A kit for allergen-specific IgE antibody detection

Abstract

The application relates to the technical field of in-vitro diagnosis, and particularly relates to a kit for detecting allergen-specific IgE antibodies. The kit comprises an anti-interference agent for mixing with a sample to be detected, an immunolabeled allergen, a complex of peroxidase capable of being combined with the immunolabel and capable of catalyzing the color development of a substrate, and a substrate capable of being catalyzed to develop color by the peroxidase; the anti-interference agent comprises the following components in parts by weight: polystyrene microspheres 3-6 parts, levogluside 12-18 parts, and casein 6-10 parts. The kit provided by the application can effectively reduce false positives and false negatives, thereby improving the accuracy of the detection result.

Description

Technical Field

[0001] This application relates to the field of in vitro diagnostics, and more particularly to a kit for detecting allergen-specific IgE antibodies. Background Technology

[0002] In the field of allergen detection, in vitro detection of specific IgE is widely used and can effectively assist in the diagnosis of allergic diseases. However, in actual testing, various interfering substances exist in the sample, which seriously affect the accuracy of the test results, leading to false positive or false negative results and interfering with clinical diagnosis.

[0003] The samples contain a wide variety of interfering substances. Heterophilic antibodies, which are antibodies against unspecified antigens present in human peripheral blood, are multispecific and have weak affinity. They can interact with labeled or capture antibodies in the detection system by binding to the Fc fragment of IgG, interfering with the detection of various indicators such as human chorionic gonadotropin (HCG) and follicle-stimulating hormone (FSH). Rheumatoid factor is an autoantibody that targets denatured IgG and can bridge antibodies in the detection system, interfering with immunoassays. The degree of interference is not clearly dose-dependent. Human anti-animal antibodies (such as the common human anti-mouse antibody HAMA), caused by iatrogenic or non-iatrogenic factors, have strong affinity, target specific antigens, and interfere with antibodies from the corresponding sources.

[0004] In addition, complement, autoantibodies, and hormone-binding proteins in the sample can also interfere with the detection. Elevated complement levels in cases of acute inflammation can lead to false positive or false negative results in enzyme-linked immunosorbent assay (ELISA) systems; autoantibodies bind to target antigens to form complexes, affecting the results of target antigen assays; and hormone-binding proteins bind to hormones, altering the concentration of the analyte in the sample and interfering with the detection.

[0005] To address the aforementioned interference issues, it is essential to incorporate blocking agents into allergen IgE detection kits. Non-specific blocking agents target a wide variety of interfering substances widely present in the sample. They prevent non-specific binding by saturating unbound binding sites, providing alternative binding sites, or creating steric hindrance through specific binding to interfering substances. This reduces the binding of interfering antibodies to the test antibody components, thereby improving the sensitivity and specificity of the detection.

[0006] Currently, although there are various blocking agents available for allergen detection kits on the market, many problems still exist. Some blocking agents are not effective against specific interfering substances and cannot comprehensively address multiple interferences in complex samples. For example, animal IgG, as a common passive blocking agent, can provide alternative binding sites to prevent interfering antibodies from binding to capture or detection antibodies, but it can only block specific types of interference. Multiple IgGs need to be used in combination, and the blocking effect depends on the affinity between the interfering antibody and the animal IgG.

[0007] Furthermore, while some blocking agents reduce interference, they can negatively impact the detection signal. For example, excessive use of passive blocking agents can lead to a decrease in the detection signal, affecting the detection of low concentrations of allergen IgE. Additionally, the applicability of existing blocking agents varies across different sample types, making it difficult to guarantee stable blocking effects in various samples such as serum, plasma, and nasal secretions.

[0008] Therefore, further optimization of the blocking agents in allergen IgE detection kits is currently needed. Summary of the Invention

[0009] This application provides a kit for detecting allergen-specific IgE antibodies. The kit provided by this application can effectively reduce false positives and false positive results, thereby improving the accuracy of the test results.

[0010] In the first aspect, this application provides a kit for detecting allergen-specific IgE antibodies, employing the following technical solution:

[0011] A kit for detecting allergen-specific IgE antibodies, the kit comprising an anti-interference agent for mixing with a sample to be tested, an immunolabeled allergen, a complex of a peroxidase capable of binding to the immunolabel and catalyzing a substrate for color development, and a substrate capable of being catalyzed by the peroxidase for color development.

[0012] The anti-interference agent comprises the following components in parts by weight: 3-6 parts polystyrene microspheres, 12-18 parts levoglucoside, and 6-10 parts casein.

[0013] Optionally, the anti-interference agent comprises the following components in parts by weight: 4-5 parts polystyrene microspheres, 14-16 parts L-glycoside, and 7-9 parts casein.

[0014] In one specific embodiment, the polystyrene microspheres are in the following weight proportions: 3 parts, 4 parts, 4.5 parts, 5 parts, and 6 parts.

[0015] In some specific embodiments, the polystyrene microspheres are in the following weight proportions: 3-4 parts, 3-4.5 parts, 3-5 parts, 4-4.5 parts, 4-5 parts, 4-6 parts, 4.5-5 parts, 4.5-6 parts, and 5-6 parts.

[0016] In one specific embodiment, the levoglucosinolate is present in parts by weight of 12, 14, 15, 16, or 18.

[0017] In some specific embodiments, the levoglucosinolate is present in parts by weight of 12-14, 12-15, 12-16, 14-15, 14-16, 14-18, 15-16, 15-18, or 16-18.

[0018] In one specific embodiment, the casein is expressed in parts by weight of 6, 7, 8, 9, or 10.

[0019] In some specific embodiments, the casein is expressed in parts by weight of 6-7, 6-8, 6-9, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10.

[0020] Optionally, the polystyrene microspheres are nanoscale microspheres with a particle size of 50 nm to 1 μm.

[0021] Optionally, the amino acid sequence of casein is the sequence shown in SEQ ID NO 1.

[0022] Optionally, the kit further includes a buffer solution; the buffer solution is a 0.01-0.1 M Tris-HCl buffer solution with a pH of 7.0-7.5.

[0023] Optionally, the Tris-HCl buffer comprises the following components: 10-100 g / L BSA, 0.5-2% Proclin-300 (by volume of buffer), 0.05-3 g / L Brij-35, and 0.1-0.3 M NaCl.

[0024] Furthermore, the concentration of BSA in the buffer solution is 10-50 g / L, for example, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, etc.

[0025] Furthermore, the volume of Proclin-300 in the buffer solution is 0.5-1.5% of the buffer solution volume, for example, 0.6%, 0.8%, 1.0%, 1.2%, etc.

[0026] Furthermore, the concentration of Brij-35 in the buffer solution is 0.05-2 g / L, for example, 0.06 g / L, 0.08 g / L, 0.10 g / L, 0.12 g / L, 0.14 g / L, 0.16 g / L, 0.18 g / L, etc.

[0027] Furthermore, the NaCl concentration in the buffer solution is 0.1-0.2M, for example, 0.12M, 0.14M, 0.16M, 0.18M, ​​etc.

[0028] Optionally, the kit further includes a stop solution, a washing solution, and standards. The stop solution is 0.05-0.5M sulfuric acid.

[0029] Optionally, the concentration of the sulfuric acid is 0.2-0.5M, such as 0.25M, 0.30M, 0.35M, 0.40M, 0.45M, etc.

[0030] Optionally, the anti-interference agent is mixed with the buffer solution, and the concentration of the anti-interference agent in the mixture is 0.2-0.8 mg / mL. For example: 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.55 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, etc.

[0031] Optionally, the anti-interference agent is mixed with the buffer solution, and the concentration of the anti-interference agent in the mixture is 0.4-0.7 mg / mL.

[0032] Optionally, the peroxidase complex capable of binding to the immunomarker and catalyzing substrate color development includes streptavidin coupled with the peroxidase.

[0033] Optionally, the peroxidase is selected from horseradish peroxidase.

[0034] Optionally, the substrate capable of being catalyzed and colored by the peroxidase is a TMB colorimetric solution.

[0035] Optionally, the immunomarker includes biotinylate markers and FITC markers.

[0036] Optionally, the allergens include ingested allergens and inhaled allergens.

[0037] Optionally, the food allergens include any one or more of eggs, dairy products, beans, seafood, and dried fruits.

[0038] Optionally, the inhaled allergens include any one or more of pollen, dust mites, animal dander, and mold.

[0039] In one specific implementation, the method of using the above-mentioned reagent kit is as follows:

[0040] (1) Mix the sample to be tested, the anti-interference agent and the solid support, incubate at room temperature and wash;

[0041] (2) Continue to add the immunolabeled allergen, incubate at room temperature and wash;

[0042] (3) Continue to add the peroxidase complex that can bind to the immunolabel and catalyze the color development of the substrate, incubate at room temperature and wash;

[0043] (4) Add a substrate that can be catalyzed by peroxidase to develop color, and then add a stop solution to terminate the reaction and detect the absorbance.

[0044] In the above preparation method, the solid support is coated with blocked anti-IgE antibody. The solid support can be any one of polystyrene solid support, magnetic nanoparticles, and nitrocellulose membrane. BSA or casein is selected for blocking.

[0045] In summary, this application includes at least one of the following beneficial technical effects:

[0046] This application utilizes a mixture of polystyrene microspheres, levoglucosinolate, and casein as an anti-interference agent, which can effectively avoid interference from other components in the test sample on the detection of specific allergens, effectively reduce false positives and false positive results, thereby improving the accuracy of the test results. Detailed Implementation

[0047] Before describing the embodiments of this application in detail, it should be understood that the terminology used herein is for the purpose of describing a particular embodiment only. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the term pertains.

[0048] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0049] The endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values, and such ranges or values ​​should be understood to include values ​​close to such ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0050] In this application, the terms "comprising" or "including" are open-ended expressions, meaning they include the content specified in this application but do not exclude other aspects.

[0051] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0052] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0053] The present application will be further described in detail below with reference to the embodiments and test results.

[0054] Unless otherwise specified, the types of antibodies and allergens mentioned in the following examples can be known or obtained by those skilled in the art through conventional technical means.

[0055] Example 1

[0056] This embodiment provides a kit for detecting allergen-specific IgE antibodies.

[0057] The above kit includes an anti-interference agent for mixing with the test sample, an immunolabeled allergen, a complex of peroxidase that binds to the immunolabel and catalyzes substrate color development, and a substrate that can be catalyzed by peroxidase for color development. Details are as follows:

[0058] (1) Preparation of anti-interference agents

[0059] The anti-interference agent is a mixture of polystyrene microspheres, L-glycoside, and casein. The polystyrene microspheres are nanoscale microspheres with a particle size of 50 nm; the amino acid sequence of the casein is as shown in SEQ ID NO 1.

[0060] Prepare the buffer solution: a 0.05M tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution containing 10 g / L BSA, 1% Proclin-300 (1% of the total buffer volume), 0.05 g / L Brij-35, 0.154 M NaCl, and pH 7.2.

[0061] The anti-interference agent was prepared into a stock solution with a concentration of 10 mg / mL using a buffer solution.

[0062] (2) Preparation of ELISA plates

[0063] Anti-IgE was dissolved in CBS coating buffer at a working concentration of 2 μg / mL. After 24 h at 4°C, the coating buffer was dried and then blocked with 5 mg / mL pH 7.4 PBS blocking buffer for 2 h. The blocking buffer was discarded, and the mixture was dried at 25°C for 8 h before being placed in an aluminum foil bag with desiccant for later use.

[0064] (3) Biotinylated anti-IgE antibody: 0.1 μg / mL Anti-IgE-biotin was dissolved in PBS buffer at pH 7.4 containing 5 mg / mL BSA, 1% Proclin-300, and 0.154 M NaCl.

[0065] (4) Preparation of biotinylated allergens: The nine biotinylated allergens were F2-Biotin, F1-Biotin, F24-Biotin, F23-Biotin, W1-Biotin, I6-Biotin, D1-Biotin, M3-Biotin, and T12-Biotin. After dilution with diluents, the working concentrations were 0.05 μg / mL, 0.003 μg / mL, 0.01 μg / mL, 0.01 μg / mL, 0.12 μg / mL, 0.34 μg / mL, 0.007 μg / mL, 0.03 μg / mL, and 0.22 μg / mL, respectively. The diluents were all in PBS buffer at pH 7.4 containing 5 mg / mL BSA, 1% Proclin-300 (1% of the total buffer volume), and 0.154 M NaCl.

[0066] (5) The enzyme conjugate is horseradish peroxidase-streptavidin conjugate (SA-HRP) with a working concentration of 0.03 μg / mL, dissolved in Tris buffer at pH 7.4. The Tris buffer also contains 10 mg / mL BSA, 0.1% Triton 100, 1% Proclin-300, and 0.154 M NaCl.

[0067] (6) Substrate solution: TMB single-component colorimetric solution purchased from Suzhou Yake Chemical Reagent Co., Ltd., product number D0022.

[0068] (7) Termination solution: 0.5M H2SO4.

[0069] (8) Standard: Human IgE full-length protein purchased from Abcam.

[0070] The specific detection method of the reagent kit provided in this embodiment includes the following steps:

[0071] (1) Dilute the anti-interference agent stock solution with buffer solution to a working solution of a certain concentration. See Table 1 for details.

[0072] (2) Add 0.5% of the volume of the anti-interference working solution to the sample to be tested.

[0073] (3) Take 50 μL of the test sample and standard containing the anti-interference agent and add them to the corresponding microwells, and incubate at room temperature for 60 min;

[0074] (4) Wash 3 times, each time with 300 μL of cleaning solution. Pat the microplate dry on absorbent paper.

[0075] (5) Add 100 μL of allergen-biotin to the sample to be tested, and add 100 μL of anti-IgE antibody-biotin to the standard. Seal the plate and incubate at room temperature for 60 min; then wash according to step (4).

[0076] (6) Add 100 μL of enzyme conjugate to each well, seal the plate, and incubate at room temperature for 30 min; then wash according to step (4).

[0077] (5) Add 100 μL of TMB substrate solution to each well, seal the plate, protect from light, and incubate at room temperature for 30 min.

[0078] (6) Add 100 μL of stop solution to each well in the order of adding substrate solution, tap the rack gently to mix the liquid in the reaction well, and read the value at 450 nm wavelength using an ELISA reader after 5 min.

[0079] Plot a standard curve based on the OD values ​​of the standards, and calculate the concentration of the sample to be tested according to the standard curve.

[0080] Example 2-18

[0081] Examples 2-18 each provide a kit for detecting allergen-specific IgE antibodies. The difference between the above examples and Example 1 is the concentration of the anti-interference agent component or the anti-interference agent working solution; all other steps are the same as in Example 1.

[0082] Table 1. The addition of anti-interference agents and the test results of the reagent kits in each example and comparative example.

[0083]

[0084] Comparative Example 1

[0085] This comparative example provides a kit for detecting allergen-specific IgE antibodies. The difference between this comparative example and Example 1 is that the kit in this comparative example does not contain an anti-interference agent.

[0086] Comparative Examples 2-7

[0087] Comparative Examples 2-7 each provide a kit for detecting allergen-specific IgE antibodies. The difference between these kits and Example 3 lies in the composition of the anti-interference agent; all other steps are consistent with Example 1.

[0088] Performance testing

[0089] The functional verification of the kits for detecting allergen-specific IgE antibodies prepared in the above examples and comparative examples was performed respectively.

[0090] In the validation experiment, all samples tested were serum samples, including all-positive samples and normal samples. All-positive samples were those that tested positive for allergens (F2 milk, W1 common ragweed, D1 house dust mite, 10T12 willow) using a commercially available allergen test kit. Normal samples were those that did not test positive for all allergens (F2 milk, W1 common ragweed, D1 house dust mite, 10T12 willow) using a commercially available allergen test kit.

[0091] Twenty all-positive samples and 30 normal samples were tested using the kits provided in the above examples and comparative examples, respectively. The 30 normal samples included 12 F2 positive samples, 10 W1 positive samples, 5 D1 positive samples, and 10 10T12 positive samples.

[0092] The test results are shown in Table 1.

[0093] As shown in Table 1, by comparing Example 3 with Comparative Examples 1-7, it can be seen that compared with not adding an anti-interference agent or adding any one or two of polystyrene microspheres, L-glucoside, and casein as an anti-interference agent, the addition of a mixture of polystyrene microspheres, L-glucoside, and casein as an anti-interference agent in this application can effectively avoid the interference of other components in the test sample on the detection of specific allergens, effectively reduce false positives and false positive results, thereby improving the accuracy of the test results.

[0094] Furthermore, this application further explored the optimal ratio of polystyrene microspheres, L-glucoside, and casein when using a mixture as an anti-interference agent. Experimental analysis showed that controlling the addition amount of polystyrene microspheres to 3-6 parts, L-glucoside to 12-18 parts, and casein to 6-10 parts can improve the accuracy of detection results to varying degrees. In particular, it can reduce false negative and false positive results when detecting F2 and 10T12, thereby improving the accuracy of the detection results.

[0095] Furthermore, this application also investigated the effect of the amount of anti-interference agent added on the detection results. Experimental analysis showed that controlling the concentration of the anti-interference agent working solution at 0.2-0.8 mg / mL yielded more accurate detection results.

[0096] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0097] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. A kit for detecting allergen-specific IgE antibodies, characterized in that, The kit includes an anti-interference agent for mixing with the sample to be tested, an immunolabeled allergen, a complex of a peroxidase that can bind to the immunolabel and catalyze the color development of the substrate, and a substrate that can be catalyzed by the peroxidase to develop color. The anti-interference agent comprises the following components in parts by weight: 4-5 parts polystyrene microspheres, 14-16 parts levoglucoside, and 7-9 parts casein; the amino acid sequence of the casein is the sequence shown in SEQ ID NO 1; the particle size of the polystyrene microspheres is 50 nm-1 μm.

2. The reagent kit according to claim 1, characterized in that, The kit also includes a buffer solution; the buffer solution is a 0.01-0.1M Tris-HCl buffer solution with a pH of 7.0-7.

5.

3. The reagent kit according to claim 2, characterized in that, The anti-interference agent is mixed with the buffer solution, and the concentration of the anti-interference agent in the mixture is 0.2-0.8 mg / mL.

4. The reagent kit according to claim 3, characterized in that, The anti-interference agent is mixed with the buffer solution, and the concentration of the anti-interference agent in the mixture is 0.4-0.7 mg / mL.

5. The reagent kit according to claim 1, characterized in that, The complex of peroxidase capable of binding to the immunomarker and catalyzing substrate color development includes streptavidin coupled with peroxidase.

6. The reagent kit according to claim 1, characterized in that, The immunomarkers include biotin and FITC markers.

7. The kit according to claim 1, characterized in that, The allergens include ingested allergens and inhaled allergens.

8. The reagent kit according to claim 7, characterized in that, The food allergens mentioned include any one or more of the following: eggs, dairy products, beans, seafood, and dried fruits.

9. The reagent kit according to claim 7, characterized in that, The inhaled allergens include any one or more of pollen, dust mites, animal dander, and mold.

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