Inhalation allergen specific IgE antibody detection kit and application thereof

Abstract

The invention relates to the technical field of allergy detection, in particular to an inhalation allergen specific IgE antibody detection kit and application thereof. The kit is a colloidal gold detection kit. The detection objects are pollen, dust mites and animal scurf; the kit comprises a colloidal gold labeled mouse anti-human IgE antibody arranged on a test strip combination pad; the pollen antigens (Art v1, Amb a1 and Bet v2), the dust mite antigens (Der f1 and Der p1) and the animal scurf antigen (Can f1) are arranged on a nitrocellulose membrane of the test strip. The kit provided by the invention can be used for rapidly identifying the allergen of the inhalation allergy symptom and classifying the severity of the allergy symptom, 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 an inhaled allergen-specific IgE antibody detection kit and its application. Background Art

[0002] With the increasing incidence of allergic diseases, approximately 20-40% of the global population suffers from them. Inhaled allergens (such as dust mites and pollen) are the main triggers of allergies. They not only seriously affect patients' quality of life but can also lead to fatal complications such as asthma and anaphylactic shock. They are particularly detrimental to children's growth, development, and learning ability. Therefore, accurate detection of inhaled allergens is extremely urgent.

[0003] Currently, inhaled allergen testing is primarily divided into in vivo and in vitro testing. While in vivo skin prick testing is simple and low-cost, it is susceptible to drug interference and carries the risk of inducing severe allergic reactions. In vitro serum-specific IgE testing, while highly safe and quantitative, is expensive, relies on specialized equipment and personnel, has a long reporting cycle, and can also result in false positive or false negative results.

[0004] In view of the limitations and shortcomings of existing detection methods, there is an urgent need to develop a colloidal gold test kit that is fast, accurate, sensitive, easy to operate and can simultaneously detect multiple inhaled allergens. Summary of the Invention

[0005] The present application provides an inhalation allergen-specific IgE antibody detection kit and its application.

[0006] The kit provided in this application is used for in vitro qualitative detection of specific IgE antibodies against inhaled allergens (pollen, dust mites and animal dander) in human serum. It can quickly identify the type of allergy and the inhaled allergens, thereby guiding the clinical diagnosis of allergic reactions and the adjustment of allergic reaction treatment plans.

[0007] 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.

[0008] In a first aspect, the present application provides an inhalation allergen-specific IgE antibody detection kit, which adopts the following technical solution: A kit for detecting inhaled allergen-specific IgE antibodies, which is a colloidal gold detection kit; the detection targets are pollen, dust mites, and animal dander; The kit includes colloidal gold-labeled mouse anti-human IgE antibodies disposed on a test strip conjugate pad; and pollen antigens, dust mite antigens, and animal dander antigens disposed on a nitrocellulose membrane of the test strip; The pollen antigens are Art v1, Amb a1, and Bet v2; the amino acid sequence of Art v1 is shown in SEQ ID NO 2, the amino acid sequence of Amb a1 is shown in SEQ ID NO 5, and the amino acid sequence of Bet v2 is shown in SEQ ID NO 8.

[0009] In this application, pollen is mainly targeted at Artemisia pollen, ragweed pollen, poplar pollen, willow pollen, and elm pollen. Among them, the antigen corresponding to Artemisia pollen is Art v1, the antigen corresponding to ragweed pollen is Amb a 1, and the antigen corresponding to poplar pollen, willow pollen, and elm pollen is the universal antigen Bet v 2.

[0010] Artemisia pollen: Artemisia belongs to the Asteraceae family. my country's Artemisia pollen, comprising 186 species and 44 varieties, is found throughout the country, with the highest concentrations in Northwest, North, Northeast, and Southwest China, and slightly less in East, Central, and South China. It is mostly found on barren slopes, in open fields, and along roadsides. Surveys show that Artemisia pollen is the most abundant pollen in the air during summer and autumn in many regions. Ragweed pollen: Ragweed belongs to the Asteraceae family. Ragweed is an important seasonal airborne allergen and a major trigger of allergic rhinitis. It can exacerbate asthma and allergic conjunctivitis, and can also induce Eustachian tube dysfunction and contact dermatitis in patients with allergic rhinitis. Poplar, elm, and willow pollen are the primary spring allergenic pollens in my country.

[0011] In this application, dust mites mainly target house dust mites and dust mites, wherein the antigen corresponding to house dust mites is Der p1, and the antigen corresponding to dust mites is Der f1.

[0012] Dust mites primarily inhabit dust, carpets, pillows, mattresses, and upholstered furniture, which are rich in biological material, particularly human dandruff. They are known to contribute to allergic diseases such as allergic rhinitis, allergic asthma, and atopic dermatitis. In much of the world, dust mites are a significant and persistent risk factor for asthma. House dust mites and dust mites, both important mite species belonging to the family Acaridae, are adapted to warm, humid environments. Compared to house dust mites, dust mites are more common in dry environments.

[0013] In this application, animal dander mainly refers to dog dander, and the antigen corresponding to dog dander is Can f1.

[0014] Dog allergens are not only detected in dog-keeping rooms but may also be present in schools and daycare centers where dogs are not frequently present. Allergens can be transmitted via clothing. Classroom furniture and textiles are important carriers of irritants and allergens, impacting school air quality. Dog allergens have been found in serum, dander, fur, hair, and saliva, with dog dander being a significant source of inhaled allergens. Numerous studies have reported that dogs frequently induce allergic symptoms in sensitized individuals. Symptoms include asthma, allergic rhinitis, and allergic conjunctivitis.

[0015] The kit provided in this application is used for in vitro qualitative detection of specific IgE antibodies against inhaled allergens (pollen, dust mites and animal dander) in human serum. It can quickly identify the type of allergy and the inhaled allergens, thereby guiding the clinical diagnosis of allergic reactions and the adjustment of allergic reaction treatment plans.

[0016] Optionally, the dust mite antigen is Der f1 or Der p1.

[0017] Optionally, the amino acid sequence of Der p 1 is shown as SEQ ID NO 10.

[0018] Optionally, the amino acid sequence of Der f 1 is shown as SEQ ID NO 13.

[0019] Optionally, the animal dander antigen is Can f1.

[0020] Optionally, the amino acid sequence of Can f 1 is shown as SEQ ID NO 15.

[0021] In a second aspect, the present application provides a use of the above-mentioned kit in rapidly identifying allergy types and rapidly identifying inhaled 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 inhaled allergens (pollen, dust mites and animal dander) in human serum. It can quickly identify the type of allergy and the inhaled allergens, 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 kit for detecting inhalant allergen-specific IgE antibodies. The test targets are pollen, dust mites, and animal dander. The kit includes colloidal gold-labeled mouse anti-human IgE antibodies disposed on a test strip conjugate pad; and pollen antigens, dust mite antigens, and animal dander antigens disposed on a nitrocellulose membrane of the test strip.

[0028] Among them, pollen antigens are Art v1, Amb a1, Bet v2; dust mite antigens are Der f1, Der p1; and animal dander antigen is Can f1.

[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 pollen antigen, dust mite antigen and animal dander 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 eight lines: seven test lines (T) and one control line (C). Seven antigens are immobilized on each of the seven 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 inhaled 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 Pollen antigens (Art v1, Amb a1, Bet v2), dust mite antigens (Derf1, Der p1), animal dander antigens (Can f1) 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 pollen antigen Art v1.

[0052] A total of two amino acid sequences expected to serve as Art v1 antigens were designed and synthesized, namely SEQ ID NO 1 and SEQ ID NO 2. Preparation Example 2

[0053] This preparation example provides the pollen antigen Amb a1.

[0054] A total of three amino acid sequences expected to serve as Amb a1 antigens were designed and synthesized, namely SEQ ID NO 3, SEQ ID NO 4 and SEQ ID NO 5. Preparation Example 3

[0055] This preparation example provides the pollen antigen Bet v 2.

[0056] A total of three amino acid sequences expected to serve as Bet v 2 antigens were designed and synthesized, namely SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8. Preparation Example 4

[0057] This preparation example provides the dust mite antigen Der p 1.

[0058] A total of three amino acid sequences expected to serve as Der p 1 antigens were designed and synthesized, namely SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11. Preparation Example 5

[0059] This preparation example provides the dust mite antigen Der f 1.

[0060] A total of three amino acid sequences expected to serve as Der f 1 antigens were designed and synthesized, namely SEQ ID NO 12, SEQ ID NO 13 and SEQ ID NO 14. Preparation Example 6

[0061] This preparation example provides the animal dander antigen Can f1.

[0062] A total of three amino acid sequences expected to serve as Can f 1 antigens were designed and synthesized, namely SEQ ID NO 15, SEQ ID NO 16 and SEQ ID NO 17. Preparation Example 1-12

[0063] Examples 1-12 provide a kit for detecting inhaled 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

[0065] The kits provided in the above examples were used to perform serum tests on 40 patients with pollen allergy, 30 patients with dust mite allergy, and 30 patients with animal dander allergy to investigate the accuracy of the kits.

[0066] The test results are shown in Table 2: ; As can be seen from Table 2, after experimental screening, the technical solution provided in Example 6 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 kit for detecting inhaled allergen-specific IgE antibodies, characterized in that: The kit is a colloidal gold detection kit; the detection objects are pollen, dust mites and animal dander; The kit includes colloidal gold-labeled mouse anti-human IgE antibodies disposed on a test strip conjugate pad; and pollen antigens, dust mite antigens, and animal dander antigens disposed on a nitrocellulose membrane of the test strip; The pollen antigens are Art v1, Amb a1, and Bet v2; the amino acid sequence of Art v1 is shown in SEQ ID NO 2, the amino acid sequence of Amb a1 is shown in SEQ ID NO 5, and the amino acid sequence of Bet v2 is shown in SEQ ID NO 8.

2. The kit according to claim 1, wherein The dust mite antigens are Der f1 and Der p1.

3. The kit according to claim 2, wherein The amino acid sequence of Der p 1 is shown in SEQ ID NO 10.

4. The kit according to claim 2, wherein The amino acid sequence of Der f 1 is shown in SEQ ID NO 13.

5. The kit according to claim 1, wherein The animal dander antigen is Can f 1.

6. The kit according to claim 5, wherein The amino acid sequence of Can f 1 is shown in SEQ ID NO 15.

7. Use of the kit according to any one of claims 1 to 6 for rapid identification of allergy types and rapid identification of inhaled allergens.