Detection kit for specific IgE (Immunoglobulin E) antibody of ingestion allergen and application of detection kit

Abstract

The invention relates to the technical field of allergy detection, in particular to an ingestion allergen specificity IgE antibody detection kit and application thereof. The kit is a colloidal gold detection kit. Detection objects are milk, eggs and soybeans; the kit comprises a colloidal gold labeled mouse anti-human IgE antibody arranged on a test strip combination pad; the milk antigens (alpha-La and beta-Lg), the egg antigens (OVA and Gal d1) and the soybean antigens (Gly m2 and Gly m5) are arranged on the nitrocellulose membrane of the test strip. The kit provided by the invention can be used for rapidly identifying the allergens of the ingestion allergy symptoms and classifying the severity of the allergy symptoms, and the identification result has relatively high accuracy.

Description

Technical Field

[0001] The present application relates to the technical field of allergy detection, and in particular to a detection kit for ingested allergen-specific IgE antibodies and its application. Background Art

[0002] Hypersensitivity refers to a specific immune response in which the immune response is enhanced when the body is exposed to the same antigen again after the initial response to certain antigens. When the amount of antigen ingested is large or the body's immunity is in a high response state, the immune response is too strong and causes tissue damage.

[0003] Allergic diseases, such as allergic rhinitis (conjunctivitis), allergic asthma, hereditary allergic dermatitis (atopic dermatitis or atopic dermatitis), severe allergic reactions, and food allergies, are caused by the immune system's overreaction after repeated exposure to normal, harmless substances in the living environment. These substances are often called allergens.

[0004] Food allergies are often caused by type I hypersensitivity reactions mediated by specific IgE antibodies, which can severely impact patients' quality of life and even be life-threatening. For those experiencing allergic symptoms like itchy skin and shortness of breath, as well as those with a family history of allergies, food allergen testing not only accurately locates allergens and helps mitigate risks, but is also a key means of preventing allergies. Its importance cannot be overstated.

[0005] Currently, there are numerous in vitro methods for detecting food allergens, such as enzyme-linked immunosorbent assay (ELISA) and immunoblotting. However, these methods generally suffer from drawbacks such as lengthy testing times, high costs, reliance on specialized instruments, and cumbersome procedures, hindering widespread adoption. In contrast, colloidal gold immunochromatography, with its advantages of ease of use, low cost, and rapid detection, has emerged as a promising method in the field of biomedical diagnostics. Summary of the Invention

[0006] The present application provides a food allergen-specific IgE antibody detection kit and its application.

[0007] The kit provided in this application is used for in vitro qualitative detection of specific IgE antibodies against food allergens (milk, eggs and soybeans) in human serum. It can quickly identify the type of allergy and the food allergen, thereby guiding the clinical diagnosis of allergic reactions and the adjustment of allergic reaction treatment plans.

[0008] The kit provided herein employs an indirect assay method, employing allergen antigens and goat anti-mouse IgG antibodies spotted on the T zone (test line) and C zone (control line) of a nitrocellulose membrane, respectively. Colloidal gold-labeled mouse anti-human IgE antibodies serve as the colorant. During testing, sample serum is dripped into the sample well. Specific IgE antibodies in the sample serum bind to the colloidal gold-labeled mouse anti-human IgE antibodies and migrate along the nitrocellulose membrane toward the test and control lines. There, they bind to the coated allergen antigen to form an "allergen antigen-IgE-mouse anti-human IgE colloidal gold" complex. When the concentration of specific IgE antibodies in the sample is sufficiently high, a purple-red band will appear on the corresponding test line. When the colloidal gold-labeled mouse anti-human IgE antibodies migrate to the control line, a "goat anti-mouse IgG-mouse anti-human IgE colloidal gold" complex will form, resulting in color development. The absence of a color band on the control line indicates a failed reaction and an inaccurate test result.

[0009] Soybeans are a popular food ingredient in Asia and are often associated with food allergies. Soybean dust, which is present during storage and transportation, and soy flour, which is consumed during consumption, have been reported to trigger asthma attacks and even worsen their severity.

[0010] Eggs are widely recognized as one of the most common foods that trigger allergic reactions in both adults and children. In infants and young children, the presence of IgE, an antibody specific for egg whey protein, is a key indicator of an allergic reaction and a precursor to the development of an allergic disease.

[0011] Milk is a major factor in adverse reactions in infants and young children. Milk-induced asthma is common among children with food allergies. Milk is a major factor in allergic rhinoconjunctivitis in infants and young children, and a major cause of severe allergic otitis media caused by food.

[0012] In a first aspect, the present application provides a food allergen-specific IgE antibody detection kit, which adopts the following technical solution: A edible allergen-specific IgE antibody detection kit, the kit being a colloidal gold detection kit; the detection objects are milk, eggs and soybeans; The kit comprises colloidal gold-labeled mouse anti-human IgE antibody disposed on a test strip conjugate pad; and milk antigen, egg antigen and soybean antigen disposed on a nitrocellulose membrane of the test strip; The milk antigens are α-La and β-Lg; the amino acid sequence of the α-La is shown in SEQ ID NO 1, and the amino acid sequence of the β-Lg is shown in SEQ ID NO 3.

[0013] The test kits in this application detect milk, eggs, and soy. Milk antigens include recombinant α-lactalbumin (α-La) and β-lactoglobulin (β-Lg), both major milk antigens with IgE binding rates exceeding 80%. Egg antigens include recombinant ovalbumin (OVA) and Gal d1. Soy antigens include recombinant soy 2S albumin (Gly m2) and glycinin (Gly m5). The former is the major allergen, with an IgE binding rate exceeding 65%, while the latter covers severe allergic cases, such as those that induce immediate anaphylaxis.

[0014] The kit provided in this application is used for in vitro qualitative detection of specific IgE antibodies against food allergens (milk, eggs and soybeans) in human serum. It can quickly identify the type of allergy and the food allergen, thereby guiding the clinical diagnosis of allergic reactions and the adjustment of allergic reaction treatment plans.

[0015] Optionally, the egg antigen is OVA or Gal d1.

[0016] Optionally, the amino acid sequence of the OVA is shown as SEQ ID NO 5.

[0017] Optionally, the amino acid sequence of the Gal d1 is shown as SEQ ID NO 8.

[0018] Optionally, the soybean antigens are Gly m2 and Gly m5.

[0019] Optionally, the amino acid sequence of Gly m2 is shown as SEQ ID NO 9.

[0020] Optionally, the amino acid sequence of Gly m5 is shown as SEQ ID NO 11.

[0021] In a second aspect, the present application provides a use of the above-mentioned kit in rapidly identifying allergy types and rapidly identifying ingested allergens.

[0022] In summary, this application includes at least one of the following beneficial technical effects: The kit provided in this application is used for in vitro qualitative detection of specific IgE antibodies against food allergens (milk, eggs and soybeans) in human serum. It can quickly identify the type of allergy and the food allergen, thereby guiding the clinical diagnosis of allergic reactions and the adjustment of allergic reaction treatment plans. DETAILED DESCRIPTION

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] This application provides a test kit for detecting specific IgE antibodies to food allergens. The test targets are milk, egg, and soybean. The kit includes colloidal gold-labeled mouse anti-human IgE antibodies placed on a test strip conjugate pad; and milk antigens, egg antigens, and soybean antigens placed on a nitrocellulose membrane of the test strip.

[0028] Among them, milk antigens are α-La and β-Lg; egg antigens are OVA and Gal d1; and soybean antigens are Gly m2 and Glym5.

[0029] The specific analysis of the above kit is as follows: a.Structure The principle of this kit is the colloidal gold method, that is, the immune colloidal gold technology.

[0030] After the colloidal gold-labeled mouse anti-human IgE antibody combines with the allergen antibody in the sample to be tested, it combines with and fixes the milk antigen, egg antigen and soybean antigen on the test line. The colloidal gold-labeled mouse anti-human IgE antibody can then aggregate on the test line to form a color development result visible to the naked eye.

[0031] The test kit is constructed as follows: the test card consists of a plastic outer shell and a test strip inside. From left to right, the strip contains the sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad. The various components of the strip are held together by a PVC backing with adhesive.

[0032] The conjugate pad is pre-sprayed with colloidal gold-labeled antibodies (referred to as gold-labeled antibodies). The nitrocellulose membrane is marked with seven lines: six test lines (T) and one control line (C). Six antigens are immobilized on each of the six test lines. A blood sample is dispersed in a buffer solution and dropped onto the sample pad. If the sample contains the corresponding antigen, it will be recognized by the gold-labeled antibodies on the conjugate pad, forming an antigen-antibody complex. This antigen-antibody complex migrates along the test strip from left to right under capillary action. Upon encountering another allergen antigen on the test line, it is recognized by the antigen-antibody complex. At this point, the gold-labeled antibodies are partially trapped and aggregated on the test line, resulting in a visible color development on the test line. If the test sample lacks antibodies, no color will develop on the test line.

[0033] The gold-labeled antibody is in excess and is not only retained at the test line but also reaches the control line, where a goat anti-mouse IgG antibody (anti-gold-labeled antibody) specifically targeting the gold-labeled antibody is immobilized. This causes the gold-labeled antibody to aggregate and develop color at the control line. If no color develops at the control line, the test result is invalid. If the nitrocellulose membrane still displays a light red background, the test is not complete.

[0034] Two groove openings can also be set on the plastic card shell, namely the sample spotting port and the color development port. The sample spotting port faces the sample pad and is used to add the sample to be tested. The color development port is the area for displaying the results and faces the test line and the quality control line. The color development results can be observed in the corresponding area.

[0035] b. Operation method During the test, a drop of blood sample diluent is added to the sample pad. The antibody to be tested in the sample diluent flows through the liquid to the conjugate pad and binds to the corresponding colloidal gold-labeled antibody there, forming an antigen-antibody complex. The antigen-antibody complex continues to flow due to capillary action. When it moves to the antibody area fixed on the nitrocellulose membrane, the antigen-antibody complex binds to the antibody corresponding to the other allergen, forming a red band, which is the band on the test line. The gold-labeled antibody that is not bound to the corresponding protein binds to the goat anti-mouse IgG antibody, forming a red band, which is the band on the quality control line, indicating that the test is positive for the allergen antibody. If no band appears on the test line but a red band appears on the quality control line, the test is negative for the allergen antibody. If no band appears on the quality control line, the test is invalid.

[0036] c. Production principle A method for preparing a kit for detecting ingested allergen-specific IgE antibodies comprises the following steps: (1) Mouse anti-human IgE antibody Mouse anti-human IgE antibody was purchased from Beijing Bersee Technology Co., Ltd. (Bersee), catalog number B2060.

[0037] (2) Preparation of colloidal gold-labeled mouse anti-human IgE antibody The pH value of the colloidal gold solution was adjusted to 9.0, and the antibody concentration was selected based on the principle of no aggregation. The mouse anti-human IgE antibody was labeled with colloidal gold. The specific process is as follows: 1) Mouse anti-human IgE antibody pretreatment: The mouse anti-human IgE antibody (primary antibody) solution was dialyzed against 0.05 mol / L NaCl, pH 7.0, at 4°C overnight to remove excess salt ions. The solution was then centrifuged at 10,000 g for 1 h at 4°C to remove aggregates.

[0038] 2) Pretreatment of the colloidal gold solution: The adsorption of colloidal gold to proteins is primarily dependent on pH. At pH values ​​close to the protein's isoelectric point or slightly alkaline, the two tend to form strong bonds. If the pH of the colloidal gold is below the protein's isoelectric point, it will aggregate and lose its binding ability. Therefore, the pH of the colloidal gold solution must be pre-adjusted to 9.0 (using 0.1 mol / L K2CO3 or 0.1 mol / L HCl).

[0039] 3) Determination of the ratio of mouse anti-human IgE antibody to colloidal gold ①After adjusting the pH value of the colloidal gold solution, divide it into 10 tubes, 1 ml per tube; ② Serially dilute the mouse anti-human IgE antibody (primary antibody) solution with 0.05 mol / L, pH 9.0 borate buffer to 5-50 mg / ml. Take 1 ml of each solution and add it to a tube of colloidal gold solution. Add 1 ml of diluent (protein-free) to the control group and mix thoroughly. ③ After standing for 5 minutes, add 0.1 ml of 10% NaCl solution to each tube, mix well, let stand for 2 hours, and observe the results; ④ The tubes without mouse anti-human IgE antibody (control group) and with insufficient amount of mouse anti-human IgE antibody to stabilize the colloidal gold showed aggregation, changing from red to blue. Tubes with mouse anti-human IgE antibody at or above the minimum stabilizing amount remained red. The tube with the lowest amount of mouse anti-human IgE antibody contains the amount of protein required to stabilize 1 ml of colloidal gold. Adding 10-20% to this amount gives the actual amount of mouse anti-human IgE antibody (primary antibody) used.

[0040] 4) Binding of colloidal gold and mouse anti-human IgE antibody ① After determining the optimal amounts of the two reagents as described above, take the required amounts of colloidal gold and the corresponding mouse anti-human IgE antibody, and adjust the pH to 9.0 with 0.1 mol / L K2CO3; ② Mix the colloidal gold solution and mouse anti-human IgE antibody protein solution under stirring; ③After 10 minutes, add a certain amount of 5% bovine serum albumin (BSA) to a final concentration of 1% to prevent the aggregation and precipitation of mouse anti-human IgE antibodies and colloidal gold.

[0041] 5) Purification of colloidal gold-labeled proteins (ultracentrifugation) ① First, centrifuge at low speed to discard the aggregated colloidal gold particles (20 nm gold colloid particles should be centrifuged at 2000 r / min for 20 min); ② Centrifuge again at high speed (20 nm colloidal gold conjugate, 14000g, 4℃ for 1h); ③ Carefully remove the supernatant and resuspend the precipitate in 0.01 mol / L, pH 7.6 PB (0.02% NaN3) containing 1% BSA to the original volume; ④ After equilibration overnight, repeat the above centrifugation twice. Finally, resuspend the mixture in 0.01 mol / L PB (pH 7.6) with 1% BSA to 1 / 10 the original volume. Aliquot and store at 4°C. Add 50% glycerol to the conjugate and it can be stored at -18°C for at least one year.

[0042] 6) Quality identification of colloidal gold-protein conjugates ① Determination of the average diameter of colloidal gold particles: Dip a nickel mesh (copper mesh can also be used) with a film-supported membrane into the gold-labeled protein reagent. Allow to dry naturally before observing directly under a transmission electron microscope. Alternatively, observe after counterstaining with uranyl acetate. Calculate the average diameter of 100 gold particles.

[0043] ②OD of colloidal gold-protein solution 520nm Determination of OD value: Colloidal gold particles have a maximum absorption peak between wavelengths of 510-50nm. Dilute the colloidal gold protein reagent 1:20 with 0.02mol / L pH8.2 PBS (containing 1% BSA, 0.02% NaN3). 520nm = About 0.25. The OD of the general application liquid 520nm It should be 0.2-0.4.

[0044] ③ Determination of the specificity and sensitivity of gold-labeled proteins: The microporous membrane immunogold-silver staining method (MF-IGSSA) is used. Soluble antigens (or antibodies) are adsorbed onto a carrier (filter paper, nitrocellulose membrane, microporous membrane). Colloidal gold-labeled antibodies (or antigens) are used for direct or indirect staining followed by silver development to detect the corresponding antigens or antibodies. The specificity and sensitivity of the gold-labeled proteins are then determined.

[0045] (3) Preparation of antibodies on test strips Milk antigens (α-La, β-Lg), egg antigens (OVA, Gal d1), soybean antigens (Gly m2, Gly m5) and goat anti-mouse IgG antibodies were immobilized on nitrocellulose membranes, and colloidal gold-labeled mouse anti-human IgE antibodies were added to the conjugate pad.

[0046] 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.

[0047] 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.

[0048] In the following examples, colloidal gold-labeled mouse anti-human IgE antibody was purchased from Beijing Bersee Technology Co., Ltd. (Bersee), catalog number B2060.

[0049] In the following examples, the diluent used to dilute the sample to be tested is a diluent commonly used in colloidal gold test kits for detecting serum, which can be known or obtained by those skilled in the art using conventional techniques in the art.

[0050] The present application is further described in detail below in conjunction with the examples and test results. Preparation Example Preparation Example 1

[0051] This preparation example provides the milk antigen α-La.

[0052] A total of two amino acid sequences expected to be used as α-La antigens were designed and synthesized, namely SEQ ID NO 1 and SEQ ID NO 2. Preparation Example 2

[0053] This preparation example provides the milk antigen β-Lg.

[0054] A total of two amino acid sequences expected to serve as β-Lg antigens were designed and synthesized, namely SEQ ID NO 3 and SEQ ID NO 4. Preparation Example 3

[0055] This preparation example provides the egg antigen OVA.

[0056] A total of two amino acid sequences expected to be used as OVA antigens were designed and synthesized, namely SEQ ID NO 5 and SEQ ID NO 6. Preparation Example 4

[0057] This preparation example provides the egg antigen Gal d1.

[0058] A total of two amino acid sequences expected to be Gal d1 antigens were designed and synthesized, namely SEQ ID NO 7 and SEQ ID NO 8. Preparation Example 5

[0059] This preparation example provides soybean antigen Gly m2.

[0060] A total of two amino acid sequences expected to serve as Gly m2 antigens were designed and synthesized, namely SEQ ID NO 9 and SEQ ID NO 10. Preparation Example 6

[0061] This preparation example provides soybean antigen Gly m5.

[0062] A total of two amino acid sequences expected to serve as Gly m5 antigens were designed and synthesized, namely SEQ ID NO 11 and SEQ ID NO 12. Preparation Examples 1-7

[0063] Examples 1-7 provide a kit for detecting ingested allergen-specific IgE antibodies. The preparation methods of the kits are described in the detailed description.

[0064] The difference between the above embodiments is that the antigen sequences of the various allergens on the test line are different, as shown in Table 1: ; Performance test results The kits provided in the above examples were used to perform serum tests on 50 patients with milk allergy, 50 patients with egg allergy, and 50 patients with soybean allergy to investigate the accuracy of the kits.

[0065] The test results are shown in Table 2: ;

[0066] As can be seen from Table 2, after experimental screening, the technical solution provided in Example 5 of the present application can more accurately identify the type of antigen in the sample to be tested and will not affect the detection results of other related competing antigens, and has higher accuracy.

[0067] 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.

[0068] 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 test kit for detecting ingested allergen-specific IgE antibodies, characterized in that: The kit is a colloidal gold detection kit; the detection objects are milk, eggs and soybeans; The kit comprises colloidal gold-labeled mouse anti-human IgE antibody disposed on a test strip conjugate pad; and milk antigen, egg antigen and soybean antigen disposed on a nitrocellulose membrane of the test strip; The milk antigens are α-La and β-Lg; the amino acid sequence of the α-La is shown in SEQ ID NO 1, and the amino acid sequence of the β-Lg is shown in SEQ ID NO 3.

2. The kit according to claim 1, wherein The egg antigens are OVA and Gal d1.

3. The kit according to claim 2, wherein The amino acid sequence of OVA is shown in SEQ ID NO 5.

4. The kit according to claim 2, wherein The amino acid sequence of the Gal d1 is shown in SEQ ID NO 8.

5. The kit according to claim 6, wherein The soybean antigens are Gly m2 and Gly m5.

6. The kit according to claim 6, wherein The amino acid sequence of Gly m2 is shown in SEQ ID NO 9.

7. The kit according to claim 6, wherein The amino acid sequence of Gly m5 is shown in SEQ ID NO 11.

8. Use of the kit according to any one of claims 1 to 7 for rapid identification of allergy types and rapid identification of ingested allergens.