Kit for detecting allergen-specific IgE (Immunoglobulin E) antibody

Abstract

The invention relates to the technical field of in-vitro diagnosis, in particular to a kit for detecting an allergen-specific IgE (Immunoglobulin E) antibody. The kit comprises an anti-interference agent used for being mixed with a to-be-detected sample, an immunolabelled allergen, a peroxidase compound capable of being combined with an immunolabel and catalyzing color development of a substrate, and the substrate capable of being catalyzed by peroxidase to develop color, the anti-interference agent is prepared from the following components in parts by weight: 3 to 6 parts of polystyrene microspheres, 12 to 18 parts of L-glucoside and 6 to 10 parts of casein. The kit provided by the invention can effectively reduce false positive and false positive results, so that the accuracy of the detection result is improved.

Description

Technical Field

[0001] The present application relates to the technical field of in vitro diagnosis, and in particular to a kit for detecting allergen-specific IgE antibodies. Background Art

[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, the presence of various interfering substances in the sample seriously affects the accuracy of the test results, leading to false positive or false negative results, and interfering with clinical diagnosis. There are many kinds of interfering substances in the samples. Heterophilic antibodies are antibodies against undefined antigens that exist in human peripheral circulating blood. They have multiple specificities and weak affinity. They can interact with the labeled antibodies or capture antibodies in the detection system by binding to the Fc segment 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 with denatured IgG as the target antigen. It can bridge the antibodies in the detection system and interfere with the immunoassay. The degree of interference has no clear dose-effect relationship with the concentration. Human anti-animal antibodies (such as the common human anti-mouse antibody HAMA) are caused by iatrogenic or non-iatrogenic factors, have strong affinity, are specific to antigens, and will interfere with antibodies from the corresponding source.

[0003] In addition, complement, autoantibodies, and hormone-binding proteins in the sample can also interfere with the test. Elevated complement levels in conditions such as 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. Hormone-binding proteins bind to hormones, altering the concentration of the analyte in the sample and interfering with the test. To address the aforementioned interference issues, it is essential to include a blocking agent in allergen IgE test kits. Nonspecific blocking agents act against the wide range of interfering substances present in the sample. By saturating unbound binding sites, providing alternative binding sites, and specifically binding to interfering substances to create steric hindrance, they prevent nonspecific binding and reduce the binding of interfering antibodies to the test antibody components, thereby improving the sensitivity and specificity of the test. Although there are a variety of blockers for allergen detection kits on the market, many problems still exist. Some blockers are ineffective in blocking specific interfering substances and cannot fully address the multiple interferences in complex samples. For example, animal IgG, a common passive blocker, can provide alternative binding sites to prevent interfering antibodies from binding to capture antibodies or detection antibodies, but it can only block specific types of interference. It requires the use of multiple IgGs, and the blocking effect depends on the affinity between the interfering antibodies and animal IgG.

[0004] Furthermore, while some blockers reduce interference, they can negatively impact the detection signal. For example, excessive use of passive blockers can reduce the detection signal, affecting the detection of low-concentration allergen IgE. Furthermore, existing blockers vary in their suitability for different sample types, making it difficult to guarantee a stable blocking effect across a variety of samples, including serum, plasma, and nasal secretions. Therefore, there is a need to further optimize the blockers in allergen IgE detection kits. Summary of the Invention

[0005] The present application provides a kit for detecting allergen-specific IgE antibodies. The kit provided in the present application can effectively reduce false positives and false negatives, thereby improving the accuracy of the test results.

[0006] In a first aspect, the present application provides a kit for detecting allergen-specific IgE antibodies, which adopts the following technical solution: A kit for detecting allergen-specific IgE antibodies, comprising an anti-interference agent for mixing with a sample to be tested, an immunolabeled allergen, a peroxidase complex capable of binding to the immunolabeled allergen and catalyzing color development of a substrate, and a substrate capable of being catalyzed by the peroxidase to develop color. The anti-interference agent comprises the following components in parts by weight: 3-6 parts of polystyrene microspheres, 12-18 parts of levoglycoside, and 6-10 parts of casein.

[0007] Optionally, the anti-interference agent comprises the following components in parts by weight: 4-5 parts of polystyrene microspheres, 14-16 parts of levodopa, and 7-9 parts of casein.

[0008] In a specific embodiment, the weight proportion of the polystyrene microspheres is 3 parts, 4 parts, 4.5 parts, 5 parts, or 6 parts.

[0009] In some specific embodiments, the weight proportion of the polystyrene microspheres is 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, or 5-6 parts.

[0010] In a specific embodiment, the weight proportion of the levodoside is 12 parts, 14 parts, 15 parts, 16 parts, or 18 parts.

[0011] In some specific embodiments, the weight proportion of the levodoside is 12-14 parts, 12-15 parts, 12-16 parts, 14-15 parts, 14-16 parts, 14-18 parts, 15-16 parts, 15-18 parts, or 16-18 parts.

[0012] In a specific embodiment, the weight parts of casein are 6 parts, 7 parts, 8 parts, 9 parts, or 10 parts.

[0013] In some specific embodiments, the weight proportion of the casein is 6-7 parts, 6-8 parts, 6-9 parts, 7-8 parts, 7-9 parts, 7-10 parts, 8-9 parts, 8-10 parts, or 9-10 parts.

[0014] Optionally, the polystyrene microspheres are nanoscale microspheres with a particle size of 50 nm-1 μm.

[0015] Optionally, the amino acid sequence of casein is the sequence shown in SEQ ID NO 1.

[0016] Optionally, the kit further comprises a buffer; the buffer is a 0.01-0.1 M Tris-HCl buffer with a pH of 7.0-7.5.

[0017] Optionally, the Tris-HCl buffer comprises the following components: 10-100 g / L BSA, 0.5-2% Proclin-300 based on the volume of the buffer, 0.05-3 g / L Brij-35, and 0.1-0.3 M NaCl.

[0018] Furthermore, the concentration of BSA in the buffer 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.

[0019] Furthermore, the volume of Proclin-300 in the buffer solution is 0.5-1.5% of the volume of the buffer solution, for example, 0.6%, 0.8%, 1.0%, 1.2%, etc.

[0020] 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.

[0021] Furthermore, the concentration of NaCl in the buffer solution is 0.1-0.2M, such as 0.12M, 0.14M, 0.16M, 0.18M, ​​etc.

[0022] Optionally, the kit further comprises a stop solution, a cleaning solution, and a standard, wherein the stop solution is 0.05-0.5 M sulfuric acid.

[0023] 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.

[0024] Optionally, the anti-interference agent is mixed with the buffer solution, and the concentration of the anti-interference agent in the mixed solution after mixing 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.

[0025] Optionally, the anti-interference agent is mixed with the buffer solution, and after mixing, the concentration of the anti-interference agent in the mixed solution is 0.4-0.7 mg / mL.

[0026] Optionally, the peroxidase complex capable of binding to the immunolabel and catalyzing substrate color development comprises streptavidin coupled to peroxidase.

[0027] Optionally, the peroxidase is selected from horseradish peroxidase.

[0028] Optionally, the substrate capable of being catalyzed by the peroxidase to develop color is TMB color development solution.

[0029] Optionally, the immunolabeling includes biotin labeling and FITC labeling.

[0030] Optionally, the allergens include ingested allergens and inhaled allergens.

[0031] Optionally, the ingested allergens include any one or more of eggs, milk, beans, seafood, and dried fruits.

[0032] Optionally, the inhaled allergens include any one or more of pollen, dust mites, animal dander, and molds.

[0033] In a specific embodiment, the method for using the above kit is as follows: (1) Mix the sample to be tested, anti-interference agent and solid phase carrier, incubate at room temperature and wash; (2) Continue adding immunolabeled allergens, incubate at room temperature, and wash; (3) Continue to add a peroxidase complex that can bind to the immunolabeled protein and catalyze the color development of the substrate, incubate at room temperature, and wash; (4) Add a substrate that can be catalyzed by peroxidase to develop color, add a stop solution to terminate the reaction, and then detect the absorbance.

[0034] In the above preparation method, the blocked anti-IgE antibody is coated on the solid phase carrier. The solid phase carrier can be any one of a polystyrene solid phase carrier, nanomagnetic particles, and a nitrocellulose membrane. Any one of BSA and casein is selected for blocking.

[0035] In summary, this application includes at least one of the following beneficial technical effects: The present application uses a mixture of polystyrene microspheres, levulin, and casein as an anti-interference agent, which can effectively avoid the interference of other components in the sample to be tested on the specific allergen detection, and can effectively reduce false positive and false negative results, thereby improving the accuracy of the test results. DETAILED DESCRIPTION

[0036] Before describing the embodiments of the present application in detail, it should be understood that the terminology used herein is only for the purpose of describing particular embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the term belongs.

[0037] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of this application, unless otherwise specified, "plurality" means two or more.

[0038] The endpoints of the ranges and any values ​​disclosed in this application are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be considered to be specifically disclosed herein.

[0039] In this application, the term "comprise" or "include" is an open expression, that is, it includes the content specified in this application, but does not exclude other aspects of the content.

[0040] To make the purpose, technical solutions, and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without making any creative work 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 understood as limiting this application.

[0041] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. All reagents or instruments used without specifying the manufacturer are commercially available conventional products.

[0042] The present application is further described in detail below in conjunction with the examples and test results.

[0043] 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 routine technical means in the art.

[0044] Example 1

[0045] This embodiment provides a kit for detecting allergen-specific IgE antibodies.

[0046] The above-mentioned kit includes an anti-interference agent for mixing with the sample to be tested, an immunolabeled allergen, a peroxidase complex capable of binding to the immunolabeled allergen and catalyzing the color development of the substrate, and a substrate capable of being catalyzed by the peroxidase to develop color. The details are as follows: (1) Preparation of anti-interference agents The anti-interference agent is a mixture of polystyrene microspheres, levoglycoside, and casein, wherein the polystyrene microspheres are nanoscale microspheres with a particle size of 50 nm, and the amino acid sequence of casein is the sequence shown in SEQ ID NO 1.

[0047] Prepare buffer: 0.05 M Tris-HCl buffer containing 10 g / L BSA, 1% Proclin-300 based on the total volume of the buffer, 0.05 g / L Brij-35, and 0.154 M NaCl, pH 7.2.

[0048] The anti-interference agent was prepared into a stock solution with a concentration of 10 mg / mL using a buffer solution.

[0049] (2) Preparation of ELISA plate Dissolve Anti-IgE in CBS coating buffer at a working concentration of 2 μg / mL. After 24 hours at 4°C, spin dry the coating buffer and block with 5 mg / mL PBS, pH 7.4, for 2 hours. Discard the blocking buffer, dry at 25°C for 8 hours, and then place in an aluminum foil bag with desiccant for later use.

[0050] (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 in the total volume of the buffer, and 0.154 M NaCl.

[0051] (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 diluent, 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 of the total buffer volume, and 0.154 M NaCl.

[0052] (5) The enzyme conjugate solution is a horseradish peroxidase-streptavidin conjugate (SA-HRP) with a working concentration of 0.03 μg / mL, dissolved in Tris buffer at pH 7.4, where the Tris buffer also contains 10 mg / mL BSA, 0.1% of the total buffer volume of Triton 100, 1% of the total buffer volume of Proclin-300, and 0.154 M NaCl.

[0053] (6) Substrate solution: TMB single-component colorimetric solution purchased from Suzhou Yaco Chemical Reagent Co., Ltd., product number D0022.

[0054] (7) Stop solution: 0.5 M H2SO4.

[0055] (8) Standard: Full-length human IgE protein purchased from Abcam.

[0056] The specific detection method of the kit provided in this embodiment includes the following steps: (1) Dilute the anti-interference agent stock solution with buffer to a working solution of a certain concentration, as shown in Table 1.

[0057] (2) Add 0.5% of the volume of the sample to be tested of the anti-interference agent working solution.

[0058] (3) Pipette 50 μL of the sample and standard containing anti-interference agent into the corresponding microwells and incubate at room temperature for 60 min; (4) Wash the microplate three times, using 300 μL of washing solution each time. Pat the remaining liquid on absorbent paper to dry the microplate.

[0059] (5) Add 100 μL of allergen-biotin to the sample to be tested and 100 μL of anti-IgE antibody-biotin to the standard. Seal the plate and incubate at room temperature for 60 min; then wash as in step (4).

[0060] (6) Add 100 μL of enzyme conjugate solution to each well, seal the plate, and incubate at room temperature for 30 min; then wash according to step (4).

[0061] (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.

[0062] (6) Add 100 μL of stop solution to each well in the order of adding substrate solution, tap the rack to mix the liquid in the reaction well, and read the results at a wavelength of 450 nm using a microplate reader after 5 minutes.

[0063] A standard curve was drawn according to the OD value of the standard, and the concentration of the sample to be tested was calculated according to the standard curve.

[0064] Example 2-18 Examples 2-18 each provide a kit for detecting allergen-specific IgE antibodies. The difference between the above examples and Example 1 lies in the concentration of the anti-interference agent component or the anti-interference agent working solution, and the remaining steps are consistent with Example 1.

[0065] Table 1 The addition of anti-interference agents in each embodiment and comparative example and the test results of the kit

[0066] Comparative Example 1 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 does not contain an anti-interference agent.

[0067] Comparative Examples 2-7 Comparative Examples 2-7 provide a kit for detecting allergen-specific IgE antibodies. The difference between the above kit and Example 3 lies in the components of the anti-interference agent, and the remaining steps are consistent with Example 1.

[0068] Performance testing Functional verification was performed on the kits for detecting allergen-specific IgE antibodies prepared in the above examples and comparative examples.

[0069] In the validation study, 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 commercially available allergen detection kits. Normal samples were those that tested negative for none of the allergens (F2 milk, W1 common ragweed, D1 house dust mite, 10T12 willow) using commercially available allergen detection kits.

[0070] The kits provided in the above examples and comparative examples were used to test 20 all-positive samples and 30 normal samples, including 12 F2-positive samples, 10 W1-positive samples, 5 D1-positive samples, and 10 10T12-positive samples.

[0071] The test results are shown in Table 1.

[0072] As can be seen from 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, levoglycoside, and casein as an anti-interference agent, the present application adds a mixture of polystyrene microspheres, levoglycoside, and casein as an anti-interference agent, which can effectively avoid the interference of other components in the sample to be tested on the specific allergen detection, and can effectively reduce false positive and false negative results, thereby improving the accuracy of the test results.

[0073] In addition, the application has further explored the optimal ratio of the three when the mixture of polystyrene microspheres, levulin and casein is an anti-interference agent. Through experimental analysis, it can be seen that the addition of polystyrene microspheres is controlled at 3-6 parts, the addition of levulin is controlled at 12-18 parts, and the addition of casein is controlled at 6-10 parts, which can improve the accuracy of test results to varying degrees. Especially when detecting F2 and 10T12, false negative and false positive results are presented, thereby improving the accuracy of test results.

[0074] In addition, the present application also explored the effect of the amount of anti-interference agent added on the test results. After experimental analysis, it was found that more accurate test results could be obtained when the concentration of the anti-interference agent working solution was controlled at 0.2-0.8 mg / mL.

[0075] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A kit for detecting allergen-specific IgE antibodies, characterized in that: The kit includes an anti-interference agent for mixing with a sample to be tested, an immunolabeled allergen, a peroxidase complex capable of binding to the immunolabeled allergen and catalyzing the color development of a substrate, and a substrate capable of being catalyzed by the peroxidase to develop color. The anti-interference agent comprises the following components in parts by weight: 3-6 parts of polystyrene microspheres, 12-18 parts of levoglycoside, and 6-10 parts of casein; the amino acid sequence of the casein is the sequence shown in SEQ ID NO 1.

2. The kit according to claim 1, wherein The anti-interference agent comprises the following components in parts by weight: 4-5 parts of polystyrene microspheres, 14-16 parts of levoglycoside, and 7-9 parts of casein.

3. The kit according to claim 1, wherein The kit further comprises a buffer; the buffer is a 0.01-0.1 M Tris-HCl buffer with a pH of 7.0-7.

5.

4. The kit according to claim 1, wherein The anti-interference agent is mixed with the buffer solution, and after mixing, the concentration of the anti-interference agent in the mixed solution is 0.2-0.8 mg / mL.

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

6. The kit according to claim 1, wherein The peroxidase complex capable of binding to the immunolabel and catalyzing substrate color development comprises streptavidin coupled to peroxidase.

7. The kit according to claim 1, wherein The immunolabeling includes biotin labeling and FITC labeling.

8. The kit according to claim 1, wherein The allergens include ingested allergens and inhaled allergens.

9. The kit according to claim 8, characterized in that The ingested allergens include any one or more of eggs, milk, beans, seafood, and dried fruits.

10. The kit according to claim 8, characterized in that The inhaled allergens include any one or more of pollen, dust mites, animal dander, and mold.

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