Flow fluorescence detection kit and detection method for allergen-specific IgE (Immunoglobulin E) antibody
Through the combination of flow fluorescence detection kit and flow cytometry, the problem of only single index detection in the prior art is solved, and high-sensitivity quantitative detection and automated detection of multiple allergens are realized, reducing costs and sample requirements.
Patent Information
- Application Number
- CN202510664741.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-22
- Publication Date
- 2025-07-25
AI Technical Summary
The existing allergen-specific IgE antibody detection methods have problems such as only single index detection, long time, high cost, low sensitivity or large sample size, and cannot achieve simultaneous quantitative detection of multiple allergens.
The allergen-specific IgE antibody flow fluorescence detection kit, including antigen-microsphere complex working fluid and PE-secondary antibody complex working fluid, was used to label APC fluorescent encoding magnetic microspheres and PE fluorescent protein-labeled antigens, and the detection was carried out by flow cytometry to achieve quantitative detection of multiple allergens.
It realizes quantitative detection with high sensitivity, can detect multiple allergic indicators simultaneously, has high degree of automation, low cost, and small sample size. It is suitable for 5-20μL samples, and has the advantages of chemiluminescence kits and reduces costs.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of biological detection, in particular to a flow fluorescence assay kit for detecting allergen-specific IgE antibodies, and also relates to a detection method for detecting allergen-specific IgE antibodies using the kit. Background Art
[0002] Allergy is a phenomenon in which the body's susceptibility to certain drugs or external stimuli is abnormally increased. Under normal circumstances, the body produces antibodies to protect itself from diseases, but in allergic individuals, the body mistakes normal harmless substances for harmful ones and produces antibodies, and such substances become "allergens". Allergic reactions often refer to immediate allergic reactions (type I) mediated by IgE. The type I allergic reaction mediated by IgE antibodies is that allergens stimulate antigen-specific B cells to differentiate into plasma cells and produce IgE antibodies. The IgE antibodies bind to the Fc receptors on the surface of basophils and mast cells through the Fc segment. When the same allergen enters the body again, the Fab segments of the IgE antibodies on the surface of basophils and mast cells bind to the allergen determinants, forming IgE receptor polymers, which leads to the reaction of basophils and mast cells degranulating and releasing bioactive substances such as histamine, and then produces typical type I allergic reaction symptoms such as measles, urticaria, dermatitis, arthritis, allergic rhinitis, conjunctivitis, asthma, and anaphylactic shock.
[0003] In the past few decades, the prevalence of IgE-related allergic diseases has increased significantly globally, with the total incidence rate in the population as high as 10% - 30%. The World Health Organization has listed allergic diseases as one of the six major diseases that need to be studied and prevented in key areas in the 21st century. With the improvement of research and attention to allergic diseases, people have a better understanding of the causes, pathogenesis, and natural development process of allergic diseases, and are still constantly seeking effective strategies for preventing and treating allergic diseases. For the detection of type I allergic reactions, the commonly used methods at present are mainly divided into two categories: in vivo and in vitro diagnosis. The former includes skin prick tests, and patients will suffer great pain from the needles. The latter includes chemiluminescence, ELISA, immunoblotting and other methodological kits, but they all have more or less disadvantages. For example, although chemiluminescence has high sensitivity, it can only detect a single index at a time, with a long time consumption and high cost; although ELISA has the characteristics of relatively high sensitivity and low cost, it can also only detect single indicators one by one, and requires a large amount of manual operation; the immunoblotting kit has the characteristics of relatively low cost, detecting multiple indicators and automation, but its sensitivity is lower than that of chemiluminescence, and it cannot perform quantitative detection, and the required sample volume is also very large, usually more than 200 μL. Summary of the Invention
[0004] In view of the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide a flow fluorescence method detection kit and detection method for allergen-specific IgE antibodies to achieve quantitative detection of multiple allergen-specific antibodies in one test.
[0005] A flow fluorescence method detection kit for allergen-specific IgE antibodies, which includes an antigen-microsphere complex working solution, a PE-secondary antibody complex working solution, and a washing solution; wherein, the working solution contains APC fluorescence-encoded magnetic microspheres labeled with different allergen antigens, and different allergy indexes are detected according to different labeled allergen antigens; the PE-secondary antibody complex working solution contains anti-human IgE antibodies labeled with PE fluorescent protein.
[0006] As a further improvement of the above solution, the washing solution adopts a PBS buffer solution containing 1% Tween 20 and 0.05% sodium azide, and the concentration of the washing solution is 0.01M, and the pH is 7.4.
[0007] As a further improvement of the above solution, the allergen antigens include natural antigens and recombinant antigens.
[0008] As a further improvement of the above solution, the APC fluorescence-encoded magnetic microspheres are carboxyl microspheres, or the fluorescence-encoded magnetic microspheres are streptavidin microspheres.
[0009] As a further improvement of the above solution, when the fluorescence-encoded magnetic microspheres adopt carboxyl microspheres, the preparation method of the antigen-microsphere complex is as follows: The carboxyl microspheres, EDC and NHS are mixed in a ratio of 1:50:100 and activated for 20 minutes to become NHS ester. The activation buffer solution adopts a MES buffer solution with a concentration of 0.05M and a pH of 5.0. The activated microspheres and allergen antigens are mixed in a ratio of 1:20 and coupled for 60 minutes to obtain the antigen-microsphere complex. In the present invention, the activated microspheres are coupled with the amino groups of the allergen antigens to form amide bonds, so that the antigens are firmly connected to the microspheres to obtain the antigen-microsphere complex.
[0010] As a further improvement of the above solution, when the fluorescently encoded magnetic microspheres are streptavidin microspheres, the preparation method of the antigen-microsphere complex is as follows: First, couple the allergen antigen and biotin for 30 minutes. The coupling buffer is PBS buffer with a concentration of 0.01M and a pH of 7.4. The molar ratio of the allergen antigen to biotin is 1:10 - 100, and the biotin is NHS-LC biotin or NHS-PEG biotin. Then, pass the biotinylated allergen antigen through a desalting column or dialysis to remove the excess biotin. Next, mix the biotinylated allergen antigen and streptavidin microspheres at a ratio of 50:1. The mixing buffer is PBS buffer with a concentration of 0.01M and a pH of 7.4. After mixing for 30 minutes, block with free LC carboxybiotin for 10 minutes to obtain the antigen-microsphere complex.
[0011] As a further improvement of the above solution, take different antigen-microsphere complexes according to an equimolar microsphere ratio, dilute with a preservation solution to obtain the working solution of the antigen-microsphere complex, and store it at 4°C. The preservation solution is PBS buffer with a concentration of 0.01M and a pH of 7.4, and the preservation solution contains 10% sucrose, 5% trehalose, and 0.05% sodium azide. In the kit of the present invention, allergen antigens with different indicators are labeled with flow cytometry fluorescently encoded microspheres with different numbers, and then a multi-index microsphere working solution, that is, a working solution of the antigen-microsphere complex, is prepared, so as to achieve the quantitative detection of multiple allergen-specific antibodies in one test.
[0012] As a further improvement of the above solution, the anti-human IgE antibody is a mouse anti-human IgE antibody, or the anti-human IgE antibody is a goat anti-human IgE antibody.
[0013] As a further improvement of the above solution, the allergy indicators include house dust mites, dust mites, cat hair, dog epithelium, mugwort, and egg white. In the kit of the present invention, different allergy indicators are detected according to different labeled allergen antigens. For example, when the house dust mite antigen is labeled, it is used to detect whether there is an allergy to house dust mites; when the dust mite antigen is labeled, it is used to detect whether there is an allergy to dust mites; when the cat hair antigen is labeled, it is used to detect whether there is an allergy to cat hair; when the dog epithelium antigen is labeled, it is used to detect whether there is an allergy to dog epithelium; when the mugwort antigen is labeled, it is used to detect whether there is an allergy to mugwort; when the egg white antigen is labeled, it is used to detect whether there is an allergy to egg white.
[0014] As a further improvement of the above solution, the PE-secondary antibody complex is diluted 1:10000 with a preservation solution to obtain the working solution of the PE-secondary antibody complex, and store it at 4°C. The preservation solution is PBS buffer with a concentration of 0.01M and a pH of 7.4, and the preservation solution contains 1% starch, 10% trehalose, 0.1% EDTA, and 0.05% sodium azide.
[0015] As a further improvement of the above solution, the preparation method of the PE-secondary antibody complex is specifically operated as follows:
[0016] (1) The anti-human IgE antibody is activated by a mercapto-reagent, and the excess activator is removed by a desalting column for standby;
[0017] (2) The PE fluorescent protein is activated by SMCC, and the excess activator is removed by a desalting column for standby;
[0018] (3) The activated anti-human IgE antibody and the activated PE fluorescent protein are subjected to a conjugation reaction for 60 min to obtain the PE-secondary antibody complex.
[0019] As a further improvement of the above solution, the anti-human IgE antibody and the mercapto-reagent are mixed at a ratio of 1:50, and the activation buffer is a PBS buffer with a concentration of 0.02 M and a pH of 7.2.
[0020] As a further improvement of the above solution, the mercapto-reagent is DTT, TCEP or 2-IT. In the present invention, the mercapto-reagent functions to activate and derivatize the anti-human IgE antibody to generate mercapto groups or break disulfide bonds.
[0021] As a further improvement of the above solution, the PE fluorescent protein and SMCC are mixed at a ratio of 1:20 - 30, and the activation buffer is a PBS buffer with a concentration of 0.02 M and a pH of 8.0.
[0022] A method for detecting allergen-specific IgE antibody uses the described kit for detection. The detection method includes the following steps:
[0023] S1 Take 10 μL of serum or plasma sample and 2 μL of the antigen-microsphere complex working solution, drop them into the micro-wells of a 96-well plate, incubate at 37 °C for 30 min on a micro-well plate mixer, then place the 96-well plate on a magnetic plate for magnetic separation for 1 min, aspirate the supernatant, and continue to wash three times with the washing solution, aspirating the supernatant each time;
[0024] S2 Add 20 μL of the PE-secondary antibody complex working solution, place the 96-well plate on a micro-well plate mixer, incubate at 37 °C for 20 min, then place the 96-well plate on a magnetic plate for magnetic separation for 1 min, aspirate the supernatant, and continue to wash three times with the washing solution, aspirating the supernatant each time;
[0025] S3 Add 100 μL of PBS buffer to resuspend the microspheres, and then place the 96-well plate on a flow cytometer for detection. In the present invention, different fluorescence-encoded microspheres are identified by the flow cytometer to determine the detected allergen index, and then the fluorescence signal of the PE-secondary antibody complex is used to quantify the corresponding allergen index, thereby realizing the quantitative detection of multiple indicators in parallel.
[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: It not only has the advantages of a chemiluminescence kit (high-sensitivity quantitative detection), but also has the characteristics of being able to detect multiple allergy indicators simultaneously, with a high degree of automation. In addition, precisely because of the ability to detect multiple indicators simultaneously, the cost is reduced, and the required sample volume is extremely low, being 5 - 20 μL. The flow-through fluorescence method kit of the present invention has excellent prospects for popularization and application in allergen detection. Detailed implementation manners
[0027] In order to make the objectives, technical solutions and advantages of the present invention clearer, the following will further elaborate on the present invention in combination with embodiments. The additional aspects and advantages of the present invention will be partially given in the following description, partially become apparent from the following description, or be understood through the practice of the present invention. It should be understood that the following description is only used to explain the present invention and is not used to limit the present invention.
[0028] As used herein, the terms "comprising", "including", "containing" or any other variation thereof are intended to cover non-exclusive inclusion. For example, a composition, step, method, article or device containing the listed elements is not necessarily limited to those elements, but may include other elements not explicitly listed or elements inherent to such composition, step, method, article or device.
[0029] When an equivalent, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper preferred values and lower preferred values, it should be understood that all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value are specifically disclosed, regardless of whether the range is separately disclosed. For example, when the range "1 to 5" is disclosed, the described range should be interpreted to include ranges "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described herein, unless otherwise specified, the range is intended to include its end values and all integers and fractions within the range.
[0030] The following will elaborate on the specific embodiments of the present invention in detail.
[0031] Example 1
[0032] The allergen antigen, fluorescently encoded microspheres, and anti-human IgE antibody used in this example were all commercially obtained. This example provides a flow cytometry fluorescence assay kit for allergen-specific IgE antibodies, which includes an antigen-microsphere complex working solution, a PE-secondary antibody complex working solution, and a washing solution. Among them, the working solution contains APC-fluorescently encoded magnetic microspheres labeled with different allergen antigens, and different allergy indicators are detected according to the different allergen antigens labeled. The PE-secondary antibody complex working solution contains an anti-human IgE antibody labeled with PE fluorescent protein. The washing solution uses a PBS buffer containing 1% Tween 20 and 0.05% sodium azide, and the concentration of the washing solution is 0.01 M and the pH is 7.4.
[0033] The allergen antigen includes natural antigen and recombinant antigen. The APC-fluorescently encoded magnetic microspheres are carboxyl microspheres, or the fluorescently encoded magnetic microspheres are streptavidin microspheres.
[0034] When the fluorescently encoded magnetic microspheres are carboxyl microspheres, the preparation method of the antigen-microsphere complex is as follows: The carboxyl microspheres, EDC, and NHS are mixed in a ratio of 1:50:100 and activated for 20 min to become NHS esters. The activation buffer uses a MES buffer with a concentration of 0.05 M and a pH of 5.0. The activated microspheres and the allergen antigen are mixed in a ratio of 1:20 and coupled for 60 min to obtain the antigen-microsphere complex.
[0035] When the fluorescently encoded magnetic microspheres are streptavidin microspheres, the preparation method of the antigen-microsphere complex is as follows: First, the allergen antigen and biotin are coupled for 30 min. The coupling buffer uses a PBS buffer with a concentration of 0.01 M and a pH of 7.4. The molar ratio of the allergen antigen to biotin is 1:10 - 100, and the biotin is NHS-LC biotin or NHS-PEG biotin. Then, the allergen antigen is passed through a desalting column or dialyzed to remove the excess biotin. Then, the biotinylated allergen antigen and the streptavidin microspheres are mixed in a ratio of 50:1. The mixing buffer uses a PBS buffer with a concentration of 0.01 M and a pH of 7.4. After mixing for 30 min, it is blocked with free LC carboxyl biotin for 10 min to obtain the antigen-microsphere complex.
[0036] Take different antigen-microsphere complexes and mix them according to the equimolar microsphere ratio, and dilute them with the preservation solution to obtain the antigen-microsphere complex working solution, and store it at 4°C. The preservation solution uses a PBS buffer with a concentration of 0.01 M and a pH of 7.4. The preservation solution contains 10% sucrose, 5% trehalose, and 0.05% sodium azide. In this example, the antigen-microsphere complexes with different allergy indicators are mixed according to the equimolar ratio of the number of microspheres to make the number of different encoded microspheres the same.
[0037] The anti-human IgE antibody is a murine anti-human IgE antibody, or the anti-human IgE antibody is a caprine anti-human IgE antibody. The allergy indicators include house dust mites, dust mites, cat hair, dog epithelium, mugwort, and egg white. The kit of this embodiment can detect different allergy indicators according to different labeled allergen antigens. For example, by labeling house dust mite antigen, it can detect whether there is an allergy to house dust mites; by labeling dust mite antigen, it can detect whether there is an allergy to dust mites; by labeling cat hair antigen, it can detect whether there is an allergy to cat hair; by labeling dog epithelium antigen, it can detect whether there is an allergy to dog epithelium; by labeling mugwort antigen, it can detect whether there is an allergy to mugwort; by labeling egg white antigen, it can detect whether there is an allergy to egg white.
[0038] The PE-secondary antibody complex is diluted 1:10000 with the preservation solution to obtain the working solution of the PE-secondary antibody complex, and it is stored at 4°C. The preservation solution is a PBS buffer solution with a concentration of 0.01M and a pH of 7.4, and the preservation solution contains 1% starch, 10% trehalose, 0.1% EDTA, and 0.05% sodium azide.
[0039] The preparation method of the PE-secondary antibody complex is specifically operated as follows:
[0040] (1) The anti-human IgE antibody is activated by a thiolation reagent, and the excess activator is removed by a desalting column and reserved. The anti-human IgE antibody and the thiolation reagent are mixed at a ratio of 1:50, and the activation buffer solution is a PBS buffer solution with a concentration of 0.02M and a pH of 7.2; the thiolation reagent is DTT, TCEP, or 2-IT.
[0041] (2) The PE fluorescent protein is activated by SMCC, and the excess activator is removed by a desalting column and reserved. The PE fluorescent protein and SMCC are mixed at a ratio of 1:20 - 30, and the activation buffer solution is a PBS buffer solution with a concentration of 0.02M and a pH of 8.0.
[0042] (3) Take the activated anti-human IgE antibody and the activated PE fluorescent protein for a coupling reaction for 60 minutes to obtain the PE-secondary antibody complex.
[0043] Example 2
[0044] In this example, taking the natural antigens of house dust mites, dust mites, dog epithelium, mugwort, and egg white labeled streptavidin fluorescent encoded microspheres as an example, an allergen-specific IgE antibody flow cytometry fluorescence detection kit is prepared.
[0045] (1) Biotinylation of natural antigen: Prepare a 10 mg / mL biotin solution with DMSO, and dilute the antigen to 1 mg / mL with PBS buffer at a concentration of 0.01 M and pH 7.4; Take 100 μL (0.1 mg) of the antigen solution, add the biotin type and its dosage calculated in Table 1, react at room temperature (25 °C) for 30 min, then pass the reaction solution through a desalting column, and the eluent is PBS buffer at a concentration of 0.01 M and pH 7.4. Collect the product to obtain biotinylated antigen.
[0046] Table 1: Biotin types and dosages used for different antigens
[0047] Antigen Biotin Antigen: Biotin Amount of antigen Amount of biotin (μL) Dermatophagoides pteronyssinus NHS-LC Biotin 1：20 0.1 mg 1.82 Dermatophagoides farinae NHS-LC Biotin 1：50 0.1 mg 4.55 Dog epithelium NHS-PEG Biotin 1：20 0.1 mg 2.35 Artemisia argyi NHS-PEG Biotin 1:50 0.1 mg 5.10 Egg white NHS-PEG Biotin 1:100 0.1 mg 12.36
[0048] (2) Preparation of antigen-microsphere complex working solution: Dilute the biotinylated antigen 50 - 1000 times with PBS buffer at a concentration of 0.01 M and pH 7.4. Take 200 μL of the diluted biotinylated antigen solution and mix it with 2 μL of streptavidin fluorescently encoded microsphere stock solution, react at room temperature for 30 min, then add 2 μL of 10 mg / mL LC carboxyl biotin and block for 10 min, perform magnetic separation, aspirate the supernatant, wash 3 times with the washing solution, and finally resuspend with 50 μL of antigen-microsphere complex preservation solution to obtain the antigen-microsphere complex working solution for a single index. Then take 10 μL each of the antigen-microsphere complex working solutions of house dust mite, dust mite, dog epithelium, mugwort, and egg white prepared separately and mix them to obtain the antigen-microsphere complex working solution for the combined index. The preservation solution is PBS buffer at 0.01 M pH 7.4, containing 10% sucrose, 5% trehalose, and 0.05% sodium azide.
[0049] (3) Preparation of PE-mouse anti-human IgE complex working solution
[0050] 3.1 Thiolation of mouse anti-human IgE: Take 0.1 mg of mouse anti-human IgE, dilute it to 1 mg / mL with PBS buffer at a concentration of 0.02 M and pH 7.2, then add 2 μL of 2-IT reagent at a concentration of 2 mg / mL, react at 37 °C for 1.5 h, and then pass through a desalting column to remove the excess activator, and collect the product.
[0051] 3.2 Activation of PE fluorescent protein with SMCC: Prepare a 5 mg / ml solution of PE fluorescent protein with PBS buffer at a concentration of 0.02 M and pH 8.0. Take 66 μL (0.33 mg) in a brown EP tube, then add 1.38 μL of cross-linking agent SMCC solution prepared with 10 mg / mL DMSO, react at 37 °C for 1 h, and then pass through a desalting column to remove the excess activator, and collect the product.
[0052] 3.3 Activation of mouse anti-human IgE and coupling with PE protein: Directly mix the activated mouse anti-human IgE and PE protein, react at 37 °C for 1 h, then add 5 μL of maleimide solution prepared with DMSO at a concentration of 10 mg / mL, continue to react at 37 °C for 20 min, and then add 10 μL of 1% ethanolamine solution prepared with PBS buffer at a concentration of 0.01 M and pH 7.4, react at 37 °C for 20 min, collect the product, dilute the product 10,000 times with the preservation solution, and store at 4 °C to obtain the working solution of the PE-mouse anti-human IgE complex. The preservation solution is PBS buffer at 0.01 M pH 7.4, containing 1% starch, 10% trehalose, 0.1% EDTA, and 0.05% sodium azide.
[0053] Example 3
[0054] On the basis of Example 2, this example adds streptavidin fluorescently encoded microspheres and carboxyl fluorescently encoded microspheres labeled with cat hair recombinant antigen to prepare a flow fluorescence assay kit for detecting allergen-specific IgE antibodies.
[0055] (1) The main allergenic proteins in cat hair include Feld1, Feld2, Feld3, and Fel d4, and recombinant antigens have been prepared for these 4 antigens. Therefore, the same numbered microspheres can be labeled with the recombinant antigens and then combined for detecting cat hair allergy indicators. Among them, Fel d1, Fel d3, and Fel d4 are labeled with streptavidin fluorescent microspheres. The types and dosages of biotin used for different antigens are shown in Table 2. Fel d2 is labeled with carboxyl fluorescent microspheres. The method of labeling with streptavidin fluorescently encoded microspheres is the same as that in Example.
[0056] Table 2: Types and dosages of biotin used for Feld1, Feld3, and Feld4 antigens
[0057]
[0058] (2) Preparation of the cat hair recombinant antigen-microsphere complex working solution
[0059] 2.1 Dilute the cat hair Fel d1, Fel d3, and Fel d4 antigens 500 times with PBS buffer at a concentration of 0.01 M and pH 7.4. Take 200 μL of the diluted biotin antigen solution and mix it with 2 μL of the streptavidin fluorescently encoded microsphere stock solution, react at room temperature for 30 min, then add 2 μL of 10 mg / mL LC carboxyl biotin to block for 10 min, perform magnetic separation, aspirate the supernatant, wash 3 times with the washing solution, and finally resuspend with 50 μL of the antigen-microsphere complex preservation solution to obtain the antigen-microsphere complex working solution for a single index.
[0060] 2.2 The Feld2 was labeled with carboxyl fluorescein - coded microspheres (the carboxyl fluorescein microspheres have the same number as the streptavidin fluorescein - coded microspheres, only with different groups): Take 50 μL of the carboxyl fluorescein microsphere stock solution, add 450 μL of MES buffer solution with a concentration of 0.05 M and pH 5.0, dilute and mix well. Then add 10 μL of NHS aqueous solution with a concentration of 5 mg / mL, mix well, and then add 5 μL of EDC hydrochloride aqueous solution with a concentration of 5 mg / mL, and immediately mix well. React at room temperature (25 °C) for 20 min, perform magnetic separation, aspirate the supernatant, wash 3 times with the washing solution. Add 100 μL (0.1 mg) of the recombinant antigen Fel d2 solution prepared with PBS buffer solution with a concentration of 0.01 M and pH 8.0, mix well, react at room temperature (25 °C) for 1 h, perform magnetic separation, aspirate the supernatant, wash 3 times with the washing solution, and finally resuspend with 50 μL of the antigen - microsphere complex storage solution to obtain the Fel d2 - microsphere complex working solution.
[0061] 2.3 Mix the Fel d1, Fel d2, Fel d3, and Fel d4 microsphere complex working solutions in equal volumes to obtain the cat hair recombinant antigen - microsphere complex working solution.
[0062] (3) Preparation of the PE - goat anti - human IgE complex working solution
[0063] 3.1 Mercapto - modification of goat anti - human IgE: Take 0.1 mg of goat anti - human IgE, dilute it to 1 mg / mL with PBS buffer solution with a concentration of 0.02 M and pH 7.2. Then add 2 μL of 2 - IT reagent with a concentration of 2 mg / mL, react at 37 °C for 1.5 h, and then pass through a desalting column to remove the excess activator, and collect the product.
[0064] 3.2 Activation of PE fluorescent protein with SMCC: Prepare the PE fluorescent protein into a 5 mg / ml solution with PBS buffer solution with a concentration of 0.02 M and pH 8.0. Take 66 μL (0.33 mg) in a brown EP tube, then add 1.38 μL of the cross - linker SMCC solution prepared with DMSO with a concentration of 10 mg / mL, react at 37 °C for 1 h, and then pass through a desalting column to remove the excess activator, and collect the product.
[0065] 3.3 Activation of Goat Anti-Human IgE and Conjugation with PE Protein: Directly mix the activated goat anti-human IgE and PE protein, react at 37 °C for 1 h, then add 5 μL of maleimide solution prepared with DMSO at a concentration of 10 mg / mL, continue to react at 37 °C for 20 min, then add 10 μL of 1% ethanolamine solution prepared with PBS buffer at a concentration of 0.01 M and pH 7.4, react at 37 °C for 20 min, collect the product, dilute the product 10,000 times with the preservation solution, and store at 4 °C to obtain the working solution of the PE-goat anti-human IgE complex. The preservation solution is PBS buffer at 0.01 M pH 7.4, containing 1% starch, 10% trehalose, 0.1% EDTA, and 0.05% sodium azide.
[0066] Use the kit of this example to detect the allergens of house dust mites, dust mites, dog epithelium, mugwort, egg white, and cat hair. The detection results are shown in Tables 3 - 9.
[0067] Table 3 Detection Results of House Dust Mite Allergens
[0068]
[0069] Table 4 Detection Results of Dust Mite Allergens
[0070]
[0071] Table 5 Detection Results of Dog Epithelium Allergens
[0072]
[0073] Table 6 Detection Results of Mugwort Allergens
[0074]
[0075] Table 7 Detection Results of Egg White Allergens
[0076]
[0077]
[0078] Table 8 Detection Results of Cat Hair Allergens
[0079]
[0080] In summary, the kit of this embodiment not only has the advantages of a chemiluminescence kit (high-sensitivity quantitative detection), but also has the characteristics of being able to detect multiple allergy indexes simultaneously, with a high degree of automation. In addition, precisely because of the ability to detect multiple indexes simultaneously, the cost is reduced, and the required sample volume is also extremely low, which is 5 - 20 μL. The flow cytometry fluorescence method kit of the present invention has excellent prospects for popularization and application in allergen detection.
[0081] Example 4
[0082] This embodiment provides a method for detecting allergen-specific IgE antibodies, which uses the kit of Example 1, Example 2 or Example 3 for detection. The detection method includes the following steps:
[0083] S1: Take 10 μL of serum or plasma sample and 2 μL of antigen-microsphere complex working solution, drop them into the micro-wells of a 96-well plate, incubate at 37 °C for 30 min on a microplate mixer, then place the 96-well plate on a magnetic plate for magnetic separation for 1 min, aspirate the supernatant, and continue to wash three times with the washing solution, aspirating the supernatant each time.
[0084] S2: Add 20 μL of PE-secondary antibody complex working solution, place the 96-well plate on a microplate mixer, incubate at 37 °C for 20 min, then place the 96-well plate on a magnetic plate for magnetic separation for 1 min, aspirate the supernatant, and continue to wash three times with the washing solution, aspirating the supernatant each time;
[0085] S3: Add 100 μL of PBS buffer to resuspend the microspheres, and then place the 96-well plate on a flow cytometer for detection.
[0086] The above embodiments are only the preferred embodiments of the present invention. Any simple modification, modification and alternative change made to the above embodiments based on the technical essence of the present invention all fall within the scope of the technical solution of the present invention.
Claims
1. Flow fluorescence immunoassay kit for detecting allergen-specific IgE antibodies, characterized in that, It includes an antigen-microsphere complex working solution, a PE-secondary antibody complex working solution, and a washing solution; wherein, the working solution contains APC fluorescence-coded magnetic microspheres labeled with different allergen antigens, and different allergy indexes are detected according to different labeled allergen antigens; the PE-secondary antibody complex working solution contains an anti-human IgE antibody labeled with PE fluorescent protein.
2. The allergen-specific IgE antibody flow fluorescence detection kit according to claim 1, wherein The washing solution uses a PBS buffer solution containing 1% Tween 20 and 0.05% sodium azide, and the concentration of the washing solution is 0.01M, and the pH is 7.
4.
3. The allergen-specific IgE antibody flow fluorescence detection kit according to claim 1, wherein The allergen antigens include natural antigens and recombinant antigens; The APC fluorescence-coded magnetic microspheres are carboxyl microspheres, or the fluorescence-coded magnetic microspheres are streptavidin microspheres.
4. The allergen-specific IgE antibody flow fluorescence detection kit according to claim 3, characterized in that, When the fluorescence-coded magnetic microspheres use carboxyl microspheres, the preparation method of the antigen-microsphere complex is as follows: The carboxyl microspheres, EDC, and NHS are mixed in a ratio of 1:50:100 and activated for 20 min to become NHS ester. The activation buffer solution uses a MES buffer solution with a concentration of 0.05M and a pH of 5.
0. The activated microspheres and the allergen antigen are mixed in a ratio of 1:20 and coupled for 60 min to obtain the antigen-microsphere complex.
5. The allergen-specific IgE antibody flow fluorescence detection kit according to claim 3, characterized in that, When the fluorescence-coded magnetic microspheres use streptavidin microspheres, the preparation method of the antigen-microsphere complex is as follows: First, the allergen antigen and biotin are coupled for 30 min. The coupling buffer solution uses a PBS buffer solution with a concentration of 0.01M and a pH of 7.
4. The molar ratio of the allergen antigen to biotin is 1:10 - 100, and the biotin is NHS-LC biotin or NHS-PEG biotin. Then, the allergen antigen is passed through a desalting column or dialyzed to remove the excess biotin. Then, the biotinylated allergen antigen and the streptavidin microspheres are mixed in a ratio of 50:
1. The mixing buffer solution uses a PBS buffer solution with a concentration of 0.01M and a pH of 7.
4. After mixing for 30 min, it is blocked with free LC carboxyl biotin for 10 min to obtain the antigen-microsphere complex.
6. The allergen-specific IgE antibody flow fluorescence detection kit according to claim 4 or 5, characterized in that Take different antigen-microsphere complexes and mix them according to an equimolar microsphere ratio, and dilute them with a preservation solution to obtain the antigen-microsphere complex working solution, and store it at 4°C. The preservation solution uses a PBS buffer solution with a concentration of 0.01M and a pH of 7.
4. The preservation solution contains 10% sucrose, 5% trehalose, and 0.05% sodium azide.
7. The allergen-specific IgE antibody flow fluorescence detection kit according to claim 1, wherein The anti-human IgE antibody is a mouse anti-human IgE antibody, or the anti-human IgE antibody is a goat anti-human IgE antibody; The allergy indexes include house dust mites, dust mites, cat hair, dog epithelium, mugwort, and egg white.
8. The allergen-specific IgE antibody flow fluorescence detection kit according to claim 1, characterized in that, The PE-secondary antibody complex is diluted 1:10000 with a preservation solution to obtain the PE-secondary antibody complex working solution, and store it at 4°C. The preservation solution uses a PBS buffer solution with a concentration of 0.01M and a pH of 7.
4. The preservation solution contains 1% starch, 10% trehalose, 0.1% EDTA, and 0.05% sodium azide; The preparation method of the PE-secondary antibody complex is as follows: (1) The anti-human IgE antibody is activated by a mercapto group reagent, and the excess activator is removed by a desalting column for standby; (2) The PE fluorescent protein was activated by SMCC, and the excess activator was removed using a desalting column for standby. (3) The activated anti-human IgE antibody and the activated PE fluorescent protein were subjected to a conjugation reaction for 60 min to obtain a PE-secondary antibody complex.
9. The allergen-specific IgE antibody flow fluorescence detection kit according to claim 8, wherein The anti-human IgE antibody and the mercapto reagent were mixed at a ratio of 1:50, and the activation buffer was a PBS buffer with a concentration of 0.02 M and a pH of 7.
2. The mercapto reagent used was DTT, TCEP or 2-IT. The PE fluorescent protein and SMCC were mixed at a ratio of 1:20 - 30, and the activation buffer was a PBS buffer with a concentration of 0.02 M and a pH of 8.
0.
10. A method for detecting allergen-specific IgE antibodies, which uses the kit described in any one of claims 1 to 9 for detection, characterized in that, The detection method includes the following steps: S1: Take 10 μL of serum or plasma sample and 2 μL of the antigen-microsphere complex working solution, drop them into the micro-wells of a 96-well plate, incubate at 37 °C for 30 min on a microplate mixer, then place the 96-well plate on a magnetic plate for magnetic separation for 1 min, aspirate the supernatant, and continue to wash three times with the washing solution, aspirating the supernatant each time. S2: Add 20 μL of the PE-secondary antibody complex working solution, place the 96-well plate on a microplate mixer, incubate at 37 °C for 20 min, then place the 96-well plate on a magnetic plate for magnetic separation for 1 min, aspirate the supernatant, and continue to wash three times with the washing solution, aspirating the supernatant each time. S3: Add 100 μL of PBS buffer to resuspend the microspheres, and then place the 96-well plate on a flow cytometer for detection.