AU2021104383A4 - KIT FOR DETECTING DUST MITE COMPONENT-SPECIFIC IMMUNOGLOBULIN G (sIgG) - Google Patents
KIT FOR DETECTING DUST MITE COMPONENT-SPECIFIC IMMUNOGLOBULIN G (sIgG) Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
- G01N2333/43552—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from insects
- G01N2333/43582—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from insects from mites
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/24—Immunology or allergic disorders
Abstract
OF THE DISCLOSURE
The present disclosure relates to a kit for detecting dust mite component-specific
immunoglobulin G (sIgG), and belongs to the technical field of antibody detection kits. The kit
of the present disclosure includes: a biotin-polystreptavidin-biotin-dust mite antigen-coated
nitrocellulose (NC) membrane, a washing solution, an alkaline phosphatase (ALP)-labeled
secondary antibody solution for dust mite component-specific antibodies sIgG, and a substrate
solution. The kit of the present disclosure has reliable performance, high sensitivity, and wide
linear range, and can be used in combination with full-automatic instruments, which can
improve detection sensitivity and reliability and reduce cost.
17896845_1 (GHMatters) P116786.AU
Description
KIT FOR DETECTING DUST MITE COMPONENT-SPECIFIC IMMUNOGLOBULIN G (sIgG)
[01] The present disclosure relates to the technical field of allergen detection kits, and in particular to a kit for detecting dust mite component-specific immunoglobulin G (sIgG).
[02] Mites are ubiquitous creatures, which cannot be seen without a microscope due to their small size. In fact, mites can be found in various natural conditions or human living environments. At present, in allergology, the most noteworthy mites are mites in the human living environment that can induce human allergic reactions. With the modernization of human living environment, an incidence of allergic diseases related to mites has increased rapidly. These mites mainly include dust mites and storage mites. The dust mites mainly include Dermatophagoides farinae, Dermatophagoides pteronyssinus, Euroglyphus maynei, and Acarus siro. The storage mites mainly include Blomia tropicalis. The most common mites in living rooms worldwide are Dermatophagoidesfarinae and Dermatophagoidespteronyssinus. Dust mite allergy is one of the most common allergic diseases, which can be manifested as allergic rhinitis, allergic asthma, atopic dermatitis, allergic conjunctivitis, gastrointestinal dysfunction, and the like.
[03] IgE-mediated type I hypersensitivity diseases (i.e., allergic diseases) are quite common. IgE plays a key role in the occurrence of allergic diseases. Allergic diseases are characterized by a higher allergen-specific IgE (sIgE) concentration in the circulating blood of a patient than that under normal conditions, and the higher the sIgE concentration, the higher the probability of suffering from allergic diseases. In recent years, allergen-specific immunotherapy (SIT) has been widely used in the treatment of allergic rhinitis, asthma, and other allergic diseases caused by dust mites. SIT is the only etiological treatment method that can affect the natural course of allergic diseases (Jean, Bousquet, and, et al. Allergen immunotherapy: Therapeutic vaccines for allergic diseases A WHO position paper [J]. Journal of Allergy & Clinical Immunology, 1998). SIT refers to a treatment method described as follows: after an allergen causing an allergic reaction in a patient is determined, a high-purity standardized allergen vaccine is used to strengthen the patient's tolerance to the antigen (with a concentration and a dosage increasing progressively from a low concentration and a small dosage), such that the patient will not have the anaphylaxis or show alleviated symptoms when exposed to the allergen once again.
17896845_1 (GHMatters) P116786.AU
[041 IgG4 is a subtype of IgG, and sIgG/sIgG4 acts as a "blocking antibody" in the therapeutic process of SIT. sIgG/sIgG4 can hinder the synthesis of IgE, reduce the amount of IgE, compete with IgE for Fc receptors on the surface of MCs, basophils, etc., prevent the occurrence of IgE-mediated MC degranulation, and reduce the release of histamine, leukotrienes, and other inflammatory mediators, such that the airway inflammation in children with asthma, lung function, and asthma symptoms can be improved and the airway hyperresponsiveness (AHR) can be reduced. After a period of SIT treatment, a long-term therapeutic effect is available for the body even if the treatment is stopped. In vitro experiments also show that the antibody can reduce allergen-specific T cell responses by inhibiting the binding of allergen-IgE complexes to antigen-presenting cells (APCs). A successful SIT is usually accompanied by a significant increase of serum sIgG/sIgG4. Studies have shown that different populations show different responses to dust mite allergen components, and different allergens lead to different clinical effects when used for SIT (M Guimares Junqueir de Queir6s, Silva D A 0, Alves R, et al. Mite-specific immunotherapy using allergen and/or bacterial extracts in atopic patients in Brazil[J]. J Investig Allergol Clin Immunol, 2007, 18(2): 84-92.).
[05] In allergen diagnosis, monomeric allergens are used to identify specific molecules causing allergies, which can improve the accuracy of allergy diagnosis and desensitization treatment. In terms of identification and molecular diagnosis of allergens, the current research and industrialization levels in China show a gap from that of developed countries in Europe and America, which is manifested in depth and breadth. So far, there are very few allergens that can be used industrially and there is no kit for allergen diagnosis in China. At present, there are few allergen diagnosis products with incomplete items worldwide, which have not been approved for marketing in China. For example, the ImmunoCAP for dust mite component detection from Thermo Fisher can only detect 4 dust mite components: Der p 1, Der p 2, Der p 10, and Der p 23, and cannot detect antibodies specific for other dust mite components. Moreover, the ImmunoCA has a low throughput, and one CAP can only detect one allergen. These are not conducive to the development of individualized dust mite component SIT.
[06] Therefore, the detection of various dust mite component-specific antibodies sIgG/sIgG4 can provide a basis for accurate immunotherapy of patients and play an indicative role in subsequent dust mite desensitization treatment. However, there is currently no kit for efficiently detecting allergen components and dust mite component-specific antibodies sIgG.
[07] The present disclosure is intended to provide a kit for detecting dust mite
2 17896845_1 (GHMatters) P116786.AU component-specific antibodies sIgG. The kit of the present disclosure has reliable performance, high sensitivity, and wide linear range, and may be used in combination with full-automatic instruments, which can improve detection sensitivity and reliability and reduce cost. Moreover, the kit of the present disclosure may efficiently detect dust mite allergen components and dust mite component-specific antibodies sIgG (including sIgG4), with high throughput.
[08] The present disclosure provides a kit for detecting dust mite component-specific antibodies sIgG, including: a biotin-polystreptavidin-biotin-dust mite antigen-coated nitrocellulose (NC) membrane, a washing solution, an alkaline phosphatase (ALP)-labeled secondary antibody solution for dust mite component-specific antibodies sIgG, and a substrate solution,
[09] where the biotin-polystreptavidin-biotin-dust mite antigen-coated NC membrane is formed by coating an NC membrane sequentially with solidified biotin, polystreptavidin, and biotin-labeled dust mite antigens; the dust mite antigens include: dust mite protein Der f 1, dust mite protein Der f 2, dust mite protein Der p 1, dust mite protein Der p 2, dust mite protein Der p 5, dust mite protein Der p 7, dust mite protein Der p 10, dust mite protein Der p 11, dust mite protein Der p 20, dust mite protein Der p 21, dust mite protein Der p 23, dust mite protein Blo t , dust mite protein Blo t 10, dust mite protein Blo t 21, Dermatophagoidespteronyssinus, Dermatophagoides farinae, and Blomia tropicalis; the dust mite antigens are coated on different positions on the NC membrane; and
[10] the sIgG includes sIgG4.
[11] Preferably, the washing solution may be a Tris-HC buffer containing Tween-20.
[12] Preferably, the Tween-20 may have a mass percentage of 0.0004% to 0.02% in the washing solution, and the washing solution may have a pH of 7.2 to 7.6.
[13] Preferably, the secondary antibody solution for dust mite component-specific antibodies may have a mass concentration of 0.1 g/mL to 10 g/mL.
[14] Preferably, the substrate solution may be a BCIP/NBT substrate solution.
[15] The present disclosure provides a kit for detecting dust mite component-specific antibodies sIgG. In the present disclosure, a variety of major standardized dust mite allergen components are used to develop a dust mite component-specific antibody (sIgG, including sIgG4) detection kit suitable for actual conditions in China. The kit can quantitatively detect dust mite component-specific antibody (sIgG, including sIgG4) levels in human serum, which clinically can play an indicative role in dust mite desensitization treatment. In the present disclosure, the high-throughput protein microarray technology is used to coat an NC membrane with dust mite allergen proteins: Der f 1, Der f 2, Der p 1, Der p 2, Der p 5, Der p 7, Der p 10,
3 17896845_1 (GHMatters) P116786.AU
Der p 11, Der p 20, Der p 21, Der p 23, Blo t 5, Blo t 10, and Blo t 21, and 3 dust mite allergens: Dermatophagoidespteronyssinus, Dermatophagoidesfarinae, and Blomia tropicalis, such that 14 dust mite allergen proteins can be detected at one time.
[16] The kit of the present disclosure adopts the amplification structure of biotin-polystreptavidin-biotin-antigen, such that an antigen can fully react with an antibody to improve the detection sensitivity. In the present disclosure, single purified natural or recombinant dust mite allergen components are used to identify specific molecules causing allergies, which can improve the accuracy of allergy diagnosis and prognosis; and a specific allergic component can be detected for a patient, which is conducive to accurate determination of dust mite allergies and provides a basis for accurate immunotherapy on patients. Moreover, the present disclosure can quantitatively detect the levels of dust mite component-specific IgG (sIgG, including sIgG4) antibodies in human serum during desensitization treatment, which may clinically be used for in vitro auxiliary diagnosis of the body's immune responses to dust mite antigen exposure; and the present disclosure may also detect the specific components causing immune responses in the patient, which may provide a basis for accurate immunotherapy of patients and play an indicative role in subsequent dust mite desensitization treatment. The kit of the present disclosure may be used subsequently in combination with scanning analysis software to accurately identify protein sites and effectively determine sIgG (including sIgG4) concentrations, which can improve the accuracy.
[17] FIG. 1 shows detection results of the sIgG4 content provided by the present disclosure.
[18] The present disclosure provides a kit for detecting dust mite component-specific antibodies sIgG, including: a biotin-polystreptavidin-biotin-dust mite antigen-coated NC membrane, a washing solution, an ALP-labeled secondary antibody solution for dust mite component-specific antibodies sIgG, and a substrate solution,
[19] where the biotin-polystreptavidin-biotin-dust mite antigen-coated NC membrane is formed by coating an NC membrane sequentially with biotin, polystreptavidin, and biotin-labeled dust mite antigens; the dust mite antigens include: dust mite protein Der f 1, dust mite protein Der f 2, dust mite protein Der p 1, dust mite protein Der p 2, dust mite protein Der p 5, dust mite protein Der p 7, dust mite protein Der p 10, dust mite protein Der p 11, dust mite protein Der p 20, dust mite protein Der p 21, dust mite protein Der p 23, dust mite protein Blo t
4 17896845_1 (GHMatters) P116786.AU
, dust mite proteinBlot10,dustmiteprotein Blo t 21, Dermatophagoidespteronyssinus, Dermatophagoides farinae, and Blomia tropicalis; the dust mite antigens are coated on different positions on the NC membrane; and
[20] the sIgG includes sIgG4.
[21] In the present disclosure, there are no special limitations on a source of the dust mite antigens, and dust mite antigens conventionally synthesized with reference to conventional technical means may be used, such as dust mite antigens obtained by genetic engineering expression and purification or natural purification. In the present disclosure, with Der p 23 as an example, total RNA of Dermatophagoidespteronyssinus is used as a template and primers are designed and synthesized according to the sequence published by GenBank to amplify a Der p 23 coding gene by RT-PCR; a prokaryotic expression plasmid is constructed and transformed into Escherichia coli (E. coli), and IPTG is used to induce expression; and a target protein is collected and purified by column chromatography and then identified by SDS-PAGE and Western blot [Zhou Ying, Wu Meili, Zhu Hanting, Cui Yubao. Preparation of allergen Der p 23 recombinant protein and establishment of Der p 23-specific IgE chemiluminescence detection method [J]. Chinese Journal of Immunology, 2020, v.36 (20): 65-68.].
[22] In the present disclosure, there are no special limitations on the method of the biotin labeling, and the dust mite antigens can be labeled with biotin according to the biotin product specification. The biotin of the present disclosure may preferably be purchased from Sigma-Aldrich, with Item No.: B4501. In the present disclosure, the polystreptavidin may preferably be purchased from Yeasen BioTechnologies co., Ltd. (Shanghai), with Item No.: 35101ES03.
[23] The biotin-polystreptavidin-biotin-dust mite antigen-coated NC membrane of the present disclosure may preferably be a biotin-polystreptavidin-biotin-dust mite antigen-coated NC membrane strip, hereinafter referred to as detection plate. In the present disclosure, a preparation method of the biotin-polystreptavidin-biotin-dust mite antigen-coated NC membrane may preferably include the following steps: coating biotin on an NC membrane, and drying; coupling polystreptavidin to the biotin, and drying; and coupling biotin-labeled dust mite antigens to the polystreptavidin. In the present disclosure, a high-accuracy automatic spotting instrument may preferably be used to coat biotin on the NC membrane. In the present disclosure, the drying may preferably be conducted at 37°C. In the present disclosure, after the biotin-labeled dust mite antigens are coated, an amplification structure of biotin-polystreptavidin-biotin-antigen is obtained. In the present disclosure, each dust mite antigen may be coated at a concentration preferably of 1 g/mL to 1,000 g/mL and more
5 17896845_1 (GHMatters) P116786.AU preferably of 20 g/mL to 400 [g/mL, which is the optimal concentration obtained through experiments to ensure the accuracy of results.
[24] In the present disclosure, the washing solution may preferably be a Tris-HCl buffer containing Tween-20, which can reduce non-specific adsorption. In the present disclosure, the Tween-20 may have a mass percentage preferably of 0.0004% to 0.02% and more preferably of 0.002% to 0.01% in the washing solution (referring to concentrated washing solution), thus ensuring the accuracy of results; and the washing solution may have a pH preferably of 7.2 to 7.6 and more preferably of 7.4. The washing solution of the present disclosure may be a concentrated liquid, with a concentration factor of 25. In the present disclosure, a Tris-HCl buffer with a pH of 7.4 may preferably be used, which is added with 0.0004% to 0.02% of Tween-20 to obtain a washing solution, namely, a concentrated washing solution. The concentrated washing solution of the present disclosure may preferably be diluted with distilled water or deionized water in a volume ratio of 1:25 before use.
[25] In the present disclosure, a preparation method of the ALP-labeled secondary antibody solution for dust mite component-specific antibodies sIgG may preferably include:
[26] mixing ALP with a secondary antibody for dust mite component-specific antibodies sIgG to obtain the ALP-labeled secondary antibody solution for dust mite component-specific antibodies sIgG. In the present disclosure, the secondary antibody for dust mite component-specific antibodies sIgG may include an anti-human IgG antibody (including anti-human IgG4 antibody). In the present disclosure, the anti-human IgG antibody may preferably be purchased from American Abcam, with Item No.: ab109489. In the present disclosure, the anti-human IgG4 antibody may preferably be purchased from American Abcam, with Item No.: ab238320. In the present disclosure, the secondary antibody solution for dust mite component-specific antibodies sIgG may have a mass concentration preferably of 0.1 pg/mL to 10 g/mL and more preferably of 0.1 g/mL to 5 g/mL, thus ensuring the accuracy of results. In the present disclosure, the anti-human IgG antibody may preferably be a rabbit anti-human IgG antibody, namely, an antibody produced in a host rabbit that can specifically bind to human IgG. In the present disclosure, the anti-human IgG4 antibody may preferably be a rabbit anti-human IgG4 antibody, namely, an antibody produced in a host rabbit that can specifically bind to human IgG4.
[27] In the present disclosure, the substrate solution may preferably be a BCIP/NBT substrate solution, which is a mixed solution of nitroblue tetrazolium and -bromo-4-chloro-3-indolyl phosphate (NBT/BCIP). In the present disclosure, there are no special limitations on the source of the substrate solution, and a conventional
6 17896845_1 (GHMatters) P116786.AU commercially-available product well known to those skilled in the art may be adopted.
[28] The kit of the present disclosure may quantitatively detect the levels of 14 dust mite component-specific IgG (including IgG4) antibodies in human serum, which clinically may play an indicative role in dust mite desensitization treatment; and the kit may also detect a specific allergic component for a patient, which provides a basis for accurate immunotherapy on the patient. The kit of the present disclosure has reliable performance, high sensitivity, and wide linear range, and may be used in combination with full-automatic instruments (Hangzhou Zheda Dixun Biological Gene Engineering Co., Ltd., full-automatic western blotting instrument, model: DX-Blot 45, DX-Blot 45 II, and DX-AutoBlot 50), which may improve detection sensitivity and reliability and reduce cost. Specifically, there is no reagent for detecting various dust mite components in China, and the detection of dust mite components can only rely on imported reagents that are expensive. Compared with imported reagents (such as products of Thermo Fisher), the kit of present disclosure has a much lower price and involves a different method. In the present disclosure, the amount of antigen coated on the NC membrane is far lower than that of Thermo Fisher's products (about 1/1,000). For the products of Thermo Fisher, the detection of one item requires one CAP and each CAP requires 40 L of test serum, while the present disclosure only requires 300 L of test serum to detect 17 items at a time, with 17.6 pL of serum for each item on average.
[29] In the present disclosure, biotin, polystreptavidin, and biotin-coupled dust mite antigens are coated on an NC membrane. When serum with dust mite-specific antibodies sIgG (including IgG4) is added to the detection plate, the antibodies sIgG (including IgG4) bind to the antigens fixed on the membrane such that an immune complex of biotin-polystreptavidin-biotin-antigen-sIgG (including IgG4) is formed and fixed on the membrane, and the remaining unbound substances are removed by washing; then an ALP-anti-human sIgG (including IgG4) antibody complex is added to the detection plate to form an immune complex of biotin-polystreptavidin-biotin-antigen-sIgG-anti-human IgG antibody-ALP (including an immune complex of biotin-polystreptavidin-biotin-antigen-sIgG4-anti-human IgG4 antibody-ALP), and the remaining unbound substances are removed by washing; and then a substrate solution is added to conduct a chromogenic reaction with the enzyme, where a color intensity of a line/dot represents an sIgG (including IgG4) concentration in the serum.
[30] A method for high-throughput detection of dust mite antigens and single-component sIgG (including IgG4) using the kit of the present disclosure may preferably include the following steps:
7 17896845_1 (GHMatters) P116786.AU
[31] wetting the detection plate with a working washing solution; adding test serum, and incubating; washing, adding the ALP-labeled secondary antibody solution for dust mite component-specific antibodies sIgG, and incubating; washing, adding the substrate solution, and incubating; and stopping the reaction, drying, and scanning with a scanner.
[32] In the present disclosure, the detection plate is wet with a working washing solution. In the present disclosure, preferably, the detection plate is taken out and added with 100 L to 500 L of a working washing solution, and then placed on a mixer for thorough wetting, and then the working washing solution is poured out. The working washing solution of the present disclosure may preferably be a working washing solution obtained by diluting the aforementioned concentrated washing solution.
[33] In the present disclosure, after the detection plate is wet, test serum is added and incubated. In the present disclosure, 100 L to 500 L of serum may preferably be added to the detection plate and then incubated on a mixer at room temperature, and the incubation may be conducted preferably for 30 min to 60 min.
[34] In the present disclosure, after the serum is added and incubated, the plate is washed, and then the ALP-labeled secondary antibody solution for dust mite component-specific antibodies sIgG is added and incubated. In the present disclosure, preferably, the liquid in the detection plate may be poured out, and the detection plate may be rinsed with a working washing solution 3 to 5 times, with 10 s to 30 s for each time. In the present disclosure, preferably, 100 L to 500 L of a secondary antibody working solution may preferably be added to the detection plate and then incubated on a mixer at room temperature for 30 min to min.
[35] In the present disclosure, after the binding of the secondary antibody, the detection plate is washed, and then the substrate solution is added and incubated; the reaction is stopped, and the plate is dried; and the plate is scanned with a scanner. In the present disclosure, preferably, the liquid in the detection plate may be poured out, and the detection plate may be rinsed with a working washing solution 3 to 5 times, with 10 s to 30 s for each time. In the present disclosure, preferably, 100 L to 500 L of a substrate solution may preferably be added to the detection plate and then incubated on a mixer at room temperature for 10 min to min. The detection plate is rinsed with running water to stop the enzyme reaction. After the detection plate is dried, the membrane strip is scanned with a scanner, and results are read with analysis software. The scanning analysis software of the present disclosure may preferably be application software that can analyze and calculate a corresponding sIgG (including IgG4) concentration according to a color intensity at a protein site (Hangzhou Zheda Dixun Biological
8 17896845_1 (GHMatters) P116786.AU
Gene Engineering Co., Ltd., allergen semi-quantitative analysis software). The protein site refers to a site coated with a dust mite antigen on the membrane strip. The concentration refers to a content level of sIgG (including IgG4) in serum, with an international unit of IU/mL or U/mL.
[36] The kit for detecting dust mite component-specific antibodies sIgG according to the present disclosure will be further described in detail below with reference to specific examples. The technical solutions of the present disclosure include, but are not limited to, the following examples.
[37] Example 1
[38] The kit included the following components:
[39] detection plate: a biotin-polystreptavidin-biotin-dust mite antigen-coated NC membrane strip;
[40] concentrated washing solution (25 x): concentrated Tris buffer;
[41] IgG secondary antibody working solution: ALP-labeled anti-human IgG antibody solution; and
[42] substrate solution: BCIP/NBT.
[43] Preparation of the detection plate:
[44] A high-accuracy automatic spotting instrument was first used to coat biotin (Sigma-Aldrich, Item No.: B4501) on an NC membrane strip, and then the strip was dried at 37°C; then polystreptavidin (Yeasen BioTechnologies co., Ltd. (Shanghai), Item No.: 35101ES3) was coated, and then the strip was dried at 37°C; and then 14 biotin-labeled dust mite proteins (Der f 1, Der f 2, Der p 1, Der p 2, Der p 5, Der p 7, Der p 10, Der p 11, Der p 20, Der p 21, Der p 23, Blo t 5, Blo t 10, and Blo t 21) and 3 dust mite allergens (Dermatophagoidespteronyssinus, Dermatophagoidesfarinae, and Blomia tropicalis) were coated to form an amplification structure of biotin-polystreptavidin-biotin-antigen. The dust mite antigens were coated at a concentration of 1 g/mL to 1,000 [g/mL.
[45] Preparation of the washing solution:
[46] A Tris-HCl buffer with a pH of 7.4 was added with 0.008% of a surfactant (Tween-20) to obtain a concentrated washing solution. The concentrated washing solution was diluted with distilled water or deionized water in 1:25 to obtain a working washing solution.
[47] Preparation of the IgG4 secondary antibody working solution:
[48] ALP was mixed with an anti-human IgG4 antibody (American Abcam, Item No.: ab238320) to obtain an ALP-labeled anti-human IgG4 antibody solution with a concentration of 1 g/mL, where the anti-human IgG4 antibody was a rabbit anti-human IgG4 antibody.
9 17896845_1 (GHMatters) P116786.AU
[49] Preparation of the substrate solution:
[50] Nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate were mixed to obtain the substrate solution (NBT/BCIP).
[51] Serum was collected from patients with allergic asthma and rhinitis that underwent dust mite SIT, and then detected by the kit of the present disclosure according to the following detection steps:
[52] 1. The detection plate was taken out and added with 300 L of the working washing solution, and then placed on a mixer for thorough wetting, and then the working washing solution was poured out.
[53] 2. 300 L of serum was added to the detection plate and then incubated for 30 min on a mixer at room temperature.
[54] 3. The liquid in the detection plate was poured out, and the detection plate was rinsed with a working washing solution 5 times, with 15 s for each time.
[55] 4. 300 L of the IgG4 secondary antibody working solution was added to the detection plate and then incubated for 30 min on a mixer at room temperature.
[56] 5. The liquid in the detection plate was poured out, and the detection plate was rinsed with a working washing solution 5 times, with 15 s for each time.
[57] 6. 300 L of the substrate solution was added to the detection plate and then incubated for 15 min on a mixer at room temperature.
[58] 7. The detection plate was rinsed with running water to stop the enzyme reaction.
[59] 8. The detection plate was dried, the membrane strip was scanned with a scanner, and results were read with analysis software.
[60] The results were shown in FIG. 1 (FIG. 1 shows sIgG4 content detection results), and it could be seen that, at the maintenance phase, the levels of Der p 1, Der p 2, Der f 1, Der f 2, Der p 5, Der p 10, and Der p 23-specific sIgG4 antibodies were higher than that before the immunotherapy. It showed that the level of dust mite sIgG4 continued to rise during the immunotherapy. Therefore, IgG4 is a useful index for evaluating the clinical responses of immunotherapy.
[61] Comparative Example 1
[62] A detection plate without the amplification structure of biotin-polystreptavidin-biotin-antigen was prepared as follows: 14 dust mite proteins and 3 dust mite antigens were directly coated on an NC membrane strip by an automatic spotting instrument. Other working solutions were prepared by the same methods as in Example 1. The detection plate prepared by this method and the detection plate of the present disclosure were
10 17896845_1 (GHMatters) P116786.AU used to test the serum of dust mite allergic patients, and results were shown in Table 4:
[63] Table 4 Serum sIgG4 detection results of dust mite allergic patients Product without the amplification structure Product of the present disclosure Item name Dust mite allergic patient Dust mite allergic patient Dust mite allergic patient Dust mite allergic patient (unit: U/mL) 1 2 1 2
Dermatophagoides 653.2 237.2 763.5 366.1 pteronyssinus
Dermatophagoidesfarinae 152 284.8 168.1 405.4
Blomia tropicalis 13.5 36.4 15.4 39.1
Derp 1 84.6 233.7 85.9 378.9
Der f 1 10.3 203.9 14.3 224.3
Der p 2 0.01 226.4 1.23 365.6
Der f 2 13.3 382.6 21.3 501.3
Der p 5 4.61 146.4 8.6 149.8
Der p 7 226.1 80.9 321.1 90.2
Der p 10 11 87.8 15.6 89.1
Der p 11 32.1 21.5 43.6 26.4
Der p 20 16.4 0.13 21.7 1.03
Der p 21 398.4 39.9 509.7 56.7
Der p 23 22 265.5 35.1 377.8
Blo t 5 0.13 15.6 0.24 16.7
Blo t 10 1.56 2.33 2.01 2.36
Blo t 21 14.6 2.11 15.5 2.99
[64] According to the ROC curve of clinical trials, 250 U/mL is determined as a threshold value to determine IgG4 negative and positive results, where a detection result > 250 U/mL indicated positive, and a detection result < 250 U/mL indicated negative. It could be seen from Table 4 that the detection results of the present disclosure with the amplification structure were improved; the membrane strip without the amplification structure showed a false-negative Der p 7 detection result for the dust mite allergic patient 1; and the membrane strip without the amplification structure showed false-negative Dermatophagoidespteronyssinus, Der p 1, and Der p 2 detection results for the dust mite allergic patient 2. Therefore, the amplification structure may enable the full reaction of an antigen with an antibody, thereby improving the
11 17896845_1 (GHMatters) P116786.AU detection sensitivity and reducing the false-negative results.
[65] The above descriptions are merely preferred implementations of the present disclosure. It should be noted that a person of ordinary skill in the art may further make several improvements and modifications without departing from the principle of the present disclosure, but such improvements and modifications should be deemed as falling within the protection scope of the present disclosure.
[661 It is to be understood that, if any prior art publication is referred to herein, such
reference does not constitute an admission that the publication forms a part of the common
general knowledge in the art, in Australia or any other country.
[671 In the claims which follow and in the preceding description of the invention, except
where the context requires otherwise due to express language or necessary implication, the
word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive
sense, i.e. to specify the presence of the stated features but not to preclude the presence or
addition of further features in various embodiments of the invention.
12 17896845_1 (GHMatters) P116786.AU
Claims (5)
1. A kit for detecting dust mite component-specific immunoglobulin G (sIgG), comprising: a biotin-polystreptavidin-biotin-dust mite antigen-coated nitrocellulose (NC) membrane, a washing solution, an alkaline phosphatase (ALP)-labeled secondary antibody solution for dust mite component-specific antibodies sIgG, and a substrate solution; wherein the biotin-polystreptavidin-biotin-dust mite antigen-coated NC membrane is formed by coating an NC membrane sequentially with solidified biotin, polystreptavidin, and biotin-labeled dust mite antigens; the dust mite antigens comprise: dust mite protein Der f 1, dust mite protein Der f 2, dust mite protein Der p 1, dust mite protein Der p 2, dust mite protein Der p 5, dust mite protein Der p 7, dust mite protein Der p 10, dust mite protein Der p 11, dust mite protein Der p 20, dust mite protein Der p 21, dust mite protein Der p 23, dust mite protein Blo t 5, dust mite proteinBlot10,dustmiteprotein Blo t 21, Dermatophagoidespteronyssinus, Dermatophagoides farinae, and Blomia tropicalis; the dust mite antigens are coated on different positions on the NC membrane; and the sIgG comprises sIgG4.
2. The kit according to claim 1, wherein the washing solution is a Tris-HCl buffer containing Tween-20.
3. The kit according to claim 2, wherein the Tween-20 has a mass percentage of 0.0004% to 0.02% in the washing solution, and the washing solution has a pH of 7.2 to 7.6.
4. The kit according to claim 1, wherein the secondary antibody solution for dust mite component-specific antibodies has a mass concentration of 0.1 g/mL to 10 g/mL.
5. The kit according to claim 1, wherein the substrate solution is a BCIP/NBT substrate solution.
13 17896845_1 (GHMatters) P116786.AU
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