A detection kit for wheat allergen-specific IgE and a detection method thereof

Abstract

The application relates to the field of biotechnology, and in particular to a wheat allergen specific IgE detection kit and a detection method thereof. The kit comprises magnetic particles, enzyme-labeled anti-human IgE antibodies and biotin-labeled wheat allergen. The wheat allergen is obtained by treating a wheat protein extract with heat treatment or a pretreatment agent. The pretreatment agent comprises one or more of sodium azide, L-cysteine and L-cysteine hydrochloride. The wheat allergen is biotin-labeled after specific pretreatment. The chemiluminescence immunoassay method uses streptavidin to prepare magnetic microspheres and horseradish peroxidase-labeled anti-human IgE antibodies. The method is fully automatic, ensures the accuracy of the detection result, and improves the test flux, the specificity and the sensitivity of the detection result.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a wheat allergen-specific IgE detection kit and a detection method thereof. BACKGROUND

[0002] The white paper on allergy (also known as allergic reaction) of the World Allergy Organization (WAO) in 2011-2012 indicates that the prevalence of allergic diseases, including allergic asthma, allergic rhinitis, drug, food and insect allergy, eczema, urticaria and angioedema, is on the rise worldwide. There are about 160 kinds of foods that cause allergy, among which wheat is one of the eight major allergenic foods identified by the FDA. A small-scale survey in China shows that the incidence of food allergy among residents in Beijing, Guangzhou and other places is 3.4%-5.0%, and more than 0.4% of the world's population is affected by wheat allergy. Studies have reported that wheat allergy accounts for about 7%, 16% and 8% of patients with urticaria, allergic rhinitis and asthma, respectively. IgE-mediated allergic reactions after ingestion of wheat include urticaria, angioedema, anaphylactic shock and wheat-dependent exercise-induced anaphylaxis, etc.

[0003] Immunoglobulin E antibody (IgE) is an important mediator of allergic diseases, which mediates type I immediate allergic reactions. IgE has cytotoxicity, and its Fc segment binds to the surface receptors of mast cells or eosinophils, sensitizing the cells. When the sensitized cells encounter the corresponding allergen, the IgE antibodies on the surface of the cells bind to the corresponding epitopes of the antigen, causing complex cascade reactions of the cell membrane, and the sensitized cells release bioactive mediators, which act on target tissues or organs to cause clinical symptoms. The level of IgE in the body can be one of the key indicators for diagnosing IgE-mediated allergic reactions. The detection of allergen-specific IgE can assist in the diagnosis of allergic diseases, and quantitative detection can more accurately assess the risk of allergy, detect sensitized children earlier, avoid risks in advance, track changes in sIgE concentration, and assist in decision-making on the timing of food reintroduction.

[0004] Currently, the kits for detecting wheat allergens mainly include immunoblotting, colloidal gold, enzyme-linked immunosorbent assay, magnetic microsphere chemiluminescence and fluorescence immunoassay. Each wheat-specific detection kit has its own advantages and disadvantages, which need to be further optimized.

[0005] The method using colloidal gold has a manual experiment, a complex operation, and a small flux; and the method is used for qualitative analysis of grain allergy, and cannot accurately quantify. Accurate quantification of grain allergy can be used for risk assessment of diseases, and a large difference in disease occurrence risk is caused by different values of allergy levels of sIgE; in addition, a quantitative concentration range of sIgE assists in decision-making of a time of food reintroduction. The sensitivity of colloidal gold is different from that of chemiluminescence due to limitations of a method. SUMMARY

[0006] Therefore, the application provides a wheat allergen-specific IgE detection kit and a detection method thereof. The wheat allergen is specifically pretreated and biotin-labeled, and a chemiluminescence immunoassay method using streptavidin to prepare magnetic microspheres and horseradish peroxidase-labeled anti-human IgE antibody is used. The method is fully automatic, ensures the accuracy of the detection result, and improves the test flux, the specificity, and the sensitivity of the detection result.

[0007] To achieve the above-mentioned application purposes, the application provides the following technical solutions.

[0008] A wheat allergen-specific IgE detection kit, characterized in that the kit comprises magnetic microspheres, enzyme-labeled anti-human IgE antibody, and biotin-labeled wheat allergen.

[0009] The wheat allergen is obtained by heat treatment or pretreatment of a wheat protein extract.

[0010] The pretreatment agent comprises one or more of sodium azide, L-cysteine, and L-cysteine hydrochloride.

[0011] In some embodiments of the application, the pretreatment agent is L-cysteine hydrochloride; and the concentration of the L-cysteine hydrochloride is preferably 0.5-100 mM, and specifically can be 0.5 mM, 2 mM, 5 mM, 10 mM, 50 mM, or 100 mM.

[0012] In the application, the temperature of the heat treatment is preferably 65-95℃, and specifically can be 65℃, 70℃, 75℃, 80℃, 85℃, or 95℃, and the time is preferably 20-60 min; and specifically can be 20 min, 30 min, 40 min, 50 min, or 60 min.

[0013] In some embodiments of the application, the wheat allergen pretreatment agent is sodium azide, and the mass fraction is preferably 1%-20%, and specifically can be 1%, 5%, 10%, 15%, or 20%.

[0014] In the present application, the concentration of the wheat allergen is preferably 0.1-40.0 μg / mL; specifically, it can be 0.1 μg / mL, 1 μg / mL, 5 μg / mL, 20 μg / mL, or 40.0 μg / mL.

[0015] In the present application, the preparation method of the biotin-labeled wheat allergen comprises mixing and reacting the wheat allergen with activated biotin, and obtaining the biotin-labeled wheat allergen by dialysis of the reactants. The activator used in the activation includes EDC and NHS, but is not limited thereto. The solvent is preferably dimethyl sulfoxide, but is not limited thereto. The reaction is preferably carried out at room temperature, and the reaction time is preferably 0.5-4 h, specifically, it can be 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, or 4 h. The molar ratio of the activated biotin to the wheat allergen is preferably 1:3-1:30, specifically, it can be 1:3, 1:5, 1:10, 1:15, 1:20, 1:25, or 1:30.

[0016] In the present application, the concentration of the streptavidin-coated magnetic microparticles is 0.1-3.0 mg / mL.

[0017] In the present application, the enzyme-labeled anti-human IgE antibody is preferably a horseradish peroxidase-labeled anti-human IgE antibody. The source of the anti-human IgE antibody is not particularly limited, and includes, but is not limited to, mouse anti-human, rabbit anti-human, and the like.

[0018] The present application also provides a method for detecting wheat allergen-specific IgE for non-diagnostic purposes, which utilizes the detection kit of the present application, and specifically comprises the following steps:

[0019] Step 1: heat treatment or pretreatment of wheat protein extract to obtain wheat allergen; mixing and reacting the wheat allergen with activated biotin, and obtaining biotin-labeled wheat allergen by dialysis of the reactants; wherein the pretreatment agent includes one or more of sodium azide, L-cysteine, and L-cysteine hydrochloride.

[0020] Step 2: mixing and incubating the sample to be tested, streptavidin-coated magnetic microparticles, and biotin-labeled wheat allergen, and then removing the supernatant;

[0021] Step 3: adding a horseradish peroxidase-labeled anti-human IgE antibody, mixing and incubating, and then removing the supernatant;

[0022] Step 4: adding a substrate solution, and measuring the chemiluminescence intensity;

[0023] Step 5: calculating the concentration of IgE in the sample to be tested according to the standard curve.

[0024] The temperature of the incubation in steps 2 and 3 of the application is preferably 15-40℃, and specifically can be 15℃, 37℃ or 40℃; the time is preferably 5-40min, and specifically can be 5min, 15min or 40min.

[0025] In step 5 of the application, a standard curve is established with the concentration value of the IgE standard as the abscissa and the corresponding chemiluminescence signal value as the ordinate.

[0026] The kit provided by the application comprises magnetic microparticles, enzyme-labeled anti-human IgE antibody, biotin-labeled wheat allergen and wheat allergen pretreatment agent; the wheat allergen pretreatment agent comprises one or more of sodium azide, L-cysteine and L-cysteine hydrochloride. The wheat allergen is optimized in the application, the wheat allergen treated with a specific pretreatment agent is biotin-labeled, and the chemiluminescence immunoassay method using streptavidin to prepare magnetic microspheres and horseradish peroxidase-labeled anti-human IgE antibody is used for full-automatic operation, thereby ensuring the accuracy of the detection result, improving the test flux and the sensitivity of the detection result, and the sensitivity is 0.1 IU / mL. DETAILED DESCRIPTION

[0027] The application provides a wheat allergen-specific IgE detection kit and a detection method thereof. Those skilled in the art can refer to the content herein and appropriately improve the process parameters. It should be particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are all regarded as included in the application. The method and application of the application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application herein without departing from the content, spirit and scope of the application, to realize and apply the technology of the application.

[0028] The test materials used in the application are all ordinary commercially available products and can be purchased in the market.

[0029] The application will be further described below in combination with examples:

[0030] Example 1 Preparation of the kit of the application

[0031] 1. Preparation of biotin-labeled wheat allergen:

[0032] (1) The wheat protein extract is treated with 0.5-100mM L-cysteine for 20-60min to obtain the wheat allergen;

[0033] (2) Biotin is dissolved with dimethyl sulfoxide, and the concentration is 2-6mg / ml;

[0034] (3) EDC and NHS are respectively dissolved with dimethyl sulfoxide, and the concentration is 2-6mg / ml;

[0035] (4) Dissolve biotin, EDC and NHS in proportion, react at room temperature, the reaction ratio is 1:1:1, and the reaction time is 1-3h;

[0036] (5) React the activated biotin in steps (1)-(3) with the wheat allergen obtained in step (1) in a molar ratio of 1:5-1:20, and the reaction time is 1-3h; and then dialyze after the reaction.

[0037] (6) Prepare a solution of 1-10 μg / mL of the biotin-labeled wheat allergen in a phosphate buffer (pH=7-8) containing 5% horse serum, 2% glycerol and 5% sucrose.

[0038] 2. Preparation of magnetic particle suspension:

[0039] Magnetic separation is performed on 100 mg / mL streptavidin-coated magnetic particles, which are resuspended in a phosphate buffer (pH=7-8) containing 5% horse serum, 2% glycerol and 5% sucrose, washed repeatedly for 2 times (5-10 min / time), magnetically separated, and finally resuspended in a phosphate buffer (pH=7-8) containing 5% horse serum, 2% glycerol and 5% sucrose to obtain a streptavidin-coated magnetic particle suspension with a concentration of 0.5-3 mg / mL.

[0040] 2. Preparation of enzyme-labeled anti-human IgE antibody:

[0041] The anti-human IgE-labeled horseradish peroxidase is prepared into a solution with a concentration of 1 mg / mL in a phosphate buffer (pH=7-8) containing 5% horse serum.

[0042] Example 2

[0043] The difference between Example 1 and Example 2 is that the preparation method of the wheat allergen is different, wherein the wheat protein extract is treated with a 1%-20% sodium azide solution for 20-60 min, and the other kit components and their preparation methods are the same.

[0044] Example 3

[0045] The difference between Example 1 and Example 3 is that the preparation method of the wheat allergen is different, wherein the wheat protein extract is heated at 65-95°C for 20-60 min, and the other kit components and their preparation methods are the same.

[0046] Example 4

[0047] The difference between Example 1 is that the preparation method of the wheat allergen is different, which is: the wheat protein extract is treated with 0.5-100 mM L-cysteine hydrochloride for 20-60 min; other kit components and their preparation methods are the same.

[0048] Example 5 Detection method of the application

[0049] Step 1: Take the wheat protein extract and pretreat it according to the methods of Examples 1-4 to obtain a wheat allergen; mix and react the wheat allergen with activated biotin, and obtain biotin-labeled wheat allergen by dialysis of the reactants;

[0050] Step 2: Mix and incubate the sample to be tested, streptavidin-coated magnetic microparticles, and biotin-labeled wheat allergen, and then remove the supernatant;

[0051] Step 3, add horseradish peroxidase-labeled anti-human IgE antibody, mix and incubate, and then remove the supernatant;

[0052] Step 4: Add substrate solution and measure the chemiluminescence intensity;

[0053] Step 5: According to the standard curve, calculate the concentration of IgE in the sample to be tested.

[0054] Test Example 1

[0055] The kit of Examples 1-4 is used for detection according to the method of Example 5, and the results are shown in Table 1.

[0056] Table 1 Effect of wheat background (RLU)

[0057]

[0058] The results show that different processing techniques show differences in the enzyme background introduced by the antigen after heat denaturation, sodium azide treatment, and L-cysteine hydrochloride process treatment. Among them, the enzyme background introduced by the antigen itself is significantly reduced after heat denaturation, sodium azide treatment, and L-cysteine hydrochloride process treatment.

[0059] Test Example 2 Effect of different wheat allergens on kit sensitivity

[0060] The kit of Examples 1-4 is used for detection according to the method of Example 5, and the results are shown in Table 2.

[0061] Table 2 Effect of kit performance (RLU)

[0062]

[0063] The results show that the different pretreated wheat allergen has no significant effect on the reactivity, the 0 value sample is significantly reduced, the signal to noise ratio is significantly increased, and the L-cysteine hydrochloride treatment has the best effect on the reaction gradient of the negative sample.

[0064] Sensitivity analysis test of test example 3

[0065] The negative sample is detected 20 times repeatedly by using the kit of the embodiments 1-4 of the present application, the average value plus 2 times standard deviation of the measurement result is the sensitivity of the kit, and the obtained sensitivity is 0.1 IU / mL-0.23 IU / mL.

[0066] Table 3: Effect of different pretreatments on the sensitivity of the kit

[0067]

[0068]

[0069] The results show that the L-cysteine hydrochloride process blank limit is significantly reduced after the heat denaturation, sodium azide treatment and L-cysteine hydrochloride process treatment, the detection ability is enhanced, and the sensitivity is obviously increased.

[0070] The above is only the preferred embodiment of the present application, and it should be pointed out that for ordinary skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should be considered as the protection scope of the present application.

Claims

1. A test kit for wheat allergen-specific IgE, characterized in that, The wheat allergen is pretreated by a pretreatment agent of wheat protein extract, and the pretreatment agent is L-cysteine hydrochloride. The wheat allergen is pretreated by a pretreatment agent of wheat protein extract, and the pretreatment agent is L-cysteine hydrochloride. The pretreatment agent is L-cysteine hydrochloride, and the concentration is 0.5-100 mM.

2. The test kit according to claim 1, characterized in that, The pretreatment agent is L-cysteine hydrochloride, and the concentration is 0.5-100 mM.

3. The test kit according to claim 1, characterized in that, The concentration of the wheat allergen is 0.1-40.0 μg / mL.

4. The test kit according to claim 1, characterized in that, The preparation method of the biotin-labeled wheat allergen comprises: The wheat allergen is mixed with activated biotin and reacted, and the reactants are dialyzed to obtain the biotin-labeled wheat allergen.

5. The test kit according to claim 4, characterized in that The activator used in the activation comprises EDC and NHS, and the solvent is dimethyl sulfoxide. The reaction is carried out at room temperature, and the reaction time is 0.5-4 h. The molar ratio of the activated biotin to the wheat allergen is 1:3-1:

30.

6. The test kit according to claim 1, characterized in that, The concentration of the streptavidin-coated magnetic particles is 0.1-3.0 mg / mL.

7. The test kit according to claim 1, characterized in that, The enzyme-labeled anti-human IgE antibody is horseradish peroxidase-labeled anti-human IgE antibody.

8. A method of detecting IgE specific for a wheat allergen for non-diagnostic purposes, characterized in that, The method comprises the following steps: Step 1: The wheat protein extract is pretreated by a pretreatment agent to obtain a wheat allergen, and the wheat allergen is mixed with activated biotin and reacted, and the reactants are dialyzed to obtain a biotin-labeled wheat allergen; wherein the pretreatment agent is L-cysteine hydrochloride; Step 2: The sample to be tested, streptavidin-coated magnetic particles and biotin-labeled wheat allergen are mixed and incubated, and then the supernatant is removed; Step 3: Horseradish peroxidase-labeled anti-human IgE antibody is added, mixed and incubated, and then the supernatant is removed; Step 4: Substrate solution is added, and the chemiluminescence intensity is determined; Step 5: According to the standard curve, the concentration of IgE in the sample to be tested is calculated.

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