Allergen-specific IgE antibody detection kit

Abstract

The present invention provides an allergen-specific IgE antibody detection kit, which includes a blocker, an immunolabeled allergen, a peroxidase complex that can bind to the immunolabel and catalyze the color development of a substrate, and a substrate that can be catalyzed by the peroxidase for color development, wherein the blocker includes one or more of inactivated thyroid peroxidase, neutrophil peroxidase, eosinophil peroxidase, horseradish peroxidase, and myelin basic protein. By utilizing the kit provided by the present invention, the interference of IgE antibodies produced by self-allergy on the detection of specific allergens can be effectively avoided, especially the interference of IgE antibodies produced by peroxidases can be blocked, and false positives caused by the interference of peroxidase IgE antibodies can be successfully eliminated, thereby improving the accuracy of the test results and having the advantages of simple operation and the like.

Description

Technical Field

[0001] The present invention specifically relates to an allergen-specific IgE antibody detection kit. Background Art

[0002] From an immunological perspective, during the inflammatory response, eosinophils and neutrophils can release major inflammatory mediators such as myelin basic protein (MBP), eosinophil peroxidase (EPX), eosinophil cationic protein (ECP), and neutrophil peroxidase (MPO). High release of eosinophil granulocyte protein and neutrophil peroxidase (MPO) or the presence of thyroid peroxidase (TPO) may induce a primary immune response, such as stimulating plasma cells to produce anti-EPX IgE. Taking urticaria as an example, urticaria is a common allergic disease, and the most common skin reaction is characterized by an expansive prurigo rash. These symptoms are caused by the activation of skin mast cells and histamine and other proinflammatory mediators subsequently released. Studies have found that patients with chronic spontaneous urticaria can produce anti-thyroid peroxidase IgE antibodies.

[0003] Since the enzyme-linked immunosorbent assay has the advantages of being mature, rapid, simple, and easy to standardize, the method has been widely used in clinical practice to detect allergen-specific IgE antibodies in the sample to be tested. The principle of the enzyme-linked immunosorbent assay kit for detecting allergen-specific IgE antibodies is that the ELISA plate is coated with Anti-IgE to capture the total IgE in the serum, and the specific IgE in the total IgE reacts with the corresponding biotinylated allergen, and then horseradish peroxidase-labeled streptavidin is added to catalyze the substrate color development, and finally sulfuric acid buffer is added to terminate the reaction and read at 450nm on the ELISA instrument. This method greatly reduces the interference of IgG and increases specificity. At the same time, it can realize the simultaneous detection of different allergens on the same ELISA plate as needed, which improves the convenience of users.

[0004] However, in clinical practice, some patients have allergic manifestations such as urticaria or other clinical symptoms. When the enzyme-linked immunosorbent assay is used to detect allergen-specific IgE antibodies in the samples, all the results are positive, which makes it impossible for doctors to make a judgment and affects the accuracy of the allergen-specific antibody detection kit. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide an allergen-specific IgE antibody detection kit which can reduce false positives and has high accuracy.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] The present invention provides a kit for detecting allergen-specific IgE antibodies, the kit comprising a blocking agent, an immunolabeled allergen, a peroxidase complex capable of binding to the immunolabel and catalyzing the color development of a substrate, and a substrate capable of being catalyzed by the peroxidase to develop color;

[0008] The blocking agent includes one or more of inactivated thyroid peroxidase, neutrophil peroxidase, eosinophil peroxidase, horseradish peroxidase and myelin basic protein.

[0009] Preferably, the blocking agent is a mixture comprising inactivated thyroid peroxidase, neutrophil peroxidase, eosinophil peroxidase, horseradish peroxidase and myelin basic protein.

[0010] Preferably, the kit further comprises a buffer, which is a 0.01-0.1M Tris-HCl buffer having a pH of 7.0-7.5, wherein the Tris-HCl buffer comprises the following components: 10-100 g / L BSA, 0.5-2% Proclin-300, 0.05-3 g / L Brij-35 and 0.1-0.3 M NaCl accounting for the volume of the buffer.

[0011] Further preferably, 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.

[0012] Further preferably, the volume of Proclin-300 in the buffer is 0.5-1.5% of the volume of the buffer, for example, 0.6%, 0.8%, 1.0%, 1.2%, etc.

[0013] Further preferably, the concentration of Brij-35 in the buffer 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.

[0014] Further preferably, the NaCl concentration in the buffer is 0.1-0.2M, such as 0.12M, 0.14M, 0.16M, 0.18M, ​​etc.

[0015] Preferably, the buffer solution is mixed with the blocking agent to form a mixed solution, and the concentration of the blocking agent in the mixed solution is 1 to 10 mg / mL, for example, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, 3.5 mg / mL, 4.0 mg / mL, 4.5 mg / mL, 5.0 mg / mL, 6.0 mg / mL, 7.0 mg / mL, 8.0 mg / mL, 9.0 mg / mL, etc.

[0016] More preferably, the concentration of the blocking agent in the mixed solution is 5-10 mg / mL.

[0017] Preferably, the immunolabeling includes biotin labeling and FITC labeling.

[0018] Preferably, the allergens include food allergens and inhalant allergens.

[0019] Further preferably, the food allergen is selected from egg white, egg yolk, milk, goat milk, yogurt, casein, buckwheat, wheat bran, corn, rice, oats, wheat, shrimp, crab, scallop, cod, hairtail, sea bass, salmon, lobster, carp, peanuts, beef, mutton, soybean, chestnut, sesame, hazelnut, almond, cashew, walnut, banana, mango, peach, kiwi, apple, pear, cherry, plum, grape, strawberry, papaya, grapefruit, orange, lemon, pineapple, cantaloupe, watermelon, melon, coconut, olive, broccoli, eggplant, bitter melon, cucumber, bell pepper, asparagus, spinach, tomato, zucchini, carrot, potato, garlic, sweet potato, radish, The inhaled allergen is selected from one or more of cat dander, dog dander, ragweed, humulus, cockroaches, house dust mites, dust mites, poplar, willow, cypress, birch, pine, elm, sycamore, dandelion, mugwort, wormwood, amaranth, tobacco scraps, mosquitoes, cotton wool, flower grass pollen, ryegrass pollen, meadow fescue pollen, timothy grass pollen, small bran grass pollen, meadow bluegrass, Penicillium, Aspergillus fumigatus, Alternaria, Mycosporium, Mucor, Rhizopus and Candida albicans.

[0020] More preferably, the food allergen is selected from one or more of egg white, egg yolk, milk, casein, buckwheat, corn, rice, oats, wheat, shrimp, crab, scallop, cod, sea bass, salmon, lobster, carp, peanut, beef, lamb, soybean, sesame, hazelnut, almond, cashew, walnut, banana, mango, peach, kiwi, apple, pear, cherry, strawberry, orange, pineapple, tomato, carrot, potato and celery, and the inhaled allergen is selected from one or more of cat dander, dog dander, ragweed, humulum, cockroach, house dust mite, dust mite, poplar, willow, cypress, birch, pine, elm, sycamore, dandelion, mugwort, wormwood, Amaranthus retroflexus, tobacco scraps, mosquito, cotton wool, Penicillium, Aspergillus fumigatus, Alternaria alternata, Mycosporium, Mucor, Rhizopus and Candida albicans.

[0021] According to a preferred embodiment of the present invention, the food allergen is selected from one or more of egg white, milk, eggs, soybeans, peanuts, apples, wheat, cod, shrimp, crab and beef, and the inhaled allergen is selected from one or more of ragweed, mugwort, humulum, cat dander, dog dander, cockroaches, house dust mites, dust mites, house dust, willow and Aspergillus fumigatus, Penicillium notatum and Alternaria alternata.

[0022] Preferably, the peroxidase complex capable of binding to an immunolabel and catalyzing the color development of a substrate comprises streptavidin coupled to peroxidase.

[0023] Preferably, the peroxidase is selected from horseradish peroxidase.

[0024] Preferably, the substrate capable of being catalyzed by the peroxidase to develop color is TMB color developing solution or OPD color developing solution.

[0025] Preferably, the blocking agent is present as a separate component and / or in a mixture with the immunolabeled allergen.

[0026] Preferably, the kit further comprises a stop solution, a cleaning solution and a standard substance, and the stop solution is 0.05-0.5M sulfuric acid.

[0027] More preferably, the concentration of the sulfuric acid is 0.2-0.5M, for example, 0.25M, 0.30M, 0.35M, 0.40M, 0.45M, etc.

[0028] Preferably, when the kit is used for detection, the blocking agent is added before the peroxidase complex capable of binding to the immunolabel and catalyzing the color development of the substrate is added to the detection system.

[0029] Preferably, the method for using the kit comprises the following steps:

[0030] (1) combining the sample to be tested, the blocking agent and the solid phase carrier, and incubating at room temperature;

[0031] (2) washing the mixed solution after incubation in step (1), washing it, mixing it with the immunolabeled allergen, and incubating it at room temperature;

[0032] (3) washing the mixed solution after incubation in step (2), adding the peroxidase complex capable of binding to the immune marker and catalyzing the color development of the substrate after washing, and incubating at room temperature;

[0033] (4) washing the mixed solution after incubation in step (3), adding the substrate capable of being catalyzed by the peroxidase to develop color after washing, adding a stop solution to terminate the reaction, and then detecting absorbance; or,

[0034] (1) combining the sample to be tested with the solid phase carrier and incubating at room temperature;

[0035] (2) washing the incubated sample, mixing it with the blocking agent and the immunolabeled allergen, and incubating it at room temperature;

[0036] (3) washing the mixed solution after incubation in step (2), adding the peroxidase complex capable of binding to the immune marker and catalyzing the color development of the substrate after washing, and incubating at room temperature;

[0037] (4) Washing step (3) After the incubation, the mixed solution is washed and a substrate that can be catalyzed by the peroxidase is added for color development. After adding a stop solution to terminate the reaction, absorbance detection is performed.

[0038] Preferably, the solid phase carrier is coated with anti-IgE antibodies and is blocked.

[0039] Further preferably, the stationary phase carrier is blocked with BSA or casein.

[0040] Preferably, the solid phase carrier comprises a polystyrene solid phase carrier, nanomagnetic particles, or nitrocellulose membrane.

[0041] Preferably, the specification of the kit is 1 to 100 servings / box, wherein, based on 1 serving / box, the amount of the blocker in the kit is 0.05 to 50 μg, the amount of the immunolabeled allergen is 0.001 to 10 μg, the amount of the peroxidase complex that can bind to the immunolabel and catalyze the color development of the substrate is 0.05 to 0.5 μg, and the amount of the substrate that can be catalyzed by the peroxidase to develop color is 50 to 200 μL.

[0042] When the kit is used for detection, the dosage of each component in the kit varies according to the type of allergen.

[0043] Further preferably, based on one serving per box, the amount of the blocker in the kit is 0.05 to 40 μg, for example, 0.5 μg, 1 μg, 5 μg, 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, etc.

[0044] Further preferably, the amount of the immune-labeled allergen in the kit is 0.001-8 μg, for example, 0.005 μg, 0.01 μg, 0.05 μg, 0.10 μg, 0.50 μg, 1.0 μg, 1.5 μg, 2.0 μg, 3.0 μg, 4.0 μg, 5.0 μg, 6.0 μg, 7.0 μg, etc., per person / box.

[0045] Further preferably, based on one serving per box, the amount of the peroxidase complex capable of binding to an immune marker and catalyzing substrate color development in the kit is 0.05 to 0.3 μg, for example, 0.08 μg, 0.10 μg, 0.15 μg, 0.20 μg, 0.25 μg, etc.

[0046] Further preferably, based on one serving per box, the amount of the substrate that can be catalyzed by the peroxidase for color development in the kit is 50 to 150 μL, for example, 55 μL, 60 μL, 70 μL, 80 μL, 90 μL, 100 μL, 110 μL, 120 μL, 130 μL, 140 μL, etc.

[0047] Compared with the prior art, the present invention has the following advantages:

[0048] In an enzyme-linked immunosorbent capture detection system using peroxidase as a marker, the allergen-specific IgE antibody detection kit provided by the present invention can be used to effectively avoid the interference of IgE antibodies produced by self-allergy on specific allergen detection by adding a blocker to the detection system, especially blocking the interference of IgE antibodies produced by peroxidases, successfully eliminating false positives caused by interference of peroxidase IgE antibodies, and when using the kit provided by the present invention for detection, adding a blocker does not affect the detection results of normal samples, thereby improving the accuracy of the detection. The kit also has the advantage of simple operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is a schematic diagram of the detection principle of the enzyme-linked immunosorbent assay for all positive samples in the present invention;

[0050] Figure 2 This is a schematic diagram of the detection principle of the enzyme-linked immunosorbent assay for normal samples in the present invention;

[0051] Wherein, before treatment refers to detection using the kit of Comparative Example 1, and after treatment refers to detection using the kit of Example 1;

[0052] Figure 3 This is the standard curve when the kit of Comparative Example 1 is used to detect all positive samples in the present invention;

[0053] Figure 4 The standard curve when the kit of Example 1 is used to detect all positive samples in the present invention;

[0054] Figure 5 The standard curve when the kit of Comparative Example 1 is used to detect normal samples in the present invention;

[0055] Figure 6 The standard curve when the kit of Example 1 is used to detect normal samples in the present invention;

[0056] Figure 7 This is the standard curve when the test samples numbered as all-positive sample 1, all-positive sample 5 and all-positive sample 10 are retested using the kit in Example 1 of the present invention. DETAILED DESCRIPTION

[0057] The present invention is further described below in conjunction with the examples. However, the present invention is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to the different requirements of specific use, and the implementation conditions not specified are conventional conditions in the industry. The technical features involved in each embodiment of the present invention can be combined with each other as long as they do not conflict with each other.

[0058] At present, peroxidase, especially horseradish peroxidase, is widely used in ELISA because of its advantages such as easy extraction, affordable price and stable properties. However, in practice, the inventor found that when the enzyme-linked immunosorbent assay using horseradish peroxidase as an enzyme marker detects allergen-specific IgE antibodies in a sample, false positives will appear in the test results. This is because horseradish peroxidase has certain cross reactions with thyroid peroxidase, myeloperoxidase and eosinophil peroxidase that can produce autoallergic IgE antibodies, that is, when the sample to be tested contains anti-peroxidase IgE antibodies, due to the overlapping protein sequences of anti-peroxidase IgE antibodies, the development of its epitope is promoted and spread between different proteins, and horseradish peroxidase will bind to the anti-peroxidase IgE antibodies fixed on the solid phase carrier, thereby causing false positive results. Therefore, the inventors have conducted a lot of research and experimental verification, and proposed an allergen-specific IgE antibody detection kit. Specifically, the kit includes a blocker, an immunolabeled allergen, a peroxidase complex that can bind to the immunolabel and catalyze the color development of the substrate, and a substrate that can be catalyzed by the peroxidase. Specifically, the blocker includes one or more of inactivated thyroid peroxidase, neutrophil peroxidase, eosinophil peroxidase, horseradish peroxidase, and myelin basic protein. When using the kit to detect allergen-specific IgE antibodies, by adding a blocker to the detection system, the interference of IgE antibodies produced by self-allergy on the specific allergen detection can be effectively avoided, especially the interference of IgE antibodies produced by peroxidases is blocked, and the false positive caused by the interference of peroxidase IgE antibodies is successfully eliminated, thereby improving the accuracy of the detection results.

[0059] The present invention is further described in detail below in conjunction with specific examples. Unless otherwise specified herein, "%" represents volume percentage. In the following examples and comparative examples, unless otherwise specified, the raw materials, reagents, etc. used are all conventional commercially available products.

[0060] Example 1

[0061] The invention provides a kit for detecting allergen-specific IgE antibodies, comprising a blocking agent, a biotin-labeled allergen, a horseradish peroxidase-labeled streptavidin, a substrate solution, a buffer solution, a stop solution, a cleaning solution and a standard product.

[0062] Preparation of each component of the kit of this embodiment:

[0063] Preparation of blocking agents

[0064] The blocking agent is a mixture of inactivated thyroid peroxidase, neutrophil peroxidase, eosinophil peroxidase, horseradish peroxidase, and myelin basic protein.

[0065] Preparation buffer: 0.05 M Tris-HCl buffer containing 10 g / L BSA, 1% Proclin-300, 0.05 g / L Brij-35, 0.154 M NaCl, pH 7.2.

[0066] The inactivated blocking agent was prepared into a stock solution with a concentration of 10 mg / mL using a buffer.

[0067] Preparation of ELISA plate: Anti-IgE was dissolved in CBS coating solution with a working concentration of 2μg / mL. After 24 hours at 4℃, the coating solution was dried and then blocked with 5mg / mL PBS blocking solution at pH 7.4 for 2 hours. The blocking solution was discarded and dried at 25℃ for 8 hours. The plate was then placed in an aluminum foil bag with desiccant for later use.

[0068] 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 accounting for the total volume of the buffer, and 0.154 M NaCl.

[0069] Preparation of biotinylated allergens: The 9 biotinylated allergens are 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 are 0.05μg / mL, 0.003μg / mL, 0.01μg / mL, 0.01μg / mL, 0.12μg / mL, 0.34μg / mL, 0.007μg / mL, 0.03μg / mL, and 0.22μg / mL, respectively; the diluents are all in PBS buffer at pH 7.4 containing 5mg / mL BSA, 1% Proclin-300 in the total volume of the buffer, and 0.154M NaCl.

[0070] The enzyme conjugate solution is SA-HRP with a working concentration of 0.03 μg / mL, dissolved in Tris buffer at pH 7.4, wherein the Tris buffer also contains 10 mg / mL BSA, 0.1% of the total volume of the buffer, 0.1% of the total volume of the buffer, Proclin-300, and 0.154 M NaCl.

[0071] Substrate solution: TMB single-component colorimetric solution purchased from Suzhou Yaco Chemical Reagent Co., Ltd., product number D0022.

[0072] Stop solution: 0.5 M H2SO4.

[0073] Standard: Full-length human IgE protein purchased from Abcam.

[0074] The detection principle of the kit for detecting allergen-specific IgE antibodies in this embodiment is shown in Figure 1 and Figure 2 , the specific detection method is as follows:

[0075] Dilute the blocking agent stock solution with buffer to a 1 mg / mL working solution.

[0076] Step 1: Add 0.5% of the volume of the blocker working solution to the sample to be tested so that the working concentration is 5 μg / mL;

[0077] Step 2: Pipette 50 μL of the sample to be tested and the standard containing the blocking agent respectively and add them into the corresponding microwells;

[0078] Step 3: Incubate at room temperature for 60 minutes;

[0079] Step 4: Wash the microplate three times using a plate washer (or manually), using 300 μL of washing solution each time. Pat the remaining liquid on the microplate dry on absorbent paper;

[0080] Step 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 minutes;

[0081] Step 6: After incubation, wash according to step 4;

[0082] Step 7: Add 100 μL of horseradish peroxidase-streptavidin binding solution to each well, seal the plate, and incubate at room temperature for 30 minutes.

[0083] Step 8: Clean according to step 4;

[0084] Step 9: Add 100 μL TMB substrate solution to each well, seal the plate, protect from light, and incubate at room temperature for 30 minutes;

[0085] Step 10: 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 450 nm (reference wavelength 620 nm or 630 nm) using a microplate reader after 5 minutes.

[0086] In the present detection method, the blocking agent is not limited to being mixed with the sample to be detected, and it is feasible to add the blocking agent before adding horseradish peroxidase-streptavidin to the detection system. For example, the blocking agent can also be mixed with the biotin-labeled allergen.

[0087] Draw a standard curve based on the OD value of the standard, and calculate the concentration of the sample to be tested according to the standard curve.

[0088] Comparative Example 1

[0089] This comparative example provides a kit for detecting allergen-specific IgE antibodies, which is basically the same as Example 1, except that the kit of this comparative example does not contain a blocking agent.

[0090] The performance of the allergen-specific IgE antibody detection kits prepared in Example 1 and Comparative Example 1 was verified. The samples in the verification experiments were all serum samples. The all-positive samples in the present invention refer to samples that are positive for any allergen item when detected by a commercially available allergen detection kit, and the normal samples refer to samples that are not fully positive for any allergen item when detected by a commercially available allergen detection kit.

[0091] (1) Verify that all positive samples contain IgE antibodies produced by autoallergy

[0092] Three experiments were set up, namely, group A, group B and group C, and 18 all-positive samples and 10 normal samples were tested respectively. The three experiments were basically carried out according to the detection method in Example 1, except that step (5) and step (6) were omitted in this verification experiment. In addition, the differences between the groups were as follows: group A did not add a blocking agent; group B added Anti-IgE to the ELISA plate, and the IgE in the sample to be tested would preferentially bind to the added Anti-IgE and be washed away in the subsequent plate washing process; group C only omitted step (5) and step (6); at the same time, other methods (nanomagnetic particle chemiluminescence platform) were used to detect the total IgE antibodies of 28 samples to be tested, and the results obtained by the detection are shown in Table 1, wherein the detection OD value was greater than 0.07, which was determined to be positive.

[0093] Table 1

[0094]

[0095]

[0096] As shown in Table 1, the total IgE antibody content in most samples is high. The experimental results of Group A show that compared with normal samples, there are substances in all-positive samples that can be captured by Anti-IgE on the ELISA plate and can bind to horseradish peroxidase-labeled streptavidin; combined with the experimental results of Group B, it is shown that the substance is IgE antibody; the experimental results of Group C further show that the IgE antibody in all-positive samples can be blocked by blockers. It can be seen that there are IgE antibodies produced by self-allergy in all-positive samples, and the blockers have a significant inhibitory effect on all-positive samples, while the test results of normal samples are not affected.

[0097] (2) Verification of the interference removal effect of the kit of the present invention

[0098] The kits of comparative example 1 and embodiment 1 were used to simultaneously detect 9 allergens in 10 positive samples with autoallergic IgE antibody interference and 10 normal samples, wherein the 10 positive samples were samples numbered 1 to 10 in the above performance verification (1), and the 10 normal samples were samples numbered 1 to 10 in the above performance verification (1), and the 9 allergens were F2 milk, F1 protein, F24 shrimp, F23 crab, W1 common ragweed, I6 cockroach, D1 house dust mite, M3 Aspergillus fumigatus, and T12 willow.

[0099] The same kit is used according to the same detection method, and the standard product detection is set on the same ELISA plate while detecting the sample to be tested. Therefore, in this embodiment, when the same kit is used to detect all positive samples or normal samples, the OD value of the corresponding calibration product will be obtained. The OD value of the measured calibration product has an error, and its error value is within a reasonable range. According to the OD value of the standard product when the kit of comparative example 1 detects all positive samples with a 96-well ELISA plate (Table 2) and the OD value of the standard product when the kit of embodiment 1 detects all positive samples with a 96-well ELISA plate (Table 3), the standard curves are drawn respectively ( Figure 3 and Figure 4 ), where the y-axis is the OD value and the x-axis is the concentration. According to the standard curve ( Figure 3 ) Calculate the concentration of all positive samples measured by the kit of Comparative Example 1 (results are shown in Table 6), according to the standard curve ( Figure 4 ) Calculate the concentration of all positive samples measured by the kit of Example 1 (results are shown in Table 6).

[0100] According to the OD values ​​of the standard products when the kit of Comparative Example 1 is used to detect normal samples using a 96-well ELISA plate (Table 4) and the OD values ​​of the standard products when the kit of Example 1 is used to detect normal samples using a 96-well ELISA plate (Table 5), standard curves are drawn respectively ( Figure 5 and Figure 6 ), where the y-axis is the OD value and the x-axis is the concentration. According to the standard curve ( Figure 5 ) Calculate the concentration of the normal sample measured by the kit of Comparative Example 1 (results are shown in Table 7), according to the standard curve ( Figure 6 ) Calculate the concentration of normal samples measured using the kit of Example 1 (results are shown in Table 7).

[0101] Table 2

[0102]

[0103] Table 3

[0104]

[0105] Table 4

[0106]

[0107] Table 5

[0108]

[0109]

[0110] Table 6

[0111]

[0112] Table 7

[0113]

[0114]

[0115] As shown in Table 6, the test results of Comparative Example 1 of all positive samples show that the sample is positive for all the detected allergens, while the results of Example 1 show that some all positive samples are positive for some allergens (such as all positive sample 2), and some all positive samples are negative for all the detected allergens (such as all positive sample 1). As shown in Table 7, for normal samples, the test results of Comparative Example 1 and Example 1 are consistent. Combining Tables 6 and 7, it can be shown that the scheme of the present application has a significant interference removal effect in all positive samples while not affecting the detection of normal samples.

[0116] In order to further verify the accuracy of the kit of the present invention, three all-positive samples numbered as all-positive sample 1, all-positive sample 5 and all-positive sample 10 were retested using the kit of Example 1 and InmmunoCAP analysis was performed at the same time. The test results are shown in Table 9.

[0117] The standard curve was drawn based on the OD values ​​of the standard sample when the above three positive samples were tested (Table 8). Figure 7 ), where the y-axis is the OD value and the x-axis is the concentration. According to the standard curve ( Figure 7 ) Calculate the concentrations of the three all-positive samples (results in Table 9).

[0118] Table 8

[0119]

[0120] Table 9

[0121]

[0122]

[0123] As can be seen from Table 9, compared with the system without adding a blocker, the correlation between the test results and phadia (gold standard) is better after adding a blocker to the system, which shows that when using the kit provided by the present invention to detect allergen-specific IgE antibodies, the interference of anti-peroxidase IgE antibodies produced by self-allergy can be significantly removed by adding a blocker to the system, thereby effectively reducing the false positive of the test results and making the test results more accurate.

[0124] The above detailed description of the present invention is intended to enable persons familiar with the art to understand the contents of the present invention and implement them. It does not limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A kit for detecting allergen-specific IgE antibodies, characterized in that: The kit comprises a blocking agent, an immunolabeled allergen, a peroxidase complex capable of binding to the immunolabel and catalyzing the color development of a substrate, and a substrate capable of being catalyzed by the peroxidase for color development; The blocking agent includes one or more of inactivated thyroid peroxidase, neutrophil peroxidase, eosinophil peroxidase, horseradish peroxidase, and myelin basic protein.

2. The kit according to claim 1, characterized in that The blocking agent is a mixture including inactivated thyroid peroxidase, neutrophil peroxidase, eosinophil peroxidase, horseradish peroxidase and myelin basic protein.

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

5. The Tris-HCl buffer solution includes the following components: 10-100g / L BSA, 0.5-2% Proclin-300, 0.05-3g / L Brij-35 and 0.1-0.3M NaCl accounting for the volume of the buffer solution.

4. The kit according to claim 3, characterized in that The buffer solution is mixed with the blocking agent to form a mixed solution, and the concentration of the blocking agent in the mixed solution is 1 to 10 mg / mL.

5. The kit according to claim 1, characterized in that The immune markers include biotin labeling and FITC labeling; the allergens include food allergens and inhaled allergens.

6. The kit according to claim 5, characterized in that The food allergens are selected from egg white, egg yolk, milk, goat milk, yogurt, casein, buckwheat, wheat bran, corn, rice, oats, wheat, shrimp, crab, scallop, cod, hairtail, sea bass, salmon, lobster, carp, peanuts, beef, lamb, soybean, chestnut, sesame, hazelnut, almond, cashew, walnut, banana, mango, peach, kiwi, apple, pear, cherry, plum, grape, strawberry, papaya, grapefruit, orange, lemon, pineapple, cantaloupe, watermelon, melon, coconut, olive, broccoli, eggplant, bitter melon, cucumber, bell pepper, asparagus, spinach, tomato, zucchini, carrot, potato, garlic, sweet potato, radish, celery The inhaled allergen is selected from one or more of cat dander, dog dander, ragweed, humulus, cockroaches, house dust mites, dust mites, poplars, willows, cypresses, birches, pine trees, elms, sycamores, dandelions, mugwort, wormwood, amaranth, tobacco scraps, mosquitoes, cotton wool, flower thatch pollen, ryegrass pollen, meadow fescue pollen, timothy grass pollen, small bran grass pollen, meadow bluegrass, Penicillium, Aspergillus fumigatus, Alternaria, Mycosporium, Mucor, Rhizopus and Candida albicans.

7. The kit according to claim 1, characterized in that The peroxidase complex capable of binding to an immune marker and catalyzing substrate color development comprises streptavidin coupled to peroxidase; and / or, the peroxidase is selected from horseradish peroxidase; and / or, the substrate capable of being catalyzed by the peroxidase for color development is TMB color developing solution or OPD color developing solution.

8. The kit according to claim 1, characterized in that The blocking agent exists as a single component and / or in the form of a mixture formed by mixing with the immunolabeled allergen; and / or, the kit further includes a stop solution, a cleaning solution and a standard substance, and the stop solution is 0.05-0.5M sulfuric acid.

9. The kit according to any one of claims 1 to 8, characterized in that When the kit is used for detection, the blocking agent is added before the peroxidase complex capable of binding to the immunolabel and catalyzing the color development of the substrate is added to the detection system.

10. The kit according to claim 1, characterized in that The method for using the kit comprises the following steps: (1) combining the sample to be tested, the blocking agent and the solid phase carrier, and incubating at room temperature; (2) washing the mixed solution after incubation in step (1), washing it, mixing it with the immunolabeled allergen, and incubating it at room temperature; (3) washing the mixed solution after incubation in step (2), adding the peroxidase complex capable of binding to the immune marker and catalyzing the color development of the substrate after washing, and incubating at room temperature; (4) washing the mixed solution after incubation in step (3), adding the substrate capable of being catalyzed by the peroxidase to develop color after washing, adding a stop solution to terminate the reaction, and then detecting absorbance; or, (1) combining the sample to be tested with the solid phase carrier and incubating at room temperature; (2) washing the incubated sample, mixing it with the blocking agent and the immunolabeled allergen, and incubating it at room temperature; (3) washing the mixed solution after incubation in step (2), adding the peroxidase complex capable of binding to the immune marker and catalyzing the color development of the substrate after washing, and incubating at room temperature; (4) Washing step (3) After the incubation, the mixed solution is washed and a substrate that can be catalyzed by the peroxidase is added for color development. After adding a stop solution to terminate the reaction, absorbance detection is performed.

11. The kit according to claim 10, characterized in that The solid phase carrier is coated with anti-IgE antibodies and is blocked; and / or, the solid phase carrier includes a polystyrene solid phase carrier, nanomagnetic particles, and a nitrocellulose membrane.

12. The kit according to claim 1, characterized in that The specification of the kit is 1 to 100 servings / box, wherein, based on 1 serving / box, the amount of the blocker in the kit is 0.05 to 50 μg, the amount of the immune-labeled allergen is 0.001 to 10 μg, the amount of the peroxidase complex that can bind to the immune label and catalyze the color development of the substrate is 0.05 to 0.5 μg, and the amount of the substrate that can be catalyzed by the peroxidase to develop color is 50 to 200 μL.

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