner diameter of 3 mm. The reaction plate 1 is made of white modified polystyrene, and the micropores 11 are formed by nanoscale surface etching. The inner surface of the micropores 11 is coated with allergens, positive controls, or negative controls.

[0060] In this embodiment, the reaction plate has 14 microwells, each coated with a different allergen and numbered sequentially, one microwell coated with a positive control and one microwell coated with a negative control. The allergens are coated in ascending order of number: corn allergen, rice allergen, milk allergen, egg allergen, wheat allergen, soybean allergen, tomato allergen, beef allergen, chicken allergen, pork allergen, crab allergen, shrimp allergen, cod allergen, and mushroom allergen.

[0061] The allergen coating method was as follows: A working solution of the corresponding working concentration was prepared using allergen diluent (1.59 g / L anhydrous sodium carbonate, 2.93 g / L sodium bicarbonate, balance water) and the allergen was applied to the wells of the reaction plate using a pipette (6 μL). The plate was then dried at 25–37 °C for 2 hours. Blocking buffer (0.1 M MES, 0.9% (w / v) sodium chloride, 1% (w / v) bovine serum albumin, balance water, pH 7.4) was added and used to block for 1 hour. The plate was then washed twice with washing buffer. Finally, it was dried at 37 °C in a forced-air drying environment for 2 hours. The washing buffer consisted of 0.01 M Trisbase, 0.9% (w / v) sodium chloride, and 0.1% (v / v) Triton X-100, pH 7.4.

[0062] The positive control coating method is the same as the allergen coating method, except that the allergen is replaced with human IgG protein.

[0063] The negative control coating method is the same as the allergen coating method, except that the allergen is replaced with an allergen dilution.

[0064] Reagent 1 is an enzyme-labeled secondary antibody solution: 4 μg / mL enzyme-labeled secondary antibody, the remainder being diluent. The diluent formula is: 20 mM HEPES, 150 mM NaCl, 5% (w / v) trehalose, 1% (w / v) bovine serum albumin, 0.05% (w / v) Pluronic F68, and the remainder being water.

[0065] The enzyme-labeled secondary antibodies are polyHRP-labeled anti-human IgG antibody and polyHRP-labeled anti-human IgE antibody produced by Suzhou Xiruimas Chemical Co., Ltd. The corresponding enzyme-labeled secondary antibody solution is selected according to the detection requirements.

[0066] Reagent 2 is the chromogenic substrate solution: 0.1M MES, 0.59mM EDTA-2Na, 0.1% (w / v) PVP K30, 0.01% (v / v) benzalkonium chloride, 0.2% (w / v) carrageenan, 0.019mM sodium thiosulfate, 0.1% (w / v) polyacrylamide, 0.04% (v / v) Proclin-300, 30% (v / v) H2O2, 2.496mM TMB, 0.3% (v / v) DMSO, with the balance being water. TMB and DMSO are added as a TMB solution, with DMSO (dimethyl sulfoxide) used to dissolve TMB and prepare a TMB stock solution.

[0067] Weigh each component according to the formula and mix them thoroughly to prepare reagent 2.

[0068] Reagent 3 is the sample diluent: 7.2 mM disodium hydrogen phosphate dodecahydrate, 2.8 mM sodium dihydrogen phosphate dihydrate, 0.9% (w / v) sodium chloride, 1% (w / v) bovine serum albumin, 0.1% (v / v) Triton X-100, 0.05% (v / v) Proclin-300, with the remainder being water, pH 7.4.

[0069] Weigh each component according to the formula and mix them thoroughly to prepare reagent 3.

[0070] Reagent 4 is the washing solution: 0.14% (w / v) trihydroxyaminomethane, 0.9% (w / v) sodium chloride, 0.002% (w / v) Triton X-100, and the balance is water.

[0071] Weigh each component according to the formula and mix them thoroughly to prepare reagent 4.

[0072] Colorimetric chart: An antibody concentration-color gradient colorimetric chart, which can be used for qualitative analysis of positive and negative or semi-quantitative assessment of antibody levels in samples. The colorimetric chart has multiple color levels, each corresponding to a concentration range, which is determined by the detection of the corresponding antibody sample.

[0073] Comparative Example 1: This comparative example provides another multiplex allergen-specific antibody (IgE / IgG) detection kit based on a small amount of fingertip blood sample.

[0074] The kit for this comparative example is basically the same as that in Example 1, except that the diluent for the enzyme-labeled secondary antibody solution in Reagent 1 is different. The diluent formulation for Reagent 1 in this comparative example is: 20 mM HEPES, 150 mM NaCl, and 1% (w / v) bovine serum albumin.

[0075] Comparative Example 2: This comparative example provides another multiplex allergen-specific antibody (IgE / IgG) detection kit based on a small amount of fingertip blood sample.

[0076] The kit for this comparative example is basically the same as that in Example 1, except that the diluent for the enzyme-labeled secondary antibody solution in Reagent 1 is different. The diluent formulation for Reagent 1 in this comparative example is: 20 mM HEPES, 150 mM NaCl, 5% (w / v) trehalose, and 1% (w / v) bovine serum albumin.

[0077] Comparative Example 3: This comparative example provides another multiplex allergen-specific antibody (IgE / IgG) detection kit based on a small amount of fingertip blood sample.

[0078] The kit for this comparative example is basically the same as that in Example 1, except that the formulation of the chromogenic substrate solution in Reagent 2 is slightly different. The formulation of the chromogenic substrate solution in Reagent 2 in this comparative example is: 0.1M MES, 0.59mM EDTA-2NA, 0.1% (w / v) PVP K30, 0.019mM sodium thiosulfate, 0.1% (w / v) polyacrylamide, 0.04% (v / v) Proclin-300, 30% (v / v) H2O2, 2.496mM TMB, 0.3% (v / v) DMSO, with the remainder being water.

[0079] Comparative Example 4: This comparative example provides another multiplex allergen-specific antibody (IgE / IgG) detection kit based on a small amount of fingertip blood sample.

[0080] The kit for this comparative example is basically the same as that in Example 1, except that the formulation of the chromogenic substrate solution in Reagent 2 is slightly different. The formulation of the chromogenic substrate solution in Reagent 2 in this comparative example is: 0.1M MES, 0.59mM EDTA-2NA, 0.1% (w / v) PVP K30, 0.01% (v / v) benzalkonium chloride, 0.019mM sodium thiosulfate, 0.1% (w / v) polyacrylamide, 0.04% (v / v) Proclin-300, 30% (v / v) H2O2, 2.496mM TMB, 0.3% (v / v) DMSO, with the remainder being water.

[0081] The usage methods of the kits in the above examples and comparative examples are as follows: Add sample: Use a capillary tube to draw 50 μL of fingertip blood into a reagent bottle containing 5 mL of reagent 3 (sample diluent), mix by inverting the bottle, and then pour it into the reaction plate; Incubation: Incubate at room temperature for 20 minutes; Washing: Add 5 mL of reagent 4 (washing solution), gently shake, pour out and pat dry. Repeat twice. Enzyme-labeled secondary antibody: Add 3 mL of reagent 1 (enzyme-labeled secondary antibody solution, IgE / IgG) and incubate at room temperature for 10 minutes; Washing: Add 5 mL of reagent 4 (washing solution), gently shake, pour out and pat dry. Repeat twice. Color development: Add 3 mL of reagent 2 (color development substrate solution) and incubate at room temperature for 2 minutes; Washing: Add 5 mL of reagent 4 (washing solution), gently shake, pour out and pat dry. Repeat twice. Interpretation: Observe the color of the reaction wells and compare it with the colorimetric card to determine the result.

[0082] Performance test comparison: After storing the kits of Example 1, Comparative Example 1, and Comparative Example 2 at 37°C for 10, 28, and 90 days, respectively, food-specific IgG antibody detection was performed on 20 clinical samples (only milk-specific IgG antibody detection was performed). The detection results of the kit stored at 4°C were used as controls. The positive and negative results of the clinical samples were determined by the commercially available food-specific IgG antibody detection kit (enzyme-linked immunosorbent assay) (Jiangsu Haooubo Biomedical Co., Ltd., Jiangsu Medical Device Registration Certificate No. 20162400480) according to its instructions. The detection results are shown in Table 1.

[0083] Table 1

[0084] Table 1 shows that the kits for Comparative Examples 1 and 2 had poor performance when stored at room temperature, resulting in varying degrees of false negatives. The kit for Example 1 showed results consistent with those obtained by enzyme-linked immunosorbent assay (ELISA) and excellent performance when stored at room temperature.

[0085] After storing the kits from Examples 1, 3, and 4 at 37°C for 90 days, food-specific IgG antibody tests were performed on 22 clinical samples and 10 physical examination samples (only milk-specific IgG antibody tests were performed). The test results of the kits stored at 4°C were used as controls. The positive or negative results of the clinical samples were determined by testing with a commercially available food-specific IgG antibody test kit (enzyme-linked immunosorbent assay) (Jiangsu Haooubo Biomedical Co., Ltd., Jiangsu Medical Device Registration Certificate No. 20162400480) according to its instructions. The test results are shown in Table 2.

[0086] Table 2

[0087] Table 2 shows that the detection performance of the kit in Comparative Example 3 was poor, with a large number of false positives and missed positives after storage at both 4°C and 37°C. The kit in Comparative Example 4 had poor performance after storage at room temperature, with varying degrees of false positives and a small number of missed positives. The kit in Example 1 showed consistent detection results with the enzyme-linked immunosorbent assay (ELISA) and excellent performance after storage at room temperature. Furthermore, the state of reagent 2 in each kit was observed before testing. Reagent 2 in Comparative Example 3 was turbid and showed precipitation regardless of whether it was stored at 4°C or 37°C, while reagent 2 in Comparative Example 4 and Example 1 was clear regardless of whether it was stored at 4°C or 37°C.

[0088] Verification of the detection results of the kit in Example 1: Methodological comparison (50 clinical cases): The kit from Example 1 was used for testing to confirm that the test results were consistent with the known values. The known values ​​were determined by testing with a commercially available food-specific IgG antibody detection kit (enzyme-linked immunosorbent assay) (Jiangsu Haooubo Biomedical Co., Ltd., Jiangsu Medical Device Registration Certificate No. 20162400480) according to its instructions.

[0089] Table 3

[0090] Stability: The kit of Example 1 was placed at 37°C for 90 days. Then, 3 negative samples and 7 positive samples were selected for each allergen and tested using the accelerated kit. 4°C was used as a control. The comparison results are shown in Table 4.

[0091] Table 4

[0092] Repeatability: One positive sample was selected for each allergen test and tested 10 times consecutively. The consistency of the results of the 10 tests was compared. The results are shown in Table 5.

[0093] Table 5

[0094] Specificity: One positive sample was selected for each allergen test. IgA, IgM and IgE were added to the sample respectively, and no addition was used as a control. The difference in test results before and after addition was compared. The results are shown in Table 6.

[0095] Table 6

[0096] As can be seen from Tables 3 to 6, the kit of Example 1 has the advantages of small sample volume, convenient sampling, high detection sensitivity, good specificity, low dependence on visualization and interpretation equipment, good kit stability, and long-term room temperature storage.

[0097] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. An allergen-specific antibody detection kit, characterized in that, It includes a reaction plate, enzyme-labeled secondary antibody solution, and chromogenic substrate solution. The reaction plate has multiple independent micropores, and the inner surface of each micropore is coated with an allergen. The enzyme-labeled secondary antibody solution is formulated as follows: 1~50 μg / mL enzyme-labeled secondary antibody, with the remainder being diluent. The diluent is formulated as follows: 10~50 mM biological buffer, 50~250 mM NaCl, 1~10% (w / v) trehalose, 0.1~5% (w / v) bovine serum albumin, 0.01~0.1% (w / v) nonionic surfactant, with the remainder being water. The enzyme-labeled secondary antibody includes polyHRP-labeled anti-human antibody. The formulation of the chromogenic substrate solution is as follows: 0.01~0.5M biological buffer, 0.1~2mM EDTA-2Na, 0.01~1% (w / v) polyvinylpyrrolidone, 0.005~0.1% (v / v) benzalkonium chloride, 0.1~0.5% (w / v) carrageenan, 0.01~0.05mM sodium thiosulfate, 0.01~0.5% (w / v) polyacrylamide, 0.01~0.1% (v / v) preservative, 20~50% (v / v) H2O2, 1~5mM TMB, 0.1~1% (v / v) DMSO, with the balance being water.

2. The allergen-specific antibody detection kit according to claim 1, characterized in that, The concentration of the enzyme-labeled secondary antibody in the enzyme-labeled secondary antibody solution is 1~10 μg / mL. And / or, the diluent is formulated as follows: 10-30 mM biological buffer, 100-200 mM NaCl, 2-8% (w / v) trehalose, 0.5-2% (w / v) bovine serum albumin, 0.03-0.08% (w / v) nonionic surfactant, with the balance being water; And / or, the enzyme-labeled secondary antibody is a polyHRP-labeled anti-human IgG antibody or a polyHRP-labeled anti-human IgE antibody.

3. The allergen-specific antibody detection kit according to claim 1, characterized in that, The formulation of the chromogenic substrate solution is as follows: 0.05~0.2M biological buffer, 0.1~1mM EDTA-2Na, 0.05~0.5% (w / v) polyvinylpyrrolidone, 0.005~0.05% (v / v) benzalkonium chloride, 0.1~0.3% (w / v) carrageenan, 0.01~0.03mM sodium thiosulfate, 0.01~0.2% (w / v) polyacrylamide, 0.01~0.05% (v / v) preservative, 25~35% H2O2, 1~4mM TMB, with the balance being organic solvent.

4. The allergen-specific antibody detection kit according to claim 1, characterized in that, The biological buffer in the diluent is HEPES, and the nonionic surfactant is Pluronic F68; And / or, the biological buffer in the chromogenic substrate solution is MES, and the organic solvent is dimethyl sulfoxide.

5. The allergen-specific antibody detection kit according to claim 1, characterized in that, The coating method for the allergen is as follows: The allergen is diluted to the working concentration using an allergen diluent, added to the microwells, dried at 25-37°C, sealed with a blocking solution, washed, and dried again. The formula for the allergen diluent is: 1~2g / L anhydrous sodium carbonate, 2~4g / L sodium bicarbonate, and the remainder water. The blocking solution is formulated as follows: 0.05~0.5M MES, 0.5~1.5% (w / v) sodium chloride, 0.5~1.5% (w / v) bovine serum albumin, and the balance being water. The pH value of the blocking solution is 7~7.

5.

6. The allergen-specific antibody detection kit according to claim 1, characterized in that, The allergens mentioned are food allergens, selected from corn allergens, rice allergens, milk allergens, egg allergens, wheat allergens, soybean allergens, tomato allergens, beef allergens, chicken allergens, pork allergens, crab allergens, shrimp allergens, cod allergens, or mushroom allergens.

7. The allergen-specific antibody detection kit according to claim 1, characterized in that, The reaction plate has multiple micropores coated with the same allergen, or the reaction plate has multiple micropores coated with different allergens. And / or, the material of the reaction plate is modified polystyrene; And / or, the reaction plate is white; And / or, the inner diameter of the micropore is 2~5mm; And / or, the reaction plate is further provided with micropores with positive quality control on the inner surface and / or micropores with negative quality control on the inner surface.

8. The allergen-specific antibody detection kit according to claim 1, characterized in that, It also includes a sample diluent, the formulation of which is: 5-10 mM disodium hydrogen phosphate dodecahydrate, 2-4 mM sodium dihydrogen phosphate dihydrate, 0.5-1.2% (w / v) sodium chloride, 0.5-1.5% (w / v) bovine serum albumin, 0.05-0.2% (v / v) nonionic surfactant, 0.01-0.1% (v / v) preservative, with the balance being water, and the pH value of the sample diluent is 7-7.

5.

9. The allergen-specific antibody detection kit according to claim 1, characterized in that, It also includes a washing liquid, the washing liquid having the following formula: 0.1~0.2% (w / v) trihydroxyaminomethane, 0.5~1.5% (w / v) sodium chloride, 0.001~0.005% (w / v) nonionic surfactant, and the balance being water.

10. The allergen-specific antibody detection kit according to claim 1, characterized in that, It also includes an antibody concentration-color gradient colorimetric chart.

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