Application of one or more of Aspergillus fumigatus allergen component and detection reagent for specific IgE of Aspergillus fumigatus allergen component in preparation of ABPA detection product, and ABPA detection product

Abstract

The invention relates to the field of biological detection, in particular to application of aspergillus fumigatus allergen components and one or more detection reagents of specific IgE of the aspergillus fumigatus allergen components in preparation of ABPA detection products and the ABPA detection products. The aspergillus fumigatus allergen component comprises a component 1, or comprises a component 1 and a component 2; the component 1 comprises one or more of Asp-f-A, Asp-f-B, Asp-f-C, Asp-f-D, Asp-f-F and Asp-f-G with amino acid sequences as shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12 and SEQ ID NO.14; and the component 2 comprises Asp-f-22 and / or Asp-f-27, and the component 2 comprises Asp-f-22 and / or According to the method, the appropriate aspergillus fumigatus allergen component is selected, and the positive condition of the aspergillus fumigatus allergen component specificity IgE in the blood sample of the patient is detected, so that the disease diagnosis of ABPA caused by aspergillus fumigatus is realized, and the method for diagnosing the allergic bronchial pulmonary aspergillosis is low in cost, simple to operate and efficient.

Description

Technical Field

[0001] This application relates to the field of biological detection, and in particular to the application of one or more of the detection reagents for Aspergillus fumigatus allergen components and their specific IgE in the preparation of ABPA detection products, and ABPA detection products. Background Technology

[0002] Aspergillus fumigatus is an opportunistic pathogenic fungus that can induce allergic reactions in humans. Allergic bronchopulmonary aspergillosis (ABPA), caused by this fungus, is a type of lung disease resulting from a severe allergic reaction to Aspergillus (primarily Aspergillus fumigatus). ABPA patients often present with poorly controlled asthma, recurrent lung infections, and varying degrees of bronchiectasis.

[0003] Currently, the clinical diagnosis of ABPA mainly relies on a combination of clinical manifestations, imaging features, and serological indicators. Among these, determining whether a patient has an Aspergillus fumigatus allergy is fundamental to the diagnosis of ABPA, and the core method for this is the specific IgE detection using crude Aspergillus fumigatus extract. However, crude Aspergillus fumigatus extract contains multiple antigenic components, which can easily induce widespread immune cross-reactions, affecting diagnostic specificity.

[0004] In terms of clinical manifestations, ABPA presents with diverse and nonspecific symptoms, overlapping with other fungal-associated lung diseases such as fungal sensitization-induced severe asthma. Clinically, both are characterized by refractory asthma, with common symptoms including cough, sputum production, and wheezing. Regarding imaging findings, ABPA patients also exhibit similar presentations to bronchiectasis and chronic pulmonary aspergillosis, such as bronchiectasis. Late-stage ABPA may also show typical changes of chronic pulmonary aspergillosis, such as cavitation or fibrosis. In terms of serological markers, in addition to ABPA patients, serum Aspergillus fumigatus-specific IgE can be detected in the serum of patients with structural lung diseases such as bronchiectasis, chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF). However, this positive result may only indicate fungal colonization or sensitization in the damaged airways, rather than active ABPA, further complicating diagnosis.

[0005] In summary, ABPA presents with diverse clinical manifestations and overlaps significantly with other fungal-associated lung diseases in terms of symptoms, imaging features, and serological markers. However, the treatment pathways and intervention strategies for ABPA differ fundamentally from those of other diseases. Diagnostic ambiguity can directly lead to deviations in treatment direction, causing patients to miss the optimal intervention window. Therefore, missed, misdiagnosed, and delayed diagnoses of ABPA are extremely common in clinical practice, a problem that has become a widespread challenge in clinical practice worldwide.

[0006] Currently, the latest diagnostic criteria for ABPA are primarily based on the diagnostic guidelines published in 2024 by the International Society for Human and Animal Mycology (ISHAM) - ABPA Expert Group. These guidelines include multiple diagnostic indicators, such as clinical manifestations, Aspergillus fumigatus-specific IgE levels, serum total IgE levels, serum IgG levels, blood eosinophil count, chest CT imaging findings, and induced sputum culture from the lungs. Therefore, for patients with Aspergillus fumigatus allergy (positive Aspergillus fumigatus-specific IgE), especially those with underlying lung diseases, a comprehensive assessment of multiple indicators is necessary to confirm a diagnosis of ABPA.

[0007] Currently, there is a lack of simple and effective ABPA diagnostic products. Therefore, developing simple and effective diagnostic products is of significant clinical importance and application value for improving the diagnostic accuracy of ABPA and simplifying the diagnostic process. Summary of the Invention

[0008] Based on this, one or more embodiments of this application provide the application of one or more of the detection reagents for Aspergillus fumigatus allergen components and their specific IgE in the preparation of ABPA detection products, and ABPA detection products. The technical solutions include the following:

[0009] One or more embodiments of this application provide the application of one or more of the detection reagents for Aspergillus fumigatus allergen components and their specific IgE in the preparation of ABPA detection products;

[0010] The Aspergillus fumigatus allergen component includes component 1, or includes component 1 and component 2;

[0011] Component 1 includes one or more of Asp-fA, Asp-fB, Asp-fC, Asp-fD, Asp-fE, Asp-fF and Asp-fG;

[0012] Component 2 includes one or more of Asp-f-22 and Asp-f-27;

[0013] The amino acid sequence of the Asp-fA comprises the amino acid sequence shown in SEQ ID NO.2, or the nucleotide sequence of the Asp-fA encoding the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO.1;

[0014] The amino acid sequence of the Asp-fB comprises the amino acid sequence shown in SEQ ID NO.4, or the nucleotide sequence of the Asp-fB encoding the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO.3;

[0015] The amino acid sequence of the Asp-fC comprises the amino acid sequence shown in SEQ ID NO.6, or the nucleotide sequence of the Asp-fC encoding the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO.5;

[0016] The amino acid sequence of the Asp-fD contains the amino acid sequence shown in SEQ ID NO.8, or the nucleotide sequence of the Asp-fD encoding nucleic acid contains the nucleotide sequence shown in SEQ ID NO.7;

[0017] The amino acid sequence of the Asp-fE comprises the amino acid sequence shown in SEQ ID NO.10, or the nucleotide sequence of the Asp-fE encoding the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO.9;

[0018] The amino acid sequence of the Asp-fF contains the amino acid sequence shown in SEQ ID NO.12, or the nucleotide sequence of the Asp-fF encoding the nucleic acid contains the nucleotide sequence shown in SEQ ID NO.11;

[0019] The amino acid sequence of the Asp-fG comprises the amino acid sequence shown in SEQ ID NO.14, or the nucleotide sequence encoding the nucleic acid of the Asp-fG comprises the nucleotide sequence shown in SEQ ID NO.13.

[0020] In some specific embodiments of this application, component 1 includes at least three of Asp-fA, Asp-fB, Asp-fC, Asp-fD, Asp-fE, Asp-fF, and Asp-fG.

[0021] In some specific embodiments of this application, component 1 includes the Asp-fC, Asp-fF and Asp-fG.

[0022] In some specific embodiments of this application, component 1 is as shown in any one of (A1) to (A7):

[0023] (A1) Component 1 includes Asp-fA, Asp-fB, Asp-fC, Asp-fD, Asp-fE, Asp-fF and Asp-fG;

[0024] (A2) Component 1 includes Asp-fB, Asp-fC, Asp-fD, Asp-fE, Asp-fF and Asp-fG;

[0025] (A3) Component 1 includes Asp-fA, Asp-fC, Asp-fD, Asp-fE, Asp-fF and Asp-fG;

[0026] (A4) Component 1 includes Asp-fD, Asp-fE, Asp-fF and Asp-fG;

[0027] (A5) Component 1 includes Asp-fB, Asp-fC, Asp-fE, Asp-fF and Asp-fG;

[0028] (A6) Component 1 includes the Asp-fC, Asp-fE, Asp-fF and Asp-fG mentioned above;

[0029] (A7) Component 1 includes Asp-fC, Asp-fD, Asp-fF and Asp-fG.

[0030] In some specific embodiments of this application, component 1 includes the Asp-fC, Asp-fE and Asp-fG.

[0031] In some specific embodiments of this application, component 1 includes Asp-fA, Asp-fB, Asp-fC, Asp-fD, Asp-fE and Asp-fG.

[0032] In some specific embodiments of this application, component 1 includes Asp-fD, Asp-fE and Asp-fF.

[0033] In some specific embodiments of this application, component 1 is as shown in any one of (B1) to (B3):

[0034] (B1) Component 1 includes Asp-fA, Asp-fB, Asp-fD, Asp-fE, Asp-fF and Asp-fG;

[0035] (B2) Component 1 includes Asp-fA, Asp-fC, Asp-fD, Asp-fE and Asp-fF;

[0036] (B3) Component 1 includes Asp-fB, Asp-fD, Asp-fE and Asp-fF.

[0037] In some specific embodiments of this application, the Aspergillus fumigatus allergen components include Asp-fA, Asp-fB, Asp-fC, Asp-fD, Asp-fE, Asp-fF, Asp-fG, Asp-f-22 and Asp-f-27 as shown.

[0038] In some specific embodiments of this application, the detection product is used to detect the specific IgE using the following methods: ELISA, protein chip method, magnetic microparticle chemiluminescence method, flow cytometry, and chemiluminescence immunoassay.

[0039] In some specific embodiments of this application, the samples tested by the testing product include blood samples.

[0040] In some specific embodiments of this application, the detection reagent includes one or more of the Aspergillus fumigatus allergen component, blocking solution, washing solution, enzyme-catalyzed colorimetric reagent, and enzyme-catalyzed reaction termination reagent; the enzyme-catalyzed colorimetric reagent includes enzyme-labeled antibody and colorimetric substrate.

[0041] One or more embodiments of this application provide an ABPA detection product, including one or more of the Aspergillus fumigatus allergen components and their specific IgE detection reagents as defined above.

[0042] In some specific embodiments of this application, the detection reagent includes one or more of the Aspergillus fumigatus allergen component, blocking solution, washing solution, enzyme-catalyzed colorimetric reagent and colorimetric termination reagent; the enzyme-catalyzed colorimetric reagent includes enzyme-labeled antibody and colorimetric substrate.

[0043] One or more embodiments of this application provide a *Aspergillus fumigatus* allergen, wherein the *Aspergillus fumigatus* allergen is a composition;

[0044] The composition comprises at least two of Asp-fA, Asp-fB, Asp-fC, Asp-fD, Asp-fE, Asp-fF, and Asp-fG as defined above; or,

[0045] The composition comprises component 1 and component 2 as defined above.

[0046] Compared with traditional technologies, this application has the following advantages:

[0047] This application enables the diagnosis of allergic bronchopulmonary aspergillosis (ABPA) caused by Aspergillus fumigatus by detecting the positivity of Aspergillus fumigatus allergen components in the blood samples of patients. The method of this application for diagnosing ABPA is low-cost, simple to operate, and efficient, and can be used for large-scale screening of ABPA. Attached Figure Description

[0048] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0049] Figure 1 Identification of seven allergen components from Aspergillus fumigatus.

[0050] Figure 2 The positive results of 7 Aspergillus fumigatus allergen components in the serum of 55 patients were statistically analyzed.

[0051] Figure 3 To evaluate the efficacy of seven single components of Aspergillus fumigatus in the diagnosis of ABPA disease.

[0052] Figure 4 This is one of the methods for evaluating the effectiveness of ABPA diagnosis based on the diversity of seven Aspergillus fumigatus positive allergen components.

[0053] Figure 5 This is the second part of an evaluation of the effectiveness of ABPA diagnosis based on the diversity of seven Aspergillus fumigatus positive allergen components.

[0054] Figure 6 This is the third part of an evaluation of the effectiveness of ABPA diagnosis based on the diversity of seven Aspergillus fumigatus positive allergen components.

[0055] Figure 7 This is the fourth part of an evaluation of the effectiveness of ABPA diagnosis based on the diversity of seven Aspergillus fumigatus positive allergen components.

[0056] Figure 8 This is the fifth in a series of evaluations of the effectiveness of ABPA diagnosis based on the diversity of seven Aspergillus fumigatus positive allergen components.

[0057] Figure 9 To evaluate the effectiveness of using a positive allergen component protein diversity method for ABPA diagnosis by combining known allergen component proteins.

[0058] Figure 10 This is a double-blind clinical result for ABPA diagnosis based on the diversity of seven Aspergillus fumigatus positive allergen components.

[0059] Figure 11 Evaluation of the effectiveness of ABPA diagnosis for common Aspergillus fumigatus allergen component protein 1-2-3-4-6.

[0060] Figure 12To evaluate the effectiveness of the diversity of seven Aspergillus fumigatus positive allergen components in the initial diagnosis of ABPA. Detailed Implementation

[0061] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0062] 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 this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0063] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0064] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0065] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0066] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0067] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0068] In this document, terms such as “preferred,” “better,” “more suitable,” and “ideal” are merely used to describe implementation methods or examples that achieve better results, and should be understood not to limit the scope of protection of this application.

[0069] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0070] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0071] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0072] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0073] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0074] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0075] In this application, %(w / w) and wt% both represent weight percentage, %(v / v) refers to volume percentage, and %(w / v) refers to mass-volume percentage.

[0076] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0077] In a first aspect of this application, one or more of the detection reagents for Aspergillus fumigatus allergen components (or Aspergillus fumigatus allergen component proteins) and their specific IgE are used in the preparation of ABPA detection products.

[0078] The Aspergillus fumigatus allergen component includes component 1, or includes component 1 and component 2;

[0079] The component 1 includes one or more of Asp-fA, Asp-fB, Asp-fC, Asp-fD, Asp-fE, Asp-fF and Asp-fG, for example, 1, 2, 3, 4, 5, 6 or 7.

[0080] Component 2 includes one or more of Asp-f-22 and Asp-f-27;

[0081] The amino acid sequence of the Asp-fA comprises the amino acid sequence shown in SEQ ID NO.2, or the nucleotide sequence of the Asp-fA encoding the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO.1;

[0082] The amino acid sequence of the Asp-fB comprises the amino acid sequence shown in SEQ ID NO.4, or the nucleotide sequence of the Asp-fB encoding the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO.3;

[0083] The amino acid sequence of the Asp-fC comprises the amino acid sequence shown in SEQ ID NO.6, or the nucleotide sequence of the Asp-fC encoding the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO.5;

[0084] The amino acid sequence of the Asp-fD contains the amino acid sequence shown in SEQ ID NO.8, or the nucleotide sequence of the Asp-fD encoding nucleic acid contains the nucleotide sequence shown in SEQ ID NO.7;

[0085] The amino acid sequence of the Asp-fE comprises the amino acid sequence shown in SEQ ID NO.10, or the nucleotide sequence of the Asp-fE encoding the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO.9;

[0086] The amino acid sequence of the Asp-fF contains the amino acid sequence shown in SEQ ID NO.12, or the nucleotide sequence of the Asp-fF encoding the nucleic acid contains the nucleotide sequence shown in SEQ ID NO.11;

[0087] The amino acid sequence of the Asp-fG comprises the amino acid sequence shown in SEQ ID NO.14, or the nucleotide sequence encoding the nucleic acid of the Asp-fG comprises the nucleotide sequence shown in SEQ ID NO.13.

[0088] In some specific embodiments of this application, component 1 includes at least three of Asp-fA, Asp-fB, Asp-fC, Asp-fD, Asp-fE, Asp-fF, and Asp-fG. For example, at least three, four, five, six, or seven.

[0089] In some of these examples, component 1 includes the Asp-fC, Asp-fF, and Asp-fG.

[0090] Optionally, component 1 is the Asp-fC, Asp-fF, and Asp-fG.

[0091] Optionally, component 1 is as shown in any one of (A1) to (A7):

[0092] (A1) Component 1 includes Asp-fA, Asp-fB, Asp-fC, Asp-fD, Asp-fE, Asp-fF and Asp-fG;

[0093] (A2) Component 1 includes Asp-fB, Asp-fC, Asp-fD, Asp-fE, Asp-fF and Asp-fG;

[0094] (A3) Component 1 includes Asp-fA, Asp-fC, Asp-fD, Asp-fE, Asp-fF and Asp-fG;

[0095] (A4) Component 1 includes Asp-fD, Asp-fE, Asp-fF and Asp-fG;

[0096] (A5) Component 1 includes Asp-fB, Asp-fC, Asp-fE, Asp-fF and Asp-fG;

[0097] (A6) Component 1 includes the Asp-fC, Asp-fE, Asp-fF and Asp-fG mentioned above;

[0098] (A7) Component 1 includes Asp-fC, Asp-fD, Asp-fF and Asp-fG.

[0099] In some of these examples, component 1 includes the Asp-fC, Asp-fE, and Asp-fG.

[0100] Optionally, component 1 is the Asp-fC, Asp-fE, and Asp-fG.

[0101] Optionally, component 1 includes Asp-fA, Asp-fB, Asp-fC, Asp-fD, Asp-fE, and Asp-fG.

[0102] In some of these examples, component 1 includes the Asp-fD, Asp-fE, and Asp-fF.

[0103] Optionally, component 1 is the Asp-fD, Asp-fE, and Asp-fF.

[0104] Optionally, component 1 is as shown in any one of (B1) to (B3):

[0105] (B1) Component 1 includes Asp-fA, Asp-fB, Asp-fD, Asp-fE, Asp-fF and Asp-fG;

[0106] (B2) Component 1 includes Asp-fA, Asp-fC, Asp-fD, Asp-fE and Asp-fF;

[0107] (B3) Component 1 includes Asp-fB, Asp-fD, Asp-fE and Asp-fF.

[0108] In some specific embodiments of this application, the Aspergillus fumigatus allergen components include Asp-fA, Asp-fB, Asp-fC, Asp-fD, Asp-fE, Asp-fF, Asp-fG, Asp-f-22 and Asp-f-27 as shown.

[0109] It is understood that this application does not impose any particular limitation on the detection methods for the detection reagents and detection products. The following methods may be used, but are not limited to, for detecting the specific IgE: ELISA, protein chip method, magnetic microparticle chemiluminescence method, flow cytometry and chemiluminescence immunoassay.

[0110] It is understandable that the components of the detection reagents and products vary depending on the detection method used. For example, in the ELISA method, the detection reagents include one or more of the following: the Aspergillus fumigatus allergen component (i.e., the coating agent), blocking buffer (which can be skim milk, such as skim milk with a skim milk powder content of 4%-6%), washing buffer (which can be phosphate buffer containing Tween 20, such as phosphate buffer containing 0.4%-0.6% Tween 20), enzyme-catalyzed colorimetric reagent (which can be a horseradish peroxidase system or alkaline phosphatase), and colorimetric termination reagent. The enzyme-catalyzed colorimetric reagent includes enzyme-labeled antibody and colorimetric substrate. In one example, the detection product may also include a multi-well plate, blocking buffer, enzyme-labeled anti-human IgE, enzyme substrate, termination buffer, and sample dilution buffer.

[0111] This application does not impose any special restrictions on the types of samples to be tested for the product; samples may be, but are not limited to, blood samples.

[0112] A second aspect of this application provides an ABPA detection product, comprising one or more of the Aspergillus fumigatus allergen components and their specific IgE detection reagents as defined above. Further, the detection product of this application is as defined in the first aspect.

[0113] It is understood that the components of the detection reagents and detection products vary depending on the detection method used. For example, in the ELISA method, the detection reagents include one or more of the following: the Aspergillus fumigatus allergen component (i.e., the coating agent), blocking buffer (which can be skim milk, such as skim milk with a skim milk powder content of 4%-6%), washing buffer (which can be phosphate buffer containing Tween 20, such as phosphate buffer containing 0.4%-0.6% Tween 20), enzyme-catalyzed colorimetric reagent (which can be horseradish peroxidase system, alkaline phosphatase), and colorimetric termination reagent; the enzyme-catalyzed colorimetric reagent includes enzyme-labeled antibody and colorimetric substrate. In one example, the detection product may also include a multi-well plate, blocking buffer, enzyme-labeled anti-human IgE, enzyme substrate, termination buffer, and sample dilution buffer. A third aspect of the embodiments of this application provides an Aspergillus fumigatus allergen, wherein the Aspergillus fumigatus allergen is a composition;

[0114] The composition comprises at least two (e.g., two, three, four, five, six, or seven) of Asp-fA, Asp-fB, Asp-fC, Asp-fD, Asp-fE, Asp-fF, and Asp-fG as defined in the first aspect above; or,

[0115] The composition comprises component 1 and component 2 as defined in the first aspect above.

[0116] A fourth aspect of this application provides a method for detecting ABPA, wherein the method determines whether the subject is an ABPA patient by detecting whether the subject has an allergic reaction to the Aspergillus fumigatus allergen component defined in the first aspect.

[0117] The Aspergillus fumigatus allergen component includes component 1, or includes component 1 and component 2;

[0118] Component 1 includes one or more of Asp-fA, Asp-fB, Asp-fC, Asp-fD, Asp-fE, Asp-fF and Asp-fG;

[0119] Component 2 includes one or more of Asp-f-22 and Asp-f-27;

[0120] If the subject has an allergic reaction to the Aspergillus fumigatus allergen component, the subject is determined to be an ABPA patient.

[0121] The detection method of this application determines whether the subject corresponding to the sample is an ABPA patient based on the positive status of the Aspergillus fumigatus allergen component in the sample.

[0122] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0123] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0124] This application relates to a strategy for diagnosing allergic bronchopulmonary aspergillosis (ABPA) based on Aspergillus fumigatus allergen component proteins. It proposes for the first time the concept of Positive Allergen Component Diversity (PACD) and utilizes PACD for a novel method of ABPA diagnosis. By detecting the positivity of Aspergillus fumigatus allergen-specific IgE in the patient's serum, the disease diagnosis of allergic bronchopulmonary aspergillosis (ABPA) caused by Aspergillus fumigatus can be achieved. Based on this method, ABPA detection products, such as diagnostic kits, can be developed using different technical approaches. This application relates to the following:

[0125] Part 1: Confirmation that 7 proteins in Aspergillus fumigatus are allergen components.

[0126] Serum testing for Aspergillus fumigatus positive patients typically uses a crude Aspergillus fumigatus allergen extract reagent. This crude extract contains crude samples of all tens of thousands of proteins from Aspergillus fumigatus. A positive serum IgE result for the crude Aspergillus fumigatus allergen extract indicates an Aspergillus fumigatus allergy. However, determining whether the patient's asthma, rhinitis, dermatitis, or ABPA is caused by Aspergillus fumigatus requires further assessment based on symptoms and other indicators.

[0127] Aspergillus fumigatus contains tens of thousands of proteins, but which protein can trigger an allergic reaction? Currently, the World Allergy Organization has reported and confirmed that more than 30 proteins in Aspergillus fumigatus (Aspergillus fumigatus allergen component proteins) can induce allergic diseases (causing patients to test positive for Aspergillus fumigatus in their serum). Among these 30+ Aspergillus fumigatus allergen component proteins, five are commercially available and commonly used: Asp-f-1, Asp-f-2, Asp-f-3, Asp-f-4, and Asp-f-6, sourced from Thermo Fisher Scientific. Of these, Aspergillus fumigatus allergen component proteins Asp-f-1, Asp-f-2, and Asp-f-4 are included in the ISHAM-ABPA expert working group's consensus criteria for diagnosing ABPA. Specifically, elevated IgE levels of recombinant Aspergillus fumigatus allergen component proteins Asp-f-1, Asp-f-2, and Asp-f-4 support the diagnosis of ABPA, serving as another indicator for diagnosing ABPA. However, when using these three Aspergillus fumigatus allergen components for ABPA diagnosis, the lack of unified standards and the differences between populations in different regions make it impossible to set a unified threshold to distinguish between ABPA disease and non-ABPA, which prevents the widespread use of methods based on Aspergillus fumigatus allergen components for ABPA diagnosis.

[0128] This application identified seven novel Aspergillus fumigatus allergen protein components, namely Asp-fA, Asp-fB, Asp-fC, Asp-fD, Asp-fE, Asp-fF, and Asp-fG. This is the first time that these seven Aspergillus fumigatus allergen protein components have been confirmed to induce allergic diseases; that is, seven novel Aspergillus fumigatus sensitizing protein components have been identified. Figure 1 ).

[0129] The nucleotide sequence of the nucleic acid encoded by Asp-fA is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; the nucleotide sequence of the nucleic acid encoded by Asp-fB is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4; the nucleotide sequence of the nucleic acid encoded by Asp-fC is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.6; the nucleotide sequence of the nucleic acid encoded by Asp-fD is shown in SEQ ID NO.7, and the amino acid sequence is shown in SEQ ID NO.8; the nucleotide sequence of the nucleic acid encoded by Asp-fE is shown in SEQ ID NO.9, and the amino acid sequence is shown in SEQ ID NO.10; the nucleotide sequence of the nucleic acid encoded by Asp-fF is shown in SEQ ID NO.11, and the amino acid sequence is shown in SEQ ID NO.12; the nucleotide sequence of the nucleic acid encoded by Asp-fG is shown in SEQ ID NO.13, and the amino acid sequence is shown in SEQ ID NO.14.

[0130] The main steps for exogenous expression of seven Aspergillus fumigatus allergy component proteins are as follows:

[0131] This application utilizes gene synthesis technology to synthesize nucleic acid fragments encoding the aforementioned seven Aspergillus fumigatus allergy component proteins and insert them into pET28a(+) plasmids to construct recombinant vectors. These recombinant vectors were then transformed into Escherichia coli BL21. The bacterial culture was plated onto LB agar plates containing kanamycin and incubated overnight at 37°C. Single colonies were picked for PCR and sequencing verification. Sequencing results, based on sequence information and molecular weight comparison, confirmed the correct insertion of the target gene. After plate selection, positive colonies were selected and incubated overnight. After a second shaking induction, IPTG at a final concentration of 0.5 mmol / L was added for induction at 16°C and 120 rpm for 16 h. The bacterial cells were then collected, sonicated, and the supernatant and precipitate were analyzed by 12% SDS-PAGE gel electrophoresis to determine protein solubility. Ni column chromatography was used for purification, including thorough washing of the column with equilibration buffer, followed by elution with imidazole equilibration buffers containing 50 mmol / L, 100 mmol / L, 200 mmol / L, and 500 mmol / L, respectively. The eluates were collected for SDS-PAGE analysis to obtain the purified expression of seven Aspergillus fumigatus allergy component proteins. Figure 1 1a).

[0132] The main steps of ELISA testing are as follows:

[0133] (1) Encapsulation and sealing

[0134] In a 96-well plate, each well was coated with 100 μL of Aspergillus fumigatus allergen protein solution (with a concentration of 2 μg / mL), and incubated overnight at 4°C. After removing the coating solution, the plates were washed five times with 0.5% PBST (phosphate buffer containing 0.5% Tween 20), and then each well was blocked with 300 μL of 5% skim milk blocking buffer diluted with PBST for 2 hours at 37°C.

[0135] (2) Add a primary antibody

[0136] After removing the blocking solution, wash five times with 0.5% PBST. Add the primary antibody and incubate at 37°C for 2 hours.

[0137] Positive group: Serum from patients allergic to Aspergillus fumigatus was diluted with 2.5% skim milk powder as blocking solution at a volume ratio of 1:10. The diluted serum was used as the primary antibody and added to the well plate.

[0138] Negative control group: Normal human serum, diluted with 2.5% skim milk powder as blocking solution, with a serum to blocking solution volume ratio of 1:10. The diluted serum was used as the primary antibody and added to the well plate.

[0139] (3) Add secondary antibody and wash

[0140] Wash 5 times with 0.5% PBST, then add 100 μL of horseradish peroxide (HRP) biotin-labeled anti-human IgE (1:5000 dilution) to each well and incubate at 37°C for 1 h.

[0141] (4) Wash 5 times with 0.5% PBST;

[0142] (5) Add 100 μL of TMB (tetramethylbenzidine) colorimetric solution (1:1) to prepare, incubate at room temperature in the dark for 15 min, and then add 100 μL of stop colorimetric solution;

[0143] (6) Use an automated microplate reader to detect its absorbance at 450 nm and analyze the data.

[0144] A positive result is defined as an absorbance value of serum IgE in patients that is higher than the cutoff value. Cutoff = mean of the negative control group + 2SD. The mean of the negative control group is the mean of the absorbance value of serum IgE in normal individuals.

[0145] The results are as follows Figure 1 The positive rates of 7 Aspergillus fumigatus allergen components in Aspergillus fumigatus-positive patients are as follows: Figure 1 As shown in 1i.

[0146] In the above procedure, a total of 40 serum samples were collected from 25 patients with Aspergillus fumigatus allergy and 15 healthy individuals. The crude extracts of Aspergillus fumigatus allergen from the patients were all IgE positive, meaning the Aspergillus fumigatus IgE content was greater than or equal to 0.35 kUA / L. The crude extracts of Aspergillus fumigatus allergen from the healthy individuals were all IgE negative, meaning the Aspergillus fumigatus IgE content was less than 0.35 kUA / L.

[0147] Part Two: Diagnosis of ABPA using seven allergen components from Aspergillus fumigatus.

[0148] Based on the above-mentioned discovery of 7 new Aspergillus fumigatus allergen component proteins, this application finds that the newly identified 7 new Aspergillus fumigatus allergen component proteins can be used for the diagnosis of ABPA.

[0149] Patients who tested positive for IgE in the crude extract of Aspergillus fumigatus allergen in clinical tests include those with asthma, dermatitis, bronchiectasis with infection, fungal infection, and COPD complicated with Aspergillus infection. Identifying which of these Aspergillus fumigatus-positive patients are ABPA patients is a clinical challenge.

[0150] In this regard, this application first tests the serum of patients who are positive for Aspergillus fumigatus to determine whether they are positive for seven Aspergillus fumigatus allergen components. Based on the positive status of the seven Aspergillus fumigatus allergen components described above, it is determined whether the patient has ABPA disease. The specific detection method (ELISA detection method) is described in Part I.

[0151] In the above procedures, a total of 55 serum samples were collected from 55 patients with Aspergillus fumigatus allergy. All patients showed positive IgE results for crude Aspergillus fumigatus allergen extracts, meaning the Aspergillus fumigatus IgE content was greater than or equal to 0.35 kUA / L. These 55 patients with positive Aspergillus fumigatus allergy included 20 ABPA patients and 35 non-ABPA patients. The 35 non-ABPA patients presented with clinical manifestations including asthma, dermatitis, bronchiectasis with infection, fungal infection, cystic fibrosis, pneumonia, and COPD complicated by Aspergillus infection.

[0152] The test results are as follows:

[0153] First, this application statistically analyzed the positive results of seven Aspergillus fumigatus allergen components in the serum of a total of 55 patients. The results showed that the detection rate of the seven Aspergillus fumigatus allergen components was significantly higher in ABPA patients than in non-ABPA patients. Figure 2 ).

[0154] Secondly, this application analyzed the efficacy of each individual component in the diagnosis of ABPA disease, and the results are shown in [the table below]. Figure 3The data analysis shows that some Aspergillus fumigatus allergen components have high potential value for the diagnosis of ABPA (P<0.01). These include components C, E, F, and G.

[0155] Furthermore, in addition to the results of predicting ABPA using a single allergen component protein as described above, this application proposes a novel method for ABPA diagnosis using Positive Allergen Component Diversity (PACD) of Aspergillus fumigatus positive allergen components. This novel method was used to detect the aforementioned 55 serum samples, and the results are shown below. Figures 4 to 8 Analysis of ABPA diagnosis based on the positive diversity of all 7 Aspergillus fumigatus allergen components showed that a positive result for at least 3 allergen components was sufficient for a definitive diagnosis of ABPA, with a diagnostic accuracy of 100%, and was able to detect 9 ABPA patients. Figure 4 In addition to using all seven Aspergillus fumigatus allergen components for ABPA diagnosis, using some of these seven allergen components as supplementary diagnostic tools still yields good results. For example, using a combination of six different Aspergillus fumigatus allergen components for positive testing, at least three positive allergen components are sufficient to confirm ABPA, with a diagnostic accuracy of 100%, capable of detecting nine ABPA patients. Figure 5 The BCDEFG and ACDEFG combinations are preferred. For example, using a combination of 5 different Aspergillus fumigatus allergen components for positive testing, at least 3 allergen components showing a positive result are sufficient to diagnose ABPA, with a diagnostic accuracy of 100%, capable of detecting 9 ABPA patients. Figure 6 The CDEFG combination is the preferred choice. For example, using a combination of four different Aspergillus fumigatus allergen components for positive testing, at least three allergen components showing a positive result are sufficient to diagnose ABPA, with a diagnostic accuracy of 100%, capable of detecting 9 ABPA patients. Figure 7 The CEFG combination is the preferred choice. For example, using a combination of three different Aspergillus fumigatus allergen components for positive testing, a positive result for all three allergen components is sufficient to diagnose ABPA, with a diagnostic accuracy of 100%, capable of detecting 8 ABPA patients. Figure 8The CFG combination is preferred. The results above show that detecting all seven Aspergillus fumigatus allergen protein components or only some of them can diagnose ABPA. Considering clinical application and the potential for positive Aspergillus fumigatus allergen components, using all seven components is recommended for ABPA diagnosis. Currently, there are no methods or products in clinical testing that can directly diagnose ABPA with a single test. Given the urgent clinical need and the complexity of ABPA diagnosis, a single test using the above method, i.e., a positive result for at least three Aspergillus fumigatus protein components, is sufficient for a definitive diagnosis of ABPA. This method, as one of the diagnostic indicators for ABPA, can significantly reduce costs and can be used for large-scale clinical screening.

[0156] In addition to using only the seven Aspergillus fumigatus allergen components mentioned above, these seven allergen components can also be used in combination with other identified Aspergillus fumigatus allergen components for the diagnosis of ABPA. Specifically, Asp-fA, Asp-fB, Asp-fC, Asp-fD, Asp-fE, Asp-fF, and Asp-fG are combined with Asp-f-22 and Asp-f-27 for diagnosis, using the ELISA detection method described in Part I. Asp-f-22: Amino acid sequence number GenBank: AAK49451.1, nucleotide sequence number GenBank: AF284645.1, nucleotide sequence. Asp-f-27: Amino acid sequence number GenBank: CAI78448.1, nucleotide sequence number GenBank: AJ937743.1. Serum sample information is the same as the 55 serum samples mentioned above in this section. The efficacy evaluation of the combined diagnostic method showed that using 7 Aspergillus fumigatus allergen components combined with 2 known Aspergillus fumigatus allergen components for a total of 9 positive tests, at least 3 positive allergen components were sufficient to diagnose ABPA, with a diagnostic accuracy of 100%. Nine ABPA patients were detected. Figure 9 .

[0157] The results above demonstrate that, compared to using a single Aspergillus fumigatus allergen component protein, using the Positive Allergen Component Diversity (PACD) method for ABPA diagnosis offers several advantages: First, the diagnostic rate for ABPA is 100% when a certain threshold is exceeded (i.e., the number of positive Aspergillus fumigatus allergen component proteins is greater than or equal to 3). Second, it eliminates the need for absolute quantification of specific IgE for Aspergillus fumigatus allergen component proteins (i.e., it avoids the problem of inconsistent thresholds caused by collecting large amounts of data from various countries and regions and setting data simulations for single-component diagnostics). Qualitative detection allows the detection of Aspergillus fumigatus allergen component proteins to be combined with methods such as protein chip assays, magnetic microparticle chemiluminescence assays, flow cytometry, and chemiluminescent immunoassay, requiring only a determination of whether a component is positive. Finally, this method avoids the problem of limited effective promotion due to differences in different countries, regions, and research methods, and can be used in conjunction with existing component products, facilitating widespread adoption.

[0158] Based on the results of using all seven Aspergillus fumigatus allergen protein components for ABPA diagnosis, the diagnostic result is 100% when three or more Aspergillus fumigatus allergen protein components are positive. Considering the current scarcity of clinical ABPA diagnostic methods and products, and the fact that no method / product worldwide can diagnose ABPA with a single test, the method and product proposed in this application are urgently needed in clinical practice. Compared to current clinical ABPA diagnostic methods (which require multiple indicators such as Aspergillus fumigatus-specific IgE content, serum total IgE content, serum IgG positivity detection, blood eosinophil count, chest CT, and fungal culture of induced sputum from the lungs for diagnosis), the method and product proposed in this application can confirm ABPA with a single positive test for three or more Aspergillus fumigatus allergen protein components, significantly reducing the cost of clinical ABPA diagnosis. It can also be used as a tool for large-scale screening to reduce the missed diagnosis rate of ABPA in clinical practice.

[0159] Part Three: Clinical Double-Blind Trial Validation of ABPA Diagnosis Based on the Diversity of Aspergillus fumigatus Positive Allergen Components

[0160] A total of 80 serum samples positive for Aspergillus fumigatus were tested in a double-blind clinical trial. All crude extracts of Aspergillus fumigatus allergens were IgE positive, meaning the Aspergillus fumigatus IgE content was greater than or equal to 0.35 kUA / L (26 cases were from ABPA patients, 31 cases from Aspergillus fumigatus infection patients, and 23 cases from patients with different types of Aspergillus fumigatus-induced asthma). Using the ELISA detection method described in Part I, all seven Aspergillus fumigatus allergen protein components mentioned in Part I were detected. A diagnosis of ABPA was made if at least three components were positive. Evaluation of the double-blind trial results showed that a diagnosis of ABPA could be made with at least three positive allergen components, with a diagnostic accuracy of 100%, detecting 14 ABPA patients, with an AUC of 0.8365 (…). Figure 10 ).

[0161] Part 4: Comparison of ABPA diagnostic efficacy with other Aspergillus fumigatus allergen component proteins

[0162] This application evaluated and compared the use of traditional common Aspergillus fumigatus allergen protein fractions and the ImmunoCAP detection method for ABPA diagnosis. A total of 92 serum samples were tested for Aspergillus fumigatus positivity. All crude extracts of Aspergillus fumigatus allergens were IgE positive, meaning the Aspergillus fumigatus IgE content was greater than or equal to 0.35 kUA / L (including 36 ABPA patients, 36 patients with Aspergillus fumigatus infection, and 20 patients with different types of asthma induced by Aspergillus fumigatus). The traditional common Aspergillus fumigatus allergen protein fractions tested were five commercially available Aspergillus fumigatus allergen fractions from Thermo Fisher Scientific: Asp-f-1, Asp-f-2, Asp-f-3, Asp-f-4, and Asp-f-6. The five protein components were detected using the ImmunoCAP assay. A positive result was defined as an IgE content greater than or equal to 0.35 kUA / L for each of the five components: Asp-f-1, Asp-f-2, Asp-f-3, Asp-f-4, and Asp-f-6.

[0163] The results are as follows Figure 11As shown in the figure, even when using the Asp-f-1, Asp-f-2, and Asp-f-4 components mentioned in the ISHAM-ABPA expert working group's consensus criteria for diagnosing ABPA, whether using a single allergen component protein for diagnosis or using the method of positive allergen component diversity of Asp-f-1, Asp-f-2, Asp-f-3, Asp-f-4, and Asp-f-6, the diagnostic effect is not as good as the ABPA diagnostic effect based on the positive diversity of 7 new Aspergillus fumigatus allergen component proteins proposed in this application. The results show that when at least 3 Aspergillus fumigatus components are positive, although 18 ABPA patients can be detected, 12 patients were still misdiagnosed as ABPA, with a diagnostic accuracy of only 78.6%. Therefore, it cannot be used for the auxiliary diagnosis of ABPA.

[0164] Part 5: Evaluation of the efficacy of seven components of Aspergillus fumigatus in the diagnosis of ABPA by integrating clinical information.

[0165] This application collected and analyzed the clinical information of all patients in Parts II, III, and IV above, totaling 46 ABPA patients and 89 non-ABPA patients who tested positive for Aspergillus fumigatus. Further analysis of the clinical sample information revealed that among the 46 patients, 26 were newly diagnosed with ABPA or met the diagnostic criteria for ABPA at the time of sample collection, and 20 patients had previously been diagnosed with ABPA but were in a stable phase after treatment. Using the ELISA detection method described in Part I, the positivity of all seven Aspergillus fumigatus allergen components was analyzed; a diagnosis of ABPA was made if at least three components were positive. Results showed that among the 26 newly diagnosed ABPA patients, 23 were diagnosed with ABPA, with a detection sensitivity of 88.46% and a diagnostic accuracy of 100%. The results are shown in [see attached table]. Figure 12 The above results indicate that the method based on the positive diversity of the seven allergen components of Aspergillus fumigatus for ABPA diagnosis has excellent performance and application value in initial patient visits or large-scale clinical screening.

[0166] Overall, this application is the first to discover and propose a novel method for ABPA diagnosis using Aspergillus fumigatus allergen components. The method utilizes the Positive Allergen Component Diversity (PACD) of Aspergillus fumigatus positive allergen components for ABPA diagnosis, significantly reducing diagnostic costs compared to traditional ABPA diagnostic methods. This application enables ABPA diagnosis with a single test, offering a significant advantage in diagnostic costs. It can be used for large-scale screening, helping to reduce the missed diagnosis rate of clinical ABPA, and can also be incorporated as an auxiliary indicator into ABPA diagnostic guidelines.

[0167] This application provides Aspergillus fumigatus allergen components and combinations thereof that can be used for ABPA diagnosis using the Positive Allergen Component Diversity (PACD) method. Different ABPA diagnostic product combinations can be developed using the provided allergen components and combinations with other allergen components.

[0168] The encoding nucleic acid sequence (1482 bp) of SEQ ID NO.1-Asp-fA

[0169]

[0170] >Amino acid sequence of SEQ ID NO.2 - Asp - f - A (493 aa)

[0171] MALFQTIVTPTVTYEQPLGLFINNEFVPGVEGKTFETINPHDEKPIVAVHEATEKDVDIAVEAARKAFKGVWRDIIPAERGRLLTRLADLLERDLDTIAAIEALDNGKAFQIAKGDVTLSANCIRYYGGWADKIVGQTIDTDPGCLTYTRHEPVGVCGQIIPWNFPLLMWAWKIGPAISTGNTVVLKTAEQTPLSALYVAKLVKEAGFPPGVINILSGFGRVAGAAIAAHMDIDKVAFTGSTLVGRQILQVAAKSNLKKVTLELGGKSPNIVFPDADLDDAIKYVNLGIYFNHGQCCAAGSRVLVHESIYDKFLALFKQRAEENKVGDPFHPETFQGPQVSQVQFDRIMGYINEGKKAGAKVVTGGARHGEKGYYIQPTIFADVHEDMKIVKEEIFGPVCTVQKFSTEEEAIEIANNTNYGLAAAVHTTNLNTAIRVSNAIRAGTVWINNYNTFLAQMPFGGFKESGLGRELGSYALDNYTQVKTVHARIKSA。

[0172] >Coding nucleic acid sequence of SEQ ID NO.3 - Asp - f - B (801 bp)

[0173] ATGCCTATCCCCGTCCCCGCCGCCTCCTCCCTCCTCGACCTCCTCAGCCTGAAGGGCAAA

[0174] ACCGTGGTGGTGACCGGCGCGTCCGGCCCCCGCGGCATGGGCATCGAAGCCGCCCGTGGCTGCGCGGAAATGGGCGCCAACATCGCCCTCACATACGCCTCCCGCCCGGAAGGCGGCGAGAAGAACGCAGCCGAGATCGCCAAAACCTACGGCGTCAAGGCCAAGGCCTACAAGTGCAACGTGGGCGACTGGGAGAGCGTCCAGAAGCTGGTCGCGGACGTGATCGCCGAGTTCGGGCAGATCGACGCCTTCATCGCGAATGCGGGGCGTACCGCCAGCGCAGGGATCCTGGATGGCTCGGTGAATGACTGGGCGGAGGTGATCCAGACGGATCTGACGGGTACGTTTTACTGCGCGAAGGCTGTCGGCCCGCACTTCAAGGAGCGGGGCAAGGGCAGCTTCGTCATCACGAGCAGTATGTCGGGCCATATTGCGAACTATCCGCAGGAGCAGACGAGCTACAATGTGGCCAAGGCGGGGTGCATTCACTTTGCCAAGTCGCTGGCGAATGAGTGGCGGGATTTTGCCAGGGTTAACAGTATCTCGCCGGGGTATATCGATACCGGCTTGTCGGACTTCGTGGACAAGAAGACGCAGGACCTGTGGTTGAGTATGATTCCCATGGGCCGCAACGGGGACGCGAAAGAGCTCAAGGGCGCGTACGTATATCTGTGCAGCGATGCCAGTTCCTACATGACGGGGACGGACCTGCTCATTGACGGGGGTTACTGCATTCGTTGA。

[0175] >SEQ ID NO.4 - Amino acid sequence of Asp - f - B (266 aa)

[0176] MPIPVPAASSLLDLLSLKGKTVVVTGASGPRGMGIEAARGCAEMGANIALTYASRPEGGEKNAAEIAKTYGVKAKAYKCNVGDWESVQKLVADVIAEFGQIDAFIANAGRTASAGILDGSVNDWAEVIQTDLTGTFYCAKAVGPHFKERGKGSFVITSSMSGHIANYPQEQTSYNVAKAGCIHFAKSLANEWRDFARVNSISPGYIDTGLSDFVDKKTQDLWLSMIPMGRNGDAKELKGAYVYLCSDASSYMTGTDLLIDGGYCIR。

[0177] >SEQ ID NO.5 - Encoding nucleic acid sequence of Asp-f-C (774bp)

[0178] ATGTCCGAGCGACCCTCCGATCTTGTAGTCAACAGGTTGGTGCTTTTCGTGGTCAAAGGTGAGTCGAGTCGACGCATCAGCAAGTCTCCTCTCATGCAGTCAACTACAGGCACCGCCACGTCCACACACAACACCGTCAAACCGTTGATTCTCCTCGAAGAGCTCGGAGTCCCTCACGATATCTATGTCGTCGAAAAGGTGTCTGCCCCCTGGTTCAGCGAGATCAATCCCCACAAGATGGTGCCCGCCATCCTGGACCGCTCCCCCGACGGCCGAGACACCCTCCGCGCCTGGGAATCGACGTCGACCCTGATGTACATTGCCGATGCATATGACAAGGACGGGACCTTTGGCGGACGCAACGTGCAAGAGAGGTCTGAGATCAACAACTGGTTGACTCTGCATACGGCGGCGCTGGGGCCCACGGCCAAGTACTGGCTGTATTTCTACAAGCTGCACCCGGAGAAGTTGCCCAAAACAATCGAGAAACTGCGCAGCAACATTACGGTGCAATATGATATTCTAGAACGTCGGCTCAACGAACCCGGCCAGCAGTACCTGGCTCTGAAGGACCGACCGACCATTGCGGATATCGCCACCTTGCCCTTTGCCATGAAGTCCACGGCGGAACTGTTCGGCCTGGAGTTTGAGAAATGGCCAAAGCTGCAGGAGTGGTCGGTTCGCATGGGTGAACGTGAGGCCGTCAAGCGGGCCTGGCAGCGCGTCGCGGGGTTTGGCCATGGCGAGAAAGAGTATGGCATGTTAGAGGCGTGA。

[0179] >SEQ ID NO.6 - Amino acid sequence of Asp - f - C (257 aa)

[0180] MSERPSDLVVNRLVLFVVKGESSRRISKSPLMQSTTGTATSTHNTVKPLILLEELGVPHDIYVVEKVSAPWFSEINPHKMVPAILDRSPDGRDTLRAWESTSTLMYIADAYDKDGTFGGRNVQERSEINNWLTLHTAALGPTAKYWLYFYKLHPEKLPKTIEKLRSNITVQYDILERRLNEPGQQYLALKDRPTIADIATLPFAMKSTAELFGLEFEKWPKLQEWSVRMGEREAVKRAWQRVAGFGHGEKEYGMLEA。

[0181] >SEQ ID NO.7 - Encoding nucleic acid sequence of Asp-f-D (750bp)

[0182] ATGCCTCGCCAATTCTTCGTCGGTGGTAACTTCAAGATGAACGGTGTCACTGACACCATCATCTCCATCGTTAAGAACCTCAACGAGGCCAAGCTCGACCCCTCCGTCGAGGTCGTCATCTCCCCCTCCGCTCTCTACCTCCTCCTCGCCCGCCAAGCTGCCGACCCCAAGATTGGCGTTGCTGCCCAGAATGTCTTCGACAAGCCCAACGGTGCTTTCACCGGTGAAATTAGCGTTGAGCAGCTCAAGGATGCTAAGATTGACTGGGTCATTATCGGACACAGCGAGCGTCGTGTCATCCTTAAGGAGACTGACGAGTTCATTGCTCGCAAGACCAAGGCTGCCATTGATGGTGGCCTGAACGTCATCCTTTGCATTGGTGAGACCCTGGAGGAGCGTGAGGCTGGCAAAACCATCGACGTCGTTAGAAGGCAGCTTACTGCTGTCGCCAAGGACCTTTCCAAGGAGCAGTGGCAGAAGGTCGTCATTGCCTATGAGCCCGTCTGGGCCATCGGCACCGGCAAGGTCGCTACCACCGAGCAGGCCCAGGAGGTCCACGCCGCTATCCGCAAGTGGCTCAGCGACGACGTCTCCGCTGAGGCCTCTGAGAACGTCCGCATCATCTACGGTGGCTCCGTCAGCGAGAAGAACTGCCGCGACCTCGCCAAGCAGCCCGATGTGGACGGTTTTCTGGTTGGCGGTGCCAGCTTGAAGCCTGCCTTCGTCGAAATTGTCAACGCCCGTCTGTAA。

[0183] >SEQ ID NO.8 - Amino acid sequence of Asp - f - D (249 aa)

[0184] MPRQFFVGGNFKMNGVTDTIISIVKNLNEAKLDPSVEVVISPSALYLLLARQAADPKIGVAAQNVFDKPNGAFTGEISVEQLKDAKIDWVIIGHSERRVILKETDEFIARKTKAAIDGGLNVILCIGETLEEREAGKTIDVVRRQLTAVAKDLSKEQWQKVVIAYEPVWAIGTGKVATTEQAQEVHAAIRKWLSDDVSAEASENVRIIYGGSVSEKNCRDLAKQPDVDGFLVGGASLKPAFVEIVNARL。

[0185] >SEQ ID NO.9 - Encoding nucleic acid sequence of Asp - f - E (1692bp)

[0186] ATGCGTTTCAACGCAGCTATCACTGGCGCTCTCGTCTCTTCGGCCACTTTGATGGGCCAA

[0187]

[0188] >Amino acid sequence of SEQ ID NO.10 - Asp - f - E (563aa)

[0189] MRFNAAITGALVSSATLMGQAHAEETEKKADATSLVEKPTFTPTTIEAPFLEQFTADWDSRWTPSHAKKEDSKSEEDWAYVGEWAVEEPTVLNGMVGDKGLVVKNVAAHHAISAKFPKKIDNKGKTLVVQYEVKPQNSLVCGGAYMKLLQENKKLHAEEFSNATPYVIMFGPDKCGATNKVHFIFRHKNPKTGEYEEKHMTAPPAARTTKLTTLYTLIVKPDQSFQILIDGEAVKNGTLLEDFAPPVNPEKEIDDPKDKKPADWVDEAKIPDPEAKKPDDWDEDAPYEIVDEEATMPEDWLEDEPTSIPDPEAEKPEDWDDEEDGDWIPPTVPNPKCNEVSGCGPWTPPMKKNPAYKGKWTAPLIDNPAYKGIWKPRKIPNPAYFEDKTPSNFEPMGAVGFEIWTMQNDILFDNIYIGHSIEDAEKLRKETFDLKHPVEVALEEASKPKLEEKAATPSVSFKEAPVTYVREKVDYFVGLAKQDPINAVKQVPEVAGGLGALLLTMILVIVGAVGASSPAPAAAAKKGKEAASAAKEKASEAVSSAADTAKGAATKRNTRSSAQ。

[0190] >Coding nucleic acid sequence of SEQ ID NO.11 - Asp - f - F (2187bp)

[0191]

[0192] >Amino acid sequence of SEQ ID NO.12 - Asp - f - F (728aa)

[0193] MRLTFIPSLIGVANAVCPYMTGELNRRDEISDGDAAAATEEFLSQYYLNDNDAFMTSDVGGPIEDQNSLSAGERGPTLLEDFIFRQKIQRFDHERVPERAVHARGAGAHGVFTSYGDFSNITAASFLAKEGKQTPVFVRFSTVAGSRGSSDLARDVHGFATRFYTDEGNFDIVGNNIPVFFIQDAILFPDLIHAVKPRGDNEIPQAATAHDSAWDFFSQQPSTMHTLLWAMSGHGIPRSFRHVDGFGVHTFRFVTDDGASKLVKFHWKSLQGKASMVWEEAQQTSGKNPDFMRQDLHDAIEAGRYPEWELGVQIMDEEDQLRFGFDLLDPTKIVPEEFVPITKLGKMQLNRNPRNYFAETEQVMFQPGHIVRGVDFTEDPLLQGRLFSYLDTQLNRHGGPNFEQLPINQPRVPVHNNNRDGAGQMFIPLNPHAYSPKTSVNGSPKQANQTVGDGFFTAPGRTTSGKLVRAVSSSFEDVWSQPRLFYNSLVPAEKQFVIDAIRFENANVKSPVVKNNVIIQLNRIDNDLARRVARAIGVAEPEPDPTFYHNNKTADVGTFGTKLKKLDGLKVGVLGSVQHPGSVEGASTLRDRLKDDGVDVVLVAERLADGVDQTYSTSDAIQFDAVVVAAGAESLFAASSFTGGSANSASGASSLYPTGRPLQILIDGFRFGKTVGALGSGTAALRNAGIATSRDGVYVAQSVTDDFANDLKEGLRTFKFLDRFPVDH。

[0194] >Coding nucleic acid sequence of SEQ ID NO.13 - Asp - f - G (966bp)

[0195] ATGAAATTCGTTGCTACTCTCATTGCCTGCGGCCTGTCTGGACTGGCCCTGGCTGCTCCT

[0196] ACCGCTACTGTCGACTCGTTGGGGAAGCGCGCCGCCGATGATGCTGCCTTTGGTTACGCCAGCTTGAACGGAGGCACCACCGGTGGTGCTGGAGGTACCACGACCACCGTCTCCTCCTACGCAGCCTTCACTGCTGCCGTCTCCAGTGATGCGAAGAAGGTGGTCTACGTCAGCGGCCCCATCAAGCAGAGCGCCAAGCAGGTCAAGGTTGGCAGCAACACCAGCATCATCGGCAAGGACTCCACCGCTGTGCTCGAGGGATTCGGCCTTCTGGTCAAGGAGAAGTCCAACGTGATCATCCGGAACCTGGGAGTCAAGAAGGTTCTGGCGGAGAATGGAGATGCCATTGGCATTCAATACTCCAACAATGTGTGGGTTGACCACGTCGATGTCTCCTCTGACCGTGACCACGACAAGGACTACTACGACGGCCTGATTGATGTCACCCACGCCGCCGACTACGTGACCATCTCCAACAGCTACATCCACGACCACTGGAAGGCGTCCCTGGTGGGCCACTCCGACAACAACGGCGATGAGGACAAGGGCCACCTGCGCGTGACGTACGCCAACAACTACTGGAGCAACATCAACTCGCGTGCTCCCTCCCTCCGCTTCGGCACCGGCCACATCTACAACAGCTACTTCGAGAACGTCAGCGACGGCATCAACACCCGCGACGGCGCCCAGGTCCTCGTCGAGTCCAACCAGTTCGTCGGCTCCAGCAAGGCCCTCTACTCCACCGACGACGGCTACGCCGTTGAGAGGGACAACGACTTTGGCGGCGCCAAGAACACTGCCCTGCAGGGTACCTTGACCACGGTTCCCTACTCTTACTCTCTGCTTGGATCCAGCAAGGTCAAGTCGGCTGTTGTTGGAGTGGCCGGTCAGACTCTGAAGTTCTAG。

[0197] >SEQ ID NO.14 - Amino acid sequence of Asp - f - G (321 aa)

[0198] MKFVATLIACGLSGLALAAPTATVDSLGKRAADDAAFGYASLNGGTTGGAGGTTTTVSSYAAFTAAVSSDAKKVVYVSGPIKQSAKQVKVGSNTSIIGKDSTAVLEGFGLLVKEKSNVIIRNLGVKKVLAENGDAIGIQYSNNVWVDHVDVSSDRDHDKDY YDGLIDVTHAADYVTISSNSYIHDHWKASLVGHSDNNGDEDKGHLRVTYANNYWSNINSRAPSLRFGTGHIYNSYFENVSDGINTRDGAQVLVESNQFVGSSKALYSTDDGYAVERDNDFGGAKNTALQGTLTTVPYSYSLLGSSKVKSAVVGVAGQTLKF.

[0199] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0200] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. The application of one or more of the Aspergillus fumigatus allergen components and their specific IgE detection reagents in the preparation of ABPA detection products; The Aspergillus fumigatus allergen component includes component 1, or includes component 1 and component 2; Component 1 includes one or more of Asp-fA, Asp-fB, Asp-fC, Asp-fD, Asp-fE, Asp-fF and Asp-fG; Component 2 includes one or more of Asp-f-22 and Asp-f-27; The amino acid sequence of the Asp-fA comprises the amino acid sequence shown in SEQ ID NO.2, or the nucleotide sequence of the Asp-fA encoding the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO.1; The amino acid sequence of the Asp-fB comprises the amino acid sequence shown in SEQ ID NO.4, or the nucleotide sequence of the Asp-fB encoding the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO.3; The amino acid sequence of the Asp-fC comprises the amino acid sequence shown in SEQ ID NO.6, or the nucleotide sequence of the Asp-fC encoding the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO.5; The amino acid sequence of the Asp-fD contains the amino acid sequence shown in SEQ ID NO.8, or the nucleotide sequence of the Asp-fD encoding nucleic acid contains the nucleotide sequence shown in SEQ ID NO.7; The amino acid sequence of the Asp-fE comprises the amino acid sequence shown in SEQ ID NO.10, or the nucleotide sequence of the Asp-fE encoding the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO.9; The amino acid sequence of the Asp-fF contains the amino acid sequence shown in SEQ ID NO.12, or the nucleotide sequence of the Asp-fF encoding the nucleic acid contains the nucleotide sequence shown in SEQ ID NO.11; The amino acid sequence of the Asp-fG comprises the amino acid sequence shown in SEQ ID NO.14, or the nucleotide sequence of the Asp-fG encoding the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO.

13.

2. The application according to claim 1, characterized in that, Component 1 includes at least three of Asp-fA, Asp-fB, Asp-fC, Asp-fD, Asp-fE, Asp-fF, and Asp-fG.

3. The application according to claim 2, characterized in that, Component 1 includes Asp-fC, Asp-fF and Asp-fG.

4. The application according to any one of claims 1 to 3, characterized in that, Component 1 is shown in any one of (A1) to (A7): (A1) Component 1 includes Asp-fA, Asp-fB, Asp-fC, Asp-fD, Asp-fE, Asp-fF and Asp-fG; (A2) Component 1 includes Asp-fB, Asp-fC, Asp-fD, Asp-fE, Asp-fF and Asp-fG; (A3) Component 1 includes Asp-fA, Asp-fC, Asp-fD, Asp-fE, Asp-fF and Asp-fG; (A4) Component 1 includes Asp-fD, Asp-fE, Asp-fF and Asp-fG; (A5) Component 1 includes Asp-fB, Asp-fC, Asp-fE, Asp-fF and Asp-fG; (A6) Component 1 includes the Asp-fC, Asp-fE, Asp-fF and Asp-fG mentioned above; (A7) Component 1 includes Asp-fC, Asp-fD, Asp-fF and Asp-fG.

5. The application according to claim 2, characterized in that, Component 1 includes Asp-fC, Asp-fE and Asp-fG.

6. The application according to any one of claims 1 to 2 and 5, characterized in that, Component 1 includes Asp-fA, Asp-fB, Asp-fC, Asp-fD, Asp-fE and Asp-fG.

7. The application according to claim 2, characterized in that, Component 1 includes Asp-fD, Asp-fE, and Asp-fF.

8. The application according to any one of claims 1 to 2 and 7, characterized in that, Component 1 is as shown in any one of (B1) to (B3): (B1) Component 1 includes Asp-fA, Asp-fB, Asp-fD, Asp-fE, Asp-fF and Asp-fG; (B2) Component 1 includes Asp-fA, Asp-fC, Asp-fD, Asp-fE and Asp-fF; (B3) Component 1 includes Asp-fB, Asp-fD, Asp-fE and Asp-fF.

9. The application according to any one of claims 1 to 3, characterized in that, The Aspergillus fumigatus allergen components include Asp-fA, Asp-fB, Asp-fC, Asp-fD, Asp-fE, Asp-fF, Asp-fG, Asp-f-22 and Asp-f-27 as shown.

10. The application according to any one of claims 1 to 3, 5 and 7, characterized in that, The specific IgE is detected using the following methods: ELISA, protein chip method, magnetic microparticle chemiluminescence method, flow cytometry, and chemiluminescence immunoassay.

11. The application according to any one of claims 1 to 3, 5 and 7, characterized in that, The samples tested by the testing product include blood samples.

12. The application according to any one of claims 1 to 3, 5 and 7, characterized in that, The detection reagent includes one or more of the Aspergillus fumigatus allergen components, blocking solution, washing solution, enzyme-catalyzed colorimetric reagent, and enzyme-catalyzed reaction termination reagent; the enzyme-catalyzed colorimetric reagent includes enzyme-labeled antibody and colorimetric substrate.

13. An ABPA testing product, characterized in that, The reagent includes one or more of the Aspergillus fumigatus allergen components and their specific IgE as defined in any one of claims 1 to 12.

14. The ABPA testing product according to claim 13, characterized in that, The detection reagent includes one or more of the Aspergillus fumigatus allergen components, blocking solution, washing solution, enzyme-catalyzed colorimetric reagent, and colorimetric termination reagent; the enzyme-catalyzed colorimetric reagent includes enzyme-labeled antibody and colorimetric substrate.

15. A type of Aspergillus fumigatus allergen, characterized in that, The Aspergillus fumigatus allergen is a composition; The composition comprises at least two of Asp-fA, Asp-fB, Asp-fC, Asp-fD, Asp-fE, Asp-fF, and Asp-fG as defined in any one of claims 1 to 12; or, The composition comprises component 1 and component 2 as defined in any one of claims 1 to 12.

Images