Quantitative detection kit for milk allergen specificity IgE (Immunoglobulin E) and application thereof
In the detection of milk allergen-specific IgE antibody, the combination of IgG antibody and HSA protective protein was used to solve the problem of inaccurate detection results of magnetic microparticle chemiluminescence, and the sensitivity and specificity of the detection are improved. It is suitable for fully automated detection. .
Patent Information
- Application Number
- CN202411451765.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-17
- Publication Date
- 2024-12-27
AI Technical Summary
The existing magnetic microparticle chemiluminescence indirect method used for the detection of milk allergen-specific IgE antibodies has the problem of inaccurate detection results, mainly due to false positives and dilution of test results caused by nonspecific binding of IgG antibodies in human serum and nonspecific binding of IgG antibodies to interfere with and free protective proteins. .
IgG antibodies were added to the biotinylated-milk allergen extract solution and human irrelevant HSA was used as a protective protein to improve detection sensitivity and specificity.
By adding IgG antibodies and HSA, the detection sensitivity and specificity are significantly improved, false positive interference is reduced, and the accuracy of the detection results is enhanced. It is suitable for the detection of fully automatic magnetic microparticle chemiluminescence instruments, and has the advantages of fast and large flux.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of in vitro detection, and in particular to a milk allergen-specific IgE quantitative detection kit and application thereof. Background Art
[0002] Milk is considered a common food that causes allergies in infants and young children worldwide, with the incidence rate ranging from 1% to 7.5% in various regions. Currently, the milk allergen-specific IgE antibody detection kits on the market mainly include immunoblotting, colloidal gold, enzyme-linked immunosorbent assay, magnetic microparticle chemiluminescence, and fluorescent immunoassay. Milk allergen-specific IgE antibody detection kits all have their own advantages and disadvantages, and need to be further optimized.
[0003] The detection method of allergen-specific IgE antibodies in human serum established by the magnetic microparticle chemiluminescence indirect method has the advantages of automated detection, rapid, sensitive, and large detection throughput. However, the detection method of allergen-specific IgE antibodies in human serum established by the magnetic microparticle chemiluminescence indirect method is not completely suitable for milk allergen-specific IgE antibody detection. At present, the commonly used protective protein in the magnetic microparticle chemiluminescence indirect method is bovine serum albumin BSA. As one of the milk allergens, the free protective protein bovine serum albumin will specifically bind to anti-bovine serum albumin IgE, reducing the detection sensitivity. Due to the interference of heterophilic antibodies that non-specifically bind to IgG in human serum, false positives will appear in the test results, and the IgE antibody content in human serum samples is low. Adding a blocking agent to the test sample will dilute the sample, resulting in a decrease in the test result, affecting the accuracy of the test result. Therefore, there is currently no better solution to the problem of inaccurate test results in the magnetic microparticle chemiluminescence detection method for milk allergen-specific IgE antibodies. Summary of the invention
[0004] In view of this, the present invention provides a milk allergen-specific IgE quantitative detection kit and its application. When preparing the kit, IgG antibodies are added to a biotinylated-milk allergen extract solution, and HSA is used as a protective protein, which can improve the detection sensitivity and specificity.
[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
[0006] The present invention provides the use of one or both of the following in preparing a kit for detecting milk allergen-specific IgE antibodies:
[0007] (i) HSA as a protective protein;
[0008] (ii) IgG as a blocking agent.
[0009] The present invention also provides a kit for detecting milk allergen-specific IgE antibodies, wherein the protective protein is HSA.
[0010] In some specific embodiments of the present invention, the above-mentioned kit includes a blocking agent, and the blocking agent is IgG.
[0011] In some specific embodiments of the present invention, the IgG in the above kit includes mouse IgG, bovine IgG and / or rabbit IgG.
[0012] In some specific embodiments of the present invention, the above-mentioned kit includes a biotinylated-milk allergen extract solution;
[0013] The biotinylated-milk allergen extract solution includes the HSA and / or the blocking agent.
[0014] In some specific embodiments of the present invention, the biotinylated-milk allergen extract solution in the above kit consists of a biotinylated-milk allergen extract and a buffer 1;
[0015] The buffer 1 is a 0.1 M phosphate buffer with a pH of 7 to 9 and containing 5 mg / mL to 30 mg / mL HSA, 0.01% to 1% Proclin 300, 0.01% to 0.05% Tween-20, 50 mg / mL to 100 mg / mL trehalose, and 0.1 mg / mL to 10 mg / mL of the blocking agent.
[0016] In some specific embodiments of the present invention, the method for preparing the biotinylated-milk allergen extract solution of the above kit comprises the following steps:
[0017] Step (1): mixing dimethyl sulfoxide and biotin at 1-10 mg / mL, preferably 2-6 mg / mL, to obtain dissolved biotin;
[0018] Step (2): reacting the dissolved biotin with the milk allergen extract at a molar ratio of 1:110 to 1:250; preferably the labeling ratio is 1:150; the reaction time is 0.5 to 4 h, preferably 1 to 3 h; after the reaction, dialyzing in phosphate buffer at 4° C. for 48 hours to obtain a biotinylated milk allergen extract;
[0019] Step (3): the biotinylated-milk allergen extract is prepared into a biotinylated-milk allergen extract solution using 0.1 M phosphate buffer (pH 7-9) containing 0.5%-3% HSA, 0.01%-1% Proclin 300, 0.01%-0.05% Tween-20, 5%-10% trehalose, and 0.01%-1% IgG blocking agent.
[0020] In some specific embodiments of the present invention, the concentration of the biotinylated-milk allergen extract solution in the above kit is 2-8 μg / mL, preferably 4.5 μg / mL.
[0021] In some specific embodiments of the present invention, the above-mentioned kit further comprises a streptavidin-modified magnetic particle suspension and an anti-human IgE enzyme-labeled antibody solution.
[0022] In some specific embodiments of the present invention, the anti-human IgE enzyme-labeled antibody solution described in the above kit consists of anti-human IgE enzyme-labeled antibody and buffer 2;
[0023] The buffer 2 is a 0.1 M phosphate buffer with a pH of 7 to 9 and containing 20 mg / mL to 50 mg / mL Casein, 0.5% to 1% Proclin300, 0.01% to 0.05% Tween-20 and 50 mg / mL to 100 mg / mL trehalose.
[0024] In some specific embodiments of the present invention, the concentration of the anti-human IgE enzyme-labeled antibody in the anti-human IgE enzyme-labeled antibody solution of the above-mentioned kit is 0.3-1.5 mg / mL; the preferred concentration is 1 mg / mL.
[0025] In some specific embodiments of the present invention, the content of magnetic particles in the streptavidin-modified magnetic particle suspension of the above kit is 1.3-2.5 mg / mL.
[0026] In some specific embodiments of the present invention, the anti-human IgE enzyme-labeled antibody in the above kit is an anti-human IgE antibody labeled with horseradish peroxidase.
[0027] The present invention also provides a method for detecting milk allergen-specific IgE antibodies, which is based on the above-mentioned kit.
[0028] In some specific embodiments of the present invention, the above-mentioned method for detecting milk allergen-specific IgE antibodies comprises the following steps:
[0029] Step (A): mixing the sample to be tested with a streptavidin-coated magnetic microparticle suspension and a biotin-labeled milk allergen extract solution, incubating, and magnetically separating to remove the supernatant;
[0030] Step (B): mixing with anti-human IgE enzyme-labeled antibody solution, incubating, and magnetically separating to remove the supernatant;
[0031] Step (C): mixing the substrate solutions and measuring the chemiluminescence intensity;
[0032] Step (D): Calculate the concentration of IgE in the sample to be tested according to the standard curve.
[0033] In some specific embodiments of the present invention, the incubation temperature in step (A) and step (B) of the above-mentioned milk allergen-specific IgE antibody detection method is 15-40°C, preferably 37°C.
[0034] In some specific embodiments of the present invention, the incubation time in step (A) and step (B) of the above-mentioned milk allergen-specific IgE antibody detection method is 5 to 40 minutes, preferably 15 minutes.
[0035] In some specific embodiments of the present invention, the anti-human IgE enzyme-labeled antibody in the above-mentioned milk allergen-specific IgE antibody detection method is an anti-human IgE antibody labeled with horseradish peroxidase.
[0036] The milk allergen-specific IgE quantitative detection kit of the present invention and its application have the following effects:
[0037] 1) The kit of the present invention can improve the detection sensitivity and specificity by adding IgG antibody to the biotinylated-milk allergen extract solution and using HSA as a protective protein.
[0038] 2) The kit of the present invention is suitable for a fully automatic magnetic microparticle chemiluminescence instrument, and has the advantages of fully automated detection, short reaction time, sensitive detection, and high throughput. DETAILED DESCRIPTION
[0039] The present invention discloses a milk allergen-specific IgE quantitative detection kit and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.
[0040] The present invention provides a quantitative detection kit for specific IgE antibodies to milk allergens. The kit adopts a streptavidin-biotin system to increase the loading capacity of a biotinylated milk allergen extract; during preparation, the milk allergen extract is biotin-labeled, IgG antibodies are added to the biotinylated milk allergen extract solution, and HSA is used as a protective protein to improve the detection sensitivity and specificity. Specifically:
[0041] The invention provides a quantitative detection kit for specific IgE antibodies of milk allergens. The kit comprises magnetic particles modified with streptavidin, biotinylated-milk allergen extracts and anti-human IgE antibodies labeled with horseradish peroxidase.
[0042] The biotinylated milk allergen extract and the solution preparation method thereof are as follows:
[0043] (1) Dissolve biotin in dimethyl sulfoxide to a concentration of 1-10 mg / mL, preferably 2-6 mg / mL;
[0044] (2) reacting the dissolved biotin with the milk allergen extract at a molar ratio of 1:110 to 1:250; the preferred labeling ratio is 1:150; the reaction time is 0.5 to 4 h, preferably 1 to 3 h; after the reaction, dialyzing in phosphate buffer at 4°C for 48 hours to obtain a biotinylated milk allergen extract.
[0045] (3) The biotinylated-milk allergen extract was prepared into a biotinylated-milk allergen extract solution using 0.1 M phosphate buffer (pH 7-9) containing 0.5%-3% HSA, 0.01%-1% Proclin 300, 0.01%-0.05% Tween-20, 5%-10% trehalose, and 0.01%-1% IgG blocker.
[0046] (4) The concentration of the biotinylated-milk allergen extract in the biotinylated-milk allergen extract solution is 2-8 μg / mL, and the preferred concentration is 4.5 μg / mL.
[0047] The concentration of the magnetic particles is 1.3-2.5 mg / mL, and the preferred concentration is 1.5 mg / mL.
[0048] The horseradish peroxidase-labeled anti-human IgE antibody and the preparation method of the solution thereof are as follows:
[0049] (1) The horseradish peroxidase-labeled anti-human IgE antibody is an anti-human IgE enzyme-labeled antibody.
[0050] (2) Dissolve the anti-human IgE enzyme-labeled antibody in 0.1 M phosphate buffer (pH 7-9) containing 2%-5% Casein, 0.5%-1% Proclin 300, 0.01%-0.05% Tween-20, and 5%-10% trehalose to prepare an anti-human IgE enzyme-labeled antibody solution;
[0051] (3) The concentration of the anti-human IgE enzyme-labeled antibody in the anti-human IgE enzyme-labeled antibody solution is 0.3-1.5 mg / mL; the preferred concentration is 1 mg / mL.
[0052] The present invention also provides a method for detecting milk allergen-specific IgE, based on the above-mentioned kit detection; preferably, the method comprises the following steps:
[0053] Step (1): incubate the sample to be tested with a suspension of magnetic microparticles coated with streptavidin and a solution of milk allergen labeled with biotin, and perform magnetic separation to remove the supernatant;
[0054] Step (2): adding horseradish peroxidase-labeled anti-human IgE antibody, incubating the mixture, and performing magnetic separation to remove the supernatant;
[0055] Step (3): adding substrate solution and measuring the chemiluminescence intensity;
[0056] Step (4): Calculate the concentration of IgE in the sample to be tested based on the standard curve.
[0057] The incubation temperature in step (1) and step (2) is 15-40°C, preferably 37°C; the incubation time in step (1) and step (2) is 5-40 min, preferably 15 min.
[0058] The present invention also provides an application of the above-mentioned kit in detecting milk allergen-specific IgE antibodies.
[0059] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.
[0060] The milk allergen extract was purchased from Zhengzhou Iminuo Biotechnology Co., Ltd.
[0061] The IgG blocker was purchased from Zhengzhou Imino Biotechnology Co., Ltd.
[0062] The present invention will be further described below in conjunction with embodiments:
[0063] Example 1
[0064] 1. Experimental Materials
[0065] A quantitative detection kit for specific IgE antibodies of milk allergens comprises magnetic particles modified with streptavidin, biotinylated milk allergen extracts, and anti-human IgE antibodies labeled with horseradish peroxidase.
[0066] The concentration of the magnetic particles is 1.3-2.5 mg / mL, and the preferred concentration is 1.5 mg / mL.
[0067] The biotinylated milk allergen extract and the solution preparation method thereof are as follows:
[0068] 1) Dissolve biotin in dimethyl sulfoxide to a concentration of 1-10 mg / mL, preferably 2-6 mg / mL;
[0069] 2) reacting the dissolved biotin with the milk allergen extract at a molar ratio of 1:110-1:250; the preferred labeling ratio is 1:150; the reaction time is 0.5-4 h, preferably 1-3 h; after the reaction, dialyzing in phosphate buffer at 4° C. for 48 hours to obtain a biotinylated milk allergen extract.
[0070] 3) The biotinylated-milk allergen extract is prepared into a biotinylated-milk allergen extract solution using 0.1 M phosphate buffer (pH 7-9) containing 1% protective protein (or no protective protein), 0.01%-1% Proclin 300, 0.01%-0.05% Tween-20, 5%-10% trehalose, and 0.01%-1% IgG blocking agent (preferably bovine IgG, 0.1 mg / mL). The protective protein is bovine serum, BSA, HSA or horse serum.
[0071] 4) The concentration of the biotinylated-milk allergen extract solution is 2-8 μg / mL, and the preferred concentration is 4.5 μg / mL.
[0072] The preparation method of the anti-human IgE antibody labeled with horseradish peroxidase and its solution is as follows:
[0073] 1) The anti-human IgE antibody labeled with horseradish peroxidase is a mouse anti-human IgE antibody labeled with horseradish peroxidase, hereinafter referred to as anti-human IgE enzyme-labeled antibody;
[0074] 2) Dissolve the anti-human IgE enzyme-labeled antibody in 0.1 M phosphate buffer (pH 7-9) containing 2%-5% Casein, 0.5%-1% Proclin 300, 0.01%-0.05% Tween-20, and 5%-10% trehalose to prepare an anti-human IgE enzyme-labeled antibody solution;
[0075] 3) The concentration of anti-human IgE enzyme-labeled antibody solution is 0.3~1.5 mg / mL; the preferred concentration is 1 mg / mL.
[0076] 2. Experimental methods
[0077] Step (1): incubate the sample to be tested with a suspension of magnetic microparticles coated with streptavidin and a solution of milk allergen labeled with biotin, and perform magnetic separation to remove the supernatant;
[0078] Step (2): adding horseradish peroxidase-labeled anti-human IgE antibody, incubating the mixture, and performing magnetic separation to remove the supernatant;
[0079] Step (3): adding substrate solution and measuring the chemiluminescence intensity;
[0080] Step (4): Calculate the concentration of IgE in the sample to be tested based on the standard curve.
[0081] The incubation temperature in step (1) and step (2) is 15-40°C, preferably 37°C; the incubation time in step (1) and step (2) is 5-40 min, preferably 15 min.
[0082] 3. Results
[0083] Table 1: Effects of different protective proteins on milk sensitivity
[0084]
[0085] The results are shown in Table 1. It can be seen that in milk allergy detection, compared with the commonly used BSA and bovine serum, the use of HSA and horse serum as protective proteins can improve the sensitivity of detection, especially HSA, which has better sensitivity and can effectively avoid the interference of free bovine live materials.
[0086] Example 2
[0087] The kit was prepared by using different IgG blockers (or without adding IgG blockers), and the experimental materials and experimental methods were the same as those in Example 1, and the protective protein was 1% HSA. The IgG blocker included mouse IgG, bovine IgG or rabbit IgG (purchased from Imeno).
[0088] result:
[0089] Table 2: Effects of different IgG antibodies on milk specificity
[0090]
[0091] The results are shown in Table 2. It can be seen that in milk allergy testing, it is necessary to add IgG as a blocking agent, which can effectively reduce the interference of heterophilic antibodies present in individual serum.
[0092] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. Use of one or both of the following in the preparation of a kit for detecting milk allergen-specific IgE antibodies: (i) HSA as a protective protein; (ii) IgG as a blocking agent.
2. A kit for detecting milk allergen-specific IgE antibodies, characterized in that: The protective protein is HSA.
3. The kit according to claim 2, characterized in that A blocking agent is included, wherein the blocking agent is IgG.
4. The kit according to claim 3, characterized in that The IgG includes mouse IgG, bovine IgG and / or rabbit IgG.
5. The kit according to claim 4, characterized in that Includes biotinylated-milk allergen extract solution; The biotinylated-milk allergen extract solution includes the HSA and / or the blocking agent.
6. The kit according to claim 5, characterized in that The biotinylated-milk allergen extract solution consists of a biotinylated-milk allergen extract and a buffer solution 1; The buffer 1 is a 0.1 M phosphate buffer with a pH of 7 to 9 and containing 5 mg / mL to 30 mg / mL HSA, 0.01% to 1% Proclin 300, 0.01% to 0.05% Tween-20, 50 mg / mL to 100 mg / mL trehalose, and 0.1 mg / mL to 10 mg / mL of the blocking agent.
7. The kit according to claim 5 or 6, characterized in that It also includes a streptavidin-modified magnetic particle suspension and an anti-human IgE enzyme-labeled antibody solution.
8. The kit according to claim 7, characterized in that The anti-human IgE enzyme-labeled antibody solution consists of anti-human IgE enzyme-labeled antibody and buffer 2; The buffer 2 is a 0.1 M phosphate buffer with a pH of 7 to 9 and containing 20 mg / mL to 50 mg / mL Casein, 0.5% to 1% Proclin 300, 0.01% to 0.05% Tween-20, and 50 mg / mL to 100 mg / mL trehalose.
9. The kit according to claim 7 or 8, characterized in that The content of magnetic particles in the streptavidin-modified magnetic particle suspension is 1.3-2.5 mg / mL.
10. A method for detecting milk allergen-specific IgE antibodies for non-diagnostic purposes, characterized in that: Detection based on the kit according to any one of claims 2 to 9.