Inhalant allergen-specific IgE antibody detection kit and application thereof

Abstract

The application relates to the technical field of allergy detection, in particular to an inhaled allergen specific IgE antibody detection kit and application thereof. The kit is a colloidal gold detection kit; the detection object is pollen, dust mites and animal dander; the kit comprises colloidal gold labeled mouse anti-human lgE antibodies arranged on a test strip combination pad; and pollen antigens (Art v1, Amb a1, Bet v2), dust mite antigens (Der f1, Der p1) and animal dander antigens (Can f1) arranged on a test strip nitrocellulose membrane. The kit provided by the application can quickly identify the allergen of inhaled allergic symptoms, classify the severity of allergic symptoms, and the identification result has a high accuracy.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of allergy detection, and particularly relates to an inhaled allergen-specific IgE antibody detection kit and application thereof. BACKGROUND

[0002] With the continuous rise of the incidence of allergic diseases, about 20-40% of the global population is affected, and inhaled allergens (such as dust mites, pollen, etc.) are the main inducement of allergies, which not only seriously affects the quality of life of patients, but also can cause fatal complications such as asthma and anaphylactic shock, especially having adverse effects on the growth and development and learning ability of children. Therefore, it is urgent to accurately detect inhaled allergens.

[0003] Currently, the detection of inhaled allergens is mainly divided into in vivo and in vitro tests. Although the in vivo skin prick test is simple to operate and low in cost, it is easily interfered by drugs and has the risk of inducing severe allergic reactions; the in vitro serum-specific IgE detection is safe, quantifiable, but high in cost, dependent on professional equipment and personnel, long in report cycle, and may have false positive or false negative results.

[0004] In view of the limitations and deficiencies of the existing detection methods, there is an urgent need to develop a colloidal gold kit which is rapid, accurate, sensitive, simple to operate and can simultaneously detect multiple inhaled allergens. SUMMARY

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

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

[0007] The kit provided in this application uses an allergen antigen and goat anti-mouse IgG antibody, respectively spotted onto the T region (detection line) and C region (control line) of a nitrocellulose membrane, and uses colloidal gold-labeled mouse anti-human IgE antibody as the chromogenic substance, prepared using an indirect method. During detection, serum sample is added to the sample well. The specific IgE antibody in the serum binds to the colloidal gold-labeled mouse anti-human IgE antibody and migrates along the nitrocellulose membrane towards the detection and control lines. On the detection line, it binds to the coated allergen antigen, forming an "allergen antigen-IgE-mouse anti-human IgE colloidal gold" complex. When the concentration of specific IgE antibody in the sample is sufficiently high, a purple-red band appears on the corresponding detection line. When the colloidal gold-labeled mouse anti-human IgE antibody moves to the control line, it forms a "goat anti-mouse IgG-mouse anti-human IgE colloidal gold" complex, resulting in color development. If no band appears on the control line, the reaction has failed, and the test result is inaccurate.

[0008] In a first aspect, this application provides a kit for detecting inhaled allergen-specific IgE antibodies, employing the following technical solution:

[0009] A kit for detecting IgE antibodies specific to inhaled allergens, wherein the kit is a colloidal gold detection kit; the detection targets are pollen, dust mites, and animal dander;

[0010] The kit includes a colloidal gold-labeled mouse anti-human IgE antibody disposed on the test strip conjugation pad; and pollen antigen, house dust mite antigen, and animal dander antigen disposed on the nitrocellulose membrane of the test strip.

[0011] The pollen antigens are Art v1, Amb a1, and Bet v2; the amino acid sequence of Art v1 is shown in SEQ ID NO2, the amino acid sequence of Amb a1 is shown in SEQ ID NO5, and the amino acid sequence of Bet v2 is shown in SEQ ID NO8.

[0012] In this application, the pollen mainly refers to 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 a1, and the antigen corresponding to poplar pollen, willow pollen, and elm pollen is the universal antigen Bet v2.

[0013] Artemisia pollen: The genus *Artemisia* belongs to the Asteraceae family. In my country, *Artemisia* pollen includes 186 species and 44 varieties, distributed throughout the country, with the highest concentrations in Northwest, North, Northeast, and Southwest China, and slightly lower concentrations in East, Central, and South China. It is mostly found on barren slopes, open fields, and 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 for allergic rhinitis. Ragweed pollen can exacerbate asthma and allergic conjunctivitis attacks, and can also induce eustachian tube dysfunction and contact dermatitis in patients with allergic rhinitis. Poplar, elm, and willow pollen are the main spring allergenic pollens in my country.

[0014] In this application, the term "dust mites" primarily refers to house dust mites and flour dust mites. Specifically, the antigen corresponding to house dust mites is Der p1, and the antigen corresponding to flour dust mites is Der f1.

[0015] Dust mites primarily inhabit dust, carpets, pillows, mattresses, and indoor decorative furniture rich in biological matter (especially human skin flakes). Dust mites easily trigger allergic rhinitis, allergic asthma, atopic dermatitis, and other allergic diseases. In most parts of the world, dust mites are a significant and persistent risk factor for asthma. House dust mites and flour mites are important mite species, belonging to the family Acaridae, and are adapted to living in warm, humid environments. Compared to house dust mites, flour mites are more common in dry environments.

[0016] In this application, animal dander primarily refers to dog dander. The antigen corresponding to dog dander is Can f1.

[0017] Dog allergens can be detected not only in petting zoos but also in schools and daycare centers where dogs are not frequently present. Allergens can be transmitted through clothing. Classroom furniture and textiles are significant carriers of irritants and allergens, impacting air quality within schools. Dog allergens have been found in serum, dander, fur, hair, and saliva, with dog dander clearly 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.

[0018] The kit provided in this application is used for the 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 allergen, thereby guiding the clinical diagnosis of allergic reactions and the adjustment of treatment plans for allergic reactions.

[0019] Optionally, the dust mite antigen is Der f1 or Der p1.

[0020] Optionally, the amino acid sequence of Der p1 is shown in SEQ ID NO 10.

[0021] Optionally, the amino acid sequence of Der f1 is shown in SEQ ID NO 13.

[0022] Optionally, the animal dander antigen is Can f1.

[0023] Optionally, the amino acid sequence of Can f1 is shown in SEQ ID NO 15.

[0024] Secondly, this application provides the application of the above-mentioned reagent kit in rapidly identifying allergy types and rapidly identifying inhaled allergens.

[0025] In summary, this application includes at least one of the following beneficial technical effects:

[0026] The kit provided in this application is used for the 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 allergen, thereby guiding the clinical diagnosis of allergic reactions and the adjustment of treatment plans for allergic reactions. Detailed Implementation

[0027] Before describing the embodiments of this application in detail, it should be understood that the terminology used herein is for the purpose of describing a particular embodiment only. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the term pertains.

[0028] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0029] The endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values, and such ranges or values ​​should be understood to include values ​​close to such ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0030] In this application, the terms "comprising" or "including" are open-ended expressions, meaning they include the content specified in this application but do not exclude other aspects.

[0031] This application provides a kit for detecting inhaled allergen-specific IgE antibodies. The targets for detection are pollen, house dust mites, and animal dander. The kit includes a colloidal gold-labeled mouse anti-human IgE antibody disposed on the conjugate pad of the test strip; and pollen antigen, house dust mite antigen, and animal dander antigen disposed on the nitrocellulose membrane of the test strip.

[0032] Among them, pollen antigens are Art v1, Amb a1, and Bet v2; dust mite antigens are Der f1 and Der p1; and animal dander antigens are Can f1.

[0033] The detailed analysis of the above reagent kit is as follows:

[0034] a. Structural Part

[0035] This kit is based on the colloidal gold method, also known as immunogold immunoassay.

[0036] After the colloidal gold-labeled mouse anti-human IgE antibody binds to the allergen antibodies in the sample to be tested, it binds to and fixes pollen antigens, dust mite antigens, and animal dander antigens on the test line. The colloidal gold-labeled mouse anti-human IgE antibody can then aggregate on the test line to form a visible colorimetric result.

[0037] The kit structure is as follows: the test card has an outer plastic casing and an inner test strip. From left to right, the test strip consists of a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad. All parts of the test strip are fixed together by a PVC backing with adhesive.

[0038] The conjugate pad is pre-coated with colloidal gold-labeled antibodies (referred to as gold-labeled antibodies). The nitrocellulose membrane has eight lines: seven test lines (T) and one control line (C). Seven antigens are immobilized on 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 moves along the test strip from left to right under capillary action. When it encounters another allergen antigen on the test line, it is recognized by the antigen-antibody complex. At this point, a portion of the gold-labeled antibody is trapped and aggregates at the test line, resulting in a visible color development. If the sample does not contain antibodies, no color will develop at the test line.

[0039] The gold-labeled antibody is in excess and is not only trapped at the test line but also reaches the control line. Here, a goat anti-mouse IgG antibody (anti-gold-labeled antibody) specifically targeting the gold-labeled antibody is immobilized, causing the gold-labeled antibody to aggregate and develop color at the control line. If there is no color development at the control line, the test result is invalid; if the nitrocellulose membrane still shows a light red background, it indicates that the test is not yet complete.

[0040] The plastic cartridge can also be equipped with two groove openings, namely a sample dispensing port and a color development port. The sample dispensing port is directly opposite the sample pad and is used to add the sample to be tested. The color development port is the area for displaying the results, which is directly opposite the detection line and the quality control line. The color development results can be observed in the corresponding area.

[0041] b. Operating method

[0042] During testing, a blood sample diluent is added to the sample pad. The antibody to be tested in the sample diluent flows to the conjugation pad and binds to the corresponding colloidal gold-labeled antibody in the conjugation pad, forming an antigen-antibody complex. The antigen-antibody complex continues to flow under capillary action. When it moves to the antibody region fixed on the nitrocellulose membrane, the antigen-antibody complex binds to the antibody corresponding to another allergen, showing a red band, which is the band on the test line. The gold-labeled antibody that does not bind to the corresponding protein binds to goat anti-mouse IgG antibody, showing a red band, which is the band on the control line. This indicates a positive result for the allergen antibody. If no band appears on the test line but a red band appears on the control line, the allergen antibody is considered negative. If no band appears on the control line, the test is invalid.

[0043] c. Manufacturing principle

[0044] A method for preparing an inhaled allergen-specific IgE antibody detection kit includes the following steps:

[0045] (1) Mouse anti-human IgE antibody

[0046] The mouse anti-human IgE antibody was purchased from Beijing Bersee Technology Co., Ltd. (Catalog No. B2060).

[0047] (2) Preparation of colloidal gold-labeled mouse anti-human IgE antibody

[0048] The pH of the colloidal gold solution was adjusted to 9.0, and the antibody concentration was selected to prevent aggregation. Mouse anti-human IgE antibody was then labeled with colloidal gold. The specific procedure is as follows:

[0049] 1) Pretreatment of mouse anti-human IgE antibody: The mouse anti-human IgE antibody (primary antibody) solution was dialyzed overnight at 4℃ with 0.05mol / L, pH7.0 NaCl to remove excess salt ions, and then centrifuged at 10000g at 4℃ for 1h to remove polymer.

[0050] 2) Pretreatment of colloidal gold solution: The adsorption of colloidal gold onto proteins mainly depends on pH value. Under pH conditions close to the isoelectric point of proteins or slightly alkaline, the two easily form a strong complex. If the pH value of colloidal gold is lower than the isoelectric point of proteins, it will aggregate and lose its binding ability. Therefore, we need to pre-adjust the pH value of colloidal gold to 9.0 (using 0.1mol / L K2CO3 or 0.1mol / L HCl).

[0051] 3) Determination of the ratio of mouse anti-human IgE antibody to colloidal gold for labeling

[0052] ① After adjusting the pH of the colloidal gold solution, dispense 10 tubes, 1 ml per tube;

[0053] ② The mouse anti-human IgE antibody (primary antibody) solution was serially diluted to 5-50 mg / ml with 0.05 mol / L, pH 9.0 borate buffer. 1 ml of each solution was added to a tube of colloidal gold solution. 1 ml of the diluent (without protein) was added to the control group. The solutions were then mixed.

[0054] ③ After standing for 5 minutes, add 0.1 ml of 10% NaCl solution to each of the above tubes, mix well, let stand for 2 hours, and observe the results;

[0055] ④ Tubes without mouse anti-human IgE antibody (control group) and those with insufficient mouse anti-human IgE antibody to stabilize colloidal gold showed a color change from red to blue due to aggregation; while tubes with mouse anti-human IgE antibody levels reaching or exceeding the minimum stabilizing amount remained red. The tube with the lowest mouse anti-human IgE antibody content represents the amount of protein required to stabilize 1 ml of colloidal gold. Adding 10-20% more to this amount gives the actual amount of mouse anti-human IgE antibody (primary antibody) used.

[0056] 4) Binding of colloidal gold to mouse anti-human IgE antibody

[0057] 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;

[0058] ② Mix the colloidal gold solution and the mouse anti-human IgE antibody protein solution with stirring;

[0059] ③ After 10 minutes, add a certain amount of 5% bovine serum albumin (BSA) to make its final concentration 1% to prevent the aggregation and precipitation of mouse anti-human IgE antibody and colloidal gold.

[0060] 5) Purification of colloidal gold-labeled proteins (ultracentrifugation method)

[0061] ① First, centrifuge at low speed to discard the aggregated colloidal gold particles (centrifuge 2000r / min for 20min for 20nm gold particles).

[0062] ② Then centrifuge at high speed (20nm colloidal gold conjugate, 14000g, 4℃ for 1h).

[0063] ③ Carefully aspirate the supernatant, and suspend the precipitate in 0.01mol / L PB (0.02% NaN3) containing 1% BSA at pH 7.6 until the original volume is reached;

[0064] ④ After equilibration overnight, repeat the centrifugation process twice. Finally, suspend the compound in 0.01 mol / L PB solution (pH 7.6) with 1% BSA to 1 / 10 of the original volume, aliquot, and store at 4°C. Adding 50% glycerol to the conjugate allows for storage at -18°C for over one year.

[0065] 6) Quality identification of colloidal gold-protein conjugates

[0066] ① Determination of the average diameter of colloidal gold particles: Dip a nickel mesh (or copper mesh) of the supporting membrane into the gold-labeled protein reagent, allow it to dry naturally, and observe it directly under a transmission electron microscope. Alternatively, observe after counterstaining with uranium acetate. Calculate the average diameter of 100 gold particles.

[0067] ② OD of colloidal gold-protein solution 520nm Determination of absorption values: Colloidal gold particles exhibit a maximum absorption peak between 510-50 nm. The colloidal gold protein reagent was diluted 1:20 with 0.02 mol / L pH 8.2 PBS (containing 1% BSA and 0.02% NaN3). OD 520nm = Approximately 0.25. The OD of commonly used liquids is... 520nm It should be 0.2-0.4.

[0068] ③ Determination of the specificity and sensitivity of gold-labeled proteins: Microporous membrane immunogold and silver staining (MF-IGSSA) was used. Soluble antigens (or antibodies) were adsorbed onto a carrier (filter paper, nitrocellulose membrane, microporous membrane), and colloidal gold-labeled antibodies (or antigens) were detected by direct or indirect staining followed by silver imaging to identify the specificity and sensitivity of the gold-labeled proteins.

[0069] (3) Preparation of antibodies on test strips

[0070] Pollen antigens (Art v1, Amb a1, Bet v2), dust mite antigens (Derf1, Der p1), animal dander antigens (Can f1), and goat anti-mouse IgG antibody were immobilized on nitrocellulose membranes, and colloidal gold-labeled mouse anti-human IgE antibody was added to the conjugate pad.

[0071] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort 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 construed as limiting this application.

[0072] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0073] In the following examples, the colloidal gold-labeled mouse anti-human IgE antibody was purchased from Beijing Bersee Technology Co., Ltd. (Catalog No. B2060).

[0074] In the following examples, the diluent used to dilute the test samples is a common diluent used in colloidal gold reagent kits that detect serum. This diluent is readily available or obtainable by those skilled in the art using conventional techniques.

[0075] The present application will be further described in detail below with reference to the embodiments and test results.

[0076] Preparation Example

[0077] Preparation Example 1

[0078] This preparation example provides pollen antigen Art v1.

[0079] Two amino acid sequences were designed and synthesized as Art v1 antigens, namely SEQ ID NO 1 and SEQ ID NO 2.

[0080] Preparation Example 2

[0081] This preparation example provides pollen antigen Amb a1.

[0082] Three amino acid sequences were designed and synthesized as the Amb a1 antigen, namely SEQ ID NO 3, SEQ ID NO 4 and SEQ ID NO 5.

[0083] Preparation Example 3

[0084] This preparation example provides pollen antigen Bet v2.

[0085] Three amino acid sequences were designed and synthesized as the Bet v2 antigen, namely SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8.

[0086] Preparation Example 4

[0087] This preparation example provides the dust mite antigen Der p1.

[0088] Three amino acid sequences were designed and synthesized as Der p1 antigens, namely SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11.

[0089] Preparation Example 5

[0090] This preparation example provides the dust mite antigen Der f1.

[0091] Three amino acid sequences were designed and synthesized as Der f1 antigens, namely SEQ ID NO 12, SEQ ID NO 13 and SEQ ID NO 14.

[0092] Preparation Example 6

[0093] This preparation example provides the animal dander antigen Can f1.

[0094] Three amino acid sequences were designed and synthesized as Can f1 antigens, namely SEQ ID NO 15, SEQ ID NO 16 and SEQ ID NO 17.

[0095] Preparation Examples 1-12

[0096] Examples 1-12 each provide a kit for detecting inhaled allergen-specific IgE antibodies. The preparation methods for these kits are detailed in the specific implementation section.

[0097] The difference between the above embodiments lies in that the antigen sequences of various allergens on the detection line are different, as shown in Table 1:

[0098] ;

[0099] Performance test results

[0100] Serum tests were performed on 40 patients with pollen allergy, 30 patients with dust mite allergy, and 30 patients with animal dander allergy using the kits provided in the above embodiments to examine the accuracy of the kits.

[0101] The test results are shown in Table 2:

[0102] ;

[0103] As shown in Table 2, after experimental screening, the technical solution provided in Example 6 of this application can more accurately identify the types of antigens in the sample to be tested without affecting the detection results of other related competing antigens, and has higher accuracy.

[0104] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this 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 targets are pollen, dust mites, and animal dander. The kit includes a colloidal gold-labeled mouse anti-human IgE antibody disposed on the test strip conjugation pad; and pollen antigen, house dust mite antigen, and animal dander antigen disposed on the nitrocellulose membrane of the test strip. The pollen antigens are Art v1, Amba1, and Bet v2; the amino acid sequence of Art v1 is shown in SEQ ID NO 2, the amino acid sequence of Amba1 is shown in SEQ ID NO 5, and the amino acid sequence of Bet v2 is shown in SEQ ID NO 8. The dust mite antigens are Der f1 and Der p1, the amino acid sequence of Der p1 is shown in SEQ ID NO 10, and the amino acid sequence of Der f1 is shown in SEQ ID NO 13; The animal dander antigen is Can f1, and the amino acid sequence of Can f1 is shown in SEQ ID NO 15.